BRORSONs works on cystic forms of Borrelia burgdorferi

Transformation of cystic forms of Borrelia burgdorferi to normal, mobile spirochetes.

Brorson O, Brorson SH. Infection 1997 Jul-Aug; 25(4): 240-6.

The purpose of this study was to evaluate the behaviour of Borrelia burgdorferi under controlled conditions. The occurrence of cystic forms of Borrelia burgdorferi in vitro was noted, and these cysts were able to be transformed to normal, mobile spirochetes. B. burgdorferi was cultivated in a commercial culture medium without serum. The spirochetes multiplied only slowly in this medium, and transformation to encysted forms was observed after 1 week. When these cysts were transferred to the same culture medium with rabbit serum, the encysted forms developed into regular, mobile spirochetes after 6 weeks, and their regeneration time was normal. Examination of these cysts in the transmission electron microscope revealed transverse fission inside the cysts. It is probable that similar phenomena may occur in vivo under conditions unfavourable for spirochetes. These observations may help to explain why diagnosis and treatment of B. burgdorferi infections in humans can be difficult.

In vitro conversion of Borrelia burgdorferi to cystic forms in spinal fluid, and transformation to mobile spirochetes by incubation in BSK-H medium.

Brorson O, Brorson SH. Infection 1998 May-Jun;26(3):144-50.

The purpose of this study was to examine the structural alterations of Borrelia burgdorferi when exposed to spinal fluid. Normal, mobile spirochetes were inoculated into spinal fluid, and the spirochetes were converted to cysts (spheroplast L-forms) after 1-24 h. When these cystic forms were transferred to a rich BSK-H medium, the cysts were converted back to normal, mobile spirochetes after incubation for 9 to 17 days. The cultures were examined by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.

A rapid method for generating cystic forms of Borrelia burgdorferi, and their reversal to mobile spirochetes.

Brorson O, Brorson SH. APMIS 1998 Dec;106(12):1131-41.

Mobile Borrelia burgdorferi were transferred to distilled water (10(6) per ml). The cultures were observed by dark field microscopy (DFM), interference contrast microscopy (ICM) and transmission electron microscopy (TEM). 95% of the spirochetes were converted to cysts after 1 min, and after 4 h no normal mobile borreliae were observed. When transferred to growth medium (BSK-H), the cysts became smaller and more irregular, and were filled with organic substances. After 1 day, 1-5 thin structures sprouted from the cysts. They continued to grow in both length and thickness until they attained a normal spirochetal structure. Finally, these new-born spirochetes detached from the cysts, by which time their mobility had become normal. The present method for producing large amounts of cystic forms of B. burgdorferi is well suited for further studies of this unique microbe.

Excerpt from discussion section:
The cysts observed in our study seem to resemble the spheroplast-L-forms observed by other researchers (11, 35), which seem to have defects in their cell wall manifested by resistance towards beta-lactam antibiotics (16). Since the conversion from normal mobile spirochetes to cystic forms occurred very rapidly, complete absence of cell wall as in L-forms is dubious, but the similarity with L-forms is shown by the inability to retain their original shape.
The biological activity of the cystic forms was confirmed by the step by step development to normal mobile spirochetes in BSK-H medium, and also indicated by the presence or RNA in 5-week-old cysts due to red-orange staining with acridine orange (pH 6.4) (Fig. 4b). This seems to be a new observation in the development of B. burgdorferi (20). Bruck et al. (35) also illustrated biological activity by incorporation or S-methionine in the cysts (spheroplast). The creation of as many as five spirochetes from each cyst may explain why the generation time was shorter for production or mobile spirochetes from cysts compared to that for normal mobile spirochetes cultivated conventionally. The generation of the normal mobile spirochetes which were converted from cysts was somewhat variable in the sense that they sometimes seemed to need a long stationary period before exponential growth occurred. Whether cysts are converted to normal mobile spirochetes or not depends strongly on the growth medium used, and possibly also the generation time.
It seems as though normal mobile spirochetes are developed from the dense core structures or the cyst by being "fed" with core substances as the "infant-spirochete" protrudes from the cyst. The development of vegetative bacteria from dense L-forms has been suggested as a method for Enterococcus faecalis to convert from L-forms to vegetative forms (40). The observation by TEM that blebs transformed into thin filaments leads us to speculate that these filaments develop to normal mobile spirochetes. If so, the blebs have to contain enough genetic material to synthesize a new bacterium (22).
Old cystic forms of B. burgdorferi require prolonged cultivation to convert to normal mobile spirochetes (4 weeks as opposed to 9 days for young cysts). Similar cystic forms may occur in the human organism (11, 14, 15), and they may explain the long periods or latency, resistance to antibiotics, negative serological results (3-7, 10, 12, 13, 25), and low PCR sensitivitv (5, 8, 10). For these reasons it is important to examine the antigens of the envelope of the cysts. DNA sequences for PCR analysis, and the cysts sensitivity to antibiotics and other chemicals. It may be hypothesized that antigenic variation in B. burgdorferi (19, 41) occurs inside the cyst while the microbe is protected against external stress, and therefore antigens detected on the cyst envelope in vitro differ from the ones present in vivo. In conclusion, we believe that the present method for rapid and easy generation of stable cysts will be a useful tool in further research on B. burgdorferi.

