INTRODUCTION

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Isolated since the mid-1980s, extended-spectrum -lactamases (ESBLs) are usually encoded by plasmid-located genes and confer resistance to penicillins, cephalosporins (except cephamycins), and monobactams. -Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) block the activity of ESBLs, and this often causes ESBL-producing organisms to appear susceptible to some -lactam-inhibitor combinations (2, 24, 27). ESBLs are derivatives of broad-spectrum penicillinases, such as TEM-1 or -2 or SHV-1, and ESBL activity is determined or enhanced by mutations at several positions, i.e., 104, 164, 237, 238, and 240, within their amino acid sequences (21, 27). Extensive use of newer-generation cephalosporins has been a strong factor selecting for ESBL variants formed de novo in a given environment (8, 32, 34), promoting their further evolution (7, 15), spread in bacterial populations by means of plasmid transmission (20, 22, 31), and clonal dissemination of producer strains (16, 30, 36), including their exportation to other health care institutions (7, 14, 38).

Another group of -lactamases demonstrating inhibitor resistance (IR) activity has been isolated since the beginning of 1990s (44, 45). These enzymes confer resistance to penicillins and their combinations with -lactamase inhibitors (24, 27, 29). The majority of IR -lactamases known to date are derivatives of TEM-1 and -2 penicillinases (IRT variants), and mutations at several amino acid positions of these, i.e., 69, 130, 244, 275, and 276, were revealed or postulated to play a role in determining IR activity (11, 21, 33). A combination of ESBL- and IR-specific mutations within a single -lactamase results in the formation of a so-called complex mutant enzyme (40). Two natural variants of such -lactamases, TEM-50/CMT-1 and SHV-10, have been studied to date and were found to express either both of the activities at a moderate level or only one of these (33, 40). Here we report a novel enzyme of this kind, TEM-68/CMT-2, presented in a context of the epidemiological study of ESBL-producing klebsiellae in a hospital in Wrocaw, Poland.

	MATERIALS AND METHODS

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Bacterial strains. Twenty-four klebsiella clinical isolates (22 of Klebsiella pneumoniae and 2 of Klebsiella oxytoca) were collected in 1996 from different patients in the neonatal ward and in the Pediatric Intensive Care Unit (PICU) of the University Hospital in Wrocaw. The isolates were cultured from various specimen types, mostly from bronchial-exudate, urine, and blood samples. Clinical data concerning the isolates is presented in Table 1. Species identification (using the ID32E ATB test; bioMerieux, Charbonnieres-les-Bains, France) and preliminary susceptibility testing were performed in the hospital microbiology laboratory. All of the isolates were identified as putative ESBL producers by the double-disk test (19).

View this table: [in this window] [in a new window]   TABLE 1.   Clinical data, RAPD patterns, PFGE types, plasmid fingerprints, IEF of -lactamases, and ESBLs identified in klebsiella isolates and E. coli transconjugants

RAPD typing. Genomic DNAs of the isolates were purified with the Genomic DNA Prep Plus kit (A & A Biotechnology, Gdask, Poland). Randomly amplified polymorphic DNA (RAPD) analysis was performed using the RAPD-7 and RAPD-1283 (35) oligonucleotides as primers. PCRs were run as described previously (15).

PFGE typing. For pulsed-field gel electrophoresis (PFGE) typing, total DNA preparations embedded in 1% agarose plugs (InCert Agarose; FMC Bioproducts, Rockland, Maine) were digested with XbaI restrictase (MBI Fermentas, Vilnius, Lithuania) and separated in 1% agarose gel (Pulsed Field-Certified; Bio-Rad, Hercules, Calif.) using a CHEF DRII PFGE system (Bio-Rad). The procedure was performed as described by Struelens et al. (41), and results were interpreted in accordance with the criteria proposed by Tenover et al. (43).

