Computational protocol: Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii

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Protocol publication

[…] Acinetobacter baumannii Ab242 is a MDR clinical strain displaying carbapenem resistance (Table ) isolated in 1997 from a clonal group disseminating in a public healthcare institution of Rosario, Argentina (Limansky et al., ; Mussi et al., ). MLST analysis assigned this strain to ST104 (CC104) in the Oxford scheme (cpn60-29, gdhB-12, gltA-12, gpi-57, gyrB-17, recA-1, rpoD-39) and to ST15 in the Pasteur scheme (cpn60-6, fusA-6, gltA-8, pyrG-2, recA-3, rplB-5, rpoB-4) ( A. nosocomialis M2 strain (Carruthers et al., ) was used in this study as a recipient for transformation assays with plasmids extracted from Ab242 (see below).The above Acinetobacter strains were routinely grown in Lysogeny Broth (LB) liquid medium at 37°C under aerobic conditions with vigorous shaking for plasmid extraction procedures, or in LB agar medium with the indicated antibiotics for transformation studies and the selection of individual colonies. Appropriate procedures for working with a level 2 pathogen were followed throughout this work (Biosafety in Microbiological and Biomedical Laboratories, 5th edition, U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, National Institutes for Health). [...] DNA sequencing of Ab242 plasmids was done at the Instituto de Agrobiotecnología Rosario (INDEAR, Rosario, Argentina) using a 454 pyrosequencing platform (Roche Diagnostics). The obtained reads were assembled in silico and the resulting sequences were refined by visual inspection. Three plasmids were inferred from this analysis and designated pAb242_25, pAb242_12, and pAb242_9, where the latter numbers indicate the approximate corresponding lengths in kbp. The circular structures of these plasmids were confirmed by PCR using specifically designed primer pairs followed by sequencing of the obtained amplicons, as well as by primer walking (see Table and Figure for details). DNA sequencing was done at the Sequencing Facility of Maine University. The nucleotide sequences of the plasmids described in this study were deposited in the GenBank nucleotide sequence database under accession numbers KY984047 (pAb242_25), KY984046 (pAb242_12), and KY984045 (pAb242_9).The Rapid Annotation using Subsystem Technology standard operating procedures (RAST, (Aziz et al., ) and the National Center for Biotechnology Information database (NCBI, U.S. National Library of Medicine, Bethesda MD, USA) were used to annotate the open reading frames (ORFs).Search for antimicrobial resistance determinants was done using ResFinder 2.1 (; Zankari et al., ). The detection of IS was done with IS Finder (Siguier et al., ) ( and ISsaga (Varani et al., ). Comparative analysis of the carbapenem and aminoglycoside adaptive module located in pAb242_25 with similar structures reported in other Acinetobacter plasmids was conducted using Mauve (Darling et al., ). […]

Pipeline specifications

Software tools BIGSdb, RAST, ISsaga, Mauve
Databases PubMLST
Applications De novo sequencing analysis, Nucleotide sequence alignment
Diseases Malocclusion, Angle Class I