Transcriptional upregulation of multiple earthworm chitinase genes following bacterial challenge suggests their implications in innate immunity.

BACKGROUND: Chitinase is a multi-functional enzyme that catalyzes the hydrolysis of ß-1,4-linkages between N-acetylglucosamines (GlcNAc) in chitin. Recent studies imply that earthworm chitinase is implicated in self-defense immunity against chitin-containing pathogens. However, a direct relationship of earthworm chitinase with innate immunity has not yet been established. OBJECTIVE: In this study, earthworm (Eisenia andrei) chitinase expression was examined following bacterial challenge by Bacillus subtilis. METHODS: RNA sequencing (RNA-seq) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to quantitatively evaluate mRNA expression changes in response to bacterial stimulation. RESULTS: Multiple chitinase-related mRNAs were found to be upregulated, among which EaChi3, EaChi4, and EaChi2 were upregulated by approximately eightfold, eightfold, and 2.5-fold, respectively. This strongly suggested that earthworm chitinases may act as inducible humoral effectors in earthworm innate immunity. The primary structures of all three chitinases contained an N-terminal glycol_18 domain with two chitin-binding and chitin-catalyzing domains, and a C-terminal proline, glycine, serine, threonine (PGST)-rich domain. In addition, EaChi2 had a chitin-binding peritrophin-A domain at the end of the C-terminus with 5 cysteine residues possibly contributing two intradomain disulfide bonds. Multiple sequence alignment of the catalytic domain centers of glycol_18 domain displayed highly conserved chitin-binding and chitin-catalyzing domains in which three essential amino acid residues (D, D, E) for catalyzing activity are well conserved except EaChi4. The critical glutamic acid (E) residue was substituted for glutamine (Q) in EaChi4 indicating that it is devoid of catalytic activity. CONCLUSIONS: To our knowledge, this is the first report providing direct evidence that multiple earthworm chitinases are bacteria-responsive, strongly suggesting that earthworm chitinases are inducible humoral effectors in earthworm innate immunity. In addition, our results possibly suggest that earthworm EaChi4 may function as a pattern recognition molecule modulating the downstream immune pathway.

Comparative evaluation of the antioxidant, antimicrobial and nutritive properties of gluten-free flours.

Gluten-free flours are interesting alternative to wheat flours. They could be by-products of oilseed processing, characterized by high content of bioactive compounds. Therefore the aim of the study was to determine the antioxidant, antimicrobial properties, amino acid and fatty acid profile of flours obtained as by-products from the oil industry. The highest total polyphenol content and antioxidant activity was found to have evening primrose flour. The widest spectrum of microbial growth inhibition was indicated for corn germ extract which showed no antimicrobial activity only against Bacillus subtilis. The highest protein content was found in pumpkin, peanut and almond flours (more than 50 g/100 g). The major abundant amino acids in all the analysed oilseed cake flours were aspartic acid, glutamic acid and arginine. The analysed gluten-free flours were found to be a good source of polyunsaturated fatty acids (PUFAs), which comprised mainly linoleic acid and α-linolenic acid, whereas the best source of PUFAs was evening primrose flour. The results suggest that the cold-pressed seed flours possess valuable chemical composition and may be considered for improvement of the nutritional properties of food products.

The use of ozone gas for the inactivation of Bacillus anthracis and Bacillus subtilis spores on building materials.

A study was conducted to assess the efficacy of ozone gas in inactivating spores of both Bacillus anthracis and Bacillus subtilis inoculated onto six building materials (glass, wood, carpet, laminate, galvanized metal, and wallboard paper). Testing conditions consisted of ozone gas concentrations ranging from 7,000-12,000 parts per million (ppm), contact times from 4 to 12 h, and two relative humidity (RH) levels of 75 and 85%. Results showed that increasing the ozone concentration, contact time, and RH generally increased decontamination efficacy. The materials in which the highest decontamination efficacy was achieved for B. anthracis spores were wallboard paper, carpet, and wood with ≥ 6 log10 reduction (LR) occurring with 9,800 ppm ozone, 85% RH, for 6 h. The laminate and galvanized metal materials were generally more difficult to decontaminate, requiring 12,000 ppm ozone, 85% RH, and 9-12 h contact time to achieve ≥6 LR of B. anthracis. Lastly, overall, there were no significant differences in decontamination efficacy between the two species.

