Metagenomic insights into the impact of litter from poultry Concentrated Animal Feeding Operations (CAFOs) to adjacent soil and water microbial communities.

In recent decades, human food consumption has led to an increased demand for animal-based foods, particularly chicken meat production. The state of Georgia, USA is one of the top broiler chicken producers in the United States, where animals are raised in Concentrated Animal Feeding Operations (CAFOs). Without proper management, CAFOs could negatively impact the environment and become a public health risk as a source of water and air pollution and/or by spreading antimicrobial resistance genes. In this study, we used metagenome sequencing to investigate the impact of the application of the CAFO's litter on adjacent soils and downstream creek waters in terms of microbial diversity and antimicrobial resistance profile changes. Our data indicate that while a few microbial groups increased in abundance within a short period of time after litter application, these populations subsequently decreased to levels similar to those found prior to the litter application or to below the detection limit of our metagenome sequencing effort. Microbial taxonomic composition analyses, relative abundance of Metagenome-Assembled Genomes (MAGs) and detection of Antimicrobial Resistance Genes (ARGs) allow us to conclude that this practice of litter application had a negligible effect on the microbiome or resistome profile of these soils and nearby waterways, likely due to its dilution in the field and/or outcompetition by indigenous microbes, revealing a minimal impact of these poultry facilities on the natural microbial communities.

Environmental Persistence and Disinfection of Lassa Virus.

Lassa fever, caused by Lassa virus (LASV), is endemic to West Africa, where ≈300,000 illnesses and ≈5,000 deaths occur annually. LASV is primarily spread by infected multimammate rats via urine and fomites, highlighting the need to understand the environmental fate of LASV. We evaluated persistence of LASV Josiah and Sauerwald strains on surfaces, in aqueous solutions, and with sodium hypochlorite disinfection. Tested strains were more stable in deionized water (first-order rate constant [k] for Josiah, 0.23 days; for Sauerwald, k = 0.34 days) than primary influent wastewater (Josiah, k = 1.3 days; Sauerwald, k = 1.9 days). Both strains had similar decay rates on high-density polyethylene (Josiah, k = 4.3 days; Sauerwald, k = 2.3 days) and stainless steel (Josiah, k = 5.3 days; Sauerwald, k = 2.7 days). Sodium hypochlorite was highly effective at inactivating both strains. Our findings can inform future risk assessment and management efforts for Lassa fever.

Persistence of Antibiotic Resistance Genes Varies with Particle Size and Substrate Conditions in Recirculating Streams.

Antibiotic resistance (AR) determinants are enriched in animal manures, a significant portion of which is land-applied as a soil amendment or as fertilizer, leading to potential AR runoff and microbial pollution in adjacent surface waters. To effectively inform AR monitoring and mitigation efforts, a thorough understanding and description of the persistence and transport of manure-derived AR in flowing waters are needed. We used experimental recirculating mesocosms to assess water-column removal rates of antibiotic resistance genes (ARGs) originating from a cow manure slurry collected from a dairy farm. We quantified the effect of three benthic (i.e., bottom) substrate variations and particle sizes of manure slurry on water column removal rates. Overall, we observed variation in ARG behavior across substrate treatments and particle sizes. For ARGs associated with small particles, removal rates were higher in mesocosms with a substrate. tetW was typically removed at the highest rates across particle size and treatment, followed by ermB and blaTEM. Our data suggests that both substrate character and particle size exert control on the fate and transport of ARGs in surface waters, laying the foundation for future research in this area to establish a predictive framework for AR persistence and fate in flowing waters.

A systematic review of enteric pathogens and antibiotic resistance genes in outdoor urban aerosols.

Aerosol transport of enteric microbiota including fecal pathogens and antimicrobial resistance genes (ARGs) has been documented in a range of settings but remains poorly understood outside indoor environments. We conducted a systematic review of the peer-reviewed literature to summarize evidence on specific enteric microbiota including enteric pathogens and ARGs that have been measured in aerosol samples in urban settings where the risks of outdoor exposure and antibiotic resistance (AR) spread may be highest. Following PRISMA guidelines, we conducted a key word search for articles published within the years 1990-2020 using relevant data sources. Two authors independently conducted the keyword searches of databases and conducted primary and secondary screenings before merging results. To be included, studies contained extractable data on enteric microbes and AR in outdoor aerosols regardless of source confirmation and reported on qualitative, quantitative, or viability data on enteric microbes or AR. Qualitative analyses and metric summaries revealed that enteric microbes and AR have been consistently reported in outdoor aerosols, generally via relative abundance measures, though gaps remain preventing full understanding of the role of the aeromicrobiological pathway in the fate and transport of enteric associated outdoor aerosols. We identified remaining gaps in the evidence base including a need for broad characterization of enteric pathogens in bioaerosols beyond bacterial genera, a need for greater sampling in locations of high enteric disease risk, and a need for quantitative estimation of microbial and nucleic acid densities that may be applied to fate and transport models and in quantitative microbial risk assessment.

