Vol. 30, No. 3ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1986, p. 414-4170066-4804/86/090414-04$02.00/0Copyright ©) 1986, American Society for Microbiology

Novel Morphological Changes in Gram-Negative Bacteria Caused byCombination of Bulgecin and Cefmenoxime

MASAFUMI NAKAO,* KOICHI YUKISHIGE, MASAHIRO KONDO, AND AKIRA IMADA

Central Research Div,ision, Takeda Chemical Indiustries, Ltd., Yodogawva-ki, Osaka 532, Japan

Received 10 March 1986/Accepted 24 June 1986

The mode of action of bulgecin was investigated by examining its bactericidal and bacteriolytic activities, itseffect on bacterial morphology, and its interaction with penicillin-binding proteins (PBPs). Bulgecin alone didnot show any antibacterial activity against Escherichia coli and Serratia marcescens, but in concert withcefmenoxime, it induced potent growth-inhibitory and bactericidal activities. Electron microscopic examina-tion of E. coli cells exposed to bulgecin combined with cefmenoxime revealed that a bulge developed in themiddle of the cell, and additional smaller bulges were formed halfway between the central bulge and the polarends. At the site of bulge development, vesicular mesosomelike structures appeared in the cytoplasm, thepeptidoglycan layer facing them became faint, and the outer membrane protruded to form blebs. Thesemorphological changes were quite different from those caused by the mecillinam-cefmenoxime combinationthat produces big bulges in E. coli. When S. marcescens was exposed to the combination of bulgecin andcefmenoxime, not only bulge formation, but also branching of the cells was observed. Bulgecin neither showedaffinity for any PBPs of E. coli nor affected the binding of cefmenoxime or mecillinam to the PBPs.

Bulgecin A, hereafter called bulgecin, is a sulfonatedglycopeptide isolated from the culture broths of Pseudomo-nas acidophila sp. nov. and P. mesoacidophila sp. nov. thatproduce sulfazecin and isosulfazecin, respectively (6, 12).Bulgecin does not show antibacterial activity by itself,however; it induces bulge formation and cell lysis in combi-nation with 1-lactam antibiotics that cause elongation ofbacterial cells (6). In gram-negative bacilli, penicillin-bindingproteins (PBPs) 2 and 3 are required for maintaining rodshape and for septum formation, respectively (8, 9, 14).Bulge formation in gram-negative bacilli is thought to be amorphological change caused by the simultaneous inhibitionof PBPs 2 and 3 by P-lactam antibiotics (14). Otsuki (11)demonstrated that the combination of mecillinam andcephalexin used against Escherichia coli causes increasedbacteriolytic and bactericidal activities and the induction ofosmotically fragile spindle cells by inhibiting PBPs 2 and 3.Fuglesang et al. (3) reported similar increased bactericidalactivity and bulge formation in several gram-negative bacte-ria with the combination of mecillinam and ampicillin.

In a previous study, we noted by phase-contrast micros-copy that the bulges formed by bulgecin combined withcefmenoxime are different in shape, size, and rate of forma-tion from those formed by mecillinam combined withcefmenoxime (6). Therefore, the bulge formation seemed toresult from different mechanisms.

In the present study, the mode of cooperative action ofbulgecin and cefmenoxime (15) against gram-negative bacte-ria such as E. c oli and Serratia marcescens was investigatedby examining the bactericidal and bacteriolytic activities, theeffect on bacterial morphology, and the binding affinities forPBPs of this combination.

MATERIALS AND METHODS

Organisms and growth conditions. The bacterial strainsemployed were E. coli NIHJ JC-2 and S. marcescens TN66.

* Corresponding author.

The cells were grown in Trypticase soy broth (BBL Micro-biology Systems, Cockeysville, Md.) at 37°C overnight andused as the inoculum.

Antibiotics and chemicals. Bulgecin, cefmenoxime, andmecillinam were prepared at the Central Research Divisionof Takeda Chemical Industries, Ltd., Osaka, Japan.["4C]benzylpenicillin (54 mCi/mmol) was purchased from theRadiochemical Centre, Amersham, England.

