The role of antibiotics and antibiotic resistance in nature

Investigations of antibiotic resistance from an environmental prospective shed new light on a problem that was traditionally confined to a subset of clinically relevant antibiotic‐resistant bacterial pathogens. It is clear that the environmental microbiota, even in apparently antibiotic‐free environments, possess an enormous number and diversity of antibiotic resistance genes, some of which are very similar to the genes circulating in pathogenic microbiota. It is difficult to explain the role of antibiotics and antibiotic resistance in natural environments from an anthropocentric point of view, which is focused on clinical aspects such as the efficiency of antibiotics in clearing infections and pathogens that are resistant to antibiotic treatment. A broader overview of the role of antibiotics and antibiotic resistance in nature from the evolutionary and ecological prospective suggests that antibiotics have evolved as another way of intra‐ and inter‐domain communication in various ecosystems. This signalling by non‐clinical concentrations of antibiotics in the environment results in adaptive phenotypic and genotypic responses of microbiota and other members of the community. Understanding the complex picture of evolution and ecology of antibiotics and antibiotic resistance may help to understand the processes leading to the emergence and dissemination of antibiotic resistance and also help to control it, at least in relation to the newer antibiotics now entering clinical practice.     

Adaptation at different points along antibiotic concentration gradients

Antibiotic concentrations vary dramatically in the body and the environment. Hence, understanding the dynamics of resistance evolution along antibiotic concentration gradients is critical for predicting and slowing the emergence and spread of resistance. While it has been shown that increasing the concentration of an antibiotic slows resistance evolution, how adaptation to one antibiotic concentration correlates with fitness at other points along the gradient has not received much attention. Here, we selected populations of Escherichia coli at several points along a concentration gradient for three different antibiotics, asking how rapidly resistance evolved and whether populations became specialized to the antibiotic concentration they were selected on. Populations selected at higher concentrations evolved resistance more slowly but exhibited equal or higher fitness across the whole gradient. Populations selected at lower concentrations evolved resistance rapidly, but overall fitness in the presence of antibiotics was lower. However, these populations readily adapted to higher concentrations upon subsequent selection. Our results indicate that resistance management strategies must account not only for the rates of resistance evolution but also for the fitness of evolved strains.     

On the evolution of functional secondary metabolites (natural products)

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern biosynthetic pathways.     

Distribution and Quantification of Antibiotic Resistant Genes and Bacteria across Agricultural and Non-Agricultural Metagenomes

There is concern that antibiotic resistance can potentially be transferred from animals to humans through the food chain. The relationship between specific antibiotic resistant bacteria and the genes they carry remains to be described. Few details are known about the ecology of antibiotic resistant genes and bacteria in food production systems, or how antibiotic resistance genes in food animals compare to antibiotic resistance genes in other ecosystems. Here we report the distribution of antibiotic resistant genes in publicly available agricultural and non-agricultural metagenomic samples and identify which bacteria are likely to be carrying those genes. Antibiotic resistance, as coded for in the genes used in this study, is a process that was associated with all natural, agricultural, and human-impacted ecosystems examined, with between 0.7 to 4.4% of all classified genes in each habitat coding for resistance to antibiotic and toxic compounds (RATC). Agricultural, human, and coastal-marine metagenomes have characteristic distributions of antibiotic resistance genes, and different bacteria that carry the genes. There is a larger percentage of the total genome associated with antibiotic resistance in gastrointestinal-associated and agricultural metagenomes compared to marine and Antarctic samples. Since antibiotic resistance genes are a natural part of both human-impacted and pristine habitats, presence of these resistance genes in any specific habitat is therefore not sufficient to indicate or determine impact of anthropogenic antibiotic use. We recommend that baseline studies and control samples be taken in order to determine natural background levels of antibiotic resistant bacteria and/or antibiotic resistance genes when investigating the impacts of veterinary use of antibiotics on human health. We raise questions regarding whether the underlying biology of each type of bacteria contributes to the likelihood of transfer via the food chain.     

