Department of Food and Environmental Hygiene
Faculty of Veterinary Medicine
University of Helsinki
Mechanisms and Development of Antimicrobial
Resistance in Campylobacter with Special
Reference to Ciprofloxacin
To be presented, with the permission of the Faculty of Veterinary Medicine, University
of Helsinki, for public examination in Hall 6, Fabianinkatu 33, Helsinki,
on June 4, 2010, at 12 noon.
Professor Marja-Liisa Hänninen, DVM, PhD
Department of Food and Environmental Hygiene
Faculty of Veterinary Medicine
University of Helsinki
Professor Séamus Fanning, PhD
School of Agriculture, Food Science & Veterinary Medicine
University College Dublin
Doctor Axel Cloeckaert, PhD
Infectiologie Animale Santé Publique
Institut National de la Recherche Agronomique
Docent Antti Hakanen, MD
National Institute for Health and Welfare
Antimicrobial Resistance Unit
ISBN 978-952-92-7360-7 (Paperback)
ISBN 978-952-10-6285-8 (PDF)
Helsinki University Print
In industrialized countries, campylobacteriosis is the most common cause of human
gastroenteric infection. It is mainly caused by Campylobacter jejuni and Campylobacter
coli, but the pathogenic role of other species, such as Campylobacter hyointestinalis,
remains unclear due to difficulties in cultivating these fastidious bacteria. Humans most
often become infected by ingesting contaminated water or food, especially undercooked
chicken. Campylobacteriosis is normally a self-limiting disease, but in severe cases the
patients are treated with antimicrobial agents. The predominant antimicrobial agents for
treating Campylobacter infections are erythromycin and ciprofloxacin. However, during
the past few decades resistant C. jejuni and C. coli strains have emerged at an alarming
rate due, at least partly, to the large-scale use of these agents in food production animals.
Understanding of the mechanisms underlying bacterial resistance can reveal the risks
involved in using antimicrobials in animal husbandry and ultimately help in preventing
antimicrobial resistance, which has life-threatening implications in human and animal
health. It was therefore the aim of this thesis to study the various resistance mechanisms of
C. jejuni, C. coli and C. hyointestinalis towards antimicrobial agents.
Finnish C. hyointestinalis strains are susceptible to most antimicrobials of veterinary
importance. When susceptibility to 12 different antimicrobials was tested, all the reindeer
strains proved susceptible, but in bovine strains resistance was observed in 32% and 24%
of the strains to streptomycin and sulphonamides, respectively. Since unlike bovines,
reindeer are very rarely treated with antimicrobials, the difference in the susceptibility
profile of the bovine and reindeer strains most probably reflects the veterinary use of these
substances in bovine husbandry, but not in reindeer.
Quinolone resistance in C. jejuni and C. coli has been associated with mutations in the
quinolone resistance-determining region (QRDR) of gyrA. Unlike these two species, C.
hyointestinalis is intrinsically resistant to nalidixic acid. To explain this phenomenon,
studies were conducted with the QRDR of C. hyointestinalis. The QRDR showed 73%
identity with that of C. jejuni, but mutations associated with nalidixic acid resistance in C.
jejuni were not found in C. hyointestinalis. However, inherent nalidixic acid resistance in
C. hyointestinalis was associated with efflux pumps since the efflux pump inhibitor,
PAβN, decreased the nalidixic acid resistance in C. hyointestinalis 2- to 8-fold. However,
susceptibility was not obtained, indicating the presence of other resistance mechanisms.
By inducing ciprofloxacin resistance in C. hyointestinalis, also the nalidixic acid MIC
values increased, indicating significant cross-resistance between these two agents. Highlevel
ciprofloxacin resistance (> 32 mg/l) in C. hyointestinalis was associated with the
same Thr86-Ile mutation in QRDR as in C. jejuni, but QRDR mutations did not explain
resistance at lower ciprofloxacin levels.
An efflux pump system, CmeABC, has been established to play an important role in
the resistance of C. jejuni and C. coli to various antimicrobial agents. When the effect of
efflux pump effectors on antimicrobial susceptibility of C. jejuni and C. coli was
evaluated, the efflux pump inhibitor PAβN emerged as the most potent. It increased the
susceptibility of C. jejuni and C. coli to erythromycin and rifampicin 8- to 32-fold and 8-
to 64-fold, respectively. Another inhibitor, NMP, decreased erythromycin, rifampicin and
tetracycline MIC values of C. jejuni and C. coli 1- to 8-fold, 2- to 8-fold and 1- to 4-fold,
respectively. The different results produced by these two inhibitors indicate a different
mode of function for these pump inhibitors. Of the two tested efflux pump inducers,
sodium salicylate produced a modest effect in the MIC values for ciprofloxacin and
rifampicin, and bile salt had no observable effect on the MIC values of the studied
antimicrobials, nor did it have any observable impact on the mutation frequencies of C.
jejuni and C. coli strains.
Since ciprofloxacin and erythromycin resistance in C. jejuni and C. coli involve a
mutation in a target gene, the mutation frequencies of C. jejuni and C. coli strains were
determined. The mutation frequency values differed strongly between different strains,
from hypomutable to strongly hypermutable. The relatively large proportion (25%) of
hypermutable strains may facilitate the adaptation of C. jejuni and C. coli to selective
environments. No correlation was seen between the mutation frequency values and
mutations in the QRDR or the promoter binding region of cmeABC after ciprofloxacin
The emergence of QRDR mutations was evaluated by subjecting originally
ciprofloxacin-susceptible C. jejuni strains to low-level (0.125 or 1 mg/l) ciprofloxacin
concentration and determining the changes in ciprofloxacin MIC values and alterations in
the QRDR sequence data. After DNA extraction and sequencing of the QRDR of the
ciprofloxacin-exposed strains, multiple peaks were observed at nucleotide positions 256
and 267 in the QRDR-sequencing chromatograms, possibly reflecting different QRDR
subpopulations. However, some variants produced ciprofloxacin MIC levels of up to
16 mg/l, even though no peaks corresponding to mutated nucleotides in the QRDR were
observed; this suggests the presence of a QRDR-independent resistance mechanism. In
subsequent persistence studies, different QRDR subpopulations were able to persist and
co-exist in both the presence and absence of low-level ciprofloxacin concentration;
however, the QRDR-independent resistance mechanism was quickly replaced by QRDR
mutations in the persistence of ciprofloxacin challenge.
This study was carried out at the Department of Food and Environmental Hygiene,
Faculty of Veterinary Medicine, University of Helsinki. The work was funded by the
Finnish Graduate School on Applied Biosciences, the Walter Ehrström Foundation and the
Academy of Finland.
My gratitude is due to my supervisor, Professor Marja-Liisa Hänninen, for her time
and guidance. Without her support and trust, this thesis may never have been written.
Professor Séamus Fanning and Doctor Axel Cloeckaert are acknowledged for
reviewing my thesis and Carol Pelli for editing the English language of the manuscript.
I gratefully acknowledge Professor Hannu Korkeala, the Head of the Department of
Food and Environmental Hygiene, for providing high-standard research facilities.
My sincere thanks to all of my colleagues at the department for creating an enjoyable
and relaxed working atmosphere. Special thanks to Urszula Hirvi and Anneli Luoti for
their help in the laboratory, and to Johanna Seppälä for her much needed help with the
I also thank Doctor Rauni Kivistö for her friendship as well as practical assistance, and
Professor Hilpi Rautelin for supporting my work as well as co-writing one of the articles.
A sincere thank you to Doctor Heidi Hyytiäinen for reviewing and giving valuable
comments on this thesis. It was a pleasure working with you, even if for only a short
A heartfelt thanks to my family for bringing so much joy and love into my life. Thank
you Jani, for being an amazing husband and a father to our gorgeous girls, Pinja and Isla. I
am also obliged to my mother and sister, two strong women who brought me up and to
whom I know I can always turn.
Finally, I dedicate this thesis to the memory of my grandmother, Liisa Nurmento, who
by her example taught me the real values of life.
List of original publications 9
1 Introduction 10
2 Review of the literature 12
2.1 Campylobacter spp. 12
2.2 Antimicrobial agents 14
2.2.1 Ciprofloxacin 17
2.2.2 Erythromycin 17
2.2.3 Tetracycline 18
2.3 Multiple drug resistance in Campylobacter 19
2.3.1 Efflux systems and efflux pump effectors 19
2.3.2 Role of the membrane proteins in Campylobacter resistance 21
2.4 Specific resistance mechanisms in Campylobacter 21
2.4.1 Resistance to fluoroquinolones 21
2.4.2 Resistance to macrolides 22
2.4.3 Resistance to tetracyclines 23
2.4.4 Resistance to other antimicrobial agents 23
2.5 Antimicrobial resistance in Campylobacter spp. 24
2.5.1 Intrinsic resistance 24
2.5.2 Incidence of resistance 25
2.5.3 Fitness and pathogenicity of resistant Campylobacter 28
2.5.4 Development of resistance 29
3 Aims of the study 31
4 Materials and methods 32
4.1 Bacterial strains and growth conditions 32
4.2 Antimicrobial agents and efflux pump effectors 33
4.3 Antimicrobial susceptibility testing 34
4.4 Estimation of mutation frequencies 35
4.5 DNA extraction, purification and sequencing 36
4.6 Development of mutations after ciprofloxacin challenge 37
5 Results 38
5.1 Susceptibility of C. hyointestinalis to antimicrobial agents 38
5.2 QRDR sequence analysis of C. hyointestinalis 39
5.3 Mutation frequency of C. jejuni and C. coli 40
5.4 cmeR–cmeA sequence analysis of C. jejuni and C. coli 41
5.5 Effect of efflux pump effectors on MIC values 42
5.6 Effect of ciprofloxacin in the development of gyrA mutations 44
6 Discussion 47
6.1 Susceptibility profile of Finnish C. hyointestinalis strains 47
6.2 Quinolone resistance mechanisms in C. hyointestinalis 48
6.3 Spontaneous mutation frequency of C. jejuni and C. coli 49
6.4 cmeR-cmeA sequence analysis of C. jejuni and C. coli 50
6.5 Effect of efflux pump effectors 51
6.5.1 Inhibitors 51
6.5.2 Inducers 52
6.6 Development and emergence of QRDR mutations 53
7 Conclusions 55
8 References 57
AAC aminoglycoside acetyltransferase
AAD aminoglycoside adenyltransferase
APH aminoglycoside phosphotransferase
ATCC American Type Culture Collection
bp base pair
cfu colony forming unit
CLSI Clinical and Laboratory Standards Institute
DDD/1000 inh/day defined daily dose/1000 inhabitants/day
IR inverted repeat
MDR multiple drug resistance
MIC minimum inhibitory concentration
MOMP major outer membrane protein
PCR polymerase chain reaction
QRDR quinolone resistance-determining region
RND resistance nodulation cell division
List of original publications
This thesis is based on the following original articles referred to in the text by Roman
numerals I to IV:
I Laatu M, Rautelin H, Hänninen ML. 2005. Susceptibility of
Campylobacter hyointestinalis subsp. hyointestinalis to antimicrobial agents
and characterization of quinolone-resistant strains. Journal of Antimicrobial
II Hänninen ML, Hannula M. 2007. Spontaneous mutation frequency and
emergence of ciprofloxacin resistance in Campylobacter jejuni and
Campylobacter coli. Journal of Antimicrobial Chemotherapy 60:1251-7.
III Hannula M, Hänninen ML. 2008. Effect of putative efflux pump inhibitors
and inducers on the antimicrobial susceptibility of Campylobacter jejuni and
Campylobacter coli. Journal of Medical Microbiology 57:851-5.
IV Hannula M, Hänninen ML. 2008. Effects of low-level ciprofloxacin
challenge in the in vitro development of ciprofloxacin resistance in
Campylobacter jejuni. Microbial Drug Resistance 14:197-201.
The original articles have been reprinted with the permission from their copyright holders:
British Society for Antimicrobial Chemotherapy (I and II), Society for General
Microbiology (III), Mary Ann Liebert, Inc. (IV). In addition, some unpublished material is
Campylobacter species are small, spirally curved and highly motile bacteria that are
commonly found in most animal species. Twenty-one Campylobacter species have
currently been validly characterized, the most important pathogenic species being
Campylobacter jejuni and to a lesser extent Campylobacter coli (Euzéby, 1997;
http://www.bacterio.cict.fr/, accessed December 2009). In humans, they cause illness,
campylobacteriosis, which is the most common cause of human gastroenteric infection in
industrialized countries and is responsible for diarrhoea in an estimated 400–500 million
people globally each year (Allos, 2001; Ruiz-Palacios, 2007). The role of other
Campylobacter species, such as Campylobacter hyointestinalis, in human and animal
disease may be greatly underestimated due to difficulties in cultivating these fastidious
and sensitive bacteria. While their impact on disease burden has earlier been thought to be
negligible, improved identification methods are likely to increase the detected numbers of
these rarer Campylobacter species (Logan et al., 2001).
Some of the Campylobacter species (see Table 1) are zoonotic pathogens, and humans
most often become infected by ingesting contaminated food or water. The most common
sources are raw or uncooked meat, especially poultry, contact with animals, unpasteurized
milk and contaminated drinking water (Harris et al., 1986; Adak et al., 1995; Olson et al.,
2008). Cases are usually sporadic, although outbreaks do occur as a result of, for example,
contamination of drinking water. Infection has been experimentally induced with as few as
500 organisms (Robinson, 1981). After an incubation period of 1–5 days, symptoms,
including diarrhoea, abdominal pain and fever, appear. Campylobacteriosis is normally a
self-limiting disease, and the symptoms usually resolve in a week. In some cases, lateonset
complications may occur, namely reactive arthritis (in 1- to 5% of Campylobacterinfected
patients) (Pope et al., 2007) and Guillain-Barré syndrome (in 0.01–0.03% of
Campylobacter enteritis patients) (McCarthy and Giesecke, 2001; Tam et al., 2006).
