Overview of Microbial Indicator Monitoring Lab Methods. Jim Ferretti, USEPA Region 2 DESA, Laboratory Branch May 23, 2018

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1 Overview of Microbial Indicator Monitoring Lab Methods Jim Ferretti, USEPA Region 2 DESA, Laboratory Branch May 23, 2018

2 Water Contamination and Public Health John Snow mapped and correlated incidence of cholera with proximity to public water pumps 1880 s- Robert Koch reported microbial disease agents Street map of area surrounding Broad Street pump London, s- Theodore Escherich suggested Bacillus coli as an indicator of fecal contamination

3 A brief history of bacterial assays 1904 assays for E. coli using glucose broths (Eijkman) Phenol red lactose broths 1920 s multiple tube fermentation with lactose broths (Leiter) 1950 s- membrane filtration (Goetz and Tsuneishi) 1988 defined substrates developed (Edberg et al.) Methods Membrane filter using m-endo broth (left) Quanti-tray using Colilert Medium (right) 2000 s- Real Time PCR

4 What is a pathogen? Pathogens are organisms capable of causing disease The following are some of the bad guys : Protozoa: Giardia, Cryptosporidium Bacteria: Salmonella typhi, Legionella, Shigella, Vibrio cholerae, Vibrio vulnificus Virus: Hepatitis, Polio How do they make you sick? They produce poisons (toxins) that result in fever, headache, vomiting, and diarrhea and destroy body tissue USDA NIFSI Food Safety in the Classroom University of Tennessee, Knoxville 2006

5 Are all bacteria pathogens? No, most are harmless Some are even helpful Examples of helpful bacteria: Lactobacillus: Pediococcus: makes cheese, yogurt, & buttermilk and produces vitamins in your intestine. makes pepperoni, salami, & summer sausage USDA NIFSI Food Safety in the Classroom University of Tennessee, Knoxville 2006

6 Example of Common Pathogens Salmonella E. coli O157:H7 Staphylococcus aureus Campylobacter jejuni

7 Examples of waterborne pathogens- Bacteria Name of micro-organisms Bacteria Major diseases Major reservoirs and primary sources Salmonella typhi Typhoid fever Human feces Salmonella paratyphi Paratyphoid fever Human feces Other Salmonella Salmonellosis Human and animal feces Shigella spp. Bacillary dysentery Human feces Vibrio cholera Cholera Human feces and freshwater zooplankton Enteropathogenic E. coli Gastroenteritis Human feces Yersinia enterocolitica Gastroenteritis Human and animal feces Campylobacter jejuni Gastroenteritis Human and animal feces Leptospira spp. Leptospirosis Animal and human urine Various mycobacteria Pulmonary illness Soil and water

8 More Examples of waterborne pathogens - Viruses Name of micro-organisms Enteric viruses Major diseases Major reservoirs and primary sources Polio viruses Poliomyelities Human feces Coxsackie viruses A Aseptic meningitis Human feces Coxsackie viruses B Aseptic meningitis Human feces Echo viruses Aseptic meningitis Human feces Norovirus Gastroenteritis Human feces to fomites and water Rotaviruses Gastroenteritis Human feces Adenoviruses Upper respiratory and gastrointestinal illness Human feces Hepatitis A virus Infectious hepatitis Human feces Hepatitis E virus Infectious hepatitis; miscarriage and death Human feces

9 More Examples of waterborne pathogens - Protozoans Name of micro-organisms Protozoa Major diseases Major reservoirs and primary sources Acanthamocba castellani Amoebic meningoencephalitis Human feces Balantidium coli Balantidosis (dysentery) Human and animal feces Cryptosporidium homonis, C. parvum Cryptosporidiosis (gastroenteritis) Water, human and other mammal feces Entamoeba histolytica Amoebic dysentery Human and animal feces Giardia lamblia Giardiasis (gastroenteritis) Water and animal feces Naegleria fowleri Helminths Primary amoebic meningoencephalitis Warm water Ascaris lumbricoides ascariosis Animal and human feces Adapted from Ashbolt, 2004

10 Protozoa Intestinal Parasites & Water Borne Diseases Giardia lamblia Cryptosporidium parvum

11 Difficulties With Routine Testing Of Pathogens in Water present in low numbers limited survival time numerous pathogens to analyze time and cost prohibitive

12 INDICATOR ORGANISM Exposure to fecal contaminated water does not always translate into infection. However, the higher the fecal bacterial levels in water, the higher the chances of pathogens to be present in significant numbers too.

