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Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 105( 1 ) Fosfomycin b- 2004 11 24 fosfomycin (FOM) FOM b- (cefazolin (CEZ), cefotiam (CTM), cefmetazole (CMZ) piperacillin (PIPC)) b - AmpC b- FOM glucose-6-phosphate (G6P) Mueller-Hinton agar (MHA) MHA 22 36 10 15 2 128 G6P nutrient agar (NA) NA 18 28 8 14 2 256 FOM G6P G6P b - 2 G6P Mueller- Hinton broth Escherichia coli, Pseudomonas aeruginosa FOM 4 MIC 2 P. aeruginosa Staphylococcus aureus 1/100 P. aeruginosa PAO1 Enterobacter cloacae ATCC13047 AmpC b - FOM 1/25 1/65 b- b - MIC FOM b - MIC b - MIC P. aeruginosa MexAB-OprM, MexCD-OprJ, MexXY/OprM MexEF-OprN

106( 2 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. FOM b- MexAB-OprM MexCD-OprJ FOM AmpC Fosfomycin (FOM) 1) 2) 20 FOM b - 3) FOM 4) FOM FOM 2 sn- -3- (G3P transport system: GlpT system) 5) (sugar phosphate transport system: UhpT system) 6) glucose-6-phosphate (G6P) G6P 7) FOM G6P 8) in vivo in vitro 1975 FOM MIC nutrient agar (NA) FOM 1) NCCLS UhpT system FOM (Mueller- Hinton agar: MHA) G6P 9,10) 11 14) FOM MIC ATCC FOM MIC 4 MIC (MHA) NCCLS (G6P MHA) FOM (NA) FOM NCCLS G6P NA b - FOM AmpC b -

Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 107( 3 ) b - MIC FOM I. 1 FOM cefazolin CEZ cefotiam CTM cefmetazole CMZ piperacillin PIPC FOM CEZ CTM CMZ PIPC 2 4 11 18 25 5 5 3 5 ) b - Staphylococcus aureus, S. epidermidis, Streptococcus pyogenes, S. pneumoniae, Enterococcus faecalis, E. faecium, E. hirae, Micrococcus luteus Escherichia coli, Citrobacter freundii Salmonella typhimurium, Shigella flexneri, Klebsiella oxytoca, K. pneumoniae, Enterobacter cloacae E. aerogenes, Serratia marcescens, Proteus mirabilis, P. vulgaris, Morganella morganii, Providencia rettgeri Pseudomonas aeruginosa, Haemophilus influenzae, Burkholderia cepacia Stenotrophomonas maltophilia Acinetobacter calcoaceticus Alcaligenes xylosoxidans A. faecalis, Neisseria gonorrhoeae, N. meningitidis Moraxella catarrhalis Peptostreptococcus anaerobius, Clostridium histolyticum, Finegoldia magna, Collinsella aerofaciens Eggerthella lenta Bacteroides fragilis, B. ovatus, B. thetaiotaomicron, Prevotella bivia, Fusobacterium varium b- S. aureus 15), E. coli 16,17), H. influenzae P. aeruginosa 18) blaz, TEM-1 Toho-1, IMP-1, AmpC, ROB-1 b - isogenic 8 12 P. aeruginosa PAO1 isogenic MexAB-OprM, MexCD-OprJ, MexXY/OprM MexEF-OprN OprD 19) isogenic ampc 3 (MIC) 20) MIC 1) 25 m g/ml G6P (Sigma) MIC 37 C Mueller-Hinton broth (MHB, Difco) 10 6 CFU/mL Mueller- Hinton agar (MHA Difco) ( ) 37 18 MIC MIC ( ) 37 C 1000 NA (Difco) MIC S. pyogenes, S. pneumoniae, M. catarrhalis MIC 5% MHA Neisseria H. influenzae MHA 5%

