Aberer E, Kersten A, Klade H, Poitschek C, Jurecka W

Heterogeneity of Borrelia burgdorferi in the skin.

Am J Dermatopathol 1996 Dec; 18(6): 571-9

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8989928

The reliability of various in vitro techniques to identify Borrelia burgdorferi infection is still unsatisfactory. Using a high-power resolution videomicroscope and staining with the borrelia genus-specific monoclonal flagellar antibody H9724, we identified borrelial structures in skin biopsies of erythema chronicum migrans (from which borrelia later was cultured), of acrodermatitis chronica atrophicans, and of morphea. In addition to typical borreliae, we noted stained structures of varying shapes identical to borreliae found in a "borrelia-injected skin" model; identical to agar-embedded borreliae; and identical to cultured borreliae following exposure to hyperimmune sera and/or antibiotics. We conclude that the H9724-reactive structures represent various forms of B. burgdorferi rather than staining artifacts. These "atypical" forms of B. burgdorferi may represent in vivo morphologic variants of this bacterium.

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p. 573
Studies with antibiotics revealed similar morphologic changes although the formation of granules of a much larger size (spheroblast-like structures) was obvious (fig. 5a).
Vibrio-like forms associated with granules were visualized in the epidermis (Fig. 3b) or short rods in perineural spaces.
Bizarre, heavily stained borreliae were visualized on serial sections (Fig. 9a-d), intracellularly in a macrophage (Fig 4b.), or in the epidermis (Fig. 3b). Large granules or spherical bodies ("gemmae") 1-3 µm were detected among collagen fibers (Fig. 5b) comparable to cysts arising after culture experiments (Fig. 5a).

p. 574
The morphological forms of borreliae seen in biopsies were correlated with clinical findings. Seropositive patients showed clumped and agglutinated borreliae in tissue (Fig. 4b), whereas seronegative patients exhibited borreliae colony formation (n=2) (Figs. 7b,8b). Neuralgies arising 6 months after ECM in spite of antibiotic therapy were evident in a seronegative patient who showed perineural rod-like borrelia structures. In ACA samples we identified agglutinated, intertwined spirochetal forms that resembled the clumped, dying borreliae seen in our culture experiments. In areas with inflammatory infiltrates, delicate dispersed, serpentine organisms were seen in degenerating collagen fibers. Also, small granular structures were evident among collagen fibers (Fig. 6b).
In biopsy sections from morphea patients, the number of borreliae was low. Yet, heavily stained intertwined forms and, in one case, clusters of delicate borreliae were seen within collagen fibers (Fig. 2d). Similarly, variant spirochetal forms were present in biopsies of three patients with plaque-like and papular eruptions arising at previous ECM sites. Histologically, two of these biopsies revealed epitheloid cell granulomas: skin sarcoidosis in one and cheilitis granulomatosa in the other patient. The third case represented lymphocytoma of the skin.
p. 576-78
The extracellular location of typical borreliae was not necessarily associated with the presence of an inflammatory infiltrate. Rather, these borrelia forms were seen in seronegative patients with uncomplicated ECM. If the extracellular borreliae were accompanied by an inflammatory infiltrate, the bacteria exhibited a heterogeneous morphology. Heavily stained, clumped, and aggregated borreliae and granules, formed by action of hyperimmune sera, were evident as were degererative changes in the connective tissue.
Whether borreliae can be located intracellularly has been heavily debated. In vitro studies demonstrated that borreliae actively penetrate endothelial cells (31) and fibroblasts, where they apparently evade eradication by antibiotics (32). The presence of borreliae in macrophages and keratinocytes, as shown in our studies and also in Berger's silver staining studies, supports the hitherto unproven concept that borreliae may survive intracellularly (33). Whether borreliae are also present in Langerhans cells could not be elucidated by this technique, although recent data suggest that borreliae invade and selectively damage them, as shown ultrastructurally (2).
The conditions necessary for the development of borrelia granules and their function are still unclear. The granules, which do not typically form under short-term culture conditions, do evolve in solid media (21), as well as in oral spirochetes (20,24), T. pallidum (23), and old acidic cultures of B. burgdorferi (34). Cystic borrelia forms have also been reported in tissue imprints of patients with Alzheimer disease (35). The development of spherical bodies or "gemmae" had been repeatedly observed in meticulous studies of spirochetal organisms over 40 years ago (20,23,24), as well as recently under varying culture conditions (26,36), in skin tissue (1,2), and after exposure to antibiotics (25,37).
Our results lead to several conclusions. First, the videomicroscopy technique described here has made possible the identification of borreliae in situ. Second, the behavior of borreliae within collagen fibers is strongly influenced by immune recognition by the patient. Borreliae may escape immune surveillance by colony formation and masking within collagen, resulting in seroregativity. Furthermore, the bacteria can survive in collagen fibers and cause tissue damage resulting in Iong-standing ACA, even in the presence of anti-Bb antibodies, which are known to kill B. burgdorferi (38). Third, whether the formation of granules or cysts represents a mode of degeneration of borreliae or their persistence is not yet clear. Nevertheless, simply knowing that B. burgdorferi are morphologically diverse may explain the large spectrum of Bb-associated diseases, may indicate a heterogenous immune responses in individuals, and may enhance future immunohistochemical studies of borreliae in animal models.

