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PubMed2001

Beyond serotypes and virulence-associated factors: detection of genetic diversity among O153:H45 CFA/I heat-stable enterotoxigenic Escherichia coli strains.

Characterization of enterotoxigenic Escherichia coli (ETEC) has been based almost exclusively on the detection of phenotypic traits such as serotypes and virulence-associated factors: heat-labile (LT) and heat-stable (ST) toxins and colonization factors (CFs). In the present work we show that the analysis of band patterns generated by randomly amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) of digested chromosomal DNA can be used to detect genetic diversity among ETEC strains expressing identical phenotypic traits. The study included 29 ETEC isolates from Latin America and Spain expressing the phenotype O153:H45 CFA/I ST plus 1 rough derivative, 2 nonmotile derivatives, and 1 O78:H12 CFA/I ST isolate, and a representative of a genetically distinct ETEC group. The results showed that the O153:H45 CFA/I ST ETEC isolates belong to a single clonal cluster whose isolates share on average, 84% of the RAPD bands and 77% of the PFGE restriction fragments, while the O78:H12 isolate shared only 44 and 4% of the RAPD bands and PFGE fragments, respectively, with the isolates of the O153:H45 group. More relevantly, RAPD and PFGE fingerprints disclosed the presence of different clonal lineages among the isolates of the O153:H45 cluster. Some of the genetic variants were isolated from defined geographic areas, while places like São Paulo City in Brazil and the middle-eastern part of Argentina were populated by several genetic variants of related, but not identical, ETEC strains. These results show that molecular biology-based typing methods can disclose strain diversity, which is usually missed in studies restricted to phenotypic typing of ETEC.

Bacterial Proteins↗
PubMed1996

Random-amplified polymorphic DNA (RAPD) analysis shows intraspecies differences among Xanthomonas albilineans strains.

Five strains of Xanthomonas albilineans, causal agent of leaf scald disease in sugarcane from various geographical regions, were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the DNA level. CsCl-purified genomic DNA from these strains were amplified by the polymerase chain reaction (PCR) using arbitrary 10-mer primers according to standard RAPD conditions and the amplification product profiles analysed by conventional agarose gel electrophoresis. Although most RAPD markers were common to all five strains, unique profiles for each strain were discernible using four 10-mer arbitrary primers individually. Reproducible DNA fingerprints indicate that RAPD analysis can be used to identify and differentiate the X. albilineans strains. This technique has the potential for use in monitoring the appearance of foreign strains of X. albilineans in various geographical regions and could be used for the construction of phylogenetic trees.

DNA Fingerprinting↗
PubMed2003

Distinction between cold-sensitive and -tolerant jute by DNA polymorphisms.

Jute is the principal coarse fiber for commercial production and use in Bangladesh. Therefore, the development of a high-yielding and environmental-stress tolerant jute variety would be beneficial for the agro economy of Bangladesh. Two molecular fingerprinting techniques, random-amplified polymorphic DNA (RAPD) and amplified-fragment length polymorphism (AFLP) were applied on six jute samples. Two of them were cold-sensitive varieties and the remaining four were cold-tolerant accessions. RAPD and AFLP fingerprints were employed to generate polymorphism between the cold-sensitive varieties and cold-tolerant accessions because of their simplicity, and also because there is no available sequence information on jute. RAPD data were obtained by using 30 arbitrary oligonucleotide primers. Five primers were found to give polymorphism between the varieties that were tested. AFLP fingerprints were generated using 25 combinations of selective-amplification primers. Eight primer combinations gave the best results with 93 polymorphic fragments, and they were able to discriminate the two cold-sensitive and four cold-tolerant jute populations. A cluster analysis, based on the RAPD and AFLP fingerprint data, showed the population-specific grouping of individuals. This information could be useful later in marker-aided selection between the cold-sensitive varieties and cold-tolerant jute accessions.

