Plasmidome AMR screening (PAMRS) workflow: a rapid screening workflow for phenotypic characterization of antibiotic resistance in plasmidomes

Background: Phenotypic characterization of antimicrobial resistance (AMR) in bacteria has remained the gold standard for investigation and monitoring of what resistance is present in an organism. However, the process is laborious and not attractive for screening multiple plasmids from a microbial community (plasmidomes). Instead, genomic tools are used, but a major bottle neck that presence of genes does not always translate into phenotypes. Methods: We designed the plasmidome AMR screening (PAMRS) workflow to investigate the presence of antibiotic resistant phenotypes in a plasmidome using Escherichia coli as a host organism. Plasmidomes were extracted from the faecal matter of chicken, cattle and humans using commercial plasmid extraction kits. Competent E. coli cells were transformed and evaluated using disk diffusion. Thirteen antibiotic resistant phenotypes were screened. Results: Here, we show that multiple antibiotic resistant phenotypes encoded by plasmids can be rapidly screened simultaneously using the PAMRS workflow. E. coli was able to pick up to 7, 5 or 8 resistant phenotypes from a single plasmidome from chicken, cattle or humans, respectively. Resistance to ceftazidime was the most frequently picked up phenotype in humans (52.6%) and cattle (90.5%), whereas in chickens, the most picked up resistant phenotype was resistance to co-trimoxazole, ceftriaxone and ampicillin (18.4% each). Conclusions: This workflow is a novel tool that could facilitate studies to evaluate the occurrence and expression of plasmid-encoded antibiotic resistance in microbial communities and their associated plasmid-host ranges. It could find application in the screening of plasmid-encoded virulence genes.

Extraction of DNA from a Wide Range of Objects by Treatment with Ammonium Salts

Currently, simple, rapid, and efficient techniques for DNA isolation from a wide range of organisms are in demand in biotechnology and bioinformatics. A key (and often limiting) step is the cell wall disruption and subsequent DNA extraction from the disintegrated cells. We have developed a new approach to DNA isolation from organisms with robust cell walls. The protocol includes the following steps: treatment of cells or tissue samples with ammonium acetate followed by cell lysis in low-salt buffer with the addition of SDS. Further DNA extraction is carried out according to standard methods. This approach is efficient for high-molecular native DNA isolation from bacteria, ascomycetes, yeast, and mammalian blood; it is also useful for express analysis of environmental microbial isolates and for plasmid extraction for two-hybrid library screening. express method for DNA isolation; ammonium salt treatment (в русских ключевых такой порядок), osmotic breakage of cells This study was financially supported by the NRC "Kurchatov Institute"-GOSNIIGENETIKA Kurchatov Genomic Center.

Selective culture enrichment and sequencing of faeces to enhance detection of antimicrobial resistance genes

ABSTRACTMetagenomic sequencing of faecal DNA can usefully characterise an individual’s intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producingEnterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes inEnterobacteriaceaeby using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer faeces were spiked with carbapenemase-producingEnterobacteriaceaeand incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction protocols increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a faecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

The prevalence and diversity of AmpC β-lactamase genes in plasmids from aquatic systems

This study aimed to investigate the presence and diversity of AmpC β-lactamase and integrase genes among DNA (genomic and plasmid) from bacterial populations in selected aquatic systems. Following an enrichment step, DNA was isolated and subjected to polymerase chain reaction (PCR) and digital droplet PCR. The intI1 gene and AmpC β-lactamase genes were present in genomic and plasmid DNA from all sites in the Mooi, Crocodile and Marico Rivers, with the exception of intI1 in the Marico River. Digital droplet PCR demonstrated that copy numbers varied considerably (0.0 to 29.38 copies per picogram of DNA). Some samples in which ampC was not detected, intI1 was present. Amplicons of ampC genes were subjected to restriction digest using HindIII. Samples where the restriction markers were absent were purified by cloning followed by plasmid extraction, PCR amplification, and sequencing of individual AmpC gene fragments. Phylogenetic analysis identified all positive AmpC genes as Class C β-lactamases, comprising of ampC, CMY- and ACT-families. Detecting AmpC and intl1 genes on plasmids suggests a high risk of horizontal gene transfer and potential dissemination of these and other antibiotic resistance genes surrounding immediate aquatic environments. Consequences of β-lactamase diversity in aquatic ecosystems are relatively unexplored in South African aquatic ecosystems.

DNA-Plasmid Extraction Of Salmonella Isolated From Raw Milk

Twenty five Isolates of Salmonella were isolated from raw milksamples which were collected from Abu-Graib village to study therelationship between their resistance to antibiotics and their plasmidscontents .Antibiotic sensitivity test for different antibiotics revealed that mostSalmonella isolates were resistant to two antibiotics .The extraction of the DNA-plasmid revealed that most Salmonellaisolates which resistant to two antibiotics or more were contained large,individual plasmids size of (55-75)Kilo Base (KB) while the othersisolates were free from such plasmid