A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method.

The increasing emergence of multi-drug resistant streptococci poses a serious threat to public health worldwide. Bacteriophage lysins are promising alternatives to antibiotics; however, their narrow lytic spectrum restricted to closely related species is a central shortcoming to their translational development. Here, we describe an efficient method for rapid screening of engineered chimeric lysins and report a unique "chimeolysin", ClyR, with robust activity and an extended-spectrum streptococcal host range against most streptococcal species, including S. pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. mutans, S. pneumoniae, S. suis and S. uberis, as well as representative enterococcal and staphylococcal species (including MRSA and VISA). ClyR is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen as well as the first lysin that kills all four of the bovine mastitis-causing pathogens. This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most streptococcal associated infections.

Effects of Mesua ferrea leaf and fruit extracts on growth and morphology of Staphylococcus aureus.

Mesua ferrea is traditionally used for treating bleeding piles, fever, and renal diseases. It has been reported to have antimircobial activity. In the present study, antibacterial efficacy of leaf and fruit extracts on the growth and morphology of Staphylococcus aureus is evaluated. Both extracts display good antibacterial activity against S. aureus with a minimum inhibition concentration of 0.048 mg/mL. Both extracts are bacteriostatic at a minimum bacteriostatic concentration of 0.39 mg/mL. The bacteriostatic activity lasts for 24 h, and then cells start to grow as normal as shown in time-kill analysis. Scanning electron microscopy study indicated potential detrimental effect of the extracts of leaf and fruits of M. ferrea on the morphology of S. aureus. The treatment with the extracts caused extensive lysis of the cells, leakage of intracellular constituents, and aggregation of cytoplasmic contents forming an open meshwork of the matrix.

Transcriptional and functional analysis shows sodium houttuyfonate-mediated inhibition of autolysis in Staphylococcus aureus.

Sodium houttuyfonate (SH), an addition compound of sodium bisulfite and houttuynin, showed in vitro antibacterial activity against 21 Staphylococcus aureus (S. aureus) strains grown in planktonic cultures. Microarray results showed decreased levels of autolysin atl, sle1, cidA and lytN transcripts in the SH-treated strain as compared to the control strain, consistent with the induction of the autolytic repressors lrgAB and sarA and with the downregulation of the positive regulators agrA and RNAIII. Triton X-100-induced autolysis was significantly decreased by SH in S. aureus ATCC 25923, and quantitative bacteriolytic assays and zymographic analysis demonstrated SH-mediated reduction of extracellular murein hydrolase activity in these cells. Anti-biofilm assay showed that SH is poorly active against S. aureus grown in biofilm cultures, whereas SH diminished the amounts of extracellular DNA (eDNA) of S. aureus in a dose-dependent manner, which suggested that SH may impede biofilm formation by reducing the expression of cidA to inhibit autolysis and eDNA release in the early phase. Some of the microarray results were confirmed by real-time RT-PCR.

Antimicrobial effect of Magnolia officinalis extract against Staphylococcus aureus.

BACKGROUND: The antimicrobial effect of Magnolia officinalis extract (MOE) against Staphylococcus aureus was investigated in a minced mutton system and the mechanisms of its antimicrobial activity were studied by light microscopy, transmission electron microscopy and scanning electron microscopy observations. RESULTS: MOE inoculation effectively inhibited the growth of S. aureus in minced mutton compared with that in control meat without MOE. The cell membrane of S. aureus treated with MOE showed structural disorganisation and cytoplasmic volume overflow. After 48 h of exposure to MOE, many S. aureus cells had completely collapsed. CONCLUSION: The antimicrobial mechanisms of MOE resulted mainly in cell membrane and wall damage, causing increased permeability of cell membranes or lysis of cell walls and loss of cellular constituents, impairment of structural components and changes in bacterial cell morphology.

Synergistic interaction of eugenol with antibiotics against Gram negative bacteria.

