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Campylobacter and its multi-resistance in the

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Trends in Food Science & Technology 22 (2011) 91e98

Review

Campylobacter and
its multi-resistance in
the food chain
Sonja Smole Mo
zina*,
Marija Kurin
ci
c, Anja Klan
cnik
and Ana Mavri
University of Ljubljana, Biotechnical Faculty,
Department of Food Science and Technology,
Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia
(e-mail: sonja.smole@bf.uni-lj.si)
Increasing antimicrobial resistance is an urgent world-wide
problem, including multidrug resistant microorganisms transmitted via the food chain. Campylobacteriosis is the leading
bacterial food-borne illness and most frequently reported zoonosis in humans. Despite the differences in results and some
insufficiency in the methodological harmonization among reports from different countries, there was an evident increase in
prevalence and multiple resistance of Campylobacter in food
production environments in central and southern EU-member
states (MSs). Non-specific efflux pumps are involved in bacterial reduced susceptibility and/or resistance against antibiotics
and other unrelated antimicrobials, including bile salts. Beside
this, the synergistic antimicrobial activity of some compounds
which could act as inhibitors of efflux pumps is a challenge to
develop more efficient protection against food-borne pathogens. Testing the activity of known and putative natural efflux
pump inhibitors (EPIs) in Campylobacter resistant isolates revealed new information about the mechanisms involved in resistance and new approaches for Campylobacter risk
management.

Introduction
Antimicrobials are extremely important for treatment
and disease prevention in animal livestock production.
For almost 50 years and until their recent withdrawal in
the EU, antibiotics at subtherapeutic levels were also used
* Corresponding author.
0924-2244/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2010.09.003

for growth promotion, improved feed efficiency and enhanced performance of livestock (Donnelly, 1999). However, it is generally acknowledged that the use of
antimicrobials in food animals drives the emergence of antimicrobial resistance and promotes its dissemination via
the food chain. The development and spread of antimicrobial resistance of zoonotic bacteria with its reservoirs in
healthy food animals, e.g. poultry, pigs and cattle, has become a global public health problem (EFSA, 2008).
Antimicrobial resistance in the food chain, including
multidrug resistance, is a complex issue. To understand
the public health concern about antimicrobial resistance
in zoonotic bacteria, not only the use of antimicrobials
but other factors, including bacterial virulence and capacity
to spread, the genetic basis of antimicrobial resistance, and
co- and cross-resistance, as well as the rate of bacterial
transfer, dependent on the animal and bacterial species,
slaughter practices and production forms should also be
considered. In a very recent joint opinion of EFSA, the
European Centre for Disease Control and Prevention
(ECDC), the European Medicines Agency (EMEA) and the
European Commissions Scientific Committee on Emerging
and Newly Identified Health Risks (SCENIHR) on antimicrobial resistance focused on zoonotic infections (EFSA,
2009a), some combinations of bacterial agent/antimicrobial/animal species are considered of the highest concern.
Among microbes these are Campylobacter and Salmonella,
and among antimicrobials quinolones, cephalosporines and
macrolides. Cephalosporins are not used for campylobacterioses treatment, so only quinolone, macrolide and multi-resistance of Campylobacter are targeted in this review. In
selecting the segment of the food chain with the highest emphasis on human health, chicken meat production was highlighted for several reasons: i) the higher prevalence of
Campylobacter in chicken than in other meats, ii) usage of
antibiotics on livestock farms is increasing in several EUMSs, especially in broiler production (MARAN, 2007), iii)
the high and still increasing consumption of chicken meat because of consumer preferences for white meat and the competitive prices of poultry with respect to other meats
(European Commission Prospects, 2009).
The aim of this review is to illustrate recent trends in the
prevalence and antibiotic (multi)resistance of Campylobacter jejuni and Campylobacter coli in the food chain. Beside
this, the role of efflux in multiple resistance of Campylobacter is highlighted, not only to antibiotics but also for

