Sandeep Garg

Biofilm-Forming Abilities of Listeria monocytogenes Serotypes Isolated from Different Sources.

A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay revealed 62 (63.26%) strains as weak, 27 (27.55%) strains as moderate, and 9 (9.18%) strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015) was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 103 cells/cm2, 33±26× 103 cells/cm2, 5±3× 103 cells/cm2, respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI) analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids.

Siderophore-Producing Bacteria from a Sand Dune Ecosystem and the Effect of Sodium Benzoate on Siderophore Production by a Potential Isolate

Bioremediation in natural ecosystems is dependent upon the availability of micronutrients and cofactors, of which iron is one of the essential elements. Under aerobic and alkaline conditions, iron oxidizes to Fe+3 creating iron deficiency. To acquire this essential growth-limiting nutrient, bacteria produce low-molecular-weight, high-affinity iron chelators termed siderophores. In this study, siderophore-producing bacteria from rhizosphere and nonrhizosphere areas of coastal sand dunes were isolated using a culture-dependent approach and were assigned to 8 different genera with the predominance of Bacillus sp. Studies on the ability of these isolates to grow on sodium benzoate revealed that a pigmented bacterial culture TMR2.13 identified as Pseudomonas aeruginosa showed growth on mineral salts medium (MSM) with 2% of sodium benzoate and produced a yellowish fluorescent siderophore identified as pyoverdine. This was inhibited above 54 μM of added iron in MSM with glucose without affecting growth, while, in presence of sodium benzoate, siderophore was produced even up to the presence of 108 μM of added iron. Increase in the requirement of iron for metabolism of aromatic compounds in ecosystems where the nutrient deficiencies occur naturally would be one of the regulating factors for the bioremediation process.

Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers for phaC Gene of Bacillus megaterium

Bacillus megaterium is gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains of B. megaterium and closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of the polyhydroxyalkanoate synthase C gene of B. megaterium in designing primers that amplify the 0.9 kb region of the phaC for its identification. The PCR method was optimized to amplify 0.9 kb region of phaC gene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only in B. megaterium and was demonstrated by several strains of B. megaterium isolated from different habitats. The use of Method I did not result in the amplification of the phaC gene with other members of Bacillales. The specificity for identification of B. megaterium was confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulating B. megaterium and members of Bacillales.

Presence of a widely disseminated Listeria monocytogenes serotype 4b clone in India.

Details about the members of the Indian Listeria Consortium are provided in the Supplementary Data. Emerging Microbes and Infections (2016) 5, e55; doi:10.1038/emi.2016.55; published online 8 June 2016

Multi-Virulence-Locus Sequence Typing of 4b Listeria monocytogenes Isolates Obtained from Different Sources in India over a 10-Year Period

Listeria monocytogenes is an emerging foodborne pathogen responsible for listeriosis. The incidence of listeriosis has increased during the last 2 decades due to the increase in consumption of ready-to-eat foods and change in food consumption habits. Outbreaks and sporadic cases of listeriosis have been reported in developed countries. These reports have helped determine the safety practices needed to control listeriosis. Although L. monocytogenes has been reported from humans, animals, and a variety of foods in India, limited data exist with respect to prevalence and distribution of L. monocytogenes in the Indian subcontinent. The Indian Listeria Culture Collection Centre in Goa maintains all of the isolates received for subtyping and molecular characterization. Of the listerial isolate collection maintained by this center, three fourths of the isolates are of 4b serotype, while the number of other serotypes is very low. Therefore, we screened L. monocytogenes serotype 4b isolates to determine their relevance to previously defined epidemics and/or outbreaks using multi-virulence-locus sequence typing (MVLST). A total of 25 isolates in serogroup 4b of L. monocytogenes were randomly selected from a repository of 156 L. monocytogenes 4b isolates obtained from different sources in India over a period of 10 years. MVLST sequence types (virulence types, VTs) were compared to known epidemic clones and other known isolates in the L. monocytogenes MVLST database. The 25 isolates were grouped into three clusters. Cluster I comprised 21 isolates including animal (n=9), human (n=4), and food (n=8), which matched Epidemic Clone I (ECI, VT20). Three isolates-two from animal and one from food-formed a cluster while a single animal isolate was placed into two novel VTs (VT98 and VT99), respectively. Based on these findings, it can be inferred that ECI has been isolated from a variety of sources and places and has persisted in India for at least 10 years.

Shake flask optimization of β-carotene production in Rhodotorula graminis RC04

The present investigation was focused on the effects and interactions of media components on the production of β-carotene by Rhodotorula graminis. Mannitol, potassium nitrate, yeast extract, monopotassium hydrogen phosphate and magnesium sulfate medium components were screened using Plackett Burman Design to identify critical factors. Initial pH of 5.5 of the medium was found to be optimum for β-carotene production. Results suggest that higher amounts of mannitol, yeast extract, potassium nitrate and magnesium sulfate would positively affect both biomass and β-carotene accumulation. Yeast extract was critical for both β-carotene and biomass accumulation. Central composite design was used to study the impact of interaction of mannitol and yeast extract on biomass accumulation and β-carotene production. Concentration of yeast extract was found more significant than mannitol for β-carotene production. From predicted values obtained using fitted binomial equations, it was seen that mannitol should be used in the range of 10 to 20 g/L and yeast extract in the range of 9.5 to 10 g/L to achieve a biomass of 3.8 to 4.3 g/L and a β-carotene content of 190 to 220 µg/L. Key words : Mannitol, yeast extract, Plackett Burman Design (PBD), central composite design, response surface methodology, yeast, β-carotene.

Mapping Anopheles stephensi midgut proteome using high-resolution mass spectrometry

Anopheles stephensi Liston is one of the major vectors of malaria in urban areas of India. Midgut plays a central role in the vector life cycle and transmission of malaria. Because gene expression of An. stephensi midgut has not been investigated at protein level, an unbiased mass spectrometry-based proteomic analysis of midgut tissue was carried out. Midgut tissue proteins from female An. stephensi mosquitoes were extracted using 0.5% SDS and digested with trypsin using two complementary approaches, in-gel and in-solution digestion. Fractions were analysed on high-resolution mass spectrometer, which resulted in acquisition of 494,960 MS/MS spectra. The MS/MS spectra were searched against protein database comprising of known and predicted proteins reported in An. stephensi using Sequest and Mascot software. In all, 47,438 peptides were identified corresponding to 5,709 An. stephensi proteins. The identified proteins were functionally categorized based on their cellular localization, biological processes and molecular functions using Gene Ontology (GO) annotation. Several proteins identified in this data are known to mediate the interaction of the Plasmodium with vector midgut and also regulate parasite maturation inside the vector host. This study provides information about the protein composition in midgut tissue of female An. stephensi, which would be useful in understanding vector parasite interaction at molecular level and besides being useful in devising malaria transmission blocking strategies. The data of this study is related to the research article “Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes”.

Variations in Stress Tolerance Abilities of Diverse Listeria monocytogenes Isolates

1 Department of Pathology, Nagpur Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur 440006, India 2 Institute of Medical Microbiology, Justus-Liebig University, 35392 Giessen, Germany 3 ICAR-National Institute of Biotic Stress Management, Baronda, Raipur, 493225, India 4 Department of Microbiology, Goa University, Taleigaon Plateau, Goa 403 206, India 5 Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, 243122, India *Corresponding author