Capillary high-performance liquid chromatography-fast atom bombardment mass spectrometry of 24 cephem antibiotics

Using capillary high-performance liquid chromatography (HPLC)-fast atom bombardment (FAB) mass spectrometry (MS), both positive and negative FAB mass spectra of 24 cephem antibiotics with diethanolamine (DEA) and glycerol (GLY) as matrices are presented. In the positive mode, an internal quasi-molecular peak together with relatively abundant fragment peaks were obtained from all 24 drugs with both matrices, though DEA provided more information on molecular mass of a compound than did GLY for some drugs. In the negative mode, the background was generally lower than that in the positive, but neither the quasi-molecular nor molecular peak was detected in several drugs with either matrix. The drugs were isolated from serum samples using an octadecyl reversed-phase cartridge; recoveries were generally over 60%. With this isolation and the capillary HPLC-FAB-MS in the positive mode, ceftriaxone and cefazolin, two of the most popular cephem antibiotics, were successfully identified in 0.5 ml of sera obtained from a clinical or an autopsy case.     

On the possible ecological roles of antimicrobials

The Introduction of antibiotics into the clinical use in the middle of the 20th century had a profound impact on modern medicine and human wellbeing. The contribution of these wonder molecules to public health and science is hard to overestimate. Much research has informed our understanding of antibiotic mechanisms of action and resistance at inhibitory concentrations in the lab and in the clinic. Antibiotics, however, are not a human invention as most of them are either natural products produced by soil microorganisms or semisynthetic derivatives of natural products. Because we use antibiotics to inhibit the bacterial growth, it is generally assumed that growth inhibition is also their primary ecological function in the environment. Nevertheless, multiple studies point to diverse nonlethal effects that are exhibited at lower levels of antibiotics. Here we review accumulating evidence of antibiosis and of alternative functions of antibiotics exhibited at subinhibitory concentrations. We also speculate on how these effects might alter phenotypes, fitness, and community composition of microbes in the context of the environment and suggest directions for future research.     

环境抗生素抗性基因研究进展

抗生素耐药性及其在全球范围内的传播已成为国际关注的热点。本文结合最新文献,综述了抗生素抗性基因在环境中的来源、传播、分布以及新型抗性基因的发现等方面的研究进展。环境中抗生素抗性基因的来源主要是环境中细菌的内在抗性基因及随人或动物粪便排到体外的抗性细菌。功能宏基因组学技术的应用极大地丰富了人们对抗生素抗性组学的认知,并已从环境中筛选到多种新型抗性基因。近年来,由于抗生素在医疗以及养殖业中的大量使用,增加了抗性基因在环境中的丰度和多样性,加速了抗性基因在环境中的传播,在多种环境介质(如养殖水域、污水处理厂、河流、沉积物和土壤等)均检测到多种高丰度的抗生素抗性基因。我们建议今后在以下方面开展深入研究:(1)抗性基因传播和扩散的机制;(2)新型抗性基因筛选和抗性机制;(3)抗生素和抗性基因环境风险评估体系等。     

Crystal structures of complexes between the R61 DD-peptidase and peptidoglycan-mimetic beta-lactams: a non-covalent complex with a "perfect penicillin"

The bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases) are the killing targets of beta-lactams, the most important clinical defense against bacterial infections. However, due to the constant development of antibiotic-resistance mechanisms by bacteria, there is an ever-present need for new, more effective antimicrobial drugs. While enormous numbers of beta-lactam compounds have been tested for antibiotic activity in over 50 years of research, the success of a beta-lactam structure in terms of antibiotic activity remains unpredictable. Tipper and Strominger suggested long ago that beta-lactams inhibit DD-peptidases because they mimic the D-alanyl-D-alanine motif of the peptidoglycan substrate of these enzymes. They also predicted that beta-lactams having a peptidoglycan-mimetic side-chain might be better antibiotics than their non-specific counterparts, but decades of research have not provided any evidence for this. We have recently described two such novel beta-lactams. The first is a penicillin having the glycyl-L-alpha-amino-epsilon-pimelyl side-chain of Streptomyces strain R61 peptidoglycan, making it the "perfect penicillin" for this organism. The other is a cephalosporin with the same side-chain. Here, we describe the X-ray crystal structures of the perfect penicillin in non-covalent and covalent complexes with the Streptomyces R61 DD-peptidase. The structure of the non-covalent enzyme-inhibitor complex is the first such complex to be trapped crystallographically with a DD-peptidase. In addition, the covalent complex of the peptidyl-cephalosporin with the R61 DD-peptidase is described. Finally, two covalent complexes with the traditional beta-lactams benzylpenicillin and cephalosporin C were determined for comparison with the peptidyl beta-lactams. These structures, together with relevant kinetics data, support Tipper and Strominger's assertion that peptidoglycan-mimetic side-chains should improve beta-lactams as inhibitors of DD-peptidases.     

Fungal biosynthesis of non-ribosomal peptide antibiotics and alpha, alpha-dialkylated amino acid constituents.

Zervamicins (Zrv) IIA and IIB are membrane modifying peptide antibiotics of fungal origin, characterized by a sequence of 15 amino acid residues. The primary structure of Zrv-IIA contains five alpha-aminoisobutyric acid residues at positions 4, 7, 9, 12 and 14 of the linear peptide. The sequence of Zrv-IIB is similar, but contains a D-isovaline at position 4. When the free amino acid Aib was added to the peptone-glucose culture medium, the fungus Emericellopsis salmosynnemata produced Zrv-IIA as the major secondary metabolite, whereas addition of DL-Iva to the culture led to a high production of Zrv-IIB. This observation is rationalized by a lack of selectivity of the non-ribosomal peptide synthetase with respect to the thiolester activated amino acid substrates during step 12 of peptide synthesis. Analysis of the configuration of the Iva residue of Zrv-IIB showed a high enantiomeric purity of the D-enantiomer, indicating a high stereoselectivity of the peptide synthetase for this substrate.When the culture was supplemented with [(15)N]DL-Iva, the nitrogen isotope was not only found at the D-Iva residue, but surprisingly also at the Aib residues as well as at the proteinogenic residues of Zrv. The partial catabolism of exogenous [(15)N]DL-Iva is explained by the assumption of a decarboxylation-dependent transamination reaction, catalysed by 2,2-dimethylglycine decarboxylase. The same enzyme might also be involved in the reversed carboxylation reactions of acetone and 2-butanone, during the anabolic biosynthesis of Aib and Iva, respectively. Zrv might possibly act as a thermodynamic sink to shift these equilibrium reactions towards the reversed side.     

The prophylactic use of antibiotics in cell culture

This article describes the historical development of the prophylactic use of antibiotics in cell culture as well as their effects on cells. The influence of antibiotics on cell morphology, cellular degeneration and cell death and cellular function is summarized. Cellular DNA as well as protein synthesis are affected which can lead to interference with, or even changes in, metabolic processes. Such effects must be considered in cell culture research. As antibiotics are used in multifold ways, the otherwise standardized conditions in cell culture are no longer comparable. The prophylactic use of antibiotics is rejected for scientific reasons.     

Synthesis and evaluation of biological activity for dual-acting antibiotics on the basis of azithromycin and glycopeptides

One of the promising directions of the combined approach is the design of dual-acting antibiotics – heterodimeric structures on the basis of antimicrobial agents of different classes. In this study a novel series of azithromycin-glycopeptide conjugates were designed and synthesized. The structures of the obtained compounds were confirmed using NMR spectroscopy and mass spectrometry data including MS/MS analysis. All novel hybrid antibiotics were found to be either as active as azithromycin and vancomycin against Gram-positive bacterial strains or have superior activity in comparison with their parent antibiotics. One compound, eremomycin-azithromycin conjugate 16, demonstrated moderate activity against Enterococcus faecium and Enterococcus faecalis strains resistant to vancomycin, and equal to vancomycin’s activity for the treatment of mice with Staphylococcus aureus sepsis.     

