PHYSICOCHEMICAL FACTORS AFFECTING THE GROWTH OF...

CHAPTER 5

PHYSICOCHEMICAL FACTORS AFFECTING THE GROWTH OF ENTEROCOCCI

5.1. INTRODUCTION

Enterococcus species are wide-spread Gram-positive bacteria and are

natural inhabitant of the gastrointestinal tract (GIT) of humans and animals.

They are also commonly found in soil, sewage, water and food. The ability

to survive in unfavorable conditions is responsible for their ubiquitous nature

and persistence in the environment. In order to survive in the human GIT,

bacteria must overcome several adverse environmental stresses such as

changes in the pH, temperature, nutrient availability, elevated osmolarity and

the deleterious actions of bile.

Another important parameter which can affect growth and survival is

disinfectant. Though disinfection procedures help to reduce the infection risk

by lowering the microbial load, many of the bacteria develop resistance

against the commercially available disinfectants. Biocide resistance will

result in inadequate decontamination of medical devices. Contaminated

environmental surfaces and medical devices can serve as a vehicle of

infectious agents (Manangan et al., 2001) and is associated with risk of

hospital-acquired infection (Boyce, 2007).

Although Enterococcus species have been reported as nosocomial

pathogens their profile of resistance to biocides has been hardly studied.

Similarly heavy metal ions of mercury, silver, copper, lead, zinc and other

metals show antimicrobial effect even in relatively low concentrations.

Studies have shown that heavy metals are inhibiting the adhesion of bacteria

98 Chapter 5

on biofilm (Kielemoes and Verstraete, 2001). In order to survive in the wild

and contaminated environment, bacteria develop different mechanisms to

confer resistance to these heavy metals (Karamanisal et al., 2008).

Since the Enterococcus is recognized as serious nosocomial

pathogens, it is of great significance to understand such responses in

enterococci which enable them to survive in a wide range of environments.

The study was done to investigate the effects of different factors like

temperature, pH, different salt concentrations and presence of bile salts on

the growth of enterococcal strains. Another aim of the work was to test the

efficacy of several commonly used disinfectants on the isolates of

Enterococcus and also to characterize the variations in the disinfectant

susceptibilities of biofilms and planktonic cells. Yet again this study was

focused to determine the effectiveness of heavy metal on Enterococcus

isolates and to find the minimum bactericidal concentration of different

heavy metals.

5.2. MATERIALS AND METHODS

Isolates from four different sources comprising water, clinical

samples, healthy human faeces and chicken faeces were subjected to study

the influence of different physicochemical parameters on the growth. Twenty

E. faecium, twenty E.faecalis and twenty other miscellaneous enterococci

including E.gallinarum, E.avium, E.raffinosus and E.durans were selected to

study the effect of physical factors on the growth.

5.2.1. Effect of incubation temperature on growth

Isolated colonies of Enterococcus strains were inoculated into brain

heart infusion broth and incubated at 37o C for 24 hours and turbidity was

adjusted to a final concentration of approximately 10 8 cells(0.5 McFarland

Physicochemical Factors Affecting the Growth of Enterococci 99

standard). 0.01 mL of the broth was inoculated into BHIB and allowed to

grow at temperatures ranging from 10o C to 40o C for 2 days and at 4o C for

one week, and optical density at a wavelength of 600 nm was checked by

using spectrophtometer (Hitachi F-2700) and was subcultured on BEA and

examined for enterococcus like colonies.

5.2.2. Effect of pH on growth

0.01 mL of Enterococcus culture in brain heart infusion broth culture

of about 108 CFU/mL was transferred into BHIB adjusted to different pH

levels. The optical density of the broth at 600 nm was determined after 48

hours and was subcultured as described above.

5.2.3. Influence of various concentrations of sodium chloride on the growth of Enterococcus

0.01 mL of Enterococcus culture in BHIB with a turbidity adjusted

to 108 CFU/mL was inoculated into BHIB containing different

concentrations of NaCl (Merck). After 48 hours, growth was monitored by

determining optical density at 600 nm and was subcultured as described

above.

