1 Department of Microbiology, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, UK; 2 Institut für Infektionsmedizin, Zentrum für Klinisch-Theoretische Medizin, Universitätsklinikum Hamburg-Eppendorf, Germany
Received 16 March 2005; returned 10 May 2005; revised 14 May 2005; accepted 24 May 2005
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Abstract |
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Methods: Isolates (n = 120) were identified to species level and antimicrobial susceptibilities were determined using agar incorporation methods and Mastascan Elite. Phenotypes were examined using an Expert System (ES) and putative genotypes were suggested using interpretative reading.
Results: Identification was correct in 119 of 120 isolates. The ES was able to identify the correct ß-lactam phenotype (as deduced from molecular methods) in a single choice in 98 of 120 (81.7%) isolates. In an additional 15 (12.5%) cases, the ES identified the correct ß-lactam phenotype within two or more choices. The detected phenotype was incorrect in seven (5.8%) isolates, but three of these were not inherent to the ES.
Conclusions: The Mastascan Elite ES is relatively inexpensive and flexible and can identify the mechanism of resistance to oxyimino-cephalosporins in the majority of Enterobacteriaceae without recourse to molecular methods.
Keywords: interpretative reading , expert system , AmpC , CTX-M , K1 , ESBLs
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Introduction |
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The requirements for interpretative reading are that organisms are identified (generally to species level), that a large battery of appropriate antimicrobial agents be tested and that one is able to apply the necessary expert knowledge. These requirements are met by automated systems such as the VITEK and VITEK2 (bioMérieux, Marcy l'Étoile, France), the Phoenix (Becton Dickinson, Sparks, MD, USA) and the Microscan Walkaway (Dade Behring Inc., Sacramento, CA, USA). However, the majority of susceptibility tests in the UK are performed using disc-diffusion methods and, despite the use of a variety of image analysers, interpretative reading is not possible because organisms are not identified fully and insufficient antimicrobials are tested.
In this study, we evaluated the performance of an agar incorporation method of susceptibility testing and identification in conjunction with a Mastascan Elite (Mast, Bootle, UK) with an Expert System (ES) and using a panel of 120 genotypically characterized organisms including those possessing: penicillinases (SHV-1, TEM-1 and -2); extended spectrum ß-lactamases (ESBLs; derived from TEM and SHV; CTX-M); chromosomal and plasmidic AmpC in the presence and absence of penicillinases and ESBLs; and also chromosomal K1 in Klebsiella oxytoca. The ES operates on a series of if-and-then rules. For example, if the organism is Klebsiella pneumoniae and it is resistant to ampicillin, cefalexin and ceftazidime, and susceptible to cefotaxime and cefotetan, then the phenotype is compatible with ESBL production and cefotaxime should be edited to resistant. As a second example, if the organism is Enterobacter cloacae and it is resistant to cefotaxime, ceftazidime and cefotetan, but susceptible to cefepime, then the phenotype is compatible with derepressed AmpC production.
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Materials and methods |
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The bacterial isolates selected for this study comprised 120 oxyimino-cephalosporin-resistant isolates of Gram-negative bacteria, the majority of which have been characterized previously.4,5 Isolates were kindly donated by: The University Medical Center, Hamburg-Eppendorf, Germany; The Antimicrobial Research Group, The University of Birmingham, UK; and The Antibiotic Resistance Monitoring & Reference Laboratory, The Health Protection Agency, Colindale, UK. Species identification was performed by routine laboratory methods including API20E and API32E (bioMérieux).
