Field-based detection of bacteria using nanopore sequencing: Method evaluation for biothreat detection in complex samples.

From pathogen detection to genome or plasmid closure, the utility of the Oxford Nanopore Technologies (ONT) MinION for microbiological analysis has been well documented. The MinION's small footprint, portability, and real-time analytic capability situates it well to address challenges in the field of unbiased pathogen detection, as a component of a security investigation. To this end, a multicenter evaluation of the effect of alternative analytical approaches on the outcome of MinION-based sequencing, using a set of well-characterized samples, was explored in a field-based scenario. Three expert scientific response groups evaluated known bacterial DNA extracts as part of an international first responder (Chemical, Biological, Radiological) training exercise. Samples were prepared independently for analysis using the Rapid and/or Rapid PCR sequencing kits as per the best practices of each of the participating groups. Analyses of sequence data were in turn conducted using varied approaches including ONTs What's in my pot (WIMP) architecture and in-house computational pipelines. Microbial community composition and the ability of each approach to detect pathogens was compared. Each group demonstrated the ability to detect all species present in samples, although several organisms were detected at levels much lower than expected with some organisms even falling below 1% abundance. Several 'contaminant' near neighbor species were also detected, at low abundance. Regardless of the sequencing approach chosen, the observed composition of the bacterial communities diverged from the input composition in each of the analyses, although sequencing conducted using the rapid kit produced the least distortion when compared to PCR-based library preparation methods. One of the participating groups generated drastically lower sequencing output than the other groups, likely attributed to the limited computer hard drive capacity, and occasional disruption of the internet connection. These results provide further consideration for conducting unbiased pathogen identification within a field setting using MinION sequencing. However, the benefits of this approach in providing rapid results and unbiased detection must be considered along with the complexity of sample preparation and data analytics, when compared to more traditional methods. When utilized by trained scientific experts, with appropriate computational resources, the MinION sequencing device is a useful tool for field-based pathogen detection in mixed samples.

A foodborne outbreak of campylobacteriosis at a wedding - Melbourne, Australia, 2022.

Campylobacter is the most common bacterial cause of foodborne gastroenteritis in Australia; however, outbreaks caused by the pathogen are relatively uncommon. In March 2022, the Victorian Department of Health was notified of a gastrointestinal illness in 20 guests following attendance at a wedding reception. Two of these individuals were notified with laboratory-confirmed campylobacteriosis, and an investigation was undertaken to identify the source of the infection and implement strategies to prevent further illness. A case-control study was conducted to determine the likely source of infection. Cases were defined as attendees of the wedding reception, with onset of diarrhoea and/or abdominal cramping 1-10 days after attending the function. Controls were randomly selected from the remaining list of non-ill guests. Cases and controls were interviewed using a standardised, menu-based questionnaire. Food preparation processes were documented, and food samples collected. A total of 29 wedding guests met the case definition. Cases reported onset of illness 2-5 days following the wedding and major symptoms included abdominal cramping (100%), diarrhoea (90%), headache (79%), and fever (62%). Two cases were hospitalised, one with ongoing secondary neurological sequelae. Illness was significantly associated with consumption of a duck breast brioche canapé containing duck liver parfait (odds ratio = 2.85; 95% confidence interval: 1.03-7.86). No leftover food samples were available for testing. The investigation found that the duck canapé was the likely vehicle of infection. Consistent with the literature on Campylobacter transmission, it is likely that inadequate cooking of the duck liver for the parfait was the contributing factor that led to illness. This highlights the risks posed by undercooked poultry dishes, and shows that education of food handlers remains a priority.

Genomic Characterisation of Vinegar Hill Virus, An Australian Nairovirus Isolated in 1983 from Argas Robertsi Ticks Collected from Cattle Egrets.

This report describes the near complete genomic sequence and subsequent analysis of Vinegar Hill virus (VINHV; tentative member of the genus Orthonairovirus, family Nairoviridae, order Bunyavirales). VINHV is the second nairovirus reported to be isolated on mainland Australia and the first to be sequenced and analysed. Our genetic analysis shows that VINHV belongs to the Dera Ghazi Khan genogroup, a group of viruses previously isolated in other parts of the world including Asia, South Africa, and the USA. We discuss possible routes of entry for nairoviruses into Australia and the need to understand the virome of Australian ticks in the context of new and emerging disease.

Identification of very small open reading frames in the genomes of Holmes Jungle virus, Ord River virus, and Wongabel virus of the genus Hapavirus, family Rhabdoviridae.

Viruses of the family Rhabdoviridae infect a broad range of hosts from a variety of ecological and geographical niches, including vertebrates, arthropods, and plants. The arthropod-transmitted members of this family display considerable genetic diversity and remarkable genomic flexibility that enable coding for various accessory proteins in different locations of the genome. Here, we describe the genome of Holmes Jungle virus, isolated from Culex annulirostris mosquitoes collected in northern Australia, and make detailed comparisons with the closely related Ord River and Wongabel viruses, with a focus on identifying very small open reading frames (smORFs) in their genomes. This is the first systematic prediction of smORFs in rhabdoviruses, emphasising the intricacy of the rhabdovirus genome and the knowledge gaps. We speculate that these smORFs may be of importance to the life cycle of the virus in the arthropod vector.

Genomic characterisation of three Mapputta group viruses, a serogroup of Australian and Papua New Guinean bunyaviruses associated with human disease.

The Mapputta serogroup tentatively contains the mosquito-associated viruses Mapputta, Maprik, Trubanaman and Gan Gan. Interestingly, this serogroup has previously been associated with an acute epidemic polyarthritis-like illness in humans; however, there has been no ensuing genetic characterisation. Here we report the complete genome sequences of Mapputta and Maprik viruses, and a new Mapputta group candidate, Buffalo Creek virus, previously isolated from mosquitoes and detected by serology in a hospitalised patient. Phylogenetic analyses indicate that the group is one of the earliest diverged groups within the genus Orthobunyavirus of the family Bunyaviridae. Analyses show that these three viruses are related to the recently sequenced Australian bunyaviruses from mosquitoes, Salt Ash and Murrumbidgee. A notable feature of the Mapputta group viruses is the absence of the NSs (non-structural) ORF commonly found on the S segment of other orthobunyaviruses. Viruses of the Mapputta group have been isolated from geographically diverse regions ranging from tropical Papua New Guinea to the semi-arid climate of south-eastern Australia. The relevance of this group to human health in the region merits further investigation.

Characterisation and evaluation of synthetic antimicrobial peptides against Bacillus globigii, Bacillus anthracis and Burkholderia thailandensis.

Antimicrobial peptides (AMPs) are produced by all forms of living organisms and represent a novel class of antibiotics to treat infectious diseases. In this study, 29 AMPs of varying length and characteristics were synthesised chemically and were evaluated for their ability to inhibit the growth of Bacillus globigii, Bacillus anthracis and Burkholderia thailandensis. Amongst the peptides tested, sheep myeloid antimicrobial peptide-29 (SMAP-29) was the most potent, inhibiting both B. globigii and B. anthracis at submicromolar concentrations. However, SMAP-29 was less effective against B. thailandensis (minimum inhibitory concentration of 71 microM). Haemolytic activity and cytotoxicity were determined using human blood cells and human embryonic kidney 293S cells, respectively. Most of the peptides tested showed varying degrees of haemolytic activity and cytotoxicity, with SMAP-29 being highly haemolytic and cytotoxic under the conditions tested. Nevertheless, strategies to reduce toxicity whilst maintaining high antimicrobial activity are worth pursuing in light of the results obtained.