Bacterial Pathogens within Persistent Algal Blooms in Southern Lake Michigan<br />Emily A. Junion<br />November 19, 2009<b...
Introduction<br />Chladophora is a native algae in the Great Lakes<br />Introduction of non-native mussels, Dreissena spp....
Algal blooms: a public health hazard?<br />Excessive algal growth and death cause areas of eutrophication<br />DO measurem...
Indicator organisms in recreational waters<br />Indicator organisms, Escherichia coli and Enterococci sp., are used to mea...
Mussel ecological impact<br />Dreissenamussel spp. increase algal growth rates<br />Appear to clean water through removing...
Hypotheses<br />There are significantly higher levels of E. coli from water within an algae mat versus water at least thre...
Hypotheses (cont.)<br />Pathogens can exist in water sampled from within an algae mat.<br />Pathogens can exist attached t...
Sample design: hypothesis 1 & 3<br />
Sample design: hypothesis 2 <br />
Geographic location within Great Lakes<br />
2007 site<br />2008 and 2009 sites<br />
Field materials<br />Cooler/ice packs<br />Sampling poles and sterile cups<br />Whirl-PacTM bags<br />Tape measurer<br />S...
E.Coli& Enterococci enumeration from algae<br />1g of algae was mixed with 9ml phosphate buffered water (PBW)<br />Vortexe...
E.colienumeration from water<br /><ul><li>All water samples were processed via membrane filtration according to EPA method...
Pathogen analysis<br /><ul><li>PBW supernatants and water from the 8L carboy were analyzed according to provided SOPs for ...
E.colianalysis of water within vs. 3m outside algae mat<br />F-test Ho: sin= soutHA: sin≠ sout<br />T-test Ho: xgin≤ xgout...
Three Year Mean Gradient (Both sides)<br />
E. coli water within mat<br />
E. coli extracted from algae<br />
Enterococci extracted from algae<br />
Pathogens<br />Results<br />
Identified pathogens within water from algae mats<br />
Identified pathogens extracted from Chladophora washings<br />
Pathogens<br />Campylobacter coli<br />Swine are the main reservoir for the bacteria <br />Birds can also be a carrier<br ...
Pathogens (cont.)<br />Plesiomonasshigelloides<br />Has been isolated from many freshwater sources<br />Infections can cau...
Potential human pathogens<br />Citrobacterfreundii<br />Found naturally in the environment<br />Normally do not cause dise...
Conclusions<br />Higher levels of indicator organisms exist within algae compared to outside algal mats at >99.95% signifi...
Recommendations<br />Research public health risks of bacterial pathogens  that may exist in algal mats<br />Research TMDLs...
Acknowledgements<br />Dr. Julie Kinzelman, City of Racine Health Department Lab<br />Jennifer Lavender, JustenePreedit, St...
Questions??<br />
Bacterial Pathogens In Algae
Bacterial Pathogens In Algae
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  • Nitrogen and phosphorus nutrients encourage growth Previous studies indicate areas with high algal growth have high levels of fecal indicator organismsused to identify surface water quality
  • E. coli for freshwater and Entero for fresh and marinePresent in feces of warm blooded animalsInfected human or animal waste can transport pathogens to the envAlgae may serve as an attachment medium and nutrient source for indicator organisms and pathogensDead decaying musselsDetached, decaying algaeOther potential dead animal species
  • Zebra mussels were first noted in the Great Lakes in the late 1980s and Quagga in the early 1990sBoth mussels have similar ecologicalExplosive population growthAn adult female can release 30,000 to 1million eggs per year, about 2% survive to adulthood600-20,000 offspring from one mussel are estimated to survive to adulthoodThought to be a carrier of type E botulismAvian, but also can infect fish sp. and humans“among highest N excretion rates of any animal”sharp shells cover beaches and possible factor in spread of waterborne diseaseZebra mussels can cover insides of pipes causing blockageCONCLUDE: Recent decades of cleared water combined with increased N and P from algae and runoff are a major source of increased algae levels
  • random and gradient samples outside of the mat were taken as grab samples in whirlpac bags and placed on ice until ready for further lab testing. DO, water temperature and air temperature were measured in the field.
