# Variability of antibiotic susceptibility and toxin production of Staphylococcus aureus strains isolated from skin, soft tissue, and bone related infections

Haziz Sina, Théodora Ahoyo, Wardi Moussaoui, Daniel Keller, Honoré Bankolé, Yves Barogui, Ymkje Stienstra, Simeon Kotchoni, Gilles Prevost,

Lamine Baba-Moussa

<figure><img src="image_1.png" /></figure>

## To cite this version:

Haziz Sina, Théodora Ahoyo, Wardi Moussaoui, Daniel Keller, Honoré Bankolé, et al.. Variability of antibiotic susceptibility and toxin production of Staphylococcus aureus strains isolated from skin, soft tissue, and bone related infections. BMC Microbiology, 2013, 13, 10.1186/1471-2180-13-188. hal-05258499

HAL Id: hal-05258499

https://univoak.hal.science/hal-05258499v1

Submitted on 15 Sep 2025

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# Variability of antibiotic susceptibility and toxin production of *Staphylococcus aureus* strains isolated from skin, soft tissue, and bone related infections

Haziz Sina¹, Théodora A Ahoyo², Wardi Moussaoui³, Daniel Keller³, Honoré S Bankolé⁴, Yves Barogui⁵, Ymkje Stienstra⁶, Simeon O Kotchoni⁷, Gilles Prévost³ and Lamine Baba-Moussa¹*

## Abstract

**Background:** *Staphylococcus aureus* is an opportunistic commensal bacterium that mostly colonizes the skin and soft tissues. The pathogenicity of *S. aureus* is due to both its ability to resist antibiotics, and the production of toxins. Here, we characterize a group of genes responsible for toxin production and antibiotic resistance of *S. aureus* strains isolated from skin, soft tissue, and bone related infections.

**Results:** A total of 136 *S. aureus* strains were collected from five different types of infection: furuncles, pyomyositis, abscesses, Buruli ulcers, and osteomyelitis, from hospital admissions and out-patients in Benin. All strains were resistant to benzyl penicillin, while 25% were resistant to methicillin, and all showed sensitivity to vancomycin. Panton-Valentine leukocidin (PVL) was the most commonly produced virulence factor (70%), followed by staphylococcal enterotoxin B (44%). Exfoliative toxin B was produced by 1.3% of the strains, and was only found in isolates from Buruli ulcers. The *tsst-1*, *sec*, and *seh* genes were rarely detected (≤1%).

**Conclusions:** This study provides new insight into the prevalence of toxin and antibiotic resistance genes in *S. aureus* strains responsible for skin, soft tissue, and bone infections. Our results showed that PVL was strongly associated with pyomyositis and osteomyelitis, and that there is a high prevalence of PVL-MRSA skin infections in Benin.

**Keywords:** *S. aureus*, MRSA, PVL, Pyomyositis, Osteomyelitis, Skin infections, Benin, Africa

## Background

*Staphylococcus aureus* is an opportunistic pathogen that mainly colonizes the nares and skin of up to 80% of the population [1]. *S. aureus* is a Gram-positive cocci that is frequently isolated in hospitals, and is responsible for diverse infections and toxicoses [2]. *S. aureus* is the most common cause of skin and soft-tissue infections (such as impetigo, furunculosis, and abscess), as well as systemic infections (such as pneumonia and endocarditis) [3]. The threat of *S. aureus* is not only due to its distribution

and pathogenicity [4,5], but also because of its ability to overcome antimicrobial agents [6-8].

Virulence factors produced by *S. aureus* render this organism highly pathogenic. The known virulence factors include exotoxins, such as exfoliative toxins (ETs), along with toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins (SEs), leukocidins (Panton-Valentine leukocidin; PVL, LukE/D), and hemolysins (α, β, γ, δ) [9]. Enterotoxins often cause food poisoning [10], while ETs (also called epidermolysins) act on the skin [11].

