Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides.

To investigate the influence of proline residues on the activity of alpha-helical peptides, variants were synthesized with insertions of proline residues to create peptides without proline, or with one or two prolines. The influence of the proline-induced bends was assessed by circular dichroism in the presence of liposomes, and the ability of the peptides to kill microorganisms, to permeabilize the outer and cytoplasmic membranes of Escherichia coli, to bind to liposomes, to form channels in planar lipid bilayers, and to synergize with conventional antibiotics. Representative peptides adopted alpha-helical conformations in phosphatidylcholine/phosphatidylglycerol (POPC/POPG, 7:3) liposomes as well as in 60% trifluoroethanol solution, as revealed by circular dichroism (CD) spectroscopy. However, the percent of helicity decreased as the number of proline residues increased. Tryptophan fluorescence spectroscopy showed that all of these peptides inserted into the membranes of liposomes as indicated by a blue shift in the emission maximum and an increase in the fluorescence intensity of the single tryptophan at residue 2. Quenching experiments further prove that the tryptophan residue was no longer accessible to the aqueous quencher KI. The peptide that lacked proline exhibited the highest activity [minimal inhibitory concentrations (MICs) of 0.5-4 microg/mL] against all tested Gram-negative and Gram-positive bacteria, but was hemolytic at 8 microg/mL. The single-proline peptides exhibited intermediate antibacterial activity. Peptides with two proline residues were even less active with moderate MICs only against E. coli. With only one exception from each group, the peptides were nonhemolytic. The ability of the peptides to demonstrate synergy in combination with conventional antibiotics increased as the antibacterial effectiveness decreased. All peptides bound to bacterial lipopolysaccharide and permeabilized the outer membrane of E. coli to similar extents. However, their ability to permeabilize the cytoplasmic membrane of E. coli as assessed by the unmasking of cytoplasmic beta-galactosidase decreased substantially as the number of proline residues increased. Correspondingly, increasing the number of proline residues caused a decreased ability to form channels in planar lipid bilayers, and the hemolytic, proline-free peptide tended to cause rapid breakage of planar membranes. Thus, the number of bends created by insertion of proline residues is an important determinant of antimicrobial, hemolytic, and synergistic activity.     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Antibacterial effect of oregano essential oil alone and in combination with antibiotics against extended-spectrum beta-lactamase-producing Escherichia coli.

In this paper, we studied the antibacterial effects of oregano essential oil (OEO) both alone, using a twofold dilution method, and combined with antibiotics, using a checkerboard microtitre assay, against extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. The result indicated that multiple drug-resistant E. coli was very sensitive to OEO and polymycin; their minimal inhibitory concentration values are 0.5 microL mL(-1) and 0.8 microg mL(-1). The antibacterial effects of OEO in combination with kanamycin were independent against E. coli, with fractional inhibitory concentration (FIC) indices of 1.5. The antibacterial effects of OEO combined with amoxicillin, polymycin, and lincomycin showed an additive effect against E. coli, with FIC indices in the range of 0.625-0.750. The antibacterial effects of OEO in combination with fluoroquinolones, doxycycline, lincomycin, and maquindox florfenicol displayed synergism against E. coli, with FIC indices ranging from 0.375 to 0.500. The combination of OEO with fluoroquinolones, doxycycline, lincomycin, and maquindox florfenicol to treat infections caused by ESBL-producing E. coli may lower, to a great extent, the effective dose of these antibiotics and thus minimize the side effects of antibiotics. This is the first report on OEO against ESBL-producing E. coli.     

Apolipoprotein A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.     

Design and synthesis of diketopiperazine and acyclic analogs related to the caprazamycins and liposidomycins as potential antibacterial agents

A systematic simplification methodology of a class of 6'-N-alkyl-5'-O-aminoribosyl-glycyluridine antibiotics was shown to produce potential antibacterial agents having a novel mechanism of action. Diketopiperazines and acyclic analogs of the caprazamycins (CPZs) and liposidomycins (LPMs) were efficiently synthesized, and their antibacterial activity was evaluated. The diketopiperazine analog 11a and the acyclic analogs 12a and 16a having a palmitoyl group as a lipophilic side chain exhibited moderate antibacterial activities with MICs of 12.5-50 microg/mL. This approach could provide ready access to a range of analogs for the development of potential antibacterial agents.     

