Passive Sampling of SARS-CoV-2 for Wastewater Surveillance.

The shedding of pathogens by infected humans enables the use of sewage monitoring to conduct wastewater-based epidemiology (WBE). Although most WBE studies use data from large sewage treatment plants, timely data from smaller catchments are needed for targeted public health action. Traditional sampling methods, like autosamplers or grab sampling, are not conducive to quick ad hoc deployments and high-resolution monitoring at these smaller scales. This study develops and validates a cheap and easily deployable passive sampler unit, made from readily available consumables, with relevance to the COVID-19 pandemic but with broader use for WBE. We provide the first evidence that passive samplers can be used to detect SARS-CoV-2 in wastewater from populations with low prevalence of active COVID-19 infections (0.034 to 0.34 per 10,000), demonstrating their ability for early detection of infections at three different scales (lot, suburb, and city). A side by side evaluation of passive samplers (n = 245) and traditionally collected wastewater samples (n = 183) verified that the passive samplers were sensitive at detecting SARS-CoV-2 in wastewater. On all 33 days where we directly compared traditional and passive sampling techniques, at least one passive sampler was positive when the average SARS-CoV-2 concentration in the wastewater equaled or exceeded the quantification limit of 1.8 gene copies per mL (n = 7). Moreover, on 13 occasions where wastewater SARS-CoV-2 concentrations were less than 1.8 gene copies per mL, one or more passive samplers were positive. Finally, there was a statistically significant (p < 0.001) positive relationship between the concentrations of SARS-CoV-2 in wastewater and the levels found on the passive samplers, indicating that with further evaluation, these devices could yield semi-quantitative results in the future. Passive samplers have the potential for wide use in WBE with attractive feasibility attributes of cost, ease of deployment at small-scale locations, and continuous sampling of the wastewater. Further research will focus on the optimization of laboratory methods including elution and extraction and continued parallel deployment and evaluations in a variety of settings to inform optimal use in wastewater surveillance.

Salmonella from a Microtidal Estuary Are Capable of Invading Human Intestinal Cell Lines.

Faecal contamination poses health risks for the recreational users of urban estuaries. However, our understanding of the potential pathogenicity of faecal microbes in these environments is limited. To this end, a study was conducted to understand the spatial and seasonal distribution of Salmonella in water and sediments of the Yarra River estuary, Melbourne, Australia. Among 210 samples in total, culturable Salmonella were recovered from 27%, 17%, and 19% of water, bank, and bed sediment samples, respectively. The combined detection increased from 15% in winter to 32% in summer (p < 0.05) indicating seasonal variation as potential part of public health risk assessments. Further, pathogenic potential of the Salmonella isolates was characterised via the quantification of attachment and invasion capacity using human epithelial colorectal cell line Caco-2 on a subset of isolates (n = 62). While all of these isolates could attach and invade Caco-2 cells, 52% and 13% of these showed greater attachment and invasiveness, respectively, than the corresponding mean values for S. Typhimurium ATCC14028 control. Isolates from winter were on average more invasive (seven out of eight isolates with the highest invasiveness recovered from the colder sampling period) than the isolates from summer, and Salmonella collected during summer showed lower invasion (p < 0.05) compared with the control. Similar low invasion compared with the same control was observed for isolates recovered from bank sediment (p < 0.05). While the higher prevalence in summer may imply higher risks during these peak recreational periods, it is essential that this information is used in combination with quantitative microbial risk assessments to fully understand the health risks posed by Salmonella in microtidal estuaries.

Plant-Microbe Interactions Drive Denitrification Rates, Dissolved Nitrogen Removal, and the Abundance of Denitrification Genes in Stormwater Control Measures.

The microbial community and function along with nitrate/nitrite (NOx) removal rates, and nitrogen (N) partitioning into "uptake", "denitrification", and "remaining" via isotope tracers, were studied in soil bioretention mesocolumns (8 unique plant species). Total denitrification gene reads per million (rpm) were positively correlated with % denitrified ( r = 0.69) but negatively correlated with total NOx removal following simulated rain events ( r = -0.79). This is likely due to plant-microbe interactions. Plant species with greater root volume, plant and microbial assimilation %, and NOx removal % had lower denitrification genes and rates. This implies that although microorganisms have access to N, advantageous functions, like denitrification, may not increase. At the conclusion of the 1.5-year experiment, the microbial community was strongly influenced by plant species within the Top zone dominated by plant roots, and the presence or absence of a saturated zone influenced the microbial community within the Bottom zone. Leptospermum continentale was an outlier from the other plants and had much lower denitrification gene rpm (average 228) compared to the other species (range: 277 to 413). The antimicrobial properties and large root volume of Leptospermum continentale likely caused this denitrification gene depression.

