Multicentre surveillance of Pseudomonas aeruginosa susceptibility patterns in nosocomial infections

J. Van Eldere*

Rega Institute and Laboratory of Microbiology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium

Received 18 September 2002; returned 23 October 2002; revised 25 November 2002; accepted 26 November 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To determine susceptibility rates and patterns in Pseudomonas aeruginosa strains isolated from nosocomial infections.

Methods: Seven hundred and sixteen P. aeruginosa isolates from 40 different hospitals in Belgium and the Grand Duchy of Luxembourg were collected in 1999.

Results: Resistance rates varied significantly between hospitals. Of the fluoroquinolones, ciprofloxacin showed least resistance (24%), levofloxacin showed 27.5% resistance and ofloxacin 37.5%. Of the aminoglycosides, amikacin was the most potent antibiotic (10.5% resistance), followed by isepamicin (12%), tobramycin (19.5%) and gentamicin (23.5%). Of the ß-lactam antibiotics, meropenem was the most active (9.5% resistance); piperacillin and piperacillin/tazobactam had, respectively, 24% and 17.5% resistance, ceftazidime 28.5%, cefepime 29.5%, ticarcillin/clavulanic acid 37% and aztreonam 55.5%. MIC distribution curves show the presence of significant subpopulations, with MICs just below breakpoint for many antibiotics.

Conclusion: Resistance of P. aeruginosa to penicillins, cephalosporins, fluoroquinolones and aminoglycosides varies between hospitals, but is increasing.

Keywords: Pseudomonas aeruginosa, nosocomial infections, susceptibility rates, surveillance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In a 1996–1998 survey of nosocomial infections in Belgian intensive care units, Pseudomonas aeruginosa was isolated in 18% of cases of nosocomial pneumonia, which made it the most frequently isolated pathogen in this type of infection.1 In primary nosocomial bacteraemias, P. aeruginosa was isolated in 6% of cases.1 The high mortality associated with P. aeruginosa infections, particularly in immunocompromised patients, and the excess morbidity and mortality associated with ineffective empirical therapy in these infections2 underscores the need for reliable data on which to base the choice of empirical therapy. In addition to its high natural resistance, resistance rates can evolve rapidly in P. aeruginosa.3,4 Substantial regional variation in resistance patterns has also been observed, and is probably related to antibiotic treatment regimens.2,5 Periodic monitoring of resistance rates is therefore mandatory. In this study, we have examined isolates collected from nosocomial infections in 40 hospitals from all over Belgium and the Grand Duchy of Luxembourg. These 40 hospitals represent roughly one in five of the general hospitals in this region.

In addition to breakpoint testing, we also determined MICs. Population MIC distributions are good indicators of the relative potency of different antibiotics against the susceptible population, and can provide a more detailed picture of ongoing susceptibility shifts. Shifts in susceptibility are also more rapidly detected by MIC testing than on the basis of breakpoint testing. There is also increasing emphasis on the use of pharmacokinetics and pharmacodynamics to guide correct dosing of antibiotics. Correct dosing includes not only clinical outcome, but also aims at preventing resistance development. In this respect, susceptibility classes alone do not provide sufficient information to guide dosing, and MIC testing is more informative.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strain collection

Different hospitals, including large tertiary-care teaching hospitals and regional hospitals from all over Belgium and the Grand Duchy of Luxembourg participated in the study. The collection period was from April to November 1999, but the majority of the isolates were collected from June to September 1999. Isolates in each hospital were consecutive, meaning that all isolates that fitted the inclusion criteria were included until a sufficient number of isolates was obtained. The inclusion criteria were as follows: only one isolate per patient was included; isolates were from inpatients with a nosocomially acquired infection; nosocomial infections were defined as infections diagnosed >72 h after the start of hospitalization. Sample collection, strain isolation and identification were carried out according to standard procedures in each participating hospital. Isolates included in the study were kept on trypticase soy agar isolation tubes at room temperature, and sent to the central test site for MIC testing. In case of doubt as to the purity of the isolate, or identity of the isolate, re-identification was performed at the central test site.

MIC testing

MICs were determined for the following antibiotics: ofloxacin, levofloxacin, ciprofloxacin, gentamicin, tobramycin, amikacin, isepamicin, piperacillin, piperacillin/tazobactam, ticarcillin/clavulanic acid, aztreonam, ceftazidime, cefepime and meropenem.

