Campylobacter Strains Research Articles

Genomic Analysis and Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli in Peru.

Campylobacter is the leading cause of human bacterial gastroenteritis worldwide and has a major impact on global public health. Whole Genome Sequencing (WGS) is a powerful tool applied in the study of foodborne pathogens. The objective of the present study was to apply WGS to determine the genetic diversity, virulence factors and determinants of antimicrobial resistance of the populations of C. jejuni and C. coli in Peru. A total of 129 Campylobacter strains (108 C. jejuni and 21 C. coli) were sequenced using Illumina Miseq platform. In silico MLST analysis identified a high genetic diversity among those strains with 30 sequence types (STs), several of them within 11 clonal complexes (CC) for C. jejuni, while the strains of C. coli belonged to a single CC with 8 different STs. Phylogeny analysis showed that Peruvian C. jejuni strains were divided into 2 clades with 5 populations, while C. coli formed a single clade with 4 populations. Furthermore, in silico analyses showed the presence of several genes associated with adherence, colonization and invasion among both species: cadF (83.7%), jlpA (81.4%), racR (100%), dnaJ (83.7%), pebA (83.7%), pldA (82.1%), porA (84.5%), ceuE (82.9%), ciaB (78.3%), iamB (86.8%), and flaC (100%). The majority (82.9%) of the Campylobacter strains carried the cdtABC operon which code for cytolethal distending toxin (CDT). Half of them (50.4%) carried genes associated with the presence of T6SS, while the frequency of genes associated with T4SS were relatively low (11.6%). Genetic markers associated with resistance to quinolones, tetracycline (tetO, tetW/N/W), beta-lactamases (blaoxa–61), macrolides (A2075G in 23S rRNA) were found in 94.5, 21.7, 66.7, 6.2, 69.8, and 18.6% of strains, respectively. The cmeABC multidrug efflux operon was present in 78.3% of strains. This study highlights the importance of using WGS in the surveillance of emerging pathogens associated with foodborne diseases, providing genomic information on genetic diversity, virulence mechanisms and determinants of antimicrobial resistance. The description of several Campylobacter genotypes having many virulence factors and resistance to quinolones and tetracyclines circulating in Peru provides important information which helps in the monitoring, control and prevention strategies of this emerging pathogen in our country.

Characterisation of burden of illness measures associated with human (Fluoro)quinolone-resistant Campylobacter spp. infections - a scoping review.

Campylobacter spp. are one of the most common causes of bacterial gastroenteritis in Canada and worldwide. Fluoroquinolones are often used to treat complicated human campylobacteriosis and strains of Campylobacter spp. resistant to these drugs are emerging along the food chain. A scoping review was conducted to summarise how human (fluoro)quinolone-resistant (FQR; quinolones including fluoroquinolones) Campylobacter spp. infections are characterised in the literature by describing how burden of illness (BOI) associated with FQR is measured and reported, describing the variability in reporting of study characteristics, and providing a narrative review of literature that compare BOI measures of FQR Campylobacter spp. infections to those with susceptible infections. The review identified 26 studies that yielded many case reports, a lack of recent literature and a lack of Canadian data. Studies reported 26 different BOI measures and the most common were hospitalisation, diarrhoea, fever and duration of illness. There were mixed results as BOI measures reported in literature were inconsistently defined and there were limited comparisons between resistant and susceptible infections. This presents a challenge when attempting to assess the magnitude of the BOI due to FQR Campylobacter spp., highlighting the need for more research in this area.

Evaluation of Three Isolation Experiments for Campylobacter Bacteriophages from Chicken Skin: A Comparative Study

