Development of Quorum-Based Anti-Virulence Therapeutics Targeting Gram-Negative Bacterial Pathogens

Quorum sensing is a cell density-dependent signaling phenomenon used by bacteria for coordination of population-wide phenotypes, such as expression of virulence genes, antibiotic resistance and biofilm formation. Lately, disruption of bacterial communication has emerged as an anti-virulence strategy with enormous therapeutic potential given the increasing incidences of drug resistance in pathogenic bacteria. The quorum quenching therapeutic approach promises a lower risk of resistance development, since interference with virulence generally does not affect the growth and fitness of the bacteria and, hence, does not exert an associated selection pressure for drug-resistant strains. With better understanding of bacterial communication networks and mechanisms, many quorum quenching methods have been developed against various clinically significant bacterial pathogens. In particular, Gram-negative bacteria are an important group of pathogens, because, collectively, they are responsible for the majority of hospital-acquired infections. Here, we discuss the current understanding of existing quorum sensing mechanisms and present important inhibitory strategies that have been developed against this group of pathogenic bacteria.     

Staphylococcus aureus enterotoxin A detection using the polymerase chain reaction (PCR) and its correlation with coagulase and thermonuclease tests

Staphylococcus aureus is a pathogenic bacterium, widely distributed on nature and associated to general infection and food borne outbreaks. The relationship between this bacterium and food borne outbreaks has been done, historically, using several tests, including coagulase, thermonuclease and actually, PCR for the genes codifying for the enterotoxin responsible of clinical symptoms. The objective of this work is to detect enterotoxin A codifying gene through PCR in a group of S. aureus strains isolated from food samples, and also to correlate the presence of this gene with the production of coagulase and thermonuclease enzymes. A total of 69 staphylococcal strains were analyzed, 58 obtained from non pasteurized milk samples from the Estación Experimental Alfredo Volio Mata and 11 from the Food and Water Microbiology Laboratory collection, Universidad de Costa Rica. Coagulase, thermonuclease and enterotoxin A were analyzed in all the strains, and a statistical correlation was performed in order to verify possible associations. Results show that there is no correlation between the three variables, nevertheless, all coagulase positive strains were thermonuclease positive, and all enterotoxin positive strains were coagulase and thermonuclease positive, but not inversely. These results show that the use of presumptive or indirect tests for establishing entorotoxigenity of S. aureus strains is not truthful, more sensible and specific analysis, as PCR, shall be performed.     

Staphylococcus aureus—A Known Opponent against Host Defense Mechanisms and Vaccine Development—Do We Still Have a Chance to Win?

The main purpose of this review is to present justification for the urgent need to implement specific prophylaxis of invasive Staphylococcus aureus infections. We emphasize the difficulties in achieving this goal due to numerous S. aureus virulence factors important for the process of infection and the remarkable ability of these bacteria to avoid host defense mechanisms. We precede these considerations with a brief overview of the global necessitiy to intensify the use of vaccines against other pathogens as well, particularly in light of an impasse in antibiotic therapy. Finally, we point out global trends in research into modern technologies used in the field of molecular microbiology to develop new vaccines. We focus on the vaccines designed to fight the infections caused by S. aureus, which are often resistant to the majority of available therapeutic options.     

Synthesis and Antibacterial Activities of 1-Alkyl-3-methacryloyl (Acryloyl) of Benzimidazolone (Thione) Derivatives

A series of (28) 1-alkyl-3-methacryloyl (acryloyl)-benzimidazolone (thione) deriv-atives were synthesized. The structures of the new derivatives were confirmed by (1)H-NMR, (13)C-NMR and ESI-MS spectral analysis. The antibacterial activities of these compounds against several strains of bacteria, such as Bacillus cereus, Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, were evaluated by methods of paper disc-diffusion and broth mciro-dilution. Methacryloyl derivatives displayed higher antibacterial activities against tested bacterial strains than those of acryloyl derivatives in in vitro tests. A structure-activity relationship (SAR) study revealed that the presences of the methacryloyl moieties is essential to the antibacterial activities of the compounds.     