In severe cases, patients are treated with antimicrobial agents (Allos, 2001). The most
common antimicrobial agents for treating Campylobacter infections are macrolides, such
as erythromycin, and fluoroquinolones, such as ciprofloxacin (Blaser and Engberg, 2008).
However, resistant Campylobacter strains have recently emerged at an elevated rate due,
at least partly, to the large-scale use of these agents in food production animals (Blaser and
Although a number of different classes of antimicrobials exist, bacteria possess and are
able to acquire many different mechanisms that allow them to evade the action of these
substances. The rate at which the pharmaceutical industry has been introducing new
antibiotics has been inadequate to guarantee successful therapy against all pathogenic
bacterial strains. Understanding of the mechanisms underlying bacterial resistance could
lead to the development of new agents to challenge bacterial resistance mechanisms.
The aim of this thesis was to study the various resistance mechanisms of C. jejuni, C.
coli and C. hyointestinalis towards antimicrobial agents. Special attention was given to
ciprofloxacin, whose role as a key antimicrobial in the treatment of campylobacteriosis
has lately been compromised due to the rapid and forceful emergence of ciprofloxacinresistant
Specifically, the susceptibility profile of Finnish C. hyointestinalis strains was established
and the effect of antimicrobial use in the host animals was discussed. Differences in
quinolone resistance between C. hyointestinalis and C. jejuni were also investigated. The
predisposition of C. jejuni and C. coli to develop resistance was investigated by
establishing mutation frequencies of these species. The effect of low-level ciprofloxacin
exposure and the development of resistance in C. jejuni and C. coli were evaluated by
observing alterations, variation and development of mutations in the gene fragment coding
for the ciprofloxacin target site subsequent to ciprofloxacin challenge. Finally, the action
of efflux pump effectors on the susceptibility of C. jejuni, C. coli and C. hyointestinalis
towards several antimicrobials was elucidated to assess the efflux mechanism in these
species and to evaluate the potential of the putative efflux pump inhibitors in therapy.
2 Review of the literature
2.1 Campylobacter spp.
Bacteria with Campylobacter-like morphology were first isolated from domestic animals
about a century ago (McFadyean and Stockman, 1913), but it was not until the 1970s that
these bacteria began to become more widely recognized as human pathogens (Dekeyser et
al., 1972, Skirrow, 1977). Today, Campylobacter are recognized as the most important
cause of bacterial gastrointestinal infection in humans. In 2008, there were 4453
laboratory-confirmed cases of Campylobacter infection in Finland (National Infectious
Disease Registry, National Institute for Health and Welfare, Finland,
http://www3.ktl.fi/stat/). Approximately 70–80% of these infections were acquired abroad
Campylobacter species belong to the epsilon class of proteobacteria. Twenty-one
validly named Campylobacter species currently exist (Euzéby, 1997;
http://www.bacterio.cict.fr/, accessed December 2009). Members of the family
Campylobacteraceae are Gram-negative, curved or spiral rods, that are 0.2–0.8 µm wide
and 0.5–5 µm long (Debruyne et al., 2008). They require a microaerobic growth
environment and are typically motile with a single unsheathed flagellum at one or both
ends of the cell (Debruyne et al., 2008). Campylobacter spp. have been isolated from a
large variety of different animals, and each Campylobacter species seems to favour certain
Campylobacter jejuni has been isolated from many different domestic and wild
animals, where it is considered a harmless commensal of the gastrointestinal tract,
although occasionally it has been associated with animal diseases (Euzéby, 1997;
http://www.bacterio.cict.fr/, accessed December 2009). The avian gut is a particularly
preferred reservoir for C. jejuni because it possesses optimal growth conditions such as a
temperature of 42°C. In humans, C. jejuni is responsible for the great majority of enteric
Campylobacter infections (80–90%) (Fitzgerald et al., 2008). Since broiler chickens are
often colonized with C. jejuni (see Table 6), eating or handling undercooked chicken meat
is considered one of the most important risk factors for human campylobacteriosis (Olson
et al., 2008).
Campylobacter coli is responsible for an estimated 5–10% of Campylobacter
infections (Fitzgerald et al., 2008). C. coli and C. jejuni are closely related and often
difficult to differentiate (Debruyne et al. 2008). Like C. jejuni, C. coli can be isolated from
many animal species, foods and environmental samples. The primary reservoir for C. coli
is pigs, up to 100% of which can be C. coli-positive (Saenz et al., 2000).
Some other Campylobacter species have also been associated with human disease
(Table 1), but in most studies the growth conditions are optimized for the selection of C.
jejuni and C. coli, leaving other Campylobacter species, including Campylobacter
hyointestinalis, often undetected and their actual prevalence underestimated.
Table 1. Campylobacter species associated with human disease (Euzéby, 1997;
http://www.bacterio.cict.fr/, accessed December 2009)
Source Human disease Animal disease
C. coli Pigs and other Gastroenteritis,
domestic animals septicaemia, abortion abortion
C. concisus* Humans Periodontal disease,
C. curvus* Humans Periodontal disease,
C. fetus Cattle, sheep Septicaemia, systemic
C. gracilis* Humans Periodontal disease, deep
empyema, ischial wound
C. hyointestinalis Cattle, pigs, deer,
C. jejuni Humans, poultry and Gastroenteritis,
other animals septicaemia, meningitis, hepatitis,
C. lari* Poultry and other Gastroenteritis,
C. rectus* Humans Periodontal disease,
abscesses, reflux disease,
C. showae* Humans Periodontal disease Not known
C. sputorum* Domestic mammals,
Abscesses, gastroenteritis Not known
C. upsaliensis Dogs, cats, humans Gastroenteritis, abortion,
* Pathogenic role unclear
C. hyointestinalis has been commonly isolated from many animals, notably cattle; 32% of
faecal samples tested positive for C. hyointestinalis in a study by Atabay and Corry
(1998), and 17.2% tested positive in a study by Inglis et al. (2005). In Finland, 15.3% of
bovine faecal samples and 6% of reindeer faecal samples have tested positive for this
species (Hänninen et al., 2002; Hakkinen and Hänninen, 2009). C. hyointestinalis has also
been associated with enteritis in pigs (Gebhart et al., 1985a), deer (Hill et al., 1987), cattle
(Diker and Ozlem, 1990) and humans (Fennell et al., 1986; Edmonds et al., 1987; Minet
et al., 1988; Salama et al., 1992; Gorkiewicz et al., 2002). The species C. hyointestinalis
includes two subspecies, namely hyointestinalis and lawsonii. The latter has been isolated
from pigs, but its clinical significance is yet unknown, whereas C. hyointestinalis subsp.
hyointestinalis has been associated with animal and human diarrhoea. At the genome
level, C. hyointestinalis is a very diverse species (On and Vandamme, 1997) and is closely
related to Campylobacter fetus, which is a major cause of septic abortion in domestic
animals and is also implicated in human illnesses (Lastovica and Allos, 2008). In this
thesis, C. hyointestinalis refers to C. hyointestinalis subsp. hyointestinalis.
2.2 Antimicrobial agents
Antimicrobial agents are defined as substances able to inhibit or kill micro-organisms.
They can be synthetic or semi-synthetic compounds or antibiotics: substances produced by
other micro-organisms. The major antibacterial classes are listed in Table 2 according to
their mode of function and chemical structure.
Table 2. Major antibacterial classes (Prescott, 2004)
Cell wall inhibitors
Exemplary drugs Target
Protein synthesis inhibitors
Aminoglycosides Gentamicin, streptomycin,
30S ribosomal subunit
Lincosamides Lincomycin, clindamycin 50S ribosomal subunit
Tetracyclines Tetracycline, doxycycline 30S ribosomal subunit
Macrolides Erythromycin, tylosin 50S ribosomal subunit
Nucleic acid inhibitors
Chloramphenicol 50S ribosomal subunit
Sulfonamides Trimethoprim, bactrim Pteroate synthetase
Nitroimidazoles Metronidazole Nucleotides/DNaseI
RNA synthesis inhibitors
Ciprofloxacin, nalidixic acid Type II topoisomerases
Rifamycins Rifampicin RNA polymerase
The discovery of penicillin in 1928 by Alexander Fleming was the beginning of modern
antibiotic medicine. Mass production started in the 1940s, and today hundreds of
antimicrobials are available on the market. An estimated half of the globally produced
antimicrobials are used for food animals (World Health Organization, 2002).
Antimicrobials are used in animals as therapeutics, prophylactics and growth promoters.
According to a European survey, in 1997, approximately 100 mg of antimicrobials were
used for every kilogram of meat destined for human consumption, and almost all
antimicrobials used in animals were identical or related to substances used in human
medicine (Committee for Veterinary Medicinal Products, 1999). Table 3 presents recent
information about the amount of meat produced and antimicrobials used in animals in four
Table 3. Meat production and animal consumption of antimicrobials in four different
European countries in 2007 or 2008
Slaughtered animals for meat
production in 2008 (Eurostat, 2009)
Finland 401 300 17.0 a
Sweden 526 200 16.4 b
Denmark 2 013 600 120.2 c
France 5 623 700 1179 d
a In 2008 (Finnish Medicines Agency, 2009b)
b In 2008 (National Veterinary Institute (SVA), 2009)
c In 2008 (DANMAP 2008, 2009), only food animals
d In 2007 (Chevance and Moulin, 2009)
antimicrobials used in
In 2006, the European Union banned the use of all antibiotics as growth promoters in its
member states. In Finland, the use of antimicrobials in animals has been monitored since
1995, at which time the total consumption was about 18 000 kg. Consumption declined to
its lowest point (about 13 000 kg) in 2003, but has since increased to 16 997 kg in 2008
(Table 3; Finnish Medicines Agency, 2009b). In Finland, β-lactams are the most
commonly used antimicrobials in animals (58%), followed by tetracyclines (18%) and
sulphonamides (17%) (Finnish Medicines Agency, 2009b).
A total of 21.68 defined daily dose/1000 inhabitants/day (DDD/1000 inh/day) of
systemic antibacterials were used in 2008 in human medicine according to Finnish sales
statistics (Finnish Medicines Agency, 2009a). Beta-lactam antibacterials were used most,
9.83 DDD/1000 inh/day, followed by tetracyclines, 4.24 DDD/1000 inh/day (Table 4).
Campylobacteriosis is usually a self-limiting disease, but in certain cases the use of
antimicrobials is justified, e.g. when the disease affects elderly people or people with
underlying illnesses or when the symptoms are prolonged or unusually severe (Allos,
2001). The drug of choice for treating campylobacteriosis is erythromycin, followed by
ciprofloxacin, and alternative therapeutic agents include tetracycline and aminoglycosides
(Blaser and Engberg, 2008; Murray et al., 2009).
Table 4. List of fluoroquinolones, macrolides and tetracyclines on the Finnish market and use of these substances in human and veterinary
medicine in 2008 (Finnish Medicines Agency, 2009a; Finnish Medicines Agency, 2009b)
Fluoroquinolones Macrolides Tetracyclines
Human medicine Veterinary medicine Human medicine Veterinary medicine Human medicine Veterinary medicine
Ciprofloxacin Danofloxacin Azitromycin Spiramycin
Claritromycin Tulatromycin Lymecycline Chlortetracycline
Moxifloxacin Enrofloxacin Erythromycin Tylosin
DDD/1000 inh/day a
90 kg b 1.45
847 kg 4.24
defined daily dose/1000 inhabitants/day
amount of active ingredient
Ciprofloxacin (Figure 1) is structurally a fluoroquinolone antimicrobial. It is a broadspectrum,
synthetic antimicrobial agent commonly used to treat many bacterial infections,
including campylobacteriosis. Nalidixic acid, a predecessor to fluoroquinolone
antimicrobials, belongs to the quinolone class of antimicrobials. Sitafloxacin is a newgeneration
fluoroquinolone that is more effective than ciprofloxacin against C. jejuni
strains, including those with a gyrA mutation (Sato et al., 1992; Lehtopolku et al., 2005),
but its approval in Europe is unlikely because in Caucasians sitafloxacin has been
associated with mild ultraviolet phototoxicity reactions (Dawe et al., 2003). In Finland,
less than 1% of antimicrobials used in animals are fluoroquinolones (Finnish Medicines
Agency, 2009b). The most commonly used fluoroquinolone in animals is enrofloxacin, the
main metabolite of which is ciprofloxacin (Küng et al., 1993). Fluoroquinolones used in
human and veterinary medicine in Finland are presented in Table 4.
Figure 1. Molecular structure of ciprofloxacin
Ciprofloxacin acts by inhibiting DNA synthesis. In a living bacterial cell, DNA is in a
negatively supercoiled form. These negative supercoils are removed by a topoisomerase II
DNA gyrase, and the daughter chromosomes are separated by topoisomerase IV, allowing
replication and transcription to take place (Drlica and Zhao, 1997). Quinolones act by
binding these topoisomerases, and in Gram-negative bacteria the quinolones interact
primarily with gyrase and DNA, thus inhibiting supercoiling and possibly forming a
cytotoxic complex leading to cell death (Maxwell, 1992).
