article: In 2015, Singapore had the first and only reported foodborne outbreak of invasive disease caused by the group B Streptococcus (GBS; Streptococcus agalactiae). Disease, predominantly septic arthritis and meningitis, was associated with sequence type (ST) 283, acquired from eating raw farmed freshwater fish. Although GBS sepsis is well-described in neonates and older adults with co-morbidities, this outbreak affected non-pregnant and younger adults with fewer co-morbidities, suggesting greater virulence. Before 2015 ST283 had only been reported from twenty humans in Hong Kong and two in France, and from one fish in Thailand. We hypothesised that ST283 was causing region-wide infection in Southeast Asia. We performed a literature review, whole genome sequencing on 145 GBS isolates collected from six Southeast Asian countries, and phylogenetic analysis on 7,468 GBS sequences including 227 variants of ST283 from humans and animals. Although almost absent outside Asia, ST283 was found in all invasive Asian collections analysed, from 1995 to 2017. It accounted for 29/38 (76%) human isolates in Lao PDR, 102/139 (73%) in Thailand, 4/13 (31%) in Vietnam, and 167/739 (23%) in Singapore. ST283 and its variants were found in 62/62 (100%) tilapia from 14 outbreak sites in Malaysia and Vietnam, in seven fish species in Singapore markets, and a diseased frog in China. GBS ST283 is widespread in Southeast Asia, where it accounts for a large proportion of bacteraemic GBS, and causes disease and economic loss in aquaculture. If human ST283 is fishborne, as in the Singapore outbreak, then GBS sepsis in Thailand and Lao PDR is predominantly a foodborne disease. However, whether transmission is from aquaculture to humans, or vice versa, or involves an unidentified reservoir remains unknown. Creation of cross-border collaborations in human and animal health are needed to complete the epidemiological picture.
  In 2015, there was an unprecedented outbreak of invasive disease due to the group B Streptococcus (GBS; Streptococcus agalactiae) in Singapore. Disease, including septic arthritis and meningitis, was associated with a GBS belonging to serotype III, subtype 4 (serotype III-4), and multilocus sequence type (MLST) 283 (ST283) [1]. Human disease was associated with consumption of raw, farmed, freshwater fish: an official public advisory was issued, and bacteraemia rates promptly fell [2,3]. Whereas GBS sepsis in neonates, post-partum adults, and older adults with co-morbidities is well known, the ST283 outbreak was different as it affected non-pregnant, younger adults with fewer co-morbidities [1], suggesting greater virulence. Although GBS colonisation has previously been associated with fish consumption [4], and the origin of GBS in late onset neonatal disease, while uncertain, may be enteric [5,6], this was the first report of an invasive GBS outbreak associated with foodborne transmission. Previous reports of ST283 were limited to 20 cases in adults in Hong Kong between 1993 and 2003 [7], two cases in a survey of 119 osteoarticular GBS infections in France between 2002 and 2007 [8], and an infected tilapia (Oreochromis sp.) amongst samples collected in Thailand between 2000 and 2010 [9]. Southeast (SE) Asia was, however, under-represented in the literature, with MLST data reported for only a handful of human GBS from SE Asia amongst thousands in global studies. We hypothesised that fish-borne ST283 might be a regional problem, causing disease in multiple SE Asian countries. As a prelude to studying transmission, we searched for ST283 and its variants, collectively called clonal complex 283 (CC283), in GBS collections from humans and aquaculture. Our aim was achieved, as we verified the wide prevalence of ST283 in Southeast Asia, though whether human disease is fish-borne outside Singapore remains an open question.
  We identified articles in English with searches of Medline, PubMed, and references from articles, with the terms “Streptococcus agalactiae” OR “ST283” AND “meningitis” OR “invasive”, up to December 2017. Institutions, selected through personal contacts and recommendations, were invited to contribute GBS and/or datasets. There were no specified criteria other than the availability of isolates or DNA from invasive GBS, or GBS from high vaginal swabs, over time, with metadata if possible. Invasive disease was defined as isolation of GBS from normally sterile sites. Indications for testing patients and accompanying characterisation data were not standardised or complete; rates of meningitis, osteoarthritis, and endocarditis may therefore be greater than recorded. Country-specific regulations resulted in some contributors sending GBS isolates to Singapore for processing while others contributed extracted DNA, whole genome sequencing (WGS) data, or other data sets. GBS were screened with a ST283-specific PCR (Lezhava A. , Sarma S. , Chen S. and Barkham T. M. S. A method for the detection of Group B Streptococcus. (2017) PCT Patent Application PCT/SG2017/050579). This PCR has been evaluated against WGS on over 660 invasive GBS representing 27 MLST types collected over 18 years in Singapore and found to be 100% sensitive and 99. 8% specific. GBS identification, determination of serotype and WGS were performed as previously described [1]. All new sequencing data were deposited in GenBank under BioProject PRJNA293392. We expressed ST283 prevalence as simple proportions of all GBS in each collection (Fig 1).
