1086 THE JOURNAL OF ANTIBIOTICS AUG.198Z

SYNERGISTIC EFFECTS OF A MACROLiDE AND A CELL WALL-AFFECTING ANTIBIOTIC ON PSEUDOMONAS AERUGINOSA

IN VITRO AND IN VIVO

3.INCORPORATION OF[14C]MIDECAMYCIN ACETATE(MOM)† INTO P.AERUGINOSA PRETREATED WITH CELL

WALL-AFFECTING. ANTIBIOTICS

TAKAo KAsAI,*TosHlo ToMITA, SHIRo KANEGAsAKI

and J, YuzuRu HoMMA**

Department of Bacteriology,Institute of Medical Science, Universityof Tokyo, Shiroganedai, Minato-ku, Tokyo lO8, Japan

(Received for publicatioll April l7,1982)

Thc occurrence inρ 一lactam treated patiellts of unstable L-forms of Pseudomonas aeruginosa hlscnsitivc to various antibiotics and synergistic effect ofcombined action of celi wa11-affecting antibiotics and macrolide on Pseudomonas infection led us to examine thc effects of macrolidc

on P.aeruginosa pretreated with cell wail-affbcting antibiotics.

The effects of macrolide antibiotics such as midecamycin acetate(MOM)on P. aeruginosa was investigated, a rapid killing effect by MOM was lloted after treatmellt wkh

suboptimal doses of ce11 wa11-affecting antibiotics such as polymyxin B, carbenicillin, dibekacill

or fosfomycin.

IIlcorporation of [1.4C]MOM lllto intact P.aeruginosa cells was not significant, but was apparent into L-form cells or cells pretreated wiIh cell wall-affecting antibiotics, The incor-

porated radioactlvity was found in thc 70 S ribosome fractio11, binding with the 50 S subunits of ribosome in both cases,

These results indicate tllat under certain conditions a macrolidc antibiotic can emer the P.aeruginosa cell,

YAMAMoTo and HoMMA1)reported isolating unstable L-forms from clinical specimens of patients

with chronic respiratory tract infection and cow mastitis due to P.aeruginosa who were undergoingβ 一

l actanl antibiotic therapy. They foしmd that these L-fbrms were resistant to almost all the antibiotics

which were effective against the parent bacillary fbrms, but were sensitive to macrolide antibiotics such

as midecamycin(MDM)and its derivative midecamycin acetate(MOM), which were completely ln-

effective against the parent bacteria.2)KAwAHARAJoρ ∫θ人3)showed that the spheroplast of P.aerugi^

nosa induced by carbellicillin become sensitive to MDM and MOM in vitro, and they aiso demonstrated in viv o higher survival rate among Inice treated with both carbenicillin alld MDM or MOM than amollg

mice treated with only one of the two above kinds ofantibiotics, KAsAI and HoMMA4・5)showed in their

previous reports the efectiveness of MDM of MOM in vitro and in vivo on P.aeruginosa in the presencc

of cell wal1-affecting antibiotics such as polymyxin B, dibekacin and fosfonlycin. t Geneticname of midecamycin acetate(MOM),9,3"-di-0-acctylmidecamycin. Present address

* Central Research Laboratories, Meiji Seika Kaislla、 Ltd.,760, Morooka-cho, Kohoku-ku, Yokohama

222,Japan ** The Kitasato Institute,5-9-l Sllirokane, Minato-ku, Tokyo 108,Japan VOL. XXXV NO.8 THE JOURNAL OF ANTiBIOTICS 1087

In this report we show that the macrohde antibiotic is actuaily incorporated into P.aeruginosa pretreated with cell wall-affectingantibiotics. We further show that the incorporated macrollde binds with the 50 S subunit of ribosome.

