Lecture 2 Microbial Indicators of Fecal and Other Types of Environmental Contamination

1. Lecture 2 Microbial Indicators of Fecal and Other Types of Environmental Contamination ENVR 421 Mark D. Sobsey

2. Microbial Indicator Concepts and Purposes • Many diverse types of pathogens can contaminate water, food, air and other environmental media; many different ones. • Measuring all of these pathogens routinely for determining presence or absence or acceptable concentration is not possible. • Methods are not available to recover and measure some, • Methods are available for others; they are technically demanding, some are slow to produce results and their costs are high. • Alternative approach: measure something other than a pathogen that is indicative of contamination, predicts pathogen presence and estimates human health risks.

3. What is Measured as Microbial Indicators and Why? • Microbial indicators have been used for >100 years (since late 1800s) to detect and quantify fecal contamination in water, food and other samples • Concerns were for bacteria causing water-and foodborne illness, such as: • Salmonella typhi: causes typhoid or enteric fever • Vibrio cholerae: causes cholera • Shigella dysenteriae and other Shigella species: dysentery • Focus was and still is on detecting primarily human (or maybe animal) fecal contamination as source of these and other enteric bacterial pathogens • Detect fecal contamination by measuring: • common enteric bacteria residing in the gut and shed fecally • Chemicals associated with the gut or with anthropogenic fecal contamination • Something else associated with and predictive of fecal contamination

4. What is Measured as Microbial Indicators and Why? • Microbial indicators are used to indicate other conditions, unrelated to fecal contamination, such as : • Food spoilage bacteria and molds • Excessive microbial growth in water and biofilms that cause appearance, taste and odor problems: • “red water” from iron biofouling • blooms of algae and cyanobacteria (blue-green algae) • Some of the organisms harbor or release toxins (harmful algal blooms or “red tides”) • Excessive bacterial release from biological filters used in water treatment

5. What is Measured as Microbial Indicators and Why? • Airborne contamination: • From wet buildings: molds and actinomycetes • From industrial processes: • bacterial endotoxins from cotton dust, solid waste and other sources • Microbial allergens from manufacturing processes (aerosols and dusts) • total airborne microbe concentrations • In health care facilities • In “clean room” manufacturing environments for electronics and pharmaceuticals • From composting operations • Salivary bacteria from dentistry activities

6. Fecal Microbial Indicators: Background and Rationale • Besides nutrients and organic matter, human and animal fecal wastes contain large numbers of microbes (~100 billion/gram). • Constitute ~1/3rd the mass of human fecal matter • Most are beneficial or essential in the gut; not pathogens • Some gut microbes are human pathogens; they cause disease. • Human pathogens can be shed in human and animal feces. • Humans and animals harbor pathogens some of the time; infection • Enteric pathogens are transmitted by the fecal-oral route; often 106-109 pathogens/gram of feces of an infected person • Ingested with fecally contaminated water, food, objects, • or by direct and indirect personal contact.

7. Limitations in Detecting Enteric (Fecal) Pathogens and their Indicators • Some enteric pathogens have environmental origins besides feces; environmental reservoirs • Some microbial indicators of fecal contamination have both fecal and non-fecal environmental sources • Some fecal indicator microbe tests also detect similar non-fecal microbes; tests are not perfect in detecting microbes of fecal origin

8. Pathogen Detection and Monitoring • Pathogen detection • technically demanding, • often tedious, • slow to produce results, • Often unreliable • expensive. • Done routinely in the health care field (clinical diagnostic microbiology): • often essential to patient treatment and care • provides national surveillance of infectious disease epidemiology

9. Pathogen Analysis, Monitoring and Surveillance • Until recently, rarely done for managing food quality • Salmonella and E. coli O157:H7 are now monitored in meat and poultry; Listeria monocytogenes monitoring also being done • Sometimes done for monitoring or managing water quality • pathogen occurrence surveys and special studies: • survey (18 months) for Giardia, Cryptosporidium and enteric viruses in larger drinking water supplies using surface water sources: ICR (Information Collection Regulation) • enteric virus survey in ground water sources of drinking water • Data base for Ground Water Disinfection Rule • investigation of waterborne outbreaks • Technology verification/performance evaluation • Pilot/in-plant studies • Pathogen monitoring sometimes done for biosolids (Class A) • Salmonella, viable Ascaris ova, culturable enteric viruses

10. Microbial Indicators of Fecal Contamination • Traditional approach to protect/assess the "sanitary" quality of water (food) with respect to fecal contamination. • Quantify bacteria commonly present in intestines of warm blooded animals • high numbers • easy to measure • surrogates for pathogens • Developed when bacterial pathogens were recognized in late 1800s and early 1900s • Salmonella, Shigella, V. cholerae, etc.

