Inability to sustain intraphagolysosomal killing of Staphylococcus aureus predisposes to bacterial persistence in macrophages

Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing of bacteria are interrelated functions essential for bacterial clearance but the rate‐limiting step when macrophages are challenged with large numbers of the major medical pathogen Staphylococcus aureus is unknown. We show that macrophages have a finite capacity for intracellular killing and fail to match sustained phagocytosis with sustained microbial killing when exposed to large inocula of S. aureus (Newman, SH1000 and USA300 strains). S. aureus ingestion by macrophages is associated with a rapid decline in bacterial viability immediately after phagocytosis. However, not all bacteria are killed in the phagolysosome, and we demonstrate reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D. This results in accumulation of viable intracellular bacteria in macrophages. We show macrophages fail to engage apoptosis‐associated bacterial killing. Ultittop mately macrophages with viable bacteria undergo cell lysis, and viable bacteria are released and can be internalized by other macrophages. We show that cycles of lysis and reuptake maintain a pool of viable intracellular bacteria over time when killing is overwhelmed and demonstrate intracellular persistence in alveolar macrophages in the lungs in a murine model.     

PINK1/Parkin-mediated mitophagy enhances the survival of Staphylococcus aureus in bovine macrophages

Mitochondria are cellular organelles that are involved in various metabolic processes, and damage to mitochondria can affect cell health and even lead to disease. Mitophagy is a mechanism by which cells selectively wrap and degrade damaged mitochondria to maintain cell homeostasis. However, studies have not focused on whether mitophagy is involved in the occurrence of Staphylococcus aureus (S. aureus)-induced mastitis in dairy cows. Here, we found that S. aureus infection of bovine macrophages leads to oxidative damage and mitochondria damage. The expression of LC3, PINK1 and Parkin was significantly increased after intracellular infection. We observed changes in the morphology of mitochondria and the emergence of mitochondrial autolysosomes in bovine macrophages by transmission electron microscopy and found that enhanced mitophagy promoted bacterial proliferation in the cell. In conclusion, this study demonstrates that S. aureus infection of bovine macrophages induces mitophagy through the PINK1/Parkin pathway, and this mechanism is used by the bacteria to avoid macrophage-induced death. These findings provide new ideas and references for the prevention and treatment of S. aureus infection.     

Enzymatic Treatment to Eliminate the Extracellular ATP for Improving the Detectability of Bacterial Intracellular ATP

A novel and effective treatment of biological samples with a combination of adenosine phosphate deaminase and apyrase was developed for reducing extracellular ATP, which has been a major problem encountered in improving the sensitivity of assays for intracellular ATP by the firefly luciferin–luciferase (L-L) method. Under the enzymatic reaction conditions, ATP and the related adenosine derivatives were converted to IMP, which are not active to the L-L system. In the model system (3.2 × 10⁻⁸mATP in 1% yeast extract solution) the treatment with adenosine phosphate deaminase resulted in the reduction of ATP to 1.3 × 10⁻¹¹m, and the concomitant use of apyrase lowered the concentration to 3.3 × 10⁻¹³m. The treatment (0.05 U/ml of adenosine phosphate deaminase and apyrase) was applied to the detection of bacteria in broth by the L-L method, affording the detection of 42 colony-forming unit (CFU)/ml ofEscherichia coliand 10 CFU/ml ofStaphylococcus aureusin the broth.     

Contribution of Catalase and Superoxide Dismutase to the Intracellular Survival of Clinical Isolates of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Murine Macrophages

The present study was performed in order to carefully investigate the interaction of Staphylococcus aureus with murine macrophages and the contribution of catalase and superoxide dismutase in intracellular persistence of Staphylococcus aureus within murine macrophages during in vitro infection. We have reported that Staphylococcus aureus internalized by murine macrophages did not appear to be rapidly killed. Data indicating the contribution of a single catalase and superoxide dismutase in intracellular survival of Staphylococcus aureus were provided using established biochemical assays. The results of the present experiment suggest that the survival of Staphylococcus aureus within phagocytic cells is facilitated by its ability to resist oxidative products. Organisms in the log phase of growth clearly demonstrate a resistance to oxidative products.     

Influence of Sae‐regulated and Agr‐regulated factors on the escape of Staphylococcus aureus from human macrophages

Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild‐type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol‐soluble modulins (PSMs) for phagosomal escape. Thus, Sae‐regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis‐related caspase 3, 7 or 8 nor to NLRP3‐dependent inflammasome activation.     

