Streptococcus mitis Induces Conversion of Helicobacter pylori to Coccoid Cells during Co-Culture In Vitro

Helicobacter pylori (H. pylori) is a major gastric pathogen that has been associated with humans for more than 60,000 years. H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors. Another factor that might influence H. pylori colonization and diseases is the gastric microbiota that was overlooked for long because of the belief that human stomach was a hostile environment that cannot support microbial life. Once established, H. pylori mainly resides in the gastric mucosa and interacts with the resident bacteria. How these interactions impact on H. pylori-caused diseases has been poorly studied in human. In this study, we analyzed the interactions between H. pylori and two bacteria, Streptocccus mitis and Lactobacillus fermentum that are present in the stomach of both healthy and gastric disease human patients. We have found that S. mitis produced and released one or more diffusible factors that induce growth inhibition and coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. Using a metabolomics approach, we identified compounds that might be responsible for the conversion of H. pylori from spiral to coccoid cells. This study provide evidences that gastric bacteria influences H. pylori physiology and therefore possibly the diseases this bacterium causes.


Introduction
Helicobacter pylori (H. pylori) is a major gastric pathogen that has been associated with humans for more than 60,000 years [1]. Most of H. pylori-infected individuals develop an asymptomatic gastritis and can harbor this bacterium for their lifetime. In a subset of hosts, H. pylori causes different gastric diseases including dyspepsia, ulcers and gastric cancers. Disease development depends on several factors including the infecting H. pylori strain, environmental and host factors [2,3]. Another factor that is emerging as playing an important role in H. pylori-caused diseases is the host gastric microbiota. The role of gastric microbiota was previously overlooked because of the belief for long that human stomach was a hostile environment that cannot support microbial life. Studies subsequent to H. pylori discovery in 1981 showed that in fact human stomach constitutes a very diverse and complex ecosystem with a bacterial density comparable to that of the duodenum [4]. Gut microbiota plays important roles in several host functions including energy harvest and storage from the diet [5], development and regulation of the gut-associated mucosal immune system [6], regulation of the central nervous system [7], detoxification of xenobiotics and carcinogens, and protection against colonization by pathogens [8]. Although the gastric microbiota has been less studied than the microbiota in other gut sites, it is obvious that its composition and diversity are crucial for gut homeostasis.
Once established, H. pylori mainly resides in the gastric mucosa, a site that has a specific microbiota closely associated with the host [9]. H. pylori continuously interacts with the resident gastric bacteria, which affect not only H. pylori colonization but also the immune response to the infection [10]. Although not formerly demonstrated, it is likely that these interactions influence H. pylori colonization and disease development. This question has been poorly addressed in humans. However, studies in animal models of H. pylori infection have provided important insights. Studies in gerbil identified gastric bacteria that inhibited H. pylori colonization while others were enriched in H. pylori-infected animals [11][12][13]. Additionally, long-term infection by H. pylori significantly modified the composition of the gastric microbiota of gerbils [14]. A similar observation was made in mice where H. pylori infection altered the composition and diversity of the gastric flora [15]. In a mouse model of gastric cancer, H. pylori-induced carcinogenesis was delayed in the absence of the microbiota suggesting a role for gastric bacteria in the development of the disease [16,17]. Studies that analyzed the gastric microbiota of humans infected with H. pylori present the limitation of including a small number of patients and have yielded conflicted results [18]. However it can be hypothesized that the modification induced by H. pylori colonization including elevation of gastric pH, destruction of epithelial cells and production of metabolites favor the development of certain bacterial species and inhibit others.
