Journal of Antimicrobial Chemotherapy (1992) 30, 633-641
Comparative in-vitro activities of newer cephalosporins cefdidin, cefepime, and cefpirome against ceftazidime- or imipenem-resistant Pseudomonas aeruginosa Nao-akl Watanabe, Ryoichl Hinmu and Kanmaai Katsn
Three hundred and thirty-four recent clinical isolates of Pseudomonas aeruginosa were examined for susceptibility to cefdidin, cefepime, and cefpirome. Of these strains, 28-7% were resistant to ceftazidime, and 23-4% were resistant to imipenem. About half of those resistant to ceftazidime were also resistant to imipcmem. Although 37-4% and 59-6% of the isolates were resistant to cefepime and cefpirome respectively, and most or all ceftazidime-resistant strains showed resistance to cefepime and cefpirome, only 3-9% were resistant to cefdidin. Ceftazidime-resistant, but cefdidin-susceptibte, P. aeruginosa produced high levels of chromosomal cephalosporinases. Of the compounds tested, cefdidin showed the lowest affinity for these enzymes, suggesting that its high activity against P. aeruginosa resistant to several ^-lactams was mainly due to its high resistance to enzymatic hydrolysis. Thirteen strains resistant to cefdidin were also resistant to both cefepime and cefpirome, and eight and three of these strains were resistant to ceftazidime and imipenem, respectively. A penicillinase, which hydrolyzed cefdidin, was detected in the cefdidin-resistant P. aeruginosa, which was similar to the OXA-1 /Mactamase.
Introduction Pseudomonas aeruginosa is a clinically significant pathogen, with poor susceptibility to many agents. Although the intrinsic resistance of P. aeruginosa to antibiotics has not yet been clearly denned, it may be due to the poor permeability of the outer membrane (Nikaido, 1985). Resistance to antipseudomonal /Mactam antibiotics has developed during the treatment of P. aeruginosa infections when these agents have been used exclusively. This resistance is caused by high levels of /Mactamase owing to the derepression of a formerly indurible, chromosomally encoded /Mactamase, as seen in ceftazidime-resistant strains (King et al,, 1983; Sanders & Sanders, 1983; Schryvers et al., 1987). In addition, carbapenem-resistance has resulted from the diminished penetration of these agents through the outer membrane owing to the lack of the outer membrane protein D2 (Quinn et al., 1986; Lynch, Drusana & Mobley, 1987; Buscher et al., 1987). These two mechanisms of resistance have been observed not only separately but simultaneously in various clinical isolates of P. aeruginosa (Satake, Yoneyama & Nakae, 1991). Three cephalosporins cefdidin (El040; Watanabe et al., 1988), cefepime (BMY-28142; Phelphs et al., 1986), and cefpirome (HR810; Kobayashi et al., 1986), have increased activity against organisms that overproduce /Mactamase. These 633 0305-7453/92/110633+09 $08.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at New York University on May 24, 2015
Department of Microbiology and Infectious Diseases, Tsukuba Research Laboratories, Eisai Co., Ltd, 1-3 Tokodai 5-chome, Tsukuba, Ibaraki 300-26, Japan
634
N. Watanabe et aL
cephalosporins contain substituents with a quaternary ammonium salt at the 3-position and methoxyimino substituents in the 7/*-side chain. They are apparently little hydrolysed by /Mactamases and have low affinity for /Mactamases and higher rates of permeation through the outer membranes of Gram-negative bacteria (Nikaido, Liu & Rosenburg, 1990). However, comparisons of their activities against ceftazidimc- or imipenem-resistant P. aeruginosa and their relationships to /Mactamase activity have not been reported. In this study, the activity of cefclidin, cefepime, and cefpiromc against clinical isolates of ceftazidime- and imipenem-resistant P. aeruginosa and /Mactamase kinetic parameters were examined.
