Colonization of the gut of the blue crab (Callinectes sapidus) by Vibrio cholerae

Attachment of Vibrio cholerae to the mucosal surface of the intestine is considered to be an important virulence characteristic. Vibrio cholerae, an autochthonous member of brackish water and estuarine bacterial communities, also attaches to crustacea, a significant factor in multiplication and survival of V. cholerae in nature. The ability of V. cholerae to attach to the gut wall of the blue crab (Callinectes sapidus) was examined, and attachment was observed only in the hindgut and not the midgut of crabs, confirming a requirement for chitin in the attachment of V. cholerae to invertebrate and zooplankton surfaces. The new finding of attachment of V. cholerae to the hindgut of crabs may be correlated with the epidemiology and transmission of cholera in the aquatic environment. The crab model may also prove useful in elucidating the mechanism(s) of ion transport in crustacea.     

Value of stool examination in patients with diarrhoea.

Findings of stool examinations in 1593 patients with diarrhoea due to a single enteric pathogen--enterotoxigenic Escherichia coli rotavirus, Shigella, Campylobacter jejuni, Vibrio cholerae 0:1, Entamoeba histolytica, or Giardia lamblia--were reviewed to determine how well they predicted the agent associated with the diarrhoea. Specimens were examined visually for blood and mucus, tested for pH, and examined under a microscope for the presence of red and white blood cells, parasites, and stool fat. Although visible blood was more common in specimens from patients infected with Shigella (51%) and Ent histolytica (39%) than in those from patients infected with other agents (6%; p less than 0.01), patients infected with Shigella were most likely to have numerous faecal leucocytes (greater than 50/high power field: 39% v 8% of all patients and 7% of patients infected with Ent histolytica, p less than 0.01 in both cases). Patients infected with enterotoxigenic E coli, rotavirus, V cholerae 0:1, or C jejuni had loose stools with fewer red or white cells. Patients infected with rotavirus and C jejuni were more likely to have acid stools with 3 to 4+ fat, but these findings were related to young age and breast feeding. Stool examination is most useful in establishing a diagnosis of dysentery and in helping to distinguish between patients infected with Shigella and Ent histolytica; it is of limited usefulness in discriminating between pathogens causing watery diarrhoea.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods.

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

A novel peptide derived from human apolipoprotein E is an inhibitor of tumor growth and ocular angiogenesis

Angiogenesis is a hallmark of tumor development and metastasis and now a validated target for cancer treatment. We previously reported that a novel dimer peptide (apoEdp) derived from the receptor binding region of human apolipoprotein E (apoE) inhibits virus-induced angiogenesis. However, its role in tumor anti-angiogenesis is unknown. This study demonstrates that apoEdp has anti-angiogenic property in vivo through reduction of tumor growth in a mouse model and ocular angiogenesis in a rabbit eye model. Our in vitro studies show that apoEdp inhibits human umbilical vein endothelial cell proliferation, migration, invasion and capillary tube formation. We document that apoEdp inhibits vascular endothelial growth factor-induced Flk-1 activation as well as downstream signaling pathways that involve c-Src, Akt, eNOS, FAK, and ERK1/2. These in vitro data suggest potential sites of the apoE dipeptide inhibition that could occur in vivo.This is the first evidence that a synthetic dimer peptide mimicking human apoE has anti-angiogenesis functions and could be an anti-tumor drug candidate.     

Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.     

The mechanoelectrical response of the cytoplasmic membrane of Vibrio cholerae

Persistence of Vibrio cholerae in waters of fluctuating salinity relies on the capacity of this facultative enteric pathogen to adapt to varying osmotic conditions. In an event of osmotic downshift, osmolytes accumulated inside the bacterium can be quickly released through tension-activated channels. With the newly established procedure of giant spheroplast preparation from V. cholerae, we performed the first patch-clamp characterization of its cytoplasmic membrane and compared tension-activated currents with those in Esherichia coli. Saturating pressure ramps revealed two waves of activation belonging to the ∼1-nS mechanosensitive channel of small conductance (MscS)-like channels and ∼3-nS mechanosensitive channel of large conductance (MscL)-like channels, with a pressure midpoint ratio p_0.5MscS/p_0.5MscL of 0.48. We found that MscL-like channels in V. cholerae present at a density three times higher than in E. coli, and yet, these vibrios were less tolerant to large osmotic downshocks. The Vibrio MscS-like channels exhibit characteristic inward rectification and subconductive states at depolarizing voltages; they also adapt and inactivate at subsaturating tensions and recover within 2 s upon tension release, just like E. coli MscS. Trehalose, a compatible internal osmolyte accumulated under hypertonic conditions, significantly shifts activation curves of both MscL- and MscS-like channels toward higher tensions, yet does not freely partition into the channel pore. Direct electrophysiology of V. cholerae offers new avenues for the in situ analysis of membrane components critical for osmotic survival and electrogenic transport in this pathogen.     

Viable but Nonculturable Vibrio cholerae O1 in the Aquatic Environment of Argentina

In Argentina, as in other countries of Latin America, cholera has occurred in an epidemic pattern. Vibrio cholerae O1 is native to the aquatic environment, and it occurs in both culturable and viable but nonculturable (VNC) forms, the latter during interepidemic periods. This is the first report of the presence of VNC V. cholerae O1 in the estuarine and marine waters of the Río de la Plata and the Argentine shelf of the Atlantic Ocean, respectively. Employing immunofluorescence and PCR methods, we were able to detect reservoirs of V. cholerae O1 carrying the virulence-associated genes ctxA and tcpA. The VNC forms of V. cholerae O1 were identified in samples of water, phytoplankton, and zooplankton; the latter organisms were mainly the copepods Acartia tonsa, Diaptomus sp., Paracalanus crassirostris, and Paracalanus parvus. We found that under favorable conditions, the VNC form of V. cholerae can revert to the pathogenic, transmissible state. We concluded that V. cholerae O1 is a resident of Argentinean waters, as has been shown to be the case in other geographic regions of the world.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

Occurrence of Vibrio cholerae serotype O1 in Maryland and Louisiana estuaries.

Vibrio cholerae serotype O1 has been isolated from Chesapeake Bay in Maryland and estuaries and sewers in Louisiana. The occurrence of V. cholerae O1 in the aquatic environment in the absence of human disease suggests that this organism survives and multiples in the natural environment.