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Immunology and Microbiology  |   May 2013
lukSF-PV in Staphylococcus aureus Keratitis Isolates and Association With Clinical Outcome
Author Affiliations & Notes
  • Henri Sueke
    St Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
    Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
  • Jayendra Shankar
    St Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
    Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
  • Timothy Neal
    Department of Medical Microbiology, Royal Liverpool University Hospital, Liverpool, United Kingdom
  • Craig Winstanley
    Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
  • Stephen Tuft
    Moorfields Eye Hospital, London, United Kingdom
  • Rosanna Coates
    Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom
  • Malcolm J. Horsburgh
    Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom
  • Stephen Kaye
    St Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
    Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom
  • Correspondence: Henri Sueke, St Paul's Eye Unit, 8z Link, Royal Liverpool Hospital, Prescott Street, Liverpool UK L7 8XP; hsueke@gmail.com
  • Footnotes
     See the appendix for the members of the Microbiology Ophthalmic Group.
Investigative Ophthalmology & Visual Science May 2013, Vol.54, 3410-3416. doi:10.1167/iovs.12-11276
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      Henri Sueke, Jayendra Shankar, Timothy Neal, Craig Winstanley, Stephen Tuft, Rosanna Coates, Malcolm J. Horsburgh, Stephen Kaye; lukSF-PV in Staphylococcus aureus Keratitis Isolates and Association With Clinical Outcome. Invest. Ophthalmol. Vis. Sci. 2013;54(5):3410-3416. doi: 10.1167/iovs.12-11276.

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Abstract

Purpose.: To determine the prevalence, genetic diversity, and clinical relevance of the lukSF-PV gene, encoding the bacterial toxin Panton-Valentine leukocidin, in Staphylococcus aureus isolates from cases of bacterial keratitis in the United Kingdom.

Methods.: Multiplex PCRs investigating carriage of lukSF-PV and mecA were performed on S. aureus isolates from patients. The lukSF-PV operon was sequenced to investigate its diversity, and multilocus sequence typing to test for a clonal relationship between lukSF-PV isolates. Antimicrobial minimum inhibitory concentrations (MICs) and clinical outcome data were compared for isolates characterized as lukSF-PV+ve, mecA+ve, and lukSF-PV/mecA-ve.

Results.: Of 95 isolates, 9 (9.5%) were lukSF-PV+ve, 9 (9.5%) mecA+ve, and 1 was positive for both. Five single nucleotide polymorphisms were found in lukSF-PV genes of seven strains. There was no significant difference between the MICs of lukSF-PV/mecA-ve and lukSF-PV+ve isolates to the antimicrobials tested, except for tigecycline (P < 0.05). The mecA+ve isolates had significantly higher mean MICs to meropenem and fluoroquinolones (P < 0.05). There were nonsignificant trends for healing and treatment times, ulcer and scar size, and overall clinical score to be greater in the lukSF-PV+ve group (P < 0.05). The proportion of patients, however, who required surgery was significantly greater among patients with lukSF-PV+ve isolates with an odds ratio of 7.8 (95% CI 1–42, P = 0.018) for patients requiring surgery.

Conclusions.: lukSF-PV+ve isolates were associated with a trend to worse clinical outcome and more surgical interventions, with an effect unrelated to MICs. This suggests that lukSF-PV may be an important virulence factor in S. aureus –associated keratitis.