Materials and Methods

Bacterial Strains

Pseudomonas aeruginosa strain IFO 3455 was klndly provided by Dr. KAzuYuK[MoRIHARA, Shiono-

gi Research Laboratory, Osaka, and strain No.5by Dr. YAsuYuKI NAKAsE, The Kitasato hstitute, Tokyo. The stable L-form of P. aeruginosa derived from strain IFO 3455 was reported previously.'.D

Culture Media alld Buffer

Nutrient broth(Difco)stlpplemellted with O.5%sucrose(NB-S)was used for propagation of baciレ Iary fbrm strains of P. aeruginosa . Brain heart infusloll broth(Difco)supplemented with 4%NaC1

(BHI-N)was used fbr propagation of L-fbrms.

Lytic buffer7) contained O.04 M tris-HCI(pH 8.0),0.02 M magnesium acetate,16mM 2-lnercapto- ethanol,1μg per ml of deoxyribonuclease(DNase, Worthington Biochemical Corp)and l%or Brij 58

〈Nakarai Chemical Co., Ltd.)was used.

Antibiotics

Dibekacin sulfate(DKB)and fosfomycin disodium(FOM)were obtained from MeijiSeika Kaisha, Ltd. Tokyo. Polymyxin B sulfate(PL)was purchased from Taito Pfizer Co., Ltd. Tokyo. Carbenicil-

Hn sodiun1(CBPC)was kindly provided by Fujisawa Pharmceutical Co., Ltd. Osaka.

Labcled Antibiotic

1℃ 一Labeled midecamycin acetate([14C]MOM)was synthesized from midecamycin(MDM)with [1-14C]acetic anhydride by Dr . ToMoKo SHoMuRA, Central Research Laboratory, Meiji Seika Kaisha,

Ltd. Yokohama, according to the nlethod previously reported.3) Radioactive label is localized only

in 9-acetyl radical in the final product(specific activity:9.461`ci/m9). The results of microbiological

assay of[14qMOM showed essentially the same as those of unlabcled MOM.[14qMOMwas dissolved

in ethanol and prepared to 5×104 counts per 111inute(cpm)per m1(2.2 nCi/m1;2.4μg/ml)wlth NB-S

or BHI-N Inedium before the experiments.

Ribosome of Escherichia coli

70Sribosome of E. co1i Q13was supplied by.Dr. SHuN NAKAMuRA of this illstitute.

Incorporation of[14C]MOM into Bacteria

One ofthe antibiotics, PL, CBPC, DKB or FOM,was added to the culture of P. aeruginosa in early logarithmic growth phase(109 cells/ml)and incubated at 37C for either l or 2 hoし1rs.011e ml[14C]MOM

solution was mixed with an equal volume of bacterial suspension(O.D600nm=1.0,109 cells/ml)thus pre-

pared. The mixture was further illcubated at 37C for the period indicated in the text. The bacterial

ce11s were chilled at 5C and washed twice with O.01 M tris-HCI buffer(pH 7.0)containing O5 M sucrose.

The L-form ceHs in early logarithmic growtll phase were concentrated to 109 cells/ml by centrifuga- tion. The cells in l ml and[14C]MOM solutioll in l ml was lnixed and incubated at 37℃ for the indi-

cated period. The cells were washed with O。01 M tris-HCI(pH 7.0)containing 4%NaCI after incubation,

and the radioactivity remaining in the pe11et was counted after washing.

Distribution of[14C]MOM Incorporated in the Cell Fractions

Bacterial cells incubated with[14C]MOM, washed and suspended in O,01 M tris-HCI(pH 8.0)were mixed with an equal volume oflytic buffer. After 30 minutes of incubation at room temperature,1ysate

was centrifuged at I2,000×g for 20 minutes and supernatant was obtained. One ml of the supernatant

was laid on 4 ml of O.4 M to 1.3 M linear sucrose gradlent in buffer A containing O.Ol M tris-HCI, pH

75,0.01Mmagnesium chloride,0.06 M potassium chloride and 5 mM 2-mercaptoethanol. The gradi-

ent was subjected to centrifugation at 240,000×g for 50 minutes in a Beckman Splnco SW50-1 R rotor

at 0C. The fractions were coHected by punching the bottom of the tubes. 1088 THE JOURNAI. OF ANTIBIOTICS AUG.1982

