Reduction of Campylobacter jejuni contamination by using UVA-LED and sodium hypochlorite on the surface of chicken meat.

Campylobacter jejuni causes gastroenteritis in humans and is a major concern in food safety. Commercially prepared chicken meats are frequently contaminated with C. jejuni, which is closely associated with the diffusion of intestinal contents in poultry processing plants. Sodium hypochlorite (NaClO) is commonly used during chicken processing to prevent food poisoning; however, its antimicrobial activity is not effective in the organic-rich solutions. In this study, we investigated the potential of a new photo-disinfection system, UVA-LED, for the disinfection of C. jejuni-contaminated chicken surfaces. The data indicated that UVA irradiation significantly killed C. jejuni and that its killing ability was significantly facilitated in NaClO-treated chickens. Effective inactivation of C. jejuni was achieved using a combination of UVA and NaClO, even in the organic-rich condition. The results of this study show that synergistic disinfection using a combination of UVA and NaClO has potential beneficial effects in chicken processing systems.

Campylobacter occurrence and antimicrobial resistance profile in under five-year-old diarrheal children, backyard farm animals, and companion pets.

Campylobacteriosis disproportionately affects children under five in low-income countries. However, epidemiological and antimicrobial resistance (AMR) information at the children-animal interface is lacking. We hypothesized that Campylobacter is a major cause of enteritis in children in Ethiopia, and contact with animals is a potential source of transmission. The objective of the study was to determine Campylobacter occurrence and its AMR in children under five with diarrhea, backyard farm animals, and companion pets. Stool from 303 children and feces from 711 animals were sampled. Campylobacter was isolated through membrane filtration on modified charcoal cefoperazone deoxycholate agar plates under microaerobic incubation, and the technique showed to be feasible for use in regions lacking organized laboratories. Typical isolates were characterized with MALDI-TOF MS and multiplex PCR. Of 303 children, 20% (n = 59) were infected, with a higher proportion in the 6 to 11-month age group. Campylobacter occurred in 64% (n = 14) of dogs and 44% (n = 112) of poultry. Campylobacter jejuni was present in both a child and animal species in 15% (n = 23) of 149 households positive for Campylobacter. MICs using the gradient strip diffusion test of 128 isolates displayed resistance rates of 20% to ciprofloxacin and 11% to doxycycline. MICs of ciprofloxacin and doxycycline varied between C. coli and C. jejuni, with higher resistance in C. coli and poultry isolates. Campylobacter infection in children and its prevalent excretion from backyard poultry and dogs is a understudied concern. The co-occurrence of C. jejuni in animals and children suggest household-level transmission As resistance to ciprofloxacin and doxycycline was observed, therapy of severe campylobacteriosis should consider susceptibility testing. Findings from this study can support evidence-based diagnosis, antimicrobial treatment, and further investigations on the spread of AMR mechanisms for informed One Health intervention.

Oral curcumin ameliorates acute murine campylobacteriosis.

Introduction: Human infections with the food-borne enteropathogen Campylobacter jejuni are responsible for increasing incidences of acute campylobacteriosis cases worldwide. Since antibiotic treatment is usually not indicated and the severity of the enteritis directly correlates with the risk of developing serious autoimmune disease later-on, novel antibiotics-independent intervention strategies with non-toxic compounds to ameliorate and even prevent campylobacteriosis are utmost wanted. Given its known pleiotropic health-promoting properties, curcumin constitutes such a promising candidate molecule. In our actual preclinical placebo-controlled intervention trial, we tested the anti-microbial and anti-inflammatory effects of oral curcumin pretreatment during acute experimental campylobacteriosis. Methods: Therefore, secondary abiotic IL-10-/- mice were challenged with synthetic curcumin via the drinking water starting a week prior oral C. jejuni infection. To assess anti-pathogenic, clinical, immune-modulatory, and functional effects of curcumin prophylaxis, gastrointestinal C. jejuni bacteria were cultured, clinical signs and colonic histopathological changes quantitated, pro-inflammatory immune cell responses determined by in situ immunohistochemistry and intestinal, extra-intestinal and systemic pro-inflammatory mediator measurements, and finally, intestinal epithelial barrier function tested by electrophysiological resistance analysis of colonic ex vivo biopsies in the Ussing chamber. Results and discussion: Whereas placebo counterparts were suffering from severe enterocolitis characterized by wasting symptoms and bloody diarrhea on day 6 post-infection, curcumin pretreated mice, however, were clinically far less compromised and displayed less severe microscopic inflammatory sequelae such as histopathological changes and epithelial cell apoptosis in the colon. In addition, curcumin pretreatment could mitigate pro-inflammatory innate and adaptive immune responses in the intestinal tract and importantly, rescue colonic epithelial barrier integrity upon C. jejuni infection. Remarkably, the disease-mitigating effects of exogenous curcumin was also observed in organs beyond the infected intestines and strikingly, even systemically given basal hepatic, renal, and serum concentrations of pro-inflammatory mediators measured in curcumin pretreated mice on day 6 post-infection. In conclusion, the anti-Campylobacter and disease-mitigating including anti-inflammatory effects upon oral curcumin application observed here highlight the polyphenolic compound as a promising antibiotics-independent option for the prevention from severe acute campylobacteriosis and its potential post-infectious complications.