Kinetics of bactericidal activity of antibiotics measured by luciferin-luciferase assay

ATP production, measured by the luciferin-luciferase assay, is an indicator of bacterial metabolic activity. This enzymatic assay yields rapid results (< 5 minutes), permitting multiple measurements and establishment of ATP growth curves in order to study the kinetics of antibiotics in bacterial populations. The measurement of free or extracellular ATP, total ATP (extra and intracellular) and the ratio of free to total ATP are additional means of studying the bacteriostatic or bactericidal activity of antibiotics. An increase in free ATP is an indicator of extracellular movement due to alteration of the cell wall. The ratio free ATP/total ATP × 100 ≥ 50%, indicates bacteriallysis. These assays were used to study the effects of 14 antibiotics on two reference strains of Escherichia coli ATCC 25922 and Staphylococcus aureus 25923.     

A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model

The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to bedue to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle.     

A Comparison of the Effects of Ocular Preservatives on Mammalian and Microbial ATP and Glutathione Levels

The aim of this study was to investigate the mechanism of action of the preservative sodium chlorite (NaClO²), and the relationship with intracellular glutathione depletion. A detailed comparison of the dose responses of two cultured ocular epithelial cell types and four species of microorganism was carried out, and comparisons were also made with the quaternary ammonium compound benzalkonium chloride (BAK), and the oxidant hydrogen peroxide (H²O²). The viability of mammalian and microbial cells was assessed in the same way, by the measurement of intracellular ATP using a bioluminescence method. Intracellular total glutathione was measured by reaction with 5,5′-dithiobis-2-nitrobenzoic acid in a glutathione reductase-dependent recycling assay. BAK and H²O² caused complete toxicity to conjunctival and corneal epithelial cells at ～25?ppm, in contrast to NaClO², where >100?ppm was required. The fungi Candida albicans and Alternaria alternata had a higher resistance to NaClO² than the bacteria Staphyloccus aureus and Pseudomonas aeruginosa, but the bacteria were extremely resistant to H²O². NaClO² caused substantial depletion of intracellular glutathione in all cell types, at concentrations ranging from <10?ppm in Pseudomonas, 25–100?ppm in epithelial cells, to >500?ppm in fungal cells. The mechanisms of cytotoxicity of NaClO², H²O² and BAK all appeared to differ. NaClO² was found to have the best balance of high antibacterial toxicity with low ocular toxicity. The lower toxicity of NaClO² to the ocular cells, compared with BAK and H²O², is in agreement with fewer reported adverse effects of application in the eye.     

The Effect of the Intra- and Extracellular Metabolites of Microorganisms Isolated from Various Ecotopes on the Catalase Activity of Staphylococcus aureus ATCC 6538 P

The cell extracts (i.e., intracellular metabolites) and culture liquids (i.e., extracellular metabolites) of microorganisms isolated from various ecotopes were found to inhibit the catalase activity of Staphylococcus aureus ATCC 6538 P, which resulted in a considerable inhibition of the growth of metabolite-treated S. aureus cells by hydrogen peroxide. The inhibitory effect of microbial metabolites on S. aureus catalase can be considered as a mechanism of intercellular interactions responsible for the formation of microbiocenoses.     

Opsonin-dependent phagocytosis of bacteria by murine macrophage-like cells

We have investigated the phagocytic properties of the macrophage-like cell line DCH-7, derived from fusion of mouse macrophages with a mouse T-lymphoma cell line. These cells phagocytosed opsonized bacteria. IgG appeared to be the major opsonin forStaphylococcus aureus Wood 46 as well as for threeEscherichia coli strains; complement components were not required as opsonins. Intracellular bacteria survived to a large extent. This model system should be a useful tool for studying the process of phagocytosis and phagocytic killing of bacteria.     

Transformation of Staphylococcus aureus by heterologous plasmids

Plasmids isolated from Bacillus subtilis and Staphylococcus epidermidis were transformed into Staphylococcus aureus. Heterologous transformation was susceptible to restriction in S. aureus but could be performed in restriction-negative mutants or in heat-treated host bacteria. Three plasmids isolated from S. epidermidis were transformed into S. aureus with this technique and characterized. Two of them, pTE109 and pCE109, appear to be similar to two tet and cml plasmids previously isolated from S. aureus. The third, pPE109, carries penicillin and cadmium resistance and shows a restriction enzyme pattern which differs from known penicillinase plasmids in S. aureus.     

