JAC Advance Access originally published online on February 21, 2006
Journal of Antimicrobial Chemotherapy 2006 57(4):747-749; doi:10.1093/jac/dkl039
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Combination of altered PBPs and expression of cloned extended-spectrum ß-lactamases confers cefotaxime resistance in Haemophilus influenzae
1 Mikrobiyoloji ve Klinik Mikrobiyoloji, Adnan Menderes Üniversitesi Hastanesi, Aydin, Turkey; 2 School of Human Life Sciences, University of Tasmania, Launceston, Tasmania, Australia; 3 Department of Pathology, Hershey Medical Centre, Hershey, PA, USA
* Corresponding author. Tel: +90-256-214-80-41; Fax: +90-256-212-31-69; E-mail: bbozdogan{at}adu.edu.tr
Received 15 November 2005; returned 19 January 2006; revised and accepted 19 January 2006
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Abstract |
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Background: Resistance to ß-lactams in Haemophilus influenzae is mostly due to the presence of TEM ß-lactamases. ß-Lactamase-negative ampicillin resistance (BLNAR) also occurs as a result of PBP3 modifications. BLNAR strains are particularly common in Japan and France, and strains with combined mechanisms of altered PBP3 and ß-lactamase (BLPACR) are emerging. Although the prevalence of ß-lactamase-positive strains is high, TEM-derived extended-spectrum ß-lactamase (ESBL)-mediated cephalosporin resistance, which is common in Enterobacteriaceae, has not been reported in H. influenzae. In this study, the ability of ESBLs TEM-3, -4 and -5 to confer ß-lactam resistance in H. influenzae strains with or without modified PBP3 was tested.
Methods: The genes encoding TEM-1, -3, -4 and -5 were cloned into plasmid pLS88 and were used to transform H. influenzae Rd and BLNAR H. influenzae Rd strains with PBP3 modifications. MICs were determined using CLSI macrobroth dilution. The ability of amoxicillin/clavulanate pre-diffusion disc tests to discriminate between ESBL-producing strains and other strains was also tested.
Results: The presence of ESBLs in BLNAR strains conferred cefotaxime resistance, but H. influenzae Rd strains expressing ESBLs and BLNAR strains expressing TEM-1 remained cefotaxime susceptible. The amoxicillin/clavulanate pre-diffusion tests were able to discriminate the ESBL-producing strains from the other strains.
Conclusions: The presence of altered PBP3 increases the effect of ESBLs in recombinant H. influenzae strains to produce cefotaxime resistance. Amoxicillin/clavulanate pre-diffusion disc tests can discriminate ESBL-producing strains from other strains.
Keywords: H. influenzae , ESBLs , ß-lactams , penicillin binding proteins
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Introduction |
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Resistance to ß-lactam antibiotics is increasing in Haemophilus influenzae, especially in Europe,1 even though a decrease has been observed in the United States in recent years.2 The most common mechanism of ß-lactam resistance is the production of ß-lactamases, mostly TEM. These enzymes confer resistance to ampicillin but not to cephalosporins or amoxicillin/clavulanate. ß-Lactamase-negative ampicillin resistance (BLNAR) also occurs as a result of amino acid substitutions in the penicillin binding proteins (PBPs), particularly in PBP3, which is encoded by ftsI.3 Such strains are resistant not only to ampicillin but also to amoxicillin/clavulanate and some of the early cephalosporins. BLNAR strains are particularly common in Japan and France, and strains with combined mechanisms of altered PBP and TEM ß-lactamase (BLPACR) are now emerging.3,4
Although the prevalence of ß-lactamase-positive strains is high, TEM-derived extended-spectrum ß-lactamase (ESBL)-mediated cephalosporin resistance, which is now common in Enterobacteriaceae, has not been detected in H. influenzae. It has been suggested that the relatively low levels of cephalosporin resistance produced by ESBLs in H. influenzae, as demonstrated in artificially produced recombinant strains, may explain the apparent failure of ESBLs to emerge naturally or be detected.5 This raises the possibility that ESBLs are more likely to emerge from BLPACR strains by mutation of the existing TEM genes, or from BLNAR strains by the acquisition of TEM ESBL genes, if the co-existing altered PBP3 augments the resistance produced by the ESBLs. If such strains do emerge, the amoxicillin/clavulanate resistance associated with the altered PBP3 may interfere with the performance of recently described screening tests for ESBLs in H. influenzae, which are based on the restoration of cephalosporin susceptibility by clavulanic acid.6
The aim of this study was to examine the levels of cephalosporin resistance in recombinant strains of H. influenzae expressing ESBLs with and without altered PBP3, and to test the ability of the screening test to detect ESBLs in H. influenzae in a background of altered PBP3.
