Methicillin Resistance in Staphylococcus aureus and ...

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which is inserted in the SCCmec cassette. This cassette is a mobile genetic element consisting of five differ- ent types and several subtypes. Oxacillin-resistant ...
Microbiol. Immunol., 51(9), 787–795, 2007

Review

Methicillin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococci: Epidemiological and Molecular Aspects André Martins, and Maria de Lourdes R.S. Cunha* Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brasil, Caixa Postal 510, CEP 18618–000 Received March 29, 2007.

Abstract: Infections caused by the genus Staphylococcus are of great importance for human health. Staphylococcus species are divided into coagulase-positive staphylococci, represented by S. aureus, a pathogen that can cause infections of the skin and other organs in immunocompetent patients, and coagulase-negative staphylococci (CNS) which comprise different species normally involved in infectious processes in immunocompromised patients or patients using catheters. Oxacillin has been one of the main drugs used for the treatment of staphylococcal infections; however, a large number of S. aureus and CNS isolates of nosocomial origin are resistant to this drug. Methicillin resistance is encoded by the mecA gene which is inserted in the SCCmec cassette. This cassette is a mobile genetic element consisting of five different types and several subtypes. Oxacillin-resistant strains are detected by phenotypic and genotypic methods. Epidemiologically, methicillin-resistant S. aureus strains can be divided into five large pandemic clones, called Brazilian, Hungarian, Iberian, New York/Japan and Pediatric. The objective of the present review was to discuss aspects of resistance, epidemiology, genetics and detection of oxacillin resistance in Staphylococcus spp., since these microorganisms are increasingly more frequent in Brazil. Key words: Staphylococcus aureus, Coagulase-negative staphylococci, Oxacillin, mecA

and birds (24). According to Kloos and Bannerman (26), the main species involved in human infections are S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, S. cohnii, S. xylosus, S. capitis, S. warneri, S. hominis, S. simulans, S. saccharolyticus, S. auricularis, S. caprae, S. lugdunensis, and S. schleiferi. The fundamental characteristics of the genus Staphylococcus compared to the genera Micrococcus are its sensitivity to 100 µg furazolidone, resistance to 0.04 U bacitracin

Introduction Among the numerous bacterial infections, those caused by the genus Staphylococcus are of great importance for human and animal health. Staphylococcus species are divided into two large groups. The first group, known as coagulase-positive staphylococci, is mainly represented by S. aureus, a pathogen that can cause a variety of infections in immunocompetent patients ranging from cutaneous to systemic infections. The second group, known as coagulase-negative staphylococci (CNS), comprises diverse species that are normally involved in infectious processes in immunocompromised patients or patients using catheters and are members of the normal flora of humans, mammals

Abbreviations: AP-PCR, arbitrarily primed PCR; CLSI, Clinical and Laboratory Standards Institute; CNS, coagulase-negative staphylococci; CPCIH, Permanent Commission for the Control of Hospital Infections; MH, Mueller Hinton agar; MIC, minimal inhibitory concentration; MLST, multilocus sequence typing; MOD-SA, modified resistance; MRSA, methicillin-resistant S. aureus; NCCLS, National Committee for Clinical Laboratory Standards; PBP, penicillin-binding proteins; PCR, polymerase chain reaction; PFGE, pulsed-field gel electrophoresis; RAPD, random amplified polymorphic DNA; REA, analysis of chromosomal DNA after enzymatic restriction; Rep-PCR, repetitive DNA sequence PCR; SCCmec, staphylococcal cassette chromosome; VRSA, vancomycin-resistant S. aureus.