  GBS ST283 accounted for 11% to 76% of invasive GBS per country, with the earliest known example collected in 1995, in Hong Kong. Five GBS ST11, single locus variants (SLV) of ST283, isolated from meningitis cases in Singapore in 1998 [28,29] were recently corrected to ST283 [30], leaving no known examples of ST11. In total, 29% of GBS were identified as ST283 (Fig 1, Table 2). The majority of patients with ST283 (345/357 (97%) were adults, and 36% to 80% did not have comorbidities. Meningitis, endocarditis, and septic arthritis were noted in 10% to 35%, 4. 5% to 10%, and 23% to 39% of adult patients with ST283, respectively (S1 Table). All newly sequenced GBS isolated from tilapia in Peninsular Malaysia and Vietnam, during outbreaks of streptococcosis, were CC283 (Fig 1, Table 3). Notably, ten of the 28 fish sampled in Malaysia did not have signs of disease. ST283 was previously reported from 17/57 (30%) GBS isolated from seven freshwater fish species, including tilapia, on sale for human consumption in Singapore in 2015: these 17 ST283 were found in only 6/586 (1%) fish from ports, but in 11/39 (28%) fish from markets [10]. Two previously reported ST283 were reported from farmed tilapia in Thailand between 2000 and 2014 [9,25]. One ST491 and one ST1311, (a new SLV of ST491, and also a double locus variant of ST283) were isolated from tilapia in Vietnam in 2006 [9] and 2016 respectively. An ST739, another SLV of ST283, was isolated in 2014 from a diseased tiger frog (Hoplobatrachus chinensis) farmed for human consumption in Guangdong, China, about 100Km from Hong Kong; although frogs are not piscine, they are often farmed in close proximity to fish and all known GBS infections in frogs are caused by clades that are also found in fishes [9]. Only four CC283, of human origin, were found: two from France, one from Britain, and one from the Netherlands (Fig 1, Table 4). All four were ST283 and none had associated epidemiological data. CC283 was otherwise absent from studies of over 4,000 human GBS from nine countries, of over 1,200 bovine GBS from five continents, and from the aquaculture literature from 6 continents, as of December 2017 (Fig 1 and S4 Table) [9,22,31–54]. Three human ST751, although an SLV of ST283 and ST10, were distinguished from ST283 by over 2,000 SNPs, based on phylogenetic analysis, and clustered outside of CC283, in CC10 (Fig 2), so ST751 is excluded from tables and figures referring to CC283. Other SLVs of ST283 listed in the MLST database are ST690, a human isolate, and ST160, of unknown origin, but we could not find WGS data for them. Three GBS serotype III-4 were not included as ST data were not available, although ST283 is the only published ST within serotype III-4: they were reported, without epidemiological data, amongst a collection from Australia and New Zealand in the original description of this subtype [55]. A previous report, using Bayesian analysis of a smaller subset of mostly Singaporean isolates, estimated the time of emergence of CC283 as 1994 (95% highest posterior density (HPD) 1991–1997) [1]. Analysis of our expanded set confirms that CC283 forms a monophyletic clade that appears to have arisen from within CC10 (Fig 2A). Bayesian analysis of our current set of CC283 and closely related CC10 strains suggests a slightly earlier predicted emergence date of 1985 (95% HPD 1980–1990). Given that the original Bayesian analysis was done on a data set that consisted largely of strains isolated from Singapore during 2015, the shift in the predicted emergence date is perhaps not surprising. However, our current data set was also not systematically collected, either by time, geography, or host, and these results should be interpreted in this context. Additional systematic sampling may provide further insights into emergence dates as well as the likely geographical and host origin of CC283. We found very little recombination in CC283 isolates, 62 kb (3. 0%) of the total chromosome, and little variation in genome content, as indicated in the phylogenetic tree (Fig 2) and single nucleotide polymorphism (SNP) distances between CC283 pairs (S5 Table).
