Effects_of_Penicillin_Ceftriaxone_and_Doxycycline_on_Morphology_of_Borrelia_brgdorefri_KERSTEN.pdf

A NTIMICROBIAL A GENTS AND C HEMOTHERAPY , May 1995, p. 1127–1133

Vol. 39, No. 5

0066-4804/95/$04.0010

Effects of Penicillin, Ceftriaxone, and Doxycycline on

Morphology of Borrelia burgdorferi

A. KERSTEN,† C. POITSCHEK, S. RAUCH, AND E. ABERER*

Department of Dermatology, Division of Immunology, Allergy and Infectious Diseases,

University of Vienna, Vienna, Austria

Received 11 October 1994/Returned for modiﬁcation 7 December 1994/Accepted 27 February 1995

Antibiotic therapy with penicillin, doxycycline, and ceftriaxone has proven to be effective for the treatment

of Lyme borreliosis. In some patients, however, it was noticed that borreliae can survive in the tissues in spite

of seemingly adequate therapy. For a better understanding of this phenomenon, we investigated the different

modes of degeneration of Borrelia burgdorferi suspensions during a 96-h exposure to various antibiotics. By

dark-ﬁeld microscopy and ultrastructural investigations, increasing blebbing and the gradual formation of

granularandcysticstructurescouldbefollowedduringtheexposuretime.Althoughantibioticconcentrations

attheMICatwhich90%oforganismsareinhibitedafter72hwere80%orevengreater,motileorganismswere

still present after incubation with penicillin and doxycycline but not after incubation with ceftriaxone. By

transmissionelectronmicroscopy,intactspirochetalparts,mostlysituatedincysts,wereseenupto96hafter

exposure with all three antibiotics tested. According to experiences from studies with other spirochetes it is

suggested that encysted borreliae, granules, and the remaining blebs might be responsible for the ongoing

antigenic stimulus leading to complaints of chronic Lyme borreliosis.

Borrelia burgdorferi , the pathogenic agent of Lyme borrelio-

sis, has been recognized as a bacterium that is susceptible to

antibiotics. The long-term persistence of these bacteria in tis-

sues,despiteadequatetreatmentofinfectedpatients,hasbeen

indicatedtoberesponsibleforlatecomplicationsandachronic

course of disease (17, 26, 28). The withdrawal of borreliae, or

partsofthem,intoprivilegedorsecludedsites,wheretheyare

further inaccessible to antibiotics, raises the question of

whether antibiotics themselves can be made responsible for

transforming the organism into a persistent, viable, or nonvi-

able but antigenically potent form.

Immobilization of bacteria has been seen as a result of

incubation with antibiotics in vitro (21). By light microscopy,

Preac-Mursic et al. (29) observed blebs, spherical structures,

and granules in B. burgdorferi cultures during incubation with

antibiotics.Inultrastrucutralstudiestheactionofpenicillinon

cultures of Borrelia hermsii has been investigated (3).

Penicillin, doxycycline, and ceftriaxone are the most pre-

ferred antibiotics for treating Lyme borreliosis. Although cer-

tain differences in the actions of these antimicrobial agents

have been evaluated, they seem to be equally effective (34).

Culture experiments were carried out to investigate the ac-

tions of these antibiotics on the motility, morphology, and

survival of B. burgdorferi during exposure to antibiotic solu-

tions. The course and mode of degeneration of these spiro-

chetes were recorded on a videomicroscope and were photo-

graphed with an electron microscope.

polyacrylamide gel electrophoresis (PAGE) and transfer to nitrocellulose by

using 22 different OspC-speciﬁc monoclonal antibodies (20). The heterogeneity

of OspC was further studied by analyzing the restriction fragment length poly-

morphismsamongPCR-ampliﬁedOspCgenes(20).Thestrainswereculturedin

BSK medium at 34

C over a period of 4 days (30).

Tubes of media containing the three drugs at 0.1-, 0.5-, 1.0-, and 2-fold the

MICs at which 90% of strains are inhibited (MIC 90 s) were used. Penicillin G

(MIC 90 ,4.0

g/ml;Biochemie,Vienna,Austria),ceftriaxone(MIC 90 ,0.06

g/ml;

Hoffmann-La Roche, Basel, Switzerland), and doxycycline (MIC 90 , 2.0

g/ml;

Pﬁzer, New York, N.Y.) (27) were inoculated with 4-day-old cultures of B.

burgdorferi to a ﬁnal cell density of 10 6 /ml, as determined in a Petroff-Hausser

countingchamber,andweresealedwithplasticcaps.Theywerekeptat34

Cfor

96 h. Before examination they were gently mixed on a vortex mixer.

