One Health Threat of Treated Wastewater Discharge in Urban Ohio Rivers: Implications for Surface Water and Fish Gut Microbiome and Resistome.

Wastewater treatment plants (WWTPs) are thought to be a major disseminating source of antibiotic resistance (AR) to the environment, establishing a crucial connection between human and environmental resistome. The objectives of this study were to determine how wastewater effluents impact microbiome and resistome of freshwater and fish, and identify potential AR-carrying clinically relevant pathogens in these matrices. We analyzed wastewater influent and effluent from four WWTPs in three metropolitan areas of Ohio, USA via shotgun metagenomic sequencing. We also sequenced river water and fish guts from three reaches (upstream, at the WWTP outfall, and downstream). Notably, we observed a decline in microbiome diversity and AR gene abundance from wastewater to the receiving river. We also found significant differences by reach and trophic level (diet) in beta-diversity of the fish gut microbiomes. SourceTracker revealed that 0.443 and 0.248 more of the of the fish gut microbiome was sourced from wastewater effluent in fish from the outfall and downstream locations, respectively, compared to upstream fish. Additionally, AR bacteria of public health concern were annotated in effluent and river water samples, indicating potential concern for human exposure. In summary, our findings show the continued role of wastewater as a significant AR reservoir and underscores the considerable impact of wastewater discharge on aquatic wildlife, which highlights the One Health nature of this issue.

Longitudinal Analysis of Urban Stormwater Microbiome and Resistome from Watersheds with and without Green Infrastructure using Long-Read Sequencing.

Since stormwater conveys a variety of contaminants into water bodies, green infrastructure (GI) is increasingly being adopted as an on-site treatment solution in addition to controlling peak flows. The purpose of this study was to identify differences in microbial water quality of stormwater in watersheds retrofitted with GI vs. those without GI. Considering stormwater is recently recognized as a contributor to the antibiotic resistance (AR) threat, another goal of this study was to characterize changes in the microbiome and collection of AR genes (resistome) of urban stormwater with season, rainfall characteristics, and fecal contamination. MinION long-read sequencing was used to analyze stormwater microbiome and resistome from watersheds with and without GI in Columbus, Ohio, United States, over 18 months. We characterized fecal contamination in stormwater via culturing Escherichia coli and with molecular microbial source tracking (MST) to identify sources of fecal contamination. Overall, season and storm event (rainfall) characteristics had the strongest relationships with changes in the stormwater microbiome and resistome. We found no significant differences in microbial water quality or the microbiome of stormwater in watersheds with and without GI implemented. However, there were differences between the communities of microorganisms hosting antibiotic resistance genes (ARGs) in stormwater from watersheds with and without GI, indicating the potential sensitivity of AR bacteria to treatment. Stormwater was contaminated with high concentrations of human-associated fecal bacterial genes, and the ARG host bacterial community had considerable similarities to human feces/wastewater. We also identified 15 potential pathogens hosting ARGs in these stormwater resistome, including vancomycin-resistant Enterococcus faecium (VRE) and multidrug-resistant Pseudomonas aeruginosa. In summary, urban stormwater is highly contaminated and has a great potential to spread AR and microbial hazards to nearby environments. This study presents the most comprehensive analysis of stormwater microbiome and resistome to date, which is crucial to understanding the potential microbial risk from this matrix. This information can be used to guide future public health policy, stormwater reuse programs, and urban runoff treatment initiatives.

Metabolic Resistance and Not Voltage-Gated Sodium Channel Gene Mutation Is Associated with Pyrethroid Resistance of Aedes albopictus (Skuse, 1894) from Cambodia.

