Campylobacter is a leading foodborne bacterial pathogen, which causes gastroenteritis in humans. This pathogenic organism is increasingly resistant to antibiotics, especially fluoroquinolones and macrolides, which are the most frequently used antimicrobials for the treatment of campylobacteriosis when clinical therapy is warranted. As a zoonotic pathogen, Campylobacter has a broad animal reservoir and infects humans via contaminated food, water or milk. Antibiotic usage in both animal agriculture and human medicine, can influence the development of antibiotic-resistant Campylobacter. This review will describe the trend in fluoroquinolone and macrolide resistance in Campylobacter, summarize the mechanisms underlying the resistance to various antibiotics and discuss the unique features associated with the emergence, transmission and persistence of antibiotic-resistant Campylobacter. Special attention will be given to recent findings and emphasis will be placed on Campylobacter resistance to fluoroquinolones...

Campylobacter jejuni , a major foodborne human pathogen, has become increasingly resistant to fluoroquinolone (FQ) antimicrobials. By using clonally related isolates and genetically defined mutants, we determined the fitness of FQ-resistant Campylobacter in chicken (a natural host and a major reservoir for C. jejuni ) in the absence of antibiotic selection pressure. When monoinoculated into the host, FQ-resistant and FQ-susceptible Campylobacter displayed similar levels of colonization and persistence in the absence of FQ antimicrobials. The prolonged colonization in chickens did not result in loss of the FQ resistance and the resistance-conferring point mutation (C257 → T) in the gyrA gene. Strikingly, when coinoculated into chickens, the FQ-resistant Campylobacter isolates outcompeted the majority of the FQ-susceptible strains, indicating that the resistant Campylobacter was biologically fit in the chicken host. The fitness advantage was not due to compensatory mutations in the genes targeted by FQ and was linked directly to the single point mutation in gyrA , which confers on Campylobacter a high-level resistance to FQ antimicrobials. In certain genetic backgrounds, the same point mutation entailed a biological cost on Campylobacter , as evidenced by its inability to compete with the FQ-susceptible Campylobacter . These findings provide a previously undescribed demonstration of the profound effect of a resistance-conferring point mutation in gyrA on the fitness of a major foodborne pathogen in its natural host and suggest that the rapid emergence of FQ-resistant Campylobacter on a worldwide scale may be attributable partly to the enhanced fitness of the FQ-resistant isolates.

CmeABC functions as a multidrug efflux pump contributing to the resistance ofCampylobacterto a broad range of antimicrobials. In this study, we examined the role of CmeABC in bile resistance and its contribution to the adaptation ofCampylobacter jejuniin the intestinal tract of the chicken, a natural host and a major reservoir forCampylobacter. Inactivation ofcmeABCdrastically decreased the resistance ofCampylobacterto various bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of thecmeABCmutants but had no effect on the growth of the wild-type strain. Bile concentration varied in the duodenum, jejunum, and cecum of chicken intestine, and the inhibitory effect of the intestinal extracts on the in vitro growth ofCampylobacterwas well correlated with the total bile concentration in the individual sections of chicken intestine. When inoculated into chickens, the wild-type strain colonized the birds as early as day 2 postinoculation with a density as high as 10⁷CFU/g of feces. In contrast, thecmeABCmutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for thecmeABCmutant was at least 2.6 × 10⁴-fold higher than that of the wild-type strain. Complementation of thecmeABCmutants with a wild-typecmeABCallele intransfully restored the in vitro growth in bile-containing media and the in vivo colonization to the levels of the wild-type strain. Immunoblotting analysis indicated that CmeABC is expressed and immunogenic in chickens experimentally infected withC. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating resistance to bile salts in the intestinal tract, is required for successful colonization ofC. jejuniin chickens. Inhibition of CmeABC function may not only control antibiotic resistance but also prevent the in vivo colonization of pathogenicCampylobacter.

