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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Background:

Intragastric growth of non-Helicobacter pylori bacteria commonly occurs during acid-suppressive therapy. The long-term clinical consequences are still unclear.

Aim:

To investigate the luminal and mucosal bacterial growth during gastric acid inhibition, in relation to the type and duration of acid-inhibitory treatment, as well as to concomitant H. pylori infection.

Methods:

A total of 145 patients on continuous acid inhibition with either proton pump inhibitors (n=109) or histamine2-receptor antagonists (H2RAs, n=36) for gastro-oesophageal reflux disease, and 75 dyspeptic patients without acid inhibition (control group) were included. At endoscopy, fasting gastric juice was obtained for pH measurement and bacteriological culture. Gastric biopsy specimens were examined for detection of H. pylori (immunohistochemistry) and of non-H. pylori bacteria (modified Giemsa stain-positive and immunohistochemistry-negative at the same location).

Results:

Non-H. pylori flora was detected in the gastric juice of 92 (41.8%) patients and in the gastric mucosa of 109 (49.6%) patients. In gastric juice, prevalence rate for non-H. pylori bacteria was higher in patients taking proton pump inhibitors than controls and those taking H2RAs (58.7% vs. 22.6% and vs. 30.6%, P < 0.0001 and P < 0.003, respectively), but did not differ statistically between H2RAs and controls. In gastric mucosa, prevalence rates for non-H. pylori bacteria were higher in patients taking proton pump inhibitors and H2RAs than in the controls (antrum: 46.9% and 48.6% vs. 25%, P < 0.05 for both; corpus: 52.2% and 56.8% vs. 23.7%, P < 0.001 for both), but did not differ between proton pump inhibitors and H2RAs. Both luminal and mucosal growth of non-H. pylori bacteria were significantly greater in H. pylori-positive than -negative patients taking proton pump inhibitors (P < 0.05 for both). Luminal growth of non-H. pylori flora increased with the intragastric pH level, whilst mucosal bacterial growth increased with the duration of acid inhibition.

Conclusions:

Non-H. pylori flora not only contaminates the gastric juice but also colonizes the gastric mucosa of a large proportion of patients treated long-term with acid inhibition. The relationship between H. pylori and non-H. pylori bacteria in the pathogenesis of atrophic gastritis and gastric cancer needs further elucidation.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

The acidic milieu of the stomach represents the first line of defence against ingested bacteria.1, 2 An intragastric pH below 4 has a bactericidal effect, whilst gastric juice with a pH above 4 enables bacterial colonization of the stomach.3[4]–5 Decreased gastric acid output may occur as a consequence of Helicobacter pylori gastritis or may be iatrogenically induced by gastric surgery or the administration of acid-suppressive therapy.6[7][8]–9 Earlier studies on intragastric bacterial overgrowth due to acid inhibitory medication brought into focus possible adverse events such as episodes of gastroenteritis, development of malabsorption syndromes, and formation of potentially carcinogenic N-nitroso compounds.10[11][12][13][14][15]–16

In recent years, considerable interest has emerged in the interactions between H. pylori, non-H. pylori bacteria, and acid-suppressive therapy. Some investigators have shown that, in H. pylori-positive patients, long-term acid suppression leads to a more rapid development of atrophic corpus gastritis, a condition associated with an elevated risk of gastric cancer.17[18][19][20]–21 Previous work of our group has indicated that this might be due to a co-infection of the stomach with non-H. pylori bacterial flora—secondary to acid inhibition.22, 23 It is therefore important to characterize more fully the determinants of non-H. pylori bacterial growth in such conditions.

In most clinical studies, the intragastric bacterial overgrowth was investigated by culture of gastric juice and only rarely of gastric mucosal tissue.13[14][15]–16, 24 Recently, our group has proposed a semi-quantitative assessment of the non-H. pylori bacteria in gastric mucosa, using a non-specific stain for total bacterial flora combined with a specific immunohistochemical stain for the detection of H. pylori.25, 26

In the present study we investigated the gastric luminal and mucosal growth of non-H. pylori bacteria, in groups of patients treated with acid inhibition compared to non-treated controls, in order to estimate their potential pathogenetic effect on the gastric mucosa. Special attention was paid to the impact of clinical features, type, degree and duration of acid-suppressive therapy, as well as concomitant H. pylori infection on the luminal and mucosal non-H. pylori bacterial growth.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Study population

