Introduction

Streptococcus suis, an important zoonotic pathogen, causes invasive infections in humans in close contact with infected pigs or contaminated pork-derived products [1]. The number of reported human cases, especially in Southeast Asian countries, has dramatically increased in the past few years [1, 2]. Currently, 29 serotypes of Streptococcus suis have been recognized [3]. Of these serotypes, serotype 2 is the most prevalent in S. suis human infections, although cases caused by serotypes 4, 5, 9, 14, 16, 21, 24, and 31 have also been reported [1, 2, 4,5,6,7,8]. In a retrospective study that included human field strains recovered from 2006 to 2008 in Thailand, clonal complexes (CC) 1, 25, 28, and 104 were shown to be responsible for human infections, with sequence type (ST) 1 (within CC1) being the most frequently found, followed by ST104 (CC104) [8].

In the present study, an additional 668 S. suis isolates recovered from human infections in Thailand between 2009 and 2012 were further characterized regarding serotype distribution, genotypic profile, and clinical manifestations.

Materials and methods

Bacterial isolates and identification

A total of 668 isolates of S. suis recovered from blood, cerebrospinal fluid (CSF), synovial fluid, or other body fluids were collected from 41 hospitals in 39 provinces distributed in 5 regions (north, northeast, central, east, and south) of Thailand between January of 2009 and December of 2012 [Supplemental file 1]. Presumptive identification and antimicrobial susceptibility of S. suis were carried out in the hospitals where the pathogen was originally isolated and sent us to confirmation. A multiplex PCR assay and the co-agglutination test were used to identify and serotype these S. suis isolates [9, 10].

Genotypic profiles

Multilocus sequence typing (MLST) was performed as described elsewhere [11, 12]. MLST alleles, resulting STs, and the CC were assigned and analyzed using the S. suis MLST database, which can be accessed at https://pubmlst.org/ssuis/. The presence of virulence-associated genes (VAG), including the extracellular protein factor (epf), the muramidase-released protein (mrp), and the suilysin (sly), was determined by PCR as described by Silva et al. [13].

Human cases and statistical analyses

The medical records of 659 cases were reviewed by attending physicians at local hospitals in Thailand using the clinical case record form approved by the Ethics Committee of the Department of Medical Sciences, Ministry of Public Health, Thailand. The medical records of the remaining nine patients were not available. The clinical manifestations of S. suis infections were classified into meningitis, sepsis, septic arthritis, infective endocarditis, and spontaneous peritonitis according to a criteria described elsewhere [8, 14,15,16,17]. Clinical isolates were categorized according to ST and VAG profiles. The association between these categories and two clinical manifestations (meningitis and non-meningitis) was analyzed by Fisher’s exact test with the Stata version 10.0 software (StataCorp, College Station, TX, USA). Data were considered significant at p < 0.01.

Results

Serotype distribution of S. suis isolates

Of the 668 isolates, multiplex PCR and the co-agglutination test with specific antisera revealed 632 isolates of serotype 2 (94.6%), 30 of serotype 14 (4.5%), three of serotype 24 (0.45%), two of serotype 5 (0.3%), and one of serotype 4 (0.15%) (Table 1).

Table 1 S. suis clonal complexes and their serotype distribution among isolates recovered from human patients in Thailand
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Genotypic profiles of S. suis isolates

MLST classified 668 isolates into seven CCs and two singleton (Table 1 and Fig. 1). The CCs were identified as follows: CC1 (56.43%), CC104 (31.74%), CC233/379 (5.4%), CC25 (4.5%), CC28 (0.9%), CC221/234 (0.6%), and CC94 (0.15%). The CC1 contained more than 50% of all serotype 2 isolates, with ST1 being the predominant sequence type. Interestingly, CC1 also contained all 30 serotype 14 isolates, with 29 being ST105 and only one belonging to ST11. CC104 was the second most important group with 212 isolates, all serotype 2, with seven different STs. CC25 was composed by 30 isolates, also all serotype 2, with 7 different STs, whereas CC233/379, a newly herein described CC responsible for human infections, included ST233 and ST379, with all isolates also belonging to serotype 2 (Table 1 and Fig. 1). Interestingly, CC233/379 was closely related to CC104, with two locus variants (gki and thrA). Indeed, the allelic profile of CC233/379 is 8-30-5-(72/97)-44-21-75, whereas that of ST104 is 8-30-5-1-44-21-4. CC28 was also composed of serotype 2 isolates belonging to either ST28 or ST382. Isolates belonging to serotypes other than 2 and 14 were distributed in CC94 (serotype 4) and CC221/234 (serotypes 5 and 24). Two isolates could not be attributed to any known CC, belonging to ST235 (serotype 5) and ST236 (serotype 2) (Table 1).

