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The effect of omeprazole treatment on the gut microflora and neutrophil function
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ORIGINAL ARTICLE
The effect of omeprazole treatment on the gut microflora and neutrophil function Maja Kostrzewska a,∗, Agnieszka ´ Swidnicka-Siergiejko a, nska b, Gra˙ zyna Jurkowska a, Marzena Garley c, Dorota Olsza´ nska c, Wioletta Ratajczak-Wrona c, Ewa Jabło´ Jacek Jamiołkowski d, Andrzej Dabrowski a a
Department of gastroenterology and internal medicine, medical university of Bialystok, ul. Sklodowska-Curie 24A, 15-276 Bialystok, Poland b Department of microbiological diagnostics and infectious immunology, university hospital of Bialystok, Bialystok, Poland c Department of immunology, university hospital of Bialystok, Bialystok, Poland d Department of public health, university hospital of Bialystok, Bialystok, Poland
Summary Background and aim: Proton pump inhibitors (PPIs) may increase the risk of Clostridium difficile infections. There are interactions between gut microbiota and innate immune cells including neutrophils. We evaluated the effect of treatment with omeprazole on the gut microflora and neutrophil function. Methods: In 50 patients, we evaluated the effect of 4-week omeprazole treatment (n = 25 with 20 mg per day and n = 25 with 20 mg twice daily) on intragastric pH, results of stool culture and lactulose hydrogen breath test (LHBT) and neutrophil function. Results: The treatment caused significant increase of the mean intragastric pH, especially in the group with 20 mg omeprazole twice daily (from 2.05 ± 0.59 to 5.06 ± 1.6, P < 0.001). In LHBT, the increase of hydrogen concentration was observed in higher percentage of patients with 20 mg of omeprazole twice daily, compared to patients with the lower dose (42.1% vs 29.4%; ns). Four weeks of omeprazole treatment have caused considerable changes in stool culture results. Patients treated with higher dose of omeprazole have had some tendency to decrease diversity of colonic microflora in comparison with patients treated with the lower dose of omeprazole. Treatment with omeprazole did not result in C. difficile positive stool culture and had no significant effect on neutrophil function.
∗
Corresponding author. E-mail address: maja [email protected] (M. Kostrzewska).
http://dx.doi.org/10.1016/j.clinre.2017.01.004 2210-7401/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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Conclusions: Omeprazole treatment have caused considerable changes in stool culture results. Patients treated with the higher dose had some tendency to decreased diversity of colonic microflora and towards changes in fermenting bacteria of the gut. The potential effect of omeprazole on gut microflora does not depend on neutrophil function deterioration. © 2017 Elsevier Masson SAS. All rights reserved.
Introduction Proton pump inhibitors (PPIs) are currently prescribed on a large scale around the world and they are considered safe and effective drugs. Additionally, many patients are treated with PPIs without approved indications. Therefore, an increasing number of patients is being exposed to side effects of this treatment [1]. The most common of side effects (headache, diarrhea, abdominal pain, nausea, vomiting, flatulence) occur in 5% of patients. Among clinically relevant side effects are: increased risk of bacterial infections [2,3], malabsorption of calcium [4], vitamin B12 [5] and iron (resulting from iatrogenic hipochlorhydria) [6] and the formation of fundic gland polyps in patients on long-term PPI therapy [7]. Some studies also suggest that PPIs can affect the neutrophil functions such as chemotaxis, adhesion to endothelial cells and phagocytosis. The effect of these drugs on neutrophils has been evaluated mostly in vitro, in animal models and in patients with Helicobacter pylori infection [8—13]. Since PPIs strongly inhibit the secretion of hydrogen ions by the gastric parietal cells, they are widely used in the treatment of gastroesophageal reflux disease and peptic ulcer disease. However, the inhibition of gastric acid secretion can also trigger pathways leading to unwanted events. Hydrochloric acid provides an important protective barrier of defense against bacterial infections and the increase of intragastric pH may facilitate colonization of the gut by microbiota normally restricted to gastrointestinal tract above the stomach. In some situations, it may predispose to small intestinal bacterial overgrowth (SIBO) or gastrointestinal infections [14]. Salmonella is an acidsensitive bacteria unable to survive in pH < 3. The increased risk of Salmonella infection has been reported not only in patients after gastrectomy but also in patients taking PPIs [15]. The drugs inhibiting gastric acid secretion have been also proven risk factor for infection with Campylobacter [16,17]. Since the greater risk of gastrointestinal infection has been associated mostly with the PPIs rather than H2-receptor antagonists treatment, it may suggest the correlation between degree of inhibition of gastric acid secretion and the incidence of gastrointestinal infection. On the other hand, although Escherichia coli is an acid-sensitive organism, infections with these bacteria have not been reported more common in patients taking PPIs [3,14]. Gastric acid suppression is considered as a risk factor of Clostridium difficile—associated diarrhea (CDAD) [14,18]. The risk of C. difficile infection (CDI) ranged from 1.4 to 3.5 times higher among patients with PPI treatment compared with those without PPI therapy [19—22]. Several
studies reported that PPI administration may lead to changes in human gastrointestinal microbiota and reduce microbial diversity. Furthermore, in the gut microbiome of PPI-users significant changes in the amount of taxa that are associated with increased risk of CDI (Enterococcaceae and Streptococcaceae) were observed [23—25]. PPIs may also bind and inhibit non-gastric H+ /K+ -ATPases in bacteria and human cells including neutrophils and thereby impair the human immune status [9]. There is an increasing body of evidence about the interactions between gut microbiota and innate immune cells including neutrophils [26,27]. Consequently, PPIs have the potential of gut microbiota modulation by two independent pathways — through inhibition of gastric acid secretion and by the impact on neutrophil function. Therefore, the aim of this study was to evaluate the effect of 4-week outpatient treatment with oral omeprazole on human gut microbiota and function of neutrophils in vivo.
Patients and methods Before enrolling to the study, all the subjects had clinical examination and laboratory blood tests: complete blood count, concentration of C-reactive protein, fibrinogen, creatinine, urea, bilirubin, glucose, sodium, potassium in serum, activity of alanine aminotransferase and aspartate aminotransferase in serum, prothrombin time and urinalysis. The study has involved patients with normal results of physical examination and laboratory tests before enrollment. Other inclusion criteria were: no PPI treatment at least 14 days before the study, indications for use of PPI (dyspepsia, gastroesophageal reflux disease), no other medications during the study. One patient in omeprazole 20 mg twice daily group was exposed to antibiotics 2 months before enrolling to the study and 9 patients (5 in omeprazole 20 mg once daily group and 4 in omeprazole 20 mg twice daily group) were exposed to PPIs 4—8 weeks before the study. Exclusion criteria were: PPI or H2 receptor antagonist treatment within 14 days before the study, antibiotic use within 4 weeks before the study, current bacterial or viral infection, diabetes, cancer, immunodeficiency in medical history. Informed consent was obtained from all of the subjects. The study was approved by the Ethics committee of medical university of Bialystok. We have prospectively enrolled 33 women and 17 men. The mean age of the subjects was 42.8 ± 16.1 years. A proton pump inhibitor was randomly selected and administered orally for 4 weeks: 25 patients were treated with omeprazole 20 mg per day (taken 30 minutes before breakfast)
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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The effect of omeprazole treatment on the gut microflora and neutrophil function and 25 patients with omeprazole 20 mg twice daily (taken 30 minutes before breakfast and before the evening meal).
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• 4: very abundant growth (equivalent to more than 1,000,000 CFU/g).
Tests for Clostridium difficile Measuring intragastric pH Twenty-four-hour intragastric pH was evaluated before and after omeprazole treatment by Sleuth pH-analyzer (Sandhill Scientific Inc). The ComfortTEC Z/pH ZAI-BG-44 (Sandhill Scientific Inc) single-use probes with two pH channels (gastric and esophageal) were calibrated before procedure using the D82-3152 buffer solution pH 7,00 (Soundhill Scientific Inc) and D82-3151 buffer solution pH 4,00 (Soundhill Scientific Inc). To analyze data of pH-monitoring BioVIEW analysis ver.5.3.4.(Sandhill Scientific Inc) software was used.
Lactulose hydrogen breath test Lactulose hydrogen breath test (LHBT) was performed in 36 out of 50 patients before treatment and after 4-week omeprazole therapy: 17 patients with the dose of 20 mg per day and 19 patients with the dose of 20 mg twice daily. We were able to perform both LBHT measurements in 36 out of 50 patients, because not all patients reapplied for the second round of this test. They were instructed to avoid food ingestion for 12 hours before the test, to avoid nicotine products and chewing gum before and during the LHBT. The tests were performed using the breath hydrogen analyzer LactoFANQuick (Fisher ANalysen Instrumente GmbH). The dose of 10 g lactulose dissolved in 150 mL of water was administered. Exhaled air samples were taken immediately before and 30, 45, 60, 75, 90, 120, 150 and 180 minutes after the lactulose solution intake. Additionally, for each measurement, we have calculated area under the curve to show total hydrogen production after a lactulose load.