The Phytochemical Tactics for Battling Antibiotic Resistance in Microbes: Secondary Metabolites and Nano Antibiotics Methods

One of the most serious threats to human health is antibiotic resistance, which has left the world without effective antibiotics. While continuous research and inventions for new antibiotics are going on, especially those with new modes of action, it is unlikely that this alone would be sufficient to win the battle. Furthermore, it is also important to investigate additional approaches. One such strategy for improving the efficacy of existing antibiotics is the discovery of adjuvants. This review has collected data from various studies on the current crisis and approaches for combating multi-drug resistance in microbial pathogens using phytochemicals. In addition, the nano antibiotic approaches, are discussed, highlighting the high potentials of essential oils, alkaloids, phenolic compounds, and nano antibiotics in combating antibiotic resistance.     

两种水生植物对抗生素污染水体的修复作用

采用水培方法,通过检测水样中氨苄青霉素（Ampicillin）、盐酸四环素（Tetracycline）、盐酸土霉素（Oxytetracycline）和盐酸金霉素（Chlortetracycline）含量的动态变化,确定水生植物大漂（Pistia stratiotes）和凤眼莲（Eichhornia crassipes）对水体中抗生素的清除作用。结果显示：①在系列高浓度（10～50 μg/mL）抗生素的条件下,凤眼莲去除水中盐酸金霉素与盐酸土霉素的效果优于大漂;②对于采集的污水（抗生素浓度＜2.5 μg/mL）,培养72 h后,大漂和凤眼莲对盐酸四环素的去除率分别达80%和90%以上,对氨苄青霉素的去除率分别达80%和70%以上。大漂和凤眼莲对4种抗生素污染的水体均表现出不同程度的修复功能,特别是凤眼莲效果更佳,可作为去除水体抗生素污染的首选材料。     

Effect of mannanoligosaccharides supplementation on caecal microbial activity of rabbits

A total of 200 weaned (35 days) hybrid Hyla rabbits were randomly divided among five groups housed in bicellular cages (20 cages per group). Between 35 and 60 days of age, the groups were submitted to the following treatments: group ANT (positive control) fed a basal diet supplemented with antibiotics (colistin sulphate, 144 mg/kg; tylosin, 100 mg/kg; and oxytetracyclin, 1000 mg/kg); groups MOS_0.5, MOS_1.0 and MOS_1.5 fed the basal diet supplemented with 0.5, 1.0 and 1.5 g/kg mannanoligosaccharides (MOS), respectively; another group fed the basal diet without antibiotics or mannanoligosaccarides supplementation (negative control). Along the trial, an episode of epizootyc rabbit enteropathy occurs so that in the control group mortality rate was very high (78%) and survivor rabbits showed severe symptoms of disease (diarrhoea). Thus, the control group was discarded from the trial. At 60 days of age, samples of caecal content were collected from 10 rabbits per group and used as inocula for an in vitro gas production trial. At the end of fermentation (120 h of incubation), organic matter digestibility (OMd), cumulative gas production, fermentation kinetics, pH, volatile fatty acid (VFA) and NH3 productions were measured. Inoculum from MOS_1.0 rabbits showed the significant higher values of OMd (64.21%, P < 0.05), gas production (262.32 ml/g, P < 0.05), acetate (96.99 mmol/g OM, P < 0.05) and butyrate (26.21 mmol/g OM, P < 0.05) than the other groups. Slight differences were recorded among the groups ANT, MOS_0.5 and MOS_1.5. In addition, branched chain acids, in proportion to total VFAs, were significantly higher in MOS_1.0 inoculum (0.04, P < 0.05). MOS are able to affect fermentation activity of caecal micro-organism, but their activities seem not proportional to their level in the diet.     

