Inactivation Of Beta-lactamase Research Articles

Determination of Beta-Lactamase Inactivation of Cephalexin by Validated RP-HPLC Method

Determination of the needed amount of liquid sterile Lactamator™ to be mixed to cephalexin, β-lactam antibiotic, for optimum deactivation of its molecule's antibacterial properties was conducted using RP-HPLC method. RP-HPLC method was validated for the parameters as linearity, accuracy, LOD, LOQ, and precision. Before the routine microbiological examination for any pharmaceutical dosage form containing β-lactam antibiotic, it is a must to make inactivate of β-lactam active pharmaceutical ingredient (API) by mixing with beta-lactamase before testing. So, the study indicated that mixing of 0.5 ml liquid sterile Lactamator™ with phosphate buffer solution pH (7.2) containing 50 mg cephalexin, with holding the test sample for 90 minutes prior to HPLC measurement will deactivation of cefalexin molecule's antibacterial properties.

Inactivation of clinically relevant pathogens by photocatalytic coatings

Novel disinfection methods are being sought to provide additional means of protection in a number of areas where disease outbreaks could lead to illness or fatalities. For example, the risk of contamination arising from contact with surfaces and medical devices has received much attention due to the rise in incidence of healthcare acquired infections. It is possible that reducing bio-burden on these sites may supplement the disinfection protocols currently in place and help reduce risk of infection. Photocatalytic surfaces offer promise as innovative and cost-effective biocidal engineering solutions which address these specific problems whilst maintaining stringent health and safety controls. A method was developed to assess the disinfection efficiency of photocatalytic surfaces allowing (a) determination of pathogen viability as a function of treatment time; (b) assessment of the surface for viable surface bound organisms following disinfection; (c) measurement of the re-growth potential of inactivated organisms. This method was used to demonstrate the inactivation of extended-spectrum beta-lactamase Escherichia coli, methicillin resistant Staphylococcus aureus, Pseudomonas aeruginosa and Clostridium difficile spores using immobilised films of commercial titania nanoparticles. 99.9% reduction in viability (a 3-log kill) was observed for all bacterial cells within 80 min photocatalytic treatment. Complete surface inactivation was demonstrated and bacterial re-growth following photocatalytic treatment was not observed. Greater than 99% inactivation (2.6-log reduction) was observed when the photocatalytic surfaces were challenged with C. difficile spores. The efficacy of photocatalytic disinfection to inactivate Staphyloccocus epidermidis cells within a biofilm was also demonstrated, with 3 h treatment rendering 96.5% ± 6 of the biofilm cells on the TiO 2 coated substrate non-viable. Disinfection of cells throughout the 3–4 μm thick biofilm was observed.

Functional diversity among metallo-beta-lactamases: characterization of the CAR-1 enzyme of Erwinia carotovora.

Metallo-beta-lactamases (MBLs) are zinc-dependent bacterial enzymes characterized by an efficient hydrolysis of carbapenems and a lack of sensitivity to commercially available beta-lactamase inactivators. Apart from the acquired subclass B1 enzymes, which exhibit increasing clinical importance and whose evolutionary origin remains unclear, most MBLs are encoded by resident genes found in the genomes of organisms belonging to at least three distinct phyla. Using genome database mining, we identified an open reading frame (ORF) (ECA2849) encoding an MBL-like protein in the sequenced genome of Erwinia carotovora, an important plant pathogen. Although no detectable beta-lactamase activity could be found in E. carotovora, a recombinant Escherichia coli strain in which the ECA2849 ORF was cloned showed decreased susceptibility to several beta-lactams, while carbapenem MICs were surprisingly poorly affected. The enzyme, named CAR-1, was purified by means of ion-exchange chromatography steps, and its characterization revealed unique structural and functional features. This new MBL was able to efficiently hydrolyze cephalothin, cefuroxime, and cefotaxime and, to a lesser extent, penicillins and the other cephalosporins but only poorly hydrolyzed meropenem, while imipenem was not recognized. CAR-1 is the first example of a functional naturally occurring MBL in the family Enterobacteriaceae (order Enterobacteriales) and highlights the extraordinary structural and functional diversity exhibited by MBLs.

