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PubMed2006

Small molecules with antimicrobial activity against E. coli and P. aeruginosa identified by high-throughput screening.

BACKGROUND AND PURPOSE: New antimicrobials are needed because of the emergence of organisms that are resistant to available antimicrobials. The purpose of this study was to evaluate a high-throughput screening approach to identify antibacterials against two common disease-causing bacteria, and to determine the frequency, novelty, and potency of compounds with antibacterial activity. EXPERIMENTAL APPROACH: A high-throughput, turbidometric assay of bacterial growth in a 96-well plate format was used to screen a diverse collection of 150,000 small molecules for antibacterial activity against E. coli and P. aeruginosa. The statistical Z'-factor for the assay was > or = 0.7. KEY RESULTS: Screening for inhibition of E. coli growth gave a 'hit' rate (> 60% inhibition at 12.5 microM) of 0.025%, which was more than 5-fold reduced for P. aeruginosa. The most potent antibacterials (EC50 < 0.5 microM) were of the nitrofuran class followed by naphthalimide, salicylanilide, bipyridinium and quinoazolinediamine chemical classes. Screening of > 250 analogs of the most potent antibacterial classes established structure-activity data sets. CONCLUSIONS AND IMPLICATIONS: Our results validate and demonstrate the utility of a growth-based phenotype screen for rapid identification of small-molecule antibacterials. The favourable efficacy and structure-activity data for several of the antibacterial classes suggests their potential development for clinical use.

Anti-Bacterial Agents↗
PubMed2005

One- and two-step hydrogen peroxide contact lens disinfection solutions against Acanthamoeba: how effective are they?

PURPOSE: Effective contact lens disinfection solutions are important to keep the storage case free of acanthamoebae and thus prevent an infection of the eye. The aim of our study was to evaluate the effectivity of two new one-step hydrogen peroxide disinfecting solutions against Acanthamoeba spp. and compare it to the effectivity of other commercially available systems. METHODS: Nine one-step 3% hydrogen peroxide systems including the new systems Silver Sept (platinum and silver disk for intensifying disinfection) and Blue Vision (newly composed catalytic tablet) and 2 two-step systems (0.6 and 3.0% H(2)O(2)) were tested for their effectivity against cysts of two Acanthamoeba keratitis isolates at different concentrations. RESULTS: After a soaking time of 8 h (overnight soaking of contact lenses) the 2 two-step systems completely destroyed the cysts of both Acanthamoeba strains, even at the highest concentration of cysts tested. The nine tested one-step systems showed weaker effects. The new Blue Vision system was able to eradicate the cysts of one strain at the low concentration of cysts. CONCLUSIONS: One-step hydrogen peroxide systems do not have sufficient effects on Acanthamoeba cysts and therefore may not protect the contact lens user from a possible infection of the eye. Further development of tablets like the ones used in the Blue Vision system may result in better cysticidal effects for one-step systems.

Acanthamoeba↗
PubMed2007

Life and death in the granuloma: immunopathology of tuberculosis.

During tuberculosis (TB) infection, the granuloma provides the microenvironment in which antigen-specific T cells colocate with and activate infected macrophages to inhibit the growth of Mycobacterium tuberculosis. Although the granuloma is the site for mycobacterial killing, virulent mycobacteria have developed a variety of mechanisms to resist this macrophage-mediated killing. These surviving mycobacteria become dormant, however, if host cellular immunity or the signals maintaining granuloma structure wane, or if mycobacteria resume replication, leading to reactivation of TB. This balance of life and death applies not only to the mycobacterium but also to the host macrophages that may undergo apoptosis or necrosis, leading to the characteristic caseous necrosis within the granuloma, and the potential spread of TB infection. The immunological factors controlling the development and maintenance of the granuloma will be reviewed.

Adaptation, Biological↗
PubMed2007

Shigella's ways of manipulating the host intestinal innate and adaptive immune system: a tool box for survival?

