Here, we evaluated Campylobacter contamination on chicken carcasses and phenotypic and genotypic profiles of antimicrobial resistance of the isolated strains. A total of 95 of samples were collected ...from 19 slaughterhouses from Minas Gerais - Brazil, and analyzed by MPN-PCR method. Campylobacter was found in 16.8% of samples with microbial load ranging from 60 to 184 MPN/carcass. All isolates were resistant to at least 5 (31.2%) of the antimicrobials screened using the disk diffusion method. Thr-86-Ile gyrA mutation, blaOxA-61 and tet(O) genes were found in 95%, 100% and 40% resistant isolates to ciprofloxacin, ampicillin and tetracycline, respectively. Almost all isolates (90%) showed the three genes required to synthesize the CmeABC efflux system. The use of efflux pump inhibitor (PAβN) resulted in a significant reduction in the MICs of antimicrobials (2–128 fold), indicating the importance of efflux systems in conferring antimicrobial resistance. Campylobacter were detected at low concentrations in Brazilian chicken carcasses. However, high-levels of antimicrobial resistance were observed and associated with several mechanisms. This study provides a baseline survey on contamination of Campylobacter in Brazilian chicken carcasses and its antimicrobial resistance, giving support for actions directed at reducing this pathogen in the food chain.
•Low prevalence and counts of Campylobacter spp. were observed in chicken carcasses slaughtered in Minas Gerais state, Brazil.•High-levels of antimicrobial resistance were observed in the Campylobacter isolates.•Several mechanisms of antimicrobial resistance were identified, including CmeABC efflux system.•The CmeABC efflux system has proven to play a key role in mediating bile salts resistance.•The knowledge of antimicrobial resistance will help reduce Campylobacter spp. in the food chain.
In this study, we aimed to determine the antibiotic resistance status of
spp. isolated from human infections in our region, including the role of mechanisms involved in erythromycin resistance. ...Standard methods were used for the isolation, identification and antibiotic susceptibility testing of
spp. isolates. Erythromycin-resistant mutants were selected from erythromycin-susceptible clinical isolates, and the erythromycin resistance mechanisms were investigated phenotypically by determining the erythromycin MICs of isolates in the presence and absence of the resistance nodulation cell division (RND) type efflux pump inhibitor, phenylalanine-arginine β-naphthylamide dihydrochloride (PAβN), and genotypically by determining ribosomal and
alterations using PCR and DNA sequence analysis.
spp., including 184
and 20
in a two-year period, were the most frequently isolated gastrointestinal bacterial pathogens in our region. However, in both
and
resistance to tetracycline and ciprofloxacin were found to be high, erythromycin resistance was especially high (20%) in
. With a ribosomal alteration, A2075G, which was found to be associated with high-level erythromycin resistance in clinical isolates, PAβN significantly reduced the erythromycin MICs in both clinical isolates and mutants. An important finding of this study, while considering
operon, is the explanation of why erythromycin resistance is more common among
than
bearing in mind the specific deletions and alterations in the intergenic region of the operon in all erythromycin-resistant
isolates. Ultimately, these findings revealed the significant role of RND-type efflux activity in increased erythromycin MICs of the isolates.
Campylobacter spp. are the leading cause of human gastroenteritis worldwide. RE-CmeABC is a newly identified resistance-enhancing multidrug efflux pump of Campylobacter spp. (C. spp.) that confers ...high-level resistance to fluoroquinolones, phenicols, macrolides, and tetracyclines (TETs), all of which are critical drugs in both human and veterinary medicine. In this study, we analyzed the presence and antimicrobial susceptibility of RE-cmeABC-positive Campylobacter isolates of food-animal origin from three representative regions (Shandong, Shanghai, and Guangdong) in China over three successive years, from 2014 to 2016. A total of 1088 Campylobacter isolates (931 C. coli and 157 C. jejuni) were recovered from the RE-cmeABC screening. We detected 122 (11.2%) RE-cmeABC-positive isolates of chicken origin, including 111 (70.7%) C. jejuni and 11 (1.2%) C. coli. This multidrug efflux pump is more prevalent among C. jejuni than C. coli. The level of resistance was significantly different in 111 RE-cmeABC-positive C. jejuni versus 46 RE-cmeABC-negative C. jejuni for florfenicol, clindamycin, and erythromycin (P < 0.05), but not for ciprofloxacin (CIP), TET, and gentamicin (GEN). However, the isolates harboring RE-cmeABC could shift the minimum inhibitory concentration distribution to the higher range for CIP and TET. Pulsed-field gel electrophoresis (PFGE) analysis suggested that horizontal transmission might be involved in the dissemination of RE-cmeABC in Shanghai and Guangdong, while clonal expansion was predominant in Shandong. Three isolates shared the indiscriminate PFGE types of RE-cmeABC-positive C. jejuni isolates in Shanghai and Guangdong, and four isolates in Shanghai and Shandong. Our study suggests the possibility of a wide dissemination of RE-cmeABC in Campylobacter of food-animal origin, which would pose a significant threat to public health.
