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The human pathogen Helicobacter pylori grows in vitro between pH 5.5 and 8.5 (10). During primary colonization, it has to overcome pH values of 1 to 2 in the gastric lumen before reaching the protective mucosa, with a pH close to neutral. It is likely that only a few cells suffice for infection and that such a low concentration of bacteria in the stomach cannot change bulk values of pH and urea concentration, which amounts to 1.7 to 3.4 mM for humans (9). H. pylori produces huge amounts of urease (11). Originally, it was thought that the enzyme is extracellular and that in the stomach protection occurs by the creation of a cloud of ammonia around the cells (3). However, urease activity is cytoplasmic, and UreI regulates its activity by mediating acid-triggered urea uptake (16). The formed NH₃ is believed to increase the buffering capacity of the periplasm (7, 17).

From previous experiments, evidence is lacking that H. pylori survives at pH 1 to 2. In situations in which cells have been suspended at high concentrations in nonbuffered, urea-containing solutions of low pH, external pH returned to neutral within minutes due to NH₃ formation, relieving the acid stress situation (1, 2, 7). In buffered solutions of low pH, cell counts decreased dramatically even in the presence of urea (1, 6, 7). Under these conditions, urease activity was abolished (17). With an improved cultivation procedure, we show that the acid tolerance of H. pylori has been widely underestimated. In the presence of urea, the pathogen survived for several hours at pH 1, and cytoplasmic pH (pH_in) remained almost neutral.

H. pylori wild-type strain DSM 4867 (NCTC 11637) was cultivated in acid-precipitated medium (APM) at 37°C by shaking at 140 rpm under microaerobic conditions in a 2.5-liter anaerobic jar using Anaerocult C (Merck). APM was prepared by supplementing brucella broth (Difco) with 5% fetal calf serum (Fisher Scientific) and titration with HCl to pH 3. Precipitated protein was removed, and the supernatant was neutralized with NaOH to pH 7. Cells grown for 24 to 48 h were subcultured at least three times in fresh prewarmed and 10% CO₂-equilibrated medium (pH 6.5, calculated optical density at 578 nm [OD₅₇₈] of inoculation of ~0.001), and finally grown to an OD₅₇₈ of ~0.2. All experiments were performed at 37°C under continuous gassing (5% O₂, 10% CO₂, 85% N₂). Acid shock was exerted by diluting the cells 50- to 100-fold in APM medium of pH 1. Under these condition, the cells did not increase external pH or exhaust urea (added at 3 mM) within 3 h. For the determination of pH_in or protein synthesis, cell suspensions of about 7 × 10⁷ CFU/ml (OD₅₇₈ ~ 0.2) were used. Urea was added at a concentration of 50 or 40 mM followed immediately by titration with HCl to pH 1 or 2, respectively. Subsequently, medium pH was kept constant by occasional titration with HCl. For the acid shock experiment in citric acid, cells were first collected by centrifugation, resuspended to an OD₅₇₈ of about 2 in 150 mM NaCl, and diluted 10-fold with 100 mM citric acid (pH 2). Urease activity was monitored by the decrease of urea concentration in the medium, determined as described elsewhere (12). CFU counts were determined by diluting samples in prewarmed brucella broth containing 5% fetal calf serum, followed by plating on prewarmed agar medium. Colonies were counted after 3 to 5 days. Protein synthesis was determined by measuring the incorporation of [³⁵S] methionine into acid-precipatable material. For this purpose, either methionine-free assay medium (Difco) supplemented with 150 mM NaCl (pH 6.5) or citric acid (pH 2) containing 40 mM urea was used. pH_in was estimated from the distribution of [¹⁴C]salicylate across the cell membrane (5, 15), using the silicone oil filtration method (8).

