Antibiotic Resistance Monitoring & Reference Laboratory, Health Protection Agency, Specialist & Reference Microbiology Division, 61 Colindale Avenue, London NW9 5HT, UK
Received 19 December 2003; returned 12 January 2004; revised 27 February 2004; accepted 27 February 2004
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Abstract |
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Methods: Standardized disc susceptibility tests were performed at sentinel laboratories in three surveys, with MIC tests performed on the collected isolates at a reference laboratory. The organisms comprised over 3300 Enterobacteriaceae, Acinetobacter spp., pseudomonads, staphylococci and enterococci, with over 29 000 antibiotic/organism tests in total.
Results: More than 90% of the antibiotic/organism combinations classed as susceptible by disc tests in the sentinel laboratories were confirmed by MIC testing. Disagreements were more frequent where disc tests indicated resistance, with half of the piperacillin/tazobactam resistance and one-third of the cephalosporin resistance found in Enterobacteriaceae by disc tests not being confirmed, and with three-quarters of teicoplanin resistance in enterococci not confirmed. None of the few apparent cases of meropenem resistance in Enterobacteriaceae or linezolid, quinupristin/dalfopristin or vancomycin resistance in staphylococci were confirmed by MIC testing. When disagreements were found between disc- and MIC-based categorization, MICs were commonly, although not invariably, one to three doubling dilutions above or below the breakpoint. However, many of the disagreements where MICs were three or more dilutions from the breakpoint were not seen when disc tests were repeated in the central laboratory.
Conclusions: The BSAC disc method seems adequate for confirming susceptibility to guide therapy and to monitor resistance trends. Nevertheless, there must be concern about the over-estimation of many resistances, and frequent zone:MIC disagreements for isolates with borderline susceptibility.
Keywords: minimum inhibitory concentration, zone diameter, standardized method
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Introduction |
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Performance data for the standardized disc testing method were published following an evaluation exercise,4 in which 19 centres each tested 240 strains distributed by the BSAC. Five of the centres were research laboratories but the remaining 14 had not previously used standardized methods. While the overall results of the study were encouraging, several organism/agent combinations (e.g. Enterobacteriaceae with cefuroxime) commonly showed discrepancies between results obtained by disc testing compared with those obtained by agar dilution.
In this study, we analysed the susceptibility categorizations obtained both by disc testing and MIC determinations for 3378 clinical isolates in three surveys.57 In each case, the BSAC disc diffusion method was used in the laboratories where the isolates were collected, and MICs for the same isolates were subsequently determined at a central reference laboratory.
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Materials and methods |
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Data were drawn from three recent surveys coordinated by the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL), in which a total of 52 British and Irish hospitals (see Acknowledgements) participated: one focused on Pseudomonas aeruginosa,5 one on Gram-positive cocci from intensive care unit infections6 and one on various species from hospitalized patients.7 In each study, the hospital microbiology departments tested consecutive clinically significant isolates from separate patients by the BSAC disc method and sent (depending on the survey) either all or a proportion of them to the ARMRL for determination of MICs.
Testing of isolates by sentinel laboratories
Identification of isolates by the sentinel laboratories was as detailed previously.57 Susceptibility testing at these laboratories was carried out using the BSAC standardized disc testing method.2 All tests were performed using Iso-Sensitest Agar from Oxoid (Basingstoke, UK). Antibiotic discs (Oxoid) were distributed by sponsors for use in two of the studies;6,7 the laboratories own discs were used in the Pseudomonas survey5 and may have varied in age and source. The antibiotics tested varied with the species and survey, and are detailed in the Results. The analysis presented here was performed using the updated zone breakpoints now advocated by the Society.3 In a few cases, these differ from those originally published2 or used in the surveys themselves.57 Each survey incorporated a quality control exercise whereby reference strains were tested by the participating centres, which only commenced testing their own isolates once they had obtained the anticipated results for these control organisms.
Testing at the reference laboratory
All isolates collected into the ARMRL had their identifications confirmed. Enterobacteriaceae were spotted on to Chromogenic UTI Medium (Oxoid), and those giving anomalous colour reactions for the reported species were re-identified using API 20E strips (bioMérieux, Basingstoke, UK). Non-fermenters were screened for production of pyocyanin on Pseudomonas P Agar (Oxoid) and for oxidase; those giving positive reactions were accepted as P. aeruginosa; those giving negative reactions in either test were examined using API 20NE strips. Enterococci were re-identified by PCR of ddI genes.8,9 Staphylococcal identifications were confirmed by colony morphology and coagulase reactions seen upon addition of culture to tubes of citrated plasma.