Introduction
The pathogenicity of Staphylococcus aureus is mediated by a multitude of secreted virulence factors, such as bacterial surface adhesins, immune evasion proteins, and toxins. Panton-Valentine leukocidin (PVL) is a pore-forming toxin produced by S. aureus . 1 It is a bicomponent toxin that consists of the polypeptides LukS-PV and LukF-PV. 2 The cognate LukS-PV and LukF-PV bind to neutrophils, monocytes, and macrophages, but not to lymphocytes. 3 Following the binding of monomers of LukS-PV and LukF-PV, further monomers attach to the cell wall forming a heptameric structure that forms a pore in the host cell surface. 2,4 This pore formation results in leukocyte cell death and the release of inflammatory cytokines.3  
The involvement, however, of PVL in the virulence of S. aureus is equivocal and its link with clinical outcome remains uncertain. In vivo studies have produced conflicting data. Murine models of S. aureus infection have shown that the absence of PVL results in an increase in virulence, 58 whereas studies in rabbits indicate that the presence of PVL increases the virulence of S. aureus . 911 These discrepancies could, however, be attributed to differences in the immunology of the models. 11 Mouse neutrophils are insensitive to the effects of PVL, whereas rabbit and human neutrophils both have sensitivity. There is also no consensus on the effect of lukSF-PV+ve S. aureus on clinical outcome in patients. Some studies suggest that PVL is not associated with clinical outcome or that the presence of PVL can reduce virulence, 5,1214 whereas other studies have found a correlation between PVL carriage and disease invasiveness. 1518  
In terms of treatment, PVL+ve S. aureus isolates are susceptible to most antimicrobials used to treat staphylococcal infections, including trimethoprim-sulfamethoxazole, glycopeptides, linezolid, and the fluoroquinolones. 1921 One particular sequence type (ST80), however, has been found to be resistant to tetracycline and fusidic acid. 19,20  
The potential involvement of PVL as a virulence factor has led to the investigation of its relevance in infectious diseases of the eye and its adnexa. Rutar et al. 22 described a case series of nine patients with severe ocular infection (including cases of orbital cellulitis and endophthalmitis) caused by the USA300 S. aureus strain that is known to carry lukSF-PV. More recently, Nadig et al. 23 reported 33 cases of ophthalmic infections caused by S. aureus taken from two centers in India. They found 17 (52%) of the isolates were lukSF-PV+ve, whereas 5 (56%) of the 9 keratitis isolates were lukSF-PV+ve. Four of the lukSF-PV+ve keratitis isolates were resistant to fluoroquinolones, but outcome data were not reported. 
To the best of our knowledge, there are no studies that have looked for an association between the lukSF-PV genotype and clinical outcome in keratitis caused by S. aureus
Community-acquired meticillin-resistant S. aureus (CA-MRSA), in particular those associated with lukSF-PV, is present in epidemic proportions in the United States. 24 This has not been noted in the United Kingdom where the vast majority of lukSF-PV+ve isolates are meticillin-sensitive S. aureus (MSSA). 21 The presence of meticillin resistance in S. aureus isolates is determined by the mecA gene that lies in the SCCmec cassette. The gene product of mecA is a penicillin-binding protein (PBP2a), which renders S. aureus refractory to the action of all available β-lactamantimicrobials. 25  
We have examined isolates from a case series of S. aureus –related keratitis to determine the prevalence of lukSF-PV and mecA. Furthermore, we have investigated the genotype of lukSF-PV+ve isolates and any association with antimicrobial minimum inhibitory concentration (MIC) and clinical outcome. 
Methods
Clinical data and isolates from 95 consecutive cases of S. aureus ulcerative keratitis were collected from seven UK centers (constituting the Microbiology Ophthalmic Group) over a 2-year period. Corneal scrape samples were placed onto agar culture plates and also into enrichment culture broth (brain heart infusion) as previously described. 26 The plates were incubated overnight at 37°C under both aerobic and enriched carbon dioxide (5%) atmospheric conditions in the local laboratory. Bacterial isolates were sent to one microbiology laboratory (Royal Liverpool University Hospital NHS Trust) where they were subcultured and stored on sterile Protect Beads (TSC Ltd., Heywood, Lancashire, UK) at −80°C. Isolates were subsequently subcultured from the beads and their identity reconfirmed by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF). 27 MICs of ciprofloxacin, ofloxacin, moxifloxacin, levofloxacin, penicillin, vancomycin, teicoplanin, chloramphenicol, gentamicin, meropenem, linezolid, and tigecycline were determined using E-Tests as previously described. 28  
DNA Extraction
Genomic DNA was prepared using QIASymphony SP (Qiagen, Manchester, UK) as per the manufacturer's instructions. Briefly, 1 mL of overnight culture was pelleted and washed in 500 μL PBS and resuspended in lysis buffer (20 mM Tris·HCl, pH 8.0; 2 mM EDTA; 1.2% [vol/vol] Triton) containing 20 μg mL−1 lysostaphin. Cells were incubated at 37°C for 1 hour before the addition of 20 μL proteinase K and 200 μL Buffer AL (Qiagen), followed by incubation at 56°C for 30 minutes. The lysate was transferred to QIASymphony-compatible tubes and DNA was purified using the Pathogen Complex 200 protocol (Qiagen). 
Detection of mecA and lukSF-PV
A multiplex PCR assay was performed using 50 to 70 ng mL−1 of the extracted DNA. Primers used were as follows: (1) Staph756F and Staph750R: amplifying the Staphylococcus genus-specific 16S rRNA gene (positive internal control); (2) Luk-PV-1 and Luk-PV-2: amplifying the lukSF-PV gene; and (3) mecA1 and mecA2: amplifying the mecA gene. PCR cycle conditions were as previously described by McLure et al. (Table 1, part a). 29  
Table 1. 
 
