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Biomedical subjects

L J Piddock

Publications and source records attributed to L J Piddock.

At least 19 recordsLinked to original sources
PubMed2004

Development of a standardized susceptibility test for campylobacter with quality-control ranges for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem.

A standardized agar dilution susceptibility testing method was developed for Campylobacter that consisted of testing on Mueller-Hinton medium supplemented with 5% defibrinated sheep blood in an atmosphere of 10% CO2, 5% O2, and 85% N2. Campylobacter jejuni ATCC 33560 was identified as a quality-control (QC) strain. Minimal inhibitory concentration (MIC) QC ranges were determined for two incubation time/temperature combinations: 36 degrees C for 48 hr and 42 degrees C for 24 hr. Quality-control ranges were determined for ciprofloxacin, doxycycline, erythromycin, gentamicin, and meropenem. For all antimicrobial agents tested at both temperatures, 95-100% of the QC MIC results fell within recommended QC ranges. Twenty-one Campylobacter clinical isolates, encompassing five species of Campylobacter (C. jejuni, C. coli, C. jejuni, subsp. doylei, C. fetus, and C. lari) were tested in conjunction with the C. jejuni QC strain. While C. jejuni and C. coli could be reliably tested under both test conditions, growth of C. jejuni subsp. doylei, C. fetus, and C. lari isolates was inconsistent when incubated at 42 degrees C. Therefore, it is recommended that these species only be tested at 36 degrees C.

Campylobacter↗
PubMed2001

Effect of hydrophobicity and molecular mass on the accumulation of fluoroquinolones by Staphylococcus aureus.

Ten novel fluoroquinolones, with similar chemical structures but differing antibacterial activities and hydrophobicities, were studied to evaluate the role of the physical properties of quinolones on their accumulation and antibacterial activity for Staphylococcus aureus. Six of the 10 agents and tosufloxacin were more active against quinolone-susceptible and -resistant S. aureus than the remaining four agents and several piperazinyl fluoroquinolones. Changes to the side chains of the pyrollidinyl substituent at the R7 position alone made little difference to the MICs. Comparison of MICs of agents that were structurally identical apart from the R1 substituents, confirmed that a t-butyl group confers enhanced activity against S. aureus over a cyclopropyl or ethyl group at this position. The steady-state concentrations (SSCs) of the 10 novel quinolones accumulated by wild-type S. aureus did not correlate with their MICs or chemical structures. There was no apparent relationship between logP of the quinolone and accumulation by S. aureus F77; however, accumulation was positively correlated with molecular mass for 9/10 agents (r = 0.745) confirming that high molecular mass is not a barrier to accumulation in S. aureus. For all 10 agents, the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased the concentration of quinolone accumulated by SA-1199, suggesting that NorA was inhibited. The fold increase of the SSC in the presence of CCCP did not correlate with hydrophobicity, but the SSC of agents with either an ethyl or cyclopropyl group at R1 was increased two- to three-fold in the presence of CCCP, suggesting that affinity for the NorA efflux pump may be influenced by quinolone structure.

Anti-Infective Agents↗
PubMed2001

Accumulation of five fluoroquinolones by Mycobacterium tuberculosis H37Rv.

The accumulation of ciprofloxacin, norfloxacin, moxifloxacin, levofloxacin and ofloxacin by Mycobacterium tuberculosis H37Rv was determined with a modified fluorescence method. The time to achieve a steady-state concentration (SSC) of each agent in M. tuberculosis was 60-240 s. Moxifloxacin was accumulated to the lowest concentration and ciprofloxacin to the highest. However, ciprofloxacin took longer to achieve an SSC than the other four agents; levofloxacin reached steady state in the shortest time. Larger fluoroquinolones accumulated to the lowest concentration and more slowly. Although all five agents had low hydrophobicity values (P(app) < or =0.11), those with the lowest values accumulated to the higher concentrations.

Anti-Infective Agents↗
PubMed2001

Absence of mutations in marRAB or soxRS in acrB-overexpressing fluoroquinolone-resistant clinical and veterinary isolates of Escherichia coli.

The amount of acrB, marA, and soxS mRNA was determined in 36 fluoroquinolone-resistant E. coli from humans and animals, 27 of which displayed a multiple-resistance phenotype. acrB mRNA was elevated in 11 of 36 strains. A mutation at codon 45 (Arg-->Cys) in acrR was found in 6 of these 11 strains. Ten of the 36 isolates appeared to overexpress soxS, and five appeared to overexpress marA. A number of mutations were found in the marR and soxR repressor genes, correlating with greater amounts of marA and soxS mRNA, respectively.

Animals↗
PubMed2000

Accumulation of rifampicin by Mycobacterium aurum, Mycobacterium smegmatis and Mycobacterium tuberculosis.

