Abstract

Fourteen patients with Helicobacter pylori infection were treated with 20 mg omeprazole, 1 g amoxycillin and 400 mg metronidazole bd for 7 days (OAM), and 16 patients were treated with 20 mg omeprazole, 250 mg clarithromycin and 400 mg metronidazole bd for 7 days (OCM). Saliva, gastric biopsies and faecal samples were collected before, during (day 7) and 4 weeks after treatment in order to analyse alterations of the normal microflora and to determine antimicrobial susceptibility. Both treatment regimens resulted in marked quantitative and qualitative alterations. A selection of resistant streptococcal strains were noticed in both treatment groups, most apparent in the OCM group where a shift from susceptible to resistant strains was recorded. In the OAM group, six patients had overgrowth of resistant enterobacteriaceae during treatment compared with none in the OCM group, in the gastric microflora. The MICs for Enterococcus spp. and Enterobacteriaceae in faeces increased significantly during treatment in both groups. Nine patients in the OAM group became intestinally colonized by yeasts during treatment. The total anaerobic microflora was strongly suppressed in both treatment groups, although most pronounced in the OCM group, where the frequency of clarithromycin-resistant bacteroides strains increased from 2 to 76% during treatment, and remained at 59% 4 weeks post-treatment. Even if the treatment outcome was better in the OCM group (100%) than in the OAM group (71%), the amoxycillin-based treatment might be preferable from an ecological point of view, since the qualitative alterations in terms of emergence and persistence of resistant strains seemed to be most pronounced in the clarithromycin-treated group.

Introduction

The treatment of Helicobacter pylori-associated diseases has been discussed during the last few years.1,2,3,4,5 Several triple therapy regimens have been proposed; proton pump inhibitor (PPI), or bismuth salt with or without acid secretion inhibition, plus two antimicrobial agents chosen from clarithromycin, metronidazole, amoxycillin and tetracycline. The administration of antimicrobial agents has been shown previously to give a number of potentially adverse effects in relation to the human oropharyngeal and intestinal microflora.6 Development of antibiotic resistance and the induction of β-lactamase production among bacteria in the normal microflora may occur, as well as overgrowth of potentially pathogenic exogenic and/or endogenous microorganisms such as yeasts, which may lead to serious infections in immunocompromised patients, and Clostridium difficile which may lead to severe diarrhoea and/or colitis. There are a number of pharmacological and pharmacokinetic factors influencing the extent to which the normal microflora will be affected. The most common is incomplete absorption of orally administered drugs. Poorly absorbed agents can reach the intestine in active form where they destroy susceptible microorganisms and change the ecological balance.

Recently, a study on the impact on the normal oral, gastric and intestinal microflora of a proton pump inhibitor alone and in combination with amoxycillin as anti-H. pylori infection regimens was performed by our group.7 Since the recommended treatment regimen today is triple therapy, there was a need for ecological studies on these new combinations.

The objectives of the present study were to investigate and compare qualitative and quantitative ecological effects of two anti-H. pylori regimens; a combination of omeprazole, amoxycillin and metronidazole, and a combination of omeprazole, clarithromycin and metronidazole on the oral, gastric and intestinal microflora. Special attempts were made to study qualitative alterations in terms of emergence and development of resistant strains due to the treatment.

Materials and methods

Patient design

Thirty dyspeptic patients with H. pylori infection were included in the study. The H. pylori infection was verified with a positive urease test (CLO, Delta West, Australia). The patients were blindly and randomly split into two treatment groups. Fourteen patients were treated with 20 mg bd omeprazole (Losec, Hässle, Mölndal, Sweden), 1 g bd amoxycillin tablets (Amoxycillin NM Pharm, NM Pharm, Stockholm, Sweden) and 400 mg bd metronidazole tablets (Flagyl, Rhône-Poulenc Rorer, Paris, France) (OAM), and 16 patients were treated with 20 mg bd omeprazole, 250 mg bd clarithromycin tablets (Klacid, Abbott Laboratories, Chicago, IL, USA) and 400 mg bd metronidazole tablets (OCM). The patients had not been treated with any antacids or antimicrobial agents 4 weeks prior to the study. There were six women and eight men in the OAM group with a mean age of 59.5 years (range 31–82 years). The OCM group consisted of 10 women and six men, with a mean age of 58.8 years (range 33–75 years). The study was approved by the local ethics committee at Huddinge University Hospital, Karolinska Institute, Sweden.

Sampling procedures

Saliva, gastric biopsies and stool samples were collected on three occasions; before treatment (day 0), during treatment (day 7) and 4 weeks after treatment (day 35). Stimulated mixed saliva samples were collected and frozen immediately in 10 mL plastic containers, and 1 mL was frozen separately in a transport medium VMG II.8 Eight biopsies were taken from both the corpus and the antrum of the stomach during gastroscopy. Two biopsies were subjected to CLO-test, four to microbial cultivation and two to histopathological examination. The biopsies for cultivation were frozen immediately in a Brain Heart Infusion medium (BHI) with glycerol 33%, and the biopsies for histopathological examination were placed in phosphate-buffered formalin (4%, pH 7.4). All samples were frozen at –72°C. C13-urea breath test9 was also performed on the same three occasions, before start of treatment, day 7 and day 35.