MIC determinations were undertaken by the BSAC agar dilution method.10 The antibiotics tested depended on the species and survey (see Results) but variously included amikacin, ampicillin, erythromycin, fusidic acid, gentamicin, penicillin, oxacillin, rifampicin and vancomycin (Sigma, Poole, UK), cefotaxime, levofloxacin, quinupristin/dalfopristin and teicoplanin (Aventis, West Malling, UK), ceftazidime and clavulanate (Glaxo SmithKline, Welwyn Garden City, UK), ciprofloxacin (Bayer, Newbury, UK), meropenem (AstraZeneca, Macclesfield, UK), piperacillin and tazobactam (Wyeth, Taplow, UK), linezolid (Pharmacia, Milton Keynes, UK) and imipenem (Merck Sharpe & Dohme, Hoddesdon, UK). Isolates were categorized as susceptible or resistant to antibiotics using the MIC breakpoints currently recommended by the BSAC.3
Data handling
For each isolate tested, the collecting laboratory completed a case record form indicating, among other things, their identification and disc zone diameters. Once received at ARMRL, these data were entered into a Microsoft Access database. Re-identification and MIC data were added, and zone:MIC categorization agreement analyses were performed once the data had been downloaded into Microsoft Excel.
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Results |
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A combined total of 3378 isolates was reviewed from the three surveys, comprising Enterobacteriaceae (1220), Acinetobacter spp. (42), pseudomonads (474), Staphylococcus aureus (840), coagulase-negative staphylococci (CoNS) (505) and enterococci (297). Four organism groups were analysed separately within the Enterobacteriaceae: (i) Escherichia coli (586), (ii) Klebsiella spp. (184), (iii) AmpC-inducible Enterobacteriaceae, comprising Enterobacter spp., Citrobacter spp., Morganella morganii and Serratia spp. (297) and (iv) Proteus mirabilis (124). Among the pseudomonads, P. aeruginosa (434) was the dominant species, and was also analysed separately.
The collection had a bias towards resistance because two of the surveys5,7 were based upon collecting all isolates with key resistances for MIC testing, together with 5%10% samples of the more susceptible bacteria; the third survey6 comprised intensive care isolates, which tend to have increased rates of resistance. Not all isolates received were accompanied by complete disc-susceptibility data, presumably because of failure or omission of individual discs, and a few isolates were unrecoverable for MIC testing when slopes were received at ARMRL. Otherwise, all results received were used in zone:MIC agreement analyses.
Overall agreement of disc and MIC test results
Tables 15 show the proportions of categorization agreements and disagreements between the hospitals disc test results and the reference laboratorys MIC results for Enterobacteriaceae, Acinetobacter spp., Pseudomonas spp., staphylococci and enterococci, respectively. In each table the most critical data are in the two right-hand columns showing, respectively, the proportion of isolates reported susceptible on the basis of disc tests that were confirmed as susceptible by MIC tests, and the proportion of those reported resistant that were confirmed as resistant.
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Most of the Klebsiella spp. (80%) and many of the AmpC-inducible Enterobacteriaceae isolates (39%) that were reported susceptible to ampicillin in disc tests proved (predictably) to be resistant to ampicillin on MIC testing, as did many of the AmpC-inducible enterics (34%) reported as susceptible to co-amoxiclav. Only 57% of Acinetobacter spp. reported as susceptible to ampicillin by disc testing were confirmed as susceptible by MIC testing, although it should be added that Acinetobacter spp. are difficult organisms to test, and that the BSAC recommends that an MIC determination is performed, or the disc test repeated, if the inoculum density is outside the acceptable range.
Disagreements between the results of disc tests at the source laboratory and reference laboratorys MIC tests were much more frequent for isolates that had been reported as resistant than for those reported as susceptible. Among Enterobacteriaceae, only 51% of those found resistant to piperacillin/tazobactam in disc tests were confirmed as resistant in MIC tests, and the situation was little better for co-amoxiclav (64%), gentamicin (65%), ceftazidime (70%) or cefotaxime (74%)with all these analyses based on at least 150 isolates reported as resistant. The 70% agreement figure for ceftazidime is based on the Societys present breakpoints for 30 µg discs, which are 22 mm for E. coli and Klebsiella spp. and 28 mm for all other Enterobacteriaceae. Had the 28 mm breakpoint been used for all Enterobacteriaceae isolates, as would be the case if these species were lumped as coliforms, the proportion of confirmed resistance falls to 59%. Among 17 Enterobacteriaceae isolates reported as meropenem-resistant by disc tests, none was confirmed as resistant in MIC tests.