Shigella, a Gram-negative invasive enteropathogenic bacterium, causes the rupture, invasion and inflammatory destruction of the human colonic epithelium. This complex and aggressive process accounts for the symptoms of bacillary dysentery. The so-called invasive phenotype of Shigella is linked to expression of a type III secretory system (TTSS) injecting effector proteins into the epithelial cell membrane and cytoplasm, thereby inducing local but massive changes in the cell cytoskeleton that lead to bacterial internalization into non-phagocytic intestinal epithelial cells. The invasive phenotype also accounts for the potent pro-inflammatory capacity of the microorganism. Recent evidence indicates that a large part of the mucosal inflammation is initiated by intracellular sensing of bacterial peptidoglycan by cytosolic leucine-rich receptors of the NOD family, particularly NOD1, in epithelial cells. This causes activation of the nuclear factor kappa B and c-JunNH(2)-terminal-kinase pathways, with interleukin-8 appearing as a major chemokine mediating the inflammatory burst that is dominated by massive infiltration of the mucosa by polymorphonuclear leukocytes. Not unexpectedly, this inflammatory response, which is likely to be very harmful for the invading microbe, is regulated by the bacterium itself. A group of proteins encoded by Shigella, which are injected into target cells by the TTSS, has been recently recognized as a family of potent regulators of the innate immune response. These enzymes target key cellular functions that are essential in triggering the inflammatory response, and more generally defense responses of the intestinal mucosa. This review focuses on the mechanisms employed by Shigella to manipulate the host innate response in order to escape early bacterial killing, thus ensuring establishment of its infectious process. The escape strategies, the possible direct effect of Shigella on B and T lymphocytes, their impact on the development of adaptive immunity, and how they may help explain the limited protection induced by natural infection are discussed.

Animals↗
PubMed2006

The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity.

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 degrees C. The bacterial membrane became permeable to the DNA-binding dye SYTOX(R) Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys(18) and Arg(4)) are distributed into three clusters on the surface of hRNase7. The first cluster, K(1),K(3),K(111),K(112), was located at the flexible coil near the N terminus, whereas the other two, K(32),K(35) and K(96),R(97),K(100), were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K(1),K(3),K(111),K(112), rather than the catalytic residues His(15), Lys(38), and His(123) or other clusters such as K(32),K(35) and K(96),R(97),K(100), is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K(1),K(3),K(111),K(112). The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 degrees C.

Amino Acid Substitution↗
PubMed2006

Phagocytosis of Giardia lamblia trophozoites by human colostral leukocytes.

AIM: To determine the phagocytic activity of the polymorphonuclear and mononuclear cells present in human colostrum, and to verify the influence of opsonins in the adherence, ingestion and killing of Giardia lamblia trophozoites. METHODS: Polymorphonuclear and mononuclear phagocytes were incubated with G. lamblia trophozoites, in the presence as well as the absence of supernatant of human colostrum (the source of opsonins) for 30, 60 and 120 min. The trophozoites/phagocytes ratio was 1:1, and the percentage of phagocytosed trophozoites was determined by microscopic examination of acridine orange-stained cells. RESULTS: The mononuclear phagocytes presented more functional activity than the polymorphonuclear. The highest indexes of adherence (77.6 +/- 5.1), ingestion (68.9 +/- 5.5) and killing (48.5 +/- 4.9) were obtained through the incubation of mononuclear cells in the presence of colostrum supernatant for 120 min. CONCLUSION: The phagocytes of human colostrum were able to ingest G.lamblia trophozoites and presented microbicidal activity in vitro, suggesting that these phagocytes may act as an additional mechanism of protection against infant giardiasis through breastfeeding.

Adolescent↗
PubMed2005

Effect of the psoralen-based photochemical pathogen inactivation on mitochondrial DNA in platelets.

