Microbiology of Lyme disease

INTRODUCTION — Lyme disease is the most common tick-borne disease in the United States, Canada, and Europe [1-3]. It is a bacterial infection caused by six species in the spirochete family Borreliaceae [4]. The taxonomy of these spirochetes has been revised [5], and the genus name is represented in the literature as either Borrelia or Borreliella. In either case, the abbreviation for the genus is "B" and stands for both terminologies in the discussion below.

In North America, infection is caused primarily by B. burgdorferi and, less commonly, by B. mayonii. In Europe and Asia, infection is caused primarily by either B. afzelii or B. garinii, less commonly by B. burgdorferi, and uncommonly by B. spielmanii or B. bavariensis.

In nature, the reservoirs for these organisms are small mammals and birds, not deer. Humans acquire the infection from the bite of an infected tick of the genus Ixodes. The infection begins in the skin at the site of the tick bite. From there, the spirochetes may disseminate in the blood to other tissues and organs. The usual manifestations of Lyme disease involve the skin, joints, heart, and nervous system.

The microbiology of Lyme disease will be reviewed here. Topics related to immunopathogenesis, epidemiology, prevention, clinical manifestations, diagnosis, and therapy of Lyme disease are discussed separately.

●(See "Epidemiology of Lyme disease".)

●(See "Immunopathogenesis of Lyme disease".)

●(See "Prevention of Lyme disease".)

●(See "Evaluation of a tick bite for possible Lyme disease".)

●(See "Clinical manifestations of Lyme disease in adults".)

●(See "Lyme disease: Clinical manifestations in children".)

●(See "Diagnosis of Lyme disease".)

●(See "Treatment of Lyme disease".)

CHARACTERISTICS OF SPIROCHETES THAT CAUSE LYME DISEASE

Classification and genome — The spirochetes that cause Lyme disease are motile, spiral bacteria that are only distantly related to gram-negative and gram-positive pathogens. Spirochetes have two cellular membranes like gram-negative bacteria, but their flagella, the organelles of motility, are uniquely located between the inner and outer membrane, rather than on the surface. B. burgdorferi is 8 to 30 microns in length and about 0.2 microns in width. Their narrowness accounts for the inability to see unstained spirochetes by standard bright field light microscopy. (See 'Culture and staining' below.)

The genomes of B. burgdorferi and the other five species of Lyme disease agents comprise small linear chromosomes of approximately 1000 kb, and up to 21 linear and circular plasmids totaling another 400 to 500 kb [6-8]. The genes of the chromosomes are more similar between species and strains than those of the more divergent plasmids. Plasmids that carry genes for infectiousness are lost during serial cultivation in the laboratory [9], thus, the preference for low-passage isolates in experiments.

The spirochetes that cause Lyme disease were originally classified as members of the genus Borrelia [10]. A new genus name, Borreliella ("borrelia-like"), distinguishes the spirochetes that cause Lyme disease from those that cause relapsing fever (which retain the genus Borrelia affiliation) within the family Borreliaceae [5,11] (see "Microbiology, pathogenesis, and epidemiology of relapsing fever").Borreliella also substitutes for the designation of "Borrelia burgdorferi sensu lato complex," which refers to a cluster of species that are closely related to B. burgdorferi. B. burgdorferi itself has commonly been distinguished in the literature from other "sensu lato" species by appending "sensu stricto," as in "B. burgdorferi sensu stricto."

Although this new classification scheme has been formally accepted for the taxonomy of Lyme disease agents and closely related species, it is not in wide usage. Thus, the use of the medical term "borreliosis" for a disease (eg, as in Lyme borreliosis) and the name "borrelia" for an individual bacterium ("borrelias"or "borreliae" for plural) of Borreliaceae still apply.

Antigens of B. burgdorferi and other Lyme disease species — Some antigens that are encoded by genes on the chromosome are important for diagnostic tests (eg, Western blots). These include flagellin (the major structural protein of flagella, with an apparent size by electrophoresis of 41 kDa), certain heat shock proteins (eg, a 60 kDa protein), an inner membrane protein of 39 kDa, an integral outer membrane protein of 66 kDa, and less-characterized proteins with apparent sizes 58 kDa and 93 kDa. (See "Diagnosis of Lyme disease", section on 'Serologic tests'.)

