Bacterial proteomics and its role in antibacterial drug discovery

Gene-expression profiling technologies in general, and proteomic technologies in particular have proven extremely useful to study the physiological response of bacterial cells to various environmental stress conditions. Complex protein toolkits coordinated by sophisticated regulatory networks have evolved to accommodate bacterial survival under ever-present stress conditions such as varying temperatures, nutrient availability, or antibiotics produced by other microorganisms that compete for habitat. In the last decades, application of man-made antibacterial agents resulted in additional bacterial exposure to antibiotic stress. Whereas the targeted use of antibiotics has remarkably reduced human suffering from infectious diseases, the ever-increasing emergence of bacteria that are resistant to antibiotics has led to an urgent need for novel antibiotic strategies. The intent of this review is to present an overview of the major achievements of proteomic approaches to study adaptation networks that are crucial for bacterial survival with a special emphasis on the stress induced by antibiotic treatment. A further focus will be the review of the, so far few, published efforts to exploit the knowledge derived from bacterial proteomic studies directly for the antibacterial drug-discovery process.     

Cell culture model for antiulcerogenic agents

To elucidate the mechanisms of antiulcerogenic agents, we established the cell culture model derived from rat gastric epithelium. The cultured cells were identified as mucus-producing cells by using histological analysis. This culture model is useful for investigating the untiulcer effect of various agents and to reveal the mechanisms of the drug action. In particular, the ulcer-healing model using the cultured monolayer is promising and convenient for the study of several growth factors such as HGF as well as antiulcerogenic agents. The effect of polaporezinc in the cultured model is introduced.     

Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus

Chitosan has been reported to be a non-toxic, biodegradable antibacterial agent. The aim of this work was to elucidate the relationship between the molecular weight of chitosan and its antimicrobial activity upon two model microorganisms, one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Atomic force microscopy (AFM) imaging was used to obtain high-resolution images of the effect of chitosans on the bacterial morphology. The AFM measurements were correlated with viable cell numbers, which show that the two species reacted differently to the high- and low-molecular-weight chitosan derivatives. The images obtained revealed not only the antibacterial effects, but also the response strategies used by the bacteria; cell wall collapse and morphological changes reflected cell death, whereas clustering of bacteria appeared to be associated with cell survival. In addition, nanoindentation experiments with the AFM revealed mechanical changes in the bacterial cell wall induced by the treatment. The nanoindentation results suggested that despite little modification observed in the Gram-positive bacteria in morphological studies, cell wall damage had indeed occurred, since cell wall stiffness was reduced after chitooligosaccharide treatment.     

Bacteria-hemocyte interactions and phagocytosis in marine bivalves

Marine bivalves (such as mussels, oysters, and clams) are widespread mollusks in coastal waters at different latitudes; due to their filter-feeding habits, they accumulate large numbers of bacteria from the harvesting waters and may act as passive carriers of human pathogens. To cope with this challenge, bivalves possess both humoral and cellular defense mechanisms with remarkably effective capabilities. The circulating cells, or hemocytes, are primarily responsible for defense against parasites and pathogens; microbial killing results from the combined action of the phagocytic process with humoral defense factors such as agglutinins (e.g., lectins), lysosomal enzymes (e.g., acid phosphatase, lysozyme), toxic oxygen intermediates, and various antimicrobial peptides. In this work, current knowledge of the mechanisms underlying the interactions between bacteria and the hemolymph components of marine bivalves is summarized. Bacterial susceptibility to hemolymph killing in different bivalve species may be a consequence of the different ability of bacterial products to attract phagocytes, the presence or absence of specific opsonizing molecules, the hemocyte capability to bind and engulf different bacteria, and the different bacterial sensitivity to intracellular killing. The role of soluble (e.g., agglutinins and opsonins) and surface-bound factors in bacterial phagocytosis by hemocytes of the most common marine bivalve species is described and the possibility that environmental temperatures and other seasonal factors may influence this process is considered. Moreover, the potential strategies used by bacteria to evade phagocytic killing by hemocytes are discussed. From the available data it is clear that several questions need further investigation; the elucidation of the factors influencing phagocytosis in bivalves and the fundamental strategies used by bacteria to escape hemolymph killing are important not only to understand bivalve immune defenses but also to explain the persistence of pathogenic bacteria in bivalve tissues and to predict the consequent impact on human health.     

Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus

Chitosan has been reported to be a non-toxic, biodegradable antibacterial agent. The aim of this work was to elucidate the relationship between the molecular weight of chitosan and its antimicrobial activity upon two model microorganisms, one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Atomic force microscopy (AFM) imaging was used to obtain high-resolution images of the effect of chitosans on the bacterial morphology. The AFM measurements were correlated with viable cell numbers, which show that the two species reacted differently to the high- and low-molecular-weight chitosan derivatives. The images obtained revealed not only the antibacterial effects, but also the response strategies used by the bacteria; cell wall collapse and morphological changes reflected cell death, whereas clustering of bacteria appeared to be associated with cell survival. In addition, nanoindentation experiments with the AFM revealed mechanical changes in the bacterial cell wall induced by the treatment. The nanoindentation results suggested that despite little modification observed in the Gram-positive bacteria in morphological studies, cell wall damage had indeed occurred, since cell wall stiffness was reduced after chitooligosaccharide treatment.     

Light-activated oxidize-mimicking nanozyme for inhibition of pathogenic Escherichia coli

Here we demonstrate the nanozyme properties of histidine-containing carbon nanodots as externally tunable antibacterial agents through irradiation with visible (VIS) light. The correlative (light and electron) microscopic analysis of treated Escherichia coli O157:H7 revealed that the positive charged carbon nanoparticles might readily adsorb at slightly acid pH on the negative charged cellular envelope of bacteria, and thus, inhibit their growth with over 80% efficiency under illumination with VIS light. The reason was that under VIS irradiation in the range 400–500 nm the adsorbed nanoparticles behaved as effective oxidase-mimicking enzymes and generated reactive oxygen species on the labeled cells. Thus, the light-activated artificial nanozyme caused serious physical damaging of bacterial envelope, which was leading to irreversible cellular inhibition. The outcomes of this study are likely to broaden the scope of designed photoactive carbon nanozymes as powerful antibacterial agents against the emergence of antibiotic and multidrug-resistant strains, as well as proposing of new strategies for infection control.     

老年患者中段尿病原菌分布及耐药性的自动化分析探讨

目的:调查老年尿液病原菌分布及其耐药情况,为选用抗菌药物控制泌尿系统感染(USI)提供依据。方法:收集我院2018年9月至2020年9月住院的1465例老年USI患者送检中段尿液标本,通过西门子SCAN4全自动细菌鉴定及药敏分析系统配套的鉴定卡、药敏卡,进行的病原菌鉴定以及分离出的可疑菌落药物敏感性试验,对病原菌分布特点和耐药情况进行分析。结果:共分离出357株病原菌,阳性检出率为24.37%。分离病原菌中G-杆菌231株、G+球菌115株、念珠菌11株,分别占比为64.71%、32.21%、3.08%。检出病原菌中以大肠埃希菌(占32.77%)、屎肠球菌(占20.45%)、铜绿假单胞菌(占13.73%)、肺炎克雷伯菌(占8.68%)和粪肠球菌(占7.56%)为常见。大肠埃希菌对头孢类、庆大霉素、氨苄西林、氨苄西林/舒巴坦、左氧氟沙星的耐药率均在50.00%以上,铜绿假单胞菌和肺炎克雷伯菌(除对阿米卡星和氨曲南耐药率<25.0%外)对大多数抗菌药物的耐药率均在45.0%以上,对氨苄西林100.0%耐药。屎肠球菌对红霉素100.0%耐药,对大多数抗菌药物的耐药率均在45.0%以上,粪肠球菌对大多数抗菌药物的耐药率均在25.0%以上。未发现亚胺培南对E.coli、阿米卡星对PA、亚胺培南和左氧氟沙星对KP、利奈唑胺对屎肠球菌和万古霉素和利奈唑胺对粪肠球菌的耐药菌株。仅检出1株万古霉素对屎肠球菌耐药,耐药率为1.37%。结论:我院住院老年USI患者的病原菌构成复杂、种类多,且对常用抗菌药物具有较为严重耐药性。临床应重视本地区老年USI患者的病原菌分布特点以及具体耐药情况,对合理选用抗菌药物以及有针对性采取预防措施有重要意义。     

