Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial Pathogen Rhodococcus equi

[EN] Rhodococcus equi is a facultative intracellular pathogen that can survive within macrophages of a wide variety of hosts, including immunosuppressed humans. Current antibiotherapy is often ineffective, and novel therapeutic strategies are urgently needed to tackle infections caused by this pathogen. In this study, we identified three mycoredoxin-encoding genes (mrx) in the genome of R. equi, and we investigated their role in virulence. Importantly, the intracellular survival of a triple mrx-null mutant (Δmrx1Δmrx2Δmrx3) in murine macrophages was fully impaired. However, each mycoredoxin alone could restore the intracellular proliferation rate of R. equi Δmrx1Δmrx2Δmrx3 to wild type levels, suggesting that these proteins could have overlapping functions during host cell infection. Experiments with the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) biosensor confirmed that R. equi was exposed to redox stress during phagocytosis, and mycoredoxins were involved in preserving the redox homeostasis of the pathogen. Thus, we studied the importance of each mycoredoxin for the resistance of R. equi to different oxidative stressors. Interestingly, all mrx genes did have overlapping roles in the resistance to sodium hypochlorite. In contrast, only mrx1 was essential for the survival against high concentrations of nitric oxide, while mrx3 was not required for the resistance to hydrogen peroxide. Our results suggest that all mycoredoxins have important roles in redox homeostasis, contributing to the pathogenesis of R. equi and, therefore, these proteins may be considered interesting targets for the development of new anti-infectivesS

Contact Effect of a Methylobacterium sp. Extract on Biofilm of a Mycobacterium chimaera Strain Isolated from a 3T Heater-Cooler System

Mycobacterium chimaera is an opportunistic slowly growing non-tuberculous mycobacteriumof increasing importance due to the outbreak of cases associated with contaminated 3T heater-cooler device (HCD) extracorporeal membrane oxygenator (ECMO). The aim of this study was to evaluate the effect of pre-treating a surface with a Methylobacterium sp. CECT 7180 extract to inhibit the M. chimaera ECMO biofilm as well as of the treatment after different dehydration times. Surface adherence, biofilm formation and treatment effect were evaluated by estimating colony-forming units (CFU) per square centimeter and characterizing the amount of covered surface area, thickness, cell viability, and presence of intrinsic autofluorescence at different times using confocal laser scanning microscopy and image analysis. We found that exposing a surface to the Methylobacterium sp. CECT 7180 extract inhibited M. chimaera ECMO biofilm development. This effect could be result of the effect of Methylobacterium proteins, such as DNaK, trigger factor, and xanthine oxidase. In conclusion, exposing a surface to the Methylobacteriumsp. extract inhibits M. chimaera ECMO biofilm development. Furthermore, this extract could be used as a pre-treatment prior to disinfection protocols for equipment contaminated with mycobacteria after dehydration for at least 96 h

<i>Enterococcus faecium</i> Bacteriophage vB_EfaH_163, a New Member of the <i>Herelleviridae</i> Family, Reduces the Mortality Associated with an <i>E. faecium</i> vanR Clinical Isolate in a <i>Galleria mellonella</i> Animal Model

The rise of antimicrobial resistant (AMR) bacteria is a major health concern, especially with regard to members of the ESKAPE group, to which vancomycin-resistant (VRE) Enterococcus faecium belongs. Phage therapy has emerged as a novel alternative for the treatment of AMR infections. This, however, relies on the isolation and characterisation of a large collection of phages. This work describes the exploration of human faeces as a source of new E. faecium-infecting phages. Phage vB_EfaH_163 was isolated and characterised at the microbiological, genomic, and functional levels. vB_EfaH_163 phage, a new member of Herelleviridae, subfamily Brockvirinae, has a dsDNA genome of 150,836 bp that does not harbour any virulence factors or antibiotic resistance genes. It infects a wide range of E. faecium strains of different origins, including VRE strains. Interestingly, it can also infect Enterococcus faecalis strains, even some that are linezolid-resistant. Its capacity to control the growth of a clinical VRE isolate was shown in broth culture and in a Galleria mellonella animal model. The discovery and characterisation of vB_EfaH_163 increases the number of phages that might be used therapeutically against AMR bacteria

Supplementary Material Enterococcus faecium Bacteriophage vB_EfaH_163, a New Member of the Herelleviridae Family, Reduces the Mortality Associated with an E. faecium vanR Clinical Isolate in a Galleria mellonella Animal Model

