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Lactam Antibiotics (lactam + antibiotics)
Selected AbstractsA Combined Theoretical and Experimental Research Project into the Aminolysis of ,-Lactam Antibiotics: The Importance of Bifunctional CatalysisEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 21 2003Natalia Díaz Abstract This paper reports the results of experimental work on the aminolysis of penicillin (6-APA) and monobactam (aztreonam) antibiotics by propylamine or ethanolamine. In general, aztreonam is slightly more reactive than 6-APA, despite the common assumption that the amide bond should be less activated in monobactams. Intriguingly, when ethanolamine acts as the nucleophile, the corresponding rate law has a kinetic term proportional to [RNH2][RNH3+]. To complement the experimental observations, the rate-determining free energy barriers in aqueous solution for various mechanistic pathways were computed by standard quantum chemical methodologies. From previous theoretical work it was assumed that the aminolysis of ,-lactams proceeds through mechanisms in which either a water molecule or a second amine molecule may act as bifunctional catalysts, assisting proton transfer from the attacking amine molecule to the leaving amino group. The energy barriers as computed have moderate values (ca. 26,34 kcal·mol,1) and reproduce most of the experimentally observed kinetic trends. Furthermore, the calculations predict that positively charged ethanolamine molecules can act as bifunctional catalysts as well, thus explaining the presence of the kinetic term proportional to [RNH2][RNH3+] in the rate law. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] Purification, crystallization and preliminary crystallographic analysis of Est-Y29: a novel oligomeric ,-lactamaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009SeungBum Kim ,-Lactam antibiotics such as penicillins and cephalosporins have a four-atom ring as a common element in their structure. The ,-lactamases, which catalyze the inactivation of these antibiotics, are of great interest because of their high incidence in pathogenic bacteria. A novel oligomeric class C ,-lactamase (Est-Y29) from a metagenomic library was expressed, purified and crystallized. The recombinant protein was expressed in Escherichia coli with an N-terminal 6×His tag and purified to homogeneity. EstY-29 was crystallized and X-ray intensity data were collected to 1.49,Å resolution using synchrotron radiation. [source] Antibiotic Resistance in Bacteria: Novel Metalloenzyme InhibitorsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 4 2009Sung-Kun Kim ,-Lactam antibiotics are among the most important drugs used to fight bacterial infection. Overuse and misuse of ,-lactam antibiotics has caused the evolution of resistance mechanisms, allowing pathogenic bacteria to survive antibiotic treatment. The major source of resistance to ,-lactam antibiotics occurs through production of enzymes called ,-lactamases capable of catalyzing hydrolysis of the ,-lactam rings in these drug compounds. The metallo-,-lactamases have become a major threat due to their broad substrate specificities; there are no clinically useful inhibitors for these metalloenzymes. We have obtained single-stranded DNA's that are potent inhibitors of the Bacillus cereus 5/B/6 metallo-,-lactamase. These are rapid, reversible, non-competitive inhibitors of the metalloenzyme, with Ki and Ki, values in the nanomolar range. The inhibition patterns and metal ion dependence of their inhibition suggest that the oligonucleotides alter the coordination of the active site metal ion(s); inhibition is efficient and highly specific. Microbiological growth experiments, using combinations of ssDNA with the ,-lactam antibiotic cephalexin, reveal that the inhibitor is capable of causing cell death in liquid cultures of both Gram-positive and Gram-negative metallo-,-lactamase producing bacteria in the micromolar concentration range. [source] Decreased virulence of a strain of Pseudomonas aeruginosa O12 overexpressing a chromosomal type 1 ,-lactamase could be due to reduced expression of cell-to-cell signaling dependent virulence factorsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2000Françoise Ramisse Abstract Pseudomonas aeruginosa produces a large variety of virulence factors and is characterized by its capacity to rapidly develop resistance when exposed to antibiotics. In order to evaluate a possible correlation between acquired resistance to antibiotics and virulence, we examined the virulence of four isogenic variants of P. aeruginosa O12 that differ in their resistance phenotypes to various ,-lactam antibiotics in a mouse model of acute pneumonia. Strains overproducing a chromosomal type 1 ,-lactamase were less virulent in both immunocompetent and immunosuppressed animals. Whereas the production of the exopolysaccharide alginate was similar between the four strains, extracellular virulence factors (elastase, rhamnolipid) that are controlled by the cell-to-cell signaling system circuit were detected