In this study, we wanted to investigate the interactions between H. pylori and two bacteria, Streptoccus mitis and Lactobacillus fermentum, which have been isolated from gastric biopsies of both healthy and gastric disease human patients including H. pyloriinfected individuals [4,9,19,20]. S. mitis is a member of the human oral microbiota [21]. Considered for long as a commensal, S. mitis is presently viewed at least as an opportunistic pathogen as evidenced by several studies that have demonstrated its involvement in oral and systemic diseases [22]. Interestingly, S. mitis was found to be significantly enriched in the stomach of atrophic gastritis and gastric cancer patients [19]. L. fermentum belongs to the group of GRAS (Generally Regarded As Safe) lactic acid bacteria. It is a member of the human gastrointestinal microbiota and strains of L. fermentum have shown probiotic properties providing protection against respiratory infections [23][24][25] or being used in functional food [26]. Probiotics have recently attracted interest for the treatment of H. pylori infection, several lactic acid bacteria showing anti-H. pylori properties and can possibly provide an alternative to address the increase of antibiotic resistance [27]. We have found that S. mitis produced and released factors that induce coccoid conversion of H. pylori cells during coculture in vitro. In contrast, both H. pylori and L. fermentum released products that improved S. mitis survival during the stationary phase of growth. These interactions possibly impact on the diseases caused by H. pylori and could explain the increase of S. mitis cells in the stomach of certain gastric disease patients.

Materials and Methods
Bacterial strains, growth conditions and co-culture assay H. pylori strain NCTC 11637, S. mitis strain ATCC 6249 and L. fermentum strain ATCC 8289 were obtained from the American Type Culture Collection (ATCC, USA). H. pylori strain UM032 is a clinical isolate from the University of Malaya Medical Centre, Kuala Lumpur, Malaysia that was previously described [28]. All the bacteria were grown on chocolate-agar plate or in Brain Heart Infusion (BHI) broth supplemented with 0.4% yeast extract and 1% b-cyclodextrin, and incubated at 37uC in a humidified incubator with 10% CO 2 . This microaerophilic condition is needed for growth of H. pylori in vitro but is not a requirement for S. mitis and L. fermentum. However, for consistency, we grew all the organisms in the same conditions throughout the study. For bacterial co-culture, we used cell culture inserts (BD Biosciences, San Jose, CA, USA) that can be placed inside the wells of a12-well plate (BD Biosciences, San Jose, CA, USA) (Fig. 1). One bacterial culture was inoculated in the 12-well plate while the other microorganism was placed inside the insert. The two co-culture compartments were separated by a polyethylene terephathalate (PTE) membrane with 0.4 mm pores that prevented physical contact between the two bacteria while allowing free diffusion of macromolecules. To verify the absence of bacterial crossing of the membrane between the 2 compartments, BHI cultures of H. pylori, S. mitis and L. fermentum were inoculated in inserts that were placed on wells of a 12-well plate containing fresh BHI broth and incubated for 5 days in an incubator as described above. Presence of bacterial cells in the wells was verified daily by plating 100 ml of the broth onto chocolate-agar plates that were incubated at 37uC in an incubator with 10% CO 2 . No bacterial growth was detected in these tests for the 5 consecutive days (not shown). For co-culture assay with H. pylori, cells from a 2-3 day old chocolate-agar plate were used to make a suspension of OD 600 ,0.02 (10 6 -10 7 cfu/ml). Two ml of suspension of strain NCTC 11637 or of strain UM032 were distributed in each well of a 12-well plate. A cell culture insert containing 0.5 ml of a suspension of S. mitis or of L. fermentum at OD 600 ,0.008 (10 5 -10 6 cfu/ml) prepared from an overnight culture in BHI broth was then placed in each well. We found these proportions between the slow growing H. pylori and the faster growing S. mitis and L. fermentum to give the most reproducible results. For co-culture of S. mitis and L. fermentum, each compartment received a bacterial suspension at OD 600 ,0.008. The co-cultured bacteria were incubated at 37uC in a humidified incubator with 10% CO 2 for 7 consecutive days. At each day, dilutions from each of the co-culture compartments were plated onto chocolate-agar plates that were incubated at 37uC as described above. The bacterial count was determined after 1 day for S. mitis and L. fermentum, and after 3 days for H. pylori. Additionally, H. pylori cells were recovered during both mono and co-culture, Gram-stained and examined at the microscope to inspect the morphology of the bacteria. Each co-culture experiment was repeated at least three times.

