Journal of Antimicrobial Chemotherapy (1992) 29, 539-546
Activity of antibiotics against resistant Pseudomonas aeruginosa Jiro Fujha, Kiyoshi Negayama, Keiichi Takigawa, Yoshifbmi Yamagishl, Aklhlto Knbo, Yasnfumi Yamaji and Jiro Takahara First Department of Internal Medicine, Clinical Laboratory, Kagawa Medical School, Kagawa, 761-07. Japan
Introduction Pseudomonas aeruginosa is currently recognized as one of the main leading causes of severe hospital infections. Although P. aeruginosa is intrinsically resistant to many antibiotics, most isolates are relatively sensitive to ureidopenicillins, some third generation cephalosporins, new quinolones, imipenem and amikacin. Nevertheless, an increased frequency of resistance among clinical isolates of P. aeruginosa to several antibiotics has been experienced in recent years. In Kagawa Medical School Hospital, Japan, P. aeruginosa is the most frequently isolated clinical species of glucose non-fermenting Gram-negative bacteria. In this study, the in-vitro activity of several antibiotics against clinical isolates of P. aeruginosa was examined to establish the appropriate treatment regimens. In particular, the efficacy of antibiotics against single-, double, or triple-drug-resistant strains was evaluated to determine the cross-resistance to each drug. Methods Kagawa Medical School Hospital is a 613-bed facility (140 beds for Internal Medicine and 100 beds for General Surgery). From June, 1990 to October 1990, 508 strains of P. aeruginosa were isolated from 120 patients. Where multiple isolates of P. aeruginosa 539 0305-7453/92/050539+08 $02.00
©1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at OCLC on March 9, 2015
The activity of 12 antibiotics, piperacillin, ccfazolin, cefotiam, ccftizoxime, latamoxef, ceftazidime, cefuzonam, amikacin, ofloxacin, imipenem, aztreonam and minocycline, against 120 isolates of Pseudomonas aeruginosa was examined. In addition, the efficacy of antibiotics against single-, double-, or triple-drug-resistant isolates of P. aeruginosa were also examined to determine the cross-resistance to each drug. There was cross-resistance between piperacillin, ceftazidime and aztreonam, but amikacin and imipenem remained effective antibiotics, especially as salvage therapy, against isolates resistant to one agent. Results also suggested that piperaciUin, ceftazidime or imipenem in combination with amikacin are effective combination regimens against most clinical isolates of P. aeruginosa. Amikacin and imipenem were also suitable antibiotics, especially as salvage therapy, against isolates of P. aeruginosa resistant to two agents. In conclusion, the results provide useful guidelines for choosing an effective treatment against clinical isolates of P. aeruginosa, and for choosing salvage therapy against resistant P. aeruginosa.
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Results Figure 1 shows the activity of the 12 antibiotics against the 120 clinical isolates of P. aeruginosa. Imipenem was the most active, inhibiting 84-2% of the isolates, followed by piperacillin (79-2%), ceftazidime (78-3%), amikacin (74-2%), aztreonam (70%), and ofloxacin (67-5%). Table I shows the activity of the six most active antibiotics against isolates resistant to one drug. There was cross-resistance between piperacillin and ceftazidime, between piperacillin and aztreonam, and between ceftazidime and aztreonam. Imipenem was the most active, inhibiting 66-9% of the isolates resistant to one drug. Ofloxacin and aztreonam were least effective inhibiting 38-0%, and 37-9% of the isolates respectively (Figure 2). Figure 3 shows the in-vitro activity of the six most active antibiotics plus another drug (data combined; activity of the combination per se not measured) against the 120
40
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Figure 1. The activity of the 12 antibiotic* against the 120 clinical isolates of P. atruglnosa, MINO, Minocycline; AZT, aztreonam; IPM, imipenem; OFX, ofloxacin; AMK, amikacin; CZON, cefuzonam; CAZ, ceftazidime; MOX, latamoxef; CZX, ceftizoxime; CTM, cefotiam; CEZ, cefazolin; PIP, piperacillin.
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were received from the same patient, only the first isolate was used, hence, 120 strains of P. aeruginosa from 120 patients were evaluated in this study. The activity of 12 antibiotics: piperacillin, cefazolin, cefotiam, ceftizoxime, latamoxcf, ceftazidime, cefuzonam, amikacin, ofloxacin, imipenem, aztreonam and minocycline was examined. The activity of each antibiotic was determined by measuring the minimum inhibitory concentration (MIC) of each agent with the MIC 2000 Plus System (Dynatech Laboratories, USA). Strains were considered resistant if the MIC of piperacillin and amikacin was > 32 mg/L, > 16 mg/L of cefazolin, cefotiam, ceftizoxime, latamoxef, ceftazidime, cefuzonam, and aztreonam, and > 8 mg/L of minocycline, ofloxacin, and imipenem. In addition, the activity of two antibiotics was also examined. Isolates which were sensitive to one drug and resistant to the other drug, or sensitive to both drugs, were designated sensitive to a combination of both drugs, although the activity of the combination itself was not measured. To evaluate the efficacy of antibiotics against isolates resistant to two or three agents, the efficacy of other antibiotics were evaluated.
Antibiotics against P. aeruginosa
541
Table I. Activity of antibiotics against P. aeruginosa resistant to one drug Number resistant
Antibiotic
25 36 31 39 19 36
Piperacillin Ceftazidime Amikacin Ofloxacin Imipenem Aztreonam Total
176-
Number sensitive to other agents (%) AMK CAZ OFX IPM
PIP
23 (74-2) — 25 (641) 18 (46-2) 9 (47-4) 11 (57-9) 13 (36-1) 26 (72-2)
10(40) 15(60) 3(12) 12 (46-2) 14 (61-5) 4 (15-4) 10 (32-2) 23 (74-2) 21 (67-7) 27 (69-2) 16 (41) — 9 (47-4) 7 (36-8) — 13 (36-1) 26 (72-2) —
73
52
3(12)
4 (15-4) 24 (77-4) 29 (74-3) 9 (47-4) 14 (38-9) 80
—
•Some (trains resistant to more than one agent PIP, Piperacillin; CAZ, ceflazidnne; AMK,
18 (72) 18 (69-2)
AZT
91
105
53
; OFX, ofloxacin; IPM imipenem; AZT, aztreonam.