The effect of mesenchymal stem cell conditioned medium incorporated within chitosan nanostructure in clearance of common gastroenteritis bacteria in-vitro and in-vivo.

Gastroenteritis infection is a major public health concern worldwide, especially in developing countries due to the high annual mortality rate. The antimicrobial and antibiofilm activity of human mesenchymal stem cell-derived conditioned medium (hMSCsCM) encapsulated in chitosan nanoparticles (ChNPs) was studied in vitro and in vivo against common gastroenteritis bacteria. The synthesized ChNPs were characterized using Zeta potential, scanning electron microscopy (SEM), and dynamic light scattering (DLS) techniques. HMSC-derived conditioned medium incorporated into chitosan NPs (hMSCsCM-ChNPs) composite was fabricated by chitosan nanoparticles loaded with BM-MSCs (positive for CD73 and CD44 markers). The antimicrobial and antibiofilm activity of composite was investigated against four common gastroenteritis bacteria (Campylobacter jejuni ATCC29428, Salmonella enteritidis ATCC13076, Shigella dysenteriae PTCC1188, and E. coli ATCC25922) in-vitro and in-vivo. Majority of ChNPs (96%) had an average particle size of 329 nm with zeta potential 7.08 mV. The SEM images confirmed the synthesis of spherical shape for ChNPs and a near-spherical shape for hMSCsCM-ChNPs. Entrapment efficiency of hMSCsCM-ChNPs was 75%. Kinetic profiling revealed that the release rate of mesenchymal stem cells was reduced following the pH reduction. The antibacterial activity of hMSCsCM-ChNPs was significantly greater than that of hMSCsCM and ChNPs at dilutions of 1:2 to 1:8 (P < 0.05) against four common gastroenteritis bacteria. The number of bacteria present decreased more significantly in the group of mice treated with the hMSCsCM-ChNPs composite than in the groups treated with hMSCsCM and ChNPs. The antibacterial activity of hMSCsCM against common gastroenteritis bacteria in an in vivo assay decreased from > 106 CFU/ml to approximately (102 to 10) after 72 h. Both in vitro and in vivo assays demonstrated the antimicrobial and antibiofilm activities of ChNPs at a concentration of 0.1% and hMSCsCM at a concentration of 1000 µg/ml to be inferior to that of hMSCsCM-ChNPs (1000 µg/ml + 0.1%) composite. These results indicated the existence of a synergistic effect between ChNPs and hMSCsCM. The designed composite exhibited notable antibiofilm and antibacterial activities, demonstrating optimal release in simulated intestinal lumen conditions. The utilization of this composite is proposed as a novel treatment approach to combat gastroenteritis bacteria in the context of more challenging infections.

Short Variable Regions flaA Gene (SVR-flaA) Diversity and Virulence Profile of Multidrug-Resistant Campylobacter from Poultry and Poultry Meat in India.

