IntroductionAcute Campylobacter jejuni infection relies on effector proteins delivered from the flagellar apparatus to gut epithelial cells. While the C. jejuni Campylobacter invasion antigen D (CiaD) secreted protein has been linked to extracellular signal-regulated kinase (ERK) activation, the contribution of ERK in establishing acute bacterial infection has not been fully explored. We hypothesized that subcellular ERK could play distinct roles in C. jejuni proinflammatory cytokine production and bacterial invasion. The objective of this study was to determine the contribution of cytoplasmic and nuclear ERKs in response to C. jejuni infection with a wild-type isolate and effector mutants.MethodsEpithelial cells were infected with C. jejuni in the presence of focal adhesion inhibitors that prevent extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and a peptide that disrupts ERK nuclear translocation. We measured markers of C. jejuni infection, including cell invasion and interleukin-8 (IL-8) cytokine secretion by gentamicin-protection assay and ELISA, respectively.ResultsOur results demonstrate that inhibitors of ERK1/2 activation drastically reduce both the C. jejuni internalization and IL-8 secretion. Moreover, ERK1/2 activation was dependent upon its association with the focal adhesion protein paxillin, which is mediated by the C. jejuni CiaD effector. Blocking ERK nuclear translocation with a phosphomimetic peptide results in a significant reduction in IL-8 secretion, and surprisingly, bacterial invasion.DiscussionWe conclude that nuclear ERK is required for C. jejuni-induced IL-8 secretion and that both cytosolic and nuclear ERK are necessary for C. jejuni maximal cell invasion. Targeting host ERK signaling represents a promising therapeutic strategy to reduce the severity of C. jejuni-mediated enteritis.

INTRODUCTION:Antimicrobial resistance (AMR) threatens food safety across the farm-to-fork continuum. Real-time surveillance is crucial to mitigate its global escalation, yet conventional antimicrobial susceptibility testing (AST) remains slow, labor-intensive, and impractical for large-scale monitoring. OBJECTIVES:We developed an Artificial Intelligence of Things (AIoT)-integrated multiplex microfluidic platform enabling automated AMR surveillance ofpathogens in food supply chain. RESULTS:Each node combines a single-board AIoT controller (Orange Pi 5B), portable incubator, chromogenic microfluidic chips, and environmental sensors, reducing costs by 98% compared with standard AST. A lightweight YOLO model embedded in the controller achieved > 99% accuracy in identifying bacterial growth and inhibition under antibiotic pressure, showing 96% and 95% agreement with standard results forSalmonella andCampylobacter, respectively. Data are synchronized to a cloud server for real-time aggregation and early resistance warning. CONCLUSION:This fully automated and low-cost system minimizes human error and workload, providing a scalable sample-to-answer solution for AMR surveillance in global agri-food system.

Campylobacter jejuni is associated with enteritis in humans and domestic animals. Acute C. jejuni-mediated enteritis requires bacterial invasion of intestinal cells with an ensuing host inflammatory response. Known is that C. jejuni invasion of human epithelial cells is dependent on host cell-focal adhesion components, which link the extracellular matrix to the actin cytoskeleton of a cell. Based on the observation that C. jejuni cell invasion engages many of the same components involved in regulating the cell cycle, we hypothesized that C. jejuni regulates the host cell cycle. Flow cytometry was used to detect the cell cycle phases (G1, S, G2 and M). Single-cell RNA-sequencing (scRNA-seq) and reverse transcriptase quantitative PCR (RT-qPCR) were used to determine the differential gene expressions of uninfected and C. jejuni-infected cells. Infection assays and confocal microscopy were employed to determine the rate of bacterial invasion and intracellular localization of C. jejuni-infected cells. Quantification of Interleukin-8 (IL-8) was determined by the ELISAs. INT 407 cells infected with C. jejuni showed a slower rate of cell cycle progression and a greater percentage of cells in the G1 cell cycle phase. scRNA-seq and RT-qPCR analysis of C. jejuni-infected cells corroborated the result, revealing host genes responsive to C. jejuni infection, including genes associated with cell cycle regulation, focal adhesions, inflammatory cytokines, and oxidative stress. Cell cycle synchronization coupled with the gentamicin-protection revealed that C. jejuni preferentially invades cells in the G1 phase. Moreover, an increase was observed in the number of bacteria colocalized with paxillin, a critical component of focal adhesion complexes, during the G1 phase. The infection of INT 407 cells in the G1 phase also increased the secretion of the proinflammatory cytokine IL-8 from cells. Based on the data, we propose that acute C. jejuni-mediated enteritis (campylobacteriosis) alters the cell cycle phase of enterocytes, cytokine production, and immune cell recruitment, disrupting the intestinal permeability barrier.

