Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories.

Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a Salmonella isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.

Mobility of ß-lactam resistance under ampicillin treatment in gut microbiota suffering from pre-disturbance.

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to dissemination of antibiotic resistance genes (ARGs) in the gut microbiota. The gut microbiota often suffers from various disturbances. It is not clear whether and how disturbed microbiota may affect ARG mobility under antibiotic treatments. For proof of concept, in the presence or absence of streptomycin pre-treatment, mice were inoculated orally with a ß-lactam-susceptible Salmonella enterica serovar Heidelberg clinical isolate (recipient) and a ß-lactam resistant Escherichia coli O80:H26 isolate (donor) carrying a blaCMY-2 gene on an IncI2 plasmid. Immediately following inoculation, mice were treated with or without ampicillin in drinking water for 7 days. Faeces were sampled, donor, recipient and transconjugant were enumerated, blaCMY-2 abundance was determined by quantitative PCR, faecal microbial community composition was determined by 16S rRNA amplicon sequencing and cecal samples were observed histologically for evidence of inflammation. In faeces of mice that received streptomycin pre-treatment, the donor abundance remained high, and the abundance of S. Heidelberg transconjugant and the relative abundance of Enterobacteriaceae increased significantly during the ampicillin treatment. Co-blooming of the donor, transconjugant and commensal Enterobacteriaceae in the inflamed intestine promoted significantly (P<0.05) higher and possibly wider dissemination of the blaCMY-2 gene in the gut microbiota of mice that received the combination of streptomycin pre-treatment and ampicillin treatment (Str-Amp) compared to the other mice. Following cessation of the ampicillin treatment, faecal shedding of S. Heidelberg transconjugant persisted much longer from mice in the Str-Amp group compared to the other mice. In addition, only mice in the Str-Amp group shed a commensal E. coli O2:H6 transconjugant, which carries three copies of the blaCMY-2 gene, one on the IncI2 plasmid and two on the chromosome. The findings highlight the significance of pre-existing gut microbiota for ARG dissemination and persistence during and following antibiotic treatments of infectious diseases.

On-Farm Anaerobic Digestion of Dairy Manure Reduces the Abundance of Antibiotic Resistance-Associated Gene Targets and the Potential for Plasmid Transfer.

The present study investigated the impact of on-farm anaerobic digestion on the abundance of enteric bacteria, antibiotic resistance-associated gene targets, and the horizontal transfer potential of extended-spectrum ß-lactamase (ESBL) genes. Samples of raw and digested manure were obtained from six commercial dairy farms in Ontario, Canada. Digestion significantly abated populations of viable coliforms in all six farms. Conjugative transfer of plasmids carrying ß-lactamase genes from manure bacteria enriched overnight with buffered peptone containing 4 mg/liter cefotaxime into a ß-lactam-sensitive green fluorescent protein (GFP)-labeled Escherichia coli recipient strain was evaluated in patch matings. Digestion significantly decreased the frequency of the horizontal transfer of ESBL genes. Twenty-five transconjugants were sequenced, revealing six distinct plasmids, ranging in size from 40 to 180 kb. A variety of ESBL genes were identified: blaCTX-M-1, blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaPER-1. blaCTX-M-15 was the most prevalent ESBL gene detected on plasmids harbored by transconjugants. Various mobile genetic elements were found located proximal to resistance genes. Ten gene targets, including sul1, str(A), str(B), erm(B), erm(F), intI1, aadA, incW, blaPSE, and blaOXA-20, were quantified by quantitative PCR on a subset of 18 raw and 18 digested samples. Most targets were significantly more abundant in raw manure; however, erm(B) and erm(F) targets were more abundant in digested samples. Overall, on-farm digestion of dairy manure abated coliform bacteria, a number of antibiotic resistance-associated gene targets, and the potential for in vitro conjugation of plasmids conferring resistance to extended-spectrum ß-lactams and other classes of antibiotics into E. coli CV601. IMPORTANCE Using livestock manure for fertilization can entrain antibiotic-resistant bacteria into soil. Manure on some dairy farms is anaerobically digested before being land applied. Recommending the widespread implementation of the practice should be founded on understanding the impact of this treatment on various endpoints of human health concern. Although lab-scale anaerobic treatments have shown potential for reducing the abundance of antibiotic resistance genes, there are very few data from commercial farms. Anaerobic digestion of manure on six dairy farms efficiently abated coliform bacteria, E. coli, and a majority of antibiotic resistance-associated gene targets. In addition, the conjugation potential of plasmids carrying ESBL genes into introduced E. coli strain CV601 was reduced. Overall, anaerobic digestion abated coliform bacteria, the genes that they carry, and the potential for ESBL-carrying plasmid transfer.

Complete Genome Sequences for 36 Canadian Salmonella enterica Serovar Typhimurium and I 1,4,[5],12:i:- Isolates from Clinical and Animal Sources.

Here, we report the complete genome sequences for 36 Canadian isolates of Salmonella enterica subsp. enterica serovar Typhimurium and its monophasic variant I 1,4,[5]:12:i:- from both clinical and animal sources. These genome sequences will provide useful references for understanding the genetic variation within this prominent serotype.

Mobility of ß-Lactam Resistance Under Bacterial Co-infection and Ampicillin Treatment in a Mouse Model.

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to the dissemination of antibiotic-resistance genes in the gut microbiota and the development of antibiotic-resistant bacterial infection, a significant threat to animal and public health. Food or water may be contaminated with multiple resistant bacteria, but animal models on gene transfer were mainly based on single-strain infections. In this study, we investigated the mobility of ß-lactam resistance following infection with single- versus multi-strain of resistant bacteria under ampicillin treatment. We characterized three bacterial strains isolated from food-animal production systems, Escherichia coli O80:H26 and Salmonella enterica serovars Bredeney and Heidelberg. Each strain carries at least one conjugative plasmid that encodes a ß-lactamase. We orally infected mice with each or all three bacterial strain(s) in the presence or absence of ampicillin treatment. We assessed plasmid transfer from the three donor bacteria to an introduced E. coli CV601gfp recipient in the mouse gut, and evaluated the impacts of the bacterial infection on gut microbiota and gut health. In the absence of ampicillin treatment, none of the donor or recipient bacteria established in the normal gut microbiota and plasmid transfer was not detected. In contrast, the ampicillin treatment disrupted the gut microbiota and enabled S. Bredeney and Heidelberg to colonize and transfer their plasmids to the E. coli CV601gfp recipient. E. coli O80:H26 on its own failed to colonize the mouse gut. However, during co-infection with the two Salmonella strains, E. coli O80:H26 colonized and transferred its plasmid to the E. coli CV601gfp recipient and a residential E. coli O2:H6 strain. The co-infection significantly increased plasmid transfer frequency, enhanced Proteobacteria expansion and resulted in inflammation in the mouse gut. Our findings suggest that single-strain infection models for evaluating in vivo gene transfer may underrepresent the consequences of multi-strain infections following the consumption of heavily contaminated food or water.

Complete Genome and Plasmid Sequences of 32 Salmonella enterica Strains from 30 Serovars.

We report here 32 completed closed genome sequences of strains representing 30 serotypes of Salmonella. These genome sequences will provide useful references for understanding the genetic variation within Salmonella enterica serotypes, particularly as references to aid in comparative genomics studies, as well as providing information for improving in silico serotyping accuracy.