PCR of ß-lactamase-encoding genes
Reference confirmation for ESBL production (CTX-M, SHV, TEM, OXA, VEB and PER) and plasmid-mediated AmpC ß-lactamase was by molecular characterization, i.e. by PCR analysis for ß-lactamase genes4 and, where applicable, by nucleotide sequencing. Nucleotide sequences were determined by bidirectional sequencing of PCR products, carried out by the Bigdye dideoxy chain termination method on an ABI Prism 310 DNA sequencer (Perkin-Elmer Corp., Foster City, CA, USA). Nucleotide sequences were analysed using commercial software (Vector NTI suite; InforMax Inc., Bethesda, MA, USA) against the GenBank database. Hyperproduction of AmpC chromosomal ß-lactamase was inferred from resistance to ureidopenicillins, oxyimino-cephalosporins and 7--methoxy-cephalosporins; hyperproduction of K1 ß-lactamase was inferred from resistance to ureidopenicillins, cefuroxime and aztreonam, resistance or intermediate resistance to cefotaxime and ceftriaxone, but susceptibility to ceftazidime.1,3 Retention of resistance mechanisms was confirmed by disc diffusion tests using the following discs: cefoxitin 30 µg, cefpodoxime 30 µg, cefpodoxime 30 µg + clavulanic acid 10 µg, and cefepime 30 µg in 25 mm proximity to a co-amoxiclav 20 + 10 µg disc. All discs were supplied by Mast and BSAC methodology6 was used. It is important to stress that results from these tests were not used to define resistance mechanisms.
The K. pneumoniae isolates (n = 43) harboured SHV-2 (n = 5), SHV2a (n = 1), SHV-3 (n = 1), SHV-4 (n = 3), SHV-5 (n = 5), SHV-2 + SHV-5 (n = 1), SHV-12 (n = 12), SHV-13 (n = 1), SHV-18 (n = 1), SHV-19 (n = 1), SHV-36 + CTX-M-9 (n = 1), SHV-2a + TEM-110 (n = 1), SHV-14 + TEM-29 (n = 1), TEM-47 (n = 1), CTX-M-26 (n = 1) and LEN type (ABL: N53S, A201P, P218A) (n = 1); five further isolates harboured plasmidic AmpC (in one case inducible) and one isolate harboured SHV-1 only. The Escherichia coli isolates (n = 28) harboured SHV-2 (n = 1), SHV-5 (n = 1), SHV-12 (n = 3), TEM-3 (n = 1), TEM-4 (n = 1), TEM-9 (n = 1), TEM-10 (n = 1), TEM-52 (n = 1), TEM-111 (n = 1), TEM-2 + CTX-M-9 (n = 1), CTX-M-1 (n = 2), CTX-M-3 (n = 1), CTX-M-14 (n = 2) and CTX-M-23 (n = 1); two isolates produced TEM-1 only. Four isolates of E. coli possessed up-regulated chromosomal versions of AmpC: two possessed plasmidic AmpC alone (one BIL-1 and one CMY-4) and two possessed CMY-4 in conjunction with either CTX-M-15 or CTX-M-33. The E. cloacae isolates (n = 19) included SHV-12 (n = 5), TEM-type (ABL: A184V) (n = 8), CTX-M-9 (n = 1), SHV-12 + CTX-M-9 (n = 1) and four isolates with derepressed chromosomal AmpC. The K. oxytoca isolates (n = 12) harboured SHV-12 (n = 1), CTX-M-1 (n = 2), plasmidic AmpC (n = 1) or hyperproduced K1 enzyme (n = 8). The Enterobacter aerogenes isolates (n = 5) possessed either CTX-M-1 (n = 2) or chromosomal AmpC (n = 3). The Proteus mirabilis isolates (n = 4) possessed TEM-92, CTX-M-1, CTX-M-15 or CTX-M-22. The Acinetobacter isolates (n = 4) did not possess ESBLs and interpretative reading was inappropriate. The Hafnia alvei and the Morganella morganii isolates and the two isolates of Citrobacter freundii produced derepressed chromosomal AmpC. The single isolate of Salmonella sp. produced SHV-12.