To avoid contamination during the observation period the tubes were opened

only once for investigations and were then discarded.

For control purposes, B. burgdorferi cultures were also transferred to medium

alone. B. burgdorferi cultures subjected to antibiotics and nonexposed cultures

were transferred to BSK medium after 96 h for subculture.

Dark-ﬁeldmicroscopy. Samplesof B.burgdorferi B31,H1,andH8inantibiotic

solutions at each antibiotic concentration were transferred to glass slides. The

movements and structures of the borreliae were examined by dark-ﬁeld micros-

copy in a Leitz Diaplan microscope connected to a Philips monitor.

Preparationforelectronmicroscopy. Electronmicroscopywasperformedwith

B.burgdorferi B31culturesexposedtotheantibioticsattheMIC 90 sfor24and96

h. Samples were centrifuged at 8,000

C. Negative-contrast

staining was done by placing 300 copper mesh grids (Balzers Union, Balzers,

F¨rstentum Liechtenstein) on the suspensions for 5 min (23). Fixation was

performed with 2.5% glutaraldehyde for 15 min; this was followed by washing

steps with distilled water and counterstaining with 1% aqueous uranyl acetate

dihydratefor30s.Fortransmissionelectronmicroscopy(TEM),thepelletswere

ﬁxed with 2% paraformaldehyde–2% glutaraldehyde dissolved in 0.1 M cacody-

latebuffer–0.1mMCaCl 2 (pH7.4)for1hat48C,rinsed,andincubatedwith1%

OsO 4 in 0.1 M buffer at room temperature for 1 h. After the pellets had again

beenrinsedinbufferanddistilledwater,theywereincubatedwithdimethoxypro-

pan (Merck, Darmstadt, Germany) and one droplet of concentrated sodium

chloride per 10 ml twice for 10 min each time. These pellets were embedded in

Spurrresin(Serva,Heidelberg,Germany)for1hatroomtemperatureandwere

allowed to polymerize for 12 h at 70

g for 20 min at 4

MATERIALS AND METHODS

Culture experiments. Three strains of B. burgdorferi sensu lato, B31 (ATCC

35219)andtwoisolatesfromerythemamigranslesionsontheskinofpatientsin

Vienna, isolates high-passage H1 and low-passage H8, were used for the exper-

iments.IsolateH1wasidentiﬁedas Borreliaafzelii ,andisolateH8wasidentiﬁed

as Borrelia garinii by Aurodye staining after sodium dodecyl sulfate (SDS)-

C. The preparations were cut into ultrathin

sections (Ultracut; Reichert), applied on 200 copper mesh grids, counterstained

with 1% uranyl acetate dihydrate in methanol, and examined with an electron

microscope (Jeol EM 100 S).

SDS-PAGE analysis. The proteins of B. burgdorferi B31 cultures were charac-

terizedbySDS-PAGEandwerecomparedwithborreliaculturesafter24and96

h of exposure to penicillin G, ceftriaxone, and doxycycline at the MIC 90 s. After

being washed twice in phosphate-buffered saline solution with 5 mM MgCl 2 ,

spirocheteswereboiledfor5minandweretestedonaverticalgel(Miniprotean

II; Bio-Rad). Gels were stained with Coomassie brillant blue R 350, and the

major proteins were compared with molecular weight standards (Bethesda Re-

*Correspondingauthor.Mailingaddress:Waehringerguertel18-20,

A-1090 Vienna, Austria. Phone: 43/1/40400/7704. Fax: 43/1/4031900.

†Present address: Institute of Pathology, University of Freiburg,

D-79104 Freiburg, Germany.

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KERSTEN ET AL.

A NTIMICROB .A GENTS C HEMOTHER .

searchLaboratories,Inc.,Gaithersburg,Md.).Quantiﬁcationoftheproteinswas

achieved by computerized analysis.

