Genomic analysis of diverse environmental Acinetobacter isolates identifies plasmids, antibiotic resistance genes, and capsular polysaccharides shared with clinical strains.

Acinetobacter baumannii, an important pathogen known for its widespread antibiotic resistance, has been the focus of extensive research within its genus, primarily involving clinical isolates. Consequently, data on environmental A. baumannii and other Acinetobacter species remain limited. Here, we utilized Illumina and Nanopore sequencing to analyze the genomes of 10 Acinetobacter isolates representing 6 different species sourced from aquatic environments in South Australia. All 10 isolates were phylogenetically distinct compared to clinical and other non-clinical Acinetobacter strains, often tens of thousands of single-nucleotide polymorphisms from their nearest neighbors. Despite the genetic divergence, we identified pdif modules (sections of mobilized DNA) carrying clinically important antimicrobial resistance genes in species other than A. baumannii, including carbapenemase oxa58, tetracycline resistance gene tet(39), and macrolide resistance genes msr(E)-mph(E). These pdif modules were located on plasmids with high sequence identity to those circulating in globally distributed A. baumannii ST1 and ST2 clones. The environmental A. baumannii isolate characterized here (SAAb472; ST350) did not possess any native plasmids; however, it could capture two clinically important plasmids (pRAY and pACICU2) with high transfer frequencies. Furthermore, A. baumannii SAAb472 possessed virulence genes and a capsular polysaccharide type analogous to clinical strains. Our findings highlight the potential for environmental Acinetobacter species to acquire and disseminate clinically important antimicrobial resistance genes, underscoring the need for further research into the ecology and evolution of this important genus.IMPORTANCEAntimicrobial resistance (AMR) is a global threat to human, animal, and environmental health. Studying AMR in environmental bacteria is crucial to understand the emergence and dissemination of resistance genes and pathogens, and to identify potential reservoirs and transmission routes. This study provides novel insights into the genomic diversity and AMR potential of environmental Acinetobacter species. By comparing the genomes of aquatic Acinetobacter isolates with clinical and non-clinical strains, we revealed that they are highly divergent yet carry pdif modules that encode resistance to antibiotics commonly used in clinical settings. We also demonstrated that an environmental A. baumannii isolate can acquire clinically relevant plasmids and carries virulence factors similar to those of hospital-associated strains. These findings suggest that environmental Acinetobacter species may serve as reservoirs and vectors of clinically important genes. Consequently, further research is warranted to comprehensively understand the ecology and evolution of this genus.

Impacts of coprophagic foraging behaviour on the avian gut microbiome.

Avian gut microbial communities are complex and play a fundamental role in regulating biological functions within an individual. Although it is well established that diet can influence the structure and composition of the gut microbiota, foraging behaviour may also play a critical, yet unexplored role in shaping the composition, dynamics, and adaptive potential of avian gut microbiota. In this review, we examine the potential influence of coprophagic foraging behaviour on the establishment and adaptability of wild avian gut microbiomes. Coprophagy involves the ingestion of faeces, sourced from either self (autocoprophagy), conspecific animals (allocoprophagy), or heterospecific animals. Much like faecal transplant therapy, coprophagy may (i) support the establishment of the gut microbiota of young precocial species, (ii) directly and indirectly provide nutritional and energetic requirements, and (iii) represent a mechanism by which birds can rapidly adapt the microbiota to changing environments and diets. However, in certain contexts, coprophagy may also pose risks to wild birds, and their microbiomes, through increased exposure to chemical pollutants, pathogenic microbes, and antibiotic-resistant microbes, with deleterious effects on host health and performance. Given the potentially far-reaching consequences of coprophagy for avian microbiomes, and the dearth of literature directly investigating these links, we have developed a predictive framework for directing future research to understand better when and why wild birds engage in distinct types of coprophagy, and the consequences of this foraging behaviour. There is a need for comprehensive investigation into the influence of coprophagy on avian gut microbiotas and its effects on host health and performance throughout ontogeny and across a range of environmental perturbations. Future behavioural studies combined with metagenomic approaches are needed to provide insights into the function of this poorly understood behaviour.

Genomic surveillance for antimicrobial resistance - a One Health perspective.

