Methicillin-resistant Staphylococcus aureus (MRSA): Microbiology and laboratory detection

INTRODUCTION — Approaches to detection of methicillin-resistant Staphylococcus aureus (MRSA) include culture methods and molecular techniques. Molecular diagnostic methods can reduce the turnaround time for detection of MRSA colonization and detection of MRSA from positive blood cultures.

Issues related to microbiology and laboratory detection of MRSA will be discussed here.

Issues related to S. aureus infection caused by vancomycin-intermediate and vancomycin-resistant isolates (vancomycin minimum inhibitory concentration ≥4) are discussed separately. (See "Staphylococcus aureus bacteremia with reduced susceptibility to vancomycin".)

MRSA DEFINITION — By definition, all MRSA isolates carry the mecA gene or a related variant known as mecC. Strains lacking a mec gene are not methicillin resistant.

Oxacillin, a semisynthetic penicillin, has supplanted methicillin since methicillin is no longer commercially available. Isolates resistant to oxacillin or methicillin are also resistant to most beta-lactam agents, including cephalosporins; exceptions include ceftaroline and ceftobiprole (fifth-generation cephalosporins).

The Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for MRSA differ:

●CLSI defines methicillin resistance as an oxacillin minimum inhibitory concentration (MIC) ≥4 mcg/mL; MICs of ≤2 mcg/mL are considered susceptible [1].

●EUCAST defines methicillin resistance as an oxacillin MIC >2 mcg/mL [2].  

Many MRSA isolates are heterogeneously resistant to methicillin. (See "Overview of antibacterial susceptibility testing", section on 'Heteroresistance'.)

MICROBIOLOGY

Genotypic and phenotypic mechanisms of resistance

●mecA gene and PBP2a – The presence of the mec gene is required for consistent expression of methicillin resistance [3-5]. The mec gene is contained within the SCCmec cassette, a mobile genetic element. Several studies point to coagulase-negative staphylococci as the origin of methicillin resistance in S. aureus [6,7].

The mec gene encodes penicillin-binding protein 2a (PBP2a). PBPs are peptidase enzymes in the bacterial membrane that catalyze transpeptidation reactions during cell wall synthesis.

In methicillin susceptible staphylococcal isolates, beta-lactams covalently bind to PBPs, leading to enzyme inactivation and prevention of transpeptidation, resulting in bacterial death. PBP2a differs from other PBPs in that it has a low affinity for beta-lactams; it can substitute for the enzymatic activity of the other PBPs, facilitating completion of cell wall assembly and therefore bacterial survival in the setting of methicillin [8].

Presence of PBP2a facilitates resistance to methicillin as well as other semisynthetic penicillinase-resistant beta-lactams: methicillin, nafcillin, oxacillin, and cephalosporins (exceptions include ceftaroline and ceftobiprole) [9,10].

●Variable phenotypic expression of resistance – Most clinical MRSA isolates are heterogeneous in their expression of methicillin resistance. Under routine growth conditions (ie, 37ºC, unsupplemented media), most isolates (≥99.9 percent) appear 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 [11]. Growth of heterogeneous strains in the presence of a beta-lactam leads to selection for a homogeneous phenotype. Conversely, serial passage of these cells in the absence of antibiotic leads to slow reversion back to the heterogeneous state.

Borderline methicillin resistance has also been described; the mechanism(s) are uncertain. (See "Overview of antibacterial susceptibility testing", section on 'Heteroresistance'.)

Some strains with borderline methicillin resistance lack the mecA gene (and therefore PBP2a); in these strains, there may be alterations in other PBPs, resulting in reduced affinity for beta-lactams or availability of more enzyme for peptidoglycan synthesis [11-13].

Virulence determinants — A number of differences have been observed between MRSA strains classified as health care-associated (HA-MRSA) or community-associated (CA-MRSA); CA-MRSA refers to MRSA infection that occurs in the absence of health care exposure. Most HA-MRSA strains carry SCCmec types I, II, and III; most CA-MRSA strains carry SCCmec type IV or V. However, these epidemiologic distinctions may be misleading, given crossover between where patients develop colonization and where they manifest infection. (See "Methicillin-resistant Staphylococcus aureus (MRSA) in adults: Epidemiology".)

CA-MRSA has been associated with enhanced virulence; the mechanisms are not fully understood and likely result from multiple contributing factors.