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole.

Brorson O, Brorson SH. APMIS 1999 Jun;107(6):566-76

The aim of this study was to examine the susceptibility of mobile and cystic forms of Borrelia burgdorferi to metronidazole. Because B. burgdorferi is a microaerobic bacterium like Helicobacter pylori, metronidazole (MZ) was chosen in the susceptibility test. For both microaerobic and aerobic incubation the normal mobile spirochetes were resistant to this antibiotic with an MBC > or = 512 microg/ml. Conversion of mobile spirochetes to cystic forms was not observed when they were incubated with MZ. When they were incubated under microaerobic conditions, the biologically active cystic forms had an MBC > or = 4 microg/ml, but the MBC was > or = 32 microg/ml with aerobic incubation at 37 degrees C. Staining with acridine orange (AO), dark field microscopy (DFM), and transmission electron microscopy (TEM) revealed that the contents of the cysts were degraded when the concentration of MZ was > or = MBC. Some cysts were also ruptured. When incubated with a sufficient concentration of MZ, core structures did not develop inside the cysts, and AO revealed less RNA in the cysts. Our observations may help efforts to treat resistant infections caused by B. burgdorferi with a combination of MZ and other antibiotics in order to eradicate both cystic and mobile forms of B. burgdorferi.

Association between multiple sclerosis and cystic structures in cerebrospinal fluid.

Brorson O, Brorson SH, Henriksen TH, Skogen PR, Schoyen R. Infection. 2001 Dec;29(6):315-9.

BACKGROUND: The aim of the study was to search for infectious agents in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS).

PATIENTS AND METHODS: CSF from ten patients with the diagnosis relapsing remitting MS and from five controls without MS were examined by transmission electron microscopy (TEM), dark field microscopy (DF), interference contrast microscopy (ICM) and UV-microscopic examination of acridine orange staining (AO). All CSF samples from patients and controls were cultured.

RESULTS: Cystic structures were observed in CSF of all ten patients by AO and TEM. DF revealed eight cyst-positive patients out of nine. One of five control persons had such structures in the CSF; this person had suffered from erythema migrans. Spirochete or rod-like structures emerged after culturing two of the MS patient CSF samples and these structures could be propagated.

CONCLUSION: A significant association of CSF cysts and MS was identified in this small study among residents in a coastal area of southern Norway. The cysts could be of spirochetal origin. Our study may encourage other researchers to study larger patient groups.

An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine.

Brorson O, Brorson SH. Int Microbiol 2002 Mar;5(1):25-31

In this work the susceptibility of mobile and cystic forms of Borrelia burgdorferi to hydroxychloroquine (HCQ) was studied. The minimal bactericidal concentration (MBC) of HCQ against the mobile spirochetes was > 32 microg/ml at 37 degrees C, and > 128 microg/ml at 30 degrees C. Incubation with HCQ significantly reduced the conversion of mobile spirochetes to cystic forms. When incubated at 37 degrees C, the MBC for young biologically active cysts (1-day old) was > 8 microg/ml, but it was > 32 microg/ml for old cysts (1-week old). Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that the contents of the cysts were partly degraded when the concentration of HCQ was > or = MBC. At high concentrations of HCQ (256 microg/ml) about 95% of the cysts were ruptured. When the concentration of HCQ was > or = MBC, core structures did not develop inside the cysts, and the amount of RNA in these cysts decreased significantly. Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ was > or = MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.

Spirochetal structures inside the cysts dissolved in the presence of high concentrations of HCQ. When the concentration of HCQ was > or = MBC, the core structures inside the cysts were eliminated. These observations may be valuable in the treatment of resistant infections caused by B. burgdorferi, and suggest that a combination of HCQ and a macrolide antibiotic could eradicate both cystic and mobile forms of B. burgdorferi.