Classification of Proteus vulgaris biogroup 3 with recognition of Proteus hauseri sp. nov., nom. rev. and unnamed Proteus genomospecies 4, 5 and 6.

Strains traditionally identified as Proteus vulgaris formed three biogroups. Biogroup 1, characterized by negative reactions for indole production, salicin fermentation and aesculin hydrolysis, is now known as Proteus penneri. Biogroup 2, characterized by positive reactions for indole, salicin and aesculin, was shown by DNA hybridization (hydroxyapatite method) to be a genetic species separate from biogroup 1 and from biogroup 3 which is positive for indole production and negative for salicin and aesculin. In this study, 52 strains were examined, of which 36 strains were Proteus vulgaris biogroup 3, which included the current type strain of the species P. vulgaris (ATCC 29905T), and compared to seven strains of Proteus vulgaris biogroup 2 and nine type strains of other species in the genera Proteus, Providencia and Morganella. By DNA hybridization, these 36 strains were separated into four distinct groups, designated as Proteus genomospecies 3, 4, 5 and 6. DNAs within each separate Proteus genomospecies were 74-99% related to each other in 60 degrees C hybridization reactions with < or = 4.5% divergence between related sequences. Proteus genomospecies 3 contained the former P. vulgaris type strain and one other strain and was negative in reactions for salicin fermentation, aesculin hydrolysis and deoxyribonuclease, unlike the reactions associated with strains considered as typical P. vulgaris which are positive in reactions for salicin, aesculin and DNase. Genomospecies 3 can be distinguished from Proteus genomospecies 4, 5 and 6 because it is negative for Jordan's tartrate. Proteus genomospecies 4, containing five strains, was differentiated from Proteus penneri, genomospecies 3 and 6 and most, but not all, strains of genomospecies 5, by its ability to ferment L-rhamnose. Proteus genomospecies 5 and 6, containing 18 and 11 strains, respectively, could not be separated from each other by traditional biochemical tests, by carbon source utilization tests or SDS-PAGE of whole-cell proteins. In an earlier publication, a request was made to the Judicial Commission that the former type strain of P. vulgaris (ATCC 13315) be replaced by P. vulgaris biogroup 2 strain ATCC 29905T, a strain considered more biochemically typical of P. vulgaris strains. This would have the effect of assigning the name P. vulgaris to P. vulgaris biogroup 2. Since this request has been acceded to, the name Proteus hauseri is herein proposed for Proteus vulgaris genomospecies 3. Its type strain is ATCC 700826T. Proteus genomospecies 4, 5 and 6 will remain unnamed until better phenotypic differentiation can be accomplished. All Proteus genomospecies were similar in their antimicrobial susceptibility patterns. Nineteen strains were isolated from urine, four from faeces, two from wounds, nine from other human sources and two from animals.

Diversity of lipopolysaccharide structures in Campylobacter jejuni.

Immune blots of electrophoresed lipopolysaccharides extracted from 38 Campylobacter jejuni serostrains suggested the presence of O chains in 16 strains and their absence in 22. Structural analysis confirmed the presence of O chains in serostrains O:19, O:23, and O:36 and the absence of O chains in serostrains O:1, O:2, and O:3. The O:19 strain has O repeat units of beta-D-glucuronic acid amidated with 2-amino-2-deoxyglycerol and N-acetylglucosamine. The 0:36 O chain has four different but closely related repeat units that each consist of N-acetylglucosamine, galactose, and a heptose that is varied in structure from one repeat unit to the next. The O:23 O chain has three different repeat units identical to three of O:36. Except for O:3, core oligosaccharides of strains with or without O chains contain sialic acid (Neu5Ac) in the terminal regions that in many cases mimic the structures of human gangliosides. Three neuropathic isolates were found to have a core terminal trisaccharide (Neu5Ac alpha2-->8Neu5Ac alpha2-->3Galbeta1) that was not found in nonneuropathic strains.

Lipopolysaccharides of Helicobacter pylori serogroups O:3 and O:6--structures of a class of lipopolysaccharides with reference to the location of oligomeric units of D-glycero-alpha-D-manno-heptose residues.

