Multilocus sequence analysis of diverse Streptococcus iniae isolates indicates an underlying genetic basis for phenotypic heterogeneity

Abstract

Streptococcus iniae is a Gram-positive, opportunistically zoonotic bacterium infective to a wide variety of farmed and wild fish species worldwide. Outbreaks in wild fish can have detrimental environmental and cultural impacts, and mortality events in aquaculture can result in significant economic losses. As an emerging or re-emerging pathogen of global significance, understanding the coalescing factors contributing to piscine streptococcosis is crucial for developing strategies to control infections. Intraspecific antigenic and genetic variability of S. iniae has made development of autogenous vaccines a challenge, particularly where the diversity of locally endemic S. iniae strains is unknown. This study genetically and phenotypically characterized 11 S. iniae isolates from diseased wild and farmed fish from North America, Central America, and the Caribbean. A multilocus sequence analysis (MLSA) scheme was developed to phylogenetically compare these isolates to 84 other strains of Streptococcus spp. relevant to aquaculture. MLSA generated phylogenies comparable to established genotyping methods, and isolates formed distinct clades related to phenotype and host species. The endothelial Oreochromis mossambicus bulbus arteriosus cell line and whole blood from rainbow trout Oncorhynchus mykiss, Nile tilapia Oreochromis niloticus, and white sturgeon Acipenser transmontanus were used to investigate the persistence and virulence of the 11 isolates using in vitro assays. In vivo challenges using an O. niloticus model were used to evaluate virulence by the intragastric route of infection. Isolates showed significant differences (p < 0.05) in virulence and persistence, with some correlation to genogroup, establishing a basis for further work uncovering genetic factors leading to increased pathogenicity.

Isolation and characterization ofLactococcus garvieaefrom rainbow trout, Onchorhyncus mykiss, from California, USA

“…The 16S rRNA gene primers and three of the nine primer sets designed for S. iniae and other closely related Streptococcus species (Heckman et al., 2020) amplified corresponding target regions in L. garvieae . The L. garvieae isolates from California had 100% pairwise identity among these housekeeping gene fragments, while heterogeneity existed between the Californian isolates and chosen reference genomes (97% pairwise identity).…”

“…Purified gDNA, as described in the REP‐PCR ( n = 12), was used for PCR amplification and sequencing of three housekeeping genes, gyrB, pheT , and rpoB , following procedures adapted from Heckman et al. (2020). Briefly, PCR of the relevant gene fragments was carried out using Phusion high‐fidelity DNA polymerase (Thermo Fischer Scientific, USA) following manufacturer's recommendations with 3% added DMSO (Thermo Fischer Scientific, USA), and annealing temperatures 6°C below the suggested melting temperature for each primer set (Table 2).…”

“…Purified gDNA, as described in the REP-PCR (n = 12), was used for PCR amplification and sequencing of three housekeeping genes, gyrB, pheT, and rpoB, following procedures adapted from Heckman et al (2020).…”

Streptococcus dysgalactiae: A Pathogen of Feral Populations of Silver Carp from a Fish Kill Event

“…Amplification and sequencing of nine housekeeping genes (arcC, glnA, groEL, gyrB, mutS, pheT, prkC, rpoB, and tkt) followed the procedures of Heckman et al (2020). Amplification reactions (50 µL) consisted of 25 µL of Phusion Green Hot Start II High-Fidelity PCR Master COMMUNICATION Mix (Thermo Scientific), 20 pmol of each respective primer, approximately 50 ng of genomic DNA, and nucleasefree water to volume.…”

“…within a discrete, well-supported phyletic clade that included three other S. dysgalactiae isolates from fish (S. dysgalactiae ssp. dysgalactiae STC3 [Heckman et al 2020]; S. dysgalactiae ssp. dysgalactiae STREP97-15 [GenBank, unpublished]; and S. dysgalactiae Kdys0611 [Nishiki et al 2019]).…”