Evolution under low antibiotic concentrations: a risk for the selection of Pseudomonas aeruginosa multidrug-resistant mutants in nature

Antibiotic pollution of non-clinical environments might have a relevant impact on human health if resistant pathogens are selected. However, this potential risk is often overlooked, since drug concentrations in nature are usually below their minimal inhibitory concentrations (MICs). Albeit, antibiotic resistant bacteria can be selected even at sub-MIC concentrations, in a range known as the sub-MIC selective window. Using short-term evolution experiments, we have determined the sub-MIC selective windows of the opportunistic pathogen Pseudomonas aeruginosa for seven antibiotics of clinical relevance, finding the ones of quinolones to be the widest, and the ones of polymyxin B and imipenem, the narrowest. Clinically relevant multidrug-resistant mutants arose within the sub-MIC selective windows of most antibiotics tested, being some of these phenotypes mediated by efflux pumps' activity. The fact that the concentration of antibiotics reported in aquatic ecosystems – colonizable by P. aeruginosa – are, in occasions, higher than the ones that select multidrug-resistant mutants in our assays, has implications for understanding the role of different ecosystems and conditions in the emergence of antibiotic resistance from a One-Health perspective. Further, it reinforces the importance of procuring accurate information on the sub-MIC selective windows for drugs of clinical value in pathogens with environmental niches.     

Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems

Multidrug efflux pumps have emerged as relevant elements in the intrinsic and acquired antibiotic resistance of bacterial pathogens. In contrast with other antibiotic resistance genes that have been obtained by virulent bacteria through horizontal gene transfer, genes coding for multidrug efflux pumps are present in the chromosomes of all living organisms. In addition, these genes are highly conserved (all members of the same species contain the same efflux pumps) and their expression is tightly regulated. Together, these characteristics suggest that the main function of these systems is not resisting the antibiotics used in therapy and that they should have other roles relevant to the behavior of bacteria in their natural ecosystems. Among the potential roles, it has been demonstrated that efflux pumps are important for processes of detoxification of intracellular metabolites, bacterial virulence in both animal and plant hosts, cell homeostasis and intercellular signal trafficking.     

Overuse of high stability antibiotics and its consequences in public and environmental health

In this paper the ecological aspects of widespread antibiotic consumption are described. Many practitioners, veterinarians, breeders, farmers and analysts work on the assumption that a antibiotics undergo spontaneous degradation. It is well documented that the indiscriminate use of antibiotics has led to the water contamination, selection and dissemination of antibiotic-resistant organisms, alteration of fragile ecology of the microbial ecosystems. The damages caused by the overuse of antibiotics include hospital, waterborne and foodborne infections by resistant bacteria, enteropathy (irritable bowel syndrome, antibiotic-associated diarrhea etc.), drug hypersensitivity, biosphere alteration, human and animal growth promotion, destruction of fragile interspecific competition in microbial ecosystems etc. The consequences of heavy antibiotic use for public and environmental health are difficult to assess: utilization of antibiotics from the environment and reduction of irrational use is the highest priority issue. This purpose may be accomplished by bioremediation, use of probenecid for antibiotic dosage reduction and by adoption of hospital infections methodology for control resistance in natural ecosystems.     

畜禽粪便中抗生素抗性基因的分布特征及消减技术研究进展

随着集约化畜禽养殖业的不断发展,兽用抗生素的长期使用导致畜禽粪便抗生素抗性基因污染日益严重,对生态环境和人类健康造成严重危害。如何有效消减畜禽粪便中的抗生素抗性基因成为当前研究热点。本文系统总结了畜禽粪便中抗生素抗性基因的产生途径、分布和影响因素,并阐述了好氧堆肥、厌氧消化及其强化工艺消减畜禽粪便抗生素抗性基因的研究进展,根据现有工艺研究存在的问题展望了今后的重点研究方向,为畜禽粪便中抗生素抗性基因的消减提供理论基础和技术支撑。     

