Gastric biopsy and juice collection from gastritis patients for isolation of clinical strains of H. pylori
Till date, there is no H. pylori strain reported from central India to the best of our knowledge. Moreover, strains from northern and southern India have been listed in previous reports . Interestingly, none of the studies from India has evaluated the association between gastritis or gastric cancer progression and pathogenic H. pylori strains. H. pylori were successfully isolated from five out of 14 biopsy samples and four out of 11 juice samples (Table 1). After observation on selective media, gram staining was performed for all isolates (Fig. 1). Further, three clinical strains of H. pylori isolated from biopsies and one from juice were subjected for the amplification of H. pylori (CagA) through qRT-PCR (data not shown). Further validation of the clinical isolates was confirmed through nucleotide sequencing (data not shown).
The growth pattern of different clinical isolates of H. pylori
Growth curve of confirmed strains of H. pylori was determined by recording OD at 600nm 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 compared to the other seven clinical and one reference strain (I10). Interestingly, HB1 and HB5 have similar but not identical growth pattern at all examined time point (fig. 2 b). It is also fascinating that the growth pattern of HB1 and HJ1 was quite different, even though they were isolated from the same gastritis patient (fig. 2 b) Other clinical strains (HB4, HJ9, HJ10, HB10, HJ14 and HB14) shows a similar growth pattern as I10 until 24 hrs.
To have a better understanding of growth pattern, we calculated fold change in growth compared to reference strain I10 (Fig. 2 b). These graphs are plotted in the form of bar chart at all recorded time points (0, 2, 6, 12, 18 and 24 hrs). Three to five folds faster growth was observed in HB1 and HB5 compared to I10 at 2 hrs and further, it is increasing up to 24 hrs. Moreover, strains such as HJ1, HB10, and HB14 were showing moderately higher growth (3 to 5 folds) compared to reference strain I10 from 6 hours onwards. Importantly, the growth of HB1 and HB5 were steadily increasing to 18 hrs. Hence, our study reflected two fast growing strains (HB1 and HB5) compared to other clinical isolates HB4, HJ9, HJ10, HB10, HJ14, HB14, HJ14 and reference strain I10.
Confirmation of chemical plaque control agents through LCMS
The chemical composition of the selected plaque control agents is reconfirmed through LCMS (Supp. Fig 2). Sophisticated Instrument Centre facility at IIT Indore was used for these two chemical identification techniques. Active components in the plaque control agents of A, B, C, D, and E were determined through LCMS. Hence, LCMS characterization validates the labeled active components in plaque control agents.
The growth pattern of selected clinical strains of H. pylori with chemical treatment
All chosen chemicals 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. 2 a, b). Interestingly, we observed that chemical A and C were not able to stop the growth of fast growing strain even after 30 sec treatment (Fig. 3 a). Although, growth of fast growing strain were accelerated after 2 hrs treatment with chemical A and C while chemical B, D, and E were able to inhibit the growth until 24 hrs post-treatment. Additionally, slow growing strain such as HJ9 and HB14 were not able to grow until 12 hrs with all used chemicals (Fig. 3 b). However, these strains start growing from 12 hrs onwards when treated with chemical C (Fig. 3 b). Notably, our reference strain, I10, has not shown any growth after treatment with all chemicals until 24 hrs (Fig. 3 b). In all these experiments, we have used untreated strains as positive controls. Also, we had included culture media as a negative control. No substantial growth was observed for I10 when bar graph was plotted (Fig. 3 c).
In this study, we have found that chemical A was least efficient in the control of HB1 growth followed by chemical C. The substantial growth of HB1 was observed from 6 hrs onwards while treated with chemical A. Furthermore, when HB1 treated with chemical C was able to grow 12 hrs post-treatment (Fig. 3 d). Moreover, the growth of another fast growing strain, HB5, treated with chemical A and C was suppressed until only 2 hrs (Fig. 3 e). We observed considerable growth of HB5, 6 hrs onwards in A and C treatment. Importantly, chemical B, D, and E were able to suppress the growth of HB1 and HB5 until 24 hrs in this study (Fig. 3 d, e).
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 these selected chemicals 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 strains with all effective chemicals alone and in combination were able to restrict the growth until 2 hrs (Fig. 4 a). Surprisingly, data recorded 6 hours post-treatment were demonstrating the growth of HB1 and HB5 with D, E and their combinations. Importantly, all groups in which chemical B is included shows to be restricting the growth of HB1 and HB5 (Fig. 4a). Interestingly slow growing strains HJ9, HB14, and reference strain I10 growth completely abolished with all above chemical combinations till 24 hrs (Fig. 4b). Moreover, when we treated these chemicals for 10 sec alone and in combination, a similar pattern was found as with 30 sec (Suppl. Fig 1). We also plotted bar graph representing fold change in growth compared to control at different time points (0, 2, 6, 12, 18 and 24 hrs) for clearer outlookFig. 4-c, d, e).