5.2.4. Heat resistance of Enterococcus

Test tubes containing sterile BHIB were inoculated with the test

strain followed by incubation at 37o C for 24 hours and turbidity was

adjusted to a concentration of approximately 108 cells/ mL (0.5 McFarland

standards). One set of tubes were kept in a water bath adjusted to 63oC for

30 minutes and others at 72oC for 20 seconds. After this the presence of

viable cells was tested by subculturing, 0.1 mL sample on to BHIA plates.

The plates were incubated at 37°C for 24h and the colonies were counted by

using colony counter (Joshibha) .Untreated tubes served as control and were

100 Chapter 5

also subcultured and counted. The log10 of number of viable count was

calculated.

5.2.5. Bile tolerance

The Isolated enterococcal colonies were inoculated into BHIA

medium containing 40% of bile salt (HiMedia) and incubated at 37°C. After

24h the plates were examined for bacterial growth.

5.2.6. Survival on dry cotton swabs

Isolates were grown overnight at 37°C in brain heart infusion broth.

After growth, 1 mL from each culture was added to a sterile centrifuge tube

and was centrifuged (10000g 5’ at 5°C). Cell pellets were resuspended in

PBS and cell density was adjusted to 108 CFU/mL by using Mc Farland

turbidity standard. Using a micropipette, cotton swabs were inoculated with

10µl of suspension. Inoculated swabs were inserted into different tubes, and

incubated at room temperature. One swab each of a strain was inoculated on

to Brain heart broth every day and after 48 hours; one loopful was

subcultured on to BEA plates and incubated at 37°C for 48 hours. This was

repeated until cultures no longer showed growth.

5.2.7. Bactericidal effect of disinfectant on planktonic cells:

The bactericidal effect of disinfectant on the isolates was measured

by suspension test (Cremieux et al., 1991). The solutions of disinfectants

(Appendix-2) at different concentrations in 1000 mL volumes were made in

distilled water. Overnight grown TSB culture of the bacteria (1mL) was

centrifuged (10000g 5’ at 5°C) and cell pellets were suspended in saline. 1

mL of the cell suspension containing approximately108 CFU/mL was added

into the 9 mL disinfectant solution. After a contact time of 1, 5 & 10

minutes, serial 1:10 dilutions were performed in neutralizing medium


(Appendix-2). 0.1 mL samples were then inoculated on TSA and the

bacterial growth was examined. A bacterial suspension treated with PBS

instead of a disinfectant was used as the control.

5.2.8 Effect of disinfectants on biofilm

Bacterial suspensions standardized to 108 CFU/mL in TSB were

prepared and 200 µl of these suspensions were taken in the sterile wells of

polystyrene micro titre plates and incubated at 37oC for 72 hours so that

biofilm was formed inside the wells. Wells were washed with PBS so as to

remove the non adherent bacteria. Uninoculated well containing trypticase

soy broth was used as control. The disinfectants at different concentrations

were added to the wells. After 1, 5 & 10 minutes, the contents were

discarded. The wells after disinfectant treatment were washed with PBS for 3

times and 200 µl of the fresh TSB was added into each well and biofilm was

detached by scraping the wells. After mixing the contents10 µl of the

suspension was then transferred to TSA and incubated at 37oC for 18 hours

and examined for bacterial growth. The microtitre plates were further

incubated overnight and subcultured to assess the viability of bacteria

remaining if any.

5.2.9. Determination of bactericidal concentration of heavy metal on Enterococcus in planktonic form

The effect of heavy metal ions on enterococci isolates was

determined by the plate-dilution method as described by Malik and Jaiswal,

(2000). Heavy metal salts like Pb (NO3)2, CoCl2, CdCl2, and ZnCl2, HgCl2

and AgNO3 (Merck) were used. Concentrations in the range of 0.1 to 20

mg/mL were used. Metal salts were dissolved in distilled water, filter

sterilized and added to MHA medium in the required concentrations. The

bacterial isolates were grown and 0.01 mL of the inoculum containing

102 Chapter 5

approximately 108enterococci cells were spot inoculated on MHA containing

graded concentrations of heavy metals. MHA medium without metal served

as control. The inoculated plates are incubated at 37oC for approximately 48

hours before reading the results. Minimum bactericidal concentration was

noted when the isolates failed to grow on plates.

