Dr Verlaine Timms

Haemophilus influenzae, a causative agent of severe invasive infections such as meningitis, sepsis and pneumonia, is classified into encapsulated or typeable (represented by serotypes A to F) and non-typeable varieties (NTHi) by the presence or absence of the polysaccharide capsule. Invasive disease caused by H. influenzae type B (HIB) can be prevented through vaccination which remains the main disease control intervention in many countries. This study examined the genomic diversity of circulating H. influenzae strains associated with invasive disease in New South Wales, Australia, before and during the COVID-19 pandemic. Ninety-six isolates representing 95 cases of invasive H. influenzae infections (iHi) diagnosed between January 2017 and September 2022 were typed and characterised using whole genome sequencing. These cases were caused by serotypes A (n=24), B (n=35), E (n=3), F (n=2) and NTHi (n=32). There was an apparent decline in the number of iHi infections during the COVID-19 pandemic, with a corresponding increase in the proportion of iHi cases caused by serotype A (HIA), which returned to pre-pandemic levels in 2022. Fifteen isolates associated with HIB or non-typeable iHi were resistant to ß-lactams due to a PBP3 mutation or carriage of blaTEM-1. Further, capsular gene duplication was observed in HIB isolates but was not found in HIA. These findings provide important baseline genomic data for ongoing iHi surveillance and control.

Cyanobacterial blooms are a concerning issue that threaten ecosystems, ecology and animal health. Bloom frequency has increased tremendously in recent times due to pollution, eutrophication of waterways, climate change, and changes in microbial community dynamics within the aquatic environment. Information about the spatiotemporal variation in microbial communities that drive a cyanobacterial bloom is very limited. Here, we analysed the spatiotemporal diversity and composition of bacterial communities, with a focus on cyanobacteria, during the bloom phase in a natural reservoir in Eastern Australia using high throughput amplicon sequencing. Sampling points and season had no influence on the richness and evenness of microbial communities during the bloom period, however some compositional differences were apparent across the seasons. Cyanobacteria were highly abundant during summer and autumn compared to winter and spring. The dominant cyanobacterial taxa were Planktothrix, Cyanobium and Microcystis and were found to be significantly abundant during summer and autumn. While cyanobacterial abundance soared in summer (25.4 %), dominated by Planktothrix (12.2 %) and Cyanobium (8.0 %), the diversity was highest in autumn (24.9 %) and consisted of Planktothrix (7.8 %), Nodularia (5.3 %), Planktothricoides (4.6 %), Microcystis (3.5 %), and Cyanobium (2.3 %). The strongly correlated non-photosynthetic Gastranaerophilales found in the sediment and water, suggested vertical transmission from the animal gut through faeces. To our knowledge, this is the first report of Planktothrix-driven toxic cyanobacterial bloom in Australia. Our study expands current understanding of the spatiotemporal variation in bacterial communities during a cyanobacterial bloom and sheds light on setting future management strategies for its control.

Bacterial antimicrobial resistance (AMR) has emerged as a significant concern worldwide. The microbial community profile and potential AMR level in aquaculture ponds are often undervalued and attract less attention than other aquatic environments. We used amplicon and metagenomic shotgun sequencing to study microbial communities and AMR in six freshwater polyculture ponds in rural and urban areas of Bangladesh. Amplicon sequencing revealed different community structures between rural and urban ponds, with urban ponds having a higher bacterial diversity and opportunistic pathogens including Streptococcus, Staphylococcus, and Corynebacterium. Despite proteobacterial dominance, Firmicutes was the most interactive in the community network, especially in the urban ponds. Metagenomes showed that drug resistance was the most common type of AMR found, while metal resistance was only observed in urban ponds. AMR and metal resistance genes were found mainly in beta and gamma-proteobacteria in urban ponds, while AMR was found primarily in alpha-proteobacteria in rural ponds. We identified potential pathogens with a high profile of AMR and metal resistance in urban aquaculture ponds. As these ponds provide a significant source of protein for humans, our results raise significant concerns for the environmental sustainability of this food source and the dissemination of AMR into the food chain.

This study was aimed to produce zinc oxide nanoparticles (ZnO-NPs) using the supernatant of Weissella confusa UPM22MT04 and assess their effectiveness in inhibiting methicillinresistant Staphylococcus aureus (MRSA). An isolate of Weissella confusa UPM22MT04 was isolated from a wastewater treatment plant in Johor, Malaysia, and was utilized to synthesize ZnO-NPs. The synthesized ZnO-NPs were characterized through several techniques, including UV-visible spectroscopy, Fourier-transform infrared spectroscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and dynamic light scattering. Monodisperse spherical ZnO-NPs of 1.7-7.9 nm were obtained with 0.1 M zinc nitrate at 80°C. The biosynthesized ZnO-NPs exhibited vigorous inhibitory activity against MRSA. Results found that ZnO-NPs inhibited MRSA at a minimum concentration of 0.625 mg/mL and were bactericidal at a minimum concentration of 1.25 mg/mL. In MTT assays, ZnO-NPs showed no toxicity to HS-27 fibroblasts. The supernatant of Weissella confusa UPM22MT04 could be used to synthesize ZnO-NPs, which are an antibacterial agent, eco-friendly and nontoxic.

