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Transcript of Div III - Final
Microbiology and Immunology Relevant to Dairy Safety and Human Health
A Critical Analysis of the Raw Milk Debate
Kevin Phillips BayDivision III, Spring 2011
Faculty Committee: Jason Tor, Associate Professor of Microbiology; Lynn Miller, Professor of Biology; and Chris Jarvis, Associate Professor of Cell Biology
A dissertation presented to the School of Natural ScienceHampshire College, Amherst, Massachusetts
In partial fulfillment of the requirements for the degree Bachelor of Arts
Table of Contents
Acknowledgements.....................................................................................................................1
Part I: Introduction to the Raw Milk Debate...........................................................................2
The Raw Milk Debate: What's at stake, what we know, and what we don't....................2
Political, Economic, and Emotional Issues Confound the Debate on Raw Milk.............2
Is There a Scientific Basis for Claims that Raw Milk Supports Health?.........................5
"Germ Theory" Dominates the Medical Paradigm of the 20th Century..........................10
Commensal Pathogens Highlight the Importance of Environment and Immunity..........12
Part II: The Enterococcus–A Probiotic Pathogen?.................................................................16
Characteristics and Identification of Enterococci...........................................................16
Habitats of Enterococci...................................................................................................17
Functionality of Enterococci in Dairy Fermentation......................................................19
Virulence Factors in Enterococci....................................................................................20
Regulation of Genes Encoding Virulence Factors..........................................................21
Diversity of Enterococcus spp. and Evolution of Virulence...........................................22
Gelatinase........................................................................................................................23
Capsule............................................................................................................................25
Biofilms...........................................................................................................................27
Aggregation Substance...................................................................................................29
Cytolysin.........................................................................................................................32
Horizontal Gene Transfer................................................................................................34
HGT, Virulence, and Antibiotic Resistance....................................................................36
Part III: Framing the Real Issues Behind the Raw Milk Debate..........................................40
What Makes an Enterococcus Pathogenic?....................................................................40
Dairy Foods and Issues of Hygiene................................................................................42
Farmstead Dairy and the Ecological Integration of Modern Communities....................48
Sources Cited (Parts I-III)........................................................................................................50
Part IV: Assessment of Gelatinase Activity in Enterococci Isolated from Local Milk.......58
Sources Cited (Part IV)............................................................................................................65
Appendix...................................................................................................................................66
Acknowledgements
I would like to offer thanks to my living family, particularly my father Keith and his wife Linda,
whose support of my pursuits has always kept me afloat. Also, to my deceased family, in particular my
mother Kathy, whose living memory has in so many ways inspired me to pursue an understanding of
what it means to be healthy.
Thanks to my friends, mod mates, and everyone in the Monday night potluck community. Thanks
to Claire Wiessbluth for helping me survive at Hampshire and find my way in the world. Thanks to
Maggie Grinnell for your loving support and perspective that has been invaluable in this past year.
Thanks to Dylan (Spring) for the light you have brought to my life through your music and friendship.
Many thanks to Luke William Gay, your persistent friendship and ability to challenge my perspectives
has been essential.
There are a number of NS faculty who are greatly deserving of thanks. Jason Tor, Lynn Miller,
and Chris Jarvis for your support, guidance, and overwhelming faith in my ability to direct my own
learning experience, even when I myself had doubts. Larry Winship for supporting my experience of
independently directed learning at Hampshire from the very beginning. Rayane Moreira for giving focus
and momentum to my fascination with the chemistry of life, and for cultivating a challenging academic
environment.
Thanks specifically to Jason for fostering the environment in which I could develop a strong
understanding of the relationship between food, bacteria, and health, and for gathering the cheese-lovers
of Hampshire together to form a community in which I was able to teach and be taught, to feed and be
fed, and to share in a feeling of togetherness with one common denominator: cultured milk.
Thanks to bare feet and dirt. Thanks to all the folks associated with FLPCI for reassuring me that
my dream of becoming an award winning cheesemaker is in fact a good idea. Thanks to Kate Clabby for
consistently engaging me in thought-provoking conversations about dairy. Thanks also to microbes
everywhere for continuing to do things that boggle my mind and giving me faith that what I can't
understand will always be central to my health and the health of the earth.
Last but certainly not least, thanks to Leslie Cox and the Dutch Belted cows at Hampshire
College Farm Center (especially Cookie) who aside from their zen-like stare, offered me an unbelievable
abundance of milk to marvel at and craft into creamy delights that could be shared with friends and
family as far away as Wisconsin. Milk is truly a sacred gift.
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Part I: Introduction to the Raw Milk Debate
The Raw Milk Debate: What's at stake, what we know, and what we don't
Modern dairy production and processing has changed significantly over the past 100 years
following technological advances that have made possible large-scale industrial agriculture and
centralized food production and distribution systems (Schmid, 2009, 203-206). In the past, it was
commonplace for minimally processed milk to be bottled on the farm and sold directly to the consumer.
Now, due to biased legislative regulations and a trend towards large-scale agriculture, small family
dairies have become increasingly rare. Farmers often have no choice but to sell their milk to large scale
processing and distribution facilities, and low milk prices have put increasing economic strain on dairy
farmers. Small-scale community and family farmers themselves express the concern that these pressures
put them in danger of being eliminated altogether (Ostrom and Buttel, 1999).
The demand for fresh minimally processed milk, however, has not gone away, and a slew of small-
scale, often unregulated farms using direct-to-consumer marketing have emerged to meet this demand,
including the demand for unpasteurized (raw) dairy products such as milk, cream, butter, and yogurt.
Pasteurization is simply a heat treatment process intended to eliminate the majority of the natural
microbiota present in milk, lowering the risk of illness outbreaks associated with milk-borne pathogens
(Gumpert, 2009, 17). The potential benefits and risks associated with consuming raw dairy products is a
hotly debated topic and an issue of increasing significance due to its involvement in a number of other
significant social, political, and health issues. These issues include disparities in power and resources;
the fundamental rights of farmers, consumers, and corporate entities; and economic, ecological, and
individual health. As a result of these complicating factors, much of the debate has been based on highly
biased interpretations of a relatively small number of controversial scientific studies. Confusion
surrounding the "true" benefits and risks associated with raw milk are amplified by the fact that the
diverse disciplines of nutrition, microbiology, biochemistry, and immunology have only begun to
unravel the mysteries of milk.
Political, Economic, and Emotional Issues Confound the Debate on Raw Milk
State and local government laws vary in regard to raw milk, but in recent years the FDA has taken
a strong stance against its production, sale, and consumption, arguing that it is inherently unsafe and
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provides no benefits over pasteurized milk (Gumpert, 2009, 103-104). Many state and local agricultural
regulators throughout the US have followed suit, and even in places where legislation allows for sale of
raw milk, fear based propaganda and strict regulations discourage the growth of the raw milk market. A
number of farms that produce raw dairy products for sale have been subject to stringent searches, sting
operations, and behaviors bordering on harassment by state and federal authorities despite the fact that in
most cases there is little to no evidence that the products produced on these farms pose a greater risk to
public health than any other food. The increased frequency of such interventions has prompted many
raw milk advocates to question the motivations of agencies such as the FDA cracking down on raw dairy
producers nationwide. Like any food, raw milk has the potential to carry food-borne pathogens,
especially if good manufacturing practices designed to decrease this risk are ignored. However, a critical
analysis of the various arguments for and against raw milk reveals that political, economic, and
emotional complications are really at the heart of what could be considered a decidedly biased and
irrational debate.
Federal guidelines for milk production are based on the Pasteurized Milk Ordinance (PMO), which
was updated last in 2007, and sets the national standards for production, processing, packaging and sale
of Grade "A" dairy products (FDA, 2007). These guidelines are an important part of food safety in the
highly automated and centralized food production system of the United States. Without them, it is likely
that negligence or ignorance regarding hygienic food production would lead to increased rates of acute
to severe cases of food-borne illness, but critical examination of this ordinance reveals that it does not
take account of consumer demands for minimally processed foods, and that its language and
interpretation is heavily influenced by the complicating factors involved in the raw milk debate.
First and foremost, these harm reduction guidelines apply only to milk intended for pasteurization,
and the document actually defines a "dairy farm" as a place with one or more lactating animal where
milk will be provided, sold, or offered for sale to a milk plant, receiving station or transfer station. That
is to say, unless you sell your milk to a processor, you don't even technically own a dairy farm according
to the PMO. The lengthy definition of "milk products" includes a wide variety of processed dairy foods,
but makes no mention of minimally processed alternatives such as non-homogenized, non-standardized,
low-heat vat pasteurized milk; nor (for obvious reasons) raw milk.
Other terms defined by the PMO bring up different issues. For example, the definition of "person...
include[s] any individual, milk plant operator, partnership, corporation, company, firm, trustee,
association or institution". This lumping together of individuals and corporate entities is indicative of the
PMO's attempt to set in place all-encompassing regulations, despite the fact that many of these
regulations are not ideal for most alternative models of dairy production. Taken in conjunction with their
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requirement that all dairy products be funneled through processing plants, it is clear that these
regulations are biased towards large-scale, centralized dairy distribution systems.
Finally, issue may be taken with the PMO's use of the term of "sanitization", which is defined as:
...the application of any effective method or substance to properly cleaned surfaces for the destruction of pathogens, and other microorganisms, as far as is practicable. Such treatment shall not adversely affect the equipment, the milk and/or milk product, or the health of consumers, and shall be acceptable to the Regulatory Agency.
This definition could be considered highly controversial from both a political and scientific standpoint.
First of all, it is important to look at the way the definition is concluded, that methods of sanitization
must be "acceptable to the Regulatory Agency". This essentially strips interpretive power from any
authority other than the FDA. A more in depth analysis of the effects of processing on milk included
later in this paper will show that the language preceding this statement is highly non-specific and the
scientific evidence supporting its conventional interpretation is inconclusive, or even contradictory.
Federal regulations disregard direct-to-consumer agricultural models as well as procedural
guidelines for hygienic milking practices on farms providing raw milk not intended for pasteurization,
and so state lawmakers as well as smaller localized political bodies and individual activists have stepped
in to fill these gaps (Schmid, 2009, 411-424). Alternative agricultural models such as cow-shares, in
which farmers and consumers organize to co-operatively distribute food within their own community,
allow conscientious individuals to opt out of regulated food systems. The observation that these markets
are then out of reach of the centralized dairy industry has led raw milk advocates to suggest that industry
profits are likely a significant motivator in the federal stance against raw milk, which is backed by
legislation that has been heavily influenced by industry lobbyists.
In spite of political considerations, though, the way that raw milk enflames the passions of parties
on both sides of the debate is indicative of the emotional issues that are at stake. For example, the
emergence of agricultural models that circumvent the authority of regulatory bodies may be personally
offensive to individuals employed by organizations such as the FDA and similar state-run organizations.
On the other hand, many consumers see interventions by regulatory bodies as an infringement of their
basic rights to consume the foods of their choice or to have access to foods they feel are healthy in what
some consider an increasingly over-processed and nutrient depleted food system.
In response to these concerns regarding consumer rights and increasing support for raw milk,
bureaucratic authorities have made statements that may be perceived as crass and personally offensive
by consumers, such as the FDA's John Sheehan, who coined the statement "drinking raw milk or eating
raw milk products is like playing russian roulette with your health" (Gumpert, 2009, 116). Such
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commentary has only exacerbated the turmoil surrounding this highly emotional issue. In an example
more deeply concerning to some consumers, a lengthy legal document prepared by FDA lawyers as part
of a suit filed by the Farm-to-Consumer Legal Defense Fund challenging the FDA ban on interstate
shipment of raw milk includes the statements "there is no absolute right to consume or feed children any
particular kind of food" and "there is no generalized right to bodily and physical health" (Sebelius and
Hamburg, 2010). The document is intended to refute claims that the ban infringes on fundamental
consumer rights, and these statements appear as headings a. and b. under section IV.–B.–4., entitled
"FDA's Regulations Do Not Infringe Upon Substantive Due Process Rights." The statements are then
followed by a detailed legal analysis defending their validity. Many consumers feel that the statements
go too far by restricting the free choices of responsible adults in the name of public health, but these
statements can actually be legally validated by the language of current federal regulations (Falkenstein,
2010).
Only time will tell whether increasing public awareness will result in legal reforms regarding the
rights of communities to produce and distribute foods outside the context of the centralized food
industry and its associated regulatory authorities. In the meantime, some community government bodies
have taken a more localized approach. Since March of 2011, several towns in Maine have voted on
ordinances that would make it unlawful for state or federal regulations to interfere with the rights of
their citizens to produce, process, sell, purchase, and consume local foods of their choice, and exempting
local food producers from licensure and inspection under the condition that their products are sold
directly to consumers for home consumption (Local Food and Community Self-Governance Ordinance,
2011). While the town of Brooksville, ME did not pass the ordinance, citing concerns that "it is
unenforceable" and "opens the town to potential liability issues and legal costs", three other towns
(Sedgewick, Penobscot, and Blue Hill) passed the ordinance unanimously (Gumpert, 2011[2 and 3]), a
powerful message to federal and state regulators that faithful consumers may not sit quietly on the
sidelines as interventions on private farm to consumer transactions threaten their trusted local food
sources.
Is There a Scientific Basis for Claims That Raw Milk Supports Health?
At the heart of the raw milk debate is the claim that it is fundamentally different from pasteurized
milk in its ability to support natural immunity and general health. Raw milk advocates expound the
miraculous health benefits of their favorite drink, citing anecdotal reports that it can help treat a variety
of serious medical issues including (but not limited to) arthritis, eczema, asthma, cancer, and diabetes
(Gumpert, 2009, 84-90). The same anecdotal reports often claim that pasteurized milk delivers no such
5
benefits, and on the contrary often causes relapse into poor health, digestive issues, and lactose
intolerance. Finally, they also claim that raw milk has natural biological defenses that confer protection
against the growth of pathogenic organisms. But if these claims are to be considered true, the question
must be asked, what is it about raw milk that makes it so different from pasteurized milk?
In order to understand the fundamental differences between raw and pasteurized milk, it is
necessary to understand the various components of milk. Unfortunately, the composition of milk
remains a larger scientific mystery that one might imagine. Although milk may seem like a homogenous
white substance, the complexity and diversity of biological structures and the various ways they can
interact with the human body has only begun to be unravelled. Most people, if they consider milk on a
molecular level at all, would probably identify the main components as protein, fat, lactose, vitamins,
and minerals. In reality, there are many other bioactive components of milk (see figure 1) including
immunoglobulins, peptides, antimicrobial factors, hormones, growth factors, and approximately 70
indigenous enzymes (Silanikove, 2008).
Milk also contains living cells, including bacteria from contamination during milking, and active
immune cells. These living cells contribute to milk their own biological structures and secretions,
including potent immune modulating substances such as cytokines (Untalan et al., 2009). In addition to
membranes associated with bacterial and somatic cells, milk contains phospholipid membranes complete
with diverse bioactive membrane proteins. These include the milk-fat globule membrane (MFGM)
which contains proteins involved in immune functioning (Cavaletto et al., 2008) due in part to the
exocytosis of milk fat globules from lacteal cells, and the milk serum lipoprotein membrane vesicles, the
origin and function of which remains a mystery (Silanikove, 2008).
6
Figure 1 - Image and text adapted from Silanikove (2008). Defines the 5 phases of milk and models their relationships and relative sizes.
All these diverse components are arranged in a highly organized fashion that can be grossly
divided into 5 distinct phases. These are displayed and defined clearly by Silanikove (2008) as shown in
Figure 1. While all the gross structural components have likely been identified, many of them have only
begun to be characterized. The functional contribution of most of these bioactive factors to milk and the
influence these may have on the health of dairy consumers is a highly complex topic that science has
barely begun to explore. It is beyond the scope of this paper to provide an in depth analysis on the
subject, but it remains a topic of great interest and a promising area of future research in dairy science.
Clearly, there is important information missing from the analysis of the biological activities of
milk. For this reason, it must be kept in mind that any scientific argument for or against the consumption
of milk (raw, pasteurized or otherwise processed) by a particular individual is vastly speculative, and
may well be a decision that is better informed by that individual's intuitive sense of which consumptive
patterns make them feel healthy. That being said, a hypothesis that raw milk may be beneficial to the
health of some individuals can be formed based on anecdotal reports, and some recent scientific findings
may support this hypothesis.
Research into the beneficial or deleterious effects of
pasteurization is heavily swayed by the agendas of its financiers,
and authorities such as the FDA vehemently deny the suggestion
that pasteurization may have an adverse effect on the health
benefits of milk, as exhibited by the chart in figure 2 taken from
the FDA's website. In an attempt to explain and validate the
deleterious effects of pasteurization, raw milk advocates most
frequently cite decreased bioavailability of nutrients,
denaturation of enzymes claimed to be vital to health, and the
elimination of naturally occurring beneficial bacteria that confer
a probiotic and immunoregulatory effect. While it is clear that
pasteurization does have some effects on these properties, these
effects are argued to be negligible (Cifelli et al., 2010).
It is true that there is not a cohesive and conclusive body of literature to support the claims of
raw milk advocates, perhaps in large part due to the fact that funding for such research is difficult to
obtain. The FDA's assertion that these claims have been conclusively refuted, however, is not accurate.
Research on this topic will probably continue to be muddled by the complicating factors of the raw milk
debate. On the other hand, research on human breast milk gives a very different and interesting
perspective on biological activities of milk, and although these results cannot be directly translated to
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Figure 2 - A side bar from the FDA's website addressing common misconceptions about pasteurization and raw milk (FDA, 2011).
consumption of cow or goat milk, they do present provocative conclusions that could inform potential
areas of interest in researching the influences of milk production and processing methods on consumer
health.
Breast milk is ubiquitously considered to be the ideal food for human infants. Some mothers,
however, may be unable to breast feed and for this purpose, "donor milk" is collected and provided at
milk banks, where it is often pasteurized to
avoid transmission of infectious disease by
microbes (Untalan et al., 2009). Viruses such
as human immunodeficiency virus (HIV) are
of particular concern (Tully et al., 2001).
While donor milk is regarded as a better
alternative than formula, the potential
deleterious effects of pasteurization on the
immune components of human milk has been
a subject of significant research. B- and T-cell
populations are entirely abolished by
pasteurization, and immunoglobulins and enzymes associated with the bacteriostatic properties of raw
milk are significantly affected as well. As a result of these effects, Tulley et al. (2001) note that
"microorganisms that could contaminate the milk after pasteurization will grow faster than they can in
raw milk". That raw milk has natural defenses against microbial growth is an argument often cited by
raw milk advocates. Although many advocates take the argument too far, asserting that raw milk actually
kills off pathogenic populations and that pathogens cannot grow in raw milk, it is clear that the natural
immune components of raw milk do have a
bacteriostatic effect.
