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Background: This study was conducted to compare the microbiomes, the levels of lipopolysaccharides (LPS), lipoteichoic acid (LTA), and cytokines (interleukin [IL]-1β and tumor necrosis factor-alpha [TNF-α]), before and after chemomechanical preparation (CMP) of the root canals (RC) and their associated periodontal pockets (PP) in teeth with combined EPL., Materials: Samples were taken from 10 RC and PP, before and after CMP. The microbiomes (next-generation sequencing, V3-V4 hypervariable region of the 16S rRNA gene), microbiome diversity (bioinformatics analyses), LPS (limulus amebocyte lysate), LTA, IL-1β, and TNF-α (ELISA) were evaluated. A statistical analysis was performed with significance level set at 5%., Results: The most abundant phyla in both sites were Firmicutes and Proteobacteria. Comparative studies of bacterial genera species revealed that some increased and others decreased after CMP at both sites. A 3% reduction in Gram-negative bacteria (RC) and a 4% increase in Gram-positive bacteria (PP) were detected. LPS levels were 4.4 times higher in PP than in the RC. LTA was detected in all samples investigated. Higher levels of IL-1β and TNF-α were detected in both sites at baseline. After CMP, LPS, LTA, IL-1β and TNF-α were reduced in both sites., Conclusion: The microbial community in the RC and PP in teeth with combined EPL indicated a similarity between both sites. CMP effectively reduced the microbial load and the LPS levels from teeth with EPL, and consequently diminished the cytokine levels. The reduction in LTA levels in the RC and PP proved challenging., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: In the lungs of cystic fibrosis patients, Pseudomonas aeruginosa is exposed to a myriad of antibiotics leading to alterations in antibiotic susceptibility. This study identifies mutations resulting in hypersusceptibility in isogenic mutants of a P. aeruginosa clinical isolate, PA34., Methods: PA34 was exposed to subinhibitory concentrations of doripenem or meropenem during growth to mid-log phase. Antibiotic susceptibility of surviving colonies was determined by agar dilution. Two carbapenem-resistant colonies hypersusceptible to non-carbapenem antibiotics were selected for further analysis. Antibiotic resistance gene expression was evaluated by RT-rtPCR and OprD production by SDS-PAGE. PA34 and isogenic mutants were evaluated with whole genome sequencing. Sequence variants were confirmed by Sanger sequencing, and cognate genes in eight carbapenem-resistant clinical isolates hypersusceptible to non-carbapenem antibiotics were sequenced. Lipopolysaccharide preparations of PA34 and hypersusceptible mutants were evaluated with ProQ-Emerald stain., Results: Isogenic mutants showed 4- to 8-fold MIC increase for imipenem, meropenem, and doripenem. However, they were hypersusceptible (≥4-fold MIC decrease) to aminoglycosides, fluoroquinolones, and non-carbapenem β-lactams. Expression of ampC or mex-opr efflux pumps was unchanged, but OprD production was decreased. Mutations causing Q86H AlgU and G77C LptG amino acid substitutions and nonsense mutations within OprD were observed in both mutants. Lipopolysaccharide modifications were observed between isogenic mutants and PA34. Non-synonymous mutations in LptF or LptG were observed in 6/8 hypersusceptible clinical isolates resistant to carbapenem antibiotics., Conclusion: Evaluation of hypersusceptible mutants identified the association between lptG and a hypersusceptible phenotype. Modifications in lipopolysaccharide profiles suggests LptG modification interferes with lipopolysaccharide transport and contributes to hypersusceptibility., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Background and Objective: Antibiotics at below minimal inhibitory concentrations (MICs) may induce various biological responses in bacteria. In this study, we hypothesized that subinhibitory concentrations (subICs) of tetracycline and doxycycline induce the shedding of lipopolysaccharide (LPS) by Porphyromonas gingivalis and, as a consequence, may contribute to enhancing the host inflammatory response associated with periodontitis., Material and Methods: A polymyxin-based enzyme-linked immunosorbent assay was used to quantify LPS shedding by P. gingivalis grown in the presence of subICs of tetracycline and doxycycline. A macrophage model was used to show that tetracycline- and doxycycline-mediated LPS shedding by P. gingivalis can induce cytokine secretion. The secretion of interleukin (IL)-1β, IL-8, and tumor necrosis factor-α was quantified by enzyme-linked immunosorbent assay., Results: LPS was shed spontaneously in a time-dependent way by P. gingivalis during growth. LPS shedding was significantly increased by growth in the presence of subICs of tetracycline and doxycycline corresponding to 1/20 of their MICs (0.025 μg/mL for tetracycline and 0.0125 μg/mL for doxycycline). This shedding was not associated with an increased rate of bacterial cell lysis. Stimulating macrophages with a P. gingivalis culture supernatant induced the secretion of IL-1β, IL-8 and tumor necrosis factor-α when the bacteria were grown in the presence of 1/20 MIC of the antibiotics., Conclusion: Our study showed that growing P. gingivalis in the presence of subICs of either tetracycline or doxycycline induces LPS shedding. Shed LPS may in turn increase cytokine secretion in a macrophage model., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: To measure the levels of hCAP18/LL-37 in gingival crevicular fluid from patients with periodontal diseases compared with healthy controls and to determine the correlation between hCAP18/LL-37 and chondroitin sulphate (CS) levels in patients with periodontitis., Material and Methods: Gingival crevicular fluid samples from 51 patients and 25 healthy volunteers were analysed for the hCAP18/LL-37 levels by immunoblotting and were determined for the CS levels by the competitive enzyme-linked immunosorbent assay., Results: Tris buffer pH 9.85 was selected to recover hCAP18/LL-37 from Periopaper strips, in which the percentages of recovery were around 70%. The median levels of hCAP18/LL-37 in the aggressive and the chronic periodontitis (CP) groups were significantly greater than those in the gingivitis and the healthy groups (p < 0.05). Significant correlations between the unprocessed 18-kDa fragment and CS levels (r = 0.650; p < 0.001) and between the mature 4.6-kDa fragment and CS levels (r = 0.502; p < 0.001) were observed only in the CP group., Conclusion: The significant correlations between the hCAP18/LL-37 and the CS levels were found in CP, but not in aggressive periodontitis. The presence versus absence of such correlations may be clinically applicable to help clinicians distinguish between two distinct types of periodontitis., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aim: Because the absorption of stimulants of Toll-like receptor (TLR)2 and TLR4 from the gastrointestinal tract into the circulation has been proposed to promote the development of atherosclerosis and insulin resistance, we aimed to quantify the abundance of stimulants of TLR2 and TLR4 in human saliva., Methods: A recently developed bioassay based upon measurement of NF-κB activation in TLR-deficient human embryonic kidney (HEK)-293 cells transfected with human TLR2 or TLR4 and calibrated with synthetic bacterial lipopeptide (Pam(3) CSK(4) ) or Escherichia coli lipopolysaccharide (LPS), was used to establish the normal range of TLR stimulants in saliva of 20 healthy subjects and 20 subjects with periodontal disease., Results: Median soluble stimulants of TLR2 and TLR4 were significantly higher in saliva of periodontitis patients compared with saliva of healthy subjects; 3450 versus 77 ng/ml Pam(3) CSK(4) equivalents (p<0.0001) and 138 versus 7 ng/ml LPS equivalents, respectively (p<0.0001). Salivary TLR stimulant levels remained relatively stable in healthy subjects over several days. Six strains of oral Gram-negative bacteria, including Tannerella forsythensis, Lysobacter enzymogenes, Prevotella intermedia, Prevotella oris and Porphyromonas gingivalis, from a panel of nine examined did not stimulate TLR4-dependent signalling., Conclusions: Elevated salivary TLR stimulants may represent a novel mechanism by which periodontitis increases the risk of developing cardiovascular disease and insulin resistance., (© 2011 John Wiley & Sons A/S.)