非特殊彩妆类化妆品的微生物检测方法的评价

对非特殊彩妆类化妆品的微生物检测方法进行评价。参考《中国药典》（2010年版）微生物限度的方法,以金黄色葡萄球菌、枯草芽孢杆菌和大肠埃希菌为试验菌株进行回收试验,同时增加阳性对照组考察控制菌的检验方法。除彩妆A的金黄色葡萄球菌、枯草芽孢杆菌和白色念珠菌外,其他所有试验菌株的回收率均大于70%,而且当采用培养基稀释法（1∶100的供试液,1 mL）时,彩妆A的金黄色葡萄球菌、枯草芽孢杆菌和白色念珠菌的回收率均大于70%;大肠埃希菌和铜绿假单胞菌的阳性生长率为100%,金黄色葡萄球菌阳性生长率为86%。目前的检测方法基本适合非特殊彩妆类化妆品的微生物检测。     

Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.     

Copper (II) Acylhydrazinates. Their Synthesis and Characterization

Acylhydrazine derived furanyl and thienyl Schiff bases and their Cu(II) complexes have been prepared and characterized on the basis of their physical, spectral and analytical data. The preferred enolic form of the Schiff base function as a tetradentate ligand during coordination to the metal ion yielding a square planar complex. The Schiff bases and their complexes with different anions were tested for their antibacterial activity against bacterial species such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa andKlebsiella pneumonae.     

Natural Quorum Sensing Inhibitors – Small Molecules,Big Messages

Due to the increasing emergence of antibiotic resistant bacterial strains within the past few decades, bacterial infections in general – and hospital-borne infections, in particular – have become increasingly difficult to fight. It is therefore crucial to find new strategies to fight pathogenic bacteria. Targeted inhibition of quorum sensing (QS) presents a promising alternative. QS is a cell density-dependent signaling pathway used for intra- and interspecies coordination of gene expression. In many bacteria, pathogenic phenotypes, as well as the expression of virulence factors, are under the control of QS regulons. A closer look at natural quorum sensing inhibitors may be helpful to identify potent compounds that can be used as alternatives to antibiotics. Moreover, it will also provide insight into the interactions between species that compete for the same habitat and resources. This review aims to summarize our current knowledge concerning natural QS inhibitors, as a starting point for the design and synthesis of new therapeutics to treat or prevent bacterial infections.     

Antibacterial Co(II), Cu(II), Ni(II) and Zn(II) Complexes of Thiadiazoles Schiff Bases

Schiff bases were obtained by condensation of 2-amino-l,3,4-thiadiazole with 5-substituted-salicylaldehydes which were further used to obtain complexes of the type [M(L)(2)]Cl(2), where M=Co(II), Cu(II), Ni(II) or Zn(II). The new compounds described here have been characterized by physical, spectral and analytical data, and have been screened for antibacterial activity against several bacterial strains such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The antibacterial potency of these Schiff bases increased upon chelation/complexation, against the tested bacterial species, opening new aproaches in the fight against antibiotic resistant strains.     

Isolation of Gram-positive bacteria from raw milk with antimicrobial residues

Two hundred samples of raw milk were collected at the receiving plants located in three areas of high milk production in Zulia state, Venezuela. The CTT test and trial disk were used in order to detect the presence of antimicrobials. The positive samples were inoculated in tripticase soy broth, human blood agar and manitol salt agar in order to isolate Gram-positive bacteria. The identification of species was performed through biochemical tests. It was found that 45 samples (22.5%) of analyzed milk contained antimicrobials, and bacterial growth was obtained in 35 of them. 100 strains were isolated namely: 44 Staphylococcus, 19 Streptococcus, 17 Enterococcus, 9 Bacillus, 4 Micrococcus, 4 Corynebacterium and 3 Lactococcus. The most frequently isolated specie was S. aureus, the main producing agent of bovine mastitis in Zulia state, a microorganism frequently associated in the country to food-borne intoxications, associated to cheese processed from raw milk. It is recommended to apply control programs for the use of antibiotics.     

