Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography

By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues. Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S. typhimurium.     

Neutralization of Shwartzman-inducing activity by antibodies recognizing the Re core or lipid A structures of lipopolysaccharide from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477

Antibodies recognizing the Re core or lipid A structures of lipopolysaccharide (LPS) derived from Salmonella minnesota R595 and Pseudomonas vesicularis JCM1477 were tested for the ability to neutralize the preparatory activity of endotoxin using the local Shwartzman reaction. Shwartzman-inducing activity of R595 LPS (Re-form) was strongly suppressed when the LPS was incubated with the rabbit anti-R595 antiserum or the purified IgG antibody which recognizes core region of the LPS. The antiserum also suppressed the preparatory activity of LPS from S. typhimurium SL1102 (Re) and Escherichia coli F515 (Re), but not that of either S. typhimurium LT-2 (S) LPS or R595 lipid A. Moreover, it was found that the murine monoclonal antibody (MAb), SmRe100G (IgG2a) which recognizes the core region of R595 LPS, significantly suppressed the preparatory activity of R595 LPS. Both conventional antibodies specific to R595 lipid A, which contains a 1,4'-bisphosphorylated beta-D-glucosaminyl-alpha-D-glucosamine disaccharide structure, and JCM1477 lipid A, which contains a monophosphorylated 3-amino-D-glucosamine disaccharide structure, neutralized the preparatory activity of homologous and a closely related lipid A, but not that of LPS. In addition, it was observed that MAb Sm5G (IgG2b) specific to enterobacterial lipid A preparations (especially R595 lipid A) neutralized the preparatory activity of R595 lipid A, although the effect was somewhat weak as compared with that of rabbit antiserum. These results suggest that anti-Re LPS antibody binding to the core of Re LPS is involved in suppressing the endotoxic activity of Re LPS, and that the direct binding of anti-lipid A antibody to some specific epitopes of lipid A is important in neutralizing the endotoxic activity.     

Immunoblotting procedure for the analysis of electrophoretically-fractionated bacterial lipopolysaccharide

A procedure is described for the efficient transfer of fractionated bacterial lipopolysaccharide (LPS) from SDS-polyacrylamide gels to nitrocellulose filters, and its subsequent display by a peroxidase-linked antibody. The method is sensitive, and reveals and resolves high molecular weight LPS molecules having side chain lengths of up to and greater than 30 repeat units. It is also useful for the rapid analysis of LPS in bacterial outer membrane preparations.     

Identification of Re lipopolysaccharide-binding protein on murine erythrocyte membrane

Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1% to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPS-responder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.     

The dispersion of gram-negative lipopolysaccharide by deoxycholate. Subunit molecular weight

The lipopolysaccharide (LPS) was isolated from three strains of Salmonella typhimurium, a "smooth" strain, STM 7, the Ra mutant, TV 119, and the Re mutant, SL 1102. The effect of depletion of divalent cations on structure and the effect of deoxycholate on hydrodynamic behavior were studied. The results confirmed previous work by others that divalent cations and hydrophobic forces are important factors influencing LPS size and morphology. The binding of deoxycholate to LPS was measured. When the weight average molecular weights of the deoxycholate-dissociated LPS were determined by sedimentation equilibrium and corrected for bound deoxycholate, the values 5,555, 10,607, and 15,592, respectively, for Re, Ra, and "smooth" LPS were in good agreement with calculated formula weights. Although others have suggested that the basic LPS subunit is a trimer, our results suggest that it exists in the dimeric form.     

Structural and immunochemical studies on the lipopolysaccharide of the ‘T-antigen’-containing mutant Proteus mirabilis R14/1959

Abstract In DOC-PAGE, lipopolysaccharide (LPS) of Proteus mirabilis R14/1959 (Rb-type) mutant showed a ladder-like migration pattern indicating the presence of a high molecular weight polysaccharide chain. The isolated polysaccharide, called T-antigen because of similarity with the T1 chain of Salmonella friedenau LPS, contained d -glucose, d -galacturonic acid ( d -GalA), and d -GlcNAc in molar ratios 2:1:1 and was structurally different from the O-antigen of the parental S-strain P. mirabilis S1959 but identical to the O-antigen of another S-strain Proteus penneri 42. The importance of a d -GalA( l -Lys)-containing epitope, most likely present in the core region of LPS, and of GalA present in the T-antigen chain in manifesting the serological specificity of P. mirabilis R14/1959 were revealed using rabbit polyclonal homologous and heterologous R- and O-specific antisera and the appropriate antigens, including synthetic antigens which represent partial structures of various Proteus LPS.     

