Repetitive Element Polymerase Chain Research Articles

Repetitive element-polymerase chain reaction for genotyping of clinical and environmental isolates of Legionella spp.

Genotyping of Legionella strains is important for the epidemiologic survey of Legionnaires' disease infections. In this study, we investigated the potential of repetitive element-polymerase chain reaction (rep-PCR) for differentiating various isolates of Legionella spp. We used 38 Legionella pneumophila isolates (collected in clinics all over Japan between 1980 and 2007), 19 environmental Legionella anisa isolates (collected in Okinawa, Nara, Osaka, and Hyogo prefecture between 1987 and 2007), and 2 Legionella-type strains. We extracted bacterial genomic DNA and applied it to rep-PCR. PCR products were then converted into bands by agarose gel electrophoresis. The L. pneumophila serogroup (SG) 1 displayed very diverse patterns. Different bands were produced for each species of Legionella, and each species was clearly distinct. Phylogenetic analysis displayed 1 cluster of L. anisa isolates, while other Legionella spp. were present at discrete levels. Our findings show that rep-PCR is an effective, rapid, and simple technique for differentiation of L. pneumophila strains as well as Legionella spp.

Repetitive element (REP)-polymerase chain reaction (PCR) analysis of Escherichia coli isolates from recreational waters of southeastern Lake Huron

Repetitive element-polymerase chain reaction (REP-PCR) DNA fingerprinting and library-based microbial source tracking (MST) methods were utilized to investigate the potential sources of Escherichia coli pollution in recreational waters of southeastern Lake Huron. In addition to traditional sources such as humans, agriculture, and wildlife, environmentally persistent E. coli isolates were included in the identification library as a separate library unit consisting of the E. coli strains isolated from interstitial water on the beach itself. Our results demonstrated that the dominant source of E. coli pollution of the lake was agriculture, followed by environmentally adapted E. coli strains, wildlife, and then humans. A similar ratio of contributing sources was observed in all samples collected from various locations including the river discharging to the beach in both 2005 and 2006. The high similarity between the compositions of E. coli communities collected simultaneously in the river and in the lake suggests that tributaries were the major overall sources of E. coli to the lake. Our findings also suggest that environmentally adapted strains (EAS) of E. coli should be included as one of the potential sources in future microbial source tracking efforts.

Genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada

Common scab is an important disease of potato caused by Streptomyces scabies and other closely related species. In this study, the genetic diversity of Streptomyces spp. causing common scab of potato in eastern Canada was for the first time investigated. Forty-one Streptomyces spp. isolates were retrieved from necrotic lesions of potato tubers harvested from different regions of the Canadian provinces New-Brunswick, Nova Scotia and Prince-Edward-Island. Most isolates were closely related to known pathogenic S. scabies strains on the basis of partial 16S ribosomal (r) RNA and rpoB gene sequence analyses. Two isolates were identified as pathogenic species of Streptomyces acidiscabies. To our knowledge, this species has never been previously isolated in these areas. Genome fingerprinting studies using repetitive elements (rep) polymerase chain reactions (PCR) revealed 10 distinct genetic groups in eastern Canada. The geographical distribution of the genetic groups was region-dependant. Pathogenicity- and virulence-related genes ( txtA, txtC, and tomA) were PCR-amplified from each isolate, and nucleotide sequence analysis of partial gene fragments revealed slight polymorphisms in both txtA and txtC genes. No genetic variation was noted in the partial tomA gene sequences.

Outbreak ofMycobacterium abscessusWound Infections among “Lipotourists” from the United States Who Underwent Abdominoplasty in the Dominican Republic

