Murine B lymphoma cell lines release functionally active interleukin 2 after stimulation with Staphylococcus aureus

In this study it is shown that an IL-2-like functional lymphokine activity derived from the murine B cell lines 2PK-3 and L10A2J after stimulation with Staphylococcus aureus can be blocked with anti-IL-2 mAb such as 9B11, DMS-1, and S4B6. Experiments demonstrate that the stimulation of the IL-2-sensitive cell line CTLL-2 by the IL-2-like functional activity derived from murine B cell tumors can also be blocked with the anti-IL-2R mAb PC61. Additionally, in RNA-RNA hybridization experiments with radiolabeled SP6-derived ssRNA probes developed from murine IL-2 genomic DNA sequences and specific for IL-2 mRNA, quantitatively significant amounts of IL-2-specific mRNA in both 2PK-3 and L10A2J are shown subsequent to stimulating the cells with S. aureus. These results suggest the murine B cell tumor lines 2PK-3 and L10A2J synthesize and release IL-2 after stimulation with selected polyclonal activators such as S. aureus.     

Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A

Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.     

Measurement of fibroblast and bacterial detachment from biomaterials using jet impingement

Fibroblast and Staphylococcus aureus detachment strength from orthopaedic alloys and a tissue culture plastic (Thermanox) have been investigated with jet impingement. For S. aureus, unlike fibroblasts, detachment is caused more by pressure than shear. For these biomaterials, detachment strength is much higher for S. aureus than fibroblasts. Comparing materials under equivalent flow conditions, S. aureus attach to stainless steel and titanium with equal strength and more strongly than to Thermanox. For fibroblasts, detachment strength from all materials was similar. Fibroblast detachment strength from these biomaterials substantially decreases with time at equal flow rates and increases with flow rate at equal exposure times. Detachment strength is very similar for 3T3 and L929 fibroblasts on Thermanox for equivalent flow rate/time combinations, though enhanced adhesion of 3T3 cells was often noted for metals. Time effects are less evident for S. aureus. S. aureus adhesion to metals is more affected by flow rate than fibroblast adhesion.     

A comparative study of the IgG-binding components of the membrane in different strains of Staphylococcus aureus using the indirect hemagglutination reaction and an immunofluorescence method

A comparative estimation of IgG-binding activity of 85 S. aureus clinical strains was carried out by the method of indirect hemagglutination reaction. The S. aureus strain selected as a result of screening was found to exceed by more than an order the Cowan I strain obtained from the L. A. Tarasevich State Institute of Standards and Control of the Medical Biological Drugs in the IgG-binding activity. It was established that the ratio of two types of IgG-binding sites located on the S. aureus surface, varied depending on the strain, composition and quality (liquid or solid) of the culture medium.     

Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB,the serine-aspartate repeat protein SdrE and protein A

The ability of Staphylococcus aureus cells to induce platelet aggregation has long been recognized. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial receptors required remains poorly understood. Using genetic manipulation, this study for the first time provides clear evidence that several S. aureus surface proteins participate in the inter-action with platelets. Mutants of S. aureus strain Newman lacking one or more surface proteins were tested for their ability to stimulate platelet aggregation. This approach was complemented by the expression of a number of candidate proteins in the non-aggregating Gram-positive bacterium Lacto-coccus lactis. S. aureus-induced aggregation was monophasic and was dependent on the platelet receptor GPIIb/IIIa. The fibrinogen-binding proteins, clumping factors A and B and the serine-aspartate repeat protein SdrE could each induce aggregation when expressed in L. lactis. Although protein A expressed in L. lactis was not capable of inducing aggregation independently, it enhanced the aggregation response when expressed on the surface of S. aureus. Thus, S. aureus has multiple mechanisms for stimulating platelet aggregation. Such functional redundancy suggests that this phenomenon may be important in the pathogenesis of invasive diseases such as infective endocarditis.     