The structure and serologic distribution of an extracellular neutral polysaccharide from Pseudomonas aeruginosa immunotype 3

Previous work has described small molecular weight neutral polysaccharides from isolates of Pseudomonas aeruginosa that appear to be associated with the lipopolysaccharide (LPS) and distributed across serologic barriers defined by antibody to the O side chain. We have isolated and characterized another of these structures obtained from culture supernatants of an immunotype 3 strain of P. aeruginosa. The isolated neutral polysaccharide has a tetrasaccharide repeat unit: (formula; see text) where Rha is rhamnose. The structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments, acid hydrolysis, methylation analysis, Smith degradation, and optical rotation determinations. Polyclonal antibodies raised to intact and alkali-treated (0.1 N NaOH, 56 degrees C, 2 h) LPS from the seven Fisher immunotype strains of P. aeruginosa bound well to the neutral polysaccharide. Antibodies affinity purified from these sera using immobilized neutral polysaccharide as well as a neutral polysaccharide-specific monoclonal antibody, E87, reacted with an antigenically similar structure found among many isolates of different LPS serotypes in a colony blot and with LPS from the seven Fisher immunotypes in an immunoblot. In an immunoblot assay, the neutral polysaccharide inhibited binding of the monoclonal antibody, E87, to material present in LPS preparations from a variety of serotypes. This structure may represent another P. aeruginosa neutral polysaccharide variant found associated with the LPS.     

Evaluation of the immunological activity of a vaccine from mutant Salmonella minnesota R 595 on a model of experimental Pseudomonas aeruginosa infection in mice

The comparative study of heated corpuscular vaccines prepared from S. minnesota mutant R 595 with defective lipopolysaccharide (LPS), chemotype Re, derived from S. minnesota strain SF 1111 with unchanged LPS, and from P. aeruginosa strain PA 103, was carried out. In contrast to the vaccine from S. minnesota strain SF 1111, the vaccine prepared from the mutant with chemotype Re induced the development of cell-mediated and humoral immunity to P. aeruginosa, and its immunogenicity was close to that of the vaccine from P. aeruginosa strain PA 103.     

The molecular heterogeneity of Shigella sonnei lipopolysaccharide based on polyacrylamide gel electrophoretic data

The molecular heterogeneity of S. sonnei lipopolysaccharide (LPS), reflecting the size of lateral O-specific polysaccharide chains, has been established by the method of electrophoresis in acrylamide gel in the presence of sodium dodecyl sulfate and urea. The dominating components fall into three types, viz. those with 0-3, 10-16 and 35-40 repeating structures, the remaining components being minor ones. The electrophoretic profile of S. sonnei LPS considerably differs from the profiles of Escherichia coli and S. flexneri LPS, but coincides with the LPS profiles of other strains with different virulence. The preparations of LPS obtained by extraction with trichloroacetic acid have the same electrophoretic profiles as LPS obtained by the method of aqueous phenol extraction. The domination of certain molecular variants reflects, seemingly, specific features of the biosynthesis of LPS, characteristic of a given strain. The mechanisms of the preferable synthesis of lateral O-specific chains of the definite size and the importance of the molecular parameters of lateral chains for the biological properties of LPS require further study.     

Fluorescence resonance energy transfer analysis of lipopolysaccharide in detergent micelles.

Bacterial endotoxins or lipopolysaccharides (LPS), cell wall components of gram-negative bacteria, are involved in septic shock. LPS consists of a lipid A tail attached to core and O-antigen polysaccharides, but little is known about the supramolecular structure of LPS in blood. We have developed an approach to locate donor and acceptor probes in sulfobetaine palmitate detergent micelles using steady-state and time-resolved fluorescence resonance energy transfer. C18-fluorescein and several LPS species of varying molecular weight labeled with fluorescein isothiocyanate (FITC-LPS) were the donor probes. Acceptor probes were 1,1-dilinoleyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (Fast C18-Dil, Ro approximately 68 A), and octadecyl B rhodamine chloride (C18-Rhd, Ro approximately 58 A). With either acceptor, the transfer was of similar high efficiency when FITC-LPS Salmonella minnesota Re 595 (2,500 mol wt, lacking both core and O-antigen) or C18-fluorescein were the fluorescent donor probes. Thus, the donor FITC-LPS with short polysaccharide chain S. minnesota Re 595 and the control donor C18-fluorescein appear to be close to the micelle surface. The transfer efficiency decreased as the molecular weight of the LPS increased. Separation distances between the longest FITC-LPS, S. minnesota (20,000 mol wt, with a long O-antigen), and the micelle were estimated to be 1.5 Ro or more (approximately 100 A), consistent with an extended conformation for the longer O-antigen polysaccharide chain in the detergent.     

