Pham, P. T. | Search bioRxiv

bioRxiv2019

Ciprofloxacin facilitates the transfer of XDR plasmids from commensal E. coli into epidemic fluoroquinolone-resistant Shigella sonnei

The global dissemination of a ciprofloxacin-resistant (cipR) S. sonnei clone outlines the mobility of this important agent of diarrheal disease, and threatens the utility of ciprofloxacin as a first-line antimicrobial for shigellosis. Here, we aimed to track the emergence of cipR S. sonnei in Vietnam to understand how novel antimicrobial resistant (AMR) Shigella clones become established in new locations. From 2014 to 2016, we isolated and genome sequenced 79 S. sonnei from children hospitalized with dysenteric diarrhea in southern Vietnam. The novel cipR S. sonnei clone displaced the resident ciprofloxacin-susceptible lineage while acquiring resistance against third-generation cephalosporins, macrolides, and aminoglycosides. This process was not the result of a single clonal expansion, as we identified at least thirteen independent acquisitions of ESBL-encoding plasmids. The frequency and diversity of the variable AMR repertoire in an expanding clonal background of S. sonnei is unprecedented and we speculated that it was facilitated by horizontal gene transfer from commensal organisms in the human gut. Consequently, we characterized non-Shigella Enterobacteriaceae from Shigella-infected and healthy children by shotgun metagenomics. We identified a wide array of AMR genes and plasmids in the commensal Enterobacteriaceae, including an E. coli isolated from a Shigella-infected child with an identical ESBL plasmid to that characterized in the infecting S. sonnei. We confirmed that these AMR plasmids could be exchanged between commensal E. coli and S. sonnei and found that supplementation of ciprofloxacin into the conjugation media significantly increased the conjugation frequency of IncI/blaCTX-M-15, IncB/O/blaCTX-M-27 and IncF/blaCTX-M-27 plasmids. In a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines. Our work highlights the role of the gut microbiota in transferring resistance plasmids into enteric pathogens and provides essential data to restrict the use of ciprofloxacin globally.

microbiology↗

bioRxiv2019

UmuD and RecA* modulate the DNA-binding activity of DNA polymerase IV in Escherichia coli

DNA polymerase IV (pol IV) is expressed at increased levels in Escherichia coli cells suffering high levels of DNA damage. In a recent single-molecule imaging study, we demonstrated that elevating the pol IV concentration is not sufficient to provide access to binding sites on the nucleoid, suggesting that other factors may recruit pol IV to its substrates once the DNA becomes damaged. Here we extend this work, investigating the proteins UmuD and RecA as potential modulators of pol IV activity. UmuD promotes long-lived association of pol IV with the nucleoid, whereas its cleaved form, UmuD, which accumulates in DNA-damaged cells, inhibits binding. In agreement with proposed roles for pol IV in homologous recombination, up to 40% of pol IV foci colocalise with a probe for RecA* nucleoprotein filaments in ciprofloxacin-treated cells. A hyperactive RecA mutant, recA(E38K), allows pol IV to bind the nucleoid even in the absence of exogenous DNA damage. In vitro, RecA(E38K) forms RecA*-like structures that can recruit pol IV, even on double-stranded DNA, consistent with a physical interaction between RecA and pol IV. Together, the results indicate that UmuD and RecA modulate the binding of pol IV to its DNA substrates, which frequently coincide with RecA* structures.

microbiology↗