Determinationofthestabilitiesoftheantibiotics. Toinvestigatethestabilities

oftheantibioticsundercultureconditions,sampleswerepreparedandincubated

in the same way as those for morphological investigations. Time points for

samplingwereat0,12,24,48,and72h.Thesamplesweretakeninduplicatefor

penicillin G, doxycycline, and ceftriaxone. The samples were stored at

until processing. Quantitative analysis was performed by high-pressure liquid

chromatography (HPLC) method. Aliquots of 0.3 ml of B. burgdorferi B31 sus-

pensionsinBSKIImediumweretakenaftervortexing.Proteinprecipitationwas

performed by adding 0.3 ml of acetonitrile and subsequent vortexing and cen-

trifugation.

For determination of the penicillin G concentration in B. burgdorferi B31

suspensions, 10

l of the clear supernatant was loaded onto the HPLC column

(an HP 1090 M chromatograph [Hewlett-Packard] with a variable UV detector

3100[LDC];wavelength,210nm;column,nucleosil1203C 18 [80by4mm,inner

diameter];mobilephase,25%acetonitrilewith75%[vol/vol]0.02Mphosphoric

acid;retentiontime,approximately3.1min).Validationofthemethoddescribed

above gave linearity in the range of 0 to 0.6

g/ml of suspension. The recovery

rate was 91 to 100%, with a coefﬁcient of variation of 0.8 to 4.8%; accuracy was

between

2.9% for the range tested.

For doxycycline concentration determinations, 200

2.2 and

l of a 1:3 dilution with

water of the clear supernatant described above was loaded onto the column

(nucleosil 100 5CN [125 by 4 mm, inner diameter]; wavelength, 350 nm; mobile

phase, 10% acetonitrile and 10% tetrahydrofuran in 80% [vol/vol] phosphate-

citrate buffer; retention time, approximately 2.1 min). The linearity of the

method was in the range of 0 to 4.1

FIG. 2. Shedding of membrane blebs is the initial morphological character-

istic of membrane alteration. Blebs tend to aggregate to pearls or tubules, as

shown by TEM after negative-contrast staining. Bar, 0.1

g/ml of suspension, with a recording rate

of between 79 and 89% (coefﬁcient of variation, 1.3 to 3.9%), the accuracy for

the range tested was 0.8 to 14.5%.

Because of the very low MIC 90 s of ceftriaxone and interference with the

culture medium, no reliable analysis method could be developed for this antibi-

otic.

development of granules, the motility of the spirochetes ap-

peared to be accelerated, reminiscent of ‘‘cramping’’ or ‘‘teta-

nia,’’ resulting in the aggregation of single or multiple organ-

isms to amorphous clusters with residual slow motility.

Formation of small colonies undergoing degeneration was ob-

served after 48 to 72 h of incubation. The motilities of altered

organisms then consecutively decreased. Singule borrelia or

immotileborreliaassociatedwith0.8-mmsphericalbodiesor2-

to 3-mm short rods, both with prominent self propelling, ap-

peared in the medium after 72 h. After 4 days of incubation

with antibiotics at concentrations greater than the MIC 90 s,

immotile aggregates or immotile granula- or vesicle-bearing

organisms were predominantly present. At less than the

MIC 90 s higher numbers of motile and morphologically intact

spirochetes were present.

The alterations in the B. burgdorferi organisms incubated

withceftriaxonewereidenticaltothoseinorganismsincubated

with penicillin. However, the onset of the alterations was al-

RESULTS

Dark-ﬁeld microscopy. There were no obvious morpholog-

icaldifferencesbetweenthethreestrainsanalyzed,strainsB31,

H1, and H8. The degree of alteration was strongly dose and

time dependent. After exposure to penicillin a few individual

motile B. burgdorferi organisms could be detected at any time

ofthe4-dayobservationperiod.Themorphologicalalterations

developedgradually;initially,after17hofincubation,granules

of up to 0.8 mm adhering to the end and/or middle regions of

the spirochetes developed in cultures incubated with concen-

trationsattheMIC 90 orgreater.Theirnumbersincreasedwith

the time of incubation, and they formed paired as well as

multiple granules after 24 h of incubation. In incubations with

concentrationslessthantheMIC 90 ,thesegranulesaroseabout

12 h later and at a lower percentage. After 48 h of incubation

with 1.0 or 2.0 times the MIC 90 , these granules were trans-

formed into up to 1.8-

m vesicle-like structures. During the

FIG. 3. Several granules along borreliae after 24 h of incubation with ceftri-

axone at the middle and end regions, as shown by TEM following negative-

contrast staining. Bar, 1.4

FIG. 1. Twomembraneblebsariseattheendofaborreliaorganisminduced

by penicillin after 24 h of incubation, as shown by TEM. Bar, 0.2

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EFFECTS OF ANTIBIOTICS ON BORRELIA MORPHOLOGY

1129

FIG. 6. Formation of vesicles by ceftriaxone after 24 h. The vesicle contains disar-

ranged ﬂagellae and residual protoplasmic cylinder, as shown by TEM. Bar, 0.6 mm.

