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PubMed1995

Randomly amplified polymorphic DNA (RAPD) for rapid typing of Lactobacillus plantarum strains.

Randomly amplified polymorphic DNA (RAPD) has been used for rapid typing of Lactobacillus plantarum strains. RAPD was used with either purified chromosomal DNA serving as template in the polymerase chain reaction, or with crude cell extracts, and using a 9-mer primer with 80% G+C content. Amplified DNA was visualized by ethidium bromide staining after separation on agarose gels. Patterns from 20 Lact. plantarum strains and two Lact. pentosus strains were analysed using the Pearson products moment correlation coefficient (r) and the unweighted pair group method with arithmetic averages (UPGMA). With some exceptions, the two sources of template DNA gave the same clusters and subclusters of strains at the similarity level of 50%. About 50% of the strains could be individually separated from all the other tested strains. The buffer brand, the amount of primer and crude cell extract used in the PCR-step were crucial for the final pattern.

Bacterial Typing Techniques↗
PubMed1998

Characterization of clinical isolates of Helicobacter pylori in Singapore.

Of the 69 Helicobacter pylori isolates analysed, 31 strains (45%) showed resistance to metronidazole, one strain (1.4%) was resistant to amoxicillin while two strains (2.9%) were resistant to clarithromycin. It was found that metronidazole resistance rates increased in Singapore from 20% to 62% between late 1995 and early 1997. By biotyping using API ZYM, a total of 80% (55/69) strains were characterized as biotype II, while the remaining 20% (14/69) strains belonged to biotype III. Interestingly, 71% (10/14) of biotype III were resistant to metronidazole compared with 38% (21/55) of biotype II. DNA profiles generated by random amplified polymorphic DNA from 69 isolates showed highly diversified DNA fingerprints allowing effective discrimination among strains. Of the 60 H. pylori isolates from peptic ulcer patients, it was found that cagA and vacA occurred in 80% (48/60) and 82% (49/60) isolates, respectively.

Amoxicillin↗
PubMed2003

[Application of randomly amplified polymorphic DNA on the DNA polymorphism of Staphylococcus aureus and Klebsiella pneumoniae].

By using short single oligomer primer with arbitrarily chosen sequences in polymerase chain reaction, randomly amplified polymorphic DNA (RAPD) was applied in differentiating clinical isolates of 52 Klebsiella pneumoniae and 28 Staphylococcus aureus. RAPD produced multiple polymorphic DNA bands when K. pneumoniae DNA was used as template. Using DNA fingerprints technique, the similarity coefficients values of 52 K. pneumoniae was measured and calculated, and dendrogram was drawn. The 52 Klebsiella pneumoniae were divided into 7 Patterns. But in the 28 Staphylococcus aureus, 3 characteristic lands were obtained. Minor differences in the RAPD fingerprints were detected. The research findings demonstrated that RAPD has a potential as a widely useful tool in providing genotypic characters for taxonomic descriptions and molecular epidemiology study.

DNA, Bacterial↗
PubMed2003

Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures.

An aberrant random amplified polymorphic DNA (RAPD) marker in genomic DNA of tissue culture plantlets was frequently observed during a comparison of DNA fingerprints derived from potato germplasm grown in tissue culture and the field. The RAPD marker was cloned, sequenced and determined to be of bacterial origin. A bacterial contaminant was isolated from the tissue culture plants and identified as a Bacillus pumilus. A set of sequence characterised amplified region (SCAR) primers were designed from the sequence of the cloned fragment and tested for the specific detection of B. pumilus. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) were also used to generate B. pumilus profiles specific to our isolate in order to test and confirm the sequence homology of amplified markers generated from a range of DNA samples isolated from tissue culture plants and pure isolates of B. pumilus-like bacteria.

Bacillus↗
PubMedDate not supplied

Evaluation of molecular typing methods for Salmonella enterica serovar Typhimurium DT104 isolated in Germany from healthy pigs.