Fig. 1
figure 1

An eBURST analysis of the entire S. suis MLST database (accessed on July 25, 2017). Clonal complexes relevant to human infection in Thailand are circled and labeled. Clonal complexes and the predicted founding STs are indicated by blue dots. The size of the dots is relative to the number of isolates with the respective ST present in the database (Color figure online)

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As shown in Table 1, eight VAG profiles were obtained by PCR: epf +/sly +/mrp + (n = 377, 56.4%), epf −/sly +/mrp + (n = 2, 0.3%), epf −/sly +/mrp s (n = 2, 0.3%), epf−/sly−/mrp + (n = 20, 3%), epf−/sly−/mrp * (n = 12, 1.8%), epf−/sly−/mrp ** (n = 4, 0.6%), epf−/sly +/mrp − (n = 248, 37.2%), and epf−/sly−/mrp− (n = 3, 0.4%). Serotype 2 showed six VAG profiles: epf+/sly +/mrp + or epf −/sly+/mrps profiles, mainly found in CC1; epf −/sly−/mrp+ or epf −/sly−/mrp* or epf −/sly−/mrp** profile associated with CC25; epf −/sly−/mrp+ profile found in CC28; and epf−/sly +/mrp− profile associated with CC104 and CC233/379. Serotype 14 (CC1) isolates presented epf +/sly +/mrp + or epf −/sly +/mrps profiles, while serotype 4 (CC94) showed epf −/sly +/mrp +. Finally, two serotype 5 isolates (ST221 and ST235) revealed a epf+/sly +/mrp +, while the VAG profile of isolates of serotype 24 (CC221/234) was epf−/sly−/mrp −.

Isolation sites and dates

Of the 668 isolates, 391 (58.53%) were recovered from patients of the northern region, 140 (20.96%) from patients of the northeastern region, 102 (15.26%) from those of the central region, 30 (4.5%) from those of the east region, and 5 (0.75%) from those of the southern region (Supplemental file 1). The dates of S. suis isolation as well as their relationship with temperature or rainfall are shown in Fig. 2. In 2009, human cases occurred more frequently in September, whereas 1 year later, the peak was between April and June. Human cases in 2011 increased from April to August and in 2012, the peaks were increased from March to May. It is interesting that the peak of human cases in 2010–2012 are, in general, in agreement with rising rainfall (Fig. 2). In general, the mean temperature in the whole of Thailand between 2009 and 2012 was about 27.2 °C (range 23.2–29.1), 27.9 °C (range 25.1–30.8), 26.8 °C (range 24.1–28.4), and 27.7 °C (range 25.4–29.4), respectively. Indeed, no significant changes of temperature during these years could be associated with the number of diagnosed cases (Fig. 2).

Fig. 2
figure 2

Seasonal distribution of the cases, temperature, and rainfall from 2009 to 2012 in Thailand

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Clinical features and relationship with the genotypic profiles of S. suis isolates

The demographic data revealed that S. suis cases were recovered from 134 females (20.3%) and 525 males (79.7%). The mean age was 56.5 years old, and median age was 56 (range 23–92 years old). The clinical features of the 659 human cases of S. suis infection are summarized in Table 2. Meningitis cases (n = 141) were mainly caused by CC1 isolates (n = 118; 83.7%), especially ST1 (n = 106), followed by CC104 (n = 21; 14.9%). Sepsis (n = 501) could be associated with isolates belonging to all CCs and two singletons (ST235 and ST236). CC1 isolates (n = 239) also accounted for 47.7% of the sepsis cases, followed by those of CC104 (n = 187; 37.3%). Infective endocarditis (n = 10) was mainly caused by CC1 isolates (n = 7). Septic arthritis cases (n = 5) were caused by CC1 isolates, whereas CC104 and CC233/379 isolates were associated with spontaneous peritonitis (n = 2) (Table 2).

Table 2 Clinical manifestations and clonal complexes of S. suis isolates recovered from human patients in Thailand
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A total of 423 out of 659 cases had a history of raw pork product consumption (64.2%), 74 cases (11.2%) had an occupation related to pig/pork products (slaughterer/farmer/butcher), and 162 cases (24.6%) did not have any information regarding pig contact or pork consumption. Eighty-four out of the 659 cases were fatal (12.7%), of which 21 cases were assigned to meningitis, 62 cases to sepsis, and one case was associated to infective endocarditis. Regarding antibiotic treatment, 598 patients (90.7%) received third-generation cephalosporin, such as ceftriaxone, 42 patients (6.4%) received penicillin, and 19 patients (2.9%) received both third-generation cephalosporin and penicillin. Susceptibility test by either disk diffusion or E-test (only penicillin) revealed all S. suis isolates including fatal 84 isolates were susceptible to antibiotics used for treatment (penicillin, ceftriaxone, cefotaxime). The characteristic symptom of hearing loss was found in 20.5% (135 cases), mostly associated with meningitis and/or sepsis. Associations of genotypic profiles with the clinical presentation showed that ST1 isolates were associated with meningitis (p < 0.001), whereas the ST104 isolates could be associated with non-meningitis cases (p < 0.001) (Table 3). In addition, VAG profile of epf +/sly+/mrp+ was also associated with meningitis whereas epf −/sly+/mrp− and epf −/sly −/mrp + were associated with non-meningitis (Table 3).

