[Plasmid-mediated AMPc producing Proteus mirabilis in the Health Care Area of Santiago de Compostela: molecular and epidemiological analysis by rep-PCR and MALDI-TOF]. / Proteus mirabilis productor de AmpC plasmídica en el Área Sanitaria de Santiago de Compostela: prevalencia y caracterización molecular por rep-PCR y MALDI-TOF MS.

INTRODUCTION: Proteus mirabilis is an important pathogen isolated from both community-acquired and health-care associated infections. Acquired AmpC-type beta-lactamases represent an important mechanism of resistance to extended-spectrum cephalosporins and are emerging in several European countries. The objective of this work was to know the prevalence of acquired AmpC beta-lactamase producing P. mirabilis over the last three years and eight months and their clonal relationships comparing MALDI-TOF and automated rep-PCR results. METHODS: P. mirabilis isolates (n= 1,396) were obtained from routine cultures at the University Hospital Complex of Santiago de Compostela from January 2006 to August 2009. Identification to the species level and antimicrobial susceptibility testing were achieved with Vitek 2. The isolates showing intermediate or total resistance to amoxicillin-clavulanic and cefoxitin, cefotaxime or ceftazidime were selected for AmpC phenotypic detection by double-disk synergy test, and molecular confirmation by multiplex PCR. Molecular typing of the isolates was performed by automated rep-PCR and MALDI-TOF. RESULTS: For the last three years and eight months, the prevalence of AmpC-producing P. mirabilis increased from 0.17% to 4.5%, mainly associated with urinary tract infection in elderly outpatients. In all cases, plasmidic AmpC belonging to LAT/CMY lineage were detected. A high genetic variability was seen with both, rep-PCR and MALDI-TOF MS. CONCLUSIONS: AmpC-producing P. mirabilis is an emergent pathogen. The high genetic variability detected suggests that the spread of the resistance mechanism is more probable than a clone dispersion. Automated rep-PCR and MALDI-TOF MS show as fast and decisive methods for bacterial strain typing in clinical microbiology laboratories.

Proteus mirabilis productor de AmpC plasmídica en el Área Sanitaria de Santiago de Compostela: prevalencia y caracterización molecular por rep-PCR y MALDI-TOF MS / Plasmid-mediated AMPc producing Proteus mirabilis in the Health Care Area of Santiago de Compostela: molecular and epidemiological analysis by rep-PCR and MALDI-TOF

Introducción: Proteus mirabilis es un patógeno de importancia creciente tanto en las infecciones nosocomiales como en las comunitarias. La producción de AmpC plasmídica es un mecanismo de resistencia frente a cefalosporinas de espectro extendido emergente en esta bacteria por lo que se consideró de gran interés estudiar su prevalencia en nuestro Área Sanitaria así como su variabilidad genética, comparando dos métodos recientemente incorporados al mercado: MALDI-TOF y rep-PCR automatizada. Métodos: Entre enero de 2006 y agosto de 2009 se recuperaron 1.396 aislamientos de P. mirabilis a partir de los cultivos de rutina realizados en Complejo Hospitalario Universitario de Santiago de Compostela. La identificación y el antibiograma se hicieron por Vitek 2. Aquellos aislamientos con sensibilidad reducida a amoxicilina-clavulánico y a cefoxitina, cefotaxima o ceftazidima fueron seleccionados para la detección fenotípica y genotípica de AmpC plasmídica mediante sinergia con doble disco y PCR múltiple, respectivamente. La tipificación molecular se llevó a cabo, comparativamente, mediante rep-PCR automatizada y MALDI-TOF. Resultados: A lo largo de tres años y ocho meses, la prevalencia de P. mirabilis productor de AmpC pasó del 0,17% al 4,5%, mayoritariamente asociado a infección urinaria en pacientes ancianos no hospitalizados. En todos los casos, AmpC plasmídica perteneció a la familia LAT/CMY. Se observó una gran variabilidad genética entre los aislamientos tanto por rep-PCR (DiversiLab) como por MALDI-TOF MS. Conclusión: P. mirabilis productor de AmpC adquirida es un patógeno emergente. La variabilidad genética de las cepas estudiadas apunta a una dispersión de este mecanismo de resistencia más que a una diseminación clonal. Rep-PCR automatizada y MALDI-TOF se muestran como métodos rápidos y resolutivos para la tipificación molecular en los laboratorios de microbiología clínica(AU)

