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PubMed1996

DNA fingerprinting by infrequent-restriction-site amplification.

Identification of bacterial strains by DNA fingerprinting facilitates epidemiologic studies and improves disease control. For some species of organisms, no typing method is available; for others, typing methods are tedious. We developed a method of amplifying DNA sequences flanking infrequent restriction sites by PCR and used the method to produce strain-specific electrophoretic patterns from crude bacterial lysates. This method of fingerprinting is rapid, sensitive, and widely applicable. Identical enzymes, adaptors, primers, and PCR conditions were used to characterize 32 Mycobacterium avium-M. intracellulare isolates, 4 Pseudomonas aeruginosa isolates, and 4 Staphylococcus aureus isolates.

DNA Fingerprinting↗
PubMed1996

Development of two species-specific fingerprinting probes for broad computer-assisted epidemiological studies of Candida tropicalis.

Candida tropicalis has emerged as the second most frequent colonizing Candida species, and it has been documented in nosocomial infections. To develop an effective fingerprinting system for this species, which is amenable to computer-assisted analyses and which provides information on the genetic relatedness of independent isolates, two DNA fragments, Ct3 (18,000 bp) and Ct14 (20,000 bp), were cloned from a genomic library of Sau3AI partial digestion products. Both probes generate complex Southern blot patterns containing 8 to 20 bands, when hybridized to EcoRI- or EcoRI-HaeIII-digested DNA of independent C. tropicalis isolates. The two probes show no cross-hybridization and are both species specific for C. tropicalis. A comparison of the capacity of the two probes to identify the same strain in different isolates, and differentiate unrelated strains, using computer-assisted computation of similarity coefficients and the genesis of dendrograms, suggests that while Ct14 is more effective in grouping independent isolates, Ct3 is more effective in discriminating small differences in the patterns of highly related isolates and is therefore a more effective probe for determining microevolution within a clonal population and substrain shuffling in recurrent infections. Because of their alternative attributes, it is suggested that both probes be used in fingerprinting studies of C. tropicalis.

Candida↗
PubMed1996

Rapid identification of Candida species by DNA fingerprinting with PCR.

DNA polymorphisms in different species and strains of the genus Candida were assessed by amplifying genomic DNA with single nonspecific primers. This PCR method employed an arbitrary primer (the 10-mer AP3), a primer derived from the intergenic spacer regions (T3B), and the microsatellite primers (GTG)5 and (AC)10. Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species. The numbers and sizes of the amplification products were characteristic for each species. All yeast species tested could be clearly distinguished by their amplification patterns. With all primers, PCR fingerprints also displayed intraspecies variability. However, PCR profiles obtained from different strains of the same species were far more similar than those derived from different Candida species. By comparing species-specific PCR fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the species level even if they could not be identified by routine biochemical methods.

Base Sequence↗
PubMed1996

DNA fingerprinting and serotyping of Campylobacter jejuni isolates from epidemic outbreaks.

The aim of the present investigation was to compare DNA fingerprinting and serotyping (heat-stabile and heat-labile antigens) of isolates from epidemic outbreaks as well as of solitary isolates. Campylobacter jejuni isolates from two epidemic outbreaks in Sweden, one milkborne (35 isolates) and one waterborne (17 isolates), and one waterborne outbreak in Norway (11 isolates), as well as 30 solitary isolates from Swedish patients with gastroenteritis, were analyzed. A total of 93 isolates were analyzed. In the waterborne outbreak in Norway, only one serotype with one DNA pattern was found. In the milkborne outbreak in Sweden, two serotypes (HS2:HL4 and HSNT:HL4) with two different DNA patterns were found. The isolates from the waterborne outbreak in Sweden were different serotypes. For two isolates of the same serotype, different DNA patterns were seen. This was also recorded for isolates from solitary cases. It was concluded that serotyping is a useful tool in most epidemiological situations but sometimes lacks sufficient discriminatory power. DNA fingerprinting can add valuable epidemiological information to that supplied by serotyping and can in some situations provide sufficient epidemiological information when used alone.

Animals↗
PubMed1996

Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting.

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.

