Vol. 58, No. 1INFECTION AND IMMUNITY, Jan. 1990, p. 197-2040019-9567/90/010197-08$02.00/0Copyright /f- 1990, American Society for Microbiology

Reduction of Wall Degradability of Clindamycin-TreatedStaphylococci within Macrophages

JORG WECKE,1* LARS JOHANNSEN,2 AND PETER GIESBRECHT'

Robert Koch-Institut, Federal Health Office, Nordufer 20, D-1000 Berlin 65, Federal Republic of Germany,' andDepartment of Physiology and Biophysics, University of Tennessee, Memphis, Tennessee 381632

Received 26 June 1989/Accepted 12 October 1989

Clindamycin treatment of Staphylococcus aureus caused a remarkable thickening of the bacterial cell walland made the bacterial wall much more resistant against lytic enzymes within bone marrow-derivedmacrophages as revealed by electron microscopy and radiolabeling experiments. This reduced wall degrad-ability resulted from an increased number of O-acetyl groups in the murein. Furthermore, such clindamycin-treated bacteria were ingested by adherent bone marrow-derived macrophages at a higher rate than untreatedbacteria. The medical aspects of these results are discussed.

Inhibitors of bacterial ribosomal protein synthesis arewidely used in the therapy of bacterial infections. One ofthese important antibiotics is clindamycin. In earlier in vitrostudies, clindamycin was reported to be more effectiveagainst staphylococci than methicillin, lincomycin, or eryth-romycin (20, 22). Since several bacterial species includingStaphylococcus aureus can survive within phagocytes, theability of clindamycin to accumulate in polymorphonuclearleucocytes or macrophages up to 40 times its extracellularconcentration (18, 23) might be an important feature in viewof its therapeutic effect against such intracellular bacteria.However, there has been a controversial discussion aboutthe intraphagosomal activity of clindamycin. The drug hasbeen reported to be inactive (5, 30), bactericidal (2, 13, 18),and bacteriostatic (1, 12).Although a number of studies were performed investigat-

ing the mode of action of clindamycin, the degradation ofclindamycin-treated staphylococci within phagolysosomeshas received little attention. Of particular interest is thecapability of the phagolysosomes to degrade walls of drug-treated bacteria because of the restricted ability of thecellular immune system to digest the walls of certain patho-genic bacteria such as streptococci, staphylococci, or gono-cocci (6, 11, 26).

In the present study, we investigated the degradation ofclindamycin-treated staphylococcal walls in bone marrow-derived macrophages.

MATERIALS AND METHODSAnimals. Female BALB/c mice (Zentralinstitut fur Ver-

suchstierzucht, Hannover, Federal Republic of Germany) 6to 8 weeks old were used in all experiments.Macrophages. Bone marrow cells were isolated from the

femur and tibia of mice. The cells were seeded on hydro-phobic Petriperm dishes (Heraeus, Hanau, Federal Republicof Germany). Dulbecco modified minimum essential medium(Biochrom, Berlin, Federal Republic of Germany) contain-ing 5% calf serum, 15% horse serum, and, as a colony-stimulating factor, 20% L cell supernatant was used. Thecells were cultivated in 5% CO2 at 37°C and refed everyother day starting at day 5.

Bacteria. S. aureus SG511 from the culture collection ofthe Robert Koch Institute was used. The bacteria were

* Corresponding author.

transferred from 1.5% peptone agar plates to bottles contain-ing 2.5% peptone water (Bacto-Peptone; Difco Laboratories,Detroit, Mich.) and 0.5% sodium chloride in water andcultivated for 16 h at 37°C (stationary-phase culture).