Oligonucleotide Forward and Reverse Primers Used in (a) Multiplex PCR Serving to Amplify the Following Genes: Staphylococcus genus-specific 16S rRNA (Staph756F and Staph750R), lukSF-PV (Luk-PV-1and Luk-PV-2), and mecA (MecA1 and 2), and (b) PCR for Full Gene Sequencing of lukSF-PV
Table 1. 
 
Oligonucleotide Forward and Reverse Primers Used in (a) Multiplex PCR Serving to Amplify the Following Genes: Staphylococcus genus-specific 16S rRNA (Staph756F and Staph750R), lukSF-PV (Luk-PV-1and Luk-PV-2), and mecA (MecA1 and 2), and (b) PCR for Full Gene Sequencing of lukSF-PV
Primer Name Sequence
(a) Staph756F 5′-AACTCTGTTATTAGGGAAGAACA-3′
Staph750R 5′-CCACCTTCCTCCGGTTTGTCACC-3′
Luk-PV-1 5′-ATCATTAGGTAAAATGTCTGGACATGATCCA-3′
Luk-PV-2 5′-GCATCAAGTGTATTGGATAGCAAAAGC-3′
MecA1 5′-GTAGAAATGACTGAACGTCCGATAA-3′
MecA2 5′-CCAATTCCACATTGTTTCGGTCTAA-3′
(b) PVL1F 5′-GGTGATGGCGCTGAGGTAGTCAAA-3′
PVL1R 5′-CTGTATGATTTTCCCAATCAACTTC-3′
PVLint2F 5′-CAACTGCAACATCAGATTCCGATAAG-3′
PVLint2R 5′-CAAATTCACTTGTATCTCCTGAGCC-3′
PVLint3F 5′-GGGACCATATGGCAGAGATAGTTATC-3′
PVLint3R 5′-GTATTGGAAAGGCCACCTCATTGC-3′
Sequencing of the PVL Gene
Isolates that were PCR positive for lukSF-PV underwent further genetic analysis. PCR amplification of an internal fragment (+108 to +1807) of the lukSF-PV operon was performed. The 25-μL PCR mixture contained 50 to 70 ng mL−1 bacterial DNA, 1.25 units Accuzyme, 12.5 μL Biomix red, 2.5 mM magnesium chloride, and 100 pmol primers (see Table 1, part b, for primer details). PCR cycle conditions were as follows: 5 minutes at 95°C, followed by 30 cycles of 1 minute at 94°C, 1 minute at 50°C, and 2 minutes at 72°C. PCR products were then sequenced (GATC Biotech, Constance, Germany) and single nucleotide sequences were built for each isolate using a multiple sequence alignment tool from Clustal Omega (provided in the public domain at http://www.ebi.ac.uk/Tools/msa/clustalo/; Hinxton, UK). 
Multilocus Sequence Typing
Multilocus sequence typing (MLST) was performed with the extracted DNA as described previously by Enright et al. 30 PCR amplicons of the seven S. aureus housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) were sequenced (GATC Biotech). The lukSF-PV locus was constructed from reads using the Clustal Omega tool (provided in the public domain, http://www.ebi.ac.uk/Tools/msa/clustalo/) as described previously. Sequence types (STs) were determined using the database provided in the public domain by http://www.MLST.net
Clinical Outcome
Data collected were ulcer size (mm2) at presentation; distance of ulcer to limbus (mm2); healing time (days), defined as interval to epithelialization; scar size (mm2) when the epithelium had healed; treatment time (days); risk factors for infection; surgical interventions (amniotic membrane grafts, penetrating keratoplasty, enucleation, or evisceration); and the ratios of ulcer size to healing time and ulcer size to treatment time as previously described. 31 For each parameter, patients were assigned a score according to the deviation parameter in SDs from the mean. For example, if the ulcer size was within 1 SD of mean they scored 0, between 1 and 2 SD they scored 1, and greater than 2 SDs they scored 2. Similarly, negative scores were given for negative SD values. Surgical events were assigned a score of 3 and loss of the eye a score of 5. Aggregate clinical scores were then calculated for all parameters. 
Statistical Analysis
Logarithms were taken for all clinical and MIC data except for discrete events, such as surgical intervention or loss of the eye. The Kruskal-Wallis test was used to test for differences between groups. In addition, logarithmic regression test was used to calculate the odds ratio (OR) and 95% confidence intervals (CIs) of surgical intervention between groups. Data analyses were undertaken by using SPSS version 20 (IBM SPSS Statistics, IBM Corporation, Chicago, IL). 
Results
PVL Presence and Type
Of the 95 S. aureus keratitis isolates, nine (9.5%) were lukSF-PV+ve, nine (9.5%) were mecA+ve, and one (1.1%) isolate was positive for both. The lukSF-PV gene was highly conserved in eight isolates (we were unsuccessful in amplifying and sequencing one of the isolates). There was, however, nucleotide variation in seven of the isolates compared with the USA300 reference strain (Figure). In total there were five nucleotide substitutions causing four amino acid changes. Two major sequence variants of lukSF-PV, the arginine (R) and histidine (H) variants, were identified, based on a substitution of adenine for guanine at nucleotide 528 that resulted in an H-to-R amino acid change. MLST typing showed that lukSF-PV+ve isolates could be classified into two diverse sequence types: three of the nine were classified as ST30 and the others were STs 12, 49, 1, 121, 1, and 8. A summary of the gene sequencing and MLST results is included in Table 2
Figure
 
