Helicobacter pylori

Analysis of the full set of PMEN1 sequences used the alignment from (26 (link)); 11 closely-related isolates were extracted as a subsample for comparison with the output of ClonalFrame. For the analysis of S. aureus ST239, 14 representatives from the South-East Asian clade were extracted from the larger alignment (49 (link)) for the equivalent comparative analysis. For the analysis of Helicobacter pylori, eight publically available complete genomes were selected from across the species that included both the most closely-related pair of isolates and the isolate most divergent from the rest of the sample, based on a previous analysis (50 (link)). These genomes were then aligned using progressiveMauve (51 (link)), generating a 1.8 Mb core genome alignment for analysis.
The resulting whole genome alignments were then analyzed using the default settings of Gubbins, except that the S. pneumoniae and S. aureus analyses were run until convergence. For S. pneumoniae and S. aureus, ClonalFrame (19 (link)) was also run using default settings, without estimating node ages, with a burn in chain length of 25 000 and a parameter estimation chain length of 25 000. For H. pylori, convergence was achieved when ClonalFrame was run without estimating node ages or theta, using a burn in chain length of 10 000 and a parameter estimation chain length of 10 000. Convergence was assessed through plotting the variation in parameter values over the course of the MCMC; these are shown in Supplementary Figures S4, S7 and S9.

A single aliquot of the mock community was used throughout the sequencing effort analyzed in this study. This mock community represented 21 strains distributed among members of the Bacteria (n = 20) and Archaea (n = 1). Among the 20 bacterial sequences, there were 6 phyla, 10 classes, 12 orders, and 18 families and genera. The aliquot of mock community DNA was prepared by mixing genomic DNA from Acinetobacter baumanii (NC_009085), Actinomyces odontolyticus (DS264586), Bacillus cereus (AE017194), Bacteroides vulgatus (NC_009614), Clostridium beijerinckii (NC_009617), Deinococcus radiodurans (NC_001263), Enterococcus faecalis (NC_004668), Escherichia coli (NC_000913), Helicobacter pylori (NC_000915), Lactobacillus gasseri (NC_008530), Listeria monocytogenes (NC_003210), Neisseria meningitidis (NC_003112), Propionibacterium acnes (NC_006085), Pseudomonas aeruginosa (NC_002516), Rhodobacter sphaeroides (NC_007493, NC_007494), Staphylococcus aureus (NC_007793), Staphylococcus epidermidis (NC_004461), Streptococcus agalactiae (NC_004116), Streptococcus mutans (NC_004350), Streptococcus pneumoniae (NC_003028), and Methanobrevibacter smithii (NC_009515). Given the low homology between the three PCR primer pairs and the M. smithii 16S rRNA gene sequence, these sequences were rarely observed and have been omitted from the analysis of this study. The proportions of genomic DNAs added were calculated to have an equal number of 16S rRNA genes represented for each species; however, the original investigators did not verify the final relative abundances.

We implemented memory-efficient indexing schemes for the classification of microbial sequences based on the FM-index, which also permits very fast search operations. We further reduced the size of the index by compressing genomic sequences and building a modified version of the FM-index for those compressed genomes, as follows. First, we observed that for some bacterial species, large numbers of closely related strains and isolates have been sequenced, usually because they represent significant human pathogens. Such genomes include Salmonella enterica with 138 genomes, Escherichia coli with 131 genomes, and Helicobacter pylori with 73 genomes available (these figures represent the contents of RefSeq as of December 2015). As expected, the genomic sequences of strains within the same species are likely to be highly similar to one another. We leveraged this fact to remove such redundant genomic sequences, so that the storage size of our index can remain compact even as the number of sequenced isolates for these species increases.
Figure 1 illustrates how we compress multiple genomes of the same species by storing near-identical sequences only once. First, we choose the two genomes (G₁ and G₂ in the figure) that are most similar among all genomes. We define the two most similar genomes as those that share the greatest number of k-mers (using k = 53 for this study) after k-mers are randomly sampled at a rate of 1% from the genomes of the same species. In order to facilitate this selection process, we used Jellyfish (Marcais and Kingsford 2011 (link)) to build a table indicating which k-mers belong to which genomes. Using the two most similar genomes allows for better compression as they tend to share larger chunks of genomic sequences than two randomly selected genomes. We then compared the two most similar genomes using nucmer (Kurtz et al. 2004 (link)), which outputs a list of the nearly or completely identical regions in both genomes. When combining the two genomes, we discard those sequences of G₂ with ≥99% identity to G₁ and retain the remaining sequences to use in our index. We then find the genome that is most similar to the combined sequences from G₁ and G₂ and combine this in the same manner as just described. This process is repeated for the rest of the genomes.
As a result of this concatenation procedure, we obtained dramatic space reductions for many species; e.g., the total sequence was reduced from 661 to 74 Mbp (11% of the original sequence size) in S. enterica and from 655 to 107 Mbp (16%) in E. coli (see Table 1). Overall, the number of base pairs from ∼4300 bacterial and archaeal genomes was reduced from 15 to 9.1 billion base pairs (Gbp). The FM-index for these compressed sequences occupies 4.2 GB of memory, which is small enough to fit into the main memory (RAM) on a conventional desktop computer. As we demonstrate in the Supplemental Methods and Supplemental Table S1, this compression operation has only a negligible impact on classification sensitivity and accuracy.

