High-throughput sequence analysis of bacterial communities and their predictive functionalities in traditionally preserved fish products of Sikkim, India.

Traditionally preserved fish products viz. suka ko maccha, a smoked fish product, sidra and sukuti, sun-dried fish products are commonly consumed in Sikkim state in India. Bacterial communities in these fish products were analysed by high-throughput sequence (HTS) method supported by bioinformatics tool. Metataxonomic of the overall bacterial communities in samples revealed the abundance of phylum Firmicutes followed by Proteobacteria. Psychrobacter was abundant genus in all traditionally preserved fish products of Sikkim, followed by Bacillus, Staphylococcus, Serratia, Clostridium, Enterobacter, Pseudomonas, Rummeliibacillus, Enterococcus, Photobacterium, Myroides, Peptostreptococcus, Plesiomonas and Achromobacter. Product-wise distribution showed that Bacillus was abundant in suka ko maacha and sidra samples, whereas Psychrobacter was abundant in sukuti samples. Unique genus to each product was observed on the basis of analysis of shared operational-taxonomic-unit (OTU) contents, Alpha diversity indices showed significantly differences among the samples, and also showed maximum coverage as per Good's coverage (0.99). Beta diversity showed clustering of bacterial compositions between suka ko maacha and sidra, whereas sukuti showed scattering pattern among the other samples, indicating a diverse population in suka ko maacha and sidra samples. Non-parametric analysis of abundant genera and predictive functionalities showed the complex bacterial inter-dependencies with predictive functionalities mostly in metabolism (79.88%).

Molecular Characterization of Bacteria, Detection of Enterotoxin Genes, and Screening of Antibiotic Susceptibility Patterns in Traditionally Processed Meat Products of Sikkim, India.

The lesser-known traditionally processed meat products such as beef kargyong, pork kargyong, satchu, and khyopeh are popular food items in the Himalayan state of Sikkim in India. The present study aimed to assess the microbiological safety of traditional meat products by identifying the potential spoilage or pathogenic bacteria, detecting the enterotoxins, and screening the antibiotic susceptibility patterns. The pH and moisture contents of the meat products varied from 5.3 to 5.9 and from 1.5 to 18%, respectively. The microbial loads of aerobic bacteria were 105 to 107 cfu/g, Staphylococcus 103 to 106 cfu/g, Bacillus 104 to 106 cfu/g, and total coliform 102 to 107 cfu/g, respectively. Based on 16S rRNA gene sequencing, the bacterial species isolated from traditionally processed meat products were Staphylococcus piscifermentans, Citrobacter freundii, Enterococcus faecalis, Salmonella enterica, Staphylococcus aureus, Citrobacter werkmanii, Klebsiella pneumoniae, Macrococcus caseolyticus, Klebsiella aerogenes, Staphylococcus saprophyticus, Pseudocitrobacter anthropi, Citrobacter europaeus, Shigella sonnei, Escherichia fergusonii, Klebsiella grimontii, Burkholderia cepacia, and Bacillus cereus. The enzyme-linked immunosorbent assay (ELISA) tests detected Salmonella spp. and enterotoxins produced by B. cereus well as Staphylococcus in a few tested samples. However, the PCR method did not detect the virulence genes of B. cereus and Salmonella in the isolates. Virulence gene (sea) was detected in S. piscifermentans BSLST44 and S. piscifermentans BULST54 isolated from beef kargyong and in S. aureus PSST53 isolated from pork kargyong. No enterotoxins were detected in khyopeh samples. The antibiotic sensitivity test showed that all bacterial strains were susceptible toward gentamicin, cotrimoxazole, norfloxacin, and trimethoprim. Gram-positive bacteria showed 100% sensitivity against clindamycin and erythromycin; however, 50% of the resistance pattern was observed against oxacillin followed by penicillin (33%) and ampicillin (27%).