Molecular identification and typing of lactobacilli isolated from kefir grains

2005 ◽  
Vol 73 (1) ◽  
pp. 20-27 ◽  
Author(s):  
Lucrecia Delfederico ◽  
Axel Hollmann ◽  
Mariano Martínez ◽  
N. Gabriel Iglesias ◽  
Graciela De Antoni ◽  
...  
Keyword(s):  
Restriction Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Restriction Enzymes ◽  
23S Rrna ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Good Tool ◽  
Rapd Pcr ◽  
Kefir Grains

Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.

Characterization of homofermentative lactobacilli isolated from kefir grains: potential use as probiotic

2008 ◽  
Vol 75 (2) ◽  
pp. 211-217 ◽  
Author(s):  
Marina A Golowczyc ◽  
Maria J Gugliada ◽  
Axel Hollmann ◽  
Lucrecia Delfederico ◽  
Graciela L Garrote ◽  
...  
Keyword(s):  
Restriction Analysis ◽  
Bacterial Species ◽  
Genotypic Diversity ◽  
23S Rrna ◽  
Pcr Analysis ◽  
High Survival ◽  
Probiotic Properties ◽  
Probiotic Potential ◽  
Rapd Pcr ◽  
Kefir Grains

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S–23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the speciesLactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2·5 was observed inLb. plantarumCIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power againstSalmonella typhimuriumandEscherichia coli. Seven out of eleven isolates showed inhibition againstSal. entericaand five isolates were effective againstSal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibitSal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.

scholarly journals Genetic Diversity of Pierce's Disease Strains and Other Pathotypes of Xylella fastidiosa

2001 ◽  
Vol 67 (2) ◽  
pp. 895-903 ◽  
Author(s):  
Mavis Hendson ◽  
Alexander H. Purcell ◽  
Deqiao Chen ◽  
Chris Smart ◽  
Magalie Guilhabert ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Xylella Fastidiosa ◽  
Random Amplified Polymorphic Dna ◽  
Consensus Sequence ◽  
23S Rrna ◽  
Pierce's Disease ◽  
Pcr Analysis ◽  
Leaf Scorch ◽  
Pierce’S Disease ◽  
Rapd Pcr

ABSTRACT Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products,NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosaat the subspecies or pathovar level.

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

1998 ◽  
Vol 64 (9) ◽  
pp. 3403-3410 ◽  
Author(s):  
Covadonga R. Arias ◽  
María Jesús Pujalte ◽  
Esperanza Garay ◽  
Rosa Aznar
Keyword(s):  
Genetic Relatedness ◽  
Vibrio Vulnificus ◽  
Genetic Relationships ◽  
Random Amplified Polymorphic Dna ◽  
Mediterranean Coast ◽  
Universal Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
Conserved Sequence ◽  
Rapd Pcr

ABSTRACT Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificusisolates.

AMPLIFIED RIBOSOMAL DNA RESTRICTION ANALYSIS (ARDRA) BAKTERI DENGAN POTENSI ANTIMIKROB YANG BERASOSIASI DENGAN SPONS Jaspis sp.

2010 ◽  
Vol 9 (1) ◽  
Author(s):  
Hermawaty Abubakar
Keyword(s):  
Genetic Diversity ◽  
Antimicrobial Activity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Ribosomal Dna ◽  
Restriction Analysis ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Dna Restriction