More recently, the effect of
pasteurization on cytokines in donor milk has
been explored, and these results have shown
that pasteurization does have significant effects
on many of these potent immunomodulating
molecules (Ewaschuk et al., 2011; Untalan et
al., 2009). Ewaschuk et al. (2011) effectively
show the differential effect of pasteurization
on a wide variety of cytokines and other
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Figure 3 - Relative concentrations of various cytokines detected in raw (black) and pasteurized (white) breast milk (Unger, 2011).
Figure 4 - Relative concentrations of Heparin-binding-EGF-like growth factor (HB-EGF), Hepatocyte growth factor (HGF) and Granulocyte colony-stimulating factor (G-CSF) in raw (black) and pasteurized (white) breast milk (Unger, 2011).
molecular immune components. Their results are currently available in an advanced online publication,
but not through research databases. Some of their results, however, are ironically available through the
FDA website as part of a slide show recently prepared in support of donor milk by Sharon Unger, MD (a
Toronto physician and member of the Ewaschuk et al. research team). These results, displayed in figures
3 and 4, show an altered cytokine profile and significant reduction in hepatocyte growth factor (HGF).
Other immune components in human milk have been described as well, although the effect of
pasteurization on these has yet to be studied. Soluble forms of toll-like receptors (TLRs; pattern
recognition receptors that play a major role in identification of pathogens by the innate immune system)
as well as TLR co-receptor CD14 are found in high levels in human milk and modulate neonatal
microbial recognition (LeBouder et al., 2006). Additionally, nanovesicles such as milk serum lipoprotein
membrane vesicles (the "lost continent" of Silanikove, (2008)) have been isolated from human milk and
are suspected to have immunological effects, in part due to the fact that they can be taken up by
macrophages and contain functional RNA that can be delivered to other cells (Lässer et al., 2011).
Research on immune components in non-human milk does exist (Mehra et al., 2006; Trujillo et
al., 2007), but it is extremely limited. However, it is still very clear that in light of the results of studies
on human breast milk, the argument that pasteurization does not have a significant effect on the
biological properties of raw milk must be called into question. Additionally, the recently proposed
"hygiene hypothesis" suggests that excessive cleanliness may contribute to higher rates of health issues
such as allergy and asthma in developed geographic regions, and one review article notes that several
studies have shown unpasteurized milk to have "a protective effect... on the development of asthma, hay
fever, allergic sensitization, and atopic dermatitis" (von Mutius and Vercelli, 2010). The same authors
also suggest that homogenization may play a role in reducing milk's "asthma- and allergy-protective
effects" due to the disruption of the MFGM, which causes adsorption of allergenic proteins onto the
newly formed milk-fat globules as a result of their increased surface area. In fact, increasing rates of
asthma and allergic disease correlate more closely to the advent of homogenization than pasteurization
(Gumpert, 2009, 111).
Other authors have also suggested that practices aiming to eliminate the natural microbiota of
foods may decrease the ability of the GI microbiota to adapt to novel foods due to decreased frequency
of horizontal gene transfer (HGT) between natural food microbiota. These indigenous food microbes
have evolved the metabolic capacity to make best use of the resources in their environment, and GI
microbiota have the potential to acquire genes encoding these metabolic pathways (Sonnenburg, 2010).
This type of HGT was demonstrated by Hehemann et al. (2010), who observed that Bacteroides plebius
populations in Japanese individuals (who typically consume large quantities of seaweed in the form of
9
sushi) had acquired genes from the marine bacterium Zobellia galactanivorans that allowed them to
digest porphyran, a polysaccharide found in red algae. These genes were not found in the microbiota of
individuals from the US. These data lend some credence to the claim that raw milk may attenuate
symptoms of lactose intolerance, while pasteurized milk provides no such benefits.
There is scientific evidence warranting further research on the potential health benefits of
consuming minimally processed milk. Unfortunately, some statements made in defense of the safety of
raw milk are detrimental to the credibility of raw milk advocates (Blum, 2010). Extremist assertions
such as "the bacteria theory's a total myth" and "everything God designed is good for you" are easily
ridiculed by authors who perceive raw milk as nothing more than the latest health fad or foodie fetish.
These statements may also be perceived as personally offensive to victims of life threatening food-borne
illness and their families who have undergone highly traumatic experiences in which microbial
pathogens do play an important role. On both sides of the debate, oversimplification and
misunderstanding of the microbiological and physiological underpinnings of disease, and far-reaching
political, social, and emotional issues surrounding raw milk cultivate an environment that is not
conducive to rational assessment of the facts.
"Germ Theory" Dominates the Medical Paradigm of the 20th Century
Since the work of Louis Pasteur (for which the process of pasteurization is named) in the 19th
century that led to technologies with miraculous efficacy in controlling or nearly eradicating many
rampant infectious diseases of his time, including debilitating milk-borne illnesses such as tuberculosis
and diptheria, much of the world has taken for granted the idea that disease is caused by microbial
pathogens (Gumpert, 2009, 42-46). Germ theory, however, was not the only theory of pathogenesis at
the time, and some of Pasteur's contemporaries and co-researchers such as Claude Bernard and Elie
Metchnikoff proposed significant theories suggesting that the internal environment of the body as well
as the body's cellular defense systems are also important factors in susceptibility to illness.
The groundbreaking work of French-American microbiologist René Dubos, which has not
received due respect from many modern microbiologists, was largely dedicated to exploring the impact
of environmental conditions on microbial growth and pathogenesis. His first wife died of tuberculosis in
1942, which ignited in him a passionate desire to understand the reasons why the illness had developed
in her at that time (Encyclopedia.com, 2003). His investigation revealed that she had been infected with
tuberculosis as a child. Despite overcoming the illness in her youth, a latent infection remained. Dubos
was personally convinced that distress surrounding World War II and her concern for her family in
France had weakened her and allowed the latent infection to again take hold. Much of his subsequent
10
work was based on the theory that diverse environmental conditions such as poor nutrition, pollution,
psychological stress, and spiritual deprivation are all important etiological factors in human disease.
These ideas eventually brought him out of the lab to work with economically depressed communities
and to speak out on important social issues such as economic disparity and environmental injustice. A
1966 study by Dubos that was republished in 2005 elegantly displays the significance of quality of life
in resistance to infectious disease (Dubos et al., 2005).
Dubos was also highly influenced by the work of Pasteur, and notes that in spite of the focus of
Pasteur's experimental research, his conception of pathogenesis was actually much more sophisticated,
and took into account the significance of environment (Dubos, 1974). Although the pressing issues of
infectious disease and vaccination came to monopolize his research efforts, Dubos suggests that
Pasteur's early discoveries could just as easily have led him into a diversity of scientific fields including
microbial physiology or the effect of environmental conditions on disease resistance. Dubos provides
compelling evidence from Pasteur's early work to support these arguments, such as the simple
observation that the gut is lined with a multitude of microbial agents that only cause illness when the
body is weakened, or that most individuals do not develop post-surgical infections despite occasional
neglect of aseptic methods. Furthermore, in Pasteur's work on flacherie (an infectious disease in
silkworms), he observes the influence of environmental conditions on resistance to the disease. Dubos
states, "Pasteur considered that excessive heat and humidity, inadequate aeration, stormy weather, and
poor food were inimical to the general physiological health of the insects. As he put it, the proliferation
of microorganisms in the intestinal tract of worms suffering from flacherie was more an effect than a
cause of the disease."
These ideas reveal a much more complex picture of pathogenesis involving several variable
factors. In contrast, germ theory involves only one variable (the presence or absence of pathogenic
microbes) and is therefore more easily studied in a scientific context, and more easily understood by the
general public. In addition, it provided a quick fix to many serious medical issues at the time of its
emergence, further contributing to its popularity in both the medical and lay communities.
Assertions by raw milk advocates that pasteurization may have negative consequences to public
health and that bacterial contamination of raw milk is actually central to its health-giving properties
challenge the deeply held views of many parties (especially within the industrial, scientific, and medical
communities) regarding the origin of disease and what steps must be taken to prevent it. Yet even
Pasteur was not so dogmatic about germ theory, and his personal conception of pathogenesis was able
to account for the observation that not all exposures to pathogenic microbes result in illness. This
concept is exemplified by Gumpert (2009, 126), who reports on a 1987 case study of campylobacter
11
associated with raw milk:
Raw milk proponents have another answer to the data and reports showing that people do become ill from raw milk. They argue that any dangers from infection by pathogens can be reduced significantly by regularly consuming raw milk, thereby building up immunity. They point to a 1987 case study of thirty-one freshmen fraternity pledges who, in the fall of 1982, went on a retreat to a large dairy farm owned by the parents of one member. Over the next ten days, nineteen of thirty-one students developed gastrointestinal illness and were found to have campylobacter, a common source of food poisoning. Three others without symptoms were also found to be infected. Interestingly, ten individuals who consumed the tainted milk and showed no signs of illness or infection–a few students and some farmhands–were found to be regular consumers of raw milk.
The authors of this study were able to directly correlate raw milk consumption with immunity to
Campylobacter jeujuni as well as levels of C. jeujuni specific antibodies (Blaser et al., 1987). This case
highlights the influence of individual variation and regular exposure to pathogens on immunity, as well
as the fact that individuals can be exposed to or even colonized by pathogenic organisms without
showing symptoms of illness. For these reasons, it provides provocative data in the debate on raw milk.
Although it is an isolated incident, and similar research has not been produced since, there are vast
bodies of scientific literature in the field of microbiology that can further inform the concepts brought to
light by this incident.
Commensal Pathogens Highlight the Importance of Environment and Immunity
It is only in recent years that technological advances have enabled scientific research to begin to
characterize the complex interactions involved in the pathogenesis of infectious disease. Bacterial
populations are transient and adaptable, and many of their phenotypic characteristics are highly
dependent on environmental conditions, which select for certain traits over time (Ehrlich et al., 2008), or
modulate genetic expression of existing populations (Hew et al., 2007). Ehrlich et al. (2008) make the
important observation that many virulence traits evolve under multiple evolutionary pressures, which
typically have nothing to do with host pathogenesis. The authors suggest it is "likely that many
pathogens did not initially evolve as pathogens, but simply take on this role as a result of a lack of
ability of the host to maintain homeostasis." The best examples of this type of evolution are the so-called
'commensal pathogens', which are typical members of the microbiota of normal healthy humans, but
under select circumstances can act as pathogens. These examples can give some insight into the
significant environmental factors associated with pathogenesis.
One such example is the bacterial genus Enterococcus, which encompasses a variety of species
that can occasionally act as pathogens, but are more commonly found as ubiquitous gastrointestinal
commensals, environmental colonizers, and food fermentors (Franz et al. 1999). Typically, enterococcal
12
infections occur in hospitals, where intrinsic and acquired antibiotic resistances harbored by many
strains of this genus give them a strong advantage over other bacteria (Gilmore and Ferretti, 2003).
Since enterococci are found ubiquitously in dairy products and other foods, and in especially high
numbers in traditional European aged raw milk cheeses, food safety concerns have been raised by the
scientific community (Franz et al., 2003). The turbulent political climate surrounding raw milk is also
the backdrop for the highly speculative debate regarding the safety of enterococci in foods. Research
directly addressing the potential risks of enterococci in raw milk and raw milk products have been
inconclusive, but continue in spite of overwhelming anecdotal evidence that their presence is not
problematic.
On the other hand, research into the probiotic capability of enterococci has been so successful
that probiotics such as Symbioflor® 1 (in which E. faecalis, the species of Enterococcus responsible for
90% of human infections (Domann et al., 2007) is the only bacterial species) have emerged for human
use. The instructions for use of Symbioflor® 1 indicate its use for "immunomodulation, chronically
recurrent infections of the upper respiratory passages, inflammations in the mouth, nose, pharynx, and
middle ear, colds, and disorders of the gastrointestinal function" (Instructions for Use Symbioflor® 1,
2002). The instructions also assert that there are no contraindications for its use nor any significant side
effects aside from isolated incidents of dry mouth, headache and stomach pain, and in the case that side
effects do occur, do not suggest discontinued use, but rather only a decrease in dosage. The product has
been on the market for over 50 years without any reported cases of infection (Vebø et al., 2010).
The vast majority of research associated with enterococci has focused on their emergence as
infectious agents in the hospital setting, where concerns surrounding their impact on health are more
than speculative (Fisher et al., 2009). There is a significant body of literature connecting the presence of
enterococci in foods and their roles in hospital acquired (nosocomial) infection and dissemination of
antibiotic resistance, and in one case a solid link between agricultural practices and antibiotic resistance
was established, resulting in far-reaching legislative and agricultural changes across Europe (van den
Bogaard et al., 2000). Critical examination of the two bodies of literature show that the complex
relationship between these two worlds is not well characterized, but that agricultural practices,
especially regarding hygiene and animal health, may be subjects of interest.
Unfortunately, studies on enterococci in food have not focused on agricultural practices and have
instead been preoccupied with characterizing the phenotypic traits associated with virulence (Semedo et
al., 2003; Lopes et al., 2006; Domann et al., 2007; Hew et al., 2007). Although life threatening cases of
enterococcal infection are rare, the very real ability of enterococci to infect hospital patients and cause
potentially fatal complications such as endocarditis (Chuang et al., 2009) stands in stark contrast to their
13
functional contribution to foods, their role as members of the commensal microbiota, and their ability to
confer a probiotic effect. For this reason, I suggest that the enterococci represent an example even more
perplexing than that of the commensal pathogens, and may more adequately be termed a 'probiotic
pathogen.'
Such apparent paradoxes have caused modern thinkers to entirely reconsider the scientific
underpinnings of what makes a pathogen pathogenic (Ehrlich et al., 2008). While the idea that pathogens
are not necessarily the root cause of disease may seem absurd to the average individual raised on the
dogma of germ theory, and government policy makers seem unwilling to even pay lip-service to its
potential validity, modern scientific research in microbiology and immunology lends support to this
newly emerging view. It may be unreasonable to claim that bacteria cannot cause illness, but it is also
unreasonable to claim that they are the sole agents of disease, as environmental, physiological and
immunological factors are now recognized to play a significant role in the pathology of most, if not all,
illnesses. An acute awareness of this neglected complexity has led some consumers to a radical line of
questioning that echoes the forgotten voices great microbiologists such as Dubos, and even Pasteur
himself, accounting for the other side of the story in examining the etiology of disease. The following
excerpt, which is contained in a legal document prepared defending accessibility of raw milk to Los
Angeles, CA consumers, is a good example of such questioning:
Are pathogens the instigators or the consequence of degenerative disease? Are they the cause or the cure? Is pointing the finger at microbes a distraction from true causes of disease? Is pollution of our food, water, air and medicine the predominant cause of disease, which then fosters bacterial growth? All hypotheses must be open to independent testing and researchers held accountable to the rules of evidence (Vonderplanitz and Douglass, 2001).
It may seem obvious that pathogens can cause disease, but generalized and oversimplified
assertions made on all sides of the raw milk debate reveal a significant gap in popular scientific
knowledge regarding dairy foods, microbiology, and pathogenesis. The general public is no longer
willing to accept the pretense that germ theory can solve all of our health problems, and rightfully so, as
the scientific community has known for some time that it is only one part of the highly complex and
diverse biological interactions that can lead to illness. Further study is necessary to develop more
inclusive models of pathogenesis, but it may be necessary to take a step back before we take a step
forward. By critical examination of current research in microbiology and immunology, we may be able
to distill more comprehensive and realistic models of pathogenesis. Such models will be of great value
in dispelling the misconceptions of the general public, of farmers, of regulatory authorities, of
legislators, and even of other scientists, so that we may all as a global community move forward to more
14
effectively and efficiently support the health of ourselves, our loved ones, and the social and ecological
systems we engage with every single day.
15
Part II: The Enterococcus–A Probiotic Pathogen?
Characteristics and Identification of Enterococci
The enterococci are Gram-positive cocci commonly found as commensal organisms in the
human gastro-intestinal (GI) tract (Tannock and Cook, 2002). They are also ubiquitous environmental
organisms and are associated with a number of fermented and processed food products (Hew, 2008).
They are facultative anaerobes displaying little to no catalase activity. Optimal growth occurs at 35C,
but growth can occur within a wide temperature range from 10 - 45C. As a result of adaptation to
environments such as fermented foods and the mammalian GI tract, enterococci can survive or grow in
challenging conditions. For example, they grow in 6.5% NaCl concentrations, and according to one
account can survive or grow in broth containing 27% NaCl (Huycke, 2002). They can also grow in up
to 40% bile salts (Facklam, et al., 2002), and in a range of acidity from pH 4.8-9.6 (Huycke, 2002). They
are reported to be highly thermotolerant and are known to withstand temperatures of 60C for up to 30
minutes (Ahmad et al., 2002). Other adverse conditions these organisms will resist include sodium
azide, detergents, sodium hypochlorite, heavy metals, ethanol, high oxidative stress, and prolonged
dessication (Huycke, 2002).
There are over 20 species currently included in the genus Enterococcus (Giraffa, 2003), but E.
faecalis, E. faecium, and to a lesser extent E. durans, are the most common to both the human GI tract
(Tannock and Cook, 2002) and dairy products (Wessels et al., 1988). Regardless of origin of isolation,
there is significant phenotypic heterogeneity within and between populations of Enterococcus, making
their identification difficult (Giraffa, 2003). Presumptive identification is relatively simple and can be
accomplished using selective growth media and basic phenotypic assays such as esculin hydrolysis
(Garg and Mital, 1991) and Gram staining. The most common species (including the most relevant dairy
organisms noted above) can then be differentiated from other Gram-positive, catalase-negative, homo-
fermentative cocci by their ability to grow at 10 and 45C, in 6.5% NaCl, in 40% bile, and at pH 9.6
(Franz et al., 2003). Other less common species, however, exhibit variation in these traits and may
require more extensive phenotypic characterization or the use of molecular methods to establish with
certainty their identity as enterococci.
Additionally, some traits that were historically used to identify enterococci are now less relevant
in modern taxonomy. For example, expression of Lancefield's group D antigen (a defining characteristic
of the enterococcal group or fecal streptococci) is not a defining characteristic of the modern genus
16
Enterococcus. Several enterococcal species do not express the group D antigen, while some bacteria
outside the genus Enterococcus, including some streptococci and leuconostocs do express the group D
antigen (Franz et al., 2003).
The enterococci were originally part of the genus Streptococcus, probably due to their
morphological similarity. In 1937, Sherman published a review classifying streptococci into 4 groups,
one of which he termed the enterococcal group. Other researchers suggested that the enterococcal group
might be sufficiently distinct as to form its own genus, but it was not until 1984, with the advent of
molecular methods to make taxonomical distinctions based on genetic similarity, that the genus
Enterococcus became formally recognized in the scientific community (Facklam et al., 2002).
