A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species

<p>Abstract</p> <p>Background</p> <p>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 <it>E. faecalis </it>and <it>E. faecium </it>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 <it>Enterococcus </it>genus, <it>ddl </it>of <it>E. faecalis </it>and <it>E. faecium</it>, <it>aac</it>A-<it>aph</it>D that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as <it>van</it>A, <it>van</it>B, <it>van</it>C and <it>van</it>D and one internal control gene.</p> <p>Results</p> <p>Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to <it>E. faecalis</it>, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases.</p> <p>Conclusion</p> <p>The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common <it>Enterococcus </it>spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.</p

Immunohistochemical, histological and ultrastructural evaluation of protection provided by cholera vaccine against V. cholerae O139

In our previous study, complete protection was observed in rabbit immunized with 1 × 1010 CFU of live attenuated VCUSM21P vaccine against challenge with 1 × 109 CFU Vibrio cholerae O139. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological, immunohistochemical and ultrastructural techniques. Severe pathology is evident in wild type injected ileum in non-immunized, showing extensive villous destruction, edema, necrosis and inflammation with infiltration of large numbers of inflammatory cells, extensive damage to the villi and microvilli with pore formation. Histology of ileum injected with wild type in immunized rabbit shows no significant pathological changes except for a few inflammatory cells in lamina propria with mild edema in mucosa and submucosa. immunohistochemical staining revealed O139 antigens of wild type are seen in the lamina propria of edematous villi, muscularis mucosa and submucosa with weak presence in the muscle coat in non-immunized rabbit after challenged with wild type in non-immunized rabbits, but in immunized rabbit localisation of the O139 LPS antigen is seen at the tips of the intact villi, within lamina propria and muscularis mucosa only. These observations suggest that the vaccine can effectively protect animals from any pathologic changes and eliminate V. cholerae O139 from the immunized animals

Soil conservation issues in India

Despite years of study and substantial investment in remediation and prevention, soil erosion continues to be a major environmental problem with regard to land use in India and elsewhere around the world. Furthermore, changing climate and/or weather patterns are exacerbating the problem. Our objective was to review past and current soil conservation programmes in India to better understand how production-, environmental-, social-, economic- and policy-related issues have affected soil and water conservation and the incentives needed to address the most critical problems. We found that to achieve success in soil and water conservation policies, institutions and operations must be co-ordinated using a holistic approach. Watershed programmes have been shown to be one of the most effective strategies for bringing socio-economic change to different parts of India. Within both dryland and rainfed areas, watershed management has quietly revolutionized agriculture by aligning various sectors through technological soil and water conservation interventions and land-use diversification. Significant results associated with various watershed-scale soil and water conservation programmes and interventions that were effective for reducing land degradation and improving productivity in different parts of the country are discussed

Soil order knowledge as a driver in soil properties estimation from Vis-NIR spectral data – Case study from northern Karnataka (India)

International audienceVisible and near-infrared (Vis-NIR, 350–2500 nm) laboratory spectroscopy has been proven to provide soil properties estimations, such as clay or organic carbon (OC). However, the performances of such estimations may be dependent on pedological and spectral similarities between calibration and validation datasets. The objective of this study was to analyze how the soil order knowledge can be used to increase regression models performance for soil properties estimation. For this purpose, Random Forest regression models were calibrated and validated from both regional database (called regional models) and subsets stratified by soil order from the regional database (called soil-order models). The regional database contained 482 soil samples belonging to four soil orders (Alfisols, Vertisols, Inceptisols and Entisols) and associated with Vis-NIR laboratory spectra and six soil properties: OC, sand, silt, clay, cation exchange capacity (CEC) and pH. First, regional models provided i) high accuracy of some soil properties estimations when considering the regional strategy in the validation step (e.g., R2val of 0.74, 0.76 and 0.74 for clay, CEC and sand, respectively) but ii) modest accuracy of these same soil properties when considering subsets stratified by soil order from the regional database in validation step (e.g., R2val of 0.48, 0.58 and 0.38 over Vertisol for clay, CEC and sand, respectively). So the estimation accuracy appreciation is highly depending on the validation database as there is a risk of over-appreciated prediction accuracies at the soil-order scale when figures of merit are based on a regional validation dataset. Second, this work highlighted that the benefit of a soil-order model compared to a regional model for calibration depends on both soil property and soil order. So no recommendations for choosing between both models for calibration may be given. Finally, while Vis-NIR laboratory spectroscopy is becoming a popular way to estimate soil physicochemical properties worldwide, this work highlights that this technique may be used discreetly depending on the targeted scale and targeted soil type