The characteristics of the accumulation of 2 mg/L [(14)C]rifampicin by wild-type strains of Mycobacterium aurum (A(+)), Mycobacterium smegmatis (mc(2)155) and Mycobacterium tuberculosis (H37Rv) were determined. After 10 min exposure M. aurum had accumulated 220 ng rifampicin/mg cells, M. smegmatis had accumulated 120 ng rifampicin/mg cells and M. tuberculosis had accumulated 154 ng rifampicin/mg cells. A steady-state concentration (SSC) of rifampicin was accumulated rapidly by M. aurum and M. tuberculosis within minutes of drug exposure, unlike M. smegmatis, which accumulated rifampicin more slowly. With an increase in the concentration of rifampicin from 0.12 mg/L to 2 mg/L there was an increase in the concentration of rifampicin accumulated by M. tuberculosis, with no detectable loss of viability over the 20 min of the accumulation experiment. With an increase in temperature there was also an increase in the concentration of rifampicin accumulated by M. tuberculosis; between 15 and 30 degrees C the increase was linear. For all three species sub-inhibitory concentrations of ethambutol increased the concentration of rifampicin accumulated. However, both growth and accumulation of rifampicin were lower in the presence of 0.05% Tween 80. Accumulation of rifampicin by M. smegmatis was unaffected by the presence of the proton motive force inhibitor, 2,4-dinitrophenol (1 mM), whether added before or after the addition of rifampicin to the mycobacterial culture. For all three species, the Gram-positive bacterial efflux inhibitor reserpine (20 mg/L) slightly increased the SSC of rifampicin, but the increase was not statistically significant. Addition of glucose to energize a putative efflux pump had little effect on the accumulation of rifampicin in the presence or absence of reserpine for M. tuberculosis; however, for M. aurum and M. smegmatis the reserpine effect was abolished by the addition of glucose. These data suggest that rifampicin may be removed from wild-type mycobacteria by efflux, but that the pump(s) is expressed at low level.

Antibiotics, Antitubercular↗
PubMed2000

Accumulation of KRM-1648 by Mycobacterium aurum and Mycobacterium tuberculosis.

After exposure to 2 mg/L (14)C-labelled KRM-1648 (a new broad-spectrum benzoxazinorifamycin antibiotic) for 5 min, a steady-state concentration of 31.3 +/- 3 ng/mg cells KRM-1648 and 12. 6 +/- 0.3 ng/mg cells KRM-1648 was accumulated by wild-type antibiotic-susceptible Mycobacterium aurum (A+) and Mycobacterium tuberculosis (H37Rv), respectively. However, 2 mg/L KRM-1648 was bactericidal for M. tuberculosis. A steady-state concentration of 3. 7 +/- 0.1 ng/mg cells KRM-1648 was accumulated after exposure to 0.5 mg/L. At pH 4 higher concentrations were accumulated than at pH 7. A sub-inhibitory concentration of ethambutol increased the concentration of KRM-1648 accumulated, but Tween 80 and reserpine had little or no effect.

Antibiotics, Antitubercular↗
PubMed2000

Activity of antibiotics used in human medicine for Campylobacter jejuni isolated from farm animals and their environment in Lancashire, UK.

A retrospective study of 96 Campylobacter jejuni isolated from farm animals and the environment showed that most were less susceptible than the NCTC type strain to nalidixic acid (MICs 4-32 mg/L), ciprofloxacin (MICs 1-2 mg/L) and erythromycin (MICs 16-64 mg/L), but had similar susceptibility to tetracycline (MICs 4-8 mg/L) and kanamycin (MICs 4-8 mg/L). None had the high MICs of ciprofloxacin (>32 mg/L) or erythromycin (1024 mg/L) typically associated with clinical resistance in this species. Some farms used antimicrobial agents, but there was no obvious association between the use of agents and the susceptibility of the isolates.

Animals↗
PubMed2000

Two efflux systems expressed simultaneously in multidrug-resistant Pseudomonas aeruginosa.

Simultaneous overexpression of MexAB-OprM and MexEF-OprN was demonstrated for a multiply antibiotic-resistant clinical isolate of Pseudomonas aeruginosa (G49). G49 also had decreased expression of OprF. No mutations in mexR or its upstream promoter region, mexT, oprM, oprF, or sigX were revealed, suggesting regulation by a hitherto undescribed locus.

Anti-Bacterial Agents↗
PubMed2000

Characterization of fluoroquinolone resistance among veterinary isolates of avian Escherichia coli.

Fluoroquinolone-resistant avian Escherichia coli isolates from northern Georgia were investigated for gyrA and parC mutations. All isolates contained a mutation in GyrA replacing Ser83 with Leu; seven isolates also contained mutations replacing Asp87 with either Gly or Tyr. Random amplified polymorphic DNA analysis revealed that quinolone-resistant E. coli isolates were genetically diverse.

