Ability of the VITEK 2 Advanced Expert System To Identify β-Lactam Phenotypes in Isolates of Enterobacteriaceae and Pseudomonas aeruginosa

Abstract

The Advanced Expert System (AES) was used in conjunction with the VITEK 2 automated antimicrobial susceptibility test system to ascertain the β-lactam phenotypes of 196 isolates of the family Enterobacteriaceae and the species Pseudomonas aeruginosa. These isolates represented a panel of strains that had been collected from laboratories worldwide and whose β-lactam phenotypes had been characterized by biochemical and molecular techniques. The antimicrobial susceptibility of each isolate was determined with the VITEK 2 instrument, and the results were analyzed with the AES to ascertain the β-lactam phenotype. The results were then compared to the β-lactam resistance mechanism determined by biochemical and molecular techniques. Overall, the AES was able to ascertain a β-lactam phenotype for 183 of the 196 (93.4%) isolates tested. For 111 of these 183 (60.7%) isolates, the correct β-lactam phenotype was identified definitively in a single choice by the AES, while for an additional 46 isolates (25.1%), the AES identified the correct β-lactam phenotype provisionally within two or more choices. For the remaining 26 isolates (14.2%), the β-lactam phenotype identified by the AES was incorrect. However, for a number of these isolates, the error was due to remediable problems. These results suggest that the AES is capable of accurate identification of the β-lactam phenotypes of gram-negative isolates and that certain modifications can improve its performance even further.

The VITEK 2 automated antimicrobial susceptibility test system is a new integrated system which automatically performs rapid identification and antimicrobial susceptibility testing after an inoculum has been prepared manually (J.-P. Gayral, R. Robinson, and D. Stamstedt, Abstr. Eur. Cong. Clin. Microbiol. Infect. Dis., abstr. P254, p. 53, 1997). Its improved performance over those of earlier rapid systems is due to the larger number of wells in its card, enhanced optics, and new algorithms based on kinetic analysis of data (S. Dib, J. Nguyen, V. Jarlier, and A. Philippon, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. D50, p. 92, 1997; Gayral et al., Abstr. Eur. Cong. Clin. Microbiol. Infect. Dis.; M. Ghanem, C. Bradford, D. Freiner, M. Ullery, and J. Gerst, Abstr. 98th Annu. Meet. Am. Soc. Microbiol. 1998, abstr. C-416, p. 200, 1998; J. H. Jorgensen, A. L. Barry, M. M. Traczewski, D. F. Sahm, M. L. McElmeel, and S. A. Crawford, Abstr. 98th Annu. Meet. Am. Soc. Microbiol. 1998, abstr. C-422, p. 201, 1998; L. A. Meeh, C. Shubert, S. Weber, P. Kim, and M. Peyret, Abstr. 98th Annu. Meet. Am. Soc. Microbiol. 1998, abstr. V-66, p. 523, 1998; E. S. Moland, K. S. Thomson, and C. C. Sanders, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. D48, p. 92, 1997; C. Shubert, R. Griffith, W. McLaughlin, M. Ullery, and M. Peyret, Abstr. 98th Annu. Meet. Am. Soc. Microbiol. 1998, abstr. C-478, p. 211, 1998). The Advanced Expert System (AES) is an expert system designed to analyze results generated by the VITEK 2 system for biologic validity and then provide comments on results (J. M. Boeufgras, A. Lazzarini, M. Peyret, and J. Zindel, Abstr. Eur. Cong. Clin. Microbiol. Infect. Dis., abstr. P299, p. 64, 1997; J. M. Boeufgras, R. Vachon, J. L. Balzer, C. Davenas, A. Rongier, M. Tarpin, and M. Peyret, Abstr. Eur. Cong. Clin. Microbiol. Infect. Dis., abstr. P300, p. 64, 1997). Unlike previous expert systems, the AES is based upon an extensive knowledge base that comprises over 2,000 phenotypes and 20,000 MIC distributions (Boeufgras et al., Abstr. Eur. Cong. Clin. Microbiol. Infect. Dis., abstr. P299). This allows it to recognize certain susceptibility patterns as indicative of specific phenotypes and interpret the results accordingly.

