Vol. 141: 53–69, 2020 DISEASES OF AQUATIC ORGANISMS Published online September 17 https://doi.org/10.3354/dao03521 Dis Aquat Org Multilocus sequence analysis of diverse Streptococcus iniae isolates indicates an underlying genetic basis for phenotypic heterogeneity Taylor I. Heckman1, Matt J. Griffin2, Alvin C. Camus3, Benjamin R. LaFrentz4, Danny Morick5, Rita Smirnov6, Tamir Ofek6, Esteban Soto1,* 1Aquatic Animal Health Laboratory, Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA 2Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Stoneville, MS 39762, USA 3College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA 4USDA-ARS, Aquatic Animal Health Research Unit, Auburn, AL 36832, USA 5Morris Kahn Marine Research Station, Department of Marine Biology, Leon H. Charney School of Marine Sciences, University of Haifa, Haifa 3498838, Israel 6Central Fish Health Laboratory, Department of Fisheries and Aquaculture, Ministry of Agriculture & Rural Development, Nir David 1080300, Israel 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 detri- mental environmental and cultural impacts, and mortality events in aquaculture can result in sig- nificant economic losses. As an emerging or re-emerging pathogen of global significance, under- standing 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 gen- erated 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 Oreo - chromis niloticus, and white sturgeon Acipenser transmontanus were used to investigate the per- sistence 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 signif- icant differences (p < 0.05) in virulence and persistence, with some correlation to genogroup, estab- lishing a basis for further work uncovering genetic factors leading to increased pathogenicity. KEY WORDS: Streptococcus iniae · Multilocus sequence analysis · MLSA · Streptococcosis · Aquaculture · Genotype Resale or republication not permitted without written consent of the publisher 1. INTRODUCTION pected to approach 10 billion by 2050 (United Na tions 2019). Infectious diseases such as strepto coc cosis Aquaculture is the world’s fastest growing food pro- have substantial economic impacts on the industry duction sector and plays a critical role in providing through diseases outbreaks, treatment ex pen ditures, livelihoods and protein sources for a population ex - and other production losses (Lucas et al. 2012, FAO *Corresponding author: [email protected] © Inter-Research 2020 · www.int-res.com 54 Dis Aquat Org 141: 53–69, 2020 2018). A major etiologic agent of pis cine streptococco- sequence analysis (MLSA) identifies variation in sis, Streptococcus iniae, is estimated to cost the global housekeeping genes in a reproducible and dissem- aquaculture industry over 100 million US$ annually inable manner. MLSA has been used successfully to (Shoemaker et al. 2001), and the number of hosts, genetically characterize a number of bacterial species habitats, and countries impacted continues to expand. (Martens et al. 2008, Macheras et al. 2011, Glaeser & Infection by S. iniae is multisystemic, but is commonly Kämpfer 2015, Whatmore et al. 2016, Liu et al. 2017), associated with menin gitis, pano phthalmitis, and sep- and publicly accessible databases facilitate inclusion ticemia. Pathogenesis varies depending on the bacte- of MLSA and whole-genome data from isolates across rial strain, host species, and environmental conditions the globe (Peacock et al. 2002, Patchanee et al. 2012, (Agnew & Barnes 2007). The host range of S. iniae in- Glaeser & Kämpfer 2015). However, no MLSA scheme cludes over 30 species of fresh, euryhaline, and salt- has been established for S. iniae. Herein, isolates pre- water fish (Agnew & Barnes 2007, Silayeva et al. viously typed by Rep-PCR (Soto et al. 2017a, T. I. 2020). Outbreaks with high mortalities occur in com- Heckman et al. unpubl. data) were used to evaluate mercially valuable fish, in cluding tilapia Oreochromis an MLSA method for S. iniae. The MLSA was de- spp. (Perera et al. 1994, Ortega et al. 2018), rainbow signed to be inclusive of other piscine streptococcal trout Oncorhynchus mykiss (Eldar et al. 1994, Erfan- pathogens, giving it broader