Methodology of stool cultures The culture of stool sample has been performed before the initiation and after completion (the next day after the last dose of the drug) of omeprazole treatment. The stool sample was inoculated into the Columbia agar of bioMerieux with 5% sheep blood, Columbia CNA agar with 5% sheep blood, MacConkey agar, Chromid VRE agar, ESBL agar and incubated for 18—24 hours at 35 ◦ C. The stool samples for Salmonella and Shigella cultures were inoculated into Selenite F Broth, incubated for 18—24 hours at 35 ◦ C afterward were seeded on Chromid Salmonella/Haectoen agar and incubated for 18—24 hours at 35 ◦ C. The cultured bacteria were identified with the GN and GP cards on the VITEK 2 XL analyzer. Bacterial counts in the colony-forming units (CFU) per 1 g of stool were estimated using traditional culturing methods. Data from stool test was described in semi-quantitative scale: • 0: no colony growth; • 1: single colonies (equivalent to 100—1000 CFU/g); • 2: scanty colonies growth (equivalent 1000—10,000 CFU/g); • 3: abundant growth (equivalent to 100,000 CFU/g);
to
The culture of stool sample has been done before the initiation and after completion (the next day after the last dose of the drug) of omeprazole treatment. It was performed on a Chromid Clostridium difficile medium by bioMerieux and incubated under anaerobic conditions (Genbag anaer) at 35 ◦ C for 48 hours. Then, in culture positive cases, the bacteria could be identified on the ANC cards with the VITEK 2XL analyzer. The presence of glutamate dehydrogenase (GDH) and toxins A and B was screened by C.diff Quick Chec Complete immunosorbent assay by TechLab. In GDH positive but C. difficile toxins negative cases, stool sample are inoculated into the Chromid C. difficile medium by bioMerieux and incubated under anaerobic conditions (Genbag anaer) at 35 ◦ C for 48 hours. The ability of C. difficile strain to produce toxins A or B is evaluated with the use C.diff Quick Chec Complete test by TechLab.
Function of polymorphonuclear neutrophils (PMNs) NBT test Nitroblue tetrazolium (NBT, Sigma) was dissolved in phosphate-buffered saline (Biomed). Spontaneous and stimulated NBT tests were performed. PMNs were isolated from peripheral blood and stimulated by latex (Sigma). The samples were incubated at 37 ◦ C for 15 minutes (NUAIRETMUS AUTOFLOW CO2 water-jacketed incubator), and then in room temperature for 15 minutes. The smear was performed and stained with a May-Grunwald-Giemsa solution (Aqua-Med). Under the 40 x magnification the number of neutrophils containing the formazan deposit has been counted among 100 encountered PMNs; the results has been shown as a percentage of NBT positive cells. Determination of total NO concentration in PMNs culture supernatants and serum NO production by PMNs was estimated using an indirect method based on the measurement of the nitrite concentration in PMNs culture supernatants and serum according to Griess’s reaction. In the analyzed samples nitrate was reduced to nitrite in the presence of cadmium and converted to nitric acid that gave a color reaction with Griess’s reagent. After 30 minutes, nitrite concentrations were determined by spectrophotometric analysis at 540 nm with reference to standard curve [28]. NO concentration in PMNs culture was expressed as M (supernatant from 106 cells suspended in 230 L of culture medium). Analysis of cGMP concentration in PMNs supernatants and serum The cGMP level in the PMNs culture supernatants and serum was measured using an enzyme-linked immunosorbent assay ® kit (R&D Systems ). The expression of iNOS and phospho-p38 MAP kinase Freshly isolated PMNs were lysed by sonification (SONICS vibra cell) in the presence of protease inhibitor cocktail
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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M. Kostrzewska et al. Table 1
Clinical characteristics of patients.
Age (mean ± SD, min-max) Male Medical history Heartburn Epigastric pain Belching Chest pain Cough Hoarseness GERD Comorbidities Arterial hypertension Coronary artery disease Medication before admission PPI H2 receptor antagonist NSAID Antihypertensive agents Statins Active smokers
Omeprazole 20 mg once daily
Omeprazole 20 mg twice daily
n = 25
n = 25
40.8 ± 15.8 (18—63) 9
44.7 ± 16.58 (20—81) 8
0.40 0.76
12 4 15 3 8 2 14
14 5 18 3 4 3 12
0.57 0.71 0.37 1.00 0.18 0.63 0.57
4 1
5 1
0.71 0.92
11 3 2 3 1 6
9 2 2 3 1 4
0.56 0.63 1.00 1.00 1.00 0.47
P-value
PPI: proton pump inhibitor; H2 receptor antagonist: histamine H2 receptor antagonist; GERD: gastroesophageal reflux disease; NSAID: nonsteroidal anti-inflammatory drugs.
(Sigma-Aldrich). The lysates were suspended in Laemmli buffer (Bio-Rad laboratories) and electrophoresed (Bio-Rad ® laboratories Mini-PROTEAN tetra cell) on sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad). The resolved protein was transferred onto nitrocellulose (Bio-Rad Laboratories ® Mini-PROTEAN tetra cell). The nitrocellulose was incubated (Milipore SNAP i.d.TM protein detection system) with the primary monoclonal anti-iNOS (1:10,000 R&D systems) or polyclonal antibody anti-phospho-p38␣ MAPK active form (1:10,000 ABR Affinity BioReagents). After washing with 0.1% TBS-T (Bio-Rad laboratories), the membrane was incubated with alkaline phosphatase-labelled anti-mouse or antirabbit IgG antibodies (Vector Laboratories). Immunoreactive protein bands were visualized using the 5-bromo-4-chloro-3indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) liquid substrate system (Sigma-Aldrich). The density of iNOS and p38 MAP kinase bands was determined using ImageJ software and quantified with arbitrary units.
Statistical analysis The results were analyzed using IBM SPSS Statistics 20.0 and GraphPad Prism 5. Data in clinical characteristics of patients have been analyzed by unpaired t-test (for age) and Chi2 test (other data). The intragastric pH is shown as mean ± SD and the differences between groups have been evaluated by t-test. In each patient, ANOVA test to analyze significant differences between LHBT before and after omeprazole treatment has been performed. The correlation between intragastric pH after treatment and change in of LHBT was evaluated by Pearson test.
Results Clinical characteristics of the patients There were no significant differences between groups before treatment (Table 1). The clinical characteristic of patients shown in Table 1 applies to the ‘‘before treatment’’ period of our study. Most of these symptoms disappeared after PPI treatment and in the course of our study, we have observed no additional symptoms or clinical signs of gastrointestinal tract infections after 4 weeks of omeprazole therapy in both groups.
Intragastric pH After omeprazole treatment, we observed significant increase of mean intragastric pH in both groups: patients treated with 20 mg per day (2.29 ± 1.05 vs. 3.8 ± 1.45; P < 0.001) and 20 mg twice daily (2.05 ± 0.59 vs 5.06 ± 1.61; P < 0.001). The effect of the higher dose of omeprazole was significantly stronger (P < 0.016) than the effect of the standard dose (Fig. 1).
Lactulose hydrogen breath test We have analyzed individual data from LHBT of 36 patients and found no change after omeprazole treatment in 15 patients, specifically: 47% (8/17) treated with 20 mg once daily and 36.8% (6/19) treated with 20 mg twice daily. The increase of hydrogen concentration in air samples was
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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Table 2 The changes in exhaled air hydrogen concentration measured by lactulose hydrogen breath test after omeprazole treatment. Exhaled air hydrogen concentration
Omeprazole 20 mg once daily n = 17
Omeprazole 20 mg twice daily n = 19
P
Increase No change Decrease
29.4% (5/17) 47.0% (8/17) 23.6% (4/17)
42.1% (8/19) 36.8% (6/19) 26.3% (5/19)
0.42 0.45 0.72
7
omeprazole 20 mg once daily
omeprazole 20 mg twice daily
omeprazole 20 mg once daily
Intragastric pH
6
omeprazole 20 mg twice daily
pH 8 7 6 5 4 3 2 1 0
5 1 vs 2 p <0.001 3 vs 4 p <0.001 2 vs 4 p <0.016
4 3 2 1 0 1 2 Before After treatment
3 4 After Before treatment
Figure 1 The effect of omeprazole in two different doses on intragastric pH. The data are shown as mean ± SD: 1: omeprazole 20 mg once daily before treatment; 2: omeprazole 20 mg once daily after treatment; 3: omeprazole 20 mg twice daily before treatment; 4: omeprazole 20 mg twice daily after treatment.
observed in 29.4% (5/17) patients with omeprazole 20 mg once daily therapy and in 42.1% (8/19) objects treated with omeprazole 20 mg twice daily, but the difference is not statistically significant (Table 2). Decrease of hydrogen concentration in air samples after omeprazole treatment was found in 23.6% (4/17) cases in the omeprazole 20 mg once daily group and in 26.3% (5/19) of 20 mg omeprazole twice daily group. The results of LBHT expressed as the area under the curve (AUC) for total hydrogen production after a
-1
pH
8 7 6 5 4 3 2 1 0
P=0.23
0
1
-1
P=0.48
0
1
Figure 3 The correlation between intragastric pH and change in exhaled air hydrogen concentration after omeprazole treatment. Change of hydrogen concentration in air samples after omeprazole treatment measured in lactulose hydrogen breath test: 0: no change; 1: increase; (−1) — decrease.
lactulose load for each patient treated with omeprazole are shown in Fig. 2.
