Methicillin-resistant Staphylococcus aureus (MRSA): Microbiology

INTRODUCTION — Methicillin is a semisynthetic beta-lactamase–resistant penicillin that was introduced in 1959; shortly thereafter, methicillin-resistant isolates of Staphylococcus aureus and coagulase-negative staphylococci were described. Outbreaks of methicillin-resistant S. aureus (MRSA) infection occurred in Europe in the early 1960s [1]. Subsequently, MRSA has emerged as major nosocomial and community-acquired pathogens.

Three pandemic MRSA clones have been traced back to the original 1959 MRSA isolates in Denmark and England [2]. In addition, molecular typing of MRSA strains collected from many geographic areas has revealed that five major MRSA clones emerged worldwide by 2002 [3]. The epidemic community-associated MRSA strains in the early 2000s appear to have emerged from earlier epidemic clones [4-6].

Our understanding of the genetic mechanisms responsible for methicillin resistance will be reviewed here, as are issues related to laboratory detection. Virulence determinants for community-acquired MRSA are discussed separately, as are mechanisms for S. aureus with reduced susceptibility to vancomycin. (See "Virulence determinants of community-acquired methicillin-resistant Staphylococcus aureus" and "Staphylococcus aureus bacteremia with reduced susceptibility to vancomycin".)

DEFINITION — Methicillin resistance requires the presence of the mec gene; strains lacking a mec gene are not methicillin resistant. Methicillin resistance is defined by the Clinical Laboratory Standards Institute (CLSI) as an oxacillin minimum inhibitory concentration (MIC) ≥4 mcg/mL; MICs of ≤2 mcg/mL are considered susceptible [7]. The EUCAST breakpoint differs from CLSI and consists of an oxacillin MIC >2 mcg/mL [8]. Oxacillin a semisynthetic penicillin that has supplanted methicillin since methicillin is no longer commercially available.

Other methods such as the use of the cefoxitin disk diffusion test or one of several polymerase chain reactions to detect the mec gene, are also commonly used. Isolates resistant to oxacillin or methicillin are also resistant to most beta-lactam agents, including cephalosporins; exceptions include ceftaroline and ceftobiprole (fifth-generation cephalosporins).

MOLECULAR MECHANISM OF METHICILLIN RESISTANCE

mec gene — The presence of the mec gene is an absolute requirement for S. aureus to express methicillin resistance. The mec gene is absent from susceptible strains and present in all resistant strains [9-11]. The structural component of the mec gene, mecA, encodes the penicillin-binding protein 2a (PBP2a) that establishes resistance to methicillin and other semisynthetic penicillinase-resistant beta-lactams.

The mechanism of oxacillin resistance may be different in borderline-resistant strains, in which the mec gene is not present or is present in a very small resistant subpopulation (referred to as heteroresistance). (See 'Borderline resistance' below.)

The complete genomes for numerous strains of MRSA have been published; three classes of pathogenicity islands and several different superantigens have been identified [12-14]. The mec gene consists of a structural component, mecA, and, in many cases, two regulatory components that control expression of the gene:

●mecR1-mecI is a negative regulator of mecA transcription. Mutations in this set of genes results in more highly resistant strains.

●The beta-lactamase genes (blaI, blaRI, and blaZ) control expression of beta-lactamase and, because of sequence similarity to the mecR1-mecI genes, also can downregulate mecA gene transcription [15]. However, beta-lactamase produces resistance by a mechanism different from mec; it hydrolyzes the beta-lactam ring. Because this negative regulation is not tightly controlled, expression of resistance following exposure to beta-lactams is relatively rapid. The looser control allows the mec gene to synthesize protein, which, under conditions of greater regulatory control, would not be permitted.

In addition to these regulatory genes, there is a series of five auxiliary genes that can modify expression of methicillin resistance. These are the fem (factor essential for the expression of methicillin resistance) A to E genes. (See 'fem genes' below.)

