Molecular characterization of Vibrio cholerae O1 biotype El Tor strains isolated between 1992 and 1995 in Calcutta, India: evidence for the emergence of a new clone of the El Tor biotype

Sixty-one clinical strains of Vibrio cholerae O1 El Tor isolated in Calcutta before, during, and after the V. cholerae O139 Bengal outbreak were examined to see if the O1 strains of the post-O139 period were different from those in existence before. Comparison of the restriction fragment length polymorphism of the rRNA genes (ribotyping) and the CTX genetic element revealed that all "before" strains except 1 belonged to a single known ribotype, whereas all "after" strains except 2 belonged to a hitherto undescribed ribotype. Also, 23 of 25 "before" strains harbored two or more copies of CTX in tandem and also a "free" RS1 element away from CTX, whereas 19 of 21 "after" strains had a single copy of CTX and no free RS1 element. CTX occupied different chromosomal locations in "before" and "after" strains. These studies clearly showed that El Tor O1 strains, which displaced V. cholerae O139 in Calcutta, belonged to a new clone and suggested that there is a continuous genetic reassortment among El Tor strains of V. cholerae O1.     

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.     

Molecular characterization of Vibrio cholerae O1 strains isolated in Romania

A collection of 89 Vibrio cholerae O1 strains, isolated in Romania between 1977 and 1994, and 6 strains from the Republic of Moldavia, was characterized by ribotyping, toxin gene restriction pattern (toxinogenotype) and distribution of cholera toxin gene (ctx), accessory toxin gene (ace) and zonula occludens toxin gene (zot). After Bg/I endonuclease restriction of chromosomal DNA, a total of 18 ribotypes and 21 toxinogenotypes were distinguished. Deletions in the core region of the toxin gene cassette were found in 20% of strains; however, with the exception of one strain, all the isolates contained the ctx gene. Used in association, the three methods of molecular typing provided an accurate characterization of V. cholerae O1 isolates.     

Phenotypic expression of a mannose-sensitive hemagglutinin by a Vibrio cholerae O1 E1Tor strain and evaluation of its role in intestinal adherence and colonization

A Vibrio cholerae O1 strain (1150) of the EIT or biotype and Ogawa serotype with haemagglutination (HA) activity was subjected to TnphoA mutagenesis. Out of several mutants isolated, one HA- and another HA+ mutant were further characterised. The HA- mutant showed about 50% reduction in its intestinal adherence capacity in vitro and about 9-fold decrease of its colonisation ability in vivo, as compared to the wild-type strain. Subsequent studies showed that the HA activity of strain 1150 was mediated by a mannose-sensitive haemagglutinin (MSHA). Thus, the phenotypic expression of MSHA appears to be partly responsible for the intestinal adherence and colonisation properties of strain 1150.     

Molecular characterization of Vibrio cholerae O1 strains isolated during cholera outbreaks in Guinea-Bissau

In the present study, 19 strains of Vibrio cholerae O1 biotype El Tor isolated during outbreaks of cholera in Guinea-Bissau in 1987, 1994, and 1995 were characterized to investigate a possible epidemiological relationship among the isolates. On the basis of ribotyping with the restriction enzyme BglI, 5 strains isolated in 1987 showed two closely related ribotypes, while 14 strains isolated in 1994 and 1995 showed the same ribotype that was distinct from the ribotypes of strains isolated in 1987. Southern blot hybridization of BglI-digested genomic DNA with a cholera toxin probe demonstrated that the strains isolated in 1987 showed an identical cholera toxin genotype, whereas O1 strains isolated in 1994 and 1995 showed the same genotype that was distinct from the genotype of strains isolated in 1987. These results were supported by the results of antibiotic susceptibility testing, in which strains isolated in 1987 showed resistance to polymyxin B only, while each of the strains from 1994 and 1995 showed resistance to polymyxin B, trimethoprim-sulfamethoxazole, and the vibriostatic agent O/129. Although our results are based on a limited number of V. cholerae O1 strains, they suggest that the epidemic in Guinea-Bissau in 1994 and 1995 was due to the introduction of a new strain to the country.     

