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Molecular characterization of methicillin-resistant Staphylococcus aureus isolated in Tunisia
Diagnostic Microbiology and Infectious Disease 55 (2006) 21 – 26 www.elsevier.com/locate/diagmicrobio
Molecular characterization of methicillin-resistant Staphylococcus aureus isolated in Tunisia Mouna Ben Nejmaa, Maha Mastourib, Sonia Frihc, Nabil Saklyd, Youcef Ben Saleme, Mohamed Nour a,4 a
Institut Supe´rieur de Biotechnologie, 5000 Monastir, Tunisia b Laboratoire de Microbiologie, 5000 Monastir, Tunisia c Faculte´ de Me´decine, 5000 Monastir, Tunisia d Laboratoire d’Immunologie, 5000 Monastir, Tunisia e Laboratoire de Microbiologie, 4000 Sousse, Tunisia Received 15 August 2005; accepted 28 October 2005
Abstract Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections are an emerging problem, especially related to the production of staphylococcal toxins. In this study we investigate the phenotypic and genotypic characteristics of 72 Tunisian MRSA. Our results revealed that these strains are multiresistant. Using multiplex polymerase chain reaction, we detected staphylococcal cassette chromosome mec (SCCmec) type IV and IVA in 66 isolates. The latter are Panton–Valentine leukocidin positive. The leukotoxin genes lukE-lukD were found in most strains (92.4%). The amplification of g-hemolysin gene was detected only in 2 MRSA isolates. Among all strains, only 1 expressed the exfoliatin A. fnbA gene was detected in 12 strains, fnbB gene in 2 strains, and both fnbA and fnbB genes in 2 other strains. The most predominant accessory gene regulator group identified was group III. Full characterization of these MRSA strains requires the association of SCCmec typing with other molecular methods such as pulsed-field gel electrophoresis, multilocus-sequence typing, and spa typing. D 2006 Elsevier Inc. All rights reserved. Keywords: MRSA; Virulence factors; agr groups; SCCmec; Characterization
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen both in nosocomial and communityacquired infections (Lowy, 1998). The emergence of MRSA was due mainly to the acquisition of the mecA gene. The latter was carried by a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec) that was integrated into methicillin-susceptible S. aureus chromosome (Katayama et al., 2000). Up to now, 5 major types of SCCmec element (I–V) and a few variants have been identified (Okuma et al., 2002; Ito et al., 2004). The types I, II, and III were described in hospital-acquired MRSA (HA-MRSA) strains. The fourth type was described in community4 Corresponding author. Institut Supe´rieur de Biotechnologie de Monastir, Unite´, 05/UR/09-11, Rue T. Haddad, 5000 Monastir, Tunisia. Fax: 216 - 73 - 465 - 404. E-mail address: [email protected] (M. Nour ). 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.10.017
acquired MRSA (CA-MRSA) isolates (Ma et al., 2002) and also in some hospital-associated MRSA (Oliveira et al., 2002). The fifth type was recently identified in the chromosome of CA-MRSA in Australia (Ito et al., 2004). The pathogenicity of S. aureus infections is linked to the expression of several virulence factors including cell wall-associated adhesins and toxins such as the staphylococcal enterotoxins, the exfoliative toxins (ETA and ETB), the toxic shock syndrome toxin 1 (TSST-1), and the leukocidins (for review, see Dinges et al., 2000). Some toxins are responsible for specific clinical syndromes such as TSST-1 associated to toxic shock syndrome and staphylococcal enterotoxins associated to staphylococcal food poisoning (Lina et al., 1997). However, most of the severe S. aureus infections are due to the cumulative effects of several virulence determinants (Peacock et al., 2002). The regulation of a number of virulence factors of S. aureus is controlled by a system called the accessory gene
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M. Ben Nejma et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 21 – 26
regulator (agr). The agr locus is characterized by a polymorphism in the amino acid sequence of the bacterial density-sensing peptide known as autoinducing peptide (AIP) and of its corresponding receptor (agr C) (Mayville et al., 1999). Based on the specificity of the AIP for its membrane receptor, the agr locus has been shown to be polymorphic and divided into 4 distinct genetic groups (I–IV) (Ji et al., 1997). Previous studies had reported associations between agr group and some staphylococcal toxins, such as the production of TSST-1 that is associated to agr group III, and the production of exfoliatin that is in turn associated to agr group IV (Jarraud et al., 2000, 2002). The association between LukE-LukD and agr group II was reported more recently (Von Eiff et al., 2004). The aims of the present study are i) to determine the SCCmec elements carried by Tunisian MRSA strains, ii) to detect some virulence factor genes, iii) and to characterize the agr groups of these isolates.