Author's personal copy

Prevalence of Campylobacter in the food chain

Several
national
(see
http://www.crl-ar.eu/206monitoring_reports.htm) and international surveillance
reports have been published recently (e.g. de Jong et al.,
2009) including Campylobacter prevalence in the food chain.
However, the results from both national and international surveys frequently suffer from insufficient harmonization between countries. Furthermore, their appearance in print is
often considerably delayed (e.g. the cited pan-European survey with five EU-MSs and 1500 samples dating from 2002 to
2003 was published only in 2009). In this respect, EFSA/
ECDC reports provide most recent data with the highest number of countries, samples and microbial isolates included. A
manual and guidance for reporting is available on-line (http://
www.efsa.europa.eu/; EFSA, 2007a, 2009c).
In the period 2005e2007, campylobacteriosis was the
most commonly reported zoonosis transmitted to humans
in the EU, with 200,507 confirmed cases reported in
2007. This was a 12% increase compared to 2006, with
most MSs reporting an increase, some as high as 27%,
e.g. Germany. Most infections are transmitted via the
food chain. In foodstuffs, Campylobacter was by far the
most commonly detected in fresh broiler meat (Figs. 1
and 2), with on average 31.6% of samples proving positive
in the EU in 2003e2007 (15,126 out of 48,243 samples
taken at slaughter, processing or at retail in 17 EU-MSs).
Other authors (Moran, Scates, & Madden, 2009; Smole
Mozina, Kurincic, Kramar, Ursic, & Katalinic, 2009;
Suzuki & Yamamoto, 2009) reported that up to 90e100%
of retail chicken meat world-wide was contaminated by
Campylobacter.
There are many fewer reports on non-broiler retail meat
contamination. For fresh non-broiler poultry meat in the EU

60
50

2003

40

2004

30

2005

20

2006

10

2007

Farm animals

Beef

Pork

Chicken

Cattle

Pigs

Broilers

Retail meat

Fig. 1. Reported prevalence of Campylobacter in animals on the farm

and in retail meat in EU-MSs in the period 2003e2007 (Source: EFSA,
2009b).

100
90
80
70
60
50
40
30
20
10
0

Broiler flocks
Chicken meat

Es
to
n
N ia
or
wa
Sw y
e
D den
en
m
ar
La k
tv
ia
Sp
ai
I n
Sw rela
itz nd
er
la
n
Au d
st
C Ge ria
ze
ch rma
Re ny
pu
Sl blic
ov
en
ia
Ita
Fr ly
an
S l ce
ov
ak
ia

bile resistance, important for pathogen host colonization

and virulence. Finally, our experimental approaches for
testing the synergistic antimicrobial activity of some natural compounds which could act as inhibitors of Campylobacter efflux pumps are presented.

S.S. Mo
z ina et al. / Trends in Food Science & Technology 22 (2011) 91e98

92

Fig. 2. The prevalence of Campylobacter in broiler flocks and fresh

broiler meat at retail (for some countries data were available for
slaughter and/or processing plants) in EU-MSs (including Switzerland
and Norway) in 2007 (Source: EFSA, 2009b and specific country
reports, 2009).

in 2007, 19% of the samples were Campylobacter positive

for turkeys (N 1574, 7 reporting MSs) and 41.2%
(N 245, 3 reporting MSs) for ducks and geese (EFSA,
2009b). In other fresh retail meat (pork, beef, see Figs. 1
and 2) and meat products, Campylobacter was much less
frequently detected. In ready-to-eat products, on average,
1.5% (N 608) of samples were positive, and for nonready-to-eat (intended to be cooked) from 0.6 to 6.3%
(N 1841). The rate of positive samples in raw milk and
dairy products (mainly soft cheeses) was very low (<1%)
(N 4678, 7 reporting MSs).
In Fig. 2, the prevalence of Campylobacter in broiler
flocks, pig and cattle herds and in retail meats for all EUMSs which reported results for Campylobacter in animals
and in broiler, pork and beef meat to EFSA in 2007, are
summarized. Interestingly, although a high prevalence of
Campylobacter was reported for pig herds (on average
47.2% were positive in 2003e2007, N 7609), Campylobacter rates decreased sharply following pig slaughter, so
retail pork meat was very rarely contaminated (e.g. 0.9%
in 2007). Thus for Spain and Germany, the only two countries with reported prevalence of Campylobacter for a number of samples higher than 25, in pig herds and fresh pork
meat in retail the respective prevalence was 71.3%
(N 230) and 0% (N 36) in Spain and 29.5%
(N 224) and 0.8% (N 123) in Germany, in pigs and retail pork meat, respectively. This sharp decrease after
slaughter is thought to be the consequence of different
slaughtering practices in pig and poultry slaughterhouses,
but, to our knowledge, there are no other explanations elucidating this difference in contamination of poultry and
pork meat. Thus there is a need to study the adaptation of
Campylobacter to poultry-meat related conditions, e.g. the
protective role of chicken meat juice (Birk, Ingmer,
Andersen, Jrgensen, & Brndsted, 2004) which was found
to be very effective in protecting Campylobacter after exposure of cells to stress, e.g. suboptimal temperature, freezing
and/or antimicrobial compounds (Piskernik et al.,
submitted for publication).

