Cholera is a secretory diarrhoeal disease caused by infection with Vibrio cholerae, primarily the V. cholerae O1 El Tor biotype. There are approximately 2.9 million cases in 69 endemic countries annually, resulting in 95 000 deaths. Cholera is associated with poor infrastructure and lack of access to sanitation and clean drinking water. The current cholera epidemic in Yemen, linked to spread of V. cholerae O1 (Ogawa serotype), is associated with the ongoing war. This has devastated infrastructure and health services. The World Health Organization had estimated that 172 286 suspected cases arose between 27th April and 19th June 2017, including 1170 deaths. While there are three oral cholera vaccines prequalified by the World Health Organization, there are issues surrounding vaccination campaigns in conflict situations, exacerbated by external factors such as a global vaccine shortage. Major movements of people complicates surveillance and administration of double doses of vaccines. Cholera therapy mainly depends on rehydration, with use of antibiotics in more severe infections. Concerns have arisen about the rise of antibiotic resistance in cholera, due to mobile genetic elements. In this review, we give an overview of cholera epidemiology, virulence, antibiotic resistance, therapy and vaccines, in the light of the ongoing epidemic in Yemen.
Anti-Bacterial Agents ; therapeutic use ; Cholera ; drug therapy ; prevention & control ; Cholera Vaccines ; therapeutic use ; DNA, Bacterial ; genetics ; Disease Outbreaks ; Drug Resistance, Multiple, Bacterial ; Humans ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Vibrio cholerae ; drug effects ; isolation & purification ; Virulence Factors ; genetics ; Yemen

OBJECTIVEA novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae.

METHODSThe Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156.

RESULTSThe capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment.

CONCLUSIONSThe Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.

Food Microbiology ; methods ; Immunomagnetic Separation ; methods ; Nanotechnology ; Real-Time Polymerase Chain Reaction ; methods ; Seafood ; analysis ; microbiology ; Vibrio cholerae ; genetics ; isolation & purification ; Vibrio parahaemolyticus ; genetics ; isolation & purification

OBJECTIVETo explore the phenetic and genetic features of clinical Vibrio parahaemolyticus strains from 2007-2009 in China.

METHODSA total of 135 clinical Vibrio parahaemolyticus strains, isolated from Zhejiang, Jiangsu, Sichuan, Guangxi, Liaoning Provinces during 2007 to 2009, were selected for the research. The occurrence of virulence genes thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh), species-specific genes thermolabile hemolysin (tlh), toxR, VPM and gyrB, the pandemic clone gene markers(GS-PCR, PGS-PCR, orf8 and HU-α) in 135 Vibrio parahaemolyticus strains was detected by PCR. The antimicrobial susceptibilities to eight antimicrobial agents of the experimental strains were determined by the broth microdilution method. All strains were serotyped and underwent the cluster analysis with pulsed-field gel electrophoreses.

RESULTSThe results of PCR methods claim that all experiment strains carry species-specific genes such as tlh, toxR, gyrB, VPM. Among clinical strains, 85.9% (116/135) carry tdh and/or trh. 85.2% (115/135) were positive for tdh, and 3.0% (4/135) were positive for trh; while 3 strains carried both.66.7% (90/135) , 80.7% (109/135) , 65.2% (88/135) , 66.7% (90/135) clinical strains carried the genes of GS-PCR, PGS-PCR, orf8, HU-α, respectively. The results of antibiotics susceptibility test showed that 8.1% (11/135) strains were resistant to at least one agent, including 9 strains were resistant to ampicillin, 2 strains were resistant to trimethoprim and sulphamethoxazole, and 1 strain were resistant to tetracycline. All clinical strains were sensitive to cefotaxime, ceftazidime, gentamicin, ciprofloxacin and chloromycetin.Serological analysis of the O and K antigens claimed that a total of 29 serotypes were identified for clinical strains, predominantly O3, O4 and O1 groups, accounting for 89.6% (121/135). O3: K6 was dominant serotype, accounting for 56.3% (76/135). The pandemic flora in China included O3: K6, O4: K68, O1: K36, O1: K25, O1: K5 and O3: K29 serotypes.Genomic DNAs of 135 clinical strains were digested with SfiI and NotI, the molecular size of PFGE restriction fragments used for analysis mainly ranged from 30-700 kb.When subjected to UPGMA clustering, 6 and 9 clusters were grouped by SfiI and NotI, and the minimal similarity was 52.6% and 58.7%, and pandemic flora were located in C groups and D group, respectively.

CONCLUSIONMost of Vibrio parahaemolyticus strains isolated from clinical sources in China were pathogenic. The pandemic clone, especially O3: K6 was prevalent. The GS-PCR and HU-α genes were reliable markers to identify the pandemic flora. The serotype by PFGE was reliable to distinguish the pandemic flora and the sporadic strains.

