Evaluation of multiplex nanopore sequencing for Salmonella serotype prediction and antimicrobial resistance gene and virulence gene detection.

In a previous study, Multiplex-nanopore-sequencing based whole genome sequencing (WGS) allowed for accurate in silico serotype prediction of Salmonella within one day for five multiplexed isolates, using both SISTR and SeqSero2. Since only ten serotypes were tested in our previous study, the conclusions above were yet to be evaluated in a larger scale test. In the current study we evaluated this workflow with 69 Salmonella serotypes and also explored the feasibility of using multiplex-nanopore-sequencing based WGS for antimicrobial resistance gene (AMR) and virulence gene detection. We found that accurate in silico serotype prediction with nanopore-WGS data was achieved within about five hours of sequencing at a minimum of 30× Salmonella genome coverage, with SeqSero2 as the serotype prediction tool. For each tested isolate, small variations were observed between the AMR/virulence gene profiles from the Illumina and Nanopore sequencing platforms. Taking results generated using Illumina data as the benchmark, the average precision value per isolate was 0.99 for both AMR and virulence gene detection. We found that the resistance gene identifier - RGI identified AMR genes with nanopore data at a much lower accuracy compared to Abricate, possibly due to RGI's less stringent minimum similarity and coverage by default for database matching. This study is an evaluation of multiplex-nanopore-sequencing based WGS as a cost-efficient and rapid Salmonella classification method, and a starting point for future validation and verification of using it as a AMR/virulence gene profiling tool for the food industry. This study paves the way for the application of nanopore sequencing in surveillance, tracking, and risk assessment of Salmonella across the food supply chain.

Effect of dietary water intake on urinary output, specific gravity and relative supersaturation for calcium oxalate and struvite in the cat.

It has been reported that daily fluid intake influences urinary dilution, and consequently the risk of urolithiasis in human subjects and dogs. The aim of the present study was to investigate the role of dietary moisture on urinary parameters in healthy adult cats by comparing nutritionally standardised diets, varying only in moisture content. A total of six cats were fed a complete dry food (6.3 % moisture) hydrated to 25.4, 53.2 and 73.3 % moisture for 3 weeks in a randomised block cross-over design. Urinary specific gravity (SG), urine volume, water drunk and total fluid intake were measured daily; relative supersaturation (RSS) for calcium oxalate (CaOx) and struvite was calculated using the SUPERSAT computer program. Cats fed the 73.3 % moisture diet produced urine with a significantly lower SG (P < 0.001) compared with diets containing 53.2 % moisture or lower. Mean RSS for CaOx was approaching the undersaturated zone (1.14 (sem 0.21); P = 0.001) for cats fed the diet with 73.3 % moisture and significantly lower than the 6.3 % moisture diet (CaOx RSS 2.29 (sem 0.21)). The effect of diet on struvite RSS was less clear, with no significant difference between treatment groups. Total fluid intake was significantly increased (P < 0.001) in the 73.3 % moisture diet (144.7 (SEM 5.2) ml, or 30 ml/kg body weight per d) compared with the 6.3 % (103.4 (SEM 5.3) ml), 25.4 % (98.6 (SEM 5.3) ml) and 53.3 % (104.7 (SEM 5.3) ml) moisture diets, despite voluntary water intake decreasing as dietary moisture intake increased. Cats fed the 73.3 % moisture diet had a higher total daily fluid intake resulting in a more dilute urine with a lower risk of CaOx when compared with the lower-moisture diets.

Nutrient intake and urine composition in calcium oxalate stone-forming dogs: comparison with healthy dogs and impact of dietary modification.

Nutrient intake and urine composition were analyzed in calcium oxalate (CaOx)stone-forming and healthy control dogs to identify factors that contribute to CaOx urolithiasis. Stone-forming dogs had significantly lower intake of sodium, calcium, potassium, and phosphorus and significantly higher urinary calcium and oxalate concentrations, calcium excretion, and CaOx relative supersaturation (RSS). Feeding a diet used in the treatment of canine lower urinary tract disease for 1 month was associated with increased intake of moisture, sodium, and fat; reduced intake of potassium and calcium; and decreased urinary calcium and oxalate concentrations, calcium excretion, and CaOx RSS. No clinical signs of disease recurrence were observed in the stone-forming dogs when the diet was fed for an additional 11 months. The results suggest that hypercalciuria and hyperoxaluria contribute to the formation of CaOx uroliths in dogs and show that dietary modifications can alter this process.

Predicting the crystallization potential of urine from cats and dogs with respect to calcium oxalate and magnesium ammonium phosphate (struvite).

The objective of this study was to validate two programs (SUPERSAT and EQUIL 2) for calculation of calcium oxalate (CaOx) and magnesium ammonium phosphate (struvite; MAP) relative supersaturation (RSS) in dog and cat urine. Healthy adult cats (n = 10) and dogs (n = 9) were fed standard diets for a 3-wk period. Urine was collected (24 h, dogs; 48 h, cats) and filtered, and the pH was measured. A 20-mL aliquot was titrated to pH 2 and frozen for analysis. Additional aliquots were incubated with 1 g seed crystals at 38 degrees C; CaOx for 24 h (cat) and 2, 6 and 9 d (dog); MAP for 48 h (dog) and 6 d (cat). Samples were analyzed for 10 substances. RSS was calculated using EQUIL 2 and SUPERSAT. CaOx RSS (SUPERSAT): dog urine was initially supersaturated, whereas cat urine was undersaturated with the diets used. Cat urine reached the solubility product (K(sp)), (RSS = 1) after 24-h incubation, whereas dog urine was still approaching K(sp) at 9 d. MAP RSS (SUPERSAT): urine from both species was undersaturated and increased toward K(sp) during incubation. Final RSS values were compared for both programs. SUPERSAT resulted in values close to 1 for both CaOx and MAP; EQUIL 2 gave similar values for CaOx RSS, although MAP RSS values were considerably higher than 1. In conclusion, EQUIL 2 and SUPERSAT both calculated reasonably accurate RSS values for CaOx, whereas only SUPERSAT provided an accurate measure of MAP RSS.