Extracellular Bactericidal Activity Research Articles

Natural antibacterial agent-based nanoparticles for effective treatment of intracellular MRSA infection.

Intracellular MRSA is extremely difficult to eradicate by traditional antibiotics, leading to infection dissemination and drug resistance. A general lack of facile and long-term strategies to effectively eliminate intracellular MRSA. In this study, glabridin (GLA)-loaded pH-responsive nanoparticles (NPs) were constructed using cinnamaldehyde (CA)-dextran conjugates as carriers. These NPs targeted infected macrophages/MRSA via dextran mediation and effectively accumulated at the MRSA infection site. The NPs were then destabilized in response to the low pH of the lysosomes, which triggered the release of CA and GLA. The released CA downregulated the expression of cytotoxic pore-forming toxins, thereby decreasing the damage of macrophage and risk of the intracellular bacterial dissemination. Meanwhile, GLA could rapidly kill intracellularly entrapped MRSA with a low possibility of developing resistance. Using a specific combination of the natural antibacterial agents CA and GLA, NPs effectively eradicated intracellular MRSA with low toxicity to normal tissues in a MRSA-induced peritonitis model. This strategy presents a potential alternative for enhancing intracellular MRSA therapy, particularly for repeated and long-term clinical applications. STATEMENT OF SIGNIFICANCE: Intracellular MRSA infections are a growing threat to public health, and there is a general lack of a facile strategy for efficiently eliminating intracellular MRSA while reducing the ever-increasing drug resistance. In this study, pH-responsive and macrophage/MRSA-targeting nanoparticles were prepared by conjugating the phytochemical cinnamaldehyde to dextran to encapsulate the natural antibacterial agent glabridin. Using a combination of traditional Chinese medicine, the NPs significantly increased drug accumulation in MRSA and showed superior intracellular and extracellular bactericidal activity. Importantly, the NPs can inhibit potential intracellular bacteria dissemination and reduce the development of drug resistance, thus allowing for repeated treatment. Natural antibacterial agent-based drug delivery systems are an attractive alternative for facilitating the clinical treatment of intracellular MRSA infections.

Inorganic arsenic administration suppresses human neutrophil function in vitro.

Arsenic, a major environmental toxicant and pollutant, is a global public health concern. Among its many adverse effects, arsenic is immunotoxic, but its effects on human neutrophil functions are not yet well-defined. In this study, we aimed to evaluate the in vitro effects of acute low-dose NaAsO2 exposure on human polymorphonuclear neutrophils (PMNs) for 12 h on the following innate defense mechanisms: formation of neutrophil extracellular traps (NETs), production of reactive oxygen species (ROS), and phagocytosis. Phorbol myristate acetate (PMA) was added to induce NETs formation, which was quantified by measuring cell-free extracellular DNA (cf-DNA), myeloperoxidase-conjugated (MPO)-DNA and neutrophil elastase-conjugated (NE)-DNA, and confirmed by immunofluorescence labeling and imaging. Extracellular bactericidal activity by NETs was evaluated by co-culturing Escherichia coli and PMNs in the presence of a phagocytic inhibitor. Levels of NETs in the culture medium after PMA stimulation was significantly lower in PMNs pre-exposed to arsenic than those not exposed to arsenic. Immunofluorescence staining and extracellular bactericidal activity by NETs revealed similar results. Phagocytosis and ROS production by PMNs were also significantly reduced by arsenic pre-exposure. Together, our findings provide new insights in arsenic immunotoxicity and suggest how it increases susceptibility to infectious diseases in humans.

Biochanin a Enhances the Defense Against Salmonella enterica Infection Through AMPK/ULK1/mTOR-Mediated Autophagy and Extracellular Traps and Reversing SPI-1-Dependent Macrophage (MΦ) M2 Polarization.

