A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria

SDS-polyacrylamide gel electrophoresis is a useful technique in bacterial differentiation and identification. A rapid, semi-automated SDS-PAGE system (Phast System) was assessed for identification of formate-fumarate-requiring, asaccharolytic, Gram-negative oral anaerobes. The system permitted loading, separation and staining of gels within 2 h. Percentage similarities between strains were determined using correlation coefficients and cluster analysis. The protein profiles were sufficiently reproducible provided that distorted profiles were disregarded. Strains were successfully separated into their species, with the exception of Bacteroides ureolyticus NCTC 10939, which appeared to be distinct from other strains of that species. Twenty-nine unidentified formate-fumarate-requiring, sub-gingival plaque strains were suitably clustered with the standard strains as verified by a series of physiological and biochemical tests.     

A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria

SDS-polyacrylamide gel electrophoresis is a useful technique in bacterial differentiation and identification. A rapid, semi-automated SDS-PAGE system (Phast System) was assessed for identification of formate-fumarate-requiring, asaccharolytic, Gram-negative oral anaerobes. The system permitted loading, separation and staining of gels within 2 h. Percentage similarities between strains were determined using correlation coefficients and cluster analysis. The protein profiles were sufficiently reproducible provided that distorted profiles were disregarded. Strains were successfully separated into their species, with the exception of Bacteroides ureolyticus NCTC 10939, which appeared to be distinct from other strains of that species. Twenty-nine unidentified formate-fumarate-requiring, sub-gingival plaque strains were suitably clustered with the standard strains as verified by a series of physiological and biochemical tests. and accepted 25 July 1989     

Characterization of Haemophilus parainfluenzae strains with low-Mr or ladder-like lipopolysaccharides

Twenty-five Haemophilus parainfluenzae strains were characterized for lipopolysaccharide (LPS) profiles, outer membrane protein profiles, serum sensitivity, plasmid profiles and DNA homology. Seventeen strains produced low-Mr LPS that did not contain O-sidechains, while the remaining eight strains contained ladder-like LPS suggestive of O-repeated units. This is the first time in the genus Haemophilus that LPS with O-repeated groups has been described. The strains producing the different types of LPS could not be distinguished from each other in outer membrane protein profiles or the other characteristics examined.     

A numerical taxonomic study of anaerobic gram-negative bacilli classified as Bacteroides ureolyticus isolated from patients with non-gonococcal urethritis

A numerical taxonomic study of 64 strains of anaerobic Gram-negative bacilli isolated from men with non-gonococcal urethritis, two unclassified laboratory strains of 'corroding bacilli', and 12 other strains of anaerobic Gram-negative bacilli, including nine received as anaerobic curved rods and three as 'Bacteroides corrodens' (B. ureolyticus), isolated from women with bacterial vaginosis, was undertaken. Seventeen reference anaerobic strains belonging to the genera Bacteroides, Fusobacterium, Mobiluncus, Mitsuokella and Wolinella were included. Morphological, biochemical and physiological characteristics were examined in 103 tests. The resemblance between the 95 strains was calculated using the SSM, SJ and DP coefficients for cluster analyses based on the UPGMA method. All three approaches gave similar groupings, and the estimated average probability of test error was 2.46%. The strains fell into 10 phenons. The unclassified strains from men and three from women with lower genital-tract infections, and the laboratory strains of 'corroding bacilli' clustered in one phenon with the reference strains of B. ureolyticus, indicating that they correspond to B. ureolyticus. The other unclassified strains of anaerobic curved rods clustered as a distinct phenon. They correspond to species of the newly described genus Mobiluncus. The taxonomic data and the compilation of diagnostic tables serve as a useful guide for the laboratory identification of clinical isolates regarded as B. ureolyticus.     

