Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, UK
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Abstract |
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Introduction |
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Despite this multiplicity of mechanisms, multicentre surveys in 19827 and 19938 found resistance to useful antibiotics was infrequent in P. aeruginosa isolates from the UK, with prevalence rates of 15% for relevant ß-lactams, aminoglycosides and ciprofloxacin. These surveys were undertaken 11 years apart and each examined 18002000 isolates from 24 centres. In principle, broader and more continuous surveillance of resistance might be achieved by compilation of routine data, but issues of data quality are a particular concern with P. aeruginosa. Central testing of isolates from the 1993 survey found that up to 73% of those reported as resistant by laboratories using routine methods were susceptible at high MIC breakpoints and up to 44% were susceptible at low MIC breakpoints, as based on central re-testing.9
Moves towards more standardized methods for routine susceptibility disc testing in the UK are now underway, with the British Society for Antimicrobial Chemotherapy (BSAC) publishing new guidelines in 2001.10 It is hoped that this change in methodology will overcome problems of data quality such as those outlined above. Accordingly, sentinel laboratories across the UK were selected to determine the prevalence of antibiotic resistance amongst their P. aeruginosa isolates and to assess whether the introduction of the BSAC standardized disc test method improved the accuracy of susceptibility testing.
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Materials and methods |
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Twenty-five sentinel laboratories with a wide geographical distribution in the UK (see membership of the Pseudomonas Study Group in Acknowledgements) were asked to collect and test 100 consecutive P. aeruginosa isolates from clinically significant infections. Duplicate isolates from the same infective episode in the same patient were excluded. For each isolate, the laboratory completed a case record form giving details on the patient's age, sex, clinical diagnosis and the site of infection, together with their own susceptibility testing results. The study was conducted from March to December, 1999.
Testing of P. aeruginosa isolates by the sentinel laboratories
P. aeruginosa isolates were identified by the sentinel laboratories using their own standard methods. Susceptibility testing was carried out using the new BSAC disc testing method.10 Antibiotic discs used were as follows: amikacin 30 µg, ceftazidime 30 µg, ciprofloxacin 1 µg, gentamicin 10 µg, imipenem 10 µg, meropenem 10 µg, piperacillin 75 µg and piperacillin/tazobactam 75/10 µg.
Quality assurance
Quality assurance was established by providing the centres with nine strains of P. aeruginosa before the commencement of the study. These comprised the standard UK control P. aeruginosa strain NCTC 10662 and eight isolates with the following resistance phenotypes: high-level enzymic gentamicin resistance, low-level impermeability-mediated aminoglycoside resistance, impermeability-type resistance to the carbapenems, partial and total derepression of AmpC ß-lactamase, PSE-3 ß-lactamase, and low- and high-level resistance to ciprofloxacin. Laboratories were asked to test these organisms according to the study protocol and to return the results for analysis. They were asked to re-test these bacteria if the inhibition zone diameters were outside one standard deviation of the mean zone diameters obtained by the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL). The sentinel laboratories were also asked to include three of these quality control (QC) strains with each batch of isolates tested.
Further QC was achieved by the sentinel laboratories sending every 10th isolate collected to the ARMRL for re-testing. Isolates with unusual resistances were also collected for re-testing: these were defined as those organisms (i) resistant to three or more antibiotics, (ii) with reduced zone diameters (15 mm) to the carbapenems or (iii) resistant to ceftazidime but susceptible to piperacillin and/or piperacillin/tazobactam.
Re-identification at the ARMRL
Isolates received at the ARMRL were examined for production of pyocyanin on Pseudomonas P agar (Oxoid, Basingstoke, UK) and for oxidase. Isolates giving positive reactions in both tests were not identified further. Oxidase-positive, pyocyanin-negative isolates were examined using API 20NE strips (bioMérieux, La Balme les Grottes, France). Oxidase-negative isolates were discarded.