cubationatconcentrationsgreaterthanMIC 90 ;after24hthere

was a loss of motility without marked morphological alter-

ations. After 4 days of incubation 90% of the bacteria were

immotile. In cultures grown in the presence of concentrations

FIG. 4. Formation of aggregated granules and vesicles of different sizes (0.4

to 1.4

m) on borreliae after exposure to penicillin for 96 h is seen. The large

vesiclecontainslooseloopsofﬂagellaeandaconvolutedspirochete,asshownby

TEM following negative-contrast staining. Bar, 0.6

ready observed after8hofincubation. The number of viable

bacteria could not be evaluated in penicillin- and ceftriaxone-

containing media because of the aggregation of motile organ-

isms and the formation of microcolonies. After 48 h no motile

borreliae were present even in the presence of concentrations

as low as 1/10 the MIC 90 , but self-propelled rods or granules

were evident, and these were sometimes associated with dead

borrelia.

In contrast, doxycycline-treated cultures revealed single or-

ganisms with gradually decreasing motilities after 18 h of in-

FIG. 5. B. burgdorferi exposed to peniciilin for 24 h. A vesicle is shown by

TEM to adhere to the outer surface of a spirochete. The outer membrane

encloses amorphous material. Bar, 0.4

FIG. 7. Cross-sectioned spherical body revealing intracellular localized,

coiled spirochetal parts. Typical unfolding of the two central cross-sectioned

cytoplasmic cylinders is shown by TEM. Bar, 0.2

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KERSTEN ET AL.

A NTIMICROB .A GENTS C HEMOTHER .

FIG. 10. Bazilla-like mesosome shown by TEM in doxycycline-incubated

borrelia culture suspensions after 4 days. Bar, 0.25

showed regular motility. In subcultures intact borreliae were

seen.

Electron microscopy. In organisms in all culture prepara-

tions exposed to penicillin and ceftriaxone, increased blebbing

was obvious after 24 h of incubation (Fig. 1). Membrane blebs

of0.05to0.2

FIG. 8. Spherical body developing after 96 h of incubation in cultures ex-

posed to doxycycline, as shown by TEM. Electron-dense material is enclosed by

an outer membrane identical to the spirochetal membrane. Bar, 0.2 mm.

mproducedbytheouterenvelopeappearedto

be either adherent to the outer surface of the spirochete or

shed off into the culture medium. Negative staining illustrated

their tendency for self-aggregation, forming pearls or even

tubules (Fig. 2). The underlying spirochetal surface was not

altered.Negativestainingrevealedgranulesandlarger,vesicle-

like structures (gemmae) ranging in size from 0.5 to 1.8

less than the MIC 90 , the proportion of motile spirochetes was

25%. Morphological alterations similar to those induced by

penicillin or ceftriaxone developed only occasionally after 4

days of incubation. Spherical bodies of 0.8 to 1.4 mm were

found free in the culture after 18 h of incubation in all culture

experiments.

Subcultures from all investigated samples containing antibi-

otics carried out on day 4 and examined by dark-ﬁeld micros-

copy once weekly for up to 2 weeks only revealed small gran-

ules and self-moving rods but no borrelia organisms with

regular morphologies.

Controls. During the period of incubation, the organisms in

untreated cultures remained morphologically unaltered and

(Fig.3and4).AsshownbyTEM,gemmaeweresurroundedby

anoutermembraneenclosingeitheramorphousmaterial(Fig.