Erythromycin is a macrolide antibiotic, produced from a strain of Saccharopolyspora
erythraea. Its molecular structure is provided in Figure 2. Macrolides used in human and
veterinary medicine are presented in Table 4. Macrolides and lincosamides represented
about 5% of antimicrobials used in animals in Finland in 2008 (Finnish Medicines
Figure 2. Molecular structure of erythromycin
The mode of action of erythromycin is inhibition of protein synthesis by binding to the
23S rRNA in the 50S ribosomal subunit (Schlunzen et al., 2001). This sterically hinders
the binding of the peptidyl transfer RNA, which results in inhibition of translocation, thus
halting the elongation of the developing peptide chain (Brisson-Noel et al., 1988).
Erythromycin is the drug of choice for treating campylobacteriosis. It is a broad-spectrum,
fairly inexpensive and well-tolerated antibiotic that is effective against Campylobacter
spp. when administered during the first days of illness (Salazar-Lindo et al., 1986).
Tetracycline (Figure 3) is a broad-spectrum antibiotic produced by bacteria in the genus
Streptomyces. Tetracyclines used in human and veterinary medicine in Finland are
presented in Table 4. Tetracyclines represent about 18% of the antimicrobials used in
animals (Finnish Medicines Agency, 2009b), but in many countries, such as France, it is
the most commonly used antimicrobial class in animals (Chevance and Moulin, 2009).
Figure 3. Molecular structure of tetracycline
Tetracycline binds to Mg
2+ cations in order to pass through outer membrane porins of
Gram-negative bacteria. In the periplasmic space, tetracycline dissociates from
magnesium and moves passively into the cytoplasm (Chopra and Roberts, 2001).
Tetracycline acts by binding to discrete sites on the ribosomal 30S subunit (Chopra et al.,
1992). Its primary antimicrobial effect takes place by direct steric hindrance by binding to
the A site in the 30S subunit, thus hindering the movement of transfer RNA (Harms et al.,
2.3 Multiple drug resistance in Campylobacter
According to definition, a bacterial strain can be considered resistant to a certain
antimicrobial agent if it is able to propagate in higher concentrations in that agent than
other strains of the same species or genus (Guardabassi and Courvalin, 2006). The
resistance level of a particular strain can be established by determining the lowest drug
concentration that completely inhibits the growth of the strain (minimal inhibitory
Multiple drug resistance (MDR) in bacteria is associated with efflux systems and
structural components of the cell membrane. These mechanisms control the transport of
structurally unrelated substances in and out of the cell.
2.3.1 Efflux systems and efflux pump effectors
Energy-dependent drug efflux pumps are used in bacteria for extruding metabolites and
toxic compounds, including antimicrobial agents (Li and Nikaido, 2004). In C. jejuni,
several putative efflux systems have been identified (Parkhill et al., 2000; Ge et al., 2005).
Of these, two systems have been characterized as conferring MDR resistance, namely
CmeABC and CmeDEF (Lin et al., 2002; Pumbwe and Piddock, 2002; Pumbwe et al.,
Homologous CmeABC system has also been characterized in C. coli (Corcoran et al.,
2005; Ge et al., 2005), and identified in C. lari, C. upsaliensis and C. fetus (Guo et al.,
2010). CmeABC is coded by an operon consisting of three genes, cmeA, cmeB and cmeC,
which code for a periplasmic fusion protein, an inner membrane drug transporter and an
outer membrane protein, respectively (Figure 4) (Lin et al., 2002). CmeB belongs to the
resistance nodulation cell division (RND) family of efflux transporters (Pumbwe and
Piddock, 2002). Inactivation of the CmeABC efflux pump by insertional inactivation of
cmeB or with efflux pump inhibitors leads to increased susceptibility to a variety of
antibiotics, bile acids and other toxic substances, including those to which Campylobacter
are intrinsically resistant, showing that CmeABC plays a key role in both intrinsic and
acquired resistance of Campylobacter (Lin et al., 2002; Pumbwe and Piddock, 2002;
Pumbwe et al., 2004; Pumbwe et al., 2005; Akiba et al., 2006). CmeABC is regulated by
CmeR, which represses the transcription of cmeABC by binding to an inverted repeat
sequence in the 97 bp intergenic (IT) region between cmeR and cmeA (Figure 4) (Lin et
al., 2005a). Upregulation of CmeABC has been associated with inactivation of the cmeR,
substitutions in the substrate binding region of CmeR, a deletion or a mutation in the
inverted repeat sequence of the promoter binding region of the cmeABC and competitive
binding of bile salts to the promoter region of the cmeABC (Pumbwe et al., 2004; Lin et
al., 2005a; Lin et al., 2005b; Cagliero et al., 2007).
Another RND system, CmeDEF, has been identified in C. jejuni. It has a narrower
substrate profile and transports, for instance, bile acids and ampicillin, but not
ciprofloxacin, erythromycin or tetracycline (Pumbwe et al., 2005; Akiba et al., 2006). In a
study comparing CmeABC and CmeDEF systems, cmeF was expressed at a much lower
level than CmeABC, but evidence exists of an interaction between these two efflux
systems that promotes viability and resistance of C. jejuni in a hostile environment (Akiba
et al., 2006).
cmeA cmeB cmeC
Figure 4. Genomic organization of the cmeABC operon in C. jejuni strain 81-176. A
part of the sequence of the intergenic (IT) region between cmeR and cmeA is shown.
The start codon (ATG) of cmeA is underlined and the inverted repeat is in bold.
Since efflux pump systems have a key role in the antimicrobial resistance of C. jejuni and
C. coli (Lin et al., 2002; Corcoran et al., 2005), inhibition of efflux would provide an
important means for preventing emerging resistance and multidrug resistance in bacteria.
To achieve this inhibition, synthetic and natural compound libraries have been screened
with the aim of finding substances capable of reversing the action of the efflux pumps.
After screening for efflux pump inhibitors and subsequently optimizing the properties of a
potential compound, Renau et al. (1999) were able to produce a substance called phenylarginine-β-naphthylamide
(PAβN), a broad-spectrum efflux pump inhibitor. It was first
characterized in Pseudomonas aeruginosa (Lomovskaya et al., 2001) and later studied in
several bacteria, including C. jejuni and C. coli (Payot et al., 2004; Cagliero et al., 2005;
Gibreel et al., 2007).
Similarly, by screening of an N-heterocyclic organic compound library for multidrug
reversal activity, Bohnert and Kern (2005) were able to characterize another putative
efflux pump inhibitor, 1-(1-naphthylmethyl)-piperazine (NMP), in Escherichia coli. It has
been shown to partially reverse multidrug resistance in some members of the
Enterobacteriaceae and in Acinetobacter baumannii (Bohnert and Kern, 2005; Pannek et
al., 2006; Schumacher et al., 2006).
Salicylate is a non-steroidal anti-inflammatory pharmaceutical agent and the active
component of aspirin, a prostaglandin synthesis inhibitor drug, commonly taken to relieve
fever and discomfort associated with infections as well as to prevent vascular diseases
(Weissmann, 1991). It is therefore of concern that salicylate has been associated with
increased resistance to multiple antibiotics in several bacteria, such as E. coli and
Salmonella serovar Typhimurium (Price et al., 2000). In E. coli, salicylic acid binds to a
repressor protein, MarR, which leads to over-expression of the global regulatory gene
marA. Subsequent to this, the outer membrane protein OmpF is down-regulated and the
synthesis of the RND multidrug efflux pump AcrAB-TolC increases, which eventually
results in decreased antimicrobial accumulation (Price et al., 2000).
Bile salts are antimicrobial compounds naturally present in the animal intestine. Multidrug
efflux pumps, such as CmeABC, contribute to the intrinsic resistance of C. jejuni to bile
salts (Lin et al., 2003). Lin et al. (2005b) have shown that bile salts increase the
expression of cmeABC in C. jejuni by preventing the binding of the transcriptional
repressor CmeR, and therefore, bile salts could be considered efflux pump inducers.
2.3.2 Role of the membrane proteins in Campylobacter resistance
Drugs can cross the bacterial membrane and enter the cell by using porin channels or by
diffusing through the lipid bilayer. Porin channels are postulated to be mainly used by
relatively small drugs, such as β-lactams, tetracycline, chloramphenicol and
fluoroquinolones, whereas large molecules, such as macrolides, diffuse slowly across the
lipid bilayer (Nikaido, 2003). In Campylobacter, two outer membrane porins have been
characterized: in C. jejuni and C. coli, the major outer membrane porin (MOMP) protein,
encoded by porA, and in C. jejuni Omp50 (Pagès et al., 1989; Bolla et al., 2000). Very
limited functional information is available about these porins, and thus far, no evidence
has emerged that they play any role in MDR of Campylobacter (Pumbwe et al., 2004).
2.4 Specific resistance mechanisms in Campylobacter
2.4.1 Resistance to fluoroquinolones
Two known resistance mechanisms that confer resistance in C. jejuni and C. coli to
fluoroquinolones are point mutations in the quinolone-resistance determining region
(QRDR) of gyrA and the function of the multidrug efflux pump CmeABC.
The target for ciprofloxacin activity in C. jejuni is gyrase, which comprises two
subunits encoded by gyrA and gyrB (Higgins et al., 1978). A single mutation in gyrA is
able to confer resistance to ciprofloxacin by reducing the affinity of gyrases to quinolones.
The most commonly found mutation in ciprofloxacin-resistant C. jejuni strains is the
Thr86-Ile mutation, which results from transition of cytosine to thymine at nucleotide 256,
conferring high-level resistance to ciprofloxacin (Charvalos et al., 1996; Mazzariol et al.,
2000; Zirnstein et al., 2000; Hakanen et al., 2002b; Piddock et al., 2003). Other reported
mutations include Thr86-Lys, Thr86-Ala, Ala70-Thr, Pro104-Ser, Asp90-Asn, Asp90-Tyr
and transitions at codon 119 (Charvalos et al., 1996; Ruiz et al., 1998; Zirnstein et al.,
1999; Bachoual et al., 2001; Hakanen et al., 2002b; Yan et al., 2006). These mutations are
less frequently observed and are associated with intermediate-level fluoroquinolone
In some instances, QRDR mutations have not been detected in ciprofloxacin-resistant C.
jejuni (Hakanen et al., 2003; Piddock et al., 2003; Pumbwe et al., 2004), suggesting
another mechanism for ciprofloxacin resistance. Studies with mutant strains where the
CmeABC efflux pump was inactivated by a cmeB insert showed a 4- to 8-fold increase in
ciprofloxacin susceptibility, and accumulation studies clearly indicated an increased
accumulation of ciprofloxacin compared with the wild-type parent strain (Lin et al., 2002;
Pumbwe and Piddock, 2002). Experiments where MIC values well below the level of
clinical significance were often obtained by insertional inactivation of cmeB in originally
resistant C. jejuni strains possessing various gyrA, suggest the existence of synergism
between these two resistance mechanisms, namely efflux and target mutations (Luo et al.,
2.4.2 Resistance to macrolides
Macrolide resistance in C. jejuni and C. coli involves two mechanisms, namely target
modification and efflux.
Mutations in 23S rRNA block the interaction of macrolide drugs with the 50S
ribosomal subunit, and thus, confer macrolide resistance. Specifically, mutations at base
positions 2074 and 2075 are associated with high-level macrolide resistance in C. jejuni
and C. coli (Jensen and Aarestrup, 2001; Vacher et al., 2003; Gibreel et al., 2005; Mamelli
et al., 2005). Mutations at these positions are likely to block the binding of macrolides to
their inhibitory site on the 23S ribosomal subunit (Pfister et al., 2004). The predominant
mutation in erythromycin-resistant strains is A2075G (Gibreel and Taylor, 2006), and in
some strains these two mutations have been found to co-exist (Vacher et al., 2003; Vacher
et al., 2005).
The chromosome of C. jejuni and C. coli contains three copies of the 23S rRNA gene
(Fouts et al., 2005). In erythromycin-resistant strains, generally all copies carry macrolide
resistance-associated mutations, but the co-existence of wild-type alleles does not seem to
affect the resistance level (Jensen and Aarestrup, 2001; Gibreel et al., 2005).
In addition to 23S RNA mutations, mutations in ribosomal proteins L4 (G74D) and
L22 (insertions at position 86 or 98) have been implicated in conferring macrolide
resistance to C. jejuni and C. coli (Cagliero et al., 2006b).
The involvement of an efflux system in the macrolide resistance of C. jejuni and C.
coli has been demonstrated in studies using the efflux pump inhibitor β-naphtylamide
(PAβN) (Lomovskaya et al., 2001; Payot et al., 2004; Cagliero et al., 2005; Martinez and
Lin, 2006; Gibreel et al., 2007) and using insertional inactivation of the cmeB gene (Payot
et al., 2004; Cagliero et al., 2005; Gibreel et al., 2007). In C. coli isolates with low-level
erythromycin resistance (MICs 8–16 mg/l), no mutations have been detected in the target
gene (Payot et al., 2004), and in these isolates the inactivation of CmeABC leads to
restored susceptibility to erythromycin, suggesting the involvement of CmeABC in the
intrinsic resistance of Campylobacter (Cagliero et al., 2005; Lin et al., 2007). In isolates
with a high erythromycin resistance level (MIC > 128 mg/l), the resistance is associated
with a mutation in the 23S rRNA gene (Payot et al., 2004). In these isolates, the
inactivation of CmeABC leads to 2- to 4-fold decrease in erythromycin resistance,
implying synergistic action with the target mutations in achieving acquired macrolide
resistance (Cagliero et al., 2005; Cagliero et al., 2006a; Lin et al., 2007).
