Click on image to view larger version.

Fig. 1. Phenotypic detection of ESBL. (a) DCDT for S. maltophilia with CTX-M-1, at 30 mm distance between the discs (centre to centre). Production of ESBL is indicated by the synergy between the CAZ, CTX and CFP discs and the AM/CL disc (arrows). In addition, the CAZ/CL zone was 5 mm larger than that of CAZ, indicating the production of ESBL. (b) DCDT for P. aeruginosa with CTX-M-1, at 20 mm distance between the discs (centre to centre). Synergy is evident between the CFP and CAZ discs and the AM/CL disc, indicating the production of ESBL. The CAZ/CL zone was 5 mm larger than that of CAZ, indicating the production of ESBL. CFP, Cefepime; CPD, cefpodoxime; AM/CL, amoxycillin/clavulanate; CTX, cefotaxime; CAZ, ceftazidime; FOX, cefoxitin; CAZ/CL, ceftazidime/clavulanate.

Simple method to determine -lactam resistance phenotypes in Pseudomonas aeruginosa using the disc agar diffusion test
G. Vedel* Laboratoire de Bactriologie (Pr Claire Poyart) Groupe Hospitalier Cochin SaintVincent-de-Paul La Roche-Guyon, 27, rue du Faubourg Saint-Jacques, 75679 Paris Cedex 14, France

Service de Bacteriologie, Groupe Hospitalier Cochin Saint-Vincent-de-Paul La Roche Guyon, 75679 Paris, France. Tel: +33-1-58-41-15-44; Fax: 33-1-58-41-15-48; E-mail: gerard.vedel@cch.ap-hop-paris.fr
Received 4 April 2005; returned 10 May 2005; revised 15 June 2005; accepted 2 August 2005

Abstract

Background: Pseudomonas aeruginosa is a major opportunistic Abstract bacterial pathogen in nosocomial infections because of the Introduction increasing prevalence of resistance to many of the commonly Materials and methods Results used antibiotics. To ensure optimal efficiency of antibiotic treatment against this species, antibiotic susceptibility tests must Discussion References be interpreted with caution. Most microbiologists now consider it essential to characterize the antibiotic resistance expressed by isolates. Particular resistance mechanisms may be suspected when the bacterium is resistant to several antibiotics in the same family (for example -lactam agents). Methods: Using the disc agar diffusion test, a simple method was developed to distinguish between the common -lactam resistance phenotypes of P. aeruginosa and, consequently, the possible resistance mechanism(s). Over a period of 5 years, we analysed 6300 P. aeruginosa strains isolated from various pathological specimens collected from different wards of Cochin Port-Royal Hospital, and reference and collection strains. Each strain had the wild-type phenotype or an acquired resistance phenotype. Eight antipseudomonal -lactams (ticarcillin, cefotaxime or moxalactam, cefepime or cefpirome, imipenem, ceftazidime, aztreonam, cefsulodin and ticarcillin + clavulanic acid) were used as phenotypic markers. Results: The following markers were sufficient to distinguish between the wild-type phenotype and the various acquired resistance phenotypes: -lactamase synthesis, reduced cell wall permeability and/or increased expression of efflux transporters (active efflux). Detection of resistance phenotypes allows interpretive reading of antibiotic susceptibility tests. Conclusions: Clearly, improved interpretation of antibiotic susceptibility tests is important for a better appreciation of the effect of antimicrobial agents on bacteria such as P. aeruginosa. Keywords: resistance phenotypes , resistance mechanisms interpretive reading , zone diameters

