Heterogeneity in Macrophage Phagocytosis of Staphylococcus aureus Strains: High-Throughput Scanning Cytometry-Based Analysis

Alveolar macrophages (AMs) can phagocytose unopsonized pathogens such as S. aureus via innate immune receptors, such as scavenger receptors (SRs). Cytoskeletal events and signaling pathways involved in phagocytosis of unopsonized bacteria likely govern the fate of ingested pathogens, but are poorly characterized. We have developed a high-throughput scanning cytometry-based assay to quantify phagocytosis of S. aureus by adherent human blood-derived AM-like macrophages in a 96-well microplate format. Differential fluorescent labeling of internalized vs. bound bacteria or beads allowed automated image analysis of collapsed confocal stack images acquired by scanning cytometry, and quantification of total particles bound and percent of particles internalized. We compared the effects of the classic SR blocker polyinosinic acid, the cytoskeletal inhibitors cytochalasin D and nocodazole, and the signaling inhibitors staurosporine, Gö 6976, JNK Inhibitor I and KN-93, on phagocytosis of a panel of live unopsonized S. aureus strains, (Wood, Seattle 1945 (ATCC 25923), and RN6390), as well as a commercial killed Wood strain, heat-killed Wood strain and latex beads. Our results revealed failure of the SR inhibitor polyinosinic acid to block binding of any live S. aureus strains, suggesting that SR-mediated uptake of a commercial killed fluorescent bacterial particle does not accurately model interaction with viable bacteria. We also observed heterogeneity in the effects of cytoskeletal and signaling inhibitors on internalization of different S. aureus strains. The data suggest that uptake of unopsonized live S. aureus by human macrophages is not mediated by SRs, and that the cellular mechanical and signaling processes that mediate S. aureus phagocytosis vary. The findings also demonstrate the potential utility of high-throughput scanning cytometry techniques to study phagocytosis of S. aureus and other organisms in greater detail.     

Adherence and intracellular survival within human macrophages of Enterococcus faecalis isolates from coastal marine sediment

Enterococcus faecalis is part of the human intestinal microbiota and an important nosocomial pathogen. It can be found in the marine environment, where it is also employed as a fecal indicator. To assess the pathogenic potential of marine E. faecalis, four strains isolated from marine sediment were analyzed for their ability to survive in human macrophages. Escherichia coli DH5α was used as a negative control. The number of adherent and intracellular bacteria was determined 2.5 h after the infection (T₀) and after further 24h (T₂₄) by CFU and qPCR counts. At T₂₄ adherent and intracellular enterococcal CFU counts were increased for all strains, the increment in intracellular bacteria being particularly marked. No CFU of E. coli DH5α were detected. In contrast, qPCR counts of intracellular enterococcal and E. coli bacteria were similar at both time points. These findings suggest that whereas E. coli was killed within macrophages (no CFU, positive qPCR), the E. faecalis isolates not only escaped killing, but actually multiplied, as demonstrated by the increase in the viable cell population. These findings support earlier data by our group, further documenting that marine sediment can be a reservoir of pathogenic enterococci.     

Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and ¹⁴C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10³ cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.     

Non-Classical ProIL-1beta Activation during Mammary Gland Infection Is Pathogen-Dependent but Caspase-1 Independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.     

Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10⁶ V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase.     

Characterization and bioactivity of hepcidin-2 in zebrafish: Dependence of antibacterial activity upon disulfide bridges

Hepcidin is an antimicrobial peptide and iron-regulatory molecule with highly conserved disulfide bridges among vertebrates, but structural insights into the function in fish remains largely missing. We demonstrate here that recombinant hepcidin-2 from zebrafish is capable of inhibiting the growth of the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 18, 15, 13 and 9 μM, respectively. We also show by TEM examination that recombinant hepcidin-2 is directly cidal to the cells of E. coli and S. aureus. Moreover, we find that hepcidin-2 displays affinity to LPS, LTA and PGN. All these data indicate that hepcidin-2 is both a pattern recognition molecule, capable of identifying LPS, LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2 depends upon the disulfide bridges.     

