Lethality for mice and chick embryos, pyrogenicity in rabbits and ability to gelate lysate from amoebocytes of Limulus polyphemus by lipopolysaccharides from Bacteroides, Fusobacterium and Veillonella

Phenol-water extracted lipopolysaccharides (LPS) from Veillonella, Fusobacterium nucleatum, Bacteroides fragilis and Bacteroides melaninogenicus were lethal for mice and 11-days-old chick embryos, pyrogenic in rabbits, and gelated Limulus amoebocyte lysate. Mouse lethality was considerably enhanced by actinomycin-D. In all test systems the endotoxin activity of Veillonella and Fusocbacterium LPS was comparable to that of LPS from Salmonella enteritidis, which was included as a reference endotoxin. The endotoxicity of the Bacteroides LPS was very low. While nanograms of the Veillonella and Fusobacterium LPS killed the chick embryos and gelated the Limulus lysates, microgram amounts of the Bacteroides LPS were needed to give positive reaction in the same test systems. As much as 74 microgram of the most active B. fragilis LPS were required to give a typical biphasic fever response in rabbits. A significant correlation was found between all test results (r = 0.90-0.98, p less than 0.001).     

Chemotaxis or migration inhibition of rabbit peritoneal polymorphonuclear leukocytes caused by chemoattractants at various concentrations

Purified lipopolysaccharide (LPS) from Veillonella incubated in normal rabbit serum was tested for chemotactic activity on rabbit polymorphonuclear leukocytes (PMNs) in modified Boyden chambers. In doses above those giving optimal response (over-optimal dose), a decrease of the PMN migration activity was found. This decrease also correlated well with an increase in the migration inhibition of the PMNs as demonstrated with the capillary tube assay. The PMN chemotactic factor isolated from LPS-induced inflammatory exudate (LPS-CF) in rabbits, produced both a decrease in chemotactic response and a migration inhibition of PMNs in over-optimal doses. This inhibitory effect was not due to cytotoxicity, proved by the trypan blue exclusion test. Also, a reduced locomotion of PMNs first preincubated with chemoattractants and then reactivated, was shown when the same PMNs were restimulated to migration using the same chemoattractants. This was interpreted as a deactivation of the cells. A cross-deactivation was demonstrated between LPS-CF and casein. The results from the experiments reported show that the Boyden chamber may be used to disciminate directional chemotaxis and migration inhibition. It may also be concluded from the study that the reduced migration activity of PMNs at over-optimal doses of chemoattractants is not due to cytotoxicity, but most probably is caused by a deactivation of the cells.     

Endotoxin-triggered haematological interactions in Fusobacterium necrophorum infections

The haematological mechanisms in the course of liver abscess formation were evaluated. They were examined by employing viable cells of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme in comparison with their endotoxins. Whole cell infection with F.n. necrophorum led to neutrophilia and to a concomitant monocytosis in parallel with those responses induced by the in vivo injection of its endotoxin. Viable infection with F.n. funduliforme was characterized by a sustained endotoxin-related monocytosis against neutropenia. The stimulatory impact of endotoxin on monocytes when released from a viable F.n. funduliforme infection suggested an inherently peculiar mechanism which differed from the induction of both neutrophilia and monocytosis when F.n. funduliforme endotoxin was administered alone. The neutrophilic inducing capacity of the F.n. necrophorum endotoxin was equally illustrated by its positive chemotactic effect on polymorphonuclear neutrophils in vitro. The data presented here emphasize the virulence of F.n. necrophorum viewed in reference to changes in leucocyte trafficking and as complemented by a relatively high endotoxin content.     

In Vitro Evaluation of Actinobolin as an Antibiotic for the Treatment of Periodontal Disease

Actinobolin was evaluated in vitro by a paper disc-agar diffusion method for inhibitory activity against mixed microbial cultures obtained from patients with periodontal disease and against pure bacterial cultures tentatively identified as strains of Bacteroides melaninogenicus, Fusobacterium fusiforme, Leptotrichia buccalis, and Veillonella parvula. Every culture tested was inhibited to some degree by actinobolin. These observations suggest that actinobolin may be effective in the treatment of periodontal disease.     

