Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance

The emergence of multi-resistant pathogenic bacteria is a worldwide health issue. Recently, clinical variants of a single antibiotic-modifying acetyltransferase, AAC(6')-Ib-a variant of aminoglycoside 6'-N-acetyltransferase-have been identified that confer extended resistance to most aminoglycosides and, more surprisingly, to structurally unrelated fluoroquinolones. The corresponding gene is carried by mobile genetic elements and is present in most multi-resistant pathogenic strains, hence making it a serious threat to current therapies. Here, we report the crystal structures of both narrow- and broad-spectrum resistance variants of this enzyme, which reveal the structural basis for the emergence of extended resistance. The active site shows an important plasticity and has adapted to new substrates by a large-scale gaping process. We have also obtained co-crystals with both substrates, and with a simple transition state analogue, which provides new clues for the design of inhibitors of this resistance mechanism.     

抗生素抗性基因在环境中的传播扩散及抗性研究方法

抗生素在医药、畜牧和水产养殖业的大量使用造成了环境中抗性耐药菌和抗性基因日益增加,抗生素抗性基因作为一种新型环境污染物引起人们的广泛关注.本文综述了近年来国内外有关抗生素抗性基因的研究进展,其在水、土壤、空气等环境介质中和动,植物体内的传播扩散,以及开展环境中抗生素抗性基因研究的必要性,重点介绍了有关抗生素抗性（包括抗性细菌和抗性基因）的研究方法,指出抗性基因研究中存在的问题,并对未来的相关研究进行了展望.     

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。     

Predicting the emergence of antibiotic resistance by directed evolution and structural analysis

Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.     

Heavy metal-induced selection and proliferation of antibiotic resistance: A review

Antibiotic resistance is recognized as a global threat to public health. The selection and evolution of antibiotic resistance in clinical pathogens were believed to be majorly driven by the imprudent use of antibiotics. However, concerns regarding the same, through selection pressure by a multitude of other antimicrobial agents, such as heavy metals, are also growing. Heavy metal contamination co-selects antibiotic and metal resistance through numerous mechanisms, such as co-resistance and cross-resistance. Here, we have reviewed the role of heavy metals as antimicrobial resistance driving agents and the underlying concept and mechanisms of co-selection, while also highlighting the scarcity of studies explicitly inspecting the process of co-selection in clinical settings. Prospective strategies to manage heavy metal-induced antibiotic resistance have also been deliberated, underlining the need to find specific inhibitors so that alternate medicinal combinations can be added to the existing therapeutic armamentarium.     

Characteristics of phenotypic antibiotic resistance of Helicobacter pylori and its correlation with genotypic antibiotic resistance: A retrospective study in Ningxia

Background

Geographic differences exist in the antibiotic resistance patterns of Helicobacter pylori. Personalized treatment regimens based on local or individual resistance data are essential. We evaluated the current status of H. pylori resistance in Ningxia, analyzed resistance-related factors, and assessed the concordance of phenotypic and genotypic resistance.

Methods

Strains were isolated from the gastric mucosa of patients infected with H. pylori in Ningxia and relevant clinical information was collected. Phenotypic antibiotic susceptibility assays (Kirby–Bauer disk diffusion) and antibiotic resistance gene detection (Sanger sequencing) were performed.

Results

We isolated 1955 H. pylori strains. The resistance rates of H. pylori to amoxicillin, levofloxacin, clarithromycin, and metronidazole were 0.9%, 42.4%, 40.4%, and 94.2%, respectively. Only five tetracycline-resistant and one furazolidone-resistant strain were identified. Overall, 3.3% of the strains were sensitive to all six antibiotics. Multidrug-resistant strains accounted for 22.9%, of which less than 20% were from Wuzhong. Strains isolated from women and patients with nonulcerative disease had higher rates of resistance to levofloxacin and clarithromycin. Higher rates of resistance to metronidazole, levofloxacin, and clarithromycin were observed in the older age group than in the younger age group. The kappa coefficients of phenotypic resistance and genotypic resistance for levofloxacin and clarithromycin were 0.830 and 0.809, respectively, whereas the remaining antibiotics showed poor agreement.