Author's personal copy

S.S. Mo
z ina et al. / Trends in Food Science & Technology 22 (2011) 91e98

Prevalence of Campylobacter resistance and

multi-resistance in the food chain
Despite efforts to harmonize protocols for monitoring
antimicrobial resistance in the EU (Anonymous, 2008),
the comparative analysis of Campylobacter antibiotic resistance is difficult, due to differences in the sampling
schemes, in the methodology of antibiotic resistance testing
and breakpoints which have also been changed recently
(EFSA, 2007b). However, despite the lack of standardization of susceptibility testing, recent comparative studies reported diffusion and dilution methods to be acceptable
(Gaudreau, Girouard, Gilbert, Gagnon, & Bekal, 2008;
Gaudreau, Girouard, Ringuette, & Tsimiklis, 2007;
McGill et al., 2009; Valdivieso-Garca et al., 2009), but
caution is necessary in interpretation of the results. While
waiting for easily applicable and reliable methods for molecular resistance testing, the use of broth microdilution remains the best method (van der Beek et al., 2010). In this
light we assessed EFSA country reports on the antimicrobial resistance of C. jejuni and C. coli in the food production/supply chain in the period 2004e2007. Their
increasing resistance to quinolones and/or macrolides, as
well as their increasing multiple drug resistance was most
evident in central and southern-European countries. In general, nearly half of retail chicken meat Campylobacter isolates in central Europe are resistant to at least two unrelated
antibiotics used in human and veterinary medicine, and one
third are resistant to several antibiotics (Fig. 3).
Excellent reviews of Campylobacter resistance to quinolones and macrolides as well as of the molecular mechanisms
of this resistance, its emergence, epidemiology and persistence have been published recently (e.g. Alfredson &
Korolik, 2007; Belanger & Shryock, 2007; EFSA, 2009b;
Gibreel & Taylor, 2006; Gyles, 2008; Luangtongkum et al.,
2009; Moore et al., 2006; Payot et al., 2006). However, because of its frequency, especially in the southern part of
Europe, it is worthwhile to emphasise the multidrug resistance of Campylobacter in the food chain.

5%

The occurrence of resistance to quinolone antibiotics,

e.g. nalidixic acid and ciprofloxacin, reflecting the same
or similar genetic mechanisms, was most often reported,
but the ranges were extremely different, from nearly 0%
in Scandinavia (e.g. Skjt-Rasmussen et al., 2009) to levels
reflecting nearly total resistance, especially for C. coli in
broilers, pigs, but also broiler meat in some EU-MSs
(EU-27 country reports, available from http://www.efsa.
europa.eu/) and from some non-EU southern-European
countries (Bostan, Aydin, & Ang, 2009; Kurincic,
Lusicky, Uzunovic-Kamberovic, & Smole Mozina, 2009;
Tambur, Miljkovic-Selimovic, Bokonjic, & Kulisic,
2009). In Slovenia, the first evidence of the increasing prevalence of Campylobacter quinolone resistance was found
10 years ago for human clinical isolates (Berce,
Sarjanovic, & Smole Mozina, 2004) but there were no
data for food isolates. In our report on chicken meat isolates
(N 55) from 2001 to 2003, on average 49.1% of them
were resistant to nalidixic acid (Kurincic, Berce, Zorman,
& Smole Mozina, 2005); in 2006 this rate had increased
to 78.6% (N 98) (EFSA, 2007c), but in 2009 it was
nearly 86.0% (Smole Mozina, unpublished).
Compared to the data for quinolones, resistance to erythromycin in the period 2004e2007 was generally low, ranging from 0 to 13% for C. jejuni. However, the prevalence of
erythromycin resistant isolates was higher for C. coli,
mainly from pigs and pork meat, probably reflecting veterinary chemotherapeutic interventions in managing infections in these food-producing animals (EFSA, 2009a).
The prevalence of erythromycin resistance was also relatively very high in some non-EU southern-European countries (Uzunovic-Kamberovic, Zorman, Berce, Herman, &
Smole Mozina, 2009; Uzunovic-Kamberovic, Zorman,
Heyndrickx, & Smole Mozina, 2007), but even more important was the fact that erythromycin resistant isolates
were very often (Bostan et al., 2009; Kurincic et al.,
2005; Kurincic, Botteldoorn, Herman, & Smole Mozina,
2007), or even always multiresistant strains (Lehtopolku

