muramidase and Escherichia-coli-Infections

The aim of this field study was to assess the impact of a single i.m. injection of lysozyme dimer and flunixin meglumine in combination with intramammary and systemic antibiotic on chemiluminescence of PMN (polymorphonuclear leucocytes) and subpopulations of lymphocyte T in blood of cows with E. coli mastitis. Examinations were performed on 30 dairy cows affected with naturally occurring acute form of E. coli mastitis. Cows were randomly divided into three groups according to the method of treatment. The first group was treated with approved intramammary antibiotic product, the same antibiotic in i.m. injection and one injection of flunixin meglumine on the first day of therapy. Next group was treated with the same antibiotic and additionally one injection of lysozyme dimer on the first day of therapy. The third one was treated only with an antibiotic and served as a control group. Blood samples were taken before treatment and on days 3 and 7. In samples haematology indices were determined, spontaneous and opsonised zymosan stimulated CL and PMA measurements were performed and the subpopulations of T lymphocyte (CD2(+), CD4(+), CD8(+)) were assayed in whole blood. There was no effect of the applied supportive treatment on the value of morphological blood indices. A significant influence of the time of sample collection on the level of CL and dynamics of lymphocytes T subpopulation was demonstrated. A single injection of flunixin meglumine or lysozyme dimer on the day of the beginning of treatment of E. coli mastitis, does not affect the level of neutrophil chemiluminescence and the percentage of T lymphocytes in the blood of mastitic cows in the analysed period of time.

Topics: Amoxicillin-Potassium Clavulanate Combination; Animals; Anti-Bacterial Agents; Cattle; Clonixin; Escherichia coli Infections; Female; Luminescence; Mastitis, Bovine; Muramidase; Neutrophils; T-Lymphocyte Subsets

The effects of treatment/prophylaxis of newborn calf colibacillosis with tryptic casein hydrolysate (TCH), recently shown to be a novel type of antimicrobial acting through stimulation of the microbial autolytic system, versus an authorized veterinary drug, Fermosorb, were evaluated. Both products showed similar high therapeutic and prophylactic efficacies, but hematological indices and daily weight gain of cured/protected animals were better with TCH. The differences in hemoglobin and hematocrit levels, total protein, gamma-globulin and sulfhydryl group quantities, bactericidal and lysozyme activities as well as daily weight gain at the end of treatment/prophylaxis were statistically significant (P<0.05-0.000005). Statistically significant differences (P<0.05-0.0005) in favor of TCH were also observed when bactericidal activity, total protein quantity of serum as well as daily weight gain of the animals were compared on the 90th day after birth. We conclude that TCH acts not only as an antimicrobial, but also as an immunostimulant (and growth promoter). The immunostimulatory activity of TCH most probably derives from a synergistic action of bioactive peptides encrypted in the preparation itself and the cell wall fragments resulting from microbial autolysis induction.

Topics: Adjuvants, Immunologic; Animals; Animals, Newborn; Anti-Bacterial Agents; Blood Protein Electrophoresis; Calcium; Caseins; Cattle; Cattle Diseases; Erythrocyte Count; Escherichia coli Infections; Hematocrit; Hemoglobins; Intestinal Diseases; Leukocyte Count; Muramidase; Phosphorus; Subtilisins

IL-18 is emerging as an IL-22-induced and epithelium-derived cytokine which contributes to host defence against intestinal infection and inflammation. In contrast to its known role in Goblet cells, regulation of barrier function at the molecular level by IL-18 is much less explored. Here we show that IL-18 is a bona fide IL-22-regulated gate keeper for intestinal epithelial barrier. IL-22 promotes crypt immunity both via induction of phospho-Stat3 binding to the Il-18 gene promoter and via Il-18 independent mechanisms. In organoid culture, while IL-22 primarily increases organoid size and inhibits expression of stem cell genes, IL-18 preferentially promotes organoid budding and induces signature genes of Lgr5

Topics: Animals; Crohn Disease; Dysbiosis; Escherichia coli; Escherichia coli Infections; Immunity, Mucosal; Interferon-gamma; Interleukin-18; Interleukin-22; Interleukins; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Knockout; Muramidase; Organoids; Paneth Cells; Promoter Regions, Genetic; STAT3 Transcription Factor; Tight Junctions

The sea urchin Lytechinus variegatus is considered a good candidate for aquaculture, but bacterial diseases are a major challenge in culture conditions. The innate immunological defenses of L. variegatus to bacterial challenges were assessed through hematology parameters, in vitro phagocytosis, lysozyme activity and total plasma protein concentrations in cell-free coelomic fluid. Adult sea urchins were inoculated with Microccocus lysodeikticus, Escherichia coli and Vibrio parahaemolyticus in the cavity coelomic. Filtrated and sterile seawater (FSW) injected and non-injected sea urchins were used as control groups. Righting time, external aspects and behavior of sea urchins were evaluated. Twenty-four hours post-inoculation, we found an increase in the population of colorless spherule cells (CLS), phagocytosis, and humoral responses in sea urchins challenged by bacterial inoculations. Righting time was not affected by the treatments and apparent external signs of disease were not observed at least during 96h post-inoculation. The immunological system of L. variegatus quickly eliminated pathogenic microorganisms. CLS and lysozyme activity cooperate in the immune defenses of L. variegatus, showing an extraordinary efficiency for adjusting the immune defenses under stress caused by microbes. We recommend that the cellular and humoral markers serve as routine tests to monitor health status in sea urchins.

