Antimicrobial Resistance In Environment Research Articles

Merging Metagenomics and Spatial Epidemiology To Understand the Distribution of Antimicrobial Resistance Genes from Enterobacteriaceae in Wild Owls.

Antimicrobial resistance (AMR) is a well-documented phenomenon in bacteria from many natural ecosystems, including wild animals. However, the specific determinants and spatial distribution of resistant bacteria and antimicrobial resistance genes (ARGs) in the environment remain incompletely understood. In particular, information regarding the importance of anthropogenic sources of AMR relative to that of other biological and ecological influences is lacking. We conducted a cross-sectional study of AMR in great horned owls (Bubo virginianus) and barred owls (Strix varia) admitted to a rehabilitation center in the midwestern United States. A combination of selective culture enrichment and shotgun metagenomic sequencing was used to identify ARGs from Enterobacteriaceae Overall, the prevalence of AMR was comparable to that in past studies of resistant Enterobacteriaceae in raptors, with acquired ARGs being identified in 23% of samples. Multimodel regression analyses identified seasonality and owl age to be important predictors of the likelihood of the presence of ARGs, with birds sampled during warmer months being more likely to harbor ARGs than those sampled during cooler months and with birds in their hatch year being more likely to harbor β-lactam ARGs than adults. Beyond host-specific determinants, ARG-positive owls were also more likely to be recovered from areas of high agricultural land cover. Spatial clustering analyses identified a significant high-risk cluster of tetracycline resistance gene-positive owls in the southern sampling range, but this could not be explained by any predictor variables. Taken together, these results highlight the complex distribution of AMR in natural environments and suggest that both biological and anthropogenic factors play important roles in determining the emergence and persistence of AMR in wildlife.IMPORTANCE Antimicrobial resistance (AMR) is a multifaceted problem that poses a worldwide threat to human and animal health. Recent reports suggest that wildlife may play an important role in the emergence, dissemination, and persistence of AMR. As such, there have been calls for better integration of wildlife into current research on AMR, including the use of wild animals as biosentinels of AMR contamination in the environment. A One Health approach can be used to gain a better understanding of all AMR sources and pathways, particularly those at the human-animal-environment interface. Our study focuses on this interface in order to assess the effect of human-impacted landscapes on AMR in a wild animal. This work highlights the value of wildlife rehabilitation centers for environmental AMR surveillance and demonstrates how metagenomic sequencing within a spatial epidemiology framework can be used to address questions surrounding AMR complexity in natural ecosystems.

From environment to clinic: the role of pesticides in antimicrobial resistance.

ABSTRACTAntimicrobial resistance (AMR) in pathogens has been associated mainly with excessive use of antibiotics. Most studies of resistance have focused on clinical pathogens; however, microorganisms are exposed to numerous anthropogenic substances. Few studies have sought to determine the effects of chemical substances on microorganisms. Exposure to these substances may contribute to increased rates of AMR. Understanding microorganism communities in natural environments and AMR mechanisms under the effects of anthropogenic substances, such as pesticides, is important to addressing the current crisis of antimicrobial resistance.This report draws attention to molecules, rather than antibiotics, that are commonly used in agrochemicals and may be involved in developing AMR in non-clinical environments, such as soil. This report examines pesticides as mediators for the appearance of AMR, and as a route for antibiotic resistance genes and antimicrobial resistant bacteria to the anthropic environment. Available evidence suggests that the natural environment may be a key dissemination route for antibiotic-resistant genes. Understanding the interrelationship of soil, water, and pesticides is fundamental to raising awareness of the need for environmental monitoring programs and overcoming the current crisis of AMR.

