Beta-lactamase characterization in Escherichia coli isolates with diminished susceptibility or resistance to extended-spectrum cephalosporins recovered from sick animals in Spain

A total of 1439 Escherichia coli isolates from sick animals were received from the Spanish Network of Veterinary Antimicrobial Resistance Surveillance (VAV) from 1997 to 2001. Antimicrobial susceptibility tests were performed and diminished susceptibility to cefotaxime and ceftazidime was identified in 2.5% and 2.8% of the isolates, respectively. Beta-lactamase characterization was carried out in the group of 20 E. coli isolates with both characteristics. The MIC ranges of different beta-lactams showed by these 20 isolates were as follows (in microg/ml): ampicillin (64-->256), amoxicillin-clavulanic acid (4-64), ticarcillin (8-->128), cefazolin (32-->256), cefoxitin (4-->128), cefotaxime (1-64), ceftazidime (2-->64), ceftriaxone (0.5-64), imipenem (< or = 0.06-0.25), and aztreonam (2-->32). TEM, SHV, CMY, and FOX beta-lactamase genes were analyzed by PCR and sequencing. The beta-lactamase genes detected were the following ones (number of isolates): bla(TEM-1b) (3), bla(TEM-1a) (1), bla(TEM-30f) (2), bla(TEM-1b) + bla(CMY-2) (2), and bla(SHV-12) (1). Sequences of the promoter and/or attenuator region of the chromosomal ampC gene were studied in all the 20 isolates. Mutations at position -42 or -32 were detected in 16 isolates and these mutations were associated with the presence of a TEM type beta-lactamase in 6 isolates. Besides, a high variety of plasmidic beta-lactamases was detected including TEM-30 and CMY-2. To our knowledge, this is the first time that TEM-30 beta-lactamase has been detected in E. coli isolates of animal origin.     

Molecular characterisation of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. isolates at a tertiary-care centre in Lebanon.

The prevalence of bla CTX-M, bla TEM and bla SHV genes among extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Escherichia coli (n = 50) and Klebsiella spp. (n = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla CTX-M-15, bla TEM-1, bla OXA-1 and six bla SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants.     

Occurrence of extended-spectrum beta-lactamases in members of the genus Shigella in the Republic of Korea

A nationwide survey was carried out in Korea to assess the prevalence of Shigella strains producing extended-spectrum beta-lactamases (ESBLs). From 1991 to 2002, 5,911 clinical strains were isolated and screened for resistance to extended-spectrum cephalosporins. Twenty of the Shigella isolates were ESBL positive, based on the synergistic effects between clavulanate and selected beta-lactams (ceftazidime and cefotaxime). Nucleotide sequence analysis of these isolates revealed that they harbored bla(TEM-19) (eight isolates), bla(TEM-15) (five isolates), bla(TEM-52) (six isolates), bla(TEM-17) (one isolate), bla(TEM-20) (one isolate), and bla(CTX-M-14) (three isolates). All the ESBL-encoding genes in this study were carried in conjugable plasmids. Thus, TEM-19, TEM-15, TEM-52, and CTX-M-14 beta-lactamases can be considered common Korean ESBL types in Shigella sonnei and are probably transmitted through interspecies spread between medical facilities and the community in Korea. This is the first report of the presence of TEM-17, TEM-19, and TEM-20 in Korea and in S. sonnei.     

Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark

The genetic background for beta-lactamase-mediated resistance to beta-lactam antibiotics was examined by PCR and sequencing in 160 ampicillin-resistant isolates (109 Escherichia coli and 51 Salmonella) obtained from healthy and diseased food animals in Denmark. Sequencing revealed three different variants of bla (TEM-1), of which bla (TEM-1b) was the most frequently detected (80 E. coli and 47 Salmonella), followed by bla (TEM-1a) (eight E. coli, one Salmonella) and bla (TEM-1c) (seven E. coli). A few isolates were found to express OXA, TEM-30, or PSE beta-lactamases. Mutations in the ampC promoter leading to increased production of the AmpC beta-lactamase were demonstrated in 11 cefoxitin-resistant or intermediate E. coli isolates. Nine of these isolates did not contain any bla (TEM) genes, whereas the remaining two did. No genes encoding SHV or extended-spectrum beta-lactamases were detected. Two new variants of bla (TEM) were detected, which have been designated bla (TEM-127) and bla (TEM-128). In TEM-127, amino acid 158 is substituted from His to Asn, whereas a substitution from Asp to Glu is seen at amino acid 157 in TEM-128. According to MIC determinations, these novel enzymes do not possess activity against extended-spectrum beta-lactams.     

Characterization of clinical isolates of Enterobacteriaceae from Italy by the BD Phoenix extended-spectrum beta-lactamase detection method

Production of extended-spectrum beta-lactamases (ESBLs) is an important mechanism of beta-lactam resistance in Enterobacteriaceae: Identification of ESBLs based on phenotypic tests is the strategy most commonly used in clinical microbiology laboratories. The Phoenix ESBL test (BD Diagnostic Systems, Sparks, Md.) is a recently developed automated system for detection of ESBL-producing gram-negative bacteria. An algorithm based on phenotypic responses to a panel of cephalosporins (ceftazidime plus clavulanic acid, ceftazidime, cefotaxime plus clavulanic acid, cefpodoxime, and ceftriaxone plus clavulanic acid) was used to test 510 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Providencia stuartii, Morganella morganii, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Citrobacter freundii, and Citrobacter koseri. Of these isolates, 319 were identified as ESBL producers, and the remaining 191 were identified as non-ESBL producers based on the results of current phenotypic tests. Combined use of isoelectric focusing, PCR, and/or DNA sequencing demonstrated that 288 isolates possessed bla(TEM-1)- and/or bla(SHV-1)-derived genes, and 28 had a bla(CTX-M) gene. Among the 191 non-ESBL-producing isolates, 77 isolates produced an AmpC-type enzyme, 110 isolates possessed TEM-1, TEM-2, or SHV-1 beta-lactamases, and the remaining four isolates (all K. oxytoca strains) hyperproduced K1 chromosomal beta-lactamase. The Phoenix ESBL test system gave positive results for all the 319 ESBL-producing isolates and also for two of the four K1-hyperproducing isolates of K. oxytoca. Compared with the phenotypic tests and molecular analyses, the Phoenix system displayed 100% sensitivity and 98.9% specificity. These findings suggest that the Phoenix ESBL test can be a rapid and reliable method for laboratory detection of ESBL resistance in gram-negative bacteria.     

Variety of TEM-, SHV-, and CTX-M-type beta-lactamases present in recent clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae from Taiwan

A total of 171 hospitals' isolates of E. coli, K. pneumoniae, and E. cloacae with a minimum inhibitory concentration (MIC) of > or =2 microg/ml for ceftazidime or cefotaxime were evaluated for the production of beta-lactamases. PCR amplification with specific primers for the bla (SHV), bla (TEM), and bla (CTX) genes revealed that a total of 53, 81, and 43 of these genes were amplified, respectively. Sequencing results confirmed that TEM-1, CTX-M-3 and -14, SHV-1, -5, -11, -12, and -33, OXY-1a, and LEN-1 were presented among these isolates. No specific large cluster of isolates carried the same beta-lactamases, indicating the wide diversity of the collected strains. Plasmid spread between E. coli and K. pneumoniae was identified in few isolates. Combinations of TEM, SHV, and CTX beta-lactamase genes, including extended-spectrum beta-lactamase genes, were observed in all three species.     

