Scottish Government

2. SUMMARY

• To detect Helicobacter pylori in water, the real time quantitative- PCR (rt Q-PCR) described by He et al. has been reproduced and validated. The use of the assay in environmental samples and enriched samples from the chemostat before spiking with H. pylori produced non-specific DNA amplification. BLAST search revealed cross-reaction between H. pylori glmM and H. acinonychis and C. jejuni.
• To improve the specificity of the real-time assay, a quantitative real-time PCR assay was developed using Taqman® technology. However, BLAST search of the targeted fragment also revealed the cross-reactions observed in the rt Q-PCR. The use of DNA from four non-pylori Helicobacter species and three Campylobacter species resulted in amplification in all cases. The exception was H. acinonychis. This confirmed the lack of specificity of the assay for H. pylori. Similar sequences to glmM or the gene itself may be present in other organisms but the data available in the GenBank is insufficient to accurately predict the specificity of the assay.
• The fluorescent in situ hybridisation assay ( FISH) described by Moreno et al. 2003 was set up and used to test for the presence of H. pylori in samples. Optimum conditions for the performance of the assay were not achieved and further optimisation is needed. The small size of H. pylori precludes the detection of individual cells at maximum magnification. However, the use of software to enhance the observed image warrant further investigation.
• A total of 20 water samples were received from Roseberry Distribution. Of these, 18 were processed and examined for the presence of H. pylori. The sample taken on 25/05/04 was positive for Helicobacter genus specific PCR but not for H. pylori specific assays. This indicates the presence of Helicobacter species in the water sample. Taqman® real-time PCR assay revealed that glmM gene was present in all samples collected between 25/05/04 and 118/11/04 and the sample collected on 19/01/05. This suggested that organisms containing glmM are present in Scottish water.
• Taqman® real-time PCR analysis of samples taken at different times from the control vessel containing Scottish water indigenous microorganisms revealed the presence of glmM gene suggesting that either Helicobacter species or microorganisms containing sequences similar to glmM gene were growing in the chemostat.
• Studies on chemostat on the survival of H. pylori in water in absence of other microorganisms revealed that H. pylori cells remained alive in water for at least 24h but their culturability was reduced by 2 log in base 2 every 4 hours.
• Observations from the survival experiments in the presence and absence of indigenous microflora also suggested that the presence of microorganisms indigenous in the Scottish water may have a positive effect on the recovery of H. pylori, which have been stressed through the transfer into a low nutrient environment at 20ºC. Due to time restraints, no attempts have been made to identify these organisms responsible.
• The percentage of cells recovered within 12h after inoculation into the chemostat main vessel containing cast iron tiles was higher than for H. pylori cells inoculated in the vessel containing indigenous microflora and glass tiles. This suggests that the conditions within the vessel containing iron tiles may be conducive to H. pylori survival.
• H. pylori cells were recovered from culture for 72h after inoculation into the chemostat in presence of background microflora and glass tiles. However, culturability of H. pylori was lost after 12h when the experiment was repeated in presence of iron tiles. This was associated with an increase in the concentration of indigenous microorganisms which, subsequently, masked the presence of H. pylori in culture
• Acid treatment before culture reduced the number of contaminant microorganisms but also the population of culturable H. pylori.
• Conventional Helicobacter genus specific PCR on samples from H. pylori survival experiments against background microflora, revealed that DNA was present in the vessel for at least 240h after inoculation. However, PCR does not distinguish between the presence of viable and non-viable cells.
• DNA quantification by Taqman® real-time PCR assay determined that H. pylori were not growing in the vessel.
• Increase in the number of glmM copies observed after 168h during H. pylori survival experiments in presence of background microflora may be contributed by microflora, other than H. pylori, which contained the glmM, whilst H. pylori numbers decrease.
• Biofilm was successfully grown on glass and iron tiles. No H. pylori were cultured from biofilms.
• General cell counts from glass tiles were higher in tiles submerged for 2weeks compared with those, which were submerged for 4 weeks. This suggests that these conditions are conducive to biofilm formation. However, bacterial cells that constitute the biofilm do not remain viable over extended periods.
• Higher cell counts were recovered from biofilm on cast iron tiles than from biofilm in glass tiles. More cells were recovered from tiles submerged for longer. The larger surface area afforded by the iron tiles is due to the accumulation of rust. The combination of this and the provision of Fe ions from these tiles, may contribute to the formation of a larger biofilm and the survival of the organisms.