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. 2021 Jul 20;16(7):e0237124. doi: 10.1371/journal.pone.0237124

DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

Adebayo Osagie Shittu 1,2,*, Tomiwa Adesoji 1, Edet Ekpenyong Udo 3
Editor: Monica Cartelle Gestal4
PMCID: PMC8291685  PMID: 34283846

Abstract

Staphylococcus aureus is an important human pathogen with an arsenal of virulence factors and a propensity to acquire antibiotic resistance genes. The understanding of the global epidemiology of S. aureus through the use of various typing methods is important in the detection and tracking of novel and epidemic clones in countries and regions. However, detailed information on antibiotic resistance and virulence genes of S. aureus, and its population structure is still limited in Africa. In this study, S. aureus isolates collected in South Africa (n = 38) and Nigeria (n = 2) from 2001–2004 were characterized by spa typing and DNA microarray. The combination of these two methods classified the isolates into seven spa types and three clonal complexes (CCs) i.e. t064-CC8 (n = 17), t037-CC8 (n = 8), t1257-CC8 (n = 6), t045-CC5 (n = 5), t951-CC8 (n = 1), t2723-CC88 (n = 1), t6238-CC8 (n = 1), and untypeable-CC8 (n = 1). A high percentage agreement (>95%) and kappa coefficient (>0.60) was largely observed with antibiotic susceptibility testing and DNA microarray, indicating substantial agreement. Some antibiotic and virulence gene markers were associated with specific clones. The detection of the collagen-binding adhesion (cna) gene was unique for t037-CC8-MRSA while the enterotoxin gene cluster (egc) and staphylococcal complement inhibitor (scn) gene were identified with t045-CC5-MRSA. Moreover, the combination of genes encoding enterotoxins (entA, entB, entK, entQ) was noted with most of the CC8 isolates. The t045-CC5-MRSA clone was positive for the mercury resistance (mer) operon. DNA microarray provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers for specific S. aureus clones in Africa.

Introduction

Staphylococcus aureus is a major human pathogen with an array of virulence factors, toxins, and a remarkable ability to acquire antibiotic resistance genes [1, 2]. This capability is further enhanced by the constant emergence of new and diverse clones within regions and countries [3]. The knowledge of the epidemiology of S. aureus, particularly of methicillin-resistant S. aureus (MRSA), is hinged on the application of various typing methods to assist in tracking newly emerging and epidemic clones [4]. Molecular epidemiological typing tools provide valuable information on the emergence of high-risk pandemic S. aureus clones, and the prevalence of antibiotic resistance mechanisms and virulence determinants. This is important in the development of intervention strategies and infection control measures in clinical and non-clinical settings [4].

The S. aureus epidemiological landscape in Africa has been described mainly through two molecular typing schemes i.e. Staphylococcus protein A (spa) typing and multilocus sequence typing (MLST) [5, 6]. These studies revealed that the most widely distributed methicillin-susceptible S. aureus (MSSA) clones in Africa include ST5, ST8, ST15, ST30, ST121, and ST152. Whereas ST5, ST30, ST121, and ST152 are predominant in Central and West Africa, ST8, ST15, ST30 are dominant in North Africa [5]. As for MRSA, ST239/241 is a major clone in many African countries, ST8 and ST88 in West, Central and East Africa, ST80 in North Africa, and ST5, ST36 and ST612 in South Africa [5, 6]. However, data on the repertoire of antibiotic resistance and virulence genes of S. aureus, and its clonal diversity in Africa are limited. In this study, we characterized archived S. aureus isolates from Nigeria and South Africa using DNA microarray. The study aimed to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of the isolates. This could provide information on antibiotic resistance and virulence genes that may represent epidemiological markers to specific S. aureus clones in Africa.

Materials and methods

Bacterial isolates

The S. aureus isolates have been described in previous investigations [7, 8] and were obtained from different clinical samples from 2001–2004. They comprised mainly archived S. aureus from South Africa (MRSA: n = 37; MSSA: n = 1) and two isolates from Nigeria were included based on their phenotypic resistance to cefoxitin and mupirocin, respectively [7]. The isolates (preserved in beads and stored at -80°C) were sub-cultured on Brain-Heart Infusion Agar (BHIA) plates, re-tested and confirmed (positive coagulase reaction) as S. aureus at the MRSA Reference Laboratory, Department of Microbiology, Faculty of Medicine, Kuwait University. Susceptibility of the isolates to penicillin, oxacillin, gentamicin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol and mupirocin was performed using the disk diffusion method according to the recommendations of the Clinical Laboratory Standards Institute [9]. S. aureus ATCC25923 was utilized as the control strain for antibiotic susceptibility testing (AST).

DNA isolation

S. aureus genomic DNA was obtained from an 18–24 hour old culture on Columbia blood agar. A pre-lysis step as described previously [10] was conducted before proceeding to the protocol of the DNeasy blood and tissue kit (Qiagen Hilden, Germany).

Spa typing and DNA microarray

Spa typing was performed by sequencing the hyper-variable region of the protein A gene (spa), as described previously [11]. The DNA microarray was performed to screen for the presence of genes for antibiotic resistance, virulence and to assign the isolates to clonal complexes (CCs). Genotyping of the isolates was performed using the S. aureus Genotyping Kit 2.0 system (Alere Technologies GmbH, Jena, Germany [now Abbott Rapid Diagnostics GmbH, Jena, Germany]) microarray-based assay. The array covers 334 different targets related to approximately 170 different genes and their allelic variants. The complete list of the target genes, sequences of probes and primers, and hybridization patterns together with the protocols have been published previously [12–14]. The DNA microarray was performed as described previously [12, 13]. S. aureus isolates were cultivated on Colombia blood agar. The DNA extraction was performed using lytic enzymes (lysostaphin, lysozyme, RNase) and buffer from the S. aureus Genotyping kit 2.0 and Qiagen DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. Thereafter, a linear amplification was performed using one primer for each target sequence. During the linear multiplex-amplification, biotin-16-dUTP was incorporated into the amplicons, which were then stringently hybridized to the specific probes on the microarray. After the washing steps, hybridization was detected using streptavidin horseradish peroxidase that triggered local precipitation at those spots where the amplicons were bound. Microarrays were photographed and analysed with a designated reader and software (IconoClust, Alere Technologies). The analysis allowed the detection of certain genes or alleles, as well as assignment to the CCs, and SCCmec types. Other target genes include species markers, capsule, agr group typing markers, common antibiotic resistance genes, toxins, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and immune evasion cluster. Isolates were assigned to CCs by automated comparison of the microarray hybridization profiles to a large database of previously characterized isolates [13]. The isolates were classified based on the spa type and clonal complexes (spa-CC).

