BACKGROUND
staphylococcus aureus is an opportunistic pathogen that mainly colonizes the nares and skin of up to 80% of the population  <cit> .
s. aureus is a gram-positive cocci that is frequently isolated in hospitals, and is responsible for diverse infections and toxicoses  <cit> .
s. aureus is the most common cause of skin and soft-tissue infections , as well as systemic infections   <cit> .
the threat of s. aureus is not only due to its distribution and pathogenicity  <cit> , but also because of its ability to overcome antimicrobial agents  <cit> .
virulence factors produced by s. aureus render this organism highly pathogenic.
the known virulence factors include exotoxins, such as exfoliative toxins , along with toxic shock syndrome toxin- <dig> , staphylococcal enterotoxins , leukocidins , and hemolysins   <cit> .
enterotoxins often cause food poisoning  <cit> , while ets  act on the skin  <cit> .
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  <cit> .
s. aureus strains that are positive for pvl are usually associated with skin and soft-tissue infections, and were first isolated in the 1960s  <cit> .
pvl-positive strains are particularly associated with furuncles, accounting for 96% of cases  <cit> , 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  <cit> , osteomyelitis  <cit> , and even cases of purpura fulminans  <cit> .
pvl toxin was recently identified in lemierre’s syndrome  <cit> , and in a case of fournier’s gangrene  <cit> .
pvl has also been associated with community-acquired necrotizing and hemorrhagic pulmonary infections affecting previously healthy children and young adults  <cit> .
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  are pervasive in the hospital environment, and have recently also caused a global epidemic of community-associated s. aureus  infections  <cit> .
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  <cit> .
vancomycin has been used successfully for over  <dig> years for the treatment of s. aureus infections, particularly those caused by mrsa strains  <cit> .
however, vancomycin-resistant s. aureus  and vancomycin-intermediate  strains have been reported, three decades after the introduction of vancomycin  <cit> .
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  <dig> 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%  of the collected strains originated from abscesses, followed by strains isolated from pyomyositis patients , furuncles , buruli ulcers , and osteomyelitis cases .
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  were active against some of the strains .
there was no significant difference in the antibiotic resistance of the strains based on their origin .
s. aureus strains originating from pyomyositis , furuncles  and osteomyelitis cases  were resistant to 4/ <dig> tested antibiotics , while strains originating from abscesses and buruli ulcer were strongly resistant to respectively 13/ <dig> and 7/ <dig> of the tested antibiotics .
of the  <dig> isolated s. aureus strains,  <dig>  were resistant to oxacillin , while none of the strains showed resistance to vancomycin .
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  <dig> toxins .
thus, a significant number of strains  were capable of producing pvl, followed by the production of staphylococcal enterotoxin b  .
none of the strains contained the genes responsible for exfoliative toxin b  or staphylococcal enterotoxin d  production, while the ability to produce staphylococcal enterotoxins c and e , as well as the toxic shock syndrome toxin , was detected in <1% of strains .
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 .
pvl toxin was particularly prevalent in strains isolated from furuncles  and pymyositis patients .
other toxins were produced in various proportions depending on the origin of the strain .
there was a significant difference in the detection of genes encoding toxins in mrsa strains .
discussion
the s. aureus stains analyzed in this study displayed a wide range of sensitivity to the  <dig> tested antibiotics.
generally, benzyl penicillin was not efficient in controlling the strains .
this is consistent with previous reports showing high rates of s. aureus resistance  to benzyl penicillin  <cit> , suggesting that this antibiotic, one of the first to be introduced, is no longer effective against s. aureus <cit> .
a very high proportion of strains showed resistance to rifampicin , tetracycline , and trimetroprim/sulfamethoxazol .
this finding is consistent with previous studies performed in africa  <cit> .
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/ <dig> tested antibiotics  were effective against all the s. aureus strains.
the collection of strains isolated from abscesses was sensitive to only four of the  <dig> tested antibiotics.
however, strains isolated from furuncles  and osteomyelitis patients  were sensitive to  <dig> antibiotics .
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  <dig> tested strains,  <dig>  were resistant to oxacillin.
this proportion of resistant strains appears to have increased steadily in benin, compared with the recorded resistance rate of  <dig> % in  <dig>  <cit> .
however, the overall proportion of oxacillin-resistant strains in benin is still low compared to surrounding countries  <cit> , even though methicillin is the preferred antibiotic for treatment of staphylococcal infections  <cit> .
to efficiently control mrsa, vancomycin is recommended  <cit> , 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 , followed by seb , seg , sea , seh , and sei .
the genes encoding etb and sed were not detected in any of the strains, while the see , sec , and tsst  genes were detected, but at a very low rate .
this high detection frequency of the gene encoding pvl  was observed throughout the analysis, regardless of the origin of the sample .
pvl appears to be a primordial toxin of s. aureus strains associated with skin, soft tissue, and bone related infections.
these results are lower than the 96% of pvl-positive production strains we observed among s. aureus isolated from furuncle  <cit> .
but the prevalence of pvl-positive s. aureus obtained in our study is higher than the  <dig> % observed in nigeria  <cit>  and in cape verdes island  <cit> .
