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Institute of Microbiology and Immunology, School of Medicine, Zaloka 4, 1000 Ljubljana, Slovenia
Department of Infectious Diseases, University Medical Centre Ljubljana, Japljeva 2, 1525 Ljubljana, Slovenia
ABSTRACT
The aim of the present study was to analyze and compare Borrelia strains isolated from two different specimens obtained simultaneously from individual patients with Lyme borreliosis. Fifty such patients and 50 corresponding pairs of Borrelia isolates (100 low-propagated strains) were subjected to genotypic and phenotypic analysis, including pulsed-field gel electrophoresis for species identification and plasmid profile determination and protein profile electrophoresis for the assessment of the presence and molecular masses of separated proteins. The strains were isolated from two distinct skin lesions (12 patients), skin and blood (28 patients), skin and cerebrospinal fluid (8 patients), and blood and cerebrospinal fluid (2 patients). Out of 100 isolates, 63 were typed as B. afzelii and 37 as B. garinii. From each individual specimen only a single Borrelia species was cultured. Comparison of 50 Borrelia strain pairs isolated from two different specimens of an individual patient revealed that 12/50 (24%) patients were simultaneously infected with two different Borrelia strains; in 3/50 (6%) patients strains differed at the species level, in 4 out of the remaining 47 (9%) patients a strain difference in plasmid profile was established, while 5 out of the remaining 43 (11%) patient strain pairs differed in regard to the protein profiles of the two concurrently isolated strains. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor different B. burgdorferi sensu lato strains.
INTRODUCTION
Lyme borreliosis in humans most frequently presents with erythema migrans, a skin lesion that usually develops at the site of a bite from an infected tick (19, 38-40). From this early localized skin infection borreliae may disseminate to other tissues, organs, and organ systems and cause different clinical manifestations (38-39). An individual patient usually presents with one clinical sign, most often erythema migrans, but in some patients several clinical manifestations can be found at the same time (1, 3, 16, 38, 39, 41).
Isolation of the etiological agent from patient material is the most reliable method for the diagnosis of borrelial infection (1, 14, 16, 23, 26, 27, 47). In addition, it also provides live microorganisms; data obtained from their further characterization provide potentially valuable information on the geographical distribution, epidemiology, and pathogenesis of borrelial infection and Lyme borreliosis.
Hundreds of Borrelia strains from human materials (skin, blood, cerebrospinal fluid, etc.), ticks, and reservoir hosts (9, 17, 25, 29, 42, 49) obtained worldwide have been analyzed by many different phenotypic methods, such as well-established protein profile and serotyping system based on the heterogeneity of OspA and OspC proteins (33-35, 42-45), and several genotypic approaches (42). Analysis of borrelial chromosomal or plasmid DNA, as performed by genotypic methods, provides more precise information on the genetic relationships among Borrelia strains (2, 5, 7, 22, 33-35, 42, 47). The results of numerous studies indicate that the B. burgdorferi sensu lato population is genetically divergent and phenotypically heterogeneous, that different Borrelia species may be associated with specific reservoir hosts, that Lyme borreliosis in humans is caused by at least three different species of the Borrelia burgdorferi sensu lato complex (B. afzelii, B. garinii, and B. burgdorferi sensu stricto), and that infection with particular strain is in some way associated with specific organothropism and results in distinct clinical manifestations (2, 4, 13, 17, 25, 30, 33-35, 42, 47).
It has not been very unusual that an individual tick or reservoir host has harbored more then one Borrelia species (30, 36, 49), but data on the presence of multiple Borrelia species in an individual human patient with Lyme borreliosis have been much more limited (11, 13, 21, 31, 35). It is of interest that the majority of such mixed infections were demonstrated by PCR, while as a rule only one species appeared after culturing of the sample (18, 24, 25, 29, 47, 49). Theoretically this discrepancy could have been explained by false-positive PCR results and/or by an overgrowth of one species over the other in culture.
The aim of the present study was to isolate strains from two different specimens obtained simultaneously from an individual patient and to analyze and compare the isolated strains regarding to their phenotypic and genotypic characteristics. We tried to find out whether a particular patient can be simultaneously infected with different Borrelia strains and to assess the existence of, type, and magnitude of the association between strain characteristics and the origin of the clinical sample or clinical manifestation.
