Inhibition of the bacteriolytic effect of β-lactam-antibiotics on Staphylococcus aureus by the polyanionic drugs suramin and Evans Blue

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<ul><li><p>APMIS 98: 7 1-8 1, 1990 </p><p>Inhibition of the bacteriolytic effect of P-lactam-antibiotics on Staphylococcus aureus by </p><p>the polyanionic drugs suramin and Evans Blue* </p><p>JORG WECKE, MARITA FRANZ and PETER GIESBRECHT </p><p>Robert Koch-Institut des Bundesgesundheitsamtes, Berlin, Germany </p><p>Wecke, J., Franz, M. &amp; Giesbrecht, P. Inhibition of the bacteriolytic effect of P-lactam-antibiotics on Staphylococcus aureus by the polyanionic drugs suramin and Evans Blue. APMIS 98: 7 1-8 1, 1990. </p><p>The anionic polyelectrolytes suramin and Evans Blue inhibited different autolytic systems involved in wall growth and wall turnover of growing staphylococci and in wall autolysis of resting bacteria. Moreover, both substances lowered the P-lactam-induced pre-lytic release of cytoplasmic constituents from staphylococci, and inhibited the P-lactam-induced bacteriolysis as well as the loss of viability. The protective effects of these sulfonated drugs against bacteriolysis were also monitored by electron microscopy. Some medical implications of our results are discussed. </p><p>Key words: Staphylococcus aureus ; bacteriolysis; inhibition; penicillin; latamoxefi suramin; Evans Blue. </p><p>Jorg Wecke, Robert Koch-Institut, Nordufer 20, D-1000 Berlin 65, Federal Republic of Germany. </p><p>In previous studies it was demonstrated that anion- ic polyelectrolytes, such as certain anticoagulants, were effective in reducing the release of a specific wall label from bacteria (Ginsburg &amp; Sela 1976). This inhibition was recently found to be caused by an interaction between the anticoagulants and the staphylococcal cell wall ( Wecke et al. 1986). It was also shown that such anionic anticoagulants were capable of suppressing the penicillin-induced bac- teriolysis ( Wecke et al. 1987). </p><p>Another substance belonging to the same group of negatively charged compounds is suramin. Apart from its therapeutic effect against try- panosomiasis and onchocerciasis, this drug repre- sents a potent competitive inhibitor of the reverse transcriptase of a number of animal retroviruses </p><p>Received December 12, 1988. Accepted August 1, 1989. *Dedicated to Prof. Dr. Georg Henneberg on the occa- sion of his 80th birthday. </p><p>(de Clercq 1979). Its virustatic activity was ascribed to its high content of sulfonic acid residues (Chan- dra et al. 1985). Recently, suramin was shown to inhibit in vitro the infectivity and the cytopathic effect of HIV (Mitsuya et al. 1984). Likewise, a virustatic in vivo effect of suramin was demon- strated in a clinical study with AIDS-patients (Broder et al. 1985). The anionic dye Evans Blue, which is structurally related to suramin, was de- scribed as an inhibitor of the cytopathic effect of HIV on ATH8 cells, too (Balzarini et al. 1986b). </p><p>So far, however, virtually nothing is known about the possible simultaneous interference of suramin or Evans Blue with the action of those antibiotics which have to be used at high doses against opportunistic bacterial infections during advanced stages of AIDS. Therefore, the aim of this study was to investigate whether suramin and Evans Blue would interact with untreated staphy- lococci and would interfere with the action of some j3-lactam antibiotics on staphylococci. </p><p>71 </p></li><li><p>WECKE et al. </p><p>MATERIALS AND METHODS </p><p>Bacterial strains and cultivation Staphylococcus aureus strain SG 5 1 1 Berlin from the </p><p>culture collection of the Robert Koch-Institute was used throughout. The bacteria were transferred from agar plates to flasks containing peptone broth (2.5% peptone) and cultivated for 22 h at 37 "C. Then the suspensions were diluted with fresh medium and, after growth for another 2 h at 37 "C under shaking, the resulting