Antimicrobial concentrations

Saliva, gastric biopsies and faecal samples were used for determination of amoxycillin and clarithromycin concentrations by the agar diffusion method. The saliva samples were assayed undiluted, the biopsies were homogenized and diluted 10-fold in the transport medium, and faecal samples were diluted 10-fold in 0.01 M phosphate buffer, pH 7.4. The indicator strain Micrococcus lutea ATCC 9431 was used for both the amoxycillin and the clarithromycin assays. Iso-agar (Oxoid, Basingstoke, UK) was used for the assay of amoxycillin and Antibiotic Medium 1 (Difco, Detroit, MI, USA) was used for the determination of clarithromycin concentrations. The agar plates were incubated at 37°C for 18 h. The concentrations of amoxycillin and clarithromycin were determined in relation to diameters of inhibition zones caused by known concentrations of amoxycillin and clarithromycin from standard series, 0.25–8 mg/L for amoxycillin and 0.5–16 mg/L for clarithromycin.

Microbial cultivation

The saliva and faecal samples were suspended in a prereduced peptone yeast glucose medium, diluted 10-fold to 10–7, and inoculated on selective agar media as described by Heimdahl & Nord.10 The aerobic agar plates were incubated for 24 h at 37°C and the anaerobic agar plates were incubated for 48 h at 37°C in anaerobic jars (Gas Pak, BBL, Cockeysville, MD, USA). The biopsies were weighed (mean weight in the OAM group, 0.012 g (s.d. 0.003), and in the OCM group 0.011g (s.d. 0.005)), homogenized, diluted 10-fold to 10–4 in BHI broth, and inoculated on different supplemented agar media. Blood agar for aerobic and anaerobic incubation and chocolate agar, incubated in CO2 atmosphere, were used for analysis of the normal gastric microflora. Two different selective agar media were used for detection of H. pylori; one containing Brucella agar (BBL, Cockeysville, MD, USA) supplemented with 5% sheep blood, vancomycin (6.0 mg/L), nalidixic acid (20 mg/L), amphotericin B (2.0 mg/L) and menadione (1.0 mg/L), and another medium containing Columbia agar base (Acumedia, Baltimore, MD, USA ), enriched with 8.5% horse blood citrate and 10% horse serum. These plates were incubated in a micro-aerobic environment (CampyPak, BBL, Cockeysville, MD, USA) for 5 days at 37°C. The detection limit was 102 cfu/mL or gram. After incubation different colony types were counted and identified to genus level by morphological, biochemical and gas-chromatographic analysis. A mean value of the numbers of microorganisms in the corpus and the antrum of the stomach was calculated.

Antimicrobial susceptibility tests

Streptococci, staphylococci, enterococci, Enterobacteriaceae and Bacteroides spp. strains from the saliva and the biopsies, and enterococci, Enterobacteriaceae and Bacteroides spp. from faeces were collected for antimicrobial susceptibility tests. Three to five isolates of each species were collected from each specimen. The MICs of amoxycillin, clarithromycin and metronidazole were determined using the agar dilution method. The agar medium used was PDM, Antibiotic Sensitivity Medium (Biodisk AB, Solna, Sweden). The inoculum was 108 cfu/mL. The aerobic agar plates were incubated for 24 h at 37°C and the anaerobic agar plates were incubated 48 h at 37°C. The reference strains were Escherichia coli ATCC 25922 and Enterococcus faecalisATCC 29212 for amoxycillin, Staphylococcus aureus ATCC 29213 for clarithromycin, and Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicronATCC 29741 for metronidazole. The MICs of amoxycillin, clarithromycin and metronidazole for H. pylori were determined by the Etest method (Etest, Biodisk AB, Solna, Sweden) on Columbia agar base plates, enriched with 8.5% horse blood citrate and 10% horse serum. The reference strain was H. pylori NCTC 11637. The MIC was defined as the lowest concentration of the drug that inhibited growth completely. MIC50s and MIC90s, the drug concentrations that inhibited the growth of 50 and 90% of the strains tested, respectively, were determined for each species and sampling occasion.

Determination of β-lactamase activity

Faecal strains belonging to the Bacteroides spp. group were assayed forβ -lactamase activity using the chromatogenic cephalosporin nitrocefin as substrate. One loop (1 µL) of fresh colonies was suspended in 0.1 mL 1 mM nitrocefin. A change of colour from yellow to red within 30 min was considered positive.

Histopathological evaluation

The biopsies for histopathological examination were fixed in phosphate-buffered formalin (4%, pH 7.4) and stained by standard methods with haematoxylin and eosin, and a modified Giemsa staining.11

C13-urea breath test

The urea breath test was performed at the Centre for Gastroenterology, Huddinge University Hospital, according to a method described by Oksanen et al.9

Statistics

Differences in numbers of a particular microorganism of two log or greater were considered to represent a significant change.12 The MIC values for each species were compared between days 0 and 7 and between days 0 and 35 using the Mann–Whitney U-test.

Results

Antimicrobial concentrations in saliva, gastric biopsies and faecal samples

The antimicrobial concentrations were determined in the saliva, biopsies and stool samples before, during and after treatment. No detectable levels of amoxycillin were found in saliva from the OAM group. All patients in the OCM group had detectable levels of clarithromycin in the saliva on day 7, median value 0.70 mg/L, (range 0.10–1.70 mg/L). In the gastric biopsies, five patients had detectable levels of amoxycillin, median value 5.03 mg/kg (range 1.18–116 mg/kg). No detectable concentrations of clarithromycin were noticed in the stomach of the patients in the OCM group. In the faecal samples, no concentrations of amoxycillin were detected in any of the patients in the OAM group. In all but one patient, clarithromycin was detected in faeces on day 7 in the OCM group. The median value of these patients was 164 mg/kg, ranging from 88.2 to 261.3 mg/kg. No patients had any detectable antimicrobial concentrations at days 0 and 35.