MATERIALS AND METHODS
Patients and Borrelia strains. Patients diagnosed with Lyme borreliosis at the Department of Infectious Diseases, University Medical Centre Ljubljana, in the period from 1993 to 2003, from whom multiple samples were obtained simultaneously and for whom cultures from specimens obtained from two different sites were positive, were the basis for the present study. Fifty such patients and 50 corresponding pairs of Borrelia isolates (100 strains) were subjected to further analysis as outlined below. In 12 patients borreliae were isolated from two distinct skin lesions, in 28 patients one strain was isolated from skin and another from blood, in 8 patients one strain was isolated from skin and another from cerebrospinal fluid (CSF), and in 2 patients one strain was isolated from blood and another from CSF. The information is summarized in Table 1.
Cultivation. Skin and CSF were inoculated into modified Kelly-Pettenkofer medium immediately after they were obtained from patients (33, 35). Citrated blood samples were transported to the laboratory where they were processed, and only plasma was inoculated in modified Kelly-Pettenkofer medium (34). All the samples were incubated at 33°C and checked for the presence of borreliae weekly (27). Isolated strains were grown in the same medium for two to four subcultures.
Identification of Borrelia species. Strain identification was done by pulsed-field gel electrophoresis (PFGE) as described previously (5, 33-35). Briefly, Borrelia strains were mixed in agarose, and whole blocks were lysed in lysis buffer containing lysozyme (1 mg/ml) and RNase (10 μg/ml) and digested in digestion buffer containing proteinase K (0.5 mg/ml). After exhaustive block washing, DNA was restricted with 30 U of MluI restriction enzyme and restriction patterns were separated for 24 h with ramping pulse times of 1 to 40 s. Molecular size markers of 50 to 1,000 kb (Sigma) were included in each electrophoresis. The Borrelia species as well as the type within the particular species were determined according to restriction fragment length polymorphism (RFLP) as described by Belfaiza et al. (2) and Picken et al. (25). Fragments interpreted as being species specific were those with molecular sizes of 440, 320, and 90 kb for B. afzelii; 220 and 80 kb for B. garinii; and 145 kb for B. burgdorferi sensu stricto. Types within species (Mla for B. afzelii, Mlg for B. garinii, and Mlb for B. burgdorferi sensu stricto) were determined according to additional fragments of different molecular sizes (2, 25).
Analysis of plasmid profile. The plasmid profile was also determined by PFGE. Blocks with borrelial DNA were prepared identically as for species identification, but electrophoresis was performed without previous restriction of DNA. Chromosomal and plasmid DNAs were separated for 37 h with ramping pulse times of 0.9 to 3 s, as described previously (5, 33-35, 48). Molecular size markers of 0.1 to 200 kb (Sigma) were added to each separation. The molecular size of a particular plasmid was calculated with the GelDoc system (Bio-Rad, Germany). To compare plasmid profiles of simultaneously isolated strains, we performed electrophoresis of both strains on the same gel.
Analysis of protein profile. For determination of protein profiles, strains were harvested, washed, and lysed in buffer containing sodium dodecyl sulfate (2.5%) and 2-mercaptoethanol (2.5%), as described elsewhere (33-35, 43). Electrophoresis was performed in a 12% polyacrylamide gel. Low-molecular-mass markers of 21 to 106 kDa (Bio-Rad, Germany) were used as size markers in each protein determination. To compare protein profiles of simultaneously isolated strains, we performed electrophoresis of both strains on the same gel.
Statistical analysis. The 2 test was used to compare differences between qualitative data. A P value of 0.05 was interpreted as being indicative of statistical significance.
RESULTS
Borrelia species. Out of 100 isolates, 63 were typed as B. afzelii and 37 as B. garinii; no B. burgdorferi sensu stricto strain was isolated. From each individual specimen only a single Borrelia species was cultured. RFLP of some of the isolated strains is shown in Fig. 1.
Borrelia species of the corresponding isolate pairs were identical in 47 out of 50 (94%) patients with B. burgdorferi sensu lato concurrently isolated from two distinct sources, while in 3/50 (6%) patients the species were divergent. In particular, in 30/50 (60%) patients (i.e., in 30/50 isolated pairs) both strains were B. afzelii, in 17/50 (34%) both isolates were B. garinii, while in 3/50 (6%) patients B. afzelii and B. garinii constituted the corresponding pair. Basic characteristics of three patients with dissimilar Borrelia species isolated from simultaneously obtained samples on two distinct sites are shown in Table 2.
