A pathogenic strain of Vibrio cholerae was attenuated by deletion of DNA sequences encoding the A1 subunit of the cholera enterotoxin. A restriction endonuclease fragment encoding the A1, but not the A2 or B sequences was deleted in vitro from cloned cholera toxin genes. The mutation was then recombined into the chromosome of V.cholerae Ogawa 395, a pathogenic strain that confers complete immunity to subsequent infection following an initial clinical infection. The resulting strain, which produces the immunogenic but nontoxic B subunit of cholera toxin and is unaltered in other critical antigens, represents a promising candidate for an attenuated live oral cholera vaccine.

An ideal vacine does not yet exist to prevent cholera, a significant health problem in many less developed countries. Vibrio cholerae, the agent of epidemic and endemic cholera, colonizes the small bowel and secretes a potent enterotoxin that consists of a single A subunit, which stimulates adenylate cyclase activity, and five identical B summits which bind to the ganglioside GM1 receptor of intestinal mucosal cells1. Previous studies in man indicate that toxoid-derived antitoxic immunity by itself is insufficient to provide effective, long-lasting protection against cholera2–4. Using recombinant DNA techniques we have now constructed a live, attenuated V. cholerae strain by deleting genes encoding the enterotoxin. Restriction enzyme fragments encoding cholera toxin were deleted in vitro from cloned vibrio chromosomal DNA and the resulting mutations introduced into the chromosome of a vibrio strain of proven immunogenicity. Recently, Mekalanos and coworkers5 have reported attenuated V. cholerae strains constructed by similar methods. It appears that recombinant DNA techniques offer a promising approach to the development of effective cholera vaccines.

We have determined the sequence of the DNA encoding the A2 (gamma) and B subunits of Vibrio cholerae enterotoxin. The order of the subunits as they would be transcribed is A2-B and the termination codon of the A2 subunit overlaps the initiation codon of the B subunit by four bases. Sequence analysis revealed a region capable of coding for a 21-amino acid leader peptide located at the NH2 terminus of the B subunit. While the nucleotide sequence homology between the cholera enterotoxin subunits and the analogous sequences of the genes for Escherichia coli heat-labile enterotoxin (LT) was 72 and 77% for the A2 and B subunits, respectively, the predicted amino acid sequences of the A2 subunits were less similar. Twenty-nine of 46 (63%) amino acids of the A2 subunits and 98 of 124 (79%) amino acids of the B subunits were identical between cholera enterotoxin and LT. The predicted amino acid sequence of the enterotoxin from a V. cholerae El Tor biotype strain reported here differs from the previously published amino acid sequences of the toxin from a classical biotype of V. cholerae at seven residues. Comparison of the A2 and B amino acid sequences among El Tor and classical biotypes of V. cholerae and E. coli LT demonstrates two regions of highly conserved sequences: 12 and 22 uninterrupted amino acids are the same among the A2 and B subunits, respectively, from the three strains.

We used numerical taxonomy procedures to analyze data obtained from 227 strains belonging to either the genus Vibrio or related genera; included were pathogenic and nonpathogenic vibrios isolated from a variety of samples and geographic locations. Each strain was tested for 150 unit characters. At a similarity coefficient of 70 to 75% or more, the 227 strains clustered into 33 phena, representing Vibrio anguillarum, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio metschnikovii, Aeromonas hydrophila, and new species of Vibrio. Non-O1 and O1 serovars of V. cholerae clustered at the species level of relationship (i.e., at a similarity coefficient of ≥75%). Furthermore, subdivision of V. cholerae into classical, El Tor, proteus, and albensis biovars was not observed. In fact, the biovar proteus was found to warrant separate species status as Vibrio proteus. Only the sucrose-negative members of Heiberg group V were distinguishable as a separate cluster and were recognized as a separate biovar. Sucrose-positive strains and urease-positive strains of V. parahaemolyticus were identified. The differences among the four groups of V. anguillarum examined did not warrant recognition of biovars.

Recombinant DNA risk assessment studies quantitated the mobilizability of "safe" plasmid pBR325, in comparison with readily mobilizable plasmid pJBK5 (chloramphenicol and tetracycline resistant). Of 15 volunteers who became colonized after ingestion of 5 X 10(10) Escherichia coli HS-4, a normal human flora strain containing pJBK5 and daily oral tetracycline, nine manifested transfer of pJBK5 to normal flora by means of triparental mating. In contrast, none of 12 other volunteers cocolonized with HS-4 bearing "safe" pBR325 and normal flora showed transfer (P = 0.001), despite ingestion of tetracycline. To accomplish transfer directly, E coli HS-4 containing both pBR325 and a derepressed, conjugative plasmid (F-amp) was fed to two groups of volunteers. Transfer of pBR325 to normal flora occurred in 13 of 18 volunteers taking daily tetracycline but in none of eight who did not (P less than 0.002). Nor were transconjugants detected, despite tetracycline ingestion, in five volunteers who ingested and excreted E coli K12 (pBR325 plus F-amp).

