Antimicrobial Transformation Products in the Aquatic Environment: Global Occurrence, Ecotoxicological Risks, and Potential of Antibiotic Resistance.

The global spread of antimicrobial resistance (AMR) is concerning for the health of humans, animals, and the environment in a One Health perspective. Assessments of AMR and associated environmental hazards mostly focus on antimicrobial parent compounds, while largely overlooking their transformation products (TPs). This review lists antimicrobial TPs identified in surface water environments and examines their potential for AMR promotion, ecological risk, as well as human health and environmental hazards using in silico models. Our review also summarizes the key transformation compartments of TPs, related pathways for TPs reaching surface waters and methodologies for studying the fate of TPs. The 56 antimicrobial TPs covered by the review were prioritized via scoring and ranking of various risk and hazard parameters. Most data on occurrences to date have been reported in Europe, while little is known about antibiotic TPs in Africa, Central and South America, Asia, and Oceania. Occurrence data on antiviral TPs and other antibacterial TPs are even scarcer. We propose evaluation of structural similarity between parent compounds and TPs for TP risk assessment. We predicted a risk of AMR for 13 TPs, especially TPs of tetracyclines and macrolides. We estimated the ecotoxicological effect concentrations of TPs from the experimental effect data of the parent chemical for bacteria, algae and water fleas, scaled by potency differences predicted by quantitative structure-activity relationships (QSARs) for baseline toxicity and a scaling factor for structural similarity. Inclusion of TPs in mixtures with their parent increased the ecological risk quotient over the threshold of one for 7 of the 24 antimicrobials included in this analysis, while only one parent had a risk quotient above one. Thirteen TPs, from which 6 were macrolide TPs, posed a risk to at least one of the three tested species. There were 12/21 TPs identified that are likely to exhibit a similar or higher level of mutagenicity/carcinogenicity, respectively, than their parent compound, with tetracycline TPs often showing increased mutagenicity. Most TPs with increased carcinogenicity belonged to sulfonamides. Most of the TPs were predicted to be mobile but not bioaccumulative, and 14 were predicted to be persistent. The six highest-priority TPs originated from the tetracycline antibiotic family and antivirals. This review, and in particular our ranking of antimicrobial TPs of concern, can support authorities in planning related intervention strategies and source mitigation of antimicrobials toward a sustainable future.

Metagenomic Analysis of the Abundance and Composition of Antibiotic Resistance Genes in Hospital Wastewater in Benin, Burkina Faso, and Finland.

Antibiotic resistance is a global threat to human health, with the most severe effect in low- and middle-income countries. We explored the presence of antibiotic resistance genes (ARGs) in the hospital wastewater (HWW) of nine hospitals in Benin and Burkina Faso, two low-income countries in West Africa, with shotgun metagenomic sequencing. For comparison, we also studied six hospitals in Finland. The highest sum of the relative abundance of ARGs in the 68 HWW samples was detected in Benin and the lowest in Finland. HWW resistomes and mobilomes in Benin and Burkina Faso resembled each other more than those in Finland. Many carbapenemase genes were detected at various abundances, especially in HWW from Burkina Faso and Finland. The blaGES genes, the most widespread carbapenemase gene in the Beninese HWW, were also found in water intended for hand washing and in a puddle at a hospital yard in Benin. mcr genes were detected in the HWW of all three countries, with mcr-5 being the most common mcr gene. These and other mcr genes were observed in very high relative abundances, even in treated wastewater in Burkina Faso and a street gutter in Benin. The results highlight the importance of wastewater treatment, with particular attention to HWW. IMPORTANCE The global emergence and increased spread of antibiotic resistance threaten the effectiveness of antibiotics and, thus, the health of the entire population. Therefore, understanding the resistomes in different geographical locations is crucial in the global fight against the antibiotic resistance crisis. However, this information is scarce in many low- and middle-income countries (LMICs), such as those in West Africa. In this study, we describe the resistomes of hospital wastewater in Benin and Burkina Faso and, as a comparison, Finland. Our results help to understand the hitherto unrevealed resistance in Beninese and Burkinabe hospitals. Furthermore, the results emphasize the importance of wastewater management infrastructure design to minimize exposure events between humans, HWW, and the environment, preventing the circulation of resistant bacteria and ARGs between humans (hospitals and community) and the environment.

Antibiotic Resistomes and Microbiomes in the Surface Water along the Code River in Indonesia Reflect Drainage Basin Anthropogenic Activities.

