International Journal of Medical and Health Research 114 International Journal of Medical and Health Research ISSN: 2454-9142 www.medicalsciencejournal.com Volume 4; Issue 2; February 2018; Page No. 114-119 Panton Valentine Leukocidin (PVL) and thermonuclease (NUC) profile in clinical isolates of S. aureus and an assessment of impact of variations in nucleotide sequences of fem A, B and X genes 1 Rosy Chikkala, 2 Karuna Deepika, 3 R Iyer, 4 KS Ratnakar, *5 V Sritharan 1 Molecular Diagnostics and Biomarkers Laboratory, Global Medical Education and Research Foundation, Hyderabad, Telangana, India 2, 5 Molecular Diagnostics and Biomarkers Laboratory Hospitals, Global Hospitals, Hyderabad, Telangana, India 4 Global Medical Education and Research Foundation, Hyderabad, Telangana, India 3 Microbiology Laboratory, Global Hospitals, Hyderabad, Telangana, India * Corresponding Author: V Sritharan Abstract Introduction: S. aureus produces more than 32 extracelleular molecules which include a number of virulence factors, toxins, enzymes and haemolysins, which allow it to adhere to surface, invade or avoid the immune system and in addition causes toxicity to the host. Virulence factors like coagulase, thermonuclease and Panton Valentine Leukocidin (PVL) are all secreted. We investigated the distribution and expression of PVL and thermonuclease in clinical isolates of S. aureus. Spontaneous sequence variations were observed in the fem genes of clinical isolates of S. aureus (data under communication) and we investigated if this would impact the secretion of thermonuclease and PVL. Materials and Methods: Genomic DNA was isolated from157 S. aureus clinical isolates and screened for fem, nuc and pvl through PCR. To evaluate whether the sequence variations in fem genes had any effect on the expression of pvl, we randomly selected 43 fem (fem A, B and X) variants from the 109 pvl positive isolates and measured pvl transcript by qPCR. The isolates were genotyped for nuc by PCR and the transcript was measured by qPCR while DNAase secretion was screened by methyl green DNA agar plate method. Results: 109 (77%) out of 157 isolates were pvl PCR positive. PVL expression was marginally down regulated when compared with the reference gene in these variants. Almost all isolates were nuc-PCR positive. No remarkable change in the expression of thermo nuclease was observed among the fem sequence variants. Conclusion: Variations in the nucelotide sequences of fem A, B and X genes were observed in several clinical isolates of S. aureus. These variations did not affect the secretion of coagulase, thermonuclease and transcription of pvl. PVL has long been known as virulence factor associated with and a molecular marker for CA-MRSA. However, our study has shown a relatively high prevalence of pvl in S. aureus isolated from hospital acquired infections. Keywords: pvl, fem (A, B and X), nuc, MRSA, MSSA Introduction Staphylococcus aureus (S. aureus) causes a range of infections (1-4) among patients in hospitals (hospital acquired infections) and people in the community. Blood stream infection and sepsis due to MRSA is life threatening unless diagnosed early and treated with appropriate antibiotics. Reliable and rapid identification of S. aureus is necessary for successful management of patients with sepsis (5, 6) and PCR based methods are increasingly being used for this (7-9). Since mecA could be carried by non-S. aureus bacteria also, it is important for any molecular method to be able to differentiate and identify S. aureus in addition to detecting mecA for the diagnosis of MRSA. Our laboratory evaluated fem and nuc for species identification and reported direct detection of MRSA in uncultured clinical samples using a simple sample processing protocol and multiplex PCR (under communication). The severity of S. aureus infection is largely attributed to the large number of virulence factors (more than 32 extracelleular products) which include toxins, enzymes, adhesins and haemolysins (10-13). It is useful to determine the virulence profile of the S. aureus simultaneously while screening for MRSA. We investigated the profile of coagulase, thermonucelase and PVL in these isolates. Coagulase, though plays an important role in the pathogenesis of S. aureus disease, is invariabley used for identification and classification of S. aureus (14, 15) as it is constitutively expressed by S. aureus. Thermonuclease gene has been widely used as a specific marker to detect S. aureus contamination, in PCR-based methods. It can resist heat upto 100 0 C for 1 h and has the ability to degrade DNA, RNA (16, 17) and it apparently plays important role in biofilms (18, 19). Besides these, one of the important exo-toxins secreted by S. aureus is Panton Valentine Leukocidin (PVL) which is encoded by two contiguous and co-transcribed genes LukS-PV and LukF-PV. PVL is known to induce cell death in soft tissue infections, pneumonia and sepsis (11, 13, 20, 21) by necrosis or apoptosis. PVL is reportedly present more often in community acquired (CA-MRSA) infections than in hospital acquired (HA-MRSA) infections and therefore it is used as molecular marker for identification of CA-MRSA (22). Prevalence of