Viable counts and turbidimetric studies. Overnight cultureswere diluted with fresh Trypticase soy broth to 10-3 andcultivated with rocking at 37°C until mid-log phase (opticaldensity at 600 nm, 0.1). Bulgecin and cefmenoxime wereadded simultaneously or separately to the cultures, and atvarious intervals a portion of them was removed. CFUswere estimated by the plate count method using Trypticasesoy agar (BBL), and turbidity was measured with a ColemanJunior II spectrophotometer.

Electron microscopy. Cells were prefixed with 2.5% glutar-aldehyde in 0.05 M cacodylate buffer (pH 7.4) for 3 h andpostfixed with cacodylate-buffered 1% osmium tetroxide for2 h. The fixed cells were dehydrated with a graded series ofethanol. For scanning electron microscopy, the specimenswere critical point dried, coated with gold, and examinedwith an ISI-DS130 scanning electron microscope. For trans-mission electron microscopy, the specimens were embeddedin Epon 812. Ultrathin sections were prepared with an LKBULTROTOME III, doubly stained with uranyl acetate andlead citrate, and examined in a JEOL JEM-1200 EX trans-mission electron microscope.

Assay of PBPs. The methods for preparing membranes ofE. coli NIHJ JC-2, binding of antibiotics, and separating anddetecting PBPs were the same as those reported by Nozakiet al. (10).

RESULTSBactericidal and bacteriolytic activities. Bulgecin alone, at

a concentration of 10 p,g/ml, showed no detectable effect onthe viability and turbidity of either E. coli NIHJ JC-2 or S.mnarcescens TN66. Cefmenoxime alone, at a concentration

414

MODE OF ACTION OF BULGECIN 415

S. marcescens TN 66

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FIG. 1. Bactericidal activitiy of bulgecin in combination withcefmenoxime against E. coli NIHJ JC-2 and S. marcescens TN66.Symbols: 0, control; A, bulgecin (10 ,ug/ml); *, cefmenoxime (0.1,ug/ml); 0, bulgecin (10 ,ug/ml) plus cefmenoxime (0.1 ,ug/ml).

of 0.1 ,ug/ml (one-fourth of the MIC), showed a slightbactericidal activity and suppressed the increase of theturbidity. By combining bulgecin and cefmenoxime, bacte-ricidal effect was potentiated (Fig. 1). The bacteriolysis of E.coli NIHJ JC-2 provoked by bulgecin became remarkable inproportion to the duration of preincubation of the cells withcefmenoxime (Fig. 2a). The degree of growth-inhibitoryeffect of cefmenoxime, on the other hand, was not affectedby the duration of preincubation of the cells with bulgecin(Fig. 2b). The results were the same with S. marcescensTN66 (data not shown).

Electron microscopy. Scanning electron micrographs of E.coli NIHJ JC-2 exposed to bulgecin and cefmenoxime aloneand in combination are shown in Fig. 3. No morphologicalchange could be noted after the cells were incubated for 2 hwith bulgecin (Fig. 3b). Mecillinam made the cells ovoid.Cell division continued for a while after the cells wereexposed to mecillinam, but the constriction proceeded asym-metrically (data not shown). Filamentous cells with smallbulges were induced by cefmenoxime (Fig. 3c). When thecells were exposed to bulgecin in combination withcefmenoxime, bulge formation became obvious (Fig. 3d). Abulge developed in the middle of the slightly elongated cells,and additional smaller bulges developed halfway betweenthe central bulge and the polar ends. A large number of blebs(spherical protrusions) were observed on the bulges. Thecombination of mecillinam and cefmenoxime, on the otherhand, induced the formation of big central bulges withoutblebs (data not shown). Bulgecin alone exhibited no mor-phological effect on S. marcescens TN66 (Fig. 4a).Cefmenoxime alone brought about filamentation of the cells(Fig. 4b). The combination of bulgecin and cefmenoximeresulted in the formation of cells with a novel morphology;i.e., not only bulge formation, but also complicated branch-ing of the cells was induced (Fig. 4c). Underneath the bulges,vesicular mesosomelike structures appeared in the cyto-plasm, and the outer membrane facing them became irregu-lar. The relationship between the development of themesosomelike structures and the morphological aberrations

of the outer membrane was examined in detail. So long asthe murein layer facing the mesosomelike structures wasvisible, the outer membrane remained intact. However,when the murein layer facing the mesosomelike structuresdisappeared, the outer membrane protruded to form blebs(Fig. 5). Cytoplasmic contents flowed from the cell at theregion where the outer membrane ruptured, and the electrondensity of the cytoplasm decreased. The combination ofmecillinam and cefmenoxime, on the other hand, causedneither emergence of the mesosomelike structures nor blebformation.

