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Phenotypic and Genotypic Identification of Bacteria Isolated From Traditionally Prepared Dry Starters of the Eastern Himalayas.

ABSTRACT: Preparation of dry starters for alcohol production is an age-old traditional technology in the Eastern Himalayan regions of east Nepal, the Darjeeling hills, Sikkim, and Arunachal Pradesh in India, and Bhutan. We studied the bacterial diversity in 35 samples of traditionally prepared dry starters, represented by marcha of Nepal, Sikkim, the Darjeeling hills, and Bhutan, phab of Bhutan, and paa, pee, and phut of Arunachal Pradesh, respectively. Populations of bacteria in these starters were 10⁵ to 10⁸ cfu/g. A total of 201 bacterial strains were isolated from starter samples, phenotypically characterized, and their identities confirmed by the 16S rRNA sanger sequencing method. The dominant phylum was Firmicutes (85%), followed by Proteobacteria (9%), and Actinobacteria (6%). Lactic acid bacteria (LAB) (59%) formed the most abundant group, followed by non-LAB (32%) and Gram-negative bacteria (9%). Based on the 16S rRNA gene sequencing result, we identified LAB: Enterococcus durans, E. faecium, E. fecalis, E. hirae, E. lactis, Pediococcus acidilactici, P. pentosaceus, Lactobacillus plantarum subsp. plantarum, Lb. pentosus, Leuconostoc mesenteroides, and Weissella cibaria; non-LAB: Bacillus subtilis subsp. inaquosorum, B. circulans, B. albus, B. cereus, B. nakamurai, B. nitratireducens, B. pseudomycoides, B. zhangzhouensis, Kocuria rosea, Staphylococcus hominis subsp. hominis, S. warneri, S. gallinarum, S. sciuri, Lysinibacillus boronitolerans, Brevibacterium frigoritolerans, and Micrococcus yunnanensis; Gram-negative bacteria: Pseudomonas putida, Klebsiella pneumoniae, Enterobacter hormaechei subsp. xiangfangensis, E. hormaechei subsp. steigerwaltii, and Stenotrophomonas maltophilia. We characterized diversity indexes of the bacterial community present in traditionally prepared dry starters. This is the first report on the bacterial diversity of traditionally dry starters of the Eastern Himalayas by sanger sequencing.

SUBMITTER: Pradhan P

PROVIDER: S-EPMC6848222 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC. A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC. RESULTS: The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster. CONCLUSIONS: The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.

Project description:BackgroundIn recent years, interest in the consumption of ready-to-eat (RTE) food products has been increased in many countries. However, RTE products particularly those prepared by meat may be potential vehicles of antibiotic-resistance foodborne pathogens. Considering kebab and hamburger are the most popular RTE meat products in Iran, this study aimed to investigate the prevalence and antimicrobial resistance of common foodborne pathogens (Escherichia coli, Salmonella spp., Staphylococcus aureus, and Listeria monocytogenes) in raw kebab and hamburger samples collected from fast-food centers and restaurants. Therefore, total bacterial count (TBC), as well as the prevalence rates and antibiogram patterns of foodborne pathogens in the samples were investigated. Also, the presence of antibiotic-resistance genes (blaSHV, blaTEM, blaZ, and mecA) was studied in the isolates by PCR.ResultsThe mean value of TBC in raw kebab and hamburger samples was 6.72 ± 0.68 log CFU/g and 6.64 ± 0.66 log CFU/g, respectively. E. coli had the highest prevalence rate among the investigated pathogenic bacteria in kebab (70%) and hamburger samples (48%). Salmonella spp., L. monocytogenes, and S. aureus were also recovered from 58, 50, and 36% of kebab samples, respectively. The contamination of hamburger samples was detected to S. aureus (22%), L. monocytogenes (22%), and Salmonella spp. (10%). In the antimicrobial susceptibility tests, all isolates exhibited high rates of antibiotic resistance, particularly against amoxicillin, penicillin, and cefalexin (79.66-100%). The blaTEM was the most common resistant gene in the isolates of E. coli (52.54%) and Salmonella spp. (44.11%). Fourteen isolates (23.72%) of E. coli and 10 isolates (29.41%) of Salmonella spp. were positive for blaSHV. Also, 16 isolates (55.17%) of S. aureus and 10 isolates (27.27%) of L. monocytogenes were positive for mecA gene.ConclusionsThe findings of this study showed that raw kebab and hamburger are potential carriers of antibiotic-resistance pathogenic bacteria, which can be a serious threat to public health.

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Phenotypic and Genotypic Identification of Bacteria Isolated From Traditionally Prepared Dry Starters of the Eastern Himalayas.

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