Contrary effects of increasing temperatures on the spread of antimicrobial resistance in river biofilms.

River microbial communities regularly act as the first barrier of defense against the spread of antimicrobial resistance genes (ARGs) that enter environmental microbiomes through wastewater. However, how the invasion dynamics of wastewater-borne ARGs into river biofilm communities will shift due to climate change with increasing average and peak temperatures remains unknown. Here, we aimed to elucidate the effects of increasing temperatures on the naturally occurring river biofilm resistome, as well as the invasion success of foreign ARGs entering through wastewater. Natural biofilms were grown in a low-anthropogenic impact river and transferred to artificial laboratory recirculation flume systems operated at three different temperatures (20°C, 25°C, and 30°C). After 1 week of temperature acclimatization, significant increases in the abundance of the naturally occurring ARGs in biofilms were detected at higher temperatures. After this acclimatization period, biofilms were exposed to a single pulse of wastewater, and the invasion dynamics of wastewater-borne ARGs were analyzed over 2 weeks. After 1 day, wastewater-borne ARGs were able to invade the biofilms successfully with no observable effect of temperature on their relative abundance. However, thereafter, ARGs were lost at a far increased rate at 30°C, with ARG levels dropping to the initial natural levels after 14 days. Contrary to the lower temperatures, ARGs were either lost at slower rates or even able to establish themselves in biofilms with stable relative abundances above natural levels. Hence, higher temperatures come with contrary effects on river biofilm resistomes: naturally occurring ARGs increase in abundance, while foreign, invading ARGs are lost at elevated speeds.IMPORTANCEInfections with bacteria that gained resistance to antibiotics are taking millions of lives annually, with the death toll predicted to increase. River microbial communities act as a first defense barrier against the spread of antimicrobial resistance genes (ARGs) that enter the environment through wastewater after enrichment in human and animal microbiomes. The global increase in temperature due to climate change might disrupt this barrier effect by altering microbial community structure and functions. We consequently explored how increasing temperatures alter ARG spread in river microbial communities. At higher temperatures, naturally occurring ARGs increased in relative abundance. However, this coincided with a decreased success rate of invading foreign ARGs from wastewater to establish themselves in the communities. Therefore, to predict the effects of climate change on ARG spread in river microbiomes, it is imperative to consider if the river ecosystem and its resistome are dominated by naturally occurring or invading foreign ARGs.

Investigating the antibiotic resistance genes and their potential risks in the megacity water environment: A case study of Shenzhen Bay Basin, China.

Antibiotic resistance genes (ARGs) constitute emerging pollutants and pose serious risks to public health. Anthropogenic activities are recognized as the main driver of ARG dissemination in coastal regions. However, the distribution and dissemination of ARGs in Shenzhen Bay Basin, a typical megacity water environment, have been poorly investigated. Here, we comprehensively profiled ARGs in Shenzhen Bay Basin using metagenomic approaches, and estimated their associated health risks. ARG profiles varied greatly among different sampling locations with total abundance ranging from 2.79 × 10-2 (Shenzhen Bay sediment) to 1.04 (hospital sewage) copies per 16S rRNA gene copy, and 45.4% of them were located on plasmid-like sequences. Sewage treatment plants effluent and the corresponding tributary rivers were identified as the main sources of ARG contamination in Shenzhen Bay. Mobilizable plasmids and complete integrons carrying various ARGs probably participated in the dissemination of ARGs in Shenzhen Bay Basin. Additionally, 19 subtypes were assigned as high-risk ARGs (Rank I), and numerous ARGs were identified in potential human-associated pathogens, such as Burkholderiaceae, Rhodocyclaceae, Vibrionaceae, Pseudomonadaceae, and Aeromonadaceae. Overall, Shenzhen Bay represented a higher level of ARG risk than the ocean environment based on quantitative risk assessment. This study deepened our understanding of the ARGs and the associated risks in the megacity water environment.

Interspecific competition can drive plasmid loss from a focal species in a microbial community.

Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids' ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species specific. Addition of growth partners increased the cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.