Functionalization of Elongated Tetrahexahedral Au Nanoparticles and Their Antimicrobial Activity Assay.

Gold nanoparticles are inert for the human body, and therefore, they have been functionalized to provide them with antibacterial properties. Here, elongated tetrahexahedral (ETHH) Au nanoparticles were synthesized, characterized, and functionalized with lipoic acid (LA), a natural antioxidant with a terminal carboxylic acid and a dithiolane ring, to generate ETHH-LA Au nanoparticles. The antioxidant activity of Au nanoparticles was investigated in vitro, showing that LA enhances the 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging and Fe3+ ion reducing activity of ETHH-LA at higher amounts. The antimicrobial propensities of the nanoparticles were investigated against Gram-positive ( Bacillus subtilis) and Gram-negative ( Escherichia coli) bacteria through propidium iodide assay as well as disk diffusion assay. ETHH-LA Au nanoparticles showed significantly higher antimicrobial activity against B. subtilis compared with E. coli. Furthermore, ETHH-LA Au nanoparticles also showed significantly better antimicrobial activity against both bacterial strains when compared with ETHH. ETHH Au nanoparticles also bring about the oxidation of bacterial cell membrane fatty acids and produce lipid peroxides. ETHH-LA showed higher lipid peroxidation potential than that of ETHH against both bacteria tested. The hemolytic potential of Au nanoparticles was investigated using human red blood cells and ETHH-LA showed reduced hemolytic activity than that of ETHH. The cytotoxicity of Au nanoparticles was investigated using human cervical cancer cells, HeLa, and ETHH-LA Au nanoparticles showed reduced cytotoxicity than that of ETHH. Taken together, LA enhances the antimicrobial activity of ETHH Au nanoparticles and Au nanoparticles interact with the bacteria through electrostatic interactions as well as hydrophobic interactions and damage the bacterial cell wall followed by oxidation of cell membrane fatty acids.

Novel O-alkyl Derivatives of Naringenin and Their Oximes with Antimicrobial and Anticancer Activity.

In our investigation, we concentrated on naringenin (NG)-a widely studied flavanone that occurs in citrus fruits. As a result of a reaction with a range of alkyl iodides, 7 novel O-alkyl derivatives of naringenin (7a⁻11a, 13a, 17a) were obtained. Another chemical modification led to 9 oximes of O-alkyl naringenin derivatives (7b⁻13b, 16b⁻17b) that were never described before. The obtained compounds were evaluated for their potential antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis. The results were reported as the standard minimal inhibitory concentration (MIC) values and compared with naringenin and its known O-alkyl derivatives. Compounds 4a, 10a, 12a, 14a, 4b, 10b, 11b, and 14b were described with MIC of 25 µg/mL or lower. The strongest bacteriostatic activity was observed for 7-O-butylnaringenin (12a) against S. aureus (MIC = 6.25 µg/mL). Moreover, the antitumor effect of flavonoids was examined on human colon cancer cell line HT-29. Twenty-six compounds were characterized as possessing an antiproliferative activity stronger than that of naringenin. The replacement of the carbonyl group with an oxime moiety significantly increased the anticancer properties. The IC50 values below 5 µg/mL were demonstrated for four oxime derivatives (8b, 11b, 13b and 16b).

Study of Bioaccumulation, Hematological Parameters, and Antioxidant Responses of Carassius auratus gibelio Exposed to Dietary Lead and Bacillus subtilis.

