JAC Advance Access originally published online on April 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):183-185; doi:10.1093/jac/dkl150
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A multiplex PCR for ß-lactamase genes of Haemophilus influenzae and description of a new blaTEM promoter variant
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 25 January 2006; returned 23 March 2006; revised 28 March 2006; accepted 30 March 2006
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Abstract |
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Objectives: To establish a multiplex PCR to detect and differentiate the ß-lactamase genes of Haemophilus influenzae.
Methods: A collection of H. influenzae isolates with a range of different ß-lactamase genes were tested in parallel with individual PCRs and the multiplex assay developed in this study.
Results: The multiplex method was found to be superior to previous methods and was able to correctly detect and differentiate blaROB-1 and the variants of blaTEM present in the strains tested, including a previously unrecognized variant associated with a 54 bp insertion in the promoter region.
Conclusions: The multiplex PCR will be a useful tool for the surveillance of ß-lactamase genes in H. influenzae.
Keywords: H. influenzae , bla genes , TEM, ROB-1
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Introduction |
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Resistance to ß-lactam antibiotics in Haemophilus influenzae is primarily mediated by the production of ß-lactamases, most commonly by the TEM enzyme and less commonly by ROB-1.1,2 In a recent global survey using Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin (PROTEKT) isolates from 1999 to 2003, the prevalence of TEM and ROB-1 in ß-lactamase-positive H. influenzae isolates was 93.7% and 4.6%, respectively, with only a single isolate out of the 2225 studied positive for both enzymes.1
There is some evidence that the ROB-1 enzyme is more active against cefaclor with ROB-1-positive isolates producing higher MICs and resistance rates compared with TEM-producing isolates, although it should be noted that the majority of ROB-1 isolates still remain susceptible to cefaclor.1–3 ß-Lactamase-associated resistance to cefaclor could have important clinical implications given that cefaclor is widely prescribed for infections with ß-lactamase-positive amoxicillin-resistant strains of H. influenzae.
Recently, it has been reported that the TEM ß-lactamase in H. influenzae can be produced from a number of different TEM genes (blaTEM) that differ in the promoter region.4,5 Different promoter sequences may have different affinities for RNA polymerase and may result in different levels of ß-lactamase expression and subsequent level of resistance to ß-lactam antibiotics. The blaTEM genes have been shown to be associated with either the P3 (blaTEM-P3), overlapping Pa/Pb (blaTEM-Pa/Pb) or the newly described Pdel (blaTEM-Pdel) promoters, the latter produced by a 135 bp deletion in the promoter region.4 Importantly, one of these studies reported an association between the presence of blaTEM-Pdel and cefaclor resistance.5
Phenotypic tests such as nitrocefin hydrolysis for ß-lactamase production in H. influenzae are useful for predicting amoxicillin resistance, but do not differentiate between TEM and ROB-1 enzymes. At present most workers perform individual PCRs for blaROB-1 and blaTEM when the nature of the ß-lactamase being produced is important, although there has been one report of an instrument-specific multiplex method using real-time PCR and fluorescent probes that will simultaneously detect both blaROB-1 and blaTEM.1 None of the current PCR methods is able to readily differentiate the blaTEM promoter variants without an additional analysis such as cutting with restriction enzymes.
A simple multiplex PCR method to detect and differentiate blaROB-1 and blaTEM, and also differentiate blaTEM-Pdel from other blaTEM promoter variants, would be useful in collecting data on ß-lactamase-mediated resistance in H. influenzae, particularly the association between cefaclor resistance and blaROB-1 or blaTEM-Pdel.
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Materials and methods |
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Bacterial strains
A collection of 91 ß-lactamase-positive strains of H. influenzae was established consisting of 89 previously described non-repeat isolates from seven geographically distinct Australian laboratories4 and 2 specifically selected isolates from the (PROTEKT) study.1 All isolates were tested for blaROB-1 and blaTEM using previously described individual PCR methods.6,7 All isolates positive for blaTEM were characterized as to promoter type using the recently described single nucleotide specific (snp) PCR to differentiate residue C or T at bp 32 (blaTEM-P3 or blaTEM-Pa/Pb) or differentiate on amplicon size for blaTEM-Pdel.4 See Table 1.