Microbial metabolism of xenobiotics: Fundamental and applied research

The ability of microorganisms to metabolise xenobiotic compounds has received much attention due to the environmental persistence and toxicity of these chemicals. The microbial degradation of xenobiotics is seen as a cost effective method of removing these pollutants from the environment by a process now known as bioremediation. Microbial treatment of industrial effluents is also possible. Fundamental work has revealed that a wide variety of microorganisms are capable of degrading an equally wide range of organic pollutants. Pure and mixed cultures of microorganisms have been studied and degradation is observed under both aerobic and anaerobic conditions. Breakdown products have been found during work on the degradative pathways involved and toxicological assessments using bacteria and higher organisms (fish, plants) have been used to determine the toxicity of these intermediates. Many of the degradative genes responsible for xenobiotic metabolism are present on plasmids, transposons or are grouped in clusters on chromosomes. This provides clues to the evolution of degradative pathways and makes the task of genetic manipulation easier such that new microbial strains capable of efficiently degrading pollutants can be developed. Several enzymes involved in xenobiotic metabolism have been isolated and factors affecting their activity investigated. Genetically manipulated strains or naturally isolated organisms may be used in the treatment of industrial wastes or as inocula to enhance degradation in the environment. Environmental factors, including pH, temperature, bioavailability, nutrient supply and oxygen availability have been shown to affect xenobiotic biodegradation. These factors must be optimised to obtain a satisfactory microbial treatment process. Using information gained from fundamental research, bioremediation technology has been used to detoxify different contaminated environments and the results of field studies are very encouraging.     

评估CHROMagar~金黄色葡萄球菌显色培养基在化妆品检验中的应用效果

目的评估CHRO Magar Staph.aureus(CSA)金葡菌显色平板和血琼脂平板(BA)用于化妆品金黄色葡萄球菌的检测效果。方法使用47株球菌菌株在CSA和BA上作典型菌落生长测试和比较CSA和血浆凝固酶反应在鉴定金黄色葡萄球菌中的敏感性和特异性;应用CSA和BA分别检测58件不同类型的化妆品样品。结果CSA和BA对所有金黄色葡萄球菌的菌株均为单一的特征性菌落生长,也有少数凝固酶阴性葡萄球菌表现有假阳性菌落特征;CSA和血浆凝固酶鉴定金黄色葡萄球菌的敏感性和特异性均为100%;CSA和BA在样品中分别检测出7株和1株金黄色葡萄球菌(X2=4.83,P<0.025),总阳性率为12.07%。结论CSA在分离实际样品过程中的筛选效率和敏感性均高于BA,且节省了鉴定菌株所用的时间和费用,建议将CSA作为检测、鉴定化妆品中金黄色葡萄球菌的常规培养基应用。     

一项微生物能力验证的多种方法检测结果分析

为确保微生物能力验证结果的准确报出,本实验室采用了多种方法进行CNAS T 0710的铜绿假单胞菌(Pseudomonas aeruginosa)和金黄色葡萄球菌(Staphylococcus aureus)的能力验证比对检验,结果表明:以上多种检测方法的测试结果相吻合,均显示5份样品中有2份检出铜绿假单胞菌,2份检出金黄色葡萄球菌。最终5份样品测试均取得满意结果,在分析比较中也总结出各种检验方法的优缺点。     

Evaluation of the antimicrobial action of honey against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, Salmonella enteritidis, Listeria monocytogenes and Aspergillus niger. Evaluation of its microbiological charge

The evaluation of the microbiological charge present in Costa Rican samples as the evaluation of its antimicrobial activity over different microorganisms, including those associated to wound infections, will allow to emit criteria referred to its use in therapeutic treatments, specially as alternative therapy for cases involving antibiotic resistant bacteria. The microbiological charge of 25 honey samples, acquired in Costa Rican markets was evaluated through several indicators including total plate aerobic count, total plate anaerobic count, total aerobic spore count, total anaerobic spore count and molds and yeast count. Also, samples were inoculated in tubes with chopped meat media and plated in egg yolk agar in order to determine the presence of Clostridium botulinum. For the antimicrobial activity evaluation, the diffusion method in Muller Hinton agar was performed, testing different honey concentrations (100, 75, 50, 25, 12,5 and 6,25 % v/v) against Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (UCR 2902), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC25922), Salmonella enteritidis (ATCC 13076), Listeria monocytogenes (ATCC 19116) and Aspergillus niger. The results obtained for the microbiological characterization of honey show that 91% of samples had counts equal or lower than 1,0 x 10(1) CFU/g. No positive result was obtained for the isolation of C. botulinum. 24 of the samples analyzed inhibited the growth of S. aureus even in a 25% v/v concentration, nevertheless, A. niger was no inhibited by any of the samples tested.     

