JAC Advance Access originally published online on September 6, 2007
Journal of Antimicrobial Chemotherapy 2007 60(5):1151-1154; doi:10.1093/jac/dkm311
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Effect of cloned inhibitor-resistant TEM ß-lactamases on the susceptibility of Haemophilus influenzae to amoxicillin/clavulanate
School of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250, Australia
* Corresponding author. Tel: +61-3-63-243323; Fax: +61-3-63-243658; E-mail: stephen.tristram{at}utas.edu.au
Received 4 May 2007; returned 3 July 2007; revised 12 July 2007; accepted 28 July 2007
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Abstract |
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Objectives: To determine the effect of cloned inhibitor-resistant TEM ß-lactamases (IRTs) on the susceptibility of Haemophilus influenzae to amoxicillin/clavulanate.
Methods: IRT-2, -4 and -5 genes with various promoters were cloned into control strains of H. influenzae and the amoxicillin/clavulanate MICs were measured using Etests.
Results: IRT enzymes were able to raise the amoxicillin/clavulanate MICs to between 0.38/0.19 and 4.0/2.0 mg/L depending on the IRT and promoter genotype, compared with MICs of 0.19/0.09 to 0.5/0.25 mg/L for the corresponding strains with TEM-1. Strains with an IRT and altered penicillin-binding proteins had amoxicillin/clavulanate MICs as high as 8.0/4.0 mg/L.
Conclusions: Cloned IRT enzymes in H. influenzae raise the amoxicillin/clavulanate MICs to an extent comparable to naturally occurring strains with decreased amoxicillin/clavulanate susceptibility.
Keywords: H. influenzae , IRTs , susceptibility
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Introduction |
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The most common mechanism of ampicillin resistance in Haemophilus influenzae is via production of TEM-1 ß-lactamase with a recent global survey reporting a prevalence of
17%.1 One strategy used to overcome this resistance is to use amoxicillin in combination with the ß-lactamase inhibitor clavulanate, and this combination is widely prescribed to treat a range of respiratory tract infections caused by ß-lactamase-producing strains of H. influenzae.2 Despite the prevalence of TEM-1 ß-lactamase in H. influenzae and the widespread use of amoxicillin/clavulanate, it has been surprising that inhibitor-resistant TEM ß-lactamases (IRTs) and associated amoxicillin/clavulanate resistance has not been reported in H. influenzae as it has in Enterobacteriaceae.3,4 This is especially so given that amoxicillin/clavulanate resistance has emerged in ß-lactamase-negative ampicillin-resistant (BLNAR) and ß-lactamase-positive amoxicillin/clavulanate-resistant (BLPACR) strains of H. influenzae as a result of altered penicillin-binding proteins (PBPs).5,6 It has been suggested that the failure of IRTs to emerge in H. influenzae might be associated with the high intrinsic activity of penicillins against the organism and relatively small amounts of ß-lactamase usually produced,3,4 such that a level of resistance sufficient to allow for selection of mutants during therapy might not be produced. In order to determine the effect that IRTs might have on the susceptibility to ampicillin and amoxicillin/clavulanate, a range of IRTs were artificially introduced into control strains of H. influenzae.
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Methods |
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Construction of blaTEM-1 and blaIRT library
Strains of TEM-1 ß-lactamase-positive H. influenzae, previously characterized with regard to promoter genotype,7,8 were used as PCR templates to clone blaTEM-1 with Pa/Pb, Pdel or Prpt promoters onto the shuttle vector pLS88 and into Escherichia coli XL1-Blue cells (Stratagene) as previously described.9
Plasmids were extracted from these clones and used as template for site-directed mutagenesis using the QuikChange Site Directed Mutagenesis Kit (Stratagene) and primers listed in Table 1 to generate blaIRT-2 and blaIRT-5 coding for IRT-2 (TEM-30) and IRT-5 (TEM-33) in E. coli XL1-Blue cells. Plasmid containing blaIRT-5 was extracted and subjected to another round of site-directed mutagenesis to generate blaIRT-4 coding for IRT-4 (TEM-35).