For pseudomonads, only 40% of reported meropenem resistance, 45% of ceftazidime resistance, and 67% of imipenem resistance was confirmed by MIC testing, whereas over 87% of aminoglycoside resistance and 96% of resistance to ciprofloxacin (using 1 µg discs) was confirmed. For S. aureus, only 54% of reported resistance to fusidic acid and 37% of rifampicin resistance was confirmed, whereas for other drugs documented, over 80% of resistance was confirmed by MIC testing whenever more than 50 isolates had been reported as resistant. For CoNS this latter criterion was met for all drugs, including fusidic acid and rifampicin. MIC tests did not confirm resistance in any of three staphylococci reported by disc testing as resistant to linezolid, or in any of four reported as resistant to quinupristin/dalfopristin. Among enterococci, only 27% (24/89) of those reported teicoplanin-resistant in disc tests were confirmed resistant in MIC determinations; for other antibiotics, over 70% of resistance reported in enterococci was confirmed. The enterococci with unconfirmed teicoplanin resistance were consistently susceptible to vancomycin, whereas those with confirmed resistance were cross-resistant to vancomycin.
Detailed analysis of major disagreements
Tables 6 and 7 show MIC distributions wherever 50 isolates had been categorized as susceptible or resistant to a drug on disc testing and where MIC tests suggested that over 10% of these had been miscategorized. Most disagreements concerned organisms with MICs within one to three doubling dilutions of the breakpoint, but a few antibiotic/organism combinations had large numbers of organisms with wider disagreements. Repeat disc tests (right-hand column of Tables 6 and 7) were performed at the central laboratory when (i) an isolate had been reported as susceptible but was found to require an MIC of
8 times the breakpoint value or (ii) when it had been reported as resistant but was found to require an MIC of
1/8 the breakpoint value. Except in the cases of Enterobacteriaceae initially miscategorized, based on disc tests, as susceptible to ampicillin or as resistant to ß-lactamase inhibitor combinations, these repeat tests very largely (>75% of cases) gave results that agreed with the ARMRL MIC data, implying that most large disagreements reflected hospitals performance of the BSAC disc test, rather than inherent problems with the method.
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Fifteen of 24 CoNS that were found oxacillin-susceptible in disc tests but resistant in MIC tests required MICs of 8 times the breakpoint value. These were all confirmed as mecA-positive by PCR11 and 12 gave zones indicating resistance in repeat disc tests on Columbia agar + 2% NaCl. Five were from one hospital, with the remaining 10 distributed among eight hospitals.
Most (580/816) of the cases where isolates were found resistant by disc tests at the source laboratory but susceptible by MIC tests at ARMRL concerned isolates with MICs on or within two doubling dilutions of the breakpoint. Other cases, however, concerned isolates that appeared fully susceptible in MIC tests, and over one-third of the Enterobacteriaceae isolates misinterpreted as resistant by disc testing required MICs of 1/8 of the breakpoint values for ciprofloxacin, cefotaxime or ceftazidime. Whereas these organisms were distributed across numerous species and hospitals, one hospital accounted for 22%, 30% and 17% of grossly discrepant results for ciprofloxacin, cefotaxime and ceftazidime, respectively. Four isolates (from separate hospitals) were interpreted as resistant to both cefotaxime and ceftazidime by disc testing but found susceptible to both in MIC tests and in repeat disc tests.
Twenty-five of the 74 (34%) P. aeruginosa isolates found to be meropenem-resistant by disc testing but susceptible by MIC testing required meropenem MICs of less than 0.5 mg/L, equating to the modal MIC for the species and to 1/8 of the susceptibility breakpoint. Of these 25, 17 (68%) were from two hospitals, with the remaining eight distributed amongst seven hospitals. When disc tests were repeated at the central laboratory, only one appeared resistant. Among the considerable numbers of S. aureus isolates reported as resistant to fusidic acid (46) and rifampicin (41) in disc tests but susceptible in MIC tests, the majority required MICs of 1/8 of the breakpoint. Repeat disc testing of these mostly gave results in agreement with MIC-based categorizations (22/24 and 32/35 susceptible, respectively). More detailed analysis showed that four hospitals were responsible for 65% (28/43) of the unconfirmed resistance to fusidic acid and 78% (31/40) of that to rifampicin.