A rapid method for preparation of rough and smooth lipopolysaccharide from Bacteroides, Porphyromonas and Prevotella

Abstract We describe a new method for lipopolysaccharide (LPS) preparation by water extraction at 100°C and subsequent digestion with proteinase K. The crude LPS could be reliably used for immunoblotting since it retained a high level of antigenicity, and was free of SDS and proteinase K, both of which can cause problems. Two monoclonal antibodies which failed to react with LPS prepared by two conventional methods reacted well with our preparation. We used the new method to prepare LPS from 44 strains of bacteria formerly classified as Bacteroides , some of which have been reclassified as Porphyromonas or Prevotella . In general, yields were good, and electrophoretic profiles obtained with SDS-PAGE and silver staining enabled strains to be rated rough, semi-rough, or smooth.     

Studies on the pathophysiological mechanism of Campylobacter jejuni-induced fluid secretion in rat ileum

Abstract Lipopolysaccharide (LPS) patterns were obtained from fast-growing Rhizobium strains after silver staining of proteinase K treated cells lysates, run in SDS-PAGE. The rhizobia came from root nodules of Acacia senegal and Prosopis chilensis , collected in differents part of the Sudan. The LPS profiles of all strains were typical of rhizobia. Two different LPS region with lower and higher electrophoretic mobility (region I and region II, respectively) coulld be dinguished in the gels, and based on the profiles the strains were divided into three groups. Strains isolated from A. senegal showed a wider range of different profiles than strains isolated from P. chilensis , even though the plants belong to the same cross-infection group. Otherwise there was no clear correlation between the taxonomic relatedness or site of isolation of the strains and their LPS profiles.     

Heterogeneity in Expression of Lipopolysaccharide and Major Outer-Membrane Proteins by Strains of Escherichia coli O157 with Different H-Serotypyes

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.     

Production of novel proteins by Bacteroides ureolyticus grown under conditions of reduced iron availability

Protein profiles of whole cells of Bacteriodes ureolyticus grown in the presence or absence of the iron chelator desferrioxamine mesylate (Desferal) were compared. Each of four strains produced novel proteins of molecular weights 19, 25 and 41 kilodaltons (kDa) under conditions of reduced iron availability. Novel proteins of molecular weights 32, 52 and 58 kDa were also detected although there was interstrain variation in their expression. Outer membranes from three of the strains grown on iron-depleted medium also contained novel proteins with molecular weights of approximately 25, 41 and 52 kDa. When organisms were grown on medium containing Desferal saturated with excess iron, the novel proteins were not detected indicating that their expression was regulated by the level of available iron in the medium.     

Staphylococcus capitis subsp. ureolyticus subsp. nov. from human skin

A new subspecies, Staphylococcus capitis subsp. ureolyticus, was isolated from human skin and is described on the basis of studies of 15 to 26 strains. DNA-DNA reassociation reactions demonstrated that these strains were closely related to Staphylococcus capitis but were significantly divergent. The strains of S. capitis subsp. ureolyticus can be distinguished from S. capitis by their positive urease activity, their ability to produce acid from maltose under aerobic conditions, their fatty acid profile, and their colony morphology. The type strain of the new subspecies is strain ATCC 49326.     

Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.     

Lipopolysaccharide heterogeneity in strains ofSerratia marcescens agglutinated by O14 antiserum

Lipopolysaccharides (LPS) from 71 strains ofSerratia marcescens that were agglutinated by O14 antiserum were examined by SDS-PAGE. Four major profiles were found, designated LPS1 to LPS4. These groups accounted for 51, 7, 5, and 3 strains respectively. Five strains were unclassified. Immunoblotting showed that O14 antibodies bound only to LPS1 and not to LPS2, 3, or 4. LPS1 also bound antibodies in O1, O4, O12, and O23 antisera. LPS2 reacted specifically with O8 antiserum, LPS3 with O6, and LPS4 with O2, O3, O6, O12, and O21 antisera. These reactions were not found in agglutination tests with boiled, whole-cell antigens. However, tests with autoclaved antigens (45 min at 121°C) corroborated the immunoblotting classifications; LPS1 strains belonged to serotype O14, LPS2 to serotype O8, LPS3 to serotype O6, and LPS4 to serotype O21. We conclude that there is a heat-stable antigen on many clinical strains ofS. marcescens that masks the expression of O-specific LPS antigens and which binds with nonspecific antibody in serum O14. We propose that O-antigens should be prepared from autoclaved cultures and that the H-reference strain O14H9 CDC 1783-57 (LPS2) should be reclassified as serotype O8.     

