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

Efficient extraction of small and large RNAs in bacteria for excellent total RNA sequencing and comprehensive transcriptome analysis.

BACKGROUND: Next-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform. RESULTS: TRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85&nbsp;&plusmn;&nbsp;0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique. CONCLUSIONS: Amresco\u27s Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run

A study on performance and emission characteristics of diesel engine using Ricinus Communis (Castor Oil) Ethyl Esters

Countries globally are focusing on alternative fuels to reduce environmental pollution. An example is biodiesel fuel, which is leading the way to other technologies. In this research, the methyl esters of castor oil were prepared using a two-step transesterification process. The respective properties of the castor oil (Ricinus Communis) biodiesel was estimated using ASTM standards. The effect of performance and emission on diesel engines was noted for four various engine loads (25%, 50%, 75%, and 100%), with two different blends (B5 and B20) and at two different engine speeds (1500 and 2000 rpm). The study determined that B5 and B20 samples at 1500 rpm engine speed obtained the same power, but diesel fuel generated greater control. The power increased at 2000 rpm for B5 samples, but B20 samples, as well as diesel, were almost the same values. In the 40–80% range, load, and load values were entirely parallel for each load observed from the engine performance of the brake power in all samples

Specific detection of fungal pathogens by 18S rRNA gene PCR in microbial keratitis

<p>Abstract</p> <p>Background</p> <p>The sensitivity and specificity of 18S rRNA polymerase chain reaction (PCR) in the detection of fungal aetiology of microbial keratitis was determined in thirty patients with clinical diagnosis of microbial keratitis.</p> <p>Methods</p> <p>Corneal scrapings from patients were used for Gram stain, culture and PCR analysis. PCR was performed with primer pairs targeted to the 18S rRNA gene. The result of the PCR was compared with conventional culture and Gram staining method. The PCR positive samples were identified by DNA sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. Main outcome measures were sensitivity and specificity of PCR in the detection of fungus in corneal keratitis.</p> <p>Results</p> <p>Combination of microscopy and culture gave a positive result in 11 of 30 samples of microbial keratitis. PCR detected 10 of 11 samples that were positive by conventional method. One of the 19 samples that was negative by conventional method was positive by PCR. Statistical analysis revealed that the PCR to have a sensitivity of 90.9% and specificity of 94.7% in the detection of a fungal aetiology in microbial keratitis.</p> <p>Conclusion</p> <p>PCR is a rapid, sensitive and useful method to detect fungal aetiology in microbial keratitis.</p

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors

Forward osmosis research trends in desalination and wastewater treatment: A review of research trends over the past decade

Issues of water scarcity and water security have driven the rapid development of various technologies to ensure water sustainability. The forward osmosis (FO) membrane process has been widely recognized as one of the more promising technologies to play an important role in alleviating the issues of water sustainability. Extensive research has been carried out worldwide to explore the potential of FO in desalination, water and wastewater treatment and reclamation. It is of the utmost importance to understand the topics of interest and research trends to further advance the development of FO process technology. In this study, a bibliometric analysis based on the Scopus database was carried out to identify and understand the global research trends of FO process based on 6 main analyses: basic growth trends, journals, countries, institutions, authors, and keywords. A total of 1462 article published between 1967-2018 were extracted from Scopus and used as the raw data for bibliometric analysis using VOSviewer software. The total number of FO articles has sharply increased since 2009 and stabilized at around 250 publications in the past three years. FO research started to diversify after the appearance of commercial FO membranes with improved characteristics, enabling the researchers to employ them for various application studies. Keywords analysis showed that the main directions of FO research could be categorized into three clusters: application of FO, membrane fouling study, and FO membrane synthesis. These bibliometric results provide a valuable reference and information on current research directions of FO for researchers and industry practitioners