222
Correspondence
could possibly be useful in widening the spectrum of mupirocin.
IOOO
MARTTI VAARA Department of Bacteriology and Immunology, University of Helsinki, Haartmaninkatu 3. 00290 Helsinki. Finland Reft
0-3 I Mupirocln(mg/U
3
10
Figure. The susceptibility of S. typhimwium SH5014 to mupirocin in the presence of PMBN. Bacteria were grown in the wells of a mkrodftution plate in L broth which contained increasing concentrations of mupirocin as well as PMBN (PMBN concentrations, 0 (D). 3 (B), 10 (O) or 30 ( • ) mg/L). The bacterial growth was quantified after an 18-h incubation by absorbance measurement.
Finland). The lowest concentration completely inhibiting visual growth was recorded and interpreted as the MIC. In the absence of PMBN, the MIC of mupirocin for SH5014 was 100 mg/L. As low a concentration of PMBN as 3 mg/L decreased the MIC of mupirocin for SH5014 by a factor of 100 (Figure). Higher concentrations of PMBN sensitized SH5014 further, and a > 1000-fold sensitization took place at a PMBN concentration of 30 mg/L. The MIC of mupirocin for PMBN-sensitized SH5014 was identical or very close to the MICs determined for mupirocin-susceptible Gram-positive cocci, such as Streptococcus pneumoniae (MIC, 0-1 mg/L), Streptococcus pyogenes (0-1 mg/L), and Staphylococcus aureus (0-02-0-12 mg/L) (see Ward & Campoli-Richards, 1986). A very similar, massive sensitization to mupirocin was observed when the other test strain, E. coli IH3080, was grown in the presence of PMBN (data not shown). Accordingly, the findings indicate that the OM of Gram-negative enteric bacteria acts as a very effective permeability barrier against mupirocin. However, this barrier can easily be disrupted by PMBN. Thus, agents which have a potent OM permeability-increasing action
Activity of snlbtctam combinations against Escherichia coE isolates with known amounts of TEM-1 0-bctamase Sir, We recently published, in this Journal, on the behaviour of combinations of clavulanate, tazobactam or BRL42715 with amoxycillin, ticarcilbn or piperacillin against Escherichia coli strains with measured amounts of the TEM-1 /Mactamase (Lrvermorc & Seetulsingh, 1991; Seetulsingh, Hall & Uvermore, 1991). The inhibitor concentrations required to
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 3, 2015
0-1
Capobianco, J. O., Doran, C. C. & Goldman, R. C. (1989). Mechanism of mupirocin transport into sensitive and resistant bacteria. Antimicrobial Agents and Chemotherapy 33, 156-63. Hughes, J. & Mellows, G. (1980). Interaction of pseudomonk acid A with Escherichia coli B isoleucyl-tRNA synthetase. Biochemical Journal 191, 209-19. Nikaido, H. & Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiological Reviews 49, 1-32. Vaara, M. (1989). Analytical and preparative highperformance liquid chromatography of the papain-deaved derivative of polymyxin B. Journal of Chromatography 441, 423-30. Vaara, M. (1990). Antimicrobial susceptibility of Salmonella typhimwium carrying the outer membrane permeability mutation SS-B. Antimicrobial Agents and Chemotherapy 34, 853-7. Vaara, M. & Jaakkola, J. (1989). Sodium hexametaphosphate sensitizes Pseudomonas aerugfnosa, several other species of Pseudomonas, and Escherichia coli to hydrophobic drugs. Antimicrobial Agents and Chemotherapy 33, 1741-7. Vaara, M. & Vaara, T. (1983). Sensitization of gram-negative bacteria to antibiotics and complement by a nontoxk oligopeptide. Nature 303, 526-8. Viljanen, P. & Vaara, M. (1984). Susceptibility of gram-negative bacteria to polymyxin B nonapeptide. Antimicrobial Agents and Chemotherapy 25, 701-5. Ward, A & Campoli-Richards, D. M. (1986). Mupirocin. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 32, 425-44.