Effect of supernatant on bacterial culture
Supernatant of H. pylori cultures were obtained by inoculating BHI broth with cells from a 2-3 day old chocolate-agar plate and incubated at 37uC as described above. After 1, 2 and 4 days of growth, the culture was centrifuged at 5,000 g for 5 min. and the supernatant recovered and filtered with a 0.22 mm syringe filter (Terumo Europe N.V., Leuven, Belgium). The filtered supernatant was used immediately. S. mitis supernatant was prepared similarly except that the BHI broth was inoculated (1/100 dilution) from an overnight liquid culture. To test the effect of the supernatants, 2 ml of H. pylori and of S. mitis suspension in BHI broth at OD 600 ,0.02 and ,0.008 respectively, prepared as described above were distributed in each well of a 12-well plate. Five hundred microliters of filtered supernatant of 1-, 2-and 4-day old cultures of the other bacterium were added to the wells and the plates incubated at 37uC as described above. At each day, dilutions of the cultures were plated onto chocolate-agar plate and incubated at 37uC and the bacterial count determined as described above. Additionally, H. pylori cells were recovered, Gram-stained and examined at the microscope to monitor the presence of coccoids.

Determination of bacterial viability
Bacterial viability was assessed by determining the metabolic activities of bacterial cells. For this purpose, the levels of cellular ATP were measured using the luciferase-based BacTiter-Glo assay kit (Promega, Madison, WI, USA) according to the manufacturer's recommendations. Briefly, 100 ml of bacterial cells from monocultures and from each compartment of co-cultures were separately mixed with 100 ml of BacTiter-Glo Reagent, incubated for five minutes at room temperature and the luminescence measured using a microplate reader (Varioskan Flash, Thermo Scientific, USA). The results were expressed as relative light units.
To determine the baseline ATP content of the bacteria (level of ATP in dead cells), cells from a bacterial culture at OD 600 ,1 were pelleted, then re-suspended in an equal volume of 10% formalin. The ATP levels in these formalin-killed bacteria were measured immediately after mixing and at different time points up to 4 hours.

Metabolite extraction
Metabolites were extracted from culture supernatants using methanol. Three hundred microliters of cold methanol was added to 100 ml of supernatant in a 1.5 ml Eppendorf tube. The sample was vortexed for 1 minute, incubated on ice for 30 minutes and then centrifuged at 8000 g, 4uC for 10 minutes. The supernatant was transferred to a new tube and vacuum-evaporated using a Labconco Refrigerated Centrivap concentrator (Kansas City, MO, USA) at 4uC. The sample was dissolved in mobile phase (95:5 acetonitrile:water) immediately before analysis.