*Address correspondence to Dr. Maria de Lourdes R.S. Cunha, Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, SP, Brasil, Caixa Postal 510, CEP 18618–000. Fax: 55–14–3815–3744. E-mail: [email protected]

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and the production of acid from glucose under anaerobic conditions (25). Staphylococcus aureus can cause food intoxication, pneumonia, bacteremia, impetigo, folliculitis and osteomyelitis in humans, and mastitis, arthritis and urinary tract infections in animals (24). Staphylococci have been described as the main cause of nosocomial bacteremias, with CNS and S. aureus being the most common causative agents of bacteremia in the United States (30). Among CNS, S. epidermidis has been the most frequently isolated species (53). S. aureus has been described as the second most frequent cause of nosocomial bacteremia in England and Wales, preceded only by Escherichia coli (28). In addition to the importance of staphylococci as nosocomial pathogens, the frequency of communityacquired infections caused by these microorganisms has increased over the last few years (1), a fact further emphasizing the importance of these pathogens. In addition, resistance to antibiotics has also increased over the years. A review on antibiotic resistance in the genus Staphylococcus reported that in 1944, when penicillin was introduced, more than 94% of isolates were susceptible to this drug (28). This number fell to half in the 1950s, and at present 80 to 90% of isolates are resistant (6). Similar data have been reported in Brazil (36). According to data from the Permanent Commission for the Control of Hospital Infections (CPCIH) of the University Hospital, Faculty of Medicine of Botucatu, 90% of S. aureus isolates are also resistant to this drug. The introduction of methicillin and other semisynthetic penicillins such as oxacillin and penicillinaseresistant methicillin in 1959 represented a significant step in antistaphylococcal therapy. The first report on methicillin resistance was published in 1961, shortly after the launching of this drug (19). In the United Kingdom, resistance to this drug was first reported in 1962, later spreading to various European hospitals and subsequently observed worldwide (43). According to these authors, the main problem of methicillin resistance lies in the fact that infections caused by methicillin-resistant S. aureus (MRSA) are difficult to treat, with these microorganisms in some cases being only susceptible to glycopeptides and experimental drugs. Epidemiological Aspects At present, resistance of Staphylococcus to methicillin is a problem of global proportions, with studies carried out throughout Europe, Africa, America and Asia demonstrating a predominance of MRSA over isolates susceptible to this drug (55). According to this

author, on the European continent the prevalence of MRSA ranges from 1% in Scandinavian countries to 80% in Italy, Greece and France. In an extensive study involving 143 hospitals in Spain, Cuevas et al. (12) evaluated the resistance of Staphylococcus spp. to different antimicrobial drugs and observed resistance to oxacillin (31.7% for S. aureus and 67% for CNS), erythromycin (31.7% for S. aureus and 63% for CNS), and gentamicin (16.9% for S. aureus and 27.8% for CNS). Among the species detected, S. aureus was isolated in 54.3% of cases and CNS in 45.7%. S. epidermidis was the most frequently isolated among CNS evaluated in that study (56%). Eighty-nine percent of the S. aureus isolates were resistant to penicillin. A study conducted in Finland reported an increase in the incidence of methicillin-resistant S. epidermidis from 28% in 1983 to 77% in 1994. A significant increase in the isolation of nosocomial MRSA strains has also been observed in other countries. For example, this percentage increased from 2.4% in 1973 to 35% in 1996 in the United States and from 1.7% in 1990 to 8.4% in 1995 in Germany (55). With respect to CNS, 65% of strains isolated from bacteremias in the United States are resistant to oxacillin (3). Several methicillin-resistant Staphylococcus spp. strains have also been isolated from catheters. Bouza et al. (4), evaluating the characteristics of the microbiota found in catheters from different regions of Europe, observed that 40% of the isolates identified as S. aureus and 63.7% of the isolates characterized as CNS were resistant to oxacillin. In Brazil, the frequency of oxacillin resistance is estimated to be high among S. aureus isolates, especially in large and university hospitals. Studies conducted at São Paulo Hospital, Escola Paulista de Medicina (54), and at the University Hospital of the Federal University of Uberlândia (27), reported incidences of the order of 50%. These strains differ from oxacillin-susceptible strains only in terms of physiological features but also in terms of their susceptibility profiles. MRSA isolates are normally resistant to other beta-lactam antibiotics, macrolides, aminoglycosides, chloramphenicol, quinolones, and tetracycline (10, 50). On the basis of in vitro observations, the CLSI (11) recommends oxacillin-resistant Staphylococcus to be reported as resistant to all beta-lactams, including cephalosporins and carbapenems. In Brazil, Cunha and Lopes (13), analyzing CNS strains isolated from newborns, observed that 71.8% of the isolates were producers of beta-lactamase and about 50% of these strains were resistant to oxacillin. In view of the emergence of vancomycin-resistant strains, together with the fact that these isolates are also

