A melioidosis case cluster of 10 blood culture-positive patients occurred in eastern Sri Lanka after an extreme weather event. Four infections were caused by Burkholderia pseudomallei isolates of sequence type 594. Whole-genome analysis showed that the isolates were genetically diverse and the case cluster was nonclonal

De Silva, Aruna

Enoka, Corea

Francis, Vaithehi R.

Jayasinghearachch, Himali S.

Krishnananthasivam, Shivankari

Masakorala, Jayanthi

Muthugama, Thilini

Sathkumara, Harindra D.

Emerging Infectious Diseases

English

A melioidosis case cluster of 10 blood culture–positive patients occurred in eastern Sri Lanka after an extreme weather event. Four infections were caused by Burkholderia pseudomallei isolates of sequence type 594. Whole-genome analysis showed that the isolates were genetically diverse and the case cluster was nonclonal

Himali S. Jayasinghearachchi

Vaithehi R. Francis

Harindra D. Sathkumara

Shivankari Krishnananthasivam

Jayanthi Masakorala

Thilini Muthugama

Aruna D. De Silva

Enoka M. Corea

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Nonclonal Burkholderia pseudomallei Population in Melioidosis Case Cluster, Sri Lanka

ResearchOnline at James Cook University

 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 27, No. 11, November 2021 2955RESEARCH LETTERSNonclonal Burkholderia  pseudomallei Population in  Melioidosis Case Cluster, Sri LankaHimali S. Jayasinghearachchi,  Vaithehi R. Francis, Harindra D. Sathkumara, Shivankari Krishnananthasivam,  Jayanthi Masakorala, Thilini Muthugama,  Aruna D. De Silva, Enoka M. CoreaAuthor affiliations: General Sir John Kotelawala Defense Univer-sity, Ratmalana, Sri Lanka (H.S. Jayasinghearachchi, T. Muthu-gama, A.D. De Silva); Eastern University, Vantharumoolai, Sri Lanka (V.R. Francis), Genetech Research Institute, Colombo, Sri Lanka (H.D. Sathkumara, S. Krishnananthasivam); University of Colombo, Colombo (J. Masakorala, E.M. Corea)DOI: https://doi.org/10.3201/eid2711.210219Melioidosis, an emerging tropical infection caused by the soil bacterium Burkholderia pseudomallei, is found most commonly in northern Australia and the tropical countries of Southeast Asia. Melioidosis is endemic in Sri Lanka and has a case-fatality rate of 24% (1). The primary route of acquisition is inocu-lation of contaminated surface water or soil through skin and mucous membranes. However, a high inci-dence of pneumonia with sepsis has been reported during extreme weather events, such as heavy rain-fall, indicating that inhalation of aerosolized bacte-ria during cyclones and typhoons is a likely mode of transmission in this setting (2).We identified a case cluster of 10 blood culture–positive cases of melioidosis in the Batticaloa District of the Eastern Province of Sri Lanka during Novem-ber–December 2015, after a flooding event; 4 case-patients died (Appendix Table, https://wwwnc.cdc.gov/EID/article/27/11/21-0219-App1.pdf). Before this case cluster was identified, 1 case had been re-ported from Batticaloa in March 2015, and after this case cluster, 5 cases were found in 2016. We confirmed isolates as B. pseudomallei by real-time PCR to detect the lpxo gene (3). We identified 6 sequence types (STs) by multilocus sequence typing (MLST) (4); of the 10 isolates, 4 were ST594, belonging to the uncommon B. thailandensis–like flagellum and chemotaxis (BTFC) gene cluster. Although the monthly average rainfall for October in the Batticaloa District is usually 160 mm, ≈331.5 mm of rain was recorded in the 48 hours ending at 8:30 a.m. Previous evidence has shown that, in this setting, infections are usually caused by diverse strains because of widespread aerosoliza-tion of multiple clones (2). We performed whole-genome