Effects of Flavonoids and Antibiotics Combination on Preformed Biofilms and Small RNA of Staphylococcus aureus.

Antibiotic resistance of Staphylococcus aureus has considerably increased among non-clinical or asymptomatic individuals. The formation of biofilms denies antimicrobial access to its targets present on the surface and inside the cell. The present study tested the effect of the combination of flavonoids and antibiotics over the preformed biofilms of S. aureus. The eradication of the preformed biofilms was analyzed using the crystal violet method. It has shown that 2500 µg mL-1 Rutin and 100 µg mL-1 Erythromycin (MIC Concentration) combination efficiently reduced the growth of the cells, which were adhered to the surfaces forming the biofilms. Fluorescence microscopic analysis indicated that the Rutin and Erythromycin (MIC value) combinations could eradicate the preformed biofilm cells more efficiently than other combinations. We found that the flavonoids and antibiotics with MIC concentration show a significant effect over the preformed biofilms cells of S. aureus. In addition, the semi-quantitative real-time PCR analysis for the sRNAs under the treatment of Rutin and Erythromycin combinations showed that few small RNAs expression (SprF, SprG, ArtR, Teg49, Teg41, and RNAIII) are getting downregulated upon the treatment; but again recovers with the incubation time interval increases. Combinations have a significant effect on Teg49 where there is a very faint intensity of the band, but for other small RNAs, there is an irregular pattern on the gel image. It has been concluded that at the initial period of incubation, the combinations have an effect on all the sRNAs but once the incubation increases, the effects have been slowly decreasing. It has been concluded that the combination has been able to reduce the doubling time of S. aureus upon treatment. Whereas, the small RNAs used in the study can be further evaluated for expression profiling through qRT-PCT.

Development of multiplex HRM-based loop-mediated isothermal amplification method for specific and sensitive detection of Treponema pallidum.

Syphilis is a sexually transmitted disease caused by the spirochaete bacterium Treponema pallidum. This study has developed a multiplex High-Resolution Melt-curve Loop-mediated isothermal amplification (multiplex HRM-LAMP) assay targeting the marker genes polA and tprL to detect T. pallidum. The multiplex HRM-LAMP assay conditions were optimized at 65 °C for 45 min. Real-time melt-curve analysis of multiplex HRM-LAMP shows two melt-curve peaks corresponding to polA and tprL with a Tm value of 80 ± 0.5 °C and 87 ± 0.5 °C, respectively. The detection limit of multiplex HRM-LAMP was found to be 6.4 × 10-4 ng/µL (3.79 copies/µL) of T. pallidum. The specificity was evaluated using seven different bacterial species, and the developed method was 100% specific in detecting T. pallidum. A total of 64 blood samples of T. pallidum suspected cases were used to validate the assay method. The clinical validation showed that the assay was 96.43% sensitive and 100% specific in detecting syphilis. Thus, the developed method was more rapid and sensitive than other available methods and provides a multigene-based diagnostic approach to detect T. pallidum.

Genome analysis of alginate synthesizing Pseudomonas aeruginosa strain SW1 isolated from degraded seaweeds.

Pseudomonas aeruginosa strain SW1 is an aerobic, motile, Gram-negative, and rod-shaped bacterium isolated from degraded seaweeds. Based on the 16S rRNA gene sequence and MALDI TOF analysis, strain SW1 exhibits 100% similarity to P. aeruginosa DSM 50,071, its closest phylogenetic neighbor. The complete genome of strain SW1 consists of a single circular chromosome with 23,258,857 bp (G + C content of 66%), including 6734 protein-coding sequences, 8 rRNA, and 63 tRNA sequences. The genome of the P. aeruginosa SW1 contains at least 27 genes for the biosynthesis of alginate and other exopolysaccharide involved in biofilm formation. KAAS and GO analysis and functional annotation by COG and CAZymes are consistent with the biosynthesis of alginate. In addition, the presence of antimicrobial resistance, multi-efflux operon, and antibiotic inactivation genes indicate a pathogenic potential similar to strain DSM50071. The high-quality genome and associated annotation provide a starting point to exploit the potential for P. aeruginosa to produce alginate.

Correction pen based paper fluidic device for the detection of multiple gene targets of Leptospira using Loop Mediated Isothermal Amplification.