Current Stormwater Harvesting Guidelines Are Inadequate for Mitigating Risk from Campylobacter During Nonpotable Reuse Activities.

Campylobacter is a pathogen frequently detected in urban stormwater worldwide. It is one of the leading causes of enteric disease in many developed countries and is the leading cause of enteric disease in Australia. Prior to harvesting stormwater, adequate treatment is necessary to mitigate risks derived from such harmful pathogens. The goal of this research was to estimate the health risks associated with the exposure to Campylobacter when harvesting urban stormwater for toilet flushing and irrigation activities, and the role treatment options play in limiting risks. Campylobacter data collected from several urban stormwater systems in Victoria, Australia, were the inputs of a Quantitative Microbial Risk Assessment model. The model included seven treatment scenarios, spanning wetlands, biofilters, and more traditional treatment trains including those recommended by the Australian Guidelines for Water Recycling. According to our modeling and acceptable risk thresholds, only two treatment scenarios could supply water of sufficient quality for toilet flushing and irrigation end-uses: (1) using stormwater biofilters coupled with UV-treatment and (2) a more conventional coagulation, filtration, UV, and chlorination treatment plant. Importantly, our modeling results suggest that current guidelines in place for stormwater reuse are not adequate for protecting against exposure to Campylobacter. However, more research is required to better define whether the Campylobacter detectable in stormwater are pathogenic to humans.

The transcriptomic response of Acinetobacter baumannii to colistin and doripenem alone and in combination in an in vitro pharmacokinetics/pharmacodynamics model.

OBJECTIVES: Colistin remains a last-line treatment for MDR Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against MDR strains. In order to understand the bacterial responses to these antibiotics, we analysed the transcriptome of A. baumannii following exposure to each. METHODS: RNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics (PK)/pharmacodynamics model to mimic the PK of both antibiotics in patients. RESULTS: After treatment with colistin (continuous infusion at 2 mg/L), >400 differentially regulated genes were identified, including many associated with outer membrane biogenesis, fatty acid metabolism and phospholipid trafficking. No genes were differentially expressed following treatment with doripenem (Cmax 25 mg/L, t1/2 1.5 h) for 15 min, but 45 genes were identified as differentially expressed after 1 h of growth under this condition. Treatment of A. baumannii with both colistin and doripenem together for 1 h resulted in >450 genes being identified as differentially expressed. More than 70% of these gene expression changes were also observed following colistin treatment alone. CONCLUSIONS: These data suggest that colistin causes gross damage to the outer membrane, facilitates lipid exchange between the inner and outer membrane and alters the normal asymmetric outer membrane composition. The transcriptional response to colistin was highly similar to that observed for an LPS-deficient strain, indicating that many of the observed changes are responses to outer membrane instability resulting from LPS loss.

Integrated conceptual modelling of faecal contamination in an urban estuary catchment.

Urban stormwater is regarded as a key input of faecal contamination in receiving water bodies and therefore, a major concern for health risks associated with aquatic recreation. Wastewater leakages, cross connections and overflows, together with faeces washed from surfaces during rainfall events, are possible origins of faecal contamination which enter these water bodies through stormwater drains. This paper applies conceptual models to a case study of the Yarra River estuary to understand the relative importance of fluxes derived from an urban creek and the 219 urban stormwater pipes which drain directly to the estuary as compared with other inputs, such as the Yarra River itself. Existing hydrologic-microorganism models were used for the estimation of the inputs from riverine and urban stormwater fluxes. These predictions were applied as boundary conditions for a new, highly simplified, model which accounts for the transport and survival of faecal microorganisms in the estuary. All models were calibrated using a rich dataset, containing over 2,000 measured Escherichia coli concentrations. Mass balances from the riverine and stormwater models indicate the limited influence of urban stormwater drains on the estuary during dry weather; less than 0.05% to 10% (5th and 95th percentile; median 0.5%) of the total daily E. coli load entering the estuary was derived from urban stormwater drains. While wet weather contributions from stormwater drains could be more significant (2% to 50%; 5th and 95th percentile), the average contribution remained marginal (median 10%). Sensitivity testing of the estuarine microorganism model by switching off stormwater boundary conditions resulted in minimal model efficiency reduction; this may reflect the low average daily contribution from urban stormwater drains. While these results confirm previous studies which show that E. coli loads derived from stormwater drains are dwarfed by other inputs, it is essential to note that these results also demonstrate that some conditions reveal the opposite; high proportions from stormwater are possible when combined with low riverine inputs and high urban rainfall. Furthermore, this study focuses on the overall impacts of direct urban stormwater inputs on the faecal contamination levels within the estuary, and localized impacts would certainly require further investigation.