AZT
IPM
OFX
AMK
CAZ
PIP
40 P*r cant Mnsltivt
Figure 2. Activity of the ax most active antibiotics against P. aeruginosa resistant to one drug. Abbreviations as in Figure 1.
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clinical isolates of P. aeruginosa. Piperacillin plus amikacin inhibited 94-2% of the isolates and amikacin plus ceftazidime or imipenem inhibited 93-3% of the isolates. Aztreonam plus ceftazidime or ofloxacin were least effective, inhibiting only 80-8% of the isolates. The activity of selected antibiotics against isolates resistant to two antibiotics is shown in Table II. Amikacin was most active, inhibiting 70-2% (113/161) of the isolates resistant to two drugs, followed by imipenem which inhibited 61-2% (101/165) of the isolates. Piperacillin, ceftazidime, ofloxacin, and aztreonam inhibited 34-5% (48/139), 33-3% (46/138), 33-8% (44/130), and 26-8% (34/127) of the isolates, respectively (Figure 4). There was cross-resistance between piperacillin, ceftazidime, and aztreonam. Table III shows the activity of antibiotics against isolates resistant to three drugs. Based on the data in Table II, the most effective antibiotic against strains resistant to two drugs was selected as the third antibiotic. The activity of amikacin could not be calculated as amikacin was included in every three-drug combination due to its high
J. Fujlta et aL
542
100
Figure 3. The activity of two drugs against the 120 clinical isolates of P. aeruginosa. Abbreviations as in Figure 1.
efficacy. Among the other five antibiotics, imipenem was most active against isolates resistant to three drugs, inhibiting 56-1% (23/41) of the strains. Ceftazidime, ofloxacin, and aztreonam inhibited 15-6% (7/45), 27-5% (11/40), and 28-3% (13/46) of the isolates, respectively. Discussion In this study, the activity of six antibiotics, piperacillin, ceftazidime, amikacin, ofloxacin, imipenem, and aztreonam against P. aeruginosa resistant to one, two or three Table II. Activity of six antibiotics to P. aeruginosa resistant to two drugs Antibiotic Piperacillin •+• ceftazidime Piperacillin + amikacin Piperacillin + ofloxacin Piperacillin + imipenem Piperacillin + aztreonam Ceftazidime + amikacin Ceftazidime + ofloxacin Ceftazidime + imipenem Ceftazidime + aztreonam Amikacin + ofloxacin Amikacin + imipenem Amikacin + aztreonam Ofloxacin + imipenem Ofloxacin + aztreonam Imipenem 4• aztreonam Total
Number resistant 22 7 15 10 22 8 14 10 23 21 8 10 12 23 10 215-
PIP
2 (25) 1 (71) 1 (10) 3 (13) 17 (81) 4 (50) 5 (50) 5 (41-7) 8 (34-8) 2 (20) 48
"Some strains resistant to more than two agents. Abbreviation* as in Table I.
Number sensitive 1:o other agents (%)i CAZ AMK OFX AZT IPM 16 (72-2) 9(40-9) 13 (591) 2(9-1) 1 (14-3) — 3 (42-9) 3 (42-9) 2 (28-6) 2 (13-3) 11 (73-3) 8 (53-3) 0(0) 1(10) 6(60) 3(30) 2(20) — 2(9-1) 17 (77-3) 7 (31-8) 14 (63-6) — — 3 (77-5) 4(50) 2(25) — 9 (64-2) 6 (42-9) 1 (71) — 6(60) 2(20) 2(20) — 17 (73-9) 10 (43-5) 15 (65-2) 16 (76-2) — — 16 (76-2) 14(66-7) 4(50) — 6(75) 3 (37-5) — 4(40) — — 3(30) 8(80) 4 (33-3) 7 (583) 3(25) — 10 (43-5) 16 (69-6) — 14 (60-9) — 2(20) 8(80) 1(10) — — 46 113 44 101 34
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60 un«itivi
543
Antibiotics against P. aervgiaosa
AZT
40
Figure 4. Activity of the m most active antibiotics against strains of P. aeruginosa resistant to two drags. Abbreviations as in Figure 1.
drugs was determined in order to evaluate cross-resistance to each drug. The differences between cross-resistance to several antibiotics seemed to be based on the differences in the major mechanism of resistance (Jacoby & Archer, 1991). The major mechanism of resistance to /J-lactams found in P. aeruginosa is the expression of /7-lactamase (Medeiros, 1989; Sanders, 1989). In P. aeruginosa, /Mactam resistance is usually mediated by a chromosomal class 1 /Mactamase and/or a plasmidmediated /Mactamase. Resistance to third-generation cephalosporins and ureidopeniT&bte m . Activity of antibiotics against P. aeruginosa resistant to three drugs
Antibiotics Piperacillin + ceftazidime Piperacillin + amikacin Piperacillin + amikacin Piperacillin + ofloxacin Piperacillin + imipenem Piperacillin + aztreonam Ceftazidime + amikacin Ceftazidime + ofloxacin Ceftazidime + imipenem Ceftazidime + aztreonam Amikacin + ofloxacin Amikacin + imipenem Amikacin + aztreonam Ofloxacin + imipenem Ofloxacin + aztreonam Imipenem + aztreonam Total Abbreviations at in Table I.
