Quality of antimicrobial susceptibility testing in the UK: a Pseudomonas aeruginosa survey revisited
David M. Livermorea,* and
Han Yuan Chenb,
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
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Abstract
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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.
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Introduction
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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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Materials and methods
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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
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Results
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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
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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
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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.
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Discussion
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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).
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Acknowledgments
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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.
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Notes
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* Tel: +44-181-200-4400; Fax: +44-181-200-7449; E-mail: DLivermore{at}phls.co.uk 
Present address. Department of Medical Microbiology, St
Bartholomew's and the Royal London School of Medicine, Turner Street, London E1
2AD 
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References
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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, 1878.[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, 287300.[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, 30917.[Abstract]
Received 3 August 1998;
accepted 7 December 1998