I have read the journal's policy and the authors of this manuscript have the following competing interests: SLC and TB are named applicants on a patent for the ST83-specific PCR test used in this study.
In 2015, Singapore had the first and only reported foodborne outbreak of invasive disease caused by the group B
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
An outbreak due to a bacterium called
In 2015, there was an unprecedented outbreak of invasive disease due to the group B
Previous reports of ST283 were limited to 20 cases in adults in Hong Kong between 1993 and 2003 [
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.
The study was approved by the Institutional Review Board of Tan Tock Seng Hospital (TTSH), Singapore, NHG DSRB 2016/00202.
We identified articles in English with searches of Medline, PubMed, and references from articles, with the terms “
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 [
This figure represents our new data, as well as the literature on
Invasive bacterial isolates are routinely saved: 331 invasive GBS isolates from 2001 to 2010 were retrieved from freezers in TTSH and Changi General Hospital. Data and sequences for 408 invasive GBS ST283 isolated from 2011–2015 were publicly available [
139 invasive GBS isolates were available from population-based surveillance for blood stream infections conducted in all 20 hospitals in Nakhon Phanom and Sa Kaeo provinces from 2007–2015; these include paediatric patients. GBS isolates from the isolate repository were screened with the ST283 PCR described above. A selection of positive isolates were subjected to WGS on the MiSeq platform using the Nextera XT DNA Library Prep Kits and the MiSeq Reagent Kit v3. In addition, sequences of seven GBS ST283 from invasive human cases in Bangkok in 2015 were publicly available [
This set comprised 38 GBS, available as isolates or as DNA extracts, obtained from blood cultures and CSF samples at the Microbiology Laboratory of Mahosot Hospital, Vientiane, Lao PDR between 2000–2017 during studies of the aetiology of fever and central nervous system infections.
13 invasive GBS isolated between 2015 and 2017 were retrieved from the Hospital for Tropical Diseases (HTD), Ho Chi Minh City (HCMC). HTD is a 550-bed hospital that serves as a main primary and secondary facility for the surrounding local population in HCMC and a tertiary referral center for infectious diseases for the southern provinces of Vietnam. Nearly 70% of HTD admissions live in HCMC, with the remainder residing in the surrounding provinces. Neonates, patients without infectious diseases, including those with surgical requirements, tuberculosis, cancer, primary hematological disorders or immunosuppression (other than HIV) are referred to other hospitals within HCMC. HIV-infected children are often referred to local paediatric hospitals. Blood cultures were performed for patients in whom an infection was suspected on the basis of a fever (>38°C) or who had evidence of sepsis on the basis of the presence of two or more of the following features: fever (>38°C) or low temperature (<36°C); tachycardia (exact level according to age); tachypnea (exact level according to age); an elevated white cell count (>12,000 cells/mm3) or depressed white cell count (<4,000 cells/mm3). There was no systematic change in the application of these criteria during the time course of the study. All data originating from consecutive patients admitted to the hospital who had a blood culture performed for suspected bloodstream infection between 2015 and 2017 were included. In addition, 38 GBS representing the most recent high vaginal swab GBS isolates from women with colpitis, three of whom were pregnant, were collected from outpatients at the National Hospital for Obstetrics & Gynaecology (Phu San Hospital), Hanoi, between September 2016 and May 2017.
DNA from eleven GBS isolated from skin and umbilical swabs collected from children presenting to Angkor Hospital for Children, a non-governmental paediatric referral hospital in Siem Reap, between 2012 and 2016 were included.
WGS data were available from 1,017 invasive human GBS submitted to the Respiratory and Vaccine Preventable Bacteria Reference Unit, Public Health England (PHE), as part of the British Society for Antimicrobial Chemotherapy Resistance Surveillance Project over a one year period bridging 2014 and 2015.
