Overview of Microbial Indicator

Monitoring Lab Methods

Jim Ferretti, USEPA Region 2

DESA, Laboratory Branch

May 23, 2018

Water Contamination and Public Health

• 1854- John Snow mapped and correlated incidence of cholera with proximity to public water pumps

• 1880’s- Robert Koch reported microbial disease agents

• 1880’s- Theodore Escherichsuggested “Bacillus coli” as an indicator of fecal contamination

Street map of area surrounding Broad Street pump

London, 1843

A brief history of bacterial assays

• 1904 – assays for E. coli using

glucose broths (Eijkman)

• 1920’s – multiple tube

fermentation with lactose

broths (Leiter)

• 1950’s- membrane filtration

(Goetz and Tsuneishi)

• 1988 – defined substrates

developed (Edberg et al.)

• 2000’s- Real Time PCR

Phenol red lactose broths

Methods

• Membrane filter using m-endo broth (left)

• Quanti-tray using Colilert Medium (right)

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

Are all bacteria pathogens?

• No, most are harmless

• Some are even helpful

• Examples of helpful bacteria:

USDA NIFSI Food Safety in the Classroom©

University of Tennessee, Knoxville 2006

makes cheese, yogurt, & buttermilk and produces vitamins in your intestine.

Lactobacillus:

makes pepperoni, salami, & summer sausage

Pediococcus:

Example of Common Pathogens

Salmonella E. coli O157:H7

Staphylococcus aureus Campylobacter jejuni

Examples of waterborne pathogens- Bacteria

Name of micro-organisms Major diseasesMajor reservoirs and primary

sources

Bacteria

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

More Examples of waterborne pathogens - Viruses

Name of micro-organisms Major diseasesMajor reservoirs and primary

sources

Enteric viruses

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

More Examples of waterborne pathogens - Protozoans

Name of micro-organisms Major diseasesMajor reservoirs and primary

sources

Protozoa

Acanthamocba castellani Amoebic meningoencephalitis Human feces

Balantidium coli Balantidosis (dysentery) Human and animal feces

Cryptosporidium homonis, C.

parvumCryptosporidiosis (gastroenteritis)

Water, human and other mammal

feces

Entamoeba histolytica Amoebic dysentery Human and animal feces

Giardia lamblia Giardiasis (gastroenteritis) Water and animal feces

Naegleria fowleriPrimary amoebic

meningoencephalitis Warm water

Helminths

Ascaris lumbricoides ascariosis Animal and human feces

Adapted from Ashbolt, 2004

Protozoa Intestinal Parasites &

Water Borne Diseases

Giardia lamblia Cryptosporidium parvum

Difficulties With Routine Testing

Of Pathogens in Water

• present in low numbers

• limited survival time

• numerous pathogens to analyze

• time and cost prohibitive

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.

Common Indicator: Bacteria

• Total Coliforms

• Fecal Coliforms

• E. coli

• Enterococci E. coli

Total

Coliforms

Fecal

Coliforms

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

Coliform bacteria are defined as rod-shaped Gram-negative non-

spore forming and motile or non-motile bacteria which can ferment

lactose with the production of acid and gas when incubated at 35–37°C

COLIFORMS…………………

Coliform Genera

►Escherichia- human and animal feces

►Enterobacter- environment, feces

►Klebsiella- environment

►Citrobacter- environment

►Serratia- environment

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 770-1500 (Fecal)

COMMON TECHNIQUES TO

MEASURE INDICATOR

BACTERIA

Approved Drinking Water Methods

[40 CFR 141.21 (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

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)

Total Coliforms (m-Endo broth)

Total Coliform

• Red colony wiih a

metallic sheen

within 24±2 hours

at 35±0.2oC

Total Coliforms(http://dl.clackamas.cc.or.us/wqt111/unit-8-coliformtest.htm)

Membrane Filtration

Fecal Coliforms (m-FC broth)

• Blue colonies

for fecal

coliforms.

Gray to cream

colored are

non fecal

coliforms

Fecal Coliform

• Blue colony

within 24±2 hours

at 44.5±0.5oCFecal Coliforms(http://dl.clackamas.cc.or.us/wqt111/unit-8-coliformtest.htm)

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.

mColiBlue24 MF Test for Total Coliforms and E. coli

Multiple Tube Fermentation Method

• Step 1 presumptive phase

• Use lauryl tryptose broth

• Grow “stressed” organisms

• Confirm positives

• Calculate MPN

Growth

Gas

http://www.bact.wisc.edu/Microtextbook/index.php?module=Book&func=displaychapter&chap_id=55&theme=printer

Lauryl Tryptose

Broth

Brilliant Green Broth

EC Media

EC w/Mug

Common Media for MTF Tests for Total, Fecal, and E. coli

Enzymatic Methods

• Coliform specific enzymes

• -Galactosidase (lacZ) –total coliforms

• Ability to breakdown lactose

• -D-Glucuronidase (uidA) –fecal coliforms

• Ability to breakdown complex sugars

Enzymatic Methods

• Presence / Absence Tests

• like MTF but with enzymes

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

Molecular Methods

• Nucleic Acid based techniques

• Polymerase Chain Reaction (PCR)

• phylogenetic primers

• enzyme specific primers

• detection limit

• viability

Thank you!