Topics: Animals; Escherichia coli; Escherichia coli Infections; Gram-Positive Bacterial Infections; Immunity, Innate; Lytechinus; Micrococcus; Muramidase; Phagocytosis; Vibrio Infections; Vibrio parahaemolyticus

Broad-Complex Z2 (Br-C Z2) is an ecdysone inducible transcription factor that regulates physiological, innate immune and developmental events in insects. Here, we identified an orthologue of Br-C Z2 from silkworm, Bombyx mori (BmBr-C Z2) to study its involvement in immune responses. The quantitative real-time PCR analysis revealed that BmBr-C Z2 was expressed ubiquitously in all tested tissues under normal physiological conditions. Further, developmental profile displayed that BmBr-C Z2 expression was detectable in different developmental stages, however the gene's expression was highest in the molting and pre-pupal stages. Administration of 20-hydroxyecdysone (20E) enhanced the expression levels of BmBr-C Z2 in hemocytes. The challenge with pathogens and pathogen associated molecular patterns (PAMPs) also upregulated the mRNA levels of BmBr-C Z2 in hemocytes when compared with the control. By contrast, the ectopic expression of BmBr-C Z2 remarkably increased the production of antimicrobial peptides, while the knock-down of this gene by double stranded RNA decreased their production. Dual-luciferase assay exhibited that BmBr-C Z2 induced the expression of lysozyme by directly binding to its promoter region. The treatment of Escherichia coli following the knock-down of BmBr-C Z2 strongly reduced the survival rate of silkworm larvae. These results suggest that BmBr-C Z2 plays an important biological role in the innate immune responses of silkworm by regulating immune-related genes.

Topics: Animals; Antimicrobial Cationic Peptides; Bombyx; Cloning, Molecular; Drosophila Proteins; Ecdysterone; Escherichia coli; Escherichia coli Infections; Gene Expression Regulation, Developmental; Immunity, Innate; Insect Proteins; Metamorphosis, Biological; Muramidase; Promoter Regions, Genetic; RNA, Small Interfering; Transcription Factors; Transcriptional Activation

Diarrhea remains one of the leading causes of morbidity and mortality globally, with enterotoxigenic Escherichia coli (ETEC) constituting a major causative pathogen. The development of alternative treatments for diarrhea that do not involve chemotherapeutic drugs or result in antibiotic resistance is critical. Considering that lysozyme is a naturally occurring antimicrobial peptide, in a previous study we developed a transgenic pig line that expresses recombinant human lysozyme (hLZ) in its milk. In the present study, we examined the protective effects of the consumption of this milk against ETEC infection in neonatal piglets. We found that consuming hLZ milk facilitated faster recovery from infection and decreased mortality and morbidity following an ETEC oral inoculation or infection acquired by contact-exposure. The protective effect of hLZ was associated with the enrichment of intestinal bacteria that improve gut health, such as Lactobacillus, and the enhancement of the mucosal IgA response to the ETEC-induced diarrhea. Our study revealed potential protective mechanisms underlying the antimicrobial activity of human lysozyme, validating the use of lysozyme as an effective preventive measure for diarrhea.

Topics: Animals; Animals, Genetically Modified; Animals, Newborn; Anti-Bacterial Agents; Diarrhea; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Female; Intestinal Diseases; Milk; Muramidase; Swine; Swine Diseases

Topics: Escherichia coli; Escherichia coli Infections; Extraintestinal Pathogenic Escherichia coli; Humans; Lipopolysaccharides; Muramidase; O Antigens

Microbes play an important function in our lives, while some pathogenic bacteria are responsible for many infectious diseases, food safety, and ecological pollution. Layered double hydroxide (LDH) is a kind of natural two-dimensional material and has been applied in many fields. Lysozyme is a green natural antibacterial agent, while the antimicrobial activity of lysozyme is not as good as antibiotics. We use a different ratio of cations to tune the morphology of LDH covered with lysozyme to enhance the antibacterial ability of lysozyme. We synthesize MgAl-LDH, ZnAl-LDH, and ZnMgAl-LDH covered with lysozyme, characterize the structure and morphology, test the antibacterial in culture media, and evaluate the biotoxicity in vitro and in vivo. The flower-like structure of ZnMgAl-LDH has a rough surface, covered with lysozyme with a perfect ring, and presents good antibaterial properties and promotes wound healing of mice. The bloom flower structure of ZnMgAl-LDH can enhance the loading rate of lysozyme; meanwhile, the rougher surface can adhere more bacteria, so lyso@ZnMgAl-LDH presents better antibacterial activity than the binary LDHs.

Topics: Aluminum; Animals; Anti-Bacterial Agents; Escherichia coli; Escherichia coli Infections; Hydroxides; Magnesium; Male; Mice; Muramidase; Staphylococcal Infections; Staphylococcus aureus; Wound Healing; Zinc

Malnutrition remains a leading contributor to the morbidity and mortality of children under the age of 5 years and can weaken the immune system and increase the severity of concurrent infections. Livestock milk with the protective properties of human milk is a potential therapeutic to modulate intestinal microbiota and improve outcomes. The aim of this study was to develop an infection model of childhood malnutrition in the pig to investigate the clinical, intestinal and microbiota changes associated with malnutrition and enterotoxigenic Escherichia coli (ETEC) infection and to test the ability of goat milk and milk from genetically engineered goats expressing the antimicrobial human lysozyme (hLZ) milk to mitigate these effects. Pigs were weaned onto a protein-energy-restricted diet and after 3 weeks were supplemented daily with goat, hLZ or no milk for a further 2 weeks and then challenged with ETEC. The restricted diet enriched faecal microbiota in Proteobacteria as seen in stunted children. Before infection, hLZ milk supplementation improved barrier function and villous height to a greater extent than goat milk. Both goat and hLZ milk enriched for taxa (Ruminococcaceae) associated with weight gain. Post-ETEC infection, pigs supplemented with hLZ milk weighed more, had improved Z-scores, longer villi and showed more stable bacterial populations during ETEC challenge than both the goat and no milk groups. This model of childhood disease was developed to test the confounding effects of malnutrition and infection and demonstrated the potential use of hLZ goat milk to mitigate the impacts of malnutrition and infection.