Lipopolysaccharides (LPS) from antigenically different strains assigned to serogroups O:3 and O:6 of Helicobacter pylori were isolated as water-soluble material of high Mr and as water-insoluble gels of low Mr. Chemical and spectroscopic analyses of the soluble LPS and oligosaccharides liberated from the water-insoluble gels led to proposed structures with Lewis (Le) antigen determinants terminating regular repeating units of different types, linked in turn to inner core regions of invariable structure. The O:6 LPS has two populations of related molecules with chains of 3-linked D-glycero-alpha-D-manno-heptose residues similar to those in the MO19 strain, one with and the other without a single terminal Lewis (Le(y)) epitope. In contrast, in the O:3 LPS, Lewis (Le(x) and Le(y)) epitopes terminate a partially fucosylated N-acetyllactosaminoglycan, but a heptan chain similar to that in the O:6 LPS was shown to connect the outer chains to the inner core. These LPS provide examples of the molecular mimicry of cell-surface glycoconjugates. Structural variations of LPS between strains, and differences in some aspects of structure within strains, between high Mr and low Mr LPS indicate a class of LPS whose mechanisms of biosynthesis lead to overall architectures different from those characteristic of most LPS from enteric bacteria.

Lipo-oligosaccharides of Campylobacter jejuni serotype O:10. Structures of core oligosaccharide regions from a bacterial isolate from a patient with the Miller-Fisher syndrome and from the serotype reference strain.

Lipo-oligosaccharide (LOSa) was obtained by phenol-water extraction of bacterial cells of an isolate PG 836, identified as Campylobacter jejuni serotype O:10, from a patient who subsequently developed the Miller-Fisher syndrome (MFS). The product was separated into a water-insoluble gel of low Mr and a water-soluble component of high Mr. The structure of the core oligosaccharide region in LOSa is reported herein for comparison with LOSb from the C. jejuni O:10 reference strain, and is based on investigations carried out on: (1) O-deacylated LOSa; (2) the core oligosaccharide (OS 1a) liberated on acetic acid hydrolysis of the ketosidic linkages to lipid A, with accompanying loss of N-acetylneuraminic acid residues; (3) the product of the removal of phosphate residues from OS 1a to give OS 2a; and (4) the Smith degradation of OS 2a to yield a mixture of Os 3a and OS 4a. The results revealed that the core oligosaccharide region in LOSa from the MFS bacterial isolate had chains (1a), of which some were terminated by an N-acetylneuraminobiose [Neu5Ac(alpha 2-8)Neu5Ac] unit in a GD3 [Neu5Ac-Neu5Ac-Gal] epitope, and the inner regions of which were different from those of other C. jejuni serotypes. Similar experiments on LOSb from bacterial cells of the C. jejuni O:10 reference strain showed that the core oligosaccharide unit [1a, R = P (phosphoric monoester)] of LOSa from the MFS isolate was more uniformly complete than that of the O:10 reference strain [1b, R = AEP (2-aminoethylphosphate)] differing in the nature of the phosphate substituent at the inner heptose residue. The close structural relationship of LOSa from the MFS associated bacterium to LOSb from the O:10 reference strain runs parallel to that of the previously studied Guillain-Barré syndrome (GBS) associated bacterium typed as C. jejuni O:19 in comparison with the lipo-oligosaccharide from the reference strain. Preliminary studies on the high Mr components showed that those from the O:10 strains were indistinguishable from each other, but were structurally unrelated to those from the GBS associated C. jejuni serotype O:19 isolates and the O:19 reference strain [G.O. Aspinall, A.G. McDonald, and H. Pang, Biochemistry, 33 (1994) 250-255].

Typing of Helicobacter pylori with monoclonal antibodies against Lewis antigens in lipopolysaccharide.

Recently, it has been shown that the lipopolysaccharide (LPS) O antigen of Helicobacter pylori contains Lewis x (Lex), Lewis y (Ley), or both Lex and Ley antigens. We applied a serotyping method for H. pylori by an enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) specific for these antigens and the related fucosylated H type 1 (H1) antigen. The selected MAbs recognized the Lex and/or Ley structures in the LPS of H. pylori. The agreement between the results of biochemical compositional analysis and the serological data validated our serotyping system. A total of 152 strains from different geographic origins (The Netherlands, Canada, Poland, Italy, and People's Republic of China) were examined for typeability based on the presence of Lewis antigens. One hundred twenty-nine (84.9%) strains were typeable, and 12 different serotyping patterns were observed; 80.9% of the strains contained Lex and/or Le(y) antigens, and 18.4% reacted with the MAb against the related H1 antigen either alone or in combination with the Lex and/or Ley antigen. Our results show that the Lex and Ley antigens are frequently encountered in the LPS of H. pylori strains from various geographic origins. This typing method is an easy-to-perform technique, which can be used for strain differentiation in epidemiological studies of H. pylori infections.

