Penicillins inhibit cell wall synthesis; therefore, Helicobacter pylori must be dividing for this class of antibiotics to be effective in eradication therapy. Identifying growth responses to varying medium pH may allow design of more effective treatment regimens.
To determine the effects of acidity on bacterial growth and the bactericidal efficacy of ampicillin.
H. pylori were incubated in dialysis chambers suspended in 1.5-L of media at various pHs with 5 mM urea, with or without ampicillin, for 4, 8 or 16 h, thus mimicking unbuffered gastric juice. Changes in gene expression, viability and survival were determined.
At pH 3.0, but not at pH 4.5 or 7.4, there was decreased expression of ~400 genes, including many cell envelope biosynthesis, cell division and penicillin-binding protein genes. Ampicillin was bactericidal at pH 4.5 and 7.4, but not at pH 3.0.
Ampicillin is bactericidal at pH 4.5 and 7.4, but not at pH 3.0, due to decreased expression of cell envelope and division genes with loss of cell division at pH 3.0. Therefore, at pH 3.0, the likely pH at the gastric surface, the bacteria are nondividing and persist with ampicillin treatment. A more effective inhibitor of acid secretion that maintains gastric pH near neutrality for 24 h/day should enhance the efficacy of amoxicillin, improving triple therapy and likely even allowing dual amoxicillin-based therapy for H. pylori eradication.
Helicobacter pylori, a gram-negative neutralophile, is the only organism known to colonise the normal acid-secreting human stomach. About 50% of the world's population is infected. Colonisation is associated with several gastric diseases, including gastritis, peptic and duodenal ulcers, gastric carcinoma and MALT lymphoma.[2-5] In 1994, the WHO classified the organism as a type 1 carcinogen. Despite recent findings that H. pylori infection may have a beneficial effect on gastroesophageal reflux disease (GERD) and other nongastric manifestations of infection, the consequences of gastric colonisation alone argue for its eradication.[7, 8]
Standard H. pylori eradication therapy involves a complicated regimen of at least two antibiotics (clarithromycin and amoxicillin or metronidazole) and a proton pump inhibitor or antibiotics, acid suppression and bismuth.[9, 10] It has never been clear as to why two antibiotics, one targeting cell wall biosynthesis and the other targeting protein synthesis, are required for eradication. Acid suppression clearly improves the efficacy of antibiotic treatment.
Emerging antibiotic resistance to clarithromycin has made successful treatment of infection progressively more difficult, with the success rate of standard triple therapy now at 70%, well below the 80% required for treatment of infectious diseases. Alternative eradication regimens have been suggested, such as sequential therapy (PPI and amoxicillin for 5 days followed by PPI, clarithromycin and metronidazole) or concomitant therapy, but the results of these regimens are not comparable to the initial eradication results with triple therapy (~95%). Quadruple therapy (addition of colloidal bismuth subcitrate to standard triple therapy regimens) has shown promise, with cure rates currently between 80% and 90%.[9, 12] However, as bismuth containing quadruple therapies also rely on antibiotics, this regimen is sensitive to and contributes to increasing antibiotic resistance. Therefore, it is likely that eradication rates will decrease with this regimen as it has for triple therapy.
There have been several hypotheses as to the role of PPIs in eradication therapy. A direct effect of the PPI on the viability of the organism in vitro has been suggested. However, this required the administration of very high doses of the PPI to show direct inhibition. At these concentrations, there is acid-independent thiol reactivity of the PPI, resulting in nonspecific effects. Another hypothesis is increased gastric bioavailability of amoxicillin after PPI administration, but it is generally agreed that gastric acidity does not influence the pharmacokinetics of this antibiotic.
