Assessing the Risk of Antibiotic Resistance Transmission from the Environment to Humans: Non-Direct Proportionality between Abundance and Risk
2016; Elsevier BV; Volume: 25; Issue: 3 Linguagem: Inglês
10.1016/j.tim.2016.11.014
ISSN1878-4380
Autores Tópico(s)Antibiotic Resistance in Bacteria
ResumoThe absence of a significant overlap of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARG) between the human microbiome and potential environmental sources should not be interpreted as an indication of risk absence. Hence, screening of ARG pools cannot be used as an accurate measure of the risk for transmission to humans. The risks of transmission of antibiotic resistance from the environment to humans must be assessed based on ARB (not only on ARG) that are able to colonize and proliferate in the human body. The risk is a function of their fitness in the human body and the presence of resistance and virulence genes. Even at extremely low abundance in environmental sources, ARB may represent a high risk for human health. The limits of quantification of methods commonly used to screen for ARG in environmental samples may be too high to allow reliable risk assessments. The past decade has witnessed a burst of study regarding antibiotic resistance in the environment, mainly in areas under anthropogenic influence. Therefore, impacts of the contaminant resistome, that is, those related to human activities, are now recognized. However, a key issue refers to the risk of transmission of resistance to humans, for which a quantitative model is urgently needed. This opinion paper makes an overview of some risk-determinant variables and raises questions regarding research needs. A major conclusion is that the risks of transmission of antibiotic resistance from the environment to humans must be managed under the precautionary principle, because it may be too late to act if we wait until we have concrete risk values. The past decade has witnessed a burst of study regarding antibiotic resistance in the environment, mainly in areas under anthropogenic influence. Therefore, impacts of the contaminant resistome, that is, those related to human activities, are now recognized. However, a key issue refers to the risk of transmission of resistance to humans, for which a quantitative model is urgently needed. This opinion paper makes an overview of some risk-determinant variables and raises questions regarding research needs. A major conclusion is that the risks of transmission of antibiotic resistance from the environment to humans must be managed under the precautionary principle, because it may be too late to act if we wait until we have concrete risk values. The risks associated with the environmental antibiotic resistome have been discussed under three different perspectives: at the microbial community level, the genome level, and the transmission of resistance. One refers to the risks of the emergence and evolution of clinically relevant antibiotic resistance from the environment and that may be enhanced by exogenous factors, such as chemical contaminants or physicochemical conditions [1Varela A.R. et al.Insights into the relationship between antimicrobial residues and bacterial populations in a hospital-urban wastewater treatment plant system.Water Res. 2014; 54: 327-336Crossref PubMed Scopus (102) Google Scholar, 2Christgen B. et al.Metagenomics shows that low-energy anaerobic-aerobic treatment reactors reduce antibiotic resistance gene levels from domestic wastewater.Environ. Sci. Technol. 2015; 49: 2577-2584Crossref PubMed Scopus (125) Google Scholar, 3Bengtsson-Palme J. et al.Elucidating selection processes for antibiotic resistance in sewage treatment plants using metagenomics.Sci. Total Environ. 2016; 572: 697-712Crossref PubMed Scopus (175) Google Scholar, 4Jutkina J. et al.An assay for determining minimal concentrations of antibiotics that drive horizontal transfer of resistance.Sci. Total Environ. 2016; 548–549: 131-138Crossref PubMed Scopus (122) Google Scholar]. This kind of insight, approached mainly at the bacterial community level, using either culture-based or culture-independent methods, is essential to understand how processes such as wastewater treatment, water disinfection, manure application in soils, or environmental pollution may contribute to enrich the environment in antibiotic-resistant bacteria (ARB). Another perspective of risk, approached essentially at the genome level, focuses on the threat imposed by genes that can confer resistance to antibiotics, but which differ on the spectrum of drugs against which they may be active and on the potential to be transferred by horizontal gene transfer. These traits affect their clinical relevance and therefore the associated human risk [5Andersson D.I. Improving predictions of the risk of resistance development against new and old antibiotics.Clin. Microbiol. Infect. 