During transmission and intestinal colonization, Campylobacter jejuni, a major foodborne human pathogen, experiences oxidative stress. CosR, a response regulator in C. jejuni, modulates the oxidative ...stress response and represses expression of the CmeABC multidrug efflux pump. CmeABC, a key component in resistance to toxic compounds including antimicrobials and bile salts, is also under negative regulation by CmeR, a TetR family transcriptional regulator. How CosR and CmeR interact in binding to the cmeABC promoter and how CosR senses oxidative stress are still unknown. To answer these questions, we conducted various experiments utilizing electrophoretic mobility shift assays and transcriptional fusion assays. CosR and CmeR bound independently to two separate sites of the cmeABC promoter, simultaneously repressing cmeABC expression. This dual binding of CosR and CmeR is optimal with a 17 base pair space between the two binding sites as mutations that shortened the distance between the binding sites decreased binding by CmeR and enhanced cmeABC expression. Additionally, the single cysteine residue (C218) of CosR was sensitive to oxidation, which altered the DNA-binding activity of CosR and dissociated CosR from the cmeABC promoter as determined by electrophoretic mobility shift assay. Replacement of C218 with serine rendered CosR insensitive to oxidation, suggesting a potential role of C218 in sensing oxidative stress and providing a possible mechanism for CosR-mediated response to oxidative stress. These findings reveal a dual regulatory role of CosR and CmeR in modulating cmeABC expression and suggest a potential mechanism that may explain overexpression of cmeABC in response to oxidative stress. Differential expression of cmeABC mediated by CmeR and CosR in response to different signals may facilitate adaptation of Campylobacter to various environmental conditions.
Sequence analysis of 79 ciprofloxacin-resistant
isolates collected in China showed resistance-related sequence variations in
and CmeR-Box. All the isolates contain an identical Thr-86-Ile ...substitution in GyrA. Several novel CmeR-Box variations, including point substitutions, deletion, and insertion, were identified. The point insertion or deletion led to dramatically reduced binding of CmeR to the
promoter, which significantly increases the expression of
and contributes to the high fluoroquinolone resistance.
We assessed the susceptibility of 182 Campylobacter jejuni isolates from patients with diarrhea to eight antibiotics and analyzed the molecular mechanisms of ciprofloxacin resistance as well as the ...genetic characteristics based on multilocus sequence typing (MLST). The C257T mutation was found on the quinolone resistance-determining region (QRDR) of the gyrA gene in all ciprofloxacin-resistant strains. Mutations on the QRDR of the gyrB gene were silent. A total of 74 strains had 7 inverted repeat (IR) (a 16-bp IR on the intergenic region between cmeR and cmeABC) mutation polymorphisms. Compared with strains without the IR mutations, strains with the IR mutations had higher resistance rates to ciprofloxacin (94.6% vs. 83.3%), nalidixic acid (94.6% vs. 83.3%), tetracycline (98.6% vs. 85.2%), doxycycline (91.9% vs. 71.3%), florfenicol (59.5% vs. 17.6%), chloramphenicol (25.7% vs. 4.6%), gentamicin (16.2% vs. 3.7%), and multidrug resistance than those without IR mutations (all p < 0.05). With C257T mutation alone, 89.9% strains with minimum inhibitory concentration (MIC) values focused on 16, 32, and 64 μg/mL, whereas strains with C257T mutation in combination with the IR mutations had a higher ciprofloxacin resistance level with 88.6% MIC values focused on 64, 128, and 512 μg/mL (p < 0.0001). The strains in this study showed a high genetic variability based on MLST with 117 sequence types (STs), 37 of which were novel. CC-21 was the most common clonal complex (CC) followed by CC-353 and CC-45. No association was found between STs and ciprofloxacin resistance. In conclusion, the C257T mutation on gyrA was the major mechanism for ciprofloxacin resistance, and the C257T mutation in combination with the IR mutations might result in more severe ciprofloxacin resistance to C. jejuni.