Some US residents travel abroad to undergo cosmetic surgery for fat removal, a practice referred to as "lipotourism." Mycobacterium abscessus can cause postsurgical wound infection. US residents who developed M. abscessus wound infection after undergoing cosmetic surgery in the Dominican Republic in 2003 and 2004 were identified using the Emerging Infections Network listserv. Twenty returning US travelers with M. abscessus infection were detected. Eight patients had matching isolates, as determined by pulsed-field gel electrophoresis and repetitive element polymerase chain reaction. All 8 patients, who had previously been healthy Hispanic women, underwent abdominoplasties at the same clinic in the Dominican Republic. Symptoms first developed 2-18 weeks after the procedure (median interval, 7 weeks). Only 2 of the 8 patients received a correct diagnosis at the initial presentation. Most patients presented with painful, erythematous, draining subcutaneous abdominal nodules. Seven patients underwent drainage procedures. Six patients received a combination of antibiotics that included a macrolide plus cefoxitin, imipenem, amikacin, and/or linezolid; 2 received clarithromycin monotherapy. All patients but 1 were cured after a median of 9 months of therapy (range, 2-12 months). Because of a lack of access to the surgical clinic, the cause of the outbreak of infection was not identified. The patients who were infected with nonmatching isolates underwent surgeries in different facilities but otherwise had demographic characteristics and clinical presentations similar to those of the 8 patients infected with matching isolates. This case series of M. abscessus infection in US "lipotourists" highlights the risks of traveling abroad for surgery and the potential role of the Internet in identifying and investigating outbreaks.

Identifying Fecal Sources in a Selected Catchment Reach Using Multiple Source‐Tracking Tools

Given known limitations of current microbial source-tracking (MST) tools, emphasis on small, simple study areas may enhance interpretations of fecal contamination sources in streams. In this study, three MST tools-Escherichia coli repetitive element polymerase chain reaction (rep-PCR), coliphage typing, and Bacteroidales 16S rDNA host-associated markers-were evaluated in a selected reach of Plum Creek in south-central Nebraska. Water-quality samples were collected from six sites. One reach was selected for MST evaluation based on observed patterns of E. coli contamination. Despite high E. coli concentrations, coliphages were detected only once among water samples, precluding their use as a MST tool in this setting. Rep-PCR classification of E. coli isolates from both water and sediment samples supported the hypothesis that cattle and wildlife were dominant sources of fecal contamination, with minor contributions by horses and humans. Conversely, neither ruminant nor human sources were detected by Bacteroidales markers in most water samples. In bed sediment, ruminant- and human-associated Bacteroidales markers were detected throughout the interval from 0 to 0.3 m, with detections independent of E. coli concentrations in the sediment. Although results by E. coli-based and Bacteroidales-based MST methods led to similar interpretations, detection of Bacteroidales markers in sediment more commonly than in water indicates that different tools to track fecal contamination (in this case, tools based on Bacteroidales DNA and E. coli isolates) may have varying relevance to the more specific goal of tracking the sources of E. coli in watersheds. This is the first report of simultaneous, toolbox approach application of a library-based and marker-based MST analyses to flowing surface water.

American foulbrood of the honey bee: Occurrence and distribution of different genotypes of Paenibacillus larvae in the administrative district of Arnsberg (North Rhine‐Westphalia)

Between March 2003 and October 2004, Paenibacillus larvae, the aetiological agent of American foulbrood disease of the honey bee, was isolated from broodcombs and honey samples of 54 apiaries in the administrative district of Arnsberg (North Rhine-Westphalia, Germany). Genotyping of 176 P. larvae isolates with repetitive element polymerase chain reaction fingerprinting (rep-PCR) using BOX A1R and MBO REP1 primers revealed five different genotypes (AB, Ab, ab, ass, Acapital BE, Cyrillic). In samples of three apiaries, more than one genotype was detected. A combination of two genotypes was isolated from honey samples of the same hive two times (ab/ass and Ab/ab). The five genotypes were not randomly distributed in the district, but revealed a certain geographical clustering. Possible factors with impact on the genotype diversity and the distribution pattern are discussed.

Mycobacterium avium restriction fragment lenght polymorphism-IS IS1245 and the simple double repetitive element polymerase chain reaction typing method to screen genetic diversity in Brazilian strains

Simple double repetitive element polymerase chain reaction (MaDRE-PCR) and Pvu II-IS1245 restriction fragment length polymorphism (RFLP) typing methods were used to type 41 Mycobacterium avium isolates obtained from 14 AIDS inpatients and 10 environment and animals specimens identified among 53 mycobacteria isolated from 237 food, chicken, and pig. All environmental and animals strains showed orphan patterns by both methods. By MaDRE-PCR four patients, with multiple isolates, showed different patterns, suggesting polyclonal infection that was confirmed by RFLP in two of them. This first evaluation of MaDRE-PCR on Brazilian M. avium strains demonstrated that the method seems to be useful as simple and less expensive typing method for screening genetic diversity in M. avium strains on selected epidemiological studies, although with limitation on analysis identical patterns except for one band.