Correlation between intragastric pH and exhaled air hydrogen concentration change in LHBT We found no correlation between intragastric pH after omeprazole treatment and lactulose hydrogen breath test results after 4 weeks of therapy (Fig. 3).
Figure 2 The results of LBHT expressed as total hydrogen production after a lactulose load in patients treated with two different doses of omeprazole. The data is shown for each patient as the area under the curve (AUC) before and after the omeprazole treatment.
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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M. Kostrzewska et al. Table 3
Bacterial strains and genera of fungi in stool cultures analysis after omeprazole treatment.
Specific taxa
Enterobacteriaceae Escherichia coli ESBL(−) Escherichia coli ESBL(+) Escherichia fergusoni Klebsiella pneumoniae Klebsiella oxytoca Proteus mirabilis Citrobacter spp Enterobacter spp Salmonella spp Shigella spp Enterococceae Enterococcus faecalis Enterococcus spp Pseudomonoaceae Pseudomonas aeruginosa Streptococceae Streptococcus spp Staphylococceae Staphylococcus CNS spp Staphylococcus aureus Anaerobes Clostridium difficile Bacteroides spp Yeasts Candida albicans Geotrichum spp
Effect after treatment
Omeprazole 20 mg once daily n = 24
Omeprazole 20 mg twice daily n = 24
Increase (new Decrease Increase (new Decrease Increase (new Decrease Increase (new Decrease Increase Decrease Increase (new Decrease Increase Decrease Increase (new Decrease
5 (1) 0 0
3 (0) 3 1 (0)
1 (1) 0 1 (1) 2 0 0 1 (1) 1 0 2 1 (1) 0 No growth No growth
0 0 2 (2) 0 0 1 1 (1) 0 0 0 0 3 No growth No growth
Increase Decrease Increase (new strain) Decrease
2 (2) 0 8 (3) 7
2 (2) 1 8 (4) 3
Increase Decrease
0 0
0 1
Increase (new strain) Decrease
2 (2) 0
2 (1) 0
Increase (new strain) Decrease Increase (new strain) Decrease
4 (4) 2 1 (1) 1
3 (3) 3 1 (1) 0
Increase Decrease
No growth 0 2
No growth 0 1
Increase (new strain) Decrease Increase (new strain) Decrease
3 (3) 3 1 (1) 2
1 (1) 4 3 (3) 0
strain) strain) strain) strain)
strain)
strain)
Increase: number of patients with higher score in the semi-quantitative scale of stool culture bacterial counts after treatment, compared to the score before treatment; Decrease: number of patients with lower score in the semi-quantitative scale of stool culture bacterial counts after treatment, compared to the score before treatment; New strain: number of patients with the new microorganism appearing in the stool culture after omeprazole treatment.
Stool cultures analysis With culturing of stool samples, we have identified 15 bacterial strains and 2 genera of yeast (Table 3). None of the cultures were C. difficile positive both before and after omeprazole treatment. The increase in colony count of
Streptococcaceace after treatment was observed in two cases in 20 mg omeprazole once daily group and in two cases of 20 mg twice daily omeprazole group. In 16 patients, the increase in the colony count of Enterococcaceae after omeprazole treatment was found: 8 patients in 20 mg once daily group and 8 patients in 20 mg twice daily group.
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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Table 4 The effect of omeprazole treatment on the NBT test results, the level of NO and cGMP in PMNs culture supernatant and serum as well as expression of inducible nitric oxide synthase (iNOS) and phospho-p38 MAP kinase in freshly isolated PMNs. Before treatment with omeprazole 20 mg once daily (n = 25)
After treatment with omeprazole 20 mg once daily (n = 25)
Before treatment with omeprazole 20 mg twice daily (n = 25)
After treatment with omeprazole 20 mg twice daily (n = 25)
1
2
3
4
NBT test spontaneous (%)
16.00 (3.00—59.00)
17.00 (5.00—51.00)
15.00 (2.00—53.00)
14.00 (3.00—48.00)
NBT test stimulated (%)
51.00 (18.00—73.00)
51.00 (18.00—69.00)
47.00 (18.00—68.00)
46.00 (14.00—68.00)
NO in PMNs culture (M)
19.32 (4.71— 60.87)
21.59 (10.8—46.02)
16.48 (8.52—45.46)
17.61 (7.39—40.34)
NO in serum (M)
30.68 (17.05—47.73)
31.25 (16.48—56.52)
31.82 (18.75—40.34)
31.82 (14.77—257.61)
cGMP in PMNs culture (pM/mL)
0.89 (0.26— 3.20)
0.96 (0.03—2.16)
0.53 (0.06—3.68)
0.85 (0.06—10.93)
cGMP in serum (pM/mL)
5.92 (1.04—41.0)
7.6 (0.03—48.5)
11.7 (1.28—46.0)
9.23 (0.06—320.69)
iNOS (arbitrary units × 103 )
70.602 (2.345—189.810)
64.030 (2.122—232.903)
82.775 (2.345—254.912)
115.821 (2.152—272.470)
p38 MAP kinase (arbitrary units × 103 )
87.475 (8.106—293.216)
80.363 (4.579—413.160)
74.356 (4.805—264.256)
63.972 (26.479—176.547)
Statistical significance
(1 vs 2) 0.516 (3 vs 4) 0.893 (2 vs 4) 0.404 (1 vs 2) 0.882 (3 vs 4) 0.326 (2 vs 4) 0.276 (1 vs 2) 0.333 (3 vs 4) 0.875 (2 vs 4) 0.130 (1 vs 2) 0.936 (3 vs 4) 0.435 (2 vs 4) 0.415 (1 vs 2) 0.300 (3 vs 4) 0.638 (2 vs 4) 0.836 (1 vs 2) 0.470 (3 vs 4) 0.272 (2 vs 4) 0.836 (1 vs 2) 0.925 (3 vs 4) 0.581 (2 vs 4) 0.093 (1 vs 2) 0.840 (3 vs 4) 0.353 (2 vs 4) 0.357
The data are shown as median (minimum-maximum); NBT: nitroblue tetrazolium; PMNs: polymorphonuclear neutrophils; NO: nitric oxide; cGMP: cyclic guanosine monophosphate; inducible nitric oxide synthase (iNOS).
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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The decrease in the colony count of Enterococcaceae was observed in 7 patients in 20 mg omeprazole once daily group and in 3 patients in 20 mg omeprazole twice daily group.
Function of PMNs NBT tests We have found no effect of omeprazole treatment on the results of both spontaneous and stimulated NBT tests (Table 4). Concentration of NO in PMNs culture supernatants and serum Treatment with omeprazole had no effect on NO concentration in the PMNs culture supernatant and serum (Table 4). Concentration of cGMP in PMNs culture supernatants and serum Treatment with omeprazole had no effect on cGMP concentration in the PMNs culture supernatant and plasma (Table 4). iNOS and phosphorylated p38 MAP kinase expression in isolated PMNs Treatment with omeprazole had no significant effect on determined by Western blotting iNOS protein expression (Table 4). Phospho-p38 MAP kinase signal in Western blotting shows only active, doubly phosphorylated forms of this kinase. Treatment with omeprazole did not stimulate or inhibit p38 MAP kinase activity in PMNs (Table 4).
