Use of Nanopore Sequencing to Characterise the Genomic Architecture of Mobile Genetic Elements Encoding blaCTX-M-15 in Escherichia coli Causing Travellers’ Diarrhoea

Abstract

Increasing levels of antimicrobial resistance (AMR) have been documented in Escherichia coli causing travellers’ diarrhoea, particularly to the third-generation cephalosporins. Diarrhoeagenic E. coli (DEC) can act as a reservoir for the exchange of AMR genes between bacteria residing in the human gut, enabling them to survive and flourish through the selective pressures of antibiotic treatments. Using Oxford Nanopore Technology (ONT), we sequenced eight isolates of DEC from four patients’ specimens who had all recently returned to the United Kingdome from Pakistan. Sequencing yielded two DEC harbouring blaCTX-M-15 per patient, all with different sequence types (ST) and belonging to five different pathotypes. The study aimed to determine whether blaCTX-M-15 was located on the chromosome or plasmid and to characterise the drug-resistant regions to better understand the mechanisms of onward transmission of AMR determinants. Patients A and C both had one isolate where blaCTX-M-15 was located on the plasmid (899037 & 623213, respectively) and one chromosomally encoded (899091 & 623214, respectively). In patient B, blaCTX-M-15 was plasmid-encoded in both DEC isolates (786605 & 7883090), whereas in patient D, blaCTX-M-15 was located on the chromosome in both DEC isolates (542093 & 542099). The two blaCTX-M-15-encoding plasmids associated with patient B were different although the blaCTX-M-15-encoding plasmid isolated from 788309 (IncFIB) exhibited high nucleotide similarity to the blaCTX-M-15-encoding plasmid isolated from 899037 (patient A). In the four isolates where blaCTX-M-15 was chromosomally encoded, two isolates (899091 & 542099) shared the same insertion site. The blaCTX-M-15 insertion site in isolate 623214 was described previously, whereas that of isolate 542093 was unique to this study. Analysis of Nanopore sequencing data enables us to characterise the genomic architecture of mobile genetic elements encoding AMR determinants. These data may contribute to a better understanding of persistence and onward transmission of AMR determinants in multidrug-resistant (MDR) E. coli causing gastrointestinal and extra-intestinal infections.

Comparison of Illumina and Oxford Nanopore Technology for genome analysis of Francisella tularensis, Bacillus anthracis, and Brucella suis

“…Similar sets of genetic markers for virulence were found, regardless of the used sequencing technology, flow cell version, or assembly approach. This is in line with other studies, focusing on genetic markers for resistance against antimicrobial agents [ 28 , 30 , 32 ]. Whether ONT data alone is sufficient to also detect mutations causing resistance against antimicrobial agents [ 45 ], needs to be further elucidated.…”

“…The PCR markers of both plasmids were found with small exceptions using data from Illumina, ONT, or their combinations. The ability of ONT to assemble closed plasmids has been demonstrated for other species in different studies [ 28 , 30 , 32 ] is a large advantage over Illumina data. A recent study has used the potential of ONT data to assemble plasmids and together with highly accurate Illumina sequencing data supported the identification of routes of infections in a hospital outbreak with clonal spread and cross-species exchange of plasmids [ 30 ].…”

“…This issue might be induced by problems within the ONT data assembly process, since the integration of Illumina data during the polishing step resulted in correct. As ST 16 is the predominant ST in Germany, the detection of other STs remains untested, while the application of ONT sequencing for MLST was successfully applied for other species [ 28 , 32 ].…”

“…These long reads simplify the assembly process and thus allow the reconstruction of complete and closed bacterial genomes [ 28 ]. Indeed, ONT data has recently been utilized for bacterial genotyping, both in combination with Illumina data [ 24 , 29 – 32 ], but also without Illumina data [ 33 , 34 ].…”

Long-Read Whole Genome Sequencing Elucidates the Mechanisms of Amikacin Resistance in Multidrug-Resistant Klebsiella pneumoniae Isolates Obtained from COVID-19 Patients

“…The diffusion of resistance genes is usually attributed to horizontal gene transfer mediated by plasmids, and conjugated plasmids are recognized as important vectors for AMR gene transmission in Gram-negative bacteria [ 12 , 13 ]. In recent years, whole genome sequencing (WGS), especially long-read sequencing, has become a powerful tool for the determination of plasmid structures and AMR gene locations [ 14 , 15 , 16 ]. WGS also allows for performing a reliable epidemiological surveillance for outbreak investigations, in which it is vitally important to determine whether particular bacterial isolates belong to an outbreak-causing strain, or not [ 17 ].…”