In vitro activity of faropenem and 20 other compounds against ß-lactamase-positive and -negative Moraxella catarrhalis and Haemophilus influenzae isolates and the effect of serum on faropenem MICs

F.-J. Schmitza,b,*, M. Boosa, S. Mayera, J. Verhoefb, D. Milatovicb and A. C. Fluitb

a Institute for Medical Microbiology and Virology, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, Geb. 22.21, D-40225 Düsseldorf, Germany; b Eijkman-Winkler-Institute for Medical Microbiology, Utrecht, The Netherlands

Sir,

Faropenem is a novel penem antimicrobial agent intended for oral administration. Preliminary reports indicate that faropenem'sin vitro activity is not only broad spectrum (active against Gram-negative, Gram-positive and some anaerobic bacteria), but also bactericidal.1,2

The purpose of the present study was to evaluate the in vitro activity of faropenem against ß-lactamase-positive and -negative Moraxella catarrhalis and Haemophilus influenzae isolates and to examine the effect, if any, that the presence of serum has on faropenem MICs for these respiratory tract pathogens.

This study compared the activities of faropenem and 20 other oral and/or parenteral antimicrobial agents against distinct recent European clinical isolates of M. catarrhalis (n = 419) and H. influenzae (n = 234). The isolates were collected from patients with bacteraemia, community-acquired respiratory tract infections or nosocomial pneumonia, from the University Hospital of Düsseldorf or during international surveillance studies.3

Cation-adjusted Mueller–Hinton broth was used for M. catarrhalis; H. influenzae was tested on Haemophilus test medium by the NCCLS broth microdilution method. To examine the effect of the presence of serum on faropenem MICs, the broth microdilution tests were also performed for 20 M. catarrhalis and 20 H. influenzae randomly selected strains in appropriate media in the presence of heat-inactivated, pooled human serum. Fifty per cent of the 40 isolates tested displayed ß-lactamase production. The serum concentrations tested were: 20, 50, 60, 70, 80 and 90% v/v.

ß-Lactamase activity was detected using the chromogenic cephalosporin disc ß-lactamase assay containing nitrocefin as the substrate (Cefinase discs; BBL Microbiology Systems, Cockeysville, MD, USA).

All of the ß-lactamase-positive M. catarrhalis isolates were selected for ß-lactamase extraction and isoelectric focusing (IEF) of the enzymes as described by Richter et al.4 IEF of ß-lactamase enzymes was performed using commercially prepared polyacrylamide gels with a pH range of 5.5–8.5.4 PCR amplification and sequencing of the putative promotor region and of a part of the bro gene of the ß-lactamase positive M. catarrhalis isolates were performed as described by Bootsma and colleagues.5 Two independent amplicons were generated and at least one strand of the second PCR product was sequenced to confirm that no mismatches were created upon amplification.

Of the 419 M. catarrhalis isolates, 385 (92%) were ß-lactamase positive. IEF and sequencing displayed corresponding results for all ß-lactamase-producing isolates. BRO-2 ß-lactamase was detected in 22 (5.7%) of the 385 isolates. As expected, all ß-lactamase-negative isolates gave negative results in the IEF as well as in PCR screening for bro genes. As described previously, we found the 21 bp deletion in all of the sequences of the bro2-containing isolates.5

Of the 234 H. influenzae isolates, 34 (15%) were ß-lactamase positive. In M. catarrhalis and H. influenzae, ß-lactamase production was detected at similar levels in both hospital and community isolates, regardless of the specimen source or the age of the subject.

The TableGo shows the MICs of faropenem and 11 other ß-lactam antibiotics for the strains studied.