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resistant to methicillin, a better understanding of the mechanisms underlying methicillin resistance is necessary. Resistance to methicillin is located on the chromosome and is encoded by a gene known as mecA, which is regulated by two other genes called mec1 and mecR1 (10). To destroy bacteria, many antibiotics bind to penicillin-binding proteins (PBP) in order to inactivate them. These proteins are involved in the assembly of the cell wall of microorganisms, with bacteria lacking a correctly assembled wall being unable to maintain their integrity and dying. Whereas S. aureus strains normally employ three PBPs (PBP 1, 2 and 3) in the synthesis of the cell wall, staphylococci resistant to methicillin or oxacillin (MRSA) possess a supplementary PBP, PBP 2' or PBP 2a. Thus, in the presence of the mecA gene the cell is able to grow in the presence of oxacillin and other beta-lactams (28, 44). Although mecA-mediated resistance is found in all cells of an intrinsically resistant population, the gene may only be expressed in a small percentage of cells, a phenomenon called heterogenous resistance. The phenotypic expression of methicillin resistance in intrinsically resistant strains has been classified into four classes (classes 1 to 4), with class 1 being the most heterogenous and class 4 being the most homogenous (47). Most cells (99.9 or 99.99%) in a culture of heterogenously resistant class 1 strains present a minimal inhibitory concentration (MIC) of 1.5 to 3 µg/ml, but this culture also contains a small number of bacteria (107 to 108) that form colonies even in the presence of 25 µg oxacillin/ml or more. In cultures of strains belonging to class 2, most cells (99.9%) present a MIC ranging from 6 to 12 µg/ml, and the frequency of highly resistant strains (able to grow in the presence of 25 µg/ml) is higher (105) in these cultures than that observed for class 1 strains (47). Cultures of strains belonging to class 3 consist of bacteria (99 to 99.9%) that present high levels of resistance to oxacillin (MIC50 to 200 µg/ml), but generally possess a subpopulation (103) of highly resistant cells able to form colonies even in the presence of 300 to 400 µg oxacillin/ml. Cultures of class 4 strains consist of homogenously resistant bacteria, with all cells showing high levels of resistance (MIC400 to 1,000 µg/ml) (47). Genetics of Methicillin Resistance Methicillin resistance in Staphylococcus spp. strains is mediated by the staphylococcal cassette chromosome (SCCmec), a mobile genetic element composed of two genetic elements: mec, which is responsible for the resistance to methicillin, and the ccr complex, which is

