Jens Blom

Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori

ABSTRACT Helicobacter pylori can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoids, with intermediate forms called U forms. The importance of nonculturable coccoid forms of H. pylori in disease transmission and antibiotic treatment failures is unclear. Metabolic activities of actively growing as well as nonculturable H. pylori were investigated by comparing the concentrations of cellular ATP and total RNA, gene expression, presence of cytoplasmic polyphosphate granules and iron inclusions, and cellular morphology during extended broth culture and nutritional cold starvation. In addition, the effect of exposing broth-cultured or cold-starved cells to a nutrient-rich or acidic environment on the metabolic activities was investigated. ATP was detectable up to 14 days and for at least 25 days after transformation from the spiral form to the coccoid form or U form in broth-cultured and cold-starved cells, respectively. mRNAs of VacA, a 26-kDa protein, and urease A were detected by using reverse transcription-PCR in cells cultured for 2 months in broth or cold starved for at least 28 months. The ATP concentration was not affected during exposure to fresh or acidified broth, while 4- to 12-h exposures of nonculturable cells to lysed human erythrocytes increased cellular ATP 12- to 150-fold. Incubation of nonculturable cold-starved cells with an erythrocyte lysate increased total RNA expression and ureA mRNA transcription as measured by quantitative real-time reverse transcription-PCR. Furthermore, the number of structurally intact starved coccoids containing polyphosphate granules increased almost fourfold (P = 0.0022) under the same conditions. In conclusion, a specific environmental stimulus can induce ATP, polyphosphate, and RNA metabolism in nonculturable H. pylori, indicating viability of such morphological forms.

Capsule and fimbria interaction in Klebsiella pneumoniae

ABSTRACT The capsular polysaccharide and type 1 fimbriae are two of the major surface-located virulence properties associated with the pathogenesis of Klebsiella pneumoniae. The capsule is an elaborate polysaccharide matrix that encases the entire cell surface and provides resistance against many host defense mechanisms. In contrast, type 1 fimbriae are thin adhesive thread-like surface organelles that can extend beyond the capsular matrix and mediate d-mannose-sensitive adhesion to host epithelial cells. These fimbriae are archetypical and consist of a major building block protein (FimA) that comprises the bulk of the organelle and a tip-located adhesin (FimH). It is assumed that the extended major-subunit protein structure permits the FimH adhesin to function independently of the presence of a capsule. In this study, we have employed a defined set of K. pneumoniae capsulated and noncapsulated strains to show that the function of type 1 fimbriae is actually impeded by the concomitant expression of a polysaccharide capsule. Capsule expression had significant effects on two parameters commonly used to define FimH function, namely, yeast cell agglutination and biofilm formation. Our data suggest that this effect is not due to transcriptional/translational changes in fimbrial gene/protein expression but rather the result of direct physical interference. This was further demonstrated by the fact that we could restore fimbrial function by inhibiting capsule synthesis. It remains to be determined whether the expression of these very different surface components occurs simply via random events of phase variation or in a coordinated manner in response to specific environmental cues.

Prevalence of cytolethal distending toxin (cdt) genes and CDT production in Campylobacter spp. isolated from Danish broilers.

The pathogenesis of campylobacter infection in man is largely unknown, although cytolethal distending toxin (CDT) has been incriminated as a virulence factor. However, little is known about the cdt genes in Campylobacter spp. isolated from broiler chickens. A total of 350 cloacal swabs was collected and tested by conventional culture and PCR. Of the 114 Campylobacter isolates obtained, 101 (88.6%) were identified as C. jejuni and 13 (11.4%) as C. coli by conventional methods. cdt genes were detected by PCR in all the isolates except one C. jejuni isolate. Cytotoxic effects were produced in a Vero cell line, by 100 of the C. jejuni isolates. In contrast, 10 C. coli isolates produced much lower levels of toxin and 3 produced no detectable toxin. These results confirm the common occurrence of campylobacter infection in chickens and indicate that cdt genes are commonly present in both C. jejuni and C. coli isolates from broilers, but that there are distinct differences in CDT production in these two closely related species.

Survival and ultrastructural changes of Helicobacter pylori after phagocytosis by human polymorphonuclear leukocytes and monocytes.

Few studies have been carried out on the phagocytosis and killing of Helicobacter pylori by both polymorphonuclear leukocytes (PMNs) and monocytes. In this study, H. pylori was incubated for up to 60 min either alone or with phagocytes in the presence or absence of human serum. Both non‐immune serum and immune serum were used. Reduction in the number of H. pylori, which corresponds to the killing of H. pylori, was analysed by a colony count and ultrastructural changes were studied by electron microscopy. No reduction in the number of H. pylori was found when the bacteria were incubated alone or with phagocytes in the absence of serum. It is remarkable that unopsonized H. pylori was phagocytosed. When immune serum was added to the suspensions of bacteria and phagocytes, the killing rate of H. pylori was found to depend on the ratio of H. pylori to phagocytes. Thus an excess of monocytes reduced the number of H. pylori, whereas an excess of PMNs resulted in complete killing of H. pylori. On incubation with PMNs and serum, ultrastructural changes were observed in the majority of the bacteria whether they were phagocytosed or not. Controls without serum did not show any changes in the morphology of H. pylori, indicating that components in the serum play an important role in the phagocytosis and killing of H. pylori. In contrast, several of the phagocytosed bacteria were found to be unaffected after incubation with monocytes and serum. Such preparations often contained large aggregates of platelets surrounding unaffected H. pylori. In the gastric mucosa, H. pylori is often found in excess as compared to the phagocytes. If these results can be compared to the situation in vivo, the phagocytes seem to be ineffective in the killing of H. pylori, and other immune mechanisms may therefore be of importance for the elimination of H. pylori from the gastric epithelium. The possible intracellular survival of H. pylori should be taken into account when treatment regimes for H. pylori infections are chosen.