Topics: Animal Feed; Animals; Animals, Genetically Modified; Body Weight; Diet; Dietary Supplements; Disease Models, Animal; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Feces; Female; Gastrointestinal Microbiome; Genotype; Goats; Intestinal Diseases; Intestines; Male; Malnutrition; Milk; Muramidase; Organ Size; Permeability; Swine; Weaning

Topics: Animal Feed; Animals; Animals, Genetically Modified; Animals, Newborn; Bacteroidetes; Diet; Disease Models, Animal; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Feces; Gastrointestinal Microbiome; Goats; Intestinal Diseases; Intestines; Milk; Muramidase; Swine; Swine Diseases

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of toxic and infectious insults. In prior studies we found that recombinant human KGF accelerates clearance of bacteria from the murine lung by augmenting the function of alveolar macrophages (AM). In this study we tested the hypothesis that endogenous KGF plays a role in the maintenance of innate pulmonary defense against gram-negative bacterial infections. KGF-deficient mice exhibited delayed clearance of Escherichia coli from the lungs, attenuated phagocytosis by AM, and decreased antimicrobial activity in bronchoalveolar lavage (BAL) fluid, due in part to reductions in levels of surfactant protein A, surfactant protein D, and lysozyme. These immune deficits were accompanied by lower alveolar type II epithelial cell counts and reduced alveolar type II epithelial cell expression of collectin and lysozyme genes on a per cell basis. No significant between-group differences were detected in selected inflammatory cytokines or BAL inflammatory cell populations at baseline or after bacterial challenge in the wild-type and KGF-deficient mice. A single intranasal dose of recombinant human KGF reversed defects in bacterial clearance, AM function, and BAL fluid antimicrobial activity. We conclude that KGF supports alveolar innate immune defense through maintenance of alveolar antimicrobial protein levels and functions of AM. Together these data demonstrate a role for endogenous KGF in maintenance of normal pulmonary innate immune function.

Topics: Animals; Cells, Cultured; Collectins; Escherichia coli Infections; Female; Fibroblast Growth Factor 7; Gene Expression; Humans; Immunity, Innate; Macrophages, Alveolar; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Muramidase; Pneumonia, Bacterial; Pulmonary Alveoli

Lysozymes are bacteriolytic peptides that are implicated in the insect nonspecific innate immune responses. In this study, a full-length cDNA encoding a c-type lysozyme from Schistocerca gregaria (SgLys) has been cloned and characterized from the fat body of immune-challenged 5(th) instar. The deduced mature lysozyme is 119 amino acid residues in length, has a calculated molecular mass of 13.4 kDa and an isoelectric point (Ip) of 9.2. SgLys showed high identities with other insect lysozymes, ranging from 41.5% to 93.3% by BLASTp search in NCBI. Eukaryotic in vitro expression of the SgLys ORF (rSgLys) with an apparent molecular mass of ∼16 kDa under SDS-PAGE is close to the calculated molecular weight of the full-length protein. rSgLys displayed growth inhibitory activity against Gram-negative and Gram-positive bacteria. 3D structure modeling of SgLys, based on comparison with that of silkworm lysozyme, and sequence comparison with the helix-loop-helix (α-hairpin) structure of hen egg white lysozyme (HEWL) were employed to interpret the antibacterial potencies. Phylogenetic alignments indicate that SgLys aligns well with insect c-type lysozymes that expressed principally in fat body and hemocytes and whose role has been defined as immune-related. Western blot analysis showed that SgLys expression was highest at 6-12 h post-bacterial challenge and subsequently decreased with time. Transcriptional profiles of SgLys were determined by semi-quantitative RT-PCR analysis. SgLys transcript was upregulated at the highest level in fat body, hemocytes, salivary gland, thoracic muscles, and epidermal tissue. It was expressed in all developmental stages from egg to adult. These data indicate that SgLys is a predominant acute-phase protein that is expressed and upregulated upon immune challenge.

Topics: Acute-Phase Proteins; Animals; Anti-Bacterial Agents; Bacteriolysis; Cloning, Molecular; Escherichia coli; Escherichia coli Infections; Fat Body; Grasshoppers; Immunity, Innate; Insect Proteins; Molecular Structure; Muramidase; Phylogeny; Transcriptome

