Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum β-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae

Mabilat, Claude; Goussard, Sylvie; Sougakoff, Wladimir; Spencer, R C; Courvalin, Patrice

doi:10.1016/0147-619x(90)90041-a

Cited by 104 publications

(76 citation statements)

References 18 publications

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“…Ampl$cation and sequencing of' b1aT-6 and of' its promoter and comparison with blaT genes We amplified by PCR (Saiki et al, 1988), using oligonucleotide primers A and B (Mabilat et al, 1990) and plasmid pIP1844 DNA as a template ( The numbering system is that of Sutcliffe (1978). P6 and P3, promoters of bluT-6 and bluT-1, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…TEM-type /?-lactamases that hydrolyse third-generation cephalosporins differ from penicillinases TEM-1 and TEM-2 by a few amino acid substitutions (Ca latz et al, 1989;Mabilat & Courvalin, 1990;Mabilat et al, 1990;Sougakoff et al, 19886, 1989). Determination of the sequence of blaT-6 encoding TEM-6 in E. coli HB251 showed that the resistance gene differed from blaT-Z specifying TEM-1 by two point mutations (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…PCR with oligonucleotides F, J and K was carried out in a final volume of loop1 containing lop1 of a 1 :I000 diluted overnight culture lysed by heating at 95 "C for 3 min. The 35 amplification cycles were performed as follows: annealing at 55 "C for 1 min, extension step at 72 "C for 30 s and denaturation at 94 "C for 30 s. The Tag DNA polymerase was purchased from PelkinElmer, Cetus Co. PCR-amplified DNA was purified (Maniatis et al, 1982) and sequenced directly with T7 polymerase (Sequenase) using the protocol of Tabor & Richardson (1987) as described previously (Mabilat et al, 1990). Oligonucleotides A to G used as amplification and/or sequencing primers were described by Mabilat et al (1990).…”

Section: Methodsmentioning

confidence: 99%

“…E. coliTHB82,87,91,102,103,107,114,136 and 137 are transconjugants derived from these strains by mating with E. coli A 15 and selecting for ampicillin and nalidixic acid resistance. Clinical isolates resistant to fl-lactams were described previously (Mabilat & Courvalin, 1990). Strains were grown in brain heart infusion broth and agar (Difco), containing ampicillin at 100 pg ml-' or chloramphenicol at 10 pg ml-' when required.…”

Section: Methodsmentioning

confidence: 99%

“…With the exception of TEM-6, all these variants have been characterized at the molecular level. They differ from TEM-1 or TEM-2 by several point mutations that are mostly involved in the extension of the substrate range of the enzymes (Collatz et al, 1989;Mabilat & Courvalin, 1990;Mabilat et al, 1990;Sougakoff et al, 19886, 1989). P-Lactamase TEM-6 was detected in Escherichia coli HB25 1, which is resistant to ceftazidime and aztreonam but susceptible to other third-generation cephalosporins (Bauernfeind & Horl, 1987).…”

Section: Courval@frpstroimentioning

confidence: 99%

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An IS1-like element is responsible for high-level synthesis of extended-spectrum -lactamase TEM-6 in Enterobacteriaceae

Goussard

Sougakoff

Mabilat

et al. 1991

Journal of General Microbiology

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Resistance of Escherichiu coli strain HB251 to the newer p-lactam antibiotics, in particular ceftazidime and aztreonam, results from production of the extended-spectrum p-lactamase TEM-6. The corresponding structural gene, bfuT-6, and its promoter region were amplified by the polymerase chain reaction. Analysis of the sequence of the amplification product showed that bluT-6 differed by two nucleotide substitutions from bluT-1, the gene encoding TEM-1 penicillinase in plasmid pBR322. The mutations led to the substitution of a lysine for a glutamic acid at position 102 and of a histidine for an arginine at position 162 of the unprocessed TEM-1 protein. The presence of a 116 bp DNA insert upstream from bluT-6 resulted in the creation of hybrid promoter P6 in which the -10 region was that of TEM-1 promoter P3 whereas the -35 canonical sequence TTGACA was provided by the right end of the insert. P6 was found to be 10 times more active than P3 and to confer higher levels of antibiotic resistance upon the host. Analysis of the sequence of the insert indicated that the 116 bp fragment is related to insertion sequence IS1 but differs from it by three internal deletions that removed regions encoding the transposase. The distribution of the ISl-like element in clinical isolates of Enterobucferiaceae was studied by the polymerase chain reaction and by DNA-DNA hybridization. The element appeared to be widespread and was detected in strains producing TEM-6 or other TEM variants.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Courval@frpstroimentioning

confidence: 99%

See 3 more Smart Citations

An IS1-like element is responsible for high-level synthesis of extended-spectrum -lactamase TEM-6 in Enterobacteriaceae

Goussard

Sougakoff

Mabilat

et al. 1991

Journal of General Microbiology

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Acyclovir-resistant Varicella-Zoster virus: Phenotypic and genetic characterization

Fillet¹,

Dumont²,

Caumes³

et al. 1998

J. Med. Virol.

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A man with acquired immunodeficiency syndrome (AIDS) developed zoster of the right arm which was resistant clinically to acyclovir. Varicella-zoster virus (VZV) was cultured from a skin biopsy performed at the beginning of acyclovir therapy (isolate 1) and after its failure (isolate 2). The emergence of acyclovir resistance during treatment was investigated by developing a simple and rapid drug sensitivity assay based on the plaque reduction reference method. This late-antigen synthesis reduction assay involved serial dilutions of cell-associated virus. The 50% inhibitory concentration (IC50) of acyclovir was 16 +/- 7.5 microM for the susceptible reference strain OKA, in agreement with published data. The acyclovir IC50 increased from 6.5 microM for isolate 1 to 100 microM for isolate 2. In comparison with the sequence of isolate 1, isolate 2 had a single mutation consisting of a C to T change at position 907 of the thymidine kinase gene, which changed a glutamine codon into a stop codon at position 303 of the thymidine kinase protein. These results show the emergence of acyclovir resistance through a single previously undescribed mutation in the thymidine kinase gene, and confirm the heterogeneity of mutations inducing acyclovir resistance.

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Applications of the Polymerase Chain Reaction

Wiedbrauk¹,

Hodinka

2002

Infectious Agents and Pathogenesis

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Direct sequencing of the amplified structural gene and promoter for the extended-broad-spectrum β-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae

Cited by 104 publications

References 18 publications

An IS1-like element is responsible for high-level synthesis of extended-spectrum -lactamase TEM-6 in Enterobacteriaceae

An IS1-like element is responsible for high-level synthesis of extended-spectrum -lactamase TEM-6 in Enterobacteriaceae

Acyclovir-resistant Varicella-Zoster virus: Phenotypic and genetic characterization

Applications of the Polymerase Chain Reaction

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