The discriminatory power of four different DNA based typing methods was tested for the molecular subtyping of Salmonella Typhimurium phage type DT104 isolates. German DT104 strains (n = 133) originating from slaughter pigs were analysed by plasmid profiling, and 32 of them by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes XbaI, SpeI or BlnI, random amplification of polymorphic DNA (RAPD) using 13 different primers and IS200 typing. A resulting subtyping scheme was obtained which is based on the most discriminatory power of the individual methods i.e. plasmid profiling and PFGE with all three enzymes. The index of discrimination obtained by the subtyping scheme was 0.909 closely approaching the maximum value of one. Although minor differences occurred in the molecular DNA pattern of single DT104 strains, a dominating subtyping pattern was observed confirming other studies which showed, that S. Typhimurium DT104 isolates are highly clonal.

Animals↗
PubMed2006

Detection of Bacillus thuringiensis kurstaki HD1 on cabbage for human consumption.

The objectives of the study were to develop a specific procedure for quantification and identification of Bacillus thuringiensis kurstaki HD1, which is used as a biopesticide, and to quantify its presence in different kinds of cabbage for human consumption. We found that B. thuringiensis kurstaki HD1 can be distinguished from other B. thuringiensis strains by its unique random amplification of polymophic DNA-PCR pattern with the OPA9 primer and the presence of the flagellin genes, as detected by the primers FLAB1 and FLAB2. We detected from one to 100 Bacillus cereus-like bacteria in 10 batches of five different cabbage products for consumption. As many as 73 out of 134 isolates (53.7%) were identical with B. thuringiensis kurstaki HD1. The results show that B. thuringiensis kurstaki HD1 from biopesticides can be found in vegetables for human consumption.

Bacillus cereus↗
PubMed2002

Typing and subtyping of 83 clinical isolates purified from surgically implanted silicone feeding tubes by random amplified polymorphic DNA amplification.

In this study, 83 clinical isolates purified from biofilms colonizing 18 silicone gastrostomy devices (12 "buttons" and six tubes converted to skin level devices) were selected for subtype characterization utilizing genetic analysis. The tubes, previously used for feeding, remained in place for 3 to 47 months (mean, 20.0 months) in children ranging in age from 6 months to 17 years. Classification of specific microbes using random amplified polymorphic DNA (RAPD) analysis revealed genetic similarities and differences among isolates belonging to the same genus. Both gram-positive and -negative bacteria were investigated, including 2 isolates of Bacillus brevis, 4 isolates of Bacillus licheniformis, 2 isolates of Bacillus pumilus, 3 isolates of Enterococcus durans, 19 isolates of Enterococcus faecalis, 8 isolates of Enterococcus faecium, 2 isolates of Enterococcus hirae, 7 isolates of Escherichia coli, 8 isolates of Lactobacillus plantarum, 19 isolates of Staphylococcus aureus, 2 isolates of Staphylococcus epidermidis, and 7 isolates of Staphylococcus saprophyticus. Amplified DNA fragments (amplicons) provided species-specific fingerprints for comparison by agarose gel electrophoresis. A total of 62 distinct RAPD types were categorized from the five genera studied. Typing analysis suggested cross acquisition of E. coli, E. faecalis, and S. aureus in three patient pairs. Genomic polymorphism detection proved efficient and reliable for classifying bacterial subtypes isolated from biofilms adhering to various portions of commonly employed enteral access tubes.

Adolescent↗
PubMed2001

Restriction endonuclease analysis of RAPD-PCR amplicons derived from Shiga-like toxin-producing Escherichia coli O157 isolates.

Shiga-like toxin-producing Escherichia coli O157 isolates were characterised by random amplification of polymorphic DNA by PCR (RAPD-PCR) analysis developed to allow robust epidemiological typing of E. coli. Amplification with primer 1247 or 1290 generated a reproducible profile, but was not capable of distinguishing sufficiently between epidemiologically unrelated strains. Subsequent digestion of the amplicons with selected restriction endonucleases improved the discriminatory ability of this method for strains showing limited differentiation following RAPD-PCR analysis alone. Restriction endonuclease analysis of RAPD-PCR fragments generated from closely related strains has the potential to provide additional discriminatory information without loss of specificity.

