Indicator Microorganisms – Chapter 23 Objectives Be able to define what an indicator microorganism is and why they are used Be able to list the criteria for an ideal indicator organism Be able to list at least three different types of indicators Be able to describe standard detection methods used to measure indicators Understand the difference between Water Quality Standards and Water Quality Guidelines

What Is an Indicator Microorganism? A nonpathogenic microorganism whose presence suggests the presence of enteric pathogens Indicator organisms are used because pathogens themselves are frequently difficult to detect in drinking water and wastewater low numbers (but still above MID) difficult, time consuming, or expensive to culture

U.S. Public Health Services adopted coliforms as indicators of fecal contamination of drinking water in 1914 coliforms are bacteria that live in the intestines of warm-blooded animals and are excreted in high numbers in feces indicate fecal contamination of drinking water presence demonstrates a breakdown of wastewater treatment processes The food industry uses other indicator microorganisms to evaluate the efficiency of food processing

Criteria for an Ideal Indicator Organism Should be useful for all types of water (drinking water, wastewater, recreational water, sea water) Should be present whenever enteric pathogens are present, and absent when pathogens are absent Should survive longer in the environment than the toughest enteric pathogen Should not grow in water Detection protocols should be easy and inexpensive Density of indicator microorganisms should correlate with the degree of fecal pollution Should be a member of the normal intestinal microflora of warm-blooded animals

Types of Indicators Total coliforms Fecal coliforms Fecal Streptococci Anaerobic bacteria Bacteriophage

Estimated levels of indicator organisms in raw sewage Organism CFU/100ml . Coliforms 107 - 109 Fecal coliforms 106 - 107 Fecal streptococci 105 - 106 Entercocci 104 - 105 Clostridium perfringens 104 Staphylococcus 103 Pseudomonas aeruginosa 105 Acid-fast bacteria 102 Coliphages 102 - 103 Bacteroides 107 - 1010

Total coliforms Most commonly used indicator for: drinking water, wastewater treatment, shellfish harvesting water, and recreational water Aerobic or facultatively anaerobic Gram negative non-spore-forming rod-shaped gas production during lactose fermentation within 48 hours at 35°C Examples: Escherichia, Citrobacter, Klebsiella and Enterobacter High numbers (2 x 109 per capita per day) in human AND animal feces < 1 coliform per 100 mL drinking water ensures the prevention of bacterial waterborne disease outbreaks

Drawbacks Coliforms may grow in aquatic environments, particularly if organic matter levels and temperatures are elevated Coliforms may form biofilms in drinking water distribution systems – this is a problem because, for example, E. coli is 2400 times more resistant to chlorine in a biofilm than when planktonic Coliforms may recover from disinfectant injury Growth of heterotrophic bacteria on media selective for coliforms can mask coliform population in water (occurs when heterotrophic counts exceed 500/mL) More vulnerable to disinfection and environmental trauma than enteric viruses or parasites Do not necessarily indicate fecal contamination

100 10 l m Coliforms 10 10 g 1 n / i n i i l a o 1 10 m c e . r E a Regrowth of coliforms and E. coli in sewage effluents after inactivation with 5 mg/L chlorine 100 10 7 l m Coliforms 10 10 6 g 1 n / i n Die-off rate depends on amount/type of organic matter present and the water temperature can lead to false positives i i l a o 1 10 5 m c e . r E a 0.1 10 4 r i o r e s t c m 0.01 10 3 a r B o f i % l 0.001 o 10 2 E. coli C 0.0001 10 1 0 1 2 3 4 5 6 7 Time (days)

Fecal coliforms Subgroup of total coliforms Able to ferment lactose and produce both acid and gas at 44.5°C in 24 hours Include Escherichia and Klebsiella, which are exclusively fecal in origin (perhaps. . .) Drawbacks same drawbacks as for total coliforms indicates fecal contamination for sure, but can’t distinguish between animal and human feces can survive and grow for extended periods of time in tropical waters may be natural inhabitants of these waters!

Fecal Streptococci do not multiply in water are more resistant to stress/disinfection last longer in the environment used as indicators of enteric viruses, and gastroenteritis for swimmers Members of the lactic acid bacteria Gram positive, non-motile, non-spore-forming, aerotolerant anaerobic bacteria that ferment sugars to lactic acid FC/FS ratio - ratio of fecal coliform counts to fecal strep counts FC/FS >4 : fecal contamination of human origin FC/FS < 0.7: fecal contamination of animal origin This relationship is only valid for recent fecal contamination (within the last 24 hours)

Anaerobic bacteria Clostridium perfringens – one example Gram positive, anaerobic spore-forming rod-shaped bacterium Spores are heat resistant (can survive 75°C for 15 min), resist disinfection, can remain viable in the environment for a long time May be used as indicator of resistant pathogens (viruses, parasites), past fecal contamination, or tracing fecal contamination in a marine environment Drawbacks A common soil bacterium; may not necessarily indicate fecal contamination Pathogenic (causes gas gangrene if it infects wounds, produces enterotoxin in small intestine causing gastroenteritis) Anaerobic culture is difficult

