JAC Advance Access originally published online on June 11, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):402-405; doi:10.1093/jac/dkm206
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Is Panton–Valentine leucocidin associated with the pathogenesis of Staphylococcus aureus bacteraemia in the UK?
1 Staphylococcus Reference Laboratory (SRL), Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK 2 Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK
* Corresponding author. Tel: +44-20-8327-7259; Fax: +44-20-8200-7449; E-mail: matthew.ellington{at}hpa.org.uk
Received 1 February 2007; returned 12 March 2007; revised 10 May 2007; accepted 14 May 2007
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Abstract |
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Background: The morbidity and mortality associated with Panton–Valentine leucocidin (PVL)-positive Staphylococcus aureus suggest that this toxin is a key marker of disease severity. Nevertheless, theimportance of PVL in the pathogenesis of primary bacteraemia caused by S. aureus is uncertain. We have determined the prevalence of PVL-encoding genes among isolates of S. aureus from bacteraemic patients.
Methods: Consecutive bacteraemia isolates of S. aureus (n = 244) from patients hospitalized in 25 centres in the UK and Ireland during 2005 were screened for PVL and mecA genes. PVL-positive isolates were characterized by toxin gene profiling, PFGE, spa-typing and MIC determinations for a range of antimicrobials.
Results: Four out of 244 isolates (1.6%) were PVL-positive and susceptible to oxacillin [methicillin-susceptibleS. aureus (MSSA)]. Eighty-eight out of 244 (36%) were oxacillin-resistant (methicillin-resistant S. aureus), but none was PVL-positive. The four patients (two males: 30 and 33 years; two females: 62 and 80 years) had infection foci of: skin and soft tissue, unknown, indwelling line, and surgical site, and were located at one centre in Wales, one in England and two in Ireland. One of four PVL-positive isolates was resistant to penicillin and fusidic acid, the remainder were susceptible to all antibiotics tested. Genotypic analyses showed that the four isolates represented three distinct strains; the two isolates from Ireland were related.
Conclusions: We found that 1.6% of S. aureus (all MSSA) from bacteraemic patients were PVL-positive. This low incidence suggests that PVL-positive S. aureus are of no particular significance as causative agents of S. aureus bacteraemia.
Keywords: PVL , virulence factors , bloodstream infections , MRSA , MSSA
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Introduction |
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The leucocidal capacity of Staphylococcus aureus was first identified in 18941 and was later found to be due to the production of the Panton–Valentine leucocidin (PVL) toxin,2 which is a bi-component non-haemolytic toxin that induces cytotoxic and cytolytic changes in human polymorphonuclear cells, monocytes and macrophages.
The PVL toxin is encoded by contiguous genes lukS-PV and lukF-PV on several temperate bacteriophage genomes and has been reported in clinical isolates of S. aureus at frequencies between 2% and 10%.3 Historically, PVL-related infections have been associated primarily with previously healthy young adults with no prior exposure to the healthcare setting or increases in other known risk factors for methicillin-resistant S. aureus (MRSA), suggesting PVL-positivity to be a key facet in the emerging threat of the so-called community-associated MRSA (CA-MRSA). Typically, individuals with PVL-positive S. aureus present with uncomplicated suppurative skin infections such as furunculosis, abscesses and boils,4 and data from more recent studies support this observation.5,6 More complicated PVL-related necrotic skin and soft tissue infections (SSTIs)3,4,6 include necrotizing fasciitis7 and purpura fulminans.8 These infection types, along with necrotizing pneumonia,6 have been associated with serious life-threatening disease demanding prompt intervention and intensive patient management.
Limited data are available on the frequency of MRSA or methicillin-susceptible S. aureus (MSSA) isolates encoding PVL across England and Wales for specific human disease aetiologies,3 and it is unclear what proportion of patients with PVL-related disease become bacteraemic. More generally, despite its association with serious invasive disease, it is unclear whether PVL plays a role in primary S. aureus bacteraemia.