relevance to fish health manesh et al. 2012), white sturgeon Acipenser trans- research and diagnostics. To further characterize montanus (Soto et al. 2017a), and others. Ornamental these isolates and improve laboratory techniques species (Russo et al. 2006), wild marine fish (Eldar et used to assess S. iniae pathogenicity, in vitro and in al. 1999, Fer guson et al. 2000, Keirstead et al. 2014, vivo assays were employed to assess isolate virulence Berzak et al. 2019), and mammalian species including in relevant fish hosts. Through the in vivo assay, this humans (Weinstein et al. 1997, Koh et al. 2004, 2009, study also validates a biologically relevant intra - Facklam et al. 2005, Lau et al. 2006, Sun et al. 2007) gastric gavage challenge model for S. iniae infections. can also be affected. As such, S. iniae is a pathogen of concern in the fields of aquaculture, conservation, and animal health. Disease transmission be tween 2. MATERIALS AND METHODS farmed and wild fish has been implicated in several outbreaks (Zlotkin et al. 1998, Colorni et al. 2002, Bro- 2.1. Bacteria mage & Owens 2002, Berzak et al. 2019), illustrating the need for a better appreciation of the disease and A total of 11 clinical isolates of Streptococcus iniae its transmission directly between hosts and indirectly from 5 wild and farmed fish species from North in the environment. Despite this, the pathogenic America, Central America, and the Caribbean were mechanisms used by S. iniae and its epidemiology re- used in all aspects of this study; 35 additional S. iniae main incompletely understood, and vaccination efforts isolates from piscine and mammalian sources, as well have been met with variable success (Bachrach et al. as representative S. agalactiae, S. dysgalactiae and 2001, Creeper & Buller 2006, Agnew & Barnes 2007, S. icta luri isolates, were included for the MLSA Eyngor et al. 2008). A contributing factor to these dif- (Table 1). Isolates were stored in 1 ml aliquots in ficulties is our incomplete knowledge of the antigenic brain heart infusion broth (BHI; MP Biomedicals) and genetic diversity of S. iniae, especially related to with 20% glycerol at −80°C. Before each assay, iso- relevant differences in pathogenesis between strains. lates revived from frozen stocks were grown at 30°C To this end, S. iniae collected from wild and for 24 h in BHI, with shaking, or for 48 h on trypticase cultured fish species in marine and freshwater envi- soy agar supplemented with 5% sheep’s blood (SBA; ronments across North America, Central America, University of California, Biological Media Services), and the Caribbean were genetically and phenotypi- unless otherwise noted. The 0.5 McFarland standard cally characterized. Genotyping schemes, such as (~1.5 × 108 CFU ml−1) used for the in vivo and in vitro repetitive sequence mediated fingerprinting (Rep- virulence assays corresponded to a bacterial suspen- PCR) and pulsed field gel electrophoresis (PFGE), sion in phosphate-buffered saline (PBS) with an opti- have been used for S. iniae typing (Weinstein et al. cal density measurement of 0.14−0.155 at 600 nm, 1997, Chou et al. 2014), but the portability of data read on a UV/Vis photometer (BioPhotometer Plus, from these techniques is cumbersome, results are of- Eppendorf AG). Isolates were phenotypically charac- ten poorly reproducible, and reliable comparisons re - terized using the API 20 STREP system following the quire isolates to be processed simultaneously. In con- manufacturer’s instructions and read at 48 h (bio- trast to these image-based approaches, multilocus Mérieux). Heckman et al.: Multilocus sequence analysis of Streptococcus iniae 55 Table 1. Streptococcus isolates used in this study. Isolates in bold were used for both multilocus sequence analysis (MLSA) typing and further phenotypic characterization Isolate Origin Geography Reference S. iniae ECO86-17 Spotted rose snapper Lutjanus guttatus Central America T. I. Heckman et al. unpubl. data S. iniae B8 Wild reef fish S. Caribbean Basin Soto et al. (2017a) S. iniae K08-409H Wild reef fish N. Caribbean Basin Soto et al. (2017a) S. iniae F15-4-3 Tilapia Oreochromis spp. California