Investigation of oxacillin-hydrolyzing beta-lactamase in borderline methicillin-resistant clinical isolates of Staphylococcus aureus.

BACKGROUND: Mechanisms of borderline resistance of Staphylococcus aureus to penicillinase-resistant penicillins (PRPs) may include hyperproduction of classical penicillinase and/or production of beta-lactamase hydrolyzing also PRPs. METHODS: beta-Lactamase activity of whole cells and purified enzymes was estimated spectrophotometrically and in isolated cytoplasmic membranes by bioassay with Bacillus subtilis as test strain. RESULTS: Out of 53 clinical isolates of S. aureus, 18 showed oxacillin MIC values from 0.5 to 2 microg/ml, which were reduced by sulbactam and/or clavulanic acid in the case of four isolates producing large quantities of inducible, type A beta-lactamase. Cytoplasmic membranes isolated from these strains showed oxacillin-hydrolyzing activity. One of these strains was grown also in the presence of globomycin, an antibiotic known to interfere with the anchorage of membrane lipoproteins; this treatment eliminated the oxacillin-hydrolyzing activity. CONCLUSIONS: The resistance in these strains was due to a membrane-bound lipoprotein with oxacillin-hydrolyzing activity.

beta-Lactam susceptibility patterns and investigation of cephalosporin hydrolysing beta-lactamases of Gram-negative extraintestinal clinical isolates.

Of more than 3500 isolates of enterobacteriaceae, 48-69% were resistant to aminopenicillins and 11-45% to amoxycillin+clavulanic acid. Resistance to second and third generation cephalosporins was present in 11-17 and 3-8% of Escherichia coli, 47-56 and 15-52% of Klebsiella-Enterobacter, 36-57 and 16-27% of Proteus, Providencia and Morganella isolates. Pseudomonas aeruginosa strains varied in their resistance to antipseudomonal beta-lactams. Isoelectric points, inhibitor profiles and substrate profiles of beta-lactamases extracted from representatives of the resistant strains indicated that the resistance was mainly due to the hyperproduction of chromosomally encoded AmpC beta-lactamases. This was confirmed by plasmid profile and PCR investigations. Extended-spectrum beta-lactamase and metallo-penicillinase producing strains were not found. One Pseudomonas maltophilia strain produced an oxacillinase.

A synthetic gamma-lactone group with beta-lactamase inhibitory and sporulation initiation effects.

Previous studies showed that some lactones have beta-lactamase inhibitory or antibacterial effects, others--like A-factor (a gamma-butyrolactone) and its derivatives--stimulate sporulation in Streptomyces griseus strains. Our experiments were aimed at exploring whether synthetic gamma-lactones had such effects. None of the seven gamma-lactones studied showed antibacterial activity, but two of them inhibited beta-lactamases isolated from various bacteria. These two gamma-lactones did not reduce colony formation of murine bone marrow cells in vitro, indicating that they were not toxic to proliferating mammalian cells. Four gamma-lactones, including the two inhibiting beta-lactamase, stimulated sporulation in the non-sporulating S. griseus bald 7 mutant. Further studies of gamma-lactones as potential inhibitors of beta-lactamase seem to be warranted.

Beta-lactam antibiotics functionalized with gem-bisphosphonates.

Acylation of amoxycillin and cephalexin with acids III, V and VII, and with isocyanate VIII furnished the corresponding beta-lactam antibiotics (X and XIII-XV, respectively). The antibacterial activity of these new antibiotic analogues against Helicobacter pylori was found to be identical with those of amoxycillin, Augmentin, erythromycin and ciprofloxacin.

Comparison of the toxicity of fluconazole and other azole antifungal drugs to murine and human granulocyte-macrophage progenitor cells in vitro.

We studied the inhibitory effects on colony formation by granulocyte-macrophage colony forming units (cfu-gm) of eight azole antifungal agents in vitro. All agents, except fluconazole, inhibited colony formation dose-dependently with 50% inhibitory concentrations (IC50) in the range of 0.78-49 micromol/L in cultures of murine and human bone marrow. For human cells, the IC50 values were 0.553 mg/L for itraconazole, 1.24 mg/L for saperconazole, 2.58 mg/L for clotrimazole, 5.33 mg/L for miconazole, 6.17 mg/L for econazole, 6.27 mg/L for ketoconazole and 8.38 mg/L for oxiconazole. The IC50 of itraconazole for human cfu-gm in vitro was similar to the plasma level of this drug recommended for systemic antifungal therapy (>0.5 mg/L) thus indicating the potential clinical relevance of our data. The IC50 of ketoconazole for human cfu-gm in vitro may be exceeded by plasma levels produced in vivo by high (> or =400 mg) doses, whereas fluconazole failed to reduce colony formation by 50% even at 100 mg/L, a concentration not reached in vivo even after extremely high doses (2000 mg/day). To most of the drugs studied, murine progenitor cells seemed to be less sensitive than the human ones. There was, however, a close correlation between the murine and human log IC50 values of the drugs (r2 = 0.964, P< 0.001), suggesting that cultures of murine bone marrow may be suitable to predict the in-vitro toxicity of azole antifungals to human cfu-gm.