2015; 21: 894-898Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 6Martínez J.L. et al.What is a resistance gene? Ranking risk in resistomes.Nat. Rev. Microbiol. 2015; 13: 116-123Crossref PubMed Scopus (530) Google Scholar]. A third perspective of risk refers to the transmission of ARB or antibiotic-resistance genes (ARG) from the environment to humans [7Ashbolt N.J. et al.Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance.Environ. Health. Perspect. 2013; 121: 993-1001Crossref PubMed Scopus (435) Google Scholar, 8Huijbers P.M. et al.Role of the environment in the transmission of antimicrobial resistance to humans: a review.Environ. Sci. Technol. 2015; 49: 11993-12004Crossref PubMed Scopus (237) Google Scholar, 9Woolhouse M. et al.Antimicrobial resistance in humans, livestock and the wider environment.Phil. Trans. R. Soc. B. 2015; 370: 20140083Crossref PubMed Scopus (339) Google Scholar]. Curiously, although the ultimate goal of most studies about the environmental antibiotic resistome is associated with human health, this is probably the least explored type of risk. This is due, in part, to some major gaps that hamper reliable risk assessments, in particular, the nonexistence of databases that cross human- and environmental-related resistome data [10Berendonk T.U. et al.Tackling antibiotic resistance: the environmental framework.Nat. Rev. Microbiol. 2015; 13: 310-317Crossref PubMed Scopus (1236) Google Scholar]. Nonetheless, the risk of transmission to humans may surpass the level of comparative analyses of epidemiological data. This paper aims to discuss critical aspects that govern the transmission of ARB and ARG from the environment to humans and proposes the identification of some key risk determinant factors. Although the pathways and modes of transmission of antibiotic resistance to humans are still poorly understood, there are multiple instances of evidence of the wide and rapid spread of both ARB clones and ARG variants. The current knowledge suggests that a complex combination of variables referring to different environmental compartments, ubiquitous bacteria, and human–bacteria interaction may rule the risks of transmission to humans [9Woolhouse M. et al.Antimicrobial resistance in humans, livestock and the wider environment.Phil. Trans. R. Soc. B. 2015; 370: 20140083Crossref PubMed Scopus (339) Google Scholar, 11Holmes A.H. et al.Understanding the mechanisms and drivers of antimicrobial resistance.Lancet. 2016; 387: 176-187Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar]. These aspects are discussed in this section. The environmental resistome comprises both the natural antibiotic-resistance pool and that resulting from human activities, herein designated contaminant (Figure 1, Key Figure). The natural resistome, where genes have been found with significant identity to those of clinically relevant multidrug-resistant (MDR) pathogens, probably represents the beginning of the whole antibiotic-resistance cycle [12D’Costa V.M. et al.Antibiotic resistance is ancient.Nature. 2011; 477: 457-461Crossref PubMed Scopus (1565) Google Scholar]. Hence, these ARB, most of which are probably strictly environmental, can be considered reservoirs [13D’Costa V.M. et al.Sampling the antibiotic resistome.Science. 2006; 311: 374-377Crossref PubMed Scopus (1098) Google Scholar, 14Riesenfeld C.S. et al.Uncultured soil bacteria are a reservoir of new antibiotic resistance genes.Environ. Microbiol. 2004; 6: 981-989Crossref PubMed Scopus (386) Google Scholar]. Reservoirs comprise phylogenetically diverse bacteria, belonging to phyla such as Actinobacteria, Proteobacteria, or Bacteroidetes, often antibiotic producers or with the capacity to transform or metabolize antibiotics [12D’Costa V.M. et al.Antibiotic resistance is ancient.Nature. 2011; 477: 457-461Crossref PubMed Scopus (1565) Google Scholar, 13D’Costa V.M. et al.Sampling the antibiotic resistome.Science. 2006; 311: 374-377Crossref PubMed Scopus (1098) Google Scholar, 14Riesenfeld C.S. et al.Uncultured soil bacteria are a reservoir of new antibiotic resistance genes.Environ. Microbiol. 2004; 6: 981-989Crossref PubMed Scopus (386) Google Scholar, 15Dantas G. et al.Bacteria subsisting on antibiotics.Science. 2008; 320: 100-103Crossref PubMed Scopus (437) Google Scholar, 16Forsberg K.J. et al.The shared antibiotic resistome of soil bacteria and human pathogens.Science. 2012; 337: 1107-1111Crossref PubMed Scopus (1031) Google Scholar]. However, it is unlikely that all ARG that have arisen in clinical pathogens and continue to spread are only due to a direct transfer from reservoirs. Thus, it can be hypothesised that some intermediary agents are required to complete this process. This role can be attributed to bacteria that are abundant in the contaminant resistome, have high genome plasticity, and are able to spread ARG over different environmental compartments and among different bacterial populations. While the transfer of ARG from reservoirs (natural) to other bacteria may be a rare and random event, contaminant ARB and ARG may be able to spread rapidly and widely (e.g., New Delhi metallo-β-lactamase, blaNDM-1; extended-spectrum beta-lactamase blaCTXM-15; methicillin-resistant Staphylococcus aureus, MRSA) [17Nicolas-Chanoine M.H. et al.Intercontinental emergence of Escherichia coli clone O25: H4-ST131 producing CTX-M-15.J. Antimicrob. Chemother. 