Alternative indicators of fecal pollution Relations with pathogens

							 Alternative indicators of fecal
pollution: Relations with
pathogens and conventional
indicators, current methodologies
for direct pathogen monitoring
and future application
perspectives
      Indicators historically used
   Total coliforms
   Fecal coliforms
   Enterococci
Limitations reported:
   Short survival in water body
   Non-fecal source
   Ability to multiply after releasing into water column
   Great weakness to the disinfection process
   Low levels of correlation with the presence of pathogens
    Correlation between conventional
        indicators and pathogens
   “Standard pathogens”: Salmonella spp.,
    Cryptospordium spp. and Giardia spp.
   Coliform group demonstrated poor correlation
    with various pathogenic microorganisms
   Streptococci had low correlation with Salmonella
    spp.
   Limited data on correlation between coliforms
    and pathogens or diarrhea diseases
      Alternative Indicators
 Fecal Anaerobes
 Viral Indicators
 Chemical Compounds
            Viral Indicators
 Bacterial indicators do not serve well as
  indicator for viruses
 Amount of phages commonly ten times
  greater than enterovirus in environmental
  water samples
         Viral Indicators
Bacteroides fragilis bacteriophage
   B. fragilis HSP 40 strain only found in human
    samples
   Multiply only in human intestinal tracts
   B. fragilis survives better than enteric virus in
    surface waters
   B. fragilis HSP 40 found in 72% of water and
    sediment samples contaminated with sewage
    pollution (56% for enterovirus)
   Difficult to detect in waters with low levels of
    fecal contamination
                  Viral Indicators
Coliphages (F-specific RNA coliphage)
   Animal and human feces contain different serotypes of
    RNA coliphage
   Male-specific (F+) coliphage are similar in size and
    shape structure and genetic makeup to human enteric
    virus
   More stable than human enterovirus in environmental
    water
   Very correlated to fecal contamination
   Less resistant in high salinity environment
   Simple methods for concentration and recovery from
    water body needed
        Chemical compounds
            Coprostanol
 Byproduct of Cholesterol
 One of the major fecal sterols excreted by
  humans and animals
 Half life of less than 10 days at 20oC
 Used as indication of fresh contamination
  in Japan and Vietnam
 Strong logarithmic correlation between the
  concentration of E. coli and coprostanol
 Simple analytical method – GC-MS
         Chemical compounds
             Coprostanol
Disadvantages
 In sediments, it is stable for 450 days
 Lack of studies related to host specificity,
  detection sensitivity and correlation with
  pathogens
    Direct Monitoring of pathogens
    Traditional methods underestimate pathogen density
   Physically injured or stressed
   VBNC state
   Non-culturable pathogens

    Molecular-based methods have stimulated interest in
    direct monitoring
    PCR-based methods were used to detect the following
    pathogens in various environmental waters with success

   Salmonella spp., Shigella spp., Campylobacter spp.,
    legionellae, Vibrio vulnificus, different pathogenic strains
    of E. coli, protozoan parasites and enteroviruses
 Direct Monitoring of pathogens
However, interpreting PCR results is
 very complicated
 Positive results ?
RNA-based PCR method
 Negative results ?
           Future Directions
 Better understanding of the source and
  fate of microbial contaminants
 Epidemiological studies are needed when
  alternative fecal indicators are assessed
 Investigate the distribution of host-specific
  genetic markers
 Development of fecal indicators suitable
  for different climatic regions
               References

   WHO (2002) Indicators (draft document).
    OECD, World Health Organization,
    Geneva.
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
    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
    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
  Nucleic Acid-based Methods
which involve library comparisons
   Repetitive element-PCR
       rapid test to ID bacteria
       geographic differences may exist, databases required
  Nucleic Acid-based Methods
which involve library comparisons
   Repetitive element-PCR
       rapid test to ID bacteria
       geographic differences may exist, databases required
   Ribotyping
       rRNA genes are amplified
       highly reproducible
       labor-intensive, reference database required
  Nucleic Acid-based Methods
which involve library comparisons
   Repetitive element-PCR
       rapid test to ID bacteria
       geographic differences may exist, databases required
   Ribotyping
       rRNA genes are amplified
       highly reproducible
       labor-intensive, reference database required
   Bacteroides-Prevotella marker
       does not require culture
       PCR method is rapid
       little is know about survival
  Nucleic Acid-based Methods
which involve library comparisons
   Repetitive element-PCR
        rapid test to ID bacteria
        geographic differences may exist, databases required
   Ribotyping
        rRNA genes are amplified
        highly reproducible
        labor-intensive, reference database required
   Bacteroides-Prevotella marker
        does not require culture
        PCR method is rapid
        little is know about survival
   PFGE (Pulse Field Gel Electrophoresis)
        bacterial DNA is isolated, digested and separated
        extremely sensitive (+/-)
        50 public labs submit data on E.coli O157:H7, Salmonella, Shigella, Listeria to a
         National database
  Nucleic Acid-based Methods
which involve library comparisons
 •Extremely valuable for:
     • identification of pathogens (especially when no antibodies exist)
     • epidemiology (to test how different isolates are related)
     • PFGE is the “gold standard”. When done correctly, 100% true
  Nucleic Acid-based Methods
which involve library comparisons
 •Extremely valuable for:
     • identification of pathogens (especially when no antibodies exist)
     • epidemiology (to test how different isolates are related)
     • PFGE is the “gold standard”. When done correctly, 100% true

 •May not necessarily be useful for routine water sampling
 under most normal circumstances
     • Require a one-time investment in equipment (PCR machines,
     gel boxes, etc etc ~ 100K) + routine molecular biology supplies
     • 41% of samples routinely tested false +/-
          • operator error (need highly skilled staff!)
          • insufficient specificity of the assay itself
          • insufficient specificity toward specific bacterial targets
          Chemical methods
   these chemicals are at ~1-100 ug/L
             Chemical methods
   these chemicals are at ~1-100 ug/L
   Detergents (fluorescent whitening agents),
    triclosan, etc
       only general human/industrial impact
              Chemical methods
   these chemicals are at ~1-100 ug/L
   Detergents (fluorescent whitening agents), triclosan,
    etc
       only general human/industrial impact
   Coprostanol
    = cholesterol in poop
     animals and humans produce it

   Bile acids
       correlates with stanols
       e.g. hyodexocholic acid is specific to pig feces
                 Chemical methods
   these chemicals are at ~1-100 ug/L
   Detergents (fluorescent whitening agents), triclosan, etc
        only general human/industrial impact
   Coprostanol
     = cholesterol in poop
      animals and humans produce it
   Bile acids
        correlates with stanols
        e.g. hyodexocholic acid is specific to pig feces
   Caffeine
        beverages
        medications