Number resistant
22 7 7 15 10 22 8 14 10 23 21 8 10 12 23 10 215
Third antibiotic Amikacin Ofloxacin Imipenem Amikacin Amikacin Amikacin Imipenem Amikacin Amikacin Amikacin Piperacillin Aztreonam Imipenem Amikacin Amikacin Amilrnpjn
Total
Number resistant
6 4 4 4 5 5 4 5 4 6 4 2 2 6 7 2 70
Number sensitive to other agents
PIP CAZ OFX IPM AZT — — — — — 1 1 1 1 — 0 0 3 3 0
0 1 0 1 0 — — — — 0 0 0 2 3 0
2 — 2 — 2 1 1 — 1 2 — 0 0 — — 0
3 2 — 2 — 3 — 2 — 4 2 — — — 5 —
1 0 2 0 2 — 2 1 2 — 0 — — 3 — —
10/38 7/45 ii/4o:23/41 13/46
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Ptr cent Mtnttlv*
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cillins usually arises via a mutation that gives rise to constitutive (stably derepressed), instead of inducible, production of chromosomal class 1 /Mactamase. Third-generation cephalosporins, such as ceftazidime, and ureidopenicillins, such as piperacillin, are labile to chromosomal /Mactamase but are weak inducers of its synthesis (Livermore & Yang, 1987), therefore stably depressed mutants from inducible populations can be selected (Livermore, 1987; Sanders & Sanders, 1987). PSE-1 is the most common plasmid-mediated /Mactamase found in P. aeruginosa (Medeiros, 1989). Recently, plasmid-mediated /Mactamases with a broad-spectrum of /Mactam resistance, including the oxyimino-/Mactams such as cefotaxime, ceftazidime, ceftriaxone, and aztreonam, have been reported in a variety of Enterobacteriaceae (Knothe et al., 1983; Kliebe et al., 1985; Jacoby & Carreras, 1990). Recently, a third type of plasmid-mediated, extended-spectrum /Mactamase has appeared (Papanicolaou, Medeiros & Jacoby, 1990). This new enzyme causes resistance to /Mactam antibiotics with a 7o-methoxy group, such as cefoxitin, cefotetan or latamoxef, as well as to oxyimino-/Mactam antibiotics. The data from this study suggest that as there was cross-resistance between piperacillin, ceftazidime, and aztreonam the isolates expressed the chromosomal /Mactamase constitutively. Alternatively, it cannot be ruled out that a plasmid-mediated, extendedspectrum /Mactamase is present in this institution. The major mechanism of resistance to imipenem in P. aeruginosa is the loss of the porin channel, Opr D2 (Trias & Nikaido, 1990) and rarely due to /Mactamase. This difference in the major mechanisms of resistance seems to explain why there was no cross-resistance between imipenem and the other drugs in this study. The major mechanisms of resistance to amikacin found in P. aeruginosa to amikacin are related to both a penetration barrier and to enzymic modification of the drug. Some Gram-negative organisms, especially P. aeruginosa, can become resistant to amikacin by non-enzymatic mechanisms that decrease the general uptake of aminoglycosides (Maloney et al., 1989), or the lipopolysaccharide structure of the cell wall (Bryan, O'Hara & Wong, 1984). Some organisms rely on enzymatic modification by 6'-acetyltransferase (Tran Van Nhieu & Collatz, 1988), 3'-phosphotransferases (Gaynes et al., 1988; Lambert, Gerbaud & Courvalin, 1988; Lambert et al., 1990), or 4'-nudeotidyltransferase (Jacoby et al., 1990) which are usually encoded on plasmid DNA. As these mechanisms of resistance do not confer cross-resistance to unrelated agents, this may explain why there was no cross-resistance between amikacin and the other drugs in this study. The major resistance mechanisms to quinolones present in P. aeruginosa are both an altered target (DNA gyrase) and reduced antibiotic uptake. Some mutants resistant to quinolones have an altered DNA gyrase (Rella & Haas, 1982; Hirai et al., 1987; Piddock & Wise, 1987; Piddock, Wijnands & Wise, 1987). In other mutants, the amount of the outer membrane porin protein is diminished and the accumulation of quinolones is decreased (Piddock et al., 1987). Mutations in DNA gyrase confer resistance only to quinolones, but alterations in the outer-membrane proteins result can cross-resistance to chemically unrelated antibiotics (Piddock et al., 1987). In this study, there was partial cross-resistance between ofloxacin and the other drugs, suggesting that both classes of resistance occurs in our institution. Clinical isolates with similar properties have been previously described (Piddock et al., 1987; Daikos, Lorans & Jackson, 1988), and resistance to quinolones is increasing among P. aeruginosa (Kreken & Wiedemann, 1988).
Antibiotics against P. aerugbtosa
545
Clinically, the choice of appropriate antibiotics for use against resistant strains of P. aeruginosa is a very important issue (Jacoby, 1986). This study clearly demonstrated cross-resistance among piperacillin, ceftazidime, and aztreonam; however, amikacin and imipenem were effective antibiotics, especially as salvage therapy, against P. aeruginosa resistant to one agent. Results also suggested that piperacillin in combination with amikacin, ceftazidime in combination with amikacin, and amikacin in combination with imipenem are effective combination antibiotic regimens against most clinical isolates of P. aeruginosa. Amikacin and imipenen were also particularly effective as salvage therapy, against P. aeruginosa resistant to two agents.
