Objectives Be able to define what an indicator microorganism is and why they are used

1. 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

2. 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

3. 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

4. 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

5. Types of Indicators • Total coliforms • Fecal coliforms • Fecal Streptococci • Anaerobic bacteria • Bacteriophage

6. 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

7. 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

8. 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

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

10. 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!

11. 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)

12. 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

13. 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

14. gas production • + gas production Detection Methods 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)

15. Sample MPN Table

16. 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

17. 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.

18. 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

20. 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

21. ONPG MUG

22. 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

23. 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

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

25. Authority Standards . U.S. E.P.A. 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

26. 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