Penicillin-binding protein 2a — Penicillin-binding proteins are peptidase enzymes located in the bacterial membrane that catalyze the transpeptidation reactions of peptidoglycan during cell wall synthesis. mecA encodes penicillin-binding protein (PBP) 2a, an inducible protein that establishes resistance to the semisynthetic penicillinase resistant beta-lactams: methicillin, nafcillin, oxacillin, and all cephalosporins [16,17]. In contrast with the other four PBPs (1-4), PBP2a has a low affinity for beta-lactam antibiotics. (See "Resistance of Streptococcus pneumoniae to beta-lactam antibiotics".)

In susceptible staphylococcal isolates, the beta-lactams covalently bind to PBPs 1-3, thereby inactivating enzyme activity, preventing transpeptidation, and ultimately contributing to bacterial death. PBP2a, with its low affinity for the beta-lactams, can substitute for the enzymatic activity of these PBPs and allow completion of cell wall assembly. The resulting peptidoglycan has a structurally different muropeptide composition, a change that does not appear to affect cellular function [18].

Staphylococcal chromosomal cassette mec (SCCmec) — The SCCmec cassette is a mobile genetic element that contains the mec gene and is found in Staphylococcal spp. There are at least 14 staphylococcal cassette chromosome (SCCmec) types that have been identified to date. The mec gene is part of a 21 to 67 kb mobile chromosomal element called the SCCmec. The majority of health care-associated (mostly nosocomial) MRSA clones is associated with SCCmec types I, II, and III and are multidrug resistant [19]. In contrast, most community-associated MRSA (CA-MRSA) strains have type IV or V SCCmec and were formerly susceptible to other antibiotic families; this is no longer the case [13,20-24].

Methicillin resistance may reduce the virulence of health care-associated MRSA by interfering with agr quorum sensing [25]. In one study, health care-associated MRSA strains carrying SCCmec type II produced reduced amounts of cytolytic toxins as measured by an in vitro T cell survival assay and in vivo murine bacteremia model. Alteration of the cell wall appeared to affect the agr quorum sensing system, resulting in diminished virulence. This effect has not been observed among CA-MRSA isolates and may help explain the failure of hospital-acquired MRSA to spread into the community.

As noted, sequencing SCCmec from many MRSA strains has resulted in identification of different SCCmec types (I-XIV) that vary in genetic makeup and size [26-28]. Transfer of SCCmec from MRSA into well-adapted strains of methicillin-susceptible S. aureus has occurred on several occasions, resulting in new MRSA isolates that spread rapidly in health care institutions. This transfer is uncommon, restricted to a limited number of staphylococcal lineages. In 2011, a novel mecA homologue mecC (SCCmec type II) was identified in isolates obtained from both cows and humans [29]. This paper (as well as others) identified livestock as a reservoir of MRSA with the potential for transmission to humans [30].

Origin in coagulase-negative staphylococci — It is believed that the mec gene was acquired from closely related staphylococcal species via a limited number of genetic events. The mec gene is essentially the same in all staphylococcal species. Several studies point to coagulase-negative staphylococci (CoNS) as the origin of methicillin resistance in S. aureus. Segments of DNA from an insertion sequence found in CoNS (IS 1272) have been identified in MRSA [31,32]. Insertion sequence elements are DNA segments that encode enzymes that allow for site-specific recombination. In addition, one study reported 88 percent amino acid homology for the MRSA mec gene in Staphylococcus sciuri or Staphylococcus fleuretti [33], another species of CoNS [34]. The presence of different insertion sequence fragments within the mec gene makes transposition a likely mechanism of transfer for the gene between species [35].

