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STABILITY AND QUANTITATIVE SURVEILLANCE OF
HELIC OBA C TER PYLORI AND CAMPYLOBACTER JEJUNI
TN ENVIRONMENTAL WATERS
BY REAL TIME qPCR

Presented by

Arun Kumar Nayak

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5/08 KlProj/Acc8iPnelelRCIDaIeDue,indd

 

STABILITY AND QUANTITATIVE SURVEILLANCE OF HELIC OBA C TER PYLORI
AND CAMPYLOBACTER JEJUNI IN ENVIRONMENTAL WATERS
BY REAL TIME qPCR

BY

Arun Kumar Nayak

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

2008

ABSTRACT
STABILITY AND QUANTITATIVE SURVEILLANCE OF HELICOBACT ER
PYLORI AND CAMPYLOBACT ER JEJUNI IN ENVIRONMENTAL WATERS BY
REAL TIME qPCR
By
Arun Kumar Nayak
Rapid and sensitive detection of infectious pathogens like H. pylori and C. jejuni is
necessary for the maintenance of a safe food or water supply. The first portion of this
research aimed to demonstrate the application of a new qPCR technique for
determination of H. pylori concentrations in water and to use this method to investigate
the occurrence of the bacteria in sewage. The other aim was to study the survival capacity
and detectability of the bacteria in artificially contaminated groundwater at different
temperatures of 4°C and 15°C. Real Time qPCR demonstrated a 100-fold greater
sensitivity for detection of H. pylori DNA in comparison to conventional PCR. SEM
observation showed that the normal spiral form changed to a coccoid form after 24hr and
72hr at 15°C and 4°C, respectively. H. pylori was found at 2 to 38 cells ml'l in sewage
and 64% (n=39) were positive for H. pylori species specific gene vacA by real time
qPCR. The second portions of this research aimed to survey and quantify C. jejuni in
sewage using real time q PCR. Twenty-two samples were analyzed and 59% of the
sewage samples were positive for the C. jejuni specific gene of gyrA by real time qPCR.
Real time qPCR assay provided a specific, sensitive and rapid method for studying the
prevalence of H. pylori and C. jejuni and can be used as required by the US EPA
Contaminant Candidate List for survey. This study demonstrates that untreated sewage is

a source of these bacteria and has the potential to contaminate other waters.

ACKNOWLEGEMENTS

I would like to thank the Homer Nowlin Endowed Chair of Water Research for funding

the research.

Many thanks to my advisor Dr. Joan B. Rose, for her support and guidance throughout
my Masters research. Also, thank you to my committee members, Dr. Tracy Dobson, Dr.

Linda S. Mansfield, and Dr. Thomas M. Schmidt, for their guidance during my research.

I would like to thank Dr. John E. Linz and Dave Wilson at Food Safety and Toxicology
for providing me guidance and for use of lab equipment and time. Also, I would like to

thank Dr. Paul Coussens giving me the opportunity to use his Laboratory.

Lastly, I would like to thank the other graduate students and employees in Dr. Rose’s Lab

for their help in many aspects of my research. Your time and effort are greatly

appreciated.

iii

TABLE OF CONTENTS

LIST OF TABLES .............................................................................. vii
LIST OF FIGURES ............................................................................ viii
CHAPTER 1 ...................................................................................... 1
INTRODUCTION ................................................................................ 1
Research Objectives .............................................................................. 6
CHAPTER2 ....................................................................................... 7

STABILITY AND QUANTITATIVE SURVEY OF HELICOBACTER PYLORI IN
WATER AND WASTE WATER BY USING REAL TIME QPCR

2.1 REVIEW OF LITERATURE .............................................................. 7
2.1.1 History, Taxonomy and Biology of Genus Helicobacter ............................ 7
2.1.2 Significant Advances in understanding of H. pylori .................................. 8
2.1.3 Infectivity and virulence activity ........................................................ 9
2.1.4 Epidemiological study .................................................................... 10
2.1.5 Detection in Environment ................................................................ 12
2.1.5.1 Culture of the organism ................................................................ 12
2.1.5.2 Viable and Nonculturable forms ....................................................... 12
2.1.5.3 Survivability in water ................................................................... 13
2.1.5.4 Molecular Approaches for Detection of H. pylori from Water .................... 15
2.2 MATERIALS AND METHODS .......................................................... 17
2.2.1 Collection of strain and culture conditions ............................................. 17
2.2.2 Culture Methods ........................................................................... 17
2.2.3 Survival Experiments ..................................................................... 17
2.2.4 Electron Microscopy Protocol ........................................................... 8
2.2.5 Immunoassay .............................................................................. 19
2.2.6 Molecular techniques ...................................................................... 20
2.2.7 Environmental Sample Processing ...................................................... 22
2.2.8 Design of Primers and Template Preparation .......................................... 22
2.2.9 Quantitative Real-Time PCR ............................................................. 23
2.2.10 Sequence analysis ........................................................................ 24
2.3 RESULTS .................................................................................... 25
2.3.1 Survival Experiment .............................................................................................. 25
2.3.2 Environmental sample analysis ............................................................................. 31
2.4 DISCUSSION ................................................................................ 34
2.5 CONCLUSION .............................................................................. 41

iv

CHAPTER 3 ...................................................................................... 42
SUVEILLANCE AND QUANTITATIVE IDENTIFICATION OF CAMPYLOBACTER
JEJUNI FROM WASTE WATER BY REAL TIME QPCR

3.1 REVIEW OF LITERTURE ............................................................... 42
3.1.1 History and Taxonomy of Campylobacter ............................................. 42
3.1.2 Biology of Campylobacter ............................................................... 42
3.1.3 Disease prevalence and transmission ................................................... 43
3.1.4 Other evidence of transmission via water .............................................. 45
3.1.5 Seasonal variation ......................................................................... 46
3.1.6 Detection of Campylobacter in water ................................................... 46
3.2 MATERIALS AND METHODS ......................................................... 48
3.2.1 Collection of strain and culture methods ............................................... 48
3.2.2 Sample collection, cultivation and extraction .......................................... 48
3.2.3 Amplification and quantification of gyrA gene ....................................... 48
3.2.4 Quantitative Real Time PCR ............................................................. 49
3.3 RESULTS .................................................................................... 50
3.4 DISCUSSION ................................................................................ 56
3.5 CONCLUSION .............................................................................. 59
CHAPTER ....................................................................................... 60
SUMMARY ....................................................................................... 60
REFERENCES ................................................................................... 64

LIST OF TABLES

Table 2-1. Examples of the evidence supporting waterborne transmission of
Helicobacter spp ............................................................... 14
Table 3-1. Some of the important waterborne outbreaks due to
C ampylobacter spp. .......................................................... 44
Table 3-2. Some of the PCR based studies for the detection of Campylobacter
spp in water ..................................................................... 47
Table 3-3. Quantitative detection Cjejum' in monthly sewage samples
from 2006 to 2007 ............................................................. 54

Table 3-4. The level of C. jejuni concentrations in wastewater in
different seasons ................................................................ 55

vi

Figure 2-1.

Figure 2-2.

Figure 2-3.

Figure 2-4.

Figure 2-5.

Figure 2-6.

Figure 2-7.

Figure 2-8.

Figure 3-1.

Figure 3-2.

Figure 3-3.

LIST OF FIGURES

Comparative evaluation of Helicobacter pylori measured

by culture techniques, Most probable number PCR and

Real Time quantitative PCR (qPCR ) from the seeded

groundwater experiment in temperature at 4 OC ........................... 26

Comparative analysis of Helicobacter pylori measured
by culture techniques, Most probable number PCR and
Real Time quantitative PCR (qPCR ) from the seeded
groundwater experiment in temperature at 15 OC ......................... 28

Scanning Electron Microscope (SEM) (X20,000) examination
of H. pylori spiral form at time Zero at 40C temperature from
survival experiment ............................................................ 29

Morphology of coccoid form H. pylori produced under 150C
temperature after 24 hr as observed by SEM (X20,000) from

survival experiment ............................................................ 30

Standard curve for serial 10 fold dilutions of H. pylori
VacA gene (copy number 2 to 2X105) ....................................... 32

Phylogram analysis of H. pylori isolates ..................................... 33

Quantitative real time PCR analysis by absolute quantification
and expressed in amounts of H. pylori per ml of water sample .......... 34

Genomic copies of H. pylori found in waste water using
Real Time PCR .................................................................. 38

Standard curve for serial 10 fold dilutions of C. jejuni fragment of
gyrA gene (copy number 24 to 2.4 X 106 in 2 ul) ........................... 52

Agarose gel showing amplified Real time PC R products of
gyrA gene ........................................................................ 53

Quantification value using Light cycler qPCR analysis for

Campy/abacterjejuni and expressed in amounts of C. jejuni
per milliliter of water sample .................................................. 55

vii

CHAPTER 1

INTRODUCTION

A large percentage of populations in the United States rely on ground water for
both their domestic and agriculture purposes. Nearly half (~45%) of Michigan's
population relies on groundwater for its domestic water; public water supplies from
groundwater serves 1.7 million people in Michigan and the majority use surface water
(MDEQ 2005). Much of the attention and management efforts for waters in the state have
focused on water withdrawals and chemical pollutants, not microbial pathogens. The Safe
Drinking Water Act Amendments of 1996 required the United States Environmental
Protection Agency (USEPA) to identify new chemicals and microorganisms for potential
regulation every five years.

The Contaminant Candidate List (CCL) is based on information about known and
suspected health risks and the occurrence of the contaminants in water. This guideline
recommends that methods be developed for the contaminants and water supplies in order
to survey to examine occurrence and potentially sources. This will then impact the type
of water treatment needed to protect public health for both groundwater and surface
water. Fecal pollution including sewage remains an important source of the microbial
pathogens which may be included in the CCL. Helicobacter pylori and Campylobacter
are included on CCL and were listed in the Federal Register in 2004 (EPA 2005) [ Just
recently C. jejuni has been included in the Contaminant Candidate List 3 by United States
Environmental Protection Agency (EPA 2008, Fact Sheet CCL 3)], however to date no

detection method has been suggested and no survey of waters has been undertaken.

H. pylori is a ubiquitous microorganism and it is estimated that about half of the
world’s population is infected (Dunn et al. 1997; Feldman et al. 1997). It is a gram-
negative, micro-aerophilic bacterium, and has been recognized as causing peptic ulcers
and chronic gastritis and may be related to stomach cancers associated with MALT
lymphomas (Atherton J.C., 2006). The World Health Organization has classified it as a
class I carcinogen (Aruin LL, 1997). In the US, 50% of the adult population is thought
to be asymptomatic carriers, however the sources of the bacteria and transmission
pathways have not been well studied (Munnangi and Sonnenberg, 1997). Fecal-oral and
oral-oral transmission have been suggested as likely (Hegarty et al. 1999). All fecal-oral
agents have some potential for waterborne disease transmission and this has been
suggested for H. pylori transmission as well (Hulten et al. 1996). Rolle-Kampczyk et a1.
(2004) found a significant correlation between the prevalence of H. pylori in well water
detected by polymerase chain reaction (PCR) and colonization of the population using
the water. Yingzhi et a1, (2002) isolated H. pylori from wastewater and evaluated the
colonies by PCR, however none of the isolates were confirmed as H. pylori in 168 rRNA
PCR assay.

Due to the slow growth of the organism and difficulties in isolating the organism,
to date, no one method currently exists for monitoring for the organism in water and this
is one of the primary problems in understanding the exposure pathways and potential for
waterborne disease. Additionally, the development of an accurate, inexpensive method to
detect pathogens would be especially useful for small communities on limited budgets or

for private well owners.

There are a number of bacteria, including H. pylori that may also enter into a
viable but nonculturable (VBNC) state in the environment and are no longer culturable
on bacteriological media (Oliver J.D., 2002). The VBNC state may be induced by a
variety of environmental conditions, such as temperature decreases or nutrient depletion
(Oliver J .D., 2002), increased oxygen tension, and/or exposure to antibiotics (Xu et al.
1999). Xu et al. (1999), reported that H. pylori changed their morphology from a spiral
form to a coccoid form after 8 days when either broth culture at room temperature and
selective media supplemented with egg and calf serum (EHP media) or Brucella broth
supplemented with calf serum were used for cultivation.The coccoid form may enable
environmental transmission (Hulten et al. 1998) but further information on detection and
survival will be necessary to address virulence in water (Engstrand L., 2001), particulary
given that lower concentrations of the bacterium may occur in the environment (Benson
et al. 2004).

