Identification of Bacteroides fragilis by co-agglutination, using a specific monoclonal antibody

A monoclonal antibody, mAbC3 (IgG(2b)), specific for the Bacteroides fragilis lipopolysaccharide (LPS) was produced and found to react with a common epitope in most strains within the species. mAbC3 was adsorbed to Staphylococcus aureus Cowan I strain and used in a co-agglutination assay for identification of B. fragilis. Almost 98% of the B. fragilis strains were positive in co-agglutination. Among the 283 non-B. fragilis only two strains showed false positive reaction. These results show that the mAbC3 has a high specificity and can be used for the rapid identification of the B. fragilis species.     

Fatty acid composition of the lipopolysaccharides of bacteria in the genera Escherichia, Shigella and Salmonella as a taxonomic trait

The comparative study of the fatty acid composition of bacterial lipopolysaccharides (LPS) in the genera Escherichia, Shigella, Salmonella has been carried out under identical experimental conditions. The LPS of the bacteria under study have been found to contain a number of saturated fatty acids and oxyacids which could not be previously detected in these bacteria in other studies. In all bacterial strains under study LPS include mainly 3-oxytetradecanoic, tetradecanoic and dodecanoic fatty acids. The essential feature of the fatty acid composition of Salmonella is the presence of 2-oxytetradecanoic acid; this acid is absent in Escherichia and Shigella, which can thus be used as a differentiating criterion. The content of other fatty acids in Salmonella is similar to that in Escherichia and Shigella. These data confirm that the genera Escherichia, Shigella and Salmonella are phylogenetically related, the relationship between Escherichia and Shigella being more close.     

The role of lipopolysaccharide in complement-killing of Aeromonas hydrophila strains of serotype O:34

The role of lipopolysaccharide (LPS) in the susceptibility of Aeromonas hydrophila strains of serotype O:34 to non-immune human serum was investigated using isogenic mutants (serum-sensitive), previously obtained on the basis of phage resistance, and characterized for their surface components. The classical complement pathway was found to be principally involved in the serum-killing of these sensitive strains. LPS preparations from serum-resistant or serum-sensitive strains, or purified core oligosaccharides (low-molecular-mass LPS) inactivated both bactericidal and complement activity of whole serum, while the O-antigen molecules (high-molecular-mass LPS) did not. The results indicate that LPS core oligosaccharide composition contributes to complement resistance of A. hydrophila strains from serotype O:34 with moderate virulence.     

Melanization and pathogenicity in the insect, Tenebrio molitor, and the crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3

Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium.     

Comparative Grouping of Mastitis Streptococci by Means of Co-Agglutination and Precipitation

Two hundred bovine mastitis streptococcal strains belonging to groups B, C, E, G and L were tested comparatively by means of the co-agglutination and precipitation technique. Identical results were obtained with the two tests in 191, or 95.5 %, of the strains. Six strains, which could not be grouped by co-agglutination, proved to belong to group B when grouped by precipitation. On the other hand, one strain which proved to belong to group G and two strains to group L when grouped by co-agglutination, could not be grouped by precipitation. Some cross-reactivity was observed between groups A and C, B and G, B and L. Only a few L strains showed marked cross-reactivity which was not easily distinguished from specific reactions. However, the cross-reactivity ought to be eliminated by dilution or adsorption. Using the precipitation test as a supplementary method, the easy and rapid co-agglutination test was found to be a suitable procedure for routine grouping of mastitis streptococci.     

Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.     

