Download Molecular mechanisms involved in the emergence and fitness of

Graduate Theses and Dissertations
Graduate College
2009
Molecular mechanisms involved in the emergence
and fitness of fluoroquinolone-resistant
Campylobacter jejuni
Jing Han
Iowa State University
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Recommended Citation
Han, Jing, "Molecular mechanisms involved in the emergence and fitness of fluoroquinolone-resistant Campylobacter jejuni" (2009).
Graduate Theses and Dissertations. Paper 10497.
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Molecular mechanisms involved in the emergence and fitness of fluoroquinoloneresistant Campylobacter jejuni
by
Jing Han
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Veterinary Microbiology
Program of Study Committee:
Qijing Zhang, Major Professor
Nancy A.Cornick
Lorraine Hoffman
Lisa K. Nolan
Edward Yu
Iowa State University
Ames, Iowa
2009
Copyright © Jing Han, 2009. All rights reserved.
ii
DEDICATION
This dissertation is dedicated to:
My beloved one-Husband, Hailin Tang
My son Eric (Jiale) H. Tang, who was born in February 2008
My Mother, Xingzhi Zhao
My Father, Futing Han:
Thanks for your love and support.
iii
TABLE OF CONTENTS
LIST OF FIGURES
v
LIST OF TABLES
vi
CHAPTER 1. GENERAL INTRODUCTION
1
1. Introduction
1
2. Dissertation Organization
2
CHAPTER 2. LITERATURE REVIEW
4
1. Campylobacter: The Organism
4
2. Epidemiologic Aspects of Campylobacter Infection
5
3. Clinical Significance of Campylobacter Infections
7
4. Virulence and Pathogenesis of Campylobacter Infection
9
5. Clinical Treatment of Campylobacter Infection in Human
15
6. Antimicrobial Resistance of Campylobacter
15
7. Prevention and Control
25
8. Reference
29
CHAPTER 3. KEY ROLE OF MFD IN THE DEVELOPMENT OF
FLUOROQUINOLONE RESISTANCE IN CAMPYLOBACTER JEJUNI
56
1. Abstract
56
2. Author Summary
57
3. Introduction
57
4. Results
60
5. Discussion
66
6. Materials and Methods
70
7. References
77
CHAPTER 4. FLUOROQUINOLONE RESISTANCE ASSOCIATED MUTATION
IN GYRA AFFECTS DNA SUPERCOILING IN CAMPYLOBACTER JEJUNI
93
1. Abstract
93
2. Introduction
94
3. Materials and Methods
97
4. Results
104
5. Discussion
109
iv
6. References
114
CHAPTER 5. TRANSCRIPTOMIC AND PROTEOMIC COMPARISON OF
CAMPYLOBACTER JEJUNI STRAINS THAT ARE RESISTANT OR
SUSCEPTIBLE TO FLUOROQUINOLONE
128
1. Abstract
128
2. Introduction
128
3. Materials and Methods
130
4. Results
135
5. Discussion
137
6. References
140
CHAPTER 6. GENERAL CONCLUSIONS
152
ACKNOWLEDGMENTS
154
v
LIST OF FIGURES
CHAPTER 3
Fig. 1 Insertional mutation of mfd and its impact on the transcription of cj1084c
88
Fig. 2 Frequencies of emergence of spontaneous FQR mutants in different
C. jejuni strains
89
Fig. 3 Growth kinetics of various C. jejuni constructs in culture media
90
R
Fig. 4 Development of FQ mutants from 11168 and JH01 grown in
MH broth supplemented with 4 µg/ml of CIPRO
91
Fig. 5 Development of FQR Campylobacter mutants in chickens initially
infected with FQ-susceptible Campylobacter, but treated with enrofloxacin
92
CHAPTER 4
Fig. 1 Pairwise competition between FQS and FQR strains in chickens
122
Fig. 2 SDS-PAGE analysis of purified recombinant GyrA and GyrB
& Supercoiling activities of the WT gyrase and the three mutant gyrases
measured by an in vitro assay
124
Fig. 3 Agarose gel electrophoresis analysis of plasmid topoisomers
extracted from different strain background
125
Fig. 4 Inhibition of DNA gyrase supercoiling activity by ciprofloxacin
as measured by an in vitro supercoiling assay
127
CHAPTER 5
Fig. 1 Proteomic analysis of protein expression in Campylobacter
150
Fig. 2 Representative DeCyder display of relative abundance of selected
protein spots in 62301R33R versus 62301R33S
151
vi
LIST OF TABLES
CHAPTER 3
Table 1 Genes differentially expressed in the presence of ciprofloxacin
85
Table 2 Oligonucleotide primers used in qRT-PCR
87
CHAPTER 4
Table 1 Bacterial strains used in this study
120
Table 2 Key PCR primers used in this study
121
CHAPTER 5
Table 1 Bacterial strains used in this study
145
Table 2 Genes upregulated in the FQR strain identified by DNA microarray
146
Table 3 Differentially expressed protein spots between 62301R33R
and 62301R33S identified by 2-D DIGE
147
Table 4 Differentially expressed proteins between 62301R33R and
62301R33S identified by 2-D DIGE
149
1
CHAPTER 1. GENERAL INTRODUCTION
1. Introduction
Since the 1970s, Campylobacter, a Gram-negative microaerobic bacterium, has
emerged as a major foodborne pathogen and a common causative agent of human
enterocolitis. Campylobacter jejuni is responsible for approximately 90% cases of human
campylobacteriosis, followed by Campylobacter coli, which accounts for approximately 10%
cases of human campylobacteriosis. Signs and symptoms of campylobacterosis often start as
a cramping pain in the abdomen, followed by watery or inflammatory diarrhea. Some
individuals may develop fever, headache, dizziness and myalgia. Campylobacter infection
causes more than 2 million cases of diarrhea each year in the U.S. alone. C. jejuni infections
in humans may also trigger a degenerative neurological disorder called Guillain-Barré
syndrome.
Majority of Campylobacter infections are mild, self-limiting and usually resolve
within a few days, no specific treatment is required for most patients with Campylobacter
enteritis. However, in specific clinical circumstances such as in severe infections, prolonged
illness or where the patient is pregnant, young, elderly, or has compromised immunity,
antimicrobial treatment is usually warranted. Fluoroquinolone (FQ) antimicrobials are one of
the antibiotics that are often prescribed for clinical treatment of diarrhea caused by enteric
bacterial pathogens including Campylobacter. However, Campylobacter is increasingly
resistant to FQ antimicrobials and FQ-resistant (FQR) Campylobacter, developed in foodproducing animals, can be transmitted to humans via the food chain, which has become a
major concern for public health. In Campylobacter, the main targets of FQs are DNA gyrases,
and resistance to FQ antimicrobials is mediated by point mutations in the quinolone
resistance-determining region (QRDR) of gyrA in conjunction with the function of the
multidrug efflux pump CmeABC. Acquisition of high-level FQ resistance in Campylobacter
does not require stepwise accumulation of point mutations in gyrA. Instead, a single point
mutation in gyrA can lead to clinically relevant levels of resistance to FQ antimicrobials.
Specific mutations at positions Thr-86, Asp-90 and Ala-70 in GyrA have been linked to FQ
2
resistance in C. jejuni. The Thr-86-Ile mutation in GyrA confers a high-level resistance to
fluoroquinolone. One unique feature of FQ resistance development in Campylobacter is the
rapid emergence of FQR mutants from a FQ-susceptible (FQS) population when treated with
FQ antimicrobials and C. jejuni possesses a high mutation rate to FQ resistance. The
rapidness and magnitude of FQ resistance development in Campylobacter in response to FQ
treatment suggest that both selective enrichment of pre-existing spontaneous mutants and
adaptive gene expression may contribute to the emergence of FQR Campylobacter, but how
Campylobacter responds to FQ treatment is unknown. Once developed, FQR Campylobacter
carrying the Thr-86-Ile change in the GyrA subunit of DNA gyrase can persist in the absence
of antibiotic selection pressure. Notably, FQR Campylobacter carrying the Thr-86-Ile
substitution in the GyrA subunit of DNA gyrase outcompeted the FQS strains in chickens,
suggesting that acquisition of FQ resistance enhances the in vivo fitness of FQR
Campylobacter. How the resistance-conferring mutation affects Campylobacter fitness
remains to be determined.
To close these important gaps in our understanding of the mechanisms underlying the
rapid emergence of FQR mutants and fitness of the FQR mutants, we conducted a series of in
vitro and in vivo studies to determine how Campylobacter responds to FQ treatment, what
facilitates the emergence of FQR mutants in Campylobacter, and what are the molecular
mechanisms contributing to the enhanced fitness in FQR Campylobacter. The findings from
this project will significantly improve our understanding of the molecular mechanisms
underlying the development of FQR Campylobacter and the fitness of the FQR
Campylobacter and will facilitate the design of strategies to reduce the emergence and spread
of FQR Campylobacter.
2. Dissertation Organization
This dissertation is organized in the alternative format with five chapters. Chapter 1
contains a general introduction. Chapter 2 contains a literature review. Chapter 3 through 5
each contains a manuscript published or to be submitted to the ASM journals. Chapter 6 is
3
the general conclusion. At the end of each chapter are appended references cited followed by
tables and figures.
4
CHAPTER 2. LITERATURE REVIEW
1. Campylobacter: The Organism
1.1 Taxonomy
The pathogenic bacteria Campylobacter spp. were first observed and described as
non-culturable spiral-shaped or Vibrio-like bacteria in 1886 by Theodor Escherich (130) and
first isolated from an aborted bovine fetus in 1913 by McFadyean and Stockman (36). At that
time, these bacteria were classified in the genus Vibrio. In 1963, based on their low G+C
composition, their microaerophilic growth requirements, and their nonfermentative
metabolism, Sebald and Veron transferred two of these Vibrio species, Vibrio fetus and
Vibrio bubulus, into a new genus Campylobacter as Campylobacter fetus and Campylobacter
bubulus, respectively. With the development of new isolation technique, additional
Campylobacter-like organisms were isolated from a variety of human, animal, and
environmental sources and some of those species were described and added to
Campylobacter genus. In 1991, Vandamme and De ley accommodated the genera
Campylobacter and Arcobacter into a new bacterial family, the Campylobacteraceae.
Currently, the genus Campylobacter includes 18 species: C. jejuni, C. coli, C. concisus, C.
curvus, C. fetus, C. gracilis, C. helveticus, C. hominis, C. hyointestinalis, C. insulaenigrae, C.
lanienae, C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, C. upsaliensis and C.
canadensis. Although most of the Campylobacter spp. have been associated with veterinary
and human infections, C. jejuni and C. coli are the most important and major cause of foodborne Campylobacter infection in humans.
1.2 General characteristics of Campylobacter
Campylobacter spp. are Gram-negative, nonspore-forming, S- or spiral-shaped rod
bacteria that are approximately 0.2 to 0.8 µm wide and about 0.5 to 5 µm long (58).
Although normally a curved-rod shape, other forms of Campylobacter such as spherical or
coccoid occur in response to stress or deleterious conditions (47, 58). Most Campylobacter
are motile with distinctive corkscrew-like darting motions, using a single polar, unsheathed
5
flagellum at one or both ends (58). Some Campylobacter species (C. gracilis) are nonmotile
while some (C. showae) have multiple flagella (58).
The members of Campylobacter spp. are slowly-growing, fastidious organisms and
require a microaerophilic atmosphere for optimal growth (47, 201). Typical Campylobacter
spp. requires an atmosphere containing approximately 3 to 15% O2 and 2 to 10% CO2 for
optimal growth. Although most Campylobacter can grow at 37 °C, thermophilic
Campylobacter spp. such as C. jejuni, C. coli, and C. lari show optimal growth at 42 °C on
artificial media (47, 221). The ideal environment for recovery of Campylobacter is at 37 °C
or 42 °C under the atmosphere containing approximately 5% O2, 10% CO2, and 85% N2.
Campylobacter spp. are unable to utilize glucose and other hexoses as carbon sources. The
main carbon and energy sources used by Campylobacter for growth are derived from the
degradation of amino acid or tricarboxylic acid cycle intermediates (123, 124). Therefore,
nutrient-rich media, such as Mueller-Hinton broth, are often used for culturing
Campylobacter.
Campylobacter spp. have a small genome of approximately 1.6-1.7 Mb with AT-rich
DNA and low G+C content ranging from 29-34 mol% (183, 191). C. jejuni NCTC 11168 has
1,643 open reading frames (ORFs) and it lacks plasmids (191). Other features found in the
C..jejuni NTCT 11168 are the lack of insertional sequences or phage-associated sequences,
and few repeat sequences (183). One of the most striking themes in the C. jejuni genome is
the presence of hypervariable sequences encoding the biosynthesis or modification of surface
structures such as flagellum, capsular polysaccharides, and lipooligosaccharides (LOS).
2. Epidemiologic Aspects of Campylobacter Infection
Campylobacter jejuni is one of the most prevalent bacterial foodborne pathogens in
the United States and many other developed countries, causing more than 2 million cases of
diarrhea each year in the U.S. alone (7, 75, 184, 214, 236). Person-to-person transmission of
Campylobacter is rare, and the main source of human Campylobacter infection is via food,
water, or milk contaminated by Campylobacter (11, 56, 75). In general, people can become
6
infected with Campylobacter spp. by four different ways: (i) by handling raw meats and meat
products, (ii) by consumption of raw or undercooked meats, unpasteurized milk and dairy
products, (iii) by consumption of foods that are usually eaten raw or without further cooking
such as salads and breads, which may become cross contaminated with raw meats in the
kitchen, or (iv) by direct contact with infected animals/pets (25, 224, 225). Domestic poultry
are considered to be a major risk factor for human Campylobacter infection (7, 229). The
correlation between the consumption of chicken meat and Campylobacter infection in
humans has been demonstrated in several studies (74, 209, 226). C. jejuni is highly prevalent
in the intestinal tract of market-age broiler chickens. Several survey studies revealed that at
least 70% of retail chickens sold in the United States are contaminated with Campylobacter
(270). The sources of C. jejuni infections in poultry have not yet been elucidated. Potential
sources for horizontal transmission on poultry farms include old litter, untreated drinking
water, other farm animals, domestic pets, wildlife species, house flies, insects, equipment and
transport vehicles (113, 174, 196, 229, 245). Besides poultry meats, other major cause of
human Campylobacter infection includes consumption of raw or unpasteurized milk,
improperly/untreated water, and contacts with pets (7, 25, 75, 126, 225).
Unlike Salmonella infection, the majority of Campylobacter infections in humans
occur as sporadic cases; outbreaks of Campylobacter infection are rare (181, 184). Sporadic
cases of Campylobacter infection have different epidemiological characteristics from
outbreaks. Most outbreak cases of Campylobacter infection are associated with raw/
unpasterized milk and untreated/contaminated water (1, 33, 70, 184), while the majority of
sporadic cases of Campylobacter enteritis result from the handling or consumption of
undercooked poultry and/or other foods that are cross-contaminated with raw poultry meat
during food preparation (7, 12, 31, 54, 108). The milk- and water- associated outbreaks
usually occur in the spring and autumn and are uncommon in the summer, while sporadic
Campylobacter infections often occur during summer (184).
Although Campylobacter enteritis is the most common enteritis worldwide, the
epidemiology of Campylobacter infections in developing countries is markedly different
7
from that in the developed countries (181). First, the incidence of campylobacteriosis in
developing countries is much higher than in developed countries. Second, in developing
countries, Campylobacter infection occurs mostly in the first few years after birth and
declines with age. Most infections become asymptomatic among adults, presumably because
of the acquisition of immunity due to multiple exposures to several serotypes of
Campylobacter in the early stages of the life. In developed countries, infections can occur at
any age and the infections are more severe. Inflammatory (bloody) diarrhea is more common
among patients in developed countries. Third, the route of transmission in developing
countries differs from that in developed countries. In developed countries, infections are
often caused by uncooked food, while in developing countries, the presence of livestock in
close proximity to living areas severely increases the risk of infection with Campylobacter.
Fourth, in developed countries, Campylobacter infections typically increase during summer
and fall. This seasonal variation is not significant in developing countries.
3. Clinical Significance of Campylobacter Infections
3.1 Campylobacter infection in human
Campylobacter species, particularly C. jejuni and C. coli, have been recognized as
important causes of bacterial enteric infections in humans since the late 1970’s (37).
C. .jejuni and C. coli account for 85-95% and 5-15% of human campylobacteriosis,
respectively (170). There is no difference between the characteristics of infections caused by
C. jejuni and C. coli. The infectious dose for humans can be as low as 500 bacterial cells (32).
The mean incubation period of the infections is 3.2 days which is longer than that of most
other intestinal infections and the range can extend from 1-8 days (31, 32, 37). Infections
with Campylobacter spp. are of variable severity ranging from asymptomatic to severe. The
main clinical symptom of campylobacteriosis in humans is acute enteritis, which is
characterized by acute diarrhea with abdominal cramps, fever, headache, and nausea. Some
patients may also vomit.
The symptoms of Campylobacter enteritis are different between developed countries
and developing countries. Patients in developed countries usually have more severe
8
symptoms than those in developing countries (181). In developed countries, the common
clinical features of Campylobacter spp. infection are acute gastroenteritis which is
characterized by diarrhea, fever, and abdominal cramps (7). The diarrhea may range in
severity from loose stools to profuse, watery stools with 10 or more bowel movements within
one day (31). The initially watery diarrhea may develop into severe bloody diarrhea with
fecal leucocytes as the disease progress. The acute diarrhea usually lasts 2-3 days, but it may
persist for 1 week or longer particularly in patients with an acute colitis. Usually the
gastroenteritis is self-limiting without the need for treatment (32). In developing countries,
the typical clinical symptom is milder form of gastroenteritis which is usually characterized
by watery, non-bloody, non-inflammatory diarrhea and symptomatic infections are extremely
common in children younger than 2 years old (45, 127, 188). In contrast, in developed
countries, infections occur in both adults and children. The highly endemic nature of
infections in developing countries result in the early exposure to Campylobacter in life and
increased Campylobacter-specific antibody levels with age which may account for the less
severe clinical symptoms in adults.
In some patients, the diarrhea is not significant, while acute abdominal pain is the
predominant feature of Campylobacter infections. Since the abdominal pain is usually
cramping in nature and most often occurs at the right lower quadrant of the abdomen, severe
abdominal pain may mimic acute appendicitis which can lead to a mistaken diagnosis (7, 3032, 37).
The symptoms of Campylobacter infection usually last for 3-4 days, and the disease
gradually resolves over a week (32). A longer relapse may occur in 15-25% of affected
patients who did not receive antibiotic treatment and last for several weeks (7, 31, 32).
Patients may shed Campylobacter in their feces for several weeks after the clinical symptoms
have disappeared unless treated with antibiotics (32).
3.2 Campylobacter infection in animals
In animals, C. jejuni and C. coli usually do not cause clinical diseases. They are
9
commensal inhabitants of the intestinal tracts of a wide range of domestic animals and pets,
as well as wild animals and birds (12, 110, 234). Commercial poultry such as broilers, layers,
turkeys, ducks and free-living birds are the natural reservoirs of C. jejuni and C. coli (220).
Recent studies indicated that C. jejuni is increasingly associated with ovine abortions and it
has replaced C. fetus as the predominant Campylobacter species causing sheep abortion in
the United States (59, 213).
3.3 Complications of Campylobacter infection in human
In general, gastrointestinal complications due to Campylobacter infections rarely
occur. However, Campylobacter spp. may spread from the gastrointestinal tract and lead to
cholecystitis, pancreatitis, or massive gastrointestinal hemorrhage (7, 37). In addition to the
gastrointestinal tract, Campylobacter also can cause some extraintestinal diseases, such as
bacteremia, meningitis, endocarditis, septic arthritis, osteomyelitis and abortion (7, 32, 60).
The most important post-infectious complication of C. jejuni infection is GuillainBarré Syndrome (GBS), an acute immune-mediated neuromuscular paralysis that may lead to
respiratory muscle compromise and death (8, 131, 172). About 20 to 50% of patients with
GBS symptoms have a preceding Campylobacter infection and symptoms of GBS typically
start 1 to 3 weeks after a Campylobacter infection (26, 37). Althought C. jejuni infection is a
common trigger of GBS, the risk of developing GBS after C. jejuni infection is actually quite
low (172). Among the different C. jejuni strains involved with GBS, Penner serotype O:19 is
most commonly associated with GBS in the United States (9, 172). It has been hypothesized
that the molecular mimicry between the peripheral nerve gangliosides and sialylated
Campylobacter lipooligosaccharide (LOS) may play a role in the pathogenesis of
Campylobacter-induced GBS (131, 267). The antibodies induced by Campylobacter LPS
cross-react to gangliosides, causing inflammation and tissue damage and eliciting the autoimmune disease GBS (7, 26, 37, 132).
4. Virulence and Pathogenesis of Campylobacter Infection
10
Although Campylobacter has emerged as a leading cause of bacterial food-borne
illness during the past decade, relatively little is known of the molecular pathogenesis and
virulence of Campylobacter infections compared to other enteric pathogens (127, 199, 247).
This is partly due to the lack of an ideal animal model to evaluate the pathogenesis and
virulence of Campylobacter (266). As a food-borne bacterial pathogen, Campylobacter
enters the human body with food or water, survives the stomach acid and highly alkaline
secretion from the bile duct, then colonizes the distal ileum and colon (109). Following
colonization of the mucus blanket and adhesion to the intestinal cell surfaces, Campylobacter
perturbs the normal absorptive capacity of the intestine by damaging epithelial cell function
through direct cell invasion and/or toxin production, or indirectly by inducing host
inflammatory reactions (260). Thus, factors involved in motility, intestinal adhesion,
colonization, invasion, protein secretion, intracellular survival and toxin production are
thought to be the major contributors to the pathogenicity of Campylobacter spp. Numerous
studies using in vitro cell cultures and in vivo model systems have been performed to identify
and characterize potential bacterial cell components involved in the pathogenesis of
Campylobacter. In C. jejuni, the bacterial factors involved in disease pathogenesis include
adhesins, flagella, capsular polysaccharides, cytolethal distending toxin (CDT) and many
others.
4.1 Adhesins
The first step involved in Campylobacter colonization and pathogenesis is adherence
to intestinal epithelial cells. The bacterial cell components involved in adhesion have been
studied extensively using in vitro cell cultures and in vivo model systems. The adhesins
identified in C. jejuni includes CadF, PEB1 (CBF 1), JIpA, and CapA.
CadF (Campylobacter adhesion to fibronectin) binds specifically to fibronectin,
which is located at the basolateral epithelial cells, and is required for maximal binding and
invasion by C. jejuni (134, 167, 168). The binding of C. jejuni to immobilized fibronectin can
be blocked by specific anti-CadF antibody (167). cadF mutants show a great reduction in
11
binding and invasion of INT407 cells and are unable to colonize chickens compared with
wild-type isolates (167, 168, 273).
PEB1 (CBF 1) is periplasmic and shares homology with the periplasmic-binding
proteins of amino acid ATP-binding cassette (ABC) transporters involved in nutrient
acquisition (78, 194). C. jejuni mutants that lack peb1 exhibit a reduction in the adherence to
HeLa cells, as well as reduced colonization of mice when compared with wild-type isolates
(194, 195).
JlpA (C. jejuni lipoprotein A) is a surface-exposed lipoprotein that binds to Hsp90α
on the surface of HEp-2 cells (115). Binding of JlpA to Hsp90α activates NF-κB and p38
mitogen-activated protein kinase, which contribute to proinflammatory responses (194).
Ablation of jlpA reduces C. jejuni adherence to HEp-2 cells by 18-19.4% compared to the
wild-type strain (115).
CapA is an autotransporter, and capA mutants show reduced adherence to Caco-2
cells and decreased colonization and persistence in chickens (17).
4.2 Flagella
Flagella are the most extensively studied virulence determinant of Campylobacter.
They are involved in several aspects of pathogenesis. Campylobacter have one or two
unsheathed polar flagella. The unique characteristics of the flagellum, combined with the
spiral shape of the bacterial cell, enable the organism to move using a darting motility. C.
jejuni flagella and flagellar motility are vital to many aspects of C. jejuni pathogenesis,
including host colonization, virulence in ferret models, protein secretion and host invasion.
