Metagenome meta-analysis reveals an increase in the abundance of some multidrug efflux pumps and mobile genetic elements in chemically polluted environments.

Many human activities contaminate terrestrial and aquatic environments with numerous chemical pollutants that not only directly alter the environment but also affect microbial communities in ways that are potentially concerning to human health, such as selecting for the spread of antibiotic-resistance genes (ARGs) through horizontal gene transfer. In the present study, metagenomes available in the public domain from polluted (with antibiotics, with petroleum, with metal mining, or with coal-mining effluents) and unpolluted terrestrial and aquatic environments were compared to examine whether pollution has influenced the abundance and composition of ARGs and mobile elements, with specific focus on IS26 and class 1 integrons (intI1). When aggregated together, polluted environments had a greater relative abundance of ARGs than unpolluted environments and a greater relative abundance of IS26 and intI1. In general, chemical pollution, notably with petroleum, was associated with an increase in the prevalence of ARGs linked to multidrug efflux pumps. Included in the suite of efflux pumps were mexK, mexB, mexF, and mexW that are polyspecific and whose substrate ranges include multiple classes of critically important antibiotics. Also, in some instances, ß-lactam resistance (TEM181 and OXA-541) genes increased, and genes associated with rifampicin resistance (RNA polymerases subunits rpoB and rpoB2) decreased in relative abundance. This meta-analysis suggests that different types of chemical pollution can enrich populations that carry efflux pump systems associated with resistance to multiple classes of medically critical antibiotics.IMPORTANCEThe United Nations has identified chemical pollution as being one of the three greatest threats to environmental health, through which the evolution of antimicrobial resistance, a seminally important public health challenge, may be favored. While this is a very plausible outcome of continued chemical pollution, there is little evidence or research evaluating this risk. The objective of the present study was to examine existing metagenomes from chemically polluted environments and evaluate whether there is evidence that pollution increases the relative abundance of genes and mobile genetic elements that are associated with antibiotic resistance. The key finding is that for some types of pollution, particularly in environments exposed to petroleum, efflux pumps are enriched, and these efflux pumps can confer resistance to multiple classes of medically important antibiotics that are typically associated with Pseudomonas spp. or other Gram-negative bacteria. This finding makes clear the need for more investigation on the impact of chemical pollution on the environmental reservoir of ARGs and their association with mobile genetic elements that can contribute to horizontal gene transfer events.

Modeling Antibiotic Concentrations in the Vicinity of Antibiotic-Producing Bacteria at the Micron Scale.

It is generally thought that antibiotics confer upon the producing bacteria the ability to inhibit or kill neighboring microorganisms, thereby providing the producer with a significant competitive advantage. Were this to be the case, the concentrations of emitted antibiotics in the vicinity of producing bacteria might be expected to fall within the ranges of MICs that are documented for a number of bacteria. Furthermore, antibiotic concentrations that bacteria are punctually or chronically exposed to in environments harboring antibiotic-producing bacteria might fall within the range of minimum selective concentrations (MSCs) that confer a fitness advantage to bacteria carrying acquired antibiotic resistance genes. There are, to our knowledge, no available in situ measured antibiotic concentrations in the biofilm environments that bacteria typically live in. The objective of the present study was to use a modeling approach to estimate the antibiotic concentrations that might accumulate in the vicinity of bacteria that are producing an antibiotic. Fick's law was used to model antibiotic diffusion using a series of key assumptions. The concentrations of antibiotics within a few microns of single producing cells could not reach MSC (8 to 16 µg/L) or MIC (500 µg/L) values, whereas the concentrations around aggregates of a thousand cells could reach these concentrations. The model outputs suggest that single cells could not produce an antibiotic at a rate sufficient to achieve a bioactive concentration in the vicinity, whereas a group of cells, each producing the antibiotic, could do so. IMPORTANCE It is generally assumed that a natural function of antibiotics is to provide their producers with a competitive advantage. If this were the case, sensitive organisms in proximity to producers would be exposed to inhibitory concentrations. The widespread detection of antibiotic resistance genes in pristine environments suggests that bacteria are indeed exposed to inhibitory antibiotic concentrations in the natural world. Here, a model using Fick's law was used to estimate potential antibiotic concentrations in the space surrounding producing cells at the micron scale. Key assumptions were that per-cell production rates drawn from the pharmaceutical manufacturing industry are applicable in situ, that production rates were constant, and that produced antibiotics are stable. The model outputs indicate that antibiotic concentrations in proximity to aggregates of a thousand cells can indeed be in the minimum inhibitory or minimum selective concentration range.