Helicobacter pylori resistance to amoxicillin is rare, suggesting it would be the antibiotic of choice as long as the bacteria remain susceptible to its action. Amoxicillin acts by inhibiting the synthesis of the bacterial cell wall. It inhibits cross-linking between the linear peptidoglycan polymer chains that make up a major component of the cell wall. Therefore, bacteria must be actively dividing and synthesising the cell envelope for this antibiotic to be effective. In the absence of urea, H. pylori enters a nonreplicative, but viable state when the environmental pH is less than 6.0 and greater than 3.0.[16, 17] In vivo gene expression of H. pylori indicates that the average pH at the site of infection in the gerbil stomach is about 3.5, placing the organism in a nondividing state, decreasing the efficacy of amoxicillin for eradication. Thus, increasing the intragastric pH to >5.0 should induce the bacteria to enter the replicative state and become more susceptible to both amoxicillin and to the protein synthesis inhibitor, clarithromycin.
Herein, we explore the effects of lengthy exposure of the organisms to pH 7.4, 4.5 and 3.0 in the presence of 5 mM urea, taking advantage of a method developed in our laboratory that exposes a small volume of the bacteria in a dialysis chamber to a large volume of pH-adjusted medium without added buffer, allowing analysis for 4, 8 or 16 h. Analysis of the bacterial transcriptome at pH 3.0 reveals down regulation of ~400 genes, including genes encoding envelope synthesis, cell division and protein synthesis. Analysis of viability and growth potential shows that cell viability is decreased at pH 3.0, but not at higher pH, and this pH also decreases the number of colony forming units (CFUs). Also, at pH 3.0, in contrast to the higher pH levels, the bactericidal effect of ampicillin disappears, indicating that the lack of growth at pH 3.0 attenuates penicillin sensitivity.
Bacterial strains and culture conditions
Helicobacter pylori strain ATCC 43504 was used. Bacteria were grown under microaerobic conditions (5% O2, 10% CO2, 85% N2) either on Trypticase Soy Agar (TSA) plates supplemented with 5% sheep blood (Gibco; Invitrogen, Carlsbad, CA, USA) or in brain heart infusion (BHI) medium (Difco; Becton Dickinson and Company, Sparks, MD, USA) supplemented with 7% horse serum (Gibco), 0.25% yeast extract (Difco) and Dent selective supplement (Oxoid Ltd, Hampshire, UK).
To investigate the effect of acid exposure on H. pylori in vitro, it is advantageous to extend the time of acid exposure to allow the organism to fully acclimate to a given environmental pH and to mimic the in vivo intragastric environment during the inter-digestive phase. It is not possible to maintain constant pH in H. pylori cultures in the presence of urea for this long a time without the use of a buffer, due to urea hydrolysis by urease, leading to an increase in pH in unbuffered solutions. To circumvent this problem and to maintain constant urea levels in unbuffered media, a system was developed using a micro-dialysis chamber (Slide-A-lyzer, MWCO 10 kD; Thermo Scientific, Rockford, IL, USA) containing H. pylori suspended in a large volume of pH-adjusted medium (1.5-L) containing 5 mM urea. This was capable of maintaining a constant pH for up to 16 h without depleting urea and allowed for analysis of bacterial status at different medium pHs (Figure S1).
Bacteria were incubated in BHI containing 7% horse serum, 0.25% yeast extract and 5 mM urea with and without 2 mg/mL ampicillin for all experiments, and pH was adjusted by the addition of HCl. Bacteria harvested from TSA plates (1 × 108) were suspended in 3 mL of BHI and injected into a sterile Slide-A-Lyzer Dialysis Chamber (Pierce), then placed in 1500 mL of media. Using this technique, it was possible to maintain constant pH and urea levels for up to at least 16 h without added buffer. Helicobacter pylori were incubated at pH 3.0, 4.5 and 7.4 for 4, 8 and 16 h. The bulk medium pH was recorded at the beginning and end of each experiment and no difference was found.
