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Isolation and characterization of lytic vibriophage against Vibrio cholerae O1 from environmental water samples in Kelantan, Malaysia

Al-Fendi A, Shueb RH, Ravichandran M, Yean CY

J Basic Microbiol, 2014 Oct;54(10):1036-43.
PMID: 24532381 DOI: 10.1002/jobm.201300458

Water samples from a variety of sources in Kelantan, Malaysia (lakes, ponds, rivers, ditches, fish farms, and sewage) were screened for the presence of bacteriophages infecting Vibrio cholerae. Ten strains of V. cholerae that appeared to be free of inducible prophages were used as the host strains. Eleven bacteriophage isolates were obtained by plaque assay, three of which were lytic and further characterized. The morphologies of the three lytic phages were similar with each having an icosahedral head (ca. 50-60 nm in diameter), a neck, and a sheathed tail (ca. 90-100 nm in length) characteristic of the family Myoviridae. The genomes of the lytic phages were indistinguishable in length (ca. 33.5 kb), nuclease sensitivity (digestible with DNase I, but not RNase A or S1 nuclease), and restriction enzyme sensitivity (identical banding patterns with HindIII, no digestion with seven other enzymes). Testing for infection against 46 strains of V. cholerae and 16 other species of enteric bacteria revealed that all three isolates had a narrow host range and were only capable of infecting V. cholerae O1 El Tor Inaba. The similar morphologies, indistinguishable genome characteristics, and identical host ranges of these lytic isolates suggests that they represent one phage, or several very closely related phages, present in different water sources. These isolates are good candidates for further bio-phage-control studies.

Matched MeSH terms: Vibrio cholerae O1/virology*
Bullous cellulitis as an extraordinary manifestation of a Vibrio cholerae O1 Ogawa infection

Ummu SF, Ding CH, Wahab AA, Tzar MN

Trop Biomed, 2023 Jun 01;40(2):170-173.
PMID: 37650403 DOI: 10.47665/tb.40.2.007

Vibrio cholerae is a gram-negative bacterium synonymous with its namesake disease, cholera. Thus, gastrointestinal symptoms are the norm and V. cholerae is very rarely associated with skin and soft tissue infections. We describe a case of a 63-year-old Chinese woman with multiple medical comorbidities on corticosteroid therapy who developed fever and a painful swelling on her left leg after being pricked by a branch while gardening. There was no abdominal pain, vomiting or diarrhea. A diagnosis of bullous cellulitis was made clinically, and blood was sent for bacteriological culture. A beta-hemolytic commashaped gram-negative bacillus was isolated from the blood. It was also oxidase-positive and produced an acid/alkaline (A/K) reaction on triple sugar iron agar. It was identified biochemically as Vibrio cholerae. After additional testing, it was found to be of the O1 serogroup and Ogawa serotype. The infection resolved following a 10-day course of high-dose co-trimoxazole therapy.

Matched MeSH terms: Vibrio cholerae O1*
Multiplex electrochemical genosensor for identifying toxigenic Vibrio cholerae serogroups O1 and O139

Yu CY, Ang GY, Yean CY

Chem Commun (Camb), 2013 Mar 11;49(20):2019-21.
PMID: 23370051 DOI: 10.1039/c3cc39144b

We developed a multiplex enzyme-based electrochemical genosensor for sequence-specific detection of multiplex linear-after-the-exponential-PCR amplicons that targeted toxigenic Vibrio cholerae O1 and O139 using novel screen-printed gold electrode bisensors.