The distribution of in Chesapeake Bay during the warmer weather of the summer months was examined. This species was found throughout the Chesapeake Bay and its tributaries, even in areas of very low salinity. Counts of this species ranged from 0.04 per 100 ml to 46 per 100 ml in the water column and 2.03 to ≥2.4×10 per 100 cc of sediment. A variety of physical, chemical and bacteriological properties associated with the incidence and distribution of were examined and salinity was found to be the major influence among the factors examined. Correlation and regression analysis showed that the population size of this species increased with increasing salinity in the estuary.

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.

A microbiological survey of Aeromonas hydrophila in Chesapeake Bay and its tributaries showed that this species is ubiquitous, occurring in numbers ranging from <0.3/l to 5 × 103/ml in the water column and ca. 4.6 × 102/g in sediment. It was recovered from water samples collected at several locations in Chesapeake Bay representing various salinity regimes, but the numbers of A. hydrophila in higher salinity water, i.e. 15O/OO, were low. Results of stepwise multiple linear regression analysis showed that concentrations of A. hydrophila were correlated with total, aerobic, viable, heterotrophic, bacterial counts, and, in addition, were inversely related to salinity and to concentration of dissolved oxygen. Seasonal occurrence was recorded, with fewer strains of A. hydrophila encountered during the winter months. The potential pathogenicity of A. hydrophila strains isolated from Chesapeake Bay was estimated by testing selected isolates for toxigenicity, using the Y‐1 adrenal cell assay. Of 116 isolates tested, 83 (71%) produced a cytotoxic response, a characteristic found to be correlated with the lysine decarboxylase and Voges‐Proskauer reactions. Eight of 11 strains tested, which elicited fluid accumulation in the rabbit ligated ileal loop assay, also provoked a cytotoxic reaction in the Y‐l adrenal cell assay. Results of the study indicate that large numbers of toxigenic A. hydrophila can be found in an estuary and such strains may be pathogenic for man and/or animals.

The distribution ofVibrio parahaemolyticus in Chesapeake Bay during the warmer weather of the summer months was examined. This species was found throughout the Chesapeake Bay and its tributaries, even in areas of very low salinity. Counts of this species ranged from 0.04 per 100 ml to 46 per 100 ml in the water column and 2.03 to ≥2.4×103 per 100 cc of sediment. A variety of physical, chemical and bacteriological properties associated with the incidence and distribution ofV. parahaemolyticus were examined and salinity was found to be the major influence among the factors examined. Correlation and regression analysis showed that the population size of this species increased with increasing salinity in the estuary.

Counts of total viable, aerobic, heterotrophic bacteria, indicator organisms, and Aeromonas spp. were made at a diver training site on the Anacostia River in Washington, D.C. The numbers of Aeromonas cells in Anacostia River sediment and water increased during periods of elevated water temperature, to maxima of 4 � 10 5 cells per g of sediment and 300 cells per ml of water. Correspondingly, Aeromonas counts dropped 2 to 4 logs as the water temperature decreased to 0 to 0.5�C. Cultures taken by sterile swabs from the ears and face masks of divers after a 30-min swim in the Anacostia River yielded bacterial types and numbers similar to those found in the river. The nasal passages of the divers apparently did not become contaminated by swimming, possibly because of the protective effect of the face masks used by the divers. Properties associated with virulence in Aeromonas hydrophila and Aeromonas sobria strains isolated from the river, sediment, and divers were investigated. Nearly 40% of the strains of both species collected during the study produced cytotoxic activity for mouse Y-1 adrenal cells, as well as elastase. Enterotoxin activity, as detected by the Y-1 assay, was observed in 3% (1 of 35) of the strains of A. sobria and in 6% (19 of 330) of the A. hydrophila strains. Fluid accumulation in rabbit ileal loops induced by both species of Aeromonas varied greatly among the 17 strains examined. Fluid accumulation of at least 0.4 ml/cm was correlated with positive cytotoxin- or enterotoxin-like response in the Y-1 tissue culture assay.

A medium was devised for the rapid presumptive identification of Aeromonas hydrophila. It also offered good differentiation of Klebsiella, Proteus, and other enteric species. Mannitol fermentation, inositol fermentation, ornithine decarboxylation and deamination, indole production, motility, and H2S production from sodium thiosulfate and cysteine could be recorded in a single tube of the medium.