Simultaneous determination of six antibiotics in human serum by high-performance liquid chromatography with UV detection.

Antibiotics are widely used in intensive care patients to treat severe infections. To avoid bacterial resistance or toxic side effects, the determination of serum concentration of ABs is advisable. Therefore, in this study, we developed and validated a simple and fast high-performance liquid chromatography method with UV detection for the simultaneous determination of four ß-lactam ABs (meropenem, imipenem, ceftazidime, and piperacillin) and two coadministered substances (cilastatin and tazobactam) in human serum. Sample preparation required a simple protein precipitation by methanol. The separation of the ABs occurred within a timeframe of 17 min. For this purpose, we used a Kinetex F5 column with a linear gradient of acetonitrile and phosphate buffer (pH 6.9). The UV detector recorded two separate chromatograms at 220 and 295 nm simultaneously. Validation has demonstrated that the method is linear, accurate, and precise within the clinically relevant range for each substance.

Magnetic solid-phase extraction of four ß-lactams using polypyrrole-coated magnetic nanoparticles from water samples by micellar electrokinetic capillary chromatography analysis.

In the present research, the application of a polypyrrole coating on Fe3O4 magnetic nanoparticles (PPy/Fe3O4 MNPs) as a sorbent was confirmed. The synthesized magnetic composites were characterized by TEM, FTIR spectroscopy, and XRD. Four ß-lactams (oxacillin (OXA), cloxacillin (CLOX), dicloxacillin (DIC), and flucloxacillin (FLU)) were selected as analytes for the experiment. The extracted ß-lactams were determined by micellar electrokinetic capillary chromatography-diode array detector (MEKC-DAD). The crucial parameters influencing the extraction efficiency and separation were studied and optimized. Under the optimal conditions, the limits of detection are 1.0 µg L-1 for OXA, CLOX, FLU, and 0.8 µg L-1 for DIC. The calibration curves are linear in the range of 2.5-200.0 µg L-1. The proposed method was applied for the determination of the ß-lactams in water samples with satisfactory results. The intra-day relative standard deviations and the inter-day relative standard deviations range from 1.09% to 4.58% and from 2.95% to 7.80%, respectively. It can be concluded that this method is sensitive, convenient, and feasible for the determination of the ß-lactams in water samples.

Re-Potentiation of ß-Lactam Antibiotic by Synergistic Combination with Biogenic Copper Oxide Nanocubes against Biofilm Forming Multidrug-Resistant Bacteria.

Biofilm-associated tissue and device infection is a major threat to therapy. The present work aims to potentiate ß-lactam antibiotics with biologically synthesized copper oxide nanoparticles. The synergistic combination of amoxyclav with copper oxide nanoparticles was investigated by checkerboard assay and time-kill assay against bacteria isolated from a burn wound and a urinary catheter. The control of biofilm formation and extracellular polymeric substance production by the synergistic combination was quantified in well plate assay. The effect of copper oxide nanoparticles on the viability of human dermal fibroblasts was evaluated. The minimum inhibitory concentration and minimum bactericidal concentration of amoxyclav were 70 µg/mL and 140 µg/mL, respectively, against Proteus mirabilis and 50 µg/mL and 100 µg/mL, respectively, against Staphylococcus aureus. The synergistic combination of amoxyclav with copper oxide nanoparticles reduced the minimum inhibitory concentration of amoxyclav by 16-fold against P. mirabilis and 32-fold against S. aureus. Above 17.5 µg/mL, amoxyclav exhibited additive activity with copper oxide nanoparticles against P. mirabilis. The time-kill assay showed the efficacy of the synergistic combination on the complete inhibition of P. mirabilis and S. aureus within 20 h and 24 h, respectively, whereas amoxyclav and copper oxide nanoparticles did not inhibit P. mirabilis and S. aureus until 48 h. The synergistic combination of amoxyclav with copper oxide nanoparticles significantly reduced the biofilm formed by P. mirabilis and S. aureus by 85% and 93%, respectively. The concentration of proteins, carbohydrates, and DNA in extracellular polymeric substances of the biofilm was significantly reduced by the synergistic combination of amoxyclav and copper oxide nanoparticles. The fibroblast cells cultured in the presence of copper oxide nanoparticles showed normal morphology with 99.47% viability. No cytopathic effect was observed. Thus, the study demonstrated the re-potentiation of amoxyclav by copper oxide nanoparticles.

