The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.
Background
Levofloxacin prophylaxis is recommended to prevent Gram-negative bloodstream infections (BSIs) in patients with prolonged chemotherapy-induced neutropenia. However, increasing fluoroquinolone resistance may decrease the effectiveness of this approach.
Methods
We assessed the prevalence of colonization with fluoroquinolone-resistant Enterobacterales (FQRE) among patients admitted for hematopoietic cell transplantation (HCT) from November 2016-August 2019 and compared the risk of Gram-negative BSI between FQRE-colonized and non-colonized patients. All patients received levofloxacin prophylaxis during neutropenia. Stool samples were collected upon admission for HCT and weekly thereafter until recovery from neutropenia, and underwent selective culture for FQRE. All isolates were identified and underwent antimicrobial susceptibility testing by broth microdilution. FQRE isolates also underwent whole-genome sequencing.
Results
Fifty-four (23%) of 234 patients were colonized with FQRE prior to HCT, including 30 (25%) of 119 allogeneic and 24 (21%) of 115 autologous HCT recipients. Recent antibacterial use was associated with FQRE colonization (P=0.048). Ninety-one percent of colonizing FQRE isolates were Escherichia coli and 29% produced extended-spectrum ß-lactamases. Seventeen (31%) FQRE-colonized patients developed Gram-negative BSI despite levofloxacin prophylaxis, compared to only two (1.1%) of 180 patients who were not colonized with FQRE on admission (P<0.001). Of the 17 Gram-negative BSIs in FQRE-colonized patients, 15 (88%) were caused by FQRE isolates that were genetically identical to the colonizing strain.
Conclusions
Nearly one-third of HCT recipients with pre-transplant FQRE colonization developed Gram-negative BSI while receiving levofloxacin prophylaxis and infections were typically caused by their colonizing strains. In contrast, levofloxacin prophylaxis was highly effective in patients not initially colonized with FQRE.
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