Objective and Background: Previous studies have described the effect of irradiation by a carbon dioxide (CO2) laser at high energy density on oral bacteria, and various side-effects have also been observed. However, no published studies have examined the effect of irradiation by a CO2 laser at low energy density on oral bacteria. The purpose of this study was to investigate the effects of super pulsed CO2 laser irradiation on periodontopathic bacteria and lipopolysaccharide (LPS)., Methods: Bacterial suspensions of two species of periodontopathic bacteria received laser irradiation at energy densities of 0-12.5 J/cm2. The suspensions were then spread over agar plates and incubated anaerobically. The bactericidal effects were evaluated based on colony formation. Samples of LPS were laser-irradiated at energy densities of 0-12.5 J/cm2. The biological activity was measured, and LPS was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)., Results: The irradiation at low energy densities of 7.5 and 12.5 J/cm2 killed more than 99.9 and 99.999% of Porphyromonas gingivalis and more than 99% of Actinobacillus actinomycetemcomitans was sterilized by the irradiation at 7.5 J/cm2. LPS biological activity was significantly decreased by laser irradiation at energy densities of more than 7.5 J/cm2 (p < 0.05), and the components of LPS analyzed by SDS-PAGE was diminished non-specifically., Conclusion: The results indicate that CO2 laser irradiation at low power is capable of bactericidal effect on periodontopathic bacteria and decreasing LPS activity.

Background: Exposure to metalworking fluid (MWF) aerosols has been shown to be associated with a variety of respiratory and skin diseases of workers., Methods: Measurements of particulate aerosols, bacterial bioaerosol, and endotoxins and 3-hydroxy fatty acids (3-OH FAs) content in water-based and straight oil MWFs were done during an 8 hr work shift in the grindery and oil basement sections of a steelworks located in Upper Silesia, Poland., Results: Particulate aerosol concentrations were below NIOSH recommended occupational exposure level. Differences in concentration and taxonomical composition of airborne bacteria between grindery and oil basement were found, due to the more aggressive creation of oil mist during machining operations and different MWF characteristics, which favor growth of different bacterial strains., Conclusions: The GC-MS analysis of 3-OH FAs as a marker of endotoxin contamination of the MWF and of the air seems to be a promising tool for evaluation of occupational exposure to bacterial bioaerosols.

Background: Lipopolysaccharide-binding protein (LBP) functions as a crucial molecule in innate host defense responses to bacterial challenge through neutralization of bacterial lipopolysaccharide (LPS) and activation of cellular responses., Objectives: This study was to investigate the expression profile and levels of LBP in gingival tissues and their associations with periodontal health and disease., Methods: Gingival biopsies were collected from 44 chronic periodontitis patients, including periodontal pocket tissues (PoTs) and the adjacent healthy gingival tissues (HT-Ps), as well as from 15 periodontally healthy subjects as controls (HT-Cs). The peptide and mRNA of LBP were detected by semi-quantitative immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR), respectively., Results: LBP peptide was detected in 90.9% of PoTs (20/22), 84.6% of HT-Ps (11/13) and all HT-Cs (7/7). The expression of LBP was mainly confined to the cytoplasm of granular and keratinized layers of gingival epithelium, spreading from the oral sulcular epithelium to oral epithelium with the expression density decreasing gradually from coronal to apical portion. LBP peptide was also found on endothelial surfaces and/or inside the lumens of blood vessels in connective tissues. The mean LBP expression levels in HT-Cs were significantly higher than those in HT-Ps and PoTs. LBP mRNA was detected in 55% of PoTs (11/20), 55% of HT-Ps (11/20) and 75% of HT-Cs (6/8)., Conclusions: We for the first time found the expression of LBP peptide and mRNA in human gingival tissues. Local expression of LBP in gingival tissues might contribute to periodontal homeostasis., (Copyright Blackwell Munksgaard, 2004)

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS-binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IRA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.