RNA isolation for transcriptomal analysis
Following incubation at various pH levels for 4 h, RNA was isolated from H. pylori using a combination of the TRIzol (Invitrogen) method and the RNeasy kit (Qiagen, Valencia, CA, USA) as described previously. RNA quality was determined using an Agilent (Santa Clara, CA, USA) 2100 bioanalyser. Only RNA with an RNA integrity number greater than 8 was used for microarray and qPCR analysis. All experiments were carried out in triplicate.
Microarray analysis was performed using a custom made 4 × 44 OligoMicroArray from Agilent. This chip contains 10 replicates of three, 60mer oligonucleotide probes to each gene of H. pylori strain ATCC 26695. Three independent microarray experiments were performed on each RNA replicate. All total RNA samples were serially diluted to a concentration of 1000 ng in 5.3 μL for use with the Quick Amp Labeling kit (Agilent). In preparation for the microarray, the Agilent One-Color Spike-In Kit was used as a positive control. cRNA labelling and microarray hybridisation were performed according to the manufacturers' protocol.
Data were analysed using Agilent's Feature Extraction Software as well as GeneSpring GX software (Agilent, Santa Clara, CA, USA). Data were filtered by expression (20–80%). All data with at least a twofold change compared with control were subjected to a t-test. Only those genes whose expression was altered by at least 1.5 fold with a P-value less than 0.05 were considered to represent significantly regulated genes.
RNA was isolated following incubation at pH 3.0, 4.5 and 7.4 as described earlier. cDNA was produced from RNA using Omniscript reverse transcriptase enzyme (Qiagen) and random primers (Invitrogen). The starting quantity of H. pylori RNA for all reverse transcriptase reactions was 6 μg. qPCR primers were designed using the help of Primer3 software. All primer sequences used are listed in Table 1 of the supplement. Primers were checked for the presence of a single product between 100–300 base pairs using standard PCR (30 cycles, 92°C denaturing, 55°C annealing, 72°C extension, 1 min each) with H. pylori gDNA as template.
Table 1. Genes involved in cell envelope biosynthesis and cell division down regulated at pH 3.0
1 μL cDNA was used for all qPCR reactions. H. pylori gDNA (isolated using the CTAB method) was serially diluted 10 fold × 4 dilutions and, including the original sample, a 5-concentration standard curve was used for all primer pairs. All reported data had standard curve efficiency within acceptable range (between 90% and 110%). qPCR was completed using the Bio-Rad CFX96 machine and Ssofast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) using hot-start (3 min × 92°C), followed by 40 cycles of 92°C × 30 s, 55°C × 30 s, 72°C × 40 s. A melt curve was completed at the end of each qPCR run and one peak was seen for all primer pairs. Fluorescence data were collected during the extension steps and data were expressed as mean starting quantity. All experiments were carried out in triplicate both in RNA isolation and in qPCR stages.
Quantitative live/dead assay
Bacterial viability was assayed using the LIVE/DEADBacLight Bacterial Viability and Counting Kit (Invitrogen) according to the manufacturer's instructions. Fluorescence intensity was measured on a Fluorolog-3 (Jobin Yvon Horiba, Edison, NJ, USA) fluorimeter. The ratio of green fluorescence intensity (Syto-9, Ex 488 nm, Em 498 nm; Invitrogen) to red fluorescence intensity (propidium iodide, Ex 488 nm, Em 617 nm) was calculated to determine changes in viability, where an increase in red fluorescence indicates cell membrane damage and loss of viability.
Measurement of survival at acidic and neutral pH
Following incubation, the bacterial suspension was removed from the Slide-A-Lyzer and added to 9 mL BHI, and serial dilutions were carried out using 1 mL into 9 mL BHI. 100 μL was plated in duplicate for each condition. Colonies were counted after 3 days.
