We isolated an imipenem-resistant strain, GN17203, of Pseudomonas aeruginosa. The strain produced a I8-lactamase that hydrolyzed imipenem. The I-lactamase was encoded by a 31-MDa plasmid, pMS350, which belongs to incompatibility group P-9. The plasmid conferred resistance to f8-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, CuS04, and HgCl2 but not by clavulanic acid or sulbactam.
We isolated spontaneous mutants from Escherichia coli K-12 with low-level resistance to norfloxacin. These mutants were classified into the following three types on the basis of their properties: (i) NorA appeared to result for mutation in the gyrA locus for the A subunit of DNA gyrase; (ii) NorB showed low-level resistance to quinolones and other antimicrobial agents (e.g., cefoxitin, chloramphenicol, and tetracycline), and the norB gene was considered to map at about 34 min on the E. coli K-12 chromosome; (iii) NorC was less susceptible to norfloxacin and ciprofloxacin but was hypersusceptible to hydrophobic quinolones such as nalidixic acid and rosoxacin, hydrophobic antibiotics, dyes, and detergents. Susceptibility to bacteriophages and the hydrophobicity of the NorC cell surface also differed from that of the parent strain. The norC gene was located near the lac locus at 8 min on the E. coli K-12 chromosome. Both NorB and NorC mutants had a lower rate of norfloxacin uptake, and it was found that the NorB mutant was altered in OmpF porin and that the NorC mutant was altered in both OmpF porin and apparently in the lipopolysaccharide structure of the outer membrane.Many new quinolones showing potent antibacterial activity against gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa, have been developed recently (12,18,33,34). They also show high antibacterial activity against nalidixic acid-resistant strains including mutational resistant strains such as gyrA and nalB mutants of Escherichia coli K-12 (14, 28). There is incomplete crossresistance between the new quinolones and nalidixic acid (14,17,28,35).The high antibacterial activity of new quinolones (e.g., norfloxacin and ciprofloxacin) might be due to their strong inhibitory action on DNA gyrase, which is the target enzyme of quinolones (K. Sato, Y. Inoue, T. Fujii, H. Aoyama, M. Inoue, and S. Mitsuhashi, personal communication). Recently, we found that the bacterial outer membrane penetration mechanisms of new quinolones differed from those of old quinolones such as nalidixic and piromidic acids (13).To study the mechanisms of resistance to norfloxacin, the first-developed new quinolone, spontaneous norfloxacinresistant mutants were isolated from E. coli K-12 and their properties were investigated. Three classes of mutants showed less susceptibility to norfloxacin; one class appeared to consist of gyrA mutants, while the other two were novel mutants that showed alteration in norfloxacin uptake. In this report we describe the biochemical and genetic properties of these new mutants in detail.( lated from E. coli KL-16 by plating on nutrient agar plates containing norfloxacin. Strain KJC-1, a gIpT derivative of JC1552, was made by fosfomycin selection as described previously (21). The phages Tula and TuIb were kindly provided by S. Mizushima of Nagoya University, Nagoya, Japan.Drugs. AM-833, ciprofloxacin, norfloxacin, ofloxacin, oxolinic acid, pipemidic acid, piromidic acid, and rosoxacin were synthesized by Central Research Laborato...
Two genetically distinct classes of norfloxacin-resistant Pseudomonas aeruginosa PA04009 mutants were isolated spontaneously. Two norfloxacin resistance genes, nfxA and nfxB, were mapped between 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.Norfloxacin and other new 4-quinolones show potent antibacterial activity against gram-negative and grampositive bacteria (11,14,20). They also have high in vitro and in vivo antibacterial activity against Pseudomonas aeruginosa strains that show a strong intrinsic resistance to various antimicrobial agents including older quinolones such as nalidixic acid (11,20,42). The high antibacterial activity of new quinolones might be due to their strong inhibitory action against DNA gyrase, a target enzyme of quinolones, which has been isolated from various bacteria including Pseudomonas aeruginosa (5,16,26,28,42).Drug resistance mediated by plasmids or transposons is a serious clinical problem. However, resistance to nalidixic acid and other quinolones in bacteria is due to chromosomal mutations (10,13,(16)(17)(18)32). Plasmids or transposons that carry quinolone resistance genes have not been found in bacteria (4). Several chromosomal mutations, gyrA, nalB, nalC (gyrB), and nalD, conferring nalidixic acid resistance were identified and mapped on the Escherichia coli K-12 chromosome (10,17,18). Recently, we (13) and Hooper et al. (16) identified norfloxacin resistance mutations (norA, norB, norC, nfxA, and nfxB) in E. coli K-12. norA and nfxA were alleles of gyrA encoding the A subunit of DNA gyrase, while norB, norC, and nfxB determined outer membrane permeability resistance to norfloxacin, were associated with a decrease in OmpF porin protein, and were mapped at 34 min, near 8 min, and at 20 to 22 min, respectively (13, 16). Two loci coding for resistance to nalidixic acid, nalA and nalB, have also been mapped on the P. aeruginosa PAO chromosome (32). It has been reported that DNA replication is resistant to nalidixic acid in permeabilized cells of nalA mutants and that nalB mutants cause a decrease of cell permeability to nalidixic acid and carbenicillin (32).To gain information on the resistance mechanisms to norfloxacin in P. aeruginosa, we isolated spontaneous...