Evaluation of frameworks proposed as protective of antimicrobial resistance propagation in the environment

Antimicrobial resistance (AMR) in the environment is a globally concerning issue. This study sought to improve the understanding of human health risks from an environmental AMR proliferation perspective. Surface water concentrations of 11 most used antibiotics in the United States were simulated for the Columbia and Sacramento River watersheds using the Pharmaceutical Assessment and Transport Evaluation (PhATE) model. The predicted environmental concentrations (PECs) and literature-reported measured environmental concentrations (MECs) of antibiotics were compared to the predicted no effect concentrations (PNECs) of three frameworks proposed as protective of AMR selection. For all of the studied antibiotics, PECs (except for moxifloxacin, a 4th generation fluoroquinolone), and at least one published MEC, were above the safe limit proposed by at least one of the three frameworks. The results indicate that a variety of different antibiotics with different mechanisms of action and physico-chemical properties are likely in environmental compartments at or above the concentrations currently proposed as safe from an AMR proliferation perspective. Understanding environmental occurrence of antibiotics is important for assessing environmental exposures and, when compared to PNECs for resistance selection, can—either alone or in combination with other methods— more specifically indicate where there are potential risks of AMR proliferation.

Seasonal variations in export of antibiotic resistance genes and bacteria in runoff from an agricultural watershed in Iowa

Seasonal variations of antimicrobial resistance (AMR) indicators in runoff water can help improve our understanding of AMR sources and transport within an agricultural watershed. This study aimed to monitor multiple areas throughout the Black Hawk Lake (BHL) watershed (5324 ha) in central Iowa during 2017 and 2018 that consists of both swine and cattle feeding operations as well as known areas with manure application. The measured indicators included plate counts for fecal indicator bacteria (FIB) E. coli, Enterococcus, antibiotic resistant fecal indicator bacteria (ARBs) tylosin resistant Enterococcus, tetracycline resistant Enterococcus, and antibiotic resistance genes (ARGs): ermB, ermF (macrolide), tetA, tetM, tetO, tetW (tetracycline), sul1, sul2 (sulfonamide), aadA2 (aminoglycoside), vgaA, and vgaB (pleuromutilin). Both the plate count and the ARG analyses showed seasonal trends. Plate counts were significantly greater during the growing season, while the ARGs were greater in the pre-planting and post-harvest seasons (Wilcoxon Rank-Sum Test p < 0.05). The ermB gene concentration was significantly correlated (p < 0.05) with E. coli and Enterococcus concentrations in 2017, suggesting a potential use of this ARG as an indicator of environmental AMR and human health risk. Flow rate was not a significant contributor to annual variations in bacteria and AMR indicators. Based on observed seasonal patterns, we concluded that manure application was the likely contributor to elevated ARG indicators observed in the BHL watershed, while the driver of elevated ARB indictors in the growing season can only be speculated. Understanding AMR export patterns in agricultural watersheds provides public health officials knowledge of seasonal periods of higher AMR load to recreational waters.

Urban informal settlements as hotspots of antimicrobial resistance and the need to curb environmental transmission

Antimicrobial resistance (AMR) is a growing public health challenge that is expected to disproportionately burden lower- and middle-income countries (LMICs) in the coming decades. Although the contributions of human and veterinary antibiotic misuse to this crisis are well-recognized, environmental transmission (via water, soil or food contaminated with human and animal faeces) has been given less attention as a global driver of AMR, especially in urban informal settlements in LMICs-commonly known as 'shanty towns' or 'slums'. These settlements may be unique hotspots for environmental AMR transmission given: (1) the high density of humans, livestock and vermin living in close proximity; (2) frequent antibiotic misuse; and (3) insufficient drinking water, drainage and sanitation infrastructure. Here, we highlight the need for strategies to disrupt environmental AMR transmission in urban informal settlements. We propose that water and waste infrastructure improvements tailored to these settings should be evaluated for their effectiveness in limiting environmental AMR dissemination, lowering the community-level burden of antimicrobial-resistant infections and preventing antibiotic misuse. We also suggest that additional research is directed towards developing economic and legal incentives for evaluating and implementing water and waste infrastructure in these settings. Given that almost 90% of urban population growth will occur in regions predicted to be most burdened by the AMR crisis, there is an urgent need to build effective, evidence-based policies that could influence massive investments in the built urban environment in LMICs over the next few decades.