New evidence for an inflammatory component in diarrhea caused by selected new, live attenuated cholera vaccines and by El Tor and Q139 Vibrio cholerae

Using a lactoferrin latex agglutination assay, we have compared the inflammatory responses to a cholera vaccine candidate, CVD 110, in which all known toxin genes have been deleted or mutated yet still produced significant diarrhea, with a less reactive vaccine strain and wild-type El Tor and 0139 Vibrio cholerae strains. Data suggest that diarrhea due to attenuated and wild-type El Tor V. cholerae, and to a lesser extent 0139 V. cholerae, involves an inflammatory response. Further study is required to further elucidate the mechanism of the process(es) involved.     

Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation

Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by V. cholerae TSI-4/R was found to have a composition of N-acetyl-D-glucosamine, D-mannose, 6-deoxy-D-galactose, and D-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.     

Molecular epidemiology of Vibrio cholerae O1 isolated from sporadic cholera cases in Okinawa, Japan

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.     

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.     

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.     

Vibrio cholerae O1 in fecal samples from the urban population of Manacapuru, AM

The study was carried out to identify asymptomatic carriers of V. cholerae O1 in Manacapuru, AM. 1249 feces samples was obtained by rectal swab and cultivated. Had no growth of V. cholerae. On the other hand were isolated and identified: V. furnissii in 12 (0.9%) samples, V. fluvialis in 4 (0.3%) and V. hollisae in 1 (0.1%).     

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.     

Preliminary evidence that cell death may contribute to separation of the trachea from the primitive foregut in the rat embryo

BACKGROUND/PURPOSE: The embryogenesis of many congenital tracheoesophageal abnormalities has been elucidated poorly, mainly because of an incomplete understanding of the normal mechanism of separation of the primitive foregut into the trachea and esophagus. There has been controversy about the existence and significance of the so-called tracheoesophageal septum. This study investigates the normal mechanism of tracheoesophageal separation in the rat. METHODS: Timed-pregnant Sprague-Dawley rats were killed on gestational days 11 to 16, respectively (the day on which the vaginal smear showed sperm was considered day 0 of gestation). Thirty-six embryos (six embryos from each age group) were fixed in 10% formalin. Fixed embryos from gestational day 13, 12, and 11 were sandwiched in melted agar to facilitate proper orientation before being processed. Half of the fetuses from each age group were sectioned serially in a transverse plane and the other half in a sagittal plane. The histological sections were stained with H&E. RESULTS: On gestational day 11, the foregut appears as a ventrodorsal slit in transverse section. Below the primitive pharynx, the epithelial cells on the ventral part of the foregut are actively proliferating, producing two lateral epithelial bulges (primitive bronchial buds). The epithelial cells of the dorsal aspect of the foregut show no sign of active growth. On day 12, the proximal trachea still shares a common lumen with the foregut. The distal trachea, the tracheal bifurcation, and both primary bronchi are recognized easily. At the point of tracheoesophageal separation, there is obvious debris of dead epithelial cells and condensed nuclei. These appearances were specifically located around the so-called tracheoesophageal septum and nearby grooves, resulting in enfolding cristae. There was no cell proliferation or inflammatory response in this region. On gestational day 13, the separation of the trachea and esophagus was almost complete. CONCLUSION: The present study demonstrates that a specific and consistent pattern of cell death in the region of the "tracheoesophageal septum" produced enfolding cristae that may be part of the mechanism of separation of the trachea and esophagus.     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)2, similar to the transposition intermediates described for a number of IS elements.     

Molecular analysis of pcc1, a gene that leads to A-regulated sexual morphogenesis in Coprinus cinereus