log-phase cultures were adjusted to an optical density at 578 nm (OD) of 0.5 by addition of fresh medium. Penicillin G was added to some of these cultures to establish a concentration of 0.1 Fg penicillin per ml. This drug concentration corresponded to 5 times the MIC and had earlier proved optimal for rapid bacteriolysis with the strain in use. Bacteriolysis was almost complete after 4 - 5 h. Bacterial growth was monitored by an "Eppendorf" photometer and the cells were harvested by a Sorvall RC 2B centrifuge ( 1 0,000 x g for 15 rnin). </p><p>Chemicals Penicillin G (Serva, FRG) amoxycillin (Becham, UK) </p><p>and latamoxef (a gift from Dr T. Nishino, Kyoto) were the p-lactam compounds used. Suramin was purchased from Bayer AG (Leverkusen, FRG), Evans Blue (EB) from Merck (Darmstadt, FRG) and lysostaphin from Sigma (Tauflcirchen, FRG). 14C-N-acetylglucosamine and 'H-uracil were obtained from Amersham/Buchler (Braunschweig, FRG). </p><p>Assay of viable counts Viability measurements were performed using a spiral </p><p>plater model CU (Spiral System Instruments Inc., USA). </p><p>Labeling of intracellular components of staphylococci Bacteria from the logarithmic growth phase were </p><p>transferred to 25 ml fresh peptone broth, containing 150 p1 (5,6-3H)-uracil (37 M Bq/ml). After four division cycles (about 120 rnin), the bacteria were harvested by centrifugation and washed with peptone broth. </p><p>Aliquots were withdrawn from the cultures and after centrifugation (12,000 x g for 5 min) supernatants were monitored by a liquid scintillation counter (Rackbeta, LKB, Sweden). </p><p>Labeling of cell walls Cell wall labeling of staphylococci with I4C-N-acetyl- </p><p>glucosamine ( I4C-NAGA) and radioactivity measure- ments were performed as described in a previous study ( Wecke et al. 1982). </p><p>Isolation and fractionation of staphylococcal cell walls After cultivation for 1 h or 22 h, bacteria were har- </p><p>vested by centrifugation at 10,000 x g for 10 min. After washing with saline, the bacterial cells were disintegrated for 3 - 6 rnin in a "Dyno-mill" TYPE KDL (Wily A. Bachofen, Basel, Switzerland) filled with glass beads. The temperature was kept below 4 "C. After separation from the glass beads by glass filters, the cell debris was shaken </p><p>72 </p><p>for 30 rnin at 60 "C in a sodium dodecylsulfate solution to remove the cytoplasmic membranes. After five wash- ings with water, the debris was incubated with trypsin (0.2 mg/ml) in 0.15 M Trisbuffer for 48 h at 37 "C. </p><p>The inhibition of lysostaphine-induced lysis of staphylo- coccal cell walls </p><p>The radiolabelled walls of staphylococci suspended in buffer (ammonium acetate pH 6.9) were pretreated with suramin (250 pg/ml) or EB (100 pg/ml) for 1 h at 4 "C. Afterwards cell walls were washed twice with ammoni- um acetate buffer. Following this treatment, the EB- treated cell walls appeared as a blue colored pellet after centrifugation (10,000 x g , 15 rnin), while the superna- tant was undyed. Then lysostaphin ( 10 pg/ml) was added at room temperature. The wall lysis was measured by determination of released wall label in the supernatant after centrifugation (10,000 x g , 15 rnin). The radioac- tivity measurements were performed as described in a previous study (Wecke et al. 1982). </p><p>Thin sectioning technique Staphylococci were prepared for thin-sectioning by </p><p>fixation with 2.5% glutaraldehyde (Serva, FRG) in 0.1 M cacodylate buffer pH 7.2 for 1 h at room temperature. After washing with cacodylate buffer the cells were postfixed with 1.5% osmiumtetroxide (Serva, FRG) + 1.65% potassium dichromate in 0.1 M cacodylate buffer for 1 h at room temperature. The fixed samples were washed again with the same buffer and poststained en bloc with 0.5% uranyl acetate in distilled water for 1 hat room temperature. After dehydration using a series of increasing ethanol concentrations, the samples were infiltrated by the LR White plastic monomer (Science