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L J Piddock

Publications and source records attributed to L J Piddock.

At least 73 records · Page 4Linked to original sources
PubMed1992

Bactericidal activities of five quinolones for Escherichia coli strains with mutations in genes encoding the SOS response or cell division.

The bactericidal effects of five quinolones (at the optimum bactericidal concentration for strain AB1157) on 15 strains of Escherichia coli with mutations in genes for the SOS response or cell division was studied by a viable-count method. The kill rate data were normalized for growth rate and compared to those for the wild type, AB1157. Similar MICs of enoxacin and fleroxacin were obtained for all mutants; however, different mutants had differing susceptibilities to ciprofloxacin, norfloxacin, and nalidixic acid. Killing kinetic studies showed that mutants with constitutive RecA expression (recA730 and spr-55 mutants) survived longer than AB1157 with all quinolones. Mutants deficient in SOS induction, e.g., recA430 and lexA3 mutants, also survived longer, suggesting that induction of the SOS response by quinolones is harmful to wild-type cells. Recombination repair-deficient mutants (recB21, recC22, and recD1009 mutants) were killed more rapidly than AB1157, as were excision repair mutants, except with nalidixic acid. Mutants which were unable to filament (sfiA11 and sfiB114 mutants) survived longer than AB1157 with all agents, but a mutant defective in the Lon protease was killed more quickly. It was concluded that (i) recombination and excision repair were involved in the repair of quinolone-damaged DNA and (ii) continuous induction (in response to exposure to quinolones) of the SOS response, and hence induction of the cell division inhibitor SfiA, causes cell filamentation and thereby contributes to the bactericidal activity of quinolones.

4-Quinolones↗
PubMed1992

A pleiotropic, posttherapy, enoxacin-resistant mutant of Pseudomonas aeruginosa.

An enoxacin-resistant Pseudomonas aeruginosa mutant (G49) isolated during patient therapy was characterized in detail. The G49 mutant was cross resistant to several classes of antibiotics including quinolones, beta-lactams, chloramphenicol, and tetracycline, but not imipenem or aminoglycosides. Compared with its paired pretherapy isolate G48, this mutant had several alterations in outer membrane proteins including a complete loss of the major porin protein OprF and a substantially altered lipopolysaccharide profile. Revertants were selected at a frequency of approximately 1% after enrichment for OprF+ cells on low-salt proteose peptone no. 2 medium. Ninety-seven of these OprF+ revertants were as susceptible to carbenicillin and norfloxacin as the pretherapy isolate. One of these revertants was characterized in more detail and shown to be indistinguishable in all properties from the pretherapy isolate. It is proposed that the multiple-antibiotic-resistance (Mar) phenotype of this mutant resulted from a single pleiotropic mutation.

Anti-Bacterial Agents↗
PubMed1991

Phenotypic characterization of quinolone-resistant mutants of Enterobacteriaceae selected from wild type, gyrA type and multiply-resistant (marA) type strains.

The NCTC type strains of Escherichia coli, Enterobacter cloacae, Serratia marcescens and Klebsiella pneumoniae were exposed to 3, 5 and 10 x MIC of nalidixic acid, enoxacin, ciprofloxacin, PD 117596 and PD 127391. From each strain a mutant with a high MIC of quinolones alone (gyrA) and a mutant with intermediate resistance to quinolones, some beta-lactams, chloramphenicol and tetracycline (multiply resistant, m-r) were selected on agar containing antibiotics. The gyrA mutants required a higher concentration of quinolone to inhibit DNA synthesis by 50% but quinolone uptake kinetics and outer membrane profile were the same as the wild type. The m-r mutants had similar DNA synthesis IC50 as the wild type, decreased quinolone uptake kinetics and had decreased expression of an OMP of approximately 40 kD. The gyrA and m-r mutants were then exposed to 3, 5 and 10 x MIC of the same quinolones and new mutants (F2) selected. The F2 mutants from the gyrA mutants displayed a further increase in quinolone MIC; the F2 mutants from the m-r mutants had several phenotypes: high quinolone MICs with cross resistance to other agents, high quinolone resistance alone, or intermediate quinolone resistance alone. Most F2 mutants had MICs above the recommended breakpoint concentrations for quinolones. The F2 mutants often had altered biochemical profiles (API 20E), however, only in the case of E. cloacae did this affect speciation with the strains being identified as Rhanella aquatalis.

