F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.134641.1 Research Article Articles Prevalence of Panton-Valentine leucocidin (pvl) and exfoliative toxin A (eta) gene within methicillin resistant and sensitive Staphylococcus aureus in an urban tertiary referral hospital: A molecular epidemiology pilot study

[version 1; peer review: 2 approved with reservations]

 Amelia Sri Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-5864-5926 a 1 Kusumawati R. Lia Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-0459-1026 1 Hasibuan Mirzan Conceptualization Formal Analysis Investigation Methodology Project Administration Resources Validation Writing – Review & Editing 2 3 Winda Lavarina Conceptualization Data Curation Formal Analysis Methodology Resources Software Supervision Validation Visualization Writing – Review & Editing https://orcid.org/0009-0000-5701-7389 4 Balatif Ridwan Conceptualization Data Curation Formal Analysis Investigation Methodology Supervision Validation Visualization Writing – Review & Editing 5 Ivander Alvin Conceptualization Data Curation Investigation Methodology Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-6263-5701 5 Department of Microbiology, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia University Hospital, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia Microbiology Laboratory, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia Central Laboratory, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia Faculty of Medicine, Universitas Sumatera Utara, Medan, North Sumatra, Indonesia sriamelia@usu.ac.id

No competing interests were disclosed.

 18 8 2023 2023 12 1002 8 6 2023 Copyright: © 2023 Amelia S et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Staphylococcus aureus is well known to cause a multitude of clinical manifestations, from mild to severe bloodstream infections that could lead to death. Infections are common, either in community-acquired or hospital-acquired settings, and treatment remains a challenge due to methicillin-resistant Staphylococcus aureus (MRSA). The pathogenesis of S. aureus is mediated by several cell-surface and secreted virulence factors. The virulence factors discussed in this study are Panton-Valentine leucocidin (pvl) and exfoliative toxin A (eta).

Our pilot study aimed to observe pvl and eta as virulence gene prevalence in a North Sumatera tertiary referral health center.

Methods: Our study was a descriptive-analytical observational study with a cross-sectional design in which we collected isolates over a single time period. The frequency of genes is reported as a percentage comparison between MRSA and methicillin-sensitive S. aureus (MSSA). Qualitative gene prevalence analysis was carried out using the polymerase chain reaction (PCR).

Results: Our results showed that from 38 MRSA sample isolates, six samples were found to be pvl-negative, or 15.7% of the total samples. From 40 MSSA sample isolates, one sample was found to be pvl-negative MSSA, or 0.025%. Regarding eta, from 38 MRSA sample isolates, 18.4% of the total sample did not have eta, while from 40 MSSA sample isolates, all samples were found to be positive for eta. We found that both pvl and eta were significantly more likely to be expressed in the MSSA strain.

Conclusions: Our study shows that pvl and eta are more likely expressed in MSSA strains than in MRSA strains in Indonesia.

 Staphylococcus aureus Virulence factor pvl eta Universitas Sumatera Utara TALENTA 393/UN5.2.3.1/PPM/KP-TALENTA/2022 This study was funded by the Universitas Sumatera Utara through the TALENTA scheme 2022 with contract No. 393/UN5.2.3.1/PPM/KP-TALENTA/2022. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Staphylococcus aureus are Gram-positive spherical bacteria, usually arranged in a grape-like manner. This bacterium is well known to cause a multitude of clinical manifestations, from mild to severe bloodstream infections that could lead to death. Infections are common, either in community-acquired or hospital-acquired settings, and treatment remains a challenge due to multi-drug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) . 1 S. aureus, including MRSA, is commonly found on the skin and mucous membranes as part of the normal flora of human bodies. 2 While usually known as a commensal bacterium, research has shown that S. aureus infection is one of the most prevalent in the world. In the industrialized world, Tong et al.’s review showed a 10 to 30 per 100,000 person-year incidence of bacteremia caused by S. aureus. While overall rates may have stabilized due to the rise of antibiotics, the contribution of MRSA has fluctuated. 3 However, while it is well observed that staphylococcal infections caused by MRSA remain varying, in which at a specific time and location MRSA prevalence might be higher while in other time and location MRSA prevalence could be lower than predicted, studies have shown an increasing amount of MRSA infections in each decade. Hasanpour et al.’s research showed that before 2000, only 441 elderlies were infected with MRSA; however, this number skyrocketed to 4,365 in the 2001–2010 period and 11,987 in 2011–2022. 4 As such, it can be said that MRSA creates a new challenge for healthcare workers and researchers to identify its pathogenesis and thus create a sound and reliable solution.