Schematic representation of sequence variance between the 8 lukSF-PV+ve S. aureus isolates sequenced. USA300 is used as a comparator reference strain at the top. The relative position of nucleotide changes from the USA300 strain are labeled in each strain and the alternative base is indicated.
Figure
 
Schematic representation of sequence variance between the 8 lukSF-PV+ve S. aureus isolates sequenced. USA300 is used as a comparator reference strain at the top. The relative position of nucleotide changes from the USA300 strain are labeled in each strain and the alternative base is indicated.
Table 2. 
 
Gene Sequencing and MLST of lukSF-PV+ve S. aureus Isolates
Table 2. 
 
Gene Sequencing and MLST of lukSF-PV+ve S. aureus Isolates
Isolate No. Location Genotype Variant MLST Type
39073 London R 30
39076 London N/A 12
39165 London H 49
39200 Manchester H 1
39348 Birmingham H 121
48016 London H 30
48135 London H 30
106030 London R 1
106035 London + H 8
Antimicrobial MIC Patterns
The MICs of the 95 isolates to the 12 antimicrobials tested are shown in Table 3. There was no significant difference in the mean MIC between the lukSF-PV/mecA-ve (isolates negative for lukSF-PV and mecA genes) and lukSF-PV+ve (isolates positive for lukSF-PV) groups, apart for tigecycline, which had a significantly lower mean MIC for the lukSF-PV+ve compared with both the mecA+ve and lukSF-PV/mecA-ve isolates. There was greater variation in the MICs of the lukSF-PV+ve isolates than that of the lukSF-PV/mecA-ve isolates to the fluoroquinolones and penicillin, as evident in the higher MIC90 (antimicrobial concentration required to inhibit the growth of 90% of organisms). In contrast, the mecA+ve isolates had significantly higher mean MICs (P < 0.05) with higher MIC90 and MIC50 to meropenem and the fluoroquinolones. 
Table 3. 
 
Antimicrobial Data for Patients With S. aureus Keratitis Who Were lukSF-PV+ve (n = 9), mecA+ve (n = 9), or Negative for Both (lukSF-PV/mecA-ve (n = 77)
Table 3. 
 