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.

Fate of Clostridia and other spore-forming Firmicute bacteria during feedstock anaerobic digestion and aerobic composting.

Pathogenic spore-forming Firmicutes are commonly present in animal and human wastes that are used as fertilizers in crop production. Pre-treatments of organic waste prior to land application offer the potential to abate enteric microorganisms, and therefore reduce the risk of contamination of crops or adjacent water resources with pathogens carried in these materials. The inactivation and reduction of gram-positive spore formers such as Clostridium spp., Clostridioides spp. and Bacillus spp. from animal and human waste can be challenging given the recalcitrance of the spores these bacteria produce. Given the significance of these organisms to human and animal health, information concerning spore-forming bacteria inactivation during anaerobic digestion (AD) and aerobic composting (AC) is required as the basis for recommending safe organic waste management practices. In this review, an assessment of the inactivation of spore-forming Firmicutes during AD and AC was conducted to provide guidance for practical management of organic matrices of animal or human origin. Temperature and pH may be the main factors contributing to the inactivation of spore-forming Firmicutes during batch lab-scale AD (log reduction <0.5-5 log). In continuous digesters, wet AD systems do not effectively inactivate spore-forming Firmicutes even under thermopholic conditions (log reduction -1.09 - 0.98), but dry AD systems could be a feasible management practice to inactivate spore-forming Firmicutes from organic materials with high solid content (log reduction 1.77-3.1). In contrast, composting is an effective treatment to abate spore-forming Firmicutes (log reduction 1.7-6.5) when thermophilic conditions last at least six consecutive days. Temperature, moisture content and composting scale are the key operating conditions influencing the inactivation of spore-forming Firmicutes during composting. Where possible, undertaking AD with subsequent composting to ensure the biosafety of digestate before its downstream processing and recycling is recommended to abate recalcitrant bacteria in digestate.

Global dispersal and potential sources of antibiotic resistance genes in atmospheric remote depositions.

Antibiotic resistance has become a major Global Health concern and a better understanding on the global spread mechanisms of antibiotic resistant bacteria (ARB) and intercontinental ARB exchange is needed. We measured atmospheric depositions of antibiotic resistance genes (ARGs) by quantitative (q)PCR in rain/snow collected fortnightly along 4 y. at a remote high mountain LTER (Long-Term Ecological Research) site located above the atmospheric boundary layer (free troposphere). Bacterial composition was characterized by 16S rRNA gene sequencing, and air mass provenances were determined by modelled back trajectories and rain/snow chemical composition. We hypothesize that the free troposphere may act as permanent reservoir and vector for ARB and ARGs global dispersal. We aimed to i) determine whether ARGs are long-range intercontinental and persistently dispersed through aerosols, ii) assess ARGs long-term atmospheric deposition dynamics in a remote high mountain area, and iii) unveil potential diffuse ARGs pollution sources. We showed that the ARGs sul1 (resistance to sulfonamides), tetO (resistance to tetracyclines), and intI1 (a proxy for horizontal gene transfer and anthropogenic pollution) were long-range and persistently dispersed in free troposphere aerosols. Major depositions of tetracyclines resistance matched with intensification of African dust outbreaks. Potential ARB mostly traced their origin back into agricultural soils. Our study unveils that air masses pathways are shaping ARGs intercontinental dispersal and global spread of antibiotic resistances, with potential predictability for interannual variability and remote deposition rates. Because climate regulates aerosolization and long-range air masses movement patterns, we call for a more careful evaluation of the connections between land use, climate change and ARB long-range intercontinental dispersal.