Impact of bare soil pixels identification on clay content mapping using airborne hyperspectral AVIRIS-NG data: spectral indices versus spectral unmixing

Hyperspectral imaging spectroscopy has facilitated the mapping of soil properties at large scales, but since the presence of photosynthetic or non-photosynthetic vegetation affects the reflectance spectra, soil properties mapping is limited to bare soil surfaces. This study analyzed the impact of bare soil pixel identification on clay content estimation using two methods (i) combination of two spectral indices, Normalized Difference Vegetation Index for identifying photosynthetic vegetation and Cellulose Absorption Index for non-photosynthetic vegetation and (ii) spectral unmixing for estimating fractions of soil, photosynthetic and non-photosynthetic vegetation. The study used AVIRIS-NG image and laboratory measured clay content of 272 soil samples acquired over Karnataka, India. Bare soil pixels were identified using the two methods and performances of partial least squares regression (PLSR) models used to estimate the clay contents and the predicted clay content maps were analyzed and compared. PLSR model based on bare soil pixels identified by unmixing provided slightly better performances (R2val of 0.61) than spectral indices (R2val of 0.46), even though the percentage of study area mapped was reduced by half. This study highlighted that an improvement in prediction performance comes at the cost of reduction in spatial coverage in mapping of clay content

Construction and evaluation of V. cholerae O139 mutant, VCUSM21P, as a safe live attenuated cholera vaccine.

Cholera is a major infectious disease, affecting millions of lives annually. In endemic areas, implementation of vaccination strategy against cholera is vital. As the use of safer live vaccine that can induce protective immunity against Vibrio cholerae O139 infection is a promising approach for immunization, we have designed VCUSM21P, an oral cholera vaccine candidate, which has ctxA that encodes A subunit of ctx and mutated rtxA/C, ace and zot mutations. VCUSM21P was found not to disassemble the actin of HEp2 cells. It colonized the mice intestine approximately 1 log lower than that of the Wild Type (WT) strain obtained from Hospital Universiti Sains Malaysia. In the ileal loop assay, unlike WT challenge, 1×10⁶ and 1×10⁸ colony forming unit (CFU) of VCUSM21P was not reactogenic in non-immunized rabbits. Whereas, the reactogenicity caused by the WT in rabbits immunized with 1×10¹⁰ CFU of VCUSM21P was found to be reduced as evidenced by absence of fluid in loops administered with 1×10²-1×10⁷ CFU of WT. Oral immunization using 1×10¹⁰ CFU of VCUSM21P induced both IgA and IgG against Cholera Toxin (CT) and O139 lipopolysaccharides (LPS). The serum vibriocidal antibody titer had a peak rise of 2560 fold on week 4. Following Removable Intestinal Tie Adult Rabbit Diarrhoea (RITARD) experiment, the non-immunized rabbits were found not to be protected against lethal challenge with 1×10⁹ CFU WT, but 100% of immunized rabbits survived the challenge. In the past eleven years, V. cholerae O139 induced cholera has not been observed. However, attenuated VCUSM21P vaccine could be used for vaccination program against potentially fatal endemic or emerging cholera caused by V. cholerae O139

Synthesis, Characterization, Cytotoxicity Analysis and Evaluation of Novel Heterocyclic Derivatives of Benzamidine against Periodontal Disease Triggering Bacteria