Antibiotic tolerance in bacteria emerges as a phenotypic variant known as persisters and has been implicated in recurrent bacterial infections. This tolerance manifests as suppressed metabolism. We demonstrate that Campylobacter jejuni readily forms persisters in response to both ampicillin and ciprofloxacin. Persisters induced by these two antibiotics with different mechanisms of actions experience consistent metabolic reprogramming, characterized by suppressed respiration and energy metabolism while conserving core metabolic processes. We reveal that the coordination of protein homeostasis underlies the metabolic reprogramming involved in persister formation. Mutating the caseinolytic protease gene clpP , a major component of the protein quality control system, compromises persister formation in vitro and impairs bacterial colonization in mouse. However, the deletion of lon , another component of the protein quality control system, or pre-activation of clpP by expressing artificial disordered protein fragments does not affect the antibiotic tolerance. This suggests that specific targets of ClpP rather than the entire quality control system are critical for persister formation. Our results reveal a convergent metabolic reprogramming in persisters, highlighting the potential to limit the emergence of antibiotic tolerance by counteracting adaptive metabolism elicited by antibiotic treatment. ### Competing Interest Statement The authors have declared no competing interest.

Campylobacter jejuni is likely the most common bacterial cause of gastroenteritis worldwide, responsible for millions of cases of inflammatory diarrhea characterized by severe abdominal cramps and blood in the stool. Further, C. jejuni infections are associated with post-infection sequelae in developed countries and malnutrition and growth-stunting in low- and middle-income countries. Despite the increasing prevalence of the disease, campylobacteriosis, and the recognition that this pathogen is a serious health threat, our understanding of C. jejuni pathogenesis remains incomplete. In this review, we focus on the Campylobacter secretion systems proposed to contribute to host-cell interactions and survival in the host. Moreover, we have applied a genomics approach to defining the structural and mechanistic features of C. jejuni type III, IV, and VI secretion systems. Special attention is focused on the flagellar type III secretion system and the prediction of putative effectors, given that the proteins exported via this system are essential for host cell invasion and the inflammatory response. We conclude that C. jejuni does not possess a type IV secretion system and relies on the type III and type VI secretion systems to establish a niche and potentiate disease.

Oxidative damage to DNA is a significant source of mutations in living organisms. While DNA damage must be repaired to maintain the integrity of the genome and cell survival, errors made during DNA repair may contribute to evolution. Previous work has revealed that Campylobacter jejuni growth in the presence of bile salt deoxycholate (DOC) causes an increase in reactive oxygen species and the occurrence of 8-oxo-deoxyguanosine (8-oxo-dG) DNA lesions. The fundamental goal of this project was to determine if C. jejuni growth in a medium containing DOC contributes to DNA mutations that provide a fitness advantage to the bacterium. Co-culture experiments revealed that C. jejuni growth in a DOC-supplemented medium increases the total number of ciprofloxacin-resistant isolates compared to C. jejuni grown in the absence of DOC. We recovered two individual isolates grown in a medium with DOC that had a point mutation in the gene encoding the EptC phosphoethanolamine transferase. Transformants harboring the EptC variant protein showed enhanced resistance to the antimicrobial agent polymyxin B and DOC when compared to an eptC deletion mutant or the isolate complemented with a wild-type copy of the gene. Finally, we found that the base excision repair (BER), homologous recombination repair (HRR), and nucleotide excision repair (NER) are involved in general oxidative damage repair in C. jejuni but that the BER pathway plays the primary role in the repair of the 8-oxo-dG lesion. We postulate that bile salts drive C. jejuni mutations (adaptations) and enhance bacterial fitness in animals.