In addition, a unique feature of B. burgdorferi and other Lyme disease species is the large number of lipoproteins, which are anchored in the outer membrane by covalently linked fatty acids at their N-terminal ends [6]. Plasmid-encoded antigens include the outer surface lipoproteins OspA (31 kDa), OspB (34 kDa) [12], OspC (23 kDa) [13], the decorin-binding proteins DbpA and DbpB (18 kDa) [14], and the antigenically variable VlsE protein (35 kDa) [15]. A conserved peptide of the VlsE proteins is the basis for the "C6" serological assay [16]. The spirochete up- or down-regulates expression of some of these proteins at different times in its life cycle, presumably to suit each environment [17,18].

Not all of the proteins are expressed in cultured organisms. For example, VlsE, a major immunogen, is poorly expressed in culture. Therefore, VlsE is produced as a recombinant protein in Escherichia coli or its "C6" peptide is synthesized for use in immunodiagnostic assays.

Culture and staining — Direct detection of the organism can be difficult and is of limited value for diagnosis [19]. (See "Diagnosis of Lyme disease".)

●Culture – Few clinical or public health laboratories perform cultures for B. burgdorferi and related species. B. burgdorferi and the other species have limited biosynthetic capabilities and require a complex medium for growth under microaerophilic conditions in vitro. Spirochetes have been isolated from skin, blood, and cerebrospinal fluid, as well as from ticks and various animals, in Barbour-Stoenner-Kelly medium or related formulations that contain multiple nutrients as well as bovine serum albumin and rabbit serum. However, under the best of circumstances, growth is slow, with generation times of six hours or longer.

●Staining – The spirochetes can be visualized with silver stains or by immunofluorescence in biopsies of affected skin, although the numbers of bacteria are low. Unlike B. burgdorferi, B. mayonii appears to reach high enough numbers in the blood of humans to be detectable in Wright- or Giemsa-stained smears of blood taken during acute infection [20]. (See 'Diversity of Lyme Disease species' below.)

The most commonly performed direct detection assay is polymerase chain reaction (PCR), but the paucity of organisms in tissues and the disappearance of the organism from the blood within two to four weeks of infection onset limit its utility. A more detailed discussion of PCR testing is found elsewhere. (See "Diagnosis of Lyme disease", section on 'Polymerase chain reaction'.)

PATHOGENESIS — Borrelias do not produce potent toxins, but cause infection by migrating through tissues, disseminating in the blood, adhering to host cells, and evading immune clearance. There are no data showing that B. burgdorferi has a prolonged or transient intracellular phase in vivo that is not treatable by antibiotics.

Under adverse conditions, spherical forms of the spirochetes may be observed. The outer membranes of the spirochetes may become detached from underlying cellular structures, but these so-called "cyst"-like forms do not represent a different stage in the life cycle of the spirochetes. There is no proven clinical significance for these "cyst"-like forms.

Ecologic niche — Borrelias are obligate parasites of arthropods and vertebrates; there are no known free-living forms [4]. The spirochetes cycle between two different environments: the tick (a poikilothermic invertebrate without an adaptive immune system) and usually a mammal or bird (homeothermic vertebrates with well-developed adaptive immune systems). The Lyme disease agents are transmitted to humans almost exclusively by ticks. They may rarely be acquired by a penetrating injury contaminated with blood or through the bite of infected rodent; however, they are not acquired through aerosols, respiratory droplets, mucosal secretions, urine, feces, food, drinking water, or fomites. In addition, they are not transmitted by bedbugs, fleas, flies, lice, or mosquitoes. Although B. burgdorferi might persist in the testes, as is the case with a relapsing fever agent [21], transmission of B. burgdorferi in the semen from a male to another person or through other forms of sexual contact has not been documented. (See "Epidemiology of Lyme disease".)

