Bases moleculares de la resistencia a fosfomicina e implicación de las subpoblaciones con sensibilidad disminuida en su actividad in vitro.

El interés global que existe actualmente, en recuperar antibióticos antiguos para el tratamiento de infecciones causadas por microorganismos multirresistentes, es fruto de la escasez de alternativas terapéuticas con las que contamos para hacer frente a este tipo de bacterias. La multirresistencia a antibióticos es un problema de salud pública internacional, y el poder rescatar herramientas eficaces como son los antibióticos antiguos, fosfomicina entre ellos, es una de las estrategias propuestas por organismos internacionales como la OMS o el ECDC para hacer frente a este problema. El objetivo de esta tesis doctoral es evaluar la contribución a la resistencia a fosfomicina de los genes implicados en el transporte intracelular de dicho antibiótico, en un entorno genético controlado, así como demostrar el impacto de las diferencias en el inóculo inicial de los métodos de sensibilidad para la determinación de la CMI a fosfomicina. Para ello se construyó una colección isogénica de mutantes de deleción simple (ΔglpT, ΔuhpT, ΔcyaA y ΔptsI) y doble deleción (ΔglpT-uhpT, ΔglpT-cyaA, ΔglpT-ptsI, ΔuhpT-cyaA, ΔuhpT-ptsI, ΔptsI-cyaA), donde se evaluó su sensibilidad a fosfomicina a la vez que se determinó la frecuencia de aparición de en dicha colección. Por otro lado, se determinó la CMI a fosfomicina de una colección de 220 asilados clínicos de E. coli y K. pneumoniae mediante dilución en agar y microdilución controlando el inóculo inicial de ambas técnicas y se analizaron los acuerdos de categoría clínica ente ambas. Los resultados de este trabajo ponen de manifiesto, que las mutaciones que causan pérdida de función de los genes codificantes para los transportadores de fosfomicina (uhpT, glpT), así como de los principales genes relacionados con la regulación de su entrada en la bacteria (cyaA y ptsI), no alcanzan valores de CMI de fosfomicina considerados como resistentes aplicando los puntos de corte de sensibilidad antibiótica de CLSI. Además, únicamente las combinaciones de dobles deleciones, que implican la inactivación del transportador UhpT, presentan CMIs a fosfomicina consideradas como resistentes aplicando los puntos de corte de CLSI o EUCAST. Por otro lado, se concluyó que la CMI a fosfomicina no es un buen predictor de la actividad de este antimicrobiano frente a E. coli y K. pneumoniae, debido a que pequeñas variaciones en la concentración del inóculo inicial utilizado para realizar los test de sensibilidad causan discrepancias entre las distintas técnicas de sensibilidad, debido a que la tasa de frecuencia de aparición de mutantes resistentes a fosfomicina observada es alta. Este hecho aumenta la probabilidad de introducir mutantes resistentes en el propio inóculo inicial durante la realización de las técnicas de sensibilidad y hace poco reproducibles estas técnica

Identification and antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures

[ES] Introducción: La espectrometría de masas Matrix-Assisted Laser Desorption-Ionization Time-of-Flight (MALDI-TOF) permite la identificación rápida de los microorganismos causantes de bacteriemia. Se requieren métodos fiables y rápidos que permitan acortar el tiempo necesario hasta disponer de los resultados de sensibilidad a antibióticos de los aislados de hemocultivos. Métodos: Se evalúa la fiabilidad de un método que combina la identificación con MALDI-TOF y el estudio de sensibilidad en paneles de microdilución inoculados a partir de un subcultivo incubado durante solo 4 h. Resultados: La concordancia de los resultados de sensibilidad a antibióticos de la técnica evaluada frente a la técnica de referencia fue del 99,3%, sin que se observaran errores máximos. Conclusión: La inoculación de paneles de microdilución a partir de un subcultivo de solo 4 h de incubación es un método fiable y fácil de realizar que permite acortar el tiempo de informe de hemocultivos positivos.[EN] Introduction: Mass spectrometry Matrix-Assisted Laser Desorption-Ionization Time-of-Flight (MALDI-TOF) helps in the rapid identification of microorganisms causing blood stream infection. Rapid and reliable methods are required to decrease the turnaround time for reporting antimicrobial susceptibility results from blood culture isolates. Methods: An evaluation was performed on the reliability of a method for antimicrobial susceptibility testing of positive blood culture isolates from briefly incubated solid medium cultures. Results: The agreement between the evaluated and standard methods was 99.3%. The major and minor error rates were 0.4% and 0.3%, respectively, and no very major errors were observed. Conclusion: The inoculation of briefly incubated solid medium cultures into antimicrobial susceptibility testing panels is an easy and reliable technique, and helps to decrease the turnaround time for reporting antimicrobial susceptibility results of positive blood cultures.Peer reviewe

Identificación bacteriana directa a partir de hemocultivos positivos usando espectrometría de masas MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight): una revisión sistemática y metaanálisis

