Methodology for a demonstration
at the 2005 Biennial Meeting of the
Interstate Shellfish Sanitation Conference
Thursday, August 11th 2005
Methods to Detect and Genotype Coliphages
in Water and Shellfish
Dr. Mark Sobsey, Dave Love, & Greg Lovelace
University of North Carolina at Chapel Hill, Chapel Hill, N.C.
AND
Dr. Jill Stewart & Brian Robinson
NOAA, Charleston, S.C
CONTENTS
Demonstration ……………………………………………………………………………………………3
TEXTUAL SUPPORT
Introduction…………………………………………………………………………………………….….4
Background on coliphage as fecal indicators…………………………………………………..4
Male-specific (F+) RNA coliphage for fecal source tracking………………………………6
Current Methods
To detect coliphage in water and shellfish……………………………………………7
To group coliphage by genetic typing………………………………….………………8
Interpretation of data
From coliphage quantification assays…………………………………………………10
From coliphage genetic typing assays……………………………….……………….12
Acknowledgements……………………………………………………………………………………13
References………………………………………………………………………………………………..14
Reprints available at: http://www.unc.edu/sobseylab/
in the “Documents” page
2
DEMONSTRATION
Goals: Describe and demonstrate the application of:
• US EPA Method 1601 (enrichment-spot plating), US EPA Method 1602
(single agar layer plaque assay) to the detection of coliphages in
shellfishing waters and shellfish tissue extracts
• current nucleic acid methods for grouping of F+ coliphages (coliphage
"genotyping").
Methods Demonstrated Through Simulation
Method 1601 in a most probable number (MPN) format
(1)
(2)
(3)
(4)
preparation of sample medium and host bacteria
spot plating of enriched samples onto medium containing host bacteria
recognition and counting of positive spots, lysis zones on plates
computation of MPN coliphage concentrations
Method 1602, the single agar layer (SAL) plaque assay method
(1)
(2)
(3)
(4)
(5)
preparation of molten agar medium
inoculation of samples with host bacteria and molten agar
pouring SAL plates
observation and counting of plaques
computation of coliphage concentration.
Confirmation Methods 1601 and 1602,
(1)"picking" material from positive lysis zones and plaques
(2) resuspending it in broth medium
(3) optional re-enrichment by overnight culture with host bacteria
(4) inoculation of resuspended or re-enriched picked material onto spot
plates to observe for appearance of lysis zones as evidence of coliphage
positivity.
Nucleic acid genotyping methods
(1)
(2)
(3)
(4)
(5)
extraction of nucleic acids from coliphage samples
PCR or RT-PCR to amplify the concentrations of coliphage nucleic acids
application of extracted or amplified nucleic acids to filters
reaction of target nucleic acids on membranes with nucleic acid probes
detection of positive nucleic acid hybrids
Positive reaction products from nucleic acid genotyping methods
(1) positive enrichment spot plates with lysis zones
(2) SAL plates with plaques
(3) positive nucleic acid hybrids on filters from coliphage genotyping
analysis
3
Introduction
Contamination of bivalve molluscan shellfish and shellfish
growing water by human and animal fecal wastes is an
important public health concern. Growing populations and
development in urban coastal areas bring increased human
waste loads that need to be treated, monitored and
managed.
Human fecal waste may harbor pathogenic
human enteric viruses such as hepatitis A virus,
enteroviruses, adenoviruses, and noroviruses, which present
as outbreaks or discrete cases of acute gastroenteritis, infectious hepatitis and
other diseases. Human enteric viruses are also known to survive sewage
treatment better than fecal indicator and pathogenic bacteria (Chung et al.,
1998), and often treatment is inadequate to prevent contamination of
estuarine/marine water and pathogen bioaccumulation in shellfish. Current
methods to detect bacterial indicators of fecal pollution (e.g. fecal coliforms,
enterococci) are slow to give results (2-4 days), do not reliably predict
pathogenic virus levels in water or shellfish, and provide no data on sources of
fecal pollution. As an alternative to bacterial indicators, coliphage are viral
indicators of fecal pollution that are cheap and easily detectable using
standardized EPA approved culture-based methods, provide fecal source
information (human/non-human), and are related in size, shape, and survival
characteristics to viral pathogens of public health concern: hepatitis A virus,
norovirus, and enteroviruses.
