Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.

Proteome profiling of Campylobacter jejuni 81-176 at 37 °C and 42 °C by label-free mass spectrometry.

BACKGROUND: The main natural reservoir for Campylobacter jejuni is the avian intestinal tract. There, C. jejuni multiplies optimally at 42 °C - the avian body temperature. After infecting humans through oral intake, the bacterium encounters the lower temperature of 37 °C in the human intestinal tract. Proteome profiling by label-free mass spectrometry (DIA-MS) was performed to examine the processes which enable C. jejuni 81-176 to thrive at 37 °C in comparison to 42 °C. In total, four states were compared with each other: incubation for 12 h at 37 °C, for 24 h at 37 °C, for 12 h at 42 °C and 24 h at 42 °C. RESULTS: It was shown that the proteomic changes not only according to the different incubation temperature but also to the length of the incubation period were evident when comparing 37 °C and 42 °C as well as 12 h and 24 h of incubation. Altogether, the expression of 957 proteins was quantifiable. 37.1 - 47.3% of the proteins analyzed showed significant differential regulation, with at least a 1.5-fold change in either direction (i.e. log2 FC ≥ 0.585 or log2 FC ≤ -0.585) and an FDR-adjusted p-value of less than 0.05. The significantly differentially expressed proteins could be arranged in 4 different clusters and 16 functional categories. CONCLUSIONS: The C. jejuni proteome at 42 °C is better adapted to high replication rates than that at 37 °C, which was in particular indicated by the up-regulation of proteins belonging to the functional categories "replication" (e.g. Obg, ParABS, and NapL), "DNA synthesis and repair factors" (e.g. DNA-polymerase III, DnaB, and DnaE), "lipid and carbohydrate biosynthesis" (e.g. capsular biosynthesis sugar kinase, PrsA, AccA, and AccP) and "vitamin synthesis, metabolism, cofactor biosynthesis" (e.g. MobB, BioA, and ThiE). The relative up-regulation of proteins with chaperone function (GroL, DnaK, ClpB, HslU, GroS, DnaJ, DnaJ-1, and NapD) at 37 °C in comparison to 42 °C after 12 h incubation indicates a temporary lower-temperature proteomic response. Additionally the up-regulation of factors for DNA uptake (ComEA and RecA) at 37 °C compared to 42 °C indicate a higher competence for the acquisition of extraneous DNA at human body temperature.

Epidemiological and molecular investigations of sequential outbreaks of Campylobacter jejuni infecting adults and schoolchildren in southeastern China, 2021-2022.

OBJECTIVES: To investigate cases of five Campylobacter jejuni outbreaks and describe laboratory characteristics of these infections. METHODS: Whole-genome sequencing and conventional methods were combined to thoroughly investigate the outbreaks, and data of contemporaneous sporadic cases was included for comparison. RESULTS: Seven sequence types (STs) of C. jejuni caused 83 cases, including ST9079 which recurred across 2 years. Trace-back investigation could not identify any food items of infection but detected identical campylobacters from food contacts. Phylogenetic analysis unveiled genetic closeness between outbreak strains and some concurrent sporadic strains, indicating local campylobacteriosis may not be wholly sporadic but rather a series of linked cases. Virulence genes disclosed species/case-specific signatures to differentiate outbreaks from truly non-outbreak strains. Resistance to fluoroquinolones and/or macrolides was prevalent (90.8%, 108/119), with a noteworthy portion exhibiting multidrug resistance (31.1%, 37/119). Five types of plasmids were harbored among outbreak isolates, of which one plasmid harboring anti-stress and resistant genes was rarely found in C. jejuni. CONCLUSIONS: This is the first reported sequential outbreak of C. jejuni in China. Our observations help to define the genomic landscape and antimicrobial resistance patterns of Campylobacter, emphasizing the need for a broader 'One Health' perspective to combat the threats posed by campylobacteriosis.

Prevalence and characterization of aminoglycoside resistance gene aph(2")-If-carrying Campylobacter jejuni.

