1 School of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250, Australia; 2 Department of Pathology, Hershey Medical Center, Hershey, PA 17033, USA
Received 24 October 2004; returned 15 December 2004; revised 20 December 2004; accepted 29 December 2004
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Abstract |
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Methods: Double disc diffusion tests using cephalosporin discs adjacent to an amoxicillin/clavulanate 3 µg disc, prediffusion tests, where an amoxicillin/clavulanate 3 µg disc was prediffused before being replaced with a cephalosporin disc, and combination cephalosporin/clavulanate discs were used to screen recombinant H. influenzae expressing cloned ESBLs. Methods initially determined to be suitable were validated by testing a range of clinical strains.
Results: Enhanced zones around cephalosporin discs due to synergy with an adjacent amoxicillin/clavulanate disc were not reliably demonstrated with the recombinant H. influenzae strains expressing ESBLs. Cefotaxime 5 µg or 30 µg and cefpodoxime 10 µg discs prediffused with an amoxicillin/clavulanate 3 µg disc, and cefotaxime 30 µg/clavulanate 10 µg and cefpodoxime 10 µg/clavulanate 1 µg combination discs all gave zone sizes 5 mm greater than the corresponding plain disc for all recombinant ESBL-producing strains. None of the clinical strains gave enhanced zones.
Conclusions: Cefotaxime 5 or 30 µg or cefpodoxime 10 µg discs prediffused with an amoxicillin/clavulanate 3 µg disc, or cefotaxime 30 µg/clavulanate 10 µg and cefpodoxime 10 µg/clavulanate 1 µg combination discs are all suitable for screening H. influenzae isolates for the presence of ESBLs. ESBL-producing strains should show an increase in zone diameter of 5 mm compared with the corresponding plain cephalosporin disc.
Keywords: H. influenzae , ESBLs , TEM
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Introduction |
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TEM-derived extended-spectrum ß-lactamases (ESBLs) and associated cephalosporin resistance are well established in Enterobacteriaceae.4 By comparison, despite the fact that non-ESBL TEM-1 ß-lactamase-positive ampicillin-resistant (BLPAR) strains of H. influenzae are common,2 ESBLs have yet to be detected clinically in this species.2,5,6 ß-Lactamase-negative ampicillin-resistant (BLNAR) strains have emerged that show reduced susceptibility to cephalosporins due to substitutions in penicillin-binding protein 3 (PBP3), and more recently strains that are both ß-lactamase (TEM-1)-positive and have altered PBP3 (BLPACR) have been described.1,3 The emergence of an ESBL in Haemophilus parainfluenzae,7 demonstrates a necessity to establish a screening test to differentiate existing H. influenzae BLPACR strains, which are ß-lactamase-positive with reduced oxyimino-cephalosporin susceptibility, from possible ESBL-producing strains.
The aim of this study was to evaluate a range of screening tests to establish their suitability for the detection of ESBLs in H. influenzae, and differentiation from strains with other ß-lactam resistance mechanisms.
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Materials and methods |
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Previously described recombinant strains of H. influenzae Rd with a range of cloned TEM-type ß-lactamases (Table 1), 5 were used to determine the ability of various screening tests to detect the presence of ESBLs. A range of clinical isolates comprising 50 BLPAR (45 TEM and 5 ROB), 10 BLNAR and 10 BLPACR (Table 1) characterized as non-ESBL-producers were used as negative controls.
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BLNAR and BLPACR strains had been previously characterized, including determination of respective PBP3 substitutions by sequencing of the ftsI gene and ß-lactamase characterization by sequence analysis.1,9
Disc diffusion methods
Unless otherwise stated, disc diffusion tests were carried out in duplicate on chocolate agar (Columbia agar base with 8% chocolatized horse blood) and incubated at 35°C in 5% CO2 for 18 h. Inocula were prepared by suspending cells from fresh overnight cultures in 0.9% saline to a turbidity equivalent to a 0.5 McFarland standard. Plates were inoculated by flooding with saline suspension and withdrawing excess. Plates were allowed to dry with the lid off for 30 min at room temperature before discs were applied.
Standard disc diffusion and double disc diffusion
NCCLS, British Society for Antimicrobial Chemotherapy (BSAC) and the Australian Calibrated Dichotomous Susceptibility (CDS) disc diffusion methods were carried out according to relevant methodologies.1012 Enhancement of the zone around the cefotaxime disc caused by synergy from clavulanate in the adjacent amoxicillin/clavulanate disc was considered suggestive of an ESBL.
