Enhancing genomics-based outbreak detection of endemic Salmonella enterica serovar Typhimurium using dynamic thresholds.

Salmonella enterica serovar Typhimurium is the leading cause of salmonellosis in Australia, and the ability to identify outbreaks and their sources is vital to public health. Here, we examined the utility of whole-genome sequencing (WGS), including complete genome sequencing with Oxford Nanopore technologies, in examining 105 isolates from an endemic multi-locus variable number tandem repeat analysis (MLVA) type over 5 years. The MLVA type was very homogeneous, with 90 % of the isolates falling into groups with a five SNP cut-off. We developed a new two-step approach for outbreak detection using WGS. The first clustering at a zero single nucleotide polymorphism (SNP) cut-off was used to detect outbreak clusters that each occurred within a 4 week window and then a second clustering with dynamically increased SNP cut-offs were used to generate outbreak investigation clusters capable of identifying all outbreak cases. This approach offered optimal specificity and sensitivity for outbreak detection and investigation, in particular of those caused by endemic MLVA types or clones with low genetic diversity. We further showed that inclusion of complete genome sequences detected no additional mutational events for genomic outbreak surveillance. Phylogenetic analysis found that the MLVA type was likely to have been derived recently from a single source that persisted over 5 years, and seeded numerous sporadic infections and outbreaks. Our findings suggest that SNP cut-offs for outbreak cluster detection and public-health surveillance should be based on the local diversity of the relevant strains over time. These findings have general applicability to outbreak detection of bacterial pathogens

Enhancing genomics-based outbreak detection of endemic salmonella enterica serovar typhimurium using dynamic thresholds

Salmonella enterica serovar Typhimurium is the leading cause of salmonellosis in Australia, and the ability to identify outbreaks and their sources is vital to public health. Here, we examined the utility of whole-genome sequencing (WGS), including complete genome sequencing with Oxford Nanopore technologies, in examining 105 isolates from an endemic multi-locus variable number tandem repeat analysis (MLVA) type over 5 years. The MLVA type was very homogeneous, with 90 % of the isolates falling into groups with a five SNP cut-off. We developed a new two-step approach for outbreak detection using WGS. The first clustering at a zero single nucleotide polymorphism (SNP) cut-off was used to detect outbreak clusters that each occurred within a 4 week window and then a second clustering with dynamically increased SNP cut-offs were used to generate outbreak investigation clusters capable of identifying all outbreak cases. This approach offered optimal specificity and sensitivity for outbreak detection and investigation, in particular of those caused by endemic MLVA types or clones with low genetic diversity. We further showed that inclusion of complete genome sequences detected no additional mutational events for genomic outbreak surveillance. Phylogenetic analysis found that the MLVA type was likely to have been derived recently from a single source that persisted over 5 years, and seeded numerous sporadic infections and outbreaks. Our findings suggest that SNP cut-offs for outbreak cluster detection and public-health surveillance should be based on the local diversity of the relevant strains over time. These findings have general applicability to outbreak detection of bacterial pathogens

Ultrasound-enhanced intrascleral delivery of protein

We aim to investigate ultrasound on enhancing protein penetration into the sclera, a non-invasive method to overcome the first barrier in taking the transscleral route for delivering therapeutics. Rabbit eyes were immersed in a fluorescein isothiocyanate conjugated bovine serum albumin solution. The distances of protein penetration, with and without ultrasound (30s continuous wave, 1MHz, 0.05W/cm 2) applied on the sclera, and at different immersion time intervals (0, 5, 15, 30 and 60min), were measured by examining the cryo-sectioned tissues under fluorescence microscope (≥60 measurements from 3 eyes for each condition). Retina was examined for structural damage by histology. It was found that ultrasound enhances the intrascleral penetration of protein, increasing the diffusivity by 1.6-folds while causing no damage to the retinal tissues. This physical modulation of the sclera is temporary, as evident by the restoration of the diffusional resistance at 15min after ultrasound treatment. The negligible effect of ultrasound-induced convection and the minimal temperature rise (<0.5°C), together with cavitation detected by acoustic emission and a decreased penetration distance at higher ultrasound frequency (30s continuous wave, 3MHz, 0.05W/cm 2), suggest that cavitation is a possible mechanism for increasing the permeability of the sclera for diffusive transport. © 2010 Elsevier B.V.link_to_subscribed_fulltex

Analysis of global Aeromonas veronii genomes provides novel information on source of infection and virulence in human gastrointestinal diseases

Background: Aeromonas veronii is a Gram-negative rod-shaped motile bacterium that inhabits mainly freshwater environments. A. veronii is a pathogen of aquatic animals, causing diseases in fish. A. veronii is also an emerging human enteric pathogen, causing mainly gastroenteritis with various severities and also often being detected in patients with inflammatory bowel disease. Currently, limited information is available on the genomic information of A. veronii strains that cause human gastrointestinal diseases. Here we sequenced, assembled and analysed 25 genomes (one complete genome and 24 draft genomes) of A. veronii strains isolated from patients with gastrointestinal diseases using combine sequencing technologies from Illumina and Oxford Nanopore. We also conducted comparative analysis of genomes of 168 global A. veronii strains isolated from different sources. Results: We found that most of the A. veronii strains isolated from patients with gastrointestinal diseases were closely related to each other, and the remaining were closely related to strains from other sources. Nearly 300 putative virulence factors were identified. Aerolysin, microbial collagenase and multiple hemolysins were present in all strains isolated from patients with gastrointestinal diseases. Type III Secretory System (T3SS) in A. veronii was in AVI-1 genomic island identified in this study, most likely acquired via horizontal transfer from other Aeromonas species. T3SS was significantly less present in A. veronii strains isolated from patients with gastrointestinal diseases as compared to strains isolated from fish and domestic animals. Conclusions: This study provides novel information on source of infection and virulence of A. veronii in human gastrointestinal diseases

Investigation of Campylobacter concisus gastric epithelial pathogenicity using AGS cells

Campylobacter concisus is an oral bacterium. Recent studies suggest that C. concisus may be involved in human gastric diseases. The mechanisms, however, by which C. concisus causes human gastric diseases have not been investigated. Here we examined the gastric epithelial pathogenicity of C. concisus using a cell culture model. Six C. concisus strains and the human gastric epithelial cell line AGS cells were used. IL-8 produced by AGS cells after incubation with C. concisus was measured using enzyme-linked immunosorbent assay (ELISA), and AGS cell apoptosis was determined by caspase 3/7 activities. The effects of C. concisus on actin arrangement in AGS cells was determined using fluorescence staining. The effects of C. concisus on global gene expression in AGS cells was determined by transcriptomic analysis and quantitative real-time PCR (qRT-PCR). The role of the upregulated CYP1A1 gene in gastric cancer survival was assessed using the Kaplan-Meier method. C. concisus induced production of IL-8 by AGS cells with strain variation. Significantly increased caspase 3/7 activities were observed in AGS cells incubated with C. concisus strains when compared to AGS cells without bacteria. C. concisus induced actin re-arrangement in AGS cells. C. concisus upregulated 30 genes in AGS cells and the upregulation of CYP1A1 gene was confirmed by qRT-PCR. The Kaplan-Meier analysis showed that upregulation of CYP1A1 gene is associated with worse survival in gastric cancer patients. Our findings suggest that C. concisus may play a role in gastric inflammation and the progression of gastric cancer. Further investigation in clinical studies is warranted