Water and sanitation are important factors in the emergence of antimicrobial resistance in low- and middle-income countries. Drug residues, metals, and various wastes foster the spread of antibiotic resistance genes (ARGs) with the help of mobile genetic elements (MGEs), and therefore, rivers receiving contaminants and effluents from multiple sources are of special interest. We followed both the microbiome and resistome of the Code River in Indonesia from its pristine origin at the Merapi volcano through rural and then city areas to the coast of the Indian Ocean. We used a SmartChip quantitative PCR with 382 primer pairs for profiling the resistome and MGEs and 16S rRNA gene amplicon sequencing to analyze the bacterial communities. The community structure explained the resistome composition in rural areas, while the city sampling sites had lower bacterial diversity and more ARGs, which correlated with MGEs, suggesting increased mobility potential in response to pressures from human activities. Importantly, the vast majority of ARGs and MGEs were no longer detectable in marine waters at the ocean entrance. Our work provides information on the impact of different influents on river health as well as sheds light on how land use contributes to the river resistome and microbiome.

Early-life formula feeding is associated with infant gut microbiota alterations and an increased antibiotic resistance load.

BACKGROUND: Infants are at a high risk of acquiring fatal infections, and their treatment relies on functioning antibiotics. Antibiotic resistance genes (ARGs) are present in high numbers in antibiotic-naive infants' gut microbiomes, and infant mortality caused by resistant infections is high. The role of antibiotics in shaping the infant resistome has been studied, but there is limited knowledge on other factors that affect the antibiotic resistance burden of the infant gut. OBJECTIVES: Our objectives were to determine the impact of early exposure to formula on the ARG load in neonates and infants born either preterm or full term. Our hypotheses were that diet causes a selective pressure that influences the microbial community of the infant gut, and formula exposure would increase the abundance of taxa that carry ARGs. METHODS: Cross-sectionally sampled gut metagenomes of 46 neonates were used to build a generalized linear model to determine the impact of diet on ARG loads in neonates. The model was cross-validated using neonate metagenomes gathered from public databases using our custom statistical pipeline for cross-validation. RESULTS: Formula-fed neonates had higher relative abundances of opportunistic pathogens such as Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, Klebsiella oxytoca, and Clostridioides difficile. The relative abundance of ARGs carried by gut bacteria was 69% higher in the formula-receiving group (fold change, 1.69; 95% CI: 1.12-2.55; P = 0.013; n = 180) compared to exclusively human milk-fed infants. The formula-fed infants also had significantly less typical infant bacteria, such as Bifidobacteria, that have potential health benefits. CONCLUSIONS: The novel finding that formula exposure is correlated with a higher neonatal ARG burden lays the foundation that clinicians should consider feeding mode in addition to antibiotic use during the first months of life to minimize the proliferation of antibiotic-resistant gut bacteria in infants.

High-Throughput Single-Cell Technology Reveals the Contribution of Horizontal Gene Transfer to Typical Antibiotic Resistance Gene Dissemination in Wastewater Treatment Plants.

The spread of antibiotic resistance genes (ARGs) has gained much attention worldwide, while the contribution of vertical gene transfer (VGT) and horizontal gene transfer (HGT) is still elusive. Here, we improved an emerging high-throughput single-cell-based technology, emulsion, paired isolation, and concatenation polymerase chain reaction (epicPCR), by lengthening the sequence of ARG in the fused ARG-16S rRNA fragments to cover the variance of both ARG and its hosts. The improved epicPCR was applied to track the hosts of a widely detected ARG, sul1 gene, in five urban wastewater treatment plants (UWTPs) during two seasons. The sul1 host bacteria were highly diverse and mostly classified as Proteobacteria and Bacteroidetes. Clear seasonal divergence of α-diversity and interaction networks were present in the host community. The consensus phylogenetic trees of the sul1 gene and their host demonstrated incorrespondence on the whole and regularity on abundant groups, suggesting the important role of both HGT and VGT, respectively. The relative importance of these two ways was further measured; HGT (54%) generally played an equal or even more important role as VGT (46%) in UWTPs. The application of the improved epicPCR technology provides a feasible approach to quantify the relative contributions of VGT and HGT in environmental dissemination of ARGs.

EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach.

EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.

Abundance and environmental host range of the SXT/R391 ICEs in aquatic environmental communities.