(1) Background: In Cambodia, Aedes albopictus is an important vector of the dengue virus. Vector control using insecticides is a major strategy implemented in managing mosquito-borne diseases. Resistance, however, threatens to undermine the use of insecticides. In this study, we present the levels of insecticide resistance of Ae. albopictus in Cambodia and the mechanisms involved. (2) Methods: Two Ae. albopictus populations were collected from the capital, Phnom Penh city, and from rural Pailin province. Adults were tested with diagnostic doses of malathion (0.8%), deltamethrin (0.03%), permethrin (0.25%), and DDT (4%) using WHO tube assays. Synergist assays using piperonyl butoxide (PBO) were implemented before the pyrethroid assays to detect the potential involvement of metabolic resistance mechanisms. Adult female mosquitoes collected from Phnom Penh and Pailin were tested for voltage-gated sodium channel (VGSC) kdr (knockdown resistance) mutations commonly found in Aedes sp.-resistant populations throughout Asia (S989P, V1016G, and F1534C), as well as for other mutations (V410L, L982W, A1007G, I1011M, T1520I, and D1763Y). (3) Results: The two populations showed resistance against all the insecticides tested (<90% mortality). The use of PBO (an inhibitor of P450s) strongly restored the efficacy of deltamethrin and permethrin against the two resistant populations. Sequences of regions of the vgsc gene showed a lack of kdr mutations known to be associated with pyrethroid resistance. However, four novel non-synonymous mutations (L412P/S, C983S, Q1554STOP, and R1718L) and twenty-nine synonymous mutations were detected. It remains to be determined whether these mutations contribute to pyrethroid resistance. (4) Conclusions: Pyrethroid resistance is occurring in two Ae. albopictus populations originating from urban and rural areas of Cambodia. The resistance is likely due to metabolic resistance specifically involving P450s monooxygenases. The levels of resistance against different insecticide classes are a cause for concern in Cambodia. Alternative tools and insecticides for controlling dengue vectors should be used to minimize disease prevalence in the country.

Household environment and animal fecal contamination are critical modifiers of the gut microbiome and resistome in young children from rural Nicaragua.

BACKGROUND: Early life plays a vital role in the development of the gut microbiome and subsequent health. While many factors that shape the gut microbiome have been described, including delivery mode, breastfeeding, and antibiotic use, the role of household environments is still unclear. Furthermore, the development of the gut antimicrobial resistome and its role in health and disease is not well characterized, particularly in settings with water insecurity and less sanitation infrastructure. RESULTS: This study investigated the gut microbiome and resistome of infants and young children (ages 4 days-6 years) in rural Nicaragua using Oxford Nanopore Technology's MinION long-read sequencing. Differences in gut microbiome diversity and antibiotic resistance gene (ARG) abundance were examined for associations with host factors (age, sex, height for age z-score, weight for height z-score, delivery mode, breastfeeding habits) and household environmental factors (animals inside the home, coliforms in drinking water, enteric pathogens in household floors, fecal microbial source tracking markers in household floors). We identified anticipated associations of higher gut microbiome diversity with participant age and vaginal delivery. However, novel to this study were the significant, positive associations between ruminant and dog fecal contamination of household floors and gut microbiome diversity. We also identified greater abundance of potential pathogens in the gut microbiomes of participants with higher fecal contamination on their household floors. Path analysis revealed that water quality and household floor contamination independently and significantly influenced gut microbiome diversity when controlling for age. These gut microbiome contained diverse resistome, dominated by multidrug, tetracycline, macrolide/lincosamide/streptogramin, and beta-lactam resistance. We found that the abundance of ARGs in the gut decreased with age. The bacterial hosts of ARGs were mainly from the family Enterobacteriaceae, particularly Escherichia coli. CONCLUSIONS: This study identified the role of household environmental contamination in the developing gut microbiome and resistome of young children and infants with a One Health perspective. We found significant relationships between host age, gut microbiome diversity, and the resistome. Understanding the impact of the household environment on the development of the resistome and microbiome in early life is essential to optimize the relationship between environmental exposure and human health. Video Abstract.

Horse Housing on Prince Edward Island, Canada: Attitudes and Experiences Related to Keeping Horses Outdoors and in Groups.

Limited research has assessed the "human dimension" of horse care. The aims of this study were to (1) understand horse owner attitudes toward horse welfare when kept outdoors versus indoors and in groups versus individually, (2) compare horse owner attitudes toward horse welfare with the ways in which they house their horses, and (3) explore horse owner reasons for and challenges with their horses' housing. Seventy-six horse owners in Prince Edward Island, Canada completed a questionnaire. Non-parametric tests and quantitative content analysis were used for data analysis. Consistent with the way horses were kept, most (82-96%) owners agreed that horses' physical health, mental well-being, and natural living were better when kept outdoors and in groups. Fewer (64-68%) participants agreed that the horses' standard of care was better when kept outdoors or in groups. Results show associations between owners whose attitudes suggest indoor and/or individual housing is better for horse welfare and keeping their horses indoors part-time and/or individually. Two overarching themes were developed from owners' responses regarding their reasons and challenges related to the ways in which horses were housed: horse-centered and owner-centered care. The results indicate that horse owners' choices about their horses' housing correspond to beliefs about improved horse welfare.

Fish as sentinels of antimicrobial resistant bacteria, epidemic carbapenemase genes, and antibiotics in surface water.

Surface waters, especially those receiving wastewater flows, can disseminate antimicrobial resistant bacteria (ARB), antimicrobial resistance genes (ARG), and antibiotics. In the Scioto River of central Ohio, United States, we evaluated fishes as potential sentinels of ARB and antimicrobial contamination and investigated the influence of antimicrobial exposure on the fish intestinal resistome. Seventy-seven fish were collected from river reaches receiving inputs from two wastewater treatment plants that serve the greater Columbus Metropolitan Area. Fish were screened for the presence of cephalosporin-resistant (CeRO) and carbapenem-resistant (CRO) organisms, epidemic carbapenemase genes, and antibiotic drugs and metabolites using culture methods, droplet digital PCR, and ultra-high performance liquid chromatography tandem mass spectroscopy (UHPLC-MS/MS). Nearly 21% of fish harbored a CeRO in their resistome, with 19.4% exhibiting bacteria expressing an AmpC genotype encoded by blaCMY, and 7.7% with bacteria expressing an extended-spectrum ß-lactamase phenotype encoded by blaCTX-M. blaKPC and blaNDM were present in 87.7% (57/65) and 80.4% (37/46) of the intestinal samples at an average abundance of 104 copies. Three antibiotics-lincomycin (19.5%), azithromycin (31.2%) and sulfamethoxazole (3.9%)-were found in hepatic samples at average concentrations between 25-31 ng/g. Fish harboring blaCTX-M and those exposed to azithromycin were at greater odds of being downstream of a wastewater treatment plant. Fish that bioconcentrated antibiotics in their liver were not at greater odds of harboring CeRO, CRO, or epidemic carbapenemase gene copies in their resistome. Our findings confirm that fishes can be effective bioindicators of surface waters contaminated with ARB, ARG, and antibiotics. Moreover, our findings highlight the varying importance of different mechanisms that facilitate establishment of ARB in aquatic ecosystems.

Comparison of environmental microbiomes in an antibiotic resistance-polluted urban river highlights periphyton and fish gut communities as reservoirs of concern.

Natural waterways near urban areas are heavily impacted by anthropogenic activities, including their microbial communities. A contaminant of growing public health concern in rivers is antibiotic resistant genes (ARGs), which can spread between neighboring bacteria and increase the potential for transmission of AR bacteria to animals and humans. To identify the matrices of most concern for AR, we compared ARG burdens and microbial community structures between sample types from the Scioto River Watershed, Ohio, the United States, from 2017 to 2018. Five environmental matrices (water, sediment, periphyton, detritus, and fish gut) were collected from 26 river sites. Due to our focus on clinically relevant ARGs, three carbapenem resistance genes (blaKPC, blaNDM, and blaOXA-48) were quantified via DropletDigital™ PCR. At a subset of nine urbanized sites, we conducted16S rRNA gene sequencing and functional gene predictions. Carbapenem resistance genes were quantified from all matrices, with blaKPC being the most detected (88 % of samples), followed by blaNDM (64 %) and blaOXA-48 (23 %). Fish gut samples showed higher concentrations of blaKPC and blaNDM than any other matrix, indicating potential ARG bioaccumulation, and risk of broader dissemination through aquatic and nearshore food webs. Periphyton had higher concentrations of blaNDM than water, sediment, or detritus. Microbial community analysis identified differences by sample type in community diversity and structure. Sediment samples had the most diverse microbial communities, and detritus, the least. Spearman correlations did not reveal significant relationships between the concentrations of the monitored ARGs and microbial community diversity. However, several differentially abundant taxa and microbial functions were identified by sample type that is definitive of these matrices' roles in the river ecosystem and habitat type. In summary, the fish gut and periphyton are a concern as AR reservoirs due to their relatively high concentration of carbapenem resistance genes, diverse microbial communities, and natural functions that promote AR.

