Lazar Ranin

Evaluation of Phenotypic and Molecular Methods for Detection of Oxacillin Resistance in Members of the Staphylococcus sciuri Group

ABSTRACT In this paper we report on an experimental evaluation of phenotypic and molecular methods as means for the detection of oxacillin resistance in members of the Staphylococcus sciuri group. A total of 109 S. sciuri group member isolates (92 S. sciuri isolates, 9 S. lentus isolates, and 8 S. vitulinus isolates) were tested by the disk diffusion method, the agar dilution method, the oxacillin salt-agar screening method, slide latex agglutination for PBP 2a, and PCR assay for mecA as the reference method. The mecA gene was detected in 29 S. sciuri isolates, and the true-positive and true-negative results of the other tests were defined on the basis of the presence or the absence of the mecA gene. For the different methods evaluated, the sensitivities and specificities were as follows: for the disk diffusion test with a 1-μg oxacillin disk, 100% and 55.9%, respectively; for the disk diffusion test with a 30-μg cefoxitin disk, 93.5% and 100%, respectively; for the agar dilution method, 100% and 50%, respectively; for the oxacillin salt-agar screen test (with 6 μg of oxacillin per ml and 4% NaCl) 100% and 100%, respectively; and for the slide latex agglutination test for PBP 2a, 100% and 100%, respectively. The disk diffusion test with various β-lactam antibiotics was performed to evaluate their use for the prediction of oxacillin resistance. The results indicate that meropenem, cefazolin, cefamandole, cefuroxime, cefotetan, cefoperazone, cefotaxime, ceftriaxone, moxalactam, cefaclor, and cefprozil may be used as surrogate markers of oxacillin resistance, although further studies of their use for the detection of oxacillin resistance are required.

Molecular characterization of macrolide resistant Streptococcus pyogenes isolates from pharyngitis patients in Serbia.

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004-2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes--emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies.

Distribution of emm types among group A streptococcal isolates from Serbia

This is the first study concerning the molecular epidemiology of group A streptococcus in Serbia and includes 145 isolates from patients with various infections during the period 2001-2007. The emm types, superantigen profile and susceptibility pattern were determined. Among 31 emm types identified, the most prevalent were emm 6, emm 12, emm 1, and emm 58. All isolates showed uniform antimicrobial susceptibility to all tested antibiotics, with the exception of tetracycline and erythromycin (41% and 0.7% resistant strains, respectively). Significant heterogeneity of emm types was found, with a high frequency of emm 6 and emm 58, as well as a considerable prevalence of tetracycline resistance, and a low level of macrolide resistance.

Antimicrobial Activity of Human Follicular Fluids

The aim of this study was to explore the antimicrobial activity of human follicular fluid (HFF), to test the hypothesis that different strains of the same bacterial species could display different patterns of susceptibility to antimicrobial action of HFF, and to preliminarily investigate the possible mechanism of antimicrobial action of this fluid. Antimicrobial activity of 60 samples of HFF toward 30 Streptococcus agalactiae strains was determined by the agar diffusion method and broth dilution method. To explore the mechanism of antimicrobial activity, biochemical analyses were performed with selected fluid samples. The obtained results indicate that 38.3% fluid samples did not inhibit bacterial growth, 53.3% showed moderate and 8.3% high antimicrobial activity. The tested effect of HFF on S. agalactiae strains was bactericidal and was not strain dependent. Lysozyme activity was detected in HFF exhibiting antimicrobial activity. There were no statistically significant differences in concentrations of estradiol, progesterone, transferrin, iron, total protein and albumin levels among tested samples regardless of the different rate of antimicrobial activity. The obtained results indicate that lysozyme is most probably a crucial antibacterial agent in this fluid; however, some other still unidentified factors may contribute to it.

Molecular epidemiology of invasive and non-invasive group B Streptococcus circulating in Serbia

Streptococcus agalactiae (group B Streptococcus, GBS) remains the leading cause of invasive diseases in neonates and an important cause of infections in the elderly. The aim of this study was to access the prevalence of GBS genito-rectal colonisation of pregnant women and to evaluate the genetic characteristics of invasive and non-invasive GBS isolates recovered throughout Serbia. A total of 432 GBS isolates were tested for antimicrobial susceptibility, capsular polysaccharide (CPS) types and the presence of the hvgA gene. One hundred one randomly selected isolates were further characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) analysis and/or multilocus sequence typing (MLST). The prevalence of GBS colonization in pregnant women was 15%. Overall, six capsular types (Ia, Ib, II to V) were identified, the most common being III (32.2%) and V (25.2%). The hiper-virulent clone type III/ST17 was present in 43.1% and 6.3% (p < 0.05) of paediatric and adults isolates, respectively. Comparative sequence analysis of the CRISPR1 spacers content indicated that a few clones comprised the vast majority of the tested GBS isolates. Thus, it was estimated that dominant clones recovered from infants were CPS III/ST17 in late-onset infections (19/23; 82.6%), and Ia/ST23 in early-onset disease (44.4%). Conversely, genotype CPS V/ST1 was the most prevalent in adults (4/9; 25.4%). All isolates were susceptible to penicillin. Macrolide resistance (23.1%) was strongly associated with the ermB gene and constitutive resistance to clindamycin (63.9%). The majority of strains was resistant to tetracycline (86.6%), mostly mediated by the tetM gene (87.7%). GBS isolates of CPS V/ST1 and CPS III/ST23 were significantly associated with macrolide and tetracycline resistance, respectively. In conclusion, hyper-virulent CPS III/ST17 and V/ST1 were recognized as dominant GBS clones in this study.

Adherence and Biofilm Production of Streptococcus pyogenes

Streptococcus pyogenes (group A streptococcus – GAS) can cause numerous human infections, varying from mild skin infections to life‐threatening, e.g. necrotizing fasciitis. Adherence and biofilm production are important in streptoccocal pathogenesis. GAS adhesins are numerous and diverse, with the ability to bind to several different receptors at the same time, which leads to difficulties in their precise identification and classifi‐ cation. Biofilm production is one of the most probable explanation for therapeutic failure in the treatment of GAS infections. Most researchers agreed that biofilm formation is a trait of individual strains rather than a general serotype attribute. The aim of our study is to investigate differences in adherence to laminin and biofilm production between invasive and non‐invasive isolates (NI) of GAS. In this study the correlation between adherence to laminin and invasiveness in GAS isolates is noticed. The strains isolated from GAS carriers and highly invasive (HI) GAS strains have excellent capacity for binding to laminin. When testing biofilm production, there was noticeable positive correlation between adherence and biofilm production among non‐invasive isolates. Non‐invasive isolates were stable biofilm productors. There was no correlation between adherence and biofilm production among invasive isolates. Invasive isolates were also unstable biofilm productors.

Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin.

This study investigated the possible relationship between the invasiveness of group A Streptococcus (GAS) strains and their abilities to adhere to laminin and assessed the effects of subinhibitory concentrations of penicillin and erythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasive and highly invasive isolates of GAS to laminin was significantly higher than the adherence displayed by isolates of low invasiveness. Antibiotic treatment caused significant reductions in adherence to laminin in all three groups of strains. Penicillin was more successful in reducing the adherence abilities of the tested GAS strains than erythromycin.