oxacillin, kanamycin, tobramycin, gentamicin, tetracycline, chloramphenicol, erythromycin, lincomycin, pristinamycin, ofloxacin, sulfamethoxazole + trimethoprim, fusidic acid, vancomycin, and teicoplanin. The MICs of oxacillin were determined by the E-test method (Biodisk, Solna, Sweden) according to the manufacturer’s guidelines. 2.4. DNA extraction Bacterial DNA was extracted using the Wizard genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer’s instructions. 2.5. SCCmec typing To determine the types of SCCmec, multiplex polymerase chain reaction (PCR) analysis was performed as described previously (Oliveira and de Lencastre, 2002). Reference strain of S. aureus CIP103514 was used as positive control for mecA gene and for SCCmec type I. 2.6. Detection of accessory genes
2. Materials and methods 2.1. Bacterial strains During 2003–2004, only 72 MRSA isolates were recovered from clinical specimens of 72 patients at the Laboratory of Microbiology from a University Hospital in Monastir. Patients are aged from 2 months to 74 years and the mean age was 34.9 F 23.6 years (mean F SD). They were 34 males and 38 females; the sex ratio (female/male) is 1:11. Strains were collected from 59 outpatients and from 13 inpatients who developed infections within the first 48 h of their admission to the hospital. The isolates were recovered from pus (75%), blood (12.5%), and other specimens such as venous catheter (2.77%), conjunctival secretion (1.4%), and pleural liquid (8.3%). These strains were associated with various community-acquired infections such as furuncles (16 cases), cutaneous abscesses (12 cases), wound infection (6 cases), impetigo (6 cases), cellulitis (1 case), superficial abscess (2 case), septicemia (6 cases), arthritis (4 cases), osteomyelitis (3 cases), otitis (2 cases), vagina infection (3 cases), catheter-related infection (2 cases), pneumonia (1 case), fistula (1 case), and conjunctivitis (1 case). 2.2. Identification of MRSA The strains were identified as S. aureus from colony and microscopic morphology, catalase test, and by their ability to coagulate rabbit plasma (BioMe´rieux, Lyon, France). 2.3. Antimicrobial susceptibility testing The determination of antibiotics susceptibility of all strains was performed by the disk diffusion test on Mueller–Hinton agar according to the recommendations of the French Society of Microbiology bComite´ de l’Antibiogramme de la Socie´te´ Franc¸aise de MicrobiologieQ (CA-SFM) (http://www.sfm. asso.fr). The antibiotics tested were penicillin, cefoxitin,
The sequences of the primers used for amplification of virulence factors genes were reported previously (Jarraud et al., 2002; Nashev et al., 2004). These primers are specific for lukS-PV, lukF-PV (encoding Panton–Valentin leukocidin [PVL]), the leukotoxin genes lukE-lukD (encoding LukE-LukD), hlg (encoding g-hemolysin), eta, etb (encoding exfoliatins ETA and ETB), and fnbA, fnbB (encoding the fibronectin-binding proteins FnbA and FnbB). Amplification of these genes was performed by multiplex PCR under the following conditions: 4 min of initial denaturation at 94 8C, followed by 30 cycles of 1 min of denaturation at 94 8C, 1 min of annealing at 46 8C, 1 min of extension at 72 8C, and 4 min of final extension at 72 8C. 2.7. agr genotyping Using multiplex PCR, we determined the presence of agr allele group (I–IV). The nucleotide sequence of each primer and the amplification conditions have been described previously (Shopsin et al., 2003). 3. Results From 2003 to 2004, 72 MRSA strains were collected from the University Hospital in Monastir. Table 1 shows the antimicrobial susceptibility pattern for all isolates. Our results revealed that MRSA strains were resistant to kanamycin (88.9%), tetracycline (61.1%), erythromycin (30.5%) fusidic acid (38.8%), and gentamicin (25%). All MRSA strains were susceptible to pristinamycin, vancomycin, and teicoplanin. MICs determination showed that 73.61% of S. aureus had oxacillin MICs of z 256 Ag/mL. 3.1. SCCmec typing Using multiplex PCR method, the 72 MRSA strains were positive for the 162-bp internal fragment of the mecA gene. According to Oliveira and de Lencastre method (2002), we
M. Ben Nejma et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 21 – 26
identified 64 strains harboring SCCmec type IV and 2 strains harboring SCCmec variant IVA. We found the SCCmec type III in 4 strains, the SCCmec variant IIIB in 1 strain, and the SCCmec type I in another one.
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detected in 12 strains, fnbB gene in 2 strains, and both fnbA and fnbB genes in 2 other strains. 3.3. agr groups The amplification of the hypervariable region of the agr locus showed that the agr group III was the most predominant, detected in 73.3% of strains, followed by agr group I (16.6%), agr group IV (6%), and agr group II (3%).
3.2. Detection of accessory genes PCR amplification revealed that all strains harboring the SCCmec type IV and SCCmec type III were PVL positive. lukE-lukD genes were found in most strains (92.4%). The g-hemolysin gene was detected only in 2 MRSA strains. The amplification of the exfoliative toxin genes showed that only 1 strain expressed the exfoliatin A. The amplification of fnbA and fnbB genes revealed that fnbA gene was
4. Discussion MRSA strains are often isolated from nosocomial isolates of S. aureus and have become widespread in hospital.