Sequencing of the MRSA strain USA300, the epidemic clone that is now the most common cause of community-acquired MRSA infections in the United States, has shown that additional virulence and resistance genes have also been acquired from CoNS [14]. These genes include molecular variants of enterotoxins Q and K and a mobile element (the arginine catabolic mobile element) that encodes an arginine deaminase pathway and an oligopeptide permease system. The authors hypothesize that these genes enable the strain to evade host immune responses and contribute to its ability to survive and spread in host tissue. The virulence of USA300 appears to be linked to the differential expression of selected virulence determinants that were already present in the progenitor strain (USA500) of USA300 [5]. There are other community-acquired MRSA strains circulating in different geographic regions [36].

EXPRESSION OF METHICILLIN RESISTANCE — Despite the presence in MRSA of the mec gene, the phenotypic expression of methicillin resistance varies. There are three different forms for the expression of methicillin resistance: homogeneous, heterogeneous, and borderline (see 'Borderline resistance' below). Most clinical isolates of MRSA from a given patient are heterogeneous in their expression of methicillin resistance.

As an example, under routine growth conditions (ie, 37ºC, unsupplemented media), ≥99.9 percent of MRSA appear to be susceptible to beta-lactams. However, if the cells are grown at 30 to 35ºC or in the presence of 6.5 percent sodium chloride, they become more homogeneously resistant and express beta-lactam resistance at a much higher frequency [35]. In addition, growth of heterogeneous strains in the presence of a beta-lactam results in the selection of a homogeneous phenotype. Serial passage of these cells in the absence of antibiotic leads to slow reversion back to the heterogeneous state.

A similar phenomenon has been observed in experimental endocarditis. Treatment of rabbits infected with MRSA with a beta-lactam results in a greater percentage of the total bacterial population being resistant than in untreated controls [37]. There was also a correlation between the potential efficacy of the antibiotic and its affinity for binding to PBP2a in vitro.

fem genes — The fem genes and other auxiliary genes are necessary for the homogeneous expression of resistance [38]. These genes affect different steps in the synthesis of peptidoglycan. Inactivation of these genes can convert a homogeneously resistant strain to a heterogeneous resistant one [39].

Borderline resistance — Borderline resistance refers to isolates that are at the margin of resistance (termed BORSA: Borderline Oxacillin Resistant S. aureus). This type of resistance may be due to one of several mechanisms:

●Some strains with borderline resistance possess the mecA gene. In these strains, the resistant subpopulation may be extremely small and therefore more susceptible to beta-lactams.

●In strains that lack the mecA gene (and therefore penicillin-binding protein [PBP] 2a), there may be alterations in or overexpression of the other PBPs, resulting in reduced affinity for beta-lactams or the availability of more enzyme for peptidoglycan synthesis. Overproduction of beta-lactamase with slow hydrolysis of the beta-lactam antibiotic has also been hypothesized as a potential mechanism for borderline resistance in mecA-negative strains [35,40,41].

LABORATORY DETECTION — There are both phenotypic and DNA-based methods for the detection of MRSA. The traditional microbiology laboratory techniques include broth microdilution, agar screening plates containing salt (4%) and oxacillin (6 mcg/mL), and cefoxitin disk diffusion tests [7]. The cefoxitin disk diffusion test was among the most reliable tests [42]. Polymerase chain reaction (PCR) assays consistently detected both genes [43]. For detection of methicillin resistance in mecC strains, phenotypic testing is needed; genotypic methods (such as PCR) may not be used given limited homology between mecA and mecC strains [44].

Rapid testing — Rapid testing for detection of MRSA is discussed in detail separately. (See "Rapid detection of methicillin-resistant Staphylococcus aureus".)

Surveillance — Surveillance cultures are performed at body sites (mostly the anterior nares and the oropharynx) that are frequently colonized with MRSA. Most patients with asymptomatic MRSA colonization will be detected by screening culture from the anterior nares (sensitivity 73 to 93 percent) [45-47].

Traditional methods used to process surveillance cultures take 48 to 72 hours to yield results. However, newer techniques shorten the amount of time required to detect MRSA in surveillance cultures. There are a variety of chromogenic agars available that can detect MRSA strains within 24 hours. One of these chromogenic selective agars contains cefoxitin and detects a majority of MRSA isolates within 24 hours, while commercially available real-time PCR tests for mecA can detect MRSA within two hours [48].