Campylobacter is one of the leading bacterial causes of gastroenteritis worldwide. As the commensal in the gastrointestinal tract of warm-blooded animals, especially food-producing animals, Campylobacter can be transmitted to humans through the food supply chain and cause human campylobacteriosis. Although Campylobacter typically causes self-limiting gastroenteritis, it can also lead to prolonged postinfectious complications, such as Guillain-Barré syndrome, reactive arthritis, and/or post infectious-irritable bowel syndrome, posing a great threat to public health. To address the potential risks associated with this disease, numerous studies have been conducted to improve our understanding of this microbe and its interaction with the agri-food system. This mini-review acts as the editorial summary of the published articles in this special issue collected in Frontiers in Microbiology and provides a brief overview of 1) improved detection methods; 2) prevalence and characterization; 3) novel intervention strategies of Campylobacter in the agri-food settings. This special issue is timely due to the increased recognition of Campylobacter organisms as a serious health threat by the World Health Organization, Centers for Disease Control and Prevention, Health Canada, and European Union.

Antimicrobial-resistant C. jejuni has been listed as a high priority of public health concern worldwide. To tackle the rapid evolution of AMR in C. jejuni , it is of great importance to understand the extent and characteristics of HGT in C. jejuni biofilms, which serve as the main survival strategy of this microbe in the farm-to-table continuum.

ABSTRACT Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblots, and super-resolution iPALM. We found that C. jejuni infection of epithelial cells results in an increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, as evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction of collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially re-establishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni -infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions.

Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins ( Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni . Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni -stimulated Rac signaling is dependent on IQGAP1.

Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblotting, and superresolution interferometric photoactivated localization microscopy (iPALM). We found that C. jejuni infection of epithelial cells results in increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction in collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of a protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially reestablishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions. IMPORTANCE Campylobacter jejuni is a major foodborne pathogen that causes severe gastritis. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected epithelial cells. Focal adhesions act as signaling complexes that connect the extracellular matrix to the intracellular cytoskeleton. The key findings of this study show that C. jejuni changes the structure (size and position), composition, and function of cellular focal adhesions using a combination of virulence factors. Mechanistically, we found that the changes in focal adhesion dynamics are dependent upon the activation of host cell signaling pathways, which affect the assembly and disassembly of cellular proteins from the focal adhesion. To summarize, we have identified a new cellular phenotype in C. jejuni-infected cells that may be responsible for the restricted intestinal repair observed in C. jejuni-infected animals.

Puroindolines are small, amphipathic, wheat proteins that determine the hardness of the wheat kernel and protect crops from different pathogens. Puroindoline A (PinA) and puroindoline B (PinB) are two major isoforms of puroindolines. These proteins have antibacterial and antifungal properties mainly attributed to their characteristic tryptophan-rich domains (TRDs). In this in vitro study, we investigated the antimicrobial effect of PinA and PinB synthetic peptides against the growth and biofilm formation of Campylobacter jejuni . C. jejuni is an important microaerobic, foodborne pathogen that causes gastrointestinal and neurological diseases in humans. Our results showed that: (1) PinA, but not PinB, has strong antimicrobial activity against C. jejuni clinical strains 81-176 and F38011, Escherichia coli O157:H7, methicillin-resistant Staphylococcus aureus , Salmonella enterica serovar Typhimurium, and Listeria monocytogenes ; (2) The substitution of two tryptophan residues to glycine (W→G) in the TRD of PinA abolishes its antimicrobial activity against these microorganisms; (3) PinA functions additively with two common antibiotics (ciprofloxacin and erythromycin) to inhibit or inactivate C. jejuni strains; (4) PinA damages the C. jejuni cellular membrane, (5) PinA is cytotoxic to human INT 407 cells at high concentrations; and (6) PinA inhibits C. jejuni biofilm formation. In summary, this study demonstrates the antimicrobial activity of PinA against C. jejuni growth and biofilm formation and further confirms the potential use of PinA as a therapeutic agent in health care or as preservatives in the agri-food industry.

