Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Parameters Characterizing the In Vitro Activity of Cefixime, a New Oral Broad Spectrum Cephalosporin, Against Respiratory and Urinary Pathogens E.A. Debbia, A. Marchese, A. Pesce, D. Saverino & G.C. Schito To cite this article: E.A. Debbia, A. Marchese, A. Pesce, D. Saverino & G.C. Schito (1992) Parameters Characterizing the In Vitro Activity of Cefixime, a New Oral Broad Spectrum Cephalosporin, Against Respiratory and Urinary Pathogens, Journal of Chemotherapy, 4:3, 131-144, DOI: 10.1080/1120009X.1992.11739153 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739153
Published online: 15 Jul 2016.
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Citing articles: 2 View citing articles
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Date: 13 September 2016, At: 00:59
Journal of Chemotherapy
Parameters Characterizing the In Vitro Activity of Cefixime, a New Oral Broad Spectrum Cephalosporin, Against Respiratory and Urinary Pathogens E.A. DEBBIA - A. MARCHESE - A. PESCE D. SAVERINO - G.C. SCHITO
Vol. 4 - n. J (131-144) - 1992
ary strains was optimal at cefixime concentrations reached in urine since eradication, except for Proteus mirabilis, was enhanced with increasing levels of the drug. The absence of an untoward paradoxic effect on the rate of cefixime bactericidal action was confirmed by employing a dynamic bladder model simulating the pharmacokinetic parameters of the drug after a single 200 mg daily dosage. Interactions of cefixime with several other drugs that may be employed in combination therapy were generally prone to provide indifference and synergism while antagonism was never observed. Favorable interactions were also registered when cefixime acted with other antibiotics on partially resistant species such as Staphylococci and Pseudomonas. The new cephem seems to provide excellent opportunities for expanding oral cephalosporin therapy to a wide range of infections produced by susceptible pathogens in the adult and pediatric populations. Key words: cefixime, in vitro activity, bactericidal effect, Eagle's effect, activity in bladder model, interaction with other drugs, cephalosporin.
Summary ----------------------------------
INTRODUCTION
The wide and potent in vitro activity of cefixime, a new oral broad spectrum cephalosporin, has been confirmed on a collection of respiratory and urinary pathogens recently isolated in Italy. The new cephem emerged as the most bactericidal of all the comparators tested against several fast as well as slowly-growing gram-negative species including Enterobacteria, Haemophilus and Moraxella, irrespective of their ability to synthetize P-lactamases. Among the gram-positive species Streptococcus pyogenes and S. pneumoniae were effectively covered. Cefixime activity was not adversely influenced by several important variables such as pH (over the range from 5 to 8), inoculum size (from 10' to 10 1 CFU per ml) and the presence of 50% human serum or urine. Time-kill tests confirmed a pronounced bactericidal potency of the drug especially towards common respiratory pathogens (H. influenzae, M. catarrhalis, S. pneumoniae and S. pyogenes). Killing of urin-
Cefixime (FK 027, FR 17027, CL 284635) is a new oral cephalosporin characterized by the presence of a vinyl group at the three position which promotes effective gut absorption, and by an aminothiazolyl substitution that confers a broad antimicrobial spectrum and a high degree of P-lactamase stability 1 • In previous studies cefixime was shown to inhibit a wide variety of gram-positive and gram-negative aerobic bacteria including members of the Enterobacteriaceae family, Hae-
Institute of Microbiology, University of Genoa Medical School, Genoa, Italy. Correspondence to: E.A. Debbia, Institute of Microbiology, University of Genoa, Medical School, Viale Benedetto XV, 10, 16132 Genoa, Italy © Edizioni Riviste Scientifiche - Firenze
mophilus in/luenzae, Streptococcus pyogenes, S. pneumoniae, and Moraxella catarrhalis. Activity against staphylococci, enterococci and Morganella morganii was poor and Pseudomonas species were generally resistant 2 • 3 • 4 • The antibacterial potency of this new oral cephem was only marginally influenced by the ISSN 1120-009X
E.A. DEBBIA - A. MARCHESE - A. PESCE - D. SAVERINO - G.C. SCHITO
132
presence of serum proteins, pH variations, media composition, bacterial inoculum size and growth conditions of the test organisms. Overall, this behavior renders the cefixime profile similar to that shown by parenteral third generation cephalosporins 3 • 5 • Given its spectrum of bactericidal activity and its pharmacokinetic parameters that allow for sustained levels in the relevant tissues and secretions, this new drug seems best suited for the treatment of respiratory and urinary tract infections both in the adult and pediatric populations 6 - 8 • This study has been designed to answer several important questions that are related to the introduction of cefixime in general therapy in this country. Due to wide geographic variations in the epidemiology of bacterial resistance it was deemed essential to confirm the in vitro efficacy of this new cephalosporin on a collection of urinary and respiratory pathogens freshly isolated from in and outpatients and representing the challenge that cefixime will meet in this region. Its bactericidal activity was also scrutinized under various experimental conditions. These tests included the assessment of the extent and rate of killing as a function of the concentration reached in the urine where cefixime becomes highly concentrated. Bactericidal potency may in fact be untowardly affected by the presence of a paradoxic Eagle's effect. The bactericidal activity of the new cephem in an in vitro model simulating its pharmacokinetics in the urinary bladder was also assessed. Finally, the outcome of the interactions of cefixime with several other drugs that may be employed on those rare but possible occasions where combination therapy may be deemed essential was determined. MATERIALS AND METHODS
Microorganisms All strains were freshly isolated from clinical specimens from patients suffering from respiratory and urinary tract infections and identified in the diagnostic laboratory of this institution by conventional methods. Though not selected, the collection employed included organisms exhibiting resistance to one or more of the antibiotics tested, and was representative of the epidemiologic situation prevailing 'in Italy.
