一株类芽孢杆菌的分离鉴定及其抗革兰氏阴性菌活性测定

【背景】随着碳青霉烯类和多粘菌素类可转移耐药基因的发现及扩散,多重耐药革兰氏阴性细菌感染更加难以治疗。【目的】筛选有效拮抗革兰氏阴性菌的菌株,为新型抗生素的发掘奠定基础。【方法】利用胰蛋白胨大豆琼脂培养基筛选土壤源细菌,通过16S rRNA基因序列鉴定其种属;通过全基因组测序,antiSMASH比对分析菌株产抗生素潜能,双层琼脂平板法验证其抗菌活性;通过甲醇萃取其次级代谢产物,高效液相色谱串联质谱(HPLC-MS/MS)进行次级代谢产物分析。【结果】从北京周边土壤样品中分离到一株类芽孢杆菌CAU136 (Paenibacillus pabuli CAU136),经过生物信息学分析和antiSMASH比对,表明该菌株有较强合成次级代谢产物的潜能,双层琼脂平板法验证其能抑制多株革兰氏阴性菌生长,HPLC-MS/MS检测结果显示其可能分泌多粘菌素E。【结论】类芽孢杆菌CAU136可能分泌多粘菌素E,能有效拮抗革兰氏阴性菌。     

Proline residue-modified polycationic analogs of gramicidin S with high antibacterial activity against both Gram-positive and Gram-negative bacteria and low hemolytic activity.

Novel polycationic analogs of the cyclic decapeptide antibiotic, gramicidin S, possessing NH(2), D/L-Phe-NH or L-Lys-NH groups at the 4alpha- or 4beta-positions of the L-Pro residues, were synthesized. While L-Pro(4alpha/beta-NH(2))-containing analogs exhibited much weaker antibacterial activity, the D/L-Phe and L-Lys-substituted analogs exhibited higher antibacterial activity against Gram-negative bacteria than the parent gramicidin S. All of these additional amino group-containing analogs showed substantially reduced toxicity against human blood cells.     

New fluorescent rosamine chelator showing promising antibacterial activity against Gram-positive bacteria

The restricted number of antibiotics to treat infections caused by common multidrug resistant bacterial pathogens in the clinical setting demands a continuous search for new molecules with antibacterial properties. Bacterial iron deprivation represents a promising alternative, being iron chelators an attractive class for drug design in which particular compounds seem to have antibacterial effect.In this work, we report the synthesis and characterization of a new fluorescent 3-hydroxy-4-pyridinone (3,4-HPO) iron chelator functionalized with a carboxyrosamine fluorophore (MRB20). The antibacterial activity of MRB20 was assessed against representative strains from clinically relevant Gram-positive and Gram-negative bacterial species and further compared with the inhibitory effect of a set of structurally related iron chelators including Deferiprone (1,2-dimethyl-3-hydroxy-4-pyridinone). Compounds exhibiting a promising minimal inhibitory concentration (MIC < 10 mg/L) were further tested against a wider range of bacterial genera and species (Staphylococcus spp. Enterococcus spp. Listeria monocytogenes, Bacillus spp.), including multidrug resistant bacteria.With the exception of the novel compound (MRB20), all chelators inhibited the strains assayed at very high concentrations [minimum inhibitory concentrations (MIC) ranging from 70 mg/L to >180 mg/L]. MRB20 revealed a good antibacterial activity (6.7–13.2 mg/L) against Gram-positive strains from different genera and species, including clinically relevant species (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis), which might be eventually compatible with a therapeutic application or as adjuvant.     