11. Criteria for Reliable Indicators of Fecal Contamination Applicable to all types of water (and other relevant samples). Present in feces, sewage and fecally contaminated samples when pathogens are present; numbers correlate with amount of fecal contamination; outnumber pathogens No "aftergrowth" or "regrowth" in the environment Survive/persist > than or = to pathogens. Easily detected/quantified by simple lab tests in a short time. Constant characteristics Harmless to humans and other animals Numbers in water (food, etc.) are associated with risks of enteric illness in consumers (dose-response relationship)

12. Dose-Response Relationship Between Indicator Density in Vehicle (Water) and Risk of Illness in Exposed Individual or Population: Hypothetical Example Illness Risks Indicator Concentration

13. Systems and Technologies for Microbial Indicator Monitoring • Static laboratories • Lab infrastructure, expensive equipment, consumable supplies and trained staff, QA/QC • Potentially long chain to get samples from field to lab • Routine in developed world but rare in developing world • Field labs / portable labs • Simpler and cheaper • Integrated systems • Colilert system, DelAgua and Millipore test kits • Still need knowledgeable and trained staff; relatively expensive • Disposable tests • Petrifilm, Presence/absence test for H2S-producing bacteria • Still relatively expensive (>$1); minimal training needed

14. Desirable Properties of Diagnostic Tests for Developing World • Affordable by those at risk • Sensitive (low false-negatives) • Specific (few false positives) • User-friendly (simple to perform) • Rapid and robust; easy to store • Equipment-free (ideally no electricity) • Delivered to those who need it (Urdea et al ‘Requirements for high impact diagnostics in the developing world’ Nature S1, 73-79 (23 Nov 06)

15. Current Options for Fecal Bacteria Testing by Culture Methods • Liquid quantal (MPN and P-A) assays: • Defined substrate technology (e.e. Colilert • Other chromogenic/fluorogenic broths • Less specific broths followed by biochemical confirmation (e.g., urease test) • Membrane filter methods • Chromogenic/fluorogenic media • Pour and spread plates • e.g., pectin gel media:Coliscan Easygel • PetriFilm and other films; as culture plates

16. Current Bacterial indicators of Fecal Contamination • Total coliforms: • drinking, bathing and shellfish water standards • not feces-specific (environmental sources). • Fecal ("thermotolerant") coliforms (FC): • detect by growing at elevated temperature of 44-45oC • ditto total coliforms, but less so • E. coli: the "fecal" coliform • Detect and distinguish from other total and fecal coliforms by Beta-glucuronidase activity • may occur naturally in tropical environments (and possibly elsewhere) • Fecal streptococci (FS): • Mostly Lancefield group D (and some group Q) streptococci and enterococci • not feces-specific. • Enterococci: • More feces-specific sub-set of FS: Enterococcus faecalis & E. faecium • EPA guideline for bathing water quality

17. Relationships among Total and Fecal Coliforms and E. coli Total Coliforms Fecal Coliforms Escherichia coli • All total and fecal coliforms and E. coli possess Beta-galactosidase; they can hydrolyze and and ferment lactose • E. coli also possesses Beta-glucuronidase and hydrolyzes glucuronide substrates

18. Some Other Bacterial Indicators of Fecal Contamination Clostridium perfringens: • spore-forming anaerobe • feces-specific? • very (too?) resistant spores (can persist for decades of centuries!) • may be an indicator for protozoan cysts Bacteroides spp. and Bifidobacteria spp.: • most plentiful in feces (100X more than FC, FS and E. coli) • strict anaerobes • poor survival in the presence of air (oxygen) • poor detection methods: requires strict anaerobic conditions • Some Bacteroides species may be human-specific Rhodococcus coprophilus: • plentiful in feces of some animals • possible animal fecal contamination indicator BACTERIA ARE NOT ALWAYS GOOD INDICATORS OF VIRUSES AND PROTOZOANS !