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Multiplex PCR
Following initial optimization of annealing temperatures of primers by gradient analysis and primer ratios by titration, the multiplex PCR protocol was established as follows. All reactions were carried out using the HotStarTaq Master Mix kit (Qiagen, Australia) in a total volume of 25 µL containing 1.25 U Taq, reaction buffer with 1.5 mM MgCl2 and 200 µM dNTP, 0.1 µL of each 20 µM TEM primer, 0.5 µL of each 20 µM ROB primer and 3 µL of water. The cycle parameters were as follows: 15 min initial activation at 95°C followed by 25 cycles of 1 min denaturation at 95°C, 1 min annealing at 50°C and 1 min 30 s extension at 72°C, followed by a final extension of 10 min at 72°C. Template DNA for all PCRs was prepared by adding a loopful of overnight growth on chocolate agar to 100 µL of distilled water, heating to 95°C for 10 min and centrifuging at high speed for 1 min. Each multiplex reaction was controlled using a blank and a positive control consisting of a mixture of 1 µL each of DNA template from individual strains positive for blaROB-1, blaTEM-Pdel and blaTEM-Pa/Pb.
Primers
Primers were modified from those used previously,6,7 to be of similar Tm and generate products of sufficiently different size to be easily differentiated on an agarose gel. Primers were MP-TEMF (–1) 5'-AATTCTTGAAGACGAAAGGG-3' and MP-TEMR (373) 5'-AAGGATCTTACCGCTGT-3' to generate an expected 375 bp amplicon from blaTEM and MP-ROBF (1) 5'-GGATCAGAGTAATAATTTCTG-3' and MP-ROBR (1289) 5'-GCCATTGAAAGCAAGTTTCAACGG-3' to generate an expected 1289 bp amplicon from blaROB-1. The numbers in parentheses represent the position of the 5' base according to the published sequences for blaTEM-1 and blaROB-1.8,9
Sequencing
Selected isolates were sequenced as described previously.6
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Results and discussion |
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In the initial optimization of the multiplex PCR, blaTEM was found to be amplified preferentially to blaROB-1 from a mixed template, necessitating the 5:1 blaTEM to blaROB primer ratio described in the method. Problems with the presence of blaTEM-P3 or blaTEM-Pa/Pb (but not blaTEM-Pdel based on size of amplicon) in the blank control occurred inconsistently during initial optimization, indicating random contamination with blaTEM in the test system. This problem continued despite replacement of all reagents and consumables and rigorous attention to technique. Problems with blaTEM contamination are not surprising given its ubiquity, both as a naturally occurring resistance gene and as a selectable marker in commercial plasmids, and contamination problems with blaTEM PCR have been reported previously.10 When the cycle number was reduced from 30 to 25, the problem of false-positive blaTEM bands was eliminated, yet the assay was still able to detect a 1 in 10 000 dilution of a standard 3 µL sample of blaTEM-containing template (data not shown).
The multiplex PCR was shown to correctly identify all isolates producing blaTEM, blaROB-1 or both compared with previously described individual methods (see Table 1).
The blaTEM primers were designed to differentiate blaTEM-Pdel from blaTEM-P3 and blaTEM-Pa/Pb based on the difference in size of the amplicons produced as a result of the 135 bp deletion in blaTEM-Pdel, and the multiplex assay achieved this. It was not expected that isolates previously characterized as blaTEM-P3 and blaTEM-Pa/Pb would be able to be differentiated as the Pa/Pb promoter only differs from the P3 promoter by a C32T substitution. However, with the multiplex assay, all isolates designated as blaTEM-P3 by the snp PCR produced longer amplicons (
430 bp) than those designated as blaTEM-Pa/Pb. When the blaTEM genes from these isolates were sequenced they all had a 54 bp insertion in the promoter region. The insertion consisted of a repeat of bp 145 to 198 inclusive and was inserted after bp 198. The problem with the previously described snp PCR method is based on the fact that the blaTEM genes were characterized as having the P3 promoter on the basis of a 32C which was confirmed with sequencing of that region of the gene and the insertion that leads to the new promoter does not alter that nucleotide. In addition, in that study the blaTEM genes were initially detected with PCR using primers to amplify the promoter region and entire open reading frame (amplicon 1000 bp depending on which blaTEM gene was present) where the 54 bp difference in size was not readily detected.4
Although little is known about the regulation of transcription in H. influenzae there is some evidence that H. influenzae RNA polymerase recognizes and transcribes from similar or identical promoters to members of the Enterobacteriaceae.11 A close examination of the sequence of the promoter with the repeat sequence (designated Prpt) reveals that it might produce an additional promoter. In the normal P3 promoter, bp 193 to 198 are TTGAAA, which are a close match for the –35 consensus sequence (TTGACA),12 but in this case are redundant because there is no accompanying –10 sequence. In the Prpt promoter, the insertion which occurs immediately downstream provides a repeat of the –10 sequence of the P3 promoter 17 bp downstream from this –35 sequence (see Figure 1). In effect the blaTEM-Prpt appears to have two promoters, with the new promoter possibly being stronger than the co-existing P3 promoter as the –35 sequence is a closer match to the consensus sequence.