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Introduction

Pseudomonas aeruginosa, produces an inducible Abstract cephalosporinase (class C enzyme), has low outer-membrane Introduction permeability1,2 and has constitutive expression of efflux Materials and methods Results transporters (MexAMexBOprM).3 It is naturally resistant to Discussion aminopenicillins, first-generation and second-generation References cephalosporins and some third-generation cephalosporins such as cefotaxime and moxalactam. However, this bacterium is susceptible to other -lactam agents including carboxypenicillins (carbenicillin, ticarcillin), ureidopenicillins (piperacillin), cephalosporins (cefsulodin, cefoperazone and ceftazidime), monobactams (aztreonam) and carbapenems (imipenem). Certain wild-type strains of P. aeruginosa may acquire resistance to -lactams during treatment. This resistance is most frequently associated with the production of enzymes. Plasmid-encoded -lactamases include oxacillinases (OXAs), Pseudomonas-specific enzymes or carbenicillinases (PSEs/CARBs), TEM-1 or TEM-2.46 Extended-spectrum lactamases (ESBLs) have been observed: (i) ESBLs derived from penicillinases (for example, TEM-4, SHV-2a, PER-1 and VEB-1);710 and (ii) OXA-type ESBLs (for example, OXA-11 and OXA-18).11,12 Metalloenzymes such as IMP and VIM1315 have also been described. Alternatively, selection of constitutively expressed cephalosporinase mutants may also lead to resistance.1,16 The level of resistance varies depending on the type of mutant. Recently, increased expression of systems of efflux transporters (active efflux) was reported to be a major determinant for the resistance of P. aeruginosa strains to -lactams.1719 Some of these mutants, showing increased expression of MexAMexB OprM active efflux system, are resistant to anti-pseudomonal -lactams, but not to imipenem.20 Strains with an increased expression of the MexCMexDOprJ active efflux system have selective cefepime and cefpirome resistance and remain susceptible to other -lactams.21 Other P. aeruginosa, with increased expression of the MexEMexFOprN active efflux system, have carbapenem resistance and remain susceptible to other lactams. MexEMexFOprN active efflux does not seem to exclude -lactams, but is regularly associated with decreased expression of OprD, leading to resistance to carbapenems.21 P. aeruginosa becomes resistant to -lactams by a decrease in its cell wall permeability.2,22 Some strains have selective imipenem resistance. This suggests that imipenem crosses the outer membrane of P. aeruginosa via a specific porin (OprD) not required for the penetration of other -lactam antibiotics.23,24 However, other mechanisms have been suggested to explain the resistance to imipenem.2,13,25 Resistance due to mutation of -lactam targets (penicillin-binding proteins) is also observed in P. aeruginosa.25 Finally, several acquired resistance mechanisms may be found simultaneously in the same strain of P. aeruginosa.

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Because P. aeruginosa is often resistant to many antibiotics, susceptibility testing for the activity of antibiotics must be viewed with caution. The most common measure of susceptibility of bacteria to antibiotics is the MIC. MICs are indirectly determined by disc diffusion tests on agar or using an automated system.26,27 These MICs can be classified into clinical categoriessusceptible (S), intermediate (I) or resistant (R), according to recommended breakpoints.28 Better understanding of antibiotic resistance mechanisms has led to the re-evaluation of bacterial susceptibility or resistance to antibiotics. Most microbiologists now consider it essential to test bacterial pathogens for antibiotic resistance mechanisms.1,29 Resistance mechanisms may be investigated by genetic methods15,29 or by biochemical methods.30 More easily, the presence of these mechanisms may be suspected if the bacteria are resistant to several antibiotics of the same family.1,31 For instance, the disc diffusion test has been used to determine -lactam resistance phenotypes of P. aeruginosa.4 The aim of this study was to develop a simple method for determining -lactam resistance phenotypes of P. aeruginosa using the disc agar diffusion test. Such analysis can indicate the possible mechanism(s) involved in resistance phenotype patterns.1

Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

Origin, identification and distribution of the strains

Over a period of 5 years, 6300 P. aeruginosa strains isolated from various pathological specimens, collected from different wards of Cochin Port-Royal Hospital were studied. The strains were identified by the API 20 NE strip method (bioMrieux, France). These P. aeruginosa strains each showed the wild-type phenotype or an acquired -lactam resistance phenotype. Reference strains (Table 1) with natural resistance phenotypes and characterized acquired resistance phenotypes were used as controls. The phenotypes of these strains are presented in Figure 1.