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  <cit> .
then, comparing with the other studies, we can say that the prevalence level of pvl ocus varies with geographical location, and clinical specimen  <cit>  in the clinical field, pvl-positive s. aureus strains are more pathogenic than pvl-negative strains  <cit> .
this is explained by the fact that the lytic activity of pvl directly affects monocytes, macrophages, polynuclear neutrophils, and metamyelocytes, although erythrocytes are not lysed by pvl  <cit> .
pvl toxin is known to have a cytolytic effect, and as such polynuclear neutrophils were identified as important indicators of staphylococcal virulence  <cit> .
moreover, the cytolytic activity of pvl is observed at high toxin concentrations, while apoptosis is observed at low concentrations  <cit> .
regarding the ets, only the gene encoding epidermolysin a  was detected, and in all cases the s. aureus strains were isolated from buruli ulcers .
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- <dig>  an important epidermal protein  <cit> .
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  were not detected at all, while three others  were detected at various proportions, regardless of the origin of the strains .
based on the homology of their sequences, the three detected enterotoxin genes belong to three different groups  <cit> .
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  <cit> .
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- <dig>  which is the most prevalent toxin in cases of food poisoning  <cit> .
our study revealed that resistance to methicillin negatively correlates with toxin production .
the difference in toxin production was extremely significant for pvl and some enterotoxins  , and we observed that mssa strains produced twice as many toxins as mrsa strains.
these results suggest that the isolated strains were in majority hospital acquired methicillin resistance s. aureus  because the community-acquired methicillin resistance s. aureus .
indeed, these ca-mrsa have an sccmec type iv cassette conferring resistance to methicillin  <cit> , and 77% of them harbor genes for panton- valentine leukocidin   <cit> .
in addition, the prevalence of the genes for some toxins is higher in ca-mrsa than in ha-mrsa, suggesting that strains circulating in the community are more toxinogenic than hospital-associated strains  <cit> .
focusing on the duality of the observed activity between the resistance to methicillin and detection of the pvl-encoding gene, we may deduce that the resistance gene has a repressive activity against pvl.
this observation was also made by baldwin and lowe  <cit> , 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  <cit> , which is not favorable for bacterial development  <cit> .
although community-acquired mrsa infections are increasingly frequent, the use of alternative antibiotics, such as vancomycin or ofloxacin/ciprofloxacin, is not appropriate because 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 prophylaxis 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, resistance 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 integrate 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.

 staphylococcus aureus is an opportunistic pathogen that mainly colonizes the nares and skin of up to 80% of the population. s. aureus is a gram-positive cocci that is frequently isolated in hospitals, and is responsible for diverse infections and toxicoses. s. aureus is the most common cause of skin and soft-tissue infections, as well as systemic infections. the threat of s. aureus is not only due to its distribution and pathogenicity, but also because of its ability to overcome antimicrobial agents. virulence factors produced by s. aureus render this organism highly pathogenic. the known virulence factors include exotoxins, such as exfoliative toxins, along with toxic shock syndrome toxin-, staphylococcal enterotoxins, leukocidins, and hemolysins. enterotoxins often cause food poisoning, while ets act on the skin. 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. s. aureus strains that are positive for pvl are usually associated with skin and soft-tissue infections, and were first isolated in the 1960s. pvl-positive strains are particularly associated with furuncles, accounting for 96% of cases, 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, osteomyelitis, and even cases of purpura fulminans. pvl toxin was recently identified in lemierre’s syndrome, and in a case of fournier’s gangrene. pvl has also been associated with community-acquired necrotizing and hemorrhagic pulmonary infections affecting previously healthy children and young adults. 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 are pervasive in the hospital environment, and have recently also caused a global epidemic of community-associated s. aureus infections. 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. vancomycin has been used successfully for over years for the treatment of s. aureus infections, particularly those caused by mrsa strains. however, vancomycin-resistant s. aureus and vancomycin-intermediate strains have been reported, three decades after the introduction of vancomycin. 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.  prevalence of s. aureus strains according to the sample origin using standard microbiological methods for identification of microorganisms; a total of 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% of the collected strains originated from abscesses, followed by strains isolated from pyomyositis patients, furuncles, buruli ulcers, and osteomyelitis cases. 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 were active against some of the strains. there was no significant difference in the antibiotic resistance of the strains based on their origin. s. aureus strains originating from pyomyositis, furuncles and osteomyelitis cases were resistant to 4/ tested antibiotics, while strains originating from abscesses and buruli ulcer were strongly resistant to respectively 13/ and 7/ of the tested antibiotics. of the isolated s. aureus strains, were resistant to oxacillin, while none of the strains showed resistance to vancomycin. 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 toxins. thus, a significant number of strains were capable of producing pvl, followed by the production of staphylococcal enterotoxin b. none of the strains contained the genes responsible for exfoliative toxin b or staphylococcal enterotoxin d production, while the ability to produce staphylococcal enterotoxins c and e, as well as the toxic shock syndrome toxin, was detected in <1% of strains. 