Emergence of a Predominant Clone of Community-Acquired Staphylococcus aureus Among Children in Houston, Texas

Community-acquired (CA), methicillin-resistant Staphylococcus aureus (MRSA) infections among children are increasing in the United States. At Texas Children's Hospital (TCH), surveillance has been in place since August 2001. The objectives of this study were to describe the distribution of CA S. aureus among patients at TCH and to study genomic relationships of isolates collected between August 2001 and July 2003. Genomic relationships were determined with repetitive element-polymerase chain reaction (PCR). Multilocus sequence typing was performed for selected strains representing major clones. Molecular characterization of CA-MRSA was performed with PCR, including staphylococcal cassette chromosome (SCCmec), pvl (lukS-PV plus lukF-PV), hla, hlb and selected microbial surface components recognizing adhesive matrix molecule genes, ie, cna, clfA, fnbA and fnbB. A 62% increase was observed in CA S. aureus infections from year 1 (2001-2002) to year 2 (2002-2003), whereas the annual number of hospital admissions was unchanged. CA methicillin-sensitive S. aureus isolates were more likely to be associated with invasive infections than were CA-MRSA isolates (P < 0.01). TCH clone A, sequence type (ST) 8, was responsible for approximately 94% of all CA-MRSA isolated from children in the greater Houston area. Clone A differed from clones B (ST30) and C (ST1) by lacking the cna gene while carrying the fnbB gene. One CA-MRSA clone, TCH clone A, has become the predominant cause of CA S. aureus infections among children in the Houston area. It causes a wide spectrum of diseases, including complicated pneumonia.

A Novel Enterohepatic Helicobacter Species ‘Helicobacter mastomyrinus’ Isolated from the Liver and Intestine of Rodents

A number of novel Helicobacter species have been isolated from both animals and humans. Many of these helicobacters colonize the lower gastrointestinal tract and hepatobiliary tract and are associated with diseases. A spiral-shaped bacterium, with bipolar single-sheathed flagella, was isolated from the liver and cecum of mastomys (the African rodent, Mastomys natalenis), from the feces and ceca of normal mice, and also from the cecum of a mouse with proctitis. 16S ribosomal RNA gene sequence analysis, restriction fragment length polymorphism (RFLP) and fluorophore-enhanced repetitive element polymerase chain reaction (FERP or rep-PCR) analysis were used to classify the organism. The bacterium grew at 37 and 42 degrees C under microaerobic conditions, rapidly hydrolyzed urea, and was catalase and oxidase positive. It did not reduce nitrate to nitrite, and was resistant to cephalothin and nalidixic acid. Like many other enterohepatic Helicobacter species, this organism expressed cytolethal distending toxin and causes cell distention. The organism was classified as a novel Helicobacter species for which we propose the name 'Helicobacter mastomyrinus'. Although 'H. mastomyrinus', like Helicobacter hepaticus and Helicobacter bilis, colonizes the liver of rodents, the pathogenic potential of this novel helicobacter is unknown.

Genetic Diversity and Pathogenic Variation of Common Blight Bacteria (Xanthomonas campestrispv.phaseoliandX. campestrispv.phaseolivar.fuscans) Suggests Pathogen Coevolution with the Common Bean

