Recircularization and Autonomous Replication of a Sheared R-Factor DNA Segment in Escherichia coli Transformants

Controlled shearing of R-factor DNA leads to formation of fragments carrying an antibiotic resistance gene present on, but not expressed by, the intact R-factor. Transformation of CaCl(2)-treated E. coli by such fragments yields an autonomously replicating tetracycline-resistance plasmid (Tc6-5) that contains only a small fraction of the genome of the parent R-factor, and lacks both its fertility functions and its other drug-resistance determinants. Although the Tc6-5 plasmid is not self-transmissible, it can interact and/or recombine with conjugally-proficient plasmids that promote its transfer to other bacteria.     

Non-Chromosomal Antibiotic Resistance in Bacteria, III: Isolation of the Discrete Transfer Unit of the R-Factor R1

A covalently-closed circular DNA species, banding at a buoyant density of rho = 1.709 g/cm(3) in CsCl, has been identified in antibiotic-sensitive colonies of E. coli strain AB2463 (rec A(-)) after mating with a Proteus mirabilis strain that carries the R-factor, R1. This plasmid, which represents a stable segregant of R1 that has lost all of the drug resistance determinants present on the parent R-factor but which has retained its ability to be transferred by conjugation, fulfills the functional definition of the R-factor transfer unit (RTF).     

Nature of R-factor replication in the presence of chloramphenicol.

Covalently closed circular deoxyribonucleic acid molecules of RSF1030, a nonconjugative R-factor, initiate and complete rounds of semiconservative replication in the absence of protein synthesis long after the bacterial chromosome has ceased its replication. RSF1030 replication under these conditions is sensitive to the inhibitor of ribonucleic acid synthesis, rifampicin. The product of this replication, a covalently closed DNA molecule, shows, in contrast to those molecules produced during the replication in a logarithmically growing culture of Escherichia coli, a transition to the open circular form upon treatment with ribonucleases of alkali. Analysis of the product resulting from alkali treatment indicates that there is a single break in one strand of the original circular duplex. This alkali-sensitive site occurs with equal probability in either of the complementary strands. These results are interpreted as a requirement for an RNA primer for the initiation of RSF1030 DNA synthesis and as showing that its removal from the covalently closed molecule is inhibited in the absence of protein synthesis.     

Antibiotic susceptibility of Helicobacter pylori in the Chinese population

AIM: To assess antibiotic susceptibility of Helicobacter pylori (H. pylori) strains to metronidazole, clarithromycin and tetracycline in the Chinese population, and to test the stability of antibiotic resistance in H. pylori 1 year after storage at -80 degrees C. METHODS: Fifty H. pylori strains isolated from patients with peptic ulcer disease were recovered from storage at -80 degrees C. Susceptibility of these strains to metronidazole, clarithromycin and tetracycline was determined by using validated disk diffusion tests, which was repeated 1 year after storage at -80 degrees C. The DNA profiles of each strain were determined by using the polymerase chain reaction-based-random amplified polymorphic DNA fingerprinting technique (PCR-RAPD). This was repeated if any change in antibiotic susceptibility pattern was noticed. RESULTS: The resistance rate was 50% to metronidazole and 8% to clarithromycin. None of the strains was resistant to tetracycline. A dual resistance to metronidazole and clarithromycin was demonstrated in three H. pylori strains. The antibiotic susceptibility test reproduced itself in 92% (36 of 39) of the strains 1 year later; the three strains with dual resistance exhibited susceptibility to both antibiotics. Variation in antibiotic susceptibility pattern in the three H. pylori strains was associated with change in the RAPD fingerprint. CONCLUSION: The prevalence of resistance in H. pylori is high to metronidazole but low to clarithromycin in the Chinese population. The disk diffusion test appears to be a simple and reliable test, while antibiotic resistance in some H. pylori strains may disappear after long-term storage at -80 degrees C.     