Numerous studies have shown that Campylobacter must be flagellated to colonize a host and
cause disease (10, 92, 102, 133, 235, 254, 264). During the initial stages of infection, the
flagella help Campylobacter overcome the clearing action of peristalsis and allow it to enter
and cross the viscous mucosal layer covering the intestinal epithelia, where it adheres to the
host cell surface.
12
Campylobacter flagellum is composed of two flagellin monomers, FlaA and FlaB,
encoded by flaA and flaB respectively (95). The flaA and flaB genes are tandemly arranged in
the Campylobacter genome and transcribed from σ28- and σ54-dependent promoters with its
own promoter respectively (10, 95, 180, 235). The major subunit FlaA and the minor subunit
FlaB share about 90% amino acide identity and are capable of assembling independently into
functional filaments (95, 180, 235). Mutagenesis studies showed that a flagellar filament
composed exclusively of the FlaA is indistinguishable in length from that of the wild type,
but shows a slight reduction in motility, while the flagellar filament composed exclusively of
the FlaB is severely truncated in length and greatly reduced in motility(95, 235). Thus, both
FlaA and FlaB are required for full-length flagella expression and motility. Study by
Wassenaar et. al. (254) demonstrated that mutants with reduced motility but intact FlaA can
colonize chickens at levels similar to the parent strain; by contrast, mutants with full motility
but inactivated flaA gene were significantly reduced in their ability to colonize chickens,
which suggests the presence of FlaA, rather than motility, is necessary for optimal bacterial
colonization of chicken intestine.
Flagella also play an active role in the process of epithelia cell invasion. Mutagenesis
and invasion studies showed the presence of a flagellum consisting of the FlaA protein is
essential for invasion of INT-407 cells, whereas FlaB protein is not required (118, 235, 253).
However, immotile mutants still penetrate at low levels, indicating that factors other than the
flagella are also involved in invasion (265).
In addition to a role in motility, C. jejuni flagella also serve as a type III secretion
system (T3SS) to secrete both flagellar and non-flagellar proteins that contribute to cell
adhesion and invasion (137, 208, 228). The protein CiaB (Campylobacter invasionassociated antigen B) secreted by the flagellar secretion system is required for the invasion of
INT-107 cells (135-137, 228). Disruption of ciaB reduces the colonization of C. jejuni in
chicken (272). The mutation of FlaC which is also secreted by the flagellar secretion system
affects Caco-2 cell invasion (228).
13
4.3 Lipooligosaccharide (LOS)/Capsular polysaccharides (CPS)
LOS and CPS ( high molecular weight lipopolysaccharides) are common features on
bacterial surfaces. Both LOS and CPS are the major surface antigens of Campylobacter and
are highly variable among different Campylobacter strains (72, 122, 190). As mentioned
earlier, the molecular mimicry between the Campylobacter LOS and human neuronal
gangliosides is thought to lead to autoimmune disorders, including GBS (131, 267).
Studies have shown that these surface polysaccharide molecules play important roles
in the interaction of the bacteria with animal/human hosts and are required for cell invasion
in vitro, chicken colonization, and full virulence in the ferret animal model (22, 23, 76, 118,
120, 122, 161). Mutations in the genes involved in LPS synthesis and transport, such as galE,
waaF and kpsE, significantly affect mutant cell adhesion to and invasion of INT-407 cells in
vitro and increase sensitivity to some antibiotics compared that of the wild-type strain (76,
120).
4.4 Cytolethal distending toxin (CDT)
One of the mechanisms involved in the virulence of Campylobacter is the production
of toxins, which are important in diarrheal diseases. C. jejuni produces cytolethal distending
toxin (CDT) (116), which is also produced by several other Gram-negative bacteria,
including E. coli, Shigella, and Helicobacter (52, 117, 182). The action mechanism of CDT
produced by C. jejuni is to cause sensitive eukaryotic cells to arrest at either the G1/S or
G2/M transition of the cell cycle, depending on the cell type (100, 101, 141, 142, 256).
The active holotoxin CDT consists of three subunits (CdtA, CdtB and CdtC) encoded
by three adjacent or slightly overlapping genes cdtABC (41, 142). CdtB is the enzymatically
active subunit of CDT and it shares homology with mammalian DNase I, however, CdtB has
weak DNase activity in vitro (141). Whether DNA damage in vivo is a direct or indirect
result of CdtB activity is still debatable (101, 159, 219, 248, 256). The exact function of
CdtA and CdtC are unclear. Studies have shown both CdtA and CdtC can bind Hela cells
14
(146). It is suggested that CdtA and CdtC may mediate the binding and subsequent
internalization of CdtB into the host cell (55, 142).
The role of CDT in C. jejuni pathogenesis remains unclear. Studies showed that CdtA,
CdtB and CdtC are all required for the induction of pro-inflammatory cytokine IL-8, a
hallmark of C. jejuni pathogenesis (103) and CDT may have a role in eliciting inflammatory
response in vivo (73). However, it seems that CDT is not required for colonization since
mutants that lack CDT colonize chickens as well as wild-type strains (29).
4.5 Glycosylation
Campylobacter expresses two glycosylation systems: a general N-linked protein
glycosylation pathway and an O-linked protein glycosylation pathway, which makes it
unique in bacteria (23). Both of these pathways are important in intestinal colonization.
The N-linked protein glycosylation pathway, which is encoded by a single gene
cluster named pgl, is responsible for post-translational modification of asparagine residues on
many surface proteins (23, 235). The N-linked glycan that is assembled by the Pgl system is
conserved in all C. jejuni and C. coli strains that have been examined (23, 235, 248).
Numerous studies haveshown that C. jejuni pgl mutants have significantly reduced ability to
adhere to and invade host cells and colonize animal models; these studies have linked Nlinked protein glycosylation to bacterial virulence (23, 102, 119, 121, 125, 143). The pglH
mutant of strain 81116 results in deficient glycosylation of a number of proteins and showed
reduced adherence and invasion of human Caco-2 cells and colonization of chickens (121).
An 81176 strain lacked glycoprotein Cj1469c was defective for both adherence to INT-407
human epithelium cells in vitro and colonization of chicken gut (119).
The O-linked protein glycosylation pathway is responsible for glycosylation of serine
or threonine residues on flagellar proteins (23). O-linked glycosylation of flagellin is
essential for the successful assembly of the flagellar filament (90). Defects in O-linked
protein glycosylation result in the loss of motility, reduced adhesion and invasion of host
15
cells, and decreased virulence in ferret (96, 239).
5. Clinical Treatment of Campylobacter Infection in Human
Since the majority of Campylobacter infections are mild, self-limiting and usually
resolve within a few days, no specific treatment is required for most patients with
Campylobacter enteritis other than the oral replacement of fluid and electrolytes that are lost
through diarrhea and vomiting (7, 32, 37).
However, in specific clinical circumstances such as in severe infections (persistent
high fever, bloody diarrhea, or more than eight bowel movements within one day), prolonged
illness (clinic symptoms last longer than 1 week), or where the patient is pregnant, young,
elderly, or has compromised immunity, antimicrobial treatment is usually warranted (7, 32,
37).
For clinical antibiotic treatment of confirmed Campylobacter infection, erythromycin
(a macrolide) is the drug of choice because of its efficacy, safety, low cost and relatively
narrow spectrum of activity (less inhibitory effect on fecal flora) (7, 31, 37, 67). In addition
to erythromycin, fluoroquinolone (FQ) antimicrobials (e.g., ciprofloxacin) have been
commonly used for the treatment of enteritis caused by Campylobacter because of their
broad spectrum activity against enteric pathogens, especially when bacterial gastroenteritis is
suspected and the cause of diarrheal illness has not yet been identified (7, 32, 37). However,
the increasing prevalence of FQ-resistant (FQR) Campylobacter and macrolide-resistant
Campylobacter has compromised the effectiveness of these treatments (67, 97, 110, 255). For
serious systemic infection, tetracyclines and aminoglycosides, such as gentamicin, are the
drugs of choice (32). Since up to 60% of strains may be resistant to tetracycline, care should
be taken before prescribing this antibiotic (32).
6. Antimicrobial Resistance of Campylobacter
As mentioned in the previous section, FQ and macrolide antibiotics are the
16
antimicrobial agents considered for treatment of human Campylobacter enteritis when
therapeutic intervention is needed (7, 45). In addition to use in human, these antibiotics have
been used in animal husbandry for therapeutic purposes or as growth promoters (4). However,
Campylobacter resistant to these clinically important antibiotics is increasingly prevalent,
which is a major public concern.
6.1 Action mode of antibiotics
FQs, such as ciprofloxacin, enrofloxacin, and levofloxacin, are a class of structurallyrelated synthetic antimicrobials that are effective against a wide range of Gram-positive and
Gram-negative bacterial and are used in both treatment and prophylaxis of bacterial
infections (16, 105, 107). FQs are bactericidal by inhibiting DNA synthesis. The main targets
of FQs in bacteria are the type II topoisomerase DNA gyrases and/or topoisomerase IV (64,
106). DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA, which is an
essential step in DNA replication, recombination, and transcription (42). Once inside
bacterial cells, FQ antimicrobials form a stable complex with the target enzymes and trap the
enzymes on DNA, resulting in double-stranded breaks in DNA and bacterial death (63, 223,
242, 259). In Gram-negative bacteria, DNA gyrase is the primary target of FQ antibiotics,
while topoisomerase IV is the main target of FQs in Gram-positive bacteria (106).
Macrolides include erythromycin, tylosin, clarithromycin, azithromycin, and
telithromycin, which have similar structures and are bacteriostatic agents that are active
against most Gram-positive and some Gram-negative bacteria. They are important drugs for
the treatment of upper and lower respiratory tract infections and gastric diseases caused by
Helicobacter pylori and Campylobacter in humans (206, 268). Macrolides are protein
synthesis inhibitors that reversibly bind to the 50S ribosome subunit and physically block
tunnel entrance of the ribosome, causing the dissociation of peptidyl-tRNAs from the
ribosome in the elongation cycle (86, 165, 186, 206, 242).
Tetracyclines are an important class of antibiotics with a broad spectrum of activity,
being active against many Gram-negative and Gram-positive bacteria and have been widely
17
used in both human and animal medicine (268). The tetracyclines can be divided into two
groups, the atypical tetracyclines (e.g. anhydrotetracycline and 6-thiatetracycline) and typical
tetracyclines (e.g. tetracycline and minocycline) (6, 48). The atypical tetracyclines function
by disrupting bacterial membranes, whereas the typical tetracyclines are bacteriostatic and
act on the ribosome as protein synthesis inhibitors. Once inside the bacteria cell, the
tetracycline binds reversibly to the 30S subunit of the ribosome, which prevents the binding
of the incoming aminoacyl-tRNA to the A site of the ribosome, resulting in elongation cycle
halt and thus inhibition of nascent polypeptide synthesis (48, 69, 268).
Aminoglycosides, such as streptomycin or gentamicin, are potent bactericidal
antibiotics that have been widely used in human and animal medicine. The bactericidal
activity of aminoglycosides is primarily exerted through irreversible binding of the 30S
ribosome subunit, which interferes with the normal proofreading activity of the ribosome and
prevents the translocation of the growing peptide from the A site to the P site of the ribosome
(114, 156, 268). As a result, the elongation of nascent proteins is disrupted.
6.2 Mechanisms of antibiotic resistance in Campylobacter
6.2.1 FQ resistance
The dominant mechanisms involved in FQ resistance are 1) target ( DNA gyrase
and/or topoisomerases IV) modification resulting in reduced affinity of these enzymes for
fluoroquinolones, 2) reduced antibiotic intracellular accumulation by increasing efflux
activity or decreasing outer membrane permeability, 3) reduced target enzyme expression,
and 4) target protection mediated by the Qnr protein which can protect DNA gyrase from
inhibition by the FQs (106, 192, 241). Of these resistance mechanisms, the first three are
encoded by chromosomal elements, while the Qnr is encoded by a plasmid-carried
quinolone-resistance gene and only confers a low level of resistance to FQs (192). In
Campylobacter, the resistance to FQs is mediated by a point mutation in the quinolone
resistance determining region (QRDR) of DNA gyrase subunit A (GyrA) (21, 81, 155, 192,
268). No mutations in DNA gyrase subunit B (GyrB) have been involved in FQ resistance in
18
Campylobacter (21, 193, 198). Unlike the FQ resistance in other enteric organisms (e.g.
Escherichia coli and Salmonella), acquisition of high-level FQ resistance in Campylobacter
does not require stepwise accumulation of point mutations in gyrA. Instead, a single point
mutation in the QRDR of gyrA is sufficient to lead to clinically relevant levels of resistance
to FQ antimicrobials (72, 155, 210). The most commonly observed mutation in FQR isolates
of Campylobacter is the C257T change in the gyrA gene which leads to the The-86-Ile
substitution in the gyrase A subunit and confers high-level resistance to ciprofloxacin(155,
268). Other mutations reported include Thr-86 Lys, Ala-70-Thr, and Asp-90-Asn, which are
less common and only confer intermediate-level FQ resistance (67, 91, 155, 210, 252).
Mutations in parC, which encodes topoisomerase IV, were reported to confer high-level FQ
resistance (84); however, subsequent studies conducted by multiple independent investigators
failed to identify the parC/parE gene in Campylobacter, which excluded the role of
parC/parE mutations in Campylobacter resistance to FQ antimicrobials (21, 155, 193, 198).
In addition to the mutations in the GyrA, the multidrug efflux pump also contributes to FQ
resistance in Campylobacter. The main efflux pump involved in FQ resistance in
Campylobacter is CmeABC, which is encoded by a three-gene operon (cmeA, cmeB, and
cmeC) located on the Campylobacter chromosome. CmeABC functions as an energydependent efflux system and contributes to both intrinsic and acquired resistance of C. jejuni
to FQ antimicrobials (81, 147, 155, 204). CmeABC plays a major role in ciprofloxacin
resistance in C. jejuni by reducing the accumulation of FQs in Campylobacter cells (81, 147,
155). Thus, CmeABC functions synergistically with the gyrA mutations in mediating FQresistance. All of the known FQ-resistance determinants in Campylobacter are
chromosomally encoded, and plasmid-mediated quinolone resistance determinants, such as
qnr, aac(6')-Ib-cr and qepA, have not been reported in Campylobacter (268).
6.2.2 Macrolide resistance
There are three mechanisms involved in macrolide resistance in bacteria: 1)
modification of the antibiotic, 2) modification of the antibiotic target, and 3) drug efflux
(144, 268). Target modification and active efflux are most commonly associated with
macrolide resistance in Campylobacter (40, 83, 148, 157, 268). Enzyme-mediated target
19
methylation and inactivation of antibiotics have not been identified in C. jejuni or C. coli (86,
268).
The most common mechanisms for macrolide resistance in C. jejuni and C. coli are
point mutations in domain V of the 23S rRNA target gene at positions 2074 and 2075, which
correspond to positions 2058 and 2059, respectively, in E. coli (53, 148, 192, 268). So far
A2074C, A2074G, A2074T, A2075G, A2075T, and A2075C mutations have been reported
in erythromycin-resistant Campylobacter (268). Among these reported mutations, the
A2074C, A2074G, and A2075G mutations all confer high-level resistance to macrolide
antibiotics with erythromycin MIC > 128 µg/ml in C. jejuni and C. coli (38, 53, 83, 148, 157,
268). But the A2075G mutation is the predominant mutation detected among the clinical- and
field- isolated C. jejuni and C. coli (86, 138, 192). There are three copies of the 23S rRNA
gene in the chromosome of Campylobacter and the resistance-associated mutations are
usually identified in all three copies of the 23S rRNA gene (71, 191, 268). However, the
wild-type and mutated alleles can coexist in some isolates and evidence suggests that at least
two mutated copies are required to confer macrolide resistance (83, 148).
In addition to the mutations in 23S rRNA, mutations affecting macrolide binding
have also been identified in the ribosome protein L4 and L22 (40, 53). The G221A change in
the L4 protein which leads to the Gly-74-Asp substitution and a insertion at position 86 or 98
of the L22 protein can result in low levels of macrolide resistance in C. jejuni (40).
As with FQ resistance, active efflux also contributes to macrolide resistance in
Campylobacter (38, 40, 138, 148, 157, 268). Studies have shown that the CmeABC efflux
pump contributes to both intrinsic and acquired resistance to erythromycin in C. jejuni and C.
coli. Inactivation of CmeABC significantly reduced the MICs of macrolides in both the
isolates with intermediate- or low-level resistance and the isolates with high-level resistance,
which indicates that CmeABC functions synergistically with target mutations to confer
resistance to macrolides in Campylobacter (40, 53, 87, 157).
20
6.2.3 Tetracycline resistance
There are four mechanisms of tetracycline resistance: 1) efflux pumps that reduce
drug accumulation, 2) protection of the ribosomal binding site of tetracycline by ribosomal
protection proteins (RPPs), 3) drug modification, and 4) ribosomal modifications that alter
the ability of the antibiotic to bind. In C. jejuni and C. coli, tetracycline resistance is mainly
conferred through the ribosomal protection protein Tet(O) and the efflux pump CmeABC
(268). The tet(O) gene is widespread in Campylobacter, and it is mainly encoded in
transferable plasmids, but the gene located on chromosome has also been reported in
tetracycline-resistant Campylobacter (56, 158, 169, 200, 238). Based on sequence homology,
G+C content (40%), codon usage and Southern blot studies, it appears that Campylobacter
acquired the tet(O) gene from Gram-positive bacteria through horizontal gene transfer (HGF)
(27, 181, 237, 268). Studies have shown that Tet(O) recognizes an open A site on
tetracycline-blocked ribosomal complexes and binds to the A site, which induces a
conformational change in the region of the decoding site and results in the release of the
bound tetracycline molecule. The conformational changes induced by the binding of the
Tet(O) can persist after the release of the Tet(O), thus allowing the aa -tRNA to bind to the
ribosomal A site (48, 49). Tet(O) functions synergistically with CmeABC to confer highlevel resistance to tetracycline (87, 147).
6.2.4 Aminoglycoside resistance
Bacterial resistance to aminoglycosides is generally mediated by four mechanisms: 1)
reduced accumulation of the drug in the intracellular environment, usually mediated by either
increased efflux activity or by decreased outer membrane permeability, 2) 16S rRNA
methylation that abolishes the intermolecular contacts between 16S rRNA and the drug, 3)
ribosomal mutations that result in decreased affinity for the drug, and 4) enzymatic
modification of the drug, one of the most important mechanisms of aminoglycoside
resistance (114, 156, 268). Based on the type of reaction they catalyze, the intracellular
aminoglycoside-modifying enzymes are classified into three different groups:
aminoglycoside phosphotransferases, aminoglycoside adenyltransferases, and
21
aminoglycoside acetyltransferases (2, 6). The modifications mediated by these enzymes
result in the loss of antibacterial activity of the drug due to abolished affinity for the bacterial
ribosomal aminoacyl-tRNA site (150). So far only 3’ aminoglycoside phosphotransferases
and aminoglycoside adenyltransferase have been described in Campylobacter; 3’
aminoglycoside phosphotransferases account for the majority of aminoglycoside-modifying
enzymes in Campylobacter spp (85, 145, 187, 268).
6.3 Emergence of antibiotic resistance in Campylobacter
As mentioned in the previous section, target mutations are the major mechanisms by
which Campylobacter develop resistance to FQs and macrolide antibiotics. Studies have
shown FQR mutants emerge rapidly from a FQS population when treated with FQ
antimicrobials (94, 155, 160, 217, 228, 244). Since there are different types of spontaneous
point mutations that may occur in the QRDR region of the gyrA gene and different point
mutations confer different levels of resistance to FQ antibiotics, the frequencies of
emergence of FQR GyrA mutants range from approximately 10-6 to 10-8 in culture media,
depending on the concentrations of ciprofloxacin used on the enumerating plates (263). The
expression level of cmeABC also influences the frequencies of emergence of spontaneous
FQR mutants and the same type of gyrA mutation confers different levels of resistance to FQs
in different genetic background with varying levels of CmeABC expression (264). In
chickens infected with FQS Campylobacter, treatment with enrofloxacin resulted in the
emergence of FQR Campylobacter mutants, detected in feces within 24-48 hours after the
initiation of treatment, and the FQR population continued to expand during the treatment and
eventually occupied the intestinal tract at densities as high as 107 CFU/g feces (155, 160,
244). Considering that FQs use in poultry promotes the development of FQR Campylobacter
species in an originally FQS population and FQR Campylobacter can be transmitted to
humans via the food chain, the Food and Drug Adminidtration (FDA) suspended all FQs use
in poultry production in 2005 in the USA. Also FQR C. jejuni could develop in
Campylobacter-infected patients treated with ciprofloxacin (66, 217, 261). These findings
suggest that Campylobacter is highly mutable to FQs treatment.
22
In contrast to FQ resistance, there are several distinct features associated with
erythromycin resistance development in Campylobacter. First, the spontaneous mutation
rates for macrolide resistance are very low in Campylobacter (approximately 10-10) (148).
Second, the development of macrolide resistance in Campylobacter is very slow. Treatment
of chickens with therapeutic doses of tylosin in drinking water for a short time did not select
for erythromycin-resistant mutants (139, 148) This finding is in clear contrast with the
emergence of FQR mutant Campylobacter in poultry, which occurred in enrofloxacin-treated
chickens rapidly after the initiation of treatment (155). However, erythromycin resistant
mutants emerged in chickens after prolonged exposure to tylosin, which suggested that
development of erythromycin-resistant mutants in Campylobacter need a prolonged exposure
to macrolide (38, 148). Third, the mutants with low to intermediate levels (MIC = 8 to 64
µg/ml) of resistance to erythromycin harbor mutations different from those found in highlevel erythromycin-resistant mutants. Low-level erythromycin-resistant Campylobacter have
mutations in the L4 and L22 proteins or no detectible mutations at all, which are not stable in
the absence of macrolides. The high-level erythromycin resistant mutants (MIC ≥ 512 µg/ml)
usually harbor a specific point mutation in the 23S rRNA gene, and most of these 23S rRNA
mutations can be stably maintained in the absence of macrolide antibiotics (38, 129, 148).
Fourth, although macrolide resistance is generally more prevalent in C. coli isolates than in C.
jejuni isolates, studies have shown there is no significant difference in the frequencies of
emergence of erythromycin-resistant mutants between these two strains, suggesting that C.
coli is not intrinsically more mutable then C. jejuni with regard to developing macrolide
resistance (148).
6.4 Prevalence of antibiotic resistant Campylobacter
Owing to the widespread use of these antibiotics in both animals and humans, a rapid
increase in the proportion of Campylobacter strains resistant to antimicrobial agents has been
reported all over the world , which has become a major public health concern in recent years
(6, 67, 97, 111, 169, 173, 255).
23
The emergence of FQR Campylobacter strains was first noticed in the early 1990s
after the approval of the use of FQs in animal production (169). In countries where FQs have
been licensed for animal use, there were increased rates of isolation of FQR Campylobacter
strains in both humans and animals following the licensure (169). Many publications have
shown that the use of FQs in poultry and livestock is associated with increased FQR strains in
humans (173, 205, 211, 227, 246).
In the United States, there were no reports of FQR Campylobacter strains isolated
from humans before 1992. However, several recent studies showed that about 19%-40% of
Campylobacter strains isolated from humans were resistant to ciprofloxacin (97, 173). In
Canada, no FQR Campylobacter strains were isolated during 1980-1986, but recent reports
indicated that ciprofloxacin resistance among C. jejuni has dramatically increased since 1992
and reached 47% in 2001 (79, 80). In Europe, there is also a clear tendency of emerging FQs
resistance in Campylobacter and the rate of FQ resistance has rapidly increased in the last
decade (6, 32, 77, 98, 152, 153, 169, 189, 197). In Asia, studies showed that FQR
Campylobacter strains isolated from humans increased rapidly since 1993 and the FQ
resistance rates have reached more than 80% in Thailand, Taiwan, and Hong Kong (44, 111,
240). In contrast, although FQ resistance in Campylobacter isolates was also been observed
in Australia and New Zealand, the rate of FQR Campylobacter isolates in these regions was
much lower than that from the other regions (6, 61, 88, 222). The lack of FQs use in
veterinary medicine probably accounts for the low frequency of FQ resistance in these
regions.
