三种头霉素类抗生素对大肠埃希菌和肺炎克雷伯菌的抗菌活性评价

目的 评价头孢美唑(CMZ)、头孢西丁(FOX)及头孢替坦(CTT)三种头霉素类抗生素对大肠埃希菌(ECO)和肺炎克雷伯菌(KPN)的体外抗菌活性.方法 对510株ECO和472株KPN,采用K-B琼脂扩散法进行CMZ、FOX及CTT的体外药敏试验,并用表型确证试验检测超广谱β-内酰胺酶(ESBLs).结果 ECO和KPN ESBLs的检出率分别为61.8％和41.3％;CMZ、FOX和CTT对ECO和KPN ESBLs阴性菌株敏感率均在96％以上;对于产ESBLs的ECO,CMZ、FOX和CTT的敏感率分别为95.6、85.7％和97.8％,经比较CMZ和CTT的敏感率均明显高于FOX的敏感率(P＜0.05);对于产ESBLs的KPN,CMZ、FOX和CTT的敏感率分别为81.0％、80.0％和100.0％,经比较CMZ和FOX的敏感率均明显低于CTT的敏感率(P＜0.05).结论 作为治疗产ESBLs菌株引起的感染头霉素类抗生素可代替三代头孢菌素作为经验治疗药物,但仍应参考药敏试验结果.CTT对产ESBLs的ECO和KPN菌株的体外抗菌活性优于CMZ和FOX.     

Fermentation mechanism of fucose and rhamnose in Salmonella typhimurium and Klebsiella pneumoniae.

An equimolar amount of 1,2-propanediol was detected in the medium when Salmonella typhimurium or Klebsiella pneumoniae fermented L-fucose or L-rhamnose. These metabolic conditions induced a propanediol oxidoreductase that converted the lactaldehyde formed in the dissimilation of either sugar into the diol. The enzyme was further identified by cross-reaction with antibodies against Escherichia coli propanediol oxidoreductase. This indicates that L-fucose and L-rhamnose fermentation takes place in these species by 1,2-propanediol production and excretion.     

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.     

Effects of Brazilian green propolis extract on planktonic cells and biofilms of multidrug-resistant strains of Klebsiella pneumoniae and Pseudomonas aeruginosa

Abstract

Propolis could represent an alternative therapeutic agent for targeting multidrug-resistant bacteria due to its antimicrobial potential. The effect of Brazilian green propolis (BGP) aqueous extract (AqExt) was evaluated on eight multidrug-resistant clinical strains of Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as on one reference strain for each bacterial species. The minimum bactericidal concentration (MBC) was determined and optimal concentrations were further evaluated in comparison with 0.12% chlorhexidine. The natural extract was chemically characterized by HPLC-DAD analysis. The MBC values ranged between 3.12 and 27.5?mg ml^?1. Analysis of bacterial metabolic activity after treatment for 5?min with BGP-AqExt revealed a strong antimicrobial potential, similar to chlorhexidine. The extract comprised several active compounds including quercetin, gallic acid, caffeic and p-coumaric acid, drupani, galangin, and artepillin C. Altogether, the findings suggest that BGP-AqExt is fast and effective against multidrug-resistant strains of K. pneumoniae and P. aeruginosa in planktonic cultures and biofilms.     

Implication of reactive oxygen species in the antibacterial activity against Salmonella typhimurium of hepatocyte cell lines