Eugenol, the principal chemical component of clove oil from Eugenia aromatica has been long known for its analgesic, local anesthetic, anti-inflammatory, and antibacterial effects. The interaction of the eugenol with ten different hydrophobic and hydrophilic antibiotics was studied against five different Gram negative bacteria. The MIC of the combination was found to decrease by a factor of 5-1000 with respect to their individual MIC. This synergy is because of the membrane damaging nature of eugenol, where 1mM of its concentration is able to damage nearly 50% of the bacterial membrane. Eugenol was also able to enhance the activities of lysozyme, Triton X-100 and SDS in damaging the bacterial cell membrane. The hydrophilic antibiotics such as vancomycin and beta-lactam antibiotics which have a marginal activity on these gram negative bacteria exhibit an enhanced antibacterial activity when pretreated with eugenol. Reduced usage of antibiotics could be employed as a treatment strategy to slow down the onset of antibiotic resistance as well as decrease its toxicity. Experiments performed with human blood cells indicated that the concentration of eugenol used for the combination studies were below its cytotoxic values. Pharmacodynamic studies of the combinations need to be performed to decide on the effective dosage.

Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins.

Plastid transformation has become an attractive tool in biotechnology. Because of the prokaryotic nature of the plastid's gene expression machinery, expression elements (promoters and untranslated regions) that trigger high-level foreign protein accumulation in plastids usually also confer high expression in bacterial cloning hosts. This can cause problems, for example, when production of antimicrobial compounds is attempted. Their bactericidal activity can make the cloning of the corresponding genes in plastid transformation vectors impossible. Here, we report a general solution to this problem. We have designed a strategy (referred to as toxin shuttle) that allows the expression in plastids of proteins that are toxic to Escherichia coli. The strategy is based on blocking transcription in E. coli by bacterial transcription terminators upstream of the gene of interest, which subsequently are excised in planta by site-specific recombination. We demonstrate the applicability of the strategy by the high-level expression in plastids (to up to 30% of the plant's total soluble protein) of 2 phage-derived protein antibiotics that are toxic to E. coli. We also show that the plastid-produced antibiotics efficiently kill pathogenic strains of Streptococcus pneumoniae, the causative agent of pneumonia, thus providing a promising strategy for the production of next-generation antibiotics in plants.

[Beta-lactam antibiotics]. / Antibióticos betalactámicos.

Beta-lactam drugs, whose mechanism of action is inhibition of the last stage of bacterial cell wall synthesis, are the largest family of antimicrobial agents and the most widely used in current clinical practice. These drugs have a slow, time-dependent bactericidal action, generally good distribution in the body, and low toxicity. Modifications of the original molecule have led to new compounds with a greater antimicrobial spectrum and activity; nonetheless, the use and efficacy of beta-lactams is limited in some clinical settings, owing to the increasing emergence of antimicrobial resistance. Despite this problem, penicillin remains the treatment of choice in a large number of infections, cephalosporins have a wide range of indications, carbapenems are used in nosocomially-acquired infection and infection caused by multiresistant microorganisms, and beta-lactam inhibitors restore the spectrum of activity of their companion penicillins (aminopenicillins, ureidopenicillins) when resistance is caused by beta lactamase production.

Differential assay for high-throughput screening of antibacterial compounds.

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.

Cell wall composition and decreased autolytic activity and lysostaphin susceptibility of glycopeptide-intermediate Staphylococcus aureus.

The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.

The bacterial multiple antibiotic resistant (Mar) phenotype leads to increased tolerance to tea tree oil.

Mutants of Escherichia coil strain AG100 exhibiting the multiple antibiotic resistance (Mar) phenotype demonstrated a greater level of tolerance to tea tree oil (TTO) compared with the parent strain. The ability of TTO to kill all E. coil strains studied was greater at 37 than at 30 degrees C. Growth of parent strain AG100 in the presence of salicylate, which induces the mar operon leading to the Mar phenotype, also increased tolerance to TTO. Escherichia coli Mar mutant YL1 demonstrated greater tolerance to antimicrobial terpenes found in TTO and did not leak K+ as rapidly in the presence of TTO when compared with its parent strain AG100. Attempts to isolate Mar mutants of Staphylococcus aureus using tetracycline gradients proved unsuccessful. However, when grown in the presence of salicylate, S. aureus strain BB255 demonstrated greater tolerance to TTO and did not leak K+ as rapidly in the presence of TTO compared with this strain grown without additions. This evidence demonstrates that bacterial Mar phenotypes increase tolerance to the killing action of TTO. This work also adds indirect evidence that the target of TTO is the cell membrane.

