A rapid method for generating cystic forms of Borrelia burgdorferi, and their
reversal to mobile spirochetes.
Brorson O, Brorson SH "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 of 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 of 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 sensitivity (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."