The pathogenesis of S. aureus is mediated by several cell-surface and secreted virulence factors. One such virulence factor is Panton-Valentine leucocidin (pvl). 5 pvl is a toxin comprising two components, LukS-PV and LukF-PV. After their secretion, both components assemble into a pore-forming heptamer on neutrophil membranes, causing neutrophil lysis. There is a significant amount of research that shows the role of pvl in pathogenesis; however, it remains unclear what the trends are for pvl-positive methicillin- sensitive S. aureus (MSSA) or MRSA. The molecular epidemiology and burden of pvl-positive MSSA or MRSA are highly variable within studies, with the US dominated by pvl-positive MRSA, while such bacteria have been found to be rare in Australia, which has a predominance of both pvl-positive MRSA and MSSA. 6 Prudent research toward pvl molecular epidemiology is vital because it is well known that pvl is associated with invasive disease and thus could be used as a gene marker for severe infection. In industrialized countries, such epidemiological studies have led to public health measures aimed at individuals infected with the pvl-producing strain. 7

Another such virulence factor was identified as exfoliative toxin A (eta). Exfoliative toxins (ETs), also known as epidermolytic toxins, are serine proteases secreted by S. aureus that recognize and hydrolyze desmosome proteins in the skin. ETs have been associated with the loss of keratinocytes and cell-cell adhesion, inducing peeling of the skin and blister formation. One of the principal isoforms of exotoxins implicated in human skin damage is eta. Recognizing eta prevalence could help distinguish the extent of the damage caused by S. aureus. 8 Research has shown that eta is a prevalent toxin. A study done by Mohseni et al. revealed that 76.7% of isolates were positive for eta in S. aureus isolates. 9 However, research is limited toward MSSA, and there is a lack of research assessing eta prevalence in MRSA strains.

Due to the need for molecular epidemiological study in defining public health measures and mapping molecular profiles, especially in Indonesia and possibly in Southeast Asia, our study aimed to measure pvl and eta gene molecular prevalence at the Adam Malik General Hospital, one of Indonesia’s main tertiary referral health centers.

 Methods Study design

Our study was a descriptive-analytical observational study with a cross-sectional design, in which we collected isolates from the North Sumatera Tertiary Referral Center over a single time point which was in January 2022. Our study was carried out after it had been approved by the ethical medical research committee of the Faculty of Medicine, Universitas Sumatera Utara, Medan, Indonesia through letter no. 540/KEPK/USU/2022. Isolates were collected in a blind manner. This method was used to prevent bias in clinical correlation due to our specific study to identify molecular epidemiology at a specific time point, not to explain a phenomenon caused by a toxin or virulence factor specifically. We determined the sample size needed using the following descriptive sample formula: n = Za 2 PQ d 2

To obtain 95% confidence interval, Za was pre-determined with 1.96 while d was 0.1. According to Mohseni’s study, 8 which found P (proportion of eta positive sample in population) to be 0.767, we found that our study needed at least a total sample of 68 isolates to obtain statistical power. Our study included samples that were as evenly divided into MRSA and MSSA as possible.

After we obtained the minimal sample needed, we collected isolates from the Adam Malik General Hospital Microbiology Installation using cluster random sampling. Sample lab numbers with MRSA and MSSA results were obtained with consecutive sampling; thus we obtained 38 MRSA samples and 40 MSSA samples without information regarding sample clinical history to prevent bias. The samples were then cultured on blood agar medium, and colonies suspected of being Staphylococcus aureus were tested using Gram staining, catalase, oxidase, and coagulase tests. Colonies suspected to be S. aureus were cultured in mannitol salt agar (MSA) medium, while antibiotic susceptibility tests were performed on Muller-Hinton agar (MHA). Antibiotic discs were placed inside MHA. after 24 hours of incubation. Suspension was transferred to the Eppendorf and stored at -80°C.

 DNA extraction

DNA extraction was done from bacterial cells using Presto Mini gDNA Bacteria (Geneaid). 1 × 10 9 colonies of bacterial cells were inserted in a 1.5 mL sterile tube and centrifuged at 13.000 rpm for 1 minute; afterwards, the supernatant was removed. Inside the tube, 200 μL of buffer (including 0.8 mL/200μL lysozyme) were vortexed. Incubation was then performed at 20°C for 5 minutes, and then 20μL of proteinase K was added and vortexed. Incubations were then performed at 70°C for 10 minutes, then added to 200 μL of absolute ethanol and vortexed. GD columns were then arranged inside the collection tube, and 400 μL of buffer W1 were centrifuged at 13,000 rpm for about 30 seconds. Then, liquids were extracted, and centrifugation was re-done with 600 μL wash buffer. After 100 μL of elution buffer were added and maintained at room temperature for 3 minutes, the tubes were filled with pure DNA. Tubes were saved in a freezer at -20°C. Our protocol was in accordance with GeneAid protocol specified inside the product kit.