Effect of Lactic Acid Bacteria on Growth of Staphylococcus aureus

Cultures of lactic acid bacteria, mostly from foods, were tested for their effect on the growth of Staphylococcus aureus in Trypticase Soy Broth (BBL). Some of the effectors, e.g., Streptococcus faecalis, S. faecium, Lactobacillus lactis, L. brevis, and Leuconostoc mesenteroides, stimulated growth of S. aureus during early hours of growth, especially at higher temperatures of incubation, but most cultures were inhibitory, and some (S. faecium and L. mesenteroides) were even killing by the time of attainment of the maximal phase of growth of the Staphylococcus. Low-temperature meat lactobacilli and Leuconostoc dextranicum inhibited S. aureus at 10, 15, 20, and 25 C throughout its growth. Streptococcus faecalis var. liquefaciens inhibited at these temperatures and at 30 and 37 C, as well. When the ratio of effectors to staphylococci in the inoculum was 100:1, the three enterococci, the meat Lactobacillus, and L. dextranicum prevented the attainment of 5 x 10(6) staphylococci per milliliter at 15 C, and all but the meat Lactobacillus did so at 22 C. A ratio of 1:1 accomplished similar results at 15 C, except that S. aureus was only delayed for 12 hr by S. faecalis. A ratio of 1:100 usually was ineffective. In general, the more effector bacteria there were in the inoculum, the greater was the overall inhibition (or stimulation) of S. aureus. Inhibition was most effective at 10 or 15 C, less so at 20 or 25 C, and least at 30 or 37 C, whereas stimulation during early growth was greater at the higher temperatures. Results with different strains of the effectors and with two strains of S. aureus were similar, for the most part.     

Immunoglobulin M synthesized by human lymphoblastoid cells: interaction with Staphylococcus aureus and protein A.

Immunoglobulin M synthesized by a human lymphoblastoid cell line, LA173, was found to bind specifically to the protein A-bearing Cowan I strain of Staphylococcus aureus. The (3H)-leucine-labeled, secreted IgM from these LA173 cells also formed precipitin complexes with purified protein A. Soluble complexes formed at high protein A/IgM ratios retained the ability to bind to the bacterial surface. Precipitin complexes also were observed in double diffusion Ouchterlony gels with a line of identity formed between the IgM, protein A, and anti-IgM in adjacent wells. Intracellular IgM species from detergent-lysed LA173 cells were bound to S. aureus. Labeled 19S pentamers, 8S monomers, and HL subunits were eluted from the bacteria and identified by velocity sedimentation and SDS agarose-acrylamide gel electrophoresis. In addition, several intermediates migrating between 8S and 19S were detected and shown to contain authentic H and L chains. Binding of the labeled IgM 19S pentamers to staphylococci was not inhibited by prior treatment of the bacteria with an excess of unlabeled human IgG. However, S. aureus saturated with unlabeled IgG did not bind either labeled IgM monomers or labeled IgG. The interaction of this human IgM with S. aureus exhibited a high degree of specificity with quantitative recovery of secreted 19S IgM. Intracellular IgM species were bound selectively by the bacteria with little if any contamination by other cytoplasmic proteins.     

嗜酸乳杆菌S-层蛋白联合抗生素对Escherichia coli和Staphylococcus aureus的抑制作用研究

旨在检测嗜酸乳杆菌S-层蛋白以及S-层蛋白与抗生素联用对大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)的抑制作用。采用液体发酵培养法获得嗜酸乳杆菌菌体,LiCl法提取S-层蛋白粗提物,凝胶过滤层析法纯化S-层蛋白,分别用E.coli和S.aureus处理Caco-2细胞2 h后,考察S-层蛋白在不同浓度和不同作用时间条件下对E.coli和S.aureus的抑制作用,并考察S-层蛋白联合抗生素对E.coli和S.aureus的抑制作用,实验分组:(1)空白对照;(2)嗜酸乳杆菌组;(3)S-层蛋白组;(4)抗生素组;(5)嗜酸乳杆菌+抗生素组;(6)S-层蛋白+抗生素组。结果显示,液体发酵得到嗜酸乳杆菌菌体,提取并纯化得到S-层蛋白;S-层蛋白对E.coli和S.aureus有很好的抑制效果,具有浓度依赖性,高浓度下抑制率达到42.2%和31.7%,差异极显著(P<0.01),且在E.coli和S.aureus作用的短时间内干预效果明显,0 h时的抑制率分别达到59.3%和48.4%;S-层蛋白联合抗生素的抑菌率分别达到81.7%和79.3%,差异极显著(P<0.01),效果优于单独使用抗生素。嗜酸乳杆菌S-层蛋白具有较强的抑菌作用,可以与抗生素联用,有望开发称为一种新型的抗菌药物。     

