Dr Verlaine Timms

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.

Background: Legionnaires' disease is a notifiable condition in New South Wales (NSW), Australia; clinicians and laboratories are required to report the disease to NSW Health. We describe the investigation of a sporadic case associated with the use of a communal spa pool in the case's apartment building complex and the use of whole genome sequencing to examine relatedness between clinical and environmental Legionella pneumophila serogroup 1 (Lp1) strains. Methods: In February 2018, a confirmed case of Lp1 infection was notified in a man in his 60s hospitalised with pneumonia. We asked the clinical team to obtain sputum in the event we found a potential source. The case described the use of the communal spa pool in his apartment building on two occasions during the putative exposure period. Environmental Health Officers from the Public Health Unit inspected the spa pool and found that the free chlorine level was well below the recommended concentration; a water sample was submitted for microbial analysis. Results: Lp1 was grown from the case's sputum and microbial analysis of the spa water sample found Lp1 at a concentration of 20 CFU/mL. The human and environmental isolates were subjected to whole genome sequencing and found to be highly genomically related. There was no other plausible environmental source of legionella. Conclusions: Whole genome sequencing of the clinical and environmental Lp1 isolates implicated a contaminated spa pool as the source of the case's exposure. This strongly supports the application of whole genome sequencing to the investigation of single cases of legionellosis. Communal spa pools in apartment buildings are not regulated in most Australian jurisdictions but must be considered to pose a potential legionella risk if improperly maintained.

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.