Gut metabolite L-lactate supports Campylobacter jejuni population expansion during acute infection.

How the microaerobic pathogen Campylobacter jejuni establishes its niche and expands in the gut lumen during infection is poorly understood. Using 6-wk-old ferrets as a natural disease model, we examined this aspect of C. jejuni pathogenicity. Unlike mice, which require significant genetic or physiological manipulation to become colonized with C. jejuni, ferrets are readily infected without the need to disarm the immune system or alter the gut microbiota. Disease after C. jejuni infection in ferrets reflects closely how human C. jejuni infection proceeds. Rapid growth of C. jejuni and associated intestinal inflammation was observed within 2 to 3 d of infection. We observed pathophysiological changes that were noted by cryptic hyperplasia through the induction of tissue repair systems, accumulation of undifferentiated amplifying cells on the colon surface, and instability of HIF-1α in colonocytes, which indicated increased epithelial oxygenation. Metabolomic analysis demonstrated that lactate levels in colon content were elevated in infected animals. A C. jejuni mutant lacking lctP, which encodes an L-lactate transporter, was significantly decreased for colonization during infection. Lactate also influences adhesion and invasion by C. jejuni to a colon carcinoma cell line (HCT116). The oxygenation required for expression of lactate transporter (lctP) led to identification of a putative thiol-based redox switch regulator (LctR) that may repress lctP transcription under anaerobic conditions. Our work provides better insights into the pathogenicity of C. jejuni.

CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.

Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Campylobacter jejuni Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.

Structural and biochemical analysis of the unique interactions of the Campylobacter jejuni CorA channel protein with divalent cations.

CorA is a Mg2+ channel that plays a key role in the homeostasis of intracellular Mg2+ in bacteria and archaea. CorA consists of a cytoplasmic domain and a transmembrane domain and generates a Mg2+ pathway by forming a pentamer in the cell membrane. CorA gating is regulated via negative feedback by Mg2+, which is accommodated by the pentamerization interface of the CorA cytoplasmic domain (CorACD). The Mg2+-binding sites of CorACD differ depending on the species, suggesting that the Mg2+-binding modes and Mg2+-mediated gating mechanisms of CorA vary across prokaryotes. To define the Mg2+-binding mechanism of CorA in the Campylobacter jejuni pathogen, we structurally and biochemically characterized C. jejuni CorACD (cjCorACD). cjCorACD adopts a three-layered α/ß/α structure as observed in other CorA orthologs. Interestingly, cjCorACD exhibited enhanced thermostability in the presence of Ca2+, Ni2+, Zn2+, or Mn2+ in addition to Mg2+, indicating that cjCorACD interacts with diverse divalent cations. This cjCorACD stabilization is mediated by divalent cation accommodation by negatively charged residues located at the bottom of the cjCorACD structure away from the pentamerization interface. Consistently, cjCorACD exists as a monomer irrespective of the presence of divalent cations. We concluded that cjCorACD binds divalent cations in a unique pentamerization-independent manner.

Structural and biochemical analysis of penicillin-binding protein 2 from Campylobacter jejuni.

Penicillin-binding protein 2 (PBP2) plays a key role in the formation of peptidoglycans in bacterial cell walls by crosslinking glycan chains through transpeptidase activity. PBP2 is also found in Campylobacter jejuni, a pathogenic bacterium that causes food-borne enteritis in humans. To elucidate the essential structural features of C. jejuni PBP2 (cjPBP2) that mediate its biological function, we determined the crystal structure of cjPBP2 and assessed its protein stability under various conditions. cjPBP2 adopts an elongated two-domain structure, consisting of a transpeptidase domain and a pedestal domain, and contains typical active site residues necessary for transpeptidase activity, as observed in other PBP2 proteins. Moreover, cjPBP2 responds to ß-lactam antibiotics, including ampicillin, cefaclor, and cefmetazole, suggesting that ß-lactam antibiotics inactivate cjPBP2. In contrast to typical PBP2 proteins, cjPBP2 is a rare example of a Zn2+-binding PBP2 protein, as the terminal structure of its transpeptidase domain accommodates a Zn2+ ion via three cysteine residues and one histidine residue. Zn2+ binding helps improve the protein stability of cjPBP2, providing opportunities to develop new C. jejuni-specific antibacterial drugs that counteract the Zn2+-binding ability of cjPBP2.

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.

Dual bacteriocin and extracellular vesicle-mediated inhibition of Campylobacter jejuni by the potential probiotic candidate Ligilactobacillus salivarius UO.C249.

Campylobacter jejuni (C. jejuni) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni. IMPORTANCE: Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni. These findings hold promise for the application of probiotic EVs for pathogen control.

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.

Antibiotic-induced microbiome depletion promotes intestinal colonization by Campylobacter jejuni in mice.

BACKGROUND: To establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion. RESULTS: Fifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes. CONCLUSIONS: Microbiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

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.

