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Heteroresistance to colistin can be defined as the presence of resistant subpopulations in an isolate that is susceptible to this antibiotic. Colistin resistance in Gram-negative bacteria is more frequently related to chromosomal mutations and insertions. This work aimed to study heteroresistance in nine clinical isolates of Klebsiella pneumoniae producing OXA-48 and to describe genomic changes in mutants with acquired resistance in vitro. Antimicrobial susceptibility was determined by broth microdilution (BMD) and heteroresistance by population analysis profiling (PAP). The proteins related to colistin resistance were analyzed for the presence of mutations. Additionally, PCR of the mgrB gene was performed to identify the presence of insertions. In the nine parental isolates, the PAP method showed colistin heteroresistance of colonies growing on plates with concentrations of up to 64 mg/L, corresponding to stable mutant subpopulations. The MICs of some mutants from the PAP plate containing 4×MIC of colistin had absolute values of ≤2 mg/L that were higher than the parental MICs and were defined as persistent variants. PCR of the mgrB gene identified an insertion sequence that inactivated the gene in 21 mutants. Other substitutions in the investigated mutants were found in PhoP, PhoQ, PmrB, PmrC, CrrA and CrrB proteins. Colistin heteroresistance in K. pneumoniae isolates was attributed mainly to insertions in the mgrB gene and point mutations in colistin resistance proteins. The results of this study will improve understanding regarding the mechanisms of colistin resistance in mutants of K. pneumoniae producing OXA-48.

Objectives To monitor quantitatively the extent of intestinal colonisation by KPC-producing Klebsiella pneumoniae (KPC-Kp) in colonised patients who receive selective digestive decontamination (SDD) with oral gentamicin. Methods We developed a real-time quantitative PCR (qPCR) method for determination of the relative load of blaKPC (RLKPC) within the gut microbiota. Clinical validation was performed using a culture method as the gold standard and receiver operating curve (ROC) analysis. Fifteen patients were observationally and prospectively followed for one year. Clinical, microbiological variables and rectal swab samples were collected at 0 (baseline), 14 and 30 days and monthly thereafter. Results Clinical validation performed on 111 rectal swab samples demonstrated that the PCR method detected 17% more positives than the culture method. ROC curve analysis documented excellent agreement between both methods (area under the curve, 0.96; 95% confidence interval 0.93–0.99). The RLKPC decreased in 6/15 (40%) and 7/12 (58.3%) patients on days 14 and 30, respectively. Persistent eradication was observed in 2/12 (16.7%), 3/9 (33.3%), 4/8 (50%) and 7/8 (87.5%) patients at 1, 3, 6 and 12 months, respectively, with a median time of 150 days (range 30–270) to persistent eradication. Gentamicin-resistant KPC-Kp isolates were identified in 4/15 (26.7%) patients. The rates of infections (57.1% vs. 12.5%, P = 0.119) and deaths (71.4% vs. 0%, P = 0.007) were higher among patients with high baseline RLKPC. Conclusion Following SDD, a rapid reduction on intestinal load is observed when the colonising KPC-Kp isolate is susceptible to gentamicin; however, persistent eradication at the end of SDD is low. Intestinal carriage of KPC-Kp persists after three months in about one third of patients. Agencia Española de Investigación and Fondo Europeo de Desarrollo Regional (FEDER) FIS PI16/01631 Ministerio de Ciencia, Innovación y Universidades RD16/0016/0008

There are scarce data on the efficacy of ertapenem in the treatment of bacteremia due to extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales (ESBL-E) in kidney transplant (KT) recipients. We evaluated the association between treatment with ertapenem or meropenem and clinical cure in KT recipients with nonsevere bacteremic urinary tract infections (B-UTI) caused by ESBL-E. We performed a registered, retrospective, international (29 centers in 14 countries) cohort study (INCREMENT-SOT, NCT02852902). The association between targeted therapy with ertapenem versus meropenem and clinical cure at day 14 (the principal outcome) was studied by logistic regression. Propensity score matching and desirability of outcome ranking (DOOR) analyses were also performed. A total of 201 patients were included; only 1 patient (treated with meropenem) in the cohort died. Clinical cure at day 14 was reached in 45/100 (45%) and 51/101 (50.5%) of patients treated with ertapenem and meropenem, respectively (adjusted OR 1.29; 95% CI 0.51 to 3.22; P = 0.76); the propensity score-matched cohort included 55 pairs (adjusted OR for clinical cure at day 14, 1.18; 95% CI 0.43 to 3.29; P = 0.74). In this cohort, the proportion of cases treated with ertapenem with better DOOR than with meropenem was 49.7% (95% CI, 40.4 to 59.1%) when hospital stay was considered. It ranged from 59 to 67% in different scenarios of a modified (weights-based) DOOR sensitivity analysis when potential ecological advantage or cost was considered in addition to outcome. In conclusion, targeted therapy with ertapenem appears as effective as meropenem to treat nonsevere B-UTI due to ESBL-E in KT recipients and may have some advantages. Instituto de Salud Carlos III RD16/0016/0001 Ministerio de Ciencias e Innovación PI18/01849

