Molecular Typing, Characterization and Pathogenicity of Clinical Multi-Drug Resistant Staphylococcus aureus isolates

Abstract

Staphylococcus aureus (S. aureus) is one of the leading causes of community and hospital-acquired infections globally. These bacteria have adapted additional resistance mechanisms against various commonly used antibiotics. The increase in resistance to multiple antibiotics by S. aureus presents a major global problem. The objectives of the study were to assess the prevalence, study the carriage of antibiotic resistance genes and evaluate the molecular typing of S. aureus isolates from a tertiary care hospital in Islamabad. In this study, 1528 staphylococci isolates were collected from tertiary care hospital, Pakistan Institute of Medical Sciences (PIMS) Islamabad. Initial confirmation was done by Gram staining and observing growth characteristics on mannitol salt agar, while further conventional biochemical tests, such as catalase, DNase, and tube coagulase were performed to confirm S. aureus. Molecular identification of S. aureus isolates was done using 16S rRNA and nuclease (nuc) gene amplification through polymerase chain reaction (PCR). These isolates were from different specimens including pus, urine, blood, and tracheal secretions, etc. Patient demographic details such as specimen, age, gender, and ward were also recorded. Antibiotic susceptibility was evaluated using Kirby-Bauer disk diffusion method and minimum inhibitory concentration (MICs) by microbroth dilution method following Clinical and Laboratory Standards Institute (CLSI) guidelines. Cefoxitin disks of 30µg (Oxoid, UK) were used to differentiate between methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates. The Double Disk Diffusion test (D-test) was performed to detect inducible clindamycin resistance. Screening of antibiotic resistance genes (ARGs) was performed using multiplex PCR. A molecular typing of study isolates was accomplished by accessory gene regulator (agr) typing, hypervariable (HVR) typing, staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing, multi locus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). The hemolysin assay was carried out for selected isolates; other virulence and regulatory factors were evaluated through multiplex PCR. Representative isolates were selected xi for expression analysis through Real-time quantitative PCR (RT-qPCR). Intracellular survival and pathogenicity of selected S. aureus strains were analyzed using in vitro Raw 264.7 cells and the in-vivo Galleria mellonella infection model. Chi-square test was applied to find correlation among phenotypic antibiotic resistance and their respective ARGs, and the association of Panton-Valentine leucocidin (PVL) toxin with SCCmec types. From a total of 1528 staphylococci isolates, 485 were characterized as S. aureus based on standard microbiological procedures. Of total 485 isolates, based on cefoxitin resistance 65% of isolates were confirmed as MRSA and 35% were MSSA. For this study, 250 isolates (200 MRSA and 50 MSSA) were further analyzed. Antibiotic susceptibility results showed that S. aureus isolates were highly resistant to clindamycin (69%), ciprofloxacin (68%), and cefoxitin (65%), followed by gentamicin (46%), erythromycin (45%), tetracycline (31%), while with lower resistance to sulfamethoxazole/trimethoprim (21%), rifampicin (15%), chloramphenicol (7%) and for linezolid and quinupristin/dalfopristin (3% each). MRSA isolates were highly resistant to ciprofloxacin (85%), and gentamicin (64%). Resistance against erythromycin was 50%, tetracycline was 36%, sulfamethoxazole/trimethoprim and clindamycin resistance level were at 26% each while rifampicin was recorded as 20%. Among MRSA isolates, high levels of susceptibility were found against linezolid (96%), Quinupristin/dalfopristin (95%), and chloramphenicol (88%). Antibiotic susceptibility results showed that MSSA isolates were highly resistant to ciprofloxacin (80%), tetracycline (72%), and to erythromycin, sulfamethoxazole/trimethoprim, and gentamicin (32% each). Low resistance was noticed to clindamycin (12%) and rifampicin (20%). All MSSA isolates were sensitive to cefoxitin, chloramphenicol, quinupristin/dalfopristin, and linezolid. Clindamycin inducible resistance was