Article

{beta}-Lactamase and the Resistance of Pseudomonas aeruginosa to Various Penicillins and Cephalosporins

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Abstract

Eight strains of Pseudomonas aeruginosa, induced by penicillin G, produced different amounts of β-lactamase. Significant enzyme activity appeared within 5 min. and its highest value was obtained 3 h. after the addition of penicillin G. The production of the enzyme was arrested shortly after removal of the inducer. High concentrations of penicillin G, other penicillins or cephalosporins were necessary for optimal induction. 6-Amino-penicillanic acid (6-APA) was the best inducer tested, while cephalothin was the worst. Most of the enzyme was intracellular after conversion of the cells to spheroplasts in the presence of carbenicillin. The structure of both the nucleus and side chain of the penicillins and cephalosporins determined the rate of their hydrolysis by the β-lactamase. Generally, the enzyme was more active against 7-aminocephalosporanic acid (7-ACA) derivatives than against 6-APA derivatives, except cephalexin and cephaloglycin, which were relatively resistant to hydrolysis. No relation was found between the sensitivity of the pseudomonads to penicillin G and the amount of the enzyme in their cell-free preparations. The sensitivity of the different antibiotics to hydrolysis by the enzyme was not a major determinant in the resistance of Pseudomonas aeruginosa 1978 to them. These results indicate that the resistance of the bacteria to the β-lactamase-sensitive penicillins and to cephalosporins is dependent on a combined effect of β-lactamase and on an intrinsic resistance, while the resistance of the bacteria to the β-lactamase-resistant penicillins depends on the intrinsic resistance alone.

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... Production of a chromosomally-mediated class Id (Richmond & Sykes, 1973) /Mactamase, the Sabath Abrahams (Sabath, Jago & Abrahams, 1965) enzyme, is universal amongst Pseudomonas aeruginosa strains. Most of the early /Mactams, for example penicillin G, ampicillin, cephaloridine and cephalothin, are hydrolysed by this /Mactamase (Sabath el al., 1965;Garber & Friedman, 1970). Carbenicillin, however, is stable and acts as an inhibitor, causing conformational changes in Id enzyme which render it inactive (Furth, 1979). ...
... Maximal Id induction requires high /Mactam concentrations, for example 5 g/1 penicillin G (Garber & Friedman, 1970). This would suggest induction is a 'rather unphysiological process' (Nordstrom & Sykes, 1974). ...
... In vitro hydrolysis studies indicate SA enzyme to be active against ampicillin, penicillin G and cephaloridine (Sabath el ai, 1965). Carbenicillin, however, is virtually SA stable and gives potent suicide inhibition of SA enzyme (Garber & Friedman, 1970;Furth, 1979). Sensitivity studies with inducibility negative mutants confirm SA enzyme is an important defence against these labile agents but not carbenicillin (Rosselet & Zimmermann, 1973;Ohmori et al, 1977;Curtis et al., 1981). ...
... Cephaloridine, which readily induces SA enzyme (Sabath et al., 1965;Garber & Friedman, 1970), anatgonized the activity of cefotaxime against the majority (12/14) Ps. aeruginosa isolates. Activity of cefsulodin was antagonised against only two strains, both of which produce exceptionally large quantities of SA enzyme (unpublished data). ...
Article
All Pseudomonas aeruginosa strains produce Sabath and Abrahams' (SA) enzyme, as inducible β-lactamase. Hydrolysis of cefotaxime by this enzyme, although slow in terms ofVmaxwas efficient when the parameter Vmax/Km (physiological efficiency (Pollock, 1965)) was considered. Hydrolysis of cefsulo–din was not detectable in assays used and enzyme binding (Ki) was poor, ensuring a very low physiological efficiency. Physiological efficiency represents a measure of enzyme function under low substrate (antibiotic) conditions, as apply in the periplasm; consequently SA enzyme might protect the cell against cefotaxime, but not cefsulodin. This depends on enzyme induction and retention within the periplasm. Agar checkerboard studies indicated the SA inducer cephaloridine antagonized the activity of cefotaxime against most (12/14) Ps. aeruginosa strains but cefsulo––din against only a minority (12/14). Cephaloridine/cefotaxime antagonism was lost in uninducible (SAI/) or constitutive (SAPcom) mutants where SA expression was independent of cephaloridine concentration. This indicated the antagonism was SA dependent. Cefotaxime plate MICs against parent SAI+/and uninducible SAI−/organisms were similar indicating cefotaxime did not induce SA enzyme in these tests. Cefotaxime was however much less active against the SAI+ organism than the SAI− when log phase broth cultures were exposed to antibiotic and incubation wasextended to 30 h. This correlated with observed SA induction in the SAI+ organism. Cefsulodin MICs against SAI+/and SAI– organisms were similar and no difference existed in activity, over long periods against broth cultures. Overall, results indicated that SA enzyme, if induced, constituted a defence against cefotaxime but not cefsulodin and this correlated with the physiological efficiency results. To what degree induction occurs in vivo during cefotaxime therapy of pseudomonal infections remains unknown.