A total of 220 consecutive dyspeptic patients referred to an open-access unit for upper gastrointestinal endoscopy were enrolled in a cross-sectional, case-control study. Case subjects were patients taking continuous acid-suppressive therapy for gastro-oesophageal reflux diseases (GERD), such as erosive and non-erosive oesophagitis and/or Barrett’s oesophagus. Continuous acid suppression was defined as at least one daily dose of either a proton pump inhibitor or a histamine2-receptor antagonist (H2RA), taken for either 6 weeks to 1 year (medium-term) or longer than 1 year (long-term), respectively. Eligible controls were dyspeptic patients with a similar distribution of age and gender, who did not receive acid-suppressive medication or antacids before referral and who had normal endoscopic findings. Exclusion criteria for all patients were: present or past history of peptic ulcer disease; previous gastric surgery and/or vagotomy; prior H. pylori eradication therapy; treatment with antimicrobial agents or prokinetic drugs within 30 days before endoscopy; chronic use of NSAIDs; and other gastrointestinal or major non-gastrointestinal pathology, including immune-compromising states. The study was approved by the Ethical Review Board of the University Hospital Maastricht and each subject gave informed oral consent before entering into the study.

Design of the study

General clinical records and a structured interview concerning the history of reflux symptoms, type, daily dose, and duration of acid-suppressive medication, any co-morbidity, co-medication or family history of gastric cancer were obtained from all patients.

Endoscopy was performed after an overnight fast using a carefully disinfected Pentax EG-2901 instrument. At the beginning of endoscopy, a 5–10 mL sample of gastric juice was obtained—through the suction channel of the endoscope into a sterile trap—for bacteriological culture. Fasting gastric juice pH was measured using pH paper strips with grading steps of 0.5 from pH 0 to pH 14 (Schleicher & Schüll GmbH; Dassel, Germany).27 During endoscopy, two antrum biopsies (2–3 cm proximal to the pylorus) and four corpus biopsies (midpart of the body mucosa, 4–5 cm above the corpus/antrum junction, along the greater curvature) were sampled for histological examination.

Culture

Samples of gastric juice were transported immediately to the laboratory and were processed within 4 h. Culture was performed according to the five segments method, inoculating 1, 10 and 100 μL onto each of the following plates: blood agar plates (Becton Dickinson 254098, Leiden, The Netherlands) for total bacterial counts; cysteine lactose electrolyte-deficient agar plates (C.L.E.D., Becton Dickinson 255529, Leiden, The Netherlands) for Gram-negative rods; anaerobic blood agar plates (Becton Dickinson 254084, Leiden, The Netherlands) for total anaerobic bacterial counts; and selective plates for H. pylori. For the latter species, Campylobacter and chocolate agar plates were used, which were incubated under microaerophilic conditions, at 37 °C, for 4 to 7 days. All other plates were incubated for a minimum of 48 h with first reading at 24 h for aerobes, and at 48 h for anaerobes. Bacterial growth was assessed semi-quantitatively as colony forming units (CFU) per mL gastric juice, with a lowest detection limit of 101 CFU/mL and an upper detection limit of 107 CFU/mL. The degree of bacterial colonization was scored as follows: grade 0, no bacteria detected; grade 1, < 103 CFU/mL; grade 2, 104–106 CFU/mL and grade 3, > 106 CFU/mL. Bacteria were identified by standard microbiological methods to species level.28

Histopathology

Serial sections of 4 μm from formalin-fixed and paraffin-embedded specimens were prepared for haematoxylin-eosin stain, modified Giemsa stain and an immunohistochemical stain (IMM). Immunohistochemistry with a purified polyclonal antiserum (DAKO B471, ITK diagnostics BV, Uithoorn, The Netherlands) in a dilution of 1:100 was applied to detect H. pylori. The presence of non-H. pylori bacterial flora was screened in the modified Giemsa stain, by morphology and location of the bacteria, as described previously.25, 26 To ascertain the presence of non-H. pylori bacteria, the immunohistochemistry stain of an adjacent section had to be H. pylori-negative on the same position. At least two adjacent sections of all biopsies were examined completely at 400 × magnification. If necessary a 1000 × magnification was used. This technique accurately detects the presence of non-H. pylori bacteria when compared to culture methods.26

The presence of both H. pylori and non-H. pylori bacteria was scored semi-quantitatively and graded as follows: grade 0, no bacteria detected; grade 1, occasionally bacteria detected; grade 2, scattered bacteria identified in several high power fields; grade 3, large amounts detected in many high power fields.29 All slides were reviewed by an experienced gastroenterological pathologist (A.B.) and by the main investigator (S.S.), without knowledge of patient identification. In the event of discordant results, the specimens were re-examined by a second pathologist and were discussed until agreement was reached.