Impact of chicken litter pre-application treatment on the abundance, field persistence, and transfer of antibiotic resistant bacteria and antibiotic resistance genes to vegetables.

Treatment of manures prior to land application can potentially reduce the abundance of antibiotic resistance genes and thus the risk of contaminating crops or water resources. In this study, raw and composted chicken litter were applied to field plots that were cropped to carrots, lettuce and radishes. Vegetables were washed per normal culinary practice before downstream analysis. The impact of composting on manure microbial composition, persistence of antibiotic resistant bacteria in soil following application, and distribution of antibiotic resistance genes and bacteria on washed vegetables were determined. A subset of samples that were thought likely to reveal the most significant effects were chosen for shotgun sequencing. The absolute abundance of all target genes detected by qPCR decreased after composting except sul1, intI1, incW and erm(F) that remained stable. The shotgun sequencing revealed that some integron integrases were enriched by composting. Composting significantly reduced the abundance of enteric bacteria, including those carrying antibiotic resistance. Manure-amended soil showed significantly higher abundances of sul1, str(A), str(B), erm(B), aad(A), intI1 and incW compared to unmanured soil. At harvest, those genes that were detected in soil samples before the application of manure (intI1, sul1, strA and strB) were quantifiable by qPCR on vegetables, with a larger number of gene targets detected on the radishes than in the carrots or lettuce. Shotgun metagenomic sequencing suggested that the increase of antibiotic resistance genes on radishes produced in soil receiving raw manure may be due to changes to soil microbial communities following manure application, rather than transfer to the radishes of enteric bacteria. Overall, under field conditions there was limited evidence for transfer of antibiotic resistance genes from composted or raw manure to vegetables that then persisted through washing.

Composting of chicken litter from commercial broiler farms reduces the abundance of viable enteric bacteria, Firmicutes, and selected antibiotic resistance genes.

We examined the ability of composting to remove ARGs and enteric bacteria in litter obtained from broiler chickens fed with a diet supplemented with Bacitracin methylene disalicylate (BDM) (conventional chicken litter), or an antibiotic-free diet (raised without antibiotic (RWA) chicken litter). This was done by evaluating the litter before and after composting for the abundance of ten gene targets associated with antibiotic resistance or horizontal gene transfer, the composition of the bacterial communities, and the abundance of viable enteric bacteria. The abundance of gene targets was determined by qPCR and the microbial community composition of chicken litter determined by 16S rRNA gene amplicon sequencing. Enteric bacteria were enumerated by viable plate count. A majority of the gene targets were more abundant in conventional than in RWA litter. In both litter types, the absolute abundance of all of the target genes decreased after composting except sul1, intI1, incW and erm(F) that remained stable. Composting significantly reduced the abundance of enteric bacteria, including those carrying antibiotic resistance. The major difference in bacterial community composition between conventional and RWA litter was due to members affiliated to the genus Pseudomonas, which were 28% more abundant in conventional than in RWA litter. Composting favoured the presence of thermophilic bacteria, such as those affiliated with the genus Truepera, but decreased the abundance of those bacterial genera associated with cold-adapted species, such as Carnobacterium, Psychrobacter and Oceanisphaera. The present study shows that chicken litter from broilers fed with a diet supplemented with antibiotic has an increased abundance of some ARGs, even after composting. However, we can conclude that fertilization with composted litter represents a reduced risk of transmission of antibiotic resistance genes and enteric bacteria of poultry origin to soil and crops than will fertilization with raw litter.

High-quality treated wastewater causes remarkable changes in natural microbial communities and intI1 gene abundance.