A novel treatment regimen for bacterial infections is the pharmacological enhancement of the host's immune defenses. We demonstrated that biochanin A (BCA), an isoflavone constituent in some plants, could enhance both intra- and extracellular bactericidal activity of host cells. First, BCA could induce a complete autophagic response in nonphagocytic cells (HeLa) or macrophages (MΦ) via the AMPK/ULK1/mTOR pathway and Beclin-1-dependent manner, and BCA enhanced the killing of invading Salmonella by autophagy through reinforcing ubiquitinated adapter protein (LRSAM1, NDP52 and p62)-mediated recognition of intracellular bacteria and through the formation of autophagolysosomes. Second, we demonstrated that BCA could enhance the release of MΦ extracellular traps (METs) to remove extracellular Salmonella also via the AMPK/ULK1/mTOR pathway, not through reactive oxygen species (ROS) pathway. Furtherly, in a Salmonella-infected mouse model, BCA treatment increased intra- and extracellular bactericidal activity through the strengthening autophagy and MET production, respectively, in peritoneal MΦ, liver and spleen tissue. Additionally, our findings showed that BCA downregulated SPI-1 (Salmonella pathogenicity island 1) expression during Salmonella infection in vitro and in vivo to reverse the MΦ M2 polarization, which was different from the MΦ M1 phenotype caused by most of bacteria infection. Together, these findings suggest that BCA has an immunomodulatory effect on Salmonella-infected host cells and enhances their bactericidal activity in vitro and in vivo through autophagy, extracellular traps and regulation of MΦ polarization.

Novel long-chain compounds with both immunomodulatory and MenA inhibitory activities against Staphylococcus aureus and its biofilm

Menaquinone (MK) biosynthesis pathway is a potential target for evaluating antimicrobials in gram-positive bacteria. Here, 1,4-dihydroxy-2-naphthoate prenyltransferase (MenA) was targeted to reduce methicillin-resistant Staphylococcus aureus (MRSA) growth. MenA inhibiting, long chain-based compounds were designed, synthesized and evaluated against MRSA and menaquinone utilizing bacteria in aerobic conditions. The results showed that these bacteria were susceptible to most of the compounds. Menaquinone (MK-4) supplementation rescued MRSA growth, suggesting these compounds inhibit MK biosynthesis. 3a and 7c exhibited promising inhibitory activities with MICs ranging 1–8 μg/mL against MRSA strains. The compounds did not facilitate small colony variant formation. These compounds also inhibited the biofilm growth by MRSA at high concentration. Compounds 3a, 6b and 7c displayed a promising extracellular bactericidal activity against MRSA at concentrations equal to and four-fold less than their respective MICs. We also observed cytokines released from THP-1 macrophages treated with compounds 3a, 6b and 7c and found decreases in TNF-α and IL-6 release and increase in IL-1β. These data provide evidence that MenA inhibitors act as TNF-α and IL-6 inhibitors, raising the potential for development and application of these compounds as potential immunomodulatory agents.

Formation of Extracellular Traps - the Effective Mechanism of Organism Protection from Pathogen

Summarized and analyzed are modern literature data on capability of cells (neutrophils, eosinophils and labrocytes) realizing the mechanisms of innate immunity, to form extracellular traps that capture and kill pathogens by secretion of DNA and antibacterial granules content into extracellular space. The efficiency of capture and bacterial clearance is higher in extracellular traps than that in phagocytosis. The mechanism of extracellular bactericidal activity is supposed to play an important role in protection of organism from bacteria which are resistant to phagocytosis by leukocytes.

Extracellular bactericidal functions of porcine neutrophils (133.20)

Abstract Neutrophils are one of the main effector cells of innate immunity and were shown to kill bacteria via phagocytosis more than 100 years ago. New developments, however, also show that neutrophils are capable of antimicrobial activity by producing extracellular structures named neutrophil extracellular traps (NETs). The antimicrobial effects of NETs can be attributed to cathelicidins, a category of antimicrobial peptides stored as inactive proforms. These proforms are cleaved by neutrophils elastase during cell activation in response to pathogens. This project focuses on the cathelicidin peptides contained within NETs and their effectiveness in killing several common bacterial pathogens of swine. Subsequent investigations address the contribution of cathelicidins to extracellular bactericidal activity of porcine neutrophils. Activated secretions of neutrophils are effective in killing 100% of Escherichia coli K12, 21% of Streptococcus suis, and 74% of Actinobacillus suis. Pasteurella multocida appears to survive in the presence of activated neutrophil secretions demonstrating a 38% increase in growth when compared to P. multocida exposed to non activated neutrophils secretions. Identification of the contribution of cathelicidins to this bactericidal activity is pending. These data suggest that activated neutrophil secretions have bactericidal activity against common pathogens in swine.