The isolates were tested using the agar dilution method recommended by the NCCLS. The reference strain P. aeruginosa ATCC 27853 was included in each test, also as recommended by the NCCLS. MIC interpretative guidelines were those advocated by the NCCLS.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Origin of the isolates

A total of 716 P. aeruginosa isolates were collected from 40 different hospitals. The number of isolates collected per hospital varied between 10 and 40. Of all the isolates, 38% originated from patients hospitalized in an intensive care unit, 21.5% were from patients in an internal medicine ward, 12% from a surgical ward and 5.5% from a haematology ward. Another 23% of isolates were from patients in other, non-specified wards. Sample origin was also checked; 41% of the strains were isolates from respiratory samples, 21% were from urinary samples, 5% were from blood and 22.5% from other sources. For 11% of the isolates, there was no information on the origin of the sample. Of all the respiratory samples, 61% were from patients hospitalized in an intensive care unit.

MIC testing results

Of all isolates taken together, 50% were susceptible to ofloxacin, 61% to levofloxacin and 71% to ciprofloxacin. Similar susceptibility percentages were found when only isolates originating from patients in intensive care units, or from the different wards, were considered, or when only isolates from specific samples (respiratory samples, blood, etc.) were considered. Only in isolates from urinary samples did we find that susceptibilities were significantly lower than in all isolates taken together (ofloxacin, 46% susceptible; levofloxacin, 55%; ciprofloxacin, 61%). This was mainly due to a higher incidence in these samples of isolates with high-level resistance.

The MIC distribution curves for all isolates taken together (Figure 1) showed that for both the susceptible and resistant subpopulations, levofloxacin was one two-fold dilution more potent than ofloxacin. For the susceptible population, ciprofloxacin was at least three two-fold dilutions more potent than levofloxacin.



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Figure 1. MIC distribution curves for ciprofloxacin (triangles), levofloxacin (squares) and ofloxacin (diamonds).

 
Susceptibility levels for aminoglycosides were: gentamicin 67%, tobramycin 80%, amikacin 85% and isepamicin 81%. We found no significant differences between isolates from patients in different wards, or samples of different origin, compared with all samples taken together. The only exception was, again, the collection of urinary isolates, which had a higher level of resistance than the other samples. Comparison of the MIC distribution curves (Figure 2) showed that tobramycin was the most active compound per unit weight, and was two two-fold dilutions more potent than gentamicin.



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Figure 2. MIC distribution curves for tobramycin (black squares), gentamicin (diamonds), amikacin (triangles) and isepamicin (white squares).

 
In the ß-lactam group of antibiotics, MIC distribution curves showed that meropenem was the most active compound, with 9.5% of the isolates fully resistant, and 9% intermediately resistant (Table 1). For meropenem-resistant isolates, aminoglycosides (amikacin, isepamicin and tobramycin) were still the most active compounds (Table 2). The distribution of the strains in the different susceptibility categories for the ß-lactam antibiotics is given in Table 1. From the MIC distribution curve (Figure 3), it was clear that per unit weight, ceftazidime and cefepime were more potent than piperacillin, ticarcillin and aztreonam. There was also a marked difference in the form of the MIC distribution curve between ceftazidime and cefepime. This is translated as a higher fraction of intermediately resistant isolates for cefepime.


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Table 1.  Results of susceptibility testing
 

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Table 2.  Cross-resistance between antibitics
 


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Figure 3. MIC distribution curves for meropenem (white squares), ceftazidime (diamonds), cefepime (circles), piperacillin/tazobactam (black squares) and ticarcillin/clavulanate (triangles).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Compared with earlier surveys that have examined susceptibility to various antibiotics in P. aeruginosa isolates from Belgian hospitals,610 there is a clear tendency towards decreased susceptibility for all groups of antibiotics. The rates at which this loss of activity proceeds, however, may vary. Susceptibility to ciprofloxacin, for example, has gradually evolved from 87% in 1991, to 82–84% in the period 1993–1995, 70% in 1998–1999, to 71% in this study. For amikacin, susceptibility was 96% in 1994–1995, 91% in 1997, 74% in 1998–1999 and 85% in this study. For cefepime, it was found that 73% of P. aeruginosa isolates were still susceptible in 1997, compared with 64% in 1998–1999 and 50% now. For meropenem, susceptibility was 86% in 1998–1999 and 81% now. In comparison with neighbouring countries such as France, we find comparable susceptibility rates for most antibiotics.11 Recent surveys in Spain and Italy give, on the whole, similar resistance rates for most antibiotics.12,13

