Origin and molecular evolution of the determinant of methicillin resistance in staphylococci

Abstract

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.

FIG. 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.

FIG. 2.

Genomic organization of the mecA_Sf gene locus of S. fleurettii. (a) Comparison to the locus-containing type II SCCmec of MRSA strain N315. The mecA_Sf-positive S. fleurettii possessed a stretch of chromosome sequence that was practically identical with that of MRSA SCCmec, spanning the class A mec gene complex and adjacent chromosomal region containing psm-mec, xylR, the genes encoding the metallo-beta-lactamase family protein, the rhodanase domain-containing protein, and other hypothetical proteins. The mvaS gene of S. fleurettii existed in intact form without insertion of IS431. Moreover, the xyl operon was identified on the S. fleurettii chromosome from which xylR of SCCmec must have originated. The entire corresponding regions of the two chromosomes are colored in pink. Arrows indicate the genes and their directions of transcription. Truncated mvaS is shown by ΔmvaS. *, The gene encoding the metallo-beta-lactamase family protein of MRSA strain N315 was a pseudogene with a nonsense mutation (ochre) incorporated next to codon 312. (b) Structural diversity observed in the mecA_Sf gene loci of S. fleurettii strains. Five types (A, B, C, D, and E) were identified. The regions with practically identical nucleotide sequences across the mva and xyl genes are colored in pink. Arrows indicate the genes and their directions of transcription.

FIG. 3.

Genomic structure analysis of the S. sciuri species group. (a) The mecA gene homologue-containing chromosomal regions of S. vitulinus, S. sciuri subsp. carnaticus, S. sciuri subsp. sciuri, and S. sciuri subsp. rodentium compared to those of S. fleurettii. The corresponding regions among the species (and subspecies) are colored in light purple. Arrows indicate the genes and their directions of transcription. Noncorresponding genes found in these regions are shown with pale blue arrows. Truncated mecR1 and mecI are shown by ΔmecR1 and ΔmecI, respectively. The positions of primers (MVAV-1, XYRV-1, XYRV-2, XYEV-1, XAS-F, and XBS-R) are shown with arrowheads. The scale bar is not necessarily correlated with the noncoding region. *, The gene encoding the APC family amino acid-polyamine-organocation transporter of S. sciuri subsp. carnaticus strain ATCC 700058^T was a pseudogene due to an early stop codon caused by a frameshift. (b) Genomic structure of the mvaSCA gene locus of S. lentus. Arrows indicate the genes and their directions of transcription.

FIG. 4.

Phylogenetic analysis of 16S rRNA, mvaS, and mecA genes. Strain names are preceded by the names of the bacterial species. The branch length indicates the distance, which is expressed as the number of substitutions per 100 bases. Numbers at the branching points represent the percent occurrence in 1,000 random bootstrap replications of neighbor-joining analyses. Values of less than 50% are not shown. The sizes of mvaS genes varied from 1,167 to 1,179 bp, except for the truncated mvaS gene (369 bp) present in SCCmec. The genes identified in SCCmec and obtained from the S. sciuri species group are colored red and blue, respectively.