Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing

Abstract

cStaphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlL R2 , atlL R3 , hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (d N /d S ) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlL R2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the d N /d S ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlL R3 , isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis.

Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and Tandem Repeat Sequence Typing (TRST), helpful tools for subtyping Staphylococcus lugdunensis

“…This latter method allowed us to define nearly the same number of profiles as MLST and was consistent with MLST, as unique STs or their single locus variants (SLVs) and double locus variants (DLVs) were found in each VT T group. Thus, this comparison confirms the coevolution of virulence-associated loci studied with housekeeping genes as shown previously 17 , and validates the use of the trilocus method as a good epidemiological tool while significantly reducing costs.…”

“…lugdunensis clinical isolates by MLST and MVLST. Data for 20 isolates were derived from our previous studies 16 , 17 , while we performed MLST and MVLST analysis of 108 additional clinical isolates for the purposes of the present study.…”

“…lugdunensis . Recently, we developed a multivirulence-locus sequence typing (MVLST) method, based on the sequence data of seven known or putative virulence-associated loci 17 . MVLST was significantly more discriminant than MLST, even allowing a trilocus sequence typing method for microepidemiological purposes.…”

Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme

“…Indeed, both housekeeping and virulence genes were found to be evolving under purifying selection as suggested by d N / d S ratios and the negative values of Tajima’s D statistic. This finding is not without precedent, since similar results have been reported for Listeria monocytogenes 36 and Staphylococcus lugdunensis 32 . Hence, aside from preserving the most fit pathotype clones, purifying selection might also contribute to maintain gene sequences that define a given pathotype.…”

“…By contrast, and as expected, the average of haplotype diversity of 0.61 found in M. hominis housekeeping genes was comparable to other mycoplasmas species, such as Mycoplasma bovis (average of 0.64) 17 and Mycoplasma hyorhinis (average of 0.651) 30 . Previous studies have confirmed that virulence gene-based typing schemes (MVLST) afford higher resolution than did MLST, making them the best surrogate approaches to carry out reliable epidemiological investigations 31 , 32 . However, neither MVLST nor MLST data could be unambiguously correlated with the clinical status of the studied strain collections.…”