Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance

Streptococcus suis is currently emerging as an important zoonotic pathogen in humans, especially in some regions of the world. The aim of our study was to provide extensive characterisation of isolates of this pathogen observed in Poland since 2000. Most of the patients affected by S. suis infection were male and middle-aged; the patient’s profession is not routinely reported to the NRCBM, but for two patients (a farmer and a butcher), infection was very likely caused by occupational exposure. Misidentification is considered a common cause for the underestimation of rates of invasive infections caused by S. suis [2]. In our study, isolates were reported to the reference laboratory as S. suis in only 11 cases (52 %); five isolates were reported as Streptococcus spp. and two as Streptococcus bovis, while the remaining three isolates were misidentified as S. agalactiae, Streptococcus anginosus and Streptococcus sanguinis.

On the basis of the serotype-specific PCR results and epidemiological information reported so far [41], all studied isolates were determined as serotype 2, which is most frequently observed in infections of humans [2, 41]. It has to be noted, however, that DNA-based methods do not allow discerning between serotypes 2 and 1/2, whose cps loci do not differ by any serotype-specific genes and whose sequences are almost identical [43]. Although strains of serotype 1/2 have not been reported from human infections [41], the presence of isolates with this serotype cannot be completely excluded in our collection due to the limitations of the method used. MLST uniformly identified all isolates as ST1. Isolates of this ST and its variants have been reported from human invasive infections worldwide, although STs from other clonal complexes have also been described, including, for example, ST20 in France and the Netherlands, STs 25 and 28 in Thailand and Japan, and ST25 in the United States (summarised in [41]). As shown recently in a Dutch study [44], the diversity of human isolates of S. suis appears generally much more limited in comparison to strains circulating in pigs in the same geographic area and a similar time span, and the vast majority of STs and serotypes found in pigs, a natural reservoir of this pathogen, have never been isolated from humans [41]. Our PFGE analysis discerned four different profiles and we additionally applied MLVA, which was suggested as a typing method allowing additional discrimination of strains, e.g. during an outbreak [26]. In the case of ST1, variability was reported only for the TR9 locus, which was selected for in our analyses. These yielded 17 variants with an even bigger range of the repeat number (8–72) than observed in the original study [26]. Such high diversity of a presumably quickly evolving typing marker is consistent with the sporadic character of all cases of human infections from which our isolates were obtained.

Biofilms play an important role in reducing bacterial susceptibility to antimicrobials and clearance by the host immune system. Formation of biofilm by S. suis required the presence of fibrinogen in the culture medium [24], as also seen in our study. We observed various levels of biofilm formation among our isolates, which was also reported by others for SS2 [45]. Some of our isolates lacked detectable DNase activity, in contrast to the findings of Haas and co-workers, who reported all ten ST1 strains in their study as DNase-positive [17]. Almost all isolates demonstrated haemolytic phenotype, associated with the presence of the sly gene. Sly, a pore-forming toxin, plays an important role in the pathogenesis of S. suis infection [46] and several studies reported all ST1 human isolates as positive for sly [47‐50], in contrast to representatives of other clonal groups found in humans [49] and isolates from non-invasive infections and carriage in pigs [30, 34]. This gene is typically well conserved in S. suis and loss of haemolytic activity is usually due to the replacement of sly by orfC encoding a product of unknown function [30]; however, one of the isolates in the current study lacked both sly and orfC. Such isolates were also observed by others [48] and our searches of the available genomic sequences of S. suis in the GenBank (as of 29th January 2016) revealed that a single strain YS56 (GenBank accession number ALMY01000022) was negative for these two genes in the corresponding position of its genome.

The isolates analysed in our study carried genes of several virulence-associated factors other than sly, such as mrp, epf, fbpS, eno, sao and ofs. The basic variant of the epf gene appears to be, similarly to sly, a marker specific for invasive ST1 strains [47‐50], while mrp, fbpS, eno, sao and ofs appear to be much more common in the whole S. suis population [29, 32, 47‐51]. In agreement with other observations, our analysis also revealed variation in the genes of some of these virulence-associated determinants, including novel alleles of mrp, sao and ofs. The mrp gene with a single 411-bp repeat in its 3′ part, most common among our isolates, is typical for ST1; a shorter version, mrp ^S, present in one isolate, was also observed for this ST [49]. Larger variants of mrp occur among representatives of other CCs associated with serotype 2, such as ST29 [49] and other serotypes [34]. Three isolates from our collection harboured new indel mutations in mrp, preventing the full-length Mrp protein synthesis. Such mutations are relatively common in S. suis [33], and isolates positive for the gene but negative for the protein expression are frequently observed [33, 34, 50]. Variability in the mrp gene may be associated with a selective pressure from the immunological system of the host [34]. The sao-M (seven repeats), observed in our study for almost all isolates, is the most common variant among various serotypes of S. suis [32], and type 1 ofs characteristic for all but one isolate is typical for ST1 [16]. In our study, all isolates belonged to the genotype A of pili and harboured the characteristic frame-shift mutation in sbp2. At least four different pili loci exist in S. suis, and the combination of presence/absence of particular genes allowed distinguishing 12 genotypes, with genotype A being characteristic for ST1 isolates from human infections and diseased pigs [15]. We did not detect sequences specific for the 89K candidate PAI, found in highly virulent strains involved in two outbreaks in China and, as yet, not observed anywhere else [31]. In summary, inclusion of all the study isolates into ST1 and SS2, together with the observed high number of established and putative virulence factors, are consistent with the features of a specific genetic cluster of S. suis, associated with human meningitis [52], described also as the epidemic and highly virulent (E/HV) group, showing resistance to phagocytosis in vivo, thus allowing bacteria persistence at high concentrations in the animal mouse model, a pre-requisite for the development of an inflammatory reaction in the host [53].

