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Thermotolerant Campylobacter spp. are the most frequent cause of foodborne bacterial diarrhea and high-priority antibiotic-resistant pathogens, according to the World Health Organization (WHO). Monitoring revealed current low prevalence of gentamicin resistance in European Campylobacter spp. isolates but substantial presence of gentamicin modifying genes circulating globally. Using a combined approach of natural transformation and whole-genome sequencing, we revealed a novel gentamicin resistance mechanism, namely the point mutation A1387G in the 16S rRNA gene, originally identified in a C. coli isolate from turkey caecal content. The transformation rate of the resistance using genomic DNA of the resistant donor to sensitive recipient C. jejuni and C. coli was ~2.5 log 10 lower compared to the control rpsL -A128G point mutation conferring streptomycin resistance. Antimicrobial susceptibility tests showed cross-resistance to apramycin, kanamycin, and tobramycin, with transformants exhibiting more than 4- to 8-fold increased MICs to apramycin and tobramycin and over 64-fold higher MICs to kanamycin compared to wild-type isolates. Although transformants showed 177-1,235 variations relative to the recipient, only the A1387G point mutation in the 16S rRNA was in common. This mutation was causal for resistance, as transformation of a 16S rRNA_A1387G PCR fragment into susceptible isolates also led to resistant transformants. Sanger sequencing of the 16S rRNA genes and Oxford nanopore whole-genome sequencing of transformants identified clones harboring either all three copies with A1387G or a mixed population of wild-type and mutated 16S rRNA gene alleles. Within 15 passages on non-selective medium, transformants with mixed populations of the 16S rRNA gene copies partially reverted to wild type, both geno- and phenotypically. In contrast, transformants harboring the A1387G point mutation in all three 16S rRNA gene copies kept full resistance within at least 45 passages. We speculate that partial acquisition and rapid loss of the point mutation limited its spread among C . spp. isolates. In-depth knowledge on resistance mechanisms contributes to optimal diagnosis and preventative measures., Competing Interests: The authors declare no conflict of interest.

Campylobacter jejuni and Arcobacter butzleri are microaerobic food-borne human gastrointestinal pathogens that mainly cause diarrheal disease. These related species of the Campylobacteria class face variable atmospheric environments during infection and transmission, ranging from nearly anaerobic to aerobic conditions. Consequently, their lifestyles require that both pathogens need to adjust their metabolism and respiration to the changing oxygen concentrations of the colonization sites. Our transcriptomic and proteomic studies revealed that C. jejuni and A. butzleri, lacking a Campylobacteria -specific regulatory protein, C. jejuni Cj1608, or a homolog, A. butzleri Abu0127, are unable to reprogram tricarboxylic acid cycle or respiration pathways, respectively, to produce ATP efficiently and, in consequence, adjust growth to changing oxygen supply. We propose that these Campylobacteria energy and metabolism regulators (CemRs) are long-sought transcription factors controlling the metabolic shift related to oxygen availability, essential for these bacteria's survival and adaptation to the niches they inhabit. Besides their significant universal role in Campylobacteria , CemRs, as pleiotropic regulators, control the transcription of many genes, often specific to the species, under microaerophilic conditions and in response to oxidative stress., Importance: C. jejuni and A. butzleri are closely related pathogens that infect the human gastrointestinal tract. In order to infect humans successfully, they need to change their metabolism as nutrient and respiratory conditions change. A regulator called CemR has been identified, which helps them adapt their metabolism to changing conditions, particularly oxygen availability in the gastrointestinal tract so that they can produce enough energy for survival and spread. Without CemR, these bacteria, as well as a related species, Helicobacter pylori , produce less energy, grow more slowly, or, in the case of C. jejuni , do not grow at all. Furthermore, CemR is a global regulator that controls the synthesis of many genes in each species, potentially allowing them to adapt to their ecological niches as well as establish infection. Therefore, the identification of CemR opens new possibilities for studying the pathogenicity of C. jejuni and A. butzleri ., Competing Interests: The authors declare no conflict of interest.

