1 Department of Pathology, Hershey Medical Center, 500 University Drive, Hershey, PA 17033; 2 Case Western Reserve University, Cleveland, OH, USA; 3 Kitasato University, Tokyo, Japan
Received 9 July 2003; returned 28 August 2003; revised 2 September 2003; accepted 15 September 2003
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Abstract |
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Keywords: H. influenzae, penicillin-binding proteins, ß-lactamases, mutations
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Introduction |
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The prevalence of resistance to penicillins in H. influenzae varies in different countries and is predominantly due to TEM-1- or ROB-1- type ß-lactamase production.4 In the USA, 41.6% of H. influenzae were found to produce ß-lactamase.5 In Japan, the incidence of ß-lactam resistance in the absence of ß-lactamase production has increased to 28.8%,6,7 but is <1% in the USA.5 This resistance occurs as a result of alterations in penicillin binding protein 3 (PBP3).7 Multiple mutations were found in the ftsI gene, which encodes PBP3, of ß-lactamase-negative, ampicillin-resistant (BLNAR) strains.7 Strains producing ß-lactamases remain susceptible to amoxicillin/clavulanate; however, clavulanate is inactive against BLNAR strains, so such strains are resistant to amoxicillin/clavulanate. In addition, very rare H. influenzae strains carrying ß-lactamase have been reported as resistant to amoxicillin/clavulanate.8,9 Isolation of such strains raises questions as to whether the ß-lactamases in these ß-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains are inhibitor-resistant, extended-spectrum ß-lactamases, which are refractory to inhibition by clavulanate, or if resistance is associated with PBP changes. This study investigated this issue.
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Materials and methods |
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Among 6382 H. influenzae strains collected for the Alexander Project (19972000), 30 were foundby microbroth dilution MICsto be resistant to ampicillin and amoxicillin/clavulanate. Four of these strains were ß-lactamase-positive (BLPACR) (Cefinase disc methodology: BBL Microbiology Systems, Cockeysville, MD, USA) and 26 were negative (BLNAR). Two of the BLPACR strains and seven BLNAR strains were available for further study; all had been isolated in Japan.
Antimicrobial susceptibility testing
MICs were determined as recommended by the NCCLS, using freshly prepared Haemophilus Test Medium with incubation for 2024 h in ambient air.10 H. influenzae ATCC 49247 and H. influenzae ATCC 49766 were used as quality-control strains for MIC testing.
Determination of resistance mechanisms
Alterations in PBP3 were investigated in the two BLPACR and seven BLNAR H. influenzae strains by amplification of the ftsI gene, as described by Ubukata et al.7 The presence of ROB-1-type and TEM-1-type ß-lactamase (blaTEM-1) was tested in ß-lactamase-positive strains by PCR amplification using primers specific for genes encoding these products.11 The PCR products, after amplification of blaTEM-1 and ftsI genes, were purified using the QIAquick PCR Purification Kit (QIAGEN, Valencia, CA, USA). Nucleotide sequences of the amplified genes were obtained by direct sequencing using the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA) and compared with ß-lactamase of pBR322 from Escherichia coli12 using ClustalW software (www.ebi.ac.uk/clustalw/).
Resistance transformation studies
Preparation of competent cells and transformation of H. influenzae Rd (ATCC 51907) with DNA derived from selected strains used in this study was carried out as described previously by Barcak et al.13 Amplicons obtained by PCR were used for transformation. Transformants were selected on Brain Heart Infusion agar (Becton Dickinson, Cockeysville, MD, USA) supplemented with haemin (2 mg/L) and NAD (2 mg/L), which contained 4/2 mg/L amoxicillin/clavulanate.
The genetic relationship of transformant and recipient H. influenzae Rd strains was tested by pulsed-field gel electrophoresis (PFGE). PFGE was performed using CHEF DR III apparatus (Bio-Rad, Hercules, CA, USA) as described previously.14
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Results and discussion |
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The transformation of the H. influenzae Rd strain with amplified ftsI gene products from two BLPACR and two BLNAR strains resulted in selection of transformants with high amoxicillin/clavulanate MICs. These had the same mutations as their parent strains (Table 1). All transformants had approximately the same level of resistance to amoxicillin and amoxicillin/clavulanate, except for amoxicillin MICs of the two BLPACR transformants as they lacked ß-lactamases. After the transformation of the H. influenzae Rd strain with an ftsI amplicon from BLPACR4 and BLPACR7, the MICs of amoxicillin and amoxicillin/clavulanate increased from 0.5 and 0.5/0.25 mg/L to 4 and 4/2 mg/L, and to 8 and 4/2 mg/L, respectively. The MICs of cefdinir and cefotaxime increased for transformants RdBLPACR4 and Rd
BLPACR7, from 0.25 to 2 and 4 mg/L, and from 0.01 to 2 and 0.25 mg/L, respectively.
We conclude that amoxicillin and amoxicillin/clavulanate resistance in the two BLPACR strains, as well as all seven BLNAR strains, was due to changes in PBP3. The two BLPACR strains had typical TEM-1 ß-lactamases, which excluded the possibility of extended spectrum ß-lactamases being associated with amoxicillin/clavulanate resistance in the two BLPACR strains. The importance of mutations in ftsI, in terms of resistance to amoxicillin and amoxicillin/clavulanate, has also been reported in BLNAR strains.7 The results of this study showed that mutations in PBP3 may cause resistance to amoxicillin/clavulanate in ß-lactamase-producing H. influenzae strains without altering ß-lactamases. Continued surveillance is necessary to find out whether the incidence of these strains is increasing.
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Footnotes |
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Corresponding author. Tel: +1-717-5313910; Fax: +1-717-531-7953; E-mail: bozdogan-b{at}psu.edu
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References |
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