Genetic Basis and Clonal Population Structure of Antibiotic Resistance in Campylobacter jejuni Isolated From Broiler Carcasses in Belgium.
ABSTRACT: Human campylobacteriosis is the leading food-borne zoonosis in industrialized countries. This study characterized the clonal population structure, antimicrobial resistance profiles and occurrence of antimicrobial resistance determinants of a set of Campylobacter jejuni strains isolated from broiler carcasses in Belgium. Minimum inhibitory concentrations (MICs) against five commonly-used antibiotics (ciprofloxacin, nalidixic acid, tetracycline, gentamicin, and erythromycin) were determined for 204 C. jejuni isolates. More than half of the isolates were resistant to ciprofloxacin or nalidixic acid. In contrast, a lower percentage of screened isolates were resistant to gentamicin or erythromycin. C. jejuni isolates resistant to ciprofloxacin and/or nalidixic acid were screened for the substitution T86I in the quinolone resistance determining region (QRDR) of the gyrA gene, while C. jejuni isolates resistant to tetracycline were screened for the presence of the tet(O) gene. These resistance determinants were observed in most but not all resistant isolates. Regarding resistance to erythromycin, different mutations occurred in diverse genetic loci, including mutations in the 23S rRNA gene, the rplD and rplV ribosomal genes, and the intergenic region between cmeR and cmeABC. Interestingly, and contrary to previous reports, the A2075G transition mutation in the 23S rRNA gene was only found in one strain displaying a high level of resistance to erythromycin. Ultimately, molecular typing by multilocus sequence typing revealed that two sequence types (ST-824 and ST-2274) were associated to quinolones resistance by the presence of mutations in the gene gyrA (p = 0.01). In addition, ST-2274 was linked to the CIP-NAL-TET-AMR multidrug resistant phenotype. In contrast, clonal complex CC-45 was linked to increased susceptibility to the tested antibiotics. The results obtained in this study provide better understanding of the phenotypic and the molecular basis of antibiotic resistance in C. jejuni, unraveling some the mechanisms which confer antimicrobial resistance and particular clones associated to the carriage and spread of resistance genes.
Project description:The aim of this study is to characterize the antimicrobial resistance of Campylobacter jejuni recovered from diarrheal patients in Belgium, focusing on the genetic diversity of resistant strains and underlying molecular mechanisms of resistance among Campylobacter jejuni resistant strains isolated from diarrheal patients in Belgium. Susceptibility profile of 199 clinical C. jejuni isolates was determined by minimum inhibitory concentrations against six commonly-used antibiotics (ciprofloxacin, nalidixic acid, tetracycline, streptomycin, gentamicin, and erythromycin). High rates of resistance were observed against nalidixic acid (56.3%), ciprofloxacin (55.8%) and tetracycline (49.7%); these rates were similar to those obtained from different national reports in broilers intended for human consumption. Alternatively, lower resistance rates to streptomycin (4.5%) and erythromycin (2%), and absolute sensitivity to gentamicin were observed. C. jejuni isolates resistant to tetracycline or quinolones (ciprofloxacin and/or nalidixic acid) were screened for the presence of the tetO gene and the C257T mutation in the quinolone resistance determining region (QRDR) of the gyrase gene gyrA, respectively. Interestingly, some of the isolates that displayed phenotypic resistance to these antimicrobials lacked the corresponding genetic determinants. Among erythromycin-resistant isolates, a diverse array of potential molecular resistance mechanisms was investigated, including the presence of ermB and mutations in the 23S rRNA gene, the rplD and rplV ribosomal genes, and the regulatory region of the cmeABC operon. Two of the four erythromycin-resistant isolates harboured the A2075G transition mutation in the 23S rRNA gene; one of these isolates exhibited further mutations in rplD, rplV and in the cmeABC regulatory region. This study expands the current understanding of how different genetic determinants and particular clones shape the epidemiology of antimicrobial resistance in C. jejuni in Belgium. It also reveals many questions in need of further investigation, such as the role of other undetermined molecular mechanisms that may potentially contribute to the antimicrobial resistance of Campylobacter.
