Comparison of phenotypic (Biolog System) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits

Cagno, Raffaella Di; Minervini, Giovanna; Sgarbi, Elisa; Lazzi, Camilla; Bernini, Valentina; Neviani, Erasmo; Gobbetti, Marco

doi:10.1016/j.ijfoodmicro.2010.08.018

Cited by 52 publications

(44 citation statements)

References 42 publications

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“…AFLP provides a high-throughput method for high-resolution genomic fingerprinting (20) that has been employed frequently for the classification of strains of various species, including lactobacilli (2,33). AFLP classification of 65 L. rhamnosus strains enabled the distinction of 11 genotypic groups at a similarity cutoff of 70%, with all L. casei strains constituting an outgroup, since they belong to a closely related but genetically distinct species.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, true niche specialization is a complex phenotype, which is unlikely to be represented by single genetic markers and probably involves both multiple discriminating genes that may be genetically unlinked and strain-specific and divergent gene regulatory patterns. Although it is among the highest-resolution methodologies for genetic fingerprinting, AFLP patterns still represent a crude way of genetic typing (2), and whole-genome sequencing and comparative genomics provide a genetic strain typing methodology of substantially higher resolution and may therefore be more appropriate for the niche-fitness correlation analyses.…”

Section: Discussionmentioning

confidence: 99%

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Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling

Ceapă

Lambert

Limpt

et al. 2015

Appl Environ Microbiol

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e Lactobacillus rhamnosus is a bacterial species commonly colonizing the gastrointestinal (GI) tract of humans and also frequently used in food products. While some strains have been studied extensively, physiological variability among isolates of the species found in healthy humans or their diet is largely unexplored. The aim of this study was to characterize the diversity of carbohydrate utilization capabilities of human isolates and food-derived strains of L. rhamnosus in relation to their niche of isolation and genotype. We investigated the genotypic and phenotypic diversity of 25 out of 65 L. rhamnosus strains from various niches, mainly human feces and fermented dairy products. Genetic fingerprinting of the strains by amplified fragment length polymorphism (AFLP) identified 11 distinct subgroups at 70% similarity and suggested niche enrichment within particular genetic clades. High-resolution carbohydrate utilization profiling (OmniLog) identified 14 carbon sources that could be used by all of the strains tested for growth, while the utilization of 58 carbon sources differed significantly between strains, enabling the stratification of L. rhamnosus strains into three metabolic clusters that partially correlate with the genotypic clades but appear uncorrelated with the strain's origin of isolation. Draft genome sequences of 8 strains were generated and employed in a gene-trait matching (GTM) analysis together with the publicly available genomes of L. rhamnosus GG (ATCC 53103) and HN001 for several carbohydrates that were distinct for the different metabolic clusters: L-rhamnose, cellobiose, L-sorbose, and ␣-methyl-Dglucoside. From the analysis, candidate genes were identified that correlate with L-sorbose and ␣-methyl-D-glucoside utilization, and the proposed function of these genes could be confirmed by heterologous expression in a strain lacking the genes. This study expands our insight into the phenotypic and genotypic diversity of the species L. rhamnosus and explores the relationships between specific carbohydrate utilization capacities and genotype and/or niche adaptation of this species.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling

Ceapă

Lambert

Limpt

et al. 2015

Appl Environ Microbiol

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“…Generally, results from DNA banding pattern recognition-based methods are simpler assays to conduct but are less reproducible and cross-comparable than sequence-based methods. In previously published studies on M13 RAPD-PCR, strains of L. plantarum clustered as expected, with an assay reproducibility of Ͼ89% (15,16). These studies utilized a built-in reference library of PCR profiles for comparison and strain identification but provided little biological relevance and limited dynamic range.…”

Section: Discussionmentioning

confidence: 83%

“…A number of methods have been examined for Lactobacillus strain typing, including multilocus sequence typing (MLST) (11)(12)(13), pulsed-field gel electrophoresis (PFGE) (11,14), amplified fragment length polymorphism analysis (AFLP) (15), random amplified polymorphic DNA PCR (RAPD-PCR) (15)(16)(17), phenotypic analysis (15), restriction fragment length polymorphism analysis (RFLP) (18), ribotyping (14,17), and microarrays (3). Despite the number of studies on this topic, consensus on the use of these methods to subtype strains of Lactobacillus remains weak, with only sporadic species-specific solutions (12,19).…”

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confidence: 99%

Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex

Ramachandran

Lacher

Pfeiler

et al. 2013

Appl Environ Microbiol

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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.

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“…For effective representation of composition and relative position of LAB, Hierarchical cluster statistical analysis was carried out to group the isolates based on the phenotypic characters (Ricciardi et al 2005;Di Cagno et al, 2010). To obtain an objective basis for grouping of the isolates, binary 0/1 matrices were created based on negative and positive results of phenotypic tests (the results were coded as 0 for tests showing negative results and 1 for tests with positive results).…”

Section: Discussionmentioning

confidence: 99%

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Maji¹,

Mohanty²,

Mahapatra³

et al. 2016

Turk. J. Fish. Aquat. Sci.

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In order to study the diversity of putative lactic acid bacteria (LAB) in freshwater fish, 76 strains of LAB were isolated from intestines and identified by phenotypic tests and 16S rDNA gene sequencing. Phenotypic characterization of the isolates allowed the identification of 18 clusters at 78% similarity level by Hierarchical cluster analysis. Functional evenness index (E value) a measure of phenotypic diversity, was found to be quite high (0.7 approximately) in most of the samples. 16S rDNA gene sequencing identified the isolates as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faeciumand Enterococcus durans. Lactobacillus plantarum was found to be the dominating strain and LAB occurred more frequently in hindgut. Most of the strains showed good survival in acid and bile tolerance tests and antimicrobial activity against fish pathogen Aeromonas hydrophila. Different bacteriocin producing genes were detected in several strains by PCR. Selective strains with probiotic attributes could be potential candidates for freshwater aquaculture practices.

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Comparison of phenotypic (Biolog System) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits

Cited by 52 publications

References 42 publications

Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling

Correlation of Lactobacillus rhamnosus Genotypes and Carbohydrate Utilization Signatures Determined by Phenotype Profiling

Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex

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