Miller-Fisher syndrome associated with Campylobacter jejuni bearing lipopolysaccharide molecules that mimic human ganglioside GD3.

A Campylobacter jejuni strain of serotype O:10 was isolated from a patient who had Miller-Fisher syndrome. In its biochemical reactions and cellular morphology, the isolate was characteristic of typical C. jejuni. Antibodies against extracted lipopolysaccharide (LPS) were detected by passive hemagglutination in the acute- and convalescent-phase patient sera. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with the O:10 antiserum, it was demonstrated that the strain possessed both low- and high-molecular-weight molecules. Chemical analysis of the LPS revealed that the core oligosaccharide has a terminal trisaccharide epitope consisting of two molecules of sialic acid linked to galactose, a structure reflecting the terminal region of human ganglioside GD3. As this trisaccharide is also present in LPS cores of serotype O:19 strains from patients with Guillain-Barré syndrome but not in cores of nonneuropathic C. jejuni, a possible role for the trisaccharide in the etiology of neuropathies is indicated, and a difference for distinguishing neuropathic strains from nonneuropathic strains may be the presence of a sialyltransferase required for the synthesis of this trisaccharide.

Lipo-oligosaccharide of Campylobacter lari strain PC 637. Structure of the liberated oligosaccharide and an associated extracellular polysaccharide.

Lipo-oligosaccharide from phenol-water extraction of cells of Campylobacter lari strain PC 637 was separated as a water-insoluble gel of low relative molecular mass (M(r)) from a water-soluble extracellular polysaccharide of high M(r). Structural investigations were performed on the lipo-oligosaccharide and the extracellular polysaccharide, variously using 1H, 13C, and 31P NMR spectroscopy, linkage analysis, and fast atom bombardment-mass spectrometry of permethylated derivatives of the glycans and their products of chemical and enzymic degradation. The following structures are proposed for the highly branched oligosaccharide region: [formula: see text] and for the tetraglycosyl phosphate repeating unit of the extracellular polysaccharide: [formula; see text]

Replacement of NCTC 4175, the current type strain of Proteus vulgaris, with ATCC 29905. Request for an opinion.

The current type strain of Proteus vulgaris, NCTC 4175 (= ATCC 13315), differs substantially from typical strains of this species both biochemically and chemotaxonomically. DNA relatedness studies revealed that strains previously classified as P. vulgaris belong to six genomospecies. One of these genomospecies contains strains that are negative in indole, salicin, and esculin reactions (biogroup 1) and has been named Proteus penneri. A second genomospecies, which is most frequently isolated from human urine, contains typical P. vulgaris strains that are positive in indole, salicin, and esculin reactions (biogroup 2). The members of the remaining four genomospecies are indole positive and negative in salicin and esculin reactions (biogroup 3). Of 36 biogroup 3 strains studied, only strain NCTC 4175T (T = type strain) and one other strain, CDC 1732-80, belong to genomospecies 3. To retain NCTC 4175 as the type strain of P. vulgaris would restrict this species to these two strains, whose origins are unknown. This would mean that hundreds of strains for which the description of P. vulgaris was written and which have been representatives of this species for the past 50 years would have to be renamed as members of a new species. To prevent this confusion, we request that biogroup 2 reference strain ATCC 29905 (= CDC PR1) replace NCTC 4175 as the type strain of P. vulgaris.

Development of resistance to macrolide antibiotics in an AIDS patient treated with clarithromycin for Campylobacter jejuni diarrhea.