2016; 61: 273-281Crossref Scopus (674) Google Scholar, 18Walsh T.R. et al.Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study.Lancet Infect. Dis. 2011; 11: 355-362Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 19Rao Q. et al.Staphylococcus aureus ST121: a globally disseminated hyper-virulent clone.J. Med. Microbiol. 2015; 64: 1462-1473Crossref PubMed Scopus (58) Google Scholar]. Therefore, the risks of transmission of ARB from the environment to humans are probably higher in the contaminant than in the natural resistome. The contaminant antibiotic resistome comprises two major types of player: (i) carriers that are ARB with a role in the spread of ARG in the environment, but which cannot colonize or infect the human body, and (ii) vectors that are ARB that can colonize and sometimes invade the human body (Figure 1). Carriers and vectors are not necessarily members of distinct taxonomic groups, and the difference between both may be at the level of ecology or at the physiological level. That is, vectors, but not carriers, have the chance to be in contact with humans; or vectors, but not carriers, have the ability to colonize the human body (Figure 2, Figure 3). According to the available literature, among the most active potential carriers and vectors seem to be members of the classes Gammaproteobacteria and Betaproteobacteria and of the phyla Actinobacteria and Firmicutes [20Vaz-Moreira I. et al.Bacterial diversity and antibiotic resistance in water habitats: Searching the links with the human microbiome.FEMS Microbiol. Rev. 2014; 38: 761-778Crossref PubMed Scopus (236) Google Scholar, 21Li B. et al.Metagenomic and network analysis reveal wide distribution and co-occurrence of environmental antibiotic resistance genes.ISME J. 2015; 9: 2490-2502Crossref PubMed Scopus (686) Google Scholar]. Members of the family Enterobacteriaceae, and of genera such as Aeromonas, Acinetobacter, Pseudomonas, Enterococcus, or Staphylococcus, have been frequently described as carriers, and some of them are also recognized vectors [20Vaz-Moreira I. et al.Bacterial diversity and antibiotic resistance in water habitats: Searching the links with the human microbiome.FEMS Microbiol. Rev. 2014; 38: 761-778Crossref PubMed Scopus (236) Google Scholar, 22World Health OrganizationAntimicrobial Resistance: Global Report on Surveillance. WHO, 2014Google Scholar]. In the conceptual model proposed, vectors would be the end-of-the line for antibiotic resistance transmission to humans. Even though carriers are not able to colonize and infect humans, their spread and proliferation in the environment would increase the abundance and diversity of ARG in vectors. Hence, it may increase the risks of transmission of ARB to humans.Figure 3Environmental Sources, Transmission and Entry of Antibiotic-Resistant Bacteria into Humans. The transmission of antibiotic-resistant bacteria from the environment to humans may occur using the portals of entry described for pathogens, in particular through exposure to water and food products, by air, or via contact. Although the exact mechanisms of invasion by antibiotic-resistant bacteria are not known, it is plausible to consider two major modes, the one typical of exogenous infections, in which the infectious agent enters the tissue or organ directly, or the endogenous infection model, in which the invasion of tissues or organs succeeds a passive colonization of the skin or mucosa. In this case, vectors may be part of the commensal microbiota for long periods of time until the host's condition allows their proliferation and/or invasion. Considering the preferential habitats of some antibiotic-resistant bacteria and the location of the infection with which they are often associated, are presented some examples of bacterial groups and potential entry portals used to enter and colonize the human body, even if they do not cause an infection. Presented in this figure are some examples of antibiotic-resistant bacteria and respective entry portals used to enter and colonize the human body, even if they do not cause an infection. These examples were chosen considering the preferential habitats of the selected bacterial groups as well as the location of the infection with which they are often associated. Abbreviations: ARB, antibiotic-resistant bacteria; ESBL, extended-spectrum beta-lactamase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The risks of transmission of antibiotic resistance from the environment to humans must be assessed based on