Campylobacter jejuni is recognized as a significant foodborne pathogen, and recent studies have indicated a rising trend of aminoglycosides resistance gene aph(2″)-If among C. jejuni isolates from food-producing animals in China. However, systematic information about aph(2″)-If-positive C. jejuni from food-producing animals and other sources worldwide based on whole-genome analysis remains a knowledge gap. In this study, we aimed to analyze the worldwide distribution, genetic environment and phylogenetic tree of aph(2″)-If by utilizing Whole Genome Sequencing (WGS) data obtained, coupled with information in the GenBank database. A total of 160C. jejuni isolates in the GenBank database and 14C. jejuni isolates in our laboratory carrying aph(2″)-If gene were performed for further analysis. WGS analysis revealed the global distribution of aph(2″)-If among C. jejuni from 6 countries. Multilocus Sequence Typing(MLST) results indicated that 70 STs were involved in the dissemination of aph(2″)-If, with ST10086 being the predominant ST. Whole-genome Multilocus Sequence Typing(wg-MLST) analysis according to times, countries, and origins of C. jejuni isolation further demonstrated a close relationship between aph(2″)-If carrying C. jejuni isolates from farm and food. The findings also revealed the existence of 32 distinct types of genetic environments surrounding aph(2″)-If among these isolates. Notably, Type 30, characterized by the arrangement ISsag10-deoD-ant(9)-hp-hp-aph(2″)-If, emerged as the predominant genetic environment. In conclusion, our analysis provides the inaugural perspective on the worldwide distribution of aph(2″)-If. This resistance gene demonstrates horizontal transferability and regional diffusion in a clonal pattern. The close association observed among aph(2″)-If-positive C. jejuni strains isolated from poultry, food, and clinical environments underscores the potential for zoonotic transmission from these isolates.

Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni.

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.

Characterization of Ssc, an N-acetylgalactosamine-containing Staphylococcus aureus surface polysaccharide.

Whole genome sequencing has revealed that the genome of Staphylococcus aureus possesses an uncharacterized 5-gene operon (SAOUHSC_00088-00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus as ssc for staphylococcal surface carbohydrate. We found that the ssc genes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed in Escherichia coli and extracted by heat treatment. Ssc was also conjugated to AcrA from Campylobacter jejuni in E. coli using protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting of N-acetylgalactosamine. We further demonstrated that the expression of the ssc genes in S. aureus affected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCE: Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide.

Epistasis, core-genome disharmony, and adaptation in recombining bacteria.

Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome's co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background-facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.

Intestinal Colonization by Campylobacter jejuni, Clostridium difficile, and Clostridium perfringens among Commensal Rattus norvegicus in the Urban Areas of Tehran, Iran.

Background: Rattus norvegicus (R. norvegicus) population plays a significant role in the spread of numerous diseases in urban environments. The present study is aimed at investigating the presence of Campylobacter jejuni (C. jejuni), C. coli, Clostridium difficile (C. difficile), C. difficile toxigenic, and C. perfringens in R. norvegicus captured from urban areas of Tehran, Iran. Methods: From October 2021 to October 2022, 100 urban rats were trapped in 5 different districts of Tehran, Iran. The genomic DNA was extracted from fecal samples, and the presence of C. jejuni, C. coli, C. perfringens, and C. difficile species was evaluated using PCR assay. Moreover, PCR was used to assess the toxicity of C. difficile isolates. Results: Overall, 30% (n = 30/100) of fecal samples were positive for zoonotic pathogens. Based on the PCR on hippuricase (hipO), glycine (gly), CIDIF, and phospholipase C (plc) genes, C. perfringens and C. difficile were isolated from 18.2% (n = 14/77) and 5.2% (n = 4/77) of male rats. The highest frequency of C. perfringens and C. jejuni was 25% (n = 5/20) related to the south of Tehran. Toxigenic C. difficile was not detected in all regions. Conclusion: According to the findings, rats are the main reservoirs for diseases. Therefore, rodent control coupled with the implementation of surveillance systems should be prioritized for urban health.

Antimicrobial resistance patterns and genes of Campylobacter jejuni isolated from chickens in Pasuruan, Indonesia.