Statistical analysis

Differences in dichotomous variables were analysed using unpaired non-parametric tests (χ2-test with Yates correction) or paired non-parametric tests (Wilcoxon’s rank-sum test). Differences in continuous variables were analysed using the Kruskal–Wallis test for multiple comparisons and the Mann–Whitney U-test for two group comparisons. Two-sided P-values < 0.05 were assumed to indicate statistical significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Study population

A total of 220 consecutive patients were enrolled in the study. The demographic and relevant clinical features are detailed in Table 1. Patients were subdivided into three groups on the basis of the type of acid-suppressive therapy they were receiving: a proton pump inhibitor group; a H2RA group; and non-treated controls. The age distribution and sex ratios did not differ statistically among the three patient groups.

Table 1.  Demographic, clinical and endoscopic characteristics of 220 patients enrolled in the study Thumbnail image of

Prevalence and density of bacterial flora in gastric juice and gastric mucosa

Overall, 56 (25.5%) patients displayed non-H. pylori bacteria simultaneously in gastric juice and in gastric mucosa; 36 (16.3%) patients had non-H. pylori flora in gastric juice only, 53 (24.1%) in gastric mucosa only. The remaining 75 (34.1%) patients had no evidence of non-H. pylori flora.

Gastric juice

Table 2 describes the prevalence and density of non-H. pylori bacteria in the gastric juice. Patients on proton pump inhibitors had higher prevalence of non-H. pylori bacteria, particularly oropharyngeal flora, compared to the controls (P < 0.0001) and to the patients on H2RAs (P < 0.003), whereas no significant differences were noticed between the H2RAs and controls. Oropharyngeal flora, including Neisseria spp., Streptococci spp., and Corynebacterium spp. was identified in 16 (21.3%) controls, in 10 (27.8%) patients on H2RAs and in 64 (58.7%) patients on proton pump inhibitors. Gram-negative Enterobacteriaceae were recovered from one (1.3%) control subject (Escherichia coli), one (2.8%) patient on H2RAs (Escherichia coli and Proteus mirabilis) and five (4.6%) patients on proton pump inhibitors (Escherichia coli in three patients, Proteus mirabilis in one patient, Enterobacter gergoviae in one patient and Klebsiella oxytoca in one patient). Anaerobes (bacteroides species) were recovered from two patients on proton pump inhibitors. In only one (1.3%) control subject and one (0.9%) patient on proton pump inhibitors, H. pylori bacteria were isolated by culture of gastric juice.

Table 2.  Prevalence and density of non-H. pylori bacterial flora in gastric juice Thumbnail image of

Gastric mucosa

Figure 1 illustrates the presence and distribution of the H. pylori and non-H. pylori bacteria in the gastric mucosa of patients receiving acid inhibition and in non-treated controls. The prevalence of H. pylori did not differ significantly between the three patient groups. However, the prevalence of non-H. pylori flora—in both antrum and corpus—was significantly higher in patients on acid suppression, either with proton pump inhibitors or with H2RAs, than in controls (antrum: P=0.003 and P=0.021, respectively; corpus: P < 0.0001 and P < 0.001, respectively). No significant difference in this respect was found between the proton pump inhibitor group and the H2RA group. The distribution of H. pylori between antral and corpus mucosa was similar in patients on H2RAs and controls, whilst patients on proton pump inhibitors had a lower prevalence of H. pylori in the antral than corpus mucosa (27.4% vs. 34.5%, P=0.033). The distribution of non-H. pylori flora between the antral and corpus mucosa did not differ significantly, within all three groups.

image

Figure 1. Prevalence and distribution of H. pylori and of non-H. pylori bacteria in gastric mucosa of patients on acid inhibition and non-treated controls.

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Figure 2 (A and B) depicts the density of H. pylori and non-H. pylori bacteria in the antral and corpus mucosa. In the antral mucosa, patients on proton pump inhibitors displayed lower density (grade 1–3) of H. pylori than controls (P=0.002), whereas no significant differences were found between patients on H2RAs and controls, or between patients on proton pump inhibitors and those on H2RAs (Figure 2A). In the corpus mucosa, a similar density of H. pylori was found among the three groups. The patients on acid suppression, either with proton pump inhibitors or with H2RAs, displayed higher density (grade 1–3) of non-H. pylori bacteria—in both the antrum and corpus mucosa—compared to controls (antrum: P=0.012 and P=0.005; corpus: P=0.05 and P=0.024, respectively, Figure 2B). No significant difference in the density of non-H. pylori flora was seen between the proton pump inhibitor group and the H2RA group. Additionally, within each group, no such differences were observed between the antral and corpus mucosa.

image

Figure 2. Density of (A) H. pylori and of (B) non-H. pylori bacteria in gastric mucosa of patients on acid inhibition and non-treated controls.