The growth pattern of H. pylori after chemical treatment on BHI Agar plate:
In addition to chemical treatment in liquid culture, we further evaluated the growth pattern on a BHI agar plate after 30 sec treatment with the selected chemicals. Representative pictures of chemical treated H. pylori strains are shown in fig. 5 a, b, c, d, e. The images were quantified using Image J (NIH) and graphs were plotted (Fig. 5 f, g, h, I, j). As expected, chemical treatment of C was not effective 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 chemical A treatment in all the strains, contrary to the growth in liquid culture (Fig. 5). As expected, no growth was observed after treatment with chemical B, D, and E at all the recorded time points.
Gene profiling of specific gastric cancer marker and H. pylori after chemical treatment
Expression of H. pylori genes namely 16s rRNA, Cag A, and Bab A were investigated in this experiment. Additionally, reported gastric cancer marker such as CCND1, CDX2, PTEN, and MMP7 were also included [25–28]. The mixed expression profile was observed on treatment with chemicals (B, C, D, and E) for 5 sec in I10 strain. On treatment with chemical B, H. pylori (16s rRNA, CagA and BabA) and GC markers (CCND1, PTEN, and MMP7) were downregulated. However, expression was higher in CDX2 with 5 sec treatment to H. pylori followed by 12 hr incubation with gastric epithelial cells (AGS). H. pylori genes, 16s rRNA and Cag A are downregulated with the treatment of C and E (except HJ9) while Bab A was downregulated with C (Fig. 6 c, e). Moreover, PTEN and MMP7 were downregulated with chemical 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 chemical 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 chemical B, C and D; C, D, and E 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 chemicals followed by incubation with AGS, we observed a mixed response in gene expression profiling. In case of PTEN; and MMP7, the expression is moderately enhanced with chemical C, D, and E; B, C, and D respectively. Whereas, chemical B and E were able to diminish the expression of PTEN and MMP7 respectively (Fig. 6 l, n). 30 sec treatment of chemical B to the same strain followed by incubation shows slight downregulation in the expression of 16s rRNA, CagA, BabA, CCND1, CDX2, and PTEN. However, expression of MMP7 was an exception (Fig. 6). Further, when we treated HB14, another slow growing strain, for 5 sec with chemicals followed by incubation with AGS, we observed that CagA, BabA, CCND1, CDX2, and MMP7 were considerably downregulated with chemical B (Fig. 6). Additionally, CCND1, CDX2, and MMP7 were minimally expressed with the treatment of B, C, and E, while PTEN is downregulated with E (Fig. 6). Moreover, when we treated HB14 for 30 sec and incubated with AGS we observed 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 varied gene expression profile with the treatment of 5 and 30 sec in fast growing strain HB1 (Fig. 6). Expression of CagA; and BabA were downregulated with the treatment of B, D, and E; and B, C, D, and E respectively (Fig. 6 c, d, e, f). Whereas, the expression of CCND1; CDX2; and MMP7 were enhanced with B, C, D and E; E; and B respectively (Fig. 6 g, h, i, j m, n). Treatment for 30 sec to HB1 shows minimal expression of CagA with chemical B, C, and D, while; MMP7 was upregulated with all the chemicals (Fig. 6 d, n).
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 30 sec chemical treated H. pylori (Fig. 7). Additionally, evaluation of apoptotic pathways such as intrinsic/extrinsic/independent was performed through qRT-PCR, after treatment with these chemicals for 5 and 30 sec (Fig. 8). 5 sec exposure of chemical D in I10 strain was able to enhance the expression of APAF1, BID and BAK (Fig. 8 c, e, g). Interestingly, I10 treated with chemical C and incubated with AGS cells were able to suppress all studied apoptotic genes (Fig. 8). Whereas, other chemicals were not able to change the expression pattern of these genes considerably. Further, 30 sec treatment of I10 and incubation with AGS cells show a different pattern (Fig. 8). I10 treated with chemical B was able to reduce the expression of all the selected genes except FADD (Fig. 8). However, treatment with chemical D and E were slightly enhancing the expression of all pro-apoptotic genes (Fig. 8).
Furthermore, when we applied these chemicals for 5 sec on HJ9 and incubated with AGS cells, we witnessed an upsurge in APAF1 expression, while BCL2 was downregulated except in chemical D (Fig. 8 c, m). Surprisingly, 30 sec treatment of HJ9 reflected a varied gene expression compared to 5 sec. Chemical C, D, and E with 30 sec exposure were able to up-regulate all apoptotic genes except NOXA and PUMA (Fig. 8). However, expression of anti-apoptotic BCL2 was reduced with chemical B (Fig. 8 n).
Application of these chemicals for 5 sec on HB14 followed by incubation with AGS cells demonstrated that chemical 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 chemical D (Fig. 8). Moreover, the expression of FADD, APAF1, and BAK, were also reduced with chemical E (Fig. 8 b, d, f).
Furthermore, fast growing strain HB1 was treated for 5 sec and incubated with AGS for the detection of apoptotic and anti-apoptotic genes. Here, chemical C and E were slightly up-regulating the expression of FADD, APAF1, BID and BAK (Fig. 8 a, c, e). In addition to this, chemical B treated cells were able to up-regulate FADD, APAF1, BID and NOXA (Fig. 8 a, c, g, k). Treatment of chemical 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 chemical treatment.