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.     

Genotypic and phenotypic characterisation of Borrelia burgdorferi sensu lato strains isolated from human blood

Lyme borreliosis often presents initially with erythema migrans. Borreliae may disseminate from the primary skin lesion, and different organs and systems could be affected. Borrelia strains were isolated from blood of 70 patients with Lyme borreliosis, including 10 patients from whom borreliae were also isolated from skin. The aim of the present study was to characterise the isolates with regard to their phenotypic and genotypic characteristics. Borreliae were cultivated in MKP medium. Species identification and plasmid profiles were determined by pulsed-field gel electrophoresis (PFGE) and protein profiles by SDS-PAGE. Digestion of Borrelia burgdorferi sensu lato DNA showed 63 (90%) B. afzelii Mla1 and 7 (10%) B. garinii Mlg2. No B. burgdorferi sensu stricto were isolated. Borreliae were isolated from both skin and blood of 10 patients, nine pairs of isolates were identical: seven B. afzelii and two B. garinii. B. afzelii was isolated from the skin and B. garinii from blood of the tenth patient. All but one isolate possessed at least one large plasmid and varying numbers of smaller plasmids. Eight (11.4%) of 70 isolates possessed an unusual plasmid profile (2 of 63 B. afzelii and 6 of 7 B. garinii). Borreliae differed in their protein profiles. OspA and OspB proteins were expressed by all B. afzelii isolates; 85.7% of B. garinii isolates expressed OspA and 71.4% expressed OspB. OspC was expressed by 65% of B. afzelii isolates and all B. garinii isolates. The ratios of B. afzelii and B. garinii isolated from blood and skin were similar. These results do not support the hypothesis that B. garinii has a higher propensity for haematogenous dissemination than B. afzelii. Antigen diversity as well as species and plasmid heterogeneity could play a role in the pathogenesis of the infection, suggesting distinctive strain organotropism.     

Phenotypic and genotypic analysis of Borrelia burgdorferi isolates from various sources.

A total of 17 B. burgdorferi isolates from various sources were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, restriction enzyme analysis, Southern hybridization with probes complementary to unique regions of evolutionarily conserved genes (16S rRNA and fla), and direct sequencing of in vitro polymerase chain reaction-amplified fragments of the 16S rRNA gene. Three groups were distinguished on the basis of phenotypic and genotypic traits, the latter traced to the nucleotide sequence level.     

Seroprotective groups among isolates of Borrelia burgdorferi.

We demonstrated that different seroprotective groups exist among isolates of Borrelia burgdorferi and Borrelia garinii. The major group was composed of isolates 297, B31, S-1-10, MMTI, IPT, and ATCC 35211 and 21 isolates obtained from California, Illinois, New York, Texas, and Wisconsin. A second group was composed of European isolates PBi and G25. A third group was composed of a single isolate, C-1-11. These groupings were supported by Western immunoblot findings. In addition, the seroprotective groups were confirmed by passive transfer of immune sera and challenge of recipient hamsters with the homologous isolate or other isolates of B. burgdorferi or B. garinii. These studies demonstrate that a monovalent vaccine will not provide complete protection against infection with all isolates of B. burgdorferi.     

Characterization of Borrelia burgdorferi isolates by restriction endonuclease analysis and DNA hybridization.

Genomes of several Borrelia burgdorferi isolates from North America and Europe were characterized by restriction endonuclease analysis and DNA hybridization using labeled B. burgdorferi whole-cell DNA (strain ATCC 35210). Several different restriction and homology patterns were observed among these isolates, indicating genotypic heterogeneity within this genus and species. It was concluded from this study that restriction endonuclease analysis of B. burgdorferi whole-cell DNA may be a reliable and accurate method for identifying strains or genotypes of the Lyme disease agent.     

Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis.

Fifty Borellia burgdorferi strains isolated from humans and ticks in Europe and the United States were analyzed by multilocus enzyme electrophoresis. Eleven genetic loci were characterized on the basis of the electrophoretic mobilities of their products. Ten loci were polymorphic. The average number of alleles per locus was 5.9, with a mean genetic diversity of 0.673 among electrophoretic types (ETs). The strains were grouped into 35 ETs constituting three main divisions (I, II, and III) separated at a genetic distance greater than 0.75. Divisions I, II, and III contained 13, 6, and 16 ETs, respectively. These findings, together with previous data from DNA hybridization and restriction enzyme analysis of rRNA genes, suggest that divisions I, II, and III may represent three distinct genomic species. All three divisions contained human clinical ETs. However, in division I, which includes the ET of the type strain of B. burgdorferi, the human pathogenic ETs constituted a single clone. The ETs of division I were from west-central Europe and the United States, whereas divisions II and III contained ETs from west-central and northern Europe but not from the United States. Finally, our data show that the genetic structure of B. burgdorferi populations is clonal.     