Features of Enterococcus faecium bacteriophage vB_EfaH_163. The orfs, gene number and gene position in the vB_EfaH_163 genome are shown, as are the predicted functions, molecular weights and isoelectric points of the encoded products. The predicted functions were assessed using RAST and PATRIC software. The top BLAST hit and E-values are also indicated.Peer reviewe

vB_EfaH_163 phage reduces the mortality of an E. faecium vanR clinical isolate in a Galleria mellonella animal model

Resumen del trabajo presentado en el I Asturias International Meeting on Clinical Microbiology and Infectious Diseases - AIMID , celebrado en Oviedo (España) , los días 22 y 23 de septiembre de 2022Introduction The rise of antimicrobial resistant (AMR) bacteria is a major health concern, especially with regard to members of the ESKAPE group, to which vancomycin-resistant Enterococcus faecium (VRE) belongs. Phage therapy has emerged as a novel alternative for the treatment of AMR infections. However, it relies on the isolation and characterization of a large collection of phages. This work describes the exploration of human faeces as a source of new E. faecium-infecting phages. Material and methods Phage vB_EfaH_163 was isolated from the faeces of a human volunteer after applying a microbiota separation method. Phage vB_EfaH_163 was then characterized at the microbiological level: Host range against strains of E. faecium from different origins -including VRE clinical isolates- capsid morphology, and one-step growth curve. The vB_EfaH_163 genome was sequenced and analysed. In addition, technological characterization-specifically pH and thermal stability- was performed. Finally, the biocontrol capacity of vB_EfaH_163 against a clinical VRE strain was assayed in broth and in a Galleria mellonella animal model. Results and discussion vB_EfaH_163 phage is a new member of Herelleviridae, subfamily Brockvirinae. Its genome consists in a dsDNA genome of 150.836 bp that do not harbours any toxin-encoding or antibiotic resistance genes. It infects a wide range on E. faecium strains of different origin, including VRE strains. Interestingly, it can also infect E. faecalis strains, even some that are linezolid-resistant. Its capacity to control the growth of a clinical VRE isolate was shown in broth culture. Moreover, the phage vB_EfaH_163 was shown to significantly reduce mortality in a Galleria. mellonella animal model of E. faecium infection. The discovery and characterization of vB_EfaH_163 increases the number of phages that might be used therapeutically against AMR bacteria

Aislamiento y caracterización de vB_EfaH_163, un fago lítico que infecta cepas de Enterococcus faecium resistentes a vancomicina

Trabajo presentado en el 6th Annual Meeting of Spanish Network of Bacteriophages and Transducer Elements (FAGOMA), celebrado en Bilbao (España), del 20 al 22 de octubre de 2021El aumento de bacterias resistentes a antimicrobianos (AMR) supone un problema de salud pública, especialmente en el caso de bacterias pertenecientes al grupo ESKAPE, al que pertenece Enterococcus faecium resistente a la vancomicina (VRE). La terapia fágica puede ser una alternativa para el tratamiento de las infecciones causadas por estas bacterias. Sin embargo, se necesita el aislamiento previo y la caracterización de una gran colección de fagos. En este trabajo exploramos el uso de heces humanas como fuente de nuevos fagos capaces de infectar a E. faecium. A partir de una muestra se aisló el fago vB_EfaH_163 que fue caracterizado a nivel microbiológico, genómico y funcional. El fago vB_EfaH_163 es un nuevo miembro de la familia Herelleviridae, subfamilia Brockvirinae y posee un genoma de dsDNA de 150.836 pb. El análisis de su genoma no reveló la existencia de genes que codifiquen toxinas o resistencias a antibióticos y además, es capaz de Infectar una amplia variedad de cepas de E. faecium de diferente origen, incluidas cepas VRE. Curiosamente, también puede infectar cepas de Enterococcus faecalis, incluyendo cepas resistentes a linezolid, un antibiótico de último recurso. Se analizó la estabilidad de los viriones a diferentes temperaturas y valores de pH. Finalmente, se ensayó su capacidad para controlar el crecimiento de un aislado clínico de E. faecium VRE, tanto en medio de cultivo como en un modelo animal de Galleria mellonella. El aislamiento y la caracterización de vB_EfaH_163 aumenta el número de fagos que podrían usarse para combatir las bacterias AMR y amplía el número de posibles fuentes de fagos con aplicaciones terapéuticas