Antimicrobial agents Cefclidin, cefepime, cefpirome, and nitrocefin were synthesized at Tsukuba Research Laboratories, Eisai Co., Ltd. All other antibacterial agents were obtained commercially. Bacterial strains The strains of P. aeruginosa except one, were originally isolated from human clinical specimens, and were obtained from various hospitals in Japan from 1988 to 1991. Strain E03441 is inducible for chromosomally mediated /Mactamase expression, and susceptible to antipseudomonal cephalosporins and carbapenems. Strain E03441 SKR2/I is a mutant selected in vitro for ceftazidime and imipenem resistance and produces high levels of derepressed /Mactamase and an undetectable amount of outer membrane protein D2. Determination of susceptibility Minimal inhibitory concentrations (MICs) were determined by an agar dilution method with Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, MD, USA). Approximately 5 x 104 cfu per spot were inoculated on to agar plates that contained two-fold serial dilutions of antibiotics. The MIC was the lowest concentration of antibiotic that completely inhibited visible growth after incubation for 18 h at 37°C. MICs > 12-5 mg/L were considered resistant p-Lactamase assay Induction of /Mactamase was achieved by exposure of logarithmic-phase cells to antibiotics for 2 h, as described by Minami et al. (1980). The /Mactamase activity of sonicated extracts prepared from the bacteria harvested from logarithmic-phase cultures was quantified by a spectrophotometric assay (Watanabe et al., 1988), with cephalothin and ampicillin as the substrates. One enzyme unit (U) is defined as the amount of enzyme that hydrolyses 1 umol of cephalothin or ampicillin per minute at 30°C. The Ki values were calculated by the method of Dixon from the rates of cephalothin hydrolysis at various concentrations of inhibitors. The protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin
Downloaded from http://jac.oxfordjournals.org/ at New York University on May 24, 2015
Materials and methods
In-rttro activity of cefdWin against P. oentgbtosa
635
as the standard. Penicillinase from strain E03402 was purified by chromatography on a CM-Sephadex C-50 (Pharmacia LKB Biotechnology, Uppsala, Sweden) ion-exchange column, by chromatofocusing on a PBE94 gel (pH 9-6; Pharmacia), and by Sephadex G-100 (Pharmacia) gel filtration. Isoelectric focusing Isoelectric focusing was performed on an Ampholinc PAG plate (Pharmacia LKB Biotechnology; pH range, 3-5-9-5). /7-Lactamase activity was visualized by overlaying the gel with 1-5% agar containing 50 fig of nitrocefin per mL. An isoekctric-focusing calibration kit (pH 3-10; Pharmacia) was used as controls.
Susceptibility of clinical isolates The in-vitro activities of seven antipseudomonal /7-lactams against 334 clinical isolates of P. aeruginosa are shown in Table I. Of these isolates, 207 (62-0%) and 199 (59-6%) were resistant to cefoperazone or cefpirome, respectively; 125 (37-4%) and 96 (28-7%) were resistant to cefpirime or ceftazidime respectively with an MIC^ of 50 mg/L of ceftazidime; and 78 (23-4%) were resistant to imipenem with an MIC*, of 25 mg/L. About 50% of the isolates resistant either to ceftazidime or to impenem showed resistance to both, and accounted for 12-3% of all isolates tested. Most of the ceftazidime-resistant isolates were also resistant to cefepime or to cefpirome. In contrast, only 13 (3-9%) P. aeruginosa were not inhibited by < 12-5 mg/L cefclidin; all 13 isolates were also resistant to cefepime and cefpirome; eight were ceftazidimeresistant; and 3/8 imipenem-resistant. Although cefclidin was more active against P. aeruginosa, with an MIC^ of 6-25 mg/L, than the other /Mactams tested, its activity was reduced four-fold against the ceftazidime-resistant strains. Characterization of cefclidin-susceptible isolates Six ceftazidime-susceptiblc P. aeruginosa, including three resistant to imipenem, were compared with six ceftazidime-resistant isolates and five isolates resistant to both antibiotics (ceftazidime-imipenem-resistant strains; Table I). The MICs of the /?-lactams tested are shown in Table II. All 17 isolates were susceptible to cefclidin; six
Table L Susceptibility of 334 clinical isolates of P. aeruginosa to seven /7-lactam antibiotics Compound Cefclidin Cefepime Cefpirome Ceftazidime Cefoperazone Cefsulodin Imipcncm
range 0-20-50 0-39-> 100 l-56->100 0-78-> 100 1-56-MOO 0-78-> 100 0-39-100
MIC (mg/L) 50% 0-78 6-25 12-5 3-13 12-5 313 3-13
The MIC breakpoints for resistance were > 12-5 mg/L.
