Journal of Antimicrobial Chemotherapy (1992) 30, 229-242
Correspondence Actirhy of amoxydHln davnlank/add against Escherichia coS in riro J Amimicrob Chemolher 1992; 30: 229
CHARLES E. CHERUBIN Department of Veterans Affairs, Medical Center, IIU East End Boulevard WilkesBarre. PA 18711, USA
References Bcale, A. S. (1992). Activity of amoxycillin/ clavulanic acid against Escherichia coli in vivo—correction. Journal of Antimicrobial Chemotherapy 30, 108. Cherubin, C. E., Eng, R. H. K., Smith, S. M. & Tan, E. N. (1991). An in-vitro and in-vivo comparison of the activity of /Mactamase inhibitor combinations with imipenem and cephalosporins against Escherichia coli producing TEM-1 or TEM-2 /7-lactamase. Journal of Antimicrobial Chemotherapy 28, 61-70. Gisby, J. &. Beale, A. S. (1988). Comparative efficacies of amoxycillin-clavulanic acid and ampicillin-sulbactam against experimental Bacleroides fragilis-Escherichia coli mixed infections. Antimicrobial Agents and Chemotherapy 32, 1830-3.
High-ferel qainolone resfatance in Pseudomtmas aermgutosa J Amimicrob Chemolher 1992; 30: 229-231 Sir, Fluorinated quinolones such as dprofloxadn achieve high concentrations in the urine and are recommended for the treatment of urinary tract infections (UT1) caused by Gram-negative bacteria including Pseudomonas aeruginosa (Wolfson & Hooper, 1985). Recently, a 54 year old woman with 25 year history of multiple sclerosis was admitted to Leeds General Infirmary. She had required a urinary catheter for 15 years and had had repeated UTls during this period.
229
Downloaded from http://jac.oxfordjournals.org/ at The Univesity of Calgary on May 27, 2015
Sir, We are distressed that we have aroused Dr Beale's criticism (Beak, 1992) because we consider her and Gisby's pap" (Gisby & Beale, 1988) a very important contribution to the evolving interpretation of our present methods of susceptibility testing. To the first point of her letter we plead nob contenaere, for which I must take full responsibility. Hers is a mouse thigh model as is ours. I wrote and intended to say although it was lost in retyping the manuscript, 'a model of a mixed aerobic/anaerobic abdominal abscesstype subcutaneous infection in mice'. On the second point, however, we must differ. In our paper (Cherubin et at., 1991) we found only minor differences between the activity of ampicillin/sulbactam and amoxycillin/clavulanic acid against strains of Escherichia coli producing large quantities of TEM-1 and 2. There was, in vitro, at most a 2 to 1 ratio in favour of amoxycillin/clavulanic acid. Similar findings were demonstrated by Gisby & Bcale (1988). With the low level /J-lactamase-producing E. coli, E 96, both E.coli and Bacteroides fragilis were nearly eliminated from the abscesses by both combination agents. With E. coli 41548, the high producer, the reduction in the abscess flora was markedly different. While both combinations reduced the B. fragilis count by 3 logs, the reduction in E. coli cfu/mL was far slower though greater with the amoxycillin/clavulanic acid combination, in particular at a high inoculum. At low inocula doubling the ampicillin/sulbactam dose rendered both combinations equivalent in terms of cfu/mL reduction for both bacterial components of the abscess. This is not too different from what was suggested from the in-vitro data and is the reason we found support in Gisby and Beale's publication. Of course in an in-vivo model the much longeT half-life of amoxycillin gives it a particular advantage. Lastly, our own mouse thigh model data showed little reduction of the high TEM-1
producing E. coli strain with either combination, especially as compared to the two cephalosporins also tested. Notwithstanding the comments of Dr Beale, we feel our data and hers (as presented in her paper of 1988) show clearly that both combination products have reduced in-vitro and in-vivo activity against high level-lactamase producing strains of Gram-negative bacteria.
230
Correspondence T«Me. Susceptibility of P. aeruginosa strains
Strain NCTC 10662 Clinical isolate (G1I0) E. coli (pNJR3-2) E. coli (pLA2917) G110xpLA2917 GI10xpNJR3-2
CIP > < < >
1024 < 012 1024 0-5
MIC (mg/L) NOR NAL 4 > 1024 < 0-12 1024 0-5
SI2 >2048 4 2 >2048 32
CAZ
GENT
4 4 0-5 0-5 4 2
4 2 0-5 05
2 1
CIP, Ciprofloxacin; OFX, ofloxacin; NOR. norfioxacin; NAL, nalidixic acid; CAZ, ceftazidime: GENT, gentamicin.
resistant isolate and the transconjugates showed that the plasmids pNJR3-2 or pLA29127 were inserted. No plasmids were detected in the original isolate. The data from this study suggest that mutations in gyrA alone were responsible for the high levels of quinolone resistance in this clinical isolate, as resistance was completely reversed by the insertion of a quinolone susceptible gyrA. Interestingly, the MICs of the quinolones for the transconjugates were lower than for a susceptible P. aeruginosa, NCTC 10662, or for a strain of P. aeruginosa with a gyrA mutation and the plasmid inserted (Robillard, 1990). Other possible mechanisms of quinolone resistance were not investigated after obtaining the above data. Resistance to fluoroquinolones is typically associated with a mutation in gyrA, but it has not been reported to give rise to the level of resistance observed in the isolate in this study (Piddock & Wise, 1989). Clearly, the appearance and development of resistance to this level in other bacteria would substantially undermine the clinical effectiveness of the quinolones. It would be interesting to sequence the DNA of gyrA from this isolate to determine whether there are multiple mutations in this gene conferring the high-level resistance. L. J. V. PIDDOCK' S. PANCHAL' T. J. J. INGLIS* P. M. HAWKEY* 'Antimicrobial Agents Research Group, Department of Infection, University of Birmingham, Birmingham, BIS 2TT; ''Department of Microbiology, The University of Leeds. Leeds LS2 9JT. UK
References Robillard, N. J. (1990). Broad host range gyrase A gene probe. Antimicrobial Agents and Chemotherapy 34, 1889-94. Piddock, L. J. V. & Wise, R. (1989). Mechanisms of
Downloaded from http://jac.oxfordjournals.org/ at The Univesity of Calgary on May 27, 2015
Before admission she had been receiving 250 mg ciprofloxacin tds and 500 mg cephradine qds for a bacteriologically unconfirmed UTI. A catheter specimen taken on admission yielded > 103 cfu/mL P. aeruginosa (G110), which was resistant to ciprofloxacin. The UTI was successfully eradicated by bladder washouts. The MICs of ciprofloxacin, ofloxacin, norfioxacin and nalidixic acid for P. aeruginosa G110 were determined both in Leeds and in Birmingham on Iso-Sensitest agar using a standard doubling dilution procedure. The isolate was found to be highly resistant to all quinolones, but retained susceptibility to ceftazidime and gentamicin (Table). Quinolone resistance in P. aeruginosa usually results in MICs of 8-64 mg/L, and so the mechanism of resistance giving rise to the exceptionally high MICs was investigated. To determine whether the isolate contained a mutation in gyrA, giving rise to decreased affinity of DNA gyrase for the quinolone, a plasmid (pNJR3-2; 'gene probe') containing wild-type quinolone-susceptible gyrA from Escherichia coli was transferred by conjugation into the resistant isolate (Robillard, 1990). When this plasmid is inserted into bacteria containing a mutation in gyrA. the plasmidcncoded DNA gyrase is produced so that the strain becomes more susceptible to quinolones; for strains with quinolone resistance due to another mechanism the susceptibility does not change. For all ten putative transconjugates of Gl 10 and the gene probe the MICs of the four quinolones were reduced, whereas when the control plasmid (pLA2917, lacking gyrA) was inserted there was no change in the MIC. All transconjugates had the typical colonial morphology of the original isolate, were oxidase-positive and carried the resistance markers encoded on the plasmid (tetr, lean1). Plasmid profiles of the DNA extracted from E. coli (pNJR3-2), E. coli (pLA2917), the
231
Correspondence resistance to quinolones and clinical perspectives. Journal of Antimicrobial Chemotherapy 23, 475-80. Wolfcon. J. S. & Hooper. D. C. (1985). The fluoroquinolones: pharmacology, clinical uses, and toxicittes in humans. Antimicrobial Agents and Chemotherapy 28, 716-21. The post-antibiotic effect of imipenem and penfcniin-bioding protein 2
J Antimicrob Chemother 1992; 30: 231-232
TiMe. MICs and PAEs of imipenem and mecillinam (in concentrations of 4 x and 8x MIC) against four strains of P- aeruginosa Imipenem
MIC Strain ATCC 27853 ATCC 94966 A 6804 A 450
PAE (h)
(mg/L)
x4'
x8
2-0 1-0 1-0 2-0
1-2 1-4 20 1-3
1-2 1-8 1-6 1-4
•Concentration of drug in multiples of MIC.
Mecillinam MIC (Mg/L) 512 32 512 512
PAE (h) x4
x8
-03 02 -02 00
-01 O6 -03 -02
Downloaded from http://jac.oxfordjournals.org/ at The Univesity of Calgary on May 27, 2015
Sir, The mechanism of the post-antibiotic effect (PAE) has not been defined, but may involve limited persistence of the antibiotic at the antimicrobial site of action, the time of re-synthesis of essential proteins (e.g. penicillin binding proteins (PBPs)) after a non-lethal drug-induced damage (Craig & Gudmundsson, 1991), or even production of endogeneous growth suppressive factors, similar to the SOS response observed in Escherichia coli after exposure to ultraviolet light (Walker, 1987). The penem antibiotics (imipenem, meropenem) are unique among the /J-lactams in inducing a PAE against Gram-negative bacilli, particularly Pseudomonas aeruginosa (Bustamante et aU 1984; Baquero el a/., 1986; Gudmundsson, Vogelman & Craig, 1986; Nadler et aU 1989; Odenholt et aU 1989). The penems bind preferentially to PBP 2 of Gramnegative bacilli (Hashizume et a/., 1984), whereas other 0-lactams bind primarily to PBP la, PBP lb and PBP 3 (Iida et al^ 1982). It has therefore been suggested that binding to PBP 2 may be involved in the mechanism of the PAE induced by these agents. Gould, Jason & Milne (1989), by measuring PAE by electrical conductance, noted that mecillinam, which also is a preferential PBP 2 binder, induced a brief PAE ( < O 6 h ) in four of six strains of E. coli tested. In contrast.
Majcherczyk & Livermore (1990) recently demonstrated no significant difference in the duration of the PAEs induced by carbapenems in a E. coli strain and its PBP 2 deficient mutant. However, P. aeruginosa was not examined in this regard by either group of investigators. We had investigated this hypothesis earlier by comparing the duration of the PAEs induced by imipenem and mecillinam against strains of P. aeruginosa (Erlendsdottir & Gudmundsson, 1988). Two standard strains (ATCC 27853 and ATCC 94966) and two clinical strains (A 6804 and A 450) were exposed to either agent for 1 h in concentrations of 4 x and 8 x MIC. The drugs were removed by 10~ 3 dilution of the exposed cultures; the control organisms were similarly diluted. The PAE was measured in standard fashion (Craig & Gudmundsson, 1991) by comparing regrowth curves determined by serial viable counts of exposed and unexposed control organisms. In each experiment, an additional 'drug control' was employed by inoculating a 10" 3 dilution of unexposed organisms into fresh broth, into which a 10" 3 dilution of the highest drug concentration had been made. A 'drug control' growing at the same rate as the unexposed control assured that the PAEs observed were not due to residual subinhibitory levels of drug. The bactericidal activity of the agents during the hour of exposure was comparable, ranging from 0-3-1-0 log, 0 cfu/mL/h, depending on bacterial strain. The MICs and the duration of the PAEs induced by the two agents are summarized in the Table. The PAEs of imipenem ranged from 1-2 to 2-0 h, whereas only short or even negative PAEs were observed after mecillinam. Similarly, the PAEs of mecillinam against E. coli observed by Gould et al. (1989) and quoted earlier were of short duration and clinically insignificant Furthermore,, the authors
Correspondence Actirhy of amoxydHln davnlank/add against Escherichia coS in riro J Amimicrob Chemolher 1992; 30: 229
CHARLES E. CHERUBIN Department of Veterans Affairs, Medical Center, IIU East End Boulevard WilkesBarre. PA 18711, USA
References Bcale, A. S. (1992). Activity of amoxycillin/ clavulanic acid against Escherichia coli in vivo—correction. Journal of Antimicrobial Chemotherapy 30, 108. Cherubin, C. E., Eng, R. H. K., Smith, S. M. & Tan, E. N. (1991). An in-vitro and in-vivo comparison of the activity of /Mactamase inhibitor combinations with imipenem and cephalosporins against Escherichia coli producing TEM-1 or TEM-2 /7-lactamase. Journal of Antimicrobial Chemotherapy 28, 61-70. Gisby, J. &. Beale, A. S. (1988). Comparative efficacies of amoxycillin-clavulanic acid and ampicillin-sulbactam against experimental Bacleroides fragilis-Escherichia coli mixed infections. Antimicrobial Agents and Chemotherapy 32, 1830-3.
High-ferel qainolone resfatance in Pseudomtmas aermgutosa J Amimicrob Chemolher 1992; 30: 229-231 Sir, Fluorinated quinolones such as dprofloxadn achieve high concentrations in the urine and are recommended for the treatment of urinary tract infections (UT1) caused by Gram-negative bacteria including Pseudomonas aeruginosa (Wolfson & Hooper, 1985). Recently, a 54 year old woman with 25 year history of multiple sclerosis was admitted to Leeds General Infirmary. She had required a urinary catheter for 15 years and had had repeated UTls during this period.
229
Downloaded from http://jac.oxfordjournals.org/ at The Univesity of Calgary on May 27, 2015
Sir, We are distressed that we have aroused Dr Beale's criticism (Beak, 1992) because we consider her and Gisby's pap" (Gisby & Beale, 1988) a very important contribution to the evolving interpretation of our present methods of susceptibility testing. To the first point of her letter we plead nob contenaere, for which I must take full responsibility. Hers is a mouse thigh model as is ours. I wrote and intended to say although it was lost in retyping the manuscript, 'a model of a mixed aerobic/anaerobic abdominal abscesstype subcutaneous infection in mice'. On the second point, however, we must differ. In our paper (Cherubin et at., 1991) we found only minor differences between the activity of ampicillin/sulbactam and amoxycillin/clavulanic acid against strains of Escherichia coli producing large quantities of TEM-1 and 2. There was, in vitro, at most a 2 to 1 ratio in favour of amoxycillin/clavulanic acid. Similar findings were demonstrated by Gisby & Bcale (1988). With the low level /J-lactamase-producing E. coli, E 96, both E.coli and Bacteroides fragilis were nearly eliminated from the abscesses by both combination agents. With E. coli 41548, the high producer, the reduction in the abscess flora was markedly different. While both combinations reduced the B. fragilis count by 3 logs, the reduction in E. coli cfu/mL was far slower though greater with the amoxycillin/clavulanic acid combination, in particular at a high inoculum. At low inocula doubling the ampicillin/sulbactam dose rendered both combinations equivalent in terms of cfu/mL reduction for both bacterial components of the abscess. This is not too different from what was suggested from the in-vitro data and is the reason we found support in Gisby and Beale's publication. Of course in an in-vivo model the much longeT half-life of amoxycillin gives it a particular advantage. Lastly, our own mouse thigh model data showed little reduction of the high TEM-1
producing E. coli strain with either combination, especially as compared to the two cephalosporins also tested. Notwithstanding the comments of Dr Beale, we feel our data and hers (as presented in her paper of 1988) show clearly that both combination products have reduced in-vitro and in-vivo activity against high level-lactamase producing strains of Gram-negative bacteria.
230
Correspondence T«Me. Susceptibility of P. aeruginosa strains
Strain NCTC 10662 Clinical isolate (G1I0) E. coli (pNJR3-2) E. coli (pLA2917) G110xpLA2917 GI10xpNJR3-2
CIP > < < >
1024 < 012 1024 0-5
MIC (mg/L) NOR NAL 4 > 1024 < 0-12 1024 0-5
SI2 >2048 4 2 >2048 32
CAZ
GENT
4 4 0-5 0-5 4 2
4 2 0-5 05
2 1
CIP, Ciprofloxacin; OFX, ofloxacin; NOR. norfioxacin; NAL, nalidixic acid; CAZ, ceftazidime: GENT, gentamicin.
resistant isolate and the transconjugates showed that the plasmids pNJR3-2 or pLA29127 were inserted. No plasmids were detected in the original isolate. The data from this study suggest that mutations in gyrA alone were responsible for the high levels of quinolone resistance in this clinical isolate, as resistance was completely reversed by the insertion of a quinolone susceptible gyrA. Interestingly, the MICs of the quinolones for the transconjugates were lower than for a susceptible P. aeruginosa, NCTC 10662, or for a strain of P. aeruginosa with a gyrA mutation and the plasmid inserted (Robillard, 1990). Other possible mechanisms of quinolone resistance were not investigated after obtaining the above data. Resistance to fluoroquinolones is typically associated with a mutation in gyrA, but it has not been reported to give rise to the level of resistance observed in the isolate in this study (Piddock & Wise, 1989). Clearly, the appearance and development of resistance to this level in other bacteria would substantially undermine the clinical effectiveness of the quinolones. It would be interesting to sequence the DNA of gyrA from this isolate to determine whether there are multiple mutations in this gene conferring the high-level resistance. L. J. V. PIDDOCK' S. PANCHAL' T. J. J. INGLIS* P. M. HAWKEY* 'Antimicrobial Agents Research Group, Department of Infection, University of Birmingham, Birmingham, BIS 2TT; ''Department of Microbiology, The University of Leeds. Leeds LS2 9JT. UK
References Robillard, N. J. (1990). Broad host range gyrase A gene probe. Antimicrobial Agents and Chemotherapy 34, 1889-94. Piddock, L. J. V. & Wise, R. (1989). Mechanisms of
Downloaded from http://jac.oxfordjournals.org/ at The Univesity of Calgary on May 27, 2015
Before admission she had been receiving 250 mg ciprofloxacin tds and 500 mg cephradine qds for a bacteriologically unconfirmed UTI. A catheter specimen taken on admission yielded > 103 cfu/mL P. aeruginosa (G110), which was resistant to ciprofloxacin. The UTI was successfully eradicated by bladder washouts. The MICs of ciprofloxacin, ofloxacin, norfioxacin and nalidixic acid for P. aeruginosa G110 were determined both in Leeds and in Birmingham on Iso-Sensitest agar using a standard doubling dilution procedure. The isolate was found to be highly resistant to all quinolones, but retained susceptibility to ceftazidime and gentamicin (Table). Quinolone resistance in P. aeruginosa usually results in MICs of 8-64 mg/L, and so the mechanism of resistance giving rise to the exceptionally high MICs was investigated. To determine whether the isolate contained a mutation in gyrA, giving rise to decreased affinity of DNA gyrase for the quinolone, a plasmid (pNJR3-2; 'gene probe') containing wild-type quinolone-susceptible gyrA from Escherichia coli was transferred by conjugation into the resistant isolate (Robillard, 1990). When this plasmid is inserted into bacteria containing a mutation in gyrA. the plasmidcncoded DNA gyrase is produced so that the strain becomes more susceptible to quinolones; for strains with quinolone resistance due to another mechanism the susceptibility does not change. For all ten putative transconjugates of Gl 10 and the gene probe the MICs of the four quinolones were reduced, whereas when the control plasmid (pLA2917, lacking gyrA) was inserted there was no change in the MIC. All transconjugates had the typical colonial morphology of the original isolate, were oxidase-positive and carried the resistance markers encoded on the plasmid (tetr, lean1). Plasmid profiles of the DNA extracted from E. coli (pNJR3-2), E. coli (pLA2917), the
231
Correspondence resistance to quinolones and clinical perspectives. Journal of Antimicrobial Chemotherapy 23, 475-80. Wolfcon. J. S. & Hooper. D. C. (1985). The fluoroquinolones: pharmacology, clinical uses, and toxicittes in humans. Antimicrobial Agents and Chemotherapy 28, 716-21. The post-antibiotic effect of imipenem and penfcniin-bioding protein 2
J Antimicrob Chemother 1992; 30: 231-232
TiMe. MICs and PAEs of imipenem and mecillinam (in concentrations of 4 x and 8x MIC) against four strains of P- aeruginosa Imipenem
MIC Strain ATCC 27853 ATCC 94966 A 6804 A 450
PAE (h)
(mg/L)
x4'
x8
2-0 1-0 1-0 2-0
1-2 1-4 20 1-3
1-2 1-8 1-6 1-4
•Concentration of drug in multiples of MIC.
Mecillinam MIC (Mg/L) 512 32 512 512
PAE (h) x4
x8
-03 02 -02 00
-01 O6 -03 -02
Downloaded from http://jac.oxfordjournals.org/ at The Univesity of Calgary on May 27, 2015
Sir, The mechanism of the post-antibiotic effect (PAE) has not been defined, but may involve limited persistence of the antibiotic at the antimicrobial site of action, the time of re-synthesis of essential proteins (e.g. penicillin binding proteins (PBPs)) after a non-lethal drug-induced damage (Craig & Gudmundsson, 1991), or even production of endogeneous growth suppressive factors, similar to the SOS response observed in Escherichia coli after exposure to ultraviolet light (Walker, 1987). The penem antibiotics (imipenem, meropenem) are unique among the /J-lactams in inducing a PAE against Gram-negative bacilli, particularly Pseudomonas aeruginosa (Bustamante et aU 1984; Baquero el a/., 1986; Gudmundsson, Vogelman & Craig, 1986; Nadler et aU 1989; Odenholt et aU 1989). The penems bind preferentially to PBP 2 of Gramnegative bacilli (Hashizume et a/., 1984), whereas other 0-lactams bind primarily to PBP la, PBP lb and PBP 3 (Iida et al^ 1982). It has therefore been suggested that binding to PBP 2 may be involved in the mechanism of the PAE induced by these agents. Gould, Jason & Milne (1989), by measuring PAE by electrical conductance, noted that mecillinam, which also is a preferential PBP 2 binder, induced a brief PAE ( < O 6 h ) in four of six strains of E. coli tested. In contrast.
Majcherczyk & Livermore (1990) recently demonstrated no significant difference in the duration of the PAEs induced by carbapenems in a E. coli strain and its PBP 2 deficient mutant. However, P. aeruginosa was not examined in this regard by either group of investigators. We had investigated this hypothesis earlier by comparing the duration of the PAEs induced by imipenem and mecillinam against strains of P. aeruginosa (Erlendsdottir & Gudmundsson, 1988). Two standard strains (ATCC 27853 and ATCC 94966) and two clinical strains (A 6804 and A 450) were exposed to either agent for 1 h in concentrations of 4 x and 8 x MIC. The drugs were removed by 10~ 3 dilution of the exposed cultures; the control organisms were similarly diluted. The PAE was measured in standard fashion (Craig & Gudmundsson, 1991) by comparing regrowth curves determined by serial viable counts of exposed and unexposed control organisms. In each experiment, an additional 'drug control' was employed by inoculating a 10" 3 dilution of unexposed organisms into fresh broth, into which a 10" 3 dilution of the highest drug concentration had been made. A 'drug control' growing at the same rate as the unexposed control assured that the PAEs observed were not due to residual subinhibitory levels of drug. The bactericidal activity of the agents during the hour of exposure was comparable, ranging from 0-3-1-0 log, 0 cfu/mL/h, depending on bacterial strain. The MICs and the duration of the PAEs induced by the two agents are summarized in the Table. The PAEs of imipenem ranged from 1-2 to 2-0 h, whereas only short or even negative PAEs were observed after mecillinam. Similarly, the PAEs of mecillinam against E. coli observed by Gould et al. (1989) and quoted earlier were of short duration and clinically insignificant Furthermore,, the authors