Antimicrobial activity of ZnII, CdII, and HgII complexes of 1,2-bis-[2-(5-R)-1H-benzimidazolyl]-1,2-ethanediols and 1,4-bis-[2-(5-R)-1H-benzimidazolyl]-1,2,3,4-butanetetraols

1,2-Bis-[2-(5-H/Me/Cl/NO2)-1H-benzimidazolyl]-1,2-ethanediols (L1-L4), 1,4-bis-[2-(5-H/Me/Cl)-1H-benzimidazolyl]-1,2,3,4-butanetetraols (L5-L7) and their complexes with ZnCl2, CdCl2 and HgCl2 were synthesized and antibacterial activity of the compounds was tested toward Staphylococcus aureus, S. epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Proteus mirabilis and antifungal activity against Candida albicans. HgII complexes have a considerably higher antimicrobial activity against all microorganisms. Some HgII complexes show higher antifungal activity than clotrimazole toward C. albicans. Zn2(L3)Cl4, Zn2(L4)Cl4, and Cd(L3)Cl2 were moderately effective against S. aureus and S. epidermidis; Cd(L4)Cl2 exhibited a weak activity only against S. epidermidis.     

Enhancement of bactericidal activity of mouse peritoneal macrophages against Staphylococcus aureus by mouse interferon preparations

The effect of mouse interferon on the bactericidal activity of macrophages against pyogenic cocci was examined. Mouse peritoneal macrophages were cultivated with Staphylococcus aureus in vitro and viable Staphylococcus was recovered by treatment of the mixed macrophage-bacteria culture with sodium dodecyl sulphate (SDS) solution. Results showed that S. aureus was phagocytized and killed by the macrophages. Mouse L cell interferon enhanced the bactericidal activity of macrophages. A mouse brain interferon preparation also enhanced this activity. However, heat-inactivated L cell interferon and heterologous rabbit RK-13 cell interferon and human leukocyte interferon did not enhance it. This suggests that interferon enhances the bactericidal activity of macrophages against S. aureus.     

Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion

Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection.     

Minimum inhibitory concentration of smoke wood extracts against spoilage and pathogenic micro-organisms associated with foods

Antimicrobial activity of seven commercial smoke preparations (four liquid and three solid) was studied. The minimum inhibitory concentration (MIC) was determined against a selection of food spoilage and pathogenic micro-organisms. The main smoke components were identified and quantified by gas chromatography/mass spectrometry. The most effective condensate was S2. All strains except Salmonella enteritidis were inhibited by S2 with an MIC <0·5–1·5%. Smoke extract L2 inhibited growth of Vibrio vulnificus, Yersinia enterocolitica, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, L. inocua, Brochothrix thermosphacta and Lactococcus lactis ssp. lactis with an MIC of <0·2–0·8%. The condensate L3 inhibited effectively V. vulnificus, B. subtilis, L. innocua and Staph. aureus. L1, L4, S1 and S3 had no inhibitory effects at levels tested against most micro-organisms. Vibrio vulnificus was the most susceptible micro-organism to test compounds. The antimicrobial activity of smoke preparations was related to the concentration of phenols.     

Conspicuous ingestion of Staphylococcus aureus organisms by murine fibroblasts in vitro.

A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described. In the present experimental system, only fibroblasts-adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophyticus. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non-professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.     