  The phylogenetic tree shows that where human and fish GBS were collected concurrently, they are intermingled, whereas separate clustering of isolates of human and piscine origin tends to reflect collections being from different countries or time periods. We found numerous examples of pairs of human and piscine GBS distinguished by few to zero SNPs. Of note, while strict use of SNP distances is not fully reliable for determination of transmission, many of the examples have SNP distances smaller than 21 and are monophyletic in the ST283 tree. These criteria both support a possible “match”, as recently defined [59], although any SNP number cutoff is arbitrary, and the fewer the SNPs, the more likely is the linkage. GBS from the Singapore outbreak cluster with GBS from multiple countries, suggesting multiple sources contributed to the outbreak; although this may not seem surprising, given that Singapore imports fish from multiple countries, it leaves an unanswered question as to why imports from across the region suddenly, and simultaneously, were associated with increased GBS incidence in 2015. Of note, the 2015/2016 El Niño broke warming records in the central Pacific [60], and higher temperatures are associated with increased GBS load [61] and outbreaks of streptococcosis [62] in fish, and increased human cases [24]. GBS is known to affect fish species other than tilapia, including farmed and wild freshwater and marine fishes [63,64]. Our detection of CC283 in fish farms was from tilapia, which were preferentially sampled because they are the most commercially important species affected by GBS in SE Asia, but the involvement of other species remains uncertain. Although the outbreak in Singapore was linked to several freshwater fish species, it is unclear whether the fish left the farms as carriers, or were contaminated during transport and handling. Cross contamination and post-contamination bacterial amplification at ambient temperature might explain the data, from Singapore, that showed that ST283 was recovered from 1% of fish taken from ports, but 28% of fish from markets [10]. Fish account for up to 37% of protein consumed in SE Asia [65], and Lao PDR and Thailand are among the top ten global tilapia producers [56]. Tilapia were introduced to SE Asia in the 1940s with repeated importations of fish fry: if these fry were contaminated at source, this could explain the finding of ST283 across the region. Alternatively, ST283 may have evolved regionally, from other animals, and acquired the ability to infect fishes through lateral gene transfer of virulence elements from other piscine GBS, followed by onward transmission [66]. Interestingly, GBS transmission from a tilapia hatchery into a new farm was reported in Malaysia where, in an effort to prevent economic losses due to streptococcosis, farms were advised to source fry from disease-free hatcheries [67]; GBS from these outbreaks in 2007/2008 were included in our study and are ST283. However, a survey of invasive GBS isolated from 13 adults in Malaysia in 2010 did not find Serotype III GBS [68]; this was a small sample and the predominant Malay population do not habitually eat raw fish, perhaps explaining this lack of human ST283. There are a limited number of GBS clades that cause streptococcosis in fish, and ST283 and its variants are the only known serotype III GBS that naturally affect tilapia [9]. This suggests that ST283 caused streptococcosis in multiple sites across Thailand between 2003/2011, when 12% to 56% of GBS isolated from diseased tilapia were serotype III [69–71]; furthermore, some were serotype III-4, and the only described example of serotype III-4 is ST283 [7]. Interestingly, untyped GBS were reported as an emerging cause of septic arthritis, in humans, in Thailand between 1990 and 2010 [72]. The three predominant clades of GBS in fish are associated with different serotypes: ST7 with serotype Ia, CC552 (including ST260) with serotype Ib, and ST283 with serotype III [9]. Commercially available vaccines cover serotype Ib only, or serotypes Ia and III, but without cross-protection between serotypes. Strain confirmation is therefore recommended prior to use of vaccination, but diagnostic infrastructure is very limited in SE Asia, so fish farmers rely on antimicrobial treatment rather than on strain typing and vaccination. Antibiotics, including tetracyclines, are commonly used in controlling streptococcosis [73]. Concentrations above maximum limits have been found in fish sold for consumption in Vietnam [74], which may explain the high prevalence of tetracycline resistance genes found in ST283 from Vietnam. However, although similar antimicrobial use is reported in Thailand [75], tetracycline resistance amongst human ST283 from Thailand disappeared after 2012: this loss was also seen in human ST283 from Lao PDR and Singapore, possibly through three separate resistance gene loss events (Fig 2). In contrast, tetracycline resistance was 88% amongst 712 non-ST283 invasive human GBS, isolated in Singapore from 2001 to 2018 (T. Barkham, personal communication). Whereas fish-associated CC7 and CC552 are geographically widespread, our literature review, up to December 