We carry out a mesocosms experiment to assess the impact of high-quality treated wastewater intended for agricultural reuse (HQWR) on freshwater bacteria seldom exposed to anthropogenic pollution. Effects were assessed by comparing the abundance and composition of bacterial communities as well as their resistance profile under control (source water from an unpolluted lake) and treatment conditions (source water mixed 1:1 with HQWR, with and without 5 µg L-1 of cefotaxime). We investigated the effect of the different conditions on the abundance of genes encoding resistance to ß-lactams and carbapenems (blaTEM, blaCTX-M, blaOXA, and blaKPC), fluoroquinolones (qnrS), tetracyclines (tetA), sulfonamides (sul2), macrolides (ermB), arsenic and cadmium (arsB and czcA, respectively), and on the gene encoding the Class 1 integron integrase (intI1). Bacterial communities exposed to HQWR showed a significant higher abundance of tetA, arsB, czcA, and intI1 genes, whereas those exposed to Cefotaxime-amended HQWR did not. Genes conferring resistance to carbapenems, ß-lactams, fluoroquinolones, and macrolides were below detection limit in all treatments. Besides, the higher availability of nutrients under treatment conditions favored bacterial growth in comparison to those exposed to control conditions. Particularly, Acinetobacter spp. and Pseudomonas spp. were significantly enriched after 22 days of treatment exposure. The presence of cefotaxime (a third generation cephalosporine) in the feeding medium caused an enrichment of bacterial communities in sequences affiliated to Acinetobacter thus suggesting that these resistant forms may possess resistance genes other than those studied here (blaCTX-M, blaOXA, and blaKPC). Although derived from a mesocosm experiment in continuous cultures, our results call attention to the need of refined regulations regarding the use of reclaimed water in agriculture since even high-quality treated wastewater may lead to undesired effects on receiving bacterial communities in terms of composition and dissemination of antibiotic resistance genes.

Does Dietary Consumption of Antibiotics by Humans Promote Antibiotic Resistance in the Gut Microbiome?

Mandated authorities have developed principles for evaluating the safety of antimicrobial residues in food and have established microbiological acceptable daily intakes (ADIs) and recommended maximum residue limits (MRLs) for antibiotic residues in food products. The evaluation of the ADI is based in relevant scientific information such as MIC data of predominant human intestinal bacteria. However, it does not include data derived from minimal selective concentration (MSC) predictions that estimate the lowest concentration of an antibiotic that will provide resistant bacteria an advantage over susceptible bacteria. Based on these insights, we sought to determine whether human exposure to selected antibiotics through ingestion of foodstuffs could result in colon concentrations exceeding apparent MSCs. Nine antibiotics-tetracycline, oxytetracycline, ciprofloxacin, sarafloxacin, erythromycin, spiramycin, tilmicosin, tylosin, and lincomycin-were selected for analysis. Dietary exposure was estimated either using published measured antibiotic concentrations in foodstuffs or using ADI values or food MRLs and a conservative diet. Using the ADI, the estimated antibiotic residue concentrations in the human colon of all antibiotics assessed may be up to a 1,000-fold greater than the predicted MSCs. When the dietary exposure assessment used MRLs or measured concentration in foodstuffs, the estimated concentrations were considerably lower but still within the MSC range for most of the foodstuffs assessed. These results suggest that the ingestion of antibiotic residues through food consumption may expose intestinal microbiota to antibiotic concentrations exceeding the MSC boundaries, thus favoring the growth of potential resistant bacteria. We suggest that MRL and ADI values be revisited in light of the recognition that antibiotic concentrations significantly below the MIC may select for resistance.

Emerging contaminants and nutrients synergistically affect the spread of class 1 integron-integrase (intI1) and sul1 genes within stable streambed bacterial communities.

