Whole-body imaging of bacterial infection and antibiotic response

We describe imaging of green fluorescent protein (GFP)-expressing bacteria from outside intact infected animals. This simple, non-intrusive technique can show in great detail the spatial-temporal behavior of the infectious process. The bacteria, expressing the GFP, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria can be whole-body imaged in most mouse organs, including the peritoneal cavity, stomach, small intestine, and colon. This imaging technology affords a powerful approach to visualizing the infection process, determining the tissue specificity of infection, the spatial migration of the infectious agents and the response to antimicrobial agents.     

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.     

Antimicrobial activity of mupirocin, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline against vancomycin-resistant enterococci (VRE) from clinical isolates in Korea (1998 and 2005)

It is a hot clinical issue whether newly approved antimicrobial agents such as daptomycin, linezolid, quinupristin/dalfopristin (synercid) and tigecycline are active enough to be used for infections caused by vancomycin resistant bacteria. We performed susceptibility tests for mupirocin, which is in widespread clinical use in Korea, and four new antimicrobials, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline, against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium isolated from Korean patients in 1998 and 2005 to evaluate and compare the in vitro activity of these antimicrobials. Among these agents, quinupristin/dalfopristin, which is rarely used in hospitals in Korea, showed relatively high resistance to several vancomycin-resistant enterococci (VRE) isolated in 2005. Likewise, daptomycin, linezolid and tigecycline have not yet been in clinical use in Korea. However, our results showed that most of the 2005 VRE isolates were already resistant to linezolid and daptomycin (highest minimum inhibitory concentration (MIC) value >100 microg/ml). Compared with the other four antimicrobial agents tested in this study, tigecycline generally showed the greatest activity against VRE. However, four strains of 2005 isolates exhibited resistance against tigecycline (MIC >12.5 microg/ml). Almost all VRE were resistant to mupirocin, whereas all E. faecium isolated in 1998 were inhibited at concentrations between 0.8 to approximately 1.6 microg/ml. In conclusion, resistances to these new antimicrobial agents were exhibited in most of VRE strains even though these new antibiotics have been rarely used in Korean hospitals.     

In vitro susceptibility of staphylococci to linezolid and other antimicrobial agents

Linezolid is a member of the new class of antibacterial agents called oxazolidinones that are active against Gram positive organisms and exert their action by protein synthesis inhibition. In this study we investigated the in vitro activity of linezolid versus the other agent against clinical strains of staphylococci: Staphylococcus aureus (n = 82) and S. epidermidis (n = 32) collected in 2002 from hospitalized patients and healthy individuals, isolated from different biological samples. Agar dilution minimum inhibitory concentrations (MICs) were determined by using Mueller-Hinton agar according to the guidelines established by the National Committee for Clinical Laboratory Standards. Linezolid demonstrated excellent in vitro activity against all isolates tested, with MICs values in the range of susceptibility (< or = 8 microg/ml). No associated resistance between linezolid and other agents tested was observed. The resistance among Gram positive bacteria continues to spread and for many patients infected with these resistant organisms antimicrobial therapy is ineffective and linezolid may be a new alternative treatment.     

Incorporation of a chiral gem-disubstituted nitrogen heterocycle yields an oxazolidinone antibiotic with reduced mitochondrial toxicity

gem-Disubstituted N-heterocycles are rarely found in drugs, despite their potential to improve the drug-like properties of small molecule pharmaceuticals. Linezolid, a morpholine heterocycle-containing oxazolidinone antibiotic, exhibits significant side effects associated with human mitochondrial protein synthesis inhibition. We synthesized a gem-disubstituted linezolid analogue that when compared to linezolid, maintains comparable (albeit slightly diminished) activity against bacteria, comparable in vitro physicochemical properties, and a decrease in undesired mitochondrial protein synthesis (MPS) inhibition. This research contributes to the structure-activity-relationship data surrounding oxazolidinone MPS inhibition, and may inspire investigations into the utility of gem-disubstituted N-heterocycles in medicinal chemistry.     

Role of amino acid residues within the disulfide loop of thanatin,a potent antibiotic peptide

Thanatin, a 21-residue peptide, is an inducible insect peptide with a broad range of activity against bacteria and fungi. It has a C-terminal disulfide loop, like the frog skin secretion antimicrobial peptides of the brevinin family. In this study, we tried to find the effect of a number of amino acids between the disulfide bond. Thanatin showed stronger antibacterial activity to Gram negative bacteria than other mutants, except Th1; whereas, the mutant peptides with deletion had higher activity to Gram positive bacteria than thanatin. An increase in the number of amino acid(s) using the alanine residue decreased the antibacterial activity in all of the bacteria. Th1 with deletion of threonine at position 15 (Thr(1)(2)) showed similar antibacterial activity against Gram-negative bacteria, but had higher activity against the Gram positive bacteria. In order to study the structure-function relationship, we measured liposome disruption by the peptides and CD spectra of the peptides. Th1 also showed the highest liposome leaking activity and alpha-helical propensity in the sodium dodecyl sulfate solution, compared with other peptides. Liposome disruption activity was closely correlated with the anti-Gram positive bacterial activity. All of the peptides showed no hemolytic activity. Th1 was considered to be useful as an antimicrobial peptide with broad spectrum without toxicity     