Among the leukocidins, PVL is an extracellular protein consisting of two subunits, F and S, which act in concert and have leucocidal and dermonecrotic functions. The PVL toxin targets the outer membrane of polymorphonuclear cells, monocytes, and macrophages [12-15]. *S. aureus*

<footnote>* Correspondence: laminesaid@yahoo.fr
¹Laboratoire de Biologie et de Typage Moléculaire en Microbiologie; Faculté des Sciences et Techniques/Université d'Abomey-Calavi, Cotonou 05 BP 1604, BENIN</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
strains that are positive for PVL are usually associated with skin and soft-tissue infections, and were first isolated in the 1960s [16-19]. PVL-positive strains are particularly associated with furuncles, accounting for 96% of cases [11,17,20], and approximately 90% of PVL-positive *S. aureus* strains were originally isolated from furuncles. PVL has also been associated with severe infections, including necrotizing pneumonia [19,21-24], osteomyelitis [25], and even cases of purpura fulminans [26]. PVL toxin was recently identified in Lemierre's syndrome [27], and in a case of Fournier's gangrene [28]. PVL has also been associated with community-acquired necrotizing and hemorrhagic pulmonary infections affecting previously healthy children and young adults [22,29].

Several antibiotics were routinely used in the treatment of *S. aureus* infections, contributing to the emergence of antibiotic-resistant strains. Widespread resistance severely complicates management of *S. aureus* infections. *S. aureus* strains that are resistant to methicillin (methicillin-resistant *S. aureus*, MRSA) are pervasive in the hospital environment, and have recently also caused a global epidemic of community-associated *S. aureus* (CA-MRSA) infections [30]. The changing trend of MRSA epidemiology, showed the use of PVL locus detection as a marker of CA-MRSA isolates, alongside with non multiresistant pattern and SCCmec type IV or V [31]. Vancomycin has been used successfully for over 50 years for the treatment of *S. aureus* infections, particularly those caused by MRSA strains [32]. However, vancomycin-resistant *S. aureus* (VRSA) and vancomycin-intermediate (VISA) strains have been reported, three decades after the introduction of vancomycin [33]. The presence of resistance genes may also affect toxin production.

The production of multiple virulence factors, as well as the presence of antibiotic resistance genes, makes *S. aureus* a highly pathogenic microorganism. The objective of this work was to study the susceptibility profile and toxin production of *S. aureus* strains isolated from various skin, soft tissue, and bone infections.

## Results

### Prevalence of *S. aureus* strains according to the sample origin

Using standard microbiological methods for identification of microorganisms; a total of 136 strains of *S. aureus* were collected during this study. The proportions of the strains varied depending on the five types of infection: furuncle, osteomyelitis, pyomyositis, abscess, and Buruli ulcer. Almost 37% (50/136) of the collected strains originated from abscesses, followed by strains isolated from pyomyositis patients (27%, 37/136), furuncles (14%, 19/136), Buruli ulcers (12%, 16/136), and osteomyelitis cases (10%, 14/136).

### Susceptibility to antibiotics

There was a wide range in the susceptibility of the isolates to the various antibiotics examined. All of the strains were resistant to benzyl penicillin, while other antibiotics (vancomycin, fusidic acid, fosfomycin, and linezolid) were active against some of the strains (Figure 1).

There was no significant difference in the antibiotic resistance of the strains based on their origin (Figure 2). *S. aureus* strains originating from pyomyositis (Figure 2a), furuncles (Figure 2b) and osteomyelitis cases (Figure 2c) were resistant to 4/17 tested antibiotics (benzyl penicillin, rifampicin, tetracycline, and trimetroprim/sulfamethoxazol), while strains originating from abscesses and Buruli ulcer were strongly resistant to respectively 13/17 and 7/17 of the tested antibiotics (Figure 2d, e). Of the 136 isolated *S. aureus* strains, 34 (25%) were resistant to oxacillin (MRSA), while none of the strains showed resistance to vancomycin (VRSA). The oxacillin-resistant strains were all isolated from abscesses and Buruli ulcers.

