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48th Interscience
Conference on
Antimicrobial Agents
and Chemotherapy
September 24-29, 2008.
Washington, USA
An Evaluation of Virulence Factor Profiles in Meticillin Resistant Staphylococcus aureus (MRSA) Isolated from Chronic Wounds
C. EMANUEL1, KE. HILL1, K. HARDING2, RA. HOWE3, DW. THOMAS1, M. WOOTTON3 1 School of Dentistry, Cardiff University, UK. 2Wound Healing Research Unit, Cardiff University, UK. 3NPHS, Wales, UK.
ABSTRACT
Background: Chronic wounds (CW) occur in around 3.5% of patients over 65 years in UK; representing a significant problem. Whilst the aetiology of CW is poorly understood they are known to support a diverse microflora, which may play a role in the non-healing phenotype. Staphylococci are commonly isolated from CW; however, it is unknown whether these show virulence factor profile alteration. This study aims to characterise the expression of virulence factors in MRSA isolates associated with carriage (C) and CW.
Methods: 17 MRSA isolated from CW and 7 C MRSA were collected from the wound healing clinic and routine nasal screens. CW originated from surgical wounds (n=8) and ulcers (n=9). PCR and Reverse transcriptase (RT) –PCR was performed using unique primers to determine presence and gene expression levels of virulence factors. All isolates were classified into agr groups. Expression was graded in intensity from I – III.
Results: Out of 17 CW isolates 15 were agr type I and 2 agr type III. 5 C isolates were classified as agr type IIIs and 1 isolate each in agr II and IV.
The lukF/lukS genes, which encode Panton Valentine Leukocidin were found in 25% of MRSA from surgical wounds compared to 89% from ulcers. Conclusions: lukF/lukS genes predominated in CW isolates, particularly those originating from ulcers. Greater cna expression was seen in CW isolates compared to C. These virulence factors may play a significant role in mediating the observed non-healing phenotype.
MATERIALS & METHODS
17 MRSA were isolated from out-patients with chronic wounds (CW) attending the Wound Healing Research Unit.
7 carrier (C.) MRSA were isolated from routine nasal swabs from a routine diagnostic microbiology laboratory.
DNA/RNA was extracted from cultures grown to mid-log and stationary phase using a Qiagen DN/RNeasy kit.
Isolates were agr typed using PCR with published primers and sequencing (Table 1).
PCR and Reverse Transcriptase (RT)–PCR was performed with specific primers to determine the presence and expression level of virulence factors (Figure 1).
Expression was graded I-III for intensity (Table 2).
Presence and expression of genes were compared with patient/wound data.
Table 1. agr typing of isolates
Ayed et al. (2006) Pathologie Biologie, 54;
Table 2. Virulence gene presence and expression intensity
INTRODUCTION
S. aureus and in particular MRSA continue to be one of the major causes of nosocomial infections in the UK, and are a frequently isolated from non-healing wounds. S. aureus expresses a number of virulence factors to enable them to succeed in their host which may contribute to the non-healing of wounds.
RESULTS
Panton-Valentine Leucocidin (PVL) genes lukF/lukS genes were found in 58.8% MRSA from CW but not from nasal isolates.
sepA was not present in any CW isolates but was found in 28.8% of C MRSA.
Expression of cna was greater in CW MRSA compared to C MRSA.
CONCLUSIONS
Panton-Valentine Leucocidin (PVL) may play a role in the non-healing of wounds.
Presence of PVL genes was associated with more severe lesions.
Greater expression of collagen binding protein gene (cna) is likely to be a factor in MRSA for the colonisation of CW.
AIMS & OBJECTIVES
To determine the virulence factor profile of MRSA isolated from chronic wounds and nasal screens.
To detect any alteration in virulence factor expression in MRSA isolated from chronic wounds compared to commensals
Figure 1. RT-PCR of cna
200 bp
500 bp
Gene: cna bp, 16S bp
Acknowledgements
Dr A Shore, Professor D Coleman of Trinity College, Dublin
Graduate School in Biomedical and Life Sciences
WM Thomas Bequest Fund
Lanes 2 & 26, 25 are negative controls H2O and Mu50 strain respectively, lane 4 is positive control NCTC 8178, lanes 3, 5 – 9 are carrier isolates, lanes 10 – 24 are chronic wound isolates.
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