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DEVELOPMENT OF A RAPID METHOD FOR A HUMAN
POLLUTION SOURCE TRACKING MARKER USING
ENTEROCOCCUS SURFACE PROTEIN (ESP) IN

E. faecium
presented by
Lekha Satheesh Kumar
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6/07 p:/ClRC/DateDue.indd-p1

DEVELOPMENT OF A RAPID METHOD FOR A HUMAN POLLUTION
SOURCE TRACKING MARKER USING ENTEROCOCCUS SURFACE
PROTEIN (ESP) IN E. faecium

By

Lekha Satheesh Kumar

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Fisheries and Wildlife

2007

ABSTRACT

Development of a rapid method for a human pollution source tracking
marker using enterococcus surface protein (esp) in E. faecium

By

Lekha Satheesh Kumar

Enumeration of fecal indicator bacteria alone will not provide information on the sources
of contamination in water, thus microbial source tracking is necessary to identify the
source of pollution. Major limitations with microbial source tracking methods are time
consumption and the cost of the technique. The objective of the first portion of the study
was to develop a rapid method for source tracking human pollution using a host specific
genetic marker “esp” gene. Methods (2 & 3) were compared to the conventional method
(1) for the detection of the esp marker in E. faecium from raw sewage. The elimination of
the DNA extraction process and the removal of the enrichment process from the
conventional method were assessed. Ninety seven membranes from 10 different raw
sewage samples were analyzed for comparing method 1 and method 2. The rapid method
limit of sensitivity was 46i27 cfu/membrane compared to the 45d:28 cfu/membrane of
the conventional method. In method 3 (without enrichment) the sensitivity was 26i40
cfu/membrane (n= 78). The second objective was to evaluate the marker in the sewage.
The marker was present in all the raw sewage samples (n=23) and in 2/10 effluent
samples. The marker was absent in all the non human samples (n=23) and was present in
2/25 environmental samples processed. In a blind study the marker correctly identified
6/7 USGS source samples from human and was absent in all the non human samples

processed (n=7) from different geographical regions.

ACKNOWLEDGEMENTS

I would like to thank the National Oceanic and Atmospheric Administration (N OAA) for
funding this research. I am very grateful to my advisor Dr. Joan B. Rose, for her guidance
and support throughout my Master’s research. I would like thank my committee
members, Dr. Darryl Warncke and Dr. Kelly Millenbah for their guidance during my

research.

Also, thank you to the other graduate students and employees in Dr. Rose’s lab for their

support and help in my research.

iii

TABLE OF CONTENTS

 

 

 

 

 

 

LIST OF TABLES ..... vi
LIST OF FIGURES - vii
CHAPTER 1 1
LITERATURE REVIEW -- - - _ - - - l
1.1 Water Pollution and Waterborne Disease Outbreaks ................................................ 1
1.2 Beach closure Days at US and Great Lakes Beaches ............................................... 3
1.3 Indicator Organisms .................................................................................................. 4
1.3.1 Michigan Water Quality Standards .................................................................... 5
1.4 Total Maximum Daily Load (TMDL) ...................................................................... 6
1.5 Microbial Source Tracking ....................................................................................... 8
1.5.1 Phenotypic Library Dependent .......................................................................... 9
1.5.2 Genotypic Library Dependent .......................................................................... 11
1.5.3 Genotypic Library Independent Methods. ....................................................... 14
1.5.4 MST using Viruses .......................................................................................... 15
1.5.5 Advantages and Disadvantages of Current Microbial Source Tracking
Methods ..................................................................................................................... 15
1.6 Characteristics of Enterococci ................................................................................ 18
1.6.1 The Enterococci Surface Protein (ESP) .......................................................... 18
1.7 Objectives of this Study .......................................................................................... 21
CHAPTER 2 - -- - - - - -- - - - -22
MATERIALS AND METHODS. - - A- - - - - - - -- 22
2.1 Introduction ............................................................................................................. 22
2.2 Sample Collection ................................................................................................... 22
2.3 Positive and Negative Controls ............................................................................... 25
2.4 Concentration of Enterococci from Liquid Samples. ............................................. 25
2.5 Concentration of Enterococci from Solid Samples ................................................. 26
2.6 Method 1- Conventional Method ............................................................................ 26
2.7 Method 2 ................................................................................................................. 27
2.8 Method 3 ................................................................................................................. 29
2.9 PCR primers and reaction condition. ...................................................................... 29
2.10 Sequencing of Amplicons ..................................................................................... 30

iv

2.1 1 Statistical Analysis ................................................................................................ 31

 

 

 

 

 

 

CHAPTER 3 - -- - -- - - -- - 32
RESULTS - - - -- _ - - - - 32
3.1 Introduction..............................................‘ ............................................................... 32
3.2.1 Detection of esp gene in naturally occurring enterococci determining the range

of the limit of detection in two methods. .................................................................. 32
3.2.2 Method Comparison using Environmental samples ........................................ 36
3.2.3 Sequencing of Amplicons ................................................................................ 37
3.2.4 Detection of esp in Naturally Occurring Enterococci using Method 3 ............ 38
3.2.5 Detection of esp Gene in Disinfected Effluent Samples .................................. 41
3.2.6 Persistence of the esp Marker in Sewage ......................................................... 42
3.2.7 Blind Analysis of the esp Gene ........................................................................ 43
3.2.8 Presence of esp Marker in Different Watersheds in US .................................. 46
3.2.9 Detection of esp Marker in Non-human Samples ............................................ 48
CHAPTER 4-- - - - 49
DISCUSSION---_-- - ............ - -_ -- - - -- .................. 49
4.1 Introduction ............................................................................................................. 49
4.2 Development of a Rapid Method ............................................................................ 49
4.2.1 Solid Samples ................................................................................................... 53
4.2.2 Presence or Absence of the Marker ................................................................. 53
4.2.3 Marker Testing ................................................................................................. 56
APPENDIX 1-- - _ - -_ -- - -- - -- - 59
APPENDIX 2- -- _- -- - - _ -- -- -- _ -- - - _ 71
REFERENCES - - -- - - - - - 72

 

LIST OF TABLES

Table 1-1. Water quality standards/criteria for recreational water (US EPA -1986,
MIDEQ- 2002) ................................................................................................... 5

Table 1- 2. Impairment of water segments due to pathogens in Michigan watersheds.
(http://oaspub.epa.gov/waters/state_rept.control?p_state=MI)* ........................ 7

Table 1-3. Comparison of Microbial Source Tracking techniques (12, 29, 40, 42) ........ 17
Table 1-4. Human sewage pollution marker validation(3 8) ............................................ 20
Table 2-1. List of total samples assayed for esp detection in this study .......................... 24

Table 3-1. Conventional method versus Rapid using raw sewage showing percentage of
agreement between the methods in each sample .............................................. 34

Table 3-2. Esp gene detection in Enterococci cultured from a variety of samples and the
comparison of method one and method two. ................................................... 36

Table 3-3. Detection of esp gene from naturally occurring enterococci from untreated

sewage using method 3. ................................................................................... 39
Table 3-4. Detection of esp gene in disinfected effluent samples .................................... 41
Table 3-5. Lowest esp positive in raw sewage using method 1, 2‘& 32 ........................... 42

Table 3-6. Enterococcus concentration and presence of E. faecium esp human pollution
marker in USGS source samples (human). ...................................................... 45

Table 3-7. Enterococcus concentration and presence of E. faecium esp human pollution
marker in USGS source samples (Non-human). .............................................. 45

Table 3-8. Detection of esp marker in non human samples‘ ............................................ 48

vi

LIST OF FIGURES

Figure 3-1. Comparison of two methods for detection of the esp gene in naturally
occurring enterococci from sewage in the range of the limit of detection‘. (N =

61)*. ................................................................................................................. 35

Figure 3-2. PCR gel image showing amplification of positive control, raw sewage and
Katrina sample. ................................................................................................ 37

Figure 3-3. Detection of esp gene in naturally occurring enterococci in the range of the
limit of detection using method 3 (N= 75)*. .................................................... 40

Figure 3-4. Presence of esp marker in different watersheds in US .................................. 47

Images in this thesis are presented in color.

vii

CHAPTER 1

LITERATURE REVIEW

1.1 Water Pollution and Waterborne Disease Outbreaks

Water quality at coastal beach areas are directly tied to the health of the people using
these waters for recreational swimming activities. Prolonged contact with the
contaminated water could seriously affect the health of the beachgoers (29). The major
sources of contamination during disease outbreaks associated with use of recreational
waters in the US during 1971-2000 were sewage discharges, wild or domestic animals
and bathers themselves (8). Beach sand also harbors bacteria that can lead to poor water
quality at beaches during rainfall or onshore winds (54). Rainfall has been associated
with discharge of untreated sewage via Combined Sewer Overflow (CSO) systems. CSOs
are overflows from older sewer systems designed to carry both domestic and storm water
loads. A discharge from a C80 occurs in response to rainfall and/or snowmelt because
the carrying capacity of the sewer system is exceeded. These discharges do not receive all
the treatment that is available and utilized under ordinary dry weather conditions.
Normally, during dry weather conditions, the wastewater is transported to a wastewater
treatment facility where it receives appropriate treatment prior to discharge. In the US,
CSOs serve an estimate of 772 communities containing about 40 million people. Most
CSOs are located in the Northeast Great Lakes region, and the Pacific Northwest, and are
considered a major contributor of untreated sewage to the waterways in Great Lakes

region (www.in.gov).

Unintentional ingestion of recreational water contaminated with pathogens from
sewage can cause digestive tract illnesses. Pathogens are disease causing microorganisms
present in high concentration in the polluted environment. Other than ingestion, full body
contact in polluted waters can also cause ear, nose and throat infections, skin rashes and
respiratory illness (52, 59). Pathogens that are abundant in the gastrointestinal tract are
shed through the feces of warm blooded animals and humans. These are found in sewage
as well as manures, animal feces, waste lagoons and wildlife (including birds). In
addition to pathogens, enteric bacteria have been traditionally used as indicators of fecal
pollution and potential waterborne disease risk. Water quality advisories or closures occur
in beaches when levels of fecal indicator organisms exceed an individual state authority’s

standards.

Prior to 1970, waterborne disease outbreaks were collected by individual agencies.
Since 1971 the Center For Disease Control (CDC) and the United States Environmental
Protection Agency (USEPA) improved the surveillance system that keeps track of the
national recreational waterborne disease outbreaks and the causative organisms (34, 58).
Summary estimates based on published epidemiological studies have shown an increase
in recreational waterborne disease outbreaks since 1993. There were 65 recreational
waterborne outbreaks reported in US during 2001 to 2002 resulting in illness among an
estimated 2,536 persons including eight fatalities. Thirty percent of the total outbreaks
involved gastroenteritis and of these 60% were related to swimming or wading pools. All

the eight fatalities were as result of swimming in lakes or rivers (30, 58).

1.2 Beach closure Days at US and Great Lakes Beaches

In 2003, in United States at least 18,284 closing and swimming advisory days were
posted at ocean and Great Lakes beaches, and of those, 46% reported sewage as a
pollution source with 54% due to non—point sources. According to data released by EPA
on July 27, 2004, the number of beaches monitored had tripled in 2004, compared with
1997, the first year when EPA began collecting beach-monitoring program data.
Nationally the number jumped 9 % in 2004 compared to 2003. Of the 3,574 beaches that
were monitored in 2004, 26 % had at least one advisory or closing during the 2004
season. Among the 4,025 beaches that were monitored in 2005, 28 % had at least one
advisory or closing during the 2005 season due to high levels of certain bacteria that

exceed standards (www.mdcorg).

 

Monitoring data published by Natural Resources Defense Council (N RDC) on July
2006 indicated that 13% of the total monitored samples (n=18,309) from 530 Great Lakes
beaches exceeded the beach water quality standards. The Beach Act suggests for E. coli a
daily maximum standard of less than 235 colony forming units (cfu) per 100ml of fresh
water. They also reported that 54 beaches situated in every Great Lakes states except
Pennsylvania had exceeded the standard at least 25% of the time. The number of closing
and/or advisory days increased 5% in 2005 compared to 2004. The increased monitoring
of the beaches could be one reason for this. In 2005, the major reason for the beach
advisory or closing was due to the elevated level of bacterial concentrations (87%).
Unknown sources of pollution caused 79% of the total days closed, which was an

increase of 403 closure days posted (some of the beaches closed more than one day per

year) from 2004. Storm water run off, sewage spills and overflows and rain fall

advisories from other sources contributed to the closures.

1.3 Indicator Organisms

Monitoring of hundreds of pathogens on a routine basis in the environment is a costly
and time-consuming process. Historically, Fecal coliform bacteria were used as indictor
organisms but complications with their use and interpretation began to emerge (47).

