Food security in many parts of the world is threatened by reduced water availability, resulting from population growth and climate change. Under these circumstances, irrigating crops with reclaimed water is becoming an increasingly important agricultural practice. A framework is proposed to address these concerns, evaluate how effectively current regulations ensure safe and sustainable water reuse in the context of antimicrobial resistance (AMR), and identify opportunities for more targeted guidance. The paper proposes indicators for water quality assessment that take into account risks specific to AMR and define primary contamination entry points, potential amplification points, and barriers (e.g., contaminant dissipation in soil, delay between irrigation and harvest, postharvest management), along the wastewater-irrigation, water-crop, and production-consumer continuum. In terms of risk management, an overview of the range of water and wastewater treatment options is provided, from secondary sewage treatments to advanced treatments, including weighing the costs and benefits of interventions in the context of local needs and constraints and the availability and quality of alternative irrigation water sources. The recommendations herein were developed through a consensus at an international multidisciplinary expert workshop and literature review.

Microbial source tracking (MST) using Bacteroidales markers provides information on faecal contamination in environmental waters. The marker BacUni targets a range of sources, while HF183 is primarily associated with human faeces. Duplex quantitative PCR (qPCR) assays allow simultaneous detection of both markers, though competition between primer-probe sets can affect accuracy. Digital PCR (dPCR) offers absolute quantification and higher sensitivity, but multiplexing can be challenging when marker concentrations differ. This study compared HF183 and BacUni quantification across singleplex and duplex qPCR formats and duplex dPCR. Synthetic standards revealed variability due to gBlock design and probe orientation, whereas environmental samples showed minimal format-dependent effects. Duplex qPCR provided comparable results to singleplex, with minor underestimation relative to dPCR. These small differences likely reflect the use of dPCR-quantified standards for qPCR calibration, rather than using DNA concentration-based calibration methods. Low-copy HF183 samples highlighted dPCR's superior sensitivity near detection limits. GoTaq qPCR was the most economical option, especially in duplex format, whereas dPCR offered competitive costs for duplexed samples with the advantage of absolute quantification. These findings demonstrate that duplex qPCR assays reliably quantify HF183 and BacUni in environmental waters, with dPCR serving as a robust complementary method for low-abundance or confirmatory analyses.

ColV/ColBM and ColIa/senB F virulence plasmids feature prominently in Escherichia coli associated with urinary tract and bloodstream infections globally. Australian-sourced E. coli that carry these plasmids were examined among 5,471 isolates (3,316 sequenced by the APG and AusGEM programmes; 2,155 from public databases) spanning years 1986-2020 from humans (n=2,996/5,471; 54.8%), wild animals (n=870/5,471; 15.9%), livestock (n=649/5,471; 11.9%), companion animals (n=375/5,471; 6.9%), environmental sources (n=292/5,471; 5.3%) and food (n=289/5,471; 5.3%). Putative plasmid reconstruction, assisted by a plasmid database comprising 23,700 complete plasmid sequences, identified 22,534 putative plasmids of which 21,814 (96.8%) represented 547 known plasmid clusters. E. coli harbouring ColV-associated putative plasmids, particularly plasmid cluster AA176 [F replicon sequence type (RST): F18:A-:B1 (repFII-18:repFIA-null:repFIB-1)] was identified among phylogenetically diverse strains from humans, livestock, particularly poultry, and food. Closely related isolates, defined as ≤10 core-genome multilocus sequence type allelic distance, that carried either ColV or ColIa/senB-associated putative (F) plasmids were identified across multiple sources and diverse phylogenetic backgrounds. ColIa/senB-associated putative (F) plasmid clusters AA337 (RST: F29:A-:B10) and AA171 (RST: F2:A1:B20) were associated with phylogenetically closely related isolates from humans, wild animals and companion animals, but their absence in E. coli sourced from food and livestock was notable. E. coli carrying ColV and ColIa/senB plasmids frequently exhibit genotypic multidrug resistance, many with critically important antimicrobial resistance genes, highlighting their role in the evolution of clinically problematic lineages. Our study has important epidemiological considerations for understanding the spread of extraintestinal pathogenic and hybrid E. coli lineages across the One Health spectrum.