Background information on coliphage fecal indicator viruses
Bacteriophages were first discovered in the early 20th century (Twort, 1915).
True to their name, they are a class of viruses that infect bacteria. Two other
classes of viruses are those that infect plants and animals. Bacteriophages have
a well-known tradition in science and health, as a proposed treatment for
bacterial diseases before the advent of antibiotics, as a model system for genetic
studies than served as the underpinnings for current molecular biology, and
currently as fecal indicators of microbial water quality, food and shellfish quality,
sewage contamination, and efficiency of water and wastewater treatment
(Furuse, 1987; Gerba 1987).
The type of bacteriophages most commonly used as indicators of fecal
contamination are male-specific coliphages with RNA genomes (or F+
RNA coliphages). The name coliphage comes from the fact that these phages
infect Escherichia coli bacteria. Figure 1 shows the two main classes of
coliphages, somatic and male-specific (F+ coliphages). Somatic coliphages infect
4
through receptors on the E. coli
host
cell
wall,
while
F+
coliphages infect through the F
pilli on E. coli hosts (Figure 2).
These two classes of coliphage
are differentiated by their ability
(or lack there of) to infect strains
of permissive host. A researcher
can selectively detect one class of
coliphage over another by using
the appropriate host strain (e.g.
E. coli K12 for somatic and E. coli
Famp for F+ coliphage). After
detection, F+ coliphage should
be further characterized using an
“RNase Test” or genetic analysis
to
determine
whether
the
genome is made from RNA or
DNA. This distinction will help
further segregate F+ coliphages
into most useful fecal indicator
groups.
F+ RNA coliphage are used
primarily as fecal indicators,
Somatic
coliphage
because these viruses fit well
with criteria for an ideal indicator
F-plasmid
of microorganisms (Table 1). F+
Somatic host
coliphages resemble many of the
E. coli C
important human enteric viruses
Somatic
F+ Coliphage
hepatitis
A
virus,
coliphage (e.g.,
enteroviruses, noroviruses) in
F+ Coliphage
size,
shape
and
general
composition (Havelaar, 1993; Hsu
Figure 2. Male-specific (F+) and Somatic Hosts and Phages
et al., 1995; Sobsey et al., 1995),
they correlate with the presence of pathogenic human viruses in water and
shellfish and an increase in viral illness (Chung et al., 1998; Havelaar, 1993;
Wade et al., 2003), and they can be grouped to differentiate human from nonhuman fecal waste as a fecal source tracking tool (discussed below) (Furuse et
al.,1981; Osawa et al., 1981, Hsu et al., 1995; Vinjé et al., 2004). F+ coliphages
are detected and quantified in water and shellfish easily and cheap using
standard media. Coliphage methods applied today have been validated by US
EPA-sponsored studies for use in groundwater (US EPA 2000a; 2000b) and by
Male-specific host
5
European Union-sponsored studies for use in bathing waters (Mooijman, et al.,
2001; 2002).
Table 1. Criteria for an Ideal Indicator of Microorganisms
(1) Indicator organism should be present when pathogen present, and absent when pathogen
absent
(2) Persistence in the environment of the pathogen and the indicator should be similar
(3) Density of the indicator organism should have a direct relationship with the density of the
pathogen(s)
(4) Indicator organism should be present in the source at levels in excess of the pathogen
concentration
(5) The indicator organism should be at least as resistant to disinfectants as the pathogen(s)
(6) The indicator organism should be non-pathogenic and easily quantifiable
(7) The test for the indicator organism should be simple, rapid and economical
Source: (Gerba, 1987)
Male-specific (F+) RNA coliphages for fecal source tracking
F+ RNA coliphage can be separated into one of four groups (I, II, III, and IV)
having their origins in either the human or animal intestinal tracts (thus human
or animal sources). Two grouping methods for F+ colipahge are serological
typing and genetic typing. Serological typing (or serogrouping) methods use a
neutralization test for coliphage isolates using rabbit antisera generated against
each of the four groups. Coliphage isolates that
flourish on plates without antisera and are inhibited
in plates with antisera are characterized by the
antisera that inhibits them. Although serogrouping is
still performed by some, genetic typing (or
genogrouping) methods are favored by most
researchers. Genetic typing methods use synthetic
complementary oligonucleotide probes which bind
with known portion of the coliphages RNA, DNA, or
cDNA genomes (cDNA refers to reverse transcribedPCR amplified products). Probes are designed to
target one of four coliphage groups and in a detector
assay to give a clear chemiluminescent, colorimetric,
or fluorescent signal for positive matches between
probe and coliphage (Figure 3).