 Identification of pvl and eta genes by PCR

In the PCR detection of the pvl and eta genes, specific primers were used after searching through the NCBI search engine to ensure their specificity. Table 1 lists the specifications for the primers used, and Table 2 describes our temperature program and volume.

The specifications for the primers.
Name Sequence Amplitude size Source publication
eta forward 5′-TTTGCTTTCTTGATTTGGATTC-3′ 464 bp Mohseni et al., 2018 9
eta reverse 5′-GATGTGTTCGGTTTGATTGAC-3′
pvl forward 5′-ACAAGCAAAAGAATACAGCG-3′ 575 bp Hesari et al., 2018 10
pvl reverse 5′-GTTTTTGGCTGCTTCTCTTG-3′
Thermal cycling device temperature program, and the amount and concentration of materials required for PCR.
Cycles Steps Temperature Time Materials
Staphylococcus aureus Materials Amounts
First step: 1 cycle Hot Start 94 5 min Master mix PCR 2x 12.5 uL
Second step: 35 cycles pvl Denaturation Annealing Extension 94 52 72 30 s 30 s 30 s Primer forward Primer reverse Nuclease free water Template DNA 1 uL 1 uL 8.5 uL 2 uL
35 cycles eta Denaturation Annealing Extension 94 53 72 30 s 30 s 30 s
Third step: 1 cycle Further extension 72 30 s

Afterward, we began PCR mastermix preparation by liquefying GoTaq Green Master Mix 2×, primers (forward and reverse), nuclease-free water and DNA template. Our primer was obtained from Integrated DNA Technologies. These were then vortexed and spun down for 10 seconds, and a PCR mix was then prepared with the materials specified below ( Table 2) to get one sample. Afterward, vortexing was done to ensure suspension homogeneity. DNA templates were then added, and the thermal cycling was carried out with the parameters listed in Table 2. In summary, for most steps, 30 seconds are sufficient, while in the first step, a 5-minute hot start at 94°C would be necessary. To detect pvl, 35 cycles consisting of 94, 52, and 72°C of denaturation, annealing, and extension were performed for 30 seconds. eta followed a similar step.

 Electrophoresis of PCR product

PCR products were electrophoresed using 2% agarose gel (Merck, Germany). A mixture of 1 λ DNA loading dye and 5 λ PCR product was loaded in the gel. Electrophoresis was performed at a voltage of 80 V.

 PCR Visualization and analysis

The electrophoresis results were obtained and visualized through a UV transilluminator. PCR results were defined as positive when a contrast streak was within the primer base pair or negative when the chamber was found to be empty after visualization. As the aim of the study was to identify specific virulence gene prevalences, quantitative PCR or RNA sampling were not performed.

 Statistical analysis

SPSS version 25 and Microsoft Office Excel were used to analyze the data. The frequency of genes was reported as a percentage frequency, and a descriptive comparison was made between MRSA strain gene prevalence and that of MSSA. A bivariate analysis was performed to determine the statistical difference between groups. Due to the categorical nature of our data data, the Chi-square test was chosen. Significance was proven with a p-value below 0.05 in the 95% confidence interval.

 Results

The results of our PCR tests showed that from 38 MRSA sample isolates, six samples were found to be pvl-negative, or 15.7% of total samples. From 40 MSSA sample isolates, one sample was found to be pvl-negative MSSA, or 0.025%. Regarding eta, of 38 MRSA sample isolates, 18.4% of the total sample did not have eta, while from 40 MSSA sample isolates, all samples were found to be positive. Afterwards, we analyzed our data with the Chi-square test to determine whether significant differences existed between pvl and eta prevalence. We found that both pvl and eta were significantly more likely to be present in the MSSA strain, with p ≤ 0.05, respectively. The results can be seen in Table 3.

pvl and eta prevalence between MRSA and MSSA.
Variables Total samples (n = 78) MRSA (n = 38) MSSA (n = 40) p-value
pvl positive 71 (91.1%) 32 (84.3%) 39 (97.5%) 0.040 *
pvl negative 7 (8.9%) 6 (15.7%) 1 (2.5%)
eta positive 71 (91.1%) 31 (81.6%) 40 (100%) 0.004 *
eta negative 7 (8.9%) 7 (18.4%) 0 (0%)

Chi-square test.