Background: Campylobacter strains are of the leading pathogens causing bacterial gastroenteritis, whose infections are generally considered to be one of the most common foodborne illnesses of animal origin. The etiology of this infection often goes back to eating contaminated raw meat or infected poultry. The bacteria are present in abundance in chicken skin. The use of appropriate bacteriophages is one of the most effective experiments in eliminating Campylobacter strains. Phage therapy refers to the use of bacteriophages to treat bacterial infections.
 Aim: Accordingly, the present study aimed to compare three experiments of bacteriophage isolation in chicken skin.
 Experiments: Thus, 15 samples of chicken skin were collected from five different fresh chicken suppliers in Ghaemshahr, Iran. The samples were transported to the laboratory aseptically in the vicinity of ice, and then cultured in blood agar medium, and the isolates were identified by various tests including gram staining, catalase and oxidase tests.
 Results: The results were compared before and after three bacteriophage isolation experiments. Out of 15 chicken skin samples tested in all three experiments, 6 (40%) strains were identified in the first experiment, 8 (53.4%) strains in the second experiment and 12 (20%) strains in the third experiment after bacteriophage therapy.
 Conclusion: The bacteriophage isolation experiments alone or in combination with other intervention strategies are recommended as promising tools for greater food safety. These experiments can be useful to increase food safety and reduce the risk of infection in humans through the consumption of potentially infected edible parts of chicken. According to the results of this study, among the three proposed experiments, the experiment of chicken skin enrichment in Bolton selective media containing target isolates was the most efficient approach, which showed a high limit of detection at low concentrations and the highest rate of phage recovery. This can be a more reliable way to isolate the Campylobacter bacteriophages and eliminate the Campylobacter strains.

Differences in Genotype and Antimicrobial Resistance between Campylobacter spp. Isolated from Organic and Conventionally Produced Chickens in Sweden.

Antibiotic resistance is a major challenge worldwide and increased resistance to quinolones in Campylobacter is being reported. Analysis of antibiotic resistance was performed on 157 Campylobacter strains (123 C. jejuni and 34 C. coli) from conventional and organic chickens produced in Sweden. Susceptibility for tetracycline, ciprofloxacin, erythromycin, nalidixic acid, streptomycin, and gentamycin was determined by microdilution. All 77 isolates from organic chickens were sensitive to all antibiotics, except two C. jejuni that were resistant to tetracycline. Of the 80 isolates from conventional chickens, 22.5% of C. jejuni and 11.1% of C. coli were resistant to quinolones and 5.6% of C. jejuni were resistant to tetracycline. Whole-genome sequencing resulted in 50 different sequence types of C. jejuni and six of C. coli. Nine sequence types were found in both organic and conventional chickens. Two of these (ST-19 and ST-257) included isolates from conventional broilers with different resistance phenotypes to the remaining isolates from conventional and organic broilers. There are management differences between the production systems, such as feed, breed, use of coccidiostats, and access to outdoor area. It is unlikely that quinolone resistance has arisen due to use of antimicrobials, since fluoroquinolones are not permitted in Swedish broiler production.

Antimicrobial Resistance and Genetic Diversity of Campylobacter spp. Isolated from Broiler Chicken at Three Levels of the Poultry Production Chain in Costa Rica

Campylobacter spp. are considered the most common bacterial cause of human gastroenteritis, one of the four main causes of diarrheal disease worldwide, and they are one of the main foodborne pathogens causing hospitalizations and deaths. Here, 148 strains of Campylobacter spp. isolated from poultry at farms, processing plants, and retail stores in Costa Rica were examined for resistance to six antibiotics. An agar dilution test was used to determine the MIC and susceptibility profiles against doxycycline, ciprofloxacin, nalidixic acid, enrofloxacin, chloramphenicol, and erythromycin. In addition, a pulsed-field gel electrophoresis analysis was carried out to determine the genotype relatedness of a representative subset of the isolates. Approximately 136 (92%) of the 148 analyzed isolates showed resistance to the tested drugs. Nalidixic acid, ciprofloxacin, and enrofloxacin were the antibiotics for which resistance occurred most frequently (91.2, 85.8, and 85.8%, respectively), followed by doxycycline (25.0%), chloramphenicol (5.4%), and erythromycin (2.7%). The profile conferring only resistance to quinolones was the most frequently found, and only 2.0% of the isolates showed resistance to quinolones and macrolides simultaneously. Results showed a high frequency of resistant Campylobacter spp. strains and evidenced the distribution, selection, and circulation of resistant strains along the poultry chain from farms to consumers. Cross-contamination and resistance seem to play important roles in the dissemination of these strains at specific points of the poultry chain, even when control measures are being taken. The establishment of effective surveillance and control strategies represents an essential tool for foodborne diseases mitigation. The rational use of antibiotics, especially those still showing efficacy, should be a priority in both human and veterinary medicine to contain the progress of this phenomenon and its consequences.