Bacterial Typing Techniques↗
PubMed2002

Comparison between Streptococcus macedonicus and Streptococcus waius strains and reclassification of Streptococcus waius (Flint et at. 1999) as Streptococcus macedonicus (Tsakalidou et al. 1998).

Two species of dairy streptococci, Streptococcus waius and Streptococcus macedonicus, were originally characterized by 16S-23S intergenic spacer sequence analysis, random amplified polymorphic DNA fingerprinting, PFGE analysis and DNA-DNA reassociation experiments. All genetic data suggested that S. waius strains belong to the previously described species S. macedonicus. Likewise, the phenotypic characterization showed that strains of S. macedonicus and S. waius were highly related and easily differentiated from the closest phylogenetic neighbour, Streptococcus bovis, principally by their failure to produce a blackening reaction in medium containing aesculin. The utilization of maltose and cellobiose by S. macedonicus/S. waius strains allowed their differentiation from the most studied dairy species, Streptococcus thermophilus. On the basis of genetic and phenotypic data S. macedonicus and S. waius species should be considered synonyms and S. macedonicus has the priority.

Bacterial Typing Techniques↗
PubMed1998

Diversity among clinical isolates of Proteus penneri detected by random amplified polymorphic DNA analysis.

DNA of thirteen haemolytic Proteus penneri strains of clinical origin, all producing calcium dependent haemolysin and having been derived from four European countries was examined for plasmid profile, and outer membrane protein profile, by random amplified polymorphic DNA-PCR (RAPD-PCR) method, and digestions with restriction endonucleases were performed. All strains contained two large plasmids of approximately 60 and 70 kilobase pairs (kb). In addition, four strains contained a small plasmid of about 6 kb. These four strains produced cell-bound haemolysin only. Outer membrane protein analysis revealed subtle differences between strains. RAPD-PCR with primer I (CCGCAGCCAA) revealed 13 types, whereas primer II (AACGCGCAAC) yielded only two main types of different patterns. Results with primer I suggests a DNA sequence diversity within this species. The RAPD-PCR method provides a fast, economical and reproducible means for the typing of P. penneri. Digestion with restriction endonucleases indicated a high level of DNA methylation in this species.

Bacterial Outer Membrane Proteins↗
PubMed1997

Genetic typing methods applied to the differentiation of clonal lines among Salmonella enterica serogroup G strains causing human salmonellosis.

In Spain, in November 1995, an epidemiological alert recommended the surveillance of Salmonella serogroup G. The nine clinical isolates collected after and the four collected before the alert in Asturias were differentiated into six clonal lines by the combination of results from HincII ribotyping, PCR ribotyping, and RAPD typing using primers named A and S. The seven Gumpensis isolates showed identical DNA fingerprinting with the four typing procedures falling into a line. Six of these were collected during May-August from people living in a single health area suggesting that they could be associated with a community outbreak. The four Worthington isolates fell into three other lines, one Poona isolate into another line and one Havana isolate into another. 100% typeability was shown with all methods. The reproducibility of HincII ribotyping was better than that of PCR-based methods, although these were less time-consuming. The highest discriminatory power was obtained with HincII ribotyping and RAPD typing using primer A.

Adult↗
PubMed1999

Variable numbers of tandem repeat loci in genetically homogeneous Haemophilus influenzae strains alter during persistent colonisation of cystic fibrosis patients.