Chloropyrimidines as a new class of antimicrobial agents

In the course of our investigations of pyrimidines as antimycotic agents, we have identified a sub-class, with significant in vitro activity against mycobacteria. The salient feature of these pyrimidine derivatives (3a-o and 7a,b) is their appended aryl, heteroaryl and alkylthio substituent at position 6 and also alkylthio substituent at position 2. The rational design, synthesis, and evaluation of the in vitro antibacterial activity against six pathogenic bacteria including virulent and non-virulent strains of Mycobacterium tuberculosis is described. Some of the synthesized compounds (3c, 3h, 3i, 3o) have displayed only potent in vitro antimycobacterial activity with MIC of 0.75 microg/mL except 3i which also demonstrated activity against Escherichia coli at 12.5 microg/mL concentration. Only two compounds, 3a and 3b, demonstrated antibacterial activity against Pseudomonas aeruginosa and E. coli with MIC 12.5 microg/mL. All the synthesized compounds were also evaluated for their antimycotic activity against five pathogenic fungi but only some of them 3j-n and 7a,b were found most potent against Aspergillus fumigatus and Trichophyton mentagrophytes.     

In vitro activity of beta-lactam antibiotics and their sensitivity to beta-lactamase

beta-lactamase production was evaluated by chromogenic cephalosporin 87/312 in 116 E. coli isolated from clinical sources. Such test revealed beta-lactamase production in 54 strains out of 116 (46%): MICs of eight beta-lactam antibiotics (Ampicillin, Piperacillin, Cefazoline, Cephaloridine, Cephalexine, Cefuroxime, Cefotaxime, Cefotaxime) were determined using a miniaturized dilution broth method. Cefotaxime and Ceftriaxome and Ceftriaxone showed the highest antibacterial activity. All beta-lactamases produced by E. coli strains under examination were isolated and purified by ultrasonic disruption and high speed centrifugation. Sensitivity of the eight antibiotics to purified beta-lactamases was assessed by a spectrophotometric method that utilizes the velocity of cytochrome c reduction. The sensitivity to beta-lactamases was reflected in the in vitro activity of the antibiotics as assessed by the determination of the MICs.     

Amino acid conjugated sophorolipids: A new family of biologically active functionalized glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).     

Antibacterial activity and chemical composition of Turkish propolis

The antibacterial activities of propolis samples have been examined in vitro, according to the principles accepted for the determination of a similar activity of antibiotics with the use of solid and liquid media. It has been found that propolis extracts showed antibacterial activity through a range of commonly encountered gram positive cocci (S. aureus, beta hem. Streptococus), but had weak activity against gram negative bacteria (E. coli, P. aeruginosa). GC/MS analysis showed that propolis samples contain a variety of chemical compounds including aromatic compounds, fatty acid esters and sesquiterpenes.     

2-Aminotetralones: novel inhibitors of MurA and MurZ

Several 2-aminotetralones were identified as novel inhibitors of the bacterial enzymes MurA and MurZ. A number of these inhibitors demonstrated antibacterial activity against Staphylococcus aureus and Escherichia coli with MICs in the range 8-128 microg/ml. Based on structure-activity relationships we propose that the alpha-aminoketone functionality is responsible for the inhibitory activity and evidence is provided to support a covalent mode of action involving the C115 thiol group of MurA/MurZ.     

The effects of charge and lipophilicity on the antibacterial activity of undecapeptides derived from bovine lactoferricin.

We have investigated the effects of charge and lipophilicity on the antibacterial activity of an undecapeptide (FKCRRWQWRMK) derived from the sequence of bovine lactoferricin. We prepared ten analogues that were modified by the incorporation of Ala, Tyr, Trp, Met and Arg residues, which are amino acids known to be important for the antibacterial activity of longer derivatives of lactoferricins. All undecapeptides contained the native Trp residues in positions 6 and 8, and the Arg residues in positions 5 and 9. Generally, the Gram-positive bacterium Staphylococcus aureus was more susceptible to these undecapeptides than the Gram-negative bacteria, and a higher antibacterial activity was observed against Escherichia coli than against Pseudomonas aeruginosa. The only exception was the peptide Undeca 9 (RRWYRWAWRMR-NH2), which was almost equally active against all three test strains, displaying minimal inhibitory concentrations of 10 microg/ml (5.8 microM), 7.5 microg/ml (4.4 microM) and 5 microg/ml (2.9 microM) against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, respectively. The peptides Undeca 6 (YRAWRWAWRWR-NH2) and Undeca 7 (YRMWRWAWRWR-NH2) were the two most active undecapeptides against Staphylococcus aureus, both displaying a minimal inhibitory concentration of 2.5 microg/ml (1.5 microM). The study showed that a level was reached in which undecapeptides having a net charge above +4 and containing three or four Trp residues all displayed a high antibacterial activity. All undecapeptides prepared were essentially non-haemolytic, but undecapeptides containing more than three Trp residues displayed 50% haemolysis of human red blood cells at concentrations above 400 microg/ml (>230 microM).     

