Whole Genome Mining and Characterization of a New Probiotic Strain Levilactobacillus brevis ILSH3 from Handia: An Ethnic Fermented Beverage of Odisha, India.

Isolation and characterization of probiotics from traditional fermented food have contributed many beneficial strains to the field of health and nutritional sciences. Handia, a traditional fermented alcoholic beverage popular in different parts of Odisha, was our source of isolation. This study characterizes one such potential bacteria, Levilactobacillus brevis ILSH3 (H3) isolated from Handia. The investigation for the probiotic attributes as per ICMR-DBT guidelines qualified the checkpoint assays like acid and bile tolerance, bile salt hydrolase activity, antimicrobial properties, and pathogen exclusion ability. The whole genome sequence of H3 (2,460,966 bp in size with GC content of 45.62%) was subjected to comparative genome analysis for its taxonomic identification and validation of probiotic potential. Various genes pertaining to its probiotic potential were identified in the genome and it showed zero matches against any pathogenic families. Metabolite profiling of cell-free supernatant using liquid chromatography-mass spectrometry revealed the presence of essential amino acids, short-chain fatty acids, antimicrobial molecules, immunomodulatory molecules, and flavor/aroma-enhancing compounds. Immunomodulatory property investigation using Bioplex and qRT-PCR showed a reduction in the levels of pro-inflammatory cytokines in L. brevis ILSH3-treated Caco-2 cells. Collectively, the results demonstrate that this Handia-origin bacteria Levilactobacillus brevis ILSH3 possesses desirable attributes of a probiotic, which is now open for nutritional and health biologists to explore. This new probiotic strain may show promising results when utilized in healthcare or functional foods.

Immuno-stimulatory effect and toxicology studies of salt pan bacteria as probiotics to combat shrimp diseases in aquaculture.

The shrimp aquaculture industry has experienced serious economic losses due to diseases caused by Vibrio species. The application of antibiotics to combat diseases has led to environmental hazards, antibiotic-resistance in pathogens and accumulation of antibiotics in tissues. This study explores the use of probiotics as an alternative to antibiotics. A probiotic consortium SFSK4 (comprising salt pan bacteria Bacillus licheniformis TSK71, Bacillus amyloliquefaciens SK27, Bacillus subtilis SK07, Pseudomonas sp. ABSK55) was used as a water additive during shrimp culture. It significantly increased shrimp (Litopenaeus vannamei) immunity i.e. total hemocyte count, phagocytosis, total plasma protein, respiratory burst and bactericidal activity as compared to the control. It also stimulated the phenoloxidase activity by two-fold. Proteomic analysis revealed the differential expression of 50 immune proteins (39 up-regulated and 11 down-regulated) in SFSK4 treated shrimps. Four major immune modulation proteins viz. Caspase2, GTPase activating protein, Hemocyanin and Glucan pattern-recognition lipoprotein involved in cell mediated immune response were identified in SFSK4 treated shrimp hemolymph. SFSK4 decreased shrimp mortality by more than 50% against pathogens. Toxicology studies revealed that administration of the highest dose of probiotic (1012 CFU/mL) showed no adverse effect on shrimp survival (LC50 analysis) and neither exhibited cytotoxicity. Genotoxicity study confirmed that the probiotic did not cause DNA damage in shrimps. The findings suggest that the probiotic SFSK4 is an eco-friendly water additive to enhance shrimp immunity against diseases in aquaculture, which could help curtail environmental hazards as an effective alternative to antibiotics.

Psychrotolerant Antarctic bacteria biosynthesize gold nanoparticles active against sulphate reducing bacteria.

This study evaluates the biosynthesis of gold nanoparticle (GNP) using Antarctic bacteria and assesses its potential antibacterial activity on sulfate-reducing bacteria (SRB). The GNPs were biosynthesized at distinct temperatures (4°, 10°, 25°, 30° and 37° C) using bacterial isolate GL1.3, obtained from Antarctic lake water. Biochemical and phylogenetic analysis concluded that the isolate GL1.3 belongs to Bacillus sp. The GNP biosynthesis was achieved at all the incubation temperatures (4°, 10°, 25°, 30° and 37° C) only during the log phase of growth. These formed nanoparticles were identified by UV-Visible spectroscopy, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), transmission electron microscopy (TEM) and X-ray diffraction (XRD) to be of size 30-50 nm. These GNPs exhibited antibacterial activity against SRB (Desulfovibrio sp.) evaluated by broth micro-dilution method. At 200 µg mL-1 GNP concentrations, being the minimal inhibitory concentration (MIC), the growth rate and sulfate reducing activity of Desulfovibrio sp. were reduced by 12% and 7% respectively. Comet assay revealed that the genotoxic effect of GNP on SRB is responsible for the inhibition of its growth and sulfide production. This showed that the Antarctic microbes could be useful for GNP synthesis even under psychrophilic conditions for various biomedical applications.