The histopathology of human gastric mucosa inhabited by Helicobacter heilmannii-like (Gastrospirillum hominis) organisms, including the first culturable case.

The aim was to determine the prevalence of Helicobacter heilmannii‐like organisms in human gastric biopsies and the associated histology compared with that of Helicobacter pylori‐bearing gastric biopsies. Furthermore, the feasibility of culturing H. heilmannii was examined. A consecutive series of 727 gastric biopsies from 650 patients were prospectively scrutinized for H. heilmannii. Their distribution pattern was recorded as well as the affiliated morphology of the gastric mucosa. Additional biopsies from some of the patients were examined microbiologically. Four cases (0.6%)(95% confidence intervals: 0.01–1.2%) of the examined material harboured H. heilmannii. The bacterial burden was graded as sparse in three cases, moderate in one case. The distribution pattern was patchy; thus, in no case did all biopsies from one endoscopy comprise H. heilmannii. Adhesion to epithelial cells was infrequent. A mild gastritis, active in three cases, characterized all biopsies. Lymphoid aggregates occurred in biopsies from three patients. Micropapillary tufting of the epithelial layer and intestinal metaplasia were not apparent. Culture studies proved successful in the one of the four cases assayed. In conclusion the morphology of H. heilmannii‐bearing mucosa deviates from that of H. pylori‐associated mucosa by the absence of epithelial damage in the former. This observation can in part be explained by the predominant location of H. heilmannii at a distance from the epithelium in contrast to the conspicuous H. pylori adhesion to epithelial cells, coupled with a usually low bacterial burden and patchy occurrence of H. heilmannii as opposed to the generally more heavy infestation with H. pylori.

A Case of Helicobacter cinaedi Bacteraemia in a Previously Healthy Person with Cellulitis.

Helicobacter cinaedi is an infrequent, but well recognized cause of gastroenteritis in immunosuppressed patients. Here we report a case of an extra-intestinal infection in a previous healthy 61-year old heterosexual male. Focus for the infection was most likely cellulitis on the lower right leg. The bacterium was cultured from blood twice within one week. Electron microscopy of the isolate visualized bipolar flagella. Partial DNA sequencing of the 16S rRNA gene and phenotypic characterization of the isolate established the species diagnosis. The patient was treated with rifampicin. After end of treatment blood cultures were negative and the cellulitis had disappeared.

Possible Clinical Importance of the Transformation of Helicobacter pylori into Coccoid Forms

Although spiral-shaped microorganisms have been observed in the stomach since the beginning of this century (1, 2), they have largely been ignored as pathogens and dismissed as contaminants (3). Not until Warren & Marshall (4, 5) culturedHelicobacter pylori (Campylobacter pylori ) in 1982 did the significance of these organisms become appreciated and their presence on the surface of the gastric mucosa linked to gastritis and peptic ulcer disease. H. pylori is a widespread common microorganism with a worldwide prevalence of 30%–90%, and it is estimated to have infected more than one billion people (6). The prevalence of H. pylori is higher in developing countries than in developed countries (7), with an increase up to 60 years of age (7, 8). No significant difference in sex has been found (7–9).H. pylori is able to convert into coccoid forms, which cannot yet be cultured and diagnosed by conventional diagnostic methods. H. pylori is found almost entirely in humans, and oral–oral transmission from parents to children and between persons in crowded institutions seems to be common (10, 11). A faecal–oral transmission through drinking water has been proposed, especially in areas with poor water supply (12–14). It has been speculated that the coccoid forms of H. pylori play a role in the survival of the bacterium outside the human host (3, 15, 16). Experimental data have shown that coccoid forms ofH. pylori are able to survive in river water for more than 1 year and subsequently be cultured (17). H. pylori may transform into coccoid forms under conditions of poor nutrition and starvation, which may contribute to the transmission ofH. pylori from an environmental source (18, 19). Coccoid forms of H. pylori have not been culturable in all studies despite prolonged culture for up to 4 weeks (19, 20). Several different antiH. pylori regimens have been established, and eradication of H. pylori infection decreases the relapses of peptic ulcers dramatically (21). H. pylori may be more easily eradicated by antibiotic treatment than other chronic infectious agents, such as Mycobacterium tuberculosis andPseudomonasin cystic fibrosis patients. However, 5%–70% treatment failures have been reported in a various studies (21–23). Some of these treatment failures are caused by poor patient compliance or by H. pylori being resistant to the antibiotics used. Some of these treatment failures remain unexplained (22). Coccoid forms of H. pylori can be induced by various compounds, including some anti-ulcer drugs and some antibiotics (24–27) and may remain in the gastric mucosa after antiH. pylori treatment of the patients in three forms: a degenerative dead form, a viable non-culturable form, and a viable culturable form (15, 20, 28, 29; H. O. Nilsson, J. Blom, M. Stollenwerk, LP. Andersen, T. Wadström. Unpublished observations). Whether the two viable forms represent different developmental stages from spiral to non-culturable coccoid forms is uncertain. The aims of this study were to critically evaluate our current understanding of the possible pathogenic potential of the coccoid forms of H. pylori in comparison with that of the spiral forms and to evaluate the clinical role of coccoid forms in transmission and relapse of H. pylori.