C. jejuni is an important food-borne pathogen displaying high genetic diversity, substantially based on natural transformation. The mechanism of DNA uptake from the environment depends on a type II secretion/type IV pilus system, whose components are partially known. Here, we quantified DNA uptake in C. jejuni at the single cell level and observed median transport capacities of approximately 30 kb per uptake location. The process appeared to be limited by the initialization of DNA uptake, was finite, and, finalized within 30 min of contact to DNA. Mutants lacking either the outer membrane pore PilQ or the inner membrane channel ComEC were deficient in natural transformation. The periplasmic DNA binding protein ComE was negligible for DNA uptake, which is in contrast to its proposed function. Intriguingly, a mutant lacking the unique periplasmic protein Cj0683 displayed rare but fully functional DNA uptake events. We conclude that Cj0683 was essential for the efficient initialization of DNA uptake, consistent with the putative function as a competence pilus protein. Unravelling features important in natural transformation might lead to target identification, reducing the adaptive potential of pathogens.

This article describes the outline and organisation of the validation of three multiplex PCR methods for species identification and/or confirmation of thermotolerant Campylobacter spp. The three PCR methods were validated against the reference method described in the EN ISO standard 10272:2017. The results of the PCR methods were compared against the reference method in a method comparison study and an interlaboratory study based on EN ISO 16140-6:2019. The performance, in terms of inclusivity and exclusivity, of each of the eight PCR targets were comparable to the performance of the reference method: close, equal, or better depending on the target. In total, all three PCR methods were concluded to be equally qualified as the reference method for molecular identification and/or confirmation of thermotolerant Campylobacter spp., C. jejuni, C. coli and C. lari isolated from the food chain and have been included in Amendment 1 of ISO 10272:2017., Competing Interests: Declaration of competing interest The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript., (Copyright © 2022. Published by Elsevier B.V.)

Campylobacter jejuni has a large adaptive potential due to enormous genetic exchange. Factors regulating natural transformation in this food-borne pathogen are largely unknown but of interest for the application of sustained reduction strategies in the food-processing industry. Using a single cell DNA uptake assay, we visualized that recognition of methylated C. jejuni DNA was essential for the first step of DNA uptake into a DNase resistant state. Transformation rates using a resistance marker correlated with the fraction of competent bacteria, harboring one to maximally four locations of active DNA uptake, not necessarily being located at the cell pole. Competence developed with rising pH between 6.5 and 7.5 under microaerobic conditions and was nearly insensitive towards growth temperatures between 32 °C and 42 °C, CO 2 concentrations ranging from 0 to 50% and growth rates. However, competence development was abolished at pH 5 or under aerobic stress conditions, in which the bacteria ceased growth but fully survived. The DNA uptake machinery in competent bacteria shut down at slightly acidic pH and was reversibly switched on upon neutralization. It was dependent on the proton motive force and, in contrast to competence development, slightly enhanced under aerobic conditions. The results suggest that natural transformation in C. jejuni occurs in the neutral and microaerobic intestinal environment for enhanced genetic diversity and pre-adaption before host switch. In addition, highly competent bacteria might be shed into the environment, still able to acquire genetic material for increased survival.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis globally, and infections are often transmitted through consumption of raw or undercooked poultry. Campylobacter jejuni ST50 is among the top ten sequence types (STs) reported in the collected isolates listed at PubMLST records from poultry, food and clinical sources for Asia, Europe, North America, Oceania and South America. This study was designed to determine the most commonly reported C. jejuni STs globally using the PubMLST database and assess similarities between genomes of C. jejuni ST50 isolates from geographically distinct locations. To gain a better understanding of C. jejuni diversity, we compared draft genome sequences of 182 ST50 isolates recovered from retail or caecal poultry samples in Oceania, Europe and North America that were collected over a period of 9 years (2010 to 2018). Overall, phylogenetic analysis revealed that isolates from geographically distinct locations tended to cluster based on the continent where the sample was collected. Among ST50 isolates from Europe and North America, we identified resistance determinants associated with phenotypic resistance to beta-lactams (EU: 55%; GB: 43.1%), tetracyclines (CA: 77.3%; EU: 37.5%; GB: 9.8%; US: 43.5%) and fluoroquinolones (EU: 60.0%; GB: 15.7%); no resistance determinants were identified in isolates from Australia. In general, the majority of the virulence genes, with rare exceptions such as wlaN, cj1138, hddA and rfbC, were evenly distributed throughout the genomes of all ST50 isolates in this study. Genomic-based characterization of C. jejuni ST50 isolates from poultry on three continents highlighted that geographically distinct isolates have evolved independently but only represent a glimpse into the diversity of C. jejuni., (© 2021 Wiley-VCH GmbH.)