Statistical analysis

In the identification of isolates susceptible and resistant to eight antibiotics, the percentage agreement between AST and DNA microarray was calculated from 2 x 2 tables. Furthermore, the level of agreement of the two methods was determined by the Cohen’s kappa (κ) test with 95% Confidence Intervals (CI) as described [15] and analyzed using GraphPad Prism (https://www.graphpad.com/quickcalcs/kappa1/). Data was interpreted as follows: no agreement (κ < 0), slight agreement (κ: 0.00–0.20), fair agreement (κ: 0.21–0.40), moderate agreement (κ: 0.41–0.60), substantial agreement (κ: 0.61–0.80), and almost perfect agreement (κ: 0.81–1.00).

Ethics statement

Ethical clearance was not necessary as archived isolates were analyzed in this study.

Results

The combination of specific S. aureus markers confirmed the identity of the isolates (n = 40) (S1 and S2 Tables). Based on the microarray data, all the isolates harboured genes encoding proteases (splA, splB, sspA, sspB and sspP), MSCRAMMs (bbp, clfA, clfB, ebpS, fib, fnbA, map, sasG, sdrC, vwB), leukocidin (lukF and lukE), haemolysin (hlgA), and intracellular adhesion (icaA). However, none possessed the exfoliative toxin (etA, etB, etD), epidermal cell differentiation (edinA, edinB and edinC), surface protein involved in biofilm production (bap), and the ACME genes (S4 Table).

AST and DNA microarray were in almost perfect agreement in the detection of isolates susceptible and resistant to chloramphenicol, erythromycin, gentamicin and mupirocin. Moreover, substantial agreement was observed between the two methods in the screening of the isolates against penicillin, oxacillin and tetracycline, while a fair agreement was noted for trimethoprim-sulfamethoxazole (Table 1).

Table 1. Percentage and level of agreement between antibiotic susceptibility testing (AST) and DNA microarray with S. aureus isolates from Nigeria and South Africa.

Penicillin DNA microarray (blaZ) Tetracycline DNA microarray (tetK, tetM)
Negative Positive Total Negative Positive Total
Antibiotic susceptibility testing Susceptible 1 0 1 Antibiotic susceptibility testing Susceptible 4 1 5
Resistant 1 38 39 Resistant 1 34 35
Total 2 38 40 Total 5 35 40
Agreement (%) 98% Agreement (%) 95%
Kappa coefficient 0.66 95% CI: 0.03 to 1.00 Kappa coefficient 0.77 95% CI: 0.47 to 1.00
Oxacillin DNA microarray (mecA) Trimethoprim-Sulfamethoxazole DNA microarray (dfrA)
Negative Positive Total Negative Positive Total
Antibiotic susceptibility testing Susceptible 2 0 2 Antibiotic susceptibility testing Susceptible 6 1 7
Resistant 1 37 38 Resistant 10 23 33
Total 3 37 40 Total 16 24 40
Agreement (%) 98% Agreement (%) 73%
Kappa coefficient 0.79 95% CI: 0.39 to 1.00 Kappa coefficient 0.37 95% CI: 0.10 to 0.64
Gentamicin DNA microarray (aacA-aphD, aphA3) Chloramphenicol DNA microarray (cat)
Negative Positive Total Negative Positive Total
Antibiotic susceptibility testing Susceptible 3 1 4 Antibiotic susceptibility testing Susceptible 33 0 33
Resistant 0 36 36 Resistant 0 7 7
Total 3 37 40 Total 33 7 40
Agreement (%) 98% Agreement (%) 100%
Kappa coefficient 0.84 95% CI: 0.55 to 1.00 Kappa coefficient 1.00 95% CI: 1.00 to 1.00
Erythromycin DNA microarray (ermA, ermC) Mupirocin DNA microarray (mupR)
Negative Positive Total Negative Positive Total
Antibiotic susceptibility testing Susceptible 6 1 7 Antibiotic susceptibility testing Susceptible 39 0 39
Resistant 0 33 33 Resistant 0 1 1
Total 6 34 40 Total 39 1 40
Agreement (%) 98% Agreement (%) 100%
Kappa coefficient 0.91 95% CI: 0.73 to 1.00 Kappa coefficient 1.00 95% CI: 1.00 to 1.00
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Corresponding antibiotic resistance genes detected by DNA microarray are indicated in parenthesis.

Molecular typing classified the isolates into seven spa types t037, t045, t064, t951, t1257, t2723 and t6238, and three clonal complexes (CCs), CC5, CC8 and CC88. The delineation of the various groups (spa-CC) and their unique characteristics are described (Fig 1).

Fig 1. Antibiotyping and molecular characterization of S. aureus isolates from Nigeria and South Africa.

Fig 1

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CC5

t045-CC5 (South German EMRSA or the South German EMRSA/Italian Clone)

Five MRSA isolates belonged to t045. They were grouped with agr group II and capsule type 5. While most of them (4/5) possessed the SCCmec II element, the cassette chromosome recombinase genes A/B-2 was not detected in one MRSA isolate and was assigned to SCCmec type I (S2 Table). All the t045 isolates harboured the resistance genes for aminoglycosides (aacA-aphD and aphA3), macrolides (ermA), fosfomycin (fosB), streptothricine (sat), and quaternary ammonium compounds (qacA). Besides, they were positive for the mercury resistance operon (mer). The hybridization signal for tetK and tetM was absent with one tetracycline-resistant MRSA (SA9). The unique features of this clone include the detection of the enterotoxin gene cluster (egc), the presence of only one of the immune evasion cluster (IEC) genes (scn), and the lack of the tetracycline resistance (tetK, tetM) and enterotoxin (entA, entB, entK, entQ) genes.

CC8

The CC8 isolates belonged to five spa types, consisting of t064 (n = 17), t037 (n = 8), t1257 (n = 6), t951 (n = 1) and t6238 (n = 1). They were associated with agr group I and capsule type 5, except those grouped with spa type t037 and assigned to capsule type 8. One MRSA each could not be characterized by spa and agr typing.

t037-CC8 (Vienna/Hungarian/Brazilian clone)

The t037 spa type was represented by eight isolates. They possessed the SCCmec type III genetic element as well as the mer (Fig 1) and the recombinase (ccrC) genes (S2 Table). However, one MRSA (SA4) lacked the hybridization signal for mecA. All the t037 isolates possessed the aphA3 and tetM genes, and those that exhibited phenotypic resistance to erythromycin (n = 8) and chloramphenicol (n = 4) were positive for the corresponding genes (ermA and cat) (Fig 1). Seven isolates harboured the fosfomycin and streptothricine resistance determinants (fosB, sat). The aacA-aphD and tetK genes were both identified in at least five isolates that were phenotypically resistant to gentamicin and tetracycline, respectively. All the trimethoprim-sulfamethoxazole-resistant MRSA were dfrA (dihydrofolate reductase) negative. The enterotoxin genes (entA, entK and entQ) were detected in at least six isolates, while the distinctive feature of this clone was the positive result for the collagen-binding adhesion (cna) gene.