Antimicrobial Data for Patients With S. aureus Keratitis Who Were lukSF-PV+ve (n = 9), mecA+ve (n = 9), or Negative for Both (lukSF-PV/mecA-ve (n = 77)
Cip Ofx Mox Lev Pen Van Tei Chl Gen Mer Lnz Tig
lukSF-PV-ve mecA-ve
 Mean MIC 0.5 0.7 0.03 0.1 0.4 1.4 0.9 3.6 0.4 0.03 0.4 0.1
 MIC50 0.4 0.5 0.03 0.1 0.8 1.5 1.0 3.0 0.4 0.02 0.5 0.1
 MIC90 1.5 1.0 0.05 0.2 1.5 2.0 1.5 8.0 1.0 0.05 0.8 0.1
lukSF-PV+ve
 Mean MIC 0.8 0.7 0.1 0.2 0.3 1.4 0.6 2.4 0.4 0.03 0.4 0.04*
 MIC50 0.4 0.5 0.1 0.1 0.5 1.5 0.8 2.0 0.4 0.03 0.5 0.1
 MIC90 32 13 1.6 25.7 7.2 1.7 1.0 4.2 0.6 0.1 0.6 0.1
mecA+ve
 Mean MIC 20* 19* 2* 12* 12* 1.2 0.4* 6.2 0.3 0.7* 0.4 0.1
 MIC50 32 32 1.0 8.0 32.0 1.3 0.3 3.5 0.4 0.9 0.4 0.1
 MIC90 32 48 4.0 38.0 32.0 1.8 1.6 131 0.5 2.3 0.6 0.1
Clinical Outcome
Ninety of the patients had received fluoroquinolone (ofloxacin or ciprofloxacin) as initial treatment. Five of the patients with isolates negative for both lukSF-PV and mecA had received chloramphenicol as initial treatment. There were no significant differences in age, sex distribution, or position of the ulcer (distance from the limbus) between the groups that were positive or negative for lukSF-PV or mecA (P = 0.40). The healing and treatment times, ulcer and scar size, and overall clinical score tended to be greater in the lukSF-PV+ve group (Table 4). The proportion of patients who required surgery as management of their keratitis was significantly greater in the lukSF-PV+ve group (three of nine cases required surgery) compared with the lukSF-PV-ve group (3 of 81 cases required surgery) or the mecA+ve group (one of nine required surgery) (P = 0.016). In comparison with patients with lukSF-PV-ve isolates, the OR for patients with lukSF-PV+ve isolates requiring surgery was 7.8 (95% CI 1–42, P = 0.018), whereas for those with mecA isolates it was 2.6 (95% CI 0.3–27.0, P = 0.42). 
Table 4. 
 
Clinical Outcome (Mean and SD) for Patients With S. aureus Keratitis; lukSF-PV+ve (n = 6), mecA+ve (n = 8), or Negative for Both (lukSF-PV/mecA-ve (n = 70)
Table 4. 
 
