Assessing the Incidence of Plasmid-borne Resistance to Clinically-Significant Antibiotics in Stream Sediments

<p>Plasmids in agriculturally-impacted bodies of water may play a significant role in the dissemination of antibiotic resistance (AR). High bacterial loads in stream sediment and selective pressures introduced by agricultural practices may facilitate the exchange and recombination of genetic material, creating reservoirs of AR genes that can potentially be accessed by fecal and other animal and human pathogens. </p> <p>  Transmissible plasmids were captured “exogenously” from stream sediment samples by conjugating sediment cells with a rifampicin-resistant strain of Escherichia coli. Transconjugants were tested for decreased antibiotic susceptibility using a modified Stokes disk diffusion method.  Twenty-three of thirty captured plasmids conferred decreased susceptibility to multiple antibiotics in addition to tetracycline. </p> <p>  One plasmid, pEG1-1, conferred resistance to tetracycline, tobramycin, kanamycin, ticarcilin, piperacillin, piperacillin-tazobactam, and cefepime. A method to sequence multi-drug resistance plasmids using both Oxford Nanopore MinION and Ion Torrent Personal Genome Machine sequencers was developed to sequence plasmid pEG1-1. A hybrid assembly generated a single 73,320 bp contig. Analysis of the genome revealed pEG1-1 to be an IncP-1β plasmid with two mobile genetic elements – a tn21-related transposon and an in104 complex integron – both of which carry multiple antibiotic resistance genes. </p> <p>  These findings suggest that plasmids in stream sediment are prone to the incorporation of mobile genetic elements that introduce a broad range of antibiotic resistance genes into their genome. This could cause serious risk to human health since IncP-1β plasmids are capable of transferring into nearly all Gram-negative bacteria, including fecal pathogens that get introduced to stream sediment.</p

Additional file 1: of Enhanced susceptibility of cancer cells to oncolytic rhabdo-virotherapy by expression of Nodamura virus protein B2 as a suppressor of RNA interference

Figure S1. B2 selectively enhances VSV∆51 replication in other cancer cells. (A) MCF7, HT-29, or SF-295 cells were infected with VSVΔ51 virus and small-RNA deep sequencing was performed. Virus-derived small RNAs have a length bias towards 22-mers. The enrichment for 22-mers is indicated for positive strand vsRNAs. (B) Fluorescence microscopy images of M14 or 786-O cells stably expressing EGFP-B2 or fluorescently-tagged empty vector (mock control). Figure S2. VSVΔ51-B2 does not enhance viral replication in non-cancer healthy cells or alter biodistribution in various organs. (A) Relative metabolic activity of GM38 fibroblasts infected with VSVΔ51-GFP or VSVΔ51-B2 for 48 h at an MOI of 1. The results are expressed as a percentage of the signal obtained compared to mock treatment. NS: P > 0.1, *P < 0.1, **P < 0.01, ***P < 0.001, using Student’s t-test. Only significantly different pairs are indicated on the fig. (B) We performed small-RNA deep-sequencing using MCF7, HT-29, or SF-295 cells infected with VSVΔ51-B2 at an MOI of 0.1 for 18 h. B2 expression in VSVΔ51 virus abrogates genomic cleavage as 22-mer vsRNAs are no longer prominent. VSVΔ51-B2 derived vsRNAs display a bias towards positive strand reads in M14 and 786-O cells. (C) Relative metabolic activity of RENCA cells infected with VSVΔ51-GFP or VSVΔ51-B2 for 48 h at an MOI of 1. The results are expressed as a percentage of the signal obtained compared to mock treatment. (D&E) Biodistribution of VSVΔ51-B2 in tumour-bearing C57BL/6 mice. Viral titers obtained from organs of tumour-bearing C57BL/6 mice, D] 24 or E] 48 hpi. Virus was administered intravenously at a dose of 1E9 pfu of VSVΔ51-GFP or VSVΔ51-B2. For organs where virus was undetectable, the titer was considered to be the value of the limit of detection of titering for this assay (5E1 pfu/organ). NS: P > 0.1, *P < 0.1, **P < 0.01, ***P < 0.001, using Student’s t-test. (PDF 5336 kb

Additional file 2: of Enhanced susceptibility of cancer cells to oncolytic rhabdo-virotherapy by expression of Nodamura virus protein B2 as a suppressor of RNA interference

Table S1. List of primers used in qRT-PCR assays. (DOCX 14 kb

Additional file 3: Figure S3. of Combination of Paclitaxel and MG1 oncolytic virus as a successful strategy for breast cancer treatment

IFNβ pre-treatment protects EMT6, 4 T1 and EO771 cells from virus-mediated killing. Coomassie Blue staining of the various breast cancer cell lines 48 h post infection with MG1-GFP with or without pre-treatment with recombinant murine IFNβ. (TIF 2149 kb

Additional file 1: Figure S1. of Combination of Paclitaxel and MG1 oncolytic virus as a successful strategy for breast cancer treatment

Murine breast cancer cell lines display different sensitivities to PAC. A EMT6, 4 T1 and EO771 cells were treated with increasing concentrations of PAC. Coomassie Blue staining was used to assess the toxic dose of the drug in the different cell lines after 72 h. B Fluorescent microscopy pictures of DAPI-stained cells with or without treatment with 0.5uM PAC for 72 h. (TIF 14849 kb