This study was conducted to investigate the effects of Clostridium butyricumon growth performance, immune function, and cecal microflora in broiler chickens challenged with Escherichia coli K88. Three hundred sixty 1-d-old broiler chickens were randomly divided into 4 treatments: negative control (NC) birds were fed a basal diet and not challenged with E. coli K88; positive control (PC) birds were fed a basal diet and challenged with E. coli K88; C. butyricum treatment (CB) birds were fed a diet containing 2 × 10(7) cfu C. butyricum/kg of diet and challenged with E. coli K88; and colistin sulfate treatment (CS) birds were fed a diet containing 20 mg of colistin sulfate/kg of diet and challenged with E. coli K88. Birds fed CB had greater (P < 0.05) BW than the PC birds from 3 to 21 d postchallenge. Birds fed CB had greater (P < 0.05) serum IgA and IgY at 14 d postchallenge, greater (P < 0.05) serum IgM at 21 d postchallenge, and greater (P < 0.05) mucosal secreted IgA at 3 and 7 d postchallenge than the PC birds. Birds fed CB had greater concentrations of serum complement component 3 at 14 d postchallenge, and greater (P < 0.05) concentrations of serum complement component 4 at 3, 7, and 14 d postchallenge than the PC birds. Birds in the CS or CB treatments had less cecal E. coli population at 3, 7, and 21 d postchallenge, and less cecal Clostridium perfringens counts at 21 d postchallenge compared with the PC birds. The CB treatment increased (P < 0.05) the population of cecal Lactobacillus at 3 d postchallenge and the number of cecal Bifidobacterium at 3, 14, and 21 d postchallenge in comparison with the PC treatment. The results indicate that dietary supplementation of CB promotes growth performance, improves immune function, and benefits the cecal microflora in Escherichia coli K88-challenged chickens.

Topics: Animals; Cecum; Chickens; Clostridium butyricum; Complement C3; Escherichia coli; Escherichia coli Infections; Immunoglobulins; Muramidase; Poultry Diseases; Probiotics

The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response.

Topics: Animals; Catechol Oxidase; Defensins; Enzyme Precursors; Escherichia coli Infections; Hemolymph; Larva; Micrococcus luteus; Monophenol Monooxygenase; Moths; Muramidase; Peptides

Despite the fact that pathogenic infections are widely treated by antibiotics in the clinic nowadays, the increasing risk of multidrug-resistance associated with abuse of antibiotics is becoming a major concern in global public health. The increased death toll caused by pathogenic bacterial infection calls for effective antibiotic alternatives. Lysozyme-coated mesoporous silica nanoparticles (MSNs⊂Lys) are reported as antibacterial agents that exhibit efficient antibacterial activity both in vitro and in vivo with low cytotoxicity and negligible hemolytic side effect. The Lys corona provides multivalent interaction between MSNs⊂Lys and bacterial walls and consequently raises the local concentration of Lys on the surface of cell walls, which promotes hydrolysis of peptidoglycans and increases membrane-perturbation abilities. The minimal inhibition concentration (MIC) of MSNs⊂Lys is fivefold lower than that of free Lys in vitro. The antibacterial efficacy of MSNs⊂Lys is evaluated in vivo by using an intestine-infected mouse model. Experimental results indicate that the number of bacteria surviving in the colon is three orders of magnitude lower than in the untreated group. These natural antibacterial enzyme-modified nanoparticles open up a new avenue for design and synthesis of next-generation antibacterial agents as alternatives to antibiotics.

Topics: Animals; Anti-Bacterial Agents; Cell Line; Cell Survival; Enzymes, Immobilized; Escherichia coli; Escherichia coli Infections; Fluorescein-5-isothiocyanate; HEK293 Cells; Hemolysis; Humans; Intestines; Mice; Mice, Inbred BALB C; Microscopy, Confocal; Muramidase; Nanoparticles; Porosity; Silicon Dioxide

Lysozyme is a low-molecular-weight protein with antimicrobial properties. An experiment was conducted to investigate the response of piglets receiving a water-soluble lysozyme supplement [Entegard (EG), Neova Technologies Inc., Abbotsford, British Columbia, Canada; 4,000 lysozyme units/mg] after oral challenge with enterotoxigenic Escherichia coli (ETEC). A total of 36 individually housed weanling pigs were randomly allotted to 1 of the 4 treatments, with 9 replicates per treatment. Treatments were a control (CONT, no additive), antibiotic (AB; 2.5 g/kg of feed of antibiotic with chlortetracycline, sulfamethazine, and penicillin), and EG delivered in the drinking water at concentrations of 0.1% (EG1) and 0.2% (EG2). All pigs received a basal diet similar in composition and nutrients, except for pigs receiving the AB diet, which had an added antibiotic. Pigs were acclimated to treatments for a 7-d period to monitor growth performance. On d 8, blood samples were collected from each pig to obtain serum, and each pig was gavaged with 6 mL (2 × 10(9) cfu/mL) of ETEC solution. Pigs were monitored for another 7 d to assess incidences of diarrhea and growth performance, and then all pigs were killed to obtain intestinal tissue and digesta samples. Treatments did not influence growth performance throughout the study. Greater ETEC counts were observed in the ileal mucosal scrapings (P = 0.001) and colonic digesta (P = 0.025) of pigs in the CONT group compared with pigs in the AB and EG1 groups. Pigs receiving AB and EG1 had greater (P < 0.05) small intestinal weights and ileal villus heights than pigs receiving CONT; however, the ileal villus height-to-crypt depth ratio was greater in pigs fed the AB diet (1.69) compared with those fed the CONT diet (1.34), whereas pigs receiving EG1 were intermediate. Pigs in the EG1 group showed greater (P < 0.001) serum tumor necrosis factor α and IL-6 concentrations before ETEC challenge; however, at 7 d postchallenge, pigs receiving EG2 showed the least (P < 0.05) circulating tumor necrosis factor α and IL-6 concentrations. Overall, better intestinal growth and development, as well as decreased ETEC counts on the intestinal mucosa and serum proinflammatory cytokines, suggest that EG can maintain gut health and function in piglets commensurate with antibiotics. However, it is noteworthy that at the largest dose tested, EG seemed to have a dramatic effect on proinflammatory cytokines but had a minimal or no effect on the other r

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Anti-Bacterial Agents; Dietary Supplements; Dose-Response Relationship, Drug; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Interleukin-6; Intestine, Small; Muramidase; Random Allocation; Swine; Swine Diseases; Tumor Necrosis Factor-alpha; Water; Weaning

