Comparison of Phenotypic and Genotypic Methods Used for the Species Identification of Lactobacillus NP51 and Development of a Strain-Specific PCR Assay

S Randhawa; M M Brashears; K W McMahon; M Fokar; E Karunasena

doi:10.1007/s12602-010-9057-0

Comparison of Phenotypic and Genotypic Methods Used for the Species Identification of Lactobacillus NP51 and Development of a Strain-Specific PCR Assay

Probiotics Antimicrob Proteins. 2010 Dec;2(4):274-83. doi: 10.1007/s12602-010-9057-0.

Authors

S Randhawa¹, M M Brashears¹, K W McMahon², M Fokar³, E Karunasena⁴

Affiliations

¹ Animal and Food Sciences, Texas Tech University, PO Box 42141, Lubbock, TX, 79409, USA.
² Internal Med Dept Lbk Genl, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 9410, Lubbock, TX, 79430, USA.
³ Center for Biotechnology Genomics, Texas Tech University, MS 3132, Lubbock, TX, 79409, USA.
⁴ Animal and Food Sciences, Texas Tech University, PO Box 42141, Lubbock, TX, 79409, USA. enusha.karunasena@ttu.edu.

PMID: 26781323
DOI: 10.1007/s12602-010-9057-0

Abstract

The species level identity of Lactobacillus NP51, a commercial direct-fed microbial previously identified as Lactobacillus acidophilus NP51, was re-evaluated to determine whether new technologies resulted in changes in the original identification. The phenotypic methods for species identification included API 50 CHL kit and two automated systems, Vitek 2 and MIDI (FAME analysis; a total of three independent FAME analyses). Discrepancies among the identification results with all methods of phenotypic analysis were reported. MicroSeqID 500 16S rRNA system (SeqWright Inc., Houston, TX), a genotypic method, identified the organism as Lactobacillus animalis. Cloning, sequencing and subsequent sequence comparison of NP51 16S-23S intergenic spacer region (ISRs) to nucleotide sequence databases using the BLAST search tool indicated that NP51 can now be named L. animalis. When NP51 was originally identified as L. acidophilus, the designation of L. animalis did not exist taxonomically. The NP51 sequence comparisons using BLAST also revealed that NP51 and a strain previously identified as L. animalis LA51 HOFG1 by Flint and Angert are identical strains under different names. A strain-specific primer pair was also identified for HOFG1 by the same research group. A primer pair (using HOFG1 forward pair) also produced an amplicon unique to NP51. These methods demonstrate the significance of genetic-based detection methods both for scientific identification of organisms from biological samples and to prevent misidentification in food and health industry related microorganisms in which proprietary considerations are an important concern.

Keywords: DFM; Escherichia coli O157:H7; Lactobacillus; NP51; Probiotics.