Services, FRG) and then polymerized for 24 h at 60 "C. </p><p>Thin sections were cut with diamond knives (Dupont, USA) on a Reichert OM U 3 ultramicrotome, and stained with lead citrate for 3 - 5 rnin (Reynolds 1963) prior to examination in a Philips 400 electron micro- scope. </p><p>RESULTS </p><p>Action of suramin or Evans Blue on staphylococci (a) Growing staphylococci. After the addition of </p><p>suramin (100 - 1000 pg/ml) to growing staphylo- cocci, no disturbance of growth, as determined by optical density measurements, could be detected. However, the release of wall label within 2 h was remarkably reduced after addition of suramin or Evans Blue to growing staphylococci (Fig. 1). This result demonstrated a suppression of the activity of wall autolytic enzymes participating in wall turnover of staphylococci. The ultrathin sections of suramin-treated bacteria revealed thickened cross walls, while the peripheral wall seemed to be unchanged (Fig. 2). Sometimes, a layering of the </p></li><li><p>INHIBITION OF BACTERIOLYSIS BY SURAMIN </p><p>Q u n t r e a t e d s t a p h y l o c o c c i ( s t a p h ) </p><p>A s t a p h + 5 0 p g / m l Evans B l u e + s t a p h + 2 5 p g / m l Evans B l u e </p><p>e s t a p h + 5 0 0 p g / m l s u r a m i n </p><p>Fig. I . Suppression of cell wall turnover of growing staphylococci by suramin or Evans Blue. </p><p>t i m e of i n c u b a t i o n I m i n ) </p><p>cross wall could be detected (Fig. 2, inset). The obviously delayed cross wall separation under this treatment could very often be observed in bacteria which possess more than one division plane; as a </p><p>result so-called pseudomulticellular bacteria (Fig. 3) were formed. Similar effects could be observed in the presence of Evans Blue. These observations indicated a partial inhibition of the autolytic wall </p><p>Fig. 2. Thin section of a staphylococcus with a thickened cross wall after addition of suramin (250 pg/ml for 2 h) to growing staphylococci (large arrows); the second division plane is alredy initiated (small arrows). Inset: layered cross wall (white arrows). </p><p>1 3 </p></li><li><p>WECKE et al. </p><p>Fig. 3. A section of staphylococcal cells with retarded cross wall separations after addition of Evans Blue (200 pg/ml, 2 h); the first division plane is marked by one arrow, the second one by two, and the third one (tangentially sectioned) by three arrows. </p><p>/&amp; pc + 100 ug/ml Suranin </p><p>0 1 2 3 4 m4 . . . . . . - . </p><p>time of incubation ( h ) </p><p>Fig. 5A. The penicillin-induced bacteriolysis of staphy- lococci (A-A) was partially suppressed by 100 pg/ml suramin (x-x) and almost completely inhibited by 250 pg/ml suramin (+-+). </p><p>CI untreated StaDhYlOCOCCl (Staph) 0 staph + 1 ug/ml Evans Blue A staph + 10 ug/ml suranin + staph + 100 ug/ml suranin </p><p>4 </p><p>a m a * d </p><p>9 </p><p>m N </p><p>0 1 2 3 4 5 m </p><p>timr o f inoubation (h) </p><p>Fig. 4. Suppressed wall autolysis of non-growing staphy- lococci (suspended in acetate buffer) by suramin or Evans Blue measured in percent release of specific wall label. </p><p>enzymes which are involved in cross wall separa- tion. </p><p>(b) Resting bacteria. The influence of the anion- ic drugs suramin and Evans Blue on the autolytic activity of resting staphlococci in buffer was </p><p>m i . . . . . . . . 4 0 1 2 3 4 </p><p>time of incubation (h) </p><p>Fig. 5B. The latamoxef-induced bacteriolysis of staphy- lococci (0-0) was weakly suppressed by 100 pg/ml suramin (+-+) and effectively inhibited by 250 pg/ml suramin (+-+). </p><p>74 </p></li><li><p>INHIBITION OF BACTERIOLYSIS BY SURAMIN </p><p>- \ pc + 100 ug/ml Suromin - - </p><p>m </p><p>m n - .- &gt; </p><p>0 , l ug/ml penicillin (pc) </p><p>0 .5 1. 0 1. 5 2.0 2.5 3.0 3. 