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At least 127 records · Page 7Linked to original sources
PubMed1968

Characteristics of penicillinase release by washed cells of Bacillus licheniformis.

Saline-washed cells of Bacillus licheniformis strain 749/C (constitutive for penicillinase) were able to release exopenicillinase in the presence of concentrations of chloramphenicol that prevented protein synthesis completely. The release reaction was strongly pH-dependent, occurring at a faster rate at alkaline pH in anionic or cationic buffers than at neutral pH. A strongly pH-dependent release reaction was noted in growing cells also. The reaction in washed cells can be stopped completely by changing the pH to 6.0. Within 30 min at pH 9.0, about 55% of the cell-bound penicillinase was released; thereafter, release continued at a greatly reduced rate. Suspensions of washed cells retained their capacity to release penicillinase at pH 9.0 for 90 min. Penicillinase released at pH 9.0 from either cells or protoplasts was not readsorbed over a 60-min period after changing the pH to 6.0. The release reaction was strongly temperature-dependent. We examined the effect of a large number of metabolic inhibitors and other compounds on the pH-dependent release phenomenon. Quinacrine hydrochloride, chloroquine diphosphate, and chlorpromazine hydrochloride reduced secretion substantially at 10(-4)m. Deoxycholate and Triton X-100 were active at 10(-3)m, but tungstate, arsenate, and molybdate had small effects at 10(-1)m. The rate of exopenicillinase release at pH 9.0 from fully stabilized protoplasts was one-half that of intact cells. Protoplasts lysed in hypotonic media or detergents showed even greater reduction in releasing activity. Penicillinase released from washed cells at pH 7.5 or 9.0 appeared to be derived from the periplasmic tubule and vesicle fraction that was released by protoplast formation.

Antimetabolites↗
PubMed1968

Localization of cell-bound penicillinase in Bacillus licheniformis.

When protoplasts are prepared from Bacillus licheniformis (strain 749/C, constitutive for penicillinase), approximately 60% of the cell-bound penicillinase is released. The remainder is retained by the protoplast and cannot be removed by washing. This release is specific, in that less than 7% of the cellular reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase and alpha-glucosidase is liberated by the treatment. The freed penicillinase is excluded from G-200 Sephadex, and it is partially sedimented with a force of 65,000 x g for 20 hr. It is probably attached to characteristic tubular and vesicular structures with single-layered membranes that are comparable to structures previously described in intact penicillinase-forming cells. The specific activity of the organelle is more than six times that of twice washed peripheral membrane; furthermore, about 8% of the protein of the structure is penicillinase. At substrate concentrations (benzylpenicillin) of about one-fifth the K(m) value, whole cells show a slight permeability restriction, although this does not occur in isolated particles and protoplasts.

Bacillus↗
PubMed1982

Failure of the fluorescent antibody reaction to identify penicillinase-producing gonococci.

The fluorescent antibody test is now widely used to confirm the identity of Neisseria gonorrhoeae but may fail to identify penicillinase-producing strains (PPNG). This problem arises when conjugates are used that incorporate only gonococci that are not penicillinase-producers. We have shown that conjugates prepared from mixtures of PPNG and non-penicillinase producing gonococci give good fluorescent reactions. This difference in the reactions of PPNG strains is clearly related to their penicillinase-producing abilities, further study of the antigenic relation between penicillinase production and the antigenic structure of N gonorrhoeae is evidently required.

Fluorescent Antibody Technique↗
PubMed1986

In vitro studies of plasmid-mediated penicillinase from Streptococcus faecalis suggest a staphylococcal origin.

A strain of Streptococcus faecalis with plasmid-mediated penicillinase production was studied further. Partially purified penicillinase from the S. faecalis strain hydrolyzed penicillin, ampicillin, and ureido-penicillins but not penicillinase-resistant semisynthetic penicillins, cephalosporins, or imipenem; hydrolysis was inhibited by clavulanic acid. Hydrolysis of a given antibiotic correlated with a marked increase in the minimal inhibitory concentration (MIC) of that drug when a high inoculum was used. As with most enterococci, the MICs of cephalosporins and penicillinase-resistant semisynthetic penicillins were too high for clinical usefulness, although these agents did not show an inoculum effect. Based upon hybridization under stringent conditions of plasmid DNA from the S. faecalis strain to cloned penicillinase genes from Staphylococcus aureus, it appears that these resistance determinants are highly homologous and suggests that this enzyme was introduced into streptococci from staphylococci.

Anti-Bacterial Agents↗
PubMed1980

[A simple method for differentiating methicillin-resistant, penicillinase-positive and -negative staphylococci by agardiffusion test (author's transl)].