Antigenic characteristics of Borrelia burgdorferi isolates from ixodid ticks in California.

Twenty (1.4%) of 1,421 adult Ixodes pacificus ticks and 2 (20%) of 10 adult Ixodes neotomae ticks collected in five counties of northern California were found to contain spirochetes by direct immunofluorescence examination of their tissues with a polyvalent conjugate. Borreliae isolated from the tissues of nine of these ticks (I. pacificus, 8; I. neotomae, 1) were identified as Borrelia burgdorferi with specific monoclonal antibodies and characterized further by polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. The isolate from I. neotomae was the first to be characterized from a tick other than I. pacificus in western North America. All strains were relatively homogeneous with respect to the kind of OspA proteins they produced, whereas they were heterogeneous with regard to their OspB proteins and to several low-molecular-weight proteins in the 21,500-to-24,000 region. Significant phenotypic variation was observed among isolates obtained within and between populations of I. pacificus. This investigation nearly doubles the number of isolates of B. burgdorferi that have been characterized from ixodid ticks in the far western United States.     

Outer surface protein C gene sequence analysis of Borrelia burgdorferi sensu lato isolates from Japan.

The nucleotide sequences of the outer surface protein C gene (ospC) from Borrelia burgdorferi sensu lato isolates representing six different restriction fragment length polymorphism (RFLP) ribotype groups were determined, and the deduced amino acid sequences were aligned in comparison with the previously published OspC protein sequences. The sequence similarity analysis revealed the high sequence variability of OspC protein, and the degree of amino acid similarity ranged from 53.8 to 100% among 25 isolates. It has been reported that the representatives belonging to the three species of B. burgdorferi sensu lato showed a species-specific amino acid sequence motif at positions 23 to 35 (B. Wilske, S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Rössler, E. Soutschek, and R.C. Johnson, J. Clin. Microbiol. 33:103-109, 1995). Alignment with the OspC sequences of RFLP ribotype group IV, V, and VI isolates revealed that a sequence motif of all the isolates was quite similar to that of Borrelia garinii. A phylogenetic analysis based on OspC protein sequences also showed that most of the Japanese isolates were closely related to the species B. garinii. THe RFLP ribotype group IV species is predominant among clinical isolates of Lyme disease patients, reservoir rodents, and adult ticks in Japan. Although the isolates differed from type strains of the three delineated genospecies in genetic and immunological characteristics, it is likely that the spirochetes diverged within the species level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain

Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic.     

Protein and antigenic analysis of Borrelia burgdorferi isolated in northern Italy: computerized analysis of phenotypic characteristics.

Four Borrelia burgdorferi strains isolated in the same restricted geographic area share different protein patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The use of polyclonal rabbit antisera and a battery of monoclonal antibodies directed toward the immunodominant proteins OspA, OspB, and pC also revealed different epitope distributions and specificities of these antigens on the strains examined. For the first time, a computerized analysis of these phenotypic characters was done mainly by cluster analysis. The computerized analysis revealed the levels of similarities among the strains and indicated, on a quantitative basis, that one of them is much closer to the American strain B31 than to the other strains.     

DNA and protein analyses of tick-derived isolates of Borrelia burgdorferi from California

Nine isolates of Borrelia burgdorferi from ixodid ticks collected in northern California were characterized. Restriction endonuclease analysis, pulsed-field gel electrophoresis, and Western blot (immunoblot) analysis were used in this study. Four isolates were very similar to each other. The others shared some similarities but were classified as having unique genotypes. A strain from an Ixodes neotomae tick displayed the greatest genetic and antigenic diversity when compared to the isolates collected from Ixodes pacificus ticks. A computerized library based on DNA banding patterns of the isolates by restriction enzyme analysis is also reported. This library was created by using a scanning laser densitometer.     

Genetic diversity among Borrelia burgdorferi isolates from wood rats and kangaroo rats in California.