Table 1 Resistance pattern and SCCmec types of 72 MRSA isolates
St
Antibiotic resistance phenotype Gm
K
Tm
C
Te
E
L
SXT
OFX
Ra
FOS
Fa
SCCmec type
Number of isolates
S R S S S R R R R R R R R R R R R R R R R R R R S S S R R R R R R S R S S S R R R R
S S S S S S S S S S S S S S S S R R R R R R S S S S S S S S S S R R R R R R R S R R
S S S S S R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R
S S S S S S R S S S S S S S S S R R R R R R R S R S S S S S S R R R R R R R R R R R
S S S S S S S S S S S S S R S S S S S S S S S S S S S S S S S S S S S S S S S S S S
S S R R R S S R S R S S R S S R R R R S S R S S S S S S S S S R R S R R R R R R R R
S S S S S S S S S S R R R R S S R S R S S R R R R S R S S R R R S R S R R R R R R S
S S S S S S S S S S S S S S S S R S R S S R S R R R R S S S R R S R S S R S S R R S
S S S S S S S S S S S S S S R S S S S S S S S S S S S R R S S S R S R S S S R S S S
S S S S S S S S S S S S S S S R S S S S S S S S S S S S S S R S S R S S S S S S R R
S S S S R S S S S S S S S S S S S S S S S S S S S S S S S S S S S R S S R S S S S S
S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S R S R S R R S S S S S S
S S S R S S S S R R S R R R S R R S S S R S S S R S S R S S R R S S S S S S S S S S
IV IV IV IV I IV IV IV IV IV IV IV IV IV IV IV IV IV III IV IV IIIB IV IV IV IV IV IV IV IV IV IV III IV III IV IV IV IVA IVA IV III
2 1 1 1 1 4 1 9 3 9 1 1 4 1 1 2 1 4 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
All isolates were susceptible to pristinamycin, vancomycin, and teicoplanin. St = streptomycin; Gm = gentamicin, K = kanamycin; Tm = tobramycin; C = chloramphenicol; Te = tetracycline; E = erythromycin; L = lincomycin; SXT = sulfamethoxazole + trimethoprim; OFX = ofloxacin; Ra = rifampicin; FOS = fosfomycin; Fa = fusidic acid; R = resistant; S = susceptible.
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M. Ben Nejma et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 21 – 26
Recently, new strains of MRSA have emerged in the community (Chambers, 2001). The term CA-MRSA is often used to refer to the detection of colonization or infection in the community rather than to actual acquisition of MRSA in the community. In the literature, different definitions were used to make a distinction between community and nosocomial MRSA. Most often, these definitions are based on the timing of isolation of MRSA in relation to the time of admission, with or without assessment of health careassociated risk factors. b Nosocomial infectionQ has been defined as an infection that develops in the hospital, but not incubating at the time of admission (Garner et al., 1988). A b community-acquired infection Q is an infection that is present or incubating at the time of admission and is not caused by an organism acquired during previous health care (Salgado et al., 2003). It has been previously reported that SCCmec type IV is a characteristic of CA-MRSA strains, and SCCmec types I, II, and III are specific to HA-MRSA (Ito et al., 2001; Oliveira and de Lencastre., 2002). Our results revealed that among 72 MRSA strains, 64 isolates contained the SCCmec type IV and 2 strains contained SCCmec variant IVA. These 66 strains were isolated from skin and invasive infections from nonhospitalized patients and hospitalized patients. The latter acquired infections within 48 h of admission to the hospital. These infections are acquired in the community, and the true site of acquisition of MRSA is unknown. The 6 remaining strains carried the SCCmec types I, III, and IIIB. This finding can be explained by the fact that these infections were caused by nosocomial strains which, in turn, have been acquired into the community. It is known that HA-MRSA strains are multiresistant because the type II and III SCCmec elements carry additional determinants, conferring resistance to non–h-lactam antibiotics (Ito et al., 2001). CA-MRSA strains harboring a type IV SCCmec element are generally susceptible to non–h-lactam antibiotics (Zetola et al., 2005). However, this does not mean that CA-MRSA strains are fully susceptible to these antibiotics (for review, see Eady and Cove 2003). Tunisian MRSA strains have high rates of resistance to kanamycin, tetracycline, and fusidic acid. Likewise, European CAMRSA isolates appeared to develop resistance to the same antibiotics (Vandenesch et al., 2003; Dufour et al., 2002). Our results revealed that resistance to erythromycin was observed in a rate of 31.8% and gentamicin in only 19.6% of the MRSA strains carrying SCCmec type IV. Conversely to our findings, the Australian and French CA-MRSA isolates were susceptible to erythromycin and gentamicin (Dufour et al., 2002). The multiresistance (resistance to more than 3 non–h-lactam antibiotics) of Tunisian MRSA isolates might be due to the antibiotic’s selective exposure. CA-MRSA strains are considered as the causative agent of skin and soft tissue infections, and some strains are particularly virulent and capable to induce life-threatening infections (for review, see Eady and Cove 2003). The detection of PVL genes demonstrated that 70 of 72 strains