t064-CC8 (USA500)

This clone comprised 16 isolates (MSSA n = 1; MRSA n = 15) from South Africa and MRSA (n = 1) from Nigeria. The SCCmec type IV was identified in all the MRSA from South Africa, while the isolate from Nigeria carried the SCCmec V element and the mer operon (Fig 1). The following genes i.e. aacA-aphD, dfrA, ermC, and tetM were detected in at least 14 of the 16 MRSA isolates. Only two MRSA were qacA-positive. One MSSA (SA17) that was susceptible (phenotypic) to gentamicin, tetracycline and trimethoprim-sulfamethoxazole was positive for the corresponding resistance genes. The combination of genes encoding enterotoxins (entA, entB, entK, entQ) was a common feature noted with most of the isolates.

t951-CC8 (Lyon Clone/UK-EMRSA-2)

The only MRSA (SCCmec IV) possessed the antibiotic resistance genes (tetM and fosB), and enterotoxin A gene. It was also positive for the immune evasion cluster genes (sak and scn).

t1257-CC8 (USA500)

The six isolates belonging to this spa type were associated with SCCmec type IV. One of the isolates (SA32) was susceptible to erythromycin but yielded a hybridization signal for the ermC gene. Moreover, the isolates exhibited similar antibiotic resistance gene profiles (aacA-aphD, dfrA, ermC, and tetM), enterotoxin (entA, entB, entK, entQ) and immune evasion (sak, scn) gene content with those assigned with t064-CC8.

t6238-CC8 (USA500)

The single isolate associated with this spa type harboured the SCCmec IV element, and the antibiotic resistance (aacA-aphD, dfrA, fosB, tetM) and enterotoxin (entA, entB, entK) genes were identified. Furthermore, the isolate was only positive for one of the IEC genes (sak). It was negative for agr types I-IV.

spa untypeable-CC8 (USA500)

The gene content of the MRSA isolate was similar to other members of CC8.

t2723-CC88

A single MSSA isolate was associated with t2723. It was assigned to agr group III and capsule type 8. Phenotypic resistance to tetracycline and mupirocin was confirmed by the detection of the tetK and mupR genes, respectively. No enterotoxin gene was detected in this isolate. However, it was positive for the IEC (chp, sak, scn) and Panton-Valentine Leukocidin (PVL) genes.

Discussion

The combination of spa typing and DNA microarray was utilized to characterize S. aureus isolates obtained in South Africa and Nigeria. The DNA microarray is a DNA-DNA hybridization method containing several probes for the rapid identification, characterization of S. aureus resistance and virulence gene profiles, and their assignment into clonal complexes [12]. In the detection of isolates susceptible and resistant to eight antibiotics, substantial to an almost perfect agreement was mainly observed between AST and DNA microarray. The results also revealed the association of some antibiotic resistance gene determinants with certain MRSA clones. Specifically, the aphA3, ermA, and mer genes were unique characteristics associated with t037-CC8-MRSA and t045-CC5-MRSA (Fig 1). Although t037-CC8-MRSA isolates exhibited resistance to trimethoprim-sulfamethoxazole, they were negative for dfrA that encode resistance to trimethoprim in S. aureus. While trimethoprim resistance in S. aureus can be due to any of three determinants, dfrA, dfrG and dfrK, the dfrG is associated with trimethoprim resistance in the majority of the trimethoprim-resistant S. aureus in Africa [16, 17]. Trimethoprim-resistant S. aureus isolates harbouring dfrG and associated with spa types t037 and t064 have also been reported in Nigeria [16], which is similar to our findings in this study. This observation suggests that dfrG was responsible for trimethoprim resistance in our dfrA-negative isolates. The DNA-microarray platform used in this study does not include probes for dfrG and explains the reason it was not detected in these isolates.

Interestingly, although t037-CC8-MRSA and t045-CC5-MRSA shared common antibiotic resistance determinants, they differed in the carriage of the tetracycline resistance determinants (tetK, tetM) that was present in t037-CC8-MRSA and not in t045-CC5-MRSA (Fig 1). MRSA is characterized by the presence of the staphylococcal cassette chromosome mec (SCCmec), a mobile 21- to 60-kb genetic element, and 13 SCCmec types have been identified [18]. The SCCmec types II (53.0 kb) and III (66.9 kb) are large elements due to the acquisition and insertion of mobile genetic elements (MBEs). The antibiotic resistance genes observed in the two clones have been identified on MBEs such as transposons including Tn554 (ermA), Tn4001 (aacA-aphD), Tn5405 (aphA3, sat), Tn916 (tetM), and plasmids i.e. pT181 (tetK), pI258 (mer) and pNE131 (ermC) [19–21]. The t037-CC8-MRSA and t045-CC5-MRSA lineages are typical hospital-associated clones, and their multi-resistant nature are attributed to the various MBEs that harbour different antibiotic resistance genes in the joining regions J1 to J3 [22].

We note with interest that all the t045-CC5-MRSA isolates possessed the mercury resistance operon, a feature also commonly present with t037-CC8-MRSA. The mechanism for the acquisition of SCCmercury by S. aureus is still unclear although two views have been postulated. The first suggests that this gene determinant may have been integrated into an SCC element with the emergence of SCCmercury in coagulase-negative staphylococci, which is subsequently transferred to S. aureus. The second opinion is that a plasmid (e.g. pI258) harbouring the resistance gene determinant to the quaternary ammonium compound could have been transferred to S. aureus and integrated into an SCC element to form SCCmercury [23]. Interestingly, a comparison of our results with a previous report consisting of a collection of CC5-MRSA isolates in the Western Hemisphere [24] revealed that the presence of mer gene in t045-CC5-MRSA is a rare feature of this clone. Therefore, future studies are to ascertain whether our observation represents a recent acquisition of the mer operon by this lineage. In this study, MRSA with SCCmec types IV and V were identified and in addition to β-lactam resistance, the isolates classified as t064/t1257/t6238-CC8 harbouring SCCmec IV also possessed genes (aacA-aphD, dfrA, ermC and tetM) mediating resistance to aminoglycosides, trimethoprim, macrolides and tetracycline, respectively. The presence of these resistance determinants in our archived isolates support existing data [25, 26] that this multi-resistant lineage is established and well adapted in the hospital environment in South Africa.