Escherichia coli O25 strains that produce heat-stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST-producing and non-producing strains of E. coli O25 was investigated. All eight ST-producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder-banding of LPS in SDS-polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed-field gel electrophoresis (PFGE). In contrast, ST-non-producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.

Background: We have investigated the possibility that the same patients may be colonized by Helicobacter pylori strains of different genotypes or phenotypes in the antrum as compared to in the duodenum. The strains were typed for DNA fingerprints, different lipopolysaccharides (LPS), and Lewis antigen expression on the O-side chains of LPS., Materials and Methods: Polymerase chain reaction (PCR) amplifications using primer sequences (i.e., the Enterobacterial Repetitive Intergenic Consensus [ERIC]) and randomly amplified polymorphic DNA (RAPD) elements were performed to asses chromosomal DNA diversity between H. pylori strains. The expression of different LPS types and Lewis antigens in the various H. pylori isolates were determined by whole bacterial enzyme-linked immunosorbent assays using monoclonal antibodies., Results: Duodenal ulcer patients had different H. pylori genotypes in the duodenum as compared to in the antrum as shown by ERIC-PCR (44%) and by RAPD-PCR (75%). Different DNA patterns were found among the strains that were isolated from various regions of the duodenum in 4 of 16 patients (25%) as shown by ERIC-PCR and in 8 of 16 patients (50%) as shown by RAPD-PCR. Sixty-three percent of the duodenal ulcer patients had H. pylori strains with a different Lewis antigen phenotype in the duodenum as compared to in the antrum, and 3 of 16 patients (19%) had strains with different Lewis antigens expressed by strains from different duodenal biopsies from the same patient., Conclusion: The results suggest that a mixed population of different H. pylori strains with marked variation, both genotypically and phenotypically, colonize the same patient.

We cloned and determined the sequence of a DNA region of approximately 15-kb containing the cluster of genes required for O17 antigen expression in the Escherichia coli K-12 strain from the chromosome of Plesiomonas shigelloides serotype O17:H2 strain. The sequencing analysis revealed that the minimum essential region of the P. shigelloides O17 antigen gene cluster had a size of approximately 11.5-kb and contained 9 contiguous open reading frames (ORFs), which were almost identical to the corresponding ORFs of Shigella sonnei form I antigen gene region, except for IS630 sequence, at the DNA as well as amino acid levels. The putative function of most of the ORFs could be determined on the basis of amino acid sequence similarities and characteristics. In addition, the G+C content of the P. shigelloides O17 antigen genes was lower than that of the chromosomal DNA of P. shigelloides and S. sonnei, suggesting that both P. shigelloides O17 and S. sonnei form I antigen genes had been derived from the same origin with a low G+C content.

Biofilms consist of microorganisms immobilized at a substratum surface embedded in an organic polymer matrix of bacterial origin. Tubing drawn from the fluid pathways within dialysis machines of various models were investigated for biofilm. Scanning electron microscopy (SEM), performed on approximately 2 cm2 samples of the tubing inner surfaces revealed that the inner surfaces of the tubing were covered with biofilms consisting of numerous deposits and glycocalix at different stages of formation with components containing bacteria and algae. Evaluations of biomass were performed from tubing sections of various lengths and inner diameters put in tubes containing water for injection and immersed in an ultrasound washtub for 1 h to ensure sloughing of the biofilm. Living bacteria were identified by plating on nutrient agar media and incubation for 48 h at 37 degrees C. Epifluorescent stains were used for the total bacteria count. Lipopolysaccharide levels were determined by the endotoxin activity measurements. Polyoside contents were determined by the colometric method, and the chemical oxygen demand was measured to evaluate the amount of organic substance. Biofilms detached from tubing samples drawn from the water path, bicarbonate path, and fresh dialysate path within dialysis machines contained approximately 1.10(3)-1.10(6) total bacteria/cm2, yet only some living bacteria were found. Endotoxin levels ranged from 1 to 12 EU/cm2. In contrast in the dialysate fluid, no bacteria were found, and the endotoxin content was under the detection level of the method. The polyoside content and chemical oxygen demand of the biomass ranged from 11 to 83 microg/cm2 and from 53 to 234 mg/cm2, respectively. It is concluded that a germ- and endotoxin-free dialysate does not exclude the risks and hazards of bacteria and endotoxin discharge from biofilm developed on the fluid pathway tubing, acting as a reservoir for continuous contamination, and efforts in the optimization of cleaning and disinfection procedures used for hemodialysis systems should aim to detach and neutralize biofilm when necessary.

A total of 11 strains of Escherichia coli (E. coli) belonging to serogroup O157 were examined for the expression of long-chain lipopolysaccharide (LPS) and major outer-membrane proteins (OMPs) by means of SDS-PAGE. The strains belonged to either one of four different flagellar (H) types or did not express flagella. Four of the eleven strains carried genes encoding Shiga-like toxins (SLTs). All the strains exhibited one of four LPS profiles, designated A, B, C or D. Electron microscopic analysis with the freeze-substitution technique demonstrated the differences in the cell surface structures of strains with each LPS profile. Strains with LPS profile A, B or C had layers of thin fibers 10, 20 and 20 nm long, respectively, on the outer membrane but strains with LPS profile D had no such structure. An analysis of the OMPs showed that all the strains had one of four OMP profiles, designated I, II, III or IV. Both LPS and OMP profiles were dependent on H-serotypes, and the combination pattern of LPS and OMP profiles of the strains was unique for each H-serotype. These data support the existence of heterogeneous groups of O157 strains.

