Galleria mellonella as an alternative model of Coxiella burnetii infection.

Coxiella burnetii is a Gram-negative intracellular bacterium and is the causative agent of the zoonotic disease Q fever. Several rodent and non-human primate models of virulent phase I C. burnetii [Nine Mile (NM)I] have been developed, and have been used to determine the efficacy of antibiotics and vaccine candidates. However, there are several advantages to using insect models to study host-microbe interactions, such as reduced animal use, lowered cost and ease of manipulation in high containment. In addition, many laboratories use the avirulent phase II C. burnetii clone (NMII) to study cellular interactions and identify novel virulence determinants using genetic manipulation. We report that larvae of the greater wax moth, Galleria mellonella, were susceptible to infection with both C. burnetii NMI and NMII. Following subcutaneous infection, we report that intracellular bacteria were present within haemocytes and that larval death occurred in a dose-dependent manner. Additionally, we have used the model to characterize the role of the type 4 secretion system in C. burnetii NMII and to determine antibiotic efficacy in a non-mammalian model of disease.

Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens.

AIM: To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and / or culture) in order to determine which of the three PCR methods (ureA, glmM and 26-kDa, SSA gene) was most appropriate in the diagnosis of Helicobacter pylori (H pylori ) infection and also to evaluate the detection of a putative virulence marker of H pylori, the cagA gene, by PCR in biopsy specimens. METHODS: One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms. The PCR methods used to detect H pylori DNA directly from biopsies were the glmM, 26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard. RESULTS: Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods, while 68% of these were positive for the cagA gene. Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened. The remaining 41% were either positive for ureA gene only, glmM only, 26-kDa only, or ureA + glmM, ureA + 26-kDa, glmM + 26-kDa. Out of the 35% positive biopsies, 41% and 82% were positive by culture and CLO respectively, while all negative biopsies were also negative by culture and cagA. Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis. CONCLUSION: This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.

Helicobacter pylori infection in patients consulting gastroenterologists in France: prevalence is linked to gender and region of residence.

BACKGROUND: Because of limited data on the epidemiology of Helicobacter pylori in France, the prevalence of this infection by region and its associated risk factors were studied between 1995 and 1997 among patients consulting a representative sample of gastroenterologists by region. METHOD: A cross-sectional study was performed. Patients consulting gastroenterologists for whatever reason were screened for H. pylori infection determined by specific salivary IgG. A questionnaire was filled out by the gastroenterologist. A multivariate analysis was performed with all relevant variables. RESULTS: 3,153 patients were included. The mean age was 48.5 years; 51.8% were women. After stratification by patients consulting for upper digestive tract (UDT) and non-UDT symptoms, H. pylori infection was found to be more prevalent, in both groups, for characteristics such as being born in a developing country, overcrowding during childhood, and primary educational level. Interestingly, gender (odds ratio OR(UDT for women) = 0.7 (95% CI 0.5-0.8] and OR(non-UDT) for women = 0.6 [95% CI 0.5-0.8]) and living in a region other than the south-west (OR(UDT) varying from 1.5 to 2.0 and OR(non-UDT) varying from 1.3 to 2.1, depending on the region) was associated with the odds of prevalent infection. CONCLUSION: These findings show (1) that gender deserves more attention in the epidemiology of H. pylori and (2) a regional disparity in France regarding H. pylori infection.

Cytoskeletal rearrangements induced by Helicobacter pylori strains in epithelial cell culture: possible role of the cytotoxin.

The relationship between Helicobacter pylori adherence, cytotoxin production, and modification of the cytoskeletal structure was investigated by studying the effects of 12 H. pylori strains cocultured with Hep-2 epithelial cells. Bacterial strains were isolated from patients with peptic ulcer disease or nonulcer dyspepsia. Presence of the cag pathogenicity island and vacA subtypes of the strains were determined as was the production of vacuolating cytotoxin. We found that cytoskeletal rearrangements, as observed by confocal microscopy after double staining of the bacteria and the cell actin with Texas red and fluorescein-conjugated phalloidin, respectively, occurred essentially when the strains were cytotoxin producers and that the supernatants alone could also lead to these modifications.

Helicobacter species colonizing pig stomach: molecular characterization and determination of prevalence.

The infection rate of 60 pigs (10 pigs from each of six farms) by Helicobacter species was studied by two techniques. Histological examination of the cardiac area of the stomach yielded a 58% positive result versus an 80% positive result by PCR with genus-specific primers. Eighty percent of the 16S rRNA gene was amplified, classified in four groups by PCR-restriction fragment length polymorphism, and sequenced. Isolates from all farms except one (farm C) were identified as Helicobacter heilmannii type 1, while those from farm C were identified as H. heilmannii type 2. Attempts to culture this organism in vitro failed. Helicobacter pylori was not found in these animals.

Is there a link between the variation in gastric cancer mortality and differences in Helicobacter pylori prevalence in different regions of France?

An ecological study was performed to correlate the cumulative gastric cancer mortality rate to the prevalence of Helicobacter pylori infection in France. National data on mortality, standardized for age and gender, and the results of a nationwide prevalence study on Helicobacter pylori infection among 1,586 patients consulting for symptoms other than upper digestive tract symptoms, in seven defined French regions were used. The correlation was described by linear regression with the standardized data and then evaluated in a linear regression model including age and gender as co-variables. The Southwest region was the least affected by the infection (15.2%) while prevalence varied from 20.5 to 25.3% for the other regions. The cumulative gastric cancer mortality rate varied from 34.4 to 51.8/100,000. The prevalence of Helicobacter pylori infection in the model explained 5% of the variability in the gastric cancer mortality. A number of biases which were difficult to control could explain the lack of association between these variables.