The zoonotic pathogen Campylobacter jejuni is among the leading causes of foodborne diseases worldwide. While C. jejuni colonises many wild animals and livestock, persistence mechanisms enabling the bacterium to adapt to host species' guts are not fully understood. In order to identify putative determinants influencing host preferences of distinct lineages, bootstrapping based on stratified random sampling combined with a k-mer-based genome-wide association was conducted on 490 genomes from diverse origins in Germany and Canada. We show a strong association of both the core and the accessory genome characteristics with distinct host animal species, indicating multiple adaptive trajectories defining the evolution of C. jejuni lifestyle preferences in different ecosystems. Here, we demonstrate that adaptation towards a specific host niche ecology is most likely a long evolutionary and multifactorial process, expressed by gene absence or presence and allele variations of core genes. Several host-specific allelic variants from different phylogenetic backgrounds, including dnaE, rpoB, ftsX or pycB play important roles for genome maintenance and metabolic pathways. Thus, variants of genes important for C. jejuni to cope with specific ecological niches or hosts may be useful markers for both surveillance and future pathogen intervention strategies.

Thermophilic Campylobacter, in particular Campylobacter jejuni, C. coli and C. lari are the main relevant Campylobacter species for human infections. Due to their high capacity of genetic exchange by horizontal gene transfer (HGT), rapid adaptation to changing environmental and host conditions contribute to successful spreading and persistence of these foodborne pathogens. However, extensive HGT can exert dangerous side effects for the bacterium, such as the incorporation of gene fragments leading to disturbed gene functions. Here we discuss mechanisms of HGT, notably natural transformation, conjugation and bacteriophage transduction and limiting regulatory strategies of gene transfer. In particular, we summarize the current knowledge on how the DNA macromolecule is exchanged between single cells. Mechanisms to stimulate and to limit HGT obviously coevolved and maintained an optimal balance. Chromosomal rearrangements and incorporation of harmful mutations are risk factors for survival and can result in drastic loss of fitness. In Campylobacter, the restricted recognition and preferential uptake of free DNA from relatives are mediated by a short methylated DNA pattern and not by a classical DNA uptake sequence as found in other bacteria. A class two CRISPR-Cas system is present but also other DNases and restriction-modification systems appear to be important for Campylobacter genome integrity. Several lytic and integrated bacteriophages have been identified, which contribute to genome diversity. Furthermore, we focus on the impact of gene transfer on the spread of antibiotic resistance genes (resistome) and persistence factors. We discuss remaining open questions in the HGT field, supposed to be answered in the future by current technologies like whole-genome sequencing and single-cell approaches.

Campylobacter is the major bacterial agent of human gastroenteritis worldwide and represents a crucial global public health burden. Species differentiation of C. jejuni and C. coli and phylogenetic analysis is challenged by inter-species horizontal gene transfer. Routine real-time PCR on more than 4000 C. jejuni and C. coli field strains identified isolates with ambiguous PCR results for species differentiation, in particular, from the isolation source eggs. K-mer analysis of whole genome sequencing data indicated the presence of C. coli hybrid strains with huge amounts of C. jejuni introgression. Recombination events were distributed over the whole chromosome. MLST typing was impaired, since C. jejuni sequences were also found in six of the seven housekeeping genes. cgMLST suggested that the strains were phylogenetically unrelated. Intriguingly, the strains shared a stress response set of C. jejuni variant genes, with proposed roles in oxidative, osmotic and general stress defence, chromosome maintenance and repair, membrane transport, cell wall and capsular biosynthesis and chemotaxis. The results have practical impact on routine typing and on the understanding of the functional adaption to harsh environments, enabling successful spreading and persistence of Campylobacter.

Campylobacter jejuni ( C. jejuni ) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa , a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni -induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10 -/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni -induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1β, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni -triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients.