The symptoms of infectious diarrheal disease are mediated by a combination of a pathogen's virulence factors and the host immune system. Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide due to its near-ubiquitous zoonotic association with poultry. One of the outstanding questions is to what extent the bacteria are responsible for the diarrheal symptoms via intestinal cell necrosis versus immune cell initiated tissue damage. To determine the stepwise process of inflammation that leads to diarrhea, we used a piglet ligated intestinal loop model to study the intestinal response to C. jejuni. Pigs were chosen due to the anatomical similarity between the porcine and the human intestine. We found that the abundance of neutrophil related proteins increased in the intestinal lumen during C. jejuni infection, including proteins related to neutrophil migration (neutrophil elastase and MMP9), actin reorganization (Arp2/3), and antimicrobial proteins (lipocalin-2, myeloperoxidase, S100A8, and S100A9). The appearance of neutrophil proteins also corresponded with increases of the inflammatory cytokines IL-8 and TNF-α. Compared to infection with the C. jejuni wild-type strain, infection with the noninvasive C. jejuni ∆ciaD mutant resulted in a blunted inflammatory response, with less inflammatory cytokines and neutrophil markers. These findings indicate that intestinal inflammation is driven by C. jejuni virulence and that neutrophils are the predominant cell type responding to C. jejuni infection. We propose that this model can be used as a platform to study the early immune events during infection with intestinal pathogens.

Prevalence of Campylobacter in raw poultry remains a major food microbiological safety challenge. Novel mitigation strategies are required to ensure the safety and quality of poultry products. Active food packaging can control pathogens without directly adding antimicrobials into the food matrix and extend the food’s shelf life. The functionalized absorbing pad with ZnO NPs developed in this study was able to inactivate C. jejuni in raw chicken meat and keep the meat free from C. jejuni contamination during shelf life without any observed migration of nanoparticles. The controllable conversion of immobilized ZnO NPs to free Zn 2+ makes this approach safe and eco-friendly and paves the way for developing a novel intervention strategy for other high-risk foods. Our study applied nanotechnology to exploit an effective approach for Campylobacter control in raw chicken meat products.

Campylobacter jejuni, a zoonotic pathogen that frequently colonizes poultry, possesses two Microbial Surface Components Recognizing Adhesive Matrix Molecule(s) (MSCRAMMs) termed CadF and FlpA that bind to the glycoprotein fibronectin (FN). Previous to this study, it was not known whether the CadF and FlpA proteins were functionally redundant or if both were required to potentiate host cell binding and signaling processes. We addressed these questions by generating a complete repertoire of cadF and flpA mutants and complemented isolates, and performing multiple phenotypic assays. Both CadF and FlpA were found to be necessary for the maximal binding of C. jejuni to FN and to host cells. In addition, both CadF and FlpA are required for the delivery of the C. jejuni Cia effector proteins into the cytosol of host target cells, which in turn activates the MAPK signaling pathway (Erk 1/2) that is required for the C. jejuni invasion of host cells. These data demonstrate the non-redundant and bi-functional nature of these two C. jejuni FN-binding proteins. Taken together, the C. jejuni CadF and FlpA adhesins facilitate the binding of C. jejuni to the host cells, permit delivery of effector proteins into the cytosol of a host target cell, and aid in the rewiring of host cell signaling pathways to alter host cell behavior.

Campylobacter jejuni, a foodborne pathogen, is one of the most common bacterial causes of gastroenteritis in the world. Undercooked poultry, raw (unpasteurized) dairy products, untreated water, and contaminated produce are the most common sources associated with infection. C. jejuni establishes a niche in the gut by adhering to and invading epithelial cells, which results in diarrhea with blood and mucus in the stool. The process of colonization is mediated, in part, by surface-exposed molecules (adhesins) that bind directly to host cell ligands or the extracellular matrix (ECM) surrounding cells. In this review, we introduce the known and putative adhesins of the foodborne pathogen C. jejuni. We then focus our discussion on two C. jejuni Microbial Surface Components Recognizing Adhesive Matrix Molecule(s) (MSCRAMMs), termed CadF and FlpA, which have been demonstrated to contribute to C. jejuni colonization and pathogenesis. In vitro studies have determined that these two surface-exposed proteins bind to the ECM glycoprotein fibronectin (FN). In vivo studies have shown that cadF and flpA mutants exhibit impaired colonization of chickens compared to the wild-type strain. Additional studies have revealed that CadF and FlpA stimulate epithelial cell signaling pathways necessary for cell invasion. Interestingly, CadF and FlpA have distinct FN-binding domains, suggesting that the functions of these proteins are non-redundant. In summary, the binding of FN by C. jejuni CadF and FlpA adhesins has been demonstrated to contribute to adherence, invasion, and cell signaling.

