Besung et al. BMC Res Notes (2019) 12:795 https://doi.org/10.1186/s13104-019-4826-7 BMC Research Notes

RESEARCH NOTE Open Access Isolation and identifcation of suis from sick in Bali, Indonesia I Nengah Kerta Besung1* , I Gusti Ketut Suarjana1, Kadek Karang Agustina2, Ida Bagus Oka Winaya3, Hamong Soeharsono4, Ni Ketut Suwiti5 and Gusti Ngurah Mahardika6

Abstract Objective: Streptococcus suis (S. suis) is a causative agent for various syndromes in pigs. It can be transmitted to humans with typical symptoms of and death. Although human infections have been confrmed at Bali Referral Hospital, Indonesia, since 2014, the have not been isolated from pigs. Here, we provide confrmation of the presence of the bacteria in sick pigs in the province. Results: Streptococcus suis was confrmed in 8 of 30 cases. The fnal confrmation was made using PCR and sequenc- ing of the glutamate dehydrogenase (GDH) and recombination/repair protein (recN) gene fragments. Upon PCR serotyping, two were confrmed to be serotype 2 or 1/2. Prominent histopathological lesions of confrmed cases were meningitis, endocarditis, pericarditis, bronchopneumonia, enteritis and glomerulonephritis. The dominant infamma- tory cells were neutrophils and macrophages. Further research is needed to understand the risk factors for human infection. Community awareness on the risk of contracting S. suis and vaccine development are needed to prevent human infections. Keywords: Glutamate dehydrogenase (GDH) gene, Recombination/repair protein (recN) gene, Streptococcus suis, Sick pigs, Bali, Indonesia

Introduction showed more than 40 confrmed human cases of S. suis Streptococcus suis (S. suis) is an important agent of meningitis since 2014 [10]. Cases of S. suis infection in emerging zoonotic, community-acquired bacterial men- pigs with encephalitis and arthritis signs have long been ingitis [1–3]. It is a global zoonotic agent originating in suspected in the province. Attempts to detect the bac- pigs [4]. Asia is a unique hotspot of S. suis infection in teria have not yet been successful. Tis is likely because humans related to traditional pork consumption customs of identifcation problems with S. suis. Its growth char- [5, 6]. Te bacteria cause signifcant economic losses acteristic of forming small colonies [11] means that this through mortality and decreased production [7–9] and bacterium might be overlooked in a culture with mixed indirect losses through decreased demand for pork if an bacterial species. Misidentifcation of S. suis is also com- outbreak occurs. mon [12–14]. Te false negative result can be solved Tis emerging zoonotic bacterium is of particular con- through a complete serotyping system [15], which is not cern in tourist destinations because it poses an infection available in Indonesia. risk to travelers and local people. Bali Province in Indo- nesia is one of world’s most prominent tourist destina- Main text tions. More than six million travelers visited Bali in 2018 Methods (https​://bali.bps.go.id). Data from Bali Referral Hospital Source of animals Sick pigs were owned by individual farmers, who con- tacted Te Animal Biomedical and Molecular Biology *Correspondence: [email protected] 1 Laboratory of Microbiology, Faculty of Veterinary Medicine, Udayana Laboratory of Udayana University reporting sickness and University of Bali, Jl. Sudirman, Denpasar 80225, Indonesia mortality in their piggery. Tey agreed for their sick and Full list of author information is available at the end of the article

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Besung et al. BMC Res Notes (2019) 12:795 Page 2 of 6