[Effect of imidazole antifungal agents on colony formation by murine bone marrow CFUc (colony forming units in culture]. / Antifungális hatású imidazol-származékok hatása egér csontvelósejtek in vitro kolóniaképzésére.

In spite of modern antifungal therapy, the prognosis of systemic mycoses in neutropenic patients is usually poor without recovery of neutrophil counts. So, even a minor myelotoxicity might be a significant disadvantage of any drug used for the treatment of neutropenic patients with fungal infections. Since "Colony Forming Units in culture" (CFUc), the common progenitors of granulocytes and macrophages, are supposed to be a major target of agents damaging bone marrow, we studied the inhibitory effect of four imidazole antifungal drugs to colony formation by murine CFUc in vitro. Clotrimazole, econazole, miconazole or ketoconazole were added to soft agar bone marrow cell cultures at final concentrations of 1 to 30 mg/l at the beginning of the 7 day culture period. A dose-dependent inhibitory effect on colony formation by CFUc was observed with all imidazole drugs studied. The 50 percent inhibitory concentrations (IC50s) were 3.54 mg/l for clotrimazole, 8.07 mg/l for econazole, 14.04 mg/l for miconazole, and 16.11 mg/l for ketoconazole. Human pharmacokinetic data available in the literature on these drugs may help to assess the potential in vivo relevance of our results. The serum levels of clotrimazole and econazole, even after oral administration, remain lower than those found to inhibit colony formation by murine bone marrow in our experiments. Taking into consideration that clotrimazole and econazole are used only topically in the clinical practice, our data do not suggest any clinically significant suppression of bone marrow by these two drugs. Intravenous administration of high doses of miconazole, however, may result in serum concentrations approaching the IC50 for colony formation by murine bone marrow cells in vitro. As for ketoconazole, it may suppress the proliferation of murine bone marrow progenitor cells in vitro at concentrations produced in vivo by high doses (12.5-18 and 30-50 mg/l after 400 or 600 mg, respectively). The serum levels produced by a daily dose of 200 mg ketoconazole (about 4 mg/l), however, did not reduce significantly the number of colonies in murine bone marrow cultures. Our present results warrant further studies of the myelotoxicity of miconazole and ketoconazole in vivo in mice with neutropenia induced by cytostatic agents.

Use of chromatofocusing for separation of beta-lactamases. IX. Analytical chromatofocusing for the separation of a chromosomal cephalosporinase from Proteus vulgaris 1028.

Simultaneous purification and isoelectric point (pI) determination was carried out at analytical scale of the chromosomal cephalosporinase from the Proteus vulgaris 1028 strain. Comparison of the enzyme to the purification results with m-aminophenylboronic acid-agarose affinity chromatography with sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that minute amounts of accompanying proteins having identical pI values but different molecular masses were found in the chromatofocused preparation. The molecular mass of the enzyme was 24,000 dalton. The pI was found to be 8.3.

Purification and some properties of Candida albicans DNA polymerases.

DNA polymerases of Candida albicans were purified to near homogeneity. Three well distinguished peaks of DNA polymerase activity (Enzyme I, II and III respectively) were obtained by DEAE-Sephacel chromatography. This purification step was followed by column chromatographies on Sepharose 6B and denatured DNA-cellulose. The enzymes' molecular mass and biochemical properties, including their inhibition by aphidicolin, were studied. Molecular mass was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and was found to be 110 kDa for Enzyme I, 80 kDa for Enzyme II and 50 kDa for Enzyme III.

In vitro investigation of BK-218, a new oral and parenteral cephalosporin.

The antibacterial activity of BK-218 was similar to that of cefamandole when it was tested against several laboratory strains. The inhibiting effect of BK-218 was greater than that of cephalexin and cefoxitin on penicillin-binding proteins of Escherichia coli HB101. This result was in close correlation with the relative inhibition of radiolabeled glucosamine incorporation (greatest with BK-218) and with the lytic effect (most intensive with BK-218). BK-218 proved to be a good inhibitor for all five of the beta-lactamases that were investigated, although two enzymes (Enterobacter cloacae P99 and Pseudomonas aeruginosa Cilote) hydrolyzed it to some extent.