禽源致病性大肠埃希菌的耐药性与中草药有效成分的抑菌活性测定

以28株合肥地区禽源致病性大肠埃希菌为实验材料,采用K-B纸片琼脂扩散法检测禽源致病性大肠埃希菌的耐药情况。同时采用平板打孔法测定盐酸小檗碱、绿原酸、靛玉红和丹参酮ⅡA 4种中草药有效成分的抑菌活性。结果表明,28株禽源致病性大肠埃希菌对17种抗菌药物均呈现不同程度的耐药性,对β-内酰胺类、氨基糖苷类、四环素类和喹诺酮类抗菌药物的耐药率分别介于0%～92.86%、14.29%～50.00%、78.57%～100%和57.14%～71.43%。中草药有效成分盐酸小檗碱和丹参酮ⅡA对大肠埃希菌具有较好的抑制活性,抑菌率分别为92.86%（26/28）和89.29%（25/28）。     

Synthesis, absolute stereochemistry and molecular design of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201

The absolute stereochemistry of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201 has been established by achieving the total synthesis of the product. A series of analogues have also been synthesized by changing the side chain and their bioactivity assessed against different microbial strains. Among them, 1e (R = C8H17) was found to be the most potent with MIC of 8 microg/mL against Mycobacterium tuberculosis, 12 microg/mL against Escherichia coli and 16 microg/mL against Bacillus subtilis 6 microg/mL against Proteus vulgaris. This was followed by 1b (R = C5H11) with MIC of 10-20 microg/mL range and 1d (R = C7H15) with MIC of 14-24 g/mL, whereas 1a (R = C4H9) and 1f (R = C18H35) were found to be completely inactive. Besides, 1c (R = C6H13) showed certain extent of antibacterial activity in the range of 24-50 microg/mL. Mycobacterium tuberculosis was very sensitive to 1e (R = C8H17) with MIC of 8 microg/mL. Antifungal activity of analogues 1d (R = C7H15) and 1e, (R = C8H17) against Fusarium oxysporum and Rhizoctonia solani were found promising with MFCs in the 15-18 microg/mL range.     

S-Euglobals: biomimetic synthesis, antileishmanial, antimalarial, and antimicrobial activities

Several new euglobal analogues (named as S-euglobals) were synthesized from phloroglucinol via a biomimetic three-component reaction involving Knoevenagel condensation followed by [4+2]-Diels-Alder cycloaddition with monoterpene. Newly synthesized euglobal analogues involve monoterpenes that have not yet been encountered in natural euglobals. S-Euglobals along with previously synthesized robustadial A and B were evaluated for in vitro antileishmanial, antimalarial, antimicrobial, and cytotoxic activities. Out of 16, nine analogues were found to exhibit antileishmanial activity against Leishmania donovani promastigotes. Analogue 7 was the most potent with IC(50) of 2.4 microg/mL and IC(90) of 8 microg/mL, followed by analogues 8 and 11 (IC(50) 5.5 and 9.5 microg/mL). Antileishmanial activity of robustadial A (5) and B (6) was moderate with IC(50) of 20 and 16 microg/mL, respectively. Robustadial A and B and S-euglobal 8 exhibited weak antimalarial activity against Plasmodium falciparum (IC(50) of 2.7-4.76 microg/mL). Few of the euglobal analogues showed antibacterial activity against methicillin-resistant Staphylococcus aureus. Amongst these, analogue 11 was the most potent with IC(50) of 1.0 microg/mL and MIC of 5.0 microg/mL. Most of the compounds were not cytotoxic up to 25 microg/mL in a panel of cell lines consisting of both cancer (SK-MEL, KB, BT-549, and SK-OV-3) as well as non-cancer kidney (Vero and LLC-PK11) cells.     

Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 microg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at <2 microg/ml for Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 mug/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.     

