Chemical, Biological, and Structural Properties of Stable Proteus L Forms and Their Parent Bacteria

Proteus L forms were disrupted by osmotic shock, and the sedimentable material present in the homogenate was further fragmented in a Sorvall pressure cell. The pressure cell was also used for disrupting normal Proteus cells. The homogenates obtained were fractionated by differential centrifugation. Purified endotoxins were isolated from the major fractions by phenol extraction. Material extracted with phenol from the membrane fraction of the L forms was about as toxic and pyrogenic on a weight basis as the typical enterobacterial endotoxins isolated from cell walls of normal bacteria. The yield of extract from L forms was about one-third of that from an equal weight of normal bacteria. No differences in the gross chemical composition of the phenol extracts from the L forms and the normal cells could be ascertained. A close serological relationship existed between extracts obtained from two L forms and their respective parent bacteria, but no such relationship was found in the case of the third L form studied and its parent bacterium. Diaminopimelic acid was not detected in the membranes of the L forms, but these membranes contained most of the succinic dehydrogenase of the organisms. Only small amounts of this enzyme were present in the wall fraction of normal bacteria. The data obtained suggest that precursors of the Proteus endotoxins are formed either in the soluble protoplasm of normal cells and L forms or at sites on the membrane from which they are readily liberated into the protoplasm, whereas the final steps of the synthesis of these toxins take place at the cytoplasmic membrane. In normal cells, much endotoxin is transported to and concentrated in the walls.     

Normalization of the increased translocation of endotoxin from gram negative enterobacteria (leaky gut) is accompanied by a remission of chronic fatigue syndrome

There is now evidence that chronic fatigue syndrome (CFS) is accompanied by an increased translocation of endotoxins from gram-negative enterobacteria through the gut wall, as demonstrated by increased prevalences and median values for serum IgM and IgA against the endotoxins of gram-negative enterobacteria. This condition can also be described as increased gut permeability or leaky gut and indicates intestinal mucosal dysfunction (IMD). Here we report a case of a 13 year old girl with CFS who showed very high values for serum IgM against the LPS of some enterobacteria and signs of oxidative and nitrosative stress, activation of the inflammatory response system, and IgG3 subclass deficiency. Upon treatment with specific antioxidants and a "leaky gut diet", which both aim to treat increased gut permeability, and immunoglobins intravenously, the increased translocation of the LPS of gram negative enterobacteria normalized and this normalization was accompanied by a complete remission of the CFS symptoms.     

A role for gastrointestinal endotoxins in enhancement of heat tolerance by physical fitness

Sakurada, Sotaro, and J. Robert S. Hales. A role forgastrointestinal endotoxins in enhancement of heat tolerance by physical fitness. J. Appl. Physiol.84(1): 207-214, 1998.To further elucidate mechanisms underlyingthe higher heat tolerance of physically fit compared with sedentarypeople, we have investigated the possibility that endotoxins (ofgastrointestinal origin) act, as in the normal development of fever, toraise body temperature and therefore reduce heat tolerance. In aninitial series of experiments, five physically fit and four sedentarysheep were exposed twice at rest to an environment of 42/35°C(dry/wet bulb temperature). When animals were given normal saline iv,rectal temperature (T_re) rose at a significantly higherrate in sedentary than in fit animals; this confirms that heattolerance is improved by physical fitness. Treatment withiv indomethacin did not affect the rate of rise of T_re infit animals. In sedentary animals, however, T_re was loweredto approximate that of fit animals. Because indomethacin blocksprostaglandin pathways involved in endotoxin-induced fever, theindomethacin-induced improvement of heat tolerance of sedentary but notfit animals supports the contention that endotoxins play a role indetermining that difference in heat tolerance. In a second series ofexperiments, quantitative cardiovascular measurements were made byusing radioactive microspheres. Under normothermic conditions, bloodflows in the brain, ileum, and diaphragm were higher in fit than insedentary animals. During hyperthermia up to T_re of42°C (in a 42/39°C environment), fit compared with sedentary animals exhibited 1) a greaterincrease in cardiac output, 2) anincrease in blood flow through arteriovenous anastomoses to higher andbetter maintained levels, 3) lessreduction in blood flow to the ileum, and4) greater increase in blood flowsto the myocardium, turbinates, nasal mucosa, and respiratorymuscles. Endotoxins are likely to come from the gut lumen,because reduction of gut blood flow forms part of the normal responseto heat stress. We suggest that improvement of heat tolerance byphysical fitness is caused by a greater cardiovascular capacity thatpermits not only greater perfusion of heat-loss tissues but themaintenance of a better gastrointestinal tract blood supply, therebybetter maintaining the normal barrier to movement of endotoxins from gut lumen to plasma. Sedentary people, with their lower cardiovascular capacity, redistribute more blood flow away from the gut during environmentally induced hyperthermia, thus allowing endotoxin-induced fever to aggravate hyperthermia.