5S ribosomal ribonucleic acid sequences in Bacteroides and Fusobacterium: evolutionary relationships within these genera and among eubacteria in general

The 5S ribosomal ribonucleic acid (rRNA) sequences were determined for Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides capillosus, Bacteroides veroralis, Porphyromonas gingivalis, Anaerorhabdus furcosus, Fusobacterium nucleatum, Fusobacterium mortiferum, and Fusobacterium varium. A dendrogram constructed by a clustering algorithm from these sequences, which were aligned with all other hitherto known eubacterial 5S rRNA sequences, showed differences as well as similarities with respect to results derived from 16S rRNA analyses. In the 5S rRNA dendrogram, Bacteroides clustered together with Cytophaga and Fusobacterium, as in 16S rRNA analyses. Intraphylum relationships deduced from 5S rRNAs suggested that Bacteroides is specifically related to Cytophaga rather than to Fusobacterium, as was suggested by 16S rRNA analyses. Previous taxonomic considerations concerning the genus Bacteroides, based on biochemical and physiological data, were confirmed by the 5S rRNA sequence analysis.     

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.     

糖尿病患者拔牙创区微生物分布的临床研究

探讨糖尿病患者拔牙术后创区微生物分布情况 ,为临床防治术后感染提供理论依据。随机选取 3 9例已确诊为糖尿病的拔牙患者 ,在术后即刻采取拔牙创的渗出物 ,按 1 0倍系列稀释 ,培养微生物 ,然后进行分离、纯化和鉴定菌。结果从标本中培养出 1 87株微生物 ,鉴定到属或种 ,共获得 1 1种不同的细菌 ,常见的 7种。研究结果表明 ,糖尿病患者拔牙创区常见微生物依次为链球菌、萘瑟氏菌、梭杆菌、产黑色素拟杆菌、拟杆菌 ,韦荣菌属、二氧化碳嗜纤维菌属等。其中 ,链球菌、萘瑟氏菌为优势菌。     

Dehydrogenase Patterns in the Study of Bacteroidaceae

Enzyme patterns were obtained by starch-gel electrophoresis of cell-free extracts from organisms in the family Bacteroidaceae. Esterases, phosphatases, and lactic and succinic dehydrogenases were detected but offered no possibility for classification purposes. Alpha-glycerophosphate dehydrogenase and 6-phosphogluconate dehydrogenase were not detected. Zymograms of malic, glutamic, and glucose-6-phosphate dehydrogenases separated Bacteroides species from Sphaerophorus and Fusobacterium species. Malic dehydrogenase and glucose-6-phosphate dehydrogenase were found in Bacteroides but not Sphaerophorus and Fusobacterium. These dehydrogenase zymograms placed three gram-negative, nonsporeforming anaerobic rod isolates with the Bacteroides species. A close correlation was found between the classification of Bacteroidaceae by zymogram analysis and a numerical taxonomy scheme previously published from this laboratory.     

Synthesis of alpha-ketoglutarate by reductive carboxylation of succinate in Veillonella, Selenomonas, and Bacteriodes species.

Evidence for reductive carboxylation of succinate to synthesize alpha-ketoglutarate was sought in anaerobic heterotrophs from the rumen and from other anaerobic habitats. Cultures were grown in media containing unlabeled energy substrates plus [14C]succinate, and synthesis of cellular glutamate with a much higher specific activity than that of cellular asparate was taken as evidence for alpha-ketoglutarate synthase activity. Our results indicate alpha-ketoglutarate synthase functions in Selenomonas ruminantium, Veillonella alcalescens, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides uniformis, Bacteroides distasonis, and Bacteroides multiacidus. Evidence for this carboxylation was not found in strains representative of 10 other species.     

Biofilm-growing intestinal anaerobic bacteria

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum.     