E. coli and enterococci, two indicators, used by States and recommended by EPA, are
considered to have a higher degree of association with disease and have been
recommended as the basis for bacterial water quality standards (6). Fecal coliforrns,

E. coli and enterococci are present in abundance in the gastrointestinal tract of warm-
blooded animals so that the presence of these bacteria is considered as a strong indication
of fecal pollution. Water containing higher levels of indicator organisms is considered
unsafe because it also indicates the possible presence of other waterborne pathogens(6).
Recreational activities in these types of impaired waters are considered to pose serious
health risks. The enterococci have been used as indicators of fecal pollution for many
years and have been valuable in the marine environment and recreational waters as
indicators of potential health risks and swimming related gastrointestinal and urinary tract
illness (51). Studies demonstrated that there was a strong correlation between
precipitation and the amount of time spent in water. Waterbome disease outbreaks, and
particularly the presence of enterococci, was directly related to the increased risk of

recreational related acquired gastrointestinal illness (4, 51, 52).

1.3.1 Michigan Water Quality Standards

Michigan uses fecal coliform and E. coli bacterial concentrations for assessing the water
quality standards for recreational waters. Michigan’s water quality standard specifies a
geometric average of < 200 cfu per 100ml of fecal coliforrns and < 130 cfu per 100ml for
E. coli (Table 1-1). For EPA, the recreational water criteria specify geometric average of
< 200 cfu per 100ml for fecal coliforms, < 126 cfu per 100ml for E. coli and less than

33cfu per 100ml for enterococci.

Table 1-1. Water quality standards/criteria for recreational water (US EPA -1986,

MIDEQ- 2002)

 

 

 

 

Indicator
Geometric Means Michigan EPA
Fecal Coliforms
CF U <200cfu/ l 00ml <200cfu/100ml
E. coli
CFU <130cfu/100m1 <126cfu/100ml
Enterococci
CFU No standard <33cfu/100ml

 

 

 

 

 

1.4 Total Maximum Daily Load (TMDL)

The Clean Water Act was passed in 1972, yet today many of the US water bodies do
not meet the water quality standards, goals and/or designated uses. Each state has
responsibility to identify highly polluted waterbodies that are not meeting water quality
standards. These impaired waters must undergo an evaluation using the Total Maximum
Daily Load (TMDL) approach according to the Clean Water Act. Microbial densities that
a particular water body can receive can be addressed partially via the TMDL because a
TMDL is defined as "the maximum pollutant load that a water body can receive and still
meet water quality standards” (50). Exceeding the TMDL criteria for indicator bacteria
generally focuses on the potential for illness in people who are in contact with such water
bodies. Water quality improvements there by also reduce the risk of infectious diseases.
As a part of this process, identifying the fecal pollution source could lead to better
management and remediation. In 2004, Michigan Department of Environmental Quality
(MDEQ) identified 580 water segments of impairment in Michigan watersheds. MDEQ
specifies that 90 of the impairments in Michigan waters were due to pathogens, which is
about 16% of the total (n= 580) impairment reported. In those 90 impairments, the
majority (n= 84) were due to pathogens and untreated sewage discharges. Bacterial
slimes, beach closures, E. coli and fecal material comprise the remainder (Table 1- 2)

(MDEQ).

Table 1- 2. Impairment of water segments due to pathogens in Michigan watersheds.

(http://oaspub.epa.gov/waters/state_rept.control?p‘state=MI)*

 

 

 

 

 

 

 

 

Cause of Impairment in MI Causes of Impairment (%)

watersheds

Bacterial Slimes 3%

Beach closures 1%

E. coli 1%

Fecal 1%

Pathogens 71%

Untreated sewage discharges, 13%
Pathogens

Total numbers of segments 90

 

 

 

 

1.5 Microbial Source Tracking

The TMDL attempts to calculate contaminant impairments and regulate the amount and
type of pollution entering the water. To correct the problem, the source of pollution must
be identified (50). Microbial Source Tracking (MST) is a developing field to help
pinpoint the source of the microbial pollution in the aquatic environment (40). For
example Hagedom et a1. (1999) were able to trace the fecal pollution in a Virginia
watershed using a MST method and were able to implement Best Management Practices
(BMP) for improving the water quality. The study was conducted for a 28 month period
where the first 12 months concentrated on identifying the source pollution. They found
that high concentration of fecal bacteria in stream areas during the summer were due to
cattle (18). By restricting the cattle access to the river they were able to reduce the
pollution in those stream areas. Traditional indicator bacteria have been used for many
years for determining the sanitary quality of surface and recreational waters. Even though
indicator organisms have been shown to relate to recreational health risks they cannot be
used to address the sources of pollution. According to the Clean Water Act, recreational
water should be protected from pathogenic microbial organisms. Thus there is a growing
need for a science - based approach to determine the pollution source to abate the

problem in a cost effective manner (38).

Various methods have been developed and used for MST (40, 43). The basic premise of
the source tracking methods is that bacteria demonstrate specific characteristics related to
host specificity and adaptation to certain environmental conditions (29, 40, 42). The

methods are classified into two main categories, Library-dependent or library-

independent methods. Library dependent methods rely on the building of a library that is
developed with bacterial isolates from known sources (40, 49). Most are culture based
and often rely on genetic characterization. Reference libraries are built using isolates,
collected directly from animals or collected directly afier excretion to ensure there is
limited contamination. Library dependent methods are based on matching the reference
libraries (phenotypic or genotypic profiles) of bacterial strains to isolates from the
environmental sample (49). Library-independent methods rely on identification of a host-

specific trait or gene. The following sections will describe some of the MST methods.

1.5.1 Phenotypic Library Dependent
1.5.1.1 Antibiotic Resistant Analysis (ARA)

Antibiotic resistance profiling is the most widely used phenotypic method for MST. A
library of antibiotic resistant genes in enterococci, fecal coliform or E. coli of known
animal sources are used to identify the source of pollution (5, 18, 55). For this method,
antibiotic resistant bacteria are isolated via inoculation onto agar plates containing
different antibiotics. The growth of the bacteria in the antibiotic plates is compared to a
control plate (without antibiotic) to develop profiles. This comparison is based on the
assumption that bacteria from human, livestock and domestic animals are more resistant
to different sets of antibiotics so that one can differentiate wildlife fecal pollution from
domestic animals and from human waste, using this technique. This requires culturing
and screening a large number of isolates against antibiotics of different concentrations to
make a large library that represents a fair number of likely sources in a given watershed.

So the accuracy of the results rely partly on the size of the library yet the heterogeneity of

the bacteria contributes to a high false positive rate (21, 31). The fingerprints are
compared to a reference database composed of bacterial isolates from known sources to
find out the Average Rate of Correct Classification (ARCC). Results from various MST
studies using antibiotic resistant genes summarized have reported ARCC ranging from 62
to 96% for this method, when classifying some subset of the library, however this rate
goes down with new isolates (42). Geary et a1. (2003) were not able to identify any
significant source of pollution when they assayed four different concentrations of four
different antibiotics in a shell fish growing area in Australia (14). The reference library
was composed of isolates of fecal streptococci from beef, dairy cattle, chickens and
humans(14). Research has shown that isolates from point sources were more diverse than
isolates from nonpoint sources and grouping is likely influenced by a strain's prior
exposure to antibiotics (31). Another draw back of this method is that antibiotic
resistance genes are carried by plasmids which may be lost upon cultivation. A study
conducted in six Virginia watersheds by Wiggins et a1. (2003) concluded that antibiotic

resistant plasmids may only be stable up to one year (55).

1.5.1.2 Carbon Source Utilization

Carbon source utilization profile is another type of phenotypic MST method. This
method is based on the BIOLOG system which has a 95 well microtitre plate that
contains different carbon substrates. The microbial isolates from known sources are
categorized in accordance with their utilization of carbon substrates. A study conducted
by Hagedom et a1. (2003) used 365 enterococci isolates of known source from four
different geographical regions using BIOLOG system. The ARCC ranged from 86.6-

92.7% for the human versus non-human two-way classification. But the ARCC was

10

lower for the three way classification system and a high percentage of false positives
(51.5%) was also noted (17). Again new isolates were not classified with high accuracy.
These phenotypic methods are not used very frequently because phenotypic characters
are not always stable and most researchers are moving toward genotypic methods. In
addition to that nutrient requirements of the microorganisms are based on environmental

factors so the carbon source utilization may not be same in all environments (17, 42).

1.5.2 Genotypic Library Dependent

Library based genotypic analyses use DNA finger prints and techniques such as rep-
PCR, ribotyping, Amplified Fragment Length Polymorphism (AF LP), and Pulse Field
Gel Electrophoresis (PFGE). For these methods DNA is extracted from bacterial cells
and restriction enzymes are used to cut the DNA into different base pair lengths. Ladder-
like patterns are visualized using gel electrophoresis and according to the size of the

DNA fragments the isolates are grouped statistically.

1.5.2.1 Rep-PCR

For these genetic methods, a specific repetitive portion of the microbial genome is
amplified by Polymerase Chain Reaction (PCR) which is visualized by electrophoresis
and pattern recognition by computer software. The three major repetitive elements in
bacteria being used to generate bacterial finger prints are REP (Repetitive Extragenic
Palindromic), ERIC (Enterobacterial Repetitive Intergenic Consenses) and BOX (154
basepare box element). A small study based on the BOX-PCR conducted by Dombeck

et.al. ((200) used154 E. coli isolates obtained from six species of animals (cows, pigs,

11

sheep, chickens, geese and ducks) and humans and revealed that 100% of the chicken and
cow isolates gave the correct classification. Between 19-29 isolates were used per source
and the correct classification of other isolates was 78 to 90% (10). In this study they also
suggested that BOX- PCR was better than REP-PCR for differentiating human from non
human E. coli isolates. While in another study conducted by Johnson et a1. (2004) using

E. coli strains from 12 different animal sources, the correct classification was only 60%

despite the relatively large size of the known source data base(23).

1.5.2.2 Ribotyping

Ribotyping includes multiple steps such as restriction enzyme digestion of genomic
DNA, fragment separation by gel electrophoresis, transfer of the DNA fragments by
Southern Transfer and hybridization using labeled rRNA probes to generate ribotype
patterns that can be used to identify bacterial strains (9, 29, 37). The rRNA (ribosomal
ribonucleic acids) is highly conserved and is an integral part of all living organisms. In a
small study 287 E. coli strains from humans, cattle, pigs, horses, chickens, turkeys,
migratory geese, and dogs were collected and riboprint patterns were generated (7). They
found this method was useful for differentiating fecal bacteria of human and non- human
origin but the Average Rate of Correct Classification (ARCC) was poor when pooled
fecal sources were analyzed. This method also required extensive manipulation of DNA
and use of labeled gene probes. However Jenkins et al. (2003) suggested that to make the
ribotyping effective one should also consider the temporal variability (40). This
observation was based on the study using wild deer and captive deer. Researchers found

that the diet was influencing the ribotype diversity of E. coli between the wild and

12

domesticated deer (22). Another draw back of this method is the necessity of a large

database that represents each watershed for the correct source identification.

1.5.2.2.1 Pulse Field Gel Electrophoresis (PFGE).

Pulse Field Gel Electrophoresis (PFGE) is another technique used for ribotyping but it
analyzes DNA rather than rRNA (ribosomal RNA). This was found to be more
discriminatory than ribotyping with pure culture bacterial cells (29, 46). However
Parveen et a1. (2001) were not able to distinguish between human versus non-human
sources when they analyzed 32 E. coli isolates using PFGE technique (33). Another draw
back of PFGE is electrophoreses time consumption (3 0-50 hours) and the specialized

equipment for the process (42).

1.5.2.3 Amplified Fragment Length Polymorphism (AFLP)

Guan et al. (2002) used 105 E. coli isolates from the feces of cattle, poultry, swine, deer,
goose, moose, human sewage, and clinical samples that were genotyped by Amplified
Fragment Length Polymorphism (AF LP)(16). These isolates were digested with
restriction enzymes to generate a subset of DNA and PCR amplification was done on that
subset. The success rate of this method solely depends on the availability of whole
genomic sequence of the particular isolate. In this study they used a smaller number of
isolates, since natural populations of E. coli comprise a large community with high
genetic variation. So it is necessary to grow a large number of isolates and the method
may take 10-12 days to complete the manual ribotyping, a large database is also needed

for comparison (32, 35).

13

1.5.3 Genotypic Library Independent Methods.

1.5.3.1 Host specific molecular markers

Host specific MST methods using genetic markers appear to have the ability to address
human versus non-human sources with a much lower false positive rates compared to
Library based methods (3, 21). Host specific methods are generally gene specific PCR
methods that have been developed by targeting specific genes, found only in certain

hosts. The prevalence of the gene determines the sensitivity and the false negative rates.

1.5.3.2 Terminal Fragment Length Polymorphism (T-RFLP)

Terminal Fragment Length Polymorphism (T-RFLP) and length-heterogeneity
polymerase chain reaction (LH-PCR) techniques use an automated sequencer for the
detection of fluorescently labeled 16S rDNA. Bernhard and Field (2000) demonstrated
that there are species composition differences between Bifidobacterium and the
Bacteroides-Prevotella groups among human and ruminants. The specific primer sets for
human and ruminant were designed (2, 3, 12). However, this method has only been tested
against a limited number of animal fecal samples to date. Other potential sources also
need to be included. Although this method was found to be helpful for MST, it is
technically very demanding and expensive equipment is required for this host specific
method for detection(42). The short term survivability of Bifidobacterium and its lower
concentration in human feces needs to be considered before applying this for routine

monitoring (41).

