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Chemotherapy 1990;36:215-221
Turbidimetric and Microscopic Analysis of Bacteroides fragilis Exposed to Tazobactam and Piperacillin Alone and in Combination E. Yourassowsky, M.P. Van der Linden, M.J. Lismont, F. Crokaert Microbiology, Brugmann University Hospital, Brussels, Belgium
Key Words. Tazobactam • Piperacillin • Bacteroides fragilis
Introduction Tazobactam (YTR-830H) is a new de rivative of penicillanic acid sulfone which has been shown to be an active P-lacta mase inhibitor [2, 6-8]. Combined with various p-lactams such as piperacillin, ta
zobactam showed a synergy against P-lactamase-producing strains including Staph ylococcus aureus, Haemophilus influenzae, Bacteroides spp. and many strains of Enterobacteriaceae. Tazobactam has a very low intrinsic activity against almost all Gram-positive and -negative organisms
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Abstract. Tazobactam, a non-amino penicillanic acid sulfone, is a new P-lactamase inhibitor and acts synergistically with piperacillin against clinical isolates of p-lactamase-producing Bacteroides fragilis. The effectiveness of this inhibitor has been demon strated by reduction of piperacillin MIC values and growth curves in two combinations (ratio 8:1 and 4:1). Tazobactam has an intrinsic activity against B.fragilis (MIC = 4-8 pg/ml). Phase contrast microscopy of treated cells showed cell-wall-deficient bacte ria, predominantly filaments for piperacillin and spheric bodies for tazobactam. The combination of these two drugs induced large bulging filaments. This would suggest that with this organism, tazobactam binds to PBP2 while piperacillin binds to PBP3. The growth curves obtained with the MS-2 system showed: (1) a significant deviation (lower OD value) from the control curve with piperacillin at lA MIC and numerous irreg ularities as compared to the control, due to the presence of filament clusters; (2) a devia tion from the control curves without irregularities, quite different that these observed with piperacillin while using tazobactam at Vs MIC, and (3) a deviation from the control curve for lower piperacillin values in piperacillin-tazobactam combinations (8:1-4:1). In conclusion, the synergistic efficiencies of piperacillin/tazobactam against B. fragi lis act in two ways, inhibition of P-lactamase and a probable competition of PBPs.
Yourassowsky/Van der Linden/Lismont/Crokaert
(MIC = 50-400 ng/ml), with the excep tion of Acinetobacter spp. and Bacteroides fragilis [1], Moosdeen et al. [8] have shown that tazobactam, despite a very poor antibacterial activity against Es cherichia coli, binds to PBP2 and at MIC values (512|ig/ml) caused rapid lysis of the formed spheroplasts. Piperacillin on the other hand, binds to PBP3. We previously showed some interesting correlations between growth curves, the presence of cell-wall-deficient bac teria, and PBPs [13, 14]. In the present study we exposed 10 strains of B. fragilis to different concentrations of tazobac tam and piperacillin alone and in com bination (ratio piperacillin: tazobac tam = 8:1 or 4:1) to define antibacterial effects on bacterial morphology and growth curve patterns. Indeed, the com bination of piperacillin and tazobactam being an association of two P-lactams, a possible synergy or antagonism for peni cillin-binding proteins may be expected. Material and Methods Bacterial Strains Ten strains of B. fragilis were obtained from clini cal specimens (pus and blood cultures). These strains were identified by methods recommended by Finegold and Edelstein [5], All isolates were ¡1-lactamase producers, detected by the chromatogenic cephalo sporin method. Antibiotics Tazobactam and piperacillin were obtained from Lederle Laboratories (Pearl River, N.Y.). Antimicrobial Susceptibility Testing Isolates were tested by methods recommanded by Sutter [11], Tubes contained doubling dilutions of piperacillin or tazobactam ranging from 0.5 to 512 pg/ml. The piperacillin-tazobactam combina