Rapid, sensitive and quantitative detection of H. pylori will aide in the
maintenance of a safe food or water supply. The present study, focused on the
development of a qPCR technique for determination of the prevalence and level of H.
pylori contamination in water. The research goal was to demonstrate the application of
this innovative approach that could be used for monitoring various sources of water
(including ground water). In addition the non cultivable nature of H. pylori in
environment may be temperature dependent and thus an alternative method for detection
of these forms is necessary. Thus a survival study was designed to evaluate the

applicability of molecular methods for rapid detection of both the spiral and coccoid

forms of H. pylori from environmental samples. Finally, it was hypothesized that sewage
or waste water was a possible source of H. pylori contamination of ambient waters.

Campylobacters are emerging infectious bacterial pathogens and one of the major
causes of diarrheal illness in humans. This pathogen is generally considered as the most
common bacterial cause of gastroenteritis worldwide (WHO, 2000). Infections of
Campylobacter in developing countries under the age of two years are more frequent,
occasionally resulting in death. The incidence of human Campylobacter infections has
been gradually increasing for several years in almost all developed countries but the
reasons are unknown. The sporadic cases of campylobacter infections are thought to be
mainly foodbome and waterborne (via undercooked meats and meat products, as well as
raw or contaminated milk). The ingestion of contaminated water, untreated water or ice is
also a recognized source of infection. (Snelling et al , 2005 ; Moreno er al, 2003b; Yang
et al ,2003).

Campylobacters are mainly gram negative, spiral-shaped, S-shaped or curved,
rod-shaped bacteria. There are several species assigned to the genus Campylobacter, of
which C. jejuni is most frequently recognized worldwide as a leading cause of human
disease predominantly associated with gastrointestinal infections (WHO, 2000). An
occasional complication of this pathogen is neuromuscular disorder that can lead to a
severe form called Guillain-Barre syndrome (Allos et al, 1998).

Campylobacter jejuni cells may enter water sources such as private wells,
(including drinking water) that have been contaminated with feces from infected animals,
birds, or humans. The source of transmission can come into the water in many different

ways from human or animal waste (i.e., sewage overflows, polluted storm water runoff,

agricultural runoff, excretion of birds, waste water from poultry houses and processing
facilities). The cells may survive and remain potentially pathogenic for long periods in
the environment and may also posseses a viable but non culturable (VBNC) form due to
starvation and physical stress (Hege et al, 2000). The presence of VBNC C. jejuni cells
even at a very low number in water could pose a health risk. Therefore, sensitive
methods are required to detect and quantify C. jejuni contamination in water sources
supplied for drinking water purposes.

Even though Campylobacter spp. are widely distributed in the environment, the
incidence and epidemiology of many cases of Campylobacter associated infections
remain unclear. This is mainly because of lack of routine monitoring, surveying of
isolates from the environment as well as parallel diagnosis of clinical cases. Moreover,
the quantitative detection of C. jejuni cells in water using different techniques (On,
S.L.W., 1996) is challenging due to inadequate methods. In fact, there is no annual
survey data for quantitative detection of C. jejuni in water.

The major data gaps are 1) lack of information on the presence of Campylobacter
infections in the population and excretion into waste water, 2) lack of quantitative
methods for accurate and rapid quantification of all forms, and 3) Indeterminate
understanding of the seasonal variability pattern for quantitative C. jejuni concentration
in water and waste water. It is particularly important to know the concentration of
Campylobacter cells in water in order to better predict the possible health risks in the
near future. To establish a better anticipated risk model for pathogen incident in certain
communities or environments, annual survey data are necessary. Thus like Helicobacler,

it is hypothesized that untreated sewage is a major source of C. jejuni contamination of

water systems and that new methods like qPCR are needed to accurately measure
Cjejuni due to VBNC form.
Research Objectives
Overall objectives of the research are in two parts, the first part of the research is directed
toward Helicobacter.
The specific research objectives for the first part are:
1. To investigate Helicobacter pylori survival capacity in artificially contaminated
groundwater at different temperatures of 4°C and 15°C.
2. To use Scanning Electron Microscopy techniques for addressing the forms of H.
pylori in water.
3. To develop and demonstrate the application of new qPCR technique for real-time
determination of H. pylori contamination in water.
The specific objectives of the second part of the research are:
4. To investigate the presence of C. jejuni in waste water.
5. To establish a rapid, sensitive real time assay targeting the 200bp fragment of
gyrA gene for quantitative detection of C .jejuni using a TaqMan Probe.
6. To conduct an annual survey for the quantitative detection of C. jejuni in waste

water using real time q PCR.

CHAPTER 2

STABILITY AND QUANTITATIVE SURVEY OF HELICOBAC T ER PYLORI IN
WATER AND WASTE WATER BY USING REAL TIME QPCR

2.1 REVIEW OF LITERATURE
2.1.1 History, Taxonomy and Biology of Genus Helicobacter

1982 H. pylori was described successfully in Australia as a spiral or curved
shaped urease producing organism found in the human stomach by Warren and
Marshall (1983). The bacterium is Gram negative, motile, and has a spiral or spherical
form. In the normal spiral morphology, the bacterium has 4-6 unipolar flagella. prlori
grows in a microaerophilic environment at an optimal temperature of 37 degree C. The
pH ranges for growth of this bacterium are from 5.5 -8.5. Important biochemical
characteristics include positive results for oxidase, catalase, urease and acid and alkaline
phosphatases tests (Northfield and Mendall, 1994).

The taxonomy of the genus Helicobacter has rapidly evolved due to mutation,
selection and evolutionary processes. Helicobacter has been suggested to be a distinct
phylum as proposed by Vandamme et al,(l99l),that is included within the class
Proteobacteria in rRNA super family VI. The nutritional requirement of the individual
species and fastidious nature of the bacterium necessitates particularly complex
requirements in order to isolate and culture these organisms (Vandamme et al, 1996). The
original taxonomy of the Helicobacter genus was particularly complex and it is now
recognized that the species have been misidentified particularly due to lack of a specific
screening method. This problem has been solved with the application of PCR technique
and 168 r DNA sequencing (Solnick et a1 , 1993). Despite the 168 rDNA sequence

analysis, taxa have been identified which have less than 97% similarity, thus limiting

resolution at the species level which makes it difficult to distinguish strains from different
sources (On S.L.W.2001).
2.1.2 Significant Advances in understanding of H. pylori

Historically, gastric ulcers were contemplated to be caused by stress and/or too
much stomach acid (Blaser M., 1996).The understanding and cause of stomach (gastric)
ulcer disease has dramatically changed since the discovery of the causative organism H.
pylori by Marshall in 1982. H. pylori is an infectious organism causing inflammation of
the stomach (gastritis), resulting in indigestion and severe abdominal pain (Braunwald er
al., 2002). As the inflammation progresses, more serious conditions such as gastric ulcers
and cancer contribute to increased morbidity (Malfertheiner and Blum, 1998). Moreover,
gastric cancer remains second among the causes of cancer deaths worldwide (Goodman
and Cockbum, 2001 ).

Multiple risk factors have been associated with prlori infection (Belkind-
Gerson et al., 2001;Czaja-Bulsa & Szymanowicz, 1995). The risk factors include
multiple allergies, contaminated food, poverty, crowded living conditions, poor hygiene,
infants delivered by vaginal birth, Latino or African American ethnicity, and
contaminated water. The prevalence of H. pylori infection in the world population is
assumed to be approximately 50%, with higher prevalence in people living in developing
than in developed countries (Dunn et al ,1997). In developed countries initially it was
thought that a clustering of H. pylori infection occurred within families, which supported
a person to person transmission pathway. On the other hand, studies from developing
countries with poor management of their water supplies have shown an association

between the prevalence of H. pylori and the source of drinking water (Klein er al , 1991).

Moreover, increased risk factors for the infection have been associated with the
consumption of vegetables irrigated with untreated sewage (Hopkins et a1 , 1993). The
economic impact of H. pylori infections in the United States is approximately $6
billion/year. Cost analysis shows that $3 billion is spent on hospitalization costs, $2
billion on physician services, and $1 billion on decreased productivity and days lost from
work (CDC, 1998).
2.1.3 Infectivity and virulence activity

She and Lin, (2003) explored the virulence and the infectivity of coccoid H.
pylori transformed from the spiral form by exposure to sterile tap water. Interestingly
they found that the urease activity and the ability to adhere to Hep-2 cells were found to
be lesser in coccoid H pylori than that in the spiral form. In the experimental infection in
mice, the positive rates of H. pylori culture were 87.5 % (14/16) in the spiral H. pylori
group and 68.8 % (11/ 16) in the coccoid H. pylori group. There was no significant
difference in either the urease test or bacterial culture rates between the groups examined
at Day 21 and Day 28 after inoculation. Moreover, they found that electron microscopic
examination of the samples taken from both groups showed the adherence of H. pylori in
spiral, bacillary and coccoid shapes to the epithelial cells of gastric wall. This was
supported by histological examination that showed the occurrence of gastric mucosa]
injury as indicated by various degrees of erosion, ulcer, and inflammatory cell
infiltration. Mucosal injury was slighter in the mice infected by the coccoid form of H.
pylori (She and Lin, 2003). Although the virulence of coccoid H. pylori induced by
contaminated water ingestion was decreased, the coccoid H. pylori still retained urease

activity and the ability to adhere to epithelial cells. Furthermore, the flagella, an

important component responsible for bacterial movement and infection, were still
observed as a cellular structure of coccoid H. pylori under the electron microscope. Thus
the evidence so far suggested that the coccoid H. pylorus induced by water is capable of
colonizing the gastric mucosa and causing gastrititis in mice.

2.1.4 Epidemiological study

H. pylori is one of the world’s most widespread microorganisms (Rolle-
Kampczyk, et al ., 2004). Its acquisition in humans remains poorly understood, however,
epidemiological studies have identified drinking water as a reservoir for the bacterium
(Reavis C., 2005). Infections occur more often in poor socioeconomic conditions and
have been consistently linked to residential crowding and human migration from high-
prevalence regions (Goodman and Correa, 1995). Food-borne, water-bome and zoonotic
transmission pathways of H. pylori have been suggested (Wesley, I.V.l997; Velazquez
and Feirtag, 1999; Baker and Hearty, 2001; Bunn et a1 2002). There is also PCR evidence
that drinking well water was contaminated with H. pylori in a German community with
H. pylori infections (Rolle-Kampczyk, et al., 2004).

As in the German study , McKeown et al, (1999) found a positive seroprevalance
of H. pylori infection in an Inuit community population in the central Canadian Artic
using polymerase chain reaction (PCR) testing of the local water supplies. It was
speculated that this organism had a natural reservoir or that there was possible
contamination from human sewage.

Studies conducted in Europe as well as in North and South America have tried to
link H. pylori colonization with the drinking water supply, especially since H. pylori is

known to survive quite well in water (Krumbiegel et al., 2004). In the city of Leipzig,

10

Germany, two counties were studied (1 and II). The PCR analyses showed H. pylori DNA
fragments in 10.8% of the wells in county I and 9.2% in county 11 and positive prlori
was found in 5.7% and 6.6% of the children in this respective cluster. The analysis found
that drinking water that was not part of the Municipal supply was associated with the
infections.

The transmission of the human gastric pathogen Helicobacter pylori has not yet
been delineated and the subject is controversial ( Hazell et al. 1994; Axon A.T.R., 1996).
Different routes of infection have been proposed and oral-oral and faecal-oral
transmission theories have gathered the most support (Shay and Axon, 1996). Zoonotic
transmission was suggested by some authors and reservoirs other than human cannot be
ruled out (Fox, 1995).