Susceptibility testing at the ARMRL
MIC determinations at the ARMRL were undertaken using the BSAC agar incorporation method on IsoSensitest agar (Oxoid). In tests with piperacillin/tazobactam, the tazobactam was used at a fixed concentration of 4 mg/L. The inocula comprised 104 cfu/spot delivered with a multipoint inoculator, and the tests were incubated overnight at 3537°C. MICs were defined as the lowest drug concentrations to prevent growth completely and isolates were categorized as susceptible or resistant based on BSAC criteria.10 The antimicrobial powders used were obtained from Sigma (Poole, Dorset, UK), with the exceptions of imipenem (Merck, Hoddesdon, UK), meropenem (AstraZeneca, Macclesfield, UK), ceftazidime (GlaxoWellcome, Uxbridge, UK) and piperacillin/tazobactam (Wyeth, Taplow, UK).
Molecular studies
Assays to determine the presence of metallo-ß-lactamases in isolates with high-level resistance to carbapenems were carried out as described previously.11 Imipenem was used as the substrate at a wavelength of 297 nm.
Statistical analyses
Statistical analysis was performed using the 2 test or, where the numbers were small, using the Fisher's exact two-tailed tests. A P value <0.05 was taken to indicate significance.
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Results |
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A total of 2229 isolates was collected and tested by the 25 sentinel laboratories between March and December, 1999. The number of isolates collected and tested by individual laboratories ranged from 22 to 100, with 84% of the hospitals testing their full complement of 100 isolates. Three hundred and twenty isolates were sent to the ARMRL for re-testing; 218 as a random 10% QC sample and 102 owing to resistance patterns perceived as unusual. Of the 320 isolates received at the ARMRL, 314 were confirmed to be P. aeruginosa: 86% of them on the basis of pyocyanin and oxidase production, and the remainder using API-20NE strips. The disc testing results from one laboratory showed poor agreement with the MICs found at the ARMRL, particularly with meropenem where it was clear there was a problem with the discs used. This laboratory's results were removed from the study, leaving a total of 2194 isolates from 24 laboratories available for analysis.
Approximately half the isolates tested were from community patients (Table I), mostly from infections of the ear (38.1%), urinary tract (26.7%) or wounds (17.2%). Of the 163 respiratory tract isolates from community patients, 98 were from individuals attending cystic fibrosis (CF) units. Isolates from hospitalized patients, excluding those in ICUs, were most frequently from the urinary tract (35.6%), wounds (24.4%) or the respiratory tract (22%). Of the 175 isolates from ICU patients, most (62.8%) were from the respiratory tract and many of these were from patients stated to be ventilated.
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Mean zone diameters and standard deviations were calculated for each organism/antibiotic combination for the nine QC strains sent to each sentinel laboratory. Coefficients of variation (standard deviation/mean) were then calculated. For 87% of the reference isolateantibiotic combinations the coefficients of variation were <0.15, indicating good inter-laboratory reproducibility. The coefficient of variation for the other 13% of combinations ranged from 0.17 to 0.29. Those strains for which problems were predominantly encountered included the organism with permeability-type resistance to the carbapenems and those with total and partial derepression of AmpC ß-lactamase. The errors mostly related to variations in the range of zone diameters but did not lead to incorrect categorization of susceptibilities.
Isolates received by the ARMRL described as unusually resistant
The isolates received by the ARMRL described as unusually resistant included 83 reported as resistant to three or more antibiotics. This degree of resistance was confirmed at ARMRL for 67% of these isolates. Nine were resistant to all ß-lactams and aminoglycosides tested, although not to ciprofloxacin. These organisms were all from CF patients, who tend to receive repeated antibiotic therapy with considerable selection pressure for resistance. Seventy-five isolates received were described as having reduced zones (15 mm) to the carbapenems33 of them as resistant to both imipenem and meropenem, 21 as resistant only to imipenem and 21 isolates as resistant to meropenem only; however, 17 of this last group were found to be susceptible on re-testing at the ARMRL, mostly with MICs of 4 mg/L. Of the 54 isolates described as resistant to imipenem, 26 were highly so, with MICs
16 mg/L. Six of these had high-level ceftazidime resistance (MIC > 32 mg/L) and these were examined for hydrolytic activity against imipenem. No such activity was found.
