S. zooepidemicus is regarded as an opportunistic pathogen in a large variety of animals, such as horses, pigs, ruminants, dogs, cats, rodents, poultry, seals, and monkeys [15]. Although S. zooepidemicus is a commensal organism in horses, several studies revealed that S. zooepidemicus was the major causative pathogen of purulent infections in horses and foals, leading to endometritis, strangle-like diseases, and severe respiratory diseases [16–17]. Genetic exchange among zoonotic streptococci of animals and humans may occur and influence host specificity or virulence [18–19].
For a further understanding of the evolution and genetic background of the pathogenic mechanism of the bacterium, S. zooepidemicus strains JMC111 and HT321, isolated from horses and donkeys in Xinjiang province, China, were chosen for genome sequencing.
In this study, using the WGS technique, we determined the complete genome sequence of S. zooepidemicus strains JMC111 and HT321, two virulent strains isolated from a horse and donkey with strangles, respectively. Subsequently, the full genomic sequences of the two isolates were compared to those of the S. zooepidemicus reference genomes H70 and ATCC35246. The JMC111 genome was found to be highly colinear with the reference genome H70, and the HT321 genome was highly colinear with the reference genome ATCC35246, and a predominant rearrangement and inversion were observed between HT321 and H70, JMC111, and ATCC35246.
The comparative genomic result provided evidence of the genetic events that have shaped the evolution and fitness of the S. zooepidemicus strains[20]. Previous studies on gene loss in the genome of S. zooepidemicus have been identified, and gene gain has been demonstrated through the acquisition of MGEs that are considered to have shaped the host tropism and pathogenic specialization of S. equi [21].
During evolution, Streptococcus acquired new traits mainly by horizontal gene transfer (HGT), which is a key driving force for expressing novel pathogenic properties, colonization niches, and metabolic adaptations [22–24]. Of the three HGT mechanisms, bacteriophage-mediated DNA transfer often provides the most profound alteration in host bacterial genomes [25]. An integrated phage is termed a prophage, and it often expresses genes that increase host cell fitness [26–27]. It has been reported that S. pyogenes is one species that has acquired strain-specific virulence genes by transduction of prophages [28–29], and the prophage regions contain variable virulence genes and confer pathogenic capacities [30], while for S. zooepidemicus MGCS10565, researchers found its genome lacking prophages and hypothesized that it might be naturally competent. [31] In the present study, four prophages were identified on the HT321 genome and two on the JMC111 genome.
Prophages can obtain a range of phenotypic effects on the host bacteria: encode toxins that increase virulence, promote binding to human platelets or cells8, evade immune defenses 9, 10, or protect from oxidative stress 11. Previous studies have assessed the effect of prophage on pneumococcal virulence, and their results showed that deletion of the whole prophage or vapE (a virulence gene carried by S. suis prophage) alone had a significant effect on S. suis virulence [32]. Here, we identified two virulence genes (hyaluronate lyases genes) on the genome of JMC111 prophages, but for HT321, this virulence gene was absent from their four prophages. Hyaluronate lyases (Hya) are secreted enzymes that degrade hyaluronic acid (HA), facilitating the invasion of bacteria and their toxins [33]. Hya, which is found active in some strains [34], is proposed to be related to increased adherence and colonization in the hosts [35–36] (Turner et al. 2015; Zhu et al. 2015). Another example reported is that S. equi 4047 has acquired a 4 bp deletion in hyaluronate lyase encoded on a prophage, and this phage-encoded enzyme exhibited a reduced substrate range and much lower activity [21]. Here, the acquired hyaluronate lyase activity of JMC111 was demonstrated by the increased recovered colony formation unit (CFU) and survivability percent in a murine infection model. Our observations of prophage and Hya virulence factors provide an alternative explanation that the increased levels of hyaluronate lyase activity may enhance the infectivity, pathogenicity, and virulence of JMC111 isolates and might act as an important factor in the evolution of the JMC111 genomes.
