Genomic Basis for Methicillin Resistance in Staphylococcus aureus

Abstract

Since the discovery of the first strain in 1961 in England, MRSA, the most notorious multidrug-resistant hospital pathogen, has spread all over the world. MRSA repeatedly turned down the challenges by number of chemotherapeutics, the fruits of modern organic chemistry. Now, we are in short of effective therapeutic agents against MRSA prevailing among immuno-compromised patients in the hospital. On top of this, we recently became aware of the rise of diverse clones of MRSA, some of which have increased pathogenic potential compared to the classical hospital-associated MRSA, and the others from veterinary sources. They increased rapidly in the community, and started menacing otherwise healthy individuals by causing unexpected acute infection. This review is intended to provide a whole picture of MRSA based on its genetic makeup as a versatile pathogen and our tenacious colonizer.

Keywords: Hetero-resistance; SCCmec; mecA; mecB; mecC; oriC environ; rpoB.

Figure 1

(A) Alignment of the 14 S. aureus chromosomes. Each chromosome is linearly represented with its origin of replication (oriC) placed at the left end. Using the Microbial Genome Database for Comparative Analysis [100, 101], we found 1887 orthologous open reading frames (orfs) shared by all the 14 chromosomes (denoted as grey vertical lines on each chromosome). The other orfs not commonly shared by the strains are denoted in red. The chromosomal regions containing a series of such non-orthologous orfs were extracted as genomic islands (GIs). sdh is a single gene encoding a 1.1-megadalton cell wall-associated fibronectin-binding protein [102], which is interrupted by insertion of multiple mobile elements in RF122 [103]. RF122 is the bovine isolate while the others are all human isolates. The integration site for νSaδ is present between the orfs SA2204 and SA2205 of N315. The corresponding sites are missing from the chromosomes of RF122, MSSA476, and MW2. φ signifies prophage. (B) Phylogenic tree of the integrases encoded by SaPI mobile genetic elements. The amino acid sequence homology of integrases encoded by the SaPI and SaRI mobile genetic elements listed in Table 1 was evaluated by using Clustal W WWW System provided by DNA Data Bank of Japan (DDBJ) (http://clustalw.ddbj.nig.ac.jp/top-j.html). The phylogenic tree was drawn by Phylodendron-Phylogenetic tree printer (http://iubio.bio.indiana.edu/treeapp/treeprint-sample1.html). The truncated integrases of SaPI6Δ were excluded from the analysis. Note that the phylogeny of integrase well correlates with the integraton sites of the mobile elements on the S. aureus chromosome. (See also Table 1).

Figure 2

Orthologous orfs commonly shared by 17 staphylococcal strains of four species. After identifying common orthologs among various species, conserved chromosomal structure ("core structure") was constructed on the basis of the consensus arrangement of the conserved orthologs [104]. An ortholog group in the resulting core structure is indicated as a colored line across horizontal black line representing a chromosome. To simplify the figure, only universally conserved, one-to-one correspondence ortholog groups are shown. To visualize chromosomal rearrangement of the core structure, color gradation is assigned according to the location on S. aureus strain N315 chromosome from red to yellow to green. The replication origins (oriC) are located at the center. Strain-specific orfs are denoted on each chromosome by grey short vertical bars. Note that the species-specific orfs are localized in the chromosome region with sparse vertical lines.

Figure 3

(A) Basic structure of SCCmec. SCCmec is bracketed by direct repeats (DRs) that contain integration site sequence (ISS) recognized by cassette chromosome recombinase (CCR). A pair of inverted repeats (IRs) are present at the termini of SCCmec. Two critical gene complexes, ccr and mec are present, and the other regions are designated J1, J2, and J3. The type of SCCmec is defined by the combination of the type of ccr-gene complex and the class of mec-gene complex. Subtype of the SCCmec is based on the difference in the J (standing for junkyard) regions. (B) Various types of SCCmec. The structures of 11 types of SCCmec are illustrated based on the nucleotide sequences deposited in the DDBJ/EMBL/GenBank databases as follows: type I, NCTC10442 (AB033763); type II, N315 (D86934); type III, 85/2082 (AB037671); type IV, CA05 (AB063172); type V, WIS [WBG8318] (AB121219); type VI, HDE288 (AF411935); type VII, JCSC6082 (AB373032); type VIII, C10682 (FJ390057); type IX, JCSC6943 (AB505628); type X, JCSC6945 (AB505630), and type XI, LGA251 (FR821779.1) Direct repeats that comprise integration site sequences of SCC are located at both extremities of SCCmec (the red arrowheads). The location of five (A-E) classes of mec-gene complexes is indicated by pink belt. The locations of ccr-gene complexes are indicated by blue belt. Insertion sequences and transposons are indicated in yellow. Representative genes related to heavy metal resistance and integrated plasmids located in the J regions are also indicated. Type XI is a newly identified SCCmec found in the MRSA strains of bovine sources.