<p><em>Sponges</em><em> are one of the components that compose coral reef which have a potential bioactive substance that has not been utilized. Sponges are generally able to survive in marine waters were nutrients are poor because of associations with other organisms, especially bacteria. This study aimed to isolate and characterize bacteria (endosymbiont and ectosimbion) that produce antimicrobial compounds, and analyze genetic diversity based on Amplified Ribosomal DNA Restriction Analysis (ARDRA). The results of isolation obtained 138 bacterial isolates, which are 70 endofit isolates and 68 surfaces isolates respectively. The results obtained, based on antimicrobial test, was 32 bacterial isolates (45.71%) of the total bacterial isolates that have endofit antimicrobial activity, whereas on the surface bacteria, 20 bacterial isolates (29.41%) of the total surface of the bacterial isolates also have antimicrobial activity. Genetic diversity was carried out on 30 isolates that has the best antimicrobial activity. Amply</em><em>fi</em><em>cation of 16S rRNA gene is done using specific primers, 63f and 1387r. The profile of 16S rRNA gene band shows a </em><em>high </em><em>diversity, which is generated after cutting with three restriction enzymes </em><em>i.e.</em><em> </em><em>RsaI</em><em>, HaeIII and HinfI. The three restriction enzymes have different cuts and properties. Construction of phylogenetic trees based on analysis of Amplified Ribosomal DNA restriction, grouped 30 isolates from the sponge Jaspis sp. which have a microbial activity on seven filotipe. This grouping is based on the similarities cuts of sites of each isolate after restriction by three different restriction enzymes.</em></p>

scholarly journals Comparison of Genospecies and Antimicrobial Resistance Profiles of Isolates in the Acinetobacter calcoaceticus-Acinetobacter baumannii Complex from Various Clinical Specimens

2012 ◽  
Vol 56 (12) ◽  
pp. 6267-6271 ◽  
Author(s):  
Ni Tien ◽  
Bang-Jau You ◽  
Hui-Lan Chang ◽  
Hsiu-Shen Lin ◽  
Chin-Yi Lee ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Acinetobacter Baumannii ◽  
Restriction Analysis ◽  
Acinetobacter Calcoaceticus ◽  
Its Region ◽  
Clinical Isolates ◽  
23S Rrna ◽  
Rrna Gene ◽  
Genotype 3 ◽  
Content Type

ABSTRACTThis study was conducted to compare the prevalences of antimicrobial resistance profiles of clinical isolates in theAcinetobacter calcoaceticus-Acinetobacter baumanniicomplex from sterile and nonsterile sites and to further study the relationship of antimicrobial resistance profiles and genospecies by amplified rRNA gene restriction analysis (ARDRA). A total of 1,381 isolates were tested with 12 different antibiotics to show their antimicrobial susceptibility profiles. A total of 205 clinical isolates were further analyzed by ARDRA of the intergenic spacer (ITS) region of the 16S-23S rRNA gene. It was found that the overall percentage of isolates from nonsterile sites (urine, sputum, pus, or catheter tip) that were resistant to the 12 antibiotics tested was significantly higher than that of isolates from sterile sites (cerebrospinal fluid [CSF], ascites fluid, and bloodstream) (46% versus 22%;P< 0.05). After ARDRA, it was found that 97% of the 62 isolates resistant to all antibiotics tested were theA. baumanniigenospecies, which was identified in only 31% of the isolates susceptible to all antibiotics tested. More genospecies diversity was identified in the isolates susceptible to all antibiotics tested, including genospecies of 13TU (34%), genotype 3 (29%), andA. calcoaceticus(5%). Furthermore, as 91% (10/11) of the isolates from CSF were susceptible to all antibiotics tested, theA. calcoaceticus-A. baumanniicomplex isolates with multidrug resistance could be less invasive than the more susceptible isolates. This study also indicated current emergence of carbapenem-, fluoroquinolone-, aminoglycoside-, and cephalosporin-resistantA. calcoaceticus-A. baumanniicomplex isolates in Taiwan.

scholarly journals Identification and Assessment of Genetic Similarity of Soil Bacterial Isolates of Pseudomonas spp. Using Molecular Techniques

2014 ◽  
Vol 63 (3) ◽  
pp. 291-298
Author(s):  
ANNA LISEK ◽  
LIDIA SAS PASZ ◽  
PAWEŁ TRZCIŃSKI
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genetic Similarity ◽  
Sour Cherry ◽  
23S Rrna ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Bacterial Strains ◽  
The 16S Rrna Gene ◽  
Selection Of