In an AIDS patient with diarrhea, identical isolates of Campylobacter jejuni susceptible and, later, resistant to macrolide antibiotics were isolated from feces before and after treatment with clarithromycin. Results of rRNA gene restriction analysis and serotyping suggest that development of resistance rather than simultaneous infection with a susceptible and a resistant strain was responsible for this phenomenon. This is the first report of in vivo development of resistance by Campylobacter jejuni in a patient treated with a macrolide for Campylobacter jejuni infection.

Lipopolysaccharides from Campylobacter jejuni associated with Guillain-Barré syndrome patients mimic human gangliosides in structure.

Lipopolysaccharides extracted from Campylobacter jejuni serostrains (serotype reference strains) for serotypes O:4 and O:19 were found to have core oligosaccharides with terminal structures resembling human gangliosides GM1 and GD1a. High-molecular-weight molecules that reflected the presence of O chains were shown in immunoblots to be immunologically specific for each serostrain. The O:19 antiserum also reacted strongly with core oligosaccharides of two isolates from patients with Guillain-Barré syndrome (GBS), but the banding patterns and molecular structures were different from those of the O:19 serostrain. A neuraminobiose disaccharide unit is attached to the terminal Gal residue in one isolate, and the other isolate lacked terminal N-acetyl glucosamine and galactose with attached sialic acid so that the sialic acid residues were present in a neuraminobiose unit linked to the only remaining galactose. Analysis of the high-M(r) lipopolysaccharides of the O:19 serostrain and the two isolates from GBS patients revealed the presence of a hyaluronic acid-like polymer with disaccharide-repeating units consisting of beta-D-glucuronic acid amidated with 2-amino-2-deoxyglycerol and N-acetyl glucosamine. The results confirm a potential role for the core oligosaccharides in the etiology of GBS but also suggest that the O-chain polysaccharide may be a contributing factor.

Lipopolysaccharides of Campylobacter jejuni serotype O:19: structures of core oligosaccharide regions from the serostrain and two bacterial isolates from patients with the Guillain-Barré syndrome.

Lipopolysaccharides from phenol-water extraction of cells of Campylobacter jejuni serotype O:19 were separated into a water-soluble gel of low M(r) and a water-soluble component of high M(r). Acetic acid hydrolysis of the ketosidic linkages to lipid A furnished respectively a core oligosaccharide, the structure of which is reported herein, and an O antigenic polysaccharide. Structural investigations were performed on the O-deacetylated lipopolysaccharide of low M(r), the liberated core oligosaccharide and the various products from removal of neuraminic acid and phosphate residues, and from the Smith degradation. It is concluded that the lipopolysaccharide from the serostrain has a core region with two types of closely related oligosaccharide chains showing striking homologies with gangliosides, the first with a single N-acetylneuraminic acid residue in an outer chain resembling GM1 and the second with two N-acetyl-neuraminic acid residues with a terminal region resembling GD1a. Similar experiments were carried out on lipopolysaccharides of low M(r) from bacterial isolates OH 4384 and OH 4382 serotyped as O:19 that had been obtained from two patients who subsequently developed the Guillain-Barré syndrome. The core oligosaccharide region of lipopolysaccharide from the former isolate differed only slightly from that of the serostrain, whereas that from the latter isolate was distinctly shorter.

An antigenic polysaccharide from Campylobacter coli serotype O:30. Structure of a teichoic acid-like antigenic polysaccharide associated with the lipopolysaccharide.

A water-soluble antigenic polysaccharide of high M(r) associated with the lipopolysaccharide has been isolated from phenol-water extraction of cells of Campylobacter coli serotype O:30. The polysaccharide and oligosaccharide degradation products formed on O-dephosphorylation and by periodate oxidation followed by reduction have been investigated by one- and two-dimensional 1H, 13C, and 31P NMR. It is concluded that the antigenic polysaccharide has a teichoic acid-like structure with a poly-Ribitol phosphate, [5-Ribitol-1-P]n, backbone with side chains at O-2 of O-(6-deoxy-beta-D-talo-heptopyranosyl)-(1-->4)-(2-acetylamino-2-deoxy-beta-D- glucopyranosyl) units. The structure is unusual in Gram-negative bacteria and is unique in possessing 6-deoxy-D-talo-heptose as a constituent sugar. Evidence for the relationship of the antigenic polysaccharide to the lipopolysaccharide of low M(r) is discussed.

Chemical structure of the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide.