isolated from various types of infection are able to produce PVL. Lina et al. (1999) have detected PVL genes in CA-MRSA associated with furuncles, cutaneous abscesses, cellulitis, as well as with necrotic hemorrhagic pneumonia, and osteomyelitis. Indeed, PVL-producing S. aureus strains cause chronic cutaneous infections (Dufour et al., 2002). These findings emphasize the importance of PVL as a virulence factor of primary necrotic infections (furuncles, cutaneous abscesses) (Couppie´ et al., 1994) and of all serious secondary skin infections (Dufour et al., 2002). PVL genes were detected also in 4 of 6 Tunisian nosocomial MRSA strains (carrying SCCmec I, III, and IIIB), whereas Dufour et al. (2002) have reported that PVL genes were never detected in MRSA strains associated with hospital-acquired infection. This result could be explained by the easy spread of the phages carrying the PVL genes among different backgrounds (Jarraud et al., 2002). In our study, lukE-lukD leukocidin genes were detected in the majority of strains (92.4%) isolated from all types of infection. Similar results have been reported by Dufour et al. (2002) in French CA-MRSA strains isolated from patients with all types of staphylococcal infections. However, Gravet et al. (2001) have reported that LukE-LukD is significantly associated with primary skin infections and revealed a direct association between LukE-LukD and other toxins. Both LukE-LukD and exfoliatin A or B were found to be produced by S. aureus associated with impetigo (Gravet et al., 2001). Furthermore, both LukE-LukD and enterotoxin A were predominantly detected in S. aureus isolated from patient with postantibiotic diarrhea (Gravet et al., 1999). The amplification of hlg gene revealed that this gene was detected only in 2 strains among all Tunisian MRSA. Indeed, Lina et al (1999) found that g-hemolysin was produced by more than 99% of S. aureus clinical strains. Generally, exfoliatin is detected in S. aureus strains, which are implicated in the case of scalded syndrome (Lina et al., 1997). Several cases of impetigo and bullous impetigo were caused by an unexpected clonal group of MRSA producing exfoliative toxin A or B. These strains were identified first in Japan and then in France (Yamaguchi et al., 2002; Liassine et al., 2004). In our study, ETA was found in the case of arthritis. It has been suggested that the production of these toxins is due to the incorporation of a phage carrying the eta gene into lysogene MRSA strains or to the acquisition of mecA by exfoliatin-producing methicillinsusceptible S. aureus strains (for review, see Eguia and Chambers 2003). The pathogenesis of S. aureus is dependent on its production of toxins as well as of staphylococcal surface adhesins. These adhesins contribute to the adherence to host extracellular matrix components such as fibronectin (Patti et al., 1994). Binding to fibronectin is mediated by 2 related proteins, FnbA and FnbB (Nashev et al., 2004). In our study the detection of fibronectin binding protein genes revealed that both FnbA and FnbB were found in cases of skin
M. Ben Nejma et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 21 – 26
infections (furuncles and wound infection) and in cases of invasive infections (otitis and osteomyelitis). These results were similar to the findings reported by Nashev et al. (2004) who demonstrated that S. aureus nasal and skin infection isolates produce FnbA and FnbB with a high frequency. Previous studies have found that FnbA and FnbB are significantly implicated in invasive diseases such as endocarditis, osteomyelitis, septic arthritis, and nosocomial pneumonia (Peacock et al., 2000; Mongodin et al., 2002). The production of some virulence factors is under the control of the agr system (Van Leeuwen et al., 2000). The most predominant agr group in MRSA isolates from worldwide collections is agr group I (Van Leeuwen et al., 2000). However, we found that agr group III was the most prevalent among the Tunisian MRSA strains. Similar results were found by Vandenesch et al (2003) in French CA-MRSA strains. In addition to agr group III, some of our strains belong to the other agr groups I, II, and IV. No relationship between agr groups and toxins were revealed, whereas previous studies have reported the association between agr group III and TSST-1, between agr group IV and exfoliatin (Jarraud et al., 2000, 2002), and between agr group II and LukE-LukD (Von Eiff et al., 2004). In conclusion, most Tunisian MRSA studied and harboring SCCmec type IV and IVA are resistant to antibiotics other than h-lactam antibiotics. The agr group III is the predominant group for these strains. The combination of the multiresistance and virulence factors may play a key role in the pathogenesis of MRSA. For best characterization and for elucidation of the genetic background of these MRSA, more studies are needed associating SCCmec typing with other molecular methods such as pulsed-field gel electrophoresis, multilocus-sequence typing, and spa typing.
