In Silico Analysis of Antibiotic Resistance Genes in the Gut Microflora of Individuals from Diverse Geographies and Age-Groups

The spread of antibiotic resistance, originating from the rampant and unrestrictive use of antibiotics in humans and livestock over the past few decades has emerged as a global health problem. This problem has been further compounded by recent reports implicating the gut microbial communities to act as reservoirs of antibiotic resistance. We have profiled the presence of probable antibiotic resistance genes in the gut flora of 275 individuals from eight different nationalities. For this purpose, available metagenomic data sets corresponding to 275 gut microbiomes were analyzed. Sequence similarity searches of the genomic fragments constituting each of these metagenomes were performed against genes conferring resistance to around 240 antibiotics. Potential antibiotic resistance genes conferring resistance against 53 different antibiotics were detected in the human gut microflora analysed in this study. In addition to several geography/country-specific patterns, four distinct clusters of gut microbiomes, referred to as ‘Resistotypes’, exhibiting similarities in their antibiotic resistance profiles, were identified. Groups of antibiotics having similarities in their resistance patterns within each of these clusters were also detected. Apart from this, mobile multi-drug resistance gene operons were detected in certain gut microbiomes. The study highlighted an alarmingly high abundance of antibiotic resistance genes in two infant gut microbiomes. The results obtained in the present study presents a holistic ‘big picture’ on the spectra of antibiotic resistance within our gut microbiota across different geographies. Such insights may help in implementation of new regulations and stringency on the existing ones.     

Transfer of transformed chloroplasts from Nicotiana tabacum to the Lycium barbarum plants

Plastid transformation is an attractive technology for obtaining crop plants with new useful characteristics and for fundamental researches of plastid functioning and nuclear-plastid interaction. The aim of our experiments was to obtain plants with Lycium barbarum nucleus and transformed Nicotiana tabacum plastids. Plastome of previously engineered transplastomic tobacco plants contains reporter uidA gene and selective aadA gene that confers resistance to antibiotics spectinomycin and streptomycin. Asymmetric somatic hybridization was performed for transferring transformed tobacco plastids from transplastomic tobacco plants into recipient L. barbarum wild type plants. Hybrid L. barbarum plants containing transformed tobacco plastome with active aadA and uidA genes were obtained as a result of the experiments. The work shows the possibility of obtaining transplastomic plants by transferring the transformed plastids to remote species by using somatic hybridization technology. The developed technique is especially effective for obtaining transplastomic plants that have low regeneration and transformation ability.     

Somatic Hybridization of Dihaploid Potato Protoplasts as a Tool for Potato Breeding*

Using electrofusion of protoplasts from different dihaploid potato breeding clones, 308 somatic hybrids, i.e. 16.7% of the totally regenerated plants, were obtained. The results indicate that factors such as ‘combining ability’ strongly influence hybrid formation in these intraspecific fusion experiments. Hybrid identification was predominantly carried out by isoenzyme analysis of the peroxidases and/or esterases in polyacrylamide-gradient gel electrophoresis. Further confirmation of the hybrid character was obtained by scoring phenotypic markers (petal colour, stem anthocyanin production, tuber characteristics, etc.) and, in some combinations, by evaluating tolerance against the herbicide metribuzin and nematode resistance against Globodera rostochiensis (Ro 1). The successful inheritance of metribuzin tolerance and nematode resistance confirm that monogenic dominant inherited traits are combined in the hybrids.     

Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea.

Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics. It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species.     

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.     