14

1.5.4 MST using Viruses

Fecal viruses also have been used to trace sources of fecal contamination in
environments. Human enteroviruses and bovine viruses were used to differentiate human
versus bovine fecal contamination. The main advantage of using viruses as indicators
includes the ability to monitor directly in water, without culturing(13). However large
volumes of water need to be screened for virus detection compared to the traditional
water quality indicator organisms. While screening larger volumes, PCR inhibitory
substances also concentrate and that may interfere with the PCR results(44). Like other
culture independent methods using viruses for source tracking will not always indicate a
recent contamination because the detection method targets viable as well as non viable

cells.

1.5.5 Advantages and Disadvantages of Current Microbial Source Tracking

Methods.

In summary different MST have their own advantages and disadvantages (Table 1-3).
Most of these methods target was E. coli, however a few of them targeted fecal
streptococci, enterococci and Bacteroides. The time for each technique and the cost are
critical factors when considering the methods applicability. For instance culture
dependent and library dependent phenotypic methods require culturing the organism and
construction of large databases for comparison. At the same time Carbon Source
Utilization is a simple process but is mostly applied to pure culture such as clinical
isolates. Rep-PCR has high discriminatory power but this method also needs a reference

library. Like Rep- PCR, ribotyping also has discriminatory power but this method is a

15

very complex and time consuming process. Despite the advantage of applicability for
most of bacterial strains, AFLP and the PF GE methods are considered less demanding for
MST because of the cost and the technical level needed. Unlike other methods RFLP
does not need a library and it is a culture independent method. However the limitations of

this method are also related to the expense and the complex processes.

16

Table 1-3. Comparison of Microbial Source Tracking techniques (12, 29, 40, 42)

 

 

 

 

 

 

 

 

 

 

 

 

Method Description Advantage Disadvantage Microbial
Target
(ARA) Distinct ARA Useful for Time Fecal
Antibiotic patterns used to smaller consuming. streptococci,
Resistant identify the watersheds Antibiotic E. coli,
analysis“ source resistant gene Enterococci
pollution. may be lost
during
cultivation.
Requires large
database
Carbon Substrate Applicable Carbon
Source utilization clinical utilization can
Utilization* differences microbiology. change and
among bacteria Simple process depends on Enterococci
indicate sources environmental
factors.
Requires large
database
Rep-PCR* PCR based High Culture
amplification discrimination dependent.
using Large database E. coli,
palindromic required
DNA sequences
Ribotyping“ DNA digestion Discriminatory Time
with restriction power and easy consuming
enzymes, interpretation complex E. coli,
hybridization procedure
using labeled
probe
AF LP & DNA Applicable in Expensive and E. coli,
PF GE* fingerprinting most strains, technically
by digestion automatable demanding
with restriction
enzymes
T-RFLP Used automated Culture Expensive
sequencer for independent equipment
the detection of Library needed and Bacteroides
fluorescently independent technically
labeled l6S demanding
rDNA

 

*Culture and Library based methods

 

1.6 Characteristics of Enterococci

The US EPA in 1986 suggested the enteococci as pollution indicators for marine
environments and both E. coli and enterococci as fecal indicators for fresh water
environments. The taxonomic group Streptococci contains both Enterococcus and
Streptococci. The enterococci sub group has the ability to grow at high and low
temperatures (IO-45°C) and at high pH (9.6). Enterococci is a gram positive roughly
spherical shaped bacteria found in the intestinal tract of warm blooded animals. The
species of Enterococcus include opportunistic pathogens such as Efaecalis and
E. faecium. Enterococci are well suited to survive in the intestine, vaginal tract and the
oral cavity. These bacteria have a natural ability to readily acquire, accumulate, and
share virulence traits (clustered on a large pathogenicity island) and/ or antibiotic
resistance genes (24, 36, 38). According to EPA criteria, a single sample maximum
concentration of Enterococci for marine water should be less than 61cfu/100ml. For fresh
water it should be less than 104/ 100ml, while a geometric mean of 33 CFU/100ml was

suggested.

1.6.1 The Enterococci Surface Protein (ESP)

Wei et a1. (2001) suggested that PCR based detection of virulence factors would be a
better indicator of the health significance and a tool for MST (53). The esp gene codes for
the enterococcus surface proteins and studies have demonstrated that the esp gene in E.
faecium is a virulence factor in this microorganism and plays a role in it’s pathogenesis
(25, 36, 45). A study conducted using 120 isolates associated with hospital patients
during outbreaks from USA, Europe and Australia reported that the esp gene in clinical

isolates of E. faecium were genetically distinct from the non epidemic isolates (n=45) as

18

well as animal isolates (n=98), it was also suggested that its presence was a marker of
increased virulence (56). Various E. faecium isolates from hospital outbreaks (11, 56)
were used to clone and sequence the esp and these revealed the presence of PAI
(pathogenicity island) related to epidemicity. Willems et a1. (2001) indicated that the esp
in E. faecium is present in the chromosome of the bacteria and had enough evidence to
believe that the presence of this gene was associated with nosocomial outbreaks of E.
faecium (24). Nosocomial outbreaks are those that originate or occur in a hospital or

hospital like settings.

Primers specific for the E. faecium esp gene developed previously were used as a
marker of human sewage (3 8). This target originated from a homologue that had been
identified in isolates of E. faecium previously, and was used to develop the primers and
the amplicon(56). A culture- PCR based method was evaluated to assess presence of the
esp gene in Enterococci from livestock, birds, other animal sources and human sewage. A
total of 167 wastewater and fecal samples were screened for the target gene and the gene
was detected in primary sewage influent (40/40), secondary sewage effluent (10/10),
filtered waste water (5/5) and septic tank effluent (8/ 10). The marker was never detected
0% (n=102) in animal waste water or fecal samples. Table 14 shown the esp marker
validation using samples from a variety of sources. Survival studies showed that the
marker was detectable in fresh water up to five days when the colony forming units
ranged between 19 and 122 and in simulated seawater up to seven days when the total

number of culturable eenterococci was more than 70 CFU (3 8).

19

Table 1-4. Human sewage pollution marker validation(3 8)

 

 

 

 

 

 

 

 

 

 

 

 

Source* No of samples positives/No of
samples tested

Primary sewage influent 40/40

Secondary sewage effluent 10/10
Filtered waste water 5/5

Septic tank 14/ 16
Waste lagoons- Poultry 0/8
Waste lagoons-Swine 0/ 12
Waste lagoons -Cattle 0/35
Feces-Canada Geese 0/ 12
Feces-Seagull 0/28
Feces-Pelican 0/7
Feces-Wild Birds 0/10

 

 

 

 

*Culture based, library independent host specific assay based on populations of
bacteria not individual isolates

20

1.7 Objectives of this Study

Based on the initial study by Scott et al. (2005) further evaluation of the esp marker in
enterococci was warranted .The overall objective of this study was to further examine the
utilization of the esp gene as human sewage marker and streamline the potential method,
allowing for more sensitive results. Specific objectives included:

1) Comparison of a rapid PCR (Direct PCR) to a conventional (DNA extraction) method.
2) Further validation of the presence or absence of the marker in various sources.
3) Persistence of the marker in raw sewage and the sensitivity of the method and

prevalence of the marker.

21

CHAPTER 2
MATERIALS AND METHODS

2.1 Introduction

Effective assays for source tracking are available, but these are often expensive, take
time and are complex (Table 1-3). Based on epidemiological studies the most serious
threat to human health comes from human and not from animal sources (52). The
elimination of human source pollution is important for managing water quality and thus
protecting public health. The source tracking method used by Scott et al. (2005) for
enterococci esp gene included cultivation followed by a three hour enrichment step,

DNA extraction procedure and PCR (3 8). One of the specific goals of this study was to
minimize the processing time without affecting the test results. Thus, procedures to
eliminate the extraction and enrichment steps were evaluated and these were termed rapid

PCR approaches.

2.2 Sample Collection

Domestic raw sewage and secondary disinfected wastewater effluents were collected
from a waste water treatment plant in East Lansing, MI from August 2005 to July 2006.
Twenty three raw sewage samples were collected in 50mL sterile centrifuge tubes and 10
secondary effluent samples were collected in one liter sterile bottles with sodium thio
sulphate to neutralize any chlorine residual. Because of the high concentrations of the
bacteria in sewage and fecal samples, numerous replicates were prepared from each

sample. In addition to waste water samples, 13 water samples from New Orleans after

22

Katrina were collected and shipped overnight to the Michigan State University and tested
for indicator bacteria and the esp gene. Four samples from animal waste water, six
samples from ditches of a near by farm and nine bird samples were also collected. A one
day beach and bird survey was conducted on July 9, 2006 at North Beach, along Lake
Michigan in Grand Haven, Michigan. The goals of this research project were to examine
bird behavior and determine the extent that bird feces impacts beach sand and water
quality at a recreational beach. Sand samples were collected that day along the beaches
and eight of the sand samples were also analyzed for esp marker. All samples were

processed within 24 hours from the time of collection.

Samples were also collected by the USGS throughout the US from human and animal
pollution sources, and duplicates of these were shipped to the MSU Environmental
Quality Laboratory. These were not identified as to the source at the time of processing
and were treated as blind samples. Samples were collected from Colorado, Texas, Iowa,
New Jersey, Minnesota, Virginia, New York, Idaho and Ohio. All liquid samples were
collected in one liter sterile bottles and the solid samples were collected in sterile 4 ounce
specimen containers. All samples were collected using aseptic techniques and were
transported to the laboratory on ice before being processed. Table 2-1 shown the total

number of samples assayed for the esp marker detection.

23

Table 2-1. List of total samples assayed for esp detection in this study

 

 

 

 

 

 

 

 

 

 

 

 

 

Total no. of Source Sample Type
samples collected
23 WWTP* Raw sewage
10 WWTP Secondary effluent
13 Katrina (New Flood water
Orleans, LA)

4 MSU (Farm) Animal waste water
9 MSU (Farm) Drainage water
9 Bird Feces
8 Beach Sand
18 USGS” Human
9 USGS Non-human

 

*waste water treatment plant

“US Geological Survey

24

 

2.3 Positive and Negative Controls

Efaecalis (ATCC# 19443) was used as negative control. E. faecium C68 strain
containing the esp gene was used as positive control (provided by Dr. Louis B. Rice of
the Cleveland Veterans Medical Center in Cleveland Ohio). The PCR products of the E.
faecium C68 were purified using QIAquick PCR Purification Kit (Qiagen, Inc.). A
TOPOTA Cloning® Kit (Invitrogen, Co.) was used to clone the PCR products and this

plasmid was used as a positive control for all conventional and rapid methods.

2.4 Concentration of Enterococci from Liquid Samples.

Enterococci were enumerated in liquid samples by membrane filtration. Serial (ten
fold) dilutions were used and lmL of the diluted solution was filtered through a 0.45 pm
pore-size 47mm nitrocellulose-mixed ester membrane filter (GE Osmonics). The first
step in making a serial dilution is to take a known volume (usually lml) of the
concentrated sample and place it into a known volume of buffer (usually 9ml). This
produces 10ml of a diluted solution. The dilute solution has lml of extract /10ml. This is
a 10 fold dilution. This process was repeated to make successive ten fold dilutions. Three
ten fold (10", 10-2, 10-3) dilutions were made from each of the samples and replicates of
each dilution were filtered as described above. The filters were then placed on
mEnterococcus (mE) agar (Difco) supplemented with indoxyl substrate (mEI) (Sigma
Inc.) according to the methodology outlined in EPA (48). Filters and media were
incubated at 413: 05°C for 24h. After 24 hour incubation, the blue halo colonies were

counted as enterococci. Filters were stored at 4°C until further analysis for the esp gene.

25

2.5 Concentration of Enterococci from Solid Samples

Three grams of each of the solid fecal sample were weighed and placed into a sterile
50ml centrifuge tube. Thirty milliliters of the 1X Phosphate Buffer Water (1 .25ml of
0.5M KH2PO4, 5mL of 0.44M MgCl2; pH 7) (PBW) were added to the tube, which was
then vortexed to suspend the sample. Serial dilutions were performed using PBW and
appropriate dilutions were filtered through 47mm nitrocellulose-mixed ester membrane
filter (GE Osmonics) placed on mEI media and incubated at 41d: 05°C for 24 hour
incubation. After 24 hour incubation Colony Forming Units (CFU) were counted and the

membrane filters on the agar plates were stored at 4° C until further analysis.

2.6 Method 1- Conventional Method

In the conventional method the E. faecium esp analysis and DNA extraction were
performed as follows:

Membranes that were positive for enterococci colonies were further evaluated to
determine the presence of the target gene esp (3 8). Membrane filters containing zero to
too numerous to count (TNTC) colony forming units (CFUs) were lifted, suspended in a
lSmL centrifuge tube containing ten mL of Tryptic Soy Broth TSB (Difco), vortexed
vigorously for 30 seconds and incubated at 41°C for three hours. The centrifuge tubes
were removed from the incubator and vortexed again. One milliliter of the resulting
culture was placed into a microcentrifuge tube and the tube was centrifuged (NapCo 2002
centrifuger) at maximum speed for five minutes. The supernatant was removed and
180uL of lysis buffer (20mg/ml lysozyme; 20mM Tris-HCL, pH8.0; 2mM EDTA; 1.2%
Triton® was added to the pellet. The pellet was resuspended and placed into a 35°C

water bath for 30 minutes. Then 20uL of Proteinase K (Qiagen, Inc) and 200uL of lysis

26

solution (Qiagen,Inc) was added to the tube and vortexed. The tube was placed into a
45°C water bath for 30 minutes. The temperature of the water bath was increased to 95°C
for 15 minutes. The microcentrifuge tube was removed from the bath and kept at room
temperature. DNA extraction was performed using the QIAamp DNA Mini Kit according
to the manufacturer’s instructions (Qiagen, Inc.). Then lul of the DNA was used as the
template for the PCR reactions from the total (400 pl) extracted DNA. The total time

consumption for the DNA extraction step was 3.5 hours.