黑大蒜提取物联合抗生素对金黄色葡萄球菌和肠埃希菌的体外抗菌作用研究

评价黑大蒜提取物分别与头孢唑林或庆大霉素联合应用,对金黄色葡萄球菌和大肠埃希菌的体外抗菌效应。采用液体稀释法分别测定黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的最低抑菌浓度（MIC）。采用棋盘法设计,微量肉汤稀释法测定黑大蒜提取物联合头孢唑林或庆大霉素对金黄色葡萄球菌和大肠埃希菌的MIC,并计算部分抑菌浓度（FIC指数）。测定黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的时间-杀菌曲线。黑大蒜提取物对金黄色葡萄球菌的MIC为256μg/mL,黑大蒜提取物对大肠埃希菌的MIC为256μg/mL。时间-杀菌曲线结果显示黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的抑菌作用呈现较强的浓度依赖性。黑大蒜提取物联合头孢唑林后对金黄色葡萄球菌的FIC指数为0.75;黑大蒜提取物联合庆大霉素后对大肠埃希菌的FIC指数为0.5。黑大蒜提取物与头孢唑林或庆大霉素联合用药,可明显降低抗生素对金黄色葡萄球菌和大肠埃希菌的MIC,表现为相加和协同效应。     

Synthesis and antibacterial activity of novel 6-O-substituted erythromycin A derivatives

A series of novel 6-O-substituted erythromycin A derivatives has been synthesized. Good in vitro antibacterial activity has been demonstrated for analogues incorporating a variety of structural features. The methodology disclosed is expected to find application in the design of future macrolide antibiotics that target the prevalent bacterial resistance problem.     

High level expression of His-tagged colicin 5 in E. coli and characterization of its narrow-spectrum bactericidal activity and pore-forming action

Since antibiotics with a broad spectrum of activity would select for resistance among the normal flora, colicins having a narrow spectrum of activity can potentially be developed as novel antibiotics. Colicin-based bactericidal proteins with modified spectra of activity might also be developed by further gene fusion or gene modification. To achieve these goals, it is necessary to first build an efficient system to produce large amounts of colicin. In the presence of an immunity gene, we successfully constructed an expression vector pQE30-cfa-cfi producing high levels of His-tagged colicin 5 (60-80 mg/L). We found that the purified His-tagged colicin 5 possesses narrow-spectrum bactericidal activity against nonimmune Escherichia coli cells. It is highly toxic to sensitive E. coli cells at a low concentration of 0.01 microg/ml, while it is nontoxic to other tested gram-negative bacteria, gram-positive bacteria and yeast at a high concentration of 1000 microg/ml. His-tagged colicin 5 kills sensitive cells by permeabilizing their cell membranes. It is not hemolytic to rabbit erythrocytes and has no obvious cytotoxicity to other nucleated mammalian cells at a high concentration of 500 microg/ml. The His-tagged colicin 5 is similar to wild-type colicin 5 in spectrum and bactericidal activity against E. coli. It is a potential novel antibiotic particularly for treating human and animal infections caused by pathogenic E. coli. Besides producing high level of colicin 5, the highly efficient expression vector constructed here might also be a useful tool to develop colicin-based artificial bactericidal proteins.     

2011～2015年医院呼吸内科常见革兰阴性菌耐药性分析与临床用药探讨

目的：分析2011～2015年我院呼吸内科常见革兰阴性菌的分布情况及耐药性特点,为规范抗生素的合理使用提供依据。方法选用法国梅里埃公司生产的ID 32GD试剂盒,应用ATB Expression全自动分析系统对细菌进行鉴定与药敏试验。结果2011～2015年在我院呼吸内科常见的病原菌分别为肺炎克雷伯菌（31．0％）、大肠埃希菌（21．2％）、铜绿假单胞菌（23．2％）与鲍曼不动杆菌（14．8％）。肺炎克雷伯菌对碳青霉烯类的耐药率几乎为0,对单纯的头孢菌素耐药率相对较高,但对含β‐内酰胺酶抑制剂的头孢菌素类以及阿米卡星有较强的抗菌活性;大肠埃希菌对哌拉西林耐药率高,但对含酶青霉素类有较强的抗菌活性,对碳青霉烯类的耐药率连续5年来均为零;铜绿假单胞菌对磺胺类耐药性较高,对头孢三代、头孢四代及含酶青霉素类与含酶头孢类均有较强的抗菌活性,但敏感性呈逐年下降趋势;鲍曼不动杆菌的耐药率呈逐年上升趋势,仅对含酶的头孢哌酮／舒巴坦敏感（约80％）。结论医院呼吸内科革兰阴性菌仍以肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌为主。鲍曼不动杆菌耐药性逐年增长的问题已日趋严重,相关部门应定期监测细菌耐药趋势,指导临床合理用药,特别是合理应用抗生素以避免细菌耐药及减少细菌耐药性的发生。