Matched MeSH terms: Vibrio cholerae O1/genetics; Vibrio cholerae O1/isolation & purification*; Vibrio cholerae O139/genetics; Vibrio cholerae O139/isolation & purification*
Fulltext Genome sequence and comparative genomics analysis of a Vibrio cholerae O1 strain isolated from a cholera patient in Malaysia

Osama A, Gan HM, Teh CS, Yap KP, Thong KL

J Bacteriol, 2012 Dec;194(24):6933.
PMID: 23209200 DOI: 10.1128/JB.01832-12

The genome sequence analysis of a clinical Vibrio cholerae VC35 strain from an outbreak case in Malaysia indicates multiple genes involved in host adaptation and a novel Na(+)-driven multidrug efflux pump-coding gene in the genome of Vibrio cholerae with the highest similarity to VMA_001754 of Vibrio mimicus VMA223.

Matched MeSH terms: Vibrio cholerae O1/genetics*; Vibrio cholerae O1/isolation & purification
Dry-reagent gold nanoparticle-based lateral flow biosensor for the simultaneous detection of Vibrio cholerae serogroups O1 and O139

Yu CY, Ang GY, Chua AL, Tan EH, Lee SY, Falero-Diaz G, et al.

J Microbiol Methods, 2011 Sep;86(3):277-82.
PMID: 21571011 DOI: 10.1016/j.mimet.2011.04.020

Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.

Matched MeSH terms: Vibrio cholerae O1/immunology; Vibrio cholerae O1/isolation & purification*; Vibrio cholerae O139/immunology; Vibrio cholerae O139/isolation & purification*
Fulltext Genetic diversity of toxigenic Vibrio cholerae O1 from Sabah, Malaysia 2015

Zaw MT, Emran NA, Ibrahim MY, Suleiman M, Awang Mohd TA, Yusuff AS, et al.

J Microbiol Immunol Infect, 2019 Aug;52(4):563-570.
PMID: 29428381 DOI: 10.1016/j.jmii.2018.01.003

BACKGROUND: Cholera is an important health problem in Sabah, a Malaysian state in northern Borneo; however, Vibrio cholerae in Sabah have never been characterized. Since 2002, serogroup O1 strains having the traits of both classical and El Tor biotype, designated as atypical El Tor biotype, have been increasingly reported as the cause of cholera worldwide. These variants are believed to produce clinically more severe disease like classical strains.
PURPOSE: The purpose of this study is to investigate the genetic diversity of V.cholerae in Sabah and whether V.cholerae in Sabah belong to atypical El Tor biotype.
METHODS: ERIC-PCR, a DNA fingerprinting method for bacterial pathogens based on the enterobacterial repetitive intergenic consensus sequence, was used to study the genetic diversity of 65 clinical V.cholerae O1 isolates from 3 districts (Kudat, Beluran, Sandakan) in Sabah and one environmental isolate from coastal sea water in Kudat district. In addition, we studied the biotype-specific genetic traits in these isolates to establish their biotype.
RESULTS: Different fingerprint patterns were seen in isolates from these three districts but one of the patterns was seen in more than one district. Clinical isolates and environmental isolate have different patterns. In addition, Sabah isolates harbor genetic traits specific to both classical biotype (ctxB-1, rstRCla) and El Tor biotype (rstRET, rstC, tcpAET, rtxC, VC2346).
CONCLUSION: This study revealed that V.cholerae in Sabah were genetically diverse and were atypical El Tor strains. Fingerprint patterns of these isolates will be useful in tracing the origin of this pathogen in the future.

Matched MeSH terms: Vibrio cholerae O1/genetics*; Vibrio cholerae O1/isolation & purification
Fulltext Molecular evidence of cholera outbreak caused by a toxigenic Vibrio cholerae O1 El tor variant strain in Kelantan, Malaysia

Ang GY, Yu CY, Balqis K, Elina HT, Azura H, Hani MH, et al.

