Correspondence
Acknowledgement. The X-ray diffraction studies were carried out by Eh- V. P. Tannincn and Dr J. Yliruusi. PEKKA T. MANNISTO MATTI VILUKSEL\ Department of Pharmacology and Toxicology, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki. Finland References Minnisto, P. T., Hanhijirvi, H.
Vuorda, A., Mantyla, R., & Buimnmi, V. (1983).
Phannacokinetics and tissue penetration of erythromycm and erythromycin-11,12-cycoc carbonate in dogs. Proceedings of the Thirteenth International Congress of Chemotherapy, Vienna. Part 107, pp. 5-10. Vihiksda, M., Hankijarvi, H., Husband, R. F. A., Kosma, V.-M., Collan, Y. & Mfinnistfi, P. T. (1988). Comparative Bver toxicity of various erythromycm derivatives in annrmlt Journal of Antimicrobial Chemotherapy 21, Suppl. D, 9-27.
Poor
of /Mactamases by tazobactun
Sir, Combination antimicrobial chemotherapy is regularly employed in the prevention and treatment of sepsis, particularly for immunocompromised patients. Such regimens can include anti-pseudomonal agents and may, for complicated infections, contain more than one class of ^-lactam compound. However, concern has been expressed about the potential for induction of Type-I chromosomallyencoded cephalosporinases in Enterobacter by the naturally occurring /f-lactamase inhibitor clavulanic acid (Minami et al., 1980). Induction of /J-lactamase by one ^-lactam could lead to a decrease in activity of the co-administered /J-lactam. In contrast, semi-synthetic /Mactamase inhibitors such as the penicillanic acid sulphones were found to produce little or no induction (Gutmann et al., 1986; Moosdeen, Keeble & Williams, 1986). We report the invitro interaction of a newly introduced irreversible 0-lactamase inhibitor, tazobactam (formerly YTR 830, Cyanamid Ltd) as measured by its effect on the activity of two anti-pseudomonal /J-lactams, aztreonam and ceftazidime, against bacteria expressing inducible chromosomal /Mactamases. The test method employed was spiral gradient endpoint (SGE) analysis (Weckbach & Staneck, 1987), but modified by the use of filter membranes to allow determination of both the MIC and the MBC. Briefly, with a spiral plater (Spiral Systems Ltd), with a modified inoculation platen (ISOmm), gradients (0-1-33-0 mg/1) of ceftazidime and aztreonam were prepared in agar, alone and combined with a fixed concentration (10 mg/1) of cefoxitin (a known potent inducer of ^-lactamase), clavulanic acid or tazobactam. The antibiotic gradients were allowed to equilibrate at 4°C overnight. The test isolates comprised Enterobacter cloacae (7), Pseudomonas aeruginosa (14) and P.maltophilia (3), and were chosen for their ability to exhibit inducible /J-lacta-
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on March 15, 2015
also used as a reference compound and, in spite of a lower dose (also in IU), it showed more toxicity than erythromycin stearate. To find out the toxic potential of test compounds, - the highest doses should be almost the maximum tolerated ones (at least in short-term toxicity studies). For example, the highest doses in our five-day dog study were selected so that they did not cause vomiting. Further, since several dose levels were studied, it is possible to find in our data nearly comparable dose levels (for acistrate, stearate and cyclic carbonate, at least) if one really wants to take the microbiological activity into consideration. Finally, Professor Bojarska-Dahlig refers to the data of Mannisto et al. (1983) on erythromycin tissue concentrations in dogs. These data were not completely relevant to our comparative discussion, because only the following derivatives were studied: erythromycin-11,12cyclic carbonate aspartate and erythromycin Iactobionate 1 h after intravenous dose, and a mixture of cyclic carbonate and L-aspartate 3 h after oral dose. In this investigation, too, Mannisto et al. (1983) referred the doses to erythromycin base or cyclic carbonate base, not to the microbiological activity. We agree with Professor Bojarska-Dahlig that we failed to discuss the importance of the long half-life of cyclic carbonate. It certainly increased the antibiotic concentration in the liver and partially contributed to the hepatotoxkaty. Shortage of space did not allow the presentation of pharmacokinetic data in animals, and a separate paper would have been needed for this purpose. In fact, it would be necessary to compare the liver toxkaties of the regular erythromycins and the new long acting macrolides (roxithromycin, clarithromycin, flurithromycin, azidomytin, etc.). No such data are available to the best of our knowledge.