The CDC (2004) reports that while the exact mechanism for H. pylori
transmission is not known, the bacteria is most likely spread from person to person
through fecal—oral or oral—oral routes. Possible environmental reservoirs may include
contaminated water. In a study of Bolivian residents with similar prevalence rates, H.
pylori infections were decreased after a program was initiated to treat the patients testing
positive for the infection and by using narrow mouth water vessels and water
disinfectants (Sivapalasingam, et al, 2000). Despite its major public health impact, the
design of prevention measures is difficult due to our limited knowledge of transmission
pathways (Goodman and Correa, 2000).

Different transmission routes may be predominant in different geographical areas
and it may be that the observed differences in prevalence can be explained by the route(s)

of infection in that area. Reports from developed countries, where sanitary procedures

11

such as water treatment and food processing are well managed, show a clustering of H.
pylori infection within families, pointing towards person-person transmission (Bamford
et al 1993).

H. prion-specific DNA was detected in water samples from middle and
downstream. but not from the upstream. reaches of four rivers in Japan. H. pylori could
not be cultured from any DNA positive water samples. The prevalence of H. pylori stool
antigen in children was 10% (6/ 61) near the middle reaches. and 24% (24/101) near the
downstream reaches. but 0% (0/62) in areas distant from the river. Hence. river water
may be a source of H. pylori infection (Fujimura et al, 2004).

2.1.5 Detection in Environment
2.1.5.1 Culture of the organism

The effect of environmental factors and substrates on survival and grth of H
pylori was reported by Jiang and Doyle (1998). They found that under microaerobic
conditions (5% 02, 10% C02, and 85% N2), the organism grew well in brain heart
infusion broth supplemented with 7% horse serum and antibiotics (BHI-HS-TVA) at
temperatures between 30 to 37 °C with agitation. The optimal NaCl concentration for
growth of H. pylori was between 0.5 to 1.0% but 2.0% NaCl inhibited growth. The
addition of urea enhanced the growth of H. pylori at both pH 4.5 and 5.5. Although H.
pylori did not grow at pH 3.5, the presence of urea in broth enhanced its survival. Growth
of H. pylori in most of the liquid media including tryptic soy broth, Mueller-Hinton
broth, brucella broth, brain heart infusion broth, and Columbia broth with supplements
was accomplished (Shahamat and Mai, 1991) when incubated in a C02 atmosphere.

2.1.5.2 Viable and Nonculturable forms

12

So far, no reports have been published on the successful culture isolation of H.
pylori from surface or ground water. Most bacillary H. pylori organisms change their
morphology upon prolonged exposure to water, and enter a viable but non-culturable
stage in which they appear as coccoid forms (N ilsson et al, 2002). It has been suggested
that this coccoid form is responsible for transmission in the environment, probably via
contaminated water (Hulten, er a1 , 1998). H. pylori’s ability to survive and maintain its
viability and virulence is still uncertain when it is in the coccoid form (Engstrand L.
2001)

The detection of this pathogen has proved difficult when H. pylori changes its cell
morphology to the coccoid form after exposure to different environmental stimuli.
Metabolism and growth patterns are also changed in the coccoid form. These VBNC
forms do not undergo cellular division and cannot be cultured by traditional methods.
Velazquez and Feirtag, (1999) used different techniques for the detection of prlori in
water and food which included filtration, immuno-separation (IMS), polymerase chain
reaction (PCR), probe hybridization, immuno-staining, autoradiography and ATP
bioluminescence. Autoradiographic methods have been developed (Shahamat and Mai.,
1993) to detect metabolic activity of viable but nonculturable cells of Helicobacter pylori
in water. Autoradiographs of tritium-labeled cells of H. pylori revealed aggregations of
silver grains associated with uptake by H. pylori of radiolabelled substrate.
2.1.5.3 Survivability in water

The survival capacity of H. pylori in artificially contaminated milk and tap water
was investigated by Fan et al,( 1998). They reported that culturable H. pylori could

survive for up to 10 days in milk at 4°C storage but only 4 days in tap water with a steady

13

decrease in colony forming units. They found approximately 2 log reduction of

organisms at 4 days in tap water. Their results showing that there is the possibility for H.

pylori to persist in water in a metabolically active stage that is not actively growing and

dividing which are intriguing and relevant to public health concern.

Table 2-1. Examples of the evidence supporting waterborne transmission of Helicobacter

 

 

 

 

 

 

 

 

 

spp.
Date/Ref Location Water type Monitoring in water Epidemiological
samples evidence
Hulten et al, Sweden Municipal water, Positive PCR for No study
1998 waste water DNA of
well water Helicobacter. spp
Mckeown er Canada Drinking water Positive H. pylori by 50% people
al, 1999 supplies PCR from two
community
positive for
H. pylori
Hegarty, et USA Surface water and Positive for No study
al, 1999 ground water Monoclonal anti-
H. pylori antibody
Bunn er al , West Drinking water H. pylori positive for Infants were
2002 Africa biofilm sample 168 r DNA by PCR positive for
H. pylori
Lu et al , Mexico Waste water Positive PCR for No study
2002 H. pylori
Moreno et al Spain River and waste Positive PCR and No study
, 2003b water FISH for H. pylori
Fujimara et Japan River water Positive PCR for Children were
al, 2004 H. pylori positive to

 

 

 

 

H. pylori
infection near
the river

 

Some of the important evidence for the presence of H. pylori in water is shown in

Table 2-1. Hulten et al., (1999) reported 9 out of 24 private wells and 3 out of 25

municipal tap water samples were positive for Helicobacter spp by PCR assay. However,

14

 

several gaps in the methodology and inadequate data have been reported which requires
further study. The major difficulty is the non-specificity of PCR methods due to presence
of unknown possibly non pathogenic speices within the genus of Helicobacter reported
by Hulten et al , (1999) and Lu et al, (2002). Moreno et al , (2003b) detected in H. pylori
in two river water and one wastewater samples using FISH. However, further studies are
necessary with water samples spiked with characterized H. pylori strains to test this
methods reliability when performed on environmental samples. Moreover, the FISH
technique needs more exhaustive evaluation. Using PCR method, Fujimara et al ,(2004)
were unable to establish a direct link or connection for the presence of prlori in river
water and the prevalence of H. pylori in children due to lack of proper epidemiological
data.
2.1.5.4 Molecular Approaches for the Detection of H. pylori from water

Unsuccessful attempts to culture H. pylori from environmental waters have led to
the use of molecular methods to detect and identify this organism. Several articles have
been published using conventional PCR to detect H. pylori in a number of types of waters
including ground water, surface water, treated and untreated wastewater, marine waters
and also biofilms (Hulten et al., 1998;; Lu et al., 2002; Watson et al., 2004; Carbone et al,
2005). Mazari-Hiriart et al., (2001) reported H. pylori and other indicator bacteria in
fresh water in the environment of Mexico City. Twenty one percent of the samples
(16/77) were positive for H. pylori ; of these, 42% (5/ 12) were confirmed for the cagA
gene by PCR hybridization. The results of their study suggested that, in Mexico City,
water used for human consumption and irrigation may play an important role as a vehicle

of H. pylori transmission as well as infection by other known enteric pathogens.

15

Helicobacter pylori isolation and genotyping from wastewater was demonstrated by Lu et
al , (2002) using a series of steps beginning with immunomagnetic separation and cell
culture. After Gram staining and three standard microbial tests, the 16S rRNA sequences
of a total of 23 out of 37 putative H. pylori isolates were verified by PCR. Eleven H.
pylori isolates were genotyped and fell into four vacA classes.

According to Moreno et al, (2003b) Fluorescence in situ hybridization (FISH)
techniques have the potential to be used as a quick and sensitive method for detection of
H. pylori in environmental samples. Using the FISH technique, H. pylori was detected in
two river water and one wastewater samples, while PCR yielded only one positive result.
Watson et al., (2004) suggested that Helicobacter specific PCR assays and direct
sequencing provide an approach to detect Helicobacter DNA in the biofilms on surfaces
and within water distribution systems. Biofilms may act either as sites for the passive
accumulation of helicobacters or as potentially important reservoirs of infection.

A rapid DNA extraction and quantitative, real time PCR (qRT-PCR) analysis
method targeting the ureA gene (using Taq Man Probe method) of Helicobacter pylori
was evaluated(McDaniels and Wymer , 2005) for the measurement of these organisms on
membrane filters at cfu levels that might be expected to be found in drinking water
samples. This assay was shown to provide sensitive (40 cells or less per membrane) and
quantitative measurements of low numbers of H. pylori cells in drinking water samples.
One liter of drinking water samples from several locations in the US were seeded with H.
pylori cells and tested; these gave measurements that were consistent with the standard
curve suggesting that these sample matrices produced no interference in the method.

However evaluation of their methods with environmental samples was not undertaken.

16

2.2 MATERIALS AND METHODS
2.2.1 Collection of strain and culture conditions

H. pylori ATCC 700329 was used in our studies. The frozen bacterial
suspension (stored at — 80 °C) was thawed and transferred into fresh Trypticase Soy
Broth (TSB) (3-5ml ), then resuscitated in biphasic slants in Trypticase Soy Agar (TSA)
(Becton Dickson, BD bioscience). Cultures were incubated under microaerophilic
conditions (5% oxygen, 10% carbon dioxide and 85% nitrogen) in anaerobic jars (Campy
Gas Pak system, Oxoid) at 37°C for 3 days. Subsequently, an aliquot of culture was
harvested and resuspended in 10ml of liquid TSB (BD bioscienee) supplemented with
10% (vol/vol) horse serum (Cambrex Bioscience) and incubated in a microaerophilic
environment at 37°C for 5 days.

2.2.2 Culture Methods

H. pylori subculture was carried out by spreading samples on Columbia blood
agar (Oxoid) plates containing 7% laked horse blood (Oxoid) and H. pylori selective
supplement Dent (Oxoid) (Moreno et al. 2003b). The plates were incubated at 37°C for 5
days in a commercial gas pack system under microaerophilic conditions in 5% oxygen,
10% carbon dioxide and 85% nitrogen. The colonies were counted after 5 days of
incubation. The mean value of three readings was calculated and reported as colony

forming units (CFU) per milliliter (CFU ml").
2.2.3 Survival Experiments

Non-chlorinated ground water was collected from a private well (Lansing, MI)
and temperature, pH, turbidity and free chlorine were recorded. The ground water was

exposed to UV (Ultra violet) light for 24 hrs to reduce naturally occurring microbes.

l7

Ultra violet light was used in order to limit the natural microflora and to reduce the risk
of predation of H. pylori. An inoculum, corresponding to approximately 1><107 CFU H.
pylori, was suspended in 50ml of water in a sterile polypropylene centrifuge tube with a
fitted cap. Tubes containing seeded ground water were maintained at 4°C and 15°C
respectively for 288 hours. The positive control was resuscitated from the original stock
bacterial culture following the same methods as described above. Samples were plated
from each set of seeded tubes of groundwater for the two temperature conditions.
Negative (unseeded groundwater) and positive controls (ATCC 700329) were also plated.
Each plate was inoculated with 100p] of the suspensions of seeded and unseeded
groundwater onto selective Columbia blood agar plates and the plates were incubated for
5 days in microaerophilic conditions at 37°C. The plating was performed for both of the
temperature conditions (4°C and 15°C) at intervals of O, 24, 48, 72, 120, 168, and 288
hours. Each time and temperature combination had three replicates (i.e, n=3 for 4°C, 0hr;
n=3 for 15°C, 0hr). After the incubation period, colonies were enumerated and the mean
was calculated as CFU ml" based on the three replicates. The study was repeated in
duplicate under the same experimental conditions.
2.2.4 Electron Microscopy Protocol

The groundwater sample with the prlori suspension, which had been kept for
288 hrs at both temperatures, was divided into two parts. One aliquot was used for PCR
analysis and the other used for analysis with scanning electron microscopy. An aliquot of
the liquid suspension was mixed with an equal quantity of 4% glutaraldehyde in 0.1M
sodium phosphate buffer at pH 7.4. Fixation proceeded for one-half hour at 4°C. One

drop of 1% Poly-L-Lysine (Sigma P1399) was placed on a plastic petri dish and a 12mm