Sequences of GBS ST283 isolated from fish in 2015 were publicly available [
We sequenced 28 GBS, isolated between 2007 and 2008, from brain, eye or kidney samples from 28 tilapia (
We sequenced 34 GBS isolated in 2016 from the brains of sick fish from five farms in the Mekong River running through An Giang Province and Can Tho District. GBS was isolated directly from the brains of red or black tilapia (
Other GBS MLST datasets were contributed by collaborators. All whole genome data sets annotated as
We performed Illumina sequencing on 145 CC283 GBS strains. All new sequencing data was deposited in GenBank under BioProject PRJNA293392. All assembled genomes (as of June 7, 2017) and short read data sets (as of November 10, 2017) annotated as
From an initial neighbour-joining tree made from all SNPs called relative to the SG-M1 reference, a set of strains containing all CC283 strains and strains from the nearest non-CC283 clade (consisting of 92 strains within CC10) was taken. We excluded those strains which had no metadata for isolation date, leaving a total of 273 strains (215 from the ST283 clade, 58 from the non-ST283 clade). We reconstructed an aligned genome sequence for each of these 273 strains by introducing that strain’s SNPs into the SG-M1 genome; gaps were also inserted where mapping coverage was below 10 as per Lofreq default parameters. The BRATNextGen [
(A) Approximately maximum-likelihood phylogenetic tree of 1,236 GBS strains. The ST283 isolate SG-M1 was used as a reference sequence. The scale bar is shown on the x-axis, in mutations/nucleotide. The bootstrap values for selected branches (supporting the difference between different CCs) are indicated by black circles; all are 1.000 except where indicated. Major clonal complexes are indicated with coloured branches and a matching coloured label. The clade containing all CC283 isolates is highlighted in blue. All ST283 variants discussed in the text (ST491, ST739, ST751, and ST1311) are highlighted with black branches and an adjacent label in black text. (B) Approximately maximum-likelihood phylogenetic tree of 227 GBS CC283 genomes. Reconstructed genome sequences (based on the SG-M1 reference sequence) of the isolates indicated as CC283 in (A) were used, after excluding redundant isolates, defined as identical sequences from the same site, based on SNP calls. Bootstrap support is indicated for selected branches by black circles. Arrows show predicted events resulting in loss of tetracycline resistance. (C) For each CC283 isolate, the metadata are indicated at the same horizontal position (i.e. directly to the right of the phylogenetic tree tip) as in panel B. Host and country are represented by coloured rectangles, as indicated in the legend. Different values for Host and Country are further offset horizontally for clarity. Asterisks to the right of the Year box indicate isolates for which individual loss of tetracycline resistance appears to have occurred.
Approximately maximum-likelihood SNP trees were created using FastTree 2.1.8 with the–gtr and–nt command line options [
This gives an overview of the origins and numbers of CC283. Further details are in Supporting Information.
Host | Country | Site or reference | Collection Year | Sample type | GBS isolates screened for ST283 (No.) | GBS isolates with WGS data (No.) | CC283 isolates with WGS data (No.) |
---|---|---|---|---|---|---|---|
Human | Singapore | Singapore General Hospital | 1998 | B | 5 | 5 | 5 |
Human | Singapore | Tan Tock Seng Hospital & Changi General Hospital | 2001–2010 | B, CSF | 331 | 331 | 21 |
Human | Thailand | Nakhon Phanom and Sa Kaeo provinces | 2007–2015 | B | 139 | 22 |
22 |
Human | Lao PDR | Mahosot Hospital, Vientiane | 2000–2017 | B, CSF | 38 | 38 | 30 |
Human | Vietnam | Hospital for Tropical Diseases, Ho Chi Minh City | 2015–2017 | B | 13 | 13 | 4 |
Human | Vietnam | Phu San Hospital, Hanoi | 2016–2017 | HVS | 38 | 0 |
0 |
Human | Cambodia | Angkor Hospital for Children, Siem Reap | 2012–2016 | S | 11 | 0 |
0 |
Human | Britain | Multiple hospitals | 2015 | B | ND | 1,017 | 1 |
Tilapia | Malaysia | Kedah and Terengganu states | 2007–2008 | Br, E, K | 28 | 28 | 28 |
Tilapia | Vietnam | An Giang and Can Tho | 2016 | Br | 34 | 34 | 34 |
Human | Singapore | [ |
2011–2015 | B, CSF | ND | 145 | 40 |
Human | Thailand | [ |
2015 | B | ND | 7 | 6 |
Human | Hong Kong | [ |
1993–2012 | SS | ND | 11 | 11 |
Human | Netherlands | GenBank | ND | ND | ND | 1 | 1 |
Fish | Singapore | [ |
2015 | S, O, M | ND | 20 | 20 |
Fish | Thailand | [ |
2000–2010 | ND | ND | 1 | 1 |
Fish | Vietnam | [ |
2006 | ND | ND | 1 | 1 |
Fish | Thailand | [ |
2012–2014 | ND | ND | 1 | 1 |
Frog | China | GenBank | 2014 | L | ND | 1 | 1 |
a = WGS was only performed on GBS positive by an ST283 specific PCR.
