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Antimicrobial Agents and Chemotherapy, May 2005, p. 1872-1880, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1872-1880.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Evolution of TEM-Type Extended-Spectrum ß-Lactamases in Clinical Enterobacteriaceae Strains in Poland

Anna Baraniak, Janusz Fiett, Agnieszka Mrówka, Jaroslaw Walory, Waleria Hryniewicz, and Marek Gniadkowski*

National Institute of Public Health, Chelmska 30/34, 00-725 Warsaw, Poland

Received 28 September 2004/ Returned for modification 5 December 2004/ Accepted 12 January 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Seventeen extended-spectrum ß-lactamase (ESBL)-producing isolates of the family Enterobacteriaceae recovered from 1998 to 2000 in hospitals of five different cities in Poland were analyzed. They expressed several TEM-type ESBLs, TEM-4, TEM-29, TEM-85, TEM-86, TEM-93, and TEM-94. TEM-85 (L21F, R164S, E240K, T265M), TEM-86 (L21F, R164S, A237T, E240K, T265M), TEM-93 (M182T, G238S, E240K), and TEM-94 (L21F, E104K, M182T, G238S, T265M) were identified for the first time. Including the enzymes described earlier, TEM-47, TEM-48, TEM-49, and TEM-68, the group of known ESBLs of the TEM family produced by enterobacteria in Polish hospitals has increased to 10 variants. Comparative sequence analysis of the genes coding for all these ß-lactamases revealed a view of their possible evolution, which, apart from the gradual acquisition of various mutations, could also have involved recombination events. Two different blaTEM-1 gene alleles were precursors of the ESBL genes: blaTEM-1A, which was the ancestor of blaTEM-93, and blaTEM-1F, from which all the remaining genes originated. The evolution of the blaTEM-1F-related genes most probably consisted of three major separate lineages, one of which, including blaTEM-4, blaTEM-47, blaTEM-48, blaTEM-49, blaTEM-68, and blaTEM-94, was highly structured itself and could have been initiated by the blaTEM-25 gene, identified exclusively in France so far. Plasmid fingerprinting analysis revealed a high degree of diversity of plasmids carrying related blaTEM genes, which suggested either the intense diversification or transposition of blaTEM genes between different plasmids or some contribution of convergent evolution. The results of this study clearly demonstrate that the environment of Polish hospitals has been highly favorable for the rapid evolution of ESBLs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ß-lactamases of gram-negative rods are responsible for the most rapidly evolving mechanisms of resistance in pathogenic bacteria under the selection pressure of antibiotic use (17, 28, 30). One of the major aspects of this evolution is the accumulation of mutations, which result, for example, in modifications of the enzymes' catalytic efficiencies, substrate spectra, and susceptibilities to inhibitors (25, 30). The gradual acquisition of mutations has recently led to a dramatic increase in the number of ß-lactamase variants observed, which is well exemplified by some families of the Ambler class A enzymes (1), namely, TEM, SHV, and CTX-M (www.lahey.org/studies/webt.htm). They include the vast majority of extended-spectrum ß-lactamases (ESBLs) that are the main source of resistance of gram-negative bacteria to oxyimino-ß-lactams (8, 28). A number of reports delivered a large amount of evidence on the direct emergence of one enzyme variant from another within these families (3, 9, 11, 32); however, wider views of the possible ß-lactamase evolution in general (16) or in hospitals of a given region (13) have been proposed in only a few papers.

