IS26-mediated amplification of blaOXA-1 and blaCTX-M-15 with concurrent outer membrane porin disruption associated with de novo carbapenem resistance in a recurrent bacteraemia cohort

Abstract

Background: Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms.

Objectives: To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population.

Methods: This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively.

Results: Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure.

Conclusions: These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.

Figure 1.

Timeline showing date of serial strain isolation from blood cultures. Patient numbers and species isolated are in the first column. The shape and colour of the isolate labels indicate potential clonality and AMR, respectively. Isolates were considered possibly clonal if they were the same ST and clustered on the phylogenetic tree presented in Figure S1. Patient subgroups (e.g. Patient 1; isolates A and B) refer to the order of isolation.

Figure 2.

PCR analysis of β-lactamase gene levels and transcript levels for Patient 4 isolates (4A–D). (a) TaqMan qPCR of genomic DNA results collected in triplicate on two separate days (n = 6) for either bla_OXA-1 or bla_CTX-M-15 relative to the endogenous control gene rpsL. Data shown are individual data points with mean ± SD superimposed. (b) bla_OXA-1 transcript level relative to endogenous control gene rpsL. RNA was collected from mid-exponential phase in triplicate on two separate days (n = 6). Data shown are mean ± SD. (c) Similar analysis to (b) except that data are for bla_CTX-M-15. P values refer to measurements in the serial isolates relative to the initial isolate using the Kruskal–Wallis test.

Figure 3.

Characterization of IS26-flanked transposon, TnMB1860, with transposition of modular TUs in Patient 4 serial isolates (i.e. isolates 4A–D). Terminal left and right inverted repeats (IR_L and IR_R, respectively) of ISs are specified by grey triangles that bracket complete and incomplete tnpA genes, respectively. ORFs are coloured as follows: non-β-lactamase AMR genes (maroon), bla_OXA-1 (blue), bla_CTX-M-15 (green), IS26 tnpA (white) and other IS/Tn elements (grey). Font colour for each serial isolate labelled represents ertapenem susceptibility (black) or ertapenem resistance (red). (a) Schematic indicates chromosomal context (GenBank accession #: CP049085) of TnMB1860 locus flanked by directly oriented IS26 transposases found in the 4A isolate. Immediately below the schematic are normalized, short-read coverage depth line graphs for the four Patient 4 serial isolates with MB1860TU_A bracketed by dotted lines. ETP-S, ertapenem susceptible; ETP-R, ertapenem resistant. (b–d) Characterization of MB1860TU_A transposition and ORF disruption events in each of the respective Patient 4 recurrent episode isolates. Black brackets beneath ORFs indicate MB168TU_A. (b) Isolate 4B chromosomal context shows an ∼10× MB1860TU_A amplification event in the original 4A chromosomal locus. (c) Isolate 4C chromosomal context additionally contains transposition and disruption of the ompC porin gene (pink) upstream of the original isolate 4A TnMB1860 locus with three copies of MB1860TU_A. (d) Isolate 4D has previous transposition events found in isolates 4B and 4C as well as another MB1860TU_A transposition, amplification and disruption of a putative glycoside hydrolase gene (i.e. GH25; yellow) downstream of the TnMB1860 locus. The TSDs created by the transposition of the TU are indicated above each respective junction site flanking the insertion and disruption of the ompC and GH25 genes, respectively.

Figure 4.

Identification and characterization of β-lactamase gene amplification following serial passaging of the index isolate 4A under ertapenem exposure. Isolate 4A was grown in the presence of ertapenem with isolates 4A_1 to 4A_4 collected during the first round of passaging and 4A_H1 and 4A_H2 collected during the second round. (a) Schematic of TnMB1860 locus from isolate 4A as detailed in Figure 3. Immediately below the schematic are normalized, short-read coverage depth line graphs for isolates 4A_1 and 4A_H2 aligned to index isolate 4A with the location of MB1860TU_A and MB1860TU_B bracketed by dotted lines. Note amplification of MB1860TU_C (i.e. MB1860TU_A and MB1860TU_C combined) in isolate 4A_1 whereas only MB1860TU_B is amplified in isolate 4A_H2. (b, c) TaqMan qPCR of genomic DNA collected in triplicate on two separate days (n = 6) for either bla_OXA-1 (b) or bla_CTX-M (c) relative to the endogenous control gene rpsL. Data shown are individual data points with mean ± SD superimposed.

Figure 5.

Genomic context of MB101 (K. pneumoniae) and MB746 (E. coli) isolates respectively. Terminal left and right inverted repeats (IR_L and IR_R, respectively) of ISs are specified by grey triangles that bracket complete and incomplete tnpA genes, respectively. ORFs are coloured as follows: AMR genes (maroon), bla_OXA-1 (blue), bla_CTX-M-15 (green), IS26 tnpA (white) and other IS/Tn elements (grey). Delta (Δ) next to an annotated gene or genetic region indicates a truncation or disruption. (a, b) Chromosomal locations of MB101TU (a) and MB101TPU (b) indicating amplification and transposition of each element, respectively. The black bracket indicates an ∼8× MB101TU repeat. Truncated ompK36 gene (b) is labelled in pink. (c, d) Genomic context of MB746TU and respective modules carrying AMR genes. Black brackets beneath (c) schematic indicate repeating MB746TU. (d) shows FIB plasmid carriage of AMR gene-containing modules. The red bracket indicates an IS26-bounded transposon structure that shares 100% coverage; 99% BLAST similarity with TnMB1860. The blue bracket indicates a small, 2× repeat structure.