Showing papers by "Ian Phillips published in 1995"

Incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli.

Abstract: Among Escherichia coli organisms isolated at St. Thomas's Hospital during the years 1990 to 1994, the frequency of resistance to amoxicillin-clavulanic acid (tested by disk diffusion in a ratio of 2:1) remained constant at about 5% of patient isolates (10 to 15% of the 41 to 45% that were amoxicillin resistant). Mechanisms of increased resistance were determined for 72 consecutively collected such amoxicillin-clavulanic acid-resistant isolates. MICs of the combination were 16-8 micrograms/ml for 51 (71%) of these and > or = 32-16 micrograms/ml for the remainder. The predominant mechanism was hyperproduction of enzymes isoelectrically cofocusing with TEM-1 (beta-lactamase activities, > 200 nmol of nitrocefin hydrolyzed per min per mg of protein) which was found in 44 isolates (61%); two isolates produced smaller amounts (approximately 150 nmol/min/mg) of such enzymes, and two isolates hyperproduced enzymes cofocusing with TEM-2. Eleven isolates produced enzymes cofocusing with OXA-1 beta-lactamase, which has previously been associated with resistance to amoxicillin-clavulanic acid. Ten isolates produced increased amounts of chromosomal beta-lactamase, and four of these additionally produced TEM-1 or TEM-2. Three isolates produced inhibitor-resistant TEM-group enzymes. In one of the enzymes (pI, 5.4), the amino acid sequence change was Met-67-->Val, and thus the enzyme is identical to TEM-34. Another (pI, 5.4) had the substitution Met-67-->Ile and is identical to IRT-I67, which we propose now be given the designation TEM-40. The third (pI, 5.2) had the substitution Arg-241-->Thr; this enzyme has not been reported previously and should be called TEM-41. The rarity and diversity of inhibitor-resistant TEM-group enzymes suggest that they are the result of spontaneous mutations that have not yet spread.

Mechanisms of hyperproduction of TEM-1 β-lactamase by clinical isolates of Escherichia coli

Abstract: The mechanisms of hyperproduction (defined as ≥ 200 nmoles nitrocefin hydrolysed per minute per mg of protein) of TEM-1 β-lactamase by 38 isolates of Escherichia coli were investigated. The copy numbers of TEM-encoding plasmids were determined for the hyperproducing isolates and for 39 TEM-1-producing isolates that did not hyperproduce the enzyme. Allele-specific PCR was used to determine if the promoter region of the TEM-I gene was of the TEM-I or TEM-2 type. Twenty three of the 38 hyperproducers had the TEM-1-type promoter but 15 had the more efficient TEM-2-type promoter ; in contrast, 37 of the 39 isolates with lower activities had the TEM-1-type promoter and only two had the TEM-2-type promoter. Many of the TEM-1-hyperproducing isolates possessed small plasmids (≤20 MDal) with high copy numbers, in some cases together with large, low copy number plasmids ; the average total copy number of TEM-encoding plasmids was 14 ; if only isolates with the TEM-1-type promoter were included, average total copy number was 22. Hyperproduction was attributed to high copy number (≥ 10) plasmids in 11 isolates ; another seven had plasmids with moderately high copy numbers (4-9). The average total copy number for isolates that produced relatively small amounts of TEM-I β-lactamase (≤100 nmoles/min/mg protein) was 2.2, and for the 12 isolates with TEM-1 activities of 101-200 nmoles/min/mg protein it was 6.8. We conclude that both high copy number plasmids and a more efficient promoter are common causes of hyperproduction of TEM-1.

Effects of the Anticonvulsant, Valproate, on the Expression of Components of the Cytochrome-P-450-Mediated Monooxygenase System and Glutathione S-Transferases

Abstract: It has been shown previously that the anticonvulsant agent, sodium valproate, induces certain cytochrome P-450 monooxygenase activities and decreases glutathione S-transferase activity. We have used Western blotting, RNase protection assays and Northern blot hybridization to determine the effects of valproate on the abundance of individual components of the cytochrome P-450 monooxygenase and of glutathione S-transferase subunits. Due to the short half-life of the drug in rats we have used an in vitro experimental system comprised of rat hepatocytes co-cultured with rat primitive biliary epithelial cells. Valproate was shown to be a potent inducer of two members of the cytochrome P-450 (CYP)2B subfamily, CYP2B1 and 2B2. The induction of the proteins was mediated at the level of the mRNAs, with the mRNA for CYP2B1 being more highly induced than that for CYP2B2. The drug also induced, but to a much lesser extent, two important components of the cytochrome-P-450-mediated monooxygenase system, NADPH-dependent cytochrome P-450 reductase and cytochrome b5, and their corresponding mRNAs. Thus, the effects of valproate on cytochromes P-450 and other components of the cytochrome-P450-mediated monooxygenase system mimic those of another, structurally diverse, antiepileptic drug, phenobarbital. However, in contrast to phenobarbital, which induces glutathione S-transferase subunits 1, 2, 3, 4 and 7, valproate selectively decreases the abundance of subunits 3 and/or 4. It has been shown previously that CYP2B1 is involved in the production of metabolites of valproate implicated in hepatotoxicity. The induction of this protein by valproate would thus contribute substantially to the hepatotoxic effects associated with the drug.