Since the 1990s, an increase in the prevalence of macrolide resistance among C jejuni
and C. coli has also been reported in both developed and developing countries (20, 24, 28, 67,
86, 211), but the rate of macrolide resistance among Campylobacter is highly variable among
different countries (13, 67, 68, 99, 104, 112, 166, 178, 212, 218). In Spain, resistance to
macrolide has been reported to be as high as 90%, but trends over time for macrolide
resistance show stable and low rates in countries such as Japan, Sweden, and Finland (99,
24
112, 178, 185, 212). Several studies showed that resistance to macrolides is more prevalent in
C. coli than C. jejuni in both human and animal isolates (67, 188).
6.5 Fitness of antibiotic resistant Campylobacter
Several studies have shown that FQR Campylobacter mutants carrying gyrA
mutations can be stably maintained in the absence of antibiotic selection pressure and FQR
Campylobacter continue to persist on poultry farms even after FQs withdrawal (15, 202, 203).
The persistence of FQR Campylobacter in the absence of antibiotic selection pressure may be
due to the enhanced fitness of the FQR Campylobacter. Recently Luo et al. examined the
persistence and biological fitness of FQR Campylobacter harboring the Thr-86-Ile mutation
in GyrA in chickens in the absence of antibiotic selection pressure by using in vivo-derived,
clonally-related isolates and defined isogenic mutants (154). Results from this study showed
that when mono-inoculated into chickens, both FQR and FQS strains were able to colonize
and persist in chickens with equal efficiency in the absence of antibiotic usage. When coinoculated into chickens as a pairwise competition, the FQR Campylobacter outcompeted the
FQS Campylobacter and quickly became the dominant population in the chicken host in the
majority of pairs, which indicated that acquisition of FQR mutants carrying the Thr-86-Ile
mutation in GyrA enhances the in vivo fitness of FQR Campylobacter instead of causing a
fitness burden. And the prolonged colonization (upto 3 months) in vivo did not result in the
loss of the resistance phenotype and the reversion/loss of the specific resistance associated
mutation in GyrA (154, 269). Since there are no compensatory mutations in GyrA and GyrB,
which are the targets of FQ antimicrobials in Campylobacter, the enhanced fitness likely is
likely due to the effect of the C257T mutation in gyrA.
For erythromycin-resistant Campylobacter, there is no published information about
the ecological fitness of the resistant mutants. One study in Denmark showed the reduction in
tylosin usage as a growth promoter in pigs has led to a significant reduction in the occurrence
of erythromycin-resistant C. coli isolated from pigs (3).
25
7. Prevention and Control
7.1 Control of Campylobacter on poultry farms
Since poultry is the major reservoir of Campylobacter and contaminated poultry meat
is considered as an important source for human campylobacteriosis, reduction of
Campylobacter contamination in poultry would have an important impact on human public
health. It is not effective to control Campylobacter during processing because of the high
prevalence of Campylobacter in poultry flocks. Therefore, control of Campylobacter
infection in poultry farm should start from the preharvest stage (249). Currently, two major
strategies have been used for control and prevention of Campylobacter infection at the farm
level: 1) biosecurity to eliminate Campylobacter from the farm; and 2) reduction/prevention
of Campylobacter colonization by competitive exclusion, phage treatment, and vaccination
(57, 151, 175, 250, 251).
7.1.1 Biosecurity
Since external environmental contamination during the life of the flocks is likely to
be the primary source of Campylobacter infection, strict hygiene and biosecurity measures
have been used in an attempt to control Campylobacter infections in chicken flocks (12, 82,
245). Empiricial biosecurity measures to prevent Campylobacter colonization of poultry
flocks include use of an all-in all-out policy, cleaning and disinfection of buildings between
flocks, maintaining buildings in good condition, controlling and keeping buildings free of
vermin and wild birds, restricting other domestic animals on the farm, proper disposal of
dead birds, along with change and disinfection of boots and outer protective clothing for
workers, restriction on staff and equipment entering broiler house, and chlorination of
drinking water (175). However, intensifying biosecurity can only reduce the colonization
level or delay the onset time of colonization of birds by Campylobacter, but can not eliminate
the introduction of Campylobacter into broiler flocks (175, 220, 249).
Because Campylobacter are commonly present in the poultry farm environment and
26
poultry flocks can be infected by multiple sources, it is unlikely that flock exposure to
Campylobacters can be prevented by use of strict biosecurity measures alone (175, 176). In
addition, stringent biosecurity measures are expensive and difficult to maintain (151).
Therefore, other supportive measures, such as competitive exclusion (CE), phage treatment,
antibacterial agents and vaccination, have also been explored.
7.1.2 Competitive exclusion (CE)
With CE, defined or undefined gut flora or probiotic anaerobic bacteria is used to
prevent target bacteria colonization. It is thought that CE works through the generation of
bactericidal metabolites, such as organic acids, or though nutritional competition. Although
CE has been developed as a successful approach for the control of organisms like Salmonella,
its efficacy for Campylobacter is variable and unpredictable (163, 177, 215, 216, 249). Some
studies showed that CE could help reduce the colonization level and prevalence of
Campylobacter in broilers, but in other experiments, these CE materials had no effect (5, 164,
177, 215, 216, 249). Since CE materials are derived from live adult birds, it is not surprising
that they are inconsistent and are influenced by the status of the donor birds and the site from
which the materials are collected (140, 164, 175, 215, 216). Recently, studies showed that
when given as feed supplements, bacteriocins extracted from Bacillus circulans,
Paneibacillus pylomyxa and Lactobacillus salivarius were highly effective in eliminating
Campylobacter colonization from broilers and young commercial turkeys (46, 232, 233).
Several probiotics from Saccharomyces boulardii, Lactobacillus acidophilus and
Streprococcus faecium have been found to be able to partially reduce Campylobacter
colonization in broiler chickens under laboratory conditions (43, 149, 171). Even though
these CE materials seem to work under experimental conditions, their abilities to control and
prevent Campylobacter colonization in chickens under production conditions remain to be
investigated. Currently none of the commercially available CE materials appear to be
effective in excluding Campylobacter from broiler flocks under production conditions (162).
7.1.3 Vaccination
27
Since Campylobacter-specific antibodies can effectively reduce Campylobacter
colonization of chickens (57, 243), vaccination seems to be a feasible approach for control
and prevention of Campylobacter. Several vaccine development strategies are currently
being investigated. These reported immunization studies have used killed whole-cell
vaccines, flagellin-based vaccines, or genetically engineered live vectors expressing
Campylobacter-specific antigens. The protective effect of these vaccines against
Campylobacter was variable. In general, the studies suggest that the Campylobacter-specific
antibodies generated provide some, but not biologically significant protective effects in
chickens (39, 57, 128, 179, 207, 249, 250, 257, 258, 271). However, a recent study reported
that oral immunization of chickens with an avirulent Salmonella strain expressing the C.
jejuni CjaA antigen (the substrate-binding component of an ABC transport system)
drastically reduced the colonization of Campylobacter in the majority of birds upon
homologous challenge (262). The encouraging results from this study suggest the possibility
of using CjaA protein in combination with the attenuated Salmonella vector as a protective
vaccine against Campylobacter colonization in poultry. Passive immunization by oral
administration of anti-Campylobacter antibodies has also been reported to have both
therapeutic and prophylactic properties in chickens (231, 243, 249). Therefore, it may be
possible to immunize parent flocks to produce passively protected offspring. Overall, the
limited results from the experimental vaccines suggest that vaccination may be a feasible
strategy to control Campylobacter infection on poultry farms.
7.1.4 Phage therapy
Bacteriophages are viruses that can infect, multiply, and kill susceptible bacteria. Like
most bacterial species, Campylobacters also have their own specific bacteriophages.
Campylobatcer-specific bacteriophages can be readily isolated from broiler chickens and the
farm environment (19, 50, 51, 65, 151, 251). Bacteriophages have shown to be a promising
approach to reduce Campylobacter colonization in broilers, but currently no commercial
product is available. Recent studies showed that bacteriophages significantly reduce the
Campylobacter number in both therapeutic and preventive treatment of chickens with
Campylobacter-specific bacteriophage. However, the level of reduction was variable and was
28
affected by many factors, such as bacteriophage type, dosage, bacteriophage dynamics and
bacteriophage ecology in chickens. More work will be needed to determine if phage therapy
is an effective and/or practical biological control measure for the reduction of Campylobacter
in poultry.
7.1.5 Other strategies
Other intervention strategies that may be used on poultry farm to control and prevent
Campylobacter colonization in broiler flocks include use of inbred chicken lines, which are
resistant to cecal colonization by Campylobacter (35, 230) or supplementation of feed with
prebiotics (e.g. fructoologosaccharide, lactose, mannose-oligosaccharide), immune response
stimulators (e.g. selenium, beta-glucan), antimicrobials (e.g. salinomycin, flavophopholipol),
or active charcoal (14, 34, 62). Similar to the strategies mentioned previously, these
strategies have shown limited success in reducing the incidence of Campylobacter
colonization in chickens.
7.2 Control of Campylobacter at the slaughterhouse during processing
Multiple measures in the slaughterhouse have been explored to reduce carcass
contamination by Campylobacter during slaughter. Currently, the most promising control
strategy is the separation of Campylobacter-positive and Campylobacter-negative flocks
during slaughter (“scheduled processing”) and decontamination of the meat from positive
flocks (250). However, one issue of the scheduled processing is the sensitivity of the on-farm
test. False-negative tests would result in misidentification of positive flocks which would be
treated as safe meat. The feasibility of scheduled processing remains an issue to the poultry
industry (249).
In addition being used to reduce Campylobacter colonization in live poultry,
bacteriophages have also been used as a disinfection agent for poultry meat in processing
facilities. Several studies investigating the direct application of bacteriophages onto raw
chicken skin showed a significant decrease in Campylobacter numbers on the surface of
29
experimentally-contaminated chicken skin (18, 89). However, these studies only involved
experimentally-infected chickens and only showed efficacy in the laboratory. Additional
studies are needed to assess the possibility of using bacteriophage to control natural
contaminants and improve food safety in a real-life setting (93).
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CHAPTER 3. KEY ROLE OF MFD IN THE DEVELOPMENT OF
FLUOROQUINOLONE RESISTANCE IN CAMPYLOBACTER JEJUNI
A paper published in PLoS Pathogens
Jing Han, Orhan Sahin, Yi-Wen Barton, and Qijing Zhang
Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary
Medicine, Iowa State University, Ames, IA 50011
1. Abstract
Campylobacter jejuni is a major foodborne pathogen and a common causative agent
of human enterocolitis. Fluoroquinolones are a key class of antibiotics prescribed for clinical
treatment of enteric infections including campylobacteriosis, but fluoroquinolone-resistant
Campylobacter readily emerges under the antibiotic selection pressure. To understand the
mechanisms involved in the development of fluoroquinolone-resistant Campylobacter, we
compared the gene expression profiles of C. jejuni in the presence and absence of
ciprofloxacin using DNA microarray. Our analysis revealed that multiple genes showed
significant changes in expression in the presence of a suprainhibitory concentration of
ciprofloxacin. Most importantly, ciprofloxacin induced the expression of mfd, which encodes
a transcription-repair coupling factor involved in strand-specific DNA repair. Mutation of the
mfd gene resulted in approximately 100-fold reduction in the rate of spontaneous mutation to
ciprofloxacin resistance, while overexpression of mfd elevated the mutation frequency. In
addition, loss of mfd in C. jejuni significantly reduced the development of fluoroquinoloneresistant Campylobacter in culture media or chickens treated with fluoroquinolones. These
findings indicate that Mfd is important for the development of fluoroquinolone resistance in
Campylobacter, reveal a previously unrecognized function of Mfd in promoting mutation
frequencies, and identify a potential molecular target for reducing the emergence of
fluoroquinolone–resistant Campylobacter.
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2. Author Summary
As a foodborne bacterial pathogen, Campylobacter jejuni is a common causative
agent of gastrointestinal illnesses in humans. Development of antibiotic resistance in
Campylobacter, especially to fluoroquinolone (a broad-spectrum antimicrobial),
compromises clinical treatments and presents a major public health threat. It is poorly
understood why Campylobacter is highly adaptable to fluoroquinolone treatment and how it
acquires mutations associated with fluoroquinolone resistance. Understanding the molecular
mechanisms involved in the resistance development will help us to reduce the emergence of
FQ-resistant Campylobacter. Using DNA microarray and other molecular methods as well as
animal studies, we uncovered the key role of Mfd in promoting spontaneous mutations and
development of fluoroquinolone resistance in Campylobacter. Mfd is a transcription-repair
coupling factor involved in DNA repair and was not previously known for its role in
promoting mutations conferring antibiotic resistance. Our findings not only reveal a novel
function of Mfd, but also provide a potential molecular target for reducing the emergence of
fluoroquinolone-resistant Campylobacter.
3. Introduction
Campylobacter jejuni, a Gram-negative microaerobic bacterium, is one of the most
prevalent bacterial foodborne pathogens in humans, causing more than 2 million cases of
diarrhea each year in the U.S. alone [1, 2, 3]. As an enteric pathogen, this organism causes
watery diarrhea and/or hemorrhagic colitis. Campylobacter infection is also the most
common antecedent to Guillain-Barre syndrome, an acute flaccid paralysis that may lead to
respiratory muscle compromise and death [4, 5]. In developed countries, person-to-person
transmission of Campylobacter is rare, and the main source of human Campylobacter
infections is via food, water, or milk contaminated by Campylobacter [6].
Fluoroquinolone (FQ) antimicrobials are often prescribed for clinical treatment of
diarrhea caused by enteric bacterial pathogens including Campylobacter [7, 8]. However,
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Campylobacter is increasingly resistant to FQ antimicrobials, which has become a major
concern for public health [9, 10, 11]. FQ-resistant (FQR) Campylobacter developed in food
producing animals can be transmitted to humans via the food chain. Poultry are considered
the major reservoir for C. jejuni and a significant source for FQR Campylobacter infections in
humans, because the majority of domestically acquired cases of human campylobacteriosis
result from consumption of undercooked chicken or food contaminated by raw chicken [2, 12,
13]. Although FQ antimicrobials have been banned since 2005 in poultry production in the
U.S., FQR Campylobacter continue to persist on poultry farms [14, 15, 16].
The main targets of FQs in bacteria are DNA gyrases and/or topoisomerase IV [17,
18]. In Campylobacter, the resistance to FQ antimicrobials is mediated by point mutation in
the quinolone resistance-determining region (QRDR) of gyrA in conjunction with the
function of the multidrug efflux pump CmeABC [10, 19, 20, 21]. Acquisition of high-level
FQ resistance in Campylobacter does not require stepwise accumulation of point mutations
in gyrA. Instead, a single point mutation in gyrA can lead to clinically relevant levels of
resistance to FQ antimicrobials [19, 20, 22]. Specific mutations at positions Thr-86, Asp-90
and Ala-70 in GyrA have been linked to FQ resistance in C. jejuni [10, 19]. When
enumerated by ciprofloxacin (CIPRO)-containing plates, spontaneous FQR Campylobacter
mutants occur at a frequency as high as 10-6 [23], suggesting that C. jejuni possess a high
mutation rate to FQ resistance. CmeABC, an energy-dependent efflux system, contributes
significantly to the intrinsic and acquired resistance to FQs in C. jejuni by reducing the
accumulation of the antibiotics within Campylobacter cells [19, 20, 24, 25]. The expression
level of cmeABC also influences the frequencies of emergence of spontaneous FQR mutants
[23].
One unique feature of FQ resistance development in Campylobacter is the rapid
emergence of FQR mutants from a FQ-susceptible (FQS) population when treated with FQ
antimicrobials. This has been observed in Campylobacter-infected animals or patients treated
with FQs [19, 26, 27, 28, 29, 30]. In chickens infected with FQS Campylobacter, treatment
with enrofloxacin resulted in the emergence of FQR Campylobacter mutants that were
59
detected in feces within 24-48 hours after the initiation of treatment, and the FQR population
continued to expand during the treatment and eventually occupied the intestinal tract at a
density as high as 107 CFU/g feces [19, 29, 30]. As shown in a comparison study, the same
FQ treatment did not result in the development and enrichment of FQR E. coli in chickens
[29], suggesting that C. jejuni has a unique ability to adapt to FQ treatment. This high
frequency of emergence of FQR Campylobacter mutants in response to the selection pressure
may have directly contributed to the global prevalence of FQR Campylobacter. For example,
multiple studies have shown the temporal link between the approval of FQ antimicrobials for
use in animal production and the rapid increase of FQR Campylobacter isolates from both
animals and humans [9, 31, 32, 33, 34, 35, 36, 37, 38, 39]. In some regions of the world, the
vast majority of Campylobacter isolates have become resistant to FQ antimicrobials [22, 40,
41].
The rapidness and magnitude of FQ resistance development in Campylobacter in
response to FQ treatment suggest that both selective enrichment of pre-existing spontaneous
mutants and adaptive gene expression may contribute to the emergence of FQR
Campylobacter, but how Campylobacter responds to FQ treatment is unknown. Within
bacterial cells, FQ antimicrobials form a stable complex with gyrases and DNA, which
generates double-stranded breaks in DNA and leads to bacterial death [18]. In other bacteria,
antibiotic treatments (including FQs) induce the SOS response, which upregulates multiple
genes involved in DNA repair, recombination, and mutation as well as other functions
[42,43,44,45]. The SOS response is controlled by LexA, a transcriptional repressor. DNA
damage triggers LexA autocleavage, which derepresses the SOS genes controlled by LexA.
Once activated, SOS response promotes the development of drug resistance, horizontal
transfer of genetic materials, and production of virulence factors [45, 46, 47]. Unlike many
other bacterial organisms, epsilonproteobacteria including Campylobacter and Helicobacter
do not have a LexA ortholog [46] and also lack many genes involved in DNA repair,
recombination, and mutagenesis, such as the mutHL genes (methyl-directed mismatch repair),
the umuCD genes (UV-induced mutagenesis), and SOS-controlled error-prone DNA
polymerases [48, 49, 50]. These observations suggest that Campylobacter may not have the
60
typical SOS response system. In light of this possibility, it is intriguing to determine how
Campylobacter copes with FQ treatment and what facilitates the emergence of FQR mutants
in Campylobacter.
In this study, we examined the gene expression profiles of C. jejuni NCTC 11168 in
response to treatment with CIPRO. Consistent with the prediction, a typical SOS response
was not observed in Campylobacter treated with CIPRO. However, 45 genes showed ≥1.5fold (p < 0.05) changes in expression when Campylobacter was exposed to a suprainhibitory
dose of CIPRO for 30 min. One of the up-regulated genes was mfd (mutation frequency
decline), which encodes a transcription-repair coupling factor involved in DNA repair. The
mfd gene in E. coli was originally linked to the phenotype of mutation frequency decline [51,
52]. Subsequently it was found that Mfd functions as a transcription-repair coupling factor
and promotes strand-specific DNA repair [53, 54]. DNA lesions stall RNA polymerase
during transcription. Mfd displaces the stalled RNA polymerase from the DNA lesions in an
ATP-dependent manner, recruits the UvrABC excinuclease complex, and enhances the repair
of the DNA lesions on the transcribed strand [54, 55]. Thus, Mfd couples transcription with
DNA repair and contributes to mutation frequency decline. Recently it was reported that
depending on the nature of DNA damage and the availability of NTPs, Mfd can also promote
the forward translocation of arrested RNA polymerase in the absence of repair, leading to
transcriptional bypass of non-repaired lesions [55]. In contrast to its previously known
function in the decline of mutation frequency in other bacterial organisms [51, 52], Mfd in
Campylobacter was found to promote the emergence of spontaneous FQR mutants and the
development of FQR mutants under FQ treatments in this study. These findings define a
novel function of Mfd and significantly improve our understanding of the molecular
mechanisms underlying the development of FQR Campylobacter.
4. Results
Transcriptional analysis of C. jejuni response to FQ treatment
To understand the adaptive response of Campylobacter to FQ treatment, DNA
microarray was used to analyze the transcriptional changes in C. jejuni NCTC 11168 after
61
exposure to CIPRO. When the Campylobacter cells were treated with a subinhibitory
concentration (0.06 µg/ml; 0.5 x the MIC) of CIPRO for 1.5 hours, no genes showed ≥1.5fold changes in expression, suggesting that the transcriptional response to the low dose of
CIPRO was very limited. When the Campylobacter cells were treated with a suprainhibitory
concentration (1.25 µg /ml; 10 x the MIC) of CIPRO for 30 min, 45 genes showed ≥1.5-fold
(p < 0.05) changes in expression (Table 1), among which 13 were up-regulated and 32 were
down-regulated. The up-regulated genes are involved in cell membrane biosynthesis, cellular
processes, and transcription-coupled DNA repair or have unknown functions, while the
majority of the down-regulated genes are involved in energy metabolisms (Table 1).
Consistent with the lack of LexA, the core genes involved in SOS responses in other bacteria,
such as recA, uvrA, ruvC, ruvA, and ruvB, did not show significant changes in expression.
The expression of other genes involved in DNA repair and recombination also did not change
significantly. These findings indicate that C. jejuni does not mount a typical SOS response or
upregulate the general DNA repair system in the early response to CIPRO treatment. Notably,
cj1085c, a homolog of mfd, was upregulated in the presence of CIPRO. Two up-regulated
genes, uppP and uppS, encode products involved in cell wall production [56,57], while pldA
encodes an outer membrane phospholipase that has been implicated in hemolysis, capsular
production, and virulence [58, 59]. According to the Q values, the identified genes would
have an estimated false discovery rate (FDR) of 20%. However, quantitative real-time RTRCR (qRT-PCR) confirmed all of the 11 genes selected from the microarray list (Table 1),
suggesting that the actual FDR is lower than the estimation.
Characteristics of Mfd
Cj1085c (978aa) was annotated as Mfd [48] and shows 31.5% amino acid identity to
the E. coli Mfd protein (1148 aa). In addition, it contains the characteristic domains
conserved in Mfd proteins, such as the ATP/GTP-binding site motif and the superfamily II
helicase motif. Mfd in other bacteria has been shown to be involved in strand-specific DNA
repair by displacing lesion-stalled RNA polymerase and recruiting enzymes involved in
recombination events [54,60]. The mfd locus is highly conserved in Campylobacter and is
present in all Campylobacter species and C. jejuni strains that have been sequenced to date.
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The Mfd proteins in different Campylobacter species share 57-79% identity to the Mfd in C.
jejuni NCTC 11168. Within C. jejuni, the Mfd proteins are 98-100% homologous among
different strains. The mfd gene is located in the middle of a gene cluster, whose transcription
is in the same direction (partially shown in Fig. 1A). The downstream gene Cj1084c encodes
a putative ATP/GTP binding protein, while the upstream gene Cj1086c encode a hypothetical
protein [48]. It is unknown if mfd and its flanking genes form an operon, but it appeared that
Cj1086c and mfd were co-transcribed because a RT-PCR product spanning both ORFs was
amplified (data not shown).
Expression levels of mfd influence the frequency of emergence of spontaneous FQresistant mutants.
Since mfd was the only DNA repair related gene that showed a significant change in
expression in the early response of C. jejuni to CIPRO treatment (Table 1), we examined its
role in the emergence of spontaneous FQR mutants in Campylobacter. Firstly, the mfd gene
was inactivated by insertional mutagenesis (Fig. 1B). As shown in Fig. 2, the mfd mutant
(JH01) showed a approximately 100-fold reduction in the frequencies of emergence of
spontaneous FQR mutants detected using plates containing three different concentrations (1, 2,
and 4 µg/ml, respectively) of CIPRO. Complementation of the mfd mutant in trans by a
plasmid-carried mfd restored the frequencies of mutant emergence to the wild-type level
(JH02 in Fig. 2). As determined by qRT-PCR, the expression level of mfd in the
complemented construct (JH02) was fully restored (1.7 x the wild-type level). pRY112 alone
(without the cloned mfd gene) did not complement the mfd mutant in the mutation frequency
(data not shown). These results indicate that Mfd contributes significantly to the rate of
spontaneous mutations to FQ resistance.
Secondly, we determined if the enhanced expression of mfd increases the mutation
frequency. For this purpose, we constructed strain JH03, which was a wild-type 11168 strain
containing an extra copy of mfd carried on a shuttle plasmid. In JH03, the mRNA of mfd
increased 3.8 times compared with that in 11168 as determined by qRT-PCR. When
compared with the wild-type 11168, the frequency of emergence of FQR mutants from JH03
63
increased about 10-fold (Fig. 2). The increase was reproducible in multiple experiments and
was statistically significant (P < 0.05). These results indicated that overexpression of mfd
increases the frequency of emergence of spontaneous FQR mutants.