Disposable bioluminescence-based biosensor for detection of bacterial count in food

A biosensor for rapid detection of bacterial count based on adenosine 5′-triphosphate (ATP) bioluminescence has been developed. The biosensor is composed of a key sensitive element and a photomultiplier tube used as a detector element. The disposable sensitive element consists of a sampler, a cartridge where intracellular ATP is chemically extracted from bacteria, and a microtube where the extracted ATP reacts with the luciferin–luciferase reagent to produce bioluminescence. The bioluminescence signal is transformed into relevant electrical signal by the detector and further measured with a homemade luminometer. Parameters affecting the amount of the extracted ATP, including the types of ATP extractants, the concentrations of ATP extractant, and the relevant neutralizing reagent, were optimized. Under the optimal experimental conditions, the biosensor showed a linear response to standard bacteria in a concentration range from 10³ to 10⁸ colony-forming units (CFU) per milliliter with a correlation coefficient of 0.925 (n = 22) within 5 min. Moreover, the bacterial count of real food samples obtained by the biosensor correlated well with those by the conventional plate count method. The proposed biosensor, with characteristics of low cost, easy operation, and fast response, provides potential application to rapid evaluation of bacterial contamination in the food industry, environment monitoring, and other fields.     

Antibacterial activity and mechanism of chloroform fraction from aqueous extract of mugwort leaves (Artemisia argyi L.) against Staphylococcus aureus

In this work, the antibacterial activity and mechanism of chloroform fraction obtained from aqueous extract of mugwort leaves against Staphylococcus aureus were investigated. The extract showed obvious antibacterial activity against S. aureus which the minimum inhibitory concentration and minimum bactericidal concentration were determined to be 3·0 and 6·0 mg ml⁻¹ respectively. The mechanism study suggested that the extract could destroy the integrity of the S. aureus cell walls and increase the permeability of cell membrane in a certain concentration, but it could not kill S. aureus in a short time. Instead, the extract could make bacteria in a state of apoptosis for a long time, interfere with the normal physiological metabolism of bacteria, and eventually make bacteria die, which was confirm by scanning electronic microscope.     

Probiotic potential of Staphylococcus hominis MBBL 2–9 as anti‐Staphylococcus aureus agent isolated from the vaginal microbiota of a healthy woman

Aims: To isolate and characterize an antagonist for use as probiotic agent in the biocontrol of Staphylococcus aureus. Methods and Results: Bacteria that exhibited antimicrobial activity against Gram‐positive bacteria including Staph. aureus were isolated from 12 healthy women, with Staphylococcus hominis MBBL 2–9 showing the strongest activity. The bacteriocin produced by Staph. hominis MBBL 2–9 was purified by 60% ammonium sulfate saturation, ultrafiltration, HLB cartridge and reverse‐phase HPLC. The molecular weight was estimated as 2038·2 Da by MALDI–TOF mass spectrometry. The antagonist survived up to 2 h in artificial gastric juice (pH 2·5) and grew in the presence of 1% porcine bile extract. In addition, Staph. hominis MBBL 2–9 adhered effectively to HT‐29 epithelial cell line. Conclusion: Staphylococcus hominis MBBL 2–9 exhibited desirable probiotic traits such as acid tolerance, bile resistance and adherence to epithelial cell line. The bacterium also produced a bacteriocin with unique molecular weight and high antimicrobial activity similar to traditional antibiotics. Significance and Impact of the Study: This study is the first report of a bacteriocin‐producing Staph. hominis MBBL 2–9 that has potential for use as a probiotic agent against Staph. aureus.     

Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane

Helveticin-M, a novel Class III bacteriocin produced by Lactobacillus crispatus exhibited an antimicrobial activity against Staphylococcus aureus, S. saprophyticus, and Enterobacter cloacae. To understand how Helveticin-M injured target cells, Helveticin-M was cloned and heterologously expressed in Escherichia coli. Subsequently, the cell wall organization and cell membrane integrity of target cells were determined. The mechanism of cellular damage differed according to bacterial species. Based on morphology analysis, Helveticin-M disrupted the cell wall of Gram-positive bacteria and disorganized the outer membrane of Gram-negative bacteria, therefore, altering surface structure. Helveticin-M also disrupted the inner membrane, as confirmed by leakage of intracellular ATP from cells and depolarization of membrane potential of target bacteria. Based on cell population analysis, Helveticin-M treatment caused the increase of cell membrane permeability, but the cytosolic enzymes were not influenced, indicating that it was the sublethal injury. Therefore, the mode of Helveticin-M action is bacteriostatic rather than bactericidal.     

Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death,and bacterial escape and dissemination

The success of Staphylococcus aureus as a pathogen is partly attributable to its ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of phagocytes. Here, we have studied the interaction of methicillin‐resistant S. aureus (MRSA) strain USA300 with murine RAW 264.7 and primary human macrophages using molecular imaging and single cell analysis to obtain an unprecedented understanding of the interaction between the macrophage and MRSA. Herein we demonstrate that macrophages fail to control intracellular infection by MRSA USA300 despite trafficking the bacteria into mature phagolysosomes. Using fluorescence‐based proliferation assays we also show that intracellular staphylococci proliferate and that replication commences while the bacteria are residing in mature phagolysosomes hours after initial phagocytosis. Finally, live‐cell fluorescence video microscopy allowed for unprecedented visual insight into the escape of MRSA from macrophages, demonstrating that the macrophages die through a pathway characterized by membrane blebbing and activation of caspase‐3 followed by acquisition of the vital dye propidium iodide. Moreover, cell death precedes the emergence of MRSA from infected macrophages, and these events can be ablated by prolonged exposure of infected phagocytes to gentamicin.     

Das alpha‐Toxin von Staphylococcus aureus

Colonisation of the body surface of healthy subjects by Staphylococcus aureus is mostly harmless because the immune system limits bacterial growth. Under as yet unknown circumstances, however, previously commensal bacteria may become pathogenic by rapid proliferation and density‐dependent generation of virulence factors that negatively affect the surrounding eukaryotic host cells. One of the most problematic virulence factors of Staphylococcus aureus is alpha‐toxin (hemolysin A, Hla). This toxin forms transmembrane pores in the plasma membranes of eukaryotic host cells. The inner diameter of the pore allows ions and small organic molecules to pass from the extracellular space to the cytosol or vice versa. The resulting dissipation of ion gradients as well as loss of energy‐rich molecules like ATP from the cells heavily disturbs host cell functions and signal transduction processes. In epithelial cells, these changes severely affect the polarized phenotype of the epithelial cells by restructuring of the actin cytoskeleton, inducing changes in cell shape and loosening cell‐cell adhesion which ultimately compromises the barrier function of the cell sheet. These effects of alpha‐toxin may provide an explanation why it is particularly Staphylococcus aureus that is involved in the onset of many cases of lung infections (pneumonia).     

Antibacterial Effect of 2R,3R-dihydromyricetin on the Cellular Functions of Staphylococcus aureus

The C-3-OH, C-4 carbonyl oxygen and hydrogenation of C2=C3 bond on the C-ring of 2R,3R-dihydromyricetin (DMY) proved to be not necessary for the antibacterial activity against Staphylococcus aureus. DMY significantly decreased the intracellular ATP of S. aureus cells but had few effects on pH_in, proline oxidation, succinate dehydrogenase activity or malate dehydrogenase activity.     

Identification of pathogenic bacteria in blood cultures: Comparison between conventional and PCR methods

Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumanii, and Klebsiella pneumoniae were found to be the most prevalent bacteremia-causing bacteria in a survey in a medical center. A PCR method for identification of these five most common pathogens in blood cultures was developed. A unique sequence was chosen for each pathogen and used for primer design. Sixty-one blood samples (from hospitalized patients) in which bacterial growth was detected were processed in parallel by conventional microbiological methods and by the PCR method. The results obtained by PCR were identical to those obtained by conventional methods in 93.4% of the cases. PCR failed to identify bacteria which were found conventionally in only 6.6% of the cases (mostly bacteria not included in the PCR cassette). Another group of eighty-eight blood samples from patients were processed immediately upon their arrival at the laboratory by taking aliquots for the PCR method. The blood sample bottles were processed in parallel by conventional methods. In 78.4% of the cases the results of both methods were identical. In 12.5% of the cases, PCR afforded identification of bacteria but conventional methods showed no bacteria in the sample. On the other hand, PCR afforded 9.1% negative results while conventional methods identified bacteria not included in the PCR cassette. It is concluded that the molecular method appears to be a specific and precise method for identifying pathogenic bacteria in blood samples.