The use of a new strain of Vibrio cholerae O139 as a producer of an enteric chemical vaccine

Amalgam remains unchallenged as a posterior restorative material. But its inability to bond to the teeth leads to some amount of microleakage at the restoration-tooth interface with associated problems such as post operative sensitivity, pulpal complications etc. Also a broken amalgam restoration requires replacement which will further weaken the tooth structure. Recently, 4-META has been introduced which can graft amalgam and composite to enamel, dentin and old amalgam restorations. In this study, the bonding and marginal sealing abilities of 4-META was assessed both at the tooth-amalgam interface and old amalgam fresh amalgam interface.     

Rapid diagnosis of cholera caused by Vibrio cholerae O139

Hybridomas secreting specific monoclonal antibodies (MAbs) to Vibrio cholerae serogroup O139 were produced. Six monoclones (hybridomas) secreting MAbs specific only to lipopolysaccharide of V. cholerae O139 strains and which did not cross-react to 137 strains of other enteric microorganisms were obtained. These clones were designated 12F5-G11, 12F5-G2, 15F5-H5, 5B9-F8, 14C9-D2, and 6D2-D8. The immunoglobulin (Ig) heavy chain isotypes secreted by these clones were IgG2b, IgG2b, IgG2b, IgM, IgG2b, and IgG3, respectively. Clone 12F5-G11 was selected for mass production of MAb, which was used as a detection reagent in the antigen detection assay for diagnosis of cholera caused by V. cholerae O139, and this assay was compared to the conventional bacterial isolation method. Five batches of rectal swab cultures in alkaline-peptone water were collected from 6,497 patients with watery diarrhea. These were 6,310 patients admitted to Bamrasnaradura Infectious Diseases Hospital, 16 patients from Krung Thon Hospital, 78 patients from Bangkok Children's Hospital, 19 patients from Karen refugee camps, and 74 Indian patients from the National Institute of Cholera and Enteric Diseases, Calcutta, India. The V. cholerae O139 isolations from the rectal swab cultures and the antigen detection assays (i.e., the MAb-based dot-blot ELISA) were performed by different persons of different laboratories, and the results were revealed after all specimens had been tested. Of the 6,497 samples tested, the dot-blot ELISA correctly identified 42 of 42 V. cholerae O139-positive samples and gave a result of positive for three samples which were culture negative for V. cholerae O139. The diagnostic sensitivity, specificity, and efficacy of the dot-blot ELISA were 100, 99.95, and 99.26%, respectively. The ELISA is easy to perform and relatively inexpensive. It can test multiple samples at a single time, does not require special equipment, and does not produce great quantities of contaminated waste. Most of all, it reduces the diagnostic time from at least 2 days for the bacterial isolation to less than 90 min. The assay is recommended as a rapid screening test of cholera cases caused by V. cholerae O139.     

The Vibrio cholerae mannose-sensitive hemagglutinin is the receptor for a filamentous bacteriophage from V. cholerae O139

We previously isolated from a 1994 isolate of Vibrio cholerae O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype V. cholerae strains in plaque assays. We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection. Spontaneous, Tn5 insertion, and mshA deletion mutants are resistant to 493 infection. Susceptibility is restored by mshA complementation of deletion mutants. Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a DeltamshA1 strain. Monoclonal antibody against MSHA inhibits plaque formation. We conclude that MSHA is the receptor for phage 493. The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493. However, mshA gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in mshA. Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays. Nevertheless, they express the mshA pilin gene. They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains. Resistance to 493 in plaque assays is thus not equivalent to resistance to infection. The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139.     

Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal

A Vibrio cholerae O139 strain, MO10-T4, lacking capsular polysaccharide, produces a short-chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high-performance anion-exchange chromatography, one- and two-dimensional 1H-, 13C-, and 31P-NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS: [structure: see text] where PEtn stands for 2-aminoethyl phosphate, Fru for fructose, Hep for L-glycero-D-manno-heptose, and Kdo for 3-deoxy-D-manno-octulosonic acid; unless otherwise stated, the monosaccharide residues are D and present in the pyranose form. An O-acetyl group is present on a secondary position, tentatively O4 of the alpha-linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown. An O139-negative mutant strain, Bengal-2R, derived from V. cholerae O139, has also been investigated and shown to produce an O-antigen-lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10-T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2-aminoethyl phosphate, the O-acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10-T4 LPS, a short O-side chain is attached at position 3 of the 7-substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.-R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43-58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59-65.]     