Oral Microbiota of the Snake Bothrops lanceolatus in Martinique.

In Martinique, Bothrops lanceolatus snakebite, although relatively uncommon (~30 cases/year), may result in serious complications such as systemic thrombosis and local infections. Infections have been hypothesized to be related to bacteria present in the snake's oral cavity. In this investigation, we isolated, identified, and studied the susceptibility to beta-lactams of bacteria sampled from the oral cavity of twenty-six B. lanceolatus specimens collected from various areas in Martinique. Microbiota from B. lanceolatus oral cavity was polymicrobial. Isolated bacteria belonged to fifteen different taxa; the most frequent being Aeromonas hydrophyla (present in 50% of the samples), Morganella morganii, Klebsiella pneumoniae, Bacillus spp., and Enterococcus spp. Analysis of antibiotic susceptibility revealed that 66.7% of the isolated bacteria were resistant to amoxicillin/clavulanate. In contrast, the majority of isolated bacteria were susceptible to the third-generation cephalosporins (i.e., 73.3% with cefotaxime and 80.0% with ceftazidime). Microbiota from B. lanceolatus oral cavity is polymicrobial with bacteria mostly susceptible to third-generation cephalosporins but rarely to amoxicillin/clavulanate. In conclusion, our findings clearly support that first-line antibiotic therapy in the B. lanceolatus-bitten patients, when there is evidence of infection, should include a third-generation cephalosporin rather than amoxicillin/clavulanate.

Determination of 21 antibiotics in sea cucumber using accelerated solvent extraction with in-cell clean-up coupled to ultra-performance liquid chromatography-tandem mass spectrometry.

An accelerated solvent extraction (ASE) with in-cell clean-up method coupled to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed to determine 21 antibiotics in sea cucumber. The analytes include 10 sulfonamides, 4 fluoroquinolones, 3 amphenicols, 2 beta-lactams, 1 lincosamide and trimethoprim. Optimal parameters of ASE method were obtained at 80 °C, 1 static cycle of 5 min with methanol/acetonitrile (1/1, v/v) using 2 g of C18 as adsorbent. Recoveries at 50.1-129.2% were achieved with RSD under 20%. Method detection limits ranged from 0.03 to 2.9 µg kg-1. Compared to the reported ultrasound-assisted extraction method, the proposed method offered comparable extraction efficiency for sulfonamides from sea cucumber, but higher for other categories of antibiotics. This validated method was then successfully applied to sea cucumber samples and 9 antibiotics were detected with the highest concentration up to 57.7 µg kg-1 for norfloxacin.

Emergencia de Providencia rettgeri NDM-1 en dos departamentos de Colombia, 2012-2013 / Emergence of Providencia rettgeri NDM-1 in two departments of Colombia, 2012-2013

Introducción: En Colombia, entre 2012 y 2013 se identificaron 19 aislamientos con NDM, de los cuales 14 correspondían a Providencia rettgeri. Métodos: Los aislamientos se identificaron con Vitek-2, y la sensibilidad antimicrobiana se evaluó por microdilución en caldo. Las carbapenemasas se determinaron fenotípicamente con test de Hodge modificado y pruebas de sinergia con EDTA/SMA y APB, genotípicamente por PCR usando iniciadores específicos para KPC, GES, IMP, VIM, OXA-48 y NDM, y las relaciones genéticas se establecieron con Diversilab. Resultados: Los aislamientos fueron resistentes a carbapenémicos, cefalosporinas de tercera generación, piperacilina-tazobactam, amicacina, gentamicina y tigeciclina, excepto a aztreonam. Todos los aislamientos fueron positivos para EDTA/SMA y NDM-1 y negativos para APB y otras carbapenemasas. Se definieron 2 grupos genéticos: grupo 1 (n = 9 aislamientos), grupo 2 (n = 4 aislamientos), y un aislamiento no relacionado genéticamente. Conclusión: Este trabajo describe la circulación de grupos de P. rettgeri productores de NDM-1 en Colombia (AU)