in reduced amounts in the supernatant of the two isolates overproducing type 1 ,-lactamase. These results suggest that strains overexpressing the chromosomal type 1 ,-lactamase could be less virulent because of a reduction of cell-to-cell signaling dependent virulence factor production. [source] Penicillin Binding Proteins: key players in bacterial cell cycle and drug resistance processesFEMS MICROBIOLOGY REVIEWS, Issue 5 2006Pauline Macheboeuf Abstract Bacterial cell division and daughter cell formation are complex mechanisms whose details are orchestrated by at least a dozen different proteins. Penicillin-binding proteins (PBPs), membrane-associated macromolecules which play key roles in the cell wall synthesis process, have been exploited for over 70 years as the targets of the highly successful ,-lactam antibiotics. The increasing incidence of ,-lactam resistant microorganisms, coupled to progress made in genomics, genetics and immunofluorescence microscopy techniques, have encouraged the intensive study of PBPs from a variety of bacterial species. In addition, the recent publication of high-resolution structures of PBPs from pathogenic organisms have shed light on the complex intertwining of drug resistance and cell division processes. In this review, we discuss structural, functional and biological features of such enzymes which, albeit having initially been identified several decades ago, are now being aggressively pursued as highly attractive targets for the development of novel antibiotherapies. [source] Identification and Characterization of an Organic Solvent Tolerance Gene in Helicobacter pyloriHELICOBACTER, Issue 1 2007Hung-Chuan Chiu Abstract Background:, Pre-cleaning and soaking in glutaraldehyde is the necessary procedure to disinfect endoscopes. However, some chemical-solvent-tolerant bacteria may survive after incomplete endoscopic disinfection. The goal of this study was to identify glutaraldehyde resistance-related genes in Helicobacter pylori. Materials and Methods:, ,-Zap phagemid expression library of H. pylori strain NTUH-C1 was selected with 0.1% glutaraldehyde. The minimal inhibitory concentration (MIC) of glutaraldehyde-resistant DNA fragments of H. pylori NTUH-C1 strain were determined. Imp/OstA recombinant protein was expressed, purified, and used to generate anti-Imp/OstA polyclonal antibody. Imp/ostA knockout, deletion, and complementation strains were constructed. The function of Imp/OstA was monitored by organic solvent tolerance assay, antibiotics susceptibility test, and n -phenylnapthylamine assay. Results:, Using Imp/ostA polyclonal antibody against cell lysate of wild-type and imp/ostA mutant showed that it is not essential in H. pylori. Organic solvent tolerance assay demonstrated the role of Imp/ostA in n-hexane tolerance. MIC test showed that the mutation of imp/ostA was susceptible to hydrophobic and ,-lactam antibiotics. NPN assay demonstrated that the level of outer membrane permeability was increased by 50% in mutant strain comparing to wild-type strain (p < .001). Conclusions:, We have identified an Imp/OstA protein that was associated with glutaraldehyde resistance in our clinical strain H. pylori NTUH-C1 by screening of ,-Zap expression library. Disruption of this protein results in altering membrane permeability, sensitivity to organic solvent, and susceptibility to antibiotics. [source] Electronic structure and physicochemical properties of selected penicillinsINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 3 2007Catalina Soriano-Correa Abstract Traditionally, penicillins have been used as antibacterial agents due to their characteristics and widespread applications with few collateral effects, which have motivated several theoretical and experimental studies. Despite the latter, their mechanism of biological action has not been completely elucidated. We present a theoretical study at the Hartree,Fock and density functional theory (DFT) levels of theory of a selected group of penicillins such as the penicillin-G, amoxicillin, ampicillin, dicloxacillin, and carbenicillin molecules, to systematically determine the electron structure of full ,-lactam antibiotics. Our results allow us to analyze the electronic properties of the pharmacophore group, the aminoacyl side-chain, and the influence of the substituents (R and X) attached to the aminoacyl side-chain at 6, (in contrast with previous studies focused at the 3, substituents), and to corroborate the results of previous studies performed at the semiempirical level, solely on the ,-lactam ring of penicillins. Besides, several density descriptors are determined with the purpose of analyzing their link to the antibacterial activity of these penicillin compounds. Our results for the atomic charges (fitted to the electrostatic potential), the bond orders, and several global reactivity descriptors, such as the dipole moments, ionization potential, hardness, and the electrophilicity index, led us to characterize: the active sites, the effect of the electron-attracting substituent properties and their physicochemical features, which altogether, might be important to understand the biological activity of these type of molecules. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007 [source] Antimicrobial therapy in DermatologyJOURNAL DER DEUTSCHEN DERMATOLOGISCHEN GESELLSCHAFT, Issue 1 2006Cord Sunderkötter Antiseptika; Antibiotika; ,-Laktam-Resistenz; Weichteilinfektion Summary The extensive and sometimes indiscriminate use of antibiotics sometimes without strict indications has led to increases in both bacterial resistance and sensitization of patients. Systemic antibiotics in skin infections are indicated when a severe local infection occurs which spreads into the surrounding tissue or when there are signs of systemic infection. There are special indications in patients with peripheral arterial occlusive disease,diabetes or immunosuppression. Topical use of antibiotics should be abandoned and replaced by antiseptics. The ,-lactam antibiotics are the antibiotics of first choice for many skin infections. They are usually effective, have a well-defined profile of adverse events and most are affordable. Penicillin G or V are the first line treatment for erysipelas. Infections with Staphylococcus aureus are usually treated with isoxazolyl penicillins or second generation cephalosporins. In mixed infections in patients with diabetes or peripheral arterial occlusive disease,the treatment of choice is metronidazole plus ,-lactam-/,-lactamase inhibitor antibiotics, but quinolones or second generation cephalosporins can also be used, once again with metronidazole. The aim of this review is to define the indications for antibiotics in dermatology, to highlight their modes of action and adverse effects and to make suggestions for rational antibiotic therapy in cutaneous infections frequently encountered in the practice of dermatology. Zusammenfassung Der bisweilen unkritische Einsatz von Antibiotika hat die Resistenzentwicklung beschleunigt und die Sensibilisierungsrate bei Patienten erhöht. Systemische Antibiotika sind bei kutanen Superinfektionen in der Regel dann indiziert, wenn eine schwere lokale Infektion mit Ausbreitung in das umgebende Gewebe vorliegt oder wenn sich gleichzeitig Zeichen einer systemischen Infektion einstellen. Bei peripherer arterieller Verschlusskrankheit, Diabetes mellitus oder Immunsuppression kann die Indikation auch früher gestellt werden. Lokale Antibiotika sollten in der Regel gemieden und durch moderne Antiseptika ersetzt werden. ,-Laktam-Antibiotika stellen für viele bakterielle Infektionserkrankungen in der ambulanten und klinischen Dermatologie die Antibiotika der ersten Wahl dar. Sie sind häufig ausreichend wirksam, besitzen ein gut definiertes Nebenwir-kungsprofil und sind zumeist preisgünstig. So wird das klassische Streptokokken-Erysipel mit Penicillin G oder V therapiert, bei Infektionen durch S. aureus kommen primär Isoxazolyl-Penicilline oder Zweit-Generations-Cephalosporine zum Einsatz. Im Falle von Mischinfektionen bei Diabetes mellitus oder pAVK sind ,-Laktam/,-Laktamaseinhibitoren indiziert, alternativ auch Chinolone oder Zweitgenerations-Cephalosporine, jeweils in Kombination mit Metronidazol. Diese Übersicht möchte die Indikationen für Antibiotika in der Dermatologie aufzeigen, das Wichtigste zu deren Wirkungsweise und Nebenwirkungen aufzählen und Therapievorschläge für häufige Infektionen der Haut in der dermatologischen Praxis geben. [source] Effect of certain bioactive plant extracts on clinical isolates of ,-lactamase producing methicillin resistant Staphylococcus aureusJOURNAL OF BASIC MICROBIOLOGY, Issue 2 2005Farrukh Aqil Ethanolic extracts and some fractions from 10 Indian medicinal plants, known for antibacterial activity, were investigated for their ability to inhibit clinical isolates of ,-lactamase producing methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). Synergistic interaction of plant extracts with certain antibiotics was also evaluated. The MRSA test strains were found to be multi-drug resistant and also exhibited high level of resistance to common ,-lactam antibiotics. These strains produced ,-lactamases, which hydrolyze one or other ,-lactam antibiotics, tested. The extract of the plants from Camellia sinensis (leaves), Delonix regia (flowers), Holarrhena antidysenterica (bark), Lawsonia inermis (leaves), Punica granatum (rind), Terminalia chebula (fruits) and Terminalia belerica (fruits) showed a broad-spectrum of antibacterial activity with an inhibition zone size of 11 mm to 27 mm, against all the test bacteria. The extracts from the leaves of Ocimum sanctum showed better activity against the three MRSA strains. On the other hand, extracts from Allium sativum (bulb) and Citrus sinensis (rind) exhibited little or no activity, against MRSA strains. The antibacterial potency of crude extracts was determined in terms of minimum inhibitory concentration (MIC) by the tube dilution method. MIC values, of the plant extracts, ranged from 1.3 to 8.2 mg/ml, against the test bacteria. Further, the extracts from Punica granatum and Delonix regia were fractionated in benzene, acetone and methanol. Antibacterial activity was observed in acetone as well as in the methanol fractions. In vitro synergistic interaction of crude extracts from Camellia sinensis, Lawsonia inermis, Punica granatum, Terminalia chebula and Terminalia belerica was detected with tetracycline. Moreover, the extract from Camellia sinensis also showed synergism with ampicillin. TLC of the above extracts revealed the presence of major phytocompounds, like alkaloids, glycosides, flavonoids, phenols and saponins. TLC-bioautography indicated phenols and flavonoids as major active compounds. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Pharmacokinetic prediction for intravenous ,-lactam antibiotics in pediatric patientsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 11 2007Kenji Shimamura Abstract A method for predicting pharmacokinetics in pediatric patients for intravenous ,-lactam antibiotics is proposed. We focused on the allometric relationships of pharmacokinetic parameters with individual body weights (BW) in human including healthy adults and pediatric patients. Drug concentration data for 15 intravenous ,-lactam antibiotics were collected retrospectively from the published articles and the individual pharmacokinetic parameters were re-calculated. A mixed effect modeling (MEM) was applied for the allometric relationship for those ,-lactam antibiotics, and mean and variances of inter-drug variability for the allometric parameters and also variance for intra-drug (residual) variability were estimated. Then drug-specific allometric parameters were estimated by an empirical Bayesian method using the pharmacokinetic parameters for a drug only in healthy adults as observations, and finally the individual pharmacokinetic parameters in pediatric patients were predicted. The predictability of the method was evaluated by the leave-one-out method. We also demonstrated a method for simulating plasma concentration,time profiles in pediatric patients, and the predicted time,course curves generally coincided well with the actual plasma concentration data for the tested drugs. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 3125,3139, 2007 [source] Pharmaceutical and pharmacological importance of peptide transportersJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2008Matthias Brandsch Peptide transport is currently a prominent topic in membrane research. The transport proteins involved are under intense investigation because of their physiological importance in protein absorption and also because peptide transporters are possible vehicles for drug delivery. Moreover, in many tissues peptide carriers transduce peptidic signals across membranes that are relevant in information processing. The focus of this review is on the pharmaceutical relevance of the human peptide transporters PEPT1 and PEPT2. In addition to their physiological substrates, both carriers transport many ,-lactam antibiotics, valaciclovir and other drugs and prodrugs because of their sterical resemblance to di- and tripeptides. The primary structure, tissue distribution and substrate specificity of PEPT1 and PEPT2 have been well characterized. However, there is a dearth of knowledge on the substrate binding sites and the three-dimensional structure of these proteins. Until this pivotal information becomes available by X-ray crystallography, the development of new drug substrates relies on classical transport studies combined with molecular modelling. In more than thirty years of research, data on the interaction of well over 700 di- and tripeptides, amino acid and peptide derivatives, drugs and prodrugs with peptide transporters have been gathered. The aim of this review is to put the reports on peptide transporter-mediated drug uptake into perspective. We also review the current knowledge on pharmacogenomics and clinical relevance of human peptide transporters. Finally, the reader's attention is drawn to other known or proposed human peptide-transporting proteins. [source] Diagnosis of nonimmediate reactions to ,-lactam antibioticsALLERGY, Issue 11 2004A. Romano Nonimmediate manifestations (i.e. occurring more than 1 h after drug administration), particularly maculopapular and urticarial eruptions, are common during , -lactam treatment. The mechanisms involved in most nonimmediate reactions seem to be heterogeneous and are not yet completely understood. However, clinical and immunohistological studies, as well as analysis of drug-specific T-cell clones obtained from the circulating blood and the skin, suggest that a type-IV (cell-mediated) pathogenic mechanism may be involved in some nonimmediate reactions such as maculopapular or bullous rashes and acute generalized exanthematous pustulosis. In the diagnostic work-up, the patient's history is fundamental; patch testing is useful, together with delayed-reading intradermal testing. The latter appears to be somewhat more sensitive than patch testing, but also less specific. In case of negative allergologic tests, consideration