Antibiotics and the resistant microbiome

Since the discovery and clinical application of antibiotics, pathogens and the human microbiota have faced a near continuous exposure to these selective agents. A well-established consequence of this exposure is the evolution of multidrug-resistant pathogens, which can become virtually untreatable. Less appreciated are the concomitant changes in the human microbiome in response to these assaults and their contribution to clinical resistance problems. Studies have shown that pervasive changes to the human microbiota result from antibiotic treatment and that resistant strains can persist for years. Additionally, culture-independent functional characterization of the resistance genes from the microbiome has demonstrated a close evolutionary relationship between resistance genes in the microbiome and in pathogens. Application of these techniques and novel cultivation methods are expected to significantly expand our understanding of the interplay between antibiotics and the microbiome.     

Crossroads of Antibiotic Resistance and Biosynthesis

The biosynthesis of antibiotics and self-protection mechanisms employed by antibiotic producers are an integral part of the growing antibiotic resistance threat. The origins of clinically relevant antibiotic resistance genes found in human pathogens have been traced to ancient microbial producers of antibiotics in natural environments. Widespread and frequent antibiotic use amplifies environmental pools of antibiotic resistance genes and increases the likelihood for the selection of a resistance event in human pathogens. This perspective will provide an overview of the origins of antibiotic resistance to highlight the crossroads of antibiotic biosynthesis and producer self-protection that result in clinically relevant resistance mechanisms. Some case studies of synergistic antibiotic combinations, adjuvants, and hybrid antibiotics will also be presented to show how native antibiotic producers manage the emergence of antibiotic resistance.     

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.     

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.     

Occurrence and patterns of antibiotic resistance in vertebrates off the Northeastern United States coast

The prevalence of antibiotic-resistant bacteria in the marine environment is a growing concern, but the degree to which marine mammals, seabirds and fish harbor these organisms is not well documented. This project sought to identify the occurrence and patterns of antibiotic resistance in bacteria isolated from vertebrates of coastal waters in the northeastern United States. Four hundred and seventy-two isolates of clinical interest were tested for resistance to a suite of 16 antibiotics. Fifty-eight percent were resistant to at least one antibiotic, while 43% were resistant to multiple antibiotics. A multiple antibiotic resistance index value ≥0.2 was observed in 38% of the resistant pathogens, suggesting exposure of the animals to bacteria from significantly contaminated sites. Groups of antibiotics were identified for which bacterial resistance commonly co-occurred. Antibiotic resistance was more widespread in bacteria isolated from seabirds than marine mammals, and was more widespread in stranded or bycaught marine mammals than live marine mammals. Structuring of resistance patterns based on sample type (live/stranded/bycaught) but not animal group (mammal/bird/fish) was observed. These data indicate that antibiotic resistance is widespread in marine vertebrates, and they may be important reservoirs of antibiotic-resistant bacteria in the marine environment.     

Microbial environments confound antibiotic efficacy

The increasing prevalence of bacteria that are insensitive to our current antibiotics emphasizes the need for new antimicrobial therapies. Conventional approaches to antibacterial development that are based on the inhibition of essential processes seem to have reached the point of diminishing returns. The discovery that diverse antibiotics stimulate a common oxidative cell-death pathway represents a fundamental shift in our understanding of bactericidal antibiotic modes of action. A number of studies, as discussed above, also provide hints about how intra- and extracellular metabolism can enable antibiotic resistance and tolerance. We have, nonetheless, just begun to understand the repertoire of tactics that bacteria use to evade antibiotics. Biosynthetic pathways for natural antibiotics are ancient, and numerous mechanisms for antibiotic resistance and tolerance are likely to have evolved over the past few million years. Unraveling these mechanisms will require concerted efforts by chemical biologists, microbiologists and clinicians. These efforts will benefit from the use of metabolic models and other network-biology approaches to guide investigation of processes that modulate antibiotic susceptibility. Importantly, by helping to identify common points of vulnerability as well as key differences between pathogens, these models may lead to the development of effective adjuvants, novel antibiotics and new antimicrobial strategies. There is also a crucial need to better understand how bacteria within a population cooperate to overcome antibiotic treatments. Such investigations may benefit from the use of novel chemical probes and experimental techniques to interrogate the physiology and functional dynamics of natural microbial communities. Insights gained from these studies will augment metagenomic models that can be used to identify biomolecules responsible for these cooperative strategies. Leveraging chemical biology methodologies and systems-biology approaches for further studies of microbial environments may reveal a wealth of untapped targets for the development of novel compounds to counter the growing threat of resistant and tolerant bacterial infections.     

