Achacin induces cell death in HeLa cells through two different mechanisms

Achacin, which belongs to the L-amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H(2)O(2). In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H(2)O(2) scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z-VAD-fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H(2)O(2) through the L-amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L-amino acids by achacin.     

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.     

Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.     

The lactoperoxidase system links anion transport to host defense in cystic fibrosis

Chronic respiratory infections in cystic fibrosis result from CFTR channel mutations but how these impair antibacterial defense is less clear. Airway host defense depends on lactoperoxidase (LPO) that requires thiocyanate (SCN-) to function and epithelia use CFTR to concentrate SCN- at the apical surface. To test whether CFTR mutations result in impaired LPO-mediated host defense, CF epithelial SCN- transport was measured. CF epithelia had significantly lower transport rates and did not accumulate SCN- in the apical compartment. The lower CF [SCN-] did not support LPO antibacterial activity. Modeling of airway LPO activity suggested that reduced transport impairs LPO-mediated defense and cannot be compensated by LPO or H2O2 upregulation.     

Antibacterial activity of highly negative charged polyoxotungstates, K27[KAs4W40O140] and K18[KSb9W21O86], and Keggin-structural polyoxotungstates against Helicobacter pylori

The antibacterial activity of polyoxometalates (PMs) against Helicobacter pylori was investigated based on determinations of minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC), time-killing of the bacteria, bacterial morphology and PM-uptake into the bacteria cell. The result of MIC values revealed that, of 13 PMs used in this study, highly negative-charged polyoxotungstates, such as K27[KAs4W40O140] and K18[KSb9W21O86], and Keggin-structural polyoxotungstates exhibited a potent antibacterial activity with the MIC values of less than 256 microg/ml. The former was the most active, and superior to metronidazole (MTZ) against MTZ-susceptible and resistant strains and also to clarithromycin (CLR) against CLR-resistant strains. In contrast, most of polyoxomolybdates showed little antibacterial activity with the MIC values of more than 256 microg/ml. The result of FIC index values indicated that the antibacterial polyoxotungstates had partially synergistic effect in combination with MTZ and CLR but indifferent effect in combination with amoxicillin (AMX). From the results of the time-killing and scanning electron microscope images, K27[KAs4W40O140] and K18[KSb9W21O86] proved the concentration-dependent bactericidal activity with the morphological change from bacillary form to coccoid form, while Keggin-structural K5[SiV(V)W11O40] showed the bacteriostatic activity with small change of morphology to coccoid form. The fluorescent X-ray analysis demonstrated that these polyoxotungstates were taken into the bacteria cell. It is pointed out that the Keggin-structure and/or high negativity polyoxotungstates are an important factor for the antibacterial activity against H. pylori.     

Effect of hydrogen peroxide on antibacterial activities of Canadian honeys

Honey is recognized as an efficacious topical antimicrobial agent in the treatment of burns and wounds. The antimicrobial activity in some honeys depends on the endogenous hydrogen peroxide content. This study was aimed to determine whether honey's hydrogen peroxide level could serve as a honey-specific, activity-associated biomarker that would allow predicting and assessing the therapeutic effects of honey. Using a broth microdilution assay, I analyzed antibacterial activities of 42 Canadian honeys against two bacterial strains: Escherichia coli (ATCC 14948) and Bacillus subtilis (ATCC 6633). The MIC90 and MIC50 were established from the dose-response relationship between antibacterial activities and honey concentrations. The impact of H2O2 on antibacterial activity was determined (i) by measuring the levels of H2O2 before and after its removal by catalase and (ii) by correlating the results with levels of antibacterial activities. Canadian honeys demonstrated moderate to high antibacterial activity against both bacterial species. Both MIC90 and MIC50 revealed that the honeys exhibited a selective growth inhibitory activity against E. coli, and this activity was strongly influenced by endogenous H2O2 concentrations. Bacillus subtilis activity was marginally significantly correlated with H2O2 content. The removal of H2O2 by catalase reduced the honeys' antibacterial activity, but the enzyme was unable to completely decompose endogenous H2O2. The 25%-30% H2O2 "leftover" was significantly correlated with the honeys' residual antibacterial activity against E. coli. These data indicate that all Canadian honeys exhibited antibacterial activity, with higher selectivity against E. coli than B. subtilis, and that these antibacterial activities were correlated with hydrogen peroxide production in honeys. Hydrogen peroxide levels in honey, therefore, is a strong predictor of the honey's antibacterial activity.     

