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10th European Congress of Clinical Microbiology and Infectious Diseases

28-31, 2000 May, Stockholm, Sweden
Poster # P278:14/1

Identification of the Naturally Occurring Variant Genes blaTEM-1d and blaTEM-70 Encoding Broad-Spectrum TEM-Type b-lactamases

M. EDELSTEIN, M. SUVOROV, I. EDELSTEIN, R. KOZLOV
Institute of Antimicrobial Chemotherapy, Smolensk, Russia

The PDF format poster (795 kb)



BACKGROUND AND OBJECTIVES

In recent years, there have been numerous reports describing the sequences of the genes for extended-spectrum and inhibitor resistant b-lactamases which belong to the TEM-family. At the same time genetic diversity of the naturally occurring parental genes encoding TEM-type penicillinases was studied to a lesser extent. Sequences of the genes blaTEM-1a, blaTEM-1b and blaTEM-2 carried by transposons Tn3, Tn2 and Tn1 were determined in the middle 1980-s, and since then it has been commonly accepted that other members of the TEM-group evolved from three distinct genetic lineages. More recently, a TEM-55 penicillinase with an altered isoelectric point (pI 5.2) was found in a single clinical isolate of E.coli using the isoelectric focusing (IEF) method, and in 1999 the third allelic variant encoding TEM-1 b-lactamase was described that received a designation blaTEM-1c.

We have used a recently developed Polymerase Chain Reaction - Single Strand Conformational Polymorphism (PCR-SSCP) method to screen for mutational events which account for the sequence diversity of parental blaTEM genes in unrelated uropathogenic strains of E.coli. We have also determined the sequences of the variant genes and characterised the properties of mutant b-lactamases.


METHODS

Selection of bacterial strains: The E.coli strains used in this study were collected from outpatients with UTI in four medical centers located in 3 different cities of Russia: Moscow (2 centers), Novosibirsk (1 center) and Smolensk (1 center) in 1998. Forty five ampicillin-resistant isolates equally representing each of the 4 participating medical centers were analysed by ERIC-PCR. For 30 strains representing the distinct molecular types the presence of a TEM b-lactamase was determined by isoelectric focusing and confirmed by PCR-amplification of the respective blaTEM gene. The amplified sequences were screened for the presence of mutations using the PCR-SSCP analysis. Mutations contributing to an unusual SSCP profile or an altered pI value of a b-lactamase were studied by direct sequencing of a PCR product.

Susceptibility testing: Susceptibility of E.coli isolates to ampicillin, gentamicin, nitrofurantoin, trimethoprim/sulphamethoxazole, nalidixic acid and ciprofloxacin was determined by standard agar dilution method on Mueller-Hinton agar (Becton Dickinson, USA) and interpreted according to the current NCCLS standards. The MICs of ticarcillin, piperacillin, cefuroxime, cefoperazone, ceftazidime, cefotaxime, cefoxitin, aztreonam and combinations of penicillins with clavulanic acid and tazobactam were additionally determined for the clinical strains and their transconjugants producing b-lactamase TEM-70. E.coli strains ATCC25922 and ATCC35218 were used for quality control.

Typing by ERIC-PCR: Template DNA was extracted from 3-4 colonies of each strain grown overnight on MacConkey agar using the InstaGene matrix (BioRad, USA). Reaction mixes were set up by adding 2ml of 25mM primer ERIC1 (5'-GTGAATCCCCAGGAGCTTACAT-3'), 2ml of template DNA and 21ml of autoclaved Milli-Q water to the 0.5ml tubes containing premixed and predispensed PCR reagents (Ready-To-Go PCR Beads; Amersham Pharmacia Biotech (APB), USA). The PCR was carried out in a PTC-200 thermocycler (MJ Research, USA) under the following conditions: initial denaturation at 94oC for 3 min followed by 35 cycles of annealing at 47oC for 1 min, elongation at 72oC for 1 min and denaturation at 94oC for 30 sec, with a final elongation step extended to 4 min. After electrophoresis in 1.3% agarose gel DNA fragments were stained with ethidium bromide and documented using a PhotoDoc-IT Link Gel Documentation System (UVP, USA). Cluster analysis of ERIC-PCR fingerprints was done using a GelCompar software (Applied Maths BVBA, Belgium) by the UPGMA method applied to densitometric tracks.