90%
No. of resistant strains' (%)
6-25 25 100 50 >100 50 25
13 (3-9%) 125 (37-4%) 199 (59-6%) 96(28-7%) 207(620%) 119 (35-6%) 78 (23-4%)
Downloaded from http://jac.oxfordjournals.org/ at New York University on May 24, 2015
Results
N. Watanabe et aL
636
Table II. MICs and /J-lactamase activity of cefclidin-susceptible P. aervginosa clinical isolates
Strain
CFCL'
MIC (mg/L) CFP CPO CAZ 3-13 1-56 1-56 1-56 1-56 078 50 25 25 25 50 25 50 25 50 25 100
/J-Lactamase pl(»)
313 1-56 313 25 12-5 12-5
0004 0O02 O001 2000 /*M) were required to inhibit the /7-lactamase than those for cefepime (A; 345-1204 ftti), cefpirome (% 75-7-179/iM), and ceftazidime (A^, 5-36-9-76/ZM). Hydrolysis of cephaloridine, cefclidin, cefepime, cefpirome, and ceftazidime by /Mactamases from these isolates was examined. No significant hydrolysis of cefclidin, cefepime, cefpirome, or ceftazidime was detected; their relative hydrolysis rates were
Comparative in-vitro activities of newer cephalosporins cefdidin, cefepime, and cefpirome against ceftazidime- or imipenem-resistant Pseudomonas aeruginosa Nao-akl Watanabe, Ryoichl Hinmu and Kanmaai Katsn
Three hundred and thirty-four recent clinical isolates of Pseudomonas aeruginosa were examined for susceptibility to cefdidin, cefepime, and cefpirome. Of these strains, 28-7% were resistant to ceftazidime, and 23-4% were resistant to imipenem. About half of those resistant to ceftazidime were also resistant to imipcmem. Although 37-4% and 59-6% of the isolates were resistant to cefepime and cefpirome respectively, and most or all ceftazidime-resistant strains showed resistance to cefepime and cefpirome, only 3-9% were resistant to cefdidin. Ceftazidime-resistant, but cefdidin-susceptibte, P. aeruginosa produced high levels of chromosomal cephalosporinases. Of the compounds tested, cefdidin showed the lowest affinity for these enzymes, suggesting that its high activity against P. aeruginosa resistant to several ^-lactams was mainly due to its high resistance to enzymatic hydrolysis. Thirteen strains resistant to cefdidin were also resistant to both cefepime and cefpirome, and eight and three of these strains were resistant to ceftazidime and imipenem, respectively. A penicillinase, which hydrolyzed cefdidin, was detected in the cefdidin-resistant P. aeruginosa, which was similar to the OXA-1 /Mactamase.
Introduction Pseudomonas aeruginosa is a clinically significant pathogen, with poor susceptibility to many agents. Although the intrinsic resistance of P. aeruginosa to antibiotics has not yet been clearly denned, it may be due to the poor permeability of the outer membrane (Nikaido, 1985). Resistance to antipseudomonal /Mactam antibiotics has developed during the treatment of P. aeruginosa infections when these agents have been used exclusively. This resistance is caused by high levels of /Mactamase owing to the derepression of a formerly indurible, chromosomally encoded /Mactamase, as seen in ceftazidime-resistant strains (King et al,, 1983; Sanders & Sanders, 1983; Schryvers et al., 1987). In addition, carbapenem-resistance has resulted from the diminished penetration of these agents through the outer membrane owing to the lack of the outer membrane protein D2 (Quinn et al., 1986; Lynch, Drusana & Mobley, 1987; Buscher et al., 1987). These two mechanisms of resistance have been observed not only separately but simultaneously in various clinical isolates of P. aeruginosa (Satake, Yoneyama & Nakae, 1991). Three cephalosporins cefdidin (El040; Watanabe et al., 1988), cefepime (BMY-28142; Phelphs et al., 1986), and cefpirome (HR810; Kobayashi et al., 1986), have increased activity against organisms that overproduce /Mactamase. These 633 0305-7453/92/110633+09 $08.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at New York University on May 24, 2015
Department of Microbiology and Infectious Diseases, Tsukuba Research Laboratories, Eisai Co., Ltd, 1-3 Tokodai 5-chome, Tsukuba, Ibaraki 300-26, Japan
634
N. Watanabe et aL
cephalosporins contain substituents with a quaternary ammonium salt at the 3-position and methoxyimino substituents in the 7/*-side chain. They are apparently little hydrolysed by /Mactamases and have low affinity for /Mactamases and higher rates of permeation through the outer membranes of Gram-negative bacteria (Nikaido, Liu & Rosenburg, 1990). However, comparisons of their activities against ceftazidimc- or imipenem-resistant P. aeruginosa and their relationships to /Mactamase activity have not been reported. In this study, the activity of cefclidin, cefepime, and cefpiromc against clinical isolates of ceftazidime- and imipenem-resistant P. aeruginosa and /Mactamase kinetic parameters were examined.
Antimicrobial agents Cefclidin, cefepime, cefpirome, and nitrocefin were synthesized at Tsukuba Research Laboratories, Eisai Co., Ltd. All other antibacterial agents were obtained commercially. Bacterial strains The strains of P. aeruginosa except one, were originally isolated from human clinical specimens, and were obtained from various hospitals in Japan from 1988 to 1991. Strain E03441 is inducible for chromosomally mediated /Mactamase expression, and susceptible to antipseudomonal cephalosporins and carbapenems. Strain E03441 SKR2/I is a mutant selected in vitro for ceftazidime and imipenem resistance and produces high levels of derepressed /Mactamase and an undetectable amount of outer membrane protein D2. Determination of susceptibility Minimal inhibitory concentrations (MICs) were determined by an agar dilution method with Mueller-Hinton agar (BBL Microbiology Systems, Cockeysville, MD, USA). Approximately 5 x 104 cfu per spot were inoculated on to agar plates that contained two-fold serial dilutions of antibiotics. The MIC was the lowest concentration of antibiotic that completely inhibited visible growth after incubation for 18 h at 37°C. MICs > 12-5 mg/L were considered resistant p-Lactamase assay Induction of /Mactamase was achieved by exposure of logarithmic-phase cells to antibiotics for 2 h, as described by Minami et al. (1980). The /Mactamase activity of sonicated extracts prepared from the bacteria harvested from logarithmic-phase cultures was quantified by a spectrophotometric assay (Watanabe et al., 1988), with cephalothin and ampicillin as the substrates. One enzyme unit (U) is defined as the amount of enzyme that hydrolyses 1 umol of cephalothin or ampicillin per minute at 30°C. The Ki values were calculated by the method of Dixon from the rates of cephalothin hydrolysis at various concentrations of inhibitors. The protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin
Downloaded from http://jac.oxfordjournals.org/ at New York University on May 24, 2015
Materials and methods
In-rttro activity of cefdWin against P. oentgbtosa
635
as the standard. Penicillinase from strain E03402 was purified by chromatography on a CM-Sephadex C-50 (Pharmacia LKB Biotechnology, Uppsala, Sweden) ion-exchange column, by chromatofocusing on a PBE94 gel (pH 9-6; Pharmacia), and by Sephadex G-100 (Pharmacia) gel filtration. Isoelectric focusing Isoelectric focusing was performed on an Ampholinc PAG plate (Pharmacia LKB Biotechnology; pH range, 3-5-9-5). /7-Lactamase activity was visualized by overlaying the gel with 1-5% agar containing 50 fig of nitrocefin per mL. An isoekctric-focusing calibration kit (pH 3-10; Pharmacia) was used as controls.
Susceptibility of clinical isolates The in-vitro activities of seven antipseudomonal /7-lactams against 334 clinical isolates of P. aeruginosa are shown in Table I. Of these isolates, 207 (62-0%) and 199 (59-6%) were resistant to cefoperazone or cefpirome, respectively; 125 (37-4%) and 96 (28-7%) were resistant to cefpirime or ceftazidime respectively with an MIC^ of 50 mg/L of ceftazidime; and 78 (23-4%) were resistant to imipenem with an MIC*, of 25 mg/L. About 50% of the isolates resistant either to ceftazidime or to impenem showed resistance to both, and accounted for 12-3% of all isolates tested. Most of the ceftazidime-resistant isolates were also resistant to cefepime or to cefpirome. In contrast, only 13 (3-9%) P. aeruginosa were not inhibited by < 12-5 mg/L cefclidin; all 13 isolates were also resistant to cefepime and cefpirome; eight were ceftazidimeresistant; and 3/8 imipenem-resistant. Although cefclidin was more active against P. aeruginosa, with an MIC^ of 6-25 mg/L, than the other /Mactams tested, its activity was reduced four-fold against the ceftazidime-resistant strains. Characterization of cefclidin-susceptible isolates Six ceftazidime-susceptiblc P. aeruginosa, including three resistant to imipenem, were compared with six ceftazidime-resistant isolates and five isolates resistant to both antibiotics (ceftazidime-imipenem-resistant strains; Table I). The MICs of the /?-lactams tested are shown in Table II. All 17 isolates were susceptible to cefclidin; six
Table L Susceptibility of 334 clinical isolates of P. aeruginosa to seven /7-lactam antibiotics Compound Cefclidin Cefepime Cefpirome Ceftazidime Cefoperazone Cefsulodin Imipcncm
range 0-20-50 0-39-> 100 l-56->100 0-78-> 100 1-56-MOO 0-78-> 100 0-39-100
MIC (mg/L) 50% 0-78 6-25 12-5 3-13 12-5 313 3-13
The MIC breakpoints for resistance were > 12-5 mg/L.
90%
No. of resistant strains' (%)
6-25 25 100 50 >100 50 25
13 (3-9%) 125 (37-4%) 199 (59-6%) 96(28-7%) 207(620%) 119 (35-6%) 78 (23-4%)
Downloaded from http://jac.oxfordjournals.org/ at New York University on May 24, 2015
Results
N. Watanabe et aL
636
Table II. MICs and /J-lactamase activity of cefclidin-susceptible P. aervginosa clinical isolates
Strain
CFCL'
MIC (mg/L) CFP CPO CAZ 3-13 1-56 1-56 1-56 1-56 078 50 25 25 25 50 25 50 25 50 25 100
/J-Lactamase pl(»)
313 1-56 313 25 12-5 12-5
0004 0O02 O001 2000 /*M) were required to inhibit the /7-lactamase than those for cefepime (A; 345-1204 ftti), cefpirome (% 75-7-179/iM), and ceftazidime (A^, 5-36-9-76/ZM). Hydrolysis of cephaloridine, cefclidin, cefepime, cefpirome, and ceftazidime by /Mactamases from these isolates was examined. No significant hydrolysis of cefclidin, cefepime, cefpirome, or ceftazidime was detected; their relative hydrolysis rates were