The assessment of the association of the Borrelia species and clinical material from which borreliae were isolated revealed that in 47 patients with congruent pairs according to species, B. garinii strain pairs predominated over B. afzelii in the group of paired isolates obtained from skin and CSF (P = 0.002), while no distinctions were established for the isolates acquired from two different skin lesions (P = 0.282) as well as for those obtained simultaneously from skin and blood samples (P = 0.581) (Table 3).
All B. afzelii species showed identical RFLP patterns and were typed as Mla1. Within B. garinii we identified five different RFLP types, Mlg1 to Mlg4, and Mlg7, but the two strains in all pairs were identical. The occurrence of a particular RFLP type is shown in Table 3.
Plasmid profile. Generally, plasmid profiles differed among the strains within a species as well as among the species. There was no specific plasmid profile that would be characteristic for a particular Borrelia species. Comparison of plasmids by electrophoresis of both strains of an individual pair on the same gel showed different plasmid profiles in seven (14%) patients, including all three patients with different Borrelia species isolated from simultaneously obtained specimens. In the group of 47 patients with matching Borrelia species, identical plasmid profiles were found in 43 (91.5%) strain pairs, while in 4 (8.5%) strain pairs the plasmid profiles differed, suggesting infection with two different strains of the same Borrelia species.
Different plasmids were therefore uncovered in 3/30 (10%) B. afzelii pairs and in 1/17 (6%) B. garinii pairs. Figure 2 shows plasmid profiles of some strain pairs: patients A to D presented with different Borrelia strains while patients E to G showed identical Borrelia strains in regard to their plasmid profile. Of the four strain pairs with different plasmid profiles, in two cases the only distinction was an additional plasmid found in one of the strains of the pair (Fig. 2, patients A and B); both patients presented with multiple erythema migrans lesions and had B. afzelii isolated from blood and CSF. The third pair, consisting of B. afzelii strains isolated from skin of two different skin lesions of a patient with multiple erythema migrans, showed six plasmids per strain; four were identical and two different, and one of the two strains lacked a large plasmid of 50 to 65 kb but possessed a plasmid dimer with a molecular size of about 126 kb (Fig. 2, patient C). The fourth pair (two B. garinii strains, isolated from skin and blood of a patient with solitary erythema migrans) showed eight and seven plasmids, respectively; five of them were identical (Fig. 2, patient D).
Protein profile. Protein profiles of the isolated strains were quite diverse; no protein profile was found to be unique for a particular Borrelia species. To assess phenotypic diversity, each strain pair was electrophoresed on the same gel and compared in regard to molecular mass. A large majority of strain pairs exhibited identical protein profiles, but as shown in Fig. 3, in several pairs distinctions were established. Different protein profiles were found in two out of three patients with different Borrelia species isolated concurrently; one strain pair differed in regard to the molecular mass of OspC protein (23 and 21 kDa in B. afzelii and B. garinii, respectively), while in the other pair B. afzelii expressed OspB protein while B. garinii did not. In the third strain pair that differed on the species level, protein profiles were identical.
Different protein profiles were found in 4 out of 4 strain pairs with congruent species but different plasmid profiles (in all these pairs one of the two strains expressed OspC protein while the other did not) and in 5 out of 43 (12%) patients with matching Borrelia species and plasmid profiles (in all of them only one of the two strains of the corresponding pair expressed OspC). All five patients of the last group presented with solitary erythema migrans, all were infected with B. afzelii, and in all of them the strains were isolated from skin and blood.
The expression of OspA, OspB, and OspC proteins has been analyzed in all 100 strains. We found differences in expression of OspB and OspC between B. afzelii and B. garinii strains, as shown in Table 4.
In total, 12/50 (24%) patients were simultaneously infected with two different Borrelia strains; in 3/50 (6%) patients strains differed on species level, in 4 out of the remaining 47 (9%) patients a strain difference in plasmid profile was established, while 5 out of remaining 43 (11%) patient strain pairs differed in regard to the protein profiles of the two concurrently isolated strains.