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. pvl toxin was particularly prevalent in strains isolated from furuncles and pymyositis patients. other toxins were produced in various proportions depending on the origin of the strain. there was a significant difference in the detection of genes encoding toxins in mrsa strains. discussion the s. aureus stains analyzed in this study displayed a wide range of sensitivity to the tested antibiotics. generally, benzyl penicillin was not efficient in controlling the strains. this is consistent with previous reports showing high rates of s. aureus resistance to benzyl penicillin, suggesting that this antibiotic, one of the first to be introduced, is no longer effective against s. aureus. a very high proportion of strains showed resistance to rifampicin, tetracycline, and trimetroprim/sulfamethoxazol. this finding is consistent with previous studies performed in africa. 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/ tested antibiotics were effective against all the s. aureus strains. the collection of strains isolated from abscesses was sensitive to only four of the tested antibiotics. however, strains isolated from furuncles and osteomyelitis patients were sensitive to antibiotics. 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 tested strains, were resistant to oxacillin. this proportion of resistant strains appears to have increased steadily in benin, compared with the recorded resistance rate of % in. however, the overall proportion of oxacillin-resistant strains in benin is still low compared to surrounding countries, even though methicillin is the preferred antibiotic for treatment of staphylococcal infections. to efficiently control mrsa, vancomycin is recommended, 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, followed by seb, seg, sea, seh, and sei. the genes encoding etb and sed were not detected in any of the strains, while the see, sec, and tsst genes were detected, but at a very low rate. this high detection frequency of the gene encoding pvl was observed throughout the analysis, regardless of the origin of the sample. pvl appears to be a primordial toxin of s. aureus strains associated with skin, soft tissue, and bone related infections. these results are lower than the 96% of pvl-positive production strains we observed among s. aureus isolated from furuncle. but the prevalence of pvl-positive s. aureus obtained in our study is higher than the % observed in nigeria and in cape verdes island. 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. then, comparing with the other studies, we can say that the prevalence level of pvl ocus varies with geographical location, and clinical specimen in the clinical field, pvl-positive s. aureus strains are more pathogenic than pvl-negative strains. this is explained by the fact that the lytic activity of pvl directly affects monocytes, macrophages, polynuclear neutrophils, and metamyelocytes, although erythrocytes are not lysed by pvl. pvl toxin is known to have a cytolytic effect, and as such polynuclear neutrophils were identified as important indicators of staphylococcal virulence. moreover, the cytolytic activity of pvl is observed at high toxin concentrations, while apoptosis is observed at low concentrations. regarding the ets, only the gene encoding epidermolysin a was detected, and in all cases the s. aureus strains were isolated from buruli ulcers. 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- an important epidermal protein. 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 were not detected at all, while three others were detected at various proportions, regardless of the origin of the strains. based on the homology of their sequences, the three detected enterotoxin genes belong to three different groups. 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. 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- which is the most prevalent toxin in cases of food poisoning. our study revealed that resistance to methicillin negatively correlates with toxin production. the difference in toxin production was extremely significant for pvl and some enterotoxins, and we observed that mssa strains produced twice as many toxins as mrsa strains. these results suggest that the isolated strains were in majority hospital acquired methicillin resistance s. aureus because the community-acquired methicillin resistance s. aureus. indeed, these ca-mrsa have an sccmec type iv cassette conferring resistance to methicillin, and 77% of them harbor genes for panton- valentine leukocidin. in addition, the prevalence of the genes for some toxins is higher in ca-mrsa than in ha-mrsa, suggesting that strains circulating in the community are more toxinogenic than hospital-associated strains. focusing on the duality of the observed activity between the resistance to methicillin and detection of the pvl-encoding gene, we may deduce that the resistance gene has a repressive activity against pvl. this observation was also made by baldwin and lowe, 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, which is not favorable for bacterial development. although community-acquired mrsa infections are increasingly frequent, the use of alternative antibiotics, such as vancomycin or ofloxacin/ciprofloxacin, is not appropriate because 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 prophylaxis in medical centers with high rates of mrsa show that the use of this antibiotic is controversial in preventing some infections.  our study showed that for s. aureus strains isolated from skin, soft tissue, and bone related infections, resistance 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 integrate 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. 
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. 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. panton-valentine leukocidin was the most commonly produced virulence factor, followed by staphylococcal enterotoxin b. a total of 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. staphylococcus aureus is an opportunistic commensal bacterium that mostly colonizes the skin and soft tissues. exfoliative toxin b was produced by % of the strains, and was only found in isolates from buruli ulcers. 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. all strains were resistant to benzyl penicillin, while 25% were resistant to methicillin, and all showed sensitivity to vancomycin. the pathogenicity of s. aureus is due to both its ability to resist antibiotics, and the production of toxins.
 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.  a total of 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 was the most commonly produced virulence factor, followed by staphylococcal enterotoxin b. exfoliative toxin b was produced by % of the strains, and was only found in isolates from buruli ulcers. the tsst- sec, and seh genes were rarely detected.  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. s. aureusmrsapvlpyomyositisosteomyelitisskin infectionsbeninafrica 