  • All water samples within the mat were taken with a specially designed sampling pole and sterile cup.Samples were taken disturbing the mat as little as possible, as specified in the procedure.DO and temperature were taken right after the water samples were taken.
  • Algae samples were collected aseptically by hand into sterile whirl pac bags and placed on ice for E.coli and pathogen analysis
  • Present in lower concentrations than E.coli and Entero, so higher volumes of water and algae must be analyzed for pathogen detection
  • Calculated geometric and geometric standard deviationUsed the T-test to test for significantly higher levels within vs outside the algae matUsed F-test to test for significant differences between the variances and they were statistically similar for each yearGeometric means for E. coli measurements taken from within vs. those take from 3 feet outside the algae mat were compared using the t-test for statistical significance
  • When averaging the three years of data for each gradient sample, there does seem to be a gradient for the concentrations of E.coliin the algae mats from 2007- 2009. The slope of the line seems to be very different near the edge of the algae mat. Looked at the gradient without measurements within 0.25m of the edge and only at measurements within 0.25m of the edge because the slope is very different in this range for some reason.
  • Slight positive trend, but not significant
  • Bacterial Pathogens In Algae

    1. 1. Bacterial Pathogens within Persistent Algal Blooms in Southern Lake Michigan<br />Emily A. Junion<br />November 19, 2009<br />Tulane University School of Public Health and Tropical Medicine<br />City of Racine Health Department<br />
    2. 2. Introduction<br />Chladophora is a native algae in the Great Lakes<br />Introduction of non-native mussels, Dreissena spp., contribute to excessivealgae blooms<br />Non-point sources of nutrient runoff into the Great Lakes are a major growth limiting factor<br />Enumeration of fecal indicator organisms<br />Detection and identification of pathogens<br />
    3. 3. Algal blooms: a public health hazard?<br />Excessive algal growth and death cause areas of eutrophication<br />DO measurements very low within mat<br />Potential survival of anaerobic pathogens<br />Attracts birds that may deposit pathogens into the water from their feces<br />Infectious bacteria may be in water or attached to algae <br />Potential to cause recreationally contracted illness<br />
    4. 4. Indicator organisms in recreational waters<br />Indicator organisms, Escherichia coli and Enterococci sp., are used to measure water quality for recreational waters<br />Increased levels indicator organisms are correlated with increased risk of infection<br />Many beaches along Great Lakes shores have closures due to high levels of E. coli<br />Mats can produce sulfur/sewage-like odors<br />
    5. 5. Mussel ecological impact<br />Dreissenamussel spp. increase algal growth rates<br />Appear to clean water through removing turbidity<br />Increase light penetration through water, increasing photosynthesis rates and algae growth<br />Produce feces and pseudofeces<br />Expel NH4+ very high N excretion rates<br />Act as significant P sink, but P is re-released with protozoa grazing<br /><ul><li>Zebra mussels cover almost any hard surface</li></ul>Quagga mussels do not need a hard substrate for growth<br />Becoming the predominant mussel sp. in lake MI<br />
    6. 6. Hypotheses<br />There are significantly higher levels of E. coli from water within an algae mat versus water at least three meters outside the mat.<br />E. coli concentrations will increase on a gradient from outside to within the mat.<br />E. coli values increase in the mat over time.<br />
    7. 7. Hypotheses (cont.)<br />Pathogens can exist in water sampled from within an algae mat.<br />Pathogens can exist attached to algae.<br />
    8. 8. Sample design: hypothesis 1 & 3<br />
    9. 9. Sample design: hypothesis 2 <br />
    10. 10. Geographic location within Great Lakes<br />
    11. 11. 2007 site<br />2008 and 2009 sites<br />
    12. 12. Field materials<br />Cooler/ice packs<br />Sampling poles and sterile cups<br />Whirl-PacTM bags<br />Tape measurer<br />Sterile 8L carboy<br />Marking flags<br />Thermometers<br />DO meter with DO/temperature probe<br />
    13. 13.