We recently described the antibacterial activity of a murine hepatocyte cell line stimulated with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and lipopolysaccharide (LPS) against intracellular Salmonella organisms. Here we show for the first time the existence of basal antibacterial activity in cultured hepatocyte cell lines. Thus treatment of resting and stimulated hepatocytes with catalase or superoxide dismutase increased bacterial number recovered per monolayer, which suggests that the mechanism involved with antibacterial activity of hepatocytes is mediated by reactive oxygen species (ROS). Also, the capacity of these cell lines to generate intracellular peroxides under resting and stimulated conditions was investigated. This revealed that IL-1 and LPS did not induce any increase in the amount of intracellular peroxides by themselves, but they primed IFN-gamma for maximal induction of peroxides. The intracellular amount of peroxides was highly increased on stimulation with IFN-gamma, IL-1, and LPS, and it was strongly inhibited by catalase. This explains that the mechanism whereby this enzyme inhibits antibacterial activity takes place by decreasing the intracellular pool of peroxides. In turn, experiments performed in the presence of several inhibitors of metabolic pathways involved in ROS generation suggested that cyclo-oxygenase are a source of these species in hepatocyte cell lines. These results attribute a prominent role to the generation of peroxides as effector molecules of antibacterial activity in hepatocyte cell lines. Thus these cells displayed a moderate basal level, which increased on stimulation with proinflammatory cytokines such as IFN-gamma, IL-1, and bacterial products such as LPS. Finally, it has been also shown for the first time that IFN-gamma stimulation induces production of peroxides in human and murine hepatocyte cell lines.     

Ginseng extract exhibits antimutagenic activity against induced mutagenesis in various strains of Salmonella typhimurium

Ginseng has been reported to exhibit antioxidant and antimutagenic activity. The present study was undertaken with a view to confirm whether the antioxidant activity of Ginseng is responsible for its antimutagenic action. The concentrated root extract of Panax ginseng (Ginseng extract I) and its lyophilized powder (Ginseng extract II) obtained from two different manufacturing houses, were tested against mutagenesis using the well-standardized Ames microsomal test system. The extracts exhibited antimutagenic effect against hydrogen peroxide induced mutagenesis in TA100 strain, and against mutagenesis produced by 4-nitroquinoline-N-oxide in both TA98 and TA100 strains of Salmonella typhimurium. Both the extracts failed to show any antimutagenic potential against tert-butyl hydroperoxide (an oxidative mutagen) in TA102 strain, a strain highly sensitive to active oxygen species. The extracts also indicated a weak antioxidant activity in a series of in vitro test systems viz., 1,1-diphenyl picryl hydrazyl (DPPH) assay, hydrogen peroxide scavenging and superoxide anion scavenging. The results indicate that the protective effects shown by ginseng extract(s) against 4-nitroquinoline-n-oxide and hydrogen peroxide induced mutagenesis in TA98 and TA100 could mainly be due to its property to initiate and promote DNA repair rather than free radical scavenging action.     

Antibacterial activity and dual mechanisms of peptide analog derived from cell-penetrating peptide against Salmonella typhimurium and Streptococcus pyogenes

A number of research have proven that antimicrobial peptides are of greatest potential as a new class of antibiotics. Antimicrobial peptides and cell-penetrating peptides share some similar structure characteristics. In our study, a new peptide analog, APP (GLARALTRLLRQLTRQLTRA) from the cell-penetrating peptide ppTG20 (GLFRALLRLLRSLWRLLLRA), was identified simultaneously with the antibacterial mechanism of APP against Salmonella typhimurium and Streptococcus pyogenes. APP displayed potent antibacterial activity against Gram-negative and Gram-positive strains. The minimum inhibitory concentration was in the range of 2 to 4 μM. APP displayed higher cell selectivity (about 42-fold increase) as compared to the parent peptide for it decreased hemolytic activity and increased antimicrobial activity. The calcein leakage from egg yolk l-α-phosphatidylcholine (EYPC)/egg yolk l-α-phosphatidyl-dl-glycerol and EYPC/cholesterol vesicles demonstrated that APP exhibited high selectivity. The antibacterial mechanism analysis indicated that APP induced membrane permeabilization in a kinetic manner for membrane lesions allowing O-nitrophenyl-β-d-galactoside uptake into cells and potassium release from APP-treated cells. Flow cytometry analysis demonstrated that APP induced bacterial live cell membrane damage. Circular dichroism, fluorescence spectra, and gel retardation analysis confirmed that APP interacted with DNA and intercalated into the DNA base pairs after penetrating the cell membrane. Cell cycle assay showed that APP affected DNA synthesis in the cell. Our results suggested that peptides derived from the cell-penetrating peptide have the potential for antimicrobial agent development, and APP exerts its antibacterial activity by damaging bacterial cell membranes and binding to bacterial DNA to inhibit cellular functions, ultimately leading to cell death.     