bacteria, rather than on their resistance genes – in particular, bacteria that are able to colonize the human body (vectors), either transiently or as residents, and, simultaneously, harbour acquired ARGs (Figure 2). To colonize humans, vector bacteria must have the chance to get in contact with the human body, which means that these bacteria must share the same environment with humans, either occasionally or on a regular basis. Indeed, according to the information available, there are multiple lineages that include bacteria that can be found in environmental sources and in the human microbiome, suggesting that vectors may belong to a wide diversity of bacterial groups [20Vaz-Moreira I. et al.Bacterial diversity and antibiotic resistance in water habitats: Searching the links with the human microbiome.FEMS Microbiol. Rev. 2014; 38: 761-778Crossref PubMed Scopus (236) Google Scholar]. However, the impact that these vector bacteria will have on the human host depends on several different circumstances, such as the health condition of the host (e.g., the natural microbiome, integrity of primary infection barriers, or the immune status) or the capability of the vector to invade and proliferate in crucial host organs or tissues. If the vector is a bacterial strain that has a high fitness in the human body, has acquired ARG conferring resistance to antibiotics of different classes, and, eventually, is able to express specific virulence factors, then it can be considered a superbug (MDR pathogens, Figure 3). The threat posed by vectors will be aggravated by the capacity to disseminate ARGs to the host microbiome via free-DNA, plasmids, or phages by horizontal gene transfer (by the processes of transformation, conjugation, or transduction) and to promote genetic recombination, for instance through transposons or integrons. The worldwide antimicrobial resistance surveillance programs agree on the identification of a few genera or species that fall within the description of widely disseminated antibiotic resistance vectors – for example, MRSA, vancomycin-resistant enterococci (VRE), extended-spectrum beta-lactamase (ESBL), carbapenemaseproducing Enterobacteriaceae, as well as multidrug-resistant Pseudomonas aeruginosa and Clostridium difficile [22World Health OrganizationAntimicrobial Resistance: Global Report on Surveillance. WHO, 2014Google Scholar]. The discussion about the risk assessment of transmission of vectors from the environment to humans cannot simply rely on the model used for pathogens. This is because even if vectors can colonize the human body, they may lack crucial virulence genes and therefore will be unable to cause disease in a healthy host. A still unanswered question, important to unveil the role of environmental sources on the transmission of ARG to humans, refers to the process that mediates between colonization and infection. While colonizing bacteria can be present on skin or mucosa, they do not have the capacity to penetrate into tissues, but infectious bacteria are able to proliferate and invade the host, causing an immune reaction typical of infectious diseases. Two major modes of transmission of antibiotic resistance to humans can be hypothesized. The first mode is a long-term and eventually cumulative silent colonization, occurring in healthy individuals. Such silent colonization may be directly caused by the vector (resident colonization) or by commensal microbiota that received ARGs from transiently colonizing vectors (Figure 2). In both cases it can evolve to an infection, but only if the host reaches some degree of debilitation, for instance due to intense antibiotherapy or an immunocompromised state. In medical microbiology this situation has been designated as an endogenous infection. The silent colonization may explain some of the infections caused by ESBL-producing or carbapenemase-producing Enterobacteriaceae, MRSA, and VRE and may be a major mode of transmission of antibiotic-resistant bacteria from the environment to humans. The second mode of transmission of ARB to humans is through an acute colonization from external sources, with the immediate invasion of the host. This process, described as an exogenous infection, is very fast and occurs mainly in debilitated individuals or is due to invasive interventions, such as surgery or catheterization. This mode of transmission is frequent in health care units, and may involve a wide diversity of nosocomial agents [23European Centre for Disease Prevention and ControlPoint Prevalence Survey of Health Care Associated Infections and Antimicrobial Use in European Acute Care Hospitals. ECDC, Stockholm2013Google Scholar], some of which may have reached the hospital environment through other patients that suffered infections with ARB that had their origin in silent colonizations. Since colonization by vector bacteria may be silent for most humans, this may lead to an underestimation of the extent