The TEM family of ESBLs constitutes the largest and widely disseminated group of these enzymes. Their evolutionary precursors are the TEM-1 and TEM-2 penicillinases (8, 28, 30), of which TEM-1 is encoded by a series of gene alleles, blaTEM-1A to blaTEM-1F, which differ from each other by specific silent mutations. Each of these genes could initiate a separate evolutionary lineage of mutant derivatives (27). In previous reports we described four TEM ESBLs, TEM-47, TEM-48, TEM-49, and TEM-68, which have been observed exclusively in Poland so far. The blaTEM-48 gene could be a direct precursor of the remaining ones, of which blaTEM-49 could emerge due a point mutation and blaTEM-47 could be due to crossing over between blaTEM-48 and a blaTEM-1B-type gene (19). Then, blaTEM-68 most probably arose from a single mutation in blaTEM-47 (15). It was also proposed that blaTEM-48 could have evolved by a point mutation from the blaTEM-25 gene (19), which had been identified in France (10). The specific set of silent mutations with respect to blaTEM-1A (27), modified in blaTEM-47 and blaTEM-68 by the putative crossing over, indicated that all these genes were descendants of blaTEM-1F (15, 19). The study reported here has been a continuation of efforts aimed at monitoring the spread and evolution of TEM ESBLs in enterobacteria in Polish hospitals.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Clinical isolates. The analysis was carried out with 17 ESBL-producing isolates of the family Enterobacteriaceae (Klebsiella pneumoniae, n = 9; Klebsiella oxytoca, n = 1; Escherichia coli, n = 7), recovered in five hospitals in different Polish cities (Table 1). Fourteen isolates from Gdansk, Kraków, and Suwalki were collected during a 1998 survey of ESBL types in enterobacteria in Poland (M. Gniadkowski, A. Baraniak, J. Fiett, and W. Hryniewicz, unpublished results) and were selected for the study as putative ESBL producers of the TEM family on the basis of the preliminary analysis of their ß-lactamase contents (by isoelectric focusing). The three remaining isolates, from Czestochowa and Bielsko-Biala (K. pneumoniae CZ9455/99 and CZ9459/99 and K. oxytoca BB1753/00), were identified in 1999 and 2000 and were sent to the National Institute of Public Health in Warsaw as a result of other multicenter studies on antimicrobial resistance. They were classified as possible TEM- and CTX-M-type double ESBL producers, and K. pneumoniae CZ9455/99 and CZ9459/99 were partially analyzed in previous work (4) with CTX-M-3-producing organisms in Poland. The isolates were recovered from various specimens, mostly urine, and were identified with the ATB ID32E test (bioMérieux, Charbonnieres-les-Bains, France). ESBL production was indicated by positive results of the double-disk synergy test (24).


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  		TABLE 1. Clinical isolates, ß-lactamases in the isolates and transconjugants, ESBLs identified by bioassay and DNA sequencing, and typing data

 
Ten additional enterobacterial isolates, characterized in earlier studies, were included in the analysis for comparative purposes (Table 1). The nine K. pneumoniae and one E. coli isolates, identified in 1995 and 1996 in four other Polish cities (Lódz, Rzeszów, Warsaw, and Wroclaw), produced four ESBL variants of the TEM family, TEM-47, TEM-48, TEM-49, and TEM-68 (15, 18, 19).

Antimicrobial susceptibility testing. The MICs of various ß-lactam antibiotics were evaluated by the agar dilution method according to the guidelines of the NCCLS (31). The following compounds were used: ampicillin and cefotaxime (Polfa Tarchomin, Warsaw, Poland), aztreonam (Bristol-Myers Squibb, New Brunswick, N.J.), cefoxitin (Sigma Chemical Company, St. Louis, Mo.), ceftazidime (GlaxoSmithKline, Stevenage, United Kingdom), cephalothin (Sigma Chemical Company), lithium clavulanate (GlaxoSmithKline, Betchworth, United Kingdom), imipenem (Merck Sharp & Dohme, Rahway, N.J.), and piperacillin and tazobactam (Wyeth, Pearl River, N.Y.). In all ß-lactam-inhibitor combinations, the constant concentrations of clavulanate and tazobactam were 2 and 4 µg/ml, respectively. E. coli ATCC 25922 was used as the reference strain.