ABSTRACT Common bacterial blight (CBB), caused by Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, is one of the most important diseases of common bean (Phaseolus vulgaris) in East Africa and other bean-growing regions. Xanthomonad-like bacteria associated with CBB in Malawi and Tanzania, East Africa, and in Wisconsin, U.S., were characterized based on brown pigment production, pathogenicity on common bean, detection with an X. campestris pv. phaseoli- or X. campestris pv. phaseoli var. fuscans-specific PCR primer pair, and repetitive element polymerase chain reaction (rep-PCR) and restriction fragment length polymorphism (RFLP) analyses. The common bean gene pool (Andean or Middle American) from which each strain was isolated also was determined. In Malawi, X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were isolated predominantly from Andean or Middle American beans, respectively. In Tanzania, X. campestris pv. phaseoli var. fuscans was most commonly isolated, irrespective of gene pool; whereas, in Wisconsin, only X. campestris pv. phaseoli was isolated from Andean red kidney beans. Three rep-PCR fingerprints were obtained for X. campestris pv. phaseoli strains; two were unique to East African strains, whereas the other was associated with strains collected from all other (mostly New World) locations. RFLP analyses with repetitive DNA probes revealed the same genetic diversity among X. campestris pv. phaseoli strains as did rep-PCR. These probes hybridized with only one or two fragments in the East African strains, but with multiple fragments in the other X. campestris pv. phaseoli strains. East African X. campestris pv. phaseoli strains were highly pathogenic on Andean beans, but were significantly less pathogenic on Middle American beans. In contrast, X. campestris pv. phaseoli strains from New World locations were highly pathogenic on beans of both gene pools. Together, these results indicate the existence of genetically and geographically distinct X. campestris pv. phaseoli genotypes. The rep-PCR fingerprints of X. campestris pv. phaseoli var. fuscans strains from East African and New World locations were indistinguishable, and were readily distinguished from those of X. campestris pv. phaseoli strains. Genetic diversity among X. campestris pv. phaseoli var. fuscans strains was revealed by RFLP analyses. East African and New World X. campestris pv. phaseoli var. fuscans strains were highly pathogenic on Andean and Middle American beans. Breeding for CBB resistance in East African beans should utilize X. campestris pv. phaseoli var. fuscans and New World X. campestris pv. phaseoli strains in order to identify germ plasm with the highest levels of resistance.

Characterization of Erwinia Populations from Nursery Retention Ponds and Lakes Infecting Ornamental Plants in Florida.

Water shortages in Florida are occurring due to intense utilization of the aquifer system by municipalities and agriculture, and because of continued deficits in annual rainfall. Water districts therefore, are, recommending the use of recycled irrigation water, stormwater runoff, reclaimed municipal sewage water, and lakes for agricultural use. With recycled water, however, there is potential for both introducing and concentrating plant pathogens. In Florida, Erwinia soft-rot bacteria (synonym Pectobacterium) cause extensive crop losses in ornamental plant production. To determine Erwinia spp. population levels, samples were taken monthly for 1 year from four hypereutropic lakes and eight nursery retention ponds. Seventy-seven Erwinia strains were collected by both direct plating and by an enrichment process. With the direct plating method, 0 to 29 CFU/ml were detected on sodium polypectate medium. Significantly higher populations of Erwinia were detected in retention ponds of nurseries that were actively reutilizing their water. Erwinia strains were identified to species by fatty acid analysis and biochemical tests. Strains were further characterized by repetitive element-polymerase chain reaction (rep-PCR) and compared with 120 strains of Erwinia collected from ornamentals over a 17-year period in Florida. Using rep-PCR, most strains were clustered into two heterogeneous populations of E. chrysanthemi and E. carotovora subsp. carotovora in a 1:2 and a 1:4 ratio for isolates from ornamentals and from water, respectively. Within each population of E. chrysanthemi and E. carotovora, genetically different subpopulations could be identified that contained high percentages of Erwinia strains from water sources. Even though genetic differences exist, 99% of the strains from water sources were found to be pathogenic on dieffenbachia. Without water treatment of irrigation and stormwater runoff, there is a potential for both introducing and concentrating Erwinia populations within these water sources.

Contributions of Genome Sequencing to Understanding the Biology ofHelicobacter pylori