Antimicrobial resistance (AMR) - the ability of microorganisms to adapt and survive under diverse chemical selection pressures - is influenced by complex interactions between humans, companion and food-producing animals, wildlife, insects and the environment. To understand and manage the threat posed to health (human, animal, plant and environmental) and security (food and water security and biosecurity), a multifaceted 'One Health' approach to AMR surveillance is required. Genomic technologies have enabled monitoring of the mobilization, persistence and abundance of AMR genes and mutations within and between microbial populations. Their adoption has also allowed source-tracing of AMR pathogens and modelling of AMR evolution and transmission. Here, we highlight recent advances in genomic AMR surveillance and the relative strengths of different technologies for AMR surveillance and research. We showcase recent insights derived from One Health genomic surveillance and consider the challenges to broader adoption both in developed and in lower- and middle-income countries.

Comparison of kits for SARS-CoV-2 extraction in liquid and passive samples.

Effective extraction and detection of viral nucleic acids from sewage are fundamental components of a successful SARS-CoV-2 sewage surveillance programme. As there is no standard method employed in sewage surveillance, understanding the performance of different extraction kits in the recovery of SARS-CoV-2 and the impact that PCR inhibitors have on quantification is essential to minimize data discrepancies caused by sample extraction. Three commercial nucleic acid extraction kits: the RNeasy PowerSoil Total RNA Kit (PS), the RNeasy PowerMicrobiome Kit (PMB), and the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (MM), with minor modifications, were evaluated. Their efficacy in recovering viral ribonucleic acid and removal of PCR inhibitors was assessed using two South Australian wastewater matrices-one from a major metropolitan site and one from a regional centre. Both had SARS-CoV-2 present due to active COVID-19 cases in these communities. Overall, the MM kit had a higher recovery of SARS-CoV-2 from the samples tested, followed by PMB and PS. The PMB kit performance was strongly influenced by the sample matrix when compared to the MM kit. It is recommended to assess the performance of extraction kits using different local wastewater matrices to ensure the accuracy and reliability of monitoring results to avoid false reporting.

Repeated applications of fipronil, propyzamide and flutriafol affect soil microbial functions and community composition: A laboratory-to-field assessment.

Pesticides play an important role in conventional agriculture by controlling pests, weeds, and plant diseases. However, repeated applications of pesticides may have long lasting effects on non-target microorganisms. Most studies have investigated the short-term effects of pesticides on soil microbial communities at the laboratory scale. Here, we assessed the ecotoxicological impact of fipronil (insecticide), propyzamide (herbicide) and flutriafol (fungicide) on (i) soil microbial enzymatic activities, (ii) potential nitrification, (iii) abundance of the fungal and bacterial community and key functional genes (nifH, amoA, chiA, cbhl and phosphatase) and (iii) diversity of bacteria, fungi, ammonia oxidizing bacteria (AOB) and archaea (AOA) after repeated pesticide applications in laboratory and field experiments. Our results showed that repeated applications of propyzamide and flutriafol affected the soil microbial community structure in the field and had significant inhibitory effects on enzymatic activities. The abundances of soil microbiota affected by pesticides recovered to levels similar to the control following a second application, suggesting that they might be able to recover from the pesticide effects. However, the persistent pesticide inhibitory effects on soil enzymatic activities suggests that the ability of the microbial community to cope with the repeated application was not accompanied by functional recovery. Overall, our results suggest that repeated pesticide applications may influence soil health and microbial functionalities and that more information should be collected to inform risk-based policy development.

Research and Innovation Opportunities to Improve Epidemiological Knowledge and Control of Environmentally Driven Zoonoses.