Given that there is only one nucleotide between the mfd gene and its downstream
gene cj1084c, it was prudent to determine if the mfd mutation resulted in a polar effect on the
expression of cj1084c. RT–PCR showed that cj1084c was transcribed at a comparable level
in both the mfd mutant and the wild-type NCTC 11168 (Fig. 1C). RT-PCR was also
performed using 10-fold serial dilutions of the RNA template, which yielded comparable
results between the two strains (data not shown). PCR without the reverse transcriptase did
not yield a product (Fig. 1C), indicating that the mRNA templates had no DNA
contamination. These results suggested that the insertional mutation in the mfd gene did not
cause an apparent polar effect on expression of the downstream gene. This finding plus the
complementation data (Fig. 2) strongly indicate that loss of Mfd is responsible for the
observed reduction in the mutation frequency in JH01.
Loss of mfd does not affect the susceptibility of C. jejuni to antibiotics.
To examine if the reduction in the emergence of spontaneous FQR mutants is caused
by the increased susceptibility of the mfd mutant to CIPRO, we compared the MICs of
several antibiotics in the mfd mutant with those in the wild type. Our results did not reveal
any differences between the mutant and the wild type in their susceptibility to the tested
antibiotics including erythromycin, ampicillin, streptomycin, and CIPRO (data not shown).
In addition, there was no apparent difference in growth kinetics between the wild-type and
the mfd mutant either in MH broth (without antibiotics) or in MH broth supplemented with a
subinhibitory concentration (0.06 µg/ml) of CIPRO (Fig.3). The growth rates of the mfd
over-expressing strain (JH03) and the complemented mutant (JH02) were also similar to that
of the wild type (Fig. 3). Thus, the reduced spontaneous mutation rate to FQ resistance in the
mfd mutant was not attributable to decreased growth rate or increased susceptibility to
antibiotics. In addition, the CIPRO-resistant colonies examined for gyrA mutations all carried
64
the C257T mutation in gyrA and had a CIPRO MIC of > 32 µg/ml regardless of the
backgrounds (11168 or JH01) from which the mutants were selected.
Mfd contributes to the emergence of FQR Campylobacter under in vitro treatment
FQR Campylobacter mutants emerge rapidly from a FQ-susceptible population once
treated with FQ antimicrobials [19,26,27,28,29,30]. To determine if Mfd influences the
development of FQR Campylobacter under selection pressure, we conducted in vitro growth
experiments, in which C. jejuni was treated with a suprainhibitory concentrations of CIPRO
(4 µg/ml). In the first treatment experiment, 109 CFU of bacterial cells were inoculated into
each flask containing 100 ml MH broth with 4 µg/ml of CIPRO, yielding an initial cell
density of 107 CFU/ml. At the beginning of the treatment, 1-3 CFU/ml of FQR mutants were
detected in the flasks inoculated with 11168, while no FQR mutants were detected in the
cultures inoculated with JH01 (Fig. 4A). One day after the initiation of the treatment, the
numbers of FQR mutants in the 11168 cultures grew to a level ranging from a few hundreds
to a few thousands CFU/ml, while no mutants or about 1 CFU/ml of FQR mutants were
detected in the cultures of JH01 (Fig. 4A). The FQR populations expanded on day 2 in both
strains, but the FQR population of JH01 was still about 1,000-fold less than that of 11168.
Due to the continued enrichment of the FQR mutants by CIPRO and the fact that the mutants
of 11168 was entering the stationary phase, the average difference between 11168 and JH01
on day 3 decreased, but was still more than one order of magnitude (Fig. 4A). In the second
experiment, 2 x 107 CFU bacterial cells of 11168 or JH01 were inoculated into each flask
containing 20 ml of MH broth with 4 µg/ml of CIPRO, yielding an initial cell density of 106
CFU/ml. At the beginning of the treatment, no FQR mutants were detected from either 11168
or JH01 (Fig. 4B). On day 1 after the initiation of the treatment, FQR Campylobacter
emerged from some of the cultures of 11168 and continued to expand in numbers on day 2
and day 3. In contrary to 11168, no FQR mutants emerged from any of the JH01 cultures
during the three-day incubation (Fig. 4B). In the third experiment, the inoculum was
decreased to 2 x 104 CFU per flask (initial cell density = 103 CFU/ml), and no FQR mutants
were detected from either 11168 or JH01 after three day’s incubation (data not shown).
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These results indicated that emergence of FQR mutants under treatment with CIPRO was
influenced by the initial bacterial cell density and facilitated by the function of Mfd.
Mfd affects the emergence of FQR mutants in vivo
To determine if Mfd influences the emergence of FQR Campylobacter during in vivo
therapeutic treatment, broiler chickens were infected with 11168 or JH01 and then treated
with enrofloxacin administered in drinking water (50 ppm). The birds in both groups were
quickly colonized by C. jejuni after inoculation (Fig. 5). Before the treatment with
enrofloxacin, all birds were colonized by Campylobacter and the colonization levels (CFU/g
feces) were similar in both groups (p > 0.05). One day after initiation of the treatment, the
number of colonized chickens and the levels of colonization decreased drastically in both
groups, with Campylobacter detectable only in three chickens that were inoculated with the
wild-type strain (Fig. 5A). After that, the numbers of Campylobacter in both groups
rebounded. On day 3 after the initiation of the treatment, all of the birds in the 11168 group
were re-colonized by Campylobacter and remained colonized until the end of the experiment.
For the group inoculated with JH01, 6 of the11 birds became positive with Campylobacter on
day 3 after initiation of the treatment (Fig. 5A) and 3 birds remained negative until the end of
the experiment. On days 3, 5 and 7 after initiation of the treatment, the average colonization
level of the JH01-inoculated group was approximately 3 log units lower than that of the
11168-inoculated group (Fig. 5A) and the differences were statistically significant (p < 0.05).
The number of FQR Campylobacter in each chicken was also monitored. Prior to the
treatment, no FQR C. jejuni was detected in any of the chickens (Fig. 5B). On day 1 after
initiation of the treatment, the three Campylobacter-positive birds of the 11168-inoculated
group still carried FQ-susceptible Campylobacter. However, FQR C. jejuni appeared on day 3
in all birds of the 11168-inoculated group and in some birds of the JH01-inoculated group
(Fig. 5B). Comparison of the total Campylobacter counts (Fig. 5A) with the numbers of FQR
Campylobacter (Fig. 5B) revealed that the birds were re-colonized by FQR mutants after
initiation of the treatment. The average numbers of FQR Campylobacter in the JH01inoculated group were approximately 3 log units lower that those of the 11168-inoculated
group (Fig. 5B) and the differences were statistically significant (p < 0.05). These results
66
indicate that loss of Mfd significantly reduced the rates of emergence of FQR Campylobacter
in enrofloxacin-treated chickens.
Representative Campylobacter isolates obtained at different sampling times from both
groups were tested for CIPRO MICs using E-test strips. The result showed that before
treatment all the tested isolates from both groups were susceptible to CIPRO (MICs = 0.094 0.125 µg/ml). The majority of the tested isolates from day 1 after initiation of the treatment
were still susceptible to CIPRO (MICs = 0.094 - 0.5 µg/ml). On day 3 after the initiation of
treatment, 21 of the 22 tested isolates (from both groups) had a CIPRO MIC of > 32 µg/ml
and the other one had an MIC of 8 µg/ml. Similarly, the majority (44 out of 49) of the tested
isolates from days 5 and 7 had a CIPRO MIC of >32 µg/ml and the rest had MICs from 1-24
µg/ml. The MIC results further confirmed the differential plating results that the chickens
were re-colonized by FQR Campylobacter.
5. Discussion
When Campylobacter cells were treated with a subinhibitory concentration (0.06
µg/ml, 0.5 x the MIC) of CIPRO for 1.5 hours, no significant changes in gene expression
were detected using the cut-off criteria defined in this study. This result was somewhat
similar to the study with Haemophilius influenzae [61] in that the treatment with a low
concentration of CIPRO induced few changes in gene expression, but was different from that
study because several genes involved in SOS response were upregulated in Haemophilius
influenzae. Prolonged treatment of Campylobacter with the subinhibitory concentration of
CIPRO may reveal noticeable changes in gene expression, but culturing Campylobacter with
0.06 µg/ml of CIPRO reduces its growth rate (Fig. 3), which will make the comparison with
the non-treated control unfeasible and complicate the interpretation of the microarray results.
To mimic clinical treatment, C. jejuni cells were exposed to a suprainhibitory dose
(1.25µg/ml, 10 x the MIC) of CIPRO. This dose is within the concentration range of CIPRO
in gut contents during FQ treatment in chickens [62]. The reason that we treated the samples
for 0.5 hour instead of a longer time was to detect the primary response triggered by CIPRO,
instead of the secondary response caused by cell death. When Campylobacter cells were
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treated with this suprainhibitory dose for 0.5 hour, the expression of multiple genes was
significantly altered (Table 1). Notably, the majority of the affected genes were
downregulated and many of them are involved in cellular processes and energy metabolism
(Table 1). This result is similar to the findings obtained with other bacteria [43, 44, 61] and
supports the notion that reducing cellular metabolism is a common strategy utilized by
bacteria to cope with antibiotic treatment.
Within bacterial cells CIPRO interacts with gyrase and DNA, blocking DNA
replication and transcription [18]. When exposed to CIPRO, the expression of gyrA and gyrB
in various bacteria was either altered or unchanged [43, 61, 63]. In this study, we found that
the expression of gyrA, gyrB, and topA was not significantly affected in Campylobacter by
CIPRO. In addition, the expression of the genes encoding enzymes involved in DNA repair,
recombination, or mutagenesis, such as recA, ruvABC, uvrABC, and mutS, did not change
significantly. Only two genes involved in DNA metabolism (mfd and dnaE) were affected by
CIPRO under the conditions used in this study (Table 1). These observations indicate that C.
jejuni does not mount a typical SOS response under the treatment with FQ. These findings
are also consistent with the fact that C. jejuni lacks LexA, the key regulator of bacterial SOS
responses [46].
In addition to transcription-coupled DNA repair, Mfd has been associated with other
functions in bacteria [64]. For example, Mfd of Bacillus subtilis is involved in homologous
DNA recombination and stationary-phase mutagenesis [65, 66]. Inactivation of the mfd gene
of B. subtilis resulted in a great reduction in the number of prototrophic revertants to Met+,
His+, and Leu+ during starvation [66], indicating that Mfd promotes adaptive mutagenesis.
This finding is in contrast to the known function of Mfd in mediating mutation frequency
decline and could be explained by the role of Mfd in promoting transcriptional bypass and
consequently increasing the adaptive mutagenesis rates [66].
In this study we found that Mfd increases the frequency of emergence of spontaneous
R
FQ mutants in Campylobacter (Fig. 2). Furthermore, the mfd mutation also decreased the
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frequency of emergence of spontaneous streptomycin-resistant mutants in Campylobacter
(data not shown). Together, the results convincingly showed that Mfd is an important player
in modulating the mutation rates in Campylobacter. To our knowledge, this is the first report
documenting the key role of Mfd in promoting spontaneous mutation rates in a bacterial
organism. How Mfd contributes to the increased mutation rates in Campylobacter is
unknown, but it can be speculated that transcriptional bypass mediated by Mfd may actively
occur in replicating non-stressed Campylobacter populations, resulting in an elevated level of
retromutagenesis (fixed changes in DNA sequence due to transcriptional mutation [67]) that
contributes to the size of the mutant pools. This possibility remains to be examined in future
studies. Although mfd contributes significantly to the mutation rate (Fig. 2), its expression
level was not precisely proportional to the mutation frequencies. For example, expression of
mfd was upregulated 3.8-fold in JH03, but its mutation frequency increased 10-fold. This
difference is probably due to the fact that emergence of spontaneous mutants is a multi-step
process and Mfd only contributes to one of the steps in the process. It is also possible that
Mfd interacts with other proteins in modulating the mutation frequency. Thus, the changes in
mfd expression level and the mutation frequency are not exactly at the same scale.
Another interesting observation of this study is the upregulation of mfd by CIPRO.
The enhanced expression may be needed for transcription repair because CIPRO treatment
causes DNA damage, which stalls RNA polymerase. Alternatively, the increased production
of Mfd may enhance transcriptional bypass of the non-repaired DNA lesions in order to
maintain cell viability and/or promote mutations for resistance. This possibility is high given
the facts that massive DNA damage incurred by a suprainhibitory dose of CIPRO may
overwhelm the DNA repair system and Campylobacter must maintain certain levels of
transcription to survive the treatment, that Mfd contributes significantly to the mutation rates
to FQ resistance (Fig. 2), and that Campylobacter does not have the error-prone DNA
polymerases, such as Pol II, Pol IV, and Pol V [48]. E. coli and other bacteria have these
error-prone DNA polymerases [68, 69], which are repressed by LexA, but upregulated by the
SOS response triggered by DNA damage. Once produced, the enzymes perform translesion
DNA synthesis, allowing replication to continue without DNA repair. This special functional
69
feature results in reduced genetic fidelity, but allows for bacterial survival under stress. The
outcome of the enhanced expression of the error-prone enzymes is the increased mutation
rates, which contribute to the emergence of drug resistance [70]. In the absence of a SOS
response and the error-prone DNA polymerases, Campylobacter may use Mfd as an
alternative pathway to increase mutation rates. Thus, enhanced expression of mfd may
represent an adaptive response of Campylobacter to the stresses imposed by CIPRO
treatment. How CIPRO upregulates Campylobacter Mfd is unknown and further work in this
direction is warranted.
FQR Campylobacter readily emerges from a FQ-susceptible population when treated
with FQ antimicrobials (Figs. 4 and 5). As shown in the in vitro experiment, the development
of FQR population under CIPRO treatment is influenced by the initial cell density (Fig. 4 and
the corresponding text) as well as the functional state of Mfd. Considering the differences in
spontaneous mutation rate between 11168 and JH01 (Fig. 2), it was likely that the 11168 and
JH01 inocula had different numbers of pre-existing FQR mutants, which were selected by
CIPRO and contributed to the differences in the FQR population detected in the cultures of
the two strains. The inoculum-dependent emergence of FQR mutants in both 11168 and JH01
suggests that development of FQR Campylobacter under FQ treatment involves selection of
preexisting mutants. However, the magnitude and dynamics of FQR development can not be
totally explained by selection. For example, in some cultures FQR mutants were not
detectible until the 2nd day of the incubation (Fig. 4). A single mutant at time zero in a culture
flask would grow to a population of more than 2,000 cells in one day (the generation time of
C. jejuni in MH broth is about 2 hours), which would be readily detected by the plating
method on day 1. Thus, if selection was the only factor in the development of FQR
Campylobacter, the latest time for detecting the pre-existing mutants in the mutant-positive
flasks would be day 1 after initiation of the treatment. Obviously, this was not the case for all
of the cultures because some of them did not show FQR mutants until day 2 (Fig. 4). In
addition, some cultures were negative with FQR mutants at time zero, but showed a large
number of mutants at day 1, which could not be easily explained by sole selection of a few
preexisting mutants from the inocula. Considering these unexplainable observations and the
70
fact that a small fraction of the FQ-susceptible inoculum survived the killing effect as long as
one day after the initiation of the treatment (data not shown), it was possible that new FQR
mutants were developed during the treatment. If this occurs, Mfd may enhance the
emergence of new mutants by promoting transcriptional bypass or other mechanisms, which
may partly explain the differences between 11168 and JH01 in the dynamics of emergence of
FQR mutants. Thus, there is a possibility that both selection of pre-existing mutants and de
nova formation of mutants are involved in the development of FQR Campylobacter during
treatment with FQ antimicrobials.
The role of Mfd in the development of FQR mutants was further shown by the in vivo
experiment, in which Campylobacter-infected chickens were treated with enrofloxacin (Fig.
5). Previous studies have shown that therapeutic use of FQ antimicrobials in chickens
promotes the emergence of FQR Campylobacter [19, 27, 28, 29, 30], which can be potentially
transmitted to humans via the food chain. In this study, we showed that inactivation of mfd
significantly reduced the development of FQR Campylobacter in chickens (Fig. 5). In fact,
several birds in the JH01-inoculated group became negative with Campylobacter once the
treatment was initiated. Since the mfd mutant did not show a growth defect in vitro (Fig. 3)
and colonized chickens as efficiently as the wild-type strain (see the colonization level before
treatment in Fig. 5), the observed differences in the development of FQR mutants were not
due to changes in growth characteristics. These in vivo results (Fig. 5) plus the in vitro
findings (Fig. 4) clearly showed that Mfd plays an important role in the development of FQR
Campylobacter mutants under the selection pressure. To our knowledge, this is the first
report that documents the role of Mfd in the development of FQ resistance in a bacterial
pathogen. Since Mfd is highly conserved in bacterial organisms [64], it would be interesting
to know if this finding applies to other bacterial pathogens. In addition, inhibition of Mfd
functions may represent a feasible approach to reducing the emergence of FQR
Campylobacter.
6. Materials and Methods
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Bacterial strains and growth conditions
C. jejuni strain NCTC 11168 (ATCC 700819) was used in this study. The strain was
routinely grown in Mueller-Hinton (MH) broth (Difco) or on MH agar at 42 °C under
microaerobic conditions (5% O2, 10% CO2, and 85% N2). The media were supplemented
with kanamycin (50 µg/ml) or chloramphenicol (4 µg/ml) as needed. Escherichia coli cells
were grown at 37 °C with shaking at 200 r.p.m. in Luria Bertani (LB) medium which was
supplemented with ampicillin (100 µg/ml) or kanamycin (30 µg/ml) when needed.
DNA microarray and qRT-PCR
DNA microarray was used to identify genes that were differentially expressed in C.
jejuni 11168 treated with CIPRO. For RNA isolation, Campylobacter cells were grown for
24 hours to the mid exponential phase (OD600 ≈ 0.1 ~ 0.15) and split into two equal portions,
one of which was treated with CIPRO and the other served as a non-treated control. A
subinhibitory concentration (0.06 µg/ml, 0.5 x the MIC) and a suprainhibitory dose (1.25
µg/ml, 10 x the MIC) of CIPRO were used in the treatments. For the treatment with 0.06
µg/ml of CIPRO, the treated and non-treated samples were incubated at 42 °C for 1.5 hours
under microaerobic conditions, while for the treatment with 1.25 µg/ml of CIPRO, the
samples were incubated at 42 °C for 30 min under microaerobic conditions. Immediately
after the incubation, RNAprotectTM Bacteria Reagent (Qiagen, Valencia, CA) was added to
the cultures to stabilize mRNA. The total RNA from each sample was extracted using the
RNeasy Mini Kit (Qiagen). The purified RNA samples were treated with On-Column DNase
Digestion Kit (Qiagen) followed by further treatments with DNase to remove residual DNA
contamination. RNA samples were extracted from 6 independent treatments with each
concentration of CIPRO. Absence of contaminating DNA in the RNA samples was
confirmed by RT-PCR. The concentration of total RNA was estimated with the NanoDrop
ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), and the integrity and size
distribution of the purified RNA was determined by denaturing agarose gel electrophoresis
and ethidium bromide staining. The quality of total RNA was further analyzed using the
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Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), which showed the
good quality and integrity of the RNA samples (Data not shown).
cDNA synthesis and labeling, microarray slide (Ocimum Biosolutions) hybridization,
Data collection and normalization, and statistical analysis were performed as described in a
previous publication [71]. For each type of treatment (0.06 µg/ml for 1.5 hours or 1.25 µg/ml
for 30min), six microarray slides were hybridized with RNA samples prepared from 6
independent experiments. For this study, we chose p-value < 0.05 and the change ≥ 1.5-fold
as the cutoff for significant differential expression between the treated and non-treated
samples. Representative genes identified by the DNA microarray were further confirmed by
qRT-PCR as described in a previous work [72]. The primers used for qRT-PCR are listed in
Table 2.
Insertional mutation of mfd
An isogenic mfd (cj1085c) mutant of strain NCTC 11168 was constructed by
insertional mutagenesis. Primers mfd-F2 (5’-TGTTGATGGAGAGTTAAGTGGTAT-3’)
and mfd-R2 (5’-AATAGCATTCATAGCGACTTCTGTT -3’) were designed from the
published genomic sequence of this strain [48] and used to amplify a 1.8-kb fragment
spanning the 5’ region of mfd. Amplification was performed with Pfu Turbo® DNA
Polymerase (Stratagene, La Jolla, CA, USA). The blunt-ended PCR product was purified
using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA), ligated to SmaI–
digested suicide vector pUC19, resulting in the construction of pUC-mfd, which was then
transformed into E. coli DH5α. Since a unique EcoRV site (which generates blunt ends)
occurs in the cloned mfd fragment, pUC-mfd was digested with EcoRV to interrupt the mfd
gene. Primers KanNco-F (5’ CTT ATC AAT ATA TCC ATG GAA TGG GCA AAG CAT
3’) and KanNco-R (5’ GAT AGA ACC ATG GAT AAT GCT AAG ACA ATC ACT AAA
3’) were used to amplify the aphA3 gene (encoding kanamycin resistance) from the pMW10
vector [73] by using Pfu Turbo® DNA polymerase (Stratagene). The aphA3 PCR product
was directly ligated to EcoRV-digested pUC-mfd to obtain construct pUC-mfd-aphA3, in
which the aphA3 gene was inserted within mfd (the same direction as the transcription of mfd)
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and the insertion was confirmed by PCR using primers mfd-F2 and Kana-intra (5’ GAA
GAA CAG TAT GTC GAG CTA TTT TTT GAC TTA 3’). The pUC-mfd-aphA3 construct,
which served as a suicide vector, was electroporated into C. jejuni NCTC 11168.
Transformants were selected on MH agar containing 10 µg/ml of kanamycin. Inactivation of
the mfd gene in the transformants by insertion of the ahpA3 gene was confirmed by PCR
using primers mfd-F2 and mfd-R2 (Fig. 1B). The mfd mutant of NCTC 11168 was named
JH01.
Complementation of the mfd mutant in trans
The entire mfd gene including its putative ribosome binding site was amplified from
strain 11168 by PCR using primers mfd-F5 (5’CGCTTCCGCGGAACTAGTAAAATTAAAGAAGATACTATC-3’) and mfd-R3 (5’GGCTTTAAATAATCTTTTCGAGCTCTATAAATT-3’). The underlined sequences in the
primers indicate the restriction sites for SacI and SacII, respectively. The PCR product was
digested with SacI and SacII, and was then cloned into the plasmid construct pRY112-pABC
to generate pRY112-mfd, in which the mfd gene was fused to the promoter of cmeABC.
pRY112-pABC was made by cloning the promoter sequence of cmeABC [74] to shuttle
plasmid pRY112 [75]. The promoter DNA of cmeABC was amplified by primers BSF (5’
AAAAGGATCCTAAATGGAATCAATAG 3’) and AR2 (5’
TGATCTAGATCATACCGAGA 3’), digested with BamHI and XbaI, and cloned into
pRY112. There were two reasons that we used the promoter of cmeABC in the expression of
mfd. First, the 5’ end of mfd overlaps with its upstream gene and the native promoter for mfd
was unknown. Second, the promoter of cmeABC is moderately active in Campylobacter [74],
preventing over- or under-expression of mfd. The constructed plasmid pRY112-mfd was
sequenced and confirmed that no mutations in the cloned sequence occurred. For
complementation, the shuttle plasmid pRY112-mfd was transferred into JH01 by conjugation.
The complemented strain was named JH02. Limited passage of JH02 in MH broth without
antibiotics indicated that the complementing plasmid was stable in the construct (data not
shown). The shuttle plasmid carrying the mfd gene was also transferred to wild-type 11168 to
generate strain JH03 for overexpression of the mfd gene.
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Growth rates in MH broth with or without CIPRO
To compare the growth kinetics of the mfd mutant with that of the wild-type, a fresh
culture of each strain was inoculated into MH broth (initial cell density of OD600 = 0.05)
and the cultures were incubated at 42 °C under microaerobic conditions. To determine if the
mutation affects C. jejuni growth with a subinhibitory concentration of CIPRO, the various
strains were grown in MH broth with 0.06 µg/ml of CIPRO (0.5 x the MIC). Culture samples
were collected and measured for OD600 at 0, 3, 6, 12, 24 and 48 hours post-inoculation.