The aim of this study is to investigate the antimicrobial synergistic effect against Campylobacter jejuni, a leading foodborne pathogen that causes human gastroenteritis, by cinnamon oil, encapsulated curcumin, and zinc oxide nanoparticles (ZnO NPs). We compared three approaches to study the antimicrobial interactions, including the time-killing method, the fractional inhibitory concentration index (FICI) method, and a mathematical concentration-effect model. Isobologram analysis was performed to evaluate the synergy in different combinations, and a median-effect equation was applied to identify the combinations of synergistic effects at median, bacteriostatic, and bactericidal reduction levels. The time-killing method overestimated the synergistic interaction between antimicrobials, while the FICI method failed to detect an existing synergistic phenomenon. This lack of accuracy and sensitivity was mainly due to combining antimicrobials without a deep understanding of their concentration-effect relationships. Our results showed that each antimicrobial had a unique concentration-effect curve. Specifically, encapsulated curcumin showed a sharp sigmoidal curve unlike cinnamon oil and ZnO NPs. A mathematical model was applied to study the interaction between antimicrobials with a different shape of concentration-effect curve. We observed an additive effect of cinnamon oil/ZnO NPs and synergistic interactions of other binary combinations (cinnamon oil/encapsulated curcumin and ZnO NPs/encapsulated curcumin). The tertiary combination of cinnamon oil/ZnO NPs/encapsulated curcumin at IC25 (additive line <1-log CFU/mL) presented the greatest synergistic effect by reducing the bacterial population over 8-log CFU/mL. This mathematical model provided an alternative strategy to develop a new antimicrobial strategy.

Major foodborne bacterial pathogens, such as Campylobacter jejuni, have devised complex strategies to establish and foster intestinal infections. For more than two decades, researchers have used immortalized cell lines derived from human intestinal tissue to dissect C. jejuni-host cell interactions. Known from these studies is that C. jejuni virulence is multifactorial, requiring a coordinated response to produce virulence factors that facilitate host cell interactions. This study was initiated to identify C. jejuni proteins that contribute to adaptation to the host cell environment and cellular invasion. We demonstrated that C. jejuni responds to INT 407 and Caco-2 cells in a similar fashion at the cellular and molecular levels. Active protein synthesis was found to be required for C. jejuni to maximally invade these host cells. Proteomic and transcriptomic approaches were then used to define the protein and gene expression profiles of C. jejuni co-cultured with cells. By focusing on those genes showing increased expression by C. jejuni when co-cultured with epithelial cells, we discovered that C. jejuni quickly adapts to co-culture with epithelial cells by synthesizing gene products that enable it to acquire specific amino acids for growth, scavenge for inorganic molecules including iron, resist reactive oxygen/nitrogen species, and promote host cell interactions. Based on these findings, we selected a subset of the genes involved in chemotaxis and the regulation of flagellar assembly and generated C. jejuni deletion mutants for phenotypic analysis. Binding and internalization assays revealed significant differences in the interaction of C. jejuni chemotaxis and flagellar regulatory mutants. The identification of genes involved in C. jejuni adaptation to culture with host cells provides new insights into the infection process.

Two metal oxide (i.e., Al2O3 and TiO2) nanoparticles and ajoene, a garlic-derived organosulfur compound, were identified to be effective antimicrobials against Campylobacter jejuni, a leading cause of human gastrointestinal diseases worldwide. A significant synergistic antimicrobial effect was observed using ajoene and Al2O3/TiO2 nanoparticles in a combined manner to cause at least 8 log10 CFU/mL reduction of C. jejuni cells. Whole transcriptome sequencing (RNA-seq) and confocal micro-Raman spectroscopic analyses revealed the antimicrobial mechanism and identified the roles of ajoene and metal oxide nanoparticles in the synergistic treatment. Ajoene and metal oxide nanoparticles mediated a two-phase antimicrobial mechanism. Ajoene served as the inducing factor at the first phase that caused injury of cell membranes and increased the susceptibility of C. jejuni to stress. Metal oxide nanoparticles served as the active factor at the second phase that targeted sensitive cells and physically disrupted cell structure. This synergistic antimicrobial treatment demonstrates a potential to reduce the prevalence of C. jejuni and other pathogens on food contact surfaces and in the food chain.