Mupirocin is a topical antibiotic that is widely used for nasal decolonization and the prevention of S. aureus infections. However, the emergence and increasing rates of resistance and treatment failure are major drawbacks [27]. Two levels of mupirocin resistance have been elucidated i.e. low-level and high-level resistance attributed to various chromosomal mutations, and the acquisition of plasmids (harbouring mupA or mupB genes), respectively [28, 29]. Decolonization is ineffective with patients and personnel colonized with high-level mupirocin resistant (HmupR) MRSA [27]. Moreover, mupirocin resistance could also facilitate the spread of multidrug resistance through co-selection with other plasmid-borne resistance genes [30, 31]. In this study, the genetic background (t2723-CC88; PVL-positive) of a HmupR MSSA was determined (Fig 1; S2 Table). Only two studies have provided information on the genetic lineage of HmupR S. aureus from clinical samples in Africa which include t127, t4805 (MSSA), and t032, t1467 (MRSA) [32, 33]. The prevalence and burden of mupirocin-resistant S. aureus are still unclear in many countries in Africa [34]. CC88-MRSA is an established lineage in West, Central and East Africa [5], and the identification of a HmupR-PVL-positive MSSA from this background is worthy of note. Prospective national and continental studies are important to evaluate the prevalence, burden and genetic background of mupR S. aureus in Africa.

S. aureus produces a range of virulence determinants including at least 23 exotoxins which are categorized into staphylococcal enterotoxins (SEs) comprising SEA-SEE, and staphylococcal enterotoxin-like (SEl) consisting of SEG-SElY [35]. They belong to the family of superantigens (SAgs) with a unique feature to act primarily on the intestine to cause enteritis characterized by emesis [36]. Our investigation indicated that some enterotoxin genes were associated with specific genetic backgrounds, which is in support of previous reports [37, 38]. The t037-CC8-MRSA was characterized by the detection of entA, entK, and entQ genes. The egc cluster (entG, entI, entM, entN, entO, and entU) were associated with t045-CC5-MRSA, while the entA, entB, entK and entQ genes were linked with t064/t1257-CC8. The SE genes are carried and disseminated through different MBEs which include prophages, plasmids, transposons, and S. aureus pathogenicity islands (SaPIs) [35]. The entA-entK-entQ genes are found on the prophage ΦSa3ms and ΦSa3mw, the egc cluster on the genomic island vSaβ and entB-entK-entQ have been identified on SaPI3 [39].

Conclusions

This study characterized archived S. aureus isolates from Nigeria and South Africa using two molecular-based typing methods (spa typing and DNA microarray). A high level of agreement was observed with AST and DNA microarray. Also, some antibiotic resistance and virulence genes were associated with specific clonal lineages. The aphA3, ermA, and mer genes were associated with hospital-associated clones (t037-CC8-MRSA and t045-CC5-MRSA), cna with t037-CC8-MRSA, the egc cluster and scn with t045-CC5-MRSA, and entA, entB, entK, entQ with most of the CC8 isolates. There are some limitations to this study. They include the small and disproportionate number of S. aureus analyzed from the two African countries. Moreover, we did not investigate factors that could be responsible for the discrepant results with some isolates based on AST and the microarray assay. The DNA microarray technology has some constraints i.e. high cost of reagents and equipment in resource-limited settings, cross-hybridization reaction, and a moderate level of reproducibility. Nevertheless, the main advantages of the technology include speed compared with procedures involving several PCR and gel electrophoresis, the diverse array of genes investigated, and the quantum of data generated. DNA microarray has provided useful information on gene determinants for antibiotic resistance and virulence, and their relationship with some S. aureus genetic background in Africa. Although the outcome of this investigation is not representative of the diverse S. aureus clonal lineages in Africa, the genetic markers noted could be a useful adjunct in the molecular typing and tracking of new and emerging S. aureus clones on the continent.

Supporting information

S1 Table. Raw data on DNA hybridization reaction (indicated as either positive, negative or ambiguous) of the S. aureus isolates.

(XLSX)

Click here for additional data file. (2.4MB, xlsx)
S2 Table. Detailed characteristics of the S. aureus isolates (n = 40) including antibiotyping, spa types, Clonal complex (CC) affiliation, and DNA microarray hybridization results of some antibiotic resistance and virulence genes.

(XLS)

Click here for additional data file. (114.5KB, xls)
S3 Table. List of target genes and DNA sequences of the probes and primers (including GenBank coordinates) for microarray in S. aureus Genotyping Kit 2.0 system.

(XLS)

Click here for additional data file. (153.5KB, xls)
S4 Table. Distribution of selected genes (DNA microarray) in S. aureus isolates from Nigeria and South Africa.

(DOC)

Click here for additional data file. (231KB, doc)

Acknowledgments

We appreciate the technical assistance of Mrs Tina Verghese, Bindu Mathew and useful comments from Oluyomi Adesoji and Busola Adebusoye.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

AOS: Alexander von Humboldt Foundation (“Georg Forster-Forschungsstipendium”) AOS: Deutsche Forschungsgemeinschaft (SCHA 1994/5-1). AOS: Open Access Publication Fund of the University of Muenster. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Monica Cartelle Gestal

26 Oct 2020

PONE-D-20-22325

DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

PLOS ONE

Dear Dr. Shittu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please revise the manuscript taking into account the suggestions and comments of the reivewers. These cahnges will substantially improve the manuscript. Please send the revised version as soon as your convenience.

Please submit your revised manuscript by Dec 10 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes the typing of isolates of Staphylococcus aureus from South Africa and Nigeria. The manuscript is a survey of historic strains from these two countries and compares spa typing to DNA microarray. Overall, the manuscript is well written. As this manuscript is mainly descriptive the analysis is limited but satisfactorily covers the results. More detail of the methods is required to allow for interpretation of the results and conclusions.

Major issues:

1. The methods section lacks the details required to understand what was done in the experiments. How were the strains grown? How was DNA obtained? Did you carry out any statistical analysis? How many biological and technical repeats were carried for each experiment?

2. You have mentioned 2 outliers. With SA32 did you confirm that ermC gene was intact by sequencing? This would tell you if it is a technical problem or not. This approach could also be used for SA17 and blaZ but a negative result would be inconclusive.

Minor:

Line 216: The abbreviation for mupirocin resistance should be MuR throughout.

Line 218: insert full stop after references.

Reviewer #2: Staphylococcus aureus is an important human pathogen with an arsenal of virulence factors and a propensity to acquire antibiotic resistance genes. The understanding of the global epidemiology of S. aureus through the use of various typing methods is important in the detection and tracking of novel and epidemic clones in countries and regions. However, detailed information on antibiotic resistance and virulence genes of S. aureus, and its population structure is still limited in Africa. The current manuscript by Shittu et al. screens S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. So while the data reported could be useful, more information and additional analyses are needed for this the report to be of value to the scientific community. Specific comments and suggestions are listed below for consideration:

1) The title is misleading. The current study only includes 40 isolates from Nigeria and only 2 from South Africa. How much value is placed on an “N” of two? To remedy this situation, the number of isolates evaluated for each location should be included in the title. For example “Microarray analysis of 40 Staphylococcus aureus isolates from Nigeria and 2 Staphylococcus aureus isolates South Africa” as one possibility for improvement.

