Mechanisms of ß-lactam resistance in Pseudomonas aeruginosa: prevalence of OprM-overproducing strains in a French multicentre study (1997)

J. D. Cavallo1,*, P. Plesiat2, G. Couetdic2, F. Leblanc3, R. Fabre and Groupe d’Etude de la Résistance de Pseudomonas aeruginosa aux Bêtalactamines (GERPB)1,§

1 Hôpital d’Instruction 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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One hundred and forty-three non-repetitive strains of Pseudomonas aeruginosa were collected in 13 French hospitals in 1997. A decreased susceptibility or resistance to ticarcillin (MIC > 16 mg/L) was found in 61 isolates (43%) and this was attributed to three major mechanisms: (i) overexpression of OprM and hence related efflux components such as MexAB or MexXY (42.6%), (ii) production of acquired ß-lactamase (29.5%) and (iii) overexpression of chromosomally encoded AmpC cephalosporinase (21.3%). Four of seven ‘intrinsically’ resistant strains (11.5%) with normal amounts of OprM were shown to produce low levels of AmpC, whereas in three isolates no resistance mechanism to ß-lactams could be identified. Overproduction of OprM thus appears as an important mechanism of ticarcillin resistance in French isolates of P. aeruginosa.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is one of the bacteria most frequently responsible for nosocomial infections. Development of resistance to ß-lactams in clinical strains has been associated with the production of acquired ß-lactamases, constitutive overproduction of the cephalosporinase AmpC or non-enzymic mechanisms such as drug efflux or outer membrane impermeability.1 In 1997, the Groupe d’Etude de la Résistance de Pseudomonas aeruginosa aux Bêtalactamines (Workgroup on Resistance of Pseudomonas aeruginosa to ß-Lactam Antibiotics) conducted a study to evaluate the contribution of OprM, and hence related efflux components such as MexAB or MexXY, to the resistance of clinical isolates in France.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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 Mueller–Hinton medium (Bio-Rad). The results were interpreted according to recommendations of the Comité de l’Antibiogramme 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic susceptibilities

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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Table 1.  Distribution of the ß-lactamase hydrolytic profiles among 61 strains with decreased susceptibility to ticarcillin compared with 20 randomly chosen susceptible strains
 
Table 2 indicates the geometric means of MICs of ß-lactams, ciprofloxacin, amikacin, choramphenicol and tetracycline with respect to the mechanisms of ß-lactam resistance reported above. Overproduction of OprM conferred low to medium levels of resistance to ß-lactams (imipenem apart) in the 23 strains devoid of significant ß-lactamase activity. Ticarcillin, aztreonam, cefepime and cefpirome were more affected than piperacillin and ceftazidime by the efflux system. Compared with susceptible strains, MICs of ciprofloxacin and, to a lesser extent, that of tetracyline and chloramphenicol were higher for the OprM overproducers.


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Table 2.  Mechanisms of ß-lactam resistance and antibiotic susceptibilities (geometric mean MIC)
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study showed that most (77%) of the clinical strains of P. aeruginosa resistant to ticarcillin by a ß-lactamase-independent mechanism were OprM overproducers. Overexpression of this active efflux system therefore represents the predominant mechanism (43%) of resistance to ß-lactams, imipenem excepted, among the 61 French isolates collected during the survey. Comparatively, overexpression of AmpC cephalosporinase (21.3%) and production of acquired ß-lactamases (29.5%) are less frequent mechanisms. Data on the expression of efflux systems in clinical strains of P. aeruginosa are still scarce despite accumulating evidence of efflux-overexpressing mutants in the hospital setting.1,5 Several studies have underlined the common occurrence of non-ß-lactamase producers among ticarcillin-resistant strains (up to 74.5%) but without providing precise information on the underlying mechanisms present in these isolates.1,6,7 OprM is the outer membrane component of both MexAB and MexXY efflux systems.8,9 Our results suggest that most of the isolates studied here are MexAB-OprM-overexpressing mutants, because of the antibiotic resistance phenotypes observed.8 This notion is supported by the observation that isolates designated in the literature as ‘intrinsically’ resistant to ticarcillin exhibit susceptibility patterns very similar to those of our OprM-overexpressing mutants.1,6 Typically, MexAB-OprM overproduction increases MICs of ticarcillin and aztreonam by a factor of four to 16, thus making bacteria intermediate or resistant to these agents (according to the recommendations of the CASFM). However, most often, drug efflux appears to be insufficient by itself to confer clinically relevant levels of resistance to other important ß-lactams such as piperacillin, ceftazidime, cefepime or meropenem. As already mentioned elsewhere,5,8 OprM overproduction is associated with cross-resistance to unrelated antibiotics such as tetracycline, chloramphenicol and quinolones. The enhanced levels of resistance to these antibiotics encountered in AmpC overproducers and penicillinase-producing strains that do not overexpress OprM are probably due to unrelated mechanisms.

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 (0.05–0.1 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.


    Acknowledgements
 
The GERPB participants are: R. Ruimy (Paris-Bichat), J. L. Avril (Rennes), C. Bébéar (Bordeaux), J. D. Cavallo (Saint-Mandé), N. Marty (Toulouse), J. Etienne (Lyon), J. Nguyen (Paris-Salpétrière), R. Leclercq (Caen), J. M. Scheftel (Strasbourg), M. H. Nicolas-Chanoine (Paris-Ambroise-Paré), A. Philippon (Paris-Cochin), P. Plésiat (Besançon), J. Sirot (Clermont-Ferrand), P. Nordmann (Le Kremlin Bicêtre) and M. Roussel-Delvallez (Lille).


    Footnotes
 
* Corresponding author. Tel: +33-1-43984734; Fax: +33-1-43985336; E-mail: hia-begin-biologie{at}worldonline.fr Back

§ The GERPB participants are listed in the Acknowledgements. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Cavallo, J. D., Fabre, R., Leblanc, F., Nicolas-Chanoine, M. H., Tabaut, A. & GERPB. (2000). Antibiotic susceptibility and mechanisms of ß-lactam resistance in 1310 strains of Pseudomonas aeruginosa: a French multicentre study (1996). Journal of Antimicrobial Chemotherapy 46, 133–6.[Abstract/Free Full Text]

2 . Comité de l’Antibiogramme 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, 248–54.

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.

5 . Ziha-Zarifi, I., Llanes, C., Köhler, T., Pechère, J. C. & Plésiat, P. (1999). In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM. Antimicrobial Agents and Chemotherapy 43, 287–91.[Abstract/Free Full Text]

6 . Williams, R. J., Livermore, D. M., Lindridge, M. A., Said, A. A. & Williams, J. D. (1984). Mechanisms of beta-lactam resistance in British isolates of Pseudomonas aeruginosa. Journal of Medical Microbiology 17, 283–93.[Abstract]

7 . Chen, H. Y., Yuan, M. & Livermore, D. (1995). Mechanisms of resistance to ß-lactam antibiotics amongst Pseudomonas aeruginosa isolates collected in the UK in 1993. Journal of Medical Microbiology 43, 300–9.[Abstract]

8 . Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, H. & Nishino, T. (2000). Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudo- monas aeruginosa. Antimicrobial Agents and Chemotherapy. 44, 3322–7.[Abstract/Free Full Text]

9 . Poole, K., Tetro, K., Zhao, Q. X., Neshats, S., Heinrichs, D. E. & Bianco, N. (1996). Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression. Antimicrobial Agents and Chemotherapy 40, 2021–8.[Abstract]