Differences in Genotype, Clinical Features, and Inflammatory Potential of Borrelia burgdorferi sensu stricto Strains from Europe and the United States

Abstract

Borrelia burgdorferi sensu stricto isolates from patients with erythema migrans in Europe and the United States were compared by genotype, clinical features of infection, and inflammatory potential. Analysis of outer surface protein C and multilocus sequence typing showed that strains from these 2 regions represent distinct genotypes. Clinical features of infection with B. burgdorferi in Slovenia were similar to infection with B. afzelii or B. garinii, the other 2 Borrelia spp. that cause disease in Europe, whereas B. burgdorferi strains from the United States were associated with more severe disease. Moreover, B. burgdorferi strains from the United States induced peripheral blood mononuclear cells to secrete higher levels of cytokines and chemokines associated with innate and Th1-adaptive immune responses, whereas strains from Europe induced greater Th17-associated responses. Thus, strains of the same B. burgdorferi species from Europe and the United States represent distinct clonal lineages that vary in virulence and inflammatory potential.

Keywords: B. afzelii; B. garinii; Borrelia burgdorferi sensu stricto; Europe; Lyme borreliosis; RNA intergenic spacer type; Slovenia; United States; bacteria; clinical features; erythema migrans; genotype; inflammatory potential; multilocus sequencing typing; outer surface protein C.

Figure 1

Distribution of Borrelia burgdorferi sensu stricto strains by outer surface protein C (OspC) and ribosomal RNA intergenic spacer type (RST). A) 29 isolates from patients with Lyme borreliosis in cental Europe (Slovenia). B) 90 isolates from patients with erythema migrans in the northeastern United States. OspC types are indicated by letters, and RSTs are indicated by colors. Red, RST1; blue, RST2; purple, RST3. *Based on previously published data (10). †Denotes OspC genotype (OspC type B) found in central Europe and the United States.

Figure 2

Phylogenetic analysis of Borrelia burgdorferi sensu stricto strains from central Europe (Slovenia) and the United States. A) Minimum spanning tree analysis of 70 isolates included in this study. Sequence types (STs) are indicated by numbers, and outer surface protein types are indicated by letters. Sizes of circles indicate ST sample sizes. Lengths of lines connecting STs indicate extent of variation (order of certainty) (no. locus variants). STs connected by the shortest black line are single-locus variants. Letters outside circles indicate OspC types. RST, ribosomal RNA intergenic spacer. B) Bayesian consensus tree resulting from simultaneous analysis of concatenated sequences of 8 housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA). Values at nodes indicate Bayesian posterior probabilities (proportion of sampled trees containing the taxon bipartition). Scale bar indicates nucleotide substitutions per site.

Figure 3

Phylogenetic comparison of 3 major pathogenic Borrelia species (Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto) that cause Lyme borreliosis. A) Minimum spanning tree analysis of 474 B. burgdorferi sensu lato human isolates. Analysis included 404 previously published datasets available in the multilocus sequence typing database (http://pubmlst.org/borrelia/) as of May 5, 2015, and 70 B. burgdorferi sensu stricto isolates from this study. Circles and numbers indicate specific sequence types (STs). Sizes of circles indicate MLST sample size and colors indicate origin of isolates. Lengths of lines connecting STs indicate order of certainty. STs connected by the shortest line are single locus-variants. B) Bayesian consensus tree resulting from simultaneous analysis of concatenated sequences of housekeeping genes of 70 B. burgdorferi sensu stricto isolates included in this study, representative strains of B. afzelii (http://pubmlst.org/borrelia/id:1546), and B. garinii (http://pubmlst.org/ borrelia/id:1829). Values at nodes indicate Bayesian posterior probabilities (proportion of sampled trees containing the taxon bipartition). Scale bar indicates nucleotide substitutions per site.

Figure 4

Frequency of symptoms in patients with erythema migrans infected with Borrelia afzelii, B. garinii, or B. burgdorferi sensu stricto from central Europe and B. burgdorferi sensu stricto from the United States. A) Any symptom, B) individual symptoms. Patients were assessed for 8 symptoms (fatigue, arthralgia, myalgia, headache, fever, chills, neck stiffness, or malaise). White bars indicate patients from Europe infected with B. afzelii, light grays bars indicate patients from Europe infected with B. garinii, dark gray bars indicate patients from Europe infected with B. burgdorferi sensu stricto, and black bars indicate patients from the United States infected with B. burgdorferi sensu stricto. Differences between strains were assessed by using the Fisher exact test. p values are indicated. There were no differences between B. afzelii, B. garinii, or B. burgdorferi sensu stricto from Slovenia.

Figure 5

Inflammatory potential of Borrelia burgdorferi sensu stricto from Europe and the United States. Levels of 22 cytokines and chemokines associated with innate (A) or adaptive (B) immune responses. Innate responses: tumor necrosis factor (TNF), interleukin-1β (IL-1β), IL-6, IL-10, granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-8, chemokine ligand 2 (CCL2), CCL3, and CCL19. Adaptive immune responses: Th1: interferon-γ (IFN-γ), IFN-α, IL-12p40, IL-12p70, cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), and CXCL10; Th17: IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, and IL-27. Immune responses were assessed in peripheral blood mononuclear cell culture supernatants after 7 days of stimulation with 14 B. burgdorferi sensu stricto isolates from Europe or 15 B. burgdorferi sensu stricto isolates from the United States (multiplicity of infection = 25) by using bead-based Luminex (EMD-Millipore, Darmstadt, Germany) multiplex assays. Each of 29 B. burgdorferi sensu stricto isolates was tested with peripheral blood mononuclear cells from each of 4 healthy donors in 4 independent experiments. Cytokine and chemokine values from the 4 experiments were averaged for analysis. Data are presented as box and whisker plots, boxes indicate interquartile ranges (1st and 3rd quartiles), lines inside boxes indicate median values, and error bars indicate 10th and 90th percentiles (value in parenthesis indicates the highest value). For comparison of B. burgdorferi sensu stricto isolates from Slovenia or the United States, *p<0.01, †p<0.001, ‡p<0.05.