Affinity for PBPs. The interaction of bulgecin alone and incombination with cefmenoxime or mecillinam with the PBPsof E. coli NIHJ JC-2 was investigated. Bulgecin showed noaffinity for any PBPs at concentrations of up to 400 pLg/ml.Cefmenoxime showed a high affinity for PBPs 3, 1A, 1B, and2, in descending order. Mecillinam showed a specificallyhigh affinity for PBP 2. Neither the degree nor the profile ofaffinity of cefmenoxime and mecillinam for PBPs was af-fected by bulgecin (data not shown).

DISCUSSIONBulgecin alone was inactive against E. coli NIHJ JC-2 and

S. marcescens TN66; however, in combination withcefmenoxime, it induced bactericidal, bacteriolytic, and

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FIG. 2. Effect of preincubation of E. coli NIHJ JC-2 with eithercefmenoxime or bulgecin on provoking bacteriolysis by addingbulgecin or cefmenoxime. Symbols: *, control; A, bulgecin (10,ug/ml); *, cefmenoxime (0.1 ,ug/ml); 0, bulgecin (10 ,ug/ml) pluscefmenoxime (0.1 ,ug/ml).

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bulge-forming effects. The bacteriolytic activity of bulgecinwas potentiated in proportion to the duration of pretreatmentof the cells with cefmenoxime. The growth-inhibitory effectof cefmenoxime was similar irrespective of the duration ofpretreatment with bulgecin. This suggests that bulgecin actson the cells, whose surface has been altered by P-lactamantibiotics such as cefmenoxime. In E. coli NIHJ JC-2, abulge began to develop in the middle of the cells about 30min after bulgecin combined with cefmenoxime was added,and additional smaller bulges were formed halfway betweenthe central bulge and the polar ends. The bulges wereobserved at these regions without exception; therefore, theyseemed to develop at the sites where cell division is expectedto take place. Besides bulge formation, the morphologicalrelationships among the emergence of the mesosomelikestructures, the disappearance of the peptidoglycan layer,and bleb formation were also noted. As the peptidoglycanlayer facing the mesosomelike structures disappeared, theexistence of some kind of lytic enzyme(s) in the mesosome-like structures is conceivable. Degradation of peptidoglycan,on which the outer membrane is thought to be anchored vialipoprotein (2, 5, 13), would cause the fragility of the outermembrane. Cell lysis by bulgecin appears to be caused byrupture of the blebs that developed from the fragile outermembrane. In S. marcescens TN66, not only bulge forma-tion but also branching of the cells was induced by bulgecincombined with cefmenoxime; therefore, some differences inthe structure or composition or both of the cell envelope mayexist between the two bacteria used in this study.

Bulge formation in gram-negative bacilli is known as amorphological change induced specifically by 1-lactam anti-biotics. Recently, bulge formation in gram-negative bacteriaby nocardicin and MT-141 was reported by Gotoh et al. (4)and Tsuruoka et al. (16), respectively. While nocardicin

FIG. 4. Scanning electron micrographs of S. marcescens TN66exposed for 2 h to (per milliliter) (a) bulgecin (10 ,ug), (b)cefmenoxime (0.1 ,ug), and (c) bulgecin (10 ,ug) plus cefmenoxime(0.1 ,ug). Bars indicate 1 p.m.

shows affinity for PBPs other than PBP 3 (1), and MT-141shows affinity for PBPs other than PBP 2 (7), bulgecinneither showed affinity for any PBPs nor affected the affin-ities of 1-lactam antibiotics such as mecillinam andcefmenoxime for PBPs of E. coli NIHJ JC-2. In addition,differences between bulgecin and nocardicin or MT-141 withrespect to the shape of bulges and the site where theydevelop were noticed. Therefore, unlike nocardicin andMT-141, bulgecin appears to have a novel mode of action inmaking gram-negative bacteria lyse by damaging their sur-face structures without affecting the PBPs.The precise mechanism of bulge formation by bulgecin is

unknown. A speculative explanation might be that the de-gree of cell elongation induced by cefmenoxime decreaseswhen this antibiotic is combined with bulgecin, but thecytoplasmic mass continues to expand and eventually causesswelling of the cell at its structurally weakest region, whichpossibly corresponds to the cell division site. However,bulgecin shows a synergistic effect specifically with 1-lactamantibiotics; no interactions were noted with other antimicro-bial agents such as nalidixic acid and mitomycin C that

