Effect of focal adhesion kinase inhibition on osteoblastic cells grown on titanium with different topographies

Abstract Objective The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS). Methodology Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively. Results FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces. Conclusions Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

La leptina promueve la expresión de Hic-5 y la formación de puntos de actina por la vía dependiente de FAK-Src en células epiteliales mamarias MCF10A / Leptin induced Hic-5 expression and actin puncta formation by the FAK/Src-dependent pathway in MCF10A mammary epithelial cells

Resumen Introducción. La leptina es una hormona secretada por los adipocitos que se ha relacionado con el proceso de la transición de epitelio a mesénquima (Epithelial- Mesenchymal Transition, EMT). Promueve la migración e invasión de las células del epitelio mamario mediante la activación de las cinasas FAK y Src, un complejo regulador de vías de señalización que favorecen la expresión de las proteínas relacionadas con la formación de estructuras proteolíticas implicadas en la invasión y progresión del cáncer. Recientemente, se ha descrito que la sobreexpresión y activación de la proteína Hic-5 durante el mencionado proceso de transición, favorece la formación de los puntos de actina (indicativa de la formación y funcionalidad de los invadopodios), lo cual promueve la degradación local de los componentes de la matriz extracelular y la metástasis del cáncer. Objetivos. Evaluar el papel de las cinasas FAK y Src sobre la expresión y localización subcelular de Hic-5 y la formación de puntos de actina inducida por la leptina en la línea celular MCF10A de epitelio mamario no tumoral. Materiales y métodos. Se utilizaron los inhibidores específicos de la FAK (PF-573228) y la Src (PP2) para evaluar el papel de ambas cinasas en los niveles de expresión y localización subcelular de la proteína Hic-5 mediante Western blot e inmunofluorescencia, así como la formación de puntos de actina mediante la tinción con faloidina-TRITC en células MCF10A estimuladas con leptina. Resultados. La leptina indujo el incremento en la expresión de Hic-5 y la formación de puntos de actina. El tratamiento previo con los inhibidores de las cinasas FAK (PF-573228) y Src (PP2), promovió la disminución en la expresión de Hic-5 y de los puntos de actina en la línea celular MCF10A de epitelio mamario no tumoral. Conclusión. La leptina indujo la expresión y la localización perinuclear de Hic-5 y la formación de puntos de actina mediante un mecanismo dependiente de la actividad de las cinasas FAK y Src en las células MCF10A.

Abstract Introduction: Leptin is a hormone secreted by adipocytes that has been associated with the epithelial-mesenchymal transition (EMT). Additionally, leptin promotes the migration and invasion of mammary epithelial cells through the activation of FAK and Src kinases, which are part of a regulatory complex of signaling pathways that promotes the expression of proteins related to the formation of proteolytic structures involved in the invasion and progression of cancer. Recently, overexpression and activation of Hic-5 during the EMT have been shown to induce the formation of actin puncta; these structures are indicative of the formation and functionality of invadopodia, which promote the local degradation of extracellular matrix components and cancer metastasis. Objective: To evaluate the role of FAK and Src kinases in the expression of Hic-5 during the epithelial-mesenchymal transition induced by leptin in MCF10A cells. Materials and methods: We used specific inhibitors of FAK (PF-573228) and Src (PP2) to evaluate Hic-5 expression and subcellular localization by Western blot and immunofluorescence assays and to investigate the formation of actin puncta by epifluorescence in MCF10A cells stimulated with leptin. Results: Leptin induced an increase in Hic-5 expression and the formation of actin puncta. Pretreatment with inhibitors of FAK (PF-573228) and Src (PP2) promoted a decrease in Hic-5 expression and actin puncta formation in the non-tumorigenic mammary epithelial cell line MCF10A. Conclusion: In MCF10A cells, leptin-induced Hic-5 expression and perinuclear localization, as well as the formation of actin puncta through a mechanism dependent on the kinase activity of FAK and Src.

Marcadores de resistencia plasmídica a quinolonas qnr en aislamientos clínicos de enterobacterias productoras de betalactamasas CTX-M en Lima, Perú / Markers of plasmid resistance to qnr quinolones in clinical isolates of CTX-M beta-lactamases-producing enterobacteria in Lima, Peru

RESUMEN Con el objetivo de reportar marcadores de resistencia plasmídica a quinolonas qnr en aislamientos clínicos de enterobacterias productoras de betalactamasas CTX-M, se realizó un estudio descriptivo, con aislamientos del cepario del proyecto TO-06/09 del Instituto Nacional de Salud del Niño. Se recuperaron 138 aislamientos. La susceptibilidad antimicrobiana se determinó por el método de disco difusión y la identificación de genes por reacción en cadena de la polimerasa. De los 138 aislados, 67 (48,5%) fueron positivos para proteínas qnr por el método genotípico. De los cuales 38 (56,7%) presentaron determinantes qnrB y 48 (71,6%) determinantes qnrS. Ningún aislado presentó determinantes qnrA. Se detectó determinantes qnr en aislamientos que presentaban betalactamasas CTX-M en una población no expuesta.

ABSTRACT Aimed at reporting markers of plasmid resistance to qnr quinolones in clinical isolates of CTX-M beta-lactamase-producing enterobacteria, a descriptive study was conducted with isolates from the strain repository of TO-06/09 project of the National Children´s Health Institute. 138 isolates were recovered. Antimicrobial susceptibility was determined by the diffusion disk method, and gene identification by polymerase chain reaction. Of the 138 isolates, 67 (48.5%) were genotypically positive for qnr proteins; of these, 38 (56.7%) had qnrB determinants and 48 (71.6%) had qnrS determinants. No isolate presented qnrA determinants. qnr determinants were detected in isolates containing CTX-M beta-lactamases in a non-exposed population.