    14. 14.
    15. 15.
    16. 16. E.Coli& Enterococci enumeration from algae<br />1g of algae was mixed with 9ml phosphate buffered water (PBW)<br />Vortexed tube for 2 min<br />Centrifuged for 45 sec at 2000 rpm<br />Bacterial enumeration was completed on the supernatant according to EPA methods 1603 (E. coli) and 1600 (Enterococci)<br />
    17. 17. E.colienumeration from water<br /><ul><li>All water samples were processed via membrane filtration according to EPA method 1603 </li></li></ul><li>Algae sample preparation for pathogens<br />5g of algae was mixed with 45ml phosphate buffered water (PBW)<br />Vortexed tube for 2 min<br />Centrifuged for 45 sec at 2000 rpm<br />
    18. 18. Pathogen analysis<br /><ul><li>PBW supernatants and water from the 8L carboy were analyzed according to provided SOPs for Campylobacter and Salmonella/Shigellaisolation</li></li></ul><li>Indicator organisms<br />Results<br />
    19. 19. E.colianalysis of water within vs. 3m outside algae mat<br />F-test Ho: sin= soutHA: sin≠ sout<br />T-test Ho: xgin≤ xgoutHA: xgin> xgout<br />
    20. 20. Three Year Mean Gradient (Both sides)<br />
    21. 21. E. coli water within mat<br />
    22. 22. E. coli extracted from algae<br />
    23. 23. Enterococci extracted from algae<br />
    24. 24. Pathogens<br />Results<br />
    25. 25. Identified pathogens within water from algae mats<br />
    26. 26. Identified pathogens extracted from Chladophora washings<br />
    27. 27. Pathogens<br />Campylobacter coli<br />Swine are the main reservoir for the bacteria <br />Birds can also be a carrier<br />Major cause of gastroenteritis in humans<br />Salmonella arizonae<br />Reptiles are the main reservoirs<br />Birds can also be a carrier <br />Has been known infect almost every human tissue, but usually children and immunosuppressed<br />
    28. 28. Pathogens (cont.)<br />Plesiomonasshigelloides<br />Has been isolated from many freshwater sources<br />Infections can cause gastroenteritis and septicemia in immunosuppressed people<br />Arcobacterbutzleri<br />Found in environmental samples and food borne<br />Originally thought to be a Campylobacter sp.<br />Accounts for about 1% of identified species from diarrheal diseases<br />
    29. 29. Potential human pathogens<br />Citrobacterfreundii<br />Found naturally in the environment<br />Normally do not cause disease<br />May cause UTIs, or infections in infants<br />Citrobacterbraaki<br />Found naturally in the environment<br />May cause mild wound infection<br />Proteus mirabilis<br />Commonly found in human intestines<br />May cause localized infections that can lead to septicemia<br />Can produce HS gas<br />
    30. 30. Conclusions<br />Higher levels of indicator organisms exist within algae compared to outside algal mats at >99.95% significance<br />There is evidence of a positive gradient of indicator organisms from 4m outside an algae mat to 4m within an algae mat<br />A slight increase in indicator organisms occurs within persistent mats over a four day period<br />Pathogens can exist in the water of algae mats and attach to the algae mat itself<br />
    31. 31. Recommendations<br />Research public health risks of bacterial pathogens that may exist in algal mats<br />Research TMDLs of rate limiting nutrients in areas of increased algal blooms<br />Continued research on interactions of invasive species in the Great Lakes<br />Research ways to reduce the introduction and spread of invasive species<br />Adopt removal of near shore and stranded algal mats as a BMP for reduction of fecal indicators<br />
    32. 32. Acknowledgements<br />Dr. Julie Kinzelman, City of Racine Health Department Lab<br />Jennifer Lavender, JustenePreedit, Stephan Kurdis, A.J. Koski, TristinBegotga, Michelle Leittl, Tamara Anan’eva<br />University of Wisconsin – Oshkosh<br />University of Wisconsin Sea Grant Research Program<br />
    33. 33. Questions??<br />
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