The ability of biofilm formation seems to play an essential role in the virulence of coagulase-negative staphylococci (CNS). The present work aimed to: (a) evaluate the biofilm-forming ability of different strains of CNS field isolates; (b) evaluate their virulence potential through the assessment of the Madin-Darby canine kidney (MDCK) cytotoxicity assay; (c) determine the frequency of biofilm-associated genes among these CNS isolates. Biofilm markers associated with biofilm formation and MDCK cells cytotoxicity were compared to find possible associations with pathogenicity. CNS isolates (n = 94) belonging to 11 different species were tested for slime production using the tube test (TA) and the Congo red agar plate test (CRA), while the presence of icaA and icaD genes were evaluated by d-PCR. Two points were addressed for the first time: (1) the specific relationship between slime phenotype and icaD gene expression; (2) the specific relationship between slime phenotype, icaAD genes, and MDCK cytotoxicity. The proportion of biofilm-positive/icaD-positive versus biofilm-positive/icaD-negative strains was 9:0 and 9:0 (81.8%) by the TA and CRA, which clearly indicates that icaD was a more reliable gene to be accounted for in the biofilm formation. MDCK recorded a higher proportion than that recorded by the CRA and TA results (MDCK-positive/icaD-positive versus MDCK-positive/icaD-negative 10:0, 90.9%). Evaluation of the ica operon, CRA plate test, TA, and MDCK can contribute to the high clinical impact in the management of antibiotic therapy, in infections associated with devices in veterinary medicine, the dairy industry, and food processing.

Aim::This study was devoted to elucidate the tetracycline resistance of coagulase-negative staphylococci (CNS) derived from normal and subclinical mastitic (SCM) buffaloes’ milk in Egypt.Materials and Methods: ::A total of 81 milk samples from 46 normal buffalo milk samples and 35 SCM buffalo milk samples at private dairy farms of Egypt were used in this study. CNS were identified using phenotypic and molecular methods (polymerase chain reaction [PCR]). CNS isolates were tested for tetracycline resistance using routine methods and multiplex PCR targeting tetracycline (tet) resistance genes followed by sequencing of positive PCR products and phylogenetic analysis.Results::Isolation and identification of 28 (34.5%) CNS from normal and SCM buffaloes’ milk, namely, Staphylococcus intermedius (39.2%), Staphylococcus xylosus (25.0%), Staphylococcus epidermidis (10.7%), Staphylococcus hominis (10.7%), and 3.5% to each of Staphylococcus sciuri, Staphylococcus hyicus, Staphylococcus lugdunensis, and Staphylococcus simulans. Using nested PCR, all the 28 CNS isolates revealed positive for 16srRNA gene specific for genus staphylococci and negative for thermonuclease (nuc) gene specific for Staphylococcus aureus species. The presence of tetracycline resistance-encoding genes (tetK, tetL, tetM, and tetO) was detected by multiplex PCR. All isolates were negative for tetL, M, and O genes while 14 (50%) CNS isolates were positive for tetK gene, namely, S. lugdunensis (100%), S. hominis (100%), S. epidermidis (66.6%), S. intermedius (45.4%), and S. xylosus (42.8%). Nucleotide sequencing of tetK gene followed by phylogenetic analysis showed the high homology between our CNS isolates genes of tetracycline resistance with S. aureus isolates including Egyptian ones. This proves the transfer of the tetracycline resistance encoding genes between coagulase-negative and coagulase positive Staphylococcus spp.Conclusion::CNS isolates have distinguishingly high resistance to tetracycline. Abundant tetracycline usage for mastitis treatment leads to the spread of genetic resistance mechanisms inside CNS strains and among all Staphylococcus spp. Consequently, tetracycline is not effective anymore.

The aim of this study was to examine the genetically mediated antimicrobial resistance in 94 coagulase-negative staphylococcal (CNS) milk isolates (buffalo, n = 88, and cow, n = 6), and to determine whether antimicrobial resistance profiles differed between bacterial species. Our analysis of 94 CNS isolates from milk confirmed the well-established multiresistant character of staphylococci in the dairy setting. Resistance against oxacillin, ciprofloxacin and cefoxitin was most frequently observed. Eleven CNS species isolated from buffalo's and cow's milk samples were 100% sensitive to gentamicin, erythromycin, clindamycin and ciprofloxacin. Resistance to oxacillin was attributed to the mecA gene in 44.7% of the oxacillin-resistant isolates. The mecA gene was detected in Staphylococcus intermedius, epidermidis, hominis, hyicus, caprae, sciuri, lugdunensis and xylosus while totally absent in chromogenes, simulans and lentus. Of the 11 CNS species, S. epidermidis, S. lugdunensis, S. hominis, S. xylosus and S. intermedius were the only species that exhibited multiple resistance.

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