356
E u r . J. Ciin. M i c r o b i a l . Infect. Dis.
References 1. Breuil J, Burnet C, Patey O, Dublanchet A: Survey of Bacteroides fragilis susceptibility patterns in France. Journal of Antirnicrobial Chemotherapy 1989, 24: 6975. 2. Cuchural G J, Tally FP, Jacobus NV, Aldridge K,
Cieary T, Finegold SM, Hill G, lannini P, O'Keefe JP, Pierson C, Crook D, Russo T, Hecht D: Susceptibility of the Bacteroides fragilis group in the United States: analysis by site of isolation. Antimicrobial Agents and Chemotherapy 1988, 32: 717-722. 3. Eley A, Greenwood D: Characterization of [~-lactamases in clinical isolates of Bacteroides. Journal of Antimicrobial Chemotherapy 1986, 18: 325-333.
4. Lamothe F, Fijalkowski C, Maiovin F, Bourgault AF, Delorme L: Bacteroides fragilis resistant to both metronidazole and imipenem. Journal of Antimicrobial Chemotherapy 1986, 18: 642. 5. Tally FP, Jacobus NV: Susceptibility of anaerobic bacteria to imipenem. Journal of Antimicrobial Chemotherapy 1983, 12, Supplement D: 47-51.
6. Duhreuil L, Derriennic M, Sedallian A, Romond C, Courtieu AL: Evolution in antibiotic susceptibility of Bacteroides fragilis group strains in France based on periodic surveys. Infection 1989, 17: 197-200. 7. Fox AR, Phillips I: The antibiotic sensitivity of the Bacteroides fragilis group in the United Kingdom. Journal of Antimicrobial Chemotherapy 1987, 20: 477488.
group. Journal of Antimicrobial Chemotherapy 1984, 14: 595--603. 16. Sorlann F, Ponte C, Wilhelmi h Increasing incidence of clinical isolates of Bacteroides fragilis resistant to clindamycin. Journal of Antimicrobial Chemotherapy 1984, 13: 395. 17. Betriu C, Campos E, Cabronero G, Rodriguez-Avial C, Picazo JJ: Susceptibilities of species of the Bacteroidesfragilis group to 10 antimicrobial agents. Antimicrobial Agents and Chemotherapy 1990, 34: 671673. 18. Scher KS: Emergence of antibiotic resistant strains of Bacteroides fragilis. Surgery, Gynaecology and Obstetrics 1988, 167: 175-179. 19. Borobio MV, Pascual A, Perea E J: Decreasing resistance of Bacteroides fragilis group strains to metronidazole in Spain between 1977 and 1982. European Journal of Clinical Microbiology and Infectious Diseases 1984, 3: 135-155. 20. Brogan O, Garnett PA, Brown R: Bacteroides fragilis resistant to metronidazole, clindamycin and cefo×itin. Journal of Antimicrobial Chemotherapy 1989, 23: 660662. 21. Eme MA, Acar .IF, Goldslein FW: Bacteroides fragilis resistant to metronid~ole. Journal of Antimicrobial Chemotherapy 1983, 12: 523-534. 22. Ingham HA, Eaton S, Venables CW, Adams PC: Bacteroides fragilis resistant to metronidazole after longterm therapy. Lancet 1978, i: 214.
8. National Committee for Clinical Laboratory Standards: Reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard: M l l - A . NCCLS, ViIlanova, PA, 1985.
9. National Committee for Clinical Laboratory Standards: Methods for antimicrobial susceptibility testing 10.
11.
12.
13.
14.
15.
of anaerobic bacteria-second edition. Approved standard: Mll-A2. NCCLS, Villanova, PA, 1990. Bourgault AM, Lamothe F: In vitro activity of amoxicillin and ticarcillin in combination with clavulanic acid compared with that of new lS-lactam agents against species of the Bacteroidesfragilis group. Journal of Antimicrobial Chemotherapy 1986, 17: 593603. Cuchural G J, Tally FP, Jacobus NV, Cleary T, Finegold SM, Hill G, Iannini P, O'Keefe JP, Pierson C: Comparative activities of newer 15-1attain agents against members of the Bacteroides fragilis group. Antimicrobial Agents and Chemotherapy 1990, 34: 479480. Garcla-Rodriguez JE, Garcla-S~inchez JE: Evolution of antimicrobial susceptibility in isolates of the Bacteroides fragilis group in Spain. Reviews of Infectious Diseases 1990, 12, Supplement 2: 142-151. Musiai CE, Rosenblatt JE: Antimicrobial susceptibilities of anaerobic bacteria isolated at the Mayo Clinic during 1982 through 1987: comparison with results from 1977 through 1981. Mayo Clinic Proceedings 1989, 64: 392-399. Lifiares J, P6rez JL, Martin A: Susceptibility studies in the Bacteroides fragilis group resistant to clindamycin. Journal of Antimicrobial Chemotherapy 1988, 12: 293. Reig M, Campello MG, Baqnero F: Epidemiology of clindamycin resistance in the Bacteroides fragilis
New High-Content Disks for Determination of High-Level Aminoglycoside Resistance in Clinical Isolates of Enterococcus
faecalis R . L e c l e r c q 1 . , R . B i s m u t h 2, J. D u v a l 1
Disks impregnated with 500 and 1000 lag of streptomycin, 1000 lag of kanamycin and 250 and 500 ~g of gentamicin were used for detection of high-level resistance to aminoglycosides in 120 clinical isolates of Enterococcus faecalis. Fiftyseven strains were highly resistant to streptomycin, 80 to kanamycin and 41 to gentamicin. Using disks containing 500 lag of streptomycin, 1000 ~g of kanamycin and 500 lag of gentamicin 1Service de Baet6riologie-Virologie-Hygi~ne, H6pital Henri Mondor, Universit6 Paris XII, 51 Avenue Mar6chal de Lattre de Tassigny, 94010 Cr6teil, France. 2Service de Bact6riologie-Virologie, Centre Hospitalier Universitaire Piti6-Salp6tri~re, 75651 Paris Cedex 13, France.
Vo1.11,1992
strains resistant to high levels of these drugs (97.9 %, 100 % and 100 %, respectively) were accurately detected. Better discrimination between high-level and low-level resistance was achieved with a 500 lag streptomycin or gentamicin disk. Zone-size breakpoints are proposed for detection of high-level resistance by disk diffusion.
Most enterococci are not killed by clinically achievable concentrations of cell-wall active drugs administered alone. Bactericidal chemotherapy is recommended in cases of severe infections due to these organisms, bactericidal activity usually being obtained by combination of cellwall active agents with aminoglycosides (1). However, the bactericidal synergy displayed by combinations is absent in the presence of high-level resistance to aminoglycosides (2). High-level resistance to streptomycin, kanamycin and gentamicin characterises the three major phenotypes of these organisms and can be routinely detected by several methods including agar dilution (3, 4), broth dilution (4-6) and agar disk diffusion (6-8). Disk diffusion is a convenient and reliable method which is easy to perform in most laboratories. The purpose of this study was to evaluate disks containing various concentrations of streptomycin, kanamycin and gentamicin for detection of enterococci showing high-level resistance to these drugs.
Materials and Methods. One hundred and twenty non-replicate strains of Enterococcus faecalis were obtained in 1988 from clinical specimens at the Henri Mondor Hospital in Cr6teil, France. Enterococci were identified by colony morphology, Gram staining and biochemical characteristics (9). The MICs of streptomycin, kanamycin and gentamicin were determined by the agar dilution technique in Mueller-Hinton agar (Diagnostics Pasteur, France) with an inoculum of 104 organisms per spot. Incubation was at 37 °C for 18 hours. Streptomycin was provided by Pfizer, France; kanamycin by Bristol Laboratories, UK and gentamicin by Schering, USA. Agar disk diffusion was performed in parallel to MIC determinations by flooding an inoculum of 5 x 106-107 cfu/ml onto Mueller-Hinton agar. Disks containing high concentrations of kanamycin (1000 ~g), gentamicin (250 and 500 lag) and of streptomycin (500 and 1000 ~g) were furnished by Diagnostics Pasteur. The perimeters of the aminoglycoside inhibition zones were clear-cut and the zone diameters could be easily measured.
357
The relationship between inhibition zone diameters and MICs was analysed with a computer programme written by Dr Le Van Thoi. For every aminoglycoside low and high MIC breakpoints were selected. Strains with aminoglycoside MICs equal to or above 2048 lag/ml were considered to have high-level resistance to this aminoglycoside. Strains with MICs of gentamicin, kanamycin and streptomycin equal to or below 64 pg/ ml, 256 lag/ml and 128 laglml, respectively, were considered to have low-level resistance to these aminoglycosides. Strains with MICs between the two breakpoints were classified as indeterminate. The zone-size breakpoints giving a minimum of very major errors (false-susceptible) and major errors (false-resistant) relative to MICs were selected (10). A minor error comprised an indeterminate result by one of the techniques only.
Results and Discussion, The results obtained for streptomycin are presented in Figure 1. With the 500 lag disk a zone-size breakpoint of 12 mm allowed discrimination of 47 of the 48 strains highly resistant to streptomycin, a single strain being categorized as indeterminate. Fifty-eight strains were resistant to low levels of streptomycin (MICs < 128 ~tg/ml) and exhibited zone diameters greater than 14 mm. The MICs of streptomycin for the 14 remaining strains were between 256 and 1024 lag/ml and corresponded to diameters ranging from 6 to 19 mm. Ten percent of minor errors and no major error were observed. A similar rate of errors was found when the 1000 lag streptomycin disk was used (Figure 1B). However, the 500 lag streptomycin disk was more convenient for detection of highly resistant enterococci since small zone diameters, nearly always _.
E u r . J. Ciin. M i c r o b i a l . Infect. Dis.
References 1. Breuil J, Burnet C, Patey O, Dublanchet A: Survey of Bacteroides fragilis susceptibility patterns in France. Journal of Antirnicrobial Chemotherapy 1989, 24: 6975. 2. Cuchural G J, Tally FP, Jacobus NV, Aldridge K,
Cieary T, Finegold SM, Hill G, lannini P, O'Keefe JP, Pierson C, Crook D, Russo T, Hecht D: Susceptibility of the Bacteroides fragilis group in the United States: analysis by site of isolation. Antimicrobial Agents and Chemotherapy 1988, 32: 717-722. 3. Eley A, Greenwood D: Characterization of [~-lactamases in clinical isolates of Bacteroides. Journal of Antimicrobial Chemotherapy 1986, 18: 325-333.
4. Lamothe F, Fijalkowski C, Maiovin F, Bourgault AF, Delorme L: Bacteroides fragilis resistant to both metronidazole and imipenem. Journal of Antimicrobial Chemotherapy 1986, 18: 642. 5. Tally FP, Jacobus NV: Susceptibility of anaerobic bacteria to imipenem. Journal of Antimicrobial Chemotherapy 1983, 12, Supplement D: 47-51.
6. Duhreuil L, Derriennic M, Sedallian A, Romond C, Courtieu AL: Evolution in antibiotic susceptibility of Bacteroides fragilis group strains in France based on periodic surveys. Infection 1989, 17: 197-200. 7. Fox AR, Phillips I: The antibiotic sensitivity of the Bacteroides fragilis group in the United Kingdom. Journal of Antimicrobial Chemotherapy 1987, 20: 477488.
group. Journal of Antimicrobial Chemotherapy 1984, 14: 595--603. 16. Sorlann F, Ponte C, Wilhelmi h Increasing incidence of clinical isolates of Bacteroides fragilis resistant to clindamycin. Journal of Antimicrobial Chemotherapy 1984, 13: 395. 17. Betriu C, Campos E, Cabronero G, Rodriguez-Avial C, Picazo JJ: Susceptibilities of species of the Bacteroidesfragilis group to 10 antimicrobial agents. Antimicrobial Agents and Chemotherapy 1990, 34: 671673. 18. Scher KS: Emergence of antibiotic resistant strains of Bacteroides fragilis. Surgery, Gynaecology and Obstetrics 1988, 167: 175-179. 19. Borobio MV, Pascual A, Perea E J: Decreasing resistance of Bacteroides fragilis group strains to metronidazole in Spain between 1977 and 1982. European Journal of Clinical Microbiology and Infectious Diseases 1984, 3: 135-155. 20. Brogan O, Garnett PA, Brown R: Bacteroides fragilis resistant to metronidazole, clindamycin and cefo×itin. Journal of Antimicrobial Chemotherapy 1989, 23: 660662. 21. Eme MA, Acar .IF, Goldslein FW: Bacteroides fragilis resistant to metronid~ole. Journal of Antimicrobial Chemotherapy 1983, 12: 523-534. 22. Ingham HA, Eaton S, Venables CW, Adams PC: Bacteroides fragilis resistant to metronidazole after longterm therapy. Lancet 1978, i: 214.
8. National Committee for Clinical Laboratory Standards: Reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard: M l l - A . NCCLS, ViIlanova, PA, 1985.
9. National Committee for Clinical Laboratory Standards: Methods for antimicrobial susceptibility testing 10.
11.
12.
13.
14.
15.
of anaerobic bacteria-second edition. Approved standard: Mll-A2. NCCLS, Villanova, PA, 1990. Bourgault AM, Lamothe F: In vitro activity of amoxicillin and ticarcillin in combination with clavulanic acid compared with that of new lS-lactam agents against species of the Bacteroidesfragilis group. Journal of Antimicrobial Chemotherapy 1986, 17: 593603. Cuchural G J, Tally FP, Jacobus NV, Cleary T, Finegold SM, Hill G, Iannini P, O'Keefe JP, Pierson C: Comparative activities of newer 15-1attain agents against members of the Bacteroides fragilis group. Antimicrobial Agents and Chemotherapy 1990, 34: 479480. Garcla-Rodriguez JE, Garcla-S~inchez JE: Evolution of antimicrobial susceptibility in isolates of the Bacteroides fragilis group in Spain. Reviews of Infectious Diseases 1990, 12, Supplement 2: 142-151. Musiai CE, Rosenblatt JE: Antimicrobial susceptibilities of anaerobic bacteria isolated at the Mayo Clinic during 1982 through 1987: comparison with results from 1977 through 1981. Mayo Clinic Proceedings 1989, 64: 392-399. Lifiares J, P6rez JL, Martin A: Susceptibility studies in the Bacteroides fragilis group resistant to clindamycin. Journal of Antimicrobial Chemotherapy 1988, 12: 293. Reig M, Campello MG, Baqnero F: Epidemiology of clindamycin resistance in the Bacteroides fragilis
New High-Content Disks for Determination of High-Level Aminoglycoside Resistance in Clinical Isolates of Enterococcus
faecalis R . L e c l e r c q 1 . , R . B i s m u t h 2, J. D u v a l 1
Disks impregnated with 500 and 1000 lag of streptomycin, 1000 lag of kanamycin and 250 and 500 ~g of gentamicin were used for detection of high-level resistance to aminoglycosides in 120 clinical isolates of Enterococcus faecalis. Fiftyseven strains were highly resistant to streptomycin, 80 to kanamycin and 41 to gentamicin. Using disks containing 500 lag of streptomycin, 1000 ~g of kanamycin and 500 lag of gentamicin 1Service de Baet6riologie-Virologie-Hygi~ne, H6pital Henri Mondor, Universit6 Paris XII, 51 Avenue Mar6chal de Lattre de Tassigny, 94010 Cr6teil, France. 2Service de Bact6riologie-Virologie, Centre Hospitalier Universitaire Piti6-Salp6tri~re, 75651 Paris Cedex 13, France.
Vo1.11,1992
strains resistant to high levels of these drugs (97.9 %, 100 % and 100 %, respectively) were accurately detected. Better discrimination between high-level and low-level resistance was achieved with a 500 lag streptomycin or gentamicin disk. Zone-size breakpoints are proposed for detection of high-level resistance by disk diffusion.
Most enterococci are not killed by clinically achievable concentrations of cell-wall active drugs administered alone. Bactericidal chemotherapy is recommended in cases of severe infections due to these organisms, bactericidal activity usually being obtained by combination of cellwall active agents with aminoglycosides (1). However, the bactericidal synergy displayed by combinations is absent in the presence of high-level resistance to aminoglycosides (2). High-level resistance to streptomycin, kanamycin and gentamicin characterises the three major phenotypes of these organisms and can be routinely detected by several methods including agar dilution (3, 4), broth dilution (4-6) and agar disk diffusion (6-8). Disk diffusion is a convenient and reliable method which is easy to perform in most laboratories. The purpose of this study was to evaluate disks containing various concentrations of streptomycin, kanamycin and gentamicin for detection of enterococci showing high-level resistance to these drugs.
Materials and Methods. One hundred and twenty non-replicate strains of Enterococcus faecalis were obtained in 1988 from clinical specimens at the Henri Mondor Hospital in Cr6teil, France. Enterococci were identified by colony morphology, Gram staining and biochemical characteristics (9). The MICs of streptomycin, kanamycin and gentamicin were determined by the agar dilution technique in Mueller-Hinton agar (Diagnostics Pasteur, France) with an inoculum of 104 organisms per spot. Incubation was at 37 °C for 18 hours. Streptomycin was provided by Pfizer, France; kanamycin by Bristol Laboratories, UK and gentamicin by Schering, USA. Agar disk diffusion was performed in parallel to MIC determinations by flooding an inoculum of 5 x 106-107 cfu/ml onto Mueller-Hinton agar. Disks containing high concentrations of kanamycin (1000 ~g), gentamicin (250 and 500 lag) and of streptomycin (500 and 1000 ~g) were furnished by Diagnostics Pasteur. The perimeters of the aminoglycoside inhibition zones were clear-cut and the zone diameters could be easily measured.
357
The relationship between inhibition zone diameters and MICs was analysed with a computer programme written by Dr Le Van Thoi. For every aminoglycoside low and high MIC breakpoints were selected. Strains with aminoglycoside MICs equal to or above 2048 lag/ml were considered to have high-level resistance to this aminoglycoside. Strains with MICs of gentamicin, kanamycin and streptomycin equal to or below 64 pg/ ml, 256 lag/ml and 128 laglml, respectively, were considered to have low-level resistance to these aminoglycosides. Strains with MICs between the two breakpoints were classified as indeterminate. The zone-size breakpoints giving a minimum of very major errors (false-susceptible) and major errors (false-resistant) relative to MICs were selected (10). A minor error comprised an indeterminate result by one of the techniques only.
Results and Discussion, The results obtained for streptomycin are presented in Figure 1. With the 500 lag disk a zone-size breakpoint of 12 mm allowed discrimination of 47 of the 48 strains highly resistant to streptomycin, a single strain being categorized as indeterminate. Fifty-eight strains were resistant to low levels of streptomycin (MICs < 128 ~tg/ml) and exhibited zone diameters greater than 14 mm. The MICs of streptomycin for the 14 remaining strains were between 256 and 1024 lag/ml and corresponded to diameters ranging from 6 to 19 mm. Ten percent of minor errors and no major error were observed. A similar rate of errors was found when the 1000 lag streptomycin disk was used (Figure 1B). However, the 500 lag streptomycin disk was more convenient for detection of highly resistant enterococci since small zone diameters, nearly always _.
