Differentiation of<i>Lactobacillus delbrueckii</i>subspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA)

Miteva, Vanya; Boudakov, Ivo; Ivanova-Stoyancheva, G.; Marinova, B.; Mitev, Vladislav; Mengaud, J

doi:10.1046/j.1365-2672.2001.01320.x

Cited by 27 publications

(19 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, several research groups have recognized that the identifications based on phenotypic and genotypic methods do not match (Giraffa et al, 2004;Miteva et al, 2001). Furthermore, although we had attempted to identify L. delbrueckii subspecies by using single housekeeping genes in the past, the resulting subspeciation was of low resolution (data not shown).…”

Section: Discussionmentioning

confidence: 96%

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii

Tanigawa

Watanabe

2011

Microbiology

View full text Add to dashboard Cite

Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080 T , was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.

show abstract

Section: Discussionmentioning

confidence: 96%

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii

Tanigawa

Watanabe

2011

Microbiology

View full text Add to dashboard Cite

show abstract

“…With 16S rRNAbased phylogenetic analysis, at least eight phylogenetic groups have been recognized (10). Investigations by many authors using several molecular approaches focused on closely related species, such as the L. casei-related taxa (6,9,12,38,44,47,57,61), the L. acidophilus group (19,32,41,45,47), the L. delbrueckii subspecies (20,35,36,56), and the L. plantarum-related species (54). Nevertheless, the discrimination of very closely related species, especially of subspecies, is often critical (10).…”

mentioning

confidence: 99%

Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation

Blaiotta¹,

Fusco²,

Ercolini

et al. 2008

Appl Environ Microbiol

View full text Add to dashboard Cite

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.Lactobacilli are some of the most important taxa involved in food microbiology and human nutrition owing to their role in food and feed production and preservation, including the probiotic properties exhibited by some strains. These traits are of increasing importance and have received attention in the food and feed industry (2).At the beginning of 2005, the genus Lactobacillus was reported to include about 100 validly described species (10). However, the number of species is continually changing due to the description of new species and/or reclassification of others: indeed, at the beginning of 2007 this genus had reached about 120 species (http://www.bacterio.cict.fr/).Their classification into three groups based on metabolism and physiological characteristics (30) is not in agreement with the results of phylogenetic studies by sequencing of 16S rRNA (7). Schleifer and Ludwig (49) reviewed the phylogeny of the genus Lactobacillus based on the 16S rRNA sequences but did not clarify the taxonomy of either the L. casei group or the L. delbrueckii subspecies. Recently, several systematic studies of lactobacilli have been car...

show abstract

“…Even with molecular methods, it is difficult to distinguish between members of this group. Methods that have been used successfully include the following: DNA-DNA hybridization and various biochemical properties (5, 11, 15); analysis of whole-cell protein profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (3,8,12,21); randomly amplified polymorphic DNA (3,8,12,24); sequencing of 16S and 23S ribosomal DNA (rDNA) and elongation factor Tu (2,14,29,30); oligonucleotide probes and primers for species-specific hybridizations and PCR, respectively (21,23,24,32); ribotyping (25); amplified fragment length polymorphism (8); and amplified ribosomal DNA restriction analysis (ARDRA) (17,23,31). There is a clear need, however, for simpler methods to differentiate the members of the L. acidophilus group to ensure that large-scale microbial ecological studies are logistically possible.…”

mentioning

confidence: 99%

Detection and Identification of Lactobacillus Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis

Guan

Hagen

Tannock

et al. 2003

Appl Environ Microbiol

112

View full text Add to dashboard Cite

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10 8 to 10 9 CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.The digestive tracts of mammals and birds are home to a diverse collection of bacterial species, collectively referred to as the gut microflora (28). From gnotobiotic animal studies, the microflora is known to influence the biochemistry, immunology, physiology, and nonspecific resistance to intestinal infection of the host (9). The impact of the gut microflora on the nutritional status of farm animals is of particular interest, especially where intensive farming practices are used (4).The crop, ileum, cecum, and colon of poultry are known to harbor bacterial populations (16,27). Recent reports have investigated the composition of the ileal (13) and cecal (35) microflora using bacteriological culture and culture-independent methods. Lactobacilli are numerous in the ileum of broilers, whereas the cecal microflora is dominated by obligately anaerobic bacteria and bacteria yet to be cultivated. From the results of culture-based studies, it has been determined that the microflora of the crop has a simple composition and is dominated by lactobacilli (16,27). Colonization of the surface of the stratified, squamous epithelium of the crop by lactobacilli has been reported by Fuller (6) and Morishita et al. (18). Lactobacillus salivarius, Lactobacillus fermentum or Lactobacillus reuteri, and Lactobacillus acidophilus were the species most commonly detected (16,27). These studies were conducted prior to the reclassification of L. acidophilus, which has been divided into two DNA homology groups containing six related species (5,11,15). DNA homology group A consists of L. acidophilus (A1), Lactobacillus crispatus (A2), Lactobacillus amylovorus (A3), and Lactobacillus gallinarum (A4); DNA homology gr...

show abstract

Differentiation ofLactobacillus delbrueckiisubspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA)

Cited by 27 publications

References 29 publications

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii

Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation

Detection and Identification of Lactobacillus Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis

Contact Info

Product

Resources

About