... Production of a chromosomally-mediated class Id (Richmond & Sykes, 1973) /Mactamase, the Sabath Abrahams (Sabath, Jago & Abrahams, 1965) enzyme, is universal amongst Pseudomonas aeruginosa strains. Most of the early /Mactams, for example penicillin G, ampicillin, cephaloridine and cephalothin, are hydrolysed by this /Mactamase (Sabath el al., 1965;Garber & Friedman, 1970). Carbenicillin, however, is stable and acts as an inhibitor, causing conformational changes in Id enzyme which render it inactive (Furth, 1979). ...
... Maximal Id induction requires high /Mactam concentrations, for example 5 g/1 penicillin G (Garber & Friedman, 1970). This would suggest induction is a 'rather unphysiological process' (Nordstrom & Sykes, 1974). ...
... As early as in 1965, Datta and Kontomichalou [11] showed the widespread transfer of penicillin resistance across Enterobacteriaceae. A marked β-lactamase activity was also measured in various Pseudomonas aeruginosa resistant strains in the early seventies [12]. More recently, acquisition of the virulence factors that distinguish Salmonella from Escherichia coli has been clearly shown as the result of horizontal gene transfer [13]. ...
Article
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The continuously improving sophistication of molecular engineering techniques gives access to novel classes of bio-therapeutics and new challenges for their production in full respect of the strengthening regulations. Among these biologic agents are DNA based vaccines or gene therapy products and to a lesser extent genetically engineered live vaccines or delivery vehicles. The use of antibiotic-based selection, frequently associated with genetic manipulation of microorganism is currently undergoing a profound metamorphosis with the implementation and diversification of alternative selection means. This short review will present examples of alternatives to antibiotic selection and their context of application to highlight their ineluctable invasion of the bio-therapeutic world.
... It therefore has the potential for widespread dissemination among pseudomonads. Susceptibility to TEM-I seriously limits the usefulness of a /Mactam antibiotic against many Gramnegative bacteria, but it has been suggested that intrinsic factors are more important than /Mactamase production in the resistance of Ps. aeruginosa to /Mactams (Garber & Friedman, 1970). Matsumoto et al. (1980) reported two treatment failures with cefoperazone and persistence of Ps. aeruginosa. ...
... Our failure to demonstrate any significant degree of ,-lactamase degradation of the cephalosporins by these resistant cultures led us to reexamine our approach to the problem and to evaluate the possible role of an inducible B-lactamase as a mechanism of resistance. Ever since Hamilton-Miller (10) demonstrated the inducibility of a B-lactamase (penicillinase) in P. morganii, this phenomenon in gram-negative bacteria has been the subject of considerable study and discussion (2,7,10,12). The need was recognized not only to test induction itself but also to test the phenomenon in whole cells as well as ruptured cells to discount permeability or crypticity of the cultures as a factor in the interpretation of results (2,11,12). ...
Article
The susceptibility to some cephalosporin antibiotics and to cephamycin C, a member of a new family of β-lactam antibiotics, was evaluated for 466 cultures representing 11 different genera or species of gram-negative clinical isolates. The susceptibility of 39 gram-negative cultures known to produce β-lactamase was also determined. The β-lactamase activity of a representative group of the clinical isolates and the 39 enzyme producers was studied with the cephalosporins (cephalothin and cephaloridine) and cephamycin C as substrates and was related to the in vitro disc susceptibility to these same antibiotics. The significant resistance to β-lactamase displayed by the cephamycins is reflected in the kinetics of enzyme activity (Km and Vmax) that are reported for the cephalosporins and the cephamycins. Resistance to β-lactamase is probably one of the reasons that many cephalosporin-resistant cultures are susceptible to cephamycin C.