Antibiotic susceptibility test
The minimum inhibitory concentration (MIC) of CIPRO was determined by using Etest strips (AB Biodisk, Solna, Sweden) as described in the manufacturer's instructions. The
detection limit of the E-test for CIPRO was 32 µg/ml. The MICs of erythromycin, ampicillin
and streptomycin for C. jejuni NCTC 11168, JH01, JH02, and JH03 were determined using a
standard microtiter broth dilution method described previously [24]. Each MIC test was
repeated at least three times to confirm the reproducibility of the MIC patterns. The
antibiotics used in this study were purchased from Sigma Chemical Co. (erythromycin,
ampicillin, streptomycin) or ICN Biomedicals Inc. (CIPRO).
Frequencies of emergence of spontaneous FQR mutants in vitro
Wild-type 11168, JH01, JH02 and JH03 were compared for the spontaneous mutation
rates to CIPRO resistance. In each experiment, each of the 4 strains was inoculated into three
flasks, each of which contained 30 ml of antibiotic-free MH broth. The cultures were
incubated to the mid logarithmic phase (OD600 ≈ 0.15) under microaerobic conditions. The
culture in each flask was collected by centrifugation and resuspended in 1 ml of MH broth.
The total CFU in each culture was measured by serial dilutions and plating on MH agar
plates, while the number of FQR mutants was detected using MH agar plates containing 1, 2
or 4 µg/ml CIPRO. The frequency of emergence of FQR mutants was calculated as the ratio
of the CFU on CIPRO-containing MH agar plates to the CFU on antibiotic-free MH agar
75
plates after 2 days of incubation at 42°C under microaerobic conditions. This experiment was
repeated five times. The mutation frequency data were log-transformed for statistical analysis.
One-Way ANOVA followed by Tukey test was used to determine the significance of
differences in the levels of spontaneous mutation rates among the strains. The data were also
analyzed by the Wilcoxon rank-sum test to allow for non-normality. For the comparisons
discussed in Results, the conclusion of the two tests was the same at significance level 0.05.
Sequence analysis of the QRDR of gyrA
Representative FQR colonies were selected for determination of the point mutations in
gyrA. The QRDR of gyrA was amplified by PCR using primer pair GyrAF1 (5'CAACTGGTTCTAGCCTTTTG-3') and GyrAR1 (5'-AATTTCACTCATAGCCTCACG-3')
[76]. The amplified PCR products were purified with the QIAquick PCR purification kit
(Qiagen) prior to sequence determination. DNA sequence analysis was carried out using an
automated ABI Prism 377 sequencer (Applied Biosystems, Foster City, CA, USA) and
analyzed by the Omiga 2.0 (Oxford Molecular Group) sequencing analysis software.
In vitro treatment with CIPRO
To determine if Mfd affects the development of FQR mutants under treatment with
CIPRO, wild-type 11168 and JH01 were treated in MH broth with 4 µg/ml (32 x the MIC) of
CIPRO. Wild-type 11168 and JH01 were grown on antibiotic-free MH agar plates under
microaerobic conditions. After 20 hours of incubation, the cells were collected and
resuspended in MH broth for inoculation. Three treatment experiments were conducted using
three different initial cell densities. In experiment 1, each strain was inoculated into 3 100-ml
flasks with MH broth containing 4 µg/ml of CIPRO and the initial cell density was 107
CFU/ml. The cultures were incubated microaerobically at 42 oC. Aliquots of the cultures
were collected at different time points (0, 1, 2, 3 days post-inoculation) and plated onto
regular MH plates for enumeration of the total bacterial number and onto MH plates
containing 4 µg/ml of CIPRO for counting FQR colonies. In experiments 2 and 3, the cultures
were treated in the same way, but the initial cell densities were 106 and 103 CFU/ml,
76
respectively. Experiment 1 was repeated three times, while experiments 2 and 3 were each
repeated twice.
The transcription level of Cj1084c
To determine if the insertional mutation in mfd affected the expression of the
downstream gene Cj1084c (encoding a possible ATP/GTP-binding protein), RT-PCR was
performed to assess the expression of Cj1084c. Total RNA was isolated from C. jejuni 11168
and JH01 using the RNeasy Kit (Qiagen). The purified RNA samples were treated with OnColumn DNase Digestion Kit (Qiagen) followed by further treatments with DNase to remove
DNA contamination. The Cj1084c-specific primers Cj1084cF (5' TTG CCT TAG CAG ATA
TCA T 3') and Cj1084cR (5' ACC ACT TCT ACT TGC TCT TA 3') were used to amplify a
430 bp region of the gene in a conventional one-step RT-PCR by using the SuperScript™ III
One-Step RT-PCR kit (Invitrogen®). An RT-PCR mixture lacking the RT was included as a
negative control.
Emergence of FQR mutants in enrofloxacin-treated chickens
To examine if Mfd plays a role in the emergence of FQR Campylobacter during in
vivo FQ treatment, a chicken experiment was performed using 11168 and JH01. Day-old
broiler chickens (Ross X Cobb) were obtained from a commercial hatchery and randomly
assigned to 2 groups (11 birds per group). Each group of chickens was maintained in a
sanitized wire-floored cage. Feed and water were provided ad libitum. Prior to inoculation
with Campylobacter, the birds were tested negative for Campylobacter by culturing cloacal
swabs. At day 3 of age, the two groups of chickens were inoculated with 11168 and JH01,
respectively, at a dose of 106 CFU/chick via oral gavage. Six days after the inoculation, the
birds were treated with 50 ppm enrofloxacin. The treatment was administered in drinking
water and lasted for five consecutive days. During the treatment, only medicated water was
given to the birds to ensure enough consumption. Cloacal swabs were collected periodically
before and after enrofloxacin treatment until the end of the experiment. Each swab was
serially diluted in MH broth and plated onto two different types of MH plates: one containing
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Campylobacter-specific growth supplements (SR 084E and SR117 E; Oxoid Ltd.,
Basingstoke, England) for the enumeration of total Campylobacter cells and the other
containing 4 µg/ml of CIPRO in addition to the same selective agents and supplements to
recover FQR Campylobacter in each chicken. At each sampling time, at least one
Campylobacter colony from each chicken were selected from the regular MH agar plates (no
CIPRO) for the determination of CIPRO MICs using the E-test (AB Biodisk). The
colonization data (CFU/g feces) were log-transformed and used for statistical analysis. The
significance of differences in the level of colonization between the two groups was
determined using Student’s t-test, Welch’s t-test to allow for non-constant variation across
treatment groups, and the Wilcoxon rank-sum test to allow for non-normality. The
conclusion of all three tests was the same at significance level 0.05.
Microarray data accession number
The microarray data have been deposited in the NCBI Gene Expression Omnibus
(GEO; http://www.ncbi.nlm.nih.gov/geo/) database and the accession number is GSE10471.
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85
TABLE 1. Genes differentially expressed in the presence of ciprofloxacin
Table 1 Genes differentially expressed in the presence of ciprofloxacin
P-Value
Q-Value
n-Fold change
qRTMicroarray
PCR
Cell membrane
Cj0205
uppP, undecaprenyl-diphosphatase
Cj0735
putative periplasmic protein
Cj0824
uppS, undecaprenyl diphosphate synthase
Cj1351
pldA, phospholipase A
Cj0033
putative integral membrane protein
Cj0179
exbB1, biopolymer transport protein
Cj0486
putative sugar transporter
Cj0553
putative integral membrane protein
Cj0834c
ankyrin repeat-containing possible periplasmic protein
Cj1013c
putative cytochrome C biogenesis protein
Cj1662
putative integral membrane protein
Cj1663
putative ABC transport system ATP-binding protein
0.0135
0.0186
0.0099
0.0046
0.0033
0.0412
0.0043
0.0106
0.0089
0.0058
0.0055
0.0002
0.130143
0.14811
0.120356
0.094812
0.086471
0.217646
0.091967
0.121714
0.110719
0.096095
0.096095
0.067622
1.59
1.70
1.52
2.02
-1.52
-1.88
-1.57
-1.59
-1.51
-1.52
-1.68
-1.75
6.1
NT*
2.1
2
NT
NT
NT
NT
NT
NT
NT
NT
DNA replication, recombination and repair
Cj1085c
mfd, transcription-repair coupling factor
Cj0718
dnaE, DNA polymerase III, alpha chain
0.0029
4.07E-05
0.082832
0.052459
1.57
-1.62
2.2
-1.98
0.0357
0.0117
0.204764
0.129371
1.93
1.54
NT
NT
0.0057
0.0252
0.0007
0.0020
0.0208
0.0125
0.0191
0.0013
0.0024
0.210454
0.170564
0.067622
0.076881
0.151677
0.130143
0.148174
0.076653
0.076881
1.54
1.50
-1.74
-1.87
-1.69
-1.52
-1.53
-1.80
-1.67
NT
NT
NT
-2.1
NT
NT
NT
NT
-2.23
0.0404
0.0116
0.0370
0.0009
0.214389
0.129371
0.207688
0.067622
-1.59
-1.97
-1.58
-2.13
NT
-1.96
NT
- 6.7
0.0016
0.0032
0.0255
0.0133
0.0039
0.0055
0.0046
0.0167
0.0021
0.0018
0.076881
0.086244
0.171473
0.130143
0.088549
0.096095
0.094812
0.142007
0.076881
0.076881
-2.12
-1.56
-1.52
-1.55
-2.03
-1.60
-1.58
-1.54
-1.63
-1.61
NT
NT
NT
NT
-2.71
NT
NT
NT
-2.15
NT
Gene ID and Functional Category
Cellular process and energy metabolism
Cj0041
putative flagellar hook-length control protein
Cj0065c
folk, putative 2-amino-4-hydroxy-6hydroxymethyldihydropteridine pyrophosphokinase
Cj1030c
lepA, GTP-binding protein homolog
Cj1280c
putative ribosomal pseudouridine synthase
Cj0009
gltd, glutamate synthase (NADPH) small subunit
Cj0123c
putative tRNA-dihydrouridine synthase
Cj0227
argD, acetylornithine aminotransferase
Cj0283c
cheW, chemotaxis protein
Cj0415
putative GMC oxidoreductase subunit
Cj0490ald, putative aldehyde dehydrogenase C-terminus
Cj0537
oorb, OORB subunit of 2-oxoglutarate:acceptor
oxidoreductase
Cj0734c
hisJ, histidine-binding protein precursor
Cj0764c
speA, biosynthetic arginine decarboxylase
Cj0767c
kdtB,3-deoxy-D-manno-octulosonic-acid transferase
Cj1264c
hydD, putative hydrogenase maturation protease
Cj1265c
hydC, Ni/Fe-hydrogenase B-type cytochrome subunit
Cj1266c
hydB, Ni/Fe-hydrogenase large subunit
Cj1364c
fumC, fumarate hydratase
Cj1476c
pyruvate-flavodoxin oxidoreductase
Cj1566c
nuoN, NADH dehydrogenase I chain N
Cj1567c
nuoM, NADH dehydrogenase I chain M
Cj1624c
sdaA, L-serine dehydratase
Cj1682c
gltA, citrate synthase
Cj1688c
secY, preprotein translocase subunit
Cj1717c
leuC, 3-isopropylmalate dehydratase large subunit
86
TABLE 1—Continued
Gene ID and Functional Category
P-Value
Q-Value
n-Fold change
Microarray
Unknown function
Cj0163c
hypothetical protein
Cj0814
hypothetical protein
Cj0959c
hypothetical protein
Cj1025c
hypothetical protein
Cj0125c
hypothetical protein Cj0125c
Cj0554
hypothetical protein
* NT: Not tested
0.0204
0.0002
0.0233
0.0389
0.0031
0.0083
0.151393
0.067622
0.160562
0.210454
0.084482
0.107589
1.60
1.99
1.50
1.54
-1.53
-1.71
qRT-PCR
NT
NT
NT
NT
NT
NT
87
TABLE 2. Oligonucleotide primers used in qRT-PCR
Table 2 Oligonucleotide primers used in qRT-PCR
Primer
16s RNA F
16s RNA R
Cj0123cF
Cj0123cR
Cj0824F1
Cj0824R1
Cj1351F1
Cj1351R1
Cj1264cF1
Cj1264cR1
Cj1085cF1
Cj1085cR1
Cj0205F1
Cj0205R1
Cj0537F1
Cj0537R1
Cj0718F
Cj0718R
Cj1688cF1
Cj1688cR1
Cj0764cF1
Cj0764cR1
Cj1566cF1
Cj1566cR1
Sequence
5’-TAC CTG GGC TTG ATA TCC TA-3’
5’-GGA CTT AAC CCA ACA TCT CA-3’
5’ CGC CTT GAT CTT TGT AGT GTT TT 3’
5’ TGA AAT CAA AAG CGG TAA AAG TG 3’
5’-CAA AGT GCG TCA CAA TGC TT-3’
5’-GAT TTA TCG CGC TTG GAA GA-3’
5’-ATC CCC TTG GCA TTA GCT CT-3’
5’-TGG AAT TTC GCC TCA CTA TT-3’
5’-GCT TAG GCG TTC ATC TTT GC-3’
5’-CAA AGC CAA AGT TCC ACC AT-3’
5’-TGT TTT GCA AAC TCC ACC AG-3
5’-ATT TTG CCC ACC ACG TCT TA-3’
5’-GAA AAG TTG CGG CTG AGT TT-3
5’-AAT TTG CAT TGC CAA GAA GC-3’
5’-GGC AAT TGG TGG AAA TCA TAC TA-3’
5’-TGG AGT AGT TGG AGA AGT TTG AGA-3’
5’-GGA CTT GGG GCT ATA AAA AGT GT 3’
5’-CGG TTT ATT TTG GTG GGA TCT AT-3’
5’-GCC TGA ATT GAT TTG TCC TAC AG-3’
5’-CGA ACA AAT CAC ACA AAG AGG TA-3’
5’-TTC AGC TGC AAT AAA GCC TAT GT-3’
5’-ATA ATA ACG AAG GCG CAC CTA TT-3’
5’-CAT AAA TTT ACC CCA AAA CAC TCC-3’
5’-GAG AGT TTA AAT GGG CTT TTG GT-3’
Gene amplified
16s RNA
cj0123c
cj0824 (uppS)
cj1351 (pldA)
cj1264c
cj1085c (mfd)
cj0205 (uppP)
cj0537
cj0718(dnaE)
cj1688c (secY)
cj0764c (speA)
cj1566c
88
Fig. 1 Insertional mutation of mfd and its impact on the transcription of cj1084c
Fig. 1. Insertional mutation of mfd and its impact on the transcription of cj1084c. (A)
Diagram depicting the genomic organization of mfd and its flanking regions. ORFs and their
directions of transcription are indicated by boxed arrows. The location of the inserted
kanamycin resistance gene (aphA3) in mfd is indicated. (B) PCR confirmation of the aphA3
insertion into the mfd gene in JH01. Lane 1 shows the PCR product from 11168, while Lane
2 shows the PCR product of JH01. The primers used in the PCR were mfd-F2 and mfd-R2.
Lane M contains 1 kb DNA size markers (Promega). (C) RT-PCR analysis of cj1084c
expression in strains 11168 and JH01. The same amount of total RNA from 11168 (Lane 1)
and JH01 (Lane 2 and 3) were used as template in the RT-PCR. Lanes 1 and 2 are normal
RT-PCR reactions. Lane 3 is a RT-PCR reaction without reverse transcriptase (DNA-free
control for the RNA preparation). In Lane 4, genomic DNA of 11168 was used as template
(positive control for PCR).
89
Fig. 2 Frequencies of emergence of spontaneous FQR mutants in different C. jejuni strains
Fig. 2. Frequencies of emergence of spontaneous FQR mutants in different C. jejuni strains
including the wild-type 11168, the mfd mutant (JH01), the complemented mfd mutant (JH02),
and the mfd-overexpressing construct (JH03). Three different concentrations of CIPRO (1, 2,
and 4 µg/ml, respectively) were used in the detection plates to count FQR colonies. Each bar
represents the mean ± standard deviation of frequencies from three independent cultures. The
bars labeled with different letters indicate that they are significantly different (P < 0.05).
90
Fig. 3 Growth kinetics of various C. jejuni constructs in culture media
Fig. 3. Growth kinetics of various C. jejuni constructs in culture media. The strains were
grown in MH broth (A) or MH broth supplemented with 0.06 µg/ml of CIPRO (B).
91
Fig. 4 Development of FQR mutants from 11168 and JH01 grown in MH broth supplemented
with 4 µg/ml of CIPRO
Fig. 4. Development of FQR mutants from 11168 (solid circle) and JH01 (triangle) grown in
MH broth supplemented with 4 µg/ml of CIPRO. In panel A, the initial cell density (at time 0)
of each culture was 107 CFU/ml, while in panel B the initial cell density was 106 CFU/ml.
Each symbol represents the number of FQR mutants in a single culture. Each horizontal bar
represents the mean log10 CFU/ml from each strain at a given time. Panel A displays the
results of 3 independent experiments, while panel B represents the results of two
independents experiments. The detection limit of the plating method is 1 CFU/ml.
92
Fig. 5 Development of FQR Campylobacter mutants in chickens initially infected with FQsusceptible Campylobacter, but treated with enrofloxacin
Fig. 5. Development of FQR Campylobacter mutants in chickens initially infected with FQsusceptible Campylobacter, but treated with enrofloxacin. (A) The level of total
Campylobacter in each chicken inoculated with the wild-type 11168 (open circle) or the mfd
mutant strain (JH01; solid circle). The treatment with enrofloxacin started on day 0 and
lasted for five consecutive days (indicated by a bracket on top of the panel). (B) The level of
FQR Campylobacter in each chicken inoculated with the wild-type (open circle) or the mfd
mutant (solid circle). In both panels, each symbol represents the number of Campylobacter in
a single bird. Each group includes eleven chickens and the mean of each group at a given
time is indicated by a horizontal bar. A chicken is considered negative if the level of
colonization was below the detection limit (100 CFU/ g of feces).
93
CHAPTER 4. FLUOROQUINOLONE RESISTANCE ASSOCIATED
MUTATION IN GYRA AFFECTS DNA SUPERCOILING IN
CAMPYLOBACTER JEJUNI
A paper to be submitted to Journal of Bacteriology
Jing Han, Yang Wang, Orhan Sahin, Baoqing Guo, and Qijing Zhang
Departments of Veterinary Microbiology and Preventive Medicine, Iowa State University,
Ames, Iowa 50011
1. Abstract
Our previous work showed that acquisition of fluoroquinolone (FQ) resistance via a
specific GyrA mutation (Thr-86-Ile) enhances the in vivo fitness of FQ-resistant mutants in
the absence of antibiotic selection pressure. In this study, we further confirmed the role of the
Thr-86-Ile mutation in modulating Campylobacter fitness by reverting the mutation.
Reversion of this mutant allele to a wild-type allele resulted in the loss of the fitness
advantage, consistent with our previous findings. To understand how the Thr-86-Ile mutation
affects Campylobacter fitness, we initiated work to determine if this mutation alters the
physiological function of DNA gyrase. Recombinant wild-type gyrase and mutant gyrases
with three different types of mutations (Thr-86-Ile; Thr-86-Lys; and Asp-90-Asn) were
generated in E. coli and compared for their supercoiling activities using an in vitro assay. The
mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity
compared with the wild-type gyrase and other mutant gyrases. In addition, we measured
DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the
results from the in vitro supercoiling assay, the FQ-resistant mutant Campylobacter carrying
the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the wild-type strain
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and the mutant strains carrying other mutations. Together, these results clearly indicate that
the Thr-86-Ile change in GyrA confers a high-level resistance to FQs and significantly
reduces the supercoiling activity of the enzyme in Campylobacter.
2. Introduction
Campylobacter jejuni, a Gram-negative microaerobic bacterium, has emerged as the
leading bacterial cause of foodborne diseases in the United States and other developed
countries (45). The estimated number of human cases of campylobacteriosis in the U.S. is
more than 2 million per year (30, 42). The medical and productivity costs resulting from C.
jejuni infection are estimated at 0.6 to 1.0 billion dollars each year in the U.S. (8). Antibiotic
treatment using fluoroquinolone (FQ) or erythromycin is recommended when the infection
by Campylobacter is severe or occurs in immunocompromised patients (12, 35). However,
Campylobacter is increasingly resistant to FQ antimicrobials, which has become a major
concern for public health (12, 16, 48). Although FQ antimicrobials have been banned since
2005 in poultry production in the U.S., FQ-resistant (FQR) Campylobacter continues to
persist on poultry farms (25, 39, 40).
The main targets of FQs in bacteria are DNA gyrases and/or topoisomerase IV (11,
18). In Gram-negative bacteria, DNA gyrase, a type II topoisomerase, is the primary target of
FQ antibiotics, while topoisomerase IV is the main target of FQs in Gram-positive bacteria
(18). Once inside bacterial cells, FQ antimicrobials form stable a complex with the target
enzymes and trap the enzymes on DNA, resulting in double-stranded breaks in DNA and
bacterial death (10, 44, 49). Bacterial DNA gyrase is essential for bacterial viability. It
catalyzes ATP-dependent negative supercoiling of DNA and is involved in DNA replication,
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recombination, and transcription (9). The enzyme consists of two subunits (subunits A and
B) that combine into the A2B2 complex to form a functional enzyme. The two subunits are
encoded by the genes gyrA and gyrB, respectively. In Campylobacter, resistance to FQ
antimicrobials is mediated by point mutation in the quinolone resistance-determining region
(QRDR) of gyrA in conjunction with the function of the multidrug efflux pump CmeABC (3,
12, 13, 27). No mutations in gyrB have been implicated in FQ resistance in Campylobacter
(3, 37, 38). Acquisition of high-level FQ resistance in Campylobacter does not require
stepwise accumulation of point mutations in gyrA. Instead, a single point mutation in gyrA
can lead to clinically relevant levels of resistance to FQ antimicrobials (13, 27, 41). Specific
mutations at positions Thr-86, Asp-90 and Ala-70 in GyrA have been linked to FQ resistance
in C. jejuni (12, 27, 47). Specifically, the Thr-86-Ile change (mediated by the C257T
mutation) is the most commonly observed mutation in FQR Campylobacter isolates and
confers high-level (ciprofloxacin MIC 16 µg/ml) resistance to FQ, whereas the Thr-86-Lys
and Asp-90-Asn mutations are less common and are associated with intermediate-level FQ
resistance (15, 27, 41, 47).
Genes targeted by antibiotics are usually essential for bacterial physiology. Thus
resistance-conferring mutations in target genes are often associated with changes in
physiological processes, resulting in reduced growth rate and fitness in the absence of
antibiotic selection (1, 2, 20, 21). However, bacteria can develop compensatory mutations to
ameliorate the fitness defect associated with antimicrobial resistance (1, 6, 7, 33, 34). In
some situations, antibiotic-resistant mutants show little or no fitness cost even without
compensatory mutations (43). A previous study by Luo et. al. showed FQR Campylobacter
carrying the C257T mutation (leading to a Thr-86-Ile substitution in the GyrA subunit) was
96
able to colonize and persist in chickens as efficiently as the FQ-susceptible (FQS) strains in
the absence of FQ. Pairwise competitions using clonally or isogenic strains further
demonstrated that the FQR isolates outcompeted the FQS strains in the majority of the
competing pairs in chickens (26), suggesting that acquisition of FQ resistance enhances the in
vivo fitness of FQR Campylobacter. How the resistance-conferring mutation affects
Campylobacter fitness is unknown. GryA plays an essential role in maintaining the
topological configuration of DNA by introducing negative superhelical turns in DNA. Work
performed with Escherichia coli and Pseudomonas revealed that antibiotic resistanceconferring mutations in GyrA reduced the supercoiling activity of the enzyme in (5, 20). It is
also known that changes in DNA supercoiling affect bacterial adaptive responses to various
environmental signals including heat shock, cold shock, osmolar stress, and oxidative stress
(24, 46). Based on the available evidence, we hypothesize that the resistance-conferring
mutations in FQR Campylobacter may affect the enzymatic function of GyrA and modulate
the DNA supercoiling status within the bacterial cells, which leads to fitness changes in FQR
Campylobacter.
