Ferenc J. Hernádi

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 β-lactamase hydrolyzing also PRPs. Methods: β-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 µg/ml, which were reduced by sulbactam and/or clavulanic acid in the case of four isolates producing large quantities of inducible, type A β-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.

β-lactam susceptibility patterns and investigation of cephalosporin hydrolysing β-lactamases of Gram-negative extraintestinal clinical isolates.

Abstract 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 β-lactams. Isoelectric points, inhibitor profiles and substrate profiles of β-lactamases extracted from representatives of the resistant strains indicated that the resistance was mainly due to the hyperproduction of chromosomally encoded AmpC β-lactamases. This was confirmed by plasmid profile and PCR investigations. Extended-spectrum β-lactamase and metallo-penicillinase producing strains were not found. One Pseudomonas maltophilia strain produced an oxacillinase.

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.

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.

Use of chromatofocusing for separation of β-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.

Use of chromatofocusing for separation of β-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.