### Toxins production and/or presence of their encoding genes

There was a significant difference in the production and/or the presence of genes encoding the 12 toxins (p < 0.0001). Thus, a significant number of strains (70.0%) were capable of producing PVL, followed by the production of staphylococcal enterotoxin B (SEB) (44.3%). None of the strains contained the genes responsible for exfoliative toxin B (ETB) or staphylococcal enterotoxin D (SED) production, while the ability to produce staphylococcal enterotoxins C and E (SEC, SEE), as well as the toxic shock syndrome toxin (TSST), was detected in

<figure><img src="image_3.png" /><figcaption>**Figure 1** Global *Staphylococcus aureus* strains isolated from primary and secondary infections resistance profile to 22 antibiotics. Benzyl penicillin (BP), oxacillin (Ox), cefoxitin screen (Cef), gentamicin (Gen), tobramycin (Tob), kanamycin (Kan), vancomycin (Van), teicoplanin (Tei), fusidic acid (FA), fosfomycin (Fos), rifampicin (Rif), trimethopim/sulfamethoxazole (T/Sul), erythromycin (Ery), lincomycin (Lin), pristinamycin (Pri), linezolid (Line), tetracyclin (Tet).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>a. Pyomyositis</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>b. Furuncle</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>c. Abscess</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>d. Osteomyelitis</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>e. Buruli ulcer</figcaption></figure>

**Figure 2** *Staphylococcus aureus* strains resistance profile to 22 antibiotics according to their origin. Benzyl penicillin (BP), oxacillin (Ox), cefoxitin screen (Cef), gentamicin (Gen), tobramycin (Tob), kanamycin (Kan), vancomycin (Van), teicoplanin (Tei), fusidic acid (FA), fosfomycin (Fos), rifampicin (Rif), trimethopim/sulfamethoxazole (T/Sul), erythromycin (Ery), lincomycin (Lin), pristinamycin (Pri), linezolid (Line), tetracyclin (Tet).*

<1% of strains (Figure 3). The observed difference was related to the origin of the *S. aureus* strains. PVL was the most commonly produced toxin, regardless of the origin of the strains (Figure 4). PVL toxin was particularly prevalent in strains isolated from furuncles (89.5%).

and pymyositis patients (89.2%). Other toxins were produced in various proportions depending on the origin of the strain (p < 0.0001). There was a significant difference in the detection of genes encoding toxins in MRSA strains (Figure 5).

<figure><img src="image_9.png" /><figcaption>**Figure 3** Toxins production by the *Staphylococcus aureus* strains isolated from primary and secondary infections.</figcaption></figure>

PVL: Panton-Valentine Leukocidin; ETA: Exfoliative Toxin A;

ETB: Exfoliative Toxin B; SEA: staphylococcal enterotoxin A;

SEB: staphylococcal enterotoxin B; SEC: staphylococcal enterotoxin C;

SED: staphylococcal enterotoxin D; SEE: staphylococcal enterotoxin E;

SEG: staphylococcal enterotoxin G; SEH: staphylococcal enterotoxin H;

SEI: staphylococcal enterotoxin I; TSST: Toxic-shock syndrome Toxin.

Means ± standard deviations (SD) for three experiments are given.

\*\*\*: p<0.0001.

## Discussion

The *S. aureus* strains analyzed in this study displayed a wide range of sensitivity to the 17 tested antibiotics. Generally, benzyl penicillin was not efficient in controlling the strains (Figure 1). This is consistent with previous reports showing high rates of *S. aureus* resistance (>90%) to benzyl penicillin [34,35], suggesting that this antibiotic, one of the first to be introduced, is no longer effective against *S. aureus* [36]. A very high proportion of strains showed resistance to rifampicin (67%), tetracycline (60%), and trimetroprim/sulfamethoxazol (57%). This finding is consistent with previous studies performed in Africa [37-40]. The high proportion of strains showing resistance to penicillin and three other antibiotics may be explained by the practice of patient self-medication in Benin, and by the availability and low price of the antibiotics. These antibiotics can be bought without prescription, especially in developing countries. In our *in vitro* study, 4/17 tested antibiotics (vancomycin, fusidic acid, fosfomycin, and linezolid) were effective against all the *S. aureus* strains.

