Fluoroquinolone-resistant and extended-spectrum beta-lactamase producing Escherichia coli isolates from free-living wild animals.

During the hunting season 2013-2014, fecal samples collected from hare, roe deer, deer and wild boars were sent to the bacteriology laboratory for the isolation of Escherichia coli and multidrug resistant isolates were characterized phenotypically and genotypically. Out of 106 fecal samples, E. coli was isolated from 101 samples. Although the majority of isolates belonged to phylogenetic groups A and B1, 14 out of 101 isolates were affiliated to group B2. A multidrug resistance phenotype was determined in 7 isolates, all of which had distinguishable genomic macrorestriction profiles. PCR analysis and sequencing revealed a variety of resistance genes, gene cassettes and cassette arrays in these multidrug resistant isolates. Resistance to fluoroquinolones was found in five E. coli isolates (two from a roe deer, one from a deer and two from a wild boar) and multiple mutations in the chromosomal topoisomerase genes were identified. In an E.coli isolate from a hare, the qnrB19 gene was detected. The same isolate carried an aadA23 gene cassette in class 1 integron. In addition, an extended- spectrum beta-lactamase blaCTX-M-1 gene was detected in an E. coli isolate from a roe deer. The gene was located on a conjugative multi resistance plasmid, which was transferable to a plasmid free E. coli recipient. In conclusion, a number of resistance genes and mobile genetic elements were detected in E. coli isolates from wildlife in Vojvodina, emphasizing the role of environmental pollution in spreading resistant bacteria.

Changes in Macrolide Resistance Among Group A Streptococci in Serbia and Clonal Evolution of Resistant Isolates.

In Serbia, the frequency of macrolide-resistant group A streptococci (MRGASs) increased significantly from 2006 to 2009. MRGAS analysis in 2008 revealed the presence of three major clonal lineages: emm75/mefA, emm12/mefA, and emm77/ermTR. The aim of the present study was to determine the prevalence of macrolide resistance and to evaluate variations in the clonal composition of MRGASs. The study included 1,040 pharyngeal group A streptococci collected throughout Serbia, which were tested for antimicrobial susceptibility. MRGAS isolates were further characterized by the presence of resistance determinants, emm typing, and pulsed-field gel electrophoresis analysis. The prevalence of macrolide resistance was 9.6%, showing a slight decrease compared with the rate of 12.5% (2008). Tetracycline resistance was present in 6% of isolates, while norfloxacin nonsusceptibility detected for the first time in Serbia was 9.8%. The M phenotype dominated (84%), followed by the constitutive macrolides, lincosamides, and streptogramin B phenotype (12%). Five emm types were detected: emm75, emm12, emm1, emm28, and emm89. The emm75/mefA (62%), emm12/mefA (14%), and emm12/ermB/tetM (6%) were predominant clones and were found in both the present and the previous study periods at different frequencies. The major change was the loss of emm77/ermTR/tetO, which contributed to 15% of MRGASs in 2008.

Molecular Characterization of Multidrug-Resistant Escherichia coli Isolates from Bovine Clinical Mastitis and Pigs in the Vojvodina Province, Serbia.

The aim of the study was to characterize multidrug-resistant (MDR) Escherichia coli isolates collected in Serbia from bovine clinical mastitis cases and diseased pigs, mainly with molecular methods. A total of 48 E. coli isolates was collected during the years 2013-2014, of which 22 were MDR and were included in further analysis. Phylogenetic typing showed that 17 isolates belonged to group A, while two isolates were classified in group B1 and a single one in group D. All isolates showed unique macrorestriction patterns. Phenotypic susceptibility testing revealed resistances of the isolates against up to 13 antimicrobial agents, including resistance to fluoroquinolones. A wide variety of resistance genes was detected by PCR amplification and sequencing of amplicons. Sequence analysis of the quinolone resistance determining regions of topoisomerase genes revealed mutations in gyrA, parC, and/or parE. Plasmid-mediated quinolone resistance genes were detected in two porcine (aac-6'-Ib-cr and qnrS, respectively) isolates and a single bovine (aac-6'-Ib-cr) isolate. Resistance genes were found to be located on conjugative plasmids in 16 cases, many of which conferred a multidrug resistance phenotype. In conclusion, the plentitude of resistance genes located on conjugative plasmids and integrons in E. coli from cows and pigs in Vojvodina, Serbia, pose a high risk for horizontal gene transfer in bacteria from livestock husbandry.

Prevalence of macrolide resistance in Streptococcus pyogenes collected in Serbia.

The purpose of our study was to determine the prevalence of macrolide resistance in 3,188 pharyngeal Streptococcus pyogenes isolates collected at the Institute of Public Health of Serbia during the period 2006-2008. The disk diffusion tests were used to determine the susceptibility of the isolates. Two hundred and sixteen S. pyogenes isolates (6.8%) were resistant to erythromycin, with 9 isolates coresistant to tetracycline: 181 isolates harbored mefA gene, 19 ermB gene, 11 ermA(TR) gene, 5 ermB and mefA genes, and 7 tetM gene. Although the prevalence of macrolide resistance in pharyngeal S. pyogenes isolates is low in Serbia, monitoring of the emergence of resistance is advisable.

Improved serodiagnosis of early Lyme borreliosis: immunoblot with local Borrelia afzelii strain.

To improve the serodiagnosis of Lyme borreliosis (LB) the performances of four tests were evaluated. An indirect immunofluorescent assay based on Borrelia burgdorferi s.s., enzyme-linked immunosorbent assay (ELISA) based on local isolates of Borrelia afzelii and B. burgdorferi s.s., and immunoblot (IB) of B. afzelii were prepared. The serum panels contained 214 serum samples: control group (n=120) and patients at different stages of LB (n=94). The specificity of IB was 96%, of in-house ELISA 93%, and of IFA 89%. In early LB the sensitivity of IFA was 36%, ELISA 67%, and IB 93%. In late-stage LB the sensitivity was: 72% for IFA, 80% for ELISA, and 94% for IB. Comparison of in-house and Behring ELISA showed that the sensitivity of the serological assay could be increased when the test was based on local Borrelia strains. IgM and IgG antibodies from sera of patients with early and late LB most frequently demonstrated reactivity to OspC. The other significant proteins in early LB were: p39, p41 in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB: p39, p41 in IgM IB, and p83/100, Osp17, p21 and p43 in IgG IB. Using IB based on local B. afzelii isolates improves the serodiagnosis of early LB in our geographical region.