In a study by Lin et al. (2005a), a deletion in the promoter region of the cmeABC operon
was spontaneously obtained by selection on a ciprofloxacin-containing medium. This
resulted in increased resistance to several antimicrobials, including erythromycin,
suggesting a possible role of promoter region mutants in the development of
erythromycin-resistant C. jejuni strains.
2.4.3 Resistance to tetracyclines
Tetracycline resistance in C. jejuni and C. coli involves ribosomal protection and efflux
The ribosomal protection is mediated by a protection protein termed Tet(O)
(Manavathu et al., 1988) that is structurally related to translational ribosome-binding
proteins (Connell et al., 2003). Tet(O) is coded by a gene located on plasmids of different
sizes and in some strains chromosomally (Lee et al., 1994; Gibreel et al., 2004). Several
studies have demonstrated that tet(O)-carrying plasmids can be transferred via conjugation
between C. jejuni and C. coli strains in vivo and in vitro (Avrain et al., 2004; Batchelor et
al., 2004; Gibreel et al., 2004; Pratt and Korolik, 2005). Tet(O) acts by removing
tetracycline bound to the A-site in the ribosomal 30S subunit (Connell et al., 2002).
Specifically, Tet(O) induces a conformational change, which is functionally opposite to
that of tetracycline and which allosterically distorts the tetracycline-binding site, resulting
in the release of tetracycline (Connell et al., 2003). Even after the Tet(O) has left the
ribosome, the induced conformation persists, thus preventing rebinding of the tetracycline
(Connell et al., 2003).
The multidrug efflux pump CmeABC has been implicated in intrinsic and acquired
tetracycline resistance (Lin et al., 2002; Pumbwe and Piddock, 2002; Gibreel et al., 2007).
Insertional inactivation of the CmeABC pump in a tetracycline-susceptible strain led to an
8-fold increase in susceptibility, and in tetracycline-resistant isolates to a 16- to 128-fold
decrease in tetracycline resistance (Lin et al., 2002; Pumbwe and Piddock, 2002; Gibreel
et al., 2007). Addition of the pump inhibitor PAβN reduced tetracycline resistance less
than 2-fold (Gibreel et al., 2007).
2.4.4 Resistance to other antimicrobial agents
Aminoglycosides act by binding to the decoding region in the A-site of the ribosomal 30S
subunit. This interaction results in aberrant proteins by interfering with accurate codonanticodon
recognition and in disruption of elongation of nascent proteins by inhibiting the
translocation of tRNA from the A-site to the P-site (Jana and Deb, 2006). Aminoglycoside
resistance is mediated by enzymatic modification that diminishes affinity of
aminoglycosides for the rRNA A-site (Llano-Sotelo et al., 2002). These enzymes fall into
three classes: aminoglycoside acetyltransferases (AAC), aminoglycoside
adenyltransferases (AAD) and aminoglycoside phosphotransferases (APH), each of which
has its own characteristic modification sites and substrates (Engberg et al., 2006). APH is
the most common enzyme found in C. jejuni and C. coli (Taylor et al., 1988; Tenover et
β-lactam molecules enter Campylobacter through outer membrane porin channels.
Once inside, β-lactams bind to enzymes involved in cell wall synthesis, preventing crosslinking
of the peptidoglycan network of the cell wall and resulting in the swelling and
eventually bursting of the bacterial cell (Siu, 2002). While Campylobacter spp. are
generally inherently resistant to many β-lactams, they remain susceptible to, for example,
amoxicillin and ampicillin (Tajada et al., 1996). According to the study of Pagès et al.
(1989), the pores of porins in C. jejuni and C. coli are cation-selective and so small that
most of the large, anionic β-lactams are unable to diffuse through them. A vast majority of
C. jejuni and C. coli isolates are also able to produce β-lactamases, which inactivate the βlactam
molecule by hydrolysing the structural lactam ring (Sykes and Matthew, 1976;
Tajada et al., 1996). A third mechanism for β-lactam resistance in C. jejuni is the action of
efflux pumps. Several recent studies have demonstrated a significant increase in
susceptibility to ampicillin in CmeABC-inactivated C. jejuni mutants and a decrease in
susceptibility in CmeABC-overexpressing mutants (Lin et al., 2002; Pumbwe and
Piddock, 2002; Pumbwe et al., 2005), but this phenomenon was less pronounced in
ampicillin-resistant and β-lactamase-positive strains (Lin et al., 2002).
Chloramphenicol inhibits bacterial protein biosynthesis by preventing peptide chain
elongation. It binds reversibly to the peptidyltransferase centre at the 50S ribosomal
subunit (Schlunzen et al., 2001). Chloramphenicol resistance is conferred by a plasmidcarried
cat gene that encodes acetyltransferase, which modifies chloramphenicol in a way
that prevents it from binding to ribosomes (Schwarz et al., 2004). Although
chloramphenicol resistance in Campylobacter is rare, a plasmid-carried chloramphenicol
resistance gene has been reported in C. coli (Wang and Taylor, 1990).
2.5 Antimicrobial resistance in Campylobacter spp.
2.5.1 Intrinsic resistance
C. jejuni, C. coli and C. hyointestinalis are considered intrinsically resistant to at least
polymyxin B, bacitracin, novobiocin, vancomycin, certain cephalosporins and penicillins,
rifampicin, trimethoprim and cycloheximide (Gebhart et al., 1985b; Ng et al., 1985).
Some of these agents are often included in Campylobacter selective media to inhibit the
growth of other bacteria, but it must be noted that not all Campylobacter species have the
same intrinsic resistance profile. For example, C. hyointestinalis is intrinsically
cephalothin-resistant and nalidixic acid-resistant, whereas C. jejuni and C. coli are
nalidixic acid-susceptible and cephalothin-resistant, and Campylobacter upsaliensis is
susceptible to both of these agents and Campylobacter lari is resistant to both of these
agents (Lastovica and Allos, 2008).
Intrinsic resistance arises from insensitivity of a bacterial species, due to a structural or
functional characteristic, to a particular antimicrobial agent. This insensitivity in
Campylobacter may be due to the following:
(1) low affinity of the antimicrobial agent for the bacterial target (e.g. trimethoprim,
(2) inability of the antimicrobial agent to cross the outer membrane (e.g. certain β-lactams)
(3) extrusion of the antimicrobial by the bacterial efflux system (e.g. rifampicin,
polymyxin B, certain cephalosporins, novobiocin)
(4) bacterial production of enzymes capable of inactivating the antimicrobial (e.g. βlactamases)
(Tajada et al., 1996; Gibreel and Skold, 1998).
2.5.2 Incidence of resistance
Geographical differences in resistance profiles of C. jejuni are significant; however,
resistance to antimicrobial agents of clinical importance has clearly increased over the last
few decades. For example, in a Canadian study of human C. jejuni samples, the frequency
of tetracycline resistance in 1980 was 6.5% and in 1999–2002 it had reached 50% (Gibreel
et al., 2004). Similarly, in an American study fluoroquinolone resistance in human C.
jejuni samples was found to increase from 0% in 1995 to 40.5% in 2001 (Nachamkin et
al., 2002) and in a Dutch study from 0% in 1982 to 32% in 2002 (van Hees et al., 2007).
In all of the above-cited studies, erythromycin resistance, on the other hand, had remained
at a low level (0–5%), although some studies report up to 18% and 60% resistance to
erythromycin in human and chicken C. jejuni and C. coli isolates, respectively (Li et al.,
Several studies have shown greater prevalence for erythromycin resistance in C. coli
than in C. jejuni (Gibreel and Taylor, 2006). This may be explained by the pig being a
natural host for C. coli and the chicken for C. jejuni, and macrolides being used more
often on pig farms than on poultry farms. In addition, pigs become more exposed to
macrolides due to their longer production cycles (Lin et al., 2007).
An overview of the studies on resistance prevalence (%) in human C. jejuni and C. coli
samples is presented in Table 5, and in chicken and pig C. jejuni and C. coli isolates in
Table 5. Resistance profiles of four antimicrobial agents for C. jejuni and C. coli isolated from human faecal samples in six different
countries, reported as percentage of resistant strains
Country Isolates tested
C. jejuni C. coli
C. jejuni C. coli
C. jejuni C. coli
C. jejuni C. coli
Finland 1 478 0.2 4.3 0.8 7
Denmark 2 185 1.6 28.1 17.3 0
Taiwan 3 93 10 50 52 75 95 85 1 15
Canada 4 203 0 2 49.8
Germany 5 430 0 29.4 46.2 41.2 38.5 35.3 0 0
France 6 5685 1.4 12.5 25.3 42.0 28.8 53.4 0 0
(Schönberg-Norio et al., 2006), domestic strains only, reported as joint values for C. jejuni and C. coli strains
(DANMAP 2008, 2009), domestic strains only
3 (Li et al., 1998)
4 (Gibreel et al., 2004)
5 (Luber et al., 2003)
6 (Gallay et al., 2007)
Table 6. Percentage of resistant C. jejuni and C. coli isolates isolated from chickens and pigs, respectively, and percentage of Campylobacterpositive
broiler flocks in four different European countries
Resistance to antimicrobials (%)
Ciprofloxacin Erythromycin Tetracycline Streptomycin Gentamicin
C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli
Finland 2 7.1 1.2 8 0 0 0 0 4.9 NA 1.2 0
Sweden 3 12.6 0 30 0 1 0 2 0 57 0 0
Denmark 4 26.8 12.2 7.1 0 15.3 9.8 5.1 4.9 51.0 0 0
France 5 80.2 30.4 55.3 0 13.2 50 78.9 3.6 15.8 0 0
In 2007 (European Food Safety Authority, 2009)
In 2007 and 2008 (FINRES-Vet 2008, 2009; FINRES-Vet 2007, 2008), 81 C. jejuni and 62 C. coli isolates tested
In 2008 (National Veterinary Institute (SVA), 2009), C. coli values are hippurate-negative thermophilic Campylobacter spp. from pigs: 38
C. jejuni and 97 C. coli isolates tested
In 2008 (DANMAP 2008, 2009), 82 C. jejuni and 98 C. coli isolates tested
In 2007 (EFSA, 2009b), C. coli values from chicken: 56 C. jejuni and 76 C. coli isolates tested
2.5.3 Fitness and pathogenicity of resistant Campylobacter
Campylobacter resistance to a particular antimicrobial is often a result of a mutation in the
gene encoding the target site of that antimicrobial. These target sites, such as DNA gyrase
and RNA polymerase, are essential for bacterial propagation, and mutations encoding
these sites may result in reduced fitness of these bacteria in the absence of selective
pressure by antimicrobials (Andersson and Levin, 1999). Contrary to this,
fluoroquinolone-resistant C. jejuni strains showed enhanced fitness and the ability to outcompete
fluoroquinolone-susceptible strains when these two strains were inoculated into
chickens (Luo et al., 2005). This finding is supported by epidemiological studies revealing
the prevalence of fluoroquinolone-resistant C. jejuni and C. coli on chicken farms for at
least 1–4 years after the farms had stopped using fluoroquinolones (Pedersen and
Wedderkopp, 2003; Price et al., 2005). Interestingly, this fitness is not explained by
compensatory mutations, but is directly linked to the Thr86-Ile mutation in gyrA (Luo et
al., 2005). Likewise, tetracycline-resistant C. jejuni strains have shown evidence of
enhanced fitness. Tetracycline resistance in C. jejuni is conferred by the tet(O)-plasmid
obtained by horizontal transfer even in the absence of antimicrobial selection pressure
(Avrain et al., 2004). In studies by Luangtongkum et al. (2008), tetracycline-resistant
Campylobacter strains were able to co-exist or replace tetracycline-susceptible strains on
organic poultry farms.
In contrast to fluoroquinolone- or tetracycline-resistant Campylobacter, erythromycinresistant
Campylobacter are less fit in the absence of the selection pressure, and hence, are
rapidly outcompeted by susceptible wild-type strains (Luangtongkum et al., 2009). Similar
conclusions can be drawn from such countries as Denmark, where by reducing the amount
of macrolides used in food animals the numbers of macrolide-resistant C. coli rapidly
decreased (Aarestrup et al., 2008).
Enhanced fitness of resistant Campylobacter strains causing campylobacteriosis may
have implications for human health. Enhanced fitness of resistant strains is likely to
increase the number of resistant strains also in human clinical samples. In addition, in
some studies fluoroquinolone-resistant Campylobacter strains have been demonstrated to
prolong disease, lengthening the duration of diarrhoea by an average of 3 days, compared
with disease inflicted by fluoroquinolone-susceptible strains (Smith et al., 1999; Engberg
et al., 2004; Nelson et al., 2004). Both quinolone- and macrolide-resistant strains have
been associated with increased risk of invasive illness or death, compared with susceptible
strains. This risk was calculated to be 6-fold greater for quinolone-resistant strains and >5fold
greater for macrolide-resistant strains (Helms et al., 2005). However, the opposite
results have also been obtained, with ciprofloxacin-susceptible isolates being associated
with more severe disease (Feodoroff et al., 2009). In a re-analysis of ciprofloxacinresistant
C. jejuni infections, Wassenaar et al. (2007) concluded that fluoroquinoloneresistant
Campylobacter infections are no more severe than infections with susceptible
2.5.4 Development of resistance
Mutations play a major role in Campylobacter resistance. Several mechanisms have been
reported to contribute to the emergence of these mutations:
Whole-genome analyses have revealed that C. jejuni lack many of the genes encoding
DNA repair molecules present in other bacteria, e.g. mutH and mutL (methyl-directed
mismatch repair), sbcB (recombination repair), phr (repair of pyrimidine dimers) and vsr
(very short patch repair), as well as genes protecting from UV-induced mutagenesis
(umuCD) and alkylating agents (ada gene) (Parkhill et al., 2000; Fouts et al., 2005; Zhang
et al., 2006), facilitating the appearance of mutations.
