Campylobacter jejuni, a gram-negative organism causing gastroenteritis in humans, is increasingly resistant to antibiotics. However, little is known about the drug efflux mechanisms in this pathogen. Here we characterized an efflux pump encoded by a three-gene operon (designated cmeABC) that contributes to multidrug resistance in C. jejuni 81-176. CmeABC shares significant sequence and structural homology with known tripartite multidrug efflux pumps in other gram-negative bacteria, and it consists of a periplasmic fusion protein (CmeA), an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily (CmeB), and an outer membrane protein (CmeC). Immunoblotting using CmeABC-specific antibodies demonstrated that cmeABC was expressed in wild-type 81-176; however, an isogenic mutant (9B6) with a transposon insertion in the cmeB gene showed impaired production of CmeB and CmeC. Compared to wild-type 81-176, 9B6 showed a 2-to 4,000-fold decrease in resistance to a range of antibiotics, heavy metals, bile salts, and other antimicrobial agents. Accumulation assays demonstrated that significantly more ethidium bromide and ciprofloxacin accumulated in mutant 9B6 than in wild-type 81-176. Addition of carbonyl cyanide m-chlorophenylhydrazone, an efflux pump inhibitor, increased the accumulation of ciprofloxacin in wild-type 81-176 to the level of mutant 9B6. PCR and immunoblotting analysis also showed that cmeABC was broadly distributed in various C. jejuni isolates and constitutively expressed in wild-type strains. Together, these findings formally establish that CmeABC functions as a tripartite multidrug efflux pump that contributes to the intrinsic resistance of C. jejuni to a broad range of structurally unrelated antimicrobial agents.Bacterial pathogens have evolved multiple mechanisms for resistance to antimicrobial agents, which has greatly compromised the effectiveness of antibiotic treatments and poses a serious threat to public health (13, 17). As one of the general resistance mechanisms, antibiotic efflux systems extrude structurally diverse antimicrobial agents out of bacterial cells (6,8,27,29,36). One important family of drug transporters that contribute to multidrug resistance (MDR) in gram-negative bacteria are the resistance-nodulation-cell division (RND) efflux systems, which consist of an inner membrane transporter, a periplasmic fusion protein, and an outer membrane protein (39). Genetically, many of the RND-type MDR efflux systems or pumps are encoded by a three-gene operon located on the bacterial chromosome (25, 39). However, some efflux pumps, such as AcrAB from Escherichia coli (19), have an outer membrane component that is encoded by a separate gene physically unattached with the other two members on the bacterial chromosome. Expression of these pumps is controlled by regulatory proteins, and their overexpression is usually mediated by mutations in the regulatory elements resulting in an MDR phenotype (27,29,36). Even without overexpression, the MDR efflux pumps ...

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 antibioticresistant Campylobacter. Special attention will be given to recent findings and emphasis will be placed on Campylobacter resistance to fluoroquinolones and macrolides. A future perspective on antibiotic resistance and potential approaches for the control of antibiotic-resistant Campylobacter, will also be discussed.

By 2030, the global population will be 8.5 billion, placing pressure on international poultry production, of which China is a key producer. From April 2017, China will implement the withdrawal of colistin as a growth promoter, removing over 8,000 tonnes per year from the Chinese farming sector. To understand the impact of banning colistin and the epidemiology of multi-drug-resistant (MDR) Escherichia coli (using bla and mcr-1 as marker genes), we sampled poultry, dogs, sewage, wild birds and flies. Here, we show that mcr-1, but not bla, is prevalent in hatcheries, but bla quickly contaminates flocks through dogs, flies and wild birds. We also screened samples directly for resistance genes to understand the true breadth and depth of the environmental and animal resistome. Direct sample testing for bla and mcr-1 in hatcheries, commercial farms, a slaughterhouse and supermarkets revealed considerably higher levels of positive samples than the bla- and mcr-1-positive E. coli, indicating a substantial segment of unseen resistome-a phenomenon we have termed the 'phantom resistome'. Whole-genome sequencing identified common bla-positive E. coli shared among farms, flies, dogs and farmers, providing direct evidence of carbapenem-resistant E. coli transmission and environmental contamination.

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 3 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.colonization ͉ gyrA mutation ͉ poultry A ntimicrobial resistance in bacterial pathogens has become a serious threat to public health. There is a general notion that the acquisition of drug resistance, particularly the resistance mediated by chromosomal mutations, entails a biological cost for pathogens, resulting in reduced fitness in the absence of antibiotic selection pressure (1, 2). However, evidence has accumulated that in vivo-selected or clinically derived isolates may develop compensatory mutations that reduce the fitness cost associated with antimicrobial resistance (3-7). Different environments (in vitro vs. in vivo) may select for different compensatory mutations and varied levels of restoration of fitness (8). Even without compensatory mutations, drug-resistant mutants may show little or no fitness cost (9). One important finding revealed by previous studies is that clinically derived drugresistant isolates display diverse fitness changes, with some showing a biological cost and others showing no cost or even enhanced fitness (4, 10). The retention of ecological fitness in resistant pathogens creates a significant barrier for the elimination of resistant organisms by natural selection.Campylobacter jejuni, a Gram-negative microaerobic bacterium, is a common causative agent of human enterocolitis (11). For antibiotic treatment of ca...

CmeABC functions as a multidrug efflux pump contributing to the resistance of Campylobacter to a broad range of antimicrobials. In this study, we examined the role of CmeABC in bile resistance and its contribution to the adaptation of Campylobacter jejuni in the intestinal tract of the chicken, a natural host and a major reservoir for Campylobacter. Inactivation of cmeABC drastically decreased the resistance of Campylobacter to various bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of the cmeABC mutants 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 of Campylobacter was 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 10 7 CFU/g of feces. In contrast, the cmeABC mutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for the cmeABC mutant was at least 2.6 ؋ 10 4 -fold higher than that of the wild-type strain. Complementation of the cmeABC mutants with a wild-type cmeABC allele in trans fully 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 with C. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating resistance to bile salts in the intestinal tract, is required for successful colonization of C. jejuni in chickens. Inhibition of CmeABC function may not only control antibiotic resistance but also prevent the in vivo colonization of pathogenic Campylobacter.