Conclusion

H. pylori antibiotic resistance is severe in Ningxia. Therefore, furazolidone, amoxicillin, and tetracycline are better choices for the empirical therapy of H. pylori infection in this region. Host sex, age, and the presence of ulcerative diseases may affect antibiotic resistance of the bacteria. Personalized therapy based on genetic testing for levofloxacin and clarithromycin resistance may be a future direction for the eradication therapy of H. pylori infection in Ningxia.     

不同环境介质中抗生素耐药性的检测方法研究进展

抗生素在医疗和畜禽养殖业的大量使用增加了环境中抗生素抗性微生物(ARB)和抗性基因(ARGs)的丰度与多样性,加速了抗生素耐药性在环境中的传播,给人类公共健康造成潜在威胁。但目前对于环境中耐药性的污染现状缺少足够的信息,相关研究方法亟待优化和完善。本文通过综述环境中抗生素耐药性的国内外研究现状,探讨了不同环境(水、土壤、空气等)样品的采集方法以及耐药性的检测方法——传统微生物培养法和分子生物学方法(如定性与定量PCR、DNA杂交及微阵列技术、宏基因组学方法等),旨在为多环境介质中抗生素耐药性的研究提供科学依据和技术支持。     

生物炭对土壤中抗生素抗性基因的阻控潜力及机制研究进展

土壤中抗生素抗性基因(ARGs)污染是全世界面临的重大环境和健康挑战,开发有效技术以减少其负面影响对维护土壤和人类健康至关重要。生物炭具有高碳含量、大表面积、良好的吸附性能和经济优势,可能是一种非常合适的阻控材料。其对ARGs的阻控作用可能归因于以下3种机制: 1) 吸附某些污染物,如抗生素和重金属,减弱ARGs的共选择性压力;2) 通过改变土壤理化特性影响微生物种群结构,从而限制细菌之间ARGs的水平转移;3) 通过吸附或破坏质粒、转座子、整合子等水平转移载体,直接减弱基因水平转移能力。但生物炭对ARGs的阻控效果取决于生物炭的物料来源、热解工艺和添加水平等。此外,生物炭的老化可能会降低其阻控ARGs的效果。生物炭的内源性污染物,如多环芳烃和重金属,也可能导致环境中特定抗生素抗性细菌的富集或诱导水平基因转移。在后续研究中,应根据土壤环境选择合适的生物炭种类,并采取生物炭老化控制措施,以进一步提高生物炭对ARGs的阻控作用。     

抗生素耐药性的研究进展与控制策略

抗生素是治疗细菌感染的有效药物,然而抗生素在人类医学及农业生产中的大规模使用催生了细菌耐药性在环境中的快速扩散和传播,特别是多种抗生素的联合使用更是促进了多重耐药性的产生,严重威胁着人类和动物健康及食品与环境安全,相关问题已经引起人们的警觉。因此新研究主要集中在以下几方面:利用组学及合成生物学等方法挖掘并合成新型抗生素;利用高通量技术等系统分析环境中耐药菌及耐药基因新的传播途径及产生的新耐药机制;减抗、替抗及控制耐药基因的策略及其相关工艺。因此,在全面认识耐药基因在环境中传播规律的基础上,如何绿色高效地切断传播途径仍是目前研究的热点。基于此,本文在细菌水平上阐述了抗生素的研发历程、耐药性的发展及控制策略,从而为有效遏制细菌耐药性的发展提供思路。     

An aminoglycoside microarray platform for directly monitoring and studying antibiotic resistance

Antibiotic resistance is a major threat to human health. Since resistance to the aminoglycoside class of antibiotics is most commonly caused by enzymatic modification, we developed a high-throughput microarray platform for directly assaying resistance enzyme activity on aminoglycosides. After modification, the array can be hybridized with the therapeutic target, a bacterial rRNA A-site mimic, to study the effect that modification has on binding. Such studies will help identify important factors that contribute to high-affinity recognition of therapeutic targets and low-affinity recognition of and modification by resistance enzymes. This platform may also be useful for screening chemical libraries to discover new antibiotics that evade resistance.     

