Characterization of Panton–Valentine leukocidin-positive Staphylococcus aureus from skin and soft tissue infections and wounds in Nigeria: a cross-sectional study

Background: Staphylococcus aureus is a significant pathogen implicated in numerous nosocomial and community-acquired infections. The Panton–Valentine leukocidin (PVL) can be associated with severe necrotizing diseases such as pneumonia, skin and soft tissue infection (SSTI). Methods: In total, 96 S. aureus isolates were obtained from patients presenting with wounds (n=48) and soft tissue infections (SSTIs, n=48). These were characterized based on their antimicrobial susceptibility profile, the possession of virulence genes (e.g. capsular type, PVL), accessory gene regulator ( agr) type, and the staphylococcal protein A ( spa) type. The production of the PVL protein was assessed by western blotting. Results: All isolates were susceptible to methicillin. The resistance was highest to penicillin (97.9%), followed by trimethoprim/sulfamethoxazole (85.4%) and tetracycline (10.4%). The PVL gene was found in 83.3% of isolates from SSTIs and in 79.2% of isolates from wound. Of these, 53 (68%) produced PVL as assessed by western blotting. The most prevalent spa type was the t084 (78.1%, n=75) and, majority of the isolates carried agr2 (82.3%, n=79). Conclusions: Prevalence of antibiotic resistant PVL-positive methicillin susceptible S. aureus strains has severe implications on PVL mediated infections.


Introduction
Staphylococcus aureus is an important human pathogen that causes significant hospital and community acquired infections 1 . S. aureus producing Panton-Valentine leukocidin (PVL) is linked to a broad array of necrotizing diseases such as pneumonia and skin and soft tissue infections (SSTIs) 2 . PVL is more frequently associated with community isolates 3 . PVL is a pore-forming toxin that can kill myeloid cells by forming channels in the plasma membrane, leading to loss of osmotic balance that ultimately lyses the cel 4 . Earlier reports have shown PVL to be one of the most important virulence determinants in S. aureus from sub Saharan Africa 5 . This study was conducted to investigate the presence of virulence genes including lukS-PV/lukF-PV, the production of the PVL protein and the antibiotic resistance in methicillin-susceptible S. aureus strains isolated from wounds and SSTIs between 2010 and 2011.

Ethical statement
Ethical approval for this study was obtained from the Ethics Committee of the Department of Biological Sciences, Covenant University, Ota, Ogun State, Nigeria (CUNG-2010-035). All participants signed a written informed consent before the commencement of the study.

Characterization of isolates
In this study we made use of an already existing database which has been published 6 . The study was conducted in four health facilities in Ogun and Lagos States of Nigeria between June 2010 and May 2011. Samples were collected from patients presenting with SSTIs and wound infections. The isolation and identification of the isolates were done by culture and genotyping. A total of 96 S. aureus isolates were obtained from wounds (n=48) and SSTIs (n=48). The Vitek automated systems (bioMérieux, Marcy L'Étoile, France) was employed to determine the antibiotic susceptibility profile. The PVL gene (lukS-PV/lukF-PV), capsular polysaccharides (cap 5, cap 8), exfoliative toxins (eta, etb), the toxic shock syndrome toxin (tst) and the agr type were detected by PCR. All amplifications was done in a thermocycler (Bio-Rad, Munich, Germany). The cycling conditions and primers used are as earlier published. Detection of the lukS-PV/lukF-PV gene was carried out using primer sequences: luk-PV-1(5'-ATCATTAGGTAAAATGTCTGGACATGATCC A-3') and luk-PV-2 (5' GCATCAASTGTATTGGATAGCAA AAGC-3') 7 . The negative control was S. aureus ATCC 49230 (MSSA) and the positive control was sta 635/636 (a PVL-positive CA-MRSA strain). Primers specific for the variable segment of the cap locus. Cap5-f: (5'-GAAAGTGAACGATTAGT AGAA-3') Cap5-r: (5'-GTACGAAGCGTTTTGATAGTT-3') Cap 8-f: (5'-GTGGGATTTTTGTAGCTTTT-3') Cap 8-r: (5'-CGC CTCGCTATATGAACTAT-3') was used for the capsular typing 8 . Sequences specific for exfoliative toxins; eta, etb and the toxic shock syndrome toxin; tst were detected by multiplex PCR 9 . The agr types of the S. aureus strains were determined by the multiplex PCR strategy 10 . Extracellular production of PVL by lukS-PV/lukF-PV -positive strains was evaluated by a Western blot using in-house antibodies raised in rabbits (anti-lukF-PV: 334 µg/ml, anti-lukS-PV: 900 µg/ml 11 . The nitrocellulose membrane (Schleicher & Schüll, Dassel, Germany) was first incubated with rabbit anti-lukS-PV/lukF-PV antibodies (in-house antibodies, 1:1000 in TBST) and later incubated with polyvalent goat alkaline-phosphatase-conjugated anti-rabbit antibodies (1:1000 in TBST, DAKO, Germany, D0487). The membranes were washed and the bands visualized using alkaline phosphatase color development substrate (BCIP/NBT, Thermo Fischer Scientific, 34042) 11 . The production of PVL was determined semi-quantatively in four categories: no PVL production; low PVL production, high and very high PVL production. The genetic diversity of all isolates was determined by the staphylococcal protein A (spa) typing 12 . The highly polymorphic region X of the protein A gene, which is composed of a variable number of 24-bp repeats, was amplified by PCR. spa types were determined with the Ridom StaphType software version 1.5 beta (Ridom GmbH, Würzburg, Germany). All statistical computations were performed in SPSS Version 25. Data is explored using relevant descriptive analysis alongside chi 2 to measure any association between antibiotic resistance, virulence genes and lukS-PV/lukF-PV. P<0.05 is deemed to be statistically significant.

