Initial expression levels of nimA are decisive for protection against metronidazole in Bacteroides fragilis

Objectives: In the genus Bacteroides, the nim genes are resistance determinants for metronidazole, a nitroimidazole drug widely used against anaerobic pathogens. The Nim proteins are considered to act as nitroreductases. However, data from several studies suggest that the expression levels of Nim do not increase with increasing resistance which is conflicting with this notion. The impact of Nim protein levels on low-level metronidazole resistance, however, representing the early stage of induced resistance in the laboratory, has not been assessed as yet.Methods: The nimA gene was cloned into two different plasmids and introduced into B. fragilis strain 638R. Expression levels of nimA mRNA were measured by RT-qPCR and compared to those in strain 638R harbouring plasmid pI417, the original clinical plasmid harbouring IS element IS1168 with the nimA gene. Further, metronidazole susceptibility was assessed by Etest and the activity of pyruvate:ferredoxin oxidoreductase (PFOR) was measured in all strains after induction of high-level metronidazole resistance.Results: The level of protection against metronidazole by nimA correleated with the level of expression of nimA mRNA. Further, the activity of PFOR in highly-resistant B. fragilis 638R was only preserved when expression levels of nimA were high.Conclusions: Although the development of high-level metronidazole resistance in B. fragilis strains with a nimA gene is not caused by an increase of nimA expression as compared to the less resistant parent strains, nimA expression levels might be of decisive importance in the early stage of resistance devel-opment. This has potential implications for metronidazole resistance in clinical isolates.(c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B . fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides , high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B . fragilis proteome and performed comparative mass-spectrometric analyses with B . fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA . In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function

Proteomic analysis of metronidazole resistance in the human facultative pathogen Bacteroides fragilis

The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function

An arginine deprivation response pathway is induced in Leishmania during macrophage invasion

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade

Twenty-three unsolved problems in hydrology (UPH) – a community perspective

This paper is the outcome of a community initiative to identify major unsolved scientific problems in hydrology motivated by a need for stronger harmonisation of research efforts. The procedure involved a public consultation through on-line media, followed by two workshops through which a large number of potential science questions were collated, prioritised, and synthesised. In spite of the diversity of the participants (230 scientists in total), the process revealed much about community priorities and the state of our science: a preference for continuity in research questions rather than radical departures or redirections from past and current work. Questions remain focussed on process-based understanding of hydrological variability and causality at all space and time scales. Increased attention to environmental change drives a new emphasis on understanding how change propagates across interfaces within the hydrological system and across disciplinary boundaries. In particular, the expansion of the human footprint raises a new set of questions related to human interactions with nature and water cycle feedbacks in the context of complex water management problems. We hope that this reflection and synthesis of the 23 unsolved problems in hydrology will help guide research efforts for some years to come

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Strengthening the health workforce to support integration of HIV and noncommunicable disease services in sub-Saharan Africa

Objective: The successful expansion of HIV services in sub-Saharan Africa has been a signature achievement of global public health. This article explores health workforce-related lessons from HIV scale-up, their implications for integrating noncommunicable disease (NCD) services into HIV programs, ways to ensure that healthcare workers have the knowledge, skills, resources, and enabling environment they need to provide comprehensive integrated HIV/NCD services, and discussion of a priority research agenda. Design and methods: We conducted a scoping review of the published and 'gray' literature and drew upon our cumulative experience designing, implementing and evaluating HIV and NCD programs in low-resource settings. Results and conclusion: Lessons learned from HIV programs include the role of task shifting and the optimal use of multidisciplinary teams. A responsible and adaptable policy environment is also imperative; norms and regulations must keep pace with the growing evidence base for task sharing, and early engagement of regulatory authorities will be needed for successful HIV/NCD integration. Ex-ante consideration of work culture will also be vital, given its impact on the quality of service delivery. Finally, capacity building of a robust interdisciplinary workforce is essential to foster integrated patient-centered care. To succeed, close collaboration between the health and higher education sectors is needed and comprehensive competency-based capacity building plans for various health worker cadres along the education and training continuum are required. We also outline research priorities for HIV/NCD integration in three key domains: governance and policy; education, training, and management; and service delivery

Identifying the Gaps: An Assessment of Nurses' Training, Competency, and Practice in HIV Care and Treatment in Kenya

Given the burden of HIV and the critical shortage of health workers in Kenya, in 2011 the National AIDS and STI Control Program recommended shifting HIV care and treatment tasks to nurses in settings without physicians and clinical officers in order to decentralize and scale-up HIV services. In September 2013, ICAP at Columbia University conducted a survey with nurses in four health facilities in eastern Kenya to assess preparedness for task shifting. Findings indicated gaps in nurses' training, perceived competency, and practice in HIV care and treatment. Further investment in nurse capacity building is needed to bridge the gaps and prepare more nurses to provide high-quality, comprehensive HIV care and treatment services to curb the epidemic in Kenya