Assessment of biofilm formation by Campylobacter spp. isolates mimicking poultry slaughterhouse conditions

This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA-restriction fragment length polymorphism (RFLP) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA-RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA-RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrug-resistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10°C, adherence levels were significantly lower than at 42°C. Under microaerobic and aerobic atmospheres, at 42°C, 3 strains (C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.

Genome and Methylome analysis of a phylogenetic novel Campylobacter coli cluster with C. jejuni introgression.

The intriguing recent discovery of Campylobacter coli strains, especially of clade 1, that (i) possess mosaic C. coli / C. jejuni alleles, (ii) demonstrate mixed multilocus sequence types (MLSTs) and (iii) have undergone genome-wide introgression has led to the speculation that these two species may be involved in an accelerated rate of horizontal gene transfer that is progressively leading to the merging of both species in a process coined ‘despeciation’. In an MLST-based neighbour-joining tree of a number of C. coli and C. jejuni isolates of different clades, three prominent Campylobacter isolates formed a seemingly separate cluster besides the previously described C. coli and C. jejuni clades. In the light of the suspected, ongoing genetic introgression between the C. coli and C. jejuni species, this cluster of Campylobacter isolates is proposed to present one of the hybrid clonal complexes in the despeciation process of the genus. Specific DNA methylation as well as restriction modification systems are known to be involved in selective uptake of external DNA and their role in such genetic introgression remains to be further investigated. In this study, the phylogeny and DNA methylation of these putative C. coli / C. jejuni hybrid strains were explored, their genomic mosaic structure caused by C. jejuni introgression was demonstrated and basic phenotypic assays were used to characterize these isolates. The genomes of the three hybrid Campylobacter strains were sequenced using PacBio SMRT sequencing, followed by methylome analysis by Restriction-Modification Finder and genome analysis by Parsnp, Smash++ and blast. Additionally, the strains were phenotypically characterized with respect to growth behaviour, motility, eukaryotic cell invasion and adhesion, autoagglutination, biofilm formation, and water survival ability. Our analyses show that the three hybrid Campylobacter strains are clade 1 C . coli strains, which have acquired between 8.1 and 9.1 % of their genome from C. jejuni . The C. jejuni genomic segments acquired are distributed over the entire genome and do not form a coherent cluster. Most of the genes originating from C. jejuni are involved in chemotaxis and motility, membrane transport, cell signalling, or the resistance to toxic compounds such as bile acids. Interspecies gene transfer from C. jejuni has contributed 8.1–9.1% to the genome of three C. coli isolates and initiated the despeciation between C. jejuni and C. coli . Based on their functional annotation, the genes originating from C. jejuni enable the adaptation of the three strains to an intra-intestinal habitat. The transfer of a fused type II restriction-modification system that recognizes the CAYNNNNNCTC/GAGNNNNNRTG motif seems to be the key for the recombination of the C. jejuni genetic material with C. coli genomes.

Genomic Characterisation of Campylobacter jejuni Isolates Recovered During Commercial Broiler Production.