Topics: Animals; Chickens; Embryo, Nonmammalian; Escherichia coli; Escherichia coli Infections; Gene Expression Regulation; Muramidase; Mutation; Poultry Diseases; Virulence; Zebrafish

Patients with acute watery diarrhea caused by Vibrio cholerae O1 or enterotoxigenic Escherichia coli (ETEC) were analyzed for innate immune factors produced by the epithelium during the disease process. Duodenal biopsies were obtained from study participants at the acute (day 2) and convalescent (day 21) stages of disease. Levels of α-defensin (HD-5 and -6), β-defensin (hBD-1-4), and cathelicidin (LL-37) mRNAs were determined by real-time qRT-PCR. hBD-2, HD-5, LL-37 peptides were analyzed in duodenal epithelium by immunomorphometry. Concentration of hBD-2 in stool was determined by ELISA. Specimens from healthy controls were also analyzed. hBD-2 mRNA levels were significantly increased at acute stage of diarrhea; hBD-2 peptide was detected in fecal specimens but barely in duodenal epithelium at acute stage. Immunomorphometry analysis showed that Paneth cells contain significantly higher amounts of HD-5 pre/propeptide at convalescence (P<0.01) and in healthy controls (P<0.001) compared to acute stage, LL-37 peptide levels also decreased at acute stage while mRNA levels remained unchanged. mRNA expression levels of the other antimicrobial peptides remained unchanged with higher levels of α-defensins than β-defensins. V. cholerae induced an innate immune response at the acute stage of disease characterized by increased expression of hBD-2, and continued expression of hBD-1, HD-5-6, and LL-37.

Topics: Adult; Animals; Antimicrobial Cationic Peptides; Cholera; Convalescence; Diarrhea; Duodenum; Enterotoxigenic Escherichia coli; Escherichia coli Infections; Female; Goats; Horses; Humans; Male; Mice; Middle Aged; Muramidase; RNA, Bacterial; RNA, Ribosomal, 18S; Vibrio cholerae O1; Young Adult

Lysozyme from egg white was modified by covalent attachment of an oleyl group to the free amino groups of lysozyme. The aim of the chemical modification was to develop an effective antimicrobial lysozyme derivative against both gram-negative and gram-positive bacteria. Lysozyme with various degrees of modification was obtained by changing oleoyl chloride/lysozyme mass ratio. Lysozyme derivatives evidently exhibited an antimicrobial effect against Escherichia coli (ATCC 29998). The modification slightly changed the antimicrobial effect of lysozyme derivative against Staphylococcus aureus (ATCC 121002). Since there was a positive correlation between the modification degree and the antimicrobial effect against E. coli, it was concluded that the change in antimicrobial behavior was due to an increase in hydrophobicity of the enzyme molecule enabling it to penetrate through the bacterial membrane of E. coli. It was also shown that oleoyl chloride with an MIC value of 10 mg/mL was effective against both E. coli and S. aureus.

Topics: Animals; Anti-Infective Agents; Chickens; Chlorides; Escherichia coli; Escherichia coli Infections; Humans; Microbial Sensitivity Tests; Muramidase; Oleic Acids; Staphylococcal Infections; Staphylococcus aureus

Lysozyme is a widely distributed antimicrobial protein having specificity for cleaving the beta-(1,4)-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (GlcNAc) of peptidoglycan of the bacterial cell walls and thus efficiently contributes to protection against infections caused mainly by Gram-positive bacteria. In the present study, we assembled a full-length cDNA of a novel invertebrate-type lysozyme from Eisenia andrei earthworm (EALys) by RT-PCR and RACE system. The primary structure of EALys shares high homology with other invertebrate lysozymes; however the highest, 72% identity, was shown for the destabilase I isolated from medicinal leech. Recombinant EALys expressed in Escherichia coli exhibited the lysozyme and isopeptidase activity. Moreover, real-time PCR revealed increased levels of lysozyme mRNA in coelomocytes of E. andrei after the challenge with both Gram-positive and Gram-negative bacteria.

Topics: Animals; Bacillus subtilis; Bacterial Adhesion; Carbon-Nitrogen Lyases; Chitinases; Cloning, Molecular; Echinodermata; Endopeptidases; Escherichia coli; Escherichia coli Infections; Glucosamine; Gram-Positive Bacterial Infections; Hirudo medicinalis; Host-Pathogen Interactions; Hydrolysis; Muramic Acids; Muramidase; Oligochaeta; Sequence Homology; Virulence

Cardiovascular dysfunction in septic shock (SS) is ascribed to the release of inflammatory mediators. Norepinephrine (NE) is often administered to treat low MAP in SS. We recently found that lysozyme c (Lzm-S) released from leukocytes was a mediator of myocardial depression in an Escherichia coil model of SS in dogs. This effect can be blocked in an in vitro preparation by chitobiose, a competitive inhibitor of Lzm-S. In the present study, we examined whether chitobiose treatment can reverse myocardial depression and obviate NE requirements in two respective canine E. coli preparations. In a 6-h study, we administered chitobiose after 3.5 h of E. coli bacteremia and compared stroke work (SW) and MAP at 6 h with a sepsis control group. In a 12-h study, we determined whether chitobiose treatment can reduce the need for NE requirements during 12 h of bacteremia. In the latter study, either chitobiose or NE was given when MAP decreased approximately 20% from the presepsis value in respective groups. In anesthetized, mechanically ventilated dogs, we monitored hemodynamic parameters during continuous E. coli infusion. In the 6-h study, chitobiose improved SW and MAP at the 6-h period as compared with the nontreated sepsis group. In the 12-h study, SW and MAP increased after chitobiose without the necessity of NE administration. These results suggest that inhibitors of Lzm-S such as chitobiose may improve myocardial depression and reduce the need for NE requirements in SS.