Emergence of mutations conferring fluoroquinolone resistance in C. jejuni has been
associated with Mfd, a transcription-repair coupling factor, as Han et al. (2008) recently
showed that Mfd increases the frequency of mutations associated with fluoroquinolone
resistance and that ciprofloxacin upregulates the mfd gene encoding this molecule.
Finally, the expression level of CmeABC has been demonstrated to affect the
frequency of spontaneous ciprofloxacin mutants in such a way that inactivation of
CmeABC decreases and overexpression of CmeABC increases the frequency of
emergence of mutations conferring ciprofloxacin resistance (Yan et al., 2006).
Mutation frequency for macrolide resistance in C. jejuni and C. coli has been revealed
to be low (~10 -10 /cell/generation) (Lin et al., 2007). This is congruent with a study
showing that a single macrolide treatment of Campylobacter-infected chickens did not
select for erythromycin resistance, in contrast to similar studies conducted with
fluoroquinolones (Lin et al., 2007). However, continuous use of tylosin in chicken feed
did result in emergence of erythromycin-resistant C. jejuni and C. coli (Ladely et al.,
2007; Lin et al., 2007).
Besides spontaneous mutations, Campylobacter are also able to acquire resistance
determinants by natural transformation, transduction or conjugation, e.g. conjugation of
tet(O)-carrying plasmids (Zhang and Plummer, 2008). In the presence of antimicrobial
selection pressure, the bacteria containing these resistance determinants out-compete the
susceptible bacteria, eventually leading to a resistant strain.
A predicted 28% of human patients treated with a fluoroquinolone will develop
resistance against fluoroquinolones (Wistrom and Norrby, 1995; Engberg et al., 2006). In
addition, the emergence of Campylobacter resistance in human clinical samples has been
shown to be closely connected to antimicrobial resistance found in animals (Smith et al.,
1999). The transmission of resistant Campylobacter strains has been contemplated in
several studies, where a temporal association between resistant animal and human strains
has been investigated (Endtz et al., 1991). Antimicrobial agents with clinical significance
to treating campylobacteriosis in humans, such as macrolides, fluoroquinolones and
tetracyclines, have all been used extensively in farm animals as therapeutic agents,
prophylactics or growth promoters. Since the beginning of large-scale use of
fluoroquinolones in the early 1990s, the number of resistant Campylobacter strains has
clearly increased in both farm animals and humans. For example, in a Dutch study,
fluoroquinolone resistance in 1982 was non-existent in broilers and clinical samples, but
by 2002–2004 it had reached 35% in broilers and 34% in human clinical samples (Endtz et
al., 1991; van Hees et al., 2007). Similarly, in a Finnish study where antimicrobial
resistance in C. jejuni and C. coli isolates from human clinical samples from 1978–1980
and 1990 were compared, ciprofloxacin resistance was observed to rise from 0% to 9%
over a decade (Rautelin et al., 1991). By 1999, 14 % of human clinical isolates were
ciprofloxacin-resistant (Rautelin et al., 2003).
A summary of characteristics involved in the emergence of fluoroquinolone, macrolide
and tetracycline resistance in C. jejuni is presented in Table 7.
Table 7. Resistance characteristics of C. jejuni to macrolides, fluoroquinolones and
Antimicrobial target 50S rRNA Gyrase 30S rRNA
Target mutation Target mutation Protein protection
Slow, multistep Rapid, single step Rapid, single step
No fitness cost/
No fitness cost/
Trends in resistance Slow increase Very rapid increase Very rapid increase
3 Aims of the study
The major aim of this thesis was to investigate development of antimicrobial resistance
and resistance mechanisms in C. jejuni, C. coli and C. hyointestinalis, especially with
respect to ciprofloxacin. Specific aims were as follows:
1. To establish the susceptibility profile of Finnish C. hyointestinalis strains to
a panel of antimicrobial agents
2. To study and compare quinolone resistance mechanisms in C. hyointestinalis
and C. jejuni
3. To assess the role of efflux in the development of resistance and the effect of
efflux pump inducers and inhibitors on the antimicrobial susceptibility of C.
coli, C. jejuni and C. hyointestinalis
4. To establish the spontaneous mutation frequencies in a group of C. jejuni
and C. coli strains
5. To characterize the development and emergence of mutations associated
with ciprofloxacin resistance in the QRDR of gyrA and in the promoter
region of the CmeABC-efflux pump
4 Materials and methods
4.1 Bacterial strains and growth conditions
The 49 C. hyointestinalis subsp. hyointestinalis strains used in Study I were isolated from
Finnish reindeer and bovine faecal samples. Twenty-four reindeer strains were isolated in
1998 from faeces contained in the rectums of slaughtered animals (Hänninen et al., 2002),
and 25 bovine isolates were obtained from rectal faecal samples collected in
slaughterhouses in Finland in 2003 (Hakkinen et al., 2007), except for strain B704, which
was isolated in 1998. C. jejuni and C. coli strains used in Studies I-IV are listed in Table 8.
Table 8. C. jejuni and C. coli strains used in Studies I–IV
Species Strain no. Source Study
C. jejuni 143483 Reference strain (Hakanen et al., 2002a) I
ATCC 33560 Reference strain (McDermott et al., 2004) II, III, IV
B1 Chicken II, IV
B5 Chicken II, IV
B42 Chicken II, III
B67 Chicken II, III, IV
B68 Chicken IV
BR1 Chicken II
Brt1 Chicken III
FB6371 Chicken II
6507 Chicken II
14/8R Chicken II
49/7R Chicken II, III, IV
49/7RAT Laboratory-induced mutant* II, III, IV
N191 Cattle II
C. coli B25 Chicken II, III
6590 Chicken II, III
6590AT Laboratory-induced mutant* II
S140R Pig II, III
72277 Pig II, III
T72498 Pig II
* Mutants 49/7RAT and 6590AT were obtained by serial passage of strains 49/7R and
6590, respectively, in increasing concentrations of tetracycline and ampicillin
All strains were grown on Mueller-Hinton (MH) agar (Oxoid Ltd., London, UK),
supplemented with 5% horse blood or without agitation in MH broth (Oxoid Ltd.) and
incubated at 37°C for 48 h under microaerobic conditions (5% O2, 10% CO2 and 85% N2).
4.2 Antimicrobial agents and efflux pump effectors
Antimicrobial discs and efflux pump effectors used in studies I-III are presented in Table
Table 9. Antimicrobial discs, efflux pump effectors and the employed concentrations used
in Studies I, II and III.
Antimicrobial agents, Study I
Amoxicillin 10 µg
Co-amoxiclav (amoxicillin + potassium clavulanate) 30 µg
Ampicillin 25 µg
Gentamicin 10 µg
Streptomycin 10 µg
Lincomycin 15 µg
Erythromycin 15 µg
Tetracycline 10 µg
Doxycycline hydrochloride 30 µg
Ciprofloxacin 5 µg
Metronidazole 5 µg
Compound S3 (sulfadiazine, sulfathiazole, sulfamerazine) 300 µg
Efflux pump effectors
Phenyl-arginine-β-naphthylamide (PAβN), Studies I, III 50 mg/l
1-(1-naphthylmethyl)-piperazine (NMP), Study III 100 mg/l
Sodium salicylate, Study III 100 mg/l
Sodium deoxycholate, Study III 1 mg/ml
Ox biles, Study II 20 mg/ml
4.3 Antimicrobial susceptibility testing
Agar dilution method
Agar dilution was used in all studies and together with broth dilution it is the only
standard susceptibility testing method for Campylobacter approved by the Clinical and
Laboratory Standards Institute (CLSI). However, for Campylobacter, no internationally
accepted minimum inhibitory concentration (MIC) interpretive criteria or resistance cutoff
values have been established for agar dilution or any of the other methods. In these
studies, we used veterinary-specific breakpoints generated for the Enterobacteriaceae by
CLSI (NCCLS, 2002) or epidemiological cut-off values recommended by EUCAST
(http://www.eucast.org) for C. jejuni or C. coli (see Table 10).
The method for agar dilution was adapted from CLSI-recommended guidelines
(NCCLS, 2002). Briefly, a stock solution of antimicrobial agent was prepared and
incorporated in doubling dilutions into Mueller-Hinton (MH) blood agar plates (for
concentration ranges for each antimicrobial agent, see Table 10). C. jejuni, C. coli and C.
hyointestinalis were incubated under microaerophilic conditions in 5 ml of MH broth and
diluted for a turbidity equivalent of 0.5 McFarland standard. Then, by using a 19-point
inoculator, bacterial suspensions from up to 19 Campylobacter strains were inoculated on
MH plates containing different concentrations of antimicrobials, and incubation was
performed at 37 °C for 48 h under microaerophilic conditions. To control contamination
and antimicrobial agent carry-over, agar plates containing no antimicrobial agent were
identically inoculated. After 48 h, for each bacterial suspension the lowest concentration
of antimicrobial agent that completely inhibited bacterial growth was recorded as the MIC
value. Quality control strains ATCC 33560 or 143483, for which quality control ranges
have been established, were included in all studies (Hakanen et al., 2002a; McDermott et
Table 10. Concentration ranges and cut-off values for MIC determination of the
antimicrobial agents used
Antimicrobial agent Concentration range (mg/l) Cut-off value (mg/l)
Ampicillin 0.125–16 8
Ciprofloxacin 0.032–128 4
Erythromycin 0.032–16 16
Kanamycin 0.5–8 64
Nalidixic acid 1–512 32
Rifampicin 2–2048 4
Tetracycline 0.125–128 16
Disc diffusion method
The agar disc diffusion method is a widely used, relatively low-cost method for screening
antimicrobial susceptibility that provides results classifying isolates as resistant,
intermediate or susceptible (Watts and Lindeman, 2006). However, breakpoint values for
resistant Campylobacter have not been established. The agar disc diffusion technique was
used in Study I to characterize the susceptibility profile of 49 C. hyointestinalis strains to
12 antimicrobial agents (Table 9). Disc diffusion testing was performed according to
CLSI-recommended guidelines (Clinical and Laboratory Standards Institute, 2004).
Briefly, fresh bacterial cultures were incubated for 48 h in microaerophilic conditions in
5 ml of MH broth and diluted for a turbidity equivalent of 0.5 McFarland standard. The
bacterial suspension was inoculated on an MH blood agar plate with a sterile swab that
was streaked over the entire agar surface to ensure even distribution of the suspension.
After the excess moisture was absorbed, up to three antimicrobial discs (Table 9) were
applied on one plate with sterile forceps. The plates were incubated at 37°C for 48 h in
microaerophilic conditions. The results were recorded as the diameters of the zones of
inhibition in millimetres. C. jejuni 143483 was used as a control strain.
4.4 Estimation of mutation frequencies
Mutation frequencies in C. jejuni and C. coli were investigated in Study II using the
methods of Björkholm et al. (2001) and Wang et al. (2001), with minor modifications. A
schematic presentation of the process is presented in Figure 5. Briefly, C. jejuni and C.
coli strains were grown in 5 ml of Brucella broth for 48 h. Then, 30 µl of bacterial
suspension was used to inoculate each of the 20 tubes containing 3 ml of a Brucella broth.
Tubes were grown for 48 h at 37°C. After incubation, 500 µl of suspension was spread on
Brucella agar plate containing 1 mg/l of ciprofloxacin. Plates were incubated for 3–4 days
in a microaerobic atmosphere at 37°C, and the colonies were counted. If the number of
colonies was too high, the experiment was repeated using 100 µl of 10-fold dilution for
the inoculation of the ciprofloxacin-containing Brucella plate.
The number of viable bacteria was determined in each experiment by plating 10 –6 , 10 –7
and 10 –8 dilutions from three tubes on non-selective Brucella blood agar plates.
The mutation frequency was calculated as the mean number of colonies found on the
selective agar plates (cfu/ml) divided by the mean of the total number of viable cells found
on the non-selective agar plates.
The effect of ox bile mixture on mutation frequency was determined by comparing two
simultaneously performed experiments using the same bacterial strain. The two
experiments were identical to the procedure presented in Figure 5, except that in one 20 g/l
of ox bile was added to the 20 tubes containing 3 ml of Brucella broth.
Figure 5. An overview of the process for calculation of mutation frequencies for a C.
jejuni or a C. coli strain
4.5 DNA extraction, purification and sequencing
DNA was extracted by using the method of Pitcher et al. (1989), modified by Hänninen et
The quinolone resistance-determining region (QRDR) of gyrA was amplified using
PCR primers and cycling conditions described by Piddock et al. (2003) (Studies I and II)
and Griggs et al. (2005) (Study IV). The intergenic region between cmeR and cmeA was
sequenced using PCR primers and cycling conditions described by Lin et al. (2005a)
PCR products were purified using the QIAquick PCR Purification Kit (Qiagen,
Valencia, CA, USA).
