Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 29, Num. 3, 2011, pp. 297-301

Indian Journal of Medical Microbiology, Vol. 29, No. 3, July-September, 2011, pp. 297-301

Original Article

Comparison of the boronic acid disk potentiation test and cefepime-clavulanic acid method for the detection of ESBL among AmpC-producing Enterobacteriaceae

RM Shoorashetty¹, T Nagarathnamma², J Prathibha²

¹ Department of Microbiology, Pondicherry Institute of Medical Sciences, Ganapathichettikulam, Village No.20, Kalapet, Pondicherry - 605 014, India
² Department of Microbiology, Victoria Hospital Campus, Fort, BMC and RI, Bangalore - 560 002, Karnataka, India
Correspondence Address: T Nagarathnamma, Department of Microbiology, Victoria Hospital Campus, Fort, BMC and RI, Bangalore - 560 002, Karnataka, India, nagarathnammat@yahoo.co.in

Date of Submission: 03-Feb-2011 
Date of Acceptance: 01-Jul-2011

Code Number: mb11071

PMID: 21860114

DOI: 10.4103/0255-0857.83917

Abstract

Keywords: AmpC β-lactamase, boronic acid, cefepime, Enterobacteriaceae, Extended spectrum β-lactamase

Introduction

β-lactamases are the important mechanism of drug resistance among the Gram-negative bacteria. Extended spectrum β-lactamases (ESBLs) belong to Group 2be of Bush′s functional classification and are derived from the point mutation in original plasmid-mediated TEM-1 and SHV-1 β-lactamases.^[1] By definition, ESBL-producing organisms confer resistance to penicillin, cephalosporins and monobactams. They cannot hydrolyse cephamycins and are inhibited by clavulanic acid (CA). ^[2],[3] AmpC β-lactamases belong to Group I of Bush′s functional classification.^[1],[4] AmpC enzymes are poorly inhibited by CA and confer resistance oxyimino-cephalosporins, α-methoxy-β-lactams (cefoxitin and cefotetan), and monobactams. ^[4] Generally, they are susceptible to advanced spectrum cephalosporins (ASCs, i.e., cefepime and cefpirome). ^[4] They can be chromosomally mediated (inducible or constitutive) as seen in Enterobacter, Citrobacter, Morganella, Serratia and Escherichia coli or plasmid mediated. ^[4] Since the first report of transferable plasmid-mediated AmpC β-lactamases in 1980, they have spread among the members of family Enterobacteriaceae . ^[4]

The ESBL confirmation method has been established by Clinical Laboratory Standards Institute (CLSI) and is used worldwide. ^[5] Currently, there are no CLSI-recommended guidelines to detect AmpC β-lactamases. Several methods of phenotypic detection of AmpC β-lactamases are described; however, these methods are labour intensive and subjective, lack sensitivity and/or specificity and cannot be adopted on a routine basis. ^{[6],[7],[8],[9],[10],[11],[12],[13]} PCR or multiplex PCR gives satisfactory results, but the test is costly and time consuming, and equipment availability is limited to few laboratories.

The CLSI-recommended phenotypic confi rmatory test (PCT) would fail to detect ESBLs in the presence of AmpC, as the latter enzyme is resistant to CA. ^[8] CA may induce high-level expression of chromosomal AmpC, masking the synergy arising from the inhibition of an ESBL.

Boronic acid (BA) derivatives were reported as reversible inhibitors of AmpC enzymes. ^[14],[15] Many studies have validated the use of BAs to detect AmpC β-lactamases among Gram-negative bacteria.^{[8],[16],[17],[18],[19]} Coudron et al. recommended the usage of BA in combination with CA for the detection of ESBLs among AmpC-producing organisms. ^[16] Derbyshire et al. described the use of cefepime with CA for the reliable detection of ESBL in the presence of AmpC enzymes. ^[20]

The aim of the present study was to evaluate the usage of BA in a PCT, to improve the ESBL detection among AmpC-producing Enterobacteriaceae, to know the efficacy of the cefepime-CA method to identify ESBLs in the presence of AmpC β-lactamases and to know the prevalence of ESBL- and/or AmpC-producing Enterobacteriaceae at our centre.

Materials and Methods

A total of 200 consecutive, nonrepetitive, clinical isolates of Enterobacteriaceae isolated from various clinical samples such as exudates (n = 118; pus, ear swab, vaginal swab, fluids), urine (n = 39), and sputum (n = 43) obtained from ICU, PICU, burns unit, inpatient, and outpatient departments between November 2009 and February 2010 were included in the study. Samples were processed and isolates were identified by standard laboratory methods.

Antibiotic susceptibility testing was done according to CLSI-recommended Kirby-Bauer disk diffusion testing. ESBL production was detected by using (i) CLSI described PCT, (ii) BA disk potentiation test and (iii) cefepime-CA method. AmpC production was detected using cefoxitin (FOX) alone and in combination with BA and confirmation was done by a modified three-dimensional (M3D) test. For the detection of iAmpC, a cephalosporin disk was kept near the imipenem disk.

