SPHEROPLAST L-FORMS (PREAC MURSIC) part 2
JWissmille
Author: RitaStanEmail:rita...@aol.comDate:1996/09/13Forums:sci.med.diseases.l
A number of investigators later began extensive studies on the
ultrastructure of spirochaetes obtained from artificial culture media and
directly from pathological lesions (36,37).
In recent studies performed by transmission and scanning electron
microscopy, the membrane structure and the presence of extracellular
vesicles of Bb were discussed by several investigators (38-40).
Three-dimensional reconstruction of Borrelia cells by serial sectioning
and transmission electron microscopy is very difficult, if not impossible;
because a high portion of the sections shows tangentially sectioned
details (small cell diameter, helical winding); severely limiting
reconstruction. Scanning electron microscopic investigations by taking
stereo pairs clearly reveal that the cell profiles are still cylindrical.
Flattening by squashing of normally growing cells is not observed
indicating that freezing of the specimen is a rather gentle and
structurally preservative method. Three-dimensional imaging shows that
the cells are spindle shaped with maximum diameter of 300 nm. The helical
winding may change within one cell from clockwise to counter-clockwise.
After destruction of the outer bacterial membrane, bundles of flagellae
become clearly visible. The real rate of destruction of Borrelia cells as
well as the form and details of blebs can be interpreted only by scanning
electron microscopy.
The blebs (cyst) forms induced with penicillin G during growth of Bb in
MKP-medium are similar to encysted Spirochaeta duttoni demonstrated by
Swain (35). In our "encysted" form we have not seen evidence of borrelia
fragments, but we found "whole" or "intact" and "empty" forms as described
by Garon et al. (38). We could not directly compare our findings of
atypical forms in Bb strains with results of other investigators because
different conditions and organisms were tested. Nevertheless, we
speculate that these atypical Borrelia forms are spheroplast-L-phase forms
as can be found in other bacterial species. It is currently held that
conversion to L-form as well as formation of spheroplasts may be a
universl property of bacteria (41, 42). Pleomorphism, the presence of
elongated forms, the inability of cells to replicate, the induction of
exposing Bb to penicillin G, the long period of adaptation to growth in
MKP-medium and the mycoplsma-like colonies after growth on PMR-agar
suggest that Bb produce spheroplast-L-phase forms (SL-forms).
Penicillin G was the most effective inducer of SL-forms. The reversion of
this form to the helical parental form was mostly achieved by cultivation
of isolated SL-colonies in penicillin G-free medium. The atypical forms
isolated from patients treated with antibiotics show similar features.
The same effect is probably obtained with all other beta-lactam
antibiotics. Furthermore, atypical forms were also induced in patient
specimens contaminated with gram-positive as well as gram-negative
bacteria and by simultaneous growth of Bb and bacteria in MKP-medium.
Here the nonmotile "rigid" form was more stable, the reversion to the
helical parental form was often impossible; the organisms were unable to
replicate. A long period of growth adaptation in modified Kelly medium
was necessary to obtain an adequate concentration of viable borreliae.
This may be the reason for negative culture results in contaminated
patient specimens with small numbers of borreliae. Our data suggest that
bacterial toxins may also play a role in the overgrowth of Bb in
contaminated cultures. The release if toxins and enzymes during growth or
after cell lysis of fast growing gram-positive and gram-negative bacteria
can induce SL-forms in the slow growing Bb culture. Obviuously, the most
important question is what the actual role of these SL-forms may be. Some
recent studies with bacterial spheroplast-L-forms in vivo suggest a role
in pathogenicity and host-parasite interaction.
Very interesting are the studies by Hoyer and King who demonstrated the
loss of a portion of the chromosomal DNA in an L-form of Enterococcus
(43).
The pathogenicity of bacterial SL-forms and their sensitivity to
antimicrobial agents have been controversially discussed. However,
findings suggest that SL-forms may play a role in the microbial
persistence of various chronic infections (31-33).
The role of spheroplast-L-forms in LB has not yet been established because
the in vitro and in vivo studies are scarce. Preliminary data about
morphological changes of cells in vitro, and isolation of persisters with
atypical form from patient specimens after treatment with antibiotics (18,
20, 34) suggest that SL-forms may be involved in LB disease.
The efficacy of induction of SL-forms in our in vitro study varied greatly
among Bb strains. In some cultures only one colony of 20 was shown to
produce SL-transformation. This transformation probably reflects
phenotypic and genetic differences among different isolates.
The Bb persisters isolated from patient specimens and two induced SL-forms
tested showed no differences in the antibiotic sensitivity pattern. The
biological functions of the membrane vesicles remain unclear. The
demonstration of DNA in blebs by Garon et al. (38, 41) has led to the
hypothesis that these structures may play a role in the protection and
transfer of genetic markers. Shoberg et al. (40) proposed that Bb
vesicles may provide an important tool for elucidation of borreliae
adhesion antigens or structures. Radolf et al. (39) support the
hypothesis of Garon and co-workers. Very interesting are the results
concerning spheroplasts demonstrated by Bruck and co-workers and Kersten
et al. (44, 45). We support their hypothesis that with the outer membrane
damage and flagellar release, spheroplasts may provide a model that mimics
cells under attack by the host's immune system during infection.