Immunostimulating effect of pule (Alstonia scholaris L. R.Br., Apocynaceae) bark extracts.

The immunostimulating effect of "Pule" (Alstonia scholaris (L.) R.Br., Apocynaceae) bark extracts was studied in BALB/c mouse. The extracts were administered orally, once a day for 7 consecutive days. The results showed that at the same doses (50, 100 and 200 mg/kg b.w.) the aqueous extract had higher phagocytic index (1.39-1.79) than the ethanolic extracts (0.81-0.93) in normal mice. The aqueous extract at 50 mg/kg b.w. also enhanced phagocytic activity of immunosuppressed mice significantly (P < 0.01). At 50 and 100 mg/kg b.w. the extract prevents the decrease of immune system induced by prednisone. The aqueous extract at 100 mg/kg b.w. increased lytic activity of peritoneal exudate cells against Escherichia coli significantly (P < 0.05). At the doses of 50 and 100 mg/kg b.w. the aqueous extract had no effect on primary antibody level. The aqueous extract at 50 mg/kg b.w. induced the cellular immune response while at 100 mg/kg b.w. inhibited the delayed type of hypersensitivity reaction.

Effects of tea tree oil on Escherichia coli.

Tea tree oil (TTO) stimulates autolysis in exponential and stationary phase cells of Escherichia coli. Electron micrographs of cells grown in the presence of TTO showed the loss of electron dense material, coagulation of cell cytoplasm and formation of extracellular blebs. Stationary phase cells demonstrated less TTO-stimulated autolysis and also had greater tolerance to TTO-induced cell death, compared to exponentially grown cells. It was also revealed that subpopulation of stationary phase cells demonstrated increased tolerance to TTO-bactericidal effects.

Inhibition of neutrophil function by human milk.

Human colostrum, the first product of lactation, has antioxidant properties and inhibits selected enzyme and bactericidal activities of human neutrophils. We examined the subsequent product of lactation, mature human milk, with respect to its antioxidant activities, its effects on neutrophil enzyme activities (myeloperoxidase, beta-glucuronidase, and lysozyme), and its effects on neutrophil bactericidal and phagocytic activities. Mature human milk displayed antioxidant characteristics similar to those of human colostrum, reducing cytochrome c and consuming H2O2. Mature milk also displayed colostrum-like characteristics in depressing neutrophil myeloperoxidase and beta-glucuronidase activities, but not in altering lysozyme activity. Neutrophil bactericidal activity against Staphylococcus aureus was depressed by both mature milk and colostrum, without dramatic effects on phagocytic activity. These data show that mature milk shares characteristics with human colostrum that may result in anti-inflammatory effects, but the magnitude of these effects is generally smaller.

Selenium effects on human neutrophilic granulocyte function in vitro.

The effects of an inorganic selenium salt on phagocytic functions of human neutrophilic granulocytes from donors with a low activity of glutathione peroxidase have been investigated. Granulocytes were exposed for 60 min in vitro to sodium selenite in two physiological concentrations (100 and 200 ng Se/ml) and one unphysiologically high concentration (2000 ng/ml). The spontaneous and chemotactic migration, the nitroblue tetrazolium reduction, the phagocytosis of fluorescein-labeled yeast particles and the intracellular killing of staphylococci were then studied in such granulocytes and compared to control cells, which had not been exposed to selenium. The migration and nitroblue tetrazolium reduction abilities of granulocytes were not affected by selenium exposure. The phagocytic and bactericidal activities were significantly increased in granulocytes exposed to selenium in physiological concentrations. However, at 2000 ng Se/ml these activities were found to be equal to or lower than control levels. Thus selenium supplementation might enhance phagocytic and bactericidal functions of human granulocytes, thereby improving the host defense against bacterial infections.

