Rintaro Nakaya

Molecular nature of the drug-resistance factors of the enterobacteriaceae

A drug-resistance episome (R-factor) carrying the determinants of resistance to streptomycin, chloramphenicol, tetracycline and sulfonamide was transferred from an Escherichia coli donor to Proteus mirabilis, E. coli and Serratia marcescens. DNA was isolated from the recombinant strains and analyzed by CsCl density-gradient centrifugation. The R-factor was shown to consist of two species of DNA, the base composition of which is 68% and 52% G + C, respectively. The molecular weight of the R-factor is 25 million daltons; the 58% component of the R-factor comprises about 80% of the episomic DNA, the other 20% being 52% in G + C content. Treatment of bacterial cells harboring this R-factor with either acridine dyes or ultraviolet light was shown to prevent the replication of the episome, so that the episomic DNA was eliminated from such “cured” cells. In addition, it was found that the E. coli K12 substrain (W677) used in these studies carried a naturally occurring DNA species of 64% G + C which was shown to be a plasmid or episome of unknown function. Evidence is presented that there is only one copy of the R-factor per chromosome in E. coli and S. marcescens. In P. mirabilis, however, there appear to be about ten copies of the R-factor per chromosome.

Cytotoxic Component(s) of Klebsiella oxytoca on HEp-2 Cells

A cytotoxic component(s) was detected in culture filtrates of Klebsiella oxytoca isolated from patients with antibiotic‐associated hemorrhagic colitis. Eleven of 12 isolates exhibited cytotoxicity on HEp‐2 cells. The cytotoxic activity of K. oxytoca strain MH43‐1 was stable for the treatment of 60 C for 30 min, but inactivated by the treatment of 100 C for 15 min. This cytotoxicity was not destroyed by the treatment with trypsin or pronase, and the component was filtrable through a membrane filter which cut off molecular weight 5,000.

Bioluminescent assay as a potential method of rapid susceptibility testing

A method of rapid susceptibility testing by bioluminescent assay was developed. Correlation between the 50% inhibition dose of antimicrobics for bacterial adenosine triphosphate measured by bioluminescent assay and the minimum inhibitory concentration obtained by the broth dilution method was satisfactory. In the bioluminescent assay the incubation time required was only 90 min.

R Plasmids among Gram-Negative Bacteria with Multiple Drug Resistance Isolated in a General Hospital

The incidence of conjugative R plasmids in multiple drug‐resistant strains of gram‐negative bacteria isolated in 1973 from patients in a 700‐bed general hospital in Tokyo and some properties of the R plasmids isolated are described. Conjugative R plasmids were found in 52 of the 96 strains (54%), from which 74 R plasmids were demonstrated. It is remarkable that the isolation frequency of R plasmids mediating quadruple‐ or five‐drug resistance was rather low, and the complete pattern of multiple resistance of the original isolates was only rarely transferred by conjugation. These results revealed the existing state of the distribution of R plasmids among hospital strains with multiple drug‐resistance.

The 106‐Kilobase Plasmid of Salmonella braenderup and the 100‐Kilobase Plasmid of Salmonella typhimurium Are Not Necessary for the Pathogenicity in Experimental Models

Among 1,041 clinical isolates (77 serovars) of Salmonella which had been derived from cases with acute enterocolitis, 601 (58%) contained one or more plasmids. Large serovar‐specific plasmids were seen in 95 of 307 isolates (31%) of Salmonella typhimurium, in 34 of 34 isolates (100%) of Salmonella enteritidis and in 36 of 38 isolates (94.7%) of Salmonella braenderup: the sizes of which were 100, 60 and 106 kilobases (kb), respectively. In order to determine the role of these plasmids in pathogenicity for enterocolitis, the plasmids were eliminated from some strains of S. braenderup and S. typhimurium and the pathogenicity of the plasmid‐less strains was compared with that of the parent strains by invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, lesion of mucosal tissue and the Sereny test. The virulence of all the plasmid‐less strains was as strong as that of the plasmid‐bearing strains in these pathogenicity assay systems. We therefore concluded that the 106‐kb plasmid of S. braenderup and the 100‐kb plasmid of S. typhimurium are not necessary for their pathogenicity in the experimental models: invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, and Sereny test.