b = Included one technical replicate. Where sequences from the same site were identical (based on SNP calls), only one was used. Abbreviations: No. = number, B = blood, CSF = cerebro-spinal fluid, HVS = high vaginal swab, S = superficial swabs, ND = not determined, SS = sterile site, Br = brain, E = eye, K = kidney, O = organs, M = muscle, L = liver.
This was performed on all CC10 and CC283 strains described above in the recombination section. Analysis of divergence times was performed using BEAST 2.4.8 [
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 [
All CC283 in this table are sequence type (ST) 283: other examples of CC283 were not found. This table shows that ST283 was found in the first year of all newly described GBS collections in Southeast Asia, so it may have predated these collections, and that both GBS numbers available and ST283 proportions vary from year to year.
Vientiane, Lao PDR | Nakhon Phanom and Sa Kaeo provinces, Thailand | Ho Chi Minh City, Vietnam | Singapore |
Total | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Year | GBS No. | CC283 No. (%) | GBS No. | CC283 No. (%) | GBS No. | CC283 No. (%) | GBS No. | CC283 No. % | GBS No. | CC283 No. (%) |
2000 | 1 | 1 (100) | ND | ND | ND | ND | ND | ND | 1 | 1 |
2001 | 0 | 0 | ND | ND | ND | ND | 16 | 3 (19) | 16 | 3 (19) |
2002 | 0 | 0 | ND | ND | ND | ND | 13 | 2 (15) | 13 | 2 (15) |
2003 | 1 | 1 (100) | ND | ND | ND | ND | 28 | 2 (7) | 29 | 3 (10) |
2004 | 0 | 0 | ND | ND | ND | ND | 32 | 7 (22) | 32 | 7 (22) |
2005 | 0 | 0 | ND | ND | ND | ND | 23 | 0 (0) | 23 | 0 (0) |
2006 | 3 | 2 (67) | ND | ND | ND | ND | 36 | 2 (6) | 38 | 4 (11) |
2007 | 5 | 5 (100) | 7 | 3 (43) | ND | ND | 41 | 2 (5) | 52 | 10 (19) |
2008 | 0 | 0 | 10 | 6 (60) | ND | ND | 46 | 1 (2) | 54 | 7 (13) |
2009 | 2 | 1 (50) | 1 | 0 | ND | ND | 53 | 1 (2) | 55 | 2 (4) |
2010 | 7 | 5 (71) | 4 | 3 (75) | ND | ND | 43 | 1 (2) | 52 | 9 (17) |
2011 | 1 | 0 (0) | 5 | 4 (80) | ND | ND | 38 | 0 (0) | 44 | 4 (9) |
2012 | 1 | 1 (100) | 13 | 8 (62) | ND | ND | 43 | 3 (7) | 57 | 12 (21) |
2013 | 3 | 3 (100) | 31 | 27 (87) | ND | ND | 58 | 9 (16) | 92 | 39 (42) |
2014 | 2 | 1 (50) | 66 | 50 (76) | ND | ND | 77 | 15 (19) | 145 | 66 (46) |
2015 | 4 | 3 (75) | 2 | 1 (50) | 4 | 1 (25) | 110 | 61 (55) |
120 | 66 (55) |
2016 | 7 | 5 (71) | ND | ND | 8 | 3 (38) | 50 | 15 (30) | 15 | 8 (53) |
2017 | 1 | 1 (100) | ND | ND | 1 | 0 (0) | ND | ND | 2 | 1 (50) |
Total | 38 | 29 (76) | 139 | 102 (73) | 13 | 4 (31) | 707 | 124 (18) | 840 | 244 (29) |
a Similarly detailed data for Hong Kong were not found.
