Gastric biopsy and juice collection from gastritis patients for isolation of H. pylori
To date, there are no H. pylori isolates reported from central India to the best of our knowledge. Moreover, isolates from northern and southern India have been listed in previous reports . H. pylori were successfully isolated from five out of 14 biopsy samples and four out of 11 juice samples (Table 1). After observation in antibiotic selective media, Gram staining was performed on all isolates (Fig. 1). Further, three clinical isolates from biopsies and one from juice were subjected for the amplification of H. pylori (16s rRNA) through qRT-PCR (data not shown). Additionally, validation of the clinical isolates was confirmed through nucleotide sequencing (data not shown). Furthermore, the growth of bacteria may be attributed to its pathogenic ability; hence we have studied the growth pattern of the isolated H. pylori and compared it with the reference strain (I10).
The growth pattern of different clinical isolates of H. pylori
The growth curve of confirmed strains of H. pylori was determined by recording OD at 600 nm at various time points (0, 2, 6, 12, 18, and 24 hrs) and a curve was plotted (Fig. 2 a). Our results revealed that the growth of two clinical strains, namely HB1 and HB5 was significantly faster (p <0.05) compared to the other seven clinical and one reference strain (I10). Interestingly, HB1 and HB5 have similar while not identical growth patterns at all examined time points (Fig. 2 a). It is also fascinating that the growth pattern of HB1 and HJ1 was quite different, even though they were isolated from the same patient (Fig. 2 a) Other clinical strains (HB4, HJ9, HJ10, HB10, HJ14, and HB14) shows a similar growth pattern as I10 until 24 hrs.
To better understand the growth pattern of isolated H. pylori, graphs were plotted in the form of a bar chart at all recorded time points (0, 2, 6, 12, 18, and 24 hrs) and were compared with the reference strain (I10) (Fig. 2b). Three to five folds faster growth was observed in HB1 and HB5 compared to I10 at 2 hrs, and further, it increased up to 24 hrs (Fig. 2b). Moreover, isolates such as HJ1, HB10, and HB14 were showing moderately higher growth (3 to 5 folds) compared to reference strain I10 from 6 hours onwards (Fig. 2b). Importantly, the growth of HB1 and HB5 was steadily increasing up to 18 hrs. Hence, our study reflected two fast-growing strains (HB1 and HB5) compared to other clinical isolates HJ1, HB4, HJ9, HJ10, HB10, HB14, HJ14, and reference strain I10 (Fig. 2b).
Confirmation of active component of oral rinses through LC-MS
The active components in the oral rinses of A, B, C, D, and E was reconfirmed through LCMS at the Sophisticated Instrument Centre facility at IIT Indore (Supp. Fig 2). The results validate the presence of labeled active components in them. We have got exact mass spectra at 304.5, 253, 205.1, 212.1, and 102.12 for CPC, chlorhexidine, clove oil, thymol, and povidone/iodine respectively (Supp. Fig 2). Further, to evaluate the efficacy of selected oral rinses H. pylori growth analysis after treatment was performed.
The growth pattern of selected clinical isolates of H. pylori after treatment with oral rinses
All chosen oral solutions recommend 30 sec oral rinsing for effective plaque control. Further, our two fast-growing (HB1 and HB5), two slow-growing (HJ9 and HB14), and a reference strain (I10) were selected for this experiment (Fig. 3 a, b, c, d). Interestingly, we observed that oral rinses A and C were not able to stop the growth of fast-growing strain after 30 sec treatment (Fig. 3 a). Although, growth of fast-growing strain was again increased after 2 hrs post-treatment with solution A and C while solution B, D, and E were able to inhibit the growth until 24 hrs post-treatment. We have found that solution A was least efficient in the control of HB1 growth followed by C. The substantial growth of HB1 was observed from 6 hrs onwards when treated with solution A and 12 hrs post-treatment when treated with oral rinse C (Fig. 3 c). Moreover, the growth of another fast-growing strain, HB5, treated with oral rinses A and C, was suppressed until only 2 hrs (Fig. 3 c). There was considerable growth of HB5; 6 hrs onwards with A, and C treatment. Importantly, oral rinses B, D, and E were able to suppress the growth of HB1 and HB5 until 24 hrs in this study (Fig. 3 a, c). Additionally, slow-growing strain such as HJ9 and HB14 were not able to grow until 12 hrs with all used oral solutions (Fig. 3 b). However, these strains start growing from 12 hrs onwards when treated with solution C (Fig. 3 b). Notably, our reference strain, I10, has not shown any growth after treatment with all oral rinses until 24 hrs (Fig. 3 b, d). In all these experiments, we have used untreated strains as positive controls and culture media as a negative control. A combination of effective oral rinses was used to evaluate their efficacy on treatment for a shorter duration.