Project description:The presence of virulence factors (VFs) and mechanisms of quinolones and macrolide resistance was analyzed in Campylobacter spp. from a pediatric cohort study in Lima. In 149 isolates (39 Campylobacter jejuni and 24 Campylobacter coli from diarrheic cases; 57 C. jejuni and 29 C. coli from controls), the presence of the cdtABC and cadF genes and iam marker was established. Nalidixic acid, ciprofloxacin, erythromycin, and azithromycin susceptibilities were established in 115 isolates and tetracycline-susceptibility was established in 100 isolates. The presence of mutations in the gyrA, parC, and 23S rRNA genes was determined. The cadF gene and all genes from the cdtABC operon were significantly more frequent among C. jejuni (P < 0.0001); the iam marker was more frequent in C. coli (P < 0.0001). No differences were observed in VFs between cases and controls. Almost all isolates were tetracycline-resistant; nalidixic acid and ciprofloxacin resistance reached levels of 90.4% and 88.7%, respectively. Resistance to macrolides was 13% (C. jejuni 4.3%; C. coli 26.1%). Resistance to ciprofloxacin was related to GyrA Thr86 substitutions, while 13 of 15 macrolide-resistant isolates possessed a 23S rRNA mutation (A2075G). Differences in the presence of VFs and alarming levels of resistance to tested antimicrobial agents were observed among C. jejuni and C. coli.
Project description:Point mutations in the topoisomerase (DNA gyrase A) gene are known to be associated with fluoroquinolone resistance in Campylobacter. Recent studies have shown that an efflux pump encoded by cmeABC is also involved in decreased susceptibilities to fluoroquinolones, as well as other antimicrobials. Genome analysis suggests that Campylobacter jejuni contains at least nine other putative efflux pumps. Using insertional inactivation and site-directed mutagenesis, we investigated the potential contributions of these pumps to susceptibilities to chloramphenicol, ciprofloxacin, erythromycin, and tetracycline in C. jejuni and Campylobacter coli. Insertional inactivation of cmeB resulted in 4- to 256-fold decreases in the MICs of chloramphenicol, ciprofloxacin, erythromycin, and tetracycline, with erythromycin being the most significantly affected. In contrast, inactivation of all other putative efflux pumps had no effect on susceptibility to any of the four antimicrobials tested. Mutation of gyrA at codon 86 (Thr-Ile) caused 128- and 64-fold increases in the MICs of ciprofloxacin and nalidixic acid, respectively. The replacement of the mutated gyrA with a wild-type gyrA allele resulted in a 32-fold decrease in the ciprofloxacin MIC and no change in the nalidixic acid MIC. Our findings indicate that CmeABC is the only efflux pump among those tested that influences antimicrobial resistance in Campylobacter and that a point mutation (Thr-86-Ile) in gyrA directly causes fluoroquinolone resistance in Campylobacter. These two mechanisms work synergistically in acquiring and maintaining fluoroquinolone resistance in Campylobacter species.
Project description:The antimicrobial susceptibilities of 378 Campylobacter isolates were determined. Resistance to tetracycline was the most common (82%), followed by resistance to doxycycline (77%), erythromycin (54%), nalidixic acid (41%), and ciprofloxacin (35%). Campylobacter coli displayed significantly higher rates of resistance to ciprofloxacin and erythromycin than Campylobacter jejuni, and Campylobacter isolates from turkey meat showed a greater resistance than those from chicken meat.
Project description:Campylobacter jejuni is one of the most common causes of human foodborne bacterial infections worldwide. The objective of this study was to assess the molecular diversity, using flaA sequencing, of 602 C. jejuni isolated from chicken food chain, i.e., chicken feces (n = 151), chicken carcasses (n = 150), chicken meat (n = 150), and from humans (n = 151) and to determine antimicrobial multiresistant profiles of the isolates as well as to analyze the relationship of the isolate genotypes with their antimicrobial resistance profiles and source of isolation. Multidrug resistant patterns were identified in 110 (18.3%) C. jejuni isolates recovered from all sources and most isolates were resistant to ciprofloxacin (CIP), nalidixic acid (NAL), streptomycin (STR), and tetracycline (TET) (92; 15.3%) or ciprofloxacin, streptomycin, and tetracycline (13; 2.2%). Only a few isolates were multiresistant to ciprofloxacin, nalidixic acid, tetracycline, and erythromycin (3; 0.5%) or ciprofloxacin, nalidixic acid, streptomycin, tetracycline, and erythromycin (2; 0.3%). A total of 79 flaA-SVR subtypes were identified, including 40 (50.6%) unique to the isolates' origins, with the most common sequence types 16, 54, 36, 34, and 287 which covered 56 (9.3%), 50 (8.3%), 48 (8.0%), 35 (5.8%), and 32 (5.3%) of C. jejuni isolates, respectively. It was found that 13 isolates had the novel flaA-SVR subtypes which were not present in the pubMLST database. These isolates were recovered from chicken feces (6 isolates), carcasses (2 isolates), meat (one isolate) and from humans (4 isolates). Multiresistant C. jejuni were classified into 26 different sequence subtypes. Among the most numerous multidrug resistant profile CIP+NAL+STR+TET 21 different flaA-SVR subtypes, with total of 92 isolates, were identified. Most of them were classified to 287 (18; 19.6% isolates), 100 (13; 14.1%), 34 (9; 9.8%), 208 (8; 8.7%), and 781 (8; 8.7%) molecular variants. Isolates resistant to CIP, STR and TET (13 isolates) were mainly from chicken feces (12 isolates) and classified into 5 flaA-SVR sequence types, with the most common 36 (8 isolates). The obtained results show a broad molecular diversity of multiresistant C. jejuni isolates and suggest chickens as a possible source of human Campylobacter infections in Poland.
