ABSTRACTMembers of theLactobacillus acidophiluscomplex 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 theL. acidophiluscomplex (L. acidophilus,L. amylovorus,L. crispatus,L. gallinarum,L. gasseri, andL. 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 thehsp60gene 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 genesfusA,gpmA,gyrA,gyrB,lepA,pyrG, andrecA, 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 theL. acidophiluscomplex 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 ofLactobacillusspecies.
Genotypic Characterization of Clostridium botulinum Strains Producing Type A Neurotoxin Complexes by Random Amplified Polymorphic DNA and PCR-RFLP Analysis