气溶胶中抗生素抗性基因研究进展

抗生素的不合理使用导致细菌耐药问题日趋严峻,给人类健康造成巨大威胁。学者们对抗生素抗性菌和抗生素抗性基因(antibiotic resistance genes, ARGs)在多种环境介质中的环境行为开展了大量研究。气溶胶作为ARGs的潜在储存库,是抗生素抗性基因在环境中的重要传播途径之一。目前缺乏对其来源、传播、人类接触和健康风险系统性的梳理。本文针对人类生活功能场所、养殖场、城市污水处理厂和医院等4类气溶胶研究的典型场所,重点综述了上述4类典型场所中气溶胶ARGs的来源、传播途径及对人体的暴露和对健康的危害,为气溶胶中ARGs的预防和控制提供参考。     

Determining the limits of the evolutionary potential of an antibiotic resistance gene

The AAC(6') enzymes inactivate aminoglycoside antibiotics by acetylating their substrates at the 6' position. Based on functional similarity and size similarity, the AAC(6') enzymes have been considered to be members of a single family. Our phylogenetic analysis shows that the AAC(6') enzymes instead belong to three unrelated families that we now designate as [A], [B], and [C] and that aminoglycoside acetylation at the 6' position has evolved independently at least three times. AAC(6')-Iaa is a typical member of the [A] family in that it acetylates tobramycin, kanamycin, and amikacin effectively but acetylates gentamicin ineffectively. The potential of the aac(6')-Iaa gene to increase resistance to tobramycin, kanamycin, or amikacin or to acquire resistance to gentamicin was assessed by in vitro evolution. Libraries of PCR mutagenized alleles were screened for increased resistance to tobramycin, kanamycin, and amikacin, but no isolates that conferred more resistance than the wild-type gene were recovered. The library sizes were sufficient to conclude with 99.9% confidence that no single amino acid substitution or combination of two amino acid substitutions in aac(6')-Iaa is capable of increasing resistance to the antibiotics used. It is therefore very unlikely that aac(6')-Iaa of S. typhimurium LT2 has the potential to evolve increased aminoglycoside resistance in nature. The practical implications of being able to determine the evolutionary limits for other antibiotic resistance genes are discussed.     

Ecogenetics of antibiotic resistance in Listeria monocytogenes

The acquisition process of antibiotic resistance in an otherwise susceptible organism is shaped by the ecology of the species. Unlike other relevant human pathogens, Listeria monocytogenes has maintained a high rate of susceptibility to the antibiotics used for decades to treat human and animal infections. However, L. monocytogenes can acquire antibiotic resistance genes from other organisms’ plasmids and conjugative transposons. Ecological factors could account for its susceptibility. L. monocytogenes is ubiquitous in nature, most frequently including reservoirs unexposed to antibiotics, including intracellular sanctuaries. L. monocytogenes has a remarkably closed genome, reflecting limited community interactions, small population sizes and high niche specialization. The L. monocytogenes species is divided into variants that are specialized in small specific niches, which reduces the possibility of coexistence with potential donors of antibiotic resistance. Interactions with potential donors are also hampered by interspecies antagonism. However, occasional increases in population sizes (and thus the possibility of acquiring antibiotic resistance) can derive from selection of the species based on intrinsic or acquired resistance to antibiotics, biocides, heavy metals or by a natural tolerance to extreme conditions. High-quality surveillance of the emergence of resistance to the key drugs used in primary therapy is mandatory.     