Borrelias are microaerophilic and grow optimally in carbon dioxide concentrations typical of the tissues of mammals and birds. These bacteria are killed by exposure to temperatures over 50°C for more than a few minutes, hypotonic or hypertonic environments, drying, common disinfectants (eg, bleach), and detergents. They do not form spores. (See "Prevention of Lyme disease".)

The small genome of borrelias encode comparatively few proteins with biosynthetic activity [6]. Thus, these organisms depend upon the animal host or a rich culture medium in the laboratory for most nutritional requirements. (See 'Culture and staining' above.)

Protein expression — B. burgdorferi expresses different repertoires of proteins to suit each environment [22-24]. To transit from the tick midgut (where the B. burgdorferi are located prior to initiation of feeding) to the tick salivary gland, the organism down-regulates outer surface protein (Osp) A and upregulates OspC. Down-regulation of OspA facilitates detachment from the tick midgut and transit to the salivary glands [25,26]. Expression of OspC facilitates invasion of bacteria into skin and subsequent dissemination [27-29]. Additional discussions of outer surface proteins are found elsewhere. (See 'Antigens of B. burgdorferi and other Lyme disease species' above and "Immunopathogenesis of Lyme disease", section on 'Outer surface protein variations'.)

Although Borrelia species do not produce lipid A-containing endotoxin [30], they do produce lipoproteins that are ligands for toll-like receptors (TLR), namely a heterodimer of TLR2 and TLR1 [31]. Binding to these receptors can lead to release of pro-inflammatory cytokines locally and systemically. A mild to moderate Jarisch-Herxheimer reaction may appear in some patients with early Lyme disease soon after the start of antibiotic treatment and the consequent lytic release from the spirochetes of lipoproteins and other constituents, such as peptidoglycan and flagella, for which there are other types of TLR receptors [32]. (See "Immunopathogenesis of Lyme disease", section on 'Toll-like receptors' and "Treatment of Lyme disease", section on 'Jarisch-Herxheimer reaction'.)

Immune response — Some lipoproteins, such as the strain-specific OspC protein, appear to be T-cell independent antigens in hosts. This would explain the antibody responses to OspC during early infection that are limited to IgM and short-lived [33]. Because B. burgdorferi do not secrete toxins, proteases, or other destructive molecules, the majority of the symptoms seen with human Lyme disease are due to the combined effects of the host innate and adaptive immune responses and lytic release of inflammatory bacterial components that follow. This is discussed in detail separately. (See "Immunopathogenesis of Lyme disease".)

DIVERSITY OF LYME DISEASE SPECIES

Overview — Three species account for most cases of Lyme disease in the world:

●B. burgdorferi causes Lyme disease in North America and less extensively in Europe.

●B. afzelii and B. garinii are the predominant species causing Lyme disease in Europe and Asia.

Different species of Ixodes ticks serve as vectors in different regions. This is discussed in detail separately. (See "Epidemiology of Lyme disease", section on 'Regional distribution'.)

Although the clinical manifestations and epidemiology of relapsing fever differ from Lyme disease, there is sufficient sequence similarity among certain Borrelia and Borreliella antigens for there to be cross-reactivity in some immunoassays for Lyme disease in persons infected with a relapsing fever agent [34]. This is also the case for Borrelia miyamotoi, which causes a relapsing fever-like illness and is transmitted by the same species of Ixodes ticks that transmits Lyme disease [35,36]. These agents are discussed in greater detail elsewhere. (See "Microbiology, pathogenesis, and epidemiology of relapsing fever" and "Clinical features, diagnosis, and management of relapsing fever" and "Borrelia miyamotoi infection".)

Geographic distribution

The Americas — In Northeastern and North-Central United States and bordering regions of Canada, B. burgdorferi is the principal cause of Lyme disease in humans [37]. The organism is transmitted by I. scapularis ticks.

In northern California, and some other coastal and foothill regions of far-western North America, human cases of Lyme disease are associated with exposure to Ixodes pacificus ticks. I. pacificus are infected with B. burgdorferi, but at a 10-fold lower frequency than occurs in the northeastern United States [38,39].