Background: Campylobacter is commonly transmitted to humans from chickens. Campylobacter jejuni is the species most frequently associated with human illness, and the most prevalent species recovered from poultry.Objective: The objective of this study was to analyse a sub-population of C. jejuni from two broiler flocks on the farm and at slaughter using whole-genome sequencing to gain insights into the changes in the Campylobacter population during broiler production, including changes in virulence and antimicrobial resistance profiles.Methods: In this study, ten composite faecal samples (n=10), obtained by pooling ten fresh faecal samples (n=10), were collected in the broiler house on two farms on days 14, 21, 28, and 34 (n=80) and ten composite (n=10) caecal samples were collected at the time of slaughter for each flock (n=20). These were tested for C. jejuni using the ISO 10272-2:2016 method. Seven isolates were randomly selected from each of the nine Campylobacter-positive sampling points (n=63) and were subjected to antimicrobial susceptibility tests. Their genomes were sequenced and the data obtained was used to characterise the population structure, virulence, antimicrobial resistance determinants and inter-strain variation.Results: The Farm 1 isolates had three MLST types (ST257-257, ST814-661 and ST48-48) while those on Farm 2 were ST6209-464 and ST9401. Interestingly, only the MLST types positive for most of the virulence genes tested in this study persisted throughout the production cycle, and the detection of antimicrobial resistance determinants (gyrA T86I and tetO) increased after thinning and at slaughter, with the detection of new strains.Conclusion: The persistence of the most virulent strains detected in this study throughout the production cycle has important implications for the risk to consumers and requires further investigation. The detection of new strains within the population corresponding with the time of thinning and transportation reflects previous reports and provides further evidence that these activities pose a risk of introducing new Campylobacter strains to broiler batches.

A REVIEW OF CURRENT KNOWLEDGE AND GAPS ABOUT CAMPYLOBACTER METHODS: FROM CULTURE TO CHARACTERIZATION

Campylobacteriosis is a zoonotic disease caused by Campylobacter, mostly associated with consumption of contaminated foodstuffs and water. Campylobacter jejuni and Campylobacter coli recognized as the leader of foodborne diarrheal illness in humans. The frequency of these microorganisms in poultry is fairly high than Salmonella and more challenging to measure, which represent an expensive burden charge on public health due to their difficulties to master them, especially with the fast increase rates of multidrug-resistant of thermophilic Campylobacter strains. It is well recognized that Campylobacter spp. is a fastidious cell, difficult to isolate in laboratories owing to their requirements and sensibility. That’s why; these factors must be taken into consideration during recovery protocols. A variety of phenotyping tests have been reported and widely used for confirmation and identification of Campylobacter species. Nonetheless, Whole Genome Sequencing (WGS) and Culture-Independent Diagnostic Tests (CIDTs) are new eras of hopeful technologies, mainly involved in the detection and characterization of threaten public health pathogens. This review aimed to describe the culture methods, phenotypic and genotypic schemes used to isolate, identify, and characterize Campylobacter isolates, through discussing the current knowledge and gaps related to the application of these techniques over others performed for typing this microaerophilic genus.

Prevalence and antimicrobial resistance of Campylobacter from wild birds of prey in Spain

Wild birds have been identified as a relevant reservoir of Campylobacter spp., therefore, a potential source of infection in humans and domestic animals. The objective of this study was to determine the occurrence of Campylobacter spp. on birds of prey in Spain. In addition, antibiotic resistance profiles of the isolates were evaluated. A total of 689 specimens of 28 raptor species were analyzed, with a resulting individual prevalence of 7.5%. C. jejuni was the most frequently isolated species (88.5%), followed by C. coli and C. lari (3.8% each). The occurrence of Campylobacter was significantly higher (p < 0.05) in nocturnal birds of prey (15.3%), in spring season (12.2%) and in carnivorous species (9.4%). Isolates displayed a remarkable resistance to nalidixic acid (69.9%), ciprofloxacin (69.9%), and tetracycline (55.6%), and a low resistance to streptomycin (6.7%).Our findings highlight the importance of birds of prey as reservoirs of Campylobacter strains and their significant role as carriers of antimicrobial resistance.

Lactic Acid Bacteria - A Promising Tool for Controlling Chicken Campylobacter Infection.