Lead (Pb) is one of the most ubiquitous and toxic elements in the aquatic environment. Bacillus subtilis (B. subtilis) is a widely used probiotic in aquaculture. The aim of this study was to explore the toxic effects on bioaccumulation, hematological parameters, and antioxidant responses of Carassius auratus gibelio (C. gibelio) exposed to dietary lead at 0, 120, and 240 mg/kg and/or B. subtilis at 109 cfu/g. At 15 and 30 days, the fish were sampled and bioaccumulation, hematological parameters, and antioxidant responses were assessed. The result showed that B. subtilis administration can provide a significant protection against lead toxicity by reducing lead bioaccumulation in tissues, increasing the antioxidant enzymes activity, recovering δ-aminolevulinic acid dehydratase activity and optimizing the hematological parameters. Our results suggested that administration of B. subtilis (109 cfu/g) has the potential to combat dietary lead toxicity in C. gibelio.

Influence of Bacillus subtilis ANSB060 on growth, digestive enzyme and aflatoxin residue in Yellow River carp fed diets contaminated with aflatoxin B1.

Aflatoxin B1 (AFB1) elicits serious threats to public health due to its widespread occurrence, as well as its teratogenic, carcinogenic and mutagenic effects. This study aimed to evaluate the toxicity of AFB1 and assess the ameliorative efficacy of Bacillus subtilis ANSB060 on aflatoxicosis in Yellow River carp. A total of 750 juvenile Yellow River carp were allocated into five dietary treatments for 60 days. Diet C0 represented for the negative control, diet M0 containing about 50 µg AFB1/kg diet represented for the positive control, and diet M0.25, M0.5 and M1.0 was diet M0 supplemented with B. subtilis ANSB060 at a dose of 0.25 × 109, 0.5 × 109 and 1.0 × 109 CFU/kg diet, respectively. The results showed that supplementation of strain ANSB060 restored the reduced body weight and enhanced feed conversion ratio of carp induced by AFB1 towards normal. ANSB060 could also relieve the alterations in hepatic morphology, improve digestive enzyme activities of hepatopancreas and intestine, as well as decrease AFB1 residues in carp's hepatopancreas and gonad. It is concluded that ANSB060 has a protective effect in carp with aflatoxicosis, with a promising potential in feed industrial applications.

Amphiphilic Peptide-Based Supramolecular, Noncytotoxic, Stimuli-Responsive Hydrogels with Antibacterial Activity.

A series of peptides with a long fatty acyl chain covalently attached to the C-terminal part and a free amine (-NH2) group at the N-terminus have been designed so that these molecules can be assembled in aqueous medium by using various noncovalent interactions. Five different peptide amphiphiles with a general chemical formula [H2N-(CH2)nCONH-Phe-CONHC12 (n = 1-5, C12 = dodecylamine)] have been synthesized, characterized, and examined for self-assembly and hydrogelation. All of these molecules [P1 (n = 1), P2 (n = 2), P3 (n = 3), P4 (n = 4), P5 (n = 5)] form thermoresponsive hydrogels in water (pH 6.6) with a nanofibrillar network structure. Interestingly, the hydrogels obtained from compounds P4 and P5 exhibit potential antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli). Dose-dependent cell-viability studies using MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by taking human lung carcinoma (A549) cells vividly demonstrates the noncytotoxic nature of these gelator molecules in vitro. Hemolytic studies show nonsignificant or little hemolysis of human erythrocyte cells at the minimum inhibitory concentration (MIC) of these tested bacteria. Interestingly, it has been found that these antibacterial noncytotoxic hydrogels exhibit proteolytic resistance toward the enzymes proteinase K and chymotrypsin. Moreover, the gel strength and gel recovery time have been successfully modulated by varying the alkyl chain length of the N-terminally located amino acid residues. Similarly, the thermal stability of these hydrogels has been nicely tuned by altering the alkyl chain length of the N-terminally located amino acid residues. In the era of antibiotic-resistant strains of bacteria, the discovery of this new class of peptide-based antibacterial, proteolytically stable, injectable, and noncytotoxic soft materials holds future promise for the development of new antibiotics.

The choice of the anchoring protein influences the interaction of recombinant Bacillus spores with the immune system.

The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently, there are no technical possibilities to predict whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study, we compared four variants of B. subtilis spores presenting a fragment of a FliD protein from Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their own phagocytosis and activate both, the J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that the observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent of its abundance and localization in the spore coat structure.