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Molecular characterization of methicillin-resistant Staphylococcus aureus isolated in Tunisia Mouna Ben Nejmaa, Maha Mastourib, Sonia Frihc, Nabil Saklyd, Youcef Ben Saleme, Mohamed Nour a,4 a
Institut Supe´rieur de Biotechnologie, 5000 Monastir, Tunisia b Laboratoire de Microbiologie, 5000 Monastir, Tunisia c Faculte´ de Me´decine, 5000 Monastir, Tunisia d Laboratoire d’Immunologie, 5000 Monastir, Tunisia e Laboratoire de Microbiologie, 4000 Sousse, Tunisia Received 15 August 2005; accepted 28 October 2005
Abstract Community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections are an emerging problem, especially related to the production of staphylococcal toxins. In this study we investigate the phenotypic and genotypic characteristics of 72 Tunisian MRSA. Our results revealed that these strains are multiresistant. Using multiplex polymerase chain reaction, we detected staphylococcal cassette chromosome mec (SCCmec) type IV and IVA in 66 isolates. The latter are Panton–Valentine leukocidin positive. The leukotoxin genes lukE-lukD were found in most strains (92.4%). The amplification of g-hemolysin gene was detected only in 2 MRSA isolates. Among all strains, only 1 expressed the exfoliatin A. fnbA gene was detected in 12 strains, fnbB gene in 2 strains, and both fnbA and fnbB genes in 2 other strains. The most predominant accessory gene regulator group identified was group III. Full characterization of these MRSA strains requires the association of SCCmec typing with other molecular methods such as pulsed-field gel electrophoresis, multilocus-sequence typing, and spa typing. D 2006 Elsevier Inc. All rights reserved. Keywords: MRSA; Virulence factors; agr groups; SCCmec; Characterization
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen both in nosocomial and communityacquired infections (Lowy, 1998). The emergence of MRSA was due mainly to the acquisition of the mecA gene. The latter was carried by a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec) that was integrated into methicillin-susceptible S. aureus chromosome (Katayama et al., 2000). Up to now, 5 major types of SCCmec element (I–V) and a few variants have been identified (Okuma et al., 2002; Ito et al., 2004). The types I, II, and III were described in hospital-acquired MRSA (HA-MRSA) strains. The fourth type was described in community4 Corresponding author. Institut Supe´rieur de Biotechnologie de Monastir, Unite´, 05/UR/09-11, Rue T. Haddad, 5000 Monastir, Tunisia. Fax: 216 - 73 - 465 - 404. E-mail address: [email protected] (M. Nour ). 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2005.10.017
acquired MRSA (CA-MRSA) isolates (Ma et al., 2002) and also in some hospital-associated MRSA (Oliveira et al., 2002). The fifth type was recently identified in the chromosome of CA-MRSA in Australia (Ito et al., 2004). The pathogenicity of S. aureus infections is linked to the expression of several virulence factors including cell wall-associated adhesins and toxins such as the staphylococcal enterotoxins, the exfoliative toxins (ETA and ETB), the toxic shock syndrome toxin 1 (TSST-1), and the leukocidins (for review, see Dinges et al., 2000). Some toxins are responsible for specific clinical syndromes such as TSST-1 associated to toxic shock syndrome and staphylococcal enterotoxins associated to staphylococcal food poisoning (Lina et al., 1997). However, most of the severe S. aureus infections are due to the cumulative effects of several virulence determinants (Peacock et al., 2002). The regulation of a number of virulence factors of S. aureus is controlled by a system called the accessory gene
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regulator (agr). The agr locus is characterized by a polymorphism in the amino acid sequence of the bacterial density-sensing peptide known as autoinducing peptide (AIP) and of its corresponding receptor (agr C) (Mayville et al., 1999). Based on the specificity of the AIP for its membrane receptor, the agr locus has been shown to be polymorphic and divided into 4 distinct genetic groups (I–IV) (Ji et al., 1997). Previous studies had reported associations between agr group and some staphylococcal toxins, such as the production of TSST-1 that is associated to agr group III, and the production of exfoliatin that is in turn associated to agr group IV (Jarraud et al., 2000, 2002). The association between LukE-LukD and agr group II was reported more recently (Von Eiff et al., 2004). The aims of the present study are i) to determine the SCCmec elements carried by Tunisian MRSA strains, ii) to detect some virulence factor genes, iii) and to characterize the agr groups of these isolates.
