Nationwide Stepwise Emergence and Evolution of Multidrug-Resistant Campylobacter jejuni Sequence Type 5136, United Kingdom.
ABSTRACT: We examined whole-genome-sequenced Campylobacter jejuni and C. coli from 2012-2015 isolated from birds and human stool samples in North East Scotland for the presence of antimicrobial resistance genes. We found that sequence type (ST) 5136 (clonal complex 464) was the most prevalent multidrug-resistant strain of C. jejuni exclusively associated with poultry host reservoirs and recovered from human cases of campylobacteriosis. Tetracycline resistance in ST5136 isolates was due to a tet(O/32/O) mosaic gene, ampicillin resistance was conferred by G???T transversion in the -10 promoter region of blaOXA-193, fluoroquinolone resistance was due to C257T change in gyrA, and aminoglycoside resistance was conferred by aac. Whole-genome analysis showed that the strain ST5136 evolved from ST464. The nationwide emergence of ST5136 was probably due to stepwise acquisition of antimicrobial resistance genes selected by high use of ?-lactam, tetracycline, fluoroquinolone, and aminoglycoside classes of drugs in the poultry industry.
Project description:Campylobacter jejuni, a common foodborne zoonotic pathogen, causes gastroenteritis worldwide and is increasingly resistant to antibiotics. We aimed to investigate the antimicrobial resistance (AMR) genotypes of C. jejuni isolated from humans, poultry and birds from wild and urban Italian habitats to identify correlations between phenotypic and genotypic AMR in the isolates. Altogether, 644 C. jejuni isolates from humans (51), poultry (526) and wild- and urban-habitat birds (67) were analysed. The resistance phenotypes of the isolates were determined using the microdilution method with EUCAST breakpoints, and AMR-associated genes and single nucleotide polymorphisms were obtained from a publicly available database. Antimicrobial susceptibility testing showed that C. jejuni isolates from poultry and humans were highly resistant to ciprofloxacin (85.55% and 76.47%, respectively), nalidixic acid (75.48% and 74.51%, respectively) and tetracycline (67.87% and 49.02%, respectively). Fewer isolates from the wild- and urban-habitat birds were resistant to tetracycline (19.40%), fluoroquinolones (13.43%), and quinolone and streptomycin (10.45%). We retrieved seven AMR genes (tet (O), cmeA, cmeB, cmeC, cmeR, blaOXA-61 and blaOXA-184) and gyrA-associated point mutations. Two major B-lactam genes called blaOXA-61 and blaOXA-184 were prevalent at 62.93% and 82.08% in the poultry and the other bird groups, respectively. Strong correlations between genotypic and phenotypic resistance were found for fluoroquinolones and tetracycline. Compared with the farmed chickens, the incidence of AMR in the C. jejuni isolates from the other bird groups was low, confirming that the food-production birds are much more exposed to antimicrobials. The improper and overuse of antibiotics in the human population and in animal husbandry has resulted in an increase in antibiotic-resistant infections, particularly fluoroquinolone resistant ones. Better understanding of the AMR mechanisms in C. jejuni is necessary to develop new strategies for improving AMR programs and provide the most appropriate therapies to human and veterinary populations.
Project description:Australian rates of campylobacteriosis are among the highest in developed countries, yet only limited work has been done to characterize Campylobacter spp. in Australian retail products. We performed whole genome sequencing (WGS) on 331 C. coli and 285 C. jejuni from retail chicken meat, as well as beef, chicken, lamb and pork offal (organs). Campylobacter isolates were highly diverse, with 113 sequence types (STs) including 38 novel STs, identified from 616 isolates. Genomic analysis suggests very low levels (2.3-15.3%) of resistance to aminoglycoside, beta-lactam, fluoroquinolone, macrolide and tetracycline antibiotics. A majority (>90%) of isolates (52/56) possessing the fluoroquinolone resistance-associated T86I mutation in the gyrA gene belonged to ST860, ST2083 or ST7323. The 44 pork offal isolates were highly diverse, representing 33 STs (11 novel STs) and harboured genes associated with resistance to aminoglycosides, lincosamides and macrolides not generally found in isolates from other sources. Prevalence of multidrug resistant genotypes was very low (<5%), but ten-fold higher in C. coli than C. jejuni. This study highlights that Campylobacter spp. from retail products in Australia are highly genotypically diverse and important differences in antimicrobial resistance exist between Campylobacter species and animal sources.
