M. Costas

Helicobacter pullorum sp. nov.-genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis.

Campylobacter-like organisms were isolated from the liver, duodenum and caecum of broiler and layer chickens, and from humans with gastroenteritis. They formed a unique DNA homology group and a polyphasic taxonomic analysis was made of 16 strains. Analysis of the nucleotide sequence of the 16S rRNA gene from seven of the strains identified them as belonging to a single species, within the genus Helicobacter. This conclusion was supported by the studies of relative DNA homology and of total protein electrophoretic patterns. The new species could be biochemically differentiated from other helicobacters and its ultrastructure in the electron microscope was typical of the genus except that the flagellum was not sheathed. We propose the name Helicobacter pullorum sp. nov. for this group. Like H. fennelliae or H. cinaedi it represents another non-gastric urease-negative Helicobacter species colonizing the lower bowel. Its isolation from the livers of chickens with vibrionic hepatitis is significant. We describe a species-specific PCR assay for H. pullorum sp. nov. which will facilitate its identification and further studies of its epidemiology.

Classification of Campylobacter sputorum and allied campylobacters based on numerical analysis of electrophoretic protein patterns

Summary A total of 28 strains of Campylobacter comprising representatives of C. sputorum subsp. sputorum , C. sputorum subsp. bubulus , C. sputorum subsp. mucosalis , C. concisus , “ C. fecalis ”, “ C. hyointestinalis ” and the CNW Campylobacter group have been characterized by SDS-PAGE of cellular proteins. The protein patterns, which contained 40 to 50 discrete bands, were highly reproducible and were used as the basis of a numerical taxonomic analysis. Eight clusters were obtained at the 55% similarity level and each corresponded to a recognized species or group. The analysis demonstrated unequivocally that C. sputorum subsp. mucosalis constituted a distinct species although the serogroup A strains differed slightly from the serogroup B and C strains. Also, it was shown that C. sputorum subsp. bubulus and “ C. fecalis ” were closely related and should be classified in the same species. As that taxon contained catalase-positive and catalase-negative strains, over-reliance on that test could lead to incorrect identification. Each of the species clusters had distinctive protein band patterns and the main bands provide useful diagnostic markers for the rapid identification of human and animal isolates.

Strain variation in Campylobacter pylori detected by numerical analysis of one-dimensional electrophoretic protein patterns

A total of 21 clinical isolates of Campylobacter pylori from Peru and the United Kingdom and two reference strains (from Australia), including the type strain (NCTC 11637T), were characterized by high resolution one-dimensional SDS-polyacrylamide gel electrophoresis of cellular proteins. The protein patterns contained more than 40 discrete bands and the approximate molecular weights of the major bands were 22, 27, 46, 57, 60, 65 and 93 kD. The total patterns were used as the basis of numerical analysis. Most strains were clustered in four phenons at 91% similarity with the exception of six ungrouped strains. Overall similarity was high with all strains linked in the phenogram at ≥81%. Variation among strains was attributable principally to qualitative and quantitative band differences in the 47 to 56 kD (hypervariable) region of the C. pylori protein profile. From the analysis, ten different electropherotypes (EP-types) were identified. We demonstrated that differences were detectable among isolates from widely separated geographical locations as well as from the same location, although multiple isolates from two Peruvian patients had the same electropherotype. Our results indicate that determination of protein profiles provides the basis of a reproducible method for characterization of C. pylori isolates.

Neisseria weaveri sp. nov. (formerly CDC Group M-5), from Dog Bite Wounds of Humans

The taxonomic relationships of strains belonging to Centers for Disease Control group M-5 were examined. Previous studies of rRNA cistron similarities placed this organism on the Neisseriaceae rRNA branch of rRNA superfamily III; the closest neighbors included the genus Neisseria and groups EF-4a and EF-4b. The group M-5 strains were characterized by a range of phenotypic tests, and their G + C contents and DNA-DNA relatedness levels were determined. In addition, a numerical taxonomic analysis of the whole-cell protein patterns (obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of group M-5 and related taxa was performed. The strains studied included 45 group M-5 strains, the type strains of six Neisseria species or subspecies, three group EF-4a reference strains, and three group EF-4b reference strains plus the type strain of the phenotypically similar organism Oligella urethralis. Our results showed that the group M-5 strains were members of a homogeneous taxon distinct from phylogenetically closely related taxa. The genomic divergence as revealed by levels of rRNA cistron similarity and phenotypic characteristics indicate that group M-5 can be considered a new species of the genus Neisseria. We therefore propose the new species Neisseria weaveri, with NCTC 12742 (= CCUG 4007 = ISL775/91 = LMG 5135) as the type strain. N. weaveri strains are strictly aerobic, gram-negative, nonmotile, rod-shaped organisms which are catalase and oxidase positive, nonsaccharolytic, and able to grow on MacConkey agar and do not reduce nitrate but generally reduce nitrite.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular Techniques for Studying the Epidemiology of Infection by Helicobacter pylori

The efficacy of one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein patterns for fingerprinting isolates of Helicobacter pylori was assessed by means of computerized numerical analysis. Virtually all strains were found to have unique, stable, and reproducible protein profiles. The application of this technique to a collection of isolates from eight patients showed clearly that each harboured a distinct strain that was present before treatment and persisted after treatment. This suggests that relapse was due to recrudescence of the same strain rather than re-infection with a different strain. Minor differences in protein banding profiles within sets of isolates from the same patient were evident, and this was confirmed by means of both two-dimensional PAGE protein patterns and restriction endonuclease analysis of DNA on the same strains.