Article
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Forty eight clinical isolates of P. aeruginosa were tested for susceptibility to seven different antimicrobial agents. When tested on Mueller-Hinton agar, the isolates were found to be resistant to ampicillin, sensitive to the antipseudomonal antibiotics, polymyxin B, gentamicin and carbenicillin. Polymyxin B inhibited all the isolates, whereas both carbenicillin and gentamicin inhibited 92.1% of the isolates. Neomycin, sulphamethoxypyridazine and chlortetracycline showed moderate activity and inhibited 50%, 28.9% and 15.8% of the isolates, respectively. However, on Mueller-Hinton agar supplemented with 0.03% cetrimide, the isolates succumbed readily to antimicrobial agents. In addition to polymyxin B, gentamicin and carbenicillin, all the strains were inhibited by neomycin and 94.7%, 92.1% and 63.6% of the isolates were inhibited by sulphamethoxypyridazine, chlortetracycline and ampicillin, respectively. Cetrimide, in concentrations ranging from 0.01 to 0.04% decreased the MIC of ampicillin against all the isolates, whereas 0.1% and 0.5% polysorbate 80 (tween 80) had no effect on the MIC. Growth inhibition studies have shown that the number of survivors was greatly reduced in presence of cetrimide and ampicillin. There was also an appreciable increase in the uptake of ampicillin by the bacterial cells in the presence of cetrimide.
Article
Thin-layer chromatogaphy procedures are described which permit the simple and rapid separation and detection of different spontaneous, chemical and enzymatic degradation products of pencillins and of two cephalosporins. The right combination of solvent system and spray reagent allows the identifation of these products, even in the presence of the present antibiotics, in aqueous preparations as well as in biological fluids and microbiological culture broths.
Article
SUMMARY Carbenicillin resistance in two strains of Pseudomonas aeruginosa has been investigated. Pseudomonas aeruginosa NCTC I 0490, a carbenicillin sensitive strain, produced a /3-lactamase when grown in the presence of carbenicillin. This enzyme although active against benzylpenicillin did not hydrolyse carbenicillin. Cell walls of P. aeruginosa NCTC 10490 habituated to the presence of carbenicillin showed a higher lipid content compared with the walls of the parent strain. Hospitalisolated, carbenicillin-resistant P. aeruginosa 69/4992 synthesized a p-lactamase which hydrolysed carbenicillin as well as benzylpenicillin. The two types of enzyme were characterized by their substrate profile, sensitivity to enzyme inhibitors and resolution by isoelectric focusing. Enzymes active against carbenicillin were differentiated by isoelectric focusing from those unable to hydrolyse carbenicillin ; both types of enzyme were shown to be complex mixtures by this technique.
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Cefoxitin is a new, cephalosporin-like antibiotic which is highly resistant to hydrolysis by β-lactamase. Ninety-one cultures were selected either for their general resistance to cephalosporin antibiotics or for their ability to produce β-lactamase. Some of these cultures were resistant to cefoxitin. The capacity of each of the 91 strains to hydrolyze cefoxitin with β-lactamase was determined. Only seven of the cultures degraded the antibiotic as determined by a general assay for β-lactamase. Several others were able to hydrolyze cefoxitin after enzyme was induced by low concentrations of the antibiotic. The role of the constitutive and inducible enzyme in bacterial resistance to the antibiotic was investigated. Enzymatic destruction of cefoxitin was found to be an important factor contributing to bacterial resistance. However, the complete and rapid degradation of cefoxitin is not essential to resistance since one strain, Enterobacter cloacae 1316, hydrolyzed the antibiotic very slowly but was able to grow unaffected in the presence of cefoxitin. The presence of the enzyme is not necessarily sufficient to confer resistance since another culture, Klebsiella D535, readily hydrolyzed the antibiotic but was susceptible to it.
Article
Ten strains of Proteus morganii were selected and the production of β-lactamase was studied. The cellular level of β-lactamase from P. morganii was regulated by the concentration of the inducer in the growth media, and the enzyme activity appeared within 10 min while its highest value was obtained 2 hr after the addition of benzylpenicillin as the inducer. The resistance level to cephaloridine was generally related to the amount of β-lactamase activity among the ten strains of P. morganii, but no relation was found between the ampicillin susceptibilities and the amounts of β-lactamase in their cell-free preparations. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by a crude enzyme was five times higher than that of benzylpenicillin. Semisynthetic penicillins were resistant to hydrolysis and exhibited competitive inhibition. Among the semisynthetic penicillins, dicloxacillin and carbenicillin were powerful competitive inhibitors of the β-lactamase from P. morganii.
