Quality of antimicrobial susceptibility testing in the UK: a Pseudomonas aeruginosa survey revisited

David M. Livermorea,* and Han Yuan Chenb,{dagger}

a Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT b Department of Medical Microbiology, King's College Hospital School of Medicine and Dentistry, Bessemer Road, London SE5 8RX, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As part of a programme to assess the usefulness of routine antimicrobial susceptibility data as a surveillance tool, we reviewed the results of a national survey of resistance in Pseudomonas aeruginosa, undertaken in 1993. Twenty-four UK laboratories contributed isolates for centralized MIC testing, indicating also their own susceptibility test data. As reported previously (Chen et al. (1995) Journal of Antimicrobial Chemotherapy 35, 521- 34), the rate of false resistance (isolates reported susceptible, but found resistant on MIC testing/all isolates reported susceptible) was 0.6- 8%, according to the antimicrobial and breakpoint. Review showed that this favourable position reflected the fact that >88% of isolates were susceptible to any given antimicrobial and— in most cases— were correctly reported as such. Reporting was more erratic for resistant isolates: for ß-lactams and amikacin, isolates resistant at the highest MIC breakpoints were equally likely to be reported as `susceptible' or `resistant'; such misreporting was less common with ciprofloxacin and gentamicin but still occurred in 9- 20% of cases. Conversely, up to 73% of the isolates reported as resistant proved to be susceptible at high breakpoints, and up to 44% were susceptible at low breakpoints. Miscategorizations did not reflect failure to detect particular mechanisms but, rather, the fact that MIC and zone breakpoints for P. aeruginosa serve to cut `tails' of resistant organisms from continuous distributions, not to distinguish discrete populations. In this situation, some disagreement between routine tests and MICs is inevitable, but the frequency at which highly resistant isolates were reported as sensitive is disturbing. For surveillance, we conclude that resistance rates based on routine tests are unreliable for P. aeruginosa. This situation may improve with greater standardization of routine testing, but the continuous susceptibility distributions without discrete resistant and susceptible populations militate against perfect agreement. Despite these deficiencies, routine data should allow trend analysis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic resistance is an increasing concern worldwide, and there is agreement that improved surveillance is needed. 1 This can entail prevalence surveys with isolates sent to a central laboratory for testing, or can be based on compilation of routine data. Centralized testing offers quality and standardization, but is constrained by throughput and sampling error. Compilation of routine results allows use of larger data sets but is beset by concerns about accuracy. Susceptibility tests at UK laboratories are performed mostly by using Stokes' method, but with variation in the medium and how the inoculum is prepared and with the possibility that their control strains may evolve apart over time. 2 A few laboratories use breakpoint tests but, with variation in conditions and, sometimes in the breakpoints themselves. This situation could improve with the adoption of standardized disc methodology, but a problem remains if future data are compared with those from the past. In this context, and as part of a programme to assess current and past UK testing, we re-analysed the results of a national Pseudomonas aeruginosa survey, 3,4 run by ourselves in 1993 from the then London Hospital Medical College (LHMC, now St Bartholomew's and the Royal London School of Medicine and Dentistry). The isolates (n = 1991) were collected at 24 UK laboratories, and MICs were determined at the LHMC. In addition, we collected the laboratories' susceptibility data and compared these with our MIC results. False susceptibility rates (isolates reported susceptible but which proved resistant on MIC testing/all isolates reported susceptible) ranged from 1.1 to 8.3%, relative to low breakpoints, but were only 0.6- 3.5% relative to high breakpoints (Table I). 3 False resistance rates (isolates reported resistant that proved susceptible on MIC testing/all isolates reported resistant) were much higher: 19- 44% of isolates reported as resistant were found susceptible at low breakpoints, and 27- 72% were susceptible at high breakpoints. 3 In the present paper we present a fuller analysis of the disagreements between the MIC results and the data from the participating laboratories, and review their implications for using of routine susceptibility data in surveillance.


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Table I. Accuracy of susceptibility testing at the participating hospitals, as analysed previously4
 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 1991 P. aeruginosa isolates were collected between February and April 1993 from 24 laboratories. For each isolate the laboratory completed a case-record form, giving source details and susceptibility results. 3,4 The antibiotics tested and the testing method were at the discretion of each laboratory. Some laboratories graded isolates as sensitive, intermediate and resistant, whereas others only recognized sensitive and resistant categories. Ceftazidime, ciprofloxacin and gentamicin had been tested against >=75% of the isolates collected; amikacin, azlocillin and imipenem had been tested against >=25%; other antibiotics were tested against <=10%, or were not retested at the LHMC.

