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Methods for Susceptibility Testing in Anaerobes: When and How they Should be Used
Anaerobe (1999) 5, 417±420 Article No. anae.1999.0257
ANTIMICROBIAL SUSCEPTIBILITY TESTING, THERAPY (FACULTY PRESENTATION)
Methods for SusceptibilityTesting in Anaerobes: When and How they Should be Used Hebe M. Bianchini* Department of Microbiology, Centro de EducacioÂn MeÂdica e Investigaciones ClõÂnicas (CEMIC), Buenos Aires, Argentina Key Words: anaerobes, susceptibility testing
Anaerobic bacteria have been established as causing a number of serious human infections, and are becoming increasingly resistant to many traditionally antianaerobic antibiotics currently in use [1,2]. Indeed, over recent years, resistance to different antimicrobial agents has been frequently described world wide and the susceptibility patterns are becoming less predictable [3±5]. Thus, the appearance of new agents with variable activity against anaerobes emphasizes the need to determine in vitro anti-anaerobic activity. Antimicrobial resistance is of major importance in some genera such as Bacteroides, especially among the B. fragilis group, i.e. B. distasonis. B. thetaiotaomicron, B. vulgatus. Furthermore, other species and genera such as Campylobacter gracilis, Fusobacterium spp., Porphyromonas spp. and Prevotella spp. have shown emerging resistance [6±9]. On the other hand, uniform
criteria on the best susceptibility testing method to be performed against anaerobes in clinical laboratories remain under discussion. Since Alexander Fleming described penicillin activity against some bacteria in 1928, many microbiologists have developed different in vitro tests to demonstrate this activity. As antimicrobial susceptibility of anaerobes had remained unchanged for years, empirical therapy of infections involving these organisms, based on in vitro surveillance studies, has long been considered appropriate. However, both emerging and unpredictable resistance among anaerobic bacteria requires that microbiologists make an effort to define a simple and cost-effective routine test for clinical purposes.
When Should Susceptibility Testing be Performed? *Corresponding author. Billinghurst 2447-1425, Buenos Aires, Argentina. Tel.: +54 11 4804 9312; Fax: +54 11 4805 3233. E-mail: [email protected]
1075±9964/99/030417 + 04 $30.00/0
Routine testing of strains is generally not required. Furthermore, determining the susceptibility of every # 1999 Academic Press
418
H.M. Bianchini
strain isolated from a mixed infection is probably not useful in deciding the therapy of an individual case, but becomes essential in detecting any regional and nosocomial resistance [10,11]. On the other hand, in some cases it is necessary to test individual isolates, such as when associated with serious infections such as brain abscess and other central nervous system infections, endocarditis, bacteremia, osteomyelitis joint, bone or prosthetic device infection, and when recovered in pure culture from patients with a poor clinical response to empirical therapy. In addition, susceptibility data for isolates from patients with mixed infections who are being treated medically, rather than surgically, may be useful for the clinician.
Which Organisms Should be Tested? In general, most variation in resistance patterns has been observed among Gram-negative rods, especially within the Bacteroides fragilis group, Fusobacterium, Prevotella, Bilophila wadsworthia and Porphyromonas spp. [8,9]. In Gram-positive anaerobes, certain Clostridium species, such as C. perfringens, C. ramosum, and C. innocuum, have demonstrated resistance to some antimicrobial agents. Finally, Campylobacter gracilis has been recovered from serious
clinical infections and is quite resistant to many antimicrobial agents.
What Drugs Should be Tested? The hospital formulary should serve as a guide in considering the agents for testing. Imipenem, chloramphenicol and metronidazole (for all but nonsporoforming Gram-positive rods) are almost uniformly active against anaerobes in the United States and in Latin-American countries at present, but not in Europe and Japan. Thus, they do not need to be tested in the U.S. and Latin America except under unusual circumstances, although antibiotic activity should be monitored periodically at reference centers to detect any emerging resistance, as has recently been found in beta-lactam/beta-lactamase inhibitor combinations. Indeed, in a preliminary study performed by our Anaerobic Bacteria Study Group of the AsociacioÂn Argentina de MicrobiologõÂa, there has been a worrying increase in the number of strains displaying decreased susceptibility to ampicillin/sulbactan. These strains represented 3% of all the Bacteroides fragilis group in 1995 and 9% in 1997 (Figure 1). Antimicrobial agents such as penicillin, clindamycin, macrolides and new quinolones are variably active against anaerobes, and to predict the susceptibility of
Figure 1. Changing ampicillin/sulbactam MIC distribution against the Bacteroides fragilis group from 1986 to 1997.
Methods for SusceptibilityTesting a particular strain on the basis of published patterns is not recommended.
Choosing the Best Susceptibility Method It is necessary to take into account the accuracy of the method and its correlation with the clinical outcome. Testing procedures include agar and broth methods: The agar dilution method is the reference procedure as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [12]. However, it is not recommended for small laboratories. This method is appropriate for laboratories doing large-scale testing of organisms for surveillance studies, for assessment of new drugs and for confirming results obtained by the screening methods. Emphasis on the proper use of this method deserves attention. For instance, at present, with the use of new macrolides for treatment of respiratory and oral infections [13], it is interesting to evaluate the effect of incubation in a CO2-containing atmosphere [8] present in the anaerobic chambers or jars, on the pH of the culture medium. We have observed in a preliminary study a significant difference in the activity of macrolides MICs performed at pH 7 or at pH 8. As shown in Table 1 (data from a unpublished preliminary study), there is at least three-fold dilution MICs difference with the pH variations. Consequently, it is of interest to standardize this method to test the activity of macrolides against anaerobic bacteria that require CO2 for growth in order to define accurately a correct interpretation of their activity. Falagas et al. [14] found that pH variation produces modifications in the activity of other antibiotics such imipenem, piperacillin±tazobactam and ciprofloxacin against anaerobic bacteria. Broth macro- and microdilution methods have similar features to the agar dilution method [15]. In addition, the MBC can also be determined and the modification of the pH within the medium by the CO2 is less important than it is in the agar medium because the contact surface is smaller in the tube. However, commercial microbroth dilution kits are not always available in many clinical laboratories and the Table 1. Increase of macrolide resistance by 58 Bacteroides fragilis group strains when the agar pH is decreased from 8 to 7 in a CO2containing atmosphere Increase in MIC (no. of strains) in terms of dilutions Macrolides Erythromycin Roxithromycin Azithromycin Clarithromycin
01 dilution
2 dilution
3 dilution
36 44 35 46
9 7 9 3
13 7 14 9
419
selection of antibiotics depends on available trays from the manufacturer, hindering antibiotic selection. Furthermore, they do not always reflect an appropriate range of drugs prescribed for anaerobic infections. The two-concentrations or break-point method is now available as commercial panels, but is only applicable to anaerobes not requiring additional nutrients. Although the correlation between the microdilution or the two-concentration panels with the agar dilution method has not been clearly defined, the method has shown good correlation in our experience [16]. The broth elution disk method is a simple, cheap and easy-to-perform test for routine susceptibility testing of anaerobic bacteria for clinical purposes. Although this method is currently questioned [17,18] and is not recommended by the NCCLS, we assessed its reliability for clinical purposes in a collaborative study [16] and found acceptable agreement between this method and the agar dilution test (98.5%). We concluded that such agreement was due to the rigorous standardization of both test conditions, including culture media, inoculum size, atmosphere and incubation temperature. On the other hand, when intra- and interlaboratory data were analysed the results were quite equivocal and were directly related with poor disk quality. Thus, as in other methods, quality control of the disks by using reference ATCC strains is essential. Taking into account that this method is available to most clinical laboratories, I believe it provides a suitable alternative that can be performed as a resistance screening test to guide therapy, although any inconsistent result must be confirmed by the reference methods. The epsilometer method (E test) is a commercial test based on the principle of a diffusion antimicrobial gradient in agar medium as a mean of determining the MIC. It is a plastic strip coated with an antibiotic gradient on one side and an interpretative scale on the other. Although acceptable correlation with the standard techniques has been reported [19,20], some discrepancies have also been described [21]. This test differs from other commercial methods in the flexibility of drug selection. However, the expense (about $3 per strip) is one of the main limitations of this test. Although we have not formally evaluated this method for anaerobic bacteria in Argentina, I think that assessment of certain drugs, such as betalactams and imidazoles should continue before this test is routinely used. With the Spiral gradient method a spiral streaker deposits an antimicrobial concentration gradient pattern on an agar plate that decreases radially from the center of the plate. This method has never been used in Argentina, even with aerobic bacteria.
420
H.M. Bianchini
Direct beta-lactamase detection is a simple and rapid test to be used as a complement to conventional susceptibility tests for beta-lactam agents and may provide the microbiologist some useful information. Beta-lactamase production in Gram-negative pigmented rods is of special importance, especially those recovered from oral or odontogenic infections. However beta-lactamase production is not the only mechanism conferring resistance to beta-lactamics [22]. The most reliable substrate for detecting betalactamase in anaerobics in nitrocefin, because acidometric tests do not detect cephaloporinases present in many anaerobic Gram-negative rods. Unfortunately, there is no general agreement about the simplest, quickest most inexpensive reliable method to be used for clinical purposes. There is agreement that physicians need to rely on data generated in their own hospital to predict whether a given antibiotic will be effective against a particular infection, especially with the plethora of new antibiotics available with variable activity against anaerobes. The susceptibility of one strain isolated from a severe infection should not be predicted on the basis of general epidemiological information collected elsewhere. Furthermore, the method should be selected carefully by a microbiologist in accordance with every clinical situation in order to give rapid and useful information to guide therapy. We should not forget that organisms harbour resistance mechanisms against antibiotics even prior to use of the drug.
References 1. Appleman M.D., Heseltine P.N.R. and Cherubin C.E. (1991). Epidemiology, antimicrobial susceptibility, pathogenicity, and significance of Bacteroides; 13: B. fragilis group organisms isolated at Los Angeles County-University of Southern California Medical Center. Rev Infect Dis 13: 12±18 2. Tamaka-Bandoh K., Kato N., Watanabe K. and Ueno K. (1995) Antibiotic-susceptibility profiles of Bacteroides fragilis and Bacteroides thetaiotaomicron in Japan from 1990 to 1992. Clin Infect Dis 20 (Suppl. 2): 352±355 3. Citron D.M., Goldstein E.J.C., Kenner M.A., Burnham L.B. and Inderleied C.B. (1995) Activity of ampicilin/sulbactam, ticarcillin/clavulanate, clarithromycin, and eleven other antimicrobial agents against anaerobic bacteria isolated from infections in children. Clin Infect Dis 20 (Suppl. a): S 356±360 4. Bianchini H., Fernandez Canjila L., Bantar C. and Smayevsky J. (1997) Trends in antimicrobial resistance of the Bacteroides fragilis group: a 20-year study at a medical center in Buenos Aires. Clin Infect Dis 25 (Suppl. 2): S268±269
5. Pestana A.C.N.R., Ribeiro R.N., Diniz C.G., Farias L.M. and Carvalho M.A.R. (1997) Resistance to metronidazole among Bacteroides fragilis group strains from human and marmosetes: comparative study. Clin Infect Dis 25 (Suppl. 2): S 270±280 6. Molitoris E., Wexler H.M. and Finegold S.M. (1997) Sources of antimicrobial susceptibilities of Campylobacter gracilis and Sutterella wadsworthensis. Clin Infect Dis 25 (Suppl. 2): 264±265 7. Arthur M., Brisson-Noel A. and Courvalin P. (1990) Origin and evaluation of specifying resistance to macrolides, lincosamine and streptogramin: data and hypothesis. J Antimicrob Chemother 20: 783±802 8. Spangler S.K., Jacobs M.R. and Applebaum P.C. (1994) Effect of CO2 on susceptibility of anaerobes to erythromycin, azithromycin, clarithromycin, and roxithromycin. Antimicrob Agents Chemother 38: 211±216 9. Jacobs M.R., Spangler S.K. and Applebaum P.C. (1992). Betalactamase production and susceptibility of U.S. and European anaerobic gram-negative bacili to beta-lactam and other agents. Eur J Clin Microbiol Infect Dis 11: 1082±1093 10. Finegold S.M. (1997) Perspective on susceptibility testing of anaerobic bacteria. Clin Infect Dis 25 (Suppl. 2): 251±253 11. Wilson S.E. and Huth J. (1997) In defence of routine antimicrobial susceptibility testing on operative site flora in patients with peritonitis. Clin Infect Dis 25(Suppl. 2): 254±257 12. National Committee for Clinical Laboratory Standards (1993) Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 3rd Edn. Approved standard M11-A3. National Committee of Clinical Standards, Villanova, PA 13. Hollander J.G., Knudsen J.D., Mouton J.W., Fuursted K., FrimotMoller N., Verbrugh H.A. and Espersen F. (1998) Comparison of pharmacodynamics of azithromycin and erythromycin in vitro and in vivo. Antimicrob Agents Chemother 42: 377±382 14. Falagas M.E., McDermott L. and Snydman D.R. (1997) Effect of pH on in vitro antimicrobial susceptibility of the Bacteroides fragilis group. Antimicrob Agents Chemother 41: 2047±2049 15. Wexler H.M. (1991) Susceptibility testing of anaerobic bacteria: myth, magic, or method? Clin Microbiol Rev 4: 470±484 16. Bianchini H., Fernandezez Canigia L., Predari S., Rollet R., Litterio M., Berestein P., Castello L., Di Martino A., Greco G. and Hardie N. (1997) Broth disk method for anaerobic bacteria: a collaborative study to assess its reliability for clinical purposes. Anaerobe 3: 225±231 17. Aldrige K.E., Henderberg A., Schiro D.D. and Sanders C.V. (1990). Discordant results between the broth disk elution and broth macrodilution susceptibility tests with Bacteroides fragilis group isolates. J Clin Microbiol 28: 375±378 18. Citron D.M. (1991) Susceptibility testing of anaerobic bacteria: a review of current methods and future prospects. The Antimicrobic Newsletter 8: 53±85 19. Citron D.M., Ostovari M.I., Katlsson A. and Goldstein E.J.C. (1991) Evaluation of the E-test for susceptibility testing of anaerobic bacteria. J Clin Microbiol 29: 2197±2203 20. Croco J.I., Erwin M.E., Jenninos J.M., Putnam L.R. and Jones R.N. (1995) Evaluation of the E-test to determinations of antimicrobial spectrum and potency against anaerobes associated with bacterial vaginosis and peritonitis. Clin Infect Dis (Suppl. 2): S339±341 21. Jorgensen J.H. and Ferraro M.J. (1998) Antimicrobial susceptibility testing: general principles and contemporary practices. Clin Infect Dis 26: 973±980 22. Hedberg M., Nagy E. and Nord C.E. (1997) Role of pencillinbinding proteins in resistance of Bacteroides fragilis group species to B-lactam drugs. Clin Infect Dis 25 (Suppl. 2): S270±271
ANTIMICROBIAL SUSCEPTIBILITY TESTING, THERAPY (FACULTY PRESENTATION)
Methods for SusceptibilityTesting in Anaerobes: When and How they Should be Used Hebe M. Bianchini* Department of Microbiology, Centro de EducacioÂn MeÂdica e Investigaciones ClõÂnicas (CEMIC), Buenos Aires, Argentina Key Words: anaerobes, susceptibility testing
Anaerobic bacteria have been established as causing a number of serious human infections, and are becoming increasingly resistant to many traditionally antianaerobic antibiotics currently in use [1,2]. Indeed, over recent years, resistance to different antimicrobial agents has been frequently described world wide and the susceptibility patterns are becoming less predictable [3±5]. Thus, the appearance of new agents with variable activity against anaerobes emphasizes the need to determine in vitro anti-anaerobic activity. Antimicrobial resistance is of major importance in some genera such as Bacteroides, especially among the B. fragilis group, i.e. B. distasonis. B. thetaiotaomicron, B. vulgatus. Furthermore, other species and genera such as Campylobacter gracilis, Fusobacterium spp., Porphyromonas spp. and Prevotella spp. have shown emerging resistance [6±9]. On the other hand, uniform
criteria on the best susceptibility testing method to be performed against anaerobes in clinical laboratories remain under discussion. Since Alexander Fleming described penicillin activity against some bacteria in 1928, many microbiologists have developed different in vitro tests to demonstrate this activity. As antimicrobial susceptibility of anaerobes had remained unchanged for years, empirical therapy of infections involving these organisms, based on in vitro surveillance studies, has long been considered appropriate. However, both emerging and unpredictable resistance among anaerobic bacteria requires that microbiologists make an effort to define a simple and cost-effective routine test for clinical purposes.
When Should Susceptibility Testing be Performed? *Corresponding author. Billinghurst 2447-1425, Buenos Aires, Argentina. Tel.: +54 11 4804 9312; Fax: +54 11 4805 3233. E-mail: [email protected]
1075±9964/99/030417 + 04 $30.00/0
Routine testing of strains is generally not required. Furthermore, determining the susceptibility of every # 1999 Academic Press
418
H.M. Bianchini
strain isolated from a mixed infection is probably not useful in deciding the therapy of an individual case, but becomes essential in detecting any regional and nosocomial resistance [10,11]. On the other hand, in some cases it is necessary to test individual isolates, such as when associated with serious infections such as brain abscess and other central nervous system infections, endocarditis, bacteremia, osteomyelitis joint, bone or prosthetic device infection, and when recovered in pure culture from patients with a poor clinical response to empirical therapy. In addition, susceptibility data for isolates from patients with mixed infections who are being treated medically, rather than surgically, may be useful for the clinician.
Which Organisms Should be Tested? In general, most variation in resistance patterns has been observed among Gram-negative rods, especially within the Bacteroides fragilis group, Fusobacterium, Prevotella, Bilophila wadsworthia and Porphyromonas spp. [8,9]. In Gram-positive anaerobes, certain Clostridium species, such as C. perfringens, C. ramosum, and C. innocuum, have demonstrated resistance to some antimicrobial agents. Finally, Campylobacter gracilis has been recovered from serious
clinical infections and is quite resistant to many antimicrobial agents.
What Drugs Should be Tested? The hospital formulary should serve as a guide in considering the agents for testing. Imipenem, chloramphenicol and metronidazole (for all but nonsporoforming Gram-positive rods) are almost uniformly active against anaerobes in the United States and in Latin-American countries at present, but not in Europe and Japan. Thus, they do not need to be tested in the U.S. and Latin America except under unusual circumstances, although antibiotic activity should be monitored periodically at reference centers to detect any emerging resistance, as has recently been found in beta-lactam/beta-lactamase inhibitor combinations. Indeed, in a preliminary study performed by our Anaerobic Bacteria Study Group of the AsociacioÂn Argentina de MicrobiologõÂa, there has been a worrying increase in the number of strains displaying decreased susceptibility to ampicillin/sulbactan. These strains represented 3% of all the Bacteroides fragilis group in 1995 and 9% in 1997 (Figure 1). Antimicrobial agents such as penicillin, clindamycin, macrolides and new quinolones are variably active against anaerobes, and to predict the susceptibility of
Figure 1. Changing ampicillin/sulbactam MIC distribution against the Bacteroides fragilis group from 1986 to 1997.
Methods for SusceptibilityTesting a particular strain on the basis of published patterns is not recommended.
Choosing the Best Susceptibility Method It is necessary to take into account the accuracy of the method and its correlation with the clinical outcome. Testing procedures include agar and broth methods: The agar dilution method is the reference procedure as recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [12]. However, it is not recommended for small laboratories. This method is appropriate for laboratories doing large-scale testing of organisms for surveillance studies, for assessment of new drugs and for confirming results obtained by the screening methods. Emphasis on the proper use of this method deserves attention. For instance, at present, with the use of new macrolides for treatment of respiratory and oral infections [13], it is interesting to evaluate the effect of incubation in a CO2-containing atmosphere [8] present in the anaerobic chambers or jars, on the pH of the culture medium. We have observed in a preliminary study a significant difference in the activity of macrolides MICs performed at pH 7 or at pH 8. As shown in Table 1 (data from a unpublished preliminary study), there is at least three-fold dilution MICs difference with the pH variations. Consequently, it is of interest to standardize this method to test the activity of macrolides against anaerobic bacteria that require CO2 for growth in order to define accurately a correct interpretation of their activity. Falagas et al. [14] found that pH variation produces modifications in the activity of other antibiotics such imipenem, piperacillin±tazobactam and ciprofloxacin against anaerobic bacteria. Broth macro- and microdilution methods have similar features to the agar dilution method [15]. In addition, the MBC can also be determined and the modification of the pH within the medium by the CO2 is less important than it is in the agar medium because the contact surface is smaller in the tube. However, commercial microbroth dilution kits are not always available in many clinical laboratories and the Table 1. Increase of macrolide resistance by 58 Bacteroides fragilis group strains when the agar pH is decreased from 8 to 7 in a CO2containing atmosphere Increase in MIC (no. of strains) in terms of dilutions Macrolides Erythromycin Roxithromycin Azithromycin Clarithromycin
01 dilution
2 dilution
3 dilution
36 44 35 46
9 7 9 3
13 7 14 9
419
selection of antibiotics depends on available trays from the manufacturer, hindering antibiotic selection. Furthermore, they do not always reflect an appropriate range of drugs prescribed for anaerobic infections. The two-concentrations or break-point method is now available as commercial panels, but is only applicable to anaerobes not requiring additional nutrients. Although the correlation between the microdilution or the two-concentration panels with the agar dilution method has not been clearly defined, the method has shown good correlation in our experience [16]. The broth elution disk method is a simple, cheap and easy-to-perform test for routine susceptibility testing of anaerobic bacteria for clinical purposes. Although this method is currently questioned [17,18] and is not recommended by the NCCLS, we assessed its reliability for clinical purposes in a collaborative study [16] and found acceptable agreement between this method and the agar dilution test (98.5%). We concluded that such agreement was due to the rigorous standardization of both test conditions, including culture media, inoculum size, atmosphere and incubation temperature. On the other hand, when intra- and interlaboratory data were analysed the results were quite equivocal and were directly related with poor disk quality. Thus, as in other methods, quality control of the disks by using reference ATCC strains is essential. Taking into account that this method is available to most clinical laboratories, I believe it provides a suitable alternative that can be performed as a resistance screening test to guide therapy, although any inconsistent result must be confirmed by the reference methods. The epsilometer method (E test) is a commercial test based on the principle of a diffusion antimicrobial gradient in agar medium as a mean of determining the MIC. It is a plastic strip coated with an antibiotic gradient on one side and an interpretative scale on the other. Although acceptable correlation with the standard techniques has been reported [19,20], some discrepancies have also been described [21]. This test differs from other commercial methods in the flexibility of drug selection. However, the expense (about $3 per strip) is one of the main limitations of this test. Although we have not formally evaluated this method for anaerobic bacteria in Argentina, I think that assessment of certain drugs, such as betalactams and imidazoles should continue before this test is routinely used. With the Spiral gradient method a spiral streaker deposits an antimicrobial concentration gradient pattern on an agar plate that decreases radially from the center of the plate. This method has never been used in Argentina, even with aerobic bacteria.
420
H.M. Bianchini
Direct beta-lactamase detection is a simple and rapid test to be used as a complement to conventional susceptibility tests for beta-lactam agents and may provide the microbiologist some useful information. Beta-lactamase production in Gram-negative pigmented rods is of special importance, especially those recovered from oral or odontogenic infections. However beta-lactamase production is not the only mechanism conferring resistance to beta-lactamics [22]. The most reliable substrate for detecting betalactamase in anaerobics in nitrocefin, because acidometric tests do not detect cephaloporinases present in many anaerobic Gram-negative rods. Unfortunately, there is no general agreement about the simplest, quickest most inexpensive reliable method to be used for clinical purposes. There is agreement that physicians need to rely on data generated in their own hospital to predict whether a given antibiotic will be effective against a particular infection, especially with the plethora of new antibiotics available with variable activity against anaerobes. The susceptibility of one strain isolated from a severe infection should not be predicted on the basis of general epidemiological information collected elsewhere. Furthermore, the method should be selected carefully by a microbiologist in accordance with every clinical situation in order to give rapid and useful information to guide therapy. We should not forget that organisms harbour resistance mechanisms against antibiotics even prior to use of the drug.
References 1. Appleman M.D., Heseltine P.N.R. and Cherubin C.E. (1991). Epidemiology, antimicrobial susceptibility, pathogenicity, and significance of Bacteroides; 13: B. fragilis group organisms isolated at Los Angeles County-University of Southern California Medical Center. Rev Infect Dis 13: 12±18 2. Tamaka-Bandoh K., Kato N., Watanabe K. and Ueno K. (1995) Antibiotic-susceptibility profiles of Bacteroides fragilis and Bacteroides thetaiotaomicron in Japan from 1990 to 1992. Clin Infect Dis 20 (Suppl. 2): 352±355 3. Citron D.M., Goldstein E.J.C., Kenner M.A., Burnham L.B. and Inderleied C.B. (1995) Activity of ampicilin/sulbactam, ticarcillin/clavulanate, clarithromycin, and eleven other antimicrobial agents against anaerobic bacteria isolated from infections in children. Clin Infect Dis 20 (Suppl. a): S 356±360 4. Bianchini H., Fernandez Canjila L., Bantar C. and Smayevsky J. (1997) Trends in antimicrobial resistance of the Bacteroides fragilis group: a 20-year study at a medical center in Buenos Aires. Clin Infect Dis 25 (Suppl. 2): S268±269
5. Pestana A.C.N.R., Ribeiro R.N., Diniz C.G., Farias L.M. and Carvalho M.A.R. (1997) Resistance to metronidazole among Bacteroides fragilis group strains from human and marmosetes: comparative study. Clin Infect Dis 25 (Suppl. 2): S 270±280 6. Molitoris E., Wexler H.M. and Finegold S.M. (1997) Sources of antimicrobial susceptibilities of Campylobacter gracilis and Sutterella wadsworthensis. Clin Infect Dis 25 (Suppl. 2): 264±265 7. Arthur M., Brisson-Noel A. and Courvalin P. (1990) Origin and evaluation of specifying resistance to macrolides, lincosamine and streptogramin: data and hypothesis. J Antimicrob Chemother 20: 783±802 8. Spangler S.K., Jacobs M.R. and Applebaum P.C. (1994) Effect of CO2 on susceptibility of anaerobes to erythromycin, azithromycin, clarithromycin, and roxithromycin. Antimicrob Agents Chemother 38: 211±216 9. Jacobs M.R., Spangler S.K. and Applebaum P.C. (1992). Betalactamase production and susceptibility of U.S. and European anaerobic gram-negative bacili to beta-lactam and other agents. Eur J Clin Microbiol Infect Dis 11: 1082±1093 10. Finegold S.M. (1997) Perspective on susceptibility testing of anaerobic bacteria. Clin Infect Dis 25 (Suppl. 2): 251±253 11. Wilson S.E. and Huth J. (1997) In defence of routine antimicrobial susceptibility testing on operative site flora in patients with peritonitis. Clin Infect Dis 25(Suppl. 2): 254±257 12. National Committee for Clinical Laboratory Standards (1993) Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria, 3rd Edn. Approved standard M11-A3. National Committee of Clinical Standards, Villanova, PA 13. Hollander J.G., Knudsen J.D., Mouton J.W., Fuursted K., FrimotMoller N., Verbrugh H.A. and Espersen F. (1998) Comparison of pharmacodynamics of azithromycin and erythromycin in vitro and in vivo. Antimicrob Agents Chemother 42: 377±382 14. Falagas M.E., McDermott L. and Snydman D.R. (1997) Effect of pH on in vitro antimicrobial susceptibility of the Bacteroides fragilis group. Antimicrob Agents Chemother 41: 2047±2049 15. Wexler H.M. (1991) Susceptibility testing of anaerobic bacteria: myth, magic, or method? Clin Microbiol Rev 4: 470±484 16. Bianchini H., Fernandezez Canigia L., Predari S., Rollet R., Litterio M., Berestein P., Castello L., Di Martino A., Greco G. and Hardie N. (1997) Broth disk method for anaerobic bacteria: a collaborative study to assess its reliability for clinical purposes. Anaerobe 3: 225±231 17. Aldrige K.E., Henderberg A., Schiro D.D. and Sanders C.V. (1990). Discordant results between the broth disk elution and broth macrodilution susceptibility tests with Bacteroides fragilis group isolates. J Clin Microbiol 28: 375±378 18. Citron D.M. (1991) Susceptibility testing of anaerobic bacteria: a review of current methods and future prospects. The Antimicrobic Newsletter 8: 53±85 19. Citron D.M., Ostovari M.I., Katlsson A. and Goldstein E.J.C. (1991) Evaluation of the E-test for susceptibility testing of anaerobic bacteria. J Clin Microbiol 29: 2197±2203 20. Croco J.I., Erwin M.E., Jenninos J.M., Putnam L.R. and Jones R.N. (1995) Evaluation of the E-test to determinations of antimicrobial spectrum and potency against anaerobes associated with bacterial vaginosis and peritonitis. Clin Infect Dis (Suppl. 2): S339±341 21. Jorgensen J.H. and Ferraro M.J. (1998) Antimicrobial susceptibility testing: general principles and contemporary practices. Clin Infect Dis 26: 973±980 22. Hedberg M., Nagy E. and Nord C.E. (1997) Role of pencillinbinding proteins in resistance of Bacteroides fragilis group species to B-lactam drugs. Clin Infect Dis 25 (Suppl. 2): S270±271