In the course of surveying for the carbapenem-hydrolyzing metallo--lactamase gene bla IMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo--lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene bla IMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of bla IMP-3 was identical to that of bla IMP-1 , except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from bla IMP-3 and bla IMP-1 by recombinant DNA techniques, and -lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the bla IMP-3 gene. This indicated that extension of the substrate profile in the metallo--lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene bla IMP-3 is an ancestor of bla IMP-1 .-Lactamases are enzymes that hydrolyze -lactam antibiotics, conferring resistance to a variety of these antibiotics for most pathogenic bacteria. These enzymes have been classified phylogenetically based on their functional and molecular characteristics (5).Molecular class B metallo--lactamases belonging to functional group 3a subclass B1 are characteristic in their broad substrate spectrum, which extends to most -lactam antibiotics, except for monobactams, and have activities as penicillinases, cephalosporinases, and carbapenemases (5,15,23). They have been reported in Bacillus cereus, alkalophilic Bacillus sp., Bacteroides fragilis, Pseudomonas aeruginosa, Serratia marcescens, and Klebsiella pneumoniae (23). Among this group of metallo--lactamases, ESP from P. aeruginosa GN17203, IMP-1 from S. marcescens TN9106, and DK4 from K. pneumoniae are all plasmid mediated and were found to be the same enzyme because the nucleotide sequences of their genes are identical (14, 22; GenBank accession number D29636).Genes bla ESP and bla IMP , respectively encoding ESP and IMP-1 -lactamase, were identified in the cassettes inserted in the integrons on plasmids (24). Both cassettes had the same nucleotide sequence, but they were found to be inserted into different integrons, class 1 integron 0 (In0) for the bla ESP cassette (2, 14) and the class 3 integron for the bla IMP cassette (1). This fact suggested that the bla IMP (bla ESP ) cassette has been disseminated among different integrons. Since In0 is reported to be widespread among clinical isolates of ...
The mechanism of penetration of quinolones through the bacterial outer membrane was studied with lipopolysaccharide-deficient and porin-deficient mutants. The data indicated that the lipopolysaccharide layer might form a permeability barrier for hydrophobic quinolones such as nalidixic acid but not for hydrophilic quinolones such as norfloxacin and ciprofloxacin. The results also showed that quinolones with a low relative hydrophobicity appeared to permeate through OmpF porin, whereas quinolones with a high relative hydrophobicity appeared to permeate through both OmpF porin and phospholipid bilayers.New quinolone derivatives which have broader and more potent antibacterial activity against gram-negative and grampositive bacteria than old quinolones, such as nalidixic acid, have recently been developed. They also have greater antibacterial activity against nalidixic acid-resistant bacteria (5,7,9,21,24,25).We previously reported that the greater antibacterial activity of norfloxacin, one of these new quinolones, might be due to its greater ability to permeate bacterial cells and its potent inhibitory action on in vivo DNA synthesis (5).The ability of drugs to pass through the bacterial outer membrane is a very important factor in their antibacterial activity and spectrum (2,10,13,14,22,23,26). There are many reports on the penetration into bacterial cells of various antimicrobial agents, especially beta-lactam antibiotics (4,8,15,22,23,26), but the penetration mechanisms of quinolones have not been studied in detail.We report here on the mechanisms of penetration of quinolones through the bacterial outer membrane with lipopolysaccharide ( ofloxacin were from Daiichi Pharmaceutical Co. Ltd.; pipemidic acid, piromidic acid, and enoxacin were from Dainippon Pharmaceutical Co. Ltd.; and ciprofloxacin was from Bayer Yakuhin Co. Ltd. The partition coefficients of the quinolones were determined by the modified method of Nikaido (13). Solutions (10 ,ug/ml) of quinolones were made in 0.1 M phosphate buffer (pH 7.2). After shaking with an equal volume of n-octanol at 25°C for 48 h and centrifuging at 1,870 x g for phase separation, the concentrations of quinolones in the aqueous phase were determined with spectrophotometric assay by measuring the A272 for enoxacin, norfloxacin, and pefloxacin, the A286 for ciprofloxacin, the A264 for pipemidic acid, the A288 for cinoxacin, the A292 for AM-833 and piromidic acid, the A294 for ofloxacin, the A264 for miloxacin, the A290 for oxolinic acid, the A258 for nalidixic acid, the A285 for rosoxacin, and the A254 for flumequine. The partition coefficients were expressed as the ratio of the amount of compound in the n-octanol phase to that in the aqueous phase.Susceptibility to quinolones was measured by the agar dilution method with Mueller-Hinton agar and an inoculum of 106 CFU per spot as described previously (7). The data were expressed as MICs.We tested the antibacterial activity of quinolones against LPS-deficient (rfa) mutants of S. typhimurium LT2 (19) ( Table 1). The suscepti...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.