2017, showed that CC283 had only been confirmed from SE Asia, although serotype III GBS had been described as the cause of disease outbreaks that occurred in 2016 in tilapia farms in Brazil [76]. Serotype III GBS isolates from Brazilian tilapia farms have now been confirmed as ST283, while this paper was in revision, and cluster with ST283 from Asia [25,77]. This observation, in combination with import records of live Nile tilapia from Singapore to Brazil in 2014, suggests that ST283 may have been introduced into South America from Southeast Asia, underlining the potential threat of Southeast Asian ST283 to expanding aquaculture worldwide. The global trade in tilapia has previously been reported to account for the dissemination of GBS CC552 (ST260/261), from Israel to Australia, Africa (Ghana), Asia (China) and America (USA) [78] but CC552 does not affect homeothermic species. If ST283 spreads in a similar manner, human disease may also occur beyond SE Asia. Our data is limited by the use of existing GBS collections. More extensive and systematic geographical sampling and transmission studies are needed. Future studies might specifically address the prevalence of GBS in fish hatcheries, animal feed, humans (including healthy carriers), and both healthy and diseased aquatic and non-aquatic animals, as well as the evolutionary origin and virulence mechanisms of ST283. Pathogenicity factors that explain the virulence of ST283 in humans are yet to be described, although a recent report identified the bceR gene as important for antibiotic resistance, biofilm formation, and lethality in a mouse model of infection [79]. Humans have previously been implicated as the source of GBS that caused a mass die-off of fish [80], so the interplay between aquaculture and human waste, which is commonly discharged into fish ponds and rivers in Asia, in part to recycle nutrients in the food production system, might also be studied. GBS ST283 is widespread in Southeast Asia, where it has been causing disease for over 25 years. Human ST283 is almost absent outside Asia but accounts for over 70% of invasive human GBS in collections from Thailand and Lao PDR. As both are significant producers of tilapia, and consumption of raw fish is common in these countries, we hypothesise that their ST283 infections are acquired from fish, as in the Singapore outbreak. ST283 has been detected in healthy and diseased farmed fish in SE Asia and is estimated to have emerged in 1985, corresponding with the beginning of the rapid expansion of the aquaculture industry. ST283 causing tilapia deaths in Brazil since 2016 are thought to have been introduced with tilapia imported from Asia in 2014. Evolutionary origins and routes of transmission within and between host species need further study, as it is possible that ST283 is transmitted from humans to aquaculture, or that there is another common source. Creation of cross-border collaborations in human and animal health are needed to complete the epidemiological picture, may lead to improved human and fish welfare, and may contribute to safer economic development in affected countries. The findings and conclusions expressed by authors contributing in this manuscript do not necessarily reflect the official position of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.
summary: An outbreak due to a bacterium called Streptococccus agalactiae in Singapore in 2015 was caused by a clone called ST283, and was associated with consumption of raw freshwater-fish. It was considered unique as it was the only reported foodborne outbreak of this bacterium. Our new data show that invasive ST283 disease is far from unique. ST283 has been causing disease in humans and farmed fish in SE Asian countries for decades. Reports of ST283 are almost absent outside Asia. We suspect that human ST283 is fishborne in other Asian countries, as it was in Singapore, but we haven’t looked at this yet. We don’t know where ST283 originally came from; it may have been transmitted from humans to fish, or come from another animal. More studies are needed to determine ST283’s geographical extent and burden of disease, as well as its origin, how it is transmitted, and what enables it to be so aggressive. We may then be able to interrupt transmission, to the benefit of fish, farmers, and the general public.
keywords: taxonomy
  fish
  medicine and health sciences
  pathology and laboratory medicine
  pathogens
  geographical locations
  microbiology
  vertebrates
  animals
  marine fish
  marine biology
  freshwater fish
  singapore
  fish farming
  phylogenetics
  data management
  phylogenetic analysis
  fisheries
  aquaculture
  bacteria
  bacterial pathogens
  streptococcus agalactiae
  computer and information sciences
  group b streptococci
  medical microbiology
  streptococcus
  microbial pathogens
  marine and aquatic sciences
  evolutionary systematics
  agriculture
  people and places
  eukaryota
  asia
  earth sciences
  biology and life sciences
  evolutionary biology
  organisms
title: One hypervirulent clone, sequence type 283, accounts for a large proportion of invasive Streptococcus agalactiae isolated from humans and diseased tilapia in Southeast Asia