CmeABC, a resistance-nodulation-division (RND) type of efflux pump, contributes to Campylobacter resistance to a broad spectrum of antimicrobial agents and is also essential for Campylobacter colonization of the animal intestinal tract by mediation of bile resistance. As one of the main systems for Campylobacter adaptation to different environments, CmeABC is likely subject to control by regulatory elements. We describe the identification of a transcriptional repressor for CmeABC. Insertional mutagenesis of cmeR , an open reading frame immediately upstream of the cmeABC operon, resulted in overexpression of cmeABC , as determined by transcriptional fusion (P _cmeABC-lacZ ) and immunoblotting with CmeABC-specific antibodies. Overexpression of the efflux pump was correlated with a moderate increase in the level of resistance of the cmeR mutant to several antimicrobials. In vitro, recombinant CmeR bound specifically to the promoter region of cmeABC , precisely, to the inverted repeat sequences in the cmeABC promoter. A single nucleotide deletion between the two half sites of the inverted repeat reduced the level of CmeR binding to the promoter sequence and resulted in overexpression of cmeABC . Together, these findings indicate that cmeR encodes a transcriptional repressor that directly interacts with the cmeABC promoter and modulates the expression of cmeABC . Mutation either in CmeR or in the inverted repeat impedes the repression and leads to enhanced production of the MDR efflux pump.

Using laboratory challenge experiments, we examined whether Campylobacter- specific maternal antibody (MAB) plays a protective role in young chickens, which are usually free of Campylobacter under natural production conditions. Kinetics of C. jejuni colonization were compared by infecting 3-day-old broiler chicks, which were naturally positive for Campylobacter -specific MAB, and 21-day-old broilers, which were negative for Campylobacter -specific MAB. The onset of colonization occurred much sooner in birds challenged at the age of 21 days than it did in the birds inoculated at 3 days of age, which suggested a possible involvement of specific MAB in the delay of colonization. To further examine this possibility, specific-pathogen-free layer chickens were raised under laboratory conditions with or without Campylobacter infection, and their 3-day-old progenies with (MAB ⁺ ) or without (MAB ⁻ ) Campylobacter -specific MAB were orally challenged with C. jejuni . Significant decreases in the percentage of colonized chickens were observed in the MAB ⁺ group during the first week compared with the MAB ⁻ group. These results indicate that Campylobacter- specific MAB plays a partial role in protecting young chickens against colonization by C. jejuni . Presence of MAB in young chickens did not seem to affect the development of systemic immune response following infection with C. jejuni . However, active immune responses to Campylobacter occurred earlier and more strongly in birds infected at 21 days of age than those infected at 3 days of age. Clearance of Campylobacter infection was also observed in chickens infected at 21 days of age. Taken together, these findings (i) indicate that anti- Campylobacter MAB contributes to the lack of Campylobacter infection in young broiler chickens in natural environments and (ii) provide further evidence supporting the feasibility of development of immunization-based approaches for control of Campylobacter infection in poultry.

CmeABC, a multidrug efflux pump, is involved in the resistance ofCampylobacter jejunito a broad spectrum of antimicrobial agents and is essential forCampylobactercolonization in animal intestine by mediating bile resistance. Previously, we have shown that expression of this efflux pump is under the control of a transcriptional repressor named CmeR. Inactivation of CmeR or mutation in thecmeABCpromoter (P_cmeABC) region derepressescmeABC, leading to overexpression of this efflux pump. However, it is unknown if the expression ofcmeABCcan be conditionally induced by the substrates it extrudes. In this study, we examined the expression ofcmeABCin the presence of various antimicrobial compounds. Although the majority of the antimicrobials tested did not affect the expression ofcmeABC, bile salts drastically elevated the expression of this efflux operon. The induction was observed with both conjugated and unconjugated bile salts and was in a dose- and time-dependent manner. Experiments using surface plasmon resonance demonstrated that bile salts inhibited the binding of CmeR to P_cmeABC, suggesting that bile compounds are inducing ligands of CmeR. The interaction between bile salts and CmeR likely triggers conformational changes in CmeR, resulting in reduced binding affinity of CmeR to P_cmeABC. Bile did not affect the transcription ofcmeR, indicating that altered expression ofcmeRis not a factor in bile-induced overexpression ofcmeABC. In addition to the CmeR-dependent induction, some bile salts (e.g., taurocholate) also activated the expression ofcmeABCby a CmeR-independent pathway. Consistent with the elevated production of CmeABC, the presence of bile salts in culture media resulted in increased resistance ofCampylobacterto multiple antimicrobials. These findings reveal a new mechanism that modulates the expression ofcmeABCand further support the notion that bile resistance is a natural function of CmeABC.