Previous analyses of the gene expression profile of H. pylori have been performed for 30 min to 1 h in the presence or absence of urea at different medium pH levels ranging from 4.5 to 7.4. Using the constant pH/urea culture system, we analysed gene expression after a period of 4 h exposed to pH 7.4 or 3.0 in the presence of 5 mM urea. Approximately 463 genes changed expression twofold or greater, of which 273 were down regulated and 190 up regulated. Among the down regulated genes, there were genes involved in protein synthesis, flagellar activity, cell division and cell wall biosynthesis, including the two penicillin-binding proteins expressed by this organism (Table 1). Three cell division genes were down regulated by at least fourfold. This indicates that, at pH 3.0, the organisms are not dividing, resulting in persistence of the infection due to antibiotic insensitivity. This finding is in contrast to previous studies performed at pH 4.5, where there was no down regulation of cell envelope or cell division genes in the presence of urea. Other genes that were down regulated, also indicating lack of growth, were several amino acid biosynthesis genes, genes involved in DNA metabolism and several histidine kinases, such as the pH sensing HP0165 and HP0244 and its cognate class 2 flagellar regulator HP0703, as well as the HP1364/1365 couple (data not shown). Table 1 shows the microarray data for several cell envelope and cell division genes. The difference in mRNA levels of several genes following 4 h of incubation at pH 3.0 and 7.4 is represented as box–whisker plots in Figure 1.
Quantitative polymerase chain reaction
To confirm the microarray results and determine the level of gene expression with increased time of exposure to acid, qPCR was performed on selected cell wall biosynthesis and cell division genes (Figure S2). The change in gene expression in the qPCR assay corresponded well with the data observed in the microarray. For example, the level of the cell division protein Ftsl (HP1556) mRNA was reduced several fold at pH 3.0 as compared with either pH 4.5 or pH 7.4, as were the penicillin-binding protein HP0155, the cell wall gene murein tranglycosylase, and the metabolic NADH oxido-reductase. These data suggest that, at pH 3.0, the organisms are nondividing and not synthesising cell wall proteins, rendering the bacteria ampicillin insensitive at pH 3.0.
Helicobacter pylori viability in the presence of ampicillin at neutral and acidic pH
To investigate the bactericidal effects of ampicillin at different pHs, a commercial quantitative viability assay was used. This assay can reveal damage to the bacterial inner membrane by permeation of the fluorescent cation, propidium iodide, and hence demonstrate the deleterious effects of either pH or ampicillin. After 8-h incubation at pH 7.4, 4.5 or 3.0 supplemented with 5 mM urea in the absence of ampicillin, the live/dead ratio was similar at pH 7.4 and pH 4.5. However, incubation at pH 3.0 very slightly decreased the ratio, showing that there was decreased survival at this pH, but with still a significant number of viable bacteria. Ampicillin significantly decreased the viability of the bacteria at both pH 4.5 and 7.4, but had no effect at pH 3.0 (Figure 2a). Sixteen-hour incubation amplified the data observed after 8-h incubation. Again, there was a decrease in the ratio of live to dead organisms at pH 3.0 as compared to pH 7.4 or 4.5 in the absence of ampicillin and a large bactericidal effect of ampicillin at pH 4.5 and 7.4, but none at pH 3.0. (Figure 2b).
Helicobacter pylori survival in the presence of ampicillin at neutral and acidic pH
The enumeration of CFUs after exposure to acid or ampicillin is another measure of bacterial survival. After 8-h incubation, there was a marked decline in survival with exposure to ampicillin at pH 4.5 or 7.4 but no change with ampicillin at pH 3.0 (Figure 3a). Similar data were obtained after 16-h (Figure 3b) incubation. Incubation at pH 7.4 or 4.5 showed a larger number of CFUs as compared with 16-h exposure at pH 3.0. Of note, there are still viable bacteria at pH 3.0. The addition of ampicillin significantly reduced the CFUs of organisms incubated at pH 7.4 or 4.5, with only a small, insignificant reduction in CFUs at pH 3.0 (Figure 3a and b).