A note on the effect of the lactoperoxidase systems on salmonellas in vitro and in vivo

The lactoperoxidase system (LPS), a natural bactericidal system in milk, was investigated for its activity against salmonellas in vivo and in vitro. In acidified raw milk, in which the LPS was supplemented with an exogenous supply of H2O2, the numbers of salmonellas decreased rapidly. Different salmonella serotypes were affected to the same extent; rough strains, however, were more susceptible than smooth strains. When calves were fed on fresh milk, containing the LPS, and challenged with Salmonella typhimurium in doses of either 10(9) or 10(10), the clinical findings and salmonella excretion patterns were similar to those of control calves fed on heated milk. It was concluded that further studies, perhaps in the field, are necessary to evaluate LPS as a possible non-antibiotic system to control salmonellosis.     

The description of strains of anaerobic 'corroding' organisms isolated from soft tissue infections in cats and dogs

Thirty-eight strains of Gram negative anaerobic 'corroding' rods were isolated from subcutaneous abscesses and pyothorax in cats and dogs. These organisms all exhibited the colony morphology and biochemical properties of organisms described as Bacteroides ureolyticus (Jackson & Goodman 1978) except that they did not hydrolyse urea and they had a guanine plus cytosine ratio of 39–44 mol%. As described for Bact. ureolyticus , 11 of the strains were non-motile. The remaining 27 strains were motile and exhibited a single polar flagellum when examined by negative staining with phosphotungstic acid in an electron microscope. The motile organisms resembled the so-called 'anaerobic vibrios' or Vibrio succinogenes except that these organisms are curved do not liquefy gelatin and are thought by some to have a G + C ratio of 49 mol%. The non-motile strains fitted no group described previously.     

Phenotypic heterogeneity of Pseudomonas corrugata strains from southern Italy

Fifty-five strains of Pseudomonas corrugata isolated in southern Italy were characterized phenotypically and compared with 23 strains of different origins. At least two main cultural types with rough or smooth colonies were observed. Strains with rough colonies produced a diffusible pigment in culture. On the basis of their nutritional profiles, Ps. corrugata strains formed a distinct phenon most closely related to fluorescent Pseudomonas spp. isolated from tomato pith necrosis-diseased plants. Three major groups of strains were differentiated within the Ps. corrugata phenon on the basis of utilization of 2-ketogluconate, meso-tartrate, hystamine, DL- glycerate and induction of a hypersensitive reaction on tobacco. Some Ps. corrugata strains belonging to group 1 and 3 which did not produce pigment in culture produced IAA in a colorimetric test. Variability in the serological reaction of the Italian strain was observed. None of the three antisera utilized reacted with all strains. Some strains isolated from diseased plants from the same greenhouse showed different nutritional profiles and reacted with different antisera. Fifteen lipopolysaccharide (LPS) patterns were observed. Strains were divided into two groups on the basis of their protein profiles. The heterogeneity which had already been observed in a world-wide study on Ps. corrugata was confirmed in strains from this restricted area.     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

Associations of Escherichia coli K-12 OmpF trimers with rough and smooth lipopolysaccharides.

The associations of both rough and smooth lipopolysaccharides (LPS) with the OmpF porin of Escherichia coli K-12 were examined in galE strains deleted for ompC. Transformation with pSS37 and growth with galactose conferred the ability to assemble a Shigella dysenteriae O antigen onto the core oligosaccharide of E. coli K-12 LPS. The association of LPS with OmpF trimers was assessed by staining, autoradiography of LPS specifically labeled with [1-14C]galactose, and Western immunoblotting with a monoclonal antibody specific for OmpF trimers. These techniques revealed that the migration distances and multiple banding patterns of OmpF porin trimers in sodium dodecyl sulfate-polyacrylamide gels were dictated by the chemotype of associated LPS. Expression of smooth LPS caused almost all of the trimeric OmpF to run in gels with a slower mobility than trimers from rough strains. The LPS associated with trimers from a smooth strain differed from the bulk-phase LPS by consisting almost exclusively of molecules with O antigen.     

