Staphylococcus aureus, a notorious human pathogen, is a major cause of the community as well as healthcare associated infections. It can cause a diversity of recalcitrant infections mainly due to the acquisition of resistance to multiple drugs, its diverse range of virulence factors, and the ability to produce biofilm in indwelling medical devices. Such biofilm associated chronic infections often lead to increase in morbidity and mortality posing a high socio-economic burden, especially in developing countries. Since biofilm formation and antibiotic resistance function dependent on each other, detection of biofilm expression in clinical isolates would be advantageous in treatment decision. In this premise, we attempt to investigate the biofilm formation and its association with antibiotic resistance in clinical isolates from the patients visiting tertiary health care hospitals in Nepal. Bacterial cells isolated from clinical samples identified as S. aureus were examined for in-vitro biofilm production using both phenotypic and genotypic assays. The S. aureus isolates were also examined for susceptibility patterns of clinically relevant antibiotics as well as inducible clindamycin resistance using standard microbiological techniques and D-test, respectively. Among 161 S. aureus isolates, 131 (81.4%) were methicillin resistant S. aureus (MRSA) and 30 (18.6%) were methicillin sensitive S. aureus (MSSA) strains. Although a majority of MRSA strains (69.6%) showed inducible clindamycin resistance, almost all isolates (97% and 94%) were sensitive toward chloramphenicol and tetracycline, respectively. Detection of in vitro production of biofilm revealed the association of biofilm with methicillin as well as inducible clindamycin resistance among the clinical S. aureus isolates.

BackgroundPersister cells comprise a phenotypic variant that shows extreme antibiotic tolerance resulting in treatment failures of bacterial infections. While this phenomenon has posed a great threat in public health, mechanisms underlying their formation in Staphylococcus aureus remain largely unknown. Increasing evidences of the presence of persister cells in recalcitrant infections underscores the great urgency to unravel the mechanism by which these cells develop. Previously, we characterized msaABCR operon that plays roles in regulation of virulence, biofilm development and antibiotic resistance. We also characterized the function of MsaB protein and showed that MsaB is a putative transcription factor that binds target DNA in response to nutrients availability.ResultsIn this study, we compared the number of persister cell in wild type, msaABCR deletion mutant and the complemented strain in two backgrounds USA300 LAC and Mu50. Herein, we report that msaABCR deletion mutant forms significantly less number of persister cells relative to wild type after challenge with various antibiotics in planktonic and biofilm growth conditions. Complementation of the msaABCR operon restored wild type phenotype. Combined antibiotic therapy along with msaABCR deletion significantly improves the killing kinetics of stationary phase and biofilm S. aureus cells. Transcriptomics analysis showed that msaABCR regulates several metabolic genes, transcription factors, transporters and enzymes that may play role in persister cells formation, which we seek to define in the future.ConclusionsThis study presented a new regulator, msaABCR operon, that is involved in the persister cells formation, which is a poorly understood in S. aureus. Indeed, we showed that msaABCR deletion significantly reduces the persister cells formation in all growth phases tested. Although, we have not yet defined the mechanism, we have shown that msaABCR regulates several metabolic, transporters, and extracellular proteases genes that have been previously linked with persister cells formation in other bacterial systems. Taken together, this study showed that inactivation of the msaABCR operon enhances the effectiveness of antibiotics for the treatment of S. aureus infections, especially in context of persister cells.Electronic supplementary materialThe online version of this article (10.1186/s12866-017-1129-9) contains supplementary material, which is available to authorized users.