Bacterial Contamination of Drinking Water Supplies in a Modern Rural Neighborhood

On six occasions during a 15-month period, the private well and spring water supplies in a modern rural neighborhood of 78 households were examined for total coliforms, fecal coliforms, Staphylococcus aureus, and standard plate count bacteria. More than one-third of the water supplies were unsatisfactory on at least one occasion as judged by standard plate counts over 10³/ml and the presence of coliforms, fecal coliforms, and/or S. aureus. Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli were the most frequently isolated total coliforms. At least 12 other genera of bacteria were identified from standard plate count agar. Coliform contamination was found to be higher after periods of rainfall, and high standard plate counts were more prevalent during warmer weather. These observations probably reflect leakage of surface water into improperly sealed wells or aquifer contamination during winter and the lack of chlorination to control microbial regrowth during the warm season. An inverse correlation was found between the presence of high standard plate counts and incidence of coliforms. Consumer education and at least a twice yearly monitoring of private water supplies (winter and summer) are suggested methods to signal that treatment may be necessary to reduce the risk of waterborne disease.     

Soft cheese supplemented with black cumin oil: Impact on food borne pathogens and quality during storage

Black cumin seed oil (BCSO) was tested for its inhibitory effect against some pathogenic bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Listeria monocytogenes Scott A and Salmonella enteritidis PT4) in Domiati cheese during cold storage. Physical, chemical and sensorial changes in cheese during storage were recorded. Pasteurized milk was inoculated before renneting with a mixed culture of bacteria at ca. 4 log CFU mL⁻¹. In vitro and in situ supplementation with BCSO showed antimicrobial impact on the growth of S. aureus, E. coli, L. monocytogenes and S. enteritidis inoculated into media and cheese samples. Supplementing of cheese with BCSO (0.1% or 0.2%, w/w) significantly reduced the counts of the inoculated pathogens by ca. 1.3 log and 1.5 log CFU g⁻¹ after 21 days of storage. In addition, BCSO controlled the development of titratable acidity, limited the changes in ripening indices, flavor components and kept considerable physicochemical and sensorial properties of cheese.     

Analogs of nitrofuran antibiotics are potent GroEL/ES inhibitor pro-drugs

In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.     

Survival of Indicator and Pathogenic Bacteria in Bovine Feces on Pasture

The survival of enteric bacteria was measured in bovine feces on pasture. In each season, 11 cow pats were prepared from a mixture of fresh dairy cattle feces and sampled for up to 150 days. Four pats were analyzed for Escherichia coli, fecal streptococci, and enterococci, and four inoculated pats were analyzed for Campylobacter jejuni and Salmonella enterica. Two pats were placed on drainage collectors, and another pat was fitted with a temperature probe. In the first 1 to 3 weeks, there were increases (up to 1.5 orders of magnitude) in the counts of enterococci (in four seasons), E. coli (three seasons), fecal streptococci (three seasons), and S. enterica (two seasons), but there was no increase in the counts of C. jejuni. Thereafter, the counts decreased, giving an average ranking of the times necessary for 90% inactivation of C. jejuni (6.2 days from deposition) < fecal streptococci (35 days) < S. enterica (38 days) < E. coli (48 days) < enterococci (56 days). The pat temperature probably influenced bacterial growth, but the pattern of increases and decreases was primarily determined by desiccation; growth occurred when the water content was greater than 80%, but at a water content of 70 to 75% counts decreased. E. coli and enterococcus regrowth appeared to result from pat rehydration. Of 20 monthly leaching losses of E. coli, 16 were <10% of the total counts in the pat, and 12 were <1%. Drainage losses of C. jejuni (generally <1%) were detected for only 1 to 2 months. Although enterococci exhibited the best survival rate, higher final counts suggested that E. coli is the more practical indicator of bovine fecal pollution.     