Results and discussion
We analyzed the characteristics of the PVL-positive S. aureus isolates as well as the relationship between antibiotic resistance, virulence genes and PVL gene (Table 1). Antibiotic resistance was highest to penicillin (100% in SSTI isolates and 94% in wound isolates), followed by trimethoprim/sulfamethoxazole (84% in SSTI isolates and 83% in wound isolates) and tetracycline (8% in SSTI isolates and 10% in wound isolates (Table 1). This is consistent with an earlier study which showed similar resistance rates for penicillin (98%), trimethoprim/sulfamethoxazole (80%) and tetracycline (18%) in Nigeria 6 . All isolates were methicillin-susceptible. The lukS-PV/lukF-PV gene was detected in 83.3% (n=40) of SSTI isolates and 79.2% (n=38) of wound isolates. Reports from other African countries have shown high rates of PVL positive MSSA ranging from 17% to 74% 5 . For example, a study in an Algiers hospital reported a prevalence of 72% among clinical isolates 13 . A multi-center study reported that deep-seated SSTIs associated with the PVL gene resulted in more hospitalizations of patients and this led more often to incision and drainage 14 . A meta-analysis showed PVL to be consistently associated with SSTIs than invasive diseases 15 . In a study carried out in Gabon, PVL-positive isolates were found to occur more in SSTIs, and PVL was also associated with resistance to trimethoprim/sulfamethoxazole 16 The presence of the PVL gene does not necessarily guarantee that the protein will be expressed and, if it is, toxin levels could vary widely from strain to strain. The production of PVL (in contrast to the sole presence of lukS-PV/lukF-PV) was observed in 75% of lukS-PV/lukF-PV SSTI isolates and 60.5% of lukS-PV/lukF-PV wound isolates. In vitro variation in the production of PVL by different strains of S. aureus has been reported and this suggests important differences in transcriptional and/or translational control of gene expression 17 . In this study, the level of PVL produced by lukS-PV/lukF-PV positive S. aureus isolates varied from strain to strain (Figure 1). It was observed in that none of the PVL-positive strains harboured other toxin genes such as eta, etb and tst. Seven different spa types were identified ( Table 1). The most prevalent spa type was t084 (78.1%, n=75). An earlier study revealed a significant association of the spa-CC 084 PVL-positive isolates with PVL-positive isolates 6 . Typing of the agr locus, which controls the expression of many S. aureus virulence factors, showed that most isolates (82.3%, n=79) possessed the agr2, while none carried agr3. Other studies have linked isolates carrying an agr4 allele to exfoliatin-related diseases and usually carry eta and/or etb 18,19 . These were absent in this study.
In conclusion, this study showed that many S. aureus isolates in Nigeria carry the PVL genes but few produced PVL in vitro. Antibiotic resistance combined with the presence of the PVL genes, has serious implications in the treatment of S. aureus infections. This study is limited by the few study locations. A larger study population is needed to provide a better understanding of the clones of S. aureus in Nigeria. The results is however significant for regional surveillance.

Introduction
Line 3, remove first `and` or recast in ``such as pneumonia and skin and soft tissue infections`S pa typing is not part of the stated objectives.

Method
Characterization of isolates`I n this study we made use of an already existing database which has been published .`` It should be clearly stated the data used from the referenced study above. The Method section should be rewritten so that all previously described method in Ayepola et al (2015) should not be rewritten in the present article but only referred to.

Results and discussion
It will be interesting to also expatiate on the proportion of isolates that are PVL positive molecularly and phenotypically i.e. the isolates with the genes and also producing PVL and those with the genes without PVL. Spa typing reported in this study should be discussed too in relation to its epidemiological significance.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? Yes 6