Discussion In our present study, treatment with the standard dose of omeprazole (20 mg) has caused a modest increase of intragastric pH with the mean pH < 4. The significant inhibition of gastric acid production (mean pH 5.06 ± 1.61) has been achieved only after treatment with a double standard dose of 40 mg of omeprazole. Since the acidity forms one of the main defenses against bacterial influx that accompanies ingesting food and oral mucus, strong inhibition of gastric acid allows more bacteria to survive this barrier and reach the gut [29]. Recently, the effect of PPI treatment on gut microflora have been described in many studies [24,25,29—35]. Among the most commonly discussed issues are the risk of SIBO and C. difficile infection in patients treated with PPI. One of the recent studies reports that 6 months lasting treatment with PPI may produce bowel symptoms and SIBO in 26% of patients [31]. In contrast, in a retrospective study including 1191 patients, PPI use was not found to be significantly associated with the presence of SIBO [32]. Hydrogen breath tests using various substrates like glucose, lactulose, lactose and fructose are being used to diagnose SIBO and lactose or fructose malabsorption [36]. Lactulose is a disaccharide composed of galactose and fructose. As there is no naturally occurring lactulose enzyme in the body to hydrolyze this sugar in the small intestine, lactulose is not broken down in the small bowel and is transported intact to the colon where it is fermented by colonic bacteria [37]. The products of its metabolism include hydrogen. LHBT is routinely
used as a simple method for estimation of orocecal transit time. However, this test has drawn our attention as a simple tool to evaluate the population of gastrointestinal tract fermenting bacteria. In our study, treatment with omeprazole had no effect on LHBT results in 47.0% and 36.8% patients treated respectively 20 mg and 40 mg per day. Interestingly, comparing to the effect of 20 mg of omeprazole once daily, treatment with 20 mg of omeprazole twice daily favored augmentation of fermenting bacteria population in the gut. There is an increasing body of evidence suggesting that treatment with PPIs can lead to changes in the composition of the gut microbiota predisposing to the development of C. difficile infection (CDI) [24,25,34]. It has been shown that PPIs alter specific taxa in the human gastrointestinal microbiome i.e. reduction of Bacteroides and Clostridium cluster IV abundance, increase of Enterococcaceae, Streptococcae, Firmicutes and Lactobacillus and thereby cause the decrease in the alpha diversity of fecal microbiota. In the large study, the effect of PPIs on the gut microbiome was investigated in 211 PPI users and 1604 non PPI-users in three cohorts: general population, patients with inflammatory bowel disease and patients with irritable bowel syndrome. Data from this report indicated that PPI treatment was associated with decrease in diversity, changes in the 20% of the identified bacteria and significant increase of oral bacteria (Rothia dentocariosa, Rothia mucilaginosa, the genera Scardovia and Actinomyces, the family Micrococcaceae) in the fecal microbiome. Genera Enterococcus, Streptococcus and Lactobacillus were also increased in PPI users [29]. A similar observation has been made in another large study with 1827 twin cohort from United Kingdom [38]. This analysis reported that PPI use is associated with lower diversity in the gut microbiome and increase in Lactobacillales, particularly Streptococcaceae. In another study involving 32 individuals, no significant change in diversity was found between longterm PPI users and controls [25]. Most of patients were treated with PPI once daily (omeprazole 53%, rabeprazole 22%, other 28%), more than 180 tablets in each of the 5 years prior to study enrolment. In PPI users, the lower abundance of Bacteroides and higher abundance of Firmicutes was demonstrated in comparison with controls. Seto et al. reported that PPI use was associated with reduced operational taxonomic unit (OTU) diversity in 10 healthy volunteers treated with 20 mg or 40 mg omeprazole for 28 days, but no difference was found between the high and low dose groups [24]. The similarity of OTU richness between PPI users and patients with first C. difficile infection was visible already after 1 week PPI therapy. Interestingly, this effect was partially reversible within 1 month after cessation of PPI usage. In a crossover trial by Freedberg et al., 12 healthy volunteers were treated with 40 mg omeprazole twice daily [34]. No significant difference in the gastrointestinal microbiome diversity was observed after 4 and after 8 weeks of treatment, compared with the baseline. However, PPI therapy induced increase in Enterococcaceae, Streptococcaceae, Microccaccea and Staphylococcaceae. It has been previously suggested that primary bile acids may predispose to CDI by transforming C. difficile spores into vegetative forms while secondary bile acids inhibit the growth of C. difficile [35]. Freedberg et al. have analyzed fecal bile acids using chromatography and found no changes after PPI treatment [34]. Very interesting observations come from
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The effect of omeprazole treatment on the gut microflora and neutrophil function the study with 45 individuals treated with lansoprazole or rabeprazole for 8 weeks and 27 healthy volunteers without PPI treatment [33]. It has been shown that microbiota of salivary and gastric fluid are similar. The bacterial count in gastric fluid evaluated by culturing methods was higher in PPI-users but the bacteria number measured by the realtime quantitative PCR was similar in treated patients and control. A correlation between the pH value and the cultured bacterial count in gastric fluid was found in all the subjects. In PPI-users group a significant increase of Streptocooccus and tendency of a reduction of Faecalibacterium at genus level in feces was found. The authors concluded that lack of killing rather than proliferation of the bacteria in the acid-suppressed stomach can provide to bacterial overgrowth in gastric fluid [33]. All the above-mentioned studies has used methods based on 16S rRNA sequencing for the analysis of the fecal microbiota composition. In the present study, we have used methods available in routine clinical practice, specifically stool culture and LHBT. Stool cultures are able to identify a limited number of microorganisms and are usually focused on detection of the most common intestinal disease-causing bacteria. With the use of routine laboratory stool culturing, we have identified 15 different bacterial and 2 fungal taxonomic units and found substantial changes in stool microbiota diversity before and after PPI treatment. The mechanism of PPI’s effect on gut microflora is still unclear. There are a few reports about the effect of PPIs on superoxide anion production by PMNs, however, their results are conflicting. Earlier in vitro study has shown, that omeprazole reduced PMNs degranulation, chemotaxis and inhibited superoxide anion generation [8]. A similar observation has been made by Capodicasa et al. who found that lansoprazole inhibited in vitro the cytotoxic activity of PMNs, natural killer cells chemotaxis and superoxide anion production [9]. In the recent study, the effect of PPIs on peripheral leukocytes function has been studied in patients with decompensated cirrhosis. Use of PPIs was associated with a decreased granulocyte and monocyte oxidative burst, but not of phagocytic activity, as compared with patients not receiving PPIs [39]. In the present study, we have evaluated the function of PMNs phagocytosis and capability to produce oxygen reactive species. Nitroblue tetrazolium (NBT) test is well-known method for evaluation of reactive oxygen species production used to screen for neutrophil dysfunction. Neutrophils reduce the dye NBT, a clear yellow water-soluble compound, to formazan upon stimulation of the respiratory burst. Formazan precipitates as a dark blue granular substance, which remains trapped in the neutrophil’s cytoplasm. Some regulatory functions of neutrophils in the immune system are mediated by the production of reactive forms of nitrogen, including nitric oxide (NO) [40,41]. NO is a signaling molecule that plays a key role in the pathogenesis of inflammation, contributes to the regulation of apoptosis, induces vasodilatation in cardiovascular system and it is also a potent neurotransmitter at the neuron synapses. Stimulation of inducible nitric oxide synthase (iNOS) expression in neutrophils by pro-inflammatory cytokines leads to the production of large quantities of nitric oxide. NO activates cytosolic guanylate cyclase (sGC), which in turn catalyzes the formation of cyclic guanosine 3 ,5 monophosphate (cGMP) from GTP. cGMP mediates a wide
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spectrum of processes in multiple cell types within the cardiovascular system, but is also involved in inflammation [42]. p38 MAP kinase is associated with immune cell activation and activated by a variety of inflammatory mediators [43]. In the present study, we have found no effect of omeprazole treatment on reactive oxygen species production in PMNs, expressed by the results of NBT test. We have also found no effect of omeprazole on determined by Western blotting iNOS protein expression in freshly isolated PMNs. Treatment with omeprazole did not influence NO and cGMP concentration in the PMNs culture supernatant or our patients’ sera as well as p38MAP kinase activity in these cells. Therefore, in our study, hypothetical omeprazole-induced PMNs function deterioration is not responsible for the potential effect of omeprazole on the gut microflora. The major weakness of this study is incomplete recruitment of patients to LBHT. Additionally, the effect of PPI treatment on gut microflora could be more pronounced if, in our study, we would include only PPI-naïve patients. In conclusion, 4 weeks of omeprazole treatment have caused considerable changes in stool culture results. Patients treated with higher dose of omeprazole have had some tendency to decrease diversity of colonic microflora and towards more changes in fermenting bacteria population in the gut, in comparison with patients treated with the lower dose of omeprazole. We suppose that the potential effect of omeprazole on gut microflora depends mostly on inhibition of gastric acid, without any significant role for neutrophil function deterioration. More studies are necessary to prove that the effect of omeprazole on the gut microflora is dose-dependent. The prescriptions of PPI are spectacularly increasing worldwide and currently significant concern is growing about overutilization of these drugs. It is suggested that in 50—70% of cases they are prescribed without indications [1]. There is growing body of evidence that PPIs may significantly affect the gut microflora and thereby predispose to C. difficile infections. Therefore, the use of these drugs should be limited to the strict and evident indications.
Disclosure of interest The authors declare that they have no competing interest.
Acknowledgements This work has been supported with grants from National Science Centre (Grant no. NN402456739).
