Bacterial Pathogens within Persistent Algal Blooms in Southern Lake Michigan
Emily A. JunionNovember 19, 2009
Tulane University School of Public Health and Tropical MedicineCity of Racine Health Department
Introduction
• Chladophora is a native algae in the Great Lakes• Introduction of non-native mussels, Dreissena
spp., contribute to excessive algae blooms• Non-point sources of nutrient runoff into the
Great Lakes are a major growth limiting factor• Enumeration of fecal indicator organisms• Detection and identification of pathogens
Algal blooms: a public health hazard?
• Excessive algal growth and death cause areas of eutrophication– DO measurements very low within mat– Potential survival of anaerobic pathogens
• Attracts birds that may deposit pathogens into the water from their feces– Infectious bacteria may be in water or attached to algae – Potential to cause recreationally contracted illness
Indicator organisms in recreational waters
• Indicator organisms, Escherichia coli and Enterococci sp., are used to measure water quality for recreational waters
• Increased levels indicator organisms are correlated with increased risk of infection
• Many beaches along Great Lakes shores have closures due to high levels of E. coli
• Mats can produce sulfur/sewage-like odors
Mussel ecological impact• Dreissena mussel spp. increase algal growth rates– Appear to clean water through removing turbidity– Increase light penetration through water, increasing
photosynthesis rates and algae growth– Produce feces and pseudofeces
• Expel NH4+
very high N excretion rates• Act as significant P sink, but P is re-released with protozoa
grazing
• Zebra mussels cover almost any hard surface• Quagga mussels do not need a hard substrate for
growth– Becoming the predominant mussel sp. in lake MI
Hypotheses
1. There are significantly higher levels of E. coli from water within an algae mat versus water at least three meters outside the mat.
2. E. coli concentrations will increase on a gradient from outside to within the mat.
3. E. coli values increase in the mat over time.
Hypotheses (cont.)
4. Pathogens can exist in water sampled from within an algae mat.
5. Pathogens can exist attached to algae.
Sample design: hypothesis 1 & 3
Sample design: hypothesis 2
Geographic location within Great Lakes
2007 site 2008 and 2009 sites
Field materials
• Cooler/ice packs• Sampling poles and
sterile cups• Whirl-PacTM bags• Tape measurer• Sterile 8L carboy• Marking flags• Thermometers• DO meter with
DO/temperature probe
E.Coli & Enterococci enumeration from algae
1. 1g of algae was mixed with 9ml phosphate buffered water (PBW)
2. Vortexed tube for 2 min3. Centrifuged for 45 sec at
2000 rpm4. Bacterial enumeration was
completed on the supernatant according to EPA methods 1603 (E. coli) and 1600 (Enterococci)
E.coli enumeration from water
All water samples were processed via membrane filtration according to EPA method 1603
Algae sample preparation for pathogens
1. 5g of algae was mixed with 45ml phosphate buffered water (PBW)
2. Vortexed tube for 2 min3. Centrifuged for 45 sec
at 2000 rpm
Pathogen analysis
PBW supernatants and water from the 8L carboy were analyzed according to provided SOPs for Campylobacter and Salmonella/Shigella isolation
INDICATOR ORGANISMS
Results
E.coli analysis of water within vs. 3m outside algae mat
F-test Ho: sin= sout HA: sin≠ sout
T-test Ho: xgin ≤ xgout HA: xgin > xgout
Test Statistic Critical Values for n1=40 and n2=40
FCV@ 0.95 ≈2.00
t@ 99.95% 3.420
t@ 99.9% 3.198
t@ 99.5% 2.640
t@ 95% 1.665
Statistic 2007 2008 2009xgin 173 5760 4152sin 5.5 2.96 2.95
xgout 46 1608 835sout 6.6 3.78 3.83
s2pooled 36.9 11.6 11.7Ftest 1.44 1.63 1.69ttest 70.15 632.6 4336.7
Three Year Mean Gradient (Both sides)
-4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 3.5 42.4
2.5
2.6
2.7
2.8
2.9
3
3.1
3.2
-4, 2.67
2.79
-1, 2.83 2.83
2.92
2.97 2.98
3.03
1, 2.95
2, 3.02
4, 3.07f(x) = 0.0533959935672871 x + 2.91356553843639R² = 0.813547488035019
