Comparison of cefotiam and cefazolin activity against Gram-negative bacilli

Transcription

1 Journal of Antimicrobial Chemotherapy (1979) 5, Comparison of cefotiam and cefazolin activity against Gram-negative bacilli Michio Ogawa, Masayoshi Hama, Goro Kosaki Second Department of Surgery, Osaka University Medical School, Fukushima-ku, Osaka 553, Japan Masahiro Shimatani and Hidekaza Snginaka Department of Microbiology and Oral Bacteriology, Osaka University Dental School, Kita-ku, Osaka 530, Japan The activity of cefotiam, a new semisynthetic cephalosporin, was compared with that of cefazolin by the agar dilution method against 289 clinical isolates of Gramnegative bacilli. The minimal inhibitory concentrations of cefotiam for Escherichia coli and Klebsiella pneumoniae were about ten times lower than those of cefazolin. Moreover, cefotiam had a potent activity against indole-positive Proteus spp., Enterobacter cloacae, E. aerogenes and Citrobacter spp., which had low susceptibility to cefazolin. In contrast, the effect of cefotiam on the inhibition of peptidoglycan synthesis in cell free enzyme system from E. coli K12 was almost identical with that of cefazolin. The possible mechanisms of the discrepancy between the antibacterial activity and the inhibition of peptidoglycan synthesis were discussed. Introduction In recent years, postoperative infections due to Gram-negative bacilli constitute a serious complication in the surgical field. Cefotiam is a new semisynthetic cephalosporin, and was reported to show potent in vitro and in vivo antibacterial activities except Pseudomonas aeruginosa (Tsuchiya et al., 1978a). Moreover, the excretion of cefotiam in the bile was two to three times higher than that of cefazolin (Tsuchiya et al., 1978Z>). In the present report, in vitro antibacterial activities of cefotiam were investigated and compared with those of cefazolin against 289 clinical isolates of Gram-negative bacilli. The effect of cefotiam on the inhibition of peptidoglycan synthesis in cell free enzyme system from Escherichia coli was also compared with that of cefazolin. Micro-organisms Materials and methods A total of 289 different strains of clinically isolated Gram-negative bacilli except Pseudomonas aeruginosa were studied. The isolates came from wound secretion, sputum, urine O /79/O6O $01.00/ The British Society for Antimicrobial Chemotherapy

2 682 M. Ogawa et al. and blood specimens at the Osaka University Hospital and referring hospitals. They consisted of 34 Escherichia coli, 28 Klebsiella pneumoniae, 30 Proteus mirabilis, 18 P. vulgaris, 21 P. rettgeri, 16 P. morganii, 21 P. inconstans, 20 Providencia spp., 18 Enterobacter cloacae, 15 E. aerogenes, 13 Citrobacter spp., 23 Serratia marcescens, 21 Acinetobacter spp. and 10 Alcaligenes spp. The organism employed for the study of the inhibition of peptidoglycan synthesis was E. coli K12. Antibiotics Cefotiam (CTM) is 70-[2-(2-aminothiazol-4-yl)ac*tarmdo]-3-{[[l-(2-dimethylaminoethyl)- lh-tetrazol-5-yl]thio]methyl]-ceph-3-em-4-carboxylic acid, and was the product of Takeda Chemical Industries, Ltd., Osaka, Japan. Cefazolin (CAZO) was commercially available material. The minimal inhibitory concentrations (MICs) of CTM and CAZO for E. coli K12 were 005 and 1-56 ng/ml, respectively. Susceptibility test The organisms were tested by the agar plate dilution method. Approximately 10* organisms grown overnight at 37 C in Trypticase Soy Broth (BBL) were inoculated onto Mueller-Hinton agar (Eiken) prepared to contain CTM or CAZO in twofold dilutions. The MIC was determined after incubation at 37 C for 18 h. Assay for peptidoglycan synthesis The cell envelope as enzyme source was prepared from the exponential growing cells after disruption with alumina-grinding and then differential centrifugation as reported previously (Izaki, Matsuhashi & Strominger, 1968). Uridine 5'-diphosphate-A^-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelyl-D-P 4 C]alanyl-D-[ 14 C]alanine (UDP- MurNAc-pentapeptide, 66 mci/mmol) and uridine 5'-diphosphate-JV-acetylglucosamine (UDP-GlcNAc) were used as substrates. Total amount of peptidoglycan was expressed by the sum of counts corresponding to the origin (peptidoglycan) and its main degradation products as reported by Izaki, Matsuhashi & Strominger (1968). Results Comparative antibacterial activity of cefotiam and cefazolin against clinically isolated Gram-negative bacilli The in vitro antibacterial activities of CTM and CAZO against 289 isolates are summarized in Table I. All strains of E. coli, K. pneumoniae and P. mirabilis were inhibited by 1-56 ng/ml of CTM. Among them, E. coli and K. pneumoniae wete quite susceptible to this antibiotic, with 98 and 96%, respectively, inhibited by 0-2 ng/ml (data not shown). At 6-25 Jig/ml of CTM, all strains of P. vulgaris, P. inconstans, Providencia spp. and E. aerogenes were inhibited. Compared with CAZO, CTM had about tenfold or more increase in activity by weight to inhibit all the isolates tested except S. marcescens and Acinetobacter spp. Acinetobacter spp. were relatively resistant to CTM and 18% of isolates was inhibited by 6-25 ug/ml of CTM. S. marcescens was resistant to CTM and only 13 % was inhibited by 25 ug/ml of CTM (data not shown). Antibacterial activities of cefotiam against cefazolin-resistant strains There wete 119 strains not inhibited by 50 ug/ml of CAZO and classified as CAZOresistant. As shown in Table n, 85 strains (71 % of CAZO-resistant strains) were in-