Background: Poultry is one of the most prominent sources of Campylobacter jejuni, which is also a major means of transmission to people. Campylobacter jejuni contamination in chicken meat comes from chicken feces because it naturally exists in the intestines of chickens. Aim: The purpose of this study is to identify the antibiotic resistance patterns and genes of C. jejuni, which was found in chickens in Pasuruan, Indonesia. Methods: The samples used in this study were 200 contents of the small intestine of broiler chickens from 40 farms in Pasuruan Regency. The enriched sample was streaked on the selective media of modified charcoal cefoperazone deoxycholate agar containing the CCDA selective supplement. Antimicrobial susceptibility test utilizing the Kirby-Bauer diffusion test method in accordance with Clinical and Laboratory Standards Institute standards. The polymerase chain reaction (PCR) method was used to detect the (hipO), which encodes the C. jejuni strain, fluoroquinolone resistance (gyrA), beta-lactam resistance (blaOXA-61), and tetracycline resistance (tetO) genes. Results: The findings revealed a 14% (28/200) prevalence of C. jejuni in the small intestine of broiler chickens. These isolates showed high resistance to enrofloxacin (92.9%). All isolates (100%) were susceptible to amoxicillin-clavulanate. The PCR results showed all C. jejuni isolates (100%) detected the gyrA gene, 96.4% detected the blaOXA-61 gene, and 50% detected the tetO gene. Conclusion: The findings of antimicrobial resistance at a high level from the small intestine of broiler chickens illustrate the potential threat to human health. To lessen the effects now and in the future, coordinated and suitable action is needed, as well as steps to guarantee the poultry industry's economic survival and public health insurance.

Meta-analyses of the global multilocus genotypes of the human pathogen Campylobacter jejuni.

Campylobacter infections are a leading cause of bacterial diarrheal illness worldwide, with increasing reports of outbreaks in both developing and developed countries. Most studies investigating strain genotypes and epidemiology of Campylobacter jejuni examined on a local scale. Using the archived multilocus sequence typing data at seven loci, and associated strain metadata from the PubMLST database, here we investigated the spatial and temporal genetic structure of the global population of C. jejuni. Our analyses revealed evidence for clonal dispersals of multiple sequence types (STs) among countries and continents. However, despite the observed clonal dispersal and that most genetic variations were found within individual geographic subpopulations, both the non-clone-corrected and clone-corrected samples showed evidence of significant genetic differentiation among national and continental subpopulations, with non-clone-corrected samples showing greater differentiation than clone-corrected samples. Phylogenetic incompatibility analyses provided evidence for recombination within each continental subpopulation. However, linkage disequilibrium analyses rejected the hypothesis of random recombination across the samples. Temporally, multiple STs were found to persist across four decades and the five globally most common STs showed relatively stable frequencies over the last two decades. We discussed the implications of our results to food security, disease transmission, and public health management.

Insights on the genomic diversity, virulence and resistance profile of a Campylobacter jejuni strain isolated from a hospitalized patient in Brazil.

Campylobacteriosis is currently recognized as one of the major causes of foodborne bacterial diseases worldwide. In Brazil, there is insufficient data to estimate the impact of Campylobacter in public health. The aim of this present study was to characterize a C. jejuni CJ-HBSJRP strain isolated from a hospitalized patient in Brazil by its ability to invade human Caco-2 epithelial cells, to survive in U937 human macrophages, and to assess its phenotypic antimicrobial resistance profile. In addition, prophages, virulence and antimicrobial resistance genes were search using whole-genome sequencing data. The genetic relatedness was evaluated by MLST and cgMLST analysis by comparison with 29 other C. jejuni genomes isolated from several countries. The CJ-HBSJRP strain showed an invasion percentage of 50% in Caco-2 polarized cells, 37.5% of survivability in U937 cells and was phenotypically resistant to ampicillin, ciprofloxacin and nalidixic acid. A total of 94 virulence genes related to adherence, biofilm, chemotaxis, immune modulation, invasion process, metabolism, motility and toxin were detected. The resistance genes blaOXA-605 (blaOXA-61), cmeB and mutations in the QRDR region of gyrA were also found and none prophages were detected. The MLST analysis showed 23 different STs among the strains studied. Regarding cgMLST analysis, the CJ-HBSJRP strain was genetically distinct and did not group closely to any other isolate. The results obtained reinforce the pathogenic potential of the CJHBSJRP strain and highlighted the need for more careful attention to Campylobacter spp. infections in Brazil since this pathogen has been the most commonly reported zoonosis in several countries worldwide.

Campylobacter jejuni extracellular vesicles harboring cytolethal distending toxin bind host cell glycans and induce cell cycle arrest in host cells.

Cytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed "surface shearing" experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewisx. Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells. IMPORTANCE: Campylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

Identification of Campylobacter jejuni and Campylobacter coli genes contributing to oxidative stress response using TraDIS analysis.

BACKGROUND: Campylobacter jejuni and Campylobacter coli are the major causative agents of bacterial gastroenteritis worldwide and are known obligate microaerophiles. Despite being sensitive to oxygen and its reduction products, both species are readily isolated from animal food products kept under atmospheric conditions where they face high oxygen tension levels. RESULTS: In this study, Transposon Directed Insertion-site Sequencing (TraDIS) was used to investigate the ability of one C. jejuni strain and two C. coli strains to overcome oxidative stress, using H2O2 to mimic oxidative stress. Genes were identified that were required for oxidative stress resistance for each individual strain but also allowed a comparison across the three strains. Mutations in the perR and ahpC genes were found to increase Campylobacter tolerance to H2O2. The roles of these proteins in oxidative stress were previously known in C. jejuni, but this data indicates that they most likely play a similar role in C. coli. Mutation of czcD decreased Campylobacter tolerance to H2O2. The role of CzcD, which functions as a zinc exporter, has not previously been linked to oxidative stress. The TraDIS data was confirmed using defined deletions of perR and czcD in C. coli 15-537360. CONCLUSIONS: This is the first study to investigate gene fitness in both C. jejuni and C. coli under oxidative stress conditions and highlights both similar roles for certain genes for both species and highlights other genes that have a role under oxidative stress.

Structural basis of DNA recognition of the Campylobacter jejuni CosR regulator.

Campylobacter jejuni is a foodborne pathogen commonly found in the intestinal tracts of animals. This pathogen is a leading cause of gastroenteritis in humans. Besides its highly infectious nature, C. jejuni is increasingly resistant to a number of clinically administrated antibiotics. As a consequence, the Centers for Disease Control and Prevention has designated antibiotic-resistant Campylobacter as a serious antibiotic resistance threat in the United States. The C. jejuni CosR regulator is essential to the viability of this bacterium and is responsible for regulating the expression of a number of oxidative stress defense enzymes. Importantly, it also modulates the expression of the CmeABC multidrug efflux system, the most predominant and clinically important system in C. jejuni that mediates resistance to multiple antimicrobials. Here, we report structures of apo-CosR and CosR bound with a 21 bp DNA sequence located at the cmeABC promotor region using both single-particle cryo-electron microscopy and X-ray crystallography. These structures allow us to propose a novel mechanism for CosR regulation that involves a long-distance conformational coupling and rearrangement of the secondary structural elements of the regulator to bind target DNA. IMPORTANCE: Campylobacter jejuni has emerged as an antibiotic-resistant threat worldwide. CosR is an essential regulator for this bacterium and is important for Campylobacter adaptation to various stresses. Here, we describe the structural basis of CosR binding to target DNA as determined by cryo-electron microscopy and X-ray crystallography. Since CosR is a potential target for intervention, our studies may facilitate the development of novel therapeutics to combat C. jejuni infection.

Gene expression profile of Campylobacter jejuni in response to macrolide antibiotics.

Campylobacter jejuni is a foodborne pathogen that causes gastroenteritis in humans and has developed resistance to various antibiotics. The primary objective of this research was to examine the network of antibiotic resistance in C. jejuni. The study involved the wild and antibiotic-resistant strains placed in the presence and absence of antibiotics to review their gene expression profiles in response to ciprofloxacin via microarray. Differentially expressed genes (DEGs) analysis and Protein-Protein Interaction (PPI) Network studies were performed for these genes. The results showed that the resistance network of C. jejuni is modular, with different genes involved in bacterial motility, capsule synthesis, efflux, and amino acid and sugar synthesis. Antibiotic treatment resulted in the down-regulation of cluster genes related to translation, flagellum formation, and chemotaxis. In contrast, cluster genes involved in homeostasis, capsule formation, and cation efflux were up-regulated. The study also found that macrolide antibiotics inhibit the progression of C. jejuni infection by inactivating topoisomerase enzymes and increasing the activity of epimerase enzymes, trying to compensate for the effect of DNA twisting. Then, the bacterium limits the movement to conserve energy. Identifying the antibiotic resistance network in C. jejuni can aid in developing drugs to combat these bacteria. Genes involved in cell division, capsule formation, and substance transport may be potential targets for inhibitory drugs. Future research must be directed toward comprehending the underlying mechanisms contributing to the modularity of antibiotic resistance and developing strategies to disrupt and mitigate the growing threat of antibiotic resistance effectively.