2) Table 1 is confusing. The percentage of selected genes from microarray is not really useful information. Either instead of or in addition to the data provided, the authors should screen the isolates for the presence of a reference nucleotide sequence for each gene. Then report the percent nucleotide identity of each gene in the isolate to the reference nucleotide sequence. This will provide much more useful data!

3) The full nucleotide sequence of each gene in the microarray needs to be provided. Currently no sequence or length of the sequence is provided. Length of the sequence is important because in some instances the length for a microarray target might be only 75 nucleotides. Any search or table for a study of this nature, needs to include the full nucleotide sequence as part of the minimum requirements for publishing!

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Jul 20;16(7):e0237124. doi: 10.1371/journal.pone.0237124.r002

Author response to Decision Letter 0

31 Dec 2020

Re: Revised manuscript: PONE-D-20-22325 – DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

On behalf of other co-authors, I forward herewith the revised manuscript for your kind consideration. It has been formatted to meet the PLOS ONE’s publication criteria. Furthermore, the comments of the reviewers have been examined and we provide a point-by-point response as indicated below.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: The revised manuscript has been formatted according to the requirements with PLOS ONE.

2. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files.

Response: Tables 1 and 2 have been included in the revised manuscript. The previous Table 1 is now Supplementary 2.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also include your statement in the online submission form via 'Edit submission'.

Response: The ethics statement has been transferred to the Methods section of the manuscript.

Reviewers' comments:

Reviewer #1: This manuscript describes the typing of isolates of Staphylococcus aureus from South Africa and Nigeria. The manuscript is a survey of historic strains from these two countries and compares spa typing to DNA microarray. Overall, the manuscript is well written. As this manuscript is mainly descriptive the analysis is limited but satisfactorily covers the results. More detail of the methods is required to allow for interpretation of the results and conclusions.

Major issues:

1. The methods section lacks the details required to understand what was done in the experiments. How were the strains grown? How was DNA obtained? Did you carry out any statistical analysis? How many biological and technical repeats were carried for each experiment?

Response: We appreciate the comments of the reviewer. Additional information on the strains including DNA isolation is provided in the revised manuscript. The tests were generally not repeated due to their high level of reproducibility. Spa typing and microarray were only repeated in a few instances where the results were ambiguous. We have provided information on some constraints in the conclusion of the manuscript, nevertheless, they do not affect the overall objective of the study.

Statistical analysis: The percentage agreement between the phenotypic (antibiotic susceptibility testing) and genotypic (microarray) methods in the identification of isolates as ‘susceptible’ and ‘resistant’ was calculated from the 2 X 2 tables. Moreover, the Cohen Kappa (κ) test was employed to determine the level of agreement of the two methods. The data is described in the revised manuscript.

2. You have mentioned 2 outliers. With SA32 did you confirm that ermC gene was intact by sequencing? This would tell you if it is a technical problem or not. This approach could also be used for SA17 and blaZ but a negative result would be inconclusive.

Response: We did not conduct a further investigation including sequencing of the blaZ and ermC genes on the isolates that were resistant to penicillin and susceptible to erythromycin, respectively. This is indicated as a limitation of the study in the conclusion. Our focus was to determine the percentage and level of agreement between antibiotic susceptibility testing and DNA microarray. We have provided data (in the revised manuscript) which showed a high level of agreement with the two methods.

Minor:

Line 216: The abbreviation for mupirocin resistance should be MuR throughout.

Response: We are grateful for the comment of the reviewer. However, we prefer the use of mupR (which is widely used) than MuR as suggested.

Line 218: insert full stop after references.

Response: The observation is noted and the amendment is in the revised manuscript.

Reviewer #2: Staphylococcus aureus is an important human pathogen with an arsenal of virulence factors and a propensity to acquire antibiotic resistance genes. The understanding of the global epidemiology of S. aureus through the use of various typing methods is important in the detection and tracking of novel and epidemic clones in countries and regions. However, detailed information on antibiotic resistance and virulence genes of S. aureus, and its population structure is still limited in Africa. The current manuscript by Shittu et al. screens S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. So while the data reported could be useful, more information and additional analyses are needed for this the report to be of value to the scientific community. Specific comments and suggestions are listed below for consideration:

1) The title is misleading. The current study only includes 40 isolates from Nigeria and only 2 from South Africa. How much value is placed on an “N” of two? To remedy this situation, the number of isolates evaluated for each location should be included in the title. For example “Microarray analysis of 40 Staphylococcus aureus isolates from Nigeria and 2 Staphylococcus aureus isolates South Africa” as one possibility for improvement.

Response: We appreciate the comments of the reviewer. The small and disproportionate number of S. aureus isolates from the two African countries is a limitation to the study, and this is highlighted in the manuscript. Nevertheless, the origin and number of the isolates are indicated in the abstract, hence we believe it is not necessary to change the title of the manuscript. Moreover, we reiterate that the main objective of the study was to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates (from Africa) based on the microarray assay. We strongly believe that despite the limitation indicated above, the investigation has fulfilled this objective.

2) Table 1 is confusing. The percentage of selected genes from microarray is not really useful information. Either instead of or in addition to the data provided, the authors should screen the isolates for the presence of a reference nucleotide sequence for each gene. Then report the percent nucleotide identity of each gene in the isolate to the reference nucleotide sequence. This will provide much more useful data!

Response: We are grateful for the comment of the reviewer. The microarray assay (Identibac S. aureus genotyping Kit 2.0) is a widely acknowledged tool specifically developed (Alere Technology, Jena, Germany [now Abbott Rapid Diagnostics GmbH, Jena, Germany]) for genotyping of S. aureus isolates. Furthermore, the list of primer/probe sequences has been published previously (Monecke et al., 2011), and this reference is provided in the revised manuscript.

Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D, Kadlec K, Kearns A, Laurent F, O'Brien FG, Pearson J, Ruppelt A, Schwarz S, Scicluna E, Slickers P, Tan HL, Weber S, Ehricht R. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011 Apr 6;6(4):e17936. doi: 10.1371/journal.pone.0017936.

Information on the proportion of isolates positive for the antibiotic resistance and virulence genes of S. aureus isolates based on the microarray assay has been described in some recent publications including:

1. Peterson JC, Durkee H, Miller D, Maestre-Mesa J, Arboleda A, Aguilar MC, Relhan N, Flynn HW Jr, Amescua G, Parel JM, Alfonso E. Molecular epidemiology and resistance profiles among healthcare- and community-associated Staphylococcus aureus keratitis isolates. Infect Drug Resist. 2019 Apr 11;12:831-843. doi: 10.2147/IDR.S190245.