Exertional heat stress promotes the presence of bacterial DNA in plasma: A counterbalanced randomised controlled trial.

OBJECTIVES: The primary aim was to explore the impact of exertional-heat stress (EHS) promoted exercise-associated bacteraemia. A secondary aim was to examine if an amino acid beverage (AAB) intervention may mitigate exercise-associated bacteraemia. DESIGN: Counterbalanced randomised control trial. METHODS: Twenty endurance trained male participants completed two randomised EHS trials. On one occasion, participants consumed a 237 mL AAB twice daily for 7 days prior, immediately before and every 20 min during EHS (2 h running at 60 % V̇O2max in 35 °C). On the other occasion, a water volume control (CON) equivalent was consumed. Whole blood samples were collected pre- and immediately post-EHS, and were analysed for plasma DNA concentration by fluorometer quantification after microbial extraction, and bacterial relative abundance by next generation 16s rRNA gene sequencing. RESULTS: Increased concentration of microbial DNA in plasma pre- to post-EHS was observed on CON (pre-EHS 0.014 ng/µL, post-EHS 0.039 ng/µL) (p < 0.001) and AAB (pre-EHS 0.015 ng/µL, post-EHS 0.031 ng/µL) (p < 0.001). The magnitude of change from pre- to post-exercise on AAB was 40 % lower, but no significant difference was observed versus CON (p = 0.455). Predominant bacterial groups identified included: phyla-Proteobacteria (88.0 %), family-Burkholderiaceae (59.1 %), and genus-Curvibacter (58.6 %). No significant variation in absolute and relative change in α-diversity and relative abundance for phyla, family, and genus bacterial groups was observed in AAB versus CON. CONCLUSIONS: The increased presence of microbial-bacterial DNA in systemic circulation in response to EHS appears positive in all participants. An amino acid beverage supplementation period prior to and consumption during EHS did not provide significant attenuation of EHS-associated bacteraemia.

Precipitation of iron on the surface of Leptospira interrogans is associated with mutation of the stress response metalloprotease HtpX.

High concentrations of free metal ions in the environment can be detrimental to bacterial survival. However, bacteria utilize strategies, including the activation of stress response pathways and immobilizing chemical elements on their surface, to limit this toxicity. In this study, we characterized LA4131, the HtpX-like M48 metalloprotease from Leptospira interrogans, with a putative role in bacterial stress response and membrane homeostasis. Growth of the la4131 transposon mutant strain (L522) in 360 µM FeSO4 (10-fold the normal in vitro concentration) resulted in the production of an amorphous iron precipitate. Atomic force microscopy and transmission electron microscopy analysis of the strain demonstrated that precipitate production was associated with the generation and release of outer membrane vesicles (OMVs) from the leptospiral surface. Transcriptional studies indicated that inactivation of la4131 resulted in altered expression of a subset of metal toxicity and stress response genes. Combining these findings, this report describes OMV production in response to environmental stressors and associates OMV production with the in vitro activity of an HtpX-like metalloprotease.

Reliability of pathophysiological markers reflective of exercise-induced gastrointestinal syndrome (EIGS) in response to 2-h high-intensity interval exercise: A comprehensive methodological efficacy exploration.