Development of a radio frequency heating system for sterilization of vacuum-packed fish in water.

We developed equipment that quickly and uniformly heats packed whole fish in circulating tap water using radio frequency (RF) heating. Four vacuumed plastic-packed Pacific sauries in tap water were set in a radial arrangement between coaxial cylindrical electrodes in a closed vessel. For sterilization testing, Bacillus subtilis spores added in the center of the sauries were counted after treatment. For quality assurance, meat color and backbone hardness were measured after treatment. The temperature at the center of the sauries was increased up to 130 °C for 19 min using 9 kW RF heating, and up to 119 °C for 45 min using conventional heating (CH) at 120 °C. B. subtilis spores were decreased by five logarithmic orders using RF heating and by four logarithmic orders using CH. The RF-treated meat was brighter than the CH-treated meat, and the RF-treated backbone was softer than CH-treated one.

Clostridium perfringens α-Toxin Impairs Innate Immunity via Inhibition of Neutrophil Differentiation.

Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system.

Mechanical, physical, antioxidant, and antimicrobial properties of gelatin films incorporated with thymol for potential use as nano wound dressing.

UNLABELLED: The aim of this study was to determine the properties of gelatin films incorporated with thymol. Gelatin films were prepared from gelatin solutions (10% w/v) containing thymol (1, 2, 4, and 8% w/w), glycerol (25% w/w) as plasticizer, and glutaraldehyde (2% w/w) as cross-linker. Cross-likened films showed higher tensile strength, higher elongation at break, lower Young's modulus, lower water solubility, lower swelling, lower water uptake, and lower water vapor permeability. Incorporation of thymol caused a significant decrease in tensile strength, increase in elongation at break, decrease in Young's modulus, increase in water solubility, decrease in swelling and water uptake, and increase in water vapor permeability slightly. The films incorporated with thymol exhibited excellent antioxidant and antibacterial properties. The antibacterial activity of the films containing thymol was greatest against Staphylucoccus aureus followed by Bacillus subtilis followed by Escherichia coli and then by Pseudomonas aeruginosa. Thus, gelatin films-containing thymol can be used as safe and effective source of natural antioxidant and antimicrobial agents with the purpose of evaluating their potential use as modern nano wound dressing. PRACTICAL APPLICATION: This study clearly demonstrates the potential of gelatin films incorporated with thymol as natural antioxidant and antimicrobial nano film. Such antimicrobial films exhibited excellent mechanical, physical, and water activities and could be used as antibacterial nano wound dressing against wounds burn pathogens.

[Influence of bacteria of Bacillus genus on the causative agent of bacterial cancer of tomatoes].

It has been shown that bacteria of the genus Bacillus inhibited the development of infection caused by Clavibacter michiganensis subsp. michiganensis, in tomatoes. Pre-sowing seed treatment with suspensions of Bacillus subtilis IMV B-7023 and Bacillus pumilus 3 enhanced resistance of plants to bacterial disease of cancer, probably due to the synthesis of biologically active substances with antimicrobial properties by these bacteria. Of the two strains of bacillus, differing by antagonist properties to C. michiganensis subsp. michiganensis, a significant stimulating effect on the growth and development of tomatoes was provided by the strain B. subtilis IMV B-7023, which is part of the bacterial preparations for crop production.

Cytotoxicity of listeriolysin O produced by membrane-encapsulated Bacillus subtilis on leukemia cells.

Encapsulation of biological material in the permiselective membrane allows to construct a system separating cells from their products, which may find biotechnological as well as biomedical applications in biological processes regulation. Application of a permiselective membrane allows avoiding an attack of the implanted microorganisms on the host. Our aim was to evaluate the performance of Bacillus subtilis encapsulated in an elaborate membrane system producing listeriolysin O, a cytolysin from Listeria monocytogenes, with chosen eukaryotic cells for future application in anticancer treatment. The system of encapsulating in membrane live Bacillus subtilis BR1-S secreting listeriolysin O was proven to exert the effective cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin O showed selective cytotoxic activity on eukaryotic cells: more human leukemia Jurkat T cells were killed than human chronic lymphocytic B cells leukemia at similar conditions in vitro. This system of encapsulated B. subtilis, continuously releasing bacterial products, may affect selectively different types of cells and may have future application in local anticancer treatment.