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ORIGINAL ARTICLE
The effect of omeprazole treatment on the gut microflora and neutrophil function Maja Kostrzewska a,∗, Agnieszka ´ Swidnicka-Siergiejko a, nska b, Gra˙ zyna Jurkowska a, Marzena Garley c, Dorota Olsza´ nska c, Wioletta Ratajczak-Wrona c, Ewa Jabło´ Jacek Jamiołkowski d, Andrzej Dabrowski a a
Department of gastroenterology and internal medicine, medical university of Bialystok, ul. Sklodowska-Curie 24A, 15-276 Bialystok, Poland b Department of microbiological diagnostics and infectious immunology, university hospital of Bialystok, Bialystok, Poland c Department of immunology, university hospital of Bialystok, Bialystok, Poland d Department of public health, university hospital of Bialystok, Bialystok, Poland
Summary Background and aim: Proton pump inhibitors (PPIs) may increase the risk of Clostridium difficile infections. There are interactions between gut microbiota and innate immune cells including neutrophils. We evaluated the effect of treatment with omeprazole on the gut microflora and neutrophil function. Methods: In 50 patients, we evaluated the effect of 4-week omeprazole treatment (n = 25 with 20 mg per day and n = 25 with 20 mg twice daily) on intragastric pH, results of stool culture and lactulose hydrogen breath test (LHBT) and neutrophil function. Results: The treatment caused significant increase of the mean intragastric pH, especially in the group with 20 mg omeprazole twice daily (from 2.05 ± 0.59 to 5.06 ± 1.6, P < 0.001). In LHBT, the increase of hydrogen concentration was observed in higher percentage of patients with 20 mg of omeprazole twice daily, compared to patients with the lower dose (42.1% vs 29.4%; ns). Four weeks of omeprazole treatment have caused considerable changes in stool culture results. Patients treated with higher dose of omeprazole have had some tendency to decrease diversity of colonic microflora in comparison with patients treated with the lower dose of omeprazole. Treatment with omeprazole did not result in C. difficile positive stool culture and had no significant effect on neutrophil function.
∗
Corresponding author. E-mail address: maja [email protected] (M. Kostrzewska).
http://dx.doi.org/10.1016/j.clinre.2017.01.004 2210-7401/© 2017 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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Conclusions: Omeprazole treatment have caused considerable changes in stool culture results. Patients treated with the higher dose had some tendency to decreased diversity of colonic microflora and towards changes in fermenting bacteria of the gut. The potential effect of omeprazole on gut microflora does not depend on neutrophil function deterioration. © 2017 Elsevier Masson SAS. All rights reserved.
Introduction Proton pump inhibitors (PPIs) are currently prescribed on a large scale around the world and they are considered safe and effective drugs. Additionally, many patients are treated with PPIs without approved indications. Therefore, an increasing number of patients is being exposed to side effects of this treatment [1]. The most common of side effects (headache, diarrhea, abdominal pain, nausea, vomiting, flatulence) occur in 5% of patients. Among clinically relevant side effects are: increased risk of bacterial infections [2,3], malabsorption of calcium [4], vitamin B12 [5] and iron (resulting from iatrogenic hipochlorhydria) [6] and the formation of fundic gland polyps in patients on long-term PPI therapy [7]. Some studies also suggest that PPIs can affect the neutrophil functions such as chemotaxis, adhesion to endothelial cells and phagocytosis. The effect of these drugs on neutrophils has been evaluated mostly in vitro, in animal models and in patients with Helicobacter pylori infection [8—13]. Since PPIs strongly inhibit the secretion of hydrogen ions by the gastric parietal cells, they are widely used in the treatment of gastroesophageal reflux disease and peptic ulcer disease. However, the inhibition of gastric acid secretion can also trigger pathways leading to unwanted events. Hydrochloric acid provides an important protective barrier of defense against bacterial infections and the increase of intragastric pH may facilitate colonization of the gut by microbiota normally restricted to gastrointestinal tract above the stomach. In some situations, it may predispose to small intestinal bacterial overgrowth (SIBO) or gastrointestinal infections [14]. Salmonella is an acidsensitive bacteria unable to survive in pH < 3. The increased risk of Salmonella infection has been reported not only in patients after gastrectomy but also in patients taking PPIs [15]. The drugs inhibiting gastric acid secretion have been also proven risk factor for infection with Campylobacter [16,17]. Since the greater risk of gastrointestinal infection has been associated mostly with the PPIs rather than H2-receptor antagonists treatment, it may suggest the correlation between degree of inhibition of gastric acid secretion and the incidence of gastrointestinal infection. On the other hand, although Escherichia coli is an acid-sensitive organism, infections with these bacteria have not been reported more common in patients taking PPIs [3,14]. Gastric acid suppression is considered as a risk factor of Clostridium difficile—associated diarrhea (CDAD) [14,18]. The risk of C. difficile infection (CDI) ranged from 1.4 to 3.5 times higher among patients with PPI treatment compared with those without PPI therapy [19—22]. Several
studies reported that PPI administration may lead to changes in human gastrointestinal microbiota and reduce microbial diversity. Furthermore, in the gut microbiome of PPI-users significant changes in the amount of taxa that are associated with increased risk of CDI (Enterococcaceae and Streptococcaceae) were observed [23—25]. PPIs may also bind and inhibit non-gastric H+ /K+ -ATPases in bacteria and human cells including neutrophils and thereby impair the human immune status [9]. There is an increasing body of evidence about the interactions between gut microbiota and innate immune cells including neutrophils [26,27]. Consequently, PPIs have the potential of gut microbiota modulation by two independent pathways — through inhibition of gastric acid secretion and by the impact on neutrophil function. Therefore, the aim of this study was to evaluate the effect of 4-week outpatient treatment with oral omeprazole on human gut microbiota and function of neutrophils in vivo.
Patients and methods Before enrolling to the study, all the subjects had clinical examination and laboratory blood tests: complete blood count, concentration of C-reactive protein, fibrinogen, creatinine, urea, bilirubin, glucose, sodium, potassium in serum, activity of alanine aminotransferase and aspartate aminotransferase in serum, prothrombin time and urinalysis. The study has involved patients with normal results of physical examination and laboratory tests before enrollment. Other inclusion criteria were: no PPI treatment at least 14 days before the study, indications for use of PPI (dyspepsia, gastroesophageal reflux disease), no other medications during the study. One patient in omeprazole 20 mg twice daily group was exposed to antibiotics 2 months before enrolling to the study and 9 patients (5 in omeprazole 20 mg once daily group and 4 in omeprazole 20 mg twice daily group) were exposed to PPIs 4—8 weeks before the study. Exclusion criteria were: PPI or H2 receptor antagonist treatment within 14 days before the study, antibiotic use within 4 weeks before the study, current bacterial or viral infection, diabetes, cancer, immunodeficiency in medical history. Informed consent was obtained from all of the subjects. The study was approved by the Ethics committee of medical university of Bialystok. We have prospectively enrolled 33 women and 17 men. The mean age of the subjects was 42.8 ± 16.1 years. A proton pump inhibitor was randomly selected and administered orally for 4 weeks: 25 patients were treated with omeprazole 20 mg per day (taken 30 minutes before breakfast)
Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004
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The effect of omeprazole treatment on the gut microflora and neutrophil function and 25 patients with omeprazole 20 mg twice daily (taken 30 minutes before breakfast and before the evening meal).
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• 4: very abundant growth (equivalent to more than 1,000,000 CFU/g).
Tests for Clostridium difficile Measuring intragastric pH Twenty-four-hour intragastric pH was evaluated before and after omeprazole treatment by Sleuth pH-analyzer (Sandhill Scientific Inc). The ComfortTEC Z/pH ZAI-BG-44 (Sandhill Scientific Inc) single-use probes with two pH channels (gastric and esophageal) were calibrated before procedure using the D82-3152 buffer solution pH 7,00 (Soundhill Scientific Inc) and D82-3151 buffer solution pH 4,00 (Soundhill Scientific Inc). To analyze data of pH-monitoring BioVIEW analysis ver.5.3.4.(Sandhill Scientific Inc) software was used.
Lactulose hydrogen breath test Lactulose hydrogen breath test (LHBT) was performed in 36 out of 50 patients before treatment and after 4-week omeprazole therapy: 17 patients with the dose of 20 mg per day and 19 patients with the dose of 20 mg twice daily. We were able to perform both LBHT measurements in 36 out of 50 patients, because not all patients reapplied for the second round of this test. They were instructed to avoid food ingestion for 12 hours before the test, to avoid nicotine products and chewing gum before and during the LHBT. The tests were performed using the breath hydrogen analyzer LactoFANQuick (Fisher ANalysen Instrumente GmbH). The dose of 10 g lactulose dissolved in 150 mL of water was administered. Exhaled air samples were taken immediately before and 30, 45, 60, 75, 90, 120, 150 and 180 minutes after the lactulose solution intake. Additionally, for each measurement, we have calculated area under the curve to show total hydrogen production after a lactulose load.
