Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the beta-lactam producer Penicillium chrysogenum

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of g-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA. (C) 2011 Elsevier Inc. All rights reserved.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene bla(CTX-M-2) was identified in 3 different enterobacteria (E. coli. Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), hut not as part of the resistance cassettes region. Multi locus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coil isolates in this study and strains associated with meningitis in the E. coil MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coil in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

Preparation and characterization of beta-lactam antibiotic as Ionic liquids and salts

With the increase of bacterial resistance a large number of therapeutic strategies have been used to fight different kind of infections. In recent years ionic liquids (ILs) have been increasing the popularity and the number of applications. First ionic liquids were used mainly as solvent in organic synthesis, but now they are used in analytical chemistry, separation chemistry and material science among others. Additional to significant developments in their chemical properties and applications, ionic liquids are now bringing unexpected opportunities at the interface of chemistry with the life sciences Ionic liquids (ILs) are currently defined as salts that are composed solely of cations and anions which melt below 100ºC. Our goal in this work is to explore the dual activity of the ionic liquids, due to the presence of two different ions, an ion with bacterial activity as a beta-lactam antibiotic and different kinds of cations. In this work the anions of ILs and salts were derived from three different antibiotics: ampicillin, penicillin and amoxicillin. The cations were derived from substituted ammonium, phosphonium pyridinium and methylimidazolium salts, such as: tetraethyl ammonium, trihexiltetradecilphosphonium, cetylpyridinium, choline (an essential nutrient), 1-ethyl-3-methylimidazolium, and 1-ethanol-3-methyl imidazolium structures. Commercial ammonium and phosponium halogen salts were first transformed into hydroxides. on ionic exchange column (Amberlite IRA-400) in methanol. The prepared hydroxides were then neutralized with beta-lactam antibiotics. After crystallization we obtained pure ILs and salts containing beta-lactam antibiotics. This work presents a novel method for preparation of new salts of antibiotics with low melting point and their characterization.

Evolution of Drug Resistance: Insight on TEM -Lactamases Structure and Activity and Beta-Lactam Antibiotics

Extended-spectrum β-lactamases (ESBLs) prevalence was studied in the north of Portugal, among 193 clinical isolates belonging to citizens in a district in the boundaries between this country and Spain from a total of 7529 clinical strains. In the present study we recovered some members of Enterobacteriaceae family, producing ESBL enzymes, including Escherichia coli (67.9%), Klebsiella pneumoniae (30.6%), Klebsiella oxytoca (0.5%), Enterobacter aerogenes (0.5%), and Citrobacter freundii (0.5%). β-lactamases genes blaTEM, blaSHV, and blaCTX-M were screened by polymerase chain reaction (PCR) and sequencing approaches. TEM enzymes were among the most prevalent types (40.9%) followed by CTX-M (37.3%) and SHV (23.3%). Among our sample of 193 ESBL-producing strains 99.0% were resistant to the fourth-generation cephalosporin cefepime. Of the 193 isolates 81.3% presented transferable plasmids harboring genes. Clonal studies were performed by PCR for the enterobacterial repetitive intragenic consensus (ERIC) sequences. This study reports a high diversity of genetic patterns. Ten clusters were found for E. coli isolates and five clusters for K. pneumoniae strains by means of ERIC analysis. In conclusion, in this country, the most prevalent type is still the TEM-type, but CTX-M is growing rapidly.

Allergie aux beta-lactamines [Allergy to beta-lactam antibiotics].

Beta-lactam antibiotics allergies are common. Up to 10% of the population describe a former allergy to penicillins. However only 10 to 15% of these individuals are actually allergic. In most cases, beta-lactam antibiotics will be avoided and replaced by other antibiotics such as quinolones. This fear of a serious allergic reaction has an economic impact and may lead to the emergence of antibiotic resistance. A thorough allergic work-up can accurately determine true allergic patients. Most of the patients with a proven allergy will be able to tolerate other antibiotics belonging to the beta-lactam family. This article focuses on the management of beta-lactam allergic patients.

Beta-lactam resistance mechanisms of methicillin-resistant Staphylococcus aureus.

In vitro and in vivo activity of amoxicillin and penicillin G alone or combined with a penicillinase inhibitor (clavulanate) were tested against five isogenic pairs of methicillin-resistant Staphylococcus aureus (MRSA) producing or not producing penicillinase. Loss of the penicillinase plasmid caused an eight times or greater reduction in the MICs of amoxicillin and penicillin G (from greater than or equal to 64 to 8 micrograms/ml), but not of the penicillinase-resistant drugs methicillin and cloxacillin (greater than or equal to 64 micrograms/ml). This difference in antibacterial effectiveness correlated with a more than 10 times greater penicillin-binding protein 2a affinity of amoxicillin and penicillin G than of methicillin and a greater than or equal to 90% successful amoxicillin treatment of experimental endocarditis due to penicillinase-negative MRSA compared with cloxacillin, which was totally ineffective (P less than .001). Amoxicillin was also effective against penicillinase-producing parent MRSA, provided it was combined with clavulanate. Penicillinase-sensitive beta-lactam antibiotics plus penicillinase inhibitors might offer a rational alternative treatment for MRSA infections.