Quantification of Campylobacter is challenging and one major reason is the fact that bacteria lose cultivability due to cold or oxygen stress during storage at retail. Alternative live/dead discriminatory qPCR currently lacks standardization and might overestimate live cells in the presence of dead cells. In this study an internal sample process control (ISPC) was developed. The ISPC consists of a specified number of peroxide-killed C. sputorum cells to be added to each sample in order to monitor (i) the level of reduction of the signal from dead cells and (ii) DNA losses during sample processing. A species-specific fragment of the 16S rRNA gene of C. sputorum was selected as real-time PCR target, based on its similar size and gene copy number compared to the C. jejuni/coli/lari target and confirmed in an exclusivity study. Extension of the amplification oligonucleotides for the target of thermotolerant Campylobacter improved real-time PCR efficiency, rendering the method suitable for quantification according to international standards. Concordant PCR signal variation of both C. jejuni and C. sputorum targets in co-inoculated chicken rinses verified the suitability of the ISPC. This provides a crucial step towards implementation of cultivation-independent quantification for improved food safety of fastidious bacteria., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria., Results: In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log10 unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log10 units., Conclusion: The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.

Campylobacter jejuni and Campylobacter coli are the predominant thermophilic species responsible for foodborne gastroenteritis worldwide. Elevated resistance to certain antibiotics was observed due to antimicrobial therapy in farm animals and humans, while reduced antimicrobial usage partially reduced antibiotic resistance. Monitoring the antimicrobial resistance demonstrated a substantial fraction of multi-resistant isolates, indicating the necessity of reliable tools for their detection. In this study, resistance determinants in 129 German and 21 Vietnamese isolates were selected to establish a novel multiplex real-time PCR (qPCR), facilitating the simultaneous detection of four resistance determinants. These comprised tet(O) gene variants associated with tetracycline resistance, point mutations GyrA_T86I and GyrA_T86V associated with ciprofloxacin resistance, and the erm(B) gene together with the point mutation A2075G in the 23S rRNA gene, associated with erythromycin resistance. Moreover, the performance of the qPCR assay was evaluated by comparing the results of qPCR to phenotypic antimicrobial resistance profiles, obtained with standardized EUCAMP3 microdilution panel, which showed 100% similarity (inclusivity and exclusivity). Variation in measurement methods, including qPCR machines and master mixes showed robustness, essential for laboratories. The assay can be used for the rapid detection of resistance determinants, and is beneficial for monitoring the spread of antibiotic resistance in C. jejuni and C. coli. [ABSTRACT FROM AUTHOR]

Campylobacter spp. cannot grow in raw milk, but it is able to transform into a viable but non‐culturable (VBNC) state enabling the survival in such harsh conditions. In this study, Campylobacter jejuni survival in raw milk was investigated taken into consideration colony‐forming units (CFUs) and VBNC cells. CFU from two different strains of C. jejuni (DSM 4688 and BfR‐CA‐18043) were enumerated at three temperatures (5°C, 8°C, and 12°C). In parallel, a viability real‐time PCR was conducted to quantify intact and putatively infectious units (IPIUs) (comprising CFU and VBNC bacteria). The data generated were used to model the viability of C. jejuni during raw milk storage. Here, a one‐step fitting approach was performed using parameter estimates from an intermediate two‐step fit as starting values to generate tertiary models. Different primary model equations (Trilinear and Weibull) were required to fit the CFU and the IPIU data. Strain‐specific linear secondary models were generated to analyze the effect of storage temperature on the maximum specific inactivation rate of the CFU data. The time of the first decimal reduction parameter of the IPIU models could be modeled by a strain‐independent linear secondary model. The developed tertiary models for CFU and IPIU differ significantly in their predictions, for example, for the time required for a one log10 reduction. Taken into consideration that VBNC could revert to a culturable state during the raw milk storage, our results underline the importance of considering IPIU and not only CFU to avoid underestimation of the survival of C. jejuni in raw milk. [ABSTRACT FROM AUTHOR]