To begin to examine our hypothesis, we conducted a series of experiments in this
study to determine the impact of various point mutations on the supecoiling activities of
GyrA. We first confirmed the specific role of Thr-86-Ile mutation of GyrA in influencing
Campylobacter fitness in chickens by reverting the mutation to a wild-type allele. We then
prepared recombinant gyrases with three different types of mutations [C257T (Thr-86-Ile),
C257A (Thr-86-Lys), and G268A (Asp-90-Asn)] and compared their supercoiling activity
with that of the wild-type gyrase in the presence or absence of ciprofloxacin using an in vitro
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supercoiling assay. Also we determined the impact of the mutations on in vivo supercoiling
(within Campylobacter cells) using a reporter plasmid. Our results consistently showed that
the Thr-86-Ile change in GyrA was directly linked to the fitness change in Campylobacter
and greatly reduced the supercoiling activitity of GyrA, while other GyrA mutations,
although reduced the susceptibility to ciprofloxacin, did not affect the supercoiling activity of
the gyrase.
3. Materials and Methods
Bacterial strains and growth conditions
The FQS strains and FQR mutant strains (carrying different point mutations in gyrA)
used in this study are listed in Table 1. The strain was routinely grown in Mueller-Hinton
(MH) broth (Difco) or agar at 42 °C under microaerobic conditions (10% CO2, 5% O2, and
85% N2). Campylobacter-specific growth supplements and selective agents (Oxoid) were
added to media when needed to recover Campylobacter from chicken feces. MH media were
supplemented with kananmycin (50 µg/ml) or chloramphenicol (4 µg/ml) as needed.
Escherichia coli (E. coli) cells were grown at 37 °C with shaking at 200 r.p.m. in LB medium
which was supplemented with ampicillin (100µg/ml) or kanamycin (30µg/ml).
Reversion of the gyrA mutation (Thr-86-Ile)
To formally define the role of the C257T mutation in influencing Campylobacter
fitness, the specific mutation (C257T) in gyrA of isolates 62301R33 was reverted to wildtype sequence by using a method reported by Ge et. al. (13). A chloramphenicol resistance
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(Cmr) cassette, which was used as a selective marker, was inserted in the cj1028c gene
immediately upstream of gyrA. Cj1028c and gyrA are tandemly positioned on the
chromosome of C. jejuni. An 850-bp fragment containing the 3' region of Cj1028c, the
intergenic region, and the gyrA sequence up to the mutation site was amplified by PCR using
C. jejuni 62301R33 as a template and gyrA1028F and gyrA257WR (corresponding to the
wild-type gyrA sequence) as primers (Table 2) and then cloned into a pGEMT-Easy
(Promega). The construct was linearized using EcoRV, which cuts once within the cloned
cj1028c sequence, ligated with a blunt-ended Cmr cassette, and electroporated into E. coli
JM109. Recombinant plasmids were purified from E. coli JM109 and used to transform the
parent strains C. jejuni 62301R33 by electroporation. Transformants were selected on MH
agar containing chloramphenicol 10 mg/L and analyzed by PCR and DNA sequencing,
confirming the insertion of the cat cassette into Cj1028c and the simultaneous replacement of
the mutant gyrA with the wild-type allele. This revertant was named 62301R33S. To make an
isogenic pair for the revertant for in vivo competition, the cat gene was also inserted into
Cj1028c of isolate 62301R33 without changing the C257T mutation in gyrA. Primers
gyrA1028F and gyrA257m(T) were used for this purpose (Table 2). The obtained construct
was named 62301R33R. The ciprofloxacin MIC in the revertant 62301R33S was restored to
the wild-type level (0.125µg/ml), while 62301R33R with the cat gene inserted into Cj1028c
retained the ciprofloxacin MIC at 32 µg/ml. S3B R33S and 62301R33R were equally motile
as determined by motility assay. Using the same strategy, a point mutation (C257T) in gyrA
was introduced into FQS NCTC 11168. The generated FQR mutant strain and its isogenic FQS
strain, both of which contained a Cmr insertion in cj1028c, were named 11168 (R) and 11168
(S), respectively (Table 1).
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In vivo colonization and pairwise competition
Chicken experiments were performed using pairwise competition as described
previously (26). Three groups (10-11 birds/group) of Campylobacter-free chickens were
inoculated with about 107 CFU of 62301R33S, 62301R33R, or a mixture (approximately 1:1)
of the two strains via oral gavage. During the entire experiment, antibiotic-free feed and
water were given to the chickens. Thus, antibiotic selection pressure was not involved in the
competition. After inoculation, cloacal swabs were collected from the chickens at days 3, 6,
and 9 for culturing Campylobacter. Each fecal suspension was serially diluted in MH broth
and plated simultaneously onto two different types of culture media: conventional
Campylobacter selective plates for recovering the total Campylobacter colonies and the
selective MH plates supplemented with 4 µg/ml ciprofloxacin for recovering FQR
Campylobacter colonies. Competion index (CI) was calculated as described in reference (26).
To confirm the results from the differential plating, 10–15 Campylobacter colonies were
selected randomly for each group from the conventional selective plates (no ciprofloxacin) at
each sampling time and tested for ciprofloxacin MICs using Etest strips (AB Biodisk, Solna,
Sweden). In the second chicken experiment, the same pairwise experiment was performed
using the isogenic pair of 11168(S) and 11168(R).
In both experiments, the detection limit of the plating methods was 100 CFU/g of
feces. The statistic analyses that were used to determined the significance of differences in
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the level of colonization between the two groups were performed as described in a previous
publication (17).
Production and purification of GyrA and GyrB
Full-length histidine (His)-tagged recombinant gyrases including wild-type (WT)
GyrA, three mutant GyrA, and GyrB were produced in E. coli by using the pQE-30 vector of
the QIAexpress expression system (Qiagen). The complete coding sequences of gyrA in
strains 62301S2 (no mutation in GyrA), 62301R33 (with the Thr-86-Ile change in GyrA),
52901-II2 (with the Thr-86-Lys change in GyrA) and 62301R37 (with the Asp-90-Asn
change in GyrA) were amplified using primers gyrA-F-2 and gyrA-R. The complete coding
sequence of gyrB in strain 62301S2 was amplified using primers gyrB-F-J2 and gyrB-R-J. A
restriction site (underlined in the primer sequences) was attached to the 5’ end of each primer
to facilitate the directional cloning of the amplified PCR product into the pQE-30 vector. The
amplified PCR product was digested with SphI and SalI, and then ligated into the pQE30
vector, which was previously digested with the same enzymes. Each plasmid in the E. coli
clone expressing a recombinant peptide was sequenced, revealing no undesired mutations in
the coding sequence. These His-tagged recombinant GyrA and GyrB proteins were expressed
and purified to near-homogeneity under native conditions by following the procedure
supplied with the pQE-30 vector. Then these proteins were analyzed by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE). To remove imidazole, the purified proteins were washed
extensively with 10 mM Tris-HCl using Centricon YM-50 (Millipore).
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In vitro supercoiling assay
DNA supercoiling activity was assayed by monitoring the conversion of relaxed
pBR322 to its supercoiled form. WT or mutant GyrA was mixed with WT GyrB in a ratio of
1:1. Each supercoiling reaction contained 500 ng of relaxed pBR322 DNA (TopoGen, Inc)
and 300 uM gyrases in 1X assay buffer (TopoGen, Inc). The reaction mixtures were
incubated at 37 °C for 1h. Assays were terminated by the addition of 0.2 volume of the stop
buffer (TopoGEN) and 1 volume of chloroform-isoamyl alcohol (24:1). The reactions were
analyzed on 1.0% agarose gels. The gels were stained with ethidium bromide for 30 min and
then destained in 1X TAE buffer for 1h.
MEC (minimum effective concentration) determinations
To determine the susceptibility of various gyrases to the inhibitory effect of
ciprofloxacin, the in vitro supercoiling assay using recombinant gyrases was also performed
with added ciprofloxacin as descried previously (28). Ciprofloxacin was added into each
reaction prior to the addition of DNA gyrases. The minimum effective concentration (MEC)
was defined as the lowest concentration of ciprofloxacin that shows an observable inhibition
on supercoiling of the plasmid DNA.
Examination of in vivo supercoiling
To determine if the mutations in GyrA changed the DNA supercoiling in
Campylobacter cells, the shuttle plasmid pRY107 was used as a reporter to monitor the
relative difference in the levels of DNA supercoiling between the FQS and FQR strains.
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pRY107, which carries a kanamycin resistance marker (50), was transferred into 62301S2,
62301R33, 52901-II2, 62301R37, 11168, and 1168CT by conjugation. The conjugates were
grown in MH broth (Difco) for one day at 42 °C under microaerobic conditions (10% CO2,
5% O2, and 85% N2), then plasmid DNA was isolated using QIAGEN Plasmid Midi kit
(Qiagen Inc.) and analyzed by agarose gel electrophoresis in the absence or presence of
chloroquine diphosphate salt, as described previously (20, 31). Agarose gels (1%) were run
for 20 h at 2 V/cm in 1 x TAE buffer containing 20 µg/ml of chloroquine and were washed
for 4 h in distilled water before staining with ethidium bromide. The topoisomers of the
plasmid DNA from each strain were visualized using a digital imaging system (Alpha
innotech). The relative amounts of supercoilied vs. relaxed DNA in each sample reflecting
the level of DNA supercoiling in each strain were determined by densitometry scanning and
were used to indicate the difference in DNA supercoiling between the FQS and FQR strains.
Cloning and sequence of topA genes
The entire topA gene from C. jejuni 62301S2 and 62301R33 were amplified by PCR
with the forward primer topA-F and reverse primer topA-R. The forward primer is 286 bp
upstream of the AUG start codon, and the reverse primer extends 239 bp beyond the UAG
stop codon of topA. The PCR was performed in a volume of 50 µl containing 100 µM each
deoxynucleoside triphosphate (dNTP), 200 nM primers, 2.5 mM MgSO4, 100 ng of
Campylobacter genomic DNA, and 5 U of Taq DNA polymerase (Promega). The cycling
conditions consisted of an initial polymerase activation step at 94 °C for 5 min, followed by
35 cycles of 94 °C for 30 seconds, 55 °C for 30 seconds and 72 °C for 3 min, with a final
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extension at 72 °C for 10 min. The amplified PCR products were purified with the QIAquick
PCR purification kit (Qiagen) and subsequently sequenced.
Real-time quantitative RT-PCR (qRT-PCR) analysis of topA transcription in FQS and FQR
Campylobacter
The transcription level of topA in C. jejuni 62301S2 and 62301R33 was compared by
qRT-PCR as described in a previous work (22). Briefly, total RNA of 62301S2 and
62301R33 was isolated from overnight cultures by using the RNasy mini kit (QIAGEN)
according to the manufacturer's instructions. Isolated RNA was further treated with TURBO
DNA-free (Ambion) to remove DNA contamination. The qRT-PCR reactions were
conducted using the iScript one-step RT-PCR kit with SYBR green (Bio-Rad). topA-specific
primers (cj1686c-F1 and cj1686c-R) and primers specific for the Campylobacter 16S RNA
gene (16SF and 16SR; for normalization) were used for qRT-PCR. Triplicate reactions, each
in a volume of 20 µl, were performed for each dilution of the RNA template. Thermal cycling
conditions were as follows: 10 min at 50 °C, 5 min at 60° C followed by 5 min at 95 °C, and
then 40 cycles of 10 s at 95 °C and 30 s at 55 °C (for 16S RNA) or 56 °C (for topA). Cycle
threshold values were determined with the MyiQ software (Bio-Rad). The relative changes
(n-fold) in topA transcription between the S3B 62301S2 and 62301R33 were calculated using
the 2−ΔΔCT method as described by Livak and Schmittgen (23).
Antibiotic susceptibility test
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The minimum inhibitory concentration (MIC) of CIPRO was determined by using Etest strips (AB Biodisk, Solna, Sweden) following the manufacturer's instructions. The
maximum detection limit of the E-test for CIPRO was 32 µg/ml.
Campylobacter motility assay
Campylobacters were grown overnight on fresh MH plates and then were collected
using MH broth. The optical density at 600 nm (OD600) was adjusted to 0.3. Approximately 1
µl of this suspension was then stabbed into a MH motility plate (MH broth+0.4%Bacto
Agar). The low density of the agar allows the bacteria to move within the agar, forming a
halo of growth around the point of inoculation. Following microaerobic growth at 42 °C for
approximately 30 h, the radius of the ring was measured relative to that of the ring produced
by the control strain.
GenBank Accession No.
The assigned GenBank accession no. for topA in strain 62301S2 and 62301R33 is
FJ795047.
4. Results
Role of the Thr-86-Ile change in enhanced fitness
Our previous study showed that FQR Campylobacter carrying the C257T mutation
(leading to a Thr-86-Ile substitution in the GyrA subunit) outcompeted its isogenic FQS
105
strains in chickens, suggesting that acquisition of FQ resistance enhances the in vivo fitness
of FQR Campylobacter. To further confirm the link of this specific mutation with the
enhanced fitness, we reverted the mutation back to the wild-type allele and conducted
pairwise competition in chickens using 62301R33S and 62301R33R. After inoculation with
either of the two isolates or mixed populations, all of the groups of birds were colonized by
Campylobacter at similar levels (Fig. 1A). When separately inoculated into chickens, the two
strains showed no significant differences (P > 0.05) in the level of colonization (the number
of Campylobacter shed in feces) (Fig. 1A). Differential plating by using ciprofloxacincontaining plates showed that the group inoculated with the FQS revertant alone shed
homogeneous FQS Campylobacter, whereas the group inoculated with FQR strain shed
homogeneous FQR Campylobacter during the entire course of the experiment. However, in
the group inoculated with mixtures of the two strains, the FQS revertant strain was
outcompeted by its parent FQR Campylobacter (P < 0.05) (Fig. 1B). Testing of randomly
selected Campylobacter colonies (10~15 colonies per group per time point) by using Etest
confirmed the results of differential plating. This result clearly indicates that once the C257T
mutation in gyrA is reverted, Campylobacter loses its fitness advantage in chickens,
confirming our previous results using clonally related and isogenic strains (26). To further
show the specific effect of the C257T mutation on fitness, we introduced this mutation into a
different strain background (NCTC 11168) and conducted chicken competition using the
isogenic pair of constructs. As shown in Fig 1C, 11168(S) was outcompeted by 11168(R) (P
< 0.05), indicating the mutation had the same impact on fitness in a C. jejuni strain different
from the previously tested ones. These new findings plus our published studies (26)
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convincingly showed that the C257T mutation in gyrA is directly responsible for the
enhanced fitness of Campylobacter in chickens.
No compensatory mutations in topA.
C. jejuni has only two types of topoisomerases, type I (TopA) and type II (gyrase).
The gyrase introduces negative supercoiling to DNA, while TopA relaxes DNA to prevent
excessive supercoiling. Thus, the two enzymes are the key proteins modulating the level of
DNA supercoiling in Campylobacter. Previously we demonstrated that isolate 62301R33
(FQR), which carried the Thr-86-Ile mutation and outcompeted FQs Campylobacter
(62301S2) in chickens, did not harbor any compensatory mutations in GyrA and GyrB (26).
In this study, we sought to determine if there were any mutations in TopA that might
potentially offset the impact of the Thr-86-Ile mutation in GyrA. The whole ORF of topA
was PCR amplified from 62301R33 and sequenced, which showed that the topA sequence
was identical to that in 62301S2. In addition, the expression level of topA in 62301R33 and
62301S2 were compared by qRT-PCR, which did not reveal a significant difference. These
results suggest that the Thr-86-Ile mutation in GyrA was not accompanied by changes in
topA sequence or expression.
Effect of GyrA mutations on in vitro supercoiling.
To determine the impact of the resistance-conferring mutations on the enzymatic
activities of GyrA, we produced the recombinant forms of various gyrases. As estimated by
SDS-PAGE, the recombinant GyrA and GyrB were 97 kDa and 87 kDa, respectively,
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comparable with the calculated molecular masses of the two proteins (Fig. 2A). The mutant
GyrA carrying Thr-86-Ile, Thr-86-Lys, or Asp-90-Asn mutation migrated at the same
position as the wild-type GyrA on the SDS-PAGE (data was not shown), indicating that the
point mutations in GyrA did not change its migration on SDS-PAGE. The supercoiling
activity of the WT and mutant DNA gyrases was analyzed in vitro by using the recombinant
proteins. Both GyrA and GyrB are required for supercoiling the plasmid DNA and no
detectable activity was observed when only GyrA or GyrB was used in the assay (data were
not shown). The mutant GyrA carrying the Thr-86-Lys or Asp-90-Asn mutation exhibited
supercoiling activities comparable to that of the WT GyrA, whereas the supercoiling activity
of the mutant GyrA carrying the Thr-86-Ile change was substantially reduced comparing to
that of the wild-type GyrA. In fact, the most supercoiled band was absent with this mutant
GyrA (Fig. 2B). The results were consistently shown in multiple experiments with different
concentrations of gyrases (up to 1200 nM; data were not shown).
Effect of the GyrA mutations on in vivo supercoiling
The recombinant mutant GyrA with the thr-86-Ile mutation showed reduced
supercoiling activity in vitro, but it is important to know if the same mutation also affects
DNA supercoiling in vivo (in Campylobacter cells). For this purpose, we used the shuttle
plasmid pRY107 as a reporter plasmid to monitor the relative levels of DNA supercoiling in
the FQS and FQR strains. The plasmid pRY107 was transferred into 62301S2 (wild-type
strain), 62301R33 (carrying the Thr-86-Ile mutation in GyrA), 52901-II2 (carrying the Thr86-Lys mutation in GyrA), and 62301R37 (carrying the Asp-90-Asn mutation), which were
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clonally related and all derived from strain S3B (26). Plasmid DNA was re-isolated from
these constructs and analyzed by agarose gel electrophoresis in the presence of chloroquine.
Under the condition utilized in this study (20 µg /ml chloroquine), negatively supercoiled
forms migrated slower than relaxed topoisomers, which was confirmed by negatively
supercoiled plasmid pBR322 and relaxed plasmid pBR322 (results not shown). Compared to
the pRY107 from 62301S2, the plasmid topoisomers from 62301R33 harboring the Thr-86Ile mutation in GyrA shifted to lower positions, indicating less supercoiling (Fig. 3A). As
determined by densitometrical analysis, the percentage of the most supercoiled DNA in the
total population of the plasmid topoisomers extracted from 62301S2 and 62301R33 was
87.4% and 20.2%, respectively. The plasmids from 52901-II2 and 62301R37 showed
topoisomer distribution patterns similar to that of 62301S2 (Fig 3A). These results indicate
that the GyrA mutant with the Thr-86-Ile mutation reduced DNA supercoiling in
Campylobacter cells, while the mutants with the Thr-86-Lys or Asp-90-Asn changes in GyrA
did not alter DNA supercoiling. To further confirm the impact of the Thr-86-Ile change on
DNA supercoiling, we introduced pRY107 to a different strain background, NCTC 11168
and 11168CT. The only known difference between this pair of isolates is the C257T mutation
in gyrA. Similar to the result from 62301R33, the plasmid topoisomers from 11168CT shifted
to lower positions compared with NCTC 11168 (Fig 3B). Together, the in vivo supercoiling
results were consistent with the findings from the in vitro supercoiling assay and indicated
that the Thr-86-Ile mutation reduced DNA supercoiling in Campylobacter.
GyrA mutations reduce the susceptibility to ciprofloxacin
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The effects of ciprofloxacin on the supercoiling activity of Campylobacter DNA
gyrases were determined using the in vitro supercoiling assay. As shown in Fig. 4, the MECs
of CIPRO to wild-type GyrA and the two mutants GyrA carrying the Thr-86-Lys or Asp-90Asn change were 32 and 1024, respectively, indicating the two mutations significantly
reduced the susceptibility of GyrA to the inhibition by CIPRO. With the mutant GyrA
carrying the Thr-86-Ile change, the inhibitory effect of CIPRO was not measurable because
the mutation itself abolished the ability of the enzyme to form the supercoiled band in the in
vitro assay (Fig. 4). These findings provide a molecular basis for the reduced susceptibility of
FQR mutants to CIPRO.
5. Discussion
Antibiotic resistance-associated mutations often affect the physiological functions in
bacteria and may impose a biological cost in the absence of antibiotics. The fitness cost
associated with topoisomerase mutations has been reported in several bacteria (4, 14, 20, 51).
In Salmonella typhimurium, the FQR mutants selected in vitro or in vivo (chicken) showed
different growth characteristics in culture medium and in the chicken host in the absence of
FQs (14). The S. typhimurium mutants selected by in vitro plating were highly resistant to
FQs, but grew significantly slower in culture medium and on solid media, and failed to
colonize chickens. On the contrary, the in vivo selected resistant isolates exhibited
intermediate susceptibility to FQs, had normal growth in liquid medium, and were able to
colonize chickens as efficiently as or lower than that of the wild-type strains (14, 51). In the
case with FQR E. coli, single mutations in DNA gyrase or topoisomerase IV confer low-level
resistance to FQs, while accumulation of multiple mutations in the enzymes result in high-
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level resistance (4, 32). Interestingly, the single mutations were not associated with fitness
reduction, but the mutants carrying multiple mutations showed a significant fitness
disadvantage when assayed in competition experiments in vitro and in a mouse model (19).
These findings suggest that resistance-conferring GyrA mutations generally impose a fitness
cost in bacterial pathogens.
In contrast to the findings in other bacteria, our previous study using clonally related
isolates and isogenic mutants generated by natural transformation showed that FQR
Campylobacter isolates outcompeted the FQS strains in chickens in the absence of FQ
antimicrobials and the enhanced fitness was linked to the single point mutation (C257T) in
gyrA, which confers on Campylobacter a high-level resistance to FQ antimicrobials (26). In
the previous study, the isogenic mutants were generated by natural transformation and
ciprofloxacin was used to select for the isogenic transformants. There was a concern that
ciprofloxacin might also have selected for spontaneous FQR mutants with unknown
compensatory mutations that could affect the fitness of Campylobacter. To address this
concern, we further defined the role of the Thr-86-Ile mutation in GyrA in enhanced fitness
by reverting the mutation back to the wild-type allele. In the creation of the revertant, a Cmr
cassette, which was inserted into cj1028c upstream of gyrA, was used as selective marker to
revert the mutant gyrA allele by homologous recombination. This strategy allowed us to test
if a compensatory mutation was involved in the enhanced fitness of the FQR mutants. The in
vivo fitness study showed that once the Thr-86-Ile mutation in GyrA of 62301R33 was
reverted, Campylobacter lost its fitness advantage in chickens, confirming the specific effect
of the point mutation on fitness. In addition, introducing the Thr-86-Ile mutation into the
111
GyrA of the wild-type strain NCTC11168, which is divergent from S3B derivatives used in
our previous work (26), also enhanced its fitness in chickens. Together, these findings
conclusively establish that the C257T mutation in gyrA enhances fitness of C. jejuni in
chickens.
How the Thr-86-Ile mutation affects Campylobacter fitness remains a question and
was the main reason for performing in this study. Gyrase introduces and maintains negative
supercoils in DNA, which is important for DNA replication and transcription. Thus,
resistance-conferring mutation in GyrA may affect its enzymatic function. Indeed, it has been
shown in other bacteria that antibiotic resistance-conferring mutations in GyrA affected the
supercoiling activity of the enzyme (5, 20). A previous study conducted by Barnard et. al.
showed that a FQR E. coli mutant carrying single (Ala87) or double mutations (Ala83 Ala87) in
GyrA exhibited reduced supercoiling compared to that of the wild type (5). In Pseudomonas
aeruginosa, it was also shown that a FQR GyrA Ile83 mutant and a Tyr87 mutant had
decreased supercoiling, which was associated with reduced growth rate (20). In this study,
we demonstrated that in Campylobacter the Thr-86-Ile change in GyrA resulted in a greatly
reduced supercoiling activity compared to that of the wild-type enzyme (Fig. 2). The change
in the enzymatic activity was further confirmed by the reduced supercoiling status in
Campylobacter cells (Fig. 3). These findings suggest that development of FQ resistance
impacts DNA supercoiling homeostasis within Campylobacter. Despite this effect, FQR
Campylobacter grow well in vitro, can colonize and persist in chickens as efficiently as the
FQS strains in the absence of antibiotic usage when monoinoculated into the chickens, and
can outcompete the FQS parent strains when coinoculated into chickens.