Metabolite profiling by mass spectrometry
Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on a 1260 Infinity Quaternary LC System coupled with a 6540 Quadrupole Time-of-Flight (Q-TOF) mass spectrometer with Dual Agilent Jet Stream Electrospray Ionization (Dual AJS ESI) ionization source (Agilent Technologies, Santa Clara, CA, USA). Samples were separated using the ZORBAX Eclipse Plus C18 Rapid Resolution High Throughput (RRHT) 2.16100 mm 1.8 mm column (Agilent Technologies) and analyzed in both positive and negative ionization modes. For positive mode experiments, the mobile phases were water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). A linear gradient was run from 2% to 98% B over 25 minutes, at 0.5 ml/min. In negative mode, the mobile phases were water with 1 mM ammonium fluoride (A) and acetonitrile (B). Similarly, a linear gradient was turn from 2% to 98% B over 25 minutes, with a flow rate of 0.5 ml/min. The injection volume was 3 mL and three injections were made for each sample. ESI conditions were spray voltage 3.0 kV, gas temperature, 300uC, drying gas, 8 L/min, nebulizer 35 psig, VCap 3500 V, fragmentor 175 V and skimmer 65 V. The instrument was set to acquire over the m/z range of 100-1700 with an acquisition rate of 1 spectra/s. Three biological replicates were analyzed for each sample. In vitro co-culturing system to study the interactions between H. pylori, S. mitis and L. fermentum. One bacterium was inoculated in a well of a 12-well plate while the other bacterium was contained in a cell culture insert placed inside the well. The two compartments were separated by a 0.4 mm membrane that prevented physical contacts between the bacteria while allowing the diffusion of macromolecules. doi:10.1371/journal.pone.0112214.g001 Data processing and statistical analysis Spectrum was extracted using the MassHunter Qualitative Analysis software (version B.06.00) (Agilent Technologies). Statistical analysis was performed by Mass Profiler Professional (MPP) software (version 3.12.61) (Agilent Technologies). For normalization, entities were baselined to the median of all samples. This entity list was used for statistical analysis by applying unpaired Ttest (one-way ANOVA, asymptotic p-value,0.05) and Benjamini-Hochberg FDR of 1.0% as multiple testing corrections. Entities were compared between samples by fold change in relative intensity.

Results
To study the interactions between H. pylori, S. mitis and L. fermentum during growth in vitro, we established a co-culture method in which 2 bacterial species were grown in 2 compartments separated by a membrane that allowed exchange of diffusible molecules produced and released by the bacteria while preventing them from making a physical contact. In these assays, we wanted the cell densities in the 2 compartments to be as close as possible. For this we had to overcome 2 constraints: (i) H. pylori slowly grows in vitro while S. mitis and L. fermentum are fast growing organisms and (ii) in our growth conditions, inocula of high density were needed for H. pylori cultures to expand. We performed preliminary tests and from the results we chose starting inocula of H. pylori at 10 6 -10 7 cfu/ml and of S. mitis and L. fermentum at 10 5 -10 6 cfu/ml and monitored the cultures from 1 to 7 days after inoculation.

Streptococcus mitis induces growth arrest of Helicobacter pylori cultures
H. pylori mono-cultures increased in cell density by ,2 logs between day 1 and day 4 following inoculation and then stabilized up to day 7 (Fig. 2a). Interestingly, when co-cultured with S. mitis, cell density of H. pylori cultures dramatically dropped from day 1 and viable cells could not be detected after two days of co-culture (Fig. 2a). This phenomenon was not strain specific since both H. pylori UM032 [28], a clinical isolate and H. pylori NCTC, a laboratory strain displayed the same behaviour (Fig. 2b). In contrast to S. mitis, co-culture with L. fermentum did not affect the growth of H. pylori cells that was comparable to that of a monoculture (Fig. 2a). These results suggest that S. mitis specifically inhibits growth of H. pylori cells in co-culture. We next analysed the effect of H. pylori on S. mitis during coculture of the two bacteria. One day after inoculation, S. mitis mono-cultures already reached the stationary phase and the cell density dropped by ,1 log per day until day 4 (Fig. 3a). From day 5, culturable cells could not be obtained anymore (Fig. 3a). In contrast, during co-culture with H. pylori, S. mitis cells were detectable until the end of the experiment at day 7, and although the cell densities continued to drop, the decrease was ,1 log and viable cells could be cultured on days 5-7 when culturable cells could not be isolated from the mono-culture (Fig. 3a). Interestingly, L. fermentum displayed the same effect as H. pylori when cocultured with S. mitis (Fig. 3a). These results suggest that both H. pylori and L. fermentum release products that promotes cultivability of S. mitis cells during the stationary phase of growth in vitro.