In vitro and in vivo activity of an antibacterial peptide analog against uropathogens

The alarming rate of bacterial resistance induction highlights the clinical need for antimicrobial agents that act by novel modes of action. Based on the activity profile, the general tissue distribution and renal clearance of peptide-based drugs, we hypothesized that our newly developed pyrrhocoricin derivative would be able to fight resistant uropathogens in vitro and in vivo. Indeed, the Pip-pyrr-MeArg dimer killed all 11 urinary tract infection-related Escherichia coli and Klebsiella pneumoniae strains we studied in the sub-low micromolar concentration range. Almost all control antibiotics, including the currently leading trimethoprim-sulfametoxazole combination for urinary tract infection, remained without considerable activity against two or more of these bacterial strains. In a mouse ascending urinary tract infection model with E. coli CFT073 as pathogen, two doses of intravenous, subcutaneous or oral treatment with the Pip-pyrr-MeArg derivative reduced the bacterial counts in the kidneys, bladder and urine to varying levels. Statistically significant elimination or reduction of bacteria compared to untreated animals was observed at dual intravenous or subcutaneous doses of 0.4 or 10mg/kg, respectively. Serial passage of the same E. coli strain in the presence of sublethal doses of the designed peptide failed to generate resistant mutants. The Pip-pyrr-MeArg dimer showed no toxicity to COS-7 cells to the highest 500microM concentration studied.     

Characterisation and in vitro activities of surface attached dihydropyrrol-2-ones against Gram-negative and Gram-positive bacteria

Bacterial infection of biomedical devices is still a major barrier to their use. This is compounded by increasing antibiotic resistance. Here, the specific covalent attachment of a series of dihydropyrrol-2-one (DHP), analogues of bacterial quorum sensing inhibitors, to surfaces via a Michael-type addition reaction is described. Differences in efficiency of attachment related to the substituent groups were found by X-ray photoelectron spectroscopy. The physical characteristics of the surfaces were further explored by atomic force microscopy and contact angle measurements. The ability of these coatings to prevent the formation of a biofilm by Pseudomonas aeruginosa and Staphylococcus aureus was examined using confocal laser scanning microscopy and image analysis. The DHP-treated surfaces showed significant reductions in bacterial adhesion without increased killing for both strains of bacteria (p < 0.001). 5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one was identified as having broad spectrum activity and consequently represents an excellent candidate for the development of novel surfaces for the prevention of biomedical device infections.     

Characterisation and in vitro activities of surface attached dihydropyrrol-2-ones against Gram-negative and Gram-positive bacteria

Bacterial infection of biomedical devices is still a major barrier to their use. This is compounded by increasing antibiotic resistance. Here, the specific covalent attachment of a series of dihydropyrrol-2-one (DHP), analogues of bacterial quorum sensing inhibitors, to surfaces via a Michael-type addition reaction is described. Differences in efficiency of attachment related to the substituent groups were found by X-ray photoelectron spectroscopy. The physical characteristics of the surfaces were further explored by atomic force microscopy and contact angle measurements. The ability of these coatings to prevent the formation of a biofilm by Pseudomonas aeruginosa and Staphylococcus aureus was examined using confocal laser scanning microscopy and image analysis. The DHP-treated surfaces showed significant reductions in bacterial adhesion without increased killing for both strains of bacteria (p < 0.001). 5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one was identified as having broad spectrum activity and consequently represents an excellent candidate for the development of novel surfaces for the prevention of biomedical device infections.     

The discovery of biaryl acids and amides exhibiting antibacterial activity against Gram-positive bacteria

Solid-phase synthetic methods for biaryl-based compounds were developed resulting in the construction of two 1000-member libraries. Numerous compounds were identified by high-throughput screening using whole cell screens to exhibit anti-microbial activity against Gram-positive bacteria. A series of biaryl compounds containing natural and unnatural amino acids were made to explore the SAR of the amino acid functionality.     