Studies on antimicrobial activity, in vitro, of Physalis angulata L. (Solanaceae) fraction and physalin B bringing out the importance of assay determination

Complex physalin metabolites present in the capsules of the fruit of Physalis angulata L. have been isolated and submitted to a series of assays of antimicrobial activity against Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, Neisseria gonorrhoeae ATCC 49226, Escherichia coli ATCC 8739; E. coli ATCC 25922, Candida albicans ATCC 10231 applying different methodologies such as: bioautography, dilution broth, dilution agar, and agar diffusion techniques. A mixture of physalins (pool) containing physalins B, D, F, G inhibit S. aureus ATCC 29213, S. aureus ATCC 25923, S. aureus ATCC 6538P, and N. gonorrhoeae ATCC 49226 at a concentration of 200 mg/microl, using agar dilution assays. The mixture was inactive against P. aeruginosa ATCC27853, E. coli ATCC 8739; E. coli ATCC 25922, C. albicans ATCC 10231 when applying bioautography assays. Physalin B (200 microg/ml) by the agar diffusion assay inhibited S. aureus ATCC 6538P by +/- 85%; and may be considered responsible for the antimicrobial activity.     

细菌L型对化学因素敏感性研究

本文研究了伤寒沙门氏菌、葡萄球菌及白喉棒状杆菌L型对化学消毒剂、细菌生长抑制剂、酸和碱的敏感性,并与其亲代细菌型作了比较。结果表明:细菌L型对表面活性剂、酚剂、重金属盐、脱水剂、卤素及氧化剂的敏感性明显(P<0.05)或不明显(P>0.05)增加,伤寒沙门氏菌L型对龙胆紫、玫瑰色酸、胆盐及酸的敏感性增高,葡萄球菌及白喉棒状杆菌L型对龙胆紫、玫瑰色酸、胆盐及酸的敏感性降低,伤寒沙门氏菌及白喉棒状杆菌L型对碱敏感性降低。     

Anti-infective properties of Lactobacillus fermentum against Staphylococcus aureus and Pseudomonas aeruginosa

Surgical wounds and implant-associated Staphylococcus aureus and Pseudomonas aeruginosa infections are often difficult to treat because of limited susceptibility of several of these strains to conventional antibiotics. As a result, there is a constant need for new alternative drugs. The aim of this study was to investigate the antimicrobial properties of Lactobacillus fermentum, a probiotic bacterium, which we have isolated from colonic biopsies. The inhibition of S. aureus and P. aeruginosa growth was evaluated by coincubating with L. fermentum strains. Growth inhibition was tested for several of their clinical isolates using agar well diffusion assays. For biofilm assay S. aureus and P. aeruginosa were grown on the glass slides and in 96-well plates in presence of 2.5 μg/ml culture filtrate of L. fermentum. Biofilms were photographed using confocal microscope or stained with 0.1% crystal violet. Reduction in the cytotoxicity of S. aureus and P. aeruginosa was observed in presence of 2.5 μg/ml L. fermentum-spent media. Using in vitroexperiments, we showed that L. fermentum-secreted compound(s) inhibits the growth, cytotoxicity and biofilm formation of several S. aureus and P. aeruginosa strains. Compound(s) present in the culture supernatant of L. fermentum may have promising applications in treating hospital-acquired infections.     

The role of human innate immune factors in nasal colonization by Staphylococcus aureus

Staphylococcus aureus colonization of the human nares predisposes to sometimes severe auto-infection. To investigate whether genetic polymorphism affects the S. aureus carriage status, sequence variation in alpha-defensin and beta-defensin, and mannose-binding lectin (MBL) genes were determined for a group of volunteers (n=109) with known S. aureus nasal carriage status. DEFA1/3 expression was measured in a subset of the volunteers (n=32). None of the single nucleotide polymorphisms studied could clearly distinguish the (non) carriage groups. S. aureus carriers differed from non-carriers in baseline level of HNP1-3 peptide production (median: 218 versus 89mug/ml, P=0.016). No association between HNP1-3 levels and the individual sequence polymorphisms was documented. The combined copy numbers of DEFA1/A3 genes ranged from 5 to 23 per diploid genome. A linear correlation between combined copy numbers and HNP1-3 peptide concentrations in nasal secretions of non-carriers was noted (r(2)=0.8991). DEFA3 gene was absent in 25% of the individuals. MBL haplotype A was overrepresented in persistent S. aureus carriers (87% vs. 67%; P=0.038). In conclusion, defensin gene polymorphism, both in sequence and in gene copy numbers, does not seem to be involved in S. aureus carriage predisposition. However, MBL haplotypes do so significantly. Baseline HNP1-3 production is more the consequence of S. aureus colonization than a reason for the (non) carrier status.     

