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Truncated Class 1 Integron Gene Cassette Arrays Contribute to Antimicrobial Resistance of Diarrheagenic Escherichia coli.

ABSTRACT: Class 1 integrons (c1-integrons) are associated with multidrug resistance in diarrheagenic Escherichia coli (DEC). However, little is known about gene cassettes located within these c1-integrons, particularly truncated c1-integrons, in DEC strains. Therefore, the aims of the present study were to reveal the relationship between antimicrobial resistance and the presence of truncated c1-integrons in DEC isolates derived from human stool samples in Japan. A total of 162 human stool-derived DEC isolates from Japan were examined by antimicrobial susceptibility testing, PCR-based gene detection, and next-generation sequencing analyses. Results showed that 44.4% (12/27) of c1-integrons identified in the DEC isolates harbored only intI1 (an element of c1-integrons) and were truncated by IS26, Tn3, or IS1-group insertion sequences. No difference in the frequency of antimicrobial resistance was recorded between intact and truncated c1-integron-positive DEC isolates. Isolates containing intact/truncated c1-integrons, particularly enteroaggregative E. coli isolates, were resistant to a greater number of antimicrobials than isolates without c1-integrons. aadA and dfrA were the most prevalent antimicrobial resistance genes in the intact/truncated c1-integrons examined in this study. Therefore, gene cassettes located within these intact/truncated c1-integrons may only play a limited role in conferring antimicrobial resistance among DEC. However, DEC harboring truncated c1-integrons may be resistant to a greater number of antimicrobials than c1-integron-negative DEC, similar to strains harboring intact c1-integrons.

SUBMITTER: Kubomura A

PROVIDER: S-EPMC7013361 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Truncated Class 1 Integron Gene Cassette Arrays Contribute to Antimicrobial Resistance of Diarrheagenic <i>Escherichia coli</i>.

Kubomura Akiko A Sekizuka Tsuyoshi T Onozuka Daisuke D Murakami Koichi K Kimura Hirokazu H Sakaguchi Masahiro M Oishi Kazunori K Hirai Shinichiro S Kuroda Makoto M Okabe Nobuhiko N

BioMed research international 20200131

Class 1 integrons (c1-integrons) are associated with multidrug resistance in diarrheagenic <i>Escherichia coli</i> (DEC). However, little is known about gene cassettes located within these c1-integrons, particularly truncated c1-integrons, in DEC strains. Therefore, the aims of the present study were to reveal the relationship between antimicrobial resistance and the presence of truncated c1-integrons in DEC isolates derived from human stool samples in Japan. A total of 162 human stool-derived D ...[more]

PMID: 32090095

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Project description:BackgroundIntegrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains.ResultsThe complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes). It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons.ConclusionOur systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene gain/loss relative to most other loci on vibrio chromosomes. We identify a relationship between the phylogenetic distance separating two species and the rate at which they exchange gene cassettes, interactions between the non-mobile portion of bacterial genomes and the vibrio gene cassette pool as well as intragenomic translocation events of integrons in vibrios.

Project description:Diarrheagenic Escherichia coli (DEC) pathotypes are the leading cause of mortality and morbidity in South Asia and sub-Saharan Africa. Daily interaction between people contributes to the spreading of Escherichia coli (E. coli), and fomites are a common source of community-acquired bacterial infections. The spread of bacterial infectious diseases from inanimate objects to the surrounding environment and humans is a serious problem for public health, safety, and development. This study aimed to determine the prevalence and antibiotic resistance of diarrheagenic E. coli found in toilets and kitchen cloths in the Vhembe district, South Africa. One hundred and five samples were cultured to isolate E. coli: thirty-five samples were kitchen cloths and seventy-five samples were toilet swabs. Biochemical tests, API20E, and the VITEK®-2 automated system were used to identify E. coli. Pathotypes of E. coli were characterised using Multiplex Polymerase Chain Reaction (mPCR). Nine amplified gene fragments were sequenced using partial sequencing. A total of eight antibiotics were used for the antibiotic susceptibility testing of E. coli isolates using the Kirby–Bauer disc diffusion method. Among the collected samples, 47% were positive for E. coli. DEC prevalence was high (81%), with ETEC (51%) harboring lt and st genes being the most dominant pathotype found on both kitchen cloths and toilet surfaces. Diarrheagenic E. coli pathotypes were more prevalent in the kitchen cloths (79.6%) compared with the toilet surfaces. Notably, hybrid pathotypes were detected in 44.2% of the isolates, showcasing the co-existence of multiple pathotypes within a single E. coli strain. The antibiotic resistance testing of E. coli isolates from kitchen cloths and toilets showed high resistance to ampicillin (100%) and amoxicillin (100%). Only E. coli isolates with hybrid pathotypes were found to be resistant to more than three antibiotics. This study emphasizes the significance of fomites as potential sources of bacterial contamination in rural settings. The results highlight the importance of implementing proactive measures to improve hygiene practices and antibiotic stewardship in these communities. These measures are essential for reducing the impact of DEC infections and antibiotic resistance, ultimately safeguarding public health.

Project description:UnlabelledIntegrons are bacterial genetic elements able to capture and express genes contained within mobile gene cassettes. Gene cassettes are expressed via a Pc promoter and can be excised from or integrated into the integron by integrase IntI. Although the mechanisms of gene cassette integration and excision are well known, the kinetics and modes of gene cassette shuffling leading to new gene cassette arrays remain puzzling. It has been proposed that under antibiotic selective pressure, IntI-mediated rearrangements can generate integron variants in which a weakly expressed gene cassette moves closer to Pc, thus leading to higher-level resistance. To test this hypothesis, we used an integron with four gene cassettes, intI1-aac(6')-Ib-dfrA15-aadA1-catB9, and applied selective pressure with chloramphenicol, resistance to which is encoded by catB9. Experiments were performed with three different Pc variants corresponding to three IntI1 variants. All three integrases, even when not overexpressed, were able to bring catB9 closer to Pc via excision of the dfrA15 and aadA1 gene cassettes, allowing their host bacteria to adapt to antibiotic pressure and to grow at high chloramphenicol concentrations. Integrase IntI1(R32_H39), reported to have the highest recombination activity, was able, when overexpressed, to trigger multiple gene cassette rearrangements. Although we observed a wide variety of rearrangements with catB9 moving closer to Pc and leading to higher chloramphenicol resistance, "cut-and-paste" relocalization of catB9 to the first position was not detected. Our results suggest that gene cassette rearrangements via excision are probably less cost-effective than excision and integration of a distal gene cassette closer to Pc.ImportanceIntegrons are bacterial genetic elements able to capture and express gene cassettes. Gene cassettes are expressed via a Pc promoter; the closer they are to Pc, the more strongly they are expressed. Gene cassettes can be excised from or integrated into the integron by integrase IntI. The kinetics and modes of gene cassette shuffling, leading to new gene cassette arrays remain puzzling. We used an integron with 4 antibiotic resistance gene cassettes and applied selective pressure with the antibiotic for which resistance was encoded by cassette 4. All IntI variants were able to bring cassette 4 closer to Pc. Rearrangements occur via excision of the previous gene cassettes instead of cut-and-paste relocalization of the fourth gene cassette.

Dataset Information

Truncated Class 1 Integron Gene Cassette Arrays Contribute to Antimicrobial Resistance of Diarrheagenic Escherichia coli.

Publications

Truncated Class 1 Integron Gene Cassette Arrays Contribute to Antimicrobial Resistance of Diarrheagenic <i>Escherichia coli</i>.

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