Bradyrhizobium japonicum mutants defective in cyclic beta-glucan synthesis show enhanced sensitivity to plant defense responses

Susceptibility of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum to inducible plant defense metabolites such as phytoalexin and H2O2, was investigated. On the wild-type strain USDA 110 the soybean phytoalexin, glyceollin, showed bacteriostatic activity. Viable bacteria isolated from intact nodules were adapted to glyceollin. H2O2 in physiological concentrations did not affect wild-type bacteria. B. japonicum mutants defective in the biosynthesis of cyclic beta-(1-->3)-(1-->6)-glucans showed higher susceptibility to both phytoalexin and H2O2.     

Direct and indirect bacterial killing functions of neutrophil defensins in lung explants

Studies of the antimicrobial activity of neutrophil defensins have mostly been carried out in microbiological media, and their effects on the host defense in physiological conditions are unclear. We examined 1) the antibacterial activity of defensins in physiological media with and without lung tissue present, 2) the effect of defensins on hydrogen peroxide (H(2)O(2)) production by lung tissue that had been exposed to bacteria, and 3) the effect of diphenyleneiodonium (DPI), an inhibitor of reactive oxygen species formation, on the antibacterial activity of defensins in the presence of lung tissue. Defensins were incubated with Escherichia coli or Pseudomonas aeruginosa in the absence or presence of primary cultured mouse lung explants. Defensins reduced bacterial counts by approximately 65-fold and approximately 25-fold, respectively, at 48 h; bacterial counts were further decreased by approximately 600-fold and approximately 12,000-fold, respectively, in the presence of lung tissue. Defensins induced H(2)O(2) production by lung tissue, and the rate of killing of E. coli by defensins was reduced by approximately 2,500-fold in the presence of 10 microM DPI. We conclude that defensins exert a significant antimicrobial effect under physiological conditions and that this effect is enhanced in the presence of lung tissue by a mechanism that involves the production of reactive oxygen species.     

Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323

Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.     

Superoxide dismutase-rich bacteria. Paradoxical increase in oxidant toxicity

Superoxide dismutase is considered important in protection of aerobes against oxidant damage, and increased tolerance to oxidant stress is associated with induction of this enzyme. However, the importance of superoxide dismutase in this tolerance is not clear because conditions which promote the synthesis of superoxide dismutase likewise affect other antioxidant enzymes and substances. To clarify the role of superoxide dismutase per se in organismal defense against oxidant-generating drugs, we employed Escherichia coli transformed with multiple copies of the gene for bacterial iron superoxide dismutase. These bacteria have greater than ten times the superoxide dismutase activity of wild-type E. coli but, importantly, are normal in other oxidant defense parameters including catalase, peroxidases, glutathione, and glutathione reductase. High superoxide dismutase and control bacteria were exposed to the O2- -generating drug paraquat and to elevated pO2. We find; high superoxide dismutase E. coli are more readily killed by paraquat under aerobic, but not anaerobic, conditions. During exposure to paraquat, high superoxide dismutase E. coli accumulate more H2O2. Coincidentally, the reduced glutathione content of high superoxide dismutase E. coli declines more than in control E. coli. E. coli with high superoxide dismutase activity are also more readily killed by hyperoxia. Interestingly, the susceptibility of the parental and high superoxide dismutase E. coli to killing by exogenous H2O2 is not significantly different. Thus, under these experimental conditions, greatly enhanced superoxide dismutase activity accelerates H2O2 formation. The increased H2O2 probably accounts for the exaggerated sensitivity of high superoxide dismutase bacteria to oxidant-generating drugs. These results support the concept that the product of superoxide dismutase, H2O2, is at least as hazardous as the substrate, O2-. We conclude that effective organismal defense against reactive oxygen species may require balanced increments in antioxidant enzymes and cannot necessarily be improved by increases in the activity of single enzymes.     

