This study aimed to improve the effectiveness of Thymus capitatus and Thymus algeriensis essential oils (EOs), as food preservatives, through their encapsulation in different delivery systems (DSs), namely nanoemulsions and biopolymeric nanoparticles. DSs’ preparation is tailored to enhance not only physical stability but also resulting Eos’ antioxidant and antibacterial activities through different fabrication methods (high-pressure homogenization emulsification or antisolvent precipitation) and using different emulsifiers and stabilizers. DSs are characterized in terms of droplet size distribution, ζ-potential, and stability over time, as well as antioxidant and antibacterial activities of encapsulated EOs. The antioxidant activity was studied by the FRAP assay; the antibacterial activity was evaluated by the well diffusion method. EOs of different compositions were tested, namely two EOs extracted from Thymus capitatus, harvested from Tunisia during different periods of the year (TC1 and TC2), and one EO extracted from Thymus algeriensis (TA). The composition of TC1 was significantly richer in carvacrol than TC2 and TA. The most stable formulation was the zein-based nanoparticles prepared with TC1 and stabilized with maltodextrins, which exhibit droplet size, polydispersity index, ζ-potential, and encapsulation efficiency of 74.7 nm, 0.14, 38.7 mV, and 99.66%, respectively. This formulation led also to an improvement in the resulting antioxidant (60.69 µg/mg vs. 57.67 µg/mg for non-encapsulated TC1) and antibacterial (inhibition diameters varying between 12 and 33 mm vs. a range between 12 and 28 mm for non-encapsulated TC1) activities of EO. This formulation offers a promising option for the effective use of natural antibacterial bioactive molecules in the food industry against pathogenic and spoilage bacteria.

Antibiotic resistance in foodborne pathogens is an emergent global health concern. The objectives of this study were to assess antimicrobial resistance (AMR) in Campylobacter isolates from chicken carcasses and to investigate the AMR molecular mechanisms as well as the presence of virulence determinants. The study was performed on 257 samples collected from abattoirs and retail shops in northeastern Tunisia. Forty-eight Campylobacter isolates were recovered and identified as C. jejuni (n = 33) and C. coli (n = 15). Antibiotic resistance was tested against eight antibiotics and high resistance rates were observed against tetracycline (100%), erythromycin (97.9%), ciprofloxacin (73%), nalidixic acid (85.4%), ampicillin (83.3%), amoxicillin/clavulanic acid (22.9%), chloramphenicol (75%), and gentamicin (27.1%). All isolates were multidrug-resistant, and 22 resistance patterns were found. All isolates were screened for AMR genes (tet(O), tet(A), tet(B), tet(L), cmeB, ermB, bla_OXA-61, and aphA-3), and for point mutations in gyrA (C257T substitution) and 23SrRNA (A2075G/A2074C) genes. All screened AMR genes, as well as the C257T and the A2075G mutations, were detected. The virulence genotypes were also determined, and all isolates carried the motility (flaA) and invasion (cadF) genes. Most of them also harbored the cdtA, cdtB, and cdtC genes, encoding the Campylobacter toxin. The screening of the cgtB and the wlaN genes, involved in Guillain-Barré Syndrome expression, revealed the presence of the cgtB in 21.2% of C. jejuni strains, whereas none of them carried the wlaN gene. Our findings highlight the emergence of Campylobacter strains simultaneously harboring several virulence and AMR determinants, which emphasizes the risk of transmission of MDR strains to humans via the food chain. Hence, controlling the dissemination of foodborne pathogens “from the farm to the fork” as well as restricting the use of antimicrobials in husbandry are mandatory to prevent the risk for consumers and to mitigate the dissemination of MDR pathogens.

Despite the importance of eggs in the human diet, and unlike other products, for which food safety risks are widely investigated, information on the occurrence of Campylobacter and antimicrobial resistance in eggs and layer hen flocks is lacking in Tunisia. This study was conducted to determine the occurrence of Campylobacter and the antimicrobial resistance in layer hens and on eggshells. Thus, 366 cloacal swabs and 86 eggshell smear samples were collected from five layer hen farms in the North-East of Tunisia. The occurrence of Campylobacter infection, and the antimicrobial resistance rates and patterns, were analyzed. The occurrence rates of Campylobacter infection in laying hens and eggshells were 42.3% and 25.6%, respectively, with a predominance of C. jejuni (68.4%, 81.9%), followed by C. coli (31.6%, 18.2%). The antimicrobial susceptibility testing revealed high resistance rates against macrolides, tetracycline, quinolones, β-lactams, and chloramphenicol, with percentages ranging from 35.5% to 100%. All isolates were multidrug resistant (MDR) and five resistance patterns were observed. These results emphasized the risk to consumer health and the need to establish a surveillance strategy to control and prevent the emergence and the spread of resistant strains of Campylobacter in poultry and humans.

During the current investigation, eight essential oils (EOs) were tested for their antimicrobial activity against six species, belonging to the genus of staphylococcus, multi-resistant to antibiotics (S. epidermidis, S. cohni, S. wareneri, S. scuiri, S. chromogenes, S. pasteuri), three methicillin-resistant Staphylococcus aureus strains (MRSA) and two strains of Escherichia coli, producing extended-spectrum β-lactamase (ESBL) responsible for bovine mastitis. Our results indicated that the antimicrobial activities of eight EOs varied significantly among the types of EOs and bacterial species. Thymus capitatus and Trachyspermum ammi EOs display important antibacterial activity against all tested strains, with the inhibition zone diameters situated between 20 and 45 mm, while EOs of Artemisia absinthium, Eucalyptus globulus, Eucalyptus camaldulensis, Myrtus communis and Mentha pulegium exerted an intermediate activity. For Cymbopogon citratus, this effect depends on bacteria species. In fact, an important effect was observed against S. warneri, S. epidermidis, S. cohenii, S. pasteuri and MRSA (EC 39+) strains. In addition, the important lytic effect was observed against MRSA strains, showing that Gram-positive bacteria were more sensitive to T. capitatus EO than Gram-negative ones. Concerning the characterization of the mode action of T. capitatus, experiments of kill-time, bacteriolytic, loss of salt tolerance and loss of cytoplasmic material showed that the used EO was able to destroy cell walls and membranes followed by the loss of vital intracellular materials. In addition, it inhibits the normal synthesis of DNA, causing the bacterial death of E. coli and MRSA strains. This study shows the potential of using of EOs, particularly T. capitaus, to inhibit the growth of Gram-positive and Gram-negative bacteria multi-resistant to antibiotics causing bovine mastitis.

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.

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.