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Finally, all constructed plasmids (pLS88 with blaTEM-1 and blaIRT-2, -4 and -5) were used to transform H. influenzae Rd. Plasmids containing blaTEM-1, blaIRT-4 and blaIRT-5 in association with the Pdel promoter were also used to transform H. influenzae Rd that had previously been transformed with ftsI gene PCR products from a BLNAR strain. All transformations were performed by electroporation as described by Ubukata et al.,5 and transformants were selected on chocolate agar supplemented with 30 mg/L kanamycin.
The presence of the respective blaTEM-1 and blaIRT genes in the E. coli strains was confirmed by appropriate MICs of ampicillin and amoxicillin/clavulanate and by bla gene sequencing. ß-Lactamase production in the H. influenzae transformants was confirmed by nitrocefin hydrolysis (Oxoid, Australia).
MICs of ampicillin, amoxicillin/clavulanate and cefotaxime (for Haemophilus only) were determined using Etest strips according to the manufacturer's instructions (Australian Laboratory Services, Melbourne). H. influenzae ATCC 49247 and ATCC 49766 were used as quality control strains.
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Results and discussion |
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The enzymes IRT-2, -4 and -5 were chosen for this study because they are representative of enzymes with substitutions at positions 69, 244 and 276 identified by computerized modelling as positions that are associated with inhibitor resistance via different molecular interactions.3 Substitutions at these positions are also those most frequently reported in clinically derived IRTs.10 Promoters Pa/Pb, Pdel and Prpt were chosen because they have been identified as the most common promoters in blaTEM-1 genes in H. influenzae.7,8
In E. coli XL-1, the ampicillin MICs for the strains expressing any of the cloned blaTEM-1 and blaIRT-2, -4 and -5 constructs were >256 mg/L, and the amoxicillin/clavulanate (2:1 ratio) MICs ranged from 8/4 to 32/16 mg/L for strains expressing blaTEM-1 and 24/12 to 128/64 mg/L for blaIRT-2, -4 and -5. These MIC data are consistent with similar naturally occurring strains,4,11 and, in conjunction with sequence data confirming the presence of appropriate promoters and IRT-related substitutions, validate the constructs as representative of naturally occurring resistance genes.
In H. influenzae, the amoxicillin/clavulanate MICs for strains expressing various cloned blaIRTs were between 2- and 10-fold higher than for strains expressing blaTEM-1 from the same promoters (Table 2). All the strains expressing cloned blaIRTs had amoxicillin/clavulanate MICs between 2- and 5-fold higher when associated with either the strong Pdel or Prpt promoters compared with the relatively weaker Pa/Pb promoters. This is consistent with what is seen in naturally occurring IRT-producing strains of E. coli, where the relatively stronger Pa/Pb or P4 promoters occur more frequently than the weaker P3 promoter.12 Stronger promoters are probably selected for because of the decreased catalytic efficiency reported for many IRT enzymes,4 where additional enzyme production is not only necessary to produce significant amoxicillin/clavulanate resistance but also to compensate for a relative loss of activity against ampicillin. In the H. influenzae strains in this study, this loss of catalytic efficiency is manifested as lower ampicillin MICs for IRT-2- compared with TEM-1-producing strains, which is consistent with the lower catalytic efficiency of IRT-2 compared with IRT-4 and IRT-5 and TEM-1.4 Such decreases are not observed in the E. coli constructs in this study, or in naturally occurring IRT-positive E. coli strains because the ampicillin MICs are usually above the highest concentrations measured.
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The cefotaxime MICs for all H. influenzae clones were between 0.008 and 0.016 mg/L with no evidence of decreased susceptibility associated with production of IRT-2, -4 or -5 enzymes compared with TEM-1.