About half of the world's population carries Helicobacter pylori, a gram-negative, spiral bacterium that colonizes the human stomach. The link between H. pylori and, ulceration as well as its association with the development of both gastric cancer and mucosa-associated lymphoid tissue lymphoma in humans is a serious public health concern. The publication of the genome sequences of two stains of H. pylori gives rise to direct evidence on the genetic diversity reported previously with respect to gene organization and nucleotide variability from strain to strain. The genome size of H. pylori strain 26695 is 1,6697,867 bp and is 1,643,831 bp for strain J99. Approximately 89% of the predicted open reading frames are common to both of the strains, confirming H. pylori as a single species. A region containing approximately 45% of H. pylori strain-specific open reading frames, termed the plasticity zone, is present on the chromosomes, verifying that some strain variability exists. Frequent alteration of nucleotides in the third position of the triplet codons and various copies of insertion elements on the individual chromosomes appear to contribute to distinct polymorphic fingerprints among strains analyzed by restriction fragment length polymorphisms, random amplified polymorphic DNA method, and repetitive element-polymerase chain reaction. Disordered chromosomal locations of some genes seen by pulsed-field gel electrophoresis are likely caused by rearrangement or inversion of certain segments in the genomes. Cloning and functional characterization of the genes involved in acidic survival, vacuolating toxin, cag-pathogenicity island, motility, attachment to epithelial cells, natural transformation, and the biosynthesis of lipopolysaccharides have considerably increased our understanding of the molecular genetic basis for the pathogenesis of H. pylori. The homopolymeric nucleotide tracts and dinucleotide repeats, which potentially regulate the on- and off-status of the target genes by the strand-slipped mispairing mechanism, are often found in the genes encoding the outer-membrane proteins, in enzymes for lipopolysaccharide synthesis, and within DNA modification/restriction systems. Therefore, these genes may be involved in the H. pylori-host interaction.

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori by REP-PCR and restriction fragment end-labelling

Genetic diversity of 32 Helicobacter pylori strains isolated from patients with gastritis, gastric or duodenal ulcer, carcinoma, or lymphoma was determined by repetitive sequence element polymerase chain reaction (REP-PCR), and by the new typing method restriction fragment end-labelling (RFEL). Furthermore, these two methods were used to investigate a possible correlation between clinical symptoms and the genetic background of Helicobacter pylori. Both REP-PCR and RFEL revealed 31 different patterns for the 32 strains tested, but the pair of isolates with identical REP-PCR patterns was not the same as the pair of isolates with identical RFEL patterns. Computer-assisted analysis of the DNA fingerprints was used to determine similarity coefficients. This analysis revealed no clustering of disease-specific strains by any of the two methods.

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori by REP-PCR and restriction fragment end-labelling.

Genetic diversity of 32 Helicobacter pylori strains isolated from patients with gastritis, gastric or duodenal ulcer, carcinoma, or lymphoma was determined by repetitive sequence element polymerase chain reaction (REP-PCR), and by the new typing method restriction fragment end-labelling (RFEL). Furthermore, these two methods were used to investigate a possible correlation between clinical symptoms and the genetic background of Helicobacter pylori. Both REP-PCR and RFEL revealed 31 different patterns for the 32 strains tested, but the pair of isolates with identical REP-PCR patterns was not the same as the pair of isolates with identical RFEL patterns. Computer-assisted analysis of the DNA fingerprints was used to determine similarity coefficients. This analysis revealed no clustering of disease-specific strains by any of the two methods.

Identification of Xanthomonas fragariae, the cause of an outbreak of angular leaf spot on strawberry in South Australia, and comparison with the cause of previous outbreaks in New South Wales and New Zealand

In February 1994, symptoms consistent with angular leaf spot were found on strawberry plants on three properties in the Kenton Valley region of South Australia. To confirm the identity of the causal organism, bacteria were isolated from affected tissue and processed for analysis using whole genome DNA fmgerprinting and repetitive element polymerase chain reaction (PCR). Whole genome and PCR fingerprints from the isolated bacteria were compared to those generated from known isolates of Xanthomonas fragariae originally collected from New South Wales and New Zealand in 1975 and 1971–2, respectively The 1994 South Australian isolates exhibited a high degree of similarity to the New South Wales and New Zealand isolates, confirming their identity as X. fragariae. The New South Wales and New Zealand isolates had identical genomic and PCR fingerprints, suggesting that they were derived from the same source, in all probability a multiplication scheme from which both areas obtained runners in the early 1970s. The differences between the 1994 outbreak and those in New South Wales and New Zealand show that a separate introduction of X. fragariae into South Australia has occurred in recent times. These three outbreaks highlight the problems of relying on passive detection of this bacterium, which requires specific environmental conditions for symptom expression.

Fingerprinting of Ehrlichia species by repetitive element polymerase chain reaction.