Susceptibilidad antimicrobiana en muestras urogenitales de ureaplasma urealyticum, el principal agente en infección intramaniótica / Antimicrobial susceptibility in urogenital samples of ureaplasma urealyticum, the main agent in intramaniotic infection

RESUMEN OBJETIVO: Ureaplasma urealyticum es el agente más frecuentemente aislado en infección intraamniótica. Los macrólidos son los antimicrobianos de primera elección en embarazadas. Se ha descrito el aumento de resistencia, pudiendo limitar las opciones terapéuticas durante la gestación. El propósito del estudio es evaluar susceptibilidad antimicrobiana de Ureaplasma urealyticum aislado en mujeres en edad fértil, que se atienden en Clínica Alemana Temuco, Araucanía, Chile. METODO: Se estudian todas las muestras de orina y flujo vaginal para cultivo de U. urealyticum, de pacientes entre 18 y 40 años, recibidas en el Laboratorio de Microbiología Clínica Alemana Temuco, en período Abril 2013 a Enero 2015. Se procesan las muestras con kit Mycoplasma IST 2 de Biomerieux. En las que resultan positivas, se estudia susceptibilidad a macrólidos, tetraciclinas y quinolonas. RESULTADOS: 426 muestras de orina y flujo vaginal (390 pacientes). 197 pacientes resultaron positivas para U. urealyticum. (50,5%). La susceptibilidad fue 88,4% (174 pctes) a Eritromicina, 87,9% (173 pctes) a Claritromicina y 91,9% (181 pctes) a Azitromicina (NS). 15 de 197 pacientes (7,6%) fueron resistentes a los 3 macrólidos. La susceptibilidad a Quinolonas fue 55,3% a Ciprofloxacino, y 94% a Ofloxacino. El 100% resultó susceptible a Tetraciclinas. CONCLUSIONES: Cerca del 10% de U. urealyticum aislados en nuestra serie son resistentes a macrólidos, contribuyendo a la no erradicación de la infección en tratamientos empíricos. Dentro de ellos, azitromicina aparece con la mayor efectividad. El aumento de resistencia limitará opciones terapéuticas, con gran impacto perinatal en futuro. La vigilancia de susceptibilidad en cada hospital es fundamental para elección terapéutica.

ABSTRACT INTRODUCTION: Ureaplasma urealyticum is the most frequently isolated microorganism in intra-amniotic infection. The macrolides are the first choice antimicrobials for treat this infection in pregnancy. The increasing resistance has been described worldwide, seriously limiting therapeutic options in pregnancy. The aim of the study is to evaluate antimicrobial susceptibility of U. urealyticum aislated in fertile-age women in Clínica Alemana Temuco, Araucania region, Chile. METHOD: Urine and vaginal samples were analyzed for U. urealyticum, from every 18 to 40 years old patients, received at Microbiology Laboratory of Clínica Alemana Temuco, between April 2013 to January 2015. The samples are processed with Mycoplasma IST 2 kit of Biomerieux. If they became positives, susceptibility to macrolides, tetracyclines and quinolones was studied. RESULTS: 426 urine and vaginal samples were collected (390 patients). 197 patients were positive for U. urealyticum (50.5%). The susceptibility was 88.4% (174 pts) to Erythromicyn, 87.9% (173 pts) to Clarithromycin and 91.9% (181 pts) to Azithromycin (NS). Resistance to all macrolides was observed in 15 out of 197 patients (7.6%). The susceptibility to Quinolones was 55.3% to Ciprofloxacin, and 94% to Ofloxacin. The 100% was susceptible to Tetracyclines. DISCUSSION: Near to 10% of isolated Ureaplasma spp in our serie were resistant to some macrolide, being a factor for failing to eradicate the infection in empirical treatment. Azithromycin was the most effective. The increasing resistance will limit therapeutic options, with great perinatal impact in the future. Susceptibility surveillance in each hospital is very important for therapeutic options.

Genes qnr en Enterobacteriaceae aisladas en un hospital de Venezuela / qnr genes in Enterobacteriaceae isolated from at a hospital in Venezuela

Resumen Introducción: La resistencia de enterobacterias a quinolonas se ha difundido por el mundo, fenómeno presente también en Venezuela. El mecanismo de esta resistencia pudiera estar mediado por genes incluidos en el cromosoma bacteriano o transmitirse en el interior de plásmidos. Objetivo: Evaluar la resistencia a quino-lonas, codificada por genes qnr, presentes en cepas de enterobacterias, aisladas en el Hospital Universitario de Cumaná, Venezuela. Métodos: A las cepas obtenidas se les realizaron pruebas de susceptibilidad antimicrobiana a quinolonas, β-lactámicos y aminoglucósidos. La presencia del gen qnr se determinó por RPC. Las enterobacterias portadoras del gen qnr fueron sometidas al proceso de conjugación bacteriana para comprobar su capacidad de transferencia. A las transconjugantes obtenidas se les realizó pruebas de susceptibilidad antimicrobiana y RPC para comprobar la transferencia de los genes. Resultados: Se encontraron elevados porcentajes de resistencia antimicrobiana a quinolonas y betalactámicos. El 33,6% de las cepas eran portadoras del gen qnrB, y 0,9% del gen qnrA. Se obtuvieron 23 cepas transconjugantes; de éstas, 20 portaban el gen qnrB, no se observó la presencia de qnrA. Discusión: En conclusión, el elevado porcentaje de genes qnr encontrado en las enterobacterias aisladas, y comprobada la presencia de éstos en plásmidos transferibles, complica la aplicación de tratamientos basados en quinolonas y fluoroquinolonas, por lo que es recomendable el uso racional de estos antimicrobianos, y proponer la rotación de la terapia antimicrobiana, a fin de evitar la selección de cepas resistentes.