土壤噬菌体及其介导的抗生素抗性基因水平转移研究进展

土壤中抗生素耐药性的扩散对全球的公共卫生和食品安全造成威胁,严重挑战人类感染类疾病的预防与治疗.噬菌体介导的抗生素抗性基因(ARGs)的水平转移是环境中抗性基因扩散的重要机制.但是,噬菌体对土壤环境中抗性基因传播的贡献尚未见报道.本文综述了土壤环境中噬菌体的分布特征与影响因子,总结了纯化和富集土壤噬菌体的主要研究方法;...     

抗生素抗性基因检测方法发展历程及应用现状

耐药性的传播已引起全球广泛关注,抗生素抗性基因(antibiotic resistance genes, ARGs)检测技术的开发是研究ARGs在环境-动植物-人群中迁移传播的关键。本文梳理了现有核酸检测技术的发展历程及首次应用于ARGs检测的时间节点,并从检测原理、应用优缺点、开发潜力等方面对各技术进行分类综述。在此基础上提出以等温扩增结合CRISPR/Cas技术为核心的ARGs原位快速检测技术的开发及应用前景展望。本综述旨在回顾各技术发展历程的基础上,为ARGs新检测技术的开发及应用提供参考,为耐药性传播的研究及控制提供技术支撑。     

A global view of antibiotic resistance

Antibiotic resistance is one of the few examples of evolution that can be addressed experimentally. The present review analyses this resistance, focusing on the networks that regulate its acquisition and its effect on bacterial physiology. It is widely accepted that antibiotics and antibiotic resistance genes play fundamental ecological roles – as weapons and shields, respectively – in shaping the structures of microbial communities. Although this Darwinian view of the role of antibiotics is still valid, recent work indicates that antibiotics and resistance mechanisms may play other ecological roles and strongly influence bacterial physiology. The expression of antibiotic resistance determinants must therefore be tightly regulated and their activity forms part of global metabolic networks. In addition, certain bacterial modes of life can trigger transient phenotypic antibiotic resistance under some circumstances. Understanding resistance thus requires the analysis of the regulatory networks controlling bacterial evolvability, the physiological webs affected and the metabolic rewiring it incurs.     

Antibiotic resistance in bacteria and its future for novel antibiotic development

Since the first introduction of the sulfa drugs and penicillin into clinical use, large numbers of antibiotics have been developed and hence contributed to human health. But extensive use of antibiotics has raised a serious public health problem due to multiantibiotic resistant bacterial pathogens that inevitably develop resistance to every new drug launched in the clinic. Consequently, there is a pressing need to develop new antibiotics to keep pace with bacterial resistance. Recent advances in microbial genomics and X-ray crystallography provide opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics respectively. To prevent and control infectious diseases caused by multiantibiotic resistant bacteria, we need to understand more about the molecular aspects of the pathogens' physiology and to pursue ways to prolong the life of precious antibiotics.     

Real-time PCR methods for monitoring antimalarial drug resistance

Drug-resistant Plasmodium falciparum is a challenge to malaria control programs. Policy makers currently depend on in vivo (and, sometimes, in vitro) resistance testing to set treatment guidelines. Molecular markers such as mutations in dhfr, dhps, pfcrt and pfmdr1 represent potential surveillance tools. In this article, we describe newer high-throughput methods for detecting these molecular markers. One method, 5' nuclease real-time polymerase chain reaction, is discussed in detail.     

生态系统完整性内涵及评价方法研究综述

生态系统完整性是资源管理和环境保护中一个重要的概念.它主要反映生态系统在外来干扰下维持自然状态、稳定性和自组织能力的程度.评价生态系统完整性对于保护敏感自然生态系统免受人类干扰的影响有着重要的意义.耗散结构理论表明,外界压力和反映系统自组织能力的生物、物理、化学完整性和生态系统功能等对生态系统的完整性有良好的指示作用.本文综述了水生生态系统和陆地生态系统完整性及其所承受的压力评价指标,并探讨了优先评价指标的筛选及综合评价方法,最后根据存在的一些问题提出今后的研究展望.