Coagulase-positive staphylococci (41 methicillin-resistant strains, 28 penicillinase-positive and 12 penicillinase-negative strains) were tested against most types of penicillines commercially available on the Austrian market using both broth-dilution test (incubated during 48 hours at 35 degrees C) and agardiffusion tests (incubated during 24 hours at 30, 35 and 37 degrees C) employing Mueller-Hinton-broth and -agar, respectively, in order to find out the most convenient way of detecting methicillin-resistant strains. Consecutively, the conclusions drawn from these experiments were verified for tests on Isosensitest-agar (Oxoid). It was demonstrated that methicillin-resistant strains could be detected easily with discs of methicillin and oxacillin at 30 degrees C (Fig. 1). At 35 degrees C this was nearly as easily possible for methicillin but oxacillin discs had to be used at amounts of 1 microgram instead of 5 microgram. Excepting penicillin G-discs with the other penicillines differing numbers of methicillin-resistant strains would have been missed at 37 degrees C (Fig. 1, 2). Only with discs containing 6 microgram penicillin G methicillin-resistant strains were unequivocally identifiable in the agardiffusion test at all 3 incubation-temperatures (Fig. 3), the largest inhibition zone diameter being 12 mm. Penicillinase-positive but methicillin-sensitive strains always produced larger inhibition zones up to 30 mm. From these strains again penicillinase-negative strains were equally well distinguishable by much larger inhibition zones. So, the conclusion was drawn that on Mueller-Hinton agar one disc loaded with 6 microgram of penicillin G allows proper distinction not only of methicillin-resistant and -sensitive but also of penicillinase-positive and -negative strains of staphylococci. On Isosensitest-agar this is true for an incubation-temperature of 35 but not 37 degrees C (Tab. 1).

Culture Media↗
PubMed1982

[Detection of penicillinase activity and its relation to other properties in staphylococci].

Comparison of the microbiological, iodometric and acidometric methods for determination of the penicillinase activity of staphylococci showed that the acidometric method was the simplest from the technical viewpoint and available for a wide range of laboratories. Penicillinase is more often produced by Staph. aureus than by Staph. epidermidis. No clear differences between production of penicillinase by various biovars of Staph. epidermidis were detected. The penicillinase activity was most often observed in the strains resistant to penicillin, erythromycin and ther antibiotics. No strains sensitive to zinc and silicon were isolated. Among Staph. aureus 96.15, 84.38, 78.18 and 64.71 per cent of the strains resistant to the ions of cadmium, mercury, plumbum and arsenic respectively produced penicillinase. No such definite results were obtained with respect to the strains of both the whole species of Staph. epidermidis and its separate biovars.

Anti-Bacterial Agents↗
PubMed1977

Penicillinase-releasing protease of Bacillus licheniformis 749 Specificity for hydroxyamino acids.

The membrane penicillinase of Bacillus licheniformis 749/C differs from the exopenicillinase in that it has an additional 24 amino acid residues and a phosphatidylserine at the NH2 terminus (Yamamoto, S., and Lampen, J.O. (1976) J. Biol. Chem. 251, 4095-4101). The conversion of the membrane penicillinase to the exo form is probably carried out by a specific penicillinase-releasing protease (PR-protease) whose properties are generally consistent with the properties of penicillinase secretion. The substrate specificity of the PR-protease was determined by identifying the NH2 and COOH termini of the peptides produced by hydrolysis of ribonuclease B and beef insulin. The enzyme hydrolyzed only peptide bonds involving the carboxyl groups of serine or thrombine. Similar bonds in synthetic di- or tripeptides of L-serine were not cleaved. The existence of seryl-lysine and threonyl-glucamic acid bonds in the protease-susceptible (phospholipopeptide) region of the membrane penicillinase and the presence of only lysine or glutamic acid at the NH2 terminus of the exoenzyme released in vivo are consistent with the specificity of PR-protease; hence, we propose that this enzyme has an essential role in the formation of exopenicillinase. The PR-protease is a potential tool for protein sequence determination because of its narrow and novel substrate specificity.

Amino Acid Sequence↗
PubMed1977

[Mechanism of penicillinase inhibition by alkyl sulfates in the presence of synthetic polyeletrolytes].

