ena-STUDY-IMGAG (Universitaetsklinikum Tuebingen)-28-03-2019-12:40:25:215-133
Tracking of antibiotic resistance transfer and rapid plasmid evolution in a hospital setting by Nanopore sequencing.
Background: Infection or colonization of patients with multidrug-resistant (MDR) bacteria often leave very limited or no treatment options. The spread of multidrug resistances carrying plasmids by horizontal gene transfer between different bacterial species represents an important mode of expansion of antimicrobial resistance genes.  At the beginning of 2009 we experienced an outbreak with an extensively multidrug resistant P. aeruginosa harbouring a carbapenemase enzyme (blaIMP-8). Interestingly, in March 2012 we detected the first Citrobacter freundii, followed by Citrobacter werkmanii, harbouring the same enzyme blaIMP-8 carbapenemase. Since this enzyme is rarely encountered in Europe we hypothesised, that horizontal gene transfer between the different Gram-negative species had occurred within our hospital. Material and Methods: All blaIMP-8 positive strains isolated in our hospital from patients or patient-related environmental water sources (n=54) over a 6 years' period were included in the study, comprising Citrobacter freundii (n=8), Citrobacter werkmanii (1) and P. aeruginosa (n=45) strains. Short-read whole genome sequences were generated from all isolates. Long read Nanopore sequencing was conducted of all Citrobacter species isolates (n=9) and selected P. aeruginosa isolates (n=5) representing different time points. In order to obtain finished genomes and circularized plasmids from the hybrid sequencing data, a new analysis pipeline was developed.  Results: We identified a 40 kb plasmid (plasmid A) harbouring blaIMP-8 in P. aeruginosa and Citrobacter freundii, indicating that plasmid transfer had occurred between the two species. Within the Citrobacter species the plasmid underwent further evolution and HGT, resulting in the detection of a blaIMP-8 harbouring 164 kb megaplasmid (plasmid C) in C. werkmanii. The mega-plasmid is most likely the result of plasmid fusion, since it contains a 40 kb region with 100% genetic homology to plasmid A, in addition to an 88 kb region highly homologous to another plasmid (plasmid B) detected in C. freundii. Moreover, changes of the multidrug resistance gene cassette on the class I integron were noted, including deletions and translocations of complete antimicrobial resistance genes.  Conclusion: The results demonstrate, that the chosen approach enabled us to track plasmid evolution dynamics during a hospital outbreak, driven by plasmid transfer between different species, plasmid fusion and evolution of the antibiotic resistance gene cassette on the plasmids.
Background: Infection or colonization of patients with multidrug-resistant (MDR) bacteria often leave very limited or no treatment options. The spread of multidrug resistances carrying plasmids by horizontal gene transfer between different bacterial species represents an important mode of expansion of antimicrobial resistance genes.  At the beginning of 2009 we experienced an outbreak with an extensively multidrug resistant P. aeruginosa harbouring a carbapenemase enzyme (blaIMP-8). Interestingly, in March 2012 we detected the first Citrobacter freundii, followed by Citrobacter werkmanii, harbouring the same enzyme blaIMP-8 carbapenemase. Since this enzyme is rarely encountered in Europe we hypothesised, that horizontal gene transfer between the different Gram-negative species had occurred within our hospital. Material and Methods: All blaIMP-8 positive strains isolated in our hospital from patients or patient-related environmental water sources (n=54) over a 6 years' period were included in the study, comprising Citrobacter freundii (n=8), Citrobacter werkmanii (1) and P. aeruginosa (n=45) strains. Short-read whole genome sequences were generated from all isolates. Long read Nanopore sequencing was conducted of all Citrobacter species isolates (n=9) and selected P. aeruginosa isolates (n=5) representing different time points. In order to obtain finished genomes and circularized plasmids from the hybrid sequencing data, a new analysis pipeline was developed.  Results: We identified a 40 kb plasmid (plasmid A) harbouring blaIMP-8 in P. aeruginosa and Citrobacter freundii, indicating that plasmid transfer had occurred between the two species. Within the Citrobacter species the plasmid underwent further evolution and HGT, resulting in the detection of a blaIMP-8 harbouring 164 kb megaplasmid (plasmid C) in C. werkmanii. The mega-plasmid is most likely the result of plasmid fusion, since it contains a 40 kb region with 100% genetic homology to plasmid A, in addition to an 88 kb region highly homologous to another plasmid (plasmid B) detected in C. freundii. Moreover, changes of the multidrug resistance gene cassette on the class I integron were noted, including deletions and translocations of complete antimicrobial resistance genes.  Conclusion: The results demonstrate, that the chosen approach enabled us to track plasmid evolution dynamics during a hospital outbreak, driven by plasmid transfer between different species, plasmid fusion and evolution of the antibiotic resistance gene cassette on the plasmids.