The presence of lipoproteins and lipooligosaccharides in Treponema denticola, an oral spirochaete associated with periodontal diseases, was investigated. T. denticola ATCC 35404 and the clinical isolate GM-1 were metabolically labeled with [3H]-cis-9-octadecenoic acid and extracted with the non-ionic detergent Triton X-114. The extract was phase separated, precipitated with acetone and delipidated to remove non-covalently bound lipid (dLPP). In T. denticola ATCC 35404, sodium dodecyl sulfate polyacrylamide electrophoretic separation followed by autoradiography showed [3H]-cis-9-octadecenoic acid incorporation in bands with apparent molecular masses of 14, 20, 26, 31, 38, 72 and 85 kDa and a broad band running from 113 kDa to the top of the gel. This last band resolved into a 53 kDa [3H]-cis-9-octadecenoic acid band upon heating for 10 min, at 100 degrees C. The structural relationship of the outer sheath major oligomeric polypeptide of strain ATCC 35404 and the 53 kDa protein was demonstrated immunologically. Antibodies against the 113 kDa component of the oligomer cross-reacted with the 53 kDa protein. Proteinase K degraded the [3H]-cis-9-octadecenoic acid bands with the exception of the 14 kDa. The 14 kDa was also the major [3H]-fatty acid labeled compound found in the water phase following phenol-water extraction of whole T. denticola ATCC 35404 cells. This compound was purified from the water phase by gel filtration followed by hydrophobic chromatography. Chemical analysis showed that hexadecanoic acid was the predominant fatty acid bound to T. denticola lipoproteins. In the GM-1 strain [3H]-cis-9-octadecenoic acid incorporation was observed in the 116 kDa and 14 kDa bands. dLPP from strain ATCC 35404 caused an enhanced (0.8-8 micrograms/ml) luminol dependent chemiluminiscence (LDCL) effect in human polymorphonuclear neutrophils (PMN) which could be related to protein concentration. The addition of dLPP to PMN together with FMLP at submaximal concentration (1 microM) resulted in a synergistic activation of LDCL. At 21 micrograms/ml, dLPP also induced lysozyme release by the PMN at approximately 30% of the release induced by the chemotactic peptide at 1 microM. In addition, dLPP (21 micrograms/ml) increased additively the release of lysozyme caused by 1 microM FMLP. The release of beta-glucuronidase was not affected. The modulation of neutrophil activity was abolished by preincubation of dLPP with proteinase K. The purified 14 kDa had no effect on either LDCL or exocytosis of lysosomal enzymes of PMN. These data strongly suggest that T. denticola possesses several lipoproteins including outer sheath major oligomeric polypeptides (113-234 kDa) and a lipooligosaccharide of molecular mass of 14 kDa. In addition, an enriched lipoprotein fraction from this oral spirochaete modulates oxygen dependent and independent mechanisms for controlling microorganisms by human PMN.

We investigated the production of interleukin (IL)-1 beta and IL-1 receptor antagonist (Ra) by peripheral blood mononuclear cell (PBMC) in vitro during hemodialysis of 7 dialysis patients using 4 differential dialysis membranes (regenerated cellulose [RC], polyamide [PA], polysulfone [PS] and AN-69). Blood sampling was performed before dialysis (0 min), 15 min after starting dialysis, and after dialysis (240 min) during the last session of each treatment. The cellular content of fresh cells and the production of IL-1 beta and IL-1Ra with and without lipopolysaccharide (LPS) stimulation of the cells were evaluated and measured by enzyme-linked immunosorbent assay (ELISA). The level of IL-1 beta with LPS stimulation using RC, PA, and PS membranes was significantly reduced at 15 min and was not changed at 240 min as compared with the level at 0 min. On the other hand, the level of IL-1 beta with LPS stimulation using an AN-69 membrane at 15 and 240 min was not significantly different from that at 0 min. Neither initial cellular content nor spontaneous production of IL-1 beta were detected at 0, 15, or 240 min in any of the membranes. The spontaneous production of IL-1Ra at 15 and 240 min was not significantly different from that at 0 min in any of the membranes. The cellular content of IL-1Ra using the RC membrane was significantly lower at 15 min and did not differ at 240 min from the level at 0 min. The cellular content of IL-1Ra using PA, PS, and AN-69 membranes was not significantly different at 15 and 240 min from that at 0 min. However, the IL-1Ra level with LPS stimulation using RC and PA membranes was significantly reduced from that at 0 min, but the level using PS and AN-69 membranes was not different from that at 0 min. Because IL-1 beta and IL-1Ra levels 15 min after starting dialysis using bioincompatible dialysis membranes were reduced from the levels at 0 min, the findings suggest that measurement of cytokines' during dialysis treatment at an early stage is a useful marker for evaluating the biocompatibility of a dialysis membrane.

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an alpha (1-->2)-linked N-(3-deoxy-L-glycero-tetronyl)-D-perosamine (4-amino-4,6-dideoxy-D-manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L-glycero-tetronyl)-2-O-methyl-D-perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the alpha (1-->2)-linked N-(3-deoxy-L-glycero-tetronyl)-D-perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).

Objective: To investigate the biologic state of Chlamydia and its surface antigen expression in the synovial membranes of patients with Chlamydia-associated reactive arthritis/Reiter's syndrome (ReA/RS)., Methods: Expression of chlamydial lipopolysaccharide (LPS), major outer membrane protein (MOMP), and elementary body (EB) antigens was studied by gold labeling immunoelectron microscopy on 6 synovial membrane and 2 synovial fluid (SF) pellet samples from 6 patients with Chlamydia-associated arthritis. The study findings were compared with 24-hour cultures of HeLa cells infected with Chlamydia trachomatis EB., Results: Persistent C trachomatis infection was found in all 6 synovial membrane samples from patients who had either early or chronic arthritis. The infection persisted despite antibiotic treatment, including a 1-month course of doxycycline therapy. Most persistent organisms were atypical reticulate bodies (RBs) found in both fibroblasts and macrophages. Specific, but weak, immunogold staining for all 3 antibodies was found on both intracellular RBs and extracellular EBs. In the SF samples, Chlamydia surface antigens were detected only in phagosomes containing degraded electron-dense materials., Conclusion: The synovial membrane biopsies conducted in this study of Chlamydia-associated ReA/RS revealed atypical RBs with diminished MOMP and LPS expression. Such altered organisms may escape immune surveillance and contribute to disease chronicity; moreover, these organisms may be difficult to detect and treat in some ReA/RS patients.