A phenotype is defined as the expression of a specific mechanism of susceptibility or resistance to a given drug class within a particular species. The word phenotype is used in this context in preference to the word genotype since categorization is often based upon tests for gene products or phenotypes that result from the expression of the gene product rather than upon tests for the actual gene or gene sequence involved. The wild-type phenotype is defined as the phenotype for that species in the “wild,” i.e., prior to any mutation of chromosomal genes or acquisition of new DNA that alters susceptibility to the drug class in question. Thus, if one considers phenotypes for Escherichia coli and β-lactam drugs, there are a number of possible phenotypes (Table 1). The wild type is devoid of any significant levels of β-lactamase and is thus susceptible to ampicillin and most other β-lactam antibiotics. Once the strain has acquired a plasmid-mediated penicillinase like TEM-1 or SHV-1, it is resistant to penicillins and perhaps cephalothin and now possesses a penicillinase phenotype (Table 1). If the strain acquires an extended-spectrum β-lactamase (ESBL) or if the gene encoding its resident TEM-1 or SHV-1 mutates to produce an ESBL derivative, the strain will have an ESBL phenotype of reduced susceptibility or resistance to expanded-spectrum cephalosporins and aztreonam, in addition to penicillins and cephalothin (Table 1). If the strain acquires a plasmid-mediated AmpC β-lactamase from an organism such as Enterobacter spp. or Citrobacter freundii or if mutations in the promoter region of its own chromosomal ampC gene occur, giving rise to elevated levels of AmpC β-lactamase, the strain will have a cephalosporinase phenotype and will display reduced susceptibility or resistance to virtually every β-lactam drug except the carbapenems (Table 1). Thus, for each species, one can prepare a list of phenotypes based upon possible mechanisms of susceptibility and resistance for each drug class, and these phenotypes can be associated with certain susceptibility patterns (3–5, 7, 12, 15, 17).

TABLE 1.

Examples of β-lactam phenotypes for E. coli^a

Phenotype	Examples of β-lactamase(s)b	Drugs affectedc
Wild type	None	None, phenotype susceptible to most β-lactams including ampicillin
Acquired penicillinase	TEM-1, SHV-1, OXA-1	Penicillins and certain older cephalosporins like cephalothin
ESBL	TEM-3, SHV-2	Same as acquired penicillinase plus newer cephalosporins and aztreonam
Cephalosporinase	Plasmid-mediated AmpC, hyperproducers of chromosomal AmpC	Same as ESBL plus inhibitor-drug combinations and cephamycins

^aFrom references 3, 4, 5, 12, and 15. 

^bExamples of β-lactamases that can produce the phenotype listed. 

^cDrugs to which an isolate with the phenotype listed shows reduced susceptibility or resistance in comparison to the susceptibility or resistance of the wild-type phenotype. 

From this association between phenotype and susceptibility patterns, the AES was developed by using a large knowledge base of MIC distributions for each of the known phenotypes. This knowledge base was obtained from published reports, human experts with their own databases on phenotypes, and in-house data at bioMérieux (Boeufgras et al., Abstr. Eur. Cong. Clin. Microbiol. Infect. Dis., abstr. P299 and P300). For each of the recognized phenotypes, the range of MICs obtained from tests with each specific drug was determined and was defined as the MIC distribution for that phenotype. For example, from data available worldwide, the MIC distribution for wild-type E. coli and ampicillin was found to be 0.5 to 16.0 μg/ml, while that for the acquired penicillinase phenotype for E. coli was found to be ≥256 μg/ml. Thus, with a large knowledge base of over 20,000 MIC distributions for over 2,000 phenotypes, it should be possible for the AES to predict a phenotype by using the susceptibility pattern obtained from tests with the VITEK 2 system.

Therefore, a study was designed to ascertain the ability of the AES to correctly identify the β-lactam phenotypes of 196 isolates of the family Enterobacteriaceae and the species Pseudomonas aeruginosa. These isolates had been collected from laboratories worldwide and had been characterized for their β-lactam phenotypes by biochemical and molecular methods.