Photochemical treatment (PCT) of platelet concentrates, using amotosalen HCl and UVA-light, inactivates pathogens by forming adducts between amotosalen and nucleic acids. The impact of the photochemical treatment on pathogens and leukocytes has been studied extensively. Yet little is known about the effect of PCT on nucleic acids in platelets. Platelets contain viable mitochondria and mitochondrial DNA (mtDNA) and this study aimed at evaluating the amotosalen modifications on platelet mtDNA. We applied two independent but complementary molecular assays to investigate qualitative as well as quantitative aspects of the psoralen-mediated DNA modifications in platelet mtDNA. The amotosalen-DNA modification density was measured using (14)C-labeled amotosalen. Amotosalen (150 microM) yielded 4.0 +/- 1.2 psoralen adducts per 1,000 bp in mtDNA after irradiation with 3 J/cm(2) UVA. Furthermore, we tested if the PCT-induced DNA modifications could be detected by a PCR assay. On the basis of PCR inhibition due to amotosalen-DNA adducts, mtDNA-specific PCR assays were developed and tested for their specificity and sensitivity. Our data revealed that mtDNA in platelets is substantially modified by PCT and that these modifications can be documented by a PCR inhibition system.

Blood Platelets↗
PubMedDate not supplied

High-pressure homogenization as a non-thermal technique for the inactivation of microorganisms.

In the pharmaceutical, cosmetic, chemical, and food industries high-pressure homogenization is used for the preparation or stabilization of emulsions and suspensions, or for creating physical changes, such as viscosity changes, in products. Another well-known application is cell disruption of yeasts or bacteria in order to release intracellular products such as recombinant proteins. The development over the last few years of homogenizing equipment that operates at increasingly higher pressures has also stimulated research into the possible application of high-pressure homogenization as a unit process for microbial load reduction of liquid products. Several studies have indicated that gram-negative bacteria are more sensitive to high-pressure homogenization than gram-positive bacteria supporting the widely held belief that high-pressure homogenization kills vegetative bacteria mainly through mechanical disruption. However, controversy exists in the literature regarding the exact cause(s) of cell disruption by high-pressure homogenization. The causes that have been proposed include spatial pressure and velocity gradients, turbulence, cavitation, impact with solid surfaces, and extensional stress. The purpose of this review is to give an overview of the existing literature about microbial inactivation by high-pressure homogenization. Particular attention will be devoted to the different proposed microbial inactivation mechanisms. Further, the different parameters that influence the microbial inactivation by high-pressure homogenization will be scrutinized.

Hydrostatic Pressure↗
PubMed2006

The effects of jet cavitation on the growth of Microcystis aeruginosa.

Different operating factors of jet cavitation method affecting one of cyanobacterial's (Microcystis aeruginosa) growths were investigated. The inhibition on M. aeruginosa growth by the jet cavitation method depended strongly on the hydraulic characteristics of cavitation tube, inlet pressure, cavitation number, light and different algae concentrations. The inhibitive efficiency of 64.58% in field water sample was obtained by this method. Moreover, the ultrastructural changes of M. aeruginosa before and after treatment were observed by the transmission electron microscope (TEM). The changes of UV-Visible spectra further demonstrated the inhibitive behaviors of jet cavitation on the growth of M. aeruginosa. The experimental results showed that jet cavitation method was a potential physical technology that could effectively inhibit the growth of M. aeruginosa.

Biomass↗
PubMed2006

Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.

Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system.

DNA, Fungal↗
PubMed1951

Studies on host-virus interactions in the chick embryo-influenza virus system. VI. Evidence for multiplicity reactivation of inactivated virus.