Diametral influence of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) on the growth of Campylobacter jejuni with consequences on the bacterial transcriptome.

BACKGROUND: Deoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing. RESULTS: The co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins. CONCLUSION: The results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens.

Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

The critical role of a conserved lysine residue in periplasmic nitrate reductase catalyzed reactions.

Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.

Epoxide-Mediated Trans-Thioglycosylation and Application to the Synthesis of Oligosaccharides Related to the Capsular Polysaccharides of C. jejuni HS:4.

The enzyme-resistant thioglycosides are highly valuable immunogens because of their enhanced metabolic stability. We report the first synthesis of a family of thiooligosaccharides related to the capsular polysaccharides (CPS) of Campylobacter jejuni HS:4 for potential use in conjugate vaccines. The native CPS structures of the pathogen consist of a challenging repeating disaccharide formed with ß(1→4)-linked 6-deoxy-ß-D-ido-heptopyranoside and N-acetyl-D-glucosamine; the rare 6-deoxy-ido-heptopyranosyl backbone and ß-anomeric configuration of the former monosaccharide makes the synthesis of this family of antigens very challenging. So far, no synthesis of the thioanalogs of the CPS antigens have been reported. The unprecedented synthesis presented in this work is built on an elegant approach by using ß-glycosylthiolate as a glycosyl donor to open the 2,3-epoxide functionality of pre-designed 6-deoxy-ß-D-talo-heptopyranosides. Our results illustrated that this key trans-thioglycosylation can be designed in a modular and regio and stereo-selective manner. Built on the success of this novel approach, we succeeded the synthesis of a family of thiooligosaccharides including a thiohexasaccharide which is considered to be the desired antigen length and complexity for immunizations. We also report the first direct conversion of base-stable but acid-labile 2-trimethylsilylethyl glycosides to glycosyl-1-thioacetates in a one-pot manner.

Campylobacter jejuni virulence factors: update on emerging issues and trends.

Campylobacter jejuni is a very common cause of gastroenteritis, and is frequently transmitted to humans through contaminated food products or water. Importantly, C. jejuni infections have a range of short- and long-term sequelae such as irritable bowel syndrome and Guillain Barre syndrome. C. jejuni triggers disease by employing a range of molecular strategies which enable it to colonise the gut, invade the epithelium, persist intracellularly and avoid detection by the host immune response. The objective of this review is to explore and summarise recent advances in the understanding of the C. jejuni molecular factors involved in colonisation, invasion of cells, collective quorum sensing-mediated behaviours and persistence. Understanding the mechanisms that underpin the pathogenicity of C. jejuni will enable future development of effective preventative approaches and vaccines against this pathogen.

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.

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.

Human Campylobacter spp. infections in Italy.

PURPOSE: Campylobacter is a frequent cause of enteric infections with common antimicrobial resistance issues. The most recent reports of campylobacteriosis in Italy include data from 2013 to 2016. We aimed to provide national epidemiological and microbiological data on human Campylobacter infections in Italy during the period 2017-2021. METHODS: Data was collected from 19 Hospitals in 13 Italian Regions. Bacterial identification was performed by mass spectrometry. Antibiograms were determined with Etest or Kirby-Bauer (EUCAST criteria). RESULTS: In total, 5419 isolations of Campylobacter spp. were performed. The most common species were C. jejuni (n = 4535, 83.7%), followed by C. coli (n = 732, 13.5%) and C. fetus (n = 34, 0.6%). The mean age of patients was 34.61 years and 57.1% were males. Outpatients accounted for 54% of the cases detected. Campylobacter were isolated from faeces in 97.3% of cases and in 2.7% from blood. C. fetus was mostly isolated from blood (88.2% of cases). We tested for antimicrobial susceptibility 4627 isolates (85.4%). Resistance to ciprofloxacin and tetracyclines was 75.5% and 54.8%, respectively; resistance to erythromycin was 4.8%; clarithromycin 2% and azithromycin 2%. 50% of C. jejuni and C. coli were resistant to ≥ 2 antibiotics. Over the study period, resistance to ciprofloxacin and tetracyclines significantly decreased (p < 0.005), while resistance to macrolides remained stable. CONCLUSION: Campylobacter resistance to fluoroquinolones and tetracyclines in Italy is decreasing but is still high, while macrolides retain good activity.

Monitoring antimicrobial resistance in Campylobacter isolates of chickens and turkeys at the slaughter establishment level across the United States, 2013-2021.