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Histologically, the non-H. pylori flora consisted mainly of cocci, although different types of rods were also detected. Most non-H. pylori bacteria were localized superficially, in the mucous layer (Figure 3A), but occasionally were also seen in the glandular lumina, on similar mucosal positions as the H. pylori (Figure 3B and C).

image

Figure 3. (A) Massive amounts of cocci present in the gastric mucous layer (1000 × objective) from a patient treated long-term with proton pump inhibitors. (B) Presence of cocci and rods in a gastric crypt from a patient with chronic atrophic gastritis and intestinal metaplasia. Bacteria were identified as non-H. pylori species by modified Giemsa stain (1000 × objective) and simultaneous negative immunohistochemistry. (C) Presence of H. pylori in a gastric crypt by immunohistochemistry (1000 × objective).

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Intragastric bacterial flora in relation to the type, degree and duration of acid-suppressive therapy

Fasting gastric juice pH values above 4 were measured more frequently in patients on proton pump inhibitors than in those on H2RAs and controls (71.6% vs. 19.4% and 14.7%, respectively, P < 0.0001 for both), whereas no significant differences were noticed between patients on H2RAs and controls.

H. pylori-positive subjects had significantly higher fasting gastric juice pH than the H. pylori-negative subjects, among all three groups (Figure 4). The highest gastric juice pH levels were observed in H. pylori-positive patients treated with proton pump inhibitors. Additionally, in patients treated long-term with proton pump inhibitors, the prevalence of non-H. pylori bacteria—in both gastric juice and gastric mucosa—was significantly higher in H. pylori-positive than -negative subjects (gastric juice: 26 out of 32, 81.2% vs. 32 out of 55, 58.2%, P=0.028; gastric mucosa: 27 out of 32, 84.3% vs. 33 out of 55, 60%, P=0.018, respectively).

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Figure 4. Median fasting gastric juice pH in H. pylori-negative (□) and H. pylori-positive (●) subjects. The error bars show the 10th and the 90th interquartile ranges.

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The patients with non-H. pylori bacteria in gastric juice were characterized by a higher gastric juice pH level than those without it (Figure 5A). No such relationship was found between the presence of non-H. pylori bacteria in the gastric mucosa and the gastric juice pH level (Figure 5B).

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Figure 5. Median fasting gastric juice pH in patients without (□) and in those with (●) non-H. pylori (nHp) bacteria in (A) gastric juice and (B) gastric mucosa. The error bars show the 10th and the 90th interquartile ranges.

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In the whole series, five categories of patients were distinguished according to the duration of acid-suppressive therapy they had undergone (Figure 6A and B). Age and gender did not differ significantly among the five categories of patients (P=0.76 and P=0.95, respectively). In gastric juice, the prevalence of non-H. pylori bacteria increased with the duration of acid suppression in patients on proton pump inhibitors, but not in those on H2RAs (P < 0.0001 and P=0.63 for patients treated > 36 months vs. those treated < 12 months, respectively; Figure 6A). In gastric mucosa, the prevalence of non-H. pylori bacteria increased with the duration of acid suppression, regardless of the type of antisecretory medication (P < 0.0001 and P < 0.05 for patients treated with proton pump inhibitors and with H2RAs > 36 months vs. < 12 months, respectively; Figure 6B).

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Figure 6. Non-H. pylori bacterial flora in (A) gastric juice and in (B) gastric mucosa in relation with the type and duration of acid inhibition.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

The current study documented a high prevalence and density of the non-H. pylori bacterial flora in the gastric mucosa of patients on acid-suppressive therapy. Previous reports have mainly focused on the luminal bacterial growth during natural and artificial hypochlorhydria and achlorhydria.3[4]–5 Hitherto, only very few studies provided insights into the mucosal bacterial growth, and none at all have investigated the relationship between the two intragastric compartments.24

Gastric luminal non-H. pylori bacterial growth

In the present study, fasting gastric juice pH levels were higher in H. pylori-positive compared to H. pylori-negative subjects, especially in the proton pump inhibitor-treated group. Additionally, H. pylori-positive patients on proton pump inhibitors were more likely to be infected with non-H. pylori bacteria than the H. pylori-negative patients. These findings are in line with those from previous studies showing a greater elevation of the intragastric pH in H. pylori-infected vs. non-infected patients treated with acid inhibition, which in turn may cause an enhanced colonization of the stomach with non-H. pylori flora.30[31]–32