2. Senok A, Somily AM, Nassar R, Garaween G, Kim Sing G, Müller E, Reissig A, Gawlik D, Ehricht R, Monecke S. Emergence of novel methicillin-resistant Staphylococcus aureus strains in a tertiary care facility in Riyadh, Saudi Arabia. Infect Drug Resist. 2019 Sep 3;12:2739-2746. doi: 10.2147/IDR.S218870.

3. Senok A, Nassar R, Celiloglu H, Nabi A, Alfaresi M, Weber S, Rizvi I, Müller E, Reissig A, Gawlik D, Monecke S, Ehricht R. Genotyping of methicillin-resistant Staphylococcus aureus from the United Arab Emirates. Sci Rep. 2020 Oct 29;10(1):18551. doi: 10.1038/s41598-020-75565-w.

4. Udo EE, Boswihi SS, Mathew B, Noronha B, Verghese T, Al-Jemaz A, Al Saqer F. Emergence of Methicillin-Resistant Staphylococcus aureus Belonging to Clonal Complex 15 (CC15-MRSA) in Kuwait Hospitals. Infect Drug Resist. 2020 Feb 21;13:617-626. doi: 10.2147/IDR.S237319.

5. Boswihi SS, Udo EE, Mathew B, Noronha B, Verghese T, Tappa SB. Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Patients Admitted to Kuwait Hospitals in 2016-2017. Front Microbiol. 2020 Jan 8;10:2912. doi: 10.3389/fmicb.2019.02912.

In our study, data on the proportion of isolates positive or negative for the antibiotic resistance genes (microarray) and antibiotic susceptibility testing (AST) was essential in determining the percentage and level of agreement of the two methods as described in Table 1 (revised manuscript). In conclusion, the previous Table 1 is now presented as Supplementary data (S2 Table).

3) The full nucleotide sequence of each gene in the microarray needs to be provided. Currently no sequence or length of the sequence is provided. Length of the sequence is important because in some instances the length for a microarray target might be only 75 nucleotides. Any search or table for a study of this nature, needs to include the full nucleotide sequence as part of the minimum requirements for publishing!

Response: We appreciate the comment of the reviewer. The list of primer/probe sequences for the microarray has been published previously (Monecke et al., 2011), and this reference is included in the revised manuscript.

Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, Chow H, Ip M, Jatzwauk L, Jonas D, Kadlec K, Kearns A, Laurent F, O'Brien FG, Pearson J, Ruppelt A, Schwarz S, Scicluna E, Slickers P, Tan HL, Weber S, Ehricht R. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011 Apr 6;6(4):e17936. doi: 10.1371/journal.pone.0017936.

Yours sincerely,

Professor Adebayo Shittu

Corresponding author

Decision Letter 1

Monica Cartelle Gestal

27 Jan 2021

PONE-D-20-22325R1

DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

PLOS ONE

Dear Dr. Shittu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Reviewer 2 still has some concerns about the methodology and results that are included in this study. Please make sure you carefully revised the manuscript and address all the reviewer comments prior to resubmission.

==============================

Please submit your revised manuscript by Mar 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed all my comments in a satisfactory manor and I have nothing further to suggest.

Reviewer #2: This is the second review for the manuscript by Shittu et al., which describes the screening of S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made some revisions to accommodate some of the suggestions posed by reviewers, overall, the authors fail to adequately address some key points raised by the reviewers. The manuscript in its current form is in need of revision to adequately address these key points to improve its suitability for publication.

Major key points:

1) The manuscript in its current form fails to meet PLoS One Data availability requirements. Specifically, “Data: PLOS journals require authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception.” Also, the instructions to authors state that all manuscripts incorporating Microarray experiments must meet MIAME (Minimum Information About a Microarray Experiment) requirements. To help aid the authors, MIAME describes the Minimum Information About a Microarray Experiment that is needed to enable the interpretation of the results of the experiment unambiguously and potentially to reproduce the experiment. [Brazma et al. (2001), Nature Genetics].

The six most critical elements contributing towards MIAME are:

a. The raw data for each hybridisation (e.g., CEL or GPR files)

b. The final processed (normalised) data for the set of hybridisations in the experiment (study) (e.g., the gene expression data matrix used to draw the conclusions from the study)

c. The essential sample annotation including experimental factors and their values (e.g., compound and dose in a dose response experiment)

d. The experimental design including sample data relationships (e.g., which raw data file relates to which sample, which hybridisations are technical, which are biological replicates)

e. Sufficient annotation of the array (e.g., gene identifiers, genomic coordinates, probe oligonucleotide sequences or reference commercial array catalog number)

f. The essential laboratory and data processing protocols (e.g., what normalisation method has been used to obtain the final processed data)

The current manuscript fails to provide any of this required information. While references are given, they do not disclose the DNA sequence information of the probes. The manuscript needs to be revised to include all of the above required information! Additionally, providing a supplementary table listing target or gene names, along with the actual DNA sequence of the probes and primers is essential along with MIAME compliance.

2) Lines 81-82: Provide the precise CLSI standard version applied in the phenotpical assay.

3) The authors claim that the objective of the current was to determine the percentage and level of agreement between antibiotic sensitive testing and DNA microarray, which alone is not suitable for publication. The authors also claim that the objective of the current was to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates from Africa. So, which is it? Please clarify! If the second objective is the correct one, then please address the original comments made both reviewers.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Jul 20;16(7):e0237124. doi: 10.1371/journal.pone.0237124.r004

Author response to Decision Letter 1

21 Feb 2021

Professor Monica Cartelle Gestal

Academic Editor

PLoS One

Dear Professor Gestal,

Re: Revised manuscript: PONE-D-20-22325-R2 – DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

On behalf of other co-authors, I forward herewith the revised manuscript for your kind consideration. The comments of reviewer 2 have been examined and we provide a point-by-point response as indicated below.

Reviewer’s comments

Reviewer #2: This is the second review for the manuscript by Shittu et al., which describes the screening of S. aureus isolates collected in South Africa (n=38) and Nigeria (n=2) from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made some revisions to accommodate some of the suggestions posed by reviewers, overall, the authors fail to adequately address some key points raised by the reviewers. The manuscript in its current form is in need of revision to adequately address these key points to improve its suitability for publication.

Major key points:

1) The manuscript in its current form fails to meet PLoS One Data availability requirements. Specifically, “Data: PLOS journals require authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception.” Also, the instructions to authors state that all manuscripts incorporating Microarray experiments must meet MIAME (Minimum Information About a Microarray Experiment) requirements. To help aid the authors, MIAME describes the Minimum Information About a Microarray Experiment that is needed to enable the interpretation of the results of the experiment unambiguously and potentially to reproduce the experiment. [Brazma et al. (2001), Nature Genetics].

