Genotypic Characterization of Clinical Isolates of Staphylococcus aureus from Pakistan

Abstract

In this study, we compared pulsed-field gel electrophoretic (PFGE), multilocus sequence typing (MLST), Staphylococcal cassette chromosome mec (SCCmec), spa typing, and virulence gene profiles of 19 Panton-Valentine leucocidin (PVL)-positive, multidrug-, and methicillin-resistant clinical Staphylococcus aureus (MRSA) isolates obtained from a hospital intensive care unit in Pakistan. The isolates exhibited 10 pulsotypes, contained eight adhesin genes (bbp, clfA, clfB, cna, fnbA, fnbB, map-eap, and spa), 10 toxin genes (hla, hlb, hld, hlg, pvl, sed, see, seg, seh, and tst), and two other virulence genes (cfb, v8) that were commonly present in all isolates. The spa-typing indicated seven known spa types (t030, t064, t138, t314, t987, t1509, and t5414) and three novel spa types. MLST analysis indicated eight ST types (ST8, ST15, ST30, ST239, ST291, ST503, ST772, and ST1413). All isolates belonged to the agr group 1. Most of the isolates possessed SCCmec type III, but some isolates had it in combination with types SCCmec IV and V. The presence of multidrug-resistant MRSA isolates in Pakistan indicates poor hygienic conditions, overuse of antibiotics, and a lack of rational antibiotic therapy that have led to the evolution and development of hypervirulent MRSA clones. The study warrants development of a robust epidemiological screening program and adoption of effective measures to stop their spread in hospitals and the community.

Keywords: MLST; SCCmec; Staphylococcus aureus; evolution; genes; methicillin resistance; virulence.

Figure 1

Virulence and antimicrobial resistance gene profile of MRSA isolates. PCR amplicons of various gene products were separated on 2% agarose gels. Lanes 1 and 16, 100-bp DNA ladder; Lanes 2 to 9 and 13, 14, 15 in gel A and lanes 2 to 15 in gel B indicate the amplicons of different virulence genes from a representative isolate 32. Lanes 10 to 12 in gel A represent virulence genes from isolate 50. In gel C, lanes 2 to 15 show the amplicons of various antimicrobial resistance genes from a representative isolate 32. PCR amplicon sizes are shown in parentheses.

Figure 2

Pulsed-field gel electrophoretic profiles of MRSA isolates in 1% Seakem gold agarose gel. Lane numbers are at the top of the gel, and isolate numbers are shown in the inset. Molecular weight marker sizes are shown on either side of the gel. The % similarity among the isolates is indicated by a dendrogram on top of the gel above the lane numbers.