7.2 Pollution of Water with Reference to Human Health: Bacterial Indicators of Fecal Pollution 169
molecular MST methods is finding these diffe-
rences against a large background of similarity.
Molecular methods are believed to be more accu-
rate for individual sources (e.g., deer, muskrat,
chickens, and horses). However, the molecular
methods are also considerably more expensive and
slower to perform in the laboratory. Only a selected
number of molecular methods will be discussed and
only the outline will be given.
(a) Repetitive PCR (Rep PCR)
Rep-PCR genomic fingerprinting makes use of
DNA primers complementary to naturally
occurring, highly conserved, repetitive DNA
sequences, present in multiple copies in the
genomes of most Gram-negative and several
Gram-positive bacteria. DNA between adjacent
repetitive extragenic elements is amplified
using PCR to produce various size DNA frag-
ments. The PCR products are then run on aga-
rose-gel electrophoresis to produce specific DNA
fingerprint patterns, which can then be analyzed
using a pattern recognition computer software.
The rep-PCR genomic fingerprints generated
from bacterial isolates permit differentiation to
the species, subspecies, and strain level.
(b) Random amplification of polymorphic DNA
(RAPD)
This is a type of PCR, but the segments of DNA
that are amplified are random. To perform
RAPD creates several arbitrary, short primers
(8–12 nucleotides) are produced, then PCR is
performed using a large template of genomic
DNA, hoping that fragments will amplify. By
resolving the resulting patterns, a semi-unique
profile can be gleaned from an RAPD reaction.
No knowledge of the DNA sequence for the
targeted gene is required, as the primers will bind
somewhere in the sequence, but it is not certain
exactly where. This makes the method popular
for comparing the DNA of biological systems
that have not had previous attention, or in a sys-
tem in which relatively few DNA sequences are
compared (it is not suitable for forming a DNA
databank). Due to the fact that it relies on a large,
intact DNA template sequence, it has some
limitations in the use of degraded DNA sam-
ples. This method requires screening primers
(there are over 1,200 commercially available) to
find sets of polymorphisms (i.e., differences in
nucleotide sequence among individuals) that are
either unique to fecal bacteria from a given
source or occur in a given source to a large and
predictable degree. Once such sets of polymor-
phisms have been found, fecal bacteria can be
“sourced” by comparison. It has been used in dif-
ferentiating human and nonhuman E. coli sources.
(c) Ribotyping
Ribotyping, also referred as molecular finger
printing, involves the bacterial genes that code
for 16S ribosomal RNA. Because such genes are
highly conserved (i.e., change very slowly evolu-
tionarily) in microorganisms, ribotyping has
been widely accepted for microbial identifica-
tion. Ribotyping involves cutting the total
genomic bacterial DNA with different DNAases,
or restriction enzymes, followed by gel electro-
phoresis. Following electrophoresis, Southern
blotting is performed to blot the DNA bands onto
nylon membranes from the gels. DNA probes
must be prepared for bacterial 16S and 23S
rRNA and labeled with some type of detection
system, such as fluorescent dye or radioactively).
Membrane hybridization is then performed to
hybridize the probes with the appropriate DNA
bands on the nylon filter. Difference in the size
and location of the ribosomal RNA bands on the
filters can then be used to differentiate between
the sources of the fecal bacteria.
Strains of E. coli are adapted to their own
specific environment (intestines of host spe-
cies), and as a result differ from other strains
found in other host species. To use this MST
method, collections of potential source material
(fecal samples of all potential sources in the
environment) must be collected and subtyped.
The genetic fingerprints of the bacterial isolates
from the water samples can then be compared
to those of the suspected source.
Ribotyping has been widely used in micro-
bial source tracking studies. It has been found
that database size, geographic distribution of
the isolated bacteria, and the presence of repli-
cate isolates in the bacterial source library
impact the ability of ribotyping to differentiate
among bacteria at the host species level
(d) Pulse-field gel electrophoresis (PFGE)
PFGE is a DNA “fingerprinting” technique that
uses restriction enzymes on the entire DNA