The six most critical elements contributing towards MIAME are:

a. The raw data for each hybridisation (e.g., CEL or GPR files)

b. The final processed (normalised) data for the set of hybridisations in the experiment (study) (e.g., the gene expression data matrix used to draw the conclusions from the study)

c. The essential sample annotation including experimental factors and their values (e.g., compound and dose in a dose response experiment)

d. The experimental design including sample data relationships (e.g., which raw data file relates to which sample, which hybridisations are technical, which are biological replicates)

e. Sufficient annotation of the array (e.g., gene identifiers, genomic coordinates, probe oligonucleotide sequences or reference commercial array catalog number)

f. The essential laboratory and data processing protocols (e.g., what normalisation method has been used to obtain the final processed data)

The current manuscript fails to provide any of this required information. While references are given, they do not disclose the DNA sequence information of the probes. The manuscript needs to be revised to include all of the above required information! Additionally, providing a supplementary table listing target or gene names, along with the actual DNA sequence of the probes and primers is essential along with MIAME compliance.

Response: We appreciate the comments of the reviewer. A description of the microarray assay including the raw data (matrix in excel) has been provided in the revised manuscript. Moreover, in response to the reviewer’s comments, we cite recent publications (kindly see below) that utilized the Staphtype genotyping kit 2.0 and refer to the methodology and data provided in the Supplementary section of these publications. These papers (including those in PLoS One) provided the raw data (in MS-Excel) and clearly showed that the methodology including the DNA sequence for the primers/probes has been published previously. Consequently, we do not agree with the reviewer that a supplementary table with the DNA sequence of the probes and primers should be provided in our manuscript. In conclusion, the description of the genes in S1 Table 1 is stated in S1 Table 3.

Monecke S, Slickers P, Gawlik D, Müller E, Reissig A, Ruppelt-Lorz A, Akpaka PE, Bandt D, Bes M, Boswihi SS, Coleman DC, Coombs GW, Dorneanu OS, Gostev VV, Ip M, Jamil B, Jatzwauk L, Narvaez M, Roberts R, Senok A, Shore AC, Sidorenko SV, Skakni L, Somily AM, Syed MA, Thürmer A, Udo EE, Vremerǎ T, Zurita J, Ehricht R. Molecular Typing of ST239-MRSA-III From Diverse Geographic Locations and the Evolution of the SCCmec III Element During Its Intercontinental Spread. Front Microbiol. 2018 Jul 6;9:1436. doi: 10.3389/fmicb.2018.01436.

Earls MR, Shore AC, Brennan GI, Simbeck A, Schneider-Brachert W, Vremerǎ T, Dorneanu OS, Slickers P, Ehricht R, Monecke S, Coleman DC. A novel multidrug-resistant PVL-negative CC1-MRSA-IV clone emerging in Ireland and Germany likely originated in South-Eastern Europe. Infect Genet Evol. 2019 Apr;69:117-126. doi: 10.1016/j.meegid.2019.01.021. Epub 2019 Jan 21.

Morach M, Käppeli N, Hochreutener M, Johler S, Julmi J, Stephan R, Etter D. Microarray based genetic profiling of Staphylococcus aureus isolated from abattoir byproducts of pork origin. PLoS One. 2019 Sep 6;14(9):e0222036. doi: 10.1371/journal.pone.0222036.

Alfouzan W, Udo EE, Modhaffer A, Alosaimi A. Molecular Characterization of Methicillin- Resistant Staphylococcus aureus in a Tertiary Care hospital in Kuwait. Sci Rep. 2019 Dec 6;9(1):18527. doi: 10.1038/s41598-019-54794-8.

Boswihi SS, Udo EE, Mathew B, Noronha B, Verghese T, Tappa SB. Livestock-Associated Methicillin-Resistant Staphylococcus aureus in Patients Admitted to Kuwait Hospitals in 2016-2017. Front Microbiol. 2020 Jan 8;10:2912. doi: 10.3389/fmicb.2019.02912.

Etter D, Corti S, Spirig S, Cernela N, Stephan R, Johler S. Staphylococcus aureus Population Structure and Genomic Profiles in Asymptomatic Carriers in Switzerland. Front Microbiol. 2020 Jun 24;11:1289. doi: 10.3389/fmicb.2020.01289.

Gawlik D, Ruppelt-Lorz A, Müller E, Reißig A, Hotzel H, Braun SD, Söderquist B, Ziegler-Cordts A, Stein C, Pletz MW, Ehricht R, Monecke S. Molecular investigations on a chimeric strain of Staphylococcus aureus sequence type 80. PLoS One. 2020 Oct 14;15(10):e0232071. doi: 10.1371/journal.pone.0232071.

Senok A, Nassar R, Celiloglu H, Nabi A, Alfaresi M, Weber S, Rizvi I, Müller E, Reissig A, Gawlik D, Monecke S, Ehricht R. Genotyping of methicillin resistant Staphylococcus aureus from the United Arab Emirates. Sci Rep. 2020 Oct 29;10(1):18551. doi: 10.1038/s41598-020-75565-w.

2) Lines 81-82: Provide the precise CLSI standard version applied in the phenotpical assay.

Response: The observation is noted and the amendment is in the revised manuscript.

3) The authors claim that the objective of the current was to determine the percentage and level of agreement between antibiotic sensitive testing and DNA microarray, which alone is not suitable for publication. The authors also claim that the objective of the current was to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates from Africa. So, which is it? Please clarify! If the second objective is the correct one, then please address the original comments made both reviewers.

Response: We appreciate the comment of the reviewer. The statement on the percentage and level of agreement between antibiotic susceptibility testing and DNA microarray has been removed in the revised manuscript (discussion section). The objective of the study is to provide detailed information on antibiotic resistance and virulence-related genes, and the population structure of S. aureus isolates (from Africa) based on the microarray assay.

Yours sincerely,

Professor Adebayo Shittu

Corresponding author

Decision Letter 2

Monica Cartelle Gestal

6 Apr 2021

PONE-D-20-22325R2

DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

PLOS ONE

Dear Dr. Shittu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

There are some minor changes that need to be address, please send us the revised version as early as your convenience

==============================

Please submit your revised manuscript by May 21 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: This is the third review for the manuscript by Shittu et al., which describes the screening of 40 S. aureus isolates collected in South Africa from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made revisions to accommodate suggestions posed by reviewers, the authors fail to accommodate the request to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers. Therefore, the manuscript in its current form fails to meet PLoS One Data availability requirements. Instead of simply complying with the policy, the authors choose to argue that 1) previous published papers have not met the policy requirements and 2) that references listed provide the requested information.