Whole genome sequence-based characterization of Campylobacter isolated from broiler carcasses over a three-year period in a big poultry slaughterhouse reveals high genetic diversity and a recurring genomic lineage of Campylobacter jejuni.

Campylobacter is among the most frequent agents of bacterial gastroenteritis in Europe and is primarily linked to the consumption of contaminated food. The aim of this study was to assess genomic diversity and to identify antimicrobial resistance and virulence genes of 155 Campylobacter isolated from broiler carcasses (neck skin samples) in a large-scale Swiss poultry abattoir over a three-year period. Samples originated from broilers from three different types of farming systems (particularly animal-friendly stabling (PAFS), free-range farms, and organic farms). Campylobacter jejuni (n = 127) and Campylobacter coli (n = 28) were analysed using a whole genome sequencing (WGS) approach (MiniSeq; Illumina). Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into complex types (CTs) using the cgMLST SeqSphere+ scheme. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI), and virulence genes were identified using the virulence factor database (VFDB). A high degree of genetic diversity was observed. Many sequence types (C. jejuni ST19, ST21, ST48, ST50, ST122, ST262 and C. coli ST827) occurred more than once and were distributed throughout the study period, irrespective of the year of isolation and of the broiler farming type. Antimicrobial resistance determinants included blaOXA and tet(O) genes, as well as the T86I substitution within GyrA. Virulence genes known to play a role in human Campylobacter infection were identified such as the wlaN, cstIII, neuA1, neuB1, and neuC1. Subtyping of the Campylobacter isolates identified the occurrence of a highly clonal population of C. jejuni ST21 that was isolated throughout the three-year study period from carcasses from farms with geographically different locations and different farming systems. The high rate of genetic diversity observed among broiler carcass isolates is consistent with previous studies. The identification of a persisting highly clonal C. jejuni ST21 subtype suggests that the slaughterhouse may represent an environment in which C. jejuni ST21 may survive, however, the ecological reservoir potentially maintaining this clone remains unknown.

Identification and functional characterization of putative ligand binding domain(s) of JlpA protein of Campylobacter jejuni.

Among the major Surface Exposed Colonization Proteins (SECPs) of Campylobacter jejuni (C. jejuni), Jejuni lipoprotein A (JlpA) plays a crucial role in host cell adhesion specifically by binding to the N-terminal domain of the human heat shock protein 90α (Hsp90α-NTD). Although the JlpA binding to Hsp90α activates NF-κB and p38 MAP kinase pathways, the underlying mechanism of JlpA association with the cellular receptor remains unclear. To this end, we predicted two potential receptor binding sites within the C-terminal domain of JlpA: one spanning from amino acid residues Q332-A354 and the other from S258-T295; however, the latter exhibited weaker binding. To assess the functional attributes of these predicted sequences, we generated two JlpA mutants (JlpAΔ1: S258-T295; JlpAΔ2: Q332-A354) and assessed the Hsp90α-binding affinity-kinetics by in vitro and ex vivo experiments. Our findings confirmed that the residues Q332-A354 are of greater importance in host cell adhesion with a measurable impact on cellular responses. Further, thermal denaturation by circular dichroism (CD) confirmed that the reduced binding affinity of the JlpAΔ2 to Hsp90α is not associated with protein folding or stability. Together, this study provides a possible framework for determining the molecular function of designing rational inhibitors efficiently targeting JlpA.

The antibacterial mechanism of (-)-epigallocatechin-3-gallate (EGCG) against Campylobacter jejuni through transcriptome profiling.