Escherichia coli populations in unpredictably fluctuating environments evolve to face novel stresses through enhanced efflux activity

There is considerable understanding about how laboratory populations respond to predictable (constant or deteriorating environment) selection for single environmental variables such as temperature or pH. However, such insights may not apply when selection environments comprise multiple variables that fluctuate unpredictably, as is common in nature. To address this issue, we grew replicate laboratory populations of Escherichia coli in nutrient broth whose pH and concentrations of salt (NaCl) and hydrogen peroxide (H₂O₂) were randomly changed daily. After ~170 generations, the fitness of the selected populations had not increased in any of the three selection environments. However, these selected populations had significantly greater fitness in four novel environments which have no known fitness‐correlation with tolerance to pH, NaCl or H₂O₂. Interestingly, contrary to expectations, hypermutators did not evolve. Instead, the selected populations evolved an increased ability for energy‐dependent efflux activity that might enable them to throw out toxins, including antibiotics, from the cell at a faster rate. This provides an alternate mechanism for how evolvability can evolve in bacteria and potentially lead to broad‐spectrum antibiotic resistance, even in the absence of prior antibiotic exposure. Given that environmental variability is increasing in nature, this might have serious consequences for public health.     

The neglected intrinsic resistome of bacterial pathogens

Bacteria with intrinsic resistance to antibiotics are a worrisome health problem. It is widely believed that intrinsic antibiotic resistance of bacterial pathogens is mainly the consequence of cellular impermeability and activity of efflux pumps. However, the analysis of transposon-tagged Pseudomonas aeruginosa mutants presented in this article shows that this phenotype emerges from the action of numerous proteins from all functional categories. Mutations in some genes make P. aeruginosa more susceptible to antibiotics and thereby represent new targets. Mutations in other genes make P. aeruginosa more resistant and therefore define novel mechanisms for mutation-driven acquisition of antibiotic resistance, opening a new research field based in the prediction of resistance before it emerges in clinical environments. Antibiotics are not just weapons against bacterial competitors, but also natural signalling molecules. Our results demonstrate that antibiotic resistance genes are not merely protective shields and offer a more comprehensive view of the role of antibiotic resistance genes in the clinic and in nature.     

肠杆菌科细菌耐药基因表达的遗传和环境调控

细菌耐药性是21世纪国际关注的重要问题,也是全球面临的重大挑战。肠杆菌科细菌是医院感染的重要病原菌之一。近年来,随着抗生素的大量使用,多种肠杆菌科耐药菌,尤其是多重耐药肠杆菌开始大量出现,对人类健康形成了日益严重的威胁。细菌可以通过耐药基因突变或水平转移的方式获得耐药性,通常情况下,可以通过已知的耐药机制预测相应的耐药表型。然而,最近有研究表明,遗传背景和环境因素能够影响耐药基因的表达,给定的基因型并不一定总是产生预期的耐药表型。这种基因型-表型分离的现象极大程度上限制了从遗传学角度预测耐药表型的能力。文中结合最新文献,从遗传背景和环境条件两个方面探讨了多种肠杆菌科细菌耐药基因的表达调控机制,以期为遗传学预测耐药表型以及临床指导用药提供一定的支持。     

Bacterial evolution of antibiotic hypersensitivity

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large‐scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole‐genome sequencing of laboratory‐evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton‐motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF‐dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade‐offs under antibiotic selection.     

畜禽养殖废物中抗生素和重金属抗性基因的产生机制和控制方法研究进展

动物饲料中常混有抗生素和重金属,导致外排的动物粪便中携带有抗生素和重金属,引发细菌产生耐药性和重金属抗性,继而产生抗生素抗性基因和重金属抗性基因。抗生素和重金属抗性基因污染已成为威胁人类身体健康及破坏生态环境的重大问题。本文从细菌进化的角度,明确了细菌的抗生素和重金属长期进化试验对抗性机制研究的重要性;抗生素抗性基因与重金属抗性基因间存在复杂的协同选择抗性,两者间相互影响,共同决定着细菌环境行为;抗性基因的水平转移增加了细菌在环境中的可变性,可移动遗传元件在抗性基因水平转移中发挥着重要作用。在抗性基因污染控制方面,高级氧化技术具有很好的抗性基因去除效果,尤其是UV/TiO₂氧化技术,能使抗生素抗性基因丰度减少4.7～5.8 log,减少率大于99.99%。其他的控制策略,如抗生素替代品博落回提取物以及噬菌体与抗生素结合使用,对于抗性基因的控制也具有重要意义。     