5.2.10 Determination of bactericidal concentration of heavy metal on Enterococcus in biofilm

Enterococcus was grown overnight in TSB at 37°C. 200µl of the

culture (108 CFU/mL) was used to inoculate sterile 96 well-polystyrene

microtitre plates. After incubating for 24 hours at 37°C, the wells were gently

washed 3 times with 200 µl phosphate buffered saline (PBS), dried in an

inverted position. The heavy metals at different concentrations (Stocks of the

metal salts prepared in distilled water) were added to the wells. After 30

minutes, the contents were discarded. The wells after heavy metal treatment

were washed with PBS for 3 times and 100 µl of the fresh PBS was added

into each well and biofilm was detached by scraping the wells.10 µl of the

suspension was then transferred on to TSA and incubated at 37oC for 18

hours and examined for bacterial growth. The microtitre plates were further

incubated overnight and subcultured to assess the viability of bacteria

remaining if any.

5.3. STATISTICAL ANALYSIS

Influence of different factors on growth of the Enterococcus species

was analyzed by Chi square test and by ANOVA by using SigmaStat

software (Sigma-Aldrich, St. Louis USA). The level of significance was set

up at P <0.05.

Physicochemical Factors Affecting

5.4. RESULTS

The growth of different species of

optical density ± SD at 4°C

Figure.5.1. Growth of

The effect of different temperatures on

studied and growth was noticed in

these temperatures affected

same manner with heavy growth

greater OD values than all

environmental sources. Error bars indicates the Standard deviation.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

E.faecium E.faecalis

Me

an

OD

60

0

Physicochemical Factors Affecting the Growth of Enterococci

of different species of Enterococcus measured as mean

4°C is given in Figure 5.1.

rowth of enterococci at low temperature

effect of different temperatures on growth of enterococci was

was noticed in all tested strains (data not shown). E

affected the growth of different enterococci species

with heavy growth except at 4°C. At 4o C, E.faecium

all other enterococci species from both human

Error bars indicates the Standard deviation.

E.faecalis E.gallinarum E.avium E.raffinosus E.durans

Human Environmental

103

measured as mean

growth of enterococci was

Each of

different enterococci species in the

yielded

human and

E.durans

104

Effect of pH 3 on the growth of enterococci is presented in figure 5.2

Figure

Enterococcal isolates from all sources

low pH conditions.

enterococcus species was

shown) except at pH 3

than all other enterococci

difference in the mean

(P>0.05).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

E.faecium

Me

an

OD

60

0

pH 3 on the growth of enterococci is presented in figure 5.2

Figure 5. 2. Growth of enterococci at pH 3

Enterococcal isolates from all sources were evaluated for

conditions. At each of this pH the growth shown by different

enterococcus species was in the same way with high OD values

except at pH 3. At pH 3 E.faecium produced a higher growth rate

enterococci species. There was no statistical

mean OD values between environmental and human strains

E.faecium E.faecalis E.gallinarum E.avium E.raffinosus

Human Environmental

Chapter 5

pH 3 on the growth of enterococci is presented in figure 5.2.

were evaluated for growth at

pH the growth shown by different

with high OD values (data not

produced a higher growth rate

no statistically significant

between environmental and human strains

E.raffinosus E.durans

Physicochemical Factors Affecting

Effect of pH 10 on the growth of enterococci is presented in

Figure 5.

Enterococcal isolates from all sources were evaluated for growth at

alkaline pH conditions. Growth was obtained up to

not shown by the isolates

shown to have a better growth in alkaline conditions than others.

slight increase in growth was observed in

E.faecium, E.faecalis and

found (P>0.05). Human isolates

more growth than environmental isolates

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

E.faecium E.faecalis

Me

an

OD

60

0

Physicochemical Factors Affecting the Growth of Enterococci

pH 10 on the growth of enterococci is presented in figure 5.3

3. Growth of enterococci at pH 10


Growth was obtained up to pH 10 and growth was

shown by the isolates at pH 13 (data not shown). E.faecium isolates were

shown to have a better growth in alkaline conditions than others. Though a

rease in growth was observed in environmental isolates

and E.durans, a significant difference could not be

Human isolates of E.avium and E.raffinosus showed slightly

than environmental isolates.

E.faecalis E.gallinarum E.avium E.raffinosus E.durans

Human Environmental

105

figure 5.3.


10 and growth was

isolates were

Though a

environmental isolates of

a significant difference could not be

showed slightly

E.durans

106

The effect of

figure 5.4.