13 Common Indicator: Bacteria Total Coliforms Fecal Coliforms E. coli Enterococci Total Coliforms Fecal Coliforms E. coli

14 Indicator Organisms Present when pathogens are present in water Absent in uncontaminated water Present in higher numbers than pathogens in contaminated water Better survival in water than pathogens Easy to analyze

15 COLIFORMS Coliform bacteria are defined as rod-shaped Gram-negative nonspore forming and motile or non-motile bacteria which can ferment lactose with the production of acid and gas when incubated at C

16 Coliform Genera Escherichia- human and animal feces Enterobacter- environment, feces Klebsiella- environment Citrobacter- environment Serratia- environment

17 Indicator Bacteria Regulated Based on Risk/Human Epidemiological Data Drinking Water 0 CFU/100 mls (E.coli) Shellfish Beds 14 CFU/100 mls (Fecal) Bathing Beaches 126 CFU/100 mls (E. coli) Secondary Contact (Fecal)

18 COMMON TECHNIQUES TO MEASURE INDICATOR BACTERIA

19 Approved Drinking Water Methods [40 CFR (f)] Membrane Filter Method - total coliforms, fecal coliforms, E. coli Multiple Tube Fermentation Method- total coliforms, fecal coliforms, E. coli Defined Substrate Tests Colilert, Colilert 18, Colisure

20 Bacterial Measurement Membrane Filtration Methods Quantify bacteria numbers by filtering water, growing bacteria on selective or deselective media, and counting Multiple Tube Fermentation Estimate bacterial numbers based upon a color change or amount of gas produced through a specific bacterial metabolic process Defined Substrate Methods Use a substrate that fluoresces and/or changes color to measure bacterial density (ex, Colilert)

21 Membrane Filtration Total Coliforms (m-endo broth) Total Coliform Red colony wiih a metallic sheen within 24±2 hours at 35±0.2 o C Total Coliforms (

22 Fecal Coliforms (m-fc broth) Blue colonies for fecal coliforms. Gray to cream colored are non fecal coliforms Fecal Coliforms ( Fecal Coliform Blue colony within 24±2 hours at 44.5±0.5 o C

23 mcoliblue24 MF Test for Total Coliforms and E. coli If Coliform bacteria are present in the sample, they will appear as red colonies and if E.coli in present in the sample, they will appear as blue colonies.

24 Multiple Tube Fermentation Method Step 1 presumptive phase Use lauryl tryptose broth Grow stressed organisms Confirm positives Calculate MPN Gas Growth

25 Common Media for MTF Tests for Total, Fecal, and E. coli Lauryl Tryptose Broth EC Media Brilliant Green Broth EC w/mug

26 Enzymatic Methods Coliform specific enzymes -Galactosidase (lacz) total coliforms Ability to breakdown lactose -D-Glucuronidase (uida) fecal coliforms Ability to breakdown complex sugars

27 Enzymatic Methods Presence / Absence Tests like MTF but with enzymes

28 Colilert - Total colifoms & E.coli ONPG (sugar analog) metabolized by coliform bacteria MUG tests for presence of enzyme found ONLY* in E. coli Results are easily read: Yellow indicates Coliform bacteria Fluorescent Blue/green indicates E. coli

29 Molecular Methods Nucleic Acid based techniques Polymerase Chain Reaction (PCR) phylogenetic primers enzyme specific primers detection limit viability

30

31 Thank you!

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