基于银纳米棒的表面增强拉曼基底探究土霉素对植物乳杆菌的影响

【背景】土霉素属四环素类抗生素,在医疗制药和动物养殖行业应用广泛。抗生素的滥用与残留极易导致环境中的益生菌产生耐药,各类益生菌通过食物进入机体后,可将耐药基因转移给致病菌。目前的耐药菌株筛选方法耗时较长,最小抑菌浓度测定结果也可能存在偏差,而且无法在抗菌机理及耐药机制方面提供线索。【目的】采用表面增强拉曼光谱（surface enhanced Raman spectroscopy,SERS）技术探究土霉素与植物乳杆菌之间的相互作用,以期为微生物耐药菌株的快速筛选提供理论基础,并为研究抗菌机理及耐药机制提供思路及方法。【方法】组装银纳米棒与石英后得到一种具有良好、稳定的SERS增强效应的固相基底;在植物乳杆菌菌液中加入不同浓度土霉素后各自分别孵育30、60和90 min,利用组装好的固相基底对细菌细胞进行SERS信号测定,拉曼光谱的出峰位置和峰强度变化可以体现不同土霉素浓度及作用时间条件下植物乳杆菌细胞成分的差异,可进一步推断土霉素对植物乳杆菌生长的影响。【结果】0.5×MIC土霉素会造成SERS光谱强度的降低;1.0×MIC土霉素作用90 min后,SERS在1 612、1 630 c...     

Metabolic scaling of antibiotics in reptiles: Basis and limitations

The allometric equation y = a · x^b has been used to scale many morphological and physiological attributes relative to body mass. For instance, in eutherian mammals, the equation P_met = 70M_b^0.75 has been used to describe the relationship between metabolic rate (P_met) and body mass (M_b). Similar equations have been derived for squamate reptiles. Recently, this relationship between metabolic rate and body mass has been used in determining appropriate dosages and dosing intervals of antibiotics both intraspecifically for different sized reptiles and interspecifically for those reptiles in which antibiotic pharmacokinetic studies have not been performed. Although this is a simple mathematical process, a number of problems surface when this approach is examined closely. First, the mass constant (a) in reptiles varies from 1–5 for snakes and 6–10 for lizards. No such information is available for chelonians or crocodilians. Unless the mass constant for the unknown species approximates that of the known species, inappropriate dosages and intervals of administration will be calculated. Second, pharmacokinetic differences may exist between widely divergent species, independent of metabolic rate. Third, all available pharmacokinetic studies and metabolic allometric equations are derived from clinically healthy reptiles. Differences more than likely exist between healthy and ill reptiles in regard to uptake, distribution, and elimination of drugs and overall metabolism. While metabolic scaling of antibiotics is a potentially useful and practical tool in drug dosing, these limitations must be considered when dosing an ill reptile. Until more scientifically derived information is available for demonstrating the accuracy of metabolic scaling of antibiotics in reptiles, the clinician will need to understand the limitations of this approach. © 1996 Wiley-Liss, Inc.     

邻氯青霉素单克隆抗体的制备及特性鉴定

采用碳化二亚胺法,将邻氯青霉素（cloxacillin）与牛血清白蛋白偶联制备免疫原,免疫BALB/c小鼠,经杂交瘤技术获得了一株能稳定分泌抗邻氯青霉素单克隆抗体的杂交瘤细胞株2H8-B1-F4。间接ELISA方法测定,抗体亚类为IgG1,其细胞上清抗体效价为1：1000,腹水效价为1：4×105。与其结构类似物苯唑青霉素、双氯青霉素的交叉反应率为21.3％和19.6％,和氨苄青霉素、羟氨苄青霉素和苄青霉素无交叉反应。体外传代培养和冻存复苏后抗体分泌稳定。为进一步研制检测邻氯青霉素的 ELISA试剂盒奠定基础。     

A simple model for the optimization of the extraction yield of antibiotics isolated from fermented broths by direct crystallization