3 Cefotiam and cefazolin in vitro activity 683 Table L Antibacterial activity of cefotiam and cefazolin against clinically isolated Gramnegative bacilli Organism No. of strains % inhibited by Concentrations (jig/ml)* for 1-56 ug/ml of 6-25 ug/ml of MIC (50) MIC (90) CTMCAZO CTMCAZO CTM CAZO CTM CAZO E.coli <0-l K. pneumoniae P.mirabitis P.vulgaris >200 P.rettgeri > 200 P.morganii >200 P. inconstans Providencia spp E. cloacae >200 E. aerogenes >200 Citrobacter spp S.marcescens >200 >200 >200 Acinetobacter spp >200 Alcaligenes spp *MIC(50) and MIQ90) represent concentrations of cefotiam and cefazolin required to inhibit the growth of 50% and 90% of isolated bacilli. Table n. Antibacterial activity of cefotiam against cefazolin-resistant Gram-negative bacilli Organism E. coli K. pneumoniae P. mirabilis P. vulgaris P. rettgeri P. morganii P. inconstans Providencia spp. E. cloacae E. aerogenes Citrobacter spp. S. marcescens Acinetobacter spp. Alcaligenes spp. No. of strains No. of CAZO" ( % of total strains) 0(0) 0(0) 1(3) 12(67) 11(52) 13(81) 7(33) 10(50) 13(72) 6(40) 3(23) 23(100) 18(82) 2(20) CAZO* represents strains not inhibited by 50 ug/ml of cefazolin. tctm' represents strains inhibited by 12-5 ug/ml or less of cefotiam. No. of CTM 1 1 (% of total CAZO* strains) 1(100) 12(100) 11(100) 13(100) 7(100) 10(100) 12(92) 6(100) 2(67) 2(9) 8(44) 1(50) hibited by 12-5 ug/ml or less of CTM, which is a clinically achievable level (unpublished data). All CAZO-resistant strains of P. mirabilis (15 strains), P. vulgaris (12 strains), P. rettgeri (11 strains), P. morganii (13 strains), P. inconstans (7 strains), Providencia spp. (10 strains) and E. aerogenes (6 strains) were susceptible to CTM at 12-5 ug/ml. On the

