Development of PCR for identifying Streptococcus parasuis, a close relative of Streptococcus suis.

Streptococcus parasuis has recently been removed taxonomically from Streptococcus suis, a zoonotic pathogen. S. parasuis has been detected in healthy pigs and in diseased pigs, which suggests that S. parasuis is involved in the normal microbiota of pigs and has potential pathogenicity. However, the pathogenicity of S. parasuis in pigs is unclear because of the lack of appropriate detection methods that discriminate S. parasuis from S. suis. In this study, we developed a PCR method that is specific for S. parasuis. The detection limit of the PCR was 350 CFU per reaction. Bacteria isolated from the saliva of eight pigs were collected and examined by PCR. Sixty-four isolates positive for PCR were obtained from the samples of all pigs. Thirteen of the 64 isolates were genetically confirmed as S. parasuis, and biologically and biochemically had nearly the same features of known S. parasuis strains, which suggested that strains positive for PCR were S. parasuis. Among the 64 isolates, 28 isolates were serotypes 20, 22, or 26 in the S. suis serotyping scheme. The remaining 36 isolates were untypeable, which suggested the presence of novel serotypes or a capsule-negative form. Therefore, the PCR method described in this study is a useful tool for identifying S. parasuis, and can be used in etiological studies on this bacterium.

Development of an appropriate PCR system for the reclassification of Streptococcus suis.

Thirty-five serotypes of Streptococcus suis (serotypes 1-34 and serotype 1/2) have so far been described on the basis of their polysaccharide capsular antigens. However, in the last decade, some serotype reference strains have been reexamined for their taxonomic status, and the reference strains of serotypes 20, 22, 26, 32, 33, and 34 may be different from taxon S. suis. In the present study, we developed a novel PCR method targeting the recombination/repair protein (recN) gene of S. suis, designated recN PCR, which corresponds to the current reclassification of this bacterium. We compared its specificity with other PCR methods for S. suis, and the results obtained confirmed its specificity. In addition, the detection limits of recN PCR were similar among all the reference strains of authentic S. suis, indicating that the recN PCR gave reliable results against bacterial strains and isolates used in this study. Therefore, recN PCR described in the present study will be a useful tool for the identification of authentic S. suis, and can also be used in epidemiological studies on this bacterium.

Development of a multilocus sequence typing scheme for Streptococcus gallolyticus.

Streptococcus gallolyticus is often found as a member of the normal gut microflora in various animals. However, it has been reported to cause mastitis in cattle, septicaemia in pigeons, and meningitis, septicaemia and endocarditis in humans. However, little is known about the epidemiology and crucial virulence factors of S. gallolyticus. To help address these issues, we developed a multilocus sequence typing (MLST) scheme for S. gallolyticus. Seven housekeeping gene fragments were sequenced from each of 58 S. gallolyticus isolates collected from diverse origins and sources. The MLST scheme had good discriminatory ability. The 63 strains, including the 5 whole genome sequenced strains examined, resolved into 57 sequence types (STs), with 52 STs represented by only a single strain. With respect to the identification of S. gallolyticus subspecies (i.e. S. gallolyticus subsp. gallolyticus, S. gallolyticus subsp. pasteurianus and S. gallolyticus subsp. macedonicus), the results of biochemical tests and DNA-DNA hybridization were in high concordance with those of the MLST scheme. The MLST scheme developed in this study may be a useful tool capable of replacing the conventional methods used for S. gallolyticus subspecies identification. The results of this study suggest that the biology and virulence of two pathogenic S. gallolyticus subspecies (i.e. S. gallolyticus subsp. gallolyticus and S. gallolyticus subsp. pasteurianus) are very different. The MLST scheme offers researchers a valuable typing tool that will promote further investigation of the epidemiology of S. gallolyticus.

Antimicrobial susceptibility of Streptococcus gallolyticus isolated from humans and animals.

Susceptibilities to some antimicrobial agents and distribution of genes associated with resistance were examined in a total of 66 Streptococcus gallolyticus isolates and reference strains from various sources. All the tested bacteria were susceptible to vancomycin, penicillin G, and ampicillin. Most of the erythromycin-resistant isolates were observed in human clinical samples. Tetracycline and doxycycline resistance was prevalent in the isolates from human patients, diseased animals, and healthy broiler chickens, while the prevalence was significantly lower in the isolates from healthy mammals. All the isolates resistant to tetracycline possessed tet(M) and/or tet(L) and/or tet(O) genes. However, most isolates from healthy animals, which were susceptible to tetracycline, possessed the above-cited resistance genes, implying the potential ability for resistance under exposure to the corresponding antimicrobial agents.

Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22, 26, and 33 based on DNA-DNA homology and sodA and recN phylogenies.

To date, Streptococcus suis was divided into thirty-three serotypes based on its polysaccharide capsular antigens. Although 16S rRNA sequence similarities of serotypes 20, 22, 26, and 33 reference strains to the type strain NCTC 10234(T) were below the threshold value of 98.5% to assign them to S. suis species, no strong evidence support to reclassification. Here, their taxonomic identities were determined by DNA-DNA hybridization assays and by partial sequencing of the sodA and recN genes. Our results confirmed that the serotype 20, 22, 26, and 33 reference strains were distantly related to the type strain NCTC 10234(T) and the whole sequence strain P1/7 of S. suis. Moreover, the reference strains of serotypes 20, 22, and 26 were closely related to each other but distinct from the serotype 33 reference strain. Sequencing analyses of sodA and recN of a total 33 serotype reference strains showed that the serotype 20, 22, and 26 reference strains and the serotype 33 reference strain did not fall with not only other serotypes of S. suis, but also other streptococcal species (63 strains of 56 species for sodA and 87 strains of 55 species for recN). The evidence further substantiates the view that the reference strains of serotypes 20, 22, 26 and 33 should be taxonomically removed from S. suis, although their taxonomic designations and determinative phenotypic characteristics are yet to be addressed.

Phenotypic and PCR-based identification of bacterial strains isolated from patients with suspected streptococcus suis infection in northern Thailand.

Twenty bacterial strains isolated from the blood of patients with suspected Streptococcus suis infection based on clinical symptoms in northern Thailand between 2009 and 2010 were subjected to phenotypic and genotypic identification. Commercial identification kits and a PCR-based assay targeting the S. suis-specific 16S rDNA sequence correctly identified S. suis isolated from patients in northern Thailand; however, there was a risk of misidentifying S. gallolyticus as S. suis using a PCR assay targeting the S. suis-specific house keeping gene encoding glutamate dehydrogenase. This is the first paper to report S. gallolyticus infection in humans in Thailand.