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Veterinary Microbiology 153 (2011) 403–406

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ort Communication

e cps locus of Streptococcus suis serotype 16: Development of arotype-specific PCR assay

icheng Wang a,b,c, Weixing Fan c, Henk Wisselink d, Chengping Lu a,b,*

Lab Animal Disease Diagnostic & Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China

llege of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

na Animal Health and Epidemiology Center, Qingdao, China

tral Veterinary Institute of Wageningen UR, Lelystad, The Netherlands

ntroduction

Streptococcus suis (S. suis) is a major zoonotic pathogen,ich infects pigs and rarely infects other animalsttschalk et al., 1989; Higgins et al., 1990). Although

otype 2 is considered to be the most pathogenic forans and pigs (Lun et al., 2007), other serotypes can

se disease in pigs and humans as well. Recently aan case of S. suis serotype 16 was described (Nghia

l., 2008). In addition, serotype 16 isolates were isolated diseased pigs in Denmark as well as from pigs in

Canada, Germany, South Korea and China (Han et al., 2001;Messier et al., 2008; Wisselink et al., 2000).

S. suis isolates are generally serotyped using a serumagglutination method with a panel of specific sera. Thistyping method is very laborious and time-consuming andcan only be performed on isolated colonies. A large panel ofsera against all serotypes is needed. Moreover, non-specificcross-reactions occur among different serotypes. Rapidand sensitive PCR assays specific for serotypes 1 (and 14), 2(and 1/2), 7 and 9 have been described. These assays werebased on serotype-specific genes in the capsular poly-saccharides synthesis locus (cps) locus (Smith et al.,1999a,b). Until now no rapid and sensitive diagnosticmethod is available to identify other S. suis serotypes. Herewe amplified, sequenced and characterized the cps locus ofS. suis serotype 16. We also describe the development of aserotype-specific PCR test for the rapid detection to S. suis

serotype 16.

T I C L E I N F O

le history:

ived 12 April 2011

ived in revised form 19 May 2011

pted 25 May 2011

ords:

ptococcus suis

type 16

sular polysaccharides

assay

A B S T R A C T

Streptococcus suis serotype 16 can infect pigs and humans. We describe the identification

and the characterization of the capsular polysaccharides synthesis locus of S. suis serotype

16. Using PCR primers flanking the capsular polysaccharides synthesis locus, a 30,101-bp

fragment was amplified. Twenty-nine open reading frames related to transcriptional

regulation, glycosyl transfer, oligosaccharide repeat unit polymerization, polysaccharide

transport, sialic acid synthesis and modification were identified. The data suggests that the

serotype 16 capsule is synthesized by a Wzy-dependent pathway. So far, no rapid and

sensitive diagnostic method is available for detection of serotype 16 isolates. A serotype

specific PCR test for the rapid and sensitive detection of S. suis serotype 16 was developed.

Cross hybridization experiments of individual cps genes with chromosomal DNAs of 33

serotypes showed that the cps16G and cps16K genes hybridized with serotype 16 only.

Primers based on cps16G were used to develop a serotype 16 specific PCR. The PCR assay

was successfully used to identify S. suis serotype 16 in the 99 Chinese S. suis clinical isolates

and 8 European isolates.

� 2011 Elsevier B.V. All rights reserved.

Corresponding author at: College of Veterinary Medicine, Nanjing

cultural University, Nanjing 210095, China.

+86 02584396517; fax: +86 02584396517.

E-mail address: lucp@njau.edu.cn (C. Lu).

Contents lists available at ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter � 2011 Elsevier B.V. All rights reserved.

10.1016/j.vetmic.2011.05.050

K. Wang et al. / Veterinary Microbiology 153 (2011) 403–406404

2. Materials and methods

2.1. Bacterial strains and growth conditions

S. suis reference strains (serotypes 1–31, 33 and 1/2)were obtained from M. Gottschalk (Faculte de MedicineVeterinaire, Universite de Montreal, Saint-Hyacinthe, Que.,Canada). Genomic DNA of 8 European S. suis serotype 16isolates was obtained from Henk Wisselink (Department ofBacteriology, Central Veterinary Institute of WageningenUR, Lelystad, The Netherlands). Ninety-nine Chinese S. suis

isolates were collected from the tonsils of pigs between2007 and 2010 (Table 1). S. suis strains were grown inTodd-Hewitt broth (code CM189, Oxoid) and plated onColumbia agar blood base (code CM331, Oxoid) containing6% (v/v) sheep blood overnight. Escherichia coli (E. coli)strains were grown in Luria broth and plated on Luria brothcontaining 1.5% (w/v) agar. If required, 50 mg/ml ofampicillin was added to the broth and plates.

2.2. DNA techniques

Routine DNA manipulations, blotting and hybridizationreactions were performed as described by Sambrook(2001). Genomic DNA of bacterial strains was isolatedand purified by Wizard Genomic DNA Purification Kit(Promega, USA).