Preparation of Ribosome and its Subunits of P. aeruginosa

Ribosome was prepared according to the method described by NIRENBERG and MATTHAEI.9)Bac- terial ce11s(2.5 g wet weight)were ground with alumilla(5 g)in ice bath fbr 15 minutes. Five ml of

buHbr B(0.02 M tris-HCI, pH 8.0,0.01 M magnesium acetate,0.02 M ammonium chloride and 5 mM 2-

mercaptoethanol)and DNase(3μg/ml)was added. After centrifugation at 12,000×g for 20 minutes,

supernatant was laid on buffer B containing L2 M sucrose. Centrifugation was perfbrmed at 210,000×

8R)r 4 hours in a Beckman Spinco type 65 rotor at OQC. The pellet containing ribosome ln the bottom

was resuspended in buffer C(0.02 M tris-HCI, pH 7.5,0.Ol M magnesium acetate,1Mammonium chloride

and 5 mM 2-mercaptoethanoD, After overnight agitation, the mixture was centrifuged at l 20,000×8

in a Beckman Spinco type 65 rotor. The procedurc was repeated three times. Purified ribosomes were

suspended in buffer B supplemented with 50%glycerol and stored at-80℃.

The ribosome丘action was Iaid either on O.4 to 1。3 M of linear sucrose density gradient in buffer Aor O.15Mto O.6 M gradjent in buffer D(0.02 M tris-HC1, pH 75,0.001 M magnesium acetate,0.1 M

amlnonium chloride and 5 mM 2-mercaptoethanol). The centrifugation was perR)rmed at 240,000×8

fo r50 minutes or at 190,000×g for 120 minutes with SW50-l R rotor.

AssaX oF Radloactivity..

The volume ofO5 ml of tlle sample was dissolved in 5 ml ofBRAゾs scintillator solution10)and radio- activity was counted using the Beckman liquid scintmator.

ResuIts

Illcorporation of[14C]MOM into LForm Cells and Parent Bacmary Ce11s Pretreated

with Cell Wa11-aHbcting Antibiotic

[14qMOM was incorporated into the L-form cells but not into the parent ce11s of P. aeruginosa

Fig. l shows the time course ofincorporation of[14C] MOM into L-form cells and its parent bacillary cells.

Incorporated radioactivity in L-fbrm cells increased up to 60 minutes and stayed at the same level fbr

120nlinutes,

The parental cells(strain IFO 3455)were pretreated with 50 units(U)/ml of PL for l hour,500μg/

ml of CBPC fr 2 hours or 3.13μg/ml of DKB R)r 2 hours. The bacterial cells treated with PL, CBPC

orDK.Bwere found to incorporatemore efficiemntly Fig.1. Incorporation of[14C]MOM into P. aerugi- as compared to cells not treated with antibiotics nosa strain IFO 3455 and its L。form.

(Fig.2A). The radioactivity increased lO to 20 One ml of logarithmic phase culture of L-form minutes after addition of[14C] MOM and retained cells(10g cells/mi)or parent cells(109g cells/ml)in BHI

medium supplemented with 4%sodlum chloride the same Ieve1 for 60 minutes. As shown in Fig. were mlxed with an equal volume of[14C]MOM 2B, the incorporation of[14C] MOM into the cells solution(5×104 cpm/m1)in the same medium. of strain No.5pretreated with 500μg/ml of FOM. After incubation at 37。C for indicated periods, bac- terial cells were removed and radioactivity counted. fo r2hours increased rapidly until lO minutes

and at a slower rate until at least 60 minutes after

addition of the isotope.

Effect of different concentrations of PL and

FOM was investigated. As shown in Fig.3A, at

concentrations above 3.13U/ml of PL, the incor-

poration of[14C] MOM into bacterial cells in-

creased linearly depending on the concentratiol1.