Examples of
standard F+ coliphage genotyping assays are (Hsu et
al., 1995) and (Figure 3 from Vinjé et al., 2004).
Genogrouped F+ coliphage can be confirmed by
comparing the sequence (of base pairs) of a portion
of the genome to other known coliphage sequences.
Figure 3. Genotyping
6
From F+ RNA grouping data beginning ~25 years ago (Osawa, et al. 1981) and
continuing today, it is thought that Group I (MS2-like) phages are associated
with animal waste or sewage, Groups II (GA-like) and III (QBeta-like) phages are
associated with human waste and some instances of hog waste and poultry
waste, and Group IV (SP-like, FI-like, and M11-like) phages are associated with
animal waste. To give a sense of how coliphage grouping works, below in Table
2 are serological results from a recent study of F+ RNA coliphages collected from
various waste sources.
Table 2. F+ RNA serogrouping from (Long et al., 2005)
Current methods to detect coliphage in water and shellfish
US EPA Method 1601 – Two-step Enrichment Procedure
“Method 1601 describes a qualitative (presence/absence) two-step enrichment
procedure for coliphage. A 100-mL or 1-L ground water sample is supplemented
with MgCl2 (magnesium chloride), log-phase host bacteria (E. coli Famp for
male-specific coliphage and E. coli CN-13 for somatic coliphage), and tryptic soy
broth (TSB) as an enrichment step for coliphage. After an overnight incubation,
samples are ‘spotted’ onto a lawn of host bacteria specific for each type of
coliphage, incubated, and examined for circular lysis zones, which indicate the
presence of coliphages.
The two-step enrichment procedure determines the presence or absence of
male-specific (F+) and somatic coliphages in ground water and other waters.
The two-step enrichment method was validated as a qualitative, presenceabsence method, and Method 1601 was written with this use in mind. The two7
step enrichment method potentially may be used as a quantitative assay of
coliphage concentrations in an MPN format, however, the method has not been
validated this way. This method is intended to help determine if ground water is
affected by fecal contamination” (USEPA 2001a).
Method 1601 is available in pdf form at the USEPA website
http://www.epa.gov/nerlcwww/1601ap01.pdf
US EPA Method 1602 – Single Agar Layer Procedure
“Method 1602 describes the single agar layer (SAL) procedure. A 100-mL ground
water sample is assayed by adding MgCl2 (magnesium chloride), log-phase host
bacteria (E. coli Famp for F+ coliphage and E. coli CN-13 for somatic coliphage),
and 100 mL of double-strength molten tryptic soy agar to the sample. The
sample is thoroughly mixed and the total volume is poured into 5 to 10 plates
(dependent on plate size). After an overnight incubation, circular lysis zones
(plaques) are counted and summed for all plates from a single sample. The
quantity of coliphage in a sample is expressed as plaque forming units (PFU) /
100 mL. For quality control purposes, both a coliphage positive reagent water
sample (OPR) and a negative reagent water sample (method blank) are analyzed
for each type of coliphage with each sample batch” (USEPA 2001b).