As in most other surveys, among the ß-lactam antibiotics, meropenem is the most potent against P. aeruginosa. In terms of percentage susceptibility, both piperacillin/tazobactam and meropenem scored best. Full resistance to meropenem was, however, lower than to piperacillin/tazobactam. In cases of resistance to other antibiotics, including the ß-lactam antibiotics, meropenem again remained the most active compound. Nevertheless, the meropenem MIC distribution curve shows a large subpopulation of isolates with meropenem MICs that are only one or two dilutions below breakpoint. The selection of these strains, and a rapid increase in the modal MIC for the susceptible population, will have to be monitored. Rapid emergence of resistance during therapy has been reported for imipenem14,15 and is apparently due to loss of OprD.16 Loss of OprD affects meropenem to a lesser extent than imipenem, and leads to borderline susceptibility. However, it has been reported that in an OprD-deficient background, meropenem selects for MexAB-OprM hyperproducers that are more resistant to meropenem.16,17 Whether this implies that high-level resistance to meropenem will develop more slowly than to imipenem remains to be established. Given the form of the MIC distribution curve, it is clear that the MIC90 of 8 mg/L of meropenem is not due to the presence of a few resistant outliers. It follows that only a high dose of 3x 2 g per day would ensure a serum concentration that is above the MIC for 50% of the dosing interval for nine out of 10 P. aeruginosa isolates. Piperacillin also has good anti-P. aeruginosa activity, although gravimetrically it is clearly less potent than ceftazidime and cefepime. The relatively high susceptibility rates of piperacillin and piperacillin/tazobactam are in part due to the high NCCLS breakpoints for these antibiotics. Several European national committees advocate lower breakpoints for piperacillin. If the breakpoint for piperacillin and piperacillin/tazobactam was defined at 64 instead of 128 mg/L, this would imply that, respectively, 30% and 27% of the strains would be classified as fully resistant.

These resistance rates are comparable to those for ceftazidime and cefepime. The difference in susceptibility rates between ceftazidime and cefepime is mainly due to a bigger subpopulation of intermediately susceptible isolates for cefepime. This has also been found in other studies.1820 It has been alleged that this difference in susceptibility rates between cefepime and ceftazidime could be due to the presence of P. aeruginosa isolates with intrinsic resistance due to reduced permeability.18 This would affect cefepime more than ceftazidime. A possible mechanism leading to this reduced permeability would be the derepression of the MexCD-OprJ efflux system, which is selective for fourth-generation cephalosporins. It has been suggested that induction of the MexCD-OprJ efflux system is particularly linked to the use of fluoroquinolones, which are also exported via this efflux system.21 This is concordant with the observation that in this study the MIC distribution profile of the fluoroquinolones is clearly shifted to the right for the cefepime intermediately susceptible strains, as compared with the ceftazidime intermediately susceptible strains. The MIC50 for the cefepime intermediately susceptible strains is 8 mg/L of ofloxacin, 4 mg/L of levofloxacin and 1 mg/L of ciprofloxacin, compared with 2, 1 and 0.5 mg/L, respectively, for the ceftazidime intermediately susceptible strains.

In another Belgian surveillance study, which examined 274 P. aeruginosa collected in 1999 from ICU units in 16 hospitals, higher susceptibility rates were found for ceftazidime (82%) and cefepime (70%) (NPRS 4 study, unpublished data). In this study, MIC distribution curves for ceftazidime and cefepime were one two-fold dilution shifted to the left compared with the present study, but the overall forms of the curves were similar, and also showed the presence of a larger subpopulation of isolates intermediately susceptible to cefepime.

In the aminoglycoside group, amikacin and isepamicin (only recently introduced in Belgium) differed only slightly. This is reflected in a slight difference in the susceptibility rates (85% susceptibility for amikacin versus 81% for isepamicin), and an MIC distribution curve that shows that for the susceptible population amikacin has a modal MIC that is one dilution lower. In our study, we confirmed the excellent anti-P. aeruginosa activity of tobramycin. Strains resistant to amikacin were nearly all resistant to fluoroquinolones and to ß-lactam antibiotics, and this was probably due to impermeability or multi-drug efflux.

