A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in <Emphasis Type="Italic">Enterococcus</Emphasis> species

Background  

Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene.     

Structural and Functional Highlights of Vacuolar Soluble Protein 1 from Pathogen Trypanosoma brucei brucei

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis, or sleeping sickness. The causative parasite, Trypanosoma, encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. The latter are acidic membrane-enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg²⁺ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from Tb_bVSP1 crystal structure is further confirmed by SAXS and TEM data. SAXS data suggest structural flexibility in EF hand domains indicative of conformational plasticity within Tb_bVSP1.     

Low risk of relapse and deformity among leprosy patients who completed multi-drug therapy regimen from 2005 to 2010: A cohort study from four districts in South India

IntroductionRelapse of leprosy among patients released from treatment (RFT) is an indicator of the success of anti-leprosy treatment. Due to inadequate follow-up, relapse in leprosy patients after RFT is not systematically documented in India. Relapsed leprosy patients pose a risk in the transmission of leprosy bacilli. We determined the incidence of relapse and deformity among the patients RFT from the leprosy control programme in four districts in South India.MethodsWe conducted two follow-up surveys in 2012 and 2014 among the leprosy patients RFT between 2005 and 2010. We assessed them for any symptoms or signs of relapse, persistence and deformity. We collected slit skin samples (SSS) for smear examination. We calculated overall incidence of relapse and deformity per 1000 person-years (PY) with 95% confidence intervals (CI) and cumulative risk of relapse.ResultsOverall, we identified 69 relapse events, 58 and 11, during the first and second follow-up surveys, respectively. The incidence of relapse was 5.42 per 1000 PY, which declined over the years after RFT. The cumulative risk of relapse was 2.24%. The rate of deformity among the relapsed patients was 30.9%. The overall incidence of deformity was 1.65 per 1000 person years. The duration of M. leprae detection in smears ranged between 2.38 and 7.67 years.ConclusionsLow relapse and deformity rates in leprosy RFT patients are indicative of treatment effectiveness. However, a higher proportion of detection of deformity among relapsed cases is a cause for concern. Periodic follow-up of RFT patients for up to 3 years to detect relapses early and ensure appropriate treatment will minimize the development of deformity among relapsed patients.     

Feeding deterrence,larvicidal and haemolymph protein profiles of an Indian traditional medicinal plant Alangium salviifolium (L.F.) Wangerin on cluster caterpillar,Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae)

Different organic solvent crude extracts from the leaves of Alangium salviifolium (L.F.) Wangerin, were tested for their feeding deterrence, larvicidal activity and protein concentrations on the fourth instar larvae of Spodoptera litura L. under laboratory conditions. Treatments were given through two different host plant leaves such as brinjal and castor. The bioassay was carried out at different concentrations viz., 0.625, 1.25, 2.5 and 5%. The maximum antifeedant activity in brinjal (52.0%) and castor (29.97%) leaf discs was recorded in ethyl acetate extract as compared with hexane and chloroform extracts at 5% concentration. The result of larvicidal activity revealed that the maximum larval mortality was registered in brinjal leaves (65.81%) and castor with (57.48%) in ethyl acetate extract as compared to hexane and chloroform extracts at 5% concentration. The ethyl acetate extract of A. salviifolium contained alkaloids, diterpenoids, and saponins. The treatment also reduced haemolymph protein concentration after 48 h.     

On the mechanism of formation of arterenone in insect cuticular hydrolyzates

Arterenone (2-amino-3′,4′-dihydroxy acetophenone) is an important hydrolytic product generated from lightly colored sclerotized cuticle that use N-acyldopamine derivatives for crosslinking reactions. It seems to arise from 1,2-dehydro-N-acetyldopamine (dehydro NADA) that has been crosslinked to the cuticular components. However, the mechanism of generation of arterenone, which has two protons on the α-carbon and no proton on the β-carbon atom from dehydro NADA crosslinks that have one proton each on these two side chain carbons, remained elusive and undetermined. To investigate the mechanism of this transformation, we synthesized specifically labeled β-deuterated dehydro NADA and incubated with Sarcophaga bullata cuticle undergoing larval puparial transformation. We also isolated the dimeric products formed during the tyrosinase-mediated oxidation of dehydro NADA. Hydrolysis of both β-deuterated dehydro NADA treated cuticle and β-deuterated dehydro NADA dimer generated arterenone as the major hydrolytic product. Liquid chromatography-mass spectrometric analysis of this arterenone revealed the retention of deuterium from the β-position of dehydro NADA at the α-carbon atom of arterenone. Hydrolysis of β-deuterated dehydro NADA also generated the labeled arterenone under oxidative conditions, but not under anaerobic conditions. These results indicate the unique hydride shift from β-carbon to α-carbon during acid hydrolysis and reveal the mechanism of liberation of arterenone and related compounds from dehydro NADA linked cuticle.     

Drosophila yellow-h encodes dopaminechrome tautomerase: A new enzyme in the eumelanin biosynthetic pathway

Melanin is a widely distributed phenolic pigment that is biosynthesized from tyrosine and its hydroxylated product, dopa, in all animals. However, recent studies reveal a significant deviation from this paradigm, as insects appear to use dopamine rather than dopa as the major precursor of melanin. This observation calls for a reconsideration of the insect melanogenic pathway. While phenoloxidases and laccases can oxidize dopamine for dopaminechrome production, the fate of dopaminechrome remains undetermined. Dopachrome decarboxylase/tautomerase, encoded by yellow-f/f2 of Drosophila melanogaster, can convert dopaminechrome into 5,6-dihydroxyindole, but the same enzyme from other organisms does not act on dopaminechrome, suggesting the existence of a specific dopaminechrome tautomerase (DPT). We now report the identification of this novel enzyme that biosynthesizes 5,6-dihydroxyindole from dopaminechrome in Drosophila. Dopaminechrome tautomerase acted on both dopaminechrome and N-methyl dopaminechrome but not on dopachrome or other aminochromes tested. Our biochemical and molecular studies reveal that this enzyme is encoded by the yellow-h gene, a member of the yellow gene family, and advance our understanding of the physiological functions of this gene family. Identification and characterization of DPT clarifies the precursor for melanin biosynthetic pathways and proves the existence of an independent melanogenic pathway in insects that utilizes dopamine as the primary precursor.     

Karyomorphological effects of two new oil formulations on Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

The cotton bollworm Helicoverpa armigera is a serious pest of many economically important crops. Since this pest has become resistant to the conventional synthetic insecticides, newer compounds and formulations are being developed against this insect pest. Many natural compounds isolated from the plants were tested against this pest. Among them Hyptis suaveolens and Melochia corchorifolia showed insecticidal properties against H. arnigera. Based on bioefficacy studies, caryophyllene and β-sitosterol were isolated from H. suaveolens and M. corchorifolia respectively. The isolated natural compounds were further developed as formulations in various combinations with neem (Azadirachta indica) and karanj (Pongamia pinnata) oils. The present study indicated that the formulations influenced the karyomorphology of H. armigera.     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.