Structure aided design of chimeric antibiotics

The rise of antibiotic resistance is of great clinical concern. One approach to reducing the development of resistance is to co-administer two or more antibiotics with different modes of action. However, it can be difficult to control the distribution and pharmacokinetics of two drugs to ensure both concentrations remain within the range of therapeutic efficacy whilst avoiding adverse effects. Hybrid drugs, where two drugs are linked together with a flexible linker, have been explored, but the resultant large, flexible molecules can have poor bioavailability. We have developed a chimeric approach using click chemistry where the pharmacophores of two drugs are overlapped into a single smaller, more drug-like molecule. Design and selection of compounds were assisted by in silico structural docking. We prepared a series of compounds that include candidates showing activity against the targets of both trimethoprim; dihydrofolate reductase, and ciprofloxacin; DNA gyrase and topoisomerase IV. The resultant triazole containing molecules show modest, but broad spectrum activities against drug sensitive and resistant Gram-negative and Gram-positive bacteria, with no observable cytotoxicity.     

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.     

Functional Characterization of Bacteria Isolated from Ancient Arctic Soil Exposes Diverse Resistance Mechanisms to Modern Antibiotics

Using functional metagenomics to study the resistomes of bacterial communities isolated from different layers of the Canadian high Arctic permafrost, we show that microbial communities harbored diverse resistance mechanisms at least 5,000 years ago. Among bacteria sampled from the ancient layers of a permafrost core, we isolated eight genes conferring clinical levels of resistance against aminoglycoside, β-lactam and tetracycline antibiotics that are naturally produced by microorganisms. Among these resistance genes, four also conferred resistance against amikacin, a modern semi-synthetic antibiotic that does not naturally occur in microorganisms. In bacteria sampled from the overlaying active layer, we isolated ten different genes conferring resistance to all six antibiotics tested in this study, including aminoglycoside, β-lactam and tetracycline variants that are naturally produced by microorganisms as well as semi-synthetic variants produced in the laboratory. On average, we found that resistance genes found in permafrost bacteria conferred lower levels of resistance against clinically relevant antibiotics than resistance genes sampled from the active layer. Our results demonstrate that antibiotic resistance genes were functionally diverse prior to the anthropogenic use of antibiotics, contributing to the evolution of natural reservoirs of resistance genes.     

Bistable Expression of Virulence Genes in Salmonella Leads to the Formation of an Antibiotic-Tolerant Subpopulation

Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.     

Somatic hybrids between transgenic <Emphasis Type="Italic">Solanum tuberosum</Emphasis> potato plants and transplastome <Emphasis Type="Italic">Solanum rickii</Emphasis> plants

Experiments on fusion of mesophyllic protoplasts of Solanum tuberosum (Lugovskoi and Slavyanka cultivars) possessing the nptII gene in the nuclear DNA with transplastome Solanum rickii plants (which possess the aadA gene) that we have derived previously, are performed. Hybrid plants with the genes aadA and nptII, the chloroplasts of S. rickii and S. tuberosum, and a hybrid nuclear genome were obtained in a selection medium containing the antibiotics kanamycin, spectomycin, and streptomycin. The result is confirmed by results of PCR analyses.     

Simplified lipid II-binding antimicrobial peptides: Design,synthesis and antimicrobial activity of bioconjugates of nisin rings A and B with pore-forming peptides

New designs of antimicrobial peptides are urgently needed in order to combat the threat posed by the recent increase of resistance to antibiotics. In this paper, we present a new series of antimicrobial peptides, based on the key structural features of the lantibiotic nisin. We have simplified the structure of nisin by conjugating the lipid II-binding motif at the N-terminus of nisin to a series of cationic peptides and peptoids with known antibacterial action and pore-forming properties. Hybrid peptides, where a hydrophilic PEG4 linker was used, showed good antibacterial activity against Micrococcus luteus.     

Foundations of antibiotic resistance in bacterial physiology: the mycobacterial paradigm

The intrinsic resistance of Mycobacterium tuberculosis and related pathogens to most common antibiotics limits chemotherapeutic options to treat tuberculosis and other mycobacterial diseases. Resistance has traditionally been attributed to the unusual multi-layer cell envelope that functions as an effective barrier to the penetration of antibiotics. Recent insights into mechanisms that neutralize the toxicity of antibiotics in the cytoplasm have revealed systems that function in synergy with the permeability barrier to provide intrinsic resistance. Here, we highlight the growing pool of information about internal, antibiotic-responsive regulatory proteins and corresponding resistance genes, and present new concepts that rationalize how they might have evolved. Pharmaceutical inhibition of these intrinsic systems could make many previously available antibiotics active against M. tuberculosis.     