Selective oral rinse are restricting H. pylori growth even at shorter exposure
Oral rinse B, D, and E were found to be effective in controlling the growth of slow as well as fast-growing strains at 30 sec treatment. Hence, we investigated the effect of these selected solutions, alone (B, D, and E) and in combination, (BD, BE, DE, and BDE) for a shorter duration of treatment (5 sec) compared to recommended 30 sec (Fig. 4 a, b). Even the 5 sec treatment to fast-growing H. pylori isolates with all efficacious oral rinses alone and in combination were able to restrict the growth until 2 hrs (Fig. 4 a, c). Surprisingly, data recorded 6 hrs post-treatment were demonstrating the growth of HB1 and HB5 with D, E, and their combinations. Importantly, all groups in which solution B is included shows to be restricting the growth of HB1 and HB5 (Fig. 4a, c). Interestingly, the growth of slow-growing strains HJ9, HB14, and reference strain I10, completely abolished with all the above solution combinations till 24 hrs (Fig. 4b). Moreover, when we treated these oral rinses for 10 sec alone and in combination, a similar pattern was established as with 30 sec (Suppl. Fig 1).
The growth pattern of H. pylori after oral rinses treatment on BHI Agar plate:
In addition to solution treatment in liquid culture, we further evaluated the growth pattern of H. pylori isolates on a BHI agar plate after 30 sec treatment with the selected oral rinse solution. Representative pictures of solution treated H. pylori strains are shown in fig. 5 a, b, c, d, and e. The images were quantified using Image J software (NIH), and graphs were plotted (Fig. 5 f, g, h, i, j). As expected, solution treatment of C was ineffective, and H. pylori growth was observed in the case of fast-growing HB1 and HB5 after 12 hrs (Fig. 5 a, b, f, g). Moreover, we also witnessed growth after 24 hrs for slow-growing strains (I10, HJ9, and HB14). Surprisingly, no growth was observed in the case of oral rinse A treatment in all the strains, contrary to the growth in liquid culture (Fig. 5). Again, as expected, no growth was detected after treatment with oral rinses B, D, and E at all the recorded time points. The solutions inhibit the H. pylori growth differentially; hence further, investigation of the known gastric cancer markers and H. pylori genes to assess the effect of oral rinses on its pathogenicity.
Gene profiling of specific gastric cancer marker and H. pylori after oral rinse treatment
Expression of H. pylori genes, namely 16s rRNA, Cag A, and Bab A, were investigated in this experiment. Additionally, reported GC markers such as CCND1, CDX2, PTEN, and MMP7 were also included [27–30]. A mixed expression profile was observed on treatment with oral rinses (B, C, D, and E) for 5 sec in the I10 strain. On treatment with solution B, H. pylori genes (16s rRNA, CagA, and BabA) and GC markers (CCND1, PTEN, and MMP7) were down-regulated. However, expression was higher in CDX2 with 5 sec treatment to H. pylori, followed by 12 hr incubation with gastric epithelial cells. H. pylori genes, 16s rRNA, and Cag A are down-regulated with the treatment of C and E (except HJ9) while Bab A was down-regulated with C (Fig. 6 c, e). Moreover, PTEN and MMP7 were down-regulated with oral rinse solutions B, C, and E (Fig. 6 k, m). Interestingly, 30 sec treatment to I10 was able to abolish the expression of 16s rRNA; CagA; and BabA with solution B; B, C, D, and E; and B, C, and E, respectively (Fig. 6 b). Our results also revealed that expression of CDX2; and MMP7 were higher with solutions C, D, E; B, C, and D respectively with the 30 sec treatment at 12 hrs time point (Fig. 6 j, n). Similarly, when we treated another slow-growing strain HJ9 for 5 sec with oral rinses followed by incubation with AGS. Strikingly a mixed response in gene expression profiling. In the case of PTEN; and MMP7, the expression is moderately enhanced with solutions C, D, and E; B, C, and D, respectively. Whereassolution B and E were able to diminish the expression of PTEN and MMP7, respectively (Fig. 6 l, n). 30 sec treatment of oral rinse B to the same strain followed by incubation shows slight downregulation in the expression of 16s rRNA, CagA, BabA, CCND1, CDX2, and PTEN, however, the expression of MMP7 was an exception (Fig. 6). Further, when we treated HB14, another slow-growing strain, for 5 sec with oral rinses solution followed by incubation with AGS, CagA, BabA, CCND1, CDX2, and MMP7 was considerably down-regulated with solution B (Fig. 6). Additionally, CCND1, CDX2, and MMP7 were minimally expressed with the treatment of B, C, and E, while PTEN is down-regulated with E (Fig. 6). Moreover, treatment of HB14 for 30 sec and incubation with AGS, also leads to the downregulation of CCND1, CDX2, and PTEN with C, D, and E, while expression of MMP7 was unregulated with C, D, and E (Fig. 6 n). However, CagA was abolished with B, C, and D (Fig. 6).