Identifying the drivers of multidrug-resistant Klebsiella pneumoniae at a European level

Beta-lactam- and in particular carbapenem-resistant Enterobacteriaceae represent a major public health threat. Despite strong variation of resistance across geographical settings, there is limited understanding of the underlying drivers. To assess these drivers, we developed a transmission model of cephalosporin- and carbapenem-resistant Klebsiella pneumoniae. The model is parameterized using antibiotic consumption and demographic data from eleven European countries and fitted to the resistance rates for Klebsiella pneumoniae for these settings. The impact of potential drivers of resistance is then assessed in counterfactual analyses. Based on reported consumption data, the model could simultaneously fit the prevalence of extended-spectrum beta-lactamase-producing and carbapenem-resistant Klebsiella pneumoniae (ESBL and CRK) across eleven European countries over eleven years. The fit could explain the large between-country variability of resistance in terms of consumption patterns and fitted differences in hospital transmission rates. Based on this fit, a counterfactual analysis found that reducing nosocomial transmission and antibiotic consumption in the hospital had the strongest impact on ESBL and CRK prevalence. Antibiotic consumption in the community also affected ESBL prevalence but its relative impact was weaker than inpatient consumption. Finally, we used the model to estimate a moderate fitness cost of CRK and ESBL at the population level. This work highlights the disproportionate role of antibiotic consumption in the hospital and of nosocomial transmission for resistance in gram-negative bacteria at a European level. This indicates that infection control and antibiotic stewardship measures should play a major role in limiting resistance even at the national or regional level.     

Antibacterial activity and carbapenem re-sensitizing ability of 1,10-phenanthroline-5,6-dione and its metal complexes against KPC-producing Klebsiella pneumoniae clinical strains

Infections caused by KPC-producing Klebsiella pneumoniae (Kp-KPC) are associated with high mortality rates due to the increased number of resistant isolates and the scarcity of therapeutic options. This scenario reinforces the urgent need for new chemotherapeutics. Herein, we investigated the effects of 1,10-phenanthroline-5,6-dione (phendione) and its metal-based complexes, [Cu(phendione)₃](ClO₄)₂.4H₂O (Cu–phendione) and [Ag(phendione)₂]ClO₄ (Ag–phendione), both alone and also combined with carbapenems (meropenem (MEM), and imipenem), against 46 clonally distinct clinical strains of Kp-KPC. All isolates were found to be multidrug resistant in accordance with their susceptibility patterns by disk diffusion method. Compounds geometric mean (GM)-MIC and GM-MBC values (μmol l⁻¹), respectively, were: phendione, 42·06 and 71·27; Cu–phendione, 9·88 and 13·75; and Ag–phendione, 10·10 and 13·06. Higher synergism rates of MEM-containing combinations were observed by the checkerboard assay, particularly with the two metal complexes. Moreover, drug combinations were able to re-sensitize 87% of the phenotypically non-susceptible strains. Time-kill studies, with MEM plus Cu–phendione or Ag–phendione, indicated that combinations with 0·5× MIC of each agent produce synergistic effects after 9–12 h. The MEM plus Ag–phendione eradicated about 10⁶ CFU per ml of bacteria. These findings support the effectiveness of the re-sensitizing combinatorial approach and provide evidence that phendione-based compounds offer real promise in the fight against Kp-KPC infections.     

Nucleotide sequences of the genes encoding type 1 fimbrial subunits of Klebsiella pneumoniae and Salmonella typhimurium.

The nucleotide sequences of the genes encoding the subunits of Klebsiella pneumoniae and Salmonella typhimurium type 1 fimbriae were determined. Comparison of the predicted amino acid sequences of the two subunits revealed domains in which the sequences were highly conserved. Both gene products possessed signal peptides, a fact consistent with the transport of the fimbrial subunit across the membrane, but these regions showed no amino acid homology between the two proteins. The predicted N-terminal amino acid sequences of the processed fimbrial subunits were in good agreement with those obtained by purification of the fimbrial subunits.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.     