Habitats of Enterococci
The enterococci are found in a diverse range of habitats including dairy products and other foods,
as well as in clinical and environmental contexts (Franz et al., 2003). E. faecalis and E. faecium are
broadly distributed and are common to most enterococcal habitats, while some of the less common
enterococci are associated with specific habitats or even specific hosts, such as E. asini, which is
specific to donkeys (Aarestrup et al., 2002). Most enterococci are considered to be of fecal origin and
are native to the GI tract of humans and many other mammals and birds.
Despite their ubiquitous nature as members of the commensal microbiota, they are typically kept
at very low levels in healthy hosts, and make up no more than 1% of the intestinal microbiota in the
average human adult (Tannock and Cook, 2002). In infants, however, their populations are much higher
and, along with lactobacilli and E. coli, enterococci form the dominant intestinal microbiota of neonates,
reaching levels of about 108 bacteria per gram of wet fecal matter of breast-fed children. Higher levels
are found in children fed infant milk formulations. A microbial succession is associated with changes in
diet from exclusive consumption of milk to the addition of solid foods. Obligate anaerobes such as
fusiforms and species of Bacteroides colonize the intestine and produce short-chain fatty acids, which
are inhibitory to facultative anaerobes under the conditions of the bowels, and contribute to the decline
in enterococcal populations.
The enterococci are occasionally pathogenic in animal hosts, and have been implicated in bovine
mastitis and some cases of diarrhea in animals such as rats, piglets, and poultry, although incidences of
enterococcal infection in animals receive less attention than human infections (Aarestrup et al., 2002).
This may be due in part to limited resources in veterinary clinics and difficulties in differentiating
enterococci from related organisms. As a result of these challenges, it is likely that many enterococcal
infections in animals are not reported. Human infection by enterococci, on the other hand, has become a
17
high profile issue in both the medical and scientific community. Enterococci are now considered
significant hospital acquired (nosocomial) pathogens (Upadhyaya et al., 2009), and special attention is
paid to their role in the acquisition and spread of antibiotic resistances in both clinical and agricultural
settings (Kak and Chow, 2002).
Interestingly, some strains of enterococci show a probiotic effect (Jansen et al., 1993), and
several commercially available probiotics contain strains of E. faecalis or E. faecium both for human
consumption and for use in animal feed (Franz et al., 1999). What factors contribute to enterococci
acting as probiotics as opposed to pathogens is debated and is currently a subject of scientific studies
(Domann et al., 2007; Veboe et al., 2010). As stated by Hew et al. (2007), "The ability of Enterococcus
to promote both health and illness at the same time is a contradiction that is currently not well
understood.” It is interesting to note that many of the same characteristics that make enterococci
potential pathogens are also important for inducing a probiotic effect. These traits include tolerance to
various adverse conditions that allow enterococci to survive in the digestive tract and the ability to
adhere to intestinal epithelial cells and effectively colonize the host intestine.
Enterococci make their way into the environment via fecal shedding (Aarestrup et al., 2002). The
use of untreated animal wastes as fertilizer may enhance the colonization of agricultural environments
by enterococci. They are often found in water samples and have been proposed as a reliable indicator of
fecal contamination. While even environmental enterococci are traditionally considered to be of fecal
origin, there are several species that appear to have adapted to the vegetative environment and
commonly colonize plants. These species are E. casseliflavus, E. mundtii, and E. sulfereus. Although
they are also found occasionally in the GI tract of animals, it is thought that they are only able to
colonize this environment transiently.
The ability of enterococci to survive on dairy equipment and in the dairy environment allows
them entry into milk, which is an ideal nutrient medium for them (Garg and Mital, 1991). Adaptation to
this niche in combination with their resistance to heat, salt, and low pH give them an edge in the cheese
environment, and they can be found during all stages of production and ripening for some types of
cheese (Manolopoulou et al., 2003). Indeed, microbiological analysis has shown enterococci to be a part
of the microbial ecology in many cheeses, particularly European artisanal cheeses, made from both raw
and pasteurized milk (Giraffa, 2003). They can colonize cheese either as contaminant during collection
and processing of milk, or sometimes by their presence in traditionally cultivated starters. Some
traditional starter cultures are made by pasteurizing raw milk and incubating it at 42 - 44C, a procedure
which strongly selects for thermophilic LAB such as enterococci and S. thermophilus.
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Functionality of Enterococci in Dairy Fermentation
The enterococci are classified as homo-fermentative lactic-acid bacteria (LAB) due to their
ability to produce L-lactic acid from carbohydrates, but their ability to colonize a variety of niches
reflects their capacity to produce a wide range of metabolites from diverse substrates. All species are
able to metabolize at least 13 sugars, and one or more species can metabolize an additional 31 sugars
(Huycke, 2002). Sugars, however, are not the only way the enterococci can extract energy from their
environment. They can also utilize such diverse energy sources as glycerol, lactate, citrate, α-keto acids,
arginine, and agmatine.
Several of these metabolic traits contribute to the functionality of enterococci in dairy ferments
including the ability to metabolize substrates that are abundant in milk, and the formation of metabolites
that lend desirable flavors to the finished product (Giraffa, 2003). For example, glycerol is a small
molecule used to bind fatty acids into triglycerides. The ability to metabolize this substrate, which is
abundant in milk fat, may provide an advantage to enterococci by allowing them access to an energy
source that may not be metabolized by competitors.
Although enterococci do produce lactic acid, they are typically much weaker acid producers than
other LAB, making them less suited as primary starter organisms (Giraffa, 2003). A diversity of other
metabolites, however, make them ideal for the development of flavor compounds during ripening,
especially due to their ability to grow in the highly inhibitory conditions of ripening cheese. Metabolism
of citrate, proteins, and lipids are traits that are well recognized as important for the ripening of cheese
and are all expressed by strains of enterococci to various degrees.
The diversity of metabolic pathways enterococci use to extract nutrients and the heterogeneity of
these metabolic traits within populations may help explain the purported functional aspects in fermented
foods. Cheese makers and connoisseurs often note the importance of "balance" in a good cheese
(Raskopf, 2010). If organisms involved in ripening have a limited number of potential metabolites
(which form the basis of flavor formation during ripening) then one flavor may become overpowering
and be considered a defect. As an extreme example, in the ripening of soft, bloomy rind cheeses,
Penicillium candidum or related molds typically produce ammonia. However, when this cheese is past
its peak, the pungent ammoniated rind can be considered undesirable. On the other hand, metabolic
diversity (such as is displayed by the enterococci) helps protect against any one metabolite or group of
metabolites becoming an overpowering off-flavor. In the same way, the presence of enterococci as part
of a diverse microbiota in fermented foods offers further protection against the dominance of one
metabolite. This may contribute to a balanced flavor palate exhibiting a diversity of subtle tastes, an
ideal in the world of cheese.
19
Another important functional characteristic of enterococci is the production of bacteriocins,
which are natural antibiotics produced by some kinds of bacteria (Giraffa, 2003). Enterococci are known
to produce a wide variety of bacteriocins, and these are sub-classified as 'enterocins.' These compounds
are relatively well characterized and have been shown to be compatible with significant starter LAB and
to be stable in the presence of rennet and under conditions of processing and fermentation such as
temperatures between 30 - 37C and low pH. Enterocins have the potential to inhibit the growth of more
vigorous pathogens such as Listeria monocytogenes and Staphylococcus aureus. The anti-Listeria
activity of enterocins is of particular relevance, as L. monocytogenes is one of the most significant
cheese-borne pathogens. Scientific studies support the use of enterococci as protective cultures, and
several experimental models have shown that enterococci can confer a protective effect on cheese
artificially inoculated with L. monocytogenes or S. aureus by inhibiting the growth of these pathogens.
The presence of enterococci in cheese, however, is not unequivocally good. Whether they
function as beneficial non-starter lactic acid bacteria (NSLAB) or as undesirable contaminants depends
largely on the microbial ecology of the cheese in question. Garg and Mital (1991) in a review on
enterococci in dairy products highlight this point by noting two significant studies. In one study, co-
culturing of E. faecalis subsp. liquefaciens with S. thermophilus significantly enhanced acid production
in the latter species. In an alternative example, the presence of high numbers of E. faecalis subsp.
liquefaciens in Swiss cheese inhibited propionibacteria, which are essential to the ripening of these types
of cheese, leading to retardation of the ripening process and formation of bitter flavors. Additionally,
recognition of the potential for enterococci to contribute to disease states either indirectly or as
opportunistic infectious agents, and the rapid development of antibiotic resistances within the genus has
raised questions regarding the safety of these organisms in food (Franz et al., 2003).
Virulence Factors in Enterococci
Due to the increasing significance of enterococci in hospital acquired infections, much scientific
research has focused on identifying traits that may contribute to their pathogenesis (Gilmore et al.,
2002). Although no single trait is ubiquitously present in pathogenic strains and absent from non-
pathogens, a number of traits have been identified that likely play a role in pathogenesis including
gelatinase, biofilm formation, polysaccharide capsule production, hemolysins, and enterococcal
aggregation substance. These traits, or "virulence factors" (Semedo et al., 2003), by definition are not
required for commensal functioning (as evidenced by their absence from some or all commensal
strains), and contribute to the severity of infection, which is usually assessed by experimental models of
infection that show the impact of knocking out a gene encoding a virulence factor (Gilmore et al., 2002).
20
These traits are often encoded on mobile genetic elements and can therefore be efficiently disseminated
throughout a population under the proper conditions.
Before the individual virulence factors are considered, it is important to understand how
virulence factors are conceptualized and take into account some complicating factors in modeling
virulence. Foremost, a strain cannot be defined as pathogenic by simply identifying the presence of
virulence factors or the genes encoding them. The textbook definition of pathogen is 'a disease causing
microorganism' (Madigan and Martinko, 2006, 3), but the presence of virulence factors in a strain of
Enterococcus does not necessarily guarantee that the strain will cause disease. This is clearly exhibited
by the presence of all of the classical virulence factors in at least some, if not most E. faecalis strains
isolated from the fecal matter of healthy Norwegian infants (Solheim et al., 2009). Genetic studies have
revealed that Symbioflor® 1, a commercially available probiotic strain of E. faecalis lacks several
virulence factors including hemolytic cytolysin, enterococcal surface protein (an adhesin), and
gelatinase; however, other virulence factors including aggregation substance, collagen adhesion protein,
ability to resist oxygen anions, and capsule formation are present in the probiotic strain (Domann et al.,
2007).
While many of the classical virulence factors are not needed to cause infection, some very basic
traits that are important for survival of enterococci in diverse niches are also important for pathogenesis.
These traits include resilience against various adverse environmental conditions needed to survive transit
through the gut, and production of adhesins allowing cells to colonize the intestinal epithelium (Hew et
al., 2007). The general hardiness of enterococci to environmental stress is advantageous while surviving
the process of cheesemaking just as much as it is advantageous while infecting a host (Giraffa, 2003).
Genes encoding adhesins are found in high proportions in populations of enterococci (Semedo et al.,
2003). They are likely advantageous in diverse environments and important for binding to both biotic
and abiotic surfaces. This has implications in both pathogenesis and in dairying. In the case of
pathogenic enterococci, adhesins that mediate binding to host cells are essential (Manley et al., 2007). In
a different scenario, adhesion to abiotic surfaces such as plastic and metal components of milking
equipment may allow enterococci entry into the milk, where they can thrive as a member of the dairy
microbiota (Giraffa, 2003).
Regulation of Genes Encoding Virulence Factors
Factors influencing genetic expression must also be considered, since an organism may present a
negative phenotype for a virulence factor even if it has the genetic potential to express that trait. Two
enterococcal mechanisms of gene regulation that are of particular significance are environmental sensing
21
and quorum sensing.
Hew et al. (2007) elegantly demonstrate that E. faecalis modulates expression of virulence genes
in response to various environmental conditions, and that some of these conditions may have
significance in food production and pathogenesis. The authors suggest that certain food processing
procedures could predispose a population of enterococci to express pathogenic traits. They also suggest
that enterococci may be able to sense the presence of host tissues and in response upregulate
transcription of traits that give them an edge in this environment (virulence factors). This type of
environmental sensing is typically mediated by two-component signal transduction systems that sense
extra-cellular conditions via membrane sensor kinases (Ma et al., 2008).
Quorum sensing (a mechanism of communication between bacterial cells) is mediated by a
similar process and appears to play an integral role in coordinating production of virulence factors
(Spoering and Gilmore, 2006). In this case, however, the critical environmental condition is cell-
density, because quorum sensing signaling molecules (typically small molecules or short-chain peptides)
are produced by the same bacterial cells capable of sensing them in their environment. As growth occurs
and cell density in a particular locality increases, a critical concentration of signaling peptide is
eventually reached, initiating significant changes in genetic expression. Since many enterococcal
virulence factors are regulated by quorum sensing (Hew et al., 2007), a critical cell density may be
requisite for pathogenesis. Considering the strict regulation of enterococcal populations in the human GI
tract (Tannock and Cook, 2002), it is possible that controlling population density of enterococci is a key
strategy in protecting against infection. Populations of enterococci in cheese, on the other hand, are less
controlled and are likely to reach cell densities required for expression of quorum mediated traits. The
implications of this, however, are unclear in terms of the risk associated with consumption of foods
containing large populations of enterococci.
Diversity of Enterococcus spp. and Evolution of Virulence
The classical virulence factors, which have been well described in E. faecalis, are much less
common in other species of Enterococcus, and although E. faecalis is responsible for the vast majority
of enterococcal infections, E. faecium is much more likely to be resistant to one or more antibiotics,
which is also significant to pathogenesis (Gilmore and Ferretti, 2003). Furthermore, less common
species of Enterococcus such as E. raffinosus, E. casseliflavus, and E. mundtii are occasionally known to
cause infection (Tannock and Cook, 2002). The latter two species just listed are native to plants, not the
GI tract of humans or even other animals, and the native habitat of E. raffinosus is unknown because it
has only been found in pathological materials.
22
That some infectious species of Enterococcus are native to plants suggests that a general
hardiness to environmental conditions may allow enterococci to persist as pathogens despite not being
specifically adapted to their host. There is some evidence to support this hypothesis. Hew et al. (2007)
performed a study in which virulence gene expression was assessed in response to various
environmental conditions. The results showed that stress related genes were typically upregulated during
exponential phase, and it is postulated that this is a preparation for entering the starvation stress induced
stationary phase, in which these genes will aid survival during nutrient sparsity. In particular, gls24
(which codes for a general stress protein) was upregulated during exponential phase in cells exposed to
almost all of the various environments, and the authors suggest that this may be a case in which food
processing conditions could contribute to the development of pathogens. As a general stress gene, gls24
may play important roles outside the context of virulence, but it has been shown to increase virulence in
multiple animal models.
It is important to stress that many pathogens evolve their pathogenic traits outside of their host
(Ehrlich et al., 2008), since this suggests that ecological and environmental conditions contribute to
pathogenesis. When conceptualizing enterococcal virulence, one must recognize that a virulence factor
may have evolved under multiple evolutionary pressures, especially considering the diverse habitats of
enterococci. A well known example of this type of evolution is E. coli 0157:H7. This human pathogen is
a native member of the bovine commensal microbiota and is not commonly transferred from person to
person, which suggests that its virulence may be subject to many evolutionary forces outside the host.
There is evidence to support this idea, at least in the case of shiga-like toxins, an important virulence
factor in E. coli. These toxins have been shown to help cells evade predation by the ubiquitous
bactivorous protozoan Tetrahymena pyriformis.
Examples like this show that microorganisms can be highly adaptable and that their multifaceted
mechanisms of survival can lend an advantage in numerous environments. While evidence strongly
suggests an etiological role for enterococcal virulence factors in pathogenesis, it is likely that these traits
perform multiple functions and are also advantageous outside the context of pathogenesis. The virulence
factors are common in clinical strains because they do lend an advantage in pathogenesis, but they are
not necessary for pathogenesis, nor do they ensure pathogenesis. Keeping that in mind, a more accurate
model of virulence can be conceptualized, and the significance of individual virulence factors can be
evaluated.
Gelatinase
Gelatinase is a multifaceted zinc metalloprotease enzyme produced by some strains of
23
enterococci that has been associated with virulence both in experimental infection models (Lopes et al.,
2006) and by prevalence in clinical isolates (Elsner et al., 2000), although conflicting data has called
into question its statistical association with clinical isolates (Semedo et al., 2003). The probiotic strain of
enterococcus marketed as Symbioflor® 1 does not produce gelatinase (Domann, 2007). Environmental
conditions can have an effect of gelatinase production, and Hew et al. (2007) found that brain heart
infusion (BHI) culture media upregulated transcription of gelE (the gene that encodes gelatinase). Dairy
enterococci commonly express gelatinase (Lopes et al., 2006), probably to extract nutrients from casein
and other mammalian proteins present in milk. To complicate accurate assessment of gelatinase
production, lab manipulation of culture isolates can cause loss of gelatinase activity.
This complication was clearly elucidated by Lopes et al. (2006) in their study on gelatinase
activity in dairy enterococci isolated from raw ewe's milk and cheese. It primarily addressed questions
regarding how conditions of culturing can influence the genetics, gene-expression, and activity of
gelatinase. They obtained 35 isolates and duplicated them, creating a total of 70 isolates. The duplicate
set of isolates underwent significantly more lab manipulation than the original set. Isolates were
screened for gelatinase activity. While in the original set, which experienced less manipulation, 33/35
isolates showed gelatinase activity, only 4/35 in the more manipulated duplicate set showed activity.
Isolates were then stored in glycerol at -80C for one year before a second set of experiments re-
assessed gelatinase activity and also collected genetic analysis to observe the presence and transcription
of gelE and the fsr operon, which contains 3 genes (fsrA, fsrB, and fsrC) that regulate expression of
various genes, including gelE. Freezing had a very large impact on gelatinase activity and only 4/70
isolates showed activity in the latter assay. The genetic data implicates deletions in the fsr operon in loss
of gelatinase activity, as at least one fsr gene was missing from 54 isolates. Some gelatinase negative
strains that do have an intact fsr operon, and further genetic testing suggests that post-translational
modification may be necessary to activate the enzyme.
This study highlights the significance of the fsr operon in regulation of gelatinase expression.
Gelatinase is probably the most thoroughly studied of the enterococcal virulence factors, and the
mechanism behind its regulation is well described (Hew, 2008). The most important thing to know about
regulation of fsr expression is that it is mediated in a quorom sensing manner. Production of gelatinase
biosynthesis activating pheromone (GBAP), an 11 amino acid cyclic lactone peptide, is encoded in the
C-terminus of the fsrB gene (Gilmore et al., 2002). This peptide has been shown to induce the
transcription of gelE and sprE (a co-transcribed serine protease), as well as to auto-induce its own
transcription creating a positive feedback loop that promotes gelatinase synthesis. According to Hew
(2008, 20), "studies have found that about 1,000 GBAP molecules are required per cell for the initiation
24
of gelE transcription."