Anti-Infective Agents↗
PubMed1991

beta-Lactamase expression and outer membrane protein changes in cefpirome-resistant and ceftazidime-resistant gram-negative bacteria.

Twenty-five strains of Enterobacteriaceae (five each of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii, and Providencia stuartii) and five strains of Pseudomonas aeruginosa were exposed to various concentrations of cefpirome or ceftazidime in agar. Mutants with a greater than four-fold increase in the MIC were examined for changes in beta-lactamase expression and outer membrane protein (OMP) profile. Both agents selected mutants with decreased susceptibility to the selecting antibiotic and other beta-lactams at a frequency of 10(-7)-10(-8). The MICs of all beta-lactams were higher for the resistant mutants of E. cloacae and P. aeruginosa than for the other species. Both agents selected mutants expressing derepressed class I beta-lactamase, but this was more common with ceftazidime. Only a few mutants of P. aeruginosa and E. cloacae had an MIC of cefpirome that was above the recommended breakpoint concentration. Some mutant strains of Enterobacteriaceae lacked an OMP of molecular size similar to OmpF, but the MIC of cefpirome was below the breakpoint concentration for all these strains.

Bacterial Outer Membrane Proteins↗
PubMed1991

Characterization of FCE 22101-resistant Enterobacteriaceae and the effect of FCE 22101 upon the activity of anti-pseudomonal beta-lactams for Pseudomonas aeruginosa.

Twenty-five strains of Enterobacteriaceae (five each of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii, and Providencia stuartii) were exposed to FCE 22101 in agar containing 3, 5, or 10 x the MIC. Any putative mutant with a greater than or equal to four-fold increase in the MIC was examined for beta-lactamase expression and outer membrane protein (OMP) profile. Mutant colonies were selected at a frequency of 10(-7)-10(-11) with decreased susceptibility to FCE 22101 and other beta-lactams, but after one subculture on antibiotic-free agar the mutants from 13 of the 25 strains reverted to wild-type. Only 19 stable mutants were selected from the other 12 wild-type strains, of which 15 lacked an OMP of similar molecular size to OmpF, and/or a low size OMP of approximately 18 kDa. None of the mutants had a significant alteration in expression of Richmond & Sykes class I beta-lactamase. In a separate section of the study in which 50 strains of Pseudomonas aeruginosa were examined, it was found that FCE 22101, at a concentration of 4 mg/L, induced beta-lactamase expression such that, after 24 h exposure, 24 of the 50 strains had a greater than or equal to four-fold rise in the MIC of several anti-pseudomonal beta-lactams.

Anti-Bacterial Agents↗
PubMed1991

A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus.

Accumulation of norfloxacin by Escherichia coli was studied with a range of published procedures that used either radioactively-labelled norfloxacin (14C and 3H) or the natural fluorescence of the quinolone for detection. All methods except bioassay generated comparable data. A method involving the detection of fluorescence was found to be the method of choice. This method was used to study the accumulation kinetics of ciprofloxacin, lomefloxacin, fleroxacin, norfloxacin, and enoxacin by several species of Gram-negative bacteria, and a Staphylococcus aureus strain. Saturation and efflux kinetics were also studied. There was no saturation at a concentration of norfloxacin less than 50 mg/L. Norfloxacin efflux was minimal during the uptake assay as the samples were withdrawn into ice-cold buffer; however, when the cells were sampled into buffer at 37 degrees C, up to 50% of cell-associated quinolone effluxed within 5 min.

Anti-Infective Agents↗
PubMed1991

Selection and characterization of cefepime-resistant gram-negative bacteria.

The NCTC type strains and four clinical isolates of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii, Providencia stuartii and Pseudomonas aeruginosa were exposed, in agar, to cefepime at 3, 5, and 10 x MIC and a breakpoint concentration of 16 mg/L. Mutants were selected at a frequency of approximately 10(-8) that had decreased susceptibility to cefepime and cefpirome, and species-dependent resistance to other beta-lactams. Any putative mutant with a greater than four-fold increase in the MIC was examined to determine its beta-lactamase expression and outer membrane protein (Omp) profile. Mutant strains of P. stuartii and M. morganii lacked an Omp of molecular mass similar to that of OmpF, and were cross-resistant to nalidixic acid. Mutant strains of E. cloacae had derepressed class I beta-lactamase production and lacked an Omp corresponding to OmpF, suggesting that in this species both parameters are necessary for decreased susceptibility. Derepressed beta-lactamases purified from mutant strains of E. cloacae, C. freundii and P. stuartii was able to hydrolyse cefepime, but not as quickly as TEM-10.