As shown Fig.3B, in bacterial ce11s pretreated with

FOMat concentrations above O.0625μg/ml, in一 VOL. XXXV NO.8 THE JOURNAL OF ANTIBIOTICS 1089

Fig.2. Incorporation of[14CjMOM into.P.aerugi- Fig.3. Incorporation of[14C]MOM into P.aerugi-

nosa cells pretreated with the cell wall-afkcting nosa cells treated with various concentrations of antibiotics. antibiotics.

P.aeruginosa cells in NB medium supplemented One ml each of.P.aeruginosa cells(10g ce11s/m1), with O5 M sucrose were incubated with the cell wall- which had been incubated with indicated concentra-

affecting antibiotics as indicated. The pretreated tions of PL or FOM in NB medium supplemented

cells were concentrated to 109 cells/ml and mixed with O.5 M sucrose, and an equal volume of[14q- with an equal volume of prewarmed[14C]MOM(5× MOM solution(5 x lO4 cpm/ml)in the same medium

104cpm/ml)in the same medium. The mixture was were mixed and incubated at 37℃ for 30 minutes.

incubated at 37。C for indicated periods. Cells were Cells were washed by centrifugation and the radioac- washed by centrifugation and the radioactivity of tivity of the precipitated cells was counted. precipitated cells were counted. Strain IFO 3455was pretreated with PL at varying A: Strain IFO 3455 concentrations for l hour(A), strain No.5was pre-

0Pretreated with PL at 50 u/ml for l hour. treated with FOM at varying concentrations for 2 △Pretreated with CBPC at 500μg/ml) for hours(B). 2hours.

口Pretreated with DKB at 3」3μg/ml for 1 hour.

●Not pretreated with antibiotic. B: Strain No.5

0Pretreated with FOM at 1,000μg/ml for 2 hours.

●Not pretreated with antibiotic.

corporation of[14C]MOM also increased witk

Concentration.

Fate of[14C]MOM Incorporated into

Bacterlal Cells Pretreated with

the Cell Wal1-affecting Antibiotics

The fate of[14C]MOM incorporated into the

cells was tested by cell fractionation. The results

of sucrose density gradient analysis of incor。

porated radioactivity are shown in Fig.4. In all

cases tested(prctreated woth one of four antibio-

tics, PL, CBPC, DKB and FOM)the maximum absorption peak of 260 nm and Inaximum radioactivity peak were observed in the same position, where 70 S ribosomes of E.coli Q13 migrated. These results suggested that MOM binds with 70 S ribosome of P.aeruginosa if the cells have been treated with cell wall-affbcting antibiotics.

Binding of [14C]MOM to Ribosome and its Subunits of P.aeruginosa

We investigated further to confirm that[14C]MOM is incorporated into 70 S ribosome of P.aerugi-

nosa.The ribosome was prepared from bacterial cells of P.aeruginosa IFO 3455 which had been in-

cubated with CBPC(1,000μg/m1)and[14C]MOM. Starting from 2.5 g wet weight cells,275 A260 units 1090 THE JOURNAL OF ANTIBIOTICS AUG.1982

Fig.4. Distribution in sucrose density gradient of [14C]MOM incorporated into P.aeruginosa cells pre-

treated with the cell wall-affヒcting antibiαics. The bacterial cells(109 ceU/ml)which had incorporated【14C】MOM as described in Materials and Methods were suspended in O.01 M tris-HCI bu恥r(pH 8.0)and mixed with an equal volume of lytic buffer(containing O.04 M tris-HC1,0.02 M magnesium acetate,16mM 2-mercaptoethanol,1μg per ml of deoxyribonuclease and l%of Briji 58 at pH 8.0). The supernatant of lysate was laid on O.4 M to 1.3 M sucrose density gradient in buffer containing O.01 M tris-HC1,0.Ol M magnesium chloride,0.06 M potas- sium chloride and 5 mM 2-mercaptoethanol at pH 7.5. Centrifugation was performed at 240,000×g for 50 minutes. The arrow indicates 70 S ribosome of E.coli Q13.