Introduction: Proteus mirabilis is an important pathogen isolated from both community-acquired and health-care associated infections. Acquired AmpC-type beta-lactamases represent an important mechanism of resistance to extended-spectrum cephalosporins and are emerging in several European countries. The objective of this work was to know the prevalence of acquired AmpC beta-lactamase producing P. mirabilis over the last three years and eight months and their clonal relationships comparing MALDI-TOF and automated rep-PCR results. Methods: P. mirabilis isolates (n= 1,396) were obtained from routine cultures at the University Hospital Complex of Santiago de Compostela from January 2006 to August 2009. Identification to the species level and antimicrobial susceptibility testing were achieved with Vitek 2. The isolates showing intermediate or total resistance to amoxicillin-clavulanic and cefoxitin, cefotaxime or ceftazidime were selected for AmpC phenotypic detection by double-disk synergy test, and molecular confirmation by multiplex PCR. Molecular typing of the isolates was performed by automated rep-PCR and MALDI-TOF. Results: For the last three years and eight months, the prevalence of AmpC-producing P. mirabilis increased from 0.17% to 4.5%, mainly associated with urinary tract infection in elderly outpatients. In all cases, plasmidic AmpC belonging to LAT/CMY lineage were detected. A high genetic variability was seen with both, rep-PCR and MALDI-TOF MS. Conclusions: AmpC-producing P. mirabilis is an emergent pathogen. The high genetic variability detected suggests that the spread of the resistance mechanism is more probable than a clone dispersion. Automated rep-PCR and MALDI-TOF MS show as fast and decisive methods for bacterial strain typing in clinical microbiology laboratories(AU)

Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery.

PURPOSE: To evaluate the acute and subacute toxicity of poly(anhydride) nanoparticles as carriers for oral drug/antigen delivery. METHODS: Three types of poly(anhydride) nanoparticles were assayed: conventional (NP), nanoparticles containing 2-hydroxypropyl-ß-cyclodextrin (NP-HPCD) and nanoparticles coated with poly(ethylene glycol) 6000 (PEG-NP). Nanoparticles were prepared by a desolvation method and characterized in terms of size, zeta potential and morphology. For in vivo oral studies, acute and sub-acute toxicity studies were performed in rats in accordance to the OECD 425 and 407 guidelines respectively. Finally, biodistribution studies were carried out after radiolabelling nanoparticles with (99m)technetium. RESULTS: Nanoparticle formulations displayed a homogeneous size of about 180 nm and a negative zeta potential. The LD(50) for all the nanoparticles tested was established to be higher than 2000 mg/kg bw. In the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw), no evident signs of toxicity were found. Lastly, biodistribution studies demonstrated that these carriers remained in the gut with no evidences of particle translocation or distribution to other organs. CONCLUSIONS: Poly(anhydride) nanoparticles (either conventional or modified with HPCD or PEG6000) showed no toxic effects, indicating that these carriers might be a safe strategy for oral delivery of therapeutics.

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF.

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.

Molecular imaging techniques to study the biodistribution of orally administered (99m)Tc-labelled naive and ligand-tagged nanoparticles.

PURPOSE: Study by molecular imaging the biodistribution of poly(anhydride) nanoparticles after oral administration. PROCEDURES: Poly (anhydride) nanoparticles (NP) and cyclodextrin-tagged nanoparticles (CD-NP) were radiolabelled with (99m)Tc. Radiochemical purity was measured with a double-solvent chromatography system and the absence of undesirable components was confirmed by size and polydispersion measurement of the technetium-labelled nanoparticles by photon correlation spectroscopy. Single photon emission computed tomography (SPECT) fused computed tomography (CT) in vivo molecular imaging was used for biodistribution studies in small animals. RESULTS: SPECT-CT images revealed activity only in the gastrointestinal tract. Thirteen percent of the given dose of CD-NP and 3% of the given dose of conventional NP were found in the stomach at 8 h. CONCLUSION: No evidence of translocation or distribution out of gastrointestinal tract was found. CD-NP moved significantly more slowly inside the gut than conventional NP, probably due to their physico-chemical structure that allows stronger interactions with the gut mucosa.

Bioadhesive properties and biodistribution of cyclodextrin-poly(anhydride) nanoparticles.

This work describes the preparation, characterization and evaluation of the nanoparticles formed by the copolymer of methyl vinyl ether and maleic anhydride (Gantrez) AN) and cyclodextrins, including beta-cyclodextrin (CD) hydroxypropyl-beta-cyclodextrin (HPCD) and 6-monodeoxy-6-monoamino-beta-cyclodextrin (NHCD). The cyclodextrin-poly(anhydride) nanoparticles were prepared by a solvent displacement method and characterized by measuring the size, zeta potential, morphology and composition. For bioadhesion studies, nanoparticles were fluorescently labelled with rhodamine B isothiocianate (RBITC). For in vivo imaging biodistribution studies, (99m)Tc-labelled nanoparticles were used. Nanoparticles displayed a size of about 150nm and a cyclodextrin content which was found optimal under the following experimental conditions: cyclodextrin/poly(anhydride) ratio of 0.25 by weight, 30min of incubation time between the cyclodextrin and the polymer. Moreover, the oligosaccharide content was higher with CD than with NHCD and HPCD. Overall, cyclodextrin-poly(anhydride) nanoparticles displayed homogeneous bioadhesive interactions within the gut. The intensity of these interactions was higher than for control nanoparticles. The high bioadhesive capacity was observed for HPCD-NP and NHCD-NP which can be related with their rough morphology and, thus, a higher specific surface than for smooth nanoparticles (CD-NP). Finally, from in vivo studies, no evidence of translocation of distribution to other organs was observed when these nanoparticles were orally administered.