of transmission of ARB from the environment to humans. However, even though risk quantification is still a challenging issue, it can hardly be assumed that such a transmission is exempt from risks. The recognition that antibiotic resistance is nowadays a major public health concern calls for the urgent implementation of effective control measures. Nevertheless, it may be difficult to act if the most relevant sources, pathways, and modes of dissemination and critical vector doses are not known. These are essential aspects to assess the risks that the environmental resistome pose to human health and are discussed in the next section. Risk is a combined measure of the probability and severity of the harm that can result to somebody from contact with a certain hazard (i.e., anything that may cause harm). The assessment of risks requires a tiered approach that involves hazard identification, hazard characterization, exposure assessment, and risk characterization (Table 1). Risk assessment applied to public health issues is often challenging, and when it involves microorganisms and their genes the accuracy of risk estimation can be seriously impaired. However, important advances in this area were made in an attempt to improve the safety of food. The establishment of adequate guidelines for the assessment of risks of transmission of vectors from the environment to humans is still in its infancy. This assessment relies on several variables, most of which are still not quantifiable. Table 1 presents an overview of factors that are critical to assess the level of risk associated with a given environmental compartment as a potential source of transmission of ARB to humans. The subsections below discuss some of the parameters that would need to be determined to establish coherent risk assessment frameworks.Table 1Critical Factors That Determine the Level of Risk Associated with antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARG) in the EnvironmentaBased on the WHO framework used for food safety purposes (http://www.who.int/topics/risk_assessment/en/).Step of risk assessmentDescription of the stepCritical factorsHazard identification (A)Is a given environmental compartment a potential source of ARB and ARG?Identification of ARB and ARG belonging to the contaminant resistome, i.e., that result from human activitiesReceptor of humans or of other animals excretaWhether the environment harbours bacteria able to proliferate and horizontally transfer genesDoes the environmental compartment contact, directly or indirectly, humans (e.g., wastewater, agriculture soil irrigated with treated wastewater, urban wildlife, recreational areas etc.)Hazard characterization (B)The potential environmental source identified in A harbours ARB able to colonize humans, i.e., vectors?The qualitative and/or quantitative evaluation of the occurrence of ARB able to colonize humans and assessment of the potential adverse associated effectsWhether ARB are able to colonize, proliferate and invade tissues or organs of the human bodyWhether ARB can resist last-defence antibiotics or is multidrug-resistant (MDR)Exposure assessment (C)How probable is colonization by the vectors identified in B?The qualitative and/or quantitative evaluation of the probability that vectors from environmental sources can affect somebodyWhether the degree of exposure to the source identified in A is very highWhether bacteria identified in B are/have:highly communicable (by food, water, air, fomites, person-to-person, pets)a high capacity to colonize humansa very low infectious doseRisk characterization (D)How dangerous is it?D = A x B x CEstimation of the adverse effects likely to occur, based on the combination of hazard identification, hazard characterization, and exposure assessmenta Based on the WHO framework used for food safety purposes (http://www.who.int/topics/risk_assessment/en/). Open table in a new tab The detection and quantification of contaminant ARB and ARG in a specific environmental compartment is a proxy of its potential as a relevant source of antibiotic resistance transmission. This first screening can be supported by well-established methods to detect and characterize specific ARB and ARG in different matrices, such as quantitative PCR (qPCR) or metagenomics analyses. However, conceptually, a given environmental compartment will represent a risk of transmission to humans only if a given vector bacterium is present and in a quantity sufficient to be able to colonize a human. Depending on the type of vector, its presence may represent a risk, even if it is at an extremely low abundance. As a complement, the degree of exposure of humans to the specific environmental compartment needs to be used as a measure of the probability that vector bacteria will colonize humans (exposure x infective dose). Vector bacteria are the other crucial component for assessing risk. A reliable characterization may