Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for both hospital- and community-onset disease.
Methicillin resistance in clinical isolated staphylococci is mostly mediated through acquisition of mecA gene encoding a mutant penicillin binding protein (PBP)2a by bacterial genome.
PBPs are the enzymes that catalyze the reaction
that crosslinks the peptidoglycan of the bacterial cell wall.
Binding of PBP to β-lactam antimicrobials
inhibits the enzyme activity and prevents
bacteria growth by interfering with cell wall formation.
In contrast to the PBPs in methicillin-susceptible strains,which have high affinity for most β-lactam antimicrobials, PBP2a has low affinity for binding β-lactams.
In methicillin-resistant strains, the essential function of PBP is undertaken by PBP2a to maintain survival of the bacterium in the presence of antimicrobials.
This mecA gene, originating from a mobile genetic element designated SCCmec (staphylococcal cassette chromosome mec), flanked by terminal inverted and direct repeats is invariably inserted into the orfX gene of S. aureus chromosome.
This element contains two site-specific cassette chromosome recombinases, ccrA and ccrB, responsible for the precise excision and integration of SCCmec within the bacterial chromosome.
To date, five differently organized SCCmec elements have been characterized.
Three types of SCCmec elements are typically found in HAMRSA strains (i) type I, a 34-kb element that was prevalent in MRSA isolates in the 1960s, (ii) type II, a 53-kb element that was identified in 1982 and is ubiquitous in Japan,Korea, and the United states, and (iii) type III, the largest (67-Kb) element, identified in 1985, currently prevalent in Germany, Austria, India, and other South Asian and Pacific areas.
In contrast to HA-MRSA, CA-MRSA isolates generally carry the SCCmec type IV element,whose size is much smaller than those of SCCmec types I, II, and III. At least four subtypes of the type IVSCCmec element, whose size varies from 20 to 24 kb, have been reported so far (IVa to IVc) and IVd. The molecular structure of the recently described type V cassette encoding a new ccrC recombinase presents some analogy with the type IV allele: both are more frequently associated with CA-MRSA, do not contain additional antibiotic-resistance determinants, and are the smallest SCCmec cassettes with a size <30.>
Molecular composition of the four SCCmec elements reveals key components useful for SCCmec typing. Variations in this gene set have allowed identifying five classes of mecA gene complexes, as discussed before.
A second essential region is the ccr gene complex. Types I and III harbor ccrA1-B1 and ccrA3-B3 recombinases,respectively; whereas Types II and IV contain ccrA2-B2 recombinases, showing some difference in the amino acid sequences. Distinct from these four types, SCCmec V strains harbor a new ccrC recombinase type.
To date, seven fully sequenced strains of Staphylococcus aureus are publicly available providing opportunities for searching conserved or variable targets within the genome of MRSA, thus allowing improved
molecular identification and characterization.
For example, the markers described above (e.g., mecA and SCCmec organization) are important epidemiological indicators of strain origin.
To screen for methicillin-resistant S. aureus, oxacillin-salt agar is very useful.
The agar is the Mueller–Hinton agar containing 4% NaCl and 6 μg/mL oxacillin. The bacteria suspension equal to 0.5 McFarland turbidity prepared from colonies grown on plate is inoculated on the agar as a spot 10 to 15 mm in diameter. After culturing in ambient air at 35◦C for 24 h, any amount of growth is considered to be resistant.
CLSI defines staphylococci with:
vancomycin MIC of ≤4 μg/mL as susceptible,
Isolates with MIC of vancomycin 8∼16 μg/mL as intermediate,
Isolates with vancomycin MIC of ≥32 μg/mL as resistant.
Accordingly, VISA and VRSA refer to S. aureus with a vancomycin MIC of 8–16 μg/mL and a MIC of ≥32 μg/mL, respectively.
Methicillin resistance in clinical isolated staphylococci is mostly mediated through acquisition of mecA gene encoding a mutant penicillin binding protein (PBP)2a by bacterial genome.
PBPs are the enzymes that catalyze the reaction
that crosslinks the peptidoglycan of the bacterial cell wall.
Binding of PBP to β-lactam antimicrobials
inhibits the enzyme activity and prevents
bacteria growth by interfering with cell wall formation.
In contrast to the PBPs in methicillin-susceptible strains,which have high affinity for most β-lactam antimicrobials, PBP2a has low affinity for binding β-lactams.
In methicillin-resistant strains, the essential function of PBP is undertaken by PBP2a to maintain survival of the bacterium in the presence of antimicrobials.
This mecA gene, originating from a mobile genetic element designated SCCmec (staphylococcal cassette chromosome mec), flanked by terminal inverted and direct repeats is invariably inserted into the orfX gene of S. aureus chromosome.
This element contains two site-specific cassette chromosome recombinases, ccrA and ccrB, responsible for the precise excision and integration of SCCmec within the bacterial chromosome.
To date, five differently organized SCCmec elements have been characterized.
Three types of SCCmec elements are typically found in HAMRSA strains (i) type I, a 34-kb element that was prevalent in MRSA isolates in the 1960s, (ii) type II, a 53-kb element that was identified in 1982 and is ubiquitous in Japan,Korea, and the United states, and (iii) type III, the largest (67-Kb) element, identified in 1985, currently prevalent in Germany, Austria, India, and other South Asian and Pacific areas.
In contrast to HA-MRSA, CA-MRSA isolates generally carry the SCCmec type IV element,whose size is much smaller than those of SCCmec types I, II, and III. At least four subtypes of the type IVSCCmec element, whose size varies from 20 to 24 kb, have been reported so far (IVa to IVc) and IVd. The molecular structure of the recently described type V cassette encoding a new ccrC recombinase presents some analogy with the type IV allele: both are more frequently associated with CA-MRSA, do not contain additional antibiotic-resistance determinants, and are the smallest SCCmec cassettes with a size <30.>
Molecular composition of the four SCCmec elements reveals key components useful for SCCmec typing. Variations in this gene set have allowed identifying five classes of mecA gene complexes, as discussed before.
A second essential region is the ccr gene complex. Types I and III harbor ccrA1-B1 and ccrA3-B3 recombinases,respectively; whereas Types II and IV contain ccrA2-B2 recombinases, showing some difference in the amino acid sequences. Distinct from these four types, SCCmec V strains harbor a new ccrC recombinase type.
To date, seven fully sequenced strains of Staphylococcus aureus are publicly available providing opportunities for searching conserved or variable targets within the genome of MRSA, thus allowing improved
molecular identification and characterization.
For example, the markers described above (e.g., mecA and SCCmec organization) are important epidemiological indicators of strain origin.
From Clinical and Laboratory Standards Institute (CLSI)
To screen for methicillin-resistant S. aureus, oxacillin-salt agar is very useful.
The agar is the Mueller–Hinton agar containing 4% NaCl and 6 μg/mL oxacillin. The bacteria suspension equal to 0.5 McFarland turbidity prepared from colonies grown on plate is inoculated on the agar as a spot 10 to 15 mm in diameter. After culturing in ambient air at 35◦C for 24 h, any amount of growth is considered to be resistant.
Detection of VISA requires MIC methods with 24 h incubation.
CLSI defines staphylococci with:
vancomycin MIC of ≤4 μg/mL as susceptible,
Isolates with MIC of vancomycin 8∼16 μg/mL as intermediate,
Isolates with vancomycin MIC of ≥32 μg/mL as resistant.
Accordingly, VISA and VRSA refer to S. aureus with a vancomycin MIC of 8–16 μg/mL and a MIC of ≥32 μg/mL, respectively.
Epidemic Strains of MRSA
Investigation of specimens for screening for MRSA
