Large Restriction Fragment Patterns Research Articles

Characterization of Borrelia burgdorferi sensu lato isolates by pulsed-field gel electrophoresis after MluI restriction of genomic DNA

At least three Borrelia species ( Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto) cause disease in humans, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported to be rare or potential causes of human disease in Europe. Pulsed-field gel electrophoresis after MluI restriction of the genomic DNA (MluI large restriction fragment patterns, LRFPs) represents one of several approaches that have been used to assess Borrelia genotypic characteristics. The aim of the present report was to analyze the value of MluI-LRFP for identification of B. burgdorferi sensu lato at a species level and for further species subtype delineation. Results of the present study are based on 1487 B. afzelii strains, 285 B. garinii strains, 29 B. burgdorferi sensu stricto strains, 23 B. valaisiana strains, 8 B. spielmanii strains and 3 B. lusitaniae strains. Using MluI-LRFP, we were able to delineate all Borrelia species included in the study. Each of the six examined Borrelia species displayed unique MluI-LRFPs that enabled straightforward separation of strains into particular species, and also of strains within species. The subtypes of B. afzelii (Mla2 and Mla3), B. spielmanii (Mls1 and Mls2) and B. lusitaniae (Mll1 and Mll2) uncovered in the present analysis have not been reported previously. MluI-LRFP represents a highly specific and reproducible method for Borrelia identification.

First characterization of Borrelia burgdorferi sensu lato from ticks and skin biopsy in Bulgaria.

Lyme borreliosis is the most frequent tick-borne disease in the Northern hemisphere. Here we describe the first isolation of Borrelia burgdorferi sensu lato in Bulgaria: the midguts of 47 Ixodes ricinus obtained by flagging from the Central park in Sofia, Bulgaria were cultivated for borreliae in BSK medium. The eight isolates obtained from the ticks and one skin isolate from a Bulgarian patient with erythema migrans were subjected to phenotypic [outer surface protein A (OspA) serotyping] and genotypic analysis (pulsed-field gel electrophoresis typing followed by large restriction fragment pattern analysis after MluI digestion, polymerase chain reaction with 16S rRNA-directed oligonucleotide probes, and restriction fragment length polymorphism analysis of rrf-rrl intergenic spacer amplicons). The skin isolate was B. burgdorferi sensu stricto, as were four of the tick isolates; the other four tick isolates were B. garinii representing three different OspA serotypes (types 3, 5 and 7). These findings confirm the wide geographic distribution of the different B. garinii-associated OspA serotypes in Europe (shown here for the first time for the Southeastern part of Europe) and of B. burgdorferi sensu stricto in the Western hemisphere. These findings have implications for development of diagnostic tests and a borrelia vaccine in Southeastern Europe.

Identification of Three Species of Borrelia burgdorferi Sensu Lato (B. burgdorferi Sensu Stricto, B. garinii, and B. afzelii) Among Isolates from Acrodermatitis Chronica Atrophicans Lesions

In Europe, at least three species of Borrelia are known to be causative agents of Lyme borreliosis: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. Observable differences in the molecular characteristics of the three species have led to speculation that they may also differ in their pathogenic potential and/or tissue tropisms. Several studies have found an association between the chronic skin manifestation of Lyme borreliosis, acrodermatitis chronica atrophicans, and infection by B. afzelii. We sought to find further evidence for such a correlation by studying the genetic profiles of 22 strains of B. burgdorferi sensu lato derived from 21 patients who presented to the University Medical Center, Ljubljana, Slovenia between 1992 and 1995. Strains were isolated in culture from skin biopsies of acrodermatitis chronica atrophicans lesions; in the case of one patient two separate acrodermatitis chronica atrophicans lesions were cultured. All 21 patients had clinically typical lesions with "classic" histopathology and high IgG antibody titers to B. burgdorferi sensu lato. Strains were characterized and typed by 16S ribosomal RNA-specific polymerase chain reaction and determination of their large restriction fragment patterns using pulsed-field gel electrophoresis of MluI-digested genomic DNA. Of the 22 isolates studied, 17 possessed the highly conserved MLa1 pattern characteristic of B. afzelii. The remaining five isolates possessed large restriction fragment patterns that were typical of B. garinii (MLg2, four isolates from three patients) and B. burgdorferi sensu stricto (MLb2, one isolate). The results of 16S ribosomal RNA-specific polymerase chain reaction were concordant with these species designations. These data show that B. afzelii is the predominant, but not the exclusive, etiologic agent of acrodermatitis chronica atrophicans.