J Clin Microbiol, 2010 Nov;48(11):3963-9.
PMID: 20826646 DOI: 10.1128/JCM.01086-10

A total of 20 Vibrio cholerae isolates were recovered for investigation from a cholera outbreak in Kelantan, Malaysia, that occurred between November and December 2009. All isolates were biochemically characterized as V. cholerae serogroup O1 Ogawa of the El Tor biotype. They were found to be resistant to multiple antibiotics, including tetracycline, erythromycin, sulfamethoxazole-trimethoprim, streptomycin, penicillin G, and polymyxin B, with 35% of the isolates being resistant to ampicillin. All isolates were sensitive to ciprofloxacin, norfloxacin, chloramphenicol, gentamicin, and kanamycin. Multiplex PCR analysis confirmed the biochemical identification and revealed the presence of virulence genes, viz., ace, zot, and ctxA, in all of the isolates. Interestingly, the sequencing of the ctxB gene showed that the outbreak strain harbored the classical cholera toxin gene and therefore belongs to the newly assigned El Tor variant biotype. Clonal analysis by pulsed-field gel electrophoresis demonstrated that a single clone of a V. cholerae strain was responsible for this outbreak. Thus, we present the first molecular evidence that the toxigenic V. cholerae O1 El Tor variant has invaded Malaysia, highlighting the need for continuous monitoring to facilitate early interventions against any potential epidemic by this biotype.

Matched MeSH terms: Vibrio cholerae O1/classification*; Vibrio cholerae O1/genetics; Vibrio cholerae O1/isolation & purification; Vibrio cholerae O1/pathogenicity*
Fulltext Outbreak-associated Vibrio cholerae genotypes with identical pulsotypes, Malaysia, 2009

Teh CS, Suhaili Z, Lim KT, Khamaruddin MA, Yahya F, Sajili MH, et al.

Emerg Infect Dis, 2012 Jul;18(7):1177-9.
PMID: 22709679 DOI: 10.3201/eid1807.111656

A cholera outbreak in Terengganu, Malaysia, in November 2009 was caused by 2 El Tor Vibrio cholerae variants resistant to typical antimicrobial drugs. Evidence of replacement of treatable V. cholerae infection in the region with antimicrobial-resistant strains calls for increased surveillance and prevention measures.

Matched MeSH terms: Vibrio cholerae O1/classification*; Vibrio cholerae O1/drug effects; Vibrio cholerae O1/genetics*; Vibrio cholerae O1/isolation & purification
Comparison of histopathological features of Vibrio cholerae O1 El Tor and O139 Bengal infections in rabbit intestinal mucosa

Amin A, Ali A, Kurunathan S, Cheong TG, Al-Jashamy KA, Jaafar H, et al.

Histol Histopathol, 2009 05;24(5):559-65.
PMID: 19283664 DOI: 10.14670/HH-24.559

Vibrio cholerae is the causative agent of the infectious disease, cholera. The bacteria adhere to the mucosal membrane and release cholera toxin, leading to watery diarrhea. There are >100 serovars of V. cholerae, but the O1 and O139 serovars are the main causative agents of cholera. The present study aimed to compare the severity of intestinal mucosal infection caused by O1 El Tor and O139 V. cholerae in a rabbit ileal loop model. The results showed that although the fluid accumulation was similar in the loops inoculated with O1 and O139 V. cholerae, the presence of blood was detected only in the loops inoculated with the O139 serovar. Serosal hemorrhage was confirmed by histopathological examination and the loops inoculated with O139 showed massive destruction of villi and loss of intestinal glands. The submucosa and muscularis mucosa of the ileum showed the presence of edema with congested blood vessels, while severe hemorrhage was seen in the muscularis propria layer. The loops inoculated with O1 El Tor showed only minimal damage, with intact intestinal villi and glands. Diffuse colonies of the O139 serovar were seen to have infiltrated deep into the submucosal layer of the intestine. Although the infection caused by the O1 serovar was focal and invasive, it was more superficial than that due to O139, and involved only the villi. These observations were confirmed by immunostaining with O1 and O139 V. cholerae-specific monoclonal antibodies. The peroxidase reaction demonstrated involvement of tissues down to the submucosal layer in O139 V. cholerae infection, while in O1 El Tor infection, the reaction was confined mainly to the villi, and was greatly reduced in the submucosal region. This is the first reported study to clearly demonstrate the histopathological differences between infections caused by the O139 Bengal and O1 El Tor pathogenic serovars of V. cholerae.