Cold Temperature↗
PubMed2002

DNA polymorphism in the living fossil Ginkgo biloba from the eastern United States.

Random amplified polymorphic DNA (RAPD) analysis is a valuable tool in studying inter- and intra-specific genetic variations, patterns of gene expression, and for the identification of specific genes using nearly isogenic variants. Here we used RAPD analysis to study the genetic variation in Ginkgo biloba grown in the eastern United States. Our results support the evidence that Southern blot hybridization of RAPD using probes made from cloned DNA fragments allows a more accurate analysis of the RAPD pattern than dye-stained gels or Southern blot hybridization of RAPD blots using probes made from purified PCR products. Using these techniques, we observed a high degree of relatedness among plants grown in certain localities although significant genetic variation may exist in the species, and could be a possible explanation for the observed variations in the efficacy of medications derived from G. biloba extract.

District of Columbia↗
PubMed2006

A high level of strain variation within the Mycoplasma ovipneumoniae population of the UK has implications for disease diagnosis and management.

Mycoplasma ovipneumoniae is one of only two mycoplasma species associated with small ruminant disease in Britain and has been associated with an increasing number of disease outbreaks since 2002. This investigation used well-defined techniques to assess the variability of UK M. ovipneumoniae isolates, in an attempt to identify strain clusters within the population. Strains received for routine diagnosis between 2002 and 2004 were analysed using random amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE). Of the 43 samples screened 40 RAPD Hum-1, 41 RAPD Hum-4 and 40 PFGE profiles were observed. Composite data analysis divided strains into 10 similarity clusters with SDS-PAGE and Western blotting indicating that this DNA variability is translated into a pattern of variable protein expression. In order to assess the strains isolated within flocks two sets of samples, from diverse locations, were included in this test panel. The presence of variable isolates existing on the same farm may reflect animal movement and the introduction of asymptomatic, carrier, animals where M. ovipneumoniae is already established within a flock. These findings have significant implications regarding disease diagnosis and management.

Animals↗
PubMed2002

A polyphasic approach to highlight genotypic and phenotypic diversities of Lactobacillus helveticus strains isolated from dairy starter cultures and cheeses.

In the present work, 67 strains of Lactobacillus helveticus isolated from whey starter cultures and cheeses were identified and grouped by genotypic and phenotypic methods. Strains were identified by sugar fermentation pattern, by cell-wall protein profile, and by probe hybridisation. Phenotypic diversity was evaluated by a chemometric model taking into account biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR, which provided stran-specific patterns and revealed the occurrence of different strains. The RAPD-PCR profiles were clustered according to their similarities: the groups obtained, together with the cell-wall protein profiling and the chemometric information, could be sometimes correlated with the type of cheese and/or dairy niches used as sources of strains. A computerised analysis of genotypic and phenotypic information could be successfully applied for rapid and reliable differentiation and characterisation of Lb. helveticus isolates occurring in different dairy products.

Animals↗
PubMed2001

Recognition of Yersinia enterocolitica multiple strain infection in twin infants using PCR-based DNA fingerprinting.

AIMS: Yersinia enterocolitica causes several syndromes in humans. The most common presentation is enterocolitis in children, presenting as fever and diarrhoea. A Y. enterocolitica multiple strain infection in twin infants was investigated. METHODS AND RESULTS: One isolate was recovered from one patient and two morphologically-different isolates were recovered from the other infant. Biochemically, all isolates were identified as Y. enterocolitica group. The genomic DNA from each strain was purified and DNA fingerprinting was performed. The banding patterns observed for Y. enterocolitica isolates 2 and 3, from patients 1 and 2, respectively, were identical when comparing the presence or absence of major bands. However, Y. enterocolitica isolate 1, from patient 1, showed a distinctive banding pattern from isolates 2 and 3. CONCLUSION: The findings indicate that one infant was colonized by more than one strain of Y. enterocolitica, demonstrating that multiple strains can colonize and invade a patient. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of multiple strain infections can be important in diagnosis, treatment and prognosis of Y. enterocolitica infections, as well as in disease epidemiology. The technique described here offers a straightforward method for strain comparison.

