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 MlnI-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) inter-genic 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. burgdoiferi sensu lato that is present in North America as well

Ecology and prevalence of Borrelia burgdorferi s.l. in Ixodes ricinus (Acari: Ixodidae) ticks

Weather conditions greatly affect tick population densities and activity, on which depends the occurrence of tick-borne diseases (TBDs). During the spring months from 2017 to 2019, 1,357 specimens of Ixodes ricinus ticks were collected at 9 localities in the vicinity of Novi Sad (Serbia). The number of collected ticks varied considerably among the different sampling sites and years. Also, a statistically significant difference was found between months and observed number of ticks for each stadium. By statistical analysis of tick activity depending on microclimatic conditions, a positive and statistically significant relationship between temperature and the number of ticks for each life stage was established, but not for humidity. Dew had a statistically significant impact only on nymphs but not on adults. The infection rate of Borrelia burgdorferi s.l. was the highest in March (46.5–51.2%) and the lowest in May (32.9–34.8%). The highest prevalence was detected in males and the lowest in nymphs. Since there is a positive and statistically significant correlation between tick number and prevalence, the ability to provide weather-based predictions of the seasonal patterns of current tick activity is important for the risk assessment of TBDs such as Lyme borreliosis

Emerging borreliae – Expanding beyond Lyme borreliosis

Lyme borreliosis (or Lyme disease) has become a virtual household term to the exclusion of other forgotten, emerging or re-emerging borreliae. We review current knowledge regarding these other borreliae, exploring their ecology, epidemiology and pathological potential, for example, for the newly described B. mayonii. These bacteria range from tick-borne, relapsing fever-inducing strains detected in some soft ticks, such as B. mvumii, to those from bat ticks resembling B. turicatae. Some of these emerging pathogens remain unnamed, such as the borrelial strains found in South African penguins and some African cattle ticks. Others, such as B. microti and unnamed Iranian strains, have not been recognised through a lack of discriminatory diagnostic methods. Technical improvements in phylogenetic methods have allowed the differentiation of B. merionesi from other borrelial species that co-circulate in the same region. Furthermore, we discuss members that challenge the existing dogma that Lyme disease-inducing strains are transmitted by hard ticks, whilst the relapsing fever-inducing spirochaetes are transmitted by soft ticks. Controversially, the genus has now been split with Lyme disease-associated members being transferred to Borreliella, whilst the relapsing fever species retain the Borrelia genus name. It took some 60 years for the correlation with clinical presentations now known as Lyme borreliosis to be attributed to their spirochaetal cause. Many of the borreliae discussed here are currently considered exotic curiosities, whilst others, such as B. miyamotoi, are emerging as significant causes of morbidity. To elucidate their role as potential pathogenic agents, we first need to recognise their presence through suitable diagnostic approaches

Genetic Diversity among Borrelia Strains Determined by Single-Strand Conformation Polymorphism Analysis of the ospC Gene and Its Association with Invasiveness

Lyme borreliosis (LB) is a tick-borne spirochetal infection caused by three Borrelia species: Borrelia afzelii, B. garinii, and B. burgdorferi sensu stricto. LB evolves in two stages: a skin lesion called erythema migrans and later, different disseminated forms (articular, neurological, cardiac…). Previous research based on analysis of ospC sequences allowed the definition of 58 groups (divergence of <2% within a group and >8% between groups). Only 10 of these groups include all of the strains isolated from disseminated forms that are considered invasive. The aim of this study was to determine whether or not invasive strains belong to restricted ospC groups by testing human clinical strains isolated from disseminated forms. To screen for ospC genetic diversity, we used single-strand conformation polymorphism (SSCP) analysis. Previously known ospC sequences from 44 different strains were first tested, revealing that each ospC group had a characteristic SSCP pattern. Therefore, we studied 80 disseminated-form isolates whose ospC sequences were unknown. Of these, 28 (35%) belonged to previously known invasive groups. Moreover, new invasive groups were identified: six of B. afzelii, seven of B. garinii, and one of B. burgdorferi sensu stricto. This study confirmed that invasive strains are not distributed among all 69 ospC groups but belong to only 24 groups. This suggests that OspC may be involved in the invasiveness of B. burgdorferi

In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy

Clinical treatment failures have been reported to occur in early Lyme borreliosis (LB) for many suitable antimicrobial agents. Investigations of possible resistance mechanisms of the Borrelia burgdorferi complex must analyze clinical isolates obtained from LB patients, despite their receiving antibiotic treatment. Here, borrelial isolates obtained from five patients with erythema migrans (EM) before the start of antibiotic therapy and again after the conclusion of treatment were investigated. The 10 isolates were characterized by restriction fragment length polymorphism analysis and plasmid profile analysis and subjected to susceptibility testing against a variety of antimicrobial agents including those used for initial chemotherapy. Four out of five patients were infected by the same genospecies (Borrelia afzelii, n = 3; Borrelia garinii, n = 1) at the site of the EM lesion before and after antimicrobial therapy. In one patient the genospecies of the initial isolate (B. afzelii) differed from that of the follow-up isolate (B. garinii). No significant changes in the in vitro susceptibilities became obvious for corresponding clinical isolates before the start and after the conclusion of antimicrobial therapy. This holds true for the antimicrobial agents used for specific chemotherapy of the patients, as well as for any of the additional agents tested in vitro. Our study substantiates borrelial persistence in some EM patients at the site of the infectious lesion despite antibiotic treatment over a reasonable time period. Borrelial persistence, however, was not caused by increasing MICs or minimal borreliacidal concentrations in these isolates. Therefore, resistance mechanisms other than acquired resistance to antimicrobial agents should be considered in patients with LB resistant to treatment

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

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