Plasmid DNA fingerprinting. Plasmid DNA was purified from bacterial cells by the alkaline lysis method (6) using the QIAGEN Plasmid Midi Kit (QIAGEN, Hilden, Germany) as previously described (4). For fingerprinting analysis, plasmid DNA was digested with the restriction enzyme PstI (MBI Fermentas) and electrophoresed in 1% agarose gels (Sigma Chemical Company, St. Louis, Mo.).

Antimicrobial susceptibility testing. The MICs of various antibiotics were determined by the agar dilution method in accordance with National Committee for Clinical Laboratory Standards (NCCLS) guidelines (28). The antibiotics used were ampicillin, cefotaxime, and gentamicin from Polfa, Tarchomin, Poland; amikacin and aztreonam from Bristol-Myers Squibb, New Brunswick, N.J.; cefoxitin from Sigma Chemical Company; ceftazidime from Glaxo Wellcome, Stevenage, United Kingdom; lithium clavulanate from SmithKline Beecham Pharmaceuticals, Betchworth, United Kingdom; imipenem from Merck, Sharp & Dohme Research, Rahway, N.J.; and piperacillin and tazobactam from Wyeth Ayerst Laboratories and Lederle Laboratories, respectively, Pearl River, N.Y. In all -lactam-inhibitor combinations, the constant concentrations of clavulanate and tazobactam were 2 and 4 µg/ml, respectively.

Resistance transfer. Ceftazidime resistance transfer was carried out as previously described (15). Transconjugants were selected on MacConkey agar (Oxoid, Basingstoke, United Kingdom) containing rifampin (128 µg/ml; Polfa) and ceftazidime (2 µg/ml).

IEF of -lactamases. Supernatants of bacterial sonicates (3) were subjected to isoelectric focusing (IEF) in accordance with the procedure by Matthew et al. (26) with modifications (3), using a model 111 Mini IEF Cell (Bio-Rad). Following IEF, -lactamase bands were visualized by staining gels with nitrocefin (Oxoid).

Bioassays for detection of ESBL activity. Following IEF, the ceftazidime- and cefotaxime-hydrolyzing activities were assigned to particular -lactamase bands by the bioassay approach described by Bauernfeind et al. (3). The concentration of both cephalosporins used in the experiment was 2 µg/ml.

PCR detection of bla_TEM and bla_SHV genes. Total DNA extracted from the isolates was used in specific PCRs for the detection of bla_TEM and bla_SHV genes. Primers TEM-A and TEM-B (25) were used for amplification of entire bla_TEM genes; primers SHV-A and SHV-C (15, 31) were used for partial amplification of bla_SHV genes. Primer SHV-C was designed to specifically amplify genes encoding SHV -lactamases with the Gly238Ser and Glu240Lys substitutions (31). PCRs were run as described previously (15).

Sequencing of bla_TEM- and bla_SHV-specific PCR products. PCR products containing the amplified bla_TEM and bla_SHV genes were purified with a QIAquick PCR Purification Kit (QIAGEN) and subjected to direct sequencing reactions (37) using an ABI PRISM 310 automatic sequencer (PE Biosystems, Foster City, Calif.). Primers TEM-A, TEM-B, TEM-C, TEM-D, and TEM-E (25) were used for sequencing of bla_TEM genes. The complete bla_SHV gene was amplified with primers SHV-D (5'-CTCAAGGATGTATTG-3') and SHV-H (5'-TTAGCGTTGCCAGTGC-3') and plasmid purified from a transconjugant strain as the template. Primers SHV-A (15), SHV-D, SHV-F (5'-TCTGGTGGACTACTC-3'), SHV-G (5'-GTTGTCGCCCATCTG-3'), and SHV-H were used for sequencing.

Cloning of bla_TEM-47 and bla_TEM-68 genes. The bla_TEM-47 and bla_TEM-68 genes were amplified together with their promoter regions using primers TEM-A/EcoRI and TEM-B/BamHI. These primers are modified versions of primers TEM-A and TEM-B (25) with the respective restriction sites added on their 5' ends. The resulting products were cut with EcoRI and BamHI and cloned into vector pGB2, which is a low-copy plasmid containing the spectinomycin-streptomycin resistance gene as a transformation marker (13). E. coli DH5 transformants were selected on tryptic soy agar (Oxoid) supplemented with streptomycin (Polfa) at 30 µg/ml and ceftazidime at 2 µg/ml. The resulting pGB2 derivatives containing the bla_TEM-47 and bla_TEM-68 genes were designated pGBT-47 and pGBT-68, respectively.