5 time o f incubation (h) </p><p>analysed by measuring the release of the specific wall label from the cells suspended in buffer. Fig. 4 shows that the bacteria were effectively protected against wall autolysis with 100 pg/ml suramin and 1 pg/ml Evans Blue, respectively. </p><p>Action of suramin and Evans Blue on p-lactam- treated staphylococci </p><p>To analyse the effect of the anionic drug suram- </p><p>1 </p><p>Fig. 6A. Enhanced viability of penicillin-treated staphy- lococci in the presence of suramin. </p><p>in during penicillin or cephalosporin treatment of staphylococci, lytic doses of penicillin G (0.1 pg/ml) and latamoxef (10 pg/ml) dissolved in peptone broth were applied. Both lytic doses corre- sponded to 5 x MIC. In suramin-free suspensions, bacteriolysis started about 60 min after addition of the drugs, while upon simultaneous addition of suramin, a dose-dependent diminution of the decline of the growth curves was observed (Fig. 5A </p><p>- C \ - - m </p><p>m </p><p>3 </p><p>LTH + 250 ug/nl Suranin </p><p>- .I </p><p>&gt; LTH + 1B0 ug/nl Suranin </p><p>10 ug/nl Lotonoxef (LTH) </p><p>0 .5 1.0 1.5 2.0 2 . 5 3.0 3.5 t ine o f incubation (h) </p><p>Fig. 6B. Enhanced viability of latamoxef-treated staph- ylococci in the presence of suramin. </p><p>75 </p></li><li><p>WECKE et al. </p><p>A 0,lpg/ml prnioillin (PO) + PO + 100pg/d muramin X po + 10p9/ml Evan. Blur </p><p>PO + 100pg/ml Evan- Blur </p><p>mJ 0 1s 30 4s 60 98 </p><p>time of incubation tmln) </p><p>Fig. 7. Suppression of the release of the cytoplasm- specific label (3H-uracil) from penicillin-treated staphy- lococci by suramin or Evans Blue. </p><p>and 5B). Basically the same inhibitory effects were observed when suramin was replaced by Evans Blue (data not shown). </p><p>It was of particular medical interest to note that the inhibition of the P-lactam-induced bacterioly- sis of staphylococci also yielded considerably higher viability rates (Fig. 6A and 6B). </p><p>In order to contribute to a better understanding of the inhibitory effects of suramin and Evans Blue on P-lactam-induced bacteriolysis, the specific cytoplasmic label 3H-uracil was introduced into staphylococcal cells and its release was followed after additon of penicillin and of penicillin plus the anionic substances respectively, to the growth medium. These experiments showed that also the penicillin-induced pre-lytic release of the cytoplas- mic label proved to be remarkably reduced in the presence of suramin or Evans Blue (Fig. 7). The most effective suppression totalling up to more than 50Yo after 60 min was observed in the pres- ence of 0.1 pg/ml penicillin + 100 pg/ml Evans Blue. </p><p>The morphogenetic consequences of the simul- taneous action of penicillin and the two anionic compounds were studied under the electron mi- croscope. It became evident that after a 4 h incubation in growth medium under the lytic dose of penicillin (0.1 pg/ml) most of the bacteria were </p><p>Fig. 8A. Thin-section of staphylococci after a 4 h incubation with 0.1 Fg/ml penicillin; typical bacterioly- sis. Fig. 8B. Thin-section of staphylococci after a 4 h incubation with penicillin (0.1 pg/ml) + suramin (200 pg/ml). Protection of the bacteria against penicillin-in- duced bacteriolysis in the presence of suramin is shown. </p><p>lysed, while in the presence of penicillin plus suramin many staphylococci remained intact (compare Fig. 8A with Fig. 8B). The protected bacteria showed some structural variations; espe- cially their cross walls were enormously thickened (Fig. 9) and appeared to consist of rather loose and fibrillar wall material, while their peripheral wall had hardly changed. Cell separation of such </p><p>76 </p></li><li><p>INHIBITION OF BACTERIOLYSIS BY SURAMIN </p><p>Fig. 9. A thin-section of a staphylococcal cell after a 4 h incubation with 0.1 Fg/ml penicillin + 100 pg/ml Evans Blue. Only one daughter cell has retained the ability to produce a new cross wall. (The first division plane is marked by one arrow, the second by two arrows). The cross walls are enormously thickened, while the periph- eral wall hardly appears to b...</p></li></ul>

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