Faecal phageome of healthy individuals: presence of antibiotic resistance genes and variations caused by ciprofloxacin treatment.

OBJECTIVES: Antimicrobial resistance genes (ARGs) can be transferred by means of mobile genetic elements, which play a critical role in the dissemination of resistance in the bacterial community. ARG transmission within mobile genetic elements has been reported in plasmids and transposons but less frequently in bacteriophages. Here, the bacteriophage fraction of seven human faecal samples was purified and deep-sequenced to detect the presence of ARGs in the phage particles. METHODS: Seven faecal samples (five from healthy individuals and two from a patient before and after receiving ciprofloxacin treatment) were used to extract phage DNA, which was purified and then sequenced in a MiSeq (Illumina). Generated reads were checked for quality and assembled, and then the generated contigs analysed with Kraken, PHASTER, VirSorter and Prokka. Some genes were also validated by quantitative PCR. RESULTS: Analysis of the purified phage DNA by Kraken identified from 4 to 266 viruses in the samples. The viral fraction corresponded mainly to the order Caudovirales, including phages from the Siphoviridae and Myoviridae families. Bacterial genes associated with antimicrobial resistance were detected in the viral DNA, as confirmed by quantitative PCR. Higher densities of ARG-carrying phage particles were observed in the post- versus pre-ciprofloxacin treatment sample. CONCLUSIONS: The finding of ARGs in phage particles supports the description of phages as mobile elements contributing to the dissemination of bacterial antibiotic resistance and suggests ciprofloxacin treatment may play a role in the release of ARG-carrying particles, thereby increasing resistance.

Prevalencia en España de mecanismos de resistencia a quinolonas en enterobacterias productoras de betalactamasas de clase C adquiridas y/o carbapenemasas / Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC Beta-lactamases and/or carbapenemases in Spain

Introducción: En los últimos años se ha observado un incremento de la resistencia a fluoroquinolonas en enterobacterias, estando asociado significativamente a la resistencia a betalactámicos. Nuestro objetivo fue conocer la prevalencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas en aislados productores de betalactamasas de claseC adquiridas y/o carbapenemasas. Métodos: Se evaluó la presencia de mecanismos cromosómicos y plasmídicos de resistencia a quinolonas [mutaciones en la región determinante de resistencia a quinolonas de gyrA y parCy genes qnr, aac(6')-Ib-cr y qepA] en 289 aislados de enterobacterias productoras de betalactamasas de claseC adquiridas y/o carbapenemasas recogidos entre febrero y julio de 2009 en 35 hospitales españoles. Resultados: Se detectaron determinantes plasmídicos en 92 aislados (31,8%); en 83 aislados (28,7%) se detectó algún gen qnr, y en 20 (7%), la variante aac(6')-Ib-cr. El gen qnr más prevalente fue qnrB4 (20%), asociado en la mayoría de los casos a DHA-1. El 14,6% de los aislados con una CMI de ciprofloxacino superior a 0,25mg/l no presentaban mutaciones en gyrA ni parC, detectándose en el 90% de los mismos algún determinante plasmídico de resistencia a quinolonas. Conclusión: qnrB4 fue el determinante plasmídico más prevalente, claramente asociado a DHA-1. Los mecanismos plasmídicos en asociación con mecanismos cromosómicos diferentes a las mutaciones en los genes de las topoisomerasas (sobreexpresión de bombas de expulsión, alteración del lipopolisacárido o disminución de porinas) pueden dar lugar a valores de CMI de ciprofloxacino que superan los puntos de corte establecidos por los principales comités internacionales de definición de puntos de corte para interpretación de datos de sensibilidad (AU)

Background: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC Beta-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates. Methods: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC Beta -lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals. Results: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates. Conclusion: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC Beta -lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints (AU)

Activity of ceftazidime-avibactam against multidrug-resistance Enterobacteriaceae expressing combined mechanisms of resistance / Actividad de ceftazidima/avibactam en enterobacterias multirresistentes con mecanismos de resistencia combinados