To facilitate identification of ehrlichial pathogens, we developed a new technique based on fingerprints resulting from repetitive element polymerase chain reaction (rep-PCR). This technique uses consensus tRNA primers to generate amplification products that reflect distance polymorphisms between adjacent tRNA genes. Species-specific fingerprint patterns were obtained for seven Ehrlichia spp., as well as the unnamed causative agent of human granulocytotropic ehrlichiosis. Bands ranged in size from approximately 50 to 1,000 base pairs. Banding patterns varied depending on dilution of template DNA, with lower dilutions giving more complex banding patterns. These preliminary data indicate that repetitive-sequence-based PCR appears to be a useful technique for identifying ehrlichial organisms to the species, and perhaps the strain level. Compared with other conventional molecular-biologic methods, rep-PCR offers the advantages of ease of performance and rapid availability of results.

Use of fatty acid profiles and repetitive element polymerase chain reaction (PCR) to assess the genetic diversity of Pseudomonas syringae pv. pisi and Pseudomonas syringae pv. syringae isolated from field peas in Australia

Isolates of Pseudomonas syringae pv. pisi and P. syringae pv. syringae obtained from both plants and seed of field peas (Pisum sativum L.) in south-eastem Australia were compared using both fatty acid analysis and genomic DNA fingerprinting by repetitive element polymerase chain reaction (PCR). Fatty acid profiling was not specific enough to distinguish isolates of P. syringae pv. syringae from isolates of P. syringae pv. pisi and could not distinguish Races 2 and 6 of P. syringae pv. pisi from each other. DNA fingerprinting, however, was able to distinguish the Australian isolates of P. syringae pv. syringae from the isolates of P. syringae pv. pisi and group the isolates of P. syringae pv. pisi on the basis of race. DNA fingerprinting successfully identified the pathovar and race of unidentified and misidentified Australian isolates of P. syringae indicating that this method could be used as a rapid laboratory-based test for race determination of isolates of P. syringae pv. pisi. The genetic diversity within the isolates of P. syringae pv. pisi present within Australia is also discussed.

Polymerase chain reaction typing of nonviable Mycobacterium tuberculosis isolates

Repetitive element polymerase chain reaction (PCR) typing was applied to two Mycobacterium tuberculosis isolates for which both viable and nonviable cultures were available. DNA extracted from the nonviable cultures and from fresh subcultures of the viable cultures was amplified with primers directed against the insertion sequence IS6110 and the polymorphic GC-rich repetitive sequence. For both isolates, the nonviable cultures generated PCR-banding patterns identical to those generated by the fresh subcultures.

Characterization of a Hybrid Hamster-Human Cell Line Complemented for the Ataxia Telangiectasia DNA Repair Defects

The fibroblast cell line AT5BIVA, from ataxia telangiectasia complementation group D, was transfected with a neo gene providing G418 resistance for subsequent selections. The G418 resistant cell line was fused with gamma-irradiated Chinese hamster ovary cells and a radioresistant hybrid, atxbc, was isolated following an X-ray selection procedure. All the cellular defects characteristic of ataxia telangiectasia were corrected to some degree; atxbc cells: (i) were confirmed to be resistant to X-rays; (ii) had regained control over DNA synthesis after DNA damage, and (iii) could overcome the radiation-induced block in the G2 phase of the cell cycle. Repetitive element polymerase chain reaction amplification of integrated hamster DNA from primers to middle repetitive elements confirmed the presence of hamster-specific sequences, suggesting that the phenotype of the cells had been corrected by integration of a normal hamster gene rather than by a reversion event.

Interspersed repetitive element polymerase chain reaction product mapping using a mouse interspecific backcross

We have developed a rapid method of generating and simultaneously mapping interrepeat polymerase chain reaction products using DNA from interspecific backcross animals derived from mating C57 BL 6 J and Mus spretus mice. This method is based on the high degree of B1, B2, and L1 dispersed repeat position polymorphism found between these two species of mouse. We have mapped 13 new loci to 9 different chromosomes and have found no evidence of clustering among these loci. The advantages of this approach are that no prior knowledge of sequence is required, a single PCR reaction generates many markers which can be mapped simultaneously, and only 50 ng of each backcross DNA (a finite resource) is required. We anticipate that many more markers remain to be characterized in this valuable new source of polymorphism.