JAC Advance Access published online on April 5, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm076
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Susceptibility of Pseudomonas aeruginosa to antimicrobials: a 2004 French multicentre hospital study
1 Ecole du Val de Grâce, Paris et Hôpital d'Instruction des Armées Bégin, St Mandé, France 2 Faculté de médecine de Besançon, Besançon, France 3 CHRU Hôpital Calmette Lille, France
* Correspondence address. Hôpital d'Instruction des Armées Bégin, 69 Avenue de Paris, 94163 St Mandé, France. Tel: +33-1-43-98-52-91; Fax: +33-1-43-98-53-36; E-mail: hia-begin-biologie{at}woldonline.fr
Received 19 December 2006; returned 29 January 2007; revised 14 February 2007; accepted 22 February 2007
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Abstract |
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Objectives: Pseudomonas aeruginosa is a major causative agent of hospital infections. The purpose of this study was to determine the antibiotic susceptibility of P. aeruginosa in a French multicentre study and to investigate the mechanisms of ß-lactam resistance.
Methods: Four hundred and fifty non-repetitive strains of P. aeruginosa were collected in 15 French university hospitals in 2004. MICs of antibiotics were measured by agar dilution methods. For all the strains with MICs of ticarcillin >16 mg/L, detection and identification of the ß-lactamases, quantitative determination of cephalosporinase and overproduction of the MexAB-OprM efflux pump were evaluated.
Results: The percentages of susceptible isolates were as follows: ticarcillin, 62%; ticarcillin+clavulanic acid, 61%; piperacillin, 78%; piperacillin+tazobactam, 80% (MICs 
16 mg/L); aztreonam, 50%; ceftazidime, 78%; cefepime, 64%; imipenem, 83%; tobramycin, 80% (MICs 4 mg/L); amikacin, 86% (MIC 8 mg/L); ciprofloxacin, 68%; and levofloxacin, 57% (MICs 1 mg/L). Decreased susceptibility to imipenem was linked in two cases to VIM-type carbapenemase production. Overexpression of the AmpC cephalosporinase, production of acquired ß-lactamases including SHV2a extended-spectrum ß-lactamase and overproduction of the MexAB-OprM efflux pump were present in 16.9%, 6.5% and 22.3% of the strains, respectively.
Conclusions: In the last decade, the overall susceptibility of P. aeruginosa hospital isolates to antibiotics has remained quite stable in France. However, the emergence of extended-spectrum ß-lactamases and carbapenemases in different locations is a matter of concern.
Key Words: Pseudomonas spp. , mechanisms of resistance , antimicrobial resistance surveillance , ß-lactams
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Introduction |
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Pseudomonas aeruginosa is one of the major species responsible for hospital infections. It is also notorious for its capability to readily develop resistance to first-line antibiotics.1 In this context, multicentre epidemiological studies have a considerable interest for empirical antibiotic treatment strategies and for the surveillance of new resistance mechanisms. This is why we conducted in 2004 a nationwide epidemiological study involving 15 microbiology laboratories from university hospitals geographically distributed in the main French areas.
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Materials and methods |
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Bacterial strains
The first 30 consecutive non-repetitive clinical strains of P. aeruginosa isolated in April and May 2004, excluding strains from cystic fibrosis patients, were collected in each of the 15 participating centres. After serotyping, all the isolates were sent to a central laboratory for investigation.
MICs of ticarcillin, ticarcillin+clavulanic acid, piperacillin, piperacillin+tazobactam, aztreonam, ceftazidime, cefepime, imipenem, amikacin, tobramycin, ciprofloxacin and levofloxacin were determined by the agar dilution method.2 Results were interpreted according to 2006 recommendations of the Comité de l'Antibiogramme de la Société Française de Microbiologie.3 P. aeruginosa ATCC 27853 was used as a control in each of the series.
Detection and identification of ß-lactam resistance mechanisms
Identification of secondary ß-lactamases, assays for AmpC cephalosporinase and determination of MexAB-OprM efflux pump expression levels were performed in all isolates presenting a decreased susceptibility or a resistance to ticarcillin (MIC > 16 mg/L).