While zoonotic diseases are defined by transmission processes between animals and humans, for many of these diseases the presence of a contaminated environmental source is the cause of transmission. Most zoonoses depend on complex environmentally driven interactions between humans and animals, which occur along an occupational and recreational environmental continuum, including farming and animal marketing systems, environmental management systems, and community leisure environments. Environmentally driven zoonoses (EDZs) are particularly challenging to diagnose and control as their reservoirs are in the natural environment and thus often escape conventional surveillance systems that rely on host monitoring. Changes in the environment as a result of climate change [1], human population density [2], and intensification of agriculture [3] have been linked to increasing transmission events for this group of infections. As such, there is a recognised need to be able to detect the presence of EDZs in the environment as a means to better anticipate transmission events and improve source attribution investigations. Finally, the recognition that a One Health approach is needed to combat these infections is signalling to governments the need to develop policy that optimises trade-offs across human, animal, and environmental health sectors. In this review, we discuss and critically appraise the main challenges relating to the epidemiology, diagnosis, and control of environmental zoonotic disease. Using a set of exemplar diseases, including avian influenza and antimicrobial resistant pathogens, we explore the epidemiological contexts (risk factors) within which these infections not only impact human health but also contribute to animal health and environmental impacts. We then critically appraise the surveillance challenges of monitoring these infections in the environment and examine the policy trade-offs for a more integrated approach to mitigating their impacts.

Maintaining a social license to operate for wastewater-based monitoring: The case of managing infectious disease and the COVID-19 pandemic.

Wastewater monitoring as a public health tool is well-established and the SARS-CoV-2 (COVID-19) pandemic has seen its widespread uptake. Given the significant potential of wastewater monitoring as a public health surveillance and decision support tool, it is important to understand what measures are required to allow the long-term benefits of wastewater monitoring to be fully realized, including how to establish and/or maintain public support. The potential for positive SARS-CoV-2 detections to trigger enforced, community-wide public health interventions (e.g., lockdowns and other impacts on civil liberties) further emphasises the need to better understand the role of public engagement in successful wastewater-based monitoring programs. This paper systematically reviews the processes of building and maintaining the social license to operate wastewater monitoring. We specifically explore the relationship between different stakeholder communities and highlight the information and actions that are required to establish a social license to operate and then prevent its loss. The paper adds to the literature on social license to operate by extending its application to new domains and offers a dynamic model of social license to help guide the agenda for researcher and practitioner communities.

The effect of ultrafiltration process on the fate of antibiotic-related microcontaminants, pathogenic microbes, and toxicity in urban wastewater.

Ultrafiltration (UF) was assessed at chemical, microbiological, genetical and toxicological level and in terms of removing specific antibiotic-related microcontaminants from urban wastewater. The UF capacity to remove various antibiotics (clarithromycin, erythromycin, ampicillin, ofloxacin, sulfamethoxazole, trimethoprim, and tetracycline; [A0] = 100 µg L-1) was optimised with respect to the feed recirculation rate (25-50%) and feed/transmembrane pressure (1.5-3/1.5-2.4 bar, respectively). Here, we tested the UF capacity to reduce the cultivable bacteria (faecal coliforms, total heterotrophs, Enterococci, Pseudomonas aeruginosa), enteric opportunistic pathogens, including antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) load. Moreover, the toxicity towards Daphnia magna and three plant species was investigated. Upon optimisation of UF, the removal of antibiotics ranged from 19% for trimethoprim to 95% for clarithromycin. The concentration of cultivable faecal coliforms in the permeate was significantly reduced compared to the feed (P < 0.001), whereas all the bacterial species decreased by more than 3 logs. A similar pattern of reduction was observed for the ARGs (P < 0.001) and enteric opportunistic pathogens (~3-4 logs reduction). A nearly complete removal of the antibiotics was obtained by UF followed by granular activated carbon adsorption (contact time: 90 min), demonstrating the positive contribution of such combination to the abatement of chemical microcontaminants.

Genomic Analysis of Carbapenem-Resistant Comamonas in Water Matrices: Implications for Public Health and Wastewater Treatments.