sequencing (WGS) to determine clonality among the isolates of identical ST. The Ethics Review Committee of the Faculty of Medicine, University of Colombo, (Colombo, Sri Lanka), approved this study.We mapped the geographic location of the cases on Google Earth using ArcGIS version 10.1 (ESRI, https://www.esri.com) (Figure). We compared WGS data of 3 ST594 B. pseudomallei isolates (114, 122, and 133) using binary alignment files with Uni-pro UGENE version 33 (5). We determined the dis-tribution of genomic islands (GIs) using the Island Viewer 4 web tool (6) and the National Center for A melioidosis case cluster of 10 blood culture–positive patients occurred in eastern Sri Lanka after an extreme weather event. Four infections were caused by Burkhold-eria pseudomallei isolates of sequence type 594. Whole-genome analysis showed that the isolates were geneti-cally diverse and the case cluster was nonclonal.Figure. Location of melioidosis cases (red arrows) in the Batticaloa District of Sri Lanka during November–December 2015. Inset shows location of Batticaloa and Ampara Districts in Sri Lanka.2956 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 27, No. 11, November 2021RESEARCH LETTERSBiotechnology Information Multiple Sequence Align-ment (https://www.ncbi.nlm.nih.gov/tools/msav-iewer) and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) tools. We used the PHAge Search Tool Enhanced Release (PHASTER) webserver to analyze the annotated bacterial genomes to locate prophage-specifying DNA regions (7). We manually searched the sequences identified by PHASTER and used the position of integrase and last phage–related genes to determine genome boundaries (8). We predicted and annotated all open reading frames (ORFs) of the prophages by PHASTER and BLASTp. We also de-termined the intracellular motility factor (bimA) allele type present in each strain as previously described (9). WGS showed genetic diversity in terms of gene con-tent, location, number and type of GIs, prophage DNA distribution, number of intact prophages and bimA al-lele type among the isolates of ST594 (Table). These re-sults show that the case cluster was nonclonal in origin.This case cluster after heavy rainfall and flooding in Sri Lanka confirmed that B. pseudomallei is present in the environment of eastern Sri Lanka. The case-fatality rate of the cluster (40%) was almost double that of sporadic infections in Sri Lanka (23%) (1). Six of the 10 isolates in our study belonged to diverse STs; 4 isolates were novel (ST1364, ST1442, ST1179, and ST1413). However, 4 isolates belonging to ST594 were from patients from different geographic locations. In addition, WGS analysis of prophage DNA distribu-tion was able to discriminate between the isolates. GI variation in B. pseudomallei is known to be asso-ciated with genomic plasticity of the organism, and GIs appear to be a main source of genomic diversity within B. pseudomallei that can be useful in identify-ing genetically diverse strains (10). The strain-specific variations in the structure and distribution of GIs in the isolates in this case cluster indicate the presence of genetically diverse strains. As weather patterns change globally and include more severe weather events and increased flooding, we may see more such case clusters of melioidosis. Physicians in tropical regions must be vigilant for such occurrences because of the high mortality rate associ-ated with melioidosis in this setting. Primary preven-tion of melioidosis is difficult because of the sapro-phytic nature of B. pseudomallei and regular flooding during the rainy season. The predominance of diabetic patients in this cluster (Appendix Table) illustrates the importance of detection and control of