Isolation and identification of bacteria in the rotifer mass culture medium
Pathogens Found Within Persistent Algal Blooms in Lake Michigan
1. Bacterial Pathogens within Persistent Algal Blooms in Southern Lake Michigan Emily A. Junion November 19, 2009 Tulane University School of Public Health and Tropical Medicine City of Racine Health Department
2. Introduction Chladophora is a native algae in the Great Lakes Introduction of non-native mussels, Dreissena spp., contribute to excessivealgae 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
3. 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
4. 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
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6. Hypotheses There are significantly higher levels of E. coli from water within an algae mat versus water at least three meters outside the mat. E. coli concentrations will increase on a gradient from outside to within the mat. E. coli values increase in the mat over time.
7. Hypotheses (cont.) Pathogens can exist in water sampled from within an algae mat. Pathogens can exist attached to algae.
12. 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
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16. E.Coli& Enterococci enumeration from algae 1g of algae was mixed with 9ml phosphate buffered water (PBW) Vortexed tube for 2 min Centrifuged for 45 sec at 2000 rpm Bacterial enumeration was completed on the supernatant according to EPA methods 1603 (E. coli) and 1600 (Enterococci)
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19. E.colianalysis of water within vs. 3m outside algae mat F-test Ho: sin= soutHA: sin≠ sout T-test Ho: xgin≤ xgoutHA: xgin> xgout
27. 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
28. Pathogens (cont.) Plesiomonasshigelloides Has been isolated from many freshwater sources Infections can cause gastroenteritis and septicemia in immunosuppressed people Arcobacterbutzleri Found in environmental samples and food borne Originally thought to be a Campylobacter sp. Accounts for about 1% of identified species from diarrheal diseases
29. Potential human pathogens Citrobacterfreundii Found naturally in the environment Normally do not cause disease May cause UTIs, or infections in infants Citrobacterbraaki 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
30. 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
31. 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
32. Acknowledgements Dr. Julie Kinzelman, City of Racine Health Department Lab Jennifer Lavender, JustenePreedit, Stephan Kurdis, A.J. Koski, TristinBegotga, Michelle Leittl, Tamara Anan’eva University of Wisconsin – Oshkosh University of Wisconsin Sea Grant Research Program
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.