Sequencing was performed using the automated cycle sequencer ABI377XL with Big
Dye terminators (PE Applied Biosystems, Foster City, CA, USA) at the Institute of
Biotechnology, University of Helsinki.
4.6 Development of mutations after ciprofloxacin challenge
In Study II, 8 C. jejuni and 2 C. coli strains were subjected to 1 mg/l of ciprofloxacin, and
DNAs from a total of 19 C. jejuni and 8 C. coli colonies were extracted. The promoter
binding region between cmeR and cmeA was amplified by PCR and sequenced.
In Study IV, each of the seven originally ciprofloxacin-susceptible C. jejuni strains
were challenged to 0.125 mg/l of ciprofloxacin in three independent experiments, after
which bacterial DNA was extracted. The seven ciprofloxacin induced strains from one of
the experiments were subcultured on non-selective plates (4 subcultures), on 0.125 mg/l
ciprofloxacin plates (7 subcultures) and on plates containing increasing concentrations of
ciprofloxacin, to a maximum of 8 mg/l. DNA was then extracted from the subcultures, and
the QRDR of gyrA was amplified with primers from Griggs et al. (2005) and sequenced.
The sequencing chromatogram was analyzed for the presence of mutated and multiple
peaks in the QRDR of gyrA.
5.1 Susceptibility of C. hyointestinalis to antimicrobial agents (I)
Susceptibility of 24 reindeer and 25 bovine C. hyointestinalis strains to 12 antimicrobial
agents (see Table 9) was evaluated by using the agar disc diffusion method. The inhibition
zones were measured in millimeters, and if no inhibition zone was detected, the diameter
value of the disc, 6 mm, was used, and the strain was considered resistant. Sulphonamides,
streptomycin, metronidazole, ciprofloxacin and lincomycin failed to produce inhibition
zones with 6, 8, 1, 1, and 1 C. hyointestinalis strains, respectively, all of which were of
bovine origin. In addition, minimum inhibitory concentration (MIC) values for
ciprofloxacin and nalidixic acid were measured for reindeer strains by using the agar
dilution method. According to the results, all reindeer strains were highly susceptible to
ciprofloxacin, with MIC values ranging from 0.064 to 0.25 mg/l. The MIC value was
32 mg/l for the one ciprofloxacin-resistant bovine strain. The results are summarized in
Table 11. Summary of inhibition zones and MIC values of 49 C. hyointestinalis bovine
and reindeer strains by using disc diffusion and agar dilution methods
(inhibition zone, mm)
ND = Not done
Gentamicin 27–34 29.6 27–42 33.4
Sulphonamides 6–42 23.7 36–62 43.8
Streptomycin 6–28 17.8 26–36 31.1
Erythromycin 25–39 31.9 28–54 34.7
Metronidazole 6–43 23.0 16–31 22.0
Ampicillin 38–56 49.8 50–69 58.8
Amoxicillin 46–56 51.9 55–72 61.4
Doxycycline 30–45 39.2 36–60 45.5
Co-amoxiclav 36–54 47.2 50–62 55.3
Tetracycline 34–49 42.3 42–57 47.6
Lincomycin 6–31 23.3 11–36 22.3
Ciprofloxacin 6–53 40.9 38–60 43.8
5.2 QRDR sequence analysis of C. hyointestinalis (I)
The QRDR of gyrA in three ciprofloxacin-susceptible C. hyointestinalis strains (PO0,
PO61 and PO271) was sequenced. The obtained sequence shared 73% identity with that of
C. jejuni and was most similar (98%) to the respective sequence in C. fetus.
PO0, PO61 and PO271 were subjected to increasing concentrations of ciprofloxacin,
and the QRDR of variants able to grow in 64 mg/l of ciprofloxacin were sequenced as was
that of C. hyointestinalis strain B704, with a ciprofloxacin MIC of 32 mg/l. Sequence
analysis showed a Thr-86–>Ile mutation in all of these four strains as a result of a singlepoint
mutation of cytosine to thymine. No other mutations were detected in the QRDR
region. When PO0, PO61 and PO271 were isolated from plates containing 16 or 32 mg/l
of ciprofloxacin, no mutations were detected in the QRDR of gyrA. When these strains
were subjected to ciprofloxacin (16 mg/l), their resistance to nalidixic acid increased from
64–128 mg/l to ≥ 512 mg/l.
5.3 Mutation frequency of C. jejuni and C. coli (II)
Mutation frequency of 11 C. jejuni and 5 C. coli strains was investigated on Brucella
blood agar plates containing 1 mg/l of ciprofloxacin. All of the strains were originally
ciprofloxacin-susceptible (MIC 0.032–0.25 mg/l). The results are summarized in Table 12.
The effect of ox bile salts on the mutation frequency of C. jejuni strains ATCC 33560,
B42, N191, 6507 and B67 and C. coli strain S140R was investigated. The presence of ox
bile salts in the culture medium had no clear effect on the mutation frequency values in
any of these strains (data not shown), compared with the experiments conducted without
Table 12. Mutation frequency of 11 C. jejuni and 5 C. coli strains obtained from agar
plates containing 1 mg/l of ciprofloxacin.
Species Strain/origin Mutation frequency Mutability a
C. jejuni ATCC 33560 / reference 2.9 ± 1.4 x 10 –6 Strongly hypermutable
FB6371 / chicken 9.8 ± 1.3 x 10 –7 Strongly hypermutable
B5 / chicken 9.4 ± 1.2 x 10 –7 Strongly hypermutable
B1 / chicken 8.1 ± 1.6 x 10 –8 Weakly hypermutable
BR1 / chicken 4.7 ± 1.2 x 10 –8 Weakly hypermutable
49/7R / chicken 1.6 ± 1.8 x 10 –8 Normomutable
B42 / chicken 1.5 ± 1.8 x 10 –8 Normomutable
N191 / cattle 1.5 ± 1.4 x 10 –8 Normomutable
B67 / chicken 1.0 ± 1.2 x 10 –8 Normomutable
6507 / chicken 9.3 ± 1.2 x 10 –9 Normomutable
14/8R /chicken 4.2 ± 1.1 x 10 –9 Hypomutable
C. coli 6590 / chicken 7.0 ± 2.0 x 10 –3 Strongly hypermutable
B25 / chicken 7.4 ± 1.2 x 10 –8 Weakly hypermutable
S140R / pig 6.2 ± 1.3 x 10 –8 Weakly hypermutable
T72277 / pig 1.5 ± 0.8 x 10 –8 Normomutable
T72498 / pig 1.3 ± 1.4 x 10 –8 Normomutable
a As defined by Baquero et al. (2004)
5.4 cmeR–cmeA sequence analysis of C. jejuni and C. coli (II)
The intergenic promoter region cmeR-cmeA (Figure 4) was sequenced in 8 C. jejuni and 2
C. coli ciprofloxacin-susceptible strains. We identified three different sequence types for
the inverted repeat (IR) sequence located in this region:
Type 1: TGTAAT AAAT ATTACA
Type 2: TGTAAT AAAA ATTACA
Type 3: TGTAAT AAA– ATTACA
The strains were transferred on Brucella blood agar plates containing 1 mg/l of
ciprofloxacin, and DNA from colonies selected from these plates was extracted and the
promoter-binding region cmeR-cmeA was amplified by PCR and sequenced. The results
are presented in Table 13.
Table 13. IR sequence types of the intergenic promoter-binding region cmeR-cmeA in
susceptible parent strains and in strains selected from Brucella agar plates containing
1 mg/l of ciprofloxacin
Number of colonies
selected from the
IR sequence type of
from the selective
ATCC 33560 / C. jejuni Type 1 4 All Type 1
N191 / C. jejuni Type 1 2 All Type 1
B67 / C. jejuni Type 2 2 Type 1, Type 2
B5 / C. jejuni Type 1 2 Type 1, Type 2
14/8R / C. jejuni Type 1 1 Type 1
FB6371 / C. jejuni Type 2 1 Type 2
49/7R / C. jejuni Type 2 2 All Type 1
49/7RAT / C. jejuni Type 3 5 All Type 3
6590 / C. coli Type 1 3 All Type 1
6590AT / C. coli Type 1 5 All Type 1
5.5 Effect of efflux pump effectors on MIC-values (I, III)
The effect of putative efflux pump inhibitors PAβN and NMP and putative efflux pump
inducers sodium salicylate and sodium deoxycholate on MIC values of ciprofloxacin,
erythromycin, kanamycin, rifampicin and tetracycline was investigated. In study III, six C.
jejuni strains and four C. coli strains were used as well as six C. jejuni and one C. coli
variants induced in the presence of 1 mg/l of ciprofloxacin. Further, the effect of PAβN on
MIC values of ciprofloxacin and nalidixic acid in 17 C. hyointestinalis strains was
evaluated (Study I). MIC values were obtained by the agar dilution method. An overview
of these results is presented in Table 14.
Table 14. Effect of putative efflux pump inducers and inhibitors on susceptibility of 6 C.
jejuni, 4 C. coli and 17 C. hyointestinalis strains to 6 antimicrobial agents. Values
represent the fold-difference between MIC values obtained in the presence and absence of
the respective efflux pump effector.
Nalidixic acid Ciprofloxacin Erythromycin Kanamycin Rifampicin Tetracycline
PAβN ND ~* 8–16X ~ 8–64X ~
NMP ND ~ 1–8X ~ 2–4X 1–4X
Salicylate ND 1–4X ~ ~ 1–2X ~
ND ~ ~ ~ ~ ~
PAβN ND ~ 16–32X ~ 16–32X ~
NMP ND ~ 2–8X ~ 2–8X 1–2X
Salicylate ND 2–4X ~ ~ 1–2X ~
ND ~ ~ ~ ~ ~
PAβN 2–8X ~ ND ND ND ND
MIC values decreased with NMP and PAβN and increased with salicylate and deoxycholate
* The ~ -sign stands for results that did not reproducibly differ from values obtained in the absence
of efflux pump effector.
ND = Not done
The MIC of kanamycin was 4–8 mg/l in C. jejuni strains and 2–8 mg/l in C. coli strains.
None of the putative efflux pump effectors used had any effect on the kanamycin MIC
The MIC of tetracycline was 0.5–8 mg/l in C. jejuni strains and 0.5–64 mg/l in C. coli
strains. In 15 of the 17 C. jejuni and C. coli strains studied, NMP produced a 2- to 4-fold
decrease in the MIC value. The other efflux pump effectors had no effect on the MIC
values of tetracycline.
Ciprofloxacin MIC values for the parent strains were 0.063–0.25 mg/l. Ciprofloxacinresistant
variants of these strains were selected on media containing 1 mg/l of
ciprofloxacin. The ciprofloxacin MIC values of these variants were 2–64 mg/l. The only
efflux pump effector producing a change in the ciprofloxacin MIC values was sodium
salicylate. It increased the MIC values of ciprofloxacin 2- to 4-fold in all C. coli strains
and in 7 of the 12 C. jejuni strains.
The effect of PAβN on ciprofloxacin and nalidixic acid MIC values of C.
hyointestinalis was evaluated. No effect was found on ciprofloxacin, but MIC values for
nalidixic acid decreased 2- to 8-fold. MIC values for nalidixic acid were from 64 mg/l to
≥ 512 mg/l, and use of PAβN decreased these values to 16–128 mg/l.
PAβN and NMP had a pronounced effect on erythromycin and rifampicin MIC values
of C. jejuni and C. coli. The results of these studies are summarized in Figures 6 and 7.
MIC mg/l *16 mg/l
Figure 6. Erythromycin MIC values in the presence and absence of PAβN or NMP
Figure 7. Rifampicin MIC values in the presence and absence of PAβN or NMP
5.6 Effect of ciprofloxacin in the development of gyrA mutations
Twelve C. jejuni and 6 C. coli ciprofloxacin-susceptible strains were challenged with
0.125 and 1 mg/l of ciprofloxacin and by serial passage in 8 mg/l of ciprofloxacin. The
resulting ciprofloxacin MIC values and corresponding QRDR mutations are presented in
Each strain was challenged with 0.125 mg/l of ciprofloxacin in three repeated
experiments, and the MIC values obtained ranged from 0.25 to 32 mg/l. The presence of
potential multiple peaks and peaks representing mutated nucleotides in the sequencing
chromatograms of QRDR of gyrA was investigated. Examination of the sequencing
chromatogram of QRDR revealed double peaks at codons 86 and 90 in almost every
chromatogram, and in one case there was a triple peak at codon 86. Only in 2/18
experiments were no multiple peaks observed. The observed peaks represented codons
ACA (Thr), ATA (Ile) and GCA (Ala) for amino acid position 86, and codons GAT (Asp),
AAT (Asn) and GGT (Gly) for amino acid position 90 (Table 16).