Determination of IC₅₀s. Supernatants of sonicates (3) of the E. coli DH5 transformants producing TEM-47 and TEM-68 were used for comparative determination of the inhibitor concentrations that reduced -lactamase activity by 50% (IC₅₀s). IC₅₀ evaluation was performed as described by Bush et al. (10). Aliquots of extracts containing about 60 µg of protein were used in reactions run in a volume of 575 µl at room temperature in a DU 640 spectrophotometer (Beckman Instruments, Fullerton, Calif.).

Nucleotide sequence accession number. The nucleotide sequence of the bla_TEM-68 gene will appear in the EMBL database under accession number AJ239002.

	RESULTS

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Typing of ESBL-producing klebsiellae from the hospital in Wrocaw. Results of PFGE and RAPD typing are shown in Table 1. PFGE analysis distinguished four different types of K. pneumoniae isolates with the most prevalent PFGE types, a and b, grouping nine and eight isolates, respectively. The remaining PFGE types, c and d, represented two and three isolates, respectively. Only the type b isolates could be further classified into subtypes b1 and b2 by a single DNA band difference in their PFGE patterns. The two K. oxytoca isolates were found to be indistinguishable in the PFGE analysis. Results of RAPD typing were very similar, with isolates producing identical or nearly identical banding patterns forming clusters, which correlated well with the distribution of PFGE types.

Resistance transfer. All of the clinical isolates were subjected to the ceftazidime-resistance transfer experiment. The majority produced transconjugants, with the exception of the K. pneumoniae isolates of PFGE subtype b2 and type c and the single isolate (3151/98) of PFGE type a.

-Lactamase contents of clinical isolates and their transconjugants and identification of ESBLs. Protein extracts of all of the clinical isolates and transconjugants were separated by IEF in order to reveal their -lactamase content. Results of the analysis are shown in Table 1. Each isolate of K. pneumoniae was found to express a -lactamase with a pI of 7.6 together with another enzyme with a pI of 5.7, 6.0, or 8.2. The pI 5.7 -lactamase was produced by the single isolate, 3151/98, belonging to PFGE type a. The pI 6.0 enzymes were expressed by all of the remaining type a isolates, two isolates of PFGE subtype b2, and all isolates of type d. The pI 8.2 -lactamases were predominant among isolates of PFGE type b (subtypes b1 and b2) and were also produced by the PFGE type c isolates and both isolates of K. oxytoca. Enzymes with a pI of 6.0 or 8.2 were identified in extracts of all of the corresponding transconjugants (Table 1). The bioassay experiment revealed that only the -lactamases with a pI of 5.7, 6.0, or 8.2 were able to hydrolyze ceftazidime and cefotaxime under the conditions used (Table 1), and so these enzymes demonstrated ESBL activity.

Plasmid fingerprinting. Results of plasmid fingerprinting analysis are presented in Table 1. All isolates producing the pI 5.7 or 6.0 ESBLs contained high-molecular-weight (high-MW) plasmids with similar PstI fingerprints, designated A1 to A4. Fingerprints A1, A2, and A3 characterized the pI 6.0 ESBLs producers, and their distribution correlated fully with the distribution of PFGE types a, b2, and d, respectively. The plasmid with fingerprint A4 was found in K. pneumoniae isolate 3151/98, which expressed the pI 5.7 enzyme and was smaller than other type A molecules. The PFGE type b (b1 and b2) K. pneumoniae and the K. oxytoca isolates producing the pI 8.2 ESBLs carried large plasmids of the same fingerprint, B1, which in several cases were copurified with molecules with lower MWs. The remaining two pI 8.2 ESBL-producing K. pneumoniae isolates of PFGE type c contained high-MW plasmids with a very similar fingerprint, B2.