Bacteria of the genus Pseudomonas are often components of bioproducts designed to enhance the condition of the soil and plants. The use of Pseudomonas bacteria in bioproducts must be preceded by the acquisition, characterization and selection of beneficial strains living in the soil. A prerequisite for the selection of bacterial strains for use in bioproducts is to be able to identify the isolates rapidly and accurately. To identify and differentiate 15 bacterial isolates obtained from the soil surrounding the roots of sour cherry trees and to assess their genetic similarity, the rep-PCR technique and restriction analysis of the 16S rRNA gene and the 16S-ITS-23S rRNA operon were used. In addition, a sequence analysis of the 16S rRNA gene was performed. The analyses made it possible to divide the isolates into four clusters and to confirm their affiliation with the Pseudomonas species. RFLP analysis of the 16S-ITS-23S rRNA operon enabled greater differentiation of the isolates than RFLP of the 16S rRNA gene. The greatest differentiation of isolates within the clusters was obtained after using the rep-PCR technique. However, none of the techniques was able to discriminate all the isolates, which indicates very high genetic similarity of the Pseudomonas isolates found in the same sample of soil from around the roots of sour cherry trees. The tests performed will find application for distinguishing and identifying Pseudomonas strains collected from the soil in order to select the most valuable bacterial strains that produce beneficial effects on plants.

scholarly journals Multiphasic Approach Reveals Genetic Diversity of Environmental and Patient Isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum Associated with an Outbreak of Bacteremias at a Texas Hospital

2008 ◽  
Vol 74 (8) ◽  
pp. 2480-2487 ◽  
Author(s):  
Robert C. Cooksey ◽  
Michael A. Jhung ◽  
Mitchell A. Yakrus ◽  
W. Ray Butler ◽  
Toidi Adékambi ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
16S Rrna ◽  
Restriction Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Bloodstream Infections ◽  
Clinical Isolates ◽  
Rrna Gene ◽  
First Case ◽  
Typing Methods ◽  
Mycobacterium Mucogenicum

ABSTRACT Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.

Characterization of two non-native invasive bark beetles, Scolytus schevyrewi and Scolytus multistriatus (Coleoptera: Curculionidae: Scolytinae)

2008 ◽  
Vol 140 (5) ◽  
pp. 527-538 ◽  
Author(s):  
Patricia L. Johnson ◽  
Jane L. Hayes ◽  
John Rinehart ◽  
Walter S. Sheppard ◽  
Steven E. Smith
Keyword(s):  
Bark Beetle ◽  
Bark Beetles ◽  
Developmental Stages ◽  
Random Amplified Polymorphic Dna ◽  
Morphological Characters ◽  
Pcr Analysis ◽  
Scolytus Multistriatus ◽  
Blind Test ◽  
Rapd Pcr ◽  
Elm Bark Beetle

AbstractScolytus schevyrewi Semenov, the banded elm bark beetle, and S. multistriatus Marsham, the smaller European elm bark beetle, are morphologically similar. Reliance on adult external morphological characters for identification can be problematic because of wide within-species variability and the need for good-quality specimens. The inability to identify developmental stages can also hamper early-detection programs. Using two character identification systems, genitalic (aedeagus) morphology, and DNA markers (random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR)) to distinguish S. schevyrewi from S. multistriatus, we examined specimens from geographically distinct populations of both species collected from infested host trees or semiochemical-baited funnel traps. We found that aedeagus morphology can be used to identify the two species. The use of two oligonucleotide primers in the RAPD-PCR analysis yielded distinct DNA banding patterns for the two species. Species identification using RAPD-PCR analysis was validated by a blind test and used to make species identifications of larval specimens. These tools improve the ability to differentiate between S. schevyrewi and S. multistriatus at immature and adult stages, and could be developed and used for other scolytines as well.

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