Mating. The isolates were subjected to ceftazidime or cefotaxime resistance transfer experiments as described previously (19), with E. coli A15, which is resistant to rifampin, used as the recipient strain. Transconjugants were selected on MacConkey agar (Oxoid, Basingstoke, United Kingdom) supplemented with 2 µg/ml ceftazidime or cefotaxime and 128 µg/ml rifampin (Polfa Tarchomin).

IEF of ß-lactamases and bioassay for oxyiminocephalosporin-hydrolyzing activities. The ß-lactamase contents of the isolates and their transconjugants were analyzed by isoelectric focusing (IEF), as described by Bauernfeind et al. (6), with a model 111 Mini IEF Cell (Bio-Rad, Hercules, Calif.). After IEF, the cefotaxime- and ceftazidime-hydrolyzing activities were assigned to particular ß-lactamases by bioassay, as described by Bauernfeind et al. (6).

PCR detection and sequencing of blaTEM genes. Total bacterial DNA was purified with a Genomic DNA Prep Plus kit (A & A Biotechnology, Gdansk, Poland). The entire blaTEM genes were amplified by PCR with primers TEM-A and TEM-B (29) under the conditions described before (19). The amplicons were purified with the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and directly sequenced with an ABI PRISM 310 sequencer (Applied Biosystems, Foster City, Calif.). Sequencing of the whole genes was performed with primers TEM-A, TEM-B, TEM-C, TEM-D, and TEM-E, whereas only the promoter regions were sequenced with primers TEM-A and TEM-G (29).

Cloning of blaTEM genes. The blaTEM-85, blaTEM-86, blaTEM-93, and blaTEM-94 genes were amplified by PCR with primers TEM-A/EcoRI and TEM-B/BamHI, as described previously (15). The resulting amplicons were cut with EcoRI and BamHI (MBI Fermentas, Vilnius, Lithuania) and cloned into the plasmid vector pGB2 (14). E. coli DH5{alpha} transformants were selected on tryptic soy agar (Oxoid) supplemented with 2 µg/ml ceftazidime and 30 µg/ml streptomycin (Polfa Tarchomin).

RAPD and PFGE typing. Randomly amplified polymorphic DNA (RAPD) analysis was carried out separately with two primers, RAPD-7 and RAPD-1283 (33), as reported previously (19). Pulsed-field gel electrophoresis (PFGE) analysis was performed as described by Struelens et al. (35) with the XbaI restriction enzyme (MBI Fermentas) and a CHEF DRIII PFGE system (Bio-Rad). The PFGE results were interpreted as described by Tenover et al. (37).

Plasmid fingerprinting. Plasmid DNA was purified with a Plasmid Midi kit (QIAGEN), according to the recommendations of the manufacturer. For the fingerprinting analysis, plasmids were digested with the PstI restriction enzyme (MBI Fermentas) and electrophoresed in 1% agarose gels (SeaKem; Cambrex, Rockland, Maine).

Nucleotide sequence accession numbers. The blaTEM sequences analyzed in this work appear in the EMBL database under the following accession numbers: blaTEM-29, AJ277416; blaTEM-85, AJ277414; blaTEM-86, AJ277415; blaTEM-93, AJ318093; and blaTEM-94, AJ318094.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ß-Lactamases of the clinical isolates. The IEF analysis demonstrated a variety of ß-lactamases in the isolates studied (Table 1). The more common types of ß-lactamases were enzymes with a pI of 5.5, expressed by four K. pneumoniae and two E. coli isolates from the hospital in Suwalki, and ß-lactamases with a pI of 6.0, observed in extracts of all the remaining isolates. Additionally, ß-lactamases with a pI of 7.6 were produced by almost all K. pneumoniae isolates, and enzymes with pIs of 8.4 and 5.4 were expressed by K. pneumoniae CZ9455/99 and CZ9459/99 and K. oxytoca BB1753/00. The extract of E. coli SU2947/98 also contained a pI 7.4 ß-lactamase. The bioassay analysis (Table 1) revealed that only the pI 5.5, 6.0, and 8.4 ß-lactamases hydrolyzed oxyiminocephalosporins under the experimental conditions used, which indicated their ESBL activities. Whereas the pI 5.5 and 6.0 enzymes hydrolyzed both ceftazidime and cefotaxime, the pI 8.4 ß-lactamases only hydrolyzed cefotaxime. Further analysis of K. pneumoniae CZ9455/99 and CZ9459/99 revealed that the pI 8.4 enzyme was CTX-M-3 (4), and the same was found for K. oxytoca BB1753/00 (data not shown).

