Molecular Epidemiology of Bulgarian Clinically Significant Staphylococcus aureus Isolates.

Severe infections due to highly virulent and resistant Staphylococcus aureus pose a serious health threat in Bulgaria and worldwide. The purpose of this study was to explore the clonal spread of recent clinically significant methicillin-susceptible S. aureus (MSSA) isolates from inpatients and outpatients treated in three university hospitals in Sofia, Bulgaria, during the period 2016-2020 and evaluate the relationship between their molecular epidemiology, virulence profiling, and antimicrobial resistance. A total of 85 isolates (invasive and noninvasive) were studied using RAPD analysis. Ten major clusters (A-K) were identified. The first major cluster A (31.8%) was found to be predominant during 2016 and 2017 and was widespread in two hospitals, unlike its case in the following years, when it was found to be replaced by newer cluster groups. All MSSA members of the second most common cluster F (11.8%) were recovered from the Military Medical Academy, mainly during 2018-2020, and were determined to be susceptible to all other groups of antimicrobials, except for penicillins without inhibitors because they harboured the blaZ gene. The newer cluster I, with 9.4% of the isolates absent in 2016-2017, showed significantly higher virulence and macrolide resistance (42.9%) due to ermB and ermC. All the isolated MSSA in groups F and I were nosocomial and mostly invasive. In conclusion, this 5-year study demonstrates the molecular epidemiology of MSSA infections in three Bulgarian hospitals. Findings can be helpful for the understanding of staphylococcal infection distribution in hospital settings and their prevention.

Molecular epidemiology, virulence and antimicrobial resistance of Bulgarian methicillin resistant Staphylococcus aureus isolates.

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.

Relationship between MLSB resistance and the prevalent virulence genotypes among Bulgarian Staphylococcus aureus isolates.

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.

Molecular-genetic Method for Fast Direct Detection of Staphylococcus Aureus and Methicillin Resistance in Blood Cultures and Punctures.

BACKGROUND: 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.