112
In contrast to the Thr-86-Ile mutation, the supercoiling activity of the mutant gyrases
harboring the Thr-86-Lys or Asp-90-Asn mutation was comparable to that of the wild-type
gyrase (Fig. 2), indicating that these two types of mutations did not affect the function of the
enzyme. Interestingly, the Thr-86-Ile and Thr-86-Lys mutations occurred at the same place,
but had a different impact on the enzyme function. This is probably due to the fact that Ile
and Lys have different chemical properties. Ile is aliphatic, hydrophobic and neutral, while
Lys is polar, hydrophilic, and positively charged. In Campylobacter GyrA, Thr-86 is
equivalent to Ser-83 in Escherichia coli GyrA(47). It has been known that Ser-83 is located
in the QRDR region , which are situated close to the catalytic cleavage residues Tyr122 , and
Ser-83 has an important role in the interaction of gyrase-quinolone-DNA complex (5). Thus
changes at Ser-83 (Thr-86 in Campylobacter) can conceivably prevent FQ binding, but is
also accompanied by altered supercoiling activities of the gyrase.
The results presented in this study strongly indicate that the Thr-86-Ile mutation
impacts DNA supercoiling in Campylobacter. It would be interesting to know if FQR mutants
acquire compensatory mutations to offset the impact of the Thr-86-Ile mutation. In
Campylobacter, the genes encoding topoisomerase IV (parC/parE) are absent (36), and
supercoiling homeostasis is controlled by topoisomerase I (TopA) and topoisomerase II
(GyrA and GyrB). Our previous study has shown that the FQR isolates had no compensatory
mutations in GyrA and GyrB (26). Since topoisomerase I, encoded by topA gene, is involved
in the relaxation of DNA supercoiling and also has an important role in the maintaining of
the topological state of DNA, it is necessary to see if there is any compensatory mutation in
113
topA and whether the expression of the topA is changed that contributes to the enhanced
fitness. Our studies showed that the primary sequences of topA gene were identical for
62301S2 and 62301R33 and there was no difference in the expression of topA between these
two strains, which excludes the possibility that mutations or altered expression of
topoisomerase I is involved in the fitness change associated with FQR Campylobacter.
The MIC is the lowest concentration of an antimicrobial that inhibits the visible
growth of a microorganism after incubation, while the MEC is the lowest concentration of a
drug that shows any inhibition of supercoiling activity (15). In this study, we demonstrated
that the mutant gyrases carrying the Thr-86-Lys or Asp-90-Asn mutation had significantly
higher MECs of CIPRO than the wild-type GyrA, indicating the mutant gyrases are more
resistant to CIPRO. Interestingly, the MECs of CIPRO with the enzymes were significantly
(at least 250 times) higher than the MICs of CIPRO in these strains. This finding was
consistent with the results from other studies (15, 29) that MECs and MICs differ by one or
two orders of magnitude. The in vitro results (Fig. 4) provide a molecular explanation for the
resistance of FQR Campylobacter mutants to FQ antimicrobials.
To our knowledge, this is the first report that a FQR-conferring mutation in gyrase
reduces its supercoiling activity, but enhances the fitness of the mutant strain. This result is in
contrast to the results from a recently published paper (20), in which reduced fitness of FQR
mutants of Pseudomonas aeruginosa was associated with decreased DNA supercoiling.
Usually decreased supercoiling causes an increased accumulation of positive supercoils in
front of a replication/transcription, which in turn reduce the growth rate (20). This apparently
114
did not happen with FQR Campylobacter because it grows as efficiently as FQS
Campylobacter in both culture media and animal hosts. The intriguing question is whether
the reduced supercoiling activity of the gyrase affects Campylobacter physiology and
directly contributes to the enhanced fitness in chickens. DNA supercoiling is important for
gene expression, and it is possible that the change in DNA supercoiling homeostasis affects
the expression of certain genes in Campylobacter. This possibility remains to be investigated
in future studies.
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120
Table 1. Bacterial strains used in this study
Table 1 Bacterial strains used in this study
FQS strains
FQR strains
Strains
62301S2
52901-I 1
62301R33S
NCTC 11168
11168 (S)
62301R33
52901-II 1
62301R37
62301R33R
11168CT
11168 (R)
aa mutation in GyrA
None
None
None
None
None
Thr-86-Ile
Thr-86-Lys
Asp-90-Asn
Thr-86-Ile
Thr-86-Ile
Thr-86-Ile
nt mutation in GyrA
None
None
None
None
None
C257T
C257A
G268A
C257T
C257T
C257T
121
Table 2. Key PCR primers used in this study
Table 2 Key PCR primers used in this study
Primer name
gyrA1028F
Sequence (5'-3')
TGCTCTGCTTTTGTGAATTA
Target gene/cluster
Cj1028c and gyrA
(ACA, Thr-86)
gyrA257WR
AAACTGCTGTATCTCCATGT
gyrA1028F
gyrA257m (T)
TGCTCTGCTTTTGTGAATTA
AAACTGCTATATCTCCATGT
gyrA-F-2
gyrA-R
ACATGCATGCGAGAATATTTTTAGCAA
ACGCGTCGACTTATTGCAAATCTAAACC
whole ORF of gyrA
gyrB-F-J2
gyrB-R-J
ACATGCATGCCAAGAAAATTACGGTGCG
ACGCGTCGACTTACACATCCAAATGCTT
whole ORF of gyrB
topA-F
topA-R
GCTGCTTTAAATCCGGACTT
CAGCTTCGCAAACATACTCA
whole ORF of topA
cj1686c-F1
cj1686c-R1
AAGCCAAAGATGCCAAAGAA
TGCGTGGTAAGGTGTTTTCA
topA
16SF
16SR
TACCTGGGCTTGATATCCTA
GGACTTAACCCAACATCTCA
16S RNA
Cj1028c and gyrA
(ATA, Ile-86)
122
(A)
(B)
(C)
Fig. 1 Pairwise competition between FQS and FQR strains in chickens
123
Fig. 1. Pairwise competition between FQS and FQR strains in chickens. (A) The level of total
Campylobacter in each chicken inoculated with 62301R33S (solid circle), 62301R33R (open
circle), or 1:1 mixture of the two stains (half solid half open circle). (B) Differential
enumeration of FQS (solid circle) and FQR (open circle) Campylobacter in the group
inoculated with a mixture of 62301R33S and 62301R33R (left panel) and competition index
of the competitive fitness (right panel). (C) The level of FQS (solid triangle) and FQR (open
triangle) Campylobacter in the second chicken experiment in which the chickens were
inoculated with a 1:1 mixture of 11168(S) and 11168(R) (left panel) and competition index
of the competitive fitness (right panel). The mean colonization level of each group at a given
time is indicated by a horizontal bar. The detection limit of the plating method is about 100
CFU/g of feces. DAI: days after inoculation.
124
(A)
M
1
2
3
kD
a
150
100
75
(B)
1
2
3
4
5
6
7
K
b
10
6
4
3
2
R
S
C
Fig. 2 SDS-PAGE analysis of purified recombinant GyrA and GyrB & Supercoiling
activities of the WT gyrase and the three mutant gyrases measured by an in vitro assay
Fig. 2. (A) SDS-PAGE analysis of purified recombinant GyrA and GyrB. Lane 1, Purified
GyrB; Lane 2, Purified WT GyrA; Lane3, Purified mutant GyrA (T86I); and Lane M, protein
size markers. (B) Supercoiling activities of the WT gyrase and the three mutant gyrases
measured by an in vitro assay. Lane 1, DNA ladder; Lane 2, relaxed pBR322 (control); Lane
3, WT GyrA+GyrB; Lane 4, mutant GyrA(T86I)+GryB; Lane 5, mutant GyrA(T86K)+GryB;
Lane 6, mutant GyrA(D90N)+GryB; and Lane 7, supercoiled pBR322 (control). SC indicates
supercoiled DNA and R represents relaxed DNA.
125
(A)
1
2
3
4
1
2
SC
3
4
87.4%
86.5%
20.2%
88.8%
(B)
1
2
1
SC
2
87.2%
25.8%
Fig. 3 Agarose gel electrophoresis analysis of plasmid topoisomers extracted from different
strain background
126
Fig. 3. Agarose gel electrophoresis analysis of plasmid topoisomers extracted from different
strain backgrounds. The plasmid DNA was run on a 1.0% agarose gel containing 20 µg /ml
chloroquine. (A) Plasmid pRY107 isolated from strain S3B derivatives. Lane1, pRY107 from
62301S; Lane 2, pRY107 from 62301R (Thr-86-Ile mutation in GyrA); Lane 3, pRY107
from 52901-II2 (T86K mutation in GyrA); and Lane 4, pRY107 from 62301R37 (D90N). (B)
Plasmid pRY107 isolated from strain 11168 backgrounds. Lane 1, pRY107 from NCTC
11168 and Lane 2, pRY107 from 11168CT (T86I mutation in GyrA). The results of
densitometric scanning are shown to the right of each gel image. The numbers on the left of
the densitometric scanning correspond to the lane numbers of the gel image. The number
below each lane indicates the percentage of the most supercoiled DNA in the total population
of plasmid topoisomers as measured by densitometry.
127
(A)
0
(B)
2
4
8
16
32
64
128
(C)
SC
SC
R
0
4
16
64 256 1024 4096
0
512 1024 2048 4096 8194
(D)
R
0
4
16
64 256 1024 4096
R
SC
Fig. 4 Inhibition of DNA gyrase supercoiling activity by ciprofloxacin as measured by an in
vitro supercoiling assay
Fig. 4. Inhibition of DNA gyrase supercoiling activity by ciprofloxacin as measured by an in
vitro supercoiling assay. A, WT GyrA+GyrB; B, mutant GyrA (Thr-86-Lys)+GyrB; C,
mutant GyrA (Asp-90-Asn)+GyrB; and D, mutant GyrA (Thr-86-Ile)+GyrB. The numbers
on the top of each panel are the concentraction of ciprofloxacin (μg/ ml) used in the reaction.
SC indicates supercoiled DNA and R represents relaxed DNA.
128
CHAPTER 5. TRANSCRIPTOMIC AND PROTEOMIC COMPARISON
OF CAMPYLOBACTER JEJUNI STRAINS THAT ARE RESISTANT OR
SUSCEPTIBLE TO FLUOROQUINOLONE
A paper to be submitted to the Journal of Bacteriology
Jing Han, Ying Wang, Eva Barton, and Qijing Zhang
Departments of Veterinary Microbiology and Preventive Medicine, Iowa State University,
Ames, Iowa 50011
1. Abstract
Our previous work showed that acquisition of fluoroquinolone (FQ) resistance via a
specific GyrA mutation (Thr-86-Ile) enhances the in vivo fitness of FQ-resistant mutants in
the absence of antibiotic selection pressure and the Thr-86-Ile change in GyrA both confers a
high-level resistance to FQs and significantly reduces the supercoiling activity of the enzyme
in Campylobacter. To understand how the reduced supercoiling activity of mutant DNA
gyrase affects Campylobacter fitness, we initiated work to determine if this altered
supercoiling activity affects the gene transcription and protein expression in the bacterial
organisms. Using both DNA-microarray and 2-D DIGE (two dimensional differential in-gel
electrophoresis) methods, we compared the transcription patterns and protein profiles
between the FQR and FQS Campylobacter grown in laboratory media. Results revealed that
the expression of multiple genes was changed in FQR strains compared to FQS stains.
Notably, the iron-response system and proteins involved in energy metabolism were
upregulated in FQR Campylobacter, which may contribute to its enhanced fitness in the
chicken host.
2. Introduction
Campylobacter jejuni is a major foodborne pathogen worldwide and causes diarrhea,
abdominal pain, colitis and other clinical diseases in humans (40). Clinically, Campylobacter
129
infections are mild, self-limiting and usually resolve within a few days. No specific treatment
is required for most patients with Campylobacter enteritis, but antibiotic treatment is needed
in severe cases or in immunocompromised patients (2, 8, 9, 11, 14, 35). Fluoroquinolones
(FQs) or erythromycin are the antibiotics of choice for treating Campylobacter infection (14,
35). However, Campylobacter has become increasingly resistant to FQs worldwide over the
past 15 years, which is a major concern for public health (14, 21, 34, 45). Although FQ
antimicrobials have been banned since 2005 in poultry production in the U.S., FQ-resistant
(FQR) Campylobacter continues to persist in poultry farms (29, 37, 38).
In Campylobacter, resistance to FQs is mediated by a point mutation in the quinolone
resistance-determining region (QRDR) of gyrA in conjunction with the function of the
multidrug efflux pump CmeABC (6, 14, 17, 31). Usually antibiotic resistance-conferring
mutations in target genes are associated with changes in physiological processes, resulting in
reduced growth rate and fitness in the absence of antibiotic selection pressure (5). However,
in Campylobacter FQR mutants carrying the Thr-86-Ile change in the GyrA subunit of DNA
gyrase were able to colonize and persist in chickens as efficiently as the FQ-susceptible (FQS)
strains in the absence of FQ and showed enhanced fitness in chickens. This altered phenotype
was directly linked to the resistance conferring mutation and was confirmed by using
isogenic mutants as well as revertants (30).
DNA gyrase plays an essential role in maintaining the topological configuration of
DNA by introducing negative superhelical turns in DNA. Our previous study has shown that
the Thr-86-Ile mutation in GyrA greatly reduced the supercoiling activity of DNA gyrase
both in vitro and in vivo (within Campylobacter cells). Whether the changed supercoiling
function of the mutant gyrase is responsible for the enhanced fitness is unknown. It has been
well established that DNA supercoiling affects gene transcription in bacterial organisms (1).
DNA supercoiling is a key player in regulating bacterial adaptive responses to environmental
challenges such as stress. A number of studies have shown that changes in environmental
conditions such as temperature, osmolarity, and oxidative stress affect DNA supercoiling and
consequently alter the expression of a number of genes involved in the adaptation to the
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changes (12, 42). DNA topological changes are also linked to intracellular adaptation,
expression of virulence factors such as surface proteins, and invasins of pathogenic
organisms (3, 15, 32). In E.coli, alteration of DNA supercoiling by mutations in TopA and
GyrB affects the relative abundance of more than 80 proteins (41). Based on the available
evidence, we hypothesize that the reduced supercoiling activity of mutant DNA gyrase (Thr86-Ile mutation in GyrA) alters gene expression in the Campylobacter cells, which
contributes to the enhanced in-host fitness.
To examine our hypothesis, we used whole-genome microarray to compare the
transcription patterns in FQS and FQR strains. Microarray results identified 23 genes with
≥1.5-fold (p <0.05) changes in expression, among which 17 were up-regulated and 5 were
down-regulated in FQR mutant (62301R33) compared with FQS (62301S2). In addition, we
conducted 2-D DIGE (two dimensional differential in-gel electrophoresis) to compare the
protein profiles of FQR (62301R33R) and FQS (62301R33S) Campylobacter. The 2-D DIGE
results revealed 42 protein spots that were significantly differentially expressed in
62301R33R compared with 62301R33S, among which 39 were up-regulated and 3 were
down-regulated. Ten spots of interest were identified by MALDI-TOF/TOF.
3. Materials and Methods
Bacterial strains and growth conditions
The various Campylobacter strains used in this study are listed in Table 1. The
Campylobacter strains were routinely grown in Mueller-Hinton (MH) broth (Difco) or on
MH agar plates at 42°C under microaerobic conditions (10% CO2, 5% O2, and 85% N2).
Bacterial RNA isolation
Campylobacter isolates 62301S2 and 62301R33 were grown in MH broth for 48
hours (OD600 ≈ 0.45) and were immediately treated with 2 volumes of RNAprotect Bacterial
Reagent (Qiagen, Valencia, CA) to stabilize the total bacterial RNA. After incubation for 10
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min at room temperature, the culture was centrifuged at 10,000 × g for 10 min. Bacterial
RNA was isolated using an RNeasy Mini Kit (Qiagen) according to the manufacturer's
instructions. Isolated RNA was treated with an on-column DNase Digestion Kit (Qiagen),
which was followed by removal of the DNase using an RNeasy Mini Kit (Qiagen). The
absence of DNA contamination in the RNA samples was confirmed by reverse transcriptionPCR (RT-PCR). The concentration of total RNA was estimated with the NanoDrop ND-1000
spectrophotometer (NanoDrop, Wilmington, DE, USA), and the integrity and size
distribution of the purified RNA was determined by denaturing agarose gel electrophoresis
and ethidium bromide staining. The quality of total RNA was further analyzed using the
Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), which showed the
good quality and integrity of the RNA samples (Data not shown). The purified RNA was
kept at −80 °C until it was used. RNA samples extracted from three independent experiments
were used for the microarray hybridization experiments.
cDNA synthesis and labeling
cDNA synthesis and fluorescence labeling were performed using ChipShot™ Indirect
cDNA Labeling and Clean-Up Protocols (Promega) according to the manufacturer's
instructions. Briefly, 5µg of total RNA was reverse transcribed using ChipShotTM reverse
transcriptase, random primers and Oligo(dT) primers in the presence of an aminoallyl-dNTP
mix together with 5X reaction. After incubation at 42°C for 2 hours, RNase H was added to
the reaction mix to degrade RNA. The synthesized aminoallyl-cDNA was purified using
ChipShot™ Membrane columns (Promega) to remove unincorporated nucleotides and eluted
with 65µl of 100mM sodium bicarbonate (pH 9.0). Sixty µl purified aminoallyl cDNA were
then labeled by coupling with one dried aliquot of post-labeling CyDye™ NHS ester, Cy3 or
Cy5 (GE Healthcare Bio-Sciences). A final purification step using ChipShot™ Membrane
Columns (Promega) removed unreacted dyes, and the amount of labeled cDNA was
estimated with the NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE,
USA). The pmol of dye incorporated and the frequency of incorporation (FOI) were
calculated accordingly. The eluted cDNA with good quality was stored in a lightproof
container at 4 °C until it was used to hybridize microarray slides.
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Microarray hybridization
C. jejuni NCTC 11168 microarray slides were purchased from Ocimum Biosolutions.
The array contained 1,632 oligonucleotide (50-mer) probes covering the entire transcriptome
of NCTC 11168. To compare the gene expression of FQR strain (C257T mutation in gyrA)
with that of FQS strain, equal volumes of Cy3- or Cy5-labeled cDNAs from the 62301S2
strain and 62301R33 strain were combined and dried with a SpeedVac and then resuspended
in 120 µl of hybridization buffer (Ocimum Biosolutions). The probes were denatured at 95°C
for 3 min, briefly cooled on ice, and then hybridized to the microarray slide using microarray
gene frames (Ocimum Biosolutions). The slide were placed in a wet hybridization chamber
and incubated on a shaker for 20 h at 42°C. After hybridization, the slide was washed
sequentially with wash buffer 1 (2x SSC, 0.1 % SDS) for 10 min, wash buffer 2 (1 x SSC)
for 10 min, and wash buffer 3 (0.1 x SSC) for 10min. Each wash step was repeated three
times. The washing buffers were prewarmed at 32°C, and all of the washing steps were
performed at 32°C. Finally, the slides were dried by centrifugation at 500 × g for 3 min. The
microarray slides were hybridized with the swaped-dye labeled cDNA samples to ensure dye
balance in the experimental design. The hybridization experiments were performed using
RNA extracted from three biological replicates and each of the experiments consisted of two
technical replicates (slides).
Data collection and analysis
Hybridized slides were scanned at a wavelength of 650 nm for Cy5 and at a
wavelength of 550 nm for Cy3 using a General Scanning ScanArray 5000 (PerkinElmer,
Boston, MA) at 10 µm resolution. The fluorescence intensities were collected with the
ImaGene software (BioDiscovery, El Segundo, CA). Genes with fluorescence signals within
less than 2 standard deviations of the background were considered insignificant. Background
subtraction, Lowess normalization, scale normalization, and median centering were
performed using the R statistical package (version 2.0.1; The R Foundation for Statistical
Computing) and the normalized data were subjected to a mixed-linear model analysis using
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the SAS statistical package. For this study, we chose a P value of < 0.05 and a change equal
to or greater than 1.5-fold as the cutoff for significant differential expression in comparisons
of the FQR and FQS strains.
Real-time quantitative PCR (qRT-PCR)
Representative genes identified by the DNA microarray were further confirmed by
Real-time quantitative PCR (qRT-PCR) as described in a previous work (27). Before being
used for quantitative reverse transcriptase PCR (RT-PCR), all RNA templates and primer
sets were tested with a conventional One-step RT-PCR Kit and a regular PCR kit (Invitrogen)
to ensure specific amplification from the target mRNA and no detectable DNA
contamination in the RNA preparation. The RT-PCRs were conducted using the iScript onestep RT-PCR kit with SYBR green (Bio-Rad). Thermal cycling conditions were as follows:
10 min at 50°C, 5 min at 60°C followed by 5 min at 95°C, and then 40 cycles of 10 s at 95°C
and 30 s at 58°C. Samples were normalized using 16S RNA as an internal standard. Cycle
threshold values were determined with the MyiQ software (Bio-Rad). The relative changes
(n-fold) of specific genes between the FQR and FQS samples were calculated using the 2−ΔΔCT
method as described by Livak and Schmittgen (28). Three independent PCR experiments
were performed to get the mean of the fold change.
Proteome analysis by 2-D DIGE
2-D DIGE was performed by Applied Biomics (Hayward, CA). Briefly, overnight
fresh bacterial cells 62301R33S and 62301R33R were harvested form MH plates and
centrifuged. The cell pellets were washed with PBS 4 times and then were dissolved in 2-D
lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS and 30 mM Tris-HCl, pH 8.5). The samples
were stored at -80 °C until they were shipped on dry ice to Applied Biomics (Hayward, CA)
for proteomic analysis. Total protein was extracted from lysed cells and equal amount of
protein extract from 62301R33S and 62301R33R were labeled with the Cy3 or Cy5 dyes,
(GE Healthcare, Piscataway, NJ) respectively. The labeled protein extracts were mixed and
subjected to a single 2-D gel electrophoresis, using isoelectric focusing (IEF) in the first
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dimension and SDS polyacrylamide gel electrophoresis (SDS-PAGE) in the second
dimension. After electrophoresis, the gel was scanned using a Typhoon Image Scanner (GE
Healthcare, Piscataway, NJ). Each scan revealed one of the CyDye signals (Cy3 and Cy5).
Scanned images were analyzed by Image Quant Software (version 5.0, GE Healthcare) and
then subjected to in-gel analysis using the DeCyder software (version 6.5, GE Healthcare).
The ratio change for differentially expressed protein spots was obtained from the in-gel
DeCyder software analysis. Protein spots of interest were picked up by Ettan Spot Picker
(GE Healthcare) following the DeCyder software analysis and spot picking design. The
selected protein spots were subjected to the in-gel trypsin digestion, peptides extraction,
desalting and followed by mass spectrometry (MALDI-TOF/TOF) (ABI 4700, Applied
Biosystems, CA). Peptide sequence information was used for protein identification in the
NCBInr databases.
SDS-PAGE and Immunoblotting
To prepare whole-cell lysates, overnight culture of 62301S2 and 62301R33 were
grown in MH broth to late logarithmic phase (about 3 x 109 cells/ml), harvested by
centrifugation, and solubilized by boiling for 10 min in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Approximately 108 whole
cells were loaded in each lane and separated by SDS-PAGE with a 12% (wt/vol)
polyacrylamide separating gel. After SDS-PAGE, the gels were equilibrated for 30 min in the
transfer buffer (0.025 M Tris base and 0.192 M glycine with 20% methanol; pH 8.3). Proteins
in the gels were then electrophoretically transferred to nitrocellulose membranes (Bio-Rad) at
60 V for 1 h on ice. The membranes were incubated with blocking buffer (5% Nestle skim
milk powder in PBS) for 4 h at 4°C prior to incubation with anti-FlaA. After incubation at
room temperature for 1 h, the blots were washed three times with PBS containing 0.05%
Tween 20 and subsequently incubated with secondary antibodies (goat anti-rabbit
immunoglobulin G-horseradish peroxidase; Kirkegaard & Perry) at room temperature for 1 h.