Environmental stress increases the invasion success of antimicrobial resistant bacteria in river microbial communities.

Environmental microbiomes are constantly exposed to invasion events through foreign, antibiotic resistant bacteria that were enriched in the anthropic sphere. However, the biotic and abiotic factors, as well as the natural barriers that determine the invasion success of these invader bacteria into the environmental microbiomes are poorly understood. A great example of such invasion events are river microbial communities constantly exposed to resistant bacteria originating from wastewater effluents. Here, we aim at gaining comprehensive insights into the key factors that determine their invasion success with a particular focus on the effects of environmental stressors, regularly co-released in wastewater effluents. Understanding invasion dynamics of resistant bacteria is crucial for limiting the environmental spread of antibiotic resistance. To achieve this, we grew natural microbial biofilms on glass slides in rivers for one month. The biofilms were then transferred to laboratory, recirculating flume systems and exposed to a single pulse of a model resistant invader bacterium (Escherichia coli) either in presence or absence of stress induced by Cu2+. The invasion dynamics of E. coli into the biofilms were then monitored for 14 days. Despite an initially successful introduction of E. coli into the biofilms, independent of the imposed stress, over time the invader perished in absence of stress. However, under stress the invading strain successfully established and proliferated in the biofilms. Noteworthy, the increased establishment success of the invader coincided with a loss in microbial community diversity under stress conditions, likely due to additional niche space becoming available for the invader.

Towards monitoring of antimicrobial resistance in the environment: For what reasons, how to implement it, and what are the data needs?

Antimicrobial resistance (AMR) is a global threat to human and animal health and well-being. To understand AMR dynamics, it is important to monitor resistant bacteria and resistance genes in all relevant settings. However, while monitoring of AMR has been implemented in clinical and veterinary settings, comprehensive monitoring of AMR in the environment is almost completely lacking. Yet, the environmental dimension of AMR is critical for understanding the dissemination routes and selection of resistant microorganisms, as well as the human health risks related to environmental AMR. Here, we outline important knowledge gaps that impede implementation of environmental AMR monitoring. These include lack of knowledge of the 'normal' background levels of environmental AMR, definition of high-risk environments for transmission, and a poor understanding of the concentrations of antibiotics and other chemical agents that promote resistance selection. Furthermore, there is a lack of methods to detect resistance genes that are not already circulating among pathogens. We conclude that these knowledge gaps need to be addressed before routine monitoring for AMR in the environment can be implemented on a large scale. Yet, AMR monitoring data bridging different sectors is needed in order to fill these knowledge gaps, which means that some level of national, regional and global AMR surveillance in the environment must happen even without all scientific questions answered. With the possibilities opened up by rapidly advancing technologies, it is time to fill these knowledge gaps. Doing so will allow for specific actions against environmental AMR development and spread to pathogens and thereby safeguard the health and wellbeing of humans and animals.

Microbiome and Resistome Profiles along a Sewage-Effluent-Reservoir Trajectory Underline the Role of Natural Attenuation in Wastewater Stabilization Reservoirs.

Antibiotic-resistant bacteria and antibiotic resistance gene (ARGs) loads dissipate through sewage treatment plants to receiving aquatic environments, but the mechanisms that mitigate the spread of these ARGs are not well understood due to the complexity of full-scale systems and the difficulty of source tracking in downstream environments. To overcome this problem, we targeted a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR), whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs and receiving aquatic ecosystems. We analyzed a large set of physicochemical measurements, concomitant with the cultivation of total and cefotaxime-resistant Escherichia coli, microbial community analyses, and quantitative PCR (qPCR)/digital droplet PCR (ddPCR) quantification of selected ARGs and mobile genetic elements (MGEs). The MABR removed most of the sewage-derived organic carbon and nitrogen, and simultaneously, E. coli, ARG, and MGE levels dropped by approximately 1.5- and 1.0-log unit mL-1, respectively. Similar levels of E. coli, ARGs, and MGEs were removed in the reservoir, but interestingly, unlike in the MABR, the relative abundance (normalized to 16S rRNA gene-inferred total bacterial abundance) of these genes also decreased. Microbial community analyses revealed the substantial shifts in bacterial and eukaryotic community composition in the reservoir relative to the MABR. Collectively, our observations lead us to conclude that the removal of ARGs in the MABR is mainly a consequence of treatment-facilitated biomass removal, whereas in the stabilization reservoir, mitigation is linked to natural attenuation associated with ecosystem functioning, which includes abiotic parameters, and the development of native microbiomes that prevent the establishment of wastewater-derived bacteria and associated ARGs. IMPORTANCE Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs. We evaluated ARB and ARG dynamics across the raw-sewage-MABR-effluent trajectory, concomitant with evaluation of microbial community composition and physicochemical parameters, in an attempt to identify mechanisms associated with ARB and ARG dissipation. We found that removal of ARB and ARGs in the MABR was primarily associated with bacterial death or sludge removal, whereas in the reservoir it was attributed to the inability of ARBs and associated ARGs to colonize the reservoir due to a dynamic and persistent microbial community. The study demonstrates the importance of ecosystem functioning in removing microbial contaminants from wastewater.