Electrophoresis visualisation for each group can be seen in Figure 1 below.

Electrophoresis of eta and pvl gene, (A) Electrophoresis of MRSA pvl DNA with product gene in 575 bp; chamber 18 shows negative expression while the rest was positive; (B) Electrophoresis of MRSA eta DNA with product gene in 464 bp; chamber 17 and 18 show absence of the target gene while the rest was positive; M = marker. Discussion

Our research was performed to identify the molecular epidemiology of pvl-positive MRSA, pvl-positive MSSA, and eta prevalence in either MRSA or MSSA, thus supporting local public health authorities in creating a sound public health measure and fulfilling data needs regarding S. aureus molecular epidemiology, especially in Southeast Asia. According to our results, most MRSA and MSSA were found positive for pvl and eta expression. We also found that MSSA was significantly more likely to encode both pvl and eta.

Our research shows very concerning new updates. In a study performed by Boan et al. in Australia during the 2007–2009 period, pvl-negative and positive samples were still equally dominating, with 141 pvl-positive S. aureus and 148 pvl-negative strains. The Boan study did find that PVL didn’t significantly affect methicillin resistance, with 56% of pvl-positive strains found to be MRSA. However, it is interesting to note that in the Boan study, which reflects Australia’s S. aureus molecular epidemiology, it was shown that pvl-positive MRSA had begun to dominate in Western Australia. 11 These results concur with our study, and by reflecting its close geographical distance, our result creates a new concern that pvl-positive MRSA and MSSA dominate most S. aureus specimens.

Recent research performed in Gambia by Darboe et al. in 2019 revealed a somewhat different result at several time points, while at the same time reflecting the similar results found in our study in another time points. While it is correct to assume that the pvl-positive samples have increased from 2005 levels to 2015 levels, their numbers has been fluctuating at best. However, it is interesting to note that in one period of the study, the pvl-positive strain was as high as 90%. Darboe’s study didn’t elaborate on whether there is methicillin resistance in pvl-positive or pvl-negative strains; however, Darboe did conclude that there was no association between pvl and antimicrobial resistance, while emphasizing the still-low antimicrobial resistance in Gambia. 12 This result brought a new perspective to our study as it showed a fluctuating trend. It might be possible that some measures were taken during the lower pvl-positive strain infection period, and it has been found that while our results show unusually high pvl-positive MRSA and MSSA, this result was not irreversible.

Jaiswal’s report in 2022 revealed another different result. Their study results were not consistent with ours, whereby Jaiswal revealed only 49 out of 162 positive pvl strains within MRSA. 13 We noted that the Jaiswal study was not consistent with our study or with other studies performed in India. A study by Kaur from India reported 85.1% pvl-positive MRSA prevalence within the country, 14 similarly to D’Souza’s study in Mumbai which revealed 64% pvl-positive MRSA. 15 This doesn’t necessarily mean that the Jaiswal study was incorrect, as it emphasized the dynamic change in MRSA genetic epidemiology. In a very large country like India, even molecular epidemiology could differ between regions and provinces.

Local studies at Andalas University performed by Linosefa et al. revealed a more balanced number. Linosefa found only two out of 19 samples to be positive for pvl. 16 While this result was encouraging, it should be noted that this research was performed nearly a decade ago, and this research recommends the importance of surveillance, as was done in our study. This result also strengthened our hypothesis regarding how varying pvl prevalence could be.

Another interesting is the research done by Bhatta et al. in 2016 in Nepal. In his study, Bhatta observed that 90.4% of MRSA acquired in the community was pvl-positive, while pvl detected in nosocomial infections was only 7.1% positive. It was also found that pvls were not associated with bloodstream infections. 17 While the number of samples makes it hard to draw any definitive scientific conclusion, there is a high possibility that Indonesian S. aureus-infected patients mostly acquire their infection from the community. Interventions toward personal hygiene or public health measures might indeed lower pvl-positive prevalence.