Human gastrointestinal infections caused by Campylobacter species is the second most important foodborne illness after salmonellosis worldwide. Poultry represent one of the main sources of Campylobacter organisms. In the present study, the short variable region of flagellin gene (SVR-flaA) typing was carried out to determine the variation among the circulating strains of Campylobacter jejuni and Campylobacter coli. The C. jejuni and C. coli isolated from poultry and poultry meat were screened for the presence of virulence determinants like cadF, flaA, cdtB, and wlaN gene. The screening for wlaN gene is crucial in view of the fact that most patients with Guillian Barre's (GB) syndrome with a preceding history of diarrheal illness have been found to harbor wlaN gene-positive C jejuni strains. Out of the 200 samples comprising poultry meat and cloacal swabs, 21.5% of samples were found to harbor Campylobacter spp. of which 2.5% were Campylobacter jejuni, and 19% were confirmed as Campylobacter coli. The cadF, flaA, cdtB virulence genes were detected in all the Campylobacter spp. isolated in the present study. The presence of the wlaN gene in the Campylobacter jejuni isolated in the present study may pose a public health threat with long-term human health implications. The SVR-flaA typing of twelve Campylobacter isolates obtained in the present study revealed that Campylobacter coli flaA sequence OL471375 is a new strain with a novel allele type 1,675 and peptide sequence 5 which stands deposited in pubMLST database for Campylobacter. The other flaA-SVR gene sequences identified in this study were OL471369, OL471370, OL471371, OL471372, OL471373, and OL471374. Among twelve Campylobacter spp., three distinct DdeI-RFLP patterns were observed, each varying in size from 100 to 1,000 base pairs. Antimicrobial profiling of the Campylobacter spp. isolated in the present study revealed that 50% of the strains were multidrug resistant. All the Campylobacter spp. were resistant to ciprofloxacin (CIP), ampicillin (AMP), penicillin (PEN), and nalidixic acid (NAL) whereas 57.1% of strains were resistant to tetracycline (TET) and erythromycin (ERY) 28% to amoxicillin (AMX) and enrofloxacin (ENO), 85% to amikacin (AMK). The high degree of resistance to fluoroquinolones observed in the present study is crucial in view of fluoroquinolones being drugs of choice for the treatment of human Campylobacter infections.

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.

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.

Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.

Campylobacter jejuni and casein hydrolysate addition: Impact on poultry in vitro cecal microbiota and metabolome.

This study investigates the impact of casein hydrolysates on the poultry ceca inoculated with Campylobacter focusing on microbial molecular preferences for different protein sources in the presence of Campylobacter jejuni. Three casein sources (intact casein (IN), casein enzyme hydrolysate (EH), and casein acid hydrolysate (AH)) were introduced to cecal contents in combination with inoculated C. jejuni in an in vitro model system incubated for 48 h at 42°C under microaerophilic conditions. Samples were collected at 0, 24, and 48 h. Genomic DNA was extracted and amplified using custom dual-indexed primers, followed by sequencing on an Illumina MiSeq platform. The obtained sequencing data were then analyzed via QIIME2-2021.11. Metabolite extracts were analyzed with ultra-high-performance liquid orbitrap chromatography-mass spectrometry (UHPLC-MS). Statistical analysis of metabolites was conducted using MetaboAnalyst 5.0, while functional analysis was performed using Mummichog 2.0 with a significance threshold set at P < 0.00001. DNA sequencing and metabolomic analyses revealed that C. jejuni was most abundant in the EH group. Microbial diversity and richness improved in casein supplemented groups, with core microbial differences observed, compared to non-supplemented groups. Vitamin B-associated metabolites significantly increased in the supplemented groups, displaying distinct patterns in vitamin B6 and B9 metabolism between EH and AH groups (P < 0.05). Faecalibacterium and Phascolarctobacterium were associated with AH and EH groups, respectively. These findings suggest microbial interactions in the presence of C. jejuni and casein supplementation are influenced by microbial community preferences for casein hydrolysates impacting B vitamin production and shaping competitive dynamics within the cecal microbial community. These findings underscore the potential of nutritional interventions to modulate the poultry GIT microbiota for improved health outcomes.

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.

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.

Combined application of bacteriophages with a competitive exclusion culture and carvacrol with organic acids can reduce Campylobacter in primary broiler production.