4 684 M. Ogawa et ai. ISO i i Ctfottam (/ig/ml) 0-3 l-o Ccfazolln (/xg/ml) Figure 1. Effects of cefotiam (left) and cefazolin (right) on peptidoglycan synthesis in cell free enzyme system from E. coli K12. Results are expressed as percent changes of control counts, o, Peptidoglycan;, released alanine. contrary, most strains of CAZO-resistant S. marcescens were also resistant to CTM, and 9 % (2 strains) of them was susceptible to CTM at this concentration. Effects of cefotiam and cefazolin on peptidoglycan synthesis in E. coli When the cell wall precursors, UDP-MurNAc-pcntapeptide and UDP-GlcNAc were incubated with the cell envelope from E. coli K12, incorporation of radioactivity into both lipid intermediate and peptidoglycan, accompanied by the release of D-alanine, was observed as previously reported (Izaki, Matsuhashi & Strominger, 1968). Various concentrations of CTM and CAZO inhibited the release of alanine and increased the amount of D-PKHJalanine incorporated into the peptidoglycan as shown in Figure 1. These indicated that CTM and CAZO inhibited cross-linking of peptidoglycan and formed partially uncross-linked peptidoglycan. The effects of CTM and CAZO on the inhibition of peptidoglycan synthesis were almost identical and the concentrations for 50 % inhibition of alanine release were in a range from 0-5 to 1-0 ng/ml. Discussion Our present investigation revealed that the excellent antibacterial activity of CTM was not parallel to the inhibition of peptidoglycan synthesis in cell free enzyme system from E. coli. CTM had a potent activity against clinically isolated Gram-negative bacilli except S. marcescens and Acinetobacter spp. Compared with CAZO, CTM had about ten fold, or even more increase in activity against these isolates. It was important that CTM was active against indole-positive Proteus spp., Citrobacter spp., E. cloacae and E. aerogenes, which had low susceptibility to CAZO. In contrast, the effect of CTM was found to be almost identical with that of CAZO on the inhibition of peptidoglycan synthesis catalyzed by the membrane system from E. coli K12. These results suggested that the stronger antibacterial activity of CTM might be attributed to: (1) its stability against hydrolysis by [J-lactamases, (2) its high permeability of the outer membrane of the Gram-negative bacilli, and/or (3) the different affinities of CTM and CAZO to penicillin binding proteins, transpeptidase and D-alanine carboxypeptidase.

5 Cefodam and cefazolin in vitro activity 685 Recently we observed that the addition of ethylenediamine-tetraacetic acid (EDTA) reduced the MIC of CAZO for E. coli K12 by two times but not the MIC of CTM. EDTA was reported to reverse the resistance of Gram-negative organisms (Hamilton- Miller, 1965; Weiser, Asscher & Wimpenny, 1968). We have reported the similar findings that the additions of EDTA caused the specific bindings of benzylpenicillin to the whole cells of P. aeruginosa (Suginaka, Ichikawa & Kotani, 1975) and reduced the MIC of cefsulodin for P. aeruginosa (Suginaka et al., 1979), indicating that EDTA increased the permeability of the outer membrane and enabled antibiotics to reach their target sites. The higher permeability of CTM through outer membrane of Gram-negative bacilli may be one of the reasons for the potent antibacterial activity of CTM. Moreover, we have found a preferential affinity of CTM to one of penicillin binding proteins of E. coli. At present, the specific mechanisms responsible for the excellent activity of CTM against Gram-negative bacilli remains unclear. Two observations mentioned above might be responsible. Details of them will be reported in a separate paper. References Hamilton-Miller, J. M. T. Effect of EDTA upon bacterial permeability to benzylpenicillin. Biochemical and Biophysical Research Communications 20: (1965). Izaki, K., Matsuhashi, M. & Strominger, J. L. Biosynthesis of the peptidoglycan of bacterial cell walls. Xin. Peptidoglycan transpeptidase and D-alanine carboxypeptidase: penicillin sensitive enzymatic reaction in strains of Escherichia coli. Journal of Biological Chemistry 243: (1968). Suginaka, H., Ichikawa, A. & Kotani, S. Penicillin-resistant mechanisms in Pseudomonas aeruginosa: binding of penicillin to Pseudomonas aeruginosa KM 338. Antimicrobial Agents and Chemotherapy 7: (1975). Suginaka, H., Shimatani, M., Kotani, S., Ogawa, M., Hama, M. & Kosaki, G. Antibacterial mechanisms of cefsulodin against Pseudomonas aeruginosa and Escherichia coli. FEMS Microbiology Utters 5: (1979). Tsuchiya, K., Kida, M., Kondo, M., Ono, H., Takeuchi, M. & Nishi, T. SCE-963, a new broad-spectrum cephalosporin: in vitro and in vivo antibacterial activities. Antimicrobial Agents and Chemotherapy 14: (1978a). Tsuchiya, K., Kondo, M., Kita, Y., Noji, Y., Takeuchi, M. & Fugono, T. Absorption, distribution and excretion of SCE-963, a new broad-spectrum cephalosporin, in mice, rats, rabbits and dogs. Journal of Antibiotics 31: 1272,-82 (19786). Weiser, R., Asscher, A. W. & Wimpenny, J. In vitro reversal of antibiotic resistance by ethylenediamine tetraacetic acid. Nature 219: (1968). {Manuscript accepted 23 March 1979)