should be given to provocation tests, and the careful administration of the suspect agents. With regard to in vitro tests, the lymphocyte transformation test may contribute to the identification of the responsible drug. Under the aegis of the European Academy of Allergology and Clinical Immunology (EAACI) interest group on drug hypersensitivity and the European Network for Drug Allergy (ENDA), in this review we describe the general guidelines for evaluating subjects with nonimmediate reactions to , -lactams. [source] The kinetic properties of the carboxy terminal domain of the Bacillus licheniformis 749/I BlaR penicillin-receptor shed a new light on the derepression of ,-lactamase synthesisMOLECULAR MICROBIOLOGY, Issue 6 2003Valérie Duval Summary To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformis,-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various ,-lactam antibiotics were studied. The second-order acylation rate constants k2/K, ranged from 0.0017 to more than 1 µM,1s,1 and the deacylation rate constants were lower than 4 × 10,5 s,1. These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR-CTD is thus one of the most sensitive penicillin-binding proteins presently described. In the light of these results, the kinetics of ,-lactamase induction in Bacillus licheniformis were re-examined. When starting with a rather high cell density, a good ,-lactamase substrate such as benzylpenicillin is too sensitive to ,-lactamase-mediated hydrolysis to allow full induction. By contrast, a poor ,-lactamase substrate (7-aminocephalosporanic acid) can fully derepress ,-lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction. [source] Incidence of ,-lactamase production and antimicrobial susceptibility of anaerobic gram-negative rods isolated from pus specimens of orofacial odontogenic infectionsMOLECULAR ORAL MICROBIOLOGY, Issue 1 2001T. Kuriyama The incidence of ,-lactamase production in anaerobic gram-negative rods isolated from 93 pus specimens of orofacial odontogenic infections and the antimicrobial susceptibility of these isolates against 11 antibiotics were determined. A total of 191 anaerobic gram-negative rods were isolated from the specimens. ,-Lactamase was detected in 35.6% of the black-pigmented Prevotella and 31.9% of the nonpigmented Prevotella. However, no strains among the other species isolated produced ,-lactamase. Ampicillin, cefazolin and cefotaxime showed decreased activity as regards ,-lactamase-positive Prevotella strains, whereas the activity of ampicillin/sulbactam, cefmetazole, and imipenem continued to be effective against such strains. All tested ,-lactam antibiotics were effective against Porphyromonas and Fusobacterium. Erythromycin showed decreased activity against nonpigmented Prevotella and Fusobacterium. Clindamycin, minocycline and metronidazole were powerful antibiotics against which anaerobic gram-negative rods could be tested. The present study showed that ,-lactamase-positive strains were found more frequently in the Prevotella strains than in any of the other species of anaerobic gram-negative rods. The effectiveness of adding sulbactam to ampicillin was demonstrated, as well as the difference in cephalosporin activity against ,-lactamase-positive strains. [source] Green tea extract weakens the antibacterial effect of amoxicillin in methicillin-resistant Staphylococcus aureus infected micePHYTOTHERAPY RESEARCH, Issue 1 2010Qing Peng Abstract Tea (Camellia sinensis) has been known for its modulation of resistance of methicillin-resistant Staphylococcus aureus (MRSA) to ,-lactam antibiotics in vitro. This study aimed to confirm the in vitro effect of green tea extracts with ,-lactams and to determine whether green tea extracts can reduce the minimum inhibitory concentrations (MICs) of amoxicillin in MRSA-infected mice. The catechins in the test tea that account for the reduced resistance to ,-lactams were quantitatively determined by high-performance liquid chromatography. The MICs of the ampicillin, cefazolin, amoxicillin, oxacillin, tea extract alone and tea extract in combination with ,-lactams were determined. Proportions of tea extracts and amoxicillin-tea extract combinations were administered to groups of mice enterally. The in vitro experiment showed that the MICs of four ,-lactams were greatly decreased in the presence of 0.25% tea extract. However, in an in vivo experiment, amoxicillin in combination with 5% tea extract conferred a higher ED50 than that of antibiotic alone. Green tea extract, alone or in combination with amoxicillin, does not have protective benefits in MRSA-infected mice. This study concluded that tea extract weakened the antibacterial effect of amoxicillin in MRSA infected mice. Tea drinking is not recommended in combination with amoxicillin treatment. Copyright © 2009 John Wiley & Sons, Ltd. [source] Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM-1 ,-lactamasePROTEIN SCIENCE, Issue 10 2009David C. Marciano Abstract A large number of ,-lactamases have emerged that are capable of conferring bacterial resistance to ,-lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM-1 ,-lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A ,-lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the ,-lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM-1 ,-lactamase to positions 220, 272, and 276 by site-directed mutagenesis. Kinetic analysis of the engineered ,-lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A ,-lactamases. [source] Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesisPROTEIN SCIENCE, Issue 9 2005Michaël Delmarcelle Abstract The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to ,-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed. [source] Crystallization and preliminary X-ray crystallographic analyses of CMY-1 and CMY-10, plasmidic class C ,-lactamases with extended substrate spectrumACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2004Sun-Joo Lee Plasmid-encoded class C ,-lactamases, including CMY-1 and CMY-10, hydrolyze the lactam bonds of ,-lactam antibiotics, inducing therapeutic failure and a lack of eradication of clinical isolates by third-generation cephalosporins or cephamycins. Therefore, the enzymes are potential targets for developing agents against pathogens isolated from patients suffering from wound infection, urinary tract infection or pneumonia. CMY-1 and CMY-10 were purified and crystallized at 298,K. X-ray diffraction data from CMY-1 and CMY-10 crystals have been collected to 2.5 and 1.5,Å resolution, respectively, using synchrotron radiation. The crystals of the two proteins are isomorphous and belong to the primitive monoclinic space group P21. [source] High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicabilityBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2006Lidija Senerovic Abstract A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPACP.rett)of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPACP.rett activity of approximately 40% (3.8 U/mL vs. 2.7U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPACP.rett purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPACP.rett expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPACP.rett. The enzyme wasimmobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPACP.rett performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g dry based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPACP.rett was finally evaluated by studying the kinetically controlled synthesis of ,-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process. © 2005 Wiley Periodicals, Inc. [source] Detection of immediate-type reaction to the epitope of ,-lactam antibiotics by the quick patch testBRITISH JOURNAL OF DERMATOLOGY, Issue 1 2003M. Oi No abstract is available for this article. [source] Antibiotic Resistance in Bacteria: Novel Metalloenzyme InhibitorsCHEMICAL BIOLOGY & DRUG DESIGN, Issue 4 2009Sung-Kun Kim ,-Lactam antibiotics are among the most important drugs used to fight bacterial infection. Overuse and misuse of ,-lactam antibiotics has caused the evolution of resistance mechanisms, allowing pathogenic bacteria to survive antibiotic treatment. The major source of resistance to ,-lactam antibiotics occurs through production of enzymes called ,-lactamases capable of catalyzing hydrolysis of the ,-lactam rings in these drug compounds. The metallo-,-lactamases have become a major threat due to their broad substrate specificities; there are no clinically useful inhibitors for these metalloenzymes. We have obtained single-stranded DNA's that are potent inhibitors of the Bacillus cereus 5/B/6 metallo-,-lactamase. These are rapid, reversible, non-competitive inhibitors of the metalloenzyme, with Ki and Ki, values in the nanomolar range. The inhibition patterns and metal ion dependence of their inhibition suggest that the oligonucleotides alter the coordination of the active site metal ion(s); inhibition is efficient and highly specific. Microbiological growth experiments, using combinations of ssDNA with the ,-lactam antibiotic cephalexin, reveal that the inhibitor is capable of causing cell death in liquid cultures of both Gram-positive and Gram-negative metallo-,-lactamase producing bacteria in the micromolar concentration range. [source] Beta lactam allergy and resensitization in children with suspected beta lactam allergyCLINICAL & EXPERIMENTAL ALLERGY, Issue 5 2009J. Hershkovich Summary Background In patients who were clinically diagnosed as having beta lactam allergy and had negative skin tests, the rates of reported resensitization to beta lactams after subsequent exposures, vary significantly. Some allergists advocate skin testing before every exposure to beta lactams. Objective We sought to determine the true rate of beta lactam allergy and of resensitization in children with a positive history for suspected beta lactam allergy. Methods The study was conducted from July 1998 to May 2004, with follow-up during 2007. Beta lactam allergy tests with the major determinant and freshly prepared minor determinant mixtures were offered to history positive children. Negative skin tests were