Discovery and characterization of antimycobacterial nitro-containing compounds with distinct mechanisms of action and in vivo efficacy.

Nitro-containing compounds have emerged as important agents in the control of tuberculosis (TB). From a whole-cell high-throughput screen for Mycobacterium tuberculosis (Mtb) growth inhibitors, 10 nitro-containing compounds were prioritized for characterization and mechanism of action studies. HC2209, HC2210, and HC2211 are nitrofuran-based prodrugs that need the cofactor F420 machinery for activation. Unlike pretomanid which depends only on deazaflavin-dependent nitroreductase (Ddn), these nitrofurans depend on Ddn and possibly another F420-dependent reductase for activation. These nitrofurans also differ from pretomanid in their potent activity against Mycobacterium abscessus. Four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and a nitrofuran (HC2250) are proposed to be inhibitors of decaprenyl-phosphoryl-ribose 2'-epimerase 1 (DprE1), based on isolation of resistant mutations in dprE1. Unlike other DprE1 inhibitors, HC2250 was found to be potent against non-replicating persistent bacteria, suggesting additional targets. Two of the compounds, HC2233 and HC2234, were found to have potent, sterilizing activity against replicating and non-replicating Mtb in vitro, but a proposed mechanism of action could not be defined. In a pilot in vivo efficacy study, HC2210 was orally bioavailable and efficacious in reducing bacterial load by ~1 log in a chronic murine TB infection model.

Risk of hospitalisation with fever following MenB vaccination: self-controlled case series analysis.

OBJECTIVE: To investigate a possible association between fever admissions and 4 component Meningococcal B (4CMenB). DESIGN: 4CMenB is given at 8 and 16 weeks in the first year of life. Self-controlled case series using linked routinely collected healthcare data, where the risk period was the 3 days immediately following receipt of a vaccine dose. PATIENTS: Children aged under 1 year in Scotland preintroduction and postintroduction of 4CMenB vaccine (pre-September 2014 to August 2015 and post-September 2015 to June 2016). MAIN OUTCOME MEASURES: Hospitalisations for fever attributable to 4CMenB vaccine. RESULTS: The postintroduction model showed an increased risk in the 3 days after dose 1 (relative incidence (RI), 10.78; 95% CI: 8.31 to 14.00) and dose 3 (RI, 9.80; 95% CI: 7.10 to 13.62), with a smaller increased risk after dose 2 (RI, 2.20; 95% CI: 1.27 to 3.82). The magnitude of these effects was greater than in the preintroduction model. The attributable fractions were 90.7%, 54.8% and 89.7%, equating to 162, 14 and 84 vaccine attributable cases per 100 000 doses, respectively.This is equivalent to 102 extra hospitalisations in Scotland annually, based on a birth cohort of 55 100 and extrapolated to 1430 across the UK based on a birth cohort of 777 165. CONCLUSION: There is an increased risk of hospital admission with fever within 3 days of the routine childhood immunisations at 8 and 16 weeks following introduction of 4CMenB vaccine. The results indicate that further understanding of the current use of prophylactic paracetamol is needed. Communication to parents and health professionals may also need to be re-examined, and guidance on the use of prophylactic paracetamol reinforced.

Changing molecular epidemiology of rotavirus infection after introduction of monovalent rotavirus vaccination in Scotland.