oxacillin, kanamycin, tobramycin, gentamicin, tetracycline, chloramphenicol, erythromycin, lincomycin, pristinamycin, ofloxacin, sulfamethoxazole + trimethoprim, fusidic acid, vancomycin, and teicoplanin. The MICs of oxacillin were determined by the E-test method (Biodisk, Solna, Sweden) according to the manufacturer’s guidelines. 2.4. DNA extraction Bacterial DNA was extracted using the Wizard genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer’s instructions. 2.5. SCCmec typing To determine the types of SCCmec, multiplex polymerase chain reaction (PCR) analysis was performed as described previously (Oliveira and de Lencastre, 2002). Reference strain of S. aureus CIP103514 was used as positive control for mecA gene and for SCCmec type I. 2.6. Detection of accessory genes
2. Materials and methods 2.1. Bacterial strains During 2003–2004, only 72 MRSA isolates were recovered from clinical specimens of 72 patients at the Laboratory of Microbiology from a University Hospital in Monastir. Patients are aged from 2 months to 74 years and the mean age was 34.9 F 23.6 years (mean F SD). They were 34 males and 38 females; the sex ratio (female/male) is 1:11. Strains were collected from 59 outpatients and from 13 inpatients who developed infections within the first 48 h of their admission to the hospital. The isolates were recovered from pus (75%), blood (12.5%), and other specimens such as venous catheter (2.77%), conjunctival secretion (1.4%), and pleural liquid (8.3%). These strains were associated with various community-acquired infections such as furuncles (16 cases), cutaneous abscesses (12 cases), wound infection (6 cases), impetigo (6 cases), cellulitis (1 case), superficial abscess (2 case), septicemia (6 cases), arthritis (4 cases), osteomyelitis (3 cases), otitis (2 cases), vagina infection (3 cases), catheter-related infection (2 cases), pneumonia (1 case), fistula (1 case), and conjunctivitis (1 case). 2.2. Identification of MRSA The strains were identified as S. aureus from colony and microscopic morphology, catalase test, and by their ability to coagulate rabbit plasma (BioMe´rieux, Lyon, France). 2.3. Antimicrobial susceptibility testing The determination of antibiotics susceptibility of all strains was performed by the disk diffusion test on Mueller–Hinton agar according to the recommendations of the French Society of Microbiology bComite´ de l’Antibiogramme de la Socie´te´ Franc¸aise de MicrobiologieQ (CA-SFM) (http://www.sfm. asso.fr). The antibiotics tested were penicillin, cefoxitin,
The sequences of the primers used for amplification of virulence factors genes were reported previously (Jarraud et al., 2002; Nashev et al., 2004). These primers are specific for lukS-PV, lukF-PV (encoding Panton–Valentin leukocidin [PVL]), the leukotoxin genes lukE-lukD (encoding LukE-LukD), hlg (encoding g-hemolysin), eta, etb (encoding exfoliatins ETA and ETB), and fnbA, fnbB (encoding the fibronectin-binding proteins FnbA and FnbB). Amplification of these genes was performed by multiplex PCR under the following conditions: 4 min of initial denaturation at 94 8C, followed by 30 cycles of 1 min of denaturation at 94 8C, 1 min of annealing at 46 8C, 1 min of extension at 72 8C, and 4 min of final extension at 72 8C. 2.7. agr genotyping Using multiplex PCR, we determined the presence of agr allele group (I–IV). The nucleotide sequence of each primer and the amplification conditions have been described previously (Shopsin et al., 2003). 3. Results From 2003 to 2004, 72 MRSA strains were collected from the University Hospital in Monastir. Table 1 shows the antimicrobial susceptibility pattern for all isolates. Our results revealed that MRSA strains were resistant to kanamycin (88.9%), tetracycline (61.1%), erythromycin (30.5%) fusidic acid (38.8%), and gentamicin (25%). All MRSA strains were susceptible to pristinamycin, vancomycin, and teicoplanin. MICs determination showed that 73.61% of S. aureus had oxacillin MICs of z 256 Ag/mL. 3.1. SCCmec typing Using multiplex PCR method, the 72 MRSA strains were positive for the 162-bp internal fragment of the mecA gene. According to Oliveira and de Lencastre method (2002), we
M. Ben Nejma et al. / Diagnostic Microbiology and Infectious Disease 55 (2006) 21 – 26
identified 64 strains harboring SCCmec type IV and 2 strains harboring SCCmec variant IVA. We found the SCCmec type III in 4 strains, the SCCmec variant IIIB in 1 strain, and the SCCmec type I in another one.
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detected in 12 strains, fnbB gene in 2 strains, and both fnbA and fnbB genes in 2 other strains. 3.3. agr groups The amplification of the hypervariable region of the agr locus showed that the agr group III was the most predominant, detected in 73.3% of strains, followed by agr group I (16.6%), agr group IV (6%), and agr group II (3%).
3.2. Detection of accessory genes PCR amplification revealed that all strains harboring the SCCmec type IV and SCCmec type III were PVL positive. lukE-lukD genes were found in most strains (92.4%). The g-hemolysin gene was detected only in 2 MRSA strains. The amplification of the exfoliative toxin genes showed that only 1 strain expressed the exfoliatin A. The amplification of fnbA and fnbB genes revealed that fnbA gene was
4. Discussion MRSA strains are often isolated from nosocomial isolates of S. aureus and have become widespread in hospital.