18

round glass coverslip was placed on top of the drop and allowed to stand for 5 minutes.
The coverslip was removed and gently washed with several drops of sterile water. The
coverslip was drained but was not allowed to dry. One drop of the fixed cells was placed
on the coverslip. The suspension was allowed to settle for five minutes. The coverslip
was then gently washed with several drops of water and placed in a graded ethanol series
(25%, 50%, 75%, 95%) for five minutes in each step and with three 5 minute exposures
to 100% ethanol. Samples were dried in a Balzers critical point dryer using liquid carbon
dioxide as the transitional fluid. After that, samples were mounted on aluminum stubs
using adhesive tabs. Samples were coated with gold (for 1 minute, 7nm thickness) in an
Emscope Sputter coater (model SC 500) purged with argon gas. Samples were examined
in a JEOL 6300 field Emission SEM manufactured by Japan Electron Optic Limited
(Mitaka,Tokyo) following the methods of Klomparens et al.(1986).
2.2.5 Immunoassay

Actively growing H. pylori was tested using a combined monoclonal mouse anti-
H. pylori immunoglobulin G (Fitzgerld Corp., Concord, MA) and an anti-mouse IgG
F ITC conjugate (Sigma). An aliquot of contaminated sample from both the experimental
sets (temperature at 4°C and 15°C ) were concentrated. Cells were fixed by drying (45°C)
and then labeled on a glass slide by blocking with goat serum buffer (composition: 10mls
of non sterile 1x PBS, 0.2m1 of goat serum and lml of 0.002% Tween-20) at room
temperature for 30 minutes. Samples were then stained with primary monoclonal mouse
anti-H. pylori immunoglobulin G (final concentration 13ugml") at room temperature for
1 hour. Next, samples were stained with a secondary antibody (anti-mouse IgG FITC

(flouorescein-S-isothioeyanate conjugate) used at a final concentration of 20 ug/ml,

l9

covered with aluminum foil, at room temperature for 1 hr. Finally, samples were washed
four times with 1X PBS and dried with absorbent paper. One drop of DABCO was added
then a cover slip was added and sealed with clear nailpolish. The slides was stored at 4 0C
until microscopic examination. Stained samples were examined under epifluorescent
illumination using a Zeiss microscope with an FITC filter. Cells that showed a bright
green fluorescence, were within the size range of 0.5 to lum and had a curved rod or
coccoid shape were considered to be H. pylori.

2.2.6 Molecular techniques

Each seeded sample from both incubation temperatures at each time interval were
analyzed by PCR. Seeded water samples were concentrated from 10m] to lml by
centrifugation (5000 rpm for 15 minutes). A lml aliquot of each concentrated sample was
resuspended and serially diluted up to 10'6 with sterile 1x PBS (Sarnbrook et al. 1989).
For the MPN PCR, serial dilutions were done before extracting DNA. DNA extraction
followed the method described in the QIAmp DNA mini kit (Valencia, CA) using
bacterial protocol D for H. pylori.

Oligonucleotide primers for H. pylori 16S rRNA and vacA have been previously
described and reported by Watson et al. (2004) and Chisholm et al. (2001) respectively.
Helicobacter detection was performed using primer 297(nucleotide position), 5’- GGC
TAT GAC GGG TAT CCG GC-3’ (Forward), and 1026(nucleotide position), 5’-GCC
GTG CAG CAC CTG TTT TC-3’ (Reverse) PCR primers to amplify a 730bp 163 r DNA
fragment from Helicobacter as described by Watson et al. (2004). Forward primer, 5’-

GAG CGA GCT ATG GTT ATG AC-3’ corresponding to nucleotide 3624, and reverse

20

primer, 5’- ACT CCA GCA TTC ATA TAG A-3’ corresponding to nucleotide 3853 were
used to amplify the H. pylori specific vacA gene of 229bp (Chisholm et al. 2001)

The amplification of prlori genomic DNA was performed with Master mix
(Qiagen) and a PCR Gradient Thermal cycler (Eppendorf, German). The reaction
mixtures used for the PCR contained (per 25 ul) 1x PCR buffer (50nmol/l KCL,10nmol/l
Tris- HCL, and 1.5 nmol/l MgClz), deoxynucleoside triphosphate at concentrations of

200umol/l and 0.25umol/l, and 2.5 U (International Unit) of Taq DNA polymerase

(Qiagen). Each extracted DNA sample of 10.5 ul was used as a template. The temperature
profile for the PCR was as follows: an initial step of 5 min at 95°C, followed by
denaturation for 30 sec, annealing at 57°C for 30 sec (165 rRNA PCR) or 54°C for 30 sec
(vacA PCR) and Primer extension at 72°C for l min (16S rRNA PCR) or 45 sec (vacA
PCR). After the 35th cycle, the extension step was prolonged for 5 min to complete
sysnthesis of all strands and then the samples were kept at 4°C until analysis. Negative
and positive controls were included in every experiment. Triplicate analyses were run for
each sample by using the respective PCR protocol for each dilution (see below for real
time PCR methods). The results were visualized with 1.8% agarose gels (stained with
Gelstain,Cambrex,East Rutherjord,NJ) UV light illumination. A 100bp DNA ladder
(Promega, Madison,WI) was included in each gel as a molecular size standard. The
quantitative results were calculated by a most probable number (MPN) program (3-
dilution, 3 tube approach; EPA software, USA). At the same time, the quantitative Real-
Time PCR was performed for the survival study. The absolute quantification was done by

real-time qPCR using SYBR Green PCR Master Mix.

21

The analytical sensitivity of the qPCR was evaluated by lO-fold serially diluting
with extracted DNA (100 to 106). Quantification of H. pylori present in bacterial
suspensions was performed by spreading on plates of H. pylori selective media as
described earlier and incubated at 37°C for five days in microaerophilic conditions. The
surviving fraction of bacteria was regressed against time. The surviving fraction was
calculated by dividing the Loglo concentration at time t (N) by the Logm initial
concentration (N 0).

2.2.7 Environmental Sample Processing

Three raw wastewater samples (50ml each) were collected from a wastewater
treatment plant (East Lansing, MI) every two weeks from June to December 2005 and
then once a month from January 2006 to October 2007. Samples were processed by two
methods: 1) an IMS (Immunomagnetic separation) protocol (Enroth and Engstrand
1995), following the IMS separation methodology as described by the manufacturer
(Dynal Biotech) with a modification using a Monoclonal rabbit anti-H. pylori and 2)
high speed centrifugation (5000rpm, 20 minutes ), followed by cell lysis (for 30 minute at
37°C) and DNA extraction using QIAmp DNA mini kit (Qiagen).

2.2.8 Design of Primers and Template Preparation

The PCR amplicon (vacA gene and 168 rRNA) was cloned into TOPO PCR 2.1
vector and transformed into the TOPOIOF’ competent cells (lnvitrogen Inc.. Carlsbad,
CA) following the manufacturer’s instructions. Plasmids were isolated following the
protocols of Wizard Mini Prep (Promega, Madison WI). Restriction digestion with EcoRI
(NEB-lab, Ipswich, MA.) was also performed to cross check the presence of inserts

inside the vector. Automated DNA sequencing (ABI, Foster city, CA) at the Michigan

22

State University Sequencing facility further confirmed that the targeted gene fragment
had been cloned. The absorbance of the DNA solution was measured three times on a
Nano drop spectrophotometer and the mean value was taken as actual absorbance. This
plasmid was used as a template for the standard curve. Serial dilutions of the plasmid
isolated from the recombinant of cloned product were prepared and standard curve were
constructed. Two sets of primers were designed using the Primer Express program
(Applied Biosystems) from the fragment of vacA gene and sequence accuracy and target
position cross checked with the H. pylori sequence found in Gene Bank. After that
analyzed the set of primers that were performed best reproducibility and reliability with
target. The primer matrix was run to find out the best primer sets and concentration
required. The primer for Forward: sequence is 5’-GCAATAGCAATCAAGTGGCTTTG-
3’andReverseisz5’GCGCGCTTCCACATTAG-C-3’are for vacA gene. The specificity of
Real-Time qPCR amplification of the vacA gene and 163 rRNA was tested with E. coli
ATCC15597 and Enteroccousfaecium ATCC19434, two common bacteria found at high
concentrations in sewage. The 16s rRNA was found not to be specific for H. pylori and
was not further evaluated. Specificity of the primers for the vacA gene was also tested
using C. jejuni (ATCC 11168) with SYBR green methods.
2.2.9 Quantitative Real-Time PCR

The quantification was done by real-time quantitative PCR using SYBR Green
PCR Master Mix (Applied Biosystems, Foster City, CA). Real-time quantitative PCR
was used for absolute quantification of the gene following the methods of Whelan et al.
2003.The primer matrix was evaluated for each gene tested to determine optimal primer

concentrations. Each reaction mixture consisted of 2 pl of DNA (maintaining the same

23

concentration of template), 1.5 pl each of forward (5 pM) and reverse primers (5 pM),
7.5 pl of nuclease free water, and 12.5 pl of SYBR Green PCR Master Mix in a total
reaction volume of 25 p1 (96-well optical plates). Reactions were performed in duplicate
for each sample in an ABI Prism 7000 Sequence Detection System (Applied
Biosystems). The thermal cycler program consisted of a preliminary step of 2 min at
50°C, denaturation for 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 sec and 60
°C for 1 min. Standard curves for each gene and controls were constructed using tenfold
serial dilutions of corresponding plasmids that were run on the same plate as samples. For
each measurement, threshold lines were adjusted by default in the software to intersect
amplification lines in the exponential portion of the amplification curve and cycles to
threshold (Ct) were recorded. For each sample the amounts of DNA (copies) for vacA
and 16S rRNA genes were analyzed and calculated from their respective standard curves.
The sensitivity of the qPCR method was compared to the detection limit of conventional
PCR methods. Percent recovery was determined by seeding known concentrations
(9X107cells/ml) of H pylori in the wastewater. The PCR product was verified on Gel
stained 1.8% agarose gels in Tris acetate- EDTA buffer. 100bp DNA marker (Promega)
was used as a size marker.
2.2.10 Sequence analysis

The Real time PCR product from the raw sewage and the positive control were
sequenced to examine the specificity of the vacA gene of prlori. The expected sizes
were isolated in a 1.75% low-melting-temperature agarose gel (SeaPlaque GTG;
Cambrex, Baltimore, Maryland) and purified with a QIAquick gel extraction kit (Qiagen,

Valencia, Calif). Then the concentration of each purified product was quantified by

24

Nanodrop (Wilmington, DE). Automated sequencing was accomplished with an AB] 377
DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.) at the Michigan
State University (MSU) Genomic Technology Support Facility (GTSF). The sequences
from the qPCR were compared with blast analysis using nucleotide Blast (Blastn). The
partial vacA gene sequences determined in this study and available in public data bases
(NCBI ) were analyzed with Squeb software, Genetics Computer Group (Madison,
Wis.). Multiple sequence alignment and phylogram analysis (Figure 2-6) of the partial
vacA gene was performed using the Pileup and GrowTree program respectively.
2.3 RESULTS
2.3.1 Survival Experiment

The viable counts of the H .pylori cultured in seeded groundwater were 1><
107CFUml'l at time zero and no colonies (<O.l CF Uml") were formed after 168 hrs when
stored at 4°C (Figure 2-1). The bacteria were no longer detectable by culture at
temperatures of 15°C after 24 hr of storage (Figure 2-2). The reduction in colony forming
units at each temperature was 0.5 and 3 logm reductions, respectively, at day] (Figure 2-1

and 2-2). H . pylori remained culturable for approximately 120 hours at 4°C.

25

Figure 2-1. Comparative evaluation of H. pylori measured by culture techniques, Most
probable number PCR and Real Time quantitative PCR (qPCR ) from the seeded
groundwater experiment for a temperature of 4 OC. (Results shown are the mean of three
replicates and average values from the two experiments).

Real Time PCR normalized data shown as (‘ ), MPN PCR normalized data shown as

M ), Colony forming Units (CF U) normalized data shown as ( 0).