Mycobacterium avium is separated into four subspecies: M. avium subspecies avium (MAA), M. avium subspecies silvaticum (MAS), M. avium subspecies hominissuis (MAH), and M. avium subspecies paratuberculosis (MAP). Understanding the mechanisms of host and tissue adaptation leading to their clinical significance is vital to reduce the economic, welfare, and public health concerns associated with diseases they may cause in humans and animals. Despite substantial phenotypic diversity, the subspecies nomenclature is controversial due to high genetic similarity. Consequently, a set of 1,230 M. avium genomes was used to generate a phylogeny, investigate SNP hotspots, and identify subspecies-specific genes. Phylogeny reiterated the findings from previous work and established that Mycobacterium avium is a species made up of one highly diverse subspecies, known as MAH, and at least two clonal pathogens, named MAA and MAP. Pan-genomes identified coding sequences unique to each subspecies, and in conjunction with a mapping approach, mutation hotspot regions were revealed compared to the reference genomes for MAA, MAH, and MAP. These subspecies-specific genes may serve as valuable biomarkers, providing a deeper understanding of genetic differences between M. avium subspecies and the virulence mechanisms of mycobacteria. Furthermore, SNP analysis demonstrated common regions between subspecies that have undergone extensive mutations during niche adaptation. The findings provide insights into host and tissue specificity of this genetically conserved but phenotypically diverse species, with the potential to provide new diagnostic targets and epidemiological and therapeutic advances.

Mycobacterium avium subspecies paratuberculosis (MAP) is the aetiological agent of Johne's disease (JD), a chronic enteritis that causes major losses to the global livestock industry. Further, it has been associated with human Crohn's disease. Several strains of MAP have been identified, the two major groups being sheep strain MAP, which includes the Type I and Type III sub-lineages, and the cattle strain or Type II MAP lineage, of which bison strains are a sub-grouping. Major genotypic, phenotypic and pathogenic variations have been identified in prior comparisons, but the research has predominately focused on cattle strains of MAP. In countries where the sheep industries are more prevalent, however, such as Australia and New Zealand, ovine JD is a substantial burden. An information gap exists regarding the genomic differences between sheep strain sub-lineages and the relevance of Type I and Type III MAP in terms of epidemiology and/or pathogenicity. We therefore investigated sheep MAP isolates from Australia and New Zealand using whole genome sequencing. For additional context, sheep MAP genome datasets were downloaded from the Sequence Read Archive and GenBank. The final dataset contained 18 Type III and 16 Type I isolates and the K10 cattle strain MAP reference genome. Using a pan-genome approach, an updated global phylogeny for sheep MAP from de novo assemblies was produced. When rooted with the K10 cattle reference strain, two distinct clades representing the lineages were apparent. The Australian and New Zealand isolates formed a distinct sub-clade within the type I lineage, while the European type I isolates formed another less closely related group. Within the type III lineage, isolates appeared more genetically diverse and were from a greater number of continents. Querying of the pan-genome and verification using BLAST analysis revealed lineage-specific variations (n = 13) including genes responsible for metabolism and stress responses. The genetic differences identified may represent important epidemiological and virulence traits specific to sheep MAP. This knowledge will potentially contribute to improved vaccine development and control measures for these strains.

Background: VRE are prevalent among patients in ICUs. Non-typeable vanA VRE, due to loss of one of the genes used for MLST (pstS), have increased in Australia, suggestive of a new, hospital-acquired lineage. Objectives: To understand the significance of this lineage and its transmission using WGS of strains isolated from patients in ICUs across New South Wales, Australia. Methods: A total of 240 Enterococcus faecium isolates collected between February and May 2016, and identified by conventional PCR as vanA positive, were sequenced. Isolates originated from 12 ICUs in New South Wales, grouped according to six local health districts, and represented both rectal screening swab (n=229) and clinical (n=11) isolates. Results: ST analysis revealed the absence of the pstS gene in 84.2% (202 of 240) of vanA isolates. Two different non-typeable STs were present based on different allelic backbone patterns. Loss of the pstS gene appeared to be the result of multiple recombination events across this region. Evidence for pstS-negative lineage spread across all six local health districts was observed suggestive of inter-hospital transmission. In addition, multiple outbreaks were detected, some of which were protracted and lasted for the duration of the study. Conclusions: These findings confirmed the evolution, emergence and dissemination of non-typeable vanA E. faecium. This study has highlighted the utility of WGS when attempting to describe accurately the hospitalbased pathogen epidemiology, which in turn will continue to inform optimal infection control measures necessary to halt the spread of this important nosocomial organism.

The association of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) with Crohn's disease is a controversial issue. M. paratuberculosis is detected by amplifying the IS900 gene, as microbial culture is unreliable from humans. We determined the presence of M. paratuberculosis in patients with Crohn's disease (CD) (n = 22), ulcerative colitis (UC) (n = 20), aphthous ulcers (n = 21) and controls (n = 42) using PCR assays validated on bovine tissue. Culture from human tissue was also performed. M. paratuberculosis prevalence in the CD and UC groups was compared to the prevalence in age and sex matched non-inflammatory bowel disease controls. Patients and controls were determined to be M. paratuberculosis positive if all three PCR assays were positive. A significant association was found between M. paratuberculosis and Crohn's disease (p = 0.02) that was not related to age, gender, place of birth, smoking or alcohol intake. No significant association was detected between M. paratuberculosis and UC or aphthous ulcers; however, one M. paratuberculosis isolate was successfully cultured from a patient with UC.We report the resistance of this isolate to ethambutol, rifampin, clofazamine and streptomycin. Interestingly this isolate could not only survive but could grow slowly at 5°C. We demonstrate a significant association between M. paratuberculosis and CD using multiple pre-validated PCR assays and that M. paratuberculosis can be isolated from patients with UC.