Background: Infections due to methicillin resistant Staphylococcus aureus (MRSA) which is the most virulent species among the staphylococci have become a global health challenge. The aim of this study was to assess the correlation of genes encoding virulence and methicillin resistance in invasive and non-invasive isolates from inpatients/outpatients with staphylococcal infections in Sofia, Bulgaria. Materials and methods: Non-duplicate S. aureus isolates were recovered from clinical samples obtained from a total of 368 in-patients with healthcare-associated infections and outpatients with community acquired infections, following overnight cultures of samples on Columbia agar with 5% sheep blood at 35°C. The isolates were presumptively identified by colony and Gram stain morphology, positive catalase reaction and plasma-coagulase test. Isolates were screened for methicillin resistance by the cefoxitin disk method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) protocol. The mecA and mecC, and 12 staphylococcal virulence genes were detected by a combination of monoplex and multiplex polymerase chain reaction (PCR) assays. Results: The prevalence of MRSA based on carriage of mecA gene was 12%; 7.7% for outpatients and 16.2% for inpatients (p<0.05). The frequency of toxin genes detection in the staphylococcal isolates were as follows; sei (72.6%), seb (59.8%), seh (41.3%), sec (38.3%), seg (37.5%), sej (32.3%), sea (26.6%), sed (10.3%), tst (6.5%), and see (4.3%). The virulence genes, tst, sea, seb, sec, seg, seh and sei were more frequently associated with MRSA than methicillin sensitive (MSSA) strains (p<0.05). About one-third of the clinical S. aureus isolates harbored seven virulence genes; sea, seb, sec, see, seg, seh and sei, that were detected significantly more among the invasive isolates (p<0.05). Conclusions: This study shows the occurrence of highly virulent staphylococcal isolates in our geographical region.

Background Severe infections of virulent methicillin-resistant Staphylococcus aureus (MRSA) are a serious health problem. The present study aimed to investigate clonal spread, virulence and antimicrobial resistance rates of Bulgarian MRSA isolates in 2016–2020. Methods Molecular identification and mecA gene detection were performed with PCR. Clonal relatedness was evaluated by RAPD PCR and MLST. MRSA epidemiology, virulence and resistance patterns were investigated by PCR. Results All 27 isolates were identified as S. aureus and were mecA positive, and all were susceptible to linezolid, tigecycline and vancomycin. The toxin genes hlg (in 92.6% of isolates), seb (77.8%), sei (77.8%), seh (59.3%), sej (55.6%), and seg (48.1%), were frequently found among the isolates. Epidemiological typing by RAPD identified 4 clones (16 isolates) and 11 were with a unique profile. MLST analysis of the same MRSA isolates showed five MLST clonal complexes and 11 ST types, including CC5 (33.3%) (ST5, ST221, ST4776), CC8 (22.2%) (ST8, ST239, ST72), CC15 (ST582), CC22 (14.8%) (ST217, ST5417), CC30 (ST30) CC398 (ST398), and CC59 (ST59). The isolates from CC5 showed higher virulence potential and almost all were macrolide resistant (ermB or ermC positive). CC8 isolates showed higher level of resistance. Conclusion To the best of our knowledge, this study is the first describing the clonal spreading of Bulgarian MRSA and the association with their virulence and resistance determinants. Monitoring of MRSA epidemiology, resistance and virulence profile can lead to better prevention and faster therapeutic choice in cases of severe infections.

The aim of this study was to investigate the rate of resistance to macrolide-lincosamide-streptogramin B (MLSB) antibiotics, the mechanisms underlying this resistance and to evaluate their relationship with virulence genes profiles of 435 Bulgarian clinical isolates Staphylococcus aureus. The highest resistance was observed to penicillin (96.09%), followed by resistance to erythromycin and clindamycin (34.02 and 22.76%, respectively). Of the tested clinical strains of S. aureus, 96.09% contained the blaZ gene associated with penicillin resistance and 11.03%, the mecA gene responsible for methicillin resistance. The most prevalent were the erm genotypes associated with the presence mainly of ermA and ermC genes followed by ermB. The frequency rates of these genes, alone or in combinations were ermA 41.89%, ermB 27.70%, ermC 43.99%. The majority of Bulgarian macrolide resistant S. aureus exhibited cMLS phenotype, in 58.78% (P = 0.0036). The following virulence genotypes were present significantly more often in the macrolide resistant S. aureus isolates among the studied ones: hlg; hlg,seb; hlg,seb,sec; hlg,seb,seh; hlg,sec; hlg,sec,sei; hlg,sec,sei; hlg,sei; hlg,sei,sej; hlg,sej. This survey found correlation between the virulence profiles with a small number of genes and macrolide resistance among Bulgarian clinical S. aureus isolates, in contrast to sensitive strains, which possessed profiles predominantly with multiple genes.

Citation: Gergova RT, Tsitou VMS, Mitov IG. Molecular-genetic method for fast direct detection of Staphylococcus aureus and methicillin resistance in blood cultures and punctures. Folia Med (Plovdiv) 2019;61(4):559-65. AbstractBackground: Invasive infections caused by methicillin resistant Staphylococcus aureus and coagulase-negative staphylococci (MRSA/ MRSCoN) require fast, adequate treatment.The aim of this study was to develop a faster protocol for direct detection of MRSA/MRSCoN in blood cultures and in abscess punctures based on mecA and species specific identification of S. aureus by polymerase-chain reaction (PCR). Materials and methods:We examined 77 growth-positive BACTEC blood cultures and 50 abscess punctures by routine microbiological assay and simultaneous PCR detection of MRSA/MRSCoN. The specificity of the PCR was evaluated by using DNA from another 15 microbial species for negative controls. We determined the minimum inhibitory concentration (MIC) of oxacillin, vancomycin, tigecycline, linezolid, levofloxacin, clindamycin, and erythromycin against the S. aureus isolates using the E-test. Results:In the blood cultures, the two methods detected 39.3% of MRSA, and 93.9% of MRCoNS. In the punctures, the PCR assay identified 20.9% of MRSA and 79.2% of MSSA. In the puncture cases, there were three PCR MRSA positive and culture negative samples. Screening for susceptibility to 14 antimicrobial agents demonstrated significantly higher (p<0.05) methicillin resistance in blood culture isolates than in the puncture ones (39.3% and 20.0%, respectively). Conclusion:The new PCR protocol was very fast and specific. It was more sensitive in detecting MRSA from abscess punctures than the routine microbiological techniques. This protocol will speed up the right choice of empirical therapy, which is extremely important for saving patients' lives.