Manel Gharbi

The increasing prevalence of multidrug-resistant (MDR) Campylobacter species poses a serious threat to food safety and public health, highlighting the urgent need for natural antimicrobial alternatives to conventional antibiotics. This study investigated the antibacterial potential and mechanism of action of seven essential oils (EOs), Cymbopogon citratus, Mentha pulegium, Artemisia absinthium, Myrtus communis, Thymus algeriensis, Thymus capitatus, and Eucalyptus globulus, against multidrug-resistant Campylobacter jejuni and Campylobacter coli. The antimicrobial activity was first assessed by the agar disk diffusion and broth microdilution methods to determine inhibition zones, minimum inhibitory concentrations (MICs), and minimum bactericidal concentrations (MBCs). The most active EOs were further evaluated through time–kill kinetics, cell lysis, salt tolerance, and membrane integrity assays to elucidate their bactericidal mechanisms. Results showed that E. globulus, T. algeriensis, and M. communis exhibited the strongest inhibitory effects, particularly against C. jejuni, with MIC values ranging from 3.125% to 6.25%, while C. coli was more resistant. Time–kill and lysis experiments demonstrated rapid bacterial reduction and significant decreases in optical density, indicating cell disruption. Additionally, EO treatments reduced salt tolerance and induced leakage of cytoplasmic materials, confirming membrane damage. Overall, these findings suggest that selected essential oils exert potent antimicrobial effects through membrane disruption and osmotic imbalance, offering promising natural strategies to control MDR Campylobacter in food systems. The application of such bioactive compounds could contribute significantly to improving food quality, extending shelf life, and enhancing food safety.

Background: Campylobacter jejuni , a major foodborne and zoonotic pathogen linked to poultry, is showing rising resistance to last‐resort antimicrobials like oxazolidinones and phenicols. Such resistance threatens both animal and human health. Its ability to acquire genes from gram‐positive bacteria highlights the need for surveillance in avian reservoirs. Aim: This study aimed to characterize oxazolidinone‐ and phenicol‐resistance determinants among 197 C. jejuni isolates from avian sources in Tunisia. Methods: Isolates were studied by determining their antimicrobial susceptibility by disk diffusion method, and resistant isolates were screened by PCR for fexA , fexB , floR , RE‐ cmeABC , cfr , and optrA genes. Results: Resistance rates were alarmingly high (57.6%–100%) across multiple antibiotic classes, and the majority were multidrug resistant. Among 155 (78.7%) chloramphenicol‐resistant isolates, the prevalence of optrA , cfr , fexA , floR , RE‐ cmeABC , and fexB was 85%, 55%, 75%, 46%, 42%, and 22%, respectively, with most isolates harboring multiple genes. Phylogenetic analysis revealed close relatedness among Tunisian optrA and fexB sequences and similarities with those reported in Enterococcus spp., suggesting intergenus gene exchange. Conclusion: This study reports for the first time the detection of fexB gene and its co‐occurrence with optrA in C. jejuni , highlighting its potential role as a reservoir of transferable resistance genes. These findings underscore the urgent need for enhanced surveillance and antimicrobial stewardship within a One Health framework.

Background/Objectives: The global emergence of carbapenem resistance is a major public health concern. Campylobacter jejuni and Campylobacter coli, key zoonotic agents causing human campylobacteriosis, are mainly isolated from poultry, their primary host. Their increasing resistance in animals and humans highlights the risk of gene transfer. This study investigates the molecular mechanisms of carbapenem resistance in 287 avian Campylobacter spp. isolates from Tunisia within a One Health approach. Methods: Antibiotic susceptibility of 287 carbapenem-resistant isolates, including 147 C. jejuni and 140 C. coli, was determined according to CLSI. All isolates were screened by PCR for genes encoding the most reported carbapenemases, including VIM, IMP, NDM and OXA-48. Eleven multidrug-resistant (MDR)/carbapenem-resistant C. coli isolates were selected to determine their clonal lineage by Multilocus sequence typing (MLST). Results: All isolates were susceptible to imipenem, but resistance to meropenem and ertapenem were observed in 60.71% and 35.71% of C. coli isolates, respectively, versus 13.6% in C. jejuni for each antibiotic. The bla_VIM, bla_NDM and bla_OXA-48 genes were detected in 15, 8, and 19 of the 20 C. jejuni isolates, respectively. However, for C. coli, 53, 12, and 15 isolates harbored bla_VIM, bla_NDM and bla_OXA-48 genes, respectively. The eleven (MDR)/carbapenem-resistant C. coli isolates belonged to a unique ST sequence type ST13450. Conclusions: We report for the first time the emergence of bla_VIM, bla_NDM, and bla_OXA-48 genes in Campylobacter spp. isolates of poultry origin highlighting possible horizontal transfer of these genes to pathogenic Gram-negative bacteria of the poultry’s microbiota.