6%

12%

21%
18%

21%

93

21%
0R

16%

1R
2R

36%

3R

23%

38%

Human isolates ,
N = 636

32%

Animal isolates,
N = 246

>3R

22%

31%

Retail chicken meat isolates,

N = 413

Fig. 3. Multiresistance of Campylobacter jejuni/coli isolates from human clinical samples, broiler flocks and retail chicken meat samples (source:
EFSA country reports for A, D, HU and SI, 2007 and 2009).

Author's personal copy

S.S. Mo
z ina et al. / Trends in Food Science & Technology 22 (2011) 91e98

94

et al., 2010). Similar results were found when we tested retail meat and human clinical samples in two geographical
areas (Slovenia and Bosnia and Herzegovina) already in
the period 2001e2003 (Kurincic et al., 2009; Smole
Mozina, Zorman, & Berce, 2005).
In Fig. 3, combined data about the multiple antibiotic resistance of C. jejuni/coli isolates from chicken meat and
other sources (animal, human isolates) reported to EFSA
in the years 2006e2007 from four central European countries are presented (Data from Austria, Germany, Hungary
and Slovenia were included). For all of them resistance to
aminoglycosides (gentamicin), fluoroquinolones (enrofloxacin), macrolides (erythromycin), quinolones (nalidixic
acid), penicillins (ampicillin) and tetracyclines (oxytetracyclin) was tested. Much higher prevalence of multiresistant
strains is again evident for the food chain, especially
meat isolates, in comparison to human isolates.

others, as well as a multidrug resistant phenotype

(Gibreel, Wetsch, & Taylor, 2007; Lin et al., 2005).
The activity of non-specific efflux pumps could be involved in bacterially acquired reduced susceptibility and/
or resistance against unrelated antimicrobials, including antibiotics but also other compounds, like bile and bile salts,
important for enteric pathogen host colonization and virulence. Studies of multidrug resistance (MDR) mediated
by efflux pumps as well as efflux pump inhibition by efflux
pump inhibitors, has recently become an important research
area, mainly in medical microbiology. Several new putative
bacterial efflux pump inhibitors (EPIs) have been described, but one of the first with known EPI activity was
Phe-Arg-b-naphthylamide (PAbN) in Pseudomonas aeroginosa (Lomovskaya et al., 2001). Several studies showed
that PAbN was also able to restore erythromycin susceptibility in Campylobacter strains (Cagliero, Mouline, Payot,
& Cloeckaert, 2005; Gibreel et al., 2005; Kurincic et al.,
2007; Payot et al., 2004). Recently, 1-(1-naphthylmethyl)piperazine (NMP) has been shown to reverse MDR first
in Escherichia coli (Bohnert & Kern, 2005) and recently
also in Campylobacter (Hannula & Hanninen, 2008).
In recent research we introduced several experimental
approaches for studying the involvement of efflux in the
multiple resistance of Campylobacter. Testing the efficiency of well-known and presumptive efflux pump inhibitors (EPIs) in combination with phenotypically and
genotypically characterized Campylobacter isolates from
the food chain, as well as with the type strains and mutants
in efflux pump genes, revealed new data about the mechanisms involved in microbial (multi)resistance. For illustration, Table 1 summarizes the results of ten C. jejuni and
C. coli food/animal/water isolates and reference strains
for their erythromycin and ciprofloxacin resistance when
determined by the broth microdilution method with or