Log 10(Nt/No) where Nt = Concentration of H. pylori cells at time (t) in hours

N0 = Starting concentration of H. pylori at time zero.

* Last two concentrations for CFU were non detectable and were the detection limits
used for the graphic.

 

 

g 1
Z
en 0 I
'30 \
o
,—-J -l .
V
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95A I
t g I
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(I) O O O O O O O O O O O
M \O 05 N 1n 00 —' <1” l\ C
Time inhours

26

H. pylori was detected in seeded groundwater for up to 12 days by conventional
most probable number (MPN) PCR. The average reduction in most probable number
(MPN) PCR at 4°C and 15°C were 1.3 and 2.3 loglO, respectively, at day 3 (Figure 2-
1&2-2). The Real-time qPCR method was also able to detect and quantify H. pylori in all
samples including those incubated for 12 days. The average reductions of copies of H.
pylori by real time qPCR at 4°C and 15°C were 1.8 and 1.2 loglO, respectively, at day 12
(Figure 2-1&2-2). The rate of reduction of H. pylori cells in culture condition occurred
more rapidly than when measured by either MPN PCR or qPCR (Figure 2-1& 2-2).
However using an MPN PCR the rate was faster than qPCR. The Real-time qPCR also
provided a 100 fold greater sensitivity for detection of H. pylori compared to MPN- PCR.
H. pylori was detected in seeded groundwater by species-specific PCR at both
temperatures tested throughout the study. We found that temperature was a significant
environmental factor associated with the culturability of H. pylori cells in groundwater.
qPCR methods allowed us to detect H. pylori in seeded ground water for 137 days even

though cells from these samples were not culturable.

27

Figure 2-2. Comparative analysis of Helicobacter pylori measured by culture techniques,
Most probable number PCR and Real Time quantitative PCR (qPCR ) from the seeded
groundwater experiment in temperature at 15 OC. (Results shown are the mean of three
replicates and average values from the two erferiments).

Real Time PCR normalized data shown as ( ), MPN PCR normalized data shown as

( I ) , Colony forming Units (CFU) normalized data shown as ( 0)

Log 10(Nt/No) where Nt = Concentration of H. pylori cells at time (t) in hours

N0 = Starting concentration of H. pylori at time zero.

* Last five concentrations for CFU were non detectable and detection limit used for the
graphic.

 

 

 

 

Surviving proportion of H.pylori (Log10 Nt/N0 value)

0 O O O O O O O O O O
m (D O) N In co ‘- V N O
‘- s- s- N N N (‘0

Time in hours

SEM examination showed normal spiral forms at time zero from the seeded
groundwater; the majority of the spiral forms were 2-4pm long and 0.5-0.8pm wide
(Figure 2-3). After 24h at 15°C, all cells observed were coccoid (Figure 2-4) and the
bacteria were non-culturable. At 4°C, the coccoid form started appearing after 72 h. Only

10 % of total observed fields showed the coccoid shape after day 3.

28

Figure 2-3. Scanning Electron Microscope (SEM) (X20,000) examination of H. pylori
spiral form at time Zero at 40C temperature from survival experiment. Scale bar
represents 1pm length.

 

 

 

 

29

 

Fig. 2-4. Morphology of coccoid form H pylori produced under 150C temperature
after 24 hr as observed by SEM (X20,000) from survival experiment. Scale bar represents
1 m length.

 

H pylori cells were detected in the slide immunoassay from the seeded
groundwater throughout the survival test study demonstrating the presumptive presence
of H pylori. H pylori occurrence was observed throughout the experiment and there was
no relation noted between the immunoassay labeled cells with temperature, thus
indicating no other major bacterial regrowth in the samples, or lysis of the cells of H
pylori.

2.3.2 Environmental sample analysis
Immunomagnetic separation and high speed centrifugation methods were

compared, followed by common DNA isolation techniques to determine which

30

processing method produced a higher quality and yield of DNA from wastewater
samples. There was no difference between the two methods based on DNA quality and
yield. Thus, DNA from all wastewater samples was isolated after high speed
centrifugation.

The specificity of Real-Time qPCR amplification of the vacA gene with E. coli
and Enteroccousfecium were tested and were PCR negative. C. jejuni amplification was
observed at higher concentrations of 3540ngpl-1 and only at high concentrations of cells,
with samples containing 108 cells, This also resulted in a poor signal only at the low end
of the standard curve. Thus H pylori amplification was readily distinguishable from this
small signal via the Tm (Melting temperature) and Ct (threshold cycle temperature) value
used in this assay. Additionally, the sequencing results from vacA (179bp) matched only
the H pylori vacA gene and no genes from any other organisms in the whole data base as
shown by blast analysis on the National Center for Biotechnology Information (NCBI)
data base. Amplified sequence of raw sewage from the qPCR had 100 % sequence
similarity with the vacuolating cytotoxin gene (vacA gene) of H pylori and all the
results or matches of the sequence blast search in the Gene Bank were specific to H
pylori strain. Multiple sequence alignment and phylogenetic of the raw sewage with
positive control of partial vacA gene was observed with 98 to 99% sequence similarity
and identity (Figure 2-6) without a single mismatch of nucleotides.

Upon optimization of real time qPCR conditions, we obtained a standard curve
with a linear range ( r2=0.991) across six logs of DNA concentrations for the threshold
cycle versus the copy number of H pylori cells (Figure 2-5). The detection limit was as

few as two bacteria in our assay (Figure 2-5).The one copies of gene considered as one

31

bacterium. Since the entire genome contains one vacA gene of H pylori. The real-time
PCR was 10 times more sensitive than the MPN PCR and 100 fold more sensitive than
conventional PCR methods. The melting curve of 39 samples showed good
reproducibility in real-time PCR and was similar to the melting curve analysis of the H
pylori vacA gene standard in each run.

Figure 2-5. Standard curve for serial 10 fold dilutions of H pylori VacA gene (copy
number 2 to 2X105) .Quantification was performed by determining the threshold cycle
(Ct) against the calculated copies of bacterial DNA. The straight line, which was
calculated by linear regression, shows an R2 of 0.991, slope: - 3.470875, intercept:
35.87289.

36

32 , 9
28 -

24 -

20

16

Cycle number

12-

0 1 2 3 4 5 6

Log concentration

32

Figure 2-6. Phylogram analysis of H pylori isolates. The phylogram is based on a partial
fragment of the vacA gene. Nodes indicate a common ancestor. The lengths of vertical
lines represent the degree of relatedness between isolates.H pyloriATTCC700329; H
pyloriWTP1(Raw sewage from May06 ); H pyloriWTP2(Raw sewage from Dec05 ),H
pylorigi3650211 (from Gene Bank data).

 

 

 

 

 

 

 

 

 

 

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As an application of the real-time PCR system, 39 raw sewage samples collected over a
one year period were screened for the numbers of H pylori cells (Fig 2-7 and Fig. 2-8).
Because there is only one copy of the vacA gene in H pylori, each gene copy was
assumed to be equal to one cell. Using qPCR 64 % of the sewage samples were positive
(25/39) for H pylori species specific gene vacA (Fig 2-7 and Fig 2-8) whereas samples

were negative by the general PCR method. Numbers ranged from 2-38 cells ml" in

33

sewage (Fig 2-7 and Fig 2-8). The maximum concentration of prlori was observed in
July 06 (Fig 2-8). Inhibition of qPCR was evaluated by seeding the control sample with
broth and sewage. Percentage of recovery of H pylori was 90% from the broth culture for
the qPCR method. The average recovery was 15% in wastewater samples.

Figure 2-7. Quantitative real time PCR analysis by absolute quantification and expressed

in amounts of H pylori per ml of water sample. Data (From Mar 05 to June 06) are
shown as the mean value of three replicates.

 

8

N
()1

N
0

Mar- Apr- May Jm Jm Jay- JUN Aug- Aug- Seo Seo Oct— Oct- Nov Nov Dec- Dec- Jar‘r Feo Mar— Apr- May Ju‘r
0505050505050505050505050505050505060606060606

Month

_.3
(’1

—§
0

Genomic DNA copies per ml

(’1

 

 

 

2.4 DISCUSSION

The efficiency of an infectious agent in spreading from one host to another is
crucial for its survival. H pylori could be considered a successful infectious agent, due to
the estimated high prevalence of chronic infection throughout most of the world (Hulten
et a1. 1998). This study provided information on the time of culturability, detection,
identity and quantification of prlori in water samples. This organism may be able to
survive for some period in water according to a number of studies; Fan et al. (1998)
reported that H pylori could survive for up to 10 days in milk at 4°C storage but only 4
days in tap water with a steady decrease of colony forming units at approximately 3 log

reductions. However, electron microsc0py clearly showed that the non-culturable coccoid

34

form was present in tap water which had been kept at 4°C for 7 days. The data in this
study were similar to theirs in that after 120 hrs (5days) the bacteria entered into a
nonculturable state in well water but remained detectable and quantifiable by MPN PCR
and qPCR assays. It is presumed that viable but nonculturable forms may be because of
changes in the membrane proteins and/or membrane integrity responsible for cell culture,
while the cells and the DNA remain present. H pylori culturability was also found to be
temperature dependent and increasing temperature enhanced the rate at which the
bacterium was no longer detected by culture methods.

The MPN-PCR demonstrated a 100-fold greater sensitivity for the detection of H
pylori compared to cultivation, while the Real-time quantitative PCR had even greater
sensitivity and had more sensitive (100 fold greater) than the MPN-PCR for screening
water samples. Our preliminary study presents a comparison of MPN PCR and Real time
PCR with a culture method and demonstrated that a stable signal for H pylori in
contaminated ground water can be found for up to 288 hours (more than 10 days). This is
one of the first studies to evaluate and contrast the three methods (cultivation,
conventional MPN PCR and qPCR).

In PCR based assays the detection of viable cells of H pylori cannot be
determined. Reports in the literature have suggested that degradation of nucleic acids
follows effects on membrane permeability and integrity, which is used to address the
loss of the potential for viability (Moreno et al, 2003b). However, quantification of the
PCR signal using qPCR provides more information on the stability and level of
contamination in water. While there have been a number of published articles regarding

detection of H pylori using conventional PCR in various water environments (Hulten et

35

01.1996; Hulten et al. 1998; Watson et al. 2004 ; Yingzhi et al. 2002), these have not
been applied to monitoring as was done in this study for wastewater and did not quantify
the level of contamination.

In this research, coexistence of spiral and coccoid forms were seen after 5 days in
ground water and different degraded forms were also present. The presence of these
degraded forms suggests that the coccoid forms were affected after prolonged exposure to
ground water, probably due to cell wall degeneration (Mizoguchi et al. 1998; Shirai et al.
2000). Some studies have shown that the coccoid form is non-culturable by ordinary
techniques but is still viable and can be converted to the spiral form under favorable
conditions (Shay and Axon 1996). From our current study, non-culturable coccoid H
pylori was present after 120 hours storage in ground water at 4°C. In this study, the
culturability of H pylori was highly correlated with the presence of spiral forms as
observed by SEM. We observed that H pylori was not culturable after 5 days but was
still detectable by both MPN PCR and qPCR. Thus, a possible VBNC (Viable but
nonculturable) form may occur after 120 hours (cells transformed to the different shape
as observed by SEM) and, in that stage, was nonculturable but quantifiable by molecular
methods. This evidence suggests that more attention should be paid to morphological
changes over time in water at that stage where it is no longer possible to culture the
organism and, also, the public health risk associated with the possibility of H pylori
retaining its infectivity should be further evaluated. Adam et a1, (2003), also found that
H pylori is able to enter the VBNC state as cells age in the laboratory or when exposed
to a natural, freshwater environment. Cells underwent a transition from culturable rods to

predominantely nonculturable cocci as they entered the VBNC state in the laboratory.

36

Reports (Cellini et al. 1994; Shay and Axon 1996) have shown that ground water may act
as vehicle for transmission of H pylori.