477
478
Correspondence
Table L In-vitro susceptibility of 24 strains of Gram-negative bacilli to aztreonam and ceftazidime, akme and combined with a fixed concentration (10 mg/1) of three /Mactam type compounds MIC (mg/1) M1C» MIC,, mean
Agents tested
Table
0-3 2-1 2-4 0-2 03 1-7 08 02
3-5 5-8 5-1 40 1-2 3-1 5-1 09
1-2 2-5
yo
1-3 05 2-3 1-6 04
< 01-51 01-5-8 < 01-16-6 < 01-5-8 01-1-7 02-14-5 01-7-5 Ol-l-O
07 5-8 4-0 04 08 5-1 21 06
2-7 7-3 5-6 2-0 1-3 6-4 4-5 1-0
< 01-21-9 O3->33 33 < 01-11-1 03-4-5 07- > 33 04-19-0 02-2-7
n. Occurrence of synergy^)*, iintagonisn^A)* and1 indifference(I)
Antibiotic
Plus cefoxitin (10 mg/1) MIC MBC I S A I S A
Plus davulanic acid (10 mg/1) MBC MIC A I S A I S
Aztreonam Ceftazidime
13 16
10 13 9 14
11 8
5-8 21-9 12-6 5-6 3-5 8-5 111 21
range
0 0
14 15
10 9
0 0
1 1
12 10
11 14
1 0
Plus tazobactam (10 mg/1) MIC MBC A I S A I S 0 1
23 22
1 1
0 0
21 23
3 1
•Defined as > four-fold increase or decrease in MIC or MBC. mases by the disc (cefoxitin 30 ng) approximation test (Sanders, Sanders & Goering, 1982). The first two species produce Class I /Mactamases; P. maltophilia produces two inducible /J-lactamases, neither of which is akin to the Class I enzymes. Sterile cellulose acetate membranes (140 mm, 0-2 micron pore size, Sartorius Ltd) were carefully overlaid on the test agars to ensure no trapping of air. Bacteria, at 10* cfu/ml were applied with sterile swabs in a radial manner giving a final inoculum of approximately lPcfu/plate and the plates were incubated overnight Antibiotic permeates the membrane and inhibits bacterial growth. Occasionally, owing to the heterogeneous nature of resistance, bacterial populations contain mixtures of sensitive and resistant cells and consequently a clear demarcation between growth and no growth is not always achieved. Therefore, the MIC (or spiral gradient endpoint, SGE) was taken as the end of the line of contiguous growth, and isolated single colonies ahead of the main body of growth were ignored. The MIC or SGE position was recorded by puncturing the membrane with a sterile needle. The membrane was then transferred to the first of two antibiotic-free agar plates for 4 h incubation before transfer to a second for a further 18 h incubation, in order to minimi™ anti-
biotic carry-over. The advance in the length of bacterial growth further up the concentration gradient on this antibiotic-free medium was taken as the MBC position. MIC and MBC were then computed mathematically, from the radial inhibition measurements, the stock concentration values of antibiotic, the depth of the agar and the liquid deposition values supplied by the manufacturer. Synergy (S) and antagonism (A) were taken to be equal to or greater than a four-fold decrease or increase in MIC or MBC and indifference (I), to be less than a four-fold change. Table I shows the in-vitro activity of aztreonam and ceftazidime against the 24 test isolates alone and combined with a fixed concentration (10 mg/1) of cefoxitin, davulanic acid or tazobactam. The table includes mean MIC, MIC,,, and MIC*,, range of MICs, mean MBC, MBC» and MBC* and range of MBCs. Applying the criterion of a four-fold increase or decrease in MIC or MBC, Table II shows the occurrence of synergy, antagonism or indifference for each of the /7-lactams alone or in combination against the 24 isolates. When the results for both MICs and MBCs and for both aztreonam and ceftazidime are considered together for each of the added agents (96 observations in each case) antagonism was found with cefoxitin in 60-4% of
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on March 15, 2015
Aztreonam Aztreonam+cefoxitin Aztreonam+clavulanate Aztreonam+Uzobactam Ceftazidime Ceftazidime+cefoxitin Ceftazidime+clavulanate Ceftazidime+tazobactam
MBC(mg/l) MBC*, mean
range
Correspondence
Royal Liverpool Hospital, University Department of Medical Microbiology, Present Street, Liverpool L7 8XP Merstyside. UK.