Mating and ß-lactamases of the transconjugants. The results for the transconjugants are shown in Table 1. Of the six isolates with pI 5.5 ESBLs, only E. coli SU3408/98 produced transconjugants, which expressed the pI 5.5 enzyme as well. Of the 11 isolates with pI 6.0 ESBLs, transconjugants were obtained for 9 isolates (including all those with the additional CTX-M-3 enzyme), and they all exclusively expressed the pI 6.0 enzymes. The results for K. pneumoniae CZ9455/99 and CZ9459/99 have been reported previously (4).

PCR detection and sequences of the blaTEM-coding regions. The total DNAs of the isolates were tested for the presence of blaTEM genes. In the case of K. pneumoniae CZ9455/99 and CZ9459/99 and K. oxytoca BB1753/00, the DNAs of their transconjugants were used, since these isolates also produced a pI 5.4 ß-lactamase, likely TEM-1. PCR with blaTEM-specific primers yielded products of about 1 kb in all cases; the results obtained for K. pneumoniae CZ9455/99 and CZ9459/99 have already been published (4).

All the amplicons were sequenced, and the results are shown in Tables 1 and 2. Analysis of the deduced amino acid sequences and comparison of the sequences with those deposited in the TEM ß-lactamase database (www.lahey.org/studies/webt.htm) revealed that the pI 5.5 TEM enzymes included three different variants, TEM-29, TEM-85, and TEM-86. TEM-29 had been identified in France before (2), whereas the last two ß-lactamases were novel TEM ESBL variants. TEM-85 was characterized by L21F, R164S, E240K, and T265M amino acid substitutions with respect to the sequence of TEM-1 (36); and TEM-86 differed from TEM-85 only by a single additional substitution, A237T. Among the pI 6.0 enzymes, four different TEM variants were found; and these were TEM-4, TEM-47, TEM-93, and TEM-94. TEM-4 and TEM-47 had originally been described in France and Poland, respectively (19, 34), but TEM-93 and TEM-94 were identified for the first time. TEM-93 carried three substitutions, M182T, G238S, and E240K, compared to the sequence of TEM-1, whereas TEM-94 was characterized by the L21F, E104K, M182T, G238S, and T265M substitutions.


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  		TABLE 2. Nucleotide sequences of blaTEM-coding regions and their promoters

 
Apart from the mutations which caused amino acid substitutions, almost all the blaTEM genes also contained silent mutations with respect to the sequence of blaTEM-1A (Table 2) (36). The full set of four such mutations that is characteristic for the blaTEM-1F gene (27), A346->G, C436->T, T682->C, and G925->A, was identified in blaTEM-4, blaTEM-29, blaTEM-85, blaTEM-86, and blaTEM-94. This indicated that of the enzyme variants that had been observed before in France, TEM-4 was encoded by an identical gene (34), whereas TEM-29 was specified by another nucleotide sequence (2). The blaTEM-47 genes were identical to those described earlier in Poland (15, 18, 19), with the silent mutations C226->T, C436->T, T682->C, and G925->A. Only the blaTEM-93 gene did not contain any silent mutations compared to the sequence of blaTEM-1A (36).