DISCUSSION
In the present study we analyzed and compared 50 Borrelia strain pairs obtained simultaneously from two different sites of an individual patient (Table 1). A large majority of these 50 patients with Lyme borreliosis included in the study presented with solitary or multiple erythema migrans (39/50, 78%), which are two main clinical signs of Borrelia infection in Slovenia (1, 40, 41) and elsewhere (3, 19, 21, 38, 39, 47). A literature search revealed very few reports on Borrelia strains simultaneously isolated from two different skin lesions, or from skin and either blood or CSF, in an individual patient; such paired isolates were limited to patients with solitary and/or multiple erythema migrans (15, 21, 31, 47).
In order to analyze the isolated Borrelia strains and to determine the degree of similarity between and within the isolated strain pairs, several typing methods were used in this study, including PFGE for species identification and plasmid profile determination and protein profile electrophoresis for the assessment of the molecular masses of separated proteins.
Regarding species identification, 63 out of 100 isolates were typed as B. afzelii and 37 as B. garinii; no B. burgdorferi sensu stricto strain was isolated. From each individual specimen only a single Borrelia species was cultured. Borrelia species of the corresponding isolate pairs were identical in 47 out of 50 (94%) patients, while in 3/50 (6%) patients the species were divergent. The concurrent presence of various Borrelia species in an individual host has been described previously (15, 21, 30, 47, 49). The majority of such mixed infections were demonstrated by PCR, which directly amplifies borrelial DNA in the sample, while as a rule only one species appeared after culturing the sample (18, 24, 25, 29-31, 47, 49). Theoretically this discrepancy could have been explained by false-positive PCR results and/or by selection during culturing.
Ticks, feeding on a variety of hosts during their life cycle, can acquire and maintain different Borrelia strains (4, 12, 36) that may be inoculated into skin at the site of the tick bite. It would be reasonable to presume that a host, including a human host, could be inoculated with all of the different strains that the tick had been infected with. However, it is far from clear how often such inoculation with multiple Borrelia species really happens in humans and how successful it is, which strains survive or perish in the new human host, which have a potential to persist in tissues and for how long, which strains disseminate, what are the ways and the target organs of such dissemination, which strains stimulate an immune response, and which evoke clinical signs. These difficult questions have not been satisfactorily answered, partly due to the methodological problems with Borrelia culturing, for example, because of the possibility of an overgrowth of one species over the other. Such an overgrowth might be a result of several factors, including a distinct support of culture media for different Borrelia species or strains, differences in the relative amounts of the species or strains present in the specimen at the very beginning (at the time when the specimen was obtained or inoculated into the medium), or intrinsic differences between the inoculated strains, such as different growth potentials and different times needed for adaptation to the new environment (10, 20, 36). Thus, during culturing one strain might overgrow the other(s), and at the final point this strain might be observed and interpreted as the sole pathogen strain. If the hypothesis of overgrowth was valid, the chances that a mixture of diverse Borrelia strains or species present in an individual specimen would generate a heterogeneous Borrelia strain (species) culture result would probably be low. However, in comparison to the culture result for an individual specimen, there might have been a higher chance of finding dissimilar Borrelia species or strains in two different tissue or body fluid samples obtained simultaneously from an individual patient.
The results of the present study supported this hypothesis. While from each individual specimen only a single Borrelia species was cultured, different Borrelia species or strains were isolated from two specimens taken simultaneously from an individual patient. Our results obviously corroborate previous reports, based predominantly on PCR findings, that some human patients with Lyme borreliosis are infected with more than one Borrelia strain or species simultaneously.
Chromosomal DNA restriction profiling using MluI digestion (e.g., PFGE) is a highly specific and reproducible approach for Borrelia typing. This method provides not only species identification but also subspecies differentiation (RFLP differentiation) within B. garinii and B. burgdorferi sensu stricto but not within B. afzelii (2, 7, 25, 33-35, 40, 42). As expected, the analysis of our strains revealed a unique Mla1 pattern within all strains of B. afzelii and different patterns within B. garinii. Comparison of strain pairs obtained simultaneously from an individual patient showed identical subspecies characteristics within all strain pairs with congruent Borrelia species but disparities in the strain pairs of three patients with different Borrelia species isolated simultaneously (Table 3).