Salmonella bacteriophage glycanases: endorhamnosidases of Salmonella typhimurium bacteriophages.

Twelve bacteriphages lysing only smooth Salmonella typhimurium strains were shown to have similar morphology--an icosahedric head to which a short, noncontractile tail carrying six spikes was attached. All phages degraded their lipopolysaccharide (LPS) receptors as shown by their ability to cleave off [14C]galactosyl-containing oligosaccharides from S. typhimurium cells labeled in their LPS. The oligosaccharides inhibited the alpha-D-galactosyl-specific Bandeiraea simplicifolia lectin agglutination of human type B erythrocytes, indicating that all 12 phage glycanases were of endorhamnosidase specificity, i.e., hydrolyzed the alpha-L-rhamnopyranosyl-(1 leads to 3)-D-galactopyranosyl linkage in the S. typhimurium O-polysaccharide chain. Two of the phages, 28B and 36, were studied in more detail. Whereas the phage 28B glycanase hydrolyzed the S. typhimurium LPS into dodeca- and octasaccharides, the phage 36 glycanase in addition cleaved off tetrasaccharides. Both phage enzymes hydrolyzed the O-polysaccharide chains of LPS from Salmonella belonging to serogroups A, B, and D1, which are built up of tetrasaccharide-repeating units identical except for the nature of the 3,6-dideoxyhexopyranosyl group (R). : FORMULA:(SEE TEXT). The phage 28B and 36 endorhamnosidases hydrolyzed also an LPS from which the 3,6-dideoxyhexosyl substituents had previously been hydrolyzed off. However, neither of the enzymes was active on LPS preparations in which the C2-C3 bond of the L-rhamnopyranosyl ring had been opened by periodate oxidation. Glucosylation at O-6 of the D-galactopyranosyl residues in the S. typhimurium LPS was found to be incompatible with hydrolysis by both enzymes. However, in an LPS glucosylated at O-4 of the D-galactopyranosyl residues, the adjacent alpha-L-rhamnopyranosyl linkages were found to be perferentially cleaved.     

Antigenic properties of LPS extracted from Bacteroides fragilis enterotoxin producing strains

Antigenic properties of enterotoxigenic Bacteroides fragilis (ETBF) strains isolated in Poland were compared with reference strains. The agglutination and passive hemagglutination, SDS-PAGE analysis and immunoblotting tests as well as analyses of sugars and fatty acids were performed with lipopolysaccharide (LPS) preparations obtained from water-phase of phenol-water extracts. Some differences in serological reactivity between ETBF antigens were observed. The antigen of the NTBF (nonenterotoxigenic) reference strain IPL E-323 expressed weak cross-reactivity with sera against whole cells of ETBF strains in serological tests. There were some differences observed between ETBF and NTBF strains in fatty acids and sugar composition. The LPS preparations probably possess a common core structure and the O-specific polysaccharides of variable chain length.     

Chemical characterization of lipopolysaccharide from Edwardsiella ictaluri, a fish pathogen

The chemical components of lipopolysaccharide (LPS) from the fish pathogen Edwardsiella ictaluri (Ed. ictaluri) were analyzed by SDS-PAGE, gas chromatography, and spectrophotometry, and compared with those of Salmonella typhimurium and Escherichia coli 0111:B4. Only four to five low molecular weight species of LPS from Ed. ictaluri were detected by silver staining after separation by polyacrylamide gel electrophoresis. The low molecular weight species, as well as a low sugar content, indicate that the LPS from Ed. ictaluri was of the rough type, compared with that of S. typhimurium and E. coli which were both of the smooth type LPS. Quantitatively, mannose was not a major sugar component in Ed. ictaluri, unlike S. typhimurium. Palmitic, palmitoleic, and cis-9,10-methylene-hexadecanoic acids were predominant fatty acids among the total cellular lipids of Ed. ictaluri. C14 fatty acids comprised 78% of the total in the LPS of this bacterium, with beta-hydroxy-myristate representing 55%. The results of this study suggest that the lipid A segment of the LPS molecule of Ed. ictaluri is similar to S. typhimurium and E. coli, at least with respect to fatty acid content; however, the core polysaccharide of E. ictaluri differs in that it has twice the heptose content.     