Proteomics in gram-negative bacterial outer membrane vesicles

Gram-negative bacteria constitutively secrete outer membrane vesicles (OMVs) into the extracellular milieu. Recent research in this area has revealed that OMVs may act as intercellular communicasomes in polyspecies communities by enhancing bacterial survival and pathogenesis in hosts. However, the mechanisms of vesicle formation and the pathophysiological roles of OMVs have not been clearly defined. While it is obvious that mass spectrometry-based proteomics offers great opportunities for improving our knowledge of bacterial OMVs, limited proteomic data are available for OMVs. The present review aims to give an overview of the previous biochemical, biological, and proteomic studies in the emerging field of bacterial OMVs, and to give future directions for high-throughput and comparative proteomic studies of OMVs that originate from diverse Gram-negative bacteria under various environmental conditions. This article will hopefully stimulate further efforts to construct a comprehensive proteome database of bacterial OMVs that will help us not only to elucidate the biogenesis and functions of OMVs but also to develop diagnostic tools, vaccines, and antibiotics effective against pathogenic bacteria.     

Interaction of bifidobacteria with the gut and their influence in the immune function

Bifidobacteria are predominant in the lumen of the large intestine and confer various health benefits on the host. They are also used in the preparation of new fermented milks (bioyogurts) or added to conventional yogurt to generate probiotic effects. The colonization of the gut by bacteria tends to be host specific due partly to the way in which bacteria adhere to the intestinal wall. Using a homologous strain of Bifidobacterium animalis in an experimental mouse model, we analyzed by immunofluorescence labelledbacteria and transmission electronic microscopy the importance of this bacterial interaction with epithelial an immune cells associated to the gut, and the effect of feeding of B. animalis in the immune response. It was able to adhere and interact with both small and large intestine. In spite of this interaction with the gut, no modifications in the immune state (secretory or systemic response) were observed.
A heterologous strain of Bifidobacterium adolescentis from human faeces, was neither uncapable of binding to the intestine, nor influence the immune system activation, when it was administered during 2, 5 or 7 consecutive days; we believe that using a homologous strain, oral tolerance is developed even when the microorganism interacts with the immune cells associated with the intestine. However, we cannot ignore the beneficial effect of these microorganisms, especially in the prevention of intestinal infections. We think that this property exerted by bifidobacteria is more related to other mechanisms such as competitive inhibition, acid production or others, than enhancement of the immune state.     

Erosion–corrosion performance of high-Cr cast iron alloys in flowing liquid–solid slurries

In many slurry transportation systems, such as in FGD (Flue Gas Desulphurization) and chemical processing applications, corrosion and erosion are the two main mechanisms of material degradation of the pump wet-end components including pump casing, impeller and liners. The performance of a selected material is mostly dependent upon its relative corrosion and erosion resistance to the service environment. In these cases erosion, corrosion and the related synergistic effects can be very complicated since they are affected by numerous factors including solid and slurry properties, chemical contents, hydraulic conditions and temperatures. In this experimental study, sliding Coriolis erosion testing has been performed with various corrosion factors such as pH value, chlorides content and temperature to evaluate the erosion–corrosion resistance of some high-alloyed white cast irons containing different levels of chromium and other elements. Optical microscope and SEM-EDS have also been used to examine microstructure and surface conditions of tested materials. Results indicated that material loss due to corrosion factors increased as acidity-chlorides and temperature increased. At relatively high corrosion intensity, the white cast irons with higher alloy content (especially chromium) clearly showed improved corrosion resistance and combined erosion–corrosion resistance over those with lower alloy content. Under certain corrosion and hydraulic conditions, particle size is perhaps the single most influential factor on erosion–corrosion rate of the high-Cr cast iron alloys. Relatively large particles are much more effective than small ones at removing both the corroded surface layer and the fresh material, causing substantially higher rate of material loss. Some other related factors have also been addressed.     

ApplicationofMassSpectrometryinPharmaceuticalDrugDiscoveryResearch

Mass spectrometry has been extensively utilized in drug discovery research and development, from the traditional medicinal chemistry support to drug metabolism and pharmacokinetic studies. As the industry faces with the challenges of tougher diseases and more challenging pathways/targets to tackle, it requires the development of innovative solutions to address needs from target discovery to biomarker identification. It also presents a great opportunity for scientists in the field for innovation, many of which often come from cross disciplinary efforts - chemistry, biology and technology. In this presentation, four areas of drug discovery research where innovative solutions have been developed will be presented: (1) target identification and validation through cellular metabolomic analysis and chemical proteomics; (2) hits discovery and validation using MS-based screening; (3) PK analysis of therapeutic monoclonal antibody; (4) MS imaging for drug tissue distribution. Investment in life saving and life style improving medicines will get more intensified as standard of living improves. Drug discovery research will continue to require and present opportunity for such application driven innovations.     