b This Singapore data is from the only two institutions that had systematically saved GBS; Tan Tock Seng Hospital and Changi General Hospital.
*The outbreak year. Abbreviations: ND = not determined.
All newly sequenced GBS isolated from tilapia in Peninsular Malaysia and Vietnam, during outbreaks of streptococcosis, were CC283 (
All these CC283 were ST283, except for two variants, as indicated.
Country/fish | Data source | Date range | Locations | Sample type | GBS No. | CC283 No. (%) |
---|---|---|---|---|---|---|
Singapore; Asian bighead carp, red tilapia, black tilapia, giant snakehead, common snakehead, grass carp and silver carp |
[ |
2015 | Ports | I, S | 27 | 6 (22) |
Markets | I, S | 30 | 11 (36) | |||
Peninsular Malaysia; red hybrid tilapia | New data | 2007–2008 | 9 farms | I | 28 | 28 (100) |
Vietnam, tilapia | [ |
2006 | ND | ND | ND | 1 |
Vietnam, Can Tho and An Giang; tilapia | New data | 2016 | 5 farms | I | 34 | 34 |
Thailand; tilapia | [ |
2000–2010 | ND | I | 8 | 1 (12·5) |
Thailand; tilapia | [ |
2012–2014 | ND | ND | ND | 1 (ND) |
a A glossary of fish scientific names is in
b This is ST491, a single locus variant of ST283.
C One of these 34 is ST1311, a double locus variant of ST283 (See
Only four CC283, of human origin, were found: two from France, one from Britain, and one from the Netherlands (
ST, place/country, isolate No. | Data source | Date range | Sample type | GBS No. | ST283 No. | ST283% |
---|---|---|---|---|---|---|
ST283, Britain; ERR1742070 | GenBank |
2014 | Invasive | 1017 | 1 | <0·1 |
ST283, France; S80 & S81 | [ |
2002–2007 | Invasive | 119 | 2 | 2 |
ST283, Netherlands; ERR1659855 | GenBank | ND | ND | ND | 1 | ND |
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) [
GBS CC283 has been widespread in SE Asia for over two decades in humans, and at least a decade in aquatic animals, but is rare outside SE Asia. As ST283 was found in the first year of every available collection of invasive GBS from SE Asia (Lao PDR, Thailand, Vietnam and Singapore), it may have been present even earlier. The estimated date of emergence, of 1985, predates the first known human cases and is roughly contemporaneous with the start of the expansion of aquaculture in SE Asia, based on reports from the Food and Agricultural Organisation of the United Nations [
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 [
GBS is known to affect fish species other than tilapia, including farmed and wild freshwater and marine fishes [
Fish account for up to 37% of protein consumed in SE Asia [
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 [
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 [
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 [
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
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.
This table shows details for each human GBS collection, showing proportion of ST283, sample origins, dates and types, and age breakdown, foci of infection and gender.
(DOCX)
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(DOCX)
The absence of CC283 in this data, of over 4,000 human and over 1,300 animal GBS, demonstrate how unusual CC283 is. The data also show that Asia is not well represented in this group, with only one study found, from China.
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Examples were selected to show minimum and maximum SNP differences found between pairs within and between countries and hosts.
(DOCX)
The authors are grateful for the support of clinical and laboratory staff involved in supplying bacteria and data. Potential conflicts of interest: TB and SLC are named applicants on a patent for the ST283-specific PCR test used in this study.