dead animals to be included in the study. Te farms were Tissue was processed and stained with hematoxylin and located across Bali Province (Table 1). eosin (H&E) staining based on a published protocol [22]. During January to July 2018, 30 suspected S. suis cases were recorded. Te inclusion criteria were acute illness Results with at least one clinical sign of neurological disorder, Out of 30 cases, 8 were indicative for S. suis and showed reddish skin discoloration and arthritis. Te animals were small and non-hemolytic colonies that were Gram posi- not treated with antibiotics. tive with coccus-chain forming appearance. Only posi- Freshly dead animals were necropsied. Organs with tive cases are described further in this manuscript. Te clear pathological lesions were collected in Stuart Trans- epidemiological and clinical data of the presumably posi- port Medium (CM0111 Oxoid) and bufered formalin. tive cases are presented in Table 1. Listed from the most Isolation and biochemical characterization were con- frequent, clinical signs were reddish skin discoloration, ducted according standard protocol [16]. Te tissues anorexia, nasal exudate, diarrhea, limping, eye exudate, from one animal were pooled and extracted. Te sus- lethargy, swollen joints, shivering, weakness, cough, lack pension was plated on a 5% defbrinated sheep blood of appetite, depression, dyspnea, tremor, fever and snot. agar plate. Te plate was incubated at 37 °C for 18–24 h. Animal were aged 3–12 months. Te cases were from Some suspected colonies were Gram stained and grown Denpasar, Gianyar, Tabanan and Karangasem regencies. in triple sugar iron agar and sulfde indole motility media. Te results of Gram staining and biochemical tests of Other tests were catalase, oxidase, citrate, methyl red, suspected colonies from the eight suspected S. suis infec- Voges–Proskauer (VP), glucose and lactose tests. Tree tions are presented in Table 2. All isolates were Gram suspected colonies were injected to separate tryptic (+), coccus form, short chained and positive in acid slant, soy broth (Sigma Aldrich MFCD00132536) and incu- catalase and lactose, but negative in VP test. bated at 37 °C for 18–24 h. DNA was isolated using 10% After electrophoresis of PCR products (not shown), chelex-100 (Biorad, CA) [17, 18]. Te glutamate dehy- positive samples showed a single band of around 700 bp drogenase (GDH) and recombination/repair protein for GDH and 350 bp for recN. Te GDH sequences of (recN) gene fragments were amplifed using polymerase fve isolates of S. suis from our study were identical. Te chain reaction (PCR) using published specifc primer sets blast result showed a percentage identity (PID) of 99.09% for S. suis [19, 20]. Te GDH and recN fragments were to human isolates from Bali Indonesia as published pre- sequenced by Apical Scientifc Sequencing (Malaysia) viously [10]. Te sequences of recN of two isolates show using an automatic chain termination method. Serotyp- a PID of 100% to human isolates, while other two each ing was conducted using PCR to detect the cps1I gene for have 98.5%. Upon PCR serotyping, two isolates were serotype 1 and 14 and the cps2I gene for 2 and 1/2 as rec- confrmed to be serotype 2 or 1/2. Two cps2I sequences ommended [21]. of animal isolates in our study are completely identical

Table 1 Clinical signs, animal age, number of animal in stall of confrmed S. suis infection in Bali, Indonesia, 2018 No Location (village and regency) Number Number Age (month) Number Clinical signs of animal of sick of death in stall animals

1 Abang, Karangasem 3 2 8 1 In-appetence, depression, reddish skin discoloration, cough 2 Sesetan, Denpasar 100 21 4 3 Fever, anorexia, lethargic, limping, paralyzed, tremor, eye and nasal exudate, erythema ear, joint enlarge- ment and bruising 3 Payangan, Gianyar 4 1 8 5 Weakness, anorectic, diarrhea, watery nose, eye exu- date, reddish skin discoloration, limping 4 Kesiman Denpasar 8 6 10 6 Diarrhea, limping, anorexia, paralyzed, snot 5 Sidakarya, Denpasar 150 14 3 3 Yellowish diarrhea, dyspnea, nasal exudate, shivering, reddish skin discoloration 6 Kediri, Tabanan 68 11 7 3 Weakness, anorexia, diarrhea, nasal and eye exudate, limping, reddish skin discoloration 7 Perean Kangin, Tabanan 12 7 6 3 Lethargic, anorexia, reddish skin discoloration, shiver- ing 8 Lod tunduh, Gianyar 24 7 12 7 Anorexia, lethargic, cough, reddish skin discoloration, swollen joint Besung et al. BMC Res Notes (2019) 12:795 Page 3 of 6

Table 2 Gram staining and biochemical properties of bacterial colonies of confrmed S. suis infection in Bali, Indonesia No Microscopic after Gram staining TSIA SIM Catalase VP Lactose

Acid slant Acid butt Gas H2S H2S Indole Motile

1 Gram positive, coccus, short chain + − − − − − − + − + 2 Gram positive, coccus no chain + − − − − − − + − + 3 Gram positive, coccus, no chain + − − − − − − + − + 4 Gram positive, coccus, no chain + − − − − − − + − + 5 Gram positive, coccus, no chain + − − − − − − + − + 6 Gram positive, coccus, no chain + − − − − − − + − + 7 Gram positive, coccus, short chain + − − − − − − + − + 8 Gram positive, coccus, no chain + − − − − − − + − + The results of oxidase, citrate, MR, and glucose are not shown; all was negative