Reduced selection for antibiotic resistance in community context is maintained despite pressure by additional antibiotics.

Selection for antibiotic resistance at very low antibiotic concentrations has been demonstrated for individual antibiotics in single species experiments. Furthermore, selection in these focal strains is reduced when taking place in complex microbial community context. However, in the environment, bacteria are rarely exposed to single, but rather complex mixtures of selective agents. Here, we explored how the presence of a second selective agent affects selection dynamics between isogenic pairs of focal E. coli strains, differing exclusively in a single resistance determinant, in the absence and presence of a model wastewater community across a gradient of antibiotics. An additional antibiotic that exclusively affects the model wastewater community, but to which the focal strains are resistant to, was chosen as the second selective agent. This allowed exploring how inhibition alters the community's ability to reduce selection. In the presence of the community, the selection coefficient at specific antibiotic concentrations was consistently decreased compared to the absence of the community. While pressure through the second antibiotic significantly decreased the activity and diversity of the community, its ability to reduce selection was consistently maintained at levels comparable to those recorded in absence of the second antibiotic. This indicates that the observed effects of community context on selection dynamics are rather based on competitive or protective effects between the focal strains and a small proportion of bacteria within the community, than on general competition for nutrients. These findings have implications for our understanding of the evolution and selection for multi-drug resistant strains.

Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool.

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode cas9 programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from Escherichia coli. Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of Pseudomonas. This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

Quantification of the mobility potential of antibiotic resistance genes through multiplexed ddPCR linkage analysis.

There is a clear need for global monitoring initiatives to evaluate the risks of antibiotic resistance genes (ARGs) towards human health. Therefore, not only ARG abundances within a given environment, but also their potential mobility, hence their ability to spread to human pathogenic bacteria needs to be quantified. We developed a novel, sequencing-independent method for assessing the linkage of an ARG to a mobile genetic element by statistical analysis of multiplexed droplet digital PCR (ddPCR) carried out on environmental DNA sheared into defined, short fragments. This allows quantifying the physical linkage between specific ARGs and mobile genetic elements, here demonstrated for the sulfonamide ARG sul1 and the Class 1 integron integrase gene intI1. The method's efficiency is demonstrated using mixtures of model DNA fragments with either linked and unlinked target genes: Linkage of the two target genes can be accurately quantified based on high correlation coefficients between observed and expected values (R2) as well as low mean absolute errors (MAE) for both target genes, sul1 (R2 = 0.9997, MAE = 0.71%, n = 24) and intI1 (R2 = 0.9991, MAE = 1.14%, n = 24). Furthermore, we demonstrate that adjusting the fragmentation length of DNA during shearing allows controlling rates of false positives and false negative detection of linkage. The presented method allows rapidly obtaining reliable results in a labor- and cost-efficient manner.

Shifts from cooperative to individual-based predation defense determine microbial predator-prey dynamics.