Table 1 Resistance pattern and SCCmec types of 72 MRSA isolates
St
Antibiotic resistance phenotype Gm
K
Tm
C
Te
E
L
SXT
OFX
Ra
FOS
Fa
SCCmec type
Number of isolates
S R S S S R R R R R R R R R R R R R R R R R R R S S S R R R R R R S R S S S R R R R
S S S S S S S S S S S S S S S S R R R R R R S S S S S S S S S S R R R R R R R S R R
S S S S S R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R
S S S S S S R S S S S S S S S S R R R R R R R S R S S S S S S R R R R R R R R R R R
S S S S S S S S S S S S S R S S S S S S S S S S S S S S S S S S S S S S S S S S S S
S S R R R S S R S R S S R S S R R R R S S R S S S S S S S S S R R S R R R R R R R R
S S S S S S S S S S R R R R S S R S R S S R R R R S R S S R R R S R S R R R R R R S
S S S S S S S S S S S S S S S S R S R S S R S R R R R S S S R R S R S S R S S R R S
S S S S S S S S S S S S S S R S S S S S S S S S S S S R R S S S R S R S S S R S S S
S S S S S S S S S S S S S S S R S S S S S S S S S S S S S S R S S R S S S S S S R R
S S S S R S S S S S S S S S S S S S S S S S S S S S S S S S S S S R S S R S S S S S
S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S R S R S R R S S S S S S
S S S R S S S S R R S R R R S R R S S S R S S S R S S R S S R R S S S S S S S S S S
IV IV IV IV I IV IV IV IV IV IV IV IV IV IV IV IV IV III IV IV IIIB IV IV IV IV IV IV IV IV IV IV III IV III IV IV IV IVA IVA IV III
2 1 1 1 1 4 1 9 3 9 1 1 4 1 1 2 1 4 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
All isolates were susceptible to pristinamycin, vancomycin, and teicoplanin. St = streptomycin; Gm = gentamicin, K = kanamycin; Tm = tobramycin; C = chloramphenicol; Te = tetracycline; E = erythromycin; L = lincomycin; SXT = sulfamethoxazole + trimethoprim; OFX = ofloxacin; Ra = rifampicin; FOS = fosfomycin; Fa = fusidic acid; R = resistant; S = susceptible.
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Recently, new strains of MRSA have emerged in the community (Chambers, 2001). The term CA-MRSA is often used to refer to the detection of colonization or infection in the community rather than to actual acquisition of MRSA in the community. In the literature, different definitions were used to make a distinction between community and nosocomial MRSA. Most often, these definitions are based on the timing of isolation of MRSA in relation to the time of admission, with or without assessment of health careassociated risk factors. b Nosocomial infectionQ has been defined as an infection that develops in the hospital, but not incubating at the time of admission (Garner et al., 1988). A b community-acquired infection Q is an infection that is present or incubating at the time of admission and is not caused by an organism acquired during previous health care (Salgado et al., 2003). It has been previously reported that SCCmec type IV is a characteristic of CA-MRSA strains, and SCCmec types I, II, and III are specific to HA-MRSA (Ito et al., 2001; Oliveira and de Lencastre., 2002). Our results revealed that among 72 MRSA strains, 64 isolates contained the SCCmec type IV and 2 strains contained SCCmec variant IVA. These 66 strains were isolated from skin and invasive infections from nonhospitalized patients and hospitalized patients. The latter acquired infections within 48 h of admission to the hospital. These infections are acquired in the community, and the true site of acquisition of MRSA is unknown. The 6 remaining strains carried the SCCmec types I, III, and IIIB. This finding can be explained by the fact that these infections were caused by nosocomial strains which, in turn, have been acquired into the community. It is known that HA-MRSA strains are multiresistant because the type II and III SCCmec elements carry additional determinants, conferring resistance to non–h-lactam antibiotics (Ito et al., 2001). CA-MRSA strains harboring a type IV SCCmec element are generally susceptible to non–h-lactam antibiotics (Zetola et al., 2005). However, this does not mean that CA-MRSA strains are fully susceptible to these antibiotics (for review, see Eady and Cove 2003). Tunisian MRSA strains have high rates of resistance to kanamycin, tetracycline, and fusidic acid. Likewise, European CAMRSA isolates appeared to develop resistance to the same antibiotics (Vandenesch et al., 2003; Dufour et al., 2002). Our results revealed that resistance to erythromycin was observed in a rate of 31.8% and gentamicin in only 19.6% of the MRSA strains carrying SCCmec type IV. Conversely to our findings, the Australian and French CA-MRSA isolates were susceptible to erythromycin and gentamicin (Dufour et al., 2002). The multiresistance (resistance to more than 3 non–h-lactam antibiotics) of Tunisian MRSA isolates might be due to the antibiotic’s selective exposure. CA-MRSA strains are considered as the causative agent of skin and soft tissue infections, and some strains are particularly virulent and capable to induce life-threatening infections (for review, see Eady and Cove 2003). The detection of PVL genes demonstrated that 70 of 72 strains