Comamonas spp. are Gram-negative bacteria that catabolize a wide range of organic and inorganic substrates. Comamonas spp. are abundant in aquatic and soil environments, including wastewater, and can cause opportunistic infections in humans. Because of their potential in wastewater bioaugmentation and bioremediation strategies, the identification of Comamonas species harboring genes encoding carbapenemases and other clinically important antibiotic resistance genes warrant further investigation. Here, we present an analysis of 39 whole-genome sequences comprising three Comamonas species from aquatic environments in South Australia that were recovered on media supplemented with carbapenems. The analysis includes a detailed description of 33 Comamonas denitrificans isolates, some of which carried chromosomally acquired blaGES-5, blaOXA, and aminoglycoside resistance (aadA) genes located on putative genomic islands (GIs). All blaGES-5- and blaOXA-containing GIs appear to be unique to this Australian collection of C. denitrificans. Notably, most open reading frames (ORFs) within the GIs, including all antimicrobial resistance (AMR) genes, had adjacent attC sites, indicating that these ORFs are mobile gene cassettes. One C. denitrificans isolate carried an IncP-1 plasmid with genes involved in xenobiotic degradation and response to oxidative stress. Our assessment of the sequences highlights the very distant nature of C. denitrificans to the other Comamonas species and its apparent disposition to acquire antimicrobial resistance genes on putative genomic islands. IMPORTANCE Antimicrobial resistance (AMR) poses a global public health threat, and the increase in resistance to "last-resort drugs," such as carbapenems, is alarming. Wastewater has been flagged as a hot spot for AMR evolution. Comamonas spp. are among the most common bacteria in wastewater and play a role in its bioaugmentation. While the ability of Comamonas species to catabolize a wide range of organic and inorganic substrates is well documented, some species are also opportunistic pathogens. However, data regarding AMR in Comamonas spp. are limited. Here, through the genomic analyses of 39 carbapenem-resistant Comamonas isolates, we make several key observations, including the identification of a subset of C. denitrificans isolates that harbored genomic islands encoding carbapenemase blaGES-5 or extended-spectrum ß-lactamase blaOXA alleles. Given the importance of Comamonas species in potential wastewater bioaugmentation and bioremediation strategies, as well as their status as emerging pathogens, the acquisition of critically important antibiotic resistance genes on genomic islands warrants future monitoring.

Assessing the Lability and Environmental Mobility of Organically Bound Copper by Stable Isotope Dilution.

The environmental mobility of Cu and therefore its potential toxicity are closely linked to its attachment to natural organic matter (NOM). Geochemical models assume full lability of metals bound to NOM, especially under strong oxidizing conditions, which often leads to an overestimation of the lability of soil metals. Stable isotope dilution (SID) has been successfully applied to estimate the labile (isotopically exchangeable) pool of soil metals. However, its application to study the lability of NOM-Cu required development of a robust separation and detection approach so that free Cu ions can be discriminated from (the also soluble) NOM-Cu. We developed a SID protocol (with enriched 65Cu) to quantify the labile pool of NOM-Cu using size exclusion chromatography coupled to a UV detector (for the identification of different NOM molecular weights) and ICP-MS (for 65Cu/63Cu ratio measurement). The Cu isotopic-exchange technique was first characterized and verified using standard NOM (SR-NOM) before applying the developed technique to an "organic-rich" podzol soil extract. The developed protocol indicated that, in contrast to the common knowledge, significant proportions of SR-NOM-Cu (25%) and soil organic-Cu (55%) were not labile, i.e., permanently locked into inaccessible organic structures. These findings need to be considered in defining Cu interactions with the reactive pool of NOM using geochemical models and risk evaluation protocols in which complexed Cu has always been implicitly assumed to be fully labile and exchangeable with free Cu ions.

Genomic analysis of Elizabethkingia species from aquatic environments: Evidence for potential clinical transmission.

Elizabethkingia species are ubiquitous in aquatic environments, colonize water systems in healthcare settings and are emerging opportunistic pathogens with reports surfacing in 25 countries across six continents. Elizabethkingia infections are challenging to treat, and case fatality rates are high. Chromosomal bla B , bla GOB and bla CME genes encoding carbapenemases and cephalosporinases are unique to Elizabethkingia spp. and reports of concomitant resistance to aminoglycosides, fluoroquinolones and sulfamethoxazole-trimethoprim are known. Here, we characterized whole-genome sequences of 94 Elizabethkingia isolates carrying multiple wide-spectrum metallo-ß-lactamase (bla B and bla GOB) and extended-spectrum serine­ß-lactamase (bla CME) genes from Australian aquatic environments and performed comparative phylogenomic analyses against national clinical and international strains. qPCR was performed to quantify the levels of Elizabethkingia species in the source environments. Antibiotic MIC testing revealed significant resistance to carbapenems and cephalosporins but susceptibility to fluoroquinolones, tetracyclines and trimethoprim-sulfamethoxazole. Phylogenetics show that three environmental E. anophelis isolates are closely related to E. anophelis from Australian clinical isolates (∼36 SNPs), and a new species, E. umeracha sp. novel, was discovered. Genomic signatures provide insight into potentially shared origins and a capacity to transfer mobile genetic elements with both national and international isolates.

