Oral rinses in growth inhibition and treatment of Helicobacter pylori infection

Abstract

Background: Helicobacter pylori (H. pylori) is well-known for its role in chronic gastritis and gastric cancer. Eradication of these carcinogenic bacteria from the gut is one of the challenges for clinicians. The complexity of treatment mainly owes to antibiotic resistance and relapse due to an additional reservoir in the oral cavity. Our study emphases the isolation of H. pylori from distinct habitats of the gut microenvironment (gastric biopsy and gastric juice) and its subsequent characterization. We have also evaluated the effect of various oral rinses on isolated H. pylori from different anatomical locations of included subjects.

Results: The possible strains isolated from two different habitats of the same subject shows a striking difference in their growth pattern. Promisingly, some of the included oral rinses are efficient in growth inhibition as per recommended 30 s treatment. The subsequent evaluation shows that oral rinse B (among A-E) is most effective and down-regulates the expression of one of the potent H. pylori gene, CagA, in the infected gastric adenocarcinoma (AGS) cells.

Conclusion: Our study, for the first time, revealed that H. pylori, isolated from the different habitat of the same subject, show a different growth pattern. The expression of H. pylori pathogenic gene (CagA) was down-regulated by the use of oral rinses. Hence, oral rinses will reduce the H. pylori in the oral cavity and help to control its migration from oral to the gastric compartment and may be used as an adjuvant treatment option for its re-infection.

Keywords: Apoptotic pathway; CagA; Gastric cancer; Gastritis; Growth curve; H. pylori; Oral rinses.

Fig. 1

Identification of bacteria through Gram staining. Gram staining of different clinical isolates of H. pylori, namely, I10, HJ9, HB10, HJ10, HB14, HJ14, HB1, HJ1, HB4, and HB5 were showing typical gram-negative bacteria

Fig. 2

Growth pattern of isolated H. pylori strains. a The growth pattern of clinical isolates (HJ1, HB1, HB4, HB5, HJ9, HJ10, HB10, HJ14, HB14) and reference strain (I10) under specific microaerophilic condition assessed at 0, 6, 12, 18 and 24 h. The data are the mean ± SD (n = 4) of two independent experiments with technical replicate. b: Graphs are plotted for relative growth in comparison to I10 at 0, 2, 6, 12, 18, and 24 h. c Plate densitometry image of selected H. pylori isolates HB1 (lane first); HB5 (lane second); HB14 (lane third); HJ9 (lane fourth); and I10 (lane fifth) are showing growth till 24 h. The experiment is performed in duplicates, and the representative images are shown. d Fold change was calculated in comparison to 0 Hr

Fig. 3

Growth pattern of H. pylori isolates after treatment with oral rinses for 30 s. Treatment of oral rinses (A, B, C, D, and E) was given to 6X10⁷ of H. pylori for 30 s, and growth was observed until 24 h compared to untreated control. Graphs reflect the growth of (a) fast (HB1 and HB5) and (b) slow-growing (HJ9, HB14, and I10) isolates. Relative growth of (c) fast (HB1 and HB5) (d) slow-growing (I10, HJ9, and HB14) was estimated compared to untreated control. The data are the mean ± SD of two independent experiments with technical replicate (n = 4, mean ± SD)

Fig. 4

Treatment with selected oral rinses for a shorter duration. 6X10⁷ of H. pylori were treated with selected oral rinse alone and in combination (B, D, E, BD, BE, DE, and BDE) for 5 s. Growth was observed until 24 h in comparison to untreated control. Growth curve of (a) fast-growing isolates (HB1 and HB5) and (b) slow-growing isolates (HJ9, HB14, and I10). Relative growth of fast HB1 and HB5 (c) and in of slow-growing I10, HJ9, and HB14 (d) compared to untreated control. The data are the mean ± SD of two independent experiments with technical replicate (n = 4, mean ± SD)

Fig. 5

Plate densitometry of H. pylori after treatment with oral rinses. Oral rinses treatment of A, B, C, D, and E was given to 1X10⁷H. pylori, followed by spreading on half of the BHI Agar plate. Representative images showing growth of (a) HB1, (b) HB5, (c) I10 (d) HJ9 and (e) HB14 till 24 h Relative growth was estimated for fast-growing isolates (f) HB1 and (g) HB5; and slow-growing isolates (h) I10, (i) HJ9, and (j) HB14. Blank plates were considered as negative control and untreated isolates as a positive control. The data are the mean ± SD of two independent experiments with technical replicate (n = 4, mean ± SD)

Fig. 6

Investigation of H. pylori and gastric cancer genes: Treatment of solution was given to 6X10⁷ of H. pylori and incubated with 0.5X10⁶ AGS cell for 12 Hrs. RNA was isolated, and transcript level was determined by qRTPCR. Experiments were performed in duplicates. Expression of 16 s rRNA (a, b); CagA (c, d); BabA (e, f); CCND1 (g, h); CDX2 (i, j); PTEN (k, l); MMP7 (m, n) was evaluated on 5 and 30 s treatment respectively. AGS cells infected with wild type H. pylori treated as control in this study

Fig. 7

EB/AO dual staining assay. 0.25X10⁶ AGS cells were plated in 6 well plates and infected with 30 s oral rinse solution (a, b, c, d, and e) treated and wild type (WT) H. pylori isolates. Furthermore, 12 h post-infection, the cells were stained with EB/AO solution, and images were acquired. (a) HB1, (b) I10, (c) HJ9 and (d) HB14 infected and uninfected AGS cells

Fig. 8

Status of apoptosis-related genes. Treatment of solution was given to 6X10⁷ of H. pylori and incubated with 0.5X10⁶ AGS cell for 12 Hrs. RNA was isolated, and the transcript level was determined by qRTPCR. Experiments were performed in duplicates. Expression of (a, b) FADD; (c, d) APAF1; (e, f) BAK; (g, h) BID; (i, j) PUMA; (k, l) NOXA; and (m, n) BCL2 was assessed after treatment for 5 and 30 s respectively. The data are the mean ± SEM of two independent experiments with technical replicate (n = 4)