Campylobacteriosis outbreaks have repeatedly been associated with the consumption of raw milk. This study aimed to explore the variation in the prevalence and concentration of Campylobacter spp. in cows' milk and feces, the farm environment and on the teat skin over an entire year at a small German dairy farm. Bi-weekly samples were collected from the environment (boot socks), teats, raw milk, milk filters, milking clusters and feces collected from the recta of dairy cows. Samples were analyzed for Campylobacter spp., E. coli, the total aerobic plate count and for Pseudomonas spp. The prevalence of Campylobacter spp. was found to be the highest in feces (77.1%), completely absent in milking equipment and low in raw milk (0.4%). The mean concentration of Campylobacter spp. was 2.43 log10 colony-forming units (CFU)/g in feces and 1.26 log10 CFU/teat swab. Only a single milk filter at the end of the milk pipeline and one individual cow's raw milk sample were positive on the same day, with a concentration of 2.74 log10 CFU/filter and 2.37 log10 CFU/mL for the raw milk. On the same day, nine teat swab samples tested positive for Campylobacter spp. This study highlights the persistence of Campylobacter spp. for at least one year in the intestine of individual cows and within the general farm environment and demonstrates that fecal cross-contamination of the teats can occur even when the contamination of raw milk is a rare event. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance remains a public health concern globally. This study presents antimicrobial resistance by microdilution and genetic diversity by the whole-genome sequencing of Campylobacter spp. from human and poultry samples isolated in Georgia in 2020/2021. The major species in poultry samples was C. coli, while C. jejuni was preferentially isolated from human samples. Resistance against tetracycline was highest (100%) in C. coli from industrial chicken and lowest in C. jejuni from clinical isolates (36%), while resistance against ciprofloxacin varied from 80% in C. jejuni from backyard chicken to 100% in C. jejuni and C. coli from industrial chicken. The point mutations in gyrA (T86I) and tet (O) genes were detected as resistance determinants for (fluoro-)quinolone or tetracycline resistance, respectively. Ertapenem resistance is still enigmatic. All isolates displayed sensitivity towards erythromycin, gentamicin and chloramphenicol. Multi-resistance was more frequently observed in C. coli than in C. jejuni, irrespective of the isolation matrix, and in chicken isolates compared to human isolates, independent of the Campylobacter species. The Georgian strains showed high variability of multi-locus sequence types (ST), including novel STs. This study provides the first antibiotic resistance data from Campylobacter spp. in Georgia and addresses the need for follow-up monitoring programs. [ABSTRACT FROM AUTHOR]

Thermotolerant Campylobacter spp. are fecal contaminants of chicken meat with serious implications for human health. E. coli is considered as hygiene indicator since, in contrast to Campylobacter. spp., the bacterium is generally present in the avian gut. Stress exposure may transiently cease bacterial division. Therefore, colony forming units (CFU) may underestimate the infection risk of pathogens. We developed a viability real-time PCR (v-qPCR) for the quantification of viable E. coli targeting the uidA gene, encoding β-glucuronidase, which is usually detected for phenotypic species identification. The short- and long-term effects of decontaminating chicken skin on the survival of both C. jejuni and an ESBL-producing E. coli were evaluated by CFU and v-qPCR. The results showed that freezing and storage in cool conditions are potentially underestimated by CFU but not by v-qPCR. The effect of treatment with peroxyacetic acid on survival was consistently detected by CFU and v-qPCR. v-qPCR analysis detected bacterial survival upon the application of lactic acid, which awaits further analysis. Interestingly, both bacteria showed similar kinetics of inactivation upon the application of reduction strategies, suggesting that E. coli might be a complementary hygiene indicator. We conclude that v-qPCR can improve food safety under the consideration of some limitations. [ABSTRACT FROM AUTHOR]

Objectives: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output.Methods: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins.Results: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance.Conclusions: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered. [ABSTRACT FROM AUTHOR]

We compared the consistency, accuracy and reproducibility of next-generation short read sequencing between ten laboratories involved in food safety (research institutes, state laboratories, universities and companies) from Germany and Austria. Participants were asked to sequence six DNA samples of three bacterial species (Campylobacter jejuni , Listeria monocytogenes and Salmonella enterica) in duplicate, according to their routine in-house sequencing protocol. Four different types of Illumina sequencing platforms (MiSeq, NextSeq, iSeq, NovaSeq) and one Ion Torrent sequencing instrument (S5) were involved in the study. Sequence quality parameters were determined for all data sets and centrally compared between laboratories. SNP and cgMLST calling were performed to assess the reproducibility of sequence data collected for individual samples. Overall, we found Illumina short read data to be more accurate (higher base calling accuracy, fewer miss-assemblies) and consistent (little variability between independent sequencing runs within a laboratory) than Ion Torrent sequence data, with little variation between the different Illumina instruments. Two laboratories with Illumina instruments submitted sequence data with lower quality, probably due to the use of a library preparation kit, which shows difficulty in sequencing low GC genome regions. Differences in data quality were more evident after assembling short reads into genome assemblies, with Ion Torrent assemblies featuring a great number of allele differences to Illumina assemblies. Clonality of samples was confirmed through SNP calling, which proved to be a more suitable method for an integrated data analysis of Illumina and Ion Torrent data sets in this study. [ABSTRACT FROM AUTHOR]

