Journal of Antimicrobial Chemotherapy (1992) 30, 149-152
Susceptibility of Pseudomonas aeruginosa tofluoroquinolonesfollowing four years of use in a tertiary care hospital Itamar Shalif, Hava Haas" and Stephen A. Berger**
Of 121 recent clinical isolates of Pseudomonas aeruginosa, 22% were resistant to ciprofloxacin, 50% to ofloxacin and 69% to pefloxacin. Resistance to these drugs was commonest among urinary isolates {P < 0-03). Cross-resistance was noted between fluoroquinolones and aminoglycosides or /f-lactams.
Introduction
Since their introduction, the fluoroquinolones have found wide usage in the therapy of infection caused by aerobic and facultative Gram-negative bacteria. Unlike other oral antimicrobial agents, their spectrum of activity includes such major pathogens as Pseudomonas aeruginosa and Acinetobacter spp. With the passage of time, reports of resistance have appeared in the medical literature, particularly with Staphylococcus aureus (Shalit et al., 1989). To date, the reported acquisition of resistance among Gram-negative bacteria has been less prominent (Kresken & Wiedeman, 1988; Michea-Hamzehpour, Lucain & Pechere, 1991). The Tel-Aviv Medical Centre is a 700-bed tertiary facility in which pefloxacin and ofloxacin have been used since 1987, and ciprofloxacin since 1988. We have examined the in-vitro activity of these drugs against clinical isolates of P. aeruginosa collected prospectively during the second half of 1990. Materiab and methods In total, 121 isolates of P. aeruginosa were recovered from blood, urine, sputum, wound and otic discharges from patients hospitalized on a variety of clinical services. In no instance was more than one strain examined from any given patient. Isolates were identified presumptively as P. aeruginosa on the basis of colonial morphology, pigment and oxidase production, with confirmation of identity in an automated microplate system (Sensititre; Radiometer Copenhagen, East Grinstead, UK). The control strain for identification and susceptibility testing was P. aeruginosa ATCC 25619. Isolates were grown overnight on Sheep Blood Agar (Hy Laboratories, Kiryat Wietzman, Israel), and were then pre-incubated in Mueller-Hinton Broth (Difco, •Corresponding author's address: Department of Microbiology, Ichilov Hospital, 6 Weitzman Street, Tel-Aviv 64239, Israel.
0305-7453/92/080149 + 04 $08.00/0
149 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
"Infectious Diseases Unit andb Department of Microbiology. Tel-Aviv Sourasky Medical Centre; and The Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
150
I. Shalit et aL Table I. In-vitro susceptibility of 121 isolates of P. aeruginosa to various antimicrobial agents Antimicrobial agent
MICM (mg/L)
MIC, (mg/L)
MIC range (mg/L)
Ciprofloxacin Ofloxacin Pefloxacin Aztreonam Ceftazidime Piperacillin Gentamidn Amikacin
05 4 4 4 2 8 32 4
16 64 64 16 16 256 64 32
006-32 025-64 1-64 025-64 0125-32 1-256 2-128 05-64
Results
Drug susceptibility data are summarized in Table I. The MIC,,, and MlCy, values indicated that isolates continued to be susceptible to ceftazidime, aztreonam and, to a lesser extent, amikacin, piperacillin and ciprofloxacin. Gentamicin was the least active drug tested. Of 121 isolates, 27 (22-3%) were resistant (MIC ^ 4 mg/L) to ciprofloxacin, 61 (50-4%) to ofloxacin and 84 (69-4%) to pefloxacin. The differences between these percentages were highly significant for each drug pair tested (P < 10~4; McNemar test for paired proportions). Twenty-six of the isolates (21-5%) were recovered from urine specimens. The percentages of urinary isolates resistant to fluoroquinolones were as follows: ciprofloxacin 61-5%; ofloxacin 80-8%; and pefloxacin 88-5%. Corresponding percentages among non-urinary isolates were 15-8%, 42-1% and 64-2%, respectively; these differences were significant for all three drugs (P < 0-03; chi-square test). It was noted that isolates resistant to non-quinolone drugs were likely to have a relatively high MIC of ciprofloxacin ( > 2 mg/L). These data are summarized in Table II. Resistance to each of the five drugs tested was found to correlate with an increased ciprofloxacin MIC (/> < 10~4-10~*). Discussion
Our data add to a growing literature describing fluoroquinolone resistance among Gram-negative bacteria (Michea-Hamzehpour et al., 1991). Resistance was most marked among urinary isolates, and this may correlate with widespread usage for
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
Detroit, MI, USA) to obtain logarithmic growth. Antibiotic stock solutions were prepared according to the manufacturers' instructions, and were maintained at — 70°C until use. MlCs were determined by an agar dilution method with Mueller-Hinton Agar (Difco), containing serial two-fold drug dilutions, and a multipoint inoculator (Cathra International, St Paul, MN, USA) delivering inocula of lO^cfu/spot (Shalit et al., 1989). Plates were read following overnight incubation in ambient air at 37°C. The MIC was defined as the lowest drug concentration that inhibited visible growth. Resistance to each of the three quinolones was defined as MIC of > 4 mg/L, based upon published breakpoint data for these drugs (Barry et al., 1985; Fuchs et al, 1986, 1989).