Correspondence
223
Table. Mean MICt of /Mactamt, and inhibitor levels required to achieve potentiation for isolates in each quartile of the /Mactamase activity distribution
Quartile
512 813 1193 2581
Geometric mean [sulbactam] (mg/L) needed to achieve ampidllin MIC cefoperazone MIC - 8 mg/L . -0-25 mg/L
1-0 1-6 2-7 80
potentiate the penicillins were shown to depend closely on the amount of TEM-1 enzyme produced. The behaviour of sulbactam combinations was investigated in a follow-up study with the same strains and methods. Briefly, MICs of ampidllin and cefoperazone alone and in combination with sulbactam (1, 2, 4, 8, 16 or 32 mg/L) were determined for 36 TEM-1 0lactamase producing E. coli isolates. The inocula comprised 104 cfu, taken from overnight nutrient broth cultures and applied to the surface of Diagnostic Sensitivity Test agar plates (Oxoid, Basingstoke, Hants) containing the antibiotics). The levels of /J-lactamase activity in these organisms was estimated from the activity of sonicates of exponential phase cells against 1 n i l benzylpenicillin at 37°C in 01 M phosphate buffer pH 7-0 (Seetulsingh et at., 1991). On the basis of the 0lactamase data the collection was divided into 4 quartiles: quartile 1 (Ql), comprised the nine isolates with lowest /Mactamase specific activities; Q2, comprised those with the 10-18th lowest (inclusive); Q3, those with the 19-27th lowest and Q4, the nine isolates with the highest specific activities. The Table shows the geometric mean MICs of ampidllin and cefoperazone for isolates in each quartile of the /Mactamase activity distribution. As with amoxycillin and piperacillin (Livermore & Seetulsingh, 1991), the geometric mean MICs of ampidllin and cefoperazone were highest for the isolates in Q4 and lowest for those in Ql. All the isolates including those with the lowest /Mactamase activities were resistant to ampidllin, with MICs of this antibiotic ranging upwards from 256 mg/L as compared to MICs of 1-4 mg/L for E. coli isolates without TEM-1 enzyme. MICs of cefoperazone for the TEM-1 producers ranged from 0-12 to 32 mg/L, as compared to O008-(M)15 mg/L for ten non-producers. Potentiation of ampidllin activity by sulbactam was weak. For 30/36 isolates, no subinhi-
13-7
2O2 32-0
471
50 101 160 400
bitory concentration of sulbactam rendered the bacteria sensitive to 2 mg/L of ampidllin, which is the MIC antidpated for TEM-non producers, and for 13/36 no subinhibitory concentration rendered the bacteria susceptible to ampidllin at the BSACs recommended breakpoint of 8 mg/L (Working Party of the British Society for Antimicrobial Chemotherapy, 1991). This was not a problem which we have encountered with other inhibitors, where subinhibitory concentrations almost invariably obviated /Mactamase activity or, at least, brought the penicillin MICs to below the breakpoint (Livermore & Seetulsingh, 1991). These weak inhibitory activity of sulbactam for TEM-1 enzyme also has been demonstrated also in assays with extracted /Mactamase. Although sulbactam acts as a suiride inhibitor of TEM-1 enzyme, the process is relatively ineffident and considerable enzymemediated hydrolysis of the inhibitor occurs for each enzyme molecule that is inactivated (Fisher et al., 1980). This hydrolytic activity was reflected in MIC data obtained with sulbactam alone. At 32 mg/L, the compound inhibited the growth of 8/9 Ql isolates, 7/9 Q2 isolates, 5/9 Q3 isolates and 0/9 Q4 isolates. By contrast MICs of clavulanate (16-64 mg/L) and tazobactam (64-256 mg/L) for these strains were not related to /Mactamase quantity (unpublished data). As with tazobactam, clavulanate and BRL42715 (Livermore & Seetulsingh, 1991), the concentrations of sulbactam required to potentiate ampidllin and cefoperazone depended on the amount of /Mactamasc produced. The Table shows the geometric mean sulbactam concentrations required to potentiate ampidllin MICs to the breakpoint of 8 mg/L. Where this level of potentiation was not achieved for an individual strain with any subinhibitory concentration of sulbactam, it was assumed, in order to calculate the geometric mean, that such potentiation would have been achieved with the next higher con-
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 3, 2015
1 2 3 4
Geometric mean MICi (mg/L) Ampkalhn Cefoperazone
224
Correspondence
DAVID M. LJVERMORE Department of Medical Microbiology, The London Hospital Medical College, Turner Street, London El 2AD, UK
References Fisher, J., Belasco, J. O., Charnas, R. L , Khosla, S. & Ksowles, J. R. (1980). 0-Lactamase inactivation by mechanism-based reagents. Philosophical Transactions of the Royal Society (London) 289B, 309-19. Livermore, D. M. & Seetnlnngh, P. (1991). Susceptibility of Escherichia coli isolates with TEM-1 0-lactamase to combinations of BRL42715, t H ^ N r " " " or clavulanate with priperacfllin or amoxycillin. Journal of Antimicrobial Chemotherapy 27, 761-7.