Functional activity of mouse peritoneal macrophages contaminated with gram-positive bacteria

The analysis of the functional and enzymatic activity of mouse peritoneal macrophages contaminated with Staphylococcus aureus and Listeria monocytogenes virulent strains is presented. The low bactericidal and digestive activity of these cells with respect to the above-mentioned microorganisms was determined. In this study a decrease in the activity of plasmatic membrane enzymes (5'-nucleotidase and ATP-ase) of macrophages contaminated with S. aureus and L. monocytogenes was observed, which was indicative of the stimulation of phagocytes. A rise in the activity of the oxygen-dependent system of macrophages contaminated with S. aureus and L. monocytogenes was detected by means of the nitro blue tetrazolium test. At the same time a decrease in the intracellular content of nitrogen oxide end metabolites in macrophages was detected with a rise in content of nitrogen oxide in the supernatants.     

Inhibition of Staphylococcus aureus by lysostaphin-expressing Lactobacillus plantarum WCFS1 in a modified genital tract secretion medium

Lactobacillus species are a predominant member of the vaginal microflora and are critical in maintaining an acidic vaginal environment thought to contribute to the prevention of a number of urogenital diseases. However, during menstruation the pH of the vaginal environment increases to neutrality, a pH conducive for Staphylococcus aureus proliferation and the production of toxic shock syndrome toxin 1 (TSST-1) in susceptible women. In order to generate Lactobacillus species capable of expressing lysostaphin (an endopeptidase that cleaves the cell wall of S. aureus) in a modified genital tract secretion medium (mGTS) under neutral-pH conditions, six prominent proteins from Lactobacillus plantarum WCFS1 spent medium were identified by mass spectrometry. Sequences for promoters, signal peptides, and mature lysostaphin were used to construct plasmids that were subsequently transformed into L. plantarum WCFS1. The promoter and signal sequences of Lp_3014 (putatively identified as a transglycosylase) or the promoter sequence of Lp_0789 (putatively identified as glyceraldehyde 3-phosphate dehydrogenase) with the signal sequence of Lp_3014 exhibited lysostaphin activity on buffered medium containing heat-killed S. aureus. The cassettes were integrated into the chromosome of L. plantarum WCFS1, but only the cassette containing the promoter and signal sequence from Lp_3014 had integrated into the appropriate site. Coculture assays using buffered mGTS showed that lysostaphin expressed from L. plantarum WCFS1 reduced the growth of TSST-1-producing strains of S. aureus under neutral-pH conditions. This study provides the basis for determining whether lysostaphin-producing Lactobacillus strains could potentially be used as a means to inhibit the growth of S. aureus during menstruation.     

Interactions of some common pathogenic bacteria with Acanthamoeba polyphaga

Protozoan grazing is a major trophic pathway whereby the biomass re-enters the food web. Nonetheless, not all bacteria are digested by protozoa and the number known to evade digestion, resulting in their environmental augmentation, is increasing. We investigated the interactions of Bacillus cereus, Enterococcus faecalis, Enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and methicillin-sensitive Staphylococcus aureus (MSSA), with the amoeba, Acanthamoeba polyphaga. There was evidence of predation of all bacterial species except L. monocytogenes and S. aureus, where extracellular numbers were significantly higher when cultured with amoebae compared with growth in the absence of amoebae. Intracellular growth kinetic experiments and fluorescent confocal microscopy suggest that S. aureus survived and may even multiply within A. polyphaga, whereas there was no apparent intra-amoebal replication of L. monocytogenes and higher numbers were likely sustained on metabolic waste products released during coculture.