Overcoming Resistance to β-Lactamase Inhibitors: Comparing Sulbactam to Novel Inhibitors against Clavulanate Resistant SHV Enzymes with Substitutions at Ambler Position 244

Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 --> <or=500). A timed inactivation-mass spectrometry analysis of SHV inhibited by sulbactam showed that SHV-1 beta-lactamase was unmodified at 15 min. A parallel experiment with R244S demonstrated 70 and 88 +/- 3 Da fragments of sulbactam covalently attached to the beta-lactamase. We also observed that the Ki values of penems 1 and 2 were increased for R244 variants compared to those for SHV; however, these inhibitors effectively restored ampicillin susceptibility in vitro. Compared to that of sulbactam, the kcat/kinact values of penems for SHV-1 and R244S were low (<or=2), and unfragmented adducts of each penem were covalently attached to SHV-1 and R244S when studied using the timed inactivation-mass spectrometry method. The R244S mutation affects beta-lactamase inactivators differently, but resistance can be overcome by designing penem inhibitors with strategic chemical properties that improve affinity and impair turnover.

Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the beta-lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. Three wild E. coli strains BDCS-N-FMu10, BDCS-N-S21 and BDCS-N-W50, producing both the enzymes PGA and beta-lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 microg ml(-1) and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 microg ml(-1). The highest concentration that gave permissible growth rate was 50 microg ml(-1). The isolated survivals were screened on the bases of PGA and beta-lactamase activities. Among the retained mutants, the occurrence of beta-lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). In seven of the mutants, PGA activity was enhanced with considerable decrease in beta-lactamase activity. One of the mutant strains (BDCS-N-M36) exhibited very negligible expression of beta-lactamase activity and twofold increase in PGA activity [12.7 mg 6-amino-penicillanic acid (6-APA) h(-1) mg(-1) wet cells] compared with that in the wild-type strain (6.3 mg 6-APA h(-1) mg(-1) wet cells). The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of beta-lactamase. These mutants could be used for industrial production of PGA.

The discovery and development of modified penicillin- and cephalosporin-derived beta-lactamase inhibitors.

While beta-lactam antibiotics remain among the most commonly prescribed pharmaceutical products, their effectiveness is currently threatened by the development of bacterial resistance. One key resistance mechanism is the ability to destroy the antibiotic through utilization of one or more types of beta-lactamase. An effective countermeasure is to employ a combination product, consisting of both a beta-lactam antibiotic and a beta-lactamase inhibitor. Unfortunately, currently available inhibitors narrowly target only class A beta-lactamases. This review will detail our research, directed toward the development of a useful broad-spectrum beta-lactamase inhibitor. In the process, we have discovered new inhibitors capable of simultaneously inactivating class A, C, and D beta-lactamase, produced conjugate siderophore/beta-lactamase inhibitors, and explored the SAR's of tunable, cephalosporin-derived beta-lactamase inactivators. Useful synthetic methodology will be described, which simplifies the large scale production of many known inhibitors and which allows the rapid preparation of libraries of prospective inhibitors.

Mechanism of inactivation of beta-lactamases by novel 6-methylidene penems elucidated using electrospray ionization mass spectrometry.

The reactions of 6-methylidene penems 4-7 with beta-lactamases (TEM-1, SHV-1, Amp-C) were characterized by electrospray ionization mass spectrometry (ESI-MS). The kinetics of the reactions were monitored, demonstrating that only one penem molecule reacts to form an acyl-enzyme complex. For penem 5, the ESI-MS/MS spectrum of the hydrolysis product produced in the reaction was identical to the spectrum generated from a synthesized dihydro[1,4]thiazepine 10, confirming the rearrangement of the penem ring system to a seven-membered dihydro[1,4]thiazepine structure. Gas-phase ESI-MS/MS fragmentation data were rationalized due to tautomerization between imine and enamine substructures. ESI-MS/MS analysis of the T-6 trypsin-digested fragments of TEM-1 and SHV-1 demonstrated that the penems were only attached to Ser-70 of these class A beta-lactamases and that the penem ring structures were rearranged to seven-membered dihydro[1,4]thiazepines.