Competing inhibition of Bacillus licheniformis 749/C penicillinase by alkylsulfates CnH2n+ 1OSO3N1 where n was 8--16 was studied. The values of the inhibition constants Ki of individual homologues were estimated. It was shown that stability of complex "enzyme-inhibitor" increased with lengthening of the hydrocarbon radical which was probably due to increased hydrophobic interaction of the alkyl radical with the lipophilic areas of the penicillinase active center. Inhibition in the presence of sopolymers of vinylpirrolidone with N-alkylvinylamine was studied with the aim of modeling the process of penicillinase inhibitions by alkylsulfates in the presence of the blood serum. It was shown that polyelectrolytes posessing hydrophobic substituents had an ability of reducing the activity of inhibited penicillinase, cooperative transfer of alkylsulfate molecules from the enzyme to polyelectrolyte being observed. The maximum effect was registered in the polyelectrolytes with substituents C12H25 and C16H33 which was confirmed in the experiments with the penicillinase-producing strains of staphylococci.

Alkanesulfonates↗
PubMed1978

Conformation of a stable intermediate on the folding pathway of Staphylococcus aureus penicillinase.

The partly unfolded intermediate (state H) of penicillinase from Staphylococcus aureus PC1 is stable in 0.8 M guanidinium chloride at pH 7.0. It has been characterized by measurements of intrinsic viscosity, sedimentation and diffusion coefficients, leading to an equivalent hydrodynamic volume of five times that of the native penicillinase molecule. Values of alpha-helix content calculated from circular dichroism were 27% for penicillinase and 16% for state H. A multi-domain model is proposed for penicillinase in which the domains can separate without appreciable change in secondary structure. This model is important in understanding the means by which the enzyme activity can be controlled, and corresponds to a kinetic pathway of folding.

Circular Dichroism↗
PubMed1992

Influence of different combinations of antibodies and penicillinase-labeled testosterone derivatives on sensitivity and specificity of immunoassays.

Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hydroxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.

Antibodies↗
PubMed1981

Bacillus licheniformis penicillinase: cleavages and attachment of lipid during cotranslational secretion.

The penicillinase of Bacillus licheniformis is shown to be secreted cotranslationally. In extracts it was formed by membrane-associated but not by free polysomes; and after extracellular labeling of cells, followed by completion of the growing chains on polysomes in vitro, labeled penicillinase could be immunoprecipitated. This product contained electrophoretic peaks of Mr 36,000, 33,000, and 29,000, which correspond to previously reported forms of the enzyme. The Mr 36,000 form exhibits moderate hydrophobicity, as expected of a precursor with an NH2-terminal signal sequence for secretion. In addition, part of the Mr 33,000 fraction evidently contains a lipid: it is even more hydrophobic, and [2-3H]glycerol was found to be incorporated into these molecules but not into the other forms of the enzyme. These findings renew the earlier, discarded suggestion that the Mr 33,000 membrane-bound penicillinase in the cells contains lipid. The incorporation of lipid and two different cleavages can evidently all occur during growth of the penicillinase chain. Moreover, the resulting terminal regions are all accessible to extracellular labeling on growing chains. Several additional, unidentified lipoproteins also incorporate lipid during chain growth.

Bacillus↗
PubMed1981

Lipoprotein nature of Bacillus licheniformis membrane penicillinase.

Membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis bears a striking resemblance to the major outer membrane lipoprotein of Escherichia coli. It can be specifically labeled in vivo with [3H]glycerol, [35S]cysteine, or [3H]palmitate but not by [32P]orthophosphate. The labeled residues are located at or near the NH2 terminus of the membrane penicillinase because they can be completely removed by trypsin which cleaves a hydrophobic peptide(s) from the NH2 terminus, thereby rendering the enzyme hydrophilic. The membrane penicillinase produced by the 749/C gene carried in E. coli on phage lambda is similar to the enzyme formed in strain 749/C itself. The peptide antibiotic globomycin, which prevents processing of the E. coli prolipoprotein, severely inhibited the attachment of [3H]palmitate or [3H]glycerol to the 749/C enzyme (either in B. licheniformis 749/C or in E. coli), blocked the accumulation of penicillinase in the plasma membrane, and enhanced the formation of exoenzyme. Under the same conditions, globomycin does not prevent the attachment of palmitate or glycerol to the E. coli prolipoprotein but inhibits processing of the modified precursor to the mature lipoprotein. These results are in contrast with the lack of effect of globomycin on the RTEM-beta-lactamase of E. coli which has no detectable hydrophobic membrane form and was not labeled with palmitate or glycerol.

Amino Acid Sequence↗
PubMed1983

Correlation of penicillinase production with phage type and susceptibility to antibiotics and heavy metals in Staphylococcus aureus.