Efflux pumps as mechanisms for the multiple

resistance of Campylobacter
Resistance to antimicrobial drugs has been mainly attributed to mutations in specific genes, e.g. in Campylobacter to quinolones and macrolides in the gyrA-encoding
subunit of the DNA gyrase (Griggs et al., 2005; Piddock,
Ricci, Pumbwe, Everret, & Griggs, 2003) and in domain
V of the 23S rRNA gene, respectively (Payot et al.,
2004). In addition to target mutations, resistance to antibiotics can be conferred by efflux pumps, limiting the access
of antibiotics to their targets by actively pumping out these
molecules, thereby preventing the intracellular accumulation necessary for lethality. A resistance-nodulation-cell division (RND) type of efflux pump CmeABC in C. jejuni
(Lin, Michel, & Zhang, 2002; Pumbwe & Piddock, 2002)
was described, which confers intrinsic resistance to several
antibiotics including erythromycin, ciprofloxacin and

Table 1. (Multi)resistance (minimal inhibitory concentration (MIC, mg/mL or mg/mL) in bold means high resistance) of ten Campylobacter isolates from the food chain and the efficiency (U) of efflux pump inhibitors (EPIs, PAbN (20 mg/mL) and NMP (80 mg/mL)) in reducing the resistance to erythromycin, ciprofloxacin, bile salts and Na-deoxycholate.
Strain

803
FC8
722
725
375
M37
171
7114
1076
140
ATCC 33559
ATCC 33560

Source

Species

Water jejuni
Poultry coli
Pig
coli
Pig
coli
Poultry jejuni
Poultry coli
Poultry coli
Pig
coli
Pig
coli
Poultry coli
Feces, coli
pig
Feces, jejuni
bovine

Erythromycin

Ciprofloxacin

Bile salts

Na-deoxycholate

MIC
mg/mL

U-PAN

U-NMP

MIC
mg/mL

U-PAN

U-NMP

MIC
mg/mL

U-PAN

U-NMP

MIC
mg/mL

U-PAN

U-NMP

1
4
8
8
64
512
512
>512
>512
>512
2

16
32
64
128
32
32
16
32
32
32
32

32
8
1
1
16
4
4
512
32
8
32

2
64
128
0.25
128
2
16
8
64
32
0.063

1
2
64
1
64
1
1
1
64
1
1

1
4
64
2
64
2
2
2
64
4
1

64
64
64
16
16
16
16
16
64
64
64

2
4
2
4
2
1
4
4
64
1
32

4
64
16
8
4
2
8
16
64
2
32

64
64
16
64
8
32
16
2
32
16
32

4
1
1
8
8
1
16
4
64
1
4

32
64
2
32
32
32
16
4
32
2
64

0.25

64

32

64

32

A four-fold or greater reduction of MIC after EPI addition is considered significant and indicated in bold.

Author's personal copy

MIC EGCG

e The efficiency was tested at the concentration of 0.5 MIC of EGCG.U-**0.25

MIC EGCG

8
16
4
8
4
64
16
64
16
32
16
1
8
1
1
8
2
32
1
1
1
128
64
8
0.25
1
1
1
1
8
2
1
1
1
4
0.5
64
4
>512
0.25
jejuni
jejuni
coli
coli
jejuni
Poultry
Poultry
Pig
Pig
Human
K49/4
375
FC8
7114
NCTC
11168

U-*0.5

8
32
16
8
32
64
8
64
2
64
4
16
16
2
16

U-0.25
U-0.5
U-0.5

e The efficiency was tested at the concentration of 0.25 MIC of EGCG.

32
32
16
8
32

MIC

U-0.25

EGCG

MIC

U-0.5

EGCG

MIC
mg/mL
EGCG
EGCG
EGCG

MIC
mg/mL

U-*

0.5 MIC

U-**

0.25 MIC

MIC
mg/mL

EGCG

MIC

U-0.25

MIC

MIC
mg/mL

EGCG

MIC

EGCG

MIC

Na-deoxycholate
Bile salts
Ciprofloxacin
Erythromycin
Species
Source

Future challenges
We are currently studying several approaches to confirm
the specific role of Campylobacter efflux pumps via testing
well-known EPIs in isolates from the food chain and in the
mutants in specific efflux pump genes. Another important
subject is the involvement of efflux in resistance to other