Introduction: In Colombia, between 2012 and 2013, 19 isolates with NDM were identified, of which 14 corresponded to Providencia rettgeri. Methods: The isolates were identified by Vitek-2, and antimicrobial susceptibility was evaluated by broth microdilution. The carbapenemase phenotypes were determined with Modified Hodge Test and synergy tests with EDTA/SMA and APB, the genotypes by PCR using specific primers for KPC, GES, IMP, VIM, OXA-48 and NDM, and genetic relationships were established with DiversiLab. Results: The isolates were resistant to carbapenems, third-generation cephalosporins, piperacillintazobactam, amikacin, gentamicin and tigecycline, except aztreonam. All isolates were positive for EDTA/SMA and NDM-1, and negative for APB and other carbapenemases. Two genetic groups, group 1 (n = 9 isolates), group 2 (n = 4 isolates) and an isolate defined as not genetically related. Conclusion: This work describes the circulating of NDM-1-producing P. rettgeri in Colombia (AU)

Validation of a new method based on salting-out assisted liquid-liquid extraction and UHPLC-MS/MS for the determination of betalactam antibiotics in infant dairy products.

In this work a new multiresidue method has been developed for the determination of 15 betalactam antibiotics in infant milk and yogurt based on follow-on milk by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The separation was achieved in 6min, using a Kinetex Biphenyl Core-Shell column (50mmx2.1mm, 1.7µm), with a mobile phase of water with 0.05% acetic acid and methanol. The analytes were detected in ESI+ with multiple reaction monitoring mode and fragmentation conditions were optimized to obtain the highest sensitivity. The use of a biphenyl bonded column allowed to obtain a satisfactory selectivity for all the studied analytes. Moreover, ion-pair salting-out assisted liquid-liquid extraction (IP-SALLE) has been proposed as sample treatment and optimized by means of experimental design. Under optimum conditions, the recoveries for fortified samples ranged from 79% to 93%, (RSD <7.5%). The limits of quantification were lower than 9.0µgkg-1, showing the high sensitivity and applicability of this fast and simple method. The results in terms of analysis time, sensitivity and accuracy showed the suitability of this procedure for the monitoring of betalactam residues in different infant foods.

Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind ß-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including ß-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like ß-lactams more efficiently.

Adsorption of Selected Antibiotics to Resins in Extracorporeal Blood Purification.

BACKGROUND/AIMS: Extracorporeal blood purification systems (EBS) use specific adsorbents for the elimination of toxins and cytokines. The aim of this study was to test different adsorbents for their ability to reduce antibiotics in parallel to extracorporeal blood purification therapy. METHODS: The in vitro adsorption experiments were carried out in human plasma with a newly established hydrophobic resin (Amberchrom CG161c) and adsorbents commercially available and approved in the clinics. The concentration of antibiotic was chosen equivalent to the recommended therapeutic dosage applied intravenously and was measured in plasma using ELISA test kits and high-performance liquid chromatography methods. RESULTS: The adsorbent that reduced all tested antibiotics in plasma close to the detection limit was the dia MARS AC250, which is an activated charcoal involved in the Molecular Adsorbents Recirculation System. CONCLUSION: For better antibiotic monitoring in sepsis treatment, further investigations have to be performed to determine the clearance rate of antibiotics by different EBS devices.

Synergistic effect of (+)-pinitol from Saraca asoca with ß-lactam antibiotics and studies on the in silico possible mechanism.

Saraca asoca bark has been used in the Ayurvedic system of medicine for female urino-genital disorders. We have recently reported the isolation and characterization of several compounds as markers to develop HPLC profiling of its methanol and aqueous methanol extracts. Now, a HPLC-PDA inactive compound, (+)-pinitol has been isolated and characterized from the bark of this medicinally important tree. Pinitol is a well known bioactive compound for a variety of biological activities, including hypoglycemic and anti-inflammatory activities. A process for the isolation of relatively good concentration of (+)-pinitol from S. asoca bark has been developed and its in vitro anti TNF-α and anti-inflammatory activities against carragenan-induced edema confirmed. While conducting experiments on the possible agonistic activity, it was found that (+)-pinitol showed up to eight fold reduction in the doses of ß-lactam antibiotics. The mechanism of its agonistic activity was studied by docking experiments which showed that different conformations of (+)-pinitol and antibiotics were actually in the same binding site with no significant change in the binding energy. These docking simulations, thus predict the possible binding mode of studied compounds and probable reason behind the synergistic effect of (+)-pinitol along with ß-lactam antibiotics.