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responsible for the integration and excision of the cassette in the bacterial genome (23, 56). Five different types of SCCmec have been characterized (SCCmec I, II, III, IV, and V). The first three types were described in detail by Ito et al. in 2001. In 2004, the same authors reported the most recent type, called type V. Ma et al. (29) described type IV, which is mainly found in community-acquired strains. SCCmec types I, II and III mainly cause nosocomial infections and are significantly larger than types IV and V. SCCmec type I has a size of 34,364 bp and is the smallest of the three types. This cassette does not carry any transposon or plasmid that confers resistance to drugs other than methicillin or to heavy metals; a subtype, known as IA, which differs from type I by the presence of an integrated plasmid (pUB110), has been described (39). The second cassette, called SCCmec type II, comprises 53,017 bp and carries, in addition to the mecA and mecRI genes that confer resistance to methicillin, transposon Tn554 which is responsible for the resistance of this type of isolate to erythromycin and streptomycin. This cassette possesses a subtype, called IIA, which is slightly smaller than type II (40 kb) (39). SCCmec type III, the largest of the five types, has a size of 66,896 bp and carries the mecA and mecRI genes, transposons Tn554 and ψTn554, and plasmid pT181, with transposon ψTn554 conferring resistance to cadmium and plasmid pT181 being responsible for resistance to tetracycline and mercury. In addition to the findings described above, Ito et al. (21) also reported differences in the ccr gene types, with the presence of a gene called ψccr in SCCmec type III which is not found in the other types. This finding suggests that some of the resistance traits present in type III might be used as selective markers. This type of cassette presents two subtypes, IIIA which does not contain plasmid pT181 or its flanking IS431 element, and IIIB which lacks copies of pT181 and Tn554 (39). SCCmec type IV is mainly responsible for community-acquired infections. The element is small and does not carry resistance genes other than mecA. In addition, this cassette presents multiple subtypes, suggesting that SCCmec type IV is highly transmissible (22). The four subtypes of SCC type IV (IVA, IVB, IVC and IVD) differ in their sequences upstream of the ccr complex, known as the L-C region (39). The most recently discovered SCCmec type is type V, which was identified in an Australian isolate by Ito et al. (22). The size of this novel cassette is 27,624 bp, slightly larger than SCCmec type IV but smaller than the other types. Like type IV, SCCmec type V possesses only genes encoding methicillin resistance; however,

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different from the other elements of this family, type V carries a new type of ccr gene, called type c, which is present as a single copy in contrast to the other elements which contain a pair of these genes. A novel element found only in type V is a restriction and modification system encoded by genes V22 and V23. At present, SCCmec type IV strains are frequent in Brazil. Trindade et al. (49) studied the prevalence of the type IV cassette in nosocomial S. aureus isolates. These strains were characterized regarding methicillin resistance by a broth microdilution technique and by the polymerase chain reaction (PCR). The latter method was also used to differentiate isolates belonging to the four different types. The authors found that type IV isolates occurred in different areas of the hospital, thus ruling out the hypothesis of an isolated infection, and observed the characteristic of susceptibility to nonbeta-lactam antibiotics predominating in communityacquired strains type IV. Another modality of methicillin resistance has been described in strains that do not carry the mecA gene, called borderline resistance. Two mechanisms have been proposed to explain this type of resistance: the first involves inactivation of oxacillin mediated by betalactamase hyperproduction (31), and the second is modified resistance, called MOD-SA, due to the production of modified intrinsic PBPs with reduced affinity for oxacillin (46). These modalities are characterized by a low level of resistance, with a MIC of 8 µg/ml (44). The mechanisms responsible for methicillin resistance in CNS are identical to those reported for S. aureus; however, mecA gene-mediated resistance is frequently expressed at lower levels than in MRSA, thus further impairing its detection (28). Genotypic Detection of Oxacillin Resistance in Staphylococcus spp. Screening tests for the detection of oxacillin-susceptible or -resistant isolates is of fundamental importance since cases of vancomycin resistance have been reported, with few antibiotics remaining for the prophylaxis of these infections. Resistance to oxacillin has been a major problem in the treatment of staphylococci, especially because of the heterogenous expression of oxacillin resistance (51). Resistance to oxacillin can be detected by several phenotypic and genotypic methods such as disk diffusion with oxacillin and cefoxitin, broth dilution, E-test, latex agglutination, and PCR, with genotypic methods being considered the gold standard for the detection of oxacillin resistance in staphylococci. At present, PCR is increasingly being used in epi-