5) or residual, coiled, intact, or unfolded protoplasmic cylin-

ders and disarranged ﬂagellae (Fig. 4, 6, and 7). In addition to

the structures described above, 0.8-

m solitary spherical bod-

iescomposedofanoutermembraneandelectron-denseinter-

nal material could be shown after 24 to 96 h. By TEM, a

ribosomalpatternofthecytoplasmiccylindersuggestedvitality

(Fig. 8). The majority of spherical bodies, however, showed

lysis after 96 h; the whole intracellular contents were released

FIG. 9. Ultrathinsectionof B.burgdorferi incubatedwithpenicillinfor4days,

as shown by TEM. Lysis of cells with residual membranes and empty protoplas-

mic cylinders and numerous membrane blebs are shown. Bar, 0.8

FIG. 11. Two cytoplasmic cylinders surrounded by a single outer membrane

arisingafter96hofincubationwithdoxycycline,asshownbyTEM.Bar,0.3

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EFFECTS OF ANTIBIOTICS ON BORRELIA MORPHOLOGY

1131

FIG. 12. Ultrathin section of B. burgdorferi in control experiments after 96 h

examined by TEM. Intact borreliae with regularly arranged ﬂagellae, axial ﬁla-

ments, and membranes are shown. Bar, 1.4

FIG. 14. Concentrations of penicillin G during incubation in BSKII medium

witha B.burgdorferi B31suspensionfor72h. r,MIC 90 ;

,twotimestheMIC 90 .

into the medium, leaving empty protoplasmic cylinders or cell

membranes (Fig. 9).

Similar to penicillin and ceftriaxone, membrane blebbing

was observed after exposure to doxycycline, but granules or

gemmae developed only occasionally after 3 to 4 days of incu-

bation.Multiplemesosome-likestructureswereapparentafter

24hofincubation.Theseovoidbodies,0.07to0.35 mminsize,

to bazilla-like bodies up to 0.4 mm in length, were located in

the center of the protoplasmic cylinder, being partially sur-

rounded by a double-layered cytoplasmic membrane (Fig. 10).

After 96 h of incubation transverse ﬁssion of the cytoplasmic

cylinder was also seen, but these parts were partly undergoing

degeneration (Fig. 11).

Controls. Besidesmembraneblebsthatwerefrequentlyseen

at any site of the organism in control cultures, no ultrastruc-

tural alterations were observed after 96 h (Fig. 12).

SDS-electrophoresis. Exposure of B. burgdorferi B31 to an-

tibiotics for 24 or 96 h revealed polypeptide patterns identical

to those of organisms in control cultures (Fig. 13).

Analysis of antibiotic concentrations. Penicillin concentra-

tions(twotimestheMIC 90 andtheMIC 90 )decreasedbyabout

20% during the ﬁrst 24 h (Fig. 14). At 48 h more than 60% of

the initial drug levels were detectable at all concentrations

tested.Theremainingpenicillinconcentrationsafter72hwere

50% of the initial concentration at two times the MIC 90 and

20% of the initial concentration at the MIC 90 .

The investigations with concentrations less than the MIC 90

presented high variabilities; interference with the culture me-

dium may have been the cause (data not shown). Doxycycline

was rather stable under the culture conditions described here.

After 48 h of incubation drug concentrations were more than

80% of the baseline levels. At 72 h drug concentrations of

between 64 and 76% of the initial values could be detected

(Fig. 15).

The biochemical analyses of ceftriaxone could not be eval-

uated because their low concentrations ranged between 0.006

and 0.12

g/ml.

DISCUSSION

Although the spectrum of antibiotics capable of eradicating

B. burgdorferi in vitro has been enlarged, treatment failures

continue to be a problem for clinicians in the management of

patientswithchronicLymeborreliosis.Beta-lactamantibiotics

such as penicillin and ceftriaxone are recommended for ther-

apy and act on the cell wall by inhibiting the assembly of

insolublepeptidoglycans,leadingtobacteriostasis(22).Studies

ingroupAstreptococcihaveshownthatpenicillinalsoinduces

aspeciﬁclossoftotalcellularRNAintheabsenceofhydrolysis

of the cell wall (22). Besides the loss of integrity of the outer

cytoplasmiccellmembrane,thislatteractionofantibioticscan

also be suspected in B. burgdorferi since several penicillin-

binding proteins have been identiﬁed (33) in association with

FIG. 13. SDS-PAGE showed identical polypeptide distributions after 24 or

96 h of exposure of B. burgdorferi B31 solutions with antibiotics. Lanes: 1,

penicillin; 2, ceftriaxone; 3, doxycyline; 4, B. burgdorferi suspension without

antibiotics.

FIG. 15. Concentrations of doxycyline during incubation in BSKII medium

witha B.burgdorferi B31suspensionfor72h. ¹,0.1timetheMIC 90 ; r,0.5time

the MIC 90 ;

, MIC 90 ; , two times the MIC 90 .

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