Antibiotic-resistant strains of Aeromonas hydrophila have been isolated from the natural environment in the Chesapeake Bay and areas surrounding Dacca and the Matlab region of Bangladesh. The Bangladesh strains carried resistance to chloramphenicol, streptomycin, and tetracycline, and 57% of them had a multiple streptomycin-tetracycline resistance phenotype correlated with the presence of a large plasmid. The Chesapeake Bay strains were resistant to polymyxin B ane tetracycline, but showed neither multiple resistance nor R-factor carriage. Twenty-five percent of the environmental strains were toxigenic in a Y-1 adrenal cell assay. Toxigenicity showed no positive correlation with drug resistance or with plasmid carriage. Environmental areas of heavy human impact appear to be associated with a higher incidence of antibiotic-resistant strains of aeromonads.

In bacterial transformation, the irreversible uptake by a cell population of DNA extracted from a variant strain is followed by integration and expression of the variant "information" carried by the transforming DNA. Little is known of the details or mechanism of transformation after uptake of the DNA by a cell, i.e., of the process of integration which results in a permanent hereditary change. One must conclude from studies on linked transforming factors, however, that some process of recombination is involved.1 3 The studies reported in this paper were designed to determine the time and some of the conditions for recombination during transformation.Goodgal and Herriott have shown that a population of cells which has irreversibly taken up transforming DNA releases a large part of this activity when lysed shortly after uptake.4 In the experiments to be described, the intracellular fate of transforming DNA has been followed quantitatively by this technique in the case of two linked markers located on the same DNA molecule. Goodgal (3) has shown that in Hemophilus influenzae the two factors responsible for resistance or sensitivity to the antibiotics streptomycin and cathomycin (Novobiocin) are linked. When DNA is extracted from donor cells resistant to both cathomycin and streptomycin and is used to transform cells sensitive to both antibiotics, some 20 per cent of the resistant clones are endowed genetically with resistance to both antibiotics. The remainder of the transformations are to streptomycin resistance alone and to cathomycin resistance alone. The degree of linkage obtained depends to some extent on the method of preparation of the DNA and the conditions of transformation, but for material treated in the same way, the fraction of linked transformations is the same. The failure to retain complete linkage may be attributed to loss or inactivation of one or the other marker in the process of extracting the DNA from the donor cells, in the process of transformation itself, or in both processes.One can transform to streptomycin or cathomycin sensitivity as well as to resistance. The frequency of transformation to sensitivity is the same as to resistance, and the same linkage relationships obtain.3 Resistance and sensitivity thus may be considered as alleles of the same locus, the frequency of genetic incorporation of either or both factors being a function of the spatial relationships between the loci and not of the particular forms utilized in the test.3 When, in the course of transformation, both streptomycin and cathomycin loci are genetically incorporated, linked transformations are obtained. When only one locus is genetically incorporated, transformations result 'which are recombinant with respect to the two markers in question, one marker supplied by the donor population and the other by the recipient population. This is the restricted manner in which the term recombination will be used in this paper, i.e., recombination between two linked loci on a single DNA molecule. 505

An F' factor, FS400, carrying the his operon, the gnd gene, and the rfb gene cluster of Salmonella typhimurium was isolated. FS400 was introduced into an Escherichia coli strain having a lengthy deletion of the his gene region. From this strain, Hfr derivatives were isolated which had the F' factor integrated in the tonB locus near the attachment site of 480. One of the Hfr strains was lysogenized with a heat-inducible, h mutant of X80, and from this strain a highfrequency transducing phage carrying the his genes and the gnd gene of Salmonella was isolated. MATERIALS AND METHODS Bacterial strains. The bacterial strains used are listed in Table 1. Strains with SB or TA numbers are from the collections of P. E. Hartman or B. N. Ames, respectively. All S. typhimurium strains are derivatives of LT2, except SB5404, which is a derivative of LT7. E. coli strain SB2201 was derived from E'36 (Table 1). E'36 was first deleted of the his genes by education with P2c5nipl (14; T. Blumenthal, Virology, in press), and then a lacderivative was selected on E M B lactose medium at 37 C. The resulting strain, SB2201, was found to be lacking hisG, hisD, hisA, hisI, and hisE gene activity by mating with hisS. typhimurium strains harboring F' E. coli his episomes mutated in these genes (8), and hence is presumably deleted for the entire his operon. It is also deleted for gnd (Table 3). SB2201, and also SB1541, contain UAG suppressors. Strains were cultured at 37 C, except for 480Ximm lysogens which were cultured at 30 C. E'36, X178, and X-33 were kindly supplied by William S. Reznikoff. Bacteriophage. P22L4 (27) was used for transduction of S. typhimurium, and P22H5 [v2 mutant of Zinder (33)] was used for testing strains for sensi-741

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Vibrio cholerae strains of the 01 serovar, isolated from both clinical and environmental sources, had a much lower frequency of plasmid carriage (2/112 = 2%) than clinical and environmental non-01 serovar isolates (46/187 = 25%). The cryptic plasmids found in non-01 strains were all of low molecular weight and were shown by hybridization analysis to consist of two unrelated subgroups. Each subgroup was observed to be present in strains isolated from both clinical and environmental sources. One 01 serovar (ATCC 14033) carried a small cryptic plasmid, belonging to one of these plasmid groups, while a second (25728) contained a high molecular weight multiple antibiotic resistance plasmid which did not hybridize to either plasmid subgroup.