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PubMed1997

A physiological and molecular analysis of the genus Nicotiana.

An analysis of the evolution of the genus Nicotiana was carried out with physiological and molecular tools. The capacity of explants from seedlings of several species of Nicotiana to differentiate roots or shoots or to habituate was used to ascertain whether the in vitro behavior of species has a nonrandom distribution in the genus. The results obtained allowed us to identify two groups of species, one root-forming prone composed of Paniculatae (subgenus Rustica) and the other composed of Alatae, Repandae, and Noctiflorae (subgenus Petunioides), with a major tendency toward the production of shoots. Habituation capacity was characteristic of species randomly distributed throughout the phylogenetic tree. These data suggest fixation throughout the evolution of coadapted gene complexes (hormone-related genes) involved in the control of developmental processes. RAPDs, on the other hand, used as molecular markers for the clustering of related species, seem entirely coherent both with classical morphological and karyological studies and with in vitro physiological methods, supporting an early subdivision of the whole genus into two diverging developmental patterns.

Adaptation, Physiological↗

PubMed2006

Phenotypic and molecular typing of Vibrio cholerae O1 and O139 isolates from India.

OBJECTIVES: To determine whether Vibrio cholerae strains with similar phage types are also genetically homogeneous or heterogeneous by molecular typing methods like PFGE and RAPD-PCR employed in this study. METHODS: A total of 26 V. cholerae O1 and O139 strains received from different parts of India were analyzed by using conventional phage typing method, antibiogram and molecular typing methods such as PFGE and RAPD-PCR. RESULTS: Both O1 and O139 strains were resistant against two antibiotics (Ampicillin and Furazolidone) were detected. All of these strains were clustered in a single phage type, i.e., Type 27 for V. cholerae O1 and Type 1 for V. cholerae O139. Extensive molecular characterization by RAPD and PFGE showed that six sets of O1 and O139 strains, each comprising two strains, had identical PFGE and RAPD profiles. Only one O139 strain (PL-4) had unique RAPD and PFGE profile among all the 26 V. cholerae strains used in this study. CONCLUSION: Apart from serology, the strains of V. cholerae can be discriminated by this conventional phage typing system that offers the basic information on identification, biotyping and discrimination of strains. But, a high level of heterogeneity was observed in RAPD and PFGE profiles indicating the clonal diversity of V. cholerae O1 and O139 strains. It was concluded that these strains were phenotypically identical through phage typing system and antibiogram but genetically dissimilar, as shown in molecular typing systems.

Anti-Bacterial Agents↗

PubMed1999

Actinobacillus actinomycetemcomitans serotype e--biotypes, genetic diversity and distribution in relation to periodontal status.

Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1-6 isolates per subject (n = 61) were genotyped using arbitrarily primed-polymerase chain reaction (AP-PCR) and apaH gene polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n = 219), localized juvenile periodontitis (n = 55) and other forms of early-onset periodontitis (n = 18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non-periodontitis subjects (n = 64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early-onset periodontitis patients and in 5 (8%) non-periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP-PCR distinguished 3 and apaH gene PCR-RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP-PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP-PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.

Adolescent↗

PubMed2003

Reclassification of Lactobacillus maltaromicus (Miller et al. 1974) DSM 20342(T) and DSM 20344 and Carnobacterium piscicola (Collins et al. 1987) DSM 20730(T) and DSM 20722 as Carnobacterium maltaromaticum comb. nov.

Phenotypic and genotypic characterizations of Lactobacillus maltaromicus strains DSM 20342(T) and DSM 20344 provided evidence for the reclassification of this species in the genus Carnobacterium. Moreover, phenotypic and genotypic comparisons made between L. maltaromicus and Carnobacterium piscicola highlighted that these two species should be considered synonyms. For these reasons, the species Carnobacterium maltaromaticum comb. nov. (type strain DSM 20342(T) = ATCC 27865(T) = CCUG 30142(T) = CIP 103135(T) = JCM 1154(T) = LMG 6903(T) = NRRL B-14852(T)) is proposed to accommodate L. maltaromicus and C. piscicola.