Seetubingh, P., Hall, L. M. C. & Livermore, D. M. (1991). Activity of clavulanate combinations against TEM-1 /)~lactamase producing Escherichia coli isolates obtained in 1982 and 1989. Journal of Antimicrobial Chemotherapy 27, 749-59. Working Party of the British Society of Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1-47.
Prediction of ofloxadn susceptibility from Inritro tests with dprofloxadn Sir, Ofloxadn is a fluoroquinolone compound that has been approved recently for use as an orally administered antimicrobial agent in the United States. An intravenous formulation will soon be available. Unfortunately, manufacturers of many broth microdilution trays and other susceptibility testing systems have not yet completed the studies that are needed in order to incorporate ofloxadn into their test systems, but dprofloxadn can be tested. Mutants that are selected in vitro for resistance to one quinolone compound show cross-resistance to other drugs in that class (Barry & Jones, 1984; King, Shannon & Phillips, 1985; Barry, Gardiner & Packer, 1987). Because of this cross-resistance, the results of susceptibility tests with dprofloxadn might be used to predict susceptibility or resistance to ofloxadn. To determine the validity of that assumption, I reviewed the results of previously reported broth microdilution tests and calculated predictive values of dprofloxadn MIC categories for five major groups of bacterial pathogens (enteric bacilli, pseudomonads, staphylococci, enterococd and streptococci). Broth microdilution tests were performed according to the procedure of the National Committee for Clinical Laboratory Standards (NCCLS) and quality control strains gave MICs within the acceptable ranges for both drugs (NCCLS, 1990). The NCCLS interpretive criteria were used to define susceptible, intermediate and resistant categories. For dprofloxadn, those MIC breakpoints were < 1-0, 20 and £ 4-0 mg/L, respectively, and for ofloxadn the MIC breakpoints were < 2-0,4-0 and > 8-0, respectively. The 32 bacterial species that were represented in the culture collection are described in Table 1. MIC^s and MIC^s of dprofloxadn and ofloxadn did not differ greatly, but for many individual strains ofloxadn MICs were two- to four-times greater than those of dpro-
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 3, 2015
centration in a doubling dilution sequence, irrespective of whether or not this level of the sulphone would itself have inhibited bacterial growth. Analysis of the cefoperazone data is problematical in that there is no obvious level of potcntiation to take as a reference point TEM-1-negative E. coli were sensitive to 0-008-0-015 mg/L of cefoperazone, but MICs of the cephalosporin for TEM-1-producers could rarely (6/36 cases) be reduced to this level with subinhibitory sulbactam concentrations. On the other hand, cefoperazone MICs only exceeded the cephalosporin's breakpoint (16 mg/L) for two isolates (both in Q3 or 4), even in the absence of sulbactam: 11/36 isolates were susceptible even at the BSACs recommended "lower breakpoint" of 2 mg/L. Included on the table are data on the sulbactam concentrations required to reduce cefoperazone MICs to 0-23 mg/L for isolates in each quartfle of the /Mactamase activity distribution. This is an arbitrary level, chosen only as being below the cefoperazone MIC for 34/36 TEM-1 producers but being achieved in the presence of sulbactam for 33/36 isolates. In summary, our results indicate two points: firstly that, as with other inhibitors, the concentrations of sulbactam required to achieve potentiation of substrate /Mactams depended on the amount of /Mactamase produced and, secondly, that the inhibitory power of sulbactam for TEM-1 was so weak that only a small minority (4/36 isolates) of the TEM producers would have counted as susceptible to the combination of 8 mg/L sulbactam plus 8 mg/L ampicillin. The cefoperazone sulbactam combination fared better, largely because TEM-1 production did not give high level resistance to this cephalosporin in E. coli.