Antioxidant enzyme activity in bacterial resistance to nicotine toxicity by reactive oxygen species.

We analyzed superoxide dismutase (SOD), catalase (CAT), and ATPase activities in the highly nicotine-degrading strain Pseudomonas sp. HF-1 and two standard strains Escherichia coli and Bacillus subtilis in an attempt to understand antioxidant enzymes in bacteria are produced in response to nicotine, which increases the virulence of the bacteria. Nicotine had different effects on different antioxidant enzymes of different bacteria. SOD plays a more important role in resistance to nicotine stress in E. coli than it does in CAT. Multiple antioxidant enzymes are involved in combating oxidative stress caused by nicotine in Pseudomonas sp. HF-1. The contribution of a particular antioxidant enzyme for protection from nicotine stress varies with the growth phase involved. The inhibition of ATPase in Pseudomonas sp. HF-1 at the stationary phase was enhanced with increasing nicotine concentration, showing a striking dose-response relationship. Nicotine probably affected the metabolism of ATP to some extent. Furthermore, different bacteria possessed distinct SOD isoforms to cope with oxidative stress caused by nicotine.

Safety evaluation of two bacterial strains used in Asian probiotic products.

Probiotics, known for their prophylactic and therapeutic properties, are routinely used by the medical community in various regions of the world. In some Asian countries, these products are controlled as pharmaceutical substances and must adhere to strict regulatory guidelines. However, outside of Europe where the European Food Safety Authority has recently adopted a Qualified Presumption of Safety approach for probiotics used in food and feed, current safety requirements do not necessitate screening for the presence of virulence and other risk factors, which may result in the inadvertent use of probiotic strains harboring harmful genes. A safety evaluation was conducted on Enterococcus faecium R0026 and Bacillus subtilis R0179 used in several commercial probiotic products marketed in Asia. Molecular techniques were used to verify the identity of each strain and antibiotic resistance profiles were determined towards clinically relevant antibiotics. Strains were subsequently screened for the presence of enterotoxins and virulence factors and were subjected to 28 days of repeated high-dose oral toxicity testing in rats. No risk factors or aberrant activities were identified using such a detailed approach. Thus, both microbes were deemed to pose low risk to the consumer and, therefore, safe for use as probiotics.

The InhA1 metalloprotease allows spores of the B. cereus group to escape macrophages.

Bacteria of the Bacillus cereus group are resistant to the immune systems of various hosts and establish potent infections, implying that bacteria circumvent the bactericidal activity of host phagocytic cells. We investigated the fate of Bacillus spores after their internalization by macrophages. We found that these spores survive and escape from macrophages, and that the bacterial metalloprotease InhA1, the major component of the exosporium, is essential for efficient spore release from macrophages. InhA1 from Bacillus thuringiensis also enables Bacillus subtilis to escape from macrophages. Analysis of membrane permeability showed that the bacteria cause alterations in the macrophage membranes and that InhA1 is involved in these processes. Thus, InhA1 contributes to protect the bacteria against the host immune system. These findings provide further insight into the pathogenicity of B. cereus group members.

Organ injury and cytokine release caused by peptidoglycan are dependent on the structural integrity of the glycan chain.