Since 2005, campylobacteriosis has been the most common zoonotic disease in Europe. The main reservoir of pathogenic Campylobacter strains is broilers, which makes raw and undercooked poultry meat two major sources of disease. Infection in chicken flocks is most often asymptomatic, despite a high level of colonization reaching 106–109cfu/g in animal ceca. It is widely believed that controlling the level of colonization of the birds’ digestive tract by pathogenic strains is a good way to increase food safety. Many treatments have been proposed to combat or at least reduce the level of colonization in animals reservoirs: probiotics, bacteriophages, vaccines, and anti-Campylobacter bacteriocins. This review focuses on the effects of Campylobacter infection on the chicken microbiome and colonization control strategies using probiotics (mostly lactic acid bacteria, LAB), which are live microorganisms included in the diet of animals as feed additives or supplements. Probiotics are not only an alternative to antibiotics, which were used for years as animal growth promoters, but they also constitute an effective protective barrier against excessive colonization of the digestive system by pathogenic bacteria, including Campylobacter. Moreover, one of the many beneficial functions of probiotics is the ability to manipulate the host’s microbiota. Recently, there have also been some promising attempts to use lactic acid bacteria as a delivery system of oral vaccine against Campylobacter. Recombinant LAB strains induce primarily a mucosal immune response against foreign antigens, accompanied by at most a low-level immune response against carrier strains. Since the main barrier against the invasion of pathogens in the gastrointestinal tract is the intestinal mucosal membrane, the development of effective oral vaccines to protect animals against enteric infection is very reasonable.

Natural Horizontal Gene Transfer of Antimicrobial Resistance Genes in Campylobacter spp. From Turkeys and Swine.

Antibiotic-resistant Campylobacter constitutes a serious threat to public health. The clonal expansion of resistant strains and/or the horizontal spread of resistance genes to other strains and species can hinder the clinical effectiveness of antibiotics to treat severe campylobacteriosis. Still, gaps exist in our understanding of the risks of acquisition and spread of antibiotic resistance in Campylobacter. While the in vitro transfer of antimicrobial resistance genes between Campylobacter species via natural transformation has been extensively demonstrated, experimental studies have favored the use of naked DNA to obtain transformants. In this study, we used experimental designs closer to real-world conditions to evaluate the possible transfer of antimicrobial resistance genes between Campylobacter strains of the same or different species (Campylobacter coli or Campylobacter jejuni) and originating from different animal hosts (swine or turkeys). This was evaluated in vitro through co-culture experiments and in vivo with dual-strain inoculation of turkeys, followed by whole genome sequencing of parental and newly emerged strains. In vitro, we observed four independent horizontal gene transfer events leading to the acquisition of resistance to beta-lactams (blaOXA), aminoglycosides [aph(2′′)-If and rpsL] and tetracycline [tet(O)]. Observed events involved the displacement of resistance-associated genes by a mutated version, or the acquisition of genomic islands harboring a resistance determinant by homologous recombination; we did not detect the transfer of resistance-carrying plasmids even though they were present in some strains. In vivo, we recovered a newly emerged strain with dual-resistance pattern and identified the replacement of an existing non-functional tet(O) by a functional tet(O) in the recipient strain. Whole genome comparisons allowed characterization of the events involved in the horizontal spread of resistance genes between Campylobacter following in vitro co-culture and in vivo dual inoculation. Our study also highlights the potential for antimicrobial resistance transfer across Campylobacter species originating from turkeys and swine, which may have implications for farms hosting both species in close proximity.

Biofilm Formation Ability of Arcobacter-like and Campylobacter Strains under Different Conditions and on Food Processing Materials.

Campylobacter jejuni is the most frequent cause of bacterial gastrointestinal food-borne infection worldwide. The transmission of Campylobacter and Arcobacter-like species is often made possible by their ability to adhere to various abiotic surfaces. This study is focused on monitoring the biofilm ability of 69 strains of Campylobacter spp. and lesser described species of the Arcobacteraceae family isolated from food, water, and clinical samples within the Czech Republic. Biofilm formation was monitored and evaluated under an aerobic/microaerophilic atmosphere after cultivation for 24 or 72 h depending on the surface material. An overall higher adhesion ability was observed in arcobacters. A chi-squared test showed no association between the origin of the strains and biofilm activity (p > 0.05). Arcobacter-like species are able to form biofilms under microaerophilic and aerobic conditions; however, they prefer microaerophilic environments. Biofilm formation has already been demonstrated at refrigerator temperatures (5 °C). Arcobacters also showed higher biofilm formation ability at the temperature of 30 °C. This is in contrast to Campylobacter jejuni NP 2896, which showed higher biofilm formation ability at temperatures of 5–30 °C. Overall, the results demonstrated the biofilm formation ability of many strains, which poses a considerable risk to the food industry, medical practice, and human health.

Assessment of Genetic Stability During Serial In Vitro Passage and In Vivo Carriage.

In this study, our objective was to evaluate the genetic stability of foodborne bacterial pathogens during serial passage in vitro and persistent in vivo carriage. Six strains of Listeria, Campylobacter, Escherichia, Salmonella, and Vibrio were serially passaged 20 times. Three colonies were picked for whole-genome sequencing (WGS) from passes P0, P5, P10, P15, and P20. In addition, isolates of Salmonella and Escherichia from three patients with persistent infections were sequenced. Genetic stability was evaluated in terms of variations detected in high-quality single-nucleotide polymorphism (hqSNP), core genome multilocus sequence typing (cgMLST), seven-gene MLST, and determinants encoding serotype, antimicrobial resistance (AMR), and virulence. During serial passage, increasing diversity was observed in Listeria, Salmonella, and Vibrio as measured by hqSNPs (from median of 0 SNPs to median of 3-5 SNPs, depending on the organism) and to a lesser extent with cgMLST (from median of 0 alleles to median of 0-5 alleles), while Escherichia and Campylobacter genomes showed minimal variation. The serotype, AMR, and virulence markers remained stable in all organisms. Isolates from persistent infections lasting up to 10 weeks remained genetically stable. However, isolates from a persistent Salmonella enterica ser. Montevideo infection spanning 9 years showed early heterogeneity leading to the emergence of one predominant genotype that continued to evolve over the years, including gains and losses of AMR markers. While the hqSNP and cgMLST variation observed during the serial passage was minimal, culture passages should be limited to as few times as possible before WGS. Our WGS data show that in vivo carriage lasting for a few weeks did not appear to alter the genotype. Longer persistent infections spanning for years, particularly in the presence of selective pressure, may cause changes in the genotype making it challenging to differentiate persistent infections from reinfections.

Editorial and News

Editorial and News

Mutagenic strategies against luxS gene affect the early stage of biofilm formation of Campylobacter jejuni.

Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species, naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81-176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated ([Formula: see text]luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.

Genomic Characterization of Fluoroquinolone-Resistant Thermophilic Campylobacter Strains Isolated from Layer Chicken Feces in Gangneung, South Korea by Whole-Genome Sequencing.

Thermophilic Campylobacter species of poultry origin have been associated with up to 80% of human campylobacteriosis cases. Layer chickens have received less attention as possible reservoirs of Campylobacter species. Initially, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of two archived Campylobacter isolates (Campylobacter jejuni strain 200605 and Campylobacter coli strain 200606) from layer chickens to five antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, tetracycline, and gentamicin) were determined using broth microdilution while the presence of selected antimicrobial resistance genes was performed by polymerase chain reaction (PCR) using specific primers. Whole-genome sequencing (WGS) was performed by the Illumina HiSeq X platform. The analysis involved antimicrobial resistance genes, virulome, multilocus sequence typing (MLST), and phylogeny. Both isolates were phenotypically resistant to ciprofloxacin (MIC: 32 vs. 32 µg/mL), nalidixic acid (MIC: 128 vs. 64 µg/mL), and tetracycline (MIC: 64 vs. 64 µg/mL), but sensitive to erythromycin (MIC: 1 vs. 2 µg/mL) and gentamicin (MIC: 0.25 vs. 1 µg/mL) for C. jejuni strain 200605 and C. coli strain 200606, respectively. WGS confirmed C257T mutation in the gyrA gene and the presence of cmeABC complex conferring resistance to FQs in both strains. Both strains also exhibited tet(O) genes associated with tetracycline resistance. Various virulence genes associated with motility, chemotaxis, and capsule formation were found in both isolates. However, the analysis of virulence genes showed that C. jejuni strain 200605 is more virulent than C. coli strain 200606. The MLST showed that C. jejuni strain 200605 belongs to sequence type ST-5229 while C. coli strain 200606 belongs to ST-5935, and both STs are less common. The phylogenetic analysis clustered C. jejuni strain 200605 along with other strains reported in Korea (CP028933 from chicken and CP014344 from human) while C. coli strain 200606 formed a separate cluster with C. coli (CP007181) from turkey. The WGS confirmed FQ-resistance in both strains and showed potential virulence of both strains. Further studies are recommended to understand the reasons behind the regional distribution (Korea, China, and Vietnam) of such rare STs.