Morphological and physical characterization of the capsular layer of Vibrio cholerae O139

Low-speed isokinetic exercise has been recommended to exert a maximal contraction and produce greater muscle torque than high-speed exercise in young adults. The purpose of this study was to compare the effectiveness of low- and high-speed isokinetic exercise programs for increasing muscle torque in young and elderly people. Twenty healthy elderly and 20 young subjects participated. The elderly subjects were divided into two groups. One group performed high-speed (300 degrees/s) isokinetic exercise training three times a week for the dominant-side knee extensor and low-speed (60 degrees/s) exercise for the non-dominant side for 6 weeks. The other group was trained using the reverse exercise regime. The training program for the young subjects was the same as that for the elderly groups. All subjects had their knee extensor torque evaluated with an isokinetic test before and at 2-week intervals during the training program. For young and elderly groups, both high- and low-speed isokinetic exercise training increased extensor torque in low- and high-speed tests. For the young group, low-speed exercise effectively improved muscle torque at low and high speeds. The improvement in slow muscle torque was significantly greater than that in fast muscle torque. For the elderly subjects, high-speed isokinetic exercise produced the greatest muscle torque at high speed in the first 2 weeks of training, and demonstrated a sharp increase in muscle torque in the final 2 weeks. Low-speed exercise frequently caused knee stress and the inability of some elder subjects to continue the exercises with maximal effort. Our findings indicate that high-speed exercise may be more appropriate for the elderly, and low-speed exercise may be more appropriate for younger people.     

Isolation and characterization of rugose form of Vibrio cholerae O139 strain MO10

An extracellular exopolysaccharide (slime) is produced by Vibrio cholerae O139 MO10 in response to nutrient starvation. The presence of this slime layer on the cell surface and its subsequent release have been shown to be associated with biofilm formation and the change from a normal smooth colony morphology to a rugose one. An immunoelectron microscopic examination demonstrated that there is an epitope common to the exopolysaccharide antigen of V. cholerae O1 and that of O139 MO10.     

Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR

In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V. cholerae O139 Bengal. PCR with the same primer pair was used to screen 180 diarrheal stool specimens. All the 67 V. cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR.     

Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V. cholerae O139 Bengal in Bangladesh

C-X-C chemokines are potent chemoattractants that are believed to mediate neutrophilic inflammation in several organs. Recent studies suggest a role for C-X-C chemokines in the pathogenesis of neutrophilic hepatitis but do not prove causation. We investigated the biological consequences of hepatic chemokine production in vivo by transiently overexpressing cytokine-induced neutrophil chemoattractant (CINC), a member of the C-X-C chemokine family, in intact rats. Rats were injected intraportally with a replication-defective recombinant adenovirus containing the CINC complementary DNA (cDNA). Within 4 days, treated animals had high levels of CINC in both liver tissue and plasma Rats overexpressing CINC exhibited an eightfold increase in circulating neutrophils; they also developed severe hepatic injury, characterized by a 6- to 25-fold increase in plasma transaminases and marked hepatic inflammation on biopsy. Liver disease in CINC-producing rats correlated positively with the number of neutrophils sequestered in the hepatic parenchyma. Tissue injury was attributed directly to chemokine overproduction, because control rats infected with adenoviruses lacking the CINC cDNA did not produce CINC and developed only minor hepatic abnormalities. These experiments provide direct evidence that C-X-C chemokines, when expressed in sufficient quantity in the liver in vivo, induce neutrophil recruitment and tissue invasion and provoke severe liver injury. The data suggest that C-X-C chemokines have important pathogenic potential in both clinical and experimental liver disease.     

Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae

Vibrio cholerae causes epidemic diarrhea throughout the world. Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics. V. cholerae, like most bacteria, has developed resistance to some antibiotics. In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics. Bengal isolates showed unique trends in antimicrobial resistance. Many clinical laboratories use automated antibiotic susceptibility testing for V. cholerae. It is important to know if automated susceptibility test results for V. cholerae coincide with reported trends in antibiotic susceptibility. In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V. cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates. Vitek susceptibilities for V. cholerae showed a good correlation with preestablished epidemiological data. Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains. Regardless of serogroup, > or = 98% of the V. cholerae isolates tested were susceptible to most antibiotics tested by us. It is important to continue susceptibility testing of all new isolates of V. cholerae because of emerging resistant strains. However, V. cholerae remains susceptible to most of the available antibiotics.     

Note: characterization of Vibrio cholerae O139 Bengal isolated from water in Malaysia

Four Vibrio cholerae O139 Bengal strains isolated from surface water were characterized by antibiotic resistance, plasmid profile, presence of cholera toxin gene and random amplification of polymorphic DNA (RAPD) analysis. All four strains exhibit multiple resistance towards the antibiotics tested with a multiple antibiotic resistance index of 0.5-0.66, and harboured a 2.0 MDa non-conjugative plasmid. The Vibrio cholerae O139 Bengal were positive for the cholera toxin gene. Antibiotyping and random amplification of polymorphic DNA analysis with four primers proved to be useful in discriminating the isolates. RAPD proved to be more sensitive. These results reveal that there is significant genetic diversity among the Vibrio cholerae O139 Bengal strains studied.     

