Origin and Molecular Evolution of the Determinant of Methicillin Resistance in Staphylococci

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant pathogens around the world. MRSA is generated when methicillin-susceptible S. aureus (MSSA) exogenously acquires a methicillin resistance gene, mecA, carried by a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec), which is speculated to be transmissible across staphylococcal species. However, the origin/reservoir of the mecA gene has remained unclear. Finding the origin/reservoir of the mecA gene is important for understanding the evolution of MRSA. Moreover, it may contribute to more effective control measures for MRSA. Here we report on one of the animal-related Staphylococcus species, S. fleurettii, as the highly probable origin of the mecA gene. The mecA gene of S. fleurettii was found on the chromosome linked with the essential genes for the growth of staphylococci and was not associated with SCCmec. The mecA locus of the S. fleurettii chromosome has a sequence practically identical to that of the mecA-containing region (∼12 kbp long) of SCCmec. Furthermore, by analyzing the corresponding gene loci (over 20 kbp in size) of S. sciuri and S. vitulinus, which evolved from a common ancestor with that of S. fleurettii, the speciation-related mecA gene homologues were identified, indicating that mecA of S. fleurettii descended from its ancestor and was not recently acquired. It is speculated that SCCmec came into form by adopting the S. fleurettii mecA gene and its surrounding chromosomal region. Our finding suggests that SCCmec was generated in Staphylococcus cells living in animals by acquiring the intrinsic mecA region of S. fleurettii, which is a commensal bacterium of animals.The first methicillin-resistant Staphylococcus aureus (MRSA) isolate was reported in England in 1961, soon after the introduction of methicillin (10). Since then, it has become an important pathogen in both health care settings and communities around the world (27). MRSA is generated when methicillin-susceptible S. aureus (MSSA) exogenously acquires a staphylococcal cassette chromosome mec (SCCmec) (9). SCCmec has been identified not only in S. aureus but also in other coagulase-positive and coagulase-negative staphylococci (8). SCCmec is speculated to be transmissible among staphylococcal species as a mobile element; however, its origin/reservoir has not been clarified yet (7). Finding the origin/reservoir of the mecA gene is important for understanding the evolution of MRSA. Moreover, it may contribute to more effective control measures for MRSA. There are various types and subtypes in SCCmec, but they are made up of the following two essential components: (i) the mec gene complex, containing the mecA gene encoding the penicillin-binding protein 2′ (PBP2′) with reduced affinity to beta-lactam antibiotics, and (ii) the ccr gene complex, encoding site-specific recombinase(s) for the movement of the element. By the action of ccr gene-encoded recombinases, SCCmec is excised from the chromosome and site-specifically integrated at the 3′ end of orfX (11), an open reading frame (ORF) of unknown function, which is located near the replication origin (oriC) on the chromosome (13). A large number of SCCmec elements have been sequenced and classified according to the combination of types of mec and ccr gene complexes (8). Although nucleotide diversity of ccr genes identified in various Staphylococcus species has been reported, mecA genes contained in SCCmec are almost identical regardless of the Staphylococcus species carrying them (8). mec gene complexes are classified into 4 classes (classes A, B, C, and D) by the presence or absence of certain insertion sequences (ISs) in the mecR1 gene, but all mec gene complexes contained IS431mec (also called IS431R) downstream of the mecA gene (Fig. ​(Fig.1)1) (8, 12). The class A mec gene complex is regarded as the prototypic structure, having the gene order IS431mec-mecA-mecR1-mecI. Class B, C, and D mec gene complexes are considered to be derived from the class A mec gene complex (8, 12). The region between the mecA gene and IS431mec is called a hypervariable region (HVR) (19), which can be used for genotyping of MRSA strains (21). The HVR contains a truncated mvaS gene encoding the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, a varied number of direct repeat units (DRUs) that is responsible for the length polymorphism of the mec gene complex (19), an intact ugpQ gene encoding glycerophosphoryl diester phosphodiesterase, and an intact maoC gene encoding the acyl dehydratase MaoC (Fig. ​(Fig.1).1). Some strains, however, lack some of the above-mentioned constituents of the HVR (16, 24).Open in a separate windowFIG. 1.Basic structure of the representative mec gene complex. The class A mec gene complex is composed of intact mecR1 and mecI, encoding the signal transducer and repressor for the mecA gene, respectively, upstream of the mecA gene without integration of an IS element. The class B and C mec gene complexes have ψIS1272 and IS431L integrated in mecR1, respectively, which results in the partial deletion of mecR1 and complete deletion of the mecI genes. Class D has no IS element, but a part of mecR1 and complete mecI genes are deleted. All mec gene complexes contained IS431mec (also called IS431R) downstream of the mecA gene.In staphylococci, previously, two mecA gene homologues with 80% and 91% nucleotide identities have been found in Staphylococcus sciuri and Staphylococcus vitulinus species, respectively (5, 22). The mecA gene homologue of S. sciuri has been considered to have a ubiquitous presence among its species and to be the evolutionary precursor of the mecA gene (5, 32). However, the mecA gene homologues found in their chromosomes were not the constituents of the mec gene complex or SCCmec. We speculated that the origin/reservoir of the mecA gene would be found in a staphylococcal species closely associated with S. sciuri and S. vitulinus. These species belong to the S. sciuri species group among the genus Staphylococcus. The group is composed of the following four species, which are resistant to novobiocin and catalase production and coagulase nonproduction: S. sciuri, S. vitulinus, Staphylococcus lentus, and Staphylococcus fleurettii (20, 25, 29, 30). They are usually isolated from a variety of animals and food products of animal origin and not frequently isolated from humans (20, 23, 25, 29, 30).We isolated staphylococcal strains of the S. sciuri species group from an animal source, tested for the presence of the mecA gene and its homologues, and determined the nucleotide sequences of the chromosome regions around the mecA gene and its homologues.