With regards to eta, our results also show a disturbing new discovery in which most MRSA and MSSA were positive for the eta gene, with MSSA being significantly more likely to have eta. However, this has actually been predicted by several studies. Mohseni, in his study conducted in Iraq, found that 87.3% of isolates, or 131 samples, were positive for at least ET genes, with eta being dominant. eta was found in 76.7% of the samples obtained by Mehsani. In the conclusion, Mehsani deemed the finding a serious problem as it may spread through gene transfer between strains. 9

A study performed by Koosha et al. in 2013 is, however, quite consistent with our study. In the Kooesha study, lack of both eta and etb genes was only detected in eight (4%) isolates, and eta dominated ET prevalence. ETs play a role in colonization and invasion of injured mucosa and skin. Koosha also found that the distribution of ETs was largely similar between the MRSA and MSSA groups. Finally, drug resistance was abundant in the analyzed population. 18 However, a study performed by Montazeri et al. in 2021 revealed that eta expression was only 23.7%. It must be emphasized that the Montazeri study was limited to cancer patients, who may have immunological disorders that could affect staphylococcal infection prevalence. 19 This result might also emphasize regional fluctuations in molecular epidemiology. The study from the Middle East showed an even more surprising result: none of the MRSA isolates expressed the eta gene, out of 76 Iraqi isolates and 49 refugee isolates. 20

The study conducted in the People’s Republic of China by Li et al. showed much more consistent results compared with our study. Li found that most MRSA and MSSA contained eta genes at rates of 61.8% and 55%, respectively. Li’s research compared the prevalence from year to year, and there was indeed an increasing prevalence of eta gene prevalence, which was 23.1% in the 2013–2014 period and increased to 80.1% in the 2018–2019 period. 21 Our results revealed that Li’s increasing trend is persisting even in Indonesia.

A systematic review was conducted regarding eta-positive S. aureus in Iran alone. It showed quite variable results. Fooladi’s study showed 92.7% Staphylococcus aureus were positive for eta. 22 On the other side of the spectrum, eta detection could be as low as 0%, as shown in the Rahimi study in 2018. 23 This systematic review showed that even between regions, the variability of the S. aureus virulence gene is quite large. 24

Lastly, a study in Indonesia done by Santosaningsih et al. in 2017 showed that only 11.3% of patients encoded eta gene. 25 This result shows the importance of our study due to the very variable and volatile molecular epidemiology of S. aureus. Modifiable factors can help reduce the burden of disease.

The limitation of our study was the blind setting. While blind research might be ideal to recognize molecular epidemiology while preventing specific clinical bias ( e.g., lower pvl prevalence in bloodstream infection), 17 a clinical-to-molecular genetics relationship study could show the magnitude of this effect. Other limitations include our single-center study. However, our hospital is the main tertiary referral center in Sumatera Island, so it could reflect Staphylococcal molecular epidemiology in all of Sumatera. It still must be emphasized that most infections occur in the acute phase, and milder strains of S. aureus might have been treated in a primary health center.

 Conclusions

Our study shows that pvl and eta are more likely expressed in MSSA strains than in MRSA strains in Indonesia. This is a relatively new finding and could have significant implications for treating MSSA, as pvl and eta have been deemed virulence factors that could worsen disease progression. Public health measures are necessary, and continued surveillance is important to ensure that pvl or eta prevalence as a virulence factor decreases with time and effort.

 Data availability Underlying data

Figshare: Electrophoresis result of Staphylococcus aureus clinical isolates from Adam Malik General Hospital, https://doi.org/10.6084/m9.figshare.22796012.v1. 26

Figshare: Result of ETA and PVL PCR and Cefoxitime Screening of Clinical Isolates, https://doi.org/10.6084/m9.figshare.22849361.v1. 27

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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Control. 2023 Jan 29;12(1):4. 36709300 10.1186/s13756-023-01210-6 PMC9884412 Darboe S Dobreniecki S Jarju S : Prevalence of Panton-Valentine leukocidin (PVL) and antimicrobial resistance in community-acquired clinical Staphylococcus aureus in an urban Gambian hospital: a 11-year period retrospective pilot study. Front. Cell. Infect. Microbiol. 2019 May 22;9:170. 31192162 10.3389/fcimb.2019.00170 PMC6540874 Health Protection Agency Guidance on the diagnosis and management of PVL-associated Staphylococcus aureus infections (PVL-SA) in England:2008. (accessed July 17, 2012). Reference Source Shallcross LJ Fragaszy E Johnson AM : The role of the Panton-Valentine leucocidin toxin in staphylococcal disease: a systematic review and meta-analysis. Lancet Infect. Dis. 2013 Jan;13(1):43–54. 23103172 10.1016/S1473-3099(12)70238-4 PMC3530297 Mariutti RB Tartaglia NR Seyffert N : Exfoliative Toxins of Staphylococcus aureus. The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus. 2017 Mar 8. 10.5772/66528 Mohseni M Rafiei F Ghaemi EA : High frequency of exfoliative toxin genes among Staphylococcus aureus isolated from clinical specimens in the north of Iran: Alarm for the health of individuals under risk. Iran J. Microbiol. 2018 Jun;10(3):158–165. 30112153 PMC6087700 Hesari MR Salehzadeh A Darsanaki RK : Prevalence and molecular typing of methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin gene. Acta Microbiol. Immunol. Hung. 2018 Mar 1;65(1):93–106. 28859499 10.1556/030.64.2017.032 Boan P Tan HL Pearson J : Epidemiological, clinical, outcome and antibiotic susceptibility differences between PVL positive and PVL negative Staphylococcus aureus infections in Western Australia: a case control study. BMC Infect. Dis. 2015 Dec;15(1):1–6. 10.1186/s12879-014-0742-6 Darboe S Dobreniecki S Jarju S : Prevalence of Panton-Valentine leukocidin (PVL) and antimicrobial resistance in community-acquired clinical Staphylococcus aureus in an urban Gambian hospital: a 11-year period retrospective pilot study. Front. Cell. Infect. Microbiol. 2019 May 22;9:170. 31192162 10.3389/fcimb.2019.00170 PMC6540874 Jaiswal R Garg A Tripathi P : Epidemiology of Panton Valentine Leukocidin in clinical Staphylococcus aureus isolates-A prospective study at a tertiary care centre in North India. Clinical Epidemiology and Global Health. 2022 May 1;15:101006. 10.1016/j.cegh.2022.101006 Kaur H : Clindamycin resistance in PVL positive isolates of Staphylococcus aureus, Belgaum, North Karnataka (India). IOSR J. Eng. 2014;4(3):31–37. 10.9790/3021-04333137 D’Souza N Rodrigues C Mehta A : Molecular characterization of methicillin-resistant Staphylococcus aureus with Emergence of epidemic clones of sequence type (ST) 22 and ST 772 in Mumbai, India. J. Clin. Microbiol. 2010;48(5):1806–1811. 20351212 10.1128/JCM.01867-09 PMC2863868 Linosefa L Lestari DC Kusumaningrum A : Prevalensi Isolat MRSA Penghasil Panton-Valentine Leukocidin Pada Pasien Icu Rumah Sakit Tersier. Majalah Kedokteran Andalas. 2016;39(1):1–10. 10.22338/mka.v39.i1.p1-10.2016 Bhatta DR Cavaco LM Nath G : Association of Panton Valentine Leukocidin (PVL) genes with methicillin resistant Staphylococcus aureus (MRSA) in Western Nepal: a matter of concern for community infections (a hospital based prospective study). BMC Infect. Dis. 2016 May 15;16:199. 27179682 10.1186/s12879-016-1531-1 PMC4867903 Koosha RZ Fooladi AA Hosseini HM : Prevalence of exfoliative toxin A and B genes in Staphylococcus aureus isolated from clinical specimens. J. Infect. Public Health. 2014 May 1;7(3):177–185. 24637043 10.1016/j.jiph.2013.11.003 Abbasi Montazeri E Khosravi AD Khazaei S : Prevalence of methicillin resistance and superantigenic toxins in Staphylococcus aureus strains isolated from patients with cancer. BMC Microbiol. 2021 Dec;21:1–9. 10.1186/s12866-021-02319-7 Rasheed NA Hussein NR : Characterization of different virulent factors in methicillin-resistant Staphylococcus aureus isolates recovered from Iraqis and Syrian refugees in Duhok city, Iraq. PLoS One. 2020 Aug 17;15(8):e0237714. 32804961 10.1371/journal.pone.0237714 PMC7430753 Li X Huang T Xu K : Molecular characteristics and virulence gene profiles of Staphylococcus aureus isolates in Hainan, China. BMC Infect. Dis. 2019 Dec;19(1):1–2. 10.1186/s12879-019-4547-5 Imani Fooladi AA Ashrafi E Tazandareh SG : The distribution of pathogenic and toxigenic genes among MRSA and MSSA clinical isolates. Microb. Pathog. 2015;81:60–66. 25778391 10.1016/j.micpath.2015.03.013 Rahimi F Shokoohizadeh L : Characterization of virulence factors and prophage profiles of methicillin-resistant Staphylococcus aureus strains isolated from a referral hospital in Tehran, Iran. Jundishapur J. Microbiol. 2018;13:e59385. 10.5812/archcid.59385 Memariani M Memariani H Shahidi-Dadras M : Contemporary systematic review and meta-analysis of exfoliative toxin-producing Staphylococcus aureus strains isolated from patients in Iran. Reviews and Research in Medical Microbiology. 2020 Jan 1;31(1):1–10. 10.1097/MRM.0000000000000177 Santosaningsih D Santoso S Setijowati N : Prevalence and characterisation of Staphylococcus aureus causing community-acquired skin and soft tissue infections on Java and Bali, Indonesia. Trop. Med. Int. Health. 2018 Jan;23(1):34–44. 29124834 10.1111/tmi.13000 Amelia S : Electrophoresis result of Staphylococcus aureus clinical isolates from Adam Malik General Hospital. 2023. 10.6084/m9.figshare.22796012.v1 Amelia S : Result of ETA and PVL PCR and Cefoxitime Screening of Clinical Isolates.Dataset. figshare. 2023. 10.6084/m9.figshare.22849361.v1 10.5256/f1000research.147714.r202379 Reviewer response for version 1 Soebandrio Amin 1 Referee https://orcid.org/0000-0002-5856-4808 Clinical Microbiology, Faculty of Medicine, University of Indonesia, Depok, West Java, Indonesia