Article
This chapter describes the process of microbial transformation of antibiotics into useful compounds, which is mostly concentrated upon the production of 6-aminopenicillanic acid (6-APA) by enzymatic hydrolysis of biosynthetic penicillins. The chapter describes biotransformation processes of penicillins into 6-APA that use, as a source of acylase, intact bacterial cells, fungal mycelium, fungal spores, crude cell extracts, purified enzyme preparations, stabilized enzymes, and supernatant liquid of extracellular acylase-producing microorganisms. The chapter examines methods to isolate and purify penicillin acylases and to stabilize these enzyme preparations. It reviews the physicochemical properties and the degradation reactions in relation to the structure of penicillins and describes the reaction of cysteine and related compounds with penicillins, resulting in a loss of antibacterial activity. 6-APA was originally produced by fermentation synthesized in a purely chemical way. Enzymatic transformation of biosynthetic penicillins into 6-APA is an industrial process of economic importance as the main source of 6-APA used in the preparation of semisynthetic penicillins.
Article
The induction of β-lactamase in Pseudomonas aeruginosa 1822s was studied using benzylpenicillin as inducer. The specific rate of β-lactamase formation was constant throughout an induction experiment. Above a threshold (20 μg/ml), the specific activity increased linearly with the concentration of the inducer. Removal of the inducer resulted in a rapid cessation of β-lactamase biosynthesis. Inhibition of protein synthesis by starvation for a required amino acid or by the addition of chloramphenicol also led to an instantaneous arrest in enzyme formation. In the absence of inducer, a basal β-lactamase activity was formed. The basal and the induced enzymes seem to be identical since they had the same substrate profile, electrophoretic mobility, and molecular weight. In all these respects, induction of β-lactamase in Pseudomonas aeruginosa is analogous to induction of the lac operon in Escherichia coli. However, there was a long, concentration-dependent lag before β-lactamase was induced. This can be explained by the outer penetration barrier decreasing the rate of inducer uptake. The lag was significantly shorter for lysozyme-ethylenediaminetetraacetic acid-produced spheroplasts than for intact cells. Induction was obtained with all β-lactam antibiotics tested, but not with other agents affecting the cell envelope.
Article
Pseudomonas aeruginosa produces a low basal level of beta-lactamase (0.002 to 0.004 IU/mg of protein when benzylpenicillin is used as substrate). The beta-lactamase specific activity can be increased several hundredfold by growing the bacteria in the presence of beta-lactam antibiotics. This induction was studied in Pseudomonas aeruginosa 1822s. The single-cell resistance to benzylpenicillin was 750 mug/ml. In liquid culture all concentrations of benzylpenicillin tested (25 to 2,000 mug/ml) affected the bacteria similarly: beta-lactamase formation was induced, the cells became cholate sensitive, growth rate decreased, filaments were formed, and beta-lactamase was excreted. The effect appeared earlier the higher the concentration of the antibiotic. Most of the effects obtained are concerned with the functioning of the outer membrane. The excretion of beta-lactamase seems to be due to an opening of the periplasmic volume rather than to lysis of the cells. Carbenicillin gave the same effects as benzylpenicillin at the same concentrations; the 10-fold lower resistance to carbenicillin than to benzylpenicillin can be explained by the inability of the inducible beta-lactamase to hydrolyze carbenicillin. The induced beta-lactamase was first cell bound and to a great extent located in the periplasmic volume, but later it was excreted into the medium. This extracellular activity was responsible for the detoxification of the medium. This is analogous to the behavior of gram-positive bacteria rather than to that of Enterobacteriaceae.
Chapter
Virtually all gram-negative bacteria studied to date produce a chromosomal betalactamase. Thus, it is not surprising that there is a great diversity among these enzymes in substrate profile, susceptibility to inhibitors, genetic control of expression and molecular makeup. Some enzymes have been studied extensively while others have been examined only superficially. Some appear to have great clinical relevance mediating resistance to a broad array of beta-lactam antibiotics. Others play little role in resistance or occur in organisms with little pathogenicity for humans. Many have been included in detailed general reviews on beta-lactamases (Richmond and Sykes 1973; Sykes and Matthew 1976; Richmond 1979; Mitsuhashi and Inoue 1981; Hamilton-Miller 1982; Bush and Sykes 1984; Medeiros 1984) and others have been examined specifically in reviews concerning the inducible chromosomal enzymes (Livermore 1987a; Sanders 1987; Sanders and Sanders 1987). The genetic control mechanisms responsible for these enzymes have also been recently reviewed in this and other publications (Lindberg and Normark 1986a; Chamberland, Chap. 7, this volume). Thus, the purpose of this chapter is to present a broad-based view of chromosomal beta-lactamases emphasizing the biochemical, physicochemical and molecular aspects as they are now known. The diverse nature of these enzymes will be presented and used in a multitiered classification system. Due to the very large number of different chromosomal enzymes known to exist and our uneven knowledge of them, this chapter will focus primarily on those enzymes of clinical importance that have been studied to the greatest extent. Thus, chromosomal beta-lactamases of gram-negative bacteria will be the major emphasis of this review.