On receipt by the LHMC, the identities of the isolates were checked, and MICs of azlocillin, carbenicillin, ceftazidime, imipenem, meropenem, amikacin, gentamicin and ciprofloxacin were determined on IsoSensitest agar (Oxoid, Basingstoke, UK) with inocula of 10 4 cfu/spot. 3 Mechanisms of resistance to ß-lactam antibiotics were analysed in isolates resistant to one or more of azlocillin 16 mg/L, ceftazidime 8 mg/L, carbenicillin 128 mg/L, imipenem 4 mg/L or meropenem 4 mg/L. 4 Isolates resistant to gentamicin but not amikacin were inferred to have enzymatic mechanisms, while those with low-level resistance to both drugs were inferred to have impermeability type resistance. 5


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Accuracy of reporting susceptibility and resistance

In earlier analysis, 3 re-summarized in Table I, we found that <4% of the P. aeruginosa isolates reported by laboratories as susceptible were resistant relative to high breakpoints, but up to 72% of those reported as resistant were susceptible. Tables II and III present a fuller analysis of the error distribution: Table II shows the laboratories' reporting of susceptibility in relation to the MICs found by ourselves, and Table III summarizes this reporting relative to the breakpoints used in the survey or presently advocated by the BSAC. It is apparent from Table II that the low rates of false susceptibility found previously (Table I) primarily reflected the fact that the great majority of isolates collected were susceptible to any given antimicrobial, and were correctly reported as such.


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Table II. Participating laboratories' reporting of susceptibility for P. aeruginosa isolates, compared with MICs found at the LHMC
 

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Table III. Laboratory reporting for isolates considered resistant at breakpoints used in the survey or currently recommended by the BSAC
 
Reporting for the minority of resistant isolates was much less satisfactory (Table III): up to 81% of those resistant to low breakpoints were reported as susceptible, as were up to 41% of those resistant at the highest breakpoints considered. For azlocillin, amikacin, ceftazidime and imipenem, an isolate resistant at the highest breakpoint was almost equally likely to be reported susceptible as resistant! Such major categorization errors were not confined to a few laboratories. Combining all antibiotics tested, there were 73 instances where isolates resistant at the highest breakpoints considered in the survey or presently advocated by the BSAC (Table III) had been reported as susceptible. These miscategorizations were from 20 of the 24 hospitals that participated in the survey and, of the four hospitals without such errors, two had contributed only small numbers of isolates (17 and 27, compared with an average of 86 isolates per centre); nevertheless, one centre was responsible for 15 (21%) of all these major miscategorizations.

Susceptibility reporting in relation to mechanism

A further analysis was possible for azlocillin and ceftazidime, since mechanisms of resistance were characterized for isolates with MICs in excess of 4 and 16 mg/L, respectively. 4 Resistance was attributable to derepression of AmpC ß-lactamase, production of plasmid type ß- lactamases or, by exclusion of other mechanisms, to reduced target accessibility arising via impermeability or increased efflux. In general, isolates with derepression of AmpC enzyme were one to two doubling dilutions more resistant to azlocillin and ceftazidime than were those in which increased efflux or impermeability was inferred; those with secondary ß-lactamases were mostly (12/14 cases) susceptible to ceftazidime at 4 mg/L, but were amongst the most resistant to azlocillin (MIC >=128 mg/L in 10/14 cases). The distribution of mechanisms in relation to the hospitals' categorization of resistant isolates is shown in Table IV. Isolates with AmpC derepression and efflux-based resistance both were prone to being misclassified, and errors were not obviously related to those with one or other mechanism. Too few isolates with secondary ß- lactamases had been tested for valid conclusions; nevertheless, one of the five producers tested with azlocillin had been reported susceptible, but was highly resistant (MIC 64 mg/L) as tested at the LHMC.


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Table IV. Hospitals' reporting of results for azlocillin and ceftazidime in relation to MICs and resistance mechanisms found at the LHMC
 
Analysis of reporting in relation to mechanism was not possible for aminoglycosides because these resistance mechanisms were not determined. However a limited analysis was undertaken for the 213 isolates with gentamicin MICs >=4 mg/L that had been tested with gentamicin at their source hospitals. Enzymatic resistance was inferred in 25 isolates, based on gentamicin:amikacin MIC ratios >2. Of these, 21 had been reported resistant and four as susceptible. The remaining 188/213 isolates with gentamicin MICs >=4 mg/L had gentamicin:amikacin MIC ratios of <=2 (<=1 in 177 cases) and were inferred to have impermeability-mediated resistance; 125 (67%) of these latter isolates had been reported gentamicin-susceptible, 17 (9%) as intermediate and 45 (24%) as resistant. The apparent conclusion that laboratories were more successful at detecting enzymatic resistance is distorted by the fact that isolates inferred to have enzymatic mechanisms were more resistant to gentamicin (MIC >32 mg/L in 23/25 cases) than those inferred to have impermeability-mediated resistance (MIC <32 mg/L, in 173/188 cases). The critical factor in detection is likely to have been the level of resistance, not its mechanism.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous analysis of the survey data for P. aeruginosa indicated that rates of false susceptibility were low, although rates of false resistance were high (Table I). 3 The present re-analysis makes the further, and key, point that the infrequency of `false susceptible' results was primarily because most (>88%) isolates were susceptible to any given antibiotic and were correctly identified as such by their source laboratories. When the reporting of resistant organisms was considered independently (Tables II and III), the picture was far bleaker: up to 81% of isolates resistant to the lowest breakpoints were reported as susceptible, as were up to 41% of those resistant at the highest breakpoints considered. For the three ß-lactams and amikacin, an isolate resistant at the highest breakpoint stood approximately equal chances of being categorized as susceptible and resistant. Together with the high frequency of `false resistance' (i.e. sensitive organisms being misreported as resistant: Table I), it is apparent that little trust can be placed on the ability of UK laboratories accurately to detect resistance in P. aeruginosa. This situation is disturbing for therapy of individual patients, let alone for surveillance of resistance and the position is only mitigated by the fact, already mentioned, that frank resistance is rare in the species.