Antimicrobial resistance situation in animal health of Bangladesh.

Antimicrobial resistance (AMR) is a crucial multifactorial and complex global problem and Bangladesh poses a regional and global threat with a high degree of antibiotic resistance. Although the routine application of antimicrobials in the livestock industry has largely contributed to the health and productivity, it correspondingly plays a significant role in the evolution of different pathogenic bacterial strains having multidrug resistance (MDR) properties. Bangladesh is implementing the National Action Plan (NAP) for containing AMR in human, animal, and environment sectors through “One Health” approach where the Department of Livestock Services (DLS) is the mandated body to implement NAP strategies in the animal health sector of the country. This review presents a “snapshot” of the predisposing factors, and current situations of AMR along with the weakness and strength of DLS to contain the problem in animal farming practices in Bangladesh. In the present review, resistance monitoring data and risk assessment identified several direct and/or indirect predisposing factors to be potentially associated with AMR development in the animal health sector of Bangladesh. The predisposing factors are inadequate veterinary healthcare, monitoring and regulatory services, intervention of excessive informal animal health service providers, and farmers’ knowledge gap on drugs, and AMR which have resulted in the misuse and overuse of antibiotics, ultimate in the evolution of antibiotic-resistant bacteria and genes in all types of animal farming settings of Bangladesh. MDR bacteria with extreme resistance against antibiotics recommended to use in both animals and humans have been reported and been being a potential public health hazard in Bangladesh. Execution of extensive AMR surveillance in veterinary practices and awareness-building programs for stakeholders along with the strengthening of the capacity of DLS are recommended for effective containment of AMR emergence and dissemination in the animal health sector of Bangladesh.

Antimicrobial resistance (AMR) and marine plastics: Can food packaging litter act as a dispersal mechanism for AMR in oceanic environments?

This study examined the carriage of antibiotic resistance in bacteria isolated from Food-related Marine Macroplastic Litter (FRMMPL) around the coastline of Northern Ireland. FRMMPL was collected from 18 coastal sites during November/December 2018 and the bacteria from the surface of the plastic examined for their susceptibility to 10 common human antibiotics. Ten bacterial genera and 13 species were identified from the plastic materials. Bacteria isolated from plastic material were most resistant to the beta-lactam antibiotics (ampicillin, ceftazidime and cefpodoxime) (98.1% resistant) and least resistant to the tetracycline group, minocycline (16.1% resistant). This study is significant as it highlights a new potential route of dispersal of such antibiotic-resistance in the environment, which may act as carriers of such bacteria by introducing them into new marine ecosystems, as well as potential pathways having impacts on animal and human health, until their final interaction with the human foodchain.

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

BackgroundShotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth (~ 200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, ‘ResPipe’.ResultsTaxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve < 1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment.ConclusionsSequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/hsgweon/ResPipe).

The Link between Antimicrobial Resistance and the Content of Potentially Toxic Metals in Soil and Fertilising Products