MATERIALS AND METHODS

Strains.

A total of 196 isolates of the family Enterobacteriaceae and the species P. aeruginosa were collected from laboratories worldwide and their β-lactam phenotypes were characterized by biochemical and molecular techniques (2, 6, 10, 11, 13, 16, 18). Many of these have been described previously (8, 13, 14, 17). β-Lactamases were identified biochemically as to their isoelectric points and inhibitor-substrate profiles. For species that produce chromosomally encoded inducible AmpC β-lactamases, basal enzyme levels and levels following induction with cefoxitin were ascertained by using cephalothin as a substrate to determine if the strain was a wild type or had a mutant phenotype. For Klebsiella oxytoca, levels of enzyme were measured by using nitrocefin as a substrate to ascertain if the strain was a wild type or a hyperproducer of the chromosomal K1 β-lactamase (8, 13, 17). Permeability mutants were determined by analysis of outer membrane porins (2). The β-lactam phenotypes and species included among the 196 isolates are shown in Table 2. These included both common and rare or atypical resistance phenotypes. For strains with more than one β-lactamase, the β-lactam phenotype produced by the dominant enzyme was used for analysis. For example, among C. freundii strains with the high-level cephalosporinase phenotype there were strains that also produced an acquired penicillinase. However, the broader substrate profile of the AmpC β-lactamase masked the presence of the acquired penicillinase. Thus, the phenotypes were the same for all high-level cephalosporinase producers regardless of the presence or absence of an acquired penicillinase, and the presence of the second enzyme was ignored for the purposes of data evaluation.

TABLE 2.

β-Lactam phenotypes included in the test panel of 196 isolates

Species (no. of isolates)	β-Lactam phenotype (no. of isolates)
E. coli (39)	Wild type (4),a acquired penicillinase (12), inhibitor-resistant penicillinase (2), cephalosporinase (7), ESBL (14)
Klebsiella pneumoniae (41)	Wild type-acquired penicillinase (20),b ESBL (16), cephalosporinase (5)
K. oxytoca (20)	Wild type (6),c acquired penicillinase (3), ESBL (6), high-level natural penicillinase (5)d
Proteus mirabilis (9)	Wild type (2),a acquired penicillinase (5), ESBL (1), impermeability (1)
Enterobacter spp. (36)e	Wild type (7),f high-level cephalosporinase (8),g acquired penicillinase (6), ESBL (13), high-level cephalosporinase + impermeability (1),h ESBL + impermeability (1)i
C. freundii (19)	Wild type (5),f acquired penicillinase (3), ESBL (3), high-level cephalosporinase (8)g
Serratia marcescens (16)	Wild type (4),f acquired penicillinase (4), ESBL (4), high-level cephalosporinase (4)g
P. aeruginosa (16)	Wild type (5),f acquired penicillinase (5), high-level cephalosporinase (3),g high-level cephalosporinase + impermeability (3)j

^aThe wild type of this species produces no significant level of β-lactamase. 

^bFor this species, the wild type may produce β-lactamase that cannot be distinguished from the acquired SHV-1 penicillinase; thus, the two phenotypes are combined. 

^cThe wild type of this species produces low levels of the chromosomal K1 β-lactamase. 

^dThe phenotype includes strains that produce high levels of the chromosomal K1 β-lactamase. 

^eIncludes 15 Enterobacter cloacae and 21 Enterobacter aerogenes isolates. 

^fThe wild type of this species produces low basal levels of AmpC β-lactamase that can be induced to higher levels. 

^gThe phenotype results from mutation in regulatory genes that control the amount of the basal level of AmpC β-lactamase and the inducibility of enzyme expression. 

^hThe phenotype involves reduced susceptibility to cefepime and imipenem as well as other β-lactam antibiotics. 

ⁱThe phenotype involves reduced susceptibility to cephamycins, cefepime, and imipenem as well as other β-lactam antibiotics. 