95

Strain

without addition of EPIs (PAbN, NMP). The strains were

mostly selected for their erythromycin resistance, but susceptible strains were included as well. Three groups of
strains were observed: susceptible with MICs up to 4 mg/
mL, low level resistant (LLR) with MICs from 8 to
16 mg/mL, and high level resistant (HLR) with
MICs  64 mg/mL (Kurincic et al., 2007). To determine
the efflux impact on the level of resistance, we determined
MICs in the presence and absence of the efflux pump inhibitors PAbN and NMP. All the strains showed a significant
increase in susceptibility to erythromycin in the presence
of PAbN. The effects of PAbN and NMP on ciprofloxacin
susceptibility in the tested Campylobacter isolates were
much limited in comparison to erythromycin. Similar or
lower effects were recently reported from some other studies (Cagliero et al., 2005; Hannula & Hanninen, 2008), although they are rarely directly comparable due to the
different conditions used, e.g. concentrations of EPIs. However, their impact on reversing resistance is dose dependent
(Kurincic et al., 2009). A significant impact of efflux inhibitors on MICs, and thus the involvement of efflux in the resistance mechanism was also confirmed in the majority of
Campylobacter strains tested in combination with bile salts.
Both inhibitors were efficient, but in the case of Na-deoxycholate and bile salts, NMP had an even stronger effect.
PAbN in the range of 10e20 mg/mL had a much stronger
effect in C. coli than in C. jejuni (Kramar, Kurincic, &
Smole Mozina, submitted for publication). However,
C. coli has been much less studied until now, although
we confirmed in our previous studies quite a high prevalence of C. coli among chicken meat and, in the Balkan
region, also among human clinical isolates (UzunovicKamberovic et al., 2007; Zorman, Heyndrickx, UzunovicKamberovic, & Smole Mozina, 2006).
The results indicate that an active efflux is involved in
the low susceptibility and cross-resistance of Campylobacter to chemically unrelated compounds, e.g. antibiotics, bile
salts and others. Thus, the inhibition of efflux pumps by inhibitors is a potential means for therapeutic interventions to
reduce Campylobacter colonizations in animal reservoirs,
as well as for more efficient strategies for MDR
Campylobacter risk management in the food chain. However, non-toxic efflux inhibitors are needed for this and
the development and/or search for natural substrates as inhibitors of efflux mediated resistance to antimicrobials is
becoming an attractive research area, with broad applications in human and veterinary medicine as well as in other
niches, including antimicrobial resistant microorganism in
the food production chain.

Table 2. The efficiency (U) of the plant phenolic compound, (L)epigallocatechin gallate (EGCG) as a putative efflux pump inhibitor in Campylobacter isolates in reducing the resistance to
erythromycin, ciprofloxacin, bile salts and Na-deoxycholate. Four-fold or greater reduction of MIC is considered significant and indicated in bold (MICs of EGCG for the isolates K49/4 and
375 were 0.078, for FC8 was 0.625, but for the isolates 7114 and 11168 were 0.313 mg/mL).

S.S. Mo
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Author's personal copy

96

S.S. Mo
z ina et al. / Trends in Food Science & Technology 22 (2011) 91e98

antimicrobials frequently used in the food chain, e.g. disinfectants, cleaning and decontamination agents, to contribute to knowledge about a possible link between the
resistance to biocides and antibiotics, which has been recently confirmed in some other enteric pathogens
(Karatzas et al., 2007; Thorrold, Letsoalo, Duse, &
Marais, 2007). However, the most challenging task is
screening for natural antimicrobial and resistance modifying agents, preferably from plants because of their GRAS
status (Klancnik, Guzej, Kolar, Abramovic, & Smole
Mozina, 2009). In Table 2, the activity of the plant phenolic
compound ()-epigallocatechin gallate (EGCG) as a putative efflux pump inhibitor and resistance modifying agent
in selected Campylobacter isolates is presented. Some
strains (e.g. C. coli VC7114 and FC8 and C. jejuni 375)
are the same as in Table 1, so also comparison with the activity of known EPIs, PAbN and NMP is possible. To our
knowledge this is the first evidence of the activity of any
plant phenolic compound as resistance modifying agent in
Campylobacter for some non-related antimicrobials. This
could have very useful applications, but should be further
studied with higher number of natural isolates and their mutants in target efflux genes both in in vitro and in vivo
systems.
Acknowledgement
We are grateful to Jasna Kovac for help in preparing
some figures and The Slovenian Research Agency
(ARRS) for PhD grants for M.K. and A.M., a post-doc
grant to A.K. and the financing of our national and bilateral
projects. This work was also partially financed by FP6036272.
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