108( 4 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. MHB McFarland 0.5 100 Proteus P. rettgeri M. morganii MHB NB 3.5% Bacto Agar (Difco) GAM ( ) GAM ( ) MIC MIC S. aureus ATCC 29213, S. aureus FDA 209P JC-1, S. pneumoniae ATCC 49619, E. coli ATCC 25922, E. coli NIHJ JC-2, B. thetaiotaomicron ATCC 29741 4. AmpC b - P. aeruginosa PAO1 E. cloacae ATCC 13047 MHA MHB 37 C MHB 20 3 FOM 1, 3, 10, 30, 100 mg/ml 3 CEZ, CTM, CMZ 1, 10, 100 mg/ml, PIPC 0.1, 1, 10 m g/ml (5,600 g, 10 minutes, 4 C) 1/15 M (ph 7.0) b - 80 C 1 ml 1/15 M (ph 7.0) (11,500 g, 60 minutes, 4 C) b - b- cephalothin (CET, Sigma) UV 21) 1 1 mmol 1 unit (unit/mg protein) DC (BioLad) b- 3 mean S.D. 0.1 m g/ml imipenem IPM E. cloacae ATCC 13047 5. S. aureus ATCC 29213 MHA G6P FOM MIC: 1 m g/ml E. coli ATCC 25922 1 m g/ml MHB 10 5 CFU/mL MHB 37 C 2 1/2, 1, 4, 16, 64 MIC FOM 25 mg/ml G6P 1, 2, 4, 6 (CFU/mL) P. aeruginosa MSC-996 32 mg/ml 2 1/2, 1, 4, 16 MIC FOM 25 m g/ml G6P 6. P. aeruginosa MSC-996 4 MIC FOM G6P 2 4 2.5% 1% Spurr EM (TEM; JEM-2000EXII, JEOL) t- (JFD-300, JEOL) (SEM; JSM-6340F, JEOL) 22)

Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 109( 5 ) II. 1 MIC 4 FOM MIC Tables 1, 2 MHA G6P 4 11 3 5 NA G6P 3 8 2 5 FOM 2 4 (Table 1) MHA G6P 18 25 7 10 NA G6P 15 20 6 9 FOM 2 128 2 256 (Table 2) FOM (8 256 ) Proteus G6P FOM G6P (Table 3) b - FOM G6P (data not shown) 2 FOM FOM S. aureus ATCC 29213 4 1/100 (Fig. 1A) E. coli ATCC 25922 2 64 MIC 6 16 MIC 2 (Fig. 1B) P. aeruginosa MSC-996 (Fig. 1C) 3 P. aeruginosa FOM 2 P. aeruginosa MSC- 996 SEM (Fig. 2A) (Fig. 2B, C) FOM 4 (Fig. 2D, E) FOM 2 TEM (Fig. 3A) FOM (Fig. 3B, C) FOM 4 (Fig. 3D, E) 4 AmpC b- P. aeruginosa PAO1 E. cloacae ATCC 13047 AmpC b - 0.007 0.004, 0.065 0.013 unit/mg protein P. aeruginosa PAO1 FOM PIPC b - b - CMZ CTM CEZ (Fig. 4A) E. cloacae ATCC 13047 50 130 b - FOM PIPC 2.0, 2.8 (Fig. 4B) 0.1 m g/ml IPM 4.28 0.51 unit/mg protein b- 5 b- E. coli c 1037 b - b - MIC FOM MIC b - MIC NA G6P MIC (Table 4) S. aureus H. influenzae P. aeruginosa b -

110( 6 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. Table 1. Drug susceptibility of aerobic Gram-positive bacteria of standard strains.

Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 111( 7 ) Table 2. Drug susceptibility of aerobic Gram-negative bacteria of standard strains.

112( 8 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. Table 3. Drug susceptibility of anaerobic bacteria of standard strains.

Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 113( 9 ) Fig. 1. Bactericidal activity of FOM against S. aureus ATCC 29213 (A) (MIC of FOM: 1 m g/ml), E. coli ATCC 25922 (B) (MIC: 1 m g/ml), and P. aeruginosa MSC-996 (C) (MIC: 32 mg/ml). : control; : 1/2 MIC; : 1 MIC; : 4 MIC; : 16 MIC; : 64 MIC. b - NA G6P FOM MHA 1024 (K. oxytoca GN10650) FOM MIC b- ampc (KG5002, 5004, 5008) MIC b- MexAB-OprM MexCD-OprJ III. 6 P. aeruginosa PAO1 MexAB-OprM, MexCD- OprJ MexXY/OprM FOM MIC PAO1 (Table 5) P. aeruginosa PAO4222 MexEF-OprN KG4001 FOM MIC PAO4222 (MHA 128 m g/ml) FOM MexEF-OprN (data not shown) isogenic ampc FOM AmpC b - FOM 1980 2,23) 2003 FOM fosfomycin tromethamine 24) 25) FOM FOM UDP- GlcNAc 3) FOM G6P FOM