This study shows that at pH 3.0, H. pylori is viable, but in a nonreplicative state, diminishing the bactericidal efficacy of penicillin antibiotics and also that of protein synthesis inhibition by clarithromycin. This is supported by the large number of cell envelope and cell division genes down regulated at pH 3.0 and the loss of ampicillin efficacy. Induction of profound acid inhibition using potent H, K-ATPase antagonists, which raise the intragastric pH to between 5.0 and 7.0, should stimulate growth of H. pylori and increase the bactericidal effect of amoxicillin, which should result in eradication.
There has been considerable controversy as to the pH of the gastric habitat of H. pylori. It has been proposed that there is a gastric barrier to proton back diffusion from the gastric lumen to the gastric surface due to both mucus and bicarbonate secretion. However, recent experiments using fluorescent dyes or microelectrodes in infected mice show that the pH gradient collapses when the luminal pH falls to <3.0.[24, 25] Also, analysis of the transcriptome of bacteria isolated from the gerbil stomach in comparison with the pH dependent transcriptome in vitro showed that average pH in their gastric habitat was about pH 3.5.
The level and duration of acid suppression affects the success of eradication with PPIs.[26-28] Nocturnal acid breakthrough is a factor in the failure of triple therapy. The major location of bacteria resistant to treatment is the more acidic fundus, and hence the lower pH in this region promotes H. pylori growth inhibition and contributes to the failure of triple therapy.
Improved inhibition of acid secretion increases the rate of eradication. Cure rates of standard triple therapy depend on the efficacy of PPI-dependent inhibition of acid secretion. Although PPIs are covalent inhibitors of the gastric H, K-ATPase, their short plasma half-life prevents adequate elevation of pH during the night, when newly synthesised pumps are not exposed to the PPI. This, coupled with the slow growth rate of H. pylori, results in insensitivity of the organisms to growth dependent antibiotics, as the pH falls frequently to <3.0. In a recent study, q.d.s. omeprazole, dosed to maintain intragastric pH above pH 5.5 for 16 h, and amoxicillin dual therapy eradicated H. pylori infection, substantiating the idea that improved acid inhibition would improve results of eradication therapy.
Further evidence that increased PPI dwell time, and therefore better acid inhibition, improves H. pylori eradication comes from studies on slow omeprazole metabolisers. PPIs are mainly metabolised by cytochrome 2C19 (CYP2C19), and in patients who are homozygous for the loss of CYP2C19, leading to slower metabolism and increased plasma dwell time of the PPI, inhibition of acid secretion is significantly increased. These patients were successfully treated for H. pylori eradication with omeprazole and amoxicillin alone.[31, 32] Recently, it was shown that 10 mg rabeprazole q.d.s. maintained plasma PPI levels above the threshold level required for H+,K+-ATPase inhibition for 24 h, resulting in a median intragastric pH of 6.6 independent of CYP2C19 status. These results show that increased acid inhibition by PPIs may enable dual therapy, and this can be achieved by increasing the amount of time that this type of drug is in the blood. However, there have been several attempts at dual therapy with lack of success, likely due to inadequate elevation of pH during the night.[34, 35]
There are several PPIs, which would likely raise intragastric pH close to neutrality, tenatoprazole, a slowly metabolised PPI, AGN904, an omeprazole prodrug with a longer dwell time than omeprazole, and the potassium-competitive acid blockers TAK-438 and AZD0865.[36-40]
The data presented herein demonstrate that an improvement in acid inhibition extending into night time hours, maintaining the intragastric pH close to neutrality without acidic excursions overnight, would greatly improve eradication rates of triple therapy and perhaps allow dual therapy with a potent H, K-ATPase inhibitor and amoxicillin. These results provide a template for new clinical studies on H. pylori eradication.
Declaration of personal interests: None.
Declaration of funding interests: Supported by NIH and USVA grants P30 DK41301 (E. A. M.), K12 HD034610 (E. A. M.), DK053462 (G. S.), 1I01BX001006 (G. S.).