Figure 5. 4

The growth of enterococci at different salt concentrations was

studied. Growth was obtained at a s

not shown). No growth was shown by the isolates at a salt concentration of

15%. At each of these

enterococci species was

E.faecium produced

isolates of all the isolates demonstrat

isolates (P>0.05)

0

0.05

0.1

0.15

0.2

0.25

0.3

E.faecium

Me

an

OD

60

0

The effect of 11% of NaCl on the growth of enterococci is shown in

4. Growth of enterococci at 11% salt concentration

growth of enterococci at different salt concentrations was

Growth was obtained at a salt concentration of 0.5 to 11% (data

No growth was shown by the isolates at a salt concentration of

At each of these concentrations the growth shown by different

enterococci species was comparable except for salt at 11%.

produced a better growth than other species.

isolates of all the isolates demonstrated slightly more growth

(P>0.05).

E.faecium E.faecalis E.gallinarum E.avium E.raffinosus

Human Environmental

Chapter 5

terococci is shown in

salt concentration

growth of enterococci at different salt concentrations was

alt concentration of 0.5 to 11% (data

No growth was shown by the isolates at a salt concentration of

the growth shown by different

11%. At 11% NaCl,

Environmental

more growth than human

E.raffinosus E.durans


The bacteria before and after heat treatment were counted and log

CFU/mL± SD formed by them are presented in table 5.1.

Table.5.1. Heat resistance of enterococcal isolates

log CFU/mL±SD

Enterococcus tested Before heating After heating

63 for 30minutes 72 for 20seconds

E.faecium 5. 6628±3.33 2.8633±1.59 1.8451±0.69

E.faecalis 5.0792±3.95 2.8062±1.64 1.6902±0.84

E,durans 5.1761±3.85 2.7993±1.36 1.5911±0.90

E.gallinarum 5.3222±3.84 2.6021±1.64 1.3010±0.34

E.avium 5.1461±3.22 2.7781±1.22 1.6021±0.48

E.raffinosus 5.0414±3.13 2.6990±1.25 1.4150±0.60

The cultures survived heat treatment at 63°C even though there was a

reduction in the viable count (P<0.05). Exposure to heat at 72 °C resulted in

a notable reduction in bacterial count than at 63°C for all isolates. E.faecium

culture produced more viable cells after heat treatment. E.gallinarum

produced least number of viable cells after heat treatment.

108 Chapter 5

The survival of enterococci strains at room temperature is presented

in table 5.2.

Table 5.2. Survival of various Enterococcus species in dry cotton swabs incubated at room temperature

Enterococcus species

No.of isolates tested

Average period (in days) of survival of the isolates from various sources

Water Chicken Human Clinical

E.faecium 8 12 10 9 13

E.faecalis 10 12 11 9 12

E.gallinarum 3 10 11 9 12

E.avium 3 11 9 9 12

E.raffinosus 3 10 11 9 12

E.durans 2 11 11 9 11

All enterococcal strains were shown to survive in dry cotton swab.

Isolates from clinical sources showed a significantly higher resistance than

the isolates from other sources (P<0.05) .The isolates from human faecal

samples showed the least days of survival.

MBCs of disinfectants against planktonic and biofilms of

E.faecium(n=51)and E.faecalis(n=31) were observed in a range and are

summarized using the box-plot method (Figures 5.5 and 5.6).


Figure 5.5. MBC of different disinfectants on biofilm and planktonic forms of E.faecium

On planktonic forms of different E.faecium isolates cresol showed

MBC in the range of 1.25- 10mg/mL (median MBC of 5mg/mL). But on

biofilms 20-30mg/mL was the MBC required (median MBC30mg/mL). The

MBC of BK was in the range of 1.25-2.5mg/mL (median MBC of

2.5mg/mL) on planktonic forms, whereas on biofilm, it was 10-50mg/mL

(median 20 mg/mL).

MBC of glutaraldehyde on planktonic forms was ranging from 1.25-

5mg/ml (median 2.5mg/mL). Nevertheless 20-30 mg/mL (median of

20mg/mL) was needed to achieve bactericicdal activity on biofilm. MBC of

NaOCl and iodine ranged from 0.3125-1.25mg/mL (median 0.3125) and

0.625-2.5(median 0.625) respectively. And on biofilms the MBC of NaOCl

were10-20mg/mL and MBC of iodine was only10mg/mL (median

10mg/mL).

