Variation in selected regions of blaTEM genes and promoters in Haemophilus influenzae

Stephen G. Tristram*, Rebecca Hawes and Juliana Souprounov

School of Human Life Sciences, University of Tasmania, Launceston, Tasmania 7250, Australia


* Corresponding author. Tel: +61-3-63-245469; Fax: +61-3-63-243658; E-mail: Stephen.Tristram{at}utas.edu.au

Received 2 March 2005; returned 26 April 2005; revised 24 May 2005; accepted 8 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To determine if the TEM ß-lactamases of Haemophilus influenzae are TEM-1 or derivatives thereof and associated exclusively with the overlapping Pa/Pb promoters.

Methods: Single nucleotide specific PCR was used to discriminate the polymorphic nucleotides at positions 32 and 317 of the blaTEM genes of a collection of TEM-positive strains.

Results: All blaTEM genes were found to be blaTEM-1 or derivatives thereof and none blaTEM-2. The blaTEM genes were associated with the P3 promoter, the Pa/Pb promoters or a novel promoter produced as a result of a 135 bp deletion and a G162T substitution.

Conclusions: The genetic features of blaTEM genes in H. influenzae are different from those in Enterobacteriaceae and more variable than previously recognized.

Keywords: H. influenzae , promoters , ß-lactamases , blaTEM genes , TEM


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The plasmid-mediated TEM-1 and TEM-2 ß-lactamases are common among Gram-negative bacteria, particularly Enterobacteriaceae. The enzymes have essentially identical enzymic properties, although TEM-2 has a Gln to Lys amino acid substitution at position 37 due to a single bp difference in the structural region of the blaTEM-2 gene (C317A).1 Another single bp difference (C32T) in the regulatory region of the blaTEM-2 gene results in stronger overlapping promoters (Pa/Pb) and produces larger amounts of the enzyme compared with the P3 promoter of blaTEM-1.2,3 The blaTEM-1 and blaTEM-2 genes are known to be transposable, with blaTEM-2 associated with transposon 1 (Tn1), and blaTEM-1 associated with a number of transposons including Tn2 and Tn3.4

Extensive sequencing of blaTEM genes in the Enterobacteriaceae over the last 15 years has revealed that there are actually seven subtypes of blaTEM-1 (blaTEM-1A to blaTEM-1G). These are defined by variations in nucleotides 32, 141, 162 or 175, which can produce one of three different promoter types (P3, P4 and P5), and by a range of silent mutations in the coding region of the gene.57 The currently accepted nomenclature schemes for blaTEM-1 do not accommodate blaTEM-1 with a Pa/Pb promoter57 even though this gene has been described in co-amoxiclav-resistant Escherichia coli isolates that hyperproduce TEM-1.8,9 Only one sequence of blaTEM-2 has been described and it is associated with the Pa/Pb promoter.5,7

In contrast, sequence data for the blaTEM genes in Haemophilus influenzae are limited, but indicate that the ß-lactamase is TEM-1,2,10 and that it is associated with the stronger Pa/Pb promoters normally associated with TEM-2.2 Sequencing of ß-lactamase plasmids from H. influenzae suggests that the blaTEM-1 gene was transposed on Tn2 from Enterobacteriacae onto cryptic plasmids already present in Haemophilus species.2 The current hypothesis is that given the relatively permeable outer membrane of H. influenzae to ß-lactam antibiotics, the C32T mutation (P3 to Pa/Pb promoters) was necessary to increase the amount of ß-lactamase produced and confer amoxicillin resistance.2 Indeed it is speculated that the need for this mutation may have been a contributing factor in delaying the emergence of TEM-mediated amoxicillin resistance in H. influenzae following the earlier emergence in Enterobacteriaceae.2

In order to more fully explore the nature and origin of the blaTEM genes in H. influenzae, single nucleotide specific PCR was used to discriminate the polymorphic nucleotides at positions 32 and 317 of the blaTEM genes of a collection of TEM-positive strains.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

A collection of 104 isolates of ß-lactamase-positive H. influenzae was established with contributions from seven geographically distinct participating Australian pathology laboratories that collected non-repeat ß-lactamase-positive clinical isolates of H. influenzae during 2001. Identification was confirmed by X and V factor testing, the presence of ß-lactamase using nitrocefin touchsticks (Oxoid, Australia) and blaTEM identified using PCR with primers A and B (see Table 1) as described below.