Development of high-level ceftazidime resistance via single-base substitutions of blaCTX-M-3 in hyper-mutable Escherichia coli

Mutations can increase the ceftazidimase activity of CTX-M-3 beta-lactamase, as seen with its widespread variant CTX-M-15. This study compared the frequencies of emerging ceftazidime resistance in isogenic wild-type and hyper-mutable mutS CTX-M-3-producing Escherichia coli strains, and sequenced the mutant bla(CTX-M) alleles selected. Ceftazidime resistance emerged more readily in the hyper-mutable background than in the wild-type strain. All selected CTX-M mutants, in both the wild-type and the mutS derivatives, had single amino-acid changes at position 167, including a novel Pro167Gln substitution. These data emphasise the potential for further diversification of CTX-M enzymes.     

Emergence of extended-spectrum beta-lactamases in Pseudomonas aeruginosa: about 24 cases at Rouen University Hospital

AIM OF THE STUDY: This retrospective study was conducted in order to characterize mechanisms of resistance to beta-lactams and clonality of 24 isolates of Pseudomonas aeruginosa. These strains were isolated from patients hospitalized in Rouen University Hospital between August 2004 and December 2006 and were resistant to cefepime and/or ceftazidime (PMR) by a mechanism not only related to overproduction of the cephalosporinase. PATIENTS AND METHODS: Clinical strains of PMR were characterized by conventional biochemical methods, antibiotic susceptibility testing by disk diffusion in agar with or without cloxacillin and RAPD for genetic comparison. Identification of beta-lactamase was performed by PCR amplification followed by sequencing of bla genes. RESULTS: All strains produced the extended-spectrum beta-lactamase (ESBL) TEM-116. Epidemiological study identified eight unrelated strains, eight related strains originating from a single unit, three related strains isolated in different wards and five related strains coproducing TEM-116 and SHV-2a but isolated in different units. Detection of ESBL in these strains was difficult due to a low level of ESBL production. CONCLUSION: This is the first report of TEM-116 in France in 24 strains of P. aeruginosa and its association with SHV-2a in five cases. SHV-2a has been described in P. aeruginosa in France but not TEM-116 which was recently reported in this species, in China and in Netherlands (in a strain coproducing SHV-12). ESBL detection in PMR remains indispensable since these strains can cause therapeutic failures.     

Identification of clinical isolates of indole-positive Klebsiella spp., including Klebsiella planticola, and a genetic and molecular analysis of their beta-lactamases.

In a collection of 43 indole-positive Klebsiella clinical isolates, which were initially identified as Klebsiella oxytoca, there were 18 isolates which exhibited a pattern characteristic of extended-spectrum beta-lactamase (ESBL) resistance. This study aimed to confirm their identity by biochemical tests and by PCR and to determine the genetic basis for their resistance to the beta-lactams and broad-spectrum cephalosporins. Chromosomal beta-lactamase genes were analyzed by PCR, and plasmid-mediated beta-lactamase genes were analyzed by conjugation and transformation. There were 39 isolates which grew on melezitose but failed to grow on 3-hydroxybutyrate, confirming them as K. oxytoca. PCR analysis of their beta-lactamase genes divided these isolates into two groups, the bla(OXY-1) group and the bla(OXY-2) group. Each group had beta-lactamases with different isoelectric points; the bla(OXY-1) group had beta-lactamases with isoelectric points at 7.2, 7.8, 8.2, and 8.8, and the more common bla(OXY-2) group had beta-lactamases with pIs at 5.2, 5.4 (TEM-1), 5.7, 5.9, 6.4, and 6.8. A pI of 5.2 was the most frequently detected and accounted for 59% of all the bla(OXY-2) beta-lactamases. Hyperproduction of clavulanate-inhibited chromosomal beta-lactamases was detected in 17 K. oxytoca isolates, resulting in an ESBL phenotype. K. oxytoca isolates having a plasmid-mediated genetic basis for their ESBL phenotype were not found, confirming that, in K. oxytoca, plasmids are rarely involved in conferring resistance to the newer cephalosporins. Four isolates proved to be isolates of K. planticola in which the beta-lactamase genes failed to react with the primers used in the PCR. One K. planticola isolate contained a transferable plasmid harboring the SHV-5 beta-lactamase gene and showed an ESBL phenotype, while the other non-ESBL K. planticola isolates contained chromosomal beta-lactamases with isoelectric points at 7.2, 7.7, and 7.9 plus 7.2.     