表面等离子共振生物传感器技术及在生命研究中的应用

表面等离子体共振（Surface Plasmon Resonance,SPR）是一种入射光照射引起金属表面产生等离子体共振而导致反射光衰减的现象,反射光衰减程度与入射角和金属表面物质质量相关。根据这一原理研制的表面等离子体传感器,在检测、分析生物分子问的相互作用等方面有广泛的应用前景。本文对表面等离子共振生物传感器的工作原理、技术参数、检测方法进行了详细介绍,同时介绍了表面等离子共振技术在免疫检测、药物代谢及其蛋白质动力学等生命研究中的应用。     

In situ analysis of microbial consortia in activated sludge using fluorescently labelled,rRNA-targeted oligonucleotide probes and confocal scanning laser microscopy

Activated sludge flocs are complex consortia of various micro-organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 μm. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.     

Non-classical use of clathrin during bacterial infections

How invasive bacteria exploit mammalian host cell components to induce their entry into cells has received a lot of attention in the last two decades. Model organisms have emerged and helped understanding the various mechanisms that are used. Among those, Listeria monocytogenes is one of the most documented organisms. It enters into cells via two bacterial proteins, internalin (also called InlA) and InlB, which interact with cell surface receptors, E-cadherin and the hepatocyte growth factor receptor, Met, respectively. These interactions initiate a series of events that leads to actin polymerization, membrane invagination and bacterial internalization. Investigations on internalin- and InlB-mediated entries have repeatedly shown that Listeria fully usurps the host cell machinery. Moreover, they have also shown that previously unknown components discovered during the study of Listeria invasion play a role either in E-cadherin-mediated cell-cell adhesion or Met signalling. Unexpectedly, recent studies have highlighted a role for clathrin in Listeria InlB-mediated actin polymerization and entry, revealing a new role for this endocytic protein, i.e. in bacterial-induced internalization. Furthermore, comparative studies have demonstrated that the clathrin-mediated endocytosis machinery is also used in the internalin-E-cadherin pathway, and for the entry of other bacteria that enter by a 'zipper' mechanism. By contrast, the clathrin-mediated endocytic machinery is not used by bacteria that inject effectors into mammalian cells via the type III secretion system and enter by the so-called trigger mechanism, characterized by enormous membrane ruflles that result in the macropinocytosis of the corresponding bacteria. Finally, adherent bacteria, for example enteropathogenic Escherichia coli (EPEC), also co-opt clathrin to induce the formation of actin-rich pedestals. Together, these new data illuminate our view on how actin rearrangements may be coupled to clathrin recruitment during bacterial infection. They also shed light on a new function for clathrin in mammalian cells, i.e. internalization of objects much larger than previously accepted.     

HPLC-ESI-LTQ-Orbitrap分析芪归银方中黄酮成分体内代谢过程

为进一步明确芪归银方的药效物质基础,采用HPLC-ESI-LTQ-Orbitrap技术对大鼠灌胃芪归银方后血清中黄酮类活性成分进行表征,并通过鉴别其尿液及粪便中的代谢物对结果进行佐证。从含药血清中鉴定出8个黄酮类化合物,其中,芹菜素-6,8-二葡萄糖苷和芹菜素以原型入血,芒柄花素、木犀草素、毛蕊异黄酮、5,7,4′-三羟基黄酮醇-7-O-β-D-葡萄糖苷、木犀草素-7-O-葡萄糖苷、山奈酚-3-O-葡萄糖苷等6个化合物以Ⅱ相代谢产物入血;从尿液中鉴定出9个黄酮类成分,包括6个入血成分的相应代谢产物;从粪便中检出3个黄酮类化合物的原型形式,未检测到与入血成分相同的成分。实验结果显示,芪归银方中的黄酮类入血成分大部分是经肾脏代谢排出,这可为阐明芪归银方延缓耐药的药效物质基础提供依据。     