2.3. Cloning and analysis of cps locus

The cps locus of the S. suis serotype 16 reference strain(2726) (Gottschalk et al., 1989) was amplified by LA-Taq(Takara, Japan), using the primers: P1(50-atgtgatggaag-catctaagtctgc), P2(50-taacccctgtgataagtctgtcgtc), P3(50-aagtttatcctgaaatacgattgtgctatggtgc) and P4(50-cttgaaa-tatctcctggaactgtaatctcttggg). The PCR fragment amplifiedby P1 and P2 was cloned into pCR-XL-TOPO vector (TOPOXL PCR Cloning Kit, invitrogen) and transformed to TOP10chemically competent E. coli cells (Invitrogen). Clones weresequenced by primer walking from each end using Big-Dyeterminator chemistry on an ABI3730 sequencing machine.The PCR fragment amplified by P3 and P4 was used directlyto construct small-insert libraries (McMurray et al., 1998),with 2- to 3-kb inserts in pUC-18. Clones from this librarywere sequenced from each end using Big-Dye terminatorchemistry (Applied Biosystems), to give a 6- to 8-foldcoverage on average. Sequence data of the two fragmentswere assembled and analyzed using the software programVector NTI. Putative promoter and terminator sequenceswere predicted by BPROM and FindTerm program onhttp://www.softberry.ru/berry.phtml, respectively.

2.4. Screening of serotype-specific gene

Cross-hybridization experiments were performed toexamine the homology between the cps16 genes and genesof all other S. suis serotypes. DNA fragments of cps16E-16Wwere amplified by PCR, labeled with 32P using DIG HighPrime DNA Labeling and Detection Starter Kit I (RocheApplied Science, Germany), and hybridized to chromoso- le

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K. Wang et al. / Veterinary Microbiology 153 (2011) 403–406 405

mbrook, 2001). As a positive control we used a probecific for S. suis 16S rRNA. The DNA on the blots wasridized at 65 8C with appropriate DNA probes. Afterridization, the membranes were washed twice with ation of 40 mM sodium phosphate (pH 7.2)–1 mMA–5% sodium dodecyl sulfate (SDS) for 30 min at 65 8C

twice with a solution of 40 mM sodium phosphate (pH)–1 mM EDTA–1% SDS for 30 min at 65 8C.

Development and application of PCR detection method

Primers (cps16G-1 and cps16G-2) used for serotype 16ection correspond to the positions 8537–8556 and5–9014 in the S. suis cps16 locus. The sequences weretgatttttgtaactgtagg and 50-ccagcttttctatttctttc, respec-ly. The reaction mixture (25 ml) contained 0.25 ml Taq

A polymerase (TaKaRa 5U/ml), 2.5 ml 10� PCR buffer0 mM Tris–HCl, pH 8.3, 500 mM KCl and 15 mM MgCl2),l dNTP mixture (0.25 mM each of the four deoxynu-tide triphosphates), 0.5 ml cps16G-1 (10 pM), 0.5 ml

16G-2 (10 pM) and 0.5 ml of cultured isolate (or 1 mg/genomic DNA). DNA amplification was carried out in akin Elmer 9600 thermal cycler and the programsisted of the incubation for 5 min at 94 8C and 30les of 30 s at 94 8C, 30 s at 43 8C and 30 s at 72 8C. Toluate the sensitivity of the PCR assay serial dilutions

107 CFU/ml to 10 CFU/ml) of cultured S. suis serotypereference strain (2726) were used in the PCR.

esults

Identification and characterization of S. suis serotype 16

cluster

The sequences obtained were assembled to a 30,101-bpment (GenBank Accession Number: HQ694980).mination of the compiled sequence revealed thesence of 31 potential open reading frames (ORFs),oding the entire cps16 locus as well as two flankinges (orf16Z and aroA). Each ORF is preceded by asome-binding site, and the majority of the ORFs were

y closely linked. The orfs of serotype 16 cps locus wereignated as orf16Y, orf16X, cps16A through cps16W and1 to tnp4, according to the polysaccharide gene

enclature of S. suis serotype 2 (Smith et al., 2000).pared to the other orfs, orf16Y was expressed in the

osite orientation. One potential promoter sequences identified (�10 box at positions 2162–2170, �35 boxositions 2141–2146). The putative transcriptional start

was located at position 2177. A putative terminatoruence forming a classical stem-loop structure wasted at positions 29078–29136. Blast analysis revealed

t the proteins expressed by orf16Y, orf16X and cps16Aough cps16E (positions 1–8076), cps16L and cps16Q-16V (sialic acid genes) showed homology to theteins translated from S. suis serotype 2 cps genes. Theels of homology differed considerably among theious proteins (62% identity for CPS16L and CPS16Q to