Wastewater effluents increase the nutrient load of receiving streams while introducing a myriad of anthropogenic chemical pollutants that challenge the resident aquatic (micro)biota. Disentangling the effects of both kind of stressors and their potential interaction on the dissemination of antibiotic resistance genes in bacterial communities requires highly controlled manipulative experiments. In this work, we investigated the effects of a combined regime of nutrients (at low, medium and high concentrations) and a mixture of emerging contaminants (ciprofloxacin, erythromycin, sulfamethoxazole, diclofenac, and methylparaben) on the bacterial composition, abundance and antibiotic resistance profile of biofilms grown in artificial streams. In particular, we investigated the effect of this combined stress on genes encoding resistance to ciprofloxacin (qnrS), erythromycin (ermB), sulfamethoxazole (sul1 and sul2) as well as the class 1 integron-integrase gene (intI1). Only genes conferring resistance to sulfonamides (sul1 and sul2) and intI1 gene were detected in all treatments during the study period. Besides, bacterial communities exposed to emerging contaminants showed higher copy numbers of sul1 and intI1 genes than those not exposed, whereas nutrient amendments did not affect their abundance. However, bacterial communities exposed to both emerging contaminants and a high nutrient concentration (1, 25 and 1 mg L-1 of phosphate, nitrate and ammonium, respectively) showed the highest increase on the abundance of sul1 and intI1 genes thus suggesting a factors synergistic effect of both stressors. Since none of the treatments caused a significant change on the composition of bacterial communities, the enrichment of sul1 and intI1 genes within the community was caused by their dissemination under the combined pressure exerted by nutrients and emerging contaminants. To the best of our knowledge, this is the first study demonstrating the contribution of nutrients on the maintenance and spread of antibiotic resistance genes in streambed biofilms under controlled conditions. Our results also highlight that nutrients could enhance the effect of emerging contaminants on the dissemination of antibiotic resistance.

Antibiotic resistance along an urban river impacted by treated wastewaters.

Urban rivers are impacted ecosystems which may play an important role as reservoirs for antibiotic-resistant (AR) bacteria. The main objective of this study was to describe the prevalence of antibiotic resistance along a sewage-polluted urban river. Seven sites along the Zenne River (Belgium) were selected to study the prevalence of AR Escherichia coli and freshwater bacteria over a 1-year period. Culture-dependent methods were used to estimate E. coli and heterotrophic bacteria resistant to amoxicillin, sulfamethoxazole, nalidixic acid and tetracycline. The concentrations of these four antibiotics have been quantified in the studied river. The antibiotic resistance genes (ARGs), sul1, sul2, tetW, tetO, blaTEM and qnrS were also quantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed an effect of treated wastewaters release on the spread of antibiotic resistance along the river. Although an increase in the abundance of both AR E. coli and resistant heterotrophic bacteria was observed from upstream to downstream sites, the differences were only significant for AR E. coli. A significant positive regression was also found between AR E. coli and resistant heterotrophic bacteria. The concentration of ARGs increased from upstream to downstream sites for both particle-attached (PAB) and free-living bacteria (FLB). Particularly, a significant increase in the abundance of four among six ARGs analyzed was observed after crossing urban area. Although concentrations of tetracycline significantly correlated with tetracycline resistance genes, the antibiotic levels were likely too low to explain this correlation. The analysis of ARGs in different fractions revealed a significantly higher abundance in PAB compared to FLB for tetO and sul2 genes. This study demonstrated that urban activities may increase the spread of antibiotic resistance even in an already impacted river.

Wastewater pollution differently affects the antibiotic resistance gene pool and biofilm bacterial communities across streambed compartments.

Wastewater discharges introduce antibiotic residues and antibiotic-resistant bacteria (ARB) into surface waters. Both inputs directly affect the streambed resistome, either by exerting a selective pressure that favour the proliferation of resistant phenotypes or by enriching the resident communities with wastewater-associated ARB. Here, we investigated the impact of raw and treated urban wastewater discharges on epilithic (growing on rocks) and epipsammic (growing on sandy substrata) streambed biofilms. The effects were assessed by comparing control and impact sites (i) on the composition of bacterial communities; (ii) on the abundance of twelve antibiotic resistance genes (ARGs) encoding resistance to ß-lactams, fluoroquinolones, sulphonamides, tetracyclines, macrolides and vancomycin, as well as the class 1 integron-integrase gene (intI1); (iii) on the occurrence of wastewater-associated bacteria, including putative pathogens, and their potential linkage to target ARGs. We measured more pronounced effects of raw sewage than treated wastewater at the three studied levels. This effect was especially noticeable in epilithic biofilms, which showed a higher contribution of wastewater-associated bacteria and ARB than in epipsammic biofilms. Comparison of correlation coefficients obtained between the relative abundance of both target ARGs and operational taxonomic units classified as either potential pathogens or nonpathogens yielded significant higher correlations between the former category and genes intI1, sul1, sul2 and ermB. Altogether, these results indicate that wastewater-associated micro-organisms, including potential pathogens, contribute to maintain the streambed resistome and that epilithic biofilms appear as sensitive biosensors of the effect of wastewater pollution in surface waters.

