Impact of Insertion Sequences and Recombination on the Population Structure of Staphylococcus haemolyticus

Abstract

Staphylococcus haemolyticus is one of the most common pathogens associated with medical-device related infections, but its molecular epidemiology is poorly explored. In the current study, we aimed to better understand the genetic mechanisms contributing to S. haemolyticus diversity in the hospital environment and their impact on the population structure and clinical relevant phenotypic traits. The analysis of a representative S. haemolyticus collection by multilocus sequence typing (MLST) has identified a single highly prevalent and diverse genetic lineage of nosocomial S. haemolyticus clonal complex (CC) 29 accounting for 91% of the collection of isolates disseminated worldwide. The examination of the sequence changes at MLST loci during clonal diversification showed that recombination had a higher impact than mutation in shaping the S. haemolyticus population. Also, we ascertained that another mechanism contributing significantly to clonal diversification and adaptation was mediated by insertion sequence (IS) elements. We found that all nosocomial S. haemolyticus, belonging to different STs, were rich in IS1272 copies, as determined by Southern hybridization of macrorestriction patterns. In particular, we observed that the chromosome of a S. haemolyticus strain within CC29 was highly unstable during serial growth in vitro which paralleled with IS1272 transposition events and changes in clinically relevant phenotypic traits namely, mannitol fermentation, susceptibility to beta-lactams, biofilm formation and hemolysis. Our results suggest that recombination and IS transposition might be a strategy of adaptation, evolution and pathogenicity of the major S. haemolyticus prevalent lineage in the hospital environment.

Fig 1. Phylogenetic analysis.

(A) Identification and characterization of a major genetic lineage. Application of eBURST algorithm to MLST data for the collection of 109 S. haemolyticus isolates obtained in this study and that of Cavanagh [11]. The relatedness between STs and clonal complexes was displayed as an eBURST diagram. Each ST is represented by a number and a node, and each line links STs that are single locus variants (SLVs). Bleu node corresponds to the founder (ST29). The size of the node corresponds to the frequency of the isolates. Clusters of linked STs correspond to clonal complexes. (B-C) Phylogenetic trees inferred from the concatenated sequences of the seven MLST loci. Maximum likelihood (ML) Phylogenetic tree was constructed based on concatenated sequences of 7 housekeeping loci for 26 STs obtained in the present study and 17 STs identified in Cavanagh study [11]. The trees were drawn to scale using MEGA 6. The ML analysis identified two groups one containing the great majority of STs that corresponded to CC29 identified by eBURST analysis, and the other containing only four STs. Asterisks indicate the STs identified in this study. STs of CC29 are underlined with red. Each red circle on phylogenetic tree (C) corresponds to an ST [The same ST in ML tree (B)].

Fig 2. Phenotypic and genotypic In vitro stability.

(A) In vitro stability over time of SmaI PFGE profiles. (B) In vitro stability over time of IS1272-hybridization patterns. Lane M: λ ladder used as a size marker. The positions of molecular markers (in kilobase pairs) are indicated by black lines. The white arrows indicates band loss/gain in PFGE and SmaI-IS1272 profiles. The arrowheads on the right of each lane correspond to the appearance of a band. The arrowheads on the left of each lane correspond to the disappearance of a band. At the top of each lane the day that the MRSHae HSM742 isolate was collected is indicated in relation to the first isolate (day 0). (C) Stability over time of phenotypic traits. Oxa, oxacillin; Fox, cefoxitin; empty set (Ø), hemolysis zone diameter; a plus sign indicates fermentation of mannitol; a minus sign indicates no fermentation of mannitol; V, variable. The asterisks indicate days that showed alteration. The presence (dark gray) and absence (light gray) of alteration are indicated by filled rectangles.