Australia↗
PubMed1996

DNA fingerprinting of Candida rugosa via repetitive sequence-based PCR.

A repetitive sequence-based PCR (rep-PCR) technique was developed to characterize the genotypic relatedness among Candida rugosa isolates. Two repetitive sequences, viz., Care-2 and Com29 from Candida albicans, were used to design primers Ca-21, Ca-22, and Com-21, respectively. When used alone or in combination, these primers generated discriminatory fingerprints by amplifying the adjacent variable regions of the genome. Twenty-three isolates from burn patients, eight from other human sources, and four C. rugosa isolates pathogenic in animals were placed into nine fingerprinting groups. Different primers placed these isolates into identical groups, indicating that rep-PCR is a specific and reproducible technique for molecular characterization of C. rugosa. Moreover, these primers unequivocally discriminated among other important Candida species such as C. albicans, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. kefyr, and C. lusitaniae. These data confirm the conservation of repetitive sequences in Candida species. Because of its ease and sensitivity, rep-PCR offers a relatively rapid and discriminatory method for molecular typing of C. rugosa in outbreaks.

Animals↗
PubMed1996

Heminested inverse PCR for IS6110 fingerprinting of Mycobacterium tuberculosis strains.

A heminested inverse PCR (HIP) for the amplification of sequences flanking the Mycobacterium tuberculosis insertion sequence IS6110 has been developed. The method depends upon primers that anneal to IS6110 at sites between its 5' end and the closest BsrFI site. The accuracy of HIP was demonstrated by the amplification of sequences within plasmid constructs carrying one or two copies of the insertion sequence IS986 in different orientations. The identities of the amplicons produced from strains carrying a single copy of IS6110 were verified by nucleotide sequencing. Analyses of 204 M. tuberculosis strains including those involved in outbreaks showed that IS6110 HIP is highly discriminatory and reproducible. HIP fingerprinting of these 204 strains generated 136 distinct types, and its discriminatory power was equivalent to that of standard restriction fragment length polymorphism analysis. The method is therefore of value for the rapid fingerprinting of M. tuberculosis strains for epidemiological purposes.

Base Sequence↗
PubMed1996

Automated DNA fingerprinting analysis of Mycobacterium tuberculosis using fluorescent detection of PCR products.

DNA fingerprints of Mycobacterium tuberculosis are produced by restriction fragment length polymorphism analysis of the insertion element IS6110. We modified a PCR-based subtyping method, mixed-linker PCR with fluorescent-labeled IS6110-specific oligonucleotides, to demonstrate rapid, automated, and unattended electrophoretic analysis. Variation in band sizing (normally occurring with fragment mobility), an artifact of lane-to-lane and gel-to-gel differences, was controlled with an internal lane standard, resulting in accurate and precise DNA sizing. By using this method, fingerprint analysis can be performed using actual fragment length rather than estimated position analysis.

Base Sequence↗
PubMed1997

Molecular fingerprinting of Mycobacterium tuberculosis on a Caribbean island with IS6110 and DRr probes.

Because of a substantial increase in human immunodeficiency virus (HIV) infection and HIV-linked tuberculosis in the Caribbean, a molecular fingerprinting study of clinical isolates of Mycobacterium tuberculosis isolated at the Pasteur Institute of Guadeloupe from 1994 to 1995 was undertaken with the insertion sequence IS6110 and the direct repeat DRr probes. We present the results for 72 isolates from 51 patients. A major cluster (cluster A) representing isolates from 12 patients (24%) was detected upon PvuII-IS6110 fingerprinting, which revealed a pattern of four bands among these isolates. Homogeneity was retained when the isolates were further analyzed with the DRr probe or further characterized by AluI and SmaI-DRr restriction fragment length polymorphism analysis. The isolates of cluster A, from 10 men and 2 women, was present in people of all ages and of different ethnic and geographical backgrounds, and infection with these isolates was independent of the HIV status of the patients (except for 2 HIV-positive patients from the same ward from whom the tubercle bacilli were isolated at the same time). The percentage of reactivation versus active transmission events could not be precisely determined in this study. These results are discussed on the basis of the genetic advantage of predominant clusters and/or specific characteristics of the settings from which a similar cluster of isolates with four bands has so far been reported, which include South Africa, French Polynesia, and Guadeloupe.

