jwissmille

from: Formation and Cultivation of Borrelia Burgdorferi Spheroplast L-Form
Variants
authors: V. Preac Mursic, et al
source: Infection 24(1996); 218-226

"...We found Bb forms in four biological variations in cultures: 1) cells
forms tending to regain formal organism status in first/second subculture and
growing in MKP-medium as well as on PMR-agar; 2) cell forms reverting to
nermal helical form of the organism after subculturing in MKP + AB (10p.c.
albumin bovine) and growing on PMR-agar; 3) cell forms of most organisms
reverting to normal (viable) after numerous subcultures in MKP + AB; THE
COLONIES MAY GROW TO RESEMBLE MYCOPLASMA; 4) cell forms with no reversion to
normal helical cells, no replication, and no growth on PMR-agar."

Taber's Medical Dictionary-"Mycoplasma- A group of bacteria that lack cell
walls and are highly pleomorphic. There are more than 70 organisms in this
group, including 12 species that infect humans.."

"pleomorphic-Having many shapes."

{REPOST: PT 1:SPHEROPLAST L-FORMS (PREAC MURSIC) }
typed by Rita Stanley
Date:1996/10/04
Forums:sci.med.diseases.lyme

FORMATION AND CULTIVATION OF BORRELIA
BURGDORFERI SPHEROPLAST-L-FORM VARIANTS

V. Preasc Mursic, G. Wanner, S. Reinhardt,
U. Busch, W. Maget

Infection 24(1996); 218-226.

SUMMARY: A clinical persistence of Borrelia burgdorferi in patients with
active Lyme borreliosis occurs despite obviously adequate antibiotic
therapy. In vitro investigations of morphological variants and atypical
forms of B. burgdorferi were undertaken. In an attempt to learn more
about the variation of B. burgdorferi and the role of atypical forms in
Lyme borreliosis, borreliae isolated from antibiotically treated and
untreated patients with the clinical diagnosis of definite and probable
Lyme borreliosis and from patient specimens contaminated with bacteria
were investigated. Furthermore, the degeneration of the isolates during
exposure to penicillin G in vitro was analyzed. Morphological analysis by
darkfield microscopy and scanning electron microscopy revealed diverse
alterations. Persistors isolated from a great number of patients (60-80%)
after treatment with antibiotics had an atypical form. The morphological
alterations in culture with penicillin G developed gradually and increased
with duration of incubation. Pleomorphism, the presence of elongated
forms and spherical structures, the inability of cells to replicate, the
long period of adaptation to growth in MKP-medium and the mycoplasma-like
colonies after growth in solid medium (PMR agar) suggest that B.
burgdorferi produce spheroplast-L-form variants. With regard to the
polyphase course of Lyme borreliosis, these forms without cell walls can
be a possible reason why Borrelia survive in the organism for a long time
and the cell-wall-dependent antibody titers disappear and emerge after
reversion.

INTRODUCTION: The etiologic agent of Lyme disease, Borrelia burgdorferi
(1) has been isolated by culture from ticks of the I. ricinus complex, and
from animals and humans. Immunological and molecular heterogeneity were
demonstrated amongst isolates from the USA and Europe (2-5). On the basis
of DNA homology, RNA-sequence analysis and r-RNA gene restriction
profiles, the isolates were recently classified into three genospecies: B
burgdorferi sensu strictu, B. garinii and B. afzelii (6-8). Lyme disease,
the most widespread disease transmitted by ticks, is characterized by
various clinical stages, including dermatologic, neurologic, cardiac, and
ocular manifestations.

Penicillin G, amoxicillin, doxycycline, the macrolides erythromycin,
azithromycin, and the cephalosporins cefotaxime, ceftriaxone, ceftazidime
and cefuroxime have been applied to prevent progressive clinical
manifestation of Lyme borreliosis. The results of comparative in vitro
and in vivo susceptibility studies of B. burgdorferi indicate efficacy of
several antibiotics including doxycycline, amoxicillin, macrolides and\0
cephalosporins (9-10). However, some patients develop late symptoms
despite apparently adequate antibiotic treatment (11-15). The persistence
of Bb even after therapy with antibiotics has been demonstrated in
cerebrospinal fluid (CSF), in skin, iris, heart and joint biopsies
(16-21).

The aim of the present study was to investigate cytomorphic variations of
Bb isolates from patients with and without antibiotic treatment.

MATERIALS AND METHODS: The Bb strains used in this study were isolated
from human CSF, skin biopsies and blood culture in MKP-medium after
incubation at 33 degrees C for 2-15 weeks as previously described (22).
The identification of isolates was performed by SDS-PAGE,
immunofluoresence and Western blot with OspA-specific monoclonal
antibodies (4).

1. The phenotypic and genotypic variations of Bb after serial in vitro
passages in MKP-medium were investigated by darkfield microscopy, by
SDS-PAGE and pulsed-field gel electrophoresis (PFGE), using strains PKo
(5th and 303rd passage) and PBi (5th and 285th passage). The virulence
and the loss of infectivity were investigated in gerbils as an animal
model. The infectivity of these strains at low passage was deomonstrated
previously (23).

2. The effect of antibiotics on the morphological form of isolates was
examined by darkfield and scanning electron microscopy during 6 days of
cultivation of Bb strains (low passage) in MKP-medium with penicillin G (3
mg/l). The methods of cultivation and in vitro and in vivo susceptibility
procedures were described elsewhere (10).

