A Multi-locus sequence typing and antimicrobial resistance profiles of equine Streptococcus equi subspecies zooepidemicus isolates

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most important veterinary pathogens frequently associated with equine endometritis, which is one of the main causes of infertility in mares [1]. In this study, we surveyed 22 strains of S. zooepidemicus collected during 2021 from cervico-uterine swabs of mares affected by infertility. The variability of the isolated strains was assessed with multi-locus sequence typing (MLST) from whole-genome sequencing (WGS) data. Sequencing libraries were prepared as reported by Bianco et al. [2]. An average of 446.061 reads (95% CI: 426.208–465.914) were produced for each isolate and quality-checked, de novo assembled and annotated as described by Trotta et al. [3]. The average length of reconstructed genomes was 2.078064 bp (95% CI: 2.055839-2.100289), which was expected for genomes of S. zooepidemicus. The assembled genomes were assigned to STs using the S. zooepidemicus scheme targeting seven loci (arcC, nrdE, proS, spi, tdk, tpi, yqiL) available in the PubMLST database. Isolates were also tested for antimicrobial resistance using the Kirby-Bauer disk diffusion method on Mueller-Hinton plates (Mueller-Hinton with 5% sheep blood for Streptococcus spp.). MLST revealed a wide variability of STs with six (27.3%) novel STs, 4/22 (18.2%) isolates assigned to ST92, 3/22 (13.6%) to ST205, and one strain (4.5%) for each of the following STs: ST10, ST30, ST39, ST49, ST101, ST132, ST147, ST314, ST369. The antibiotic resistance pattern of the three ST205 strains (resistance to amoxicillin-clavulanate, ampicillin, amikacin, gentamicin, streptomycin, enrofloxacin, sulfamethoxazole-trimethoprim, tetracycline, oxytetracycline) was the most common profile observed in half of the studied S. zooepidemicus strains. In addition, antibioticresistant phenotypes of the four ST92 isolates resulted to be conserved only across two of them. However, high resistance rates to tested antibiotics were always observed, enough to classify all the strains as multidrug-resistant. Precisely, a very high proportion (>95%) of the isolates was resistant to penicillins, fluoroquinolones, aminoglycosides, sulfonamides and tetracyclines. Our study provides a comprehensive insight into the epidemiology, antibiotic resistance profile and ST diversity of S. zooepidemicus strains causing ipofertility problems in mares. To our knowledge, this report represents the first equine ST identification of S. zooepidemicus in Italy. Thus, considering that little information is currently available on the genetic strains and clonal spread of equine S. zooepidemicus, these findings justify the need for wider epidemiological monitoring of S. zooepidemicus in mares.