To complete the analysis of the effects of the three bacteria on each other during co-culture, we monitored the growth of L. fermentum in the presence of H. pylori and of S. mitis. L. fermentum co-cultured with either species displayed a growth pattern similar to that of mono-cultures of the bacterium (Fig. 3b). All together these results suggest that H. pylori and L. fermentum released diffusible factors that promoted survival of S. mitis during the stationary phase of in vitro culture.

Streptococcus mitis induces conversion of Helicobacter pylori to coccoid
We wanted to further investigate the growth arrest of H. pylori cells co-cultured with S. mitis. We first verified whether the H. pylori cells were still alive or were killed by measuring their level of ATP, an indicator of metabolic activity. H. pylori cells in monocultures or co-cultured with L. fermentum had similar levels of ATP (Fig. 4a). When co-cultured with S.mitis, H. pylori cells displayed levels of ATP significantly lower than in mono-cultures at days 1, 2, 4 and 5 the times points tested in this experiment (Fig. 4a). However, these levels of ATP were significantly higher than the ones found in formalin-killed bacteria (Fig. 4d) suggesting that H. pylori cells in co-culture with S. mitis were alive. As controls, we measured the ATP contents of S. mitis (Fig. 4c) and L. fermentum (Fig. 4b) that, contrary to H. pylori, did not experience growth arrest. In both mono-and co-culture conditions, the bacteria displayed similar levels of ATP that were comparable to that in H. pylori cells in monoculture (Fig. 4a) but higher that ATP levels in H. pylori co-cultured with S. mitis. Collectively, these results showed that H. pylori cells that experienced growth arrest when co-cultured with S. mitis were alive though with a reduced Induction of Helicobacter pylori coccoid by Streptococcus mitts PLOS ONE | www.plosone.org metabolic activity. H. pylori is known to convert from a spiral to a coccoid shape in adverse conditions such as nutrient limitation, environmental stress, or presence of antibacterial compounds [29]. Since coccoid H. pylori cells are alive but non-culturable, we wanted to verify whether the presence of S. mitis induced this morphological change. For this, we performed microscopic examination of Gram-stained H. pylori cells grown alone or cocultured with S. mitis. H. pylori cells in monoculture appeared in bacillary form at days 1, 2 and 4 (Figs. 5a, 5c and 5e), while coccoid cells were detected at Day 6 ( Fig. 5g) likely indicating nutrient limitation in the medium. In contrast, during co-culture with S. mitis, coccoid H. pylori cells were predominant at Day 2 (Fig. 5d), a time point that corresponded to failure to obtain culturable bacteria (Fig. 2a), and were exclusively present at days 4 and 6 ( Figs. 5f and 5h). These results clearly showed that S. mitis induced morphological conversion of H. pylori to coccoid cells.
Coccoid conversion of Helicobacter pylori and extended survival of Streptococcus mitis during co-culture are mediated by diffusible factors released by the two bacteria The effects exerted by H. pylori and S. mitis on each other during co-culture might result from diffusible factors or metabolites the two bacteria produced. Alternatively, these effects could be explained by the difference in growth speed between the two organisms. S. mitis growing faster, it may deplete the medium in H. pylori compartment thus acquiring more nutriment while applying a stress on H. pylori that converts to coccoid. To differentiate between these possibilities, we compared the growth of each bacterium in the absence or in the presence of supernatants from 1-, 2-or 4-day old culture of the other organism. H. pylori cells supplemented with a 1-day S. mitis supernatant grew similarly as non-supplemented bacteria until day 5, then culturable cells could not be obtained at later time points (Fig. 6a). In contrast, in the presence of a supernatant from a 2-day old S. mitis culture, H. pylori cells experienced a growth arrest after two days of culture (Fig. 6a). The growth arrest was even more dramatic in the presence of a 4-day S. mitis supernatant that resulted in a failure to obtain culturable H. pylori cells at day 1 (Fig. 6a). These results indicated that growth arrest and coccoid conversion in H. pylori was mediated by a factor(s) released or a metabolite(s) produced by S. mitis that accumulate(s) during the stationary phase. Similarly to co-culture, survival of S. mitis cells was improved by addition of supernatants from H. pylori cultures (Fig. 6b). We did not observe differences in S. mitis survival depending on the age of the H. pylori culture. These results indicated that the benefit conferred to S. mitis is mediated by a diffusible product(s) present in H. pylori supernatant before the stationary phase of growth.