To explore these aspects, we sought to determine the prevalence of the PVL genes among MSSA and MRSA bacteraemia isolates collected during 2005 as part of the BSAC bacteraemia survey (http://www.bsacsurv.org) for the UK and Ireland. Such an initiative would also help estimate the extent to which PVL-associated disease is currently unrecognized resulting in under-ascertainment of cases. We present data demonstrating the low prevalence of PVL among isolates from a dedicated, contemporaneous, collection of bacteraemia isolates and detail three distinct PVL-positive MSSA strains present in the UK and Ireland.
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Bacteria
Consecutive S. aureus (n = 244) isolated from hospitalized bacteraemic patients at 25 UK and Irish centres (approximately 10 isolates per centre) as part of the 2005 survey for the BSAC (http://www.bsacsurv.org, date last accessed 10 May 2007) were included. The survey collected information regarding patient age, sex, probable focus of infection, the specialty to which the patient was admitted and the location of the referring centre. The isolates were identified using tube coagulase testing.
MICs were determined by the Etest (AB Biodisk, Solna, Sweden) or agar dilution using Iso-Sensitest agar (Oxoid, Basingstoke, UK) according to the BSAC method.9 The antimicrobials tested werepenicillin, oxacillin, ciprofloxacin, tetracycline, erythromycin, gentamicin, fusidic acid, clindamycin, rifampicin, teicoplanin and vancomycin.
Detection of methicillin resistance and PVL-encoding genes
Three primer pairs lukS-PV and lukF-PV, mecAP4 and mecAP7, and 16SF and 16SR3,4 were used in a triplex PCR format to detect PVL-encoding genes, the mecA gene associated with methicillin resistance and, as an internal control, the staphylococcal 16S rDNA gene, respectively. Reactions were performed essentially as described previously,3 using 0.1 µM of each primer, and cycling as follows: 5 min at 95°C, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 2 min, followed by 72°C for 5 min. Isolates found positive for the PVL genes were further characterized for toxin gene complement, as described by Holmes et al.3
Multiplex PCR was used to determine the accessory gene regulator (agr) allotype of the PVL-positive isolates.3 Single locus spa sequence typing was carried out.10 DNA sequencing template was prepared using EXOSAP.IT (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions and sequenced on both strands. Data were analysed using Ridom StaphType® software (Ridom GmbH, Wurzburg, Germany).
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The sex and age range of the 244 patients with S. aureus bacteraemia are shown in Table 1. Overall, 12 (5%) were
16 years, 28 (11%) were between 17 and 40 years, 49 (20%) were between 41 and 60 years and 154 (63%) 
61 years. A single isolate was from an individual of unknown age. Of 244 isolates tested, 88 (36%) were MRSA (mecA-positive), the majority of which (69 or 28%) were from individuals 61 years. All isolates from paediatric patients were mecA-negative MSSA. Forty-one (17%) of the 244 isolates were from bacteraemiaoriginating from SSTIs and 20 (8%) were from bacteraemia related to respiratory tract infections; these are the major aetiologies for PVL-producing strains.
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Four of 244 isolates (1.6%) harboured the lukS-PV and lukF-PV genes, indicating a low prevalence. Of these four, one was from Wales (case 1), one from England (case 2) and two (cases 3 and 4) from geographically separate centres in Ireland (Table 2). Clinical and patient demographic data are shown in Table 2. Case 2 was associated with an SSTI and was isolated within 48 h of patient hospitalization, suggesting community acquisition; the remaining three were associated with infections that developed after 48 h of hospital care. No isolates from patients
16 years (n = 12) or older adults (41–60 years, n = 49) were PVL-positive. All four PVL-positive S. aureus were susceptible to oxacillin and were negative for mecA by PCR. The isolate from case 2 was resistant to fusidic acid (MIC = 8 mg/L) and penicillin (MIC = 16 mg/L) only, which is consistent with the presence of fusB and increased expression of the ß-lactamase genes, respectively. The remaining three isolates were fully susceptible to all antibiotics tested (Table 2). Among all 244 isolates tested for PVL, only 28 (11%) were susceptible to penicillin; the three penicillin-susceptible PVL-positive isolates thus represent 11% of all penicillin-susceptible isolates.