In the fluoroquinolone group, ciprofloxacin clearly had better anti-Pseudomonas activity than levofloxacin or ofloxacin. MIC distribution curves show that the MIC range for the susceptible population is very narrow for all fluoroquinolones. For levofloxacin, the susceptible population is situated within two dilutions of the susceptibility breakpoint. Given the possibility of rapid selection of resistant mutants if insufficiently high concentrations (<10 x MIC) are used, this implies that levofloxacin serum levels of >5 and >10 mg/L are needed in cases of P. aeruginosa infection.

The incidence of resistance among isolates from different centres was compared. In general, there were differences between centres, probably reflecting the varying patterns of antibiotic usage in the hospitals. Surprisingly, no significant differences were found between resistance rates in isolates from intensive care units compared with isolates from other wards or units.

In conclusion, resistance of P. aeruginosa to penicillins, cephalosporins, fluoroquinolones and aminoglycosides varies between hospitals, but on the whole is increasing in Belgium. Our data question the use of monotherapy in cases of suspected P. aeruginosa infection. MIC distribution curves show the presence of a significant section of the P. aeruginosa population with MICs just below breakpoint. This suggests the possibility of rapid evolution towards higher resistance rates, and supports the need for correct, high dosing and combination therapy to minimize the risk of resistance development in cases of P. aeruginosa infection.


    Acknowledgements
 
The following colleagues are gratefully acknowledged for providing the isolates that were used in this study: M. Delmée, Cliniques Universitaires St Luc, Bruxelles; I. Grosjean, C.H. ‘Peltzer-La Tourelle’, Verviers; F. Meunier, C.H. Jolimont-Lobbes, Haine St Paul; S. Waegemans, Sint Vincentius ziekenhuis, Antwerpen; K. Magerman, Virga Jesse ziekenhuis, Hasselt; D. Govaerts, C.H.U. A. Vésale, Montigny-le-Tilleul; G. Vanderstocken, Hôpital Marie Astrid, Niederkorn (Luxembourg); P. Gabriels, Regionaal ziekenhuis St Trudo, St Truiden; J.-M. Hubrechts, C.H. Joseph Bracops, Bruxelles; M. Van De Vijvere, Stuyvenbergziekenhuis, Antwerpen; D. Fameree, CHU de Charleroi Site de Jumet, Jumet; L. Van Nimmen, A.Z. Jan Palfijn, Gent; C. Potvliege, CHU Tivoli, La Louvière; M. Fossion, St Joseph, Gilly; S. Uyttendaele, A.Z. Sint Maarten Campus Duffel, Duffel; R. Reybrouck, H. Hartkliniek, Tienen; C. Sion, C.H. Huttois, Huy; Ch. Trolin, Centre Hospitalier, Etterbeek-Ixelles; G. Coppens, Ziekenhuis Oost Limburg, Genk; B. Van Bosterhaut, Site Refuge, Mouscron; L. Waumans, Salvatorkliniek, Hasselt; D. Allemeersch, Clinique Ste Elisabeth, Uccle; J. Hoegaerts, H. Hartkliniek, Eeklo; J. Wautelet, Clinique St Pierre, Ottignies; J. Van Schaeren, A.Z. St Camillus/St Augustinus, Wilrijk; C. Coppens, A.S. Ziekenhuis, Aalst; J.-F. Marchal, Clinique La Dorcas, Tournai; A. Dediste, CHU Brugmann, Bruxelles; R. Van Noyen, Imeldaziekenhuis, Bonheiden; P. Brouart, Clinique Sacré Coeur, Luxembourg Ville; F. Trigaux, C.H. Frameries/Hornu, Hornu; A.-M. Van den Abbeele, A.Z. St Lucas, Gent; J. Sentrie, St Elisabeth ziekenhuis, Sijsele Damme; A. Verhaeghe, St Augustinuskliniek, Veurne; B. Masschelin, Algemeen Ziekenhuis St Jozef, Turnhout; M. Struelens, Hôpital Erasme, Bruxelles; H. Goossens, U.Z. Antwerpen, Edegem; J. Colaert, C.A.Z.K. Groeninghe, Kortrijk, Philippart, Hôpital St Joseph, Mons/Hôpital de Warquignies, Boussu. This study was supported by a financial grant from Bayer, Brussels, Belgium.

Part of this work was presented at the Eleventh European Congress of Clinical Microbiology and Infectious Diseases, Istanbul, Turkey, 1–4 April 2001, abstract P1373.


    Footnotes
 
* Tel: +32-16-34-79-02; Fax: +32-16-34-79-31; E-mail: johan.vaneldere{at}uz.kuleuven.ac.be Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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