Identification of small moleculs involed in interaction of H. pylori and S. mitis by LCMS
To identify the factors that mediate coccoid conversion of H. pylori and survival of S. mitis during late exponential growth phase, we determined the metabolite profiles of supernatants from 1-, 2-and 4-day old monocultures and co-cultures of the two bacteria by LC/MS (see materials and methods). Metabolites that were statistically significantly up-regulated by more than two folds at any time point in triplicated experiments could be classified into three groups (Table 1). Group 1 comprises metabolites that were detected in the co-culture and in either of the mono-cultures (Table 1). Group 2 was constituted by compounds found only in the co-culture while Group 3 contained products from S. mitis and/or H. pylori mono-culture that were absent in the co-culture (Table 1). Metabolites involved in H. pylori conversion to coccoid or the extended survival of S. mitis are likely to belong to Group 1 while compounds in groups 2 and 3 mainly demonstrate interactions between H. pylori and S. mitis during co-culture. We reasoned that a compound that induced H. pylori conversion to coccoid should be present in supernatant from S. mitis and possibly from co-culture. Additionally, this compounds should start accumulating at day 2 since supernatant from this time point and not from a 1-day old culture induced coccoid conversion (Fig. 6a). Only one compound, a Tenovin-6-like molecule, that belongs to group 1 fulfilled these requirements ( Table 1). The Tenovin-6-like compound was significantly up-regulated in S. mitis supernatant at day 2 (8019 fold change) and drastically increased at day 4 (21234 fold change). This molecule was also significantly up-regulated in supernatants from co-culture at the three time points tested (Table 1).Three other metabolites could theoretically be involved in H. pylori conversion to coccoid. The first of these metabolites (KDNa2-3Galb1-3(KDNa2-6)Gal-NAcb1-4Galb1-4Glcb-Cer) was similar to a compound that belongs to the class of gangliosides and, similarly to the Tenovin-6-like molecule, was significantly up-regulated in S. mitis supernatant at day 2 and increased at day 4 (Table 1). However, this metabolite was absent in supernatants from the co-culture. The two other compounds were similar to Sulfoglycolithocholate, a secondary bile salts metabolite and to Caracurine V, a plant derived antimicrobial, respectively. These two compounds were detected in the co-culture but were absent in the mono-cultures (Table 1). To identify metabolites involved in S. mitis survival at the exponential growth phase, we reasoned that such compounds were to be expected in supernatants from H. pylori cultures at the three time points since these supernatants equally conferred the extended survival phenotype to S. mitis (Fig. 6b). None of the metabolites were up-regulated at least 2 folds in the three time points in H. pylori supernatant. A total of six compounds were Figure 5. Morphology of H. pylori cells in mono and co-culture. H. pylori NCTC 11637 cells in monoculture ((a), (c), (e) and (g)) and in co-culture with S. mitis ((b), (d), (f) and (h)) were recovered after 1 ((a) and (b)), 2 ((c) and (d)), 4 (e) and (f)) and 6 ((g) and (h)) of growth. The bacteria were Gramstained and microscopically examined with a 1000X magnification. doi:10.1371/journal.pone.0112214.g005   (Table 1). Although these compounds could theoretically play a role in S. mitis survival we believe their involvement in this phenomenon to be unlikely.