Anti-Bacterial Agents↗
PubMed2002

Use of predictive modeling for Propionibacterium strain classification.

Computed data analysis of biochemical or molecular profiles is currently used in studies of microbial taxonomy, epidemiology, and microbial diversity. We assessed the use of Partial Least Squares (PLS) regression for multivariate data analysis in bacteriology. We identified clear relationships between RAPD profiles of propionibacteria strains and their species classification, autolytic capacities, and their origins. The PLS regression also predicted species identity of some strains with RAPD profiles partially related to those of reference strains. The PLS analysis also allowed us to identify key characteristics to use to classify strains. PLS regression is particularly well adapted to i) describing a collection of bacterial isolates, ii) justifying bacterial groupings using several sets of data, and iii) predicting phenotypic characters of strains that have been classified by routine typing methods.

Autolysis↗
PubMed2002

Correlation of phylogenetic lineages of group B Streptococci, identified by analysis of restriction-digestion patterns of genomic DNA, with infB alleles and mobile genetic elements.

Phylogenetic lineages of pathogenic Streptococcus agalactiae (group B streptococci [GBS]) can be identified by analysis of restriction-digestion patterns (RDPs) of chromosomal DNA. The purpose of the present study was to correlate GBS RDP types and (1) alleles of the highly conserved gene encoding translation-initiation factor IF2, infB, and/or (2) the inserted elements IS1548 and GBSi1. Only 1 combination of serotype and infB allele was found within each RDP type. Strains within a particular RDP type also tend to have the same inserted elements in each of 3 loci examined. A novel insertion sequence, designated "IS1563," was found within all RDP type II-2 strains. Most RDP types could be identified by a combination of serotype, infB allele, and inserted elements at each of the loci. These molecular markers can be used to identify GBS populations and to correlate RDP types and phylogenetic lineages identified by different methods.

Alleles↗
PubMed2002

Escalating association of Vibrio cholerae O139 with cholera outbreaks in India.

Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other. In two of these outbreaks each, only V. cholerae O1 biotype ElTor or V. cholerae O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated. The interesting feature is the escalating association of V. cholerae O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139. The O139 strains from the six different outbreaks were genotypically closely related. These trends indicate a shift in the outbreak propensity of V. cholerae O139.

Bacterial Typing Techniques↗
PubMed2001

[Analysis of Leishmania species and strains from China by RAPD technique].

OBJECTIVE: To analyze genetic relationship of Leishmania species and strains from China by RAPD technique. METHODS: DNAs from Leishmania strains, including L. donovani (L. d.) isolates from patients, dogs and sandflies of three different foci in China and international reference strains, were amplified by seven random primers. The DNA polymorphic bands detected were analyzed by clustering analysis with SPSS software. RESULTS: 1. L. d. isolates from hill and plain foci in China were divided into two groups. The genetic distance of L. d. isolates is distant between them. 2. L. d. XJ771, L. d. XJ901, L. d. XJ801 from desert, vicinity of desert, and plain regions in Xinjiang were in the same group. It indicated that the genetic distance among L. d. isolates from the three regions is close. 3. L. d. isolated from VL patients and dogs in hill foci could not be discriminated distintly, showing high homology between them. 4. L. d. DD8 from India, the reference strain of plain type, was clustered with L. d. isolates from plain foci in China. It provided scientific basis for the viewpoint "Kala Azar from east area of China is similar to that from India". 5. L. infantum and L. d. isolates from hill foci in China were clustered into different groups. 6. The genetic distance is close between L. d. isolates from plain foci in China and L. d. Jed; 7. L. infantum and L. tropica showed the closest genetic distance. CONCLUSION: Differences at genetic level exist in Leishmania isolates from different foci in China.