A chlorinated phenylpyrrole antibiotic fromActinoplanes

Two chlorophenyl-containing antibiotics have been isolated from a strain ofActinoplanes (ATCC 33002). Antibiotic A 15104 Y is a chlorinated phenylpyrrole compound whose structure has been confirmed by chemical synthesis. Antibiotic A 15104 Z is a chlorophenol derivative for which a structural formula is proposed on the basis of its physicochemical properties. A 15104 Y shows antimicrobial activity against Gram-positive, Gram-negative, and acid-fast bacteria, yeasts, fungi, and protozoa, while A 15104 Z possesses a low activity against Gram-positive bacteria andTrichophyton. A 15104 Y has a weak activity in curing experimentally infected mice, at a dose that is one-fifth the LD₅₀ for the same species. This is the first example of a chloropyrrole derivative isolated from an actinomycete.     

Protein aggregation as an antibiotic design strategy

Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation‐prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin‐resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation‐prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.     

Synthesis and antibacterial activity of 5-substituted oxazolidinones

A series of 5-substituted oxazolidinones with varying substitution at the 5-position of the oxazolidinone ring were synthesized and their in vitro antibacterial activity was evaluated. The compounds demonstrated potent to weak antibacterial activity. A novel compound (PH-027) demonstrated potent antibacterial activity, which is comparable to or better than those of linezolid and vancomycin against antibiotic-susceptible standard and clinically isolated resistant strains of gram-positive bacteria. Although the presence of the C-5-acetamidomethyl functionality at the C-5 position of the oxazolidinones has been widely claimed and reported as a structural requirement for optimal antimicrobial activity in the oxazolidinone class of compounds, our results from this work identified the C-5 triazole substitution as a new structural alternative for potent antibacterial activity in the oxazolidinone class.     

中华蜂体内放线菌的分离、多样性及抗菌活性研究

【目的】探索药用昆虫中华蜂(Apis cerana cerana Fabricius)体内可培养放线菌的分离方法,研究其物种多样性及抗菌活性,挖掘更多微生物资源。【方法】选用7种分离培养基对中华蜂样品体内放线菌进行分离;通过采用16S rRNA PCR-RFLP和16S rRNA基因序列分析方法研究其多样性;选用4种致病菌对菌株进行抗菌活性初探。【结果】共分离得到180株放线菌,根据菌落的形态和细胞特征观察结果,从中选取84株作为代表菌株,其分属于3个目、4个科中的4个属,其中6株为潜在新种;最适的表面消毒方法:浓度为0.2%的Cl O2(二氧化氯)作为消毒剂,作用60 s;拮抗实验显示,31.0%、48.8%、27.4%、16.7%的代表菌株分别对大肠杆菌、枯草芽孢杆菌、玉米弯孢病菌、西瓜枯萎病菌有不同程度的抗菌活性,71.4%的代表菌株对至少一种病原菌有拮抗作用。【结论】分离方法的选择对昆虫体内放线菌的分离效果影响较大;中华蜂体内放线菌具有丰富的多样性,展现出很好的抗菌活性,表明其在发现新型生物活性物质中具有很大的潜力。     

EcDBS1R6: A novel cationic antimicrobial peptide derived from a signal peptide sequence

BackgroundBacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm.MethodsAntimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure.ResultsSignal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide.ConclusionThe strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents.General significanceThe process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.     

Exometabolome analysis identifies pyruvate dehydrogenase as a target for the antibiotic triphenylbismuthdichloride in multiresistant bacterial pathogens

The desperate need for new therapeutics against notoriously antibiotic-resistant bacteria has led to a quest for novel antibacterial target structures and compounds. Moreover, defining targets and modes of action of new antimicrobial compounds remains a major challenge with standard technologies. Here we characterize the antibacterial properties of triphenylbismuthdichloride (TPBC), which has recently been successfully used against device-associated infections. We demonstrate that TPBC has potent antimicrobial activity against many bacterial pathogens. Using an exometabolome profiling approach, a unique TPBC-mediated change in the metabolites of Staphylococcus aureus was identified, indicating that TPBC blocks bacterial pyruvate catabolism. Enzymatic studies showed that TPBC is a highly efficient, uncompetitive inhibitor of the bacterial pyruvate dehydrogenase complex. Our study demonstrates that metabolomics approaches can offer new avenues for studying the modes of action of antimicrobial compounds, and it indicates that inhibition of the bacterial pyruvate dehydrogenase complex may represent a promising strategy for combating multidrug-resistant bacteria.     