2.7 Method 2

The main difference between method 2 and method 1 was in the preparation of the
template for rapid PCR reaction where by no extraction was used (Figure 2-1). For the
rapid PCR lml of the TSB was transferred after the two hour incubation to a 1.5m]

centrifuge tube.

27

Figure 2-1. Schematic diagram showing the sample analysis for detecting enterococci

and the esp gene using three methods

 

Sample Collection

 

 

 

 

 

Membrane Filtration

 

 

 

 

 

Incubation

 

 

 

 

 

Enumeration (cfu)

 

 

 

 

 

 

 

V I

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Method 1 Method 2 Method 3
Enrichment (TSB) Enrichment Cell concentration from the
(3 hours) (TSB) (2 hours) membrane filter (30 minutes)

DNA Extraction Cell concentration
(3.5 hours) from TSB
' PCR

 

 

 

28

The cells were spun down by centrifuging five minute at maximum (14000 rpm) speed
(N apCo 2002 centrifiiger). The supernatant was removed without disturbing the pellet
using a micropipette. This step was repeated one more time. Finally the pellet was
resuspended with 500ul of molecular grade water (DNase/RNase free water) and placed
into a -20°C freezer until analyzed by PCR. Two micro liters of the cell suspension was

placed into PCR reaction tube as the template for the marker detection.

2.8 Method 3

In method 3 the preparation of the template was done without the enrichment step
(Figure 2-1). After 24 hours of incubation the blue halo colonies were counted and the
cells were washed off the filters with lml of molecular grade water (Sterile water) using a
sterilized plastic cell spreader. The cell suspension was spun down by centrifuging five
minutes at the maximum speed (14000 rpm) (NapCo 2002 centrifuger). The pellet was
resuspended with 500 pl of molecular grade water and washed via centrifugation. Finally
the cell suspension was placed into a -20°C freezer until analyzed by PCR. Two micro
liters of the cell suspension were placed into a PCR reaction tube as the template for esp

detection.

2.9 PCR primers and reaction condition.

The primers specific for the esp gene in Efaecium previously developed and examined
for specificity to human fecal pollution were used for detecting the esp marker (28). The
forward primer: (5’-TAT GAA AGC AAC AGC ACA AGT-3’) and the conserved

reverse primer (5’ —ACG TCG AAA GTT CGA TTT CC-3’), were used for all reactions.

29

PCR reactions were performed in ZOuL reaction mixtures containing 1X PCR buffer,
1.5mM MgClz, ZOOuM of each of the four deoxyribonucleotides, 0.3 uM of each primer
0.5Unit of HotStarTaq DNA polymerase (Qiagen) and lul of template DNA.
Amplification was performed in Eppendorf Mastercycler gradient (Eppendorf scientific
inc. Westbury, New York) with an initial step at 95°C for 15 minutes (to activate Taq
polymerase) , followed by 35 cycles of 94 °C for 1 min,58 °C for 1 min, and 72 °C for 1
min. The PCR products were separated on a 1.5% agarose gel stained with Gelstar
nucleic acid stain (Cambrex Bio Science, Inc.) and viewed under ultraviolet (UV) light.

The PCR product is 680 base pairs in length.

2.10 Sequencing of Amplicons

The amplified products were isolated in a 1.75% low melting temperature agarose gel
(Sea Plaque GTG; Cambrex Bio Science Baltimore Inc. Baltimore, MD) and the product
was purified with a QIAquick gel extraction kit (Qiagen, Valencia, CA). The same
primers for esp gene were used for dye terminator cycle sequencing of each amplicon.
Sequencing was carried out with an ABI 377 DNA sequencer (Perkin-Elmer Applied
Biosystems, Foster City, CA) at the Michigan State University (MSU) sequencing
facility. The multiple sequence alignment of the Enterococcus esp gene was executed
using the pileup program of genetics Computer group (Madison, Wis.) Squeb software.
The sequence analysis was also performed by the blast program on the National Center

for Biotechnology Information.

30

2.11 Statistical Analysis

A paired t-test was performed at the average cfu/membrane limit of detection on a log
transformed data to address the statistical difference between method 1 and method 2 and
a chi-square test was done to test the significant association between method 1 and
method 2 in the relative presence versus absence of the marker per membrane. All
statistical analysis (Chi-squared, t-tests) were performed using SPSS for windows,

Rel.12.0.0.2003.Chicago: SPSS Inc.

31

CHAPTER 3
RESULTS

3.1 Introduction

Microbial source tacking (MST) is becoming an accepted field of study because of its
applicability to better define the relationship between environmental quality, source of
pollution and human health (40, 42, 51). However the MST field needs to be improved
with regard to affordability to the communities using them and accuracy of the results.
This research focuses on improving a host specific test and addressing the accuracy for

identification of improperly treated sewage.

3.2.1 Detection of esp gene in naturally occurring enterococci determining the range

of the limit of detection in two methods.

Ninety seven membranes from ten different samples of raw sewage were analyzed by
method 1 and 2. Method Us a conventional method which includes membrane filtration,
incubation on mEI media, recovery of all colonies from membrane, enrichment and DNA
extraction followed by PCR for esp detection where as the method 2 excludes the DNA
extraction and is considered rapid. Membranes from zero to TNTC were assayed for the
comparison. Results are shown in Table 3-1. All four membranes assayed from sample 1
with cfu ranging between 29 and TNTC were esp positive in method 1 and in method 2.
Sample 2 had cfu between zero and 48 per membrane and of the six membranes all were
negative by both methods. In samples 3, 4, and 5, 13 membranes with cfu from 4 to 560
were compared and six were positive and seven were negative by both methods. There

was 100% agreement between the two methods in these samples. Forty seven membranes

32

from sample 6 were assayed and comparative results in 40/47 were obtained using the
two methods. There were five esp positive membranes from the conventional method and
two positives from the rapid method that did not agree each other (85% agreement).
Figure 3-1 shows the results graphically for each membrane. Agreement in sample 6, 7, 8
was 88% to 95 %. In all other samples (sample 9 & 10) the results by the two methods
were in 100% agreement.

Overall the two methods agreed 91% of the time (88/97). The sensitivity of method 1
was 45128 cfu/membrane and the sensitivity of the method 2 was 46i27 cfu/membrane.
The sensitivity was calculated by taking the geometric average of all counts
cfu/membrane with esp negative results. Positive esp membranes had cfu ranging from
21 to TNTC in method 1 and method 2 where as the negative esp membranes had cfu
range from zero to 117 in method 1 and and zero to 106 in method 2.

The paired t-test performed showed that there was no difference between the two
methods ((p = 0.53) chosen level of p = 0.05). Hence the paired t-test failed to reject the

null hypothesis at the 95% confidence limit.

33

Table 3-1. Conventional method versus Rapid using raw sewage showing percentage of

agreement between the methods in each sample

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

No cfu No.0f Method 1 Mehod 2 Method 1 Method 2 Agreement in
(Range)* membranes esp +ve esp +ve esp -ve esp -ve both methods
assayed (%)
1 29-TNTC 4 4 4 0 0 100
2 0-48 6 O O 6 6 100
3 19-560** 4 3 3 l 1 100
4 12-500" 4 1 1 3 3 100
5 4-280 5 3 3 2 2 100
6 45-120 47 23 21 24 26 85
7 36-65 14 4 3 10 ll 93
8 39-100 5 4 5 l O 80
9 58-140 4 4 4 0 0 100
10 44-160 4 l 1 3 3 100
Positive Negative Total
Method 1*“ 47 50 97
Method 2 45 52 97

 

 

 

 

 

*cfu/membrane cultured on mEl
** Higher values extrapolated from plates in countable range.
***Chi-square test showed no significant difference in the results obtained by method 1
versus method 2. (p< 0.001)

34

 

Figure 3-1. Comparison of two methods for detection of the esp gene in naturally

occurring enterococci from sewage in the range of the limit of detection'. (N= 61)*.

 

. ti“ :17 In :21 I nnnl 010 DIIIJCIZII

ESP No

000:. ‘0 [[0 ‘0I0 0!:10020 0 00 CI] 0 0 0 I

ESP -vo

 

CFU I Membrane

 

 

 

‘Sensitivity was 45:1:28 cfu/membrane in method 1 & 463:27 cfu/membrane in method 2
‘No. of membranes in this figure is 61.

35

3.2.2 Method Comparison using Environmental samples

A total of 36 membranes from 23 different environmental samples were processed and
compared using method 1 and method 2. All of the results between the two methods were
in 100% agreement. Results are shown in Table 3-2. The majority of the samples were
from the New Orleans Katrina affected area, but the cfu range was generally below the
detection limit of the methods (30). Only one sample came up as positive for the esp
marker in Katrina where the membrane had 121 cfu. The rest of the samples (n= 12) were
esp negative by both methods. Nine water samples from ditches near the MSU farm were
assayed for the esp marker. One sample was esp positive by method one and two. It was
speculated that a leaky sewage line or an older septic field could have contributed to the
finding of the human marker. The membranes processed for the esp marker from the
MSU samples had a cfu range between 10 and TNTC where the total membranes assayed
was 14. A ground water sample was also processed from a private house well which had
a cfu ranging between 67 and 189 per membrane. All three membranes assayed for the

esp marker were esp negative by both methods in this sample.

Table 3-2. Esp gene detection in Enterococci cultured from a variety of samples and the

comparison of method one and method two.

 

 

 

 

Membranes No. of Source Esp + Esp - Agreement
assayed samples (%)
14 9 MSU Farm 1 8 100
3 1 House Well 0 1 100
19 13 New Orleans 1 12 100

 

 

 

 

 

 

 

 

36

3.2.3 Sequencing of Amplicons

The PCR product from the positive control, one sample from raw sewage and one from
the Katrina affected area which were positive for the esp marker in method 2 previously,
were sequenced to examine the specificity of the esp marker in the samples (Figure 3-2).
The lanes were loaded with Marker (lkb), positive control, negative control, raw sewage,
and Katrina sample respectively except lane five which was empty. The remaining lanes
were loaded alternatively with samples that had been previously negative for esp. The
lanes between the samples were empty. The three amplified PCR products were used for

the sequencing.

Figure 3-2. PCR gel image showing amplification of positive control, raw sewage and

Katrina sample.

Marker

Positive control

  

Raw sewage

-
:0 I! G ""'—‘i —¥'#‘ -_,_ Katrina sample

37

3.2.3.1 Sequencing Results

Multiple sequence alignment of the three selected isolates was undertaken. A total of
612 base pairs (bp) of the esp marker was assessed (appendix 1). Alignment of Esp gene
from the two environmental samples (isolates from Katrina samples and raw sewage)
matched the positive control with 100% sequence similarity and without a single
mismatch of nucleotides. This genetic evidence supports that the human marker esp gene
present in raw sewage and in Katrina samples were the same as that of the positive

control.

3.2.4 Detection of esp in Naturally Occurring Enterococci using Method 3

In an attempt to reduce the time taken for the esp analysis, the next step was to
eliminate the enrichment step. A total of 78 membranes from 12 different samples were
processed by method 3 for the detection of esp marker. All samples were raw sewage
collected from February 2005 to July 2006 from East Lansing waste water treatment
plant. Of the total 78 membranes processed, 38 were esp positive with cfu ranging
between 1 and TNTC per membrane. Results are shown in Table 3-3 and in Figure 3-3.
Positive esp membranes had cfu ranging fiom 2 to TNTC while negative esp membranes
had cfu ranging from 1 to 140 in method 3. Over all method 3 was found to be more
sensitive but had greater variability than method 1 and method 2. The sensitivity of
method 3 was 26i40 cfu/membrane at the 95% confidence limit. Unlike method 1 and

method 2, the positive esp was detected on membranes with lower cfu (< 10).

38

Table 3-3. Detection of esp gene from naturally occurring enterococci from untreated

sewage using method 3.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

No No. of cfu range/membrane Esp+ Esp- Total
membranes esp+
analyzed (%))‘k

1 14 15-53 4 10 27

2 6 51-1 15 6 0 100

3 5 63-150 3 2 60

4 5 47- 1 98 4 l 80

5 3 125-140 1 2 33

6 7 1-36 4 3 57

7 5 5-70 4 1 80

8 4 . 13-64 2 2 50

9 8 10-1 13 1 7 12

10 5 4-48 3 2 60

1 1 6 4-142 3 3 50

12 5 4-149 2 3 40

13 5 4-TNTC 5 0 100

 

 

 

 

 

 

 

*percentage of membranes positive for esp

39

Figure 3-3. Detection of esp gene in naturally occurring enterococci in the range of the

limit of detection using method 3 (N= 75)‘.