P. aeniginosa and
fluoroquinolones
151
Table II. Correlation between resistance of P. aeniginosa to ciprofloxacin and five non-quinolone antimicrobial agents Antimicrobial agent Gentamicin Amikacin Aztreonam
Number of isolates with ciprofloxacin MIC >4mg/L
R-87 S = 33 R = 32 S = 88 R-26 S = 79 R - 14 S = 107
Piperacillin S = 98
P
29 (33-3%) 2 (61%)
< 0-001*
20 (62-5%) 11 (12-5%) 18 (69-2%) 12 (15-2%)
< lO"6*
11 (78-5%) 20(18-7%) 19 (86-4%) 12 (12-2%)
< 10"" < 0-00002* < 10"6*
"Fisher's exact test; *chi-square test. R. Resistant; S, sensitive.
therapy of both community and hospital-acquired urinary tract infection. The influence of increased usage on fluoroquinolone resistance has been demonstrated in a multivariate analysis of hospital-acquired Gram-negative bacteria (Muder et al., 1991). Although the incidence of ciprofloxacin resistance among our non-urinary isolates of P. aeruginosa is still small, ofloxacin and pefloxacin have proven to be increasingly less active toward this species. These trends may influence the choice of such drugs for presumed or confirmed infection caused by P. aeruginosa. Previous studies have demonstrated an association between resistance of klebsiella, serratia, acinetobacter and staphylococcal isolates to quinolones and a variety of other antibiotic agents (Sanders et al., 1984; Gutmann et al., 1985; Shalit et al., 1989, 1990). Resistance mechanisms in these bacteria may involve reduced outer membrane permeability or altered DNA gyrase (Sanders et al., 1984; Chamberland et al., 1989; Michea-Hamzehpour et al., 1991), and may also explain our findings with respect to P. aeruginosa. Based on our data, we would advise careful control of quinolone usage in an attempt to decrease the selection of resistance among strains of pseudomonas. Continuous monitoring of resistance patterns among such bacteria should enable the clinician to choose the appropriate agent for treatment of P. aeruginosa infection. References Barry, A. L., Fass, R. J., Anhalt, J. P., Neu, H. C , Thornsberry, C, Tilton, R. C. et al. (1985). Ciprofloxacin disk susceptibility tests: interpretive zone size standards for 5-/jg disks. Journal of Clinical Microbiology 21, 880-3. Chamberland, S., Bayer, A. S., Schollaardt, T., Wong, S. A. & Bryan, L. E. (1989). Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis. Antimicrobial Agents and Chemotherapy 33, 624-34. Fuchs, P. C , Barry, A. L., Jones, R. N. & Thornsberry, C. (1986). Tentative disk diffusion susceptibility interpretive criteria forpefloxacin. Journal of Clinical Microbiology 24,448-50. Fuchs, P. C, Jones, R. N., Barry, A. L., Gavan, T. C. & the Collaborative Antimicrobial
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
Ceftazidime
Susceptibility pattern
152
I. Shallt et at.
(Received 18 December 1991; revised version accepted 4 March 1992)
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
Susceptibility Test Group. (1989). Ofloxacin susceptibility testing quality control parameters for microdilution and disk diffusion, and confirmation of disk diffusion interpretive criteria. Journal of Clinical Microbiology 27, 49-52. Gutmann, L., Williamson, R., Moreau, N., Kitzis, M. D., Collatz, E., Acar, J. F. et al. (1985). Cross-resistance to nalidixic acid, trimethoprim, and chloramphenicol associated with alterations in outer membrane proteins of Klebsiella. Enierobacter. and Serraiia. Journal of Infectious Diseases 151, 501-7. Kresken, M. & Wiedemann, B. (1988). Development of resistance to nalidixic acid and the fluoroquinolones after the introduction of norfloxacin and ofloxacin. Antimicrobial Agents and Chemotherapy 32, 1285-8. Michea-Hamzehpour, M., Lucain, C. & Pechere, J.-C. (1991). Resistance to pefloxacin in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 35, 512-8. Muder, R. R., Brennen, C , Goetz, A. M., Wagener, M. M. & Rihs, J. D. (1991). Association with prior fluoroquinolone therapy of widespread ciprofloxacin resistance among Gram-negative isolates in a Veterans Affairs medical center. Antimicrobial Agents and Chemotherapy 35, 256-8. Sanders, C. C , Sanders, W. E., Goering, R. V. & Werner, V. (1984). Selection of multiple antibiotic resistance by quinolones, 0-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes. Antimicrobial Agents and Chemotherapy 26, 797-801. Shalit, I., Berger, S. A., Gorea, A. & Frimerman, H. (1989). Widespread quinolone resistance among methicillin-resistant Staphylococcus aureus isolates in a general hospital. Antimicrobial Agents and Chemotherapy 33, 593-4. Shalit, I., Dan, M., Gutman, R., Gorea, A. & Berger, S. A. (1990). Cross resistance to ciprofloxacin and other antimicrobial agents among clinical isolates of Acinetobacter calcoaceticus biovar anitratus. Antimicrobial Agents and Chemotherapy 34, 494-5.
Susceptibility of Pseudomonas aeruginosa tofluoroquinolonesfollowing four years of use in a tertiary care hospital Itamar Shalif, Hava Haas" and Stephen A. Berger**
Of 121 recent clinical isolates of Pseudomonas aeruginosa, 22% were resistant to ciprofloxacin, 50% to ofloxacin and 69% to pefloxacin. Resistance to these drugs was commonest among urinary isolates {P < 0-03). Cross-resistance was noted between fluoroquinolones and aminoglycosides or /f-lactams.
Introduction
Since their introduction, the fluoroquinolones have found wide usage in the therapy of infection caused by aerobic and facultative Gram-negative bacteria. Unlike other oral antimicrobial agents, their spectrum of activity includes such major pathogens as Pseudomonas aeruginosa and Acinetobacter spp. With the passage of time, reports of resistance have appeared in the medical literature, particularly with Staphylococcus aureus (Shalit et al., 1989). To date, the reported acquisition of resistance among Gram-negative bacteria has been less prominent (Kresken & Wiedeman, 1988; Michea-Hamzehpour, Lucain & Pechere, 1991). The Tel-Aviv Medical Centre is a 700-bed tertiary facility in which pefloxacin and ofloxacin have been used since 1987, and ciprofloxacin since 1988. We have examined the in-vitro activity of these drugs against clinical isolates of P. aeruginosa collected prospectively during the second half of 1990. Materiab and methods In total, 121 isolates of P. aeruginosa were recovered from blood, urine, sputum, wound and otic discharges from patients hospitalized on a variety of clinical services. In no instance was more than one strain examined from any given patient. Isolates were identified presumptively as P. aeruginosa on the basis of colonial morphology, pigment and oxidase production, with confirmation of identity in an automated microplate system (Sensititre; Radiometer Copenhagen, East Grinstead, UK). The control strain for identification and susceptibility testing was P. aeruginosa ATCC 25619. Isolates were grown overnight on Sheep Blood Agar (Hy Laboratories, Kiryat Wietzman, Israel), and were then pre-incubated in Mueller-Hinton Broth (Difco, •Corresponding author's address: Department of Microbiology, Ichilov Hospital, 6 Weitzman Street, Tel-Aviv 64239, Israel.
0305-7453/92/080149 + 04 $08.00/0
149 © 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
"Infectious Diseases Unit andb Department of Microbiology. Tel-Aviv Sourasky Medical Centre; and The Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
150
I. Shalit et aL Table I. In-vitro susceptibility of 121 isolates of P. aeruginosa to various antimicrobial agents Antimicrobial agent
MICM (mg/L)
MIC, (mg/L)
MIC range (mg/L)
Ciprofloxacin Ofloxacin Pefloxacin Aztreonam Ceftazidime Piperacillin Gentamidn Amikacin
05 4 4 4 2 8 32 4
16 64 64 16 16 256 64 32
006-32 025-64 1-64 025-64 0125-32 1-256 2-128 05-64
Results
Drug susceptibility data are summarized in Table I. The MIC,,, and MlCy, values indicated that isolates continued to be susceptible to ceftazidime, aztreonam and, to a lesser extent, amikacin, piperacillin and ciprofloxacin. Gentamicin was the least active drug tested. Of 121 isolates, 27 (22-3%) were resistant (MIC ^ 4 mg/L) to ciprofloxacin, 61 (50-4%) to ofloxacin and 84 (69-4%) to pefloxacin. The differences between these percentages were highly significant for each drug pair tested (P < 10~4; McNemar test for paired proportions). Twenty-six of the isolates (21-5%) were recovered from urine specimens. The percentages of urinary isolates resistant to fluoroquinolones were as follows: ciprofloxacin 61-5%; ofloxacin 80-8%; and pefloxacin 88-5%. Corresponding percentages among non-urinary isolates were 15-8%, 42-1% and 64-2%, respectively; these differences were significant for all three drugs (P < 0-03; chi-square test). It was noted that isolates resistant to non-quinolone drugs were likely to have a relatively high MIC of ciprofloxacin ( > 2 mg/L). These data are summarized in Table II. Resistance to each of the five drugs tested was found to correlate with an increased ciprofloxacin MIC (/> < 10~4-10~*). Discussion
Our data add to a growing literature describing fluoroquinolone resistance among Gram-negative bacteria (Michea-Hamzehpour et al., 1991). Resistance was most marked among urinary isolates, and this may correlate with widespread usage for
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
Detroit, MI, USA) to obtain logarithmic growth. Antibiotic stock solutions were prepared according to the manufacturers' instructions, and were maintained at — 70°C until use. MlCs were determined by an agar dilution method with Mueller-Hinton Agar (Difco), containing serial two-fold drug dilutions, and a multipoint inoculator (Cathra International, St Paul, MN, USA) delivering inocula of lO^cfu/spot (Shalit et al., 1989). Plates were read following overnight incubation in ambient air at 37°C. The MIC was defined as the lowest drug concentration that inhibited visible growth. Resistance to each of the three quinolones was defined as MIC of > 4 mg/L, based upon published breakpoint data for these drugs (Barry et al., 1985; Fuchs et al, 1986, 1989).
P. aeniginosa and
fluoroquinolones
151
Table II. Correlation between resistance of P. aeniginosa to ciprofloxacin and five non-quinolone antimicrobial agents Antimicrobial agent Gentamicin Amikacin Aztreonam
Number of isolates with ciprofloxacin MIC >4mg/L
R-87 S = 33 R = 32 S = 88 R-26 S = 79 R - 14 S = 107
Piperacillin S = 98
P
29 (33-3%) 2 (61%)
< 0-001*
20 (62-5%) 11 (12-5%) 18 (69-2%) 12 (15-2%)
< lO"6*
11 (78-5%) 20(18-7%) 19 (86-4%) 12 (12-2%)
< 10"" < 0-00002* < 10"6*
"Fisher's exact test; *chi-square test. R. Resistant; S, sensitive.
therapy of both community and hospital-acquired urinary tract infection. The influence of increased usage on fluoroquinolone resistance has been demonstrated in a multivariate analysis of hospital-acquired Gram-negative bacteria (Muder et al., 1991). Although the incidence of ciprofloxacin resistance among our non-urinary isolates of P. aeruginosa is still small, ofloxacin and pefloxacin have proven to be increasingly less active toward this species. These trends may influence the choice of such drugs for presumed or confirmed infection caused by P. aeruginosa. Previous studies have demonstrated an association between resistance of klebsiella, serratia, acinetobacter and staphylococcal isolates to quinolones and a variety of other antibiotic agents (Sanders et al., 1984; Gutmann et al., 1985; Shalit et al., 1989, 1990). Resistance mechanisms in these bacteria may involve reduced outer membrane permeability or altered DNA gyrase (Sanders et al., 1984; Chamberland et al., 1989; Michea-Hamzehpour et al., 1991), and may also explain our findings with respect to P. aeruginosa. Based on our data, we would advise careful control of quinolone usage in an attempt to decrease the selection of resistance among strains of pseudomonas. Continuous monitoring of resistance patterns among such bacteria should enable the clinician to choose the appropriate agent for treatment of P. aeruginosa infection. References Barry, A. L., Fass, R. J., Anhalt, J. P., Neu, H. C , Thornsberry, C, Tilton, R. C. et al. (1985). Ciprofloxacin disk susceptibility tests: interpretive zone size standards for 5-/jg disks. Journal of Clinical Microbiology 21, 880-3. Chamberland, S., Bayer, A. S., Schollaardt, T., Wong, S. A. & Bryan, L. E. (1989). Characterization of mechanisms of quinolone resistance in Pseudomonas aeruginosa strains isolated in vitro and in vivo during experimental endocarditis. Antimicrobial Agents and Chemotherapy 33, 624-34. Fuchs, P. C , Barry, A. L., Jones, R. N. & Thornsberry, C. (1986). Tentative disk diffusion susceptibility interpretive criteria forpefloxacin. Journal of Clinical Microbiology 24,448-50. Fuchs, P. C, Jones, R. N., Barry, A. L., Gavan, T. C. & the Collaborative Antimicrobial
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
Ceftazidime
Susceptibility pattern
152
I. Shallt et at.
(Received 18 December 1991; revised version accepted 4 March 1992)
Downloaded from http://jac.oxfordjournals.org/ at University of Michigan on June 12, 2015
Susceptibility Test Group. (1989). Ofloxacin susceptibility testing quality control parameters for microdilution and disk diffusion, and confirmation of disk diffusion interpretive criteria. Journal of Clinical Microbiology 27, 49-52. Gutmann, L., Williamson, R., Moreau, N., Kitzis, M. D., Collatz, E., Acar, J. F. et al. (1985). Cross-resistance to nalidixic acid, trimethoprim, and chloramphenicol associated with alterations in outer membrane proteins of Klebsiella. Enierobacter. and Serraiia. Journal of Infectious Diseases 151, 501-7. Kresken, M. & Wiedemann, B. (1988). Development of resistance to nalidixic acid and the fluoroquinolones after the introduction of norfloxacin and ofloxacin. Antimicrobial Agents and Chemotherapy 32, 1285-8. Michea-Hamzehpour, M., Lucain, C. & Pechere, J.-C. (1991). Resistance to pefloxacin in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 35, 512-8. Muder, R. R., Brennen, C , Goetz, A. M., Wagener, M. M. & Rihs, J. D. (1991). Association with prior fluoroquinolone therapy of widespread ciprofloxacin resistance among Gram-negative isolates in a Veterans Affairs medical center. Antimicrobial Agents and Chemotherapy 35, 256-8. Sanders, C. C , Sanders, W. E., Goering, R. V. & Werner, V. (1984). Selection of multiple antibiotic resistance by quinolones, 0-lactams, and aminoglycosides with special reference to cross-resistance between unrelated drug classes. Antimicrobial Agents and Chemotherapy 26, 797-801. Shalit, I., Berger, S. A., Gorea, A. & Frimerman, H. (1989). Widespread quinolone resistance among methicillin-resistant Staphylococcus aureus isolates in a general hospital. Antimicrobial Agents and Chemotherapy 33, 593-4. Shalit, I., Dan, M., Gutman, R., Gorea, A. & Berger, S. A. (1990). Cross resistance to ciprofloxacin and other antimicrobial agents among clinical isolates of Acinetobacter calcoaceticus biovar anitratus. Antimicrobial Agents and Chemotherapy 34, 494-5.