Finally, 12 isolates received were described as resistant to ceftazidime but susceptible to piperacillin and piperacillin/tazobactam; the ARMRL was unable to confirm ceftazidime resistance in any of these organisms.
Prevalence of resistance
Zone distributions for all 2194 isolates (based on the sentinel laboratories' results) are shown in the Figure, and the resistance rates relative to the BSAC zone breakpoints are indicated. The resistance rate exceeded 10% only for gentamicin (11%). Most zone distributions were unimodal, with some degree of negative skew reflecting the inclusion of isolates with low-level resistance or reduced susceptibility. Exceptions to these unimodal distributions were ciprofloxacin, where there was a substantial secondary cluster of isolates giving no zones, and imipenem, where there was a small secondary cluster with a modal zone of 1015 mm compared with 2536 mm for the main group of isolates. Zone distributions for piperacillin and piperacillin/ tazobactam were essentially superimposable.
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Accuracy of susceptibility testing
Table III shows the reporting by the sentinel laboratories compared with the MICs obtained at the ARMRL for the 297 P. aeruginosa isolates that were collected as part of the QC (as already noted, widely discrepant data from one laboratory were discarded and are omitted). Of the isolates reported as susceptible to ß-lactams and aminoglycosides, most (83100%, depending on the antibiotic), were confirmed as such, with a further 315% found to be intermediate. Only 02% of the isolates reported as susceptible to these drugs were found resistant on MIC testing at the ARMRL. In the case of ciprofloxacin, 11/16 isolates with MICs of 2 mg/L had been reported susceptible; ciprofloxacin MIC of 2 mg/L counts as intermediate on BSAC breakpoint criteria, but no intermediate category is recognized in the BSAC disc test. Other cases of false susceptibility concerned mostly isolates with MICs only one dilution above the breakpoint; 35 isolates with gentamicin MICs of 4 mg/L reportedly gave zones of 2024 mm, compared with zone and MIC breakpoints of 19 mm and 2 mg/L, respectively.
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Table IV compares the sensitivity and specificity of the testing in the 1999 survey, when the sentinel laboratories used the new BSAC method, with the 1993 survey,8,9 when most laboratories used variants of Stokes' method. The sensitivity of the test (i.e. the proportion of resistant isolates correctly identified as resistant by the sentinel laboratories) was significantly improved in the 1999 survey, except for the aminoglycosides at high breakpoints, where improvements were apparent but insignificant. The specificity of the test (i.e. the proportion of susceptible isolates correctly identified as susceptible by the sentinel laboratories) was high in both surveys, ranging from 86 to 98%. A significant difference in the specificity between the two surveys was only seen for gentamicin at the low breakpoint and for amikacin at both low and high breakpoints.
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Discussion |
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Like previous UK surveys in 19827 and 1993,8 the present investigation found that resistance was infrequent in P. aeruginosa isolates, with rates below 12% for all the antibiotics considered. Similar studies conducted in Europe12 have shown little variation in the rates of resistance seen in P. aeruginosa although isolates from northwestern Europe tended to be more susceptible than those from southeastern Europe. In general, resistance rates in the UK were found to be similar to Europe for amikacin, meropenem, gentamicin and piperacillin/tazobactam, but UK isolates were less susceptible to ciprofloxacin. In all these instances, precise comparison with these and previous UK surveys is difficult since the centres and their collection strategies differed; for example, the present study included isolates from CF patients with increased resistance, whereas CF isolates were not obtained in some European studies nor in the 1993 UK study. Testing in the present study was carried out primarily by the sentinel laboratories, whereas testing in the 1982 and 1993 UK surveys was largely centralized. Comparison with previous UK studies is easier, and one potentially significant change is the apparent increase in imipenem resistance, from 2.5% in 19938 to 8.1% in 1999, with the resistant isolates now forming a small but discrete cluster on the zone distribution histogram (Figure). Imipenem is well known to select for OprD permeability mutants; these are resistant to imipenem itself and have reduced susceptibility to meropenem. Carmeli et al.13 recently suggested that imipenem selected resistance in P. aeruginosa more often than ciprofloxacin, piperacillin or ceftazidime; moreover, in a recent European study,12 most imipenem-resistant isolates were not found to be clonally related, implying repeated selection of resistance. Despite this rise in resistance to imipenem, the actual rates of resistance to carbapenems among P. aeruginosa nevertheless remained well below 10% and, although an increasing number of P. aeruginosa isolates with metallo-ß-lactamases have been reported from around the world,6,14,15 no producers were found in this survey.