BACKGROUND: Rotaviruses (RV) are the leading cause of gastroenteritis in children less than five years of age worldwide. Rotarix®, a live attenuated monovalent vaccine containing a RV strain of G1P[8] specificity has been included in the childhood immunisation schedule from June 2013 in Scotland. This study aimed to characterise the prevalent RV strains in Scotland before and after the introduction of the RV vaccine. METHODS: RV positive faecal samples from Scottish virology laboratories covering the years 2012-2015 were genotyped. Viral RNA was extracted from faecal suspensions. VP7 and VP4 gene specific primers were used for multiplex hemi-nested PCRs and sequencing. Mann-Whitney U test and Chi-square test were used for statistical comparison. RESULTS: There was a decrease in RV positive samples from the Scottish virology laboratories from 7409 samples in the pre-vaccination years (2009-2013) to 760 in 2014-2015, with an annual reduction of RV infections by 74.4% (RR-3.95; 95%-CI, 3.53-4.42, p<0.001). 362 samples from the pre-vaccination period and 278 samples from the post-vaccination were genotyped. There was a drop in prevalence of G1P[8] strains (72.1%, 95%-CI, 67.42-76.33 to 15%, 95%-CI, 11.38-19.79) after introduction of the vaccine. In the post-vaccination period G2P[4] was the dominant strain in Scotland (21.9%, 95%-CI, 17.48-27.17) with increase in G9P[8] (12.9%, 95%-CI, 9.50-7.41), G12P[8] (12.2%, 95%-CI, 8.89-16.60) and G3P[8] (11.9%, 95%-CI, 8.58-16.20) infections. Phylogenetic analysis of the VP7 and VP4 genes showed no major differences between the pre and post-vaccination G1P[8] strains. CONCLUSION: This laboratory based surveillance study shows significant reduction in reported RV cases and a shift in proportion from G1P[8] to G2P[4] strains after introduction of RV vaccination in Scotland. The genotyping data from a subset of the total reported RV cases will be used to ascertain cross protection against strains and identify vaccine induced RV strain shifts in the years to come.

Control of Clostridium difficile infection in the hospital setting.

Clostridium difficile infection (CDI) has emerged as a leading challenge in the control of healthcare-associated infection (HCAI). The epidemiology of CDI has changed dramatically, this is associated with emergence of 'hypervirulent' strains, particularly PCR ribotype 027. Despite the epidemic spread of these strains, there are recent reports of decreasing incidence from healthcare facilities where multi-facetted targeted control programs have been implemented. We consider these changes in epidemiology and reflect on the tools available to control CDI in the hospital setting. The precise repertoire of measures adopted and emphasis on different interventions will vary, not only between healthcare systems, but also within different institutions within the same healthcare system. Finally, we consider both the sustainability of reductions already achieved, and the potential to reduce CDI further. This takes account of newly emerging data on more recent changes in the epidemiology of CDI, and the potential of novel interventions to decrease the burden of disease.

Minimising risk in the use of central lines.

Central lines are inserted for a number of clinical reasons, including to measure central venous pressure and the regular administration of intravenous drugs. One of their benefits is that several treatments can be given simultaneously. However, they are invasive and tend to leave patients prone to infection, so careful management is vital. This article focuses on the risk of infection, particularly of the bloodstream.

Importance of hand hygiene during invasive procedures.

Invasive devices, such as urinary catheters and peripheral and central venous catheters, can form part of essential patient care and may provide life-saving support and treatment. However, the invasive nature of these devices and the vulnerability of patients can increase the risk of acquiring a healthcare-associated infection (HCAI). This article highlights the importance of best practice in relation to insertion and management of invasive devices, incorporating hand hygiene, to reduce the risk of HCAI. Although the information can be applied to invasive devices in general, the focus is on urinary catheters.

Design of stable nanocrystalline alloys.

Nanostructured metals are generally unstable; their grains grow rapidly even at low temperatures, rendering them difficult to process and often unsuitable for usage. Alloying has been found to improve stability, but only in a few empirically discovered systems. We have developed a theoretical framework with which stable nanostructured alloys can be designed. A nanostructure stability map based on a thermodynamic model is applied to design stable nanostructured tungsten alloys. We identify a candidate alloy, W-Ti, and demonstrate substantially enhanced stability for the high-temperature, long-duration conditions amenable to powder-route production of bulk nanostructured tungsten. This nanostructured alloy adopts a heterogeneous chemical distribution that is anticipated by the present theoretical framework but unexpected on the basis of conventional bulk thermodynamics.

Howardella ureilytica gen. nov., sp. nov., a Gram-positive, coccoid-shaped bacterium from a sheep rumen.