Immunological analysis of extracellular products and cell surface components of motile Aeromonas isolated from fish

The present work describes the characterization of antigens present in the extracellular products (ECP) and cell wall of strains of motile Aeromonas isolated from rainbow trout culture systems. The relationships among virulence for fish, O-serogroup and profile of LPS were also examined. The slide agglutination test showed that most of the virulent strains of motile Aeromonas (72%) were included in the serotypes O3, O6, O11 and O19 (Guinée and Jansen System). However, there were also non-pathogenic strains within these groups. Electrophoretic analysis of lipopolysaccharides (LPS) and proteins from cell envelope and ECP showed heterogeneity not only among the different serogroups but also within the same serotype. Immunoblot assays of cell envelope components, and of LPS present in the ECP demonstrated a close relationship among Aeromonas strains from the same serotype, while strains from different serotypes were not immunologically related. Moreover, this assay showed that motile Aeromonas belonging to distinct serotypes produced extracellular proteins immunologically related. On the other hand, antigenic cross reactivity was observed between the LPS obtained from cell envelope and those obtained from the ECP. The present results point out the need to include strains representative of each of the serotypes which predominates in a particular area and their ECPs in the formation of vaccines against motile Aeromonas septicaemia.     

Relationship between immune system and gram-negative bacteria. IV. T lymphocytes from Lpsd mice possess binding site(s) for Rb Salmonella

We have previously shown that Salmonella minnesota R345 (Rb) spontaneously binds to 50 to 55% of human peripheral blood mononuclear cells (PBMC). In the present study, we have compared Rb cytoadherence to lymphoid cells from various tissues of lipopolysaccharide (LPS) hyporesponsive (Lpsd) and LPS responsive (Lpsn) mouse strains. A higher number of spleen cells from Lpsd mice (C3H/HeJ and C57BL/10ScN) bound Rb bacteria (22 to 30%) than cells from Lpsn mice (4 to 9%). Rb bound mainly to T cells, and cytoadherence occurred in both Lyt-1+ and Lyt-2+ T cell subsets. By contrast, purified splenic B cells from Lpsd and Lpsn mice gave less than 4% Rb cytoadherence. In both mouse strains, cytoadherence was mediated by the homologous LPS structure, because purified Rb-LPS blocked Rb Salmonella binding to T cells. On the other hand, smooth Salmonella typhimurium LT-2 LPS (S-LPS) and Salmonella R595 (Re) LPS (Re-LPS), which contain mainly lipid A, were without effect on Rb binding. Increased Rb binding was seen with T cells from Peyer's patches (PP), mesenteric lymph nodes (MLN), and peripheral blood than from spleen of C3H/HeN (Lpsn) mice; however, greater cytoadherence was always seen with T cells of these tissues from C3H/HeJ mice. Interestingly, treatment of whole spleen or purified T cells from C3H/HeN mice with neuraminidase enhanced cytoadherence to levels seen with C3H/HeJ cells. The observed Rb binding to PP, MLN, and PBMC cells in both mouse strains suggests that gut microbial environment may play an important role in Rb cytoadherence. This is also supported by the evidence that when spleen cells of germfree and conventional mice were tested for Rb binding, higher cytoadherence was observed in conventional mice only. Taken together, these results indicate that T cells of Lpsd mice express binding site(s) for Salmonella, whereas Lpsn mice have T cells with these structure(s) in a cryptic configuration.     

Characteristics of lipopolysaccharides ofSalmonella typhi isolated from carriers and patients suffering from typhoid fever

Lipopolysaccharides (LPS) ofSalmonella typhi strains, isolated from carriers and patients suffering from typhoid fever, were characterised according to their biochemical properties, morphological structure and degree of aggregation of complexes. All preparations of LPS, regardless of their origin, were morphologically heterogeneous. Free electrophoresis and immunoelectrophoresis demonstrated that LPS preparations were composed of components possessing different mobilities in electric fields. LPS of bacterial strains isolated from both carriers and patients, split upon reaction in immunoelectrophoresis with specific antiserum 73, rabbit antiserum toSalmonella typhi Vi Bhatnagar and 0–901 split into anodic and cathodio fractions. The anodic fraction reacted similarly as Vi antigen. LPS fromSalmonella typhi Ty-2 yielded only the cathodic fraction, typical for O antigen. LPS from strains whioh were passaged twice in nutritional medium possessed identical properties as LPS from fresh cultures ofSalmonella typhi. Electron microscopy revealed that LPS appears as long bands, rods, ellipsoid forms and amorphous material. Contrary to amorphous material, the bands, rods and ellipsoid forms possessed three-layer structure.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses.