PCR detection and sequencing of ESBL-encoding genes. Specific PCRs were run in order to identify the ESBL types produced by the clinical isolates studied. Results of the analysis are presented in Table 1. Total DNA preparations of the isolates expressing the pI 5.7 or 6.0 ESBLs were tested for the presence of bla_TEM genes. Amplification products of the expected size of about 1 kb were obtained with primers TEM-A and TEM-B (25) for all of these isolates. Detection of bla_SHV genes encoding SHV -lactamases that contain the Gly238Ser and Glu240Lys ESBL-specific substitutions was carried out on DNAs purified from isolates producing the pI 8.2 enzymes. PCR products of the expected size of about 220 bp were identified for all of the isolates in this group.

Comparative typing and plasmid fingerprinting of TEM-47-producing K. pneumoniae isolates from different hospitals. The group of representative K. pneumoniae TEM-47-producing isolates from the hospitals in ód (15) and Warsaw (14) were typed along with the TEM-47 producers from the Wrocaw hospital. Results of PFGE and RAPD analyses are shown in Table 1. Wrocaw isolates of PFGE subtype b2 were found to be indistinguishable by both approaches from the L-267 strain from ód, in which the TEM-47 enzyme was originally identified, and closely related to the set of TEM-47-expressing isolates from Warsaw. Plasmids purified from the ód and Warsaw isolates produced PstI fingerprints very similar to the type A molecules carrying the bla_TEM-47 genes in isolates from Wrocaw (Table 1).

Antimicrobial susceptibility testing of clinical isolates and transconjugants strains. Table 3 shows the antimicrobial susceptibility data presented with regard to the ESBL types and the PFGE types of the isolates. Increased MICs of the majority of -lactams tested characterized all of the clinical isolates; however, K. pneumoniae isolates demonstrated a remarkably higher level of resistance than K. oxytoca isolates. MICs of ceftazidime and aztreonam were significantly higher than those of cefotaxime. For some of the K. pneumoniae isolates, the MICs of cefoxitin were elevated (16 to 32 µg/ml) and all of the isolates were fully susceptible to imipenem. For TEM-68-producing K. pneumoniae strain 3151/98, the MICs of inhibitor combinations (e.g., piperacillin-tazobactam, >512 µg/ml; ceftazidime-clavulanate, 128 µg/ml) were very high; in all other cases, -lactamase inhibitors efficiently restored the activity of -lactam antibiotics. Susceptibility patterns of transconjugants correlated with the data obtained for the corresponding clinical isolates.

Comparative analysis of TEM-47 and TEM-68 activities. In order to compare activities of TEM-47 and TEM-68, genes coding for both -lactamases were cloned and expressed in an isogenic system. The cloned DNA fragments containing the bla_TEM-47 and bla_TEM-68 genes together with their promoters differed from each other only by a single mutation specifying the Arg275Leu substitution in the TEM-68 enzyme. The pGB2 plasmid, which was used as a vector, does not contain any other -lactamase-encoding gene.

	DISCUSSION

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Twenty-four ESBL-producing klebsiella isolates were collected in 1996 from patients in two wards of the University Hospital in Wrocaw. Molecular typing has revealed a remarkable clonal diversity within the group, with four distinct clusters of related K. pneumoniae isolates and one of K. oxytoca. The most prevalent were K. pneumoniae isolates of PFGE types a and b, and these most probably represented two strains with relatively high epidemic potential, clonally spread in the neonatal ward and the PICU, respectively.