Competing interests: No competing interests were disclosed.

 24 11 2023 Copyright: © 2023 Soebandrio A 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Methodology:

Please clarify the followings:

 Antibiotic discs were placed inside or on MHA? 

What does it mean "after 24 hours of incubation"?

What suspension was transferred?  

DNA extraction

​Bacterial cells should be first suspended before centrifugation. In what solution or buffer the  bacteria was suspended.

Buffer contained Lysozyme was then added and vortexed. What buffer?

200uL of absolute ethanol was then added and vortexed.

Sentences in this paragraph should be rewritten to improve clarity or the procedures.

Discussion.

The percentage of PVL gene in community-acquired and hospital-acquired MRSA and MSSA should be described and discussed. More references should be included in the discussion and conclusion development. Among others, Melles DC, van Leeuwen WB, Boelens H, Peeters JK, Verbrugh HA, van Belkum A. Panton-Valentine Leukocidin Genes in Staphylococcus aureus. Emerg Infect Dis. 2006;12(7):1174-1175. https://doi.org/10.3201/eid1207.050865.

Others.

Any abbreviation should be described when it appears for the first time.

Example: ET.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Clinical Microbiology, Immunology, Molecular Biology.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References : Panton-Valentine Leukocidin Genes inStaphylococcus aureus. Emerging Infectious Diseases .2006;12(7) : 10.3201/eid1207.050865 1174-1175 10.3201/eid1207.050865 Amelia Sri Microbiology Department Faculty Medicine Universitas Sumatera Utara, Indonesia

Competing interests: No competing interest

 29 12 2023

Methodology:

Please clarify the followings:

 Antibiotic discs were placed inside or on MHA? 

What does it mean "after 24 hours of incubation"?

What suspension was transferred?  

Thank you for the correction all unclear parts in methodology has been corrected.

DNA extraction

​Bacterial cells should be first suspended before centrifugation. In what solution or buffer the  bacteria was suspended.

Buffer contained Lysozyme was then added and vortexed. What buffer?

200uL of absolute ethanol was then added and vortexed.

Sentences in this paragraph should be rewritten to improve clarity or the procedures.

Thank you for the correction all unclear parts in methodology has been corrected.

Discussion.

The percentage of PVL gene in community-acquired and hospital-acquired MRSA and MSSA should be described and discussed. More references should be included in the discussion and conclusion development. Among others, Melles DC, van Leeuwen WB, Boelens H, Peeters JK, Verbrugh HA, van Belkum A. Panton-Valentine Leukocidin Genes in Staphylococcus aureus. Emerg Infect Dis. 2006;12(7):1174-1175. https://doi.org/10.3201/eid1207.050865.

We found this research interesting and we had added this research in our discussion. Thank you

Others.

Any abbreviation should be described when it appears for the first time.

Example: ET.

Thank you for the correction. We had followed the review as best as we could.

10.5256/f1000research.147714.r221584 Reviewer response for version 1 Santosaningsih Dewi 1 Referee Department of Clinical Microbiology, Faculty of Medicine, Universitas Brawijaya, Malang, East Java, Indonesia

Competing interests: No competing interests were disclosed.

 24 11 2023 Copyright: © 2023 Santosaningsih D 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

Review

Manuscript title: Prevalence of Panton-Valentine leucocidin (pvl) and exfoliative toxin A (eta) gene within methicillin resistant and sensitive Staphylococcus aureus in an urban tertiary referral hospital: A molecular epidemiology pilot study

Aim: to observe pvl and eta as virulence gene prevalence in a North Sumatera tertiary referral health center.