Gelatinase is capable of hydrolyzing a variety of substrates including fibrin, fibrinogen, collagen,
casein, and its arbitrary namesake gelatin (Hew, 2008; Lopes et al., 2006). This suggests that gelatinase
is a multifunctional enzyme, and that it may be advantageous in more than one setting. It addition to it's
role in pathogenesis, it could be useful in extracting nutrients from casein when growing in milk. During
fermentation, especially the prolonged fermentation of aged cheeses, hydrolysis of casein by proteolytic
enzymes is an important functional aspect of enterococci contributing to flavor development (Giraffa,
2003). Considering its ability to hydrolyze casein, gelatinase has the potential to contribute to this flavor
development.
Gelatinase is considered an extra-cellular enzyme (Lopes et al., 2006), but it is also highly
hydrophobic (Makinen et al., 1989) a characteristic that could cause it to associate with the bacterial cell
surface, capsule, or substrate surfaces. Considering its ability to extract nutrients from the environment
(Lopes et al., 2006), and defend the cell against host immune response (Thurlow, 2009), close proximity
to the cell may be advantageous, allowing the cell to get the most benefit out of the enzyme.
Capsule
Of enterococcal serotypes A-D, only C and D produce a capsular polysaccharide (Thurlow et al.,
2009). This variable molecule forms a thick mucoid layer that surrounds the bacterium and is significant
to pathogenesis because it can help enterococci and other bacteria evade the immune response.
At least two mechanisms of immune evasion are described by Thurlow et al. (2009), both of
which rely on interfering with binding of molecular identity markers to receptors on the surface of
immune cells. In the first mechanism, capsular polysaccharide inhibits recognition of surface bound C3,
a protein present in the blood that is part of the complement system of innate immunity. The protein acts
somewhat like an antibody, but is less specific and binds to common pathogens marking them for
phagocytosis; however, the capsule interferes with recognition of C3 bound to encapsulated cells,
allowing them to resist opsonization by white blood cells, accounting for their enhanced survival in
serum and resistance to phagocytosis.
In addition to receptors that recognize proteins like C3, immune cells express pathogen
recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). For
Gram-positive bacteria, one of the most common PAMPs is lipoteichoic acid (LTA), which binds
specifically to immune cell receptors such as Toll-like receptor, allowing the recognition of pathogens
(Ginsburg, 2002). This binding induces production of cytokines, stimulating an immune response. If
LTA is covered by a layer of capsular polysaccharide, however, the immune response is attenuated. See
25
figure 5 for a detailed model of the organization of structures in the E. faecalis cell wall.
Thurlow et al. (2009) show experimentally that enterococcal capsules interfere with LTA binding
and alter cytokine production in cultured macrophages. Significantly, production of tumor necrosis
factor alpha (TNF-α), a transcription factor that induces expression of genes involved in the immune
response, was much higher when macrophages were exposed to unencapsulated strains as opposed to
encapsulated strains, which actually did not produce significantly greater TNF-α production than when
macrophages were not stimulated.
Coyette and Hannock (2002) note that capsule formation by enterococci is growth-phase
dependent, but a quorum sensing mechanism of regulation has not been identified. The synthesis of
capsular polysaccharides by Gram-positive organisms is, however, highly regulated (Hancock et al.,
2003), and quorum sensing regulates capsule formation in a strain of S. aureus via AI-2, a universal
bacterial signaling molecule (Zhao et al., 2010). This evidence, along with the prevalence of quorum
sensing mechanisms in regulation of other virulence genes, suggests that synthesis of capsular
26
Figure 5 - "Model for the organization of cell wall polymers in the cell wall of E. faecalis. The lipid-anchored lipoteichoic acid, also known as the streptococcal group D antigen, is shown protruding into the cell wall peptidoglycan. Shown anchored to N-acetylmuramic acid (MNAc) residues of the peptidoglycan are the integral cell wall teichoic acids and [a] hypothesized enterococcal species antigen. Anchored to the N-acetylglucosamine (GNAc) reisdues in the peptidoglycan and protruding out from the peptidoglycan is the serotype-specific capsular polysaccharide. (Coyette and Hancock, 2002)"
polysaccharide production is also influenced by quorum sensing mechanisms. Capsule production is
encoded by nine genes, cpsC-cpsK (Gilmore et al., 2002).
Capsules can be observed in enterococci using a simple india ink stain, which stains the
background of the slide but not the capsule (Bottone et al., 1998). Cell bodies can then be counter
stained with crystal violet, and the capsule appears as a transparent halo surrounding cells. This stain has
been used to detect capsules in isolates from patients with persistent urinary tract infections. Other
studies using india ink staining have found about 20% of isolates from the mammary secretions of
mastitic cows to be encapsulated (Coyette and Hancock, 2002). Like gelatinase production, detection of
capsule by india ink staining was also found to be highly dependent on culture and storage conditions
and about 80% of encapsulated strains had no identifiable capsule after storage in skim milk at -80C.
Studies specifically addressing the influence of environmental conditions on capsule production
in enterococci have been sparse as of yet, but initial findings suggest that capsule production is slightly
downregulated in both serum and urine as opposed to enriched microbiological media (Coyette and
Hancock, 2002). It is interesting to note that culture broth made from skim milk powder is used to
induce capsule production for capsule staining procedures (Smith and Hughes, 2010). Aside from
helping evade the immune system, capsules are thought to protect from desiccation, promote adhesion,
and biofilm formation. All these qualities either directly enhance virulence, or could potentially allow
virulent strains to persist in the environment and spread from one host to another. As a somewhat
counterintuitive twist in the story of enterococcal virulence, however, the probiotic strain Symbioflor® 1
does produce a capsule (Domann et al., 2007).
Biofilms
A biofilm is a population of cells encased in a hydrated mixture of polysaccharides, proteins, and
nucleic acids (Mohamed and Huang, 2007). The formation of biofilms is significant in both the dairy
and hospital environments. Since they allow adherence to abiotic surfaces, biofilms give enterococci the
potential to colonize surfaces such as milking equipment, allowing entry into milk, or surfaces in
hospitals allowing clinical strains adapted to human hosts to persist in the environment and spread.
Biofilm formation has been associated with the ability of Enterococci grow on various medical devices
such as silicone gastrostomy devices and intravascular catheters. Adherence to medical devices that are
inserted into patient bodies is a potential method of infection.
Of perhaps greater concern is the difficulty in eradicating bacterial biofilms, and the increased
resistance of biofilms to antibiotics, which can be 10-1000 times greater than that of planktonic bacteria
(Mohamed and Huang, 2007). Biofilms are no doubt one of the traits that allows enterococci to be such
27
hardy environmental organisms. They are also associated with pathogenesis, but prevalence and degree
of biofilm formation have been shown to vary, and one study found that 93% of clinical and faecal
enterococcal isolates produced biofilm (Mohamed et al., 2004). Despite variation, trends suggest that
biofilms are more common in E. faecalis than in E. faecium, and that biofilms may be significant in
enterococcal pathogenesis.
Biofilm formation is associated with gelatinase and is regulated by quorom sensing as shown by
recent studies investigating the role of DNA in biofilm structure (Thomas et al., 2009). Their findings
implicate gelatinase in a fratricidal mechanism of DNA release, lending evidence to a model of biofilm
formation in which gelatinase biosynthesis-activating pheromone (GBAP) mediates the production of
gelatinase via quorum sensing, which kills cells that do not respond to gelatinase regulating quorum
sensing oligopeptide GBAP, since they will also not express the gene SprE, which codes for a serine
protease that is co-transcribed with gelatinase and provides immunity to gelatinase mediated killing.
The killing action of gelatinase releases DNA into the environment where it can be used for
biofilm structure. It is interesting to note in this model that in order to provide immunity to a particular
cell, the extra-cellular protease SprE would provide a more reliable advantage if it associated with the
cell surface, as may be the case with hydrophobic gelatinase (Makinen et al., 1989), although evidence
to support this theory has not yet been gathered.
Various environmental conditions have been shown to influence biofilm formation in enterococci
(Mohamed and Huang, 2007). One of these factors is the presence of glucose, which reliably enhances
biofilm formation in several studies. Considering the environments of interest to this paper, it is
significant that glucose would be available both in milk (by hydrolysis of lactose) and in human tissues,
where it is used as a basic unit of energy. Of further significance to enterococcal infection, human serum
also enhances biofilm production.
Enterococcal surface protein (Esp), another purported virulence factor, is associated with biofilm
formation (Gilmore et al., 2002). The probiotic strain Symbioflor® 1 probably does not produce
biofilms since its genome does not code for gelatinase or Esp (Domann et al., 2007). The interconnected
functionality of virulence factors supports the idea that they are multi-functional traits. Looking at
biofilms through the multi-functional lens, it is significant to emphasize that biofilm formation is a
highly advantageous trait commonly expressed by bacteria to help cope with stressful environmental
conditions in a variety of settings, including pathogenesis.
On the other hand, exposure to 2-3% NaCl completely stops biofilm formation (Mohamed and
Huang, 2007), suggesting that salting of foods such as cheese may be a processing step that inhibits the
formation of biofilms and reduces virulence. In contrast to brain heart infusion, which upregulated
28
gelatinase production, Hew et al. (2007) found that in vitro environments mimicking food processing
conditions universally downregulated expression of gelatinase, which may be needed for biofilm
formation (Thomas et al., 2009). These data suggest that biofilm production is regulated in response to
environmental conditions, that enterococci may be able to sense host tissues, and that processing of food
may actually reduce virulence.
Aggregation Substance
Enterococcal aggregation substance (EAS) is a multifunctional surface protein expressed by
many enterococci. EAS is actually a group of related proteins sharing 90% sequence similarity with the
exception of a variable central domain where sequence similarity is only 30 to 40% (Chuang et al.,
2009). Although traditionally associated with E. faecalis, PCR amplification using primers designed for
agg (a gene that encodes for the most common EAS protein produced by E. faecalis) shows that highly
similar genes can be found in most if not all strains of Enterococcus (Semedo et al., 2003). This suggests
that homologous proteins exist in other species.
The various aggregation proteins are encoded on plasmids capable of sex-pheromone mediated
conjugative transfer (Wirth, 1994). This specialized quorum-mediated interaction requires both donor
cells and a recipient cells. Donor cells have the plasmid. Recipient cells lack the plasmid, but produce a
chromosomally encoded signal peptide
(sex-pheromone) that can be considered
similar to other quorum signal peptides
such as GBAP. When a critical
concentration is reached, the pheromone
induces transcription of genes encoding
EAS proteins, which subsequently coat
the surface of the donor cell (see figure 6).
The function of EAS proteins is
what makes this quorum sensing
mechanism unique. These proteins bind to
components of the bacterial surface such
as lipoteichoic acids and enterococcal
binding substance (EBS), genes for which
are encoded on the chromosome and
expressed by recipient cells (Wirth, 1994).
29
Figure 6 - Image produced using field emission scanning electron microscopy showing expression of surface proteins encoded on pCF10 pheromone responsive plasmid. Cells in both images carry a shuttle vector, but for cells on the left Asc10 and Sec10 (surface protein genes encoded on pCF10) have been spliced into the vector. These proteins are clearly visible as discrete globular entities on the surface of the cell. This image was retrieved from Clewell and Dunny (2002).
This binding causes the formation of cellular aggregates, or clumps, bringing donors and recipients in
close enough proximity to ensure the conjugative transfer of EAS encoding plasmids. Although there is
not yet research to support the hypothesis, clumping could have the added benefit of increasing local
concentrations of quorum signaling molecules and inducing the expression of other virulence factors and
otherwise advantageous quorum regulated traits. In this sense, EAS could be considered a meta-
virulence factor, promoting the proliferation of other virulence factors by forming aggregates of cells
with diverse traits and inducing the sharing of these traits. Harsh environmental conditions would then
drive the selection of only the most advantageous traits, enhancing adaptation.
EAS is perhaps the most well studied enterococcal virulence factor next to gelatinase, and many
studies have investigated its role in pathogenesis and its structural and functional properties. One such
study, performed by Chuang et al. (2009), assessed the virulence of chromosomally identical strains with
variations on the pCF10 plasmid. Variations included mutations of the prgB gene that encodes for Asc10
(mutations ranged from insertions and deletions of large sections of amino acid sequences to more subtle
alterations such as single amino acid substitutions), deletion of the prgB gene, and absence of the pCF10
plasmid. Virulence was assessed in a rabbit endocarditis model and was quantified based on the bacterial
load of infected tissue and weight of bacterial vegetative growths. The results showed that deletion of
the prgB gene resulted in greatly decreased virulence. Mutations of the prgB gene also resulted in
decreased virulence, with the more subtle changes such as amino acid substitutions resulting in some of
the greatest decreases in virulence. Interestingly, strains without the pCF10 plasmid did not show a
significant decrease in virulence compared to strains with wild-type pCF10, while strains with mutations
in the plasmid did show decreased virulence. The authors suggest that pCF10 may suppress the
expression of homologous, chromosomally encoded traits, or that proteins encoded on the plasmid may
coat the cell surface in such a way as to obscure other functional proteins, such that the presence of a
plasmid with non-functional proteins may inhibit the expression or functioning of otherwise significant
traits.
Gene transfer can still occur without EAS or EBS, but it is much less efficient in a liquid
environment, where conjugative transfer between these primarily non-motile cells is reliant on random
collisions. EAS/EBS deficient enterococci, however, still efficiently transfer genes on solid surfaces
(Clewell and Dunny, 2002). Biofilms, such as those that form on abiotic surfaces or on heart tissues
during endocarditis may speculatively be implicated in gene transfer in enterococci that do not express
EAS, but EAS has also been shown to enhance biofilm formation in experimental models of
endocarditis (Chuang et al., 2010). This could be a result of aggregation enhancing the efficacy of
quorum induced trait expression. While biofilms may facilitate gene transfer on medical equipment or
30
milking equipment, both serum and milk are liquid media, and EAS mediated clumping may be
important for enhancing genetic exchange and facilitating adaptation of enterococcal populations in
these environments.
In addition to their role in clumping and genetic exchange, EAS proteins also mediate adhesion
to eukaryotic cells including neutrophils (a type of immune cell), kidney cells, and intestinal cells
(Chuang et al., 2009). Their role in adhesion to eukaryotic cells was discovered when sequence
similarity was found between amino acid motifs in EAS and motifs in fibronectin, which mediates
binding between eukaryotic cells (Gilmore et al., 2002). Their ability to adhere to eukaryotic cells
suggests that these proteins may be important for mediating interactions between enterococcal cells and
host cells.
There is substantial evidence to support the role of EAS proteins in modulation of immune
function, which could contribute to pathogenesis (Gilmore et al., 2002). EAS proteins mediate binding
with immune cells such as neutrophils and macrophages, promoting phagocytosis of bacterial clumps.
EAS proteins also promote survival of cells after phagocytosis. Resistance to phagocytic killing is
strongly adaptive for pathogenic strains which must avoid the immune response both on mucosal
surfaces, or in serum and infected tissues.
EAS may also be expressed in response to environmental conditions such as exposure to serum,
and in contrast to scenarios in which EAS proteins attenuate the immune response, EAS and EBS
together can act as a 'super-antigen', stimulating T-cell proliferation and production of inflammatory
cytokines (Kayaoglu and Oerstavik, 2004). It is clear that EAS proteins can have varied effects on the
immune system, but that in general it provides an advantage to infectious populations.
EAS proteins are strongly implicated in pathogenesis due to their various interactions with host
tissues and fluids as well as their ability to act as a nexus for other virulence factors. Their association
with clinical isolates (Elsner et al., 2000) and ability to enhance virulence in experimental models
(Chuang et al., 2009) lend further support to this hypothesis. EAS proteins, however, appear to be very
common amongst isolates from diverse sources (Semedo et al., 2003) including strains isolated from
healthy infants (Solheim et al., 2009) and the probiotic strain Symbioflor® 1 (Domann et al., 2007). In
addition, the results of experimental models of pathogenesis sometimes suggest that EAS proteins may
not enhance virulence (Johnson et al., 2004).
It seems plausible that the probiotic efficacy of some strains of enterococci is due in part to
virulence traits like EAS protein and capsular polysaccharide that stimulate and/or modulate immune
function. Considering that the immune system is highly co-evolved with this ubiquitous organism, it
makes sense that exposure to enterococci in the right context may be useful or even necessary to
31
maintain balance in the immune system. Scientific research is beginning to show that the commensal
microbiota are essential in training the immune system and in maintaining healthy immune function
(Tlaskalova-Hogenova et al., 2004). It is likely that depending on environmental conditions, immuno-
modulating virulence factors like EAS protein can be involved in mechanisms that promote either
pathogenesis or healthy immune responses to enterococci or perhaps other microbes as well.
Cytolysin
Hemolysins are a common type of bacterial toxin that bind to the membrane of host cells forming
pores and resulting in cellular lysis (Bhakdi et al., 1996). Pore-forming toxins have been described as
"potent and versatile weapons with which invading microbes damage the host macroorganism," and they
are recognized as virulence factors in pathogens such as S. aureus and E. coli. They have also been
identified as virulence factors in enterococci, and studies on enterococci as early as 1921 utilized
hemolytic assays (Gilmore et al., 2002). Of the enterococcal hemolysins, the most well studied is
cytolysin, a unique hemolysin that also has bactericidal activity, perhaps due to distant relations to other
bacteriocins. This dual activity is a clear example of multifunctionality in enterococcal virulence traits.
Hemolytic activity can be assayed by culturing isolates with red blood cells (erythrocytes).
Isolates are grown on blood agar and colonies able to lyse erythrocytes are surrounded by a transparent
halo (Gaspar et al., 2009). Critical examination of this methodology, however, along with the advent of
genetic data, has revealed some discrepancies in this type of testing. These discrepancies may be due to
variables such as media contents, incubation conditions, or type of erythrocytes. In addition, genetic data
may conflict with phenotypic data because 8 different genes are required to express a positive
phenotype. In order to develop an accurate phenotypic assay for cytolysin, Gaspar et al. (2009) used
PCR specific to all 8 cytolysin genes to test the efficacy of various hemolysin assay procedures. The
researchers found that the assay was most accurate using Colombia blood agar supplemented with horse
erythrocytes and incubating anaerobically at 37C for 24-48 hours.
Some studies note a high incidence of hemolytic activity in clinical isolates (Ike et al., 1987;
Semedo et al., 2003). Others show the effect of its absence in experimental models of infection (Gilmore
et al., 2002). It may play a role in survival in the GI tract and translocation across intestinal epithelial
cells (IECs), and it is more certain to aid survival in the bloodstream. Cytolysin has killing action against
macrophages, PMNs, erythrocytes, and Gram-positive, but not Gram-negative, bacteria (Kayaoglu and
Oerstavik, 2004). It is easy to imagine a role in immune evasion considering its activity against
phagocytic immune cells, but in vitro experiments designed to test this hypothesis have so far been
inconclusive (Gilmore et al., 2002). The traditional perspective on hemolysins suggests that they may
32
increase fitness by releasing otherwise unavailable nutrients via cellular lysis. Lending support to an
etiological role of cytolysin in enterococcal pathogenesis, the probiotic strain Symbioflor® 1 does not
produce cytolysin (Domann et al., 2007).
