Routine full characterization of Mycobacterium tuberculosis is culture based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near the point of care. We demonstrate a low-cost method of DNA extraction directly from patient samples for M. tuberculosis WGS. We initially evaluated the method by using the Illumina MiSeq sequencer (40 smear-positive respiratory samples obtained after routine clinical testing and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction were obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. With Illumina MiSeq/MiniSeq, the workflow from patient sample to results can be completed in 44/16 h at a reagent cost of £96/£198 per sample. We then employed a nonspecific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis strain BCG DNA and to combined culture-negative sputum DNA and BCG DNA. For flow cell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 h, with full susceptibility results 5 h later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis DNA in direct samples.

The discovery of antibiotics in the previous century lead to reduction in mortality and morbidity due to infectious diseases but their inappropriate and irrational use has resulted in emergence of resistant microbial populations. Alteration of target sites, active efflux of drugs and enzymatic degradations are the strategies employed by the pathogenic bacteria to develop intrinsic resistance to antibiotics. This has led to an increased interest in medicinal plants since 25-50% of current pharmaceuticals are plant derived. Crude extracts of medicinal plants could serve as an alternate source of resistance modifying agents owing to the wide variety of secondary metabolites. These metabolites (alkaloids, tannins, polyphenols etc.) could act as potentials for antimicrobials and resistance modifiers. Plant extracts have the ability to bind to protein domains leading to modification or inhibition protein-protein interactions. This enables the herbals to also present themselves as effective modulators of host related cellular processes viz immune response, mitosis, apoptosis and signal transduction. Thus they may exert their activity not only by killing the microorganism but by affecting key events in the pathogenic process, thereby, the bacteria, fungi and viruses may have a reduced ability to develop resistance to botanicals. The article is meant to stimulate research wherein the cidal activity of the extract is not the only parameter considered but other mechanism of action by which plants can combat drug resistant microbes are investigated. The present article emphasizes on mechanisms involved in countering multi drug resistance.

Background: Aegle marmelos(L.) Correa has been widely used in indigenous systems of Indian medicine due to its various medicinal properties. However, despite its traditional usage as an anti-diarrhoeal there is limited information regarding its mode of action in infectious forms of diarrhoea. Hence, we evaluated the hot aqueous extract (decoction) of dried unripe fruit pulp ofA. marmelosfor its antimicrobial activity and effect on various aspects of pathogenicity of infectious diarrhoea.Methods: The decoction was assessed for its antibacterial, antigiardial and antirotaviral activities. The effect of the decoction on adherence of enteropathogenicEscherichia coliand invasion of enteroinvasiveE. coliandShigella flexnerito HEp-2 cells were assessed as a measure of its effect on colonization. The effect of the decoction on production ofE. coliheat labile toxin (LT) and cholera toxin (CT) and their binding to ganglioside monosialic acid receptor (GM1) were assessed by GM1-enzyme linked immuno sorbent assay whereas its effect on production and action ofE. coliheat stable toxin (ST) was assessed by suckling mouse assay.Results: The decoction showed cidal activity againstGiardiaand rotavirus whereas viability of none of the six bacterial strains tested was affected. It significantly reduced bacterial adherence to and invasion of HEp-2 cells. The extract also affected production of CT and binding of both LT and CT to GM1. However, it had no effect on ST.Conclusion: The decoction of the unripe fruit pulp ofA. marmelos, despite having limited antimicrobial activity, affected the bacterial colonization to gut epithelium and production and action of certain enterotoxins. These observations suggest the varied possible modes of action ofA. marmelosin infectious forms of diarrhoea thereby validating its mention in the ancient Indian texts and continued use by local communities for the treatment of diarrhoeal diseases.

Cells of Sphingomonas sp. strain BSAR-1 constitutively expressed an alkaline phosphatase, which was also secreted in the extracellular medium. A null mutant lacking this alkaline phosphatase activity was isolated by Tn 5 random mutagenesis. The corresponding gene, designated phoK , was cloned and overexpressed in Escherichia coli strain BL21(DE3). The resultant E. coli strain EK4 overexpressed cellular activity 55 times higher and secreted extracellular PhoK activity 13 times higher than did BSAR-1. The recombinant strain very rapidly precipitated >90% of input uranium in less than 2 h from alkaline solutions (pH, 9 ± 0.2) containing 0.5 to 5 mM of uranyl carbonate, compared to BSAR-1, which precipitated uranium in >7 h. In both strains BSAR-1 and EK4, precipitated uranium remained cell bound. The EK4 cells exhibited a much higher loading capacity of 3.8 g U/g dry weight in 7 h in BSAR-1. The data demonstrate the potential utility of genetically engineering PhoK for the bioprecipitation of uranium from alkaline solutions.

Psidium guajava L., Myrtaceae, is used widely in traditional medicine for the treatment of diarrhoea, dysentery, gastroenteritis, stomachaches, and indigestion. However, the effect of the leaf extract of P. guajava on the pathogenesis of infectious diarrhoea has not been studied. The present study evaluates the effect of a hot aqueous extract (decoction) of dried leaves of P. guajava on parameters associated with pathogenicity of infectious diarrhoea. The aim was to understand its possible mechanism(s) of action in controlling infectious diarrhoea and compare it with quercetin, one of the most reported active constituents of P. guajava with antidiarrhoeal activity.