Alterations in the oral microflora

The alterations in the oral microflora during the study period are shown in Table I. In the OAM group, two patients became colonized by enterobacteria (Klebsiella oxytoca,Klebsiella pneumoniae, Enterobacter agglomeransand E. coli) during treatment. The median number of yeasts (mainly Candida albicans) increased significantly during OAM treatment. In the OCM group, one patient was newly colonized by enterobacteria on day 7 (K. pneumoniae, Enterobacter cloacaeand E. coli). Three patients became colonized with yeasts during treatment. In the anaerobic microflora, very similar results were seen between the two groups.

Alterations in the gastric microflora

More alterations were seen in the corpus than in the antrum of the stomach although the differences were not significant. A mean value of the alterations in the microflora of the corpus and the antrum is shown in Table II. Greater alterations were seen in the aerobic gastric microflora than in the anaerobic microflora. Patients treated with the OAM regimen experienced the most pronounced disturbances. Six patients become colonized by different enterobacteria during treatment. No patients were colonized with yeasts before treatment but four patients became colonized with yeasts (mainly C. albicans) during treatment, while only one of them still had detectable levels 4 weeks after treatment. The anaerobic microflora in the OAM group showed minor alterations. In the OCM group significant increases were seen in the numbers of Streptococcus mitior, Haemophilus spp. and Neisseria spp. during treatment, which all returned to pretreatment levels 4 weeks after the end of treatment. Three patients were colonized with low numbers of yeasts at day 7. Bacteroides spp. decreased in numbers after but not during treatment.

Alterations in the intestinal microflora

Marked ecological disturbances were seen in the intestinal microflora during treatment (Table III). In the OAM group, the numbers of enterococci, enterobacteria (except E. coli) and peptostreptococci increased significantly during treatment. Eight patients became newly colonized by Klebsiella spp. and Citrobacter freundii during treatment. The number of patients colonized with yeasts (mostly C. albicans) increased from zero to nine during treatment; two patients were still colonized with yeasts after treatment. All alterations in the OAM group were normalized 4 weeks after treatment. In the OCM group, the numbers of Bifidobacterium, Clostridium and Bacteroides spp. were significantly decreased in numbers at day 7, while the numbers of enterococci were significantly increased. Four weeks after treatment, a significant suppression was still seen in the levels of Bifidobacterium and Bacteroides spp. No significant alterations in the numbers of yeasts were noticed in the OCM group. No patients were colonized with Clostridium difficile at any time.

Antimicrobial susceptibility

The MICs of amoxycillin, clarithromycin or metronidazole against 1523 isolated strains, determined by the agar dilution method, are shown in Tables IVVI. Among the saliva isolates from both groups (OAM/OCM), a significant increase in the MIC values of amoxycillin and clarithromycin, respectively, were seen on day 7 against Streptococcus spp. in both groups according to the Mann–Whitney U-test (P= 0.001). The increase remained significant at day 35. Table VII shows the distribution of the different resistant isolates in the susceptibility groups according to the National Committee for Clinical Laboratory Standards (NCCLS).13,14 The breakpoints used for amoxycillin resistance were: R = 8 mg/L for streptococci, R= 0.5 mg/L for staphylococci, R = 32 mg/L for enterobacteria and R = 0.5 mg/L for bacteroides. The breakpoints used for clarithromycin were 1 mg/L for streptococci and 8 mg/L for other tested bacterial species.13,14 None of the streptococcal isolates was classified as resistant to amoxycillin, while 74% of the streptococci from the OCM group became resistant to clarithromycin during treatment and 26% were still resistant day on 35 (Table VII). Streptococcus spp. isolated from the gastric mucosa showed increased MIC values during (P =0.001) and after (P =0.05) treatment in the OAM group. In the OCM group, the MIC values for gastric Streptococcus spp. were also increased during treatment (P> 0.001). The proportion of clarithromycin-resistant streptococci in the OCM group increased from 6% on day 0 to 70% on day 7 (Table VII). The MIC for Staphylococcus spp. increased significantly (P > 0.05) in the OAM group during treatment, whereas in the OCM group the alterations in MIC values for staphylococci were not significant (Table V). In faeces, Enterococcus spp. showed a significant increase (P> 0.001) in MIC values during treatment in both groups which remained high in the OAM group, but returned to pretreatment levels in the OCM group (Table VI). The MIC values for Enterobacteriaceae increased significantly during treatment in both treatment groups (P > 0.001). The frequency of clarithromycin- resistant Enterococcus spp. increased from 2% at day 0 to 92% during treatment, and were still 29% after treatment (Table VII). The MIC values for Bacteroides spp. increased significantly from day 0 in the OCM group during and after treatment (P = 0.001), while no significant alterations were seen among Bacteroides spp. in the OAM group (Table VI). In the OCM group, the frequency of resistant Bacteroides spp. increased from 2% pretreatment to 76 and 59% on days 7 and 35, respectively (Table VII). No significant alterations in MIC values of metronidazole against Bacteroides spp. were noticed in either of the treatment groups. All H. pylori strains were susceptible (>0.016 mg/L) to amoxycillin and clarithromycin. The MICs of metronidazole varied, but no differences were seen between the two treatment groups; the MIC50 was 3 mg/L and MIC90 was 32 mg/L.

β-Lactamase activity

The frequency of β-lactamase-producing bacteroides strains increased from 52% on day 0 to 82% on day 7 in the amoxycillin-treated group (OAM). Four weeks after the treatment, 68% of the isolated bacteroides strains were positive in the β-lactamase assay. Among patients not treated with β-lactam agents (OCM), the proportion of β-lactamase-producing bacteroides at days 0, 7 and 35 were 44, 50 and 45%, respectively.