Large-restriction-fragment analysis of Mycobacterium kansasii genomic DNA and its application in molecular typing.

Large-restriction-fragment (LRF) polymorphisms in Mycobacterium kansasii isolates from 84 patients with bronchopulmonary infections in Japan between the 1960s and 1995 were studied by pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with VspI were most suitable for PFGE separation of 16 to 21 fragments of between 40 and 550 kbp. All 84 isolates and the type strain M. kansasii ATCC 12478 were successfully typed by LRF analysis with VspI digestion. Twenty-one distinctive LRF types were identified, and the LRF patterns tested over time were reproducible and stable. A computer-assisted dendrogram of the percent similarity demonstrated that isolates of 18 LRF types had relatively close genetic relatedness, while isolates of the remaining 3 types showed divergence. Sequence analysis of the 16S rRNA gene in the isolates showing divergent genetic relatedness revealed a sequence identical to that of a previously reported subspecies of M. kansasii. In the Chugoku district of Japan, 11 cases of M. kansasii infection which occurred in workers in a coastal industrial zone between 1982 and 1993 were caused by one particular strain tentatively named LRF type M. When both detailed demographic data for the patients and ecologic data for the M. kansasii isolates are obtained, LRF typing may be of potential use for investigating the source and transmission of M. kansasii infection.

Molecular Subtyping of Borrelia burgdorferi sensu lato Isolates from Five Patients with Solitary Lymphocytoma

Solitary lymphocytoma is a rare cutaneous manifestation of Lyme borreliosis that has been reported almost exclusively from Europe. This suggests that its etiologic agent may be absent or extremely rare on the North American continent. All three species of B. burgdorferi sensu lato known to be associated with human Lyme borreliosis (B. burgdorferi sensu stricto, B. garinii, and B. afzelii have been isolated in Europe, whereas only B. burgdorferi sensu stricto has been found in North America. This suggests that either B. garinii or B. afzelii might be the etiologic agent of borrelial lymphocytoma. To investigate this hypothesis we characterized five strains of B. burgdorferi sensu lato isolated from lymphocytoma lesions of patients residing in Slovenia. The methods used included: large restriction fragment pattern analysis of restriction enzyme MluI-digested genomic DNA, plasmid profiling, protein profiling, ribotyping using 5S, 16S, and 23S rDNA probes, and polymerase chain reaction amplification of the rrf (5S)-rrl (23S) intergenic spacer region. Molecular subtyping showed that four of the five isolates belonged to the species B. afzelii; however, this species is the predominant patient isolate in Slovenia and, therefore, may not represent a preferential association with lymphocytoma. The fifth isolate appeared to be most closely related to the DN127 genomic group of organisms. Further characterization of the isolate revealed that it possessed a unique molecular "fingerprint." The results not only show that borrelial lymphocytoma can be caused by B. afzelii but also demonstrate an association with another genomic group of B. burgdorferi sensu lato that is present in North America as well.

Three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B afzelii, and B. garinii) identified from cerebrospinal fluid isolates by pulsed-field gel electrophoresis and PCR.

A total of 36 European Borrelia burgdorferi sensu lato cerebrospinal fluid isolates (mainly from southern Germany) were analyzed by pulsed-field gel electrophoresis (PFGE) for large restriction fragment pattern (LRFP) and linear plasmid profiles. Analyzing this large panel of isolates, we detected all three species of B. burgdorferi sensu lato pathogenic for humans in cerebrospinal fluid from patients with Lyme neuroborreliosis by PFGE typing after MluI digestion: 21 B. garinii (58%), 10 B. afzelii (28%), and 4 B. burgdorferi sensu stricto (11%) strains as well as 1 isolate with bands characteristic of both B. afzelii and B. garinii. Species classification by PFGE typing was confirmed by 16S rRNA-specific PCR. Eighteen isolates (11 B. garinii, 6 B. afzelii, and 1 B. burgdorferi sensu stricto isolate) were further characterized by LRFP with four different restriction enzymes (ApaI, KspI, SmaI, and XhoI). All B. afzelii isolates showed identical patterns for each restriction enzyme group. Considerable heterogeneity was demonstrated within the B. garinii group. Subsequent analysis of plasmid profiles revealed only marginal differences for B. afzelii strains but different patterns for B. garinii isolates. In one B. afzelii strain we found a linear plasmid of about 110 kbp not described before. LRFP analysis by PFGE is a suitable tool for the molecular characterization of B. burgdorferi sensu lato strains and allows determination not only of the species but also of the subtypes within B. garinii.