Eleven enterococci were reported as ampicillin resistant, but were found to be susceptible, with MICs of 1/41/8 of the breakpoint. More striking was the high rate of unconfirmed teicoplanin resistance (73%, Table 5). Of 65 such isolates, 51 (78%) required MICs 1/8 of the breakpoint. Forty-two of the 65 (65%) were from five hospitals, whereas the remaining 23 were distributed among 16 hospitals. When re-tested, only six of 50 isolates with teicoplanin MICs of
1/8 of the breakpoint value gave zone diameters that implied resistance, whereas the other 45 gave zone diameters indicating susceptibility, agreeing with the MIC-based categorizations.
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Discussion |
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The disc breakpoints of the BSAC method are based on the zone distributions for isolates with known MICs, so one would expect good categorization agreement between zone and MIC testing by the Societys methods. In practice, as this analysis of over 3000 isolates, 29 000 tests and 50 laboratories shows, the great majority of isolates categorized as susceptible in BSAC disc tests were indeed confirmed as susceptible in MIC tests, but there were more disagreements for isolates categorized as resistant in disc tests. These disagreements were particularly frequent for those antibiotics detailed in Table 6 and, in many cases (966/1268), concerned isolates with MICs close to breakpoints. This is not unexpected and reflects the facts that zone:MIC correlations inevitably have some scatter and that, for most antibiotics, the BSACs method does not have an intermediate category to act as a buffer zone between susceptible and resistant as does the NCCLS method. It is debatable whether or not this is a deficiency. While the absence of an intermediate category exaggerates the number of categorization disagreements, there are many who argue that intermediate reports are profoundly unhelpful to clinicians. For this reason the BSAC uses an intermediate category only in specially defined circumstances, such as where a whole species has inherently borderline susceptibility (as with pneumococci and ciprofloxacin), or if the infection may respond to an increased drug dosage. Where an intermediate category was allowed by the BSAC, e.g. with P. aeruginosa and aminoglycosides, it included many of the isolates where disc/MIC disagreements arose (Table 6). Of the remaining discrepancies, (302/1268) related to cases where isolates had been found susceptible in disc tests at the sentinel sites, but were then found to require MICs of 8 times the breakpoint value, or had been found resistant and then proved to require MICs of
0.125 times the breakpoint. Among these, 44/65 cases of unconfirmed susceptibility and 199/232 of the unconfirmed resistance in disc tests were discounted when the disc tests were repeated at the ARMRL (five isolates failed to grow), suggesting that many such disagreements reflected local problems, not fundamental flaws in the BSAC disc test.
Unconfirmed resistance might arise, for example, (i) from disc failure at the sentinel site, (ii) as a result of plasmid loss, (iii) too heavy an inoculum, or (iv) as a result of overgrowth, by the majority population, of resistant variants that were present initially. For important resistances that are currently rare (e.g. glycopeptide resistance in S. aureus) microbiologists may consider it prudent to follow-up disc testing results that indicate resistance with MIC testing (e.g. by Etest) or by referral to a reference laboratory. Unconfirmed susceptibility might arise if (i) the wrong organism was submitted or (ii) if resistant mutants were more evident in the re-test. Plasmid loss before re-testing at ARMRL is an especially plausible explanation for four Enterobacteriaceae isolates that were found resistant to both cefotaxime and ceftazidime in disc tests at their source hospital but to neither agent in MIC tests at the ARMRL, nor in repeat disc tests. In other cases, for example the failure to confirm much of the reported meropenem resistance in P. aeruginosa, plasmid loss seems an unlikely explanation, as such resistance is rarely plasmid mediated.12
Some of the antibiotic/organism combinations that gave frequent categorization disagreements between disc and MIC tests were surprising, others not. The large number of Enterobacteriaceae isolates found to be ampicillin susceptible in disc tests (and often in repeats of these at the ARMRLTable 6) but resistant in MIC tests was surprising, to the extent that one-third (33/93) of these were E. coli, a species where most ampicillin resistance is anticipated to be high-level and due to TEM enzymes.13 However, other discrepancies with ampicillin and Enterobacteriaceae were predictable, since the BSACs recent reduction in the zone breakpoint to 14 mm leads to numbers of isolates of Klebsiella spp. and AmpC-inducible species being captured within the borders of the susceptible category despite their possession of ampicillin-degrading chromosomal ß-lactamases. Perhaps the best advice here is that any finding of ampicillin susceptibility in members of these species should be treated with scepticism, as advised by Livermore et al.,14 or suppressed. The desirability of providing this type of advice underscores the need for accurate species identification in the clinical diagnostic laboratory.
