John L. Penner

Serotyping of Campylobacter jejuni and Campylobacter coli on the basis of thermostable antigens.

Hippurate hydrolysis tests performed on the serotype reference strains of the serotyping scheme based on thermostable antigens under development forCampylobacter jejuni showed that 42 strains wereCampylobacter jejuni and 17 wereCampylobacter coli. Moreover, only four (0.2 %) of 2025 hippurate positiveCampylobacter jejuni isolates reacted inCampylobacter coli antisera and 12 (4.3 %) of the 282Campylobacter coli reacted inCampylobacter jejuni antisera. Evidently each species has its own array of antigenic specificities. Separate schemes for serotypingCampylobacter jejuni andCampylobacter coli are advocated.

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 Jordans 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.

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].

Cloning and sequence analysis of the flagellin gene ofCampylobacter jejuni TGH9011

Flagella are essential for motility and have been implicated to be one of the pathogenic determinants. The flagellum ofCampylobacter jejuni is a polymeric structure of a 62-kd protein. Using a high-affinity flagellin antibody to screen a lambda gt 11 phage genomic expression library ofC. jejuni strain TGH9011 (Serotype LIO36), a recombinant phage clone lambda gt 11RK that expresses theC. jejuni flagellin protein was isolated. The recombinant lambda gt 11 RK produced a 56-kd protein upon induction with isopropylthiogalactoside, which reacted specifically with anti-flagellin antibody. The flagellin gene was sequenced, and comparative analysis of the nucleotide and amino acid sequence identified a region of the flagellin that shows hypervariability among differentCampylobacter species and strains.

Clinical and serological manifestations in patients during a waterborne epidemic due to Campylobacter jejuni

A clinical and serological investigation of an epidemic due to Campylobacter jejuni in a community with a population of 1026 is presented. Altogether, 22 faecal samples from 27 patients were positive, with serotypes O 2 (n = 21) and O 6, 7 (n = 1) being identified. Serotype O 19, 21 was isolated from drinking water which had been consumed by 89.5% households answering a questionnaire, thereby indicating an attack rate of 66.5% (i.e. 680 persons). Mean duration of illness was 6.5 +/- 4.6 days. Diarrhoea (82.3%), abdominal pains (62.8%) and fever (41.8%) were the most common symptoms. Acute stage samples of serum from Campylobacter-positive patients had lower concentrations of IgG antibodies against the most common serotype (O 2) than against serotype O 6, 7 (P = 0.05), which had previously been implicated in epidemics in the region. More than 80% samples drawn after 1-2 weeks of illness were positive for either IgA, IgM or IgG antibodies to serotype O 2 with a dominance of IgA. In the convalescent group (n = 24), serum from only one patient who developed a long-lasting reactive arthritis had antibodies to all serotypes.

Distribution of sero- biotypes of Campylobacter jejuni and C. coli isolated from paediatric patients

During a one-year period, 258 isolates of Campylobacter jejuni and C. coli were obtained from children with gastroenteritis or bacteraemia at the Red Cross Childrens Hospital, Cape Town, South Africa. These isolates were biotyped by hippurate hydrolysis, H2S production and tolerance to 2,3,5-triphenyltetrazolium chloride (TTC). Our study indicated that 95.4% of the isolates were C. jejuni biotype 1, 1.5% were C. jejuni biotype 2 and 3.1% were C. coli; 70% of the isolates were resistant to TTC. Serotyping on the basis of soluble, thermostable antigens detected by a passive-haemagglutination technique revealed that 79% of the Cape Town isolates were typable and that the most common serotypes, in order, were: 4, 2, 12, 23/36 and 19, together comprising 25% of the isolates. About 37% of the typable isolates belonged to nine serotypes. The finding that 21% of the isolates were non-typable suggests the existence of antigenic specificities different from those defined by the 60 antisera in current use.

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]

Development of resistance to macrolide antibiotics in an AIDS patient treated with clarithromycin forCampylobacter jejuni diarrhea

In an AIDS patient with diarrhea, identical isolates ofCampylobacter 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 byCampylobacter jejuni in a patient treated with a macrolide forCampylobacter jejuni infection.

Cloning and expression of the Campylobacter jejuni glyA gene in Escherichia coli

Genetic studies of Campylobacter jejuni are greatly hampered by the lack of genetic markers and an established classical gene transfer mechanism between strains of this species. To facilitate future genetic studies and to provide a recombinant DNA approach for analyzing genes of C. jejuni, we constructed an extensive genomic library of a pathogenic C. jejuni strain TGH9011 (serotype 0:3) using pBR322. We report the isolation of a number of recombinant plasmids containing the complete structural gene of glyA, that encodes serine hydroxymethyltransferase (SHMT) of C. jejuni. Escherichia coli cells containing this multicopy recombinant plasmid with the glyA gene produce high levels of SHMT. The SHMT-encoding fragment was identified by subcloning and functional complementation. The expression of the C. jejuni glyA gene was probably via transcription initiated from its own promoter.