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PubMed1998

Taxonomy and physiology of probiotic lactic acid bacteria.

The current taxonomy of probiotic lactic acid bacteria is reviewed with special focus on the genera Lactobacillus, Bifidobacterium and Enterococcus. The physiology and taxonomic position of species and strains of these genera were investigated by phenotypic and genomic methods. In total, 176 strains, including the type strains, have been included. Phenotypic methods applied were based on biochemical, enzymatical and physiological characteristics, including growth temperatures, cell wall analysis and analysis of the total soluble cytoplasmatic proteins. Genomic methods used were pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA-PCR (RAPD-PCR) and DNA-DNA hybridization for bifidobacteria. In the genus Lactobacillus the following species of importance as probiotics were investigated: L. acidophilus group, L. casei group and L. reuteri/L. fermentum group. Most strains referred to as L. acidophilus in probiotic products could be identified either as L. gasseri or as L. johnsonii, both members of the L. acidophilus group. A similar situation could be shown in the L. casei group, where most of the strains named L. casei belonged to L. paracasei subspp. A recent proposal to reject the species L. paracasei and to include this species in the restored species L. casei with a neotype strain was supported by protein analysis. Bifidobacterium spp. strains have been reported to be used for production of fermented dairy and recently of probiotic products. According to phenotypic features and confirmed by DNA-DNA hybridization most of the bifidobacteria strains from dairy origin belonged to B. animalis, although they were often declared as B. longum by the manufacturer. From the genus Enterococcus, probiotic Ec. faecium strains were investigated with regard to the vanA-mediated resistance against glycopeptides. These unwanted resistances could be ruled out by analysis of the 39 kDa resistance protein. In conclusion, the taxonomy and physiology of probiotic lactic acid bacteria can only be understood by using polyphasic taxonomy combining morphological, biochemical and physiological characteristics with molecular-based phenotypic and genomic techniques.

Bifidobacterium↗

PubMed2002

Genetic instability in cancer tissues analyzed by random amplified polymorphic DNA PCR.

OBJECTIVE: To detect DNA and chromosomes instabilities during the progression of tumors and screen new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. METHODS: Five kinds of tumors, in a total of 128 specimens, were analyzed by random amplified polymorphic DNA (RAPD) PCR. Bands representing instabilities were recovered, purified, and cloned, labeled as probes for Southern and Northern blot analysis and DNA sequencing. RESULTS: Sample 5 and 3 of the gastric cancer tissues showed the highest genomic changes and the average detectability in five cancers was up to at least 40% (42.2% - 49.4%). There were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% (primer 8) to 68% (primer 2). Band B is a single copy fragment, and was found to be an allelic loss in gastric and colon cancers. It is a novel sequence and was registered in GenBank with Accession Number AF151005. Further analysis revealed that it might be part of a cis-regulatory element of a new tumor suppressor gene, containing a promoter of cis-action "CACA" box, an enhancer of "GATA" family and a start codon. CONCLUSIONS: It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

Base Sequence↗

PubMed2002

Molecular characterization of methicillin-resistant coagulase-negative staphylococci from a neonatal intensive care unit.

