Invasive methods requiring general anaesthesia are needed to sample the lung microbiota in young children who do not expectorate. This poses substantial challenges to longitudinal study of paediatric airway microbiota. Non-invasive upper airway sampling is an alternative method for monitoring airway microbiota; however, there are limited data describing the relationship of such results with lung microbiota in young children. In this study, we compared the upper and lower airway microbiota in young children to determine whether non-invasive upper airway sampling procedures provide a reliable measure of either lung microbiota or clinically defined differences. The microbiota in oropharyngeal (OP) swabs, nasopharyngeal (NP) swabs and bronchoalveolar lavage (BAL) from 78 children (median age 2.2 years) with and without lung disease were characterised using 16S rRNA gene sequencing. Permutational multivariate analysis of variance (PERMANOVA) detected significant differences between the microbiota in BAL and those in both OP swabs (p = 0.0001, Pseudo-F = 12.2, df = 1) and NP swabs (p = 0.0001; Pseudo-F = 21.9, df = 1) with the NP and BAL microbiota more different than the OP and BAL, as indicated by a higher Pseudo-F value. The microbiota in combined OP and NP data (upper airways) provided a more comprehensive representation of BAL microbiota, but significant differences between the upper airway and BAL microbiota remained, albeit with a considerably smaller Pseudo-F (PERMANOVA p = 0.0001; Pseudo-F = 4.9, df = 1). Despite this overall difference, paired BAL and upper airway (OP and NP) microbiota were >50 % similar among 69 % of children. Furthermore, canonical analysis of principal coordinates (CAP analysis) detected significant differences between the microbiota from clinically defined groups when analysing either BAL (eigenvalues >0.8; misclassification rate 26.5 %) or the combined OP and NP data (eigenvalues >0.8; misclassification rate 12.2 %). Upper airway sampling provided an imperfect, but reliable, representation of the BAL microbiota for most children in this study. We recommend inclusion of both OP and NP specimens when non-invasive upper airway sampling is needed to assess airway microbiota in young children who do not expectorate. The results of the CAP analysis suggest lower and upper airway microbiota profiles may differentiate children with chronic suppurative lung disease from those with persistent bacterial bronchitis; however, further research is needed to confirm this observation.

Darwin Harbour in northern Australia is an estuary in the wet-dry tropics subject to increasing urbanisation with localised water quality degradation due to increased nutrient loads from urban runoff and treated sewage effluent. Tropical estuaries are poorly studied compared to temperate systems and little is known about the microbial community-level response to nutrients. We aimed to examine the spatial and temporal patterns of the bacterial community and its association with abiotic factors. Since Darwin Harbour is macrotidal with strong seasonal patterns and mixing, we sought to determine if a human impact signal was discernible in the microbiota despite the strong hydrodynamic forces. Adopting a single impact–double reference design, we investigated the bacterial community using next-generation sequencing of the 16S rRNA gene from water and sediment from reference creeks and creeks affected by effluent and urban runoff. Samples were collected over two years during neap and spring tides, in the dry and wet seasons. Temporal drivers, namely seasons and tides had the strongest relationship to the water microbiota, reflecting the macrotidal nature of the estuary and its location in the wet-dry tropics. The neap-tide water microbiota provided the clearest spatial resolution while the sediment microbiota reflected current and past water conditions. Differences in patterns of the microbiota between different parts of the harbour reflected the harbour's complex hydrodynamics and bathymetry. Despite these variations, a microbial signature was discernible relating to specific effluent sources and urban runoff, and the composite of nutrient levels accounted for the major part of the explained variation in the microbiota followed by salinity. Our results confirm an overall good water quality but they also reflect the extent of some hypereutrophic areas. Our results show that the microbiota is a sensitive indicator to assess ecosystem health even in this dynamic and complex ecosystem.

"The Expanding Global Footprint of Burkholderia pseudomallei and Melioidosis" published on 09 May 2023 by The American Society of Tropical Medicine and Hygiene.

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314’s sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314’s airways over a >16-year period, and also sampled the patient’s home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host. Pathogens frequently jump between different hosts, and associated adaptation may lead to the emergence of new infectious agents. Such host-jumping evolution is witnessed through endpoint analyses but these cannot capture genetic changes in lineages that have gone extinct. In this study, we have identified and monitored an example of the evolution of a bacterium often deadly to its mammalian host, in an unprecedented case whereby disease lessened through time and the pathogen became a part of the commensal human flora. We used genomic analyses to characterize more than 16 years of this evolutionary process and the stepwise mutations that control pathogen interactions with the patient. Soon after infection, mutational changes occurred that allowed the bacterium to remain in the airways without causing disease. This shift towards avirulence was determined based on clinical data and virulence testing in an animal model. In addition, mutations occurred that contributed to the persistence of the bacteria in the patient's lungs. Finally, we found evidence for the evolutionary emergence and persistence of two distinct lineages of the bacterium over the last 13 years, presenting interesting questions about niche utilization. Bacteria are ubiquitous in the human body and almost all are beneficial or benign. In this study, we document the evolutionary conversion of a normally deadly bacterium towards a commensal.

