Lecture 2
Microbial Indicators of
Fecal and Other Types of
Environmental Contamination
ENVR 421
Mark D. Sobsey
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.
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
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
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
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.
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
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
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
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.
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)
Dose-Response Relationship Between Indicator Density in Vehicle
(Water) and Risk of Illness in Exposed Individual or Population:
Hypothetical Example
Illness
Risks
Indicator Concentration
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
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)
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
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 Betaglucuronidase 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
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 Betaglucuronidase and hydrolyzes
glucuronide substrates
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 !
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
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
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; hostdependent 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
Male-specific and Somatic Coliphage Hosts and Phages
Male-specific host
E. coli Famp
F+
Somatic
host
E. coli C
F+ Coliphage
Somatic
Coliphage
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.
Single Agar Layer Plaque Assay
Combine:
Sample
+
Host
+
Molten Agar
Mix
Pour in plate
Incubate
Count plaques
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
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, sourcespecific 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.