Project description:The gyrA gene of Campylobacter jejuni UA580, which encodes the A subunit of DNA gyrase, was cloned and its nucleotide sequence was determined. An open reading frame of 2,589 nucleotides was identified, which could code for a polypeptide of 863 amino acids with a M(r) of 97 kDa. Both the nucleotide sequence and the putative amino acid sequence show ca. 50% identity with those of other gyrA genes from gram-positive and gram-negative bacteria. The locations of the gyrA gene on genome maps of both C. jejuni UA580 and Campylobacter coli UA417 were determined. Six nalidixic acid-resistant isolates of C. jejuni were shown to carry mutations in gyrA. Three clinical isolates had Thr-86-to-Ile substitutions. Three laboratory mutants had substitutions of Thr-86 to Ile, Asp-90 to Ala, and Ala-70 to Thr, respectively. The mutation at Thr-86, which is homologous to Ser-83 in Escherichia coli, was associated with high-level resistance to ciprofloxacin in C. jejuni.
Project description:BACKGROUND:Thermophilic Campylobacter species are a major cause of bacterial foodborne diarrhoea in humans worldwide. Poultry and their products are the predominant source for human campylobacteriosis. Resistance of Campylobacter to antibiotics is increasing worldwide, but little is known about the antibiotic resistance in Campylobacter isolated from chicken in Kenya. In this study, 35 suspected Campylobacter strains isolated from faeces and cloacal swabs of chicken were tested for their susceptibility to seven antibiotics using a broth microdilution assay and molecular biological investigations. RESULTS:Overall, DNA of thermophilic Campylobacter was identified in 53 samples by PCR (34 C. jejuni, 18 C. coli and one mix of both species) but only 35 Campylobacter isolates (31 C. jejuni and 4 C. coli) could be re-cultivated after transportation to Germany. Isolates were tested for their susceptibility to antibiotics using a broth microdilution assay. Additionally, molecular biological detection of antibiotic resistance genes was carried out. C. jejuni isolates showed a high rate of resistance to nalidixic acid, tetracycline and ciprofloxacin of 77.4, 71.0 and 71.0 %, respectively. Low resistance (25.8 %) was detected for gentamicin and chloramphenicol. Multidrug resistance in C. jejuni could be detected in 19 (61.3 %) isolates. Resistance pattern of C. coli isolates was comparable. Resistance to ciprofloxacin was confirmed by MAMA-PCR and PCR-RFLP in all phenotypically resistant isolates. The tet(O) gene was detected only in 54.5 % of tetracycline resistant C. jejuni isolates. The tet(A) gene, which is also responsible for tetracycline resistance, was found in 90.3 % of C. jejuni and in all C. coli isolates. Thirteen phenotypically erythromycin-resistant isolates could not be characterised by using PCR-RFLP and MAMA-PCR. CONCLUSIONS:To the best of our knowledge, this study is the first report about resistance to antibiotics in thermophilic Campylobacter originating from chicken in Kenya. Campylobacter spp. show a high level of resistance to ciprofloxacin, nalidixic acid and tetracycline but also a remarkable one to chloramphenicol and gentamicin and they are multidrug resistant. Resistance to antibiotics is a global public health concern. In Kenya, resistance surveillance needs further attention in the future. Efforts to establish at least a National Laboratory with facilities for performing phenotypic and genotypic characterization of thermophilic Campylobacter is highly recommended.
Project description:The gyrA quinolone resistance determining region was sequenced from 13 ciprofloxacin-resistant and 20 ciprofloxacin-susceptible Campylobacter jejuni isolates. All isolates resistant to ciprofloxacin had Thr-86-to-Ile mutations, a mutation frequently associated with the acquisition of resistance to fluoroquinolones. A mismatch amplification mutation assay (MAMA) PCR protocol was developed that detects this gyrA mutation in quinolone-resistant isolates. The MAMA PCR provides a means for routine detection of the gyrA mutation without the need for sequencing the gyrA gene.