After washing, the blots were developed with the 4 CN Membrane Peroxidase Substrate
System (Kirkegaard & Perry). Prestained molecular mass markers (Bio-Rad) were
coelectrophoresed and blotted to allow estimation of the sizes of the proteins.
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4. Results
DNA microarray identified multiple genes differently expressed in the FQR mutant
To compare the gene transcription patterns in FQS and FQR (Thr-86-Ile) strains, DNA
microarray was used to analyze the differences in transcriptoms between 62301S2 and
62301R33. The two strains are clonally related and were well characterized in terms of
genotypic and phenotypic characteristics (30). Microarray results showed 23 genes with
≥1.5-fold (p <0.05) changes in expression, among which 17 were up-regulated (Table 2) and
5 were down-regulated in FQR mutant (62301R33) compared with FQS (62301S2).
Notably, 11 (indicated by an asterisk in Table 2) of the 17 upregulated genes are
known Campylobacter genes whose expression is increased in iron-limited conditions (24,
36). ChuA (Cj1614) which functions as a haemin receptor involved in iron uptake (43).
Cj1613c encodes an orthology (39% aa identity) of HugZ, which is involved in heme iron
utilization (20, 23, 43). ExbB1 (Cj0179) is a biopolymer transport protein. Cj1661 and cj
0176c encode the ferric-binding protein of a putative ABC transport system. Hypothetical
protein Cj0427 co-transcribed with Cj0426, which is a putative ABC transporter ATPbinding protein. Ahpc (Cj0334) is an alkyl hydroperoxide reductase. Cj1664 encodes
putative periplasmic thioredoxin. Both Cj0334 and Cj1664 are involved in the oxidative
stress response of Campylobacter. Cj1383c and cj1384c are tandemly positioned on the
Campylobacter chromosome and encode hypothetical proteins of unknown function. Cj0089
encodes a putative lipoprotein. The other upregulated genes include flaA, which encodes the
major subunit of flagellum and has an important role in the motility of Campylobacter (18,
44). Cj1002c encodes a putative phosphoglycerate/ bisphosphoglycerate mutase, which
catalyzes reactions involving the transfer of phospho groups between the 3 carbon atoms of
phosphoglycerate. Cj0843c encodes a putative secreted transglycosylase. Cj1626c and
cj0200c encode putative periplasmic protein. The transcription of 7 genes selected from the
microarray list was further confirmed by three independent qRT-PCR experiments (Table 2).
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The down-regulated genes include cj0262c, cj0246c, cj1127c, cj1139c, and cj1180c.
pglJ (Cj1127c) is a GalNAc transferase involved in the N-glycan biosynthesis in C. jejuni
(26). Cj0262c encodes a putative methyl-accepting chemotaxis signal transduction protein.
Cj0246c encodes a putative MCP (methyl-accepting chemotaxis protein)-domain signal
transduction protein. Cj1139c encodes a putative galactosyltransferase. Cj1180c encodes a
putative ABC transporter ATP-binding protein. None of the five down-regulated genes is
among the ones known to be regulated by iron concentration.
Proteomic analysis of 62301R33S and 62301R33R
2-D DIGE was used to investigate the difference in the protein profiles between
62301R33S and 62301R33R. Using fold change ≥1.5-fold as cutoff, the Decyder analysis of
2D-DIGE image revealed 42 protein spots that were significantly differentially expressed in
62301R33R compared with 62301R33S (Figure 1 and 2), among which 39 were up-regulated
and 3 were down-regulated (Table 3). Ten spots of interest were identified by MALDITOF/TOF using MASCOT search engines on NCBInr sequence database (Table 4). These
proteins include flavodoxin (FldA), flagellar hook protein (FlgE), translation elongation
factor Tu (Tuf), HAD-superfamily hydrolase subfamily IA variant 1 family protein,
ribosomal proteins L23 and L7/L12, DNA-binding protein HU, F0F1 ATP synthase subunit
epsilon, and a hypothetical protein. Flavodoxins are electron-transfer proteins that function in
various electron transport systems. FlgE is a flagellar hook subunit protein. The function of
the flagellar hook is to connect the filament to the basal body and acts as a joint to transmit
the rotation of the rod of the basal body to the filament. Elongation factor Tu promotes the
GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein
biosynthesis. HAD-superfamily hydrolase, subfamily IA, variant 1 represents part of one
structural subfamily of the HAD-superfamily of aspartate-nucleophile hydrolases. The
function of F0F1-ATP synthase, which is composed of membrane-embedded F0-portion and
hydrophilic catalytic F1-portion, is to couple ATP synthesis/hydrolysis with transmembrane
proton transport. HU is a histone-like DNA-binding protein that plays an important role in
bacterial nucleoid organization and is involved in numerous processes including transposition,
recombination and DNA repair (10, 25). Ribosomal proteins L23 and L7/L12 are 50S
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ribosomal proteins. L23 is one of the proteins that surrounds the polypeptide exit tunnel on
the outside of the ribosome and forms the main docking site for trigger factor binding to the
ribosome. L7/L12 is a stalk proteins that implicated in the ribosomal interaction with the
translocase (33).
5. Discussion
DNA gyrase plays an essential role in maintaining the topological configuration of
DNA, which affects gene transcription in bacterial organisms and plays an important role in
regulating bacterial adaptive responses to environmental challenges such as stress (1). Our
previous study showed that the FQR-associated Thr-86-Ile mutation in GyrA greatly reduced
the supercoiling activity of DNA gyrase both in vitro and in vivo (within Campylobacter
cells). Whether the change in DNA supercoiling homeostasis alters the expression of certain
genes in Campylobacter cells is unknown. To examine our hypothesis, we conducted
transcriptome analysis to compare the mRNA level in FQS and FQR strains. One of the
unregulated genes identified by DNA microarray is flaA, which encodes the major subunit of
flagellum and is essential for the motility and colonization of Campylobacter (18, 44).
Although 62301R33 and 62301S2 have similar motility, and both have intact flagellar
structures when observed by electron microscopy, it is still possible that the increased
expression of flaA may have functional consequences in vivo by facilitating the adaptation of
62301R33 in the intestine, contributing to the enhanced fitness. However, the difference in
the expression level of FlaA was be detected using immunoblotting (data not shown). This
suggests that the transcriptional change is not reflected at the translational level.
One interesting finding from the microarray results is that among the 17 upregulated
genes in 62301R33, 11 are known to be controlled by the Fur regulator and their expression
is increased in iron-limited conditions (24, 36). One of the upregulated genes is ChuA
(Cj1614) which functions as a haemin receptor involved in iron uptake. Inactivation of the
chuA gene rendered Campylobacter unable to grow in media in which haemin was the sole
iron source (43). Cj1613c, which is immediately upstream of chuA and divergently
transcribed, encodes an orthology (39% aa identity) of HugZ, which is involved in haeme
138
utilization (20, 23, 43). Recently, Pfam domain pyridoxamine 5'-phosphate oxidase, which is
a flavin mononucleotide (FMN) flavoprotein that catalyses the oxidation of pyridoxamine-5P (PMP) and pyridoxine-5-P (PNP) to pyridoxal-5-P (PLP), was identified within CDS of
Cj1613 (19). ExbB1 (Cj0179) is a biopolymer transport protein and it also involved in
transport of iron. Cj1661 encodes the ferric-binding protein of a putative ABC transport
system. The protein encoded by Cj0426 and Cj0427, which overlap 10 nucleotides and are
co-transcribed, are likely to form an ABC transport system. Although cj0426 is not on the list
of known genes regulated by iron stress, expression of cj0427 was increased under ironlimited conditions (24), suggesting that cj0426 might also be subject to iron regulation.
Cj0334 and Cj1664 encode proteins involved in oxidative stress response, which is known to
be regulated by iron concentration (7, 43). AhpC (Cj0334) is an important determinant of the
ability of Campylobacter to survive oxidative and aerobic stress (7). Insertional mutagenesis
of the ahpC gene resulted in an increased sensitivity to oxidative stresses. The iron-regulated
expression of AhpC might also be significant in survival of Campylobacter in the intestinal
epithelium of infected chickens (7). These results strongly suggest that the iron-response
system is enhanced in FQR Campylobacter. Since iron is essential for microbial physiology
and free iron is limited to the bacterial organism in animal hosts (24, 36), it is possible that
the changed DNA supercoiling homeostasis in FQR Campylobacter increases the expression
of the genes involved in iron stress response, which facilitate the iron acquisition and
subsequently contributes to the enhanced in-host fitness.
We also used 2-D DIGE method to compare the protein profiles between the FQR and
FQS Campylobacter grown in laboratory media. 2-D DIGE is an advanced version of
classical two-dimensional gel electrophoresis (2-D PAGE). Compared to classical 2-D PAGE,
2-D DIGE has many advantages. First, in 2-D DIGE, the protein samples are pre-labeled
with fluorescent dyes (CyDye) and then separated by 2D-PAGE. Different protein samples
are labeled with different fluorescent dyes, mixed together and separated by the identical gel.
Analyzing multiple samples on one 2-D DIGE rather than running single samples on
individual 2D-PAGE gels reduces experimental gel-to-gel variations, which are the most
severe problem in the gel-based proteomics. Second, fluorescent dyes (CyDye technique )
139
used in 2-D DIGE have much more sensitivity than Coomassie blue and silver staining used
in traditionally 2D-PAGE gels. Third, with silver and Coomassie staining, the gels need to be
fixed and stained / destained using time-consuming techniques. Gels are then compared using
computer software for changes in protein expression. However with DIGE, immediately after
completion of electrophoresis each gel is scanned multiple times by laser scanners with
different wavelengths for different CyDye used. The multiple images obtained by 2-D DIGE
are analyzed by the image software such as DeCyder software. The normalization of Cy5
intensity with Cy3 intensity is automatically achieved for all protein spots by the software
and subtle changes in protein expression detected very accurately. Using proteomics, we
identified 42 protein spots that were differentially present between 62301R33R and
62301R33S. Among the 10 proteins that were upregulated in 62301R33R, 3 are involved in
energy metabolism and 5 are involved in macromolecule metabolism. Two of the most
upregulated proteins were both identified as flavodoxin (FldA), which is a small FMNcontaining electron transferases (16). It functions as the electron acceptor of the pyruvateoxidoreductase complex (POR) that catalyzes pyruvate-oxidative decarboxylation to produce
NADPH (39). Several studies have shown that flavodoxin is an essential protein for the
survival of bacteria. Inactivation of FldA precludes the survival of Helicobacter pylori,
which is a microaerophlic pathogen that is a close relative to C.jejuni (13). fldA is a vital
gene in E. coli and a FldA mutation is lethal for E. coli under both aerobic and anaerobic
stress conditions (16). In Campylobacter, the expression of fldA is also upregulated in ironlimited conditions (24). This result plus the results from DNA microarray strongly suggest
that the iron-response system is enhanced in FQR Campylobacter.
No significant correlation between the microarray data and proteomic data was found
in this study. Since posttranscriptional mechanisms control the turnover of proteins and the
posttranscriptional modifications of proteins, it is not surprising to see the inconsistency
between the mRNA and protein data. Similar findings have been reported in other studies (4,
22). Since the whole genome sequence of C.jejuni S3B (from which all of the isolates used in
this study were derived) is not available, we used the DNA microarray designed from strain
NCTC 11168. Thus there is a possibility that the genes that are uniquely present in the S3B
140
series may not be detected by the microarray even though their expression is altered in
62103R33. On the other hand, proteomics is capable of identifying all protein spots that show
differential production between the paired strains. Due to budget constraints, we were not
able to identify all of the 42 protein spots. Further work is needed to identify the remaining
spots, which might reveal genes that were detected by the DNA microarray. In addition,
whole cell proteins were used in the proteomics work, which might reduce the sensitivity of
detecting expression changes of low-abundance proteins. Thus, fractioning cell proteins prior
to 2-D DIGE may be needed to further enhance the detection sensitivity of the proteomics
method. Nevertheless, our DNA microarray and proteomics studies revealed multiple
changes in gene expression and protein production in FQR Campylobacter carrying the Thr86-Ile mutation in GyrA. Future research will be needed to assess the contribution of the
altered gene expression to the enhanced fitness of FQR Campylobacter in chickens.
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Table 1. Bacterial strains used in this study
Table 1 Bacterial strains used in this study
Strains
S3B
S
FQ
62301S2
strain 62301R33S
FQR
62301R33
strains 62301R33R
nt mutation
in gyrA
None
None
C257T
C257A
aa mutation
in GyrA
None
None
Thr-86-Ile
Thr-86-Ile
Genotype
gyrA (Thr-86)
Cj1028c::Cmr; gyrA (Thr-86)
gyrA (Ile-86)
Cj1028c::Cmr gyrA (Ile-86)
MIC
(µg/ml)
0.125
0.125
>32
>32
146
Table 2. Genes upregulated in the FQR strain identified by DNA microarray
Table 2 Genes upregulated in the FQR strain identified by DNA microarray
Gene ID
Description
Bayesian P Fold change Fold
(Microarray) Change
(RT-PCR)#
Cj1339c
flaA; encoding the major
9.75E-05
3.4
24
flagellin subunit
*Cj1613c Ortholog of hugZ involved in
1.42E-03
2.9
3.9
haemin utilization
Cj0426
Probable ABC transporter
5.83E-03
2.0
4.2
ATP-binding protein
*Cj0427
Hypothetical protein; co4.34E-03
1.7
3.0
transcribed with Cj0426
*Cj1614
chuA, Haemin uptake system
6.34E-03
2.1
2.3
outer membrane receptor
*Cj1384c Hypothetical protein
6.99E-03
2.2
2.0
Cj1002c
Phosphohistidine phosphatase
9.95E-03
2.4
2.5
SixA
*Cj1383C Hypothetical protein
1.58E-02
2.1
NT
*Cj0176C Ferric-binding protein
1.32E-02
2.0
NT
*Cj0334
AhpC (involved in oxidative
4.76E-02
1.6
NT
stress)
*Cj0179
ExbB1; transport protein
1.25E-02
1.9
NT
*Cj1661
Putative ABC transporter
2.61E-02
1.8
NT
protein; ferric-binding protein
*Cj1664
Thioredoxin
3.14E-02
1.9
NT
*Cj0089
Probable lipoprotein
4.52E-02
1.6
NT
Cj1626c
Probable periplasmic protein
2.23E-02
1.8
NT
Cj0200c
Probable periplasmic protein
4.34E-02
1.7
NT
Cj0843c
Probable secreted
4.7-E-02
1.6
NT
transglycosylase
* Genes whose expression is known to be upregulated under iron-limited conditions.
# Means of three independent PCR experiments. NT, not tested.
147
Table 3. Differentially expressed protein spots between 62301R33R and 62301R33S
identified by 2-D DIGE
Table 3 Differentially expressed protein spots between 62301R33R and 62301R33S identified by 2-D DIGE
Assigned spot No.
Volume Ratio (62301R33R/62301R33S)
1
1.66
2
1.68
3
-2.83
4
1.56
5
1.73
6
1.84
7
1.8
8
1.84
9
2.45
10
3.89
11
5.92
12
9.53
13
3.65
14
1.44
15
2.73
16
1.6
17
1.64
18
-2.22
19
-1.69
20
1.48
21
1.77
22
1.51
23
2.42
24
1.91
25
3.96
26
3.76
27
3.1
28
17.52
29
16.84
30
8.77
31
12.75
32
4.92
33
3.29
34
2.72
35
3.58
36
3.81
37
3.39
38
2.69
148
Table 3. Differentially expressed protein spots between 62301R33R and 62301R33S
identified by 2-D DIGE (Continued)
Assigned spot No.
Volume Ratio
(62301R33R/62301R33S)
39
40
41
42
43
1.6
1.72
1.6
1.82
1.48
149
Table 4. Differentially expressed proteins between 62301R33R and 62301R33S
identified by 2-D DIGE
Table 4 Differentially expressed proteins between 62301R33R and 62301R33S identified by 2-D DIGE
Assigin
Top Ranked Protein
NBCInr
Protein
Protein
No. of
Protien
ed Spot Name(Species)
accession No.
MW
PI
matched
Score
No.
peptides
flagellar hook protein FlgE
gi|57236913
89851
4.9
25
352
2
[C. jejuni RM1221]
translation elongation factor
gi|57504721
43580.2
5.11
22
505
13
Tu
[C. coli RM2228]
hypothetical protein CJE0800
gi|57237003
25979.7
4.44
12
498
20
[C.jejuni RM1221]
HAD-superfamily hydrolase,
gi|57238105
23722.1
6.08
11
229
subfamily IA, variant 1 family
23
protein
[C. jejuni RM1221]
flavodoxin FldA
gi|57238425
17124
3.89
7
486
28
[C.jejuni RM1221]
flavodoxin FldA
gi|57238425
17124
3.89
4
168
31
[C.jejuni RM1221]
ribosomal protein L23
gi|86152332
10547.7
9.78
3
98
32
[C.jejuni subsp. jejuni 260.94]
F0F1 ATP synthase subunit
gi|57237115
13683
4.85
6
311
41
epsilon
[C.jejuni RM1221]
DNA-binding protein HU
gi|88596497
6393.3
6.07
3
220
42
[C.jejuni subsp. jejuni 84-25]
50S ribosomal protein L7/L12
gi|57237531
13022.9
4.7
7
255
43
[C.jejuni RM1221]
Protein
Score
C.I.%
100
Fold
Change
100
3.65
100
1.48
100
2.42
100
17.52
100
12.75
99.934
4.92
100
1.60
100
1.82
100
1.68
1.68
150
pH 3-10
S
D
S
P
A
G
E
Fig. 1 Proteomic analysis of protein expression in Campylobacter
Fig.1. Proteomic analysis of protein expression in Campylobacter. The image shows the
overlay of 2-D DIGE patterns of 62301R33R versus 62301R33S. 62301R33R is in red and
62301R33S is in green. The 42 differentially expressed protein spots were circled. Red
indicates increased expression in 62301R33 and green indicates decreased expression in
62301R33 in comparison to 62301R33S. Yellow indicates similar expression levels of
between the two strains.
151
(A)
(C)
(B)
(D)
Fig. 2 Representative DeCyder display of relative abundance of selected protein spots in
62301R33R versus 62301R33S
Fig.2. Representative DeCyder display of relative abundance of selected protein spots in
62301R33R versus 62301R33S (fold change in parentheses). A: FldA (17.52); B: translation
elongation factor Tu (3.65); C: F0F1 ATP synthase subunit epsilon (1.60); and D: DNAbingding protein HU (1.82).
152
CHAPTER 6. GENERAL CONCLUSIONS
The increasing FQ resistance rate of Campylobacter isolated from food-producing
animals is a continuing concern for food safety and public health. How to reduce the
occurrence and spread of FQR Campylobacter has become more and more important in the
control of foodborne campylobacteriosis. Understanding the mechanisms underlying the
rapid emergence of FQR and the enhanced fitness of FQR mutants in Campylobacter will help
to reduce the occurrence and spread of FQR Campylobacter.
In this project, we conducted a series of studies to determine how Campylobacter
responds to FQ treatment, what facilitates the emergence of FQR mutants in Campylobacter,
and what are the molecular mechanisms contributing to the enhanced fitness in FQR
Campylobacter. In the first study, we examined the gene expression profiles of C. jejuni
NCTC 11168 in response to treatment with ciprofloxacin (CIPRO) using microarray and
found that 45 genes showed ≥1.5-fold (p <0.05) changes in expression when exposed to a
suprainhibitory dose of CIPRO for 30 min. Most of the differentially-expressed genes
involved in cell membrane biosynthesis and DNA repair are up-regulated, while the genes
associated with cellular processes and energy metabolism are down-regulated following
exposure to CIPRO. These findings suggest that C. jejuni modulates membrane biosynthesis,
increases spontaneous mutation rates, and decreases metabolism in response to FQ treatment.
One of the up-regulated genes was mfd (mutation frequency decline), which encodes a
transcription-repair coupling factor involved in DNA repair. We found that in this study Mfd
promotes the emergence of spontaneous FQR mutants and the development of FQR mutants
under FQ treatments. These findings define a novel function of Mfd and significantly
improve our understanding of the molecular mechanisms underlying the development of FQR
Campylobacter. In the second study, we formally defined the role of the Thr-86-Ile mutation
of GyrA in the enhanced fitness FQR Campylobacter in chickens by reverting the mutant
allele. Then we conducted in vitro supercoiling assay using recombinant gyrases to assess the
impact of various resistance-associated mutations on the enzymatic activities of DNA gyrase.
We found that compared with that of the wild-type gyrase, the mutant gyrase with the Thr-
153
86-Ile change showed greatly reduced supercoiling activity, while other GyrA mutations,
although reduced the susceptibility of Campylobacter to ciprofloxacin, did not affect the
supercoiling activity of the gyrase. Subsequently we determined the impact of the GyrA
mutations on in vivo supercoiling (within Campylobacter cells) using a reporter plasmid. The
in vivo supercoiling result was consistent with the in vitro supercoiling findings and revealed
that the Thr-86-Ile mutation altered the DNA supercoiling state in FQR Campylobacter. In
the third study, we examined if the altered function of mutant gyrase affected gene and
protein expression in Campylobacter. We compared the differences in transcriptomes and
protein profiles between FQR and FQS strains using DNA microarray and 2-D DIGE. The
microarray data and 2-D DIGE data showed that the expression of multiple genes was altered
between the FQR strains and FQS strains. Especially, the iron-response system and proteins
involved in energy metabolism were upregulated in FQR Campylobacter, which may
contribute to its enhanced fitness in the chicken host. Together, these findings from this
project have significantly improved our understanding of the molecular mechanisms
underlying the development of FQR Campylobacter and the fitness of the FQR
Campylobacter. Findings from these studies will facilitate the design of strategies to reduce
the emergence and spread of FQR Campylobacter.
154
ACKNOWLEDGMENTS
First of all, I wish to express my deep appreciation to my major professor, Qijing Zhang for
giving me the opportunity to pursue my Ph.D. degree abroad, and for his intellectual
guidance, support, and encouragement, which made this dissertation possible. I am very
fortunate to have such a talented, patient, caring and courageous person as my mentor. The
experiences and lessons I have learned here have been invaluable to my future career.
Special thanks also go to my dissertation committee members Drs. Lisa K. Nolan, Nancy
A.Cornick, Lorraine Hoffman, and Edward Yu for their kindness and willingness to serve on
the committee, as well as for their valuable advice and contribution on this dissertation.
I appreciate the previous and current members of Dr. Zhang’s lab for their technical
assistance and companionship. Thanks Drs. Jun Lin and Ying Wang for their help in the
beginning of my Ph.D. study. I appreciated their continuous support and friendship. Thanks
Dr. Orhan Sahin for his generous advice, his technical assistance in chicken experiments, and
a lot of fun stories sharing with me. I own Dr. Byeong Hwa Jeon many thanks for answering
my continous flow of questions and his helpful advice for my MFD paper. I also thank Pam
Vardaxis and Rachel Sippy for helping me order reagents and getting them ready for
experiment. Special thanks to Rachel Sippy for her help with my writing. I would like to
thank my other co-workers: Dr. Baoqing Guo, Dr. Paul Plummer, Dr. Taradon
Luangtongkum, Eva, Zhangqi, Yang, Haihong, Rocky, Wayne and Tara for all assistance in
my research.
Special thanks are given to Vern Hoyt and Liz Westberg for their coordination efforts which
made my graduate studies going more smoothly. I am thankful to everyone at the Department
of Veterinary Microbiology and Preventive Medicine for their friendship and kind
assiatance.
I am grateful to Dr. Fuyong Chen, my previous advisor at China Agricultural University, for
his guidance and encouragement that brought me to scientific research.
155
Finally, my heartfelt gratitude goes to those who are the most important persons in my life,
my husband, Hailin Tang, my son, Eric (Jiale) H. Tang, my parents Xingzhi Zhao and Futing
Han , and my brother Jie (Jason) Han. Thanks my husband for his enduring love, patience,
and encouragement, and helping me get through the tough, difficult, and stressful times
during my studies. Thanks our son for all the great pleasure he has brought during this
challenging time. Thanks my parents for their unconditional love, life-long support, and
believing in me. I am forever indebted to my parents for taking care of my son so that I can
focus on my research. Thanks my brother for always encouraging me and supporting me
during my studies. Without them, I could not have imagined the completion of this work.