     

The Injured Liver Induces Hyperimmunoglobulinemia by Failing to Dispose of Antigens and Endotoxins in the Portal System

Hyperimmunoglobulinemia is frequently observed in patients with chronic liver diseases. However, the exact mechanism underlying the high level of antibody formation is not fully understood. In our study, we provide evidence for the functional role of the liver and the stimulation of plasma cell proliferation in hyperimmunoglobulinemia. We collected sera from patients with chronic liver diseases, and the level of serum immunoglobulins in patients was examined; this was also investigated in animal models of liver cirrhosis and hepatocellular carcinoma. An end-to-side microsurgical portacaval shunt was used to mimic liver dysfunction in rats. We used portal vein serum and inferior vena cava serum to immunize healthy rats and mice in order to confirm the function of the healthy liver in disposing of antigens and endotoxins from the gut. For the analysis of the state of plasma cell activation, plasma cells from mice were stained with PE-conjugated anti-CD138 and FITC-conjugated anti-BrdU for flow cytometry analysis. Hyperimmunoglobulinemia was observed both in patients with chronic liver diseases and in related animal models, and high plasma LPS levels were also observed. There was a significant increase in the activation and proliferation of plasma cell in mice immunized with antigens or LPS-positive serum compared with controls that were immunized with antigens and LPS-negative serum. We confirmed that the healthy liver plays an important role in disposing of antigens and endotoxins derived from the gut. Hyperimmunoglobulinemia in chronic liver diseases mainly arises due to the collateral circulation secondary to portal hypertension, gut antigens and endotoxins that bypass the liver and reach the antibody-producing cells.     

Preparation of endotoxin-free bacteriophages

Bacteriophages (phages) are bacterial viruses that interact with bacterial walls and invade bacterial cells. Moreover, they disturb bacterial metabolism and lead to bacteria lysis. In the case of Gram-negative bacteria crude phage cultures, apart from the phages themselves, the bacterial debris, bacterial proteins and nucleic acids contain endotoxins. These endotoxins (lipopolysaccharides) posses a high degree of toxicity in vitro and in vivo, and their removal is essential for safety in antibacterial bacteriophage therapy. An effective, scaleable purification of bacteriophages from endotoxins was accomplished by sequential ultrafiltration through polysulfone membrane (30 nm) followed by chromatography on sepharose 4B and Matrex Cellulofine Sulfate. The phage fraction after gel filtration chromatography routinely contained endotoxins in the 150-2500 EU/ml range. The procedure yielded bacteriophages contaminated with as little as 0.4-7 EU/ml (Limulus assay). This value lies within the permitted level for intravenous applications (5 EU/kg/h by European Pharmacopoeia, 1997).     

Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

Structural changes of gut microbiota in Parkinson's disease and its correlation with clinical features

The aim of this study was to compare the structure of gut microbiota in Parkinson's disease(PD) patients and healthy controls;and to explore correlations between gut microbiota and PD clinical features. We analyzed fecal bacterial composition of 24 PD patients and 14 healthy volunteers by using 16 S rRNA sequencing. There were significant differences between PD and healthy controls, as well as among different PD stages. The putative cellulose degrading bacteria from the genera Blautia(P=0.018),Faecalibacterium(P=0.048) and Ruminococcus(P=0.019) were significantly decreased in PD compared to healthy controls.The putative pathobionts from the genera Escherichia-Shigella(P=0.038), Streptococcus(P=0.01), Proteus(P=0.022), and Enterococcus(P=0.006) were significantly increased in PD subjects. Correlation analysis indicated that disease severity and PD duration negatively correlated with the putative cellulose degraders, and positively correlated with the putative pathobionts. The results suggest that structural changes of gut microbiota in PD are characterized by the decreases of putative cellulose degraders and the increases of putative pathobionts, which may potentially reduce the production of short chain fatty acids, and produce more endotoxins and neurotoxins; and these changes is potentially associated with the development of PD pathology.     

