Contribution of ß-lactamase and PBP amino acid substitutions to amoxicillin/clavulanate resistance in ß-lactamase-positive, amoxicillin/clavulanate-resistant Haemophilus influenzae

Vlatka Matic1,*, Bülent Bozdogan1,§, Michael R. Jacobs2, Kimiko Ubukata3 and Peter C. Appelbaum1

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


    Abstract
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
The roles of ß-lactamase and alterations in penicillin-binding protein in the development of amoxicillin and amoxicillin/clavulanate resistance in two ß-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains of Haemophilus influenzae were investigated. Seven ß-lactamase-negative, ampicillin-resistant (BLNAR) strains were also studied for comparison of their resistance mechanisms. All strains had been recovered from patients in Japan. The TEM type ß-lactamase of the two BLPACR strains had 100% homology with the amino acid sequences of published TEM-1 ß-lactamase, showing that amoxicillin/clavulanate resistance was not associated with mutations in this ß-lactamase. However, these strains, as well as the seven BLNAR strains, had multiple mutations in ftsI, which encodes penicillin binding protein 3 (PBP3). The transformation of H. influenzae Rd strain with amplified ftsI genes from two BLPACR and two BLNAR strains enabled the selection of amoxicillin/clavulanate-resistant transformants with the same mutations as their parent strains. We concluded that amoxicillin/clavulanate resistance in the two BLPACR strains was due to changes in PBP3. The possibility of the presence of an extended spectrum ß-lactamase was excluded in the BLPACR strains studied.

Keywords: H. influenzae, penicillin-binding proteins, ß-lactamases, mutations


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Haemophilus influenzae is a commensal of the nasopharynx in humans and causes community-acquired infections in children and adults, including pneumonia, acute exacerbations of chronic bronchitis, sinusitis and otitis media.1,2 The majority of H. influenzae infections are caused by non-typeable H. influenzae strains.3

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.


    Materials and methods
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
Bacteria

Among 6382 H. influenzae strains collected for the Alexander Project (1997–2000), 30 were found—by microbroth dilution MICs—to 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 20–24 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


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The MICs of amoxicillin, amoxicillin/clavulanate, cefdinir and cefotaxime against two BLPACR and seven BLNAR strains, and H. influenzae Rd strain, are shown in Table 1. The MIC range of antibiotics tested against the seven BLNAR and two BLPACR were as follows: amoxicillin 8–>64 mg/L, amoxicillin/clavulanate 8–16 mg/L, cefdinir 2–8 mg/L, cefotaxime 0.25–2 mg/L. No clear-cut differences were observed in the MICs of amoxicillin/clavulanate, cefdinir and cefotaxime between BLNAR and BLPACR strains; however, differences in amoxicillin MICs were obvious between BLNAR and BLPACR strains. The BLNAR strains had relatively low MICs of amoxicillin (8–32 mg/L) compared with BLPACR strains, which had high resistance levels (>64 mg/L).


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Table 1. MICs of amoxicillin and amoxicillin/clavulanate against ß-lactamase-negative, ampicillin-resistant (BLNAR) and ß-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains
 
The two BLPACR strains produced TEM-type ß-lactamase, the deduced amino acid sequences of which had 100% homology with ß-lactamase encoded by blaTEM-1.12 However, two silent mutations were detected in blaTEM-1 genes from both strains—C to T substitutions at positions 18 and 228—but these mutations did not affect the amino acid sequence of the ß-lactamase. In all nine strains, multiple mutations detected in the ftsI gene caused alterations in the deduced amino acid sequences of PBP3, but we found no correlation between the number of mutations and the amoxicillin MIC. However, high MICs of cefotaxime (1 or 2 mg/L) were associated with L389F point mutations. In all BLNAR and BLPACR strains, arginine was substituted by lysine at position 526. Mutations found in the two BLPACR strains were not identical: both had mutations at positions 350, 357, 385, 526, 547 and 569, and BLPACR4 had additional mutations at positions 377, 389 and 562. BLPACR7 had additional mutations at positions 280 and 502 (Table 1).

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 Rd{Omega}BLPACR4 and Rd{Omega}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.


    Footnotes
 
* Present address: PLIVA Inc., Research Institute, Prilaz baruna Filipovica 25, 10000 Zagreb, Croatia Back

§ Corresponding author. Tel: +1-717-531–3910; Fax: +1-717-531-7953; E-mail: bozdogan-b{at}psu.edu Back


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 Materials and methods
 Results and discussion
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