The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in Campylobacter coli isolates collected from broilers, laying hens and poultry farm environments. One hundred and thirty-nine C. coli isolates were isolated from broilers (n = 41), laying hens (n = 53), eggs (n = 4) and the environment (n = 41) of 23 poultry farms located in northeastern of Tunisia. Antimicrobial susceptibility testing was performed on all isolates according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing guidelines. The detection of PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6)-Ib gene was performed using polymerase chain reaction (PCR) and specific primers. aac(6′)-Ib amplicons were further analyzed by digestion with BtsCI to identify the aac(6′)-Ib-cr variant. Mutations in GyrA and the occurrence of RE-CmeABC efflux pump were determined by mismatch amplification mutation assay (MAMA) PCR and PCR, respectively. In addition, eleven isolates were selected to determine their clonal lineage by MLST. The 139 C. coli isolates were resistant to ciprofloxacin, and 86 (61.8%) were resistant to nalidixic acid. High rates of resistance were also observed toward erythromycin (100%), azithromycin (96.4%), tetracycline (100%), chloramphenicol (98.56%), ampicillin (66.1%), amoxicillin-clavulanic acid (55.39%), and kanamycin (57.55%). However, moderate resistance rates were observed for gentamicin (9.35%) and streptomycin (22.3%). All quinolone-resistant isolates harbored the Thr-86-Ile amino acid substitution in GyrA, and the RE-CmeABC efflux pump was detected in 40.28% of isolates. Interestingly, the qnrB, qnrS, qepA, and aac(6′)-Ib-cr were detected in 57.7%, 61.15%, 21.58%, and 10% of isolates, respectively. The eleven isolates studied by MLST belonged to a new sequence type ST13450. This study described for the first time the occurrence of PMQR genes in C. coli isolates in Tunisia and globally.

Campylobacter species, especially C. coli and C. jejuni, have been associated with a range of human gastrointestinal diseases. During the last two decades, due to the irrational use of antibiotics in poultry farms, high rates of antimicrobial resistance have been globally reported in C. coli and C. jejuni isolates. Recently, acquired linezolid‐resistance mechanisms have been reported in Campylobacter spp. isolates, which is a cause of concern to human health. In this study, we performed a retrospective analysis of 139 C. coli isolates previously collected from broilers (n = 41), laying hens (n = 53), eggs (n = 4), and environment (n = 41) to detect acquired genes implicated in linezolid resistance. Isolates were tested for their susceptibility to antimicrobial agents using the Kirby–Bauer disk diffusion assay. Chloramphenicol‐ and linezolid‐resistant isolates were subjected to PCR screening for the following genes: fexA, fexB, floR, RE‐cmeABC, cfrA, and optrA. The genetic relatedness of eight multidrug‐resistant isolates was determined by multilocus sequence typing (MLST). Among the 139 C. coli isolates, high rates of resistance (57.55%–100%) were detected toward nalidixic acid, ciprofloxacin, erythromycin, azithromycin, ampicillin, chloramphenicol, linezolid, and kanamycin. Among 135 chloramphenicol‐resistant isolates, the optrA, cfr, fexA floR, RE‐cmeABC, and fexB genes were detected in 124 (124/135, 91.85%), 108 (80%), 105 (77.7%), 64 (47.4%), 56 (41, 48%), and 27 (20%) isolates, respectively. In addition, the majority of isolates harbored more than one of these genes. The selected eight isolates belonged to the same sequence type ST13450, which is a new sequence type (ST), not belonging to ST828 and ST1150 complexes. In conclusion, the emergence of optrA gene in Campylobacter spp. isolates makes this genus an optrA reservoir and vector to other pathogens such as Staphylococcus aureus and Enterococcus spp., which is a cause of concern for human and animal health.

The aac(6′)-Ib gene is the most widespread gene encoding aminoglycoside-modifying enzyme and conferring resistance to tobramycin, streptomycin and kanamycin. The variant aac(6′)-Ib-cr gene confers resistance to both aminoglycosides and fluoroquinolones (FQ). A total of 132 Campylobacter isolates, including 91 C. jejuni and 41 C. coli, were selected from broiler hens isolates. The aac(6′)-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6′)-Ib-cr. Among these isolates, 31 out of 41 C. coli (75.6%) and 1 (0.98%) C. jejuni were positive for the aac(6′)-Ib gene, which was identified as the aac(6′)-Ib-cr variant in 10 (32.25%) C. coli isolates. This variant was correlated with mutations in gyrA (Thr-86-Ile), as well as resistance to FQs. This study is the first report in Tunisia on Campylobacter coli strains harboring both the aac(6′)-Ib and aac(6′)-Ib-cr variants. These genes were present in Campylobacter isolates exhibiting resistance to multiple antibiotics, which restricts the range of available treatments.