Bacteriophage Coliphage – one example bacteriophage that infect coliforms, particularly E. coli similar to enteric viruses in size, morphology, and performance in environment found in higher numbers than enteric viruses in wastewater and other waters rapid and easy detection methods available survive for 7 days in shellfish without increasing in numbers routinely used as indicator microorganisms to determine the effectiveness of wastewater treatment processes resistant to disinfection

Most Probable Number (MPN) Detection Methods gas production + gas production Most Probable Number (MPN) Used to detect coliforms This test consists of two to three steps: Presumptive test Confirming test Completed test Presumptive test: dilute water sample Inoculate 3 or 5 tubes of lauryl sulfate-tryptose-lactose broth containing upside-down Durham tubes with water dilutions Incubate at 35°C for 48 hours Determine number of tubes at each dilution that are positive for gas production (contain bubble in Durham tube)

Sample MPN Table

Confirming test – select a positive tube and inoculate a Levines EMB agar and Endo Agar plate Completed test – inoculate a colony back into MPN media and confirm acid and gas production. (Not always performed) Levines EMB agar Endo Agar + - + - Coliforms produce “nucleated” colonies Coliforms and surrounding medium turn red

What would you do to detect fecal coliforms instead of coliforms ???? Drawback to MPN test: HPC can outcompete coliforms and fecal coliforms for nutrients in the environment and mask their detection by this method.

Membrane Filter Test Used to detect coliforms Filter 100 mL water through a 0.45 m filter Incubate filter on pad soaked with a differential medium (Endo medium; contains lactose and Basic Fuchsin dye) at 35°C for 18-24 hours Count colonies that grow on filter coliforms will be dark red with metallic gold sheen To enumerate Fecal Streptococci, grow on Streptococcus agar at 37°C for 24 hours. Fecal streptococci reduce 2,4,5-triphenyltetrazolium chloride to formazan, which makes colonies appear red Much quicker and easier than MPN method

Presence-Absence Tests, e.g., Colilert Test Qualitative NOT quantitative Used to detect total coliforms and E. coli Add packet of salts and nutrients to water sample and incubate 24 hours Total coliforms can convert o-nitrophenyl--D-galactopyranoside (ONPG) to yellow nitrophenol with -galactosidase E. coli can metabolize 4-methylumbelliferone glucuronide (MUG) to a molecule that fluoresces under UV light with glucuronidase May not detect up to 1/3 of E. coli strains (including pathogenic ones!) Broth and agar plate techniques involving ONPG and MUG also exist

ONPG MUG

Heterotrophic Plate Counts (HPC) Enumeration of all aerobic and facultative anaerobic chemoheterotrophs in water includes Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Enterobacter, Citrobacter, Serratia, Acinetobacter, Proteus, Alcaligenes, and Moraxella Varies from 1 to 104 CFU/mL, and depends on temperature, residual chlorine concentration, and availability of organic nutrients Indicates general quality of water (particularly levels of organic matter in water) HPC > 500 CFU/mL indicates poor water quality

Plaque Assay Used to detect bacteriophage Filter phage from water with charged membrane filter Elute with beef extract, pH 9.0 Flocculate solids (including phage) with HCl. Centrifuge. Remove supernatant and resuspend pellet in beef extract. Neutralize solution. Inoculate 4 mL loose (0.7%) agar with host bacterial culture and 100 L phage concentrate. Pour loose agar onto a solid agar plate. Incubate for 8-18 hours Host bacteria will form lawn on plate. Bacteriophage will lyse small holes in the lawn (plaques) Count plaques and compare to the volume of filtered water to determine bacteriophage population in the water sample

Water Quality Standards and Guidelines Regulated at both the Federal and State levels Water quality standards are legally enforceable!!

Authority Standards . U.S. E.P.A. California Safe Drinking Water Act 0 coliforms/100ml Clean Water Act Wastewater discharges 200 fecal coliforms/100 ml Sewage sludge <1000 fecal coliforms/4 g <3 Salmonella/4 g <1 enteric virus/4 g <1 helmintha ova/4 g California Wastewater reclamation <2.2 MPN coliforms for irrigation Arizona Wastewater reclamation 25 fecal coliforms/100ml for irrigation of golf 125 enteric virus/40 L courses No detectable Giardia/40 L

Guidelines for Recreational Water Quality Standards Water Quality Criteria and Guidelines Comprise recommendations for acceptable levels of indicator microorganisms NOT legally enforceable!!! Guidelines for Recreational Water Quality Standards Regime Country (samples/time) Criteria or standard U.S.EPA 5/30 days 200 fecal coliforms/100ml <10% to exceed 400/ml Fresh water 33 enterocci/100 ml 126 fecal coliforms/100 ml Marine water 35 enterococci/100 ml