0

5

10

15

20

25

30

35

40

45

50

55

60

MB

C (

mg

/mL)

P-Plaktonic, B-biofilm

Q1 Min Median Max Q3

110 Chapter 5

Figure 5.6. MBC of different disinfectants on biofilm and planktonic forms of E.faecalis

On planktonic forms of E.faecalis, cresol showed MBC in the range

of 1.25-10 mg/mL (median 5mg/mL). And on biofilm bacteria 30mg/ml was

required to produce a 100% kill. MBC shown by BK was in the range of

1.25-2.5 mg/mL (median 1.25mg/mL). MBC demonstrated by NaOCl ranged

from 0.3125-0.625 mg/mL (median 0.625mg/mL). Iodine showed a very

high bactericidal activity even at a low MBC ranging from .3125-2.5 mg/mL

(median 0.3125mg/mL). 10 mg/mL concentration of Iodine, NaOCl and BK

killed biofilm of E.faecalis. For planktonic forms glutaraldehyde MBC was

in the range of 1.25-5 mg/mL (2.5 median). Glutaraldehyde of 20mg/mL was

required to produce a 100% kill of biofilm bacteria.

Comparison of action of disinfectants on planktonic and biofilm

forms of other Enterococcus species is shown in Table 5.3.

0

5

10

15

20

25

30

35

MB

C (

mg

/mL)

P-planktonic, B-biofilm

Q1 Min Median Max Q3


Table 5.3. MBC of various disinfectants in respect of planktonic and biofilm forms of miscellaneous Enterococcus

Range of MBC (mg/mL) of disinfectants

Species tested* Cresol BK NaOCl Iodine Glutaraldehyde

E.gallinarum (4) planktonic 1.25 1.25 0.3125 0.3125 1.25

Biofilm 20 10 10 10 20

E.durans (4) planktonic 1.25 1.25 0.3125 0.3125 1.25

Biofilm 20 10 10 10 20

E.avium (5) planktonic 2.5 1.25 0.3125 0.3125 1.25

Biofilm 20 10 10 10 20

E.hirae (1) planktonic 1.25 1.25 0.3125 0.3125 1.25

Biofilm 20 10 10 10 20

E.raffinosus (5) planktonic 1.25 1.25 0.3125 0.3125 1.25

Biofilm 20 10 10 10 20

E.mundtii (1) planktonic 1.25 1.25 0.3125 0.3125 1.25

Biofilm 20 10 10 10 20

*No. of strains tested is given in brackets

Among the miscellaneous bacteria E.avium planktonic form was

found to be more resistant when treated with cresol. Biofilm bacteria were

killed by 20 mg/mL of cresol and glutaraldehyde respectively. All the

miscellaneous bacteria exhibited similar pattern of resistance. When BK,

iodine and NaOCl were used, biofilm forms of all these bacteria were

removed by 10 mg/mL of concentration.

The resistance of enterococcal strains to six different heavy metals

such as cobalt, lead, zinc, cadmium, silver and mercury were determined.

112 Chapter 5

Comparative analysis of MBCs on planktonic and biofilm E. faecium is

shown in tables 5.4.

Table 5.4: Minimum bactericidal concentrations of heavy metals required to kill 100% of the tested isolates of E.faecium (n=5)

Metal

Concentration (mg/mL) of heavy metal required to kill 100% of the tested isolates

Planktonic Biofilm

Mercury 0.1-0.25 0.25

Silver 0.1-0.25 0.25

Zinc 0.5 5-20

Cadmium 0.5 2.5-10

Lead 0.5 5-20

Cobalt 0.5 5-20

Silver and mercury were highly effective against E.faecium and 80%

of them were removed at 0.1mg/mL (data not shown). Though 80% of

bacteria in biofilm were killed by 5% of zinc, lead and cobalt, resistant strain

was eliminated only after treating with 20mg/mL of these metals. Ag and Hg

were found to be more effective on planktonic and biofilm than all metals

tested (P<0.05).

Comparative analysis of MBCs on planktonic and biofilm of E.

faecalis are shown in tables 5.5.


