Can genomics and meteorology predict outbreaks of legionellosis in urban settings?

Legionella pneumophila is ubiquitous and sporadically infects humans causing Legionnaire's disease (LD). Globally, reported cases of LD have risen fourfold from 2000 to 2014. In 2016, Sydney, Australia was the epicenter of an outbreak caused by L. pneumophila serogroup 1 (Lpsg1). Whole-genome sequencing was instrumental in identifying the causal clone which was found in multiple locations across the city. This study examined the epidemiology of Lpsg1 in an urban environment, assessed typing schemes to classify resident clones, and investigated the association between local climate variables and LD outbreaks. Of 223 local Lpsg1 isolates, we identified dominant clones with one clone isolated from patients in high frequency during outbreak investigations. The core genome multi-locus sequence typing scheme was the most reliable in identifying this Lpsg1 clone. While an increase in humidity and rainfall was found to coincide with a rise in LD cases, the incidence of the major L. pneumophila outbreak clone did not link to weather phenomena. These findings demonstrated the role of high-resolution typing and weather context assessment in determining source attribution for LD outbreaks in urban settings, particularly when clinical isolates remain scarce.IMPORTANCEWe investigated the genomic and meteorological influences of infections caused by Legionella pneumophila in Sydney, Australia. Our study contributes to a knowledge gap of factors that drive outbreaks of legionellosis compared to sporadic infections in urban settings. In such cases, clinical isolates can be rare, and thus, other data are needed to inform decision-making around control measures. The study revealed that core genome multi-locus sequence typing is a reliable and adaptable technique when investigating Lpsg1 outbreaks. In Sydney, the genomic profile of Lpsg1 was dominated by a single clone, which was linked to numerous community cases over a period of 40 years. Interestingly, the peak in legionellosis cases during Autumn was not associated with this prevalent outbreak clone. Incorporating meteorological data with Lpsg1 genomics can support risk assessment strategies for legionellosis in urban environments, and this approach may be relevant for other densely populated regions globally.

Genome-wide comparison of Corynebacterium diphtheriae isolates from Australia identifies differences in the Pan-genomes between respiratory and cutaneous strains.

BACKGROUND: Corynebacterium diphtheriae is the main etiological agent of diphtheria, a global disease causing life-threatening infections, particularly in infants and children. Vaccination with diphtheria toxoid protects against infection with potent toxin producing strains. However a growing number of apparently non-toxigenic but potentially invasive C. diphtheriae strains are identified in countries with low prevalence of diphtheria, raising key questions about genomic structures and population dynamics of the species. This study examined genomic diversity among 48 C. diphtheriae isolates collected in Australia over a 12-year period using whole genome sequencing. Phylogeny was determined using SNP-based mapping and genome wide analysis. RESULTS: C. diphtheriae sequence type (ST) 32, a non-toxigenic clone with evidence of enhanced virulence that has been also circulating in Europe, appears to be endemic in Australia. Isolates from temporospatially related patients displayed the same ST and similarity in their core genomes. The genome-wide analysis highlighted a role of pilins, adhesion factors and iron utilization in infections caused by non-toxigenic strains. CONCLUSIONS: The genomic diversity of toxigenic and non-toxigenic strains of C. diphtheriae in Australia suggests multiple sources of infection and colonisation. Genomic surveillance of co-circulating toxigenic and non-toxigenic C. diphtheriae offer new insights into the evolution and virulence of pathogenic clones and can inform targeted public health actions and policy. The genomes presented in this investigation will contribute to the global surveillance of C. diphtheriae both for the monitoring of antibiotic resistance genes and virulent strains such as those belonging to ST32.

Relentless spread and adaptation of non-typeable vanA vancomycin-resistant Enterococcus faecium: a genome-wide investigation.

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 hospital-based pathogen epidemiology, which in turn will continue to inform optimal infection control measures necessary to halt the spread of this important nosocomial organism.

Genome Sequencing Links Persistent Outbreak of Legionellosis in Sydney (New South Wales, Australia) to an Emerging Clone of Legionella pneumophila Sequence Type 211.