to those of human isolates [10]. Te GenBank Acc. clinical signs of S. suis infection in the literature can No. of GDH, recN and cps2I for serotype 2 or 1/2 are indeed be multi-organ. Te signs can be pyrexia, lack of MN334770–MN334774, MN520294–MN520297, and appetite, depression, nasal discharge, dyspnea, tremors, MN520292–MN520293, respectively. seizures, incoordination, unusual stances (such as sit- A panel of histological pictures of confrmed cases ting like a dog), inability to stand, paddling, opisthoto- is depicted in Fig. 1. Te histopathologies of confrmed nos, convulsions, nystagmus, skin disease, swollen limbs cases were similar with varying severities. Te prominent and death [26]. In some cases, the disease goes per-acute pictures were congestion in the brain, meningitis, bron- and ends with sudden death without obvious signs [26]. chopneumonia, endocarditis, myocarditis, pericarditis, Although septicemia and meningitis are the most strik- erosion and enteritis along the gastrointestinal tract with ing manifestations of the disease, endocarditis, pneumo- obvious depletion of Payer patches, hemorrhagic hepa- nia and arthritis have been reported [27]. Another review titis and glomerulonephritis, as well as lymphoid deple- article also reported that the disease syndromes caused tion, hemorrhage and accumulation of infammatory cells by S. suis in swine include arthritis, meningitis, pneu- in the spleen. Dominant infammatory cell infltration in monia, septicemia, endocarditis, polyserositis, abortion those tissues was neutrophil and macrophage. and abscesses [28]. In a recent experimental infection, afected pigs presented clinical signs of anorexia, depres- Discussion sion, fever, glazed eyes, reddened mucous membranes, We confrmed that S. suis does present and cause illness severe nervous symptoms (incoordination, lateral pros- in pigs in Bali. Human cases confrmed at Bali Referral tration, paddling, opisthotonos, convulsions and lame- Hospital [10] must have originated from pigs. Strepto- ness in the posterior limbs) [29]. coccus suis meningitis is a global zoonotic community- Our suspected cases were from all regencies in Bali acquired bacterial meningitis [1, 2, 4]. Te bacterium is of Province. However, the confrmed cases were from four extra importance in Asia as human outbreaks are related regencies, namely Tabanan, Denpasar, Gianyar and to traditional pork consumption practices [5, 6]. Te Karangasem. Considering that Bali is a small island of practice of consuming a delicacy of raw pork with raw 5.600 km2 with a high population density (http://www. blood is also common in Bali. balip​rov.go.id/v1/geogr​aphi) and understanding the free In this study, we carefully selected suspected cases, movement of animals in the province, we assume that especially those of acute cases with no history of anti- S. suis is distributed throughout the province. Te mor- biotic medication. Te cases occurred in pigs under bidity, mortality and case fatality rates in our study were 1 year old. Although S. suis can be isolated from sick and 18.7%, 8.4% and 44.9%, respectively. Morbidity, mortality healthy pigs at various ages [23], clinical manifestation and case fatality rates of S. suis in pigs vary [30]. Tere- seems more frequent in young animals [24]. A study in fore, we assumed that our observation on the case epide- Canada [25] showed that S. suis was more frequently iso- miology is plausible. lated from pigs aged between 5 and 10 weeks. Our microbiological data confrmed S. suis. Gram We recorded clinical signs in our cases involving many staining, chain formation and biochemical characteriza- organs of central nervous, respiratory, urogenital and tion shows that identifed isolates were Gram (+), coc- circulatory systems and gastrointestinal tract. Recorded cus with grape-like or short chain. All were acid slant, Besung et al. BMC Res Notes (2019) 12:795 Page 4 of 6

Fig. 1 Histopathological pictures of various tissues of confrmed S. suis infection in cases in Bali, Indonesia, 2018. Panel a1–2 are brain and meninges showing meningitis; a3–4 are lung tissues showing bronchopneumonia; b1–2 are myocardium showing endocarditis; b3–4 are myocardium showing pericarditis; c1–2 are liver showing congestion and hepatitis; c3–4 are intestine showing hemorrhagic enteritis with depletion of Payer patches; d1–2 are kidney showing hemorrhagic glomerulonephritis; d3–4 are spleen showing perifollicular infltration of infammatory cells and hemorrhage; H&E stained. Magnifcations in column 1 and 3 are 100; Column 2 and 4 are 400 × × catalase and lactose positive, while VP test was negative. detection from human and animal samples at Udayana S. suis is an encapsulated gram-positive bacterial coccus University, Bali. Te GDH blast result showed a PID of that occurs singly, frequently in pairs, or occasionally 99.09%, while four recN sequences showed PIDs of 98.5% in short chains [3]. Most strains are alpha-hemolytic on and 100% to human isolates from Bal [10]. Upon PCR bovine and sheep blood agar plates after 24 h of incuba- serotyping, two isolates were positive with primer sets for tion at 37 °C [3]. Four tests are used for a presumptive serotype 2 or 1/2 but not those of serotype 1 and 14. Only identifcation of S. suis, that is, no growth in 6.5% NaCl two primer sets were available in our lab. Te readable agar, a negative VP test and production of acid in treha- sequences were identical to the S. suis serotype 2 and 1/2 lose and salicin broths [25, 31]. Te VP test is critical in Cps2I gene Ref. [21]. diferentiating S. suis from other Streptococcus species Although we carefully selected the cases with inclu- [15]. sion criteria of suspected cases that were acute and had Final confrmation was made using PCR of GDH and at least one clinical signs of neurological disorder, red- recN. Both gene fragments are proposed as a system for dish skin discoloration and arthritis, as well as no his- reclassifying S. suis or as a specifc PCR system for S. tory of antibiotic treatment, only 8 of 30 suspected cases suis [19, 20]. We have established the system for S. suis were confrmed as S. suis infection. Te animals were not Besung et al. BMC Res Notes (2019) 12:795 Page 5 of 6