Topics: Animals; Bacteremia; Cardiomyopathies; Disaccharides; Dogs; Enzyme Inhibitors; Escherichia coli; Escherichia coli Infections; Humans; Inflammation Mediators; Male; Muramidase; Norepinephrine; Shock, Septic; Stroke Volume; Time Factors; Vasoconstrictor Agents

Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause disease by invading normally sterile niches within the host body, e.g., urinary tract, blood and cerebrospinal fluid. Infections due to ExPEC strains, in particular urinary tract infections, cause considerable morbidity and significant health-care costs. The goal of our study is to evaluate whether Caenorhabditis elegans can be used as a model to study phenotypic and genetic virulence determinants of ExPEC strains. For this purpose, we used a collection of 31 E. coli strains isolated during acute extra-intestinal infections or from the feces of healthy individuals. For all strains, the phylogeny, the presence of ExPEC virulence factors, the resistance to biologically relevant stressors (bile, human serum and lysozyme), the motility, the growth rate, the virulence in C. elegans and in a murine septicaemia model has been established. The results show that there is a strong link between virulence in C. elegans and certain phenotypic and genetic virulence predictors of ExPEC strains determinable in vitro. Furthermore, there is a significant correlation between virulence of different ExPEC strains in C. elegans and in the murine model. Therefore, our results suggest that C. elegans can be used as a model to study virulence determinants of ExPEC strains.

Topics: Animals; Anti-Bacterial Agents; Bile; Caenorhabditis elegans; Escherichia coli; Escherichia coli Infections; Growth Inhibitors; Humans; Mice; Models, Animal; Movement; Muramidase; Sepsis; Serum; Survival Analysis; Virulence; Virulence Factors

Mechanisms for protecting spermatozoa, and the testes that produce them, from infection are essential, given the importance of these cells and organs for the fertility of the individual and perpetuation of the species. This is borne out by the publication of numerous papers on this subject over the last 50 years. We extended our work and that of others on the anti-infectious defense system of the male genital tract, using a new strategy for the direct identification of antibacterial molecules in human seminal plasma. We subjected a liquefied seminal plasma cationic fraction to reversed-phase HPLC, monitored microbicidal activity by gel overlay and radial diffusion assays, and identified the proteins and/or peptides present in each active fraction by mass spectrometry. In addition to proteins with known potent microbicidal activity--phospholipase A2, lactoferrin, and lysozyme--we also found that peptides produced by cleavage of semenogelin I, the predominant human semen coagulum protein, had high levels of antibacterial activity.

Topics: Amino Acid Sequence; Cations; Cell Fractionation; Chromatography; Chromatography, High Pressure Liquid; Electrophoresis; Escherichia coli Infections; Humans; Lactoferrin; Male; Molecular Sequence Data; Muramidase; Peptide Fragments; Phospholipases A; Phospholipases A2; Semen; Seminal Vesicle Secretory Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Reversible myocardial depression in sepsis has been ascribed to the release of inflammatory mediators. We recently found that lysozyme c (Lzm-S), consistent with that originating from the spleen, was a mediator of myocardial depression in an Escherichia coli model of septic shock in dogs. We further showed in a right ventricular trabecular (RVT) preparation that Lzm-S's depressant activity could be blocked by N,N',N" triacetylglucosamine (TAC), a competitive inhibitor of Lzm-S. We hypothesized that Lzm-S binds to or cleaves a cardiac membrane glycoprotein, thereby interfering with myocardial contraction in sepsis. In the present study, we examined whether TAC could prevent myocardial depression in an in vivo preparation and whether other related N-acetylglucosamine (NAG) structures could also inhibit Lzm-S's effect in RVT.. Randomized experimental study.. University laboratory.. Anesthetized, mechanically ventilated dogs.. We produced sepsis by infusion of E. coli over an approximately 6-hr period.. We examined the effect of TAC on stroke work, our primary index of myocardial function, when treatment was administered before sepsis (pretreatment) and after 1.5 hrs (early treatment study) and 3.5 hrs of sepsis (late treatment study; LTS). In the pretreatment study and early treatment study, myocardial depression would have not yet occurred but would have already been present in the late treatment study. In RVT, we assessed the effect of other NAG oligosaccharides and variants to the NAG structure on Lzm-S's depressant activity. In pretreatment and the early treatment study, TAC prevented the reduction in stroke work observed in nontreated septic groups but did not reverse the reduction found in the late treatment study. In RVT, of the compounds tested, only N,N'-diacetylglucosamine showed an inhibitory effect.. We found that TAC, a competitive inhibitor of Lzm-S, prevented myocardial depression in experimental sepsis. Only specific NAG structures are inhibitory to Lzm-S's depressant activity. TAC may be useful in attenuating cardiovascular collapse in sepsis.