^jThe phenotype produces reduced susceptibility or resistance to imipenem as well as all other antipseudomonal β-lactams. 

Susceptibility tests.

Antibiotic susceptibilities were determined according to the manufacturer's recommendations by using the VITEK 2 instrument. The cards used for the test were standard European cards and contained the following antibiotics and concentration ranges: (i) AST-N009 for members of the family Enterobacteriaceae (ampicillin, 2 to 32 μg/ml; amoxicillin-clavulanate [2:1 ratio], 2-1 to 32-16 μg/ml; cephalothin, 2 to 64 μg/ml; cefoxitin, 4 to 64 μg/ml; cefotaxime, 1 to 64 μg/ml; ceftazidime, 1 to 64 μg/ml; ticarcillin, 8 to 128 μg/ml; ticarcillin-clavulanate [clavulanate at 2 μg/ml with ticarcillin at a twofold dilution], 8-2 to 128-2 μg/ml; piperacillin-tazobactam [tazobactam at 4 μg/ml with piperacillin at a twofold dilution], 4-4 to 128-4 μg/ml; and imipenem, 0.5 to 16 μg/ml) and (ii) AST-N008 for P. aeruginosa (cefepime, 1 to 64 μg/ml; ceftazidime, 1 to 64 μg/ml; piperacillin, 4 to 128 μg/ml; pipercillin-tazobactam [tazobactam at 4 μg/ml with piperacillin at a twofold dilution], 4-4 to 128-4 μg/ml; ticarcillin, 8 to 128 μg/ml; ticarcillin-clavulanate [clavulanate at 2 μg/ml with ticarcillin at a twofold dilution], 8-2 to 128-2 μg/ml; imipenem, 0.5 to 16 μg/ml; meropenem, 0.25 to 16 μg/ml; and aztreonam, 1 to 64 μg/ml). Quality control was performed with each run by using E. coli ATCC 25922 and P. aeruginosa ATCC 27853.

Data analysis.

Since this study was not designed to assess the ability of the VITEK 2 system to identify these gram-negative species, the species name of the strain was manually entered into the instrument and only susceptibility tests were performed. (It is acknowledged that the interpretive abilities of the AES are dependent on the accuracy of the organism identification and that the fact that different laboratories use different identification systems is a relevant issue.) The results were then analyzed by the AES with a test version of software, and a hard-copy report of that analysis was obtained. The β-lactam phenotype identified by the AES was then compared to the phenotype that had been identified by biochemical and molecular methods. If the MIC distributions of a given phenotype were unique for the drugs tested, then a single phenotype was identified by the AES. However, if the MIC distributions for the drugs tested overlapped for several phenotypes, the AES would list all of the possible phenotypes. The AES was considered to have correctly identified the β-lactam phenotype of a strain if it listed the same β-lactam phenotype identified by biochemical and molecular methods (i) in a single choice or (ii) in one of several possibilities.

In tests with some strains, the AES could not identify a β-lactam phenotype. For these isolates, the AES suggested that there was either an error in the identification of the strain or an error in an MIC obtained with the VITEK 2 system or that there were so many inconsistencies that the test should be repeated. For these strains, data were analyzed to determine the precise cause of the problem, and tests were repeated for some isolates.

RESULTS

Overall, the AES was able to identify a β-lactam phenotype for 183 of the 196 (93.4%) isolates tested. The correct phenotype was identified by the AES in one or more choices for 157 of the 183 (85.8%) isolates, and for 111 of the 183 (60.7%), the correct phenotype was identified in a single choice. The 13 isolates for which a β-lactam phenotype could not be identified are described below.

Species.

The performance of the AES by species is shown in Table 3. The percentage of strains tested for which the AES correctly identified the β-lactam phenotype in one or more choices varied from a high of 92% for Enterobacter spp. to a low of 74% for E. coli and C. freundii.

TABLE 3.