The present study used a novel disk placement pattern which includes BA disk potentiation test which uses cefotaxime (CTX), cefotaxime plus clavulanic acid (CTX/CA), cefotaxime plus boronic acid (CTX/BA) and cefotaxime plus clavulanic acid with boronic acid (CTX/CA/BA), CPM and CPM-CA disk test, in a single plate with imipenem at the centre [Figure - 1a]. Briefly, 5 μl of the freshly prepared clavulanic acid (2 g/l of PBS at pH 6; Medrich Pharmaceuticals, Bangalore, Karnataka, India) was added to cefotaxime (30 μg), ceftazidime (CAZ; 30 μg), and cefepime (30 μg) disks (HiMedia Laboratories, Mumbai, Maharashtra, India). Then 5 μl of a 3-aminophenyl boronic acid (BA; Sigma Aldrich, India) stock solution (60 g/l of DMSO) was added to cefotaxime and CAZ disks with or without CA and also to the cefoxitin disk. The final concentration of BA and CA on the disks was 300 μg and 10 μg, respectively. The disks were allowed to dry for 60 min and used immediately. A lawn of test organism was made on the Mueller-Hinton agar (MHA) after adjusting the inoculum to 0.5 McFarland unit and disks were placed as shown in [Figure - 1a] and incubated at 35 C for 18-24 h in ambient air. The interpretation of results is as follows:

1. A ≥5 mm increase in the zone diameter of the CTX (and/or CAZ) alone and in combination with CA or BA was indicative of ESBL or AmpC production, respectively [Figure - 1b and c].
2. A ≥5 mm increase in the zone diameter of CTX/BA (and/or CAZ/BA) and CTX/CA (and/or CAZ/CA) versus CTX/CA/BA (and/or CAZ/CA/BA) was indicative of combined ESBL and AmpC production [Figure - 1d].
3. A ≥5 mm increase in the zone diameter of the CPM alone and in combination with CA was indicative of ESBL production [Figure - 1b].
4. A ≥5 mm increase in the zone diameter of the FOX alone and in combination with BA was considered positive for AmpC production [Figure - 2].
5. Blunting of the zone of the cephalosporin disk towards the imipenem disk was considered as iAmpC production [Figure - 1e].
6. AmpC production was confirmed by using the M3D test. Quality control was achieved using Klebsiella pneumoniae ATCC 700603 and E. coli ATCC 25922 (HiMedia Laboratories).

Results

Of a total of 200 clinical isolates of Enterobacteriaceae, 82 (41%) and 12 (6%) were only ESBL and only AmpC producers, respectively; 55 (27.5%) were combined ESBL and AmpC producers and 14 (7%) isolates showed inducible AmpC (iAmpC) β-lactamases. Rest of the isolates did not produce any type of enzymes [Table - 1]. None of the isolates were carbapenamase producers.

The CLSI described PCT detected all (82/82, i.e., 100%) ESBL producers correctly but in combined (ESBL + AmpC) producers, the method failed to detect 20 of the 55 (36.3%) ESBL producers [Table - 2]. The CLSI method could detect a total 117 of 137 ESBL-producing isolates, while it detected all the negatives correctly. The cefepime plus CA disk potentiated method detected all the ESBL-producing Enterobacteriaceae correctly (P = 0.00).

Discussion

Cephalosporins are the first-line drugs used in the treatment of infections caused by members of family Enterobacteriaceae. The extensive use of third-generation Cephalosporins has resulted in the increased prevalence of ESBL and plasmid-mediated AmpC among these organisms. An indiscriminate administration of betalactams also increases the risk of colonization of hospitalized patients with ESBL-producing Enterobacteriaceae. Such organisms are usually derived from colonized health care settings. ^[2] Studies have shown the prior gastrointestinal carriage of ESBL-producing K. pneumoniae as an independent variable associated with such infections. ^[2] It is important to recognize patients with asymptomatic colonization of ESBL-producing organisms as they serve as an important reservoir for other hospitalized patients. Recent studies showed an increased prevalence of community-acquired infections with ESBL-producing organisms. ^[2],[6] The cause of this sudden upsurge is not yet clear.

The occurrence of multiple β-lactamases among bacteria not only limits the therapeutic options but also poses a challenge for microbiology laboratories to identify them. ^[21] The detection of the co-production of ESBL and AmpC is essential for enhanced infection control and effective anti-microbial therapy. The CLSI described phenotypic ESBL confirmatory test is in routine use, but no guidelines are available for the detection of AmpC β-lactamases or multiple β-lactamases. Several studies have been done on the phenotypic detection of AmpC β-lactamases, such as the three-dimensional test using enzyme extracts, disk potentiation method, and cefoxitin agar medium-based tests.^{[7],[8],[9],[10],[11],[12],[13]} However, these tests are intricate and need to be interpreted carefully.