DNA Fingerprinting↗
PubMed1997

Identification and DNA fingerprinting of Legionella strains by randomly amplified polymorphic DNA analysis.

The randomly amplified polymorphic DNA (RAPD) technique was used in the development of a fingerprinting (typing) and identification protocol for Legionella strains. Twenty decamer random oligonucleotide primers were screened for their discriminatory abilities. Two candidate primers were selected. By using a combination of these primers, RAPD analysis allowed for the differentiation between all different species, between the serogroups, and further differentiation between subtypes of the same serogroup. The usefulness of RAPD analysis was also evaluated with outbreak-related clinical and environmental isolates previously typed by the restriction fragment length polymorphism technique. RAPD analysis proved to be as accurate as other genotypic methods, reproducible, and highly discriminatory and is a valuable new alternative to traditional fingerprinting and identification of Legionella species and strains.

Animals↗
PubMed1998

DNA fingerprinting of Mycobacterium tuberculosis complex culture isolates collected in Brazil and spotted onto filter paper.

The usefulness of filter paper for preservation of bacterial cells was shown by mixed-linker DNA fingerprint analysis of Mycobacterium tuberculosis isolates from 77 Brazilian patients. DNA fingerprints of samples spotted onto filter paper and conventional culture material were identical. Thus, filter paper specimens analyzed by an amplification-based typing method provide a new resource for epidemiological studies of infectious diseases.

Brazil↗
PubMed1998

Comparison of amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and amplified fragment length polymorphism fingerprinting for identification of Acinetobacter genomic species and typing of Acinetobacter baumannii.

Thirty-one strains of Acinetobacter species, including type strains of the 18 genomic species and 13 clinical isolates, were compared by amplified ribosomal DNA restriction analysis (ARDRA), random amplified polymorphic DNA analysis (RAPD), and amplified fragment length polymorphism (AFLP) fingerprinting. ARDRA, performed with five different enzymes, showed low discriminatory power for differentiating Acinetobacter at the species and strain level. The standardized commercially available RAPD kit clearly enabled the discrimination of all Acinetobacter genomic species but showed great polymorphism between isolates of Acinetobacter baumannii. AFLP fingerprinting with radioactively as well as fluorescently labelled primers showed high discriminatory power for the identification of 18 Acinetobacter genomic species and typing of 13 clinical Acinetobacter isolates. Compared to radioactive AFLP, fluorescent AFLP was technically fast and simple to perform, and it permitted analysis with an automated DNA sequencer. Fluorescent AFLP seems particularly well suited for studying the epidemiology of nosocomial infections and outbreaks caused by Acinetobacter species.

Acinetobacter↗
PubMed1999

Amplified-fragment length polymorphism fingerprinting of Mycoplasma species.

Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including Mycoplasma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneumoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5). AFLP templates were prepared by the digestion of mycoplasmal DNA with BglII and MfeI restriction endonucleases and subsequent ligation of corresponding site-specific adapters. The amplification of AFLP templates with a single set of nonselective primers resulted in reproducible fingerprints of approximately 60 to 80 fragments in the size range of 50 to 500 bp. The method was able to discriminate the analyzed strains at species and intraspecies levels as well. Each of the tested Mycoplasma species developed a banding pattern entirely different from those obtained from other species under analysis. Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma species analyzed. The extent of polymorphism varied markedly between the analyzed mycoplasmas, comprising pattern similarity levels from 61.7% detected among M. dispar strains to 95.9% detected among M. genitalium strains. The results of the present study provide evidence of the high discriminatory power of AFLP analysis, suggesting the possible applicability of this method to the molecular characterization of mycoplasmas.

Animals↗
PubMed2001

Bacterial genetic fingerprint: a reliable factor in the study of the epidemiology of human campylobacter enteritis?