Methodology of stool cultures The culture of stool sample has been performed before the initiation and after completion (the next day after the last dose of the drug) of omeprazole treatment. The stool sample was inoculated into the Columbia agar of bioMerieux with 5% sheep blood, Columbia CNA agar with 5% sheep blood, MacConkey agar, Chromid VRE agar, ESBL agar and incubated for 18—24 hours at 35 ◦ C. The stool samples for Salmonella and Shigella cultures were inoculated into Selenite F Broth, incubated for 18—24 hours at 35 ◦ C afterward were seeded on Chromid Salmonella/Haectoen agar and incubated for 18—24 hours at 35 ◦ C. The cultured bacteria were identified with the GN and GP cards on the VITEK 2 XL analyzer. Bacterial counts in the colony-forming units (CFU) per 1 g of stool were estimated using traditional culturing methods. Data from stool test was described in semi-quantitative scale: • 0: no colony growth; • 1: single colonies (equivalent to 100—1000 CFU/g); • 2: scanty colonies growth (equivalent 1000—10,000 CFU/g); • 3: abundant growth (equivalent to 100,000 CFU/g);
to
The culture of stool sample has been done before the initiation and after completion (the next day after the last dose of the drug) of omeprazole treatment. It was performed on a Chromid Clostridium difficile medium by bioMerieux and incubated under anaerobic conditions (Genbag anaer) at 35 ◦ C for 48 hours. Then, in culture positive cases, the bacteria could be identified on the ANC cards with the VITEK 2XL analyzer. The presence of glutamate dehydrogenase (GDH) and toxins A and B was screened by C.diff Quick Chec Complete immunosorbent assay by TechLab. In GDH positive but C. difficile toxins negative cases, stool sample are inoculated into the Chromid C. difficile medium by bioMerieux and incubated under anaerobic conditions (Genbag anaer) at 35 ◦ C for 48 hours. The ability of C. difficile strain to produce toxins A or B is evaluated with the use C.diff Quick Chec Complete test by TechLab.
Function of polymorphonuclear neutrophils (PMNs) NBT test Nitroblue tetrazolium (NBT, Sigma) was dissolved in phosphate-buffered saline (Biomed). Spontaneous and stimulated NBT tests were performed. PMNs were isolated from peripheral blood and stimulated by latex (Sigma). The samples were incubated at 37 ◦ C for 15 minutes (NUAIRETMUS AUTOFLOW CO2 water-jacketed incubator), and then in room temperature for 15 minutes. The smear was performed and stained with a May-Grunwald-Giemsa solution (Aqua-Med). Under the 40 x magnification the number of neutrophils containing the formazan deposit has been counted among 100 encountered PMNs; the results has been shown as a percentage of NBT positive cells. Determination of total NO concentration in PMNs culture supernatants and serum NO production by PMNs was estimated using an indirect method based on the measurement of the nitrite concentration in PMNs culture supernatants and serum according to Griess’s reaction. In the analyzed samples nitrate was reduced to nitrite in the presence of cadmium and converted to nitric acid that gave a color reaction with Griess’s reagent. After 30 minutes, nitrite concentrations were determined by spectrophotometric analysis at 540 nm with reference to standard curve [28]. NO concentration in PMNs culture was expressed as M (supernatant from 106 cells suspended in 230 L of culture medium). Analysis of cGMP concentration in PMNs supernatants and serum The cGMP level in the PMNs culture supernatants and serum was measured using an enzyme-linked immunosorbent assay ® kit (R&D Systems ). The expression of iNOS and phospho-p38 MAP kinase Freshly isolated PMNs were lysed by sonification (SONICS vibra cell) in the presence of protease inhibitor cocktail
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M. Kostrzewska et al. Table 1
Clinical characteristics of patients.
Age (mean ± SD, min-max) Male Medical history Heartburn Epigastric pain Belching Chest pain Cough Hoarseness GERD Comorbidities Arterial hypertension Coronary artery disease Medication before admission PPI H2 receptor antagonist NSAID Antihypertensive agents Statins Active smokers
Omeprazole 20 mg once daily
Omeprazole 20 mg twice daily
n = 25
n = 25
40.8 ± 15.8 (18—63) 9
44.7 ± 16.58 (20—81) 8
0.40 0.76
12 4 15 3 8 2 14
14 5 18 3 4 3 12
0.57 0.71 0.37 1.00 0.18 0.63 0.57
4 1
5 1
0.71 0.92
11 3 2 3 1 6
9 2 2 3 1 4
0.56 0.63 1.00 1.00 1.00 0.47
P-value
PPI: proton pump inhibitor; H2 receptor antagonist: histamine H2 receptor antagonist; GERD: gastroesophageal reflux disease; NSAID: nonsteroidal anti-inflammatory drugs.
(Sigma-Aldrich). The lysates were suspended in Laemmli buffer (Bio-Rad laboratories) and electrophoresed (Bio-Rad ® laboratories Mini-PROTEAN tetra cell) on sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad). The resolved protein was transferred onto nitrocellulose (Bio-Rad Laboratories ® Mini-PROTEAN tetra cell). The nitrocellulose was incubated (Milipore SNAP i.d.TM protein detection system) with the primary monoclonal anti-iNOS (1:10,000 R&D systems) or polyclonal antibody anti-phospho-p38␣ MAPK active form (1:10,000 ABR Affinity BioReagents). After washing with 0.1% TBS-T (Bio-Rad laboratories), the membrane was incubated with alkaline phosphatase-labelled anti-mouse or antirabbit IgG antibodies (Vector Laboratories). Immunoreactive protein bands were visualized using the 5-bromo-4-chloro-3indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) liquid substrate system (Sigma-Aldrich). The density of iNOS and p38 MAP kinase bands was determined using ImageJ software and quantified with arbitrary units.
Statistical analysis The results were analyzed using IBM SPSS Statistics 20.0 and GraphPad Prism 5. Data in clinical characteristics of patients have been analyzed by unpaired t-test (for age) and Chi2 test (other data). The intragastric pH is shown as mean ± SD and the differences between groups have been evaluated by t-test. In each patient, ANOVA test to analyze significant differences between LHBT before and after omeprazole treatment has been performed. The correlation between intragastric pH after treatment and change in of LHBT was evaluated by Pearson test.
Results Clinical characteristics of the patients There were no significant differences between groups before treatment (Table 1). The clinical characteristic of patients shown in Table 1 applies to the ‘‘before treatment’’ period of our study. Most of these symptoms disappeared after PPI treatment and in the course of our study, we have observed no additional symptoms or clinical signs of gastrointestinal tract infections after 4 weeks of omeprazole therapy in both groups.
Intragastric pH After omeprazole treatment, we observed significant increase of mean intragastric pH in both groups: patients treated with 20 mg per day (2.29 ± 1.05 vs. 3.8 ± 1.45; P < 0.001) and 20 mg twice daily (2.05 ± 0.59 vs 5.06 ± 1.61; P < 0.001). The effect of the higher dose of omeprazole was significantly stronger (P < 0.016) than the effect of the standard dose (Fig. 1).
Lactulose hydrogen breath test We have analyzed individual data from LHBT of 36 patients and found no change after omeprazole treatment in 15 patients, specifically: 47% (8/17) treated with 20 mg once daily and 36.8% (6/19) treated with 20 mg twice daily. The increase of hydrogen concentration in air samples was
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Table 2 The changes in exhaled air hydrogen concentration measured by lactulose hydrogen breath test after omeprazole treatment. Exhaled air hydrogen concentration
Omeprazole 20 mg once daily n = 17
Omeprazole 20 mg twice daily n = 19
P
Increase No change Decrease
29.4% (5/17) 47.0% (8/17) 23.6% (4/17)
42.1% (8/19) 36.8% (6/19) 26.3% (5/19)
0.42 0.45 0.72
7
omeprazole 20 mg once daily
omeprazole 20 mg twice daily
omeprazole 20 mg once daily
Intragastric pH
6
omeprazole 20 mg twice daily
pH 8 7 6 5 4 3 2 1 0
5 1 vs 2 p <0.001 3 vs 4 p <0.001 2 vs 4 p <0.016
4 3 2 1 0 1 2 Before After treatment
3 4 After Before treatment
Figure 1 The effect of omeprazole in two different doses on intragastric pH. The data are shown as mean ± SD: 1: omeprazole 20 mg once daily before treatment; 2: omeprazole 20 mg once daily after treatment; 3: omeprazole 20 mg twice daily before treatment; 4: omeprazole 20 mg twice daily after treatment.
observed in 29.4% (5/17) patients with omeprazole 20 mg once daily therapy and in 42.1% (8/19) objects treated with omeprazole 20 mg twice daily, but the difference is not statistically significant (Table 2). Decrease of hydrogen concentration in air samples after omeprazole treatment was found in 23.6% (4/17) cases in the omeprazole 20 mg once daily group and in 26.3% (5/19) of 20 mg omeprazole twice daily group. The results of LBHT expressed as the area under the curve (AUC) for total hydrogen production after a
-1
pH
8 7 6 5 4 3 2 1 0
P=0.23
0
1
-1
P=0.48
0
1
Figure 3 The correlation between intragastric pH and change in exhaled air hydrogen concentration after omeprazole treatment. Change of hydrogen concentration in air samples after omeprazole treatment measured in lactulose hydrogen breath test: 0: no change; 1: increase; (−1) — decrease.
lactulose load for each patient treated with omeprazole are shown in Fig. 2.