Mobile genetic elements (MGEs) such as plasmids or integrative conjugative elements (ICEs) are widely involved in the horizontal transfer of antibiotic resistant genes (ARGs), but their environmental host-range and reservoirs remain poorly known, as mainly assessed through the analysis of culturable and clinical bacterial isolates. In this study, we used a gradual approach for determining the environmental abundance and host-range of ICEs belonging to the SXT/R391 family, otherwise well known to bring ARGs in Vibrio spp. epidemic clones and other pathogens. First, by screening a set of aquatic bacteria libraries covering 1794 strains, we found that almost 1% of the isolates hosted an SXT/R391 element, all belonging to a narrow group of non-O1/non-O139 Vibrio cholerae. However, when SXT/R391 ICEs were then quantified in various aquatic communities, they appeared to be ubiquitous and relatively abundant, from 10-6 to 10-3 ICE copies per 16 S rDNA. Finally, the molecular exploration of the SXT/R391 host-range in two river ecosystems impacted by anthropogenic activities, using the single-cell genomic approach epicPCR, revealed several new SXT/R391 hosts mostly in the Proteobacteria phylum. Some, such as the pathogen Arcobacter cryaerophilus (Campylobacteraceae), have only been encountered in discharged treated wastewaters and downstream river waters, thus revealing a likely anthropogenic origin. Others, such as the non-pathogenic bacterium Neptunomonas acidivorans (Oceanospirillaceae), were solely identified in rivers waters upstream and downstream the treated wastewaters discharge points and may intrinsically belong to the SXT/R391 environmental reservoir. This work points out that not only the ICEs of the SXT/R391 family are more abundant in the environment than anticipated, but also that a variety of unsuspected hosts may well represent a missing link in the environmental dissemination of MGEs from and to bacteria of anthropogenic origin.

Unraveling the diversity of sedimentary sulfate-reducing prokaryotes (SRP) across Tibetan saline lakes using epicPCR.

Sulfate reduction is an important biogeochemical process in the ecosphere; however, the major taxa of sulfate reducers have not been fully identified. Here, we used epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) technology to identify the phylogeny of sulfate-reducing prokaryotes (SRP) in sediments from Tibetan Plateau saline lakes. A total of 12,519 OTUs and 883 SRP-OTUs were detected in ten lakes by sequencing of 16S rRNA gene PCR amplicons and epicPCR products of fused 16S rRNA plus dsrB gene, respectively, with Proteobacteria, Firmicutes, and Bacteroidetes being the dominant phyla in both datasets. The 120 highly abundant SRP-OTUs (> 1% in at least one sample) were affiliated with 17 described phyla, only 7 of which are widely recognized as SRP phyla. The majority of OTUs from both the whole microbial communities and the SRPs were not detected in more than one specific lake, suggesting high levels of endemism. The α-diversity of the entire microbial community and SRP sub-community showed significant positive correlations. The pH value and mean water temperature of the month prior to sampling were the environmental determinants for the whole microbial community, while the mean water temperature and total nitrogen were the major environmental drivers for the SRP sub-community. This study revealed there are still many undocumented SRP in Tibetan saline lakes, many of which could be endemic and adapted to specific environmental conditions.

Critical knowledge gaps and research needs related to the environmental dimensions of antibiotic resistance.

There is growing understanding that the environment plays an important role both in the transmission of antibiotic resistant pathogens and in their evolution. Accordingly, researchers and stakeholders world-wide seek to further explore the mechanisms and drivers involved, quantify risks and identify suitable interventions. There is a clear value in establishing research needs and coordinating efforts within and across nations in order to best tackle this global challenge. At an international workshop in late September 2017, scientists from 14 countries with expertise on the environmental dimensions of antibiotic resistance gathered to define critical knowledge gaps. Four key areas were identified where research is urgently needed: 1) the relative contributions of different sources of antibiotics and antibiotic resistant bacteria into the environment; 2) the role of the environment, and particularly anthropogenic inputs, in the evolution of resistance; 3) the overall human and animal health impacts caused by exposure to environmental resistant bacteria; and 4) the efficacy and feasibility of different technological, social, economic and behavioral interventions to mitigate environmental antibiotic resistance.1.

Antimicrobial resistance and the environment: assessment of advances, gaps and recommendations for agriculture, aquaculture and pharmaceutical manufacturing.