The presence of H. pylori

The patients were included on the basis of the CLO-test of biopsies from corpus and antrum. All results were later confirmed with C13-UBT, cultivation and histology. H. pylori was, however, not cultivable in biopsies from two patients on day 0. During treatment, H. pylori was not detected with any of the tests, except in two patients where H. pylori was detected with C13-UBT. After treatment, four patients in the OAM group had detectable levels of H. pylori, while no patients in the OCM group were colonized with H. pylori on day 35. These findings were verified with all tests in three patients and only with cultivation and histology in one patient. One of the two patients with detectable H. pylori during treatment was still colonized with H. pylori after treatment. The four patients with recurrence were shown 4 weeks after treatment to be recolonized with the same strain as before treatment, verified by fingerprinting with PCR methods (data not shown).

Discussion

It has been argued whether all H. pyloriinfections should be treated or not.15 Non-ulcer dyspeptic patients with H. pylori infection is a large patient group and, according to recommendations made by the National Institutes of Health in 1994, this patient group should not be treated.16 However, in the Maastricht Consensus Report made by the European Helicobacter pylori Study Group in 1997, this patient group is suggested for treatment, especially if the patient has a family history of gastric cancer.5 The recommended treatment regimen today for H. pylori includes a proton pump inhibitor, mainly omeprazole or lanzoprazole, in combination with antimicrobial agents. The reason why proton pump inhibitors enhance the effect of antimicrobial agents on H. pylori is not fully understood. It is generally believed that the elevation of the pH in the stomach is a crucial factor and the antimicrobial activity has been shown to be greater at a higher pH.17,18,19 The higher gastric pH also promotes overgrowth of other microorganisms which might compete with H. pylori for survival.7 A proton pump inhibitor produces a higher pH level than an H2 receptor antagonist, which is why a proton pump inhibitor is used in combination with antimicrobial agents when eradicating H. pylori today.20 Another reason could be that omeprazole has been shown to have an in-vitro activity against H. pylori.4 A large proportion of the population will be considered for treatment of H. pylori infection. Since administration of many antimicrobial agents has been shown to lead to adverse effects on the normal microflora, with possible emergence of resistance, it is important to study and evaluate different treatment regimens regarding this aspect.

Both treatment regimens (OAM and OCM) studied in the present investigation resulted in marked quantitative alterations in the oral microflora. Streptococci and anaerobic microorganisms were suppressed in numbers, indicating a decrease in the colonization resistance. This finding was also verified in some patients by new colonization by enterobacteria and yeasts. A selection of resistant streptococcal strains was noticed in both treatment groups, most apparent in the OCM group where a shift from susceptible to resistant strains was recorded. In the stomach, ecological disturbances were more pronounced in the OAM group where six of the 14 patients had overgrowth of resistant Enterobacteriaceae in contrast to none in the OCM group. However, as in the oral microflora, the OCM treatment lead to a selection of clarithromycin-resistant streptococci. An overall increase in the numbers of microorganisms during treatment was noticed, probably as a result of the acid suppression caused by omeprazole, as shown in a previous study.21 In the faecal microflora both treatment regimens were associated with large quantitative and qualitative changes. The MICs for Enterococcus spp. and Enterobacteriacae increased significantly in both groups, and the increase in MICs for enterobacteria was mainly represented by Klebsiella spp. This finding has been previously noticed in a study by Edlund et al.,22 where an overgrowth of Klebsiella spp. was observed during amoxycillin treatment, together with an increase in faecal β-lactamase activity. Nine patients in the OAM group, including those with yeasts in the gastric mucosa, were intestinally colonized by yeasts during treatment. The anaerobic intestinal microflora is considered to be mainly responsible for the colonization resistance in the intestinal tract.23 In the present study, the total anaerobic microflora was strongly suppressed in both treatment groups, although most pronounced in the OCM group. This finding is in accordance with a previous study where oral administration of clarithromycin given to healthy volunteers was shown to suppress the anaerobic microflora.24 Administration of amoxycillin as a single agent has been shown not to affect the normal anaerobic intestinal microflora.6 The suppressive effects of the treatment regimens against anaerobic bacteria in the present study could also be due to metronidazole, a finding which is in accordance with a study by Kager et al.25 High concentrations of metronidazole have previously been measured in colon tissues, although in faecal samples, low or non-detectable levels of nitroimidazoles have been reported.25,26

In the previous study, where omeprazole and placebo were compared with omeprazole plus amoxycillin, alterations in the oral microflora were less in the omeprazole/placebo group than in the omeprazole/amoxycillin group, although the two triple therapies used in the present study caused the most pronounced disturbances in the oral microflora.7 However, in the gastric microflora, more alterations were seen in the omeprazole/placebo group than in the triple therapy treatment groups, with an increase in both aerobic and anaerobic microorganisms during treatment, probably due for the most part to the increase in pH produced by the proton pump inhibitor. In the intestinal microflora, very few alterations were seen during treatment with omeprazole/placebo and omeprazole/amoxycillin compared with the marked ecological disturbances produced by the triple therapies.

In the OAM group, the proportion of β-lactamase-producing Bacteroides spp. isolates increased during treatment, although 100% of these strains were classified as resistant to amoxycillin at all sampling occasions. On the other hand, only 2% of the isolated bacteroides strains from the OCM group were resistant to clarithromycin before treatment, while 76% were resistant on day 7. Even 4 weeks post-treatment, 59% of the bacteroides isolates were clarithromycin resistant according to the NCCLS.13,14 A prolonged ecological disturbance in patients receiving the OCM treatment regimen may be serious, since Bacteroides spp. is the dominant species in the intestinal microflora, and also mainly responsible for serious anaerobic intra-abdominal infections.27 Resistance genes from members of the normal microflora might also be spread on transposable elements to other potentially pathogenic microorganisms as well as to other hosts, which might result in serious treatment failures.