Effects of phenotype and genotype on methods for detection of extended-spectrum-beta-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway

Consecutive clinical isolates of Escherichia coli (n = 87) and Klebsiella pneumoniae (n = 25) with reduced susceptibilities to oxyimino-cephalosporins (MICs > 1 mg/liter) from 18 Norwegian laboratories during March through October 2003 were examined for bla(TEM/SHV/CTX-M) extended-spectrum-beta-lactamase (ESBL) genes, oxyimino-cephalosporin MIC profiles, ESBL phenotypes (determined by the ESBL Etest and the combined disk and double-disk synergy [DDS] methods), and susceptibility to non-beta-lactam antibiotics. Multidrug-resistant CTX-M-15-like (n = 23) and CTX-M-9-like (n = 15) ESBLs dominated among the 50 ESBL-positive E. coli isolates. SHV-5-like (n = 9) and SHV-2-like (n = 4) ESBLs were the most prevalent in 19 ESBL-positive K. pneumoniae isolates. Discrepant ESBL phenotype test results were observed for one major (CTX-M-9) and several minor (TEM-128 and SHV-2/-28) ESBL groups and in SHV-1/-11-hyperproducing isolates. Negative or borderline ESBL results were observed when low-MIC oxyimino-cephalosporin substrates were used to detect clavulanic acid (CLA) synergy. CLA synergy was detected by the ESBL Etest and the DDS method but not by the combined disk method in SHV-1/-11-hyperproducing strains. The DDS method revealed unexplained CLA synergy in combination with aztreonam and cefpirome in three E. coli strains. The relatively high proportion of ESBL-producing E. coli organisms with a low ceftazidime MIC in Norway emphasizes that cefpodoxime alone or both cefotaxime and ceftazidime should be used as substrates for ESBL detection.     

Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR

We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants.     

Characterization of the first extended-spectrum beta-lactamase-producing Salmonella isolate identified in Canada

A single Salmonella enterica serovar Typhimurium isolate with an UT2 phage type producing an extended-spectrum beta-lactamase (ESBL) was identified in Canada in 2000. The isolate harbored two plasmids, one containing a bla(TEM-1) gene and the other containing a bla(SHV-2a) gene. The ESBL gene was located on a 70-kb transferable plasmid which also carried tetracycline and trimethoprim resistance elements.     

Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE

To determine whether confirmatory tests for extended-spectrum beta-lactamase (ESBL) production in Escherichia coli are necessary, we selected 131 E. coli isolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was >/=2 micro g/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by >/=3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by >/=5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genes bla(TEM), bla(SHV), and bla(OXA), and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one beta-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC = 2 to 4 micro g/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 micro g/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC beta-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131 E. coli isolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to >/=8 micro g/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents.     

Effects of inoculum and beta-lactamase activity in AmpC- and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using NCCLS ESBL methodology

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum beta-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing beta-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no beta-lactamases gave consistent MIC results with inocula of 10(5) and 10(6) CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total beta-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.     

Prevalence and characterization of multidrug-resistant and extended-spectrum beta-lactamase-producing Escherichia coli from pediatric wards of a Malaysian hospital