The World Health Organization identified Stenotrophomonas maltophilia as an underestimated, multi-drug-resistant and opportunistic nosocomial pathogen. It poses a threat to patients who are immunocompromised, suffering chronic disease, requiring indwelling catheters and undergoing mechanical ventilation, as well as victims of trauma and burns. Here, whole-genome sequences of 266 Stenotrophomonas spp. from diverse environmental sources in Fiji and Australia were generated and used to improve understanding of the phylogeny of the genus. Isolates were sourced from freshwater, soil, waste biosolids, wastewater and animal agriculture using selective differential growth media. Phylogenomic analysis identified eight species, plus a singular novel Australian isolate most closely related to Stenotrophomonas indicatrix and Stenotrophomonas lactitubi. The Fijian isolates were primarily S. maltophilia (Sm), with isolates belonging to 13 of the previously recognized 19 Sm subgroups, while most Australian isolates were notably non-maltophilia. A genotypic characterization of the collections was performed, with a focus on well-characterized antimicrobial resistance and virulence genes identified in S. maltophilia, highlighting the presence of these key genes within other Stenotrophomonas species, particularly S. indicatrix, S. lactitubi and Stenotrophomonas rhizophila. Allelic analysis of chromosomal β-lactamase bla L2 revealed its presence across the entire Stenotrophomonas genus, while carbapenemase gene bla L1 was restricted to S. maltophilia and its closest relatives. These data represent a significant contribution of non-maltophilia genome sequences to the public domain, and the first dataset representing Stenotrophomonas from Fiji.

The first therapeutic use of antimicrobial agents initiated their endless arms race with antimicrobial resistance (AMR). Although the genes encoding antimicrobial resistance are ancient and ubiquitous in various environmental compartments, including aquatic environments, over eight decades of exposure to selective pressure has changed the way antimicrobial resistance genes (ARGs) emerge and transmit among the three One Health sectors (that is, the intersected sectors of humans, animals and the environment). The dissemination of ARGs has been facilitated by the widespread use of antimicrobials, along with direct and secondary pollution pathways. Current global consensus dictates that AMR should be addressed under a One Health framework. AMR National Action Plans have frequently been formulated. However, the capacity for implementation is not ready in most countries, especially in low- and middle-income regions. This is in part due to the substantial challenges in documenting and controlling cross-sector AMR connectivity. Here we describe the past and current status of AMR, emphasizing the contribution of connectivity to global AMR burden. We discuss connectivity at ecological, microbial and genetic levels; propose an approach based on genomics and metagenomics to assess connectivity; and finally advocate for cross-sector studies to better understand AMR connectivity and mitigate dissemination. We believe that such harmonized connectivity studies will facilitate coordinated actions and investments across sectors and regions to scale up AMR management globally. Antimicrobial resistance has evolved over decades due to widespread antimicrobial use, with resistance genes now circulating across humans, animals and the environment, creating complex cross-sector connectivity challenges. This Perspective advocates for genomics-based studies of AMR connectivity to enable coordinated global action and investment under the One Health framework.

Legionella spp. are Gram-negative bacteria present in natural and engineered water systems that can cause legionellosis (Legionnaires' disease and Pontiac fever). When present in biofilms of healthcare facilities, they are a likely source of legionellosis for immunocompromised patients. The objective of this study was to evaluate an electrochemical water disinfection system to produce and dose hypochlorous acid (HOCl) to reduce this risk in a hospital with systemic Legionella spp. contamination. Furthermore, Legionella spp. colony counts were compared to quantitative polymerase chain reaction (qPCR) results. Before and after implementing the disinfection system, tap water and pipe biofilms were analysed for microbial contamination. Post-implementation monitoring continued for over six months, assessing microbial quality using heterotrophic colony counts, Legionella serotyping, and qPCR targeting total bacteria (16S rRNA) and Legionella spp. By the third sampling event (22 days post-commissioning), water quality consistently improved, with no culture-positive Legionella counts observed thereafter. qPCR analysis confirmed these results, proving more sensitive and reliable than traditional methods. The qPCR assays for Legionella spp. and bacterial 16S rRNA were also cost-effective for system optimisation and diagnostics.