1272例尿液标本细菌培养结果及耐药性分析

目的调查临床送检中段尿标本菌群分布及体外抗生素耐药性,为临床合理使用抗生素提供依据。方法回顾性分析2010年1月至2012年6月住院患者送检中段尿标本中,分离的1272株细菌（剔除重复菌株）临床分布及耐药性。采用美国BDphoenix100全自动细菌鉴定药敏分析仪进行菌种鉴定及药敏试验,结合相关临床资料,应用WHONET5．6分析软件进行耐药性分析。结果从分离到的l272株细菌中,检出前5位的细菌是大肠埃希菌、肺炎克雷伯菌、屎肠球菌、粪肠球菌和铜绿假单胞菌,构成比分别为20．3％、14．4％、14．2％、9．4％和7．4％。肠杆菌对氨苄西林的耐药率最高为93．9％,耐药率较低的有亚胺培南、美洛培南、头孢哌酮／舒巴坦和多粘菌素B,它们的耐药率在10．5％～15．9％。非发酵菌对氯霉素、氨苄西林、头孢唑啉和阿莫西林／克拉维酸的耐药率最高在90．1％～100％,对多粘菌素B和头孢哌酮／舒巴坦的耐药率较低,分别为7．7％和16．9％。肠球菌对利奈唑胺、万古霉素、替考拉宁和呋西地酸的耐药率最低,分别为2．6％、7．2％、8．8％和8．8％;葡萄球菌对万古霉素的耐药率为0,对喹奴普汀-达福普汀的耐药率为0．8％,对呋哺妥因、替考拉宁、夫西地酸、利奈唑胺、利福平的耐药率在2．5％～10．7％。结论泌尿系统感染主要病原菌为肠杆菌和肠球菌,由于各病原菌均存在不同程度的耐药性,临床医师应根据尿液细菌培养结果,科学合理使用抗菌药物。     

Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic

The growing public health problem of infections caused by multiresistant Gram-positive bacteria, in particular Staphylococcus aureus, prompted us to screen human epithelia for endogenous S. aureus-killing factors. A novel 5-kDa, nonhemolytic antimicrobial peptide (human beta-defensin-3, hBD-3) was isolated from human lesional psoriatic scales and cloned from keratinocytes. hBD-3 demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes including multiresistant S. aureus and vancomycin-resistant Enterococcus faecium. Ultrastructural analyses of hBD-3-treated S. aureus revealed signs of cell wall perforation. Recombinant hBD-3 (expressed as a His-Tag-fusion protein in Escherichia coli) and chemically synthesized hBD-3 were indistinguishable from naturally occurring peptide with respect to their antimicrobial activity and biochemical properties. Investigation of different tissues revealed skin and tonsils to be major hBD-3 mRNA-expressing tissues. Molecular cloning and biochemical analyses of antimicrobial peptides in cell culture supernatants revealed keratinocytes and airway epithelial cells as cellular sources of hBD-3. Tumor necrosis factor alpha and contact with bacteria were found to induce hBD-3 mRNA expression. hBD-3 therefore might be important in the innate epithelial defense of infections by various microorganisms seen in skin and lung, such as cystic fibrosis.     

Conformation-dependent antibiotic activity of tritrpticin,a cathelicidin-derived antimicrobial peptide

Tritrpticin, a Trp-rich cationic antimicrobial peptide with a unique amino acid sequence (VRRFPWWWPFLRR), is found in porcine cathelicidin cDNA. Tritrpticin has a broad spectrum of antibacterial and antifungal activities and hemolytic activity comparable to that of indolicidin. To investigate the mechanism of the bacterial killing action of tritrpticin and to identify structural features important for bacterial cell selectivity, we designed several tritrpticin analogs with amino acid substitutions of the Pro and Trp residues. Circular dichroism studies revealed that the substitution of Pro-->Ala (TPA) or Trp-->Phe (TWF) leads to significant conformational changes in SDS micelles, converting the beta-turn to alpha-helix or to poly-L-proline II helix, respectively. Compared to tritrpticin, TPA retained most of its antimicrobial activity, but showed enhanced hemolytic and membrane-disrupting activities. In contrast, TWF showed a 2-4-fold increase in antimicrobial activity against Gram-negative bacteria, but a marked decrease in both hemolytic and membrane-disrupting activities. Taken together, our findings suggest that compared with the beta-turn and alpha-helical structures, the poly-L-proline II helix is crucial for effective bacterial cell selectivity in tritrpticin and its analogs.     