Article
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Chapter
Development of β-lactam antibiotics over the past 40 years represents an unparalleled effort in the history of antimicrobial chemotherapy. The continual emergence of new compounds, natural, semisynthetic, and synthetic, is a tribute to the research programs being carried out around the world. Alongside the intensive search for new and improved β-lactam antibiotics has been the study of enzymes that interact with these molecules.
Article
One-hundred clinical isolates of Serratia marcescens were tested for susceptibility to cephalothin, carbenicillin, ticarcillin, ampicillin, and cefoxitin. The majority of the 100 isolates (≥70%) were susceptible to carbenicillin, ticarcillin, and cefoxitin; less than one-half were susceptible to ampicillin; none were susceptible to cephalothin. Ten isolates from the 100 organisms tested were selectively assayed for their β-lactamase activity. Enzyme activity was measured using either iodometric or spectrophotometric methods, and the microbiological assay technique. It was concluded that β-lactamase production was not the sole determinant in β-lactam antibiotic resistance. Resistance without demonstrable β-lactamase was evident in strains for cephalothin, ampicillin, and cefoxitin. In addition, one strain which was susceptible to all antibiotics except cephalothin, elaborated considerable β-lactamase activity.
Article
This chapter discuses the assay method and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Enzyme activity can be quantitated by either measuring (1) the chloramphenicol (CM)-dependent disappearance of acetyl-S-CoA, (2) the appearance of 3-O-acetoxy derivative of CM, or (3) the formation of reduced (unesterified) CoA. In practice, the choice between these alternatives is determined by the level of sensitivity required and by complications created by interfering substances. It has been suggested that conventional techniques of protein fractionation have been adequate for the purification of chloramphenicol acetyltransferase (CAT) from (1) R factor-bearing strains of E. coli, (2) Staphylococci harboring CM plasmids, (3) CM-resistant mutants of Proteus mirabilis in which the CAT gene is probably chromosomal, and (4) selected strains of Agrobacterium tumefaciens. The procedure described in the chapter gives a homogeneous CAT product from R factor-containing strains of E. coli and isolates of S. aureus harboring a CM plasmid. The ease with which such results can be achieved is because of the fact that fully induced S. aureus cultures and virtually all R+E. coli cultures synthesize CAT to levels approximating 0.5–1% of the soluble cell protein in stationary phase.
Article
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Article
1. The beta-lactamase (penicillin amido-beta-lactamhydrolase EC 3.5.2.6) appeared to be periplasmic rather than truly intracellular, since it was released by freeze-thawing without gross morphological changes in the cell. 2. The partially purified enzyme had pI between 5.0 and 5.5, mol. wt 32 000 and a broad pH vs activity profile with a maximum at pH 8. 3. The cephalosporins tested were hydrolysed less rapidly than most of the penicillins, and the Km values for penicillins were lower than for cephalosporins. However cloxacillin was hydrolysed very slowly although it was strongly bound. The substrate-induced inactivation common to many beta-lactamases was particularly marked with cephaloridine and cloxacillinmthe cloxacillin-induced inactivation was shown to be reversible.
Article
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Article
Bacterial suspensions to be used as an enzyme source were prepared by diluting non-induced cultures of the strains after overnight growth in brain heart infusion broth to 5xl09 cell/ml. The ß-lactamase was assayed by the iodometric method described by Perret8 and the reaction mixtures were prepared by mixing samples of the bacterial suspensions with a 5 mM solution of the antibiotic used as potential substrate at pH 8 (ref.
Chapter
The remarkable growth in the development of the cephalosporin group of antibiotics stems from the isolation of cephalosporin C in 19551. The recognition of the active agents, however, preceded this by some years. In 1945, Brotzu, at that time Rector in the Sicilian University of Cagliari, noted that the seawater near a sewage outlet in that town appeared to be self-purifying and proposed that this might be due in part to bacterial antagonism. Brotzu examined the microbial flora of the seawater and isolated a fungus which he concluded to be similar to Cephalosporium acremonium. This fungus appeared to have an inhibitory effect on certain bacteria in vitro and Brotzu used the crude products of its growth with some success for treating typhoid fever and brucellosis. In 1948 he published his findings in a journal that he founded for the purpose since no other learned journal would accept the work2. He also sent a culture of his fungus to Oxford in England.