Selected References

1 Hulinska D, Jirous J, Valesova M, Herzogova J. Ultrastructure of Borrelia burgdorferi in tissues of patients with Lyme disease. J Basic Microbiol 1989; 29(2): 73-83

2 Hulinska D, Bartak P, Hercogova J, Hancil J, Basta J, Schramlova J. Electron microscopy of Langerhans cells and Borrelia burgdorferi in Lyme disease patients. Int J Med Microbiol Virol Parasitol Infect Dis 1994 Jan; 280(3): 348-59

20 Hampp EG, Scott DB, Wyckoff RWG. Morphologic characteristics of certain cultured strains of oral spirochetes and Treponema pallidum as revealed by the electron microscope. J Bacteriol 1948;56:755-69. Excerpt

21 Kurtti TJ, Munderloh UG, Johnson RC, Ahlstrand GG. Colony formation and morphology in Borrelia burgdorferi. J Clin Microbiol 1987 Nov; 25(11): 2054-8

23 Mudd S, Polevitzky K, Anderson TF. Bacterial morphology as shown by the electron microscope. J Bact 1943, 46:15-24

24 De Lamater ED, Haanes M, Wiggall RH, Pillsbury DM. Studies on the life cycle of spirochetes. J Invest Dermatol 1951; 16:231-56

25 Barbour AG, Todd WJ, Stoenner HG. Action of penicillin on Borrelia hermsii. Antimicrob Agents Chemother 1982 May; 21(5): 823-9

26 Barbour AG, Hayes SF. Biology of Borrelia species. Microbiol Rev 1986 Dec; 50(4): 381-400

32 Georgilis K, Peacocke M, Klempner MS. Fibroblasts protect the Lyme disease spirochete, Borrelia burgdorferi, from ceftriaxone in vitro. J Infect Dis 1992 Aug; 166(2): 440-4

33 Berger BW, Clemmensen OJ, Ackerman AB. Lyme disease is a spirochetosis. A review of the disease and evidence for its cause. Am J Dermatopathol 1983 Apr; 5(2): 111-24

34 Pillot J, Ryter A. Structure des spirochetes. 1. Etude des genres treponema, borrelia et leptospira au microscope electronique. Ann Inst Pasteur Paris 1965 Jun; 108(6): 791-804

35 MacDonald AB. Concurrent neocortical borreliosis and Alzheimer's disease. Demonstration of a Spirochetal Cyst Form. Ann N Y Acad Sci 1988:468-70

36 Hayes SF, Burgdorfer W, Barbour AG. Bacteriophage in the Ixodes dammini spirochete, etiological agent of Lyme disease. J Bacteriol 1983 Jun; 154(3): 1436-9

37 Kersten A, Poitschek C, Rauch S, Aberer E. Effects of penicillin, ceftriaxone, and doxycycline on morphology of Borrelia burgdorferi. Antimicrob Agents Chemother 1995 May; 39(5): 1127-33. PDF

38 Kochi SK, Johnson RC. Role of immunoglobulin G in killing of Borrelia burgdorferi by the classical complement pathway. Infect Immun 1988 Feb; 56(2): 314-21