Synthesis of cytolysin is complex, and requires the presence of 8 different genes (Gaspar et al.,
2009). These genes are typically encoded on plasmids, but occasionally are found on the chromosome
(Kayaoglu et al., 2004). The toxin itself actually consists of two separate peptides (CylLs" and CylLL"),
which are both required to elicit activity, and go through an elaborate modification process to achieve
their final active conformation (Gilmore et al., 2002). When first transcribed, the peptides are called
CylLs and CylLL. They are then post translationally modified within the cell to form CylLs* and CylLL*.
During secretion, a cysteine protease associated with cytolysin's membrane transporter cleaves leader
sequences from each of the peptides, at which point they are named CylLs' and CylLL'. Finally, the
peptides must be activated outside of the cell by proteolytic cleavage to form CylL s" and CylLL". A
membrane associated immunity protein is transcribed along with cytolysin to protect cells from their
own bactericidal toxin.
In addition to its pore-forming action, CylLs" also plays a role in quorum sensing, acting as an
auto-inducing peptide that regulates cytolysin expression via a novel mechanism that is not yet
characterized, but involves two regulatory components (CylR1 and CylR2) that suppress expression of
cytolysin at subthreshold levels of CylLs" auto-inducing peptide (Gilmore et al., 2002). Lending further
support to this model, Hew et al. (2007) found that cytolysin production correlates to cell density. The
presence of host cells also influences this regulation, as CylLL" binds preferentially to host cells, leaving
CylLs" free to function as an auto-inducer, stimulating high level cytolysin production (Coburn et al.,
2004).
Cytolysin is associated with EAS and pheromone mediated conjugative plasmids (Tanimoto,
1993), and Huycke et al. (1992) use a syrian hamster model show in vivo that pheromone-inducible
conjugation can effectively transfer hemolytic activity (along with antibiotic resistance) horizontally on
plasmids between E. faecalis strains. Genes for cytolysin and EAS are invariably located near one
another, such as in the case of pheromone responsive plasmids that encode for both (Gilmore et al.,
2002). Experimental models of pathogenesis also suggest that they may have a synergistic effect, as
strains expressing both traits were found to be 8 times more virulent than strains expressing either one
trait. This synergism may be due to bacterial clumping and its influence on quorum sensing; since
cytolysin, like the other virulence factors, is mediated by a quorum sensing mechanism, and requires a
critical local concentration of an inducer peptide in order to be expressed.
Gilmore et al. (2002) note that due to the nature of cytolysin auto-regulation, it is unlikely that
33
free bacterial cells in the bloodstream will produce high levels of cytolysin, since diffusion into vast
volumes of blood will inhibit auto-induction. However, microenvironments such as cellular aggregates,
biofilms, heart valve vegetations, and sites of intravascular coagulation may provide the conditions
necessary for local accumulation of auto-inducing peptide and high-level expression of cytolysin.
Curiously, isogenic mutants of the quorum sensing locus fsr (which is associated with biofilm
formation) show enhanced hemolytic activity in comparison to wild type cells (Pillar et al., 2003),
suggesting that while sex-pheromone mediated quorum sensing can enhance cytolysin expression,
GBAP mediated fsr quorum sensing actually attenuates this virulence factor. This relationship exhibits
the complexity of interactions between virulence factors, as their activities can be synergistic,
antagonistic, or independent of one another.
Horizontal Gene Transfer
Horizontal gene transfer (HGT) is the process by which mobile genetic elements are exchanged
between bacterial cells. Modern research in microbial genetics has elucidated the highly significant role
it plays in bacterial evolution, mediating processes such as "the rapid spread of antibiotic resistance
genes... [and] the evolution of pathogenic potential, metabolic diversity, and perhaps even the operon
structure of the genome itself " (Weaver et al., 2002). While it is not a virulence factor per se, its central
role in the evolution of bacterial populations make it a key factor in adaptation to the host environment
(as a commensal or as a pathogen), persistence in food production and health care environments, and
resistance to antimicrobial agents. Therefore, a basic understanding of HGT is requisite when discussing
enterococci and food safety.
Scientific research has identified three classes of HGT called transformation, transduction and
conjugation (Madigan and Martinko, 2006, 257-298). Transformation is the process by which free DNA
from lysed cells is randomly encountered by a recipient cell, taken up, and incorporated into
chromosome resulting in a genetic change. However, there are significant limitations on this type of
genetic exchange. First, because of the instability of long prokaryotic DNA molecules, outside the cell
they typically break into pieces about 10 kbp (about the length of 10 average genes). Although this is
enough to transfer significant traits (enterococcal cytolysin, for example, requires 8 genes for
expression), it does theoretically limit the amount of genetic data that can be transferred by this
mechanism. More significantly, not all cells have the capacity to 'be transformed'. A cell that is able to
take up free DNA and be transformed is said to be competent. Competence is regulated by special
proteins that uptake and process DNA. The percentage of cells that become competent and the length of
time that they remain competent during growth cycles differ widely between organisms. These factors
34
contribute to the relative inefficiency of transformation in comparison to other types of HGT.
Transduction occurs by incorporation of bacterial host DNA into the DNA of a bacteriophage
(bacterial viruses that commonly occur in microbial ecosystems), which can 'infect' other cells allowing
the transfer and incorporation of DNA into the new host (Madigan and Martinko, 2006, 257-298). These
phages, however, are typically not infectious in the traditional sense, since viral genes have been
replaced by bacterial genes. Transduced genes must be incorporated into the host chromosome or else
they will be lost because they cannot replicate independently.
Conjugation is the transfer of plasmids or chromosomally encoded transposable elements
through cell-to-cell contact, and is sometimes described as bacterial mating (Madigan and Martinko,
2006, 257-298). Plasmids are circular or linear segments of DNA that are not associated with the
chromosome. Plasmids range in size from 1 kbp to 1 Mbp in length, but most rolling circle replicating
(RCR) plasmids, which are the most significant in the context of gram-positive virulence and antibiotic
resistance, are less than 10 kbp in size, which is hypothesized to be the result of limitations imposed by
their mechanism of replication (Weaver et al., 2002).
Many bacteria, including enterococci, can contain multiple plasmids. In some cases, when a
plasmid that is taken up by a cell already containing a plasmid, the new plasmid will not be maintained
or replicated (Madigan and Martinko, 2006, 257-298). This is called incompatibility. Research has
identified that related plasmids sometimes cannot co-exist with one another within the same cell.
Groups of plasmids that cannot co-exist are called 'incompatibility groups'. Plasmids within an
incompatibility group exclude each other from replicating, but can coexist with plasmids from different
groups. This occurs because plasmids in the same incompatibility groups share mechanisms of
replication. They essentially compete for the resources required for replication, and only the plasmid that
confers the greatest adaptive advantage will remain. Plasmids are considered 'conjugative' if they encode
for their own transfer. When plasmid genes can be expressed by a recipient cell, it then becomes a donor,
which allows conjugative plasmid genetics to spread rapidly throughout populations. Plasmid encoded
traits include the ability to metabolize nutrients, bacteriocin production/resistance, virulence factors such
as hemolysins, and antibiotic resistances. A plasmid will typically persist in a population only if
selective pressure justifies the use of resources required to maintain it.
Transformation has not been a subject of discussion in the scientific literature, probably because
its inefficiency and inability to transfer large nucleotide sequences make its contribution to the transfer
of pertinent traits such as virulence factors and antibiotic resistances negligible. Although bacteriophage
based transduction likely does play a significant role in the transfer of these traits, this subject too has
not been the focus of scientific studies (Weaver et al., 2002). The conjugative transfer of mobile genetic
35
elements by plasmids and transposons, however, has received a lot of attention, and studies on this
subject have elucidated some unique and significant aspects of enterococcal evolution, ecology, and
pathogenesis.
HGT, Virulence, and Antibiotic Resistances
Enterococcal isolates typically possess from 1-7 plasmids (Palmer et al., 2010). Mobile genetic
elements in enterococci contribute to rapid adaptation and may also influence evolution through
mutation or hybridization of mobile elements. Many elements encoded on the chromosome are only
mobile via plasmid mediated horizontal transfer, and as noted above in discussions on EAS, species
specific pheromone responsive plasmids transfer by conjugation with great efficiency. Palmer et al.
(2010) speculate that the mechanism of HGT by pheromone-inducible conjugation "evolved to shuttle
niche specialization traits as E. faecalis strains from prey comingled with E. faecalis strains from
predators, allowing E. faecalis as a species to readily adapt to the dietary habits and other peculiarities of
particular hosts." Similar speculation could be extrapolated to suggest that this mechanism allows them
to adapt quickly to a variety of transient environmental factors, contributing to their ubiquitous nature
and their ability to colonize diverse niches. Whatever the evolutionary history of HGT in enterococci, in
the modern world its role in the transfer of antibiotic resistances has attracted much attention from the
scientific community and is at the heart of the debate on enterococci and food safety.
Experimental models show that conjugative plasmids containing virulence traits and antibiotic
resistances can occur during fermentation (Cocconcelli et al., 2003), although this occurs much more
efficiently in fermented sausages (10-3 transfers/recipient) than in cheese (10-6). For HGT to be a
significant factor in food safety, however, plasmids must also be able to transfer from populations in
food to populations in the consumer GI tract. Although a study by Huycke et al. (1992) exhibits the in
vivo capacity of E. faecalis to transfer a pheromone-inducible plasmid encoding erythromycin resistance
and hemolysin in the hamster GI tract, their experimental design does not reflect likely real-life
circumstances. Enterococcal overgrowth was induced by feeding antibiotics and subsequently
inoculating with 109-1010 CFU of streptomycin-spectinomycin resistant enterococci by orogastric
gavage. Three days later, hamsters were inoculated with 109 CFU of donor enterococci containing one of
three plasmids encoding erythromycin resistance and hemolysin production. Hamster stool was then
quantitatively tested for the presence of transconjugants (cells containing the donor plasmid). This
model was designed to mimic conditions experienced by hospital patients being treated with broad-
spectrum antibiotics that have little enterococcal activity, but even the authors note that the high levels
of inoculum used for orogastric gavage are unrealistic.
36
On the other hand, in traditional raw milk cheeses with relatively high levels of enterococcal
growth, populations after ripening generally range between 105-107 CFU/g (Franz et al., 1999),
indicating that in some cases, a 100 gram serving of cheese could provide as many as 109 CFU.
Therefore, the model used by Huycke et al. (1992) could be compared to a scenario in which a
hospitalized patient receiving antibiotic therapy eats a medium-large sized portion of cheese (aside from
differences in the anatomy of hamsters and humans and differences in environmental conditions of lab
animals and hospitalized humans). This suggests that cheese could potentially act as a carrier for
antibiotic resistances or virulence factors and that consumption of cheese by hospitalized patients
receiving broad-spectrum antibiotic therapy may increase the risk of enterococcal infection or facilitate
the spread of antibiotic resistances originating from agricultural environments.
These risks, however, may not be long term due to the fact that plasmids can be spontaneously
lost by a population in absence of selective pressures to justify energy expenditure on maintenance of
the plasmid (Madigan and Martinko, 2006, 279). For example, antibiotic resistance can be lost if no
antibiotics are present in environment. Indeed Huycke et al. (1992) find that transconjugants drop to
nearly undetectable levels within 7 days of inoculation, but more recent studies using similar methods
have produced conflicting results (Licht et al., 2002), one of which even demonstrates intergeneric
exchange of antibiotic resistance encoding plasmids from lactobacilli to enterococci (Jacobsen et al.,
2007). This study used a strain of Lactobacillus plantarum isolated from traditionally fermented
sausage.
The lactobacilli have not elicited the same concern regarding food safety in the scientific
literature as have the enterococci. They have a reputation as beneficial microorganisms with a protective
effect on fermented dairy products and possess the FDA designation "generally recognized as safe"
(GRAS) (Chung and Yousef, 2005) like most lactic acid bacteria. The Jacobsen et al. (2007) study,
however, suggests that even these benign organisms may play a role in compromising public health via
intergeneric dissemination of antibiotic resistances. This is a role which has also been attributed to the
enterococcus, which is not designated GRAS although it is a member of the lactic acid bacteria
(Fracalanzza et al., 2007). Safety concerns surrounding enterococci stem from its occurrence, if
infrequent, as an infectious agent, its capacity to harbor a number of intrinsic and acquired antibiotic
resistances, including resistance to penicillins, streptogramins, aminoglycosides, macrolides,
tetracyclines, ansamycins, phenicols, fluoroquinolones, nitrofurantoins, fosfomycins, and significantly,
the glycopeptides vancomycin and teicoplanin (Facklam et al., 2002).
Enhancing the level of concern is the ability of enterococci to mobilize its virulence and
resistance traits within its own genus and beyond. One of the most prevalent concerns regarding
37
horizontal transfer of enterococcal antibiotic resistances is the transfer of vancomycin resistance genes
to strains of methicillin-resistant Staphylococcus aureus (MRSA), a closely related but much more
virulent pathogen with a significantly larger impact on public health. Although the replication and
establishment of pheromone responsive plasmids is not observed in non-enterococcal hosts, pheromones
may induce the transfer of non-conjugative plasmids (Palmer et al., 2010). Although the exact
mechanism of transfer is unclear, 10 accounts in the US since 2002 document the transfer of
vancomycin resistance from enterococci to MRSA, and a 1992 study by Noble et al. shows the in vitro
transfer of vancomycin resistance from E. faecalis to S. aureus.
Since vancomycin represents the last line of
defense in antibiotic therapy of some infections
such as MRSA (Palmer et al., 2010), the
emergence of 'super-bugs' such as VRE and
vancomycin-resistant S. aureus (VRSA) is of
significant concern in the medical community,
and has prompted some to ask if we are not on
the verge of a re-emergence of untreatable and
fatal bacterial infections (Livermore, 2009).
Increases in prevalence of antibiotic resistance,
however, are associated with the frequency of
antibiotic use both in hospitals and agricultural
settings. For example, increased use of vancomycin as an antibiotic therapy in the US has been linked to
increasing rates of vancomycin resistance (Malani et al., 2002).
Similar phenomena have been observed with regard to casual antibiotic use in animal agriculture,
such as the high profile discovery that prevalence of VRE in european products of agriculture is
statistically correlated to the use of the aminoglycoside antibiotic avoparcin (which is cross-tolerant with
glycopeptides vancomycin and teicoplanin) as growth promoters (van den Bogaard et al., 2000). In
geographic regions where such practices are not authorized, VRE is not found in the food supply
(Fracalanzza et al., 2007), and following European bans on the use of avoparcin as a growth promoter,
rates of VRE have been found to decline sharply within the span of a decade (van den Bogaard et al.,
2000).
When populations of enterococci are exposed to antibiotics, the more antibiotic resistant variants
have a greater chance for survival, and this selective phenomenon fuels the evolution of resistant strains
38
Figure 7 - Electron micrograph image of S. aureus growing in a biofilm on the luminal surface of an indwelling catheter. An erythrocyte is also visible. Similar biofilms including both staphylococcal and enterococcal cells may be the site of intergeneric transfer of vancomycin resistance genes. Image retrieved from Stokowski (2008).
(Goldman, 2004). This is a straightforward concept backed up by modern understanding of microbial
evolution, yet despite pleas from the scientific community that the emergence of new antibiotic
resistance traits are "a cause for worldwide concern" (Moellering, 2010) and that "strong
epidemiological evidence [supports] a link between the use of antibiotics in human medicine and animal
husbandry and the emergence, spreading and persistence of resistant strains in animal products"
(Fracalanzza et al., 2007), irresponsible use of antibiotics remains a major global issue. The issue,
however, is becoming harder to ignore, and we may soon be forced re-evaluate protocols for the use of
antibiotics in both hospitals and agricultural settings if we hope to maintain the efficacy of antibiotic
therapy for when we really need it.
39
Part III: Framing the Real Issues Behind the Raw Milk Debate
What Makes an Enterococcus Pathogenic?
It is clear that the virulence traits cannot be considered in isolation, and that complex interactions
between these and other traits, environmental conditions, and host conditions all influence one another
in the evolution of an enterococcal population. The diverse distribution of adaptive traits in populations
of enterococci, along with the ability of these populations to efficiently exchange genetic material
through horizontal gene transfer probably contribute to their hardy, flexible nature, which allows them to
colonize a variety of niches and makes them ubiquitous organisms. Given specific permutations of
environmental conditions, these traits can work in concert to achieve a variety of goals, from surviving
on abiotic surfaces, to fermentation of dairy, to colonizing a host in either a commensal or pathogenic
manner.
An important question, however, remains inconclusive: do agricultural practices and food
processing practices provide the conditions in which virulent enterococci can emerge? Food production
environments have in the past been proposed as a stage for the evolution of virulence, as in the case of
the high profile pathogen E. coli O157:H7. Like the enterococcus, E. coli is typically a commensal
organism inhabiting the GI tract of humans and other animals. E. coli O157:H7, however, is a serotype
that has become a great concern to food safety due to the severity of illness that it causes in humans
(FDA, 2009). It has been suggested that the practice of feeding grain to cattle contributes to the
emergence or perpetuation of the pathogen in food products, although evidence to the contrary exists as
well (Hancock and Besser, 2006).
The fact that enterococci are not a cause of food-borne illness suggests that agriculture and
traditional processing practices such as fermentation do not fuel the evolution of particularly virulent
traits, and that their presence in cheese is not of great concern. Despite their inability to become
significant food-borne pathogens, their incidence as infectious agents in hospitals has been an important
subject of discussion in the medical and scientific communities. Scientific analysis of clinical isolates
suggests that certain strains of enterococci do possess enhanced virulence. Genetic analysis has led to
the identification of an enterococcal pathogenicity island, a group of over 100 genes including several
recognized virulence factors that can be mobilized for horizontal transfer as an integrated unit (Manson
et al., 2010). Phylogenetic analysis provides some evidence regarding the origin of these extra-virulent
strains, and the existence of a "hospital clade" of enterococci has been suggested (Ehrlich et al., 2008).
40
The hospital clade represents a lineage of enterococci that evolved to exploit a very specific, and
historically novel niche: the intensive care unit.