The effects of hypoxia and pH on phenoloxidase activity in the Atlantic blue crab, Callinectes sapidus

In its natural coastal and estuarine environments, the blue crab, Callinectes sapidus, often encounters hypoxia, accompanied by hypercapnia (increased CO2) and an associated decrease in water pH. Previous studies have shown that exposure to hypercapnic hypoxia (HH) impairs the crab's ability to remove culturable bacteria from its hemolymph. In the present study we demonstrate that the activity of phenoloxidase (PO), an enzyme critical to antibacterial immune defense in crustaceans, is decreased at the low levels of hemolymph O2 and pH that occur in the tissues of blue crabs exposed to HH. Hemocyte PO activity was measured at tissue O2 levels that occur in normoxic (5% and 15% O2, approximate venous and arterial hemolymph, respectively) and hypoxic (1% O2) crabs and compared to PO activity in air-saturated conditions (21% O2). PO activity decreased by 33%, 49% and 70% of activity in air at 15%, 5% and 1% O2, respectively. When O2 was held at 21% and pH lowered within physiological limits, PO activity decreased with pH, showing a 16% reduction at pH 7.0 as compared with a normoxic pH of 7.8. These results suggest that decreased PO activity at low tissue O2 and pH compromises the ability of crustaceans in HH to defend themselves against microbial pathogens.     

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking. The full-length DNA of the scallop H2A was 696bp long, including a 5'-terminal untranslated region (UTR) of 90bp, a 3'-terminal UTR of 228bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated.     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.     

Pore-forming properties and antibacterial activity of proteins extracted from epidermal mucus of fish.

Among several biological functions, the epidermal mucus of fish may play an important role in host defense, particularly in the prevention of colonization by parasites, bacteria and fungi. In previous work, two hydrophobic proteins of 27 and 31 kDa were isolated from carp mucus. This study identified a strong antibacterial activity (0.16-0.18 microM) well correlated with pore-forming properties. Here this work was extended to other fish species, four fresh water fish and one sea water fish. After a first step of purification, water-soluble and hydrophobic material were separated, and both fractions were analyzed by SDS-PAGE and capillary electrophoresis. Only the hydrophobic component induced pore-forming activity, when reconstituted in planar lipid bilayers. This pore-forming activity was well correlated to a strong antibacterial activity against several bacteria strains. These results suggest that fish secrete antibacterial proteins able to permeabilize the membrane of the target cell and thus act as a defense barrier.     

Solution structure of a cathelicidin-derived antimicrobial peptide, CRAMP as determined by NMR spectroscopy.

CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram-positive and gram-negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H2O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly alpha-helical conformation in TFE/H2O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H2O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic alpha-helices from Leu4 to Lys10 and from Gly16 to Leu33. These two helices are connected by a flexible region from Gly11 to Gly16. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18-residue fragment with the sequence from Gly16 to Leu33 was found to retain antibacterial activity. Therefore, the amphipathic alpha-helical region from Gly16 to Leu33 of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Activity of dermaseptin K4-S4 against foodborne pathogens

Dermaseptin S4 and its substituted derivative K(4)-S4 were investigated against various food-related pathogenic bacteria in culture media. K(4)-S4, but not the native peptide displayed significant growth inhibitory activity against all bacteria tested. Next, activity of K(4)-S4 against Escherichia coli O157:H7 was defined in terms of milieu dependencies. Salt-dependent kinetic studies in growth medium indicated that the peptide's antibacterial activity is maintained at fairly high (up to 600mM) NaCl concentrations but inhibited at higher concentrations. Similarly, antibacterial activity was reduced at high but not low pH conditions. Importantly, antibacterial activity was significantly maintained at temperatures lower than 37 degrees C and significantly enhanced at 42 degrees C. With respect to bactericidal kinetics, negative cultures were obtained in LB as well as in commercial apple juice, respectively, within 1 and 2h treatment, at twice the minimal inhibitory concentration (MIC) value. Overall, the data collected is indicative of a certain interest for dermaseptin derivatives as potential food preservatives.     