An unidentified obligately anaerobic, fastidious, Gram-positive, non-motile, non-spore-forming, non-fermentative coccoid-shaped bacterium (designated strain GPC 589(T)) was isolated from the rumen fluid of a sheep. The major fatty acid constituents (>5 %) were C(16 : 0) (29.2 %), C(18 : 0) (40.7 %) and an unidentified compound (19.7 %) with an equivalent chain-length of 13.523. The G+C content of the DNA was 34 mol%. The organism was strongly ureolytic and generated ATP through the hydrolysis of urea. Comparative 16S rRNA gene sequence analysis demonstrated that strain GPC 589(T) was far removed, phylogenetically, from the ruminococci and related Gram-positive anaerobic cocci but exhibited a phylogenetic association with Clostridium rRNA cluster XIVa [as defined by Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). Int J Syst Bacteriol 44, 812-826]. Sequence divergence values of 12.5 % or more were observed between strain GPC 589(T) and all other recognized species within this and related rRNA clostridial clusters. Phylogenetic analysis showed that strain GPC 589(T) represents a new genus within cluster XIVa. On the basis of both phylogenetic and phenotypic evidence, it is proposed that strain GPC 589(T) should be classified as representing a new genus and novel species, Howardella ureilytica gen. nov., sp. nov. The type strain is strain GPC 589(T) (=DSM 15118(T)=JCM 13267(T)).

Proteolytic inactivation of the bovine spongiform encephalopathy agent.

Thermostable proteases have been investigated for their ability to provide a novel biological solution to decontamination of prion agents responsible for transmissible spongiform encephalopathies (TSEs). Proteases were identified that digested total mouse brain homogenate (MBH) protein from uninfected mice. These proteases were then evaluated for digestion of BSE (301V) infectious MBH over a range of pH and temperatures, screened for loss of anti-prion antibody 6H4 immunoreactivity and protease-treated infectious MBH assessed in mouse bioassay using VM mice. Despite a number of proteases eliminating all 6H4-immunoreactive material, only the subtilisin-enzyme Properase showed a significant extension in incubation period in mouse bioassays following a 30-min incubation at 60 degrees C and pH 12. These results demonstrate the potential of the method to provide a practical solution to the problems of TSE contamination of surgical instruments and highlight the inadequacy of using Western blot for assessment of decontamination/inactivation of TSE agents.

Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954.

A nitroreductase with distinct properties that can activate the prodrug 5-aziridinyl-2,4-dinitrobenzamide (CB 1954) was isolated from Bacillus amyloliquefaciens. The encoding gene was identified as a homologue of the ywrO of Bacillus subtilis, and was obtained as a PCR product by reverse genetics, cloned and the entire nucleotide sequence determined. The gene was found to reside between homologues of the B. subtilis alsD and yswB genes; however, the ywrO and yswB genes of B. amyloliquefaciens were not separated by a fourth gene, ywsA. The B. amyloliquefaciens ywrO gene was overexpressed, the recombinant protein purified and its properties were compared with those of two CB 1954-activating enzymes, Escherichia coli B nitroreductase (NTR) and Walker DT-diaphorase (DTD). In common with these enzymes menadione was an electron acceptor (K(m) 3 microM) and activity with this substrate was inhibited by the presence of dicoumarol (K(i) 1.0 microM). In contrast, YwrO showed a marked preference for NADPH as a cofactor (K(m) 40 microM) and therefore could not be classified as a DTD (EC 1.6.99.2). The flavin FMN was an acceptor with high affinity. B. amyloliquefaciens YwrO was shown to be a flavoprotein with a monomeric molecular mass of 21.5 kDa by calculation and SDS-PAGE. The cytotoxic 4-hydroxylamine derivative was the single CB 1954 reduction product, but B. amyloliquefaciens YwrO was inactive with the bischloroethyl analogue of CB 1954, SN 23862. In both of these properties B. amyloliquefaciens YwrO more closely resembles DTD than NTR. Its K(m) for CB 1954 was lower than that of NTR (617 microM compared to 862 microM). Enhanced in vitro cytotoxicity of CB 1954 was demonstrated on incubation of V79 cells with prodrug, NADPH and B. amyloliquefaciens YwrO. The work has led to the identification of a previously unknown nitroreductase, B. amyloliquefaciens YwrO, with distinct properties which will aid the rational selection of appropriate genes for applications in directed enzyme prodrug therapy (DEPT).