OBJECTIVE: To evaluate clonal dissemination of methicillin-resistant coagulase-negative staphylococci (CNS). SETTING: Neonatal intensive care unit of a 180-bed, university-affiliated general hospital. PATIENTS: Neonates admitted to the neonatal intensive care unit between March 1999 and October 2000, from whom CNS were isolated as a unique pathogen. Patients from other wards from whom epidemiologically unrelated staphylococci strains were obtained served as control-patients. METHODS: Conventional methods were used for phenotypic characterization of CNS. Methicillin resistance was determined by mecA polymerase chain reaction (PCR) amplification. Genotypic characterization was done by random amplification of DNA with degenerated primers (RAPD) and repetitive element sequence-based PCR (rep-PCR). RESULTS: Forty methicillin-resistant CNS isolates obtained from neonates were characterized as Staphylococcus epidermidis (33), S. hominis (5), S. warneri (1), and S. auricularis (1). Both RAPD and rep-PCR indicated the presence of 4 different clones among the 33 S. epidermidis isolates. In turn, the 4 randomly selected, epidemiologically unrelated methicillin-resistant CNS strains obtained from control-patients showed 3 new profiles by RAPD and 2 by rep-PCR, which differed from the corresponding patterns mentioned earlier. Persistence of S. hominis in a neonate could be assessed by both genotypic techniques. CONCLUSIONS: The molecular characterization of the methicillin-resistant CNS studied indicated dissemination of one particular methicillin-resistant CNS clone among the neonates in the ward studied. Although RAPD showed a superior power to discriminate among methicillin-resistant CNS isolates, both RAPD and rep-PCR detected intraspecific and interspecific genomic diversity.

Argentina↗

PubMed2006

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups.

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.

Animals↗

PubMed1999

A polyphasic approach towards the identification of strains belonging to Lactobacillus acidophilus and related species.

A set of 98 strains belonging to nine species of the Lactobacillus acidophilus rRNA-group have been analysed by SDS-PAGE of cellular proteins, RAPD-PCR and AFLP with fluorescently labeled primers in order to find improved methods for their identification. Strains of the following phenotypically highly similar species were examined: L. acidophilus, L. amylovorus, L. crispatus, L. johnsonii, L. gasseri, L. gallinarum, L. helveticus, L. iners and L. amylolyticus. Although the majority of the species can be differentiated by SDS-PAGE of whole-cell proteins, the latter technique showed poor discrimination between L. gasseri and L. johnsonii strains and between some strains of L. amylovorus and L. gallinarum. However, this study shows that the RAPD-PCR (using at least 3 different primers followed by numerical analysis of the combined patterns) and AFLP are most suitable genomic fingerprinting techniques for the differentiation of all the species listed above, and that databases for identification can be constructed, particularly when commercially available molecular tool-kits are used. The separate species status of the recently described L. amylolyticus and L. iners was fully confirmed.

Bacterial Proteins↗

PubMed1998

Physiological and molecular techniques for the study of bacterial community development in sausage fermentation.

The development of the microbial community involved in the production process of Italian dry sausage was investigated using physiological analysis and molecular techniques for strain typing and taxonomical identification. A cycle of sausage production was followed collecting samples during the 2 months of ripening process. Microbiological analysis allowed the identification of the main bacterial groups responsible for the fermentation process as lactobacilli and coagulase-negative staphylococci. The use of a polymerase chain reaction-based technique of strain typing, RAPD fingerprinting, demonstrated that the environmental parameters interact to select a limited number of strains that dominate the fermentation process. The staphylococcal populations were characterized for their physiological properties and the two dominant strains were identified as Staphylococcus xylosus and Staph. sciuri. The use of 16S rDNA sequencing allowed the definition of the taxonomical position of the two dominant strains of lactic acid bacteria, as belonging to Lactobacillus sake and Lact. plantarum.

Bacterial Typing Techniques↗

PubMed2000

Evaluation of numerical analyses of RAPD and API 50 CH patterns to differentiate Lactobacillus plantarum, Lact. fermentum, Lact. rhamnosus, Lact. sake, Lact. parabuchneri, Lact. gallinarum, Lact. casei, Weissella minor and related taxa isolated from kocho and tef.

AIM: The study was carried out to assess the agreement of API 50 CH fermentation data of food lactobacilli with their RAPD profiles to determine whether the system could be used alone as a reliable taxonomic tool for this genus. METHODS AND RESULTS: API 50 CH, RAPD and DNA:DNA reassociation data for 42 lactobacilli from tef and kocho were compared with 30 type strains. Discrepancies were observed between the three methods in assigning strains of Lactobacillus plantarum, Lact. fermentum, Weissella minor and Lact. gallinarum, and Lact. fermentum, Lact. amylophilus, Lact. casei subsp. pseudoplantarum and Lact. rhamnosus. DNA reassociation data agreed well with RAPD results. CONCLUSIONS: API 50 CH profiles should be complemented with molecular genetic results for effective identification in Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggested less dependability of metabolic data alone as an identification tool.

