Sarita G. Bhat

Isolation and partial characterization of ΦSP-1, a Salmonella specific lytic phage from intestinal content of broiler chicken

Salmonella enterica subsp. enterica serovar Enteritidis is a major causative agent of gastroenteritis with contaminated eggs and chicken meat being the major source of infection. Phages are seriously being considered as a safe and cheaper alternative to antibiotics. The intestinal content of chicken was used as source for isolating phages. Phage designated as ΦSP‐1 was selected for the study. Transmission electron microscopy (TEM) of phage ΦSP‐1 revealed that it belonged to family Podoviridae. The optimal multiplicity of infection (MOI) was 5 phages/cell. Latent and rise period were calculated to be 30 and 55 minutes respectively, while burst size was 44 phages/bacterial cell. The genome size of ΦSP‐1 was estimated to be 86 kb from pulsed‐field gel electrophoresis analysis (PFGE). The effect of different physical and chemical parameters like temperature, pH, salinity and CaCl2 were analyzed to optimize the conditions for large scale production of phages and to check the viability of ΦSP‐1 under different physiochemical conditions. A temperature of 40 °C, pH 8 and 0.25 M NaCl were found to be optimum for phage adsorption and it was able to survive up to a temperature of 50 °C for 3 min. Capability to survive under hostile environmental conditions, absence of virulence genes in genome and genus specificity suggest suitability of ΦSP‐1 to be used as a biocontrol agent.

Evaluation of five in situ lysis protocols for PCR amenable metagenomic DNA from mangrove soils

Highlights • Five different metagenomic DNA extraction methods were compared on mangrove soils from three islands.• Quantity and quality of the total community DNA were analysed.• PCR amenability of isolated DNA was evaluated employing amplification of 16S rRNA gene.• One among the protocols yielded PCR amenable DNA, while the protocol yielding highest concentration of DNA contained residual humic substance.

Amplification and sequence analysis of phaC gene of polyhydroxybutyrate producing Vibrio azureus BTKB33 isolated from marine sediments

Vibrios have shown their potential as polyhydroxyalkanoates (PHAs) producers, but until recently little information was available about their PHA-related genes. The present study attempts to characterize the phaC genes from a potent PHA-accumulating bacterium, Vibrio azureus strain BTKB33, isolated from marine sediments. The molecular detection of class I PHA synthase gene in the V. azureus strain BTKB33 gave the required amplicon and was confirmed by subsequent seminested PCR; however, a class II PHA synthase gene was not detected. The in silico characterization of the PCR product helped to deduce the presence of class I PHA synthase, particularly a polyhydroxybutyrate polymerase. Sequence alignment of the nucleotide sequence of class I PHA synthase of strain BTKB33 and other related Vibrio sp. showed intra-generic variation within genus Vibrio and this is revealed in the dendrogram. The multiple sequence alignment of the in silico-translated phaC gene of BTKB33 with the protein sequence of PHA synthase of related organisms showed the conserved regions of protein sequences within the genus Vibrio and the dendrogram constructed showed the relatedness based on the deduced amino acid sequences.

Biocontrol of Salmonella Enteritidis in spiked chicken cuts by lytic bacteriophages ΦSP-1 and ΦSP-3

The ability of host specific bacteriophages ΦSP‐1 and ΦSP‐3 to lyse Salmonella in artificially contaminated cuts of pressure cooked chicken meat was evaluated at different temperatures −4 °C, room temperature (28 ± 0.5 °C) and 37 °C applying low and high multiplicity of infection (MOI). Bacteriophages were able to significantly reduce the bacterial counts at all the temperatures studied. At 4 °C, individual application of Φ SP‐1and Φ SP‐3 resulted in significant drop in bacterial counts (log10 2.46 and 2.1 CFU/ml, respectively) at high MOI and (log10 0.98 and 0.52 CFU/ml, respectively) at low MOI, when compared to the untreated control on day 3. Similarly at room temperature the drop was log10 3.99 and 3.46 CFU/ml at high MOI and log10 2.51 and 2.3 CFU/ml at low MOI. At 37 °C the drop was log10 1.98 and 2.38 at high MOI and at low MOI it was log101.52 and 1.98 CFU/ml. Increased efficiency was observed when phages where applied as cocktail at high MOI as the bacterial counts at the end of day 3 dropped by log10 3.52 CFU/ml at 37 °C and to beyond detectable level at 4 °C and room temperature. The average reduction of bacterial load in the same group was −4 °C (79%), room temperature (92%) and 37 °C (78%).