Correspondence
could possibly be useful in widening the spectrum of mupirocin.
IOOO
MARTTI VAARA Department of Bacteriology and Immunology, University of Helsinki, Haartmaninkatu 3. 00290 Helsinki. Finland Reft
0-3 I Mupirocln(mg/U
3
10
Figure. The susceptibility of S. typhimwium SH5014 to mupirocin in the presence of PMBN. Bacteria were grown in the wells of a mkrodftution plate in L broth which contained increasing concentrations of mupirocin as well as PMBN (PMBN concentrations, 0 (D). 3 (B), 10 (O) or 30 ( • ) mg/L). The bacterial growth was quantified after an 18-h incubation by absorbance measurement.
Finland). The lowest concentration completely inhibiting visual growth was recorded and interpreted as the MIC. In the absence of PMBN, the MIC of mupirocin for SH5014 was 100 mg/L. As low a concentration of PMBN as 3 mg/L decreased the MIC of mupirocin for SH5014 by a factor of 100 (Figure). Higher concentrations of PMBN sensitized SH5014 further, and a > 1000-fold sensitization took place at a PMBN concentration of 30 mg/L. The MIC of mupirocin for PMBN-sensitized SH5014 was identical or very close to the MICs determined for mupirocin-susceptible Gram-positive cocci, such as Streptococcus pneumoniae (MIC, 0-1 mg/L), Streptococcus pyogenes (0-1 mg/L), and Staphylococcus aureus (0-02-0-12 mg/L) (see Ward & Campoli-Richards, 1986). A very similar, massive sensitization to mupirocin was observed when the other test strain, E. coli IH3080, was grown in the presence of PMBN (data not shown). Accordingly, the findings indicate that the OM of Gram-negative enteric bacteria acts as a very effective permeability barrier against mupirocin. However, this barrier can easily be disrupted by PMBN. Thus, agents which have a potent OM permeability-increasing action
Activity of snlbtctam combinations against Escherichia coE isolates with known amounts of TEM-1 0-bctamase Sir, We recently published, in this Journal, on the behaviour of combinations of clavulanate, tazobactam or BRL42715 with amoxycillin, ticarcilbn or piperacillin against Escherichia coli strains with measured amounts of the TEM-1 /Mactamase (Lrvermorc & Seetulsingh, 1991; Seetulsingh, Hall & Uvermore, 1991). The inhibitor concentrations required to
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 3, 2015
0-1
Capobianco, J. O., Doran, C. C. & Goldman, R. C. (1989). Mechanism of mupirocin transport into sensitive and resistant bacteria. Antimicrobial Agents and Chemotherapy 33, 156-63. Hughes, J. & Mellows, G. (1980). Interaction of pseudomonk acid A with Escherichia coli B isoleucyl-tRNA synthetase. Biochemical Journal 191, 209-19. Nikaido, H. & Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiological Reviews 49, 1-32. Vaara, M. (1989). Analytical and preparative highperformance liquid chromatography of the papain-deaved derivative of polymyxin B. Journal of Chromatography 441, 423-30. Vaara, M. (1990). Antimicrobial susceptibility of Salmonella typhimwium carrying the outer membrane permeability mutation SS-B. Antimicrobial Agents and Chemotherapy 34, 853-7. Vaara, M. & Jaakkola, J. (1989). Sodium hexametaphosphate sensitizes Pseudomonas aerugfnosa, several other species of Pseudomonas, and Escherichia coli to hydrophobic drugs. Antimicrobial Agents and Chemotherapy 33, 1741-7. Vaara, M. & Vaara, T. (1983). Sensitization of gram-negative bacteria to antibiotics and complement by a nontoxk oligopeptide. Nature 303, 526-8. Viljanen, P. & Vaara, M. (1984). Susceptibility of gram-negative bacteria to polymyxin B nonapeptide. Antimicrobial Agents and Chemotherapy 25, 701-5. Ward, A & Campoli-Richards, D. M. (1986). Mupirocin. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 32, 425-44.
