Distinction of gastric Helicobacter spp. in humans and domestic pets by scanning electron microscopy

Background. A number of different Helicobacter spp. can colonize the stomach of humans and domestic pets. Difficulties encountered with primary isolation of these spiral microorganisms and their unusual inertia with respect to biochemical reactions still represent considerable obstacles to their characterization with classic tools. In addition, the high degree of similarity in the 16S rRNA sequence hampers differentiation of Helicobacter spp. using routine molecular biological assays.
Materials and Methods. Samples from experimentally monoinfected mice, of naturally infected hosts, and of cultured strains were examined by scanning electron microscopy (SEM). In parallel, all samples were analyzed by molecular techniques to ascertain the Helicobacter spp. involved.
Results. Using the mouse samples as a reference, microorganisms found in naturally infected hosts were identified by SEM as belonging to H. pylori , H. felis , or a group consisting of H. bizzozeronii and H. heilmannii. A further spiral microorganism with unique morphology was found in a dog that was positive for H. salomonis , but the organism could not be recovered from experimentally infected mice. In culture, most Helicobacter strains lost their ultrastructural characteristics.
Conclusions. When gastric Helicobacter spp. were collected from their natural habitat and examined by SEM, relevant differences could be detected between H. felis , H. bizzozeronii and H. heilmannii , and H. salomonis , respectively. SEM, therefore, seems to be a useful auxillary tool for the distinction of various gastric Helicobacter spp. as based on their ultrastructure.     

Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni

AIMS: To develop a method for assessing the relative epidemiological significance of possible infection sources for human campylobacteriosis. METHODS AND RESULTS: Using fluorescent amplified fragment length polymorphism (AFLP), 243 apparently epidemiologically unrelated Campylobacter jejuni isolates were genotyped (77 human, 46 cattle, 49 pet and 71 poultry isolates). In total 136 different phena were identified, of which 48 were clusters grouping at least two isolates. Isolates from different sources were frequently clustered together, underlining the high degree of source mixing and the lack of host specificity of C. jejuni. The phena were classified into different phenon types according to the sources of the isolates they contained. The occurrence of these phenon types was analysed using an area-proportional Euler diagram to describe epidemiological relatedness among C. jejuni isolates. Group separation statistics revealed that 43% of analysed human isolates expressed maximum similarity to other human isolates, 9% to cattle isolates, 21% to pet isolates and 27% to poultry isolates; these results were in accordance with the pattern observed in the phenon cluster analysis. CONCLUSIONS: Based on the grouping of strains into molecular similarity clusters, ecological patterns between sources can be investigated. Significance and IMPACT OF THE STUDY: This approach is a new methodological contribution to establish the relative epidemiological significance of concurrent infection sources.     

Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes.

The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif. Most of its members were shown to have cytolytic activity. By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species. The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica. A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes. The probes detected all known genes for RTX toxins. Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet. This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria. The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step. This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested. The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications. Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance.     

Campylobacter helveticus sp. nov., a new thermophilic species from domestic animals: characterization, and cloning of a species-specific DNA probe.

An atypical group of thermophilic catalase-negative Campylobacter strains, the 'CH' (Swiss) group, can be recovered from faeces of domestic cats and dogs after selection by filtration, or with the antibiotic cefoperazone. This group of strains shows no relative DNA homology with any species in rRNA superfamily VI (Vandamme et al., 1991, International Journal of Systematic Bacteriology 41, 88-103) except with four thermophilic Campylobacter species, notably C. upsaliensis. The group is homogeneous and possesses a DNA base composition, cellular morphology at the electron microscope level and phenotypic properties characteristic of Campylobacter. Nonetheless it is distinct from known species of Campylobacter in terms of conventional bacteriological tests, total cellular protein profile, rRNA gene profile, and genomic DNA homology. On the basis of an integrated study of phenotype and genotype, we conclude that these bacteria constitute a previously undescribed species for which we propose the name Campylobacter helveticus sp. nov. A species-specific recombinant DNA probe was cloned from the designated type strain (NCTC 12470) for use in identification and further analysis of the epidemiology, pathogenicity and transmission of C. helveticus.     

Cloning and characterization of two bistructural S-layer-RTX proteins from Campylobacter rectus

Campylobacter rectus is an important periodontal pathogen in humans. A surface-layer (S-layer) protein and a cytotoxic activity have been characterized and are thought to be its major virulence factors. The cytotoxic activity was suggested to be due to a pore-forming protein toxin belonging to the RTX (repeats in the structural toxins) family. In the present work, two closely related genes, csxA and csxB (for C. rectus S-layer and RTX protein) were cloned from C. rectus and characterized. The Csx proteins appear to be bifunctional and possess two structurally different domains. The N-terminal part shows similarity with S-layer protein, especially SapA and SapB of C. fetus and Crs of C. rectus. The C-terminal part comprising most of CsxA and CsxB is a domain with 48 and 59 glycine-rich canonical nonapeptide repeats, respectively, arranged in three blocks. Purified recombinant Csx peptides bind Ca2+. These are characteristic traits of RTX toxin proteins. The S-layer and RTX domains of Csx are separated by a proline-rich stretch of 48 amino acids. All C. rectus isolates studied contained copies of either the csxA or csxB gene or both; csx genes were absent from all other Campylobacter and Helicobacter species examined. Serum of a patient with acute gingivitis showed a strong reaction to recombinant Csx protein on immunoblots.     

Discrimination of Helicobacter pullorum and Campylobacter lari by analysis of whole cell fatty acid extracts

Helicobacter pullorum and Campylobacter lari are rarely isolated from humans with acute enteritis. Hitherto the two species could only be identified by genotypic techniques. Gas liquid chromatography of whole cell fatty acid extracts is described as the first phenotypic method for discrimination of the two species. Cholesteryl glucoside, a characteristic feature of the genus Helicobacter, but seldom found in other bacteria, could not be detected in Helicobacter pullorum. Therefore, rapid determination of this glycolipid may serve as a discrimination marker for Helicobacter pullorum from most other Helicobacter species.     

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains.

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.     

The insertion sequence IS200 fingerprints chromosomal genotypes and epidemiological relationships in Salmonella heidelberg.

In Salmonella heidelberg the copy number of the Salmonella-specific insertion element IS200 was found to vary from four to six. All strains tested contained at least one common insertion site which was serovar specific, and most strains contained three common sites. Concurrent analysis of plasmids indicated that all insertion sequence copies were chromosomally located, and also supported the equivalence of an IS200 fingerprint and clonality. Seven intra-serovar clonal lines were thereby identified. One of these was associated with human infections, including septicaemias. Another was associated with chicken as a host: all these strains also carried a unique plasmid of 23 MDa, which was typed as a member of the IncX group. The chromosomal fingerprint of a third clone showed it to be a descendant of the chicken line marked by a single IS200 transposition. One or two representatives of four other clonal lines were identified. These lines of S. heidelberg could be related by divergent evolution, and the most recent relatives conformed to a continuous branching process model of IS200 transposition. This insertion sequence provided a highly discriminatory molecular marker of the S. heidelberg chromosome, and two of the seven clonal lines so identified were associated with distinct clinical/epidemiological contexts.