Fig.5. Incorporation of[14qMOM into ribosomes of P.aeruginosa IFO 3455. The ribosome obtained from P.aeruginosa IFO 3455 which had incorporated[14]MOM as described in Materials and Methods was Iaid either on O.4 M to l.3 M sucrose density gradient in buffer containing O.Ol M tris-HC1,0.01 M magnesium chloride,α06 M potassium chloride, and 5 mM 2-mercaptoethanol at pH 7.5(A)or O.15Mto O.6Msucrose density gradient in buffer containing O.02 M trls-HC1,0.001 M mag- nesium acetate,0.1 M ammonium chloride, and O.05 M 2-mercaptoethanol at pH 7.5(B). Centrifuga- tion was performed at 240,000×8 for 50 minutes and at I90,000×gfor 120 mlnutes, respectively. VOI.. XXXV NO.8 THE JOURNAL OF ANTIBIOTICS 1091

of purified ribosomes were obtained. A portion of the purified ribosomes were laid on 5 ml of O.4 M to 1.3Mlinear sucrose density gradient in buffer A(pH 7.5)and centrifuged at 240,000×8fbr 50 minutes.

As shown in Fig.5A, thc radloactivity was found to migrate to where 70 S ribosomes migrated. Using

5ml ofO」5Mto O.6 M linear sucrose density gradient in buffer D(pH 7.5),70 S ribosomes were dissociat- ed into 50 S and 30 S subunits(Fig.5B). The radioactivity was fbund to migrate to where 50 S ribo- somal subunits migrated. No radioactMty was found by the 30 S ribosomal subunits.

Discussion

The results of the present study indicate that[14C]MOM is able to penetrate into the cytoplasm of P. aeruginosa wh en the cells have been treated with PL, DKB, CBPC or FOM. The incorporated radioac- tivity was fbund in the 70 S ribosome fraction. Further analysis revealed that the[1℃]MOM was speci- fically bound to 50 S ribosomal subunits. There are reports which suggest that sensitivity to antibotics

are due to modification ofthe cell wall including the outer membrane. For example, lipopolysaccharide- deficient(rough)mutants of E. co1i and Salmonella typhimurium were more sensitive to macrolide and aminoglycoside antibiotics than their smooth strains.11・12) The mutants of.P.aeruginosa deficient in hpopolysaccharides were more sensitive to CBPC than smooth strains.13) The outer membrane per-

meability ofcephalosporin in porin-deficient mutants of Gram-negative organisms was shown to be lowe .r than that ofpenici11in.14) However, YAMAMoTo and HoMMA2)demonstrated that incorporation of DKB into the Lfbrm cells of、P.aeruginosa was very slight as compared with that of the parent bacillary form.

This result indicates that even when the cell wall(outer membrane and peptideglycan Iayer)of P.aerugi-

〃nosa is affected or completely defective, the bacteria is not necessarily more susceptible to all antibiotics.

The results obtained in the prescnt study indicate that both L-form cells and bacterium pretreated with ce11 wall-affecting antibiotics incorporate MOM and as a result, protein synthesis is inhibited.

Unstable L-fbrms were isolated from specimens of patients with chronic respiratory tract infection, urinary tract infection and cow mastitis due to P.aeruginosa who had been administeredβ-lactam anti- biotics.1) Osmolarity of the specimens of these patients was sufHcient enough to allow the L-forms to live. In order to stop recurring infbction, it is also necessary to eliminate spheroplasts and/or L-forms of

P.aeruginosa.Based on an ecological viewpoint of P.aeruginosa and its L-forms or spheroplasts ill patients, we demonstrated in vitro and in vivo experiments that the combined use of a macrolide and a ceU wall-affecting antibiotic, PL, DKB, CBPC or FOM, is effective.4・5)

Acknowledgment

This study was supported in part by a grant from the Ministry of Education, Science and Culture, Japan.