View this table: Table 1.. Reference strains of P. aeruginosa used as controls [in this window] [in a new window]

View larger version (348K): [in this window] [in a new window]

Figure 1.. (a) P. aeruginosa ATCC 27853 (Table 1) with the wild-type phenotype. Note the antagonistic effect of imipenem on cefsulodin and cefepime (revealed by the blunting of the cefsulodin and cefepime zones adjacent to the imipenem disc). The contents of the numbered discs were as follows: 1, piperacillin + tazobactam; 2, piperacillin; 3, aztreonam; 4, ceftazidime; 5, cefotaxime; 6, ticarcillin; 7, ticarcillin + clavulanic acid; 8, imipenem; 9, cefsulodin; 10, cefoperazone; 11, moxalactam; 12, cefpirome; 13, cefepime. Zone diameters (mm): piperacillin + tazobactam, 32 3; piperacillin, 30 3; aztreonam, 32 3; ceftazidime, 32 3; cefotaxime, 24 4; ticarcillin, 28 3; ticarcillin + clavulanic acid, 29 3; imipenem, 31 2; cefsulodin, 27 3; cefoperazone, 28 3; moxalactam, 25 4; cefpirome, 29 3; cefepime, 31 3. (b) P. aeruginosa PA038 (pMG90) (Table 1) with the low-level penicillinase phenotype (OXA-4). Note the antagonistic effect of imipenem on cefsulodin. (c) P. aeruginosa Dalgleish (Table 1) with the high-level penicillinase phenotype (PSE-4/CARB-1). Note the potentiation of piperacillin and piperacillin + tazobactam by clavulanic acid. Note also the antagonistic effect of imipenem on cefepime. (d) P. aeruginosa RNL-1 (Table 1) with the ESBL phenotype (PER-1). Note the potentiation of cefotaxime, ceftazidime and aztreonam by clavulanic acid. (e) P. aeruginosa. Momon (Table 1) with the high-level cephalosporinase phenotype. Note the activity of imipenem and ceftazidime zone diameter < aztreonam zone diameter. (f) P. aeruginosa. CN17203 (Table 1) with the metallo--lactamase phenotype. Note the resistance to imipenem and ceftazidime zone diameter < aztreonam zone diameter. (g) P. aeruginosa PAO4098E (Table 1) with the MexAMexBOprM active efflux phenotype. Note ceftazidime zone diameter > aztreonam zone diameter. Note also the antagonistic effect of imipenem on cefsulodin and cefepime. (h) P. aeruginosa PAO1 (ERYR) (Table 1) with the MexC MexDOprJ active efflux phenotype. Note the antagonistic effect of imipenem on cefsulodin. (i) P. aeruginosa H729 or P. aeruginosa PAO 7H (Table 1) with the selective impermeability to imipenem phenotype and/or the MexEMexFOprN active efflux phenotype. Note the antagonistic effect of imipenem on