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Table 1. Primer details and annealing temperatures for single nucleotide specific PCR

 
Susceptibility testing

All isolates were tested for susceptibility to amoxicillin, co-amoxiclav and cefaclor using Etests (Australian Laboratory Services, Melbourne) according to the manufacturer's instructions and interpreted using NCCLS breakpoints.11

Single nucleotide specific PCR

TEM-positive strains were subjected to four different PCR reactions to detect the presence of either of two nucleotides at bp 32 (C or T) and 317 (C or A). All PCR reactions in the study were carried out with the HotStarTaq Master Mix Kit (Qiagen, Australia) according to the manufacturer's instructions. Briefly, reactions were carried out in a total volume of 25 µL containing 1.25 U of Taq DNA polymerase, 0.5 µM (each) primer, reaction buffer with 1.5 mM MgCl2, 200 µM dNTP and 1 µL of DNA. An initial inactivation step of 15 min at 95°C was followed by 30 cycles of 1 min of denaturation at 95°C, 2 min at the relevant annealing temperature (see Table 1) and 2 min of primer extension at 72°C, followed by a final extension for 10 min at 75°C. The specificity of the PCR reactions was controlled using isolates with previously sequenced TEM genes with known nucleotides at positions 32 and 317,12 and specific annealing temperatures determined by gradient analysis. Template DNA for all PCR reactions 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.

Further characterization of blaTEM genes from selected strains

Five of the blaTEM genes characterized as 32C and 317C, five of the genes characterized as 32T and 317C and five of the genes characterized as neither 32T nor C and 317C in the initial single nucleotide specific PCR were sequenced (up to bp 325) as previously described13 to confirm the specificity of the PCR. Strains chosen for this further characterization were selected so as to include strains from five different contributing laboratories for each of the three genotypes. Following identification of a deletion in the promoter region in the five sequenced genes that were initially characterized as neither 32T nor C, a segment of the gene was amplified for all 63 such strains using primers A and G for size estimation on an agarose gel.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All nucleotide substitutions and deletions are described in relation to blaTEM-1A. Using single nucleotide specific PCR, all 97 blaTEM genes were characterized as 317C (consistent with blaTEM-1) and none as 317A (blaTEM-2), 17 as 32C (P3 promoter), 17 as 32T (Pa/Pb promoter) and 63 gave no PCR product with P1/F or P2/F primers discriminating bp 32. All of these 63 blaTEM genes produced a shortened product of ~250 bp (379 bp expected from known blaTEM sequence) when amplified with primers A and G and were represented in all collection centres. When the gels for the initial primer A and B PCR products were retrospectively reviewed for these strains, the bands had migrated slightly faster than those from the strains without deletions.

Of the 10 sequenced blaTEM genes with either the P3 or Pa/Pb promoter, three of each had G175/T226, with the remaining two of each having A175/C226. Of the five sequenced strains that failed to amplify with P1/F or P2/F and showed a shortened PCR product with primers A and G, all had a 135 bp deletion from bp G23 through to C157 inclusive, all had a G162T substitution and all had G175/T226. All 15 sequenced strains had C317.

The presence of blaTEM with G175/T226 is consistent with an origin from Tn2 as originally described by Chen and Clowes,2 but the presence of genes with A175/C226 is consistent with an origin from Tn3.3

This is the first time that the presence of the P3 promoter has been demonstrated with the blaTEM in H. influenzae, and challenges the hypothesis that a C32T mutation and resulting Pa/Pb promoter was necessary to provide sufficient levels of resistance for the TEM ß-lactamase to become established in this organism.2 Chen and Clowes2 also predicted that the level of amoxicillin resistance in H. influenzae with TEM transcription initiated from Pa/Pb would be ~10-fold higher than that expected from P3.2 This has not been demonstrated with the isolates tested in this study, with both promoter types associated with a wide and similar range of amoxicillin MICs (see Table 2).


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Table 2. MICs (mg/L) of amoxicillin, co-amoxiclav and cefaclor according to promoter type detected

 
The most significant finding of the study was the large number of isolates with a new promoter region produced by a 135 bp deletion and G162T mutation compared with blaTEM-1A. The deletion brings together the –35 region of the Pa promoter with the –10 region of the P3 promoter, and the mutation makes the –10 region more closely approximate the –10 consensus sequence to produce a stronger promoter (Figure 1).14–16 This promoter has previously been described only in Enterobacteriaceae in the extended-spectrum ß-lactamases (ESBLs) TEM-20 and TEM-92,14,17 where it was associated with high-level ß-lactamase production and high levels of cephalosporin resistance.14 The G162T substitution alone produces the P4 promoter and has been associated with co-amoxiclav resistance due to hyperproduction of TEM-1 in Enterobacteriaceae.6 A similar finding of a deletion in the promoter region of blaTEM in H. influenzae in a recent study by Molina et al.,18 differed in that a 137 bp deletion was reported without the G162T substitution. In that study, the novel promoter was associated with increased cefaclor resistance.