 

N

E§P we

.5

 

,ESP -v.e

CFU I Membrane

 

 

 

*Number of membranes in this figure is 75

4o

3.2.5 Detection of esp Gene in Disinfected Effluent Samples

Ten disinfected effluent samples were collected from the waste water treatment plant in
East Lansing. The first three samples were processed by method 1 and 2 where as the
remaining samples were processed by method 3. The colony forming units were below
the detection limit of the esp marker for majority of the samples due to the disinfection.
Results are shown in Table 3-4. Less volume was filtered through the membrane for
samples one two and three, hence the cfu range was below the detection limit (30). For
samples four thru ten the volume filtered through the membrane was increased to one
liter. Samples six and eight were esp positive with a cfu range between 68 and TNTC
when assayed from a total of 10 membranes. Sample seven was esp negative despite
higher enterococci counts where as samples nine and ten were esp negative with cfu

range below the detection limit (30).

Table 3-4. Detection of esp gene in disinfected effluent samples.

 

 

 

 

 

 

 

 

Method used Membranes cfu range Results Esp +
assayed (%)*
1& 2 5 0-17 - 0
1&2 6 10-16 - 0
3 9 1-21 - 0
3 4 68-100 + 100
3 4 . 54-139 - 0
3 6 >100 + 67

 

 

 

 

 

 

*percentage of membranes positive for esp

41

3.2.6 Persistence of the esp Marker in Sewage

Twenty three samples were assayed for the detection of the esp marker over a year from
August 2005 to March 2006 using method 1 and 2. Raw sewage samples were collected
twice a month. The marker was detected in 100% of the samples processed (22/22) when
the cfu range was above the detection limit (45:1:28). This indicates that the marker was
persistent over the time. All the four membranes were esp negative in only one sample.
The lowest positive membrane fell within the limit of detection for all methods

previously described (Table 3-5).

Table 3-5. Lowest esp positive in raw sewage using method 1, 2‘& 32

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Method Month Lowest positive Range
(cm/membrane) (cfu/membrane)
1&2 Aug-05 23 23-TNTC
1&2 Sept-05 21 0-560
1&2 Nov-05 144 4-500
1&2 Dec-05 1 12 1 12-260
1&2 Jan-06 45 45-120
1&2 Feb-06 37 37-100
3 Feb-06 19 17-1 15
1&2 Mar-06 58 44-160
3 Mar-06 47 63-198
3 Apr-06 2 2-140
3 May-06 4 4-1 1 3
3 June-06 4 4-142
3 J uly-06 4 4-TNTC

 

 

 

 

 

‘Number of membranes tested in method 1&2 was 97
2 Number of membranes tested in method 3 was 78

42

3.2.7 Blind Analysis of the esp Gene
3.2.7.1 Introduction

Blind wastewater and fecal samples were used to evaluate the predictive ability of the
host specific esp marker in conjunction with a 2004 project run by the United States
Geological Survey (USGS) on source sampling for pharmaceuticals. The 2004 USGS
source sampling was conducted to provide a refined estimate on the relative potential
environmental contributions of Organic Wastewater Contaminants (OWC’s) from a
variety of urban and agricultural waste sources. This study was part of the USGS Toxic
Substances Hydrology Program - Source Characterization (2004). The MSU
Environmental Quality Laboratory assisted in the study by analyzing the samples
collected from various animal and human sources by the USGS for the abundance of
Enterococci and for the presence of an E. faecium esp marker (30). The sources were

revealed after the analysis.

3.2.7.2 Results

Method 1 was used to analyze for the esp marker in the USGS Blind samples except for
one sample. Results are shown in Table 3-6 and Table 3-7. Enterococcus concentrations
ranged from <3 to >103 cfu/ lOOmL. The E. faecium esp human marker was found in 6/7
of the human sourced samples with high enterococci counts and 0/6 animal source
samples examined. The human sourced samples were from septic tanks of an elementary
school, restaurant, multi-family home, convenience store, single family household and

from a waste water treatment plant. The non-human sources were from a veterinary

43

hospital, antibiotic-free swine operation, antibiotic intense swine operation, cottage
cheese factory, cattle operation (ranch) and antibiotic free poultry operations.

All of the six septic tank effluent samples analyzed were esp positive (100%).The solid
form of sample was esp negative in 4/6 septic tank samples and 2/2 municipal pond
samples due to the low concentrations of enterococci. Less volume was filtered for the
solid samples and this may be one reason for the low cfu count and hence the absence of
the esp marker. One solid sample with high enterococci counts was also esp negative.
Only one septic tank sample was esp positive for both solid and liquid portions with
TNTC enterococci using method 3. Enterococcus concentrations in three samples from
animal sources were also found below the detection limit (45i28) for the esp marker and
the remaining six samples were negative for the esp marker. The presence of antibiotics

may account for the low microbial numbers in these samples.

44

Table 3-6. Enterococcus concentration and presence of E. faecium esp human pollution

marker in USGS source samples (human).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Source State No. of Esp Esp

samples + -

Septic tank CO 5 5 0

Septic tank NJ 1 1‘ 0
Municipal pond CO 1* NA NA
Municipal pond MN 1* NA NA
Municipal pond IA 1* NA NA

Biosolid NY 1 0 1

‘Analyesd in method 3

* Not analyzed as number of cfu/membrane was below detection limit (45i28).

Table 3-7. Enterococcus concentration and presence of E. faecium esp human pollution

marker in USGS source samples (Non-human).

 

 

 

 

 

 

 

Source State No. of Esp Esp
samples + -
Hog lagoon IA 4 O 4
Cheese factory ID 1* NA NA
Poultry litter VA 1 0 1
Poultry litter OH 2* NA NA
Biosolid (Ranch) TX 1 0 1

 

 

 

 

 

 

*Not analyzed as number of cfu/membrane was below detection limit (45:1:28).

45

3.2.8 Presence of esp Marker in Different Watersheds in US

The marker was tested for in samples collected from different watersheds in US during
the USGS source sample analysis. The marker was present in human samples from
Colorado and New Jersey and the marker was absent in non-human samples from Texas,
Virginia, and Iowa. One sample from Florida, (Katrina area) was esp positive by PCR
and in the sequencing confirmed the presence of the marker. Despite high enterococci
count the marker was absent in the Waste Water Treatment Plant (WWTP) solid sample

from New York. Figure 3-4 shows the different watersheds evaluated and the results.

46

Figure 3-4. Presence of esp marker in different watersheds in US

 

+ States in which samples were positive for esp
. States in which samples were negative for esp.

47

3.2.9 Detection of esp Marker in Non-human Samples

The esp marker was tested using 29 membranes from numerous non-human sources.
Three manure and one swine slurry samples were collected from animal waste points at
the MSU farm. All four samples were negative. Nine bird samples assayed from parks
and lakes were also negative. The bird samples included 2 gulls, 1 goose and 6 duck
feces. All samples were assayed from numerous membranes and some membranes with
cfu well above the detection limit. Eight sand samples collected from the North Beach,
along Lake Michigan in Grand Haven, M1, were processed and were esp negative. These
came from the same area where bird samples were also negative for the esp marker.

Results are shown in Table 3-8.

Table 3-8. Detection of esp marker in non human samples‘

 

 

 

 

 

 

 

No of samples Method Source cfu Range Esp+/-
analyzed
3 1&2 Manure 250-TNTC -
1 1&2 Swine slurry 156 -
2 1&2 Sea gulls 28-TNTC -
6 3 Duck 12-TNTC -
1 3 goose 16-90 -
8 3 Beach sand* 6-125 -

 

 

 

 

 

 

 

‘ 29 membranes tested for esp marker.
* Sand collected from beaches. High enterococci count possibly due to fecal
pollution from birds or wild life.

48

 

CHAPTER 4
DISCUSSION

4.1 Introduction

Microbial Source Tracking is becoming widely used but factors like library size, cost
and complexity of the procedures are limiting application in environmental samples (49).
Understanding the potential of each method before use is also challenging. The recently
published MST guide document by EPA (2005) addresses these difficulties (49).The
literature addressed all source tracking methods that are currently available and compared
the methods with respect to their applicability, methodology and cost. According to the
EPA an ideal MST method should be able to differentiate all the sources of pollution
using a single approach, but none of the current methods meet that criterion (5, 18, 42,
55). So EPA has suggested that approaches involving multiple techniques are suitable for
source tracking, particularly host specific methods that target genes which are highly
specific, having the greatest potential in the future compared to the library based
methods. The EPA guide document can be used as a reference for community managers
to learn about MST methods for addressing the pollution issues, prioritization and
remediation. However because this field is developing rapidly this EPA guide may be out

of date quickly.

4.2 Development of a Rapid Method

The first portion of this research addressed a rapid method for detecting a human

pollution source tracking marker using the enterococcus surface protein (esp). The

49

 

method that was previously developed by Scott et a1. (2005) included a two day
incubation at 41°C and an enrichment step lasting three hours followed by a 3.5 hour
DNA extraction method and then the PCR step for the esp detection (3 8). The time from
sample collection to result was about 3.5 days. The rapid method that was developed in
this study compared to the conventional method obtained results in 1.5 days. By applying
this method modification in the future, labor costs and the cost for manufactured DNA

extraction kits would be reduced.

Developing a rapid method was also seen as important for identifying the sources in a
timely manner, hence community managers could take corrective actions to protect
human health (51). Presence of a sewage marker in a particular water body is a strong
indication of sewage discharges or leaking septic systems in the surrounding area (26,
28). If sewage is the likely source of pollution this suggests a more serious health, alert
should be given to the people engaged in recreational activities in those waters,
particularly young children. The method may be useful for indicating when to close a
beach as opposed to just posting advisories and may be useful for indicating when to
reopen a beach. In addition, by routinely monitoring suspected areas using rapid and
affordable methods, one could address future water quality degradation and prioritize
beachsheds for remediation. The enterococci is currently being used as an indicator
organism and the method for detecting the esp gene allows the laboratories to implement

this method easily complementing what they are already doing (48).

50

There were 97 membranes assayed for the comparison of method 1 and 2 taken from 12
different raw sewage samples. The cfu ranged from zero to TNTC. When the results were
compared there were slightly more positive results from the conventional method
(method 1) compared to the rapid method (method 2). The rapid method (method 2) used
whole bacterial cells as a template for PCR, which is simpler and faster than using
extracted DNA. However, DNA extraction is seen as a critical step for getting a high
quality DNA by removing inhibitory substances like humic acids (42). Thus the negative
esp by the rapid method may be a result of interferences from the material in the mEI
media or from the increased number of cells in the PCR reaction. Some of the esp
positives in the rapid method also showed faint bands indicating that the enzyme

concentration (Taq polymerase) may need to be increased per reaction (9).

While the marker is a clear indication of human fecal contamination, it is also clear that
the marker containing strain is not always excreted by healthy human beings (24, 56, 57).
Thus more membranes or larger volumes of water samples from the suspected areas may
need to be processed with mixtures of enterococci. This study was mainly based on the
raw sewage sample, hence the bacterial concentrations and the presence of the marker in
the natural aquatic environment should be lower because of dilution (54). Thus as with

many environmental monitoring programs proving the negative is difficult.
Molecular sequencing of positive samples allowed for greater confidence that the PCR

reaction was specific and that human sewage was present. This was done with the raw

sewage and the positive environmental sample from the Katrina affected area. A similar

51

approach will be important proof for other samples in the future for further identification
of the specificity of the marker. Some of the host specific markers that have been used for
MST methods previously have shown geographic and host- impacted diet variations (20,
22, 40). The esp has been detected from numerous areas across the US. In future
sequence analysis in a national survey would be beneficial to examine the distribution in

different geographic regions.

Method 3 concentrated on removing the enrichment step by taking viable cells from the
membrane and using this as a template for the PCR. This variation in the method
increased the sensitivity, and detection of the esp marker was below the detection limits
(26i40) of methods 1 and 2. The marker was detected from membranes containing as
few as 2 cfu. However this method failed to detect the marker in one membrane from
sewage with enterococci counts of 140. One reason for this result could be the PCR
inhibitors which may have prevented the amplification. Removing PCR inhibitors from
environmental samples is challenging without DNA extraction. By selecting optimizing

enzyme concentrations inhibitors may overcome this to some extent (1).

Faster methods for MST targeting specific genes include Quantitative PCR (QPCR) and
do not require live cells for cultivation. The advantage of this approach is that this
method could detect past contamination in the environment (51) and possibly provide
results within several hours. A study conducted by Lleo et a1. (2005) pointed out that 50
% of the ground water samples tested (n=30) contained nonculturable cells for E. faecalis
(27). Thus molecular tests are able to detect the nonculturable cells along with the viable

cells. In this study the method targeted the human pollution in viable cells only because

52

the objective of the research was to identify recent pollution and address adequately
treated and disinfected sewage. Wastewater that was adequately treated contained low
enterococci counts and no esp marker was found using the methods in this study unless

the numbers of cfu increased.