Co-selection of multi-antibiotic resistance in bacterial pathogens in metal and microplastic contaminated environments: An emerging health threat.

Misuse/over use of antibiotics increases the threats to human health since this is a main reason behind evolution of antibiotic resistant bacterial pathogens. However, metals such as mercury, lead, zinc, copper and cadmium are accumulating to critical concentration in the environment and triggering co-selection of antibiotic resistance in bacteria. The co-selection of metal driven antibiotic resistance in bacteria is achieved through co-resistance or cross resistance. Metal driven antibiotic resistant determinants evolved in bacteria and present on same mobile genetic elements are horizontally transferred to distantly related bacterial human pathogens. Additionally, in marine environment persistent pollutants like microplastics is recognized as a vector for the proliferation of metal/antibiotics and human pathogens. Recently published research confirmed that horizontal gene transfer between phylogenetically distinct microbes present on microplastics is much faster than free living microbes. Therefore, microplastics act as an emerging hotspot for metal driven co-selection of multidrug resistant human pathogens and pose serious threat to humans which do recreational activities in marine environment and ingest marine derived foods. Therefore, marine environment co-polluted with metal, antibiotics, human pathogens and microplastics pose an emerging health threat globally.

Native hypersaline sulphate reducing bacteria contributes to iron nanoparticle formation in saltpan sediment: A concern for aquaculture.

A hypersaline dissimilatory sulphate reducing bacterium, strain LS4, isolated from the sediments of Ribander saltpan, Goa, India was found to produce (Fe2O3) maghemite nanoparticles. The presence of maghemite nanoparticles was also detected in the same sediment. Strain LS4 was isolated anaerobically on modified Hatchikian's media at 300 psu, growing optimally at 30 °C, 150 psu salinity and pH 7.8. Based on biochemical characteristics and 16S rRNA sequence analysis, the strain LS4 belongs to genus Desulfovibrio. This isolate synthesized iron oxide nanoparticles in vitro when challenged with FeCl3 & FeSO4 in the growth medium. The biological nanoparticles were characterized to be Fe2O3 nanoparticle of 19 nm size by X-ray diffraction, transmission electron microscopy, fourier transform infrared spectroscopy, scanning electron microscopy and energy-dispersive x-ray spectroscopy. Maghemite nanoparticles (5.63 mg g-1) were isolated from the saltpan sediment by magnetic separation which showed similar characteristic features to the Fe2O3 nanoparticle produced by strain LS4 with an average size of 18 nm. Traditionally Goan saltpans were used for aquaculture during the non-salt making season, thus effects of these nanoparticles on Zebra fish embryo development were checked, which resulted in developmental abnormalities and DNA damage in a dose dependent manner. With the increasing nanoparticle concentration (0.1 mg.L-1 to100 mg.L-1), the mortality rate increased with a decrease in the hatching rate (93.05 ± 2.4 to 25 ± 4.16%) and heart rate (150-120 beats per minute). The nanoparticle exposed embryos developed malformed larvae with a characteristic of pericardial edema, curved body, curved notochord, curved tail and curved tail tip. These results suggest that strain LS4 might be playing a role as a contributor in the formation of iron oxide nanoparticle in the Ribander saltpan sediment, however; its high concentration will have a negative impact on aquaculture in these saltpans.

Effects of iron nanoparticles on iron-corroding bacteria.

The toxicological effects of Fe3O4 nanoparticles were evaluated with an iron-corroding bacterium (ICB) for preventing the biocorrosion of iron. Fe3O4 nanoparticles of 18 nm were successfully prepared and characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD) patterns and scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS). A halophilic ICB strain L4 was isolated from Ribandar saltpan Goa, India and identified biochemically and by 16S rRNA gene sequence analysis as Halanaerobium sp. The Fe3O4 nanoparticles in increasing doses (0.1-100 mg/L) caused transformation in growth and sulfide production of ICB strain L4. SEM-EDS analysis revealed a deformed cell structure with adsorption of nanoparticle on the cell surface and increased cell size. Comet assay revealed genotoxic effect of Fe3O4 nanoparticles on strain L4 which resulted in dose-dependent DNA damage by increasing percentage tail DNA from 5 to 88% with increasing Fe3O4 nanoparticles concentration. Furthermore, sulfide production rate was reduced to 11.8% in presence of 100 mg/L Fe3O4 nanoparticles which reduced the corroding property of ICB strain L4; thus, it was unable to corrode the iron nail in presence of Fe3O4 nanoparticle. This work suggests the possible application of Fe3O4 nanoparticle in addressing biocorrosion problems faced by different industries.