Method 1602 is available in pdf form at the USEPA website
http://www.epa.gov/nerlcwww/1602ap01.pdf
Current methods to group coliphage by genetic typing (genogrouping)
Genetic typing (genogrouping) of F+ coliphages can be performed using a solid
or liquid format with a variety of detector signals (e.g. chemiluminescent,
colorimetric, or fluorescent). The approach we recommend is a published
method termed “Reverse Line Blot (RLB) Hybridization” and has been
validated using environmental isolates of F+RNA coliphage (Vinjé et al., 2004;
Long et al., 2005). As described in Figure 4, RLB hybridization involves binding
biotin-labeled RT-PCR amplified cDNA products of F+ coliphage onto solid phase
nylon membranes. The membranes are pre-labeled with short sequences of
oligonucleotide probes which are complementary to and target each of the four
F+RNA groups (Groups I, II, III, IV) (Figure 4- step 1). The F+ coliphage cDNA
products are hybridized to the membrane probes at 48°C and excess product is
washed free using a mild detergent (2% SSPE - 0.5% SDS) (Figure 4- step 2). A
streptavidin-labeled antibody conjugate is washed over the membrane and binds
to RT-PCR products, with excess streptavidin removed from the membrane off
with subsequent washes using a 2% SSPE - 0.5% SDS solution (Figure 4- step
8
3). A film detector solution is applied to the nylon membrane, and after 30 min
of exposure to film in a dark room, the film is developed and black spots indicate
where F+ coliphage hybrids occur (Figures 3 and 4- step 4). Because the
membrane is labeled with probes targeting each of the four F+RNA groups,
genogrouping is possible by comparing unknown samples (Figure 3; # 21-38) to
positive controls (Figure 3; # 15-20).
The principal article on coliphage RLB hybridization is available online in pdf form
at http://www.unc.edu/~janvinje For a detailed laboratory protocol of this
method, please contact Dr. Jan Vinjé, (janvinje@email.unc.edu).
Coliphage grouping with hybridization
4
1. Labeled nylon membrane with probes
2. Run Biotin labeled RT-PCR product of
environmental isolates across membrane
3
3. Attach Streptavidin conjugate to bound
RT-PCR product
2
4. Positive detection (hybrids; black spots)
after 30 min as on developed film exposure
(Vinjé et al., 2004)
1
2
4
1
Figure 4. Coliphage grouping with hybridization
9
Interpretation of data from coliphage quantification assays
Single Agar Layer (SAL) plate with coliphage plaques (zones of lysis) in
host lawn
-
count phage plaques (zones of host lysis) on each plate
pick a representative number of plaques for further characterization by RNase testing
and/or genotyping
Spot-plating enriched water samples in a 3 x 3 MPN matrix
S: # positive = 3-3-0; MPN = 2.33 pfu / 100 mL
I: # positive = 3-1-0; MPN = 0.94 pfu / 100 mL
B: # positive = 3-2-0; MPN = 1.46 pfu / 100 mL
-
Samples enriched overnight at 37ºC in 3 x3 MPN format, then 5 ul of enrichment is
spotted the next day on plates with permissive host lawn.
Spot plates incubated overnight at 37ºC and detected the next day as presence/absence
of plaques (host lysis).
calculate MPN based on # positive & negative samples and inoculum volumes
10
F+ Coliphage Characterization: RNase Test
No RNAse
DNA phage
-
-
RNA phage
With RNAse
DNA phage RNA phage
spot plates with permissive host made with and without RNase
5 ul of enriched samples spotted on plates and incubated overnight at 37ºC
Compare coliphage plaques (zones host lysis) for each samples
A sample with plaques on both RNase and RNase free plates signifies a DNA phage or a
mixed sample (RNA and DNA phages)
(hint: look for faint plaques on RNase plates and intense plaques on RNase free plates as
a sign of an RNA phage)
A sample with plaques only on RNase free plates signifies a RNA phage
A sample with no plaques on either plate is negative for FRNA or FDNA coliphages
11
Interpretation of data from genetic typing assays (RLB hybridization)
F+ RNA Coliphage grouping for
source tracking in environmental isolates
Group I (MS2 like)
Hybridization
by genogroup
# I II III IV
Group II (GA like)
Group III (Qß like)
Group IV (Sp/Fi like)
Excel Spreadsheet
-
-
The image (seen on right) depicts part of a Reverse Line Blot hybridization of 27
coliphage isolates # 452-479. Isolates are from shellfish homogenates and estuarine
water samples.
In the image, genogroups I, II, III, & IV read vertically and coliphage isolates read
horizontally. In this case, the isolates are either positive for Group I (MS2-like)
coliphages or Group III (Qbeta-like) coliphages.
Coliphage isolate # and genogroup are recorded in an excel spreadsheet, and a hard
copy of the hybridization film can be saved in a lab notebook.
12
Acknowledgement
This work was funded by a grant from The Cooperative Institute for Coastal
Estuarine and Environmental Technology and a grant from North Carolina Sea
Grant.
13
References
Chung, H., L.-A. Jaykus, G. Lovelace and M.D. Sobsey (1998) Bacteriophages and bacteria as
indicators of enteric viruses in oysters and their harvest waters. Wat. Sci. Tech., 38(12):37-44.
Furuse, K. (1987) Distribution of coliphages in the environment: general considerations. In:
Phage Ecology. Eds: Goyal, SM, Gerba, CP, and G Bitton. Wiley-Interscience Publication. New
York.1987. pp. 87-120.
Furuse, K., Ando, A., Osawa, S., Watanabe, I. (1981). Distribution of ribonucleic acid coliphages
in raw sewage from treatment plants in Japan. Appl Environ Microbiol. 41(5):1139-43
Havelaar, A.H. (1993). Bacteriophages as models of human enteric viruses in the environment.
ASM News. 59(12):614-619.
Hsu, F.-C., Y.-S.C. Shieh, J. van Duin, M.J. Beekwilder and M.D. Sobsey (1995) Genotyping
male-specific RNA coliphages by hybridization with oligonucleotide probes. Appl. Environ.
Microbiol., 61(11): 3960-3966.
Gerba, CP. (1987). Phage as indicators of fecal pollution. In: Phage Ecology. Eds: Goyal, SM,
Gerba, CP, and G Bitton. Wiley-Interscience Publication. New York.1987. pp. 197-205.
IAWPRC. (1991). Water Res. 25(5):529-545.
Long, SC, El-Khoury, SS, Oudejans, S, Sobsey, MD and J. Vinjé (2005). Assessment of Sources
and Diversity of Male-Specific Coliphages for Source Tracking. Eng. Eng. Sci. 22(3): 367-377.
Osawa, S., Furuse, K., Watanabe, I. (1981). Distribution of ribonucleic acid coliphages in animals.
Appl Environ Microbiol.41(1):164-8.
Sobsey, M.D., D.A. Battigelli, T.R. Handzel and K.J. Schwab (1995) Male-specific Coliphages as
Indicators of Viral Contamination of Drinking Water, 150 pp. American Water Works Association
Research Foundation, Denver, Co.
Sobsey, M.D., Yates, M.V., Hsu, F.C., Lovelace, G., Battigelli, D., Margolin, A., Pillai, S.D., and N.
Nwachuku (2004) Development and evaluation of methods to detect coliphages in large volumes
of water. Water Sci Technol. 2004;50(1):211-7.
Twort, FW, 1915. An investigation on the nature of the ultramicroscopic viruses. Lancet.
189(2):1241-1243.
US EPA. (2000a). Part II, Environmental Protection Agency 40 CFR Parts 141 and 142. National
Primary Drinking Water Regulations: Long Term 1 Enhanced Surface Water Treatment and Filter
Backwash Rule: Proposed Rule. Federal Register, Vol. 65, No. 69: 19045- 19094.
US EPA. (2000b). Part II, Environmental Protection Agency 40 CFR Parts 141 and 142. National
Primary Drinking Water Regulations: Ground Water Rule: Proposed Rule. Federal Register, Vol.
65, No. 91: 30193- 30274.
USEPA (2001a) Method 1601: Male-specific (F+) and Somatic Coliphage in Water by Two-step
Enrichment Procedure. EPA Number: 821-R-01-030, April, 2001, Washington, DC.
14
USEPA (2001b) Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single Agar
Layer (SAL) Procedure. EPA Number: EPA 821-R-01-029, April, 2001, Washington, DC.
USEPA. (2002) Method 1600: Enterococci in Water by Membrane Filtration Using membraneEnterococcus Indoxyl-Beta-D-Glucoside Agar (mEI). EPA 821-R-02-022 Office of Water,
Washington.
Vinje J, Oudejans SJ, Stewart JR, Sobsey MD, Long SC. (2004) Molecular detection and
genotyping of male-specific coliphages by reverse transcription-PCR and reverse line blot
hybridization. Appl Environ Microbiol.70(10):5996-6004.
Wade, T.J., Pai, N., Eisenberg, J.N.S, Colford, J.M. Jr. (2003). Do U.S. Environmental Protection
Agency Water Quality Guidelines for Recreational Waters Prevent Gastrointestinal Illness? A
Systematic Review and Meta-analysis. Environmental Health Perspectives. 111(8):1102-1109.
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