The study aimed to determine the test-retest reliability of exercise-induced gastrointestinal syndrome (EIGS) biomarkers, and assess the association of pre-exercise short chain fatty acid (SCFA) concentration with these biomarkers in response to prolonged strenuous exercise. Thirty-four participants completed 2 h of high-intensity interval training (HIIT) on two separate occasions with at least 5-days washout. Blood samples were collected pre- and post-exercise, and analysed for biomarkers associated with EIGS [i.e., cortisol, intestinal fatty-acid binding protein (I-FABP), sCD14, lipopolysaccharide binding protein (LBP), leukocyte counts, in-vitro neutrophil function, and systemic inflammatory cytokine profile]. Fecal samples were collected pre-exercise on both occasions. In plasma and fecal samples, bacterial DNA concentration was determined by fluorometer quantification, microbial taxonomy by 16S rRNA amplicon sequencing, and SCFA concentration by gas-chromatography. In response to exercise, 2 h of HIIT modestly perturbed biomarkers indicative of EIGS, including inducing bacteremia (i.e., quantity and diversity). Reliability analysis using comparative tests, Cohen's d, two-tailed correlation, and intraclass correlation coefficient (ICC) of resting biomarkers presented good-to-excellent for IL-1ra (r = 0.710, ICC = 0.92), IL-10 (r = 0.665, ICC = 0.73), cortisol (r = 0.870, ICC = 0.87), and LBP (r = 0.813, ICC = 0.76); moderate for total (r = 0.839, ICC = 0.44) and per cell (r = 0.749, ICC = 0.54) bacterially-stimulated elastase release, IL-1ß (r = 0.625, ICC = 0.64), TNF-α (r = 0.523, ICC = 0.56), I-FABP (r = 0.411, ICC = 0.21), and sCD14 (r = 0.409, ICC = 0.38), plus fecal bacterial α-diversity; and poor for leukocyte (r = 0.327, ICC = 0.33) and neutrophil (r = 0.352, ICC = 0.32) counts. In addition, a medium negative correlation was observed between plasma butyrate and I-FABP (r = -0.390). The current data suggest a suite of biomarkers should be used to determine the incidence and severity of EIGS. Moreover, determination of plasma and/or fecal SCFA may provide some insight into the mechanistic aspects of EIGS instigation and magnitude in response to exercise.

The Impact of a 24-h Low and High Fermentable Oligo- Di- Mono-Saccharides and Polyol (FODMAP) Diet on Plasma Bacterial Profile in Response to Exertional-Heat Stress.

Exertional-heat stress (EHS) compromises intestinal epithelial integrity, potentially leading to the translocation of pathogenic agents into circulation. This study aimed to explore the impact of EHS on the systemic circulatory bacterial profile and to determine the impact of a short-term low (LFOD) and high (HFOD) fermentable oligo- di- mono-saccharide and polyol dietary intervention before EHS on this profile. Using a double-blind randomized cross-over design, thirteen endurance runners (n = 8 males, n = 5 females), with a history of exercise-associated gastrointestinal symptoms (Ex-GIS), consumed a 24 h LFOD and HFOD before 2 h running at 60% V.O2max in 35.6 °C. Blood and fecal samples were collected pre-EHS to determine plasma microbial DNA concentration, and sample bacteria and short chain fatty acid (SCFA) profiles by fluorometer quantification, 16S rRNA amplicon gene sequencing, and gas chromatography, respectively. Blood samples were also collected post-EHS to determine changes in plasma bacteria. EHS increased plasma microbial DNA similarly in both FODMAP trials (0.019 ng·µL-1 to 0.082 ng·µL-1) (p < 0.01). Similar pre- to post-EHS increases in plasma Proteobacteria (+1.6%) and Firmicutes (+0.6%) phyla relative abundance were observed in both FODMAP trials. This included increases in several Proteobacteria genus (Delftia and Serratia) groups. LFOD presented higher fecal Firmicutes (74%) and lower Bacteroidota (10%) relative abundance pre-EHS, as a result of an increase in Ruminococcaceae and Lachnospiraceae family and respective genus groups, compared with HFOD (64% and 25%, respectively). Pre-EHS plasma total SCFA (p = 0.040) and acetate (p = 0.036) concentrations were higher for HFOD (188 and 178 µmol·L-1, respectively) vs. LFOD (163 and 153 µmol·L-1, respectively). Pre-EHS total fecal SCFA concentration (119 and 74 µmol·g-1; p < 0.001), including acetate (74 and 45 µmol·g-1; p = 0.001), butyrate (22 and 13 µmol·g-1; p = 0.002), and propionate (20 and 13 µmol·g-1; p = 0.011), were higher on HFOD vs LFOD, respectively. EHS causes the translocation of whole bacteria into systemic circulation and alterations to the plasma bacterial profile, but the FODMAP content of a 24 h diet beforehand does not alter this outcome.

Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-ß-1,6-N-acetylglucosamine.

We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-ß-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

Interactive effect of temperature and plant species on nitrogen cycling and treatment in stormwater biofiltration systems.

