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.