Temporal detection of Cry1Ab-endotoxins in coccinellid predators from fields of Bacillus thuringiensis corn

The area planted to genetically engineered crops has increased dramatically in the last ten years. This has generated many studies examining non-target effects of bioengineered plants expressing Bacillus thuringiensis endotoxins. To date, most have focused on population-level effects in the field or laboratory evaluation of specific plant-herbivore or plant-herbivore-predator trophic pathways. Using a post-mortem enzyme-linked immunosorbent assay, we examined the uptake of Cry1Ab-endotoxins by predatory coccinellids and the importance of anthesis to this trophic pathway. Adult Coleomegilla maculata, Harmonia axyridis, Cycloneda munda and Coccinella septempunctata contained low, but detectable, quantities of Bt-endotoxin when screened by ELISA. This was most evident in C. maculata, with 12.8% of 775 individuals testing positive for Cry1Ab-endotoxins. Interestingly, the presence of endotoxins in gut samples was not confined to periods around anthesis, but coccinellid adults tested positive two weeks before and up to ten weeks after pollen was shed, suggesting tri-trophic linkages in their food chain facilitates the transfer of endotoxins into higher-order predators. This contrasts with adult Coleomegilla maculata entering overwintering sites where Bt-endotoxins were not detected in gut samples, indicating low levels of persistence of Cry1Ab-endotoxins within coccinellid predators. This study enhances our understanding of complex interactions between transgenic crops and non-target food webs, but further research is required to quantify the significance of specific trophic linkages in the field.     

Biological health risk associated with resource recovery,sorting of recycle waste and composting

Numbers of airborne microorganisms, fungi, Gram-negative bacteria, thermophilic microorganisms, endotoxins and dust have been monitored in resource recovery plants and composting plants. The work is still in progress, so this paper decribes only preliminary results. Only low levels (< 15 ng m^?3) of endotoxins were found at all locations. Levels of microorganisms, fungi, Gram-negative bacteria and dust changed with quality of waste, activity in the plant etc. Levels of airborne microorganisms, endotoxins and dust could be considerably decreased in resource recovery plants if only waste of good quality, e.g. presorted materials, is handled. For composting plants the highest levels of airborne microorganisms were found during aeration, especially by indoor composting where levels of 8.3 × 10⁵ CFU of mesophilic microorganisms were found.     

Induction of fever in a teleost fish (Lepomis gibbosus, L.)

Although fishes are ectotherms they are nevertheless able to thermoregulate behaviorally by selecting appropriate water temperatures (1). In a temperature gradient fish will congregate to a species-specific range of preferred temperature (“final thermal referendum”) which is unaffected by previous thermal history of the individual (2,3). Several aquatic (and terrestrial) ectothermic vertebrates have been found to exhibit “behavioral fever” which is manifested as an increase in preferred temperature above the final thermal preferendum (4). Fever can be elicited by pyrogens: whole bacteria (alive or killed), components of bacterial cellwall (endotoxins), endogenous pyrogens, prostaglandins or from several other sources (5). Since the results with fever induction in fish using whole bacteria or endotoxins are very scarce the aim of the present work was to compare possible thermoregulatory effects of endotoxins and prostaglandins in the same species (Lepomis gibbosus, L.) by means of identical methods.     

Microbiological contamination of raw materials for large-volume parenterals.

A wide range of raw materials used for the production of large-volume parenterals were tested for their content of bacteria, molds, yeasts, and endotoxins. All raw materials were relatively free of microorganisms, although some mannitol samples contained relatively high amounts of endotoxin. The low endotoxin content of the other raw materials did not require the use of depth filters to remove endotoxins to prepare pyrogen-free infusion fluids.     