Stormwater biofiltration systems (also known as biofilters, bioretention, rain gardens) are engineered nature-based solutions, which help mitigate aquatic nitrogen pollution arising from storm runoff. These systems are being increasingly used in a range of climates across the world. A decline in treatment performance is frequently observed in cold weather conditions. While plant species comprise an important design factor influencing system performance, the effect of temperature on the fate of dissolved nitrogen forms, namely ammonium (NH4+) and nitrate (NO3-), in the presence of different plant species in these systems remains unclear. A large scale laboratory experiment was undertaken that measured potential rates of nitrification, denitrification and dissimilatory nitrate reduction to ammonium (DNRA) as well as the microbial community structure to investigate nitrogen fate and hence removal under two different temperature conditions (2 °C and 15 °C) in the presence of four distinct plant species. The results indicate that lower nitrification rates (reduced by a factor of 4) coupled with potential media NH4+ desorption could be contributing to reduced NH4+ removal during cold conditions. Planting with species exhibiting good nutrient uptake capacity can reduce the extent of this performance decline. While NO3- reduction generally remains problematic during cold weather (<0 to 55% reduction), which may not be significantly different from warmer periods, the study demonstrated that the denitrification potential and gene abundance (nap, nar, NirS, norB, nosZ) to be higher than those of nitrification (amoA). Denitrification may not proceeding at optimal rates due to lack of conducive environmental conditions. Nitrogen transformation via DNRA was found to be relatively insignificant. Future studies should investigate the potential of employing cold-resilient plant species to maintain both NH4+ and NO3- removal in cold weather conditions.

Riparian buffers: Disrupting the transport of E. coli from rural catchments to streams.

High levels of E. coli and associated faecal microbes in waterways as a result of agricultural and residential land use can pose environmental, human health, and economic risks. This study aims to understand the impacts of land use, climatic variables, and riparian buffers on in-stream E. coli concentrations. Flow, temperature, and E. coli were monitored during three sampling campaigns within eleven independent catchments. These catchments have varying land use and extents of riparian buffer coverage. Results showed that catchments with predominantly agricultural and residential land uses (average = 349.7 MPN/100 mL) had higher E. coli concentrations than predominantly forested catchments (average = 111.8 MPN/100 mL). However, there were no statistically significant differences in E. coli concentrations between the agricultural and residential land uses. Riparian buffers appear to reduce E. coli concentrations in streams, as indicated by significant negative correlations between in-stream E. coli concentrations with the riparian buffer areal coverage (Pearson's r = -0.95, Spearman's ρ = -0.90) and the ratio of buffer length to stream length (Pearson's r = -0.87, Spearman's ρ = -0.90). We find that riparian buffers potentially disrupt transport pathways that govern E. coli movement, which in-turn can affect the concentration-discharge relationship. This reinforces the importance of protecting and restoring riparian buffers along drainage lines in agricultural and rural-residential catchments to improve downstream microbial water quality.

Mutations affecting Leptospira interrogans lipopolysaccharide attenuate virulence.

Leptospira interrogans is the causative agent of leptospirosis. Lipopolysaccharide (LPS) is the major outer membrane component of L. interrogans. It is the dominant antigen recognized during infection and the basis for serological classification. The structure of LPS and its role in pathogenesis are unknown. We describe two defined mutants of L. interrogans serovar Manilae with transposon insertions in the LPS locus. Mutant M895 was disrupted in gene la1641 encoding a protein with no known homologues. M1352 was disrupted in a gene unique to serovar Manilae also encoding a protein of unknown function. M895 produced truncated LPS while M1352 showed little or no change in LPS molecular mass. Both mutants showed altered agglutination titres against rabbit antiserum and against a panel of LPS-specific monoclonal antibodies. The mutants were severely attenuated in virulence via the intraperitoneal route of infection, and were cleared from the host animal by 3 days after infection. M895 was also highly attenuated via the mucosal infection route. Resistance to complement in human serum was unaltered for both mutants. While complementation of mutants was not possible, the attenuation of two independently derived LPS mutants demonstrates for the first time that LPS plays an essential role leptospiral virulence.

Draft Genome Sequences of Eight Campylobacter volucris Isolates from Freshwater Sources in Victoria, Australia.

Campylobacter spp. can survive and be transmitted from a range of environments. Here, we examine eight draft genome sequences of Campylobacter volucris, identified as part of an examination of waterborne Campylobacter species. This is the first report of environmental survival of C. volucris outside gull species.