References Gutmann, L., Kitzis, M. D., Yamabe, S. & Acar, J. F. (1986). Comparative evaluation of a new /Jlactamase inhibitor, YTR 830, combined with different /Mactam antibiotics against bacteria harboring known ^-lactamases. Antimicrobial Agents and Chemotherapy 29, 955-7. Jacobs, M. R., Aronoff, S. C , Johenning, S., Shlaes, D. M. & Yamabe, S. (1986). Comparative activities of the 0-lactamase inhibitor! YTR 830, davulanate and sulbactam combined with ampicillin and broad spectrum penicillins against defined /7-lactamase producing gram-negative bacilli. Antimicrobial Agents and Chemotherapy 29,980-5. Mmami, S., YoUuji, A., Inoue, M. & Mitsuhashi, S. (1980). Induction of /MacUmase by various /Jlactam antibiotics in Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 18, 382-5. Moosdeen, F., Keeble, J. & Williams, J. D. (1986). Induction/inhibition of chromosomal filactamases by ^-lactamase inhibitors. Reviews of Infectious Disease 8, Suppl. 5. S562-«.
Sanders, C. C , Sanders, W. E. & Goering, R. V. (1982). In-vitro antagonism of beta-lactam antibiotics by cefoxitin. Antimicrobial Agents and Chemotherapy 21, 968-75. Weckbach, L. S. & Staneck, J. L. (1987). Examination of the spiral gradient endpoint (SGE) for the determination of MICs. In Abstracts of the Annual Meeting of the American Society for Microbiology, Atlanta, Ga, USA, 1987. Abstract C-65. p. 334. American Society for Microbiology.
Enhancement of the activity of cefotaxbne by iron-binding proteins Sir, Recent studies have shown that cefotaxime has greater antibacterial activity when tested in heat-inactivated serum than in buffer alone (Jones & Barry, 1987). The mechanism by which activity is enhanced has not been determined, although the findings suggest an interaction with serum proteins. Work in our laboratory has shown that the iron-binding proteins transferrin and lactoferrin can damage the outer membrane of Gram-negative bacteria, increasing its permeability (Ellison, Giehl & LaForce, 1988). This effect is associated with an increase in bacterial susceptibility to the hydrophobk antibiotic, rifampicin, which is normally excluded by the bacterial outer membrane (Ellison et al., 1988). Since the activity of cefotaxime is dependent on its ability to penetrate the outer membrane of Gram-negative bacteria, we considered that the observed enhancement of cefotaxime's activity by serum could be due to an effect of serum transferrin on outer membrane permeability. To test this possibility, we have evaluated the activity of cefotaxime, and its metabolite desacetylcefotaxime (both provided by Hoechst-Roussel Pharmaceuticals Inc.), against six randomly selected clinical Escherichla coli isolates in the presence of either serum transferrin or human milk lactoferrin (purchased from Sigma Chemical Company, St Louis MO or CalBiochem Corporation, La Jolla CA). MICs were determined in triplicate by a microtitre broth-dilution method, in MuellerHinton broth (National Committee for Clinical Laboratory Standards, 1985). Tunekill studies were performed in a modified WMS broth supplemented with (M)3 mM galactose (Ellison et al., 1988) with a 5 x 105 cfu/ml inoculum of exponentially growing cells. Bacteria were exposed to transferrin
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on March 15, 2015
observations, with clavulanic acid in 42-7% and with tazobactam in 1-0%. On no occasion was synergy exhibited in the presence of cefoxitin. Synergy was observed with tazobactam in 6-2% of observation and with clavulanic acid in 3 1 % . Interestingly, addition of tazobactam, particularly to ceftazidime, slightly reduced both MIC and MBC values. This observation suggests that perhaps tazobactam also reduces the pre-formed low basal level of chromosomally-encoded /J-lactamase produced by the test strains. Other workers have demonstrated that against Enterobacteriaceae producing Class I chromosomal /Mactamases, combinations of tazobactam and /J-lactams were more synergistic than combinations containing clavulanic acid (Gutmann et al., 1986; Jacobs el al., 1986). At the test concentration (10mg/l), the level of /Mactamase induction by cefoxitin and clavulanic acid but not by tazobactam was sufficient to reduce the antibacterial activity of ceftazidime and aztreonam. Continued caution is required in the clinical use of /Mactam combinations. The data presented do not indicate that either agent will select for 'stably' derepressed mutants. J. E. CORKILL C. A. HART
479
Acknowledgement. The X-ray diffraction studies were carried out by Eh- V. P. Tannincn and Dr J. Yliruusi. PEKKA T. MANNISTO MATTI VILUKSEL\ Department of Pharmacology and Toxicology, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki. Finland References Minnisto, P. T., Hanhijirvi, H.