Transmission of COVID-19 and other infectious diseases in public washrooms: A systematic review.

BACKGROUND: The risk of infectious disease transmission in public washrooms causes concern particularly in the context of the COVID-19 pandemic. This systematic review aims to assess the risk of transmission of viral or bacterial infections through inhalation, surface contact, and faecal-oral routes in public washrooms in healthcare and non-healthcare environments. METHODS: We systematically reviewed environmental sampling, laboratory, and epidemiological studies on viral and bacterial infection transmission in washrooms using PubMed and Scopus. The review focused on indoor, publicly accessible washrooms. RESULTS: Thirty-eight studies from 13 countries were identified, including 14 studies carried out in healthcare settings, 10 in laboratories or experimental chambers, and 14 studies in restaurants, workplaces, commercial and academic environments. Thirty-three studies involved surface sampling, 15 air sampling, 8 water sampling, and 5 studies were risk assessments or outbreak investigations. Infectious disease transmission was studied in relation with: (a) toilets with flushing mechanisms; (b) hand drying systems; and (c) water taps, sinks and drains. A wide range of enteric, skin and soil bacteria and enteric and respiratory viruses were identified in public washrooms, potentially posing a risk of infection transmission. Studies on COVID-19 transmission only examined washroom contamination in healthcare settings. CONCLUSION: Open-lid toilet flushing, ineffective handwashing or hand drying, substandard or infrequent surface cleaning, blocked drains, and uncovered rubbish bins can result in widespread bacterial and/or viral contamination in washrooms. However, only a few cases of infectious diseases mostly related to faecal-oral transmission originating from washrooms in restaurants were reported. Although there is a risk of microbial aerosolisation from toilet flushing and the use of hand drying systems, we found no evidence of airborne transmission of enteric or respiratory pathogens, including COVID-19, in public washrooms. Appropriate hand hygiene, surface cleaning and disinfection, and washroom maintenance and ventilation are likely to minimise the risk of infectious disease transmission.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

Pesticide effects on nitrogen cycle related microbial functions and community composition.

The extensive application of pesticides in agriculture raises concerns about their potential negative impact on soil microorganisms, being the key drivers of nutrient cycling. Most studies have investigated the effect of a single pesticide on a nutrient cycling in single soil type. We, for the first time, investigated the effect of 20 commercial pesticides with different mode of actions, applied at their recommended dose and five times their recommended dose, on nitrogen (N) microbial cycling in three different agricultural soils from southern Australian. Functional effects were determined by measuring soil enzymatic activities of ß-1,4-N-acetyliglucosaminidase (NAG) and l-leucine aminopeptidase (LAP), potential nitrification (PN), and the abundance of functional genes involved in N cycling (amoA and nifH). Effects on nitrifiers diversity were determined with amplicon sequencing. Overall, the pesticides effect on N microbial cycling was dose-independent and soil specific. The fungicides flutriafol and azoxystrobin, the herbicide chlorsulfuron and the insecticide fipronil induced a significant reduction in PN and ß-1,4-N-acetylglucosaminidase activity (P < 0.05) (NAG) in the alkaline loam soil with low organic carbon content i.e. a soil with properties which typically favors pesticide bioavailability and therefore potential toxicity. For the nitrifier community, the greatest pesticide effects were on the most dominant Nitrososphaeraceae (ammonia-oxidizing archaea; AOA) whose abundance increased significantly compared to the less dominant AOA and other nitrifiers. The inhibiting effects were more evident in the soil samples treated with fungicides. By testing multiple pesticides in a single study, our findings provide crucial information that can be used for pesticide hazard assessment.

Genomic comparisons of Escherichia coli ST131 from Australia.

