ASM summer student research awards: 2023

Optimisation and evaluation of bait capture approaches for genomic surveillance of arboviruses

Sarah Smith^A, Cheryl Toi^B, Cameron Webb^A,B,C, Stephen Doggett^A,B, Jen Kok^A,B,C, Vitali Sinchenko^A,B,C, Rebecca J Rockett^A,C and Tanya Golubchik^C

^ACentre for Infectious Diseases and Microbiology – Public Health, Westmead Hospital, WSLHD, Westmead, NSW, Australia.

^BCentre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, NSW Health Pathology, Westmead, NSW, Australia.

^CSydney Institute of Infectious Diseases, The University of Sydney, Camperdown, NSW, Australia.

Abstract: Genomics has revolutionised public health surveillance programs enabling high resolution detection of point source outbreaks. To generate whole viral sequences directly from clinical specimens whole-genome amplification methods are commonly used. These methods rely on tiling primers to amplify small overlapping fragments of the target genome. However, these primers require >98% nucleotide homology to the target genome and a new primer set needs to be developed for each pathogen. Primer target regions are sensitive to mutations within the primer binding region that reduce or eliminate amplification. This can be overcome by employing the novel technique of hybridisation capture probes, utilising probes that tile the entire genome of the pathogen of interest and has the advantage of capturing sequences with >80% nucleic homology to the probes. This method can be difficult to control however as it captures a wide range of viral genomes, hence a synthetic control is needed to ensure capture efficiency over the spectrum viruses targeted. The synthetic control will enable intra-assay capture efficiency to be determined and controlled for novel pathogens or infectious threats. This is additionally useful where obtaining positive clinical material is difficult, particularly at the time of emergence or diagnosis. This is especially relevant due to the recent emergence of previously undetected arboviruses in NSW Australia, Japanese encephalitis virus and Murray valley encephalitis virus. As such it is more important than ever to detect and monitor viruses present within mosquito populations.

The sterilisation of Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus in hospital waste into carbon material by microwave pyrolysis system

Laura Tengzelius, Catherine Rush, Mohan Jacob and Jeffery Warner

College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, Qld, Australia.

Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, Qld, Australia.

Abstract: Hospital waste has increased in drastic numbers and has had a large impact on hospital departments around the world especially in underdeveloped countries. The waste is contaminated with bacteria and other microorganisms increasing hospital acquired infections. This investigation aims to identify and prove the efficiency of the wavelength, heat and pressure (1 kPa, 400°C & 60 min) from the microwave pyrolysis system (MPS) to kill the organisms Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus on different hospital waste into sterile carbon material. Based on the extreme heat generated from the MPS it is hypothesised that it will inactivate the microorganisms. The result revealed that there was no bacterial growth on nutrient agar plates after the treatment of MPS and after 72 h of incubation. These findings confirmed the efficiency of the MPS system to sterilise bacteria from contaminated hospital waste.

Characterisation of the polysialic acid O-acetyltransferase NeuO in E. coli

Lachlan Walker, Duy Phan and Mark Schembri

School of Chemistry and Molecular Biosciences, The University of Queensland, Qld, Australia.

Institute of Molecular Bioscience, The University of Queensland, Qld, Australia.

Abstract: Uropathogenic Escherichia coli (UPEC) strains with K1 capsule type are associated with severe disease including pyelonephritis, neonatal meningitis, and sepsis. O-acetylation of the K1 capsule, regulated by the phase variable gene neuO, is proposed as a virulence factor of pathogenic K1 UPEC. In this study we successfully deleted neuO from the neuO phase ‘ON’ strain MS7163, resulting in increased susceptibility to K1 phage killing. Complementation of three wild type (WT) neuO phase ‘OFF’ strains with a NeuO-expression vector (pSU2718-neuO^ON) led to protection of 1/3 strains against K1 phage killing. Overall, this study has provided a foundation for future work to examine the role of neuO as a virulence factor of K1 UPEC that cause severe human disease.

Study of virulence factors for the rationale design of a non-typeable Haemophilius influenzae vaccine

Megan Young and John M Atack

Institute for Glycomics, Griffith University, Gold Coast, Qld, Australia.

School of Environment and Science, Griffith University, Gold Coast, Qld, Australia.

Abstract: Non-typeable Haemophilus influenzae (NTHi) is a bacterial pathogen of global importance. NTHi colonises the human nasopharynx asymptomatically but is an important pathogen in middle ear infection (otitis media) in children, exacerbations in bacterial bronchitis, chronic obstructive pulmonary disease, and bronchiectasis, and community-acquired pneumonia, in adults. NTHi also causes invasive infections, and these are fatal in ~10% of children <1 year, and in ~25% of adults aged >80-years. No vaccine yet exists due to the extreme variability of this organism. Understanding the stably expressed antigenic repertoire of NTHi is crucial for the rational design of a protein subunit vaccine. Finding suitable antigens for pathogens that exhibit extreme variability like NTHi is incredibly challenging as many proteins are not stably expressed, not conserved, and not present in all strains. NTHi proteins that are not conserved, but highly immunogenic, include the major outer membrane adhesins HMW, Hia, and Lav. Although each of these proteins are not present in all strains, combining them in a single formulation would cover all strains of NTHi, and potentially generate a universally protective vaccine against this pathogen. This research cloned the main ligand binding domain, binding domain 1 (BD1) of the NTHi adhesin Hia fused to a solubility NEXT tag. Future studies will investigate the precise ligands of BD1, and its potential use as an immunogen for inclusion in the rational design of a NTHi vaccine.