Background: Enterobacteria resistant to quinolones is increasing worldwide, including Venezuela. The mechanism for this resistance could be due to genes included in the chromosome or in transmissible plasmids. Aim: To evaluate the resistance to quinolones, coded by qnr genes present in enterobacteria species, isolated in the University Hospital of Cumana, Venezuela. Methods: Antimicrobial susceptibility tests to quinolones, beta-lactams and aminoglycosides were carried out to all the isolates. The presence of qnr genes were determined by PCR. The isolates carrying the qnr genes were used for bacterial conjugation tests to determine the presence of transferable plasmids. Antimicrobial susceptibility tests and PCR were carried out in the transconjugants to verify the transfer of the genes. Results: High levels of antimicrobial resistance to quinolones and beta-lactams were found among the isolates. We found that 33.6% of the isolates carry the qnrB gene and 0.9% qnr A gene. Of the 23 transconjugants, 20 showed to have qnrB gene, but none qnrA. Discussion: We concluded that the high frequency of qnr genes found in the enterobacteria isolates and their presence on transferable plasmids, complicate the use of quinolones for the treatment of bacterial infections, thus, a treatment plan should be designed with the rational use and the rotation of different types of antimicrobials, in order to avoid the selection of increasingly resistant strains.

Primer relevamiento de marcadores de resistencia a antibióticos en Enterobacteriaceae en Cochabamba, Bolivia / First survey on antibiotic resistance markers in Enterobacteriaceae in Cochabamba, Bolivia

A molecular survey was conducted in Cochabamba, Bolivia, to characterize the mechanism involved in the resistance to clinically relevant antibiotics. Extended Spectrum β-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) markers were investigated in a total of 101 oxyimino-cephalosporin-resistant enterobacteria recovered from different health centers during four months (2012-2013). CTX-M enzymes were detected in all isolates, being the CTX-M-1 group the most prevalent (88.1%). The presence of blaOXA-1 was detected in 76.4% of these isolates. A high quinolone resistance rate was observed among the included isolates. The aac(6′)-Ib-cr gene was the most frequent PMQR identified (83.0%). Furthermore, 6 isolates harbored the qnrB gene. Interestingly, qepA1 (6) and oqxAB (1), were detected in 7 Escherichia coli, being the latter the first to be reported in Bolivia. This study constitutes the first molecular survey on resistance markers in clinical enterobacterial isolates in Cochabamba, Bolivia, contributing to the regional knowledge of the epidemiological situation. The molecular epidemiology observed herein resembles the scene reported in South America.

Se llevó a cabo un relevamiento molecular de la resistencia a antibióticos de importancia clínica en aislamientos recuperados en Cochabamba, Bolivia. Se estudiaron los genes codificantes de β-lactamasas de espectro extendido y de resistencia a quinolonas de localización plasmídica (PMQR) en un total de 101 aislamientos de enterobacterias resistentes a oximinocefalosporinas recuperados en distintos centros de salud, durante 4 meses (2012-2013). En todos ellos se detectó la presencia de cefotaximasas, las CTX-M grupo 1 fueron las más prevalentes (88,1%). La presencia de blaOXA-1 se detectó en el 76,4% de estos aislamientos. Se observó una elevada proporción de aislamientos resistentes a quinolonas. El gen aac(6′)-Ib-cr fue el determinante PMQR más frecuentemente identificado (83%). Además, 6 aislamientos resultaron ser portadores de qnrB. Por otro lado, cabe remarcar que 7 Escherichia coli presentaron qepA1 (6) y oqxAB (1); se documenta así por primera vez la presencia de oqxAB en Bolivia. Este estudio constituye el primer relevamiento de marcadores de resistencia en aislamientos clínicos de enterobacterias en Cochabamba, Bolivia; de este modo se contribuye al conocimiento regional de la situación epidemiológica, la cual presenta un escenario similar al observado en el resto de Latinoamérica.

Prevalencia de la resistencia de Mycobacterium tuberculosis a quinolonas y fármacos inyectables en Colombia, 2012-2013 / Prevalence of Mycobacterium tuberculosis resistance to quinolones and injectables in Colombia, 2012-2013

Resumen Introducción: La tuberculosis es un problema de salud pública a nivel mundial. En 2014, la Organización Mundial de la Salud estimó que se habían presentado 9,6 millones de casos nuevos y 480.000 multirresistentes. La evaluación de la resistencia a fármacos inyectables y a quinolonas se introdujo hace pocos años, por lo cual no se conoce su prevalencia. Objetivo: Determinar la prevalencia de la resistencia a amicacina, kanamicina, capreomicina y ofloxacina en casos de tuberculosis resistente a isoniacida, rifampicina o a ambas, entre 2012 y 2013. Materiales y métodos: Se hizo un estudio de corte transversal con 489 aislamientos resistentes a isoniacida o rifampicina. Las pruebas de sensibilidad se hicieron con la técnica Bactec MGITTM. Para el análisis de la proporción de la resistencia, los casos se agruparon según el antecedente de tratamiento con medicamentos de segunda línea. Resultados: En los 438 casos nuevos, la resistencia global a la kanamicina fue mayor (7,1 %; IC95% 4,6-9,6); en los 51 casos previamente tratados, dicha resistencia fue de 27,5 % (IC95% 14,2-40,7). La resistencia global fue mayor en casos con antecedentes de tratamiento con quinolonas y fármacos inyectables. Se encontraron siete casos de tuberculosis extremadamente resistente. Conclusión: El estudio evidenció la presencia de resistencia a fármacos de segunda línea en personas con tuberculosis farmacorresistente sin tratamiento previo o tratadas previamente con quinolonas o fármacos inyectables, estos últimos con mayor porcentaje de resistencia. En consecuencia, es esencial practicar rutinariamente las pruebas de sensibilidad y el análisis de esta información.

