9260

Introduction To Detecting Pathogenic Bacteria

Approved by Standard Methods Committee, 2007. Editorial revisions, 2021. Joint Task Group: Nelson P. Moyer (chair), Edward J. Bottone, Joseph O. Falkinham, III,
J.J. Farmer, III, Barry S. Fields, Mark W. LeChevallier.

9260
1. General Discussion
One purpose of drinking water and wastewater treatment is to
reduce the numbers of viable organisms to acceptable levels, and
to remove or inactivate all pathogens capable of causing human
disease. Despite the remarkable success of water treatment and
sanitation programs in improving public health, sporadic cases
and point-source outbreaks of waterborne diseases continue to
occur. Water and wastewater may contain a wide variety of bacte-
ria that cause intestinal or extra-intestinal infections. Waterborne
pathogens enter human hosts through intact or compromised
skin, inhalation, ingestion, aspiration, and direct contact with the
mucous membranes of the eye, ear, nose, mouth, and genitals.
This section provides an introduction to bacterial agents respon-
sible for diseases transmitted by drinking and recreational waters
in the United States.
The Centers for Disease Control and Prevention (CDC) and
the US EPA maintain a collaborative system for collecting and
reporting waterborne disease outbreak data for both micro-
bial and chemical agents; however, detection and investiga-
tion of waterborne outbreaks is primarily the responsibility of
the local, state, and territorial public health departments with
A. Introduction
v­ oluntary reporting to the CDC.1 The average number of water-
borne disease outbreaks per year between 1971 and 2013 was
46.2 From 1971 to 1998, 691 drinking-waterborne disease out-
breaks due to a microbiological, chemical, or unknown etiology
were reported in the United States (see Figure 9260:1). In the
years 1972 through 1994, 291 outbreaks associated with drink-
ing water were attributed to microorganisms as follows: Giardia
lamblia, 109 (37.5% of total); Shigella, 34 (11.7%); nontyphoidal
Salmonella serotypes, 13 (4.5%); Salmonella serotype Typhi, 5
(1.7%); Vibrio cholerae, 2 (0.7%), enterotoxigenic Escherichia
coli, 1 (0.3%); E. coli O157, 1 (0.3%). Community waterborne
outbreaks have declined since the mid-1980s (see Figure 9260:1),
largely because of the promulgation of more stringent drinking
water standards, including the Surface Water Treatment Rule,3 the
Total Coliform Rule,4,5 and other regulations.6–8 In addition, many
water utilities have made voluntary improvements.
The agents responsible for reported outbreaks are predom-
inantly unidentified, microbial (parasitic, bacterial, or viral),
or chemical (Figure 9260:2). Large numbers of parasitic out-
breaks in the early 1980s were caused mostly by Giardia; these
outbreaks were reduced by the implementation of the Surface
Water Treatment Rule.3 Relatively few outbreaks due to viruses

Figure 9260:1. Number of drinking water-related disease outbreaks in the United States, 1971–1998. Individual—private or individual water sys-
tems (9% of U.S. population, or 24 million users); community—systems that serve > 25 users year-round (91% of U.S. population, or 243 million users);
noncommunity—systems that serve < 25 users and transient water systems, such as restaurants, highway rest areas, and parks (millions of users yearly).
Adapted from Barwick RS, Levy DA, Craun GF, Beach MJ, Calderon RL. 2000. Surveillance for waterborne disease outbreaks—United States 1997–1998.
Morbid Mortal Week Rep. 49(SS-4):1.

