Molecular characterisation of Panton-Valentine Leukocidin (PVL)-positive Staphylococcus aureus from Wales N. Bome-Mannathoko1, M. Wooton3, L. G. Harris1, R. Howe3, D. Mack1,2 1Medical Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine, Swansea University 2NPHS Microbiology Swansea, Singleton Hospital, Abertawe-Bro Morgannwg University NHS Health Board, Swansea 3Specialist Antimicrobial Chemotherapy Unit, NPHS Microbiology Cardiff Introduction PVL-positive S.aureus typically cause primary skin and soft-tissue abscesses but have also been implicated in life threatening infections i.e. haemorrhagic pneumonia and necrotising fasciitis. Recent widespread dissemination of PVL-positive community-acquired MRSA clones, i.e. USA300 and USA400 in the USA, has heightened the question of the clinical relevance of PVL. Although the prevalence of PVL-positive S. aureus in the UK is low (<2%), the enhanced virulence and transmissibility of USA300 in the USA accentuates the need for continuous surveillance of PVL-positive strains in the UK. Of 61 SACU isolates, 16 MRSA had the USA300-0114 pulsotype, ACME and the MRSAIVa-t008 genotype. Most of the remaining MRSA belonged to MRSAIVc-t044 and MRSAIVc-t002 genotypes (Table 3). Table 3. Molecular epidemiology of SACU S. aureus isolates spa type No. SCCmec ACME PFGE MRSA t008 (t024, t1578, t1624, t2743) 16 (26.2%) IVa POS USA300-0114 t008 1 (1.6%) IVc NEG Unique t044 (t4725) 5 (8.2%) B1 t002 3 (4.9%) C1 t127 2 (3.2%) USA400 (MW2) Miscellaneous* 8 (13.1%) IVMISC Miscellaneous USA300 reference strains FPR3857 n/a SF8300 MSSA t159 H1 t211 K1 t2271 G1 Miscellaneous** 18 (29.6%) Total 61 Methods 560 consecutive S. aureus wound isolates from NPHS Microbiology Swansea, Singleton Hospital (ABMU) were screened for PVL by real-time PCR and 19 (3.4%) were positive. This non-selected PVL-positive ABMU cohort (n=19) and a PVL-positive cohort (n=61) from the Specialist Antimicrobial Chemotherapy Unit, NPHS Microbiology Cardiff (SACU) were evaluated. The 80 isolates were characterised by: detection of mecA and the arginine catabolic mobile element (ACME) by PCR; pulsed-field gel electrophoresis (PFGE); spa and SCCmec typing. Susceptibility testing was performed with BD Phoenix PMIC/ ID-67 panels. Results – genotypic characterisation Table 1. Prevalence of MRSA and MSSA per location Location MRSA MSSA Total SACU, Cardiff 35 (57.4%) 26 (42.6%) 61 ABMU, Swansea 2 (10.5%) 17 (89.5%) 19 ** t019, t105, t138, t160, t162, t177, t189, t314, t345, t1425, t1635, t1869, t2393, t3204, 2xnew(c), 2xnew(d) *t005, t202, t223, t275, t311, 2xnew (a&b),1-untypeable Results- antibiotic resistance All 80 PVL-positive S. aureus were susceptible to: vancomycin, daptomycin, mupirocin, rifampin, teicoplanin and had varying resistance to other antibiotics (Table 4). p =0.0004:Fisher’s 2-tailed exact test 17/19 of the ABMU isolates were MSSAs and only 2 were MRSAs. spa types t021 and t314 were exhibited by 3 strains, and the remaining isolates had various spa types (Table 2). Table 4. Antibiotic resistance of PVL-positive S. aureus All (n=80) SACU (n=61) ABMU (n=19) Penicillin 98.8% (79) 100% (61) 94.7% (18) Oxacillin 45.0% (36) 57.4% (35) 5.3% (1)* Gentamicin 5.0% (4) 1.6% (1) 15.8% (3)* Tobramycin 36.3% (29) 41.0% (25) 21.1% (4) Erythromycin 28.8% (23) 31.1% (19) Clindamycin 1.3% (1) 0.0% (0) 5.3% (1) Tetracycline 17.5% (14) 9.8% (6) 42.1% (8)* Trimethoprim 63.8% (51) 67.2% (41) 52.6% (10) TMP-SMZ 14.8% (9) 26.3% (5) Ciprofloxacin 4.9% (3) Fusidic acid 8.8% (7) Table 2. Molecular epidemiology of ABMU S. aureus isolates spa type No. SCCmec ACME PFGE MRSA t019 1 (5.3%) IVd NEG Unique t437 V MSSA t021 3 (15.8%) n/a M1 t314 L1 Miscellaneous* 11 (57.8%) Miscellaneous Total 19 *t002, t005, t084, t159, t645, t891, t903, t917, t4363, t4791, t3011 *Oxacillin p=0.0001; Gentamicin p=0.04; Tetracycline p=0.003: Fisher’s 2-tailed exact test 89.5% of the ABMU cohort comprised extensively diverse MSSA, 57.4% of the SACU isolates were MRSA and PFGE revealed that these isolates belonged to several clones (Figure 1). Conclusion 3.4% of unselected S. aureus from ABMU were PVL-positive. Distinct differences in MRSA prevalence and clonal composition of the PVL-positive cohorts from ABMU and the SACU referral unit. USA300 was predominant and strains affiliated to other clones i.e. European clone (MRSAIVc-t044), USA800 (MRSAIVc-t002) were present in the SACU cohort but not in the ABMU cohort. PVL genes are present in MSSA strains of diverse genotypes and not limited to specific MRSA clones. Molecular epidemiology of S. aureus strains submitted to referral units is valuable but is not necessarily representative of strains of a geographical location. Verification by evaluation of unselected isolates from the same area is vital. USA300-0114 B1 C1 USA400 Unique 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 485kb 388kb 242.5kb 145.5kb 97kb References Ellington et al. 2009. Clin Microbiol Infect . 28 :1113–1121 Ellington et al. 2008. J Antimicrob Chem . 61:73- 77 Holmes et al. 2005. J Clin Microbiol. 43: 2384-90. Strommenger et al. 2006. J Clin Microbiol. 44: 2533–2540 Deurenberg et al. 2004. FEMS Microbiol Let. 240: 225-28 Acknowledgements Thanks to Binh A. Diep for provision of the USA300 reference strains. Our gratitude to the staff at NPHS Microbiology Swansea, Singleton Hospital for their support and collaboration 48.5kb Figure 1. PFGE of 20 PVL-positive MRSA from SACU