1 Hôpital dInstruction des Armées Bégin, 69 Avenue de Paris, 94163 St Mandé; 2 Faculté de Médecine de Besançon, Besançon; 3 Laboratoire Glaxo-Wellcome, 100 route de Versailles, 78163 Marly le Roi, France
Received 3 April 2002; returned 13 June 2002; revised 12 July 2002; accepted 23 July 2002
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Abstract |
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Introduction |
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Materials and methods |
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The first 11 non-repetitive isolates of P. aeruginosa, isolated in May 1997 in the 13 collecting centres, were sent to a central laboratory for investigation.
Determination of MICs
MICs of 14 antibiotics were determined by the agar dilution method in MuellerHinton medium (Bio-Rad). The results were interpreted according to recommendations of the Comité de lAntibiogramme de la Société Française de Microbiologie (CASFM).2
Detection and identification of ß-lactamases and cephalosporinase assays
ß-Lactamase identification assays were performed on isolates presenting decreased susceptibility or resistance to ticarcillin (MIC > 16 mg/L) and cephalosporinase assays were performed only for the non-penicillinase producers. Isoelectrofocusing and determination of hydroelectrolytic activity by a macro-acidimetric technique were used for ß-lactamase identification and have been described previously.1
Cephalosporinase AmpC activities were determined for strains by a spectrophotometric assay (UV) on lysate supernatants. Changes in absorbance resulting from enzymic hydrolysis of cefaloridine were recorded at 255 nm. ß-Lactamase activities were expressed in µmoles of cefaloridine hydrolysed per minute and per milligram of protein. Protein concentrations were estimated by the method of Bradford.3 Preliminary assays showed that cephalosporinase levels are always <0.1 µmol/min/mg protein in wild-type susceptible strains. Isolates producing higher levels of enzyme were thus considered as cephalosporinase overproducers.4
Detection of OprM in outer membrane extracts
OprM overproduction was determined in all clinical strains with decreased susceptibility or resistance to ticarcillin, except for those with acquired ß-lactamase. This overproduction was assessed by immunodetection of OprM, the outer membrane component of the efflux system, using a specific, polyclonal antibody.5 Preparation of bacterial outer membranes and the conditions of western blotting have been described in detail previously.5 Since OprM levels only increase two to three times in OprM-overexpressing mutants, western blots of isolates showing intrinsic resistance to ticarcillin (AmpC activity <0.1 µmol/min/mg protein) were compared with those of 20 randomly chosen ticarcillin-susceptible strains used as negative controls. OprM bands on immunoblots were estimated semi-quantitatively from computerized pictures generated by the Gel Doc program (Bio-Rad). Resistant strains were considered as OprM overproducers when the amounts of OprM in their outer membranes were at least twice that of the control strains.
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Results |
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Forty-six per cent of the 143 strains collected were found to be susceptible to cefpirome, 57% to ticarcillin and aztreonam, 64% to cefepime, 71% to piperacillin, 78% to ceftazidime and 91% to imipenem. In addition, 74% of the isolates were susceptible to amikacin and 61% to ciprofloxacin.
Mechanisms of resistance to ß-lactams
Mechanisms of resistance to ß-lactams were analysed in the 61 ticarcillin-resistant strains (43% of the isolates) (Table 1). Eighteen isolates (29.5%) were found to produce an acquired ß-lactamase, 13 (21.3%) overproduced the chromosomally encoded cephalosporinase AmpC (0.1 µmol/min/mg protein), whereas the remaining 30 strains (49.2%) did not display significant ß-lactamase activities. Overexpression of OprM, the outer membrane component of the MexAB and MexXY efflux system, was frequent in this latter group (23 of 30 isolates), but was also detected in three AmpC-overproducing strains. This overexpression was not found for the 20 control strains susceptible to ticarcillin. The ß-lactamase isoelectric points and hydrolytic profile were indicative of a carbenicillinase in 17 of 18 isolates with acquired ß-lactamase. One strain turned out to produce an oxacillinase enzyme (Table 1).
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Discussion |
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In this study, seven intrinsically ticarcillin-resistant strains were found to contain normal amounts of OprM in their outer membranes. Four of them produced borderline levels of AmpC cephalosporinase (0.050.1 µmol/min/mg protein) that could account for their lower susceptibility to ticarcillin. The remaining three isolates displayed wild-type ß-lactamase activities (<0.04 µmol/min/mg protein). However, since AmpC is inducible in P. aeruginosa, the ß-lactamase activity may not be a true reflection of the level of expression during ß-lactam challenge. The induced level of expression may be sufficient to synergize with pump overexpression and cause resistance. Furthermore, resistance could be due to porin impermeability, reduced affinity of penicillin-binding proteins or production of other efflux mechanisms (MexCD-OprJ, MexEF-OprN).
In conclusion, overproduction of OprM is frequently involved in the resistance of French P. aeruginosa isolates to ß-lactams. Further investigations are now needed to clarify the therapeutic relevance of this commonly occurring mechanism.
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Acknowledgements |
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Footnotes |
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The GERPB participants are listed in the Acknowledgements.
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References |
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2 . Comité de lAntibiogramme de la Société Française de Microbiologie. (1999). Communiqué 1999. Société Française de Microbiologie. [Online.] http://www.sfm.asso.fr/
3 . Bradford, M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Annals of Biochemistry 72, 24854.
4 . Thabaut, A., Girardet M., Bercion, R. & Labia, R. (1993). Expression of the chromosomal class I ß-lactamase in 1508 clinical isolates of Pseudomonas aeruginosa. In Program and Abstracts of the Eighteenth International Congress of Chemotherapy, Stockholm, Sweden, 1993. Abstract 922, p. 276.
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