114( 10 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. Fig. 2. Scanning electron micrographs illustrating morphological changes in P. aeruginosa MSC-996 after treatment with FOM at four times the MIC. A: control; B and C: 2 hours after treatment; D and E: 4 hours after treatment. Bars indicate 200 nm. FOM MIC 5 mg/ml G6P S. aureus, E.coli, Shigella MIC 1) TSUBOI 40 G6P 4.0 1.0 m g/ml 8) NCCLS G6P (25 mg/ml) 9) FOM NA G6P MIC 2 (B. cepacia, A. xylosoxidans) 32 mg/ml MHA MIC in vivo 26) MHA MIC in vivo 27) FOM (BP) MHA 25mg/mL G6P 64 mg/ml 28) FOM

Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 115( 11 ) Fig. 3. Transmission electron micrographs illustrating morphological changes in P. aeruginosa MSC-996 after treatment with FOM at four times the MIC. A: control; B and C: 2 hours after treatment; D and E: 4 hours after treatment. Bars indicate 200 nm. MIC BP 4 FOM MIC 4 BP BP FOM BARRY A. fosfomycin tromethamine E. coli, Citrobacter spp., Klebsiella spp., Enterobacter spp. G6P 29) Staphylococcus spp. Enterococcus spp. Pseudomonas spp. S. marcescens G6P FOM 4 G6P b -

116( 12 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. Fig. 4. AmpC b -lactamase expression in P. aeruginosa PAO1 (A) and E. cloacae ATCC 13047 (B) induced by FOM, CEZ, CTM, CMZ, and PIPC. Data were expressed as mean S.D. of three independent experiments. : FOM; : CEZ; : CTM; : CMZ; : PIPC. P. aeruginosa PAO1 E. cloacae ATCC13047 AmpC b- FOM 2 REGUERA J.A. b - cefoxitin 12.5 b - FOM 2.5 30,31) b- b - FOM (GlpT UhpT ) (mura ) FOM ( fosa fosb fosc) 23) 4 MexAB-OprM MexCD-OprJ MexXY/OprM MexEF-OprN 19) FOM FOM MIC b - MexAB-OprM MexCD-OprJ 32) b- 33) 1980

Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 117( 13 ) Table 4. Drug susceptibility of isogenic mutants carrying b-lactamase coded plasmid and clinical isolates.

118( 14 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. Table 5. Drug susceptibility of isogenic efflux mutants of P. aeruginosa PAO1.

Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 119( 15 ) MRSA 1999 (Centers for Disease Control and Prevention: CDC) surgical site infection (SSI) 13) Class II/Clean-Contaminated operation Class I/Clean operation 11,12) ampicillin (ABPC) CEZ FOM FOM CMZ flomoxef b - FOM clindamycin FOM S. aureus, E. coli, K. pneumoniae, Proteus spp. Enterococcus spp., P. aeruginosa Bacteroides. spp. 11 14) 5 FOM 1g 1.5 34) 11) 35) FOM 1 g 1 2 4 37.2 12.0, 18.3 6.1 mg/ml 34) 5 36) G6P 3 FOM MIC 100, 230 FOM 0.56% FOM 3 FOM 5 (0.005%) 2) E. coli CEZ MIC 128 m g/ml 37) 38) 11) FOM FOM MIC b - b - FOM b -

