Clonal complex 398 methicillin susceptible Staphylococcus aureus: a frequent unspecialized human pathogen with specific phenotypic and genotypic characteristics

Abstract

Clonal complex 398 livestok-associated-MRSA (CC398 LA-MRSA) clone is described as a major animal pathogen that can also colonize and infect humans. CC398 methicillin susceptible Staphylococcus aureus (CC398 MSSA) is less described. We identified 126 CC398 MSSA strains of human origin within 6380 S. aureus isolates gathered between 2009 and 2011, from the French National Reference Centre for Staphylococci. They were characterized using antimicrobial susceptibility testing, spa typing, DNA microarrays (Identibac S. aureus Genotyping ®, Alere), CC398-specific sequence PCR, ermT (encoding macrolides résistance) PCR. Fifty-three CC398 LA-MRSA collected from French pigs and veal were used as comparators, and phylogenetic relations between human CC398 MSSA and animal CC398 MRSA populations were explored on the basis of spa-typing and DNA microarrays. CC398 MSSA were able to induce a large spectrum of infections (especially skin, bloodstream, and pneumonias). The prevalence rate of this clone was high in MSSA population, i.e., 24.7% in a local prospective study on nasal colonization, and 7.5% in a national prospective study on infective endocarditis. CC398 MSSA isolates were frequently (89%) erythromycin resistant, due to the presence of the ermT gene, a gene not detected in erythromycin resistant CC398 LA-MRSA strains. Expression of staphylococcal complement inhibitor (scn) and the chemotaxis inhibitory protein (chp), was also specific to this population. The CC398 MRSA signature included also a panel of antibiotic resistance genes, especially a type IV or V cassette mec and tetM. CC398 MSSA and CC398 LA-MRSA populations were closely related based on spa-typing and DNA microarrays, with the MRSA strains forming the most derived lineage in phylogenic trees. Both MSSA and MRSA populations may come from common ancestors, which would have evolved in the settings of different selective pressures, explaining the acquisition of ermT, chp and scn for MSSA, and antibiotic resistance genes for MRSA.

Figure 1. Nature and number of infections caused by 89 MSSA CC398 strains collected in mainland France between 1999 and 2011.

Disseminated infections are defined by the presence of septic metastasis in at least 2 noncontiguous organs (by opposition to bloodstream infections). Infections classified as infective endocarditis correspond to bloodstream infections with infective endocarditis and without any other septic metastasis.

Figure 2. Dendrogram (UPGMA method, squared representation) based on the homology degree of the spa-types of the 105 MSSA CC398 strains and the 53 MRSA CC398 isolated in France.

The homology between the repeats of the strains is of 76%. spaCC: spa clonal complex. spa types corresponding to MRSA strains are colored in red.

Figure 3. Phylogenic tree with 158 S. aureus CC398 strains (modified Parsimony method, circular representation), based on the analysis of 319 genes and alleles by DNA microarrays.

Since the PCR analysis of the agr alleles confirmed that all the strains were agr 1, the analysis of the results of DNA microarrays concerned only 319 genes and alleles. MSSA group is subdivided in bla− and bla+ clusters, and MRSA group in SCCmec IV and SCCmec V clusters.

Figure 4. Phylogenic tree with 158 S. aureus CC398 strains (modified Parsimony method, circular representation), based on the analysis of 274 genes and alleles by DNA microarrays, after exclusion of the 45 antibiotic resistance genes, and the agr variants, since the PCR analysis of the agr alleles confirmed that all the strains were agr 1.

Human MSSA are in light grey and animal MRSA in black. The Z branch groups 7 strains presenting a more frequent equipment in virulence genes and alleles than the others (lukE, sak, splA, splE, ssl07/set1 (MRSA252), hysA2 (All Other Than COL+USA300+NCTC)), but also 2 hyaluronate lyase genes (hysA2 (AllOtherThan COL+USA300+NCTC)). The Y branch gathers 8 strains with some of the virulence genes (lukS (ST22+ST45), lukX, chp, scn, set6-var4_11, ssl07/set1, ssl11/set2 (MRSA252)) and one of an adhesine (clfA (MRSA252)) less frequent.

Figure 5. Dendrogram (UPGMA method, circular representation) based on the results of DNA microarrays study (172 genes only) of the 105 MSSA CC398 strains and the 53 MRSA CC398 isolated in France.

For each of the 172 genes, the average of the results of all the strains corresponding to the same spa type was calculated. Each group of strains is represented by the corresponding spa type. spa types corresponding to MRSA strains are colored in red.