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  Table of Contents  
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 489-491

First report of Vibrio cholerae O9, novel st520, isolated from a child with bacteraemia-associated sepsis

1 Department of Microbiology, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Cochin, Kerala, India
2 Cholera and Biofilm Research Laboratory, Rajiv Gandhi Center for Biotechnology, Trivandrum, Kerala, India
3 Department of Neurosurgery, Amrita Institute of Medical Sciences, Amrita Vishwa Vidyapeetham, Cochin, Kerala, India

Date of Submission22-Jun-2020
Date of Decision14-Jul-2020
Date of Acceptance30-Jul-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Anil Kumar
Department of Microbiology, Amrita Institute of Medical Sciences, Amrita Vishwavidyapeetham, Cochin - 682 041, Kerala
Dr. Sabu Thomas
Cholera and Biofilm Research Lab, Rajiv Gandhi Centre for Biotechnology, Poojappura, Thycaud P.O, Trivandrum - 695 014, Kerala
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_20_283

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 ~ Abstract 

Vibrios have been identified to cause extra-intestinal complications apart from the occasional cholera-like diarrhoeal outbreaks. The non-O1/O139 Vibrio cholerae strains are ubiquitous in environmental water bodies and hence pose a threat to people even without obvious risk factors. We describe a case of sepsis in a child with spinal dysraphism caused by a V. cholerae O9 strain belonging to a novel sequence type (ST520). The present case highlights the need of considering V. cholerae non-O1/O139 as one of the pathogens while dealing with sepsis cases, and also, the study expounds the importance of proper characterisation of the pathogen for an effective treatment.

Keywords: Non-O1/O139 Vibrio cholerae, sepsis, sequence type, spinal dysraphism

How to cite this article:
Shashindran N, Narendrakumar L, Udayakumaran S, Vijayakumar DM, Thomas S, Kumar A. First report of Vibrio cholerae O9, novel st520, isolated from a child with bacteraemia-associated sepsis. Indian J Med Microbiol 2020;38:489-91

How to cite this URL:
Shashindran N, Narendrakumar L, Udayakumaran S, Vijayakumar DM, Thomas S, Kumar A. First report of Vibrio cholerae O9, novel st520, isolated from a child with bacteraemia-associated sepsis. Indian J Med Microbiol [serial online] 2020 [cited 2021 Jan 27];38:489-91. Available from:

 ~ Introduction Top

Extra-intestinal diseases due to Vibrio cholerae are almost exclusively caused by the non-O1/O139 serogroups. Predominantly associated with sporadic diarrhoeal illness, non-O1/O139 V. cholerae are also known to cause bacteraemia, meningitis, ear infections and cellulitis.[1],[2] Bacteraemia due to these strains usually occurs among patients with immunosuppression, liver cirrhosis and diabetes.[3],[4] In most of the cases, non-O1/O139 V. cholerae-associated bacteraemia has been reported to be fatal as it leads to sepsis.[5] Wound exposure to marine water or consumption of undercooked seafood often precedes these infections. However, in 75% of the cases, the source of infection remains unidentified.[1] Since non-O1/O139 infections are less common than the cholera caused by toxigenic O1/O139 V. cholerae, antibiotic guidelines for the treatment of bacteraemia are not well defined. However, except for sparse cases, non-O1/O139 V. cholerae has been identified to be sensitive to most antibiotics. Yet, successful recovery predominantly depends on the early diagnosis and timely start of antibiotics.

 ~ Case Report Top

We describe a case of sepsis due to non-O1/O139 V. cholerae in a 2-year-old male child with spinal dysraphism hospitalised for a neurosurgical procedure that involved detethering of the spinal cord. Two weeks before the scheduled date of surgery, the child had spikes of high-grade fever. Peripheral blood smear showed leucocytosis (13,800/μL) with neutrophil preponderance (69.9%). C-reactive protein levels were elevated. A blood sample was sent for culturing while empiric antibiotic therapy with intravenous meropenem was started. There was no history of diarrhoea.