Genetic relatedness of antibiotic resistance determinants in multiply resistant Hemophilus influenzae

Antimicrobial resistance determinants and plasmids present in 10 multiply antibiotic-resistant strains of Hemophilus influenzae isolated from patients in different geographic regions of Spain were characterized. Conjugative plasmids with molecular sizes of 38-50 MDa encoded resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfamethoxazole, and tetracycline. Trimethoprim resistance was not linked to the other antibiotic resistance determinants and trimethoprim-resistant transconjugants and transformants lacked detectable plasmid DNA, suggesting that this determinant is chromosomal. Restriction endonuclease analysis revealed similarities among the plasmids, but several restriction patterns could be distinguished. Three hybridization patterns were found with DNA probes coding for H. parainfluenzae beta-lactamase and chloramphenicol-acetyltransferase. Resistance to kanamycin was due to drug modification by aminoglycoside-phosphotransferase (3')I. In Spain, it appears that multiple antibiotic resistance phenotypes in H. influenzae did not arise from acquisition of a single R plasmid; rather, both plasmid and chromosomal resistance evolved independently from several sources.     

Genetic and phenotype changes following in vitro interactions between Helicobacter pylori strains

BACKGROUND: The purpose of the present paper was to determine whether in vitro interaction between different Helicobacter pylori strains leads to changes in antibiotic susceptibility, cagA, vacAM2 and DNA fingerprint patterns. METHODS: Three H. pylori strains with known antibiotic susceptibility, cagA, vacAM2 status and polymerase chain reaction-random amplified polymorphic DNA (PCR-RAPD) fingerprint analysis were suspended in phosphate buffered saline (PBS pH 7.0), and the suspensions were mixed in equal proportion prior to culture on chocolate agar plates. Subcultures were performed five times every 3 days. As a control, each of the three strains was also subcultured separately. Antibiotic susceptibility testing, PCR for cagA, vacAM2 and PCR-RAPD analysis were done. RESULTS: Surviving strain of the two H. pylori strains in each combination demonstrated change in resistance to both antibiotics but no change in sensitivity. CagA status of the surviving strain varied as compared to the vacAM2 status, which did not change. The PCR-RAPD fingerprint showed unique band pattern. CONCLUSION: DNA transformation follows in vitro interaction. Helicobacter pylori strain with antibiotic resistance is likely to dominate in such in vitro interactions between various strains.     

Molecular mechanisms for transposition of drug-resistance genes and other movable genetic elements

Transposition is proposed to be responsible for the rapid evolution of multiply drug-resistant bacterial strains. Transposons, which carry the genes encoding drug resistance, are linear pieces of DNA that range in size from 2.5 to 23 kilobase pairs and always contain at their ends nucleotide sequences repeated in inverse order. In some transposons the terminal inverted repeat sequences are capable of independent movement and are called insertion sequences. Transposons carry a gene that encodes transposase(s), the enzyme(s) responsible for recombination of the transposon into another DNA molecule. Studies on transposable genetic elements in bacteria have not only given insight into the spread of antibiotic resistance but also into the process of DNA movement.     

Transformation of Pseudomonas putida and Escherichia coli with plasmid-linked drug-resistance factor DNA.

Conditions optimal for the transformation of Pseudomonas putida and E. coli with a drug-resistance factor (RP 1) DNA, which specifies resistance to carbenicillin, tetracycline, kanamycin, and neomycin, are described. The transformants retain all the fertility, incompatibility, and drug-resistance characteristics present in the parent. Covalently-closed circular molecules of almost identical contour lengths have been isolated from the parent and the transformants. The frequency of transformation is drastically reduced by treatment of RP 1 DNA with DNase and by denaturation or sonication. Shearing of RP 1 DNA in vitro and their subsequent introduction in P. putida cells, by transformation, produces transformants that exhibit a wide range of drug-resistant phenotypes, including those which are resistant to neomycin but sensitive to kanamycin. Isolation of such neomycin-resistant but kanamycin-sensitive transformants indicates that there might be two separate mechanisms specified by RP 1 for resistance to the two antibiotics.