Piperacillin and piperacillin/tazobactam were not tested in the 1993 survey;8 however, in a study conducted in 1991 by Chen et al.,16 the prevalence of resistance to piperacillin in P. aeruginosa was 5% and to piperacillin/tazobactam was 3%; thus the present study indicated no significant change in the prevalence of these resistances (P > 0.5).
An analysis of the patient population represented in this survey showed that resistance was more prevalent amongst isolates from CF patients than among general isolates. Nine isolates from CF patients were resistant to all agents apart from ciprofloxacin. Recent studies17 have shown that this high degree of resistance seen in P. aeruginosa from these patients may involve the selection of hypermutable (mutator) strains that can readily develop mutational resistance to multiple antibiotics.
The prevalence of resistance was also higher among isolates from in-patients than out-patients, although this difference was only statistically significant for the ß-lactams. In contrast to the 1993 survey,8 we did not find higher resistance rates among isolates from ICU patients except in the case of imipenem.
Aside from surveying the prevalence of resistance, a key objective was to test the accuracy of susceptibility testing, with sentinel laboratories using the new BSAC disc testing method. For this purpose the accuracy of reporting in the present study was compared with that of the 1993 survey, when most laboratories used Stokes' method, in which each isolate is compared with a sensitive control on the same plate. This controls disc quality, but the definition of resistance is arbitrary and, to complicate matters, laboratories use various modifications of the method in terms of media and inocula. The BSAC method standardizes the media and inocula and interpretive criteria for discs in relation to MIC breakpoints.
In the 1993 study, laboratories mostly detected susceptibility accuratelya performance partly reflecting the fact that most isolates are susceptible. Reporting for the minority of resistant isolates was less satisfactory, since up to 72% of the isolates reported as resistant proved susceptible on re-testing and susceptibility was recorded by the sentinel laboratories for up to 41% of isolates with high-level resistance (ceftazidime) and 74% of those with low-level resistance (amikacin). Incorrect reports of resistant isolates as susceptible in the present survey were much rarer, and mostly concerned isolates with MICs only one dilution above the breakpoint. Thus, the accuracy of reporting resistance for resistant isolates ranged from 61 to 100% (depending on the antibiotic), compared with 2673% for the 1993 survey (Table IVa). Since incorrectly reporting a resistant isolate as susceptible is the more serious error, potentially leading to inappropriate therapy, the BSAC disc test offers a significant advantage. The proportion of susceptible isolates incorrectly reported as resistant was under 10%, for all the antibiotics tested, except for aminoglycosides interpreted against the high breakpoints (Table IVb
). This level of accuracy was generally equivalent to the 1993 survey, apart from gentamicin at the low breakpoint and for amikacin at both low and high breakpoints.
Although the BSAC method achieved a valuable reduction in the incorrect reporting of resistant isolates as susceptible, there remains the problem that much reported resistance was not confirmed on MIC testing. In the worst case, with meropenem, 21/43 (49%) of isolates that had been reported as resistant were found to be inhibited by the compound at 4 mg/L or less. Among the other agents, the proportion of reported resistance that was not confirmed (even as intermediate resistance) on MIC testing ranged from 7% for ciprofloxacin to 34% for ceftazidime. These proportions are an improvement compared with the 1993 survey; moreover, the MICs of over half of the isolates reported as resistant but then found susceptible at the ARMRL were exactly at the breakpoint (Table III), indicating that susceptibility was marginal. Nevertheless, the proportion of isolates in which resistance was reported but could not be confirmed was still disturbingly high. Although such inconsistencies do not compromise patient therapy, they interfere with surveillance of resistance based on routine data. In this context the rates of resistance found in this study and detailed in the Figure
and Table II
should be seen as upper limits.