Bacterial Outer Membrane Proteins↗
PubMed1991

Mechanism of action of sparfloxacin against and mechanism of resistance in gram-negative and gram-positive bacteria.

The inhibition of DNA synthesis by sparfloxacin; accumulation of sparfloxacin into members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and staphylococci; induction of recA in Escherichia coli; and the optimum bactericidal concentration (OBC) were measured, and killing kinetics at the OBC were estimated. The OBC and maximum recA-inducing concentration in E. coli were both 1 microgram of sparfloxacin per ml. Accumulation was rapid; two- to threefold more sparfloxacin than ciprofloxacin accumulated in staphylococci and more sparfloxacin accumulated in staphylococci than in gram-negative bacteria. Laboratory mutants with decreased susceptibilities to quinolones alone or multiply resistant were selected from the Enterobacteriaceae and Staphylococcus aureus by using sparfloxacin.

Anti-Bacterial Agents↗
PubMed1990

Cloning, sequencing and analysis of the structural gene and regulatory region of the Pseudomonas aeruginosa chromosomal ampC beta-lactamase.

The chromosomal gene from Pseudomonas aeruginosa encoding beta-lactamase has been cloned, and the sequence determined and compared with corresponding sequences of beta-lactamases from members of the enterobacteriaceae. Upstream of the beta-lactamase gene is an open reading frame which we postulate encodes a regulatory protein, AmpR. We identified a helix-turn-helix region in AmpR and a putative AmpR-binding site.

Amino Acid Sequence↗
PubMed1990

The accumulation of five quinolone antibacterial agents by Escherichia coli.

The accumulation of five radiolabelled quinolone antibacterial agents by Escherichia coli KL16 was examined using a vacuum filtration method. Preliminary experiments were performed to determine the optimum quinolone concentration, inoculum of cells, filter washing regimen and filter type. All five quinolones showed a similar biphasic pattern of accumulation with high radioactive counts cell-associated during the first ten seconds of the assay, followed by steadily increasing accumulation over 30 min. Analysis of the mean accumulation after 30 min for each quinolone showed that there was no direct relationship between quinolone accumulation and antibacterial activity (as quantified by the MIC or bactericidal activity). Mechanistic investigations showed that accumulation was decreased by low reaction temperatures, acid pH and the presence of the metabolic inhibitors 2,4-dinitrophenol, potassium cyanide and sodium azide. These results suggest that quinolone accumulation by E. coli KL16 is partly dependent on cell metabolism and may proceed by an active transport mechanism. Treatment of cells with EDTA was found not to increase quinolone accumulation, suggesting that the outer membrane of E. coli KL16 does not act as a permeability barrier to these quinolones. The implication of these results in terms of possible mechanisms of bacterial resistance to quinolones is discussed.

4-Quinolones↗
PubMed1990

A comparison of the mechanisms of decreased susceptibility of aztreonam-resistant and ceftazidime-resistant Enterobacteriaceae.

Twenty five strains of Enterobacteriaceae (five each of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii and Providencia stuartii) were exposed to aztreonam and ceftazidime at 1/2 and 1 x MIC in liquid medium for 22 h, and also to 3, 5 and 10 x MIC and 16 mg/l of each agent in agar. Any putative mutant with an increase in the MIC of greater than or equal to 4 fold was examined for beta-lactamase expression and outer membrane protein profile. Mutants were selected on agar at approximately 10(7); however in liquid medium not all strains yielded mutants. Mutants lacking an outer membrane protein (OMP) with a molecular weight of 40,000 (+/- 5000) were selected with both agents, as were mutants expressing constitutive Richmond and Sykes Class 1 beta-lactamase. For the Ent. cloacae mutants increased beta-lactamase gave rise to MICs above the breakpoint of both agents, whereas with the other species ceftazidime susceptibility was more affected. Strains that were OMP- rarely had MICs above the breakpoint, unless there was also increased beta-lactamase expression, as in species such as M. morganii. Hence the major mechanism of resistance in these strains would appear to be beta-lactamase mediated rather than due to altered expression of outer membrane proteins.

Aztreonam↗