Characterization of a Multiresistant Mosaic Plasmid from a Fish Farm Sediment Exiguobacterium sp. Isolate Reveals Aggregation of Functional Clinic-Associated Antibiotic Resistance Genes

The genus Exiguobacterium can adapt readily to, and survive in, diverse environments. Our study demonstrated that Exiguobacterium sp. strain S3-2, isolated from marine sediment, is resistant to five antibiotics. The plasmid pMC1 in this strain carries seven putative resistance genes. We functionally characterized these resistance genes in Escherichia coli, and genes encoding dihydrofolate reductase and macrolide phosphotransferase were considered novel resistance genes based on their low similarities to known resistance genes. The plasmid G+C content distribution was highly heterogeneous. Only the G+C content of one block, which shared significant similarity with a plasmid from Exiguobacterium arabatum, fit well with the mean G+C content of the host. The remainder of the plasmid was composed of mobile elements with a markedly lower G+C ratio than the host. Interestingly, five mobile elements located on pMC1 showed significant similarities to sequences found in pathogens. Our data provided an example of the link between resistance genes in strains from the environment and the clinic and revealed the aggregation of antibiotic resistance genes in bacteria isolated from fish farms.     

New strategies for combating multidrug-resistant bacteria

Antibiotic resistance is a problem that continues to challenge the healthcare sector. In particular, multidrug resistance is now common in familiar pathogens such as Staphylococcus aureus and Mycobacterium tuberculosis, as well as emerging pathogens such as Acinetobacter baumannii. New antibiotics and new therapeutic strategies are needed to address this challenge. Advances in identifying new sources of antibiotic natural products and expanding antibiotic chemical diversity are providing chemical leads for new drugs. Inhibitors of resistance mechanisms and microbial virulence are orthogonal strategies that are also generating new chemicals that can extend the life of existing antibiotics. This new chemistry, coupled with a growing understanding of the mechanisms, origins and distribution of antibiotic resistance, position us to tackle the challenges of antibiotic resistance in the 21st century.     

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.     

Heteroresistance to beta-lactam antibiotics may often be a stage in the progression to antibiotic resistance

Antibiotic resistance is a growing crisis that threatens many aspects of modern healthcare. Dogma is that resistance often develops due to acquisition of a resistance gene or mutation and that when this occurs, all the cells in the bacterial population are phenotypically resistant. In contrast, heteroresistance (HR) is a form of antibiotic resistance where only a subset of cells within a bacterial population are resistant to a given drug. These resistant cells can rapidly replicate in the presence of the antibiotic and cause treatment failures. If and how HR and resistance are related is unclear. Using carbapenem-resistant Enterobacterales (CRE), we provide evidence that HR to beta-lactams develops over years of antibiotic usage and that it is gradually supplanted by resistance. This suggests the possibility that HR may often develop before resistance and frequently be a stage in its progression, potentially representing a major shift in our understanding of the evolution of antibiotic resistance.

A study of heteroresistance to broad range of beta-lactam antibiotics in clinical isolates of E. coli suggests that it may be an intermediate stage in the development of full antibiotic resistance, representing a shift in our understanding of the evolution of antibiotic resistance.     