Quantitative microbial risk assessment of outdoor aerosolized pathogens in cities with poor sanitation.

The aeromicrobiological transmission pathway of enteric pathogens in places with unsafe sanitation services is poorly understood. In an attempt to partly fill this knowledge gap, we assessed the potential public health impact of bioaerosols near open waste canals (OWCs) using Quantitative Microbial Risk Assessment (QMRA). We used data acquired in La Paz, Bolivia to characterize the risk of disease that aerosolized enteric pathogens may pose through food, fomites and inhalation (all followed by ingestion). Three reference pathogens were selected to conduct the assessment: enterotoxigenic Escherichia coli (ETEC), Shigella flexneri, and Campylobacter jejuni. Inhalation followed by ingestion had the highest median infection risk per event i.e. 3 × 10-5 (3 infections for every 100,000 exposures), compared to contaminated food e.g. 5 × 10-6 and fomites e.g. 2 × 10-7, all for C. jejuni infections. Our sensitivity analysis showed that bacterial fluxes from the air were the most influential factor on risk. Our results suggest that fecal bacterial aerosols from OWCs present non-negligible risks of infection in La Paz, with median annual infection risks by C. jejuni being 18 (food), and 100 (inhalation) times greater than the EPA's standard for drinking water (1 × 10-4). We included two of the QMRA models presented here in a novel web application we developed for user-specified application in different contexts.

Detection and Quantification of Enteric Pathogens in Aerosols Near Open Wastewater Canals in Cities with Poor Sanitation.

Urban sanitation infrastructure is inadequate in many low-income countries, leading to the presence of highly concentrated, uncontained fecal waste streams in densely populated areas. Combined with mechanisms of aerosolization, airborne transport of enteric microbes and their genetic material is possible in such settings but remains poorly characterized. We detected and quantified enteric pathogen-associated gene targets in aerosol samples near open wastewater canals (OWCs) or impacted (receiving sewage or wastewater) surface waters and control sites in La Paz, Bolivia; Kanpur, India; and Atlanta, USA, via multiplex reverse-transcription qPCR (37 targets) and ddPCR (13 targets). We detected a wide range of enteric targets, some not previously reported in extramural urban aerosols, with more frequent detections of all enteric targets at higher densities in La Paz and Kanpur near OWCs. We report density estimates ranging up to 4.7 × 102 gc per mair3 across all targets including heat-stable enterotoxigenic Escherichia coli, Campylobacter jejuni, enteroinvasive E. coli/Shigella spp., Salmonella spp., norovirus, and Cryptosporidium spp. Estimated 25, 76, and 0% of samples containing positive pathogen detects were accompanied by culturable E. coli in La Paz, Kanpur, and Atlanta, respectively, suggesting potential for viability of enteric microbes at the point of sampling. Airborne transmission of enteric pathogens merits further investigation in cities with poor sanitation.

Open Waste Canals as Potential Sources of Antimicrobial Resistance Genes in Aerosols in Urban Kanpur, India.

Understanding the movement of antimicrobial resistance genes (ARGs) in the environment is critical to managing their spread. To assess potential ARG transport through the air via urban bioaerosols in cities with poor sanitation, we quantified ARGs and a mobile integron (MI) in ambient air over periods spanning rainy and dry seasons in Kanpur, India (n = 53), where open wastewater canals (OWCs) are prevalent. Gene targets represented major antibiotic groups-tetracyclines (tetA), fluoroquinolines (qnrB), and beta-lactams (blaTEM)-and a class 1 mobile integron (intI1). Over half of air samples located near, and up to 1 km from OWCs with fecal contamination (n = 45) in Kanpur had detectable targets above the experimentally determined limits of detection (LOD): most commonly intI1 and tetA (56% and 51% of samples, respectively), followed by blaTEM (8.9%) and qnrB (0%). ARG and MI densities in these positive air samples ranged from 6.9 × 101 to 5.2 × 103 gene copies/m3 air. Most (7/8) control samples collected 1 km away from OWCs were negative for any targets. In comparing experimental samples with control samples, we found that intI1 and tetA densities in air are significantly higher (P = 0.04 and P = 0.01, respectively, alpha = 0.05) near laboratory-confirmed fecal contaminated waters than at the control site. These data suggest increased densities of ARGs and MIs in bioaerosols in urban environments with inadequate sanitation. In such settings, aerosols may play a role in the spread of AR.

Antimicrobial resistance genes are enriched in aerosols near impacted urban surface waters in La Paz, Bolivia.