Escherichia coli ST131 is a globally dispersed extraintestinal pathogenic E. coli lineage contributing significantly to hospital and community acquired urinary tract and bloodstream infections. Here we describe a detailed phylogenetic analysis of the whole genome sequences of 284 Australian ST131 E. coli isolates from diverse sources, including clinical, food and companion animals, wildlife and the environment. Our phylogeny and the results of single nucleotide polymorphism (SNP) analysis show the typical ST131 clade distribution with clades A, B and C clearly displayed, but no niche associations were observed. Indeed, interspecies relatedness was a feature of this study. Thirty-five isolates (29 of human and six of wild bird origin) from clade A (32 fimH41, 2 fimH89, 1 fimH141) were observed to differ by an average of 76 SNPs. Forty-five isolates from clade C1 from four sources formed a cluster with an average of 46 SNPs. Within this cluster, human sourced isolates differed by approximately 37 SNPs from isolates sourced from canines, approximately 50 SNPs from isolates from wild birds, and approximately 52 SNPs from isolates from wastewater. Many ST131 carried resistance genes to multiple antibiotic classes and while 41 (14 %) contained the complete class one integron-integrase intI1, 128 (45 %) isolates harboured a truncated intI1 (462-1014 bp), highlighting the ongoing evolution of this element. The module intI1-dfrA17-aadA5-qacEΔ1-sul1-ORF-chrA-padR-IS1600-mphR-mrx-mphA, conferring resistance to trimethoprim, aminoglycosides, quaternary ammonium compounds, sulphonamides, chromate and macrolides, was the most common structure. Most (73 %) Australian ST131 isolates carry at least one extended spectrum ß-lactamase gene, typically blaCTX-M-15 and blaCTX-M-27. Notably, dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a unique feature of clade C ST131 isolates, were identified in some clade A isolates. The results of this study indicate that the the ST131 population in Australia carries diverse antimicrobial resistance genes and plasmid replicons and indicate cross-species movement of ST131 strains across diverse reservoirs.

Progressing Antimicrobial Resistance Sensing Technologies across Human, Animal, and Environmental Health Domains.

The spread of antimicrobial resistance (AMR) is a rapidly growing threat to humankind on both regional and global scales. As countries worldwide prepare to embrace a One Health approach to AMR management, which is one that recognizes the interconnectivity between human, animal, and environmental health, increasing attention is being paid to identifying and monitoring key contributing factors and critical control points. Presently, AMR sensing technologies have significantly progressed phenotypic antimicrobial susceptibility testing (AST) and genotypic antimicrobial resistance gene (ARG) detection in human healthcare. For effective AMR management, an evolution of innovative sensing technologies is needed for tackling the unique challenges of interconnected AMR across various and different health domains. This review comprehensively discusses the modern state-of-play for innovative commercial and emerging AMR sensing technologies, including sequencing, microfluidic, and miniaturized point-of-need platforms. With a unique view toward the future of One Health, we also provide our perspectives and outlook on the constantly changing landscape of AMR sensing technologies beyond the human health domain.

Inactivation, removal, and regrowth potential of opportunistic pathogens and antimicrobial resistance genes in recycled water systems.

With two thirds of the global population living in areas affected by water scarcity, wastewater reuse is actively being implemented or explored by many nations. There is a need to better understand the efficacy of recycled water treatment plants (RWTPs) for removal of human opportunistic pathogens and antimicrobial resistant microorganisms. Here, we used a suite of probe-based multiplex and SYBR green real-time PCR assays to monitor enteric opportunistic pathogens (EOPs; Acinetobacter baumannii, Arcobacter butzlieri, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Legionella spp., Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Enteritidis, Streptococcus spp.) and antimicrobial resistance genes (ARGs; qnrS, blaSHV, blaTEM, blaGES, blaKPC, blaIMI, blaSME, blaNDM, blaVIM, blaIMP, blaOXA-48-like, mcr-1 and mcr-3) of key concern from an antimicrobial resistance (AMR), waterborne and foodborne disease perspective. The class 1 integron-integrase gene (intl1) was quantified as a proxy for multi-drug resistance. EOPs, intl1 and ARGs absolute abundance (DNA and RNA) and metabolic activity (RNA) was assessed through three RWTPs with differing treatment trains. Our results indicate that RWTPs produced high quality recycled water for non-potable reuse by removing >95% of EOPs and ARGs, however, subpopulations of EOPs and ARGs survived disinfection and demonstrated potential to become actively growing members of the recycled water and distribution system microbiomes. The persistence of functional intl1 suggests that significant genetic recombination capacity remains in the recycled water, along with the likely presence of multi-drug resistant bacteria. Results provide new insights into the persistence and growth of EOPs, and prevalence and removal of ARGs in recycled water systems. These data will contribute towards the emerging evidence base of AMR risks in recycled water to inform quantitative risk-based policy development regarding water recycling schemes.