Differential drug susceptibility of intracellular and extracellular tuberculosis, and the impact of P-glycoprotein

If tuberculosis therapy is to be shortened it is imperative that the sterilising activity of current and future anti-tuberculosis drugs is enhanced. Intracellular Mycobacterium tuberculosis (MTB) phagocytosed by macrophages may be a key subpopulation of bacteria that are less readily eliminated by therapy. Here we investigate whether macrophages provide MTB with a pharmacological sanctuary site, making them less susceptible to chemotherapy than extracellular bacilli. Intracellular drug activity was determined by a novel colorimetric method that measures the ability of a drug to protect A-THP1 cells from infection-mediated cell death by H37Rv. Extracellular bactericidal activity was determined by the microplate alamar blue assay (MABA). Further, the effect of P-glycoprotein (P-gp) expressed on macrophages on the intracellular kill of H37Rv was assessed. To screen the anti-tuberculosis drugs for P-gp substrate specificity, their toxicity and cellular accumulation were determined in CEM and CEM(VBL100) cells. Intracellular and extracellular anti-tuberculosis drug activity following 7-day treatment with isoniazid (mean EC(50)+/-SD: 36.7+/-2.2 and 57.2+/-2.5 ng/mL, respectively) and ethambutol (243+/-95 and 263+/-12 ng/mL, respectively) were similar. However, for rifampicin a higher concentration was required to kill intracellular (148+/-32 ng/mL) versus extracellular (1.27+/-0.02 ng/mL) bacilli. The P-gp inhibitor tariquidar, significantly increased intracellular kill of H37Rv by ethambutol and rifampicin and both of these drugs were shown to be substrates for P-gp using the P-gp overexpressing CEM(VBL100) cells. We observed a large discrepancy between intracellular and extracellular activity of rifampicin (but not with isoniazid or ethambutol). Several factors could have accounted for this including inoculum size, media and cell-mediated metabolism. These factors make the comparison of intracellular and extracellular drug activity complex. However, the intracellular assay described here has potential for studying the impact of host proteins (such as drug transporters) on the intracellular activity of drugs, and has been used successfully here to demonstrate that both rifampicin and ethambutol are substrates for P-gp.

Phagolysosomal Alkalinization and Intracellular Killing of Staphylococcus aureus by Amikacin

The aminoglycosides are ineffective against intracellular Staphylococcus aureus, which resides within lysosomes, despite a strong extracellular bactericidal activity. Since they are slowly concentrated within lysosomes it was hypothesized that acidity within these cell compartments might impair antibiotic activity. The bactericidal activity of amikacin alone and combined with lysosomotropic alkalinizing agents (LAA), which can alkalinize acidic cell compartments, was evaluated. Before antibiotic challenge, some cells were preincubated with amikacin for 3 days to allow intracellular accumulation of drug. No intracellular killing activity was shown when non-preincubated cells were used. Conversely, with preincubated cells, S. aureus was killed within 4 h when LAA were incorporated into the incubation media but not with amikacin alone. Enhanced antimicrobial activity correlated with increase in lysosomal pH. Intracellular accumulation of amikacin was not changed by LAA. These results provide evidence that acidic pH within lysosomes impairs amikacin in killing S. aureus.

Intraphagocytic bioactivity of lomefloxacin against Pseudomonas aeruginosa.

The kinetics of extracellular bactericidal activity and the intraphagocytic bioactivity of lomefloxacin at concentrations 2 mg/L (1/2 x MIC), 4 mg/L (MIC) and 8 mg/L (2 x MIC) against Pseudomonas aeruginosa were measured, the first in 40% human serum and the second in polymorphonuclear leucocytes (PMNLs), by the colony-counting method. The PMNLs were treated with phenylbutazone to prevent them killing the bacteria during the ingestion period. The kinetics of extracellular killing were biphasic with an initial rapid phase lasting 30 min when lomefloxacin was used at concentrations equal to the MIC and 2 x MIC, during which a decrease of 3 log of cfu/mL occurred. Lomefloxacin showed intraphagocytic activity at the three concentrations assayed. With non-treated PMNLs the percentages of intraphagocytic killing after 30 min of incubation were 93.5, 91.3 and 95.5% with 2, 4 and 8 mg/L, respectively, and 79.8, 65.0 and 93.3% for the same lomefloxacin concentrations with phenylbutazone-treated PMNLs, suggesting a synergic interaction between the antibacterial activity of PMNLs and lomefloxacin.

Electron-autoradiographic demonstration of the intra-and extracellular bactericidal activity of neutrophils

Electron-autoradiographic demonstration of the intra-and extracellular bactericidal activity of neutrophils