The molecular epidemiology of the highly virulent ST93 Australian community Staphylococcus aureus strain

Abstract

In Australia the PVL-positive ST93-IV [2B], colloquially known as "Queensland CA-MRSA" has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S. aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known.

Figure 1. Dendrogram of the 58 pulsed-field gel electrophoresis patterns (PFGE) of ST93 (13 MSSA and 45 MRSA).

Sequenced JKD6159 strain was used as the ST93 control. S. aureus strain NCTC 8325 was used as the reference strain.

Figure 2. Minimum spanning tree (MST) of the seven ST93 spa types.

Cluster analysis was performed using the spa typing plug-in tool of the BioNumerics program. spa types separated by an MST distance of ≤1 (i.e., if they were ≥98% similar) were considered closely related and assigned to the same cluster. MSSA and MRSA spa types are designated in red and green print respectively. Pulsed-field gel electrophoresis (PFGE) pulsotypes and dru types (dt) are recorded for each spa type.

Figure 3. Relative LukF-PV expression in ST93 isolates.

All isolates were tested for LukF-PV expression using western blot and LukF-PV specific antibody. Results are expressed as optical density of test strain relative to a 50 µg control of rLukF-PV that was run on every gel. All experiments were performed with multiple replicates and mean and range is shown. Positive control, rLUKF-PV; negative control, RN4220.

Figure 4. Minimum spanning tree (MST) of the six ST93 dru types.

Cluster analysis was performed using the Polymorphic VNTR plug-in tool of the BioNumerics program. dru types separated by an MST distance of ≤1 (i.e., if they were ≥98% similar) were considered closely related and assigned to the same cluster. Pulsed-field gel electrophoresis (PFGE) pulsotypes and spa types are recorded for each dru type.