Coxiella burnetii was isolated from raw milk (36/214, 16.8%) and uterus swab samples (13/61, 21.3%) originating from dairy cattle with reproductive disorders, aborted bovine fetus samples (2/4, 50%), mammary gland samples (4/50, 8%) originating from healthy dairy cattle, and tick samples (4/15, 26.7%) originating from 2 pastures. Fifty-nine strains had various degrees of pathogenicity, high (8; 13.6%), moderate (28; 47.5%) and low (23; 39%), for guinea pigs. The results of isolation suggested a high prevalence of Coxiella infection in dairy cattle with reproductive problems in Japan. Twelve strains (7, 2 and 3 strains from cattle, ticks and humans, respectively) and the reference Nine Mile strain of phases I and II were propagated in both yolk sacs of embryonated hen eggs and Buffalo green monkey (BGM) cell cultures. Protein profiles of these strains were similar to those of the reference strain of phase I. Lipopolysaccharide (LPS) profiles of 12 strains were similar to those of the reference strain of phase I and different from those of the reference strain of phase II. The LPS profiles of 12 strains suggested that these strains are associated with an acute form of Q fever.

Various chemotypes (Re, Rd2, Rd1P-, Rd1, RcP-, Rc, Rb3, Rb2, Rb1, and Ra) of R-form lipopolysaccharides (LPSs) of Salmonella spp. were crystallized by treatment with 70% ethanol containing 250 mM MgCl2, and crystals of the LPSs were observed electron microscopically and analyzed by electron diffraction and synchrotron X-ray diffraction. All the LPSs tested formed three-dimensional crystals showing very similar shapes; hexagonal plate, solid column, discoid, square or rectangular plate, lozenge plate and truncated hexangular or rectangular pyramid forms. Electron diffraction patterns from the hexagonal plate crystals of all these LPSs obtained by electron irradiation from the direction perpendicular to the basal plane showed that they consist of hexagonal lattices with the lattice constant of 4.62 A. The crystals of all the LPSs thus formed gave ring-like X-ray diffraction patterns because of their small sizes. The long-axis values were calculated from the X-ray diffraction patterns from crystals of all the LPSs in the low-angle region and they corresponded roughly to the length of the proposed primary chemical structures of the R cores of the LPSs. The volume occupied by a single molecule of all the LPSs were calculated from the molecular weights based on the proposed structures and the crystallographic data obtained by electron diffraction, X-ray diffraction, and density determination.

This investigation assessed the effectiveness of an in vitro ultrasonic debridement regime on periodontally involved teeth with visually-detectable calculus deposits, using the LAL assay for lipopolysaccharides (LPS) as a marker of bacterial products. 34 single rooted teeth with extensive amounts of calcified accretions were selected, of which 10 served as uninstrumented controls for the estimation of existing LPS levels. The periodontally involved roots of the 24 experimental teeth were debrided with a Cavitron TF-10 tip for a limited time using light pressure and overlapping strokes which ensured complete surface instrumentation without reference to the presence of calculus. Marked reductions in root surface area coverage by calculus were nevertheless achieved as assessed planimetrically from photographic records of the teeth before and after debridement. The root surfaces of each tooth were then stripped away and the harvested material subjected to 'Westphal' extraction and LAL assay for LPS. The resulting LPS yields from the experimental teeth ranged from less than 0.08 to 22.387 ng compared to 1,900 to 29,200 ng from the uninstrumented controls. The extremely small residual LPS yields from the experimental teeth not only confirm the efficacy of ultrasonic debridement but, when coupled with the lack of a relationship with the widely varying amounts of the remaining calculus, support the contention that bacterial plaque rather than calculus has the greater pathogenic potential. These results reinforce the growing impression of the superficial location of bacterial toxic products associated with periodontally involved root surfaces.

ELISA was used to measure the amount and avidity of IgG antibodies to exotoxin A (ExA) and 7 Fisher's immunotypes of lipopolysaccharide (LPS) in the sera of 13 patients with mild or moderate Pseudomonas aeruginosa infections. Changes in the specificity of tested sera during the course of infection were demonstrated. A statistically significant increase was seen in the amount and avidity of the antibodies to ExA in a majority of the sera, and an increase was seen in amount of antibodies to LPS immunotype 4 in the sera of patients with moderate infections.

Recent demonstrations of the superficial nature and ease of removal of root-surface-associated cytotoxic materials suggest that extensive root-surface instrumentation is not warranted clinically. This in vitro investigation determined the detoxifying effects of a conservative regime of ultrasonic root debridement, using the Limulus amoebocyte lysate assay for lipopolysaccharide (LPS) as an indication of the presence or absence of cytotoxic materials. 20 extracted periodontally-involved single-rooted teeth, with no clinically detectable calculus deposits, were debrided with a Cavitron TF-10 tip. Light pressure, resulting from a force application of approximately 50 g, lasting for 0.8 s/mm2 of root surface was used and complete overlapping instrumentation ensured. The finding of LPS levels of less than 2.5 ng per root in 19 out of 20 teeth after such debridement was comparable to LPS levels found on healthy, uninvolved control teeth, and thus endorses the growing belief that root surface cleanliness can be readily achieved.

