1. Lecture 2 Microbial Indicators of Fecal and Other Types of Environmental Contamination
ENVR 133
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 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
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. 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

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

15. Candidate 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 !

16. 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

17. 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

18. 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

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

20. Methods to Detect Coliphages in Water
Direct Plating Methods: water + host + medium
Enumerative: Double or Single Agar Layer : ml (DAL) or 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 s 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.

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

22. 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

23. 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.