Detection and identification of the ß-lactamases were obtained by isoelectric focusing on polyacrylamide gels4,5 associated with detection of the genes encoding ß-lactamase by real-time PCR using the LightCycler instrument (Roche Diagnostics, Meylan, France). The identification was completed by sequencing the amplification products on a CEQ 8000 Genetic Analysis System (Beckman Coulter). The forward and reverse primer sequences used for PCR were, respectively, as follows: pse, ACC GTA TTG AGC CTG ATT TA and ATT GAA GCC TGT GTT TGA GC; tem, ATA AAA TTC TTG AAG ACG AAA and GAC AGT TAC CAA TGC TTA ATC A; shv, TCA GCG AAA AAC ACC TTG and TCC CGC AGA TAA ATC ACC A; veb, AAA ATT GGG CAA CCC CAA CA and GTG GCT GCT GCA ATT CCA TT; ges, CGT CGA TCC CCA AGG AGA GA and GCT GCC TCG AGA ACC GTC AT; per, ATG AAT GTC ATT ATA AAA GC and AAT TTG GGC TTA GGG CAG AA; vim, GCA CTT CTC GCG GAG AT and ACT GGA CCG AAG CGC ACT G; imp, CTA CCG CAG CAG AGT CTT TG and AAC CAG TTT TGC CTT ACC AT; oxa-10 group, TCA ACA AAT CGC CAG AGA AG and TCC CAC ACC AGA AAA ACC AG; oxa-1 group, TAT CTA CAG CAG CGC CAG TG and CGC ATC AAA TGC CAT AAG TG; and oxa-2 group, CGA TAG TTG, TGG CAG ACG AA and CAT TGC TGA TCG CAA ATC TT.
AmpC cephalosporinase activities were determined in all strains by a spectrophotometric analysis using nitrocefin as a substrate, except in those producing acquired ß-lactamases.4 Enzymatic activity was expressed in milli-units (mU) per mg of protein. A unit is defined as the quantity of enzyme hydrolysing 1 µmol of substrate per minute at pH 7 at 37°C. The average of 10 determinations for reference strain PAO1 was 20 mU/mg with a range of 11–33 mU/mg. Preliminary assays on a large panel of other wild-type strains showed that cephalosporinase production never exceeded 100 mU/mg protein. Strains producing >100 mU (an increase of 5-fold with respect to PAO1) were thus classified as cephalosporinase overproducers.
Overproduction of the MexAB-OprM efflux pump was assessed in all strains with ticarcillin MICs >16 mg/L (n = 171) by quantitative real-time PCR after RT–PCR, as described previously.6 Expression levels of the gene mexB in the strains were further normalized by using the housekeeping gene rpsl. Results were compared with those of reference strain PAO1 and of the MexAB-OprM-overproducing mutant PT629. Strains with mexB expression at least two times higher than in PAO1 were considered as mexAB-oprM-overexpressing isolates.
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Results |
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Strains were isolated from the following sources: urine [n = 142 (31.6%)], sputum [n = 91 (20.2%)], skin/soft tissue [n = 86 (19.1%)], bronchoalveolar lavage fluid [n = 34 (7.5%)], blood cultures [n = 16 (3.5%)], other sterile body sites [n = 22 (4.9%)], vascular prosthetic device [n = 10 (2.2%)], faeces [n = 17 (3.8%)], otitis [n = 12 (2.7%)], genital source [n = 6 (1.3%)] and other sources [n = 14 (3.1%)]. Among the 450 collected strains 88% were serotypable. The distribution of the serotypes was as follows: O:6, 25%; O:11, 16%; O:4 and O:1, 10%; O:3 and O:10, 6%; O:12, 4%; O:2, 3%; O:8 and O:9, 2%; O:5, O:7 and O:16, 1%; and <1% for O:13, O:14 and O:15. Table 1 shows the overall susceptibility data for the 450 strains of P. aeruginosa and according to the main serotypes. Among the ß-lactam antibiotics tested, imipenem, piperacillin+tazobactam and ceftazidime showed the best in vitro activities with susceptibility rates of 83%, 80% and 78% respectively. Aztreonam was the least active ß-lactam antibiotic with a susceptibility rate of only 50%. The less susceptible serotype was O:12, followed by O:11 and O:4. The urinary tract isolates were less susceptible to ciprofloxacin (56% versus 74%; P < 0.001) and those from the respiratory tract were less susceptible to ticarcillin, ticarcillin+clavulanic acid and cefepime (respectively, 53%, 53% and 55% versus 65%, 65% and 67%; P = 0.02), ceftazidime (70% versus 82%; P = 0.06) and imipenem (73% versus 87%; P < 0.001).