Clinical Outcome (Mean and SD) for Patients With S. aureus Keratitis; lukSF-PV+ve (n = 6), mecA+ve (n = 8), or Negative for Both (lukSF-PV/mecA-ve (n = 70)
TT, d HT, d UA, mm2 SA, mm2 UA/SA HT/UA Clinical Score
lukSF-PV-ve and mecA-ve
 Mean 20.16 9.71 3.06 1.63 0.83 3.43 2.06
 SD 26.25 12.62 11.79 7.55 1.08 5.55 2.76
lukSF-PV+ve
 Mean 20.61 13.54 5.30 3.37 0.73 2.37 3.57
 SD 31.68 27.01 5.88 4.26 0.41 2.81 3.74
mecA+ve
 Mean 15.54 6.15 2.17 0.99 0.66 3.42 1.33
 SD 20.31 3.35 3.69 4.17 0.29 5.34 1.86
Discussion
Interest in PVL toxin (lukSF-PV+ve) as an S. aureus virulence factor began after it was associated with fatal cases of community-acquired pneumonia in previously healthy children. 32  
Yoong et al. 5 identified that PVL induces proinflammatory cytokines and a moderate TNF-alpha response in pulmonary infections in a murine model, indicating a possible immunomodulatory role. The same group had previously found that mutant PVL strains with isogenic lukS and lukF deletions were more virulent in a murine skin abscess model compared with wild type. 6 Further studies using murine models of sepsis, abscess, skin infection, and pneumonia showed similar results. 7,8 As previously mentioned, there has been much discussion about the suitability of using mice to study PVL toxicity, due to the differences in mouse and human immunology, in particular, the insensitivity of mouse neutrophils to the effects of the leukocidin. 11 There is a growing consensus that a rabbit model might yield more meaningful results. In vivo studies in rabbits indicate that the presence of PVL increases the virulence of S. aureus strains. 9,10 Diep et al. 10 identified a role for PVL in the virulence of S. aureus USA300 and USA400 strains during the early, acute stages of bacteremia in rabbits, when lysis of Polymorphonuclear leukocytes (PMNs) might allow colonization to be established. These findings were supported by Lipinska et al., 11 who noted that during the early course of skin infection in a rabbit model, a PVL+ve USA300 isolate of S. aureus produced larger lesions and more skin necrosis, compared with PVL knockout strains. 
There is also debate on the role of PVL in S. aureus infection in patients. There are several studies that suggest there is no difference in clinical outcome with PVL+ve MRSA infections or that the presence of PVL can reduce virulence. 5,1214 The presence, however, of PVL in the virulent CA-MRSA clone USA300 33 lends epidemiological support that the toxin influences the virulence of S. aureus . Indeed, a correlation between PVL carriage and invasiveness of disease, as well as virulence, has been found not only in USA300, but also in other community-acquired strains. 1517 Although these hypervirulent lineages are known to be MRSA, attention must also be paid to PVL+ve MSSA, which represents the vast majority of UK PVL-containing clones. 21  
In our case series of 95 patients with S. aureus keratitis, 9.5% of isolates were lukSF-PV+ve and these cases tended to have larger ulcers and worse outcomes, in particular a significantly higher incidence of cases requiring surgical intervention. Our findings reflect those of Muttaiyah et al., 18 who found a statistically significant correlation between PVL+ve MSSA and the need for surgical intervention among 411 isolates taken from patients with a variety of S. aureus infections. Our data do not suggest a link between a particular allele of lukSF-PV and keratitis, as we identified six different lukSF-PV alleles, including both H and R variants. The predominant subgroup was the H variant, which is consistent with the findings of Wolter et al., 34 who identified the H variant as the most common form in S. aureus isolates from Europe. 
It is of note that eight of the nine lukSF-PV+ve isolates in our study were mecA-ve. As mentioned previously, the MRSA status is determined by the mecA gene that lies in the SCCmec cassette. It has been proposed that the presence of mecA may impose a fitness cost on isolates in environments such as the community with limited antimicrobial use. This may result in evolutionary pressure for some CA-MRSA strains to lose the SCCmec cassette while retaining key virulence or fitness factors, such as lukSF-PV. 35 Alternatively, the MSSA strains may have never acquired the SCCmec cassette. The small number of lukSF-PV+ve isolates in our study makes it difficult to speculate why only one of nine lukSF-PV+ve isolates in our study was mecA+ve. 