19. Methods to Detect Bacterial Indicators: Quantal Methods • Inoculate multiple sample volumes usually into broth culture medium, incubate and score each volume as positive or negative for indicator growth • Positive cultures in differential/selective medium or based on characteristic appearance • Results for numbers of positive and negative cultures are used to estimate bacterial density • Usually based on a maximum likelihood estimate • Expressed as a Most Probable Number or MPN

20. Methods to Detect Bacterial Indicators: Enumerative Methods • Agar media methods • Inoculate sample onto/into differential/selective agar medium • Pour, spread or spot plate methods • Membrane filter methods • Filter a sample volume through a membrane filter that retain bacteria and place membrane on a differential/selective medium • Incubate • Count numbers of characteristic bacterial colonies on/in agar plates or on membrane filters and compute colony forming units (CFU) per unit volume

21. CANDIDATE VIRAL INDICATORS OF FECAL CONTAMINATION OF WATER Coliphages: viruses (bacteriophages) infecting E. coli and perhaps other coliforms; attach directly to cell wall (somatic) heterogeneous group; may not be feces-specific; host-dependent detection Male-specific (F+) coliphages: coliphages infecting "male" strains of E. coli (posses pili); may be feces-specific May distinguish human from animal fecal contamination by group classification (II & III human; I & IV animal); but, pigs may harbor groups II & III, too Bacteroides fragilis phages: may be human feces specific; USA studies do not show human-specificity; concentrations too low Salmonella phages: in human and animal feces; may indicate presence of Salmonella bacteria; concentrations too low

22. Male-specific and Somatic Coliphage Hosts and Phages Male-specific host Somatic host E. coli Famp F+ E. coli C F+ Coliphage Somatic Coliphage

23. Methods to Detect Coliphages in Water • Direct Plating Methods: water + host + medium • Enumerative: Double or Single Agar Layer : 0.1-1 ml (DAL) or 10-100 ml SAL • “pour plate” or plaque assay for development of phage “plaques” • Quantal: Enrichment culture: 100-1,000 ml; confirm by lysis of host on “lawn”. • Filter Adsorption-Elution Methods: • filter 100-1000s of mls through electropositive filter • (or add divalent cations and filter through cellulose membrane) • elute adsorbed phages • plaque assay or other assay on host bacteria by other methods • Direct Membrane Filter Method: • add divalent cation to water; filter through cellulose ester membrane • place membrane face down on agar medium-host lawn • incubate, and count plaques.

24. Single Agar Layer Plaque Assay Combine: Sample + Host + Molten Agar Mix Pour in plate Incubate Count plaques

25. Indicators of Protozoan Parasites • Currently, there is no universally reliable indicator of enteric protozoan parasites • Candidate indicators are bacterial spores: • Spores of Clostridium perfringens (a gut anaerobe); fecal indicator; also used as enteric virus indicator; indicator of occurrence, survival and treatment • Aerobic endospores (primarily spores of Bacillus species); ubiquitous environmental indicators of water treatment efficacy for Giardia, Cryptosporidium • Microalgae: candidate water treatment indicators; some are the same size and shape as protozoan pathogens; some also have similar surface properties

26. Microbial Source Indicators: Microbial Source Tracking Indicators and Methods • Standard indicators and methods are unable to identify specific fecal sources impacting water and other environmental media; need advanced methods • Phenotypic: antimicrobial resistance patterns, source-specific microbes (Rhodococcus coprophilus for animals vs. Bacteroides/Prevotella species like fragilis, thetaiotaomicron, etc. for humans, fecal sterols, etc. • Molecular methods: human vs. animal F+ RNA coliphages, Bacteroides/Prevotella species (16S rRNA), multilocus PCR and nucleotide sequencing of specific bacteria, virus or parasite genes or loci, ribotyping, pulse field gel electrophoresis, etc.