Mundy et al. (2000) note in a review on enterococcal virulence that most enterococci occur as
commensal organisms or environmental contaminants, and a relatively miniscule number undergo
natural selection as human pathogens. Since hospitals provide the unique conditions under which
selection for human pathogens can occur, it could be argued that this environment is a breeding ground
for the evolution and rapid horizontal transmission of virulence factors. However, I have argued in this
dissertation that many virulence factors emerge under multiple evolutionary pressures and are useful
outside the context of pathogenesis. In all likelihood, both scenarios occur, which can account for the
existence of virulence factors throughout diverse populations of enterococci as well as the increased
prevalence of virulence factors in clinical isolates.
At any rate, it appears that the clinical environment is much more significant than the agricultural
environment in the emergence of enterococci with enhanced virulence, although attenuated virulence
may be an intrinsic characteristic of all enterococci. From this perspective, enterococcal colonization of
the gut could be considered an unavoidable infection that impacts 100% of the population shortly after
birth. It is an infection, though, that is actually essential for health, and this is where a close examination
of the enterococcus really begins to turn traditional conceptions of virulence inside out. I propose that it
is not in spite of the presence of virulence factors, but because of them that this organism is essential for
health. Early and consistent challenge by enterococci with attenuated virulence may provide a safe
mechanism for training the innate immune system to recognize and control potentially invasive bacterial
pathogens.
Breast feeding could also be an integral aspect in encouraging growth of the proper enterococcal
populations and/or stimulating immune functioning in order to maintain a balanced microbiota.
Enterococcal populations in formula fed infants are on average 109.6 CFU/g of feces as opposed to breast
fed infants with an average of 106.3 CFU/g of feces (Tannock and Cook, 2002). Formula is designed
using processed cow's milk and supplemented to have a nutritional profile similar to human milk, but it
lacks many of the unique immune factors present in mother's milk. These factors likely play an
important role in establishing a balanced microbiota, especially in infants with developing immune
systems. Enterococci are also found in human milk and one study screening for virulence factors in
breast milk enterococcal isolates states:
The high concentration of enterococci in milk from healthy mothers strongly suggests that they may play an important biological role during the first months of life. Work is in progress to elucidate their potential to protect the newborn against infectious diseases and their role in the maturation of the infant gut-associated lymphoid tissue (Reviriego et al., 2005).
41
The authors could not find any virulence determinants in breast milk isolates, but virulence assays were
conducted 3 years after isolation of strains, and strains were routinely grown in brain heart infusion
broth. This suggests that virulence traits such as gelatinase could have been present in initial populations
and lost during lab handling, as has been shown in dairy enterococci (Lopes et al., 2006).
The use of enterococci as probiotics suggests they may have significant health benefits to adults
as well. Additionally, the current model of enterococcal infection discussed earlier in this dissertation
suggests enterococcal overgrowth is a crucial risk factor for enterococcal infection. Given these
observations as well as anecdotal reports of the health benefits of raw milk, an intriguing question arises.
Could unpasteurized cow's milk for adults act in a similar way to mother's milk for infants? As discussed
previously, scientific evidence to support or refute the health benefits of raw milk consumption is
lacking, but further studies on the interactions between the human gut microbiota, milk, and the immune
system could offer insight into this complex question.
Dairy Foods and Issues of Hygiene
Putting aside the potential immune benefits of exposure to low levels of bacteria, the
omnipresent risk of dairy products (or any food) causing food-borne illness can be drastically reduced
by using hygienic production and distribution practices. In this way, farms are much like hospitals,
where the single greatest tool for effective control of infectious disease is basic hygiene (Manchester,
2005). In the clinical setting, where patients are at high risk for enterococcal infection and strains with
enhanced virulence and antibiotic resistance are common, the spread of enterococci is a significant risk
factor for infection and the spread of antibiotic resistances. Controlling this transmission is an immense
challenge due to their ubiquitous nature. Enhancing the challenge is the fact that even when strict
standards are set in place, the tendency of some health care workers to resist compliance with standards
or to inaccurately report compliance makes monitoring and enforcing the standards difficult (Bay, L.M.,
MSN, RN, ACNS-BC, CCRN, Adult Health Clinical Nurse Specialist, personal correspondence).
In the dairy environment, effective implementation of hygienic standards is the single most
important factor in limiting the risk of food-borne illness outbreaks. Dairy producers, however,
encounter the same challenges as health care professionals: ubiquitous environmental contaminants and
difficulty enforcing protocols. This is why pasteurization is an important safety net when good hygiene
cannot be ensured, but for this same reason the practice of pasteurization may take the emphasis off of
strict hygienic standards. CDC data on the prevalence of Listeria monocytogenes in milk samples shows
that 5% of bulk milk samples prior to pasteurization are culture positive for the food-borne pathogen and
42
that 2% of pasteurized milk samples from over 700 U.S. dairy plants were culture positive for listeria
species, primarily L. monocytogenes (CDC, 1988). This calls into question both the hygienic standards
of dairy producers that intend to sell their milk to processing plants for pasteurization, as well as the
efficacy of pasteurization in protecting against food-borne illness.
Pasteurization is not the same as sterilization. It does not entirely eliminate any bacterial
populations, but only reduces them to levels that are very low and often undetectable using standard
microbiological plating methods. Although the most likely source of L. monocytogenes in pasteurized
milk is officially considered to be improper processing or post-pasteurization contamination, scientific
studies have raised questions regarding the efficacy of pasteurization in eliminating L. monocytogenes
(Doyle et al., 1987). Could remaining populations evolve high heat resistance just as many pathogens
have evolved resistances to multiple antimicrobial agents? This phenomenon has not yet been observed,
but the continuing efficacy of pasteurization as an effective food safety practice is challenged by the
theoretical possibility that pasteurization could select for pathogens with high heat tolerance, or that
heat-resistant microbes could, for any number of reasons, evolve pathogenic traits. While this scenario
may be unlikely, the chances of selecting for a novel trait are increased if pathogens are pooled together
and subsequently exposed to consistent selective pressures, as in both the case of injudicious
antimicrobial use in hospitals and compulsory pasteurization of bulk raw milk. Additionally, the effect of
heat shock on pathogenic populations may enhance their ability to survive stressful conditions, thus
enhancing virulence (Wong, 2010), although this hypothesis remains speculative.
Farmstead dairy products are by definition processed on the farm where the milk is produced.
This model affords some benefits to food safety as there is a shorter transportation and distribution chain
and the production and processing is monitored in a more personal way. Although they are found with
lower frequency than enterococci and other commensals such as fecal coliforms, food-borne pathogens
including L. monocytogenes, Salmonella, and Campylobacter are common residents of agricultural
environments (Pradhan et al., 2009). In light of this, the FDA's zero-tolerance policy on L.
monocytogenes might appear unrealistic, especially when applied to farmstead dairy operations. Other
countries have different policies, such as Canada, where up to 100 organisms per gram are allowed in
foods that have not been associated with an illness outbreak and do not foster the growth of L.
monocytogenes over a 10-day period of refrigeration (FDA, 2010). The Danish policy is much more
nuanced and includes several categories of food, but in raw ready-to-eat foods allows 2 of 5 samples to
contain between 10-100 organisms per gram, while no individual sample may exceed 100 organisms per
gram.
The zero-tolerance policy has been at the forefront of recent confrontations between the FDA and
43
farmstead cheesemakers such as the highly publicized case of Estrella Creamery in Washington State. In
this case, the FDA ordered the destruction of product representing countless hours of hard labor and
approximately $100,000 worth of sales (Neuman, 2010) based on the results of routine sampling which
identified L. monocytogenes in environmental samples and one product sample, despite the fact that no
illnesses have been associated with the farm's products. It is hard to say whether or not these tests
actually indicate a significant risk associated with the consumption of Estrella Creamery products. More
reliable data on the prevalence of pathogens in food and incidence of food-borne illness, as well as
continuing studies on the interaction between food-borne pathogens and the immune system are needed
to provide a realistic assessment of such risks.
While emphasizing the significance of hygiene in dairy environments is always of highest
priority, it must also be recognized that the production and processing of milk is not sterile even when
the most strict hygienic standards are applied. In cheese-making, these unavoidable environmental
contaminants are called non-starter lactic acid bacteria (NSLAB), and in spite of the addition of starter
cultures, the NSLAB will eventually become the dominant cultures responsible for the ripening of
cheese (Williams et al., 2002). The tenacity of the NSLAB is illustrated well by the following excerpt
from the text Fundamentals of Cheese Science:
Using aseptic conditions, it is relatively easy to produce curd free of NSLAB, but in our experience NSLAB always grow in such cheese, sometimes only after a long lag period (eg. 100 days). A cocktail of antibiotics (penicillin, streptomycin, and nisin) extends the lag period and reduces the final number of NSLAB (Fox et al., 2000).
The authors' language is initially confusing. While they claim to have produced "curd free of NSLAB",
the eventual growth of these organisms indicates that NSLAB were present in the initial curd, but
perhaps at levels so low as to be undetectable by typical microbiological techniques. Thus, even when
using modern materials such as milking machines, and metal or plastic implements that help maintain an
aseptic environment, milk collection and processing is not a sterile. Additionally, that NSLAB were able
to grow in the presence of multiple antibiotics indicates that these populations were diverse and included
individuals or populations harboring some degree of resistance to common antibiotics. As enterococci
are common members of the NSLAB in cheese and also typically harbor intrinsic and acquired
antibiotic resistances, it is likely that they are part of the tenacious populations described by Fox et al.
(2000).
It is also important to emphasize that the presence of enterococci in milk is not a reliable
indication of fecal contamination. Enterococci are considered a reliable index for the degree to which
water sources are contaminated with the feces of warm-blooded animals (EPA, 2002). To use this same
index for milk, however, is problematic, as fecal contamination is not likely the reason enterococci are
44
so common to dairy products. Enterococci, like other bacteria, are capable of colonizing abiotic surfaces
such as milking equipment and storage tanks, and surface contamination is well recognized as the
primary source of bacteria in modern milking systems (Akam et al., 1999). Cleaning of teats before
milking and use of milking systems that minimize exposure to the air render these sources of
contamination inconsequential. Regular cleaning of milking equipment minimizes surface
contamination, but very low levels of contamination will occur even under the most hygienic conditions.
These contaminants, however, will be of a defined and established microbial ecology so long as more
variable sources of contamination such as animal feces are not predominant.
One very interesting study summarized by Franz et al. (2003) provides evidence for the existence
of a defined dairy microbiota and gives some insight into the nature of this unique microbial ecology.
The study gives insight into sources of enterococcal contamination in raw milk cheddar cheese produced
on a small family farm in Ireland. The researchers used RAPD-PCR and PFGE to type enterococcal
isolates from dairy equipment, dairy products, and human fecal isolates. Their analyses showed that 3
clones (one of E. faecalis and two of E. casseliflavus) predominated in isolates from milk, cheese, and
human feces, and that the same clones were also isolated from bulk tanks and milking equipment. They
were able to show conclusively that enterococcal contamination was not of bovine fecal origin, as only
E. faecium and Streptococcus bovis strains were isolated from this source.
It is interesting to note that of the more diverse enterococcal populations colonizing the abiotic
surfaces of dairy equipment, only a specific subset were found in frequently in both dairy products and
human feces. It is also interesting to note that two of the strains were of the species E. casseliflavus,
which is considered native to plants and not the GI tract (Tannock and Cook, 2002). The researchers
speculate that the same enterococcal clones were found in the feces of the farmers because they
consistently ate the cheese containing these bacteria, not because the farmer's feces consistently
contaminated the cheese-milk. More studies of a similar nature are needed to better characterize the
nature of enterococcal contamination in traditional cheesemaking environments, but it is interesting to
consider that it is more likely a circular relationship than a linear one. The microbial ecology of the farm
environment, the processing environment, the farmers, and the consumers of the eventual dairy product
are all connected in complex ways that feed back on one another.
The microbial ecology associated with these relationships could be called 'the dairy microbiome',
and considering the integral connection between microbial ecologies and health, the factors influencing
the dairy microbiome could have far-reaching impacts on the health of both individuals and ecological
systems. The effects of different processing methods on this ecological relationship is another area
where continuing study is warranted, as it would appear that the act of processing milk products in any
45
way may fundamentally alter the ecology of its dairy microbiome.
Even in farmstead dairying, these relationships are highly complex. As dairy systems scale up,
become more centralized, and produce products that encounter a greater number of environments via
numerous production, processing, and distribution facilities, these relationship become even more mind-
boggling. While a circular relationship of some kind is still likely to exist, the impact of individual
actions are lost in a sea of multi-systemic interactions that in the modern age are not confined to
geographic localities, but are part of a global food system. Additionally, each environment encountered
by a dairy product has the potential to influence its hygienic quality, and as the number of check-points
increases, the likelihood that a product will encounter compromised hygiene at one or more of these
check-points and present an enhanced food safety risk is also increased.
Enterococci give interesting insight into the sources of bacterial contamination in dairy products.
This same issue, however, has been brought to the forefront of more significant political battles by
another gut-associated bacterium: coliforms. In 2006, six children in California became ill with E. coli
O157:H7 that was circumstantially linked to raw milk from Organic Pastures Dairy Co., the nation's
largest and most lucrative raw milk dairy (Gumpert, 2009, 4). Although the pathogen was never isolated
directly from any Organic Pastures (OP) products, the incident provoked considerable investigation into
OP dairy by state and federal authorities. It also ignited a political debate that would eventually result in
the proposal of AB 1735, a controversial California state law that would restrict the sale of raw milk
containing more than 10 coliforms per mL on the basis that levels higher than this would indicate that
the milk had been contaminated with feces (Gumpert, 2009, 187).
Like enterococci, coliforms can colonize surfaces and do not necessarily indicate fecal
contamination. Frustrated with publicly asserted accusations by the California Department of Food and
Agriculture (CDFA), California raw milk farmer Ronald Garthwaite exclaimed, "I am so sick and tired
of the CDFA telling people that our milk is contaminated with feces. It is not true. Our milk is not
contaminated with feces." As often seems to be the case when raw milk is the topic of discussion, this
exchange would scarcely seem out of place on an elementary school playground. The language of this
dissertation attempts to encompass the complexity of the microbiological world as it relates to dairy
safety and human health, from a molecular to an ecological level. In contrast, petty arguments remain at
the forefront of the discourse between farmers and regulators on highly significant topics such as food-
borne illness and fecal contamination of milk. This clearly indicates the inability of regulatory
authorities to apply basic concepts in dairy microbiology and engage in sophisticated discourse on the
subject.
In contrast to the idea that the presence of gut-associated bacteria in milk indicates a high risk of
46
that milk causing food-borne illness, enterococcal populations may confer protection against common
milk-borne pathogens such as L. monocytogenes through the production of bacteriocins (Giraffa, 2003;
Domann et al., 2007). Protection could occur on a more general level as well. With the application of
effective hygienic practices, the dairy microbiome becomes a mostly closed loop between the farmers
and the dairy products (with a minimal amount of milking and processing equipment in between),
environmental contaminants containing the occasional pathogen are kept to minimal levels and are
unable to compete with the predominant populations of the microbial ecosystem. If hygienic milk is
intentionally allowed to sour and is repeatedly used to inoculate fresh milk, the protective effect could
become yet more defined in subsequent cultured milk products. Occasionally, pathogens native to the
farm environment are likely to contaminate the milk at very low levels. Such low level exposure to
pathogens could over time cultivate enhanced immunity in consumers of raw dairy products.
Furthermore, since the pathogens that do make their way into the milk are the same ones that are
common to the local geographic region, raw milk may function as a tailor-made source of immunity that
confers the greatest benefits to local consumers.
Claims that pasture based dairy farms do not harbor pathogens or that raw milk cannot foster the
growth of pathogens are no more accurate than the claim that bacteria in milk are invariably indicative
of fecal contamination, and neither one of these extremist attitudes contributes to an earnest effort to
maintain public health. Human pathogens are inherent in natural ecosystems, especially those that
include animals. Hygienic milking practices and proper waste management are truly the best protection
against contamination by food-borne pathogens. Hygiene is easier to implement in small-scale pasture
based dairy farms due to the personalized nature of milking and even distribution of waste that occurs
naturally as cows are rotated throughout pastures. As opposed to being a health hazard, this waste is a
valuable and rapidly utilized fertilizer that leads to healthier pastures, healthier cows, and healthier milk.
Furthermore, conscientious raw milk advocates take hygiene very seriously, and detailed
resources on hygienic practices for the safe production of raw milk have been made openly available
(FTCLDF, 2008). Most raw dairy farmers are aware of the risk associated with unpasteurized milk and
are dedicated to producing a safe product, as both their reputations and the health of their consumers
(who they may know personally) are at stake. For these reasons, raw milk produced on small-scale
pasture based farms is typically of the highest hygienic quality. Nevertheless, it is still best for the
consumer to know the farmer, to have the opportunity to ask questions and/or directly experience the
conditions on the farm and the procedures implemented to minimize risk. Centralized dairy systems,
however, afford no such opportunity, and therefore the practice of compulsory pasteurization, even if
problematic and occasionally ineffective, is truly a necessity for consumer safety.
47
Farmstead Dairy and the Ecological Integration of Modern Communities
As exhibited by the circular nature of enterococcal contamination, it appears that dairy
enterococci are actually part of a unique ecosystem involving a diversity of microbes, plants, and
animals, in addition to human stewardship and dairy processing practices. It also appears that processing
milk on the farm using traditional fermentation practices can cultivate an intimate and integrated
relationship between the dairy microbiome, the farmer, and the consumers. Finally, it seems that
ecologically minded stewardship practices also contribute to the integrity of this relationship, and may
help to build ecological health, which then extends to the health of individuals participating in that
ecosystem.
Considering the most significant issues at stake in the raw milk debate, it could be highly
beneficial to move away from the emotionally charged term 'raw milk', and move towards the use of a
term such as 'farmstead milk' that more accurately reflects the values and ecological stewardship
practices supported by advocates of raw milk. Farmstead milk products could be raw, heat-treated,
cultured, or otherwise processed on the farm according to the needs and standards of local consumers.
Let us also keep in mind that milk could be boiled at home by the consumer. This accessible, low-tech
practice is the predominant mode of protection from pathogens in other parts of the world (Gumpert,
2011[1]), and actually offers greater protection by limiting the potential for post-heat-treatment
contamination.
Farmstead dairy offers a much more intimate integration between human beings and the
ecological systems they inhabit. This does not, however, suggest the plausibility of an idealistic world
filled with farmstead milk that is healthy for all people all the time, and two important caveats must also
be taken into account. First of all, individual heterogeneity in sensitivity to foods and susceptibility to
illnesses due to both environmental and inherited factors suggests that individuals must be conscious of
how their food choices impact their personal health, and not assume that the dietary choices of a healthy
individual will also benefit their own health. Secondly, it must be considered that the act of
consumption, or any other type of interaction with the outside world, brings with it some risk of
exposure to pathogens.