Vuorda, A., Mantyla, R., & Buimnmi, V. (1983).
Phannacokinetics and tissue penetration of erythromycm and erythromycin-11,12-cycoc carbonate in dogs. Proceedings of the Thirteenth International Congress of Chemotherapy, Vienna. Part 107, pp. 5-10. Vihiksda, M., Hankijarvi, H., Husband, R. F. A., Kosma, V.-M., Collan, Y. & Mfinnistfi, P. T. (1988). Comparative Bver toxicity of various erythromycm derivatives in annrmlt Journal of Antimicrobial Chemotherapy 21, Suppl. D, 9-27.
Poor
of /Mactamases by tazobactun
Sir, Combination antimicrobial chemotherapy is regularly employed in the prevention and treatment of sepsis, particularly for immunocompromised patients. Such regimens can include anti-pseudomonal agents and may, for complicated infections, contain more than one class of ^-lactam compound. However, concern has been expressed about the potential for induction of Type-I chromosomallyencoded cephalosporinases in Enterobacter by the naturally occurring /f-lactamase inhibitor clavulanic acid (Minami et al., 1980). Induction of /J-lactamase by one ^-lactam could lead to a decrease in activity of the co-administered /J-lactam. In contrast, semi-synthetic /Mactamase inhibitors such as the penicillanic acid sulphones were found to produce little or no induction (Gutmann et al., 1986; Moosdeen, Keeble & Williams, 1986). We report the invitro interaction of a newly introduced irreversible 0-lactamase inhibitor, tazobactam (formerly YTR 830, Cyanamid Ltd) as measured by its effect on the activity of two anti-pseudomonal /J-lactams, aztreonam and ceftazidime, against bacteria expressing inducible chromosomal /Mactamases. The test method employed was spiral gradient endpoint (SGE) analysis (Weckbach & Staneck, 1987), but modified by the use of filter membranes to allow determination of both the MIC and the MBC. Briefly, with a spiral plater (Spiral Systems Ltd), with a modified inoculation platen (ISOmm), gradients (0-1-33-0 mg/1) of ceftazidime and aztreonam were prepared in agar, alone and combined with a fixed concentration (10 mg/1) of cefoxitin (a known potent inducer of ^-lactamase), clavulanic acid or tazobactam. The antibiotic gradients were allowed to equilibrate at 4°C overnight. The test isolates comprised Enterobacter cloacae (7), Pseudomonas aeruginosa (14) and P.maltophilia (3), and were chosen for their ability to exhibit inducible /J-lacta-
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on March 15, 2015
also used as a reference compound and, in spite of a lower dose (also in IU), it showed more toxicity than erythromycin stearate. To find out the toxic potential of test compounds, - the highest doses should be almost the maximum tolerated ones (at least in short-term toxicity studies). For example, the highest doses in our five-day dog study were selected so that they did not cause vomiting. Further, since several dose levels were studied, it is possible to find in our data nearly comparable dose levels (for acistrate, stearate and cyclic carbonate, at least) if one really wants to take the microbiological activity into consideration. Finally, Professor Bojarska-Dahlig refers to the data of Mannisto et al. (1983) on erythromycin tissue concentrations in dogs. These data were not completely relevant to our comparative discussion, because only the following derivatives were studied: erythromycin-11,12cyclic carbonate aspartate and erythromycin Iactobionate 1 h after intravenous dose, and a mixture of cyclic carbonate and L-aspartate 3 h after oral dose. In this investigation, too, Mannisto et al. (1983) referred the doses to erythromycin base or cyclic carbonate base, not to the microbiological activity. We agree with Professor Bojarska-Dahlig that we failed to discuss the importance of the long half-life of cyclic carbonate. It certainly increased the antibiotic concentration in the liver and partially contributed to the hepatotoxkaty. Shortage of space did not allow the presentation of pharmacokinetic data in animals, and a separate paper would have been needed for this purpose. In fact, it would be necessary to compare the liver toxkaties of the regular erythromycins and the new long acting macrolides (roxithromycin, clarithromycin, flurithromycin, azidomytin, etc.). No such data are available to the best of our knowledge.