Early production of a chemotactic factor to T lymphocytes by peritoneal macrophages

Supernatant fluids (SNF) were obtained from peritoneal exudate adherent cells stimulated in vitro with sheep red blood cells (SRBC) or BCG, and SNF collected at 6 and 24 hr were able to induce the migratory responses of rat leukocytes from the spleen and peripheral blood. The production of these SNF was dependent on protein active synthesis upon in vitro antigenic stimulation. The chemotactic activity from 6-hr SNF was inhibited by using several proteolytic enzymes and temperatures. We found the macrophages to be the producer cell of this activity, while the T cells were the target cells. The chemotactic activity from 6-hr SNF was found not to be due to IL-1. Six-hour chemotactic activity has not been reported previously.     

Identification of fusobacteria in a routine diagnostic laboratory

A scheme for differentiating Fusobacterium spp. and Leptotrichia spp. from Bacteroides spp. was devised after examining 114 strains of fusobacteria and asaccharolytic bacteroides (17 reference strains and 97 clinical isolates). Sensitivity to a 300 micrograms/ml plate of phosphomycin and an acid reaction on a lysine plate were found to be reliable for differentiating Fusobacterium spp. and L. buccalis from Bacteroides. Using a short set of simple cultural and biochemical tests, isolates could be identified as F. necrophorum, F. necrogenes, F. nucleatum, F. varium or L. buccalis. These tests were: indole, lecithinase, phosphatase, DNase and gas production, aesculin and casein hydrolysis, greening of casein/methylene blue agar, nitrite reduction, bile tolerance and haemolysis on horse blood agar.     

Antagonism among the normal anaerobic bacteria of the mouse gastrointestinal tract determined by immunofluorescence.

Strictly anaerobic Bacteroides sp., Eubacterium sp., and Fusobacterium sp. were isolated from the cecum of a conventional mouse. An immunofluorescent method utilizing rabbit antisera specific for each of these three strains was developed to determine their population levels in the gastrointestinal tracts of gnotobiotic mice. Population levels of these anaerobes in groups of gnotobiotic mice colonized with either Bacteroides, Eubacterium, or Fusobacterium were compared with those of gnotobiotes colonized with all three strains. Bacteroides population levels in gnotobiotes colonized with all three strains were 100-fold less than the Bacteroides population level in gnotobiotes colonized with only the Bacteroides strain. Eubacterium or Fusobacterium population levels were not reduced by the presence of the other anaerobic strains. Thus, strictly anaerobic Eubacterium sp. and Fusobacterium sp. that colonized gnotobiotic mice caused a reduction in the in vivo population levels of a strictly anaerobic Bacteroides sp.     

Antagonism among the normal anaerobic bacteria of the mouse gastrointestinal tract determined by immunofluorescence.

Strictly anaerobic Bacteroides sp., Eubacterium sp., and Fusobacterium sp. were isolated from the cecum of a conventional mouse. An immunofluorescent method utilizing rabbit antisera specific for each of these three strains was developed to determine their population levels in the gastrointestinal tracts of gnotobiotic mice. Population levels of these anaerobes in groups of gnotobiotic mice colonized with either Bacteroides, Eubacterium, or Fusobacterium were compared with those of gnotobiotes colonized with all three strains. Bacteroides population levels in gnotobiotes colonized with all three strains were 100-fold less than the Bacteroides population level in gnotobiotes colonized with only the Bacteroides strain. Eubacterium or Fusobacterium population levels were not reduced by the presence of the other anaerobic strains. Thus, strictly anaerobic Eubacterium sp. and Fusobacterium sp. that colonized gnotobiotic mice caused a reduction in the in vivo population levels of a strictly anaerobic Bacteroides sp.     

