Sequencing and comparative genomics analysis of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA
Introduction
Plasmids of the H incompatibility (IncH) complex can be divided into two major Inc groups, IncHI and IncHII [1]. Incompatibility interactions between IncHI and IncHII can be explained by a negative control model: the IncHII repressor binds more strongly to the IncHI operator site than the IncHI repressor binds to the IncHII operator; replication of the IncHI plasmid is usually suppressed, whereas IncHII replication continues [1]. Three different subgroups, namely IncHI1–3 [2], [3], have been further discriminated for IncHI plasmids based on their nucleotide sequence homology, but the incompatibility interactions between these subgroups are unclear.
IncHI plasmids were originally isolated from the major causal agents of enteric fever, namely Salmonella enterica serovars Typhi and Paratyphi A, and are linked to the persistence and re-emergence of typhoid fever outbreaks [4]. IncHI plasmids have also been widely isolated from other enterobacterial species both of environmental and clinical origin, such as Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae [5].
IncHI plasmids are typically large, often >200 kb in size. Comparative analysis of some of the IncHI1 plasmids shows that these plasmids are very diverse in terms of genetic structure and have accumulated a number of accessory resistance regions [6], [7]. These plasmids carry the determinants for resistance not only to heavy metals such as mercuric ions, copper, silver ions, tellurite, arsenate and arsenite, but also to antibiotics such as β-lactams (including carbapenems), quinolones, aminoglycosides, tetracyclines, amphenicols and fosfomycin [6], [7].
IncHI plasmids carry multiple genes involved in the initiation of replication, but none of these genes are universally present in all sequenced IncHI plasmids. IncHI plasmids contain the conjugal transfer gene regions tra1 and tra2 and are characteristic of a thermosensitive mode of transfer—the transfer efficiency is optimal between 22–30 °C but is inhibited at 37 °C [8]. Functional studies on the IncHI plasmid R27 showed that the tra1 and tra2 regions encode a mating-pair formation apparatus essential for conjugal transfer [9] and that the expression of tra genes is transcriptionally regulated by temperature [10]. The advantage conferred by temperature-dependent conjugative transfer has implicated IncHI plasmids as important vectors in the dissemination of antibiotic resistance among pathogenic and indigenous bacterial species in water and soil environments [11].
This study proposes a scheme for typing IncHI plasmids into five subgroups (IncHI1–5) and presents the complete nucleotide sequences of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA from clinical E. cloacae, Citrobacter freundii and Raoultella ornithinolytica isolates, respectively. Further genomic comparison identified complex chimeric structures among these multidrug-resistant (MDR) plasmids, which mainly resulted from the acquisition of complex combinations of accessory regions especially including those encoding a series of antibiotic resistance genes.
Section snippets
Bacterial strains
Enterobacter cloacae T5282 was isolated in 2012 from a sputum specimen of a 62-year-old female with advanced lung adenocarcinoma and pulmonary infection in a teaching hospital in Chongqing City, China. Citrobacter freundii 112298 was isolated in 2013 from a mid-stream urine specimen from a 56-year-old male with septic shock in a teaching hospital in Guangzhou City, China [12]. Raoultella ornithinolytica YNKP001 was recovered in 2010 from a blood specimen from a 4-year-old child with acute
Subgrouping of IncHI plasmids
To infer the evolutionary history of the 21 representative IncHI plasmids, three phylogenetic trees (Fig. 1) were constructed from the individual sequences of two arbitrarily selected genes traI and trhC (located in the tra1 and tra2 regions, respectively) as well as the concatenated sequences of these two genes (Supplementary Table S1), respectively. These three trees were topologically very similar and thus the conflicting phylogenetic signals between these two genes and the resulting
Discussion
The IncHI plasmids could be divided into five separately clustering subgroups (IncHI1–5). Genetic conservation of the plasmid backbone sequences within each subgroup was much higher than between different subgroups. Acquisition of a number of accessory regions, likely through horizontal gene transfer, has resulted in the complex chimeric structure of the three IncHI2 plasmids pT5282-mphA, p112298-catA and R478 and the two IncHI5 plasmids pYNKP001-dfrA and pKOX_R1. Accessory genetic elements
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These two authors contributed equally to this work.