followed by oral challenge. The tests were performed again 1,5 months later in order to address the possibility of resensitization. Results Tests were performed on 166 children: 150 for penicillins alone, 14 for penicillin in combination with cephalosporins, and an additional 2 patients solely for cephalosporins. Only 10 children (6%) were positive in the initial evaluation, four by skin test and six by oral challenge. A second set of tests was performed in 98 children with a negative initial evaluation; only two children (2%) were resensitized. On a follow-up survey of 71 of the 96 patients, 59 (83%) had received beta lactams; only one had developed a minor rash after subsequent exposure to amoxicillin. Conclusions Most children with suspected beta lactam allergy were not allergic to beta lactams. Resensitization to beta lactam antibiotics in children in this study was infrequent. In children with a clinical diagnosis of beta lactam allergy and negative skin tests, repeated skin testing before every exposure is usually unnecessary. [source] Drug allergy claims in children: from self-reporting to confirmed diagnosisCLINICAL & EXPERIMENTAL ALLERGY, Issue 1 2008E. Rebelo Gomes Summary Background Poorly documented self-reported drug allergy (DAll) is a frequent problem in daily clinical practice and has a considerable impact on prescription choices. The diagnostic work-up of drug hypersensitivity (DHs) allows a better classification of the reactions and provides patients with more reliable information and recommendations for future treatments. Objective To assess the prevalence of self-reported adverse drug reactions (ADRs) and DAll in a paediatric population and to investigate children reporting suspected DAll in order to achieve a firm diagnosis. Design The first phase was based on a cross-sectional survey assessing the life occurrence of ADRs and self-reported DAll carried out at the outpatient clinic of a paediatric hospital. The second phase was based on the diagnostic work-up in children with parent-reported DAll, including detailed anamnesis and in vitro and in vivo investigations (skin and provocation tests). Participants One thousand four hundred and twenty-six parents responded to the initial survey. Sixty of the 67 patients with reported DAll were evaluated at the allergy clinic. Results The prevalences of self-reported ADRs and DAll were 10.2% and 6.0%, respectively. Most of the suspected allergic reactions were non-immediate cutaneous events attributable to ,-lactam antibiotics and occurred in very young children. Thirty-nine of the 60 patients consulting for evaluation had a plausible clinical history and were recommended further investigation. DHs was diagnosed in three children only, based on positive responses in skin (n=1) and oral provocation (n=2) tests. Conclusion ADRs are frequently reported in children, and many children are classified as having a DAll. After complete evaluation, only a few of these reactions can be attributed to DHs and DAll. Most of the patients (94% in this study) could actually tolerate the initially suspected drug. [source] Tigecycline: in-vitro performance as a predictor of clinical efficacyCLINICAL MICROBIOLOGY AND INFECTION, Issue 4 2007P. Hawkey Abstract The incidence of nosocomial disease caused by Gram-negative pathogens is increasing, and infections caused by Enterobacter, Klebsiella, Acinetobacter, Escherichia coli and Pseudomonas aeruginosa are more commonly refractive to traditional antimicrobial agents, including aminoglycosides, fluoroquinolones and broad-spectrum cephalosporins. The most important mechanism of resistance to ,-lactam antibiotics among Gram-negative bacilli involves the production of ,-lactamases. Extended-spectrum ,-lactamases are particularly worrisome, since they are often associated with multidrug resistance phenotypes, which can pose a significant therapeutic challenge. Novel agents for the treatment of Gram-negative infections are uncommon, as recent emphasis has been placed on the development of agents targeting drug-resistant strains of Gram-positive bacteria, e.g., streptococci, enterococci and staphylococci. Tigecycline, a semi-synthetic derivative of minocycline, has a unique and novel mechanism of action, which not only allows this agent to overcome the well-known tet gene-encoded resistance mechanisms, but also maintains its activity against Gram-negative pathogens producing a broad array of extended-spectrum ,-lactamases. Tigecycline is the first example of a new class of glycylcyclines with activity against a wide range of clinically important Gram-negative pathogens. Tigecycline has potent antimicrobial activity, and has been associated with an excellent therapeutic response in animal infection models and recently reported clinical trials, which reflect the effectiveness of tigecycline against pathogens causing intra-abdominal, skin and soft-tissue infections, including susceptible or multidrug-resistant strains of most Enterobacteriaceae, as well as anaerobic pathogens. [source] | ||||||||||||||||||||||||||||