The rate of human intestinal infections with more than a single Campylobacter strain was determined and the genetic variabilities of Campylobacter strains throughout an infection episode were investigated by means of pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). For 48 and 49 of 50 patients, all isolates from one sample showed identical patterns by PFGE and ERIC-PCR, respectively. Throughout an infection episode in 47 of 52 patients, the PFGE fingerprints of the isolates remained stable, while in 1 patient two different species were observed and in 4 patients different patterns were observed. Therefore, ERIC-PCR proved less discriminative than PFGE. These findings suggest that human infection with more than one Campylobacter strain is rare and should not significantly impair epidemiologic analyses. However, changes in the genetic fingerprint throughout an infection should be considered in the assessment of epidemiologic studies of Campylobacter spp.

Bacterial Typing Techniques↗
PubMed2003

Tandem tetramer-based microsatellite fingerprinting for typing of Proteus mirabilis strains.

Two microsatellite tandem repeated tetramers, (GACA)(4) and (CAAT)(4), were used for Proteus mirabilis strain differentiation. The microsatellite-based PCR tests were applied for the examination of interstrain diversity for 87 P. mirabilis strains. Forty-six of the investigated strains were clinical isolates (5 were hospital isolates and 39 were outpatient clinic isolates); 42 strains were derived from the Kauffmann-Perch collection of laboratory strains. Fingerprinting done with the tetramers had a high discrimination ability [0.992 and 0.940 for (GACA)(4) and (CAAT)(4), respectively]. The distributions of clinical isolates among well-defined laboratory strains, determined by numerical analysis (unweighted pair-group method with arithmetic averages; Dice similarity coefficient), proved their genetic similarity to reference strains in the Kauffmann-Perch collection. This analysis also indicated that it is possible to estimate some phenotypic properties of P. mirabilis clinical isolates solely on the basis of microsatellite fingerprinting.

Bacterial Typing Techniques↗
PubMed2004

Multilocus sequence typing is a reliable alternative method to DNA fingerprinting for discriminating among strains of Candida albicans.

Multilocus sequence typing (MLST) has emerged as a powerful new DNA-typing tool for the evaluation of intraspecies genetic relatedness. This method relies on DNA sequence analysis of nucleotide polymorphisms in housekeeping genes and has shown a high degree of intraspecies discriminatory power for bacterial and fungal pathogens. However, the results of the MLST scheme for Candida albicans have heretofore never been formally compared to those of other established typing techniques. To assess the value of MLST relative to those of other DNA fingerprinting tools for discriminating among strains of C. albicans, we applied it to a previously well-characterized set of 29 C. albicans isolates evaluated by the random amplified polymorphic DNA (RAPD), multilocus enzyme electrophoresis (MLEE), and Ca3 Southern hybridization probe techniques. MLST identified three clusters of genetically related isolates, with 82.3% direct concordance with MLEE, 82.7% with RAPD analysis, and 86.2% with the Ca3 Southern hybridization technique. When MLST was applied to a subset of 22 isolates of unrelated origins, it identified 21 independent diploid sequence types (DSTs), resulting in a discriminatory power of 99.6%. These DSTs were 96.9, 99.6, and 99.6% concordant with the genotypes identified by RAPD analysis, MLEE, and Ca3 Southern hybridization, respectively. These results demonstrate that MLST is a highly effective technique that performs at least comparably to other established DNA fingerprinting techniques.

Candida albicans↗
PubMed2005

Molecular fingerprinting of Leishmania infantum strains following an outbreak of visceral leishmaniasis in central Israel.

Human and canine visceral leishmaniasis caused by Leishmania infantum emerged in central Israel after an absence of over 30 years. The origin of this outbreak was investigated by examining genetic polymorphisms in 37 strains isolated from dogs and patients with visceral leishmaniasis in the continuously active northern Israeli and West Bank foci and in a new Israeli focus using DNA fingerprinting with the human multilocus minisatellite probe 33.15. Analysis of the patterns obtained by DNA fingerprinting separated the strains geographically into northern (clade B) and central (clades A and C) genotypic groups. These results suggest that the emergence of visceral leishmaniasis in central Israel is due not to parasite spread from northern Israel to the new focus but rather to increased parasite transmission in central Israel and the West Bank coupled with changes in the ecoepidemiology of this region.

Animals↗
PubMed2005

Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii.

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.

Acinetobacter Infections↗