DNA microarray analysis of Staphylococcus aureus from Nigeria and South Africa

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 nonclinical 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.

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][13][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).

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

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) 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 trimethoprimsulfamethoxazole 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).
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.
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].

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.