Antibiotics Cefixime was obtained from Menarini, Firenze, Italy. Cefuroxime, amoxicillin, amoxicillin-clavulanic acid (amox-clav), cefaclor, fosfomycin, netilmicin, ciprofloxacin, vancomycin, teicoplanin, rifampin, and clarithromycin were supplied from their respective manufacturers. Sterile stock solutions of the antibiotics were prepared from the standard reference powders in accordance with the instructions provided. For the detection of beta-lactamase activity nitrocefin paper disks (BBL) were used.
Susceptibility tests under various conditions Minimal inhibitory concentrations (MICs) of cefixime and of the other drugs were determined by using the methods suggested by the NCCLS (National Committee for Clinical Laboratory Standards) 9 , and as previously described 10 - 12 • Organisms for quality control were included in each assay. Exponentially growing bacteria (5 X 10 5 cells per ml, final inoculum) were added to the various concentrations of antibiotics, diluted in cation-supplemented Mueller-Hinton broth and distributed on a microplate by using the automatic Biomek 1000 dilutor (Beckman, USA) . After 18-24 hours of incubation at 37°C the antibiotic concentration which prevented a visible bacterial growth was recorded as the MIC. For tests performed with Streptococcus strains, 5% defibrinated horse blood was added to the Mueller-Hinton medium, whereas for evaluation of Haemophilus strains, Haemophilus Test Medium (HTM) 13 was employed. M. catarrhalis was tested on chocolate agar medium and incubated in an atmosphere of 5% carbon dioxide . The effect of serum on the activity of cefixime was evaluated by employing pooled normal human serum combined with Mueller-Hinton medium to yield a 50% final concentration. To assess possible changes in the activity of cefixime due to modification of the pH of the medium, Mueller-Hinton broth was buffered with 0.01 M phosphate (pH 7 and pH 8) or 0.01 M citrate-phosphate (pH 5). Inoculum effect studies were carried out with 10' and 10 8 cells per ml in addition to the standard inoculum density of 5 X 10 5 cells per
mi. Pooled human urine samples were filtered
PARAMETERS CHARACTERIZING THE IN VITRO ACTIVITY OF CEFIXIME, ETC .
through 0.45j.l Millipore filters, and the pH was adjusted to 6.5-7 to determine the activity of cefixime in this environment. Minimal bactericidal concentrations (MBCs) were determined by drawing three 0.01 ml samples from each of the MIC wells showing no growth and streaking them onto the surface of Mueller-Hinton agar plates. After 24 h at 37°C the MBC was read as the lowest concentration of antibiotic which resulted in a 99.9% reduction in viable cells of the original inoculum. As suggested by Pearson et al., this corresponds to a rejection value of regrowing colonies of 25 14 • Time-kill experiments were performed by ' adding cefixime to log-phase bacterial cultures diluted to 10 6 -10' CFU/ml at a concentration corresponding to 4 X MIC and growing in 500 ml flasks at 37°C. Just before the antibiotic was added (zero time) and at 2, 6 and 24 h thereafter the viable number of organisms was determined by serial 10-fold dilutions made in physiologic saline. A calibrated micropipetter was used to deliver 100 j.tl portions from each dilution onto a Mueller-Hinton agar plate. Because of the drug carry-over effect only 10 3 CFU/ml could be accurately counted and no data below this level of detection were reported.
Determination of a paradoxic bactericidal (Eagle's) eHect Exponentially growing bacteria in broth and in urine were exposed to increasing concentrations of cefixime (from 1 to 500 X MIC). After 2 hours at 3 7°C, the survivors were evaluated by determining the colony forming unit (CFU) on Mueller-Hinton agar plates. The colonies were scored after 48 hours of incubation at 37 °C.
Model for the in vitro simulation of the urinary bladder The model for in vitro simulation of the human vesica was essentially that described by Greenwood and O'Grady 1 s. This device reproduces the predicted mean urine concentrations of the antibiotic after an oral dose of 200 mg 6 • 8 • The apparatus consists of 3 vessels kept in agitation in a water-bath at 37°C, and containing in flask A the bacterial culture, in recipient B the drug-free medium, and in vessel C broth
133
contammg the antibiotic. A peristaltic pump (Micro Perpex, LKB 2132) conveyed, through a T-valve, the diluent medium from flask B or the drug-containing broth from recipient C to that of the test culture (A). Liquid flow was regulated to 1 ml per minute to simulate the secretion of ureteric urine. At 2 h intervals the accumulated medium was removed leaving a 20 ml residuum (micturition). To start the experiment an overnight culture of the organism to be assayed was diluted 1:100 in 20 ml of pre-warmed · broth and transferred to flask A. After 1h of incubation the concentration of cefixime was allowed to rise gradually to reach a peak of 30 j.tgfml after 2h, and 100 j.tgfml after 4h. This level was maintained for 2h and thereafter the concentration of cefixime was allowed to fall gradually to 5 j.tgfml over a further 6 hours, and finally to zero. Bacterial counts were assessed every 2 h during the first 12 h of the experiment. A final count was performed at 24 h. The amount of antimicrobial agent in the test culture was controlled by a microbiological method using Bacillus subtilis ATCC 6633, as described elsewhere 16 •
Interaction of cefixime with other drugs The results of combining cefixime with other molecules on the antibacterial activity of the drug were assessed by standard checkerboard titration and timed-kill curves. Cefixime was tested against 3 isolates of Escherichia coli,
Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Proteus mirabilis and Pseudomonas aeruginosa in combination with ciprofloxacin, netilmicin and fosfomycin, while H. influenzae, M. catarrhalis, S. pyogenes and S. pneumoniae were assayed in combination with clarithromycin and rifampin in addition to the above mentioned antibiotics. Staphylococcus aureus, S. epidermidis, S. saprophyticus and Enterococcus faecalis were studied in combination with the already listed antibiotics plus vancomycin and teicoplanin. Checkerboard studies were performed in microdilution trays, as previously described 10 • 12 • Antibiotic interactions were evaluated using as guideline the fractional inhibitory concentrations (FIC indices) calculated by the following formula: MIC of antibiotic A in the presence of antibiotic B/MIC of A alone + MIC of Bin the presence of A/MIC of B alone 1 ' . Synergism is present if the FIC index
134
E.A. DEBBIA • A. MARCHESE • A. PESCE - D. SAVERINO - G.C. SCHITO
is s 0.5 and antagonism is the result when the index values exceed 4. Indifference is represented by an index of 0.5 to 4. Time-kill studies of drug combinations were performed as described before except that cefixime and all other drugs were used at concentrations corresponding to one-half their MICs. Antibiotic interactions were interpreted as synergistic or antagonistic if the combinations, compared with the most effective single antibiotic, caused at least a 100-fold reduction or increase, respectively, in the CFU of the survivors at 24 h. Intermediate results were defined as indifference.
RESULTS AND DISCUSSION
In vitro activity of cefixime under various experimental conditions The in vitro susceptibilities to cefixime and other comparative drugs of 289 gram-negative strains belonging to several Enterobacteriaceae TABLE
and other gram-negative species are reported in Table 1. Cefixime emerged as one of the more potent antibiotics among those studied both in terms of MIC9o values and in percentage of strains inhibited. In particular, it was more effective on a weight for weight basis (MIC9o in l!g/ml), than the other agents against E. coli (0.5), K. pneumoniae (1), K. oxytoca (0.25), K. planticola (0.06), Serratia spp. (0.125), P. mirabilis (1), and P. vulgaris (1). The other antibiotics showed values that were generally from 4 to over 128fold higher than those registered with cefixime. Enterobacter spp. other than E. cloacae, S. marcescens, P. rettgeri, P. stuartii, M. morganii and C. freundii all exhibited MIC9o values to cefixime of 8 l!g/ml, a value at least 4-fold lower than that registered with cefuroxime, the most active comparative agent. When the approved breakpoints for susceptibility were considered 9, the percentage of strains sensitive to cefixime spanned from 31 (C. freundii) to 100% (K. planticola and Serratia spp.). Cefuroxime showed a similar pattern of
1 - Minimal inhibitory concentrations of cefixime and other comparative drugs against gram-negative pathogens. MIC (1-!g/ml) range
50%
90%
% susceptible at breakpoint
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.125->64 0.5- > 128 2- >32 0.125 - >64 2- >64
0.25 16 8 16 4
0.5 > 128 >32 >64 16
94 45 49 49 69
K. pneumoniae (26)
cefixime amoxicillin amox-clav cefaclor cefuroxime
64 2- > 128 2- >32 8- >64 2- >64
128 >32 >64 64
92 8 12 23 57
K. oxytoca (20)
cefixime amoxicillin amox-clav cefaclor cefuroxime
64 2- > 128 8- >32 0.25- >64 2- >64
128 >32 >64 64
95 5 10 30 60
K. planticola (10)
cefixime amoxicillin amox-clav cefaclor cefuroxime
128 2- >32 0.5-1 2-32
0.06 16 16 0.5 8
0.125 > 128 >32 1 32
100 20 40 100 80
E. cloacae (28)
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.25- >64 64- > 128 16- > 32 >64 0.5- >64
64 > 128 >32
>64 > 128 >32
>64
>64
43 0 0 0 25
Organism (No. tested)
Antibiotics
E. coli (67)
(continued)
135
PARAMETERS CHARACTERIZING THE IN VITRO ACTIVITY OF CEFIXIME, ETC .
TABLE
1 - Continued.
MIC (l!g/ml) range
50 %
90%
% susceptible at breakpoint
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.5- >64 32- > 128 16- >32 2- >64 1- >64
2 64 32 64 64
8 128 >32 >64 >64
36 0 0 14 27
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06 -· 8 64- > 128 16- >32 >64 0.5- >64
4 > 128 >32
8 > 128 >32
>64
>64
44 0 0 0 19
Serratia spp. (10)
cefixime amoxicillin amox-clav cefaclor cefuroxime
128 16- > 32 0.25-32 0.5- >64
0.06 > 128 >32 0.25 >64
0.125 > 128 >32 8 >64
100 0 0 90 40
P. mirabilis (30)
cefixime amoxicillin amox-clav cefaclor cefuroxime
64 0.25-8 0.06-8 0.5->128 2-32
0.125 1 1 32 2
1 4 2 64 4
90 100 97 47 93
P. vulgaris (24)
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06- >64 0.25- > 128 0.06-16 0.5- > 128 8- > 128
0.125 1 1 8 16
1 128 2 64 > 128
96 75 83 54 71
P. rettgeri (16)
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16- > 32 >64 0.5 ->64
4 > 128 >32
8 > 128 >32
>64
>64
44 0 0 0 44
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16 - > 32 >64 0.5 - >64
4 > 128 >32
8 > 128 >32
>64
>64
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16 - >32 >64 0.5 ->64
4 > 128 >32
8 > 128 >32
>64
>64
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16- > 32 >64 0.5->64
4 > 128 >32
8 > 128 >32
>64
>64
cefixime amoxicillin amox-clav cefaclor cefuroxime
2- > 128 > 128 > 128 > 128 32- > 128
64
> 128
> 128
> 128
Organism (No. tested)
Antibiotics
Enterobacter spp (22)
S. marcescens (16)
P. stuartii (14)
M. morganii (14)
C. /retmdii (16)
Pseudomonas spp. (19)
* Breakpoints for susceptibility (in 1-!g/ml): cefixime 1, amoxicillin 8, amox-clav 4, cefaclor 8, cefuroxime 8.
43 0 0 0 43 43 0 0 0 29 31 0 0 0 25 0 0 0 0 0
136
E.A. DEBBIA - A. MARCHESE - A. PESCE - D. SAVERINO - G.C. SCHITO
TABLE 2 - Minimal inhibitory concentrations of ce/ixime and other comparative drugs against M. catarrhalis, H. influenzae, S. pneumoniae and S. pyogenes.
range
50%
90%
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.03 - 0.125 0.06-0.25 0.03-0.25 0.125-16 0.03-0 .25
0.06 0.125 0.03 0.25 0.03
0.125 0.25 0.25 0.5 0.125
100 100 100 92 100
cefixime amoxicillin amox-clav cefaclor cefuroxime
32 0.03 - 1 0.5-32 0.03-0.25
0.125 32 0.25 4 0.03
0.5 >32 0.5 32 0.25
100 20 100 60 100
cefixime amoxicillin amox-clav cefaclor cefuroxime
< 0.03- 0.125 0.125 - 1 0.25-1 32 0.125-0.5
0.03 >32 0.25 8 0.25
0.06 >32 1 >32 0.5
100 27 100 27 100
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.03 - 0.5
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Parameters Characterizing the In Vitro Activity of Cefixime, a New Oral Broad Spectrum Cephalosporin, Against Respiratory and Urinary Pathogens E.A. Debbia, A. Marchese, A. Pesce, D. Saverino & G.C. Schito To cite this article: E.A. Debbia, A. Marchese, A. Pesce, D. Saverino & G.C. Schito (1992) Parameters Characterizing the In Vitro Activity of Cefixime, a New Oral Broad Spectrum Cephalosporin, Against Respiratory and Urinary Pathogens, Journal of Chemotherapy, 4:3, 131-144, DOI: 10.1080/1120009X.1992.11739153 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739153
Published online: 15 Jul 2016.
Submit your article to this journal
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [University of Minnesota Libraries, Twin Cities]
Date: 13 September 2016, At: 00:59
Journal of Chemotherapy
Parameters Characterizing the In Vitro Activity of Cefixime, a New Oral Broad Spectrum Cephalosporin, Against Respiratory and Urinary Pathogens E.A. DEBBIA - A. MARCHESE - A. PESCE D. SAVERINO - G.C. SCHITO
Vol. 4 - n. J (131-144) - 1992
ary strains was optimal at cefixime concentrations reached in urine since eradication, except for Proteus mirabilis, was enhanced with increasing levels of the drug. The absence of an untoward paradoxic effect on the rate of cefixime bactericidal action was confirmed by employing a dynamic bladder model simulating the pharmacokinetic parameters of the drug after a single 200 mg daily dosage. Interactions of cefixime with several other drugs that may be employed in combination therapy were generally prone to provide indifference and synergism while antagonism was never observed. Favorable interactions were also registered when cefixime acted with other antibiotics on partially resistant species such as Staphylococci and Pseudomonas. The new cephem seems to provide excellent opportunities for expanding oral cephalosporin therapy to a wide range of infections produced by susceptible pathogens in the adult and pediatric populations. Key words: cefixime, in vitro activity, bactericidal effect, Eagle's effect, activity in bladder model, interaction with other drugs, cephalosporin.
Summary ----------------------------------
INTRODUCTION
The wide and potent in vitro activity of cefixime, a new oral broad spectrum cephalosporin, has been confirmed on a collection of respiratory and urinary pathogens recently isolated in Italy. The new cephem emerged as the most bactericidal of all the comparators tested against several fast as well as slowly-growing gram-negative species including Enterobacteria, Haemophilus and Moraxella, irrespective of their ability to synthetize P-lactamases. Among the gram-positive species Streptococcus pyogenes and S. pneumoniae were effectively covered. Cefixime activity was not adversely influenced by several important variables such as pH (over the range from 5 to 8), inoculum size (from 10' to 10 1 CFU per ml) and the presence of 50% human serum or urine. Time-kill tests confirmed a pronounced bactericidal potency of the drug especially towards common respiratory pathogens (H. influenzae, M. catarrhalis, S. pneumoniae and S. pyogenes). Killing of urin-
Cefixime (FK 027, FR 17027, CL 284635) is a new oral cephalosporin characterized by the presence of a vinyl group at the three position which promotes effective gut absorption, and by an aminothiazolyl substitution that confers a broad antimicrobial spectrum and a high degree of P-lactamase stability 1 • In previous studies cefixime was shown to inhibit a wide variety of gram-positive and gram-negative aerobic bacteria including members of the Enterobacteriaceae family, Hae-
Institute of Microbiology, University of Genoa Medical School, Genoa, Italy. Correspondence to: E.A. Debbia, Institute of Microbiology, University of Genoa, Medical School, Viale Benedetto XV, 10, 16132 Genoa, Italy © Edizioni Riviste Scientifiche - Firenze
mophilus in/luenzae, Streptococcus pyogenes, S. pneumoniae, and Moraxella catarrhalis. Activity against staphylococci, enterococci and Morganella morganii was poor and Pseudomonas species were generally resistant 2 • 3 • 4 • The antibacterial potency of this new oral cephem was only marginally influenced by the ISSN 1120-009X
E.A. DEBBIA - A. MARCHESE - A. PESCE - D. SAVERINO - G.C. SCHITO
132
presence of serum proteins, pH variations, media composition, bacterial inoculum size and growth conditions of the test organisms. Overall, this behavior renders the cefixime profile similar to that shown by parenteral third generation cephalosporins 3 • 5 • Given its spectrum of bactericidal activity and its pharmacokinetic parameters that allow for sustained levels in the relevant tissues and secretions, this new drug seems best suited for the treatment of respiratory and urinary tract infections both in the adult and pediatric populations 6 - 8 • This study has been designed to answer several important questions that are related to the introduction of cefixime in general therapy in this country. Due to wide geographic variations in the epidemiology of bacterial resistance it was deemed essential to confirm the in vitro efficacy of this new cephalosporin on a collection of urinary and respiratory pathogens freshly isolated from in and outpatients and representing the challenge that cefixime will meet in this region. Its bactericidal activity was also scrutinized under various experimental conditions. These tests included the assessment of the extent and rate of killing as a function of the concentration reached in the urine where cefixime becomes highly concentrated. Bactericidal potency may in fact be untowardly affected by the presence of a paradoxic Eagle's effect. The bactericidal activity of the new cephem in an in vitro model simulating its pharmacokinetics in the urinary bladder was also assessed. Finally, the outcome of the interactions of cefixime with several other drugs that may be employed on those rare but possible occasions where combination therapy may be deemed essential was determined. MATERIALS AND METHODS
Microorganisms All strains were freshly isolated from clinical specimens from patients suffering from respiratory and urinary tract infections and identified in the diagnostic laboratory of this institution by conventional methods. Though not selected, the collection employed included organisms exhibiting resistance to one or more of the antibiotics tested, and was representative of the epidemiologic situation prevailing 'in Italy.
Antibiotics Cefixime was obtained from Menarini, Firenze, Italy. Cefuroxime, amoxicillin, amoxicillin-clavulanic acid (amox-clav), cefaclor, fosfomycin, netilmicin, ciprofloxacin, vancomycin, teicoplanin, rifampin, and clarithromycin were supplied from their respective manufacturers. Sterile stock solutions of the antibiotics were prepared from the standard reference powders in accordance with the instructions provided. For the detection of beta-lactamase activity nitrocefin paper disks (BBL) were used.
Susceptibility tests under various conditions Minimal inhibitory concentrations (MICs) of cefixime and of the other drugs were determined by using the methods suggested by the NCCLS (National Committee for Clinical Laboratory Standards) 9 , and as previously described 10 - 12 • Organisms for quality control were included in each assay. Exponentially growing bacteria (5 X 10 5 cells per ml, final inoculum) were added to the various concentrations of antibiotics, diluted in cation-supplemented Mueller-Hinton broth and distributed on a microplate by using the automatic Biomek 1000 dilutor (Beckman, USA) . After 18-24 hours of incubation at 37°C the antibiotic concentration which prevented a visible bacterial growth was recorded as the MIC. For tests performed with Streptococcus strains, 5% defibrinated horse blood was added to the Mueller-Hinton medium, whereas for evaluation of Haemophilus strains, Haemophilus Test Medium (HTM) 13 was employed. M. catarrhalis was tested on chocolate agar medium and incubated in an atmosphere of 5% carbon dioxide . The effect of serum on the activity of cefixime was evaluated by employing pooled normal human serum combined with Mueller-Hinton medium to yield a 50% final concentration. To assess possible changes in the activity of cefixime due to modification of the pH of the medium, Mueller-Hinton broth was buffered with 0.01 M phosphate (pH 7 and pH 8) or 0.01 M citrate-phosphate (pH 5). Inoculum effect studies were carried out with 10' and 10 8 cells per ml in addition to the standard inoculum density of 5 X 10 5 cells per
mi. Pooled human urine samples were filtered
PARAMETERS CHARACTERIZING THE IN VITRO ACTIVITY OF CEFIXIME, ETC .
through 0.45j.l Millipore filters, and the pH was adjusted to 6.5-7 to determine the activity of cefixime in this environment. Minimal bactericidal concentrations (MBCs) were determined by drawing three 0.01 ml samples from each of the MIC wells showing no growth and streaking them onto the surface of Mueller-Hinton agar plates. After 24 h at 37°C the MBC was read as the lowest concentration of antibiotic which resulted in a 99.9% reduction in viable cells of the original inoculum. As suggested by Pearson et al., this corresponds to a rejection value of regrowing colonies of 25 14 • Time-kill experiments were performed by ' adding cefixime to log-phase bacterial cultures diluted to 10 6 -10' CFU/ml at a concentration corresponding to 4 X MIC and growing in 500 ml flasks at 37°C. Just before the antibiotic was added (zero time) and at 2, 6 and 24 h thereafter the viable number of organisms was determined by serial 10-fold dilutions made in physiologic saline. A calibrated micropipetter was used to deliver 100 j.tl portions from each dilution onto a Mueller-Hinton agar plate. Because of the drug carry-over effect only 10 3 CFU/ml could be accurately counted and no data below this level of detection were reported.
Determination of a paradoxic bactericidal (Eagle's) eHect Exponentially growing bacteria in broth and in urine were exposed to increasing concentrations of cefixime (from 1 to 500 X MIC). After 2 hours at 3 7°C, the survivors were evaluated by determining the colony forming unit (CFU) on Mueller-Hinton agar plates. The colonies were scored after 48 hours of incubation at 37 °C.
Model for the in vitro simulation of the urinary bladder The model for in vitro simulation of the human vesica was essentially that described by Greenwood and O'Grady 1 s. This device reproduces the predicted mean urine concentrations of the antibiotic after an oral dose of 200 mg 6 • 8 • The apparatus consists of 3 vessels kept in agitation in a water-bath at 37°C, and containing in flask A the bacterial culture, in recipient B the drug-free medium, and in vessel C broth
133
contammg the antibiotic. A peristaltic pump (Micro Perpex, LKB 2132) conveyed, through a T-valve, the diluent medium from flask B or the drug-containing broth from recipient C to that of the test culture (A). Liquid flow was regulated to 1 ml per minute to simulate the secretion of ureteric urine. At 2 h intervals the accumulated medium was removed leaving a 20 ml residuum (micturition). To start the experiment an overnight culture of the organism to be assayed was diluted 1:100 in 20 ml of pre-warmed · broth and transferred to flask A. After 1h of incubation the concentration of cefixime was allowed to rise gradually to reach a peak of 30 j.tgfml after 2h, and 100 j.tgfml after 4h. This level was maintained for 2h and thereafter the concentration of cefixime was allowed to fall gradually to 5 j.tgfml over a further 6 hours, and finally to zero. Bacterial counts were assessed every 2 h during the first 12 h of the experiment. A final count was performed at 24 h. The amount of antimicrobial agent in the test culture was controlled by a microbiological method using Bacillus subtilis ATCC 6633, as described elsewhere 16 •
Interaction of cefixime with other drugs The results of combining cefixime with other molecules on the antibacterial activity of the drug were assessed by standard checkerboard titration and timed-kill curves. Cefixime was tested against 3 isolates of Escherichia coli,
Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Proteus mirabilis and Pseudomonas aeruginosa in combination with ciprofloxacin, netilmicin and fosfomycin, while H. influenzae, M. catarrhalis, S. pyogenes and S. pneumoniae were assayed in combination with clarithromycin and rifampin in addition to the above mentioned antibiotics. Staphylococcus aureus, S. epidermidis, S. saprophyticus and Enterococcus faecalis were studied in combination with the already listed antibiotics plus vancomycin and teicoplanin. Checkerboard studies were performed in microdilution trays, as previously described 10 • 12 • Antibiotic interactions were evaluated using as guideline the fractional inhibitory concentrations (FIC indices) calculated by the following formula: MIC of antibiotic A in the presence of antibiotic B/MIC of A alone + MIC of Bin the presence of A/MIC of B alone 1 ' . Synergism is present if the FIC index
134
E.A. DEBBIA • A. MARCHESE • A. PESCE - D. SAVERINO - G.C. SCHITO
is s 0.5 and antagonism is the result when the index values exceed 4. Indifference is represented by an index of 0.5 to 4. Time-kill studies of drug combinations were performed as described before except that cefixime and all other drugs were used at concentrations corresponding to one-half their MICs. Antibiotic interactions were interpreted as synergistic or antagonistic if the combinations, compared with the most effective single antibiotic, caused at least a 100-fold reduction or increase, respectively, in the CFU of the survivors at 24 h. Intermediate results were defined as indifference.
RESULTS AND DISCUSSION
In vitro activity of cefixime under various experimental conditions The in vitro susceptibilities to cefixime and other comparative drugs of 289 gram-negative strains belonging to several Enterobacteriaceae TABLE
and other gram-negative species are reported in Table 1. Cefixime emerged as one of the more potent antibiotics among those studied both in terms of MIC9o values and in percentage of strains inhibited. In particular, it was more effective on a weight for weight basis (MIC9o in l!g/ml), than the other agents against E. coli (0.5), K. pneumoniae (1), K. oxytoca (0.25), K. planticola (0.06), Serratia spp. (0.125), P. mirabilis (1), and P. vulgaris (1). The other antibiotics showed values that were generally from 4 to over 128fold higher than those registered with cefixime. Enterobacter spp. other than E. cloacae, S. marcescens, P. rettgeri, P. stuartii, M. morganii and C. freundii all exhibited MIC9o values to cefixime of 8 l!g/ml, a value at least 4-fold lower than that registered with cefuroxime, the most active comparative agent. When the approved breakpoints for susceptibility were considered 9, the percentage of strains sensitive to cefixime spanned from 31 (C. freundii) to 100% (K. planticola and Serratia spp.). Cefuroxime showed a similar pattern of
1 - Minimal inhibitory concentrations of cefixime and other comparative drugs against gram-negative pathogens. MIC (1-!g/ml) range
50%
90%
% susceptible at breakpoint
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.125->64 0.5- > 128 2- >32 0.125 - >64 2- >64
0.25 16 8 16 4
0.5 > 128 >32 >64 16
94 45 49 49 69
K. pneumoniae (26)
cefixime amoxicillin amox-clav cefaclor cefuroxime
64 2- > 128 2- >32 8- >64 2- >64
128 >32 >64 64
92 8 12 23 57
K. oxytoca (20)
cefixime amoxicillin amox-clav cefaclor cefuroxime
64 2- > 128 8- >32 0.25- >64 2- >64
128 >32 >64 64
95 5 10 30 60
K. planticola (10)
cefixime amoxicillin amox-clav cefaclor cefuroxime
128 2- >32 0.5-1 2-32
0.06 16 16 0.5 8
0.125 > 128 >32 1 32
100 20 40 100 80
E. cloacae (28)
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.25- >64 64- > 128 16- > 32 >64 0.5- >64
64 > 128 >32
>64 > 128 >32
>64
>64
43 0 0 0 25
Organism (No. tested)
Antibiotics
E. coli (67)
(continued)
135
PARAMETERS CHARACTERIZING THE IN VITRO ACTIVITY OF CEFIXIME, ETC .
TABLE
1 - Continued.
MIC (l!g/ml) range
50 %
90%
% susceptible at breakpoint
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.5- >64 32- > 128 16- >32 2- >64 1- >64
2 64 32 64 64
8 128 >32 >64 >64
36 0 0 14 27
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06 -· 8 64- > 128 16- >32 >64 0.5- >64
4 > 128 >32
8 > 128 >32
>64
>64
44 0 0 0 19
Serratia spp. (10)
cefixime amoxicillin amox-clav cefaclor cefuroxime
128 16- > 32 0.25-32 0.5- >64
0.06 > 128 >32 0.25 >64
0.125 > 128 >32 8 >64
100 0 0 90 40
P. mirabilis (30)
cefixime amoxicillin amox-clav cefaclor cefuroxime
64 0.25-8 0.06-8 0.5->128 2-32
0.125 1 1 32 2
1 4 2 64 4
90 100 97 47 93
P. vulgaris (24)
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06- >64 0.25- > 128 0.06-16 0.5- > 128 8- > 128
0.125 1 1 8 16
1 128 2 64 > 128
96 75 83 54 71
P. rettgeri (16)
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16- > 32 >64 0.5 ->64
4 > 128 >32
8 > 128 >32
>64
>64
44 0 0 0 44
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16 - > 32 >64 0.5 - >64
4 > 128 >32
8 > 128 >32
>64
>64
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16 - >32 >64 0.5 ->64
4 > 128 >32
8 > 128 >32
>64
>64
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.06-8 64- > 128 16- > 32 >64 0.5->64
4 > 128 >32
8 > 128 >32
>64
>64
cefixime amoxicillin amox-clav cefaclor cefuroxime
2- > 128 > 128 > 128 > 128 32- > 128
64
> 128
> 128
> 128
Organism (No. tested)
Antibiotics
Enterobacter spp (22)
S. marcescens (16)
P. stuartii (14)
M. morganii (14)
C. /retmdii (16)
Pseudomonas spp. (19)
* Breakpoints for susceptibility (in 1-!g/ml): cefixime 1, amoxicillin 8, amox-clav 4, cefaclor 8, cefuroxime 8.
43 0 0 0 43 43 0 0 0 29 31 0 0 0 25 0 0 0 0 0
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E.A. DEBBIA - A. MARCHESE - A. PESCE - D. SAVERINO - G.C. SCHITO
TABLE 2 - Minimal inhibitory concentrations of ce/ixime and other comparative drugs against M. catarrhalis, H. influenzae, S. pneumoniae and S. pyogenes.
range
50%
90%
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.03 - 0.125 0.06-0.25 0.03-0.25 0.125-16 0.03-0 .25
0.06 0.125 0.03 0.25 0.03
0.125 0.25 0.25 0.5 0.125
100 100 100 92 100
cefixime amoxicillin amox-clav cefaclor cefuroxime
32 0.03 - 1 0.5-32 0.03-0.25
0.125 32 0.25 4 0.03
0.5 >32 0.5 32 0.25
100 20 100 60 100
cefixime amoxicillin amox-clav cefaclor cefuroxime
< 0.03- 0.125 0.125 - 1 0.25-1 32 0.125-0.5
0.03 >32 0.25 8 0.25
0.06 >32 1 >32 0.5
100 27 100 27 100
cefixime amoxicillin amox-clav cefaclor cefuroxime
0.03 - 0.5