Biofilms in Food Industry: Mitigation Using Bacteriophage

Abstract Phage therapy adds a new dimension to biofilm control, and can advance to future therapeutic approaches. The study focuses on Pseudomonas aeruginosa, a common pathogen capable of causing not only food borne diseases, but nosocomial infections. The sources for isolation of host and phage were milk samples from the local markets in Kochi, Kerala. P. aeruginosa isolates were identified and characterized using 16S rDNA sequence analysis, following amplification, sequencing, and BLAST. Phage was designated as ΦPAP-1. Transmission electron microscopy of phage ΦPAP-1 indicated characteristics of family Tectiviridae. The optimal multiplicity of infection was 5phages/cell. Latent and rise period were determined to be 30 and 60 min, respectively, while the burst size was found to be 60phages/bacterial cell. The effect of different physicochemical parameters showed that at 50°C, pH 8, and 0.1M NaCl were optimum, while only mannitol caused the least phage inactivation. The capability of ΦPAP-1 to survive under hostile environmental conditions and broad antibiofilm activity irrespective of narrow host range suggests the suitability of ΦPAP-1 as a biocontrol agent especially in food industry.

Metabarcoding data of bacterial diversity of the deep sea shark, Centroscyllium fabricii

This data article describes the bacterial diversity of the deep sea shark, Centroscyllium fabricii. The data was acquired by metabarcoding using 16S rDNA. Centroscyllium fabricii, a deep sea shark found at depths below 275 m was sampled during Sagar Sampada cruise no 305 in the Indian Ocean and metagenomic DNA was isolated from the gut contents using QIAamp DNA stool minikit. V3 region of 16S rDNA region was amplified and the amplicons were sequenced on Illumina MiSeq system using 151 bp × 2 paired end reads. The data of this metagenome is available in the BioSample Submission Portal as Bio-Project PRJNA431407and Sequence Read Archive (SRA) accession number SRR6507004.

Data on the characterization of non-cytotoxic pyomelanin produced by marine Pseudomonas stutzeri BTCZ10 with cosmetological importance

The article focuses on data dealing with characterization of black brown melanin produced by marine bacteria Pseudomonas stutzeri BTCZ10. Figures deal with the production of melanin by strain BTCZ10 and characterization of the pigment using biophysical techniques. Table presents the data on photo-protective ability of melanin when blended with commercial sunscreens.

PCR in Metagenomics

Metagenomics approach involves direct genetic analysis of environmental samples, evading the tedious culturing process. Polymerase chain reaction is one invaluable tool used for such analyses. Here, we describe one protocol for metagenomic DNA isolation that gives inhibitor-free DNA suitable for PCR and other genetic manipulations. Subsequently, the chapter describes the use of PCR as an indicator of quality of DNA and to amplify a marker of phylogeny. Further, the application of PCR for detection of specific genes and screening of metagenomic libraries is outlined.

Application of ΦSP-1 and ΦSP-3 as a therapeutic strategy against Salmonella Enteritidis infection using Caenorhabditis elegans as model organism.

The potential of Salmonella-specific phages ΦSP-1 and ΦSP-3 as biocontrol agents was studied in vitro, employing host cell lysis test and in vivo, using Caenorhabditis elegans as a model organism. For in vivo testing, stage 4 C. elegans larvae were experimentally infected with the pathogen Salmonella. Worm mortality was scored for 10 days. TD50 (the time required for 50% of the nematodes to die) of infected worms in the presence of bacteriophages was comparable to uninfected worms, and the two phages provided an increased protection than each one. This study in addition demonstrated the simplicity, elegance, and the cost effectiveness of the C. elegans model for in vivo validation.

Effect of Cobalt Stearate and Vegetable Oil on Uv and Biodegradation of Linear Low-Density Poly(Ethylene)-Poly(Vinyl Alcohol) Blends

The main objective of this study was to enhance the rate of UV and biodegradation of polyethylene by incorporating biodegradable materials and prooxidants. Prooxidants such as transition metal complexes are capable of initiating photooxidation and polymer chain cleavage, rendering the product more susceptible to biodegradation. In this work, the effect of (1) a metallic photoinitiator, cobalt stearate, and (2) different combinations of cobalt stearate and vegetable oil on the photooxidative degradation of linear low-density poly(ethylene)-poly(vinyl alcohol) (LLDPE/PVA) blend films has been investigated. For this, film-grade LLDPE was blended with different proportions of PVA. PVA is widely used in the industrial field, and recently it has attracted increasing attention as a water-soluble biodegradable polymer. Cobalt stearate and vegetable oil were added to the blends as prooxidants. The blends were prepared by melt mixing in a Thermo HAAKE Polylab system. Thin films containing these additives were prepared by a subsequent compression moulding process. The effect of UV exposure on LLDPE/PVA films in the presence as well as absence of these additives was investigated. Tensile properties, FTIR spectra, and scanning electron microscopy (SEM) were employed to investigate the degradation behaviour. It was found