References

.1)Y AMAMoTo, A.&J. Y、 HoMMA:Isolatlon of unstable L-forms ffom cllnical speclmens with Pseudomonas

aeruginosa infection during antibiotic therapy. Jap. J. Exp. Med.49:361～364,1979 2) YAMAMoTo, A.&J. Y. HoMMA:L-Form of Pseudomonas aeruginosa.2, Antibiotic sensitivity or L-forms

and their parent forms. Jap, J. Exp. Med.48:355～362,1978 3) KAwAHARAJo, K.;T. KAsAI&J. Y. HoMMA:Combined action of semisynthetlc penicillln and macroHde

antibiotic on.Pseudomonas aeruginosa in vitro ans in vivo. Jap. J. Exp. Med.49:331～336,1979 4)KAsAI, T.&J. Y. HoMMA:Synergistic effects of a macrolide and a cell wall-affecting antibiotic on Pseudo-

monas aeruginosa in vitro and in vivo. Comblned effects ofamacrolide and a peptide antibiotic. J. Anti- biotics 35:343～348,1982 5) KAsAI, T.&J. Y. HoMMA:Synergistic effects of a macrolide and a cell wall-affecting antibiotic on Pseudo-

monas aeruginosa in vitro and in vivo.2. Combined effects of a macrolide with a fosfomycin and an amino- glycoside antibiotic. J. Antibiotics 35:858～865,1982 6)YAMAMoTo, A.&J. Y. HoMMA:L-Form of Pseudomonas aeruginosa. 1. effective method for the pro-

duction of stable L-fbrm. Jap. J. Exp. Med.48:219～226,1978 1092 THE JOURNAL OF ANTIBIOTICS AUG.1982

7 .)GoDosoN, G. N.&R, SINsHE1MER:Lysis of Escherichia coli with a neutral detergent. Biochem. Biophys. Acta 149: 476～488,1967 8) NAKAMuRA, T.;S. FuKATsu, S. SEKI&T. NlIDA: Aconvenient method fbr the preparation of the acylated

macrolide antibiotic midecamycin using molecular sieves and acylchloride. Chem. Letter 1978:1293～

1296,1978 9)NIRENBERG, M. W,&J. H. MATrHAEI:The dependence of cell一free protein synthesis in E coli upon natur.

ally occurring or synthetic polyribonucleotides. Proc. Nat. Acad. Sci. U. S.47:1588～1602,1961 10)BRAY, G, A.:Asimple efficient Iiquid scintillator fbr counting aqueous solution in a Iiquid scintnlation

counter. AnaL Biochem.1:279～285,1960

11) SANDERsoN, K. E.;T. MAcAusTER&J. W. CosTERToN:Permeability of Iipopolysaccharide-deficient (rough)mutants of Salmonella typhimurium to antibiotics, Iysozyme, and other agents. Can, J. Microbio1.

20:1135～1145,1974 12)TAMAKI, S.&M. MATsuHAsHI:Increase in sensitivity to antibiotics and Iysozyme on deletion of lipopoly-

saccharides in Escherichia aoli strains. J. Bacteriol. l l 4:453～454,1973 13)KovAL, S. F.&P. M. MEADow:The isolation and characterization of lipopolysaccharide-defective mu-

tants of Pseudomonas aeruginosa PACL J. Gen. Microbio1.98:387～398,1977 14)SAwAI, T.;R。 HIRuMA, M. SoNoDA&N. KAwANA:Outer-layer permeability ofβ 一lactam antibiotics in

Gram-negative bacteria。3rd Tokyo Symp. Microb..Drug Resistance. p.42,0ct.23～25,198 i 15, MAsHIMo, K.;J. Y. HoMMA, Y. IKI, K.1NAToMI, T. KAsAI,0. KuNn, K. SHIMADA, H. TANIMoTo, T. TAKI-

GAMI&Y. NIsHIMuRA:Combined therapy using 9,3”-di-0-acetylmidecamycin withβ 一1actam antibiotic against Pseudomonas aeruginosa infection. Jap.J. Exp. Med。51:271～285,1981