Potentially toxic metals (PTM), along with PTM-resistant bacteria and PTM-resistance genes may be introduced to soil and water through sewage systems, direct excretion, land application of biosolids (organic matter recycled from sewage, especially for use in agriculture) or animal manures as fertilisers, and irrigation with wastewater or treated effluents.&#x0D; The Norwegian Food Safety Authority (NFSA) asked the Norwegian Scientific Committee for Food Safety (Vitenskapskomiteen for mattrygghet, VKM) for an assessment of the link between antimicrobial resistance (AMR) and potentially toxic metals (PTM) in soil and fertilising products.&#x0D; The NFSA would like VKM to give an opinion on the following question related to the influence of potentially toxic metals on antimicrobial resistance:&#x0D; &#x0D; Can the content of arsenic (As), cadmium (Cd), chromium (CrIII + CrVI), copper (Cu), lead (Pb), mercury (Hg), nickel (Ni), and zinc (Zn) in soil and fertilising products that are relevant for Norway play a role in the development, spreading, and persistence of bacterial resistance to these elements, as well as cross or co-resistance to antimicrobial agents?&#x0D; &#x0D; VKM appointed a working group, consisting of two members of the Panel on Biological Hazards, to prepare a draft Opinion document and answer the questions. The Panel on Biological Hazards has reviewed and revised the draft prepared by the working group and approved the Opinion document “The link between antimicrobial resistance and the content of potentially toxic metals in soil and fertilising products”.&#x0D; In this report we assess the following PTM: arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), mercury (Hg), lead (Pb), and zinc (Zn), because of their possible presence in fertilisers and their potential to induce AMR in bacteria.&#x0D; This assessment is based on internationally published data. There is no systematic monitoring for toxic metals in soils in Norway, and the levels are expected to be highly variable depending on the input sources, previous and current agricultural practices, and the characteristics of the soil. Data on PTM in fertilising products added to soil are also fragmented and limited. Fertilising materials, in the form of sewage sludge or livestock manure, will add toxic metals to the existing levels in soil, and in areas of intensive agriculture, the levels will be expected to be highest. The additive effect of toxic metals in fertilising materials must be assessed from a long-term perspective, as these metals accumulate in the environment.&#x0D; Development of AMR can be partly regarded as a dose- and time-dependant response to exposure to different drivers for resistance. There is an indication that the presence of potentially toxic metals is a driver for development of AMR in exposed bacteria, but the dose and time exposure most likely to cause this effect is not known. Investigation of PTM-driven co-selection of AMR in environments impacted by agriculture and aquaculture should focus especially on Cu and Zn, which are added to animal feed, and on Cd because of its high concentration, in comparison with other PTM, in inorganic fertilising products. The naturally occurring background resistance in environmental bacteria complicates the estimation of the effect of PTM exposure on development of resistance. In addition, it is difficult to distinguish between the natural resistome and an elevated abundance of AMR in environmental samples.&#x0D; Spreading of resistance towards the PTM evaluated in this assessment involves cross- and co-resistance to antimicrobial agents used in prophylaxis and therapy in animals and people. Most important are those cases where toxic metal resistance is coupled to resistance towards highly important and critically important antibiotics. This has been described in some of the published articles included in this assessment. We do not fully understand the mechanisms behind persistence of AMR, and removing drivers for development and spread of resistance may result in a decrease in the levels of resistance, but not necessarily full disappearance.&#x0D; There is lack of knowledge regarding links between the level and concentration of PTM in fertilising products and soil and development of resistance in bacteria. Data regarding the routes and frequencies of transmission of AMR from bacteria of environmental origin to bacteria of animal and human origin were lacking in the published articles reviewed here. Due to the lack of such data, it is difficult to estimate the probability of development, transmission, and persistence of PTM resistance in the Norwegian environment. More research is needed to explain the relationship between development of resistance against potential toxic metals and resistance toward antimicrobial agents in bacteria.