Microbiological contamination of raw materials for large-volume parenterals

A wide range of raw materials used for the production of large-volume parenterals were tested for their content of bacteria, molds, yeasts, and endotoxins. All raw materials were relatively free of microorganisms, although some mannitol samples contained relatively high amounts of endotoxin. The low endotoxin content of the other raw materials did not require the use of depth filters to remove endotoxins to prepare pyrogen-free infusion fluids.     

The Contribution of Gut-Derived Endotoxins to Liver Injury

The liver serves as the key organ for the removal and detoxification of bacterial endotoxins that are continously absorbed in small amounts from the gastrointestinal tract. This paper postulates that liver injury impairs this detoxification process leading to further liver damage and systemic effects as well. Evidence is reviewed to support the contention that endotoxin may be a major common pathway for liver injury by a variety of agents, and methods of reducing endotoxicity of gut origin are proposed. Finally, a new solid phase radioimmunometric assay for E. coli 026 is described and its usefulness as a gut marker suggested.     

Endotoxins from Cyanobacteria and Gram-negative Bacteria as the Cause of an Acute Influenza-like Reaction after Inhalation of Aerosols

Endotoxins (lipopolysaccharides) in aerosols, originating from cyanobacteria and gram-negative bacteria, were the likely etiological agent behind outbreaks of a transient, flu-like syndrome, described from four Scandinavian towns and Harare, Zimbabwe. The syndrome with fever, malaise, muscle pains, tightness of the chest and respiratory-tract symptoms, also known as toxic pneumonitis, occurred 1.5–6 hours after taking a bath or shower. The outbreaks were associated with mass developments of cyanobacteria in the drinking water reservoirs. Cyanobacterial cells and elevated levels of endotoxins were detected in the Harare tap water when human subjects reported symptoms. In a field study of 21 water bodies, the concentrations of endotoxins were much higher in water dominated by cyanobacteria, compared to water with dominance of eukaryotic algae. This observation may partially be explained by the fact that cyanobacteria possess endotoxins and partially by our findings that endotoxin-possessing bacteria inhabit the mucilage of several mass developing cyanobacterial taxa.     

七氟菊酯和溴氰菊酯对棉铃虫肠道菌群的影响

【目的】本研究旨在探究拟除虫菊酯类杀虫剂对棉铃虫Helicoverpa armigera幼虫肠道菌群结构及代谢的影响,丰富对杀虫剂作用机理的认识。【方法】分别对棉铃虫2和3龄幼虫饲喂普通人工饲料(对照组, SS)、含2%七氟菊酯(Ⅰ型拟除虫菊酯)粉剂饲料(七氟菊酯处理组,Te)和含2.5%溴氰菊酯(Ⅱ型拟除虫菊酯)乳油饲料(溴氰菊酯处理组, DM),然后提取3龄幼虫肠道菌群基因组DNA;利用Illumina MiSeq二代高通量测序技术对肠道细菌的16S rDNA的V3-V4变异区进行测序,分析其肠道细菌的多样性和丰富度;利用qPCR验证16S rDNA测序分析结果。取2和3龄幼虫肠道,匀浆后进行Biolog-Eco实验,分析肠道细菌对Eco板上31种碳源的代谢情况。【结果】16S rDNA测序结果表明,棉铃虫3龄幼虫肠道细菌主要是厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和蓝藻菌门(Cyanobacteria)。与对照组相比,溴氰菊酯处理组和七氟菊酯处理组的棉铃虫幼虫肠道细菌的α多样性指数没有显著性改变,但是菌群结构发生了变化：在门水平,拟杆菌门的相对丰度减少,厚壁菌门和蓝藻菌门的相对丰度增加,qPCR验证结果亦支持16S rDNA测序分析的这个结果;在属水平,拟杆菌属Bacteroides、普氏菌属Prevotella和假单胞菌属Pseudomonas等的相对丰度降低,狭义梭菌属Clostridium sensu stricto 1、埃希菌属-志贺氏菌属Escherichia-Shigella和盐单胞菌属Halomonas等的相对丰度增加,其中盐单胞菌属Halomonas的相对丰度显著增加。Biolog-Eco结果表明,与对照组相比,溴氰菊酯处理组中2龄幼虫对羧酸类碳源的代谢能力下降;溴氰菊酯处理组和七氟菊酯处理组中3龄幼虫对DL-α-磷酸甘油、肝糖和L-苯丙氨酸等碳源的利用能力下降。【结论】结果显示,拟除虫菊酯类杀虫剂对棉铃虫肠道菌群的结构和代谢能力有明显影响,拟除虫菊酯类杀虫剂使棉铃虫肠道有益菌的相对丰度下降,而使致病菌的相对丰度增加。短时间拟除虫菊酯处理未造成抗药性菌群的丰度增加。qPCR检测结果与16S rDNA测序分析结果相似。Ⅰ型和Ⅱ型拟除虫菊酯类杀虫剂对棉铃虫肠道菌群结构和代谢功能的影响不同。     