When IRT enzymes were expressed in BLNAR strains of H. influenzae, the amoxicillin/clavulanate MICs were 2–5-fold higher than those for otherwise identical non-BLNAR strains. A similar finding was observed when TEM-type extended-spectrum ß-lactamases (ESBLs) were cloned into H. influenzae, with significantly higher MICs of cefotaxime observed when the ESBL was expressed in a BLNAR background.13 Significantly, the only report of a naturally occurring ESBL in Haemophilus has been in two strains of Haemophilus parainfluenzae with TEM-16 and altered PBPs similar to those found in BLNAR strains of H. influenzae,14 so a background of altered PBPs in BLNAR/BLPACR strains of H. influenzae might favour the emergence of IRT enzymes.
It is difficult to gauge the significance of the decreased susceptibility to amoxicillin/clavulanate of the IRT-producing H. influenzae strains in this study because the MICs cluster around the breakpoints for amoxicillin/clavulanate used by various regulatory bodies. All the strains are susceptible according to CLSI breakpoints (S 
4, R 
8 mg/L) but some would be considered resistant according to either the BSAC breakpoints (S 1, R > 1 mg/L) or the recently proposed PK/PD breakpoints (S 2, R 4 mg/L).2,15,16 The significance of the decreased susceptibility might best be considered in the context of comparisons to other naturally occurring strains. The strains produced in this study had amoxicillin/clavulanate MICs ranging from 0.38/0.19 to 4.0/2.0 mg/L, whereas the amoxicillin/clavulanate MIC90 for 8500 strains from the Alexander study 1998–2000 was 1.0/0.5 mg/L,17 and the MIC90 was 2.0/1.0 for 2000 ß-lactamase-positive strains surveyed by Farrell et al. in 2000–03.18 In addition, the amoxicillin/clavulanate MICs for 108 BLNAR and BLPACR strains characterized by Dabernat et al.6 ranged from 0.12/0.06 to 4.0/2.0 mg/L. On this basis it can be concluded that IRT enzymes in H. influenzae could produce resistance to amoxicillin/clavulanate significantly greater than baseline susceptible strains, and equivalent to that shown by currently existing BLNAR and BLPACR strains.
If IRTs in H. influenzae can produce a similar decrease in amoxicillin/clavulanate susceptibility to that seen in naturally occurring BLPACR and BLNAR strains, then why have IRTs not been detected? One possible reason is that the decreased amoxicillin/clavulanate susceptibility in BLNAR and BLPACR strains is not actually an evolutionary response to selective pressure of amoxicillin/clavulanate use, but rather an incidental finding associated with the altered PBPs and decreased cephalosporin susceptibility selected for by widespread cephalosporin use. This is consistent with the observation that strains with altered PBPs are more common in Japan where oral cephalosporin use is favoured over amoxicillin and amoxicillin/clavulanate compared with the United States and Europe.2
It is also possible that IRT-mediated decreased amoxicillin/clavulanate susceptibility has emerged and gone undetected, particularly given that the MICs will not exceed the current CLSI resistance breakpoint.
A lack of consensus on breakpoints and incomplete correlation between phenotypic and genotypic characterization has created difficulties and inconsistencies in detecting currently existing BLNAR and BLPACR strains.2 In this context, detecting the possible emergence of IRT-producing strains might also be difficult. A clinical isolate that is ß-lactamase-positive with reduced amoxicillin/clavulanate susceptibility or resistance is most probably a BLPACR strain, but could be a strain producing an IRT. The major differentiating characteristic would be that as a result of the altered PBP3, BLPACR strains would usually also show reduced susceptibility or resistance to cephalosporins whereas IRT-producing strains in this study did not. Confirmation of IRT production would require sequencing of the blaTEM gene.
In conclusion, IRT enzymes in H. influenzae could cause a significant decrease in amoxicillin/clavulanate susceptibility but would be difficult to detect using current CLSI breakpoints. Laboratories should consider the presence of an IRT in ß-lactamase-positive strains of H. influenzae with decreased susceptibility to amoxicillin/clavulanate without a concomitant decrease in cephalosporin susceptibility.
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None to declare.
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Funding |
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This research was funded by the School of Human Life Sciences, University of Tasmania.
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References |
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