The location of lipopolysaccharide (LPS) was studied by immunofluorescence and immunoelectron microscopy in macrophages infected with a non-invasive Shigella dysenteriae 1 strain. Bacterial degradation began only 3 h after the end of infection. The first visible sign of degradation was detected by immunogold labelling at the level of LPS which detached from the bacterial surface and was transferred to the perinuclear lysosomes. After a few hours, it was found in small vesicles spread over the whole macrophage cytoplasm in which it remained visible for 72 h. These vesicles seemed to belong to a compartment in which slowly or non-degradable compounds are stored. LPS separation from the bacterial surface was immediately followed by the degradation of the intrabacterial constituents. The long lag period observed before initiation of bacterial degradation was not due to a lack of phagosome acidification, since DAMP, a lysosomotropic drug was found in all phagosomes at the end of the ingestion period. The frequency of phagosome-lysosome fusion was 30% for S dysenteriae and 72% for B subtilis used as a reference of high fusion frequency. The low frequency of fusion of S dysenteriae may play an important role in the survival of the virulent strains in macrophage by providing bacteria enough time to lyse the phagosome membrane before lysosome fusion occurs.

Previously we showed that Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates or solid columns, when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2, and stored at 4 C for 10 days. Later, Escherichia coli K-12 LPS thus treated was found to form discoid crystals as well as hexagonal plate crystals and solid column crystals. Analysis by electron diffraction of the discoid crystals from the direction perpendicular to the basal plane showed that they consisted of hexagonal lattices with the a axis of 4.62 A. This result was quite the same as that of the hexagonal plate crystals. Electron micrographs of the edges of the discoid crystals revealed stacked sheets of the hexagonal plate crystals. From these results it was concluded that formation of the discoid crystals results from irregular overlapping of the hexagonal plate crystals.

The relationship between formation of the Mg2(+)-induced hexagonal lattice structure by R-form lipopolysaccharides (LPS) and chemical structure of their R-cores was investigated using different kinds of R-form LPS from a series of mutants of Salmonella minnesota or S. typhimurium. The optimal experimental condition for formation of the hexagonal lattice structure was to suspend LPS preparations, from which cationic material was removed by electrodialysis, in 50 mM tris (hydroxymethyl) aminomethane buffer at pH 8.5 containing 10 mM MgCl2. Under this experimental condition, Rb1 LPS formed the hexagonal lattice structure with the lattice constant of 14.0 +/- 0.2 nm. Ra LPS, which possesses the full length of R-core, also formed the hexagonal lattice structure but its lattice constant was larger (18.1 +/- 0.2 nm) than that of Rb1 LPS (the lattice structure by Ra LPS was looser than that by Rb1 LPS). All the other R-form LPS preparations tested, RcP+, PcP-, Rd1P-, and Re LPS, whose R-cores are shorter than that of Rb1 LPS, did not form the hexagonal lattice structure, but formed membranous structures showing various shapes which consisted of multiple bilayer structures. Failure to form the hexagonal lattice structure was the common feature of these kinds of R-form LPS irrespective of temperature at which the LPS suspensions in 10 mM MgCl2-50 mM Tris buffer were incubated. From the results of the present study it was concluded that capability of R-form LPS to form the hexagonal lattice structure has a close correlation with the chemical structure of their R-cores.

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil-Felix (WF) test, were performed by quantitative agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS, and the reaction was inhibited by the O-polysaccharide fraction isolated from the homologous LPS except OX19-LPS, which lacked O-polysaccharide moiety. The immunological data support the findings that the O-polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121-133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK-WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK-WC and OXK-LPS independently of the titers of WF or IP tests.

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.

We examined Escherichia coli K-12 lipopolysaccharide (LPS), which is known to be an R-form LPS, for its ability to form a hexagonal lattice structure in vitro. The LPS from E. coli K-12 strain JE1011 did not form a hexagonal lattice structure when it was precipitated by addition of two volumes of 10 mM MgCl2-ethanol, but it did form such a structure when it was electrodialyzed and then converted to the magnesium or calcium salt form. The lattice constant of the magnesium salt form was 15.2 +/- 0.3 nm and that of the calcium salt form 18.5 +/- 0.3 nm. Since prior treatment of the LPS with proteinase K in the presence of sodium dodecyl sulfate did not affect its capability of hexagonal assembly, the lattice formation by the LPS does not require the presence of proteins.

The antitumor activity of lipopolysaccharide (LPS) and radio-detoxified LPS of Vibrio parahaemolyticus was tested against S180 cells in Swiss mice. The toxicity of the LPS was 200 times less than that of Salmonella typhimurium LPS. The V. parahaemolyticus LPS could be detoxified by exposure to gamma radiation. Both LPS and the irradiated LPS exhibited antitumor activity, though the irradiated LPS was less effective than the native LPS. These observations indicated that exposure to gamma radiation caused significant detoxification of V. parahaemolyticus LPS and the detoxified LPS still possessed considerable antitumor activity.

A new approach has been developed to circumvent the problems of false positive reactions in the Limulus Amoebocyte Lysate (LAL) assay for lipopolysaccharide (LPS) in root surface materials. These LAL-reactive materials include thrombin, thromboplastin, ribonuclease, ribonucleic acid, lipoteichoic acid and peptidoglycan fragments. In the present study, hot phenol/water extraction of these substances followed by ultracentrifugation of the resulting aqueous phases reduced their concentrations to very low levels. Furthermore, the application of Polymyxin B/Sepharose 4B affinity chromatography to these extracts enabled their intrinsic LAL-activity to be determined. Use of these techniques to assay root surface materials has identified LPS as being the major LAL-reactive material present. The mean LPS yield for the periodontally involved teeth was 4.13 micrograms/tooth, representing 2.82 micrograms/root. In contrast, the mean yield of LPS for the periodontally uninvolved teeth was 3.12 ng/tooth.