Individual heterogeneity and risk are fundamental attributes of biological and ecological systems
and their eradication does not seem likely nor on the whole desirable. In order to make an informed
decision, consumers must be conscious of the multitude of environmental and individual factors that
influence the potential health impacts of dairy consumption. These include (but are not limited to) the
agricultural practices of the farm from which the milk originated and the way the milk is subsequently
48
processed, epidemiological trends of infectious diseases relevant to the geographic region, and the
resilience afforded by an individual's immune system. The acquisition of such specific information relies
on access to local foods of which the agricultural production practices can be identified, decentralized
acquisition of and access to information on the health of local populations, and individual awareness of
one's own state of health. In contrast, the federal campaign against raw milk production decreases access
to local foods and offers highly centralized national epidemiological data, the statistical analysis of
which is questionable (Gumpert, 2009, 122-123). Furthermore, it encourages consumers to assume that
all foods are safe if they are endorsed by the FDA, produced by regulated and centralized food systems,
and available at authorized retail outlets. It does not serve to encourage the challenging and often
unpleasant process of personal self-reflection that is necessary to cultivate awareness of one's state of
health. Instead, it offers consumers a convenient, but highly ineffective cop-out. While such a campaign
may be perpetuated in the name of public health, it seems detrimental to its own cause in light of the
basic requirements for informed decision making.
Unfortunately, avoiding emotionally charged terminology on this topic is a much greater
challenge, as it is a topic that is inextricably connected to the very realistic human fear that good health
is transient, and could at any moment be taken away. That this fear is realistic, though, does not justify
its Orwellian exploitation by the interconnected systems of the dairy industry, the pharmaceutical
industry, and government regulatory authorities. There is an immense amount of individual profit to be
had in the business of centralized food distribution, and perhaps even more in protecting the public from
the perils of such a system. The pursuit of individual profit, however, is not a motive that contributes to
increased health on a population level. To be clear, making war with microbes and farmers is not a
productive approach to public health.
What is needed are not systems that allow a select few individuals to gain wealth, but systems
that allow the ecological community to maintain health. Observation of natural ecologies indicates that
such systems involve sacrifice, imperfection, and risk on an individual level. Current systems protect the
public from food-borne illness in the short-term, but in doing so perpetuate centralized systems of food
production and distribution that cannot fit within an ecological framework. This is ultimately the big
issue behind the raw milk debate, and the fact that many brush off raw dairy as simply the latest "foodie
fetish" is indicative both of public ignorance regarding the current state of global food systems, and the
inability of raw milk advocates to accurately articulate the various and complex issues that are actually
at stake.
Supporters of sustainable agriculture clearly cannot make the vastly idealistic argument that
returning to the ecological framework that will make pathogens disappear, nor that such a framework
49
will bring about the end of human suffering. Instead, we must find the words to articulate a much more
difficult and realistic argument. Although modern civilizations can lay claim to many great
accomplishments, particularly in the realm of individual freedom and prosperity, these accomplishments
have often been achieved by flouting a fundamental ecological truth. If we hope to establish a truly
sustainable human culture, we must integrate within ourselves the understanding that individual
sacrifice, imperfection, and risk, ubiquitous as they are to the human experience of suffering, are also
vital to ecological health.
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Part IV: Assessment of Gelatinase Activity in Enterococci Isolated from Local Milk
Abstract
The enterococci are Gram-positive, facultative anaerobes, and are ubiquitous colonizers of the
gastro-intestinal (GI) tract of humans and other animals, an assortment of processed and fermented
foods, as well as hospitals, farms, and other domestic and wild environments. Within the last 20 years,
they have emerged as significant hospital acquired infectious agents. Within the same time frame,
research has elucidated their presence in traditional foods, reaching high levels in some kinds of
traditional cheese with no apparent ill effects, and several strains have even been tested and marketed as
probiotics. The aim of this research was to isolate naturally occurring enterococci from local dairy
products (including both raw and pasteurized milk), and screen isolates for the production of the
virulence-related factor gelatinase, which was assayed based on the ability of isolates to hydrolyze
gelatin. Isolates were successfully obtained from raw milk, while levels of viable cells in pasteurized
milk were too low to do so. Gelatinase could not be identified in isolated strains, but previous research
suggests that lab handling may silence expression of this trait despite its presence in the original isolates.
The results of this study elucidate some significant issues and challenges in food microbiology research.
In addition, this field of research poses many provocative questions in relation to hygiene, health, and
the politics of food.
Introduction
Gelatinase is a hydrolytic enzyme that can degrade a variety of substrates including gelatin,
collagen, and casein (Lopes et al., 2006). It is considered to be a virulence factor in enterococci due to
its association with clinical isolates, but it also occurs in food isolates, probably due to its ability to
break down proteins and aid in nutrient extraction in a variety of environments. The dualistic nature of
enterococci as etiological factors in both health and disease is increasingly recognized in the scientific
community, and questions regarding the conditions influencing enterococcal virulence have been raised
(Hew et al., 2007).
Enterococci are common to dairy products and are found in particularly high levels in
unpasteurized dairy products (Lopes et al., 2006) such as traditional European raw milk cheeses, in
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which their presence is considered to have a beneficial effect on cheese ripening (Giraffa, 2003).
Enterococci have also been found in pasteurized milk (Fracalanzza et al., 2007). Contrary to their
beneficial effect on traditional cheeses, in pasteurized industrial cheeses their presence is considered an
indication of poor hygiene and is associated with undesirable effects on the sensory aspects of these
products (Giraffa, 2003). This suggests that pasteurization or factors associated with it may alter
attributes of enterococcal populations. More likely, pasteurization may have broad effects on the
microbial ecology that alter the influence of enterococcal populations. In the context of questions of
food safety, and considering the fact that the expression of virulence factors may be affected by food
processing conditions (Hew et al, 2007), it is important to characterize the effect of pasteurization on
virulence determinants such as gelatinase. Therefore, this study seeks to confirm the presence of
enterococci in local raw and pasteurized dairy products and to collect data regarding the prevalence of
gelatinase in enterococcal isolates from these populations. The goal of this research is to contribute to an
understanding of virulence factors in enterococcal populations from raw and heat-treated dairy products.
In an attempt to accomplish this goal, samples of raw and pasteurized milk were plated on
selective growth media to isolate presumptive enterococci. However, only raw milk samples yielded any
growth. In order to provide evidence that isolates were indeed enterococci, they were tested for their
ability to grow in the presence of bile salts, and to hydrolyze esculin, which are both characteristic traits
of the genus. Isolates were then stored on agar slants under refrigeration for later testing. After reviving
cultures in BHI broth, isolates were tested for their ability to grow in the presence of 6.5% NaCl
(another characteristic trait of the genus), and to hydrolyze gelatin.
Materials and Methods
Preparation of Growth Media
Slanetz and Bartley (SB) growth media (Oxoid) and Bile Esculin Azide (BEA) growth media
(Acumedia) were obtained for the purposes of this experiment and prepared according to manufacturer's
instructions. Detailed preparation protocols as well as a comparative analysis of media composition are
included in the appendix. All other media utilized Brain Heart Infusion (BHI; Oxoid) as a nutrient
source. BHI storage slants were made with 1 g agar in 80 mL medium (protocol detailed in appendix).
For NaCl assays, 3 grams of agar and 13 grams of NaCl were used for 200 mL of medium. For
gelatinase assays, 3 grams of agar and 6 grams of gelatin were used for 200 mL of medium.
Obtaining and Culturing Milk Samples
Milk was collected using a mobile milking machine that had been routinely sanitized by the
59
farmer. The milk was filtered into a metal container and then poured into a heat sterilized glass jar. The
jar was immediately closed and transported by foot directly to the lab (approx. a 15 minute walk) where
it was stored at 3C. After 3 days of refrigeration a serial dilution was prepared with the milk. 1 mL
aliquots of undiluted milk (0), 1/10 diluent (-1), 1/100 diluent (-2), and 1/1000 diluent (-3) were each
plated in duplicate on previously prepared 20 mL SB agar plates. Two plates were left empty to check
for contamination. Plates were then incubated at 37C and checked daily for growth until they were
removed on for observation after 70 hours of incubation. Detailed protocols can be found in the
appendix.
100 mL SB growth media was prepared for isolation of enterococci from pasteurized milk. Aside
from the difference in volume, the mixing protocol was the same as outline in the appendix. Pasteurized
milk was purchased from a local grocer on the day of the experiment. The milk was contained in a
factory sealed plastic bottle, was stored at 3C while in the lab, and was not opened until time of
sampling. 1 mL aliquots of pasteurized milk were deposited into pour plates in several evenly distributed
droplets using a sterile pipet tip. One plate was left empty to check for contamination. Cooled but still
liquid media was then poured into the plates. Plates were then incubated at 37C and checked for growth
daily until after 4 days it became apparent that no growth would appear.
Storing Cultures on BHI Agar Slants
BHI agar slants were prepared and cultures from BEA plates were streaked onto slants and stored
under refrigeration for storage. Detailed protocol is included in the appendix. Cultures were revived in
BHI broth for gelatinase and NaCl tolerance assays.
Assays for Esculin Hydrolysis, Gelatinase Hydrolysis, and NaCl Tolerance
For all assays, isolates were streaked onto agar plates containing their respective growth media
and amendments and incubated at 37C for 24-48 hours. A detailed protocol for streaking on BEA agar
plates is included in the appendix. All other assays utilize this streaking procedure aside from the fact
that in subsequent assays, each isolate was streaked on an individual plate, instead of splitting plates in
half as in the case of BEA. Criteria for assay interpretation are as follows: Isolates were considered
positive for esculin hydrolysis if a black halo appeared around colonies after incubation. Isolates were
considered positive for gelatinase if a clear halo appeared around colonies after incubation. Isolates were
considered NaCl tolerant if colonies appeared after incubation.
Results
60
Only undiluted samples displayed any growth, one of which grew 2 colonies while the other
grew 6. One of the 1/10 plates had a very small black dot that could potentially be a colony, but this is
unclear as it does not look similar to the colonies on the 0 plates. These colonies are a very deep red
color and are ringed in pinkish white. The shape of the colonies was diverse, ranging from relatively
uniform circles, to indented patterns reminiscent of flower petals, to more irregular splotchy shapes. One
colony showed a particularly interesting distinguishing feature. It had grown in a visible film of dried
milk solids and there appeared to be a semi-translucent halo ringing the colony, which could be a
preliminary indication of gelatinase activity. Attempted isolation of enterococci from pasteurized milk
using SB growth media yielded no results.
Substantial growth was apparent on BEA agar
plates 24 hours after inoculation with SB isolates.
Large black splotches appeared on all of the plates.
Some colonies were successfully isolated in more
dilute streaks and all of these were surrounded by
black halos. This indicates that all of the colonies on
the initial SB plates were capable of hydrolyzing
esculin. While this evidence is not conclusive, it does
support the hypothesis that colonies isolated from SB
agar are of the genus Enterococcus.
Only 7 of the 10 isolates showed turbidity after inoculation in BHI broth. Samples of broth from
all tubes were still plated, but indeed the 3 isolates that did not show turbidity also did not show growth
on agar, aside from two individual colonies on two separate gelatin agar plates, which were determined
to be contaminants. Of the isolates that were successfully recovered, all of them tolerated 6.5% NaCl.
However, no zones of clearance whatsoever were observed around any of the colonies growing on
gelatin agar. The results of all 3 assays are summarized in table 1.
Discussion
In order to interpret the results, an understanding of the composition and function of the media
used is necessary. A detailed list of the contents of the two media is included in the appendix as a
reference.
In some ways, SB agar and BEA agar are very similar in content, but there are distinct
differences as well. Both contain some form of enzymatically digested proteins and yeast extract, which
are typical staples in growth media as sources of nitrogen and various other nutrients. In SB medium,
61
Isolate Esculin 6.5% NaCl GelRME1 + + - RME2 no growth no growth contaminatedRME3 no growth no growth contaminatedRME4 + + - RME5 + + - RME6 + + - RME7 + + - RME8 no growth no growth no growthRME9 + + -
RME10 + + - Table 1: Raw Milk Enterococcus (RME) 1-10 were assayed for esculin hydrolysis, NaCl tolerance, and gelatinase activity. Positive assay (+), negative assay (-), inability to revive cultures after storage ("no growth"), and suspected contamination ("contaminated") are indicated.
tryptose is the main nitrogen source. Tryptose is considered superior to meat peptone as a single source
of nitrogen for fastidious organisms (Bacto™ Tryptose Product Information). This may be why BEA
medium contains two major protein sources. One of these is an enzymatic digest of casein, or milk
protein. While this may not be nutritionally sufficient for the diversity of fastidious organisms that
tryptose is, it makes sense that it would be used for culturing enterococci, which have metabolic
capabilities that are well adapted to the dairy environment. BEA medium also contains yeast enriched
meat peptone as a secondary source of nitrogen as well as a variety of other nutrients associated with
yeast. Perhaps most importantly are B-complex vitamins, which are abundant in yeast extract and are
essential nutrients for the growth of enterococci. SB medium contains yeast extract as a separate
addition. The total amount of protein and yeast extract in SB medium is 25 g/L, while BEA contains
34.5 g/L, making it a more nutrient rich medium.
SB medium also contains 2 g/L glucose, a common monosaccharide that is used by many
microorganisms as a source of carbon and energy. Using this specific monosaccharide and excluding
others might also help to select for certain organisms. BEA medium does not contain any sugars, but it
does contain both sodium citrate (1 g/L) and ferric ammonium citrate (0.5 g/L), which could potentially
serve as a source of carbon and energy, as enterococci are capable of metabolizing citrate. These salts
also perform other functions, though. They can act as a buffer to maintain the medium at a stable pH. SB
medium also includes 4 g/L of buffer salts in the form of di-potassium hydrogen phosphate.
Ferric ammonium citrate performs a unique function in BEA medium. It reacts with hydrolyzed
esculin forming a black pigment which can be observed as a halo around colonies. In this way colonies
that can hydrolyze esculin (a distinguishing feature of enterococci) can be differentiated from those that
do not. This is the main function that esculin serves in BEA medium. SB medium contains a visual
enhancer as well. Tetrazolium chloride (0.1 g/L) is added because it is reduced inside of bacterial cells
forming a red pigment. This coloration is apparent in the pink-dark red appearance of colonies that form
on this medium and makes them easier to see, especially if many need to be counted for enumeration.
The other main components of these media (besides agar, which is used simply to keep the medium
solid at room temperature) serve as environmental stressors or toxins to select for enterococci. Both
media contain sodium azide, which is highly toxic to aerobic organisms and helps select for enterococci,
which are facultative anaerobes. SB medium, however, contains 0.4 g/L; this is nearly twice as much as
BEA medium, which contains 0.25 g/L. Additionally, much of the sodium azide in BEA medium is
inactivated in the autoclave, where high temperature and pressure cause it to react with other
components in the medium. Thus, in reality, the active amount of sodium azide in BEA is significantly
less than that reported in its initial contents.
62
The heat susceptibility of sodium azide is the main reason why SB medium is gently heated
instead of being autoclaved, since SB medium is designed for enumeration of enterococci from water
samples, which could contain a vast diversity of microbes both aerobic and anaerobic. Therefore
stronger selection against aerobes might be beneficial. Interestingly, this is the only selective agent in the
medium. Perhaps the specificity and sparsity of nutrients in SB media acts sufficiently to select for
enterococci. In addition to sodium azide, BEA medium contains oxbile. This helps select for enteric
organisms, which must be able to tolerate environmental stressors in the GI tract such as bile salts and
acids. Finally, BEA medium contains 5 g/L of NaCl, which selects for enterococci due to their
remarkable salt tolerance. At approximately 7.2 and 7.1 respectively, these media have very similar pH
profiles, both being relatively neutral.
The results of the serial dilution suggest a very low level of enterococcal contamination in this
sample of fresh raw milk. The deep red coloration and white border of colonies is consistent with
expectations and indicates that these are likely colonies of Enterococcus species. A simple average of the
number of colonies suggests that there are approximately 4 CFUs of enterococci per mL of milk in this
sample. However, the number of colonies on both plates was so low that these estimates are not really
statistically valid. Furthermore, the variation in colony shape may suggest that one "colony forming
unit" could actually consist of two or more aggregated cells, especially given the tendency of
enterococci to from pairs, chains, and clumps.
That enterococci were undetectable in pasteurized milk samples suggests that low levels of
contamination in raw milk are reduced further by the process of pasteurization. This alone is an
interesting result, given the hypothesis that low level contamination of raw milk is in part responsible for
its health benefits. The capacity of pasteurization to reduce low level contamination to undetectable
contamination could therefore have a significant effect on its heath benefits. Reduced exposure to low
levels of enterococci may reduce the ability of the immune system to keep these ubiquitous infectious
agents in check, and pre-dispose individuals consuming pasteurized milk to enterococcal infection.
Colonies isolated from raw milk on SB agar and transferred to BEA agar did have the ability to
hydrolyze esculin. This is another positive indicator that enterococci were successfully isolated. In
routine enumerations, such as in water quality testing, these parameters are considered enough to assume
that the isolates are enterococci (Condalab, 2011). More in depth identification methods, such as
metabolic and serological assays, or protein-based and genetic fingerprinting, are used in scientific
studies to identify further to the species and strain level (Domig et al., 2003). Whether or not any of
these methods will be necessary will depend on future experimental goals.
The follow-up assay provides some interesting results as well. The ability of all isolates to
63
tolerate 6.5% NaCl is further evidence indicating their identity as enterococci, and suggests that they
would likely persist in cheeses produced with this milk. If aged sufficiently, enterococci could
potentially become a dominant organism. The results of the gelatinase assay did not indicate any
gelatinase activity in these isolates, but for now these results must be considered inconclusive. Although
the Gel+ phenotype is more prevalent in clinical strains than in food strains (Semedo et al., 2003), there
is strong evidence to suggest that enterococci in dairy products do produce gelatinase to some extent,
and that this expression my be silenced as a result of lab handling (Lopes et al., 2006). It is therefore
possible that some of the isolates obtained in this study had a Gel+ phenotype when they were initially
isolated, but lost this phenotype upon repeated sub-culturing and prolonged storage. This hypothesis is
supported by the observation that one of the colonies isolated on SB agar had grown in a film of dried
milk solids and formed a zone of clearance, which could be an indicator of proteinase activity,
potentially gelatinase. Finally, with regards to the results of this and other assays, the presence of
suspected contaminants on two separate gelatin agar plates suggests the potential for other unknown
contaminants, which must be considered when interpreting the results.
Gelatinase has been identified in both food and clinical isolates (Semedo et al, 2003), and is one
of the most well studied virulence factors in enterococci. Other virulence factors may provide more
promising areas of research. Although the production of capsular polysaccharide has been identified in
some enterococci, there is far less data in the primary literature on this subject. In particular, the
prevalence of capsular polysaccharide in food isolates does not appear to be addressed. Since capsule
production helps enterococci evade opsonization by phagocytes, a hypothesis could be formed that this
trait would be less common in isolates obtained from products made with pasteurized milk, as opposed
to raw milk, which still contains living phagocytes (Silanikove, 2008).