Differentiation among closely related organisms of the Actinobacillus-Haemophilus-Pasteurella group by means of lysozyme and EDTA.

Bacteriolysis in Tris-maleate buffer (0.005 M, pH 7.2) supplemented with EDTA (0.01 M) and hen egg white lysozyme (HEWL, 1.0 microgram/ml) was set up to assist differentiation between the taxonomically closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus. A. actinomycetemcomitans was more sensitive to lysis in this system than H. aphrophilus. The standard method for bacteriolysis separated the 10 tested strains of A. actinomycetemcomitans into two groups (I and II) based on their lysis patterns, whereas the 7 strains of H. aphrophilus examined were homogeneous. In group I of A. actinomycetemcomitans, EDTA displayed a considerable lytic effect, which was not increased by supplementation with HEWL. In group II, the lytic effect of EDTA was much less, but HEWL had a considerable supplementary lytic effect. When the turbidity of A. actinomycetemcomitans (ATCC 29522) or H. aphrophilus (ATCC 33389) suspended in Tris buffer was monitored at close pH intervals (0.2) from pH 5.2 to 9.2, maximal lysis of ATCC 29522 occurred with EDTA at pH 8.0 and with EDTA-HEWL at pH 7.6, while ATCC 33389 lysed with EDTA at pH 9.0 and with EDTA-HEWL at pH 9.2. When other members of the family Pasteurellaceae (Haemophilus influenzae type b, Haemophilus paraphrophilus, Pasteurella multocida, Pasteurella haemolytica, and Pasteurella ureae) were included for comparison, the group I strains of A. actinomycetemcomitans were the most rapidly lysed by EDTA. H. paraphrophilus was the least sensitive of the gram-negative strains tested, but not as resistant as Micrococcus luteus (control). M. luteus was the organism most sensitive to lysozyme, followed by P. ureae and the group II strains of A. actinomycetemcomitans, while the group I strains of A. actinomycetemcomitans, H. paraphrophilus, and P. haemolytica were the least sensitive organisms.

Autolysis of Caulobacter crescentus grown in the presence of glycine.

Caulobacter crescentus was grown in complex medium supplemented with low (0.05%) concentration of glycine, a component of the murein peptide side chains of this bacterium. Murein synthesized in the presence of glycine was poorly crosslinked and the rate of its synthesis was slowed down compared to the control cells. The glycine-grown cells were considerably more sensitive to the chelating agent EDTA and Tris buffer than the control cells and also lysed faster when incubated with beta-lactam antibiotics. No changes in phospholipid composition in the presence of glycine were observed and the outer membrane protein composition of the glycine-grown cells was altered only in the amount of 130 000 protein which forms the surface array of C. crescentus. The effects of glycine can thus be tentatively put down to the reduced crosslinkage of murein synthesized in its presence.

Determination of phagocytosis of 32P-labeled Staphylococcus aureus by bovine polymorphonuclear leukocytes.

A procedure for the measurement of phagocytosis by bovine polymorphonuclear leukocytes (PMN) of 32P-labeled Staphylococcus aureus was modified so that a larger number of samples could be compared in a single run, and smaller volumes of sample, PMN, and 32P-labeled S aureus could be used. Results were highly reproducible, with a coefficient of variation between duplicate determinations of less than or equal to 2%. Lysostaphin was prepared from the supernatant of S staphylolyticus and was compared with a commercially available preparation. Effects of lysostaphin on PMN and influence of incubation media on release of 32P from 32P-labeled S aureus by lysostaphin were examined.

Lysozyme and its presence in bovine milk and serum.

The literature on the presence of lysozyme in various biological fluids in physiological and pathological conditions is reviewed. Preliminary results of lysozyme estimations in bovine milk show that lysozyme levels are definitely higher in colostrum and mastitis milk than in normal milk. The biological role of the enzyme in the milk still has to be defined. On the other hand, lysozyme may interfere with microbiological screening techniques for penicillin in cow's milk; the lytic effect of purified human lysozyme on B. stearothermophilus var. calidolactis was demonstrated microscopically.