tions were used in 8:1 and 4:1 ratios. The medium presently used was a Wilkin-Chalgreen broth. The in itial bacterial density was I06 CFU/ml. Synergy was defined as a 4-fold or greater decrease on the MIC of piperacillin in the presence of tazobactam compared with piperacillin alone. Growth Curves Growth curves were performed with the MS-2 system [10]. This system was originally not designed for anaerobic studies. The technical modification of this system, previously published [12], has been elab orated in the way to obtain an anaerobic environ ment in multichamber cuvettes. The inoculum was prepared in an anaerobic chamber (Forma Scientific Anaerobic System, Model 1024, Marietta, Ohio) us ing Wilkin-Chalgreen broth. A growing culture was adjusted to a 0.5 MacFarland turbidity and diluted 10 times. This culture in growth phase contained ap proximately 107 CFU/ml. Transfer cartridges (Ab bott Laboratories, Diagnostic Division, Dallas, Tex.) were used. Discs impregnated with piperacillin or ta zobactam determining concentrations decreasing from 256 to 0.03 mg/1, alone and in combination (ra tio = 4:1 or 8:1) were introduced into the lower chambers of the cartridges. The bottom of the car tridges were sealed with varnisch. The air-entry ori fice remained open. Cartridges so prepared were stocked in an anaerobic chamber before use. 16 ml of the microbial suspension were introduced into the upper chamber of the cartridge using an anaerobic glove box. The transfer of the culture to the lower chambers was done in the interchange (air-lock) of the Forma anaerobic vessel where it was possible to achieve a vacuum of -20 in. of mercury. When the normal pressure was restored, the liquid was trans ferred from the upper part of the cartridge to the lower chambers. The cartridge was removed from the anaerobic glove box chamber and the air-entry open ing of the transfer cartridge (diameter = 1 mm) im mediately coated with plasticine. The cartridges were placed in the MS-2 analysis module, and recordings of optical densities started at once. At time zero, all the organisms were in the growth phase as assessed by the control curves. Recordings were performed every 5 min for 22 h. Growth Curves Signification The problem encountered with the MS-2 system is the mixing procedure which was not perfect. How-
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Fig. 1. MS-2 growth curve pat terns of B.fragilis submitted to pip eracillin. The antibiotic was intro duced at time zero in a log phase culture. C - Control curve; D = deviation from control curve; ROV = rapid OD variations observed at concentrations near the MIC value and due to clusters of filaments; I = irregularities due to clusters of filaments observed at all piperacil lin concentrations from 2 to 256 mg/ml.
Table 1. Antibacterial effects (pg/ml) of piperacillin and tazobactam alone and in combination against 10 strains of B.fragilis Antibiotic
MIC
MBC
Deviationb
ROVc
Piperacillin Tazobactam Combination 8:1“ Combination 4:1
2(1-4) 8(4-8) 0.5 (0.5-1) 0.5 (0.5-1)
4(2-8) 16(4-8) 1(0.5-2) 1 (0.5-1)
0.5(0.12-1) 0.8(0.12-4) 0.5 0.5
2(1-8) none 1(1-2) 1 (0.5-2)
ever, the reproducibility of the growth curves proved to be excellent. A growth curve, 5-fold performed, showed OD values of 0.29 ±0.012 or 4% and 1.21 ±0.064 or 5%, respectively, after 3 and 6 h of incubation. We considered that the growth curves obtained using the MS-2 system represent a combi nation of bacterial multiplication and other factors responsable for biomass increase (particularly cellwall-deficient bacteria) combined with sedimenta tion. In these experimental conditions some particu larities may be detected in the growth curve which were not seen using conventional growth curves, constructed point by point with a vigorous mixing between each measure. Each particularity that was not observed in the control curve has to be taken into
consideration to evaluate the antibacterial effect of antibiotics. Normal growth curves of B. fragilis pre sent as classic S-shape curves. In the presence of some antibiotics, curves present numerous indenta tions after a few hours. The mean magnitude of these OD variations depends on the time of contact be tween the culture and the antibiotic. These rapid OD variations (ROV) are interesting because they corre spond to clusters of filament formations observed by phase-contrast microscopy. Figure 1 shows the different particularities of the growth curve using the MS-2 system which were taken into consideration to make a comparative eval uation of tazobactam and piperacillin, separately and in combination.
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“ Expressed in piperacillin concentration. b Lowest antibiotic concentration inducing a minimum reduction of 0.1 OD at 3 h. c Lower antibiotic concentration inducing ROV at 6 h.
Results Antimicrobial Susceptibility Testing and Synergy Table 1 presents the MIC and MBC val ues obtained with 10 strains of B.fragilis using the macrodilution method. The mean values and ranges were 2 pg/ml (1-4 pg/ml) for piperacillin and 8 pg/ml (4-8 pg/ml) for tazobactam. Synergy was demonstrated against the 10 strains. A 4-fold decrease and even more in the MIC value was observed with piperacillin in the presence of tazobactam for the ra tios 1:4 or 1:8. Expressed as MIC or MBC values, the differences between 1:4 and 1:8 ratios were not significant. Growth Curves Some characteristics of the growth curves as detailed in figure 1 are presented in table 1. A deviation from the control curve was observed with piperacillin alone for 0.5 pg/ml. We considered these con centrations as MAC values which were found for lA MIC values. For tazobactam these deviations were observed at 0.8 pg/ ml or Vio MIC value. Expressed in pipera cillin concentration, the combination of the two drugs did not reduce the MAC val ue (0.5 pg/ml) of piperacillin alone when the ratios were 8:1 or 4:1. Rapid OD varia tion was detected with piperacillin at con centrations near the MIC value. This phenomenon was not observed with ta zobactam. When the two drugs acted to gether, the ROV was observed at concen trations of piperacillin which produced this phenomenon when used alone. Figure 2 shows phase contrast micros copy of B.fragilis submitted to piperacil lin and tazobactam alone and in combina
Yourassowsky/Van der Linden/Lismont/Crokaert
tion. This photography has been taken after 2, 6 and 24 h of contact between the antibiotics and the microorganisms. Piper acillin induced filament formation at all concentrations used. Tazobactam induced round forms. When the two drugs acted together we observed large bulging fila ments. The overall result showed a pre dominant activity of piperacillin.
Discussion As previously pointed out, tazobactam reduces the MIC value of piperacillin against B.fragilis to 'A MIC. In our study, the mean MIC value for piperacillin was 2 pg/ml. In combination with tazobactam at the ratio 8:1 and 4:1, the MIC value for piperacillin was reduced to 0.5 pg/ml. The in vitro synergistic activity was due to the p-lactamase inhibitory effect of tazobac tam. Tazobactam has nevertheless an in trinsic activity against B.fragilis with a MIC value of 8 pg/ml [1, 3]. The morpho logical responses caused by piperacillin and tazobactam showed at their MIC threshold filament formation for piper acillin and round bodies for tazobactam. These observations suggest two different binding sites on PBPs for piperacillin and tazobactam. The exact PBPs of B.fragilis
Fig. 2. Phase contrast microscopy of B.fragilis submitted to piperacillin and tazobactam alone and in combination. A = Control: B = tazobactam 8 pg/ ml, 2 h; C = piperacillin 2 pg, 6 h; D = piperacillin 2 pg/ml, 24 h; E = piperacillin 2 pg + tazobactam 0.5 pg/ml, 24 h; F = piperacillin 2 pg/ml + tazobac tam 0.12 pg/ml, 24 h.
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Tazobactam against Bacteroidesfragilis 219
which represent the target of P-lactams and related compounds remain controversal. For Piddock and Wise [9], the com pound of PBP1 is involved in cell elonga tion while PBP2 is involved in septum for mation and PBP3 in maintenance of cell shape (i.e. BPB2 in B.fragilis = PBP3 in E. coli, and PBP3 in Bacteroides spp. = PBP2 in E. coli). Growth curves performed with the MS-2 system showed an interest ing phenomenon due to the formation of clusters of filaments in the cuvette. ROV appear at lower piperacillin concentra tions, near the MIC value and was absent from tazobactam-treated cultures. In com bination, the ROV phenomenon persists at a concentration near the MIC value of piperacillin alone. When the tazobactam concentration increased to a more ele vated concentration, the ROV persists. On the other hand a deviation from the con trol curve, which may be considered as the MAC value, was observed for piperacillin at '/4 MIC and for tazobactam at V io MIC. The MAC values of piperacillin remained unchanged with lower concentrations of tazobactam. The overall results of our study showed that growth curves might be used to define the MAC values and an interesting correlation between some particularities (ROV) and morphology of bacteria submitted to the cell wall active substance. ROV phenomenon was only taken into consideration when it was not present in the control curve. Indeed, the morphology of B.fragilis might be greatly influenced by the medium composition [4], In our study, we used Wilkin-Chalgreen broth as recommanded for anaerobes. In conclusion, tazobactam, a (3-lactamase inhibitor, is mainly used in combina
Yourassowsky/Van der Linden/Lismont/Crokaert
tion with a second P-lactam antibiotic pri marily to inhibit the p-lactamase. They may nevertheless exert some intrinsic ac tivity at propriate concentrations, reached in patients or in susceptibility tests. References 1 Appelbaum, P.C.; Jacobs, M.R.; Spangler, S.K.; Yamabe, S.: Comparative activity of the beta-lactamase inhibitors YTR 830, clavulanate and sul bactam combined with beta-lactams against betalactamase-producing anaerobes. 86th Annu. Meet. Am. Soc. Microbiol., Abstract A88, p. 15 (1988). 2 Aronoff, S.C.; Jacobs, M.R.: Johenning, S.; Ya mabe, S.: Comparative activities of the beta-lactamase inhibitors YTR 830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin. Antimicrob. Agents Chemother. 26: 580-582(1984). 3 American Cyanamid Company: Brochure for clinical investigators CL298.741 beta-lactamase inhibitor. (Lederle Laboratories, Pearl River 1987). 4 Eley, A.; Greenwood, D.; O'Grady, F.: Compara tive growth of Bacteroides species in various an aerobic culture media. J. med. Microbiol. 19: 195-201 (1985). 5 Finegold, S.M.; Edelstein, M.C.: Gram-negative, nonsporeforming anaerobe bacilli; in Lennette, Balows, Flausler, Jr., Shadomy, Manual of clini cal microbiology: 4th ed., pp 450-460 (American Society for Microbiology, Washington 1985). 6 Gutman, L.; Kitzis, M.D.: Yamabe, S.; Acar, J.F.: Comparative evaluation of the new beta-lacta mase inhibitor, YTR 830, combined with dif ferent beta-lactam antibiotics against bacteria harboring known beta-lactamases. Antimicrob. Agents Chemother. 29: 955-957 (1986). 7 Jacobs, M.R.: Aronoff, S.C.; Johenning, S.; Ya mabe, S.: Comparative activities of the beta-lactamase inhibitors YTR 830, clavulanate and sul bactam combined with extended-spectrum peni cillins against ticarcillin-resistant Enterobacteriaceae and pseudomonads. J. antimicrob. Chemo ther. 18: 177-18(1984). 8 Moosdeen, F.; Williams, J.D.; Yamabe, S.: Anti
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11
12
13 Yourassowsky, E.; Van der Linden, M.P.; Lismont, M.J.; Crokaert, F.; Glupczynski, Y.: Effect on growth curve patterns of brief exposure of bacteria to different concentrations of beta-lactam antibiotics. J. antimicrob. Chemother. 15: suppl. A, pp. 27-36 ( 1985). 14 Yourassowsky, E.; Van der Linden, M.P.; Lismont, M.J.; Crokaert, F.; Glupczynski, Y.: Corre lation between growth curve and killing curve of Escherichia coli after a brief exposure to suprainhibitory concentration of ampicillin and piper acillin. Antimicrob. Agents Chemother. 28:756760(1985).
Prof. E. Yourassowsky Microbiology Brugman University Hospital Place A. Van Gehuchten 4 B -1020 Bruxelles (Belgium)
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bacterial characteristics of YTR 830, a sulfone beta-lactamase inhibitor, compared with those of clavulanic acid and sulbactam. Antimicrob. Agents Chemother. 32: 925-927(1988). Piddock, L.J.V.; Wise, R.: Properties of the peni cillin-binding proteins of four species of the ge nus Bacteroides. Antimicrob. Agents Chemother. 29: 825-832(1986). Spencer, H.J.; Stockert, J.; Welaj, P.; Wilborn, R.; Price, B.: Automated antibiotic susceptibility test ing with the MS-2 system; in Johnston, Newsom, 2nd Int. Symp. Rapid Methods and Automation in Microbiology, pp. 272-275 (Learned Informa tion, Oxford 1976). Sutter, V.L.: Susceptibility testing of anaerobes; in Lennette, Balows, Hausler, Jr., Shadomy, Manual of clinical microbiology; 4th ed., pp. 988-990 (American Society for Microbiology, Washington 1985). Yourassowsky, E.; Van der Linden, M.P.; Lismont, M.J.; Labbe, M.; Crokaert, F.: The effect of beta-lactam antibiotics on the growth curves of Bacteroides fragilis obtained with the MS-2 system. J. antimicrob. Chemother. 8: 219-224 (1981).
Chemotherapy 1990;36:215-221
Turbidimetric and Microscopic Analysis of Bacteroides fragilis Exposed to Tazobactam and Piperacillin Alone and in Combination E. Yourassowsky, M.P. Van der Linden, M.J. Lismont, F. Crokaert Microbiology, Brugmann University Hospital, Brussels, Belgium
Key Words. Tazobactam • Piperacillin • Bacteroides fragilis
Introduction Tazobactam (YTR-830H) is a new de rivative of penicillanic acid sulfone which has been shown to be an active P-lacta mase inhibitor [2, 6-8]. Combined with various p-lactams such as piperacillin, ta
zobactam showed a synergy against P-lactamase-producing strains including Staph ylococcus aureus, Haemophilus influenzae, Bacteroides spp. and many strains of Enterobacteriaceae. Tazobactam has a very low intrinsic activity against almost all Gram-positive and -negative organisms
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Abstract. Tazobactam, a non-amino penicillanic acid sulfone, is a new P-lactamase inhibitor and acts synergistically with piperacillin against clinical isolates of p-lactamase-producing Bacteroides fragilis. The effectiveness of this inhibitor has been demon strated by reduction of piperacillin MIC values and growth curves in two combinations (ratio 8:1 and 4:1). Tazobactam has an intrinsic activity against B.fragilis (MIC = 4-8 pg/ml). Phase contrast microscopy of treated cells showed cell-wall-deficient bacte ria, predominantly filaments for piperacillin and spheric bodies for tazobactam. The combination of these two drugs induced large bulging filaments. This would suggest that with this organism, tazobactam binds to PBP2 while piperacillin binds to PBP3. The growth curves obtained with the MS-2 system showed: (1) a significant deviation (lower OD value) from the control curve with piperacillin at lA MIC and numerous irreg ularities as compared to the control, due to the presence of filament clusters; (2) a devia tion from the control curves without irregularities, quite different that these observed with piperacillin while using tazobactam at Vs MIC, and (3) a deviation from the control curve for lower piperacillin values in piperacillin-tazobactam combinations (8:1-4:1). In conclusion, the synergistic efficiencies of piperacillin/tazobactam against B. fragi lis act in two ways, inhibition of P-lactamase and a probable competition of PBPs.
Yourassowsky/Van der Linden/Lismont/Crokaert
(MIC = 50-400 ng/ml), with the excep tion of Acinetobacter spp. and Bacteroides fragilis [1], Moosdeen et al. [8] have shown that tazobactam, despite a very poor antibacterial activity against Es cherichia coli, binds to PBP2 and at MIC values (512|ig/ml) caused rapid lysis of the formed spheroplasts. Piperacillin on the other hand, binds to PBP3. We previously showed some interesting correlations between growth curves, the presence of cell-wall-deficient bac teria, and PBPs [13, 14]. In the present study we exposed 10 strains of B. fragilis to different concentrations of tazobac tam and piperacillin alone and in com bination (ratio piperacillin: tazobac tam = 8:1 or 4:1) to define antibacterial effects on bacterial morphology and growth curve patterns. Indeed, the com bination of piperacillin and tazobactam being an association of two P-lactams, a possible synergy or antagonism for peni cillin-binding proteins may be expected. Material and Methods Bacterial Strains Ten strains of B. fragilis were obtained from clini cal specimens (pus and blood cultures). These strains were identified by methods recommended by Finegold and Edelstein [5], All isolates were ¡1-lactamase producers, detected by the chromatogenic cephalo sporin method. Antibiotics Tazobactam and piperacillin were obtained from Lederle Laboratories (Pearl River, N.Y.). Antimicrobial Susceptibility Testing Isolates were tested by methods recommanded by Sutter [11], Tubes contained doubling dilutions of piperacillin or tazobactam ranging from 0.5 to 512 pg/ml. The piperacillin-tazobactam combina
tions were used in 8:1 and 4:1 ratios. The medium presently used was a Wilkin-Chalgreen broth. The in itial bacterial density was I06 CFU/ml. Synergy was defined as a 4-fold or greater decrease on the MIC of piperacillin in the presence of tazobactam compared with piperacillin alone. Growth Curves Growth curves were performed with the MS-2 system [10]. This system was originally not designed for anaerobic studies. The technical modification of this system, previously published [12], has been elab orated in the way to obtain an anaerobic environ ment in multichamber cuvettes. The inoculum was prepared in an anaerobic chamber (Forma Scientific Anaerobic System, Model 1024, Marietta, Ohio) us ing Wilkin-Chalgreen broth. A growing culture was adjusted to a 0.5 MacFarland turbidity and diluted 10 times. This culture in growth phase contained ap proximately 107 CFU/ml. Transfer cartridges (Ab bott Laboratories, Diagnostic Division, Dallas, Tex.) were used. Discs impregnated with piperacillin or ta zobactam determining concentrations decreasing from 256 to 0.03 mg/1, alone and in combination (ra tio = 4:1 or 8:1) were introduced into the lower chambers of the cartridges. The bottom of the car tridges were sealed with varnisch. The air-entry ori fice remained open. Cartridges so prepared were stocked in an anaerobic chamber before use. 16 ml of the microbial suspension were introduced into the upper chamber of the cartridge using an anaerobic glove box. The transfer of the culture to the lower chambers was done in the interchange (air-lock) of the Forma anaerobic vessel where it was possible to achieve a vacuum of -20 in. of mercury. When the normal pressure was restored, the liquid was trans ferred from the upper part of the cartridge to the lower chambers. The cartridge was removed from the anaerobic glove box chamber and the air-entry open ing of the transfer cartridge (diameter = 1 mm) im mediately coated with plasticine. The cartridges were placed in the MS-2 analysis module, and recordings of optical densities started at once. At time zero, all the organisms were in the growth phase as assessed by the control curves. Recordings were performed every 5 min for 22 h. Growth Curves Signification The problem encountered with the MS-2 system is the mixing procedure which was not perfect. How-
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Fig. 1. MS-2 growth curve pat terns of B.fragilis submitted to pip eracillin. The antibiotic was intro duced at time zero in a log phase culture. C - Control curve; D = deviation from control curve; ROV = rapid OD variations observed at concentrations near the MIC value and due to clusters of filaments; I = irregularities due to clusters of filaments observed at all piperacil lin concentrations from 2 to 256 mg/ml.
Table 1. Antibacterial effects (pg/ml) of piperacillin and tazobactam alone and in combination against 10 strains of B.fragilis Antibiotic
MIC
MBC
Deviationb
ROVc
Piperacillin Tazobactam Combination 8:1“ Combination 4:1
2(1-4) 8(4-8) 0.5 (0.5-1) 0.5 (0.5-1)
4(2-8) 16(4-8) 1(0.5-2) 1 (0.5-1)
0.5(0.12-1) 0.8(0.12-4) 0.5 0.5
2(1-8) none 1(1-2) 1 (0.5-2)
ever, the reproducibility of the growth curves proved to be excellent. A growth curve, 5-fold performed, showed OD values of 0.29 ±0.012 or 4% and 1.21 ±0.064 or 5%, respectively, after 3 and 6 h of incubation. We considered that the growth curves obtained using the MS-2 system represent a combi nation of bacterial multiplication and other factors responsable for biomass increase (particularly cellwall-deficient bacteria) combined with sedimenta tion. In these experimental conditions some particu larities may be detected in the growth curve which were not seen using conventional growth curves, constructed point by point with a vigorous mixing between each measure. Each particularity that was not observed in the control curve has to be taken into
consideration to evaluate the antibacterial effect of antibiotics. Normal growth curves of B. fragilis pre sent as classic S-shape curves. In the presence of some antibiotics, curves present numerous indenta tions after a few hours. The mean magnitude of these OD variations depends on the time of contact be tween the culture and the antibiotic. These rapid OD variations (ROV) are interesting because they corre spond to clusters of filament formations observed by phase-contrast microscopy. Figure 1 shows the different particularities of the growth curve using the MS-2 system which were taken into consideration to make a comparative eval uation of tazobactam and piperacillin, separately and in combination.
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“ Expressed in piperacillin concentration. b Lowest antibiotic concentration inducing a minimum reduction of 0.1 OD at 3 h. c Lower antibiotic concentration inducing ROV at 6 h.
Results Antimicrobial Susceptibility Testing and Synergy Table 1 presents the MIC and MBC val ues obtained with 10 strains of B.fragilis using the macrodilution method. The mean values and ranges were 2 pg/ml (1-4 pg/ml) for piperacillin and 8 pg/ml (4-8 pg/ml) for tazobactam. Synergy was demonstrated against the 10 strains. A 4-fold decrease and even more in the MIC value was observed with piperacillin in the presence of tazobactam for the ra tios 1:4 or 1:8. Expressed as MIC or MBC values, the differences between 1:4 and 1:8 ratios were not significant. Growth Curves Some characteristics of the growth curves as detailed in figure 1 are presented in table 1. A deviation from the control curve was observed with piperacillin alone for 0.5 pg/ml. We considered these con centrations as MAC values which were found for lA MIC values. For tazobactam these deviations were observed at 0.8 pg/ ml or Vio MIC value. Expressed in pipera cillin concentration, the combination of the two drugs did not reduce the MAC val ue (0.5 pg/ml) of piperacillin alone when the ratios were 8:1 or 4:1. Rapid OD varia tion was detected with piperacillin at con centrations near the MIC value. This phenomenon was not observed with ta zobactam. When the two drugs acted to gether, the ROV was observed at concen trations of piperacillin which produced this phenomenon when used alone. Figure 2 shows phase contrast micros copy of B.fragilis submitted to piperacil lin and tazobactam alone and in combina
Yourassowsky/Van der Linden/Lismont/Crokaert
tion. This photography has been taken after 2, 6 and 24 h of contact between the antibiotics and the microorganisms. Piper acillin induced filament formation at all concentrations used. Tazobactam induced round forms. When the two drugs acted together we observed large bulging fila ments. The overall result showed a pre dominant activity of piperacillin.
Discussion As previously pointed out, tazobactam reduces the MIC value of piperacillin against B.fragilis to 'A MIC. In our study, the mean MIC value for piperacillin was 2 pg/ml. In combination with tazobactam at the ratio 8:1 and 4:1, the MIC value for piperacillin was reduced to 0.5 pg/ml. The in vitro synergistic activity was due to the p-lactamase inhibitory effect of tazobac tam. Tazobactam has nevertheless an in trinsic activity against B.fragilis with a MIC value of 8 pg/ml [1, 3]. The morpho logical responses caused by piperacillin and tazobactam showed at their MIC threshold filament formation for piper acillin and round bodies for tazobactam. These observations suggest two different binding sites on PBPs for piperacillin and tazobactam. The exact PBPs of B.fragilis
Fig. 2. Phase contrast microscopy of B.fragilis submitted to piperacillin and tazobactam alone and in combination. A = Control: B = tazobactam 8 pg/ ml, 2 h; C = piperacillin 2 pg, 6 h; D = piperacillin 2 pg/ml, 24 h; E = piperacillin 2 pg + tazobactam 0.5 pg/ml, 24 h; F = piperacillin 2 pg/ml + tazobac tam 0.12 pg/ml, 24 h.
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Tazobactam against Bacteroidesfragilis 219
which represent the target of P-lactams and related compounds remain controversal. For Piddock and Wise [9], the com pound of PBP1 is involved in cell elonga tion while PBP2 is involved in septum for mation and PBP3 in maintenance of cell shape (i.e. BPB2 in B.fragilis = PBP3 in E. coli, and PBP3 in Bacteroides spp. = PBP2 in E. coli). Growth curves performed with the MS-2 system showed an interest ing phenomenon due to the formation of clusters of filaments in the cuvette. ROV appear at lower piperacillin concentra tions, near the MIC value and was absent from tazobactam-treated cultures. In com bination, the ROV phenomenon persists at a concentration near the MIC value of piperacillin alone. When the tazobactam concentration increased to a more ele vated concentration, the ROV persists. On the other hand a deviation from the con trol curve, which may be considered as the MAC value, was observed for piperacillin at '/4 MIC and for tazobactam at V io MIC. The MAC values of piperacillin remained unchanged with lower concentrations of tazobactam. The overall results of our study showed that growth curves might be used to define the MAC values and an interesting correlation between some particularities (ROV) and morphology of bacteria submitted to the cell wall active substance. ROV phenomenon was only taken into consideration when it was not present in the control curve. Indeed, the morphology of B.fragilis might be greatly influenced by the medium composition [4], In our study, we used Wilkin-Chalgreen broth as recommanded for anaerobes. In conclusion, tazobactam, a (3-lactamase inhibitor, is mainly used in combina
Yourassowsky/Van der Linden/Lismont/Crokaert
tion with a second P-lactam antibiotic pri marily to inhibit the p-lactamase. They may nevertheless exert some intrinsic ac tivity at propriate concentrations, reached in patients or in susceptibility tests. References 1 Appelbaum, P.C.; Jacobs, M.R.; Spangler, S.K.; Yamabe, S.: Comparative activity of the beta-lactamase inhibitors YTR 830, clavulanate and sul bactam combined with beta-lactams against betalactamase-producing anaerobes. 86th Annu. Meet. Am. Soc. Microbiol., Abstract A88, p. 15 (1988). 2 Aronoff, S.C.; Jacobs, M.R.: Johenning, S.; Ya mabe, S.: Comparative activities of the beta-lactamase inhibitors YTR 830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin. Antimicrob. Agents Chemother. 26: 580-582(1984). 3 American Cyanamid Company: Brochure for clinical investigators CL298.741 beta-lactamase inhibitor. (Lederle Laboratories, Pearl River 1987). 4 Eley, A.; Greenwood, D.; O'Grady, F.: Compara tive growth of Bacteroides species in various an aerobic culture media. J. med. Microbiol. 19: 195-201 (1985). 5 Finegold, S.M.; Edelstein, M.C.: Gram-negative, nonsporeforming anaerobe bacilli; in Lennette, Balows, Flausler, Jr., Shadomy, Manual of clini cal microbiology: 4th ed., pp 450-460 (American Society for Microbiology, Washington 1985). 6 Gutman, L.; Kitzis, M.D.: Yamabe, S.; Acar, J.F.: Comparative evaluation of the new beta-lacta mase inhibitor, YTR 830, combined with dif ferent beta-lactam antibiotics against bacteria harboring known beta-lactamases. Antimicrob. Agents Chemother. 29: 955-957 (1986). 7 Jacobs, M.R.: Aronoff, S.C.; Johenning, S.; Ya mabe, S.: Comparative activities of the beta-lactamase inhibitors YTR 830, clavulanate and sul bactam combined with extended-spectrum peni cillins against ticarcillin-resistant Enterobacteriaceae and pseudomonads. J. antimicrob. Chemo ther. 18: 177-18(1984). 8 Moosdeen, F.; Williams, J.D.; Yamabe, S.: Anti
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13 Yourassowsky, E.; Van der Linden, M.P.; Lismont, M.J.; Crokaert, F.; Glupczynski, Y.: Effect on growth curve patterns of brief exposure of bacteria to different concentrations of beta-lactam antibiotics. J. antimicrob. Chemother. 15: suppl. A, pp. 27-36 ( 1985). 14 Yourassowsky, E.; Van der Linden, M.P.; Lismont, M.J.; Crokaert, F.; Glupczynski, Y.: Corre lation between growth curve and killing curve of Escherichia coli after a brief exposure to suprainhibitory concentration of ampicillin and piper acillin. Antimicrob. Agents Chemother. 28:756760(1985).
Prof. E. Yourassowsky Microbiology Brugman University Hospital Place A. Van Gehuchten 4 B -1020 Bruxelles (Belgium)
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bacterial characteristics of YTR 830, a sulfone beta-lactamase inhibitor, compared with those of clavulanic acid and sulbactam. Antimicrob. Agents Chemother. 32: 925-927(1988). Piddock, L.J.V.; Wise, R.: Properties of the peni cillin-binding proteins of four species of the ge nus Bacteroides. Antimicrob. Agents Chemother. 29: 825-832(1986). Spencer, H.J.; Stockert, J.; Welaj, P.; Wilborn, R.; Price, B.: Automated antibiotic susceptibility test ing with the MS-2 system; in Johnston, Newsom, 2nd Int. Symp. Rapid Methods and Automation in Microbiology, pp. 272-275 (Learned Informa tion, Oxford 1976). Sutter, V.L.: Susceptibility testing of anaerobes; in Lennette, Balows, Hausler, Jr., Shadomy, Manual of clinical microbiology; 4th ed., pp. 988-990 (American Society for Microbiology, Washington 1985). Yourassowsky, E.; Van der Linden, M.P.; Lismont, M.J.; Labbe, M.; Crokaert, F.: The effect of beta-lactam antibiotics on the growth curves of Bacteroides fragilis obtained with the MS-2 system. J. antimicrob. Chemother. 8: 219-224 (1981).