Mass Spectral Profile for Rapid Differentiating Beta-Lactams from Their Ring-Opened Impurities.

High performance liquid chromatography tandem mass spectrometry (HPLC MS) has been widely used for ß-lactam antibiotics determination. However, its application to identify impurities of these frequently used drugs is not sufficient at present. In this job, characteristic profiles of the collision induced dissociation (CID) spectra of both ß-lactams and ring-opened ß-lactams were extracted from the MS data of six ß-lactam antibiotics and their forty-five impurities, and were confirmed by the MS data reported in the literature. These characteristics have been successfully applied to rapid differentiation of ß-lactam and ring-opened ß-lactam impurities in cefixime, cefdinir, and cefaclor. However, these characteristic profiles can only be obtained under low activating voltage. They did not display in the high energy activated CID spectra. Diagnostic fragmentations for determining the localization of double bond and substituents on the thiazine ring and the side chain were also observed. In addition, several characteristic fragmentations are hopeful to be used to differentiate the configurations of C-2 on the thiazine ring of ring-opened impurities, which is generally disadvantageous of mass spectrometry. Taken together, forty-five impurities were identified from the capsules of cefixime, cefdinir, and cefaclor.

A novel ß-lactam derivative, albactam from the flowers of Albizia lebbeck with platelets anti-aggregatory activity in vitro.

A novel ß-lactam derivative, albactam, was isolated from the alcoholic extract of the flowers of Albizia lebbeck. It showed a significant anti-aggregatory activity against adenosine diphosphate and arachidonic acid induced guinea-pigs' platelets aggregation in vitro. Six more known compounds were also isolated and fully characterized by measuring 1D and 2D NMR, two of them are the triterpenes ß-amyrin and 11α, 12α-oxidotaraxerol, two ceramide derivatives and two flavonoids, kampferol 3-O-rutinoside and rutin.

Lajollamycins, nitro group-bearing spiro-ß-lactone-γ-lactams obtained from a marine-derived Streptomyces sp.

Lajollamycins (1-4), each of which bears a spiro-ß-lactone-γ-lactam ring and a nitro-tetraene moiety, were obtained from a marine-derived Streptomyces strain isolated from the southern area of Jeju Island, Republic of Korea. The planar structures of the lajollamycins were elucidated on the basis of spectroscopic analyses by NMR, UV, IR, and MS. The absolute configuration of lajollamycin (1), the planar structure of which has been previously reported, was determined using J-based configuration analysis based on (1)H-(1)H and (1)H-(13)C coupling constants, as well as ROESY correlations, followed by the modified Mosher's method. The absolute configurations of lajollamycins B-D (2-4) were established by comparing their CD spectra with that of 1. The lajollamycins exhibited moderate inhibitory activity toward Candida albicans isocitrate lyase.

The selection of suitable columns for a reversed-phase liquid chromatographic separation of beta-lactam antibiotics and related substances via chromatographic column parameters.

The selection of RP-LC columns suitable for a particular analysis in official compendia is difficult as only a general description of the stationary phase in the description of a LC method is given. General methods to characterize RP-LC columns often assume that each of the column parameters is equally important. This can cause the user to select columns inappropriate for particular analyses. This paper focuses on the relationship between the critical peak pairs and the column parameters (H, S, A, B, and C) in the Snyder/Dolan column characterization methodology to find the key parameters influencing real separations. Some varieties of ß-lactam antibiotics and their related compounds were used as test compounds. We found column parameter A to be the most important factor affecting their separation. Parameters B and C also played an important role in some separation processes. This indicated that the hydrogen bonding of column and solute can directly affect the separation of ß-lactam antibiotics. Choosing columns for which column parameter A is near 0.1 can facilitate the ideal separations of impurities from ß-lactam antibiotics. The most suitable column for any common pharmaceutical analysis could be selected easily if the key column parameters would be given in the description of the chromatographic method. For these reasons, key column parameters should be listed in the monographs of official compendia.