require the identification of the species or even strain level, which supports the prediction of some relevant characteristics. For instance, relevant characteristics for assessing risk include the recognized capacity of members of that species to colonize and infect the human body (a specific tissue or organ), the common modes of transmission to humans, and the portal of entry into the host (Figure 3). Traits related to the genetic background may also vary among different strains, including the potential to acquire and mobilize ARG (phage recombination sites, plasmids, integrons, or transposons) and the ARG pool already present. However, all of this information may be of limited value if the dose of vector bacteria that may be able to colonize or infect humans is not known. For pathogens, this amount is designated as the infective dose and corresponds to the number of bacterial cells that are required to infect a host [24Leggett H.C. et al.Mechanisms of pathogenesis, infective dose and virulence in human parasites.PLoS Pathog. 2012; 8: e1002512Crossref PubMed Scopus (86) Google Scholar]. In spite of the importance that these parameters have for reliable risks assessment models, the truth is that the infective dose and modes of transmission of most of the ARB of relevance (e.g., Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, or Enterococcus faecalis) are still unknown. Even when it is known for some bacterial groups, as for example for Staphylococcus aureus (>105 cells), for Mycobacterium tuberculosis (>10 cells), or for enterohemorrhagic strains of Escherichia coli (>10 cells) [25Schmid-Hempel P. Frank S.A. Pathogenesis, virulence, and infective dose.PLoS Pathog. 2007; 3: e147Crossref Scopus (156) Google Scholar, 26Fewtrell L. Kay D. Recreational water and infection: a review of recent findings.Curr. Environ. Health. Rep. 2015; 2: 85-94Crossref PubMed Scopus (85) Google Scholar], it is not known if the infective dose will be identical for antibiotic-resistant clones of the same species. Nonetheless, the values available suggest that very low doses of some vectors may be able cause harm to humans. In addition, considering the silent colonization model, which may be progressive and cumulative, the dose of vector bacteria that may start an ARB colonization in an individual may be indeed extremely low. Therefore, while the infective dose depends on the pathogenicity mechanisms used to evade the immune system [25Schmid-Hempel P. Frank S.A. Pathogenesis, virulence, and infective dose.PLoS Pathog. 2007; 3: e147Crossref Scopus (156) Google Scholar], in the case of ARB vectors, other factors such as the capacity to establish a resident colonization, or the high potential for horizontal gene transfer, may also be a determinant. Moreover, it must be emphasized that, often, people with the highest risk of an infection due to ARB have their primary and immune barriers compromised. In the face of these arguments, it is possible to conclude that, at least for some vectors, the dose that can endanger human health, immediately or in the long term, can be extremely low. Imagining that the infective dose for all clinically relevant vectors was known, the next question would be how, based on such a value, one could infer the risks associated with a given source of ARB. As a first step it would be necessary to gather quantitative data on the absolute abundance of the ARB of interest in the environmental source, as this would be the basis for estimating the exposure assessment value (Table 1). One of the shortcomings in this aspect is that the specificity and sensitivity of currently used methods (qPCR and metagenomics) may be insufficient to detect a specific vector. Eventually, the use of culture-based or selective probing (e.g., fluorescence in situ hybridization (FISH) techniques) may be required as complementary approaches. Another concern refers to the fact that, for these kinds of assessment, it is insufficient to have prevalence or relative abundance values, as, most of the time, it is expressed in metagenomics-based or quantitative PCR-based studies [2Christgen B. et al.Metagenomics shows that low-energy anaerobic-aerobic treatment reactors reduce antibiotic resistance gene levels from domestic wastewater.Environ. Sci. Technol. 2015; 49: 2577-2584Crossref PubMed Scopus (125) Google Scholar, 27Narciso-Da-Rocha C. et al.blaTEM and vanA as indicator genes of antibiotic resistance contamination in a hospital-urban wastewater treatment plant system.J. Glob. Antimicrob. Resist. 2014; 2: 309-315Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 28Munck C. et al.Limited dissemination of the wastewater treatment plant core resistome.Nat. Commun. 