Investigation of biofilm formation and antibiotic resistance in Streptococcus pyogenes strains isolated from clinical samples

Streptococcus pyogenes (Group A streptococcus, GAS) is an important bacterial pathogen that can cause a wide range of clinical conditions, particularly in the pediatric population, including pharyngitis, tonsillitis, and skin and soft tissue infections. The pathogenic potential of GAS depends on the combined effects of multiple virulence components, such as surface proteins, DNases, superantigens, and adhesion-related factors. As the distribution of these virulence factors may vary considerably among strains, the generation of regional molecular epidemiological data is important for clinical management and antimicrobial resistance surveillance. The aim of this study was to investigate the antimicrobial resistance profiles, biofilm-forming capacities, and the presence of various virulence factor genes, including prtF1, prtF2, speA, speB, speC, and DNase genes, in GAS strains isolated from clinical specimens. A total of 100 GAS strains were included in the study. Phenotypic biofilm formation was evaluated using the microtiter plate method, and the presence of virulence factor and antibiotic resistance genes was investigated by in-house PCR. Of the isolates, 64 were obtained from upper respiratory tract (URT) samples and 36 from non-upper respiratory tract (non-URT) samples. It was determined that %87.5 of the patients from whom the isolates were obtained were in the 5-15 age group. Phenotypically, all strains were susceptible to penicillin. Erythromycin, clindamycin, and tetracycline resistance rates were %6, %1, and %9, respectively. Phenotypic analysis revealed inducible clindamycin resistance (iMLSB) in two strains, constitutive MLSB (cMLSB) in one strain, and the M phenotype in three strains. At least one of the ermB, ermTR, or mefA genes was detected genotypically in eight (%12.5) of the 64 URT isolates and in one (%2.8) of the 36 non-URT isolates. All six phenotypically erythromycin-resistant isolates carried a macrolide resistance gene, while macrolide resistance genes were also detected in three phenotypically erythromycin-susceptible isolates. The ermB gene was detected in the single isolate showing phenotypic resistance to clindamycin. At least one tetracycline resistance gene was detected in 10 (%15.6) URT isolates and in two (%5.6) non-URT isolates, with tetM being the most frequently identified gene. No statistically significant difference was observed between URT and non-URT isolates in terms of antimicrobial resistance rates. Overall, %71 of the isolates were found to produce biofilm. Biofilm formation was detected in 49 (%76.5) URT isolates and 22 (%61.1) non-URT isolates. Strong biofilm production was observed in 24 (%37.5) URT isolates and 11 (%30.6) non-URT isolates, resulting in a total of 35 isolates. No statistically significant difference was found between URT and non-URT isolates with respect to biofilm positivity or biofilm intensity. The prtF1 gene was detected in 37 (%57.8) URT isolates and 21 (%58.3) non-URT isolates, resulting in a total of 58 isolates, whereas the prtF2 gene was detected in 25 (%39.1) URT isolates and 25 (%69.4) non-URT isolates, for a total of 50 isolates. The positivity rate of prtF2 was significantly higher in non-URT isolates than in URT isolates (p=0.007). No statistically significant differences were observed between prtF1 or prtF2 positivity and biofilm positivity or biofilm levels. prtF1 positivity was significantly higher in strains showing phenotypic and genotypic erythromycin resistance and genotypic tetracycline resistance compared to susceptible strains (p=0.038, p=0.007, and p=0.012, respectively). In addition, prtF2 positivity was significantly higher in phenotypically tetracycline-resistant strains than in susceptible strains (p=0.036). Among the DNase genes, sdaB was detected in all isolates. The positivity rates of sdc, sdaD, and spd3 genes in URT isolates were %29.7, %7.8, and %85.9, respectively, while the corresponding rates in non-URT isolates were %30.6, %16.7, and %66.7. The spd3 gene was detected at a significantly higher rate in URT isolates compared to non-URT isolates (p=0.023). No statistically significant differences were found between DNase gene positivity and antimicrobial resistance or biofilm positivity (p>0.05). The positivity rates of speB, speC, and speA genes were %82.8, %60.9, and %14 in URT isolates, and %86.1, %66.7, and %13.9 in non-URT isolates, respectively. When evaluated together, at least one of these genes was detected in %91 of the isolates. No statistically significant differences were observed between URT and non-URT isolates regarding spe gene positivity rates (p>0.05). Similarly, no significant differences were found between spe gene positivity and biofilm positivity or biofilm levels (p>0.05). In conclusion, this study provides comprehensive data on antimicrobial resistance profiles, biofilm-forming capacities, and the distribution of various virulence genes among GAS isolates obtained from different clinical specimens. The high rate of biofilm production observed in this study highlights its potential role as an important virulence factor contributing to bacterial persistence in host tissues. Although antimicrobial resistance rates were low, the detection of resistance genes in a small number of phenotypically susceptible isolates indicates the need for caution regarding silent gene regions or differences in gene expression. Future studies incorporating larger sample sizes, multicenter designs, clinical data, and gene expression analyses may contribute to a better understanding of the pathogenesis and resistance dynamics of S. pyogenes. Keywords: Streptococcus pyogenes, antimicrobial resistance, biofilm, virulence factors