cefsulodin and cefepime. (j) P. aeruginosa (clinical isolate) with the high-level penicillinase phenotype and with the MexAMexBOprM active efflux phenotype. Note the resistance to ticarcillin. Note also ceftazidime zone diameter > aztreonam zone diameter. (k) P. aeruginosa (clinical isolate) with the high-level cephalosporinase (without antagonistic effect) phenotype and with the selective impermeability to imipenem phenotype and/or the MexEMexFOprN active efflux phenotype. Note ceftazidime zone diameter < aztreonam zone diameter. Note also the low activity of imipenem. (l) P. aeruginosa (clinical isolate) with the MexAMexBOprM active efflux phenotype and with the selective impermeability to imipenem phenotype and/or the MexEMexFOprN active efflux phenotype. Note ceftazidime zone diameter > aztreonam zone diameter. Note also the low activity of imipenem. Antibiotic susceptibility testing Susceptibility to -lactams was determined by the disc diffusion test26,28 using Mueller Hinton medium (Bio-Rad, France) and an inoculum of 106 cfu/mL. The following lactam discs (Bio-Rad) were tested: ticarcillin (75 g) ticarcillin + clavulanic acid, (75 + 10 g), piperacillin (75 g), piperacillin + tazobactam (75 + 10 g), cefoperazone (30 g), cefsulodin (30 g), cefotaxime (30 g), moxalactam (30 g), cefepime (30 g), cefpirome (30 g), ceftazidime (30 g), aztreonam (30 g) and imipenem (10 g). The ticarcillin + clavulanic acid disc was placed in the centre of the plate (Figure 1) to visualize putative synergy. Detection of -lactam resistance phenotypes P. aeruginosa showing resistance to several -lactams in disc diffusion tests may be suspected of harbouring resistance mechanisms to -lactams. Each mechanism of resistance shows a specific phenotypic pattern. Eight -lactams may be used as phenotypic detection markers (Figure 1). The four major markers include ticarcillin, cefotaxime (or moxalactam), ceftazidime and imipenem. Other -lactams used were cefsulodin, cefepime (or cefpirome) and aztreonam to differentiate certain phenotypes, clavulanic acid combined with ticarcillin to visualize possible synergy (Figure 1c and d) and imipenem to detect cephalosporinase from an inducible chromosomal gene (see antagonistic effects, Figure 1a). Acquired resistance mechanisms in P. aeruginosa increase the MICs of the antibiotics. Consequently, growth inhibition zone diameters around the antibiotic discs may be decreased. Using phenotypic detection markers, the comparison between zone diameters for a resistant strain (Figure 1bl) and reference zone diameters for wild-type strains

(Figure 1a) can indicate the resistance phenotype. For a marker, a zone smaller than the wild-type zone signifies an inhibition of this marker.

Results
Top Abstract Introduction Materials and methods Results Discussion References

Using the disc diffusion test, the eight phenotypic detection marker antibiotics were sufficient to identify the wild-type phenotype and the common -lactam resistance phenotypes of P. aeruginosa previously described.4 Wild-type phenotype

Wild-type strains of P. aeruginosa (Figures 1a and 2) having natural resistance mechanisms were susceptible to the phenotypic detection markers including ticarcillin, ceftazidime, aztreonam, ticarcillin + clavulanic acid, imipenem, cefsulodin and cefepime. With these antibiotics, the zone diameters were 30 mm. However, cefotaxime (or moxalactam), which give a smaller zone diameter, was less active against these strains. Imipenem antagonized the activity of other -lactams (particularly cefsulodin and cefepime).

Figure 2.. Flow diagram to determine -lactam resistance phenotypes in P. aeruginosa. (mm), zone diameters; Tic. + clav. ac., ticarcillin + clavulanic acid; 3GC, third-generation cephalosporins. Major marker antibiotics are shown boxes. Reference zone diameters for wild-type View larger version (19K): strains (see legend to Figure 1). Wild-type, wildtype phenotype; MexEFOprN, MexEMexF [in this window] OprN active efflux phenotype; OprD, selective [in a new window] impermeability to imipenem phenotype; MexCD OprJ, MexCMexDOprJ active efflux phenotype; Low-level Pase, low-level penicillinase phenotype; High-level Pase, high-level penicillinase phenotype; ESBL, extended-spectrum -lactamase phenotype; MBL, metallo--lactamase phenotype; High-level Case, high-level cephalosporinase phenotype;