This reviewer’s response to these points are:

1) This reviewer was not part of the peer-review process of the noncompliant previous published papers. In contrast, this reviewer is part of the peer-review process for the manuscript by Shittu et al., which in its current form, is NOT compliant.

2) The authors are correct in that one of the cited references provides the required information however, it is provided in a convoluted style. For example, the gene aacA-aphD, a bifunctional enzyme Aac/Aph conferring gentamicin resistance, the probe name is “hp_aacA-59243_PM1” and the probe sequence is “AB049452.1[5151:5178]”.

This reviewer is unsure why the authors persist to refuse to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers, which would fulfill PLoS One Data availability requirements. These requirements are not meant to be a cumbersome obstacle. They are meant to help make all aspects of the scientific process accessible to everyone.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Jul 20;16(7):e0237124. doi: 10.1371/journal.pone.0237124.r006

Author response to Decision Letter 2

24 Apr 2021

Re: Revised manuscript: PONE-D-20-22325-R3 – DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

On behalf of other co-authors, I forward herewith the revised manuscript for your kind consideration. The Journal requirements and comments of reviewer 2 have been examined and we provide a point-by-point response (in italics) as indicated below.

Journal Requirements

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: Based on comments/suggestions from the reviewers, the under-listed references were included in the revised manuscript which was not in the original version.

9. Franklin R. Cockerill, III M, Jean B. Patel, PhD D. M100-S25 Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. Clin Lab Stand Inst. 2015.

10. Al-Haqan A, Boswihi SS, Pathan S, Udo EE. Antimicrobial resistance and virulence determinants in coagulase-negative staphylococci isolated mainly from preterm neonates. PLoS One. 2020. doi:10.1371/journal.pone.0236713.

13. Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, et al. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011. doi:10.1371/journal.pone.0017936.

14. Monecke S, Jatzwauk L, Müller E, Nitschke H, Pfohl K, Slickers P, et al. Diversity of SCCmec elements in staphylococcus aureus as observed in south-eastern Germany. PLoS One. 2016. doi:10.1371/journal.pone.0162654.

15. Landis JR, Koch GG. The Measurement of Observer Agreement for Categorical Data. Biometrics. 1977. doi:10.2307/2529310.

The reference number 10 in the original manuscript: Monecke S, Jatzwauk L, Weber S, Slickers P, Ehricht R. DNA microarray-based genotyping of methicillin-resistant Staphylococcus aureus strains from Eastern Saxony. Clinical Microbiology and Infection. 2008. doi:10.1111/j.1469-0691.2008.01986.x was replaced with the reference below.

12. Monecke S, Slickers P, Ehricht R. Assignment of Staphylococcus aureus isolates to clonal complexes based on microarray analysis and pattern recognition. FEMS Immunol Med Microbiol. 2008. doi:10.1111/j.1574-695X.2008.00426.x.

The reference list is complete and correct in the revised manuscript.

Reviewer’s comments

Reviewer #2: This is the third review for the manuscript by Shittu et al., which describes the screening of 40 S. aureus isolates collected in South Africa from 2001-2004 for a variety of virulence factors and antimicrobial elements using DNA microarray analysis. This paper basically summarizes the findings of the screen and the concludes that “DNA microarray assay provides information on antibiotic resistance and virulence gene determinants and can be a useful tool to identify gene markers of specific S. aureus clones in Africa”. While the authors have made revisions to accommodate suggestions posed by reviewers, the authors fail to accommodate the request to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers. Therefore, the manuscript in its current form fails to meet PLoS One Data availability requirements. Instead of simply complying with the policy, the authors choose to argue that 1) previous published papers have not met the policy requirements and 2) that references listed provide the requested information.

This reviewer’s response to these points are:

1) This reviewer was not part of the peer-review process of the noncompliant previous published papers. In contrast, this reviewer is part of the peer-review process for the manuscript by Shittu et al., which in its current form, is NOT compliant.

2) The authors are correct in that one of the cited references provides the required information however, it is provided in a convoluted style. For example, the gene aacA-aphD, a bifunctional enzyme Aac/Aph conferring gentamicin resistance, the probe name is “hp_aacA-59243_PM1” and the probe sequence is “AB049452.1[5151:5178]”

This reviewer is unsure why the authors persist to refuse to provide a list of all target or gene names, along with the actual DNA sequence of the probes and primers, which would fulfill PLoS One Data availability requirements. These requirements are not meant to be a cumbersome obstacle. They are meant to help make all aspects of the scientific process accessible to everyone.

Response: We have included the primer and probe names, GenBank coordinates and the corresponding DNA sequences in the revised manuscript (Supplementary data: S1 Table 3). The DNA sequences for the primers and probes were retrieved from the NCBI website https://www.ncbi.nlm.nih.gov/nuccore through their accession numbers and coordinates, as published previously.

Monecke S, Coombs G, Shore AC, Coleman DC, Akpaka P, Borg M, et al. A field guide to pandemic, epidemic and sporadic clones of methicillin-resistant Staphylococcus aureus. PLoS One. 2011. doi: 10.1371/journal.pone.0017936.

Monecke S, Jatzwauk L, Müller E, Nitschke H, Pfohl K, Slickers P, et al. Diversity of SCCmec elements in Staphylococcus aureus as observed in south-eastern Germany. PLoS One. 2016. doi: 10.1371/journal.pone.0162654.

Yours sincerely,

Professor Adebayo Shittu

Corresponding author

Attachment

Submitted filename: Response to Reviewers_R3.doc

Click here for additional data file. (87.5KB, doc)

Decision Letter 3

Monica Cartelle Gestal

18 Jun 2021

DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

PONE-D-20-22325R3

Dear Dr. Shittu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Monica Cartelle Gestal

24 Jun 2021

PONE-D-20-22325R3

DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

Dear Dr. Shittu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Monica Cartelle Gestal

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Raw data on DNA hybridization reaction (indicated as either positive, negative or ambiguous) of the S. aureus isolates.

    (XLSX)

    Click here for additional data file. (2.4MB, xlsx)
    S2 Table. Detailed characteristics of the S. aureus isolates (n = 40) including antibiotyping, spa types, Clonal complex (CC) affiliation, and DNA microarray hybridization results of some antibiotic resistance and virulence genes.

    (XLS)

    Click here for additional data file. (114.5KB, xls)
    S3 Table. List of target genes and DNA sequences of the probes and primers (including GenBank coordinates) for microarray in S. aureus Genotyping Kit 2.0 system.

    (XLS)

    Click here for additional data file. (153.5KB, xls)
    S4 Table. Distribution of selected genes (DNA microarray) in S. aureus isolates from Nigeria and South Africa.

    (DOC)

    Click here for additional data file. (231KB, doc)
    Attachment

    Submitted filename: Response to Reviewers_R3.doc

    Click here for additional data file. (87.5KB, doc)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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