Modified general affinity adsorbent for large-scale purification of penicillinases.

N-Acetyl-D-(-)-penicillamine as a stable second-generation biospecific affinity ligand has previously been suggested for purification of Bacillus cereus 569/H beta-lactamase I. A complex spacer arm is coupled with the matrix by using epichlorohydrin and phloroglucinol doubly activated with divinyl sulphone in the meta position. Coupling of D-(-)-penicillamine ligand resulted in an active affigel. However, we found that two affinity ligands in close proximity prevents simultaneous binding of two penicillinase molecules, therefore one ligand is superfluous. Our results show that: (1) shortening the spacer arm by direct activation of the matrix with divinyl sulphone is satisfactory to produce the affinity material with N-acetyl-D-(-)-penicillamine; (2) incorporation of 15 mumol of N-acetyl-D-(-)-penicillamine per ml of wet Sepharose 4B satisfies the maximum binding capacity requirements of the affigel (about half of the originally incorporated amount of ligand); (3) our simplified affinity adsorbent is generally applicable for large-scale purification of penicillinases to homogeneity from various bacterial sources by the convenient batch method without prior concentration of these enzymes; (4) reacetylation for four/five times can regenerate the original binding capacity of the affigel.

Curing of 60Co gamma-resistance inducing effect of R46 R-factor by 5-fluorouracil.

The 60Co gamma-resistance inducing effect of R46 factor, and its elimination by 5-fluorouracil were studied. R46 increased the survival of the wild-type strain and its rec- mutants. After treatment with 5-fluorouracil (1 g/liter) the clones lost not only antibiotic resistance, but the 60Co gamma-radioresistance as well, encoded by R46 R-factor.

Use of chromatofocusing for separation of beta-lactamases. VIII. Analytical chromatofocusing of chromosomal cephalosporinases from four Klebsiella strains.

Although still there are Klebsiella strains which do not harbour plasmids and produce constitutive chromosomal beta-lactamases, recently clinical isolates were found in ever increasing numbers carrying mainly TEM-, CARB- and OXA type R-factors. We selected four chromosomal cephalosporinase producing Klebsiella strains to study the pI values of the enzymes and their simultaneous separability from accompanying proteins by chromatofocusing techniques. We compared pI values of the pure and the crude preparations: K. pneumoniae K1 SC 10436: pIpure = 6.4, pIcrude = 6.42; K. aerogenes K1 1082 E: pIpure = 6.5, pIcrude = 6.5; K. oxytoca 1082 E: pIpure = 6.42, pIcrude = 6.4; K. oxytoca 20: pIpure = 7.62, pIcrude = 7.6. Excellent agreement of the pI values among each other, but occasional differences with those obtained by analytical isoelectrofocusing are attributed to methodological diversities and to the presence of satellite enzymes, known to exist in Klebsiella.

Elimination of UV-resistance inducing effect of R46 R factor by 5-fluorouracil.

R-factor curing capacity of 5-fluorouracil (5-FU) was studied. Well detectable increase in UV-resistance was found in E. coli K12 AB1157 strain and its recB-, recC-, recF- mutants harbouring R46 R-factor. After treatment with 5-FU, these strains lost not only the antibiotic resistance coded for R46 R-factor but their UV-radioresistance, as well.

Effect of the pKM101 plasmid on the repair of single-strand breaks in DNA induced by ionizing irradiation in Escherichia coli.

The effect of pKM101 plasmid on repair of single-strand breaks in DNA induced by 60Co-gamma irradiation in E. coli K12 AB1157 (wild type) and in its recA- and recB- mutant cells was studied by alkaline sucrose gradient sedimentation method. For quantitative analysis of sedimentation profiles we calculated the S1/2 values described by Veatch and Okada. The S1/2 values of unirradiated cells were 21.10, and after 200 Gray irradiation 11.35, due to the original incidence of single-strand breaks. The presence of pKM101 did not influence these values in either cases. This means that pKM101 had no effect on the rise of single-strand breaks in DNA. During a post-irradiation incubation period at 37 degrees C for 60 min the S1/2 value of the wild type strain increased from 11.35 to 19.22, that of the recB- from 11.50 to 15.23, while the S1/2 value of the recA- mutant did not change owing to the lack of repair of single-strand breaks. pKM101 plasmid markedly increased the S1/2 value in wild type strain and in recB- mutant, while it had no effect on S1/2 in recA- cells, during this post-irradiation incubation period. Thus the effect of pKM101 on the repair of single-strand breaks in DNA proved to be dependent on recA+ genotype. Nalidixic acid at 100 micrograms/ml concentration inhibited the repair of single-strand breaks in both wild type and recB- mutant cells harbouring pKM101 plasmid.