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Use of inverse PCR for analysis of class 1 integrons carrying an unusual 3' conserved segment structure.

ABSTRACT: By using inverse PCR and DNA sequencing, 13 sul3-associated mutational integrons, 2 defective class 1 integrons, and 1 qnrB2-associated complex sul1-type class 1 integrons were identified in Salmonella enterica serovar Choleraesuis, Pseudomonas aeruginosa, and Enterobacter cloacae, respectively. In addition, conjugation and Southern hybridization demonstrated that unusual class 1 integrons were located on plasmids or integrated into chromosomal DNA. Thus, an inverse PCR assay can be a valuable tool for the analysis of unusual structures of the 3' conserved region of class 1 integrons.

SUBMITTER: Lee MF

PROVIDER: S-EPMC3028803 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Use of inverse PCR for analysis of class 1 integrons carrying an unusual 3' conserved segment structure.

Lee Mei-Feng MF Peng Chien-Fang CF Hsu Hui-Jine HJ Toh Han-Siong HS

Antimicrobial agents and chemotherapy 20101122 2

By using inverse PCR and DNA sequencing, 13 sul3-associated mutational integrons, 2 defective class 1 integrons, and 1 qnrB2-associated complex sul1-type class 1 integrons were identified in Salmonella enterica serovar Choleraesuis, Pseudomonas aeruginosa, and Enterobacter cloacae, respectively. In addition, conjugation and Southern hybridization demonstrated that unusual class 1 integrons were located on plasmids or integrated into chromosomal DNA. Thus, an inverse PCR assay can be a valuable t ...[more]

PMID: 21098251

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Project description:Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI) encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination), a recombination site (attI1), and a promoter (Pc) responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought.

Project description:BackgroundShigella spp. are major cause of diarrhoeal disease in both developing and developed countries. Shigella sonnei is the serogroup of Shigella most frequently responsible for sporadic and epidemic enteritis in developed countries. In recent years the emergence and spread of S. sonnei biotype g carrying class 2 integron have been frequently reported in many countries. Recently, S. sonnei has been reported as the prevalent serogroup of Shigella in Iran. The present study was carried out to investigate phenotypic and genetic characteristics of Shigella sonnei isolates identified in the years 2002 and 2003 in Tehran, Iran.MethodsBiotyping, drug susceptibility testing, pulsed field gel electrophoresis (PFGE) and analysis of class 2 integrons have been carried out on 60 S. sonnei isolates, including 57 sporadic isolates from paediatric cases of shigellosis occurring in 2002 and 2003, two sporadic isolates recovered in 1984 and the ATCC 9290 strain.ResultsBiotype g and resistance to streptomycin, sulfamethoxazole-trimethoprim and tetracycline were exhibited by 54 of the 57 recent isolates. Of the 54 biotype g isolates, 28 exhibited a class 2 integron of 2161 bp, and 24 a class 2 integron of 1371 bp, respectively. Class 2 integrons were not detected in four isolates only, including the two endemic isolates recovered in 1984 and two strains from recent sporadic cases. PFGE divided the strains into eight pulsotypes labeled A to H, three major pulsotypes - A to C - including the large majority of the recent sporadic S. sonnei isolates. Pulsotypes A and C were the most prevalent groups, accounting for 41.6% and 35.0%, respectively, of the isolates under study.ConclusionThe results suggest that biotype g, class 2 integron carrying S. sonnei are prevalent in our geographic area. S. sonnei isolated in the years 2002 and 2003 could be attributed to a few predominant clusters including, respectively, strains with pulsotypes B and C carrying a 2161 bp class 2 integron, and those having pulsotype A and a 1371 bp class 2 integron. A few epidemic clones are responsible for the apparently endemic occurrence of shigellosis in Tehran, Iran.

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Use of inverse PCR for analysis of class 1 integrons carrying an unusual 3' conserved segment structure.

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Use of inverse PCR for analysis of class 1 integrons carrying an unusual 3' conserved segment structure.

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