To investigate the persistence risk of Campylobacter spp. in poultry farms, and to study the virulence and antimicrobial resistance characteristics in the recovered strains, we collected 362 samples from breeding hen flocks, before and after disinfection. The virulence factors were investigated by targeting the genes; flaA, cadF, racR, virB11, pldA, dnaJ, cdtA, cdtB, cdtC, ciaB, wlaN, cgtB, and ceuE by PCR. Antimicrobial susceptibility was tested and genes encoding antibiotic resistance were investigated by PCR and MAMA-PCR. Among the analyzed samples, 167 (46.13%) were positive for Campylobacter. They were detected in 38.7% (38/98) and 3% (3/98) of environment samples before and after disinfection, respectively, and in 126 (75.9%) out of 166 feces samples. In total, 78 C. jejuni and 89 C. coli isolates were identified and further studied. All isolates were resistant to macrolids, tetracycline, quinolones, and chloramphenicol. However, lower rates were observed for beta-lactams [ampicillin (62.87%), amoxicillin-clavulanic acid (47.3%)] and gentamicin (0.6%). The tet(O) and the cmeB genes were detected in 90% of resistant isolates. The bla_OXA-61 gene and the specific mutations in the 23S rRNA were detected in 87% and 73.5% of isolates, respectively. The A2075G and the Thr-86-Ile mutations were detected in 85% and 73.5% of macrolide and quinolone-resistant isolates, respectively. All isolates carried the flaA, cadF, CiaB, cdtA, cdtB, and cdtC genes. The virB11, pldA, and racR genes were frequent in both C. jejuni (89%, 89%, and 90%, respectively) and C. coli (89%, 84%, and 90%). Our findings highlight the high occurrence of Campylobacter strains exhibiting antimicrobial resistance with potential virulence traits in the avian environment. Thus, the improvement of biosecurity measures in poultry farms is essential to control bacterial infection persistence and to prevent the spread of virulent and resistant strains.

Hackathons are collaborative events that bring together diverse groups to solve predefined challenges. The COVID‐19 pandemic caused by SARS‐CoV‐2 has emphasized the need for portable and reproducible genomics analysis pipelines to study the genetic susceptibility of the human host and investigate human‐SARS‐CoV‐2 protein interactions. To build and strengthen institutional capacities in OMICS data analysis applied to host‐pathogen interaction (HPI), the PHINDaccess project organized two hackathons in 2020 and 2021. These hackathons are aimed at developing bioinformatics pipelines related to the SARS‐CoV‐2 viral genome, its phylodynamic transmission, and the identification of human genome host variants, with a focus on addressing global health challenges, particularly in low‐ and middle‐income countries (LMIC). This paper outlines the preparation, proceedings, and lessons learned from these hackathons, including the challenges faced by participants and our recommendations based on our experience for organizing hackathons in LMIC and beyond.

Globally, Campylobacter is a significant contributor to gastroenteritis. Efficient pathogens are qualified by their virulence power, resistance to antibiotics and epidemic spread. However, the correlation between antimicrobial resistance (AR) and the pathogenicity power of pathogens is complex and poorly understood. In this study, we aimed to investigate genes encoding virulence and AR mechanisms in 177 Campylobacter isolates collected from layer hens and eggs in Tunisia and to assess associations between AR and virulence characteristics. Virulotyping was determined by searching 13 virulence genes and AR-encoding genes were investigated by PCR and MAMA-PCR. The following genes were detected in C. jejuni and C. coli isolates: tet(O) (100%/100%), bla_OXA-61 (18.82%/6.25%), and cmeB (100%/100%). All quinolone-resistant isolates harbored the Thr-86-Ile substitution in GyrA. Both the A2074C and A2075G mutations in 23S rRNA were found in all erythromycin-resistant isolates; however, the erm(B) gene was detected in 48.38% and 64.15% of the C. jejuni and C. coli isolates, respectively. The machine learning algorithm Random Forest was used to determine the association of virulence genes with AR phenotypes. This analysis showed that C. jejuni virulotypes with gene clusters encompassing the racR, ceuE, virB11, and pldA genes were strongly associated with the majority of phenotypic resistance. Our findings showed high rates of AR and virulence genes among poultry Campylobacter, which is a cause of concern to human health. In addition, the correlations of specific virulence genes with AR phenotypes were established by statistical analysis.