isolated from various types of infection are able to produce PVL. Lina et al. (1999) have detected PVL genes in CA-MRSA associated with furuncles, cutaneous abscesses, cellulitis, as well as with necrotic hemorrhagic pneumonia, and osteomyelitis. Indeed, PVL-producing S. aureus strains cause chronic cutaneous infections (Dufour et al., 2002). These findings emphasize the importance of PVL as a virulence factor of primary necrotic infections (furuncles, cutaneous abscesses) (Couppie´ et al., 1994) and of all serious secondary skin infections (Dufour et al., 2002). PVL genes were detected also in 4 of 6 Tunisian nosocomial MRSA strains (carrying SCCmec I, III, and IIIB), whereas Dufour et al. (2002) have reported that PVL genes were never detected in MRSA strains associated with hospital-acquired infection. This result could be explained by the easy spread of the phages carrying the PVL genes among different backgrounds (Jarraud et al., 2002). In our study, lukE-lukD leukocidin genes were detected in the majority of strains (92.4%) isolated from all types of infection. Similar results have been reported by Dufour et al. (2002) in French CA-MRSA strains isolated from patients with all types of staphylococcal infections. However, Gravet et al. (2001) have reported that LukE-LukD is significantly associated with primary skin infections and revealed a direct association between LukE-LukD and other toxins. Both LukE-LukD and exfoliatin A or B were found to be produced by S. aureus associated with impetigo (Gravet et al., 2001). Furthermore, both LukE-LukD and enterotoxin A were predominantly detected in S. aureus isolated from patient with postantibiotic diarrhea (Gravet et al., 1999). The amplification of hlg gene revealed that this gene was detected only in 2 strains among all Tunisian MRSA. Indeed, Lina et al (1999) found that g-hemolysin was produced by more than 99% of S. aureus clinical strains. Generally, exfoliatin is detected in S. aureus strains, which are implicated in the case of scalded syndrome (Lina et al., 1997). Several cases of impetigo and bullous impetigo were caused by an unexpected clonal group of MRSA producing exfoliative toxin A or B. These strains were identified first in Japan and then in France (Yamaguchi et al., 2002; Liassine et al., 2004). In our study, ETA was found in the case of arthritis. It has been suggested that the production of these toxins is due to the incorporation of a phage carrying the eta gene into lysogene MRSA strains or to the acquisition of mecA by exfoliatin-producing methicillinsusceptible S. aureus strains (for review, see Eguia and Chambers 2003). The pathogenesis of S. aureus is dependent on its production of toxins as well as of staphylococcal surface adhesins. These adhesins contribute to the adherence to host extracellular matrix components such as fibronectin (Patti et al., 1994). Binding to fibronectin is mediated by 2 related proteins, FnbA and FnbB (Nashev et al., 2004). In our study the detection of fibronectin binding protein genes revealed that both FnbA and FnbB were found in cases of skin
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infections (furuncles and wound infection) and in cases of invasive infections (otitis and osteomyelitis). These results were similar to the findings reported by Nashev et al. (2004) who demonstrated that S. aureus nasal and skin infection isolates produce FnbA and FnbB with a high frequency. Previous studies have found that FnbA and FnbB are significantly implicated in invasive diseases such as endocarditis, osteomyelitis, septic arthritis, and nosocomial pneumonia (Peacock et al., 2000; Mongodin et al., 2002). The production of some virulence factors is under the control of the agr system (Van Leeuwen et al., 2000). The most predominant agr group in MRSA isolates from worldwide collections is agr group I (Van Leeuwen et al., 2000). However, we found that agr group III was the most prevalent among the Tunisian MRSA strains. Similar results were found by Vandenesch et al (2003) in French CA-MRSA strains. In addition to agr group III, some of our strains belong to the other agr groups I, II, and IV. No relationship between agr groups and toxins were revealed, whereas previous studies have reported the association between agr group III and TSST-1, between agr group IV and exfoliatin (Jarraud et al., 2000, 2002), and between agr group II and LukE-LukD (Von Eiff et al., 2004). In conclusion, most Tunisian MRSA studied and harboring SCCmec type IV and IVA are resistant to antibiotics other than h-lactam antibiotics. The agr group III is the predominant group for these strains. The combination of the multiresistance and virulence factors may play a key role in the pathogenesis of MRSA. For best characterization and for elucidation of the genetic background of these MRSA, more studies are needed associating SCCmec typing with other molecular methods such as pulsed-field gel electrophoresis, multilocus-sequence typing, and spa typing.
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