An observational study was conducted between September 2014 and May 2016. The study population comprised patients who presented with gastric symptoms and were diagnosed with H. pylori infection based on positive rapid urease tests conducted on gastric biopsy specimens. All were outpatients who presented at the General Surgery Clinic of Fukuoka Dental College Medical and Dental Hospital. The inclusion criteria were as follows: 20 years or older, having own teeth, and no oral symptoms that require immediate treatment. Those with drug allergies and pregnant women were excluded. Twelve patients (5 men, 7 women; mean age 59.5 ± 15.3 years) were analyzed (Additional file 1; Table S1). They were prescribed a 1-week course of a triple-drug regimen (30 mg lansoprazole, 750 mg amoxicillin, and 200 mg clarithromycin, twice daily) as the primary eradication regimen. In cases in which the primary treatment failed, 250 mg metronidazole was used instead of clarithromycin as the secondary regimen.
Oral malodor and oral clinical symptoms were evaluated on the date of initiation of H. pylori eradication treatment and 1 and 7 weeks thereafter. The severity of oral malodor was determined by OLT  and GC. A gas chromatograph (model GC2014; Shimazu Works, Kyoto, Japan) was used to assay the concentrations of H2S, CH3SH, and CH3SCH3 in mouth air. Periodontal health, plaque control assessed with the plaque index (PlI) , and the degree of tongue coating measured using the tongue coating score (TCS)  were evaluated. Periodontal health was assessed using the average probing pocket depth (PPD) and the percentage of bleeding on probing (BOP) values. The ammonia concentration was measured using a portable ammonia-monitoring device (ATTAIN; Taiyo, Osaka, Japan) according to the manufacturer’s instructions. Saliva samples were obtained by stimulation using chewing gum (CheckBuf; Morita, Osaka, Japan) and then subjected to sequencing of the 16S rRNA gene amplicons and quantitative analysis of bacterial species. These examinations were conducted at 9 am; the subjects were prohibited from eating, drinking, chewing, smoking, brushing teeth, or rinsing their mouth after waking up. The subjects were also instructed not to undergo dental treatment during the observation period.
Quantitative analysis of bacterial species in saliva
Quantitative analysis of bacterial species in saliva was performed using the fibrous DNA chip Genopal® (Mitsubishi Chemical, Tokyo, Japan). The copy numbers of all oral bacteria and 12 bacterial species were determined. According to the Socransky pyramid classification , the bacterial species were classified into the red (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola), orange (Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, and Prevotella nigrescens), green (Aggregatibacter actinomycetemcomitans and Capnocytophaga gingivalis), and yellow (Streptococcus gordonii, Streptococcus intermedius, and Streptococcus mutans) groups, and the proportions of these groups among the total oral bacteria were evaluated.
Sequencing of 16S rRNA gene amplicons
DNA was extracted from each saliva sample as described previously . The V1-V2 regions of the 16S rRNA gene were amplified by PCR using the universal bacterial primers 8F and 338R with adaptor and sample-specific 8-base tag sequences as described previously . Equal amounts of the purified PCR amplicons were pooled. Following emulsion PCR, sequencing was performed on the Ion PGM using an Ion PGM Hi-Q View sequencing kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions. The raw sequencing reads were quality-filtered as described previously . The quality-checked reads were assigned to the appropriate sample by examining the tag sequence. Similar sequences were assigned to operational taxonomic units (OTUs) using UPARSE , with a minimum pairwise identity of 97%. The taxonomy of each representative sequence was determined using blast against oral bacterial 16S rRNA gene sequences in the Human Oral Microbiome Database . Nearest-neighbor species with ≥98.5% identity were selected as candidates for each OTU. The taxonomy of an OTU without any hits was determined to the genus level using the Ribosomal Database Project classifier with a minimum support threshold of 80%. The alpha diversity index and UniFrac distances  were calculated following rarefaction to 5,000 reads per sample.
The Wilcoxon signed-rank test was used to compare malodor, clinical, and bacterial parameters among baseline, 1 week, and 7 weeks. The relative abundance of bacterial genera after 1 and 7 weeks were compared with those at baseline using Dunnett’s test. Statistical analyses were conducted using R software, version 3.6.1 . A P-value of <0.05 was considered to reflect statistical significance.