The collection of strains isolated from abscesses was sensitive to only four of the 17 tested antibiotics. However, strains isolated from furuncles (Figure 2b) and
<figure><img src="image_10.png" /><figcaption>a. *Pyomyositis*</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>b. *Furuncle*</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>c. *Abscess*</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>d. *Osteomyelitis*</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>e. *Buruli ulcer*</figcaption></figure>

**Figure 4 Specificity of the toxins production by the S. aureus strains isolated from primary and secondary infections.** PVL: Panton-Valentine Leukocidin; ETA: Exfoliative Toxin A; ETB: Exfoliative Toxin B; SEA: staphylococcal enterotoxin A; SEB: staphylococcal enterotoxin B; SEC: staphylococcal enterotoxin C; SED: staphylococcal enterotoxin D; SEE: staphylococcal enterotoxin E; SEG: staphylococcal enterotoxin G; SEH: staphylococcal enterotoxin H; SEI: staphylococcal enterotoxin I; TSST: Toxic-shock syndrome Toxin. Means ± standard deviations (SD) for three experiments are given.

<figure><img src="image_15.png" /></figure>

**Figure 5 Effect of Methicillin resistance on the encoding toxins genes presence.** PVL: Panton-Valentine Leukocidin; ETA: Exfoliative Toxin A; ETB: Exfoliative Toxin B; SEA: staphylococcal enterotoxin A; SEB: staphylococcal enterotoxin B; SEC: staphylococcal enterotoxin C; SED: staphylococcal enterotoxin D; SEE: staphylococcal enterotoxin E; SEG: staphylococcal enterotoxin G; SEH: staphylococcal enterotoxin H; SEI: staphylococcal enterotoxin I; TSST: Toxic-shock syndrome Toxin. Means ± standard deviations (SD) for three experiments are given. ***: P<0.001; the other differences were not statistically significant (P<0.05).

osteomyelitis patients (Figure 2d) were sensitive to 13 antibiotics (Figure 2). This result can likely be attributed to the origin of the strains. In our study, the samples collected from furuncles and osteomyelitis patients were from an extra-hospital community origin. Indeed, the selection pressure observed when using antibiotics in a hospital environment causes nosocomial strains to develop multi-resistance, in contrast to strains of community origin.

Of the 136 tested strains, 34 (25%) were resistant to oxacillin. This proportion of resistant strains appears to have increased steadily in Benin, compared with the recorded resistance rate of 11.6% in 1999 [40]. However, the overall proportion of oxacillin-resistant strains in Benin is still low compared to surrounding countries [41], even though methicillin is the preferred antibiotic for treatment of staphylococcal infections [42]. To efficiently control MRSA, vancomycin is recommended [43], and in our study we observed no resistance to vancomycin. Fortunately, vancomycin remains active against methicillin resistant strains of S. aureus.

In the current study of S. aureus strains isolated from skin, soft tissue, and bone related infections, PVL was the most prevalent toxin in our collection (70.0% of strains), followed by SEB (44.3%), SEG (35.5%), SEA (32.0%), SEH (28.8%), and SEI (28.9%). The genes encoding ETB and SED were not detected in any of the
strains, while the SEE (0.8%), SEC (0.6%), and TSST
(1%) genes were detected, but at a very low rate (Figure 3).
This high detection frequency of the gene encoding
PVL (p < 0.0001) was observed throughout the ana-
lysis, regardless of the origin of the sample (Figure 4).
PVL appears to be a primordial toxin of S. aureus strains
associated with skin, soft tissue, and bone related infec-
tions. These results are lower than the 96% of PVL-
positive production strains we observed among S. aureus
isolated from furuncle [20]. But the prevalence of PVL-
positive S. aureus obtained in our study is higher than
the 52.1% observed in Nigeria [44] and in cape Verdes
Island [45]. The observe differences observed can be
explain by the fact that in our study, we use various
kind of strains. However, our result is close to the 72%
obtained in Algeria [46]. Then, comparing with the
other studies, we can say that the prevalence level of PVL
ocus varies with geographical location, and clinical spe-
cimen [47] In the clinical field, PVL-positive S. aureus
strains are more pathogenic than PVL-negative strains
[22]. This is explained by the fact that the lytic activity
of PVL directly affects monocytes, macrophages, poly-
nuclear neutrophils, and metamyelocytes, although
erythrocytes are not lysed by PVL [48]. PVL toxin is
known to have a cytolytic effect, and as such polynuclear
neutrophils were identified as important indicators of
staphylococcal virulence [16]. Moreover, the cytolytic ac-
tivity of PVL is observed at high toxin concentrations,
while apoptosis is observed at low concentrations [49].