MexABOprM, MexAMexBOprM active efflux phenotype. Acquired resistance phenotype due to plasmid-encoded penicillinase production Two phenotypes were observed. (i) A low-level penicillinase phenotype (Figures 1b and 2), often due to production of OXAs (e.g. OXA-1 and OXA-4) and TEM-1 or TEM-2: these strains were intermediate or resistant to ticarcillin and showed slightly reduced susceptibility to cefsulodin. The activities of cefotaxime, ceftazidime, aztreonam and imipenem against these strains were similar to those observed for wild-type phenotype (Figures 1a and 2). Imipenem antagonized cefsulodin and/or cefepime activity. When OXA-type enzymes were produced, clavulanic acid did not totally restore susceptibility to ticarcillin. Consequently, the zone diameter of ticarcillin + clavulanic acid was generally smaller than that for the wild-type. No synergic effect was observed between clavulanic acid and other -lactams. Finally, the activity of cefepime and cefpirome was reduced. (ii) A high-level penicillinase phenotype, caused by the production of PSE/CARB enzymes (Figures 1c and 2), was differentiated from the first penicillinase phenotype by a highlevel of resistance to ticarcillin and cefsulodin. Clavulanic acid generally failed to increase the susceptibility of -lactamase producers to ticarcillin. However, except for the penicillinase hyperproducers, synergy was observed between ticarcillin + clavulanic acid and other -lactams (such as piperacillin and piperacillin + tazobactam). Acquired resistance phenotype due to ESBL production P. aeruginosa isolates with the extended-spectrum -lactamase phenotype (Figures 1d and 2) were resistant to ticarcillin, cefotaxime, ceftazidime, aztreonam, cefsulodin and all other -lactams studied, but were susceptible to imipenem. Clavulanic acid did not always restore susceptibility to ticarcillin. Nevertheless, a synergic effect was usually observed between ticarcillin + clavulanic acid and cefotaxime, ceftazidime or aztreonam; when ESBLs derived from penicillinases (for example, PER-1) were produced. Acquired resistance phenotype due to constitutive cephalosporinase production The cephalosporinase constitutive mutants (derepressed mutants) gave high-level cephalosporinase phenotype (Figures 1e and 2). This phenotype was primarily differentiated from the penicillinase phenotypes (Figures 1b, c and 2) by a total lack of susceptibility to cefotaxime, whereas ticarcillin generally retained an effect. The hydrolysis of cefsulodin, ceftazidime and aztreonam was variable, but ceftazidime was always less active than aztreonam (zone diameter of ceftazidime < zone diameter of aztreonam). Only imipenem was active against these strains. In addition, the presence of irregular, crenellated, zone edges, consisting of scatter colonies were often observed with strains of this phenotype. Two different phenotype patterns and levels of resistance were observed. (i) A high-level cephalosporinase (with an antagonistic effect) phenotype: in this case, the antagonistic effects of imipenem on other -lactams were slight. Clavulanic acid did not restore susceptibility to ticarcillin. In contrast, the zone diameter of ticarcillin + clavulanic acid was frequently lower than that of ticarcillin alone.