For reducing Campylobacter (C.) in the food production chain and thus the risk to the consumer, the combined application of different measures as a multiple-hurdle approach is currently under discussion. This is the first study to investigate possible synergistic activities in vivo, aiming at reducing intestinal C. jejuni counts by administering (i) bacteriophages (phages) in combination with a competitive exclusion (CE) product and (ii) carvacrol combined with organic acids. The combined application of the two selected phages (Fletchervirus phage NCTC 12673 and Firehammervirus phage vB_CcM-LmqsCPL1/1) and the CE product significantly reduced C. jejuni loads by 1.0 log10 in cecal and colonic contents as well as in cloacal swabs at the end of the trial (33 and 34 days post hatch). The proportion of bacterial isolates showing reduced phage susceptibility ranged from 10.9% (isolates from cecal content) to 47.8% (isolates from cloacal swabs 32 days post hatch) for the Fletchervirus phage, while all tested isolates remained susceptible to the Firehammervirus phage. The use of carvacrol combined with an organic acid blend (sorbic acid, benzoic acid, propionic acid, and acetic acid) significantly reduced Campylobacter counts by 1.0 log10 in cloacal swabs on day 30 only.

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.

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.

Development of Fluoroquinolone Resistance through Antibiotic Tolerance in Campylobacter jejuni.

Antibiotic tolerance not only enables bacteria to survive acute antibiotic exposures but also provides bacteria with a window of time in which to develop antibiotic resistance. The increasing prevalence of Campylobacter jejuni isolates resistant to clinically important antibiotics, particularly fluoroquinolones (FQs), is a global public health concern. Currently, little is known about antibiotic tolerance and its effects on resistance development in C. jejuni. Here, we show that exposure to ciprofloxacin or tetracycline at concentrations 10 and 100 times higher than the MIC induces antibiotic tolerance in C. jejuni, whereas gentamicin or erythromycin treatment causes cell death. Interestingly, FQ resistance rapidly develops in C. jejuni after tolerance induction by ciprofloxacin and tetracycline. Furthermore, after tolerance is induced, alkyl hydroperoxide reductase (AhpC) plays a critical role in reducing FQ resistance development by alleviating oxidative stress. Together, these results demonstrate that exposure of C. jejuni to antibiotics can induce antibiotic tolerance and that FQ-resistant (FQR) C. jejuni clones rapidly emerge after tolerance induction. This study elucidates the mechanisms underlying the high prevalence of FQR C. jejuni and provides insights into the effects of antibiotic tolerance on resistance development. IMPORTANCE Antibiotic tolerance compromises the efficacy of antibiotic treatment by extending bacterial survival and facilitating the development of mutations associated with antibiotic resistance. Despite growing public health concerns about antibiotic resistance in C. jejuni, antibiotic tolerance has not yet been investigated in this important zoonotic pathogen. Here, our results show that exposure of C. jejuni to ciprofloxacin or tetracycline leads to antibiotic tolerance development, which subsequently facilitates the emergence of FQR C. jejuni. Importantly, these antibiotics are commonly used in animal agriculture. Moreover, our study suggests that the use of non-FQ drugs in animal agriculture promotes FQ resistance development, which is crucial because antibiotic-resistant C. jejuni is primarily transmitted from animals to humans. Overall, these findings increase our understanding of the mechanisms of resistance development through the induction of antibiotic tolerance.

Antimicrobial Resistance and Antibiogram of Thermotolerant Campylobacter Recovered from Poultry Meat in Baghdad Markets, Iraq.

Antimicrobial resistance is a critical public health issue that affects people all over the world. Since bacteria have a proclivity for rapidly acquiring and propagating the resistance gene, antimicrobial-resistant Campylobacter has a negative impact on public health. As a result, the creation of new and highly pathogenic clones is facilitated, making antimicrobial treatment more challenging. This study aimed to determine the antimicrobial resistance pattern (ARP) models, multiple drug resistance (MDR) models, and multiple antibiotic resistance (MAR) index of Campylobacter species isolated from poultry meat sold in Baghdad markets, Iraq. By employing the disc diffusion test, 30 Campylobacter strains from chicken meat, including C. jejuni (n=10) and C. coli (n=20), were exposed to tetracycline (TET), erythromycin, Gentamicin, nalidixic acid, ciprofloxacin, and norfloxacin (NOR). The ARP of the Campylobacter isolates revealed up to five antibiotypes for two species, which revealed resistance to one or more antimicrobials, and 67% of them had MDR resistance to two or more experienced antimicrobials. The NOR-TET model is the most common MDR, having a prevalence of 30% among experienced isolates. In addition, the MAR index, equal to and lower than one, was found in 87% of the isolates. Antibiotic resistance in Campylobacter raises the probability of treatment failure in humans and animals, as well as the propagation of antimicrobial resistance genes. As a result, the presence of Campylobacter in meat could pose a risk of human infection and pollution of the environment.

