Comparative Genomic Reveals Clonal Heterogeneity in Persistent Staphylococcus aureus Infection

Abstract

Persistent infections caused by Staphylococcus aureus remain a clinical challenge. Adaptational mechanisms of the pathogen influencing infection persistence, treatment success, and clinical outcome in these types of infections by S. aureus have not been fully elucidated so far. We applied a whole-genome sequencing approach on fifteen isolates retrieved from a persistent S. aureus infection to determine their genetic relatedness, virulome, and resistome. The analysis of the genomic data indicates that all isolates shared a common clonal origin but displayed a heterogenous composition of virulence factors and antimicrobial resistance. This heterogeneity was reflected by different mutations in the rpoB gene that were related to the phenotypic antimicrobial resistance towards rifampicin and different minimal inhibitory concentrations of oxacillin. In addition, one group of isolates had acquired the genes encoding for staphylokinase (sak) and staphylococcal complement inhibitor (scn), leading to the truncation of the hemolysin b (hlb) gene. These features are characteristic for temperate phages of S. aureus that carry genes of the immune evasion cluster and confer triple conversion by integration into the hlb gene. Modulation of immune evasion mechanisms was demonstrated by significant differences in biofilm formation capacity, while invasion and intracellular survival in neutrophils were not uniformly altered by the presence of the immune evasion cluster. Virulence factors carried by temperate phages of S. aureus may contribute to the course of infection at different stages and affect immune evasion and pathogen persistence. In conclusion, the application of comparative genomic demonstrated clonal heterogeneity in persistent S. aureus infection.

Keywords: Staphylococcus aureus; bloodstream infection; comparative genomic; device related infection; immune evasion cluster; phage; sak; scn.

Figure 1

Minimum spanning tree on the sequences of S. aureus isolates. Blood culture isolates (B1-B9) are displayed in red and isolates from the internal cardioverter defibrillator (device, D1-6) in blue. Distance between nodes in SNPs. According to results of the WGS, isolates with an * present an insertion of a prophage within the gene hlb.

Figure 2

Heatmap of detected virulence genes. Virulence factors that are present in the respective isolates are black while white represents absence. Grey shading represents a truncated gene.

Figure 3

UPGMA phylogenetic tree based on the nucleotide sequence of the prophage and the surrounding genomic context. UPGMA phylogenetic tree based on the nucleotide sequence of the prophage and the hlb genetic environment containing the integration site of the prophage based on reads mapping analysis. dapE, probable succinyl-diaminopimelate desuccinylase; hlb, hemolysin b; xerC, tyrosine recombinase; lexA, LexA repressor; ssbA Staphylococcus aureus, single-stranded DNA-binding protein; clpP, ATP-dependent Clp protease proteolytic subunit; sak, staphylokinase; scn, staphylococcal complement inhibitor; map, Major histocompatibility complex class II analogue protein; lucC, siderophore biosynthesis protein.

Figure 4

RT-qPCR for the circular form of the bacteriophage and the phage-free chromosomal integration site (attB). The circular form (cf) of the bacteriophage and phage-free chromosomal site (attB) were detected by PCR. Isolates are regarded as IEC⁺ and IEC^- according to WGS data. Results of three independent experiments performed as duplicates. The comparison of two data groups were analyzed by Mann–Whitney U test (one-tailed, confidence intervals 95%) with **P < 0.01.

Figure 5

Invasion and intracellular survival in differentiated neutrophils and biofilm production of clinical isolates. Capacity of S. aureus clinical isolates to (A) invade neutrophils and survive intracellular and (B) form biofilm. (A) For measurements of invasion and intracellular survival, differentiated HL-60 were incubated with S. aureus at a MOI of 100 2h at 37°C. Extracellular bacteria were killed using gentamicin and cells were lysed to detect intracellular bacteria following incubation for 1h to allow intracellular killing. The outliers are isolates B3 and D6. Results of three independent experiments. (B) Biofilm formation was measured using a crystal violet stain, experiments performed as triplicates. The comparison of two data groups were analyzed by Mann– Whitney U test (one-tailed, confidence intervals 95%) with ***P < 0.001.