Overall mean
Distance from mat edge
Log
E.co
li (C
FU/1
00m
l)
0 1 2 3 40
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
f(x) = 0.204494390682192 x + 2.69427653165617R² = 0.0651805586252722
2007-2009 four day trend log E. coli (CFU/100ml)
Sampling Day
Log
E.co
li (C
FU/1
00m
l)E. coli water within mat
E. coli extracted from algae
0 1 2 3 40
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
f(x) = 0.178832212095708 x + 3.19938610312623R² = 0.0677957714045125
2007-2009 four day trend of log E. coli (CFU/g)
Sampling Day
Log
E. c
oli (
CFU
/g)
Enterococci extracted from algae
0 1 2 3 40
0.5
1
1.5
2
2.5
3
3.5
4
4.5
f(x) = 0.232083735534842 x + 2.53104598238795R² = 0.121767184848736
2007-2009 four day trend of log Enterococci (CFU/g)
Sampling Day
Log
Ente
ro (C
FU/1
00m
l)
PATHOGENS
Results
Identified pathogens within water from algae mats
Pathogens and other identified microorganisms found in water within algae mats2007 None found
2008 6-Aug1000ml filtered for
Campylobacter analysisCampylobacter
coli
2009 3-Aug 4-Aug 5-Aug 6-Aug1000ml S./Shigella enrichment broth
Salmonella arizonae
Salmonella arizonae
Citrobacter freundii
Proteus mirabilis
100ml S./Shigella enrichment broth
Plesiomonas shigelloides
Identified pathogens extracted from Chladophora washings
Pathogens and other identified microorganisms extracted from algae2007 None found
2008 None found
2009 3-Aug 4-Aug 5-Aug 6-Aug
Algae 1Salmonella
arizonae(10ml)
Algae 2Citrobacter braakii
(100ml)Salmonella
arizonae(100ml)
Algae 3Salmonella arizonae
(100ml)Salmonella
arizonae (10ml)Plesiomonas
shigelloides (100ml)
Algae 4Salmonella arizonae
(100ml)
Arcobater butzleri (Campylobacter analysis ~120ml)
Pathogens• Campylobacter coli– Swine are the main reservoir for the bacteria – Birds can also be a carrier– Major cause of gastroenteritis in humans
• Salmonella arizonae– Reptiles are the main reservoirs– Birds can also be a carrier – Has been known infect almost every human
tissue, but usually children and immunosuppressed
Pathogens (cont.)
• Plesiomonas shigelloides– Has been isolated from many freshwater sources– Infections can cause gastroenteritis and
septicemia in immunosuppressed people• Arcobacter butzleri– Found in environmental samples and food borne– Originally thought to be a Campylobacter sp.– Accounts for about 1% of identified species from
diarrheal diseases
Potential human pathogens
• Citrobacter freundii– Found naturally in the environment– Normally do not cause disease– May cause UTIs, or infections in infants
• Citrobacter braaki– Found naturally in the environment– May cause mild wound infection
• Proteus mirabilis– Commonly found in human intestines– May cause localized infections that can lead to septicemia– Can produce HS gas
Conclusions
• Higher levels of indicator organisms exist within algae compared to outside algal mats at >99.95% significance
• There is evidence of a positive gradient of indicator organisms from 4m outside an algae mat to 4m within an algae mat
• A slight increase in indicator organisms occurs within persistent mats over a four day period
• Pathogens can exist in the water of algae mats and attach to the algae mat itself
Recommendations• Research public health risks of bacterial
pathogens that may exist in algal mats• Research TMDLs of rate limiting nutrients in
areas of increased algal blooms• Continued research on interactions of invasive
species in the Great Lakes• Research ways to reduce the introduction and
spread of invasive species• Adopt removal of near shore and stranded algal
mats as a BMP for reduction of fecal indicators
Acknowledgements
• Dr. Julie Kinzelman, City of Racine Health Department Lab
• Jennifer Lavender, Justene Preedit, Stephan Kurdis, A.J. Koski, Tristin Begotga, Michelle Leittl, Tamara Anan’eva
• University of Wisconsin – Oshkosh• University of Wisconsin Sea Grant Research
Program
Questions??
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