Identification of β-lactam phenotypes by the AES with the data analyzed by species

Species	No. (%) of strains tested	No. (%) of strains
		Correct 1+a	Correct 1b	Incorrectc	Noned
E. coli	39	29	23	4	6
K. pneumoniae	41	35	30	6	0
K. oxytoca	20	15	10	4	1
P. mirabilis	9	7	4	1	1
Enterobacter spp.	36	33	21	3	0
C. freundii	19	14	10	0	5
S. marcescens	16	12	4	4	0
P. aeruginosa	16	12	9	4	0
Total	196 (100)	157 (80)	111 (57)	26 (13)	13 (7)

^aNumber of strains for which the correct β-lactam phenotype was identified by the AES in one or more choices. 

^bNumber of strains for which the correct β-lactam phenotype was identified by the AES in one choice. 

^cNumber of strains for which β-lactam phenotype was incorrectly identified by the AES. 

^dNumber of strains for which a phenotype could not be ascertained by the AES. 

Phenotypes.

Certain phenotypes were more difficult than others for the AES to identify (Table 4). Among the wild-type phenotypes, 10 were incorrectly identified by the AES. Many of these were incorrectly identified as having an acquired penicillinase phenotype, and none of these errors was due to incorrect MICs obtained with the VITEK 2 instrument (Table 5). One wild-type Enterobacter aerogenes strain was identified by the AES as an ESBL or acquired penicillinase producer due to a falsely elevated ceftazidime MIC. One of the three problems with the wild-type–acquired penicillinase and acquired penicillinase phenotypes was due to a falsely elevated cefotaxime MIC (Table 5). The elevated ceftazidime and cefotaxime MICs reverted to the correct result of susceptible on repeat testing. The phenotypes of all five of the K. pneumoniae strains with a plasmid-mediated cephalosporinase (AmpC) were incorrectly identified as ESBL plus impermeability by the AES because the correct phenotype was not in the database (Table 5). Although for the purposes of this study this was considered an incorrect identification, the MIC distributions for the two phenotypes are similar. Had the cephalosporinase phenotype been in the database for this species, the AES would have listed cephalosporinase or ESBL plus impermeability as the two phenotypes possible. Two E. coli strains that produced ESBLs were incorrectly identified as acquired penicillinase producers by the AES (Table 5). These two strains were unusual in that the MICs of cefotaxime and ceftazidime, the two expanded-spectrum cephalosporins on the test card, for the strains were below 1.0 μg/ml. For the current ESBL-producing indicator strains (9), only cefpodoxime MICs are ≥2.0 μg/ml in tests with these strains. Thus, the inability of the AES to ascertain the ESBL phenotypes for these strains was due to the absence of cefpodoxime on the test card. Two high-level cephalosporinase-producing Serratia marcescens strains were incorrectly identified by the AES as belonging to more susceptible phenotypes (Table 5). These errors were due to the fact that the MICs of a variety of drugs for these strains are uncharacteristically low. Thus, any MIC-based system would incorrectly categorize these strains. The failure of the AES to identify an impermeability phenotype for a single strain of P. mirabilis was due to the absence of the phenotype in the database (Table 5). However, when the AES identified the strain as having an acquired penicillinase phenotype, it did note that the MIC pattern was highly unusual.

TABLE 4.

Identification of β-lactam phenotypes by the AES with the data analyzed by phenotype

Phenotypea	No. (%) of strains tested	No. (%) of strainsb
		Correct 1+	Correct 1	Incorrect	None
Wild type	33	19	15	10	4
Wild type-acquired penicillinase	20	19	17	1
Acquired penicillinase	38	34	25	2	2
Inhibitor-resistant penicillinase	2	2
Cephalosporinase	12	4	2	5	3
High-level cephalosporinase	23	21	15	2
ESBL	57	48	32	5	4
High-level natural penicillinase	5	5	1
Impermeability ± β-lactamasec	6	5	4	1
Total	196 (100)	157 (80)	111 (57)	26 (13)	13 (6)

^aSee footnote c of Table 1 and footnotes to Table 2 for descriptions of phenotypes. 

^bSee footnotes a to d of Table 3 for descriptions of Correct 1+, Correct 1, Incorrect, and None, respectively. 

^cIncludes high-level cephalosporinase plus impermeability, ESBL plus impermeability, and impermeability. 