Disagreements for oxacillin against CoNS (where many isolates were found susceptible in disc tests but resistant by MIC, and by detection of mecA) were also unsurprising. The problems of detecting oxacillin resistance in CoNS are well known and have been discussed elsewhere.15 Currently there is optimism that cefoxitin will prove a much better screening agent than methicillin or oxacillin for mecA-associated resistance in staphylococci, but this method post-dates the surveys on which this analysis is based; otherwise molecular detection of mecA or its product must remain the reference method.
Frequent zone:MIC categorization disagreements (in both directions) for ß-lactamase inhibitor combinations against Enterobacteriaceae are also unsurprising. The susceptibility distributions for these combinations notoriously straddle breakpoints, increasing the likelihood of categorization errors.7 Moreover, in the case of piperacillin/tazobactam, the MIC:zone correlation line for the standard 75+10 µg discs has a very shallow gradient, and there is an anomaly in standardizing zones from discs, which deliver a drug ratio, against MICs determined with a fixed inhibitor concentration of 4 mg/L tazobactam. We have explored the particular problems of testing with piperacillin/tazobactam discs elsewhere, recommending that Etests should be used for isolates that give borderline results.7 Another unsurprising case of frequent categorization disagreements was teicoplanin against enterococci, where many teicoplanin-susceptible isolates were graded as resistant in the hospitals disc tests but were found fully susceptible in MIC tests (and, mostly, in repeat disc tests at the ARMRL). Teicoplanin is a large molecule that diffuses poorly in agar, perhaps underlying these problems.16 Microbiologists should be sceptical of any enterococcus that appears to be susceptible to vancomycin but resistant to teicoplanin in disc tests. Such a phenotype has never been confirmed in the genus.
Despite the quality assurance procedures adopted in the surveys,57 it seems that particular problems arose at several testing hospitals, judging from the disproportionate number of disagreements between their disc categorizations and the MIC determinations and repeat disc tests at the ARMRL. One hospital in particular categorized 12% of its 137 Enterobacteriaceae incorrectly with respect to ampicillin (9% unconfirmed resistance, 16% unconfirmed susceptibility). The same hospital also misreported Enterobacteriaceae as resistant to ciprofloxacin, cefotaxime and ceftazidime to a greater extent than other hospitals, although the absolute numbers of resistant organisms were small. Whereas the total numbers of S. aureus reported as resistant to fusidic acid or rifampicin by disc test were low, the majority of isolates (63/76) reported as resistant by four laboratories proved susceptible by MIC testing, and all the isolates reported by one laboratory as resistant to fusidic acid (10/10) or rifampicin (8/8) proved to be susceptible. Five laboratories also accounted for 42/65 of the unconfirmed reports of teicoplanin resistance in enterococci.
It is useful to compare the present findings with those of the BSACs own evaluation (Table 8), which was based on the distribution of reference strains to 19 hospital laboratories comparing the zones that they recorded with centrally determined MICs. Caution is needed in comparing the absolute rates of disagreements between the two studies, as both used different but non-random sets of strains biased towards those with resistance. Nevertheless, it is notable that both analyses showed higher disagreement rates for P. aeruginosa, where most resistance is mutational and low level, than for Enterobacteriaceae, where more resistance is plasmid-mediated and high level. Moreover, both studies found more categorization errors with CoNS than with S. aureus, and both encountered particularly high disagreement rates with teicoplanin against enterococci.
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Acknowledgements |
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Footnotes |
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Present address. Healthcare-Associated Infection and Antimicrobial Resistance Department, HPA Communicable Disease Surveillance Centre, Colindale, London NW9 5EQ, UK.
¶ Present address. Wells Healthcare Communications Ltd., Speldhurst Place, Speldhurst Road, Tunbridge Wells, Kent, TN4 0JB, UK
Present address. St. Georges Healthcare NHS Trust, Blackshaw Road, London SW17 0QT, UK.
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References |
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2
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