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The isolate from case 1 was distinct from the other three by PFGE (data not shown) and spa type (t383) (Table 2) and harboured six staphylococcal enterotoxin genes (sea, sec, sed, seg, sei and sej) in addition to PVL. The isolate from case 2 was also distinct from the other three by PFGE (data not shown) and spa-typing (t127) and had a unique enterotoxin gene profile (sea and seh). The two isolates from Ireland (cases 3 and 4) were closely related by PFGE (data not shown) and shared identical toxin gene profiles (seg, sei and PVL) and the same spa type (t021), indicating clonality.
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Although primarily a cause of uncomplicated SSTI, PVL-positive S. aureus has also been associated with serious life-threatening disease worldwide, leading to a notion that S. aureus from patients with more serious, invasive disease, perhaps with contingent bacteraemia, are more likely to be PVL-positive. We therefore hypothesized that S. aureus from patients with bacteraemia might provide a source of unrecognized PVL-related disease, with potential for under-ascertainment of cases. Among the 244 S. aureus isolates from the BSAC annual bacteraemia survey that we tested, 88 (36%) were MRSA and 156 (64%) were MSSA. We found no PVL-positive MRSA and a PVL-positive MSSA rate of 2.5% among the 156 MSSA isolates. This compares with a previous broader study in which 2% of all PVL-positive S. aureus in England and Wales referred to the Staphylococcus Reference Laboratory were associated with bacteraemia.3 Collectively, these data suggest that PVL is of no particular significance as a causative agent of S. aureus bacteraemia in the UK and Ireland. Interestingly, Etienne11 noted that patients with pneumonia due to CA-MRSA do not readily develop an associated bacteraemia, providing further evidence that PVL isolates are not closely associated with the pathogenesis of bloodstream infections per se. In sharp contrast, studies in the USA have shown that up to 20% of bloodstream infections are due to the PVL-positive CA-MRSA strain USA300,12 but it is worth noting that this higher prevalence has occurred against a background of escalating CA-MRSA rates in both community and healthcare settings. Moreover, at present, the USA300 lineage is not prevalent in the UK or elsewhere in Europe, with only isolated cases having been identified. The low prevalence of PVL observed in this study suggests that other factors contribute more significantly to the pathogenesis of bacteraemia, and moreover, it seems likely that a complex combination of host- and/or pathogen-related factors ultimately contributes to S. aureus bacteraemia.
The small number of PVL-positive isolates found makes further conclusions about associations of PVL with bacteraemia in different (primary) disease origins and patient age groups impossible. However, one point of note is that the PVL-positive isolate from case 3 in this study was associated with an infectionat a surgical site, correlating with a recent case report ofaPVL-positive CA-MRSA associated with a surgical site infection in the USA.13 The observation of PVL in 11% of the penicillin-susceptible isolates suggests over-representation among this group in the UK and Ireland, but this conclusion is tenuous because of the low numbers of PVL-positive isolates found. Indeed, although the analysis of this isolate set, which is unspecified for PVL-related disease, is useful for providing a broad clinical picture, a more systematic, prospective survey collecting isolates from suspected PVL-related disease may gain larger numbers of PVL-positive isolates. A targeted study, collecting larger numbers of isolates from each centre, may better indicate the contribution that PVL makes to pathogenesis and disease and would enable better assessment of the contribution that PVL makes to disease in the UK.
In summary, these findings do not support the concept that PVL-positive S. aureus are over-represented among isolates from bloodstream infections or are of any particular significance for causing bacteraemia. Focusing solely on bloodstream infections will significantly underestimate the full extent of PVL-related infections occurring in the UK.2 To improve our understanding of PVL-related disease, and attempt to maximize case ascertainment, it is important to note that skin infections, complicated or not, remain the most likely reservoir of PVL-positive disease.3,4
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None to declare.
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Acknowledgements |
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We thank Rosy Reynolds and the BSAC for kindly providing access to the isolates tested in this study.
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References |
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