The results from subsequent studies of the dynamics of the QRDR subpopulations
after subcultivation in either the presence or absence of ciprofloxacin are presented in
Table 15. Observed QRDR mutations in gyrA and corresponding MIC ranges and median MIC values after challenging C. jejuni and C. coli
strains with 0.125, 1 or 8 mg/l of ciprofloxacin
Alterations in amino acid positions 86 and 90 in QRDR of gyrA
No mutations Thr86-Ile Asp90-Asn Other mutations
C. jejuni 0.125 6/21 14 0.25-16 2 1 8 8 3 8-16 16 3 b 8-32 8
C. jejuni 1 12/40 9 1-16 1 23 1-16 16 7 2-16 4 1 c 2 2
C. coli 1 6/17 5 2-16 2 9 4-8 8 3 4 4 0 0 0
C. jejuni 8 7/7 0 0 0 5 16-64 32 2 16-32 24 0 0 0
Number of colonies in this group
Other mutations in this group were Thr86-Ala x 2 and Asp90-Gly
A double mutation Thr86-Ile + Asp90-Asn
Table 16. Chromatogram analysis of QRDR sequence data of gyrA in six C. jejuni strains after ciprofloxacin challenge (0.125 mg/l)
No challenge 0.125 mg/l challenge 4 passages in MH plates 7 passages in selective plates Serial passage up to 8 mg/l
ACA GAT 0.125
B1 ACA GAT 0.064 ACA GAT 8 ACA GAT 0.5
B5 ACA GAT 0.125 ACA GAT 16 ACA GAT 0.5
B67 ACA GAT 0.125
B68 ACA GAT 0.125
49/7R ACA GAT 0.064
GAT 16 ATA GAT 16
GAT 16 ATA GAT 32
GAT 8 ATA GAT 8 ATA GAT 32
GAT 8 ATA GAT 64
*Codon represented by the predominant peak is indicated in normal type; codons represented by weaker peaks appear in italics.
6.1 Susceptibility profile of Finnish C. hyointestinalis strains (I)
C. hyointestinalis is a common Campylobacter species in cattle and reindeer in Finland,
representing 15.3% and 6% of positive faecal samples, respectively (Hänninen et al.,
2002; Hakkinen and Hänninen, 2009). Since these animals are used for food production
and C. hyointestinalis has been associated with human disease, it was deemed important to
establish the susceptibility profile of this species to antimicrobial agents.
Susceptibility of 49 Finnish C. hyointestinalis strains to 12 antimicrobial agents was
investigated. According to the results, Finnish C. hyointestinalis strains are susceptible to
most antimicrobials of veterinary importance, with the exception of sulphonamides (12%
resistant) and streptomycin (16% resistant). In comparison, in a Canadian study involving
tetracycline, erythromycin, ampicillin and ciprofloxacin, 10.7%, 10.1%, 1.1% and 0% of
the 1500 bovine C. hyointestinalis isolates were resistant, respectively (Inglis et al., 2005).
Interestingly, all of the Finnish resistant C. hyointestinalis strains were of bovine origin,
and the reindeer strains in general had larger inhibition zones in disc diffusion tests than
the bovine isolates, suggesting higher susceptibility (Table 11). The mean diameter was at
least 8 mm larger in reindeer strains than in bovine strains for sulphonamides,
streptomycin, ampicillin, co-amoxiclav and amoxicillin. This could be explained by
reindeer living and feeding freely as semi-domesticated animals and only exceptionally
being treated with antimicrobials. Cattle, on the other hand, are treated, when necessary,
mostly with penicillins, but also with e.g. tetracyclines, macrolides, lincosamides,
sulphonamides and aminoglycosides. The overall use of antimicrobials in veterinary
medicine in Finland is moderate (Table 3). A possible explanation for the high resistance
rates for tetracycline and erythromycin in the Canadian study could be the use of
tetracyclines as growth promoters and prophylactics and tylosin as a prophylactic in
Canadian food animal production (Inglis et al., 2005).
C. jejuni was isolated from the same bovine faecal samples as C. hyointestinalis, and
results from the susceptibility determinations are presented in our previous study
(Hakkinen et al., 2007). Congruently with the bovine C. hyointestinalis isolates, the
overall prevalence of antimicrobial resistance in bovine C. jejuni isolates was low, as only
9% of the isolates were resistant to any of the antimicrobial agents tested, most commonly
to nalidixic acid (6%) (Hakkinen et al., 2007).
6.2 Quinolone resistance mechanisms in C. hyointestinalis (I)
C. hyointestinalis is inherently nalidixic acid-resistant, but ciprofloxacin-susceptible. Our
studies showed 73% identity of the QRDR in C. hyointestinalis with that of C. jejuni.
Nalidixic acid resistance in C. jejuni has been associated with mutations at Ala-70, Thr-86
or Asp-90 in the QRDR (Wang et al., 1993). However, the mutations associated with
nalidixic acid resistance in C. jejuni were not present in C. hyointestinalis, and thus, did
not explain the mechanism of nalidixic acid resistance in C. hyointestinalis. In C. jejuni
and C. coli, ciprofloxacin resistance is often associated with nalidixic resistance and vice
versa, although exceptions do occur (Bachoual et al., 2001). Similar cross-resistance was
observed in our studies, where challenging originally ciprofloxacin-susceptible C.
hyointestinalis strains with up to 16 mg/l of ciprofloxacin increased the nalidixic acid
resistance of these same strains from 64–128 mg/l to ≥ 512 mg/l.
Possible mechanisms for nalidixic acid resistance include decreased antimicrobial
uptake in the form of increased impermeability of the membrane and action of the efflux
pumps. The latter was supported by our study, where the presence of efflux pump
inhibitor PAβN decreased the MIC of nalidixic acid 2- to 8-fold. However, the pump
inhibitor did not establish complete susceptibility, which could be due to inefficiency of
the pump inhibitor, function of secondary pump systems or presence of other resistance
mechanisms, such as those involved in impermeability of the membrane. Thus far, the
CmeABC-efflux pump has not been characterized in C. hyointestinalis.
By comparing the QRDR sequence of gyrA in ciprofloxacin-susceptible C.
hyointestinalis and respective ciprofloxacin-induced variants, we observed that high-level
ciprofloxacin resistance was associated with the Thr–>Ile mutation in the position
corresponding to codon 86 in C. jejuni. Therefore, the mechanism of high-level
ciprofloxacin resistance was the same in C. hyointestinalis as in C. jejuni and C. coli.
However, this mutation was not observed in variants with MIC of ciprofloxacin < 32 mg/l,
which could indicate the presence of alternative resistance mechanisms for this lower level
ciprofloxacin resistance, such as those discussed for nalidixic acid in the previous
paragraph. The prevalence of this phenomenon was not assessed and possibly Thr86-Ile
mutations could emerge later, as in Study IV conducted with C. jejuni variants. No other
mutations in the QRDR of gyrA associated with ciprofloxacin resistance in C. jejuni or C.
fetus were observed in the C. hyointestinalis strains of this study. Similarly to studies
conducted with C. jejuni and C. coli, the presence of PAβN did not affect the MIC of
ciprofloxacin in C. hyointestinalis.
6.3 Spontaneous mutation frequency of C. jejuni and C. coli (II)
Since the majority of resistance mechanisms are associated with target mutations, the rate
at which mutations occur in bacteria has an impact on the emergence of bacterial
resistance. Previous to our study, mutation frequency has been characterized in, for
example, Helicobacter pylori strains and in single C. jejuni and C. coli strains (Taylor et
al., 1985; Gootz and Martin, 1991; Björkholm et al., 2001; Yan et al., 2006). It is difficult
to compare results obtained from different studies for several reasons: 1) the same
antimicrobial is not used in all mutation frequency studies due to different species having
different inherent resistance profiles, 2) different concentrations of selective agents have
been used, although this may not always influence the results (Yan et al., 2006), 3) for
reliable results, the MIC of the bacteria studied to the antimicrobial agent employed
should always be at least 4-fold smaller than the concentration used for selection (Chopra
et al., 2003), 4) different methods have been used to conduct and calculate mutation
Our mutation frequency determinations revealed that mutation frequencies of
fluoroquinolone-susceptible C. jejuni and C. coli strains vary considerably, from
hypomutable to strongly hypermutable. Genetic diversity in general among C. jejuni
strains is extensive (Dorrell et al., 2001), which may facilitate the adaptation of bacteria to
the selective environment. Large variance in mutation frequencies has also been observed
in H. pylori (Björkholm et al., 2001). However, in studies conducted with E. coli, as few
as 0.7% of strains were of the hypermutable type (Baquero et al., 2004). The high number
of hypermutable strains in C. jejuni and C. coli could be explained by the lack of such
repair systems as SOS response and functional methyl-directed mismatch repair (Parkhill
et al., 2000; Fouts et al. 2005; Gaasbeek et al., 2009). In a recent study of a single C.
jejuni strain, three functional DNA repair mechanisms, namely nucleotide excision repair
(NER), base excision repair (BER) and RecA-dependent recombinational repair, were
identified. None of these affected repair of point mutations, which was analysed by
determining mutation frequency for mutations conferring nalidixic acid resistance
(Gaasbeek et al., 2009). Another gene involved in DNA repair in C. jejuni is mfd, which
encodes a transcription-repair coupling factor. In addition, Mfd increases the rate of
spontaneous mutation to ciprofloxacin resistance, and ciprofloxacin induces the expression
of mfd (Han et al., 2008). It is therefore likely that the mutation frequency values obtained
in our study were influenced by the expression of ciprofloxacin-induced mfd.
The obtained mutation frequency values were compared with QRDR and intergenic
promoter binding cmeR-cmeA sequences derived by sequencing these regions from single
colonies collected from selective plates used for mutation frequency calculations.
However, no particular mutation pattern was associated with any of the mutability classes.
6.4 cmeR-cmeA sequence analysis of C. jejuni and C. coli (II)
The transcriptional repressor CmeR represses the expression of the CmeABC efflux pump
by directly binding to a 97-bp cmeR-cmeA intergenic (IT) region (Lin et al., 2005a).
Previously, mutations in cmeR have been shown to inhibit the binding of CmeR, resulting
in overexpression of CmeABC and increased resistance to multiple antimicrobials,
including ciprofloxacin (Pumbwe et al., 2004; Lin et al., 2005a). CmeR binding can also
be inhibited by inducing ligands, such as bile salts, which interact with CmeR and cause a
conformational change that makes it unable to bind DNA (Lin et al., 2005b). In a study by
Lin et al. (2005), a spontaneous deletion, and in a study by Cagliero et al. (2007), a
spontaneous transition, in the inverted repeat sequence located in IT, were associated with
increased resistance (see Table 17).
In Study II, sequence Types 1 and 2 in the inverted repeat of IT were identified, as well
as a previously unreported Type 3 (Table 17), both in the original ciprofloxacinsusceptible
C. jejuni and C. coli strains, and in variants selected from plates containing
1 mg/l of ciprofloxacin. The different sequence types were compared with mutations in the
QRDR region and MIC values obtained for ciprofloxacin, but no relationship was
detected. However, sequence Type 3 was exclusively and consistently found in the
49/7RAT strain, a tetracycline- and ampicillin-induced variant of 49/7R, which is of
sequence Type 1. The difference in MIC of ciprofloxacin between these strains
(0.032 mg/l in 49/7R and 0.5 mg/l in 49/7RAT) could be explained by this deletion in the
A CmeR structural study revealed that CmeR binds the inverted repeat in a relatively
weak fashion and that CmeR has a very flexible DNA-binding domain, permitting
recognition of several DNA sites (Routh et al., 2009). This is concordant with the role of
CmeR as a pleiotropic regulator (Guo et al., 2008). Taking these observations into
consideration, possibly not all alterations in the inverted repeat sequence bear significance
in the binding of CmeR.
Table 17. IR sequence types observed in different studies of the cmeR-cmeA IT region
IR sequence Reference
TGTAAT AAAT ATTACA (Lin et al., 2005a)
TGTAAT AA–T ATTACA (Lin et al., 2005a)
TGTAAT AAAA ATTACA (Cagliero et al., 2007)
TGTAAT AAAA ATTATA (Cagliero et al., 2007)
TGTAAT AAAT ATTACA Study II (Type 1)
TGTAAT AAAA ATTACA Study II (Type 2)
TGTAAT AAA– ATTACA Study II (Type 3)
6.5 Effect of efflux pump effectors (I, III)
Of all the putative efflux pump effectors employed in Studies I and III, PAβN had the
most important effect. It was able to decrease the intrinsic resistance of C. hyointestinalis
to nalidixic acid 2- to 8-fold, intrinsic resistance of C. jejuni and C. coli to rifampicin 8- to
64-fold and 16- to 32-fold, respectively, and increase the inherent erythromycin
susceptibility of C. jejuni and C. coli 8- to 16-fold and 16- to 32-fold, respectively. No
reproducible effect on the MIC values of ciprofloxacin, kanamycin and tetracycline was
Earlier studies of PAβN on resistant and susceptible erythromycin strains have resulted
in an 8- to 128-fold decrease in erythromycin MIC levels (Payot et al., 2004; Cagliero et
al., 2005; Martinez and Lin, 2006; Gibreel et al., 2007). No high-level erythromycinresistant
C. jejuni were included in our investigation, but according to the study of Gibreel
et al. (2007), PAβN failed to decrease resistance of high-level erythromycin-resistant C.
jejuni, which would constitute a major drawback when considering this substance as a
potential drug candidate against development of resistance. Contrary to this, Martinez and
Lin (2006) reported the susceptibility to erythromycin to be restored in resistant strains in
the presence of PAβN.