The primary therapeutic modalities were determined using the Lugano and Paris staging system (Online Resource 1) and the HPI status. H. pylori eradication was performed in all patients with HPI and localized stage gastric MALT lymphoma. For first-line eradication therapy, a proton pump inhibitor (PPI)-based triple therapy regimen was administered for 2 weeks: PPI (standard dose twice a day), clarithromycin (0.5 g twice a day), and amoxicillin (1 g twice a day). 13C urea breath tests were performed in all patients for 3 months or at least 8 weeks after treatment completion, and at least 2 weeks after PPI withdrawal to confirm HPI eradication. For patients who failed first-line triple therapy, a second-line quadruple-therapy regimen consisting of PPI (standard dose twice a day), tripotassium dicitrato bismuthate (300 mg four times a day), metronidazole (500 mg thrice a day), and tetracycline (500 mg four times a day) was administered for 1–2 weeks.
Patients received radiotherapy, chemotherapy, or chemoradiotherapy if they did not achieve lymphoma regression following first- and second-line HPI eradication therapy, or were at the localized stage without initial HPI, or had advanced-stage gastric MALT lymphoma. For radiotherapy, the clinical target volume included the entire stomach and regional lymph nodes and was prescribed as 30.6 Gy over 17 fractions on the stomach [20 (link)]. The internal target volume (ITV) and planning target volume were set using the motion information obtained from the 4-dimensional CT for assessment of breathing motions and defined as an expansion of 5 mm from the ITV considering the set-up error of the patient [20 (link)]. Patients with the involvement of ≥ 2 organs were excluded from radiotherapy. The R-CVP was the primary systemic chemotherapy regimen, consisting of rituximab 375 mg/m², cyclophosphamide 750 mg/m², and vincristine 1.4 mg/m² on day 1, and prednisolone 60 mg/m² on days 1–5 every 21 days. Localized stage lesions involving small-sized mucosal layers in patients with initial HPI-negative findings could be selectively treated by endoscopic mucosal resection (EMR) and close observation. In the case of chemoradiotherapy, we only used additional radiotherapy for consolidation purposes after chemotherapy by the physicians’ decision. To investigate the side effects of each treatment modality, we reviewed the medical records following the National Cancer Institute’s Common Terminology Criteria for Adverse Events version 5.0.

Helicobacter pylori culture filtrate (Hpcf) was prepared as described by Cuomo et al. [36 (link)]. In detail, culture filtrate of H. pylori P12 strain was prepared by culturing the bacterium on selective Columbia agar (Oxoid, Basingstoke, Hampshire, UK) supplemented with 7% (v/v) of defibrinated horse serum (Oxoid, Basingstoke, Hampshire, UK) and antibiotic mix (DENT; Oxoid). Bacteria plates were incubated for 3–4 days at 37°C in a 10% CO₂ atmosphere. After growth, bacteria were scratched using brain heart infusion (BHI; Oxoid, Basingstoke, Hampshire, UK) and measured. 2 x 10⁷ bacteria were cultured in DMEM (Microtech, Naples, Italy) supplemented with 10% FBS (Microtech, Naples, Italy) and incubated at 37°C in a 10% CO₂ atmosphere for 24 hours. Finally, bacterial suspension was centrifuged at 10,000 g for 10 minutes and the supernatant was filtered by using a 0.22 μm filter.

Data on socio-demographic and behavioral characteristics of the study participants were collected using structured pre-tested interviewer administered questionnaire. Approximately 2 gram of stool sample was collected from each participant in a clean container. H. pylori stool antigen was detected by Wondfo one step H. pylori feces test kit (Guangzhou Wondfo Biotech, China) according to the manufacture`s instruction.