Animals↗
PubMed2001

Characterisation of lab in typical Salento Pecorino cheese.

Twenty-nine strains of Lactic Acid Bacteria isolated from the typical Pecorino cheese of the Salento area of Italy, were identified and grouped according to their genetic similarity. A preliminary characterisation of the strains was conducted by means of morphological and biochemical analysis, but molecular approaches were necessary for the clear identification of the species. For the species detection, the amplification and sequencing of the 16S rDNA gene was employed In addition, restriction analysis of amplified rDNA (ARDRA) and PCR and AFLP fingerprinting enabled inter- and intra-specific variation to be estimated UPGMA cluster analysis was used to divide the strains into distinct clusters which corresponded with the species delineation obtained by molecular identification. The data obtained show that the community of lactobacilli responsible for the fermentation and aging of Pecorino cheese is composed of a limited number of species. The main identified strains were Lactobacillus brevis, L. plantarum, L. casei, L. sakei, L. pentosus, L. farciminis and Leuconostoc mesenteroides.

Bacterial Typing Techniques↗
PubMed2004

Trichomonas vaginalis: random amplified polymorphic DNA analysis of isolates from symptomatic and asymptomatic women in India.

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a protozoan parasite. Trichomoniasis is the most common non-viral sexually transmitted disease. The infection in women may be asymptomatic or may lead to severe vaginitis, cervicitis and severe sequelae. Despite its high prevalence, the genetic variability and factors leading to symptomatic infection have been poorly understood. One thousand women in childbearing age group were screened for the presence of T vaginalis. Thirty-eight women were found positive for T vaginalis and out of these 22 (57.9%) were having symptomatic infection and 16 (42%) were asymptomatic. Fresh isolates from 15 symptomatic and 15 asymptomatic women were axenised and subjected to random amplified polymorphic DNA (RAPD) analysis with the use of five different random primers (OPD 1-OPD 5). The isolates with similar banding pattern were assigned as a single type. OPD 3 indicated least (nine types) while OPD 4 indicated highest typing (18 types) ability. Phylogenetic analysis using RAPD distance software indicated two distinct lineages; upper branch consisting of only seven symptomatic isolates while lower branch consisting of all the 15 asymptomatic isolates the other eight symptomatic isolates were recorded in separate cluster. The study indicated that RAPD technique might be helpful to delineate the pathogenic mechanism(s) for its virulence; however, further studies on large number of isolates are desired to elucidate the findings.

Animals↗
PubMed2005

RecA-mediated multistrand formation for cloning PCR products into vectors: simplified process for 5'-rapid amplification of cDNA ends.

I have developed a novel rapid amplification of cDNA ends (RACE) technology that uses multistranded DNA formation mediated by the RecA protein. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary single-stranded DNA in the presence of RecA and exonuclease I. The possibility of applying this finding to the direct cloning of a 5'-RACE product onto a cDNA fragment, which does not require the use of restriction endonucleases, was explored. The results show that the terminal multistranded structure formed by the RecA-mediated reaction can be applied to RACE systems. Modifications to the RACE protocol to improve the effectiveness of the technique are also suggested.

Base Pair Mismatch↗
PubMed2000

The Prevotella intermedia group organisms in young children and their mothers as related to maternal periodontal status.