Dihydroxy-Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance.

There is an urgent global need for the development of novel therapeutics to combat the rise of various antibiotic-resistant superbugs. Enzymes of the branched-chain amino acid (BCAA) biosynthesis pathway are an attractive target for novel anti-microbial drug development. Dihydroxy-acid dehydratase (DHAD) is the third enzyme in the BCAA biosynthesis pathway. It relies on an Fe-S cluster for catalytic activity and has recently also gained attention as a catalyst in cell-free enzyme cascades. Two types of Fe-S clusters have been identified in DHADs, i.e. [2Fe-2S] and [4Fe-4S], with the latter being more prone to degradation in the presence of oxygen. Here, we characterise two DHADs from bacterial human pathogens, Staphylococcus aureus and Campylobacter jejuni (SaDHAD and CjDHAD). Purified SaDHAD and CjDHAD are virtually inactive, but activity could be reversibly reconstituted in vitro (up to ∼19,000-fold increase with kcat as high as ∼6.7 s-1 ). Inductively-coupled plasma-optical emission spectroscopy (ICP-OES) measurements are consistent with the presence of [4Fe-4S] clusters in both enzymes. N-isopropyloxalyl hydroxamate (IpOHA) and aspterric acid are both potent inhibitors for both SaDHAD (Ki =7.8 and 51.6 µM, respectively) and CjDHAD (Ki =32.9 and 35.1 µM, respectively). These compounds thus present suitable starting points for the development of novel anti-microbial chemotherapeutics.

Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia).

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C.jejuni for hippuricase, GenBank: Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.

Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA.

The combined effects of ethylenediaminetetraacetic acid (EDTA) and bacteriophage (phage) treatment of foodborne pathogens were investigated. Although viable counts for Campylobacter jejuni decreased by 1.5 log after incubation for 8 h in the presence of phage PC10, re-growth was observed thereafter. The combination of phage PC10 and 1 mM EDTA significantly inhibited the re-growth of C. jejuni. The viable counts for C. jejuni decreased by 2.6 log (P < 0.05) compared with that of the initial count after 24 h. Moreover, EDTA at 0.67 or 1.3 mM, combined with the specific lytic phages, also effectively inhibited the re-growth of phage-resistant cells of Campylobacter coli, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Typhimurium. In addition, the combined effects of lytic phages and EDTA were investigated on the viability of Campylobacter in BHI broth at low temperatures followed by the optimum growth temperature. The re-growth of C. coli was significantly inhibited by the coexistence of 1.3 mM EDTA, and the viable counts of surviving bacteria was about the same as the initial viable count after the incubation. This is the first study demonstrating the combined use of lytic phages and EDTA is effective in inhibiting the re-growth of phage-resistant bacteria in Gram-negative bacteria.

Pharmacokinetics of tulathromycin in pregnant ewes (Ovis aries) challenged with Campylobacter jejuni.

The purpose of this study was to evaluate the pharmacokinetics of tulathromycin in the plasma and maternal and fetal tissues of pregnant ewes when administered within 24 hours of a single, IV Campylobacter jejuni (C. jejuni) challenge. Twelve, pregnant ewes between 72-92 days of gestation were challenged IV with C. jejuni IA3902 and then treated with 1.1 ml/45.36 kg of tulathromycin subcutaneously 18 hours post-challenge. Ewes were bled at predetermined time points and euthanized either at a predetermined time point or following the observation of vaginal bleeding or abortion. Following euthanasia, tissues were collected for bacterial culture, pharmacokinetics and histologic examination. The maximum (geometric) mean tulathromycin plasma concentration was estimated at 0.302 µg/mL, with a peak level observed at around 1.2 hours. The apparent systemic clearance of tulathromycin was estimated at 16.6 L/h (or 0.28 L/kg/h) with an elimination half-life estimated at approximately 22 hours. The mean tissue concentrations were highest in the uterus (2.464 µg/g) and placentome (0.484 µg/g), and were lowest in fetal liver (0.11 µg/g) and fetal lung (0.03 µg/g). Compared to previous reports, results of this study demonstrate that prior IV administration of C. jejuni appeared to substantially alter the pharmacokinetics of tulathromycin, reducing both the peak plasma concentrations and elimination half-life. However, additional controlled trials are required to confirm those observations.