Periodontal disease (PD) is multifactorial oral disease that damages tooth-supporting tissue. PD treatment includes proper oral hygiene, deep cleaning, antibiotics therapy, and surgery. Despite the availability of basic treatments, some of these are rendered undesirable in PD treatment due to side effects and expense. Therefore, the aim of the present study is to develop novel molecules to combat the PD triggering pathogens. The study involved the synthesis of 4-((5-(substituted-phenyl)-1,3,4-oxadiazol-2-yl)methoxy)benzamidine (5a-e), by condensation of 2-(4-carbamimidoylphenoxy)acetohydrazide (3) with different aromatic acids; and synthesis of 4-((4-(substituted benzylideneamino)-4H-1,2,4-triazol-3-yl)methoxy)benzamidine (6a-b) by treatment of compound 3 with CS2 followed by hydrazination and a Schiff reaction with different aromatic aldehydes. Synthesized compounds were characterized based on the NMR, FTIR, and mass spectrometric data. To assess the effectiveness of the newly synthesized compound in PD, new compounds were subjected to antimicrobial evaluation against P. gingivalis and E. coli using the micro-broth dilution method. Synthesized compounds were also subjected to cytotoxicity evaluation against HEK-293 cells using an MTT assay. The present study revealed the successful synthesis of heterocyclic derivatives of benzamidine with significant inhibitory potential against P. gingivalis and E. coli. Synthesized compounds exhibited minimal to the absence of cytotoxicity. Significant antimicrobial potential and least/no cytotoxicity of new heterocyclic analogs of benzamidine against PD-triggering bacteria supports their potential application in PD treatment

Synthesis, Characterization, Cytotoxicity Analysis and Evaluation of Novel Heterocyclic Derivatives of Benzamidine against Periodontal Disease Triggering Bacteria

Periodontal disease (PD) is multifactorial oral disease that damages tooth-supporting tissue. PD treatment includes proper oral hygiene, deep cleaning, antibiotics therapy, and surgery. Despite the availability of basic treatments, some of these are rendered undesirable in PD treatment due to side effects and expense. Therefore, the aim of the present study is to develop novel molecules to combat the PD triggering pathogens. The study involved the synthesis of 4-((5-(substituted-phenyl)-1,3,4-oxadiazol-2-yl)methoxy)benzamidine (5a-e), by condensation of 2-(4-carbamimidoylphenoxy)acetohydrazide (3) with different aromatic acids; and synthesis of 4-((4-(substituted benzylideneamino)-4H-1,2,4-triazol-3-yl)methoxy)benzamidine (6a-b) by treatment of compound 3 with CS2 followed by hydrazination and a Schiff reaction with different aromatic aldehydes. Synthesized compounds were characterized based on the NMR, FTIR, and mass spectrometric data. To assess the effectiveness of the newly synthesized compound in PD, new compounds were subjected to antimicrobial evaluation against P. gingivalis and E. coli using the micro-broth dilution method. Synthesized compounds were also subjected to cytotoxicity evaluation against HEK-293 cells using an MTT assay. The present study revealed the successful synthesis of heterocyclic derivatives of benzamidine with significant inhibitory potential against P. gingivalis and E. coli. Synthesized compounds exhibited minimal to the absence of cytotoxicity. Significant antimicrobial potential and least/no cytotoxicity of new heterocyclic analogs of benzamidine against PD-triggering bacteria supports their potential application in PD treatment

Not Available

Not AvailableGroundwater sources are drastically changing in their quantity and quality depending on local and regional level natural andanthropogenic factors, influencing their suitability for drinking and irrigation purposes. The objective of this study is to characterizethe hydrochemistry and assess the groundwater quality in the fluvial deltaic plains of Cauvery river basin, Tamil Nadu,India. A total of 50 georeferenced groundwater samples were collected across Needamangalam block of Thiruvarur district and analyzed for major ions and hydrochemical processes. The results showed an ionic sequence of Cl− > Na+ > HCO3− > Mg2+ >Ca2+ > CO32− > SO42− > K+ based on their relative proportions. The scatter diagram indicated that groundwater chemistry wasmostly influenced by weathering dominance followed by evaporation and silicate weathering. The dominant hydro-chemical facies were Na+-Cl−-HCO3− type, Na+-Mg2+-Cl−-HCO3− type, Na+-Cl−-HCO3−-CO3 2− type and Na+-Mg2+-Cl−-HCO3−-CO3 2−type influenced by the ion-exchange reaction. Most of the groundwater samples are suitable for drinking and irrigation except few with higher Na+ and Cl− content caused by the mixing of salt from fluvio-marine sources or agriculture return flow. The high sodium content in irrigation water may affect the soil hydraulic and nutrient properties in the long runNot Availabl