Molecular diversity and antimicrobial susceptibility of Streptococcus equi ssp. equi isolates from equines

Abstract

Streptococcus equi subspecies equi (See) is the causative agent of strangles, a highly infectious disease of equines worldwide. This study utilized various molecular typing methods, including analysis of whole genome sequence (WGS) data, to enlighten the phylogenetic relationships of See isolates obtained from equines in Germany and other countries, as well as to examine the associations between genotype, virulence-associated genes (VAGs), and epidemiological data. Additionally, antimicrobial susceptibilities, biofilm formation in vitro, and the relationship between genotype and biofilm formation were investigated. This study examined 628 non-duplicate putative S. equi isolates obtained from equines between 2001 and 2020, which were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as See (n = 265) and Streptococcus equi subspecies zooepidemicus (Sez) (n = 363), respectively. A published Se-mPCR successfully detected the sodA gene in all tested isolates and confirmed their assignment to the species S. equi. All but one isolates identified as See by MALDI-TOF MS (99.2 %) also harboured the See signature locus ICESe2, while none of them carried the ICESz1 locus of Sez. In contrast, only 44.6 % of the Sez isolates harboured the respective PCR target ICESz1. Among the 265 See isolates, analysis of their seM gene revealed a total of 47 different alleles, including 30 novel ones. The seM-9 allele was the most prevalent (87.9%). Multilocus sequence typing (MLST) of 191 See isolates selected for whole genome sequencing confirmed a low phylogenetic diversity of See since only two highly related sequence types (STs) were detected: ST-151 (73.8 %) and ST-179 (26.2 %). The core genome MLST (cgMLST) analysis exhibited 159 core genome (cg)-geno¬types affiliated with three globally recognized BAPS clusters of See: BAPS-2 (94.8 %), BAPS-5 (4.7 %), and BAPS-6 (0.5 %). Spatial-temporal analysis of See isolates from Germany identified some phylogenetically closely related or identical strains within clusters and beyond, suggesting modern horse transportation promotes the distribution of See strains. Comparison of the cgMLST results with those publicly available for 759 other See isolates from around the world revealed that all genotypes of this study fit perfectly into the general phylogenetic tree of See, supporting the hypothesis of a common See ancestor strain and global expansion of its descendants since the late 19th or early 20th century. In silico screening of WGS data from See isolates for known virulence-associated genes (VAGs) revealed 38 VAGs consistently present in all 191 tested isolates, with an additional 29 VAGs occurring only in 5 % to 99 % of the isolates. Differences in VAG combinations made it possible to distinguish 107 virotypes. The study also revealed that all antigens used in the commercial Strangvac® vaccine were highly-conserved in the investigated See isolates, suggesting the efficacy of Strangvac® against the See strains represented. All See isolates proved susceptible to tested beta-lactam antimicrobials, including penicillin G. Thus, penicillin G can be recommended as the first-line antimicrobial for strangles cases where antimicrobial therapy is clinically indicated. Biofilm assays indicated enhanced or diminished biofilm formation by certain See STs and seM allele groups, respectively, implying a role for the SeM protein in biofilm assembly in vitro.