Evidence has been presented that influenza viruses both of type A and B partially inactivated by ultraviolet irradiation may regain their capacity to propagate in the allantoic membrane of the chick embryo. In using such irradiated preparations as inocula for growth curve experiments it could be shown that the development of hemagglutinins as well as of infectivity preceded at rates resembling those noted with more than 10 times the amount of infective virus actually found in the irradiated seed. Partial inactivation of the inocula by heating to 56 degrees C. gave similar results. The phenomenon was observed only with seed irradiated for short periods of time so that the virus particles sustained only few hits of radiation. On prolonged exposure resulting in numerous hits per virus particle the capacity of reactivation was lost. Likewise, an irradiated preparation capable of reactivation in the allantoic membrane, could not be diluted more than about 30-fold and still clearly produce this phenomenon. This indicated that reactivation is obtained only when one host cell adsorbs more than one non-infective virus particle but not upon adsorption of a single particle. These data are in striking agreement with the phenomenon of "multiplicity reactivation" observed in the bacteriophage-E. coli system by Luria and Dulbecco.

Escherichia coli↗
PubMed1952

The antistreptococcal property of milk. I. Some characteristics of the activity of lactenin in vitro; the effect of lactenin on hemolytic streptococci of the several serological groups.

The measurement in vitro of lactenin, the antistreptococcal substance of milk, is affected by the size of the inoculum, the temperature of incubation, and the type of medium employed. Hemolytic streptococci belonging to the several serological groups vary in susceptibility to lactenin. All group A streptococci, regardless of type, are highly sensitive to it, and milk receiving a small inoculum sterilizes itself within 48 hours or less. By contrast, most strains of groups B, C, D, and E, although they may temporarily be inhibited, ultimately achieve full growth. Strains belonging to groups F, G, H, K, and L vary in sensitivity, some being fully inhibited and others achieving full growth. When streaked on the surface of milk-agar plates and examined at the end of 24 hours the streptococci fall into two classes: sensitive strains which do not produce visible colonies on the plate, and resistant strains which grow excellently. Very few strains show an intermediate degree of sensitivity. Human and goat milk contain an antistreptococcal principle which appears to be the same as the lactenin of cow milk, since streptococci which are inhibited by milk from one species are inhibited by milk from the others, and vice versa.

Animals↗
PubMed1952

The antistreptococcal property of milk. II. The effects of anaerobiosis, reducing agents, thiamine, and other chemicals on lactenin action.

Lactenin is reversibly inactivated by the exclusion of atmospheric oxygen. It is also inactivated by the sulfur-containing reducing agents cysteine, glutathione, thioglycollic acid, and BAL. Group A streptococci which have been acted upon by lactenin have been killed, and not merely prevented from multiplying, since they cannot be revived by inactivating lactenin through the addition of a reducing agent. Thiamine in great excess inactivates lactenin. The mechanism by which it accomplishes this has not been discovered, but it suggests that the mode of action of lactenin may be to deny thiamine to the lactenin-sensitive cell. Lactenin sensitivity is not, however, related to a requirement for exogenous thiamine, nor does lactenin appear to function by binding environmental thiamine in a form unavailable to the sensitive cell.

Anaerobiosis↗
PubMed1953

The protection of intracellular brucella against therapeutic agents and the bactericidal action of serum.

A method for the in vitro study of intracellular brucella has been described. Exudative leukocytes containing intracellular brucella have been maintained in vitro in a synthetic tissue culture medium or in human or animal serum. Intracellular brucella are protected in vitro against the lethal action of therapeutic agents or the bactericidal action of serum. This protection of intracellular brucella is dependent upon the presence of an intact, viable host cell. None of the currently available therapeutic agents, whether used alone or in combinations, were capable of killing all intracellular brucella in vitro in 24 hours. A remarkable protection of intracellular brucella against streptomycin has been demonstrated. The most effective reduction in the number of viable intracellular brucella was accomplished by exposure of the host cells to streptomycin plus aureomycin, terramycin, or chloramphenicol. The available evidence suggests that the ability of brucella to localize and remain viable within the cells of an infected host is an important biologic factor in establishing and perpetuating brucella infections, despite therapeutic measures or the operation of the host's humoral defense mechanisms. Reduction of neotetrazolium by leukocytes and brucella in vitro provides a method for assessing the metabolic status of the host cell, but does not discriminate with any degree of certainty a viable from a non-viable intracellular organism.