Abstract Introduction: Tuberculosis is a health problem worldwide. The World Health Organization estimated 9.6 million new cases and 480,000 multirresistant cases for 2014. The assessment of resistance to quinolones and injectables was implemented only a few years ago, so its prevalence is not known. Objective: To determine the prevalence of resistance to amikacin, capreomycin and ofloxacin in cases of tuberculosis resistant to isoniazid and/or rifampin during 2012-2013. Materials and methods: This was a cross-sectional study of 489 isolates resistant to isoniazid and/or rifampin. We used the Bactec MGITTM technique for susceptibility tests. For analyzing the rate of resistance, we grouped cases according to the history of treatment with second line drugs. Results: In the 438 new cases, the drug that showed greater overall resistance was kanamycin with 7.1 % (95% CI: 4.6 to 9.6). In 51 previously treated cases, this highest resistance was 27.5 % (95% CI: 14.2 to 40.7). The overall resistance was higher in cases with a history of treatment with quinolones and injectables. We found seven cases of extremely resistant tuberculosis. Conclusion: This study demonstrates the presence of resistance to second line drugs in people with drug-resistant tuberculosis with and without previous treatment with quinolones and/or injectables, these latter having a higher percentage of resistance. For that reason, it is essential to perform susceptibility testing and analyze this information routinely.

Effects of indacaterol versus tiotropium on exercise tolerance in patients with moderate COPD: a pilot randomized crossover study / Efeitos do indacaterol versus tiotrópio na tolerância ao exercício em pacientes com DPOC moderada: estudo cruzado randomizado piloto

Abstract Objective: To compare a once-daily long-acting β2 agonist (indacaterol 150 µg) with a once-daily long-acting anticholinergic (tiotropium 5 µg) in terms of their effects on exercise endurance (limit of tolerance, Tlim) in patients with moderate COPD. Secondary endpoints were their effects on lung hyperinflation, exercise-related dyspnea, and daily-life dyspnea. Methods: This was a randomized, single-blind, crossover pilot study involving 20 patients (mean age, 60.9 ± 10.0 years; mean FEV1, 69 ± 7% of predicted). Spirometric parameters, Transition Dyspnea Index scores, Tlim, and exertional dyspnea were compared after three weeks of each treatment (with a one-week washout period between treatments). Results: Nineteen patients completed the study (one having been excluded because of COPD exacerbation). Improvement in Tlim from baseline tended to be greater after treatment with tiotropium than after treatment with indacaterol (96 ± 163 s vs. 8 ± 82 s; p = 0.06). Tlim significantly improved from baseline after treatment with tiotropium (having increased from 396 ± 319 s to 493 ± 347 s; p = 0.010) but not after treatment with indacaterol (having increased from 393 ± 246 to 401 ± 254 s; p = 0.678). There were no differences between the two treatments regarding improvements in Borg dyspnea scores and lung hyperinflation at "isotime" and peak exercise. There were also no significant differences between treatments regarding Transition Dyspnea Index scores (1.5 ± 2.1 vs. 0.9 ± 2.3; p = 0.39). Conclusions: In patients with moderate COPD, tiotropium tends to improve Tlim in comparison with indacaterol. No significant differences were observed between the two treatments regarding their effects on lung hyperinflation, exercise-related dyspnea, and daily-life dyspnea. Future studies, including a larger number of patients, are required in order to confirm our findings and explore mechanistic explanations. (ClinicalTrials.gov identifier: ...

RESUMO Objetivo: Comparar um β2-agonista de longa duração administrado uma vez por dia (indacaterol 150 µg) a um anticolinérgico de longa duração administrado uma vez por dia (tiotrópio 5 µg) quanto a seus efeitos na resistência ao exercício (limite de tolerância, Tlim) em pacientes com DPOC moderada. Os desfechos secundários foram seus efeitos na hiperinsuflação pulmonar, na dispneia causada pelo exercício e na dispneia na vida diária. Métodos: Estudo piloto randomizado cruzado e simples cego com 20 pacientes (média de idade: 60,9 ± 10,0 anos; média do VEF1: 69 ± 7% do previsto). Parâmetros espirométricos, pontuação no Transition Dyspnea Index, Tlim e dispneia aos esforços foram comparados após três semanas de cada tratamento (com uma semana de intervalo entre os tratamentos). Resultados: Dezenove pacientes completaram o estudo - um foi excluído por causa de exacerbação da DPOC. A melhora no Tlim tendeu a ser maior com tiotrópio do que com indacaterol (96 ± 163 s vs. 8 ± 82 s; p = 0,06). Em comparação com os valores basais, o Tlim melhorou significativamente com tiotrópio (aumentando de 396 ± 319 s para 493 ± 347 s; p = 0,010), mas não com indacaterol (aumentando de 393 ± 246 para 401 ± 254 s; p = 0,678). Não houve diferença entre os tratamentos quanto à melhora na pontuação na escala de dispneia de Borg e na insuflação pulmonar no "isotempo" e no pico do exercício. Também não houve diferenças significativas entre os tratamentos quanto à pontuação no Transition Dyspnea Index (1,5 ± 2,1 vs. 0,9 ± 2,3; p = 0,39). Conclusões: Em pacientes com DPOC moderada, o tiotrópio tende a melhorar o Tlim em comparação com o indacaterol. Não houve diferenças significativas entre os tratamentos quanto a seus efeitos na insuflação pulmonar, na dispneia durante o exercício e na dispneia na vida diária. São necessários mais estudos, com um número maior de pacientes, para confirmar nossos achados e explorar explicações mecanicistas. (ClinicalTrials.gov ...

