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??