Comparative genomics of Streptococcus parauberis: new target for molecular identification of serotype III

Abstract

This paper describes the predicted structure for the cps loci involved in capsule biosynthesis for Streptococcus parauberis serotypes III, IV, and V. Based on the specific serotype regions I, II, and III, a multiplex PCR protocol (mPCR) was designed to differentiate the main serotypes causing fish diseases. A real-time PCR method (qPCR) is also described to identify S. parauberis of serotype III in bacterial cultures and fish tissues. In silico and in vitro analyses revealed that both methods have a 100% specificity. The mPCR assay was optimized for the detection of S. parauberis strains of subtypes Ia (amplicon size 213 bp), subtypes Ib and Ic (both amplicon size 303 bp), serotype II (amplicon size 403 bp), and serotype III (amplicon size 130 bp) from bacterial cultures. The qPCR assay was optimized for the identification and quantification of S. parauberis serotype III strains in bacterial cultures and fish tissues. This assay achieved a sensitivity of 2.67 × 10² gene copies (equivalent to 3.8 × 10^-9 ng/μl) using pure bacterial cultures of S. parauberis serotype III and 1.76 × 10² gene copies in fish tissues experimentally and naturally infected with S. parauberis of the serotype III. The specificity and sensitivity of the protocols described in this study suggest that these methods could be used for diagnostic and/or epidemiological purposes in clinical diagnostic laboratories. KEY POINTS: • Structure of loci cps for S. parauberis of serotypes III, IV and V was described. • mPCR to differentiate S. parauberis serotypes causing disease in fish was optimized. • qPCR assay to quantify strains of S. parauberis serotype III in fish tissues.

Keywords: Genomic analyses; IV and V; Real-time PCR; Serotypes III; Streptococcus parauberis; Typing.

Fig. 1

Structure of loci cps of S. parauberis detected in this study. In black are represented conserved regions of the cps locus of S. parauberis, in gray are represented genes that are present in two or more serotypes, in white are represented serotype-specific regions

Fig. 2

Multiplex PCR products of S. parauberis strains. Lanes: MM, Generuler DNA ladder 1 kb (Fermentas); 1, (subserotype Ia); 2, subserotype Ib; 3, subserotype Ic; 4, serotype II; 5, serotype III; 6 and 7, non-typeable strains; 8, NCDO2020 (serotype IV)

Fig. 3

Melting curve analysis using qPCR and SP3-130F and SP3-130R primers, showing a specific melting peak at Tm 73 ± 0.40 °C using bacterial DNA from strains of Streptococcus parauberis serotype III (n = 11 strains) and the lack of amplification obtained using DNA from strains of other serotypes of S. parauberis or unrelated bacteria and negative control

Fig. 4

Standard curves obtained by qPCR from the amplification of 10-fold dilutions of purified amplicon obtained using SP3-130F and SP3-130R primers and DNA from the reference strain S. parauberis NCIMB 703043 (black circles) and a clinical isolate of S. parauberis (SK451/04) (gray squares) (a) and the corresponding electrophoresis gel showing the band intensities correlating with the number of copies of the cps3K gene (b). Lanes: MM, Generuler DNA ladder 1-kb (Fermentas); 1, 2.67 × 10⁸ copies; 2, 2.67 × 10⁷ copies; 3, 2.67 × 10⁶ copies; 4, 2.67 × 10⁵ copies; 5, 2.67 × 10⁴ copies; 6, 2.67 × 10³ copies; 7, 2.67 × 10² copies