Quantification of beta-lactam antibiotics in veterinary drugs: amoxicillin and ampicillin determination by high performance liquid chromatography

This work focused on the development and validation of an RP-HPLC-UV method for quantification of beta-lactam antibiotics in three pharmaceutical samples. Active principles analyzed were amoxicillin and ampicillin, in 3 veterinary drugs. Mobile phase comprised 5 mmol L-1 phosphoric acid solution at pH 2.00, acetonitrile with gradient elution mode and detection wavelength at 220 nm. The method was validated according to the Brazilian National Health Surveillance regulation, where linear range and linearity, selectivity, precision, accuracy and ruggedness were evaluated. Inter day precision and accuracy for pharmaceutical samples 1, 2 and 3 were: 1.43 and 1.43%; 4.71 and 3.74%; 2.72 and 1.72%, respectively, while regression coefficients for analytical curves exceeded 0.99. The method had acceptable merit figure values, indicating reliable quantification. Analyzed samples had active principle concentrations varying from -12 to +21% compared to manufacturer label claims, rendering the medicine unsafe for administration to animals.

Effect of hexadecyltrimethylammonium bromide-based microemulsions on the rate of decomposition of the beta-lactam antibiotic cephaclor

Microemulsions of hexadecyltrimethylammonium bromide (HTAB)/n-butanol/hexadecane/water catalyze the intramolecular degradation of cephaclor. The rate increase is a sensitive function of the microemulsion volume fraction and salt concentration. The effects of microemulsions, analyzed quantitatively using a pseudophase ion-exchange model, assumed that the extent of ion dissociation from the microemulsions varies with volume fraction. Comparison of micellar and microemulsion effects on the same reaction shows that microemulsions are less effective catalysts. Acceleration decreased significantly by increasing the relative proportion of n-butanol ratio in microemulsions and by addition of n-butanol in HTAB micelles. Comparison of the activation parameters of the reaction in aqueous solution, microemulsions, and micelles suggests that catalysis by both aggregates is driven mainly by entropic contributions.

Hypersensitivity reactions to non beta-lactam antimicrobial agents, a statement of the WAO special committee on drug allergy

Antibiotics are used extensively in the treatment of various infections. Consequently, they can be considered among the most important agents involved in adverse reactions to drugs, including both allergic and non-allergic drug hypersensitivity [J Allergy Clin Immunol 113:832–836, 2004]. Most studies published to date deal mainly with reactions to the beta-lactam group, and information on hypersensitivity to each of the other antimicrobial agents is scarce. The present document has been produced by the Special Committee on Drug Allergy of the World Allergy Organization to present the most relevant information on the incidence, clinical manifestations, diagnosis, possible mechanisms, and management of hypersensitivity reactions to non beta-lactam antimicrobials for use by practitioners worldwide.

Antibacterial activity of beta-lactam antibiotics in experimental meningitis due to Streptococcus pneumoniae

In order to define the characteristics of the antibacterial activity of beta-lactam antibiotics in the treatment of bacterial meningitis, the relationship between cerebrospinal fluid (CSF) drug concentrations and the rate of bacterial killing was investigated for penicillin G and four new cephalosporins in an animal model of meningitis due to Streptococcus pneumoniae. All five drugs showed a significant correlation between increasing drug concentrations in CSF and increasing bactericidal rates. Minimal activity was observed in CSF at drug concentrations of approximately the broth minimal bactericidal concentration (MBC). Maximal activity occurred with CSF concentrations 10-30 times higher. In vitro tests did not reproduce the unique correlation of increasing drug concentrations and killing activity found in vivo. When evaluating new beta-lactam antibiotics for the treatment of bacterial meningitis, it is reasonable to establish a minimum standard of CSF drug concentrations of greater than or equal to 30 times the MBC against the infecting organism.

Beta-lactam allergy in adults with cystic fibrosis

Background: Allergic reactions to one or more beta-lactam antibiotic can pose a management problem in patients with cystic fibrosis (CF), and may limit antibiotic choice. Method: The aim of this study was to assess the prevalence of allergy to anti-pseudomonal beta-lactam antibiotics in an adult CF centre and to assess variables, which may contribute to the development of allergic reactions. A questionnaire-based interview and a review of medical records were performed. Results: Of the 150 patients, 54 (36%) had allergic reactions to one or more beta-lactam antibiotics and 20 (19%) had allergic reactions to multiple beta-lactam antibiotics. The proportion of patients allergic to specific beta-lactam antibiotics varied from 10% to 26%. Rates of reactions were highest for penicillins and cephalosporins, intermediate for carbepenems and lowest for aztreonam. Of all reactions, 40% occurred within 24 h of the commencement of an individual antibiotic course. Patients with one or more beta-lactam allergic reactions had received greater cumulative exposure (p < 0.0001), were older (p=0.016) and had lower lung function (p=0.037) than patients without a history of beta-lactam allergy. Cystic Fibrosis transmembrane regulator (CFTR) status, gender, peripheral blood eosinophil count and total IgE concentrations were not different in patients with allergic reactions. Conclusions: This study demonstrates that the prevalence of allergic reactions to beta-lactam antibiotics is high in adults with CF. Increasing age; cumulative exposure and decreasing FEV1 were associated with the development of allergy. (C) 2006 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.