Dynamic changes in microbial community structure in farming pond water and their effect on the intestinal microbial community profile in juvenile common carp (Cyprinus carpio L.)

Water quality parameter dynamics, gut, sediment and water bacteria communities were studied to understand the environmental influence on the gut microbial community of a new strain of Huanghe common carp. A total of 3,384,078 raw tags and 5105 OTUs were obtained for the gut, water and sediment bacteria. The water quality had a stronger influence on the water bacteria community than gut and sediment bacteria communities. The ambient water quality parameters also significantly influenced the water and sediment bacteria communities. Comparing the gut, sediment, and water microbial communities, a relationship was found among them. However, gut bacteria were more closely related to sediment bacterial communities than to water bacteria communities. The results showed that the top three bacterial taxa were identical in gut and sediment samples in the early days of rearing. Interestingly, bacterial communities in the carp gut, water, and sediment had different adaptabilities to variations in environmental factors.     

Endotoxins in commercial vaccines.

Twenty samples of commercial vaccines intended for administration to humans were assayed for the presence of bacterial endotoxins by using the Limulus amebocyte lysate test. Sixteen of the vaccines contained more than 0.1 ng of endotoxin per ml (which corresponds to 103 bacterial cell wall equivalents per ml in the undiluted vaccines). These results suggest that at some stage of preparation, the vaccines have contained varying amounts of gram-negative bacteria and may indicate the presence of other bacterial products as well. It might be useful to list the level of endotoxins, phage, and other contaminants on each vaccine lot to facilitate studies on any side effects of these contaminants. Selection of vaccine lots with the least endotoxin might reduce some of the adverse effects of vaccinations.     

肠道微生物菌群对脑及行为的影响

肠道微生物菌群组成的变化对正常生理的影响及其在疾病中的作用逐渐成为研究热点。肠道微生物菌群通过脑肠轴影响宿主生理学的各个方面,包括脑-肠交流、脑功能甚至行为。对无菌动物、被致病细菌感染的、使用益生菌或用抗生素药物的动物研究表明,肠道微生物菌群可以调节宿主焦虑样症状及行为。研究表明对肠道微生物菌群的调节可能是治疗复杂中枢神经系统失调症的新策略。     

Detection of endotoxins of Gram-negative bacteria on the basis of electromagnetic radiation frequency spectrum

Method of Gram-negative bacteria endotoxins detection on the basis of their own spectrum of electromagnetic radiation frequency was developed. Frequency spectrum typical for chemotype Re glycolipid, which is a part of lypopolysaccharides in the majority of Gram-negative bacteria, was used. Two devices--"Mini- Expert-DT" (manufactured by IMEDIS, Moscow) and "Bicom" (manufactured by Regumed, Germany)--were used as generators of electromagnetic radiation. Detection of endotoxin using these devices was performed by electropuncture vegetative resonance test. Immunoenzyme reaction with antibodies to chemotype Re glycolipid was used during analysis of preparations for assessment of resonance-frequency method specificity. The study showed that resonance-frequency method can detect lypopolysaccharides of different enterobacteria in quantities up to 0.1 pg as well as bacteria which contain lypopolysaccharides. At the same time, this method does not detect such bacteria as Staphylococcus aureus, Bifidobacterium spp., Lactobacillus spp., and Candida albicans. The method does not require preliminary processing of blood samples and can be used for diagnostics of endotoxinemia, and detection of endotoxins in blood samples or injection solutions.