demiological studies and laboratory routine for the identification of methicillin resistance genes, with satisfactory results. Several studies have described the use of PCR for the identification of the SCCmec element. Real-time PCR has been proposed by Huletsky et al. (20) for the detection of MRSA in clinical material without the need for previous isolation of the bacteria, an important fact in routine laboratory practice. PCR has also been applied in epidemiological studies, with good results. Using multiplex PCR, isolates carrying all SCCmec types described can be detected and differentiated in a single assay, thus providing a rapid and practical method for this type of study (56). Zhang et al. (56) used eight different primer pairs for the detection of the five SCCmec types and three subtypes, with good results, with the method permitting discrimination between the different types. To validate the study, 453 MRSA isolates already identified by standard PCR were typed by the new technique and the results were compared to those obtained previously, with the new method showing good results in SCCmec typing. However, the authors emphasized that in the future this assay should be adapted to new types and subtypes that may emerge. Although the application of PCR has increased over the last few years, the technique used as the gold standard in epidemiological studies continues to be pulsedfield gel electrophoresis (PFGE), which has been considered to be superior to PCR by some investigators (48). In this technique, isolates are typed regarding their electrophoretic DNA profile after enzymatic digestion. Trindade et al. (48) compared the main molecular typing techniques for the identification of isolates such as MRSA, including plasmid analysis, Southern blot, PFGE, repetitive DNA sequence PCR (rep-PCR), arbitrarily primed PCR (AP-PCR), Random Amplified Polymorphic DNA (RAPD), multilocus sequence typing (MLST) and analysis of chromosomal DNA after enzymatic restriction (REA) by analyzing the results of numerous scientific articles. This analysis revealed that a variation of PCR, called rep-PCR, presents a good discriminatory power and good reproducibility. RepPCR uses primers complementary to highly conserved repetitive DNA sequences that are present as multiple copies in the genomes of most Gram-negative and various Gram-positive bacteria (48). Soares et al. (42) compared variations of PFGE using different probes for the identification of 84 MRSA isolates belonging to the Brazilian clone and observed that the IS256 probe presented a higher discriminatory power and reproducibility than the other probes. The importance of that study lies in the fact that it was car-

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ried out with isolates belonging to the Brazilian clone, thus reflecting our reality in terms of the identification of MRSA isolates. Another technique used in epidemiological studies on MRSA isolates is MLST. This technique consists of the sequencing of fragments of seven housekeeping genes, with each isolate being characterized by a pattern of a numerical sequence of genes which is then identified by the letters ST followed by a number (15). Pandemic Clones of MRSA Isolates Staphylococcus spp. isolates have been typed in numerous epidemiological studies using the three techniques described above, which led to the identification of five large pandemic clones (Fig. 1). These clones include the Brazilian clone characterized by an SCCmec type IIIA, PFGE pattern B and MLST 2-3-1-14-4-3 (ST 239); the Iberian clone which possesses an SCCmec type IA element, PFGE pattern A and MLST 3-3-1-1/12-4-4-16 (ST 247); the New York/Japan clone characterized by an SCCmec type II element, PFGE pattern C and MLST 1-4-1-4-12-1-10 (ST 05); the Hungarian clone which possesses an SCCmec type III element, PFGE pattern A and MLST 2-3-1-1-4-4-3 (ST 239), and the last large clone known as Pediatric, which possesses an SCCmec type IV element, PFGE pattern A (with subtypes) and MLST 1-4-1-4-12-1-10 (ST 05) (32, 35, 37, 52). In Brazil, the predominant MRSA isolates belong to

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the Brazilian clone (36). The occurrence of this clone was first reported in 1992/1993 in different Brazilian hospitals. The isolates were characterized as belonging to the same clone by techniques such as PFGE and analysis of Tn554 patterns and mecA gene polymorphism (45). The Brazilian clone is also found in other countries such as the Czech Republic where it corresponded to 80% of all MRSA strains isolated between 1996 and 1997 (32). The Brazilian clone has also been detected in two hospitals in the Bangalore region, India, together with the Hungarian clone (SCCmec III) (2). The other clones are distributed throughout various parts of the world. The Iberian clone was first described in isolates from hospitals in Barcelona and Madrid, Spain, and Lisbon, Portugal. These isolates were typed by PFGE and probe hybridization, which produced a pattern that characterized them as belonging to the same clone. This clone is currently also found in other countries such as the Czech Republic (32). The New York/Japan clone was first isolated in the United States between 1994 and 1998, and then in Japan in 1997–1998 (35). This clone also predominated in a study on 98 MRSA isolates from Mexico, replacing the local clone, known as Mexican (PFGE M, SCCmec type IV), in nosocomial infections (52). The Hungarian clone, identified for the first time in Hungary in 1993–1994, was characterized by the same techniques as used for the clones described above in a study including MRSA isolates from six provincial hospitals in the country (14). The last of the large pandem-