Detection of plasmids in strains of Bacteroides ureolyticus isolated from the genital tract

Plasmids were detected in 17 of 24 clinical isolates of Bacteroides ureolyticus. All 17 strains harboured a single large plasmid of ≥24·5 MD but two also had smaller plasmids of 2·3 and 3·9 MD, respectively. The function of the plasmids is unknown.     

Serological Characterization of Actinobacillus pleuropneumoniae Biotype 1 Strains Antigenically Related to both Serotypes 2 and 7

Nine Danish Actinobacillus pleuropneumoniae biotype 1 isolates were shown by latex agglutination and indirect haemagglutination to possess capsular polysaccharide epitopes identical to those of serotype 2 strain 1536 (reference strain of serotype 2) and strain 4226 (Danish serotype 2 strain). Immunodiffusion confirmed the antigenic relationship with serotype 2 and further demonstrated an antigenic relationship with strain WF83 (reference strain of serotype 7). SDS-PAGE with LPS from strains 1536, 4226, WF83 and strain 7317 (representative of the 9 isolates examined) showed that strains WF83 and 7317 had an identical smooth ladder pattern whereas LPS from strains 1536 and 4226 showed a distinctly different pattern. The antigenic similarities of the LPS of strains WF83 and 7317 were confirmed by immunoblots using rabbit or pig antisera prepared against the 3 strains. No antigenic similarities in the LPS of strains 1536 and 7317 were revealed. Since an antigenic determinant specific for the 9 isolates could not be demonstrated with the methods used, the strains are proposed to be designated K2:O7.     

Separation and characterization of two chemically distinct lipopolysaccharides in two Pectinatus species.

Lipopolysaccharides (LPS) from the type strains of the anaerobic beer spoilage bacteria Pectinatus cerevisiiphilus and P. frisingensis were extracted with the 5:5:8 volume ratio modification of the phenolchloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982). Sequential precipitations of LPS with water and acetone from the phenol phase yielded LPS which differed in that water-precipitable material (LPS-H2O; 0.1 to 0.4% of the dry weight of the cells) was rough-type LPS, whereas acetone-precipitable material (LPS-Ac; 4.6 to 5.8% of the dry weight) contained both rough-type LPS and high-molecular-weight material resembling smooth LPS. The LPS were chemically characterized, and they contained D-glucosamine, 4-amino-4-deoxy-L-arabinose, 3-deoxy-D-manno-2-octulosonic acid, D-fucose, D-galactose, D-glucose, D-mannose, and phosphate. D-Fucose was present mostly in LPS-Ac, suggesting that it is a constituent of the O antigen. The major fatty acids were ester- and amide-linked (R)-3-hydroxytridecanoic and ester-linked undecanoic acids, with minor amounts of ester-linked tridecanoic and (R)-3-hydroxyundecanoic acids. The chemical compositions of LPS-H2O and LPS-Ac suggested that they differ not only in their smooth or rough nature but also in the structure of their core regions. This may explain their different precipitabilities from the extraction mixture. The extraction method was also shown to be applicable to the isolation of smooth-type LPS from Salmonella enterica serovar Typhimurium. Extraction of two Typhimurium strains carrying chemically different O antigens resulted in high yields (8% of the dry weight) of LPS. Strain SH2183, which contains the relatively hydrophobic O-4,5,12 antigen yielded almost exclusively LPS-Ac, whereas the LPS of strain SH5770, which has a hydrophilic O-6,7 antigen, was exclusively LPS-H2O. No fractionation to smooth and rough LPS occurred with the Typhimurium strains.     

Clinical and veterinary isolates of Salmonella enterica serovar enteritidis defective in lipopolysaccharide O-chain polymerization

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.