Antibiotic resistance poses a major global health threat. Understanding emergence and dissemination of antibiotic resistance in environmental media is critical to the design of control strategies. Because antibiotic resistance genes (ARGs) may be aerosolized from contaminated point sources and disseminated more widely in localized environments, we assessed ARGs in aerosols in urban La Paz, Bolivia, where wastewater flows in engineered surface water channels through the densely populated urban core. We quantified key ARGs and a mobile integron (MI) via ddPCR and E. coli spp. as a fecal indicator by culture over two years during both the rainy and dry seasons in sites near wastewater flows. ARG targets represented major antibiotic groups-tetracyclines (tetA), fluoroquinolines (qnrB), and beta-lactams (blaTEM)-and an MI (intI1) represented the potential for mobility of genetic material. Most air samples (82%) had detectable targets above the experimentally determined LOD: most commonly blaTEM and intI1 (68% and 47% respectively) followed by tetA and qnrB (17% and 11% respectively). ARG and MI densities in positive air samples ranged from 1.3 × 101 to 6.6 × 104 gene copies/m3 air. Additionally, we detected culturable E. coli in the air (52% of samples <1 km from impacted surface waters) with an average density of 11 CFU/m3 in positive samples. We observed decreasing density of blaTEM with increasing distance up to 150 m from impacted surface waters. To our knowledge this is the first study conducting absolute quantification and a spatial analysis of ARGs and MIs in ambient urban air of a city with contaminated surface waters. Environments in close proximity to urban wastewater flows in this setting may experience locally elevated concentrations of ARGs, a possible concern for the emergence and dissemination of antimicrobial resistance in cities with poor sanitation.

Detection and assessment of the antibiotic resistance of Enterobacteriaceae recovered from bioaerosols in the Choqueyapu River area, La Paz - Bolivia.

As a highly contaminated waterway flowing through a densely populated urban area, microbiological pollution associated with the Choqueyapu River and the absence of a wastewater treatment plant in La Paz city threatens public health. We collected air samples adjacent to this river using impingement. Laboratory analyses identified the presence of Enterobacteriaceae, reporting a maximum concentration of 86,11 CFU/m3 of sampled air. Positive samples were tested for antibiotic susceptibility against the antibiotics amoxicillin-clavulanic acid, ciprofloxacin, gentamicin, meropenem, sulfamethoxazole-trimethoprim and tetracycline via disk diffusion. The highest percentages of antibiotic resistance were registered for tetracycline (50% of isolates) and sulfamethoxazole-trimethoprim (38,9%), while the lowest resistance profile was reported for meropenem (5,6%). A comparison of results obtained on the pilot studies [elaborated during the wet season of 2018 by Chavez, 2019 and Salazar et al., 2020] and the present study has been done, highlighting seasonal effects over airborne Enterobacteriaceae concentration. Also, it was determined an increase of antibiotic resistance for tetracycline, gentamicin and ciprofloxacin; and a reduction for sulfamethoxazole-trimethoprim, meropenem and amoxicillin-clavulanic acid.

Extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli presence in urban aquatic environments in Kanpur, India.

In India, high rates of antibiotic consumption and poor sanitation infrastructure combine to pose a significant risk to the public through the environmental transmission of antimicrobial resistance (AMR). The WHO has declared extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli a key indicator for the surveillance of AMR worldwide. In the current study, we measured the prevalence of AMR bacteria in an urban aquatic environment in India by detecting metabolically active ESBL-positive E. coli. Water samples were collected in duplicate from 16 representative environmental water sources including open canals, drains, and rivers around Kanpur, Uttar Pradesh. We detected culturable E. coli in environmental water at 11 (69%) of the sites. Out of the 11 sites that were positive for culturable E. coli, ESBL-producing E. coli was observed at 7 (64%). The prevalence of ESBL-producing E. coli detected in the urban aquatic environment suggests a threat of AMR bacteria to this region.

Bioaerosol sampling optimization for community exposure assessment in cities with poor sanitation: A one health cross-sectional study.

Evidence of exposure to enteric pathogens through the air and associated risk of infection is scarce in the literature outside of animal- or human-waste handling settings. Cities with poor sanitation are important locations to investigate this aerial exposure pathway as their rapid growth will pose unprecedented challenges in waste management. To address this issue, simple surveillance methods are needed. Therefore, the objectives of this study were to optimize a community exposure bioaerosol surveillance strategy for urban outdoor locations with poor sanitation, and to determine which bioaerosols could contribute to exposure. Passive and active bioaerosol sampling methods were used to characterize the fate and transport of sanitation-related bioaerosols during the rainy and dry seasons in La Paz, Bolivia. Median coliform bacteria fluxes were 71 CFU/(m2 × h) during the rainy season and 64 CFU/(m2 × h) during the dry season, with 38% of the dry season samples testing positive for E. coli. Wind speed, relative humidity and UVB irradiance were identified as significant covariates to consider in bioaerosol transport models in La Paz. Active sampling yielded one positive sample (10%) for human adenovirus (HadV) and one sample (10%) for influenza A virus during the rainy season. HadV was detected at the site with the highest bacterial flux. Four samples (8%) were positive for influenza A virus in the dry season. These findings suggest that aerosols can contribute to community exposure to potentially pathogenic microorganisms in cities with poor sanitation. The use of passive sampling, despite its limitations, can provide quantitative data on microorganisms' viability within realistic timeframes of personal exposure.