Analysis of Small RNAs in Streptococcus mutans under Acid Stress—A New Insight for Caries Research

Streptococcus mutans (S. mutans) is the major clinical pathogen responsible for dental caries. Its acid tolerance has been identified as a significant virulence factor for its survival and cariogenicity in acidic conditions. Small RNAs (sRNAs) are recognized as key regulators of virulence and stress adaptation. Here, we constructed three libraries of sRNAs with small size exposed to acidic conditions for the first time, followed by verification using qRT-PCR. The levels of two sRNAs and target genes predicted to be bioinformatically related to acid tolerance were further evaluated under different acid stress conditions (pH 7.5, 6.5, 5.5, and 4.5) at three time points (0.5, 1, and 2 h). Meanwhile, bacterial growth characteristics and vitality were assessed. We obtained 1879 sRNAs with read counts of at least 100. One hundred and ten sRNAs were perfectly mapped to reported msRNAs in S. mutans. Ten out of 18 sRNAs were validated by qRT-PCR. The survival of bacteria declined as the acid was increased from pH 7.5 to 4.5 at each time point. The bacteria can proliferate under each pH except pH 4.5 with time. The levels of sRNAs gradually decreased from pH 7.5 to 5.5, and slightly increased in pH 4.5; however, the expression levels of target mRNAs were up-regulated in acidic conditions than in pH 7.5. These results indicate that some sRNAs are specially induced at acid stress conditions, involving acid adaptation, and provide a new insight into exploring the complex acid tolerance for S. mutans.     

Eradicating Biofilms of Carbapenem-Resistant Enterobacteriaceae by Simultaneously Dispersing the Biomass and Killing Planktonic Bacteria with PEGylated Branched Polyethyleneimine

Carbapenem-resistant Enterobacteriaceae (CRE) are emerging pathogens that cause variety of severe infections. CRE evade antibiotic treatments because these bacteria produce enzymes that degrade a wide range of antibiotics including carbapenems and β-lactams. The formation of biofilms aggravates CRE infections, especially in a wound environment. These difficulties lead to persistent infection and non-healing wounds. This creates the need for new compounds to overcome CRE antimicrobial resistance and disrupt biofilms. Recent studies in our lab show that 600 Da branched polyethyleneimine (BPEI) and its derivative PEG350-BPEI can overcome antimicrobial resistance and eradicate biofilms in methicillin-resistant S. aureus, methicillin-resistant S. epidermidis, P. aeruginosa, and E. coli. In this study, the ability of 600 Da BPEI and PEG350-BPEI to eradicate carbapenem-resistant Enterobacteriaceae bacteria and their biofilms is demonstrated. We show that both BPEI and PEG350-BPEI have anti-biofilm efficacy against CRE strains expressing Klebsiella pneumoniae carbapenemases (KPCs) and metallo-β-lactamases (MBLs), such as New Delhi MBL (NDM-1). Furthermore, our results illustrate that BPEI affects planktonic CRE bacteria by increasing bacterial length and width from the inability to proceed with normal cell division processes. These data demonstrate the multi-functional properties of 600 Da BPEI and PEG350-BPEI to reduce biofilm formation and mitigate virulence in carbapenem-resistant Enterobacteriaceae.     