The other species known to cause Lyme disease in North America is B. mayonii, which is transmitted by I. scapularis ticks in north-central United States [40]. B. mayonii has been distinguished from B. burgdorferi by its ability to achieve higher concentrations in the blood in some patients. (See 'Culture and staining' above.)

Several other Borreliella species in North America have life cycles involving Ixodes ticks; these include B. americana, B. andersoni, B. bissettii, B. californiensis, B. carolinensis, B. kurtenbachii, B. lanei, and B. maritima [41]. However, to date, none of these species have been established as causes of human disease (ie, they have not been isolated in culture from patients). The usual vectors of most of these other types of spirochetes are Ixodes species of ticks that humans would seldom encounter or be bitten by.

In South America, B. chilensis was isolated from Ixodes ticks of that continent [42]. Another novel species of Borreliella was identified in an Ixodes tick of birds in Brazil [43]. However, the risk of either of these organisms to humans has not been established.

Europe and Asia — In Europe and Asia, there are four species known to cause Lyme disease. (See "Epidemiology of Lyme disease", section on 'Regional distribution'.)

●In Europe, B. afzelii and B. garinii commonly infect humans [44,45]. B. burgdorferi is a less common cause in Europe and has not yet been found in Asia. Strains of B. burgdorferi in Europe and North America are unique to each continent [46].

●In Asia, B. afzelii and B. garinii are also the most common causes of Lyme disease. Unlike North America and western Europe, where most infections are acquired from the bite of a nymphal tick, in Asia and Asian Russia, transmission to humans is mainly by the adult stage of the tick [47].

●B. bavariensis is closely related to B. garinii [48]. While B. bavariensis is distributed across the Eurasian continent, the infrequent cases of human disease have only been reported from Europe.

●B. spielmanii has been infrequently isolated from patients with erythema migrans in Europe. In a survey from Germany, this species occurred less frequently in ticks than B. garinii, B. afzelii, or B. burgdorferi [49].

There are differences in the ecology of the species, especially between the two most prevalent species, B. afzelii and B. garinii. B. afzelii is mainly associated with rodents as reservoirs, whereas B. garinii is mainly associated with birds [50,51]. This pattern of reservoir hosts for these spirochetes is attributed wholly, or in part, to the relative susceptibility of specific host complement lysis [52]. (See 'Ecologic niche' above and "Immunopathogenesis of Lyme disease", section on 'Evasion of complement-mediated killing'.)

There are several other varieties of Borreliella (B. burgdorferi sensu lato complex) species that have been identified in Europe and/or Asia in either Ixodes species ticks or vertebrate hosts. An example is B. valaisiana, first identified in Switzerland and subsequently in other parts of Europe and in Asia, but this and other species have not been documented as causes of human infections [41,53].

Relationship to clinical manifestations

Differences among species — Biologic differences have been observed among the three predominant species of Lyme disease agents. In an in vitro study that included representatives of the three species, B. burgdorferi stimulated macrophages to secrete higher levels of cytokines and chemokines than did B. afzelii or B. garinii [54]. (See "Immunopathogenesis of Lyme disease".)

These biologic differences are likely to account for some of the differences in clinical manifestations in patients in Europe compared with North America. As an example, although all three species have been recovered from various sites in patients (eg, skin, blood, and cerebrospinal fluid), infection with B. afzelii is associated with a lower risk of neurologic disease than infection with either B. garinii or B. burgdorferi [55]. The different clinical manifestations seen with each of the three species is not attributable to the higher frequency of bacteremia observed in B. burgdorferi infections [56]. More detailed discussions of the clinical differences between Lyme disease in Europe and the United States are presented elsewhere. (See "Clinical manifestations of Lyme disease in adults", section on 'United States versus Europe'.)

Differences among strains — In Lyme disease-endemic regions of North America, there are usually 10 to 15 genetically distinct strains of B. burgdorferi circulating among wildlife and ticks in a given area [37,38]. Strain typing by DNA sequence (genotyping) has been by outer surface protein C gene, a set of conserved chromosomal genes, and the spacer sequence between the 16S and 23S ribosomal RNA genes. The last typing scheme has been abbreviated "RST," and there are three major RST types: 1, 2, and 3 [57]. However, these methods are being supplanted by whole-genome sequencing.

