Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes

Abstract

Staphylococcus aureus strains often carry in their genomes virulence genes that are not found in all strains and that may be carried on discrete genetic elements. Strains also differ in that they carry one of four classes of an accessory gene regulator (agr) locus, an operon that regulates virulence factor expression and that has been proposed to be a therapeutic target. To look at their distribution among hospital strains, we investigated 38 methicillin-sensitive S. aureus isolates, classifying the isolates by agr class and screening them for the presence and restriction fragment length polymorphisms (RFLPs) of 12 core and 14 accessory virulence genes. Twenty-three (61%) were agr class I, 10 (26%) were agr class II, and 5 (13%) were agr class III. None were agr class IV. The S. aureus strains had distinguishable RFLP profiles, although clusters of isolates with clearly related core gene profiles were found among our strains, including all five agr class III strains, two sets of six strains within agr class I, and six strains within agr class II. Within these clusters there was evidence of horizontal acquisition and/or loss of multiple accessory virulence genes. Furthermore, two isolates from the same patient were identical except for the presence of the sea gene, indicating that movement of mobile elements may occur in vivo. Several strong correlations with the carriage of virulence genes between strains were seen, including a positive correlation between tst and agr class III and negative correlations between tst and lukE-splB and between lukE-splB and seg-sei. This suggests that the core genome or the presence of accessory genetic elements within a strain may influence acquisition and loss of other elements encoding virulence genes.

FIG. 1

BURST analysis of core genotypes of MSSA isolates. The relatedness of strains was determined by BURST analysis, based on core gene RFLP values (16). Numbers refer to the MSSA isolate numbers listed in Table 1. Core genotypes were defined by RFLP values for core genes rnaIII (including agr class), sarA, ssp, spa, icaA, fnbA, and hlg. Isolates in each central ring cluster have identical core genomes. Isolates in the first concentric ring have a core genome RFLP fingerprint that differs by one of the seven core gene RFLPs compared to those for isolates in the central ring. Isolates in the second concentric ring (dotted circle) differ by two core gene RFLPs from those for isolates in the central ring. Similarly, the solid lines connect rings of isolates that differ by a single RFLP, whereas dotted lines connect rings of isolates that differ by two RFLPs. At least four clusters of related strains can be identified and are called agr class III, SGH-1, SGH-2, and SGH-3 (Table 1).

FIG. 2

Membrane array results for PM152. The array was manufactured as described in Materials and Methods. Each gene was spotted four times. (A) PM152; (B) order of genes spotted onto the membrane. agr I, II, and III refer to the agr class I-, II-, and III-specific probes listed in Table 2, respectively. The results illustrate the ability of membrane arrays to identify differences in accessory gene carriage in different strains. We have no explanation for the variable sizes of the spots on the arrays.