Genotyping of dairy Bacillus licheniformis isolates by high resolution melt analysis of multiple variable number tandem repeat loci

• Published in: Food microbiology 2013-06, Vol.34 (2), p.344-351
• Main Authors: Dhakal, Rajat, Chauhan, Kanika, Seale, R. Brent, Deeth, Hilton C., Pillidge, Christopher J., Powell, Ian B., Craven, Heather, Turner, Mark S.
• Format: Article
• Language: English
• Subjects: Animals; Bacillus - classification; Bacillus - genetics; Bacillus - isolation & purification; Bacillus licheniformis; Bacterial Proteins - genetics; Bacterial Typing Techniques - methods; Biological and medical sciences; Cattle; compliance; Dairy; dried milk; Food Contamination; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; Genotype; Genotyping; HRMA; loci; melting; Milk - microbiology; Minisatellite Repeats; MLVA; monitoring; Multilocus Sequence Typing - methods; new methods; Phylogeny; random amplified polymorphic DNA technique; spoilage
• ISSN: 0740-0020; 1095-9998
• DOI: 10.1016/j.fm.2013.01.006

In dairy foods, the sporeformer Bacillus licheniformis can be the cause of spoilage or specification compliance issues. Currently used methods for genotyping B. licheniformis have limited discrimination with only 2 or 3 different subgroups being identified. Here, we have developed a multi-locus variable number tandem repeat analysis (MLVA) method and combined it with high resolution melt analysis (MLV-HRMA) for genotyping B. licheniformis. Five repetitive loci were identified and used as markers for genotyping 52 isolates from two milk powder processing plants and retail samples. Nineteen genotypes could be identified using both MLVA and MLV-HRMA leading to Hunter–Gaston discrimination indices (D-value) of 0.93 each. It was found that all 5 MLVA loci were stable following 10 days of sub-culturing of 8 representative isolates. All isolates were also genotyped using previously used methods including randomly amplified polymorphic DNA-PCR (RAPD) and partial rpoB sequencing. Five different RAPD profiles and 5 different partial rpoB sequence types were identified resulting in corresponding D-values of 0.6 and 0.46, respectively. Analysis of the genotypes from dairy samples revealed that dairy B. licheniformis isolates are more heterogeneous than previously thought and that this new method can potentially allow for more discriminatory tracking and monitoring of specific genotypes. ► A new MLVA genotyping method for Bacillus licheniformis based on 5 loci was developed. ► Analysis can be done using agarose gels or high resolution melt curve comparisons. ► MLVA was more discriminatory than previously used RAPD and rpoB gene sequencing methods. ► Groupings determined using the 3 genotyping methods were found to be consistent. ► B. licheniformis isolates from milk powder samples were found to be heterogeneous.