Isolation and characterization of Paenibacillus amylolyticus strain NCT146 from a human microbiome

Abstract

As a part of academic lab in microbiology, human skin microbiome was sampled and swabbed onto a nutrient agar plates. Morphologically distinct colonies were purified using quadrant streaking. The pure culture was preserved using glycerol stocking in triplicates. One of these glycerol stocks was revived and used for further morphological and molecular characterization.

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National College Culture Collection Centre www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
1
N4C
Isolation and characterization of Paenibacillus amylolyticus strain NCT146 from a human
microbiome
Background
As a part of academic lab in microbiology, human skin microbiome was sampled and swabbed
onto a nutrient agar plates. Morphologically distinct colonies were purified using quadrant
streaking. The pure culture was preserved using glycerol stocking in triplicates. One of these
glycerol stocks was revived and used for further morphological and molecular characterization.
Work plan
Bergey’s manual was used to determine the lowest possible taxonomic hierarchy and the strain
was commercially outsourced for bidirectional 16S rRNA gene sequencing for species level
resolution.

National College Culture Collection Centre www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
2
N4C
Strain description: NCT146 grows stringently in nutrient agar plate at 37°C in 24 hours. Colonies
are small, round, white, shiny with rough edges and are 4mm in diameter (last quadrant metrics).
Cells are Gram positive, sporulative, non-motile rods. Biochemical reaction implies Indole
negative, methyl red positive, voges proskauer negative and citrate negative. Triple Sugar Iron
implies acid butt and alkaline slant with no gas production. Catalase and oxidase negative.
Amylase and lipase positive. Sedimentation type of growth was observed in nutrient broth. The
strain NCT146 was weakly sensitive to rifampicin, penicillin and streptomycin and erythromycin.
16S rRNA gene sequencing:
Based on the colony, biochemical and molecular characteristics, the strain NCT146 was identified
as Paenibacillus amylolyticus. The 16S rRNA gene sequences of NCT146 was 99.58% similar
to Paenibacillus amylolyticus (GenBank accession number: NR_025882) and can be globally
accessed through genbank accession number OQ255787.
The Neighbor-Joining method tree with percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (500 replicates) are shown. The tree is drawn to scale,
with branch lengths in the same units as those of the evolutionary distances used to infer the
phylogenetic tree. The evolutionary distances were computed using the Maximum Composite
Likelihood method and are in the units of the number of base substitutions per site.

National College Culture Collection Centre www.ncccc.in
PG & Research Department of Biotechnology and Microbiology
National College, Tiruchirappalli- 620 001, Tamil Nadu, India
3
N4C
This analysis involved 11
nucleotide sequences. All
ambiguous positions were
removed for each sequence pair
(pairwise deletion option).
There were a total of 1424
positions in the final dataset.
The strain NCT146 was
deposited in National College
Culture Collection Centre
(ncccc) and are freely available
for academic and research
purposes. The strain can be
obtained here www.ncccc.in