High prevalence of plasmid-mediated quinolone resistance determinants in Enterobacter cloacae isolated from hospitals of the Qazvin, Alborz, and Tehran provinces, Iran

Abstract: INTRODUCTION: Plasmid-mediated quinolone resistance (PMQR) is a growing clinical concern worldwide. The main aims of this study were to detect qnr-encoding genes and to evaluate the clonal relatedness of qnr-positive Enterobacter cloacae isolates. METHODS: A total of 116 E. cloacae isolates that were not susceptible to quinolone were obtained from seven hospitals in Tehran, five hospitals in Qazvin, and two hospitals in Karaj (Iran). Bacterial identification was performed using standard laboratory methods and API 20E strips. Quinolone resistance was determined using the Kirby-Bauer disk diffusion method according to the Clinical Laboratory Standards Institute (CLSI) guidelines. PCR and sequencing were employed to detect qnrA, qnrB, and qnrS genes, and clonal relatedness was assessed using the enterobacterial repetitive intergenic consensus (ERIC)-PCR method. RESULTS: In total, 45 (38.8%) and 71 (61.2%) of isolates showed high- and low-level quinolone resistance, respectively, and qnr-encoding genes were detected in 70 (60.3%) of them. qnrB1 [45 (38.8%) isolates] was the most commonly detected gene, followed by qnrS1 [28 (24.1%) isolates] and qnrB4 [18 (15.5%) isolates] either alone or in combination with other genes. The results of the ERIC-PCR revealed that 53 (75.7%) qnr-positive isolates were genetically unrelated. CONCLUSIONS: This study describes, for the first time, the high prevalence of the qnrB1, qnrS1, and qnrB4 genes among E. cloacae isolates in Iran. The detection of qnr genes emphasizes the need for establishing tactful policies associated with infection control measures in hospital settings in Iran.

Characterization of quinolone resistance in Salmonella spp. isolates from food products and human samples in Brazil

Abstract Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance in Salmonella spp. and its association with fluoroquinolone susceptibility in Brazil. A total of 129 NTS isolates (samples from human origin, food from animal origin, environmental, and animal) grouped as from animal (n = 62) and human (n = 67) food were evaluated between 2009 and 2013. These isolates were investigated through serotyping, antimicrobial susceptibility testing, and the presence of plasmid-mediated quinolone resistance (PMQR) genes (qnr, aac(6')-Ib) and associated integron genes (integrase, and conserved integron region). Resistance to quinolones and/or fluoroquinolones, from first to third generations, was observed. Fifteen isolates were positive for the presence of qnr genes (8 qnrS, 6 qnrB, and 1 qnrD) and twenty three of aac(6')-Ib. The conserved integron region was detected in 67 isolates as variable regions, from ±600 to >1000 pb. The spread of NTS involving PMQR carriers is of serious concern and should be carefully monitored.

Resistencia bacteriana en infecciones del tracto urinario de origen comunitario / Bacterial resistance in urinary tract infections of community origin

Antecedentes: Las infecciones del tracto urinario (ITU) representan una de las principales causas de morbilidad. Recientemente ha surgido preocupación por la creciente tasa de resistencia en los patógenos causantes, y el origen comunitario de la infección ya no garantiza susceptibilidad antibiótica. Objetivo: Determinar la tasa de los diferentes patrones de resistencia en infecciones de vías urinarias comunitarias, junto a los principales factores de riesgo asociados. Metodología: Estudio descriptivo prospectivo donde se incluyó a todos los pacientes que consultaron a la emergencia de Medicina Interna con síndrome clínico de ITU y en quienes se demostró la etiología infecciosa mediante urocultivo. Se documentaron las comorbilidades y el consumo previo de antibióti-cos y se buscó la significancia estadística de esto sobre el desarrollo de resistencia.Resultados: Se incluyeron 100 sujetos a estudio, 90% de las infecciones se debieron a E coli y K pneu-moniae, se documentó resistencia en 68% de los casos, 41% resistentes a quinolonas, 27% ESBL(+) y 17% a cefalosporinas de 3ra generación. Se encontró que la DM era la comorbilidad más común (46%) y representó un factor de riesgo para el desarrollo de resistencia a cefalosporinas (p=0.031) y cepas ESBL(+) (p=0.045). El consumo previo de aminopenicilinas, cefalosporinas y quinolonas condicionó para el desarrollo de diferentes resistencias.Conclusiones: Se encontró relación significativa entre la DM y el desarrollo de resistencia bacteriana, al igual que el uso previo de quinolonas, cefalosporinas y aminopenicilinas. El 68% de las cepas mos-tró alguna resistencia. Hay elevada tasa de resistencia a quinolonas, cefalosporinas y cepas ESBL(+). (AU)