The different strains can impact:

●Immunity from infection – Immunity from infection is usually strain specific, and infection with one strain generally does not confer protection against another strain [58]. However, more broadly protective immunity may develop across strains, especially after long-standing infections [59]. As an example, reinfection has not been observed in patients with Lyme arthritis who typically have expanded antibody responses to many spirochetal proteins that persist for years [60].

●Risk of dissemination – Some strains of B. burgdorferi are associated with a higher frequency of disseminated infection in humans and other mammals compared with others [61-67]. In particular, RST1 strains have been associated with more severe early disease [68] and more frequent antibiotic-refractory Lyme arthritis [68,69]. However, the basis for differences in pathogenicity is not known. Whether identification of the strain of an infection will lead to more effective management of the patient (eg, a longer course of antibiotics for some strains) has not been evaluated.

●Inflammatory potential – Strains of B. burgdorferi from the United States and Europe were found to differ in their inflammatory potential. In general, United States strains generally elicit higher levels of cytokines and chemokines associated with innate and Th1-adaptive immune responses than B. burgdorferi strains from Europe [70].

There are some strains of B. burgdorferi in the United States that occur in all three regions where the microbe is endemic: the Northeast, North-Central, and Far West. One of these is strain B31, which was the first laboratory isolate of this pathogen, and one of the most common. Other strains have more limited distributions (eg, occurring in the Midwest but not in the Northeast or the Far West) [71]. The coexistence in an area of both local strains and widely distributed strains characterizes populations of B. afzelii and B. garinii in Europe as well [46].

The mix of strains in a given area is probably determined in part by the adaptive immune responses, including strain-specific antibodies, of host animals in the population over time [58]. There may also be strain differences in host associations, such as different patterns of susceptibility to complement of various vertebrate species [51,52]. Susceptibility of B. burgdorferi to the nonimmune, bactericidal effects of the serum of lizards is one of the explanations for the lower prevalence of B. burgdorferi in Ixodes ticks in the far-western and southeastern United States. Although lizards are common hosts for I. pacificus and I. scapularis ticks in these respective regions [72], when an infected tick feeds on a lizard, the blood meal has a sterilizing action on spirochetes present in the gut of the feeding tick. (See "Immunopathogenesis of Lyme disease".)

SUMMARY

●Lyme disease is the most common tick-borne disease in the United States, Canada, and Europe. It is a bacterial infection caused by six species in the spirochete family Borreliaceae. The taxonomy of these spirochetes is undergoing revision, and the genus name may be represented as either Borrelia or Borreliella. (See 'Introduction' above.)

●Lyme disease spirochetes are motile, spiral bacteria that are only distantly related to gram-negative and gram-positive pathogens. Their genomes comprise small linear chromosomes and linear and circular plasmids. (See 'Classification and genome' above.)

●Direct detection of the organism can be difficult. B. burgdorferi and the other species have limited biosynthetic capabilities and require a complex medium and microaerophilic conditions for growth in vitro. Under the best of circumstances, growth is slow. The spirochetes can be visualized with silver stains or by immunofluorescence in biopsies of affected skin; however, the numbers of bacteria are low. (See 'Culture and staining' above.)

●B. burgdorferi and related species do not produce toxins but cause infection by migrating through tissues, adhering to host cells, and evading immune clearance. Illness also results from the inflammatory responses to the spirochete's presence. (See 'Pathogenesis' above.)

●In North America, infection is caused primarily by B. burgdorferi. In Europe and Asia, B. burgdorferi is less common or absent, and infection is caused primarily by either B afzelii or B. garinii. (See 'Geographic distribution' above.)

●Biologic differences have been observed between different species of Lyme disease agents and among strains within a given species. These are likely to account for some of the differences in clinical manifestations in patients in Europe and Asia compared with North America. (See 'Relationship to clinical manifestations' above.)