This article is concerned with the development of a model to maximize the overall yield of isolation of antibiotics recovered from fermented broths by the so-called direct precipitation method. In this process, as in most antibiotics isolation processes, a second filtration of the semiexhaust mycellium from the first filtration, after slurrying it in water at the suitable pH, is required. The maximization of the overall yield of isolation implies the use of an optimal amount of water, measured as a volume to fermented broth mass ratio, which can be calculated by using the model derived here. The model also allows for the calculation of the partial and overall yields of the isolation process, and its validity is demonstrated by its ability to describe reasonably well the isolation data of two different tetracycline-fermented broths produced according to two different technologies.The application of the model requires only the knowledge of easily obtainable fermented broth parameters and is illustrated for two different types of tetracycline-fermented broths. Although the model had been derived for the optimization of the overall yield of isolation of antibiotics recovered by direct precipitation, it can easily be adapted to be used for the optimization of the overall yield of isolation of antibiotics recovered by other isolation processes. (c) 1993 John Wiley & Sons, Inc.     

基因工程在β-内酰胺药物研制上的应用

本文综合国内外有关文献介绍了青酶素Ｇ酰化酶的改造,异青霉素Ｎ合成酶和头孢菌素Ｃ水解酶的克隆,以及细菌血红蛋白基因克隆进丝状真菌。比较了化学法和两步酶法转化ｃｐｃ（头孢菌素Ｃ）成７－ＡＣＡ的优缺点。还讨论了基因工程β－内酰胺抗生素研制的发展方向。     

Kinetics of bactericidal activity of antibiotics measured by luciferin-luciferase assay

ATP production, measured by the luciferin-luciferase assay, is an indicator of bacterial metabolic activity. This enzymatic assay yields rapid results (< 5 minutes), permitting multiple measurements and establishment of ATP growth curves in order to study the kinetics of antibiotics in bacterial populations. The measurement of free or extracellular ATP, total ATP (extra and intracellular) and the ratio of free to total ATP are additional means of studying the bacteriostatic or bactericidal activity of antibiotics. An increase in free ATP is an indicator of extracellular movement due to alteration of the cell wall. The ratio free ATP/total ATP × 100 ≥ 50%, indicates bacteriallysis. These assays were used to study the effects of 14 antibiotics on two reference strains of Escherichia coli ATCC 25922 and Staphylococcus aureus 25923.     

Involvement of phenazines and lipopeptides in interactions between Pseudomonas species and Sclerotium rolfsii, causal agent of stem rot disease on groundnut

Aims: To determine the role of phenazines (PHZ) and lipopeptide surfactants (LPs) produced by Pseudomonas in suppression of stem rot disease of groundnut, caused by the fungal pathogen Sclerotium rolfsii. Methods and Results: In vitro assays showed that PHZ‐producing Pseudomonas chlororaphis strain Phz24 significantly inhibited hyphal growth of S. rolfsii and suppressed stem rot disease of groundnut under field conditions. Biosynthesis and regulatory mutants of Phz24 deficient in PHZ production were less effective in pathogen suppression. Pseudomonas strains SS101, SBW25 and 267, producing viscosin or putisolvin‐like LPs, only marginally inhibited hyphal growth of S. rolfsii and did not suppress stem rot disease. In contrast, Pseudomonas strain SH‐C52, producing the chlorinated LP thanamycin, inhibited hyphal growth of S. rolfsii and significantly reduced stem rot disease of groundnut in nethouse and field experiments, whereas its thanamycin‐deficient mutant was less effective. Conclusions: Phenazines and specific lipopeptides play an important role in suppression of stem rot disease of groundnut by root‐colonizing Pseudomonas strains. Significance and Impact of the Study: Pseudomonas strains Phz24 and SH‐C52 showed significant control of stem rot disease. Treatment of seeds or soil with these strains provides a promising supplementary strategy to control stem rot disease of groundnut.