Discussion
In this study, we analyzed the interactions, during growth in vitro, between H. pylori and two bacteria, S. mitis and L. fermentum that have been isolated from the stomach of both healthy and H. pylori-infected gastric disease patients [4,9,19,20]. Using a co-culture method, we found that S. mitis produced and released one or more diffusible factors that directly or indirectly induce coccoid conversion of H. pylori cells. In contrast, both H. pylori and L. fermentum secreted factors that promote survival of S. mitis during the stationary phase of growth. We did not find any effect of H. pylori or S. mitis on the growth of L. fermentum during co-culture. To identify the factors responsible for coccoid conversion of H. pylori and for survival of S. mitis in the stationary phase, we performed metabolomics analysis of supernatants from mono-and co-cultures of H. pylori and S. mitis. We detected a few compounds that could possibly be involved in H. pylori's morphological conversion while we did not find molecules that match the phenotype conferred to S. mitis by co-culture or H. pylori supernatant supplementation. It should be noted that both H. pylori conversion to coccoid and S. mitis survival in the stationary phase could be mediated by proteins secreted by these bacteria. Such factors could not be detected by the LC/MS approach we used in this study.
One of the compounds we detected was similar to Tenovin-6 an anticancer molecule that was first identified in a screen for p53 activators (Lain et al 2008). p53 is a tumor suppressor encoded by a gene that is the most mutated gene in cancer. Tenovin-6 is currently subjected to intensive studies because of promises this molecule held in cancer treatment [30][31][32]. The Tenovin-6-like molecule identified in our metabolic profiling was the compound that best matched the induction of coccoid conversion of H. pylori. It was not significantly increased in supernatant from a 1-day old S. mitis mono-culture but was highly induced from day 2 and drastically increased at day 4 (Table 1). Coccoid conversion of H. pylori during co-culture with S. mitis was detected from day 2. Furthermore, 2-day old but not 1-day old S. mitis supernatants induced H. pylori coccoid conversion and this phenotype was more pronounced with supernatant from a 4 day old culture (Fig. 6a). Whether the effect of the Tenovin-6-like molecule has any significance during colonization of the stomach by H. pylori and S. mitis is unknown. However, the anticancer properties of Tenovin-6 and the possible role of coccoid H. pylori in tumorisation deserve much attention. Chan et al. analyzed gastrectomy specimens from cancer and peptic ulcer patients and found that coccoid H. pylori cells were enriched in adenocarcinoma compared to peptic ulcer samples [33]. Consistent with this finding, another study that compared the effects of spiral and coccoid H. pylori cells on gastric epithelial cells reported that coccoid H. pylori exerted a stronger effect on proliferation and a weaker effect on apoptosis than did spiral form [34]. These observations suggest an involvement of coccoid H. pylori in carcinogenesis. It is tempting to hypothesize from these observations that the Tenovin-6-like molecule produced by S. mitis antagonizes coccoid cells during colonization of the stomach by the two bacteria. Interestingly, during co-culture, the Tenovin-6like molecule started accumulating at day 1 while it was detected at day 2 in S. mitis mono-culture (Table 1). This observation suggests that spiral H. pylori but not coccoid cells stimulates the production of the compound by S. mitis. Further investigation are needed to elucidate the effect of Tenovin-6-like molecule on H. pylori both in vitro and in vivo.
The findings in this study reflect the numerous interactions that take place between the members of the gastric microbiota. These interactions contribute to shaping the composition of the gastric microbiota and indirectly influence the pathogenesis of bacteria like H. pylori. H. pylori is known to undergo a morphological change from spiral to coccoid form in adverse conditions [35][36][37][38][39]. Coccoid cells are more resistant to different stresses and represent a survival form of the bacterium [40,41]. However, how spiral H. pylori cells convert to coccoid in vivo and the role of coccoid cells in H. pylori pathogenesis are still unclear. Our findings point to a possible mechanism in which members of the gastric microbiota secrete factors that induce coccoid conversion of spiral H. pylori cells. These bacteria by this means indirectly influence H. pylori pathogenesis and disease outcome in infected individuals.