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Unusual expression of lipopolysaccharide by strains of Salmonella enteritidis phage type 30

An investigation of 83 strains of Salmonella enteritidis phage type 30 showed that these bacteria were unusual in expressing either trace amounts of, or no, lipopolysaccharide (LPS). Regardless of whether strains expressed LPS or not, or carried a 38 MDa mouse virulence' plasmid, these strains were avirulent for BALB/c mice.     

蒙脱石对细菌黏附Caco-2细胞的影响

采用Caco-2细胞培养模型,观察两歧双歧杆菌、嗜酸乳杆菌、嗜水气单胞菌、副溶血弧菌、大肠杆菌、鼠伤寒沙门菌的黏附率,并在培养液中加入蒙脱石,计算蒙脱石对细菌黏附的阻断率,探讨蒙脱石对上述细菌黏附作用的影响。结果表明:所试菌与Caco-2细胞均有不同程度的黏附作用;蒙脱石对细菌黏附Caco-2细胞均有不同程度的阻断作用,对病原菌黏附Caco-2细胞的阻断作用要明显大于其对益生菌的阻断效果,其中对大肠杆菌、鼠伤寒沙门菌、嗜水气单胞菌、副溶血弧菌黏附的阻断率分别为54.22%、48.41%、60.53%、50.64%,而对两歧双歧杆菌、嗜酸乳杆菌黏附的阻断率分别为25.64%和21.49%。结果提示蒙脱石可有效阻断病原菌黏附,从而防治肠道细菌感染和细菌移位。     

Lipopolysaccharide expression and virulence in mice of Australian isolates of Salmonella enteritidis

The lipopolysaccharide (LPS) of 54 Australian isolates, nine isolates acquired or isolated overseas, and two reference strains of Salmonella enteritidis was studied to assess its relation to pathogenicity. LPS was extracted by proteinase K digestion of whole cells, and analysed by polyacrylamide gel electrophoresis. All isolates possessed an LPS structure identical to that of a reference strain of Salm. enteritidis phage type 4. Representative strains of the clinically prevalent phage types 4, 14 and 26, which express long chain LPS, were assessed for their pathogenicity in mice. Salmonella enteritidis phage type 4 produced a lethal infection in BALB/c mice, but not in C3H/HeJ or Quackenbush (outbred) strains. Phage types 14 and 26 did not produce an obvious infection in any mice, suggesting Australian strains of phage type 4 are more virulent than phage types 14 and 26.     

Lipid A, the active part of bacterial endotoxins in inducing serum colony stimulating activity and proliferation of splenic granulocyte/macrophage progenitor cells.

An analysis of which component of lipopolysaccharides (LPS), the lipid or the polysaccharide (PS), is active in stimulating the murine granulopoietic system has been performed. LPS with different structures, isolated from different mutant strains of Salmonella and chemical degradation products of lipopolysaccharides have been used. Lipid A obtained by acid hydrolysys of the LPS and complexed to bovine serum albumin (BSA) (lipid A-BSA) was shown to be active in generating serum colony stimulating factor (CSF) and in increasing the splenic colony forming cells (CFC) levels, although it was less active than the parent LPS. The polysaccharide (PS) showed no significant activity at the concentrations used. LPS (glycolipids) from R mutants of Salmonella minnesota were active to the same extent as the LPS. The fact that even the most defective LPS from the R mutant R595 which contains lipid A and KDO only is a potent endotoxin, points unequivocally, to lipid A, as the active principle in stimulating the granulopoietic system.     