In conclusion, the findings about atypical forms allow us to speculate
about the formation of spheroplast-L-forms in Bb.
Further in vitro and in vivo studies with respect to Bb protoplasts,
spheroplast-L-forms, biochemical and genetic composition, and the role in
LB disease are necessary. Of particular importance are their persistence
and significance for immune response and treatment of the disease. It
would be important to find different antibiotic groups or combinations in
respect to spheroplast-L-form production.
ACKNOWLEDGEMENTS
We are greateful to Cecilia Teufel and Ilka Durr for technical assistance.
We also thank Hans Nitschko for his critical review of the manuscript.
Prof. Vera Preac Mursic, Ph. D. Sylvia Reinhardt, Bettina Wilske, M.D., U.
Busch. Dipl., Max von Pettenkofer-Institut, Ludwig-Maximilians-Universitat
Munchen, Pettenkoferstr. 9a, D-80336 Munchen; G. Wanner, Ph. D.,
Botanisches Institut, Universitat Munchen, Menzinger Str. 67, D-80638
munchen; Prof. Dr. med. W. Marget, kinderklinik, Universitat Munchen,
Lindwurmstr. 4, D-80337 Munchen, Germany.
REFERENCES
1. Burgdorfer, W. et al.: Lyme disease - a tick-borne spirochetosis?
Science 216 (1982) 1317-1319.
2. Barbour, A.G. et al.: Heterogeneity of major proteins in Lyme disease
borreliae: a molecular analysis of North American and European isolates.
J. Infect. Dis. 152 (1985) 478-484.
3. Marconi, R.T., Garon, C.F.: Phylogenetic analysis of the genus
Borrelia: a coparison of N. American and European isolate of Bb.
J.Bacteriol. 174(1992) 241-244.
4. Wilske B. et al.: An OspA serotyping system for Bb based on reactivity
with monoclonal antibodies and OspA sequence analysis. J. Clin. Microbiol.
31 (1993) 340-350.
5. Wallich R. et al.: Evaluation of genetic divergence among Bb isolates
by use of OspA, fla, HSP60, and HSp70 gene probes. Infect. Immun. 60
(1992) 4856-4866.
6. Belfaiza, J. et al.: Genomic fingerprinting of Bb sensu lato by
pulsed-field gel electrophoresis. J. Clin. Microbiol. 31 (1993) 2873-2877.
7. Marconi, R.T., Garon, C.F.: Identification of a third genomic group of
Bb through signature nucleotide analysis and 16s rRNA sequence
determination. J. Gen. Microbiol. 138(1992) 533-536.
8. Baranton, G. etal.: Delineation with Bb sensu stricto, B garinii sp.
nov., and group VS461 associated with Lyme borreliosis. Int. J. Syst.
Bacteriol. 42 (1992) 378-383.
9. Johnson, R.C. et al.: Comparative in vitro and in vivo
susceptabilities of the Lyme disease spirochete Bb to cefuroxime and other
antimicrobial agents. Antimicrob. Agents Chemother. 34 (1990) 2133-2136.
10. Preac Mursic, V.: Antibiotic susceptability of Bb in vitro and in
vivo. In Weber, K. (eds): Aspects of Lyme borreliosis. Springer-Berlin,
Heidelberg, NY 1993, pp. 301-311.
11. Asch, E.S., et al.: Lyme disease: an infectious and postinfectious
syndrome. J. Rheumatol. 21 (1994) 454-461.
12. Dattsyler, R.J., Halperin, J.J.: Failure of tetracycline therapy in
early Lyme disease. Arthrits Rheum. 30 (1987) 448-450.
13. Pal G.S. et al.: Penicillin-resistent Borrelia encephalitis respondinf
to cefotaxime. Lancet 1 91988) 50-51.
14. Liegner, K.B. et al.: Recurrent erythema migrans despite extended
antibiotic treatment with minocycline ina patient with persisting Bb
infection. J. Am. Acad. Dermatol. 28 (1993) 312-314.
15. Hassler, D. et al.: Nodulare Panniculitis eine Verlaufsform der Lyme
Borreliose? Hautarzt 43 (1992) 134-138.
16. Haupl, T. et al.: Persistence of Bb in ligamentous tissue from a
patient with chronic Lyme borreliosis. Arthritis Rheum. 36 (1993)
1621-1626.
17. Schmidll, J. et al.: Cultivation of Bb from joint fluid three months
after treatment of facial palsy due to Lyme borreliosis. J. Infect. Dis.
158 (1988) 905-906.