Bacterial Typing Techniques↗

PubMed2000

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci.

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.

Animals↗

PubMed2002

Subtype distribution of Haemophilus influenzae isolates from north India.

A total of 120 Haemophilus influenzae isolates from blood, cerebrospinal fluid, sputum and throat swabs of patients and carriers in North India was characterised by biotyping, ribotyping and random amplification of polymorphic DNA (RAPD)-PCR. Of these, 77 isolates (64%) were serotype b; the other 43 (36%) were non-typable. Biotype I was the most predominant among the typable strains and biotype II among the non-typable strains. Ribotyping with restriction endonucleases HaeIII and EcoRI differentiated the isolates into three and six ribotypes, respectively. However, RAPD fingerprints generated by the application of arbitrary primers AP1 and AP2 provided a higher level of discrimination. RAPD typing revealed distinct polymorphism among the serologically typable isolates. This study is the first report that stratifies the subtypes of H. influenzae strains from India by molecular techniques.

Adolescent↗

PubMed1998

Molecular epidemiology: assumptions and limitations of commonly applied methods.

An understanding of the epidemiology of a disease (i.e. its aetiology, transmission patterns) is crucial for the development and implementation of effective management practices. This requires sound epidemiological data. It is therefore important that scientists understand the assumptions and limitations of the methods used to gather such data. The aim of this paper is to discuss some of the assumptions and limitations of PCR-based methods used in studies of epidemiology. Since its development, PCR has had a major impact in the biological sciences. The ability to selectively amplify a specific region of the genome from a small amount of DNA makes this technique particularly useful as a diagnostic tool. A variety of PCR-based methods are available which can be used to identify strains and species of parasites. Some of these methods, such as random amplification of polymorphic DNA, have intrinsic properties which can limit their application. Other methods, such as PCR-restriction fragment length polymorphism, require the availability of a sound taxonomic or genetic framework for the development of any diagnostic system for a particular organism. The problems encountered developing diagnostic probes in the absence of such a framework will be discussed using Giardia intestinalis as an example.

Animals↗

PubMed2003

Culture-induced variation in plants of Coffea arabica cv. caturra rojo, regenerated by direct and indirect somatic embryogenesis.

Amplified fragment-length polymorphism (AFLP) was used to evaluate the stability of DNA in regenerated plantlets of Coffea arabica obtained by direct (DSE) and indirect somatic embryogenesis (ISE). Cluster analysis using the unweighted pair-group method (UPGMA), showed no specific grouping pattern related to the type of embryogenesis. These results suggest that the somatic embryogenesis (SE) process has a mechanism for the selection of normal and competent cells. Bulked DNA from regenerated plants obtained by DSE and ISE, and from the mother plants, was used to characterize specific AFLP fragments associated with each SE process. Twenty-three primer combinations were tested. A total of 1446 bands were analyzed, with 11.4% being polymorphic and 84% being specific for regenerated plants. Furthermore, specific bands were detected for DSE, ISE, and the mother plants. These results indicate that the SE process induces rearrangements at the DNA level and demonstrates discrepancies between the mechanisms involved in each SE process. Coffea arabica breeding programs that involve DSE and ISE can use AFLP as an additional tool for assessing DNA stability.

Breeding↗

PubMed1999

Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.

Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences.

Animals↗

PubMed2000

Detection and molecular characterization of Vibrio vulnificus from coastal waters of Malaysia.

A total of 57 Vibrio vulnificus isolates from coastal water were characterized for their antimicrobial resistance, plasmid profiles and were typed by the PCR-based techniques: a random amplification of polymorphic DNA (RAPD) method and the enterobacterial repetitive intergenic consensus sequence (ERIC) method. All isolates were susceptible to chloramphenicol, nalidixic acid, tetracycline and trimethoprim-sulfamethoxazole. Fifty-one isolates were resistant to one or more of the other antibiotics tested. Plasmid analysis indicated that only 18 isolates carried small plasmids of 1.6 to 16 megadaltons. Analysis of the RAPD and ERIC DNA fingerprints of the V. vulnificus isolates with Gel Compare and cluster analysis software revealed significant genetic heterogeneity among these isolates. The combination of RAPD and ERIC analysis allowed us to distinguish all isolates. Thus, the combination of the two techniques is recommended for epidemiological investigation.