AbstractGenomics is a cornerstone of modern pathogen epidemiology yet demonstrating transmission in a One Health context is challenging, as strains circulate and evolve within and between diverse hosts and environments. To identify phylogenetic linkages and better define relevant measures of genomic relatedness in a One Health context, we collated 5471 Escherichia coli genome sequences from Australia originating from humans (n = 2996), wild animals (n = 870), livestock (n = 649), companion animals (n = 375), environmental sources (n = 292) and food (n = 289) spanning over 36 years. Of the 827 multi-locus sequence types (STs) identified, 10 STs were commonly associated with cross-source genomic clusters, including the highly clonal ST131, pandemic zoonotic lineages such as ST95, and emerging human ExPEC ST1193. Here, we show that assessing genomic relationships at ≤ 100 SNP threshold enabled detection of cross-source linkage otherwise obscured when applying typical outbreak-oriented relatedness thresholds ( ≤ 20 SNPs) and should be considered in interrogation of One Health genomic datasets.

Safe, clean water is vital to maintain healthy societies and socioeconomic development, yet providing it remains an evolving challenge for water managers. Among the numerous contaminants that need to be managed, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are recognized globally as a growing priority, and the development of new and improved disinfection methods to remove them and their drivers (e.g., antibiotics) from treated wastewater effluents for recycling or discharge to water bodies is a matter of increasing interest. This study investigated the disinfection efficacy of hydrogen peroxide (H2O2), chlorination (Cl-2), ultraviolet light at 254 nm (UV-254), UV-254/H2O2, ultraviolet light at 369 nm (UV-369) and UV-369/H2O2 treatments against several bacterial strains (i.e., Escherichia coli, Salmonella enteritidis 11RX, Listeria innocua, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus). Disinfection efficacy was assessed in multiple sample matrices, including ultrapure water, wastewater influent, and effluent waters. Irrespective of the bacterial strain, the removal efficacy followed the order: UV-254/H2O2 > UV-254 > Cl-2 > UV-369/H2O2 > UV-369 > H2O2. 4-log reduction (99.99 % removal) of E. coli was achieved at 0.5, 1, 2, 20 and 30 min by UV-254/H2O2, UV-254, Cl-2, UV-369/H2O2 and UV-369 treatments, respectively, whereas H2O2 alone only achieved 97.6 % removal of E. coli after 30 min of treatment. UV-254 and UV-254/H2O2 were also able to remove antibiotic resistance genes with an observed rate constant ranging from 0.3445 to 0.6122 min(-1). The findings of this study suggest that coupling H2O2 with solar light is a promising alternative approach for reducing the quantity of opportunistic human pathogens in reclaimed water.

Effective management of water resources is crucial for global food security and sustainable development. In this Review, we explore the potential benefits and challenges associated with treated wastewater (TW) reuse for irrigation. Currently, 400 km(3) yr(-1) of wastewater is generated globally, but <20% is treated, and of that TW, only 2-15% is reused for irrigation depending on region. The main limitation of TW for irrigation is the inability of current treatment technologies to completely remove all micropollutants and contaminants of emerging concern, some of which have unknown impacts on crops, environment and health. However, advanced water treatment and reuse schemes, supported by water quality monitoring and regulations, can provide a stable water supply for agricultural production, as demonstrated in regions such as the USA and Israel. Such schemes could potentially serve a net energy source, as the embedded energy in wastewater exceeds treatment needs by 9 to 10 times. Agriculturally useful nutrients such as nitrogen, phosphorus and potassium could be also recovered and reused. TW reuse for irrigation could act as a major contributor to a circular economy and sustainable development, but the first steps will be funding and implementation of advanced and sustainable treatment technologies and social acceptance.

ABSTRACT 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 p dif 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 p dif 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. IMPORTANCE Antimicrobial 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 p dif 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.

Abstract Efforts have been made to strengthen national and global antimicrobial resistance (AMR) surveillance programs by integrating data collection across the human, animal, and environmental sectors. We searched the literature to review published studies reporting the implementation of integrated AMR surveillance approaches, and we identified a total of 96 articles from 36 countries published from 2000 to 2022, which met our inclusion criteria. Standard review protocols were applied in our study. Out of 96 studies, 47 (49%) articles integrated AMR analysis from human and animal (HA) populations, 24 (25%) considered human, animal, and environmental (HAE) samples together, 11 (12%) studies included animal and environment (AE) samples, and 12 (13%) integrated human and environmental (HE) surveillance. Human isolate-based and animal isolate-based surveillance were the most common study designs (38, 52.8%), with the remainder applying human case-based and isolate-based for animal populations (32, 44.4%). Finally, our results demonstrate that AMR studies on E. coli revealed a lower correlation between human and animal AMR prevalence compared to Salmonella spp. and Campylobacter spp. studies. Different aspects of surveillance design were associated with the level of correlation of AMR prevalence between sectors. Our study found that while global efforts for integrated AMR surveillance have increased in the past 10 years, significant variation exists between studies with regard to the epidemiological and laboratory aspects of their surveillance designs. Our findings indicate that to enable the generation of comparable epidemiological data across countries and sectors, there is a need for the development of a global protocol to support the design of surveillance programs that aim to conduct integrated surveillance of AMR.