Relationship between immune system and gram-negative bacteria: monocyte chemotaxis induced by supernatants from human peripheral blood OKT8+ lymphocytes stimulated with smooth and rough Salmonella strains

It has been recently reported that smooth (S) Salmonella typhimurium LT-2 and rough (R) mutants, S. minnesota R345 (Rb) and R595 (Re), spontaneously adhere to human peripheral blood mononuclear cells (PBMC). The binding is confined to T cells and is mediated by the lipopolysaccharide (LPS) moiety of the bacteria outer membrane. In this study, we have investigated the monocyte chemotactic responsiveness induced by supernatants recovered from human PBMC stimulated with either S or R Salmonella strains. All supernatants were able to trigger monocyte chemotaxis, even if to a different degree according to the bacterial strain used. These data were further supported by experiments which showed that stimulation of PBMC by purified homologous LPS led to the release of a chemotactic lymphokine (CLK) for human monocytes. Moreover, this CLK seems to be released by T cells, and in particular OKT8+ cells, since OKT8- PBMC failed to produce CLK.     

内源性硫化氢在脂多糖引起的肺动脉高压中的作用

为观察硫化氢(hydrogen sulfide,H2s)在脂多糖(1ipopolysaccharide,LPS)引起的肺动脉高压中的作用,应用离体血管环张力测定方法测定肺动脉反应性,采用生物化学方法测定肺动脉组织中H2S产出率和胱硫醚-γ-裂解酶(cystathionine γ-lyase,CSE)活性,定量PCR方法测定肺动脉组织中CSE表达水平.结果如下:(1)与对照组相比,LPS可显著升高肺动脉平均压(mean pulmonary arterial pressure,mPAP)[(1.82±0.29)kPa vs(1.43±0.26)kPa,P<0.01],降低肺动脉组织中H2S产出率[(26.33±7.84)vs(42.92±8.73)pmoFg wet tissue per minute,P<0.01]和ACh诱导的肺动脉内皮依赖性舒张反应[(75.72±7.22)%vs(86.40±4.40)%,P<0.01];(2)NariS可部分逆转上述变化,而PPG加剧上述变化;(3)CSE活性和CSE mRNA表达的变化与H2S产出率的变化相同.结果提示,LPS对内皮依赖性舒张反应的抑制导致肺动脉高压的发生,此作用可能与H2S有关.     

Analysis of unmodified endotoxin preparations by 252Cf plasma desorption mass spectrometry. Determination of molecular masses of the constituent native lipopolysaccharides

Nine unmodified endotoxin preparations constituted of Re-, Rd-, and Rc-type lipopolysaccharides (2 to 5 glycoses), representing four species of enterobacteria were analyzed by 252Cf plasma desorption mass spectrometry. The constituent lipopolysaccharides were characterized by the ion pair: (M-H)- and its corresponding lipid fragment ion. The lipid fragment ion is produced by cleavage of the glycosidic bond of the 3-deoxy-D-manno-oct-2-ulosonic acid unit that substitutes O-6' of the glucosamin beta 1'-6glucosamine ("lipid A backbone") disaccharide of the lipid A moiety. These lipid fragment ions were identical to the (M-H)- ions seen in the spectra of homologous isolated lipid A preparations that were obtained by hydrolysis (pH 4.5, 100 degrees C) promoted by sodium dodecyl sulfate. Since the molecular components present in the endotoxin preparations analyzed are known, the ion pair (M-H)(-)-lipid fragment ion defines the molecular compositions of each individual lipopolysaccharide. Heterogeneity of the R-type endotoxin preparations analyzed was due almost exclusively to differing lipid A moieties. In three Salmonella minnesota 595 Re endotoxin preparations 10 different lipopolysaccharides were identified, only two of which were common to all three preparations. Of the nine lipopolysaccharides identified in two S. minnesota R7 endotoxin preparations, only two were present in both.     

Interactions between magainin 2 and Salmonella typhimurium outer membranes: effect of lipopolysaccharide structure

The role of the outer membrane and lipopolysaccharide (LPS) in the interaction between the small cationic antimicrobial peptide magainin 2 and the Gram-negative cell envelope was studied by FT-IR spectroscopy. Magainin 2 alters the thermotropic properties of the outer membrane-peptidoglycan complexes from wild-type Salmonella typhimurium and a series of LPS mutants which display differential susceptibility to the bactericidal activity of cationic antibiotics. These results are correlated with the LPS phosphorylation pattern and charge (characterized by high-resolution 31P NMR) and outer membrane lipid composition, and are compared to the bactericidal susceptibility. LPS mutants show a progressive loss of resistance to killing by magainin 2 as the length of the LPS polysaccharide moiety decreases. Disordering of the outer membrane lipid fatty acyl chains by magainin 2, however, depends primarily upon the magnitude of LPS charge rather than the length of the LPS polysaccharide, contradicting the proposal by Weiss et al. [Weiss, J., Beckerdite-Quagiata, S., & Elsbach, P. (1980) J. Clin. Invest. 65, 619-628] that the sugar side chain of LPS shields the negative charges of the outer membrane surface. While disruption of outer membrane structure most likely is not the primary factor leading to cell death, the susceptibility of Gram-negative cells to magainin 2 is associated with factors that facilitate the transport of the peptide across the outer membrane, such as the magnitude and location of LPS charge, the concentration of LPS in the outer membrane, outer membrane molecular architecture, and the presence or absence of the O-antigen side chain.     