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Figure 1. Promoter region (bp 1–180) of blaTEM gene with P3, Pa/Pb or 135 bp deletion. Boxes represent –35 and –10 regions of respective promoters. Bold and large font base pairs represent those where substitution occurs between promoter types. Shaded area represents 135 base pairs (G23 to C157 inclusive) deleted in new promoter type. E. coli consensus sequence is –35 TTGACA and –10 TATAAT. Figure modified with permission from Lartigue et al.6

 
These and other previously reported associations between promoter types and levels of resistance in clinical isolates of Enterobacteriaceae2,6,8,9,14 and H. influenzae18 were not demonstrated in H. influenzae in this study where all isolates were resistant to amoxicillin and susceptible to co-amoxiclav and cefaclor according to NCCLS breakpoints11 (see Table 2). It is difficult to compare the effect of a blaTEM promoter on resistance levels in E. coli with H. influenzae because of the different permeability of the outer membrane to ß-lactams.12 In addition, any realistic study of the effects attributable to the promoter type alone would need to be carried out on recombinant strains with the respective blaTEM genes expressed in an isogenic background, to remove the influence of many other factors known to influence levels of ß-lactam resistance.12

From this limited number of strains and method of collection it is not possible to describe the absolute frequency with which the promoter types detected in the blaTEM genes in this study occur. It is possible to conclude that both the Pa/Pb promoter and the P3 promoter occur, and that the promoter associated with the 135 bp deletion is relatively common in the blaTEM genes in this collection of strains. The detection of these different blaTEM promoter types in strains from multiple geographically distinct collection centres indicates that the variation is not limited to a successful local clone but that it is spread throughout the areas from which strains were collected.

If the 135 bp (or similar) deletion in the blaTEM gene occurs as frequently in H. influenzae as this study and that of Molina et al.18 indicate, it is surprising that it has not been detected before, particularly since a number of large studies have used gel visualization of blaTEM PCR products to confirm the presence of a TEM ß-lactamase in H. influenzae.10 The difference in size of the PCR products is clearly visible, although as occurred in this study, it may be missed initially if the primers used generate large products where the effect of the 135 bp deletion is relatively small. Alternatively, some large studies on H. influenzae use primer sets that amplify a region of the blaTEM gene internal to the deletion and hence would not detect it.19

TEM-1 has been termed a fully efficient enzyme in relation to catalytic efficiency, and in many of the ESBLs detected in Enterobacteriaceae, the increased spectrum of activity has come at a cost of decreased catalytic efficiency.20 One way of overcoming this loss of efficiency is to increase the amount of enzyme produced, and it is notable that although TEM-2 is much less common than TEM-1 in ampicillin-resistant E. coli, blaTEM-2 is over represented as a progenitor in ESBLs.20 This is probably due to the more efficient Pa/Pb promoter in blaTEM-2. Furthermore, many ESBLs that have arisen from blaTEM-1 have a C32T substitution to produce the Pa/Pb promoter.20 Similarly, the strong promoter associated with the 135 bp deletion and G162T substitution has only been described in Enterobacteriaceae expressing ESBLs.14,17 In this context the relatively antibiotic-permeable outer membrane of H. influenzae could be thought of as creating a similar need for, or benefit from, increased amounts of ß-lactamase, leading to the occurrence of the Pa/Pb and 135 bp G162T promoters in the blaTEM genes in this organism. Little is known about the regulation of transcription in H. influenzae,21 and although there is some evidence that H. influenzae recognizes and transcribes from similar or identical promoters to Enterobacteriaceae,2,21 it is important to consider that the relative strengths of various promoters may not be the same for both organisms.

The results of this study show that selective pressures have influenced the evolution of blaTEM genes in H. influenzae differently compared with Enterobacteriaceae. Further studies and more extensive sequencing will be required to fully elucidate the diversity of blaTEM genes in H. influenzae and current nomenclature schemes for parental blaTEM genes5–7 may have to be modified or expanded to accommodate the findings of this study.


    Acknowledgements
 
This work was supported by a grant from the Clifford Craig Medical Research Trust, Launceston, Tasmania.