Escherichia coli portador de betalactamasas de espectro extendido. Resistencia / Extended spectrum beta-lactamase - positiveEscherichia coli. Resistance

Introducción: Escherichia coli es el microorganismo que con más frecuencia se encuentra implicado en infecciones nosocomiales y comunitarias, patógeno responsable en la etiología de infecciones de vías respiratorias altas, infecciones del tracto urinario, heridas quirúrgicas, sangre o gastroenteritis. En los últimos años ha experimentado importantes cambios encontrándose un aumento de infecciones por cepas de éstos microorganismos productores de betalactamasas de espectro extendido. Objetivos: Se decide hacer este estudio retrospectivo de las muestras procesadas en el Laboratorio de Microbiología del Hospital Básico de la Defensa San Carlos (San Fernando), para conocer la frecuencia y el patrón de sensibilidad en nuestra población por gérmenes productores de betalactamasas de espectro extendido en este caso por Escherichia coli, dada la importancia de las infecciones causadas por esta bacteria y la repercusión que tiene por todo el mundo los mecanismos de resistencia. Material y Método: Se recogieron los datos de resultados obtenidos en las muestras procesadas en el Laboratorio de Microbiología durante 36 meses (Enero 2009 a Diciembre 2011), en las que se hubieran identificado cepas de Escherichia coli y de éstas las productoras de betalactamasas de espectro extendido. Resultados: Se aislaron 34 cepas de Escherichia coli productoras de betalactamasas de espectro extendido lo que supone una tasa del 5,10%. Se encontró una frecuencia mayor en el año 2010 (6,9%) que en el 2009 (2,61%), pero similar al 2011 (5,98%). Conclusión: La frecuencia de cepas Escherichia coli con betalactamasas de espectro extendido encontrada es similar a la de otros estudios realizados en España, pero la tasa de resistencia de algunos antimicrobianos como Amoxicilina/clavulánico, Cotrimoxazol y Fluorquinolonas en nuestra población es elevada (AU)

Introduction: Escherichia coli is the microorganism most frequently found in nosocomial and community infections, responsible for upper airway infections, urinary tract infections, surgical wounds, blood infections or gastroenteritis. In the last years there have been important changes with a significant increase of infections caused by these extended spectrum beta-lactamase - positive germs. Objectives: It was decided to carry out this retrospective study of the samples processed in the microbiology laboratory of the Defense Basic Hospital «San Carlos» (San Fernando) in order to determine the frequency and sensitivity pattern of extended spectrum beta-lactamase - positive strains of E. coli, given the importance of the infections produced by this bacteria and the impact worldwide of the resistance mechanisms. Material and Method: The data obtained from the samples processed in the microbiology laboratory, and in which extended spectrum beta-lactamase - positive strains of E. coli were identified, were collected during 36 months (January 2009 to December 2011). Results: 34 extended spectrum beta-lactamase- positive strains of E. coli were isolated, that is a rate of 5.10%. A higher frequency was found in the year 2010 (6.9%) than in 2009 (2.61%) but similar to 2011 (5.98%). Conclusion: The frequency of extended spectrum beta-lactamase- positive strains of E. coli was similar to that found in other studies in Spain, but for some antimicrobials as amoxicillin/ clavulanic, cotrimoxazol, fluorquinolones, in our population the resistance rate is high (AU)

Development of a direct ELISA based on carboxy-terminal of penicillin-binding protein BlaR for the detection of ß-lactam antibiotics in foods.