Regarding the ETs, only the gene encoding epidermolysin A (ETA) was detected, and in all cases the *S. aureus* strains were isolated from Buruli ulcers (Figure 4e). Such specific production of ETA by Buruli ulcers may be explained by the fact that ETs are known to be serine active proteases, with their activity highly specialized for desmoglein-1, an important epidermal protein [50,51]. Therefore, the production of ETA by *S. aureus* strains from Buruli ulcers indicates a secondary role for *S. aureus* in the development of these ulcers, which are predominantly caused by *Mycobacterium ulcerans*.

Of the eight genes encoding enterotoxins, three (SEC, SED, and SEE) were not detected at all, while three others (SEA, SEB, and SEH) were detected at various proportions, regardless of the origin of the strains (Figure 4). Based on the homology of their sequences, the three detected enterotoxin genes belong to three different groups [52]. This explains the diversity of enterotoxins produced by *S. aureus* isolated from skin infections. Those toxins associated with food poisoning have antigenic and emetic activities [53-55]. The presence of enterotoxin genes in the strains isolated from skin, soft tissue, and bone related infections may be explained by human or environmental contamination, through the presence of open wounds. Similar

observations are reported for TSST-1, which is the most
prevalent toxin in cases of food poisoning [56].

Our study revealed that resistance to methicillin nega-
tively correlates with toxin production (Figure 5). The dif-
ference in toxin production was extremely significant for
PVL and some enterotoxins (B, G, and I) (p < 0.0001), and
we observed that MSSA strains produced twice as many
toxins as MRSA strains. These results suggest that the iso-
lated strains were in majority Hospital acquired methicillin
resistance S. aureus (HA-MRSA) because the community-
acquired methicillin resistance S. aureus (CA-MRSA). In-
deed, these CA-MRSA have an SCCmec type IV cassette
conferring resistance to methicillin [57], and 77% of them
harbor genes for Panton-Valentine leukocidin (PVL)
[58,59]. In addition, the prevalence of the genes for some
toxins is higher in CA-MRSA than in HA-MRSA, sugges-
ting that strains circulating in the community are more
toxinogenic than hospital-associated strains [60]. Focusing
on the duality of the observed activity between the resist-
ance to methicillin and detection of the PVL-encoding
gene, we may deduce that the resistance gene has a repres-
sive activity against PVL. This observation was also made
by Baldwin and Lowe [61], and mostly relates to HA-
MRSA strains. In addition, we found that the presence of
the methicillin resistance gene negatively impacts the
expression of the gene encoding PVL. The emergence of
MRSA in the hospital acquired strains may be viewed as
disadvantageous in the selection of strains producing
toxins, notably PVL. Indeed, *mecA*-encoded methicillin
resistance involves β-lactamase production [62], which is
not favorable for bacterial development [63]. Although
community-acquired MRSA infections are increasingly
frequent, the use of alternative antibiotics, such as vanco-
mycin or ofloxacin/ciprofloxacin, is not appropriate be-
cause of the risk of the development of resistance to these
antibiotics. Vancomycin is usually not available because of
high costs and the necessity for assessing drug levels in
the blood. Studies on the use of vancomycin for prophy-
laxis in medical centers with high rates of MRSA show
that the use of this antibiotic is controversial in preventing
some infections.

Conclusions

Our study showed that for *S. aureus* strains isolated
from skin, soft tissue, and bone related infections, resist-
ance to antibiotics depends on the origin of the strain.
We attempted to perform a correlation analysis between
toxin production, resistance to antibiotics, and the origin
of samples. The *S. aureus* strains examined in this study
produced a variety of toxins, with PVL, one of the most
severe *S. aureus* toxins, being the most common
amongst all of the strains. Overall, it is desirable to inte-
grate to the current morphological and biochemical
diagnostic analysis with virulence factor screening to
accurately diagnose infectious disease mediated by S. aureus. This integrated diagnostic strategy will help to efficiently treat patients affected by pathogenic S. aureus strains. This study concerning skin, soft tissue, and bone related infections should be extended to include other types of infections in Benin.