The complete structure for the core region of Campylobacter jejuni serotype O:2 lipopolysaccharide (LPS) was assigned through studies on derivatives of the liberated oligosaccharide (OS 2) and the intact LPS. Structure determinations were performed using 1H-NMR spectroscopy, methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analysis by gas chromatography/mass spectrometry, Smith degradation, and oxidation with chromium trioxide. It was concluded that complete oligosaccharide chains had the following structure: [formula: see text]

Chemical structures of the core regions of Campylobacter jejuni serotypes O:1, O:4, O:23, and O:36 lipopolysaccharides.

Complete structures, including the location of N-acetylneuraminic acid (Neu5Ac) residues, were assigned for the core regions of Campylobacter jejuni serotypes O:1, O:4, and O:23 and O:36 lipopolysaccharides (LPS). In continuation of earlier studies, structure determinations of liberated oligosaccharides and, where necessary, of intact LPS, were by 1H-NMR spectroscopy, Smith degradation, chromium trioxide and enzymic degradations, in conjunction with methylation studies supported by fast-atom-bombardment mass spectrometry and linkage analyses by gas chromatography/mass spectrometry. It was concluded on the basis of the following structures, in which each was linked 1-->5 to a terminal 3-deoxy-D-manno-octulosonic acid residue, that the core regions with qualitatively similar sugar compositions showed serotypic differences in one or more of their sequences, linkage types, and anomeric configurations: [formula: see text] [corrected]. The outer regions of each structure carry Neu5Ac residues linked 2-->3 to available beta-D-Galp residues and show striking similarities with various glycosphingolipids of the ganglioside family. However, Neu5Ac epitopes are not apparently involved in determining serospecificity.

Lipopolysaccharide of Campylobacter coli serotype O:30. Fractionation and structure of liberated core oligosaccharide.

Lipopolysaccharide from phenol-water extraction of cells of Campylobacter coli serotype O:30 was separated as a water-insoluble gel of low M(r) from a water-soluble antigenic polysaccharide of high M(r). Acetic acid hydrolysis of the ketosidic linkages to lipid A in the lipopolysaccharide furnished a core oligosaccharide. Structural investigations were performed using 1H and 13C NMR, fast atom bombardment-mass spectrometry of permethylated derivatives, and methylation linkage analysis on the core oligosaccharide and the products of two successive Smith degradations. It is concluded that the highly branched 3-deoxy-D-manno-octulosonic acid-terminated oligosaccharide chains carried at the nonreducing end disaccharide units of beta-D-Qui3NAc-(1-->2)-beta-D-Qui3NAc (where Qui3NAc represents 3-acylamino-3,6-dideoxy-D-glucose), in which N-acyl residues were either both (R)-3-hydroxybutanoyl or both 3-hydroxy-2,3-dimethyl-5-oxoprolyl. The demonstration of these unusual features provides further evidence for a wide variety of structures within the core oligosaccharide region of lipopolysaccharides from Campylobacter sp.

Antigenicity of Helicobacter pylori lipopolysaccharides.

An investigation of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of lipopolysaccharides (LPSs) extracted from seven strains of Helicobacter pylori revealed that these molecules were silver stainable and exhibited a high degree of variability in their patterns. Two strains synthesized a variety of sizes of LPS molecules such that fractionation by SDS-PAGE resulted in a stepwise gradation of bands which extended from the top to the bottom of the silver-stained gel. The LPSs from the remaining five strains were made up of molecules which were more homogeneous in size and clustered around two separate areas of the gel. Antigenic analyses of phenol-water-extracted LPSs by immunoblotting and the passive hemagglutination assay suggested that, in addition to strain-specific antigens, all of the LPSs carried a common antigen. Antibodies to this common antigen could be removed from antisera by absorption, and the resulting antisera were used to differentiate strains on the basis of their O antigens by the passive hemagglutination assay technique. The finding that LPSs from 3 of 10 clinical isolates reacted specifically in one or two of the typing antisera suggested that the development of a scheme for differentiating H. pylori on the basis of O antigens is feasible.

Comparison of a blood-free medium and a filtration technique for the isolation of Campylobacter spp. from diarrhoeal stools of hospitalised patients in central Australia.

Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p less than 0.001; chi 2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all "C. upsaliensis" except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.