Staphylococcus aureus has evolved a complex regulatory network that controls a multitude of defense mechanisms against the deleterious effects of oxidative stress stimuli, subsequently leading to the pathogen’s survival and persistence in the hosts. Previously, we characterized the msaABCR operon as a regulator of virulence, antibiotic resistance, and the formation of persister cells in S. aureus. Deletion of the msaABCR operon resulted in the downregulation of several genes involved in resistance against oxidative stress. Notably, those included carotenoid biosynthetic genes and the ohr gene, which is involved in resistance against organic hydroperoxides. These findings led us to hypothesize that the msaABCR operon is involved in resisting oxidative stress generated in the presence of both H2O2 and organic hydroperoxides. Here, we report that a protein product of the msaABCR operon (MsaB) transcriptionally regulates the expression of the crtOPQMN operon and the ohr gene to resist in vitro oxidative stresses. In addition to its direct regulation of the crtOPQMN operon and ohr gene, we also show that MsaB is the transcriptional repressor of sarZ (repressor of ohr). Taken together, these results suggest that the msaABCR operon regulates an oxidative stress defense mechanism, which is required to facilitate persistent and recurrent staphylococcal infections. Moving forward, we plan to investigate the role of msaABCR in the persistence of S. aureus under in vivo conditions. IMPORTANCE This study shows the involvement of the msaABCR operon in resisting oxidative stress by Staphylococcus aureus generated under in vitro and ex vivo conditions. We show that MsaB regulates the expression and production of a carotenoid pigment, staphyloxanthin, which is a potent antioxidant in S. aureus. We also demonstrate that MsaB regulates the ohr gene, which is involved in defending against oxidative stress generated by organic hydroperoxides. This study highlights the importance of msaABCR in the survival of S. aureus in the presence of various environmental stimuli that mainly exert oxidative stress. The findings from this study indicate the possibility that msaABCR is involved in the persistence of staphylococcal infections and therefore could be a potential antimicrobial target to overcome recalcitrant staphylococcal infections.

ObjectiveStaphylococcus genus comprising both Staphylococcus aureus and coagulase negative staphylococci (CoNS) are widely distributed in nature and can infect diversity of hosts. Indeed, staphylococci are the major pathogens causing biofilm associated infections caused by contaminated hospital indwelling devices. These infections are persistent in nature being highly refractory to various stresses including antibiotics. Implementation of efficient diagnostic techniques for the biofilm production would help minimize the disease burden. Thus, early detection of pathogenic strains producing biofilms warrant the utmost importance in diagnostic laboratories especially in resource limited settings.ResultAmong 375 isolates collected from different clinical specimens, 214 (57%) were identified as coagulase negative staphylococci and 161 (43%) S. aureus. Detection of In-vitro biofilm formation in these isolates were carried out by three commonly used phenotypic assays and a genotypic assay. While evaluating the results, tissue-culture method with supplemented glucose and sucrose showed the best correlation with the results of genotypic assay.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3820-9) contains supplementary material, which is available to authorized users.

Introduction: Staphylococcus aureus is found to be a major source of community as well as hospital acquired infection. Staphylococcal isolates from tertiary care hospital are found to be resistant to commonly used antimicrobial agents. Methicillin resistant S. aureus (MRSA) with intrinsically developed antimicrobial resistance has been associated with an increase in morbidity and mortality of the patients in the hospital. This study was undertaken to know the antibiotic sensitivity pattern of staphylococcal isolates with special reference to Methicillin resistant S. aureus. Methods: Clinical specimens received from July 2009 to July 2010 in Kathmandu Medical college-Teaching Hospital were processed and all S. aureus isolates were included in the study. The isolates were identified by standard laboratory procedure. The antibiotic susceptibility pattern of all staphylococcal strain was determined by modified Kirby Bauer antibiotic sensitivity method. Results: Of 111 S .aureus isolates 29(26.12%) were identified to be MRSA. The rate of multi drug resistance was 75.86% for MRSA and 6.09% for MSSA. All the staphylococcal isolates were resistant to penicillin. However, all strains were sensitive to vancomycin. Conclusions: This study showed a high prevalence of MRSA in tertiary care hospital of Kathmandu valley. Regular surveillance of hospital-associated infection and monitoring of antibiotic sensitivity pattern is mandatory to reduce MRSA prevalence in hospital and its spread to community as well. Present study conclusively shows that vancomycin remains the first choice of treatment for MRSA infection. To preserve its value, use of vancomycin should be limited to those cases where there are clearly needed. DOI: http://dx.doi.org/10.3126/joim.v34i1.9117 Journal of Institute of Medicine, April, 2012; 34:1 13-17