A homokaryotic strain (5337) in our culture stock of Coprinus cinereus produced fertile fruit bodies after prolonged culture. Microscopic examination revealed that hyphae dedifferentiated from the tissues of one of the fruit bodies, as well as all basidiospore derivatives from the fruit body, exhibited pseudoclamps, whereas vegetative hyphae of 5337, from which the fruit body developed, had no clamp connections. Genetic analysis showed that the formation of pseudoclamps results from a recessive mutation in a gene designated pcc1 (pseudoclamp connection formation), which is distinct from the A and B mating type genes. Cloning and sequencing of the pcc1 gene and cDNA identified an ORF of 1683 bp interrupted by one intron. Database searches revealed that pcc1 encodes an SRY-type HMG protein. The HMG box shared 44, 41, and 29% sequence identities (>80 amino acids) to those of FPR1 of Podospora anserina, MAT-Mc of Schizosaccharomyces pombe, and prf1 of Ustilago maydis, respectively. Northern analysis revealed that the level of pcc1 expression is higher in the dikaryon, in homokaryons in which the A and B mating type developmental sequences are individually activated, than in the homokaryon in which these sequences are not active. Sequencing of the pcc1-1 mutant allele revealed that the mutant carries a nonsense mutation at serine 211, a residue located between the HMG box and the C terminus. Based on these results, possible roles of the pcc1 gene in the sexual development of homobasidiomycetes are discussed.     

Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, was initiated in January 1994. The incidence of cases of V. parahaemolyticus infection suddenly increased in February 1996 and has remained high since then. One hundred thirty-four strains of V. parahaemolyticus isolated from January 1994 to August 1996 were examined for serovar, the presence of the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin genes (trh1 and trh2), production of urease, and antibiogram. Strains of the O3:K6 serovar appeared for the first time in February 1996. The O3:K6 serovar strains accounted for 50 to 80% of the strains isolated during the high-incidence period (February to August 1996). All of the serovar O3:K6 strains carried the tdh gene but not the trh genes and did not produce urease. All of the isolates except two were sensitive to all of the antibiotics tested. These and the results of analysis by an arbitrarily primed PCR method indicated that the O3:K6 serovar strains belong to a unique clone. When the O3:K6 serovar strains, isolated from travelers arriving in Japan from Southeast Asian countries, were compared by the arbitrarily primed PCR method, the strains isolated between 1982 and 1993 were distinct from Calcutta O3:K6 while the strains isolated in 1995 and 1996 were indistinguishable from the Calcutta O3:K6 strains. The results suggest that this unique O3:K6 clone may have become prevalent not only in Calcutta but also in Southeast Asian countries very recently. Not only the O3:K6 strains but also the non-O3:K6, tdh-bearing strains isolated in 1996 produced thermostable direct hemolysin at high levels, and thus the level of hemolysin produced does not appear to have influenced the high incidence of serovar O3:K6 strains.     

Viral determinants of HIV-1 sufficient to extend tropism to macrophages are distinct from the determinants that control the cytopathic phenotype in HL-60 cells

DESIGN: Infection of the human promyelocytic cell line HL-60 with NL4-3, a molecularly cloned HIV-1 strain that productively infects T cells, results in adaptation of the virus and production of a variant, NL4-3(M). Unlike NL4-3, NL4-3(M) has a rapid cytopathic effect in HL-60 and other myeloid cell lines. OBJECTIVE: To demonstrate that the tropism of NL4-3(M) is extended to primary monocyte-derived macrophages (MDM), and to determine whether the envelope gene, env, of NL4-3(M) is responsible for cytopathicity in HL-60 cells and replication in MDM. METHODS: A chimeric virus (NL4-3envA) containing the majority of env of NL4-3(M) was generated, and tested for virus replication and cytopathic effect in H9 and HL-60 cells, as well as for virus replication in primary MDM. To assess virus replication, the cultures were analyzed for expression of viral envelope glycoproteins on the infected cells and production of extracellular HIV-1 p24 antigen. Cytopathic effect on HL-60 cells was evaluated by monitoring the viabilities of the cultures. In addition, the majority of env of NL4-3envA was sequenced. RESULTS: The biological phenotypes of NL4-3, NL4-3(M), and NL4-3envA are distinctly different. Although both NL4-3(M) and NL4-3envA replicate in MDM, only NL4-3(M) is rapidly cytopathic in HL-60 cells. Nine amino-acid changes were identified within the envelope glycoproteins of NL4-3envA compared with NL4-3. CONCLUSIONS: The viral determinants of NL4-3(M) sufficient to extend the tropism of this virus to MDM reside, in part, in env. These genetic determinants are distinct from the viral determinants that control the cytopathic phenotype of this virus in HL-60 cells.     