Molecular basis of antibiotic resistance and beta-lactamase inhibition by mechanism-based inactivators: perspectives and future directions.

Antibacterial chemotherapy is particularly striking in the family of penicillins and cephalosporins. Over 40 structurally different beta-lactam molecules are available in 73 formulations and the majority of them are currently prescribed for medical use in hospitals. beta-Lactams are well tolerated by humans with few side effects. They interact very specifically with their bacterial target, the D-alanyl-D-alanine carboxypeptidase-transpeptidase usually referred to as DD-peptidase. The outstanding number of beta-lactamases produced by bacteria represent a serious threat to the clinical utility of beta-lactams. The discovery of beta-lactamase inhibitors was thought to solve, in part, the problem of resistance. Unfortunately, bacteria have evolved new mechanisms of resistance to overcome the inhibitory effects of beta-lactamase inactivators. Here, we summarize the diversified mechanistic features of class A beta-lactamases interactions with mechanism-based inhibitors using available microbiological, kinetic and structural data for the prototype TEM beta-lactamases. A brief historical overview of the strategies developed to counteract beta-lactamases will be presented followed by a short description of the chemical events which lead to the inactivation of TEM beta-lactamase by inhibitors from different classes. Finally, an update on the clinical prevalence of natural and inhibitor-resistant enzyme mutants, the total chemical synthesis to design and synthesize a new structure and produced a broad spectrum beta-lactamase inhibitor that mimics the beta-lactam ring, but does not contain it is discussed.

X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.

Combination beta-lactam and beta-lactamase-inhibitor therapy: pharmacokinetic and pharmacodynamic considerations.

Pharmacokinetic and pharmacodynamic considerations in in vitro susceptibility testing are described, and the integration of pharmacokinetic and pharmacodynamic concepts in dosage-regimen design is explored. Both the amount of beta-lactamase produced per unit of time and the amount of beta-lactamase inhibitor supplied greatly influence susceptibility to beta-lactam antibiotics. The goal should be to supply enough beta-lactamase to render the bacteria functionally beta-lactamase negative. In susceptibility testing, the amount of inhibitor may be more important than the ratio between the beta-lactam antibiotic and the inhibitor. Irreversible inactivation of beta-lactamases by inhibitors allows for a period of killing of bacteria by beta-lactams, even when concentrations of the inhibitor fall to concentrations below those tested in vitro. The integration of pharmacokinetic and pharmacodynamic concepts allows for comparisons between in vitro and in vivo drug exposure. With some antibiotic-inhibitor combinations, it may be possible to extend the dosage interval if an adequate amount of inhibitor is provided over the course of therapy.

Site-directed mutagenesis and substrate-induced inactivation of beta-lactamase I.

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

Clinical pharmacokinetics of enzyme inhibitors in antimicrobial chemotherapy.