Antimicrobial susceptibility testing — Antibiotic susceptibility testing for methicillin resistance has been modified to enhance the detection of these isolates; many are heterogeneously resistant to methicillin. Susceptibility testing now includes use of the more stable oxacillin rather than methicillin disk, incubation at ≤35ºC for 24 rather than 18 hours, and the incorporation of 6.5 percent sodium chloride into the media. Cefoxitin disk diffusion tests have proven a reliable method for detection of MRSA because cefoxitin is a better inducer of the mecA gene than oxacillin. This is especially true for the detection of SCCmec II. It is important to remember that isolates resistant to oxacillin are also resistant to all beta-lactam agents including cephalosporins.

In the past, most health care-associated MRSA strains were multidrug resistant. Isolates that are resistant to oxacillin but remain susceptible to most non-beta-lactam agents (eg, trimethoprim-sulfamethoxazole, clindamycin, and ciprofloxacin) are often community-associated MRSA; however, these strains have also become increasingly antibiotic resistant.

Elevated vancomycin MIC — Increased mortality and treatment failure have been reported in patients treated with vancomycin for MRSA bacteremia when their isolates have elevated minimum inhibitory concentrations (MICs). Risk factors that predict a vancomycin MIC of 4 mcg/mL include age >50 years, administration of vancomycin for >48 hours in the week prior to bacteremia, chronic liver disease, history of MRSA bacteremia, and presence of a central line [49]. (See "Staphylococcus aureus bacteremia with reduced susceptibility to vancomycin".)

Additional data suggest that an increased risk of treatment failure among patients with hospital-acquired MRSA infections compared with community-acquired MRSA may be independent of the vancomycin MIC. There may be an intrinsic strain-specific defect [44]. The perceived risk of treatment failure may be independent of antibiotic choice, as patients with methicillin-susceptible S. aureus bacteremia and a high vancomycin MIC have an equal risk for treatment failure as patients with MRSA bacteremia and a high vancomycin MIC [50]. A systematic review and meta-analysis of 38 studies of S. aureus bacteremia concluded that patients with S. aureus bacteremia and a vancomycin MIC ≥1.5 had no difference in outcomes compared with patients with S. aureus bacteremia and vancomycin MIC <1.5, regardless of assay type [51].

SUMMARY

●Shortly after the introduction of methicillin in 1959, isolates resistant to this agent were reported. Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections occurred in Europe in the early 1960s. Since these original descriptions, MRSA as well as coagulase-negative staphylococci, which are commonly resistant to methicillin, have emerged as major nosocomial and, in the case of MRSA, community-acquired pathogens. (See 'Introduction' above.)

●Methicillin resistance is defined by the Clinical Laboratory Standards Institute as an oxacillin minimum inhibitory concentration (MIC) ≥4 mcg/mL. Isolates resistant to oxacillin or methicillin are also resistant to all beta-lactam agents, including cephalosporins; exceptions include ceftaroline and ceftobiprole. (See 'Definition' above.)

●Methicillin resistance requires the presence of the mec gene; strains lacking a mec gene are not methicillin resistant. The structural component of the mec gene, mecA, encodes the penicillin-binding protein 2a (PBP2a) that establishes resistance to methicillin and other semisynthetic penicillinase resistant beta-lactams. (See 'Definition' above and 'mec gene' above.)

●The most accurate methods to detect MRSA are polymerase chain reaction for detection of the mecA gene and latex agglutination tests for the protein product of mecA, PBP2a. When these tests are not available, traditional microbiology laboratory techniques are acceptable, such as oxacillin-salt agar screening plates and cefoxitin disk diffusion tests. Rapid testing for detection of MRSA is discussed in detail separately. (See 'Laboratory detection' above and "Rapid detection of methicillin-resistant Staphylococcus aureus".)