Animals↗
PubMed2000

Evidence for an efflux pump mediating multiple antibiotic resistance in Salmonella enterica serovar Typhimurium.

The mechanism of multiple antibiotic resistance in six isolates of Salmonella enterica serovar Typhimurium recovered from a patient treated with ciprofloxacin was studied to investigate the role of efflux in the resistance phenotype. Compared to the patient's pretherapy isolate (L3), five of six isolates accumulated less ciprofloxacin, three of six isolates accumulated less chloramphenicol, and all six accumulated less tetracycline. The accumulation of one or more antibiotics was increased by carbonyl cyanide m-chlorophenylhydrazone to concentrations similar to those accumulated by L3 for all isolates except one, in which accumulation of all three agents remained approximately half that of L3. All isolates had the published wild-type sequences of marO and marR. No increased expression of marA, tolC, or soxS was observed by Northern blotting; however, three isolates showed increased expression of acrB, which was confirmed by quantitative competitive reverse transcription-PCR. However, there were no mutations within acrR or the promoter region of acrAB in any of the isolates.

Bacterial Proteins↗
PubMed2000

Accumulation of norfloxacin by Bacteroides fragilis.

The accumulation of norfloxacin by Bacteroides fragilis NCTC 9343 was determined by the modified fluorescence method. The time required to achieve a steady-state concentration (SSC) after allowing B. fragilis to accumulate norfloxacin in an aerobic or an anaerobic environment was approximately 2 min; the SSC achieved in air was 90.28 +/- 9.32 ng of norfloxacin/mg (dry weight) of cells, and that achieved anaerobically was 98.45 +/- 3.7 ng of norfloxacin/mg (dry weight) of cells. Initial rates of accumulation were determined with a range of external concentrations, as up to 8 microg/ml the concentration of norfloxacin accumulated increased proportionally to the external concentration, 12.13 ng/mg (dry weight) of cells per microg of exogenous norfloxacin per ml. At concentrations above 10 microg/ml no increase in the rate of norfloxacin accumulation was observed. From the kinetic data, a Lineweaver-Burk plot calculated a K(m) of 5.03 microg/ml and a V(max) of 25.1 ng of norfloxacin/s. With an increase in temperature of between 0 and 30 degrees C, the concentration of norfloxacin accumulated also increased proportionally at 4.722 ng of norfloxacin/mg (dry weight) of cells/ degrees C. At low concentrations of glucose (<0.2%; 11 mM), the concentration of norfloxacin accumulated was decreased. With the addition of 100 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP) the mean SSC of norfloxacin was increased to 116 +/- 7.01 ng of norfloxacin/mg (dry weight) of cells; glucose had no significant effect in the presence of CCCP. Magnesium chloride (20 mM) decreased the SSC of norfloxacin to 40.5 +/- 3.76 ng of norfloxacin per mg (dry weight) of cells. These data suggest that the mechanism of accumulation of norfloxacin by B. fragilis is similar to that of aerobic bacteria and that the fluoresence procedure is suitable for use with an anaerobic bacterium.

Anti-Infective Agents↗
PubMed1999

Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.

The accumulation of nalidixic acid and 14 fluoroquinolones over a range of external drug concentrations (10-100 mg/L; c. 25-231 microM) into intact cells of Escherichia coli KL-16, Staphylococcus aureus NCTC 8532, Pseudomonas aeruginosa NCTC 10662 and spheroplasts of E. coli was investigated. The effect of 100 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) upon the concentration of quinolone accumulated by intact cells and spheroplasts of E. coli was also determined. Except for pefloxacin, there was an increase in the concentration of the six quinolones examined accumulated by E. coli, despite a reduction in fluorescence at alkaline pH. For ciprofloxacin the partition coefficient (P(app)) was constant despite an increase in the pH; however, the P(app) for nalidixic acid decreased significantly with an increase in pH. The concentration of nalidixic acid, ciprofloxacin and enrofloxacin accumulated by E. coli and S. aureus increased with an increase in temperature up to 40 degrees C and 50 degrees C, respectively. Above these temperatures the cell viability decreased. With an increase in drug concentration there was, for intact E. coli and 12/15 agents, and for S. aureus and 10/15 agents, a linear increase in the concentration of drug accumulated. However, for P. aeruginosa and 13/15 agents there was apparent saturation of an accumulation pathway. Assuming 100% accumulation into intact cells of E. coli, for 10/14 fluoroquinolones < or = 40% was accumulated by spheroplasts. CCCP increased the concentration of quinolone accumulated but the increase varied with the agent and the bacterial species. The variation in the effect of CCCP upon accumulation of the different quinolones into E. coli could result from chemical interactions or from different affinities of the proposed efflux transporter for each quinolone. Overall, these data suggest that accumulation of most quinolones into E. coli and S. aureus proceeds by simple diffusion, but that P. aeruginosa behaves differently.

