Pooled Plasmid Sequencing Reveals the Relationship Between Mobile Genetic Elements and Antimicrobial Resistance Genes in Clinically Isolated Klebsiella pneumoniae

Abstract

Plasmids remain important microbial components mediating the horizontal gene transfer (HGT) and dissemination of antimicrobial resistance. To systematically explore the relationship between mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), a novel strategy using single-molecule real-time (SMRT) sequencing was developed. This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant (MDR) Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period. The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates. The plasmid pool was then subjected to SMRT sequencing, and 82 assembled plasmid fragments were obtained. In total, 124 ARGs (responsible for resistance to β-lactam, fluoroquinolone, and aminoglycoside, among others) and 317 MGEs [including transposons (Tns), insertion sequences (ISs), and integrons] were derived from these fragments. Most of these ARGs were linked to MGEs, allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, and ISKpn19. As the most predominant IS in the network, a typical IS26-mediated multicopy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications (TSDs). The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs, underpinning the clinical value of dominant HGT profiles.

Keywords: Horizontal transfer profile; Insertion sequence; Plasmid; Single-molecule real time sequencing; Transconjugant.

Figure 1

Overall strategy and workflow of our study The strategy developed in our study is shown, from extracting the pooled plasmids of E. coli transconjugants to plasmid sequence analysis including ARG and MGE relationship network construction. MDR, multidrug resistance; MGE, mobile genetic element; ARG, antimicrobial resistance gene.

Figure 2

ARGs identified in the plasmid sequence and the related antimicrobial agent classes aac(6′)-Ib-cr (labeled by *) encodes an aminoglycoside acetyltransferase which can also inactivate fluoroquinolones, and thus it also belongs to fluoroquinolone resistance determinants.

Figure 3

Network of MGEs and ARGs for their relationships The oval-shaped nodes represent MGEs; the diamond-shaped nodes represent ARGs. Nodes belonging to the same IS family or resistant to the same class of antimicrobial agents are presented in the same color. The more frequent the gene relationships are, the thicker the line is. The colored lines represent MGE or ARG clusters in which the relationships occur more frequently (more than twice per pair). The network was constructed by using Cytoscape v. 3.2.1. IS, insertion sequence; Tn, transposon.

Figure 4

The entire process of multicopy IS26 composite transposon generation deduced based on tracing the 8-bp TSD distribution A. First, an IS26 intermolecular replicative transposition event occurs, whereby IS26 from the donor plasmid attacks the target site “1” in another plasmid near bla_SHV-11, resulting in duplication of IS26 and the 8-bp TSD1 (GGGGCTCG). B. Second, IS26 with TSD1 launches another intramolecular attack and copies itself to the other side of bla_SHV-11, leading to the duplication of the 8-bp (TSD2, AACGCCGG) at the target site “2”. C. The unequal crossover combination occurs, producing a copy of the IS26 composite transposon. D. The formation of the IS26 composite transposon with multiple copies in a row. E. Finally, the multicopy IS26 composite transposon attacks the target site “0” within the transposase (tnpA) gene of the Tn3 transposon and inserts the 27-kb fragment along with the duplication of the 8-bp TSD0 (TTTCACCT). TSD, target site duplication.