Evaluation of plant-derived lactic acid bacteria to acidify and produce diacetyl flavour in plant-based dairy alternative milks

Melody Yap Rui Zhen, Wenkang Huang and Mark Turner

School of Agriculture and Food Sustainability, Faculty of Science, The University of Queensland, Qld, Australia.

Abstract: This project was completed by Melody Yap with guidance from Shuyu Yang and lead by Wenkang Huang and Mark Turner under the University of Queensland. The fermented plant-based milk (PBM) has the potential to cater to wider consumers with improved flavour than dairy milk. However, dairy-adapted lactic acid bacteria (LAB) do not perform well in plant-based milk (PBM) due to their inability to utilise plant sugars, e.g. sucrose. We investigated if 15 plant-derived LAB that were previously identified to produced diacetyl in de Man Rogosa Sharpe broth could acidify and produce diacetyl in soy, coconut, oat, cashew, rice and almond milks. This was done through pH monitoring and using an adapted version of Voges-Proskauer test on 24-h fermented PBMs by LAB. All LABs could acidify and grow in PBMs but only 40% could produce diacetyl in all PBM. The strongest acidifying LAB was Lactiplantibacillus plantarum 881 and the strongest diacetyl producing LAB was Lactococcus raffinolactis 126. Coconut milk was acidified to the lowest pH and supported the greatest diacetyl production by LAB. Since all LAB were able to produce diacetyl in at least two PBMs, it was concluded that all LAB have potential for development of plant-based alternative protein products, such as a cheese analog, by producing acid and an important flavourant.

Assessment of the replication of enteric viruses in the human salivary gland and pancreatic enterochromaffin cell lines

Valeria Sanders and Jillian M. Carr

Microbiology and Infectious Diseases, College of Medicine and Public Health, Flinders University, Adelaide, SA, Australia.

Abstract: Adenoviruses, particularly enteric strains AdV-F40/41, contribute significantly to acute gastroenteritis and, occasionally, severe acute hepatitis in children. AdV is common, but enteric strains are difficult to study in the laboratory. This study investigated the susceptibility of different cell lines to AdV-F40/41 replication. Two cell lines, a pancreatic carcinoma with enterochromaffin cell-like properties, (QGP-1), and the salivary gland cell line, A253, were inoculated with AdV-F40/41 viral stocks prepared from clinical samples. DNA was extracted from the supernatant and cell lysates at different time points following infection and viral loads were determined using qPCR. The number of AdV-F40/41 genome copies in QGP-1 supernatant displayed a statistically significant increase, peaking at 24 h post infection and subsequently diminishing at 48 h post infection, before stabilising between 72 and 96 h post infection. Comparing the DNA quantity of AdV-F40/41 in the cell lysates at 96 h post infection to the initial time point revealed no significant differences. HSG and A253 cell lines did not support AdV-F40/41 replication as no increase in AdV DNA was observed. In conclusion, the QGP-1 EC-like cell line shows promise as an alternative in vitro model for studying AdV-F40/41 infections. Utilising QGP-1 cells may advance our understanding of AdV-F40/41 replication and host responses to infection.

Isolation of microbial isolates from Australian spider gastrointestinal tracts

Lachlan Duell and Anya Shindler

Applied and Environmental Microbiology Laboratory, La Trobe University, Melbourne, Vic., Australia.

Abstract: Very little previous research has investigated the microbiome of Australian spiders. The purpose of this project was to isolate microbes from the spider microbiome for further testing and sequencing. The Australian spiders used were Badumna insignis (black house spider) and Badumna longinqua (grey house spider), super optimal broth and malt extract broth were inoculated using the internal organs from the spider’s abdomen, the growth in the broth was then streaked out onto respective their respective agar plates. Bacteria and fungi were taken from the plates and gram stained and stained using methylene blue respectively. The results showed that the spider microbiome contains multiple different bacterial species including an unknown gram-negative filamentous bacterium, a bacterium suspected to be either Staphylococcus aureus or an unknown Streptococcus, there was also an unknown species of Bacillus. Alongside the different bacteria there were also multiple unknown species of fungi. The isolated microbes created from this project would complement future testing and sequencing work by Dr Anya Shindler to find the specific species of bacteria and fungi present.

Isolation of desert cyanobacteria

Montgomery Hall, Pok Man Leung and Chris Greening

Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Vic., Australia.