Panton-Valentine leukocidin (PVL) is among the best studied virulence factors; it is a cytotoxin that causes leukocyte destruction and tissue necrosis [14]. It is often found in S. aureus strains with mobile genetic elements SCCmec type IV and V, and observed less frequently among strains with SCCmec types I, II, and III [15-23]. Uncommonly, PVL has also been observed in some methicillin-susceptible strains of S. aureus. PVL-containing S. aureus strains have been most frequently associated with skin and soft tissue infection (SSTI) and necrotizing pneumonia [24-30].

The role of PVL in the pathogenesis and spread of infection is controversial [17,31-35]. PVL expression may be variable; in one study including 31 S. aureus strains from patients with infections of varying severity; the quantity of PVL produced in vitro did not correlate with severity of infection [35]. The most compelling clinical data include association of PVL with pyomyositis and necrotizing pneumonia following influenza infection [19,20,36].

DIAGNOSTIC LABORATORY TOOLS — Tools for detection of methicillin-resistant Staphylococcus aureus (MRSA) include genotypic methods (such as polymerase chain reaction [PCR] and other molecular tools) and phenotypic methods.

Molecular methods — Molecular methods utilize target deoxyribonucleic acid (DNA) sequences that include regions of the SCCmec mobile genetic element (which carries the mecA gene and is common in S. aureus as well as coagulase-negative staphylococci), together with additional nucleic sequences specific for S. aureus. Different assays use different combinations of targets (table 1).

Several commercial molecular methods have been described using techniques such as conventional polymerase chain reaction (PCR), multiplex PCR, real time PCR (RT-PCR), and gene-probe hybridization [37]. These techniques allow detection of MRSA within two to six hours.

Given the large number of SCCmec elements and variants, the performances of the different assays vary depending on the MRSA strain present in any given sample.

In general, PCR assays reliably detect the mecA gene [38]. However, not all molecular assays detect the mecC variant; in some circumstances, phenotypic testing may be needed [39-41]. In one study including 308 clinical isolates, the Xpert MRSA Gen 3 PCR assay accurately detected mecA and/or mecC in all cases [38]. The choice of assay for a particular laboratory setting depends on several factors including assay performance characteristics as well as local MRSA epidemiology and laboratory volume.

Phenotypic methods — Phenotypic methods for detection of MRSA include conventional culture techniques, cefoxitin disk diffusion, and rapid culture with chromogenic agar [1,42].

●Conventional culture – Issues related to conventional techniques for blood and sputum cultures are discussed in detail separately. (See "Detection of bacteremia: Blood cultures and other diagnostic tests" and "Sputum cultures for the evaluation of bacterial pneumonia".)

●Cefoxitin disk diffusion – The cefoxitin disk diffusion test is the most accurate phenotypic assay for detecting the presence of the mecA or mecC gene. Cefoxitin is used because it is a more potent inducer of mecA/mecC gene expression than other agents such as oxacillin. The test involves incubating a lawn of the test isolate on Mueller Hinton agar +2 percent sodium chloride under standardized conditions with a cefoxitin disk (30 mcg). The test requires overnight incubation [1].

According to the Clinical Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST), a zone of growth inhibition around the cefoxitin disk of ≥22 mm rules out MRSA; a zone size <22 mm indicates that the mecA gene is present and the isolate should be reported as MRSA [2].

●Rapid culture with chromogenic agar – Rapid culture uses chromogenic agar containing media substrates that change color in the presence of S. aureus; selectivity for MRSA is achieved by incorporation of antibiotics into the agar. Use of such agar allows identification of MRSA from primary isolation plates within 24 to 48 hours, reducing need for further subculture or biochemical testing [43].

Chromogenic media may be used for detection of MRSA from surveillance specimens (nasal swabs and swabs of the throat, groin, and rectum) as well a blood cultures [44]. Chromogenic media has also been evaluated for identification of MRSA in blood cultures with high sensitivity and specificity at 18 to 24 hours (97.6 and 100 percent, respectively) [45].

There are several commercially available chromogenic media [43]. The reported sensitivities of these media vary widely (50 to 100 percent) and depend on the duration of incubation and the "gold standard" comparator [43,46-53]. However, the specificities of these media are consistently high (90 to 100 percent) after 24 hours of incubation; prolonging the incubation time to 48 hours improves sensitivity but reduces specificity [43].

Many laboratories use a combination of chromogenic agar (read at 24 hours) and conventional culture methods. This approach allows early detection of many MRSA-colonized patients at 24 hours, with the remainder detected within the conventional 48- to 72-hour timeframe [43].

CLINICAL APPROACH

Evaluation of symptomatic illness

●Obtaining clinical specimens – The approach to collection of clinical samples should be guided by clinical circumstances; common specimens include blood cultures, wound cultures, and respiratory cultures. Assays are summarized in the table (table 1).

Issues related to blood and sputum cultures are discussed further separately. (See "Detection of bacteremia: Blood cultures and other diagnostic tests" and "Sputum cultures for the evaluation of bacterial pneumonia".)

●Clinical application of laboratory assays  

•Blood cultures – Molecular assays can reduce the time to identify MRSA in positive blood cultures with gram-positive cocci in clusters seen on Gram stain. There are numerous assays available; the appropriate choice depends on test performance, clinical use, and how the assay fits laboratory workflow (table 1).

•Respiratory specimens – Rapid diagnostic tools are emerging for diagnosis of lower respiratory tract infection due to MRSA on broncho-alveolar lavage (BAL) specimens [54]. In one study including 45 ventilated patients with suspected pneumonia, use of a rapid test on BAL specimens facilitated reduction in use of unnecessary treatment with antibiotic therapy targeting MRSA [55].

Active surveillance and infection control

●Obtaining clinical specimens – For purposes of MRSA surveillance for infection control, the anterior nares are the most common MRSA carriage site. Most patients with MRSA colonization may be identified by anterior nares screening (sensitivity 73 to 93 percent) [56-58].

Other carriage sites include the throat, axilla, groin, and perineum [44,59,60]. Lower sensitivities have been observed for these sites compared with the nares, perhaps due to lower colonization rates or higher quantities of competing flora at these sites.

Sampling multiple sites improves MRSA detection. In one study including more than 12,000 MRSA screenings, culture sensitivity increased from 48 percent (nares alone) to 79 percent (nares and groin) to 96 percent (nares, groin, and throat); PCR sensitivity also increased (62 to 92 to 99 percent, respectively) [61].

The gastrointestinal tract is increasingly recognized as a MRSA carriage site [62]; however, the utility of rectal swabs (if they contain polymerase chain reaction inhibitors) with molecular methods may be limited [63].

●Assay selection and interpretation – Tools for MRSA surveillance include traditional culture (48 to 72 hours), molecular tests (2 hours), and rapid culture with chromogenic agar (24 hours) [64]. (See 'Diagnostic laboratory tools' above.)

The predictive value of molecular testing for detection of MRSA colonization depends on the prevalence of MRSA in the population. In high-prevalence settings, an assay with high specificity may be used to definitively establish MRSA colonization. In low-prevalence settings, an assay with high sensitivity may be used for exclusion of MRSA colonization; however, positive results may require culture confirmation before MRSA colonization can be established definitively (table 1).

Issues related to prevention and control of MRSA are discussed further separately. (See "Methicillin-resistant Staphylococcus aureus (MRSA) in adults: Prevention and control".)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Management of Staphylococcus aureus infection".)

SUMMARY AND RECOMMENDATIONS

●MRSA definition – By definition, all methicillin-resistant Staphylococcus aureus (MRSA) isolates carry the mecA gene or a related variant known as mecC. Strains lacking a mec gene are not methicillin resistant. MRSA breakpoints are outlined above. (See 'MRSA definition' above.)

●Microbiology – The mec gene is contained within the SCCmec cassette, a mobile genetic element. The mec gene encodes penicillin-binding protein 2a (PBP2a). PBPs are peptidase enzymes in the bacterial membrane that catalyze transpeptidation reactions during cell wall synthesis. (See 'Genotypic and phenotypic mechanisms of resistance' above.)

●Diagnostic tools

•Molecular methods – Molecular methods utilize target DNA sequences that include regions of the SCCmec mobile genetic element (which carries the mecA gene and is common in S. aureus as well as coagulase-negative staphylococci), together with additional nucleic sequences specific for S. aureus. Different assays use different combinations of targets (table 1). (See 'Molecular methods' above.)

•Phenotypic methods – Phenotypic methods for detection of MRSA include conventional culture techniques, cefoxitin disk diffusion, and rapid culture with chromogenic agar. The cefoxitin disk diffusion test is the most accurate phenotypic assay for detecting the presence of the mecA or mecC gene. (See 'Phenotypic methods' above.)

●Clinical approach — Rapid detection of MRSA in blood cultures may be helpful for antimicrobial stewardship and to facilitate early implementation of effective therapy. (See 'Clinical approach' above.)