Abundance of carbapenemase genes (blaKPC, blaNDM and blaOXA-48) in wastewater effluents from Tunisian hospitals.

Carbapenems are ß-lactam antibiotics with a broad spectrum of activity and are usually considered the last resort for the treatment of severe infections caused by multidrug-resistant pathogens. The clinically most significant carbapenemases are KPC, NDM, and OXA-48-like enzymes, whose genes have been increasingly reported worldwide in members of the family Enterobacteriaceae. In this study, we quantified the abundance of these genes in wastewater effluents from different Tunisian hospitals. The blaNDM and blaOXA-48-like genes were detected at similar concentrations in all hospital wastewater effluents. In contrast, the blaKPC gene was detected at lower concentration than other genes and it was only detected in three of the seven effluents analyzed. To the best of our knowledge, this study quantified for the first time the abundance of blaKPC, blaNDM, and blaOXA-48-like genes in wastewater effluents from Tunisian hospitals, highlighting the widespread distribution of these carbapenemase genes.

Abundance and Co-Distribution of Widespread Marine Archaeal Lineages in Surface Sediments of Freshwater Water Bodies across the Iberian Peninsula.

Archaea inhabiting marine and freshwater sediments have a relevant role in organic carbon mineralization, affecting carbon fluxes at a global scale. Despite current evidences suggesting that freshwater sediments largely contribute to this process, few large-scale surveys have been addressed to uncover archaeal diversity and abundance in freshwater sedimentary habitats. In this work, we quantified and high-throughput sequenced the archaeal 16S rRNA gene from surficial sediments collected in 21 inland waterbodies across the Iberian Peninsula differing in typology and trophic status. Whereas methanogenic groups were dominant in most of the studied systems, especially in organic-rich sediments, archaea affiliated to widespread marine lineages (the Bathyarchaeota and the Thermoplasmata) were also ubiquitous and particularly abundant in euxinic sediments. In these systems, Bathyarchaeota communities were dominated by subgroups Bathyarchaeota-6 (87.95 ± 12.71%) and Bathyarchaeota-15 (8.17 ± 9.2%) whereas communities of Thermoplasmata were mainly composed of members of the order Thermoplasmatales. Our results also indicate that Archaea accounted for a minor fraction of sedimentary prokaryotes despite remarkable exceptions in reservoirs and some stratified lakes. Copy numbers of archaeal and bathyarchaeotal 16S rRNA genes were significantly different when compared according to system type (i.e., lakes, ponds, and reservoirs), but no differences were obtained when compared according to their trophic status (from oligotrophy to eutrophy). Interestingly, we obtained significant correlations between the abundance of reads (Spearman r = 0.5, p = 0.021) and OTU richness (Spearman r = 0.677, p < 0.001) of Bathyarchaeota and Thermoplasmata across systems, reinforcing the hypothesis of a potential syntrophic interaction between members of both lineages.

Real-time PCR assays for the detection and quantification of carbapenemase genes (bla KPC, bla NDM, and bla OXA-48) in environmental samples.