Abstract: Cyanobacteria mediate numerous key processes in desert ecosystems, and display remarkable desiccation resistance. High affinity hydrogenase enzymes are widespread in bacteria and act as a lifeline by extracting energy and water from atmospheric hydrogen (H₂). Many cyanobacteria encode hydrogenases, but their capacity to oxidise atmospheric H₂ remains untested. This study isolated desert cyanobacteria for use in future experiments to test their ability for atmospheric H₂ oxidation. Three filamentous cyanobacterial strains were isolated and morphologically characterised. We propose the ‘surface-sterilisation’ method of isolating motile cyanobacteria to reduce contaminants. In future, the isolated strains will be identified by sequencing and may be used to investigate hydrogenase activity.

Investigation of genetic determinants associated with serotype 6B persistence following pneumococcal vaccine introduction

Paige Skoko^A, Laura Boelsen^A, Claire von Mollendorf^B and Catherine Satzke^A,C,D

^ATranslational Microbiology, Murdoch Children’s Research Institute, Parkville, Vic., Australia.

^BNew Vaccines, Murdoch Children’s Research Institute, Parkville, Vic., Australia.

^CDepartment of Paediatrics, The University of Melbourne, Parkville, Vic., Australia.

^DDepartment of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Vic., Australia. 

Abstract: Streptococcus pneumoniae is a major cause of pneumonia. Following introduction of the pneumococcal conjugate vaccine in Mongolia from 2016, there were substantial declines in the serotypes included in the vaccine. However, there was persistence of vaccine-serotype 6B. Previously, serotype 6B was isolated from the nasopharynx of 17 children with pneumonia in Mongolia between 2015 and 2021 and were whole genome sequenced. Preliminary analysis of these genomes identified a change in lineage composition of 6B (characterised using Global Pneumococcal Sequence Cluster, GPSC), where the dominant lineage pre-vaccine introduction (GPSC23) was overtaken by GPSC6 post-vaccine introduction. There was also evidence that the replacement lineage GPSC6 was less virulent in vivo. In the current study, we aimed to identify differences in key virulence genes between GPSC23 and GPSC6 using comparative genomics. To do this, genome assemblies were annotated, and a pan-genome analysis was conducted using Roary. There were 1697 genes detected in all 17 serotype 6B genomes (core genes) and 1060 genes detected in a subset of the sampled genomes (accessory genes). There were five virulence genes exclusively detected among the GPSC23 pan-genome, which we hypothesise may contribute to the difference in virulence phenotype between lineages. Future experiments will investigate these genes to better inform our understanding of pneumococcal pathogenesis.

Phenotypic resistance/genotypic susceptibility to erythromycin and clindamycin in Clostridium difficile

Angelina Cheng, Dan Knight and Thomas Riley

School of Biomedical Science, University of Western Australia, Perth, WA, Australia.

Abstract: Macrolide, lincosamide and streptogramin B (MLSB) is an important antimicrobial class of protein synthesis inhibitors that often promote Clostridium difficile infection. Their frequent use has resulted in an accumulation of antimicrobial resistance (AMR) in C. difficile. MLSB resistance (MLSB+) is typically encoded by ermB genes, however, MLSB-resistant strains lacking any erm genes (erm-) have been observed and the reasons for this have not been elucidated. Hence, this study aimed to gain comprehensive genomic insights into this phenomenon with a focus of clindamycin and erythromycin as MLSB agents. The strains were characterised using multi-locus sequence typing, tested for susceptibility to two antimicrobials by E-test if they had yet to be phenotyped, and pan-GWAS analysis used to identify AMR genes other than erm. In total, 103 sequence types (STs) were identified across 1532 whole-genome sequenced C. difficile strains isolated worldwide. E-tests showed that erythromycin inhibited slightly a greater number of strains than clindamycin (16.7 v. 13.3%, n = 30). No novel genes were identified, therefore, MLSB resistance in M-strains might be associated with intrinsic causes mediated by chemical molecules or epigenetic factors. Moving forward, the focus should be on understanding non-genetic causes of MLSB resistance to reduce AMR in C. difficile.

Generating tools for understanding the role of Mip in C. burnetii

Benjamin Dumbrek and Aleksandra Debowski

School of Biomedical Sciences, University of Western Australia, WA, Australia.

Abstract: Coxiella burnetii is a highly infectious bacterium and causes the disease known as Q fever. Current treatments for C. burnetii have limited effectiveness and thus treatment avenues are sought. One avenue involves inhibiting Macrophage infectivity potentiator (Mip) protein which has been shown to result in decreased virulence in several pathogens. C. burnetii is known to also express a Mip protein however its role in pathogenesis is unknown. Consequently, we aimed to clone the Cbmip gene into a C. burnetii expression vector to generate tools to study its function during infection. The gene encoding Mip was successfully cloned into the vector pJB-KAN-3xFLAG with and without a FLAG tag. One expression plasmid was transformed into C. burnetii and isolated clones were analysed by western blot. They were shown to express Cbmip; however, further experimental optimisation is required to confirm CbMip overexpression in these clones. Once validated, these clones can be used to study CbMip during infection.