Disc diffusion-based screening tests for extended-spectrum ß-lactamases in Haemophilus influenzae

Stephen G. Tristram1,*, Bülent Bozdogan2 and Peter C. Appelbaum2

1 School of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250, Australia; 2 Department of Pathology, Hershey Medical Center, Hershey, PA 17033, USA


* Corresponding author. Tel: +61-3-63-243323; Fax: +61-3-63-243658; Email: stephen.tristram{at}utas.edu.au

Received 24 October 2004; returned 15 December 2004; revised 20 December 2004; accepted 29 December 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To establish a suitable screening test for the detection of extended-spectrum ß-lactamases (ESBLs) in Haemophilus influenzae.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Non-encapsulated Haemophilus influenzae strains are key pathogens in a range of community-acquired respiratory tract infections.1,2 Antibiotic resistance in H. influenzae is increasing,13 and continued surveillance for emerging resistance mechanisms in H. influenzae is warranted.

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

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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Table 1. MIC data and standard disc diffusion and prediffusion test results for recombinant ESBL-producing H. influenzae and selected clinical isolates

 
BLPAR strains were confirmed as ß-lactamase-positive using nitrocefin touchsticks (Oxoid, Australia) and typed as TEM or ROB using PCR as previously described.1 MICs were determined using NCCLS macrobroth dilution using freshly prepared Haemophilus test medium.8

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MICs showed that all strains tested, including the recombinant ESBL-producing H. influenzae, were susceptible to cefotaxime by NCCLS breakpoints (Table 1).

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.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Initial work on recombinant ESBL-producing H. influenzae reported that despite raised MICs of cefotaxime, organisms were categorized as susceptible according to NCCLS criteria.5 In this study, BSAC and CDS disc diffusion methods, which are both based on breakpoints different from NCCLS (MICs for susceptible strains ≤ 1, ≤ 0.25 and ≤ 2 mg/L, respectively), also categorized recombinant ESBL-producing strains as susceptible. This is consistent with the findings with clinical ESBL-producing Enterobacteriaceae, where the MICs are often within susceptibility breakpoints.4

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.


    Acknowledgements
 
The assistance of Dr Kimiko Ubukata (Kitasato Institute for Life Sciences, Kitasato University, Japan) and Dr Henri Dabernat (Laboratoire de Microbiologie, Centre National de Reference des Haemophilus influenzae, France) in providing some of the BLPACR and BLNAR strains is gratefully acknowledged. The work was supported by the Clifford Craig Medical Research Trust, Launceston, Tasmania, Australia.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dabernat, H., Delmas, C., Seguy, M. et al. (2002). Diversity of ß-lactam resistance-conferring amino-acid substitutions in penicillin-binding protein 3 of Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 46, 2208–18.[Abstract/Free Full Text]

2 . Hoban, D. & Felmingham, D. (2002). The PROTEKT surveillance study: antimicrobial susceptibility of Haemophilus influenzae and Moraxella catarrhalis from community-acquired respiratory tract infections. Journal of Antimicrobial Chemotherapy 50, Suppl. S1, 49–59.[Abstract/Free Full Text]

3 . Hasegawa, K., Yamamoto, K., Chiba, N. et al. (2003). Diversity of ampicillin-resistance genes in Haemophilus influenzae in Japan and the United States. Microbial Drug Resistance 9, 39–46.[ISI]

4 . Bradford, P. (2001). Extended-spectrum ß-lactamases in the 21st century: characterisation, epidemiology, and detection of this important resistance threat. Clinical Microbiology Reviews 14, 933–51.[Abstract/Free Full Text]

5 . Tristram, S. (2003). Effect of extended-spectrum ß-lactamases on the susceptibility of Haemophilus influenzae to cephalosporins. Journal of Antimicrobial Chemotherapy 51, 39–43.[Abstract/Free Full Text]

6 . Galan, J.-C., Morosini, M.-I., Baquero, M.-R. et al. (2003). Haemophilus influenzae blaROB-1 mutations in hypermutagenic {Delta}ampC Escherichia coli conferring resistance to cefotaxime and ß-lactamase inhibitors and increased susceptibility to cefaclor. Antimicrobial Agents and Chemotherapy 47, 2551–7.[Abstract/Free Full Text]

7 . Pitout M., Macdonald K., Musgrave H. et al. (2002) Characterisation of extended spectrum ß-lactamase (ESBL) activity in Haemophilus parainfluenzae. In Abstracts of the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 2002. Abstract C2-645, p. 96. American Society for Microbiology, Washington, DC, USA.

8 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7–A5. NCCLS, Villanova, PA, USA.

9 . Matic, V., Bozdogan, B., Jacobs, M. et al. (2003). Contribution of ß-lactamase and PBP amino acid substitutions to amoxicillin/clavulanate resistance in ß-lactamase-positive, amoxicillin/clavulanate-resistant Haemophilus influenzae. Journal of Antimicrobial Chemotherapy 52, 1018–21.[Abstract/Free Full Text]

10 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility Tests—Sixth Edition: Approved Standard M2–A7. NCCLS, Villanova, PA, USA.

11 . Andrews, J. M. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.[Abstract/Free Full Text]

12 . Bell, S., Gatus, B., Pham, J., et al. (2002). Antibiotic Susceptibility Testing by the CDS Method: a Manual for Medical and Veterinary Laboratories 2002. Arthur Productions Pty Ltd, Sydney, Australia.