The influence of cationic dendrimers on antibacterial activity of phage endolysin against P. aeruginosa cells

Nowadays, the researchers make a big effort to find new alternatives to overcome bacterial drug resistance. One option is the application of bacteriophage endolysins enable to degrade peptidoglycan (PG) what in consequence leads to bacterial cell lysis. In this study we examine phage KP27 endolysin mixed with poly(propyleneimine) dendrimers to evaluate an antimicrobial effect against Pseudomonas aeruginosa. Polycationic compounds destabilize bacterial outer membrane (OM) helping endolysins to gain access to PG. We found out that not only bacterial lipopolysaccharide (LPS) is the main hindrance for highly charged cationic dendrimers to disrupt OM and make endolysin reaching the target but also the dendrimer surface modification. The reduction of a positive charge and concentration in maltose poly(propyleneimine) dendrimers significantly increased an antibacterial effect of endolysin. The application of recombinant lysins against Gram-negative bacteria is one of the future therapy options, thus OM permeabilizers such as cationic dendrimers may be of high interest to be combined with PG-degrading enzymes.     

Design and expression of peptides with antimicrobial activity against Salmonella typhimurium

We showed previously that insertion of Synechocystis Δ¹²‐desaturase in salmonella's membrane alters membrane physical state (MPS), followed by the expression of stress genes causing inability to survive within murine macrophages (MΦ). Recently, we showed that expression of one membrane lipid domain (MLD) of Δ¹²‐desaturase (ORF200) interferes with salmonella MPS, causing loss of virulence in mice and immunoprotection. Here, we postulate that an α‐antimicrobial peptide (α‐AMP) intercalates within membrane lipids, and depending on its amino acid sequence, it does so within specific key sensors of MLD. In this study, we choose as target for a putative synthetic AMP, PhoP/PhoQ, a sensor that responds to low Mg²⁺ concentration. We synthesised a modified DNA fragment coding for an amino acid sequence (NUF) similar to that fragment and expressed it in salmonella typhimurium. We showed that the pattern of gene expression controlled by PhoP/PhoQ highlights dysregulation of pathways involving phospholipids biosynthesis, stress proteins and genes coding for antigens. RNA‐Seq of strain expressing ORF200 showed that the pattern of those genes is also altered here. Accumulation of NUF conferred temporary immunoprotection. This represents a powerful procedure to address synthetic α‐AMPs to a specific MLD generating live non‐virulent bacterial strains.     

Mutagenicity of diphenylhydantoin and some of its metabolites towards Salmonella typhimurium strains

The mutagenicities of 5,5-diphenylhydantoin (DPH) and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were tested in vitro using different Salmonella strains (TA1535, TA100, TA1537, TA1538, TA98). Experiments were carried out at various concentrations in the absence and in the presence of an activating system consisting of hepatic S9 fraction from control rats and from rats pretreated with phenobarbital (PB), β-napthoflavone (BNF), 3-methylcholanthrene (3-MC) and Aroclor 1254 (PCB).DPH slightly increased the number of revertants per plate only after incubations with TA1538 in the presence of the S9 fraction from the liver of 3-MC- and PCB-pretreated animals. A similar but more significant frameshift mutation was observed for HPPH on both TA98 and TA1538 strains and in conditions of metabolic activation by the liver microsomal fractions of rats after pretreatment with BNF, 3-MC and especially PCB.Parallel experiments on the metabolism of DPH to HPPH and of HPPH to the catechol derivative in vitro support the hypothesis of an involvement of epoxide intermediates in the mutagenic activity of DPH.     

Studies on the structure of lipopolysaccharides of Salmonella minnesotta and Salmonella typhimurium R strains