Animals↗
PubMed2006

Bactericidal activity and target preference of a piperazinyl-cross-linked ciprofloxacin dimer with Staphylococcus aureus and Escherichia coli.

BACKGROUND: Previous work showed that piperazinyl-cross-linked ciprofloxacin dimer exhibits good bacteriostatic activity with Streptococcus pneumoniae and Staphylococcus aureus; lethal activity was not measured. Subsequently, the dimer failed to kill Mycobacterium smegmatis but blocked growth. Whether the compound is lethal with non-mycobacterial species is not known. METHODS: Bacteriostatic and bactericidal activities were measured with wild-type cells and topoisomerase mutants of S. aureus and Escherichia coli for ciprofloxacin and a dimer of ciprofloxacin. Spontaneous resistance mutants were selected with S. aureus for both compounds, followed by target identification by nucleotide sequence determination of the quinolone-resistance-determining-region of gyrA (gyrase) and parC (topoisomerase IV). RESULTS: The dimer was lethal, in some cases exhibiting more activity than ciprofloxacin (particularly with wild-type cells and a parC mutant of S. aureus). Dimerization affected target preference with S. aureus but not with E. coli. Resistance mutations in either gyrA or parC of S. aureus raised the MIC of the dimer, but only a parC mutation raised the MIC of ciprofloxacin. With S. aureus, the dimer selected spontaneous resistant gyrA mutants, whereas ciprofloxacin selected a parC mutant. With E. coli, a gyrA, but not a parC, mutation raised the MIC of both compounds. CONCLUSION: The dimer readily killed S. aureus and E. coli, representative gram-positive and gram-negative bacteria. In both cases the preferred target was DNA gyrase. The switch in target preference may be responsible for the greater lethality of the dimer seen with S. aureus.

Anti-Bacterial Agents↗
PubMed2006

Efficacy of daptomycin in the treatment of experimental endocarditis due to susceptible and multidrug-resistant enterococci.

OBJECTIVES: Daptomycin was tested in vitro and in rats with experimental endocarditis against the ampicillin-susceptible and vancomycin-susceptible Enterococcus faecalis JH2-2, the vancomycin-resistant (VanA type) mutant of strain JH2-2 (strain JH2-2/pIP819), and the ampicillin-resistant and vancomycin-resistant (VanB type) Enterococcus faecium D366. METHODS: Rats with catheter-induced aortic vegetations were treated with doses simulating intravenously kinetics in humans of daptomycin (6 mg/kg every 24 h), amoxicillin (2 g every 6 h), vancomycin (1 g every 12 h) or teicoplanin (12 mg/kg every 12 h). Treatment was started 16 h post-inoculation and continued for 2 days. RESULTS: MICs of daptomycin were 1, 1 and 2 mg/L, respectively, for strains JH2-2, JH2-2/pIP819 and D366. In time-kill studies, daptomycin showed rapid (within 2 h) bactericidal activity against all strains. Daptomycin was highly bound to rat serum proteins (89%). In the presence of 50% rat serum, simulating free concentrations, daptomycin killing was maintained but delayed (6-24 h). In vivo, daptomycin treatment resulted in 10 of 12 (83%), 9 of 11 (82%) and 11 of 12 (91%) culture-negative vegetations in rats infected with strains JH2-2, JH2-2/pIP819 and D366, respectively (P < 0.001 compared to controls). Daptomycin efficacy was comparable to that of amoxicillin and vancomycin for susceptible isolates. Daptomycin, however, was significantly (P < 0.05) more effective than teicoplanin against the glycopeptide-susceptible strain JH2-2 and superior to all comparators against resistant isolates. CONCLUSIONS: These results support the use of the newly proposed daptomycin dose of 6 mg/kg every 24 h for treatment of enterococcal infections in humans.

Amoxicillin↗