Predation defense is an important feature of predator-prey interactions adding complexity to ecosystem dynamics. Prey organisms have developed various strategies to escape predation which differ in mode (elude vs. attack), reversibility (inducible vs. permanent), and scope (individual vs. cooperative defenses). While the mechanisms and controls of many singular defenses are well understood, important ecological and evolutionary facets impacting long-term predator-prey dynamics remain underexplored. This pertains especially to trade-offs and interactions between alternative defenses occurring in prey populations evolving under predation pressure. Here, we explored the dynamics of a microbial predator-prey system consisting of bacterivorous flagellates (Poteriospumella lacustris) feeding on Pseudomonas putida. Within five weeks of co-cultivation corresponding to about 35 predator generations, we observed a consistent succession of bacterial defenses in all replicates (n = 16). Initially, bacteria expressed a highly effective cooperative defense based on toxic metabolites, which brought predators close to extinction. This initial strategy, however, was consistently superseded by a second mechanism of predation defense emerging via de novo mutations. Combining experiments with mathematical modeling, we demonstrate how this succession of defenses is driven by the maximization of individual rather than population benefits, highlighting the role of rapid evolution in the breakdown of social cooperation.

Does Environmental Exposure to Pharmaceutical and Personal Care Product Residues Result in the Selection of Antimicrobial-Resistant Microorganisms, and is this Important in Terms of Human Health Outcomes?

The environment plays a critical role in the development, dissemination, and transmission of antimicrobial resistance (AMR). Pharmaceuticals and personal care products (PPCPs) enter the environment through direct application to the environment and through anthropogenic pollution. Although there is a growing body of evidence defining minimal selective concentrations (MSCs) of antibiotics and the role antibiotics play in horizontal gene transfer (HGT), there is limited evidence on the role of non-antibiotic PPCPs. Existing data show associations with the development of resistance or effects on bacterial growth rather than calculating selective endpoints. Research has focused on laboratory-based systems rather than in situ experiments, although PPCP concentrations found throughout wastewater, natural water, and soil environments are often within the range of laboratory-derived MSCs and at concentrations shown to promote HGT. Increased selection and HGT of AMR by PPCPs will result in an increase in total AMR abundance in the environment, increasing the risk of exposure and potential transmission of environmental AMR to humans. There is some evidence to suggest that humans can acquire resistance from environmental settings, with water environments being the most frequently studied. However, because this is currently limited, we recommend that more evidence be gathered to understand the risk the environment plays in regard to human health. In addition, we recommend that future research efforts focus on MSC-based experiments for non-antibiotic PPCPS, particularly in situ, and investigate the effect of PPCP mixtures on AMR. Environ Toxicol Chem 2022;00:1-14. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Deciphering chloramphenicol biotransformation mechanisms and microbial interactions via integrated multi-omics and cultivation-dependent approaches.

BACKGROUND: As a widely used broad-spectrum antibiotic, chloramphenicol is prone to be released into environments, thus resulting in the disturbance of ecosystem stability as well as the emergence of antibiotic resistance genes. Microbes play a vital role in the decomposition of chloramphenicol in the environment, and the biotransformation processes are especially dependent on synergistic interactions and metabolite exchanges among microbes. Herein, the comprehensive chloramphenicol biotransformation pathway, key metabolic enzymes, and interspecies interactions in an activated sludge-enriched consortium were elucidated using integrated multi-omics and cultivation-based approaches. RESULTS: The initial biotransformation steps were the oxidization at the C1-OH and C3-OH groups, the isomerization at C2, and the acetylation at C3-OH of chloramphenicol. Among them, the isomerization is an entirely new biotransformation pathway of chloramphenicol discovered for the first time. Furthermore, we identified a novel glucose-methanol-choline oxidoreductase responsible for the oxidization of the C3-OH group in Sphingomonas sp. and Caballeronia sp. Moreover, the subsequent biotransformation steps, corresponding catalyzing enzymes, and the microbial players responsible for each step were deciphered. Synergistic interactions between Sphingomonas sp. and Caballeronia sp. or Cupriavidus sp. significantly promoted chloramphenicol mineralization, and the substrate exchange interaction network occurred actively among key microbes. CONCLUSION: This study provides desirable strain and enzyme resources for enhanced bioremediation of chloramphenicol-contaminated hotspot sites such as pharmaceutical wastewater and livestock and poultry wastewater. The in-depth understanding of the chloramphenicol biotransformation mechanisms and microbial interactions will not only guide the bioremediation of organic pollutants but also provide valuable knowledge for environmental microbiology and biotechnological exploitation. Video Abstract.