The effectiveness of some antimicrobial agents can be enhanced by using them in combination; such combinations are termed synergistic. Where one compound potentiates the effect of a second drug they may be coformulated. Inhibition of the bacterial degradation of an active antimicrobial is the basis of clavulanate and sulbactam-potentiated penicillin combinations, and inhibition of degradative pathways in the host is the rationale behind imipenem/cilastatin therapy. Trimethoprim/sulphonamide combinations depend on the maintenance of an effective ratio for synergistic action. In order to achieve potentiation the coformulated drugs should have similar pharmacokinetics. Trimethoprim was originally matched with sulphamethoxazole, since these two drugs have similar elimination half-lives, but the significantly poorer penetration of sulphonamides, their greater non-renal clearance, the emergence of resistance, and the adverse reactions attributable to them argue against the rationale that underlies their coformulation. Time-dependent inhibition of bacterial beta-lactamases by clavulanic acid and sulbactam has extended the use of penicillins which are highly susceptible to beta-lactamase inactivation. The beta-lactamase inhibitors must penetrate to the same extent as the penicillin used with them, and be present long enough to effect inhibition; thus, rapid penetration, similar or slower elimination and equivalent volume of distribution are necessary. These requirements are met for amoxycillin/clavulanic acid, ticarcillin/clavulanic acid and ampicillin/sulbactam combinations. Clavulanic acid is absorbed orally and is given with amoxycillin. However, since sulbactam is labile by this route, the combination of sulbactam with ampicillin to form the prodrug sultamicillin has been necessary to enable an oral form to be developed. Imipenem is metabolised by renal brush-border dehydropeptidases, and may cause proximal tubular necrosis. Cilastatin was designed to inhibit this metabolism, which it effectively does, thereby both potentiating the effect of imipenem and avoiding toxicity. Appropriate matching of the kinetics of coformulated drugs is intended to maximise potentiation and minimise the risk of emergent resistance. The kinetics of the above combinations are discussed in the light of these requirements and the effects of age and disease.

Sulbactam/ampicillin, a new beta-lactamase inhibitor/beta-lactam antibiotic combination.

Sulbactam/ampicillin is a combination of a beta-lactamase inhibitor with minimal intrinsic antibacterial activity (sulbactam sodium), and an aminopenicillin (ampicillin sodium). The addition of sulbactam to ampicillin has no effect on the chemical stability of ampicillin in aqueous solution, and the administration guidelines of the combination are the same as for ampicillin alone. Sulbactam acts primarily by irreversible inactivation of beta-lactamases from most beta-lactamase-producing organisms. The pharmacokinetics of sulbactam are similar to those of ampicillin with an elimination half-life of about one hour in most patients. One difference is that serum and tissue concentrations of sulbactam are usually twice those of ampicillin, at equivalent doses. The sulbactam/ampicillin combination has been approved for the treatment of adults with intraabdominal, skin and skin structure, and gynecological infections due to beta-lactamase-producing bacteria such as Staphylococcus aureus, Escherichia coli, and species of Klebsiella and Bacteroides. Clinical studies to date have also shown the combination to be effective for the treatment of meningitis, pneumonia, gonorrhea, epiglottis, urinary tract infections, cervical adenitis, and as prophylaxis for abdominal and gynecological surgeries. Many of these studies, however, have included small numbers of patients and/or had design flaws. Adverse effects have been minor with most being attributed to the ampicillin component. Sulbactam/ampicillin compares favorably with other antibiotic regimens in terms of acquisition costs and ease of administration.

Inactivation of beta-lactamases from Enterobacter cloacae by monophosphams.

Amongst the monocyclic beta-lactam antibiotics, selected monophosphams were potent mechanism-based inactivators of the P99 and E2 cephalosporinases of Enterobacter cloacae. Inhibition of these enzymes was time-dependent with second order rate constants for inactivation of 100,000 to 20,000,000 l/mol/min. After incubation for 24 h at least 99% of the enzymatic activity was inhibited when enzyme was exposed to a ten-fold excess of inactivator. Amongst the monophosphams three classes of inhibitors were seen: irreversible inactivators as described above, transient inactivators and competitive (inhibitory) substrates.