Table 5.5. Minimum bactericidal concentrations of heavy metals required to kill 100% of the tested isolates of E.faecalis (n=5)

Metal

Concentration (mg/mL) of heavy metal required to kill 100% of the tested isolates

Planktonic Biofilm

Mercury 0.1 0.25

Silver 0.1 0.25

Zinc 0.5 5

Cadmium 0.25 5

Lead 0.5 5

Cobalt 0.1—0.25 5

Silver and mercury were very effective on E.faecalis. Mercury and

silver removed all planktonic bacteria at 0.1mg/mL concentration. Zinc and

lead were less effective and all cells were removed only at 0.5mg/mL

concentration. Resistance of biofilm was more compared to planktonic forms

of bacteria. A concentration of 5mg/mL of zinc, lead, cadmium and cobalt

was required to kill E.faecalis biofilm.

5.5. DISCUSSION

While investigating the growth and survival strategies of enterococci,

the growth of Enterococcus was obtained in all the temperatures tested.

When growth was analyzed at 4o C in this study all isolates were found to be

growing. At this low temperature the total count was less in all the isolates

except in E. faecium. E. faecium was shown to have more growth rate than

other enterococci. Minimum growth temperature for E. faecium was reported

to be 0.1o C (Zanoni, 1993). Earlier studies support this by stating E faecium

114 Chapter 5

as a psychrotolerant species (Biomine news). It can be assumed that from

food microbiology point of view, Enterococcus species are important

because of its growth potential at refrigeration temperature.

The findings of this study show the ability of enterococci to grow in

different pH conditions. Growth of enterococci in a wide range of pH was

observed. At pH 3 all species survived and yielded growth and at pH 2, none

of the isolates survived except one strain of E.faecium [data not shown in the

table]. Growth of enterococci at pH 3 was observed in an earlier study (Galaz

et al., 2004). Acid resistance in enterococci helps to establish Enterococcus

of animal origin in the human gut. Enterococcal growth was also obtained at

pH 10 in this study. This is in conformity with a previous study by Flahaut et

al., (1996). Many factors contribute to the tolerance of alkaline pH found in

enterococci, including activation of specific proton pumps and specific

enzyme or buffer devices that helps to keep internal pH constant (Nagao et

al., 1990). This help in the survival of enterococci in alkaline environments

resulting from pollution of waters by industrial effluents or agricultural

wastes and in some foods (Rowbury et al., 1996).

Up to a concentration of 11% of NaCl, all enterococci produced

growth. And E.faecium was the more salt tolerant species. Though there is a

lack of earlier reports in this regard, Enterococcus is well known for its

extraordinary ability to survive in salt water. Taking this into consideration,

EPA recommends enterococci as the best indicator of health risk in salt water

used for recreation. NaCl has a role in the induction of general stress protein

in Enterococcus ( Rincé et al.,2002). Enterococcs has a cation homeostasis

which is thought to be contributing to its resistance to pH, salt, metals and

desiccation.


Enterococci, showed the ability to resist pasteurization temperatures

in this study. Consistent results have been reported in previous studies

(Ahmad et al., 2002). E.faecium was more heat resistant than other

enterococci. Similar results were found in many other studies (Kearns et al.,

1995; Bradley and Fraise, 1996). These results confirm that enterococci are

highly thermo resistant bacteria as mentioned in a previous study by Perez et

al., (1982), Heat resistance property is attributed to be responsible for

contamination and spoilage of meat products (Gordon &Ahmed, 1991).

There are several explanations regarding the temperature resistance.

Temperature resistance depends on the membrane structure and has been

related to lipid and fatty acid content (Ivanov, et al., 1999). Enterococci have

been found to respond to sudden increases in temperature by synthesizing the

heat shock proteins (Parsell & Lindquist, 1993).

In this study all enterococci survived and growth occurred in the

presence of 40% bile salts (not shown in table). This capability of

enterococci shows that this pathogen is adapted to grow in the

gastrointestinal tract.

The length of survival of these organisms in a non nutrient medium

may have significant infection control implications. Up to 13 days,

Enterococcus survived on cotton swabs. This is supported by the results

obtained by Wendt et al., (1998) and Neeley and Maley, (2000) who found

similar survival on various dry surfaces (Wendt et al., 1998; Neeley and

Maley, 2000). All enterococcal species had more or less similar survival

period. Compared to other sources, isolates from clinical sources had more

ability to survive in non nutrient dry surface. This obviously contributes to

the enterococcal prevalence and their dissemination in hospital environments

to cause nosocomial infection.