Molecular characterization ofBorrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates

One hundred twenty-nine Slovenian isolates of Borrelia burgdorferi sensu lato derived from patients (69 strains) or Ixodes ricinus ticks (60 strains) were characterized. All of the strains were first- or second-passage isolates obtained in 1992 and 1993 from the same endemic region. The techniques used for the molecular analysis of strains included species-specific polymerase chain reaction (PCR) typing, and pulsed-field gel electrophoretic separation of undigested and MluI-digested genomic DNA. Isolates were identified to the species level by large restriction fragment pattern (LRFP) analysis and the results compared with the species-specific PCR result. Fifty-two patient isolates (75%) were typed as Borrelia afzelii (LRFP MLa1), 6 (9%) as Borrelia garinii (LRFPs MLg1-4), and 11 (16%) as Borrelia burgdorferi sensu stricto. The latter included 9 isolates (13%) with a new LRFP that is not typical of Borrelia burgdorferi sensu stricto and for which the designation MLx is suggested. In contrast, only 32 of 60 (53%) tick isolates were typed as Borrelia afzelii, while 20 strains (33%) were typed as Borrelia garinii and 8 strains (13%) as Borrelia burgdorferi sensu stricto. Three new LRFPs were found among the Borrelia garinii (MLg5 and 6) and Borrelia burgdorferi sensu stricto (MLb15) tick isolates. Large restriction fragment pattern analysis identified new groups of Borrelia burgdorferi sensu lato and revealed an apparent difference in the isolation frequency of different species from patients and ticks in the same endemic region.

Borrelia burgdorferi sensu lato Strains Isolated from Cutaneous Lyme Borreliosis Biopsies Differentiated by Pulsed-field Gel Electrophoresis

25 skin biopsy isolates of Borrelia burgdorferi sensu lato, mainly from German patients with erythema migrans [EM], borrelial lymphocytoma [BL] and acrodermatitis chronica atrophicans [ACA], were species-differentiated using pulsed-field gel electrophoresis (PFGE). The isolates revealed 15 B. afzelii, 8 B. garinii and 2 B. burgdorferi sensu stricto species according to Miu I digestion. All 6 ACA isolates were identified as B. afzelii species, whereas the 2 borrelial lymphocytoma isolates were grouped into B. garinii species. 12 B. afzelii strains, including 4 ACA isolates, were further investigated by digestion with 5 restriction enzymes. Most of the strains revealed an individual pattern. No specific characteristically large restriction fragment pattern (LRFP) could be detected within the ACA and/or EM group. The ACA isolates could not be clearly differentiated from the EM isolates according to the LRFP patterns. PFGE is thus a highly effective method for detecting differences among B. afzelii isolates.

Infection rate ofIxodes ricinus ticks withBorrelia afzelii, Borrelia garinii, andBorrelia burgdorferi sensu stricto in Slovenia

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.

Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois.

We have characterized 33 isolates of Borrelia burgdorferi from northern Illinois (32 isolates) and Wisconsin (1 isolate) representing the largest series of midwestern isolates investigated to date. The techniques used for molecular analysis of strains included (i) genospecies typing with species-specific PCR primers, (ii) plasmid profiling by pulsed-field gel electrophoresis of total genomic DNA, (iii) large-restriction-fragment pattern (LRFP) analysis by pulsed-field gel electrophoresis of MluI-digested genomic DNA (J. Belfaiza, D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons, J. Clin. Microbiol. 31:2873-2877, 1993), (iv) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins, (v) microsequencing of high-performance liquid chromatography-purified peptides derived from proteins showing high levels of expression, (vi) amino acid composition analysis of proteins, and (vii) immunological analysis of proteins with a polyclonal antiserum of human origin. Five reference strains as well as two atypical tick isolates from California (DN127) and New York (25015) were included for comparison. All of the Illinois and Wisconsin isolates were typed as B. burgdorferi sensu stricto with genospecies-specific PCR primers. The isolates were found to be heterogeneous with regard to their plasmid and protein profiles. One isolate from Illinois possessed two large-molecular-size plasmids instead of the usual 49-kb plasmid. Fragment patterns resulting from MluI digestion of genomic DNA from the 33 isolates and strains DN127 and 25015 were separable into six distinct LRFPs, five of which have not previously been described. Strain 25015 and an isolate from Illinois (CT39) shared an unusual LRFP that is not typical of other B. burgdorferi sensu stricto strains, suggesting that they may represent a fifth species of B. burgdorferi sensu lato. Five of the 33 isolates and strains DN127 and 25015 showed high-level expression of proteins with molecular masses of approximately 22 kDa. Investigation of these proteins by microsequencing of individual peptides and total amino acid composition analysis indicated that the 22-kDa proteins expressed by the seven strains were polymorphic OspC proteins. By using a polyclonal serum of human origin, expression of OspC could be detected in all 33 Illinois and Wisconsin isolates.