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. The stability of this hexagonal lattice structure in long-term incubation at 4 C was investigated. The hexagonal lattice structure was stable for at least 220 days when the LPS was suspended in distilled water, but it had been disintegrated into a rough mesh-like structure when the LPS was suspended in 50 mM tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.5, at 4 C for 60 days. Half of the Mg bound to the LPS was released when the LPS was suspended in Tris buffer for 60 days, whereas Mg was not released when it was suspended in distilled water even for 220 days. By contrast, it was stable for at least 220 days in Tris buffer containing 5 mM MgCl2. The LPS suspended in Tris buffer for 60 days, at which time the structure had been disintegrated, could be restored to the original hexagonal lattice structure within 24 hr by addition of 5 mM MgCl2. From these results it is concluded that the hexagonal lattice structure of the LPS retains long-range stability if Mg bound to the LPS is not released from the LPS.

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1-) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.

The occurrence of uronic acid as a sugar constituent of lipopolysaccharides (LPS) in Vibrionaceae was demonstrated for the first time. More than 100 strains were examined. Of five genera constituting Vibrionaceae, i.e., Vibrio, Aeromonas, Plesiomonas, Photobacterium, and Lucibacterium, the latter three contained uronic acid in LPS of all of their constituting members examined, while it was totally lacking in Aeromonas LPS so far tested. Only the members of genus Vibrio were found to be divided into uronic acid-containing and -lacking groups; V. parahaemolyticus, V. alginolyticus, V. fisheri, V. costicola, Beneckea ('Vibrio'), and V. fluvialis belonged to the former, while all four biotypes of V. cholerae regardless of their serotypes, V. vulnificus and V. anguillarum, belonged to the latter group. The uronic acid content of V. parahaemolyticus O1 to O12 LPS ranged from 1.6 to 4.2%. The uronic acid residue released from V. parahaemolyticus O1, O4, O10, and O12 LPS by heating in 5% acetic acid at 100 C for 2 hr was identified as galacturonic acid; in particular, that from O12 LPS was characterized as D-galacturonic acid.

The chemical and biological properties of lipopolysaccharides (LPS) in five strains of symbiotic luminous bacteria isolated from four species of luminous marine fishes, Coelorhynchus kishinouyei (CK-1), Chlorophthalmus albatrossis (CA-1), Ventrifossa garmani (VG-1), and Acropoma japonicum (AJ-1b), as well as from a luminous squid, Doryteuthis kensaki (DK-1) were examined. The LPS isolated from these symbiotic luminous bacteria were characterized by the absence of 2-keto-3-deoxyoctonate, known to be a basic component of the usual gram-negative bacterial LPS. All LPS from these symbiotic luminous bacteria upon electrophoresis in sodium dodecylsulfate polyacrylamide gel exhibited one or two clear main bands with high mobility, and one or two obscure minor bands with low mobility when stained with periodate-Schiff reagent. LPS from CA-1 and VG-1 exhibited similar electrophoretic patterns, whereas the electrophoretic patterns of the LPS from CK-1, AJ-1b, and DK-1 were easily distinguishable from each other. All these LPS also had similarly potent and diverse biological activities in regard to their adjuvanticity, immunosuppression, polyclonal effect, B-cell mitogenicity, and activation of the phagocytic function of macrophages.

A chemotaxonomic study was carried out on Vibrio fluvialis and V. vulnificus on the basis of the sugar composition of the polysaccharide portion of their lipopolysaccharides (LPS). A previously developed rapid method of preparing samples for compositional sugar analysis was employed. Nineteen O-serogroups of V. fluvialis were divided into 14 chemotypes while seven O-serogroups of V. vulnifucus were divided also into seven chemotypes since the polysaccharide portion of LPS of each serogroup has a different sugar composition from that of the other serogroups. Close similarities in the sugar composition of the same portion were demonstrated between serologically cross-reacting non-O1 group V. cholerae and V. fluvialis, and non-O1 V. cholerae and V. mimicus.

The chemical and biological properties of the lipopolysaccharide (LPS) isolated from a marine bacterium, Photobacterium phosphoreum PJ-1, were studied. This LPS consists of 40.6% carbohydrate, 27.3% fatty acid, 0.2% 2-keto-3-deoxyoctonate (KDO) and other components. One characteristic of this LPS is its small amount of KDO, the basic component of the usual LPS. Electrophoresis in sodium dodecylsulfate polyacrylamide gel revealed at least two staining bands for carbohydrates. These bands were continuous and broad, and showed rapid electrophoretic mobility which corresponded closely to the fastest moving band of LPS from Salmonella typhimurium. This LPS preparation had adjuvant activity, lethality for ddY mice, and the ability to gel Limulus amebocyte lysate, and the strength of these activities corresponded closely to those of LPS preparations from Escherichia coli 0111:B4 and S. typhimurium. In the test for lethality of the LPS for ddY mice, the lethal action appeared in two phases depending on the dose used for intravenous (i.v.) injection: the early lethal action appeared within 30 min after injection of 250 micrograms or less, and the late lethal action occurred gradually after 16 hr at doses of 500 microgrms or more. The total (both phases) LD50 of this LPS (i.v.) for ddY mice was 265 micrograms per mouse and in only the late phase it was 500 micrograms. These results show that in spite of structural differences in regard to KDO content, LPS from P. phosphoreum PJ-1 has some biological properties similar to those of LPS from E. coli 0111:B4 and S. typhimurium but it shows no immunological cross-reaction with other LPS.