Serial sputum isolates of Haemophilus influenzae (n = 69) were obtained from eight patients suffering from cystic fibrosis. For two of these patients all strains were analysed for polymorphism in the major outer membrane protein profile. For all patients the strains were genetically characterised by random amplification of polymorphic DNA analysis. All strains were included in a survey for polymorphism in regions containing moieties of repetitive DNA as well. A single locus containing trinucleotide repeat units, three loci harbouring tetranucleotides, one region comprising pentanucleotide units and two hexanucleotide repeat unit-containing loci were analysed for repeat number variability. Most of the regions were previously shown to be directly adjacent to or even within virulence genes. All regions behaved as genuine variable number of tandem repeat loci in the sense that genetic polymorphism based on the presence of varying numbers of repeat units could be demonstrated among different strains. Interestingly, several of the repeats showed variation in the absence of the variability as assessed by major outer membrane protein or random amplification of polymorphic DNA analysis. These observations indicate that the repeat loci may vary independently from major chromosomal polymorphism. Consequently, H. influenzae appears to modify its virulence gene regions of the chromosome during persistent colonisation of the lung in cystic fibrosis patients.

Bacterial Typing Techniques↗
PubMed1999

Legionella taurinensis sp. nov., a new species antigenically similar to Legionella spiritensis.

A group of 42 Legionella-like organisms reacting specifically with Legionella spiritensis serogroup 1 antisera were collected throughout Europe by the Centre National de Référence (French National Reference Centre) for Legionella. This group of isolates differed somewhat from L. spiritensis in terms of biochemical reactions, ubiquinone content and protein profile. The latter two analyses revealed that one of these L. spiritensis-like isolates, Turin I no. 1T, was highly related, but not identical to any of the red autofluorescent species of Legionella. In fact, this strain was the first of these particular isolates recognized to emit a red autofluorescence when exposed to UV light. Profile analysis of randomly amplified polymorphic DNA established that the red autofluorescent L. spiritensis-like isolates constituted a homogeneous group distinct from Legionella rubrilucens and Legionella erythra. DNA-DNA hybridization studies involving the use of S1 nuclease confirmed that the indicated group of isolates are a new species of Legionella, for which the name Legionella taurinensis is proposed with strain Turin I no. 1T (deposited as ATCC 700508T) as the type strain.

Antigens, Bacterial↗
PubMed2004

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park.

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.

Animals↗
PubMed2006

Molecular and phenotypic characterisation of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L.

Twelve Bacillus thuringiensis strains were isolated from intestinal tracts of Cydia pomonella larvae during epizootics in different laboratory insect culture lines. Phenotypic and genetic similarity of these isolates, together with two cultured from Leucoma salicis larvae and 14 reference B. thuringiensis strains were determined. The epizootic bacteria did not form a single group based on numerical analysis of biochemical properties. Simple RAPD method with only one primer does not allow estimating the genetic similarity of B. thuringiensis strains. We propose a novel strategy based on combining several DNA patterns obtained by RAPD technique with different primers for B. thuringiensis typing. Majority of infections in the C. pomonella culture lines were caused by bacteria with different genotypes. However, two isolates cultured from infected insects at different time (one strain was isolated in 1990 and the other in 1992) had identical DNA fingerprint that suggested a possibility of these bacteria to survive in the laboratory and to cause infections in different years. The results of SDS-PAGE of whole-cell proteins revealed a possibility to apply protein profile analysis in epidemiological investigations of infections caused by B. thuringiensis. Strains with identical DNA patterns had very similar whole-cell protein profiles.

Animals↗
PubMed2003

Identification of lactic acid bacteria isolated from human vaginal secretions.

A total of 57 lactic acid bacteria were isolated from the vaginal secretions of 259 patients. Of these strains, 37 were isolated from patients attending pre-natal clinics and the remaining strains from patients attending post-natal clinics. The strains were identified by using simple physiological and biochemical tests and their phenotypic relatedness determined by numerical analysis of total soluble cell protein patterns. The genotypic relatedness of representative strains selected from each of the protein profile clusters was determined by numerical analysis of the DNA banding patterns obtained from RAPD-PCR. The majority of lactobacilli isolated belonged to the species Lactobacillus pentosus, Lactobacillus fermentum and Enterococcus faecium. A few strains of Lactobacillus plantarum and Weissella viridescens were also isolated. One strain, TV 1029, grouped into the same protein profile cluster as E. faecium, but revealed a DNA banding pattern closer related to Enterococcus faecalis. This is the first report of W. viridescens associated with the human vagina.

Bacterial Proteins↗