title: Development of a tiered multilocus sequence typing scheme for members of the Lactobacillus acidophilus complex.
abstract: Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.
authors.affiliation: Division of Molecular Biology, Center for Food Safety and Applied Nutrition (CFSAN), United States Food and Drug Administration, Laurel, Maryland, USA.
authors.forename: Padmini
authors.initials: P
authors.lastname: Ramachandran
authors.forename: David W
authors.initials: DW
authors.lastname: Lacher
authors.forename: Erika A
authors.initials: EA
authors.lastname: Pfeiler
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Elkins
mesh_terms: D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005516:Food Microbiology / Q000379:methods*; D020043:Genes, Essential; D005838:Genotype; D007779:Lactobacillus acidophilus / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D058885:Multilocus Sequence Typing / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial
references.citation: J Dairy Sci. 2001 Feb;84(2):319-31
references.pmid: 11233016
references.citation: Mol Phylogenet Evol. 2003 Jan;26(1):1-7
references.pmid: 12470932
references.citation: Appl Environ Microbiol. 2003 Nov;69(11):6908-22
references.pmid: 14602655
references.citation: Mol Biol Evol. 2006 Feb;23(2):254-67
references.pmid: 16221896
references.citation: Microbiology (Reading). 2006 Jan;152(Pt 1):85-93
references.pmid: 16385118
references.citation: J Microbiol Methods. 2005 Nov;63(2):135-44
references.pmid: 15893395
references.citation: Curr Microbiol. 2000 Jan;40(1):40-6
references.pmid: 10568802
references.citation: Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2512-7
references.pmid: 14983040
references.citation: Curr Microbiol. 1995 Aug;31(2):114-8
references.pmid: 7606186
references.citation: J Hered. 2001 Jul-Aug;92(4):371-3
references.pmid: 11535656
references.citation: Res Microbiol. 2006 Nov;157(9):803-10
references.pmid: 16919915
references.citation: J Bacteriol. 2007 Feb;189(4):1311-21
references.pmid: 17142402
references.citation: Int J Food Microbiol. 2010 Oct 15;143(3):246-53
references.pmid: 20850884
references.citation: J Clin Gastroenterol. 2008 Sep;42 Suppl 3 Pt 2:S160-2
references.pmid: 18685516
references.citation: Microbiology (Reading). 2007 Aug;153(Pt 8):2655-2665
references.pmid: 17660430
references.citation: FEMS Microbiol Rev. 2009 Sep;33(5):892-916
references.pmid: 19453749
references.citation: Appl Environ Microbiol. 2007 Oct;73(20):6601-11
references.pmid: 17704267
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):3906-12
references.pmid: 15671160
references.citation: Curr Issues Intest Microbiol. 2007 Sep;8(2):44-61
references.pmid: 17542335
references.citation: Mol Biol Evol. 2011 Oct;28(10):2731-9
references.pmid: 21546353
references.citation: FEMS Microbiol Rev. 2010 Mar;34(2):199-230
references.pmid: 20088967
references.citation: Clin Infect Dis. 2008 Feb 1;46 Suppl 2:S58-61; discussion S144-51
references.pmid: 18181724
references.citation: Int J Syst Bacteriol. 1992 Jul;42(3):487-91
references.pmid: 1503977
references.citation: J AOAC Int. 2010 May-Jun;93(3):974-84
references.pmid: 20629403
references.citation: J Food Prot. 2002 Nov;65(11):1811-29
references.pmid: 12430709
references.citation: Antonie Van Leeuwenhoek. 2010 Jun;98(1):85-92
references.pmid: 20361257
references.citation: Microbiology (Reading). 2011 Mar;157(Pt 3):727-738
references.pmid: 21178164
references.citation: Appl Environ Microbiol. 2008 Jan;74(1):208-15
references.pmid: 17993558
references.citation: Appl Environ Microbiol. 2008 May;74(9):2679-89
references.pmid: 18326671