London Calling 2019
The second speaker in this breakout session, Séverine Rangama from the University of Warwick, gave an overview of her work utilising long-read nanopore sequencing to assemble a large environmental blaCTX-M-15 harbouring plasmid. Opening her presentation, Séverine underlined the growing threat of antimicrobial resistance, which she described as a ‘global health emergency’. For example, in Europe alone, over 25,000 people die each year from five key antimicrobial resistant (AMR) bacteria. Alarmingly, it has been estimated that the failure to address the problem of antibiotic resistance could lead to 10 million deaths worldwide by 2050.
Séverine described how, through the use of antibiotics in human medicine, animal husbandry, plant production, and aquaculture may be a key driver for AMR. For example, resistance can be disseminated into the environment in waste water and manure, from where it spreads to lakes, rivers, and soil. Using next generation sequencing of whole sediment and cultured fractions, the team at the University of Warwick have identified waste water treatment plants (WWTPs) as key hotspots for this environmental dissemination of antimicrobial resistance genes/bacteria (ARG/ARB).
Anaerobic digesters are one potential solution to dealing with animal, food, and crop waste, producing slurry for fields and biomethane for energy; however, it is not known if such digesters can break down antimicrobial resistance bacteria/genes. The aim of this study is to test if environmentally isolated AMR bacteria can survive the conditions within these digesters.
The team isolated two E. coli strains from the River Sowe (UK), just downstream of a WWTP. The first isolate carried no mobile genetic elements or AMR genes; however, the second isolate included multiple mobile genetic elements and a number of resistance genes - including the betalactamase genes blaOXA, blaTEM, and blaCTX-M-15. The latter of which produces an enzyme that can break down the third-generation antibiotic cefotaxime.
Next the team set out to determine if the blaCTX-M-15 is secreted in anaerobic conditions. The first experiment they monitored the growth of both isolates in the presence of different concentrations of cefotaxime. As expected the susceptible strain did not grow but the blaCTX-M-15 isolate grew at all antibiotic concentrations. They were also able to see that the blaCTX-M-15 enzyme was secreted using nitrocefin - a chromogenic cephalosporin that changes colour when hydrolysed by the enzyme.
The next step in the study was to further characterise the plasmid within which the blaCTX-M-15 gene resides. Due to the high level of repeats, this was not possible using short-read sequencing technology; however, the long reads provided by nanopore sequencing in conjunction with the Canu tool enabled the generation of a complete, closed assembly. The 100 kb plasmid was revealed to be a classic conjugative plasmid exhibiting just one AMR gene (blaCTX-M-15), which is positioned near to a transposase element.
They then set out to determine whether this enzyme, if secreted in anaerobic conditions, could protect susceptible strains. By placing a culture of the susceptible strain in a previously grown culture of resistant strain, in the presence of cefotaxime, they were able to show that protection was indeed conferred.
In summary, Séverine stated nanopore sequencing ‘permitted accurate de novo assembly and has helped further resolve the AMR genes location, composition, order, function and putative mechanism of transposition’. She further commented that ‘such assembly has been previously unachievable using our existing short-read sequence data set’.
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27 май 2019