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Table. Susceptibility of 419 M. catarrhalis and 234 H. influenzae isolates to 11 ß-lactam antibiotics and faropenem
 
Against M. catarrhalis, cefixime proved to be the most active cephalosporin, based on MIC90 values. Furthermore, we found no resistance to cephalosporins even though increased MICs of cephalosporins and resistance to cefaclor have been reported previously.6 Our BRO-2 isolates often showed only slight differences in MIC when compared with strains that did not produce ß-lactamase. Faropenem exhibited good in vitro activity with MIC90 values of 1 mg/L for BRO-1 and, notably, 0.25 mg/L for BRO-2 and ß-lactamase-negative isolates. All of the five quinolones tested (ciprofloxacin, clinafloxacin, levofloxacin, gatifloxacin, gemifloxacin) exhibited good in vitro activity, with a maximal MIC of 1 mg/L of ciprofloxacin (data not shown). Although resistance to tetracycline and macrolides has been reported previously,6 none of the European isolates tested showed resistance (data not shown).

Against H. influenzae the activity of faropenem was comparable to that of co-amoxiclav, whereas the newer cephalosporins were more active than faropenem. Faropenem exhibited good MIC50 and MIC90 values of 0.5 and 1 mg/L, respectively, for ß-lactamase-positive and -negative isolates. All of the isolates tested were susceptible to the quinolones tested, with a maximal MIC of 1 mg/L of ciprofloxacin, whilst resistance rates to tetracyclines and macrolides were <1% (data not shown).

The faropenem MICs for all 40 isolates tested in the presence of heat-inactivated human serum were within one (34/40) or two (6/40) doubling dilution of the MIC in the absence of serum, without any influence of ß-lactamase production. At 70% v/v addition of serum, all 40 isolates grew, but at 80% v/v addition of serum, two of 20 M. catarrhalis isolates and 11 of 20 H. influenzae isolates failed to grow.

In summary, the data indicate that faropenem was one of the most potent agents with an MIC90 of 1 mg/L for all isolates tested. The addition of serum, in concentrations ranging from 20 to 70%, did not result in markedly higher MICs. The highest MIC observed for M. catarrhalis isolates was 1 mg/L, indicating that all isolates tested, including those with ß-lactamase activity, were susceptible to the drug based on a provisional breakpoint of <1 mg/L. In H. influenzae only five of 234 (2.1%) isolates displayed MICs of faropenem of >=2 mg/L. Based on the in vitro data and owing to its oral bioavailability, faropenem appears to be a promising new antimicrobial agent in respiratory tract infections and warrants further clinical investigation.

Notes

* Corresponding author. Tel/Fax: +49-2132-72040; E-mail: schmitfj{at}uni-duesseldorf.de Back

References

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2 . Boswell, F. J., Andrews, J. M. & Wise, R. (1997). Pharmacodynamic properties of faropenem demonstrated by studies of time–kill kinetics and postantibotic effect. Journal of Antimicrobial Chemotherapy 39, 415–8. [Abstract]

3 . Milatovic, D., Schmitz, F. J., Brisse, S., Verhoef, J. & Fluit, A. C. (2000). In vitro activities of sitafloxacin (DU-6859a) and six other fluoroquinolones against 8,796 clinical bacterial isolates. Antimicrobial Agents and Chemotherapy 44, 1102–7. [Abstract/Free Full Text]

4 . Richter, S. S., Winokur, P. L., Brueggemann, A. B., Huynh, H. K., Rhomberg, P. R., Wingert, E. M. et al. (2000). Molecular characterization of the beta-lactamases from clinical isolates of Moraxella (Branhamella) catarrhalis obtained from 24 U.S. medical centers during 1994–1995 and 1997–1998. Antimicrobial Agents and Chemotherapy 44, 444–6. [Abstract/Free Full Text]

5 . Bootsma, H. J., Van Dijk, H., Verhoef, J., Fleer, A. & Mooi, F. R. (1996). Molecular characterization of the BRO beta-lactamase of Moraxella (Branhamella) catarrhalis. Antimicrobial Agents and Chemotherapy 40, 966–72. [Abstract]

6 . McGregor, K., Chang, B. J., Mee, B. J. & Riley, T. V. (1998). Moraxella catarrhalis: clinical significance, antimicrobial susceptibility and BRO beta-lactamases. European Journal of Clinical Microbiology and Infectious Diseases 17, 219–34. [ISI][Medline]