Borrelial linear plasmids were described in many previous reports, always separated by PFGE (11, 32, 48). This method enables the comparison of the identity of plasmids according to their number and molecular mass but not in regard to the plasmid structure, content of genes, repeated patterns, and virulence sequences (11, 28). Plasmid profiling represents an excellent tool for strain discrimination, particularly when comparing strains within the same species (11, 32-35, 42, 48). However, there is no specific plasmid profile that would be characteristic for particular Borrelia species; e.g., there is no plasmid profile typical for B. afzelii or B. garinii strains. In our study distinct plasmid profiles were found in all three patients with different Borrelia species isolated simultaneously and in 4 out of 47 (9%) patients with matching Borrelia species (in 3/30 B. afzelii pairs and in 1/17 B. garinii pairs), suggesting infection with two different strains of the same Borrelia species. Many conditions can influence borrelial plasmid content, including the possibility that plasmids can be lost upon cultivation. Because in our study all strains were subjected to identical circumstances (they were isolated, multiplied, and analyzed using equal approaches and procedures), we believe that their plasmid content represents the natural situation. Thus, different plasmid profiles of simultaneously isolated strains subjected to identical manipulations may be quite safely interpreted as an infection with two different Borrelia clones of the same species with identical RFLPs.
Similar, but probably somewhat less reliable, conclusions could have been valid also for distinctions in Borrelia protein expression, because protein expression is less stable then plasmid content. Specific changes in protein profile are usually but may not always be the result of specific changes in plasmid profile. For example, while a large plasmid encoding OspA and OspB was present in all our isolated strains, only OspA and not OspB was expressed by all strains. Several factors may influence the expression of antigens of the emerging strain population, such as medium components, pH, temperature, biological source, etc. (6, 8, 36, 37). Almost all of the observed changes affect membrane proteins (8, 36). To show the protein content of Borrelia strains, we performed electrophoresis of the whole-cell lysate, a standard approach that enables detection of phenotypic differences in the isolates (33-35, 43). In the present study we found differences in protein profiles in 5 out of 43 (12%) Borrelia strain pairs that were identical in species and plasmid content and in all but one (6/7) strain pairs composed of distinct Borrelia species and/or with different plasmid profiles. The large majority of the strain pairs with distinct protein profiles differed only in the expression of OspC protein. We are aware of the ability of Borrelia to respond to environmental signals with an adjustment in expression of proteins, but again, the conditions under which all our strains were analyzed in this study were the same. Therefore, we believe that proteins expressed by the strains presented in this study most probably reflect their own basic status. The fact that each strain has its own potential to express (or not express) particular proteins is supported by the finding that besides adaptive mechanisms of the spirochete that influence a variety of protein expression, the population of B. afzelii strains and the population of B. garinii strains differ generally in their potential to express some important proteins, such as OspB and OspC (33-35, 43, 46). This finding was also corroborated in the present study (Table 4).
Comparing the origin of Borrelia isolates with the species of isolated strain pairs in the present study, a significant difference was established only for simultaneously obtained skin and CSF samples, from which B. garinii strain pairs were more frequently isolated than B. afzelii strain pairs (P = 0.002); no equivalent distinctions for B. afzelii and B. garinii strain pairs concurrently isolated from two different skin lesions and for those obtained simultaneously from skin and blood samples were established (Table 3). These findings indicate that while the potentials of B. afzelii and B. garinii strains for the dissemination from skin into blood are similar, their proclivities to involve the central nervous system differ; B. garinii strains must have some additional factor(s) with a potential to infect the central nervous system that is as a rule not present in B. afzelii strains. However, our findings do not inevitably indicate a more efficient spread of B. garinii in comparison to B. afzelii from blood to central nervous system. Differences at the species level according to clinical manifestation of Lyme borreliosis and clinical samples from which borreliae were isolated have been described previously (25, 33-35, 38, 39). In the United States, where B. burgdorferi sensu stricto has been the only species of the B. burgdorferi sensu lato complex causing disease in humans, Wormser et al. reported that specific genetic subtypes of B. burgdorferi sensu stricto were significantly associated with spirochetemia in patients with erythema migrans (47), but no corresponding data for the involvement of the central nervous system have been published.
In conclusion, the results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor different B. burgdorferi sensu lato strains.
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