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1. Goussard S, Courvalin P. Sequences of the genes blaT-1B and blaT-2. Gene 1991; 102: 71–3.[CrossRef][ISI][Medline]

2. Chen S, Clowes R. Nucleotide sequence comparisons of plasmids pHD131, pJB1, pFA3, and pFA7 and ß-lactamase expression in Escherichia coli, Haemophilus influenzae, and Neisseria gonorrhoeae. J Bacteriol 1987; 169: 3124–30.[ISI][Medline]

3. Chen S, Clowes R. Variations between the nucleotide sequences of Tn1, Tn2, and Tn3 and expression of ß-lactamase in Pseudomonas aeruginosa and Escherichia coli. J Bacteriol 1987; 169: 913–16.[ISI][Medline]

4. Levesque RC, Jacoby GA. Molecular structure and interrelationships of multiresistance ß-lactamase transposons. Plasmid 1998; 19: 21–9.

5. Pomba-Feria C, Canica M. A novel sequence framework (blaTEM-1G) encoding the parental TEM-1 ß-lactamase. FEMS Microbiol Lett 2003; 220: 177–80.[CrossRef][ISI][Medline]

6. Lartigue M, Leflon-Guibout V, Poirel, L et al. Promoters P3, Pa/Pb, P4 and P5 upstream from blaTEM genes and their relationship to ß-lactam resistance. Antimicrob Agents Chemother 2002; 46: 4035–7.[Abstract/Free Full Text]

7. Leflon-Guibot V, Heym B, Nicolas-Chanoine M-H. Updated sequence information and proposed nomenclature for blaTEM genes and their promoters. Antimicrob Agents Chemother 2000; 44: 3232–4.[Abstract/Free Full Text]

8. Wu P, Shannon K, Phillips I. Mechanisms of hyperproduction of TEM-1 ß-lactamase by clinical isolates of Escherichia coli. J Antimicrob Chemother 1995; 36: 927–39.[Abstract]

9. Leflon-Guibot V, Speldooren V, Heym B et al. Epidemiological survey of amoxicillin-clavulanate resistance and corresponding molecular mechanisms in Escherichia coli isolates in France: new genetic features of blaTEM genes. Antimicrob Agents Chemother 2000; 44: 2709–14.[Abstract/Free Full Text]

10. Scriver S, Walmsley S, Kau C et al. Determination of antimicrobial susceptibilities of Canadian isolates of Haemophilus influenzae and characterization of their ß-lactamases. Antimicrob Agents Chemother 1994; 38: 1678–80.[Abstract]

11. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved standard M7-A5. NCCLS, Villanova, PA, USA, 2000.

12. Tristram S. Effect of extended-spectrum ß-lactamases on the susceptibility of Haemophilus influenzae to cephalosporins. J Antimicrob Chemother 2003; 51: 39–43.[Abstract/Free Full Text]

13. Chanal C, Poupart M, Sirot D et al. Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 ß-lactamase genes. Antimicrob Agents Chemother 1992; 36: 1817–20.[Abstract]

14. Arlet G, Goussard S, Courvalin P et al. Sequences of the genes for TEM-20, TEM-21, TEM-22 and TEM-29 extended spectrum ß-lactamases. Antimicrob Agents Chemother 1999; 43: 969–71.[Abstract/Free Full Text]

15. Speldooren V, Heym B, Labia R et al. Discriminatory detection of inhibitor-resistant ß-lactamases in Escherichia coli by single-strand conformation polymorphism-PCR. Antimicrob Agents Chemother 1998; 42: 879–84.[Abstract/Free Full Text]

16. Xu J, McCabe B, Koudelka G. Function-based selection and characterization of base pair polymorphisms in a promoter of Escherichia coli RNA polymerase-{sigma}70. J Bacteriol 2001; 183: 2866–73.[Abstract/Free Full Text]

17. De Champs C, Monne C, Bonnet R et al. New TEM variant (TEM-92) produced by Proteus mirabilis and Providencia stuartii isolates. Antimicrob Agents Chemother 2001; 45: 1278–80.[Abstract/Free Full Text]

18. Molina J, Cordoba J, Monsoliu A et al. Haemophilus influenzae and ß-lactam resistance: description of blaTEM gene deletion. Rev Esp Quimioterap 2003; 16: 195–203.

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21. Zulty J, Barcak G. Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene. J Bacteriol 1993; 175: 7269–81.[Abstract]





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