Molecular Indicators Used in the Development

of Predictive Models for Microbial Source

Tracking

Aisha Akram
Roll # 20
 INTRODUCTION
• Fecal pollution represents serious health
problems associated with the pathogens from
the infected animals and humans.
• Fecal coliforms indicate the presence of fecal
matter but does not identify the source of fecal
contamination.
• A number of chemical, microbial, and
eukaryotic indicators have been proposed as
indicators of fecal pollution sources in water
bodies.
• Microbial source tracking (MST) provide
rapid fecal source determination and facilitate
remediation.
o Several Bifidobacterium species are human
specific such as Bifidobacterium adolescentis
and Bifidobacterium dentium and the
molecular markers used for their identification
are (ADO)and(DEN) respectively.
o Bacteriodetes markers are based on host-specific
oligonucleotide i.e.HF-134 and HF183F for
human Bacteriodetes and CF128F and CF193F
for ruminant Bacteriodetes.

o The gene esp. of Enterococcus which encodes an

enterococcal surface protein has also been
proposed as an indicator of human fecal
pollution.
o Other MST eukaryotic markers used were host
specific mitochondrial DNA associated with
humans, cattle and pigs (Humito,Bomito and
Pomito respectively).
• Predictive models to distinguish between
human and non human fecal pollution have
been developed by combining indicators.
 MATERIALS AND METHODS
Sample collection
• 144 samples of wastewater, feces and slurry
were collected from human and animal source.
DNA extraction
• Genomic DNA from 200 µl of the sample was
extracted using Q1A amp DNA blood extraction
minikit.
Bacteriodetes group detection

• Specific primers were used to discriminate

bacteriodetes from human and ruminant sources
of fecal pollution.

• Human HF134F, HF183F

• Ruminants CF128F,CF193F
• The 30 cycle PCR reaction was performed in
Perkin Elmer thermocycler.
 Initial denaturation 94°C for 2 min
 Denaturation 94°C for 1 min
 Annealing temperature 62°C - 63°C for 1 min
 Extraction 72°C for 1.5 min
 Final 7-min extension at 72°C .
Detection of gene esp. of E.faecium
• The method for detecting the gene esp requires
a previous cultivation stage
• Samples were first processed by 0.45-µm
porous membrane filters
• Filters were incubated on m-Enterococcus agar
and incubated for 48 h at 37°C
• Filters were suspended on tryptic soy broth and
incubated for 3 h at 41°C. Then, DNA
extraction was performed using a QIAamp
DNA blood minikit
• The gene was amplified using a forward primer
that is specific for the protein esp.
Mitochondrial DNA
• Nested-PCR assays were performed on each
sample to detect mitochondrial host-specific DNA
associated with humans, cattle, and pigs
Statistical analysis
• Sensitivity (r) and specificity (s) were
calculated according to the following formulas:
r = [TP/(TP + FN)]
s = [TN/(TN + FP)]
Where,
• TP is the number of samples that were positive for
the PCR marker of their own species (true
positive)
• FN is the number of samples that were negative for
a PCR marker of their own species (false negative)
• TN is the number of samples that were negative for
a PCR marker of another species (true negative)
• FP is the number of samples that were positive for
a PCR marker of another species (false positive).
Predictive models
• Linear and quadratic discriminant analysis
(LDA and QDA) are widely used
 RESULTS
Assay validation
• The sensitivity and specificity of the markers
were tested against DNA extracted from host-
specific samples.
Comparisons between the positive samples for
each marker are shown in Table 1
The values obtained for the sensitivity,
specificity, and conditional probability of each
marker are shown in Table 2.
• ADO ------95.6% human samples positive
• DEN ------ 64.4% human samples positive
• HF134 ------ 30% human Bacteroidetes marker
• HF183 ------ 50% human Bacteroidetes marker
• CF128 ------ 26% ruminant Bacteroidetes
marker
• esp gene ------ 77% sensitivity for human
• esp gene ------ 68% sensitivity for cows and
pigs
Three mitochondrial DNA markers
• Humito -------- 84.4% sensitivity for human
• Bomito -------- 84.2% sensitivity for bovine
• Pomito -------- 87.9% sensitivity for porcine
Predictive models
• Several models were developed to distinguish
between the four possible fecal pollution
sources (labeled HPBP: human, porcine,
bovine, poultry) (Table 3).
• Models for discriminating human and
nonhuman sources (labeled H-NH) are also
shown in Table 3.
• The predictive model for HPBP

seven markers (HF134, HF183, ADO, DEN,

Humito, Bomito, and Pomito)
• Correctly classified 79.5% sample source.
• Other methods used less number of markers
• Decision tree models correctly classified :
DTa 4 markers (ADO, HF183, Bomito, 71.3%
and Pomito)
DTb 3markers (ADO, Bomito, and 75.7%
Pomito)
DTa 3 markers (ADO, HF183, and 87.5%
Pomito)
DTa 4 markers (ADO, DEN, HF134, and 89.7%
Humito )
DTb 2 markers (ADO and Pomito) 84.6
Decision trees
 DISCUSSION
• B. adolescentis has been reported as one of the
most abundant species of Bifidobacterium in
the human microbiota. B. dentium is less
abundant, but is also described in the
composition of normal human microbiota.
• Therefore, both species have been described as
possible markers of fecal human pollution in
the environment .The detection of both species
by an ADO-DEN multiplex PCR
• ADO had the highest sensitivity and DEN had
the highest specificity.
• Bacteroidetes markers have been used in many
different locations around the world to identify
the source of the fecal pollution.
• The esp marker is related to human pathogenic
strains of E. faecium.
• The method for detecting the esp gene requires
an enrichment step before PCR. Without this
step, the technique is not sensitive enough to
detect the potential marker.
• Large amounts of exfoliated epithelial cells are
removed with feces. Every cell has a high
number of mitochondria, and every
mitochondrion has many copies of
mitochondrial DNA.
• The use of nested PCR increases the sensitivity
of the technique.
• The percentage of correct identification
increased with the number of markers
analyzed.
• The best predictive model for distinguishing
human from nonhuman fecal sources was
based on 5 molecular markers (HF134, ADO,
DEN, Bomito, and Pomito) and provided
90.1% correct classification.
 CONCLUSION
• Some molecular markers could be considered
potential MST indicators.
• They could be used as new parameters in
combination with other culture-dependent
MST indicators (host specific or unspecific)
for the development of feasible universal
predictive models in order to determine fecal
pollution sources in water bodies.