Furthermore, results reflect different gene expression profiles with the treatment of 5 and 30 sec in fast-growing strain HB1 (Fig. 6). Expression of CagA; and BabA were down-regulated with the treatment of B, D, and E; and B, C, D, and E, respectively (Fig. 6 c, e). Whereas the expression of CCND1; CDX2; and MMP7 were enhanced with B, C, D and E; E; and B, respectively (Fig. 6 g, i, m). Treatment for 30 sec to HB1 shows a minimal expression of CagA with solution B, C, and D, while; MMP7 was up-regulated with all the oral rinses (Fig. 6 d, n). Induction of apoptosis in the cancer cell is one of the widely used treatment regimes against cancer . Hence we have assessed apoptotic pathways that may be induced after growth inhibition of H. pylori due to the treatment of oral rinses.
Status of apoptotic gene expression
Earlier studies have classified cells as live, apoptotic, and necrotic after EB/AO staining . We investigated these cells (live, apoptotic, and necrotic) on infection with H. pylori treated with oral rinses for 30 sec (Fig. 7). Additionally, the evaluation of apoptotic pathways, such as intrinsic/extrinsic/independent, was performed after treatment of H. pylori isolates with these solutions for 5 and 30 sec through qRT-PCR (Fig. 8). 5 sec exposure of solution D in I10 strain was able to enhance the expression of APAF1, BID, and BAK (Fig. 8 c, e, g). Interestingly, I10 treated with solution C and incubated with AGS cells were able to suppress all studied apoptotic genes (Fig. 8). Whereas, other solutions were not able to change the expression patterns of these genes considerably. Furthermore, 30 sec treatment of I10 and incubation with AGS cells show a different pattern (Fig. 8). I10 treated with solution B was able to reduce the expression of all the selected genes except FADD (Fig. 8). However, treatment with solution D and E were slightly enhancing the expression of all pro-apoptotic genes (Fig. 8).
Furthermore, when we applied these solutions for 5 sec on HJ9 and incubated with AGS cells, an upsurge in APAF1 expression, while BCL2 found down-regulated except in solution D (Fig. 8 c, m). Surprisingly, 30 sec treatment of HJ9 reflected a varied gene expression compared to 5 sec. Oral rinse C, D, and E with 30 sec exposure were able to up-regulate all apoptotic genes except NOXA and PUMA (Fig. 8). However, the expression of anti-apoptotic BCL2 was reduced with oral rinse B (Fig. 8 n).
Application of these oral rinses for 5 sec on HB14 followed by incubation with AGS cells demonstrated that oral rinse D up-regulates all pro-apoptotic genes (Fig. 8). Interestingly, treatment with B was able to enhance extrinsic apoptotic regulator FADD and reduce the expression of all used intrinsic markers. It also diminished the expression of BCL2 (Fig. 8), fascinatingly, all apoptotic markers except PUMA were considerably down-regulated with the 30 sec treatment of solution D (Fig. 8). Moreover, the expression of FADD, APAF1, and BAK, were also reduced with solution E (Fig. 8 b, d, f).
Furthermore, in the case of fast-growing strain, HB1, solution C and E were slightly up-regulating the expression of FADD, APAF1, BID, and BAK (Fig. 8 a, c, e). In addition to this, solution B treated cells were able to up-regulate FADD, APAF1, BID, and NOXA (Fig. 8 a, c, g, k). Treatment of solution C for 30 sec to the same strain showed up-regulation of FADD, BID, and PUMA (Fig. 8 b, h, j). In contrary to this, slightly up-regulation was observed for BCL2 in all used solutions.