Currently, the Prevotella intermedia group includes three biochemically and phylogenetically related species: Prevotella intermedia, Prevotella nigrescens, and the newly described Prevotella pallens. The two first-named species are mentioned with varying emphasis in connection with periodontal diseases, while such a connection of P. pallens is not known. Mothers serve as a plausible source of bacteria to their children, and conceivably, a mother with periodontitis as a recurrent reservoir of periodontally infecting organisms. In the present study, 23 mothers and their young children were examined for the presence of the P. intermedia group organisms in relation to maternal periodontal status (I: periodontal health, II: initial periodontitis, and III: advanced periodontitis). Species differentiation was based on established biochemical methods, electrophoretic mobility patterns, SDS-PAGE, and DNA hybridization. P. intermedia was not recovered from children but nearly exclusively from mothers in group III, thus confirming its association with periodontitis. P. nigrescens and P. pallens were frequently found in mothers and children. To determine bacterial transmission between a mother and her child, 72 isolates from 13 mother-child pairs were analyzed by arbitrarily primed PCR (AP-PCR). Similar AP-PCR types of P. nigrescens and/or P. pallens were recovered from 3/4 pairs in group I, 2/5 pairs in group II, and none in group III. Our results indicate that different species within the P. intermedia group have a different colonization pattern in childhood and that the periodontal status reflects qualitatively their presence in maternal saliva. Intra-familial transmission of P. nigrescens and P. pallens can occur in early childhood, however similar AP-PCR types were most obvious within periodontally healthy mother-child pairs.

Adult↗
PubMed2003

Typing of Listeria monocytogenes isolates by random amplification of polymorphic DNA.

BACKGROUND & OBJECTIVES: Listeria monocytogenes is an important food-borne pathogen causing meningitis and septicaemia in newborns and immunocompromised persons, abortion and preterm labour in pregnant women. Though various methods are available for typing L. monocytogenes, RAPD analysis has been used for epidemiological purposes in developed countries due to its greater discriminating ability. However, as there are no published reports from India on the typing of L. monocytogenes by RAPD technique the present study was undertaken to type isolates of L. monocytogenes from clinical, food and veterinary samples. METHODS: Isolates of L. monocytogenes were subjected to RAPD using four decamer random primers R1, R2, R3 and R4. Amplified products were analysed by agarose gel electrophoresis. RESULTS: Eight strains of L. monocytogenes on RAPD analysis generated 4 distinct profiles each with R1 and R4 primers and 3 different profiles with R2 and R3 primers. The isolates from fish, clinical and veterinary samples showed different profiles with respect to each other. Isolate from flat fish (serovar 4) showed a different profile from that of clams (serovar 1). Two isolates from placenta (serovar 1) showed similar profiles and all the isolates from veterinary samples generated similar profiles. INTERPRETATION & CONCLUSION: RAPD analysis in the present study allowed discrimination of isolates among the same serotype but from different sources. Since RAPD is a rapid technique and offers greater discrimination of strains, this method may be used for typing L. monocytogenes in India.

Animals↗
PubMed2002

Persistence of Lactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 but not L. rhamnosus GG in the human vagina as demonstrated by randomly amplified polymorphic DNA.

Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 are well-characterized probiotic strains with efficacy in the prevention and treatment of urogenital infections in women. The aim of the present study was to apply a molecular biology-based methodology for the detection of these strains and L. rhamnosus GG (a commercially available intestinal probiotic) in the human vagina in order to assess probiotic persistence at this site. Ten healthy women inserted vaginally a capsule containing either a combination of strains GR-1 and RC-14 or the GG strain for 3 consecutive nights. Vaginal swabs taken before and at various time points after probiotic insertion were analyzed, and the Lactobacillus flora was assessed by randomly amplified polymorphic DNA (RAPD) analysis. This method generated discrete DNA fingerprints for GR-1, RC-14, and GG and enabled successful detection of these strains in the vagina. Strain GR-1 and/or strain RC-14 was found to persist in the vaginal tract for up to 19 days after vaginal instillation, while L. rhamnosus GG was detectable for up to 5 days postadministration. In conclusion, the fates of probiotic L. rhamnosus and L. fermentum strains were successfully monitored in the human vagina by RAPD analysis. This technique provides molecular biology-based evidence that RC-14 and GR-1, strains selected as urogenital probiotics, persist in the human vagina and may be more suited to vaginal colonization than L. rhamnosus GG. This highlights the importance of proper selection of strains for urogenital probiotic applications.

Adult↗