General comments:

This study is important to predict the clinical manifestation of Staphylococcus aureus infection based on the properties of virulence genes including pvl and eta.

According to the previous studies, pvl and eta genes were more likely found among Staphylococcus aureus isolated from community setting. In this study, the authors have evaluated the prevalence of the pvl and eta genes among clinical Staphylococcus aureus isolated from a tertiary care hospital in Indonesia. The reasons or background for evaluating the prevalence of pvl and eta genes in the hospital setting have not been described yet (both in the abstract and body text).

The academic merit of this study have not been explained yet.

English grammar issue should be improved.

Title:

The genes should be written in italic (e.g pvl, eta)

Please use methicillin- susceptible Staphylococcus aureus instead of methicillin- sensitive Staphylococcus aureus

A tertiary care hospital in Indonesia indicates a referral hospital, therefore the title contains redundancy.

Abstract:

The abstract was not described the study clearly, it should be re-arranged. The abstract would be better after the authors revised the body text (introduction, materials and methods, results, and discussion)

Introduction:

The authors should be described the purpose of the study clearly. The urgency of the study was not described in the background yet.

Materials and methods:

The type of specimens of clinical isolates Staphylococcus aureus collected in this study were not mentioned yet.

The method of identification and antibiotic susceptibility test of the clinical isolates Staphylococcus aureus were not described clearly yet.

Results:

The description of results section should be re-arranged to be clearer and more understandable. The authors can start by explaining the prevalence of Staphylococcus aureus (overall) and then divide it into MRSA (%) and MSSA (%). Furthermore, the authors could describe the prevalence of pvl and eta genes among either MSSA or MRSA clinical isolates based on the type of specimen. How many pvl gene was positive among either MSSA or MRSA isolates from blood, sputum, and wound? Then, the severity of disease could be predicted.

Discussion:

The authors should elaborate the discussion section based on the results section, therefore the conclusion will be drawn adequately supported by the results.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Antimicrobial resistance; infection control; Staphylococcus aureus

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Amelia Sri Microbiology Department Faculty Medicine Universitas Sumatera Utara, Indonesia

Competing interests: No competing interest

 29 12 2023

General comments:

This study is important to predict the clinical manifestation of Staphylococcus aureus infection based on the properties of virulence genes including  pvl and  eta.

According to the previous studies,  pvl and  eta genes were more likely found among  Staphylococcus aureus isolated from community setting. In this study, the authors have evaluated the prevalence of the  pvl and  eta genes among clinical  Staphylococcus aureus isolated from a tertiary care hospital in Indonesia.  The reasons or background for evaluating the prevalence of pvl and eta genes in the hospital setting have not been described yet (both in the abstract and body text).

The academic merit of this study have not been explained yet.

English grammar issue should be improved.

 We had attempted to fix this issue, hopefully while minor, this could improve our manuscript.

Title:

The  genes should be written in  italic (e.g  pvl,  eta)

Please use methicillin- susceptible  Staphylococcus aureus instead of methicillin- sensitive Staphylococcus aureus

A tertiary care hospital in Indonesia indicates a referral hospital, therefore the title contains redundancy.

 Thank you for the correction. We had fixed the issue.

Abstract:

The abstract was not described the study clearly, it should be re-arranged. The abstract would be better after the authors revised the body text (introduction, materials and methods, results, and discussion)

Thank you for the correction. We had fixed the issue.

Introduction:

The authors should be described the  purpose of the study clearly. The  urgency of the study was not described in the background yet.

Thank you for the correction. We had fixed the issue.

Materials and methods:

The  type of specimens of clinical isolates  Staphylococcus aureus collected in this study were not mentioned yet.

The  method of identification and antibiotic susceptibility test of the clinical isolates  Staphylococcus aureus were not described clearly yet.

Thank you for the correction. We had fixed the issue.

Results:

The description of results section should be re-arranged to be clearer and more understandable. The authors can start by explaining the prevalence of  Staphylococcus aureus (overall) and then divide it into MRSA (%) and MSSA (%). Furthermore, the authors could describe  the prevalence of  pvl and  eta genes among either MSSA or MRSA clinical isolates based on the type of specimen. How many  pvl gene was positive among either MSSA or MRSA isolates from blood, sputum, and wound? Then, the severity of disease could be predicted.

We couldn't fix this issue, all samples were obtained from bacteremia patient through blood collection. No other samples were obtained through other methods. However this may be the limitation of our studies thus we had described this in limitation.

Discussion:

The authors should elaborate the discussion section based on the results section, therefore the conclusion will be drawn adequately supported by the results.

We had attempted to fix this issue.