Chapter
The effectiveness of an anti-bacterial agent against strains of Ps. aeruginosa is dependent on three main factors: (1) activity at the active site (preferably bactericidal), (2) penetration into the active site, and (3) resistance to enzymatic modification. Of the effective compounds available now, the β-lactam antibiotics, the aminoglycosides, and the polymyxins are all bacteriocidal. Antibiotic resistance in clinical isolates of Ps. aeruginosa is generally because of the presence of chromosomal mediated genes determining the synthesis of enzymes that specifically modify and inactivate antibiotics. A number of new antipseudomonas β-lactam antibiotics are being developed. The β-lactam antibiotics hold great promise because of their low toxicity. Biochemical studies of the substrate specificities of the aminoglycoside inactivating enzymes have revealed the functional groups in these antibiotics important for enzyme recognition. A number of compounds lacking certain of these groups have been synthesized because of such studies.
This chapter discusses the cephalosporin group of antibiotics. Two sites in the cephalosporin molecule are: (1) the 7-acyl side chain, leading to the production of 7-acylaminocephalosporanic acids—for example, ring-substituted phenylacetylcephalosporanic acids (analogous to benzylpenicillin) and ring-substituted phenoxyacetylcephalosporanic acids members of both types being active mainly against gram-positive bacteria, and (2) the 3-acetoxymethyl side chain leading to the production of deacetylcephalosporins and deacetoxylcephalosporins. The former have about half the activity of the parent compounds against gram-positive bacteria, such as staphylococci, but much reduced activity against gram-negative bacteria. Removal of the acetoxy group gives 3-hydroxyl derivatives,which are less antibacterial than the parent acetoxy compounds. Cephalosporin C, cephalothin, and cephaloglycin all lose antibacterial activity rapidly when incubated with rat liver homogenate, because of removal of the acetoxy group; however, cephaloridine and cephalexin do not possess these labile ester linkages and are thus, not inactivated.
Article
Kinetics of induction of several enzymes of Escherichia coli have been investigated under conditions where non-specific nutrient effects and permeability mechanisms are not important. Measurements made over time intervals of a few minutes permitted detection of initial events brought about by addition or removal of inducers or inhibitors.With each enzyme a lag of about 3 min between addition of inducer and appearance of enzyme at 37° was noted. This lag was dependent on temperature and independent of inducer concentration, in contrast to an inhibitor which showed the opposite behavior. The induction lag, therefore, does not represent the time required for penetration of the inducer. Studies with inhibitors suggest that some metabolism involving the synthesis of a ribonucleic acid is required before enzyme synthesis can proceed. The formation of enzyme ceased about 5 min after inducer was removed or after glucose was supplied to the culture, suggesting that the enzyme-forming system is unstable.A model for the enzyme forming apparatus of the cell is suggested which is consistent with the kinetic data and with other information regarding induction. A repressor prevents function of the genetic material of the bacteria. The inducer is thought to prevent the repressor from acting. This freed genetic material forms a special, unstable ribonucleic acid which interacts with the ribosomes to provide an active template for enzyme synthesis. The time required for enzyme formation to commence is attributed principally to the interval during which the repressor is lost and the unstable ribonucleic acid is formed. A possible similarity between repressor and unstable ribonucleic acid is suggested. Instability appears to be spontaneous at temperatures where metabolism goes on, rather than being caused by use of the system for enzyme production.
Article
The beta-lactamase activity in 10 bacterial species from different genera were evaluated where direct evidence and quantitative data were lacking. A quantitative iodometric method and infrared spectrophotometry were used for the determination of the beta-lactamase activity. The organisms tested were shown to have enzyme activity directed against the beta-lactam ring, and on the basis of the activity on two members of the beta-lactam group of antibiotics, penicillin G and cephalosporin C, a particular ratio was obtained for each species. This report supports the fact of the widespread distribution of beta-lactamase and reopens the question of its significance.
Article
Speroplasts of Pseudomonas aeruginosa were induced with carbenicillin. Morphological changes were observed as early as 1.5 hr after the initial exposure to carbenicillin.
Article
Examination of the substrate profile of a number of beta-lactamases synthesized by strains of Pseudomonas aeruginosa has shown the presence of at least two distinct types of enzyme, one of which is more active against carbenicillin than against benzyl penicillin.
Article
1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.