Mitogen-activated protein kinase (MAPK) cascades mediate cellular responses to environmental signals. Previous studies in the fungal pathogen Fusarium oxysporum have revealed a crucial role of Fmk1, the MAPK orthologous to Saccharomyces cerevisiae Fus3/Kss1, in vegetative hyphal fusion and plant infection. Here, we genetically dissected the individual and combined contributions of the three MAPKs Fmk1, Mpk1 and Hog1 in the regulation of development, stress response and virulence of F. oxysporum on plant and animal hosts. Mutants lacking Fmk1 or Mpk1 were affected in reactive oxygen species (ROS) homeostasis and impaired in hyphal fusion and aggregation. Loss of Mpk1 also led to increased sensitivity to cell wall and heat stress, which was exacerbated by simultaneous inactivation of Fmk1, suggesting that both MAPKs contribute to cellular adaptation to high temperature, a prerequisite for mammalian pathogens. Deletion of Hog1 caused increased sensitivity to hyperosmotic stress and resulted in partial rescue of the restricted colony growth phenotype of the mpk1Δ mutant. Infection assays on tomato plants and the invertebrate animal host Galleria mellonella revealed distinct and additive contributions of the different MAPKs to virulence. Our results indicate that positive and negative cross-talk between the three MAPK pathways regulates stress adaptation, development and virulence in the cross-kingdom pathogen F. oxysporum.

Durante la interacción planta-patógeno tiene lugar un reconocimiento mutuo entre el hongo y la planta que comienza con la producción de señales por parte de la planta. Estas señales son percibidas por el hongo y procesadas por las rutas de transducción de señales que ponen en funcionamiento la maquinaria génica necesaria para el proceso de patogénesis. Las cascadas de proteínas quinasas activadas por mitógenos (cascadas MAPK) son rutas de traducción de señales altamente conservadas en organismos eucariotas. Se caracterizan por un tener módulo central de tres proteínas quinasas secuenciales, una MAPKKK activadora de una MAPKK, que a su vez activa la última MAPK del módulo, fosforilando los residuos conservados tirosina y treonina. Actualmente se desconoce en gran parte la naturaleza de las señales que participan en la activación de estas rutas y de los procesos que median el reconocimiento de dichas señales durante la infección. En F. oxysporum, la ruta de la MAPK Fmk1 es ortóloga a la ruta Kss1 de levadura y regula la infección de la planta en F. oxysporum. Los mutantes fmk1 son incapaces de infectar la planta de tomate la cual permanece sin síntomas de marchitez vascular frente a las plantas infectadas por la cepa silvestre que comienzan a mostrar síntomas a los 10 días tras la infección y mueren aproximadamente 20 días después. Hasta ahora se desconoce cuáles son los receptores que regulan esta ruta MAPK de virulencia. Basados en la evidencia aportada por los estudios en la levadura, nos planteamos si proteínas receptoras de tipo mucina podrían estar regulando la ruta Fmk1 a nivel en F. oxsyporum. Para abordar esta hipótesis, se identificó en F. oxysporum una posible proteína ortóloga de Msb2 de levadura mediante búsqueda por homología de secuencia y análisis de predicción bioinformática. Una vez seleccionado el gen candidato, se generaron mutantes por eliminación del gen en la cepa silvestre y en la cepa carente de la MAPK Fmk1. Nuestro trabajo confirma la hipótesis de Msb2 como componente aguas arriba de la ruta MAPK Fmk1 en F. oxysporum, directamente implicado en la fosforilación de Fmk1 y la regulación de la patogénesis. Los resultados obtenidos además sugieren que Msb2 y Fmk1 tienen papeles independientes pero coordinados en la regulación de otras funciones celulares, como la respuesta a estrés de pared.