Production and cross-reactivity patterns of a panel of high affinity monoclonal antibodies to Vibrio cholerae O139 Bengal

Abnormal brain stem auditory-evoked responses (BAER) were recorded on 14 dogs with brain lesions confirmed by necropsy (n = 13) or magnetic resonance imaging and surgical biopsy (n = 1). Lesions included brain stem or cerebellar tumors (6 dogs), brain stem trauma (1 dog), forebrain tumors (3 dogs), hydrocephalus (2 dogs), granulomatous meningoencephalitis (1 dog), and meningoencephalitis (1 dog). Five affected dogs were comatose at the time of recording. BAER abnormalities could be classified as (1) absence of some or all of waves I to V, (2) increased latencies, with wave V being most frequently affected, or (3) a reduction in the amplitude ratio of waves V/I.     

Isolation of sucrose late-fermenting and nonfermenting variants of Vibrio cholerae O139 Bengal: implications for diagnosis of cholera

The sucrose-containing selective medium thiosulfate-citrate-bile salt-sucrose agar missed a sucrose nonfermenting and four sucrose late-fermenting variant strains of Vibrio cholerae O139 Bengal from diarrheal stools. These strains were, however, correctly identified as V. cholerae O139 on a sucrose-deficient selective medium, taurocholate-tellurite-gelatin agar.     

Resurgent Vibrio cholerae O139: rearrangement of cholera toxin genetic elements and amplification of rrn operon

PURPOSE: The objective of this work is to prepare microspheres by the emulsion-solvent evaporation process using MPOE-PLA copolymers as the matrix material and/or the surfactant. This preparation was studied in order to avoid the use of PVA as the surfactant in the process. METHODS: Two series of MPOE-PLA copolymers were synthesised. The first, with a long and constant length PLA chain (45,000 g.mol(-1), was used as the matrix material, the second, with short PLA chains (< or = 2.200 g.mol(-1)), and different HLB as the surfactant. Microspheres were prepared by the "simple" and "double" emulsion methods. The steric stabilization effect of the copolymers was investigated using confocal microscopy and X-ray photoelectron spectroscopy (XPS). RESULTS: Confocal microscopy and XPS analysis showed that the microspheres prepared using MPOE5K-PLA0.5K as the surfactant and MPOE-PLA45K copolymers as the matrix material had an MPOE coating present at the microsphere surface. This hydrophilic layer ensures steric stabilization of the particles. CONCLUSIONS: MPOE-PLA copolymers can be used for the preparation of particles instead of PVA and their use can be extended whenever a biocompatible and bioeliminable surfactant is required for biological or medical applications.     

Isolation of Vibrio cholerae O139 from the drinking water supply during an epidemic of cholera

In mid-1994, the public water supply was investigated in a medium-sized town in south India during an epidemic of cholera due to Vibrio cholerae O139. Vibrio cholerae O139 was isolated from the public water supply including one of the wells supplying the town, the central overhead tank, and domestic taps connected to the public supply. Following chlorination, the organism was no longer isolated from the water supply and the epidemic subsided. This demonstration of V. cholerae O139 in the drinking water supply of a town underlines the need for adequate treatment of the water supply.     

Structure of the O-specific polysaccharide of an Aeromonas trota strain cross-reactive with Vibrio cholerae O139 Bengal

The O-specific polysaccharide of an Aeromonas trota strain was isolated by hydrolysis of the lipopolysaccharide at pH 4.5 followed by gel-permeation chromatography and found to consist of hexasaccharide repeating units containing D-galactose, L-rhamnose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose in the ratios 1:1:2:1:1. Partial hydrolysis of the polysaccharide with 48% hydrofluoric acid resulted in selective removal of colitose to give a modified polysaccharide containing the other four sugar constituents. On the basis of methylation analysis and NMR spectroscopic studies of the initial and modified, colitose-free polysaccharide, it was concluded that the repeating unit of the O-specific polysaccharide has the following structure [sequence: see text] The known cross-reactivity between the strain studied and Vibrio cholerae O139 Bengal is substantiated by the presence of a common colitose-containing epitope shared by the O-specific polysaccharide of A. trota and the capsular polysaccharide of V. cholerae, which is thought to carry determinants of O-specificity.     