Within 48 h, blood culture flagged positive and yielded Gram-negative bacilli. The isolate was identified as V. cholerae by the automated Vitek 2 Compact System (bioMérieux, Marcy l'Etoile, France) and by MALDI-TOF using Vitek MS (bioMérieux, Marcy l'Etoile, France). Antibiotic susceptibility testing by Kirby–Bauer disc-diffusion assay in accordance with the Clinical and Laboratory Standards Institute guidelines[6] showed that the isolate was susceptible to co-trimoxazole, ampicillin, ceftazidime, tetracycline, meropenem, gentamicin, chloramphenicol and azithromycin and intermediate to ciprofloxacin, kanamycin, erythromycin and norfloxacin. The isolate was resistant to nalidixic acid and polymyxin B.

The isolate was confirmed to be V. cholerae by species-specific amplification of 16S-23S intergenic spacer region. Failure to agglutinate with O1 and O139 antisera and lack of amplification of O1rfb and O139rfb genes confirmed the isolate to be non-O1/O139 V. cholerae. Serogrouping performed according to the protocol developed by the National Institute of Infectious Diseases (Tokyo, Japan) at the National Institute of Cholera and Enteric Diseases, Kolkata, identified the strain as V. cholerae O9. Screening for toxin genes (ctx, hlyA, tcp, ace, zot, sto, rtxA and toxR) by polymerase chain reaction detected toxR and hylA (hemolysin) genes. The hylA gene appeared to belong to the El Tor rather than the classical biotype.

Multilocus sequence typing (MLST) was performed to phylogenetically analyse the clinically isolated V. cholerae O9 strain. Seven house-keeping genes (adk, mdh, pntA, gyrB, purM, pyrC and metE) were amplified and sequenced.[7] The gene sequences were analysed using Sequence Scanner Software v2 and aligned by BioEdit Sequence Alignment Editor v7.2.5. Allele numbers of each of the seven loci were obtained using the PubMLST database. The test isolate was obtained the name VCAR1. A novel sequence type (ST520) was assigned on submission of sequences to pubMLST, as the allelic combination of the strain was new ( Phylogenetic comparison of VCAR1 to non-O1/O139 V. cholerae strains from the pubMLST database was done using PHYLOViZ v2 software. MLST (ST520) demonstrated that VCAR1 was different from the dominant clones of non-O1/O139 V. cholerae. Phyloviz analysis of the strain revealed a genetic relatedness of up to four loci to other clinically isolated non-O1/O139 strains. Furthermore, the strain clustered separately from the environmental non-O1/O139 V. cholerae used for the analysis [Figure 1].
Figure 1: Dendrogram of hierarchical clustering of non-O1/O139 Vibrio cholerae attained using unweighted pair grouping with mathematical averaging

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 ~ Discussion Top

The most common reason for non-O1/O139 V. cholerae-associated septicaemia has been exposure to marine or brackish water. Septicaemia caused due to the consumption of raw or undercooked seafood has also been reported.[8] Like the V. cholerae O1 infection that causes cholera, there has been a seasonal pattern of non-O1/O139 bacteraemia identified.[9] However, most of the cases had underlying medical conditions leading to immunosuppression. The child in the present case had spinal dysraphism but was not immunosuppressed. The source of the infection could not be identified, as there was no history of exposure to marine and freshwater bodies, bacteria-contaminated potable water or consumption of raw seafood. The patient responded well to meropenem and a subsequent blood culture sent a week later was negative. A stool sample was sent for culture despite the absence of gastrointestinal symptoms in view of sepsis caused by V. cholerae. However, no V. cholerae was isolated from the stool.

A non-toxigenic V. cholerae non-O1/O139 strain with a similar toxin profile (hlyA and toxR positive and ctxAB negative) was isolated in 2012 in the same centre from a patient with cirrhotic liver disease who suffered a fatal case of septicaemia.[8] Another case of meningitis and septicaemia in a neonate caused by a non-O1/O139 V. cholerae strain was reported from Qingdao, China, in 2015 which was assigned a novel sequence type (ST188). The strain also lacked the ctxAB gene but showed high haemolysin, protease and cytotoxin activity.[10] There was also a recent report of isolation of non-O1/O139 V. cholerae carrying the hypervirulent Haitian-type cholera toxin gene (ctxB7) and other major virulence genes such as tcpA, zot and rtxC from a child with persistent diarrhoea in Ghaziabad, India.[11]V. cholerae O9 serogroup has been previously reported to be widely isolated from chironomid egg mass.[12] In many cases, serogrouping and MLST profiling of the non-O1/O139 isolate that has been isolated from the patient remain to be difficult, as not all hospitals/laboratories are competent for it.