Importantly, the investigated isolates of S. suis from our collection retained susceptibility to agents recommended in Poland for empirical therapy of community-acquired bacterial meningitis in adults, such as cefotaxime and vancomycin (http://​www.​koroun.​edu.​pl, accessed 17th December 2015). As yet, no resistance to these compounds has been reported for S. suis isolated from humans [54‐56]. We observed concomitant resistance to erythromycin and tetracycline for 24 % of isolates. Resistance to tetracycline appears to be common, or even very common, in S. suis from human infections, reaching 100 % among 114 strains of ST7 in China [54], 100 % among 33 isolates in Hong Kong [55] and 89 % among 175 isolates in Vietnam, with a clear increase over the period 1997–2008 [56]. Resistance rates to erythromycin were 21 % and 22 % in Hong Kong and Vietnam, respectively [55, 56], similar to our observations. The acquisition of resistance determinants by S. suis occurs chiefly in its animal host under the selective pressure exerted by the use of antimicrobial agents in animal production. In Poland, according to the European Surveillance of Veterinary Antimicrobial Consumption (ESVAC) report for 2011, the sales of lincosamides, macrolides and tetracyclines for food-producing animals amounted to 4.1, 25.6 and 179.6 tonnes, respectively [57], and resistance to erythromycin and tetracycline among pig isolates reached 31 % and 64 %, respectively, in 2004 [58]. In our collection, isolates resistant to tetracycline and erythromycin/clindamycin carried the tet(O) and erm(B) genes. The presence of tet(O) was reported for a small number of human isolates from Europe and North America [54]. The tet(M) gene, associated with Tn916, was the most widespread determinant of tetracycline resistance in human S. suis in China and Vietnam [54, 56]. In Vietnam, tet(O) was the second most common gene responsible for tetracycline resistance; tet(L) and tet(W) were also observed in that study [56]. The erm(B) gene was present in 95 % of erythromycin-resistant human S. suis in Vietnam [56], while in Hong Kong, most of the isolates carried mef(A) [55]. Thus, there are obvious differences in the local epidemiology of tetracycline and erythromycin resistance genes among human S. suis. Additionally, one of the resistant isolates in our collection carried the lsa(E) and lnu(B) genes. The lsa(E) gene encodes an ABC transporter that confers resistance to lincosamides, streptogramin A and pleuromutilins (LS_AP phenotype) by efflux of the drugs from the bacterial cell [59], while lnu(B) (formerly linB) inactivates lincosamides by adenylation [60]. These two genes are found in several Gram(+) bacteria, e.g. E. faecalis (AF408195), the C2944 strain of S. aureus, the SGB76 strain of S. agalactiae, the pDX5 plasmid of E. faecium and the Ery-11 strain of the pig pathogen Erysipelothrix rhusiopathiae [61‐64], but, to our knowledge, have not yet been reported in S. suis. Our search of genomic sequences of 375 pig and human isolates of S. suis [65], deposited at the European Nucleotide Archive (http://​www.​ebi.​ac.​uk/​ena, accessed 2nd February 2016), revealed the presence of lsa(E) and lnu(B) in four pig isolates, associated with serotype 2 from Vietnam and in two pig isolates of serotypes 7 and 14 from the United Kingdom, suggesting independent acquisition of these resistance genes by different clones in various parts of the world. The macrolide, lincosamide and tetracycline resistance determinants detected among isolates from our collection were likely carried by S. suis-specific ICEs [18], consistent with the concomitant presence of their repA gene and absence of genes characteristic for other mobile elements, such as Tn916 and broad-host plasmids.

In conclusion, we performed a detailed phenotypic and genotypic analysis of S. suis isolates obtained in Poland from invasive human infections. While all these isolates belonged to a single clonal complex associated with considerable morbidity and mortality in humans worldwide, diversification of this complex was observed, including the presence of novel variants of virulence determinants and the acquisition of antimicrobial resistance genes. The human invasive S. suis infections in Poland very likely remain underestimated, considering the fact that, in 2012, the number of professionally active farmers amounted to 2.1 million and pig livestock exceeded 10 million heads (http://​www.​stat.​gov.​pl, accessed 17th December 2015), which indicates an existence of a significant at-risk human population and a need for improved surveillance of invasive human infections, caused by this pathogen in our country.