Multidrug resistance in hydrocarbon-tolerant Gram-positive and Gram-negative bacteria

New Gram-positive and Gram-negative bacteria were isolated from Poeni oily sludge, using enrichment procedures. The six Gram-positive strains belong to Bacillus, Lysinibacillus and Rhodococcus genera. The eight Gram-negative strains belong to Shewanella, Aeromonas, Pseudomonas and Klebsiella genera. Isolated bacterial strains were tolerant to saturated (i.e., n-hexane, n-heptane, n-decane, n-pentadecane, n-hexadecane, cyclohexane), monoaromatic (i.e., benzene, toluene, styrene, xylene isomers, ethylbenzene, propylbenzene) and polyaromatic (i.e., naphthalene, 2-methylnaphthalene, fluorene) hydrocarbons, and also resistant to different antimicrobial agents (i.e., ampicillin, kanamycin, rhodamine 6G, crystal violet, malachite green, sodium dodecyl sulfate). The presence of hydrophilic antibiotics like ampicillin or kanamycin in liquid LB-Mg medium has no effects on Gram-positive and Gram-negative bacteria resistance to toxic compounds. The results indicated that Gram-negative bacteria are less sensitive to toxic compounds than Gram-positive bacteria, except one bacteria belonging to Lysinibacillus genus. There were observed cellular and molecular modifications induced by ampicillin or kanamycin to isolated bacterial strains. Gram-negative bacteria possessed between two and four catabolic genes (alkB, alkM, alkB/alkB1, todC1, xylM, PAH dioxygenase, catechol 2,3-dioxygenase), compared with Gram-positive bacteria (except one bacteria belonging to Bacillus genus) which possessed one catabolic gene (alkB/alkB1). Transporter genes (HAE1, acrAB) were detected only in Gram-negative bacteria.     

Functional analysis of antibacterial activity of Bacillus amyloliquefaciens phage endolysin against Gram-negative bacteria.

To analyze the antibacterial activity of Bacillus amyloliquefaciens phage endolysin, nine deletion derivatives of the endolysin were constructed. Each deletion mutant was overexpressed, purified and characterized. The catalytic domain was located on the N-terminal region and the C-terminus had an affinity with the bacterial envelope. The enzymatic activity remained in spite of the deletion of the C-terminal 116-amino acid region; however, the antibacterial activity was lost. These results indicate that antibacterial action requires both the C-terminal cell-binding and the N-terminal enzymatic activities.     

Water-soluble phosphate prodrugs of pleuromutilin analogues with potent in vivo antibacterial activity against Gram-positive pathogens

A phosphate prodrug strategy was investigated to address the problem of poor aqueous solubility of pleuromutilin analogues. Water-soluble phosphate prodrugs 6a, 6b and 6c of pleuromutilin analogues were designed and synthesized. Three compounds all exhibited excellent aqueous solubility (>50 mg/mL) at near-neutral pH and sufficient stability in buffer solution. In particular, the phenol pleuromutilin prodrug 6c displayed favourable pharmacokinetic profiles and comparable potency with vancomycin against MSSA and MRSA strains in vivo.     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

In vitro antibacterial activity of Lactobacillus helveticus strain KS300 against diarrhoeagenic, uropathogenic and vaginosis-associated bacteria

AIMS: The purpose of this study was to investigate in vitro the antibacterial activity of the Lactobacillus helveticus strain KS300 against vaginosis-associated bacteria including Gardnerella vaginalis and Prevotella bivia, uropathogenic Escherichia coli, and diarrhoeagenic Salmonella enterica serovar Typhimurium. METHODS AND RESULTS: The KS300 strain inhibited the growth of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. After direct co-culture, data show that the Lactobacillus strain decreased the viability of G. vaginalis, P. bivia, S. typhimurium, and pathogenic E. coli. The adhering KS300 strain inhibited the adhesion of G. vaginalis DSM 4944 and uropathogenic Dr-positive E. coli IH11128 onto HeLa cells. Moreover, the KS300 strain inhibited the internalization of uropathogenic Dr-positive E. coli IH11128 within HeLa cells and S. typhimurium SL1344 within Caco-2/TC7 cells. CONCLUSIONS: The findings demonstrate that L. helveticus strain KS300 is adhesive onto cultured human cells and has antagonistic activities against vaginosis-associated, uropathogenic and diarrhoeagenic pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Adhering L. helveticus strain KS300 is a potential probiotic strain displaying a strain-specific array of in vitro antibacterial activities.     