TABLE 5.

Strains incorrectly identified by AES^a

Phenotype (no.)b	Species (no.)c	Incorrect AES phenotype (no.)d	Causee
Wild type (10)	K. oxytoca (3)	AcPenase (3)	Database
	E. cloacae (1)	AcPenase (1)	Database
	E. aerogenes (1)	+ (1)f	Error in ceftazidime MIC
	S. marcescens (1)	AcPenase (1)	Database
	P. aeruginosa (4)	AcPenase (2)	Database
		+ (2)g	Database
Wild type, AcPenase (1)	K. pneumoniae (1)	+ (1)h	Database
AcPenase (2)	K. oxytoca (1)	Wild type (1)	Database
	S. marcescens (1)	+ (1)i	Error in cefotaxime MIC
Cefase (5)	K. pneumoniae (5)	ESBL + imperm (5)	No phenotype
HiCefase (2)	S. marcescens (2)	Wild type (1)	Strain
		AcPenase (1)	Strain
ESBL (5)	E. coli (4)	AcPenase (2)	Configuration
		AcPenase + Cefase (2)	Database
	E. cloacae (1)	HiCefase	Database
Imperm ± βlase (1)	P. mirabilis (1)	AcPenase (1)	No phenotype

^aAbbreviations: AcPenase, acquired penicillinase; Cefase, cephalosporinase; HiCefase, high-level cephalosporinase; ESBL + imperm, ESBL plus impermeability; Imperm ± βlase, impermeability with or without a β-lactamase. 

^bPhenotype (number of strains incorrectly identified). 

^cSpecies (number of strains) incorrectly identified. 

^dIncorrect phenotype (number of strains) identified by the AES. The symbol + indicates that two or more incorrect phenotypes were listed by the AES. 

^eCauses for the error in identification of phenotypes included general problems with the database for the AES (database), absence of a discriminating drug on the card used to test the strain (configuration), absence of the correct phenotype in the database (no phenotype), or peculiarities in the strain itself leading to atypical MICs which would prevent correct phenotype identification by any MIC-based system (strain). 

^fWild-type E. aerogenes (n = 1) ESBL, acquired penicillinase. 

^gWild-type P. aeruginosa (n = 2), one with high-level resistance and acquired penicillinase and one with high-level resistance, acquired penicillinase plus resistance to imipenem, and high-level resistance plus resistance to imipenem. 

^hWild-type and acquired penicillinase phenotype with ESBL, acquired penicillinase plus impermeability, impermeability, ESBL plus impermeability phenotype. 

ⁱAcquired penicillinase phenotype with ESBL and high-level cephalosporinase phenotype. 

Unidentifiable phenotypes.

The AES was unable to identify a phenotype for 13 of the 196 strains tested (Table 6). In most instances, there were either real errors in the MICs that made it impossible for the AES to match the susceptibility pattern to a phenotype for the species or the AES indicated that there were errors in MICs that made it impossible for it to match the pattern to a phenotype. For example, the AES could not identify a phenotype for three cephalosporinase-producing E. coli strains because the susceptibility pattern (which was in fact accurate) looked similar to that expected of an organism like Enterobacter or Citrobacter with an inducible AmpC β-lactamase (Table 6). True errors in the MICs of ampicillin, amoxicillin-clavulanate, cephalothin, and/or cefoxitin led the AES to suggest that three wild-type C. freundii strains were in fact Escherichia, Citrobacter youngae, or Citrobacter braakii. Repeat testing of most of these isolates did not resolve these problems.

TABLE 6.