Article
THE series of iodometric methods 1 for assaying penicillinase reported here permits enzyme activity to be measured easily over wide ranges of pH and temperature, with higher speed, accuracy or sensitivity than were previously attainable 2. Although designed for the penicillinase of B. cereus NRRL-569, it appears likely that they may be equally suitable in principle for penicillinase from other sources. 'Unit rate of penicillin destruction' is used here to mean a destruction-rate of 1 mol./hr. Since the amount of a penicillinase which will give unit rate of destruction depends on the nature of the enzyme and the conditions to which it is exposed, a 'unit dose' (U.D.) of B. cereus NRRL-569 penicillinase is here defined as that amount of the enzyme which gives unit rate of destruction when exposed to 0.005 M sodium benzyl penicillin in 0.2 M potassium phosphate buffer at pH 6.5 and 30°C. The commercial crystalline sodium penicillin G of Glaxo, Ltd., is used in the following assays; its potency is stated to be 1,650 I.U./mgm.
Article
The development of resistance to ampicillin (aminophenylacetamidopenicillanic acid) and penicillin G was investigated by selecting resistant variants of strains of Escherichia coli. Resistance occurred in stepwise manner. The parent strains contained minute amounts of penicillinase and production of the enzyme by resistant variants increased in relation to increase in resistance. Evidence was obtained that production of penicillinase was responsible for this increase in resistance. By using resistant variants and naturally occurring ampicillin-resistant organisms, it was found that production of only small amounts of penicillinase was sufficient to confer a high degree of resistance to the Gram-negative bacteria examined. In contrast, Gram-positive bacteria required much greater quantities of penicillinase for high grade resistance. It is suggested that an important reason for this difference is the complex lipid-containing cell wall of Gram-negative bacteria, which slows penetration by penicillin and enables small amounts of penicillinase to protect the cell. The penicillinase of all the Gram-negative organisms investigated was found to be a β-lactamase and to be more active against penicillin G than against ampicillin. This provides one explanation for the greater activity of ampicillin against Gram-negative bacteria.
Article
CRUDE preparations of penicillinase from Bacillus cereus N.R.R.L. 569, unlike the purified penicillinase from this organism or crude penicillinase from Staphylococcus aureus R1, have been shown to catalyse the hydrolysis of the beta-lactam ring of cephalosporin C and other derivatives of 7-aminocephalosporanic acid at a significant rate. The enzyme responsible for this hydrolysis was termed a cephalosporinase1,2. The maximum rate of hydrolysis (Vmax) of cephalosporin C obtained with a crude enzyme from B. cereus was only 5 per cent of that of benzyl-penicillin, but this value increased to 33 per cent after selective inactivation of penicillinase in aqueous solution at 60° C. Subsequent studies were made with a crude enzyme from Bacillus subtilis N.C.T.C. 6346, induced with cephalosporin C and freed from the cells by the action of lysozyme3. With this enzyme preparation Vmax for cephalosporin C was about 10 per cent that for benzyl-penicillin, but attempts to separate a cephalosporinase from a penicillinase, or selectively inactivate a penicillinase, were unsuccessful.
Article
SUMMARY The insensitivity of Gram-negative bacteria towards different penicillins has been correlated with the inactivation of these compounds by the penicillinases produced by these bacteria. Penicillins such as phenethi- cillin, propicillin, methicillin, and cloxacillin were relatively inactive against Gram-negative bacteria, but resistance was not accompanied by inactivation of these compounds. With ampicillin and benzylpenicillin, however, bacterial resistance was associated with destruction of the penicillins. Ampicillin was more stable to the penicillinases produced by certain Gram-negative bacteria than was benzylpenicillin and was corre- spondingly more active against these organisms. Gram-negative bacteria which produced little or no penicillinase were two to four times more sensitive to ampicillin than to benzylpenicillin, but ampicillin was at least ten times more active than benzylpenicillin against several penicillinase- producing coliform organisms. All strains of bacteria which were resistant to both ampicillin and benzylpenicillin were capable of inactivating both compounds, but the insensitivity of these bacteria was not necessarily due solely to penicillinase production. Strains of Pseudomonas aeruginosa and certain strains of Escherichia coli and Protew mirabilis were able to grow in high concentrations of these antibiotics, whereas with the strains of Aerobacter aerogernes, Protew morgartii and Proteus vulgaris examined, the resistance appeared to be due solely to inactivation of the penicillins.