In vitro cytotoxicity of lipopolysaccharides for chinese hamster ovary cells

Abstract From our survey of various lipopolysaccharide (LPS) preparations, we demonstrated that three out of five commercial LPS preparations of Salmonella typhimurium were not cytotoxic for Chinese hamster ovary (CHO) cell monolayers at a concentration of 1000 μg/ml. One commercial LPS preparation produced cellular damage at a concentration of 1000 μg/ml and another at 400 μg/ml. Two S. typhimurium LPS preparations made in our laboratory were also cytotoxic at a concentration of 1000 μg/ml but not at lower concentrations. Cell-free sonic lysates of S. typhimurium TML R66 were cytotoxic when tested undiluted and up to a dilution of 1:20. Based on the 2-Keto-3-deoxyoctonate (KDO) content of all preparations, sonic lysateas were cytotoxic at KDO concentrations of 0.42 μg/ml while the KDO content of the most cytotoxic LPS preparation was 15.2 μg/ml. There was no apparent correlations between KDO content of the LPS preparations and cell detachment, leading to the conclusion that cell detachment activity of Salmonella cell lysates cannot be attributed to their LPS content.     

Protective artificial polysaccharide-protein antigens made from the O-specific polysaccharides of Pseudomonas aeruginosa bacteria of the immunotypes 1, 2 and 7

O-specific polysaccharide (PS) obtained from P. aeruginosa lipopolysaccharide (LPS), immunotypes 1, 2 and 7, were condensed in the presence of aminated bovine serum albumin (amBSA). As a result, artificial polysaccharide-protein antigens were synthesized and their protective properties were studied by the subcutaneous immunization of mice with their subsequent challenge with the cells of the homologous strain injected intraperitoneally in a dose of 3-5 LD50. The most effective immunization was achieved by using homologous LPS, taken in a subimmunogenic dose, as adjuvant. Heterologous LPS, taken in the same doses, showed no stimulating effect. The protective properties of the conjugates were also found to depend on the length of the PS chain. The conjugate of amBSA and PS of immunotype 2 with a molecular weight of about 10 KD was found to possess the highest protective potency. The decrease of the molecular weight of PS of immunotype 7 from 50 KD to 10 KD by selective acid hydrolysis led to obtaining the conjugate with high protective potency. The possibility of using the structurally linked pair antigen-adjuvant for the development of artificial vaccines is discussed.     

Lipopolysaccharide O-chain microheterogeneity of Salmonella serotypes Enteritidis and Typhimurium

Variability in the lipopolysaccharide (LPS) of the two most prevalent Salmonella serotypes causing food-borne salmonellosis was assessed using gas chromatography analysis of neutral sugars from 43 Salmonella enterica serovar Enteritidis ( S . Enteritidis) and 20 Salmonella enterica serovar Typhimurium ( S . Typhimurium) isolates . Four substantially different types of O-chain chemotypes were detected using cluster analysis of sugar compositions; these were low-molecular-mass (LMM) LPS, glucosylated LMM LPS, high-molecular-mass (HMM) LPS and glucosylated HMM LPS. Nineteen out of 20 S . Typhimurium isolates yielded glucosylated LMM . In contrast, S . Enteritidis produced a more diverse structure, which varied according to the source and history of the isolate: 45.5% of egg isolates yielded glucosylated HMM LPS; 100% of stored strains lacked glucosylation but retained chain length in some cases; and 83.3% of fresh isolates from the naturally infected house mouse Mus musculus produced glucosylated LMM LPS. A chain length determinant ( wzz ) mutant of S . Enteritidis produced a structure similar to that of S . Typhimurium and was used to define what constituted significant differences in structure using cluster analysis. Fine mapping of the S . Enteritidis chromosome by means of a two-restriction enzyme-ribotyping technique suggested that mouse isolates producing glucosylated LMM LPS were closely related to orally invasive strains obtained from eggs, and that stored strains were accumulating genetic changes that correlated with suppression of LPS O-chain glucosylation. These results suggest that the determination of LPS chemotype is a useful tool for epidemiological monitoring of S . Enteritidis , which displays an unusual degree of diversity in its LPS O-chain.