116 Chapter 5

This study evaluated the efficacy of conventional antimicrobial

products like povidone iodine, sodium hypochlorite, cresol, glutaraldehyde

and benzalkonium choride on enterococci from different sources in

planktonic and biofilm forms. MBCs of disinfectants did not vary with

respect to source. There are also some reports with results demonstrating

similar observation (Anderson, 1975).

Chlorine is used extensively in the disinfection of drinking waters to

reduce the problem of bacterial growth in distribution systems and in health

care environments. In this study low concentrations of NaOCl and iodine

effectively killed enterococci. Effectiveness of povidone iodine was

previously proved against vancomycin resistant and sensitive enterococci

(Block et al., 2000). Similarly the efficacy of sodium hypochlorite in

eradicating enterococci has already been reported by Abdulla et al., (2005).

In contrast, there is a report on Enterococcus exhibiting resistance to sodium

hypochlorite (Kearns et al., 1995).

Benzalkonium chloride and glutaraldehyde are common hospital

disinfectants. In this study benzalkonium chloride at a low concentration

(1.25 mg/mL) eradicated most strains of enterococci. The resistant strains

were inhibited, at 2.5mg/mL concentration. It is appealing to note that there

are also reports on benzalkonium chloride resistant genes in enterococci

(Braga et al., 2010). It has been suggested that the widespread use of

quaternary ammonium compounds [QAC] may impose a selective pressure

and contribute to the emergence of QAC resistance. In this study

glutaraldehyde at a low concentration (1.25 mg/mL) eradicated most of the

strains of E.faecalis and E.faecium and all other enterococci. Those resistant

strains of E .faecalis and E.faecium were killed, at a higher concentration.

The effectiveness of glutaraldehyde was reported in another study by


Bovallius and Anas, (1977). There is even report on the intrinsic resistance to

glutaraldehyde in enterococci (Fraise, 2002). All enterococcal strains showed

identical elevated pattern of resistance to Cresol. Phenolics were found to be

resistant in many of the studies (Bean and Walters, 1961).

As explained by other authors, this study also supports the

observation that the bacteria sequestrated in biofilms are shielded and are

often harder to be killed than their free floating counterparts (Gilbert et al.,

2002). It was observed in this study that sodium hypochlorite and iodine

were found to be effective in eradicating the bacteria in the biofilm. Paulson,

(2005), was also of opinion that enterococcal biofilm could be removed by

the action of povidone iodine and alcohol. And the usefulness of sodium

hypochlorite against enterococcal biofilm was reported by Dunavant et al.,

(2006). In the present study on planktonic and biofilm, povidone-iodine

treatment even at low concentrations resulted in the elimination of a massive

number of bacteria. Thus, the use of povidone-iodine solution for local

treatment of biofilm may be effective.

Benzalkonium chloride and glutaradehyde are often used in hospitals

for disinfection purpose. They were effective against E.faecium biofilm, at

higher concentrations than the recommended in use concentrations in this

study.

The mechanism of biofilm resistance may be due to the delayed

penetration of disinfectants through the biofilm matrix or the reaction

between the two. Other factors that may be responsible for the increased

resistance of the biofilm cells are the presence of extracellular polymeric

substances, alteration of the target in the bacterial cell, enzymatic

modification or active efflux mechanisms. In order to increase the efficacy

118 Chapter 5

of antiseptic and disinfectants on bacterial biofilms, Takeo et al., (1994)

recommended an increase in the concentration and contact time. But in this

study contact time did not have much influence in the MBC of disinfectant.

Only a slight decrease in the count could be noticed after 1 minute contact

time (data not shown).

In the present study E.faecalis and E.faecium strains showed high

resistance to the disinfectants, while other species were annihilated at lower

concentrations of the disinfectants. Since there is species wise difference in

susceptibility to disinfectants, it is possible that the mechanism of resistance

can also be different in different species. It can be inferred from the

observations in this study that sodium hypochlorite and iodine are the most

effective disinfectants on all enterococcal strains in comparison with other

disinfectants tested. Laboratory experiments have demonstrated that

biocides, present at low concentrations in the environment due to discharge

after use, may lead to an increased selective pressure towards disinfectant

and antibiotic resistance. These observations raise concerns over the

indiscriminate and often inappropriate use of biocides, whereby contributing

to the development of microbial resistance. There are reports suggesting a

possible link between the disinfectant resistance and antibiotic resistance at

the genetic level Codling et al., (2004).