Isoelectric focusing: Crude sonic extracts containing b-lactamases were examined using a PhastSystem apparatus and precast IEF 5-8 pI gels (APB, USA) as previously described, and b-lactamases with known pIs (TEM-37 (pI 5.2), TEM-1 (pI 5.4), TEM-2 (pI 5.6) and TEM-3 (pI 6.3)) were used as standards.

Amplification of blaTEM genes and SSCP-analysis: A pair of primers (A: 5-ATAAAATTCTTGAAGACGAAA-3 and B: 5-GACAGTTACCAATGCTTAATCA-3) was used to amplify a 1080-bp fragment that covers the entire blaTEM gene sequence. The PCR mixture contained: 12.5mM Tris-HCl (pH 8.3), 62.5mM KCl, 2mM MgCl2, 200mM of each dNTP, 0.25mM of each primer, 1.25U AmpliTaq DNA polymerase (Perkin-Elmer, USA) and 20ml of template DNA in a total volume of 50ml. The PCR was carried out in a PTC-200 thermocycler (MJ Research, USA) under the following protocol: initial denaturation at 94oC for 2 min followed by 35 cycles of annealing at 54oC for 10 sec, elongation at 72oC for 45 sec and denaturation at 94oC for 10 sec, with a final elongation step at 72oC for 3 min. 20ml of amplified DNA was digested with either 1U Taq I and 10U Pst I or 1U Taq I and 4U Ava II restriction endonucleases (APB, USA) for 2 h at 37oC and 1 h at 65oC. Restriction fragments were then denatured to single-stranded (ssDNA) form by by mixing 2ml of digest with double volume of SSCP buffer (98% formamide, 2% glycerol, 0.05% bromphenol blue, 10mM EDTA ) and heating the mixture at 98oC for 10 min with subsequent cooling at 0oC in a thermocycler. ssDNA fragments were separated on a PhastSystem (APB, Sweden) using a PhastGel Homogeneous 12.5 system with PhastGel Native Buffer Strips. The program had three steps as follows: 1) Pre-run step at 400V, 5mA, 2W, 15oC for 70Vh; 2) Sample loading step at 400V, 1mA, 2W, 15oC for 2Vh; 3) Separation step at 400V, 5mA, 2W, 15oC for 350Vh in the case of Taq I - Pst I digests or for 230Vh in the case of Taq I - Ava II digests. The gels were stained with a PhastGel DNA Silver Staining Kit (Pharmacia Biotech, Sweden) as recommended by manufacturer.

DNA sequencing: Sequencing of the mutant blaTEM genes was performed on 1080-bp PCR-products using a dideoxy-chain termination method with a Cycle Reader DNA Sequencing Kit (Fermentas, Latvia), with primers A, B and additional internal primers (5'-ATTCTCAGAATGACTTGGTTGAG-3' and .5'-TTACTGTCATGCCATCCGTAAG-3').

Kinetic Analysis of b-lactamases: The genes coding for b-lactamase TEM-70 in clinical isolates NS13 and SM91 were transferred by conjugation and expressed in E.coli C600. Kinetic parameters of TEM-70 were determined by using partially purified extracts obtained from transconjugants and compared with those of TEM-1 (E.coli C600, blaTEM-1b). The complete time course of hydrolysis of benzylpenicillin, ampicillin, ticarcillin, cephalothin and cefoperazone was recorded with an Ultrospec 3000 spectrophotometer (APB, Sweden) with two different starting concentrations of each substrate. The Km and Vmax values were calculated using the results 3 independent measurements fitted to the Hanes plot.