Base Sequence↗

PubMed2005

Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains.

An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene.

Bacillus cereus↗

PubMed1998

Outbreak of Staphylococcus schleiferi wound infections: strain characterization by randomly amplified polymorphic DNA analysis, PCR ribotyping, conventional ribotyping, and pulsed-field gel electrophoresis.

Within a 1-year period, six surgical-site infections (SSI) caused by Staphylococcus schleiferi were observed in the department of cardiac surgery of Ignatius Hospital, Breda, The Netherlands. Since outbreaks caused by this species of coagulase-negative staphylococci have not been described before, an extensive environmental survey and a case control study were performed in combination with molecular typing of the causative microorganism in order to identify potential sources of infection. Variability, as detected by four different genotyping methods (random amplification of polymorphic DNA [RAPD], conventional and PCR-mediated ribotyping, and pulsed-field gel electrophoresis [PFGE] of DNA macro restriction fragments), appeared to be limited both among the clinical isolates and among several control strains obtained from various unrelated sources. Among unrelated strains, RAPD and PCR-mediated ribotyping identified two types only, whereas seven different types were identified in a relatively concordant manner by conventional ribotyping and PFGE. The latter two procedures proved to be the most useful tools for tracking the epidemiology of S. schleiferi. Four of the outbreak-related strains were identical by both methods, and two isolates showed limited differences. In the search for a potential source of S. schleiferi infection, two slightly different PFGE types were encountered on several occasions in the nose of a single surgeon. These strains were, however, clearly different from the outbreak type. In contrast, S. schleiferi cultures remained negative for two persons identified on the basis of case control analysis. It was demonstrated that SSI caused by S. schleiferi had a clinical impact for patients comparable to that of a wound infection caused by Staphylococcus aureus. This report describes the first well-documented outbreak of S. schleiferi infection. A source of the outbreak was not detected.

Aged↗

PubMed2000

Genotypic characterization of xanthomonad strains isolated from passion fruit plants (Passiflora spp.) and their relatedness to different Xanthomonas species.

The genetic diversity of 55 xanthomonad strains isolated from passion fruit plants (Passiflora spp.) and identified as Xanthomonas campestris pv. passiflorae was initially assessed by randomly amplified polymorphic DNA (RAPD) analysis. The strains showed a high level of polymorphism with almost unique fingerprints. Fifteen clusters with a similarity of approximately 70% were identified, three of which were prevalent. There was a correlation between the clusters and the geographic origin of the strains. A representative strain of each cluster, together with the pathovar reference strain, were used to verify the relationships of these strains to 18 Xanthomonas species and Pseudomonas syringae pv. passiflorae. All Xanthomonas species yielded a unique RAPD profile and no consistent relatedness to the X. campestris pv. passiflorae strains was observed. Amplification products were also analysed by repetitive (rep) primers (BOX, ERIC and REP), RFLP of the 16S-23S rDNA intergenic spacer and SDS-PAGE of whole-cell proteins. All of these approaches generated profiles characteristic for each Xanthomonas species but the taxonomic position of the X. campestris pv. passiflorae strains could not be unequivocally assigned. Finally, DNA-DNA hybridization allowed a sound taxonomic allocation of the strains to Xanthomonas axonopodis pv. passiflorae.

Bacterial Typing Techniques↗

PubMed2002

Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity.

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.

Animals↗

PubMed2004

Comparative assessment of genotyping methods for study genetic diversity of Fusarium oxysporum isolates.

In this study, we evaluated three PCR-based methods for the molecular typing of nonpathogenic Fusarium oxysporum isolates: random amplified polymorphic DNA (RAPD), polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and amplified fragment length polymorphism (AFLP). The analyses were performed using 64 isolates of F. oxysporum collected from cotton-producing areas in Egypt. A number of polymorphic RAPD, PCR-RFLP and AFLP bands were scored in all isolates and the genetic similarity among them was assessed. Clustering analysis separated the isolates into two main groups, with similarities ranging from 87 to 100% for RAPD, 80 to 100% for PCR-RFLP and 88 to 97% for AFLP, respectively. The obtained data suggested that all three types of markers are equally informative, but the three assays differed in the amount of detected polymorphic bands. AFLP fingerprinting was also found to be more differentiating than other techniques for the typing of F. oxysporum populations.