Correlation between intragastric pH and exhaled air hydrogen concentration change in LHBT We found no correlation between intragastric pH after omeprazole treatment and lactulose hydrogen breath test results after 4 weeks of therapy (Fig. 3).
Figure 2 The results of LBHT expressed as total hydrogen production after a lactulose load in patients treated with two different doses of omeprazole. The data is shown for each patient as the area under the curve (AUC) before and after the omeprazole treatment.
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M. Kostrzewska et al. Table 3
Bacterial strains and genera of fungi in stool cultures analysis after omeprazole treatment.
Specific taxa
Enterobacteriaceae Escherichia coli ESBL(−) Escherichia coli ESBL(+) Escherichia fergusoni Klebsiella pneumoniae Klebsiella oxytoca Proteus mirabilis Citrobacter spp Enterobacter spp Salmonella spp Shigella spp Enterococceae Enterococcus faecalis Enterococcus spp Pseudomonoaceae Pseudomonas aeruginosa Streptococceae Streptococcus spp Staphylococceae Staphylococcus CNS spp Staphylococcus aureus Anaerobes Clostridium difficile Bacteroides spp Yeasts Candida albicans Geotrichum spp
Effect after treatment
Omeprazole 20 mg once daily n = 24
Omeprazole 20 mg twice daily n = 24
Increase (new Decrease Increase (new Decrease Increase (new Decrease Increase (new Decrease Increase Decrease Increase (new Decrease Increase Decrease Increase (new Decrease
5 (1) 0 0
3 (0) 3 1 (0)
1 (1) 0 1 (1) 2 0 0 1 (1) 1 0 2 1 (1) 0 No growth No growth
0 0 2 (2) 0 0 1 1 (1) 0 0 0 0 3 No growth No growth
Increase Decrease Increase (new strain) Decrease
2 (2) 0 8 (3) 7
2 (2) 1 8 (4) 3
Increase Decrease
0 0
0 1
Increase (new strain) Decrease
2 (2) 0
2 (1) 0
Increase (new strain) Decrease Increase (new strain) Decrease
4 (4) 2 1 (1) 1
3 (3) 3 1 (1) 0
Increase Decrease
No growth 0 2
No growth 0 1
Increase (new strain) Decrease Increase (new strain) Decrease
3 (3) 3 1 (1) 2
1 (1) 4 3 (3) 0
strain) strain) strain) strain)
strain)
strain)
Increase: number of patients with higher score in the semi-quantitative scale of stool culture bacterial counts after treatment, compared to the score before treatment; Decrease: number of patients with lower score in the semi-quantitative scale of stool culture bacterial counts after treatment, compared to the score before treatment; New strain: number of patients with the new microorganism appearing in the stool culture after omeprazole treatment.
Stool cultures analysis With culturing of stool samples, we have identified 15 bacterial strains and 2 genera of yeast (Table 3). None of the cultures were C. difficile positive both before and after omeprazole treatment. The increase in colony count of
Streptococcaceace after treatment was observed in two cases in 20 mg omeprazole once daily group and in two cases of 20 mg twice daily omeprazole group. In 16 patients, the increase in the colony count of Enterococcaceae after omeprazole treatment was found: 8 patients in 20 mg once daily group and 8 patients in 20 mg twice daily group.
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Table 4 The effect of omeprazole treatment on the NBT test results, the level of NO and cGMP in PMNs culture supernatant and serum as well as expression of inducible nitric oxide synthase (iNOS) and phospho-p38 MAP kinase in freshly isolated PMNs. Before treatment with omeprazole 20 mg once daily (n = 25)
After treatment with omeprazole 20 mg once daily (n = 25)
Before treatment with omeprazole 20 mg twice daily (n = 25)
After treatment with omeprazole 20 mg twice daily (n = 25)
1
2
3
4
NBT test spontaneous (%)
16.00 (3.00—59.00)
17.00 (5.00—51.00)
15.00 (2.00—53.00)
14.00 (3.00—48.00)
NBT test stimulated (%)
51.00 (18.00—73.00)
51.00 (18.00—69.00)
47.00 (18.00—68.00)
46.00 (14.00—68.00)
NO in PMNs culture (M)
19.32 (4.71— 60.87)
21.59 (10.8—46.02)
16.48 (8.52—45.46)
17.61 (7.39—40.34)
NO in serum (M)
30.68 (17.05—47.73)
31.25 (16.48—56.52)
31.82 (18.75—40.34)
31.82 (14.77—257.61)
cGMP in PMNs culture (pM/mL)
0.89 (0.26— 3.20)
0.96 (0.03—2.16)
0.53 (0.06—3.68)
0.85 (0.06—10.93)
cGMP in serum (pM/mL)
5.92 (1.04—41.0)
7.6 (0.03—48.5)
11.7 (1.28—46.0)
9.23 (0.06—320.69)
iNOS (arbitrary units × 103 )
70.602 (2.345—189.810)
64.030 (2.122—232.903)
82.775 (2.345—254.912)
115.821 (2.152—272.470)
p38 MAP kinase (arbitrary units × 103 )
87.475 (8.106—293.216)
80.363 (4.579—413.160)
74.356 (4.805—264.256)
63.972 (26.479—176.547)
Statistical significance
(1 vs 2) 0.516 (3 vs 4) 0.893 (2 vs 4) 0.404 (1 vs 2) 0.882 (3 vs 4) 0.326 (2 vs 4) 0.276 (1 vs 2) 0.333 (3 vs 4) 0.875 (2 vs 4) 0.130 (1 vs 2) 0.936 (3 vs 4) 0.435 (2 vs 4) 0.415 (1 vs 2) 0.300 (3 vs 4) 0.638 (2 vs 4) 0.836 (1 vs 2) 0.470 (3 vs 4) 0.272 (2 vs 4) 0.836 (1 vs 2) 0.925 (3 vs 4) 0.581 (2 vs 4) 0.093 (1 vs 2) 0.840 (3 vs 4) 0.353 (2 vs 4) 0.357
The data are shown as median (minimum-maximum); NBT: nitroblue tetrazolium; PMNs: polymorphonuclear neutrophils; NO: nitric oxide; cGMP: cyclic guanosine monophosphate; inducible nitric oxide synthase (iNOS).
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M. Kostrzewska et al.
The decrease in the colony count of Enterococcaceae was observed in 7 patients in 20 mg omeprazole once daily group and in 3 patients in 20 mg omeprazole twice daily group.
Function of PMNs NBT tests We have found no effect of omeprazole treatment on the results of both spontaneous and stimulated NBT tests (Table 4). Concentration of NO in PMNs culture supernatants and serum Treatment with omeprazole had no effect on NO concentration in the PMNs culture supernatant and serum (Table 4). Concentration of cGMP in PMNs culture supernatants and serum Treatment with omeprazole had no effect on cGMP concentration in the PMNs culture supernatant and plasma (Table 4). iNOS and phosphorylated p38 MAP kinase expression in isolated PMNs Treatment with omeprazole had no significant effect on determined by Western blotting iNOS protein expression (Table 4). Phospho-p38 MAP kinase signal in Western blotting shows only active, doubly phosphorylated forms of this kinase. Treatment with omeprazole did not stimulate or inhibit p38 MAP kinase activity in PMNs (Table 4).