Non-antibiotic pharmaceuticals promote conjugative plasmid transfer at a community-wide level.

BACKGROUND: Horizontal gene transfer (HGT) plays a critical role in the spread of antibiotic resistance and the evolutionary shaping of bacterial communities. Conjugation is the most well characterized pathway for the spread of antibiotic resistance, compared to transformation and transduction. While antibiotics have been found to induce HGT, it remains unknown whether non-antibiotic pharmaceuticals can facilitate conjugation at a microbial community-wide level. RESULTS: In this study, we demonstrate that several commonly consumed non-antibiotic pharmaceuticals (including carbamazepine, ibuprofen, naproxen and propranolol), at environmentally relevant concentrations (0.5 mg/L), can promote the conjugative transfer of IncP1-α plasmid-borne antibiotic resistance across entire microbial communities. The over-generation of reactive oxygen species in response to these non-antibiotic pharmaceuticals may contribute to the enhanced conjugation ratios. Cell sorting and 16S rRNA gene amplicon sequencing analyses indicated that non-antibiotic pharmaceuticals modulate transconjugant microbial communities at both phylum and genus levels. Moreover, microbial uptake ability of the IncP1-α plasmid was also upregulated under non-antibiotic pharmaceutical exposure. Several opportunistic pathogens, such as Acinetobacter and Legionella, were more likely to acquire the plasmid conferring multidrug resistance. CONCLUSIONS: Considering the high possibility of co-occurrence of pathogenic bacteria, conjugative IncP1-α plasmids and non-antibiotic pharmaceuticals in various environments (e.g., activated sludge systems), our findings illustrate the potential risk associated with increased dissemination of antibiotic resistance promoted by non-antibiotic pharmaceuticals in complex environmental settings. Video abstract.

Biogeographical Patterns of Bacterial Communities and Their Antibiotic Resistomes in the Inland Waters of Southeast China.

Freshwater ecosystems are important sources of drinking water and provide natural settings for the proliferation and dissemination of bacteria and antibiotic resistance genes (ARGs). However, the biogeographical patterns of ARGs in natural freshwaters and their relationships with the bacterial community at large scales are largely understudied. This is of specific importance because data on ARGs in environments with low anthropogenic impact is still very limited. We characterized the biogeographical patterns of bacterial communities and their ARG profiles in 24 reservoirs across southeast China using 16S rRNA gene high-throughput sequencing and high-throughput-quantitative PCR, respectively. We found that the composition of both bacterial communities and ARG profiles exhibited a significant distance-decay pattern. However, ARG profiles displayed larger differences among different water bodies than bacterial communities, and the relationship between bacterial communities and ARG profiles was weak. The biogeographical patterns of bacterial communities were simultaneously driven by stochastic and deterministic processes, while ARG profiles were not explained by stochastic processes, indicating a decoupling of bacterial community composition and ARG profiles in inland waters under relatively low-human-impact at a large scale. Overall, this study provides an overview of the biogeographical patterns and driving mechanisms of bacterial community and ARG profiles and could offer guidance and reference for the control of ARGs in drinking water sources. IMPORTANCE Antibiotic resistance has been a serious global threat to environmental and human health. The "One Health" concept further emphasizes the importance of monitoring the large-scale dissemination of ARGs. However, knowledge about the geographical patterns and driving mechanisms of bacterial communities and ARGs in natural freshwater environments is limited. This study uncovered the distinct biogeographical patterns of bacterial communities and ARG profiles in inland waters of southeast China under low-anthropogenic impact at a large scale. This study improved our understanding of ARG distribution in inland waters with emphasis on drinking water supply reservoirs, therefore providing the much-needed baseline information for future monitoring and risk assessment of ARGs in drinking water resources.