Antibiotic Resistance and Sewage-Associated Marker Genes in Untreated Sewage and a River Characterized During Baseflow and Stormflow.

Since sewage is a hotspot for antibiotic resistance genes (ARGs), the identification of ARGs in environmental waters impacted by sewage, and their correlation to fecal indicators, is necessary to implement management strategies. In this study, sewage treatment plant (STP) influent samples were collected and analyzed using quantitative polymerase chain reaction (qPCR) to investigate the abundance and correlations between sewage-associated markers (i.e., Bacteroides HF183, Lachnospiraceae Lachno3, crAssphage) and ARGs indicating resistance to nine antibiotics (belonging to aminoglycosides, beta-lactams, sulfonamides, macrolides, and tetracyclines). All ARGs, except bla VIM, and sewage-associated marker genes were always detected in untreated sewage, and ermF and sul1 were detected in the greatest abundances. intl1 was also highly abundant in untreated sewage samples. Significant correlations were identified between sewage-associated marker genes, ARGs and the intl1 in untreated sewage (τ = 0.488, p = 0.0125). Of the three sewage-associated marker genes, the BIO-ENV procedure identified that HF183 alone best maximized correlations to ARGs and intl1 (τ = 0.590). Additionally, grab samples were collected from peri-urban and urban sites along the Brisbane River system during base and stormflow conditions, and analyzed for Escherichia coli, ARGs, the intl1, and sewage-associated marker genes using quantitative polymerase chain reaction (qPCR). Significant correlations were identified between E. coli, ARGs, and intl1 (τ = 0.0893, p = 0.0032), as well as with sewage-associated marker genes in water samples from the Brisbane River system (τ = 0.3229, p = 0.0001). Of the sewage-associated marker genes and E. coli, the BIO-ENV procedure identified that crAssphage alone maximized correlations with ARGs and intl1 in river samples (τ = 0.4148). Significant differences in E. coli, ARGs, intl1, and sewage-associated marker genes, and by flow condition (i.e., base vs. storm), and site types (peri-urban vs. urban) combined were identified (R = 0.3668, p = 0.0001), where percent dissimilarities between the multi-factorial groups ranged between 20.8 and 11.2%. Results from this study suggest increased levels of certain ARGs and sewage-associated marker genes in stormflow river water samples compared to base flow conditions. E. coli, HF183 and crAssphage may serve as potential indicators of sewage-derived ARGs under stormflow conditions, and this merits further investigation. Data presented in this study will be valuable to water quality managers to understand the links between sewage pollution and ARGs in urban environments.

Plastics in biosolids from 1950 to 2016: A function of global plastic production and consumption.

Plastics are ubiquitous contaminants that leak into the environment from multiple pathways including the use of treated sewage sludge (biosolids). Seven common plastics (polymers) were quantified in the solid fraction of archived biosolids samples from Australia and the United Kingdom from between 1950 and 2016. Six plastics were detected, with increasing concentrations observed over time for each plastic. Biosolids plastic concentrations correlated with plastic production estimates, implying a potential link between plastics production, consumption and leakage into the environment. Prior to the 1990s, the leakage of plastics into biosolids was limited except for polystyrene. Increased leakage was observed from the 1990s onwards; potentially driven by increased consumption of polyethylene, polyethylene terephthalate and polyvinyl chloride. We show that looking back in time along specific plastic pollution pathways may help unravel the potential sources of plastics leakage into the environment and provide quantitative evidence to support the development of source control interventions or regulations.

Making Waves: Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.