Even if this were the case, however, it does not necessarily suggest that enterococci from heat
treated cultured products are less dangerous. Although capsule is well defined as a factor that aids
immune evasion and therefore contributes to virulence, the expression of capsular polysaccharide by the
probiotic strain Symbioflor® 1 (Domann et al., 2007) indicates that capsule production and probiotic
effect are not mutually exclusive phenomena. On the contrary, it suggests that the presence of capsule in
naturally occurring populations of enterococci may actually be a boon to health. Further studies
regarding the interaction between encapsulated enterococci and the immune system both in vitro and in
vivo could be useful in characterizing the factors influencing the effect of enterococci on health.
Gelatinase, on the other hand, is absent from Symbioflor® 1, and there is evidence to suggest (as
argued in the preceding dissertation on enterococcal virulence) that capsule and gelatinase could have a
synergistic effect. Therefore, an interesting follow-up study might involve obtaining enterococcal
64
isolates from both raw and pasteurized dairy products that have been cultured at 37C and testing for the
prevalence of both capsule and gelatinase production. Points of interest in this study would include
comparing the prevalence of these traits in populations cultured in raw and pasteurized milk, and
analyzing the relationship between the two traits. Do the traits ever occur in the same isolates? If so, is
there any statistical correlation between the two traits? All of these questions could be answered with
more extensive follow-up studies, indicating the need for further research on the subject.
Sources Cited
Bacto™ Tryptose Product Information. Accessed on 4/27/2011 at http://www.bd.com/ds/technicalCenter/inserts/Bacto_Tryptose.pdf.
Condalab catalogue. Slanetz-Bartley Medium: For the Detection and Enumeration of Enterococci by the Membrane Filtration Technique. Accessed on 4/17/2011 at http://www.condalab.com/pdf/1109.pdf.
Domann, E., Hain, T., Ghai, R., Billion, A., Kuenne, C., Zimmermann, K., and Chakraborty, T. (2007). Comparative Genomic Analysis for the Presence of Potential Enterococcal Virulence Factors in the Probiotic Enterococcus faecalis Strain Symbioflor 1. International Journal of Medical Microbiology 297: 533-539.
Domig, K.J., Mayer, H.K., and Kneifel, W. (2003). Methods Used for the Isolation, Enumeration, Characterisation and Identification of Enterococcus spp. 2. Pheno- and Genotypic Criteria. International Journal of Food Microbiology 88: 165-188.
Fracalanzza S.A.P., Scheidegger, E.M.D., Santos, P.F., Leite, P.C., and Teixeira, L.M. (2007). Antimicrobial Resistance Profiles of Enterococci Isolated from Poultry Meat and Pasteurized Milk in Rio de Janeiro, Brazil. Memórias do Instituto Oswaldo Cruz 102(7): 853-859.
Giraffa, G. (2003). Functionality of Enterococci in Dairy Products. International Journal of Food Microbiology 88: 215-222.
Hew, C.M., Korakli, M., and Vogel, R.F. (2007). Expression of Virulence-Related Genes by Enterococcus faecalis in Response to Different Environments. Systematic and Applied Microbiology 30: 257-267.
Lopes, M.F.S., Simoes, A.P., Tenreiro, R., Marques, J.J.F., and Crespo, M.T.B. (2006). Activity and Expression of a Virulence Factor, Gelatinase, in Dairy Enterococci. International Journal of Food Microbiology 112: 208-214.
Semedo, T., Santos, M.A., Lopes, M.F.S., Marques, J.J.F., Crespo, M.T.B., and Tenreiro, R. (2003). Virulence Factors in Food, Clinical and Reference Enterococci: A Common Trait in the Genus? Systematic and Applied Microbiology 26: 13-22.
Silanikove, N. (2008). Milk Lipoprotein Membranes and Their Imperative Enzymes. From Bioactive Components of Milk. Bosze, Z. (ed). Springer: 143-161.
65
Appendix
Mixing SB agar selective growth medium for isolation and enumeration of enterococci Materials: latex gloves, mask, goggles, hot plate, stir bar, metal spatula, thermometer, 500 mL graduated cylinder, 500 mL beaker, bunsen burner, striker, Slanetz and Bartley (SB) pre-mixed selective growth media, scale, weigh paper, ultra-pure water (UPW), aluminum foil
For 200 mL SB growth medium:1. Put on gloves, a mask and goggles to avoid exposure to sodium azide. 2. Set up hot plate underneath a vented hood, turn on the hood. 3. Light the bunsen burner. 4. Gather stir bar, a metal spatula, a thermometer, a 500 mL graduated cylinder, and a 500 mL beaker. Briefly flame to sterilize. 5. Using the spatula, gently spoon 10.5 g of dry SB mixed media onto a scale with weigh paper. Take care to do this gently, producing as little dust as possible. 6. Dump the dry mix into a 500 mL beaker, taking care to cover the beaker for a moment with the weigh paper and a gloved hand while the dust settles. 7. Carefully transfer the beaker into the hood. 8. Measure 250 mL UPW in a graduated cylinder and pour into the beaker under the hood. 9. Stir to mix with the metal spatula. 10. Place the spatula on used weigh paper to minimize spread of toxic SB media. 11. Place the stir bar in the beaker and place the beaker on the hot plate. 12. Take a piece of aluminum foil and use it to cover the beaker. 13. Turn the heat on high and use the magnetic stir mechanism to create a vortex in the fluid. 14. Monitoring temperature with the thermometer, heat until there is no more visible undissolved powder and bubbles begin to appear at the bottom of the beaker (about 90C). 15. Remove from heat and let stand uncovered with a thermometer, checking the temperature periodically until the it reaches approximately 40C. 16. Arrange 10 petri plates in stacks of 2 or 3 underneath the hood. 17. Pour liquid SB media into petri plates, taking care to not lift the lids for very long.18. Make sure to leave a note indicating the toxicity of the media. 19. Allow to stand and cool until agar has firmly set.
Mixing Phosphate Buffered Saline (PBS) for serial dilutions of refrigerated raw milk Materials: NaCl, KCl, Na2HPO4 , KH2PO4, scale, weigh paper, ultra-pure water (UPW), HCl solution, NaOH solution, 500 mL graduated cylinder, 500 mL pyrex bottle with cap, pH meter, pipette
1. Using a scale and weigh paper, measure 8.00 g of NaCl, 0.20 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4, mixing them together in a 500 mL pyrex bottle. 2. Measure 400 mL of UPW in a graduated cylinder and pour into the bottle to dissolve buffer salts. Use a plastic stir rod to break up chunks if needed. 3. Measure pH and adjust to 7.4 using a pipette and solutions of HCl or NaOH. 4. Pour buffered solution into a graduated cylinder and add UPW to 500 mL 5. Return buffered solution to the bottle. Cap, label, and store under refrigeration. Autoclave before use.
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Serial Dilution of Raw Milk from Hampshire College Farm Center Materials: phosphate buffered saline (PBS), 6 test tubes with caps, test tube rack, 50 mL beaker, micropipette, 1 mL sterile pipette tips, bunsen burner, striker, glass hockey stick spreader, 10 petri plates prepared with Slanetz and Bartley selective growth media, marker for labeling, ethanol, kimwipes, parafilm, raw milk (collected from HCFC and stored under refrigeration)
1. Place 6 test tubes each filled with 9 mL PBS in a rack and lightly place the caps on top. 2. Autoclave this along with a 50 mL beaker to sterilize. 3. Begin to prepare the workspace by lighting the bunsen burner. 4. Label tubes 3 tubes: -1, -2, -3. Label the other 3 tubes: -1*, -2*, -3*. 5. Label 5 SB agar plates: raw milk, -1, -2, -3, and control. Do the same for the other 5 plates, differentiating with * in the same fashion as the second set of tubes. 6. Arrange the plates in front of the test tube rack so that all are easily accessible. 7. Fill the beaker with refrigerated milk and place it next to the test tube rack. 8. Take the pipette with a sterile tip and transfer 1 mL raw milk from the beaker onto each of the corresponding petri plates. 9. Uncap and flame tube -1, then transfer 1 mL raw milk into the tube. Flame, recap, swirl to mix, and place in rack. 10. Repeat step 9 for tube -1*. 11. Discard the used pipette tip and replace with a new sterile tip. 12. Uncap and flame tube -1 and place in rack uncapped. 13. Transfer 1 mL diluent from tube -1 onto the corresponding petri plate. 14. Uncap and flame tube -2, then transfer 1 mL diluent from tube -1 into tube -2. Flame, recap, swirl to mix and place in rack. 15. Recap tube -1. 16. Repeat steps 11-15 using tubes -1* and -2*; tubes -2 and -3; and tubes -2* and -3*. 17. Repeat steps 11-13 using tubes -3 and -3*. 18. Surface sterilize the hockey stick spreader first with a kimwipe and ethanol, and then by briefly flaming and waiting a moment for it to cool. 19. Use the sterilized hockey stick to spread the sample over plates labeled raw milk and raw milk*. 20. Repeat steps 18 and 19 for plates -1 and -1*; plates -2 and -2*; plates -3 and -3*; and both control plates. 21. Seal petri plates with parafilm and incubate upside down at 37C for 70 hours.
Improved Raw Milk Serial Dilution Procedure Proposal Materials: see above procedure
1. Place 3 test tubes each filled with 9 mL PBS in a rack and lightly place the caps on top. 2. Autoclave this along with a 50 mL beaker to sterilize. 3. Begin to prepare the workspace by lighting the bunsen burner. 4. Label the tubes -1, -2, and -3. 5. Label 5 SB agar plates: 0, -1, -2, -3, and C. Label the other 5 plates: 0*, -1*, -2*, -3*, and C*. 6. Arrange the plates in front of the test tube rack so that all are easily accessible. 7. Fill the beaker with refrigerated milk and place it next to the test tube rack. 8. Take the pipette with a sterile tip and transfer 1 mL raw milk from the beaker onto petri plates 0 and 0*.
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9. Uncap and flame tube -1, then transfer 1 mL raw milk into the tube. Flame, recap, swirl to mix, and place in rack. 10.Discard the used pipette tip and with a sterile tip, transfer one mL of diluent from tube -1 to tube -2 using aseptic technique. 11. Repeat step 10 using tubes -2 and -3 to finish the dilution series. 12.Discard the pipette tip used for the dilution series and with a sterile tip transfer 1 mL of diluent from tube -3 onto both plate -3 and -3*. 13.Repeat step 12 for tubes -2 and -1, transferring onto their respective plates. 14.Surface sterilize the hockey stick spreader first with a kimwipe and ethanol, and then by briefly flaming and waiting a moment for it to cool. 15.Starting with plates C and C* (which contain no sample), and working up through the dilution series, use the sterilized hockey stick to evenly spread the samples over the agar media. 16.Seal petri plates with parafilm and incubate upside down at 37C for 70 hours.
Mixing Bile Esculin Azide agar for confirmation of suspected enterococcal colonies on SB mediumMaterials: gloves, goggles, bile esculin azide (BEA) premixed growth media, metal spatula, scale, weighing paper, hot plate, magnetic stir bar and magnetic rod for removal, 500 mL beaker, distilled water, aluminum foil, bunsen burner, striker, plastic petri plates
For 100 mL BEA growth media: 1. Assemble materials underneath a vented hood. 2. Weigh out 5.6 g premixed BEA media. Use a metal spatula to gently spoon the mix onto weighing paper. 3. Transfer the mix into a 500 mL beaker. 4. Use a volumetric flask to measure 100 mL of distilled water and add this to the beaker. 5. Use the metal spatula to stir and try to break up any large chunks. 6. Place the beaker on a hot plate and turn the heat on high. 7. Place a magnetic stir bar in the beaker and turn on the stir function to create a vortex. 8. Cover the beaker with aluminum foil. 9. Allow to heat to a boil, periodically turning off the stir function in order to check for the formation of bubbles indicating boiling temperature. 10.Continue heating at a boil for 1 minute or until there is no more undissolved powder remaining in the solution. 11. Remove the magnetic stir bar and replace aluminum foil. 12.Autoclave the beaker for 15 minutes at 121C. 13.Wait for the mixture to cool so it won't melt the plastic petri plates. 14.Before pouring the plates, light a bunsen burner. 15.Working around the flame, remove the aluminum foil from the beaker. Pour the molten media into 4 plastic petri plates, taking care not to lift the lids for very long. 16.Leave a note to indicate toxicity of the media. Allow to cool until set.
Streaking BEA agar plates to isolate colonies and confirm esculin hydrolysis Materials: previously prepared BEA agar plates, inoculating loop, enterococcal colonies previously cultured on SB agar, bunsen burner, striker, marker, parafilm
1. Set up the work station underneath the hood by assembling a bunsen burner, previously cultured SB agar plates, previously prepared BEA agar plates, and an inoculating loop.
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2. Use a marker to divide the BEA agar plates in half, drawing a line down the center. Label the plates with initials, date, and media type. 3. Light the bunsen burner and turn on the hood. 4. Wash hands. 5. Thoroughly flame the inoculating loop to sterilize and set aside to cool. 6. Remove parafilm from colonized SB agar plates. 7. Briefly flame inoculating loop to sterilize and wait a moment for it to cool. 8. Prod a colony with the inoculating loop, making sure some of the sample sticks to the loop. 9. Streak the sample over half of a BEA agar plate using the following procedure. 10.At the top of the semi-circular plate section, thoroughly smear the sample back and forth over a small area. 11. Briefly flame the loop to sterilize and allow to cool. 12.Pull the loop through a section of the previously smeared sample, then thoroughly smear this diluted sample back and forth over a small area. 13.Briefly flame the loop to sterilize and allow to cool. 14.Pull the loop through a section of the second smear, then utilizing the rest of the space available, gently streak the loop back and forth across the media, taking care NOT to overlap with any previously smeared sections. 15.Repeat steps 7-14 until all desired colonies from SB agar plates have been smeared on BEA agar plates. 16.Wrap BEA agar plates in a double layer of parafilm. 17.Incubate upside down for 24 hours at 37C.
Preparing and Streaking BHI Agar Slants to Store Enterococcal CulturesMaterials: 8 screw cap tubes, test tube rack, slant boards, brain heart infusion (BHI), agar, ultra-pure water (UPW), graduated cylinder, 500 mL beaker, hot plate, magnetic stir bar and magnetic rod for removal, scale, weighing paper, metal spatula, marker, tape, inoculating loop, bunsen burner, striker, previously prepared enterococcal cultures on BEA agar plates, parafilm
1. Weigh out 2.96 g BHI and 1 g agar and place in a 250 mL beaker.2. Add 80 mL UPW.3. Add a magnetic stir bar and place on a hot plate.4. Turn the hot plate on medium-high heat and initiate the stir mechanism to form a vortex.5. Allow to heat with agitation until agar is melted and BHI is completely dissolved.6. While you wait Arrange 8 screw cap tubes in a test tube rack.7. Using a graduated cylinder, measure 10 mL of UPW and pour it into the first screw cap tube.
Then use a marker to indicate approximately 10 mL of volume.8. Transfer this water into the next tube, again using a marker to indicate the 10 mL mark.9. Repeat step 9 until all the tubes have been marked and dispose of the 10 mL of water.10. Now remove the beaker from the hot plate and remove the magnetic stir bar.11. Decant approximately 10 mL of the liquid BHI agar into each of the 8 tubes.12. Loosely cap the tubes and autoclave to sterilize.13. Place the tubes on a slant board and allow to cool until fully set.14. Label the tubes (initial, date, 'BHI - enterococci').15. Assemble a bunsen burner, an inoculating loop, the cultured BEA agar plates, and the BHI agar
slants underneath the hood and light the bunsen burner.16. Thoroughly wash and dry hands.17. Thoroughly flame the inoculating loop to sterilize, then set aside to cool.18. Remove the parafilm from the BEA agar plates and place them in an accessible area.
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19. Briefly flame the first tube and remove the cap.20. Briefly flame the inoculating loop and allow to cool before using it to pick a colony from one
half of a BEA agar plate.21. Use the inoculating loop to streak the culture across the agar.22. Briefly flame the tube and recap. Place the tube back on the slant board.23. Repeat step 19-22 for subsequent tubes until all have been streaked.24. Thoroughly parafilm the tubes.25. Incubate for 24-48 hours at 37 C.26. Place in a labelled container (initials, date, 'potential pathogens') and store at 4 C.
Enterococcal Growth Media - Constituents
Slanetz and Bartley - Formula / Liter
Tryptose..................................................................................20 g An enzymatic digest of protein useful for culturing microbes that, like enterococci, can be difficult to grow due fastidious nutrient requirements. Yeast extract.............................................................................5 g Prepared from autolyzed yeast cells. Provides a variety of common microbial nutrients such as nitrogen, carbon, amino acids and vitamins. An especially good source of B-complex vitamins, some of which are essential to enterococcal growth. Glucose.....................................................................................2 g A common monosaccharide. Source of energy and carbon. Di-potassium hydrogen phosphate...........................................4 g Probably added as a pH buffer. May also serve as a nutrient source. Sodium Azide.........................................................................0.4 g Highly toxic to aerobic organisms, selects for anaerobic organisms. Tetrazolium chloride...............................................................0.1 g Reduction of this compound withing bacterial cells results in red coloration of colonies, making them easier to identify. Agar........................................................................................10 g Polysaccharide isolated from algal growth. Allows media to solidify at room temperature. Final pH 7.2 ± 0.2 at 25°C
Bile Esculine Azide - Formula / Liter
Enzymatic Digest of Casein....................................................25 g Nutrient source for bacterial growth. Since enterococci are commonly found in dairy environments, casein probably meets their nutritional needs quite well. Yeast Enriched Meat Peptone...............................................9.5 g Meat peptone is also an enzymatic digest of protein derived from animal tissues that serves as a nutrient source. It is likely enriched with yeast extract in order to provide essential nutrients such as B- complex vitamins. Oxbile........................................................................................1 gDehydrated fresh bile, selects for bile tolerant enteric organisms. Sodium Chloride........................................................................5 g Selects for NaCl tolerant organisms.Sodium Citrate..........................................................................1 g May be added as a pH buffer as well as a source of energy and carbon, as enterococci are able to
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metabolize citrate. Ferric Ammonium Citrate.......................................................0.5 g May be added as a pH buffer as well as a source of iron and citrate. Reacts with hydrolyzed esculin to produce the black halo useful for identifying colonies able to hydrolyze esculin. Esculin.......................................................................................1 g An organic compound found in plants. Esculin hydrolysis is a useful trait in differentiating enterococci from related organisms. A black halo is formed around colonies that hydrolyze esculin. Sodium Azide.......................................................................0.25 g Agar.........................................................................................14 g Final pH: 7.1 ± 0.2 at 25°C
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