16S rRNA dataset of non-lactic acid bacteria cultures from lait caillé in Burkina Faso reveals predominantly bacteria associated with emerging risks to public health

Description

The objective of this investigation was to provide information on the identity of non-lactic acid bacteria originating from traditional lait caillé and capable of growing abundantly on the standard media used for dairy LAB isolation. Specifically, Gram positive catalase positive and Gram negative bacteria growing on MRS and M17 agar were considered for molecular identification MRS and M17 agar plates previously inoculated with traditional lait caillé were used to screen for Gram positive catalase positive and Gram negative bacteria. Successive streaking were performed to purify colonies. Culture purity, cells morphology were analysed by microscopy. Gram and catalase tests were done to select Gram negative and Gram positive catalase positive bacteria. (GTG)5-based rep-PCR fingerprint followed by cluster analysis, sequencing of 16S rRNA gene and database search in GenBank were performed to identify the isolates. Data showed a predominance of Klebsiella pneumoniae/K. quasipneumoniae/K. variicola among Gram negative bacteria and a variety of species among Gram positive catalase positive bacteria such as Kocuria spp., Bacillus cereus group species, Macrococcus caseoliticus, Clostridium bifermentans, Staphylococcus hominis. These data are useful as they gave some information on the presence of bacteria associated with emerging public health risks such as Klebsiella pneumonia species complex, Bacillus cereus group species, Kocuria spp. occurring in a traditional spontaneously fermented milk product. They also gave further insights for a broader investigation on non-lactic acid bacteria occurring in traditional fermentation of unpasteurised milks and for evaluation and development of selective media for Lactic acid bacteria growth.

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Origin of samples The traditional lait caillé was previously collected in different studies from two sites, Tolotama (in the south-west area) and Katre-Yaar (in the central area) of Burkina Faso and inoculated on MRS and M17 agar plates. In total 04 and 09 samples were collected during lait caillé fermentation in Katre-Yaar and Tolotama respectively. MRS agar plates were incubated anaerobically for 48h while M17 agar plates were incubated aerobically for 48h. The agar plates were used to screen for Gram positive catalase positive and Gram negative bacteria. Colonies isolation and purification Colonies were isolated and purified through successive streaking. Gram test with 3% KOH and catalase test with 3% H2O2 were performed. Cells morphology and culture purity were analysed by using the hanging drop technique with a contrast phase microscope. DNA of bacteria isolates retrieved from MRS and M17 agar plates were extracted using InstaGene (Bio-Rad Laboratories, Hercules, CA, USA) following the instructions of the manufacturer. The PCR reaction was carried out in a SureCycler 8800 thermal cycler (Agilent Technologies, Malaysia). Primer (GTG)5 was used for all bacteria. The cycling program was as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 1 min and elongation at 65 °C for 8 min. The PCR ended with a final elongation step at 65 °C for 16 min and cooling of the amplified products at 4 °C. The PCR products were separated by 1.5% agarose gel electrophoresis (5 h, 120 V) in Tris-Borate-EDTA buffer (0.5 X TBE) with 1-kb DNA ladder as the size marker. Afterward, gels were stained with ethidium bromide and documented using an Alphalmager gel imaging system (Alpha Innotech, San Francisco, CA, USA). The cluster analysis was performed by combined Bionumerics 7.1 system (Applied Maths, Sint-Martens-Latem, Belgium) and visual observation. Bacteria showing similar DNA profiles were clustered in the same group. Sequencing of genes and species identification Bacteria isolates were identified after the sequencing of the 16S rRNA gene of representatives picked from the clusters. Primers 27f and 1540r were used for amplification at the following conditions: initial denaturation at 95 °C for 5 min, then 35 cycles at 95 °C for 30 s, 60 °C for 30 s and 72 °C for 120 s . The final extension was carried out at 72 °C for 10 min and the products were cooled at 4°C. The PCR products from gene amplification were sent to a commercial facility (MacroGen, Germany) for purification, set up of sequencing reaction, purification and analysis of sequencing products. Subsequently, forward and reverse reads were trimmed and assembled to produce a consensus 16S rRNA sequence with CLC Genomics Workbench 8.5.1 (CLC bio, Aarhus, Denmark). A database search was performed in NCBI Genbank and in EzBioCloud. The corrected sequences were aligned to 16S rRNA (Tables 2, 3) gene sequences.

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