Correspondence
223
Table. Mean MICt of /Mactamt, and inhibitor levels required to achieve potentiation for isolates in each quartile of the /Mactamase activity distribution
Quartile
512 813 1193 2581
Geometric mean [sulbactam] (mg/L) needed to achieve ampidllin MIC cefoperazone MIC - 8 mg/L . -0-25 mg/L
1-0 1-6 2-7 80
potentiate the penicillins were shown to depend closely on the amount of TEM-1 enzyme produced. The behaviour of sulbactam combinations was investigated in a follow-up study with the same strains and methods. Briefly, MICs of ampidllin and cefoperazone alone and in combination with sulbactam (1, 2, 4, 8, 16 or 32 mg/L) were determined for 36 TEM-1 0lactamase producing E. coli isolates. The inocula comprised 104 cfu, taken from overnight nutrient broth cultures and applied to the surface of Diagnostic Sensitivity Test agar plates (Oxoid, Basingstoke, Hants) containing the antibiotics). The levels of /J-lactamase activity in these organisms was estimated from the activity of sonicates of exponential phase cells against 1 n i l benzylpenicillin at 37°C in 01 M phosphate buffer pH 7-0 (Seetulsingh et at., 1991). On the basis of the 0lactamase data the collection was divided into 4 quartiles: quartile 1 (Ql), comprised the nine isolates with lowest /Mactamase specific activities; Q2, comprised those with the 10-18th lowest (inclusive); Q3, those with the 19-27th lowest and Q4, the nine isolates with the highest specific activities. The Table shows the geometric mean MICs of ampidllin and cefoperazone for isolates in each quartile of the /Mactamase activity distribution. As with amoxycillin and piperacillin (Livermore & Seetulsingh, 1991), the geometric mean MICs of ampidllin and cefoperazone were highest for the isolates in Q4 and lowest for those in Ql. All the isolates including those with the lowest /Mactamase activities were resistant to ampidllin, with MICs of this antibiotic ranging upwards from 256 mg/L as compared to MICs of 1-4 mg/L for E. coli isolates without TEM-1 enzyme. MICs of cefoperazone for the TEM-1 producers ranged from 0-12 to 32 mg/L, as compared to O008-(M)15 mg/L for ten non-producers. Potentiation of ampidllin activity by sulbactam was weak. For 30/36 isolates, no subinhi-
13-7
2O2 32-0
471
50 101 160 400
bitory concentration of sulbactam rendered the bacteria sensitive to 2 mg/L of ampidllin, which is the MIC antidpated for TEM-non producers, and for 13/36 no subinhibitory concentration rendered the bacteria susceptible to ampidllin at the BSACs recommended breakpoint of 8 mg/L (Working Party of the British Society for Antimicrobial Chemotherapy, 1991). This was not a problem which we have encountered with other inhibitors, where subinhibitory concentrations almost invariably obviated /Mactamase activity or, at least, brought the penicillin MICs to below the breakpoint (Livermore & Seetulsingh, 1991). These weak inhibitory activity of sulbactam for TEM-1 enzyme also has been demonstrated also in assays with extracted /Mactamase. Although sulbactam acts as a suiride inhibitor of TEM-1 enzyme, the process is relatively ineffident and considerable enzymemediated hydrolysis of the inhibitor occurs for each enzyme molecule that is inactivated (Fisher et al., 1980). This hydrolytic activity was reflected in MIC data obtained with sulbactam alone. At 32 mg/L, the compound inhibited the growth of 8/9 Ql isolates, 7/9 Q2 isolates, 5/9 Q3 isolates and 0/9 Q4 isolates. By contrast MICs of clavulanate (16-64 mg/L) and tazobactam (64-256 mg/L) for these strains were not related to /Mactamase quantity (unpublished data). As with tazobactam, clavulanate and BRL42715 (Livermore & Seetulsingh, 1991), the concentrations of sulbactam required to potentiate ampidllin and cefoperazone depended on the amount of /Mactamasc produced. The Table shows the geometric mean sulbactam concentrations required to potentiate ampidllin MICs to the breakpoint of 8 mg/L. Where this level of potentiation was not achieved for an individual strain with any subinhibitory concentration of sulbactam, it was assumed, in order to calculate the geometric mean, that such potentiation would have been achieved with the next higher con-
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 3, 2015
1 2 3 4
Geometric mean MICi (mg/L) Ampkalhn Cefoperazone
224
Correspondence
DAVID M. LJVERMORE Department of Medical Microbiology, The London Hospital Medical College, Turner Street, London El 2AD, UK
References Fisher, J., Belasco, J. O., Charnas, R. L , Khosla, S. & Ksowles, J. R. (1980). 0-Lactamase inactivation by mechanism-based reagents. Philosophical Transactions of the Royal Society (London) 289B, 309-19. Livermore, D. M. & Seetnlnngh, P. (1991). Susceptibility of Escherichia coli isolates with TEM-1 0-lactamase to combinations of BRL42715, t H ^ N r " " " or clavulanate with priperacfllin or amoxycillin. Journal of Antimicrobial Chemotherapy 27, 761-7.