A roundtable discussion held at the fourth International Symposium on the Environmental Dimension of Antibiotic Resistance (EDAR4) considered key issues concerning the impact on the environment of antibiotic use in agriculture and aquaculture, and emissions from antibiotic manufacturing. The critical control points for reducing emissions of antibiotics from agriculture are antibiotic stewardship and the pre-treatment of manure and sludge to abate antibiotic-resistant bacteria. Antibiotics are sometimes added to fish and shellfish production sites via the feed, representing a direct route of contamination of the aquatic environment. Vaccination reduces the need for antibiotic use in high value (e.g. salmon) production systems. Consumer and regulatory pressure will over time contribute to reducing the emission of very high concentrations of antibiotics from manufacturing. Research priorities include the development of technologies, practices and incentives that will allow effective reduction in antibiotic use, together with evidence-based standards for antibiotic residues in effluents. All relevant stakeholders need to be aware of the threat of antimicrobial resistance and apply best practice in agriculture, aquaculture and pharmaceutical manufacturing in order to mitigate antibiotic resistance development. Research and policy development on antimicrobial resistance mitigation must be cognizant of the varied challenges facing high and low income countries.

Antibiotic-Resistance Genes in Waste Water.

Waste water and waste water treatment plants can act as reservoirs and environmental suppliers of antibiotic resistance. They have also been proposed to be hotspots for horizontal gene transfer, enabling the spread of antibiotic resistance genes between different bacterial species. Waste water contains antibiotics, disinfectants, and metals which can form a selection pressure for antibiotic resistance, even in low concentrations. Our knowledge of antibiotic resistance in waste water has increased tremendously in the past few years with advances in the molecular methods available. However, there are still some gaps in our knowledge on the subject, such as how active is horizontal gene transfer in waste water and what is the role of the waste water treatment plant in the environmental resistome? The purpose of this review is to briefly describe some of the main methods for studying antibiotic resistance in waste waters and the latest research and main knowledge gaps on the issue. In addition, some future research directions are proposed.

Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers.

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.

Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells.

Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

Cd-specific mutants of mercury-sensing regulatory protein MerR, generated by directed evolution.

The mercury-sensing regulatory protein, MerR (Tn21), which regulates mercury resistance operons in Gram-negative bacteria, was subjected to directed evolution in an effort to generate a MerR mutant that responds to Cd but not Hg. Oligonucleotide-directed mutagenesis was used to introduce random mutations into the key metal-binding regions of MerR. The effects of these mutations were assessed using a vector in which MerR controlled the expression of green fluorescent protein (GFP) and luciferase via the mer operator/promoter. An Escherichia coli cell library was screened by fluorescence-activated cell sorting, using a fluorescence-based dual screening strategy that selected for MerR mutants that showed GFP repression when cells were induced with Hg but GFP activation in the presence of Cd. Two Cd-responsive MerR mutants with decreased responses toward Hg were identified through the first mutagenesis/selection round. These mutants were used for a second mutagenesis/selection round, which yielded eight Cd-specific mutants that had no significant response to Hg, Zn, or the other tested metal(loid)s. Seven of the eight Cd-specific MerR mutants showed repressor activities equal to that of wild-type (wt) MerR. These Cd-specific mutants harbored multiple mutations (12 to 22) in MerR, indicating that the alteration of metal specificity with maintenance of repressor function was due to the combined effect of many mutations rather than just a few amino acid changes. The amino acid changes were studied by alignment against the sequences of MerR and other metal-responsive MerR family proteins. The analysis indicated that the generated Cd-specific MerR mutants appear to be unique among the MerR family members characterized to date.

Quantification of ecotoxicological tests based on bioluminescence using Polaroid film.

Assays based on the measurement of bacterial luminescence are widely used in ecotoxicology. Bacterial strains responding either to general toxicity or specific pollutants are rapid, cost-effective and easy to use. However, quantification of the signal requires relatively expensive instrumentation. We show here that the detection of luminescence of BioTox, a Vibrio fischeri-based toxicity test, and of a specific recombinant bacterial strain for arsenic determination, is possible using common Polaroid film. The exposed films can be used for visual or computer-assisted quantification of the signal. Qualitative visual comparison to standards can be used in the rapid and relatively accurate estimation of toxicity or pollutant concentration. The computer-assisted method significantly improves the accuracy and quantification of the results. The results obtained by computer-assisted quantification were in good agreement with the values obtained with a luminometer.