2015; 6: 8452Crossref PubMed Scopus (146) Google Scholar]. It is the absolute and not the relative abundance value that is required to estimate risks. Moreover, it may be important to bear in mind that any enumeration of microbial cells in a given environment is expressed as an average value. However, bacterial cells often occur in aggregates where they can reach clinically relevant doses, even if their average abundance in a given source is apparently low, below the infectious dose. These arguments suggest that the risks posed by environmental sources can be higher than apparently could be anticipated. According to the conceptual model proposed, the risks of transmission of antibiotic resistance from environmental sources to humans cannot be estimated based simply on the analysis of the antibiotic-resistance pool, using the current state-of-the-art screening techniques. A bacterial strain with a low infective dose, harboring a gene conferring resistance to a last-resource antibiotic, and with a high potential to multiply in the human body, will be among the ARB classified at the top level of risk. However, these harmful bacteria might not be detected using currently used methods, such as metagenomics or qPCR. Clearly, there is a non-direct proportionality between the abundance of antibiotic resistance and the associated risks. The non-detection of given ARB and ARG, based on state-of-the-art methodologies, cannot be interpreted as the absence of risks. ARB and ARG that are below the limits of detection and quantification can, in fact, represent a significant risk for human health. The high limits of detection and quantification of microorganisms and genes in environmental sources (latus sensus, e.g., water, food products, or clinical samples) is still a technical shortcoming of microbiology and it impairs reliable risk assessments (see Outstanding Questions). Therefore, at the moment, it is necessary to bring together different approaches (e.g., culture-dependent, metagenomics, and immunological) to assess the risks associated with antibiotic-resistant bacteria in the environment. While risks cannot be quantified accurately, it is necessary to observe the precautionary principle and not disregard the potential risks associated with some environmental sources and practices.Outstanding QuestionsIs the silent process of colonization, supposedly cumulative and progressive, the major mode of transmission of antibiotic-resistant bacteria from environmental sources to humans?Which are the main mechanisms that mediate between colonization and infection by antibiotic-resistant bacteria that have originated from environmental sources? Is it the transfer of antibiotic-resistance genes to commensal bacteria and/or virulent pathogens? The debilitation of the host defences (e.g., microbiome or primary barriers in the immune system)? Or, is it a combination of these and other events?How can currently available techniques and modelling tools be used to determine the doses of antibiotic-resistant bacteria of clinical relevance that are necessary to cause an infection?How can currently available techniques be improved, aiming at a significant lowering of the limits of detection and quantification of antibiotic-resistant bacteria and genes in environmental sources? Is the silent process of colonization, supposedly cumulative and progressive, the major mode of transmission of antibiotic-resistant bacteria from environmental sources to humans? Which are the main mechanisms that mediate between colonization and infection by antibiotic-resistant bacteria that have originated from environmental sources? Is it the transfer of antibiotic-resistance genes to commensal bacteria and/or virulent pathogens? The debilitation of the host defences (e.g., microbiome or primary barriers in the immune system)? Or, is it a combination of these and other events? How can currently available techniques and modelling tools be used to determine the doses of antibiotic-resistant bacteria of clinical relevance that are necessary to cause an infection? How can currently available techniques be improved, aiming at a significant lowering of the limits of detection and quantification of antibiotic-resistant bacteria and genes in environmental sources? This opinion paper is framed by the objectives of the research projects supported by National Funds from FCT – Fundação para a Ciência e a Tecnologia through projects UID/Multi/50016/2013-CBQF and WaterJPI/0001/2013 STARE – “Stopping Antibiotic Resistance Evolution”, European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 675530 ANSWER – “ANtibioticS and mobile resistance elements in WastEwater Reuse applications: risks and innovative solutions” and COST-European Cooperation in Science and Technology, to the COST Action ES1403: New and emerging challenges and opportunities in wastewater reuse (NEREUS). Disclaimer: The content of this article is the authors’ responsibility and neither the financing entities nor any person acting on their behalf is responsible for the use, which might be made of the information contained in it. 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