The diversity in the sequence types observed in this study is in agreement with the clonal diversity observed in MSSA isolated from skin and soft tissue infections in the United States. 35 The presence of multiple PVL alleles within the same genetic lineage, for example the presence of both H variants and R variants, as well as three different single nuclear polymorphism profiles among the ST30 isolates, could indicate horizontal gene transfer of the PVL phage between different clonal complexes. 
Identification of particular subsets of bacteria associated with keratitis paves the way for detecting bacterial factors that may determine clinical outcome. For example, a recent study by Shankar et al. 36 investigated the genetic characteristics of Pseudomonas aeruginosa isolates collected from patients with keratitis. They analyzed 13 single nucleotide polymorphisms based on the conserved genome, as well as three variable markers (flagellin types a or b and the mutually exclusive type III secretion exotoxin genes exoU or exoS). They found that when the isolates were compared to a population of nonkeratitis isolates, 71% of the keratitis isolates clustered together. Our study is potentially another example of an association involving a particular genotypic subset of a pathogen causing keratitis. Early detection of potential virulence factors could therefore provide information to enable treatment of bacterial keratitis to be modified if necessary. Multiplex PCR, as described by McClure et al, 29 is a relatively quick and inexpensive way to screen for both lukSF-PV and mecA using the 16S rRNA gene locus as a positive control. 
The higher MICs seen to the fluoroquinolones for mecA isolates reflects that reported in previous studies on patients with keratitis. 28,37 In contrast, the mean MICs for lukSF-PV and lukSFPV-ve groups were similar for the antimicrobials tested. Of note, however, was the lower mean MIC of lukSF-PV to tigecycline compared with both the lukSF-PV-ve and mecA+ve S. aureus isolates. In this study, we have not used systemic breakpoints to calculate the proportion of susceptible and resistant isolates to a particular antimicrobial. The absence of established breakpoint concentrations for antimicrobials when they are used topically limits the interpretation of susceptibility data. The susceptibility criteria used to select an antimicrobial for treatment are based on the anticipated response of the bacteria against concentrations of the antimicrobial that can be achieved in serum. Topical application of an antimicrobial to the cornea may achieve a very different concentration and bioavailability in the tissue than the serum levels. 38 Although the appropriate disc susceptibility breakpoint for each antimicrobial and bacterial isolate combination has not yet been determined, there is good evidence demonstrating the relationship between the MIC of topically applied antimicrobials and clinical outcome in bacterial keratitis. 31,39  
In conclusion, we found patients with lukSF-PV+ve S. aureus were associated with a trend to worse clinical outcome and more surgical interventions, with an effect unrelated to MICs. This suggests that lukSF-PV may be an important virulence factor in S. aureus –associated keratitis. A larger study would be needed to verify this, especially in light of previous epidemiological studies that suggest that the presence of PVL is not associated with a worse clinical outcome in S. aureus infection in other organs. 12,13,40  
Acknowledgments
The authors thank Gabriela Czanner, Department of Eye and Vision Science, University of Liverpool, for advice on statistical aspects of study. 
Supported by grants from Fight for Sight and the Royal College of Surgeons Edinburgh. 
Disclosure: H. Sueke, None; J. Shankar, None; T. Neal, None; C. Winstanley, None; S. Tuft, None; R. Coates, None; M.J. Horsburgh, None; S. Kaye, None 
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Appendix
Members of The Microbiology Ophthalmic Group
Stephen Kaye, Craig Winstanley, Malcolm Horsburgh, Amanda Hall, Henri Sueke, and Timothy Neal (Royal Liverpool University Hospital); Stephen Tuft (Moorfields Eye Hospital); Derek Tole and John Leeming (Bristol Eye Hospital); Peter McDonnell (Birmingham and Midlands Eye Hospital); Francisco Figueiredo and Manjusha Narayanan (Royal Victoria Infirmary, Newcastle); Andrew Tullo, Fiona Carley, Hannah Lloyd, and Malcolm Armstrong (Manchester Royal Eye Hospital); Colin Willoughby, Johnny Moore, and Grace Ong (Royal Victoria Hospital, Belfast). 
Figure
 