Matched MeSH terms: Vibrio cholerae O1/pathogenicity*; Vibrio cholerae O139/pathogenicity*
Laboratory study of Vibrio cholerae O1 survival on three types of boiled rice (Oryza sativa L.) held at room temperature

Huat JT, Leong YK, Lian HH

J Food Prot, 2008 Dec;71(12):2453-9.
PMID: 19244898

This study examined whether the survival of Vibrio cholerae O1 on contaminated cooked rice was influenced by the type of rice. Vibrios survived unchanged on clumps of glutinous white rice (wet, grains adhered) held at room temperature for 24 h. On nonglutinous white rice (slightly moist, grains separate), 30% viable vibrios remained at 24 h. On nonglutinous brown rice (moist, separate, covered with a mucus-like substance), the number of vibrios increased 2.7-fold at 24 h. Survival rates of vibrios on the surfaces of a row of five cooked rice grains after 2 h of exposure at room temperature were 86, 29, 12, and 4% for glutinous rice, white rice, and the endosperm and pericarp of brown rice, respectively. (Each boiled brown rice grain surface was partly pericarp and partly endosperm, which became exposed by a rupture of the pericarp.) Covering each inoculated grain with a similar cooked rice grain surface increased the corresponding figures to 93, 99, 60, and 94%. Scanning electron microscopy revealed that each type of cooked grain surface possessed a distinct microtopography. For example, the surfaces of glutinous rice grains consisted of separated overlapping strips with many holes, while the pericarps of brown rice were flat interspersed with small pits. In conclusion, each type of boiled rice produced a distinct survival pattern of V. cholerae O1 caused by both the distinct gross features and the fine surface characteristics of the rice. The significance of this finding is that the type of rice consumed can be a factor in cholera transmission by contaminated rice.

Matched MeSH terms: Vibrio cholerae O1/growth & development*
Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity

Zamri HF, Shamsudin MN, Rahim RA, Neela V

Vaccine, 2012 May 2;30(21):3231-8.
PMID: 22426330 DOI: 10.1016/j.vaccine.2012.02.012

A gene associated with lipopolysaccharide (LPS) transport was cloned from a local clinical Vibrio cholerae O1 strain of the Ogawa serotype by using the Lactococcus lactis nisin-controlled expression (NICE) system. The V. cholerae wzm gene, which codes for an integral membrane transporter protein, was expressed and targeted to the cytoplasmic membrane, and was crudely isolated through simple centrifugation and SDS solubilization. To examine seroreactivity of this construct, rabbits were orally fed with 10(9) cfu/ml of live, recombinant L. lactis carrying the wzm gene, induced with nisin prior to administration. Recombinant plasmids were retrieved from L. lactis cultured directly from stool samples of inoculated rabbits. Reverse-transcriptase PCR of wzm using the retrieved plasmids confirmed transcription of this gene, indicating viability and stability of the recombinants in vivo. The L. lactis-Wzm construct elicited substantial levels of IgG and sIgA, and challenge with virulent V. cholerae O1 evoked severe diarrhoea in the naive, non-immunised control group, but not in those fed with either recombinant or non-recombinant L. lactis. Oral administration with recombinant L. lactis expressing the V. cholerae wzm gene increases both systemic and mucosal immunity, whereas L. lactis itself appears capable of protecting against the diarrhoeal symptoms caused by V. cholerae. Wzm is a conserved membrane protein associated with the LPS endotoxin, and together with the food-grade L. lactis, represent an attractive target for the development of a safer, live anti-infective therapy against V. cholerae.

Matched MeSH terms: Vibrio cholerae O1/genetics; Vibrio cholerae O1/immunology*

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