The emergence of Escherichia coli resistant to extended-spectrum cephalosporins (ESCs) is of concern as ESC is often used to treat infections by Gram-negative bacteria. One-hundred and ten E. coli strains isolated in 2009-2010 from children warded in a Malaysian tertiary hospital were analyzed for their antibiograms, carriage of extended-spectrum beta-lactamase (ESBL) and AmpC genes, possible inclusion of the beta-lactamase genes on an integron platform, and their genetic relatedness. All E. coli strains were sensitive to carbapenems. About 46% of strains were multidrug resistant (MDR; i.e., resistant to ≥3 antibiotic classes) and almost half (45%) were nonsusceptible to ESCs. Among the MDR strains, high resistance rates were observed for ampicillin (98%), tetracycline (75%), and trimethoprim/sulfamethoxazole (73%). Out of 110 strains, bla(TEM-1) (49.1%), bla(CTX-M) (11.8%), and bla(CMY-2) (6.4%) were detected. Twenty-one strains were ESBL producers. CTX-M-15 was the predominant CTX-M variant found and this is the first report of a CTX-M-27-producing E. coli strain from Malaysia. Majority (3.1%) of the strains harbored class 1 integron-encoded integrases with a predominance of aadA and dfr genes within the integron variable region. No gene cassette encoding ESBL genes was found and integrons were not significantly associated with ESBL or non-ESBL producers. Possible clonal expansion was observed for few CTX-M-15-positive strains but the O25-ST131 E. coli clone known to harbor CTX-M-15 was not detected while CMY-2-positive strains were genetically diverse.     

Prolonged outbreak of infection due to TEM-21-producing strains of Pseudomonas aeruginosa and enterobacteria in a nursing home

Over a 6-year period, 24 extended-spectrum beta-lactamase (ESBL)-producing isolates of Pseudomonas aeruginosa were collected from 18 patients living in a nursing home. These isolates had a delayed development of a red pigment and exhibited a similar antibiotype (resistance to all beta-lactams except for imipenem and to gentamicin, tobramycin, netilmicin, ciprofloxacin, and rifampin) associated with the production of the TEM-21 beta-lactamase and a type II 3'-N-aminoglycoside acetyltransferase [AAC(3)-II] enzyme. Surprisingly, serotyping showed that these isolates belonged to four successive serotypes (P2, P16, P1, and PME), although molecular typing by PCR methods and pulsed-field gel electrophoresis yielded identical or similar profiles. Moreover, in all isolates the bla(TEM-21) gene was part of a chromosomally located Tn801 transposon truncated by an IS6100 element inserted within the resolvase gene, and the aac(3)-II gene was adjacent to this structure. During the same period, 17 ESBL-producing isolates of enterobacteria were also collected from 10 of these patients. These isolates harbored a similar large plasmid that contained the bla(TEM-21) and the aac(3)-II genes and that conferred additional resistance to sulfonamides and chloramphenicol, as well as to kanamycin, tobramycin, netilmicin, and amikacin, conveyed by an AAC(6')-I enzyme. The bla(TEM-21) gene was part of the Tn801 transposon disrupted by IS4321. Thus, a single clone of P. aeruginosa that had undergone a progressive genetic drift associated with a change in serotype appeared to be responsible for an outbreak of nosocomial infections in a nursing home. This strain has probably acquired the bla(TEM-21)-encoding plasmid that was epidemic among the enterobacteria at the institution, followed by chromosomal integration and genomic reorganization.     

First characterization in Tunisia of a TEM-15, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolate

Klebsiella pneumoniae CH0905 strain exhibiting high-level cefotaxime resistance was isolated from a stool culture in the intensive care unit. The resistance gene responsible was shown to be located on a conjugative 60-kb plasmid designated pCH0905. The minimum inhibitory concentration (MIC) values for cefotaxime and ceftazidime of the original isolate and the transconjugates were 256 mug/ml. Isoelectric focusing of a protein preparation from the K. pneumoniae strain showed beta-lactamases with the pI values of 7.6 and 6.3. A 1,080-bp fragment amplified with PCR was cloned into the pGEM-T Easy vector. The nucleotide sequence of the complete 1,080 bp was determined. Sequence analysis revealed that the bla(TEM) gene of pCH0905 differed from bla(TEM-1) by two mutations, leading to the following amino acid substitutions: the glutamic acid residue at position 104 by lysine and the glycine residue at position 238 by serine (Ambler numbering). The association of these two mutations was described previously in TEM-15 beta-lactamase, but this is the first detection of this enzyme in Tunisia.