FIG. 3. Scanning electron micrographs of E. coli NIHJ JC-2 induce filamentation of E. (oli (6). Thus, bulgecin appears toexposed for 2 h to (per milliliter) (a) nothing, (b) bulgecin (10 j.g), (c) recognize cellular structures damaged by 1-lactam antibiot-cefmenoxime (0.1 ,ug), and (d) bulgecin (10 ,ug) plus cefmenoxime ics as the target of its action. The action mechanisms of(0.1 ,ug). Bars indicate 1 p.m. ,B-lactam antibiotics, though extensively studied, remain

416 NAKAO ET AL.

MODE OF ACTION OF BULGECIN 417

FIG. 5. Transmission electron micrographs showing cell surface structures of E. coli NIHJ JC-2 exposed for 2 h to bulgecin (10 ,ug/ml) pluscefmenoxime (0.1 jig/ml), Note that the outer membrane facing mesosomelike structures (M) protrudes to make blebs (B), and thepeptidoglycan layer (PG) is obscure at the region where bleb formation is seen. Bars indicate 0.1 ,um.

obscure; further investigation on the mode of interaction ofbulgecin with P-lactam antibiotics might contribute to thebetter understanding of these mechanisms.

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Interaction of nocardicin A with the penicillin-binding proteinsof Escherichia coli in intact cells and in purified cell envelopes.Eur. J. Biochem. 126:155-159.

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6. Imada, A., K. Kintaka, M. Nakao, and S. Shinagawa. 1982.Bulgecin, a bacterial metabolite which in concert with ,B-lactamantibiotics causes bulge formation. J. Antibiot. 35:1400-1403.

7. Inoue, S., T. Tsuruoka, H. Goi, K. Iwamatsu, K. Miyauchi, T.Ishii, A. Tamura, Y. Kazuno, and M. Matsuhashi. 1984. Struc-ture-activity relationships on the terminal D-amino acid moietyof a novel cephamycin MT-141. J. Antibiot. 37:1403-1413.

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9. Ishino, F., S. Tamaki, B. G. Spratt, and M. Matsuhashi. 1982. Amecillinam-sensitive peptidoglycan cross-linking reaction inEscherichia coli. Biochem. Biophys. Res. Commun. 109:689-696.

10. Nozaki, Y., A. Imada, and M. Yoneda. 1979. SCE-963, a newpotent cephalosporin with high affinity for penicillin-bindingproteins 1 and 3 of Escherichia coli. Antimicrob. Agents Che-mother. 15:20-27.

11. Otsuki, M. 1981. Synergistic effect of cephalexin with mecil-linam. J. Antibiot. 34:739-752.

12. Shinagawa, S., M. Maki, K. Kintaka, A. Imada, and M. Asai.1985. Isolation and characterization of bulgecins, new bacterialmetabolites with bulge-inducing activity. J. Antibiot. 38:17-23.

13. Sonntag, I., H. Schwarz, Y. Hirota, and U. Henning. 1978. Cellenvelope and shape of Escherichia coli: multiple mutants miss-ing the outer membrane lipoprotein and other major outermembrane proteins. J. Bacteriol. 136:280-285.

14. Spratt, B. G. 1975. Distinct penicillin binding proteins involvedin the division, elongation, and shape of Escherichia coli K12.Proc. Natl. Acad. Sci. USA 72:2999-3003.

15. Tsuchiya, K., M. Kondo, M. Kida, M. Nakao, T. Iwahi, T. Nishi,Y. Noji, M. Takeuchi, and Y. Nozaki. 1981. Cefmenoxime(SCE-1365), a novel broad-spectrum cephalosporin: in vitro andin vivo antibacterial activities. Antimicrob. Agents Chemother.19:56-65.

16. Tsuruoka, T., Y. Yamada, H. Goi, K. Miyauchi, A. Miyata, andS. Inoue. 1985. The bacteriolytic action of MT-141, a newcephamycin antibiotic, on gram-negative bacteria. J. Antimi-crob. Chemother. 15:159-171.

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