Introduction: Antimicrobial resistance in Enterobacteriaceae is increasing worldwide and is making treating infections caused by multidrug-resistant Enterobacteriaceae a challenge. The use of Beta -lactam agents is compromised by microorganisms harboring extended-spectrum Beta -lactamases (ESBLs) and other mechanisms of resistance. Avibactam is a non Beta -lactam agent that inhibits clinically relevant Beta -lactamases, such as ESBL and AmpC. The ceftazidime-avibactam combination (CAZ-AVI) was recently approved for use in certain complicated infections, and may provide a therapeutic alternative for infections caused by these microorganisms. Methods: The in vitro activity of CAZ and CAZ-AVI (AVI at a fixed concentration of 4mg/L) was tested against 250 clinical isolates of Enterobacteriaceae using broth microdilution. EUCAST breakpoint criteria were used for CAZ, and FDA criteria for CAZ-AVI. Clinical isolates included bacteria producing extended-spectrum Beta -lactamases (ESBLs) and acquired AmpC Beta -lactamases (AACBLs). Porin loss in Klebsiella pneumoniae was also evaluated. Results: The combination of AVI with CAZ displayed excellent activity against clinical isolates of ESBL-producing Escherichia coli and Klebsiella pneumoniae, rendering all the ceftazidime-resistant isolates susceptible to ceftazidime. CAZ-AVI retained activity against porin-deficient isolates of K. pneumoniae producing ESBLs, AACBLs, or both, although MIC values were higher compared to porin-expressing isolates. CAZ-AVI rendered all the ceftazidime-resistant AACBL-producing Enterobacteriaceae tested susceptible to ceftazidime. Conclusion: CAZ-AVI showed potent in vitro activity against clinical isolates of Enterobacteriaceaeproducing ESBLs and/or AACBLs, including K. pneumoniae with loss of porins (AU)

Introducción: La resistencia antibiótica en enterobacterias está en aumento y el tratamiento de infecciones producidas por enterobacterias multirresistentes supone un reto terapéutico. El uso de betalactámicos se afecta con la producción de betalactamasas de espectro extendido (BLEE) y otros mecanismos de resistencia. Avibactam es un compuesto no betalactámico que inhibe betalactamasas como BLEE o AmpC. La combinación ceftazidima-avibactam (CAZ-AVI) ha sido aprobada recientemente para el tratamiento de infecciones complicadas y puede ser una alternativa terapéutica en estas infecciones. Métodos: La actividad in vitro de CAZ y CAZ-AVI (AVI, concentración fija de 4mg/mL) fue determinada en 250 aislamientos clínicos de enterobacterias mediante microdilución en caldo. Los puntos de corte de EUCAST fueron utilizados para CAZ, y los criterios de FDA se utilizaron para CAZ-AVI. Las enterobacterias estudiadas producían BLEE y/o AmpC adquiridas (BLAA). El papel de la pérdida de porinas en Klebsiella pneumoniae también fue evaluado. Resultados: CAZ-AVI demostró una excelente actividad en Escherichia coli y Klebsiella pneumoniaeproductoras de BLEE, devolviendo la sensibilidad a CAZ en todos los aislamientos resistentes a CAZ. CAZ-AVI mantuvo su actividad en aislamientos de K. pneumoniae deficientes en porinas productoras de BLEE y/o BLAA, aunque los valores de CMI fueron más altos comparados con las cepas que expresaban porinas. En todas las enterobacterias resistentes a ceftazidima productoras de BLAA analizadas en este estudio CAZ-AVI devolvió la sensibilidad a ceftazidima. Conclusión: CAZ-AVI demostró una potente actividad in vitro en aislamientos clínicos de enterobacterias productoras de BLEE y/o BLAA, incluyendo K. pneumoniae con pérdida de porinas (AU)

[Infections caused by Clostridium difficile]. / Infecciones producidas por Clostridium difficile.

The epidemiology of Clostridium difficile infections (CDIs) has dramatically changed over the last decade in both North America and Europe, and it has become more frequent, more severe, more refractory to standard therapy, and more likely to relapse. These changes have been associated with the emergence of a "hypervirulent" strain known as BI/NAP1/027 which has become endemic in some areas, although, other hypervirulent genotypes (e.g. PCR ribotype 078) have also been described. To reduce the incidence of CDIs, the diagnostic guidelines on diagnosis and treatment methods have been recently updated. The aim of this review is to highlight the recent epidemiological data on CDIs and to provide an overview of the pathogenicity of the infection, diagnostic approaches, old and new treatment options, and current knowledge of infection control measures.