In large part the high rate of miscategorizations reflects the fact that most resistance in P. aeruginosa accrues by stepwise mutation. Such resistance to ß-lactams arises via increased impermeability, increased efflux or via derepression of the AmpC ß-lactamase; to aminoglycoside via transport lesions; or to quinolones via impermeability, efflux and DNA gyrase mutation. 4,5,6 The MIC distributions for all these groups of antimicrobials are unimodal for UK isolates of the species, with some skew to an excess of resistance. 3 Thus, the breakpoints listed in Table III all cut tails of resistant organisms from majority sensitive populations and do not, as for example with ampicillin against Escherichia coli, divide the discrete populations of bimodal distributions. Resistance of this type is prone to be detected erratically especially when, as now, susceptibility test methods vary much in detail from laboratory to laboratory. In this context it is worth noting the findings of Limb et al., 7 who distributed a P. aeruginosa strain with low-level gentamicin resistance (MIC, 4 mg/L) to 31 hospitals participating in the Microbe-Base surveillance scheme. Only nine laboratories (29%) reported the isolate resistant, whereas far greater accuracy in reporting (87- 100%) was seen for other species with high-level forms of resistance to various antimicrobials. Increased standardization of disc testing, now in prospect in the UK (Working Party of the British Society for Antimicrobial Chemotherapy, personal communication), should ensure less laboratory-to-laboratory variation in methods and results and, hopefully, should also reduce the number of grossly resistant isolates misclassified as susceptible. Whether or not this standardization with improve agreement with MIC tests for isolates with low-level resistance is much less certain and, in studies with ciprofloxacin and P. aeruginosa, Ibrahim-Elmagboul & Livermore 8 showed that disagreements between disc and MIC categorizations could be minimized but not eliminated by the choice of disc content and medium, even when all the tests were performed in a single centre.

In conclusion, P. aeruginosa presents a case where detection of resistance by routine tests agrees poorly with MIC data, but that this problem is disguised by the fact that most UK isolates are susceptible to relevant drugs, and are recognized as such in routine tests. It follows that compilation of routine data for surveillance would give only a very approximate picture of the incidence of resistance; nevertheless, trends in resistance should be detected, since there is an approximately 50% chance that a resistant isolate will be categorized as such, but only a 1- 2% chance that a susceptible isolate will be mis-classified as resistant! In the medium term, greater standardization of susceptibility testing should give an improvement, but precise agreement with MIC data is unlikely. This situation is in contrast to E. coli, where most resistance to several key antimicrobials, including trimethoprim and ampicillin, is high-level and is readily distinguished in disc tests, which consequently agree well with MIC data (unpublished observations).


    Acknowledgments
 
We are grateful to the hospitals that contributed isolates and data to the survey. These are listed in references 4 and 5. We are grateful also to Zeneca Pharmaceuticals for financial support.


    Notes
 
* Tel: +44-181-200-4400; Fax: +44-181-200-7449; E-mail: DLivermore{at}phls.co.uk Back

{dagger} Present address. Department of Medical Microbiology, St Bartholomew's and the Royal London School of Medicine, Turner Street, London E1 2AD Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . House of Lords Select Committee on Science and Technology. (1998). Resistance to Antibiotics and other Antimicrobial Agents. The Stationery Office, London.

2 . Andrews, J. M., Brown, D. & Wise, R. (1996). A survey of antimicrobial susceptibility testing in the United Kingdom. Journal of Antimicrobial Chemotherapy 37, 187–8.[ISI][Medline]

3 . Chen, H. Y., Yuan, M., Ibrahim-Elmagboul, I. B. & Livermore, D. M. (1995). National survey of susceptibility to antimicrobials amongst clinical isolates of Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 35, 521– 34.[Abstract]

4 . Chen, H. Y., Yuan, M. & Livermore, D. M. (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]

5 . Shannon, K. & Phillips, I. (1982). Mechanisms of resistance to aminoglycosides in clinical isolates. Journal of Antimicrobial Chemotherapy 9, 91– 102.[ISI][Medline]

6 . Cullmann, W. (1989). Mode of action and development of resistance to quinolones. Antibiotics and Chemotherapy 42, 287–300.[Medline]

7 . Limb, D. I., Winstanley, T. G. & Wheat, P. F. (1995). Quality assessment of Microbe Base antimicrobial susceptibility data. Journal of Clinical Pathology 48, 1122– 5.[Abstract]

8 . Ibrahim-Elmagboul, I. B. & Livermore, D. M. (1997). Sensitivity testing of ciprofloxacin for Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 39, 309–17.[Abstract]

Received 3 August 1998; accepted 7 December 1998