In a study involving two C. jejuni strains, rifampin resistance decreased considerably,
1024- to 2048-fold, and tetracycline resistance decreased up to 512-fold in the presence of
PAβN (Martinez and Lin, 2006). In our study, no effect of PAβN on tetracycline
susceptibility was observed. The fact that all strains used in our study had low MIC values
for tetracycline, unlike the strain evaluated by Martinez and Lin, may explain this
The effect of PAβN on ciprofloxacin and nalidixic acid resistance in C. jejuni and C.
coli has been investigated previously. In a study by Corcoran et al. (2005), PAβN at a
concentration of 20 mg/l produced no effect on the MIC of ciprofloxacin or nalidixic acid
in the ciprofloxacin- and nalidixic acid-resistant strain, but in the two evaluated
susceptible strains, it produced a 2-fold and a 2.5- to 4-fold increase in ciprofloxacin and
nalidixic acid susceptibility, respectively. The results obtained by Martinez and Lin (2005)
by the broth dilution method in the presence of 10 µg/ml of PAβN showed up to a 2-fold
and a 4-fold increase in ciprofloxacin and nalidixic acid susceptibility, respectively.
Cagliero et al. (2006b) reported that of the 22 ciprofloxacin-resistant and -susceptible C.
jejuni and C. coli strains only 3 were able to decrease ciprofloxacin MIC values by more
than 2-fold. In studies I and III, PAβN had no effect on the ciprofloxacin MIC values of
resistant and susceptible C. jejuni, C. coli and C. hyointestinalis. It did, however, lower the
nalidixic acid resistance of C. hyointestinalis 2- to 8-fold, indicating different resistance
mechanisms for these two related antimicrobials in C. hyointestinalis.
To date, PAβN remains the only broad-spectrum efflux pump inhibitor extensively
characterized in C. jejuni. Unfortunately, its unfavourable pharmacokinetic and
toxicological profiles prohibit its clinical use (Lomovskaya and Bostian, 2006).
Studies with NMP inhibitor showed 1- to 16-fold, 2- to 8-fold and 1- to 4-fold decreases in
erythromycin, rifampicin and tetracycline MIC levels, respectively, for C. jejuni and C.
coli. According to these results, NMP is less effective in decreasing MICs of erythromycin
and rifampicin than PAβN, which is congruent to studies conducted with E. coli and
Acinetobacter baumannii (Bohnert and Kern, 2005; Pannek et al., 2006). This also
suggests that the effects conferred by NMP to antimicrobial susceptibility differ from
those conferred by PAβN. Moreover, neither of these substances renders the strains
completely susceptible. In addition, inactivation of the CmeABC efflux pump at the
molecular level has a stronger effect on antimicrobials and impacts a wider range of
antimicrobials than the use of PAβN or the NMP in C. jejuni and C. coli (Cagliero et al.,
2006b). The mechanism underlying the ability of NMP to increase antimicrobial
susceptibility warrants further investigations, and, to our knowledge, the suitability of
NMP for therapeutic use has not been determined.
In Study III, sodium salicylate increased ciprofloxacin and rifampicin resistance 1- to 4fold
and 1- to 2-fold, respectively, in C. coli and C. jejuni. A similar small effect on
ciprofloxacin resistance in C. jejuni and C. coli was reported earlier by Randall et al.
(2003). In the same study, Randall et al. observed an increase in tetracycline and
erythromycin MIC levels in the presence of sodium salicylate, but these findings were not
confirmed in our study. Salicylate has been shown to decrease the susceptibility of E. coli
by altering membrane proteins and inducing efflux pumps (Price et al., 2000). This is of
concern since salicylates are common constituents of non-steroidal anti-inflammatory
drugs, which are used in combination with antimicrobials to treat microbial diseases.
The CmeABC efflux pump has been established to confer resistance to bile salts present in
the intestinal tract of the host animal, and bile salt resistance is a prerequisite for
successful colonization of C. jejuni (Lin et al., 2003). Further, various bile salts, including
deoxycholate, strongly increase the expression of CmeABC, mainly by inhibiting the
binding of CmeR to the promoter region of cmeABC (Lin et al., 2005b). The presence of a
bile salt, taurocholate, has been shown to moderately decrease the susceptibility of C.
jejuni to, for instance, ciprofloxacin and erythromycin (2-fold change) (Lin et al., 2005b).
Bile salts were included in Studies II and III. In Study II, the effect of ox bile on mutation
frequencies of five C. jejuni and one C. coli strain was investigated by including 20 mg/ml
of ox bile in the culture media, but no marked impact emerged relative to studies
conducted without bile salts. In Study III, the effect of 1 mg/ml of sodium deoxycholate
on the susceptibility of five C. jejuni and four C. coli strains to various antimicrobials was
evaluated. Apart from variable 2-fold increases in rifampicin MIC values found in each
round of testing, deoxycholate had no influence on antimicrobial susceptibility of the
strains examined. Interestingly, although bile salts included in the culture medium of C.
jejuni resulted in a 6- to 16-fold increase in the expression of cmeABC (Lin et al., 2005b),
their effect on ciprofloxacin MIC values was negligible.
6.6 Development and emergence of QRDR mutations (IV)
In Studies II and IV, MIC values and QRDR alterations were investigated after subjecting
originally ciprofloxacin-susceptible C. jejuni and C. coli strains to 1 mg/l or 0.125 mg/l of
ciprofloxacin, respectively. In Study IV, the experiment was extended by investigating the
persistence of these alterations in the presence and absence of ciprofloxacin and also at a
higher concentration of ciprofloxacin (8 mg/l).
Initial ciprofloxacin challenge of C. jejuni with 0.125 mg/l produced MIC values of 0.25–
32 mg/l, and challenge with 1 mg/l produced MICs of 1–16 mg/l in C. jejuni and 2–
16 mg/l in C. coli. Pumbwe et al. (2005) obtained similar results with a C. jejuni strain
challenged with 0.5 and 1 mg/l of ciprofloxacin, giving rise to MIC levels of 32 mg/l.
Surprisingly, of the 78 colonies studied from 12 C. jejuni and 6 C. coli strains subjected to
0.125 or 1 mg/l of ciprofloxacin, 28 (36%) had no mutation in their QRDR region despite
elevated MIC values from 0.25 to 16 mg/l, the median being 2 mg/l. Resistant strains
without QRDR mutations have previously been reported in clinical and animal isolates
(Piddock et al., 2003; Hakanen et al., 2003). The proportion of colonies not possessing a
QRDR mutation was highest (67%) in colonies subjected to 0.125 mg/l of ciprofloxacin.
The most common mutation type was Thr86-Ile (42%), followed by Asp90-Asn (17%).
Other observed mutation types were Thr86-Ala, Asp90-Gly and the double mutation
Thr86-Ile + Asp90-Asn. Repeated challenges or different colonies of the same strain often
produced different QRDR profiles and MIC values.
In Study IV, primary challenge with 0.125 mg/l of ciprofloxacin generated multiple
peaks in the sequencing chromatogram at mutation-prone codons 86 and 90 in QRDR.
This is indicative of different bacterial subpopulations with variable QRDR sequences that
are most likely present in the susceptible strain already prior to the ciprofloxacin
challenge. When subjected to ciprofloxacin challenge, the QRDR subpopulations
conferring ciprofloxacin resistance became significantly amplified. These findings suggest
that the sequencing results of a QRDR of a ciprofloxacin-resistant strain may, in some
cases, be interpreted as not containing a mutation, although actually the sample contains
both QRDR-mutated and unmutated bacteria.
After the preliminary exposure of strains to 0.125 mg/l of ciprofloxacin, the strains were
either subcultured seven times on plates containing 0.125 mg/l of ciprofloxacin or
transferred on plates without a selective agent and subcultured four times. The presence of
multiple peaks at codons 86 and 90 persisted during several subcultures, both after the
removal of selection pressure and on the selective plates. These results indicate that both
mutated and unmutated bacteria are able to persist in the absence and presence of
0.125 mg/l of ciprofloxacin. The persistence of ciprofloxacin-resistant strains in the
absence of selection pressure has also been reported in vivo (Luo et al., 2005). Apart from
C. jejuni strains B1 and B5, the high MIC values obtained during the preliminary exposure
persisted or increased.
A QRDR-independent resistance mechanism was also activated by the presence of low
ciprofloxacin concentration. The C. jejuni strains B1 and B5 behaved differently from the
other strains. After the primary challenge with ciprofloxacin, they presented wild-type
QRDR sequences and had no multiple peaks at codons 86 and 90, but nevertheless
expressed ciprofloxacin MIC values of 8–16 mg/l. When the selection pressure was
removed, their MIC values decreased to 0.5 mg/l (original MIC 0.125 mg/l), whereas in
other strains showing multiple peaks in the QRDR sequencing chromatogram, the high
MIC values obtained during preliminary exposure persisted or increased. On the other
hand, when the subcultivation of strains B1 and B5 was continued in the presence of
ciprofloxacin, secondary peaks appeared in the sequencing chromatogram, and the MIC of
ciprofloxacin remained at 16 mg/l. The QRDR-independent resistance mechanism may be
speculated to involve the action of an efflux pump, which is then replaced or
supplemented by resistance conferring mutations when selection pressure is prolonged.
The experiment was continued by serial passage of the strains on plates containing
increasing concentrations of ciprofloxacin, until 8 mg/l was attained. This resulted in
strains with either the Thr86-Ile or Asp90-Asn mutation and notably reduced the number
of double peaks in the sequencing chromatogram. The measured ciprofloxacin MIC values
were 16–64 mg/l. The appearance of different mutant types after low-level
fluoroquinolone challenge has also been reported for Mycobacterium by Zhou et al.
(2000). Concordant with our study, low-level fluoroquinolone challenge selected for
QRDR-independent resistant mutants, and increasing the fluoroquinolone concentration
subsequently led to QRDR mutants and reduced the number of different mutant types.
1. Finnish C. hyointestinalis strains are susceptible to most antimicrobials of veterinary
importance. However, 32% and 24% of bovine strains were resistant to streptomycin and
sulphonamides, respectively. Reindeer strains remained susceptible to all tested
antimicrobials. The difference in the susceptibility profile of bovine and reindeer strains
most probably reflects the veterinary use of these substances in bovine husbandry, but not
2. The QRDR of gyrA in C. hyointestinalis showed 73% identity with that of C. jejuni.
Mutations associated with nalidixic acid resistance in C. jejuni were not found in the
QRDR of C. hyointestinalis, and thus, do not explain the inherent nalidixic acid resistance
of C. hyointestinalis. Inherent nalidixic acid resistance is partly associated with efflux
pumps since PAβN, an efflux pump inhibitor, decreased the nalidixic acid resistance in C.
hyointestinalis 2- to 8-fold. However, low nalidixic acid MIC values were not obtained,
which indicates the presence of other resistance mechanisms. Cross-resistance between
nalidixic acid and ciprofloxacin was observed in C. hyointestinalis, as induced
ciprofloxacin resistance also increased MIC values for nalidixic acid. High-level
ciprofloxacin resistance (> 32 mg/l) in C. hyointestinalis was associated with the same
Thr86-Ile mutation as in C. jejuni, but no mutations in the QRDR were detected at lower
ciprofloxacin MIC levels.
3. The impact of efflux pump effectors on erythromycin, ciprofloxacin, tetracycline,
rifampicin and kanamycin MIC values in C. jejuni and C. coli was investigated. Efflux
pump inhibitor PAβN increased the susceptibility of C. jejuni and C. coli to erythromycin
and rifampicin 8- to 32-fold and 8- to 64-fold, respectively. In addition, it decreased the
nalidixic acid MIC values of C. hyointestinalis 2- to 8-fold. Another inhibitor, NMP,
produced 1- to 8-fold, 2- to 8-fold and 1- to 4-fold decreases, respectively, in
erythromycin, rifampicin and tetracycline MIC levels for C. jejuni and C. coli. Neither of
these inhibitors had an effect on ciprofloxacin MIC values. The different results produced
by these inhibitors indicate different modes of function for these pump inhibitors.
An efflux pump inducer, sodium salicylate, was only able to increase the MIC values
of one of the antimicrobials evaluated, ciprofloxacin (2- to 4-fold). Bile salts, which are
putative efflux pump inducers, had no observable effect on the MIC values of any of the
antimicrobials, nor did it have a notable impact on the mutation frequencies of C. jejuni
and C. coli strains.
4. Mutation frequencies of C. jejuni and C. coli strains varied considerably, from strongly
hypermutable (2.9 ± 1.4 x 10 –6 ) to hypomutable (4.2 ± 1.1 x 10 –9 ). The proportion of
hypermutable strains was much higher than previous reports for E. coli, which may
facilitate the adaptation of C. jejuni and C. coli to a selective environment. No correlation
was found between mutation frequency and mutations in the QRDR or cmeR-cmeA region.
5. Low-level ciprofloxacin concentration (0.125 mg/l) in a medium of originally
ciprofloxacin-susceptible C. jejuni strains gave rise to multiple peaks in the sequencing
chromatograms of QRDR, which may reflect different QRDR subpopulations. Exposure
to 0.125 mg/l of ciprofloxacin resulted in C. jejuni variants with MIC values ranging from
0.25 to 16 mg/l. Mutations in codon 86 (ACA, Thr) of QRDR included ATA (Ile) and
GCA (Ala), and mutations in codon 90 (GAT, Asp) included AAT (Asn) and GGT (Gly).
In two strains, MIC levels of up to 32 mg/l for ciprofloxacin were obtained, but no
mutations or multiple peaks in the sequencing chromatograms of QRDR were observed.
This suggests the presence of a QRDR-independent resistance mechanism. The strains
expressing the resistance mechanism, which involves different QRDR subpopulations, are
able to persist in both the presence and absence of low-level ciprofloxacin concentration.
However, the QRDR-independent resistance mechanism is quickly replaced or
supplemented by the appearance of QRDR mutations if ciprofloxacin challenge persists.
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