Urinary tract infections (UTI's) represent one of the leading causes of disease worldwide. In recent years, great concern has emerged regarding the growing resistance rate among the different bacteria responsible for these infections, and community acquired infections no longer guarantee antibiotic susceptibility. Objective: To determine the rate of antibiotic resistance patterns in community acquired UTI's, and to identify the associated risk factors.Methods: This was a prospective study performed in the Emergency Room of a Reference Hospital in Guatemala City. Every patient that consulted with urinary symptoms and whose diagnosis was con-firmed by urinary culture was included. The patients were questioned about past medical history and previous antibiotic use. The statistical analysis was done using the IBM SPSS ™ software.Results: One-hundred patients were included. 90% were caused by E coli and K pneumoniae. Any resistance was detected in 68% of the cases, 41% were resistant to fluoroquinolones, 27% had ESBL enzymes, and 17% were found to be resistant to 3rd generation cephalosporines. Diabetes was pre-sent in 46% of the patients and prooved to be an important risk factor for the development of cepha-losporines (p=0.031) and ESBL(+) (p=0.045) resistance. Previous use of fluoroquinolones, cephalos-porines and aminopenicillins determined the development of certain resistance patterns.Conclusion: A statistical significant relationship was found between diabetes and ATB resistance as well as with previos ATB consumption and the latter. 68% showed any resistance. A high resistance pattern to quinolones, cephalosporins ESBL+ was documented.(AU)

Susceptibility to β-lactams and quinolones of Enterobacteriaceae isolated from urinary tract infections in outpatients

Abstract The antibiotic susceptibility profile was evaluated in 71 Enterobacteriaceae isolates obtained from outpatient urine cultures in July 2010 from two health institutions in Santa Fe, Argentina. The highest rates of antibiotic resistance were observed for ampicillin (AMP) (69%), trimethoprim/sulfamethoxazole (TMS) (33%), and ciprofloxacin (CIP) (25%). Meanwhile, 21% of the isolates were resistant to three or more tested antibiotics families. Thirty integron-containing bacteria (42.3%) were detected, and a strong association with TMS resistance was found. Third generation cephalosporin resistance was detected in only one Escherichia coli isolate, and it was characterized as a blaCMY-2 carrier. No plasmid-mediated quinolone resistance (PMQR) was found. Resistance to fluoroquinolone in the isolates was due to alterations in QRDR regions. Two mutations in GyrA (S83L, D87N) and one in ParC (S80I) were observed in all CIP-resistant E. coli. It was determined to be the main phylogenetic groups in E. coli isolates. Minimum Inhibitory Concentration (MIC) values against nalidixic acid (NAL), levofloxacin (LEV), and CIP were determined for 63 uropathogenic E. coli isolates as MIC50 of 4 μg/mL, 0.03125 μg/mL, and 0.03125 μg/mL, respectively, while the MIC90 values of the antibiotics were determined as 1024 μg/mL, 64 μg/mL, and 16 μg/mL, respectively. An association between the phylogenetic groups, A and B1 with fluoroquinolone resistance was observed. These results point to the importance of awareness of the potential risk associated with empirical treatment with both the families of antibiotics.

Quinolonas: Perspectivas actuales y mecanismos de resistencia / Quinolones: Nowadays perspectives and mechanisms of resistance

Quinolones are a family of synthetic broad-spectrum antimicrobial drugs whose target is the synthesis of DNA. They directly inhibit DNA replication by interacting with two enzymes; DNA gyrase and topoisomerase IV. They have been widely used for the treatment of several community and hospital acquired infections, in the food processing industry and in the agricultural field, making the increasing incidence of quinolone resistance a frequent problem associated with constant exposition to diverse microorganisms. Resistance may be achieved by three non-exclusive mechanisms; through chromosomic mutations in the Quinolone Resistance-Determining Regions of DNA gyrase and topoisomerase IV, by reducing the intracytoplasmic concentrations of quinolones actively or passively and by Plasmid-Mediated Quinolones-Resistance genes, [Qnr determinant genes of resistance to quinolones, variant gene of the aminoglycoside acetyltransferase (AAC(6')-Ib-c)] and encoding genes of efflux pumps (qepA and oqxAB)]. The future of quinolones is uncertain, however, meanwhile they continue to be used in an irrational way, increasing resistance to quinolones should remain as an area of primary priority for research.

Las quinolonas son un grupo de antimicrobianos sintéticos de amplio espectro, cuyo objetivo es la síntesis del ADN. Inhiben directamente su replicación al interactuar con dos enzimas; ADN girasa y topoisomerasa IV. Se han utilizado ampliamente para el tratamiento de infecciones intra y extra-hospitalarias, en el campo de la agricultura y en el procesamiento de alimentos, lo que hace que el incremento de resistencia a quinolonas sea un problema cada vez más frecuente, asociado a la constante exposición de diversos microorganismos. La resistencia puede alcanzarse mediante tres mecanismos no excluyentes entre sí; a través de mutaciones cromosómicas en genes codificantes que afectan las regiones determinantes de resistencia a quinolonas de ADN girasa y topoisomerasa IV, al reducir las concentraciones intracitoplásmicas de quinolonas de manera activa o pasiva y por genes de resistencia a quinolonas mediados por plásmidos [genes de resistencia a quinolonas determinates de qnr, gen variante de la aminoglucósido acetil transferasa (AAC(6’)-lb-cr) y genes codificadores de bombas de eflujo (qepAy oqxAB)]. El futuro de las quinolonas es incierto; sin embargo, mientras continúen empleándose para el manejo de infecciones en el ser humano, el incremento de resistencia a quinolonas debe permanecer como un área de importancia primaria para la investigación.

Risk factors associated with structural postural changes in the spinal column of children and adolescents / Fatores de risco associados a alterações posturais estruturais da coluna vertebral em crianças e adolescentes

OBJECTIVE: To investigate the association between behavioral risk factors, specifically postural habits, with the presence of structural changes in the spinal column of children and adolescents. METHODS: 59 students were evaluated through the self-reporting Back Pain and Body Posture Evaluation Instrument and spinal panoramic radiographic examination. Spine curvatures were classified based on Cobb angles, as normal or altered in the saggital plane and as normal or scoliotic in the frontal plane. Data were analyzed using SPSS 18.0, based on descriptive statistics and chi-square association test (a=0,05). RESULTS: The prevalence of postural changes was 79.7% (n=47), of which 47.5% (n=28) showed frontal plane changes and 61% (n=36) sagital plane changes. Significant association was found between the presence of thoracic kyphosis and female gender, practice of physical exercises only once or twice a week, sleep time greater than 10 hours, inadequate postures when sitting on a seat and sitting down to write, and how school supplies are carried. Lumbar lordosis was associated with the inadequate way of carrying the school backpack (asymmetric); and scoliosis was associated wuth the practice of competitive sports and sleep time greater than 10 hours. CONCLUSIONS: Lifestyle may be associated with postural changes. It is important to develop health policies in order to reduce the prevalence of postural changes, by decreasing the associated risk factors. .

OBJETIVO: Verificar se existe associação de fatores de risco comportamentais, especificamente hábitos posturais, com a presença de alteração postural estrutural na coluna vertebral de crianças e adolescentes. MÉTODOS: Foram avaliados 59 crianças e adolescentes, que responderam ao questionário auto-aplicável Back Pain and Body Posture Evaluation Instrument e fizeram o exame radiográfico panorâmico da coluna vertebral. De acordo com o ângulo de Cobb, as curvaturas sagitais da coluna vertebral foram classificadas como normais ou alteradas e, no plano frontal, como escolioses ou normais. Os dados foram analisados no SPSS 18.0, a partir de estatística descritiva e do teste de associação qui-quadrado (a=0,05). RESULTADOS: A prevalência de alterações posturais foi de 79,7% (n=47), 47,5% (n=28) apresentavam alteração no plano frontal e 61% (n=36) no sagital. Foi encontrada associação entre cifose torácica e sexo feminino, prática de exercício físico apenas uma ou duas vezes na semana, tempo de sono superior a 10 horas, posturas inadequadas para sentar no banco e sentar para escrever e o meio de transporte do material escolar. Para lordose lombar, observou-se associação com o transporte da mochila escolar de modo inadequado (assimétrico). Houve associação significativa entre a presença de escoliose com a prática de esporte competitivo e o tempo de sono superior a 10 horas. CONCLUSÕES: Hábitos de vida podem estar associados a alterações posturais, é importante o desenvolvimento políticas de saúde a fim de reduzir a prevalência de alterações posturais por meio da redução dos fatores de risco associados. .

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.

Background & objectives: There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Methods: Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥0.25 μg/ml), SFM2 (≥4 μg/ml) and SFM3 (≥32 μg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥0.25 μg/ml) and SDM2 (≥4 μg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Results: Mutations were observed in gyrA Ser83→Leu, Asp87→Asn/Gly, Val196→Ala and in parC Phe93→Val, Ser80→Ile, Asp101→Glu and Asp110→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance (p<0.05); while tolC and acrR expression levels did not. Interpretation & conclusions: Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val196→Ala in gyrA in clinical isolates and Phe93→Val, Asp101→Glu, Asp110→Glu and in parC in majority of laboratory-grown mutants.

Effect of Farnesyltransferase Inhibitor R115777 on Mitochondria of Plasmodium falciparum

The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (DeltaPsim) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.

Risk factors for infection with carbapenem-resistant Klebsiella pneumoniae: a case-case-control study / Factores de riesgo para infección con Klebsiella pneumoniae resistente a carbapenémicos: estudio caso-caso-control

Objective: To evaluate the association between quinolone exposure and the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and to estimate CRKP-specific mortality. Methods: Case-case-control study implemented in a tertiary care institution. Three groups of patients were analyzed: 61 consecutive cases of infection with CRKP (Group I); 61 randomly chosen cases of patients infected with carbapenem-sensitive Klebsiella pneumoniae (CSKP; Group II); and 122 randomly chosen controls without CRKP or CSKP infection. Matching was based on the length of stay in intensive care unit and the date of bacterial isolation. An active search was performed for patients with CRKP and CSKP infection, and prospective cases were included in the study. We compared the results for Groups I and II against those for the controls by using two conditional logistic regression analyses that included infection as the dependent variable and controlled for time at risk and co-morbidities. Results: Exposure to quinolones was not associated with CRKP infection: no association was found in the analysis of CRKP with the controls (OR = 1.7; 95% CI: 0.2-6.5) or in the analysis of CSKP against the controls (OR = 0.6; 95% CI: 0.2-1.6). Use of carbapenems (OR = 3.3; 95% CI: 1.2-9.3) and colonization with CRKP (OR = 3.3; 95% IC: 1.2-9.3) were specific risk factors for infection with CRKP. Mortality associated with CRKP was 61.3%. Conclusion: No association was found between exposure to quinolones and infection with CRKP; however, colonization by CRKP and use of carbapenems are risk factors for infection with CRKP.

Resumen Objetivo: Evaluar la asociación entre la exposición a quinolonas y la aparición de infección por Klebsiella pneumoniae resistente a Carbapenémicos (CRKP) y estimar la mortalidad específica por CRKP. Métodos: Estudio caso-caso-control realizado en una institución de tercer nivel de atención. Se adelantó búsqueda activa y prospectiva de los casos. Se analizaron tres grupos: 61 casos consecutivos de infección por CRKP, 61 casos elegidos al azar de los pacientes con infección por Klebsiella pneumoniae sensible a Carbapenémicos (CSKP) y 122 controles sin infección por CRKP ni por CSKP, elegidos al azar. Se realizó emparejamiento por estancia en unidad de cuidados intensivos y fecha del aislamiento bacteriano. Los datos se extractaron de la historia clínica electrónica. Se comparó los casos CRKP y los casos CSKP contra los controles mediante dos análisis de regresión logística condicional, con la infección como variable dependiente y controlando por el tiempo en riesgo y la comorbilidad. Resultados: La exposición a quinolonas no se asoció a infección con CRKP: no se encontró asociación en el análisis de CRKP contra controles (OR 1.7 IC 95%: 0.2-6.5) ni en el análisis de CSKP contra controles (OR 0.6 IC 95%: 0.2-1.6). El uso de carbapenémicos (OR 3.3 IC 95%: 1.2-9.3) y la colonización por KPRC (OR 16,2 IC 95%: IC 95%: 3.3-79.1) fueron factores de riesgo específicos para infección por CRKP. La mortalidad específica asociada a CRKP fue de 61.3%. Conclusión: No se encontró asociación entre la exposición a quinolonas y la infección por KPRC, pero la colonización por CRKP y el uso de carbapenémicos son factores de riesgo asociados a la infección por KPRC.

A Prospective Korean Multicenter Study for Infectious Complications in Patients Undergoing Prostate Surgery: Risk Factors and Efficacy of Antibiotic Prophylaxis

This multicenter study was undertaken to determine the efficacy of antibiotic prophylaxis and identify the risk factors for infectious complications after prostate surgery in Korean patients. A total of 424 patients who underwent surgery of the prostate were reviewed. All patients underwent urinalysis and urine culture preoperatively and postoperatively. Efficacy of antibiotic prophylaxis and risk factors for infectious complications were investigated. Infectious complications were observed in 34.9% of all patients. Factors independently associated with infectious complications were diabetes mellitus (adjusted OR, 1.99; 95% CI, 1.09-3.65, P=0.025) and operation time (adjusted OR, 1.08; 95% CI, 1.03-1.13, P=0.004). Clinicians should be aware of the high risk of infectious complications in patients with diabetes and those who undergo a prolonged operation time. Neither the type nor duration of prophylactic antibiotics resulted in differences in infectious complications.

Future Trends of Helicobacter pylori Eradication Therapy in Korea / 대한소화기학회지

The prevalence of Helicobacter pylori infection in Korea shows a decreasing trend and has changed to that of developed country, especially for those below 30 years old. However, the primary antibiotic resistance rates are higher than those of developed countries. The reason for the decrease in the efficacy of standard triple therapy is mainly due to the increase in the resistance against clarithromycin. Sequential therapy seems to be more effective than the standard triple therapy, but the intention-to-treat eradication rate of sequential therapy in Korea, which is mostly under 80.0%, is still not satisfactory. Therefore, a promising regimen is needed. Recently, the Japanese health insurance system admitted 'H. pylori-infected gastritis' as an indication of eradication. Furthermore, the Kyoto Consensus Meeting on H. pylori Gastritis held from January 30th to February 1st, 2014, proposed that 'all H. pylori positive patients should be offered to receive H. pylori eradication'. This suggests that the concept of eradication has been changed from 'treatment' to 'prevention'. Various individualized tailored therapy based on the polymorphism, age and other demographic factors and antibiotic resistance has been attempted to maximize H. pylori eradication therapy. The aim of this article is to review the current epidemiology, H. pylori resistance state, treatment guideline, and to assess the possible future strategy and treatment for H. pylori infection in Korea.

Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil

The objective of this study was to identify mutations in the Quinolone Resistance Determining sources Regions (QRDR) of the gyrA, gyrB, parC, and parE genes and to determine if any of the qnr variants or the aac(6')-Ib-cr variant were present in strains of Salmonella spp. isolated in Brazil. A total of 126 Salmonella spp. strains from epidemic (n = 114) and poultry (n = 12) origin were evaluated. One hundred and twelve strains (88.8%) were resistant to nalidixic acid (NAL) and 29 (23.01%) showed a reduced susceptibility to ciprofloxacin (Cip). The mutations identified were substitutions limited to the QRDR of the gyrA gene in the codons for Serine 83, Aspartate 87 and Alanine 131. The sensitivity to NAL seems to be a good phenotypic indication of distinguishing mutated and nonmutated strains in the QRDR, however the double mutation in gyrA did not cause resistance to ciprofloxacin. The qnrA1 and qnrB19 genes were detected, respectively, in one epidemic strain of S. Enteritidis and one strain of S. Corvallis of poultry origin. Despite previous detection of qnr genes in Brazil, this is the first report of qnr gene detection in Salmonella, and also the first detection of qnrB19 gene in this country. The results alert for the continuous monitoring of quinolone resistance determinants in order to minimize the emergence and selection of Salmonella spp. strains showing reduced susceptibility or resistance to quinolones.