Staphylococcus aureus is recognized worldwide as one of the major agents of dairy cow intra-mammary infections. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host. The present study evaluated 120 isolates from eight different countries that were genotyped by RS-PCR and investigated for 26 different virulence factors to increase the knowledge on the circulating genetic lineages among the cow population with mastitis. New genotypes were observed for South African strains while for all the other countries new variants of existing genotypes were detected. For each country, a specific genotypic pattern was found. Among the virulence factors, fmtB, cna, clfA and leucocidins genes were the most frequent. The sea and sei genes were present in seven out of eight countries; seh showed high frequency in South American countries (Brazil, Colombia, Argentina), while sel was harboured especially in one Mediterranean country (Tunisia). The etb, seb and see genes were not detected in any of the isolates, while only two isolates were MRSA (Germany and Italy) confirming the low diffusion of methicillin resistance microorganism among bovine mastitis isolates. This work demonstrated the wide variety of S. aureus genotypes found in dairy cattle worldwide. This condition suggests that considering the region of interest might help to formulate strategies for reducing the infection spreading.

Fifty-five Escherichia coli isolates were acquired from chicken and turkey meat obtained from two slaughterhouses in Tunis. Eighty-nine percent, 80%, 78%, 67%, 45%, 27%, 7%, 4%, and 2% of these isolates showed resistance to tetracycline, trimethoprim/sulfamethoxazole, streptomycin, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, colistine, and gentamicin, respectively. No resistance was detected to cefotaxime, ceftazidime, or amikacin. bla _TEM gene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored bla _OXA-1 gene. Tetracycline resistance was predominately mediated by the tetA gene. The sul1 , sul2 , and sul3 genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1 and sul3 were included in class 1 integrons in some cases. Sixty percent of isolates harbored integrons (class 1, 30 isolates; class 2, 5 isolates). Class 2 integrons contained in all cases the dfrA1 - sat1 - aadA1 - orfX gene cassette arrangement. Nine gene cassette arrangements have been detected among class 1 integrons, containing different alleles of dfrA (five alleles) and aadA (2 alleles) genes, which encode trimethoprim and streptomycin resistance, respectively. An uncommon gene cassette array ( sat - psp - aadA2 - cmlA1 - aadA1 - qacH -IS 440 - sul3 ) has been identified in three class 1 integron-positive isolates, and one additional isolate had this same structure with the insertion of IS 26 inside the aadA1 gene (included in GenBank with accession no. FJ160769). The 55 studied isolates belong to the four phylogenic groups of E. coli , and phylogroups A and D were the most prevalent ones. At least one virulence-associated gene ( fimA , papC , or aer ) was detected in 44 of the 55 (80%) studied isolates. E. coli isolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.

Objectives: The epidemiology of Enterococcus resistant to priority antibiotics including linezolid has mainly been investigated in developed countries and especially in hospitals. We aimed to evaluate the contribution of different non-human reservoirs for the burden of MDR enterococci in Tunisia, where scarce data are available.Methods: Samples (n = 287) were collected from urban wastewater (n = 57), retail meat (n = 29; poultry/bovine/ovine), milk (n = 89; bovine/ovine), farm animal faeces (n = 80; poultry/bovine/ovine) and pets (n = 32; rabbit/dogs/cats/birds) in different Tunisian regions (2014–17). They were plated onto Slanetz–Bartley agar after pre-enrichment without antibiotics. Standard methods were used for bacterial identification and characterization of antibiotic resistance and virulence genes (PCR), antibiotic susceptibility testing (disc diffusion/broth microdilution; EUCAST/CLSI) and clonality (SmaI-PFGE/MLST).Results: All samples carried Enterococcus (n = 377 isolates) resistant to antibiotics considered to be critical or highly important by WHO. Even without antibiotic selection, 38% of Enterococcus faecalis (Efs) and 22% of Enterococcus faecium (Efm) were identified as MDR. Linezolid-resistant isolates (5%; MIC = 8 mg/L) comprised six poxtA-carrying Efm (cow milk), seven optrA-carrying Efs (chicken faeces/meat) and five Efm lacking cfr/optrA/poxtA (poultry/bovine/ovine/wastewater). Clinically relevant Efm clones (clade A1) were identified in animal/meat sources. Ampicillin resistance (1%) was confined to ST18/ST78-like MDR Efm clones from bovine meat/milk samples carrying relevant virulence markers (e.g. ptsD/IS16).Conclusions: This study provides evidence of the contribution of livestock and foodstuffs to the dispersal of acquired linezolid resistance genes including poxtA and optrA. We report the first poxtA-carrying Efm in Tunisia, and for the first time in bovine samples, stressing the urgent need for alternative measures to counteract the spread of linezolid-resistant enterococci globally.

Oxazolidinone resistance is a serious limitation in the treatment of MDR Enterococcus infections. Plasmid-mediated oxazolidinone resistance has been strongly linked to animals where the use of phenicols might co-select resistance to both antibiotic families. Our goal was to assess the diversity of genes conferring phenicol/oxazolidinone resistance among diverse enterococci and to characterize the optrA genetic environment. Chloramphenicol-resistant isolates (>16 mg/L, n = 245) from different sources (hospitals/healthy humans/wastewaters/animals) in Portugal, Angola and Tunisia (1996–2016) were selected. Phenicol (eight cat variants, fexA, fexB) or phenicol + oxazolidinone [cfr, cfr(B), optrA] resistance genes were searched for by PCR. Susceptibility (disc diffusion/microdilution), filter mating, stability of antibiotic resistance (500 bacterial generations), plasmid typing (S1-PFGE/hybridization), MLST and WGS (Illumina-HiSeq) were performed for optrA-positive isolates. Resistance to phenicols (n = 181, 74%) and phenicols + oxazolidinones (n = 2, 1%) was associated with the presence of cat(A-8) (40%, predominant in hospitals and swine), cat(A-7) (29%, predominant in poultry and healthy humans), cat(A-9) (2%), fexB (2%) and fexA + optrA (1%). fexA and optrA genes were co-located in a transferable plasmid (pAF379, 72 918 bp) of two ST86 MDR Tunisian Enterococcus faecalis (wastewaters) carrying several putative virulence genes. MICs of chloramphenicol, linezolid and tedizolid were stably maintained at 64, 4 and 1 mg/L, respectively. The chimeric pAF379 comprised relics of genetic elements from different Gram-positive bacteria and origins (human/porcine). To the best of our knowledge, we report the first detection of optrA in an African country (Tunisia) within a transferable mosaic plasmid of different origins. Its identification in isolates from environmental sources is worrisome and alerts for the need of a concerted global surveillance on the occurrence and spread of optrA.