Clindamycin treatment. Stationary-phase suspensions ofstaphylococci were diluted with fresh peptone medium togive an optical density of 1.0 at 578 nm. Dalacin C (clinda-mycin hydrochloride; The Upjohn Co., Kalamazoo, Mich.)was added to the bacterial suspension (final concentration ofclindamycin, 1 mg/liter). After growth for 4 h at 37°C underconstant shaking, the bacteria were harvested by centrifu-gation (RC 2B; Ivan Sorvall, Inc., Norwalk, Conn.) at 10,000x g for 15 min.Opsonization. Before phagocytosis, staphylococci were

opsonized with bacterial wall-specific antiserum producedby rabbits (25% antiserum in phosphate buffer; incubationfor 20 min at 20°C).

Labeling experiments. N-acetyl-D-Cl-[14C]glucosamine(Amersham-Buchler, Braunschweig, Federal Republic ofGermany) with a specific activity of 2.17 GBq/mmol and aradioactive concentration of 7.4 M Bq/ml was used. To a1.3-ml suspension of staphylococci, 60 ,ul of N-acetyl[14C]glucosamine was added and overnight cultivationfollowed. After that, one-half of this suspension was mixedtogether with fresh medium, clindamycin (final concentra-tion, 1 ,ug/ml), and fresh label. During this drug treatment,the control cells were kept under stationary-phase growthconditions. After 4 h, the control cells and the clindamycin-treated cells were centrifuged and the pellets were washed(three times). The total label incorporated into untreatedstaphylococci was 82.9% (overnight), and during clindamy-cin treatment the total label incorporated was 73.1% (4 h).About 95% of the incorporated activity was found in thestaphylococcal cell wall (3). Half of both labeled sampleswere de-O-acetylated.

De-O-acetylation. O-Acetyl groups were removed with0.05 N NaOH for 1 h at 37°C (15, 19), and the removal wasverified by FT-IR spectroscopy (21).

Phagocytosis. Phagocytosis was performed with bone mar-row-derived macrophages 10 to 11 days old. The opsonizedstaphylococci were added to macrophages that were adher-ent to the biofoil of Petriperm dishes (approximately 100bacteria per macrophage). After 30 min of ingestion, themacrophages were washed three times with Dulbecco mod-ified minimal essential medium. To determine the amount of

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FIG. 1. (a) Thin section of a staphylococcal cell. (b) Staphylococcal cell after clindamycin treatment (1 ,ug/ml, 4 h); formation of a thickcell wall (compare with panel a). The thin outer layer of the cell wall having lower contrast is marked by arrowheads.

incorporated radioactivity, samples were removed from themedium after ingestion and from the washing liquids andcounted in a liquid scintillation counter (Rackbeta; LKB/Wallac, Torku, Finland). Scanning electron microscopy per-formed after the washings revealed that the macrophagesurface was virtually free of attached bacteria (data notshown).

After 3 h of digestion, the label released to the supernatantwas measured. The medium was then completely replacedby a medium containing antibiotics (100 U of penicillin perml plus 100 ,ug of streptomycin per ml) to prevent secondaryinfections. Further digestion lasted for 1, 2, 3, or 4 days. Foreach digestion period of any phagocytosis experiment, threeor four dishes were run in parallel. The same dishes wereused in both the radiolabeling experiments and the electronmicroscopic analysis.

Electron microscopy. All steps of fixation, staining, dehy-dration, and Epon embedding were applied to adherentmacrophages in Petriperm dishes. The adherent macro-phages were fixed with 2.5% glutardialdehyde (Serva,Heidelberg, Federal Republic of Germany) in 0.1 M cacody-late buffer (pH 7.2) for 16 h at 4°C. After being washed withcacodylate buffer, the macrophages were postfixed with1.5% osmium tetroxide (Serva), plus 1.65% potassium di-chromate (E. Merck AG, Darmstadt, Federal Republic ofGermany) in 0.1 M cacodylate buffer (pH 7.2) for 1 h at 20°C.After being washed again with the same buffer, the macro-phages were poststained with 1% uranyl acetate in distilledwater for 1 h at 20°C. All further steps for embedding weredone as described earlier (34).Thin sectioning technique. Blocks of Epon were sawed out

on Petriperm dishes. After the hydrophobic biofoil wasremoved, ultrathin sections of macrophages were preparedby cutting in parallel to the adherent side of macrophageswith a diamond knife (Du Pont, Bad Nauheim, FederalRepublic of Germany). Thin sections were stained as de-scribed earlier (34). The ultrathin sections were examined ina Philips EM 400 electron microscope.

