The aim of the current study is to assess the prevalence ofCampylobacterinfection in broiler chickens, raised in intensive production conditions, and to evaluate the antimicrobial susceptibility of recoveredCampylobacterisolates. A total of 590 cloacal swab samples were taken from 13 broiler chicken flocks in the North East of Tunisia. All samples were tested for the presence of thermophilicCampylobacterby culture and PCR, targeting themapAandceuEgenes, respectively. Susceptibility to antimicrobial drugs was tested against 8 antibiotics. Prevalence ofCampylobacterinfection, relationship with geographic origins and seasons, antimicrobial resistance rates and patterns were analyzed. Total prevalence ofCampylobacterinfection in broiler flocks was in the range of 22.4%, with a predominance ofC. jejuni(68.9%), followed byC. coli(31.1%). Positive association was highlighted between the infection level and the season (P< 0.001), but no link was emphasized considering the geographic origin. Antimicrobial susceptibility testing revealed very high resistance rates detected against macrolide, tetracycline, quinolones, and chloramphenicol, ranging from 88.6% to 100%. Lower resistance prevalence was noticed forβ-lactams (47% and 61.4%) and gentamicin (12.9%). 17 R-type patterns were observed, and a common pattern was found in 30.3% of isolates. This study provides updates and novel data on the prevalence and the AMR of broiler campylobacters in Tunisia, revealing the occurrence of high resistance to several antibiotics and emphasizing the requirement of better surveillance and careful regulation of antimicrobials use.

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.

The aim of this study was to determine the species distribution of Staphylococcus, Gram negative bacteria (GNB) and the occurrence of Methicillin Resistant Staphylococci (MRS) and Extended-Spectrum β-lactamase- (ESBL-) producing GNB. Bacterial culture of 300 clinical mastitis milk samples from 30 different farms across different regions of Tunisia during four seasons was realized. The obtained results showed the presence of high frequency of the tested samples with a positive growth for bacteria (64%). In addition a high recovery rate of Staphylococci and/or GNB in these clinical mastitis milk samples (87%) was detected. In addition, a high percentage of GNB (68.2%) compared to Staphylococcus species (32%) was noted. Moreover, a significant variation of the number of these bacteria according to the farm location, the seasons, and cows age was detected. The highest percentage was observed in the North of Tunisia during the winter and the spring seasons in adult cows with a dominance of GNB growth. Coagulase negative Staphylococci (CNS) (n=11) and GNB (n=16) species were identified. Escherichia coli (E. coli) was the most frequently found bacterium followed by Klebsiella pneumoniae. The dominant Staphylococcus isolates was S. xylosus followed by S. aureus the major pathogen isolated. Methicillin resistance was confirmed by the presence of the mecA gene in 3 S. aureus and 14 CNS isolates; all of these isolates were lacking the mecC gene. Various species of GNB, resistant to cefotaxime, were detected (n=15). ESBLs were detected on selective medium in 10 E. coli and 4 K. pneumoniae. All ESBL producers strains carry the blaCTX-M. The presence of different resistant mastitis pathogens in dairy farms may complicate therapeutic options and contaminated animals could become zoonotic agent reservoir for human.

Circulation of a multi-resistance clone of bacteria associated with genetic elements in diseased animals constitutes a global public health problem. Our study focused on the characterization of the support of ESBL in cefotaxime resistant E. coli (CTX^R) isolates recovered from poultry with diarrhea, analysis of their clonal lineage, and virulence-associated genes. The study was carried out on 130 samples of chickens with diarrhea, collected in 2015 from poultry farms in Tunisia. Isolates of 20 CTX^R E. coli strains were identified as ESBL and AmpC β- lactamase producers. The following β-lactamase genes (number of isolates) were detected: bla_CTX-M-15+ bla_OXA1 (4), bla_CTX-M-15 + bla_OXA1 + bla_TEM-1b (2), bla_CTX-M-1 + bla_TEM-1b (9), bla_CTX-M-1 (2), bla_CMY2 + bla_TEM-1b (3). Six E. coli harboring bla_CTXM-15 were allocated to ST131-B2-O25b-; six and three bla_CTX-M-1 were grouped in ST155, ST10, and ST58, respectively, related to the phylogroup D and A. The qnrB gene, the variant aac(6′)-Ib-cr, and the class 1 integrons with different gene cassettes, were detected amongst our 20 isolated strains, which were classified as ExPEC and aEPEC. Our findings highlighted the emergence of the human pandemic ST131-CTX-M-15-O25-B2 clone and the high risk of such clonal lineage strains in diarrheic poultry, in Tunisia, which could constitute a risk of their transfer to healthy animals and humans.

Marek's disease (MD) is a contagious avian viral disease that is responsible for large economic losses to farmers. The disease is caused by Marek's disease virus (species Gallid alphaherpesvirus 2), which causes neurological lesions, immune suppression, and tumor proliferation of lymphoid cells that invade a large number of organs and tissues. Despite widespread vaccination, Marek's disease virus (MDV), has shown a continuous increase in its virulence and has acquired the ability to overcome immune responses induced by vaccines. In the present study, the oncogenic serotype MDV-1 was detected by real-time PCR in DNA samples extracted from organs developing tumor infiltrations. Identification of the pathotype based on a 132-bp tandem repeat and sequencing and phylogenetic analysis of the Meq gene and its encoded protein allowed classification of the isolated viruses as "very virulent", with two new and unique mutations in the Meq gene resulting in amino acid substitutions. Sequencing of pp38, vIl-8, UL1 and UL44 genes did not reveal any new mutations that were characteristic of the Tunisian isolates or correlated with virulence. These results raised concerns about the ability of HVT and CVI988 vaccines, which are currently used in Tunisia and other countries, to protect chickens against highly virulent virus strains.