## Methods

### Ethics statement

Ethical clearance was obtained from the Ministry of Public health of Benin Republic under protocol number: 2959/MSP/DC/SG/DRH/SPREA-05-2002. But it was important to notice that, the strains were de-identified and analyzed anonymously and the strains, not a human, were studied.

### Samples collection

Clinical *Staphylococcus aureus* samples were collected from patients with skin, soft tissue at the National University Hospital of Cotonou (Benin) for various bacteriological screenings in routine, from November 2009 to March 2011. The incidence of secondary infections in Burili ulcer is unknown; antibiotics may be frequently

prescribed for this indication. It is equally unknown which bacteria these antibiotics should target and what the sensitivity of these bacteria is. So the samples from Burili ulcer were screened for S. aureus. Theses samples were carried out during a prospective study made in a village of Lalo in Benin. Osteomyelitis and pyomyositis samples were collected during a prospective study made in a Hlagba Ouassa village in Benin. So these strains are considered as community strains and the others sample were isolated in hospital as stated previously.

### S. aureus' identification

Standard microbiological methods for identification of microorganisms were applied. All swabs were inoculated onto mannitol salt agar, incubated at 37°C and inspected visually for three days. Any suspected colony was subcultured on tryptic soy agar (bioMérieux) and identified by subsequent Gram staining, catalase test and Slidex Staph Plus (bioMérieux) and the coagulase test with the rabbit plasma [64]. Bacterial identification was performed by colony isolation on sheep blood agar plates and the automated Vitek 2 system.

<table><caption>Table 1 Primers sequences used in this study for the detection of genes encoding toxins</caption><thead><tr><th>Primers</th><th>Sequences</th><th>Polarity</th><th>Expected fragment (base pair)</th><th>Accession N°</th></tr></thead><tbody><tr><td rowspan="2">eta</td><td>5'-GCAGGTGTTGATTTAGCATT-3'</td><td>Sens</td><td rowspan="2">93</td><td>A8658-043</td></tr><tr><td>5'-AGATGTCCCTATTTTTGCTG-3'</td><td>Antisens</td><td>A8658-044</td></tr><tr><td rowspan="2">etb</td><td>5'-ACCCCTGTTCCTTATCATC-3'</td><td>Sens</td><td rowspan="2">226</td><td>A8659-036</td></tr><tr><td>5'-GTTTTTGGCTGCTTCTCTTG-3'</td><td>Antisens</td><td>A8659-037</td></tr><tr><td rowspan="2">lpv</td><td>5'-AAAATGCCACTGTTATCCAGAGGTA-3'</td><td>Sens</td><td rowspan="2">433</td><td>A8658-029</td></tr><tr><td>5'-TTTGCAGCGTTTTGTTTTTCG-3'</td><td>Antisens</td><td>A8658-030</td></tr><tr><td rowspan="2">sea</td><td>5'-ATGGTTATCAATGTGCGGGTGIIIIICCAAACAAAAC-3'</td><td>Sens</td><td rowspan="2">520</td><td>A8658-031</td></tr><tr><td>5'-TGAATACTGTCCTTGAGCACCAIIIIATCGTAATTAAC-3'</td><td>Antisens</td><td>A8658-032</td></tr><tr><td rowspan="2">seb</td><td>5'-TGGTATGACATGATGCCTGCACIIIIIGATAAATTTGAC-3'</td><td>Sens</td><td rowspan="2">163</td><td>A8658-033</td></tr><tr><td>5'-AGGTACTCTATAAGTGCCTGCCTIIIIIACTAACTCTT-3'</td><td>Antisens</td><td>A8658-034</td></tr><tr><td rowspan="2">sec</td><td>5'-GATGAAGTAGTTGATGTGTATGGATCIIIIIACTATGTAAAC-3'</td><td>Sens</td><td rowspan="2">283</td><td>A8658-035</td></tr><tr><td>5'-AGATTGGTCAAACTTATCGCCTGGIIIIIGCATCATATC-3'</td><td>Antisens</td><td>A8658-036</td></tr><tr><td rowspan="2">sed</td><td>5'-CTGAATTAAGTAGTACCGCGCTIIIIIAATGAAAC-3'</td><td>Sens</td><td rowspan="2">384</td><td>A8658-037</td></tr><tr><td>5'-TCCTTTTGCAAATAGCGCCTTGIIIIIGCATCTAATTC-3'</td><td>Antisens</td><td>A8658-038</td></tr><tr><td rowspan="2">see</td><td>5'-CTTACCGCCAAAGCTGTCG-3'</td><td>Sens</td><td rowspan="2">159</td><td>A8658-039</td></tr><tr><td>5'-GTCCACTTGTAAATGGTAGCGAGAA-3'</td><td>Antisens</td><td>A8658-040</td></tr><tr><td rowspan="2">seg</td><td>5'-AATTATGTGAATGCTCAACCCGAT-3'</td><td>Sens</td><td rowspan="2">408</td><td>HA01804615</td></tr><tr><td>5'-CTTTAGTGAGCCAGTGTCTTGCTTTG-3'</td><td>Antisens</td><td>HA01804616</td></tr><tr><td rowspan="2">seh</td><td>5'-CATCTACCCAAACATTAGCACC-3'</td><td>Sens</td><td rowspan="2">222</td><td>HA01804617</td></tr><tr><td>5'-AGAAATCAAGGTGATAGTGGCAA-3'</td><td>Antisens</td><td>HA01804618</td></tr><tr><td rowspan="2">sei</td><td>5'-AGGCGTCACAGATAAAAAACCTACCIIIIICAAATCAACTC-3'</td><td>Sens</td><td rowspan="2">454</td><td>HA01804619</td></tr><tr><td>5'-ACAAGGACCATTATAATCAATGCCIIIIITATCCAGTTTC-3'</td><td>Antisens</td><td>HA01804620</td></tr><tr><td rowspan="2">tsst-1</td><td>5'-ACCCCTGCCTTTCCATCATC-3'</td><td>Sens</td><td rowspan="2">209</td><td>A8658-041</td></tr><tr><td>5'-TTTTCAGTATTTGTAACGCC-3'</td><td>Antisens</td><td>A8658-042</td></tr></tbody></table>
Antibiotic susceptibility