Prior to this study, none of the isolates had been made available to the testing laboratory. For phenotypic detection of resistance mechanisms, antimicrobial susceptibilities were determined by an agar incorporation breakpoint method based on that described by Faiers et al.7 Antibiotics were incorporated into Iso-Sensitest agar (Oxoid, Basingstoke, UK) at single breakpoint concentrations recommended by the BSAC6 using Adatabs (Mast). Antibiotic plates comprised: ampicillin (16 mg/L), ceftazidime (2 mg/L), cefuroxime (8 mg/L), cefotaxime (1 mg/L), meropenem (4 mg/L), piperacillintazobactam (16 mg/L), cefotetan (4 mg/L), aztreonam (1 mg/L), piperacillin (16 mg/L) and cefepime (1 mg/L). Identification was by multipoint inoculation onto agar media containing substrates (Mast) to detect: fermentation of cellobiose, inositol, maltose, rhamnose, sorbitol and sucrose; hydrolysis of aesculin; presence of tryptophan deaminase, lysine and ornithine decarboxylase and urease; the ability to grow on citrate as sole carbon source; and the production of acetoin. The presence of ß-galactosidase and production of indole were determined in liquid media. Methods have been published previously.8,9 The inoculum for both susceptibility and metabolic tests was 104 cfu/spot and plates were read using a Mastascan Elite image analyser (Mast). Resistance phenotypes were determined using an ES running on Microsoft Access and populated with published rules.3,10,11 The ES is now integral to the Mastascan Elite and is completely configurable by the user.
Data analysis
Initially, the identity and genotypic mechanism of resistance of the isolates was known only to workers in Hamburg, Germany (81 isolates) and Birmingham, UK (39 isolates). Isolates were assigned accession numbers and were processed blindly in Sheffield, UK. For each isolate, the Sheffield evaluators completed a data sheet indicating the identity of the isolate and the ES's interpretation of the phenotype. These records were sent to Germany, where they were merged and analysed using Microsoft Excel tools.
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Results |
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The ES provided an interpretation of the ß-lactam phenotype for all 120 (100%) isolates tested (Table 1). Overall, the ES was able to identify the correct ß-lactam phenotype (as deduced from molecular methods) in a single choice in 98 of the 120 (81.7%) isolates. In an additional 15 (12.5%) cases, the ES identified the correct ß-lactam phenotype within two or more choices. The detected phenotype was incorrect in seven (5.8%) isolates, which are discussed below: three of these were not inherent to the ES.
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The performance of the ES by species is shown in Table 1. The percentage of isolates for which the ES correctly identified the correct phenotype in one or more choices varied from a high of 100% for C. freundii (two of two isolates), E. aerogenes (five of five), H. alvei (one of one), K. oxytoca (12 of 12), M. morganii (one of one), Salmonella sp. (one of one) and P. mirabilis (four of four), to lows of 79% for E. cloacae (four of 19 isolates misinterpreted) and 75% for Acinetobacter spp. (one of four isolates misinterpreted).
Phenotypes
Certain phenotypes were more difficult than others for the ES to identify (Table 2). Among the ESBL-producing E. cloacae isolates (genotype composition: chromosomal AmpC + ESBL), four were incorrectly identified by the ES as having only chromosomal AmpC: these isolates were susceptible to cefepime. One isolate of Acinetobacter sp. was interpreted by the ES as an L2 or L1 + L2 ß-lactamase producer, because initially it was misidentified as S. maltophilia. When the correct species name was entered into the ES on repeat testing, interpretation of the resistance mechanism was correct (Table 3). One K. pneumoniae isolate that produced SHV-2a was identified by the ES as having a wild-type-acquired penicillinase phenotype (SHV-1). Repeat testing of the genotype composition revealed that this isolate had, indeed, lost its ESBL plasmid (Table 3). The inability of the ES to ascertain a definite phenotype to one isolate of E. coli (genotype composition: chromosomal AmpC ß-lactamase) was due to a mixed culture with an ESBL-producing isolate. Retesting resolved the problem and the ES suggested chromosomal AmpC production (Table 3).