Fig. 1. Schematic presentation of the different types of the staphylococcal cassette chromosome (SCCmec). Mec region: encodes resistance to oxacillin; ccr region: site of excision of the cassette. pUB110: encodes aminoglycoside resistance; Tn554: transposon encoding resistance to erythromycin and streptomycin; ΨTn554: encodes resistance to cadmium.

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ic clones, known as Pediatric, was isolated in large numbers in Colombia between 1996 and 1998, and also in Argentina and Poland between 1994 and 1998 and between 1990 and 1998, respectively (35). Phenotypic Detection of Resistance Genotypic methods have been used over the last few years in various epidemiological studies and have shown high sensitivity and specificity. These methods are able to discriminate various strains and clones and can also be applied to laboratory diagnosis. However, these tests are not available in all microbiology laboratories, with application of phenotypic tests thus being required. Reference methods recommended by the NCCLS or CLSI for the detection of oxacillin-resistant S. aureus include the determination of the MIC by the drug dilution method on agar or in broth, disk diffusion, screening on Mueller-Hinton (MH) agar supplemented with 4% NaCl and 6 µg oxacillin and, more recently, the cefoxitin disk diffusion test (11, 33, 34). The NCCLS (33) does not recommend screening on MH agar with 4% NaCl and 6 µg oxacillin for the detection of methicillin-resistant CNS due to the large number of falsenegative results. However, different phenotypic methods employing oxacillin for the evaluation of resistance of S. aureus and CNS strains have been used for a long time. Ferreira et al. (18) analyzed methicillin resistance in 132 CNS isolates by disk diffusion, E-test, latex agglutination and PCR. Sensitivity and specificity were 94.2% and 91.8% for the disk diffusion method, 100% and 71.4% for the E-test, and 97.1% and 98% for the latex agglutination technique, respectively. In the study by Tveten et al. (51), the agar dilution test presented 97.6% sensitivity and 95.4% specificity, whereas the Etest showed 100% sensitivity and 95.4% specificity, demonstrating that the E-test is a good phenotypic test for the determination of methicillin resistance in S. aureus and CNS. In a review of new techniques for the detection of methicillin resistance, Swenson (44) reported that phenotypic methods show high sensitivity which, however, does not reach 100%. The authors also observed that disk diffusion tests present a low sensitivity ranging from 61 to 88.5%. Studies evaluating the performance of disk diffusion for the detection of MRSA showed that this method is less reliable in the case of heterogenous strains. Studies analyzing known heterogenous isolates reported a sensitivity of 61% among a total of 80 mecA-positive isolates. In another study, sensitivity was 88.5% for class 1 isolates (extremely heterogenous) and 96.4% for class 2 isolates (16). The performance of the screening test on MH agar