Schematic representation of sequence variance between the 8 lukSF-PV+ve S. aureus isolates sequenced. USA300 is used as a comparator reference strain at the top. The relative position of nucleotide changes from the USA300 strain are labeled in each strain and the alternative base is indicated.
Figure
 
Schematic representation of sequence variance between the 8 lukSF-PV+ve S. aureus isolates sequenced. USA300 is used as a comparator reference strain at the top. The relative position of nucleotide changes from the USA300 strain are labeled in each strain and the alternative base is indicated.
Table 1. 
 
Oligonucleotide Forward and Reverse Primers Used in (a) Multiplex PCR Serving to Amplify the Following Genes: Staphylococcus genus-specific 16S rRNA (Staph756F and Staph750R), lukSF-PV (Luk-PV-1and Luk-PV-2), and mecA (MecA1 and 2), and (b) PCR for Full Gene Sequencing of lukSF-PV
Table 1. 
 
Oligonucleotide Forward and Reverse Primers Used in (a) Multiplex PCR Serving to Amplify the Following Genes: Staphylococcus genus-specific 16S rRNA (Staph756F and Staph750R), lukSF-PV (Luk-PV-1and Luk-PV-2), and mecA (MecA1 and 2), and (b) PCR for Full Gene Sequencing of lukSF-PV
Primer Name Sequence
(a) Staph756F 5′-AACTCTGTTATTAGGGAAGAACA-3′
Staph750R 5′-CCACCTTCCTCCGGTTTGTCACC-3′
Luk-PV-1 5′-ATCATTAGGTAAAATGTCTGGACATGATCCA-3′
Luk-PV-2 5′-GCATCAAGTGTATTGGATAGCAAAAGC-3′
MecA1 5′-GTAGAAATGACTGAACGTCCGATAA-3′
MecA2 5′-CCAATTCCACATTGTTTCGGTCTAA-3′
(b) PVL1F 5′-GGTGATGGCGCTGAGGTAGTCAAA-3′
PVL1R 5′-CTGTATGATTTTCCCAATCAACTTC-3′
PVLint2F 5′-CAACTGCAACATCAGATTCCGATAAG-3′
PVLint2R 5′-CAAATTCACTTGTATCTCCTGAGCC-3′
PVLint3F 5′-GGGACCATATGGCAGAGATAGTTATC-3′
PVLint3R 5′-GTATTGGAAAGGCCACCTCATTGC-3′
Table 2. 
 
Gene Sequencing and MLST of lukSF-PV+ve S. aureus Isolates
Table 2. 
 
Gene Sequencing and MLST of lukSF-PV+ve S. aureus Isolates
Isolate No. Location Genotype Variant MLST Type
39073 London R 30
39076 London N/A 12
39165 London H 49
39200 Manchester H 1
39348 Birmingham H 121
48016 London H 30
48135 London H 30
106030 London R 1
106035 London + H 8
Table 3. 
 
Antimicrobial Data for Patients With S. aureus Keratitis Who Were lukSF-PV+ve (n = 9), mecA+ve (n = 9), or Negative for Both (lukSF-PV/mecA-ve (n = 77)
Table 3. 
 
Antimicrobial Data for Patients With S. aureus Keratitis Who Were lukSF-PV+ve (n = 9), mecA+ve (n = 9), or Negative for Both (lukSF-PV/mecA-ve (n = 77)
Cip Ofx Mox Lev Pen Van Tei Chl Gen Mer Lnz Tig
lukSF-PV-ve mecA-ve
 Mean MIC 0.5 0.7 0.03 0.1 0.4 1.4 0.9 3.6 0.4 0.03 0.4 0.1
 MIC50 0.4 0.5 0.03 0.1 0.8 1.5 1.0 3.0 0.4 0.02 0.5 0.1
 MIC90 1.5 1.0 0.05 0.2 1.5 2.0 1.5 8.0 1.0 0.05 0.8 0.1
lukSF-PV+ve
 Mean MIC 0.8 0.7 0.1 0.2 0.3 1.4 0.6 2.4 0.4 0.03 0.4 0.04*
 MIC50 0.4 0.5 0.1 0.1 0.5 1.5 0.8 2.0 0.4 0.03 0.5 0.1
 MIC90 32 13 1.6 25.7 7.2 1.7 1.0 4.2 0.6 0.1 0.6 0.1
mecA+ve
 Mean MIC 20* 19* 2* 12* 12* 1.2 0.4* 6.2 0.3 0.7* 0.4 0.1
 MIC50 32 32 1.0 8.0 32.0 1.3 0.3 3.5 0.4 0.9 0.4 0.1
 MIC90 32 48 4.0 38.0 32.0 1.8 1.6 131 0.5 2.3 0.6 0.1
Table 4. 
 
Clinical Outcome (Mean and SD) for Patients With S. aureus Keratitis; lukSF-PV+ve (n = 6), mecA+ve (n = 8), or Negative for Both (lukSF-PV/mecA-ve (n = 70)
Table 4. 
 
Clinical Outcome (Mean and SD) for Patients With S. aureus Keratitis; lukSF-PV+ve (n = 6), mecA+ve (n = 8), or Negative for Both (lukSF-PV/mecA-ve (n = 70)
TT, d HT, d UA, mm2 SA, mm2 UA/SA HT/UA Clinical Score
lukSF-PV-ve and mecA-ve
 Mean 20.16 9.71 3.06 1.63 0.83 3.43 2.06
 SD 26.25 12.62 11.79 7.55 1.08 5.55 2.76
lukSF-PV+ve
 Mean 20.61 13.54 5.30 3.37 0.73 2.37 3.57
 SD 31.68 27.01 5.88 4.26 0.41 2.81 3.74
mecA+ve
 Mean 15.54 6.15 2.17 0.99 0.66 3.42 1.33
 SD 20.31 3.35 3.69 4.17 0.29 5.34 1.86
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