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 9260 INTRODUCTION TO DETECTING PATHOGENIC BACTERIA - A. Introduction
Figure 9260:2. Agents responsible for drinking water-related disease outbreaks. Adapted from Blackburn BG, Craun GF, Yoder JS, Hill V, Calderon
RL, N. Chen, Lee SH, Levy DA, Beach MJ. 2004. Surveillance for waterborne-disease outbreaks associated with drinking water—United States, 2001–
2002. Morbid Mortal Week Rep. 53(SS-8):23.
have been reported, in part because the detection methodologies
have difficulty attributing an outbreak to a specific virus. To
better address the occurrence of microbial pathogens in drink-
ing water, the US EPA has issued a Contaminant Candidate List
that includes 11 microbes for methods development and potential
future ­regulation.9,10
Water contamination and disease transmission may result from
conditions generated at overloaded or malfunctioning sanitary
waste disposal and potable water treatment systems. In addi-
tion, common outdoor recreational activities, such as swimming
(including pools and hot tubs), wind surfing, and waterskiing, all
place humans at risk of waterborne diseases from ingestion or
direct contact with contaminated water.11 Outbreaks of gastro-
enteritis, pharyngoconjunctivitis, folliculitis, otitis, and pneumo-
nia are associated with these recreational activities. Overcrowded
parks and recreational areas contribute to the contamination of
surface and groundwater.
The laboratory diagnosis of infectious disease depends on the
detection or isolation of an etiologic agent or demonstration of
antibody response in a patient. Environmental microbiological
examinations are conducted for compliance monitoring of the
environment, to troubleshoot problems in treatment plants and
distribution systems, and in support of epidemiological investi-
gations of disease outbreaks. Ideally, the public health microbiol-
ogist can contribute expertise in both clinical and environmental
microbiology, thereby facilitating epidemiological investigations.
When testing for pathogens in environmental samples, include
analyses for indicator organisms. Routinely, coliforms (total coli-
form, thermotolerant coliform, and E. coli) are used as water
quality indicators. Fecal streptococci, enterococci, Clostridium
perfringens, Bacteroides, Bifidobacterium, and bacteriophages
have been proposed as water quality indicators. No single indi-
cator provides assurance that water is pathogen-free. The choice
of monitoring indicators presupposes an understanding of the
https://doi.org/10.2105/SMWW.2882.201
parameters to be measured and the relationship of the indicators to
the pathogens. Some bacterial pathogens, such as Pseudomonas,
Aeromonas, Plesiomonas, Yersinia, Vibrio, Legionella, and Myco-
bacterium, may not correlate with coliform indicators. Traditional
bacterial indicators also may not correlate with viruses or parasites
in pristine waters or groundwaters, and they may be of limited
utility in estuarine and marine waters. Nevertheless, tests for total
and fecal bacteria and E. coli are useful, because it is rare to isolate
bacterial enteric pathogens in the absence of fecal contamination.
Other more general indicators may be of value also for assess-
ing the potential for pathogen contamination and interpreting culture
results. Heterotrophic plate count provides information about the total
numbers of aerobic organotrophic bacteria and an indication of the
total organic composition of the aquatic environment. Physicochem-
ical factors, such as turbidity, pH, salinity, temperature, assimilable
organic carbon, dissolved oxygen, biochemical oxygen demand, and
ammonia, may provide useful information about contamination or
the potential of water to support bacterial growth. For treated waters,
chlorine residual should be measured at the sample collection point.
The following sections contain methods for Aeromonas (Sec-
tion 9262), Campylobacter (Section 9264), diarrheagenic E. coli
(Section 9266), Legionella (Section 9268), Leptospira (Sec-
tion 9270), Mycobacterium (Section 9272), Salmonella (Sec-
tion 9274), Shigella (Section 9276), Vibrio (Section 9278), and
Yersinia entercolitica (Section 9280). Methods for isolation and
enumeration of P. aeruginosa are found in Sections 9213 E and F.
Methods for other pathogens are found elsewhere.12
The methods outlined below may be used to analyze samples
associated with disease outbreaks, or in other studies concerned
with the occurrence of pathogens in water and wastewater. (The
methods presented below are not standardized, and may need
modification to fit a particular set of circumstances. No single
procedure is available for reliable detection of any pathogen or
group of pathogens.)
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 9260 INTRODUCTION TO DETECTING PATHOGENIC BACTERIA - A. Introduction
Because the presence of pathogens is intermittent and the sur-
vival times in the environment are variable, routine examination
of water and wastewater for pathogenic bacteria is not recom-
mended. Even in outbreak situations, the recovery of pathogens
from water and wastewater may be limited by lack of facilities,
untrained personnel, inadequate methods, and high costs. Despite
these constraints, it is important to recover a substantial number
of isolates, especially if molecular fingerprinting methods will be
used during outbreak investigations.
A negative result by these methods for pathogenic bacteria
does not conclusively prove their absence. Consider the concept
of viable but nonculturable versus injured organisms in relation to
a negative culture result.13,14
All microbes described in Section 9262 through Section 9280
are human pathogens, and they pose an infectious threat to per-
sons involved in sample collection and laboratory analyses. Bio-
safety Level 2 precautions are required for all pathogens in this
section except the mycobacteria, which require Biosafety Level 3
precautions.15-17
References
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Published Online: August 27, 2018
Revised: March 31, 2022
https://doi.org/10.2105/SMWW.2882.201
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bacterial autoinducer. Appl Environ Microbiol. 2003;68:4788–
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publications/bmbl5/
16. Laboratory Biosafety Manual, 3rd edition. World Health Organiza-
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int/csr/resources/publications/biosafety/Biosafety7.pdf
17. Laboratory Safety Guidance. OSHA 2404-11R. Occupational Health
and Safety Administration, U.S. Department of Labor; 2009.
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