Myeloperoxidase-Halide-Hydrogen Peroxide Antibacterial System

An antibacterial effect of myeloperoxidase, a halide, such as iodide, bromide, or chloride ion, and H(2)O(2) on Escherichia coli or Lactobacillus acidophilus is described. When L. acidophilus was employed, the addition of H(2)O(2) was not required; however, the protective effect of catalase suggested that, in this instance, H(2)O(2) was generated by the organisms. The antibacterial effect was largely prevented by preheating the myeloperoxidase at 80 C or greater for 10 min or by the addition of a number of inhibitors; it was most active at the most acid pH employed (5.0). Lactoperoxidase was considerably less effective than was myeloperoxidase when chloride was the halide employed. Myeloperoxidase, at high concentrations, exerted an antibacterial effect on L. acidophilus in the absence of added halide, which also was temperature- and catalase-sensitive. Peroxidase was extracted from intact guinea pig leukocytes by weak acid, and the extract with peroxidase activity had antibacterial properties which were similar, in many respects, to those of the purified preparation of myeloperoxidase. Under appropriate conditions, the antibacterial effect was increased by halides and by H(2)O(2) and was decreased by catalase, as well as by cyanide, azide, Tapazole, and thiosulfate. This suggests that, under the conditions employed, the antibacterial properties of a weak acid extract of guinea pig leukocytes is due, in part, to its peroxidase content, particularly if a halide is present in the reaction mixture. A heat-stable antibacterial agent or agents also appear to be present in the extract.     

三种消毒剂对大黄鱼致病性弧菌的体外杀灭作用研究

为了解不同消毒剂对大黄鱼中致病性弧菌的抑菌效果。采用纸片扩散法比较了聚维酮碘(PVP-I)、聚六亚甲基双胍(PHMB)和过氧化氢(H2O2)对溶藻弧菌和副溶血性弧菌的抑菌效果,再以肉汤稀释法测定了三种消毒剂的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)及MBC/MIC比值,在此基础上,获得了抑菌效果相对较优的H2O2、PHMB对溶藻弧菌、副溶血性弧菌及其混菌的杀菌动力学曲线。结果表明,H2O2(3%)对两种弧菌具有最大的抑菌圈直径(19~21mm),PHMB(30mg/mL)次之(约为15mm),PVP-I(10%)的抑菌效果最弱(小于10mm)。3种消毒剂的MIC值由小到大依次为PHMB相似文献   

革兰氏阳性菌苏云金芽孢杆菌和革兰氏阴性菌大肠杆菌诱导的柞蚕蛹生理指标变化

【目的】明确柞蚕Antheraea pernyi对外源微生物防御性生理变化规律,为柞蚕的病害防治和合理饲养提供理论依据。【方法】本研究选用革兰氏阳性菌苏云金芽孢杆菌Bacillus thuringeinsis(Bt)和革兰氏阴性菌大肠杆菌Escherichia coli(Ec)为外源诱导微生物,调整至10~6～10~8cfu/m L菌液,灭活后处理柞蚕蛹,诱导24,48,72和96 h后不同时间测定血淋巴蛋白含量、PO活性、CAT活性、抗菌活性和溶菌酶活性等生理指标。【结果】Ec和Bt诱导柞蚕蛹导致各生理指标出现显著变化,但两种菌株诱导生理指标变化规律差异明显,Ec高浓度诱导72 h会增加血淋巴蛋白含量,而Bt各浓度诱导会在24,48和96 h增加血淋巴蛋白含量。免疫防御关键酶系PO和CAT活性变化规律在不同菌株诱导后差异更明显,Ec诱导后,PO活性随着时间增加表现为先升高后降低的趋势,CAT活性呈现"升高-降低-升高"的规律;而Bt诱导后PO活性表现为"升高-降低-升高"的规律,CAT活性随诱导时间增加变化规律不明显,但有随菌液浓度增加而降低的趋势。对抗菌活性测定表明,Ec和Bt诱导都会显著增加蛹粗酶液抗菌活性,溶菌酶活性也会极显著增加,但2个指标高峰值出现的时间会有明显差别。【结论】本研究结果表明Ec和Bt不同处理均可诱导柞蚕蛹产生明显防御反应,但柞蚕蛹生理指标变化规律与不同种类微生物及处理时间和浓度有关,推测革兰氏阳性菌和革兰氏阴性菌具有不同的诱导防御反应机制。研究结果可以为外源微生物侵染柞蚕后的免疫防御反应规律提供理论指导。     

Local bacteriophage isolates showed anti- Escherichia coli O157:H7 potency in an experimental ligated rabbit ileal loop model

Escherichia coli O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiotics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reaction for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacterial activity (P?< 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth (r = 0.992). Combined application of the phages was found most satisfactory, reducing seven?log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E.?coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages.