Molecular diagnostics and ITS-based phylogenic analysis of Streptococcus suis serotype 2 in central Vietnam

Introduction: Streptococcus suis (S. suis) serotype 2 has recently become the most prevalent cause of meningitis in adults in many areas of Vietnam. This study provides data on S. suis molecular diagnosis in central Vietnam using a real-time polymerase chain reaction (PCR) assay targeting the S. suis serotype 2 cps2J gene. Additionally, 16S-23S rDNA intragenic spacer (ITS)-based phylogenic analysis of strains isolated from cerebrospinal fluid (CSF) in Thua Thien Hue Province, Vietnam, is presented and discussed. Methodology: Pathogenic bacteria were isolated from 40 CSF samples, and 18 were identified as S. suis by culture-dependent methods. Capsular serotyping was assessed by real-time PCR. ITS sequences were obtained after traditional PCR and were used in phylogenic analyses. Results: Pathogenic bacteria were isolated from 36 out of 40 CSF samples. A total of 18 S. suis strains were isolated and assigned to serotype 2 by real-time PCR. One CSF sample, negative when tested by culture-dependent methods, was positive to S. suis serotype 2 by real-time PCR. Pairwise alignments of the 18 ITS sequences did not reveal any variable nucleotide position, and resulted in a single sequence type. Sequences were similar to S. suis serotype 2 reference ITS sequences (> 98.1%), and there was no lack of an ITS spacer region in the isolates. Conclusions: S. suis serotype 2 is the most prevalent serotype in central Vietnam. Real-time PCR assay proved to be a reliable diagnostic method for early detection of S. suis 2 in CSF samples.


Introduction
Streptococcus suis (S. suis) is a Gram-positive bacterium and, with clinical relevance in pigs, is emerging in humans [8,9].Based on differences in antigenic properties of the polysaccharide capsule, 35 S. suis serotypes have been distinguished to date, of which serotype 2 is most commonly associated with disease in humans and pigs worldwide [14,15].S. suis was first reported in 1954 during outbreaks of meningitis, septicemia, and purulent arthritis in piglets.Since then, this pathogen has been reported in nearly all countries that have extensive pig industries.S. suis has been isolated from the upper respiratory tract, nasal cavities, and palatine tonsils of clinically healthy pigs, which therefore can be considered to be carriers.Recently, S. suis has emerged as a zoonotic agent in humans in contact with infected pigs or with their products [28].In 2006, a major S. suis outbreak resulted in more than 200 human cases with a fatality rate of nearly 20% in China [30].More recently, several studies from Thailand, Hong Kong, Taiwan, Singapore, and Vietnam indicated that S. suis is an important cause of adult endocarditis, septicemia, arthritis, and especially meningitis, with high fatality rates and severe neurological sequelae [5,6,12].S. suis serotype 2 has been reported as the most virulent and is the most common type isolated from diseased pigs and meningitis patients [2].For diagnosis, S. suis serotype 2 is isolated from cerebrospinal fluid (CSF) or blood samples in blood agar, and identified by morphological and biochemical characteristics of colonies and by agglutination with specific antisera.In some cases, S. suis serotype 2 could be identified as alpha-hemolytic, viridans Streptococcus species [4].Culture results can be negative in infected subjects treated with antimicrobial agents before sampling.
Development and evaluation of a specific and sensitive PCR assay for rapid detection of S. suis serotype 2 is crucial to meningitis treatment.
Multilocus enzyme electrophoresis, restriction endonuclease analysis with HaeIII, ribotyping, repetitive extragenic palindromic (REP) or enterobacterial repetitive intergenic consensus (ERIC), arbitrarily primed PCR, and pulsed-field gel electrophoresis (PFGE) have been used to determine S. suis strains epidemiological relationships [1,2,18,25].Characterization of the 16S-23S ribosomal (r) DNA intergenic spacer region (ITS) has also been used to compare bacterial strains and to identify species within genus Streptococcus, including S. suis.This paper reports the development of a molecular approach based on S. suis serotype 2 cps2J gene for diagnosis in meningitis patients hospitalized in Thua Thien Hue, Vietnam.The ITS was amplified by traditional PCR from strains obtained in this study and sequenced.ITS-based phylogenetic analysis is also presented and discussed.

Ethical approval
The study was approved by the Scientific and Ethics Committee of Hue University of Medicine and Pharmacy.

Clinical samples
Forty cerebrospinal fluid (CSF) samples were collected between July 2013 and July 2014 by clinicians from hospitalized patients with purulent meningitis before antimicrobial therapy was started.Clinical specimens were transported to the microbiological laboratory within two hours of collection for microbiological analysis.CSF samples were processed for bacterial isolation immediately after arrival in the laboratory.Also, aliquots of CSF samples were used for DNA extraction.DNA samples were stored at -80°C until use.

Bacterial strain, culture, and isolation
At laboratory arrival, CSF samples were centrifuged at 2,000 × g for 10 minutes.Pellets were cultured on blood agar (BA) and chocolate agar (CA) at 37°C, 5% CO 2 , for 24 hours.Samples were also cultured in nutrition agar and brain-heart infusion broth (BHI) at 37°C for 24 hours.Identification of bacterial isolates was performed following standard procedures [27].Briefly, S. suis was identified on the basis of colony morphology, Gram stain, microscopic examination, and biochemical tests such as negative catalase reaction, optochin resistance, esculine hydrolysis, and negativity for Voges-Proskauer test with API 20 Strep (BioMérieux SA, Marcy l'Etoile, France) [24].

DNA extraction
The iVApDNA Extraction Kit (Viet A Technology Corporation, Ho Chi Minh City, Vietnam) was used for DNA extraction.Briefly, 200 μL of bacterial suspension or CSF were treated as recommended by the manufacturer.DNA was resuspended in a final volume of 50 μL.Concentration and purity of total DNA were evaluated using a spectrophotometer (NanoDrop 2000, Thermo Scientific, Waltham, USA).

Capsular serotyping confirmation
Real-time PCR assay was performed by using forward primer (5'-GGT TAC TTG CTA CTT TTG ATG GAA ATT-3'), reverse primer (5'-CGC ACC TCT TTT ATC TCT TCC AA-3'), and the TaqMan probe (5' FAM-TCA AGA ATC TGA GCT GCA AAA GTG TCA AAT TGA-TAMRA 3'), which specifically targets 85 bp of the cps2J gene, a gene involved in the biosynthesis of serotype 2-specific S. suis polysaccharide capsule [15,21,22].Ten ng DNA extractions, 0.4 µM for each primer, 0.1 µM of probe, 0.2 mM for each NTPs, and 0.5 units of Platinum Taq DNA Polymerase (Invitrogen, Thermo Fisher Scientific, Massachusetts, USA) were combined in a 25 µL total volume reaction.PCR amplification was profiled as follows: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds in Mx3000P qPCR System (Agilent Technologies Inc, Santa Clara, USA).Negative controls (no-template control) and other pathogenic bacteria (S. pneumoniae, N. meningitides, H. influenzae, S. suis serotype 1) were coupled with samples in each experiment to confirm specificity of primers and probes.A DNA sample extracted from the reference strain of S. suis serotype 2, provided by the Department of Microbiology, Hue Central Hospital, was used as positive control.PCR was considered positive if the negative controls were all negative and a FAM signal with a cycle threshold value (Ct) of ≤ 38 was obtained.

16S-23S rRNA intergenic spacer fragment amplication
Genomic DNA samples obtained from 18 S2 isolates confirmed by real-time PCR were used for amplifying ITS fragments by traditional PCR.PCR analysis was accomplished using a newly designed forward primer (5'-GCT GCA ACT CGC CTA CAT GA-3') located at position 1259 of 16S rDNA and reverse primer (5'-ACT TAC AGC TCC CCA AGG CA-3') located at position 93 of 23S rDNA, which specifically amplify approximately a 729 bp amplicon including full-length ITS fragment.
A total of 100 ng genomic DNA, 0.4 µM for each primer, 0.2 mM for each dNTP, 0.5 units of Platinum Taq DNA Polymerase (Invitrogen, Thermo Fisher Scientific, Inc.) were combined in a 50 µL total volume reaction.PCR amplification was performed as follows: initial denaturation at 95°C for 10 minutes, followed by 36 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, then a final extension at 72°C for 5 minutes in a Veriti Thermal Cycler (Applied Biosystems, Carlsbad, USA).PCR products were separated by electrophoresis on 1% agarose gel with 1X GelRed (Biotium Inc., Hayward, USA) [13].

Gene profile analysis
PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.Ten ng of purified ITS fragments and 0.32 µM of primer were used for direct sequencing.To sequence both strands, two specific PCR primers were run for each ITS sample.Chromatograms were analyzed with Geneious software version 8.1 and compared with ITS sequence data strains available in the GenBank (http://www.ncbi.nlm.nih.gov/genbank/) using the BLASTn plugin of Geneious software [9].All sequences were aligned using ClustalX [10].The ITS sequences of S. suis serotype 2 from different countries (China, Taiwan, Germany, Denmark, France, and Canada) were obtained from GenBank for comparisons and construction of phylogenetic trees.
Phylogenetic and molecular evolutionary analyses were performed using the MEGA6 program.Phylogenies were reconstructed using the maximum likelihood method with bootstrap values calculated over 100 replicate runs [23].

Results
Forty CSF samples were processed for bacterial isolation.A total of 36 bacterial isolates were isolated and identified by culture-dependent methods.Four (10%) samples tested negative when seeded in cultures.Among bacterial isolates, 18 CSF samples (45%) were identified as S. suis by API20 STREP with 85%-90% confidence, while 2 CSF samples (5%) were identified as S. pneumoniae.H. influenzae and N. meningitidis were detected in 6 CSF samples (15%) samples, and L monocytogenes was detected in 1 sample (2.5%).Nine CSF sample (22.5%) isolates were identified as A. baumannii, E. coli, E. faecium, K. pneumoniae ss.pneumonia, coagulase-negative Staphylococcus, and S. mitis.Co-infections with two bacterial species were not detected (Table 1).
Real-time PCR assay for capsular serotype 2 was positive when all the 18 S. suis strains (45%) were tested (30-33 Ct value range on three replicates).Additionally, a DNA sample extracted from a CSF that was negative in isolation tests was positive by real-time PCR with a Ct value ranging from 33 to 37. All negative controls (non-template controls, other bacterial pathogens such as S. pyogenes, S. pneumonia, S. aureus, Enterococcus, E. coli were negative, see Table 2).
Amplification of the 16S-23S intergenic spacer was confirmed by gel electrophoresis of the amplified fragments.The expected size of about > 700 bp was obtained (lanes 3-7) with all S. suis serotype 2 tested (Figure 1).A sequence including the full-length ITS region (about 455 bp) was obtained from each of the 18 amplicons of S. suis serotype 2. Alignments of 18 sequences allowed for their grouping into a single sequence type (100% pairwise identities).This sequence type was named HUE_286_2013 (GenBank accession number KR779926) and corresponds to nucleotides 327857-328585 of the complete genome of S. suis 98HAH33 (GenBank accession number CP000408).
Maximum likelihood trees based on the Kimura two-parameter model obtained by aligning the HUE_286_2013 ITS sequence with 23 Streptococcus species showed that HUE_286_2013 clusters with S. suis together with S. parasanguinis in a monophyletic clade (Figure 2).

Discussion
Recently, S. suis infection has become the most common cause of meningitis in adults in Vietnam and in other Asian countries such as China (including Hong Kong) and Thailand.In Thailand, S. suis infection was mostly reported in northern provinces.Between 2006 and 2012, 38 patients with S. suis infection were hospitalized in Nakhon Phanom.Deafness developed in 12 patients, and none died [19].In China, a large outbreak of S. suis serotype 2 emerged in summer 2005 in Sichuan, with 38 fatalities and 215 infected people [7].S. suis serotype 2 was also reported to cause sporadic illness in humans in various European countries (France, Germany, Denmark, United Kingdom, the Netherlands), in Australia, and in the United States [11,20,25,26,29].In Vietnam, S. suis serotype 2 infection is the most common cause of meningitis in adults.Approximately 40% of all acute bacterial meningitis cases in adults in Ho Chi Minh City and in Hanoi were attributed to S. suis serotype 2 infection [15,16,17].
Results obtained in this study showed that S. suis serotype 2 was detected in 47.5% (19 positive of 40 CSF samples) of all meningitis cases.It can be speculated that in Vietnam, S. suis serotype 2 infection frequencies can be higher.Indeed, only a few local laboratories are able to confirm S. suis serotype 2 infection by PCR.For this reason S. suis can be considered an overlooked and underdiagnosed pathogen in central Vietnam.A great proportion of humans infected by S. suis serotype 2 were found to be associated with the habit of farming domestic animals at home, in which pigs are raised in closed small piggeries with unhygienic conditions and are slaughtered without being quarantined, which results in contamination of raw pork products [15,16,17].Moreover, the unsafe habit of purchasing pork in flea markets and the consumption of undercooked animal products are well-established risk factors for acquiring many infectious diseases, especially S. suis serotype 2 from pigs [6,8].A recent study from Hong Kong reported heavy contamination of raw pork meat at local supermarkets or wet markets with S. suis; therefore, hot and humid climates may facilitate the growth of S. suis in raw pork products [3].
We detected one CSF positive for S. suis serotype 2 by real-time PCR in four CSF samples that were negative by bacterial isolation.CSF was significantly more often negative in culture in patients who received antimicrobial treatment before hospitalization.Realtime PCR might be a more sensitive and reliable method for S. suis serotype 2 detection and early diagnosis in clinical samples [15].
Amplification and sequencing resulted in the same product size of the 18 ITS sequences obtained in this study (455 bp).Gel electrophoresis of the 18 amplified ITS constantly revealed only one band, and sequence data were compatible with the presence of a single template.
Surprisingly, ITS sequences of the 18 isolates obtained in this study showed 100% homology when aligned, although strains were isolated from 18 CSF samples of 18 patients with meningitis at different times of hospitalization, and infected subjects were from different districts in the Thua Thien Hue province.Isolates of S. suis serotype 2 in Thua Thien Hue could be under constant selection pressure, resulting in stability of ITS gene sequences, as also suggested by the lack of a spacer region in the isolates sequenced in this study.

Conclusions
This study confirms the importance of S. suis serotype 2 as a prominent bacterium causing purulent meningitis in adults in Vietnam.Real-time PCR may represent a sensitive method for detection of S. suis serotype 2. Local isolates are genetically stable, and this could promote the development of specific molecular assays for diagnosis.

Figure 1 .
Figure 1.Amplification of ITS fragments of S. suis 2 by PCR

Figure 2 .
Figure 2. Molecular phylogenetic analysis by maximum likelihood method based on the Kimura two-parameter model of HUE_286_2013 and 23 Streptococcus species

Figure 3 .
Figure 3. Molecular phylogenetic analysis of ITS sequences of S. suis serotype 2 strains.A: maximum parsimony analysis of taxa; B: maximum likelihood method based on the Tamura three-parameter model of S. suis serotype 2 strains

Table 1 .
Bacterial pathogens identified by culture-dependent methods from CSF samples

Table 2 .
Specificity of S. suis serotype 2 real-time PCR on culture-confirmed CSF samples and no growth samples