Paper-based nucleic acid testing techniques are increasingly in demand. Hence, we have developed a simple and cheap paper fluidic device to detect multiple gene targets in Leptospira. Fluidic channels of the penta-clover device are drawn using a correction pen on Whatman filter paper 1. The fluid blocks the pores of the paper, avoiding leakage and ensuring the equal flow of sample to the reaction pads. The target genes are amplified by performing Loop-Mediated Isothermal Amplification (LAMP) with dry reaction components. Thecolor change of leuco crystal violetallows real-time monitoring of a positive amplification. The difference in color intensity is captured with a smartphone and analyzed using image processing software. The device amplifies the target within 15 min, detects the pathogen at a concentration as low as 50 attogram µL-1, detects Leptospira in blood samples without prior treatment and differentiates the Leptospira species even after 21 days of storage at room temperature.

Detection of microcystin-producing cyanobacteria in water samples using loop-mediated isothermal amplification targeting mcyB gene.

Microcystin toxin-producing cyanobacteria are known to have harmful effects on humans and animals. We have developed a loop-mediated isothermal amplification (LAMP)-based detection method by targeting the microcystin synthetase B gene (mcyB), the gene responsible for the production of microcystin. The sensitivity of the method was found to be 1 fg per reaction, and it was 1000-fold higher than the conventional PCR. The LAMP method was able to amplify the target gene with a minimum amount of dNTP (0.4 mM), which further reduces the cost of reaction. The improved LAMP assay could detect the presence of the toxin-producing cyanobacteria in water samples within 2 h of time, which demonstrates the rapidness of the method. Freshwater samples were screened using the developed LAMP, and seven water samples collected from lakes and a bird sanctuary tested positive for mcyB gene harboring cyanobacteria, and negative in all other drinking waters. Hence, the developed LAMP could be a possible alternative to the existing molecular methods for screening for microcystin in environmental samples with greater sensitivity.

Genome-wide DNA polymorphisms in Kavuni, a traditional rice cultivar with nutritional and therapeutic properties.

Although rice genome was sequenced in the year 2002, efforts in resequencing the large number of available accessions, landraces, traditional cultivars, and improved varieties of this important food crop are limited. We have initiated resequencing of the traditional cultivars from India. Kavuni is an important traditional rice cultivar from South India that attracts premium price for its nutritional and therapeutic properties. Whole-genome sequencing of Kavuni using Illumina platform and SNPs analysis using Nipponbare reference genome identified 1 150 711 SNPs of which 377 381 SNPs were located in the genic regions. Non-synonymous SNPs (62 708) were distributed in 19 251 genes, and their number varied between 1 and 115 per gene. Large-effect DNA polymorphisms (7769) were present in 3475 genes. Pathway mapping of these polymorphisms revealed the involvement of genes related to carbohydrate metabolism, translation, protein-folding, and cell death. Analysis of the starch biosynthesis related genes revealed that the granule-bound starch synthase I gene had T/G SNPs at the first intron/exon junction and a two-nucleotide combination, which were reported to favour high amylose content and low glycemic index. The present study provided a valuable genomics resource to study the rice varieties with nutritional and medicinal properties.

Whole genome sequencing and analysis of Swarna, a widely cultivated indica rice variety with low glycemic index.

Swarna is a popular cultivated indica rice variety with low glycemic index (GI) but its genetic basis is not known. The whole genome of Swarna was sequenced using Illumina's paired-end technology, and the reads were mapped to the Nipponbare reference genome. Overall, 65,984 non-synonymous SNPs were identified in 20,350 genes, and in silico analysis predicted that 4,847 of them in 2,214 genes may have deleterious effect on protein functions. Polymorphisms were found in all the starch biosynthesis genes, except the gene for branching enzyme IIa. It was found that T/G SNP at position 246, 'A' at position 2,386, and 'C' at position 3,378 in the granule bound starch synthase I gene, and C/T SNP at position 1,188 in the glucose-6-phosphate translocator gene may contribute to the low GI phenotype in Swarna. All these variants were also found in the genome of another low GI indica rice variety from Columbia, Fedearroz 50. The whole genome analysis of Swarna helped to understand the genetic basis of GI in rice, which is a complex trait involving multiple factors.