Promoter regions of the blaTEM genes. In order to identify the promoters of the blaTEM genes, the sequences located upstream from their coding regions were compared with that of the blaTEM-1A gene, which is driven by the promoter P3 (27). Moreover, the promoter sequences of the previously identified blaTEM-47, blaTEM-48, blaTEM-49, and blaTEM-68 genes were determined for 10 selected isolates from earlier work (15, 18, 19). The results are shown in Table 2. The sequences of all but one of the promoter regions differed from that of P3 only by the presence of a T instead of a C at position 32; therefore, they were identified as the double overlapping promoter Pa/Pb (27). The only exception was the promoter of blaTEM-49 from the E. coli L-867 isolate (19), whose sequence was identical to that of P3. Additionally, the sequences of the 5' regions of all blaTEM-47 and blaTEM-68 genes differed from that of blaTEM-1A by a G instead of an A at position 175.

Typing. The E. coli and K. pneumoniae isolates were typed by RAPD analysis, as were nine representative TEM-47-, TEM-48-, and TEM-68-producing K. pneumoniae isolates studied before (15, 18, 19). The results (Table 1) confirmed our previous observations of the similarity of the older TEM-47-producing K. pneumoniae isolates from Lódz, Warsaw, and Wroclaw (RAPD type F) and of the other, older TEM-47 and TEM-68 producers from Wroclaw (RAPD type G) (15, 18). The RAPD patterns of all of the newer K. pneumoniae isolates differed from those of the older ones; moreover, the only similarities among them were observed within groups from a single center. The four TEM-85- and TEM-86-producing K. pneumoniae isolates from Suwalki were indistinguishable from each other, and this was the case for the two TEM-4 (and CTX-M-3)-producing K. pneumoniae isolates from Czestochowa. Similarly, the two TEM-94-producing E. coli isolates from Gdansk were characterized by identical RAPD patterns.

The TEM-47-producing K. pneumoniae isolates from Gdansk, together with the older TEM-47-, TEM-48-, and TEM-68-producing isolates of this species, were also typed by PFGE. The results (Table 1) were in concordance with the RAPD data and the partial earlier observations (15). The clonal structure of the group of older isolates consisted of four clones. The first of these included TEM-47-producing isolates from Warsaw, Lódz, and Wroclaw (PFGE type b), whereas two others, with TEM-47 or TEM-68 or only TEM-47, were present only in Wroclaw (types a and d, respectively). The TEM-48-producing K. pneumoniae isolate from Rzeszów (19) represented a separate clone (PFGE type h). The newer TEM-47-producing isolates from Gdansk were not related to each other or to any of the clones identified before (PFGE types e, f and g).

Plasmid fingerprinting. Plasmid DNA was purified from the transconjugants or clinical isolates and subjected to fingerprinting analysis. Plasmids specific for the older isolates producing TEM-47, TEM-48, TEM-49, and TEM-68 (15, 18, 19) were also fingerprinted. The results are shown in Table 1. In general, the plasmids were highly diverse; however, some clear similarities in their PstI fingerprints could be observed. The four TEM-47-producing K. pneumoniae and E. coli isolates from Gdansk contained large plasmids that were similar to each other and to plasmids carried by the older TEM-47- and TEM-68-producing isolates from Lódz, Warsaw, and Wroclaw (fingerprints A1 to A11). Plasmids present in the three TEM-4-producing K. pneumoniae and K. oxytoca isolates from Czestochowa and Bielsko-Biala were indistinguishable from each other (fingerprint C), and this was also the case for the two TEM-94-producing E. coli isolates from Gdansk (fingerprint D), TEM-85- or TEM-86-producing K. pneumoniae and E. coli isolates from Suwalki (fingerprints E1 and E2), and two TEM-93-producing E. coli isolates from Kraków (fingerprint G). The older TEM-48-producing K. pneumoniae isolate from Rzeszów and the TEM-29-producing E. coli isolate from Suwalki possessed plasmids with unique restriction patterns.