Bacteria from the Vibrio genus are a ubiquitous component of coastal and estuarine ecosystems with several pathogenic Vibrio species displaying preferences for warm tropical waters. We studied the spatial and temporal abundance of three key human potential pathogens V. parahaemolyticus, V. cholerae and V. vulnificus in northern tropical Australia, over the wet and dry seasons, to identify environmental parameters influencing their abundance. Quantitative PCR (qPCR) analysis revealed that V. parahaemolyticus occurred more frequently and in higher abundance than V. cholerae and V. vulnificus across all locations examined. All three species were more abundant during the wet season, with V. parahaemolyticus abundance correlated to temperature and conductivity, whereas nutrient concentrations and turbidity best explained V. vulnificus abundance. In addition to these targeted qPCR analyses, we assessed the composition and dynamics of the entire Vibrio community using hsp60 amplicon sequencing. Using this approach, 42 Vibrio species were identified, including a number of other pathogenic species such as V. alginolyticus, V. mimicus and V. fluvialis. The Vibrio community was more diverse in the wet season, with temperature and dissolved oxygen as the key factors governing community composition. Seasonal differences were primarily driven by a greater abundance of V. parahaemolyticus and V. vulnificus during the wet season, while spatial differences were driven by different abundances of V. harveyi, V. campbellii, V. cholerae and V. navarrensis. When we related the abundance of Vibrio to other bacterial taxa, defined using 16S rRNA gene amplicon sequencing, V. parahaemolyticus was negatively correlated to several taxa, including members of the Rickettsiales and Saccharimonadales, while V. vulnificus was negatively correlated to Rhobacteriaceae and Cyanobiaceae. In contrast, V. alginolyticus, V. harveyi and V. mediterranei were all positively correlated to Cyanobacteria. These observations highlight the dynamic nature of Vibrio communities and expands current understanding of the processes governing the occurrence of potentially pathogenic Vibrio spp. in tropical coastal ecosystems.

In the wet-dry tropics of Northern Australia, drinking water in remote communities is mostly sourced from bores accessing groundwater. Many aquifers contain naturally high levels of iron and some are shallow with surface water intrusion in the wet season. Therefore, environmental bacteria such as iron-cycling bacteria promoting biofilm formation in pipes or opportunistic pathogens can occur in these waters. An opportunistic pathogen endemic to northern Australia and Southeast Asia and emerging worldwide is Burkholderia pseudomallei. It causes the frequently fatal disease melioidosis in humans and animals. As we know very little about the microbial composition of drinking water in remote communities, this study aimed to provide a first snapshot of the microbiota and occurrence of opportunistic pathogens in bulk water and biofilms from the source and through the distribution system of three remote water supplies with varying iron levels. Using 16s-rRNA gene sequencing, we found that the geochemistry of the groundwater had a substantial impact on the untreated microbiota. Different iron-cycling bacteria reflected differences in redox status and nutrients. We cultured and sequenced B. pseudomallei from bores with elevated iron and from a multi-species biofilm which also contained iron-oxidizing Gallionella, nitrifying Nitrospira and amoebae. Gallionella are increasingly used in iron-removal filters in water supplies and more research is needed to examine these interactions. Similar to other opportunistic pathogens, B. pseudomallei occurred in water with low organic carbon levels and with low heterotrophic microbial growth. No B. pseudomallei were detected in treated water; however, abundant DNA of another opportunistic pathogen group, non-tuberculous mycobacteria was recovered from treated parts of one supply. Results from this study will inform future studies to ultimately improve management guidelines for water supplies in the wet-dry tropics. Water providers in the wet-dry tropics of Northern Australia face additional challenges to keep drinking water microbiologically safe. The source water is often rich in iron-cycling bacteria leading to excessive biofilm formation in pipes and it can also contain the emerging opportunistic pathogen Burkholderia pseudomallei causing the severe disease melioidosis in humans and animals. We know very little about the ecology of microbes in remote community water supplies, so to start to fill this gap we assessed the microbial composition from the source to the distribution of three remote water supplies. We not only found that the geochemistry of the source water had a substantial impact on the composition of the iron-cycling bacteria but B. pseudomallei was cultured from source water with low organic carbon but elevated iron levels and from a multi-species biofilm linked to iron bacteria. No B. pseudomallei were detected in treated water; however, abundant DNA of another opportunistic pathogen group, non-tuberculous mycobacteria, was recovered from treated parts of one water supply. This work lays the foundation for future studies to ultimately improve management guidelines for water supplies in the wet-dry tropics.