Amoxicillin/clavulanate prediffusion disc replacement
A 2/1 µg amoxicillin/clavulanate disc was placed on an inoculated plate and allowed to diffuse for 60 min at room temperature before being removed and replaced with a cefotaxime disc (30, 5 or 0.5 µg) or a cefpodoxime disc (10 µg). Another cefotaxime or cefpodoxime disc of equal strength and a 2/1 µg amoxicillin/clavulanate disc were included on plates for comparison. An increase in zone diameter 5 mm for discs prediffused with a 2/1 µg amoxicillin/clavulanate disc compared with both plain cephalosporin or plain amoxicillin/clavulanate disc was considered suggestive of an ESBL.
Combination discs
Commercially available discs (Oxoid, Victoria, Australia) containing a combination of a cephalosporin and clavulanic acid were tested in conjunction with a corresponding plain cephalosporin disc. Discs tested were cefpodoxime/clavulanic acid 10/1 µg and cefpodoxime 10 µg, and cefotaxime/clavulanic acid 30/10 µg and cefotaxime 30 µg. An increase in zone diameter 5 mm more for clavulanic acid supplemented discs compared with plain discs was considered suggestive of an ESBL.
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Results |
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Standard disc diffusion and double disc diffusion test
None of the recombinant ESBL-producing strains were resistant to cefotaxime using standard disc diffusion (Table 1), and no enhanced inhibition zones were demonstrated by adjacent amoxicillin/clavulanate discs (data not shown).
Amoxicillin/clavulanate prediffusion disc replacement
All recombinant ESBL-producing strains, but none of 50 BLPAR strains showed increases in zones 5 mm for cephalosporin discs prediffused with amoxicillin/clavulanate compared with plain cephalosporin discs. However, four of 10 BLNAR strains and one of 10 BLPACR strains repeatedly showed an increase of
5 mm for the zone around the cefotaxime 0.5 µg disc prediffused with a 3 µg amoxicillin/clavulanate disc compared with the plain cefotaxime disc. This increase was not demonstrated with higher strength cefotaxime discs (Table 1).
Combination discs
All recombinant ESBL-producing strains, but none of 70 control strains showed increases in zones of inhibition of 5 mm for combination cephalosporin/clavulanate discs compared with the plain cephalosporin discs.
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Discussion |
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Although useful for the Enterobacteriaceae,4 adjacent positioning of cephalosporin and amoxicillin/clavulanate discs on a standard disc diffusion susceptibility test is unlikely to detect ESBLs in H. influenzae. This is primarily because at the disc spacing usually used in a standard test, there is significant overlap in inhibition zones that may mask zone enhancement by the ß-lactamase inhibitor. This overlap occurs with H. influenzae but not Enterobacteriaceae, because recombinant ESBL-producing H. influenzae have relatively low cefotaxime MICs with large zone sizes, even with low-content discs. A similar situation exists with amoxicillin/clavulanate discs, with relatively large zone sizes produced to the amoxicillin component even with plain TEM-1-producing strains.
The amoxicillin/clavulanate prediffusion and disc replacement method has been successfully used with Enterobacteriaceae to overcome the problem of disc spacing with various double disc diffusion tests.4 In this study, only amoxicillin/clavulanate 2/1 µg discs were evaluated to avoid potential for interference from amoxicillin in the higher strength discs.
Both amoxicillin/clavulanate prediffusion disc replacement and combination disc methods successfully detected recombinant ESBL-producing strains of H. influenzae. False positive tests with the amoxicillin/clavulanate prediffusion test (0.5 µg cefotaxime only) observed with one BLPACR and four BLNAR strains were probably a result of interference from amoxicillin in the amoxicillin/clavulanate disc.
In summary, this study has shown that NCCLS, BSAC and CDS disc diffusion methods may fail to detect ESBLs in H. influenzae, even with adjacent placement of cefotaxime and amoxicillin/clavulanate discs. The amoxicillin/clavulanate prediffusion disc replacement method with the exclusion of cefotaxime 0.5 µg discs and use of commercially available combination discs are recommended as useful screening tests. Based on previous experience with ESBLs in Enterobacteriaceae, it would seem prudent to use both cefpodoxime and cefotaxime as substrates for whichever test is used.4 Susceptibility testing of H. influenzae is highly method dependent, and workers should avoid deviating from the methods described if results are to reflect these findings. These recommendations are interim and based on analysis of a small number of artificially introduced ESBLs, and would be expected to evolve as and when naturally occurring ESBL-producing strains are detected and characterized.
Laboratories should be alert for H. influenzae showing decreased susceptibility to ESBLs. In the first instance, they should carry out a nitrocefin-based test for ß-lactamase as a BLNAR strain is still the most likely cause of reduced susceptibility. If the isolate is ß-lactamase-positive, one of the recommended screening tests should be carried out. Isolates that are positive with the screening test should be referred to a reference laboratory for characterization of the ß-lactamase.
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Acknowledgements |
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