In conclusion, both treatment regimens studied in the present investigation caused marked ecological disturbances in the oral, gastric and intestinal microflora that may lead to decreased colonization resistance and overgrowth of potentially pathogenic microorganisms. This aspect should be taken into consideration when an antimicrobially based treatment for H. pylori infections is planned. Although the treatment outcome was better in the OCM group than in the OAM group, the amoxycillin-based treatment might be preferable from an ecological point of view since the qualitative alterations in terms of emergence and persistence of resistant strains seemed to be most pronounced in the clarithromycin-treated group.

Table I.

Distribution of the saliva microflora in patients receiving omeprazole–amoxycillin–metronidazole and in patients receiving omeprazole–clarithromycin–metronidazole

 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Microorganism na day 0  day 7  day 35  na day 0  day 7  day 35 
aNumber of patients with detectable levels of microorganisms within the sampling period. 
bMedian (minimum–maximum) log values of the number of microorganisms/mL saliva. 
cNumber of patients with detectable levels of microorganisms at the actual sampling day. 
Streptococcus salivarius 14 6.3 (8.0–5.7)b 14c <2.0 (<2.0–7.8)b 5c 6.4 (<2.0–7.5)b 13c 16 7.1 (5.5–8.3)b 16c 5.1 (<2.0–8.0)b 9c 6.7 (<2.0–8.2)b 14c 
Streptococcus mitior 13 4.0 (<2.0–7.0) 5.8 (<2.0–7.0) 10 6.3 (<2.0–7.9) 15 5.6 (<2.0–8.3) 6.3 (<2.0–7.3) 10 7.2 (<2.0–8.2) 14 
Streptococcus intermedius 3.1 (<2.0–6.9) <2.0 (<2.0–8.7) 5.9 (<2.0–6.7) 6.7 (<2.0–7.9) 5.4 (<2.0–7.2) <2.0 (<2.0–7.4) 
Streptococcus sanguis 5.8 (<2.0–7.0) <2.0 (<2.0–6.1) 4.0 (<2.0–7.2) 5.6 (<2.0–7.9) <2.0 (<2.0–7.2) 6.5 (<2.0–8.2) 
Staphylococcus spp. 3.2 (<2.0–4.0) 2.3 (<2.0–2.7) 3.2 (<2.0–4.8) 11 3.0 (<2.0–5.9) <2.0 (<2–4.4) 3.0 (<2.0–4.7) 
Haemophilus spp. 10 <2.0 (<2.0–6.8) 5.3 (<2.0–7.4) 6.0 (<2.0–6.7) 13 5.7 (<2.0–6.5) 5.3 (<2.0–6.8) 11 <2.0 (<2.0–7.8) 
Neisseria spp. 13 5.4 (<2.0–6.8) 12 6.0 (<2.0–7.2) 11 6.1 (3.0–7.0) 13 15 5.7 (<2.0–6.7) 14 5.9 (<2.0–7.3) 12 6.0 (<2.0–7.3) 14 
Enterobacteriaceae 2.6 (<2.0–3.3) 2.6 (<2.0–4.9) 2.3 (<2.0–3.4) 2.3 (<2.0–2.7) 2.8 (2.7–3.0) <2.0 
Yeasts 2.7 (<2.0–7.8) 4.8 (2.7–6.8) 3.3 (<2.0–6.6) 2.4 (<2.0–6.2) 4.3 (<2.0–6.3) 3.2 (<2.0–6.3) 
Peptostreptococcus spp. 2.8 (<2.0–6.2) 3.2 (<2.0–6.6) 3.7 (<2.0–7.0) <2.0 (<2.0–5.8) <2.0 (<2.0–8.0) 5.7 (3.5–7.0) 
Lactobacillus and Bifidobacterium spp. 14 5.0 (<2.0–7.8) 12 5.4 (3.8–7.7) 14 5.2 (3.2–7.8) 14 16 5.0 (3.6–7.7) 16 4.0 (<2.0–7.8) 14 5.1 (2.7–7.9) 16 
Anaerobic Gram-positive bacilli 2.5 (<2.0–5.2) <2.0 (<2.0–4.0) 5.3 (<2.0–6.7) 11 5.0 (<2.0–5.7) <2.0 5.0 (<2.0–6.0) 