The Impact of Pipe Material on the Diversity of Microbial Communities in Drinking Water Distribution Systems.

As many cities around the world face the prospect of replacing aging drinking water distribution systems (DWDS), water utilities must make careful decisions on new pipe material (e.g., cement-lined or PVC) for these systems. These decisions are informed by cost, physical integrity, and impact on microbiological and physicochemical water quality. Indeed, pipe material can impact the development of biofilm in DWDS that can harbor pathogens and impact drinking water quality. Annular reactors (ARs) with cast iron and cement coupons fed with chloraminated water from a municipal DWDS were used to investigate the impact of pipe material on biofilm development and composition over 16 months. The ARs were plumbed as closely as possible to the water main in the basement of an academic building to simulate distribution system conditions. Biofilm communities on coupons were characterized using 16S rRNA sequencing. In the cast iron reactors, ß-proteobacteria, Actinobacteria, and α-proteobacteria were similarly relatively abundant (24.1, 22.5, and 22.4%, respectively) while in the cement reactors, α-proteobacteria and Actinobacteria were more relatively abundant (36.3 and 35.2%, respectively) compared to ß-proteobacteria (12.8%). Mean alpha diversity (estimated with Shannon H and Faith's Phylogenetic Difference indices) was greater in cast iron reactors (Shannon: 5.00 ± 0.41; Faith's PD: 15.40 ± 2.88) than in cement reactors (Shannon: 4.16 ± 0.78; Faith's PD: 13.00 ± 2.01). PCoA of Bray-Curtis dissimilarities indicated that communities in cast iron ARs, cement ARs, bulk distribution system water, and distribution system pipe biofilm were distinct. The mean relative abundance of Mycobacterium spp. was greater in the cement reactors (34.8 ± 18.6%) than in the cast iron reactors (21.7 ± 11.9%). In contrast, the mean relative abundance of Legionella spp. trended higher in biofilm from cast iron reactors (0.5 ± 0.7%) than biofilm in cement reactors (0.01 ± 0.01%). These results suggest that pipe material is associated with differences in the diversity, bacterial composition, and opportunistic pathogen prevalence in biofilm of DWDS.

Monitoring of diverse enteric pathogens across environmental and host reservoirs with TaqMan array cards and standard qPCR: a methodological comparison study.

BACKGROUND: Multiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmited via several pathways through human, animal, and environmental reservoirs. Individual qPCR assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan array card (TAC), which allows simultaneous detection of multiple enteropathogens, has only previously been validated in human clinical samples. METHODS: In this methodological comparison study, we compared the performance of a custom 48-singleplex TAC relative to standard qPCR. We established the sensitivity and specificity of each method for the detection of eight enteric targets, by using spiked samples with varying levels of PCR inhibition. We then tested the prevalence and abundance of pathogens in wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji using both TAC and qPCR. FINDINGS: Both methods exhibited similarly h specificity (TAC 100%, qPCR 94%), sensitivity (TAC 92%, qPCR 100%), and quantitation accuracy (TAC 91%, qPCR 99%) in non-inhibited sample matrices with spiked gene fragments. PCR inhibitors substantially affected detection via TAC, though this issue was alleviated by ten-fold sample dilution. Among samples from informal settlements, the two techniques performed similarly for detection (89% agreement) and quantitation (R2 0·82) for the eight enteropathogen targets. The TAC additionally included 38 other enteric targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR. INTERPRETATION: The two techniques produced similar results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel enabling a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs could allow better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load. FUNDING: Wellcome Trust Our Planet, Our Health programme.

Study design, rationale and methods of the Revitalising Informal Settlements and their Environments (RISE) study: a cluster randomised controlled trial to evaluate environmental and human health impacts of a water-sensitive intervention in informal settlements in Indonesia and Fiji.