The ability of β-lactam antibioties to select mutants with derepressed β-lactamase synthesis from Citrobacter freundii

Abstract: The ability of beta-lactam compounds to induce synthesis of the class C beta-lactamase of Citrobacter freundii was assessed both directly and indirectly, the latter by measurement of the different susceptibilities of strains with inducible and depressed beta-lactamase synthesis and those of a beta-lactamase-negative strain. Most beta-lactam compounds were poor inducers but ampicillin, amoxycillin and cephalexin were moderately good inducers and cefoxitin, imipenem and meropenem were strong inducers. The ability of the compounds to select mutants in which the synthesis of the beta-lactamase was derepressed was also assessed. Imipenem and temocillin, which had a high degree of stability to the beta-lactamase failed to select such mutants or were very poor selectors. In contrast, most other compounds showed considerable lability to the beta-lactamase and readily selected derepressed mutants, whether they were strong inducers of beta-lactamase synthesis (for example cefoxitin) or poor inducers (cefotaxime, ceftazidime and many other compounds). Thus it appears that lability to the beta-lactamase is a more important factor in determining whether or not a compound will select derepressed mutants than the power of the compound as an inducer of beta-lactamase synthesis, since induction results in the production of less beta-lactamase than derepression as a result of mutation.

DNA sequence differences of ampD mutants of Citrobacter freundii

Abstract: Three groups of mutants with increased levels of beta-lactamase synthesis were selected from Citrobacter freundii 382010 by beta-lactam antibiotics at concentrations just above the MIC. Uninduced cultures of the hyperinducible group had 3- to 5-fold more beta-lactamase activity than the parent strain, with one mutant (termed type b) expressing 19 times the activity of the parent strain; the partially derepressed group had a relative 55-fold increase, while fully derepressed strains exhibited a 460-fold increase. Upon induction by growth in the presence of cefoxitin (32 micrograms/ml) for 2 h, the hyperinducible and derepressed groups had similar relative beta-lactamase activities of 650 and 725, respectively. Induction of beta-lactamase activity from partially derepressed mutants resulted in a relative activity of only 240. The ampD gene including its promoter region was amplified from the parent strain and the mutant strains by PCR. The sequence of ampD from the parent strain showed only three nucleotide changes from a previously published sequence, none of which resulted in a change to the deduced amino acid sequence. Hyperinducible mutant strains of type a had an amino acid change of either a tryptophan in codon 95 to an arginine (Trp-95-->Arg) (three mutants) or Ala-158-->Asp (one mutant). The hyperinducible type b strain had the change Tyr-102-->Asp. The derepressed strains had the following changes: Val-33-->Gly (one mutant), Asp-164-->Glu (one mutant), and Trp-95-->termination codon (two mutants). We infer that the amino acid changes in the hyperinducible mutants result in altered AmpD activity, whereas, in contrast, they lead to an inactive protein in derepressed mutants. No nucleotide differences were found in the ampD gene from partially derepressed strains.

Resistance to imipenem in Pseudomonas aeruginosa

Abstract: There was a slow increase in imipenem resistance in Pseudomonas aeruginosa at St. Thomas' Hospital in the 1990s, reaching 3% in 1994. Of the 94 imipenem-resistant strains, 69% were susceptible to all the other agents tested; 15% were also resistant to one other antibiotic, 5.3% to two, 6.4% to three, 4.3% to four or more compounds. 16% of strains were resistant to at least one other beta-lactam in addition to imipenem. Resistance to other antibiotics was more common among the strains for which imipenem MICs were 4 mg/L than among either imipenem-susceptible or imipenem-resistant (MICs > or = 8 mg/L) strains.

Evidence for the importance of weakly bound water for matrix metalloproteinase activity.

Abstract: The effects of organic cosolvents on the kinetic characteristics of two matrix metalloproteinases, gelatinase A and stromelysin 1, were investigated In each case, addition of the cosolvent resulted in a decrease in the apparent kcat/Km for the catalyzed hydrolysis of fluorogenic peptide substrates Two factors were identified as being responsible for this decrease in catalytic activity: hydrophobic partitioning of the substrate in favor of the bulk solvent and decrease in the water content of the enzyme The former reflects the hydrophobic nature of the enzyme-substrate interaction and the effect can be corrected for by using the solvent to water partition coefficient of the substrate in the mixed solvent systems The catalyzed hydrolysis of substrate, corrected for the effect of hydrophobic partitioning, was demonstrated to be sixth order in water for gelatinase A and third order in water for stromelysin 1 Variation in water concentration did not produce saturation even at concentrations close to 555 M The results indicate that weakly bound water molecules are essential to mediate the interaction between substrate and enzyme The sensitivity of these enzymes to water concentration could be an important mechanism for regulating catalytic activity in vivo

In-vitro activity of purpuromycin and MDL 63, 604 against microorganisms that cause vaginitis and vaginosis

Abstract: Purpuromycin and its semi-synthetic derivative MDL 63,604 had in-vitro activity similar to that of amphotericin B against isolates of Candida albicans. MDL 63,604 had activity similar to that of metronidazole against Trichomonas vaginalis. Both compounds were very active against most species of Gram-positive and Gram-negative anaerobes and against Gardnerella vaginalis. MDL 63,604 had significantly lower MICs than purpuromycin against T. vaginalis and most of the bacteria, probably due to antagonism of purpuromycin's activity by medium supplements (blood or serum). Purpuromycin or related compounds may have a potential role in the topical treatment of vaginitis and vaginosis.