All Staphylococcus aureus bacteraemia-inducing strains can cause infective endocarditis: Results of GWAS and experimental animal studies

Abstract

Objectives: We aimed at determining whether specific S. aureus strains cause infective endocarditis (IE) in the course of Staphylococcus aureus bacteraemia (SAB).

Methods: A genome-wide association study (GWAS) including 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients from international cohorts was conducted, and a subset of strains was tested with two experimental animal models of IE, one investigating the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically-damaged rabbit valves.

Results: The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences, and k-mers analysed in GWAS. In the murine inflammation-induced IE model, no difference was observed between IE and non-IE SAB strains both in terms of adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding the vegetation size and CFU.

Conclusion: All strains of S. aureus isolated from SAB patients must be considered as capable of causing this common and lethal infection once they have accessed the bloodstream.

Keywords: Bacteraemia; Experimental animal model; Genome-wide association study; Infective endocarditis; Staphylococcus aureus.

Fig. 1.

Rooted Phylogenetic tree of all the 15,436 SNPs from the 924 S. aureus strains. The tree is rooted by the TCH60 S. aureus strain (ST30; GenBank accession nos. NC_017342.1) using a maximum likelihood method and a generalised time-reversible model after repeated region and recombination corrections. The inner circle represents IE (red) and non-IE SAB (blue) strains. The six largest clonal complexes and five largest sequence types are coloured as follows: CC/ST1, orange; CC/ST15, yellow; CC/ST30, dark green; CC/ST45, light green; CC/ST5, purple; CC/ST8, light blue; other CCs or STs are pooled together (grey). The outer strip refers to the four geographical origins of the samples (AUS/NZ: beige; DK: brown; FR: turquoise; US: pink).

Fig. 2.

Presence (dark grey) and absence (light grey) hierarchical clustering of the 4757 coding sequences and non-coding RNAs for the 924 S. aureus strains. The blue/red stripe on top shows the infection type, either IE (red) or non-IE SAB (blue). The 2 strips below correspond to the six major clonal complexes and the five major sequence types, respectively (CC/ST1: orange; CC/ST15: yellow; CC/ST30: dark green; CC/ST45: light green; CC/ST5: purple; CC/ST8: light blue; Others: grey). The last strip refers to the four geographical origins of the samples (AUS/NZ: beige; DK: brown; FR: turquoise; US: pink).

Fig. 3.

Adhesion of endocarditis and bacteraemia CC5 strain pools on inflamed cardiac valves. Staphylococcus aureus CC5 strain pool (2.10⁷ CFU/mouse) were intravenously injected in C57BL/6 mice. Subsequently, via a transaortic catheter, valves were inflamed (5 min of histamine infusion). Mice were immediately sacrificed and adhesion was quantified. Adhesion on inflamed valves of a S. aureus CC5 IE strain pool (12 CC5 IE strains, number of mice = 11) was compared with a S. aureus CC5 non-IE SAB strain pool (12 CC5 non-IE SAB strains, number of mice = 11, p-value = 0.5619). Results represent log-transformed vegetation volumes in single mice. Median values ± interquartile range are represented; *P < 0.05; Mann-Whitney Wilcoxon test.

Fig. 4.

Propensity of endocarditis and bacteraemia CC5 and CC45 strain pools to cause IE on inflamed cardiac valves. Staphylococcus aureus CC5 or CC45 strain pools (2.10 ⁶ CFU/mouse) were intravenously injected in C57BL/6 mice. Subsequently, via a transaortic catheter, valves were inflamed (5 min of histamine infusion). Afterwards, the catheter was removed, mice were monitored for three days to see if endocarditis developed. The proportion of mice that developed endocarditis (i) in the CC5 strain pool vs. no endocarditis (IE strain pool: 12 IE strains, number of mice = 25; non-IE SAB strain pool: 12 non-IE SAB strains, number of mice = 24; OR [95% CI]: 5.0117 [0.8466; 54.4250], p-value = 0.0738); (ii) in the CC45 strain pool vs. no endocarditis (IE strain pool: 10 IE strains, number of mice = 15; non-IE SAB strain pool: 9 non-IE SAB strains, number of mice = 16; OR [95% CI]: 2.2475 [0.1055; 144.9300], p-value = 0.5996).

Fig. 5.

Propensity of endocarditis and bacteraemia single strains of CC5 to cause IE on inflamed cardiac valves. S. aureus IE and non-IE SAB strains (2–4.10⁶ CFU/mouse) were perfused via the tail vein catheter (strain 101,291 and 136,123; low severity model, LS) or via a transaortic catheter (increasing the model severity, HS; strain 77,521 and 136,107) in C57BL/6 mice. Valves were inflamed by histamine infusion (5 min) via a transaortic catheter. Afterwards, the catheter was removed and mice were monitored for one (strain 77,521 and 136,107) or three days (strain 101,291 and 136,123) to see whether endocarditis developed. Proportions of endocarditis in mice infected with an IE strain (strain 77,521, number of mice = 23) vs a non-IE SAB strain (strain 136,107, number of mice = 21; HS OR [95% CI]: 2.1427 [0.5509; 8.8883], p-value = 0.2387) and with an IE strain (101,291, number of mice = 11) vs a non-IE SAB strain (strain 136,123, number of mice = 9; LS OR [95% CI]: Inf. [0.0210; Inf.], p-value = 1). Fisher’s exact tests (P* <0.05).

Fig. 6.

Native valve, infective endocarditis in rabbits with individual bacteraemia and infective endocarditis strains from the CC5 clonal group. Rabbits were infected intravenously with 1 × 10⁷ - 5 × 10⁸ CFUs/rabbit after mechanical damage to the aortic valves, and infection was allowed to progress for a maximum of 4 days. The doses of S. aureus used were previously determined to ensure vegetation development within 24 h, increases in vegetation size occurring over the 4-day test period, and to prevent early lethality. (A) Total weight of vegetations dissected from aortic valves; (B) bacterial counts recovered from aortic valve vegetations shown in panel A; (C) bacterial counts per millilitre of blood at the end of experimentation. Horizontal lines and error bars represent median values with interquartile ranges. There is no statistical difference between strains in terms of vegetation weight, vegetation CFU, and blood CFU. Kruskal-Wallis tests (P* <0.05; A, p-value = 0.0699; B, p-value = 0.1822; C, p-value = 0.7356).