Evaluation of the effect of probiotic cultures on two different yogurt brands over a known population of Staphylococcus aureus and the production of thermonuclease

The effect of probiotic cultures over known populations of Staphylococcus aureus inoculated in yogurt was studied; also the production and stability of its thermonuclease during yogurt storage was evaluated. In three different occasions, two different yogurt brands, one with additional probiotic cultures (Lactobacillus casei and L. acidophilus), were inoculated with known populations of S. aureus in high and low concentration (10(9) CFU/g and 10(7) CFU/g), respectively. These samples were stored for 28 days at 5 degrees C. Every four days the count of lactic bacteria, S. aureus and pH were evaluated, according to the methodology described in the Compendium of Methods for the Microbiological Examination of Foods, Vanderzant & Splittstoesser. The presence of thermonuclease was determined using petrifilm for S. aureus from 3M company. The pH and lactic bacteria population were constant during the testing period. Yogurt with additional probiotic cultures (high and low concentration) lowered the population of S. aureus to non detectable levels in 8 days; but, S. aureus could be cultured from yogurt without probiotics even after 24 days of incubation. Same time, the presence of thermonuclease was positive in all tests; it was not affected by probiotics. The presence of thermonuclease is related to the production of S. aureus enterotoxin. This work emphasizes again the beneficial effects of probiotic cultures in yogurt over bacteria and the importance of keeping hygienic practices in order to avoid the contamination of food with S. aureus and the eventual production of its enterotoxin, since it is not affected by probiotics.     

Structures of the N-Terminal Domain of PqsA in Complex with Anthraniloyl- and 6-Fluoroanthraniloyl-AMP: Substrate Activation in Pseudomonas Quinolone Signal (PQS) Biosynthesis

Pseudomonas aeruginosa, a prevalent pathogen in nosocomial infections and a major burden in cystic fibrosis, uses three interconnected quorum-sensing systems to coordinate virulence processes. At variance with other Gram-negative bacteria, one of these systems relies on 2-alkyl-4(1H)-quinolones (Pseudomonas quinolone signal, PQS) and might hence be an attractive target for new anti-infective agents. Here we report crystal structures of the N-terminal domain of anthranilate-CoA ligase PqsA, the first enzyme of PQS biosynthesis, in complex with anthraniloyl-AMP and with 6-fluoroanthraniloyl-AMP (6FABA-AMP) at 1.4 and 1.7 Å resolution. We find that PqsA belongs to an unrecognized subfamily of anthranilate-CoA ligases that recognize the amino group of anthranilate through a water-mediated hydrogen bond. The complex with 6FABA-AMP explains why 6FABA, an inhibitor of PQS biosynthesis, is a good substrate of PqsA. Together, our data might pave a way to new pathoblockers in P. aeruginosa infections.     

Antibacterial activity of copolymers of trialkyl(4-vinylbenzyl)ammonium chlorides with acrylonitrile

Homopolymers of cetyldimethyl(4-vinylbenzyl)ammonium chloride (VB16), lauryl-dimethyl(4-vinylbenzyl)ammonium chloride (VB12), and trimethyl (4-vinylbenzyl)ammonium chloride (VB1), and copolymers of these compounds with acrylonitrile were examined for antibacterial activity using B. subtilis, S. aureus, E. coli, and P. aeruginosa. All homopolymers and copolymers showed germicidal action to the bacterias, especially strong action to gram-positive B. subtilis and S. aureus. The strength of the germicidal action of the quaternary ammonium branches was in the order: VB16 > VB12 ? VB1.     

Bacterial Community Structure on Membrane Surface and Characteristics of Strains Isolated from Membrane Surface in Submerged Membrane Bioreactor

Abstract

In order to investigate the bacterial community structure and the characteristics of bacteria on the membrane surface, a submerged membrane bioreactor treating municipal wastewater was continuously operated under two different conditions. Bacterial community structures were examined by PCR‐denaturing gradient gel electrophoresis and PCR cloning of 16S rRNA genes. Bacterial strains isolated from membrane surface were identified and their growth curve, EPS concentration and hydrophobicity were measured. The structures of bacterial communities in the suspended solids and on the membrane surface were obviously different, and γ‐Proteobacteria more selectively adhere and grow on the membrane surface than other microorganisms. Most of the membrane isolates grew slowly as compared with the strains isolated from the suspended solids. Also, the membrane isolates were higher cell surface hydrophobicities, higher EPS concentrations, and higher ratios of protein to carbohydrate within the EPSs than the isolates from suspended solids.