Using 50- to 100-fold-diluted cell suspensions (equivalent to a calculated OD₅₇₈ of 0.004 to 0.002), which mimics the situation in the stomach most closely, about 45% H. pylori cells survived acid shock from pH 6.5 to pH 1 for 3 h in the presence 3 mM urea (Fig. 1). However, such a low cell concentration was not suitable for measuring urease activity, pH_in, or protein synthesis of H. pylori. Thus, those experiments were performed at about 7 × 10⁷ CFU/ml (OD₅₇₈ ~ 0.2) and started with 40 to 50 mM urea to guarantee urea supply for at least 1 h after acid shock to pH 2 or 1. Under these conditions, H. pylori survived for 1 h without any significant reduction of CFU (Fig. 2A). In the absence of urea no viable cells, were detected after 30 min of exposure to pH 1 or 2 (data not shown). Urease activity amounted to 0.63 ± 0.07 and 0.38 ± 0.07 µmol of urea · min¹ · 10⁸ CFU at 1 and pH 2, respectively.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Survival of H. pylori at pH 1 in the presence of 3 mM urea. Cells grown to an OD578 of ~0.2 were diluted 50-fold () or 100-fold (). Mean values and standard deviations were calculated from results of two different cultures.

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Survival (A) and pHin (B) of H. pylori after acid shock. (A) CFU determined by plating diluted samples; (B) pHin calculated from the distribution of [14C]salicylate across the cytoplasmic membrane. Urea (40 and 50 mM for pH 2 and 1, respectively) and 1 mM 2, 4-dinitrophenol (2,4-DNP) were present as indicated. Bar symbols: , cells in APM at pH 6.5 prior to acid shock; , cells in APM at pH 2; , cells in APM at pH 1; , cells in citric acid at pH 2; , cells in APM at pH 2 supplemented with chloramphenicol (50 µg/ml); , cells in APM at pH 1 supplemented with chloramphenicol (50 µg/ml). Mean values and standard deviations were taken from two to four independent experiments.

At pH 6.5, prior to acid shock the pH_in of H. pylori was 6.9 (Fig. 2B), confirming previous results (5). After acid shock to pH 2 or 1, pH_in remained almost neutral (5.8) in the presence of urea (Fig. 2B). In citric acid of pH 2, pH_in was 5.5 and the cells also survived for at least 1 h (Fig. 2). Addition of 1 mM of the uncoupler 2, 4-dinitrophenol lowered the pH_in to 4.1. Similar results have been obtained with acidophilic organisms at low pH (8). In the absence of urea pH_in was also close to 4 (Fig. 2B), suggesting that H. pylori was not capable of maintaining a physiologically relevant pH gradient under these conditions. Like others (13), we observed that in the presence of uncoupler, urease activity was completely abolished at low pH. Our data suggest that at a pH_in of 4.1 urease becomes inactive, which is in accord with in vitro data (17).

Addition of of chloramphenicol (50 µg/ml) 15 min prior to acid shock did not reduce the viability of H. pylori at pH 1 or 2 (Fig. 2A). At pH 6.5, protein synthesis was inhibited by more than 95% by the inhibitor. Compared to pH 6.5, incorporation of [³⁵S]methionine at pH 2 without inhibitor was only 0.5% and even less when chloramphenicol was present. These results suggest that protein synthesis during acid shock was not essential for the survival of H. pylori at low pH.

In conclusion, first, we demonstrated a much higher level of acid tolerance of H. pylori than reported previously. Most likely, the use of rapidly growing cultures (with a doubling time of about 2.7 h) and as little stress and manipulation of the cells during the acid shock procedure as possible were essential for our success. Second, urease activity appears to trigger pH_in homeostasis. We favor the mechanism proposed in reference 18 for other bacteria in which the NH₃ formed in the cytoplasm buffers away protons leaking in through the cytoplasmic membrane and NH₄⁺ leaves the cells electrogenically. Finally, our data also indicate that H. pylori can effectively transit the gastric acid barrier without any previous acid adaptation or the formation of coccoid forms as has been postulated by others (4, 7, 14). Apparently, cells grown at pH 6.5 are already fully adapted to long-term survival at low pH.