Antimicrobial susceptibility was determined by the disc
diffusion method of Kirby-Bauer on agar Mueller-Hinton
(bioMérieux, Marcy l'Etoile, France) as recommended by
the Antibiogram Committee of the French Microbiology
Society (CASFM) [65]. After 24 h at 37°C, the zone of in-
hibition was measured. Susceptibility to vancomycin was
confirmed by broth microdilution methodology as de-
scribed by the Clinical and Laboratory Standards Institute
(CLSI) [66]. For susceptibility to oxacillin, an inoculum of
10⁷ CFU/ml was prepared and the plate was incubated at
37°C for 24 hours on Mueller-Hinton agar + 2% NaCl.
Antibiotic disks were obtained from Biorad, Marne la
Coquette, France.

The 17 tested antibiotics were: benzyl penicillin (10 UI), oxacillin (5 µg), cefoxitin screen (30 µg), gentamicin (10 UI), tobramycin (10 µg), kanamycin (30 µg), vancomycin (30 µg), teicoplanin (30 µg), fusidic acid (10 µg), fosfomycin (50 µg), rifampicin (30 µg), trimethoprim/ sulfamethoxazole (1.25/23.75 µg), erythromycin (15 µg), lincomycin (30 µg), pristinamycin (15 µg), linezolid (30 µg) and tetracyclin (30 UI).

Toxin detection

Phenotypic detection of toxins

For the phenotypic detection of toxins radial gel immu-
nodiffusion was performed. The production of Panton-
Valentine Leukocidin (PVL) and epidermolysins A (ETA)
and B (ETB) were evidenced from culture supernatants
after 18 h of growth in Yeast Casamino-acid Pyruvate
(YCP) medium [67] by radial gel immunodiffusion in
0.6% (wt/vol) agarose with component-specific rabbit
polyclonal and affinity-purified antibodies [68,69].