477
478
Correspondence
Table L In-vitro susceptibility of 24 strains of Gram-negative bacilli to aztreonam and ceftazidime, akme and combined with a fixed concentration (10 mg/1) of three /Mactam type compounds MIC (mg/1) M1C» MIC,, mean
Agents tested
Table
0-3 2-1 2-4 0-2 03 1-7 08 02
3-5 5-8 5-1 40 1-2 3-1 5-1 09
1-2 2-5
yo
1-3 05 2-3 1-6 04
< 01-51 01-5-8 < 01-16-6 < 01-5-8 01-1-7 02-14-5 01-7-5 Ol-l-O
07 5-8 4-0 04 08 5-1 21 06
2-7 7-3 5-6 2-0 1-3 6-4 4-5 1-0
< 01-21-9 O3->33 33 < 01-11-1 03-4-5 07- > 33 04-19-0 02-2-7
n. Occurrence of synergy^)*, iintagonisn^A)* and1 indifference(I)
Antibiotic
Plus cefoxitin (10 mg/1) MIC MBC I S A I S A
Plus davulanic acid (10 mg/1) MBC MIC A I S A I S
Aztreonam Ceftazidime
13 16
10 13 9 14
11 8
5-8 21-9 12-6 5-6 3-5 8-5 111 21
range
0 0
14 15
10 9
0 0
1 1
12 10
11 14
1 0
Plus tazobactam (10 mg/1) MIC MBC A I S A I S 0 1
23 22
1 1
0 0
21 23
3 1
•Defined as > four-fold increase or decrease in MIC or MBC. mases by the disc (cefoxitin 30 ng) approximation test (Sanders, Sanders & Goering, 1982). The first two species produce Class I /Mactamases; P. maltophilia produces two inducible /J-lactamases, neither of which is akin to the Class I enzymes. Sterile cellulose acetate membranes (140 mm, 0-2 micron pore size, Sartorius Ltd) were carefully overlaid on the test agars to ensure no trapping of air. Bacteria, at 10* cfu/ml were applied with sterile swabs in a radial manner giving a final inoculum of approximately lPcfu/plate and the plates were incubated overnight Antibiotic permeates the membrane and inhibits bacterial growth. Occasionally, owing to the heterogeneous nature of resistance, bacterial populations contain mixtures of sensitive and resistant cells and consequently a clear demarcation between growth and no growth is not always achieved. Therefore, the MIC (or spiral gradient endpoint, SGE) was taken as the end of the line of contiguous growth, and isolated single colonies ahead of the main body of growth were ignored. The MIC or SGE position was recorded by puncturing the membrane with a sterile needle. The membrane was then transferred to the first of two antibiotic-free agar plates for 4 h incubation before transfer to a second for a further 18 h incubation, in order to minimi™ anti-
biotic carry-over. The advance in the length of bacterial growth further up the concentration gradient on this antibiotic-free medium was taken as the MBC position. MIC and MBC were then computed mathematically, from the radial inhibition measurements, the stock concentration values of antibiotic, the depth of the agar and the liquid deposition values supplied by the manufacturer. Synergy (S) and antagonism (A) were taken to be equal to or greater than a four-fold decrease or increase in MIC or MBC and indifference (I), to be less than a four-fold change. Table I shows the in-vitro activity of aztreonam and ceftazidime against the 24 test isolates alone and combined with a fixed concentration (10 mg/1) of cefoxitin, davulanic acid or tazobactam. The table includes mean MIC, MIC,,, and MIC*,, range of MICs, mean MBC, MBC» and MBC* and range of MBCs. Applying the criterion of a four-fold increase or decrease in MIC or MBC, Table II shows the occurrence of synergy, antagonism or indifference for each of the /7-lactams alone or in combination against the 24 isolates. When the results for both MICs and MBCs and for both aztreonam and ceftazidime are considered together for each of the added agents (96 observations in each case) antagonism was found with cefoxitin in 60-4% of
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on March 15, 2015
Aztreonam Aztreonam+cefoxitin Aztreonam+clavulanate Aztreonam+Uzobactam Ceftazidime Ceftazidime+cefoxitin Ceftazidime+clavulanate Ceftazidime+tazobactam
MBC(mg/l) MBC*, mean
range
Correspondence
Royal Liverpool Hospital, University Department of Medical Microbiology, Present Street, Liverpool L7 8XP Merstyside. UK.
References Gutmann, L., Kitzis, M. D., Yamabe, S. & Acar, J. F. (1986). Comparative evaluation of a new /Jlactamase inhibitor, YTR 830, combined with different /Mactam antibiotics against bacteria harboring known ^-lactamases. Antimicrobial Agents and Chemotherapy 29, 955-7. Jacobs, M. R., Aronoff, S. C , Johenning, S., Shlaes, D. M. & Yamabe, S. (1986). Comparative activities of the 0-lactamase inhibitor! YTR 830, davulanate and sulbactam combined with ampicillin and broad spectrum penicillins against defined /7-lactamase producing gram-negative bacilli. Antimicrobial Agents and Chemotherapy 29,980-5. Mmami, S., YoUuji, A., Inoue, M. & Mitsuhashi, S. (1980). Induction of /MacUmase by various /Jlactam antibiotics in Enterobacter cloacae. Antimicrobial Agents and Chemotherapy 18, 382-5. Moosdeen, F., Keeble, J. & Williams, J. D. (1986). Induction/inhibition of chromosomal filactamases by ^-lactamase inhibitors. Reviews of Infectious Disease 8, Suppl. 5. S562-«.
Sanders, C. C , Sanders, W. E. & Goering, R. V. (1982). In-vitro antagonism of beta-lactam antibiotics by cefoxitin. Antimicrobial Agents and Chemotherapy 21, 968-75. Weckbach, L. S. & Staneck, J. L. (1987). Examination of the spiral gradient endpoint (SGE) for the determination of MICs. In Abstracts of the Annual Meeting of the American Society for Microbiology, Atlanta, Ga, USA, 1987. Abstract C-65. p. 334. American Society for Microbiology.
Enhancement of the activity of cefotaxbne by iron-binding proteins Sir, Recent studies have shown that cefotaxime has greater antibacterial activity when tested in heat-inactivated serum than in buffer alone (Jones & Barry, 1987). The mechanism by which activity is enhanced has not been determined, although the findings suggest an interaction with serum proteins. Work in our laboratory has shown that the iron-binding proteins transferrin and lactoferrin can damage the outer membrane of Gram-negative bacteria, increasing its permeability (Ellison, Giehl & LaForce, 1988). This effect is associated with an increase in bacterial susceptibility to the hydrophobk antibiotic, rifampicin, which is normally excluded by the bacterial outer membrane (Ellison et al., 1988). Since the activity of cefotaxime is dependent on its ability to penetrate the outer membrane of Gram-negative bacteria, we considered that the observed enhancement of cefotaxime's activity by serum could be due to an effect of serum transferrin on outer membrane permeability. To test this possibility, we have evaluated the activity of cefotaxime, and its metabolite desacetylcefotaxime (both provided by Hoechst-Roussel Pharmaceuticals Inc.), against six randomly selected clinical Escherichla coli isolates in the presence of either serum transferrin or human milk lactoferrin (purchased from Sigma Chemical Company, St Louis MO or CalBiochem Corporation, La Jolla CA). MICs were determined in triplicate by a microtitre broth-dilution method, in MuellerHinton broth (National Committee for Clinical Laboratory Standards, 1985). Tunekill studies were performed in a modified WMS broth supplemented with (M)3 mM galactose (Ellison et al., 1988) with a 5 x 105 cfu/ml inoculum of exponentially growing cells. Bacteria were exposed to transferrin
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on March 15, 2015
observations, with clavulanic acid in 42-7% and with tazobactam in 1-0%. On no occasion was synergy exhibited in the presence of cefoxitin. Synergy was observed with tazobactam in 6-2% of observation and with clavulanic acid in 3 1 % . Interestingly, addition of tazobactam, particularly to ceftazidime, slightly reduced both MIC and MBC values. This observation suggests that perhaps tazobactam also reduces the pre-formed low basal level of chromosomally-encoded /J-lactamase produced by the test strains. Other workers have demonstrated that against Enterobacteriaceae producing Class I chromosomal /Mactamases, combinations of tazobactam and /J-lactams were more synergistic than combinations containing clavulanic acid (Gutmann et al., 1986; Jacobs el al., 1986). At the test concentration (10mg/l), the level of /Mactamase induction by cefoxitin and clavulanic acid but not by tazobactam was sufficient to reduce the antibacterial activity of ceftazidime and aztreonam. Continued caution is required in the clinical use of /Mactam combinations. The data presented do not indicate that either agent will select for 'stably' derepressed mutants. J. E. CORKILL C. A. HART
479