The city of Sydney, Australia, experienced a persistent outbreak of Legionella pneumophila serogroup 1 (Lp1) pneumonia in 2016. To elucidate the source and guide public health actions, the genomes of clinical and environmental Lp1 isolates recovered over 7 weeks were examined. A total of 48 isolates from human cases and cooling towers were sequenced and compared using single-nucleotide polymorphism (SNP)-based core-genome multilocus sequencing typing (MLST) and pangenome approaches. All three methods confirmed phylogenetic relatedness between isolates associated with outbreaks in the Central Business District (CBD) in March and May and those in suburb 1. These isolates were designated the "main cluster" and consisted of isolates from two patients from the CBD March outbreak, one patient and one tower isolate from suburb 1, and isolates from two cooling towers and three patients from the CBD May outbreak. All main cluster isolates were sequence type 211 (ST211), which previously has only been reported in Canada. Significantly, pangenome analysis identified mobile genetic elements containing a unique type IV A F-type secretion system (T4ASS), which was specific to the main cluster, and cocirculating clinical strains, suggesting a potential mechanism for increased fitness and persistence of the outbreak clone. Genome sequencing enabled linking of the geographically dispersed environmental sources of infection among the spatially and temporally coinciding cases of legionellosis in a highly populated urban setting. The discovery of a unique T4ASS emphasizes the role of genome recombination in the emergence of successful Lp1 clones.IMPORTANCE A new emerging clone has been responsible for a prolonged legionellosis outbreak in Sydney, Australia. The use of whole-genome sequencing linked two outbreaks thought to be unrelated and confirmed the outliers. These findings led to the resampling and subsequent identification of the source, guiding public health actions and bringing the outbreak to a close. Significantly, the outbreak clone was identified as sequence type 211 (ST211). Our study reports this ST in the Southern Hemisphere and presents a description of ST211 genomes from both clinical and environmental isolates. A unique mobile genetic element containing a type IV secretion system was identified in Lp1 ST211 isolates linked to the main cluster and Lp1 ST42 isolates that were cocirculating at the time of the outbreak.

Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity.

BACKGROUND: A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen. RESULTS: This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes. CONCLUSIONS: Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies.

Characterisation of IS1311 in Mycobacterium avium subspecies paratuberculosis genomes: Typing, continental clustering, microbial evolution and host adaptation.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a global burden for livestock producers and has an association with Crohn's disease in humans. Within MAP there are two major lineages, S/Type I/TypeIII and C/Type II, that vary in phenotype including culturability, host preference and virulence. These lineages have been identified using the IS1311 element, which contains a conserved, single nucleotide polymorphism. IS1311 and the closely related IS1245 element belong to the IS256 family of insertion sequences, are dispersed throughout M. avium taxa but remain poorly characterised. To investigate the distribution and diversity of IS1311 in MAP, 805 MAP genomes were collated from public databases. IS1245 was absent, while IS1311 sequence, copy number and insertion loci were conserved between MAP S lineages and varied within the MAP C lineage. One locus was specific to the S strains, which contained nine IS1311 copies. In contrast, C strains contained either seven or eight IS1311 loci. Most insertion loci were associated with the boundaries of homologous regions that had undergone genome rearrangement between the MAP lineages, suggesting that this sequence may be a driver of recombination. Phylogenomic geographic clustering of MAP subtypes was demonstrated for the first time, at continental scale, and indicated that there may have been recent MAP transmission between Europe and North America, in contrast to Australia where importation of live ruminants is generally prohibited. This investigation confirmed the utility of IS1311 typing in epidemiological studies and resolved anomalies in past studies. The results shed light on potential mechanisms of niche/host adaptation, virulence of MAP and global transmission dynamics.

Prevalence of opportunistic pathogens and anti-microbial resistance in urban aquaculture ponds.

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.

Global Phylogeny of Mycobacterium avium and Identification of Mutation Hotspots During Niche Adaptation.

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.

Legionella pneumophila serogroup 1 infection associated with the use of an apartment building spa pool.

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.

Comparative Genomics of Mycobacterium avium Subspecies Paratuberculosis Sheep Strains.

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.

Complete microbial genomes for public health in Australia and the Southwest Pacific.

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific; enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.

Genome-wide analysis of Streptococcus pneumoniae serogroup 19 in the decade after the introduction of pneumococcal conjugate vaccines in Australia.

The decline in invasive pneumococcal disease (IPD), following the introduction of the 7-valent pneumococcal conjugate vaccination (PCV-7), was tempered by emergence of non-vaccine serotypes, particularly 19A. In Australia, three years after PCV-7 was replaced by PCV-13, containing 19A and 19F antigens, serogroup 19 was still a prominent cause of IPD in children under five. In this study we examined the evolution of serogroup 19 before and after introduction of paediatric vaccines in New South Wales (NSW), Australia. Genomes of 124 serogroup 19 IPD isolates collected before (2004) and after introduction of PCV-7 (2008) and PCV-13 (2014), from children under five in NSW, were analysed. Eleven core genome sequence clusters (cgSC) and 35 multilocus sequence types (ST) were identified. The majority (78/124) of the isolates belonged to four cgSCs: cgSC7 (ST199), cgSC11 (ST320), cgSC8 (ST63) and cgSC9 (ST2345). ST63 and ST2345 were exclusively serotype 19A and accounted for its predominantly intermediate penicillin resistance; these two clusters first appeared in 2008 and largely disappeared after introduction of PCV-13. Serogroup 19 was responsible for the highest proportion of vaccine failures in NSW. Relatively low immunogenicity of serogroup 19 antigens and Australia's three-dose vaccine schedule could affect the population dynamics of this serogroup.

Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance.

Candida glabrata can rapidly acquire mutations that result in drug resistance, especially to azoles and echinocandins. Identification of genetic mutations is essential, as resistance detected in vitro can often be correlated with clinical failure. We examined the feasibility of using whole genome sequencing (WGS) for genome-wide analysis of antifungal drug resistance in C. glabrata. The aim was torecognize enablers and barriers in the implementation WGS and measure its effectiveness. This paper outlines the key quality control checkpoints and essential components of WGS methodology to investigate genetic markers associated with reduced susceptibility to antifungal agents. It also estimates the accuracy of data analysis and turn-around-time of testing. Phenotypic susceptibility of 12 clinical, and one ATCC strain of C. glabrata was determined through antifungal susceptibility testing. These included three isolate pairs, from three patients, that developed rise in drug minimum inhibitory concentrations. In two pairs, the second isolate of each pair developed resistance to echinocandins. The second isolate of the third pair developed resistance to 5-flucytosine. The remaining comprised of susceptible and azole resistant isolates. Single nucleotide polymorphisms (SNPs) in genes linked to echinocandin, azole and 5-flucytosine resistance were confirmed in resistant isolates through WGS using the next generation sequencing. Non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgPDR1, CgCDR1 and FCY2 were identified. Overall, an average of 98% of the WGS reads of C. glabrata isolates mapped to the reference genome with about 75-fold read depth coverage. The turnaround time and cost were comparable to Sanger sequencing. In conclusion, WGS of C. glabrata was feasible in revealing clinically significant gene mutations involved in resistance to different antifungal drug classes without the need for multiple PCR/DNA sequencing reactions. This represents a positive step towards establishing WGS capability in the clinical laboratory for simultaneous detection of antifungal resistance conferring substitutions.

The Association of Mycobacterium avium subsp. paratuberculosis with Inflammatory Bowel Disease.

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.

Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium avium subsp. paratuberculosis.

The aim of this study was to investigate DNA extraction methods and PCR assays suitable for the detection of Mycobacterium paratuberculosis in bovine tissue. The majority of methods currently used to detect M. paratuberculosis have been developed using bovine samples, such as faeces, blood or tissue and, in many cases, have been based on detection from pooled samples from a herd. However most studies have not compared PCR results to culture results. In order to address this problem, four DNA extraction protocols and three PCR assays were employed to detect M. paratuberculosis in bovine tissue. Given that culture is reliable from cows, the results were then compared with the known M. paratuberculosis culture status. The following DNA extractions were included, two commercial kits, a boiling method, an in house extraction based on a published method and enrichment by sonication. The three PCR assays used included single round IS900 and f57 assays and a nested IS900 assay. In addition, another PCR assay was validated for the detection of any Mycobacterial species and a universal bacterial 16S rRNA gene assay was used to detect sample inhibition. The in-house DNA extraction was the most consistent in extracting good quality DNA compared to all other methods. The use of two PCR markers, IS900 and f57, and a universal PCR enabled the correct samples to be identified as M. paratuberculosis positive. In addition, when compared to the culture result, false-positives did not occur and PCR inhibition was readily identified. Using an in house DNA extraction coupled with the IS900 and f57 PCR markers, this study provides a reliable and simple method to detect M. paratuberculosis in both veterinary and spill over infections.

How accurately can we detect Mycobacterium avium subsp. paratuberculosis infection?

Mycobacteria have thwarted detection by scientists for centuries. Mycobacterium paratuberculosis is one of the most fastidious of the Mycobacteriaceae, and has been implicated in both animal and human diseases. In domestic livestock, M. paratuberculosis has been associated with Johne's disease, which given its increasing incidence, is currently a cause for concern, due to the potential for M. paratuberculosis to enter our food chain. In addition, a tenuous link has been reported between M. paratuberculosis and Crohn's disease, however evidence to support this link is hampered by the lack of accurate methodologies for detection of M. paratuberculosis in humans. This review compares the sensitivity and specificity of traditional and more recent techniques to the culture and molecular detection of M. paratuberculosis. While serology and culture are popular choices for the livestock industry they have not produced useful data for human infection. Although the advent of molecular biology has enabled faster diagnosis of M. paratuberculosis in human infection, there is currently no gold standard such as culture on which to validate these findings. Even with DNA/RNA detection methods, there is the ever present issue of the genetic relatedness of M. paratuberculosis to other mycobacteria of the Mycobacterium avium complex, some of which also infect humans with very different pathological outcomes. Recent developments in this field include more rapid methods of M. paratuberculosis culture as well as the development of more accurate and sensitive PCR assays. The application of these techniques should offer a greater insight as to the role of M. paratuberculosis in human gastrointestinal diseases.