Synthesis and evaluation of a class of new coumarin triazole derivatives as potential antimicrobial agents

A series of new coumarin-based 1,2,4-triazole derivatives were designed, synthesized and evaluated for their antimicrobial activities in vitro against four Gram-positive bacteria (Staphylococcus aureus, MRSA, Bacillus subtilis and Micrococcus luteus), four Gram-negative bacteria (Escherichia coli, Proteus vulgaris, Salmonella typhi and Shigella dysenteriae) as well as three fungi (Candida albicans, Saccharomyces cerevisiae and Aspergillus fumigatus) by two-fold serial dilution technique. The bioactive assay showed that some synthesized coumarin triazoles displayed comparable or even better antibacterial and antifungal efficacy in comparison with reference drugs Enoxacin, Chloromycin and Fluconazole. Coumarin bis-triazole compounds exhibited stronger antibacterial and antifungal efficiency than their corresponding mono-triazole derivatives.     

Bactericidal effect of Naja nigricollis toxin γ is related to its membrane-damaging activity

The aim of the present study is to investigate the causal relationship between membrane-damaging activity and bactericidal activity of Naja nigricollis toxin γ. Toxin γ showed a similar inhibitory activity on the growth of Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria). Antibacterial activity of toxin γ correlated positively with increase in membrane permeability of bacterial cells. Morphological examination showed that toxin γ disrupted the integrity of bacterial membrane. Toxin γ showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased bactericidal effect of toxin γ on E. coli and S. aureus, respectively. Although the potency of toxin γ on permeabilzing model membrane of E. coli and S. aureus was similar, the mode of interaction between toxin γ and model membrane of E. coli and S. aureus differed. Membrane-damaging activity of toxin γ was inhibited by either LPS or LTA. Nevertheless, LPS and LTA altered differently membrane-bound conformation of toxin γ. Taken together, our data suggest that bactericidal activity of toxin γ depends on its ability to induce membrane permeability, and that LPS and LTA structurally suppresses bactericidal effect of toxin γ.     

Differences in Toll-like receptor expression and cytokine production after stimulation with heat-killed gram-positive and gram-negative bacteria

Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognize conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. The aim of this study was to compare the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed gram-negative and gram-positive human pathogens: Neisseria meningitidis, Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli, and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of gram-positive bacteria, whereas higher concentrations of gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase when compared to low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, gram-negative pathogens induced higher cytokine levels when compared to gram-positive bacteria at the same density. These results indicate that gram-positive and gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not cytokine expression.     

Diversity of antagonistic bacteria isolated from medicinal plant Peganum harmala L.

The antimicrobial activity of plant extract of Peganum harmala, a medicinal plant has been studied already. However, knowledge about bacterial diversity associated with different parts of host plant antagonistic to different human pathogenic bacteria is limited. In this study, bacteria were isolated from root, leaf and fruit of plant. Among 188 bacterial isolates isolated from different parts of the plant only 24 were found to be active against different pathogenic bacteria i.e. Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, Enterococcus faecalis and Pseudomonas aeruginosa. These active bacterial isolates were identified on the basis of 16S rRNA gene analysis. Total population of bacteria isolated from plant was high in root, following leaf and fruit. Antagonistic bacteria were also more abundant in root as compared to leaf and fruit. Two isolates (EA5 and EA18) exhibited antagonistic activity against most of the targeted pathogenic bacteria mentioned above. Some isolates showed strong inhibition for one targeted pathogenic bacterium while weak or no inhibition for others. Most of the antagonistic isolates were active against MRSA, following E. faecium, P. aeruginosa, E. coli and E. faecalis. Taken together, our results show that medicinal plants are good source of antagonistic bacteria having inhibitory effect against clinical bacterial pathogens.     