Topics: Acetylglucosaminidase; Animals; Cardiac Output; Disease Models, Animal; Dogs; Escherichia coli Infections; Female; Male; Muramidase; Myocardial Contraction; Myocardial Depressant Factor; Probability; Random Allocation; Reference Values; Sensitivity and Specificity; Shock, Septic; Stroke Volume; Ventricular Dysfunction, Left

Topics: Acetylglucosaminidase; Animals; Disease Models, Animal; Dogs; Escherichia coli Infections; Muramidase; Myocardial Contraction; Risk Assessment; Sensitivity and Specificity; Shock, Septic

The objective of the present study was to identify the nature of a filterable cardiodepressant substance (FCS) that contributes to myocardial dysfunction in a canine model of Escherichia coli septic shock. In a previous study, it was found that FCS increased in plasma after 4 h of bacteremia (Am J Physiol 1993;264:H1402) in which FCS was identified by a bioassay that included a right ventricular trabecular (RVT) preparation. In that study, FCS was only partially identified by pore filtration techniques and was found to be a protein of molecular weight between 10 and 30 K. In the present study, FCS was further purified by size exclusion high-pressure liquid chromatography, until a single band was identified on one-dimensional gel electrophoresis. This band was then subjected to tandem mass spectrometry and protein-sequencing techniques and both techniques identified FCS as lysozyme c (Lzm-S), consistent with that originating from the canine spleen. Confirmatory tests showed that purified Lzm-S produced myocardial depression in the RVT preparation at concentrations achieved during sepsis in the in vivo preparation. In addition, Lzm-S inhibited the adrenergic response induced by field stimulation and the beta- agonist isoproterenol in in vitro preparations, these results suggesting that Lzm-S may inhibit the sympathetic response in sepsis. The present findings indicate that Lzm-S originating from disintegrating leukocytes from organs such as the spleen contributes to myocardial dysfunction in this model. The mechanism may relate to its binding or hydrolysis of a cardiac membrane glycoprotein thereby interfering with myocardial excitation-contraction coupling in sepsis.

Topics: Adrenergic Antagonists; Adrenergic beta-Agonists; Animals; Dogs; Escherichia coli Infections; Heart; Isometric Contraction; Isoproterenol; Muramidase; Myocardial Contraction; Shock, Septic; Spleen; Trisaccharides

Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection. Proof for this concept is limited and was addressed during in vivo and in vitro studies. Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli. Control animals had 1,000-fold more colony-forming units of E. coli per milliliter of blood than treated animals (P < 0.001). Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02). Oral therapy with rh-LF + FeSO(4) did not alter the protective effect. In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages. An in vitro assay showed that rh-LF did not kill E. coli, but a combination of rh-LF + lysozyme was microbicidal. In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF. The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E. coli in vivo.

Topics: Animals; Animals, Newborn; Cell Death; Colony Count, Microbial; DNA-Binding Proteins; Drug Combinations; Early Growth Response Protein 1; Escherichia coli; Escherichia coli Infections; Female; Humans; Immediate-Early Proteins; Intestines; Lactoferrin; Liver; Macrophages; Muramidase; NF-kappa B; Nitric Oxide; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Severity of Illness Index; Transcription Factors; Tumor Necrosis Factor-alpha

Endotoxemia is considered to be associated with the high mortality of gram-negative septic patients. Increasing evidence shows that beta-lactam antibiotics have a propensity to induce endotoxin release from the bacterial outer membrane while killing bacteria. We have recently found that egg white lysozyme (EW-LZM) shows strong inhibition of beta-lactam induced bacteriolysis and lipopolysaccharide (LPS) release from Escherichia coli O111, resulting in reduction of the LPS-initiated inflammatory response. In this study, we compared the effect of EW-LZM on E. coli J5, which possesses rough-type LPS (RaLPS), in order to demonstrate the effect of O-antigenic polysaccharide on endotoxin neutralizing activity of EW-LZM and on inhibition of beta-lactam induced lysis by LZM. Both of the beta-lactam induced bacterial lysis and subsequent LPS release were almost completely inhibited by EW-LZM. The effect was more potent than that of wild-type LPS as assessed by released LPS concentration and LPS induced cytokine syntheses. In addition, EW-LZM was effective against lethal infection of E. coli J5 in cyclophosphamide induced leukopenic mice. These facts strongly suggested that O-antigenic polysaccharide negatively modulates LPS neutralizing activity of EW-LZM.

Topics: Animals; Anti-Bacterial Agents; Bacteriolysis; Carrageenan; Cell Line; Cyclophosphamide; Disease Models, Animal; Endotoxemia; Endotoxins; Escherichia coli; Escherichia coli Infections; Galactose; Lactams; Leukopenia; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred ICR; Monokines; Muramidase; O Antigens

Topics: Animals; Armin; Blood Bactericidal Activity; Cholinergic Agents; Cholinesterase Inhibitors; Escherichia coli Infections; Immunity, Cellular; Mice; Muramidase; Neutrophils; Peritonitis; Quinuclidines

Previous studies have shown that the decreased neutrophil migratory responsiveness seen in burned patients correlates with the extent of thermal injury and the extent of the neutrophil-specific granule deficiency. To understand better the relationship between the neutrophil dysfunction, degranulation, and thermal injury, a rabbit model was studied. Eighteen rabbits were burned over 20% of their surface area. Assay of peripheral blood heterophils disclosed decreased migratory activity compared with preburn levels and decreased lysozyme content vs preburn levels, but no change in the beta-glucuronidase content. The specific binding of tritiated formyl-methionyl-leucyl-phenylalanine to peripheral blood heterophils was increased fivefold over that of control cells. These studies indicate that, following thermal injury, there is a selective decrease of specific granule contents and an increase in chemoattractant binding to the cell and also suggest an abnormality in chemoattractant receptor processing. The rabbit provides a convenient model for the study of compromised host defenses following thermal injury.

