Microbial indicators of water quality

1. Microbial indicators of water quality

2. (Total) Heterotrophic Count any bacteria growing in water indicate poor quality (100-500 cfu/ml is acceptable, depending on the country) not suitable for more complex samples the cheapest and easiest technique of all good survey technique detects only bacteria which grow fast (48 hrs at 30-37oC) on common rich media won�t detect anaerobes won�t detect bacteria with specialized growth requirements (e.g. Campylobacter, Legionella, Listeria) tells little about the identity of cultured bugs. not a Coliform test

3. WHO criteria for microbial indicators An indicator should be: absent from unpolluted water

4. WHO criteria for microbial indicators An indicator should be: absent from unpolluted water present when the source of pollution is present

5. WHO criteria for microbial indicators An indicator should be: absent from unpolluted water present when the source of pollution is present easy (and inexpensive) to isolate, identify and enumerate

6. WHO criteria for microbial indicators An indicator should be: absent from unpolluted water present when the source of pollution is present easy (and inexpensive) to isolate, identify and enumerate present in higher numbers than pathogens

7. WHO criteria for microbial indicators An indicator should be: absent from unpolluted water present when the source of pollution is present easy (and inexpensive) to isolate, identify and enumerate present in higher numbers than pathogens respond to treatment and environmental conditions similarly to the pathogens of concern

8. WHO criteria for microbial indicators An indicator should be: absent from unpolluted water present when the source of pollution is present easy (and inexpensive) to isolate, identify and enumerate present in higher numbers than pathogens respond to treatment and environmental conditions similarly to the pathogens of concern not be a pathogen

9. WHO criteria for microbial indicators An indicator should be: absent from unpolluted water present when the source of pollution is present easy (and inexpensive) to isolate, identify and enumerate present in higher numbers than pathogens respond to treatment and environmental conditions similarly to the pathogens of concern not be a pathogen should not multiply in the environment

10. E. coli Advantages: easy to identify fast and cheaply normally non-pathogenic present in higher concentrations than pathogens Disadvantages: survives and multiplies in contaminated water, soils (esp. in warmer climates) isolated from pristine waters not unique to humans

11. Coliforms as indicators Coliforms: Escherichia, Citrobacter, Enterobacter and Klebsiella Defined as: gram-negative not sporulating oxidase-negative optional aerobic or anaerobic able to multiply in the presence of bile salts ferment lactose with acid and gas production in 48 h at the temperature of 44C False positives: many strains of Enterobacter and Klebsiella are strictly plant-associated. ID�s of high loads of these bugs in tea, sprouts caused media frenzy but no outbreaks

12. Enterococcus spp. Advantages: E. faecalis, E. faecium most frequently found in humans esp. reliable for marine water samples (grows fine in 6.5% NaCl, pH 9.6) Disadvantages: can re-grow once introduced into soil, water doesn�t persist in soils and vegetables as long as E.coli

13. Clostridium perfingens Controversial indicator. Hawaii. G+ Advantages fairly specific to human feces sometimes recommended as a predictor of remote pollution or viral contamination Disadvantages anaerobic spore-forming pathogenic re-grows in the environment

14. if these microbial indicators are so controversial�.. �. then WHAT?

15. Risk assessment analysis Other microbial indicators (none without +&-) Direct monitoring for Cryptosporidium, Giardia, viruses Fecal coliforms/fecal streptococcus ratio >4 (human pollution), <0.7 (nonhuman) fast relies on existing methods

16. ASM Recommendations In 2001, American Society for Microbiology was asked to comment on Total Coliform Rule ASM recommends: Use E. coli as the primary indicator of fecal contamination Employ other indicators (e.g. Clostridium or bacteriophages) of viral and protozoan contamination. WHY? Use the total coliform group as a secondary standard of treatment optimization Adopt the comprehensive approach for risk-based distribution system monitoring Encourage the development of automated and advanced analytical techniques Streamline and simplify the TCR to reduce monitoring and reporting problems

17. Other microbial indicators Bifidobacterium major component of human intestine, abundant in feces (+) rarely found in animals (+) only human isolates ferment sorbitol (+) Human bifid sorbitol agar (HBSA) by Mara and Oragui obligate anaerobe (+/-) not trivial to culture won�t multiply in most environments, but doesn�t survive well either (+/-) indicates recent contamination

18. Other microbial indicators Phage (virus) of Bacteroides fragilis B. fragilis is obligate anaerobe in high numbers in human & animal intestines 10% of all humans carry strain HSP40 phage, specific to HSP40, is the indicator phage don�t replicate in the environment some highly polluted waters don�t contain the phage

19. Other microbial indicators F-specific RNA coliphage coliphages are viruses of E.coli F-specific is specific to F+ E.coli variety of enumeration methods exist low number of the phages in the environment only a small %% of human feces contain this phage

20. Other microbial indicators Human enteric viruses direct measurement! 100 viruses live in human GI tract bacterial indicators are un-reliable when enteroviruses are present direct PCR monitoring of drinking water for enteric viruses is recommended hard to ID non-culturable viruses, RT-PCR may be an option viable vs non-viable viruses cell culture infections

21. Other microbial indicators Indicators of non-human contamination: Streptococcus bovis. Cattle Rhodococcus coprophilus. Specific to graizing animals bovine enteroviruses host-adapted strains of Giardia and Cryptosporidium (need Nucleic Acid assays)

22. Phenotypic tracking methods MAR analysis E.coli (or Enterococcus) isolates are tested for antibiotic resistance differentiate between human and non-human isolates based on a/b �fingerprints� 60-80% accurate in assigning bacterial origin, BUT always informative regarding resistance to particular antibiotics

23. Phenotypic tracking methods MAR analysis E.coli (or Enterococcus) isolates are tested for antibiotic resistance differentiate between human and non-human isolates based on a/b �fingerprints� 60-80% accurate in assigning bacterial origin, BUT always informative regarding resistance to particular antibiotics CUP (carbohydrate utilization profile) rate of false-positives is similar to the rate of correct positive IDs

24. Phenotypic tracking methods MAR analysis E.coli (or Enterococcus) isolates are tested for antibiotic resistance differentiate between human and non-human isolates based on a/b �fingerprints� 60-80% accurate in assigning bacterial origin, BUT always informative regarding resistance to particular antibiotics CUP (carbohydrate utilization profile) rate of false-positives is similar to the rate of correct positive IDs Serotyping human and animal serotypes are somewhat different

25. Enumeration of bio-indicators in water Membrane filtration Most probably number (MPN) For phages: plaques

26. Phages, plaques, etc Life cycle of a phage

27. Phages, plaques, etc Plaque formation

28. Phages, plaques, etc Plaque formation

29. Plaque MPN

30. Detection of coliforms: