Activity of faropenem and imipenem for ciprofloxacin-resistant bacteria

Laura J. V. Piddock*, M. M. Johnson and Mark A. Webber

Antimicrobial Agents Research Group, Division of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, UK

Received 28 March 2003; returned 22 April 2003; revised 13 June 2003; accepted 18 June 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Aim: To determine whether an association exists between ciprofloxacin and faropenem resistance in bacteria including multiply drug-resistant isolates.

Methods: The MICs were determined for 150 fluoroquinolone-resistant bacteria, plus 20 nalidixic acid-resistant strains of Salmonella enterica serovar Typhimurium.

Results: Faropenem was very active against Escherichia coli and Streptococcus pneumoniae, but 5/31 Staphylococcus aureus and 2/26 Bacteroides fragilis required >=16 mg/L for inhibition. Of 30 multiply drug-resistant isolates with a phenotype suggestive of enhanced efflux, only for one strain (a Bacteroides fragilis) was the faropenem MIC higher than that associated with the other isolates of the same species.

Conclusions: Faropenem was in general as active as imipenem. There was no association between resistance to ciprofloxacin and faropenem or imipenem resistance.

Keywords: carbapenem, fluoroquinolone, MIC


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Owing to the increasing numbers of ampicillin-resistant ß-lactamase-producing Haemophilus influenzae and Moraxella catarrhalis, and penicillin-resistant Streptococcus pneumoniae, physicians have been increasing the use of fluoroquinolones.1 However, resistance to these agents has also emerged. In recent years, there have not been many new agents of classes other than fluoroquinolones developed and agents with different modes of action have been sought.

Faropenem, a novel broad-spectrum ß-lactam (penem) has been shown to have good activity for pathogens of the respiratory tract.25 Faropenem has also been shown to have good activity for Enterobacteriaceae2,6,7 and anaerobes.7,8 Therefore the aim of this study was to determine the activity of faropenem, compared with those of ciprofloxacin and imipenem, for fluoroquinolone-resistant bacteria (ciprofloxacin MIC >= 2 mg/L) of species commonly isolated from the respiratory and digestive tracts and from the skin. This set of isolates was chosen to compare in vitro activities of different drug classes against selected clinical isolates or laboratory-generated strains. The overall objective of this study was to determine whether there was any association between fluoroquinolone resistance and carbapenem resistance.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The minimum inhibitory concentration (MIC) of each agent was determined with the appropriate media and incubation conditions according to the guidelines of the British Society for Antimicrobial Chemotherapy.9 In brief, for Escherichia coli, Salmonella enterica and Staphylococcus aureus Iso-Sensitest media were used; for Streptococcus pneumoniae, agar was supplemented with 5% defibrinated horse blood. For the enterococci and Campylobacter, Mueller–Hinton agar was supplemented with 5% defibrinated horse blood. For Bacteroides fragilis, Wilkins and Chalgren medium was used. All cultures were incubated at 37°C for 24 h. S. pneumoniae were incubated in 5% CO2 at 37°C, Campylobacter spp. were incubated in 7.5% CO2 at 37°C and B. fragilis were incubated in an anaerobic cabinet with an atmosphere of 80% CO2, 10% N2 and 10% H2. All antibiotics were made up and used according to the manufacturer’s instructions. The DNA sequences of gyrA, gyrB, parC/grlA and parE/grlB were obtained from the EMBL database and oligonucleotide primers synthesized to amplify the quinolone resistance determining regions (QRDRs) of each gene of each species. The QRDR of all four genes was amplified by PCR from the DNA of each strain and the PCR amplimers were screened for mutations conferring resistance by the new method of WAVE D-HPLC (Transgenomic Limited).10 The DNA sequences of each novel WAVE pattern were determined by cycle sequencing the PCR amplimers and analysis on an ABI Prism 3700 DNA analyser (Functional Genomics Laboratory, University of Birmingham, Birmingham, UK). Strains over-expressing specific efflux pump genes were also examined, and the multidrug resistance phenotype confirmed by determining the MICs of ethidium bromide, acriflavine, tetracycline and chloramphenicol.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The MICs of faropenem were similar for strains with different mutations in topoisomerase genes encoding ciprofloxacin resistance.

For the 21 laboratory mutants and clinical isolates of S. pneumoniae, the MICs of ciprofloxacin were 4–>32 mg/L (Table 1). The control strains were NCTC 7466, R6 and its pmrA mutant R6N. Nine strains had at least one of seven substitutions in ParC: Ser-16 to Gly (n = 1), Asp-78 to Asn (n = 1), Ser-79 to Phe (n = 5), Ser-80 to Pro (n = 1), Asp-83 to Tyr (n = 1) Arg-95 to Cys (n = 1) and Lys-137 to Asn (n = 3). Five of the strains had two of these mutations. Thirteen strains had substitutions at Ser-79 or Ser-81 in GyrA. Irrespective of susceptibility to fluoroquinolones or resistance mechanism, including over-expression of the efflux pump gene pmrA, both faropenem and imipenem had good activity.


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Table 1. Susceptibility (mg/L) of strains to ciprofloxacin, faropenem and imipenem
 
For the 31 laboratory mutants and clinical isolates of S. aureus, the MICs of ciprofloxacin were 4–>32 mg/L. The control strain was NCTC 8532. The S. aureus strains also had varied mutations within grlA and gyrA: GrlA Ser-80 to Phe (n = 3) or Tyr (n = 5); GrlA Glu-84 to Lys (n = 4); GrlA Ala-116 to Val (n = 1). Six strains contained a substitution in GyrA alone: Glu-88 to Lys (n = 3), Ser-84 to Leu (n = 3). Two strains also contained a GrlA substitution plus GyrA Ser-84 to Leu or Glu-88 to Gly. Faropenem had good activity for 29/31 of the fluoroquinolone-resistant S. aureus (Table 1). Three strains required 16–32 mg/L faropenem or imipenem for inhibition. Over-expression of norA had no effect upon faropenem susceptibility.

The vancomycin-resistant (MICs >= 2 mg/L) enterococci (17 Enterococcus faecium, three Enterococcus faecalis) comprised laboratory mutants and human clinical isolates. Eighteen of these strains were also ciprofloxacin-resistant (MIC >= 2 mg/L). The control strains were E. faecalis NCTC 775 and E. faecium NCTC 7171. We were unable to determine the DNA sequences of the QRDRs of the topoisomerase genes for these strains as there was insufficient sequence information in the EMBL database to design primers. Faropenem inhibited 10/22 of the enterococci at <=4 mg/L. Imipenem was less active than faropenem.

For the 27 laboratory mutants and clinical isolates of E. coli, the MICs of ciprofloxacin were 2–>32 mg/L. The control strain was E. coli NCTC 10418. All of the E. coli had a substitution in GyrA of Ser-83 to Leu. Twenty-five strains also had substitutions at Asp-87, the most common being with Asn (n = 16). Twenty-three strains also had substitutions within the QRDR of ParC, the most common being at Ser-80 (n = 21; two at Glu-84). Faropenem and imipenem were active against all the ciprofloxacin-resistant E. coli including 11 strains that over-expressed acrB, six strains that over-expressed soxS and four strains that over-expressed marA.

For the 20 laboratory mutants and clinical isolates of S. enterica serovar Typhimurium, the MIC of nalidixic acid was >=128 mg/L and for ciprofloxacin 0.06–0.5 mg/L. The control strain was S. Typhimurium NCTC 74. Eight S. Typhimurium contained substitutions in GyrA at Ser-83 to Phe or Asp-87 to Gly. Faropenem was active against the nalidixic acid-resistant S. Typhimurium including three strains that over-expressed acrB. Imipenem was also active but MICs were generally one-fold higher than those for faropenem.

For the 12 clinical isolates of Pseudomonas aeruginosa, the MIC of ciprofloxacin was 2–64 mg/L. The control strain was NCTC 10662. One isolate had a substitution in GyrA at Thr-83 to Ile, and two had substitutions at Arg-87 to Ile. The remaining nine isolates had a wild-type gyrA, and mutation in parC was not investigated. Only 6/12 of the P. aeruginosa were inhibited by <=8 mg/L faropenem, however 11/12 were inhibited by the same concentration of imipenem. The one strain that over-expressed both mexABoprM and mexEFoprN was susceptible to faropenem.

For the 26 laboratory mutants and clinical isolates of B. fragilis strains, the MIC of ciprofloxacin was 4–>32 mg/L. The control strain was NCTC 9343. None of the B. fragilis had mutations in gyrA or gyrB. Twenty-five of the 26 B. fragilis were inhibited by <=8 mg/L faropenem and imipenem (Table 1). One multiply antibiotic-resistant strain (mechanism unknown) was resistant to faropenem.

For the 13 clinical isolates of Campylobacter jejuni, the MIC of ciprofloxacin was 16–64 mg/L. The control strain was C. jejuni ATCC 33560. All 13 isolates had substitutions in GyrA at Thr-86 to Ile, and 4/13 also had a silent mutation at His-81. Despite repeated attempts, no PCR amplimers for parC were obtained. Faropenem and imipenem had good activity against the ciprofloxacin-resistant campylobacteria (Table 1).

In summary, faropenem shows excellent activity against ciprofloxacin-resistant bacteria, and was as potent as imipenem in this study. The MICs of faropenem were similar for strains with different mutations in the topoisomerase genes encoding ciprofloxacin resistance, suggesting that there was no effect upon faropenem activity. There was no relationship between ciprofloxacin resistance and faropenem resistance regardless of the level of ciprofloxacin resistance. As may have been anticipated, these data indicate that the mechanisms of fluoroquinolone and faropenem resistance are distinct. Finally, faropenem activity was unaffected by over-expression of efflux genes.


    Acknowledgements
 
This study was supported by Bayer AG.


    Footnotes
 
* Corresponding author. Tel: +44-121-414-6966; Fax: +44-121-414-3599; E-mail: l.j.v.piddock{at}bham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Davidson, R., Cavalcanti R., Brunton J. L. et al. (2002). Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. New England Journal of Medicine 346, 747–50.[Free Full Text]

2 . Cormican, M. G. & Jones, R. N. (1995). Evaluation of the in-vitro activity of faropenem (SY 5555 or SUN 5555) against respiratory tract pathogens and ß-lactamase producing bacteria. Journal of Antimicrobial Chemotherapy 35, 535–9.[Abstract]

3 . Inoue, E. & Mitsuhashi, S. (1994). In vitro antibacterial activity and ß-lactamase stability of ST5555, a new oral penem antibiotic. Antimicrobial Agents and Chemotherapy 38, 1974–9.[Abstract]

4 . Mortensen, J. E. & Egleton, J. H. (1995). Comparative activity of faropenem against aerobic bacteria isolated from pediatric patients. Diagnostic Microbiology and Infectious Disease 22, 301–6.[CrossRef][ISI][Medline]

5 . Woodcock, J. M., Andrews, J. M., Brenwald, N. P. et al. (1997). The in-vitro activity of faropenem, a novel oral penem. Journal of Antimicrobial Chemotherapy 39, 35–43.[Abstract/Free Full Text]

6 . Sewell, D., Barry, S., Allen, S. et al. (1995). Comparative antimicrobial activities of the penem WY-49605 (SUN 5555) against recent clinical isolates from five US centers. Antimicrobial Agents and Chemotherapy 39, 1591.

7 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1994). In vitro susceptibilities of 185 penicillin susceptible and -resistant pneumococci to WY-49605 (SUN/SY 5555), a new oral penem, compared with those of penicillin G, amoxicillin, amoxicillin-clavulanate, cefixime, cefaclor, cefpodoxime, cefuroxime and cefdinir. Antimicrobial Agents and Chemotherapy 38, 2902–4.[Abstract]

8 . Fuchs, P. C., Barry, A. L. & Sewell, D. L. (1995). Antibacterial activity of WY-49605 compared with those of six other oral agents and selection of disk content for disk diffusion susceptibility testing. Antimicrobial Agents and Chemotherapy 39, 1472–9.[Abstract]

9 . Andrews, J. M. (2000). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[CrossRef][ISI]

10 . Eaves, D. J. & Piddock, L. J. V. (2002). Denaturing HPLC, a rapid method to detect novel and multiple mutations in bacterial genes: detection of gyrA mutations in quinolone-resistant Salmonella enterica. Journal of Clinical Microbiology 40, 4121–5.[Abstract/Free Full Text]





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