The presence of bacterial biofilms is one of the main challenges in

terms of antimicrobial resistance and is of public health concern, particularly

when dealing with medical isolates (Donlan and Costerton, 2002) Most

laboratories do not use biofilm disinfection tests to assess the efficacy of

biocides and no standards for the testing of disinfectants against biofilms in

health care applications exist (Cookson, 2005). Resistance or increased

tolerance to disinfectants shown by the biofilms in the present study could


explain the ability of Enterococcus to persist in the environment and even in

the body. All these difficulties emphasize the importance of regular and

effective disinfection.

Heavy metal resistance is a widespread attribute among

microorganisms isolated from different environments. Enterococcal growth

was not affected much in the presence of low concentrations of zinc. The

pattern of zinc tolerance in the different enterococcal isolates was identical in

the present study. In agreement with the present finding, a high zinc

tolerance was reported by da Silva et al., (2012) in enterococci.

The tolerance of cadmium by the Enterococcus species was not high.

The observations in this study show that enterococci were inhibited by low

concentration of cadmium. But there are reports indicating the requirement

of higher concentration of cadmium for the bactericidal action against

enterococci (da Silva et al., 2012). Furthermore a high natural resistance to

cadmium exhibited by E. faecalis has been reported by Laplace et al.,

(1996).

Even though several studies revealed the effectiveness of cobalt as a

potent antimicrobial agent (Kimiran-Erdem et al.,2007), this study shows

cobalt resistance in enterococci and is possibly due to variation in isolates

from different geographical areas.

Silver ions are widely used as bactericide in catheters, burn wounds

and dental work (Margaret et al., 2006). Silver salts can still be used as

topical antimicrobial agents. In this study silver killed enterococci at a very

low concentration. The effectiveness of silver on enterococci has been

reported by Day and Russell, (1999) & Jones et al., (2004).

120 Chapter 5

Mercury has no beneficial biological role, and is highly toxic to all

forms of life. The results of the study have shown it was active against all

planktonic and biofilms of enterococci. At an incredibly low concentration, it

inhibited enterococci in a study by da Silva et al., (2012). There are reports

on the plasmid borne resistance to mercury transferred by conjugation or

transduction (Ghosh et al., 2000).

E.faecium demonstrated resistance against most of the heavy metal

tested. All other species were relatively less resistant to the heavy metals.

Biofilms were observed to be more resistant to heavy metals than the

planktonic bacteria. Mercury and silver were found to have more action than

the other heavy metals studied.

Generally, the resistance to heavy metals could be due to plasmid

(Bruins et al., 2000). Several other metal resistance mechanisms have been

identified which include exclusion by permeability barrier, intra and extra

cellular sequestration, active transport, efflux pumps, enzymatic

detoxification, and reduction in the sensitivity of the cellular targets to metal

ions (Poole and Gadd, 1989).

Many investigators have also reported the association between heavy

metal and antibiotic resistance (Bhattacherjee et al., 1988 & Lawrence,

2000). The genes encoding heavy metal resistance are located in plasmids

together with antibiotic resistance genes (Dalsgarrd and Guardbassi, 2002) .

It is therefore, likely that selective pressure by any of these indirectly selects

for the whole set of resistance. Since the resistance to different heavy metals

is brought about by similar mechanism, multiple tolerances are common

phenomena among heavy metal resistant bacteria.


This study has demonstrated the amazing ability of enterococci

particularly E.faecium to grow at a wide range of temperatures, pH shifts and

high salt and its survival for extended periods in unfavorable environmental

conditions. These properties in turn may favor its growth and survival. The

biofilm formation capability should be taken into account while carrying out

the disinfection. Disinfectant and heavy metal resistance allows enterococci

to survive in the environments where antimicrobial agents are heavily used

such as a hospital environment. This may result in the selective proliferation

and dissemination of the drug resistant enterococci capable of infections.

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