基于荧光显微拉曼光谱的细菌快速解耦检测方法

在细菌检测环境中往往存在多种活细菌、死菌以及非生物微颗粒,它们形态和大小相近且耦合寄居在一起,这使得普通的菌类检测方法在检测细菌的种类和含量时很难奏效。提出一种细菌快速解耦检测方法,该方法采用荧光微视技术与拉曼光谱分析技术相结合,利用荧光微视技术实现活菌与非生物微颗粒及死菌的解耦检测,并且实现活菌的定位;利用拉曼光谱技术对定位活菌进行数据采集并结合Ward聚类分析法构建各种活菌的解耦检测模型。实验证明所提出解耦方法的对活菌的解耦检测精度达到了94%以上,且检测时间仅为普通显微拉曼光谱法的1/9。适用于烧伤伤口细菌感染检测等要求检测精度高、速度快的场合。     

Plant growth-promoting rhizobacteria (PGPR) and its mechanisms against plant diseases for sustainable agriculture and better productivity

Plant growth-promoting rhizobacteria (PGPR) are specialized bacterial communities inhabiting the root rhizosphere and the secretion of root exudates helps to, regulate the microbial dynamics and their interactions with the plants. These bacteria viz., Agrobacterium, Arthobacter, Azospirillum, Bacillus, Burkholderia, Flavobacterium, Pseudomonas, Rhizobium, etc., play important role in plant growth promotion. In addition, such symbiotic associations of PGPRs in the rhizospheric region also confer protection against several diseases caused by bacterial, fungal and viral pathogens. The biocontrol mechanism utilized by PGPR includes direct and indirect mechanisms direct PGPR mechanisms include the production of antibiotic, siderophore, and hydrolytic enzymes, competition for space and nutrients, and quorum sensing whereas, indirect mechanisms include rhizomicrobiome regulation via. secretion of root exudates, phytostimulation through the release of phytohormones viz., auxin, cytokinin, gibberellic acid, 1-aminocyclopropane-1-carboxylate and induction of systemic resistance through expression of antioxidant defense enzymes viz., phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenyloxidases (PPO), superoxide dismutase (SOD), chitinase and β-glucanases. For the suppression of plant diseases potent bio inoculants can be developed by modulating the rhizomicrobiome through rhizospheric engineering. In addition, understandings of different strategies to improve PGPR strains, their competence, colonization efficiency, persistence and its future implications should also be taken into consideration.

     

Fungal mediated synthesis of silver nanoparticles and evaluation of antibacterial activity

Nanoparticles as biomedicine has made a crucial role in health biotechnology. Different transition metals in various forms playing role in nanotechnological advances and biological applications. Silver as one of the nontoxic, safe inorganic antibacterial agents and can serve as replacement of antibiotics. Present research is based on biogenic synthesis of silver nanoparticles (Ag‐NPs) as potential antibiotics from fungal metabolites of Penicillium oxalicum. We used different analytical techniques X‐ray diffraction (XRD) and scanning electron microscopy (SEM) for characterization of biosynthesized silver nanoparticles. Furthermore, the antibacterial activity of biosynthesized silver nanoparticles was checked against Staphylococcus aureus, S. dysenteriae, and Salmonella typhi by using well diffusion method and UV visible spectrophotometer. Maximum zone of inhibition recorded against S. aureus, Shigella dysenteriae was 17.5 ± 0.5 mm (mm) for both species and 18.3 ± 0.60 mm for Salmonella typhi. The biosynthesized silver nanoparticles of P. oxalicum showed excellent antibacterial activity. It was concluded from our results that biosynthesized silver nanoparticles have significant potential and might be useful for a wide range of biological applications such as bactericidal agent against resistant bacteria, preventing infections, healing wounds, and anti‐inflammation.     

Current engineering and clinical aspects of needle-free injectors: A review

Needle-free injectors can be used to achieve non-invasive drug delivery by impregnating biological barriers. They are considered as the future of drug delivery and therapeutic applications. The history of needle-free injectors dates back to the 1940s and these devices have been constantly evolving since then. Their operating principles and applications have been improved over the years. Herein, we review the current engineering mechanisms and clinical aspects of needle-free microjet injectors. The present study focuses on using engineering approaches to deal with various factors that affect the penetration and dispersion characteristics of the microjet.