Fast and Effective Photodynamic Inactivation of Multiresistant Bacteria by Cationic Riboflavin Derivatives

Photodynamic inactivation of bacteria (PIB) proves to be an additional method to kill pathogenic bacteria. PIB requires photosensitizer molecules that effectively generate reactive oxygen species like singlet oxygen when exposed to visible light. To allow a broad application in medicine, photosensitizers should be safe when applied in humans. Substances like vitamin B2, which are most likely safe, are known to produce singlet oxygen upon irradiation. In the present study, we added positive charges to flavin derivatives to enable attachment of these molecules to the negatively charged surface of bacteria. Two of the synthesized flavin derivatives showed a high quantum yield of singlet oxygen of approximately 75%. Multidrug resistant bacteria like MRSA (Methicillin resistant Staphylococcus aureus), EHEC (enterohemorrhagic Escherichia coli), Pseudomonas aeruginosa, and Acinetobacter baumannii were incubated with these flavin derivatives in vitro and were subsequently irradiated with visible light for seconds only. Singlet oxygen production in bacteria was proved by detecting its luminescence at 1270 nm. After irradiation, the number of viable bacteria decreased up to 6 log₁₀ steps depending on the concentration of the flavin derivatives and the light dosimetry. The bactericidal effect of PIB was independent of the bacterial type and the corresponding antibiotic resistance pattern. In contrast, the photosensitizer concentration and light parameters used for bacteria killing did not affect cell viability of human keratinocytes (therapeutic window). Multiresistant bacteria can be safely and effectively killed by a combination of modified vitamin B2 molecules, oxygen and visible light, whereas normal skin cells survive. Further work will include these new photosensitizers for topical application to decolonize bacteria from skin and mucosa.     

Killing of Staphylococcus aureus via Magnetic Hyperthermia Mediated by Magnetotactic Bacteria

Staphylococcus aureus is a common hospital and household pathogen. Given the emergence of antibiotic-resistant derivatives of this pathogen resulting from the use of antibiotics as general treatment, development of alternative therapeutic strategies is urgently needed. Here, we assess the feasibility of killing S. aureus cells in vitro and in vivo through magnetic hyperthermia mediated by magnetotactic bacteria that possess magnetic nanocrystals and demonstrate magnetically steered swimming. The S. aureus suspension was added to magnetotactic MO-1 bacteria either directly or after coating with anti-MO-1 polyclonal antibodies. The suspensions were then subjected to an alternating magnetic field (AMF) for 1 h. S. aureus viability was subsequently assessed through conventional plate counting and flow cytometry. We found that approximately 30% of the S. aureus cells mixed with uncoated MO-1 cells were killed after AMF treatment. Moreover, attachment between the magnetotactic bacteria and S. aureus increased the killing efficiency of hyperthermia to more than 50%. Using mouse models, we demonstrated that magnetic hyperthermia mediated by antibody-coated magnetotactic MO-1 bacteria significantly improved wound healing. These results collectively demonstrated the effective eradication of S. aureus both in vitro and in vivo, indicating the potential of magnetotactic bacterium-mediated magnetic hyperthermia as a treatment for S. aureus-induced skin or wound infections.     

Serine-based surfactants as effective antimicrobial agents against multiresistant bacteria