treated with antibiotics. Te negative cases might have Authors’ contributions INKB and GNM designed the study and were major contributor in writing the been caused by other infectious agents such as Haemo- manuscript. IGKS conducted the microbiology work. KKA, HS, and NKS col- philus parasuis, pseudorabies or Escherichia coli [32]. lected feld samples and data. IBOW conducted and judged the pathological Prominent histologic pictures were congestion in the examination. All authors read and approved the fnal manuscript. brain and meningitis, bronchopneumonia, myocarditis, Funding erosion and enteritis along the gastrointestinal tract, The study was funded by Directorate General of Research and Development, hemorrhagic liver and kidney. Dominant infammatory The Ministry of Research, Technology, and Higher Education of Indonesia, with research contract no. 171.53/UN.14.4.A/LT/2018 dated 19 February 2018. The cell infltration in those tissues was neutrophilic. Gross funding body had no role in the design of the study and collection, analysis, and microscopic fndings of S. suis include one or more interpretation of data, and in writing the manuscript. of fbrinous polyserositis, fbrinous or hemorrhagic Availability of data and materials bronchopneumonia, purulent meningitis, myocardial The GenBank Acc. No. of GDH, recN and cps2I for serotype 2 or 1/2 are necrosis, focal myocarditis and valvular endocarditis MN334770–MN334774, MN520294–MN520297, and MN520292–MN520293, [30]. Moreover, meningoencephalitis, a striking lesion, respectively. The sequence data are in the process to be made available in dryad (https​://datad​ryad.org). has been found in China [29]. Meningitis histology in one case (Fig. 1, panel A1–2) resembles meningitis as Ethics approval and consent to participate described by that group. The study was not dealing with life animal. The ethical approval of this study was issued by The Ethical Committee on the use of animal in research of the Tis study was the result of passive surveillance in Faculty of Veterinary Medicine Udayana University of Bali. The owners signed which the owners contacted our laboratory report- informed consent to use of the sick and dead animals in the study. ing sickness and mortality in their piggeries. Te study Consent to publish did not investigate the link of animal cases to human Not applicable. infections. Active surveillance should be conducted to determine the overall risk factors and elucidate the link Competing interests The authors declare that they have no competing interests. between animal isolates and human cases. Tat the GDH sequences of animal isolates are not identical to those of Author details 1 human isolates and the variation of recN and the con- Laboratory of Microbiology, Faculty of Veterinary Medicine, Udayana University of Bali, Jl. Sudirman, Denpasar 80225, Indonesia. 2 Department frmed serotype of 2 or 1/2 in two out of eight isolates of Veterinary Public Health, Faculty of Veterinary Medicine, Udayana University expands our knowledge that the circulating bacteria of Bali, Jl. Sudirman, Denpasar 80225, Indonesia. 3 Laboratory of Pathology, might vary in the province. Tis should be mapped as it Faculty of Veterinary Medicine, Udayana University of Bali, Jl. Sudirman, Denpasar 80225, Indonesia. 4 Laboratory of Biochemistry, Faculty of Veterinary will beneft understanding of human transmission. Medicine, Udayana University of Bali, Jl. Sudirman, Denpasar 80225, Indonesia. In conclusion, Streptococcus suis has been confrmed 5 Department of Basic Veterinary Science, Faculty of Veterinary Medicine, 6 in sick pigs in Bali, Indonesia. Two isolates were con- Udayana University of Bali, Jl. Sudirman, Denpasar 80225, Indonesia. 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