RESULTS

According to IEF, 28 isolates produced a single b-lactamase with pI 5.4, corresponding to a TEM-1 enzyme. Two strains (SM91 and NS13) isolated in different cities expressed a b-lactamase with pI 5.2 (Fig.1). DNA sequencing revealed that the blaTEM genes of the last-named isolates were identical and differed from blaTEM-1b by a single G813A transition leading to a substitution Arg204Gln in the deduced amino acid sequence. Until now this mutation has not been described in TEM b-lactamases and the new enzyme containing a glutamine residue at position 204 received a designation TEM-70 (Tab.1). Comparative susceptibility testing of E.coli strains producing b-lactamases TEM-1 and TEM-70 has demonstrated that the MICs of different b-lactams, including ceftazidime, cefotaxime and inhibitor-protected penicillins, were not affected by mutation, although, the MIC of cefoperazone for E.coli C600 encoding TEM-70 was slightly high.(2 mg/ml) in comparison with that of the same strain producing TEM-1.(0.5 mg/ml). The Km and relative Vmax values were very similar for both enzymes except for the Km value of cefoperazone (143 for TEM-70 and 205 for TEM-1).

Another variant gene was identified by PCR-SSCP analysis in 3 unrelated strains (CH380, MST450 and MST813) producing a b-lactamase with pI 5.4 (Fig. 12). As determined by sequencing, the gene was associated with P3 promotor and resembled blaTEM-1a at nucleotides 175, 226, 317 but contained 4 silent mutations at positions 346, 436, 682 and 925 which were previously found in blaTEM-2 (Tab. 1). The new gene, therefore, encodes a TEM-1 penicillinase and could be designated blaTEM-1d. It may be proposed, that blaTEM-1d could result from recombination of blaTEM-1a and blaTEM-2 between positions 317 and 346. Interestingly, the SSCP profiles and sequencing data suggested that MST813 strain carried two genes (blaTEM-1c and blaTEM-1d) encoding TEM-1 penicillinase.

The nucleotide sequence data for blaTEM-70 and blaTEM-1d have been submitted to the GenBank under accession no. AF188199 and AF188200, respectively.


Table 1. Substitutions in blaTEM genes and derived penicillinases.

Nucleotide no.1 32 175 226 317 346 436 604 682 813 925 pI
Amino acid substitution2
Q39-
K
R204-
Q
blaTEM-1a C A C C A C G T G G 5.4
blaTEM-1b G T T T 5.4
blaTEM-1c T 5.4
blaTEM-2 T A G T C A 5.6
blaTEM-1d3 G T C A 5.4
blaTEM-703 G T T T A 5.2

1 Numbering is according to Sutcliffe (1978).
2 Numbering is according to Ambler et al. (1991). Q - glutamine; K - lysine; R - arginine.
3 Genes identified in this study.
pI - isoelectric point of a corresponding b-lactamase.



UPGMA clustering of ERIC-PCR fingerprints showing the distribution of mutant blaTEM genes in unrelated E.coli strains

Figure 1: UPGMA clustering of ERIC-PCR fingerprints showing the distribution of mutant blaTEM genes in unrelated E.coli strains.

1 Letters are used to encode the medical center from which the isolate was obtained.
2 Co-resistance is defined as an MIC in the resistant range by NCCLS criteria.
G - gentamycin, F - nitrofurantoin, T - trimethoprim/sulphamethoxazole, N - nalidixic acid, C - ciprofloxacin.



Selected SSCP profiles of blaTEM genes obtained with the combinations of restriction endonucleases Taq I - Pst I (A) and Taq I - Ava II (B).

Figure 2: Selected SSCP profiles of blaTEM genes obtained with the combinations of restriction endonucleases Taq I - Pst I (A) and Taq I - Ava II (B).

Lane 1: Standard profile of blaTEM-1a; Lanes 2-4: SSCP profiles corresponding to the clinical isolates E.coli MST450, E.coli CH380 and E.coli MST813, respectively.


CONCLUSIONS

Although, the sequences coding for TEM penicillinases were generally conserved among the E.coli strains used in this study, new mutant genes have been identified and even more variants may be expected in future if the appropriate genetic methods, like PSR-SSCP, will be used to screen for mutations. The identification of new parental blaTEM genes may provide further insight into the understanding of heterogeneity and evolution of TEM-type b-lactamases.



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