China ; epidemiology ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Molecular Epidemiology ; Vibrio Infections ; epidemiology ; microbiology ; Vibrio parahaemolyticus ; genetics ; isolation & purification ; pathogenicity ; Virulence ; genetics

When cholera was first detected in Papua New Guinea (PNG) in mid-2009, national diagnostic capacity faced many challenges. This was in part due to the non-endemic status of the outbreak, resulting in few local staff experienced in Vibrio cholerae detection and poor access to the required consumables. The PNG Institute of Medical Research conducted culture on specimens from suspected cholera patients in Madang Province, with presumptive V. cholerae isolates sent to Goroka for confirmation. Of 98 samples analysed 15 were culture positive, with V. cholerae detected by polymerase chain reaction (PCR) in an additional 3 samples. Further analyses were conducted to identify other pathogenic bacteria from thiosulphate citrate bile salt sucrose (TCBS) agar. Molecular-based assays detected enteropathogenic (n = 1) and enterotoxigenic (n = 1) strains of Escherichia coli. No other major enteric pathogens were detected. The low detection rate of V. cholerae at the provincial level reflects challenges in the laboratory diagnosis of cholera and in-country challenges in responding to an outbreak of a non-endemic disease, such as lack of in-country diagnostic expertise and available consumables in the early stages. It also suggests that full aetiological investigations are warranted in future outbreaks of acute watery diarrhoea in PNG to fully elucidate the potentially complex aetiology, which could in turn guide diagnostic, treatment and prevention measures.
Cholera/*epidemiology/*microbiology ; *Disease Outbreaks ; Enterobacteriaceae/isolation & purification ; Feces/microbiology ; Humans ; Immunoassay ; Papua New Guinea/epidemiology ; Polymerase Chain Reaction ; Vibrio cholerae/*isolation & purification

OBJECTIVETo isolate novel actinomycetes and to evaluate their antibacterial activity.

METHODSThree soil samples were collected from Vengodu (village) in Kanchipuram district, Tamil Nadu, India. Actinomycetes were isolated using serial dilution and plating method on actinomycetes isolation agar.

RESULTSTotally 35 isolates were obtained on the basis of colony characteristics on actinomycetes isolation agar. All the isolates were screened for antibacterial activity by cross streak method. Medium and optimization of day were done for the potent strains using Nathan's agar well diffusion method. Isolation of bioactive compounds from significant active isolates was done by using different media. The most active isolate VAS 10 was identified as Actinobacterium Loyola PBT VAS 10 (accession No. JF501398) using 16s rRNA sequence method. The hexane, ethyl acetate, dichloromethane and butanol extracts of VAS 10 were tested against bacteria. The maximum antibacterial activity was observed in dichloromethane and ethyl acetate; maximum zones of inhibition were observed against Enterococcus durans. The rRNA secondary structure and the restriction sites of Actinobacterium Loyola VAS 10 were predicted using Genebee and NEBCutter online tools respectively.

CONCLUSIONSThe present study showed that among the isolated actinomycetes, Actinobacterium Loyola PBT VAS 10 (accession No. JF501398) showed good antibacterial activity against the tested bacteria.

Actinobacteria ; chemistry ; isolation & purification ; physiology ; Animals ; Anti-Bacterial Agents ; pharmacology ; Antibiosis ; physiology ; Bacillus subtilis ; drug effects ; Enterobacter aerogenes ; drug effects ; Escherichia coli ; drug effects ; India ; Microbial Sensitivity Tests ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Soil Microbiology ; Species Specificity ; Vibrio parahaemolyticus ; drug effects

OBJECTIVEThis study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of Vibrio parahaemolyticus (V. parahaemolyticus).

METHODSThe specificity of this assay was evaluated by using a panel of 33 strains of V. parahaemolyticus and 22 strains of other species bacteria. The sensitivity was determined by using serial dilutions of V. parahaemolyticus (ATCC 17802) chromosomal DNA (5×10(0) - 5×10(5) copies/µl). The samples were also tested by using qualification PCR assay and Taqman real-time PCR assay in parallel for comparison with LAMP.

RESULTSBoth sensitivity and specificity of LAMP assay, PCR assay and Taqman real-time PCR assay were 100% (22/22, 33/33, respectively). The detection limits of above three methods assay were 5×10(1) copies/µl, 5×10(3) copies/µl and 5×10(2) copies/µl, respectively. The reaction period of time needed of the above three assays was 22 min, 3 h, 50 min, respectively.

CONCLUSIONCompared to qualification PCR assay and Taqman real-time PCR assay, the established LAMP assay was better in low detection limit and less reaction time, which made it an ideal method for quick detection of V. parahaemolyticus.