Topics: Animals; Body Weight; Burns; Cell Migration Inhibition; Chemotaxis, Leukocyte; Disease Models, Animal; Endotoxins; Escherichia coli Infections; Muramidase; Neutrophils; Oligopeptides; Rabbits; Receptors, Formyl Peptide; Receptors, Immunologic; Staphylococcal Infections; Time Factors

Topics: Blood Bactericidal Activity; Complement System Proteins; Escherichia coli Infections; Humans; Immunoglobulins; Leukocyte Count; Muramidase; Species Specificity; Staphylococcal Infections; Surgical Wound Infection

In the course of 4 years we isolated 193 E. coli strains of 55 patients with chronic pyelonephritis. In patients with obstructive chronic pyelonephritis the mean value of the immunofluorescence titre (in the serum) to the E. coli strain excreted in the urine as well as the total complement were significantly increased, the serum lysozyme was significantly lower than in patients with non-obstructive chronic pyelonephritis. A relation to the activity of the disease was existing only in the non-obstructive chronic pyelonephritis, where in the active stage the total complement was significantly decreased, the complement factors C3 and C4 as well as the urine lysozyme were significantly increased in comparison to the inactive stage. 94.64% of all immunofluorescence titres obtained to the homologous strain in the patients' serum were above the border of the normal area of 1:40. A relation between level of the titre and activity of the disease could not be established. No significant differences could be proved between the titres taken to serum-sensitive and serum-resistant strains. In 32.73% of the patients we observed disturbances of the serum bactericidia against the homologous serum-sensitive E. coli urine strain at one or several points. They fall to equal shares to patients with obstructive and non-obstructive chronic pyelonephritis and were found at 66.67% in the active stage of the two forms of the disease. In patients with and without disturbances of bactericidia no significant differences in the total complement, in the complement factors C3 and C4, the C3-activator, the serum lysozyme and the immunofluorescence titres could be proved.

Topics: Antibody Formation; Bacteriuria; Blood Bactericidal Activity; Chronic Disease; Complement System Proteins; Escherichia coli; Escherichia coli Infections; Fluorescent Antibody Technique; Humans; Immunocompetence; Muramidase; Pyelonephritis

Topics: Animals; Antibodies, Bacterial; Cattle; Complement System Proteins; Escherichia coli; Escherichia coli Infections; Female; Lactoferrin; Mastitis, Bovine; Muramidase; Properdin; Vaccination

Topics: Animals; Bacteriuria; Clinical Enzyme Tests; Escherichia coli Infections; Humans; Leukocytes; Muramidase; Pyelonephritis; Rabbits; Time Factors

Phagocytic activity of leukocytes, as well as the complement, properdin, and lysozyme levels in the blood serum of miniature piglets, germfree and monocontaminated with E. coli 055 and E. coli 083, were studied. E. coli 055 phagocytosis was decreased in the presence of autologous serum and complement and increased under the effect of specific opsonins (antibodies to E. coli 055). Complement, properdin, and lysozyme levels were decreased in the germfree, in comparison with conventional animals. In the E. coli contaminated piglets properdin and complement production was stimulated most, and lysozyme formation--less. No antibodies to E. coli 055 were revealed in monocontaminated piglets. The highest lysozyme levels were found in the ex-germfree animals, this indicating the participation of factors other than E. coli contamination in lysozyme stimulation. It is concluded that microbial contamination played an important role in the development of cellular and humoral factors of the organism resistance.

Topics: Animals; Animals, Newborn; Antibodies, Bacterial; Complement System Proteins; Escherichia coli Infections; Germ-Free Life; Immunity; Leukocytes; Muramidase; Opsonin Proteins; Phagocytosis; Properdin; Swine

Topics: Animals; Antibody Formation; Blood; Blood Bactericidal Activity; Cell Movement; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Estradiol; Female; Hemagglutination Tests; Injections, Intramuscular; Leukocytes; Liver; Monocytes; Muramidase; Phagocytosis; Pyelonephritis; Rats; Spleen

Topics: Abortion, Septic; Adolescent; Adult; Anti-Bacterial Agents; Antitoxins; Carbenicillin; Cephalosporins; Complement System Proteins; Endometritis; Escherichia coli Infections; Female; Humans; Methicillin; Muramidase; Penicillin G; Pregnancy; Staphylococcal Infections; Streptococcal Infections; Tetracycline; Tetracyclines

Topics: Animals; Antigens, Bacterial; Escherichia coli; Escherichia coli Infections; Female; Immunosuppressive Agents; Male; Mercaptopurine; Mice; Muramidase

Topics: Adjuvants, Immunologic; Animals; Animals, Newborn; Antibodies; Bacterial Vaccines; Colostrum; Complement System Proteins; Diarrhea; Escherichia coli; Escherichia coli Infections; Feces; Female; Formaldehyde; Muramidase; Pregnancy; Swine; Swine Diseases; Vaccination

Topics: Abortion, Septic; Antitoxins; C-Reactive Protein; Clostridium Infections; Escherichia coli Infections; Female; gamma-Globulins; Humans; Muramidase; Peritonitis; Pregnancy; Sepsis; Staphylococcal Infections

Topics: Adolescent; Adult; Age Factors; Aged; Antibodies; Antineoplastic Agents; Blood Bactericidal Activity; Burns; Child; Complement System Proteins; Drug Resistance, Microbial; Escherichia coli Infections; Female; Humans; Immunity; Middle Aged; Muramidase; Parotitis; Phagocytosis; Pneumonia; Polysaccharides, Bacterial; Pseudomonas Infections; Sepsis; Serratia marcescens; Staphylococcal Infections; Stimulation, Chemical

Topics: Animals; Anti-Bacterial Agents; Erythromycin; Escherichia coli Infections; Fluorescent Antibody Technique; Immune Sera; L Forms; Muramidase; Osmotic Fragility; Osmotic Pressure; Pyelonephritis; Rats