Acute cyanotoxin poisoning reveals a marginal effect on mouse gut microbiome composition but indicates metabolic shifts related to liver and gut inflammation.

Freshwater harmful algal blooms (HABs) are a major environmental health problem worldwide. HABs are caused by a predominance of cyanobacteria, some of which produce potent toxins. The most ubiquitous cyanotoxin is microcystin (MC) and the congener MC-LR is the most studied due to its toxicity. Short-term exposure to toxins can cause gut microbiome disturbances, but this has not been well described with MC-LR exposure. This study investigated the gut microbial communities of mice from a prior study, which identified significant liver toxicity from ingestion of MC-LR daily for 8 days. CD-1 mice were divided into three dosage groups: control, low exposure (sub-lethal MC-LR concentration), and high exposure (near-lethal MC-LR concentration). Fecal samples were analyzed using 16S rRNA sequencing. Results revealed that at population level, there were no significant shifts in bacterial diversity or the microbial community structure over the exposure period. However, there were significant differences between male and female mice. Predictive functional gene analysis indicated that several metabolic pathways were significantly different in the high dose group before exposure and following 7 doses of MC-LR, as well as between the control and high dose groups on Day 8. Significant differentially abundant taxa were also identified contributing to these pathways. Several pathways, including superpathway of N-acetylneuraminate degradation, were related to liver and gut inflammation. The outcome of this study suggests a need for in-depth investigation of metabolic activity and other functions in the gut in future studies, as well as potential consideration of the role of sex in MC-LR toxicity.

The threat of carbapenem-resistant bacteria in the environment: Evidence of widespread contamination of reservoirs at a global scale.

Environmental reservoirs of antibiotic resistance (AR) are a growing concern that are gathering more attention as potential sources for human infection. Carbapenem-resistant Enterobacteriaceae (CRE) are extremely dangerous, as carbapenems are often drugs of last resort that are used to treat multi-drug resistant infections. Among the genes capable of conferring carbapenem resistance to bacteria, the most transferrable are those that produce carbapenemase, an enzyme that hydrolyzes carbapenems and other ß-lactam antibiotics. The goal of this review was to comprehensively identify global environmental reservoirs of carbapenemase-producing genes, as well as identify potential routes of transmission to humans. The genes of interest were Klebsiella pneumoniae carbapenemase (KPC), New Delhi Metallo-ß-lactamase (NDM), Oxacillinase-48-type carbapenemases (OXA-48), and Verona Integron-Mediated Metallo-ß-lactamase (VIM). Carbapenemase genes have been reported in the environment on almost every continent. Hospital and municipal wastewater, drinking water, natural waterways, sediments, recreational waters, companion animals, wildlife, agricultural environments, food animals, and retail food products were identified as current reservoirs of carbapenemase-producing bacteria and genes. Humans have been recorded as carrying CRE, without recent admittance to a hospital or long-term care facility in France, Egypt, and China. CRE infections from the environment have been reported in patients in Montpellier, France and Cairo, Egypt. This review demonstrates the need for 1) comprehensive monitoring of AR not only in waterways, but also other types of environmental matrices, such as aerosol, dusts, periphyton, and surfaces in indoor environments; and 2) action to reduce the prevalence and mitigate the effects of these potentially deadly resistance genes. In order to develop an accurate quantitative model for environmental dimensions of AR, longitudinal sampling and quantification of AR genes and bacteria are needed, using a One Health approach.