El proceso de infección del hongo Fusarium oxysporum es complejo y requiere algunos mecanismos bien regulados: 1) el reconocimiento de señales de la planta, 2) la adhesión a la superficie de la raíz y la diferenciación de hifas de penetración, 3) la invasión del córtex de la raíz y la degradación de barreras físicas hasta llegar al tejido vascular, 4) adaptación al entorno adverso del tejido vegetal, incluyendo la tolerancia a compuestos antifúngicos, 5) la proliferación de las hifas y producción de conidios en los vasos del xilema y, 6) la secreción de factores de virulencia tales como enzimas, péptidos o fitotoxinas

[Objectives] To monitor quantitatively the extent of intestinal colonisation by KPC-producing Klebsiella pneumoniae (KPC-Kp) in colonised patients who receive selective digestive decontamination (SDD) with oral gentamicin., [Methods] We developed a real-time quantitative PCR (qPCR) method for determination of the relative load of blaKPC (RLKPC) within the gut microbiota. Clinical validation was performed using a culture method as the gold standard and receiver operating curve (ROC) analysis. Fifteen patients were observationally and prospectively followed for one year. Clinical, microbiological variables and rectal swab samples were collected at 0 (baseline), 14 and 30 days and monthly thereafter., [Results] Clinical validation performed on 111 rectal swab samples demonstrated that the PCR method detected 17% more positives than the culture method. ROC curve analysis documented excellent agreement between both methods (area under the curve, 0.96; 95% confidence interval 0.93–0.99). The RLKPC decreased in 6/15 (40%) and 7/12 (58.3%) patients on days 14 and 30, respectively. Persistent eradication was observed in 2/12 (16.7%), 3/9 (33.3%), 4/8 (50%) and 7/8 (87.5%) patients at 1, 3, 6 and 12 months, respectively, with a median time of 150 days (range 30–270) to persistent eradication. Gentamicin-resistant KPC-Kp isolates were identified in 4/15 (26.7%) patients. The rates of infections (57.1% vs. 12.5%, P = 0.119) and deaths (71.4% vs. 0%, P = 0.007) were higher among patients with high baseline RLKPC., [Conclusion] Following SDD, a rapid reduction on intestinal load is observed when the colonising KPC-Kp isolate is susceptible to gentamicin; however, persistent eradication at the end of SDD is low. Intestinal carriage of KPC-Kp persists after three months in about one third of patients., This work was supported by research funds ‘FIS PI16/01631’ granted to EPN from Plan Estatal de I+D+I 2013-2016, co-financed by the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación and the Fondo Europeo de Desarrollo Regional (FEDER); and Plan Nacional de I+D+i 2013‐2016 and Instituto de Salud Carlos III (ISCIII), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (RD16/0016/0008) ‐ co‐financed by European Development Regional Fund ‘A way to achieve Europe’, operative program Intelligent Growth 2014–2020. EPN holds a research contract from Consejería de Salud y Familias, Junta de Andalucía (RH-0065-2020I).

Background The best available treatment against carbapenemase-producing Enterobacteriaceae (CPE) is unknown. The objective of this study was to investigate the effect of appropriate therapy and of appropriate combination therapy on mortality of patients with bloodstream infections (BSIs) due to CPE. Methods In this retrospective cohort study, we included patients with clinically significant monomicrobial BSIs due to CPE from the INCREMENT cohort, recruited from 26 tertiary hospitals in ten countries. Exclusion criteria were missing key data, death sooner than 24 h after the index date, therapy with an active antibiotic for at least 2 days when blood cultures were taken, and subsequent episodes in the same patient. We compared 30 day all-cause mortality between patients receiving appropriate (including an active drug against the blood isolate and started in the first 5 days after infection) or inappropriate therapy, and for patients receiving appropriate therapy, between those receiving active monotherapy (only one active drug) or combination therapy (more than one). We used a propensity score for receiving combination therapy and a validated mortality score (INCREMENT-CPE mortality score) to control for confounders in Cox regression analyses. We stratified analyses of combination therapy according to INCREMENT-CPE mortality score (0–7 [low mortality score] vs 8–15 [high mortality score]). INCREMENT is registered with ClinicalTrials.gov, number NCT01764490. Findings Between Jan 1, 2004, and Dec 31, 2013, 480 patients with BSIs due to CPE were enrolled in the INCREMENT cohort, of whom we included 437 (91%) in this study. 343 (78%) patients received appropriate therapy compared with 94 (22%) who received inappropriate therapy. The most frequent organism was Klebsiella pneumoniae (375 [86%] of 437; 291 [85%] of 343 patients receiving appropriate therapy vs 84 [89%] of 94 receiving inappropriate therapy) and the most frequent carbapenemase was K pneumoniae carbapenemase (329 [75%]; 253 [74%] vs 76 [81%]). Appropriate therapy was associated with lower mortality than was inappropriate therapy (132 [38·5%] of 343 patients died vs 57 [60·6%] of 94; absolute difference 22·1% [95% CI 11·0–33·3]; adjusted hazard ratio [HR] 0·45 [95% CI 0·33–0·62]; p