observed in 4% of isolates. In all isolates, 85% were recorded as MDR, within MRSA 83% were confirmed as MDR MRSA, while in MSSA 20% were MDR-MSSA. MICs against vancomycin confirmed that 5% were vancomycin-intermediate S. aureus (VISA), and 28% MRSA isolates were fosfomycin resistant. Molecular detection of ARGs showed xii that in MRSA isolates, mec(C) gene had a low prevalence of 3%, and only 1 MRSA had both mec(A) and mec(C) genes in combination. Clinical MRSA carrying mec(C) alone and mec(A)+mec(C) in combination was also detected for the first time from Pakistan. Prevalence of ARGs in MRSA was recorded as 80% for dfr(B), 87% tetK, 81% tetM, 54% mec(A), 3% mec(C), 100% bla(Z), 95% gyr(A), 33% gyr(B), 9% grl(A), 6% grl(B), 80% rpo(B), 75% aacphD1, 13% erm(A), 81% fos(A), 56% vat(C), 29% vat(B), 4% vat(A), and 100% erm(C). While the prevalence of ARGs in MSSA were noticed as 75% dfr(B), 31% tetK, 52% gyr(A), 67% rpo(B), 75% aacphD1, 75% erm(A), 50% vat(C), 2% vat(B), and 100% erm(C). In MRSA isolates agrI was the most prevalent group (22%), while in MSSA agrIII (16%) was the dominant group. Among SCCmec types, SCCmec III was the most prevalent (86%) one. Other prevalent mec types included type I (14%), II (23%), IV (40%), and VI (23%), while 7.1% were non-typeable (NT), whereas type V was not detected in any of the isolates. The study reports SCCmec type VI that has not been reported previously from Pakistan. The PVL toxin was observed in 24% MRSA and 10% in MSSA of the study isolates with distribution in all prevalent SCCmec types but was strongly associated with types VI, IV and II. In MRSA isolates, 42% were found to be positive for the HVR whereas 58% of isolates did not reveal the presence of HVR. In MRSA isolates 5 HVR types were observed, the most dominant HVR type was type 6 (60%), followed by type 5 (15%), type 7 (10%), type 3 (10%), and type 4 (5%). In the present study, a total of 50 isolates (25 MRSA and 25 MSSA) were processed for spa typing, 10 different spa types were found in MRSA with t657 (24%) as the dominant spa type while 15 spa types were found in MSSA with t021 (24%) with the highest prevalence. Among these, four spa types (t657, t314, t632, and t5598) were common among MRSA and MSSA. One NEW spa type was identified in MSSA isolates. The current study advances information of spa types of local clinical S. aureus isolates as out of 21 spa types in this study, 17 are reported first time from Pakistan. A subset of 5 representative isolates (n=4 MRSA and n=1 MSSA) were further processed for MLST and PFGE analysis. Combining MLST and spa typing, xiii three genotypic patterns were identified; ST772-t657, ST1-t127 in MRSA, and ST1535-t3214 in MSSA. Among multi-locus sequence types, the MSSA with ST1535-t3214 has not been previously reported from Pakistan. Clonal analysis of representative isolates showed that two isolates were of same clone (100% similarity), 2 isolates showed 85% similarity and one isolate had 75% similarity index. The alpha-hemolysin (45%, 60%) was more dominant as compared to beta hemolysin (28%, 28%) and gamma-hemolysin (27%, 12%) in MRSA, and MSSA respectively. Among MRSA, alpha-hemolysin gene (hla) was detected in 80% of phenotypically positive isolates, followed by 65% for beta-hemolysin (hlb) and 58% were positive for gamma-hemolysin gene (hlg), and hld gene was found in 3.2% isolates. Prevalence of virulence and regulatory factors in MRSA isolates were recorded as 58%, 86%, 87%, 86%, 55%, 57%, 52%, 68%, 74%, 78%, 10%, 5%, 2%, 79% and 24%, for sigB, codY, fnbA, fnbB, clfA, saeS, sarA, arlS, yycG, spoVG, sea, seb, sec, rot, and PVL genes respectively. The percentages were 62%, 84%, 86%, 82%, 52%, 56%, 50%, 64%, 72%, 70%, 6%, 4%, 4%, 76% and 10%, for sigB, codY, fnbA, fnbB, clfA, saeS, sarA, arlS, yycG, spoVG, sea, seb, sec, rot and PVL genes respectively in MSSA isolates. Similar virulence factors were observed both in MRSA and MSSA. Macrophages induced intracellular killing effect was not observed after 20 hours of incubation for agr groups II, III, and agr mutant strain RN4220. Survival assay in G. mellonella infection model showed that HA-MRSA was more virulent than VISA, CA-MRSA, and MSSA. Hemocyte density experiment demonstrated that the CA-MRSA provoked a more pronounced immune response