Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: a case study of an outbreak in a rural Ethiopian hospital

Abstract

Objectives MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements. We believe that this method has the potential to be a tool of pivotal importance for MDR control. Methods Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria. Results ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles. Conclusions ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low- and middle-income countries.

Identity of blaCTX-M Carrying Plasmids in Sequential ESBL-E. coli Isolates from Patients with Recurrent Urinary Tract Infections

“…This was detectable thanks to the ODM method that gives a detailed fingerprint for each plasmid that can be compared between isolates. A further advantage of the ODM method is the ability to overcome the plasticity of plasmids when analyzing plasmid identity over time, with insertions and deletions, as demonstrated also in our previous studies [18,20,21].…”

“…In combination with CRISPR/Cas9 restriction, we can obtain information about plasmid size, resistance gene location, and barcode in the same experiment [17]. For the characterization of plasmids, we have previously demonstrated that the ODM results correlate very well with NGS analysis [14,[18][19][20]. We have also showed its usefulness in tracking possible transmission routes in nosocomial outbreak investigations, in a neonatal polyclonal outbreak due to CTX-M-producing bacteria in Sweden, and in an ESBL E. coli ST410 outbreak in a clinical setting in Ethiopia [12,20].…”

Polyphasic characterization of carbapenem-resistant Klebsiella pneumoniae clinical isolates suggests vertical transmission of the blaKPC-3 gene

“…In addition, the dynamic nature of plasmidomes, including repetitive sequences, presence of multiple replicons on a single plasmid, and presence of multiple plasmids of similar or different sizes in a cell, provide challenges to all of the mentioned techniques and might contribute to confusing or inconclusive results, giving room for the development of novel plasmid characterization methods. Optical DNA mapping (ODM), based on direct visualization of individual intact plasmid molecules using fluorescence microscopy, has shown great potential as a rapid and simple method to analyse and compare plasmids [15][16][17][18]. In ODM, large DNA molecules are labelled in a sequence specific fashion and upon stretching in nanofluidic channels and imaging by fluorescence microscopy [19], a pattern reflecting the underlying DNA sequence, a barcode, is obtained [20,21].…”

“…In ODM, large DNA molecules are labelled in a sequence specific fashion and upon stretching in nanofluidic channels and imaging by fluorescence microscopy [19], a pattern reflecting the underlying DNA sequence, a barcode, is obtained [20,21]. The method can distinguish different plasmids in a sample, based on their sizes and barcodes, which can be used for identification [17] and tracing [15,16,18], for example during a resistance outbreak. Furthermore, using the CRISPR/Cas9 system makes it possible to, in the same experiment, identify a specific (resistance) gene and determine its location along the plasmid sequence [22].…”

Optical DNA Mapping of Plasmids Reveals Clonal Spread of Carbapenem-Resistant Klebsiella pneumoniae in a Large Thai Hospital

“…We have applied the method extensively to plasmids and in combination with Cas9 restriction, the gene causing the resistance can be identified [17]. The barcodes can be used to compare plasmids between different isolates and to compare with a theoretical sequence obtained from other sequencing techniques [18,19]. The ODM method has been successfully used in several studies of clinical relevance, including identifying potential conjugation during a resistance outbreak [20,21], or in patients with recurring urinary tract infections [22,23] as well as identifying clonal spread of bacteria in a hospital setting [19].…”