Article
A strain of Pseudomonas aeruginosa was obtained which was able to grow on acetamide or propionamide as sole source of carbon and nitrogen. When grown on these amides, whole bacteria and cell-free extracts rapidly hydrolysed acetamide, glycollamide, acrylamide and propionamide and slowly hydrolysed formamide and butyramide. N-Methylformamide, N-methylacetamide, N-ethylacetamide, N-acetylacetamide, N-methylpropionamide, N-ethylpropionamide, lactamide and methyl carbamate were found to be non-substrate inducers of the amidase when the organism was grown in succinate + ammonium chloride medium. N-Methylformamide, N-methylacetamide, lactamide and methyl carbamate did not inhibit propionamide hydrolysis by whole bacteria, but under the same conditions glycine amide, iodoacetamide and urea were effective inhibitors of amidase activity. N-Phenylacetamide, cyanoacetamide, glycine amide, sarcosine amide, β-hydroxy-propionamide and thioacetamide were neither substrates nor inducers of the amidase in this strain, but inhibited amidase induction by N-methylacetamide in succinate + ammonium chloride medium. Formamide also inhibited amidase induction under the same conditions.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
Induction of spheroplasts of Pseudomonas aeruginosa by carbenicillin Cephalosporinase and penicillinase activities of
  • Ch Watanaktjnakorn
  • M L D Hamburger
  • M Jaw
  • E P Abraham
WATANAKTJNAKORN, CH. & HAMBURGER, M. (1969). Induction of spheroplasts of Pseudomonas aeruginosa by carbenicillin. Applied Microbiology 17, 935. L. D., JAW, M. & ABRAHAM, E. P. (1965). Cephalosporinase and penicillinase activities of
Iodometric assay of penicillinase Induction of cephalosporinase and penicillinase in Proteus species Examination of various Gram-negative bacteria for beta-lactamase R. Observations on the /3-lactamase system of 23-2 r352 POLLOCK, absence of free substrate
  • C J G A J Perret
PERRET, C. J. (1954). Iodometric assay of penicillinase. Nature, London 174, 1012. G. A. J. (1964). Induction of cephalosporinase and penicillinase in Proteus species. Nature, M. (I 968). Examination of various Gram-negative bacteria for beta-lactamase R. (1969). Observations on the /3-lactamase system of 23-2 r352 POLLOCK, absence of free substrate. British Journal of Experimental Pathology 31, 739
Purification et caractkristiques physiquochimiques de la pknicillinase de Bacillus cereus Comptes rendus des siances de 1’ Acadernie des sciences Penicillinase adaptation in B. cereus: Adaptive enzyme formation in the SABATH, a /3-lactamase from Pseudomonas pyocyanea
  • M R Pollock
  • A M N Torriani
  • N Garber
POLLOCK, M. R. & TORRIANI, A. M. (1953). Purification et caractkristiques physiquochimiques de la pknicillinase de Bacillus cereus. Comptes rendus des siances de 1’ Acadernie des sciences, Paris 237, 276. N. GARBER A N D J. FRIEDMAN M. R. (1950). Penicillinase adaptation in B. cereus: Adaptive enzyme formation in the SABATH, a /3-lactamase from Pseudomonas pyocyanea. Biochemical Journal 96, 739
Observations on the /3-lactamase system of Pseudomonas aeruginosa
  • N Garber
  • J Friedman
  • R Rozansky
GARBER, N., FRIEDMAN, J. & ROZANSKY, R. (1969). Observations on the /3-lactamase system of Pseudomonas aeruginosa. Israel Journal of Chemistry 7, I 34p.
Protein measurement with the Folin phenol reagent
  • N J Farr
  • A L Randall
LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265.
Purification et caractkristiques physiquochimiques de la pknicillinase de Bacillus cereus Comptes rendus des siances de 1' Acadernie des sciences Cephalosporinase and penicillinase activities of a /3-lactamase from Pseudomonas pyocyanea
  • M R Torriani
  • A M Paris
  • L D Jaw
  • M Abraham
POLLOCK, M. R. & TORRIANI, A. M. (1953). Purification et caractkristiques physiquochimiques de la pknicillinase de Bacillus cereus. Comptes rendus des siances de 1' Acadernie des sciences, Paris SABATH, L. D., JAW, M. & ABRAHAM, E. P. (1965). Cephalosporinase and penicillinase activities of a /3-lactamase from Pseudomonas pyocyanea. Biochemical Journal 96, 739.
Purification et caractkristiques physiquochimiques de la pknicillinase de Bacillus cereus
  • M R Pollock
  • A M Torriani
POLLOCK, M. R. & TORRIANI, A. M. (1953). Purification et caractkristiques physiquochimiques de la pknicillinase de Bacillus cereus. Comptes rendus des siances de 1' Acadernie des sciences, Paris