identity for ORF16Y). But the identities of cps16E,16L, cps16Q-cps16V and the genes in the serotype 2 cps

s are very low. So there was no positive reaction

between some cps2 genes (cps2E, sialic acid genes) andserotype 16 reference strain in previous hybridization data(Smith et al., 1999b, 2000). The protein CPS16W is similarto the UDP-galactopyranose mutase (positions 590317–591429) outside the cps locus of S. suis P1/7. CPS16G,CPS16I-K and CPS16M-N proteins showed homology toproteins involved in capsular synthesis of Streptococcus

thermophiles or Streptococcus agalactiae, whereas CPS16H,and CPS16P showed homology to hypothetical proteins innon-streptoccocal species. CPS16O did not show homologyto any of the proteins present in the data libraries.

3.2. Serotype 16-specific cps genes

The 16S rRNA probe hybridized with almost equalintensities to all serotypes tested. Most cps16 geneshybridized with the genomic DNAs of serotype 16 as wellas to a selected number of other serotypes (serotypes 5, 6,8, 15, 17, 20 and 25–28). None of the cps16 genes usedhybridized to all S. suis serotypes. The cps16G and cps16Kgenes hybridized with the genomic DNA of serotypes 16only. These data clearly indicate that the cps16G andcps16K genes are specific for serotype 16. Remarkably, aconsiderably number of the cps16 genes (cps16L-cps16W)hybridized to serotype 6 as well.

3.3. Serotype-specific PCR

We selected oligonucleotide primers within the cps16Ggene to generate a serovar 16-specific amplicon of 487 bp.The results show that the serotype 16 reference strain(2726) was positive in PCR, whereas all other S. suis

serotypes remained negative (Fig. 1A). In addition to thereference strains, 8 European S. suis serotype 16 strainswere included into the analyses. A fragment of approxi-mately 500 bp was amplified from all serotype 16 strainsstudied (Fig. 1B). The sensitivity of the PCR assay was atleast 5 CFU (Fig. 1C). Among the 99 Chinese isolates, 5isolates which were identified as serotype 16 by serumagglutination, were positive in the PCR assay (selectionshown in Fig. 1D).

4. Discussion

In the present paper, we describe the identification andcharacterization of the cps genes specific for S. suis serotype16. The cps locus revealed 29 putative ORF. According tothe genes involved in capsular synthesis of S. suis serotype16, its capsule is synthesized by Wzy(oligosacchariderepeat unit polymerase)-dependent mechanism (Guoet al., 2008), in which individual repeat units assembledon undecaprenyl phosphate on the inner face of thebacterial membrane are polymerized on the outermembrane surface. The Wzy-dependent pathway is alsofound in several other streptococcal species, such as S.

agalactiae and Streptococcus pneumoniae (Llull et al., 2001).Cross-hybridization experiments with the individual

cps16 genes as probes showed that cps16G and cps16Kspecifically hybridized with serotype 16 strain. Based onthese data, the cps16G gene was selected to develop rapidand sensitive PCR. The PCR assay confirmed the traditional

K. Wang et al. / Veterinary Microbiology 153 (2011) 403–406406

serum agglutination results when used on the referencestrain, as well as on clinical isolates. The serotype 16-specificPCR assay is more rapid and sensitive than regularbacteriological examination. It is able to detect 5 CFU strainsin one reaction at least. All cps16 probes except 16F, G, H, andK hybridized to serotype 6. These data can explain the two-way cross-agglutination observed between serotype 6 and16 isolates (Higgins and Gottschalk, 1990). Smith et al.(1999b) showed that the serotypes 1, 2 and 9-specifc PCRassays could easily be performed on clinical samples withoutprior isolation. Although this has not been tested yet, similarresults are expected for the serotype 16-specifc PCR test. Ifso, this assay will be an important diagnostic tool to detectpigs or human carrying serotype 16 strains.

Acknowledgement

This work was supported by Special Fund for PublicWelfare Industry of Chinese Ministry of Agriculture(200803016).

References

Gottschalk, M., Higgins, R., Jacques, M., Mittal, K.R., Henrichsen, J., 1989.Description of 14 new capsular types of Streptococcus suis. J. Clin.Microbiol. 27, 2633–2636.

Guo, H., Yi, W., Song, J.K., Wang, P.G., 2008. Current understanding onbiosynthesis of microbial polysaccharides. Curr. Top. Med. Chem. 8,141–151.

Han, D.U., Choi, C., Ham, H.J., Jung, J.H., Cho, W.S., Kim, J., Higgins, R., Chae,C., 2001. Prevalence, capsular type and antimicrobial susceptibility of

Streptococcus suis isolated from slaughter pigs in Korea. Can. J. Vet.Res. 65, 151–155.