Seetubingh, P., Hall, L. M. C. & Livermore, D. M. (1991). Activity of clavulanate combinations against TEM-1 /)~lactamase producing Escherichia coli isolates obtained in 1982 and 1989. Journal of Antimicrobial Chemotherapy 27, 749-59. Working Party of the British Society of Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1-47.
Prediction of ofloxadn susceptibility from Inritro tests with dprofloxadn Sir, Ofloxadn is a fluoroquinolone compound that has been approved recently for use as an orally administered antimicrobial agent in the United States. An intravenous formulation will soon be available. Unfortunately, manufacturers of many broth microdilution trays and other susceptibility testing systems have not yet completed the studies that are needed in order to incorporate ofloxadn into their test systems, but dprofloxadn can be tested. Mutants that are selected in vitro for resistance to one quinolone compound show cross-resistance to other drugs in that class (Barry & Jones, 1984; King, Shannon & Phillips, 1985; Barry, Gardiner & Packer, 1987). Because of this cross-resistance, the results of susceptibility tests with dprofloxadn might be used to predict susceptibility or resistance to ofloxadn. To determine the validity of that assumption, I reviewed the results of previously reported broth microdilution tests and calculated predictive values of dprofloxadn MIC categories for five major groups of bacterial pathogens (enteric bacilli, pseudomonads, staphylococci, enterococd and streptococci). Broth microdilution tests were performed according to the procedure of the National Committee for Clinical Laboratory Standards (NCCLS) and quality control strains gave MICs within the acceptable ranges for both drugs (NCCLS, 1990). The NCCLS interpretive criteria were used to define susceptible, intermediate and resistant categories. For dprofloxadn, those MIC breakpoints were < 1-0, 20 and £ 4-0 mg/L, respectively, and for ofloxadn the MIC breakpoints were < 2-0,4-0 and > 8-0, respectively. The 32 bacterial species that were represented in the culture collection are described in Table 1. MIC^s and MIC^s of dprofloxadn and ofloxadn did not differ greatly, but for many individual strains ofloxadn MICs were two- to four-times greater than those of dpro-
Downloaded from http://jac.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on June 3, 2015
centration in a doubling dilution sequence, irrespective of whether or not this level of the sulphone would itself have inhibited bacterial growth. Analysis of the cefoperazone data is problematical in that there is no obvious level of potcntiation to take as a reference point TEM-1-negative E. coli were sensitive to 0-008-0-015 mg/L of cefoperazone, but MICs of the cephalosporin for TEM-1-producers could rarely (6/36 cases) be reduced to this level with subinhibitory sulbactam concentrations. On the other hand, cefoperazone MICs only exceeded the cephalosporin's breakpoint (16 mg/L) for two isolates (both in Q3 or 4), even in the absence of sulbactam: 11/36 isolates were susceptible even at the BSACs recommended "lower breakpoint" of 2 mg/L. Included on the table are data on the sulbactam concentrations required to reduce cefoperazone MICs to 0-23 mg/L for isolates in each quartfle of the /Mactamase activity distribution. This is an arbitrary level, chosen only as being below the cefoperazone MIC for 34/36 TEM-1 producers but being achieved in the presence of sulbactam for 33/36 isolates. In summary, our results indicate two points: firstly that, as with other inhibitors, the concentrations of sulbactam required to achieve potentiation of substrate /Mactams depended on the amount of /Mactamase produced and, secondly, that the inhibitory power of sulbactam for TEM-1 was so weak that only a small minority (4/36 isolates) of the TEM producers would have counted as susceptible to the combination of 8 mg/L sulbactam plus 8 mg/L ampicillin. The cefoperazone sulbactam combination fared better, largely because TEM-1 production did not give high level resistance to this cephalosporin in E. coli.