DNA, Ribosomal Spacer↗

PubMedDate not supplied

Epidemiological survey of an outbreak of multiresistant Serratia marcescens by PCR-fingerprinting.

During an outbreak of Serratia marcescens from May to November 1993 43 strains obtained from 27 ICU patients infected or colonized with multiresistant S. marcescens were genotypically characterized with random amplified polymerase chain reaction (RAPD-PCR)-fingerprinting. In addition, 43 epidemiologically unrelated control isolates were selected. PCR-fingerprinting identified ten different genotypes of S. marcescens among the outbreak related strains. One predominant genotype was demonstrated in 21/43 isolates of 11/27 patients. A cluster of this genotype was found in seven/eight patients on the cardiosurgical ICU. The epidemiologically unrelated strains all showed different genotypes as compared to the predominant type. This survey proved RAPD-PCR to be a highly discriminatory and reproducible method for epidemiological studies of S. marcescens strains in nosocomial outbreaks.

Bacterial Typing Techniques↗

PubMed2005

Escherichia coli strains belonging to phylogenetic group B2 have superior capacity to persist in the intestinal microflora of infants.

Escherichia coli strains segregate into 4 phylogenetic groups, designated "A," "B1," "B2," and "D." Pathogenic strains belong to group B2 and, to a lesser extent, group D, which more frequently carry virulence-factor genes than do group A strains and group B1 strains. This study investigated whether the capacity of E. coli to persist in the human intestine is related to its phylogenetic type. Resident (n=58) and transient (n=19) commensal E. coli strains isolated during a longitudinal study of 70 Swedish infants and previously tested for virulence-factor-gene carriage were tested for phylogenetic type. Of the strains resident in the intestinal microflora, 60% belonged to group B2, compared with only 21% of the transient strains (P=.004). In logistic regression, group B2 type predicted persistence in the intestinal microflora, independent of carriage of all investigated virulence-factor genes, including genes for P fimbriae (P=.03). Thus, group B2 strains appear to possess yet unidentified traits that enhance their survival in the human intestine.

Adhesins, Escherichia coli↗

PubMed1998

Evaluation of PCR-based methods and ribotyping performed with a mixture of PstI and SphI to differentiate strains of Salmonella serotype Enteritidis.

The capacity to differentiate Salmonella serotype Enteritidis strains by PCR ribotyping; RAPD typing with three arbitrary primers and ribotyping with a mixture of PstI and SphI or 'PS ribotyping', was evaluated on a series of 65 strains associated with human infections and 11 reference strains. The series had been analysed previously by phage typing and ribotyping performed with PstI and SphI, separately. All methods typed all the strains; however, only ribotyping showed good reproducibility and sensitivity. Twenty-two PS ribotypes (discrimination index = 0.74) were identified, differentiating strains ascribed to seven phage types (PTs 1, 4, 6, 6a, 7, 8 and RDNC) as well as phage untyped strains. Conversely, some strains of PTs, 1, 4, 5a, 6, 6a, 7, 34 and RDNC showed the most frequent PS ribotype. By PCR ribotyping a single profile was found; while by RAPD typing, one, two or three RAPD types were identified with the primers MK22, OPB6 and OPB17, respectively. All Spanish strains were assigned to a single combined RAPD type, except PT11 strains which showed a different and specific RAPD type with OPB17. The banding patterns defining the PS ribotypes were interpreted more easily and the patterns could be compared more accurately than the banding patterns defining RAPD types. A similarity dendrogram generated from the 22 PS ribotypes was traced and compared with RAPD types and phage types. Data from this work indicated that 'PS ribotyping' was the most useful genetic procedure to differentiate Enteritidis strains, and, therefore, it can be used as a complementary or alternative typing method to phage typing within this serotype.

Bacterial Typing Techniques↗