Bacterial Typing Techniques↗

PubMed1999

Strain typing among enterococci isolated from home-made Pecorino Sardo cheese.

Three molecular techniques (RAPD-polymerase chain reaction analysis, plasmid profile and pulsed-field gel electrophoresis) were used for a preliminary approach to type, at strain level, enterococci isolated from a 24-h-old home-made Pecorino Sardo (protected designation of origin) cheese. A high genetic polymorphism was found. Clusters obtained by the RAPD technique and plasmid profile analysis contained different strains. Pulsed-field gel electrophoresis proved to be an efficient and highly reproducible typing method. In addition, by combining the results from plasmid profile analysis and pulsed-field gel electrophoresis, it was possible to identify closely related strains probably belonging to the same clonal lineage.

Bacterial Typing Techniques↗

PubMed2000

Molecular characterisation of emergent multiresistant Salmonella enterica serotype [4,5,12:i:-] organisms causing human salmonellosis.

Salmonella multidrug-resistant clinical organisms identified as serotype [4,5,12:i:-] were typed using selected genetic procedures and compared with typhimurium organisms collected in the same Spanish region. Results showed a low genetic heterogeneity among [4,5,12:i:-] organisms, which generated identical ribotypes and similar but not identical XbaI PFGE, RAPD, and plasmid profiles. Multidrug resistance could be eliminated by curing and seems to be mediated by 140-kb (spvC+) and 120-kb (spvC-) non-self-transferable plasmids. The [4,5,12:i:-] organisms fall into a single genetic lineage, which emerged in 1997 and presents a different degree of genetic relationship with typhimurium lineages.

Anti-Bacterial Agents↗

PubMed2001

Binary typing of Staphylococcus aureus strains through reversed hybridization using digoxigenin-universal linkage system-labeled bacterial genomic DNA.

A novel binary typing (BT) procedure, based on reversed hybridization of digoxigenin-universal linkage system-labeled bacterial DNA to strip-immobilized probes, is presented. Chromogenic detection of hybrids was performed. Staphylococcus aureus isolates (n = 20) were analyzed to establish the feasibility of BT. A technically simple and fast procedure has been developed for application in routine microbiology laboratories.

Bacterial Typing Techniques↗

PubMed1998

Transmission and persistence of Actinobacillus actinomycetemcomitans in twins with advanced periodontitis.

The arbitrarily primed polymerase chain reaction technique (AP-PCR) was used to fingerprint Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis isolates in 44-year-old African American male and female twins who had not lived in the same household for 26 years. Both twins exhibited severe loss of periodontal attachment on several maxillary and mandibular teeth. All isolates of A. actinomycetemcomitans yielded the same AP-PCR banding pattern, whereas the P. gingivalis isolates from each twin showed different AP-PCR profiles. The finding of the same amplitype of A. actinomycetemcomitans in both twins suggests a single source of the organism and possibly a persistence of the organism in each twin for at least 26 years.

Actinobacillus Infections↗

PubMed1999

Molecular epidemiological investigation using a randomly amplified polymorphic DNA assay of Burkholderia cepacia isolates from nosocomial outbreaks.