Structural basis for the antibiotic activity of ketolides and azalides

The azalide azithromycin and the ketolide ABT-773, which were derived by chemical modifications of erythromycin, exhibit elevated activity against a number of penicillin- and macrolide-resistant pathogenic bacteria. Analysis of the crystal structures of the large ribosomal subunit from Deinococcus radiodurans complexed with azithromycin or ABT-773 indicates that, despite differences in the number and nature of their contacts with the ribosome, both compounds exert their antimicrobial activity by blocking the protein exit tunnel. In contrast to all macrolides studied so far, two molecules of azithromycin bind simultaneously to the tunnel. The additional molecule also interacts with two proteins, L4 and L22, implicated in macrolide resistance. These studies illuminated and rationalized the enhanced activity of the drugs against specific macrolide-resistant bacteria.     

Eosinophil cationic protein high-affinity binding to bacteria-wall lipopolysaccharides and peptidoglycans

The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. The protein displays antimicrobial activity against both Gram-negative and Gram-positive strains. The protein can destabilize lipid bilayers, although the action at the membrane level can only partially account for its bactericidal activity. We have now shown that ECP can bind with high affinity to the bacteria-wall components. We have analyzed its specific association to lipopolysaccharides (LPSs), its lipid A component, and peptidoglycans (PGNs). ECP high-affinity binding capacity to LPSs and lipid A has been analyzed by a fluorescent displacement assay, and the corresponding dissociation constants were calculated using the protein labeled with a fluorophor. The protein also binds in vivo to bacteria cells. Ultrastructural analysis of cell bacteria wall and morphology have been visualized by scanning and transmission electron microscopy in both Escherichia coli and Staphylococcus aureus strains. The protein damages the bacteria surface and induces the cell population aggregation on E. coli cultures. Although both bacteria strain cells retain their shape and no cell lysis is patent, the protein can induce in E. coli the outer membrane detachment. ECP also activates the cytoplasmic membrane depolarization in both strains. Moreover, the depolarization activity on E. coli does not require any pretreatment to overcome the outer membrane barrier. The protein binding to the bacteria-wall surface would represent a first encounter step key in its antimicrobial mechanism of action.     

A new antitumor antibiotic, chounghwamycin A. I. Taxonomy, isolation and characterization

Chounghwamycin A, a new antitumor antibiotic produced by a strain of Streptomyces sp. No. PL-D-5, was isolated and characterized. It appeared to belong to the actinomycin group of antibiotics from physico-chemical studies and has an empirical formula of C63H88N11O21. The antibiotic is extractable into an organic solvent from the fermentation broth, possessing potent antileukemic activity against P388 mouse leukemia in mice and antimicrobial activity against Gram-positive bacteria with MIC values about 0.1-0.4 microgram/ml, but showed no activity on Gram-negative bacteria, yeast and fungi tested.     

In vitro activity of linezolid and eperezolid, two novel oxazolidinone antimicrobial agents, against anaerobic bacteria

Linezolid (formerly U-100766) and eperezolid (formerly U-100592) are novel oxazolidinone antimicrobial agents that are active against multi-drug-resistant staphylococci, streptococci, enterococci, corynebacteria, and mycobacteria. Preliminary studies also demonstrated that the compounds inhibited some test strains of anaerobic bacteria. Therefore, we extended the in vitro evaluation of these agents to include a total of 54 different anaerobic species. Minimal inhibitory concentration (MIC) values were determined using a standard agar dilution method for 143 anaerobic bacterial isolates. Eperezolid and linezolid demonstrated potent activity against the anaerobic Gram-positive organisms with most MIC values in the range of 0.25-4 microg/mL. Viridans streptococci demonstrated MICs of 1-2 microg/mL; Peptostreptococcus species and Propionibacterium species were inhibited by 相似文献   

Antimicrobial activity of surface attached marine bacteria in biofilms

Relatively little is known about the microbial ecology of biofilm communities or the diversity of antimicrobial molecules that they produce to regulate these communities. This study tested whether the production of antimicrobial activity in biofilm cultures is enhanced towards competing bacteria found in those biofilms. First, the production of antimicrobial activity of marine bacteria grown in biofilms was tested. Fourteen of the 105 marine isolates tested were found to produce antimicrobial factors when grown in biofilms. The antimicrobial activity produced by these isolates in biofilms was more potent and inhibited a broader range of target bacteria grown in biofilms compared to shaken liquid cultures. In a separate experiment, we found that cultivation in biofilms containing produced metabolites from an ‘inducer’ bacterium stimulated the production of antimicrobial molecules by ‘producer’ bacteria that were active against the ‘inducer’ bacterium. Overall, the study suggests that surface attached marine bacteria can target their antimicrobial activity towards competing bacteria in biofilms.