Twenty-nine Borrelia burgdorferi isolates, obtained from dusky-footed wood rats (Neotoma fuscipes) and California kangaroo rats (Dipodomys californicus) in California, were analyzed genetically. Chromosomal DNA was examined by restriction endonuclease analysis (REA) and gene probe restriction fragment length polymorphism. Pulsed-field gel electrophoresis was used to analyze the plasmid profiles of the isolates. REA, the method with the greatest discrimination, disclosed 24 distinct restriction patterns among the 29 isolates. These restriction patterns were sorted into four restriction fragment length polymorphism groups on the basis of their gene hybridization patterns. Results of the REA and plasmid profile analysis supported this grouping. The degree of genetic diversity among Californian isolates demonstrated by our findings is greater than that previously reported among other groups of North American isolates and is similar or greater than the diversity reported among European isolates.     

Genotypic and phenotypic characterization of Pseudomonas aeruginosa isolates from urinary tract infections

Pseudomonas aeruginosa is one of the most frequent agents of urinary tract infections especially in patients with indwelling urethral catheters. A total of 30 P. aeruginosa isolates from urinary tract infections was investigated for their genotypic and phenotypic characteristics. 'Single Nucleotide Polymorphism' chip typing experiments in combination with bioinformatical cluster analyses allowed genotypic grouping of the isolates. Some similarities to strains from lung infections but also to environmental strains were observed. Finally, several urinary tract-specific groups were identified indicating a strong heterogeneity of the urethral isolates. Pyoverdin, protease, and phospholipase A production in combination with quorum sensing activity and biofilm formation were common phenotypic characteristics of these strains. In contrast, swarming phenotypes, the production of pyocyanin, and the extracellular enzymes phospholipase C and elastase were rarely observed. Interestingly, strains isolated from catheter-associated infections showed significantly enhanced biofilm formation, decreased motility, and a slightly increased expression of virulence factors in relation to isolates from acute urinary tract infections.     

Genotypic and phenotypic heterogeneity among Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients

Although the heterogeneity of Mycobacterium tuberculosis populations and the existence of mixed infections are now generally accepted, systematic studies on their relative importance are rare. In the present study, 10 individual colonies of each M. tuberculosis isolate (primary isolate) from 97 tuberculosis patients in a primarily human immunodeficiency virus-negative population were screened for heterogeneity and detectable mixed infections by spoligotyping, IS6110-based restriction fragment length polymorphism analysis, and mycobacterial interspersed repetitive unit-variable number of tandem repeat typing. The MICs of antituberculosis drugs for colonies with divergent fingerprints were determined. Infections with different bacterial subpopulations were detected in the samples from eight patients (8.2%), and the frequency of detectable mixed infections in the study population was estimated to be 2.1%. Genotypic variations were found to be independent of the drug susceptibilities, and the various molecular markers evolved independently in most cases. The predominant strains and the primary isolates always had concordant drug susceptibility and MIC testing results. These findings have implications on the interpretation of molecular epidemiology results for patient follow-up and in transmission studies.     

Genotypic and phenotypic diversity of Anabaena isolates from diverse rice agro-ecologies of India

The genus Anabaena is one of the commonly observed genera in the rice fields in South-east Asia. Diversity analyses of a set of 70 Anabaena strains (including 67 strains isolated from diverse rice agro ecologies of India, and three International Reference/Type strains), was carried out using morphological and molecular datasets. The pattern of growth in liquid and solid medium and microscopic observations revealed tremendous diversity in the Anabaena germplasm analysed. The species wise distribution in different soil types and soil pH revealed that Anabaena iyengarii was present at pH ranging from 5.5-8.5 and all the species of Anabaena except A. oscillarioides were present in alluvium soils. Molecular profiling using primers based on HipTG, STRR(mod) and STRR1A sequences generated specific fingerprints for individual isolates. STRR1A was observed to be the most informative and useful for differentiating the isolates. Analyses of a combined dataset, including both morphological and molecular data, proved highly effective in discerning the genetic relationships among the 70 Anabaena strains. The present study provided useful information for the development of a comprehensive database based on the distribution of Anabaena strains in diverse agro ecologies of India and identified useful primers for PCR based differentiation of isolates.     

Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions.

Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E. coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid. These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro. Nine of these 13 isolates were found to be eaeA positive by PCR: four of these nine were eaeB positive. The 5' end, but not the 3' end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used. Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively. The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present. Only one canine E. coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test. The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement. However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract. Our results indicate that canine E. coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group.     