ß-Lactam antibiotics, including penicillins and cephalosporins, are commonly used in veterinary medicine. Illegal use and abuse of ß-lactams could cause allergy and selected bacterial resistance. BlaR-CTD, the carboxy-terminal of penicillin-recognizing protein BlaR from Bacillus licheniformis ATCC 14580, was utilized in this study to develop a receptor-based ELISA for detection and determination of ß-lactam antibiotics in milk, beef, and chicken. This assay was based on directly competitive inhibition of binding of horseradish peroxidase-labeled ampicillin to the immobilized BlaR-CTD by ß-lactams. The assay was developed as screening test with the option as semiquantitative assay, when the identity of a single type of residual ß-lactam was known. The IC50 values of 15 ß-lactam antibiotics, including benzylpenicillin, ampicillin, amoxicillin, dicloxacillin, oxacillin, nafcillin, cefapirin, cefoperazone, cefalotin, cefazolin, cefquinome, ceftriaxone, cefotaxime, cefalexin, ceftiofur and its metabolite desfuroylceftiofur were evaluated and ranged from 0.18 to 170.81 µg L(-1). Simple sample extraction method was carried out with only phosphate-buffered saline, and the recoveries of selected ß-lactam antibiotics in milk, beef, and chicken were in the range of 53.27 to 128.29 %, most ranging from 60 to 120 %. The inter-assay variability was below 30 %. Limits of detection in milk, beef, and chicken muscles with cefquinome matrix calibration were 2.10, 30.68, and 31.13 µg kg(-1), respectively. This study firstly established a rapid, simple, and accurate method for simultaneous detection of 15 ß-lactams in edible tissues, among which 11 ß-lactams controlled by European Union could be detected below maximum residue limits.

Ultrasound-assisted matrix solid phase dispersive extraction for the simultaneous analysis of ß-lactams (four penicillins and eight cephalosporins) in milk by high performance liquid chromatography with photodiode array detection.

The application of ultrasound-assisted matrix solid phase dispersive extraction for the confirmatory analysis of 12 ß-lactam antibiotics in milk by high performance liquid chromatography with photodiode array detection has been proposed herein. Four penicillins (cloxacillin, dicloxacillin, oxacillin, and amoxicillin) and eight cephalosporins (cefaclor, cefadroxil, ceftiofur, cefuroxime, cefoperazone, cefazolin, cephalexin, and cefotaxime) are effectively extracted using a mixed sorbent of Quick Easy Cheap Effective Rugged Safe technique and OASIS HLB providing a matrix free from any endogenous interference. Examined analytes were well resolved on an Inertsil ODS-3 analytical column with a mobile phase of CH(3)COONH(4) (0.05 M) and acetonitrile delivered under a gradient program. 1,7-Dimethyl-xanthine was used as internal standard. The method was validated meeting the European Legislation determining linearity, selectivity, stability, decision limit, detection capability, accuracy, precision, and ruggedness according to the Youden approach. Recoveries of all antibiotics rated from 85.0 to 115.7%, while RSD values were <12.7%. Finally, the method was successfully applied to milk samples purchased from local market.

Cationic polyelectrolyte copolymer modified polyurethane foam for flow injection preconcentration and separation of trace amounts of ß-lactam antibiotics.

A more sensitive flow injection preconcentration method has been developed for the determination of four ß-lactam antibiotics (BLAs) namely cefaclor, cefotaxime, amoxicillin and ampicillin in urine, pharmaceuticals and milk. A mini-column packed with PUF functionalized with the cationic polyelectrolyte, poly(N-chloranil N,N,N',N'-tetramethylethylene diammonium dichloride) PCTDD, was utilized for selective preconcentration. The detection limits with this method were 3.3, 3.8, 5.1 and 7.0 ng mL(-1) and enrichment factors were 38, 21, 39, and 36 for cefaclor, cefotaxime, amoxicillin and ampicillin, respectively with a sample throughput of 12 h(-1) for all BLAs. Moreover, the BLAs were successfully separated by isocratic elution using a micellar mobile phase. Application of the method developed has resulted in recovery values in the range 95%-109% (RSD≤8.7), 83%-99% (RSD≤9.7) and 91%-103% (RSD≤4.0) for urine, pharmaceuticals and milk samples, respectively.

