Abstract
Four plasmids ranging in size from 4.7 to 44.7 kb found in the extensively antibiotic resistant Acinetobacter baumannii isolate D36 that belongs to lineage 2 of global clone 1 were examined. D36 includes two cryptic plasmids and two carrying antibiotic resistance genes. The smallest plasmid pD36-1 (4.7 kb) carries no resistance genes but includes mobA and mobC mobilisation genes related to those found in pRAY* (pD36-2, 6,078 bp) that also carries the aadB gentamicin, kanamycin and tobramycin resistance gene cassette. These two plasmids do not encode a Rep protein. Plasmid pRAY* was found to be mobilised at high frequency by the large conjugative plasmid pA297-3 but a pRAY* derivative lacking the mobA and mobC genes was not. The two larger plasmids, pD36-3 and pD36-4, encode Rep_3 family proteins (Pfam1051). The cryptic plasmid pD36-3 (6.2 kb) has RepAci1 and pD36-4 (44.7 kb) encodes two novel Rep_3 family proteins suggesting a co-integrate. Plasmid pD36-4 includes the sul2 sulfonamide resistance gene, the aphA1a kanamycin/neomycin resistance gene in Tn4352::ISAba1 and a mer module in a hybrid Tn501/Tn1696 transposon conferring resistance to mercuric ions. New examples of dif modules flanked by pdif sites (XerC-XerD binding sites) that are part of many A. baumannii plasmids were also identified in pD36-3 and pD36-4 which carry three and two dif modules, respectively. Homologs of three dif modules, the sup sulphate permease module in pD36-3, and of the abkAB toxin-antitoxin module and the orf module in pD36-4, were found in different contexts in diverse Acinetobacter plasmids, consistent with module mobility. A novel insertion sequence named ISAba32 found next to the pdif site in the abkAB dif module is related to members of the ISAjo2 group which also are associated with the pdif sites of dif modules. Plasmids found in D36 were also found in some other members of GC1 lineage 2.
Highlights
Acinetobacter baumannii is a Gram negative opportunistic pathogen and a member of the ESKAPE group of bacteria that are the leading cause of difficult to treat nosocomial infections throughout the world [1]
We showed that D36 is a carbapenem resistant A. baumannii isolate that belongs to lineage 2 and ST81 (Institut Pasteur MLST scheme), which is a single locus variant (SLV) of the predominant GC1 sequence type, ST1 [8]
The copy number of pD361, pRAYÃ and pD36-3 was estimated to be between 11–13 copies/cell while that of the largest plasmid, pD36-4, was 2–3 copies/cell
Summary
A range of bioinformatics tools was used to annotate the sequences of the three novel plasmids, pD36-1, pD36-3 and pD36-4 found in D36 (GenBank accession numbers CP012953, CP012955 and CP012956, respectively). The outputs of the automatic annotation program Prokka [18] were manually modified where further information such as assigned gene names was available. Antibiotic resistance genes were identified using ResFinder Dk/services/ResFinder/) [19] and ISFinder (https://www-is.biotoul.fr/) was used to identify insertion sequences. Sequence repeats including iterons were found using Unipro UGENE v1.29.0 (http://ugene.net/). Pfam searches (http://pfam.xfam.org/) were used to identify possible protein functions. The copy number of each plasmid was estimated by dividing the coverage of contigs containing plasmid sequences by the coverage of chromosomal contigs
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