One hundred and thirty-nine bacteraemia strains of Staphylococcus aureus, representing different combinations of phage type and susceptibility to antibiotics and to cadmium (Cd), arsenate (As) and mercury (Hg), were investigated for penicillinase production. The determination of enzyme activity in induced and uninduced conditions was performed by iodometric titration. The amount of penicillinase produced could be correlated with phage pattern. Epidemically occurring strains of the 94,96 and the 83A complexes produced the largest amount of penicillinase, whereas strains of the 52,52A,80,81 complex were weaker producers. Group-II and group-III strains produced the smallest amount. Susceptibility to antibiotics and to Cd, As and Hg could not be correlated with enzyme activity, but strains resistant to penicillin plus tetracyclines and strains resistant only to Cd did produce less enzyme than strains with other resistance patterns. The percentage mean values than strains with other resistance patterns. The percentage mean values of extracellularity of the enzyme was highest amongst strains of the 94,96 complex and of type 95. Four strains had constitutive production, one being macro-constitutive and three micro-constitutive. All four strains represented rare combinations of the above properties but were susceptible to fusidic acid. The importance of penicillinase production by epidemically occurring strains is discussed.

Acetates↗
PubMed1976

Inducible oxacillin-hydrolyzing penicillinase in Aeromonas hydrophila isolated from fish.

An inducible penicillinase was shown to be present in a strain of Aeromonas hydrophila subsp. hydrophila isolated from freshwater fish. Enzyme induction was observed with benzylpenicillin or 6-aminopenicillanic acid, and the enzyme was cell bound. The penicillinase was purified 50-fold from a crude cell extract. The molecular weight was estimated to be 23,000 by gel filtration. The pH and temperature optima for the enzyme activity were 8.0 and 35 degrees C, respectively. The penicillinase showed a unique substrate profile by hydrolyzing oxacillin about twice as rapidly as benzylpenicillin. The enzyme activity was weakly inhibited by sodium chloride but was not affected by p-chloromercuribenzoate. The property of penicillinase production by the A. hydrophila strain could not be transferred to Escherichia coli and also could not be eliminated from the bacteria by ethidium bromide treatment.

Aeromonas↗
PubMed1973

Screening and isolation of penicillinase inhibitor, KA-107.

It is known that penicillin resistance of bacteria is mainly caused by the inactivation of penicillin by penicillinase derived from such strains. We have developed a screening procedure for penicillinase inhibitors. Several microorganisms were found to produce such inhibitors, and from the culture filtrate of Streptomyces gedanensis ATCC 4880 a penicillinase inhibitor, named KA-107, was isolated. The characteristics of this inhibitor were revealed by an in vitro test by using penicillinase derived from penicillin resistant Staphylococcus aureus, FS-1277. When KA-107 was used in combination with penicillin-G, ampicillin, d- or l-phenethicillin, the growth inhibitory activity of these penicillins was maintained.

Anti-Bacterial Agents↗
PubMed2004

Amoxicillin-clavulanate therapy increases childhood nasal colonization by methicillin-susceptible Staphylococcus aureus strains producing high levels of penicillinase.

We examined factors associated with penicillinase production by nasal carriage Staphylococcus aureus strains in 648 children aged 3 to 6 years attending 20 randomly sampled playschools. The children were prospectively monitored for drug use and medical events for 6 months and were then screened for S. aureus carriage. Isolates were tested for their susceptibility to penicillin G and methicillin, and penicillinase production by methicillin-susceptible, penicillin-resistant strains was quantified. S. aureus was isolated from 166 children (25.6%). Exposure to amoxicillin-clavulanate during the previous 3 months was associated with higher penicillinase production by penicillin-resistant, methicillin-susceptible strains (odds ratio, 3.6; P = 0.03). These results suggest that use of the amoxicillin-clavulanate combination could induce a herd selection process of S. aureus strains producing higher levels of penicillinase.

Amoxicillin-Potassium Clavulanate Combination↗
PubMed1969

Periplasmic structure of frozen-etched and negatively stained cells of Bacillus licheniformis as correlated with penicillinase formation.

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.

Bacillus↗
PubMed1970

Phospholipid metabolism during penicillinase production in Bacillus licheniformis.

During membrane-bound penicillinase production, Bacillus licheniformis forms vesicles and tubules that do not appear in the absence of penicillinase production. The major lipids of B. licheniformis were shown to be phospholipids. The proportions, metabolism, and the total phospholipid per cell were shown to be essentially the same in the uninduced control, induced and constitutive penicillinase forming cells during both the exponential and stationary growth phases. Membrane phospholipids were not secreted into the medium during penicillinase formation. In the shift from the exponential to the stationary growth phase, there was an accumulation of phosphatidyl glycerol and a marked decrease in cardiolipin. These two lipids had the most active turnover of their phospholipid phosphate of all the lipids studied.

Bacillus↗