Antimicrobial susceptibility testing of the clinical isolates and their transconjugants. The clinical isolates and their transconjugants were subjected to susceptibility testing (Table 3). All the organisms analyzed demonstrated MIC patterns that are typical for ESBL producers, with significantly elevated MICs of the majority of ß-lactams tested and the remarkable effect of inhibitors on the MICs of selected compounds. TEM-29-producing isolate E. coli SU2947/98 in general exhibited low-level resistance to ß-lactam antibiotics. TEM-85 producers were characterized by relatively low MICs of cephalothin (MICs, 16 to 64 µg/ml) compared to those of ceftazidime (MICs, 64 to >512 µg/ml). In the majority of isolates the MICs of ceftazidime were clearly higher than the cefotaxime MICs (TEM-29, TEM-47, TEM-85, TEM-86, and TEM-93 producers), and it was especially profound in the case of TEM-85-producing isolates (cefotaxime MICs, 1 to 4 µg/ml). The opposite situation was observed in TEM-4-producing K. pneumoniae CZ9455/99 and CZ9459/99 and K. oxytoca BB1753/00; however, apart from TEM-4, these isolates also expressed the cefotaxime-hydrolyzing CTX-M-3 enzyme (4). Only TEM-94 producers showed fully comparable levels of resistance to ceftazidime and cefotaxime.


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  		TABLE 3. Antimicrobial susceptibilities of the clinical isolates; their E. coli A15 transconjugants; and the TEM-85-, TEM-86-, TEM-93-, and TEM-94-producing E. coli DH5{alpha} transformants

 
In general, the MICs of the transconjugants reflected well the patterns observed in clinical isolates. Transconjugants of isolates K. pneumoniae CZ9455/99 and CZ9459/99 and K. oxytoca BB1753/00 expressed only TEM-4 and, therefore, were characterized by comparable MICs of cefotaxime and ceftazidime (MICs, 4 to 8 µg/ml).

Cloning of blaTEM genes and characterization of the E. coli transformants. In order to characterize the new TEM variants, the blaTEM-85, blaTEM-86, blaTEM-93, and blaTEM-94 genes were cloned together with their promoters and expressed in the isogenic E. coli background. The only nucleotide differences between the DNA fragments cloned were those located in the coding regions of the genes. The resulting constructs were designated pGBT-85, pGBT-86, pGBT-93, and pGBT-94, respectively. The susceptibility profiles of the transformants were characterized by evaluation of the MICs (Table 3). The TEM-85-producing strain was characterized by much higher MICs of ceftazidime and aztreonam (MICs, 256 and 128 µg/ml, respectively) than those of cefotaxime and cephalothin (MICs, 2 and 16 µg/ml, respectively). In the case of the TEM-86-producing transformant, the MICs of ceftazidime and aztreonam were significantly diminished (MICs, 64 and 8 µg/ml, respectively) and the cephalothin MIC was much increased (MIC, 256 µg/ml) compared to those for the TEM-85 producer. The resistance levels of the TEM-93- and TEM-94-producing strains were very similar to each other, and they demonstrated almost even MICs of cefotaxime, ceftazidime, and aztreonam (MICs, 2 to 8 µg/ml).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The analysis of 17 K. pneumoniae, K. oxytoca, and E. coli isolates collected from five hospitals in Poland from 1998 to 2000 has much enriched the view of the possible evolution of TEM ESBLs in Poland, as proposed in our previous studies (15, 19). They expressed seven TEM ESBL variants, of which only TEM-47 had been observed before (15, 18, 19). The other variants were TEM-4 and TEM-29, which were originally described in France (2, 34), and four novel enzymes, TEM-85, TEM-86, TEM-93, and TEM-94. The blaTEM-4 gene was identical to that identified in France (34), which suggested that it could have appeared in Poland due to importation, although one also cannot exclude the possibility that it emerged independently. On the other hand, the blaTEM-1F-specific silent mutations clearly differentiated the blaTEM-29 gene reported here from its blaTEM-1B-derived "French" counterpart (2). This indicated that blaTEM-29 emerged in Poland due to convergent evolution.