Project description:Campylobacter jejuni was longitudinally isolated from beef cattle housed in four confined feeding operations (CFOs) in Southern Alberta, Canada, over 18 months. All of the cattle were administered a variety of antimicrobial agents (AMAs) nontherapeutically and metaphylactically during their time in the CFOs. In total, 7,966 C. jejuni isolates were recovered from cattle. More animals were colonized by the bacterium after >60 days in the CFO (interim) than were individuals upon entry at the CFO (arrival). Subtyping and resistance to seven AMAs were determined for 1,832 (23.0%) and 1,648 (20.7%) isolates, respectively. Increases in the proportion of isolates resistant to tetracycline were observed at all four CFOs between sample times and to ciprofloxacin and nalidixic acid at one or more CFOs. The vast majority of isolates resistant to tetracycline carried tetO, whereas ciprofloxacin resistance was predominantly attributed to mutations in the gyrA gene. Although considerable diversity was observed, a majority of C. jejuni isolates belonged to one of five predominant subtype clusters. There was no difference in subtype diversity by CFO, but the population structure differed between sample times. Selection for resistance to ciprofloxacin and nalidixic acid was subtype dependent, whereas selection for resistance to tetracycline was not. The findings indicate that a proportion of cattle entering CFOs carry resistant C. jejuni subtypes, and the characteristics of beef cattle CFOs facilitate transmission/proliferation of diverse subtypes, including those resistant to AMAs, which coupled with the densities of CFOs likely contribute to the high rates of cattle-associated campylobacteriosis in Southern Alberta.IMPORTANCE A small proportion of cattle entering a CFO carry Campylobacter jejuni, including subtypes resistant to AMAs. The large numbers of cattle arriving from diverse locations at the CFOs and intermingling within the CFOs over time, coupled with the high-density housing of animals, the high rates of transmission of C. jejuni subtypes among animals, and the extensive use of AMAs merge to create an ideal situation where the proliferation of diverse antimicrobial-resistant C. jejuni subtypes is facilitated. Considering that Southern Alberta reports high rates of campylobacteriosis in the human population and that many of these clinical cases are due to C. jejuni subtypes associated with cattle, it is likely that the characteristics of beef cattle CFOs favor the propagation of clinically relevant C. jejuni subtypes, including those resistant to medically important AMAs, which constitute a risk to human health.
Project description:Campylobacter species are zoonotic pathogens and the leading cause of bacterial enteritis worldwide. With the increase of antimicrobial resistance to fluoroquinolones and macrolides, they have been identified by the World Health Organization (WHO) as high-priority antimicrobial-resistant pathogens. There is currently little known about the prevalence and antimicrobial resistance characteristics of Campylobacter species in Beijing. In this study, we performed a 2-year surveillance of Campylobacter in Beijing, China. We used multilocus sequence typing (MLST) and antimicrobial susceptibility testing to analyze 236 Campylobacter isolates recovered from 230 clinical infectious cases in Beijing between 2017 and 2018. The Campylobacter isolation rate in diarrhea patients was 7.81%, with higher isolation rates in male patients than female patients and in autumn compared with other seasons. We identified 125 sequence types (STs) of 23 cloning complexes (CCs) among the 236 isolates, including four new alleles and 19 new STs. The most commonly isolated STs of Campylobacter jejuni were ST-22 and ST-760 (4.50%), and the most commonly isolated ST of Campylobacter coli was ST-9227 (16.67%). We also compared our isolates with clinical Campylobacter isolates from other countries in Asia, CC-353 of Campylobacter coli was found in eight countries, CC-1034 and CC-1287 of Campylobacter coli were found only in China. All C. jejuni isolates were resistant to at least one antimicrobial. C. jejuni showed the highest rate of resistance toward ciprofloxacin (94.50%), followed by tetracycline (93.50%), and nalidixic acid (92.00%), while C. coli showed highest resistance toward ciprofloxacin (94.44%) and tetracycline (94.44%) followed by nalidixic acid (88.89%). The most commonly observed MDR combination of C. jejuni were quinolone, phenicol and tetracycline (11.50%), while the most commonly observed MDR combination of C. coli were macrolide, quinolone, phenicol, tetracycline and lincosamide (30.56%). Surveillance of molecular characterization will provide important information for prevention of Campylobacter infection. This study enhances insight into Campylobacter infections in diarrheal patients, with relevance for treatment regimens in Beijing.