Table 3 The association between sequence type or virulence-associated gene profile (VAG) and clinical manifestations
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Discussion

Our present data indicate that Thai patients are uniquely infected with a large variety of serotypes, differently from what has been described in other countries/regions [4, 5, 8]. Serotype 2 remained the main serotype causing human infections in Thailand during the period 2009–2012. This study confirms that serotype 14 is also more prevalent in Thailand than in other countries/regions [2, 6]. We also report here a second case of human infection due to a serotype 4 (Tables 1 and 2), this serotype having been described 30 years ago in a case of meningitis in the Netherlands [18]. The serotype 4 isolate in this study was recovered from a sepsis case and belongs to ST94 of CC94, while no information is available for the previous Dutch isolate [18].

MLST genotyping of S. suis collected between 2009 and 2012 also revealed a higher diversity than that observed in our previous study [8]. That study had shown four CCs (CC1 CC25, CC28, and CC104) while this study revealed seven CCs as described previously. CC94 and CC233/379 are newly emerging human infectious clones that have not been reported elsewhere before [2]. To the best of our knowledge, CC104, CC221/234, and CC233/379 are CCs exclusively found in Thailand. The unusual high number of isolates included in this study allowed us to note that S. suis serotype 2 strains isolated from humans in Thailand are much more diverse than previously thought [1, 2]. However, the major S. suis CCs caused human infection in worldwide are restricted to well-recognized CCs, for example, CC1 and CC20 are found in the Netherlands [19]; CC1, CC25, CC28, CC104, and CC221/234 in certain Asian countries (China, Vietnam, Thailand, Japan, Hong Kong, and Cambodia) [2, 5,6,7,8, 20]; CC1 in South America (Argentina) [21]; CC25 in North America (Canada and USA); and CC28 in North America and Japan [2, 20].

Our previous study also revealed that peaks of S. suis human cases occurred during the rainy season between 2006 and 2008 [8], which had also been described in China [22] and Northern Vietnam [23]. The peak in this study changed between years with no clear pattern; however, human cases seem to increase in accordance with rising rainfall, although no cause-effect explanation of such observations could be found. A study in Vietnam also showed a clear association between porcine reproductive and respiratory syndrome virus (PRRSv) outbreaks in pigs and S. suis infection in humans [24]. A highly virulent PRRSv strain was introduced in Thai swine herds in 2008 causing major outbreaks, and the virus was first isolated in 2010 [25]. More studies are needed to evaluate if the introduction of such a virus may have influenced the pattern of S. suis isolation from humans in Thailand.

Oral consumption of raw pork products is the major transmission route of this disease in Thailand. The existence of deep-rooted habitual consumption of raw pork products mainly in the northern part of Thailand may explain the higher number of isolates from these areas. In fact, this is another factor that may influence the peak of infection during the years, since there is, in general, an increase in the frequency of raw pork products consumed by local people during traditional Thai festivals. A recent study confirmed the effectiveness of a food safety campaign for controlling S. suis infection in humans in the Phayao Province of Thailand between January 2011 and December 2011 [26]. The trends of incidence proportion before (2008–2010) and after (2011–2013) the campaign revealed a 3.94/100,000 persons decrease in the trend of incidence proportion after campaign (p < 0.001), with the cost to reduce to an incidence proportion of 1.0/100,000 persons around US$380 [26]. Therefore, a continuous campaign including introducing educational programs in childhood or improve the sanitation of pork processing or additional and alternative public health interventions are needed to decrease or eliminate the disease.

The findings reported herein confirm the association between ST1 strains and meningitis cases, whereas ST104 strains are associated with non-meningitis cases as previously reported [8]. This may indicate that ST1 strains might have an advantage in bacterial survival resisting host immunity, with rapid multiplication in the bloodstream, and high levels of bacteremia, which was already associated with the development of meningitis [27]. In addition, the suilysin, a S. suis hemolysin typically present in ST1 strains, was confirmed to contribute to the development of bacterial meningitis in a mouse model [28].

This study revealed a higher mortality (12.7%) of S. suis cases than in a retrospective study from 2006 to 2008 in Thailand (9.5%) [8]; indeed, a population-based study in Phayao Province in Thailand revealed 16.1% of the case fatality rate, which is even higher [29]. In other countries, the case fatality rates were 2.6% in Vietnam, 18.6% in China, 5% in Hong Kong, and 6.7% in the Netherlands [23, 30]. Globally, the fatality rate of S. suis infection is around 12% [31]. Regarding antibiotic treatment, no relationship between the treatment used and the fatality could be found. Since all S. suis isolates in this study revealed susceptible to antibiotics used for treatment, the cause of death in our patients remains uncertain; however, this may be due to delayed admission, underlying diseases, rapid disease progression, or infection with higher virulence strains of S. suis, although the latter cause remains to be confirmed.

In conclusion, this study revealed a relatively large number of CCs of S. suis causing human infection in Thailand. Among them, CC1 followed by CC104, with serotype 2 isolates, are predominant. Food safety campaigns and public health interventions would be important for controlling the S. suis infection in humans.