4.2.1 Solid Samples

The majority of the solid samples tested were esp negative. Most of the time the
concentration of the enterococci was below detection limits for the esp. The only solid
sample that was esp positive was a sample from a septic tank (Table 2-7) from the USGS
source. That sample was tested using method 3. While processing sediment/solid type
samples, the presence of a dark background due to the solids in the membrane may
prevent the correct enumeration of CFU. This can be avoided by dilution and filtering
smaller volumes for the enumeration and filtering larger volumes in replicate for testing
the sample for the esp marker. The survival of the bacteria containing the marker has
been evaluated in only one experiment to date in fresh and marine water. Further studies
are also needed to better understand the persistence of the sub-population of enteroccci

containing the marker particularly in solids and sediments (3 8).

4.2.2 Presence or Absence of the Marker

This study reconfirmed the absence of the marker in animal wastes. However,
interpretation of the esp result in the environment should be done with the concept that a
negative result does not guarantee a true negative, where as a positive indicates that at

least some of the pollution is from humans. The reasons for obtaining negative results

53

may be a low concentration of enterococci in that particular sample or the absence of
marker in the sample. Absence of the marker may indicate that there are other pollution
sources other than from humans in the area, so further investigation needs to be done to
pinpoint the sources. There are no methods that warrant complete resolution for tracking
all the sources of contamination. Forensic approaches which match exactly isolates
would mean thousands of samples and isolates using methods like QPCR, AF LP, PFGE,
T- RF LP which is not affordable for most of the communities(49). Many samples and
more than one test need to be included in any source tracking study. A “toolbox approach
“(EPA 2005) has been recommended. The success of the application of library dependent
and library independent source tracking methods depends on the geographic stability of
the marker and the size of the library. Several studies were able to pinpoint the source of
pollution in smaller watersheds, where as the accuracy of the method was lower when
compared to broad geographic regions(19, 20, 39). Thus more studies are needed to
quantify the relative numbers of esp positive samples to esp negative in various types and
sizes of water bodies. A combination of different and inexpensive methods is
approachable for the community managers when they deal with environmental quality
issues and it is concluded by this study that the Rapid esp method can be included as one

of those.

Unlike contamination from other sources, human fecal contamination in the
environment has a direct and adverse impact on human health(15). Human fecal
contamination and sewage are generally considered to be of a higher risk because it is

possible that the wastes contain more bacteria, viruses and parasitic pathogens. However,

54

wastewater treatment can be implemented, changed and optimized, correcting the
problem effectively compared to control of birds or wildlife fecal contamination. The
EPA source tracking guide compared 8 field applications ((1) Targeted sampling and
Enterococcus speciation- Georgia, (2) ARA with fecal colifonns, ribotyping with E. coli,
and human pathogenic enterovirus detection-Tampa, Forida.(3),Rep-PCR with E. coli,-
Minnesota, (4) ARA and PFGE with enterococci- Maryland,(5) Two-enzyme ribotyping
with E. coli.- Virginia, (6) Host-specific Bacteroides/Prevotella markers and human-
pathogenic enterovirus detection- California, (7) ARA with E. coli- Virginia, (8) F+
RNA coliphage genotyping- Florida) using MST methods, but follow up studies and
remediation steps were undertaken only with three studies, where the major contributor
of the pollution was human and cattle(49). In other case studies the major contributors of
the pollution were birds and wildlife, so no implementation was taken in any of these
watersheds. One major implementation based on the Virginia study (case no.5) was
fencing along the river side where the main source of pollution was found to come from
cattle. A corrective measurement of slip-lining the sewer lines that run along the beach
were taken based on the Host-specific Bacteroides/Prevotella markers and human-
pathogenic enterovirus detection study in Avalon Bay, California. In the Virginia study
(case no.7) human sources were the dominant cause of the pollution; hence a septic
system maintenance project was undertaken because all of the homes used septic systems
and wells in the area where the study was conducted. These case studies demonstrate the

usefulness of the MST tool box.

55

4.2.3 Marker Testing

The lack of a single genetic marker to differentiate all possible sources of pollution, and
the geographic variability of many markers are some of the limitations when considering
MST. The esp marker has been tested in samples from different geographical regions. All
liquid wastewater samples from humans thus far tested from Colorado, Florida and
Michigan were also esp positive. The marker was absent in the non human sources from
Idaho, Ohio, Virginia and Iowa. McDonald et al. (2006) were able to detect the esp
marker in environmental samples from Georgia watershed (28). More samples from other

geographical regions need to be evaluated.

This study focused on a rapid method for the evaluation of raw sewage. More
environmental samples need to be tested for the esp marker using methods 2 and 3. When
dealing with samples of low concentrations of enterococci, method 3 may be the most
appropriate because methods 1 and 2 failed to detect positives from membranes with <20
cfu. More membranes with higher cfu (>100) need to be evaluated in method 3 to explore
the role of inhibitors in preventing the amplification. The esp marker should be evaluated
in more geographical regions. More sediment samples also need to be processed and the
stability of the marker in the environment including ponds, lakes and sludge need to be

further investigated.

Sewage discharges including CSOs are still one of the leading causes of human
pathogens in recreational waters and adversely affecting the people’s health who engage

activities in those waters. This research targeted development of a method which is

56

comparatively faster than a conventional method developed for detecting the human
pollution marker esp (3 8). The research concentrated on the esp marker present in sewage
to better define the prevalence of this gene in the human population and in community
sewage. Additional research needs to be undertaken to examine how the esp marker
behaves in the environment. The esp positive environmental sample from New Orleans
was collected two months after Hurricane Katrina and demonstrated that the marker can
persist up to two months in the environment. The absence of the marker in all non-human
sources and the geographic stability of the marker are promising. A more comprehensive
study with targeted sampling in problem areas would increase the understanding of the
survival of the marker in the environment and the effectiveness of the rapid test. Case

studies before and after remediation should be undertaken.

Routine monitoring and targeted sampling are ways to identify a water pollution
problem before affecting public health. The MST methods have been found to be very
helpful for finding the root cause of the pollution. Enterococci is already being used as a
water quality indicator by EPA and by many States. By adding the esp detection test after
assessing the water quality one can begin to address more definitively the role of sewage
as a source of waterbody impairment. Based on this study, the rapid method is as
effective as the conventional method, is cheaper and less time consuming. Using the
traditional indicator enterococci for water quality and then testing membranes with high
enterococci counts (:20) for the esp marker using the rapid method is a simple approach
for tracking a human source of contamination. Public health agencies that monitor

beaches, as well as water utilities that are concerned with drinking water supplies should

57

find this method useful. In addition such as the case in New Orleans, the recovery and
identification of hot spots of areas where sewage contamination remained risky could be

monitored for.

58

Table A-1. Method Comparison using raw sewage

APPENDIX 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Filtered esp
No ID Date Volume cfu‘ +/- Method
1 RW1-1 8/10/2005 10p| 23 + 1
2 RW 1-1 8/10/2005 10pl 23 + 2
3 RW 1-2 8/10/2005 50pl 136 + 1
4 RW 1-2 8/10/2005 50pl 136 + 2
5 RW 1-3 8/10/2005 100111 270 + 1
6 RW 1-3 8/10/2005 100p| 270 + 2
7 RW 14 8/10/2005 1 ml TNTC + 1
8 RW 1-4 8/10/2005 1 ml TNTC + 2
9 RW 2-1 9/7/2005 10111 38 - 1
10 RW 2-1 9/7/2005 10111 38 - 2
11 RW 2-2 9/7/2005 10p| 44 - 1
12 RW 2-2 9/7/2005 10111 44 - 2
Dilution2
1 3 RW 2-3 9/7/2005 103 9 - 1
14 RW 2-3 9/7/2005 10'3 9 - 2
15 RW 2-4 9/7/2005 10'3 6 - 1
16 RW 2-4 9/7/2005 10'3 6 - 2
17 RW 2-5 9/7/2005 10'4 0 - 1
18 RW 2-5 9/7/2005 10'4 0 - 2
19 RW 2-6 9/7/2005 10'5 1 - 1
20 RW 2-6 9/7/2005 10'5 1 - 2
Volume
21 RW 3-1 9/21/2005 5011' 560 + 1
22 RW 3-1 9/21/2005 50111 560 + 2
23 RW 3-2 9/21/2005 10pI 121 + 1
24 RW 3-2 9/21/2005 1Cyl 121 + 2
Dilution
25 RW 3-3 9/21/2005 10'3 19 - 1
26 RW 3-3 9/21/2005 10'3 19 - 2
27 RW 3-4 9/21/2005 103 21 + 1
28 RW 3-4 9/21/2005 10'3 21 + 2
Volume
29 RW 4-1 1 1/2/2005 50111 500 + 1
30 RW 4-1 1 1/2/2005 50p| 500 + 2
31 RW 4-2 1 1/2/2005 10p| 98 - 1
32 RW 4-2 1 1/2/2005 10p| 98 — 2
Dilution
33 RW 4-3 1 1/2/2005 10'3 22 - 1
34 RW 4-3 1 1/2/2005 10'3 22 - 2
35 RW 4-4 11/2/2005 10'4 12 - 1
36 RW 4-4 1 1/2/2005 10'4 12 - 2
37 RW 5-1 11/15/2005 10p| 280 + 1

 

59

 

Table A-1 continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

38 RW 5-1 11/15/2005 10p| 280 + 2
39 RW 5-2 1 1/15/2005 10pI 280 + 1
40 RW 5-2 1 1/15/2005 10p| 280 + 2
41 RW 5-3 11/15/2005 10'2 144 + 1
42 RW 5-3 1 1/15/2005 10"2 144 + 2
43 RW 54 1 1/15/2005 10'3 20 - 1
44 RW 5-4 1 1/15/2005 10'3 20 - 2
45 RW 5-5 1 1/15/2005 10'4 4 - 1
46 RW 5-5 1 1/15/2005 10'4 4 - 2
47 RW 10-1 1/18/2006 10'2 50 - 1
48 RW 10-1 1/18/2006 10'2 50 - 2
49 RW 10-2 1/18/2006 10'2 51 - 1
50 RW 10-2 1/18/2006 10'2 51 - 2
51 RW 10-3 1/18/2006 10'2 52 - 1
52 RW 1 0-3 1/18/2006 10'2 52 - 2
53 RW 10-4 1/18/2006 10'2 49 + 1
54 RW 10-4 1/18/2006 102 49 + 2
55 RW 10-5 1/1 8/2006 10'2 60 + 1
56 RW 1 0-5 1l18/2006 102 60 + 2
57 RW 10-6 1/18/2006 10'2 57 - 1
58 RW 10-6 1/18/2006 10'2 57 - 2
59 RW 10-7 1/18/2006 10'2 54 + 1
60 RW 10-7 1/1 8/2006 10'2 54 + 2
61 RW 10-8 1/18/2006 10'2 58 - 1
62 RW 10-8 1 I 1 8/2006 10'2 58 - 2
63 RW 1 0-9 1/1 8/2006 102 56 - 1
64 RW 1 0-9 1/1 8/2006 102 56 - 2
65 RW 10—1 0 1l18/2006 10'2 45 - 1
66 RW 10-10 1/18/2006 10'2 45 + 2
67 RW 10-1 1 1/18/2006 10'2 46 + 1
68 RW 10-1 1 1/18/2006 10'2 46 + 2
69 RW 10-12 1l18/2006 10'2 59 + 1
70 RW 10-12 1/18/2006 10'2 59 + 2
71 RW 10-13 1l18/2006 10’2 69 - 1
72 RW 10-13 1/18/2006 10'2 69 - 2
73 RW 10-14 1l18/2006 102 64 - 1
74 RW 10-14 1/18/2006 102 64 - 2
75 RW 10-15 1/18/2006 10'2 62 - 1
76 RW 10-15 1/18/2006 10'2 62 - 2
77 RW 10-16 1/18/2006 10'2 63 + 1
78 RW 10-16 1/18/2006 10'2 63 - 2
79 RW 10-17 1/18/2006 102 65 - 1
80 RW 10-17 1l18/2006 10'2 65 - 2

 

 

 

 

 

 

 

60

 

