594
Correspondence
M. TEDCELL* "Institute Municipal de Investigaclon Midica, Paseo Maritime*, 25-28. 08003-Barcelona; 'Microbiologia-MetUdna Unhersidad Autdnoma de Barcelona, BeUaterra. Barcelona, Spain References Cooksey, R., Swenson, J., Clark, N., Gay, E. & Thonuberry, C. (1990), Patterns and mechanisms of 0-lactam resistance among isolates of Escherichia coll from hospitals in the United States. Antimicrobial Agents and Chemotherapy 34, 739-45. Huovinen, S., Huovinen, P., Torniainen, K. & Jacoby, O. A. (1988). Evaluation of plasmidencoded beta-lactamase resistance in Escherichia coli blood culture isolates. European Journal of Clinical Microbiology and Infectious Diseases 7, 651-5. Roy, C , Tirado, M., Morilla, A. & Esteva, C. (1990). Detection de hiperproducti6n de /(-lactamasa cromosomka. Enfermedadei Infeeciosas y Microbiologla Clinica 8, 123-4. Sousa, J. C , Carneiro, G., Peixe, M. L., Quetros, M. L. & Rebelo, I. (1991). Characterization of Blactamases encoded by pathogenic strains of Escherichia coli from Portugal. Journal of Antimicrobial Chemotherapy 27, 437-40.
Effect of pH on antibiotics nsed to treat anaerobic Infection Sir, The antibiotic treatment of intra-abdominal sepsis must provide cover against anaerobic
bacteria. Antimicrobial agents are generally designed to work at near neutral pH values, as the pH of body fluids normally ranges from 7-2 to 7-4. Indeed in-vitro susceptibility tests are performed at neutral pH. A wide pH range (5-5-6-8) is, however, found in intra-abdominal abscesses (Bryant, 1984). It is important, therefore, to establish that antibiotics which demonstrate anti-anaerobe activity in vitro do so throughout the pH range to which they may be exposed in vivo. Tally, DePasqua & Jacobus (1986) studied 75 isolates of the Bacteroides fragilis group and found that the in-vitro activity of imipenem, cefoxitin, clindamycin and piperacillin was significantly reduced in 100%, 49%, 26% and 11% of strains, respectively, at pH 5-5 compared to pH 7-0. We have studied the effect of pH on the in-vitro activity of cefoxitin, clindamycin, imipenem, metronidazole and piperacUlin/tazobactam (8:1) against seven clinical isolates and the type strain (NCTC 9343) of B. fragilis. MICs were determined using a micrc-dihition method with antibiotics made up in Fastidious Anaerobe Broth (FAB, Oxoid) prepared in citrate-phosphate buffer (pH 5-5-7-0). End-points were determined after incubation for 48 h in anaerobic conditions. Citrate-phosphate buffer was chosen after preliminary experiments demonstrated that the use of unbuffered FAB resulted in marked decreases in broth pH during growth. Citrate-phosphate buffer maintained the starting pH within 0-1 during growth. It is noteworthy that Tally et al. (1986) used unbuffered broth in their studies. Results are shown in the Table. The activity of metronidazole and piperacillin/tazobactam was not adversely affected by a pH as low as 5-5. However, when the pH was decreased from 7-0 to 5-5, one, six and eight strains were less susceptible (four-fold or greater increase in MIC) to cefoxitin, clindamycin and impipenem, respectively. Killing curve experiments performed with strain 30, using clinically achievable antibiotic concentrations (2 x MIC, as determined at pH 7-0), showed that imipenem did not exhibit bactericidal activity after incubation for 24 h at pH 5-5 in contrast to all other antibiotic/pH combinations tested (data not shown). Although Tally et al. (1986) found that the activity of cefoxitin was markedly inhibited at pH 5-5, their use of unbuffered broth may have inadvertently provided a much lower pH challenge. Our results, obtained using stable pH conditions, indicate that both metronidazole
Downloaded from http://jac.oxfordjournals.org/ at Simon Fraser University on June 12, 2015
1984-86 and those of Sousa et al. in 19881990. Furthermore we did not identify TEM-2 /Mactamases in any of the strains, whereas we detected strains with a loss of susceptibility to /Mactam antibiotics due to non-enzymatic mechanisms. The strains described by Sousa et al. also were less likely to be TEM-1 producing (78-2% against our 93%) but were more frequently SHV-1 producing (7-9% vs 2-8%) and also hyperproducing chromosomal /?lactamases (4-7% VJ 0-7%). Our results were similar to those reported by Houvinen et al. (1988), and Cooksey et al. (1990). Occasionally the hyperproduction of chromosomal ^-lactamase alterations in the permeability of the outer membrane has been involved as a cause of resistance (Houvinen et al., 1988), 0-4% and 1-2% respectively, of our strains showed resistance attributable to these mechanisms. C. ROY* D. TERUEL' R. RHG** M. HERMIDA*
595 Table. Inflaence of pH on antibiotic MIC for seven clinical isolates and a NCTC strain of B. fragiiis
PH Antibiotic
Microorganism (all B. fragiiis)
Ccfoiitin
NCTC 9343
Oindamycin
NCTC 9343
15 87 49 30 64 37 99 Inupenem
NCTC 9343
15 87 49 30 64 57 99 Metronidazoie
NCTC 9343
15 87 49 30 64 57 99 Piperaaflin +tazobactam
NCTC 9343
IS 87 49 30 64 57 99
and piperacjllin/tazobactam should retain useful activity in the relatively acidic conditions found in anaerobic infections in vivo. T. G. WINSTANLEY M. H. WILC0X R. C. SPENCER Department of Bacteriology and Experimental and Clinical Microbiology, Royal Hallamshire Hospital and Unbersity of Sheffield Medical School. Sheffield S10 2JF, UK
6-5
6
*5
(MICmg/L)
8 8 8 4 8 8 32 8 64 16 8 4 16 16 16 32 4 0125
16 0-5 0-25
1 1 2 05 05 1 1 1 1 05 2-0 025 05 4 1 025 025 1 4
8 16 16 8 8 16 32 16 256 32 8 8 64 32 8 64 8
8 32 8 8 16 16 8 8 8 16 16 16 32 32 8 16 256 256 32 64 16 32 16 16 64 128 64 32 16 64 256 128 64 >64 0125 64 >64 16 >64 64 05 4 2 1 32 32 1 >8 >8 1 4 2 8 64 32 025 05 05 025 025 05 025 O25 05 1 05 05 025 025 05 1 1 1 025 025 025 1 1 1 025 025 025 05 05 05 8 8 4 05 05 05 05 05 05 I 05 05 1 1 1 8 4 4
Reft Bryant, R. E. (1984). Effect of the supparative environment on antibiotic activity. In New Dimensions of Antimicrobial Therapy (Root, R. K. & Sande, M. A., Eds), pp. 313-57. Churchill Livingstone, New York. Tally, F. P., DePasqua, J. & Jacobus, N. V. (1986). Effect of pH on the in vitro activity of selected antimicrobial agents against Bacteroides fragiiis. ASM Annual General Meeting. Washington, DC, Abstract A60.
Downloaded from http://jac.oxfordjournals.org/ at Simon Fraser University on June 12, 2015
15 87 49 30 64 57 99
7
Correspondence
M. TEDCELL* "Institute Municipal de Investigaclon Midica, Paseo Maritime*, 25-28. 08003-Barcelona; 'Microbiologia-MetUdna Unhersidad Autdnoma de Barcelona, BeUaterra. Barcelona, Spain References Cooksey, R., Swenson, J., Clark, N., Gay, E. & Thonuberry, C. (1990), Patterns and mechanisms of 0-lactam resistance among isolates of Escherichia coll from hospitals in the United States. Antimicrobial Agents and Chemotherapy 34, 739-45. Huovinen, S., Huovinen, P., Torniainen, K. & Jacoby, O. A. (1988). Evaluation of plasmidencoded beta-lactamase resistance in Escherichia coli blood culture isolates. European Journal of Clinical Microbiology and Infectious Diseases 7, 651-5. Roy, C , Tirado, M., Morilla, A. & Esteva, C. (1990). Detection de hiperproducti6n de /(-lactamasa cromosomka. Enfermedadei Infeeciosas y Microbiologla Clinica 8, 123-4. Sousa, J. C , Carneiro, G., Peixe, M. L., Quetros, M. L. & Rebelo, I. (1991). Characterization of Blactamases encoded by pathogenic strains of Escherichia coli from Portugal. Journal of Antimicrobial Chemotherapy 27, 437-40.
Effect of pH on antibiotics nsed to treat anaerobic Infection Sir, The antibiotic treatment of intra-abdominal sepsis must provide cover against anaerobic
bacteria. Antimicrobial agents are generally designed to work at near neutral pH values, as the pH of body fluids normally ranges from 7-2 to 7-4. Indeed in-vitro susceptibility tests are performed at neutral pH. A wide pH range (5-5-6-8) is, however, found in intra-abdominal abscesses (Bryant, 1984). It is important, therefore, to establish that antibiotics which demonstrate anti-anaerobe activity in vitro do so throughout the pH range to which they may be exposed in vivo. Tally, DePasqua & Jacobus (1986) studied 75 isolates of the Bacteroides fragilis group and found that the in-vitro activity of imipenem, cefoxitin, clindamycin and piperacillin was significantly reduced in 100%, 49%, 26% and 11% of strains, respectively, at pH 5-5 compared to pH 7-0. We have studied the effect of pH on the in-vitro activity of cefoxitin, clindamycin, imipenem, metronidazole and piperacUlin/tazobactam (8:1) against seven clinical isolates and the type strain (NCTC 9343) of B. fragilis. MICs were determined using a micrc-dihition method with antibiotics made up in Fastidious Anaerobe Broth (FAB, Oxoid) prepared in citrate-phosphate buffer (pH 5-5-7-0). End-points were determined after incubation for 48 h in anaerobic conditions. Citrate-phosphate buffer was chosen after preliminary experiments demonstrated that the use of unbuffered FAB resulted in marked decreases in broth pH during growth. Citrate-phosphate buffer maintained the starting pH within 0-1 during growth. It is noteworthy that Tally et al. (1986) used unbuffered broth in their studies. Results are shown in the Table. The activity of metronidazole and piperacillin/tazobactam was not adversely affected by a pH as low as 5-5. However, when the pH was decreased from 7-0 to 5-5, one, six and eight strains were less susceptible (four-fold or greater increase in MIC) to cefoxitin, clindamycin and impipenem, respectively. Killing curve experiments performed with strain 30, using clinically achievable antibiotic concentrations (2 x MIC, as determined at pH 7-0), showed that imipenem did not exhibit bactericidal activity after incubation for 24 h at pH 5-5 in contrast to all other antibiotic/pH combinations tested (data not shown). Although Tally et al. (1986) found that the activity of cefoxitin was markedly inhibited at pH 5-5, their use of unbuffered broth may have inadvertently provided a much lower pH challenge. Our results, obtained using stable pH conditions, indicate that both metronidazole
Downloaded from http://jac.oxfordjournals.org/ at Simon Fraser University on June 12, 2015
1984-86 and those of Sousa et al. in 19881990. Furthermore we did not identify TEM-2 /Mactamases in any of the strains, whereas we detected strains with a loss of susceptibility to /Mactam antibiotics due to non-enzymatic mechanisms. The strains described by Sousa et al. also were less likely to be TEM-1 producing (78-2% against our 93%) but were more frequently SHV-1 producing (7-9% vs 2-8%) and also hyperproducing chromosomal /?lactamases (4-7% VJ 0-7%). Our results were similar to those reported by Houvinen et al. (1988), and Cooksey et al. (1990). Occasionally the hyperproduction of chromosomal ^-lactamase alterations in the permeability of the outer membrane has been involved as a cause of resistance (Houvinen et al., 1988), 0-4% and 1-2% respectively, of our strains showed resistance attributable to these mechanisms. C. ROY* D. TERUEL' R. RHG** M. HERMIDA*
595 Table. Inflaence of pH on antibiotic MIC for seven clinical isolates and a NCTC strain of B. fragiiis
PH Antibiotic
Microorganism (all B. fragiiis)
Ccfoiitin
NCTC 9343
Oindamycin
NCTC 9343
15 87 49 30 64 37 99 Inupenem
NCTC 9343
15 87 49 30 64 57 99 Metronidazoie
NCTC 9343
15 87 49 30 64 57 99 Piperaaflin +tazobactam
NCTC 9343
IS 87 49 30 64 57 99
and piperacjllin/tazobactam should retain useful activity in the relatively acidic conditions found in anaerobic infections in vivo. T. G. WINSTANLEY M. H. WILC0X R. C. SPENCER Department of Bacteriology and Experimental and Clinical Microbiology, Royal Hallamshire Hospital and Unbersity of Sheffield Medical School. Sheffield S10 2JF, UK
6-5
6
*5
(MICmg/L)
8 8 8 4 8 8 32 8 64 16 8 4 16 16 16 32 4 0125
16 0-5 0-25
1 1 2 05 05 1 1 1 1 05 2-0 025 05 4 1 025 025 1 4
8 16 16 8 8 16 32 16 256 32 8 8 64 32 8 64 8
8 32 8 8 16 16 8 8 8 16 16 16 32 32 8 16 256 256 32 64 16 32 16 16 64 128 64 32 16 64 256 128 64 >64 0125 64 >64 16 >64 64 05 4 2 1 32 32 1 >8 >8 1 4 2 8 64 32 025 05 05 025 025 05 025 O25 05 1 05 05 025 025 05 1 1 1 025 025 025 1 1 1 025 025 025 05 05 05 8 8 4 05 05 05 05 05 05 I 05 05 1 1 1 8 4 4
Reft Bryant, R. E. (1984). Effect of the supparative environment on antibiotic activity. In New Dimensions of Antimicrobial Therapy (Root, R. K. & Sande, M. A., Eds), pp. 313-57. Churchill Livingstone, New York. Tally, F. P., DePasqua, J. & Jacobus, N. V. (1986). Effect of pH on the in vitro activity of selected antimicrobial agents against Bacteroides fragiiis. ASM Annual General Meeting. Washington, DC, Abstract A60.
Downloaded from http://jac.oxfordjournals.org/ at Simon Fraser University on June 12, 2015
15 87 49 30 64 57 99
7