Understanding the acylation mechanisms of active-site serine penicillin-recognizing proteins: a molecular dynamics simulation study

Beta-lactam antibiotics inhibit enzymes involved in the last step of peptidoglycan synthesis. These enzymes, also identified as penicillin-binding proteins (PBPs), form a long-lived acyl-enzyme complex with beta-lactams. Antibiotic resistance is mainly due to the production of beta-lactamases, which are enzymes that hydrolyze the antibiotics and so prevent them reaching and inactivating their targets, and to mutations of the PBPs that decrease their affinity for the antibiotics. In this study, we present a theoretical study of several penicillin-recognizing proteins complexed with various beta-lactam antibiotics. Hybrid quantum mechanical/molecular mechanical potentials in conjunction with molecular dynamics simulations have been performed to understand the role of several residues, and pK(a) calculations have also been done to determine their protonation state. We analyze the differences between the beta-lactamase TEM-1, the membrane-bound PBP2x of Streptococcus pneumoniae, and the soluble DD-transpeptidase of Streptomyces K15.     

Microbial metabolism of chloroanilines: enhanced evolution by natural genetic exchange

2-, 3-, and 4-chloroaniline degrading bacteria were obtained by natural genetic exchange between an aniline or toluidine degrading Pseudomonas strain and the chlorocatechol assimilating Pseudomonas sp. B13. Hybrid organisms were isolated through cocultivation of the parent strains in the chemostat as well as through conjugation on solid media in presence of chloroanilines as the selective substrates. Biochemical analysis of the gene products in the hybrid strains clearly showed that the genes coding for the aniline dioxygenase or the genes for the chlorocatechol assimilatory sequence had been transferred.     

基于cDNA-AFLP技术的玉米种子劣变相关基因分析

以人工控制劣变处理（温度45℃,相对湿度100%）3 d的玉米杂交种‘郑单958’种子为材料,通过cDNA-AFLP技术,共获得约5 400个转录本。对差异片段进行测序分析,最终获得122个种子劣变相关基因序列（DRS）,其中,上调表达74个,下调表达48个,功能涉及初生代谢、次生代谢、蛋白代谢、转录与调控、胁迫响应、信号转导和运输。采用qRT-PCR技术分析了11个上调表达差异基因的表达模式,结果显示不同基因的表达模式存在较大差异,说明不同基因在种子劣变中可能扮演不同角色。     

Production of neutralizing antibody to rabies and vesicular stomatitis viruses by hybrid cell lines

Hybrid cell lines were obtained following fusion of P 3 × 63 Ag-8 myeloma cells with spleen cells derived from BALB/c mice immunized either with rabies virus or with vesicular stomatitis virus. Hybrid cell lines were selected which continued to secrete rabies virus or vesicular stomatitis virus neutralizing antibody specifically directed against coat glycoprotein of respective viruses.     

Optimizing future treatment of enterococcal infections: attacking the biofilm?

Enterococcus faecalis and Enterococcus faecium are among the leading causative agents of nosocomial infections and are infamous for their resistance to many antibiotics. They cause difficult-to-treat infections, often originating from biofilm-mediated infections associated with implanted medical devices or endocarditis. Biofilms protect bacteria against antibiotics and phagocytosis, and physical removal of devices or infected tissue is often needed but is frequently not possible. Currently there are no clinically available compounds that disassemble biofilms. In this review we discuss all known structural and regulatory genes involved in enterococcal biofilm formation, the compounds directed against biofilm formation that have been studied, and potentially useful targets for future drugs to treat enterococcal biofilm-associated infections.