Bacteriophages as antibiotic resistance genes carriers in agro-food systems

Antibiotic resistance genes (ARGs) are a global health concern. Antibiotic resistance occurs naturally, but misuse of antibiotics in humans and animals is accelerating the process of antibiotic resistance emergency, which has been aggravated by exposure to molecules of antibiotics present in clinical and agricultural settings and the engagement of many countries in water reuse especially in Middle East and North Africa region. Bacteriophages have the potential to be significant actors in ARGs transmission through the transduction process. These viruses have been detected along with ARGs in non impacted habitats and in anthropogenic impacted environments like wastewater, reclaimed water and manure amended soil as well as minimally processed food and ready to eat vegetables. The ubiquity of bacteriophages and their persistence in the environment raises concern about their involvement in ARGs transmission among different biomes and the generation of pathogenic-resistant bacteria that pose a great threat to human health. The aim of this review is to give an overview of the potential role of bacteriophages in the dissemination and the transfer of ARGs to pathogens in food production and processing and the consequent contribution to antibiotic resistance transmission through faecal oral route carrying ARGs to our dishes.     

土壤中抗生素的环境风险及污染土壤的生物修复技术

抗生素的广泛使用导致其在环境中普遍存在,所引发的抗性基因问题已对全球公共卫生构成重大威胁。土壤是环境中抗生素的重要汇,抗生素暴露会对土壤生物带来危害,甚至会间接对人体健康造成潜在风险,因此需采取有效手段修复抗生素污染的土壤。文中综述了抗生素对土壤植物表型生长指标、土壤动物生理特征及群落分布、微生物群落组成与功能的影响,以及抗生素抗性基因在土壤生物间的传播风险等;总结了利用耐受土壤植物、动物、微生物以及其互作关系修复抗生素污染土壤的潜力与前景,指出了已有土壤中抗生素环境风险和生物修复研究中尚存在的问题,展望了未来的研究方向。     

Metabolic constraints on the evolution of antibiotic resistance

Despite our continuous improvement in understanding antibiotic resistance, the interplay between natural selection of resistance mutations and the environment remains unclear. To investigate the role of bacterial metabolism in constraining the evolution of antibiotic resistance, we evolved Escherichia coli growing on glycolytic or gluconeogenic carbon sources to the selective pressure of three different antibiotics. Profiling more than 500 intracellular and extracellular putative metabolites in 190 evolved populations revealed that carbon and energy metabolism strongly constrained the evolutionary trajectories, both in terms of speed and mode of resistance acquisition. To interpret and explore the space of metabolome changes, we developed a novel constraint‐based modeling approach using the concept of shadow prices. This analysis, together with genome resequencing of resistant populations, identified condition‐dependent compensatory mechanisms of antibiotic resistance, such as the shift from respiratory to fermentative metabolism of glucose upon overexpression of efflux pumps. Moreover, metabolome‐based predictions revealed emerging weaknesses in resistant strains, such as the hypersensitivity to fosfomycin of ampicillin‐resistant strains. Overall, resolving metabolic adaptation throughout antibiotic‐driven evolutionary trajectories opens new perspectives in the fight against emerging antibiotic resistance.     

细菌耐药影响肠道菌群及其宿主免疫调控

抗生素在养殖业、医疗业及制药业的广泛应用导致环境中的细菌耐药性日益严重,环境中的抗生素及耐药细菌一旦进入人体肠道,将破坏肠道菌群稳态,对人体健康造成威胁,而残存于饮食中的环境污染物则加剧了细菌耐药造成的人体健康影响。文中在总结大量文献的基础上,阐述了细菌耐药对人体和动物肠道菌群的影响机制及其相关的机体免疫调控,以环境中影响人体肠道菌群获得耐药性的来源作为切入点,阐述抗生素和耐药细菌进入人体肠道后对人体肠道菌群结构和耐药基因组成的影响,以及与人体免疫和免疫调节相关疾病之间的相关机制,并对今后的研究方向进行了展望。