3. The degeneration of 18 Bb persistors, isolated from patients with
definite and probable Lyme borreliosis after antibiotic therapy, was
investigated by darkfield (18) and scanning electron microscopy (3) after
growth in MKP-medium. We also investigated 20 isolates from untreated
patients by darkfield microscopy (20) and by scanning electron microscopy
(3). Case histories will be taken into consideration in a later
publication.

4. The cytomorphic change from atypical nonmotile, "rigid" form to motile
helical form of Bb was studied on specimens (2 CSF, 2 skin biopsies) from
four patients primarily contaminated with bacteria. To eliminate
bacterial contamination, cultures were filtered by 0.45 micometer filter;
the recoverable borreliae were subcultivated in MKP-medium at 33 degrees C
and passaged repeatedly until motility. The origin of the "rigid" form
was studied in vitro using an experimentally contaminated PKo strain.
Cultures of this PKo strain (used as control strain) were contaminated
with Acinetobacter sp. (1000/ml) and cultured in MKP-medium until the
"rigid" form was observed. Cultures of "rigid" forms were manipulated as
described above for patient strains. Darkfield microscopy and scanning
electron microscopy were used to investigate the motility, morphology and
structure of Bb cells.

The cell concentration of Bb was determined by total counts and by viable
counts using counting chamber and darkfield microscopy (22). cultures
were rountinely monitored for bacterial contamination. Specimens were
prepared from 2-, 4- and 6-day cultures. Drops of culture medium were
either placed on slides for darkfield microscopy or prepared for scanning
electron microscopy. For colony morphology analysis, cultures on PMR-agar
were done on the basis of information presented in a previous study (24).
Subcultures from agar to agar were made by loop inoculation and by the
"push block" technique used for mycoplasma. Dienes staining technique was
used for staining agar colonies (25).
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NOTE: SEE ORIGINAL FOR REST OF M & M, PHOTOS AND RESULTS
---------------------------------------
DISCUSSION: The role of different atypical bacterial forms, spheroplasts
and L-forms in infectious disease is considered an important problem in
microbiology. spheroplasts and growth into L-forms occur in many
bacterial species in liquid and on various solid media. These forms are
products of partial or complete removal of the cell wall by enzymatic
digestion (27) and partial or incomplete inhibition of cell wall synthesis
(28). The morphology and reproductive processes of bacteria with
defective cell walls was studied extensively by numerous investigators
(29, 30, 25).

Many factors have been shown to induce the formation of these fragile
bacterial forms in vitro: inadequate culture media, alteration of pH,
enzymatic activity (lysozymes), antibodies and complement as well as
antibiotics. The morphology of bacteria, the mode of multiplication and
the structure of colonies may be altered.

An important discovery was that L-forms can be induced in vivo by
treatment with penicillins and that microbial variants may persist in vivo
and may thereby contribute to the establishment of subclinical or chronic
infection. The recovery of spheroplasts, L-forms and other bacterial
variants was reported from blood, body fluid and tissues of humans and
animals. Dolman et al. (31) described the isolation of the L-form of
Streptobacillus moniliformis fromt he blood of one patient with rat bite
fever.

The recurrence of endocarditis and the persistence of L-forms of
Corynebacterium sp. was reproted by Wittler et al. (29): the persistent
and antibiotic resistant L-form was asociated with the latent stage of
infection. Furthermore, the chronicity of Brucella infection may be due to
Brucella L-forms (32). Demonstration of atypical forms of Haemophilus
influenzae, Listera sp., Streptococcus sp., Staphylococcus sp., E. coli,
Klebsiella sp. in human disease was made by Charache (33).

About atypical forms of Bb and their role in Lyme borreliosis infection
there is scarcely any information. As persistence of Bb and recurrence of
the disease occur despite adequate antibiotic therapy, in vitro
investigations of Bb morphological variants and atypical forms were
undertaken by the present authors.

We found Bb forms in four biological variations in cultures: 1) cell
forms tending to regain formal organism status in first/second subculture
and growing in MKP-medium as well as on PMR-agar; 2) cell forms reverting
to normal helical form of the organism after subculturing in MKP + AB (10
p.c albumin bovine) and growing on PMR-agar; 3) cell forms of most
organisms reverting to normal (viable) after numerous subcultures in MKP +
AB; the colonies may grow to resemble mycoplasma; 4) cell forms with no
reversion to normal helical cells, no replication, and no growth on
PMR-agar.

In older cultures we found mostly type 1 and 2 cells, in patients treated
with antibiotics type 2 and 3, and after growth of cells in the presence
of penicillin G in vitro we found type 1, 2 and 3. In contaminated
patient specimens and in the experimentally contaminated strain PKo
incubated with Acinetobacter sp. we found type 3 and 4. Furthermore, the
colony morphology on PMR-agar varied. We obtained smooth, rough,
rough-diffused and spherical-granular forms.

Very interesting are the membrane blebs which have been observed and
described earlier by Barbour and Hayes (34) and Preac Mursic et al. (22).
Many years ago, Swain (35) demonstrated the encysted form of pathogenic
spirochetes by electron microscopy. Transmission electron microscopic
(TEM) studies showed the presence of large bubbles (1.0-1.6 micrometers)
and encysted forms of leptospirae and borreliae. Encysted forms were
occasionally adherent to the end of the middle of the organism or were
separated (Leptospira).

SPHEROPLAST L-FORMS (PREAC MURSIC) part 2

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 preervative 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.

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