Kuster er al , (1997) reported that culturability of H pylori suspension was lost
when almost 50% of cells were still in a bacillary form. A decrease in nucleic acid
content and loss in membrane potential were also observed. So, these authors considered
coccoid forms as a manifestation of bacterial degeneration. Other authors have found
great diversity in metabolic features of these coccoid forms, suggesting that only some of
these forms may be viable cells while most of them may be non revivable (Mizoguchi et
al, 1998). Animal infectivity studies will be needed to clarify the risk under these
conditions.

The US EPA is considering the regulation of prlori in drinking waters and as a
part of the risk assessment framework needs to obtain more information on the
occurrence of the bacterium in water supplies in the US as part of CCL (Contaminant
Candidate List) (McDaniels et al. 2005). Currently, isolation of H pylori cells from
environmental and drinking water samples by cultivation methods is rare, which may be
due to their transformation into the coccoid from (Kuster et al. 1997) and or the
unavailability of an appropriate growth medium. Thus, we believe that meeting the
requirements of the CCL (Contaminant Candidate List) for conducting a national study
on the occurrence of H pylori in water and potential for exposure cannot be
accomplished without molecular techniques. A Quantitative Real Time PCR method for
quantifying H pylori has been applied in a clinical setting (He et al. 2002; Mikula et al.

2003) but has not yet been used for any environmental study.

37

Figure 2-8. Genomic copies of H pylori found in waste water using Real Time PCR.

copiesperml
a. a a B a: 8 8 8

r l c . . l v I -

July- Aug Sep- Oct- Nov Dec- Jan- Feb- Mar- Apr- May- Jun- July- Aug- Sep- Oct-
06060606060607070707070707070707

Months

Genomic DNA

0

This is the first Real Time qPCR assessment of environmental waters where
quantification of H pylori is combined with melting curve analysis (by SYBR green
assay).The advantage of melting curve is that it provides primer and target reliability,
accuracy and it is observed that target (vacA gene) consistently amplified at same
dissociation temperature.This method is also easy and cost effective in comparison with
the Taqman Assay. Primers pairs were specific in our qPCR assay, since it amplified only
H pylori partial vacA gene sequences which were then proved by the sequence analysis
and from the Blast results. This genetic evidence supports that the vacA gene of H pylori
was present in raw sewage samples. The Real-Time qPCR was able to detect as low as
two copies/reaction of the plasmid containing the vacA gene and the efficiency of
performance was confirmed in 10 different runs with the same reaction conditions. The
standard curves indicated a good correlation between the amount of template (Log

copies) and the amount of product (represented by Ct ) (r2 = 0.991). The linearity of the

38

standard curves and the fact that the PCR operates with constant efficiency confirmed
that the assay was well suited to quantitative measurements of H pylori from
environmental samples.

This present study examined the presence of H pylori in waste water samples by
Real Time PCR in a temporal survey obtaining quantitative assessment of H pylori
during 1 year. It was found that sewage is a source of H pylori, and the bacterium is
commonly found in community sewage at levels between 2 and 38 cells/ml. This has also
demonstrated that there is variability of infection and/or variability in excretion rates in
the community. The novel aspects of this work focuses on several areas A) assessment
of environmental samples with adequate sensitivity and specificity (in terms of accuracy
and reproducibility), B) first paper to use a SYBR green method for quantification of H
pylori from environmental water samples (which demonstrated a greater reliable and.
cheaper method than the TaqMan Assay), C) A novel primer set for environmental
applications, and D) demonstration of the method for routine monitoring of sewage for
Helicobacter. There has only been one paper to our knowledge which examined real
time PCR for drinking water but failed to detect naturally occurring Helicobacter
(McDaniels et al. 2005). There are only a few papers published so far that used PCR not
qPCR for detection of H pylori. in sewage (Yingzhi et al. 2002) and this was not used
for routine monitoring.

The low recovery in waste water influences the detection limit when using the qPCR
method. Recovery could be influenced by different factors such as the presence of
inhibitors in the sample. The Tm value determined how well the sequence of primers or

probes matched the sequence of template DNA, and it will decrease if mismatched DNA

39

isa

up

to

is amplified (Baczynska er a1. 2004). Single mismatches can decrease the Tm from 1 oC
up to 30 °C (Aboul-ela et al. 1989; Ke et al. 1993) depending on many factors, such as
pH, duplex length and G+C content. In the present study, the Tm of the standard
template and positive environmental samples was very consistent and showed a similar
melting cure with single peak at 77.9 0C temperatures.

One of the most striking results was that most of the samples were negative for H
pylori by general PCR, but contained H pylori DNA when analyzed by qPCR. A
possible explanation for this is that higher sensitivity and a lower detection limit are
obtained using qPCR. 16s rRNA is a conserved sequence for Helicobacter; therefore,
there was little variation among the samples. This is a promising result because H pylori
are found in the environment at very low numbers. In an earlier study in lakes and ground
water Benson et al. (2004) reported a small number of H pylori cells by MPN DPCR
which is similar to the findings reported here.

Helicobacter pylori may have developed a mechanism to resist adverse effects of
aquatic environments by entering a persistent or dormant coccoid form and may remain
viable for some period. This work suggests that cultivation methods are not applicable to
environmental samples. This supports the work published by McDaniel er al. 2005.
Results presented show low concentrations of H pylori cells in raw sewage samples;
these cells were not detectable by cultivation or by routine PCR. We think this is
evidence in support of the hypothesis that H pylori represent a waterborne disease risk
due to untreated fecal or raw sewage contamination of the aquatic environment. This
hypothesis is also supported by Benson et al. (2004). Helicobacter may be similar to

viruses such as noroviruses and other non-cultivatable pathogens in that molecular tools

40

will be the methods of choice to characterize their occurrence in and “potential” risk for
water. Thus, qPCR allows for vulnerability assessment and relative quantification from
environmental samples. The H pylori seasonal assay for sewage demonstrated the
variability of infection in the community and the possible source of water contamination.
The results indicated that wastewater was commonly contaminated with H pylori and
could serve as a source for potential human health risks via water borne transmission.

2.5 CONCLUSION

This study showed that H pylori culturability is temperature dependent. In this results,
the culturability of H pylori was highly correlated with the presence of spiral forms as
observed by SEM. We have established a Real Time assay targeting the vacA gene
(vacuolating cytotoxin gene) for quantitative detection of H pylori using the SYBR
Green method. When comparing traditional PCR protocols with the newly developed
real-time protocol, the new method offers interesting advantages such as rapidity,
sensitivity, a closed system (avoid contaminaition), and quantification of amplification
product. To test the new PCR assay, raw water from one wastewater treatment plant was
screened for the presence of H pylori. The development of these tools and techniques
will further serve the water industry, regulatory community, and public health
community. Future goals for the estimation and protection of drinking water quality are
moving toward the ability to help forecast the potential for risk and mitigation before
outbreaks or disease transmission occur. The health risks of ulcers and cancer from H
pylori infections suggest that it will be important to reduce exposure via water. H pylori
seasonal occurrence in sewage demonstrated the variability of infection in the population

and suggests sewage may be the possible source of water contamination.

41

CHAPTER 3

SUVEILLANCE AND QUANTITATIVE IDENTIFICATION OF
CAMPYLOBA C T ER JEJUNI FROM WASTE WATER BY REAL TIME QPCR

3.1 REVIEW OF LITERTURE
3.1.1 History and Taxonomy of Campylobacter

In 1963 the genus Campylobacter was first proposed by Sebald and Veron (1963),
however the taxonomic determination of Campylobacter spp was unclear due to
phenotypical mis-identification and lack of molecular and genetic analysis. Gradually
with increased interest due to the high prevalence of the bacteria associated with human
diarrhoea in the study of Butzler er al, (1973) and the availability of adequate isolation
procedures, Campylobacter research advanced during 1981 and certainly Campylobacter
like organisms were isolated from human, animal and environmental sources. The
phylogenetic relationships using the potential of the 16s rRNA gene (Woese, 1987)
played a major role in an extensive rearrangement of Campylobacter taxonomy. At
present, the genus Campylobacter contains 16 species distributed among six subspecies
and belongs to the group referred to as the epsilon division of the class Proteobacteria.
Many of these species and their taxonomic diversity match the diverse habitats and the
wide range of diseases that their are associated (On S.L.W.,2001).
3.1.2 Biology of Campylobacter

Campylobacter are Gram negative, small, motile, spirally curved, rod shaped,
microaerophilic bacteria with a polar flagellum at one or both ends of the cell. The
organisms are catalase and oxidase positive and urease negative. The favorable

temperature for growth ranges from 33-44 °C, and the optimal temperature required for

42

growth is 42 °C. Campylobacter spp require complex growth media, and are unable to
ferment carbohydrate due to fastidious nature of the organisms. The genome size is
approximately 1600 kb for Cy'ejuni which is relatively small compared to Escherichia
coli (Chang and Taylor, 1990) and the G+C content of Campylobacter is low (on an
average 30-35%) which can elicit difficulties when cloning the A+T rich sequences.
C. jejuni is in nature transforrnable and able to carry out conjugation naturally.

Campylobacter changes shapes from spiral to coccoid forms on prolonged
exposure to environmental stressors (e.g. temperature, oxygen, nutrient depletion etc)
(McKay, A.M., 1992) (Vilet and Ketley, 2001). These coccoid forms are referred to as a
viable but non-culturable state (VBNC), and it is suggested that this form is in a
dormant state, which is not actively growing (Rollins and Colwell 1986). However the
pathogenicity of Campylobacter remains unclear in the VBNC form (Cappelier et al,
1999 and Jones et al, 1991).
3.1.3 Disease prevalence and transmission

Each year in the United States, an estimated 2.1 to 2.4 million cases of human
Campylobacteriosis (illnesses ranging from loose stools to dysentery) occurs (Tauxe
R.V., 1992 and Vilet and Ketley, 2001). Disease associated with Campylobacter usually
leads to gastrointestinal illness and diarrhoea, which is often more noticeable in young
children and young adults in industrialized countries (Vilet and Ketley, 2001). There is a
high rate of asymptomatic carriage, and milder clinical symptoms of watery non-
inflammatory diarrhea are also seen in developing countries (Ketley, 1997). Generally the
disease is limited to a period of 5-8 day, but may continue longer. The symptoms

progress to profuse diarrhoea which later contains mucus and blood. C. jejuni infection

43

has also been associated with a neuromuscular disorder Guillian Barre Syndrome (GBS)
(Allos er al, 1998).

Campylobacters are regarded as normal intestinal flora of a wide range of
domestic and wild animals (Vilet and Ketley, 2001). Fecal contamination of meat,
uncooked meat or other cross-contaminated food products, and animal products, notably
broiler chickens, are the main source of Campylobacters in food (Vilet and Ketley, 2001).
Most infections are sporadic and believed to be food borne and water borne; large
outbreaks are infrequent and mostly due to the consumption of raw milk or unchlorinated
contaminated water (Skirrow M.B.,l991 and Clark et al ,2003). Other risk factors
accounting for a smaller proportion of sporadic illnesses include drinking untreated
water; traveling abroad; or consumption of sausage. It is suggested that person-to-person
transmission is uncommon (Norkrans and Svedhem, 1982).

Table 3-1. Some of the important waterborne outbreaks due to Campylobacter spp.