1. The composition of the lipopolysaccharides and the corresponding lipid-free polysaccharides from four R-mutants of Salmonella has been studied. All the lipopolysaccharides, from RI and RII serotypes contained d-glucose, d-galactose, heptose, N-acetylglucosamine and 3-deoxy-2-oxo-octonate. The polysaccharide obtained from the RII lipopolysaccharides also contained all these sugars. The polysaccharides from RI lipopolysaccharides lacked N-acetylglucosamine. 2. From partial hydrolysates of the lipopolysaccharides, a number of oligosaccharides have been isolated and partially characterized. Oligosaccharides containing N-acetylglucosamine or glucosamine were obtained only from RII lipopolysaccharides. Several oligosaccharides composed of glucose and galactose were common to RI and RII preparations. 3. A structural unit, based on the oligosaccharides found, is proposed for the RII lipopolysaccharide. It contains the sequence: alpha-N-acetylglucosaminyl- alpha-glucosyl-alpha-galactosyl-glucosyl.... A second alpha-galactosyl residue is bound to position 6 of the last glucosyl group. The complete unit is believed to to be attached to a polyheptose phosphate backbone in the RII antigen. 4. The RI lipopolysaccharide of Salmonella minnesota contains an analogous structure lacking the terminal N-acetylglucosamine residue. 5. A basal structure common to the lipopolysaccharides of several Salmonella species is proposed.     

Natural and antibody-dependent cell-mediated activity against Salmonella typhimurium by peripheral and intestinal lymphoid cells in mice

Cell-mediated immune responses were assessed employing a 2-hr in vitro cytotoxicity assay against S. typhimurium. It was observed that lymphocytes from GALT as well as from peripheral lymphoid organs possessed natural antibacterial activity, whereas macrophages were devoid of this spontaneous activity. The distribution of this newly described natural activity was PPL greater than MnL greater than IEL = SpL = PBL greater than PoL; this did not correlate with the organ distribution of NK activity against YAC-1 tumor cells, which was PBL greater than SpL = IEL greater than MnL = PoL = PPL. Moreover, the phenotype of the splenic effector cell of the natural activity against S. typhimurium showed some differences from that of NK activity. In fact, both these cells were asialo GM1+, Fc-receptor+, nonadherent, and nonphagocytic, but the former was Thy-1.2- and the latter Thy-1.2+. The effector cell of the natural antibacterial activity in the Peyer's patches had the same phenotype as the splenic one. It was then observed that the antibacterial activity could be augmented by the addition of immune antibodies against S. typhimurium. This was particularly evident employing IEL, SpL, and PBL as effector cells, whereas PPL and MnL did not show any antibody-dependent antibacterial activity. Furthermore, these last two populations could not mediate ADCC against CRBC. Employing selective methods to deplete cell populations, we observed that, at least at the splenic level, there is also a cell that differs in its phenotypic characteristics from that mediating natural antibacterial activity but that plays a role in the antibody-dependent reactions. In conclusion, these results suggest that natural and antibody-dependent antibacterial mechanisms might be important in defense against S. typhimurium, particularly at the gastrointestinal level, where many bacterial infections first take place and begin to interact with the host immune system.     

New short cationic antibacterial peptides. Synthesis,biological activity and mechanism of action

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.     

Sanfetrinem, the member of trinems--in vitro activity against Klebsiella pneumoniae producing ESBL

Sanfetrinem is the first member of tricyclic beta-lactams (trinems) which can be administered orally as a hexatil ester. His chemical structure is related to carbapenems. High stability to many beta-lactamases and human renal dehydropeptydase were described. The investigation was performed on 43 strains of Klebsiella pneumoniae producing ESBL isolated from hospitalized patients. The MICs of sanfetrinem and imipenem were determined by E-test. Additionaly, susceptibility to antibiotics was tested by disc diffusion method. Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922 and Escherichia coli ATCC 35218 were used as a control strains. MIC50 and MIC90 of sanfetrinem and imipenem amounted respectively 0,38/3 mg/1 and 0,19/0,25 mg/l. Range of MIC value was from 0,064 mg/l to 4 mg/l for sanfetrinem, and from 0,094 mg/l to 0,38 mg/l for imipenem. Additionaly, geometric and aritmetic means were calculated to both antibiotics. All results of study were compared using correlation factor and "Student" t-test. None of these 43 Klebsiella pneumoniae strains was resistant to imipenem and cefepime. Majority of isolates demonstrated susceptibility to ciprofloxacin and cefoxitin--90,7% and 74,4% respectively All strains were resistant to gentamicin, amikacin and trimethoprim/sulfamethoxazole.