Induction of the lysogenic phage encoding cholera toxin in naturally occurring strains of toxigenic Vibrio cholerae O1 and O139

In toxigenic Vibrio cholerae, the CTX genetic element which carries the genes for cholera toxin (CT) is the genome of a lysogenic bacteriophage (CTXPhi). Clinical and environmental strains of V. cholerae O1 or O139 and stools that were culture positive for cholera were analyzed to study the induction and transmission of CTXPhi. To our knowledge, this is the first report of the examination of CTXPhi in clinical materials and in naturally occurring strains. DNA probe analysis revealed that 4.25% (6 of 141) of the isolated V. cholerae strains spontaneously produced a detectable level of extracellular CTXPhi particles in the culture supernatants whereas another 34.04% (48 of 141) produced CTXPhi particles when induced with mitomycin C. CTXPhi isolated from 10 clinical or environmental strains infected a CT-negative recipient strain, CVD103, both inside the intestines of infant mice and under laboratory conditions. All culture-positive stools analyzed were negative for the presence of CTXPhi both in the DNA probe assay and by in vivo assay for the infection of the recipient strain in infant mice. These results suggested that naturally occurring strains of toxigenic V. cholerae are inducible lysogens of CTXPhi but that cholera pathogenesis in humans is not associated with the excretion of CTXPhi particles in stools, indicating that induction of the phage may not occur efficiently inside the human intestine. However, in view of the efficient transmission of the phage under conditions conducive to the expression of toxin-coregulated pili, it appears that propagation of CTXPhi in the natural habitat may involve both environmental and host factors.     

Epidemiology and transmission of V. cholerae O1 and V. cholerae O139 infections in Delhi in 1993

In 1993, rectal swabs from clinically suspected cases of cholera admitted to the Infectious Diseases Hospital (IDH), Delhi were examined for Vibrio cholerae O1 and O139. Epidemiological data of 396 cholera cases were collected before the patients' discharge from IDH. Of the 1528 laboratory-confirmed cholera cases, 46% and 54% were caused by serotype O1 and O139 respectively. Both serotypes appeared and disappeared simultaneously, and peaked during the same time of the year. However, the two serotypes affected persons of different age groups; about 65% of the O1 cases occurred in children aged less than 10 years, whereas this age group accounted for 40% of the cases due to V. cholerae O139. Although there were some focal outbreaks due to serotype O139, both serotypes had almost similar geographical distributions. Important risk factors for transmission of cholera were almost equally prevalent in the majority of both types of cholera cases. Since the seasonality, geographical distribution, and risk factors for transmission were similar for both serotypes, the study indicates that the preventive and control measures are also likely to be similar. The study also shows that the emergence of V. cholerae O139 in 1993 did not affect the incidence, seasonality, and epidemiology of endemic V. cholerae O1 E1 Tor strains in Delhi.     

Unique organization of the CTX genetic element in Vibrio cholerae O139 strains which reemerged in Calcutta, India, in September 1996

We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional.     

Comparison of Vibrio cholerae O1 isolates by polymerase chain reaction fingerprinting and ribotyping

AIM: To determine reproducibility and accuracy of the Nikon Retinomax autorefractor when used with children who were made cycloplegic. METHODS: Autorefraction and retinoscopy or subjectively refined retinoscopy (where, under the patient's direction, the refraction was varied until the best visual acuity was achieved) were performed on the right eye of 47 children, age 11-93 months. Autorefraction was performed using the Nikon Retinomax, which provides up to eight measured values of refractive error and one representative measurement of refractive error. RESULTS: Autorefractor measurements were successfully obtained from 7/9 children age 3 years or younger, and from all older children. Vector methods were used to calculate differences. Retinomax reproducibility averaged 0.43 D. Unbiased Retinomax and retinoscopy measurements differed by an average of 0.82 D. Unbiased Retinomax and subjectively refined retinoscopy differed by an average of 1.03 D. CONCLUSIONS: Reproducibility of Retinomax measured values in children is comparable with reproducibility of retinoscopy, subjective refraction, and autorefraction measurements in adults. Agreement between Retinomax and retinoscopy and agreement between Retinomax and subjective refinement in children is comparable with agreement between autorefraction and subjective refraction in adults. The study indicates that the Retinomax is a useful instrument for measuring refractive errors in young children.     

Isolation of a new variant of Vibrio cholerae O1: V. cholerae O1 ribotype B27 toxinogenotype TB31 during the last cholera epidemic in Senegal

A total of 205 Vibrio cholerae O1 isolates from recent cholera epidemic in Senegal were analyzed by conventional methods, polymerase chain reaction (PCR) for genes encoding cholera toxin (ctx A), zonula occludens toxin (zot) and accessory cholera enterotoxin (ace), ribotyping and toxinogenotyping. Ribotyping after Bg1 I digestion of total DNA revealed that ribotype B5a, the predominant ribotype of the seventh pandemic in Africa and Asia, was not isolated. A new ribotype designated B27 in our database is predominant and was associated with a new toxinogenotype designated TB31.