Fitness effects of plasmids shape the structure of bacteria-plasmid interaction networks.

Antimicrobial resistance (AMR) genes are often carried on broad host range plasmids, and the spread of AMR within microbial communities will therefore depend on the structure of bacteria­plasmid networks. Empirical and theoretical studies of ecological interaction networks suggest that network structure differs between communities that are predominantly mutualistic versus antagonistic, with the former showing more generalized interactions (i.e., species interact with many others to a similar extent). This suggests that mutualistic bacteria­plasmid networks­where antibiotics are present and plasmids carry AMR genes­will be more generalized than antagonistic interactions, where plasmids do not confer benefits to their hosts. We first develop a simple theory to explain this link: fitness benefits of harboring a mutualistic symbiont promote the spread of the symbiont to other species. We find support for this theory using an experimental bacteria­symbiont (plasmid) community, where the same plasmid can be mutualistic or antagonistic depending on the presence of antibiotics. This short-term and parsimonious mechanism complements a longer-term mechanism (coevolution and stability) explaining the link between mutualistic and antagonistic interactions and network structure.

Multiwalled Carbon Nanotubes Promote Bacterial Conjugative Plasmid Transfer.

Multiwalled carbon nanotubes (MWCNTs) regularly enter aquatic environments due to their ubiquity in consumer products and engineering applications. However, the effects of MWCNT pollution on the environmental microbiome are poorly understood. Here, we evaluated whether these carbon nanoparticles can elevate the spread of antimicrobial resistance by promoting bacterial plasmid transfer, which has previously been observed for copper nanomaterials with antimicrobial properties as well as for microplastics. Through a combination of experimental liquid mating assays between Pseudomonas putida donor and recipient strains with plasmid pKJK5::gfpmut3b and mathematical modeling, we here demonstrate that the presence of MWCNTs leads to increased plasmid transfer rates in a concentration-dependent manner. The percentage of transconjugants per recipient significantly increased from 0.21 ± 0.04% in absence to 0.41 ± 0.09% at 10 mg L-1 MWCNTs. Similar trends were observed when using an Escherichia coli donor hosting plasmid pB10. The identified mechanism underlying the observed dynamics was the agglomeration of MWCNTs. A significantly increased number of particles with >6 µm diameter was detected in the presence of MWCNTs, which can in turn provide novel surfaces for bacterial interactions between donor and recipient cells after colonization. Fluorescence microscopy confirmed that MWCNT agglomerates were indeed covered in biofilms that contained donor bacteria as well as elevated numbers of green fluorescent transconjugant cells containing the plasmid. Consequently, MWCNTs provide bacteria with novel surfaces for intense cell-to-cell interactions in biofilms and can promote bacterial plasmid transfer, hence potentially elevating the spread of antimicrobial resistance. IMPORTANCE In recent decades, the use of carbon nanoparticles, especially multiwalled carbon nanotubes (MWCNTs), in a variety of products and engineering applications has been growing exponentially. As a result, MWCNT pollution into environmental compartments has been increasing. We here demonstrate that the exposure to MWCNTs can affect bacterial plasmid transfer rates in aquatic environments, an important process connected to the spread of antimicrobial resistance genes in microbial communities. This is mechanistically explained by the ability of MWCNTs to form bigger agglomerates, hence providing novel surfaces for bacterial interactions. Consequently, increasing pollution with MWCNTs has the potential to elevate the ongoing spread of antimicrobial resistance, a major threat to human health in the 21st century.

Municipal Wastewaters Carry Important Carbapenemase Genes Independent of Hospital Input and Can Mirror Clinical Resistance Patterns.

