Study of a Natural Mutant SHV-Type ß -Lactamase, SHV-104, from Klebsiella pneumoniae.

Klebsiella pneumoniae ML2011, a multiresistant isolate, was isolated from the Military Hospital of Tunis (Tunisia). The determination of the minimal inhibitory concentrations exhibited by K. pneumoniae ML2011 was performed by Etest. The crude extract of the isolates contains four different ß -lactamases with pI 5.5, 7.3, 7.6, and 8.6. Only the ß -lactamases with pI 7.3 and pI 8.6 were transferred by transformation and conjugation experiment. Molecular characterization of these genes was performed by PCR and sequencing. The chromosomal ß -lactamases are TEM (pI 5.5) and SHV-1 (7.6). CTX-M-28 (pI 8.6) and the novel variant of SHV named SHV-104 (pI 7.3) were encoded by bla gene located on a 50 kb highly conjugative plasmid. The SHV-104 ß -lactamase was produced in E. coli and purified. Its profile of activity was determined. Compared to SHV-1, SHV-104 contains one mutation, R202S. Their kinetic parameters were similar except for cefotaxime. The analysis of the predicted structure of SHV-104 indicated that the R202S mutation suppresses a salt bridge present in SHV-1. Therefore, the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime. We can conclude that the multiresistant phenotype of K. pneumoniae ML2011 strain is mainly linked to the production of CTX-M-28 since SHV-104 possesses a narrow spectrum of activity.

Characterization of a novel extended-spectrum TEM-type beta-lactamase, TEM-164, in a clinical strain of Klebsiella pneumoniae in Tunisia.

Klebsiella pneumoniae ML1708 exhibited a multiresistance phenotype, including resistance to all beta-lactams tested, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline, and streptomycin. The double-disk synergy test was positive. ML1708 harbored a 50 kb conjugative plasmid that encoded a beta-lactamase of pI 5.5. The corresponding bla gene was identified by polymerase chain reaction and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new variant of TEM-1 beta-lactamase designated TEM-164. TEM-164 contains the unusual following mutations: L40V and I279T. These modifications may result in a change of the pI to 5.5 and hydrolyze cefotaxime and ceftazidime.

Cefotaxime and ceftazidime-resistant Escherichia coli isolate producing TEM-15 beta-lactamase from a Tunisian hospital.

A clinical isolate of Escherichia coli LBT04 was found to have a high-level resistance to broad-spectrum beta-lactams. Analysis of this strain by the disk diffusion test revealed synergies between clavulanic acid and ceftazidime, cefotaxime. Clavulanic acid decreased the MICs of ceftazidime, cefotaxime, and ceftriaxone, which suggested that LBT04 produced an extended-spectrum beta-lactamase. These resistances were carried by a 1080-bp chromosomal gene that encoded a beta-lactamase with a pI of 6.3. Cloning and sequencing experiments showed that this beta-lactamase revealed identity with the bla(TEM-1) gene encoding the TEM-1 beta-lactamase, except for a replacement of the Glu residue at position 104 by Lys, and of the Gly residue at position 238 by Ser. These two mutations were encountered in TEM-15 beta-lactamase, but this is the first description of this enzyme in the E. coli species in Tunisian hospitals.

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.

Cloning and sequencing of the class A beta-lactamase gene (blaTEM-15) located on a chromosomal Tn801 transposon.

Escherichia coli CA0210 was identified in a stool culture of a 03-month-neonate in Tunisia. This strain was resistant to beta-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefpirome, ceftazidime, and cefotaxime, but it remained susceptible to imipenem and cefoxitin. The beta-lactam-hydrolyzing beta-lactamase gene of E. coli CA0210 and the upstream and downstream regions were cloned, sequenced, and expressed in E. coli DH5alpha. These resistances were carried by a 1080-bp chromosomal gene that encoded a beta-lactamase with a pI of 6.3. Cloning and sequencing experiments showed that the corresponding blaTEM-15 gene was part of a chromosomally located Tn801 transposon.