MGEs are ubiquitous in streptococci and the most significant driving force of gene transfer, resulting in the dissemination of antimicrobial resistance and virulence determinants. Streptococci contain various types of MGEs, including transposons, insertion sequences (IS), integrative conjugative elements, and bacteriophages. Several MGEs have been found in many streptococci genomes that contain ARGs and virulence genes, like secretion systems, adhesins, and exotoxins [37], while IS and transposons were found in S. mutans, which contribute to its genetic diversity [38–39]. Recently, components of a T4SS have been found in C. difficile genomes within transposon 2 (CTn2), CTn4, and CTn5 [40–42]. In our current study, it is interesting that a type-IV secretion system virD4 gene cluster has been identified on IS of JMC 111, which is absent in strain HT321. In general, Type-IV secretion systems (T4SSs) in many gram-negative and gram-positive bacteria were acquired and/or evolved during their long co-evolution with their host [43][44] and are molecular transport systems capable of the transportation of DNA and various protein effectors into target cells during infection, horizontal transfer of MGEs, and exchange of DNA with the outer space [45]. T4SSs comprise 11 structural protein subunits, called VirB1 to VirB11, the coupling protein VirD4/TraG, and the DNA processing enzyme VirD2. In particular, DNA transfer mediated by T4SSs commonly requires additional DNA processing enzymes such as VirD2 relaxases and VirD4 (TraG) coupling proteins. It has been shown that VirD4-like proteins (TraG) exhibit NTPase activity, which interacts with both dsDNA and ssDNA [46–48] and binds DNA without sequence specificity[49–50].
Recently, T4SS has attracted intense interest due to its importance in the virulence and even survival of some bacterial species. Zhang et al. reported that the T4SS secretion system is located in the 89K pathogenicity island of S. suis type 2 strains and has been proposed as a new T4SS subgroup (Type-IVC secretion system) [51].
VirD4 may act as an adapter protein, guiding CagA from Helicobacter pylori into the transport channel and presumably helping induce host proinflammatory responses in both a VirD4-CagA-dependent and a VirD4-CagA-independent manner [52]. Jiang showed that deletion of the virD4 gene decreased SS2 virulence in mice, and virD4 plays an important role in antiphagocytosis and increased release of proinflammatory cytokines [53]. Here, we show that the JMC 111 strain containing the virD4 gene exhibited higher survival and a higher bacterial load in organs than that of HT321 in a mouse infection model. These virulence phenotypes were similar to the previous findings in SS2 strains [53–54].
WGS has become an essential tool to elucidate the mechanisms used by bacteria to resist various antibiotics. In the present study, we have investigated the genomic characteristics among genes associated with virulence and antimicrobial resistance in two clinical isolates of S. zooepidemicus strains JMC111 and HT321 based on their susceptibility profiles against 21 antibiotics. Concerning antimicrobial resistance, we observed that JMC 111 carries more antimicrobial resistance genes but exhibits lower antimicrobial resistance ability. The observations argue against the idea that the antimicrobial resistance gene is a key factor affecting bacterial susceptibility profiles.
For some bacterial pathogens, biofilm production can significantly modulate their virulence and antimicrobial resistance [55]. The formation of biofilms allows microbial pathogens to create a safe, self-produced matrix in which sessile cells remain in a protected environment to resist stress factors (e.g., antibiotics, nutrient deprivation, and host immune defenses). Cells within a biofilm may have more benefits for colonization and persistence in the host [56–58].
Our results are in agreement with those described by Hall [59], who reported that biofilm helps resist both host immune responses and antibiotic treatment. Our results suggest that the degree of biofilm produced in vitro correlates well with antimicrobial resistance because strong biofilm formers HT321 showed greater antimicrobial resistance ability than weak biofilm formers JMC111.
On the other hand, biofilm formation is considered a virulence phenotype in bacteria [60–61]. Our results differ from those reported by other researchers, who suggested a link between biofilm formation and bacterial virulence [62–64]. In our study, the avirulent strain HT321 had a greater ability to form biofilms than the virulent strain JMC111, and we found no correlation between virulence and biofilm formation in the two strains tested.
In addition, antimicrobial therapy is one of the principal choices in the treatment of strangles on horse and donkey farms. Although an antimicrobial susceptibility assay was used, this technique is not representative of what happens locally in affected tissues. Since infections are often associated with biofilm-grown bacteria, they will display an inherent lack of susceptibility to antimicrobials. An example of antibiotic treatment failure in S. pyogenes infections has been demonstrated to be associated with biofilm formation. The strain may use it as a barrier against the antimicrobials and survive in the host [65]. Therefore, our result suggests it is important to carry out antimicrobial therapy and susceptibility assays based on biofilm production.