Investigation of multidrug-resistant plasmids from carbapenemase-producing Klebsiella pneumoniae clinical isolates from Pakistan

Abstract

Objectives: The study aim was to investigate multidrug-resistant (MDR) plasmids from a collection of 10 carbapenemase-producing Klebsiella pneumoniae clinical isolates identified within the same healthcare institution in Pakistan. Full characterization of the MDR plasmids including structure, typing characteristics, and AMR content as well as determination of their plasmid-based antimicrobial susceptibility profiles were carried out.

Methods: Plasmids were isolated from 10 clinical isolates of Klebsiella pneumoniae, and from a corresponding set of Escherichia coli transconjugants, then sequenced using Nanopore/Illumina technology to generate plasmid hybrid assemblies. Full characterization of MDR plasmids, including determination of next generation sequencing (NGS)-based AMR profiles, plasmid incompatibility groups, and types, was carried out. The structure of MDR plasmids was analyzed using the Galileo AMR platform. For E. coli transconjugants, the NGS-based AMR profiles were compared to NGS-predicted AMR phenotypes and conventional broth microdilution (BMD) antimicrobial susceptibility testing (AST) results.

Results: All carbapenemase-producing K. pneumoniae isolates (carrying either bla_NDM-1, or/and bla_OXA-48) carried multiple AMR plasmids encoding 34 antimicrobial resistance genes (ARGs) conferring resistance to antimicrobials from 6 different classes. The plasmid incompatibility groups and types identified were: IncC (types 1 and 3), IncFIA (type 26) IncFIB, IncFII (types K1, K2, K7, and K9), IncHI1B, and IncL. None of the bla_NDM-1 and bla_ESBL-plasmids identified in this study were previously described. Most bla_NDM-1-plasmids shared identical AMR regions suggesting potential genetic material/plasmid exchange between K. pneumoniae isolates of this collection. The majority of NGS-based AMR profiles from the E. coli transconjugants correlated well with both NGS-based predicted and conventional AST results.

Conclusion: This study highlights the complexity and diversity of the plasmid-based genetic background of carbapenemase-producing clinical isolates from Pakistan. This study emphasizes the need for characterization of MDR plasmids to determine their complete molecular background and monitor AMR through plasmid transmission between multi-resistant bacterial pathogens.

Keywords: AMR data analysis; AMR determinants; Klebsiella pneumoniae; MDR plasmids; nanopore hybrid assemblies.

Figure 1

Comparison of plasmid-sequence assembly by Miniasm, WTDBG2, Canu, and Flye. Three plasmids were isolated from each of two K. pneumoniae strains: BL12456 (pKpBL12456-1, −2 and −3; A), and MS84 (pKpMS84-1, −2 and −3; B). Assembled was considered a complete sequence with the same nucleotide order as the reference sequence (navy); partially assembled represented a sequence that was assembled, but not as a single contig (green); misassembled included a sequence that was assembled, but the bases were in a different order than the reference sequence, or a sequence assembled multiple times resulting in an incomplete assembly (yellow); unassembled refers to a sequence that was not present in the reference assembly (orange).

Figure 2

Annotation Diagram of (A) pB557-NDM_KX786648, (B) MS84_pNDM, and (C) Ec_MS84_pNDM using Galileo AMR platform. Gaps >50 (base pairs) bp are indicated by dashed red lines and the length in bp is given. Genes features (e.g., bla_CMY-6, sul1, rmtC, bla_NDM-1) are shown by arrows; gene cassettes (e.g., bla_OXA-1/aacA4.2) by pale blue boxes; the CS of integrons as orange boxes; and IS (e.g., ISEc23) as white block arrows labelled with the IS number/name and the pointed end indicating IR_R. Unit transposons (e.g., Tn1696) are shown as boxes of different colors and their IR are shown as flags, with the flat side at the outer boundary of the transposon. Truncated features (e.g., Tn1696) are shown with a jagged edge on the truncated side(s). Direct repeats flanking ISs are shown as ‘lollipops’ of the same color ISEcp1 (white) and ISEc23(green). Here, an ISEc23 has been inserted in MS84_pNDM and Ec_MS84_pNDM compared to pB557-NDM-KX786648.

Figure 3

Annotation Diagram of (A) pKPX-1-NDM_AP012055, (B) BL13802_pNDM, and (C) Ec_BL13802_pNDM using Galileo AMR platform. Gaps >50 (base pairs) bp are indicated by dashed red lines and the length in bp is given. Genes features (e.g., catB12, rmtF, bla_NDM-1) are shown by arrows; gene cassettes (catB3/aacA4/arr2) by pale blue boxes; the CS of integrons as orange boxes; and IS (e.g., IS3000) as white block arrows labelled with the IS number/name and the pointed end indicating IR_R. Unit transposons are shown as boxes of different colors (e.g., Tn5403, blue, Tn40, dark purple) and their IR are shown as flags, with the flat side at the outer boundary of the transposon. Truncated features (e.g., 3’-CS) are shown with a jagged edge on the truncated side(s). Direct repeats flanking ISs are shown as ‘lollipops’ of the same color ISKpn21 (green) and IS903B (yellow). Here, a 46-kb region containing aacA4, catB3, and arr2 cassettes is found in pKPX-1-A012055 upstream of bla_NDM-1 both in BL13802_pNDM, and Ec_BL13802_pNDM.

Figure 4

Representation of AMR modules A-D described in this study using Galileo AMR platform.

Figure 5

Representation of AMR modules N0-N3 described in this study using Galileo AMR platform.

Figure 6

Annotation Diagrams of pNDM-plasmids (A) BL849_pNDM, (B) BL12125/456_pNDM, (C) BU19801_pNDM, and (D) BA3783_pNDM using Galileo AMR platform. Gaps >50 bp are indicated by dashed red lines and the length in bp is given. Genes features (e.g., armA, sul1, bla_NDM-1) are shown by arrows; gene cassettes (arr2/OXA-10/catB3) by pale blue boxes; the CS of integrons as orange boxes; and IS (e.g., IS3000) as white block arrows labelled with the IS number/name and the pointed end indicating IR_R. Unit transposons are shown as boxes of different colors (e.g., Tn5393, green) and their IR are shown as flags, with the flat side at the outer boundary of the transposon. Truncated features (e.g., 3’-CS) are shown with a jagged edge on the truncated side(s). Direct repeats flanking ISs are shown as ‘lollipops’ of the same color ISKpn21 (green). Modules A, B, C and D are represented by yellow, blue, purple, and light green boxes. Modules N1, N2 and N3 comprising bla_NDM-1 are represented by red boxes.

Figure 7

Annotation Diagrams of OXA-48-plasmids (A) pRJ119-1-OXA-48_KX636096, (B) pBA2664-NDM, (C) pBL8800_OXA-48, (D) pBABL2880_OXA-48, (E) pBA3783-OXA-48, and (F) pBL13802_OXA-48 using Galileo AMR platform. Gaps >50 bp are indicated by dashed red lines and the length in bp is given. Genes features (i.e., bla_OXA-48) are shown by arrows, and IS (e.g., IS1999, IS1) as white block arrows labelled with the IS number/name and the pointed end indicating IR_R. Truncated features (e.g., IS1999) are shown with a jagged edge on the truncated side(s). Direct repeats flanking ISs are shown as ‘lollipops’ of the same color IS1R (white). Here, the bla_OXA-48 module found in all isolates are the same and identical to the module identified in pRJ119-2_KX636096.