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Discussion |
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Laboratory staff must thus be aware of an increasing array of different resistance mechanisms and phenotypes. One answer is to use computerized ESs. These operate on the tenet that resistances to multiple related antimicrobial agents often depend on single mechanisms, analyse susceptibility patterns rather than results for individual antimicrobials and predict underlying resistance mechanisms using interpretative reading.
In this paper, we evaluated a rule-based ES that can accurately predict the ß-lactamase phenotype of Enterobacteriaceae isolates on the basis of routine agar dilution tests. This system has several advantages over existing ESs. First, it is relatively inexpensive and operates on qualitative data: in this study we analysed agar incorporation susceptibility results, but the system is equally applicable to results derived from disc diffusion tests. The system is also extremely flexible regarding selection of antimicrobial agents and choice of expert rules: most existing expert systems are typically built-in components of complex AST systems, such as VITEK2, Phoenix or MicroScan Walkaway. One must always realize, of course, that choice of antibiotics impacts on performance of the ES. The ES used in the current study did not make use of testing for synergy between oxyimino-cephalosporins and clavulanic acid, primarily because this latter compound was too unstable for use,7 rather it analysed the susceptibility pattern of a wide range of ß-lactam antibiotics using if-and-then rules. Also, other workers have pointed out the disadvantages of using clavulanic acid to detect ESBL production in isolates that simultaneously possess AmpC ß-lactamase.16
Accurate speciation forms the basis on which interpretative reading operates. The agar incorporation identification scheme used in this study has been evaluated previously,9 and in the present study, correctly identified 119 out of 120 isolates. It misidentified one isolate of Acinetobacter sp. as S. maltophilia, but this was re-identified correctly on retesting at the end of the study.
The ES performed remarkably well, demonstrating 82% interpretative agreement with genotype composition data. In a further 15 (12%) cases the resistance profile of the antimicrobials tested overlapped several phenotypes and the correct phenotype was identified by the ES in two or more choices. Although not included in the present evaluation, the ES is also perfectly able to detect other enzymes commonly found in the UK, e.g. CTX-M-15, or in continental Europe, e.g. TEM-24.
The VITEK2 Advanced Expert System (AES) has been shown to be an impressive system in some studies19 but has performed less well in others.20 The results of the present study suggest that the Mastascan ES can offer comparable or even better sensitivity and performance than the VITEK2 AES when tested in a similar study design.20 In this study, the AES was able to correctly and accurately identify the ß-lactam phenotype in only 111 of 196 (56.6%) isolates. In an additional 46 (23.5%) isolates, the AES listed several possible mechanisms but did not specify the most likely one. The phenotype detected was incorrect in 26 (13.2%) other cases, and no phenotype could be identified in the remaining 13 (6.6%) cases.
However, it is only possible to make fair comparisons between methods when the same set of challenge organisms has been used. Stürenburg et al.21 compared the ability of Phoenix and VITEK2 to detect ESBLs in 34 clinical isolates of E. coli and Klebsiella spp. Phoenix correctly identified ESBLs in all isolates, whereas VITEK2 failed to detect ESBLs in five isolates (85% detection rate). Using the same strains, the Mastascan ES detected ESBLs in 33 of 33 isolates (one isolate was not examined). On the other hand, VITEK2 correctly identified K. oxytoca chromosomal ß-lactamase in all eight isolates studied, whereas Phoenix misidentified seven out of eight as ESBLs. The Mastascan ES correctly identified K1 ß-lactamase in three of the four isolates examined.
Secondly, Stürenburg et al.16 evaluated the ability of cefepimeclavulanate ESBL Etests to detect ESBLs in 54 clinical isolates of Enterobacteriaceae. Overall, the ESBL Etest was 98% sensitive with cefepimeclavulanate, 83% with cefotaximeclavulanate and 74% with ceftazidimeclavulanate. In the present study, using the same isolates, the Mastascan ES detected ESBLs in 44 of 47 isolates (94% sensitivity). With Enterobacter isolates, the cefepimeclavulanate Etest confirmed ESBL production in 13 of 13 isolates compared with four of 13 with cefotaximeclavulanate or ceftazidimeclavulanate Etests. The Mastascan ES detected ESBLs in 10 of 13 Enterobacter isolates. In terms of specificity, the cefepimeclavulanate Etest misidentified four of six K1 ß-lactamases as ESBLs, whereas the Mastascan ES correctly identified K1 ß-lactamase in three of the four isolates examined.