 

 

 

 

 

 

 

Year of Study Water type Source of References
outbreak Location contamination
2004 USA Well water Waste water Fong et al,
2007
2000 Canada Town water Cattle farm Clark et al ,
supply 2003
2000 Finland Drinking water Ground water Hanninen et
* al, 2003
1997 England Drinking water School camp Frost et al ,
2002
1990 New Zealand Drinking water Run off spring MMWR ,
water 1991

 

 

 

 

 

 

Some of the important waterborne outbreak associated with Campylobacter spp. Are
listed in table (3-1).The most notable Canadian waterborne outbreak involving

Campylobacter in recent history occurred in Walkerton, Ontario, in May 2000 (Clark et

44

al., 2003). This outbreak was linked to faecally (cattle farm) contaminated well water that
was not properly treated before consumption. Other important water borne outbreaks
associated with Campylobacter isolated from surface and well waters have been reported
in the literature (MMWR, 1991; Frost et' al, 2003). Recently Fong et al,(2007) reported
that Campylobacter-like isolates were detected from groundwater in the vicinity of a
waterborne disease outbreak on South Bass Island, Ohio. A case-control study was
conducted by the CDC and found significant associations between gastroenteritis
symptoms and well water consumption. and C. jejuni was detected in human stools of the
human cases. They suggested that this waterborne outbreak was caused by transport of
microbiological contaminants from sewage discharges. The water related outbreaks
signify how the survival characteristics of C. jejuni for extended periods in a aqueous
environments occur at infectious concentrations, perhaps even in a VBNC state (Rollins
and Colwell, 1986).
3.1.4 Other Evidence of transmission via water

One of the most important reservoirs of Campylobacter species may be surface
water which is a source associated with infections in humans as well as for poultry and
live stock (Refregier—Petton et al, 2001). Campylobacter species have been detected in
contaminated drinking water, ground water, rivers, lake, and marine water and run off
worldwide. (Hanninen et al , 2003; Arvanitidou, M. et al , 1995 ; Alonso and Alonso,
1993). The transport of Campylobacter into surface water is due to: 1) excretion of the
organism from waterfowl and gulls into water, 2) flushing of the bacteria into water

supplies during heavy rainfall and flooding or runoff from agricultural and residential

45

land, 3) waste water releases from poultry and meat processing facilities and 4)
contamination of water with municipal sewage. (Bolton et al, 1987).
3.1.5 Seasonal variation

The seasonal patterns of Campylobacter in surface water have reported that
during the late autumn and winter, the prevalence of Campylobacter in surface waters is
highest and lowest in spring and summer (Bolton et al, 1987 and Carter et al, 1987). The
seasonality may arise because Campylobacters survive for longer periods in relatively
cold water. Unlikely in natural waters, Campylobacters in sewage effluent may peak in
summer months in response to infection rates in the human community and level of
contamination in slaughter house waste (Jones, et al , 1990). However an unusual
pattern for Campylobacter spp in sewage effluent was observed in the Netherlands
where the highest levels were observed at different periods of the year. Lesser abundance
was reported during June —August (Koenraad er al, 1994).
3.1.6 Detection of Campylobacter in water

The detection of stressed Campylobacter cells in water, presents several
challenges. The number of cells in naturally contaminated water often is quite low and
the slow grth rate against a large background of native bacterial flora means that a
sensitive recovery method is required for monitoring water. The conventional culturing
methods usually involve filtration to concentrate the cells followed by enrichment and
plating (Salis et al, 2002). The standard culture methods only measure viable cells but
recovery can be lower and the VBNC cells may not be detected. However, water has
been reported to retain a high population of VBNC Campylobacter (Rollins and Colwell,

1986). Standardization of culture method for the distinction of Campylobacter spp in

46

aquatic samples has not yet been achieved. Therefore, detection methods that attempt to
identify all water-bome Campylobacter are generally polymerase chain reaction (PCR)
based.

Table 3-2. Some of the PCR based studies for the detection of Campylobacter spp in
water.

 

 

 

 

 

 

 

References Location Water type Target gene

Waage et al, 1999 Norway Sewage water flaA and flaB

Salis et al , 2002 United kingdom Pond, lake, canals, ORF-C gene
coastal water species specific

motifs

Moreno et al, 2003a Spain River water and 168 r RNA
waste water fragment

Yang et al , 2003 China Surface water and VS] sequences
ground water

Walters et al , 2007 Canada River water 168 r RNA, MapA

 

 

 

 

and CeuE gene

 

A number of PCR assays have been developed for the detection of C. jejuni in
water (Table 3-2) and employ enrichment and or sample filtration (Waage et al , 1999
Sails et al , 2002; Moreno et al , 2003a). Waage et al, 1999 reported the level of
sensitivity to detect C. jejuni was between 3 to 30 cells / 100ml of water by qPCR assay.
A variety of PCR protocols using various primers have been used to detect
Campylobacters in water and waste water but analysis focused only on presence or
absence of Campylobacters in water and lacked quantitative assessment (Krik and Rowe,
1994; Wage er al, 1999). The usefulness of real-time PCR using a TaqMan probe with
the aim of obtaining for quantitative detection of Cjejuni in poultry, milk and
environmental water has been established by Yang et al, (2003). Report on naturally

contaminated environmental water samples analyzed by the real-time PCR assay without

47

prior enrichment showed that 13.6% (41/300) of natural surface and ground water
samples were positive for the presence of C. jejuni (Yang et al, 2003) .
This research focus of my study was to advance further the use of qPCR to investigate the
presence of C. jejuni in waste water. In this case, the goal was to establish a rapid,
sensitive real time assay targeting approximately 200 bp fragment of gyrA gene for
quantitative detection of C .jejuni using a TaqMan Probe and to use this assay to conduct
annual surveys for the quantitative detection of C. jejuni in waste water.
3.2 MATERIALS AND METHODS
3.2.1 Collection of strain and culture methods

A pure culture of Campylobacter jejuni ATCC11168 was obtained from the
laboratory of Dr John E. Linz , at the National Food Safety and Toxicology Center,
Michigan State University. The isolates were plated on Bolton Selective Enrichment
Agar - BSEA (Oxoid) and flooded with 10 ml of Bolton broth supplemented with 5%
sheeps blood (Cleveland Scientific, Bath, Ohio). The plates were placed in an anaerobic
jar and incubated at 37°C, with 40 rpm agitation under an atmosphere of 10% C02, 10%
H2, 80% N2. After 48 hr the colonies were harvested with a sterile cotton swab and were
suspended in 3ml of Bolton Broth. Bacterial cultures were pelleted and DNA was
extracted.
3.2.2 Sample collection, cultivation and extraction

For sample collection, a grab sample protocol was used (Standard methods 1995).
Aseptic techniques were followed while sampling. Three raw wastewater samples (50ml
each) were collected from a wastewater treatment plant (East Lansing, MI) once a month

from January 2006 to October 2007. Samples were processed by high speed

48

centrifugation (5000 rpm, 20 minutes ), followed by cell lysis (for 30 minute at 37°C) and
DNA extraction using QIAmp DNA mini kit (Valencia, CA, USA). Volumes of lml
were concentrated and DNA extracted to 400 pl for PCR.
3.2.3 Amplification and quantification of gyrA gene

A gyrA fragment (420bp) of Campylobacter strain was amplified with primers
previously described by Husmann et a1 (1997) by general PCR. Reagent concentrations
in the PCR reactions were as follows: 5 - 10 ng/pl templates, 0.2 mM each dNTP, 1X
PCR buffer, 0.5 pmole/pl each primer, approximately 1.5mM MgCl2 and 2.5U of Taq
polymerase (lnvitrogen, CA). Thermocycler parameters were as follows: 1 min at 94°C
for denaturing, 1 min at 50°C for annealing, and 30 sec at 72°C for extension for 32
cycles. The amplified PCR product was placed on a agarose gel (1.8%, w/v)
electrophoresis, purified the target product by QIAmp PCR purification kit (Valencia,
CA, USA). After that, the purified target of part of gyrA gene was cloned into plasmid
vector (pCR® 2.1 TOPO®) and transformed into TOPlO Competent cells (One Shot®
Chemical Transformation) based on TOPO TA Cloning Instruction Manual (Invitrogen,
Carlsbad, CA). Plasmid DNA was purified using a DNA purification kit (Wizard® Plus
SV Minipreps DNA Purification System, Promega, Madison, WI). Purification
procedures were based on Technical Bulletin of Wizard® Plus SV Minipreps DNA
Purification System. Automated DNA sequencing was accomplished with an ABI Prism
3100 Genetic Analyzer (Applied Biosystems, Foster City, CA), at the GTSF at MSU
further confirmed the targeted gyrA fragment had been cloned.

3.2.4 Quantitative Real Time PCR

49

The primer and TaqMan Probe (5’ TTT GCT TCA GTA TAA CGC ATC GCA
GC 3’ ) (IDT INC, coralville, IA) were used following the real-time PCR methods of
Wilson et al (2000). The primers included (JL 238 (F): 5’ TGG GTG CTG TTA TAG
GTC GT 3’and JL 239 (R): 5’ GCT CAT GAG AAA GTT TAC TC 3’) published by
Wilson et a1 (2000). Each reaction mixture consisted of 2 pl of DNA (maintaining the
same concentration of template), 1.0 pl each of forward (5 pM) and reverse primers (5
pM), 1 pl of TaqMan probe (lOpM), 11 pl of nuclease free water, and 4 pl of LightCycler
Master Mix (5X) (Roche,Indianapolis,IN). PCR master mix in a total reaction volume of
20 pl reactions were performed in duplicate for each sample in a LightCycler® (Roche,
Indianapolis, IN). PCR programming included denaturation of 95°C for 10 min,
amplification at 95°C for 10 sec, 60°C for 30 sec and 72°C for 5 sec for 50 cycles, and
finally cooling for 30 sec at 40 °C. A Standard curve for the gyrA gene was constructed
using tenfold serial dilutions of plasmid DNA. The run was carried out on six dilutions
(2. 4 x 10 to 2. 4 x 106 copies) in duplicate with each 2ul volume. The threshold cycle
temperatures were observed according to the corresponding standard dilution. Real time
PCR was used for absolute quantification of the gene following the methods of Whelan et
al , (2003)

The Real time PCR product from the raw sewage and positive control were
sequenced to examine the specificity of the gyrA gene of Cjejuni. The expected sizes
were analyzed in a 1.8% agarose gel and purified with a QIAquick PCR purification kit

(Qiagen, Valencia, CA.).

50

3.3 RESULTS

The target gyrA fragment of Cjeuni (cloned product) was confirmed in sequence
analysis and used as a template for standardization of real time PCR. Additionally, the
sequencing results matched the Cjejum' gyrA region but no gene from any other
organism (from whole blast assemble of microbes data base), as shown by blast analysis
on the National Center for Biotechnology Information web site.

To determine the linear range of quantification, a standard curve of the template
DNA genome equivalent copy numbers and threshold cycles Ct was automatically
generated by the instrument for three replicate sets of controls in the real-time PCR assay.
The assay had a linear range of quantification of between 2.4 x 101 and 2.4 X 106 genome
equivalents per PCR, and the limit of detection was approximately 24 genome
equivalents per reaction. The standard curve based on the dilutions of DNA showed a
linear relationship between log concentration and threshold cycles (Ct) (Figure 3-1). The
linear regression was 0.991 and the mean square error was 0.045 (The standard value of
mean square level should be less than 0.1).The reproducibility of the standard curve was

observed to be very consistent in each replicate run which were almost identical.

51

Figure 3-1. Standard curve for serial 10 fold dilutions of Cjejuni fragment of gyrA gene
(copy number 24 to 2.4 X 106 in 2 pl). Quantification was performed by determining the
threshold cycle (Ct) against the calculated copies of bacterial DNA. The straight line,
which was calculated by linear regression, shows an r2 of 0.991 and mean square error =

0.045

50*
45 .
4o -
35 _
30 y
25 .
20 .
15
10 .

0.

Threshold cycle (Ct)

0 1 I 2 3 4 5 6 7
Log Concentration
A qualitative verification of Light Cycler qPCR with the amplification of primers
was performed for the selected samples. The results confirmed that amplification using
the designed primers and probes produced the exact fragment of gyrA approximately
200bp for C. jejuni. The PCR products were analyzed on 1.8% agarose gels. In addition,

no non specific PCR products were detected (Figure 3-2).