Strains for which no phenotype could be identified by AES

Species (no. of strains)	Phenotype (no. of strains)	AES recommendationa
E. coli (6)	Cefase (3)	Correct BE in ceftazidime or change ID to Enterobacter, Citrobacter (2)
		Change ID to Enterobacter, Citrobacter or retest (1)
	ESBL (3)	Change ID to Citrobacter or retest (1)
		Correct BE in cefoxitin or change ID to Citrobacter (1)
		Retest (1)
K. oxytoca (1)	Wild type (1)	Correct BE in cefoxitin or change ID to K. pneumoniae (1)
P. mirabilis (1)	AcPenaseb (1)	Retest (1)
C. freundii (5)	Wild type (3)	Change ID to Escherichia or other Citrobacter species or retest (3)
	AcPenase (1)	Retest (1)
	ESBL (1)	Retest (1)

^aExplanation given by AES for its inability to select a phenotype. Number in parentheses is number of strains. Abbreviations: BE, biologic error in MIC of drug named; ID, identification. 

^bAcPenase, acquired penicillinase. 

DISCUSSION

Overall, the AES performed well in this validation study, identifying correctly the β-lactam phenotypes of 157 of the 196 isolates tested in one or more choices. It should be noted that for the purposes of this study, the isolate panel selected included many strains with phenotypes rarely or infrequently encountered in the clinical laboratory (e.g., ESBL- and cephalosporinase-producing E. coli) as well as strains with rarely encountered phenotypes that gave atypical results in susceptibility tests (i.e., ESBL-producing E. coli and K. pneumoniae for which ceftazidime MICs were <1 μg/ml). Thus, the overall performance of the AES reflects the strains and phenotypes tested and is not a reflection of overall performance in the average clinical laboratory.

Examination of the causes for the incorrect phenotypes identified by the AES revealed the need for several improvements. First, the AES, like any database system, needs to be updated often to ensure that all known phenotypes and MIC distributions are in the database. The occurrence of plasmid-mediated AmpC β-lactamases in clinical isolates of E. coli and K. pneumoniae was extremely rare during the time when the database for the AES was being developed. Thus, these phenotypes were not in the database and strains with these phenotypes were either unidentifiable or incorrectly identified by the AES.

Certain errors in identification of the cephalosporinase and ESBL phenotypes highlighted the need for a specific ESBL test on the card. At this time, ESBL producers are recognized by MIC distributions which cannot always distinguish between the cephalosporinase and ESBL phenotypes. Cefoxitin resistance in E. coli and K. pneumoniae may be due to impermeability rather than to the cephalosporinase. Thus, an isolate of these species with a cephalosporinase will have a susceptibility pattern similar to that of a porin mutant with an ESBL. The use of a specific test for ESBL production that compares the MICs of certain drugs in the presence and absence of clavulanic acid may improve discrimination between these phenotypes (13, 17, 18); M. M. Traczewski, A. L. Barry, S. D. Brown, J. A. Hindler, D. A. Bruckner, and D. F. Sham, Abstr. 97th Gen. Meet. Am. Soc. Microbiol. 1998, abstr. C-37, p. 137, 1998.

A final problem concerns the difficulty with identifying the impact of permeability changes on β-lactam susceptibility. For most species, the impact of permeability changes even on the wild-type phenotype has not been studied adequately to provide a database for phenotype recognition. Furthermore, certain susceptibility patterns can arise from the presence of β-lactamase, altered permeability to a drug, or a combination of the two factors. In only a few instances, e.g., resistance to imipenem in P. aeruginosa, can the role of altered permeability be clearly defined and predicted by use of MIC-based tests. In most instances, e.g., cefoxitin resistance in E. coli and K. pneumoniae or ceftazidime resistance in S. marcescens, MICs alone are inadequate for identification of the actual mechanism responsible for the resistance. Thus, it is likely that the problem of recognition of most impermeability phenotypes will not be resolved in the near future. However, tests for recognition of phenotypes such as ESBL plus impermeability in E. coli and acquired cephalosporinase in K. pneumoniae will be added to the commercial version of the software.

In summary, the AES was able to provide correct the β-lactam phenotypes of 157 of the 196 gram-negative isolates tested, including strains with phenotypes rarely encountered in the routine clinical laboratory, by using the card configurations in this study. Certain remediable problems with the system were identified, and remediation of these problems should lead to improved performance in the future. These results suggest that the AES should be very useful for the identification of the β-lactam phenotypes of gram-negative isolates and that further study of its utility for the clinical laboratory is warranted.