Mapping of the gene specifying aminoglycoside 3'-phosphotransferase II on the Pseudomonas aeruginosa chromosome.

We examined the aminoglycoside inactivation enzymes in Pseudomonas aeruginosa strains, seven clinical isolates and seven laboratory strains without plasmids. All strains were found to possess the enzyme aminoglycoside 3'-phosphotransferase II [APH(3')-II]. We isolated an APH(3')-II-deficient mutant from a PAO strain by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. By plasmid (FP5 or R68.45)-mediated conjugation, we determined the locus of the gene specifying the APH(3')-II between trp-6 and pro-82 on the PAO chromosome and designated this gene aphA. It was concluded that the intrinsic resistance of P. aeruginosa to kanamycins, neomycins, paromomycins, ribostamycin, and butirosins was due to this newly determined gene

Sex Pili Mutants Isolated by Macarbomycin Treatment

Macarbomycin (MM) preferentially kills Escherichia coli carrying episomes derepressed for sex pili formation (drd episomes). Using MM, we have isolated various types of mutants from both F-lac.tet and R100-1 episomes and have classified them into two types by sex pili formation. Among 26 mutants, 20 could not produce pili and had lost both the ability for conjugal transmission and sensitivity to male phages. From 12 of these 20 mutants, revertants capable of pili formation could be obtained. Most of these had simultaneously regained susceptibility to MM, indicating that the MM susceptibility of E. coli carrying drd episomes is due to pili formation. Three of the revertants could not confer MM susceptibility on their host and two of them could not produce normal pili, indicating that there are some types of pili insensitive to MM. The six MM-resistant mutants could produce pili, but the frequency of pili formation was slightly lower than that of their parent episomes. Mutants of this type exhibited various degrees of decrease in conjugal transferability or male phage sensitivity, and one mutant produced pili that were altered in shape. It is suggested that this latter mutant became MM-resistant as a result of changes in the chemical composition of its pili

Tn2001, a transposon encoding chloramphenicol resistance in Pseudomonas aeruginosa.

We isolated a new transposon, Tn2001, from the group P-2 plasmid Rms159-1 in Pseudomonas aeruginosa. Tn2001-encoded chloramphenicol resistance did not result from the formation of chloramphenicol acetyltransferase. Tn2001 was transposable between temperate phages and conjugative and nonconjugative plasmids belonging to various incompatibility groups, including P-1, P-3, P-4, P-5, P-7, and P-8 in P. aeruginosa. Transposition occurred independently of the general recombination ability of the Pseudomonas host, and its frequency varied between 10(-1) and 10(-8), depending upon the donor and recipient replicons. Tn2001 transposition also occurred in a recombination-deficient strain of Escherichia coli. Agarose gel electrophoresis and electron microscopic observations revealed that Tn2001 could transpose to different sites in the RP4 replicon and that the transposed deoxyribonucleic acid fragment was 2.1 kilobases long

Transferable imipenem resistance in Pseudomonas aeruginosa.

We isolated an imipenem-resistant strain, GN17203, of Pseudomonas aeruginosa. The strain produced a beta-lactamase that hydrolyzed imipenem. The beta-lactamase was encoded by a 31-MDa plasmid, pMS350, which belongs to incompatibility group P-9. The plasmic conferred resistance to beta-lactams, gentamicin, and sulfonamide and was transferable by conjugation to P. aeruginosa but not to Escherichia coli. The molecular weight of the purified enzyme was estimated to be 28,000, and the isoelectric point was 9.0. The enzyme showed a broad substrate profile, hydrolyzing imipenem, oxyiminocephalosporins, 7-methoxycephalosporins, and penicillins. The enzyme activity was inhibited by EDTA, iodine, p-chloromercuribenzoate, CuSO4, and HgCl2 but not by clavulanic acid or sulbactam

Mechanisms of high-level resistance to quinolones in urinary tract isolates of Pseudomonas aeruginosa.

Twenty-eight strains of Pseudomonas aeruginosa with various degrees of norfloxacin resistance were isolated from patients with urinary tract infections. P. aeruginosa strains (norfloxacin MICs, 3.13 to 200 micrograms/ml) were transformed by either pPAW207 or pNF111 plasmid DNA, which included either the gyrA or nfxB gene, respectively. For transformants with pPAW207, norfloxacin MICs decreased 8- to 128-fold. It was suggested that moderate and high degrees of resistance to norfloxacin were expressed as a result of alterations in gyrA. No strain manifesting only an alteration in nfxB permeability was observed. The MICs of norfloxacin (200 micrograms/ml) for two P. aeruginosa strains, GN17605 and GN17434-2, were decreased following transformation not only by pPAW207 but also by pNF111. Analysis of outer membrane proteins disclosed the presence of a 54,000-Da protein in these parent strains that was not expressed in the pNF111 transformants. The level of accumulation of norfloxacin by the pNF111 transformant of GN17605 was higher than that by the parent strain. The norfloxacin susceptibility of DNA gyrase subunit A purified from GN17605 was only 1/35th that of the gyrase containing a subunit A from P. aeruginosa PAO1. These findings suggest that GN17605 is a gyrA-nfxB double mutant and that strain GN17434-2 possesses double mutations in both nfxB and some unknown gene

Plasmid-mediated gentamicin resistance of Pseudomonas aeruginosa and its lack of expression in Escherichia coli.