Several studies have implicated a role of peptidoglycan (PepG) as a pathogenicity factor in sepsis and organ injury, in part by initiating the release of inflammatory mediators. We wanted to elucidate the structural requirements of PepG to trigger inflammatory responses and organ injury. Injection of native PepG into anesthetized rats caused moderate but significant increases in the levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, and bilirubin (markers of hepatic injury and/or dysfunction) and creatinine and urea (markers of renal dysfunction) in serum, whereas PepG pretreated with muramidase to digest the glycan backbone failed to do this. In an ex vivo model of human blood, PepG containing different amino acids induced similar levels of the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), IL-8, and IL-10, as determined by plasma analyses (enzyme-linked immunosorbent assay). Hydrolysis of the Staphylococcus aureus cross-bridge with lysostaphin resulted in moderately reduced release of TNF-alpha, IL-6, IL-8, and IL-10, whereas muramidase digestion nearly abolished the ability to induce cytokine release and IL-6 mRNA accumulation in CD14(+) monocytes compared to intact PepG. However, additional experiments showed that muramidase-treated PepG synergized with lipopolysaccharide to induce TNF-alpha and IL-10 release in whole blood, despite its lack of inflammatory activity when administered alone. Based on these studies, we hypothesize that the structural integrity of the glycan chain of the PepG molecule is very important for the pathogenic effects of PepG. The amino acid composition of PepG, however, does not seem to be essential for the inflammatory properties of the molecule.

Endospores of B subtilis are pyrogenic and activate Mono Mac 6 cells: importance of the CD14 receptor.

The monocytic cell line Mono Mac 6 is sensitive to pyrogens and interleukin-6 secretion is induced after exposure to pyrogens. The aim of this study is to examine the pyrogenic activity and the interleukin-6-inducing capacity of the Gram-positive B. subtilis bacteria, endospores and isolated cell wall components. Furthermore the involvement of CD14 in activation of interleukin-6 release is investigated. All test substances are pyrogenic in the rabbit pyrogen test. The test substance is incubated with monocytic cells (Mono Mac 6) for 24 h and the secreted interleukin-6 is determined in a sandwich immunoassay. B. subtilis bacteria and endospores induce interleukin-6 in a dose-dependent manner. Endospores are less potent than bacteria. Lipoteichoic acid (LTA) isolated from B. subtilis induces interleukin-6 in a dose-dependent manner, whereas muramyl dipeptide (MDP) is unable to induce interleukin-6. Lipopolysaccharides (LPS) dose-dependently induce interleukin-6 release, but the curve differs from that of LTA both in shape and offset. The interleukin-6 secretion induced by LPS, LTA and B. subtilis bacteria can be blocked by 73-85% by an antibody directed against CD14, whereas the antibody only blocks 25% of B. subtilis endospores-induced interleukin-6 release. The results might indicate that B. subtilis endospores use an additional pathway to CD14 to activate mononuclear cells.

A cell wall component from pathogenic and non-pathogenic gram-positive bacteria (peptidoglycan) synergises with endotoxin to cause the release of tumour necrosis factor-alpha, nitric oxide production, shock, and multiple organ injury/dysfunction in the rat.

The incidence of sepsis and septic shock due to gram-positive organisms has increased dramatically over the last two decades. Interestingly, many patients with sepsis/septic shock have both gram-positive and gram-negative bacteria present in the bloodstream and these polymicrobial or "mixed" infections often have a higher mortality than infection due to a single organism. The reason for this observation is unclear. The aim of this study was to investigate whether cell wall fragments from gram-positive and gram-negative bacteria could synergise to cause the release of cytokines, shock, and organ injury/ dysfunction in vivo. Male Wistar rats were anaesthetised and received an intravenous bolus of vehicle (saline), lipopolysaccharide (LPS) from Escherichia coli (0.1 mg/kg), peptidoglycan (Pep G) from Staphylococcus aureus (S10 mg/kg), co-administration of LPS (0.1 mg/kg) and PepG from S. aureus (10 mg/kg), LPS (10 mg/kg), PepG from Bacillus subtilis, or co-administration of LPS and PepG from B. subtilis. Blood pressure and heart rate were monitored for 6 h before plasma samples were taken for the measurement of TNF-alpha, total nitrite, and biochemical indices of organ injury. Peptidoglycan from both pathogenic (S. aureus) and non-pathogenic (B. subtilis) gram-positive bacteria synergised with endotoxin to cause formation of TNF-alpha, nitrite, shock, and organ injury. Synergism between PepG and LPS may partly explain the high mortality associated with mixed bacterial infections, as well as the deleterious effects of translocation of bacteria, or their cell wall components from the gut lumen in patients with sepsis.