RESULTSUltrastructure of clindamycin-treated staphylococci. After

the addition of 1 ,ug of clindamycin per ml to staphylococci,the cell wall was markedly thickened (Fig. lb) comparedwith untreated bacteria (Fig. la). In some cases, the thick-ened wall seemed to be layered: a small distinct peripheralpart of the wall revealed a lower contrast than the underlyingmajor portion of the wall (Fig. lb). After de-O-acetylation,the wall ultrastructure of clindamycin-treated bacteria ap-peared unchanged from the thickened appearance.

Ingestion of staphylococci by macrophages. The ingestionof untreated staphylococci amounted to 23.6% as calculated

FIG. 2. Higher magnification of clindamycin-pretreated staphy-lococcus after 1 day of digestion by the macrophages; the unde-graded bacterial cell wall reveals a differentiation into two layersseparated by a small layer of higher contrast (arrowheads).

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DEGRADATION OF STAPHYLOCOCCAL WALL 199

FIG. 3. Macrophage after 3 days of digestion of clindamycin-pretreated staphylococci; most bacteria appear undegraded.

from radiolabeling experiments. After 4 h of clindamycintreatment, the amount of staphylococci ingested was 28.5%.After de-O-acetylation, however, the corresponding amountsof radioactivity ingested increased up to 34.3 and 39.5%,respectively.

Digestion of clindamycin-treated staphylococci. The prog-

ress of bacterial wall degradation within macrophages was

examined by electron microscopy at different times. After 1day of digestion, no changes in the bacterial ultrastructurecould be detected (Fig. 2). The staphylococcal wall repre-sented a highly resistant part of the ingested bacteria.Compared with clindamycin-treated bacteria before phago-cytosis, the thickened cell wall of ingested staphylococciclearly showed a layered structure, with an intensely stainedsmall layer separating thicker ones from each other (Fig. 2).

Also, after 3 days, in later stages of digestion of clinda-mycin-pretreated staphylococci, a high resistance of thebacteria to degradation could be demonstrated (Fig. 3). Onlyin few bacteria was a certain localized degradation processof the cell wall visible (Fig. 4). In some phagolysosomes,myelinlike structures located close to the staphylococcicould be identified (Fig. 5).

In another series of experiments, clindamycin was addedto adherent macrophages after ingestion of untreated staph-

ylococci. After 16 h, only a few bacteria showed a thickeningof wall structures, especially of their cross wall (Fig. 6),while the cell wall of most bacteria appeared unchanged. Thewall material produced during clindamycin treatment couldbe clearly identified by its lower contrast compared with thepreviously produced wall.To remove a possible cause for the high degradation

resistance of bacterial walls, we hydrolyzed the O-acetylgroups from the murein before phagocytosis. After thistreatment, both cytoplasm and cell wall were already exten-sively degraded after 3 h of digestion (Fig. 7).

Different stages of degraded bacteria could be identifiedwithin the same phagolysosome (Fig. 8). Beside bacteriashowing only minor signs of degradation, others revealedextensive degradation, the walls of such bacteria havingoften a spongelike appearance. At higher magnification, thedegradation process could be observed in more detail. Theperipheral part of the layered cell wall then showed anextensive degradation, while the inner part appeared to beunaffected (Fig. 9), indicating an attack on the cell wall fromthe outside. Both layers were separated by a thin layer ofhigh contrast. After 1 day of digestion within macrophages,any remnants of de-O-acetylated bacteria were hardly de-tectable.

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FIG. 4. Thin section of the periphery of the bacterial cell walldemonstrating some local degradation of a clindamycin-pretreatedstaphylococcal cell wall after 3 days of digestion (arrows).

While electron microscopy contributed to the understand-ing of the morphological aspects of bacterial wall degrada-tion within macrophages, labeling experiments helped togather further information about the quantitative aspects.The release of the specific bacterial wall label from themacrophages was measured after different digestion times.Data indicate that macrophages fed with clindamycin-pre-

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FIG. 5. Clindamycin-pretreated staphylococcal cell after 3 daysof digestion by the macrophage, the staphylococcus is surroundedby a phagolysosomal membrane (arrowheads). Lipidlike structures(L) are located in close connection to the bacterium.

FIG. 6. Phagocytosed S. aureus. The bacteria were ingestedbefore the addition of clindamycin (10 ,ug/ml) to the medium with thephagocytes. The drug, apparently having penetrated the phagoly-sosome, induced no detectable changes in the peripheral bacterialcell wall except a localized thickening of the cross wall (arrow-heads).

treated staphylococci released considerably less labeled wallmaterial to the medium than did those fed with untreatedbacteria (Fig. 10, compare C with A). However, afterremoval of O-acetyl groups from the murein of untreatedbacteria, the release of wall label increased remarkably (Fig.10, compare A with B). Also, de-O-acetylation of clindamy-cin-pretreated bacteria resulted in a dramatic increase ofdegradation within macrophages (Fig. 10, compare C withD). The degradation of these staphylococci amounted to fivetimes that of the respective 0-acetylated clindamycin-pre-treated bacteria.

DISCUSSION

Treatment of S. aureus with clindamycin yielded a re-markably thickened cell wall which had been describedearlier (27). These data are in accordance with the effects ofother inhibitors of the ribosomal protein synthesis, such aschloramphenicol or erythromycin, on S. aureus (8, 32).A certain degree of wall thickening could also be demon-

strated after the addition of clindamycin to macrophagescontaining ingested untreated bacteria. However, in contrastto bacteria treated with clindamycin outside of macro-phages, the ingested bacteria within macrophages revealedonly cross wall thickening. This phenomenon could beexplained as decoupling of different bacterial wall growthprocesses (9) obviously caused by special conditions withinmacrophages. This wall thickening of staphylococci withinmacrophages is evidence of the earlier-described ability ofclindamycin to penetrate into phagocytes (23). Since onlyviable intraphagolysosomal staphylococci are able to reactto clindamycin, this wall thickening of ingested bacteriacould be considered as an indicator for the number of livingstaphylococci within phagocytes. As to the amount of radio-

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DEGRADATION OF STAPHYLOCOCCAL WALL 201

FIG. 7. Macrophage with ingested clindamycin-pretreated staphylococci followed by de-O-acetylation of their cell walls; after 3 h ofdigestion, the staphylococci already exhibit a high degree of degradation (arrowheads).

activity, clindamycin-pretreated staphylococci were in-gested slightly faster than untreated bacteria. The reasonsfor this increase are not yet understood. Clindamycin treat-ment might remove part of the protein A or parts of the fuzzycoat (28) on the bacterial cell surface, which would rendersuch cells more susceptible to phagocytic uptake (7, 29).Another possible explanation may be found in the particularconditions of the phagocytosis system employing adherentmacrophages. Since the clindamycin-treated staphylococcihad a higher sedimentation velocity owing to their thickerwalls, they had the chance to be ingested more rapidly thanuntreated bacteria.

Concerning the bactericidal mechanisms within phagoly-sosomes, our observation of myelinlike structures located inclose connection to staphylococci (Fig. 5) might be of

particular importance. In a previous study, it was suggestedthat staphylococci were killed by a "cidal" fatty acid gen-erated within lesions of experimentally infected mice (17).Polymorphonuclear leukocytes and macrophages are cer-tainly candidates to produce such compounds, and themyelinlike structures mentioned above might be connectedto the bactericidal fatty acids.The impressively increased degradation resistance after

pretreatment with clindamycin may be ascribed to an in-creased O-acetyl substitution of the murein after clindamy-cin treatment. In earlier studies, it was demonstrated thattreatment of S. aureus with antibiotics of a similar mode ofaction, such as chloramphenicol or erythromycin, increasedthe number of O-acetyl groups of the murein (4, 16, 31, 32)and the degradation resistance of their cell walls against

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FIG. 9. Ingested clindamycin-pretreated staphylococcus afterde-O-acetylation of its cell wall; 3 h of digestion. The peripheral partof the layered wall reveals extensive degradation, while the innerpart seems to be hardly degraded (arrowheads).

FIG. 8. Higher magnification of a macrophage with ingestedclindamycin-pretreated staphylococci and after de-0-acetylation oftheir cell walls; 3 h of digestion. Different stages of wall degradationwithin the same phagolysosome can be identified. Besides bacteriawith minor changes (large arrowheads), others show completelydegraded cytoplasm and different stages of wall degradation (smallarrowheads).

lysozyme in vitro (14). The importance of O-acetyl groups ofmurein for the degradation resistance was also demonstratedwith certain strains of Neisseria gonorrhoeae (24) showing adegradability by human polymorphonuclear leukocytes thatwas only one-fourth that of non-O-acetylated murein (25).Concerning the mechanisms of bacterial cell wall degra-

dation, one has to differentiate between two processes, (i)degradation by the activity of bacterial wall autolysinslocated in the region between the cytoplasmic membraneand cell wall which have been shown to attack the cell wallsfrom the inside (33) and (ii) degradation by the wall lyticcapacity of the macrophage enzymes, which can be expectedto degrade the bacterial wall primarily from the outside ofthe bacteria. Our electron micrographs indicated that afterphagocytosis, the wall degradation was predominantly per-formed from the outside of the bacteria because no lyticactivity was detectable between the cytoplasmic membrane

and cell wall of bacteria. These data point to the involvementof the lytic activity of the macrophage in these wall-de-grading processes. This conclusion was confirmed by phago-cytosis experiments with staphylococci whose autolytic en-zymes had previously been inactivated by thermic treatment(J. Wecke, manuscript in preparation).The evidently different stages of degradation of the two

wall layers of de-O-acetylated bacteria during phagocytosis(Fig. 9) indicate the existence of two different wall qualitieswithin the staphylococcal cell wall. The observed differentdegradabilities are evidence for possible different chemicalstructures of the wall layers. Since the outer layer appearedto be distinctly thinner than the inner one, we suggest thatthe outer layer represents the normal cell wall, while theunderlying thicker portion is produced by apposition growthof wall material in the presence of clindamycin.From the therapeutic point of view, the reduced degrad-

ability of staphylococcal cell walls after clindamycin treat-ment must be taken seriously, because undegradable wallmaterial set free from dead phagocytes is known to bereingested by other phagocytes, which would result inanother unsuccessful attempt at wall digestion and which, atthe same time, would weaken the overall defensive force ofthe cellular immune system. Moreover, such undegradablewall material is considered to induce chronic inflammatoryprocesses like some types of arthritis owing to being depos-ited in joint regions. The pathogenetic importance of thiseffect was earlier demonstrated in an animal model (10).Since the latter study was performed with bacteria untreatedwith bacteriostatic antibiotics, it cannot be excluded thatclindamycin treatment would enhance this arthropathic ef-fect of staphylococcal cell walls.

ACKNOWLEDGMENTS

The skillful technical assistance of E. Albrecht, C. Han, G.Kniffke, E. Mertens, and B. Nurnberg is much appreciated.

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DEGRADATION OF STAPHYLOCOCCAL WALL 203

D

-D~~~~~~~~~~~~D

D A~~D

A A

3 h 24h 48h 96hFIG. 10. Wall degradation of clindamycin-pretreated staphylococci within macrophages in terms of release of specific bacterial wall label

after different digestion periods. (A) S. aureus control cells; (B) S. aureus with de-O-acetylated cell walls; (C) Clindamycin-pretreatedstaphylococci; (D) Clindamycin-pretreated staphylococci with de-O-acetylated cell walls.

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