52

Figure 3-2. Agarose gel showing amplified Realtime PCR products of gyrA gene A)
Lane M is a 100 bp marker, Lane P- Positive control , Lanes Sl — S4 isolates positive
from waste water, Lanes SS —Negative from waste water and Lane N- Water (Negative
control)

M PS1SZS3S4S5N

 

Since there is only one copy of the gyrA gene in each cell of C. jejuni (Wang et
al , 1993), the gyrA gene copy number can in theory be used to determine the number of
C. jejuni in an unknown sample. Upon optimization of Light cycler qPCR conditions,
twenty two raw sewage samples collected over a period of 22 consecutive months were
assayed for C. jejuni (Table 3-3 and Figure 3-3). Thirteen samples (59 %) were positive
for the C. jejuni partial fragment gyrA gene when examining raw sewage samples. The
concentrations of C. jejuni in positive sewage samples ranged from 1.7 x103 to 9.9 X 104

cells ml". The maximum concentration of C. jejuni was observed in September 2007

53

(Table 3-3 and Figure 3-3). In 2006, a high concentration of C. jejuni was found in July
which was 5.5 X 104 cells ml". Seven of the months were positive in 2006 (Table 3-3 and
Figure 3-3), while six samples were positive during 10 months of sampling in 2007.
Inhibition of the reaction in the light cycler qPCR was evaluated by seeding a known
concentration of control sample into broth and raw sewage. The recovery percentage of
C. jejuni was 90% from the broth culture for the TaqMan qPCR assay. The average
recovery was 6 % in raw sewage samples. The recovery concentration result was
consistent in duplicate experiments.

Table 3-3. Quantitative detection Cjejuni in monthly sewage samples from 2006 to
2007. N= Total sample test per month. For Average calculation included the zero value.

 

 

 

Month Mean values of DNA copies per ml
Jan 06 0

Feb 06 1.7lx103
Mar 06 1.8x 103
Apri 06 3.03 ><104
May 06 0

June 06 0

July 06 5.56x104
Aug 06 3.36 ><103
Sept 06 0

Oct 06 0

Nov 06 6.48x 103
Dec 06 2.29><104
Jan 07 2.26x103
Feb 07 6.5><103
Mar 07 2.95x103
April 07 3.87x103
May 07 0

June 07 0

July 07 3.56><103
Aug 07 0

Sept 07 9.94x104
Oa07 0

N=22 Average =1 .lzsxlo4

54

Table 3-4 The level of C. jejuni concentrations in waste water in different seasons .
n = Total number of sample tested in that season. * For Average calculation included the
all the non detected samples value as zero.

 

 

 

 

 

 

 

 

Season Winter Spring Summer Fall
(Dec,Jan,Feb) (Mar,April,May) (June,July,Aug) (Sept, Oct, Nov)

Percentage 80% 66% 50% 40%

tested positive n=5 n=6 n=6 n=5

Average

concentrations 8.14><103 6.48X103 1.04><104 2.1l><104

(cells/ml)

 

Eighty percent of C. jejuni positive samples were detected in waste water during

the winter as shown in Table (3-4), however the concentrations of C. jejuni were ten-fold

higher in summer despite a smaller percentage of positive samples (40% to 50%). The

average concentration observed in the fall was 2.11X104 cells /ml.

55

 

Figure 3-3. Quantification value by using quantitative Light cycler qPCR analysis and
expressed in amounts of Campylobacterjejuni per milliliter of water sample. Data shown
as mean value of three replicates.

120000

100000

2

:mm

:W'

3

o- -1, - ,--- -13-. ,I 1J1} l
JarFeb-IuH/pn-May-Jnfly-Mg-Sept-OctNov-Dec-Jan-Feb-Iar/pri-Ity-M-My-Mg-Sept-Oct-
00000000060000060000060607070701010707010707
month
3.4 DISCUSSION

C. jejuni is recognized as a leading human food-borne and water borne pathogen.
Recently USEPA announced the CCL 3 in which C. jejuni was included as an
unregulated contaminant that may require a national drinking water regulation in the
future. However, the detection of Campylobacter from environmental and drinking water
samples by culture methods is challenging, (Yang et a1 , 2003) and more over, it is now

understood that the detection of C. jejuni VBNC cells is important as they may remain

56

infectious (Hege er al , 2000). In order to meet the requirements of the CCL for
conducting a quantitative screening survey of C. jejuni in water and more importantly to
address the potential for exposure molecular techniques should be used. Application of
real-time PCR for quantitative detection C. jejuni has been evaluated in environmental
waters (Yang et al , 2003) but has not yet been used for yearly monitoring of waste water
samples which could indicate both community infection and potential for environmental
health risk.

This current study that investigated the presence of C. jejuni in waste water
samples by Real Time PCR is the first annual quantitative assessment of C.jejuni in
waste water. The detection limit of the light cycler qPCR was estimated at 24 copies per
reaction of the fragment of gyrA gene and the reproducibility of the accuracy was
confirmed in three different runs with the same reaction conditions. The detection limit in
our study was efficient compared to LightCycler qPCR method reported by Ridley et al
(2008). They reported the detection limit for C. jejuni strain in the TaqMan assay was
estimated to be 3.7X102 CFU/ml .The standard curve (R2=O.991) in our assay
represented a good correlation between the amounts of template (log copies) and the
amount of product (represented by Ct ).

The current study established a yearly quantity survey of C. jejuni from raw
sewage. However, previously various PCR based studies have been employed to detect
Campylobacters in water and waste water but mostly the studies reported the presence or
absence of Campylobacter (Krik and Rowe, 1994; Salis et a1 , 2002). In the present
study, both VBNC and normal forms of Cjejuni were quantified. Waage er a1 , (1999)

detected Campylobacters from water and waste water after culture enrichment culture

57

and reported 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a
background flora of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of
coliforrn bacteria per 100 ml. Thus this study detected 1000x higher concentrations

(102 /ml). It has been suggested by other studies that all forms of C. jejuni, including
VBNC forms should be detected and considered infectious and pathogenic (Cappelier et
al, 1999). Yang et al, (2003) suggested that they had detected C. jejuni in naturally
contaminated environmental water by direct PCR without need for prior enrichment
culture of samples but their experiment did not report the yearly quantitative detection.
They reported that using real time PCR detection system 13.6% were found to be positive
for C. jejuni, whereas in API Campy system 8% were positive for C. jejuni from the
water samples.

In this study C. jejuni excretion by the population into sewage was observed
throughout the year (Figure 3-3 and Table 3-1). Thus infection rates and or excretion
rates in that community were temporally heterogenous. In this study the overall average
concentration detected in sewage was 1.128X104cells/ml which is much higher (10x to
100x) than other studies and the average value included the sample detected zero.
Diergaardt et al., (2004) found Campylobacter numbers in the surface water samples
ranged from less than 10 cfu/ 100 ml to more than 102 cfu/ 100 ml on modified blood-free
charcoal cefoperazone deoxycholate agar (mCCDA) and more than 103 cfir/ 100 ml on
blood agar. This demonstrated the culture method is less efficient than real time PCR
assay for the quantitative detection of C.jejuni from water. Koenraad et al, (1994) also
examined Campylobacter spp occurrence in raw sewage effluent in the Netherlands.

They reported that at three municipal wastewater plants the seasonal variation of

58

Campylobacter spp in the numbers observed in the sewage of the activated sludge
system; during the end of the summer the numbers were on average 1 loglo unit smaller
than during the rest of the year. Others have reported the prevalence of Campylobacter in
surface water highest in late autumn and winter and lowest in spring a summer (Carter et
al, 1987).

3.5 CONCLUSION

This study presents the first quantitative seasonal investigation of C. jejuni in sewage
samples with real-time PCR, and it represents a considerable advancement in the ability
to make a rapid assessment of pathogen quality of waters. The study shows sewage is a

source of C. jejuni contamination to water ways.

59

CHAPTER 4

SUMMARY

Tracking trends of new and reemerging infectious diseases will continue to increase
in near future due to mutation, selection, and evolutionary changes influenced by the
environment and genetic nature of host. Recently US EPA has placed C. jejuni and H
pylori on the Candidate Contaminant List (CCL) which contains new contaminants that
are known or anticipated to occur in public water systems. One of the problems in
understanding the source of contamination is the difficulty in isolating pathogenic
organisms from the environment. Unfortunately, there are several constraints concerning
detection of H pylori and C. jejuni in food or water using culture methods. The three
most important problems are the small numbers in the environment, slow growth rate and
the possibility that these bacteria can enter a viable but nonculturable (VBNC) state. The
major goal of this research was to develop rapid and sensitive methods that could be used
for the detection and quantification of H pylori and C. jejuni which will aid in
maintaining a safe food or water supply.

Rapid methods for the quantitative analysis of H pylori and Cjejuni abundance in
water were developed. These techniques offered a significant improvement over other
available methods for detecting H pylroi and C. jejuni from environmental samples. One
of the most significant results was that most of the samples that were negative for H
pylori by general PCR, yet contained H pylori DNA when analyzed by real-time qPCR.
The possible explanation for this is that a high sensitivity (low level detection limit) is

obtained using the Real-time q PCR method.

60

The main advantage of Real-Time qPCR is that the amplification of PCR during the

early phases of the reaction. Measuring the kinetics of the reaction provides a distinct

advantage over traditional PCR (which only measures the end point of amplification by

agarose gel). In real-time PCR, reactions are characterized by the point in time during

cycling when amplification of a target is first detected rather than the amount of target

accumulated after a fixed number of cycles. This quantifies the amount of PCR product

at each cycle and uses a threshold detection for relative abundance.

The important findings are summarized as below

This study showed that H pylori culturability is temperature dependent.

The preliminary work here presents the first comparison of three different
methods (MPN PCR, Real time PCR and culture method) and demonstrated the
assessment of the stability of H pylori in ground water.

Real time PCR was demonstrated to be significantly more (100-fold greater)
sensitive for detection of H pylori compared to MPN PCR.

The culturability of H pylori was highly correlated with the presence of spiral
forms as observed by SEM. It was observed that H pylori were not culturable
after 5 days but were still detectable by both MPN PCR and qPCR.

This is the first Real Time qPCR assessment of environmental waters where
quantification of H pylori is combined with melting curve analysis (by SYBR
green assay).

Real Time qPCR assay provided a specific, sensitive and rapid method for

quantification of H pylori in waste water.

61

I The results indicated that wastewater was commonly contaminated with H pylori
and could serve as a source for potential human health risks via water borne
transmission.

I In a survey of H pylori annually in sewage, it was demonstrated that there was a
variability of infection in the community and in the excretion rates.

I The results in my study also indicated that wastewater was consistently
contaminated with C. jejuni, showing variable excretion rates from the population
or community after a one year survey.

This study verified the development of novel technologies for detection of emerging
waterborne pathogens. The development of these tools and techniques will further serve
the water industry, regulatory community and public health. The future goals for the
estimation and protection of drinking water quality are moving toward the ability to help
forecast the potential for risk and mitigation before outbreaks or disease transmission
occur.

Many bacteria may transform to VBNC (viable and non culturable ) stage under
stress in the environment or under nutrient deficient conditions. In this stage, the
bacteria are not cultivable by routine laboratory procedures but are quantifiable by
molecular methods. There is some evidence that this stage is more infectious for H
pylori (She and Lin, 2003). Therefore further research should be undertaken to study the
morphological changes over time in water and evaluate to what end point H pylori
retains its infectivity. These studies will likely require an infectivity assay with an animal

model experiment.

62

A low recovery in waste water was found (6 % for C. jejuni and 15% for H pylori) and
this influenced the detection limit when using the qPCR method. Low recovery could be
caused by different factors such as the presence of inhibitors in the sample. New
approaches are needed to reduce inhibitors in sewage samples without the loss of the
target. These could include chemical flocculation where multivalent cations were
investigated as a potential method for eliminating soil-based inhibitors during the
extraction process ( Braid et al, 2003). Braid et al reported that the addition of
AINH4(SO4)2 during extraction significantly reduced the co-purification of PCR
inhibitors with minimal loss of DNA yield.

This research was targeted and only one location (waste water treatment plant) was
used for the survey of the occurrence of bacterial pathogens. Other facilities should be
studied from different geographical locations from various populations, in order to
identify comprehensive infection and or excretion rates. More annual assessment is
required to understand if any statistical significance of a seasonal variability exists.
However, Bolton et al, (1987) reported a seasonal effect on the isolation of
Campylobacter in surface waters which was highest in late autumn and winter and lowest
in spring and summer. It is probable that the differences in the concentrations of
Campylobacter in waste water versus natural waters may be due to environmental factors

effecting survival and growth (Koenraad et al, 1997).

63

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