Discussion In our present study, treatment with the standard dose of omeprazole (20 mg) has caused a modest increase of intragastric pH with the mean pH < 4. The significant inhibition of gastric acid production (mean pH 5.06 ± 1.61) has been achieved only after treatment with a double standard dose of 40 mg of omeprazole. Since the acidity forms one of the main defenses against bacterial influx that accompanies ingesting food and oral mucus, strong inhibition of gastric acid allows more bacteria to survive this barrier and reach the gut [29]. Recently, the effect of PPI treatment on gut microflora have been described in many studies [24,25,29—35]. Among the most commonly discussed issues are the risk of SIBO and C. difficile infection in patients treated with PPI. One of the recent studies reports that 6 months lasting treatment with PPI may produce bowel symptoms and SIBO in 26% of patients [31]. In contrast, in a retrospective study including 1191 patients, PPI use was not found to be significantly associated with the presence of SIBO [32]. Hydrogen breath tests using various substrates like glucose, lactulose, lactose and fructose are being used to diagnose SIBO and lactose or fructose malabsorption [36]. Lactulose is a disaccharide composed of galactose and fructose. As there is no naturally occurring lactulose enzyme in the body to hydrolyze this sugar in the small intestine, lactulose is not broken down in the small bowel and is transported intact to the colon where it is fermented by colonic bacteria [37]. The products of its metabolism include hydrogen. LHBT is routinely
used as a simple method for estimation of orocecal transit time. However, this test has drawn our attention as a simple tool to evaluate the population of gastrointestinal tract fermenting bacteria. In our study, treatment with omeprazole had no effect on LHBT results in 47.0% and 36.8% patients treated respectively 20 mg and 40 mg per day. Interestingly, comparing to the effect of 20 mg of omeprazole once daily, treatment with 20 mg of omeprazole twice daily favored augmentation of fermenting bacteria population in the gut. There is an increasing body of evidence suggesting that treatment with PPIs can lead to changes in the composition of the gut microbiota predisposing to the development of C. difficile infection (CDI) [24,25,34]. It has been shown that PPIs alter specific taxa in the human gastrointestinal microbiome i.e. reduction of Bacteroides and Clostridium cluster IV abundance, increase of Enterococcaceae, Streptococcae, Firmicutes and Lactobacillus and thereby cause the decrease in the alpha diversity of fecal microbiota. In the large study, the effect of PPIs on the gut microbiome was investigated in 211 PPI users and 1604 non PPI-users in three cohorts: general population, patients with inflammatory bowel disease and patients with irritable bowel syndrome. Data from this report indicated that PPI treatment was associated with decrease in diversity, changes in the 20% of the identified bacteria and significant increase of oral bacteria (Rothia dentocariosa, Rothia mucilaginosa, the genera Scardovia and Actinomyces, the family Micrococcaceae) in the fecal microbiome. Genera Enterococcus, Streptococcus and Lactobacillus were also increased in PPI users [29]. A similar observation has been made in another large study with 1827 twin cohort from United Kingdom [38]. This analysis reported that PPI use is associated with lower diversity in the gut microbiome and increase in Lactobacillales, particularly Streptococcaceae. In another study involving 32 individuals, no significant change in diversity was found between longterm PPI users and controls [25]. Most of patients were treated with PPI once daily (omeprazole 53%, rabeprazole 22%, other 28%), more than 180 tablets in each of the 5 years prior to study enrolment. In PPI users, the lower abundance of Bacteroides and higher abundance of Firmicutes was demonstrated in comparison with controls. Seto et al. reported that PPI use was associated with reduced operational taxonomic unit (OTU) diversity in 10 healthy volunteers treated with 20 mg or 40 mg omeprazole for 28 days, but no difference was found between the high and low dose groups [24]. The similarity of OTU richness between PPI users and patients with first C. difficile infection was visible already after 1 week PPI therapy. Interestingly, this effect was partially reversible within 1 month after cessation of PPI usage. In a crossover trial by Freedberg et al., 12 healthy volunteers were treated with 40 mg omeprazole twice daily [34]. No significant difference in the gastrointestinal microbiome diversity was observed after 4 and after 8 weeks of treatment, compared with the baseline. However, PPI therapy induced increase in Enterococcaceae, Streptococcaceae, Microccaccea and Staphylococcaceae. It has been previously suggested that primary bile acids may predispose to CDI by transforming C. difficile spores into vegetative forms while secondary bile acids inhibit the growth of C. difficile [35]. Freedberg et al. have analyzed fecal bile acids using chromatography and found no changes after PPI treatment [34]. Very interesting observations come from
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The effect of omeprazole treatment on the gut microflora and neutrophil function the study with 45 individuals treated with lansoprazole or rabeprazole for 8 weeks and 27 healthy volunteers without PPI treatment [33]. It has been shown that microbiota of salivary and gastric fluid are similar. The bacterial count in gastric fluid evaluated by culturing methods was higher in PPI-users but the bacteria number measured by the realtime quantitative PCR was similar in treated patients and control. A correlation between the pH value and the cultured bacterial count in gastric fluid was found in all the subjects. In PPI-users group a significant increase of Streptocooccus and tendency of a reduction of Faecalibacterium at genus level in feces was found. The authors concluded that lack of killing rather than proliferation of the bacteria in the acid-suppressed stomach can provide to bacterial overgrowth in gastric fluid [33]. All the above-mentioned studies has used methods based on 16S rRNA sequencing for the analysis of the fecal microbiota composition. In the present study, we have used methods available in routine clinical practice, specifically stool culture and LHBT. Stool cultures are able to identify a limited number of microorganisms and are usually focused on detection of the most common intestinal disease-causing bacteria. With the use of routine laboratory stool culturing, we have identified 15 different bacterial and 2 fungal taxonomic units and found substantial changes in stool microbiota diversity before and after PPI treatment. The mechanism of PPI’s effect on gut microflora is still unclear. There are a few reports about the effect of PPIs on superoxide anion production by PMNs, however, their results are conflicting. Earlier in vitro study has shown, that omeprazole reduced PMNs degranulation, chemotaxis and inhibited superoxide anion generation [8]. A similar observation has been made by Capodicasa et al. who found that lansoprazole inhibited in vitro the cytotoxic activity of PMNs, natural killer cells chemotaxis and superoxide anion production [9]. In the recent study, the effect of PPIs on peripheral leukocytes function has been studied in patients with decompensated cirrhosis. Use of PPIs was associated with a decreased granulocyte and monocyte oxidative burst, but not of phagocytic activity, as compared with patients not receiving PPIs [39]. In the present study, we have evaluated the function of PMNs phagocytosis and capability to produce oxygen reactive species. Nitroblue tetrazolium (NBT) test is well-known method for evaluation of reactive oxygen species production used to screen for neutrophil dysfunction. Neutrophils reduce the dye NBT, a clear yellow water-soluble compound, to formazan upon stimulation of the respiratory burst. Formazan precipitates as a dark blue granular substance, which remains trapped in the neutrophil’s cytoplasm. Some regulatory functions of neutrophils in the immune system are mediated by the production of reactive forms of nitrogen, including nitric oxide (NO) [40,41]. NO is a signaling molecule that plays a key role in the pathogenesis of inflammation, contributes to the regulation of apoptosis, induces vasodilatation in cardiovascular system and it is also a potent neurotransmitter at the neuron synapses. Stimulation of inducible nitric oxide synthase (iNOS) expression in neutrophils by pro-inflammatory cytokines leads to the production of large quantities of nitric oxide. NO activates cytosolic guanylate cyclase (sGC), which in turn catalyzes the formation of cyclic guanosine 3 ,5 monophosphate (cGMP) from GTP. cGMP mediates a wide
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spectrum of processes in multiple cell types within the cardiovascular system, but is also involved in inflammation [42]. p38 MAP kinase is associated with immune cell activation and activated by a variety of inflammatory mediators [43]. In the present study, we have found no effect of omeprazole treatment on reactive oxygen species production in PMNs, expressed by the results of NBT test. We have also found no effect of omeprazole on determined by Western blotting iNOS protein expression in freshly isolated PMNs. Treatment with omeprazole did not influence NO and cGMP concentration in the PMNs culture supernatant or our patients’ sera as well as p38MAP kinase activity in these cells. Therefore, in our study, hypothetical omeprazole-induced PMNs function deterioration is not responsible for the potential effect of omeprazole on the gut microflora. The major weakness of this study is incomplete recruitment of patients to LBHT. Additionally, the effect of PPI treatment on gut microflora could be more pronounced if, in our study, we would include only PPI-naïve patients. In conclusion, 4 weeks of omeprazole treatment have caused considerable changes in stool culture results. Patients treated with higher dose of omeprazole have had some tendency to decrease diversity of colonic microflora and towards more changes in fermenting bacteria population in the gut, in comparison with patients treated with the lower dose of omeprazole. We suppose that the potential effect of omeprazole on gut microflora depends mostly on inhibition of gastric acid, without any significant role for neutrophil function deterioration. More studies are necessary to prove that the effect of omeprazole on the gut microflora is dose-dependent. The prescriptions of PPI are spectacularly increasing worldwide and currently significant concern is growing about overutilization of these drugs. It is suggested that in 50—70% of cases they are prescribed without indications [1]. There is growing body of evidence that PPIs may significantly affect the gut microflora and thereby predispose to C. difficile infections. Therefore, the use of these drugs should be limited to the strict and evident indications.
Disclosure of interest The authors declare that they have no competing interest.
Acknowledgements This work has been supported with grants from National Science Centre (Grant no. NN402456739).
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Please cite this article in press as: Kostrzewska M, et al. The effect of omeprazole treatment on the gut microflora and neutrophil function. Clin Res Hepatol Gastroenterol (2017), http://dx.doi.org/10.1016/j.clinre.2017.01.004