Table A-1 continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

81 RW10-18 1/18/2006 10'2 68 + 1
82 RW 10-18 1/18/2006 10’2 68 + 2
83 RW 10-19 1/18/2006 10-2 67 - 1
84 RW 10-19 1/18/2006 10"2 67 - 2
85 RW 10-20 1/18/2006 10'2 66 + 1
86 RW 10-20 1/1 8/2006 10-2 66 + 2
87 RW 10-21 1/18/2006 10'2 75 + 1
88 RW 10-21 1/18/2006 10'2 75 - 2
89 RW 10-22 1/18/2006 10'2 72 - 1
90 RW 10-22 1/18/2006 102 72 - 2
91 RW 10-23 1/18/2006 10'2 71 + 1
92 RW 10-23 1l18/2006 10’2 71 - 2
93 RW 10-24 1/18/2006 10'2 70 + 1
94 RW 10-24 1/1 8/2006 10'2 70 + 2
95 RW 10-25 1/18/2006 10'2 74 + 1
96 RW 10-25 1/18/2006 102 74 + 2
97 RW 10-26 1/1 8/2006 10'2 78 + 1
98 RW 10-26 1/18/2006 10'2 78 + 2
99 RW 10-27 1/18/2006 10'2 77 - 1
100 RW 10-27 1/18/2006 102 77 - 2
101 RW 10-28 1/18/2006 10"2 73 - 1
102 RW 10-28 1/18/2006 10"2 73 - 2
103 RW 10-29 1/18/2006 10'2 84 + 1
104 RW 1029 1/18/2006 10'2(2ml) 84 + 2
105 RW 10-30 1/18/2006 10'2(2ml) 85 + 1
106 RW 10-30 1l18/2006 10'2(2ml) 85 + 2
107 RW 10-31 1/18/2006 10'2(2mI) 83 - 1
108 RW 10-31 1/18/2006 10'2(2m|) 83 - 2
109 RW 1 0-32 1/18/2006 10'2(2m|) 88 + 1
1 10 RW 10-32 1l18/2006 10'2(2m|) 88 + 2
1 11 RW 10-33 1/18/2006 10'2(2m|) 81 - 1
112 RW 10—33 1/18/2006 10'2(2ml) 81 - 2
113 RW 10-34 1/18/2006 10'2(2ml) 89 - 1
1 14 RW 10-34 1/18/2006 1072(2ml) 89 - 2
1 15 RW 10-35 1/18/2006 10'2(2mI) 86 + 1
1 16 RW 10-35 1/18/2006 10'2(2m|) 86 + 2
117 RW 10-36 1/18/2006 10'2(2ml) 82 + 1
1 18 RW 10-36 1/18/2006 10'2(2m|) 82 + 2
119 RW 10-37 1/18/2006 10'2(2m|) 96 + 1
120 RW 10-37 1/18/2006 10'2(2m|) 96 - 2
121 RW 10-38 1/18/2006 10'2(2ml) 91 - 1
122 RW 10-38 1/18/2006 10'2(2ml) 91 - 2
123 RW 10-39 1/18/2006 10'2(2ml) 90 + 1
124 RW 10-39 1/18/2006 10‘2(2ml) 90 - 2

 

 

 

 

 

 

 

 

61

 

Table A-1 continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

125 RW 1040 1/18/2006 10'2(3m|) 93 + 1
126 RW 1040 1718/2006 10'2(3ml) 93 + 2
127 RW 1041 1/18/2006 10‘2(3ml) 92 - 1
128 RW 1041 1/18/2006 10'2(3m|) 92 + 2
129 RW 1042 1718/2006 10'2(4m|) 97 - 1
130 RW 1042 171872006 101ml) 97 - 2
131 RW 1043 171872006 10‘2(5ml) 106 - 1
132 RW 1043 171872006 10'2(5m|) 106 - 2
133 RW 1044 1/18/2006 10'2(5ml) 110 + 1
134 RW 1044 171872006 10'2(5m1) 110 + 2
135 RW 1045 1/18/2006 10'2(5ml) 101 — 1
136 RW 1045 1/18/2006 10'2(5ml) 101 - 2
137 RW 1046 171872006 10’2(5ml) 117 - 1
138 RW 1046 171872006 10’2(5m|) 117 + 2
139 RW 1047 171872005 10'2(5ml) 120 + 1
140 RW 1047 1/18/2006 10'2(5ml) 120 + 2
141 RW 11-1 271372006 102 41 - 1
142 RW 114 271372006 102 41 - 2
143 RW 11.2 271372006 102 52 - 1
144 RW 11.2 271372006 102 52 - 2
145 RW 11-3 271372006 102 65 - 1
146 RW 11-3 271372006 102 65 - 2
147 RW 114 271372006 102 54 + 1
148 RW 114 271372006 102 54 + 2
149 RW 1 15 271372006 102 53 + 1
150 RW 115 271372006 102 53 - 2
151 RW 11-6 271372006 102 48 - 1
152 RW 115 271372006 102 48 - 2
153 RW 11-7 271372006 102 58 - 1
154 RW 1 1-7 271372006 102 58 - 2
155 RW 11-8 271372006 102 51 - 1
156 RW 11-8 271372006 102 51 - 2
157 RW 11-9 271372006 102 37 + 1
158 RW11-9 271372006 102 37 + 2
159 RW 1 1-10 271372006 102 42 - 1
160 RW 1 1-10 271372006 102 42 - 2
161 RW 1141 271372006 102 44 - 1
162 RW 1 1-1 1 271372006 102 44 - 2
163 RW 11-12 271372006 102 38 + 1
164 RW 11-12 271372006 102 38 + 2
165 RW 11-13 271372006 102 43 - 1
166 RW 1143 2/13/2006 102 43 - 2
167 RW 11-14 2/13/2006 102 36 - 1
168 RW 1 144 271372006 102 36 - 2

 

 

 

 

 

 

 

62

 

Table A-1 continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

169 RW12-1 2/24/2006 10'2(1.7mD 100 + 1
170 RW12-1 2/24/2006 10'2(1.7m|) 100 + 2
171 RW12-2 2/24/2006 10'2 60 + 1
172 RW12-2 2/24/2006 10'2 60 + 2
173 RW12-3 2/24/2006 10'2 48 - 1
174 RW12-3 2/24/2006 102 48 + 2
175 RW12-4 2/24/2006 10'2 39 + 1
176 RW12-4 2/24/2006 10'2 39 + 2
177 RW12-5 2/24/2006 10'2 70 + 1
178 RW12-5 2/24/2006 10'2 70 + 2
179 RW13-1 3/7/2006 10'2(2ml) 126 + 1
180 RW13-1 3/7/2006 10'2(2m|) 126 + 2
181 RW13-2 3/7/2006 10'242ml) 140 + 1
182 RW13-2 3/7/2006 10'2(2ml) 140 + 2
183 RW13-3 3/7/2006 10'2 73 + 1
184 RW1 3-3 3/7/2006 10"2 73 + 2
185 RW13-4 3/7/2006 10'2 58 + 1
186 RW13-4 3/7/2006 10'2 58 + 2
187 RW14-1 373172006 10'2(2ml) 44 - 1
188 RW14-1 3/31/2006 10'2le) 44 - 2
189 RW14-2 3/31/2006 10'2(2m|) 75 - 1
190 RW14-2 3/31/2006 10'2le) 75 - 2
191 RW14-3 3/31/2006 10'2(2m|) 92 - 1
192 RW14-3 3/31/2006 10'2(2ml) 92 - 2
193 RW14-4 3/31/2006 10'1(2m|) 160 + 1
194 RW14-4 3/31/2006 10'1(2m|) 160 + 2
‘cfu/membrane

2 In all diluted samples lmL of the volume was filtered unless noted otherwise

63

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table A-2. Method 2 only
Sample Filtered
No ID Date Volume cfu‘ Esp+/- Method
1 RW 6-1 11/30/2005 101.11 117 + 2
2 RW 6-2 11/30/2005 10111 109 + 2
Dilution2
3 RW 6-3 11/30/2005 103 17 - 2
4 RW 6-4 1 1/30/2005 103 26 - 2
5 RW 7-1 12/21/2005 101 260 + 2
6 RW 7-2 12/21/2005 10'2 151 + 2
7 RW 7-3 12/21/2005 102 1 12 + 2
8 RW 8-1 1/3/2006 10'2 124 + 2
9 RW 8-2 1/3/2006 10'3 34 - 2
1O RW 8-3 1/3/2006 10'3 19 - 2
11 RW 9-1 1/12/2006 10'2 29 + 2
12 RW 9-2 1/12/2006 10'2 30 - 2
Volume
13 RW 9-3 1/12/2006 50111 31 + 2
14 RW 9-4 1/12/2006 50111 34 - 2
Dilution
15 RW 9-5 1/12/2006 10'1 77 - 2
16 RW 9-6 1/12/2006 10'1 90 + 2
‘cfu/membrane

2 In all diluted samples lmL of the volume was filtered unless noted otherwise

64

 

Table A-3. Method Comparison using environmental samples

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Filtered
No Sample ID Date Volume cfu‘ Method esp+/-
1 MSU Farm 12 9/27/2005 50ml TNTC 1 +
2 MSU Farm 12 9/27/2005 50ml TNTC 2 +
MSU Farm
3 12(duplicate) 9/27/2005 50ml TNTC 1 +
MSU Farm
4 12(duplicate) 972772005 50ml TNTC 2 +
5 MSU Farm 15 9/27/2005 50ml TNTC 1 --
6 MSU Farm 15 9/27/2005 50ml TNTC 2 -
7 MSU Farm 1 9/29/2005 60ml 33 1 --
8 MSU Farm 1 9/29/2005 60ml 33 2 --
9 MSU Farm WSP 15 11/9/2005 10ml 84 1 -
10 MSU Farm WSP 15 11/9/2005 10ml 84 2 -
11 MSU Farm WSP 1 11/9/2005 50ml 115 1 -
12 MSU Farm WSP 1 11/9/2005 50ml 115 2 -
13 MSU Farm WSP 15 11/17/2005 10ml >250 1 --
14 MSU Farm WSP 15 11/17/2005 10ml >250 2 -
15 MSU Farm WSP 1 11/17/2005 10ml 58 1 -
16 MSU Farm WSP 1 11/17/2005 10ml 58 2 -
17 MSU Farm WSP 12 A 01/12/06 150ml 15 1 --
18 MSU Farm WSP 12 A 01/12/06 150ml 15 2 -
19 MSU Farm WSP 12 A 01/12/06 100ml 11 1 -
20 MSU Farm WSP 12 A 01/12/06 100ml 11 2 --
21 MSU Farm WSP 12 A 01/12/06 100ml 10 1 —
22 MSU Farm WSP 12 A 01/12/06 100ml 10 2 -
23 MSU Farm WSP 12 B 01/12/06 10ml 48 1 -
24 MSU Farm WSP 12 B 01/12/06 10ml 48 2 -
25 MSU Farm WSP 12 3 01/12/06 50ml 150 1 -
26 MSU Farm WSP 12 3 01/12/06 50ml 150 2 -
27 MSU Farm WSP 12 8 01/12/06 100ml TNTC 1 -
28 MSU Farm WSP 12 8 01/12/06 100ml TNTC 2 -
29 MSU Farm WSP 12 3 01/12/06 150ml TNTC 1 --
30 MSU Farm WSP 12 3 01/12/06 150ml TNTC 2 -
31 Randall House 9/26/2005 350ml 189 1 -
32 Randall House 9/26/2005 350ml 189 2 -
33 Randall House 9/26/2005 250ml 131 1 -
34 Randall House 9/26/2005 250ml 131 2 -
35 Randall House 9/26/2005 100ml 67 1 --
36 Randall House 9/26/2005 100ml 67 2 --
‘cfu/membrane

65

 

Table A-4. Method Comparison using Environmental samples

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Filtered
No Sample ID Date Volume cfu‘ Method esp+/-
1 Gulf sample 9/30/2005 160ml 37 1 -—
2 Gulf sample 9/30/2005 160ml 37 2 -
3 NO#1 Water 10l19/2005 100ml 44 1 -
4 NO#1 Water 10/19/2005 100ml 44 2 -
5 NO#2 Water 10/19/2005 100ml 222 1 -
6 NO#2 Water 10/19/2005 100ml 222 2 -
7 NO#3 Water 10/19/2005 100ml 11 1 -
8 NO#3 Water 10/19/2005 100ml 11 2 --
9 NO#4 Water 10/19/2005 100ml 22 1 -
1O NO#4 Water 10/19/2005 100ml 22 2 --
11 NO#5 Water 10l19/2005 100ml 16 1 -
12 NO#5 Water 10/19/2005 100ml 16 2 --
13 NO#8 Water 10l19/2005 100ml 29 1 --
14 NO#B Water 10/19/2005 100ml 29 2 -
15 NO#9 Water 10/19/2005 100ml 289 1 -
16 NO#9 Water 10/19/2005 100ml 289 2 -
17 NO#1O Water 10/19/2005 100ml 121 1 +
18 NO#10 Water 10/19/2005 100ml 121 2 +
NO#1O Water
19 (duplicate) 10/19/2005 100ml 121 1 +
NO#1O Water
20 (duplicate) 10/19/2005 100ml 121 2 +
21 NO#11 Water 10/19/2005 100ml 245 1 -
22 NO#11 Water 10/19/2005 100ml 245 2 --
23 LPOO4Sediments 10/19/2005 100ml 42 1 -
24 LPOO4Sediments 10/19/2005 10ml 42 2 -
25 LP005 NIA 10ml 1 1 --
26 LP005 NIA 10ml 1 2 --
‘cfu/membrane

66

 

