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Bacillus subtilis strain NRRL B-67989

Compositional Guideline for Bacillus subtilis strain NRRL B-67989 permitted for use in listed medicines.

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Description 

Bacillus subtilis species is a gram-positive, spore-forming, rod-shaped bacterium. The Bacillus subtilis DE111 strain was originally soil extracted from a site in Kennesaw, Georgia, United States. It is deposited in the Agricultural Research Culture Collection under the accession number NRRL B-67989. The ingredient is manufactured by aerobic fermentation using animal component-free fermentation media. It is then pasteurised, separated, and dried to the desired concentration of 100-200 billion CFU/g.

TestMethod referenceAcceptance criteria
Characterisation 
General properties
Appearance Visual & Organoleptic evaluationLight brown to brown powder
Water activityUSP <922><0.5 aw at 25oC
pHUSP <791>5.5-8.5 at 25oC
MacroscopyVisualApproximately 1.5 mm in diameter, circular, fuzzy, white to slightly yellow in colour with jagged edges colonies
MicroscopyUSP <776>2 µm long, rod-shaped
Gram staining

USP <1113>

 

Gram-positive
Water solubility Ph. Eur. 04/2022:51100Very soluble

Antimicrobial resistance and susceptibility

 

Antimicrobial susceptibility test[1]

Susceptible to antibiotics: Vancomycin, Gentamicin, Kanamycin, Streptomycin, Erythromycin, Clindamycin, and Tetracycline

 

Whole genome screening using, at least 80% ID and 70% length threshold levels, ResFinder[2] and Resistance Gene Identifier[3] databases

 

Absence of acquired resistance genes
Absence of virulence factorsWhole genome screening for the potential virulent factors using (at least 80% ID and 70% length threshold levels) PathogenFinder[4] and ToxFinder[5] databases

Absent

 

Identity
Polymerase Chain Reaction (PCR)USP <1127>Matches Bacillus subtilis (NRRL B-67989)
Assay

Total Viable cell count

 

In-house method[6]100-200 Billion CFU/g
Impurities and incidental constituents
Elemental impurities                                                                 
LeadUSP <2232><1 ppm
CadmiumUSP <2232><1 ppm
MercuryUSP <2232><1 ppm
ArsenicUSP <2232><1 ppm
Microbial contaminants 
Yeast and MoldsBP: SC I M. Microbial contamination<300 CFU/g
ColiformsBP: SC I M. Microbial contamination<100 CFU/g
EnterobacteriaceaeBP: SC I M. Microbial contamination<100 CFU/g
E. coliBP: SC I M. Microbial contaminationNegative
SalmonellaBP: SC I M. Microbial contaminationNegative
StaphylococcusBP: SC I M. Microbial contaminationNegative

Footnotes

  1. Cockerill, F. R., et al. (2012). Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Eleventh Edition. Clinical and Laboratory Standards Institute M02-A11, 32(1).
  2. Florensa A. F., et al. (2022). ResFinder - an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes. Microbial Genomics, 8(1):000748.
  3. Alcock B. P., et al. (2020). CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Research, 48(D1):D517–D525.
  4. Cosentino S., et al. (2013). PathogenFinder - distinguishing friend from foe using bacterial whole genome sequence data. PLoS one, 8(10):e77302.
  5. Centre for Genomic Epidemiology, ToxFinder 1.0. 
  6. B. subtilis was grown in a soybean-casein digest agar and broth at 37 ± 2°C for 18-24 hours prior to diluting and plating approximately 25-250 colonies per plate. The cell count was conducted manually under a light microscope.
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