Beta-lactamase inhibitors from laboratory to clinic.

beta-Lactamases constitute the major defense mechanism of pathogenic bacteria against beta-lactam antibiotics. When the beta-lactam ring of this antibiotic class is hydrolyzed, antimicrobial activity is destroyed. Although beta-lactamases have been identified with clinical failures for over 40 years, enzymes with various abilities to hydrolyze specific penicillins or cephalosporins are appearing more frequently in clinical isolates. One approach to counteracting this resistance mechanism has been through the development of beta-lactamase inactivators. beta-Lactamase inhibitors include clavulanic acid and sulbactam, molecules with minimal antibiotic activity. However, when combined with safe and efficacious penicillins or cephalosporins, these inhibitors can serve to protect the familiar beta-lactam antibiotics from hydrolysis by penicillinases or broad-spectrum beta-lactamases. Both of these molecules eventually inactivate the target enzymes permanently. Although clavulanic acid exhibits more potent inhibitory activity than sulbactam, especially against the TEM-type broad-spectrum beta-lactamases, the spectrum of inhibitory activities are very similar. Neither of these inhibitors acts as a good inhibitor of the cephalosporinases. Clavulanic acid has been most frequently combined with amoxicillin in the orally active Augmentin and with ticarcillin in the parenteral beta-lactam combination Timentin. Sulbactam has been used primarily to protect ampicillin from enzymatic hydrolysis. Sulbactam has been used either in the orally absorbed prodrug form as sultamicillin or as the injectable combination ampicillin-sulbactam. Synergy has been demonstrated for these combinations for most members of the Enterobacteriaceae, although those organisms that produce cephalosporinases are not well inhibited. Synergy has also been observed for Neisseria gonorrhoeae, Haemophilus influenzae, penicillinase-producing Staphylococcus aureus, and anaerobic organisms. These antibiotic combinations have been used clinically to treat urinary tract infections, bone and soft-tissue infections, gonorrhea, respiratory infections, and otitis media. Gastrointestinal side effects have been reported for Augmentin and sultamicillin; most side effects with these agents have been mild. Although combination therapy with beta-lactamase inactivators has been used successfully, the problem of resistance development to two agents must be considered. Induction of cephalosporinases can occur with clavulanic acid. Permeability mutants could arise, especially with added pressure from a second beta-lactam.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and mechanism of inactivation of the RTEM-2 beta-lactamase by phenylpropynal. Identification of the characteristic chromophore.

beta-Lactamases of all three classes, A, B, and C, are inactivated by phenylpropynal and p-nitrophenylpropynal. The inactivation of RTEM-2 beta-lactamase and of Bacillus cereus beta-lactamase I is accelerated in the presence of A type substrates such as dicloxacillin, quinacillin, and cefoxitin, which are thought to expand or loosen the conformation of these enzymes. In the presence and absence of cefoxitin the inactivation of the RTEM-2 beta-lactamase is first and second order, respectively, in phenylpropynal concentration. The additional phenylpropynal molecule in the latter case may serve the same function as cefoxitin, viz. catalyze access to sensitive functional groups. Correlation of the loss of activity of the RTEM-2 enzyme with the extent of modification suggests that the modification of any one of about four kinetically equivalent groups leads to inactivation. Modification of all of the above mentioned enzymes leads to formation of a characteristic chromophore of unusual stability to nucleophiles, which absorbs maximally between 315 and 320 nm. A consideration of the properties of model compounds demonstrated that the protein-bound chromophore is that of a 1-phenyl-3-imino-1-propen-1-ammonium ion (Formula: see text), formed by reaction of phenylpropynal with two enzymic amine groups, and thus cross-linking the enzyme intramolecularly. Phenylpropynal may be a convenient general reagent for rapid and stable intramolecular cross-linking of proteins through lysine.

Lincomycin stimulates synthesis of TEM-2 beta-lactamase by Escherichia coli.