Table A-5. esp validation on non human samples

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

No Source Date cfu‘ Method esp+/-
MSU Farm
1 swine slurry 10/27/2005 1 56 1 --
2 MSU Farm swine slurry 10/27/2005 156 2 —
3 MSU Farm Manure 10/27/2005 250 1 -
4 MSU Farm Manure 10/27/2005 250 2 --
5 Manure S P 11/3/2005 TNTC 1 -
6 Manure S P 11/3/2005 TNTC 2 -
7 Manure C P 11/3/2005 TNTC 1 -
8 Manure C P 11/3/2005 TNTC 2 -
9 008 sea gull feces 12/16/05 38 2 -
10 008 sea gull feces 12/15/05 45 2 -
11 008 sea Mfeces 12/ 15105 TNTC 2 -
12 005 sea gull feces 12/15/05 28 2 -
13 005 sea gull feces 12/16/05 41 2 -
14 005 sea gull feces 12/ 16105 TNTC 2 -
1 5 Goose feces 3/1 5/06 90 3 -
16 Goose feces 3/1 5/06 82 3 -
17 Goose feces 3/ 1 5/06 16 3 -
18 F/D/002 Duck feces 3/3/2006 133 2 -
19 FID/002 Duck feces 3I3/2006 143 1 -
20 F/D/002 Duck feces 3/3/2006 143 2 --
21 F/D/002 Duck feces 3/3/2006 12 1 —
22 F/D/002 Duck feces 3/3/2006 12 2 -
23 F/D/006 Duck feces 3/3/2006 135 3 -
24 F/D/008 Duck feces 3/3/2006 103 3 --
25 F/D/007 Duck feces 3/3/2006 44 3 -
26 F/D/010 Duck feces 3/3/2006 100-200 3 -
26 F/D/004 Duck feces 3/3/2006 TNTC 3 --
27 SU-U2-4 Sand 7/10/2006 45 3 -
28 SU-L-12 Sand 7l10/2006 125 3 -
29 SU-U2-12 Sand 7/10/2006 35 3 --
30 SU-B1-12 Sand 7/10/2006 25 3 -
31 SU-L-8 Sand 7/10/2006 6 3 -
32 SU-B1-12 Sand 7/10/2006 25 3 -
33 SU-U2-4 Sand 7/10/2006 47 3 --
34 SU-B2-12 Sand 7l10/2006 20 3 -
‘cfu/membrane

67

 

Table A-6. Preliminary data without enrichment step

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Filtered
No Sample ID Date Volume cfu‘ esp +/- Method
1 RW11-15 2/14/2006 250111 6+5+9 - 3
2 RW 1 1 -16 2/14/2006 400111 15+6 + 3
3 RW 11-17 2/14/2006 250g 8+4+3 - 3
4 RW 11-18 2/14/2006 500111 17 - 3
5 RW 11 -1 9 2/14/2006 400111 19 + 3
6 RW 11-20 2/14/2006 400111 18 - 3
7 RW 11-21 2/14/2006 750111 35 - 3
8 RW 1 1-22 2/14/2006 750111 32 - 3
9 RW 11-23 2/14/2006 7501.11 22 - 3
10 RW 11-24 2/14/2006 750111 22 - 3
11 RW 11-25 2/14/2006 750111 28 - 3
12 RW 11-26 2/14/2006 750111 31 - 3
Dilution2
13 RW 11-27 2/14/2006 10'2 53 + 3
14 RW 11-28 2/14/2006 10'2 38 + 3
15 RW12-1 2/24/2006 10'2 73 + 3
16 RW12-2 2/24/2006 10'2 57 + 3
17 RW12-3 2/24/2006 10'2 51 + 3
18 RW12-4 2/24/2006 10'2 63 + 3
19 RW12-5 2/24/2006 10'2(1.7m1) 110 + 3
20 RW12-6 2/24/2006 10'2(1.7m|) 115 + 3
21 RW13-1 3/7/2006 10'2 63 + 3
22 RW13-2 3/7/2006 10'2 72 - 3
23 RW13-3 3/7/2006 10'2 80 - 3
24 RW13-4 37772006 10'2(2m1) 150 + 3
25 RW13-5 3/7/2006 10'2(2m|) 161 + 3
26 RW14-1 3/31/2006 10’2(2ml) 47 + 3
27 RW14-2 3/31/2006 10'2(2m|) 71 + 3
28 RW14-3 3/31/2006 10'1(2ml) 198 + 3
29 RW14-4 3/31/2006 10'1(2ml) 180 + 3
30 RW14-5 3/31/2006 10‘2(2ml) 67 - 3
31 RW15-1 4/18/2006 10'2(25011|) 125 + 3
32 RW15-3 4/18/2006 10'2(25011|) 126 - 3
33 RW15-4 4/18/2006 10'2(25011|) 140 - 3
34 RW16-1 4/19/2006 10'21500111) 36 - 3
35 RW16-2 4/19/2006 10'3 3 + 3
36 RW16-3 4/19/2006 10'3 9 + 3
37 RW16-4 4/19/2006 10'2(50011|) 28 + 3
38 RW16-5 4/19/2006 103 2 - 3
39 RW16-6 4/19/2006 10'3 1 - 3
40 RW16-7 4/19/2006 10'3 6 + 3
41 RW17-1 4/20/2006 10'A3ml) 70 + 3

 

68

 

Table A-6 continued

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

42 RW17-2 4/20/2006 10'2 55 + 3
43 RW17-3 4/20/2006 10'3 2 + 3
44 RW17-4 4/20/2006 10'2 43 + 3
45 RW17-5 4/20/2006 103 5 - 3
46 RW18-1 4/21/2006 10'3(2ml) 13 - 3
47 RW18-2 4/21/2006 10'2 64 + 3
48 RW18-3 4/21/2006 10'3(2m|) 14 - 3
49 RW18-4 4/21/2006 1 O'2(50011|) 39 + 3
50 RW19-1 5/25/2006 10'3(2m|) 1 1 - 3
51 RW19-2 5/25/2006 1073(3ml) 25 - 3
52 RW19-3 5/25/2006 102 69 - 3
53 RW19-4 5/25/2006 10'3 5 - 3
54 RW19-5 5/25/2006 10'3(3ml) 22 - 3
55 RW19—6 5/25/2006 10’2(2ml) 113 - 3
56 RW19-7 5/25/2006 10'2(2m1) 89 + 3
57 RW19-8 5/25/2006 10'3(2m|) 10 - 3
58 RW 20-1 5/31/2006 10'2(2m1) 42 - 3
59 RW 20-2 5/31/2006 10’3fiml) 7 - 3
60 RW 20-3 5/31/2006 10'2(2ml) 48 + 3
61 RW 20-4 5/31/2006 10'3(3ml) 4 + 3
62 RW 20-5 5/31/2006 10'2(2m|) 19 + 3
63 RW 21 -1 6/8/2006 10'3(1 ml) 4 + 3
64 RW 21-2 6/8/2006 101(500119 120 - 3
65 RW 21 -3 6/8/2 006 10'2 43 + 3
66 RW 21 -4 6/8/2006 10'1(500111) 142 + 3
67 RW 21 -5 6/8/2 006 1072 49 - 3
68 RW 21 -6 6/8/2006 10'3(2m|) 19 — 3
69 RW 22-1 6/1 312006 10'1 (500111) 106 - 3
70 RW 22-2 6/13/2006 10'2 45 - 3
71 RW 22-3 6/13/2006 102 29 + 3
72 RW 22-4 6/13/2006 10'3(1 ml) 4 + 3
73 RW 22-5 6/13/2006 10'1 (500111) 1 18 - 3
74 RW 23-1 7/25/06 10'2 101 + 3
75 RW 23-1 7/25/06 10'2 102 + 3
76 RW 23-1 7/25/06 10'1 TNTC + 3
77 RW 23-1 7/25/06 1m| TNTC + 3
78 RW 23-1 7/25/06 10'4 4 + 3
‘cfu/membrane

2 In all diluted samples lmL of the volume was filtered unless noted otherwise

69

 

Table A-7. Detection of esp gene in disinfected Effluent samples.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sample Filtered Date cfu/100mL esp Method
No volume
1A 50ml 8/11/05 4 - 1&2
lB 20ml 8/1 1/05 1 - 1&2
2A 100ml 09/08/05 0 - 1&2
ZB 200ml 09/08/05 0 - 1&2
3A 100ml 09/30/05 4 - 1&2
3B 200ml 09/30/05 9 - 1&2
3C 200ml 09/30/05 17 - 1&2
4A 1L 09/07/06 17 - 2
48 IL 09/07/06 14 - 2
4C 1L 09/07/06 19 - 2
4D 1L 09/07/06 15 - 2
4E 1L 09/07/06 13 - 2
4F 1L 09/07/06 21 - 2
5A 1L 09/08/06 10 - 2
5B 1L 09/08/06 14 - 2
5C 1L 09/08/06 21 - 2
5D 1L 09/08/06 1 1 - 2
5E 1L 09/08/06 16 - 2
SF 1L 09/08/06 10 - 2
6A 2L 09/09/06 TNTC + 2
6B 2L 09/09/06 TNTC + 2
6C 2L 09/09/06 TNTC + 2
6D 1.5L 09/09/06 TNTC + 2
6B 1 .5L 09/09/06 TNTC - 2
6B 1 .5L 09/09/06 TNTC - 2
7A 1L 09/25/06 1 - 2
8A 1L 09/26/06 8 - 2
88 IL 09/26/06 16 - 2
9A 1L 10/03/06 83 + 2
9B 1L 10/03/06 78 + 2
9C 1L 10/03/06 68 + 2
9D 1L 10/03/06 100 + 2
10A 1.5L 10/04/06 139 - 2
108 1L 10/04/06 56 - 2
10C 1L 10/04/06 65 - 2
10D 1L 10/04/06 54 - 2

 

70

 

APPENDIX 2

Esp gene sequence of positive control, Katrina sample and raw sewage.

EspATCC
EspKatrtna
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatr1na
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawSewage

EspATCC
EspKatrina
EspRawaewage

EspATCC
EspKatrtna
EspRawSewage

1

Cttfgattct
crr:gar:cc
Ctttgatfc:

51

atggaacagg
atggaacagg
atggaacagg

101

gacgataaag
gacgaraaag
gacgaraaag

151

attagaagaa
attagaagaa
attagaagaa

201

'ccacgagtt
tccacgagft
accacgagit

251

ggtacrgtat
ggractgzat
ggtacrgtat

301

:ccaatttca
tccaarttca
iccaattzca

351

aa*'aatcgc
aattdatcgc
aattaatcgc

401

garaaaccag
gazaaaccag
gataaaccag

451

tgataaacaa
:gaiaaacaa
tgataaacaa

501

rtcggaaraa
ttcggaaraa
ttcggaazaa

551

gagcctgaia
gagcc:gaza
gagcc:gata

601

raagatcgga
taaga"cgga
raagarcgga

rgg'tgtcgg
tggttgtcgg
tggitgtcgg

t' '.7 81' 'Ta'.
’Q’.a1.'_.at
[Laltat

1:.a
Eta
L a

o
I

aagagagcgg
aagagagcgg
aaQaQaQCQQ

gtarargarg
gtaratgaig
gtata*9aig

agcgggaaca
agcgggaaca
agcgggaaca

tagcagaaaa
tagcagaaaa
ragcagaaaa

gatactgtag
gatactgtag
gazacrgrag

gaaagargaa
gdaagatgaa
gaaagargaa

aagaaggt'c
aagaagg'rc
aagdaggitc

accgtggtag
accgzggtag
accgaggtag

taaaaaC'ta
taaaaac:fia
:aaaaaczta

Ct‘C"aacgfi
c:;c;aacgt
CZ’CZaacg:

612

atacgaaiaa
atacgaataa
atacgaataa

tfgcaaga'a
rtgcaagata
ttgcaagata

agacacgaat
agacacgaat
agacacgaat

ttgacacaac
ttgacacaac
tcgacacaac

ggtcacaaag
gg'cacaaag
ggzcacaaag

aacratcgct
aacra'cgc1
aac:atcgcr

aatttgaat:
aatttgaatt
aattigaat:

‘C T. aaaaat CC
L 1. 61616161111"- CC
t 7. aaaaat' CC

aaccgtrart
aaccgiraflt
aaccgiaazr

ttggatrtaa
riggaittaa
ttggarttaa

CCtgaagatg
ccrgaagatg
eczgaagazg

tactgacagt
Lac:garag:
tac;gatagt

71

816867. T. aat C
aaaat taarc
8.651137. F 6161' C

rigatgg'ga
r39131991113
ttgatggzga

ccaiararcg
ccatatarcg
ccazatatcg

agtraagggg
agitaagggc
agitaagggg

cccaaCt'gt
cccaacrrgr
cccaaCZtgt

ccaaatgaaa
ccaaa'gaaa
ccaaatgaaa

racgggtgta
racgggtgta
tacgggtgta

aaatcgirtc
addLCQLLLC
aaatcgtttc

aaggaarcac
aaggaaicac
aaggaaicac

accagatgcr
accagatgc:
accagatgcc

cagagtaffc
cagag:a::c
cagagtaiic

acgaaaggfia
acgaaagg:a
acgaaaggta

50
cagaacactt
cagaacaCtt
cagaacacrt

100
zggaaaccc:
tggaaaccct
tggaaaCCCt

150
ggaaaCC1ga
ggaaacc:ga
ggaaaccrga

200
aaagtattca
aaag'attca
aaagtattca

250
tgaraaagaa
tgataaagaa
agazaaagaa

300
aagatggggc
aagarggggc
aagatggggc

350
gattcaagta
gattcaagta
gattcaagta

400
tccaggtttt
Lccaggnztt
tccaggtttt

450
caaaagcggt
caaaagcggt
caaaagcggt

500
aaagaafcaa
aaagaaicaa
aaagaatcaa

550
atggaaaaca
arggaaaaca
atggaaaaca

600
ttgtaactg:
t:g:aactgt
t:gzaac:g:

10.

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