V. cholerae non-O1/O139 are ubiquitous in marine and brackish water bodies. However, they also thrive in fresh water. From a public health perspective, increased awareness and hygiene can help to prevent these sporadic, potentially serious infections. This is the first report of V. cholerae O9 ST520 causing sepsis. Unlike the toxigenic O1/O139 V. cholerae strains, in-depth studies on non-O1/O139 V. cholerae infections are limited. Considering the increasing cases of non-O1/O139 V. cholerae-associated sepsis, we recommend the pathogen to be accorded more consideration while dealing with cases of sepsis worldwide. Such cases also highlight the need of understanding the pathogenicity and invasiveness of non-O1/O139 strains for the better management of the infection.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.


The authors are thankful to the National Institute of Cholera and Enteric Diseases, Kolkata, India, for serotyping of the vibrio isolate. The authors acknowledge the contribution of the Department of Microbiology, Kasturba Medical College, Manipal, India, in identifying the isolate using MALDI-TOF MS (bioMérieux).

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 ~ References Top

Deshayes S, Daurel C, Cattoir V, Parienti JJ, Quilici ML, de La Blanchardière A. Non-O1, non-O139 Vibrio cholera bacteraemia: Case report and literature review. Springerplus 2015;4:575.  Back to cited text no. 1
Chowdhury G, Joshi S, Bhattacharya S, Sekar U, Birajdar B, Bhattacharyya A, et al. Extraintestinal Infections caused by Non-toxigenic Vibrio cholerae non-O1/non-O139. Front Microbiol 2016;7:144.  Back to cited text no. 2
Janda JM, Powers C, Bryant RG, Abbott SL. Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin Microbiol Rev 1988;1:245-67.  Back to cited text no. 3
Daniel D, Kumar S. Rare strain of Vibrio cholerae septicemia in a patient with multiple myeloma. Case Rep Crit Care 2015. pii: 596906.  Back to cited text no. 4
Patel NM, Wong M, Little E, Ramos AX, Kolli G, Fox KM, et al. Vibrio cholerae non-O1 infection in cirrhotics: Case report and literature review. Transpl Infect Dis 2009;11:54-6.  Back to cited text no. 5
CLSI: Methods for Antimicrobial Dilution and Disc Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria, Approved Guideline. Document M45-A2. 2nd ed., Vol. 1. Wayne, PA: Clinical Laboratory Standards Institute; 2010. p. 56238-732.  Back to cited text no. 6
Octavia S, Salim A, Kurniawan J, Lam C, Leung Q, Ahsan S, et al. Population structure and evolution of Non-O1/Non-O139 Vibrio cholerae by multilocus sequence typing, PLos One 2013;8:65342.  Back to cited text no. 7
Khan S, Kumar A, Meparambu D, Thomas S, Harichandran D, Karim S. Fatal non-O1, non-O139 Vibrio cholerae septicaemia in a patient with chronic liver disease. J Med Microbiol 2013;62:917-21. doi: 10.1099/jmm. 0.049296-0.  Back to cited text no. 8
Rajendran K, Sumi A, Bhattachariya MK, Manna B, Sur D, Kobayashi N, et al. Influence of relative humidity in Vibrio cholerae infection: A time series model. Indian J Med Res 2011;133:138-45.  Back to cited text no. 9
[PUBMED]  [Full text]  
Hao Y, Wang Y, Bi Z, Sun B, Jin Y, Bai Y, et al. A case of non-O1/non-O139 Vibrio cholerae septicemia and meningitis in a neonate. Int J Infect Dis 2015;35:117-9.  Back to cited text no. 10
Kumar P, Karmakar S, Prasad R, Chopra R, Khandelwal S, Gupta S, et al. Persistent diarrhoea in a 5-month-old baby carrying Vibrio cholerae nonO1/nonO139 producing Haitian cholera toxin. New Microbes New Infect 2018;21:72-4.  Back to cited text no. 11
Halpern M, Gancz H, Broza M, Kashi Y. Vibrio cholerae hemagglutinin/protease degrades chironomid egg masses. Appl Environ Microbiol 2003;69:4200-4.  Back to cited text no. 12


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