In vivo evidence that the stromelysin-3 metalloproteinase contributes in a paracrine manner to epithelial cell malignancy

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699-704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3-/-) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7, 12-dimethylbenzanthracene-induced tumorigenesis in ST3-/- mice. Moreover, ST3-/- fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.     

Interleukin (IL)-1beta increases glucose uptake and induces glycolysis in aerobically cultured rat ovarian cells: evidence that IL-1beta may mediate the gonadotropin-induced midcycle metabolic shift

This communication explores the possibility that interleukin (IL)-1beta, a putative intermediary in the ovulatory process, may take part in the gonadotropin-driven midcycle diversion of ovarian carbohydrate metabolism toward glycolysis. We examined the effect of treatment with IL-1beta on glucose metabolism in aerobically cultured whole ovarian dispersates from immature rats. Treatment with IL-1beta increased cellular glucose consumption/uptake, stimulated extracellular lactate accumulation and media acidification, and decreased extracellular pyruvate accumulation in a receptor-mediated, time-, dose- and cell density-dependent manner. Endogenous IL-1beta-like bioactivity was shown to mediate the ability of gonadotropins to exert these same metabolic effects. The IL-1beta effect was also (1) apparent over a broad range of glucose concentrations, inclusive of the putative physiological window; (2) relatively specific, because tumor necrosis factor-alpha and insulin were inactive; (3) contingent upon cell-cell cooperation (4) and reliant on de novo protein synthesis. Comparison of the molar ratios of lactate accumulation to glucose consumption in IL-1beta-replete vs. IL-1beta-deplete cultures suggests that IL-beta promotes the conversion of all available glucose to lactate but that other substrates for lactate production may also exist. However, no lactate was generated by cells grown under glucose-free conditions. Taken together, our data suggest that IL-1beta may act as a metabolic hormone in the ovary. Subject to the limitations of the in vitro paradigm, our data also suggest that IL-1beta may mediate the gonadotropin-associated midcycle shift in ovarian carbohydrate metabolism. By converting the somatic ovarian cells into a glucose-consuming glycolytic machinery, IL-1beta may establish glycolysis as the main energy source of the relatively hypoxic preovulatory follicle and the resultant cumulus-oocyte complex. The consequent oxygen sparing may conserve the limited supply of oxygen needed for vital biosynthetic processes such as steroidogenesis. This adaptational response may also provide the glycolytically incompetent oocyte with the obligatory tricarboxylic cycle precursors it depends on to meet the increased energy demands imposed upon it by the resumption of meiosis.     

Metaphyseal chondrodysplasia type Schmid mutations are predicted to occur in two distinct three-dimensional clusters within type X collagen NC1 domains that retain the ability to trimerize

Metaphyseal chondrodysplasia type Schmid (MCDS) is caused by mutations in COL10A1 that are clustered in the carboxyl-terminal non-collagenous (NC1) encoding domain. This domain is responsible for initiating trimerization of type X collagen during biosynthesis. We have built a molecular model of the NC1 domain trimer based on the crystal structure coordinates of the highly homologous trimeric domain of ACRP30 (adipocyte complement-related protein of 30 kDa or AdipoQ). Mapping of the MCDS mutations onto the structure reveals two specific clusters of residues as follows: one on the surface of the monomer which forms a tunnel through the center of the assembled trimer and the other on a patch exposed to solvent on the exterior surface of each monomeric unit within the assembled trimer. Biochemical studies on recombinant trimeric NC1 domain show that the trimer has an unusually high stability not exhibited by the closely related ACRP30. The high thermal stability of the trimeric NC1 domain, in comparison with ACRP30, appears to be the result of a number of factors including the 17% greater total buried solvent-accessible surface and the increased numbers of hydrophobic contacts formed upon trimerization. The 27 amino acid sequence present at the amino terminus of the NC1 domain, which has no counterpart in ACRP30, also contributes to the stability of the trimer. We have also shown that NC1 domains containing the MCDS mutations Y598D and S600P retain the ability to homotrimerize and heterotrimerize with wild type NC1 domain, although the trimeric complexes formed are less stable than those of the wild type molecule. These studies suggest strongly that the predominant mechanism causing MCDS involves a dominant interference of mutant chains on wild type chain assembly.