18. Preac Mursic, V. et al.: Survival of Bb in antibiotically treated
patients with Lyme borreliosis. Infection 17 (1989) 355-359.
19. Stanek, G. et al.: Isolation of Bb from the myocardium of a patient
with longstanding cardiomyopathy. N. Engl. j. Med. 322 (1990) 249-252.
20. Prea Mursic, V. et al.: First isolation of Bb from an iris biopsy. J.
Clin Neuro. Ophthalmol. 13 (1993) 155-161.
21. Preac Mursic, V. et al.: Kill kinetics of Bb and bacterial findings
in relation to the treatment of Lyme borreliosis. Infetion 24 (1996) 9-16.
22. Preac Mursic V. et al.: European Bb isolated form humans and ticks:
culture conditions and antibiotic suseptibility. Zbl. Bakt. Hyg. A 263
(1986) 112-118.
23. Preac Mursic, V. et al: Persistence of Bb and histopathological
findings in human Lyme disease. Infection 18 (1990) 332-341.
24. Preac Mursic, V. et al.: Culture of Bb on six solid media. Eur. J.
Mcirobiol. Infect. dis. 10 (1991) 1076-1079.
25. Dienes, L., Bullivant, S.: Morphology and reproductive processes of
the L-forms of bacteria.II: comparative study of L-forms and mycoplasma
with the electron microscope. J. Bacteriol 95 (1968) 672-682.
26. Casjens, S., Huang, W.M.: Linear chromosomal physical and genetic map
of Bb, the Lyme diseas agent. Molec. Microbiol. (1993) 967-980.
27. Brenner, S. et al.: Bacterial protoplasts. Nature 181 (1958)
1713-1716.
Park, J.T.: Some observations on murein synthesis and the action of
penicillin G. Symp. Soc. Gen. Microbiol. 16(1966) 70.
29. Wittier, R.G.: Protoplasts, protoplasts, spheroplasts and L-forms.
Williams and Wilkins, Baltimore 1968, p. 200.
30. Lederberg, J.: Bacterial protoplasts induced by penicillin. Proc.
Natl. Acad. Sci. USA 42 (1957) 574.
31. Dolman, C.D., Two cases of a rat-bite fever due to Streptobacillus
moniliformis. Can J. Publ. Health 42 (1951) 228.
32. Nelson, E.L., Pickett, M.J.: The recovery of L-forms of Brucellia and
their relation to Brucella phage. J. Infect. dis. 89 (1951) 226.
33. Charache, P.: Atypical bacterial forms in human disease. In: Guze,
l.B. (ed): Microbial protoplasts, protoplasts, spheroplasts and L-forms.
Williams and Wilkins, Baltiore 1968, p. 484.
34. Barbour, A.G., Hayes, S.F.: Biology of Borrelia species. Microbiol.
Rev. 50 (1986) 381-400.
35. Swain, R.H.A.: Electron microscopic studies of the morphology of
pathogenic spirochaetes. J. Path. Bact. 69 (1955) 117-128.
36. Umemoto, T. Namikawa,I.: Electron microscopy of the spherical body of
oral spirochetes in vitro. Microbiol. Immunol. 4 (1980) 321-334.
37. Gebbers, J.O., Marder, H.P.: Unusual in vitro formation of cyst-like
structures associated with human intestinal spirochaetosis. Eur. J. Clin
Microbiol. Infect. Dis. 8 (1989) 302-306.
38. Garon, C.F. et al.: Structural features of Bb- the Lyme disease
spirochete: silver staining for nucleic cids. Scanning Microsc. (suppl. 3)
(1989) 109-115.
39. Radolf, J.D. et al.: Analysis of Bb membrane architecture by
freeze-fracture electron microscopy. J. Bacteriol. 176 (1994) 21-31.
40. Shoberg, R.J., Thomas, D.D.: Specific adherence of Bb extracellular
vesicles to human endothelial cells in culture. Infect. Immun. 61(1993)
3892-3900.
41. Dorward, D.W. , Garon, C.F.: DNA is packaged within membrane-derived
vesicles of gram-negative but not gram-positive bacteria. Appl. Environ.
Microbiol. 56 (1990) 1960-1962.
42. Madoff, S., Lawson, J.W.: The L-forms of bacteria. In Balows, A.
(ed): The prokaryots. Springer, NY 1992, p. 4068.
43. Hoyer, B.H., King, J.R.: Desoxyribonucleic acid sequence losses in a
stable streptococcal L-form. J. Bacteriol. 97 (1969) 1516-1517.
44. Bruck, D.K. et a.: Ultrastructural characteristics ofthe stages of
spheroplast preparation of Bb. J. Microbiol. Methods 23 (1995) 219-228.
45. Kerste, A. et al. Effects of penicillin, ceftriaxone and doxycycline
on morphlogy of Bb. Antimicrob. Agents Chemother. 39 (1995) 1127-1133.