Variation in Salmonella core lipopolysaccharide as detected by the monoclonal antibody M105

L.P. MANSFIELD, E. BILLETT, E. OLSEN AND S.J. FORSYTHE. 1996. The lipopolysaccharide antigenicity of 22 Salmonella strains (representing nine serogroups) and four non-salmonellae Enterobacteriaceae to the Salmonella genus specific monoclonal antibody M105 was analysed. The monoclonal antibody M105 reacted with all 22 Salmonella strains. Probing SDS-PAGE separated LPS molecules with MAb M105 revealed that the antibody reacted with the core region of all Salmonella serovars. However, no reaction was obtained to the long-chain LPS of serovars O ( Salm. adelaide and Salm. ealing ), C₁ ( Salm. infantis, Salm. livingstone and Salm. virchow ) or Salm. arizonae . It is plausible that the presence of a second core antigenic type results in the lack of reaction between long-chain LPS and the Salmonella genus specific monoclonal antibody M105.     

Properties of monoclonal antibodies to the genus-specific antigen of Chlamydia and their use for antigen detection by reverse passive haemagglutination

The chlamydial genus-specific antigen was extracted with phenol/chloroform/petroleum ether (PCP) from preparations of Chlamydia trachomatis and C. psittaci, and quantities measured using an assay for lipopolysaccharide (LPS). The LPS from C. trachomatis contained 2.2% (w/w) of ketodeoxyoctanoic acid. Five IgG monoclonal antibodies reacted in an ELISA with LPS from both species, the antigen being periodate-sensitive and heat-resistant, confirming that all antibodies were against the genus-specific antigen. All the antibodies bound to the PCP extract of C. trachomatis on an immunoblot, at a position corresponding to the periodate-Schiff-stained bands of both C. trachomatis extract and Salmonella Re-LPS. When linked to trypsin-treated sheep erthrocytes and used in reverse passive haemagglutination tests, all antibodies gave indicator cells capable of detecting chlamydial LPS or crude preparations of chlamydiae grown in McCoy cells, the sensitivity varying with the antibody used. The antibodies varied in IgG subclass (either IgG2a or IgG3), and in ability to precipitate in immunodiffusion tests. Two antibodies cross-reacted with one strain of Acinetobacter in ELISA and with Salmonella Re-LPS in both ELISA and immunodiffusion tests. The other three did not react in ELISA with Acinetobacter strains or Salmonella Re-LPS, and none of the five reacted with LPS of E. coli or Pseudomonas morsprunorum.     

Control of lipopolysaccharide biosynthesis and release by Escherichia coli and Salmonella typhimurium.

The influence of the relA gene on lipopolysaccharide (LPS) biosynthesis and release by Escherichia coli and Salmonella typhimurium was investigated. Similar results were obtained with both species. The incorporation of [3H]galactose into LPS by galE mutants was inhibited by at least 50% (as compared with normal growing controls) during amino acid deprivation of relA+ strains. This inhibition could be prevented by the treatment of the amino acid-deprived relA+ bacteria with chloramphenicol, a known antagonist of the stringent control mechanism. Furthermore, LPS biosynthesis was not inhibited during amino acid deprivation of isogenic relA mutant strains. These results indicate that LPS synthesis is regulated by the stringent control mechanism. Normal growing cells of both relA+ and relA strains released LPS into the culture fluid at low rates. Amino acid deprivation stimulated the rate of LPS release by relA mutants but not by relA+ bacteria. Chloramphenicol treatment markedly stimulated the release of cell-bound LPS by amino acid-deprived relA+ cells. Thus, a low rate of LPS release was characteristic of normal growth and could be increased in nongrowing cells by relaxing the control of LPS synthesis.     

应用MUCAP试剂快速检测沙门氏菌

报告了用4-甲基伞形酮辛酯(4-Methylumbelliferyl-caprylate, MUCAP)快速检测沙门氏菌的特异性、敏感性和实用性。经HE,DHL,SS和麦康凯琼脂平板分离的65株沙门氏菌标准菌株和48株从食品中分离的沙门氏菌,用MUCAP测试均呈阳性反应;394株非沙门氏菌中呈阳性反应的假单胞菌、气单胞菌、邻单胞菌可通过氧化酶试验与沙门氏菌区分开;与粘质沙雷氏菌的交叉反应改用加1％蔗糖的分离平板也可排除。此方法的敏感性和特异性均达到97％以上,而且操作简便、快速,数分钟内即可完成。     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

Abstract We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21–28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.