Virulence Determinants in Staphylococcus aureus Clones Causing Osteomyelitis in Italy

Abstract

Staphylococcus aureus is the most common pathogen causing osteomyelitis (OM). The aim of this study was to explore the clonal complex (CC) distribution and the pattern of virulence determinants of S. aureus isolates from OM in Italy. Whole-genome sequencing was performed on 83 S. aureus isolates from OM cases in six hospitals. Antibiotic susceptibility tests showed that 30.1% of the isolates were methicillin-resistant S. aureus (MRSA). The most frequent CCs detected were CC22, CC5, CC8, CC30, and CC15, which represent the most common lineages circulating in Italian hospitals. MRSA were limited in the number of lineages (CC22, CC5, CC8, and CC1). Phylogenetic analysis followed the sequence type-CC groupings and revealed a non-uniform distribution of the isolates from the different hospitals. No significant difference in the mean number of virulence genes carried by MRSA or MSSA isolates was observed. Some virulence genes, namely cna, fib, fnbA, coa, lukD, lukE, sak, and tst, were correlated with the CC. However, different categories of virulence factors, such as adhesins, exoenzymes, and toxins, were frequently detected and unevenly distributed among all lineages. Indeed, each lineage carried a variable combination of virulence genes, likely reflecting functional redundancy, and arguing for the importance of those traits for the pathogenicity in OM. In conclusion, no specific genetic trait in the most frequent lineages could explain their high prevalence among OM isolates. Our findings highlight that CCs detected in OM isolates follow the epidemiology of S. aureus infections in the country. It is conceivable that any of the most common S. aureus CC can cause a variety of infections, including OM.

Keywords: Staphylococcus aureus; antibiotic resistance; clones; osteomyelitis; virulence genes; whole genome sequencing.

Figure 1

Clonal complex (CC) and sequence type (ST) distribution of 83 Staphylococcus aureus isolates from OM. CC22 (ST22, n = 20; ST3863, n = 1), CC5 (ST5, n = 9; ST105, n = 2; ST228, n = 2), CC8 (ST8, n = 7; ST72, n = 1; ST368, n = 1; ST789, n = 1), CC30 (ST30, n = 7; ST34, n = 1; ST4391, n = 1; ST7297, n = 1), CC15 (ST15, n = 5; ST582, n = 3), CC398 (ST398, n = 5), CC45 (ST45, n = 4), CC97 (ST97, n = 3), CC1 (ST1, n = 1; ST6927, n = 1), ST20 (n = 2), ST7 (n = 1), ST26 (n = 1), ST96 (n = 1), and ST101 (n = 1).

Figure 2

Neighbor joining (NJ) tree based on the allelic profiles of the cgMLST target genes (n = 1,861) of 83 Staphylococcus aureus isolates from osteomyelitis in Italy, and associated heat-map of in silico detected virulence genes (indicated on top).

Figure 3

Virulence-related genes detected in isolates belonging to the most frequent Staphylococcus aureus clonal complexes (CC) causing OM. All virulence genes (A), adhesin genes (B), exoenzymes genes (C), and superantigen genes (D). CC22, n = 21; CC5, n = 13; CC8, n = 10; CC30, n = 10; CC15, n = 8. The significance of the differences in the number of virulence factors between CCs was assessed using the Mann–Whitney-Wilcoxon’s test (non-normally distributed data) for all pairwise comparisons with the exception of CC15 vs. CC30, for which Student’s t-test was employed (normally distributed data). Boxes denote the second and third quartiles, vertical lines (whisker) the smallest and largest values of the first and fourth quartiles, with outliers marked by dots. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.