The use of saliva as a source of components that may identify subjects at risk of developing destructive periodontitis, or provide markers of disease potential or activity, has been reviewed. It was concluded that bacteria, their constituents or products are unlikely to be rewarding and that host-derived salivary factors such as enzymes cannot identify risk, as deficiency states for these do not exist. Secretory IgA, plasma IgA and IgG isotype levels and specific antibodies may be associated with risk, but probably only if levels fall below those which are protective or a specific antibody response is absent. More work is needed to distinguish between monomeric and dimeric IgA antibodies and to identify IgG antibodies in longitudinal clinical studies. In general, although saliva may prove to be useful as a source of indicators of current disease activity or as a means of assessing responses to treatment, it is unlikely to provide evidence for the existence of risk factors.

The aqueous layer was isolated from Leptospira interrogans serovar canicola strain Moulton by the hot phenol-water method. After ultracentrifugation, the precipitate was designated as lipopolysaccharide-like substance (LLS) fraction and the chemical composition was compared with that of bacterial LPS. The LLS fraction consists of 35.2% carbohydrate, 3.8% amino sugar, 36.4% lipid, 15.2% protein, and 0.3% phosphorus. Neutral sugars were detected as rhamnose, arabinose, xylose, 4-O-methylmannose, mannose, galactose, and a small amount of erythrose, fucose and glucose by gas-liquid chromatography (GLC), but 2-keto-3-deoxyoctonic acid was not detected in the LLS by thiobarbituric acid test and high voltage paper electrophoresis. Fatty acids detected by GLC were decanoic acid (C10: 0), dodecanoic acid (C12: 0), dodecenoic acid (C12: 1), tridecenoic acid (C13: 1), tetradecanoic acid (C14: 0), hexadecanoic acid (C16: 0), hexadecenoic acid (C16: 1), and octadecenoic acid (C18: 1). With SDS-polyacrylamide gel electrophoresis, bacterial LPS showed many orderly bands, while the banding pattern of the leptospiral LLS was very simple. These findings demonstrate that the physicochemical properties and chemical composition of LLS fraction from Leptospira are different from those of LPS extracted from gram-negative bacteria such as Enterobacteriaceae, and suggesting that Leptospira has no typical LPS.

The distribution of lipopolysaccharide (LPS) in periodontally involved root surface associated materials has been investigated using improved methods of identification and quantification. 39% of the LPS could be removed by gently washing in water for 1 min and 60% by brushing for 1 min with a slowly rotating bristle brush. The finding that 99% of the LPS can be removed by comparatively gentle procedures suggests that effective root surface debridement may be achieved by methods other than traditional hand instrumentation.

Vibrio parahaemolyticus, a causative bacterium of food poisoning unique for its particular primary association with sea products, is now divided serologically into 11 or 12 O-forms based on agglutination and agglutinin-absorption tests. We determined the sugar composition of the somatic O-antigens, i.e., lipopolysaccharides (LPS), of representative strains of each O-form. Of particular interest is the absence of evidence for the presence of 2-keto-3-deoxy-octonic acid (KDO), a regular sugar component of gram-negative bacterial LPS, in any LPS examined, with the exception of 06. Furthermore, 07 and 012 LPS contained a KDO-like compound that is, however, not identical with KDO. Glucose, glucosamine, and L-glycero-D-mannoheptose were found as common sugar constituents. Three unidentified amino sugars, designated here as P1, P2, and P3, were found. Various combinations of each of these unidentified amino sugars, and of galactose, fucose, arabinose, D-glyucero-D-mannoheptose, galactosamine, KDO, and the KDO-like substance were detected in accordance with the O-form of LPS. On the basis of the sugar composition, LPS of the 12 O-forms of V. parahaemolyticus can be classified into nine chemotypes, because 03, 05, and 011 LPS belong to the same chemotype and 07 and 012 to another chemotype.

A chemotaxonomic study was carried out on 31 strains of non-O1 Vibrio cholerae bio-serogroup Hakata, isolated in Japan, which possesses the Inaba antigen C of O1 V. cholerae. On the basis of the compositional sugar pattern of the polysaccharide portion of their lipopolysaccharides, the 23 strains isolated from the environment were separated into two groups, one (20 strains) containing mannose, glucose, fructose, L-glycero-D-mannoheptose, glucosamine, perosamine, quinovosamine, and an unidentified amino sugar AS, and the other (3 strains) containing two additional sugars, galactose and a trace amount of galactosamine. All of the eight strains isolated from imported seafoods belonged to the former group.

The R-form lipopolysaccharide (LPS) from Klebsiella strain LEN-111 (O3-:K1-) forms a hexagonal lattice structure with a lattice constant of 14 to 15 nm when it is precipitated by addition of two volumes of 10 mM MgCl2-ethanol. When the LPS was suspended in various buffers (50 mM) at pH 2 to 12 for 24 hr at 4 C, at pH 2 and 3 pits of the hexagonal lattice structure markedly disappeared, at pH 4 to 8.5 the lattice structure was stable, and at pH 9 to 12 it tended to loosen somewhat. The LPS from which cations were removed by electrodialysis retained the ability of hexagonal assembly, although the lattice constant of the hexagonal lattice of the electrodialyzed LPS was large. The lattice structure of the electrodialyzed LPS was much more labile than that of the non-electrodialyzed LPS at alkaline pH levels and the former was completely disintegrated into ribbon-like structures when the LPS was suspended in 50 mM Tris buffer at pH 7.7 or higher. However, the electrodialyzed LPS formed a hexagonal lattice structure in Tris buffer at pH 8.5 containing 0.1 to 100 mM MgCl2. The lattice constants of the hexagonal lattice formed by the electrodialyzed LPS at 10 or 100 mM MgCl2 were very similar to that of the lattice of the non-electrodialyzed LPS. From these results it is concluded that the lability of the hexagonal lattice structure of the electrodialyzed LPS at alkaline conditions is due to removal of Mg2+ by electrodialysis.