Figure 4

The mecA gene originated from Staphylococcus fleurettii genome. Practically identical mecA gene and its surrounding region of SCCmec were identified on the chromosomes of S. fleurettii, a member of the sciuri group of staphylococcal species. The mecA-gene orthologs found in other staphylococcal species at the corresponding chromosomal loci (mvaS, a vital gene for life, serves as a landmark for the locus) are either deleted or degenerated presumably due to disuse of their function. Two mobile elements IS431 and Tn554 seem to have excised this part of the chromosome and inserted it into an SCC to form the historically first SCCmec element.

Figure 5

Novel mecA gene homologs mecB and mecC. mecB (formerly mecAm) and mecC, are found linked with blaZ homolog encoding beta-lactamase. mecB-gene complex is associated with transposase genes and transmitted as transposon Tn6045. mecA- and mecC-gene complexes are transmitted as SCCmec elements.

Figure 6

mecB transposon Tn6045 is carried by a big R plasmid. Physical map of the Macrococcus caseolyticus plasmid pMCCL2 is illustrated [18]. BlastP analysis was performed on the protein product of each orf. The bacterial genus of the top-hit entry for each orf is denoted by coloration. Those having no entry with e-value of 1 × 10^-6 or above, the orfs are colored in grey. The plasmid contain antibiotic resistance genes such as erm and aac/aph genes besides mecB transposon. Note that the plasmid contains orfs whose top-hit entry convers as many as 13 different genera.

Figure 7

Phylogenic tree of Penicillin-Binding Protein (PBP) genes homologous to the Macrococcus caseolyticus PBP genes. M. caseolyticus carries four PBP genes, pbpA-D on the chromosome and mecB on the plasmid pMCCL2. BlastX was performed with each of the five PBP genes as query. The genes with lower than 55% similarity or 50% query coverage to each of the PBP genes of M. caseolyticus were declined for subsequent analysis. All the used nucleotide sequences were obtained from GenBank database (http://www.ncbi.nlm.nih.gov/Genbank/). The phylogenetic tree was generated using the neighbor joining method in the ClustalW program (http://clustalw.ddbj.nig.ac.jp/top-j.html). The tree was visualized in the Phylodendron Web Form (http://iubio.bio.indiana.edu/treeapp/treeprint-form.html). To simplify the tree, we adopted the pbp homologs of strain N315 as the representatives of S. aureus pbps, all the pbp homologs of non-aureus staphylococcal species, and the most homologous pbp from each of the non-staphylococcal bacterial families. Macrococcus genes are depicted in red. Note that pbp1-3 genes of various gram-positive bacteria are ordered in a similar phylogenetic relationship, whereas the entry of bacterial families or species carrying mecA and pbp4 homologs and their relative phylogenetic distance are significantly different from the former. This indicates that mecA and pbp4 are laterally transmissible genes.

Figure 8

Relative position of pbp gene homologs on the chromosome of staphylococcal species. The figure shows relative location of the pbp genes on each chromosome. The arrow on the chromosome denotes the pbp gene and its direction of transcription. The relative placement of the chromosomes and black arrows represent a rough phylogenic tree of staphylococcal species based on 16S ribosome sequence. The distance between each species is not to the scale. Only the order of branching (speciation) from ancestral bacteria is shown. The relative locations and directions (synteny) of pbp1-3 genes are well conserved among staphylococcal species. Note that synteny is incomplete with pbp4 and mecA. They are located around the oriC (the oriC environ). In S. carnosus, two types of pbp4 genes, denoted as pbp4a and pbp4b, are noticed [31].

Figure 9

Four phenotype expressions of methicillin resistance. Symbols: open circle, pre-MRSA strain N315 (mecA gene is only weakly induced in N315, because it is strongly repressed by the repressor protein encoded by an intact copy of mecI gene. Spontaneous mutational inactivation of mecI gene makes the cell express methicillin resistance by allowing constitutive production of the mecA gene transcript [48]); open square, hetero-MRSA strain N315ΔIP (mecI gene-deletant of N315 expressing hetero-methicilin resistance); closed triangle, homo-MRSA strain N315ΔIP-H5 (obtained by selecting N315ΔIP with 8 mg/L of imipenem. Single mutation rpoB(N967I) is responsible for the 'hetero-to-homo conversion'); closed circle, strain N315rpoB(N967I) (the chr^* mutation rpoB(N967I) was introduced into N315 by gene replacement procedure). Note that chr^* causing hetero-to-homo conversion confers 'Eagle-phenotype' on the pre-MRSA [97].

Figure 10

rpoB gene mutation as the 'regulatory mutation'. Those rpoB gene mutations found in-vitro-derived mutant strains and the phenotype of the strains are listed above the rpoB gene figure. Those in clinical strains that express altered susceptibilities to vancomycin, ß-lactam, daptomycin and linezolid, but susceptible to rifampin (MIC_RIF < 1.0 mg/L) are listed under the rpoB figure. The mutations in the clinical strains remain to be demonstrated for their direct contribution to the phenotypes.