supplemented with 4% NaCl and 6 µg oxacillin also depends on the degree of heterogeneity of the isolates analyzed, with a lower sensitivity (95%) being reported in studies analyzing a larger number of heteroresistant isolates, whereas other investigations found sensitivities of 97% (16, 38, 44). Recent studies evaluating cefoxitin disk diffusion for the detection of methicillin resistance obtained good results, with a sensitivity of about 100% and a specificity of 99% (5, 17, 40, 41). Cauwelier et al. (5) evaluated methicillin resistance in 155 clinical MRSA isolates using different methods such as oxacillin disk diffusion, latex agglutination and a screen test. Sensitivity and specificity were 100% and 99% for the cefoxitin disk diffusion test, respectively, whereas sensitivity fell to 91.7% in the oxacillin disk diffusion tests. Vancomycin-Resistant S. aureus (VRSA) The growing number of methicillin-resistant staphylococcal strains has left few alternatives for the treatment of infections caused by these pathogens. One of the drugs of choice has been vancomycin; however, cases of reduced susceptibility and resistance to this drug have been reported, a fact emphasizing the importance for the detection of methicillin-resistant strains. The first VRSA strain (MIC 16 µg/ml) was reported in the State of Michigan, U.S.A., in 2002, and was isolated from the catheter of a patient with diabetes, renal failure and vascular disease. The MIC for this isolate was 128 µg/ml, a value much higher than the levels accepted by the NCCLS as susceptible. In addition to vancomycin resistance, the isolate was also resistant to oxacillin, but was susceptible to chloramphenicol, linezolid, minocycline, quinupristin/dalfopristin, tetracycline and sulfamethoxazole/trimethoprim (7). In another case report from Pennsylvania, U.S.A., a VRSA was isolated from a patient with foot ulcerations and osteomyelitis. The isolate presented a MIC of 64 µg/ml and carried the vanA and mecA genes encoding vancomycin and methicillin resistance, respectively. The isolate was susceptible to chloramphenicol, linezolid, minocycline, quinupristin-dalfopristin, rifampicin and sulfamethoxazole/trimethoprim (7). Two years later, a third VRSA strain was isolated, this time from a urine culture (8). The isolate presented a MIC of 64 µg/ml and was again resistant to oxacillin and susceptible to rifampicin, chloramphenicol, sulfamethoxazole/trimethoprim, linezolid and minocycline. With respect to CNS, in a survey conducted in a neonatal intensive care unit, Center et al. (9) observed reduced susceptibility to vancomycin in 3.9% of the

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isolates, with 75% being identified as S. warneri. Conclusions and Perspectives With the emergence of vancomycin-resistant staphylococcal strains, which in the future may become a serious problem in the treatment of infections caused by Staphylococcus spp., further studies are necessary to improve the detection of methicillin-resistant strains since the number of nosocomial and communityacquired S. aureus and CNS isolates resistant to this drug is increasing. In particular, investigations involving CNS are required due to the lack of studies on these species. Epidemiological studies employing increasingly more precise tools will be necessary for the detection of different clones and because characteristics such as antibiotic resistance vary among different subtypes. Over the last few years, studies on the genetics of methicillin resistance in Staphylococcus spp. have provided valuable data for the scientific community; however, continuation of these studies is necessary since the prevalence of these strains in the hospital environment is still high in many regions of the world. References 1) Almer, L.S., Shortridge, V.D., Nilius, A.M., Beyer, J.M., Soni, N.B., Bui, M.H., Stone, G.G., and Flamm, R.K. 2002. Antimicrobial susceptibility and molecular characterization of community-acquired methicillin-resistant Staphylococcus aureus. Diagn. Microbiol. Infect. Dis. 43: 225–232. 2) Arakere, G., Nadig, S., Swedberg, G., Macaden, R., Amarnath, S.K., and Raghunath, D. 2005. Genotyping of methicillin-resistant Staphylococcus aureus strains from two hospitals in Bangalore, South India. J. Clin. Microbiol. 43: 3198–3202. 3) Baquero, F. 1997. Gram-positive resistance: challenge for the development of new antibiotics. J. Antimicrob. Chemother. 39 (Suppl A): 1–6. 4) Bouza, E., San Juan, R., Munoz, P., Pascau, J., Voss, A., and Desco, M. 2004. Cooperative Group of the European Study Group on Nosocomial Infections (ESGNI). A European perspective on intravascular catheter-related infections: report on the microbiology workload, aetiology and antimicrobial susceptibility (ESGNI-005 Study). Clin. Microbiol. Infect. 10: 838–842. 5) Cauwelier, B., Gordts, B., Descheemaecker, P., and Van Landuyt, H. 2004. Evaluation of a disk diffusion method with cefoxitin (30 µg) for detection of methicillin-resistant Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 23: 389–392. 6) CDC. 2005. Laboratory detection of: oxacillin/methicillinresistant Staphylococcus aureus. http://www.cdc.gov/ ncdod/hip/Lab/FactSheet/mrsa.htm 7) CDC. 2002. Staphylococcus aureus resistant to vancomycin —United States. MMWR 51: 565–567.

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8) CDC. 2004. Vancomycin-resistant Staphylococcus aureus — New York. MMWR 53: 322–323. 9) Center, K.J., Reboli, A.C., Hubler, R., Rodgers, G.L., and Long, S.S. 2003. Decreased vancomycin susceptibility of coagulase-negative staphylococci in a neonatal intensive care unity: evidence of spread of Staphylococcus warneri. J. Clin. Microbiol. 41: 4660–4665. 10) Chambers, H.F. 1997. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin. Microbiol. Rev. 10: 781–791. 11) CLSI. 2005. Performance standards for antimicrobial susceptibility testing. CLSI approved standard M100-S15, Clinical and Laboratory Standards Institute, Wayne, Pa., U.S.A. 12) Cuevas, O., Cercenado, E., Vindel, A., Guinea, J., SanchezConde, M., Sanchez-Somolinos, M., and Bouza, E. 1999. Evolution of the antimicrobial resistance of Staphylococcus spp. in Spain: five nationwide prevalence studies, 1986 to 2002. Antimicrob. Agents Chemother. 48: 4240–4245. 13) Cunha, M.L.R.S., and Lopes, C.A.M. 2002. Estudo da produção de β-lactamase e sensibilidade às drogas em linhagens de estafilococos coagulase-negativos isolados de recém-nascidos. J. Bras. Patol. Med. Lab. 38: 281–290. 14) De Lencastre, H., Severina, E.P., Milch, H., Thege, M.K., and Tomasz, A. 1997. Wide geographic distribution of a unique methicillin-resistant Staphylococcus aureus clone in Hungarian hospitals. Clin. Microbiol. Infect. 3: 289–296. 15) Enright, M.P., Day, N.P.J., Davies, C.E., Peacock, S.J., and Spratt, B.G. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38: 1008–1015. 16) Felten, A., Grandry, B., Lagrange, P.H., and Casin, I. 2002. Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test. J. Clin. Microbiol. 40: 2766–2771. 17) Fernandes, C.J., Fernandes, L.A., and Collingnon, P. 2005. Cefoxitin resistance as a surrogate marker for the detection of methicillin-resistant Staphylococcus aureus. J. Antimicrob. Chemother. 55: 506–510. 18) Ferreira, R.B.R., Iorio, N.L.P., Malvar, K.L., Nunes, A.P.F., Fonseca, L.S., Bastos, C.C.R., and Santos, K.R.N. 2003. Coagulase-negative Staphylococci: comparison of phenotypic and genotypic oxacillin susceptibility tests and evaluation of the agar screening test by using different concentrations of oxacillin. J. Clin. Microbiol. 41: 3609–3614. 19) Hiramatsu, K., Chui, L., Kuroda, M., and Ito, T. 2001. The emergence and evolution of methicillin resistant Staphylococcus aureus. Trends. Microbiol. 9: 486–493. 20) Huletsky, A., Giroux, R., Rossbach, V., Gagnon, M., Vaillancourt, M., Bernier, M., Gagnon, F., Truchon, K., Bastien, M., Picard, F.J., Van Belkum, A., Ouellette, M., Roy, P.H., and Bergeron, M.G. 2004. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J. Clin. Microbiol. 42: 1875–1884. 21) Ito, T., Katayama, Y., Asada, K., Mori, N., Tsutsumimoto,

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