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The 171 ticarcillin-resistant strains were analysed for their resistance mechanisms to ß-lactam antibiotics. Among those strains, 29 isolates (17.0%) produced an acquired ß-lactamase and 76 (44.4%) overproduced the chromosomally encoded cephalosporinase AmpC, whereas 71 strains (41.5%) did not display significant ß-lactamase activities. Overproduction of the MexAB-OprM active efflux system was frequent in this latter group (44 isolates out of 71), but was also detected in 36 of the 76 AmpC-overproducing strains and 20 of the 29 isolates producing an acquired ß-lactamase (Table 2). The resistance mechanisms were frequently concomitant in the strains. Carbenicillinase PSE-1 was by far the most prevalent acquired ß-lactamase, present in 16 out of 29 isolates (55%). Six strains turned out to produce TEM-2 enzyme, four (located in two centres) produced the extended-spectrum ß-lactamase SHV2a and one strain an OXA-10 enzyme. Among the 76 strains with decreased susceptibility to imipenem, only two strains from two centres were VIM-2 producers.
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Table 2 summarizes the distribution of the resistance mechanisms to ß-lactams, the impact on the resistance level to ticarcillin and ciprofloxacin and the cross-susceptibility to imipenem, ciprofloxacin, amikacin and tobramycin. The lowest levels of susceptibility were encountered in strains with acquired penicillinases (frequently serotype O:12) or with high levels of AmpC (frequently serotype O:11).
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Discussion |
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When compared with previous studies conducted by our group in 1998 and 1999,7,8 serotypes O:6, O:11 and O:1 were still the most frequent in France. The frequency of serotype O:4 has increased (10% in 2004 versus 5% in 1998 and 1999), whereas the multiresistant serotype O:12 has decreased (4% in 2004 versus 6% in 1999 and 8% in 1998).
The susceptibility rates of P. aeruginosa strains remain stable for ß-lactams and are slightly higher for aminoglycosides and ciprofloxacin, compared with the end of the 1990s.8 The low rates of susceptibility of the urinary tract isolates to ciprofloxacin and of the respiratory tract isolates to ß-lactam antibiotics have been reported previously8 and are probably correlated with the strong antibiotic pressure in French hospitals. The frequency of acquired ß-lactamases has decreased in 2004 when compared with 1996 and 1999 (6% in 2004 versus 9.1% in 1999 and 12.9% in 1996). The PSE-1 carbenicillinase, frequently produced by serotype O:12,9 was still the most prevalent acquired ß-lactamase, but its frequency occurrence tends to decrease (from 90% of acquired ß-lactamases in 1996 to 72% in 1999 and 55% in this study).7,8 This evolution is closely correlated with the serotype O:12. If extended-spectrum ß-lactamases and carbapenemases were very rare in the past decade,7,8 they were detected in 4 out of 15 centres in 2004 and 3.5% of ticarcillin-resistant strains. Among the non-enzymatic ß-lactam resistance mechanisms, overproduction of MexAB-OprM was still the most frequent in France.10 Acquired ß-lactamases and/or high-level production of cephalosporinase AmpC are frequently associated with decreased susceptibility to aminoglycosides and ciprofloxacin.11
In conclusion, in the last decade, the overall susceptibility of P. aeruginosa hospital isolates to antibiotics has remained quite stable in France. However, the emergence of extended-spectrum ß-lactamases and carbapenemases in different locations is a matter of concern. This highlights the necessity to conduct close surveillance in order to detect these new mechanisms and to reinforce prevention measures in French hospitals.
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None to declare.
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Footnotes |
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Groupe d'Etude de la Résistance de Pseudomonas aeruginosa aux antibiotiques (Workgroup on the resistance of Pseudomonas aeruginosa to antibiotics), Paris, France 
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Acknowledgements |
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This work was supported by a grant from Wyeth-Lederle Pharmaceuticals. The other members of the GERPA (Groupe d'Etude de la Résistance de Pseudomonas aeruginosa) in this study are C. Bébéar (CHR Pellegrin, Bordeaux), R. Bismuth (Pitié-Salpétrière, Paris), P. Brisou (HIA St Anne, Toulon), J. Caillon (CHU Laennec, Nantes), C. Chanal (CHU Gabriel-Montpied, Clermond-Ferrand), M. Chomarat (CHU Lyon Sud, Pierre-Bénite), R. Leclercq (CHRU Côte-de-Nacre, Caen), H. Marchandin (CHU Arnaud-de-Villeneuve, Montpellier), C. Muller (CHU Bichat-Claude-Bernard, Paris), C. Poyart (CHU Cochin, Paris), F. Delpierre (CHRU Calmette, Lille), C. Segonds (CHU Rangueil, Toulouse) and J. M. Scheftel (Hôpitaux universitaires, Strasbourg).
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References |
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3 . Comité de l'Antibiogramme de la Société Française de Microbiologie. Communiqué 2006 http://www.sfm.asso.fr.
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