In this study, we have developed real-time PCR assays using SYBR Green chemistry to detect all known alleles of bla KPC, bla NDM, and bla OXA-48-like carbapenemase genes in water, sediment, and biofilm samples collected from hospital and wastewater treatment plant (WWTP) effluents and rivers receiving chronic WWTP discharges. The amplification of bla KPC, bla NDM, and bla OXA-48 DNA was linear over 7 log dilutions (R 2 between 0.995 and 0.997) and showing efficiencies ranging from 92.6% to 100.3%. The analytical sensitivity indicated that the reaction for bla KPC, bla NDM, and bla OXA-48-like genes was able to detect 35, 16, and 19 copy numbers per assay, respectively. The three carbapenemase genes were detected in hospital effluents, whereas only the bla KPC and bla NDM genes were detected in biofilm and sediment samples collected from wastewater-impacted rivers. The detection of bla KPC, bla NDM, and bla OXA-48-like genes in different matrices suggests that carbapenem-resistant bacteria occur in both planktonic and benthic habitats thus expanding the range of resistance reservoirs for last-resort antibiotics. We believe that these real-time PCR assays would be a powerful tool for the rapid detection and quantification of bla KPC, bla NDM, and bla OXA-48-like genes in complex environmental samples.

Exploring the contribution of bacteriophages to antibiotic resistance.

Bacteriophages (phages) are the most abundant and diverse biological entities in our planet. They infect susceptible bacterial hosts into which they either multiply or persist. In the latter case, phages can confer new functions to their hosts as a result of gene transfer, thus contributing to their adaptation (short-term) and evolution (long-term). In this regard, the role of phages on the dissemination of antibiotic resistance genes (ARGs) among bacterial hosts in natural environments has not yet been clearly resolved. Here, we carry out a comprehensive analysis of thirty-three viromes from different habitats to investigate whether phages harbor ARGs. Our results demonstrate that while human-associated viromes do not or rarely carry ARGs, viromes from non-human sources (e.g. pig feces, raw sewage, and freshwater and marine environments) contain a large reservoir of ARGs, thus pointing out that phages could play a part on the spread of antibiotic resistance. Given this, the role of phages should not be underestimated and it should be considered when designing strategies to tackle the global crisis of antibiotic resistance.

Metagenomic analysis reveals that bacteriophages are reservoirs of antibiotic resistance genes.

A metagenomics approach was applied to explore the presence of antibiotic resistance genes (ARGs) in bacteriophages from hospital wastewater. Metagenomic analysis showed that most phage sequences affiliated to the order Caudovirales, comprising the tailed phage families Podoviridae, Siphoviridae and Myoviridae. Moreover, the relative abundance of ARGs in the phage DNA fraction (0.26%) was higher than in the bacterial DNA fraction (0.18%). These differences were particularly evident for genes encoding ATP-binding cassette (ABC) and resistance-nodulation-cell division (RND) proteins, phosphotransferases, ß-lactamases and plasmid-mediated quinolone resistance. Analysis of assembled contigs also revealed that blaOXA-10, blaOXA-58 and blaOXA-24 genes belonging to class D ß-lactamases as well as a novel blaTEM (98.9% sequence similarity to the blaTEM-1 gene) belonging to class A ß-lactamases were detected in a higher proportion in phage DNA. Although preliminary, these findings corroborate the role of bacteriophages as reservoirs of resistance genes and thus highlight the necessity to include them in future studies on the emergence and spread of antibiotic resistance in the environment.

The role of biofilms as environmental reservoirs of antibiotic resistance.

Antibiotic resistance has become a significant and growing threat to public and environmental health. To face this problem both at local and global scales, a better understanding of the sources and mechanisms that contribute to the emergence and spread of antibiotic resistance is required. Recent studies demonstrate that aquatic ecosystems are reservoirs of resistant bacteria and antibiotic resistance genes as well as potential conduits for their transmission to human pathogens. Despite the wealth of information about antibiotic pollution and its effect on the aquatic microbial resistome, the contribution of environmental biofilms to the acquisition and spread of antibiotic resistance has not been fully explored in aquatic systems. Biofilms are structured multicellular communities embedded in a self-produced extracellular matrix that acts as a barrier to antibiotic diffusion. High population densities and proximity of cells in biofilms also increases the chances for genetic exchange among bacterial species converting biofilms in hot spots of antibiotic resistance. This review focuses on the potential effect of antibiotic pollution on biofilm microbial communities, with special emphasis on ecological and evolutionary processes underlying acquired resistance to these compounds.