The aim of this study was to investigate the presence of tetracycline resistance in lactobacilli isolated from traditional Serbian white brined raw milk cheeses (Homolje, Sjenica, Zlatar). Isolation of presumptive lactobacilli was initially performed using MRS-S agar without tetracycline, or supplemented with 16 and 64 µg/mL of tetracycline. Rep-PCR (GTG) genotyping showed a high diversity of the isolates obtained, as examination of 233 isolates resulted in 156 different Rep-PCR fingerprints. Ninety out of 156 (57.69%) of the strains, representatives with different (GTG) fingerprints, were identified by MALDI-TOF MS as lactobacilli, while 66 out of 156 (42.31%) strains were identified as members of other LAB genera. All except one out of 90 isolates further tested by microdilution method, demonstrated unimodal distribution of tetracycline MIC values which were equal to or lower from the breakpoint MIC values (EFSA in EFSA J 10: 1-10, 2012. 10.2903/j.efsa.2012.2740). Only one isolate showed the presence of (M) gene, while the other analyzed genes [(A)(B)(C) (K),(L), (O) and(W)] were not detected in any of the isolates. The results of this study indicates that lactobacilli from traditional Serbian raw milk cheeses do not present considerable tetracycline resistance reservoirs. For final conclusions about the safety of these autochthonous cheeses regarding the possible tetracycline resistance transferability, the assessment of the entire cheese microbiota is needed.

This study was undertaken to determine the prevalence of coagulase positive staphylococci (CPS) by examining a total of 71 raw milk cheeses. Additionally, enterotoxigenicity, antimicrobial susceptibility and the presence of mecA and mecC genes in the staphylococcal isolates were investigated. The isolation and enumeration procedure of CPS followed the International Organization for Standardization (ISO) standard. The presumptive staphylococci were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the VITEK MS system. VIDAS® Staph enterotoxin II assay was used for the detection of classical enterotoxins. Antimicrobial susceptibility testing (AST) was accomplished performing the disk diffusion method. All suspected methicillin resistant staphylococci were investigated for the presence of mecA and mecC genes by PCR assay. A high prevalence (87.32%) of CPS was detected in the cheeses at contamination levels up to 5.58 log CFU g. Among 47 staphylococcal isolates screened for enterotoxin production, only one isolate, identified as S. hyicus, was confirmed as being enterotoxigenic. Resistance to penicillin (63.70%) was the most common resistance among the tested Staphylococcus aureus isolates. The dominant phenotypic resistance patterns in coagulase negative staphylococci (CNS) were resistance to ofloxacin and fusidic acid. All CNS isolates were susceptible to the clinically important antibiotics clindamycin, chloramphenicol, gentamicin, linezolid, rifampicin and trimethoprim-sulfamethoxazole. The mecA and mecC genes were not detected. To the best of our knowledge, this is the first study concerning evaluation of the presence of methicillin resistant staphylococci (MRS) in dairy products in Serbia.

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The objective of the study was to determine the frequency of spontaneous acquisition of resistance to select antibiotics by Salmonella Typhimurium (ST) when grown in glucose amended continuous flow culture at slow (D = 0.025 h(-1)) or fast (D = 0.27 h(-1)) dilution rates. The bacterium was grown in LB minimal medium (pH 6.25) containing no antibiotics. Upon achieving steady state, samples were plated to tryptic soy agar (TSA) alone or supplemented (per ml) with 2 and 16 microg oxytetracycline, 4 and 16 microg tetracycline, 2 and 64 microg kanamycin, and 0.25 and 2 microg enrofloxacin. Regardless of growth rate, CFU of resistant ST from the TSA containing antibiotics was less than 2 x 10(1) except for 2 microg kanamycin and 0.25 microg enrofloxacin treatments (higher than 1 x 10(9) and 4 x 10(7) CFU of resistant ST for trials 1 and 2, respectively). Frequency of recovering resistant ST from the TSA containing the higher antibiotic concentrations was less than 1 in 10(9) for all antibiotics, but was higher on the media containing 2 microg kanamycin and 0.25 microg enrofloxacin at both slow and fast growth rates. In general, minimal susceptibility differences were detected for isolates from slow and fast dilution rates.

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