Objectives To investigate the molecular mechanisms involved in the high-level erythromycin resistance of clinical Spanish Campylobacter jejuni and Campylobacter coli strains. Methods Overall ...susceptibilities of 678 C. jejuni and 119 C. coli strains, collected from 10 Spanish provinces during 2006 and 2007, were determined by Etest. In high-level erythromycin-resistant strains, molecular determinants were studied. The analysis was focused on region V of the 23S rRNA gene, the rplD and rplV ribosomal genes, and the regulatory region of the CmeABC efflux pump. Results The global resistance rate to erythromycin was 3.8%. Among the resistant strains, 93% were C. coli and 7% were C. jejuni. The A2075G mutation in the 23S rRNA gene was detected in all of the resistant strains except for two, which carried the A2074G mutation. None of the ribosomal rplD and rplV genes harboured the described mutations that confer resistance to macrolides. Different mutations affecting the regulatory region of the CmeABC efflux pump were also found. Conclusions C. coli strains are clearly more resistant to erythromycin than C. jejuni. The mutation A2075G in the 23S rRNA gene was responsible for the resistance in most of the strains; A2074G was only found in two strains. Further studies are required to ascertain the effect of mutations in the regulatory region of cmeABC. Our data indicate that the rate of resistance was similar to that of other European countries.
•Identification of C−32→T mutation in the cmeABC operon of avian Campylobacter jejuni isolates.•The mutation is associated with overexpression of the CmeABC efflux system.•The clonal population ...harbouring the mutation is multidrug-resistant (MDR).•We describe the mechanisms of resistance for the antimicrobials tested.•Description of a MDR C. jejuni clone without previous antimicrobial treatment.
The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, β-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, β-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed β-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C−32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C−32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.
Objectives We analysed the contribution of spontaneous mutation frequency to the evolution of ciprofloxacin resistance and the diversity of mutations in the quinolone resistance-determining region ...(QRDR) of gyrA and in the intergenic region, cmeR–cmeA, of the CmeABC efflux pump in Campylobacter jejuni and Campylobacter coli. Methods The mutation frequency was measured in 11 quinolone-susceptible C. jejuni and 5 C. coli strains, with and without ox bile. MICs and target-specific and efflux-associated mutations were studied for a number of colonies of each strain selected from plates containing 1 mg/L ciprofloxacin. Results The spontaneous mutation frequency level among susceptible C. jejuni and C. coli strains ranged from hypomutable (∼4 × 10−9) to strongly mutable (∼7 × 10−3). Ox bile had no effect on mutation frequency. Pre-existing ampicillin and tetracycline resistance increased the MICs of ciprofloxacin for two strains from 0.032–0.125 to 0.5 mg/L. MICs of ciprofloxacin for the colonies selected from plates containing 1 mg/L ciprofloxacin varied from 1 to 16 mg/L, with the Thr-86→Ile or Asp-90→Asn mutation detected in the QRDR of gyrA. In 21.5% (14/65) of the selected colonies, no specific mutations existed. Two types of mutations in CmeR promoter-binding inverted sequences were identified both in the parent strains and in the colonies selected from ciprofloxacin plates. Conclusions The variation in mutation frequencies between most C. jejuni and C. coli strains was up to 700-fold. Mutation in the QRDR of gyrA or in the intergenic region was not associated with differences in spontaneous mutation frequencies. Previously acquired resistance to tetracycline and ampicillin predisposed strains to high-level ciprofloxacin resistance and to multiple antibiotic resistance.
The multidrug efflux pump CmeABC described in the food-borne pathogen
Campylobacter jejuni was shown to be an important element of bile resistance and significant for successful colonization of ...chicken intestines. Recently, another gene (
Cj0561c) strongly suppressed by the same repressor (CmeR) that regulates expression of CmeABC was identified in
C. jejuni NCTC 11168. Initial data suggested that, similarly to
cmeABC,
Cj0561c could be induced by bile salts. In the present study, the occurrence of the
Cj0561c gene and bile-salt-dependent induction was investigated in ten poultry and eight human
C. jejuni strains. The
Cj0561c gene was present in all strains. When cultured without addition of bile salts, the transcription level of that gene was about tenfold higher than that of
cmeABC. Bile salts cholate and taurocholate induced transcription of
cmeABC 1.66-fold and 2.71-fold and that of
Cj0561c 3.71-fold and 10.99-fold, respectively. Thus
Cj0561c was more effectively induced by bile salts than
cmeABC and taurocholate was superior to cholate as an inducer of transcription. More efficient induction of both
cmeABC and
Cj0561c by taurocholate might be the reason for the higher minimum inhibitory concentrations (MICs) observed with taurocholate than with cholate (100 mg/ml vs. 10 mg/ml). There was no significant difference between poultry and human
C. jejuni strains concerning the transcription levels of
cmeABC and
Cj0561c in cultures without bile salts and concerning bile-salt-induced changes in transcription of
cmeABC and
Cj0561c. Thus, higher MIC values observed for taurocholate in human strains than in poultry strains (200 mg/ml vs. 75 mg/ml) could not be explained by different responses of
cmeABC and
Cj0561c to bile salts. Therefore, they must be due to another mechanism.
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