(ii) A high-level cephalosporinase (without antagonistic effect) phenotype: for these derepressed mutants, no antagonistic effect of imipenem on cefsulodin or cefepime was observed. Acquired resistance phenotype due to metallo--lactamase production P. aeruginosa of this phenotype (Figures 1f and 2) were resistant to ceftazidime, while their susceptibilities to aztreonam, piperacillin and carbapenems appeared to be diverse. However, some of these strains remained susceptible to aztreonam. Acquired resistance phenotype due to increased expression of MexAMexBOprM active efflux system In this case, many different phenotype patterns and levels of resistance were observed. The phenotype most prevalent among our samples (Figures 1g and 2) was characterized by a decrease in susceptibility to ticarcillin and cefotaxime. The activities of other antipseudomonal -lactams were variable. The activity of ceftazidime was higher than that of aztreonam (zone diameter ceftazidime > zone diameter aztreonam); thereby differentiating this phenotype from high-level cephalosporinase phenotype, for which ceftazidime zone diameters was smaller than that of aztreonam (Figures 1e and 2). Moreover, imipenem antagonized cefsulodin and cefepime. Acquired resistance phenotype due to increased expression of MexCMexDOprJ active efflux system P. aeruginosa of this phenotype (Figures 1h and 2) showed reduced susceptibility to cefepime (or cefpirome). With this exception this phenotype was similar to the wild-type phenotype (Figures 1a and 2). Acquired resistance phenotypes due to selective impermeability to imipenem (OprD) and/or to increased expression of MexEMexFOprN active efflux system P. aeruginosa of these phenotypes (Figures 1i and 2) were poorly or not susceptible to imipenem. With this exception these phenotypes were similar to the wild-type phenotype (Figures 1a and 2). Complex phenotypes Many P. aeruginosa strains isolated from various pathological specimens expressed two or more acquired resistance mechanisms and gave complex phenotypes (Figure 1j, k and l). These mechanisms were predicted using Figure 2.

Discussion

Emergence of P. aeruginosa clinical isolates exhibiting acquired resistance mechanisms is a problem when choosing therapy. In particular the prevalence of resistance to -lactams, the most important group of antiTop Abstract pseudomonal agents, is growing. In France, 42% of hospital Introduction clinical isolates of P. aeruginosa are resistant or presented an Materials and methods intermediate susceptibility to ticarcillin. Mechanisms are as Results follow: 14.5% non-enzymatic mechanism, 12.5% Discussion overproduction of constitutive cephalosporinase, 7.1% References transferable -lactamase and 6.9% a combination of these mechanisms. The prevalence among transferable -lactamases is: PSE-1, 71.6%; TEM-2, 19.4% and OXA, 7.5%.32 Moreover, 19% of P. aeruginosa clinical isolates are resistant to imipenem.32 During a 5 year period, we analysed 6300 P. aeruginosa strains isolated from various pathological specimens collected from different wards of Cochin Port-Royal Hospital. The distribution of the strains showing each type of resistance mechanism was in agreement with the above results. Most microbiologists now consider it useful to analyse the type of resistance by susceptibility testing.1,31 Thus using the disc diffusion test, resistance phenotypes indicate which resistance mechanisms are expressed by the infecting organism. With our simple method, the -lactam agents, ticarcillin, ticarcillin + clavulanic acid, cefotaxime (or moxalactam), ceftazidime, cefsulodin, cefepime (or cefpirome), aztreonam and imipenem used as phenotypic detection markers were necessary and sufficient to determine -lactam resistance phenotypes of P. aeruginosa. Ticarcillin was the best indicator of penicillinase phenotypes, and cefsulodin distinguished the low-level penicillinase phenotype from the high-level penicillinase phenotype. Either cefotaxime or moxalactam could be used to detect the high-level cephalosporinase phenotype. The relative zone diameters of ceftazidime and aztreonam were useful to differentiate the preceding phenotypes from the MexAMexBOprM active efflux phenotype. Cefepime or cefpirome were the markers of MexCMexDOprJ active efflux phenotype. Similarly, the use of imipenem detected the imipenem-specific impermeability phenotype or the MexEMexFOprN active efflux phenotype and revealed any antagonistic effect of this carbapenem on other -lactams in P. aeruginosa. Ticarcillin + clavulanic acid detected the effect of a -lactam inhibitor on the -lactamase activity. Optimal determination of phenotypes requires comments. For instance, cefotaxime and moxalactam had a lower activity than ceftazidime against the strains of the wild-type phenotype; this is due to the natural low permeability of the outer membrane and to the constitutive MexAMexBOprM active efflux system of P. aeruginosa. Within the phenotype groups, resistance phenotype patterns differed, as a consequence of the type of mutant, the type of penicillinase, the amount of enzyme produced or the amount of lactam agent bound to -lactam targets.1,4 P. aeruginosa of the high-level cephalosporinase phenotype constitutively expresses a cephalosporinase. The strains