The detailed comparative analysis of the coding regions of all the blaTEM genes identified in Poland revealed a relatively compact, putative view of their evolutionary tree (Fig. 1). It could consist of two major parts that originated from blaTEM-1A and blaTEM-1F precursor genes, respectively. The blaTEM-1A branch has led to the blaTEM-93 gene, and it could have proceeded through the stepwise acquisition of three nonsynonymous mutations, with the intermediary variants not yet identified in Poland. The nine blaTEM-1F-related variants have most probably evolved along three separate branches. One of these has included only blaTEM-29, which differs from blaTEM-1F by a single mutation. The second lineage has contained two genes, blaTEM-85 and blaTEM-86, of which blaTEM-86 most likely emerged from blaTEM-85 by acquisition of a single mutation. blaTEM-85 differs from blaTEM-1F by four mutations, and none of the intermediates has been observed in Poland to date. The structure of the third hypothetical branch has been the most complex, and its view has developed gradually with time (15, 19). It may consist of the genes described earlier, blaTEM-47, blaTEM-48, blaTEM-49, and blaTEM-68, and the blaTEM-4 and blaTEM-94 genes identified in this work. The blaTEM-94 gene is a single-point mutant of blaTEM-4, which suggests direct evolution. On the other hand, blaTEM-4 differs from blaTEM-25 by only one mutation (10), which indicates that the latter gene could have been a possible precursor of blaTEM-4. As mentioned above, blaTEM-25 has not been identified in Poland to date; but it was proposed to be the ancestor of the blaTEM-47, blaTEM-48, blaTEM-49, and blaTEM-68 genes (15, 19). Therefore, it is possible that blaTEM-25 gave rise to two separate subbranches in the evolution of the blaTEM-1F-derived ESBL genes observed in Poland, which could have proceeded in part in France.



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  		FIG. 1. Possible view of the evolution of TEM ESBL variants observed in Poland so far. A part of the scheme containing the TEM-47, TEM-48, TEM-49, and TEM-68 variants has been proposed earlier (15, 19). Asterisks indicate the enzymes (TEM-24) or the enzymes encoded by genes (TEM-1 encoded by blaTEM-1A [Pa/Pb] and blaTEM-1F [Pa/Pb]) that have not yet been identified in Poland or not at all, respectively. c/o blaTEM-48 + blaTEM-1B, the hypothetical crossing-over event that could give rise to the blaTEM-47 sequence (19).

 
Analysis of the sequences located upstream from the blaTEM-coding regions has confirmed in general the evolutionary hypotheses drawn from the comparison of the coding parts. Almost all the genes were driven by the strong overlapping promoter Pa/Pb (26, 27), which suggested that the blaTEM-1A and blaTEM-1F alleles, which could have been the precursors of "Polish" blaTEM genes, were driven by Pa/Pb rather than by P3 and P4, respectively, as reported to date (27). The only exception was blaTEM-49, which was found to have the P3 promoter (26, 27). This indicated that the particular allele of this gene identified in one of our previous studies had not been a direct, one-step descendant of blaTEM-48 from the same study (19) and, for example, could have acquired its promoter due to homologous recombination.

The scheme for the evolution of the blaTEM gene indicates the existence of genetic links between bacterial isolates producing different but closely related TEM variants in terms of the similarities of their PFGE patterns and/or plasmid fingerprints. However, the only clear examples of such links were observed within a single hospital, for example, between TEM-47- and TEM-68-producing K. pneumoniae isolates in Wroclaw (15) and between K. pneumoniae with TEM-85 and TEM-86 in Suwalki. On the other hand, significant genetic diversity was observed among the isolates from different centers, exemplified mostly by the diversity of the plasmids carrying the blaTEM-48 and blaTEM-47 genes or the blaTEM-4 and blaTEM-94 genes. These data might indicate an important contribution of convergent evolution; however, blaTEM genes could have also been transposed between different plasmids and/or the plasmids could have diversified due to multiple recombination events. The center-to-center transmission of TEM ESBL-producing strains was documented only by similarities between the isolates that expressed the same enzyme variant. For example, the clonal relatedness of TEM-47-producing K. pneumoniae from Lódz, Warsaw, and Wroclaw was demonstrated in our earlier work (15, 18); and this study showed the similarities of their blaTEM-47-carrying plasmids to those present in isolates from Gdansk. Considering the dynamics of bacterial evolution under the pressure of antibiotic use, one must realize that a study of a relatively small group of isolates collected over a longer period in several hospitals usually results in only a fragmentary view of their epidemiology.