Anti-Infective Agents↗
PubMed1999

Antimicrobial activity and accumulation of moxifloxacin in quinolone-susceptible bacteria.

The antibacterial activity of moxifloxacin, compared with that of ciprofloxacin, was determined for five strains of Staphylococcus aureus, including one NorA-overproducing strain, two quinolone-susceptible strains of Streptococcus pneumoniae, four quinolone-susceptible strains of Haemophilus influenzae, and one strain each of quinolone-susceptible Escherichia coli, Pseudomonas aeruginosa and Moraxella catarrhalis. In addition, the accumulation of moxifloxacin and ciprofloxacin by the NCTC type strain of S. pneumoniae, H. influenzae, S. aureus, E. coli and P. aeruginosa was determined by a fluorescence method. For all strains, moxifloxacin accumulated to a lower concentration than ciprofloxacin. The concentrations of moxifloxacin accumulated ranged from 12 to 44 ng/mg dry cells. The lowest concentration was accumulated by S. pneumoniae NCTC 7465 and the highest concentration by S. aureus NCTC 8532. Increased expression of norA in S. aureus had no effect on the accumulation of moxifloxacin. Despite differences in the concentration of moxifloxacin accumulated by the different species, there was little difference between the MICs of this agent for each strain (0.06-0.5 mg/L), suggesting that the concentration accumulated by wild-type bacteria has little effect on the MIC.

Anti-Infective Agents↗
PubMed1999

Effect of carbon dioxide on testing of susceptibilities of respiratory tract pathogens to macrolide and azalide antimicrobial agents.

The in vitro activities of erythromycin, azithromycin, and clarithromycin against 178 clinical isolates from the lower respiratory tract of patients with chronic obstructive pulmonary disease were determined by an agar dilution method. The plates were incubated in air alone or in 5% carbon dioxide. The MICs measured in air alone were lower for most isolates than those measured in 5% carbon dioxide, illustrating the "pH effect" of incubation in carbon dioxide. Testing of isolates in 5% carbon dioxide on pH-adjusted medium (pH 8.4) resulted in MICs of one or two doubling dilutions lower than those obtained on agar with a neutral pH. A bioassay of the three agents incubated in air and in 5% carbon dioxide resulted in a significant loss of activity of all three agents in the carbon dioxide-enriched atmosphere. However, this loss-of-activity effect was significantly reduced when the bioassay medium was adjusted to pH 8.4 prior to incubation in 5% carbon dioxide.

Anti-Bacterial Agents↗
PubMed1999

Mechanisms of fluoroquinolone resistance: an update 1994-1998.

Fluoroquinolone resistance is mediated by target changes (DNA gyrase and/or topoisomerase IV) and/or decreased intracellular accumulation. The genes (gyrA/gyrB/parC/parE) and proteins of DNA topoisomerase IV show great similarity, both at the nucleotide and amino acid sequence level to those of DNA gyrase. It has been shown that there are hotspots, called the quinolone resistance determining region (QRDR), for mutations within gyrA and parC. Based on the Escherichia coli co-ordinates, the hotspots most favoured for giving rise to decreased susceptibility and/or full resistance to quinolones are at serine 83 and aspartate 87 of gyrA, and at serine 79 and aspartate 83 for parC. Few mutations in gyrB or parE/grlB of any bacteria have been described. Efflux of fluoroquinolones is the major cause of decreased accumulation of these agents; for Staphylococcus aureus, the efflux pump involved in norfloxacin resistance is NorA, and for Streptococcus pneumoniae, PmrA. By analysis of minimum inhibitory concentration (MIC) data derived in the presence and absence of the efflux inhibitor reserpine, it has been shown that up to 50% of ciprofloxacin-resistant clinical isolates of S. pneumoniae may possess enhanced efflux. This suggests that efflux may be an important mechanism of clinical resistance in this species. In Pseudomonas aeruginosa, several efflux operons have been demonstrated genetically and biochemically. These operons are encoded by mex (Multiple EffluX) genes: mexAmexB-oprM, mexCD-OprJ system and mexEF-oprN system. The E. coli efflux pump is the acrAB-tolC system. Both the mar operon and the sox operon can give rise to multiple antibiotic resistance. It has been shown that mutations giving rise to increased expression of the transcriptional activators marA and soxS affect the expression of a variety of different genes, including ompF and acrAB. The net result is that expression of OmpF is reduced and much less drug is able to enter the cell; expression of acrAB is increased, enhancing efflux from the cell.

Amino Acid Sequence↗