exhibiting the other phenotypes have inducible cephalosporinase activities, and thus imipenem (an inducer) antagonized other -lactams. Clavulanate had inhibitory effects on plasmid-encoded -lactamases. Unfortunately, P. aeruginosa isolates producing plasmidencoded -lactamases were often resistant to inhibitor -lactam associations. This does not seem to reflect the resistance of the enzyme to inhibition but may be due to the impermeability of the organism or a -lactam efflux mechanism. Nevertheless, the OXAtype enzymes have been reported to be less susceptible to clavulanic acid in other species.1 Clavulanic acid is not a good inhibitor of cephalosporinase and, consequently, it cannot restore the activity of ticarcillin. In contrast, it is a stronger inducer than is ticarcillin, which can then be hydrolysed by the induced enzyme.1 Consequently, for the high-level cephalosporinase phenotype ticarcillin + clavulanic acid was frequently less active than unprotected ticarcillin. Finally, to differentiate the metallo--lactamase phenotype from other phenotypes, such as ESBL and high-level cephalosporinase phenotypes, two methods of the double-disc synergy test using a different combination of substrates and inhibitors (ceftazidime-2-mercaptopropionic acid and imipenem-EDTA) have been described.33,34 Many P. aeruginosa isolates express two or more acquired resistance mechanisms (plasmid-encoded -lactamase production + selective impermeability to imipenem, constitutive cephalosporinase production + selective impermeability to imipenem, plasmid-encoded -lactamase production + active efflux + selective impermeability to imipenem, etc.). These mechanisms may act in synergy and give complex phenotypes. Combining the effect of each mechanism, the complex phenotypes resulting from the presence of more than one mechanism may be predicted using Figure 2. 35 However, complex phenotypes are sometimes poorly differentiated by the disc diffusion test. In these cases, further investigations using physicochemical and genetic methods should be undertaken to identify the resistance mechanisms.15,29,30 Finally, some strains of P. aeruginosa isolated from chronic infections (for example, in patients with cystic fibrosis) give indeterminate phenotypes. This property is due to this organism's ability to synthesize a mucoid shield (exopolysaccharide coat). Detection of resistance phenotypes of P. aeruginosa requires disc agar diffusion tests to be performed meticulously; in particular, the inoculum density must be carefully standardized. The density of viable cells in the inoculum is one of the most important variables that influence the results of susceptibility testing.4 Heavy inocula tend to give small zones of inhibition. Thus, an inoculum of 106 viable cells is generally recommended. The results of disc diffusion tests may also be influenced by the type of equipment and reagents. Detection of resistance phenotypes allows interpretive reading of the antibiotic susceptibility test results.1,36 It may modify the classification indicated by antibiotic susceptibility tests based on breakpoints.28 It is essential to avoid clinical failures, as observed with other bacteria (e.g. Enterobacteriaceae).37,38 This interpretive reading is, moreover, recommended by the French Antibiotic Sensitivity Testing Committee (CASFM).28 Optimal reading of antibiotic susceptibility tests requires a thorough knowledge of resistance mechanisms. Thus identification of resistance mechanisms can

be laborious, although expert systems for determining resistance mechanisms using automated susceptibility tests are now available and facilitate the microbiologist's work.27 Clearly interpretive reading of antibiotic susceptibility tests is essential for a satisfactory understanding of the action of antibacterial agents. Our method, using the disc agar diffusion test and only eight anti-pseudomonal -lactams as phenotypic detection markers, simplifies the detection of -lactam resistance phenotypes of P. aeruginosa and the interpretive reading of their susceptibility tests.

Acknowledgements
I thank J. D. Cavallo, R. Fabre, R. Labia, G. Paul, A. Philippon and P. Plesiat for generously providing reference strains.

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