Genotype detection of toxins

Presence of genes encoding for the 12 toxins, for which
we don't have antibody, was detected by Multiplex PCR
using specific primers (Table 1) previously used for [70].
Then, the genes encoding for enterotoxins A (sea), B
(seb), C (sec), D (sed), E (see), G (seg), H (seh), I (sei) and
tsst were analyzed. Additionally, genes encoding PVL,
ETA and ETB were also detected. Briefly, total DNA was
purified using QIAamp® DNA Mini Kit (Qiagen, GmbH,
Germany) with a Gene Amp® PCR System 9700 (Perkin-
Elmer, Norwalk, USA) and amplified in a total volume of
50 µl containing 25 pmoles of each primer, 50 ng of total
DNA, 1.5 mM MgCl2, 200 µM of dNTP mixture, 1x
PCR reaction Buffer and 5 units of Taq™ DNA polymer-
ase (Invitogen™). The thermal cycling conditions in-
cluded an initial denaturation step (2 min at 92°C)
followed by 35 cycles of amplification comprising three
steps: 2 min denaturation for 92°C, 1 min annealing at
50°C, 2 min extension at 72°C. The reaction was termi-
nated with 3 min extension at 72°C. PCR products were

analysed by electrophoresis through 1.4% (wt/vol) agar-
ose gel (Euromedex, Mundolsheim, France).

Staphylococcal enterotoxins production (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI) by Bio-Plex Assay (xMAP Multiplex assay)

The centrifuged supernatant (3ml) of *S. aureus* grown
on BHI at 37°C (night) was recovered and diluted 1/2 in
TBS-Tween20-IgG rabbit nonspecific at 100µg/mL, and
incubated for 30 min at room temperature. The Bio-Plex
assays consisted of three incubation steps that were
performed into flat-bottom Multiscreen microplates
(pores diameter = 1.2 µm, Millipore) according to the
previous describe method [71]. Any steps were separated
by three washes into TBS-Tween 20.

Statistical analysis

Microsoft Excel Spreadsheet has been used for data pro-
cessing. For comparison tests of positive isolates of each
patient group, we used the Student T test, and the
Fischer's test for lower number series (GraphPad Prism
5). P < 0.05 was considered statistically significant.

Competing interests

Authors declare no conflict of interest.

Authors’ contributions

Conception and design of the study: LB-M and GP. Acquisition of data: HS, AT-A, WM, YB, HB. Analysis and interpretation of data: LB, GP, YS. Drafting the article: LB-M, SOK, and HS. Revising it critically for important intellectual content: LB-M, GP, SOK, YS. Final approval of the version to be submitted: All the co-authors. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by Benin Government which granted Haziz Sina and by grant EA-4438 from "Direction de la Recherche et des Etudes Doctorales" of Unversity of Strasbourg in France.

Author details

¹Laboratoire de Biologie et de Typage Moléculaire en Microbiologie; Faculté des Sciences et Techniques/Université d'Abomey-Calavi, Cotonou 05 BP 1604, BENIN. ²EPAC/ Université d'Abomey-Calavi, Cotonou 01 BP 526, BENIN.

³Unité: EA-4438 Physiopathologie et Médecine, Institut de Bactériologie, 3 rue Koeberlé, Strasbourg F-67000, France. ⁴Laboratoire National de Santé Publique, Cotonou, Bénin. ⁵Programme National de Lutte contre l'Ulcère de Buruli, Cotonou, Benin. ⁶Department of Internal Medicine/Infectious Diseases, University of Groningen, University Medical Center Groningen, P.O. Box 30.001, Groningen 9700 RB, The Netherlands. ⁷Department of Biology and Center for Computational and Integrative Biology, Rutgers University, Camden NJ 08102, USA.

Received: 20 March 2013 Accepted: 7 August 2013
Published: 8 August 2013

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doi:10.1186/1471-2180-13-188

Cite this article as: Sina et al.: Variability of antibiotic susceptibility and toxin production of *Staphylococcus aureus* strains isolated from skin, soft tissue, and bone related infections. *BMC Microbiology* 2013 13:188.

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