At least three different ESBLs of the TEM (pI, 5.7 or 6.0) and SHV (pI, 8.2) families were produced by the isolates. The pI 6.0 -lactamases were identified in 13 K. pneumoniae isolates of three different PFGE types (a, b2, and d), all of which contained plasmids with very similar restriction patterns (type A plasmids). Sequencing of bla_TEM genes amplified from representative isolates revealed that these were bla_TEM-47 genes with the same sequence. An analogous situation was observed in the case of isolates producing the pI 8.2 SHV ESBLs. These enzymes were identified in eight K. pneumoniae isolates differing by PFGE (b1, b2, and c) and in the isolates of K. oxytoca, all with large plasmids of the same or very similar fingerprints (type B plasmids). PCR with the use of specifically designed primers revealed that all of the SHV enzymes contained the Gly238Ser and Glu240Lys ESBL-specific substitutions; sequencing of the bla_SHV gene from a single isolate demonstrated that it codes for SHV-5. It is likely that all of the pI 6.0 and 8.2 ESBLs analyzed were TEM-47 and SHV-5, respectively, and that genes coding for both kinds of enzymes were disseminated among nonrelated klebsiellae by parallel plasmid transfer events.

Identification of TEM-47-producing K. pneumoniae isolates in the hospital in Wrocaw raised the question of their possible relatedness to the K. pneumoniae clinical isolates expressing the same enzyme and recovered in pediatric hospitals in ód in 1995 (15) and in Warsaw in 1996 (14). The comparative typing of isolates representing all of the identified PFGE types and subtypes of TEM-47 producers from the three institutions revealed that the fraction of isolates from Wrocaw (subtype b2) was very closely related to isolates from the other cities. This data indicated a very probable transfer of the epidemic K. pneumoniae strain between the pediatric centers, leading to its spread over the country.

A novel variant of a TEM -lactamase, TEM-68 (pI 5.7), was identified in the single K. pneumoniae isolate 3151/98 recovered from a patient in the neonatal ward. This isolate was indistinguishable by PFGE and RAPD from the PFGE type a TEM-47-producing isolates. The bla_TEM-68 gene, together with its promoter region, differs by only a single point mutation from bla_TEM-47 and was carried by the nonconjugative plasmid (fingerprint A4), which very likely has emerged by recombination from the transferable type A1 molecule. All of this data indicated that the bla_TEM-68 gene has most probably evolved from bla_TEM-47 in the genetic background of the PFGE type a K. pneumoniae strain. This finding has extended our view of TEM-2-related ESBL evolution in Poland (Table 2). In the previous study, it was postulated that genes coding for TEM-47 and TEM-49 have emerged independently by single genetic events from the TEM-48-encoding sequence (15).

TEM-68 represents a new variant of complex mutant -lactamases which combine ESBL-specific mutations with those determining IR activity. Two -lactamases of this kind have been identified in clinical isolates to date, and these were TEM-50 (40) and SHV-10 (33). Two ESBL-specific mutations, Glu104Lys and Gly238Ser, and two IR-type substitutions, Met69Leu and Asn276Asp, are known to characterize the TEM-50 enzyme. The SHV-10 -lactamase combines the Gly238Ser and Glu240Lys (both ESBL-type) substitutions and the Ser130Gly (IR-specific) substitution. Activities of the complex mutant -lactamases were compared with those of the corresponding ESBL and IR enzymes. It was shown that the SHV-10 -lactamase manifested increased resistance to inhibitors and no ESBL activity compared to SHV-9 (33). On the other hand, the TEM-50 enzyme retained both activities; however, the ESBL activity was found to be weaker than that of the TEM-15 -lactamase and the IR activity was reduced compared with that of TEM-35/IRT-4 (40).

TEM-68 contains the Gly238Ser and Glu240Lys ESBL-type substitutions and the Arg275Leu mutation, which up to now has been identified once, in the TEM-38/IRT-9 -lactamase (18). Another substitution at this site, the Arg275Gln mutation, was found in the TEM-45/IRT-14 enzyme (11). Both TEM-38 and TEM-45 are enzymes with strong IR activity; however, the Arg275 mutations are accompanied by well-characterized IR-type Met69 substitutions (21) within their sequences. The Arg275 substitutions have never been observed separately until now, and so their contribution to the IR phenotype, even if postulated, was not clear (11, 18). Comparative analysis of the TEM-47 and TEM-68 -lactamases, which differ only by the Arg275Leu substitution and are expressed at the same level in an isogenic background, has provided an opportunity to study the influence of this substitution on -lactamase activity.