Effects of antibiotics and anti-bacterial components of preparations for local treatment of suppurative wounds on pathogens of odonto- genic infections]

The results of identification and sensitivity assay of 156 strains of pathogens causing odontogenic infections are presented. In the sensitivity assay antibacterial drugs were used. 42.3 percent of the strains were obligate anaerobes belonging to Bacteroides, Fusobacterium, Peptococcus, Peptostreptococcus, Veillonella and Actinomyces. Significant differences in the microbial sensitivity to the drugs used for general and local therapy were detected. There was observed high sensitivity of the obligate anaerobes to gramicidin (0.02 micrograms/ml), nitazol (10 micrograms/ml), levomycetin and tetracycline (60 micrograms/ml). Antiseptics such as dioxidine and chlorhexidine used locally showed satisfactory results. The above mentioned drugs and especially levomycetin were also rather active against facultative organisms in associations of pathogens causing odontogenic infections: Bacillus coagulans, B. licheniformis, Pseudomonas sp., Acinetobacter calcoaceticus, Staphylococcus sp. and Streptococcus sp.     

Endotoxin in germfree, gnotobiotic, or conventional-flora Sprague-Dawley rats.

The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans. Plasma from animals monoassociated with the gram-negative bacteria or C. albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E. agglomerans were negative. Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS). Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups. The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts. Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts.     

胃癌及癌旁组织中无芽孢厌氧菌相关性的研究

在实验研究过程中发现 ,不同的消化道疾病患者 ,胃黏膜微生物群的检出率、菌种、数量均有不同 ,其中胃癌组微生物群改变最明显。为了解微生物与胃癌的相关性 ,对 5 7例胃癌患者的癌组织和癌旁组织进行了无芽孢厌氧菌的检测 ,实验结果表明 ,胃癌病变的中心部位与癌旁组织的无芽孢厌氧菌的检出情况不同。胃癌组织检出的主要为革兰氏阳性无芽孢厌氧菌 (优杆菌、丙酸杆菌、消化链球菌等 ) ,占检出菌株的 6 4 .86 %(48/74 ) ,其中硝酸盐还原实验阳性菌株为 5 9.4 6 % (44 /74 )。癌旁组织检出的主要为革兰氏阴性无芽孢厌氧菌 (类杆菌、梭杆菌、紫单胞菌、韦荣球菌 ) ,占检出细菌的 5 7.14 % (2 4 /42 ) ,硝酸盐还原实验阳性菌株为16 .6 7% (7/42 )。胃癌的病因受多种因素的影响 ,在胃癌组织中检测到无芽孢厌氧菌对胃癌的形成起到的作用 ,有待进一步研究。     

Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1)

LPS stimulated human blood mononuclear leukocytes to produce a chemotactic factor for human neutrophils. The effect of LPS was dose-dependent; 10 micrograms/ml was optimal for production of chemotactic factor. Chemotactic activity was detected 3 hr after LPS stimulation, and reached its peak at 12 hr. No activity was detected in culture supernatants of unstimulated cells, provided LPS-free media were selected. Isoelectric point of the factor, determined by chromatofocusing, was approximately 8 to 8.5. Molecular weight was approximately 10 kilodaltons by Sephacryl S-200 gel filtration or by HPLC gel filtration on TSK-2000 and -3000 columns in succession. The gel filtration fractions were also assayed for IL 1 activity. The elution position of IL 1 activity corresponded to a m.w. of 18. There was no chemotactic activity in the IL 1 activity peak. Furthermore, highly purified natural Il 1 alpha and -beta and recombinant Il 1 alpha and -beta did not exhibit chemotactic activity for neutrophils in our assay. Among mononuclear leukocytes, the monocyte was the principal producer of neutrophil chemotactic factor. These results suggest that a chemotactic factor for neutrophils, different from IL 1, is produced by LPS-stimulated blood monocytes.     

Chemotypes of Veillonella lipopolysaccharides

Lipopolysaccharides (LPS) were isolated by phenol-water extraction from 34 strains of Veillonella, and examined by paper chromatography and colorimetric methods for the presence of neutral sugars, amino sugars and 2-keto-3-deoxy-octonate (KDO). Several preparations were also examined for neutral sugars by gas liquid chromatography. The LPS had in common glucosamine, galactosamine, L-glycero-D-manno-heptose glucose and KDO. Most LPS contained galactose, and a few rhamnose. D-glycero-D-manno-heptose was found in LPS from one of the strains. Based on the sugar composition of the LPS, the Veillonella strains could be classified into four chemotypes.