[Introduction] The rapid identification of bacteraemia-causing pathogens could assist clinicians in the timely prescription of targeted therapy, thereby reducing the morbidity and mortality of this infection. In recent years, numerous techniques that rapidly and directly identify positive blood cultures have been marketed, with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) being one of the most commonly used.[Methods] The aim of this systematic review and meta-analysis was to evaluate the accuracy of MALDI-TOF (Bruker) for the direct identification of positive blood culture bottles.[Results] A meta-analysis was performed to summarize the results of the 32 studies evaluated. The overall quality of the studies was moderate. For Gram-positive bacteria, overall rates of correct identification of the species ranged from 0.17 to 0.98, with a cumulative rate (random-effects model) of 0.72 (95% CI: 0.64–0.80). For Gram-negative bacteria, correct identification rates ranged from 0.66 to 1.00, with a cumulative effect of 0.92 (95% CI: 0.88–0.95). For Enterobacteriaceae, the rate was 0.96 (95% CI: 0.94–0.97).[Conclusion] MALDI-TOF mass spectrometry shows high accuracy for the correct identification of Gram-negative bacteria, particularly Enterobacteriaceae, directly from positive blood culture bottles, and moderate accuracy for the identification of Gram-positive bacteria (low for some species).[Introducción] La identificación rápida de los patógenos causantes de bacteriemia orienta a los clínicos a prescribir con mayor celeridad un tratamiento dirigido y reducir así la morbimortalidad de dicha infección. Durante los últimos años, han aparecido en el mercado numerosas técnicas con la intención de cubrir esta necesidad, que logran una identificación rápida y directa a partir de los frascos de hemocultivos positivos. La espectrometría de masas matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) es una de las tecnologías más utilizadas en este campo.[Métodos] El objetivo de ese estudio es realizar una revisión sistemática y metaanálisis que evalúe la precisión de MALDI-TOF MS (Bruker) para la identificación directa a partir de frascos de hemocultivos positivos.[Resultados] El metaanálisis fue realizado para sintetizar los resultados de los 32 estudios evaluados. La calidad total de los estudios fue moderada. Para las bacterias grampositivas, el ratio total de identificaciones correctas a nivel de especie fue del 0,17 al 0,98 con un ratio acumulativo (modelo de efectos aleatorios) de 0,72 (IC 95%: 0,64-0,80). Para las bacterias gramnegativas, el rango de identificaciones correctas fue del 0,66 al 1,00 con un efecto acumulativo de 0,92 (IC 95%: 0,88-0,95), llegando a un 0,96 (IC 95%: 0,94-0,97) en Enterobacteriaceae.[Conclusiones] La espectrometría de masas MALDI-TOF muestra una alta precisión para la correcta identificación de bacterias gramnegativas realizada directamente a partir de los frascos de hemocultivos positivos, siendo mayor en el grupo de las enterobacterias. Para grampositivas, la precisión es moderada, llegando a ser baja para alguna especie.Peer Reviewe

Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae.

Fosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies. Fosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n=81) and Klebsiella pneumoniae (n=139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC=1mg/L) and 21 K. pneumoniae (MIC=16mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC=1mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5×105-3.91×103 CFU/well, 1 : 2 dilutions). For E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1-log2) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05×10-5 (4×MIC) to 5.59×10-7 (256×MIC) for E. coli, and 1.49×10-4 (4×MIC) to 1.58×10-5 (16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×104 CFU/well. The higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method

Molecular insights into fosfomycin resistance in Escherichia coli.

Fosfomycin activity in Escherichia coli depends on several genes of unknown importance for fosfomycin resistance. The objective was to characterize the role of uhpT , glpT , cyaA and ptsI genes in fosfomycin resistance in E. coli. WT E. coli BW25113 and null mutants, Δ uhpT , Δ glpT , Δ cyaA , Δ ptsI , Δ glpT-uhpT , Δ glpT-cyaA , Δ glpT-ptsI , Δ uhpT-cyaA , Δ uhpT-ptsI and Δ ptsI-cyaA , were studied. Susceptibility to fosfomycin was tested using CLSI guidelines. Fosfomycin mutant frequencies were determined at concentrations of 64 and 256 mg/L. Fosfomycin in vitro activity was tested using time-kill assays at concentrations of 64 and 307 mg/L (human C max ). Fosfomycin MICs were: WT E. coli BW25113 (2 mg/L), Δ glpT (2 mg/L), Δ uhpT (64 mg/L), Δ cyaA (8 mg/L), Δ ptsI (2 mg/L), Δ glpT-uhpT (256 mg/L), Δ glpT-cyaA (8 mg/L), Δ glpT-ptsI (2 mg/L), Δ uhpT-cyaA (512 mg/L), Δ uhpT-ptsI (64 mg/L) and Δ ptsI-cyaA (32 mg/L). In the mutant frequency assays, no mutants were recovered from BW25113. Mutants appeared in Δ glpT , Δ uhpT , Δ cyaA and Δ ptsI at 64 mg/L and in Δ uhpT and Δ cyaA at 256 mg/L. Δ glpT-ptsI , but not Δ glpT-cyaA , Δ uhpT-cyaA or Δ uhpT-ptsI , increased the mutant frequency compared with the highest frequency found in each single mutant. In time-kill assays, all mutants regrew at 64 mg/L. Initial bacterial reductions of 2-4 log 10 cfu/mL were observed for all strains, except for Δ uhpT-ptsI , Δ glpT-uhpT and Δ uhpT-cyaA . Only Δ glpT and Δ ptsI mutants were cleared using 307 mg/L. Fosfomycin MIC may not be a good efficacy predictor, as highly resistant mutants may appear, depending on other pre-existing mutations with no impact on MIC

Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae

[Objectives] Fosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies.[Methods] Fosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n = 81) and Klebsiella pneumoniae (n = 139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC = 1 mg/L) and 21 K. pneumoniae (MIC = 16 mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC = 1 mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5 × 105−3.91 × 103 CFU/well, 1 : 2 dilutions).[Results] For E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1–log2) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05 × 10–5 (4 × MIC) to 5.59 × 10–7 (256 × MIC) for E. coli, and 1.49 × 10–4 (4 × MIC) to 1.58 × 10–5 (16 × MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13 × 104 CFU/well.[Conclusions] The higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method.This work was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (PI13/01885 and PI13/01282) and the Consejería de Salud, Junta de Andalucía (PI-0044-2013), Spain. It was partially supported by Plan Nacional de I+D+i and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015)—co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF. Fernando Docobo-Pérez is supported by a VPPI-US fellowship from the University of Sevilla. JRB acts as scientific advisor for AstraZeneca, Merck and Achaogen, and was speaker for AstraZeneca, Merck, Pfizer and Astellas. He received funding for research from 115523 COMBACTE-NET, 115620 COMBACTE-CARE, and 115737 COMBACTE-MAGNET projects, Innovative Medicines Initiative (IMI), funded by the European Union and EFPIA companies

Molecular insights into fosfomycin resistance in Escherichia coli

[Objectives] Fosfomycin activity in Escherichia coli depends on several genes of unknown importance for fosfomycin resistance. The objective was to characterize the role of uhpT, glpT, cyaA and ptsI genes in fosfomycin resistance in E. coli.[Methods] WT E. coli BW25113 and null mutants, ΔuhpT, ΔglpT, ΔcyaA, ΔptsI, ΔglpT-uhpT, ΔglpT-cyaA, ΔglpT-ptsI, ΔuhpT-cyaA, ΔuhpT-ptsI and ΔptsI-cyaA, were studied. Susceptibility to fosfomycin was tested using CLSI guidelines. Fosfomycin mutant frequencies were determined at concentrations of 64 and 256 mg/L. Fosfomycin in vitro activity was tested using time–kill assays at concentrations of 64 and 307 mg/L (human Cmax).[Results] Fosfomycin MICs were: WT E. coli BW25113 (2 mg/L), ΔglpT (2 mg/L), ΔuhpT (64 mg/L), ΔcyaA (8 mg/L), ΔptsI (2 mg/L), ΔglpT-uhpT (256 mg/L), ΔglpT-cyaA (8 mg/L), ΔglpT-ptsI (2 mg/L), ΔuhpT-cyaA (512 mg/L), ΔuhpT-ptsI (64 mg/L) and ΔptsI-cyaA (32 mg/L). In the mutant frequency assays, no mutants were recovered from BW25113. Mutants appeared in ΔglpT, ΔuhpT, ΔcyaA and ΔptsI at 64 mg/L and in ΔuhpT and ΔcyaA at 256 mg/L. ΔglpT-ptsI, but not ΔglpT-cyaA, ΔuhpT-cyaA or ΔuhpT-ptsI, increased the mutant frequency compared with the highest frequency found in each single mutant. In time–kill assays, all mutants regrew at 64 mg/L. Initial bacterial reductions of 2–4 log10 cfu/mL were observed for all strains, except for ΔuhpT-ptsI, ΔglpT-uhpT and ΔuhpT-cyaA. Only ΔglpT and ΔptsI mutants were cleared using 307 mg/L.[Conclusions] Fosfomycin MIC may not be a good efficacy predictor, as highly resistant mutants may appear, depending on other pre-existing mutations with no impact on MIC.This work was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (PI13/01885 and PI 13/01282) and the Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI-0044-2013), Spain. It was partly supported by the Plan Nacional de I + D+i and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD12/0015)—co-financed by the European Development Regional Fund ‘A way to achieve Europe’ ERDF. F. D.-P. is supported by a VPPI-US fellowship from the University of Sevilla.Peer reviewe