Compared with the PFGE type a TEM-47 producers (e.g., isolate 3144/98), for the TEM-68-expressing K. pneumoniae 3151/98 isolate, the MICs of penicillins, ceftazidime, and aztreonam were similarly high and the MICs of the inhibitor combinations studied were much increased (Table 4). For the TEM-68-producing isolate, the MIC of cefoxitin was also significantly higher than for the TEM-47-expressing isolate. Since both of these isolates belonged to the same PFGE and RAPD types, it could be suggested that the differences in the MICs of -lactams were due mostly to the diverse activities of their -lactamases. In order to check this hypothesis, the bla_TEM-47 and bla_TEM-68 genes were cloned together with their original, identical promoters in the E. coli laboratory strain. The resulting transformants were characterized by MIC evaluation, and protein extracts of the recombinant strains were used for determination of IC₅₀s for the TEM-47 and TEM-68 enzymes. Surprisingly, for the TEM-47- and TEM-68-producing E. coli transformants, the MICs of all of the -lactams tested were identical, including inhibitor combinations in which clavulanate and tazobactam were at fixed routine concentrations of 2 and 4 µg/ml, respectively (Table 4). The inhibitors efficiently reduced the MICs of piperacillin, cefotaxime, ceftazidime, and aztreonam. Nevertheless, when inhibitor concentrations were reduced, remarkable differences between TEM-47 and TEM-68 recombinant producers were revealed, demonstrating the IR activity of TEM-68, which affected the inhibition by tazobactam more than that by clavulanate (Table 5). Both effects were reflected by kinetic data; the IC₅₀s of the inhibitors were significantly higher for TEM-68 than for TEM-47, and this difference was more explicit in the case of tazobactam (about 10 times) than in that of clavulanate (about 5 times). All of the results indicated that TEM-68 is a complex mutant -lactamase which, similar to the TEM-50 enzyme, combines ESBL and IR activities. In contrast to TEM-50, its ESBL activity is high and fully comparable to that of TEM-47, the corresponding ESBL variant. The IR activity of TEM-68 is determined by the Arg275Leu mutation alone and is relatively weak since this could be demonstrated only at reduced concentrations of inhibitors in the E. coli laboratory strain. However, the effect of the IR activity was strongly enhanced when the enzyme was produced by the original wild-type strain of K. pneumoniae, which was most probably of lower permeability for antibiotics than the E. coli laboratory strain. It is possible that the permeability of the TEM-68-producing isolate, 3151/98, was additionally reduced by a mutation, as suggested by the raised MIC of cefoxitin. The ESBL and IR activities of TEM-68, when expressed in the context of the relatively low permeability of K. pneumoniae cells, together conferred on the clinical isolate a remarkably high level of resistance to a wide spectrum of -lactam antibiotics, including combinations of piperacillin with tazobactam and oxyimino -lactams with clavulanate. TEM-68-producing isolate 3151/98 is the first reported strain of K. pneumoniae expressing a complex mutant -lactamase and one of the very few examples of non-E. coli isolates producing a class A -lactamase with IR activity (9, 23, 39).

This work allowed us to document several concurrent epidemiological phenomena concerning ESBL-mediated resistance in a single medical center. Five different ESBL-expressing klebsiella strains were clonally disseminated at the same time. They produced at least three different ESBL variants, and two of these were spread due to plasmid transfer among the nonrelated strains. ESBL-producing strains must have been transmitted between the two wards and were undergoing evolutionary diversification. Two different plasmids carrying different ESBL genes occurred alternatively in cells of a single epidemic strain, and one of these strain variants was spread to other hospitals located in distant cities. The ongoing evolution of ESBLs has led to a new complex mutant enzyme conferring resistance to a very wide variety of -lactam antibiotics, including inhibitor combinations.