diabetes in high-risk communities for primary prevention. Secondary prevention requires close coordination between clini-cians and microbiologists to encourage patients to seek medical advice early in illness and to transfer febrile patients promptly to centers with blood culture facili-ties. Early suspicion of melioidosis will enable timely, effective antimicrobial treatment.AcknowledgmentsWe thank K. Arulmoly, P. Mayurathan, S.A. Jauffar, S. Prasath, V. Jeyathas, and M.T.A. Rikarz, and other clinical staff of the Teaching Hospital, Batticaloa. We thank the staff of the Genetech Research Institute and the Department of Microbiology, Faculty of Medicine, University of Colombo, for laboratory assistance. We thank Sameera Fernando, Anjana Rahubaddha, Rajitha Fernando, Kasun de Soyza, Thanuka Gunawardana, and Shehan Dayarathna for  assistance with sample and data collection. This study was funded by US Army Medical Research Acquisition Activity (grant no. W81XWH14C0071) and partially funded by National Research Council, Sri Lanka (grant no. NRC 18079 to A.D.D.S.).  Table. Characteristics of Burkholderia pseudomallei isolates from cluster cases of melioidosis after heavy rain, Sri Lanka* Characteristic Isolate 114 Isolate 122 Isolate133 Sequence type 594 594 594 Clade BTFC BTFC BTFC BimA allele type  BimABm BimABp BimABP No. prophages in chromosome 1 6 1 6 No. prophages in chromosome 2 1 1 1 No. intact phages 0 2 0 Presence or absence of intact prophage PHAGE_Burkho_phiE202_NC_009234 Absent Present Absent GIs identified GI1, GI4, GI5, GI6, GI7, GI8, GI9, GI8.i, GI8.ii, GI11, GI12, GI13, GI14, GI16 GI1. GI2, GI4, GI6, GI7, GI8, GI8.i, GI8.ii, GI11, GI12, GI13, GI14, GI16 GI1, GI4, GI6, GI7, GI8, GI8.i, GI8.ii, GI11, GI12, GI13, GI14, GI16 GIs located next to tRNA genes GI7 GI2, GI4 GI7, GI8 GIs containing phage or site-specific integrases GI4, GI7, GI15i GI7, GI15i GI4, GI7, GI15i GIs containing prophage DNA GI7, GI4, GI15i, GI13 GI2, GI7, GI15i, GI13 GI4, GI7, GI15i, GI13 No. transposable elements in GI8 7 6 6 New GI15i GI15i GI15i GI15i No. integrases 2 2 2 *BTFC, B. thailandensis–like flagellum and chemotaxis; GI, genomic island.     Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 27, No. 11, November 2021 2957RESEARCH LETTERSAbout the AuthorDr. Jayasinghearachchi is a senior lecturer at General Sir John Kotelawala Defense University, Ratmalana. Her research interests include molecular evolution of human pathogens and their epidemiology.References  1. Corea EM, de Silva AD, Thevanesam V. Melioidosis in Sri Lanka. Trop Med Infect Dis. 2018;3:22. https://doi.org/ 10.3390/tropicalmed3010022  2. Ko WC, Cheung BM, Tang HJ, Shih HI, Lau YJ, Wang LR, et al. Melioidosis outbreak after typhoon, southern Taiwan. Emerg Infect Dis. 2007;13:896–8. https://doi.org/10.3201/eid1306.060646  3. Merritt A, Inglis TJ, Chidlow G, Harnett G. PCR-based  identification of Burkholderia pseudomallei. Rev Inst Med  Trop São Paulo. 2006;48:239–44. https://doi.org/10.1590/ S0036-46652006000500001  4. Jayasinghearachchi HS, Corea EM, Krishnananthasivam S, Sathkumara HD, Francis VR, Abeysekere TR, et al.  Whole-genome sequences of eight clinical isolates of  Burkholderia pseudomallei from melioidosis patients in eastern Sri Lanka. Microbiol Resour Announc. 2019;8:e00645–19. https://doi.org/10.1128/MRA.00645-19  5. Okonechnikov K, Golosova O, Fursov M; UGENE team.  Unipro UGENE: a unified bioinformatics toolkit.  Bioinformatics. 2012;28:1166–7. https://doi.org/10.1093/bioinformatics/bts091  6. Bertelli C, Laird MR, Williams KP, Lau BY, Hoad G,  Winsor GL, et al.; Simon Fraser University Research  Computing Group. IslandViewer 4: expanded prediction  of genomic islands for larger-scale datasets. Nucleic  Acids Res. 2017;45(W1):W30–5. https://doi.org/10.1093/nar/gkx343  7. Arndt D, Grant JR, Marcu A, Sajed T, Pon A, Liang Y,  et al. PHASTER: a better, faster version of the PHAST  phage search tool. Nucleic Acids Res. 2016;44(W1):W16–21. https://doi.org/10.1093/nar/gkw387  8. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious Basic: an integrated and  extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;15;28(12):1647–9. https://doi.org/10.1093/ bioinformatics/bts199  9. Sitthidet C, Stevens JM, Chantratita N, Currie BJ,  Peacock SJ, Korbsrisate S, et al. Prevalence and sequence diversity of a factor required for actin-based motility  in natural populations of Burkholderia species. J Clin  Microbiol. 2008;46:2418–22. https://doi.org/10.1128/JCM.00368-0810. Holden MT, Titball RW, Peacock SJ, Cerdeño-Tárraga AM, Atkins T, Crossman LC, et al. Genomic plasticity of the caus-ative agent of melioidosis, Burkholderia pseudomallei.  Proc Natl Acad Sci U S A. 2004;101:14240–5.  https://doi.org/10.1073/pnas.0403302101Address for correspondence: Enoka M. Corea, Department of Microbiology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka; email: enokac@micro.cmb.ac.lk; Aruna D.  De Silva, Department of Paraclinical Sciences, Faculty of Medicine, General Sir John Kotelawala Defense University, Ratmalana, Sri Lanka; email: dharshan_fom@kdu.ac.lkReal-Time Genomic  Surveillance for SARS-CoV-2 Variants of Concern, UruguayNatalia Rego,1 Alicia Costábile,1 Mercedes Paz,1 Cecilia Salazar,1 Paula Perbolianachis,1 Lucía Spangenberg, Ignacio Ferrés, Rodrigo Arce,  Alvaro Fajardo, Mailen Arleo, Tania Possi,  Natalia Reyes, Ma Noel Bentancor, Andrés Lizasoain, María José Benítez, Viviana Bortagaray, Ana Moller, Gonzalo Bello, Ighor Arantes,  Mariana Brandes, Pablo Smircich, Odhille Chappos, Melissa Duquía, Belén González, Luciana Griffero, Mauricio Méndez, Ma Pía Techera, Juan Zanetti, Bernardina Rivera, Matías Maidana, Martina Alonso, Cecilia Alonso, Julio Medina, Henry Albornoz, Rodney Colina, Veronica Noya, Gregorio Iraola, Tamara Fernández-Calero, Gonzalo Moratorio,  Pilar MorenoAuthor affiliations: Institut Pasteur, Montevideo, Uruguay (N. Rego, A. Costábile, M. Paz, C. Salazar, P. Perbolianachis,  L. Spangenberg, I. Ferres, R. Arce, A. Fajardo, M. Brandes,  B. Rivera, M. Maidana, M. Alonso, G. Iraola, T. Fernandez-Calero, G. Moratorio, P. Moreno); Universidad de la República,  Montevideo, Uruguay (A. Costabile, P. Perbolianachis, R. Arce,  A. Fajardo, A. Lizasoain, M.J. Benítez, V. Bortagaray, A. Moller,  O. Chappos, M. Duquía, B. González, L. Griffero, M. Méndez, M.P. Techera, J. Zanetti, C. Alonso, R. Colina, G. Moratorio,  P. Moreno); Sanatorio Americano, Montevideo (M. Arleo, T. Possi, N. Reyes, M.N. Bentancor, V. Noya); Instituto Oswaldo Cruz–Fiocruz, Rio de Janeiro, Brazil (G. Bello, I. Arantes); Instituto de Investigaciones Biológicas Clemente Estable, Montevideo  (P. Smirnich); Ministerio de Salud Pública, Montevideo (J. Medina, H. Albornoz)DOI: https://doi.org/10.3201/eid2711.211198By late 2020, because of natural viral evolution, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants emerged, some of which show increased transmissibility and cause more severe coronavirus disease (COVID-19) (1). In We developed a genomic surveillance program for real-time monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARS-CoV-2 VOCs, the surveillance workflow, and multiple in-dependent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay.1These authors contributed equally to this article.

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Nonclonal Burkholderia pseudomallei Population in Melioidosis Case Cluster, Sri Lanka

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