Detección fenotípica de metalobetalactamasas en aislados clínicos de Pseudomonas aeruginosa / Phenotypic detection of metallo-beta-lactamase in clinical isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa, es considerado uno de los más importantes gérmenes hospitalarios, siendo común su aislamiento en pacientes hospitalizados, adicionalmente, este microorganismo presenta una marcada multiresistencia, lo que incrementa la mortalidad. El tratamiento de estos pacientes suele ser difícil, ya que además de su resistencia natural, Pseudomonas puede adquirir mecanismos de resistencia para prácticamente la totalidad de los antimicrobianos disponibles para su tratamiento, por lo que cada vez es más frecuente y necesario el empleo de antibióticos como los carbapenems, lo que facilita la adquisición de mecanismos de resistencia a estas drogas. El presente estudio intenta determinar la producción de metalobetalactamasas (MBL) en aislados clínicos de Pseudomonas aeruginosa, utilizando para ello dos métodos fenotípicos. Se utilizó el método del doble disco (MDD) y el test de Hodge modificado (MHT). Se analizaron 726 aislados clínicos de P. aeruginosa, el 20,11% (146) de estos fueron resistentes a imipenem (IPM) y meropenem (MEM), por lo que se les realizaron los dos métodos fenotípicos, de los 146 aislados resistentes a carbapenems, 139 fueron positivas para el MDD, mientras que 144 lo fueron para el MHT, estos dos métodos permitieron confirmar la presencia de una carbapenemasa tipo MBL en el 98,63% de los aislados de P. aeruginosa, por otra parte, cinco aislados no fueron positivos para el MDD pero si para el MHT, lo que indicaría la presencia de carbapenemasas no MBL en estos aislado, también se obtienen 2 aislados que a pesar de ser IPM y MEM resistentes fueron negativos por los dos métodos fenotípicos utilizados, esto indicaría la presencia de un mecanismo de resistencia no enzimático que confiere resistencia a carbapenems...

Pseudomonas aeruginosa is considered one of the most important hospital germs; its isolation is common in hospitalized patients. In addition, this microorganism has a marked multi-resistance, which increases mortality. Treatment of these patients is often difficult, since in addition to its natural resistance, Pseudomonas can obtain resistance mechanisms to virtually all antimicrobial drugs available for its treatment; due to this, its appearance is increasingly frequent and necessitates the use of antibiotics such as carbapenems, which facilitates the acquisition of resistance mechanisms to these drugs. This study attempts to determine the production of metallo-beta-lactamase (MBL) in clinical isolates of Pseudomonas aeruginosa, utilizing two phenotypic methods: the double disc method (MDD) and the modified Hodge test (MHT). 726 clinical isolates of P. aeruginosa were analyzed; 20.11% (146) of these were resistant to imipenem (IPM) and meropenem (MEM); 139 were positive for the MDD, while 144 were positive for the MHT. These two methods permitted confirming the presence of an MBL-type carbapenemase in 98.63% of P. aeruginosa isolates; five isolates were negative for the MDD but positive for the MHT, indicating the presence of non-MBL-type carbapenemase in these isolates. Also, 2 isolates were obtained that, despite being resistant to IPM and MEM, were negative according to the two phenotypic methods used; this would indicate the presence of a non-enzymatic resistance mechanism conferring resistance to carbapenems. The use of phenotypic methods for detecting MBL in P. aeruginosa isolates is quite an acceptable option for use in routine laboratories where specialized molecular biology tests are not available.

Assessment of ß-lactams retention in hydrophilic interaction chromatography applying Box-Behnken design.

In this paper, the retention prediction models for mixture of ß-lactam antibiotics analyzed by hydrophilic interaction chromatography (HILIC) are presented. The aim of the study was to investigate the retention behavior of some organic acids and amphoteric compounds including cephalosporins (cefotaxime, cefalexin, cefaclor, cefuroxime, and cefuroxime axetil) and penicillins (ampicillin and amoxicillin). Retention of substances with acidic functional group in HILIC is considered to be interesting since the majority of publications in literature are related to basic compounds. In the beginning of the study, classical silica columns were chosen for the retention analysis. Then, preliminary study was done and factors with the most significant influence on the retention factors were selected. These factors with the impact on the retention factors were investigated employing Box-Behnken design as a tool. On the basis of the obtained results the mathematical models were created and tested using ANOVA test and finally verified. This approach enables the presentation of chromatographic retention in many ways (three-dimensional (3-D) graphs and simple two-dimensional graphical presentations). All of these gave the possibility to predict the chromatographic retention under different conditions. Furthermore, regarding the structure of the analyzed compounds, the potential retention mechanisms in HILIC were suggested.