Staphylococcus aureus enhances biofilm formation, aerotolerance, and survival of Campylobacter strains isolated from retail meats

In retail meat products, Campylobacter jejuni, C. coli, and Staphylococcus aureus have been reported in high prevalence. The polymicrobial interaction between Campylobacter and other bacteria could enhance Campylobacter survival during the adverse conditions encountered during retail meat processing and storage. This study was designed to investigate the potential role of S. aureus from retail meats in enhancing the survival of Campylobacter exposed to low temperature, aerobic conditions, and biofilm formation. Results indicated that viable S. aureus cells and filter-sterilized cell-free media obtained from S. aureus prolonged the survival of Campylobacter at low temperature and during aerobic conditions. Biofilm formation of Campylobacter strains was significantly enhanced in the presence of viable S. aureus cells, but the results were inconclusive when extracts from cell-free media were used. In conclusion, the presence of S. aureus cells enhances survivability of Campylobacter strains in adverse conditions such as low temperature and aerobic conditions. Further investigations are warranted to understand the interaction between Campylobacter and S. aureus, and effective intervention strategies are needed to reduce the incidence of both foodborne pathogens in retail meat products.

Whole genome sequencing data used for surveillance of Campylobacter infections: detection of a large continuous outbreak, Denmark, 2019.

Background Campylobacter is one of the most frequent causes of bacterial gastroenteritis. Campylobacter outbreaks are rarely reported, which could be a reflection of a surveillance without routine molecular typing. We have previously shown that numerous small outbreak-like clusters can be detected when whole genome sequencing (WGS) data of clinical Campylobacter isolates was applied.AimTyping-based surveillance of Campylobacter infections was initiated in 2019 to enable detection of large clusters of clinical isolates and to match them to concurrent retail chicken isolates in order to react on ongoing outbreaks.MethodsWe performed WGS continuously on isolates from cases (n = 701) and chicken meat (n = 164) throughout 2019. Core genome multilocus sequence typing was used to detect clusters of clinical isolates and match them to isolates from chicken meat.ResultsSeventy-two clusters were detected, 58 small clusters (2–4 cases) and 14 large clusters (5–91 cases). One third of the clinical isolates matched isolates from chicken meat. One large cluster persisted throughout the whole year and represented 12% of all studied Campylobacter cases. This cluster type was detected in several chicken samples and was traced back to one slaughterhouse, where interventions were implemented to control the outbreak.ConclusionOur WGS-based surveillance has contributed to an improved understanding of the dynamics of the occurrence of Campylobacter strains in chicken meat and the correlation to clusters of human cases.

The diversity of Campylobacter spp. throughout the poultry processing plant

Campylobacteriosis is the leading food-borne disease in developed countries, and poultry are a major source for human infection. The diversity of Campylobacter on chicken carcasses during processing may lead to isolates that are able to survive abattoir processing. This has important implications for public health and adds a further layer to the complexity of the epidemiology of campylobacteriosis. The diversity of the Campylobacter spp. populations on broiler carcasses was studied at three different stages of processing (post-bleed, post-scald and post-chill) in three UK processing plants, using the pulsed-field gel electrophoresis (PFGE) KpnI enzyme. One hundred and sixty Campylobacter strains from 3 processing plants were identified as C.jejuni (92.3%) with 27 PFGE subtype profiles recovered from carcasses at the post-bleed point. Change in populations was identified when carcasses move towards the end of poultry processing. Seven C.jejuni genotypes were able to survive the scalding tank stage process, and 5 genotypes surviving the entire poultry process. Confirmation by PFGE gives information on the genotypic profiles of C.jejuni on chicken carcasses and how they change according to the temperatures exposed to during processing. Diversity within C.jejuni populations produces genotypes that adapt to tolerate the processing environment, and these may be capable of causing human disease. Understanding more about the genotypes that survive the processing will have important implications for public health.