Temperature sensitive R plasmid originated from Salmonella typhimurium

Summary Tetracycline(TC) resistance in a strain of Salmonella typhimurium was easily transfered to an Escherichia coli K12 substrain at 30 C, but rarely at 37 C. Whereas, transfer of the other concomitant resistances, sulfathiazole(SA) and streptomycin(SM) were not restricted at 37 C. In addition, these drug resistances easily segregated either to (SA SM) or (TC) exclusively on the conjugal transfer. These indicate that (SA SM) and (TC) resistances are carried separately in two independent R plasmids in this S. typhimurium strain. Our additional transfer experiments between E. coli strains suggest that these R plasmids exist as seperate replicon also in E. coli , but they tend to associate on conjugal transfer. In addition to the temperature sensitive transfer, TC resistance was specifically eliminated either from S. typhimurium or E. coli host cell at 42 C.

Electron Microscopic Observation of New Transposable Elements Inserted into P22 Phage Genome from R Plasmids

By using phage P22spl, a deletion mutant of phage P22, the structures of two new transposons on P22 genomes were studied by the electron microscopic heteroduplex method. One of these was the Cm (chloramphenicol) transposon derived from an R plasmid, NR1, and the other the Km (kanamycin) transposcn from pNR502. The heteroduplex between P22 phage DNAs with and without the Cm transposon revealed that the Cm transposon was similar in structure to the Tn9 element, a well‐known Cm transposon derived from the R plasmid pMS14. On the other hand, the Km transposon of pNR502 was quite different in structure from other Km transposons reported previously. This transposon consists of a 6.8 kilo‐base (kb) segment of DNA, in which a short inverted repeat is contained. The heteroduplex experiments showed that a 4.5 kb segment of DNA was deleted from the P22 genome in the P22spl genome. Because of a shorter unit length of the genome, phage P22spl is considered to be useful for assaying various kinds of transposable elements.

R plasmids with thermosensitive transferability in Salmonella strains isolated from humans.

Temperature dependence of transfer was examined with ten R plasmids originating from clinical isolates of Salmonella. Six of the plasmids were thermosensitive upon transfer, five of which were originally harbored in S. typhimurium and the remaining one in S. derby. One of these plasmids, pNR502, which conferred resistance to kanamycin, streptomycin (Sm) and tetracycline (Tc) on its host was stably maintained both in Salmonella and Escherichia coli at either 30, 37, or 43 C. Another plasmid, pNR516, which was resistant to chloramphenicol, sulfathiazole, Sm and Tc, was slightly unstable only at 43 C. The remaining four plasmids, pNR503, pNR510, pNR512 and pNR514, conferred resistance to Sm and Tc. Of these plasmids, the former two were stably maintained at both 30 and 37 C, but were unstable at 43 C. The latter two were slightly unstable at the lower temperatures and considerably unstable at 43 C. Kinetics of the transfer of the plasmid pNR503 revealed that the efficiency of transfer of the plasmid between E. coli strains was affected not only by the temperature of the conjugation but also by the preincubation temperature of the donor culture before the conjugation.

Abnormalities in the Fecal Flora of Subacute Myelo-Optic Neuropathy (SMON) Patients and Effects of Clioquinols on Human Intestinal Flora

The fecal flora of 51 SMON patients were cotpared with those of 35 healthy adult subjects. Marked abnormalities were found in the flora of SMON patients, who had received clioquinols for a long period. The total counts and the counts of anaerobic bacterial species in the feces of SMDN patients were found to be markedly reduced. Sixteen cases of the patients were reexamined on their flora one year after discontinuation of clioquinol, and nine of them shewed inprovement. These results suggested that the administration of clioquinols was closely associated with the abnormalities. When the flora of seven healthy adults were examined in the presence or absence of clioquinol in the plating media, similar changes were observed. Sensitivity test to clioquinol with 26 strains of several predominant bacterial species revealed that most anaerobes except bifidobacteria were sensitive, while aerobes were insensitive. It was shown that clioquinols did not inhibit the intracellular multiplication of virulent Shigella flexneri and enteropathogenic Escherichia coli in the in viro experimental infection model. Clioquinol proved to exhibit both growth inhibition and cytotoxicity to cultured cells at the concentrations of 8 µg per ml and higher.

Interactions between two heterogenic R plasmids: Cointegrative suppression of the thermosensitive replication of Rts1 by a nonconjugative derivative of NR1

Abstract A recombinant plasmid, R428, between a temperature-sensitive R plasmid, Rts1, and a transfer-deficient mutant of NR1, NR1–4, was isolated. Experiments on stability at higher temperature, transfer frequency, and density profiles of DNAs suggested that R428 is a cointegrate and that the thermosensitivity of Rts1 replication was suppressed.