Genetic differences between BCG substrains

Setting: University-affiliated Mycobacteriology Reference Laboratory. Objective: To determine the genetic differences of 25 BCG isolates representing 16 referenced substrains. Design: Non-randomized, observational study based on the visual comparison of the large restriction fragment (LRF) patterns created by digesting each BCG isolate's DNA with an infrequent cutting restriction endonuclease (DraI, AsnI, XbaI or Spel) and separating the resultant DNA fragments with pulsed field gel electrophoresis. Results: The 25 BCG isolates gave 13 different Dral LRF patterns, 11 different XbaI LRF patterns, 11 different AsnI LRF patterns, and 15 different SpeI LRF patterns. Examples of the same BCG substrains from different sources produced the same LRF patterns for only 2 of 6 substrains studied. These findings suggest a significant degree of genetic diversity in this group of isolates despite a common origin. Four clinical BCG isolates gave LRF patterns identical to BCG Tice, BCG Connaught or BCG Glaxo. The BCG LRF patterns more closely resembled patterns of Mycobacterium bovis than M. tuberculosis. Conclusions: LRF patterns can accurately identify specific BCG substrains and will be useful in epidemiologic studies, monitoring vaccine production and studies of BCG vaccine efficacy.

Molecular characterization of Borrelia burgdorferi sensu lato strains by pulsed-field gel electrophoresis.

The large restriction fragment patterns (LRFP) and linear plasmid profiles of eight tick isolates of Borrelia burgdorferi sensu lato were investigated with pulsed-field gel electrophoresis (PFGE). The whole cell lysate was examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The MluI LRFP differentiates two species of Borrelia burgdorferi sensu lato (Borrelia burgdorferi sensu stricto and Borrelia garinii). LRFP is a suitable method to demonstrate genetic hetero- or homogeneity of isolates within one species without subsequent hybridization utilizing diverse probes. Different strains with similar or identical LRFP can be further discriminated by plasmid profile analysis. Our results show that each strain analyzed had a different plasmid profile. Therefore the linear plasmid profile has a potential application as a strain typing procedure. SDS-PAGE of whole-cell lysate supports the findings of homology within the B. burgdorferi sensu stricto species and the heterology within the B. garinii species.

Genomic fingerprinting of Borrelia burgdorferi sensu lato by pulsed-field gel electrophoresis.

A total of 46 Borrelia burgdorferi sensu lato isolates that were isolated from patients with Lyme borreliosis and infected animals or were extracted from ticks of the genus Ixodes were analyzed. Large restriction fragment patterns obtained after cleavage of genomic DNAs with MluI were analyzed by pulsed-field gel electrophoresis (PFGE). To eliminate the contribution of plasmid DNA, only fragments greater than 70 kb were used for the analysis. The results indicated that each of the 14 B. burgdorferi sensu stricto isolates were recognized by a band at 135 kbp, each of the 12 Borrelia garinii isolates by two bands (220 and 80 kbp), and each of the 20 Borrelia afzelii isolates by three bands (460, 320, and 90 kbp). Whereas differences in the PFGE patterns among B. burgdorferi sensu stricto isolates and B. garinii isolates were noted, B. afzelii isolates were all similar. Identification of isolates by PFGE correlates with their belonging to a given species within B. burgdorferi sensu lato.

DNA large restriction fragment patterns of sporadic and epidemic nosocomial strains of Mycobacterium chelonae and Mycobacterium abscessus