We experienced two Burkholderia cepacia outbreaks over a 1-year period. During this period, 28 B. cepacia isolates were obtained from clinical specimens, and 2 were obtained from environmental specimens (i.e., from a nebulizer solution and a nebulizer tube). These 30 isolates were subjected to the PCR-based randomly amplified polymorphic DNA (RAPD) assay as well as to pulsed-field gel electrophoresis (PFGE). In the first outbreak, in which eight patients hospitalized in the Trauma and Critical Care Center were involved, the RAPD assay revealed that all 20 isolates obtained from clinical specimens and the 2 isolates from environmental specimens had identical DNA profiles. These RAPD data enabled us to pinpoint a possible source and to take countermeasures to prevent further spread of the epidemic-causing strain. In the second outbreak, two consecutive B. cepacia infection/colonization cases were seen in the surgery ward. The RAPD profiles of four isolates obtained were again identical, but they were distinct from those seen in the first outbreak, clearly indicating that the second outbreak was not related to the first. Thus, our experience demonstrated that the RAPD assay is a useful and reliable tool for epidemiological studies of B. cepacia isolates from nosocomial outbreaks. Since the RAPD assay could provide discriminatory potential and reproducibility comparable to those of the widely used PFGE assay with less complexity and in a shorter time, the introduction of the RAPD assay into hospital microbiology laboratories as a routine technique may help prevent nosocomial outbreaks.

Bacterial Typing Techniques↗

PubMed2001

[Genetic instability in cancer tissues analyzed by RAPD PCR].

In five kinds of tumors, total 128 specimens were analyzed by RAPD (random amplified polymorphic DNA) PCR with nine 10-base arbitrary primers for detecting instabilities of DNA and chromosome and screening new molecular markers coupled to putative or unknown oncogenes and/or tumor suppressor genes. Bands representing instabilities have been recovered and purified from agarose and cloned into pCAPs vector, and further labeled by DIG as probes for analysis of Southern blot, Northern blot and Sequencing. Results revealed that sample 5 and 3 of the gastric cancers showed the highest genomic changes and the average detectability in five sorts of cancers was up to at least 40% (42.2%-49.4%), and that there were significant differences in the ability of each primer to detect genomic instability, which ranged from 27% to 68%. Despite the highest detectability of genetic instability (68%) in tumor tissues, primer 2 could produce stable profiles of DNA bands in normal tissue genome with good reproducibility. On the contrary, primer 8 was of the lowest one (27%). Band B of single copy found to be allelic losses in gastric and colon cancers according to RFLP analysis was of a novel sequence and registered by Gen-Bank (Accession Number AF151005). Therefore the genetic instabilities often concentrated on some special locuses of chromosome e.g. repetitive sequences etc. and coupled to carcinogenesis. It was impossible or difficult to get great achievements for cancer treatments with the procedure of gene therapy only to one oncogene or one tumor suppressor gene because the extensive DNA variations occurred during the progression of tumor. RAPD assay connected with other techniques was a good tool for the detection of genomic instabilities and direct screening of some new molecular markers related to tumor suppressor genes or oncogenes.

Base Sequence↗

PubMed1996

DNA profiling and plant variety registration: 1. The use of random amplified DNA polymorphisms to discriminate between varieties of oilseed rape.

Before they can be marketed in the UK, newly bred varieties of crop species have to undergo a process of statutory testing, part of which involves the examination of the distinctness, uniformity and stability (DUS) of the variety. DUS testing is also used as the basis for the award of Plant Breeders' Rights. This paper examines the potential of DNA polymorphisms, amplified using arbitrary primers (RAPDs) for use in DUS testing of varieties of oilseed rape. RAPDs using suitable primers can produce high levels of discrimination (> 95%) between varieties, although there are certain problems in gel 'scoring' that are only partially resolved by computerised gel scanning/evaluation techniques. Varieties of oilseed rape are also heterogeneous in their RAPD profiles using certain primers, which could cause problems in the DUS testing context. DNA profiling with RAPDs could be used for discrimination between and identification of oilseed rape varieties, but its use for DUS testing needs to be considered carefully.

Base Sequence↗

PubMed1998

Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods.

Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) represent an increasing problem in hospitals. Quick and reliable typing methods are required to obtain information about the relatedness of MRSA isolates and to allow faster implementation of appropriate infection control measures. This investigation describes the distribution of MRSA isolates from 11 hospitals in the Düsseldorf region of Germany, and the ability of six different genotypic typing techniques -- pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), 16S-23S rDNA spacer amplification, protein A-gene PCR, PCR characterisation of the hypervariable region (HVR) adjacent to mecA, and coagulase gene-PCR -- to detect different unrelated types. Of 7814 S. aureus isolates tested, 489 (6.3%) were MRSA, of which 183 were selected for subsequent molecular analyses on the basis of being the first MRSA isolated from colonised or infected patients. Larger hospitals had a higher incidence of MRSA and a greater variability in genotypes than smaller hospitals. All methods confirmed the presence of two main clonal types. The ability of techniques to detect different unrelated types was found to be as follows: PFGE, 28 types; 16S-23S rDNA spacer-amplification, 10 types; RAPD, nine types; protein A-gene PCR, five types; HVR-PCR, five types; and coa gene-PCR, two types. Combination of PFGE and one other PCR-based method (spacer-amplification, RAPD or protein-A gene PCR) provided the best resolution of types and allowed the identification of subtypes. Similar molecular types were identified with international MRSA isolates. Although PCR-based techniques have the advantage of rapid performance and easy handling, their discriminatory capacity is inferior compared to the more labour intensive PFGE.

Bacterial Typing Techniques↗

PubMed1999

Phenotypic and genotypic typing of food and clinical isolates of Enterobacter sakazakii.

Enterobacter sakazakii, designated a unique species in 1980, has been implicated as the causative organism in a rare but severe form of neonatal meningitis. Dried infant formula milk has been identified as a potential source of the organism. E. sakazakii isolates from dried infant formula available in Canada and clinical isolates obtained from Canadian hospital culture collections were characterised by phenotypic (biotype and antibiograms) and genotypic (ribotyping, random amplification of polymorphic DNA and pulsed-field gel electrophoresis) methods. Three biotypes and four antibiogram patterns were observed in the 18 isolates examined. Ribotyping with the Dupont Riboprinter microbial identification system divided the 18 isolates into 10 ribotypes. Three isolates from the same hospital had indistinguishable ribotyping patterns although each was isolated in a different year, as did three food isolates from one company. Pulsed-field gel electrophoresis (PFGE) and random amplification of polymorphic DNA (RAPD) profiles indicated minor differences between the isolates that were indistinguishable by ribotyping. PFGE (with the restriction endonucleases Xba1 and Spe1) and RAPD gave discrete patterns that enabled easy comparison of E. sakazakii isolates, with a high degree of discrimination. The discriminatory index showed RAPD and PFGE were shown to be the most discriminatory typing schemes for E. sakazakii, followed by ribotyping, biotyping and antibiograms.

Bacterial Typing Techniques↗

PubMed1996

Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli.

Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species. This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains. Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones. The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis. Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes. As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse. Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named alpha and beta. Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers. Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the alpha rRNA spacer group. The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization. The alpha and beta clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution.

Adhesins, Bacterial↗

PubMed2000

Genetic diversity of the fish pathogen Aeromonas salmonicida demonstrated by random amplified polymorphic DNA and pulsed-field gel electrophoresis analyses.

The current taxonomy of Aeromonas salmonicida includes 4 subspecies. A. salmonicida subsp. salmonicida is associated with salmonid furunculosis, and A. salmonicida subsp. achromogenes, A. salmonicida subsp. masoucida, and A. salmonicida subsp. smithia are strains that show variation in some biochemical properties. This classification does not readily encompass isolates from a wide range of fish hosts currently described as atypical A. salmonicida. This study examined 17 typical strains, 39 atypical strains and 3 type A. salmonicida subspecies strains for genetic similarity using the random amplified polymophic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) techniques. On the basis of RAPD- and PFGE-derived profiles, similarity matrices and dendrograms were constructed. The results showed that species A. salmonicida constituted a genetically heterogeneous group of strains, encompassing within an homogeneous or clonal lineage comprised solely of typical strains and the A. salmonicida subsp. salmonicida type strain.

Aeromonas↗