The behaviour of ciprofloxacin deserves particular comment. Past studies have shown high rates of false resistance using disc diffusion methods.9,18 Using ARMRL MIC results as the reference standard, this study showed that only a few susceptible isolates had been reported as resistant by disc tests (1.6%) and a much higher proportion of resistant isolates had been reported as susceptible (30%). The BSAC zone breakpoint divides resistant and susceptible isolates on the basis of zone diameters of 9 and
10 mm and does not recognize an intermediate category, whereas in MIC testing, values
1 mg/L are taken as susceptible, 24 mg/L as intermediate and
8 mg/L as resistant. Isolates for which the ARMRL MICs were 2 mg/L were mostly found to give zone diameters of 10 17 mm (mean 12.5 mm) to ciprofloxacin 1 µg discs, and therefore susceptible. A similar range of zone sizes for isolates with MICs of 2 mg/L was found by Ibrahim-Elmagboul & Livermore19 using a disc diffusion method closely resembling the BSAC test. Moreover, both these studies found that the zone ranges for isolates with MICs of 1 and 2 mg/L overlapped considerably, so it is unlikely that any arbitrary zone breakpoint could reliably differentiate these groups.
In summary, resistance rates for P. aeruginosa isolates appear to be stable and low in the UK, except amongst isolates from patients with CF and, to some extent, those in ICUs where heavy use of antibiotics may select resistance. The BSAC standardized disc testing method significantly reduced the proportion of resistant isolates incorrectly reported as susceptible. This is valuable for patient care. Nevertheless, for some antibiotics, notably meropenem, resistance detected in the disc tests was often not confirmed by MIC tests. This continuing problem underscores and reflects the difficulty of setting breakpoints for organism/ antibiotic combinations where the distributions are unimodal and do not divide into discrete resistant and susceptible populations.
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Acknowledgments |
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Members of the Pseudomonas Study Group: I. Gould, K. Milne (Aberdeen Royal Infirmary, Aberdeen); A. M. Walker, K. T. Dunkin (Bangor Hospital, Bangor); P. Rooney, I. Craig (Belfast City Hospital, Belfast); J. Andrews (City Hospital, Birmingham); R. C. Spencer, G. Wilson (Bristol Royal Infirmary, Bristol); D. F. J. Brown, E. Walpole (Addenbrooke's Hospital, Cambridge); I. Hosein, A. Paull (University Hospital of Wales, Cardiff); J. Grierson, S. Grundy (Cumberland Royal Infirmary, Carlisle); L. Teare, C. Purton (Chelmsford PHL); P. T. Mannion, S. B. Fraser (Countess of Chester Hospital, Chester); I. Thangkhiew, G. Ackland (Coventry & Warwickshire Hospital, Coventry); M. Logan, A. Burris (Gloucestershire Royal Hospital); R. Kent, R. Westacott (Ipswich Hospital); E. Youngs, J. McCluskie (Lincoln County Hospital); B. Oppenheim, C. Thornhill (Withington Hospital, Manchester); E. Mckay-Ferguson (South Cleveland Hospital, Middlesbrough); D. A. B. Dance, N. Cooper (Derriford Hospital, Plymouth); A. Pritchard (Glan Clwyd District General Hospital); K. Al Shafi, P. James (Royal Gwent Hospital); M. Yuan, A. Sefton (Royal London Hospital, London); A. King, A. Blake (St Thomas' Hospital, London); S. A. Howe, D. S. Gilbert (Royal Shrewsbury Hospital, Shrewsbury); K. Bowker (Southmead Hospital, Bristol); A. Tuck, E. Bartlett (Southampton General Hospital, Southampton); J. Bates, P. Kennedy (Worthing & Southlands Hospital, Worthing).
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Notes |
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Members of the Pseudomonas Study Group are listed in the Acknowledgements.
Deceased. This paper is dedicated to his memory.
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References |
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Received 25 August 2000; returned 11 December 2000; revised 5 January 2001; accepted 18 January 2001