The spatiotemporal variation of several carbapenemase-encoding genes (CRGs) was investigated in the influent and effluent of municipal WWTPs, with or without hospital sewage input. Correlations among gene abundances, bacterial community composition, and wastewater quality parameters were tested to identify possible predictors of CRGs presence. Also, the possible role of wastewaters in mirroring clinical resistance is discussed. The taxonomic groups and gene abundances showed an even distribution among wastewater types, meaning that hospital sewage does not influence the microbial diversity and the CRG pool. The bacterial community was composed mainly of Proteobacteria, Firmicutes, Actinobacteria, Patescibacteria, and Bacteroidetes. Acinetobacter spp. was the most abundant group and had the majority of operational taxonomic units (OTUs) positively correlated with CRGs. This agrees with recent reports on clinical data. The influent samples were dominated by blaKPC, as opposed to effluent, where blaIMP was dominant. Also, blaIMP was the most frequent CRG family observed to correlate with bacterial taxa, especially with the Mycobacterium genus in effluent samples. Bacterial load, blaNDM, blaKPC, and blaOXA-48 abundances were positively correlated with BOD5, TSS, HEM, Cr, Cu, and Fe concentrations in wastewaters. When influent gene abundance values were converted into population equivalent (PE) data, the highest copies/1 PE were identified for blaKPC and blaOXA-48, agreeing with previous studies regarding clinical isolates. Both hospital and non-hospital-type samples followed a similar temporal trend of CRG incidence, but with differences among gene groups. Colder seasons favored the presence of blaNDM, blaKPC and blaOXA-48, whereas warmer temperatures show increased PE values for blaVIM and blaIMP. IMPORTANCE Wastewater-based epidemiology has recently been recognized as a valuable, cost-effective tool for antimicrobial resistance surveillance. It can help gain insights into the characteristics and distribution of antibiotic resistance elements at a local, national, and even global scale. In this study, we investigated the possible use of municipal wastewaters in the surveillance of clinically relevant carbapenemase-encoding genes (CRGs), seen as critical antibiotic resistance determinants. In this matter, our results highlight positive correlations among CRGs, microbial diversity, and wastewater physical and chemical parameters. Identified predictors can provide valuable data regarding the level of raw and treated wastewater contamination with these important antibiotic resistance genes. Also, wastewater-based gene abundances were used for the first time to observe possible spatiotemporal trends of CRGs incidence in the general population. Therefore, possible hot spots of carbapenem resistance could be easily identified at the community level, surpassing the limitations of health care-associated settings.

Elevated levels of antibiotic resistance in groundwater during treated wastewater irrigation associated with infiltration and accumulation of antibiotic residues.

Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

Corrigendum: Distribution and Diversity of Comammox Nitrospira in Coastal Wetlands of China.

[This corrects the article DOI: 10.3389/fmicb.2020.589268.].

Simultaneous estimation of parameters governing the vertical and horizontal transfer of antibiotic resistance genes.

The accelerated spread of antibiotic resistance genes (ARG) in the environment occurs mainly through plasmid transfer facilitated via bacterial conjugation. To predict and efficiently counteract the problems associated with ARG transmission, it is important to estimate conjugation rates under different experimental conditions. The classical models typically used to estimate parameters for mating experiments, while pragmatic in calculating growth and plasmid transfer, often ignore processes such as the reduction in growth due to plasmid bearing costs and are non-inclusive of environmental influences like temperature effects. Here, we present a process-based numerical model taking into account the fitness cost associated with plasmid carriage and temperature dependencies in vertical and horizontal gene transfer processes. Observations from liquid culture conjugation experiments using Escherichia coli and the plasmid pB10 were used to validate our proposed model. We present a comparison between the parameters estimated using the existing and the proposed model. Uncertainties in the estimated parameters were quantified using classical and advanced Bayesian methods. For our mating experiments, we found that at temperatures between 20 and 37 °C, the plasmid bearing costs reduced the growth rates by > 35%. The temperature dependency model of conjugation showed a good fit (mean absolute percentage error < 10%) independent of the bacteria and the plasmid under study. The proposed model simultaneously estimates growth and plasmid transfer rate constants for all three strains (donor, recipient, and transconjugant). Simultaneous estimation of growth and conjugation parameters is particularly useful to estimate the spread of ARG when one of the mating partners inhibits the growth of the other, which is common in multi-species mating or when the incurred plasmid costs are situation dependent (e.g., increased plasmid cost in a mating environment) as observed in this study.