INTRODUCTION: Increasing urban populations have led to the growth of informal settlements, with contaminated environments linked to poor human health through a range of interlinked pathways. Here, we describe the design and methods for the Revitalising Informal Settlements and their Environments (RISE) study, a transdisciplinary randomised trial evaluating impacts of an intervention to upgrade urban informal settlements in two Asia-Pacific countries. METHODS AND ANALYSIS: RISE is a cluster randomised controlled trial among 12 settlements in Makassar, Indonesia, and 12 in Suva, Fiji. Six settlements in each country have been randomised to receive the intervention at the outset; the remainder will serve as controls and be offered intervention delivery after trial completion. The intervention involves a water-sensitive approach, delivering site-specific, modular, decentralised infrastructure primarily aimed at improving health by decreasing exposure to environmental faecal contamination. Consenting households within each informal settlement site have been enrolled, with longitudinal assessment to involve health and well-being surveys, and human and environmental sampling. Primary outcomes will be evaluated in children under 5 years of age and include prevalence and diversity of gastrointestinal pathogens, abundance and diversity of antimicrobial resistance (AMR) genes in gastrointestinal microorganisms and markers of gastrointestinal inflammation. Diverse secondary outcomes include changes in microbial contamination; abundance and diversity of pathogens and AMR genes in environmental samples; impacts on ecological biodiversity and microclimates; mosquito vector abundance; anthropometric assessments, nutrition markers and systemic inflammation in children; caregiver-reported and self-reported health symptoms and healthcare utilisation; and measures of individual and community psychological, emotional and economic well-being. The study aims to provide proof-of-concept evidence to inform policies on upgrading of informal settlements to improve environments and human health and well-being. ETHICS: Study protocols have been approved by ethics boards at Monash University, Fiji National University and Hasanuddin University. TRIAL REGISTRATION NUMBER: ACTRN12618000633280; Pre-results.

A planetary health model for reducing exposure to faecal contamination in urban informal settlements: Baseline findings from Makassar, Indonesia.

BACKGROUND: The intense interactions between people, animals and environmental systems in urban informal settlements compromise human and environmental health. Inadequate water and sanitation services, compounded by exposure to flooding and climate change risks, expose inhabitants to environmental contamination causing poor health and wellbeing and degrading ecosystems. However, the exact nature and full scope of risks and exposure pathways between human health and the environment in informal settlements are uncertain. Existing models are limited to microbiological linkages related to faecal-oral exposures at the individual level, and do not account for a broader range of human-environmental variables and interactions that affect population health and wellbeing. METHODS: We undertook a 12-month health and environmental assessment in 12 flood-prone informal settlements in Makassar, Indonesia. We obtained caregiver-reported health data, anthropometric measurements, stool and blood samples from children < 5 years, and health and wellbeing data for children 5-14 years and adult respondents. We collected environmental data including temperature, mosquito and rat species abundance, and water and sediment samples. Demographic, built environment and household asset data were also collected. We combined our data with existing literature to generate a novel planetary health model of health and environment in informal settlements. RESULTS: Across the 12 settlements, 593 households and 2764 participants were enrolled. Two-thirds (64·1%) of all houses (26·3-82·7% per settlement) had formal land tenure documentation. Cough, fever and diarrhoea in the week prior to the survey were reported among an average of 34.3%, 26.9% and 9.7% of children aged < 5 years, respectively; although proportions varied over time, prevalence among these youngest children was consistently higher than among children 5-14 years or adult respondents. Among children < 5 years, 44·3% experienced stunting, 41·1% underweight, 12.4% wasting, and 26.5% were anaemic. There was self- or carer-reported poor mental health among 16.6% of children aged 5-14 years and 13.9% of adult respondents. Rates of potential risky exposures from swimming in waterways, eating uncooked produce, and eating soil or dirt were high, as were exposures to flooding and livestock. Just over one third of households (35.3%) had access to municipal water, and contamination of well water with E. coli and nitrogen species was common. Most (79·5%) houses had an in-house toilet, but no houses were connected to a piped sewer network or safe, properly constructed septic tank. Median monthly settlement outdoor temperatures ranged from 26·2 °C to 29.3 °C, and were on average, 1·1 °C warmer inside houses than outside. Mosquito density varied over time, with Culex quinquefasciatus accounting for 94·7% of species. Framed by a planetary health lens, our model includes four thematic domains: (1) the physical/built environment; (2) the ecological environment; (3) human health; and (4) socio-economic wellbeing, and is structured at individual, household, settlement, and city/beyond spatial scales. CONCLUSIONS: Our planetary health model includes key risk factors and faecal-oral exposure pathways but extends beyond conventional microbiological faecal-oral enteropathogen exposure pathways to comprehensively account for a wider range of variables affecting health in urban informal settlements. It includes broader ecological interconnections and planetary health-related variables at the household, settlement and city levels. It proposes a composite framework of markers to assess water and sanitation challenges and flood risks in urban informal settlements for optimal design and monitoring of interventions.

Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production.

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.