Antimicrobial resistance (AMR) is a major public health concern, and environmental bacteria have been recognized as important reservoirs of antimicrobial resistance genes (ARGs). Citrobacter, a common environmental bacterium and opportunistic pathogen in humans and other animals, has been largely understudied interms of its diversity and AMR potential. Whole-genome (short-read) sequencing on a total of 77 Citrobacter isolates obtained from Australian silver gull (Chroicocephalus novaehollandiae) (n = 17) and influent wastewater samples (n = 60) was performed, revealing a diverse Citrobacter population, with seven different species and 33 sequence types, 17 of which were novel. From silver gull using non-selective media we isolated a broader range of species with little to no mobilised ARG carriage. Wastewater isolates (selected using Carbapenem-Resistant Enterobacterales (CRE) selective media) carried a heavy burden of ARGs (up to 21 ARGs, conferring resistance to nine classes of antibiotics), with several novel multidrug-resistant (MDR) lineages identified, including C. braakii ST1110, which carried ARGs conferring resistance to eight to nine classes of antibiotics, and C. freundii ST1105, which carried two carbapenemase genes, blaIMP-4 in class 1 integron structure, and blaKPC-2. Additionally, we identified an MDR C. portucalensis isolate carrying blaNDM-1, blaSHV-12, and mcr-9. We identified IncC, IncM2, and IncP6 plasmids as the likely vectors for many of the critically important mobilised ARGs. Phylogenetic analyses were performed to assess any epidemiological linkages between isolation sources, demonstrating low relatedness across sources beyond the ST level. However, these analyses did reveal some closer relationships between strains from disparate wastewater sources despite their collection some 13,000 km apart. These findings support the need for future surveillance of Citrobacter populations in wastewater and wildlife populations to monitor for potential opportunistic human pathogens.

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. Antimicrobial resistance (AMR) is an important public health issue that affects human, animal and environmental sectors worldwide. The authors review the role of genomics in AMR surveillance using a One Health approach, and how genomic approaches can help mitigate the spread of AMR to improve global health.

Antimicrobial resistance (AMR) is a complex challenge that poses a critical threat to food and water safety and security as well as to human, animal and environmental health. It is projected to cost the global economy US$100 trillion by 2050. Australia’s new Cooperative Research Centre (CRC) for Solving Antimicrobial Resistance in Agribusiness, Food and Environments (SAAFE) is part of Australia’s One Health approach to mitigating AMR. SAAFE’s 10-year, A$150-million industry-led program will help protect Australia’s food and agribusiness industries, and the environments in which they operate, from the growing threat of AMR. Through its research programs, CRC SAAFE uses a partner-based approach to assist industries to monitor, analyse and mitigate AMR, with projects spanning horticulture, viticulture, aquaculture, animal industries, water and waste.

Flocs produced during drinking water treatment play a key role in particle separation and removal. Floc characteristics affect both sedimentation and filtration efficiency, however, the methods used to optimise the coagulant dose during treatment do not usually take these into account. This study investigated the feasibility of using the Flocculation Index (FI), obtained from a photometric dispersion analyser (PDA) during flocculation, as a control parameter to optimise turbidity removal. Five waters, natural and synthetic, with different turbidity levels (from 6 to -120 NTU) and low DOC (i.e., <5 mg/L) were used to study the effects of various alum doses on floc characteristics [i.e., initial floc aggregation (IFA), plateau (P) and variance (VAR)]. No correlation was found between the IFA and VAR values and the alum dose achieving the greatest turbidity removal and relative settling factor (RSF). However, P followed a similar pattern to the RSF, and was negatively correlated with the residual turbidity. An optimisation model was developed using response surface methodology and the effects of two independent variables (i.e., raw turbidity and alum dose) on P values were investigated. The model achieved a high correlation (R2 of -98 %, p-value <0.05) and was validated using waters with different turbidity levels. It predicted the alum dose within 8 % error. The results indicate that the FI can be used as an alternative parameter for optimising the coagulation process.

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.

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.

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.