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The finding that the blaTEM-P3 genes initially characterized in this collection of organisms are actually blaTEM-Prpt is consistent with the initial hypothesis of Chen and Clowes that greater permeability of the outer membrane of H. influenzae to ß-lactam antibiotics requires blaTEM genes with promoters stronger than P3 to be successful.11
There is no direct evidence that the proposed Prpt promoter described in this work or the recently described Pdel promoter is the actual site of initiation of transcription in H. influenzae or that isolates in which the blaTEM is associated with these different promoters produce different amounts of ß-lactamase compared with those with the Pa/Pb promoter. Further work is required to definitively establish the proposed –35 and –10 regions of both promoters as sites of the initiation of transcription in H. influenzae and to determine their effect on the expression of the gene.
In conclusion, this multiplex PCR protocol has been shown to reliably detect and differentiate blaROB-1, blaTEM-Pdel, blaTEM-Prpt and blaTEM-Pa/Pb genes in ß-lactamase-positive strains of H. influenzae. This will be a useful tool in determining how widespread these new promoters are, and it will simplify surveillance studies on ß-lactamase-mediated resistance in H. influenzae, particularly with respect to examining the association between specific ß-lactamase genes and resistance phenotypes.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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The work was supported by a grant from the Clifford Craig Medical Research Trust, Launceston, Tasmania.
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References |
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1 Farrel D, Morrissey I, Bakker S, et al. (2005) Global distribution of TEM-1 and ROB-1 ß-lactamases in Haemophilus influenzae. J Antimicrob Chemother 56:773–6.
2
Karlowsky J, Verma G, Zhanel G, et al. (2000) Presence of ROB-1 ß-lactamase correlates with cefaclor resistance among recent isolates of Haemophilus influenzae. J Antimicrob Chemother 45:871–5.
3
Scriver S, Walmsley S, Kau C, et al. (1994) Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their ß-lactamases. Antimicrob Agents Chemother 38:1678–80.
4
Tristram S, Hawes R, Souprounov J. (2005) Variation in selected regions of blaTEM genes and promoters in Haemophilus influenzae. J Antimicrob Chemother 56:481–4.
5 Molina J, Cordoba J, Monsoliu A, et al. (2003) Haemophilus influenzae and ß-lactam resistance: description of blaTEM gene deletion. Rev Esp Quimioter 16:195–203.[Medline]
6
Chanal C, Poupart M, Sirot D, et al. (1992) Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 ß-lactamase genes. Antimicrob Agents Chemother 36:1817–20.
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Galan J, Morosini M, Baquero M, et al. (2003) Haemophilus influenzae blaROB-1 mutations in hypermutagenic 
ampC Escherichia coli conferring resistance to cefotaxime and ß-lactamase inhibitors and increased susceptibility to cefaclor. Antimicrob Agents Chemother 47:2551–7.
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Sutcliffe J. (1978) Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci USA 75:3737–41.
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Juteau JM and Levesque R. (1990) Sequence analysis and evolutionary perspectives of ROB-1 ß-lactamase. Antimicrob Agents Chemother 34:1354–9.
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Chiang C, Liu C, Weng L, et al. (2005) Presence of ß-lactamase gene TEM-1 DNA in commercial Taq DNA polymerase. J Clin Microbiol 43:530–1.
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Chen S and Clowes R. (1987) Nucleotide sequence comparisons of plasmids pHD131, pJB1, PfA3, and pFA7 and ß-lactamase expression in Escherichia coli, Haemophilus influenzae, and Neisseria gonorrhoeae. J Bacteriol 169:3124–30.
12
Xu J, McCabe B, Koudelka G. (2001) Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-
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