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Apr. THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 121( 17 ) anaerobic condition. J. Antimicrob. Chemother. 24: 657 666, 1989 28) GILBERT, D. N.; R. C. MOELLERING, Jr., G. M. ELIOPOULOS, et al.: The Sanford Guide to Antimicrobial Therapy 34 th edition, 2004, Antimicrobiology Therapy, Inc. Hyde Park, Vt, USA 29) BARRY, A. & P. C. FUSHS: In vitro susceptibility testing procedures for fosfomycin tromethamine. Antimicrob. Agents Chemother. 35: 1235 1238, 1991 30) REGUERA, J. A.; F. BAQUERO, J. BERENGUER, et al.: b -Lactam-fosfomycin antagonism involving modification of penicillin-binding protein 3 in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 34: 2093 2096, 1990 31) SUN, D.; S. COHUN, N. MANI, et al.: A pathway-specific cell based screening system to detect bacterial cell wall inhibitors. J. Antibiotics 55: 279 287, 2002 32) MASUDA, N.; E. SAKAGAWA, S. OHYA, et al.: Substrate specifities of MexAB-OprM, MexCD-OprJ, and MexXY- OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44: 3322 3327, 2000 33) NAKAE, T.; A. NAKAJIMA, T. ONO, et al.: Resistance to b -lactam antibiotics in Pseudomonas aeruginosa due to interplay between the MexAB-OprM efflux pump and b -lactamase. Antimicrob. Agents Chemother. 43: 1301 1303, 1999 34) Fosfomycin-Na Jpn. J. Antibiotics 31: 549 560, 1978 35) OHGE, H.; Y. TAKESUE, T. YOKOYAMA, et al.: An additional dose of cefazolin for intraoperative prophylaxis. Surg. Today 29: 1233 1236, 1999 36)Fosfomycin ( ) Chemotherapy 23: 3389 3395, 1975 37) 2002 Jpn. J. Antibiotics 57: 33 69, 2004 38) 27: 2358 2367, 1994 ANTIMICROBIAL ACTIVITY OF FOSFOMYCIN UNDER VARIOUS CONDITIONS AGAINST STANDARD STRAINS, b -LACTAM RESISTANT STRAINS, AND MULTIDRUG EFFLUX SYSTEM MUTANTS TAKESHI MIKUNIYA, TORU HIRAISHI, KAZUNORI MAEBASHI, TAKASHI IDA, TOSHIHIKO TAKATA and MUNEO HIKIDA Infectious Disease Research Laboratories, Meiji Seika Kaisha, LTD., 760 Morooka-cho, Kohoku-ku, Yokohama 222 8567, Japan SAKUO YAMADA Department of Microbiology, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701 0192, Japan NAOMASA GOTOH and TAKESHI NISHINO Department of Microbiology, Kyoto Pharmaceutical University, 5 Misasaginakauchi-cho, Yamashina-ku, Kyoto 607 8414, Japan The purpose of this study was to evaluate the possible benefit of fosfomycin (FOM) as prophylactic antibiotic in terms of antimicrobial activity and the potential of inducibility of b -lactamase, compared with cefazolin, cefotiam, cefmetazole, and piperacillin that are commonly used as perioperative agents. The in vitro activity of FOM against aerobic Gram-negative bacteria using Mueller-Hinton agar or nutrient agar supplemented with glu-

122( 18 ) THE JAPANESE JOURNAL OF ANTIBIOTICS 58 2 Apr. cose-6-phosphate (G6P) as tested medium increased within a range from 2 to 256 times the activity in the medium without G6P. However, the susceptibility of Gram-positive bacteria to FOM remained largely unchanged with or without G6P. There was no aerobic- or anaerobic-bacteria which changed susceptibility against b -lactam antibiotics under various tested medium conditions. FOM demonstrated strong bactericidal activity against Escherichia coli and Pseudomonas aeruginosa in a dose dependent manner, and decreased viable cell counts of Staphylococcus aureus. In the case of P. aeruginosa, transmission electron micrographs study revealed that numerous lysed cells were present 2 hours after treatment with FOM at four times the MIC. First and second generation cephalosporins induced AmpC-type b -lactamase in a dose dependent manner among b -lactamase inducible strains of P. aeruginosa and Enterobacter cloacae. On the other hand, inducible activity of FOM on b -lactamase production was less than 1/25 to 1/65 compared with those of cephalosporins. In addition, FOM maintained strong antimicrobial activity for over then 20 years after marketing, because of the excellent stability against various types of b -lactamase produced by plasmid-carrying bacteria and clinical isolates. FOM was not extruded by four types of efflux systems, such as MexAB-OprM, MexCD-OprJ, MexXY/ OprM and MexEF-OprN, however b -lactam antibiotics were substrates of MexAB-OprM and MexCD-OprJ. In conclusion, FOM provides adequate coverage for both aerobic Gram-positive and Gram-negative bacteria causing postoperative infections. Further, FOM would not select/concentrate b -lactamase producing bacteria in the clinical fields and would not be a substrate for multidrug efflux system of P. aeruginosa.

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