New ionic liquids and salts derived from β-Lactam antibiotics

In recent years ionic liquids (ILs) have been increasing the popularity and the number of applications. Ionic liquids were used mainly as solvent in organic synthesis, but in recent years they are also used in analytical chemistry, separation chemistry and material science. 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 anion with bacterial activity as β-lactam antibiotics 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 β-lactam antibiotics. After crystallization we obtained pure ILs and salts containing β-lactam antibiotics. This work presents a novel method for preparation of new salts of antibiotics with low melting point and their chemistry and microbiological characterization.

Fast screening procedure for antibiotics in wastewaters by direct HPLC-DAD analysis

Growing concern about the contamination of wastewaters by antibiotics demands fast but sensitive analytical methodologies, for the screening of a large number of samples. The purpose of this work was to develop a simple methodology, using direct injection of the samples, by HPLC with diode array detection (DAD), for a multiresidue analysis of five antibiotics of different classes. Wastewater from an urban water treatment plant was selected as a model to study possible coelution of interfering compounds. The linearity interval ranged from 40 to 400 µg/L for amoxicillin (Amox), metronidazole (Metro), cefazolin (Cefa), and chloramphenicol (Chloram) and from 20 to 200 µg/L for sulfamethoxazole (Sulfa), with LODs lower than 14 µg/L. Repeatability, expressed by the CV of six repeated injections, ranged from 1 to 8%, while the intermediate precision varied between 2 and 11%. The recovery ranged from 90 to 109%. This method enables the fast screening of a large number of samples, with an expanded uncertainty in the 1–22% range. The advantage of the proposed method is to significantly reduce the number of samples to be analyzed by more complex methods.

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.

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

Since the discovery of the first penicillin bacterial resistance to β-lactam antibiotics has spread and evolved promoting new resistances to pathogens. The most common mechanism of resistance is the production of β-lactamases that have spread thorough nature and evolve to complex phenotypes like CMT type enzymes. New antibiotics have been introduced in clinical practice, and therefore it becomes necessary a concise summary about their molecular targets, specific use and other properties. β-lactamases are still a major medical concern and they have been extensively studied and described in the scientific literature. Several authors agree that Glu166 should be the general base and Ser70 should perform the nucleophilic attack to the carbon of the carbonyl group of the β-lactam ring. Nevertheless there still is controversy on their catalytic mechanism. TEMs evolve at incredible pace presenting more complex phenotypes due to their tolerance to mutations. These mutations lead to an increasing need of novel, stronger and more specific and stable antibiotics. The present review summarizes key structural, molecular and functional aspects of ESBL, IRT and CMT TEM β-lactamases properties and up to date diagrams of the TEM variants with defined phenotype. The activity and structural characteristics of several available TEMs in the NCBI-PDB are presented, as well as the relation of the various mutated residues and their specific properties and some previously proposed catalytic mechanisms.

Role of oxidative stress-induced systemic and cavernosal molecular alterations in the progression of diabetic erectile dysfunction

Background Erectile dysfunction (ED) is a prevalent complication of diabetes, and oxidative stress is an important feature of diabetic ED. Oxidative stress-induced damage plays a pivotal role in the development of tissue alterations. However, the deleterious effects of oxidative stress in the corpus cavernosum with the progression of diabetes remain unclear. The aim of this study was to evaluate systemic and penile oxidative stress status in the early and late stages of diabetes. Methods Male Wistar streptozotocin-diabetic rats (and age-matched controls) were examined 2 (early) and 8 weeks (late) after the induction of diabetes. Systemic oxidative stress was evaluated by urinary H2O2 and the ratio of circulating reduced/oxidized glutathione (GSH/GSSG). Penile oxidative status was assessed by H2O2 production and 3-nitrotyrosine (3-NT) formation. Cavernosal endothelial nitric oxide synthase (eNOS) was analyzed by quantitative immunohistochemistry. Dual immunofluorescence was also performed for 3-NT and α-smooth muscle actin (α-SMA) and eNOS–α-SMA. Results There was a significant increase in urinary H2O2 levels in both diabetic groups. The plasma GSH/GSSG ratio was significantly augmented in late diabetes. In cavernosal tissue, H2O2 production was significantly increased in late diabetes. Reactivity for 3-NT was located predominantly in cavernosal smooth muscle (SM) and was significantly reduced in late diabetes. Quantitative immunohistochemistry revealed a significant decrease in eNOS levels in cavernosal SM and endothelium in late diabetes. Conclusions The findings indicate that the noxious effects of oxidative stress are more prominent in late diabetes. Increased penile protein oxidative modifications and decreased eNOS expression may be responsible for structural and/or functional deregulation, contributing to the progression of diabetes-associated ED.