Nucleic Acid Amplification Techniques ; methods ; Sensitivity and Specificity ; Vibrio parahaemolyticus ; genetics ; isolation & purification

Vibrios are universal conditioned-pathogenic bacteria in marine culture environment, and the outbreak of vibrio disease resulted in a serious damage to aquaculture. Considering that vibrio disease in aquatic species, especially fishes, usually originated from mixed infection of different species (serotypes or subspecies) of vibrios, it is important to select the potential cross-protective protein antigens as candidates of polyvalent or combined vaccines. In present research, several strains of vibrios were isolated from infected large yellow croaker (Pseudosciaena crocea) and subsequently identified as six strains of V. harveyi, one V. parahaemolyticus and one V. alginolyticus by physiological, biochemical and molecular biological methods. Their outer membrane proteins (OMPs) were extracted and the SDS-PAGE and Western blotting results show that three immuno-blots with common molecular weight presented at approximate 45 kDa, 35 kDa and 22 kDa on their OMP electrophoretogram, indicating the existence of antigens with cross-protection in their OMPs. With the aids of combination of two-dimensional electrophoresis (2-D) and Western blotting and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), a deduced porin (GenBank Accession No. ZP_01260407) from V. alginolyticus and a maltoporin precursor (GenBank Accession No. NP_801154) from V. parahaemolyticus were able to react with polyclonal antibody to whole V. harveyi, suggesting these two proteins could act as the cross-protective antigens and the vaccines prepared with these porins would be probable to bring cross protection to three different vibrios.
Animals ; Antigens, Bacterial ; immunology ; Bacterial Outer Membrane Proteins ; immunology ; Cross Reactions ; Fish Diseases ; microbiology ; Perciformes ; microbiology ; Vibrio ; classification ; immunology ; isolation & purification ; pathogenicity ; Vibrio Infections ; microbiology

A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V. parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 10(7) CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.
ATP-Binding Cassette Transporters ; genetics ; DNA Primers ; chemistry ; metabolism ; Food Microbiology ; methods ; Genome, Bacterial ; Real-Time Polymerase Chain Reaction ; Seafood ; microbiology ; Vibrio ; genetics ; isolation & purification ; Vibrio parahaemolyticus ; genetics ; isolation & purification

Vibrio cholerae non-O1 have caused several well-studied food-borne outbreaks of gastroenteritis and also have been responsible for sporadic cases of otitis media, wound infection, and bacteremia. Few cases of liver abscess caused by Vibrio cholerae non-O1 have been reported. A 73-year-old man with underlying diabetes mellitus was admitted with nausea, vomiting, dyspepsia and febrile sensation. We identified Vibrio cholerae non-O1 in his blood cultures and multiple hepatic microabscess on abdominal computed tomography. He was treated with systemic antibiotics and fluid therapy, but died due to septic shock on sixth day. We report here, a case of liver abscess with bacteremia due to Vibrio cholerae non-O1 in a patient with diabetes mellitus.
Aged ; Anti-Bacterial Agents/therapeutic use ; Bacteremia/drug therapy/*microbiology ; Ceftriaxone/therapeutic use ; Humans ; Liver Abscess/*diagnosis/drug therapy/microbiology ; Male ; Metronidazole/therapeutic use ; Shock, Septic/diagnosis ; Tomography, X-Ray Computed ; Vibrio Infections/drug therapy/*microbiology ; Vibrio cholerae non-O1/*isolation & purification

OBJECTIVETo understand the genetic structures and variations of the superintegron (SI) in Vibrio cholerae isolated in the seventh cholera pandemic.

METHODSPolymerase chain reaction scanning and fragment sequencing were used. Sixty toxigenic V. cholerae O1 El Tor strains isolated between 1961 and 2008 were analyzed.

RESULTSSome variations were found, including insertions, replacements, and deletions. Most of the deletions were probably the result of recombination between V. cholerae repeat sequences. The majority of the variations clustered together. The SIs of the strains isolated in the 1960s and 1970s showed more diversity, whereas SI cassette variations in strains isolated in the 1990s and after were lower, with ∼24 kb signature sequence deletion. This indicates the predominant SI in the host during the epidemic in the 1990s and after. The insertion cassettes suggested the mobilization from the SIs of other V. cholerae serogroups and Vibrio mimicus.

CONCLUSIONThe study revealed that structural variations of SIs were obvious in the strains isolated in epidemics in different decades, whereas the divergence was based on syntenic structure of SIs in these El Tor strains. Also, the continuing cassette flows in the SIs of the host strains during the seventh cholera pandemics were displayed.

Cholera ; epidemiology ; microbiology ; Chromosomes, Bacterial ; genetics ; ultrastructure ; Cluster Analysis ; DNA, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Gene Deletion ; Gene Flow ; Genetic Variation ; Humans ; Integrons ; genetics ; Mutagenesis, Insertional ; Open Reading Frames ; genetics ; Polymerase Chain Reaction ; Tandem Repeat Sequences ; Vibrio cholerae O1 ; classification ; genetics ; isolation & purification ; ultrastructure