Anthropogenic environmental drivers of antimicrobial resistance in wildlife

The isolation of antimicrobial resistant bacteria (ARB) from wildlife living adjacent to humans has led to the suggestion that such antimicrobial resistance (AMR) is anthropogenically driven by exposure to antimicrobials and ARB. However, ARB have also been detected in wildlife living in areas without interaction with humans. Here, we investigated patterns of resistance in Escherichia coli isolated from 408 wild bird and mammal faecal samples. AMR and multi-drug resistance (MDR) prevalence in wildlife samples differed significantly between a Sewage Treatment Plant (STP; wastes of antibiotic-treated humans) and a Farm site (antibiotic-treated livestock wastes) and Central site (no sources of wastes containing anthropogenic AMR or antimicrobials), but patterns of resistance also varied significantly over time and between mammals and birds. Over 30% of AMR isolates were resistant to colistin, a last-resort antibiotic, but resistance was not due to the mcr-1 gene. ESBL and AmpC activity were common in isolates from mammals. Wildlife were, therefore, harbouring resistance of clinical relevance. AMR E. coli, including MDR, were found in diverse wildlife species, and the patterns and prevalence of resistance were not consistently associated with site and therefore different exposure risks. We conclude that AMR in commensal bacteria of wildlife is not driven simply by anthropogenic factors, and, in practical terms, this may limit the utility of wildlife as sentinels of spatial variation in the transmission of environmental AMR.

The widespread dissemination of integrons throughout bacterial communities in a riverine system

Anthropogenic inputs increase levels of antimicrobial resistance (AMR) in the environment, however, it is unknown how these inputs create this observed increase, and if anthropogenic sources impact AMR in environmental bacteria. The aim of this study was to characterise the role of waste water treatment plants (WWTPs) in the dissemination of class 1 integrons (CL1s) in the riverine environment. Using sample sites from upstream and downstream of a WWTP, we demonstrate through isolation and culture-independent analysis that WWTP effluent significantly increases both CL1 abundance and antibiotic resistance in the riverine environment. Characterisation of CL1-bearing isolates revealed that CL1s were distributed across a diverse range of bacteria, with identical complex genetic resistance determinants isolated from both human-associated and common environmental bacteria across connected sites. Over half of sequenced CL1s lacked the 3′-conserved sequence ('atypical’ CL1s); surprisingly, bacteria carrying atypical CL1s were on average resistant to more antibiotics than bacteria carrying 3′-CS CL1s. Quaternary ammonium compound (QAC) resistance genes were observed across 75% of sequenced CL1 gene cassette arrays. Chemical data analysis indicated high levels of boron (a detergent marker) downstream of the WWTP. Subsequent phenotypic screening of CL1-bearing isolates demonstrated that ~90% were resistant to QAC detergents, with in vitro experiments demonstrating that QACs could solely select for the transfer of clinical antibiotic resistance genes to a naive Escherichia coli recipient. In conclusion, this study highlights the significant impact of WWTPs on environmental AMR, and demonstrates the widespread carriage of clinically important resistance determinants by environmentally associated bacteria.

Low-Concentration Ciprofloxacin Selects Plasmid-Mediated Quinolone Resistance Encoding Genes and Affects Bacterial Taxa in Soil Containing Manure.

The spread of antimicrobial resistance in environment is promoted at least in part by the inappropriate use of antibiotics in animals and humans. The present study was designed to investigate the impact of different concentrations of ciprofloxacin in soil containing manure on the development of plasmid-mediated quinolone resistance (PMQR) – encoding genes and the abundance of soil bacterial communities. For these studies, high-throughput next-generation sequencing of 16S rRNA, real-time polymerase chain reaction and standard microbiologic culture methods were utilized. We demonstrated that the dissipate rate of relative abundances of some of PMQR-encoding genes, such as qnrS, oqxA and aac(6′)-Ib-cr, were significantly lower with ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared to no-ciprofloxacin control and ciprofloxacin 4 mg/kg exposure during 2 month. Also, the number of ciprofloxacin resistant bacteria was significantly greater in ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared with no-ciprofloxacin control and the ciprofloxacin 4 mg/kg exposure. In addition, lower ciprofloxacin concentration provided a selective advantage for the populations of Xanthomonadales and Bacillales in orders while Agrobacterium, Bacillus, Enterococcus, and Burkholderia in genera. These findings suggest that lower concentration of ciprofloxacin resulted in a slower rate of PMQR-encoding genes dissipation and selected development of ciprofloxacin-resistant bacteria in soil amended with manure.