Use of Deep-Amplicon Sequencing (DAS), Real-Time PCR and In Situ Hybridization to Detect H. pylori and Other Pathogenic Helicobacter Species in Feces from Children.

BACKGROUND: Detecting Helicobacter pylori in fecal samples is easier and more comfortable than invasive techniques, especially in children. Thus, the objective of the present work was to detect H. pylori in feces from children by molecular methods as an alternative for diagnostic and epidemiological studies. METHODS: Forty-five fecal samples were taken from pediatric patients who presented symptoms compatible with H. pylori infection. HpSA test, culture, real-time quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), direct viable count associated with FISH (DVC-FISH), and Illumina-based deep-amplicon sequencing (DAS) were applied. RESULTS: No H. pylori colonies were isolated from the samples. qPCR analysis detected H. pylori in the feces of 24.4% of the patients. In comparison, DVC-FISH analysis showed the presence of viable H. pylori cells in 53.3% of the samples, 37% of which carried 23S rRNA mutations that confer resistance to clarithromycin. After DAS, H. pylori-specific 16S rDNA sequences were detected in 26 samples. In addition, DNA from H. hepaticus was identified in 10 samples, and H. pullorum DNA was detected in one sample. CONCLUSION: The results of this study show the presence of H. pylori, H. hepaticus, and H. pullorum in children's stools, demonstrating the coexistence of more than one Helicobacter species in the same patient. The DVC-FISH method showed the presence of viable, potentially infective H. pylori cells in a high percentage of the children's stools. These results support the idea that fecal-oral transmission is probably a common route for H. pylori and suggest possible fecal-oral transmission of other pathogenic Helicobacter species.

Optimization of pre- treatments with Propidium Monoazide and PEMAX™ before real-time quantitative PCR for detection and quantification of viable Helicobacter pylori cells.

Accurate detection of H. pylori in different environmental and clinical samples is essential for public health strtdudies. Now, a big effort is being made to design PCR methodologies that allow for the detection of viable and viable but non-culturable (VBNC) H. pylori cells, by achieving complete exclusion of dead cells amplification signals. The use of DNA intercalating dyes has been proposed. However, its efficacy is still not well determined. In this study, we aimed to test the suitability of PMA and PEMAX™ dyes used prior to qPCR for only detecting viable cells of H. pylori. Their efficiency was evaluated with cells submitted to different disinfection treatments and confirmed by the absence of growth on culture media and by LIVE/DEAD counts. Our results indicated that an incubation period of 5 min for both, PMA and PEMAX™, did not affect viable cells. Our study also demonstrated that results obtained by using intercalating dyes may vary depending on the cell stress conditions. In all dead cell's samples, both PMA and PEMAX™ pre-qPCR treatments decreased the amplification signal (>103 Genomic Units (GU)), although none of them allowed for its disappearance confirming that intercalating dyes, although useful for screening purposes, cannot be considered as universal viability markers. To investigate the applicability of the method specifically to detect H. pylori cells in environmental samples, PMA-qPCR was performed on samples containing the different morphological and viability states that H. pylori can acquire in environment. The optimized PMA-qPCR methodology showed to be useful to detect mostly (but not only) viable forms, regardless the morphological state of the cell.

Evaluation of different culture media for detection and quantification of H. pylori in environmental and clinical samples.

The objective of the present study was to establish the most suitable culture medium for the isolation of H. pylori from environmental and clinical samples. Ten different culture media were compared and evaluated. Four of them had been previously described and were modified in this study. The rest of the media were designed de novo. Three different matrices, tap water, wastewater, and feces, were inoculated with serial dilutions of H. pylori NCTC 11637 strain at a final concentration of 104 and 103 CFU/ml and the recovery rates were calculated. From inoculated tap water and wastewater samples, H. pylori colonies were recovered from four out of the analyzed culture media. When fecal samples were analyzed, the isolation of the pathogen under study was only possible from two culture media. Different optimal media were observed for each type of sample, even for wastewater and stool samples. Nevertheless, our results indicated that the combination of Dent Agar with polymyxin B sulfate did not inhibit the growth of H. pylori and was highly selective for its recovery, regardless of the sample origin. Thus, we propose the use of this medium as a diagnostic tool for the isolation of H. pylori from environmental and clinical samples, as well as for epidemiological studies.

Deep-amplicon sequencing (DAS) analysis to determine the presence of pathogenic Helicobacter species in wastewater reused for irrigation.

Wastewater has become one of the most important and least expensive water for the agriculture sector, as well as an alternative to the overexploitation of water resources. However, inappropriate treatment before its reuse can result in a negative impact on the environment, such as the presence of pathogens. This poses an increased risk for environmental safety, which can subsequently lead to an increased risk for human health. Among all the emerging wastewater pathogens, bacteria of the genus Helicobacter are some of the most disturbing ones, since they are directly related to gastric illness and hepatobiliary and gastric cancer. Therefore, the aim of this study was to determine the presence of potentially pathogenic Helicobacter spp. in treated wastewater intended for irrigation. We used a next generation sequencing approach, based on Illumina sequencing in combination with culture and other molecular techniques (qPCR, FISH and DVC-FISH), to analyze 16 wastewater samples, with and without an enrichment step. By culture, one of the direct samples was positive for H. pylori. FISH and DVC-FISH techniques allowed for detecting viable Helicobacter spp., including H. pylori, in seven out of eight samples of wastewater from the tertiary effluents, while qPCR analysis yielded only three positive results. When wastewater microbiome was analyzed, Helicobacter genus was detected in 7 samples. The different molecular techniques used in the present study provided evidence, for the first time, of the presence of species belonging to the genus Helicobacter such as H. pylori, H. hepaticus, H. pullorum and H. suis in wastewater samples, even after disinfection treatment.

Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoebae in lettuce samples by next generation sequencing and other molecular techniques.

Vegetables are one of the sources from which Helicobacter pylori can be acquired. This bacterium infects >50% of the global population and is a recognized type I human carcinogen. H. pylori enters into the viable but non-culturable state when it is in the environment, and therefore the use of molecular techniques is much convenient for its detection. Free-living amoebae (FLA) are protozoans found in vegetables. They are transmission vehicles for amoeba-resistant bacteria, among which H. pylori is included. The aim of this study is to study the occurrence and viability of H. pylori from lettuce samples, H. pylori internalized into FLA and the microbiome of FLA isolated from these samples. Special focus was pointed to human pathogenic bacteria. H. pylori was not directly detected in any lettuce sample by means of molecular techniques and neither by culture. However, intra-amoebic H. pylori DNA was detected by means of PMA-qPCR in 55% of the samples and viable intra-amoebic H. pylori cells in 25% of the samples by means of DVC-FISH technique. When FLA microbiome was studied, 21 bacterial genera were part of FLA microbiome in all samples. Helicobacter genus was detected as part of the FLA microbiome in two samples. Other bacteria of public health interest such as Aeromonas sp., Arcobacter sp., Legionella sp., Mycobacterium sp., Pseudomonas sp. and Salmonella sp. were detected as part of FLA microbiome along the analysed samples. This study demonstrates for the first time that H. pylori is internalized as well as alive inside FLA isolated from vegetables. Moreover, this study shows that FLA promote H. pylori detection in environmental samples. In addition, as far as we are aware, this is the first study which studies the microbiome of FLA isolated from vegetables. Among the FLA microbiome, bacteria of public health interest were detected, pointing out that FLA are carriers of these pathogens which can reach humans and cause a public health concern.