Raw milk is a frequent vehicle for transmission of thermophilic Campylobacter , leading to reported outbreaks. Milk is a challenging food matrix for pathogen detection, due to its high protein and lipid content. Limited detection of Campylobacter colony-forming unit (CFU) in raw milk might underestimate the pathogen's infectious potential. We optimized a viability real-time PCR (qPCR) for application with raw milk. The procedure was robust against variations of milk lots and different Campylobacter strains. Various DNA-intercalating dyes were evaluated for their ability to reduce the PCR signal of dead cells. Only propidium monoazide (PMA) and PMAxx qualified for diagnostic use. Different sedimentation properties of viable and dead Campylobacter jejuni and Campylobacter coli strains in 10-fold diluted milk enhanced viable/dead differentiation. The new method enabled to review survival of Campylobacter spp. in raw milk based on viable cells harboring an intact cell membrane. The data were compared to culturability according to ISO10272-2:2017. A difference of up to 4.5 log10 between viable Campylobacter counts and CFU values became apparent. Relevance of viability qPCR values was corroborated by full recovery of CFU under extremely reduced oxygen concentration in the presence of hydrogen. Recovery of CFU was limited, however, upon prolonged exposure in raw milk. The data confirm that Campylobacter survival in raw milk can be largely underestimated when relying on CFU data only. We conclude that raw milk led to oxidative stress-induced growth arrest in thermophilic Campylobacter , which was reversible by reduction of the oxygen partial pressure in a time-limited way. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is the leading bacterial cause of foodborne infections worldwide. However, our understanding of its cell cycle is poor. We identified the probable C. jejuni origin of replication (oriC) – a key element for initiation of chromosome replication, which is also important for chromosome structure, maintenance and dynamics. The herein characterized C. jejuni oriC is monopartite and contains (i) the DnaA box cluster, (ii) the DnaA-dependent DNA unwinding element (DUE) and (iii) binding sites for regulatory proteins. The cluster of five DnaA boxes and the DUE were found in the dnaA-dnaN intergenic region. Binding of DnaA to this cluster of DnaA-boxes enabled unwinding of the DUE in vitro. However, it was not sufficient to sustain replication of minichromosomes, unless the cluster was extended by additional DnaA boxes located in the 3′ end of dnaA. This suggests, that C. jejuni oriC requires these boxes to initiate or to regulate replication of its chromosome. However, further detailed mutagenesis is required to confirm the role of these two boxes in initiation of C. jejuni chromosome replication and thus to confirm partial localization of C. jejuni oriC within a coding region, which has not been reported thus far for any bacterial oriC. In vitro DUE unwinding by DnaA was inhibited by Cj1509, an orphan response regulator and a homolog of HP1021, that has been previously shown to inhibit replication in Helicobacter pylori. Thus, Cj1509 might play a similar role as a regulator of C. jejuni chromosome replication. This is the first systematic analysis of chromosome replication initiation in C. jejuni, and we expect that these studies will provide a basis for future research examining the structure and dynamics of the C. jejuni chromosome, which will be crucial for understanding the pathogens’ life cycle and virulence. [ABSTRACT FROM AUTHOR]

Background: The incidence of human Campylobacter jejuni infections is progressively increasing worldwide. Probiotic compounds might open up valuable tools to decrease pathogen burden and subsequent pro-inflammatory immune responses, but in vivo data are scarce. Methods and results: Secondary abiotic mice generated by broad-spectrum antibiotic treatment were perorally challenged with the commercial probiotic compound VSL#3 consisting of Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus delbrueckii ssp. bulgaricus) either 5 days before (i.e. prophylactic regimen) or after (i.e. therapeutic regimen) peroral C. jejuni strain 81-176 infection, and analyzed 3 weeks following the initial bacterial reassociation. Upon challenge, mice were colonized with the probiotic bacteria and/or C. jejuni at comparable intestinal loads, but co-colonization did not result in reduction of the pathogen burden. Remarkably, prophylactic as well as therapeutic VSL#3 treatment of C. jejuni infected mice ameliorated intestinal apoptosis and pro-inflammatory immune responses as indicated by lower numbers of innate and adaptive immune cell populations in the murine colon upon probiotic prophylaxis or treatment and reduced colonic concentrations of pro-inflammatory mediators including IL-6 and MCP-1. Importantly, concentrations of anti-inflammatory mediators such as IL-10 were significantly elevated in the colon of probiotics treated mice as compared to untreated controls. Strikingly, prophylactic VSL#3 treatment attenuated C. jejuni induced systemic pro-inflammatory responses as indicated by less TNF and IL-12p70 secretion in the spleen of VSL#3 pre-treated as compared to non-treated mice. Conclusion: Administration of probiotic formulations such as VSL#3 might open up valuable strategies for prophylaxis and/or treatment of C. jejuni induced intestinal and systemic sequelae in vivo by the suppression of pro-inflammatory and induction of anti-inflammatory responses. [ABSTRACT FROM AUTHOR]

A screen of bacteriophages infecting a panel of Campylobacter jejuni PT14 gene knock-out mutants identified a role for the minor flagellin encoded by the flaB gene, in the defense of the host against CP8unalikevirus bacteriophage CP_F1 infection. Inactivation of the flaB gene resulted in an increase in the susceptibility of PT14 cultures to infection by CP_F1 and an increase in bacteriophage yields. Infection of wild type PT14 with CP_F1 produces turbid plaques in bacterial lawns, from which 78% of the resistant isolates recovered exhibit either attenuation or complete loss of motility. CP_F1 produces clear plaques on the flaB mutant with no regrowth in the lysis zones. Complementation of the mutant restored overgrowth and the development of resistance at the expense of motility. Further analyses revealed an increase in bacteriophage adsorption constant of nearly 2-fold and burst-size 3-fold, relative to the wild type. Motility analysis showed no major reduction in swarming motility in the flaB mutant. Thus, we propose a new role for FlaB in the defense of campylobacters against bacteriophage infection. [ABSTRACT FROM AUTHOR]

Campylobacteriosis is the leading zoonosis in the European Union with the majority of cases attributed to Campylobacter jejuni. Although the disease is usually selflimiting, some severe cases need to be treated with antibiotics, primarily macrolides and quinolones. However, the resistance to the latter is reaching alarming levels in most of the EU countries. To shed light on the expansion of antibiotic resistance in central Europe, we have investigated genetic similarity across 178 ciprofloxacin-resistant C. jejuni mostly isolated in Slovenia, Austria and Germany. We performed comparative genetic similarity analyses using allelic types of seven multilocus sequence typing housekeeping genes, and single nucleotide polymorphisms of a quinolone resistance determining region located within the DNA gyrase subunit A gene. This analysis revealed high genetic similarity of isolates from clonal complex ST-21 that carry gyrA allelic type 1 in all three of these central-European countries, suggesting these ciprofloxacin resistant isolates arose from a recent common ancestor and are spread clonally. [ABSTRACT FROM AUTHOR]

This is the first study on campylobacteriosis carried out in Georgia. It targeted 382 hospitalized children with acute inflammatory diarrhea. The study was conducted between July 2020 to July 2021 based on the main infection clinic of the capital city. Culture-based bacteriological methods were followed by phenotypic and Real-time PCR tests for bacterial confirmation and identification. The data revealed recent epidemiologic prevalences of the three main causative bacteria in the target population. Shigella sonnei with 19.1% (95% CI: 15.2–23.4%) was the most frequently detected pathogen followed by Campylobacter spp. with 12.3% (95% CI: 9.2–16.0%) and Salmonella spp. with 4.9% (95% CI: 3.0–7.6%). However, in 63.6% of the samples, the causative agent remained unknown. Species differentiation of Campylobacter spp. revealed 81% Campylobacter jejuni and 19% Campylobacter coli. An epidemiological pyramid with estimated magnification factors may give more insights into the burden of campylobacteriosis among the studied population, resulting in a putative annual incidence of 6 per 1000 children in Tbilisi. Children with campylobacteriosis were younger (median age 40 months (interquartile range (IQR) 22−95)) than with shigellosis (median age 92 months (interquartile range (IQR) 52−140)). However, no statistically significant difference was found with the age range of patients with campylobacteriosis and salmonellosis as well as with salmonellosis and shigellosis. In conclusion, Campylobacter spp. may be suspected to be the second most frequent bacterial causative agent of acute inflammatory diarrhea in hospitalized children and the primary cause in the 0–3 age group in Georgia. In addition, Campylobacter CROMagar showed better selectivity in comparison to mCCDA selective agar of stool samples in our study. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter , the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log 10 Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log 10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens. • The fastidious nature of Campylobacter poses problems for quantification by CFU. • The new viability (v-)qPCR method reliably differentiates between live and dead Campylobacter. • We validated the alternative v-qPCR method according to ISO 16140-2:2016(E). • International ring trial suggests v-qPCR fit for purpose with enhanced performance. • The v-qPCR can serve as alternative for CFU for quality control of meat rinses. [ABSTRACT FROM AUTHOR]

We investigated trends in antimicrobial resistance (AMR) in Campylobacter jejuni and Campylobacter coli in poultry between 2010 and 2016 in Germany and their association with antimicrobial use. Campylobacter had been isolated from the caeca of broilers and turkeys at slaughter by regional laboratories according to current ISO methods in the framework of a national monitoring program. Isolates were submitted to the National Reference Laboratory for Campylobacter and tested for AMR using broth microdilution methods. Minimum inhibitory concentrations were evaluated according to epidemiological cut-off values. Antimicrobial use (AMU) data from 2014 to 2016 were taken from a government report. AMR was higher in C. coli than in C. jejuni and higher in turkeys than in broilers. AMR was highest to tetracycline and the tested (fluoro)quinolones while it was rare to gentamicin in both bacterial species, infrequent to erythromycin in C. jejuni, and moderate in C. coli. AMR to tetracycline and erythromycin decreased over time while it increased to (fluoro)quinolones. An association of AMU and AMR was observed for resistance to tetracycline and erythromycin, while it was not observed for the aminoglycosides. Resistance to nalidixic acid and ciprofloxacin increased despite a decrease of fluoroquinolone use between 2014 and 2016, indicating that other factors have a strong influence on resistance to (fluoro)quinolones in Campylobacter. [ABSTRACT FROM AUTHOR]

A total of 502 Campylobacter jejuni isolates from poultry in 12 different European countries (10 of them the largest poultry production countries in Europe) were whole genome sequenced to examine the genomic diversity of fluoroquinolone resistant (FQ‐R) and susceptible (FQ‐S) C. jejuni across the poultry producing European countries and to determine whether the emergence of fluoroquinolone resistance among C. jejuni is related to the transmission through countries or to the selection through fluoroquinolone use in the individual countries. A high genomic diversity was observed. The isolates clustered in four main clusters. All trees revealed that the isolates were clustered according to the presence/absence of the gyrA mutations causing fluoroquinolone resistance and ST‐types. The cgMLST trees of only FQ‐R and FQ‐S isolates showed that isolates from the same country of origin were distributed into multiple clusters similarly to the trees combining FQ‐R and FQ‐S isolates. The different phylogenetic methods, ranging from single nucleotide polymorphisms analysis to gene‐by‐gene approaches such as rMLST, cgMLST, wgMLST and core genome tree, provided concordant results, but it is not known which is the most accurate method for identifying the country of origin of the isolates. Allele frequency analysis of isolates under this study and a selection of previously published C. jejuni genomes in ENA showed association of geographical origin of poultry C. jejuni populations between Romania‐Poland, Italy‐Germany‐England, Portugal‐The Netherlands and USA‐Luxemburg. Allele frequency and phylogenetic analysis indicated that the isolates from Finland were genetically different from C. jejuni populations from other European countries included in this study. Trade pattern and antimicrobial use in livestock were not significantly associated with allele frequency or populations of C. jejuni, but data available to investigate these associations were limited. [ABSTRACT FROM AUTHOR]