Evaluation of PCR Methods for Rapid Identification and Differentiation of Streptococcus uberis and Streptococcus parauberis

Abstract

Streptococcus uberis and Streptococcus parauberis reference strains and isolates obtained from routine diagnostics were investigated by PCR with oligonucleotide primers designed according to species-specific parts of the 16S rRNA gene, the 23S rRNA gene, and the 16S-23S rRNA intergenic spacer region of both species. All three primer pairs allowed an identification of 67 isolates as S. uberis and 4 isolates as S. parauberis.The use of PCR as a diagnostic tool for the detection of bacterial pathogens has become more frequent during the past few years. The PCR technique allows the amplification of preselected, species-specific DNA regions which can be used for genotypic identification of bacteria. A molecule most suited for these purposes appears to be the gene encoding the 16S rRNA. According to Bentley et al. (4) and Bentley and Leigh (6), the sequence variability of the V2 region of the 16S rRNA allows a differentiation of 31 species of the genus Streptococcus, including the species S. uberis and S. parauberis. Both species, formerly classified as S. uberis types I and II (19), are well known as causative agents of bovine mastitis. According to biochemical and serological characteristics, the two species are almost indistinguishable (19). A molecular identification of both species can be performed by analysis of restriction fragment length polymorphisms (RFLPs) of the 16S rRNA gene (13,14,18) or by the use of species-specific oligonucleotide probes (5, 6). A major disadvantage of these procedures is the additional manipulation of the samples subsequent to the PCR and the time-consuming preparation of the gene probes. In the present study, species-specific regions of the 16S and 23S rRNA genes and the 16S-23S rRNA intergenic spacer region of S. uberis and S. parauberis were used to construct speciesspecific oligonucleotide primers. These oligonucleotide primers were used to identify and differentiate both species.A total of 17 S. uberis and 2 S. parauberis strains were used in this study, all of which were obtained from the Institute's strain collection. The cultures were identified and differentiated into both species as described previously (15, 18). In addition, 51 streptococci obtained from routine mastitis diagnostics and also S. parauberis strain 94/16 were included. The latter, kindly obtained from J. F. Fernández-Garayzábal (Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain), was originally isolated from a diseased turbot (9). The cultures isolated from routine diagnostics were preliminarily identified as S. uberis by using conventional methods (15, 16). These methods included carbohydrate fermentation tests for arabinose, fructose, inulin, lactose maltose, mannitol, raffinose, ribose, saccharose, salicin, sorbitol, and trehalose; esculin and sodium hippurate hydrolysis; determination of arginine hydrolysis; and analysis of the enzymes ␤-D-glucuronidase, pyrrolydonylaminopeptidase, and hyaluronidase. The ␤-D-glucuronidase and pyrrolydonylaminopeptidase enzyme activities were inves...

Virulence-associated genes and molecular typing of Streptococcus uberisassociated with bovine mastitis in northern Thailand

“…However, an additional subsequent technique is usually required to confirm its identification. We used a species- This technique can rapidly provide unambiguous results to detect S. uberis and can distinguish S. uberis from another phenotypically identical species, S. parauberis (20). This PCR protocol has been widely used to identify S. uberis isolated from mastitis cases prior to further genotyping methods (25,26).…”

“…All streptococcal isolates were identified using conventional PCR, which is specific to amplify a region in the 16S rRNA gene of S. uberis (20) …”

Genetic diversity and virulence genes in Streptococcus uberis strains isolated from bovine mastitis

“…This involved performing S. uberis-specific PCR according to Hassan et al (2001). The strains confirmed to be S. uberis by genotyping were screened for the most relevant virulence genes (pauA, skc, and sua) using primers described by Luther et al (2008), Johnsen et al (1999) and Rosey et al (1999) (Table 1).…”

Molecular Characterization of 16S rRNA and Internal Transcribed Spacer (ITS) Regions of Aeromonas spp. Isolated from Cultured Freshwater Fishes in Malaysia

“…Kong et al, (1999) also have stated that in his study that, the highest similarity was observed amongst three Aeromonas hydrophila (95 % -99 %). This can be refer to the Berridge et al (2001) and Hassan et al (2001), the ITS region has been useful to identified the species level as in instance relative genus analysis of the Streptococcus and Staphylococcus.…”