Foodborne infections with antimicrobial-resistant Campylobacter spp. remain an important public health concern. Publicly available data collected by the National Antimicrobial Resistance Monitoring System for Enteric Bacteria related to antimicrobial resistance (AMR) in Campylobacter spp. isolated from broiler chickens and turkeys at the slaughterhouse level across the United States between 2013 and 2021 were analysed. A total of 1,899 chicken-origin (1,031 Campylobacter coli (C. coli) and 868 Campylobacter jejuni (C. jejuni)) and 798 turkey-origin (673 C. coli and 123 C. jejuni) isolates were assessed. Chicken isolates exhibited high resistance to tetracycline (43.65%), moderate resistance to ciprofloxacin (19.5%), and low resistance to clindamycin (4.32%) and azithromycin (3.84%). Turkey isolates exhibited very high resistance to tetracycline (69%) and high resistance to ciprofloxacin (39%). The probability of resistance to all tested antimicrobials, except for tetracycline, significantly decreased during the latter part of the study period. Turkey-origin Campylobacter isolates had higher odds of resistance to all antimicrobials than isolates from chickens. Compared to C. jejuni isolates, C. coli isolates had higher odds of resistance to all antimicrobials, except for ciprofloxacin. The study findings emphasize the need for poultry-type-specific strategies to address differences in AMR among Campylobacter isolates.

High molecular/low acetylated chitosans reduce adhesion of Campylobacter jejuni to host cells by blocking JlpA.

Infections caused by Campylobacter spp. are a major cause of severe enteritis worldwide. Multifactorial prevention strategies are necessary to reduce the prevalence of Campylobacter. In particular, antiadhesive strategies with specific inhibitors of early host-pathogen interaction are promising approaches to reduce the bacterial load. An in vitro flow cytometric adhesion assay was established to study the influence of carbohydrates on the adhesion of C. jejuni to Caco-2 cells. Chitosans with a high degree of polymerization and low degree of acetylation were identified as potent antiadhesive compounds, exerting significant reduction of C. jejuni adhesion to Caco-2 cells at non-toxic concentrations. Antiadhesive and also anti-invasive effects were verified by confocal laser scanning microscopy. For target identification, C. jejuni adhesins FlpA and JlpA were expressed in Escherichia coli ArcticExpress, and the influence of chitosan on binding to fibronectin and HSP90α, respectively, was investigated. While no effects on FlpA binding were found, a strong inhibition of JlpA-HSP90α binding was observed. To simulate real-life conditions, chicken meat was inoculated with C. jejuni, treated with antiadhesive chitosan, and the bacterial load was quantified. A strong reduction of C. jejuni load was observed. Atomic force microscopy revealed morphological changes of C. jejuni after 2 h of chitosan treatment, indicating disturbance of the cell wall and sacculi formation by electrostatic interaction of positively charged chitosan with the negatively charged cell surface. In conclusion, our data indicate promising antiadhesive and anti-invasive potential of high molecular weight, strongly de-acetylated chitosans for reducing C. jejuni load in livestock and food production. KEY POINTS: • Antiadhesive effects of chitosan with high DP/low DA against C. jejuni to host cells • Specific targeting of JlpA/Hsp90α interaction by chitosan • Meat treatment with chitosan reduces C. jejuni load.

Effect of WQ-3334 on Campylobacter jejuni carrying a DNA gyrase with dominant amino acid substitutions conferring quinolone resistance.

INTRODUCTION: Campylobacteriosis stands as one of the most frequent bacterial gastroenteritis worldwide necessitating antibiotic treatment in severe cases and the rise of quinolones-resistant Campylobacter jejuni poses a significant challenge. The predominant mechanism of quinolones-resistance in this bacterium involves point mutations in the gyrA, resulting in amino acid substitution from threonine to isoleucine at 86th position, representing more than 90% of mutant DNA gyrase, and aspartic acid to asparagine at 90th position. WQ-3334, a novel quinolone, has demonstrated strong inhibitory activity against various bacteria. This study aims to investigate the effectiveness of WQ-3334, and its analogues, WQ-4064 and WQ-4065, with a unique modification in R1 against quinolones-resistant C. jejuni. METHODS: The structure-activity relationship of the examined drugs was investigated by measuring IC50 and their antimicrobial activities were accessed by MIC against C. jejuni strains. Additionally, in silico docking simulations were carried out using the crystal structure of the Escherichia coli DNA gyrase. RESULT: WQ-3334 exhibited the lowest IC50 against WT (0.188 ± 0.039 mg/L), T86I (11.0 ± 0.7 mg/L) and D90 N (1.60 ± 0.28 mg/L). Notably, DNA gyrases with T86I substitutions displayed the highest IC50 values among the examined WQ compounds. Moreover, WQ-3334 demonstrated the lowest MICs against wild-type and mutant strains. The docking simulations further confirmed the interactions between WQ-3334 and DNA gyrases. CONCLUSION: WQ-3334 with 6-amino-3,5-difluoropyridine-2-yl at R1 severed as a remarkable candidate for the treatment of foodborne diseases caused by quinolones-resistant C. jejuni as shown by the high inhibitory activity against both wild-type and the predominant quinolones-resistant strains.