Large restriction fragment (LRF) pattern analysis of genomic DNA using pulsed-field gel electrophoresis was performed on three reference strains, 32 sporadic isolates, and 92 nosocomial isolates from 12 epidemics of Mycobacterium chelonae and Mycobacterium abscessus. Only 17 of 30 (57%) unrelated strains of M. abscessus, compared with 10 of 11 (91%) of M. chelonae strains, gave satisfactory DNA extractions, with the remainder resulting in highly fragmented DNA. DraI, AsnI, XbaI, and SpeI gave satisfactory LRF patterns. Sporadic isolates of the two species had highly variable LRF patterns, except for one reference strain and one sporadic isolate of M. chelonae that differed by only two to five bands. Evaluation of repeat isolates from five patients monitored for 8 months to 13 years (mean, 5.8 years) revealed LRF patterns to be stable, with changes of not more than two bands. LRF analysis of the seven nosocomial outbreaks with evaluable DNA revealed identical patterns in most or all of the patient isolates and in three outbreaks revealed identity with environmental isolates. These outbreaks included endoscope contamination, postinjection abscesses, and surgical wound infections. LRF analysis of genomic DNA is a useful technique for epidemiologic studies of M. abscessus and M. chelonae, although improved technology is needed for the approximately 50% of strains of M. abscessus with unsatisfactory DNA extractions.

Large DNA restriction fragment polymorphism in the Mycobacterium avium-M. intracellulare complex: a potential epidemiologic tool

Mycobacterium avium-M. intracellulare complex (MAI) isolates were studied by comparing the large restriction fragment (LRF) patterns produced by digesting their DNAs with infrequently cutting restriction endonucleases and separating the resultant large fragments by pulsed-field gel electrophoresis. Four reference strains and 35 randomly selected clinical MAI isolates gave highly diverse LRF patterns when their DNAs were digested with XbaI or AsnI. The LRF patterns of random isolates identified to be the same species by DNA probe analysis were not similar. The LRF patterns of random isolates of the same serotype were also different. In contrast, all isolates recovered from the same patient gave identical patterns. This included 28 isolates from nine patients. One isolate from sputum, one isolate from bone marrow, and two isolates from blood recovered over a 27-month period from a patient with AIDS were identical. Seven isolates recovered from the sputum of a second patient over 37 months also had identical patterns. The LRF patterns of unrelated MAI strains are highly polymorphic, appear to be strain specific, are relatively stable, and offer exciting promise as epidemiologic markers for the study of MAI infections.

DNA polymorphisms in strains of Mycobacterium tuberculosis analyzed by pulsed-field gel electrophoresis: a tool for epidemiology.

Mycobacterium tuberculosis isolates were studied by comparing large restriction fragment (LRF) patterns produced by digestion of chromosomal DNA with infrequent-cutting endonucleases and pulsed-field gel electrophoresis. Four cultures of H37Rv and 36 clinical isolates of M. tuberculosis were compared by using DraI, AsnI, XbaI, and SpeI. DraI and AsnI allowed easy visual separation of 18 of 21 epidemiologically unrelated strains. XbaI and SpeI allowed discrimination of all 21 unrelated strains, including the 3 strains inseparable with DraI and AsnI, but comparison of LRF patterns was more tedious because of overlapping fragments. A total of 26 isolates belonging to 10 clusters of related isolates were compared by pulsed-field gel electrophoresis, with all related isolates giving identical LRF patterns. These included multiple isolates from the same patient or the same family. The same grouping of clustered isolates was obtained when BamHI DNA digests were hybridized with two probes from the insertion sequence IS6110. Long-term laboratory passage of H37Rv produced minimal detectable changes in LRF patterns. LRF patterns are useful tools for epidemiologic studies of tuberculosis without the need for radioactive or specific DNA probes.

Large restriction fragment patterns of genomic Mycobacterium fortuitum DNA as strain-specific markers and their use in epidemiologic investigation of four nosocomial outbreaks.

Pulsed-field gel electrophoresis and restriction endonucleases with rare recognition sites were used to generate large restriction fragment (LRF) patterns of genomic DNA from 48 isolates of Mycobacterium fortuitum biovariant fortuitum. Epidemiologically unrelated isolates gave highly diverse patterns when AsnI, HpaI, AflII, DraI, NdeI, XbaI, SpeI, or SspI was used. Epidemiologically related isolates produced identical or minimally different LRF patterns. Minor variations in LRF patterns were seen in two epidemic isolates digested with XbaI, suggesting that genetic alteration had occurred. LRF patterns were used to study three cardiac surgery wound infection outbreaks and one respiratory disease nosocomial outbreak. In two outbreaks, LRF patterns confirmed the reported clustering of isolates on the basis of multiple phenotyping methods. In the remaining two outbreaks, isolates which could not be separated by prior typing methods were easily distinguished by LRF pattern analysis. Environmental water isolates from two outbreaks had LRF patterns identical to those of the disease-producing strains, confirming that the local environment was the source of infection. Pulsed-field gel electrophoresis of LRFs of genomic DNA offers great promise as an epidemiologic tool for the study of M. fortuitum.