The study showed that 41% of isolates were only ESBL producers while 27.5% of isolates were both ESBL and AmpC producers. If PCT was used alone, 14.6% of ESBL-producing organisms were missed which is comparable to the findings of Song et al.^[17] With the addition of CA to CTX, pure ESBL producers showed an average zone enhancement of 17.1 mm while combined enzyme producers showed only 8.1 mm. This was overcome by using the BA method.

The BA disk test in combination with PCT could detect only ESBL, only AmpC and both ESBL and AmpC-producing isolates correctly. But the method failed to detect iAmpC [Table - 2] which could be easily detected by placing the imipenem disk at the center. Blunting of the CTX/CTZ zone toward the imipenem disk was considered as iAmpC production. The inhibition of blunting of the CTX/CTZ zone with the addition of BA further confirmed iAmpC production. If the BA disk potentiation test was done without placing imipenem at the centre, the test would have missed all iAmpC producers giving false negative results.

Various BA derivatives were used to detect class C enzymes but 3-aminophenylboronic acid gave most convincing results. ^[8] Yagi et al. reported that CTZ showed the best performance in combination with APB than CTX in the detection of ESBL. ^[8] But the present study did not show any difference in ESBL and/or AmpC detection using either CTX or CTZ in combination with BA and/or CA. Yagi et al. evaluated the double disk synergy test using CTZ or CTX kept at a distance of 12-18 mm from the plain BA disk and zone enhancement was considered AmpC production. But we adapted the disk potentiation test using BA and CA, as it was more convenient, easy to interpret, gave consistent results and did not require the adjustment of the inter-disk distance for ESBL and AmpC co-producers.

All AmpC enzymes can hydrolyse cephamycins except the ACC-1 type of enzyme; ^[4] this makes these drugs better screening agents for AmpC production. But some of the studies have shown that cefoxitin is a poor screening agent for AmpC production because mechanisms other than AmpC such as porin channel mutation may lead to cefoxitin resistance leading to false positive interpretation. ^[22] In the present study, cefoxitin resistance was seen in 91 (45.5%) of 200 of Enterobacteriaceae [Table - 2]. A ≥ 5 mm increase in the zone size of FOX with the addition of BA was considered as AmpC production. The method detected 8 of 12 AmpC producers, 50 AmpC out of 55 combined AmpC plus ESBL producers and 12 of 14 iAmpC producers. Overall, the method detected 70 (86.4%) of total 81 AmpC-producing isolates. Though FOX resistance was seen in 91 isolates, only 70 showed ≥5 mm enhancement with the addition of BA. FOX resistance in isolates that did not show any enhancement with the addition of BA may be due to a mechanism other than AmpC production. In such isolates, the M3D test could differentiate AmpC from non-AmpC producers. Song et al.^[18] reported that the FOX-BA method was 97.7% sensitive for AmpC detection while the present study showed it to be only 86.4%. All the cefoxitin-sensitive isolates were non-AmpC producers. The FOX-BA method could not differentiate between plasmid-mediated AmpC, iAmpC or derepressed AmpC enzymes.

AmpC enzymes cannot hydrolyse advanced spectrum cephalosporins (ASC; cefepime, cefpirome) while ESBLs have extended their spectrum towards these drugs. ^[1],[4] Sturenburg et al.^[23] reported that the cefepime-clavulanic acid (CPM-CA) method could reliably detect ESBL in the presence of AmpC. In the present study, the CPM-CA method detected all ESBL production whether alone or in combination with AmpC correctly (P = 0.000), making it a better alternative for CLSI described PCT [Table - 2]. Average zone enhancement of cefepime with the addition of CA was 9.7 mm.

The high prevalence of ESBL plus AmpC co-production indicates the inappropriate use of extended spectrum cephalosporins. ESBL and/or AmpC production among Enterobacteriaceae is shown in [Table - 1]. Antibiotic co-resistance was high among ESBL, AmpC and co-producers when compared to non-producers and iAmpC. This may be due to the fact that plasmids carrying these enzymes may carry co-resistance genes for other antibiotics.

To conclude, the BA disk test in combination with the CLSI phenotypic confirmatory test was very simple, highly sensitive and specific for the identification of ESBL and/or AmpC among Enterobacteriaceae. The test showed a 100% positive and negative predictive value for ESBL and/or AmpC detection. The BA disk test requires the addition of two more disks to the CLSI described PCT, making it more simple and easy to adapt on a routine basis by a clinical microbiology laboratory. The test results were reproducible when carried out in duplicate. The CPM-CA disk test detected all the ESBLs correctly but another test for AmpC detection like the M3D test has to be incorporated into the susceptibility testing. The cefoxitin-boronic acid method is reliable for AmpC detection but may give false negative results when other mechanisms for cefoxitin resistance are at play.

Acknowledgements

We gratefully thank Mr. Sridhar A. (Laboratory technician, Department of Microbiology, Victoria Hospital, BMCRI, Bangalore, Karnataka, India) for technical assistance. We thank Medrich Pharmaceuticals Pvt. Ltd., Bangalore, Karnataka, India, for providing the pure form of potassium clavulunate for conducting the study.

References

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