Isoprenoid quinone composition of representatives of the genus Campylobacter.

The isoprenoid quinone composition of 17 strains representing nine species or sub-species of the genus Campylobacter was investigated. All strains produced similar respiratory quinone patterns consisting of unsaturated menaquinones with six isoprene units and a novel unidentified quinone. Mass spectral analysis indicate the unknown compound has six isoprene units and a formula C42H58O2. The present study indicates respiratory quinones may be useful generic markers for Campylobacter.

Differentiation of Helicobacter Species by Numerical Analysis of their One-Dimensional Electrophoretic Protein Patterns

Summary The suitability of sodium dodecyl sulfate-polyacrylamide gel electrophoretic protein profiles coupled to a computer-assisted numerical analysis for the differentiation of helicobacters was evaluated. Fifty-three strains representing most of the known and available species of Helicobacter and reference strains of allied taxa were included. The results indicate, (i) that the majority of the species examined by this technique could be clearly differentiated, including H. cinaedi and H. fennelliae , which can be difficult to separate by conventional bacteriological methods; two species, H. acinonyx and H. pylori constitute a single phenon at the 69% similarity level and cannot be distinguished at this level, (ii) that gastric helicobacters were more closely clustered to each other than they were to those of enteric origin, (iii) that within several species, different electrophoretic types could be distinguished and that, in the case of the H. pylori/H. acinonyx complex, there appeared to be clustering based on the host source of the strains. Two minor groups (one of human and one of canine origin) were identified and these may merit further phylogenetic analysis.

Evaluation of numerical analysis of SDS-PAGE of protein patterns for typing Enterobacter cloacae.

Twenty cultures comprising 13 clinical isolates of Enterobacter cloacae from two hospitals, the type and another reference stain of E. cloacae and the type strains of four other Enterobacter sp. and of Escherichia coli, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. The protein patterns were highly reproducible and were used as the basis of a numerical analysis which divided the clinical isolates into nine clearly defined protein types. Comparison with established typing methods indicated that the discrimination of SDS-PAGE was similar to that achieved with conventional typing methods and all strain groups recognized by combined sero/phage typing were also found by SDS-PAGE. In addition, protein typing sub-divided a group of four serotype O3 isolates that were difficult to distinguish by phage typing. We conclude that high-resolution SDS-PAGE of proteins provides an effective method of typing isolates of E. cloacae.

Numerical analysis of SDS-PAGE protein patterns of Serratia marcescens: a comparison with other typing methods.

Twenty-five cultures comprising 18 clinical isolates of Serratia marcescens from two hospitals, the type strain of S. marcescens, two reference strains of S. marinorubra, the type or a reference strain of three other Serratia species and a reference strain of undetermined species, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. The protein patterns were highly reproducible and were used as the basis of a numerical analysis which divided the clinical isolates into eight protein types. Comparison with O-serotyping indicated that the level of discrimination by SDS-PAGE was similar. As with O-serotyping, a secondary scheme, such as phage typing, is necessary to differentiate strains of the same protein type. We conclude that high-resolution SDS-PAGE of proteins provides an effective adjunct to other methods for typing isolates of S. marcescens.

Classification and Identification of Campylobacter sputorum using Numerical Analyses of Phenotypic Tests and of One-dimensional Electrophoretic Protein Profiles

Summary The application of numerical analyses of phenotypic tests and sodium-dodecyl sulphate polyacrylamide gel electrophoretic protein profiles in the classification and identification of Campylobacter sputorum was examined. Thirty-eight strains received, or isolated as, representatives of each of the defined biovars of Campylobacter sputorum (i.e. bubulus, fecalis, and sputorum), along with seventeen type or reference strains of other Campylobacter, Arcobacter , and Helicobacter species, were studied. The results from both analyses indicated, (i) that thirty-two strains formed a distinct cluster which included the type strain of C. sputorum , confirming their identity as members of this species, (ii) that catalase-positive strains of C. sputorum are associated with faeces, (iii) that the normal subdivision of catalase-negative C. sputorum strains into two source-specific biovars is invalid. Thus, catalase-positive strains of C. sputorum should remain as biovar fecalis and catalase-negative strains (i.e. biovars sputorum and bubulus) should be unified as a single biovar sputorum and the biovar bubulus discarded. Five oral isolates initially believed to be C. sputorum biovar sputorum were identified as potentially novel campylobacters. Numerical analysis of both types of data used were shown to be effective methods of classifying and identifying campylobacters and allied taxa.