(-)-Epigallocatechin-3-gallate (EGCG) has been shown antibacterial activity against Campylobacter jejuni; however, the relevant antibacterial mechanism is unknown. In this study, phenotypic experiments and RNA sequencing were used to explore the antibacterial mechanism. The minimum inhibitory concentration of EGCG on C. jejuni was 32 µg/mL. EGCG-treated was able to increase intracellular reactive oxygen species levels and decline bacterial motility. The morphology and cell membrane of C. jejuni after EGCG treatment were observed collapsed, broken, and agglomerated by field emission scanning electron microscopy and fluorescent microscopy. The RNA-seq analysis presents that there are 36 and 72 differential expressed genes after C. jejuni was treated by EGCG with the concentration of 16 and 32 µg/mL, respectively. EGCG-treated increased the thioredoxin expression, which was a critical protein to resist oxidative stress. Moreover, downregulation of the flgH and flgM gene in flagellin biosynthesis of C. jejuni was able to impair the flagella, reducing cell motility and virulence. The primary antibacterial mechanism revealed by RNA-seq is that EGCG with iron-chelating activity competes with C. jejuni for iron, causing iron deficiency in C. jejuni, which potentially impacts the survival and virulence of C. jejuni. The results suggested a new direction for exploring the activity of EGCG against C. jejuni in the food industry. PRACTICAL APPLICATION: A deeper understanding of the antibacterial mechanism of EGCG against C. jejuni was more beneficial in improving the food safety, eliminating concerns about human health caused by C. jejuni in future food, and promoting the natural antibacterial agent EGCG application in the food industry.

Added insult to injury? The response of meat-associated pathogens to proposed antimicrobial interventions.

Modern requirements for 'green label' meat products have led to the design of novel antimicrobial innovations which prioritise quality, safety and longevity. Plasma-functionalised water (PFW), ultraviolet light and natural antimicrobial compositions have been investigated and optimised for control of foodborne pathogens like Campylobacter jejuni and Salmonella enterica serovar Typhimurium. However, given the adaptive mechanisms present in bacteria under external stresses, it is imperative to understand the effect that sublethal treatment may have on the bacterial transcriptome. In this study, Salmonella Typhimurium and C. jejuni were treated with sublethal doses of ultraviolet light, a citrus juice/essential oil marinade, and 'spark' or 'glow' cold plasma generation system-produced PFW. Immediately after treatment, cells were lysed and RNA was extracted and purified. mRNA was converted to cDNA by reverse transcription-PCR and sequenced by an Illumina MiSeq® system. Sequences were filtered and analysed using the Tuxedo workflow. Sublethal treatment of Campylobacter jejuni and Salmonella Typhimurium led to increased immediate cellular and metabolic activity, as well as diversification in protein and metabolic functioning. There was further expression of pathogenesis and virulence-associated traits associated with spark PFW and marinade treatment of Salmonella Typhimurium. However, similar concerns were not raised with glow PFW or UV-treated samples. This study provides science-based evidence of the efficacy of multi-hurdle antimicrobial system using green-label marinades and PFW or UV to inactivate pathogens without upregulating virulence traits in surviving cells. This study will inform policymakers and food industry stakeholders and reinforces the need to incorporate in-line novel technologies to ensure consumer safety. KEY POINTS: • Salmonella and C. jejuni showed increased cell activity in immediate response to stress. • Virulence genes showed increased expression when treated with natural antimicrobials and sPFW. • Reduced immediate transcriptomic response to gPFW and UV treatment indicates lower risk.

Structural analysis of the YqeY proteins from Campylobacter jejuni and Vibrio parahaemolyticus.

YqeY is a functionally and structurally uncharacterized protein that is ubiquitously expressed in bacteria. To gain structural insights into the function of YqeY, we determined the crystal structures of the Campylobacter jejuni and Vibrio parahaemolyticus YqeY proteins (cjYqeY and vpYqeY, respectively) and analyzed the structural and functional roles of conserved residues via a mutational study. Both cjYqeY and vpYqeY were found to adopt a two-domain structure consisting of an N-terminal four-α-helix domain and a C-terminal three-α-helix domain, with a relatively flexible interdomain orientation. The YqeY structure is unique in its linkage of the two α-helix domains although the C-terminal YqeY domain is structurally homologous to the terminal appendages of glutaminyl-tRNA synthetase and tRNA-dependent amidotransferase. We identified six conserved YqeY residues (Y67, R72, E82, Y89, P91, and G119) and evaluated their roles in protein stability via alanine mutation using a thermal shift assay. Residues Y67, R72, Y89, and P91 were shown to be required to maintain the structural integrity of YqeY. In contrast, residues E82 and G119 were not found to be essential for protein stability and are highly likely to contribute to the biological function of YqeY.