Project description:Campylobacteriosis is the leading cause of human bacterial gastroenteritis, very often associated with poultry consumption. Thermophilic Campylobacter (Campylobacter jejuni and Campylobacter coli) isolates (n = 158) recovered from broiler neck skin and caecal contents in Ireland over a one-year period, resistant to at least one of three clinically relevant antimicrobial classes, were screened for resistance determinants. All ciprofloxacin-resistant isolates (n = 99) harboured the C257T nucleotide mutation (conferring the Thr-86-Ile substitution) in conjunction with other synonymous and nonsynonymous mutations, which may have epidemiological value. The A2075G nucleotide mutation and amino acid substitutions in L4 and L22 were detected in all erythromycin-resistant isolates (n = 5). The tetO gene was detected in 100% (n = 119) of tetracycline-resistant isolates and three of which were found to harbour the mosaic tetracycline resistance gene tetO/32/O. Two streptomycin-resistant C. jejuni isolates (isolated from the same flock) harboured ant(6)-Ib, located in a multidrug resistance genomic island, containing aminoglycoside, streptothricin (satA) and tetracycline resistance genes (truncated tetO and mosaic tetO/32/O). The ant(6)-Ie gene was identified in two streptomycin-resistant C. coli isolates. This study highlights the widespread acquisition of antimicrobial resistance determinants among chicken-associated Campylobacter isolates, through horizontal gene transfer or clonal expansion of resistant lineages. The stability of such resistance determinants is compounded by the fluidity of mobile genetic element.
Project description:In a structured survey of all major chicken-meat producers in Australia, we investigated the antimicrobial resistance (AMR) and genomic characteristics of Campylobacter jejuni (n?=?108) and C. coli (n?=?96) from cecal samples of chickens at slaughter (n?=?200). The majority of the C. jejuni (63%) and C. coli (86.5%) samples were susceptible to all antimicrobials. Fluoroquinolone resistance was detected among both C. jejuni (14.8%) and C. coli (5.2%), although this only included three sequence types (STs) and one ST, respectively. Multidrug resistance among strains of C. jejuni (0.9%) and C. coli (4.1%) was rare, and fluoroquinolone resistance, when present, was never accompanied by resistance to any other agent. Comparative genome analysis demonstrated that Australian isolates were found dispersed on different branches/clusters within the international collection. The major fluoroquinolone-resistant STs of C. jejuni (ST7323, ST2083, and ST2343) and C. coli (ST860) present in Australian chickens were similar to those of international isolates and have been reported previously in humans and animals overseas. The detection of a subpopulation of Campylobacter isolates exclusively resistant to fluoroquinolone was unexpected since most critically important antimicrobials such as fluoroquinolones are excluded from use in Australian livestock. A number of factors, including the low level of resistance to other antimicrobials, the absence of fluoroquinolone use, the adoption of measures for preventing spread of contagion between flocks, and particularly the genomic identities of isolates, all point to humans, pest species, or wild birds as being the most plausible source of organisms. This study also demonstrates the need for vigilance in the form of surveillance for AMR based on robust sampling to manage AMR risks in the food chain.IMPORTANCE Campylobacter is one of the most common causes of gastroenteritis in humans, with infections frequently resulting from exposure to undercooked poultry products. Although human illness is typically self-limiting, a minority of cases do require antimicrobial therapy. Ensuring that Campylobacter originating from meat chickens does not acquire resistance to fluoroquinolones is therefore a valuable outcome for public health. Australia has never legalized the use of fluoroquinolones in commercial chickens and until now fluoroquinolone-resistant Campylobacter has not been detected in the Australian poultry. This structured survey of meat chickens derived from all major Australian producers describes the unexpected emergence of fluoroquinolone resistance in Campylobacter jejuni and C. coli Genetic characterization suggests that these isolates may have evolved outside the Australian poultry sector and were introduced into poultry by humans, pest species, or wild birds. The findings dramatically underline the critical role of biosecurity in the overall fight against antimicrobial resistance.
Project description:Campylobacter jejuni is a zoonotic pathogen and the most common bacterial cause of human gastroenteritis worldwide. With the increase of antibiotic resistance to fluoroquinolones and macrolides, the drugs of choice for treatment, C. jejuni was recently classified as a serious antimicrobial resistant threat. Here, we characterized 94 C. jejuni isolates collected from patients at four Michigan hospitals in 2011 and 2012 to determine the frequency of resistance and association with phylogenetic lineages. The prevalence of resistance to fluoroquinolones (19.1%) and macrolides (2.1%) in this subset of C. jejuni isolates from Michigan was similar to national reports. High frequencies of fluoroquinolone-resistant C. jejuni isolates, however, were recovered from patients with a history of foreign travel. A high proportion of these resistant isolates were classified as multilocus sequence type (ST)-464, a fluoroquinolone-resistant lineage that recently emerged in Europe. A significantly higher prevalence of tetracycline-resistant C. jejuni was also found in Michigan and resistant isolates were more likely to represent ST-982, which has been previously recovered from ruminants and the environment in the U.S. Notably, patients with tetracycline-resistant C. jejuni infections were more likely to have contact with cattle. These outcomes prompt the need to monitor the dissemination and diversification of imported fluoroquinolone-resistant C. jejuni strains and to investigate the molecular epidemiology of C. jejuni recovered from cattle and farm environments to guide mitigation strategies.
Project description:Antimicrobial resistance in Campylobacter, common in poultry, is a global public health issue. The emergence and spread of antimicrobial resistant Campylobacter has been linked to the use of antimicrobials in food animals. Small poultry flocks are becoming increasingly popular not only as a source of food but also as pets, yet not all small flock owners are aware of proper antimicrobial use practices and safe food handling protocols. This trend could contribute to antimicrobial resistance. In order to determine the prevalence of antimicrobial resistance in Campylobacter in small poultry flocks, we analyzed data from birds that had been submitted to a diagnostic laboratory in Ontario between October 2015 and September 2017. A pooled cecal sample was obtained from each submission and cultured for Campylobacter jejuni and Campylobacter coli. Three isolates were recovered from each positive sample and tested for susceptibility to nine antimicrobials using a broth microdilution method. Overall, 176 isolates were recovered (141 chicken, 21 turkey, 6 duck, and 8 game bird). A high frequency of resistance to tetracycline was observed in the C. jejuni isolates from chickens (77%) and turkeys (100%), and in the C. coli isolates from turkeys (50%) and game birds (40%). Campylobacter jejuni isolates had higher odds of resistance to tetracycline (OR = 3.54, P ? 0.01) compared to C. coli isolates. Overall, there was a low frequency of resistance to quinolones and a very low frequency of resistance to macrolides. Multidrug resistance was uncommon. The high prevalence of tetracycline resistance emphasizes the importance of prudent antimicrobial use in small flocks. Although low, the presence of resistance to macrolides and quinolones, which are used to treat campylobacteriosis in humans, highlights the need for proper food safety and infection control practices by small flock owners to prevent exposure to antimicrobial resistant Campylobacter.
Project description:Of 203 human clinical isolates of Campylobacter jejuni from Alberta, Canada (1999 to 2002), 101 isolates (50%) were resistant to at least 64 microg of tetracycline/ml, with four isolates exhibiting higher levels of tetracycline resistance (512 microg/ml). In total, the MICs for 37% of tetracycline-resistant isolates (256 to 512 microg/ml) were higher than those previously reported in C. jejuni (64 to 128 microg/ml). In the tetracycline-resistant clinical isolates, 67% contained plasmids and all contained the tet(O) gene. Four isolates resistant to high levels of tetracycline (MIC = 512 microg/ml) contained plasmids carrying the tet(O) gene, which could be transferred to other isolates of C. jejuni. The tetracycline MICs for transconjugants were comparable to those of the donors. Cloning of tet(O) from the four high-level tetracycline-resistant isolates conferred an MIC of 32 microg/ml for Escherichia coli DH5alpha. In contrast, transfer to a strain of C. jejuni by using mobilization conferred an MIC of 128 microg/ml. DNA sequence analysis determined that the tet(O) genes encoding lower MICs (64 to 128 microg/ml) were identical to one other, although the tet(O) genes encoding a 512-microg/ml MIC demonstrated several nucleotide substitutions. The quinolone resistance determining region of four ciprofloxacin-resistant isolates (2%) was analyzed, and resistance was associated with a chromosomal mutation in the gyrA gene resulting in a Thr-86-Ile substitution. In addition, six kanamycin-resistant isolates contained large plasmids that carry the aphA-3 marker coding for 3'-aminoglycoside phosphotransferase. Resistance to erythromycin was not detected in 203 isolates. In general, resistance to most antibiotics in C. jejuni remains low, except for resistance to tetracycline, which has increased from about 8 to 50% over the past 20 years.
Project description:Campylobacter jejuni is one of the most prevalent causes of bacterial gastroenteritis worldwide, and it is largely associated with consumption of contaminated poultry. Current Campylobacter control measures at the poultry production level remain insufficient, and hence there is the need for alternative control strategies. We evaluated the potential of the monoterpene (-)-?-pinene for control of C. jejuni in poultry. The antibacterial and resistance-modulatory activities of (-)-?-pinene were also determined against 57 C. jejuni strains. In addition, the anti-quorum-sensing activity of (-)-?-pinene against C. jejuni NCTC 11168 was determined for three subinhibitory concentrations (125, 62.5, 31.25 mg/L) over three incubation times using an autoinducer-2 bioassay based on Vibrio harveyi BB170 bioluminescence measurements. The effects of a subinhibitory concentration of (-)-?-pinene (250 mg/L) on survival of C. jejuni, and in combination with enrofloxacin on fluoroquinolone resistance development in C. jejuni, were determined in a broiler chicken model, by addition of (-)-?-pinene to the broiler water supply. The reduction of C. jejuni numbers by (-)-?-pinene was further determined in broiler chickens that were colonized with either fluoroquinolone-susceptible or -resistant strains, by direct gavage treatment. We observed weak in vitro antimicrobial activity for (-)-?-pinene alone (MIC >500 mg/L), but strong potentiating effects on antibiotics erythromycin and ciprofloxacin against different Campylobacter strains (>512 fold change). After 24 h of treatment of C. jejuni with (-)-?-pinene, its quorum-sensing signaling was reduced by >80% compared to the untreated control. When given in the drinking water, (-)-?-pinene did not show any significant inhibitory effects on the level of C. jejuni in the colonized chickens, and did not reduce fluoroquinolone resistance development in combination with enrofloxacin. Conversely, when (-)-?-pinene was administered by direct gavage, it significantly reduced the number of fluoroquinolone susceptible C. jejuni in the colonized broiler chickens. These results demonstrate that (-)-?-pinene modulates quorum-sensing in Campylobacter, potentiates antibiotics against different Campylobacter strains, and reduces Campylobacter colonization in broiler chickens.
Project description:The objective of this study was to test the prevalence of virulence-associated markers and antimicrobial resistance in 624 C. jejuni isolated from poultry food chain, i. e., chicken feces (n = 160), poultry carcasses (n = 157), poultry meat (n = 152) and from humans (n = 155). All human strains were positive for 9 out of 13 putative virulence genes responsible for expression of pathogenic factors involved in different stages of the infection. The presence of all markers was also high in strains from chicken feces, carcasses and meat although not all of them were identified in 100% of the isolates. On the other hand, the virB11, wlaN, and iam putative pathogenic genes were detected in only 1.9, 15.2, and 20.5% of strains, respectively. C. jejuni isolates, irrespective of the origin, were highly resistant to ciprofloxacin (92.5% isolates), followed by nalidixic acid (88.9%) and tetracycline (68.4%). In case of ciprofloxacin, significantly more isolates from poultry feces, carcasses and meat were resistant than those obtained from humans and the same relationship was observed for tetracycline where the isolates from chicken feces were more often resistant than C. jejuni of carcasses and meat origin. A low number of strains was resistant to streptomycin (18.4% isolates) and only 5 strains (0.8%) displayed resistance to erythromycin. A relationship between resistance to fluoroquinolones and presence of selected pathogenic markers was observed, e.g., from 83.3% strains with the virB11 to 93.4% with the docA genes were resistant to ciprofloxacin. The isolates that did not possess any of the pathogenic traits were also mainly resistant to this antimicrobial, although the number of such strains was usually low, except virB11 (612 isolates), wlaN (529 strains), and iam (496 isolates). Furthermore, resistance to tetracycline was somehow associated with the presence of the virulence associated genes wlaN and virB11 (56.8 and 75.0% isolates, respectively). The present study shows a high antimicrobial resistance to quinolones and tetracycline of C. jejuni isolated along poultry food chain and from patients with diarrhea, which was closely correlated with the presence of several virulence genes playing a role in the pathogenesis of Campylobacter infection.
Project description:Hawaii has one of the highest incidences of Campylobacteriosis in the United States, but there remains little published data on circulating strains or antimicrobial resistance. We characterized 110 clinical Campylobacter isolates (106?C. jejuni, 4?C. coli) processed at Tripler Army Medical Center in Honolulu, HI from 2012-2016. Twenty-five percent of C. jejuni isolates exhibited fluoroquinolone (FQ) resistance, compared with 16% for tetracycline (TET), and 0% for macrolides. Two of the four C. coli isolates were resistant to FQ, TET, and macrolides. C. jejuni isolates further underwent multilocus sequence typing, pulsed-field gel electrophoresis, and molecular capsular typing. Nineteen capsule types were observed, with two capsule types (HS2 and HS9) being associated with FQ resistance (p?<?0.001 and p?=?0.006, respectively). HS2 FQ-resistant isolates associated with clonal complex 21, possibly indicating clonal spread in FQ resistance. Macrolides should be considered for treatment of suspect cases due to lack of observed resistance.