Higgins, R., Gottschalk, M., 1990. An update on Streptococcus suis identi-fication. J. Vet. Diagn. Invest. 2, 249–252.

Higgins, R., Gottschalk, M., Fecteau, G., Sauvageau, R., De Guise, S., DuTremblay, D., 1990. Quebec. Isolation of Streptococcus suis fromcattle. Can. Vet. J. 31, 529.

Llull, D., Lopez, R., Garcia, E., 2001. Genetic bases and medical relevance ofcapsular polysaccharide biosynthesis in pathogenic streptococci.Curr. Mol. Med. 1, 475–491.

Lun, Z.R., Wang, Q.P., Chen, X.G., Li, A.X., Zhu, X.Q., 2007. Streptococcus suis:an emerging zoonotic pathogen. Lancet Infect. Dis. 7, 201–209.

McMurray, A.A., Sulston, J.E., Quail, M.A., 1998. Short-insert libraries as amethod of problem solving in genome sequencing. Genome Res. 8,562–566.

Messier, S., Lacouture, S., Gottschalk, M., 2008. Distribution of Strepto-coccus suis capsular types from 2001 to 2007. Can. Vet. J. 49,461–462.

Nghia, H.D., Hoa, N.T., Linh le, D., Campbell, J., Diep, T.S., Chau, N.V., Mai, N.T.,Hien, T.T., Spratt, B., Farrar, J., Schultsz, C., 2008. Human case ofStreptococcus suis serotype 16 infection. Emerg. Infect. Dis. 14, 155–157.

Sambrook, J.R.D.W., 2001. Molecular Cloning: A laboratory Manual, 3rded. Cold Spring Harbor Laboratory Press.

Smith, H.E., van Bruijnsvoort, L., Buijs, H., Wisselink, H.J., Smits, M.A.,1999a. Rapid PCR test for Streptococcus suis serotype 7. FEMS Micro-biol. Lett. 178, 265–270.

Smith, H.E., Veenbergen, V., van der Velde, J., Damman, M., Wisselink, H.J.,Smits, M.A., 1999b. The cps genes of Streptococcus suis serotypes 1, 2,and 9: development of rapid serotype-specific PCR assays. J. Clin.Microbiol. 37, 3146–3152.

Smith, H.E., de Vries, R., van’t Slot, R., Smits, M.A., 2000. The cps locus ofStreptococcus suis serotype 2: genetic determinant for the synthesis ofsialic acid. Microb. Pathogenesis 29, 127–134.

Wisselink, H.J., Smith, H.E., Stockhofe-Zurwieden, N., Peperkamp, K.,Vecht, U., 2000. Distribution of capsular types and production ofmuramidase-released protein (MRP) and extracellular factor (EF) ofStreptococcus suis strains isolated from diseased pigs in seven Eur-opean countries. Vet. Microbiol. 74, 237–248.

Fig. 1. (A) Ethidium bromide-stained agarose gel showing PCR products obtained with chromosomal DNA of S. suis reference strains. 1–31: S. suis serotypes

1–31; 32: S. suis serotype 33; 33: S. suis serotype 1/2; M: molecular size marker (DL2,000 DNA Marker, Takara). (B) PCR detection results to the European

serotype 16 strains. 1: 2921; 2: 5686; 3: 7125; 4: 6228; 5: 6509; 6: 2846; 7: 1061; 8: 3520; 9: reference strain 2726; 10: negative control, no DNA present;

M: molecular size marker (DL2,000 DNA Marker, Takara). (C) Sensitivity of the PCR assay. 1: 5 � 103 CFU; 2: 5 � 102 CFU; 3: 5 � 101 CFU; 4: 5CFU; 5:

5 � 10�1 CFU; 6: 5 � 10�2 CFU; 7: 5 � 10�3 CFU; M: molecular size marker (DL2,000 DNA Marker, Takara). (D) PCR detection results to some Chinese

isolates. 1: HeN5 (serotype 1); 2: GX19 (serotype 2); 3: GX24 (serotype 3); 4: 090318 (serotype 5); 5: FJ110 (serotype 6); 6: YN13B (serotype 7); 7: 090843

(serotype 8); 8: TL1 (serotype 9); 9: SH13 (serotype 10); 10: NP40 (serotype 12); 11: SPB31 (serotype 15); 12: TL41 (serotype 16); 13: FL38 (serotype 16);

14: TL13 (serotype 16); 15: DDK10 (serotype 16); 16: 451 (serotype 16); 17: 090202 (serotype 20); 18: HT50 (serotype 24); 19: 090416 (serotype 29); 20:

07-SS-RG01 (serotype 30); 21: FL42 (serotype 1/2); 22: RC24 (untypable by serum agglutination); 23: serotype 16 reference strain 2726; 24: negative

control, no DNA present; M: molecular size marker (DL2,000 DNA Marker, Takara).