Many of the substitutions identified in the TEM enzymes analyzed here were those that significantly affect ß-lactamase activity and that confer a selective advantage to bacterial strains. First, these were the mutations responsible for ESBL activity, R164H, R164S, and G238S (25). Second, the E104K or E240K substitution, which enhances enzyme interactions with ceftazidime and aztreonam (25), was observed in all the variants except TEM-29. Third, the intragenic suppressor mutation M182T (23) was found in TEM-93 and TEM-94. Fourth, the R275L substitution, identified in TEM-68, diminishes the effects of ß-lactamase inhibitors (15, 22). Finally, the A237T mutation was observed in TEM-86, and TEM-85 and TEM-86 have been the third pair of natural TEM ß-lactamase variants that differ from each other only by this mutation. In the comparative analysis of TEM-46 and TEM-24, the stimulatory effect of A237T on their catalytic efficiencies against cephems (cephalothin and cefotaxime) and negative effect on catalytic efficiencies against penams were observed (12). In the comparison of TEM-10 and TEM-5, the clear increases in the cephalothin and cefotaxime MICs were accompanied by decreases in the MICs of amoxicillin, ceftazidime, and aztreonam; therefore, this mutation was described as modulating the activities of the enzymes against various substrates (7). In the analysis of TEM-85 and TEM-86, the A237T mutation exerted a strong positive effect on the cephalothin MIC, while decreases in resistance to piperacillin, ceftazidime, and aztreonam were observed. No significant differences in the cefotaxime MIC were found; however, the cefotaxime MIC for the TEM-85 producer was already high compared to those for the TEM-46 and TEM-10 producers (7, 12). Our data confirm the hypothesis of the modulating effect of the A237T mutation (7).

In the 1998 survey of ESBL types among the enterobacteria in Polish hospitals, ß-lactamases of the TEM family were produced by 20.1% of the isolates collected over a 4-month period. Their frequency was similar to that of the CTX-M ß-lactamases (18.8%) but far below that of the SHV-type enzymes (60.4%) (Gniadkowski et al., unpublished). The data presented in this report and in our earlier reports (3, 5, 15, 19, 20) clearly show that although TEM ß-lactamases are not the most prevalent, they have certainly been the most diversified ESBLs. Using large amounts of expanded-spectrum cephalosporins and often lacking proper infection control measures, Polish hospitals have created good selective conditions for the rapid evolution of ß-lactamase-mediated resistance.


   ACKNOWLEDGMENTS
 
We thank J. Empel for critical reading of the manuscript and J. Kedzierska, J. Kurlenda, E. Mackowiak, H. Maslanka, and M. Pilarska for providing bacterial isolates.

This work was partially financed by a grant from the Polish Committee for Scientific Research (KBN; grant 6P05A 029 21) and grant 6 PCRD LSHM-CT-2003-503-335 from the European Commission.


   FOOTNOTES
 
* Corresponding author. Mailing address: National Institute of Public Health, ul. Chelmska 30/34, 00-725 Warsaw, Poland. Phone: 48 22-851 43 88. Fax: 48 22-841 29 49. E-mail: gniadkow{at}cls.edu.pl. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, May 2005, p. 1872-1880, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1872-1880.2005
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