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Materials and methods |
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Strains and plasmids
Various blaTEM genes which encode for wild-type ß-lactamase TEM-1 and ESBLs TEM-3, -4 and -5 on plasmid pLS885 were used to transform H. influenzae Rd (ATCC 51907) and strains BLNAR1, BLPACR4, BLNAR5 and BLPACR7, which represent BLNAR H. influenzae Rd strains previously transformed to express various PBP3 modifications.7 This established a panel of 25 organisms, as detailed in Table 1.
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Antimicrobial susceptibility testing
MICs for all strains were determined by macrobroth dilution using freshly prepared Haemophilus test medium according to CLSI methodology.8 All strains were screened for ESBLs using the amoxicillin/clavulanate pre-diffusion screening test.6 Chocolate agar plates were inoculated with a 0.5 McFarland suspension of cells prepared from a fresh overnight growth. A 2/1 µg amoxicillin/clavulanate disc was placed on the plate for 60 min before being replaced with a cefotaxime disc (5 or 30 µg) or cefpodoxime 10 µg disc, and another cefotaxime or cefpodoxime disc of equal strength was included on the plate for comparison. All three discs were tested for all 25 strains. An increase in zone diameter of 
5 mm for the pre-diffused disc relative to the plain disc was considered suggestive of an ESBL.
Genetic techniques
Plasmid DNA from the respective TEM-producing strains5 was extracted using the QIAprep Spin Miniprep Kit as recommended by the manufacturer (Qiagen Inc.). Transformation of the H. influenzae Rd, BLNAR1, BLPACR4, BLNAR5 and BLPACR7 strains7 with each of the plasmids was as described previously by Barcak et al.9 Transformants were selected on brain heart infusion agar (Becton Dickinson, Cockeysville, MD, USA), supplemented with haemin (2 mg/L) and NAD (2 mg/L) (sBHI), that contained kanamycin (30 mg/L) to select for the pLS88 plasmid carrying the respective TEM genes.
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Results and discussion |
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There is great diversity in the mutations in the ftsI genes and associated amino acid substitutions in the PBP3 of BLNAR and BLPACR strains, and the MICs of cefotaxime can range from as low as 0.06 to 2 mg/L compared with a maximum of 0.06 mg/L for ß-lactamase-positive amoxicillin-resistant strains or strains without any ß-lactam resistance mechanisms.3,4 The BLNAR strains used in this study represent those at the higher end of the range, with cefotaxime MICs from 0.25 to 1.0 mg/L.
All the strains with a combination of an altered PBP and an ESBL gave significantly higher cefotaxime MICs than either mechanism alone, whereas the presence of an altered PBP3 and TEM-1 did not increase the cefotaxime MICs above that for the strain expressing just the altered PBP. All the combinations involving either TEM-3 or -4 (cefotaximases) had cefotaxime MICs above the CLSI10 susceptibility breakpoint of 
2 mg/L, whereas those involving TEM-5, which is primarily a ceftazidimase, had raised MICs but not above the breakpoint (Table 1). This is an encouraging observation which suggests that if strains with altered PBPs and ESBLs emerged in a natural way, most of them would be categorized as resistant by laboratory susceptibility tests, which has not always been the case with ESBLs in Enterobacteriaceae.11
Detecting the presence of ESBLs in Enterobacteriaceae that also produce AmpC-type enzymes has been problematic because of the resistance of AmpC-type enzymes to inhibition by clavulanate.11 All the ESBL-producing and non-ESBL-producing strains of H. influenzae used in this study were correctly categorized by all the substrate discs (cefotaxime 5 and 30 µg and cefpodoxime 10 µg) used in the amoxicillin/clavulanate pre-diffusion screening test, and the concomitant presence of a clavulanate-resistant mechanism (altered PBP) with an ESBL did not interfere with the ability of the test to differentiate ESBL-mediated resistance from other types of resistance.
We conclude that a combination of modified PBPs and a TEM-type ESBL in recombinant strains of H. influenzae Rd produces significantly raised cefotaxime MICs that exceed those produced by either mechanism alone; for TEM-3 or TEM-4 ESBLs, the MICs would be categorized as resistant by CLSI criteria. The amoxicillin/clavulanate pre-diffusion disc screening test reliably differentiated ESBL-producing recombinant strains with or without concomitant altered PBP3 from strains exhibiting a range of other ß-lactam resistance mechanisms comprising TEM-1 ß-lactamase alone, a range of altered PBPs alone or a range of altered PBPs and TEM-1.
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Transparency declarations |
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None to declare.
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References |
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