References Bryan, L. E., O'Hara, K. & Wong, S. (1984). lipopolysaccharide changes in impermeability-type aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 26, 250-5. Daikos, G. L., Lolans, V. T. & Jackson, G. G. (1988). Alterations in outer membrane proteins of Pseudomonas aeruginosa associated with selective resistance to quinolones. Antimicrobial Agents and Chemotherapy 32, 785-7. Gaynes, R., Groisman, E., Nelson, E., Casadaban, M. & Lerner, S. A. (1988). Isolation, characterization, and cloning of a plasmid-borne gene encoding a phosphotransferase that confers high-level amikacin resistance in enteric bacilli. Antimicrobial Agents and Chemotherapy 32, 1379-84. Hirai, K., Suzue, S., Irikura, T., Iyobe, S. & Mitsuhashi, S. (1987). Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 30, 248-53. Jacoby, G. A. (1986). Resistance plasmids of Pseudomonas. In The Bacteria: A Treatise on Structure and Function, vol. 10. The Biology o/Pseudomonas (Sokatch, J. R., Ed.), pp. 26593. Academic Press, San Diego. Jacoby, G. A. & Archer, G. L. (1991). New mechanisms of bacterial resistance to antimicrobial agents. New England Journal of Medicine 324, 601-12. Jacoby, G. A., Blaser, M. J., Santanam, P., Hachler, H., Kayser, F. H. & Hare, R. S. (1990). Appearance of ntniVorin and tobramycin resistance due to 4'-aminoglycoside nudeotidyltransferase in Gram-negative pathogens. Antimicrobial Agents and Chemotherapy 34, 2381-6. Jacoby, G. A. & Carreras, I. (1990). Activities of 0-lactam antibiotics against Escherichia coli strains producing extended-spectrum /Mactamases. Antimicrobial Agents and Chemotherapy 34,858-62. Kliebe, C , Nies, B. A. & Meyer, J. F. Tolxdorff-NeutzKng, R. M. & Wiedemann, B. (1985). Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 28, 302-7. Knothe, H., Shah, P., Krcmery, V., Antal, M. & Mitsuhashi, S. (1983). Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection 11, 315-7. Kresken, M. & Wiedemann, B. (1988). Development of resistance to nalidixtc acid and the fluoroquinolones after the introduction of norfloxacin and ofloxarin. Antimicrobial Agents and Chemotherapy 32, 1285-8. Lambert, T., Gerbaud, G., Bouvet, P., Vieu, J.-F. & Courvalin, P. (1990). Dissemination of
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Based on the results of this study, the best therapy seemed to be a combination of amikacin and imipenem (efficacy rate 93-3%), salvaged by aztreonam (efficacy rate 98-3%). These three drugs in combination covered 118 of the 120 strains of clinically isolated P. aeruginosa. In conclusion, our results seem helpful in providing useful guidelines for choosing an effective treatment against clinical isolates of P. aeruginosa, and for choosing salvage therapy against resistant strains of P. aeruginosa.
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{Received 24 July 1991; revised version accepted 19 December 1991)
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amikacin resistance gene aphA6 in Acinetobacter spp. Antimicrobial Agents and Chemotherapy 34, 1244-8. Lambert, T., Gerbaud, G. & Courvalin, P. (1988). Transferable amikacin resistance in Acinetobacter spp. due to a new type of 3'-aminoglycoside phosphotransferase. Antimicrobial Agents and Chemotherapy 32, 15-9. Livermore, D. M. (1987). Clinical significance of beta-lactamase induction and stable derepression in Gram-negative rods. European Journal of Clinical Microbiology 6, 439—45. Livermore, D. M. & Yang, Y.-J. (1987). ^-Lactamase lability and inducer power of newer /J-lactam antibiotics in relation to their activity against /Mactamase-inducibility mutants of Pseudomonas aeruginosa. Journal of Infectious Diseases 155, 775-82. Maloney, J., Rimland, D., Stephens, D. S., Terry, P. & Whitney, A. M. (1989). Analysis of amikacin-resistant Pseudomonas aeruginosa developing in patients receiving amikacin. Archives of Internal Medicine 149, 630-4. Medeiros, A. A. (1989). Plasmid-detcrmined beta-lactamases. In Microbial Resistance to Drugs (Bryan, L. E., Ed.), pp. 101-27. Springer-Verlag, Berlin. Papanicolaou, G. A., Medeiros, A. A. & Jacoby, G. A. (1990). Novel plasmid-mediated /f-lactamase (MIR-1) conferring resistance to oxyimino and ct-methoxy /Mactams in clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 34, 2200-9. Piddock, L. J. V., Wijnands, W. J. A. & Wise, R. (1987). Quinolone/ureidopcnicillin crossresistance. Lancet ii, 907. Piddock, L. J. V. & Wise, R. (1987). Characterization of post-therapy isolates of Pseudomonas aeruginosa with decreased susceptibility to enoxacin: evidence for two mechanisms. In Program and Abstracts of the Twenty-Seventh Intersdence Conference on Antimicrobial Agents and Chemotherapy, New York, 1987. Abstract 468. American Society for Microbiology, Washington, DC. Rella, M. & Haas, D. (1982). Resistance of Pseudomonas aeruginosa PAO to nalidixic acid and low levels of /Mactam antibiotics: mapping of chromosomal genes. Antimicrobial Agents and Chemotherapy 22, 242-9. Sanders, C. C. (1989). The chromosomal beta-lactamases. In Microbial Resistance to Drugs (Bryan, L. E., Ed.), pp. 129-49. Springer-Verlag, Berlin. Sanders, C. C. & Sanders, W. E. (1987). Conical importance of inducible beta-lactamase in Gram-negative bacteria. European Journal of Clinical Microbiology 6, 435-8. Tran Van Nhieu, G. & Collatz, E. (1988). Heterogeneity of 6'-N-acetyltransferases of type 4 conferring resistance to amikacin and related aminoglycosides in members of the family Enterobacteriaceae. Antimicrobial Agents and Chemotherapy 32, 1289-91. Trias, J. & Nikaido, H. (1990). Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeurginosa. Antimicrobial Agents and Chemotherapy 34, 52-7.
Activity of antibiotics against resistant Pseudomonas aeruginosa Jiro Fujha, Kiyoshi Negayama, Keiichi Takigawa, Yoshifbmi Yamagishl, Aklhlto Knbo, Yasnfumi Yamaji and Jiro Takahara First Department of Internal Medicine, Clinical Laboratory, Kagawa Medical School, Kagawa, 761-07. Japan
Introduction Pseudomonas aeruginosa is currently recognized as one of the main leading causes of severe hospital infections. Although P. aeruginosa is intrinsically resistant to many antibiotics, most isolates are relatively sensitive to ureidopenicillins, some third generation cephalosporins, new quinolones, imipenem and amikacin. Nevertheless, an increased frequency of resistance among clinical isolates of P. aeruginosa to several antibiotics has been experienced in recent years. In Kagawa Medical School Hospital, Japan, P. aeruginosa is the most frequently isolated clinical species of glucose non-fermenting Gram-negative bacteria. In this study, the in-vitro activity of several antibiotics against clinical isolates of P. aeruginosa was examined to establish the appropriate treatment regimens. In particular, the efficacy of antibiotics against single-, double, or triple-drug-resistant strains was evaluated to determine the cross-resistance to each drug. Methods Kagawa Medical School Hospital is a 613-bed facility (140 beds for Internal Medicine and 100 beds for General Surgery). From June, 1990 to October 1990, 508 strains of P. aeruginosa were isolated from 120 patients. Where multiple isolates of P. aeruginosa 539 0305-7453/92/050539+08 $02.00
©1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at OCLC on March 9, 2015
The activity of 12 antibiotics, piperacillin, ccfazolin, cefotiam, ccftizoxime, latamoxef, ceftazidime, cefuzonam, amikacin, ofloxacin, imipenem, aztreonam and minocycline, against 120 isolates of Pseudomonas aeruginosa was examined. In addition, the efficacy of antibiotics against single-, double-, or triple-drug-resistant isolates of P. aeruginosa were also examined to determine the cross-resistance to each drug. There was cross-resistance between piperacillin, ceftazidime and aztreonam, but amikacin and imipenem remained effective antibiotics, especially as salvage therapy, against isolates resistant to one agent. Results also suggested that piperaciUin, ceftazidime or imipenem in combination with amikacin are effective combination regimens against most clinical isolates of P. aeruginosa. Amikacin and imipenem were also suitable antibiotics, especially as salvage therapy, against isolates of P. aeruginosa resistant to two agents. In conclusion, the results provide useful guidelines for choosing an effective treatment against clinical isolates of P. aeruginosa, and for choosing salvage therapy against resistant P. aeruginosa.
540
J. Fujit» et aL
Results Figure 1 shows the activity of the 12 antibiotics against the 120 clinical isolates of P. aeruginosa. Imipenem was the most active, inhibiting 84-2% of the isolates, followed by piperacillin (79-2%), ceftazidime (78-3%), amikacin (74-2%), aztreonam (70%), and ofloxacin (67-5%). Table I shows the activity of the six most active antibiotics against isolates resistant to one drug. There was cross-resistance between piperacillin and ceftazidime, between piperacillin and aztreonam, and between ceftazidime and aztreonam. Imipenem was the most active, inhibiting 66-9% of the isolates resistant to one drug. Ofloxacin and aztreonam were least effective inhibiting 38-0%, and 37-9% of the isolates respectively (Figure 2). Figure 3 shows the in-vitro activity of the six most active antibiotics plus another drug (data combined; activity of the combination per se not measured) against the 120
40
60
80
100
P»r ewit wmltlv*
Figure 1. The activity of the 12 antibiotic* against the 120 clinical isolates of P. atruglnosa, MINO, Minocycline; AZT, aztreonam; IPM, imipenem; OFX, ofloxacin; AMK, amikacin; CZON, cefuzonam; CAZ, ceftazidime; MOX, latamoxef; CZX, ceftizoxime; CTM, cefotiam; CEZ, cefazolin; PIP, piperacillin.
Downloaded from http://jac.oxfordjournals.org/ at OCLC on March 9, 2015
were received from the same patient, only the first isolate was used, hence, 120 strains of P. aeruginosa from 120 patients were evaluated in this study. The activity of 12 antibiotics: piperacillin, cefazolin, cefotiam, ceftizoxime, latamoxcf, ceftazidime, cefuzonam, amikacin, ofloxacin, imipenem, aztreonam and minocycline was examined. The activity of each antibiotic was determined by measuring the minimum inhibitory concentration (MIC) of each agent with the MIC 2000 Plus System (Dynatech Laboratories, USA). Strains were considered resistant if the MIC of piperacillin and amikacin was > 32 mg/L, > 16 mg/L of cefazolin, cefotiam, ceftizoxime, latamoxef, ceftazidime, cefuzonam, and aztreonam, and > 8 mg/L of minocycline, ofloxacin, and imipenem. In addition, the activity of two antibiotics was also examined. Isolates which were sensitive to one drug and resistant to the other drug, or sensitive to both drugs, were designated sensitive to a combination of both drugs, although the activity of the combination itself was not measured. To evaluate the efficacy of antibiotics against isolates resistant to two or three agents, the efficacy of other antibiotics were evaluated.
Antibiotics against P. aeruginosa
541
Table I. Activity of antibiotics against P. aeruginosa resistant to one drug Number resistant
Antibiotic
25 36 31 39 19 36
Piperacillin Ceftazidime Amikacin Ofloxacin Imipenem Aztreonam Total
176-
Number sensitive to other agents (%) AMK CAZ OFX IPM
PIP
23 (74-2) — 25 (641) 18 (46-2) 9 (47-4) 11 (57-9) 13 (36-1) 26 (72-2)
10(40) 15(60) 3(12) 12 (46-2) 14 (61-5) 4 (15-4) 10 (32-2) 23 (74-2) 21 (67-7) 27 (69-2) 16 (41) — 9 (47-4) 7 (36-8) — 13 (36-1) 26 (72-2) —
73
52
3(12)
4 (15-4) 24 (77-4) 29 (74-3) 9 (47-4) 14 (38-9) 80
—
•Some (trains resistant to more than one agent PIP, Piperacillin; CAZ, ceflazidnne; AMK,
18 (72) 18 (69-2)
AZT
91
105
53
; OFX, ofloxacin; IPM imipenem; AZT, aztreonam.
AZT
IPM
OFX
AMK
CAZ
PIP
40 P*r cant Mnsltivt
Figure 2. Activity of the ax most active antibiotics against P. aeruginosa resistant to one drug. Abbreviations as in Figure 1.
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clinical isolates of P. aeruginosa. Piperacillin plus amikacin inhibited 94-2% of the isolates and amikacin plus ceftazidime or imipenem inhibited 93-3% of the isolates. Aztreonam plus ceftazidime or ofloxacin were least effective, inhibiting only 80-8% of the isolates. The activity of selected antibiotics against isolates resistant to two antibiotics is shown in Table II. Amikacin was most active, inhibiting 70-2% (113/161) of the isolates resistant to two drugs, followed by imipenem which inhibited 61-2% (101/165) of the isolates. Piperacillin, ceftazidime, ofloxacin, and aztreonam inhibited 34-5% (48/139), 33-3% (46/138), 33-8% (44/130), and 26-8% (34/127) of the isolates, respectively (Figure 4). There was cross-resistance between piperacillin, ceftazidime, and aztreonam. Table III shows the activity of antibiotics against isolates resistant to three drugs. Based on the data in Table II, the most effective antibiotic against strains resistant to two drugs was selected as the third antibiotic. The activity of amikacin could not be calculated as amikacin was included in every three-drug combination due to its high
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100
Figure 3. The activity of two drugs against the 120 clinical isolates of P. aeruginosa. Abbreviations as in Figure 1.
efficacy. Among the other five antibiotics, imipenem was most active against isolates resistant to three drugs, inhibiting 56-1% (23/41) of the strains. Ceftazidime, ofloxacin, and aztreonam inhibited 15-6% (7/45), 27-5% (11/40), and 28-3% (13/46) of the isolates, respectively. Discussion In this study, the activity of six antibiotics, piperacillin, ceftazidime, amikacin, ofloxacin, imipenem, and aztreonam against P. aeruginosa resistant to one, two or three Table II. Activity of six antibiotics to P. aeruginosa resistant to two drugs Antibiotic Piperacillin •+• ceftazidime Piperacillin + amikacin Piperacillin + ofloxacin Piperacillin + imipenem Piperacillin + aztreonam Ceftazidime + amikacin Ceftazidime + ofloxacin Ceftazidime + imipenem Ceftazidime + aztreonam Amikacin + ofloxacin Amikacin + imipenem Amikacin + aztreonam Ofloxacin + imipenem Ofloxacin + aztreonam Imipenem 4• aztreonam Total
Number resistant 22 7 15 10 22 8 14 10 23 21 8 10 12 23 10 215-
PIP
2 (25) 1 (71) 1 (10) 3 (13) 17 (81) 4 (50) 5 (50) 5 (41-7) 8 (34-8) 2 (20) 48
"Some strains resistant to more than two agents. Abbreviation* as in Table I.
Number sensitive 1:o other agents (%)i CAZ AMK OFX AZT IPM 16 (72-2) 9(40-9) 13 (591) 2(9-1) 1 (14-3) — 3 (42-9) 3 (42-9) 2 (28-6) 2 (13-3) 11 (73-3) 8 (53-3) 0(0) 1(10) 6(60) 3(30) 2(20) — 2(9-1) 17 (77-3) 7 (31-8) 14 (63-6) — — 3 (77-5) 4(50) 2(25) — 9 (64-2) 6 (42-9) 1 (71) — 6(60) 2(20) 2(20) — 17 (73-9) 10 (43-5) 15 (65-2) 16 (76-2) — — 16 (76-2) 14(66-7) 4(50) — 6(75) 3 (37-5) — 4(40) — — 3(30) 8(80) 4 (33-3) 7 (583) 3(25) — 10 (43-5) 16 (69-6) — 14 (60-9) — 2(20) 8(80) 1(10) — — 46 113 44 101 34
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60 un«itivi
543
Antibiotics against P. aervgiaosa
AZT
40
Figure 4. Activity of the m most active antibiotics against strains of P. aeruginosa resistant to two drags. Abbreviations as in Figure 1.
drugs was determined in order to evaluate cross-resistance to each drug. The differences between cross-resistance to several antibiotics seemed to be based on the differences in the major mechanism of resistance (Jacoby & Archer, 1991). The major mechanism of resistance to /J-lactams found in P. aeruginosa is the expression of /7-lactamase (Medeiros, 1989; Sanders, 1989). In P. aeruginosa, /Mactam resistance is usually mediated by a chromosomal class 1 /Mactamase and/or a plasmidmediated /Mactamase. Resistance to third-generation cephalosporins and ureidopeniT&bte m . Activity of antibiotics against P. aeruginosa resistant to three drugs
Antibiotics Piperacillin + ceftazidime Piperacillin + amikacin Piperacillin + amikacin Piperacillin + ofloxacin Piperacillin + imipenem Piperacillin + aztreonam Ceftazidime + amikacin Ceftazidime + ofloxacin Ceftazidime + imipenem Ceftazidime + aztreonam Amikacin + ofloxacin Amikacin + imipenem Amikacin + aztreonam Ofloxacin + imipenem Ofloxacin + aztreonam Imipenem + aztreonam Total Abbreviations at in Table I.
Number resistant
22 7 7 15 10 22 8 14 10 23 21 8 10 12 23 10 215
Third antibiotic Amikacin Ofloxacin Imipenem Amikacin Amikacin Amikacin Imipenem Amikacin Amikacin Amikacin Piperacillin Aztreonam Imipenem Amikacin Amikacin Amilrnpjn
Total
Number resistant
6 4 4 4 5 5 4 5 4 6 4 2 2 6 7 2 70
Number sensitive to other agents
PIP CAZ OFX IPM AZT — — — — — 1 1 1 1 — 0 0 3 3 0
0 1 0 1 0 — — — — 0 0 0 2 3 0
2 — 2 — 2 1 1 — 1 2 — 0 0 — — 0
3 2 — 2 — 3 — 2 — 4 2 — — — 5 —
1 0 2 0 2 — 2 1 2 — 0 — — 3 — —
10/38 7/45 ii/4o:23/41 13/46
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Ptr cent Mtnttlv*
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cillins usually arises via a mutation that gives rise to constitutive (stably derepressed), instead of inducible, production of chromosomal class 1 /Mactamase. Third-generation cephalosporins, such as ceftazidime, and ureidopenicillins, such as piperacillin, are labile to chromosomal /Mactamase but are weak inducers of its synthesis (Livermore & Yang, 1987), therefore stably depressed mutants from inducible populations can be selected (Livermore, 1987; Sanders & Sanders, 1987). PSE-1 is the most common plasmid-mediated /Mactamase found in P. aeruginosa (Medeiros, 1989). Recently, plasmid-mediated /Mactamases with a broad-spectrum of /Mactam resistance, including the oxyimino-/Mactams such as cefotaxime, ceftazidime, ceftriaxone, and aztreonam, have been reported in a variety of Enterobacteriaceae (Knothe et al., 1983; Kliebe et al., 1985; Jacoby & Carreras, 1990). Recently, a third type of plasmid-mediated, extended-spectrum /Mactamase has appeared (Papanicolaou, Medeiros & Jacoby, 1990). This new enzyme causes resistance to /Mactam antibiotics with a 7o-methoxy group, such as cefoxitin, cefotetan or latamoxef, as well as to oxyimino-/Mactam antibiotics. The data from this study suggest that as there was cross-resistance between piperacillin, ceftazidime, and aztreonam the isolates expressed the chromosomal /Mactamase constitutively. Alternatively, it cannot be ruled out that a plasmid-mediated, extendedspectrum /Mactamase is present in this institution. The major mechanism of resistance to imipenem in P. aeruginosa is the loss of the porin channel, Opr D2 (Trias & Nikaido, 1990) and rarely due to /Mactamase. This difference in the major mechanisms of resistance seems to explain why there was no cross-resistance between imipenem and the other drugs in this study. The major mechanisms of resistance to amikacin found in P. aeruginosa to amikacin are related to both a penetration barrier and to enzymic modification of the drug. Some Gram-negative organisms, especially P. aeruginosa, can become resistant to amikacin by non-enzymatic mechanisms that decrease the general uptake of aminoglycosides (Maloney et al., 1989), or the lipopolysaccharide structure of the cell wall (Bryan, O'Hara & Wong, 1984). Some organisms rely on enzymatic modification by 6'-acetyltransferase (Tran Van Nhieu & Collatz, 1988), 3'-phosphotransferases (Gaynes et al., 1988; Lambert, Gerbaud & Courvalin, 1988; Lambert et al., 1990), or 4'-nudeotidyltransferase (Jacoby et al., 1990) which are usually encoded on plasmid DNA. As these mechanisms of resistance do not confer cross-resistance to unrelated agents, this may explain why there was no cross-resistance between amikacin and the other drugs in this study. The major resistance mechanisms to quinolones present in P. aeruginosa are both an altered target (DNA gyrase) and reduced antibiotic uptake. Some mutants resistant to quinolones have an altered DNA gyrase (Rella & Haas, 1982; Hirai et al., 1987; Piddock & Wise, 1987; Piddock, Wijnands & Wise, 1987). In other mutants, the amount of the outer membrane porin protein is diminished and the accumulation of quinolones is decreased (Piddock et al., 1987). Mutations in DNA gyrase confer resistance only to quinolones, but alterations in the outer-membrane proteins result can cross-resistance to chemically unrelated antibiotics (Piddock et al., 1987). In this study, there was partial cross-resistance between ofloxacin and the other drugs, suggesting that both classes of resistance occurs in our institution. Clinical isolates with similar properties have been previously described (Piddock et al., 1987; Daikos, Lorans & Jackson, 1988), and resistance to quinolones is increasing among P. aeruginosa (Kreken & Wiedemann, 1988).
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Clinically, the choice of appropriate antibiotics for use against resistant strains of P. aeruginosa is a very important issue (Jacoby, 1986). This study clearly demonstrated cross-resistance among piperacillin, ceftazidime, and aztreonam; however, amikacin and imipenem were effective antibiotics, especially as salvage therapy, against P. aeruginosa resistant to one agent. Results also suggested that piperacillin in combination with amikacin, ceftazidime in combination with amikacin, and amikacin in combination with imipenem are effective combination antibiotic regimens against most clinical isolates of P. aeruginosa. Amikacin and imipenen were also particularly effective as salvage therapy, against P. aeruginosa resistant to two agents.
References Bryan, L. E., O'Hara, K. & Wong, S. (1984). lipopolysaccharide changes in impermeability-type aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 26, 250-5. Daikos, G. L., Lolans, V. T. & Jackson, G. G. (1988). Alterations in outer membrane proteins of Pseudomonas aeruginosa associated with selective resistance to quinolones. Antimicrobial Agents and Chemotherapy 32, 785-7. Gaynes, R., Groisman, E., Nelson, E., Casadaban, M. & Lerner, S. A. (1988). Isolation, characterization, and cloning of a plasmid-borne gene encoding a phosphotransferase that confers high-level amikacin resistance in enteric bacilli. Antimicrobial Agents and Chemotherapy 32, 1379-84. Hirai, K., Suzue, S., Irikura, T., Iyobe, S. & Mitsuhashi, S. (1987). Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 30, 248-53. Jacoby, G. A. (1986). Resistance plasmids of Pseudomonas. In The Bacteria: A Treatise on Structure and Function, vol. 10. The Biology o/Pseudomonas (Sokatch, J. R., Ed.), pp. 26593. Academic Press, San Diego. Jacoby, G. A. & Archer, G. L. (1991). New mechanisms of bacterial resistance to antimicrobial agents. New England Journal of Medicine 324, 601-12. Jacoby, G. A., Blaser, M. J., Santanam, P., Hachler, H., Kayser, F. H. & Hare, R. S. (1990). Appearance of ntniVorin and tobramycin resistance due to 4'-aminoglycoside nudeotidyltransferase in Gram-negative pathogens. Antimicrobial Agents and Chemotherapy 34, 2381-6. Jacoby, G. A. & Carreras, I. (1990). Activities of 0-lactam antibiotics against Escherichia coli strains producing extended-spectrum /Mactamases. Antimicrobial Agents and Chemotherapy 34,858-62. Kliebe, C , Nies, B. A. & Meyer, J. F. Tolxdorff-NeutzKng, R. M. & Wiedemann, B. (1985). Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrobial Agents and Chemotherapy 28, 302-7. Knothe, H., Shah, P., Krcmery, V., Antal, M. & Mitsuhashi, S. (1983). Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection 11, 315-7. Kresken, M. & Wiedemann, B. (1988). Development of resistance to nalidixtc acid and the fluoroquinolones after the introduction of norfloxacin and ofloxarin. Antimicrobial Agents and Chemotherapy 32, 1285-8. Lambert, T., Gerbaud, G., Bouvet, P., Vieu, J.-F. & Courvalin, P. (1990). Dissemination of
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Based on the results of this study, the best therapy seemed to be a combination of amikacin and imipenem (efficacy rate 93-3%), salvaged by aztreonam (efficacy rate 98-3%). These three drugs in combination covered 118 of the 120 strains of clinically isolated P. aeruginosa. In conclusion, our results seem helpful in providing useful guidelines for choosing an effective treatment against clinical isolates of P. aeruginosa, and for choosing salvage therapy against resistant strains of P. aeruginosa.
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{Received 24 July 1991; revised version accepted 19 December 1991)
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amikacin resistance gene aphA6 in Acinetobacter spp. Antimicrobial Agents and Chemotherapy 34, 1244-8. Lambert, T., Gerbaud, G. & Courvalin, P. (1988). Transferable amikacin resistance in Acinetobacter spp. due to a new type of 3'-aminoglycoside phosphotransferase. Antimicrobial Agents and Chemotherapy 32, 15-9. Livermore, D. M. (1987). Clinical significance of beta-lactamase induction and stable derepression in Gram-negative rods. European Journal of Clinical Microbiology 6, 439—45. Livermore, D. M. & Yang, Y.-J. (1987). ^-Lactamase lability and inducer power of newer /J-lactam antibiotics in relation to their activity against /Mactamase-inducibility mutants of Pseudomonas aeruginosa. Journal of Infectious Diseases 155, 775-82. Maloney, J., Rimland, D., Stephens, D. S., Terry, P. & Whitney, A. M. (1989). Analysis of amikacin-resistant Pseudomonas aeruginosa developing in patients receiving amikacin. Archives of Internal Medicine 149, 630-4. Medeiros, A. A. (1989). Plasmid-detcrmined beta-lactamases. In Microbial Resistance to Drugs (Bryan, L. E., Ed.), pp. 101-27. Springer-Verlag, Berlin. Papanicolaou, G. A., Medeiros, A. A. & Jacoby, G. A. (1990). Novel plasmid-mediated /f-lactamase (MIR-1) conferring resistance to oxyimino and ct-methoxy /Mactams in clinical isolates of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 34, 2200-9. Piddock, L. J. V., Wijnands, W. J. A. & Wise, R. (1987). Quinolone/ureidopcnicillin crossresistance. Lancet ii, 907. Piddock, L. J. V. & Wise, R. (1987). Characterization of post-therapy isolates of Pseudomonas aeruginosa with decreased susceptibility to enoxacin: evidence for two mechanisms. In Program and Abstracts of the Twenty-Seventh Intersdence Conference on Antimicrobial Agents and Chemotherapy, New York, 1987. Abstract 468. American Society for Microbiology, Washington, DC. Rella, M. & Haas, D. (1982). Resistance of Pseudomonas aeruginosa PAO to nalidixic acid and low levels of /Mactam antibiotics: mapping of chromosomal genes. Antimicrobial Agents and Chemotherapy 22, 242-9. Sanders, C. C. (1989). The chromosomal beta-lactamases. In Microbial Resistance to Drugs (Bryan, L. E., Ed.), pp. 129-49. Springer-Verlag, Berlin. Sanders, C. C. & Sanders, W. E. (1987). Conical importance of inducible beta-lactamase in Gram-negative bacteria. European Journal of Clinical Microbiology 6, 435-8. Tran Van Nhieu, G. & Collatz, E. (1988). Heterogeneity of 6'-N-acetyltransferases of type 4 conferring resistance to amikacin and related aminoglycosides in members of the family Enterobacteriaceae. Antimicrobial Agents and Chemotherapy 32, 1289-91. Trias, J. & Nikaido, H. (1990). Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeurginosa. Antimicrobial Agents and Chemotherapy 34, 52-7.