We isolated 11 nonconjugative plasmids mediating resistance to aminoglycoside antibiotics, including gentamicin, from Pseudomonas aeruginosa strains. Their genetic properties were investigated in both P. aeruginosa and Escherichia coli transformants. The plasmid molecular weights ranged from 11 x 10(6) to 24 x 10(6). A low level or complete absence of gentamicin resistance was observed when these plasmids were introduced into E. coli, but gentamicin resistance was restored when the plasmids were transferred back to P. aeruginosa from E. coli. Aminoglycoside-modifying enzyme activity was detected in P. aeruginosa harboring these plasmids, but was absent or greatly reduced in E. coli strains. This lack of expression may explain the observed decrease in aminoglycoside resistance

Gentic properties of an R factor carrying resistance to aminolgycoside antibiotics.

R factor Rms 151 is an fi+ R factor and belongs to a incompatibility group FII. It carries the genes governing resistance to various aminoglycoside antibiotics, i.e., kanamycin (KM), lividomycin (LV), gentamicin C complex (GM), and 3',4'-dideoxykanamycin B (DKB), in addition to those governing to tetracycline (TC), chloramphenicol (CM), sulfanilamide (SA), and ampicillin (APC). Electron microscopy observation disclosed that the Rms151 deoxyribonucleic acid was a circular form with length of 31.2 mum. A probable circular genetic map of Rms151 was proposed by genetic and biochemical studies, the genes being in the order of -tet-tra-amp-aad-sul-aph-cml-, in which aad and aph confer resistance to KM.GM.DKB by adenylytransferase or resistance to KM.LV by phosphotransferase, respectively

In vitro antibacterial activity of DU-6859a, a new fluoroquinolone.

The in vitro antibacterial activity of DU-6859a, a new fluoroquinolone, against a wide variety of clinical isolates was evaluated and compared with those of tosufloxacin, ofloxacin, ciprofloxacin, and sparfloxacin. DU-6859a showed potent broad-spectrum activity against gram-positive, gram-negative, and anaerobic bacteria, and its activity was greater than those of the control quinolones. By comparison of MICs at which 90% of strains are inhibited, DU-6859a had potent activity against bacteria resistant to the control quinolones. The time-killing curves of quinolones showed that the number of viable cells decreased rapidly during 2 to 4 of incubation, and regrowth was not seen even after 8 h incubation. At a concentration of four times the MIC, the frequencies of appearance of spontaneous mutants of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa resistant to DU-6859a were < or = 4.0 x 10(-9) to 1.9 x 10(-8). The 50% inhibitory concentrations of DU-6859a were 0.86 and 1.05 micrograms/ml for the supercoiling activities of DNA gyrases isolated from E. coli and P. aeruginosa, respectively. The rank order of the 50% inhibitory concentrations observed for both DNA gyrases roughly paralleled the MICs

Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa.

Two genetically distinct classes of norfloxacin-resistant Pseudomonas aeruginosa PAO4009 mutants were isolated spontaneously. Two norfloxacin resistance genes, nfxA and nfxB, were mapped hex-9001 and leu-9005 and between pro-9031 and ilv-9023, respectively, on the P. aeruginosa PAO chromosome. The nfxA gene was shown to be an allele of nalA by transductional analysis with bacteriophage F116L. The nfxB mutant showed a 16-fold increase in resistance to norfloxacin and a slight increase in resistance to nalidixic acid. The nfxB mutant was unique in that it showed hypersusceptibility to beta-lactam and aminoglycoside antibiotics. This mutant had about a threefold-lower rate of norfloxacin uptake than that of the wild-type strain or nfxA mutant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins demonstrated the appearance of a 54,000-dalton protein in the nfxB mutant. These findings suggested that the norfloxacin resistance mechanism in the nfxB mutant might be an alteration in outer membrane permeability to norfloxacin

New norfloxacin resistance gene in Pseudomonas aeruginosa PAO.

A new type of norfloxacin-resistant mutant of Pseudomonas aeruginosa PAO was isolated. This mutant showed cross resistance to imipenem and chloramphenicol and hypersusceptibility to beta-lactam and aminoglycoside antibiotics. The new norfloxacin resistance gene nfxC was mapped near catA (46 min) on the PAO chromosome. Norfloxacin accumulation was decreased in the nfxC mutant; furthermore, the rate of imipenem diffusion through the outer membrane of the nfxC mutant was lower than that of the parent strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins showed a decrease of a 46-kilodalton protein and an increase of a 50-kilodalton protein in the nfxC mutant. We conclude the nfxC is a new norfloxacin resistance gene that affects outer membrane permeability to quinolones and other antimicrobial agents