Finally, Stürenburg et al.22 evaluated the MicroScan ESBL-plus confirmation panel for detection of ESBLs in clinical isolates of oxyimino-cephalosporin-resistant Gram-negative bacteria. The system correctly classified 50 of 57 ESBL producers as ESBL positive (88% sensitivity) and 25 of 30 non-ESBL producers as ESBL negative (83% specificity). In the present study, the Mastascan ES correctly classified 47 of 50 of the same ESBL producers as ESBL positive (94% sensitivity). The Mastascan ES identified 18 of 27 non-ESBL (predominantly AmpC-producing) isolates as ESBL negative (67% specificity). This apparent lack of specificity was caused by the system suggesting more than one possible resistance mechanism for a particular phenotype. As with all phenotypic assays, a primary limitation of the ES was its inability to reliably discern multiple resistance mechanisms affecting the same class of antibiotics, particularly when one trait was dominant. The ES does not indicate the most likely genotype and, if deemed appropriate, the 15 of 120 isolates for which the ES suggested more than one possible resistance mechanism could be subjected to molecular examination. The organisms used in this evaluation were a particularly challenging set and it is likely that use of the ES would negate the need for molecular characterization in the majority of routine clinical isolates.
Any phenotypic method of interpreting antibiograms may also fail to detect resistance mechanisms that are poorly expressed in vitro. For example, as seen in four Enterobacter isolates tested in this study, there was little to reliably distinguish the resistance pattern of an ESBL in the presence of a derepressed AmpC (genetic characterization) from that of AmpC cephalosporinase production alone. In three of these isolates, the organism was resistant to cefotetan (suggesting derepressed AmpC production) but susceptible to oxyimino-cephalosporins and monobactams by agar incorporation testing at a fixed breakpoint concentration of antibiotic (suggesting the absence of an ESBL). These isolates were examined by disc diffusion tests. With one isolate, an ESBL-producing subpopulation was detected within the inhibition zone of a cefepime disc; in a second, a subpopulation around a ceftazidime disc was inhibited by clavulanate; and in a third isolate, resistance to ceftazidime was partially reduced by clavulanate. Interestingly, these three isolates had TEM-type (ABL: A184V) enzymes; the fourth isolate possessed SHV-12 in the presence of AmpC. In the UK, failure to detect ESBLs in Enterobacter spp. should not compromise individual patient management, as cephalosporins should always be avoided when treating AmpC-producing organisms. However, this may not always be the case in countries where cefepime is widely used, e.g. USA and Belgium. Similarly, the presence of plasmidic AmpC in three isolates of K. pneumoniae (genotypic characterization) mimicked the phenotypic pattern of an ESBL in the presence of AmpC: indeed, two of these isolates were resistant to cefepime and demonstrated synergy with clavulanate, a classical ESBL picture. Again, in terms of individual patient management, this would be unimportant but may be important in terms of infection control and molecular testing may occasionally be warranted.
The current ES operated using therapeutic antimicrobial breakpoints; tailoring the system to use epidemiological breakpoints would increase its sensitivity. Furthermore, exercises such as this permit the rule base to be continually expanded and developed to recognize unusual and novel resistance mechanisms. Antimicrobial resistance mechanisms are becoming increasingly complex1218 and, with staffing problems in clinical laboratories and a general loss of expertise, the requirement for inexpensive, flexible expert systems may be met by systems such as the Mastascan Elite.
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References |
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Livermore DM, Struelens M, Amorim J et al. Multicentre evaluation of the VITEK2 Advanced Expert System for interpretive reading of antimicrobial resistance tests. J Antimicrob Chemother 2002; 49: 289300.
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