伤口分泌物的病原菌分布及耐药性分析

目的调查福建省龙岩市第二医院伤口分泌物病原菌的分布及耐药情况,为合理应用抗生素提供依据。方法收集2012年1月至2013年5月患者伤口分泌物标本,采用常规方法进行分离培养,用VITEK-2 Compact全自动微生物分析仪系统进行鉴定及药敏分析。结果送检503份标本,培养阳性272份,阳性率为54. 1% ;病原菌检出343株,其中革兰阴性菌189株占55. 1%,革兰阳性菌151株占44.0%,真菌3株占0.9% ;前5位的病原菌分别为金黄色葡萄球菌、凝固酶阴性葡萄球菌、铜绿假单胞菌、大肠埃希菌、鲍曼不动杆菌。耐甲氧西林金黄色葡萄球菌（MRSA）和耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的检出率分别是30.4%、82. 1%。粪肠球菌中耐高浓度氨基糖苷类肠球菌（HLAR）的检出率为55%。革兰阳性菌对万古霉素、替加环素、利奈唑胺未出现耐药菌株。铜绿假单胞菌、肠杆菌科细菌对亚胺培南的耐药率分别为5. 6%、0。大肠埃希菌中超广谱P-内酰胺酶（ESBL）的检出率是42.9%。鲍曼不动杆菌对头孢哌酮/舒巴坦的耐药率最低为25.0%,对其他抗菌药物耐药严重,多数抗菌药物的耐药率均〉45%。结论伤口感染的主要病原菌是革兰阴性菌,多重耐药菌株比例较高,临床应根据药敏结果合理选用抗生素,减少新的耐药菌株出现。     

A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria

The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic-contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi-drug resistance among bacteria living under an extreme antibiotic selection pressure.     

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.     

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.     

Determinants of resistance to chlortetracycline and other antibiotics in chlortetracycline-producing strain of Streptomyces aureofaciens

Data are presented on resistance of Streptomyces aureofaciens strain TB-633 FU--the producer of chlortetracycline (CTC) to autogenous antibiotics and a number of other antibiotics. It is demonstrated that resistance to CTC is specified by ctr genes of constitutive expression as well as by inducible genes. CTC and ethidium bromide may serve as efficient inductors of inducible ctr genes. The induction process is accompanied by increase in antibiotic biosynthesis level. Genes responsible for strain resistance to a number of macrolide antibiotics and thiostrepton are inducible and only function in the presence of appropriate antibiotics in the medium. The action of inducible mtr gene(s) is described in detail. The gene(s) simultaneously ensure increase in resistance to CTC and a number of macrolide antibiotics in the presence of exogenous inductors in media, such as both CTC and macrolide antibiotics. Mutants have been isolated which provide constitutive level of resistance to these antibiotics. A series of ctr and mtr mutants have increased CTC biosynthesis as compared to the initial level. Data on comparative analysis of the results obtained from hybridization of fragments of S. aureofaciens and S. rimosus DNAs to actI and actIII genes, responsible for polyketide synthases' synthesis, demonstrate that genes for CTC and OTC biosynthesis are situated on DNA fragments of similar size. This determines the strategy for cloning ctr and mtr genes as well as genes for CTC biosynthesis from S. aureofaciens.     

Combination drugs, an emerging option for antibacterial therapy

The emerging and sustained resistance to antibiotics and the poor pipeline of new antibacterials is creating a major health issue worldwide. Bacterial pathogens are increasingly becoming resistant even to the most recently approved antibiotics. Few antibiotics are being approved by regulatory organizations, which reflects both the difficulty of developing such agents and the fact that antibiotic discovery programs have been terminated at several major pharmaceutical companies in the past decade. As a result, the output of the drug pipelines is simply not well positioned to control the growing army of resistant pathogens, although academic institutions and smaller companies are trying to fill that gap. An emerging option to fight such pathogens is combination therapy. Combinations of two antibiotics or antibiotics with adjuvants are emerging as a promising therapeutic approach. This article provides and discusses clinical and scientific challenges to support the development of combination therapy to treat bacterial infections.     

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.     

Short peptides conferring resistance to macrolide antibiotics

Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases.     

水产品中嗜水气单胞菌耐药性研究进展

嗜水气单胞菌Aeromonas hydrophila具有广泛的致病性,是水生动物最常见的致病菌之一。由于抗生素不合理使用、质粒以及耐药基因的水平转移等因素,来自零售市场、超市和餐厅的即食海鲜产品中,均可分离出大量的嗜水气单胞菌耐药菌株及其耐药基因。因此,探明关键控制点、寻求有效缓解抗生素耐药性的防控策略至关重要。文中介绍了我国嗜水气单胞菌的耐药现状,嗜水气单胞菌的主要侵染及耐药机制,以及目前削减和防控耐药性的主要手段和策略,并对水产品耐药性研究方向和重点作出展望。