Linear and circular plasmid content in Borrelia burgdorferi clinical isolates

The genome of Borrelia burgdorferi, the etiologic agent of Lyme disease, is composed of a linear chromosome and more than 20 linear and circular plasmids. Typically, plasmid content analysis has been carried out by pulsed-field gel electrophoresis and confirmed by Southern hybridization. However, multiple plasmids of virtually identical sizes (e.g., lp28 and cp32) complicate the interpretation of such data. The present study was undertaken to investigate the complete plasmid complements of B. burgdorferi clinical isolates cultivated from patients from a single region where early Lyme disease is endemic. A total of 21 isolates obtained from the skin biopsy or blood samples of Lyme disease patients were examined for their complete plasmid complements by Southern hybridization and plasmid-specific PCR analysis. All clinical isolates harbored at least six of the nine previously characterized cp32s. Fourteen isolates harbored all B31-like linear plasmids, and seven isolates simultaneously lacked lp56, lp38, and some segments of lp28-1. The distinctive plasmid profile observed in these seven isolates was specific to organisms that had ribosomal spacer type 2 and pulsed-field gel type A, which implies a clonal origin for this genotype. The presence of nearly identical complements of multiple linear and circular plasmids in all of the human isolates suggests that these plasmids may be particularly necessary for infection, adaptation, and/or maintenance in the infected host.     

Genotypic comparison of clinical Legionella isolates and patient-related environmental isolates in The Netherlands, 2002-2006.

This study investigated the hypothesis that the genotype distribution of Legionella isolates from sporadic patients with Legionnaires' disease differs from that of Legionella strains in the environment. An amplified fragment-length polymorphism (AFLP) assay was used to genotype patient-derived and environmental Legionella isolates. The three Legionella pneumophila genotypes isolated most frequently from human respiratory secretions were AFLP types 004 Lyon, 010 London and 006 Copenhagen. These genotypes were cultured significantly less frequently from environmental samples (50% vs. 4%; p <0.001). The most frequently observed L. pneumophila serogroup 1 genotype among patient-derived isolates was 004 Lyon (32%). This genotype was cultured from only one of 6458 environmental samples. The positive sample contained 1.26 x 10(6) CFU/mL and originated from a whirlpool spa that had not been disinfected and had been maintained at 36 degrees C for several months. Overall, the distribution of genotypes differed significantly among patient and environmental isolates. A possible explanation is that virulent strains may exist in potential environmental sources at undetectable concentrations.     

Genotypic and phenotypic analysis of the env gene from South African HIV-1 subtype B and C isolates

The objective of the study was to assess the genotypic and phenotypic properties of 18 viral strains from human immunodeficiency virus-1 (HIV-1) positive patients and to identify subtype C isolates for vaccine design strategies. All the isolates were non-syncytium-inducing (NSI) in both the primary and MT-2 cell cultures. The amino acid charge of the V3 loop correlated with the NSI phenotype of the strains. The V3 competitive peptide enzyme immunoassay and DNA sequencing of the partial gp120 region gave concordant results on the 15 subtype C strains, whereas the three B genotypes gave a positive to B, a nonreactive to B, and a dual reaction to the B-D peptides, respectively. Sixteen of the isolates used only CCR5 as coreceptor whereas two isolates made use of additional coreceptors including CXCR4. In summary, all our subtype C isolates are NSI phenotypically and almost all of them use CCR5 exclusively as their coreceptor.     

Identification of a transferrin-binding protein from Borrelia burgdorferi.

Bacterial pathogens have evolved various strategies to acquire iron from the iron-restricted environment found in mammalian hosts. Borrelia burgdorferi should be no different with regard to its requirement for ferric iron, and previous studies have suggested that transferrin (Tf) may be a source of iron in vivo. By probing blots with Tf conjugated to horseradish peroxidase, we have identified an outer membrane protein (28 kDa) from B. burgdorferi B31 that bound holo-Tf but not apo-Tf. The 28-kDa protein bound human, rat, or mouse Tf and was produced only by low-passage (less than passage 5), virulent isolates of strain B31. In addition, the Tf-binding protein (Tbp) from strain B31 retained the ability to bind Tf after treatment with 2% sodium dodecyl sulfate-1% beta-mercaptoethanol and heating to 100 degrees C for 5 min. These properties are remarkably similar to those of the Tbp of Staphylococcus aureus and Tbp2 from Neisseria meningitidis. B. burgdorferi Sh-2-82 produced an outer membrane protein different in size, i.e., 26 kDa, but with properties similar to those of to the protein from strain B31, suggesting variation in B. burgdorferi Tbps. The exact role of the 28-kDa protein in iron acquisition by B. burgdorferi remains to be determined.