The antimicrobial activity of two serine derived gemini cationic surfactants, amide (12Ser)₂CON12 and ester (12Ser)₂COO12, was tested using sensitive, E. coli ATCC 25922 and S. aureus ATCC 6538, and resistant, E. coli CTX M2, E. coli TEM CTX M9 and S. aureus ATCC 6538 and S. aureus MRSA ATCC 43300 Gram-positive and Gram-negative bacteria strains. Very low MIC values (5 μM) were found for the two resistant strains E.coli TEM CTX M9 and S. aureus MRSA ATCC 43300, in the case of the amide derivative, and for S. aureus MRSA ATCC 43300, in the case of the ester derivative. The interaction of the serine amphiphiles with lipid-model membranes (DPPG and DPPC) was investigated using Langmuir monolayers. A more pronounced effect on the DPPG than on the DPPC monolayer was observed. The effect induced by the surfactants on bacteria membrane was explored by Atomic Force Microscopy. A clear disruption of the bacteria membrane was observed for E. coli TEM CTX M9 upon treatment with (12ser)₂CON12, whereas for the S. aureus MRSA few observable changes in cell morphology were found after treatment with either of the two surfactants. The cytotoxicity of the two compounds was assessed by hemolysis assay on human red blood cells (RBC). The compounds were shown to be non-cytotoxic up to 10 μM. Overall, the results reveal a promising potential, in particular of the amide derivative, as antimicrobial agent for two strains of antibiotic resistant bacteria.     

Two major medicinal honeys have different mechanisms of bactericidal activity

Honey is increasingly valued for its antibacterial activity, but knowledge regarding the mechanism of action is still incomplete. We assessed the bactericidal activity and mechanism of action of Revamil® source (RS) honey and manuka honey, the sources of two major medical-grade honeys. RS honey killed Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa within 2 hours, whereas manuka honey had such rapid activity only against B. subtilis. After 24 hours of incubation, both honeys killed all tested bacteria, including methicillin-resistant Staphylococcus aureus, but manuka honey retained activity up to higher dilutions than RS honey. Bee defensin-1 and H₂O₂ were the major factors involved in rapid bactericidal activity of RS honey. These factors were absent in manuka honey, but this honey contained 44-fold higher concentrations of methylglyoxal than RS honey. Methylglyoxal was a major bactericidal factor in manuka honey, but after neutralization of this compound manuka honey retained bactericidal activity due to several unknown factors. RS and manuka honey have highly distinct compositions of bactericidal factors, resulting in large differences in bactericidal activity.     

Differences in Toll-like receptor expression and cytokine production after stimulation with heat-killed Gram-positive and Gram-negative bacteria

Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognise conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. Here, we compared the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed Gram-negative and Gram-positive human pathogens Neisseria meningitidis, Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of Gram-positive bacteria, whereas higher concentrations of Gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase compared with low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, Gram-negative pathogens induced higher cytokine levels than Gram-positive ones at same concentrations. These results indicate that Gram-positive and Gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not in cytokine expression.     

Modification of membrane properties and fatty acids biosynthesis-related genes in Escherichia coli and Staphylococcus aureus: Implications for the antibacterial mechanism of naringenin

In this work, modifications of cell membrane fluidity, fatty acid composition and fatty acid biosynthesis-associated genes of Escherichia coli ATCC 25922 (E. coli) and Staphylococcus aureus ATCC 6538 (S. aureus), during growth in the presence of naringenin (NAR), one of the natural antibacterial components in citrus plants, was investigated. Compared to E. coli, the growth of S. aureus was significantly inhibited by NAR in low concentrations. Combination of gas chromatography–mass spectrometry with fluorescence polarization analysis revealed that E. coli and S. aureus cells increased membrane fluidity by altering the composition of membrane fatty acids after exposure to NAR. For example, E. coli cells produced more unsaturated fatty acids (from 18.5% to 43.3%) at the expense of both cyclopropane and saturated fatty acids after growth in the concentrations of NAR from 0 to 2.20 mM. For S. aureus grown with NAR at 0 to 1.47 mM, the relative proportions of anteiso-branched chain fatty acids increased from 37.2% to 54.4%, whereas iso-branched and straight chain fatty acids decreased from 30.0% and 33.1% to 21.6% and 23.7%, respectively. Real time q-PCR analysis showed that NAR at higher concentrations induced a significant down-regulation of fatty acid biosynthesis-associated genes in the bacteria, with the exception of an increased expression of fabA gene. The minimum inhibitory concentration (MIC) of NAR against these two bacteria was determined, and both of bacteria underwent morphological changes after exposure to 1.0 and 2.0 MIC.