Testing of different antibiotics against Gram-positive and Gram-negative bacteria isolated from plant tissue culture

Different Gram-positive and Gram-negative bacteria (Staphylococcus xylosus, S. aureus, S. cohnii, Bacillus sp., Corynebacterium sp., Pseudomonas vesicularis) were isolated from homogenized shoot tips of Drosera rotundifolia, Spatiphyllum sp., Syngonium cv. White butterfly, Nephrolepis exaltata cv. Teddy Junior. Growth inhibition of selected bacterial strains was examined using 28 different single antibiotics and 7 antibiotic mixtures. It was found that with the two mixtures Imipenem/Ampicillin and Imipenem/Penicillin G at concentrations of 5 mg l^–1 each, bacterial growth inhibition was most effective. Because of the lack of toxic effects on in vitro plants of 7 species it was proposed that these antibiotic mixtures can be applied advantageously to inhibit bacterial growth in tissue culture.     

Combination studies between polycationic peptides and clinically used antibiotics against Gram-positive and Gram-negative bacteria

The in vitro interaction between five polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, and ranalexin, and several clinically used antimicrobial agents was evaluated against several clinical isolates of Gram-positive and Gram-negative aerobic bacteria, using the microbroth dilution method. The combination studies showed synergy between ranalexin and polymyxin E, doxycycline and clarithromycin. In addition, magainin II was shown to be synergic with betalactam antibiotics.     

Mechanism of chlorpromazine binding by Gram-positive and Gram-negative bacteria

Chlorpromazine forms charge-transfer complexes with xanthene dyes in bacteria. These complexes permit the differentiation of Gram-positive and Gram-negative bacteria in both light and polarization microscopy. The birefringence induced by the charge-transfer complex might explain the molecular basis of bacterial staining.The charge-transfer complexes formed between chorpromazine and xanthene dyes accumulate in the bacterial cell, mainly inside the bacterial cell wall. The complexes give the cells a color, which depends on the chemical composition of the staining structure, and in particular the polysaccharides of the cell wall in bacteria.Metachromatic granules were seen inside Gram-positive bacteria after chlorpromazine and rose bengal staining. Although the nature of these granules remains unclear, this type of binding may have a role in the inhibition of biochemical processes in the bacterial cells.     

LOX-1 supports adhesion of Gram-positive and Gram-negative bacteria

Adhesion of bacteria to vascular endothelial cells as well as mucosal cells and epithelial cells appears to be one of the initial steps in the process of bacterial infection, including infective endocarditis. We examined whether lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), a member of scavenger receptor family molecules with C-type lectin-like structure, can support adhesion of Gram-positive and Gram-negative bacteria. Chinese hamster ovary-K1 (CHO-K1) cells stably expressing LOX-1 can support binding of FITC-labeled Staphylococcus aureus and Escherichia coli, which was suppressed by poly(I) and an anti-LOX-1 mAb. Adhesion of these bacteria to LOX-1 does not require divalent cations or serum factors and can be supported under both static and nonstatic conditions. Cultured bovine aortic endothelial cells (BAEC) can also support adhesion of FITC-labeled S. aureus, which was similarly suppressed by poly(I) and an anti-LOX-1 mAb. In contrast, binding of FITC-labeled E. coli to BAEC was partially inhibited by the anti-LOX-1 mAb, and poly(I) did not block FITC-labeled E. coli adhesion to BAEC, but, rather, enhanced it under a static condition. TNF-alpha increased LOX-1-dependent adhesion of E. coli, but not that of S. aureus, suggesting that S. aureus adhesion to BAEC may require additional molecules, which cooperate with LOX-1 and suppressed by TNF-alpha. Taken together, LOX-1 can work as a cell surface receptor for Gram-positive and Gram-negative bacteria, such as S. aureus and E. coli, in a mechanism similar to that of class A scavenger receptors; however, other unknown molecules may also be involved in the adhesion of E. coli to BAEC, which is enhanced by poly(I).