Use of DNA fingerprinting determines multiple sources of coliform contamination
By Garry Palmateer,
GAP EnviroMicrobial Services
As a result of the E. coli
O157:H7 and Campylobacter
jejuni outbreak in Walkerton,
Ontario, all aspects of drinking
water have come under careful
scrutiny by Ontario Ministries of
Environment, Health, Agriculture and
Food, as well as the Ontario Chief
Medical Officer of Health. The necessity
to provide safe drinking water has
opened doors to new methods of detecting
and tracing sources of microbial
contamination in distribution systems.
The use of DNA fingerprinting to
trace multiple sources of coliform
contamination was used by GAP
EnviroMicrobial Services to investigate
a large distribution system serving
four communities, 48,000 citizens,
in Ontario. The system had been subjected
to fifteen Boil Water Advisories
over a two-year period. Coliform bacteria
were repeatedly detected at various
locations in all four of the municipalities
and background bacteria levels
were in excess of the standard set
by the Safe Drinking Water Act, of
200 non-coliform colonies per 100
mL of water.
A thorough investigation of the distribution
system revealed that significantly
low water pressures were experienced
in various areas over the twoyear
period, that sporadic detection of
coliform bacteria was consistent with
biofilm accumulation and periodic
sloughing into watermains, and that
elevated nutrient levels in the system
were observed. The predominant coliform
bacteria detected were identified
as Enterobacter and Klebsiella spp.,
and the absence of E. coli suggested a
common origin of soil and vegetation.
A comprehensive sampling schedule
was devised to cover a wide variety
of sample locations. Phase I sampling
included filter effluent, plant effluent,
and various distribution water locations
in each of the municipalities.
Phase II sampling was conducted at
various potential source locations.
Nearby lake water and surface water
from watermains below the water table
where intrusion may have occurred as
well as watermains in heavy clay
where surface water would collect
around pipe connections after heavy
rainfall, were potential sources. In
addition, large water users such as
greenhouse water systems, where
water service met private distribution
systems at a backflow preventor, were
suspected.
Figure 1. Banding patterns (fingerprints) of several coliform bacteria. Lanes 1,
6, 11 and 16 are marker lanes for size comparison. Lane 5 is a blank control.
The identified bacteria were subsequently
analyzed by a technique known
as rep-PCR that produces specific
DNA fingerprints for each bacterial
isolate and provides sufficient data for
tracking the sources and movement of
bacterial contamination. Figure 1 provides
an image of DNA fingerprints or
the banding pattern that results from
DNA fragments being separated
according to size. Each bacterial isolate
has a unique fingerprint that permits
differentiation between closely related
bacterial strains. The DNA fingerprints
of the coliform and non-coliform
(background) bacteria obtained from
Phase I were compared to fingerprints
obtained during Phase II.
The results showed that fingerprints
of bacteria obtained during Phase I
occasionally matched the fingerprints
of bacteria obtained during Phase II.
For example, a fingerprint match
occurred between a sample collected
from an apartment building (a large
water user) and distribution samples
collected from three separate locations
in three separate months. This suggested
that coliform bacteria had migrated
through the distribution system,
becoming attached to the inside surface
of the pipes at various locations.
Because sporadic coliform occurrences
matched isolates obtained at
different times and from distant areas
of the distribution system, it was
unlikely that a direct input of bacteria
from a large amount of untreated water
was the cause of the contamination.
The matching of DNA fingerprints
of coliform and non-coliform bacteria
with fingerprints of bacteria from the
potential sources proved to be a powerful
tool in establishing which of the
many possible sources were actual
sources of bacterial contamination. In
addition, DNA fingerprinting provided
valuable information to the water purveyors
as to the cause of frequent
events in the decline of microbial
water quality.
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