Genomic Investigation of Methicillin-Resistant Staphylococcus aureus ST113 Strains Isolated from Tertiary Care Hospitals in Pakistan

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant and opportunistic pathogen. The emergence of new clones of MRSA in both healthcare settings and the community warrants serious attention and epidemiological surveillance. However, epidemiological data of MRSA isolates from Pakistan are limited. We performed a whole-genome-based comparative analysis of two (P10 and R46) MRSA strains isolated from two provinces of Pakistan to understand the genetic diversity, sequence type (ST), and distribution of virulence and antibiotic-resistance genes. The strains belong to ST113 and harbor the SCCmec type IV encoding mecA gene. Both the strains contain two plasmids, and three and two complete prophage sequences are present in P10 and R46, respectively. The specific antibiotic resistance determinants in P10 include two aminoglycoside-resistance genes, aph(3')-IIIa and aad(6), a streptothrin-resistance gene sat-4, a tetracycline-resistance gene tet(K), a mupirocin-resistance gene mupA, a point mutation in fusA conferring resistance to fusidic acid, and in strain R46 a specific plasmid associated gene ant(4')-Ib. The strains harbor many virulence factors common to MRSA. However, no Panton-Valentine leucocidin (lukF-PV/lukS-PV) or toxic shock syndrome toxin (tsst) genes were detected in any of the genomes. The phylogenetic relationship of P10 and R46 with other prevailing MRSA strains suggests that ST113 strains are closely related to ST8 strains and ST113 strains are a single-locus variant of ST8. These findings provide important information concerning the emerging MRSA clone ST113 in Pakistan and the sequenced strains can be used as reference strains for the comparative genomic analysis of other MRSA strains in Pakistan and ST113 strains globally.

Keywords: ST113; antibiotic-resistance; comparative genome analysis; methicillin-resistant Staphylococcus aureus; multi-locus sequence type; whole-genome sequencing.

Figure 1

Circular visualization of P10 and R46 genomes via CGViewer, showing antibiotic resistance determinants (labeled in purple) and virulence genes (labeled in black).

Figure 2

A Maximum Likelihood tree based on variations in housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqi) of the 254 (test strains n = 2, global strains n = 252) MRSA genomes. The evolutionary history was inferred by using the Maximum Likelihood method and the General Time Reversible model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The tree also shows sequence type (ST) assignment and antibiotic resistance genes. The test strains are highlighted in red, boldfaced, and yellow background. The strains (M92 and V605) for which new ST numbers (ST-5354, and ST-5355) were assigned are boldfaced and highlighted in blue background. The rectangular tree is given in Additional file 1 for better presentation.

Figure 3

Circularized SNP tree of 253 (test strains n = 2, and global strains n = 251) MRSA genomes. The Maximum Likelihood tree generated by the single nucleotide polymorphisms (SNPs) with the reference genome USA300_FPR3757, which is not shown in the tree. The test strains are colored in red, boldfaced, and highlighted with a yellow background. Color keys indicating ST numbers are provided alongside the circularized tree.

Figure 4

Comparison of clusters of orthologous groups (COGs) of P10 and R46 with their phylogenetically related strains M51 ST1516, SVH7513 ST612, and USA300_2014.C02 ST8. The Venn diagram represents the numbers of unique and shared COGs, while the bar chart represents the number of clusters in each strain.