The bacterial species isolated from the gastric juice were mainly of oropharyngeal origin, with Streptococci, Neisseria and Corynebacterium as the most common species. Only a few patients harboured faecal-type flora, in particular, Escherichia coli, Proteus and Klebsiella species. The growth of faecal-like bacteria in gastric juice during pharmacological acid inhibition describes large variations among studies, ranging from very few to as many as 30% or more of the treated patients.15, 16, 33 These discrepancies may reflect differences in population samples and methodologies. As an alternative or additional explanation, a higher susceptibility of such flora to acid—compared to the oropharyngeal-like bacterial species—may prevent its isolation.33

Gastric mucosal non-H. pylori bacterial growth

In the current study, gastric mucosal bacterial growth was detected in about 50% of the patients on acid inhibition, mainly after long-term treatment. Of particular note was the observation that non-H. pylori bacteria developed with a similar prevalence and density in the gastric mucosa of patients treated either with proton pump inhibitors or with H2RAs. This finding adds support to previously published data showing that intragastric bacterial growth may occur during acid inhibition with any type of effective antisecretory drug.12[13][14][15]–16

Interestingly, nearly half of the patients with histological evidence of non-H. pylori bacteria, had a ‘sterile’ gastric juice at the time of examination. As illustrated in Figure 6 (A and B), this was particularly evident in patients treated with H2RAs. It can be reasoned therefore that the fluctuations in the intragastric pH—which are far more commonly encountered during H2RA therapy—dynamically influence the luminal bacterial growth. In contrast, the gastric mucosa during acid inhibition seems to behave as a constant ‘bacterial reservoir’.

The discrepancy between luminal and mucosal growth of non-H. pylori flora also suggests that although hypochlorhydria (with subsequent colonization of gastric juice) is a prerequisite for the colonization of the mucosa, the latter may depend upon additional factors. It can be speculated that on a background of compromised gastric mucosal barrier (secondary to the H. pylori infection) the resident non-H. pylori bacteria develops pathogenic properties adhering to and even penetrating into the mucosa. As illustrated in Figure 3 (B and C), the identification of non-H. pylori bacterial species on similar mucosal positions to the H. pylori supports this hypothesis.

Unfortunately, in the present study we did not assess the bacterial composition in the gastric mucosa by means of microbiological culture. However, Dolby et al. have found a similar spectrum of micro-organisms in cultures of gastric juice and of gastric tissue.24 In view of this finding and of our own observations, it is conceivable that the similar composition of the two intragastric compartments reflects a two–way interaction:26 not only can the gastric juice non-H. pylori bacteria colonize the gastric mucosa, but with the constant renewal of the mucosa, the bacteria might flow back into the gastric lumen, as well.

General considerations and conclusions

The results of our study may trigger the question: Is the mucosal non-H. pylori bacterial growth of pathogenetic significance? It has been largely debated whether bacterial colonization of the stomach, during several conditions associated with hypochlorhydria, increases the risk of gastric cancer through nitrosamine synthesis.34, 35 Additionally, it has been suggested that non-H. pylori bacteria and their by-products may act as a persistent antigenic stimulus, and thereby augment the inflammatory response induced by an H. pylori infection.22, 23 The resulting cumulative damage to the mucosa caused by a double infection of the stomach with H. pylori and non-H. pylori bacteria may, in the long term, lead to development of atrophic gastritis and ultimately of gastric cancer.

The results of this study may also have relevance for clinical bacteriological research. We have shown that the sole assessment of luminal bacterial growth by culturing gastric juice—as performed in earlier studies—seems to underestimate the changes in the intragastric milieu. In addition, the semi-quantitative histological evaluation of the non-H. pylori flora used in this study provides information regarding the morphology and location of the bacteria, thus being a useful complement to the standard microbiological methods.

In summary, our study clearly demonstrates that the non-H. pylori bacterial flora not only contaminates the gastric juice but also colonizes the gastric mucosa of a large proportion of patients on continuous acid inhibitory medication. This seems to be largely influenced by the infection with H. pylori and the length of antisecretory therapy, either with H2RAs or with proton pump inhibitors. Further studies are needed to elucidate the role of the double infection of the stomach with H. pylori and non-H. pylori bacteria in the pathogenesis of atrophic gastritis and of gastric cancer.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

This study was supported by an educational grant from Astra Pharmaceutica B.V., The Netherlands.

Bibliography

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. PATIENTS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography
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