Lincomycin increased the TEM-2 beta-lactamase activity of Escherichia coli K-12 cells carrying plasmid RP4 at a concentration which slightly inhibited cell growth. In a control culture beta-lactamase activity reached its maximal level in late log phase, whereas when lincomycin was present beta-lactamase activity continued to increase into the stationary phase. Lincomycin (100 micrograms/ml) inhibited both cell growth and protein synthesis by about 35% but stimulated beta-lactamase activity 2.5-fold per ml of culture and about 4-fold per cell after 20 h of growth. The amount of beta-lactamase produced in each culture was also compared by densitophotometry of a stained sodium dodecyl sulfate-polyacrylamide gel. The relative values were in good agreement with the relative enzyme activities, indicating that the stimulatory effect of lincomycin was due to an increase in the amount of beta-lactamase protein. Inactivation of beta-lactamase appeared to be faster when lincomycin was present. This was determined by measuring the decrease in beta-lactamase activity when phenethyl alcohol was present to prevent maturation of the enzyme. There was no significant difference in plasmid copy number between the cells grown in the presence or absence of lincomycin. These results indicate that lincomycin stimulates transcription, translation, or translocation of beta-lactamase.

Mechanism of inactivation of TEM-1 beta-lactamase by 6-acetylmethylenepenicillanic acid.

The interaction between 6-acetylmethylenepenicillanic acid (compound Ro 15-1903; AMPA) and TEM-1 beta-lactamase was investigated in order to elucidate the mechanism of action of AMPA. Formation of the enzyme-inhibitor complex (EA) was accompanied by a shift of the absorbance maximum from 292 nm to 303 nm and an increase in the absorption. Regeneration of activity was very slow and incomplete, reaching about one-third of the initial activity after 48 h at 37 degrees C. This behaviour indicated a branched pathway of the decay of the inactivated enzyme. Kinetic and isoelectric-focusing experiments proved this assumption. The first-order constant of regeneration of active enzyme was 6 X 10(-6)-10 X 10(-6) s-1, whereas the rate constant leading to inactive enzyme (EA') was 10 X 10(-6)-15 X 10(-6) s-1 at pH 7.0. Both constants became larger at higher pH. Inactive enzyme (EA') consisted of two major species, with pI 5.36 (EA'1) and 5.30 (EA'2). The former increased at the beginning of incubation but decreased after prolonged incubation. From consideration of these results and previous data [Arisawa & Then (1983) Biochem. J. 209, 609-615], a likely mechanism of inactivation of TEM-1 beta-lactamase by AMPA is discussed.

Current problems in the treatment of infective endocarditis due to Pseudomonas aeruginosa.

Mortality from pseudomonas infective endocarditis remains high despite optimal use of available antibacterial agents. Infection of the tricuspid valve is subacute, but involvement of the mitral or aortic valve precipitates more serious disease. Most valvular infections are due to a single pseudomonad immunotype, but 20% of cases are mixed infections. Antimicrobial susceptibility tests and tests of synergy by beta-lactam and aminoglycoside antibiotics in combination were performed on 30 isolates of Pseudomonas aeruginosa. Azlocillin was the most effective beta-lactam in combination with an aminoglycoside; MKO 787 was least effective. Among the aminoglycosides tested, netilmicin was the most effective. Medical treatment combined with valvulectomy (without valve replacement) is now standard treatment for refractory right-sided endocarditis at this medical center. A high dose of aminoglycoside in combination with a beta-lactam has proved efficacious. For left-sided infection, immediate valve replacement accompanied by a six-week course of the high dose-combined drug regimen is recommended. Newer beta-lactam antibiotics, such as piperacillin, may be limited in usefulness due to beta-lactamase inactivation.

Moxalactam: an oxa-beta-lactam antibiotic that inactivates beta-lactamases.

Moxalactam, like cefoxitin, cefuroxime, and cefotaxime, shows little or no interaction with penicillinases. Resistance of moxalactam to cephalosporinases, however, appears to be considerably greater than that of these other recently discovered beta-lactam antibiotics. Moxalactam is a beta-lactamase inactivator and inactivates most of the cephalosporinases produced by gram-negative bacteria. The process of inactivation is specific and follows time-dependent or progressive kinetics, as is the case for a class of enzyme inhibitors called "suicide substrates." Moxalactam is the first beta-lactam antibiotic shown to possess this property of inactivation of beta-lactamases, a property that explains its excellent resistance to beta-lactamases.