Different patterns of Helicobacter pylori adherence to gastric mucosa cells in children and adults. An ultrastructural study.

BACKGROUND Infection with Helicobacter pylori in childhood may be the initiation of a lifelong coexistence between microorganisms and epithelial cells resulting in chronic inflammation. The adhesion pattern of H. pylori found in antral biopsies from a group of H. pylori-infected children with recurrent abdominal pain was compared with a group of H. pylori-infected adults suffering from dyspepsia, in an attempt to reveal differences in the type of adhesion. METHODS The histology of antrum biopsies and the ultrastructure of adherent H. pylori in biopsies from 26 children (median age, 10.1 years) were compared with organisms in biopsies from 19 adults (median age, 54.4 years). RESULTS More than 1000 adherent H. pylori were studied and divided into four types of adhesion: 1) contact to microvilli; 2) connection to the plasma membrane via filamentous material; 3) adhesive pedestal formation; and 4) abutting or making a depression in the plasma membrane. Contact to microvilli was significantly higher (69% versus 39%; P = 0.002) in children compared with adults and comprised two-thirds of all adherent organisms in children. The more intimate adhesion types as abutting or adhesive pedestals dominated in adults. CONCLUSIONS These results indicate a change in contact types between H. pylori and gastric epithelial cells in adults compared with children and this may be a natural development in the lifelong infection of humans.Background: Infection with Helicobacter pylori in childhood may be the initiation of a lifelong coexistence between microorganisms and epithelial cells resulting in chronic inflammation. The adhesion pattern of H. pylori found in antral biopsies from a group of H. pylori-infected children with recurrent abdominal pain was compared with a group of H. pylori-infected adults suffering from dyspepsia, in an attempt to reveal differences in the type of adhesion. Methods: The histology of antrum biopsies and the ultrastructure of adherent H. pylori in biopsies from 26 children (median age, 10.1 years) were compared with organisms in biopsies from 19 adults (median age, 54.4 years). Results: More than 1000 adherent H. pylori were studied and divided into four types of adhesion: 1) contact to microvilli; 2) connection to the plasma membrane via filamentous material; 3) adhesive pedestal formation; and 4) abutting or making a depression in the plasma membrane. Contact to microvilli was significantly higher (69% versus 39%; P = 0.002) in children compared with adults and comprised two-thirds of all adherent organisms in children. The more intimate adhesion types as abutting or adhesive pedestals dominated in adults. Conclusions: These results indicate a change in contact types between H. pylori and gastric epithelial cells in adults compared with children and this may be a natural development in the lifelong infection of humans.

Confirmation of electron microscopy results by direct testing of viruses adhered to grids using nucleic acid amplification techniques.

Abstract It is possible to visualize rapidly viral particles by electron microscopy (EM) in patient samples and in cell cultures, and characterize the particles on the basis of their size and morphology. In many instances, EM has contributed to the diagnosis of specific infectious agents. Four different types of viruses with different characteristics of particle size, capsid structure, the presence or absence of an envelope, genomic content and stability outside the host were screened and diagnosed by EM at the level of family/genus. The results were confirmed at the species level by elution of the sample material from the grids used for EM examination and nucleic acid amplification. This approach could be valuable in situations where the immediate diagnosis is unclear, or when new infectious agents appear.

Aerococcus urinae: polyphasic characterization of the species

A polyphasic characterization of Aerococcus urinae is presented. In this study the intraspecies relationships between 26 strains of varying geographical origin were examined by phenotypic tests, ribotyping and multilocus enzyme electrophoresis. The results demonstrated two main phenotypic patterns that could be distinguished in tests for hydrolysis of aesculin, and acid production from amygdalin and salicin. Strains were either negative (n=19) or positive (n=6) in these tests. One strain had a deviating pattern. Heterogeneity within the 19 pattern I strains was demonstrated especially by phenotypic tests (acid production from ribose, mannitol, sorbitol, sucrose and D‐arabitol) and by multilocus enzyme electrophoresis. However, DNA sequence analysis of the 16S rRNA (n=7) and gyrB genes (n=3) from strains representing the two main patterns showed no variation in sequences among strains. Comparison of A. urinae and representatives of related taxa by 16S rDNA sequence analysis showed that the taxon is related to, but distinct from, other Aerococcus spp.