Veillonella spp. 14 5.2 (<2.0–6.2) 13 <2.0 (<2.0–6.8) 5.7 (3.4–7.0) 14 15 6.2 (4.0–7.0) 15 <2.0 (<2.0–3.9) 6.0 (<2.0–7.6) 14 
Prevotella spp. and Bacteroides spp. 13 6.1 (<2.0–6.7) 12 <2.0 (<2.0–6.2) 6.0 (<2.0–7.7) 12 15 5.8 (3.4–7.1) 15 <2.0 (<2.0–3.4) 5.0 (<2.0–7.3) 12 
Fusobacterium spp. 10 3.2 (<2.0–6.2) <2.0 4.0 (3.0–6.0) 10 11 3.1 (<2.0–4.4) <2.0 3.2 (<2.0–6.1) 
Leptotrichia spp. 3.7 (<2.0–5.0) <2.0 3.6 (<2.0–6.2) 12 3.4 (<2.0–4.7) 11 <2.0 (<2.0–3.3) 3.3 (<2.0–5.1) 
 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Microorganism na day 0  day 7  day 35  na day 0  day 7  day 35 
aNumber of patients with detectable levels of microorganisms within the sampling period. 
bMedian (minimum–maximum) log values of the number of microorganisms/mL saliva. 
cNumber of patients with detectable levels of microorganisms at the actual sampling day. 
Streptococcus salivarius 14 6.3 (8.0–5.7)b 14c <2.0 (<2.0–7.8)b 5c 6.4 (<2.0–7.5)b 13c 16 7.1 (5.5–8.3)b 16c 5.1 (<2.0–8.0)b 9c 6.7 (<2.0–8.2)b 14c 
Streptococcus mitior 13 4.0 (<2.0–7.0) 5.8 (<2.0–7.0) 10 6.3 (<2.0–7.9) 15 5.6 (<2.0–8.3) 6.3 (<2.0–7.3) 10 7.2 (<2.0–8.2) 14 
Streptococcus intermedius 3.1 (<2.0–6.9) <2.0 (<2.0–8.7) 5.9 (<2.0–6.7) 6.7 (<2.0–7.9) 5.4 (<2.0–7.2) <2.0 (<2.0–7.4) 
Streptococcus sanguis 5.8 (<2.0–7.0) <2.0 (<2.0–6.1) 4.0 (<2.0–7.2) 5.6 (<2.0–7.9) <2.0 (<2.0–7.2) 6.5 (<2.0–8.2) 
Staphylococcus spp. 3.2 (<2.0–4.0) 2.3 (<2.0–2.7) 3.2 (<2.0–4.8) 11 3.0 (<2.0–5.9) <2.0 (<2–4.4) 3.0 (<2.0–4.7) 
Haemophilus spp. 10 <2.0 (<2.0–6.8) 5.3 (<2.0–7.4) 6.0 (<2.0–6.7) 13 5.7 (<2.0–6.5) 5.3 (<2.0–6.8) 11 <2.0 (<2.0–7.8) 
Neisseria spp. 13 5.4 (<2.0–6.8) 12 6.0 (<2.0–7.2) 11 6.1 (3.0–7.0) 13 15 5.7 (<2.0–6.7) 14 5.9 (<2.0–7.3) 12 6.0 (<2.0–7.3) 14 
Enterobacteriaceae 2.6 (<2.0–3.3) 2.6 (<2.0–4.9) 2.3 (<2.0–3.4) 2.3 (<2.0–2.7) 2.8 (2.7–3.0) <2.0 
Yeasts 2.7 (<2.0–7.8) 4.8 (2.7–6.8) 3.3 (<2.0–6.6) 2.4 (<2.0–6.2) 4.3 (<2.0–6.3) 3.2 (<2.0–6.3) 
Peptostreptococcus spp. 2.8 (<2.0–6.2) 3.2 (<2.0–6.6) 3.7 (<2.0–7.0) <2.0 (<2.0–5.8) <2.0 (<2.0–8.0) 5.7 (3.5–7.0) 
Lactobacillus and Bifidobacterium spp. 14 5.0 (<2.0–7.8) 12 5.4 (3.8–7.7) 14 5.2 (3.2–7.8) 14 16 5.0 (3.6–7.7) 16 4.0 (<2.0–7.8) 14 5.1 (2.7–7.9) 16 
Anaerobic Gram-positive bacilli 2.5 (<2.0–5.2) <2.0 (<2.0–4.0) 5.3 (<2.0–6.7) 11 5.0 (<2.0–5.7) <2.0 5.0 (<2.0–6.0) 
Veillonella spp. 14 5.2 (<2.0–6.2) 13 <2.0 (<2.0–6.8) 5.7 (3.4–7.0) 14 15 6.2 (4.0–7.0) 15 <2.0 (<2.0–3.9) 6.0 (<2.0–7.6) 14 
Prevotella spp. and Bacteroides spp. 13 6.1 (<2.0–6.7) 12 <2.0 (<2.0–6.2) 6.0 (<2.0–7.7) 12 15 5.8 (3.4–7.1) 15 <2.0 (<2.0–3.4) 5.0 (<2.0–7.3) 12 
Fusobacterium spp. 10 3.2 (<2.0–6.2) <2.0 4.0 (3.0–6.0) 10 11 3.1 (<2.0–4.4) <2.0 3.2 (<2.0–6.1) 
Leptotrichia spp. 3.7 (<2.0–5.0) <2.0 3.6 (<2.0–6.2) 12 3.4 (<2.0–4.7) 11 <2.0 (<2.0–3.3) 3.3 (<2.0–5.1) 

Table II.

Distribution of the gastric mucosal microflora in patients receiving omeprazole–amoxycillin–metronidazole and in patients receiving omeprazole–clarithromycin–metronidazole

 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Microorganism na day 0  day 7  day 35  na day 0  day 7  day 35 
aNumber of patients with detectable levels of microorganisms within the sampling period. 
bMedian (minimum–maximum) of log values of the number of microorganisms/g biopsy. 
cNumber of patients with detectable levels of microorganisms at the actual sampling day. 
S. salivarius 12 4.3 (<2.0–6.6)b 11c <2.0 (<2.0–4.3)b 3c 2.7 (<2.0–5.2)b 7c 11 3.3 (<2.0–6.3)b 7c 3.2 (<2.0–6.7)b 6c <2.0 (<2.0–6.0)b 4c 
S. mitior 14 4.3 (<2.0–6.5) 12 5.0 (<2.0–6.0) 12 3.4 (<2.0–5.8) 12 14 3.4 (<2.0–7.2) 5.4 (3.0–6.6) 14 2.7 (<2.0–6.4) 
S. intermedius 12 <2.0 (<2.0–5.6) 2.7 (<2.0–5.2) 3.0 (<2.0–5.7) <2.0 (<2.0–4.4) 3.1 (<2.0–6.4) <2.0 
S. sanguis 4.1 (<2.0–5.2) <2.0 (<2.0–5.4) <2.0 (<2.0–4.0) <2.0 (<2.0–3.7) 3.0 (<2.0–5.6) <2.0 (<2.0–5.3) 
Staphylococcus spp. 11 2.3 (<2.0–4.0) 2.7 (<2.0–3.0) <2.0 (<2.0–4.6) 10 <2.0 (<2.0–4.7) 2.7 (<2.0–4.7) 2.3 (<2.0–5.9) 
Micrococcus 4.7 (3.4–5.3) 3.3 (<2.0–4.7) 2.7 (<2.0–4.5) <2.0 (<2.0–4.2) <2.0 (<2.0–3.9) 2.7 (<2.0–4.3) 
Haemophilus spp. 10 <2.0 (<2.0–4.8) 4.5 (3.2–5.2) 10 <2.0 (<2.0–4.0) 14 <2.0 (<2.0–3.5) 4.2 (2.7–5.7) 14 <2.0 (<2.0–5.6) 
Neisseria spp. 12 2.8 (<2.0–5.4) 4.0 (2.7–6.3) 12 3.2 (<2.0–5.0) 14 <2.0 (<2.0–6.0) 4.5 (<2.0–5.8) 13 <2.0 (<2.0–5.2) 
Enterobacteriaceae <2.0 (<2.0–4.7) 3.6 (2.7–4.6) <2.0 (<2.0–2.7) 3.3 (<2.0–4.6) 2.5 (<2.0–3.0) 3.7 (<2.0–5.4) 
Yeasts <2.0 4.7 (2.7–4.7) <2.0 (<2.0–3.0) <2.0 2.7 (<2.0–4.2) <2.0 (<2.0–3.7) 
Peptostreptococcus spp. 10 4.4 (<2.0–5.4) <2.0 (<2.0–5.5) 3.0 (<2.0–5.2) 11 3.2 (<2.0–4.8) 3.7 (<2.0–5.0) <2.0 (<2.0–4.7) 
Lactobacilli and Bifidobacterium spp. 4.0 (<2.0–5.8) 3.0 (<2.0–5.2) <2.0 (<2.0–3.7) 2.7 (<2.0–4.1) <2.0 (<2.0–4.7) <2.0 (<2.0–4.0) 
Actinomyces 11 <2.0 (<2.0–5.6) 2.8 (<2.0–4.9) 2.8 (<2.0–5.3) 2.4 (<2.0–5.3) <2.0 (<2.0–4.6) 2.8 (<2.0–4.7) 
Veillonella spp. 11 3.2 (<2.0–5.5) <2.0 (<2.0–5.3) 2.7 (<2.0–5.0) 3.0 (<2.0–6.3) <2.0 (<2.0–4.0) 2.7 (<2.0–5.8) 
Prevotella spp. and Bacteroides spp. <2.0 (<2.0–4.3) 4.0 (<2.0–5.6) <2.0 4.4 (<2.0–6.8) 3.8 (<2.0–4.4) <2.0 (<2.0–3.2) 
Fusobacterium spp. 4.0 (<2.0–4.7) <2.0 <2.0 (<2.0–4.2) 3.7 <2.0 <2.0 
 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Microorganism na day 0  day 7  day 35  na day 0  day 7  day 35 
aNumber of patients with detectable levels of microorganisms within the sampling period. 
bMedian (minimum–maximum) of log values of the number of microorganisms/g biopsy. 
cNumber of patients with detectable levels of microorganisms at the actual sampling day. 
S. salivarius 12 4.3 (<2.0–6.6)b 11c <2.0 (<2.0–4.3)b 3c 2.7 (<2.0–5.2)b 7c 11 3.3 (<2.0–6.3)b 7c 3.2 (<2.0–6.7)b 6c <2.0 (<2.0–6.0)b 4c 
S. mitior 14 4.3 (<2.0–6.5) 12 5.0 (<2.0–6.0) 12 3.4 (<2.0–5.8) 12 14 3.4 (<2.0–7.2) 5.4 (3.0–6.6) 14 2.7 (<2.0–6.4) 
S. intermedius 12 <2.0 (<2.0–5.6) 2.7 (<2.0–5.2) 3.0 (<2.0–5.7) <2.0 (<2.0–4.4) 3.1 (<2.0–6.4) <2.0 
S. sanguis 4.1 (<2.0–5.2) <2.0 (<2.0–5.4) <2.0 (<2.0–4.0) <2.0 (<2.0–3.7) 3.0 (<2.0–5.6) <2.0 (<2.0–5.3) 
Staphylococcus spp. 11 2.3 (<2.0–4.0) 2.7 (<2.0–3.0) <2.0 (<2.0–4.6) 10 <2.0 (<2.0–4.7) 2.7 (<2.0–4.7) 2.3 (<2.0–5.9) 
Micrococcus 4.7 (3.4–5.3) 3.3 (<2.0–4.7) 2.7 (<2.0–4.5) <2.0 (<2.0–4.2) <2.0 (<2.0–3.9) 2.7 (<2.0–4.3) 
Haemophilus spp. 10 <2.0 (<2.0–4.8) 4.5 (3.2–5.2) 10 <2.0 (<2.0–4.0) 14 <2.0 (<2.0–3.5) 4.2 (2.7–5.7) 14 <2.0 (<2.0–5.6) 
Neisseria spp. 12 2.8 (<2.0–5.4) 4.0 (2.7–6.3) 12 3.2 (<2.0–5.0) 14 <2.0 (<2.0–6.0) 4.5 (<2.0–5.8) 13 <2.0 (<2.0–5.2) 
Enterobacteriaceae <2.0 (<2.0–4.7) 3.6 (2.7–4.6) <2.0 (<2.0–2.7) 3.3 (<2.0–4.6) 2.5 (<2.0–3.0) 3.7 (<2.0–5.4) 
Yeasts <2.0 4.7 (2.7–4.7) <2.0 (<2.0–3.0) <2.0 2.7 (<2.0–4.2) <2.0 (<2.0–3.7) 
Peptostreptococcus spp. 10 4.4 (<2.0–5.4) <2.0 (<2.0–5.5) 3.0 (<2.0–5.2) 11 3.2 (<2.0–4.8) 3.7 (<2.0–5.0) <2.0 (<2.0–4.7) 
Lactobacilli and Bifidobacterium spp. 4.0 (<2.0–5.8) 3.0 (<2.0–5.2) <2.0 (<2.0–3.7) 2.7 (<2.0–4.1) <2.0 (<2.0–4.7) <2.0 (<2.0–4.0) 
Actinomyces 11 <2.0 (<2.0–5.6) 2.8 (<2.0–4.9) 2.8 (<2.0–5.3) 2.4 (<2.0–5.3) <2.0 (<2.0–4.6) 2.8 (<2.0–4.7) 
Veillonella spp. 11 3.2 (<2.0–5.5) <2.0 (<2.0–5.3) 2.7 (<2.0–5.0) 3.0 (<2.0–6.3) <2.0 (<2.0–4.0) 2.7 (<2.0–5.8) 
Prevotella spp. and Bacteroides spp. <2.0 (<2.0–4.3) 4.0 (<2.0–5.6) <2.0 4.4 (<2.0–6.8) 3.8 (<2.0–4.4) <2.0 (<2.0–3.2) 
Fusobacterium spp. 4.0 (<2.0–4.7) <2.0 <2.0 (<2.0–4.2) 3.7 <2.0 <2.0 

Table III.

Distribution of the intestinal microflora in patients receiving omeprazole–amoxycillin–metronidazole and in patients receiving omeprazole–clarithromycin–metronidazole

 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Microorganism na day 0  day 7  day 35  na day 0  day 7  day 35 
aNumber of patients with detectable levels of microorganisms within the sampling period. 
bMedian (minimum–maximum) log values of the number of microorganisms/g faeces. 
cNumber of patients with detectable levels of microorganisms on the actual sampling day. 
dEnterobacteriaceae except E. coli. 
Enterococcus spp. 13 5.4 (<2.0–6.7)b 12c 7.7 (3.3–10.0)b 13c 6.5 (2.9–10.3)b 13c 16 4.5 (<2.0–6.1)b 14c 6.6 (<2.0–10.6)b 13c 5.2 (2.5–9.6)b 16c 
E. coli 13 5.3 (2.8–8.3) 13 4.8 (<2.0–9.7) 11 5.7 (3.7–8.6) 11 15 5.1 (<2.0–7.8) 12 <2.0 (<2.0–5.9) 5.0 (<2.0–7.7) 14 
Enterobacteriaceaed 14 <2.0 (<2.0–5.2) 6.2 (2.5–9.3) 14 <2.0 (<2.0–6.7) 15 <2.0 (<2.0–8.0) 3.8 (<2.0–6.6) 10 3.0 (<2.0–7.6) 10 
Yeasts 10 <2.0 3.9 (<2.0–5.4) <2.0 (<2.0–3.4) <2.0 (<2.0–3.5) 3.1 (<2.0–3.9) 3.0 (<2.0–4.0) 
Peptostreptococcus spp. <2.0 (<2.0–6.0) 5.5 (<2.0–7.0) <2.0 (<2.0–8.3) <2.0 2.5 (<2.0–7.7) 5.3 (<2.0–8.0) 
Lactobacillus spp. 13 4.9 (<2.0–6.7) 11 4.5 (<2.0–6.9) 11 5.8 (<2.0–8.0) 13 14 4.8 (<2.0–9.2) 13 3.2 (<2.0–7.8) 11 5.3 (<2.0–9.2) 13 
Bifidobacterium spp. 12 6.2 (4.8–7.8) 12 <2.0 (<2.0–8.7) 6.3 (<2.0–8.4) 11 13 7.1 (5.2–8.5) 12 <2.0 (<2.0–5.3) 3.9 (<2.0–8.3) 10 
Clostridium spp. 11 5.0 (<2.0–7.0) 11 <2.0 (<2.0–4.9) 5.7 (<2.0–8.0) 11 15 5.5 (<2.0–6.1) 13 <2.0 (<2.0–8.7) 4.8 (<2.0–7.9) 13 
Veillonella spp. 2.3 (<2.0–5.3) <2.0 3.6 (<2.0–7.1) 2.7 (<2.0–4.4) <2.0 3.0 (<2.0–4.2) 
Bacteroides spp. 14 7.3 (5.6–9.7) 14 6.0 (<2.0–9.5) 7.3 (3.0–9.9) 14 16 7.6 (<2.0–9.1) 14 2.6 (<2.0–8.1) 5.4 (<2.0–9.8) 11 
 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Microorganism na day 0  day 7  day 35  na day 0  day 7  day 35 
aNumber of patients with detectable levels of microorganisms within the sampling period. 
bMedian (minimum–maximum) log values of the number of microorganisms/g faeces. 
cNumber of patients with detectable levels of microorganisms on the actual sampling day. 
dEnterobacteriaceae except E. coli. 
Enterococcus spp. 13 5.4 (<2.0–6.7)b 12c 7.7 (3.3–10.0)b 13c 6.5 (2.9–10.3)b 13c 16 4.5 (<2.0–6.1)b 14c 6.6 (<2.0–10.6)b 13c 5.2 (2.5–9.6)b 16c 
E. coli 13 5.3 (2.8–8.3) 13 4.8 (<2.0–9.7) 11 5.7 (3.7–8.6) 11 15 5.1 (<2.0–7.8) 12 <2.0 (<2.0–5.9) 5.0 (<2.0–7.7) 14 
Enterobacteriaceaed 14 <2.0 (<2.0–5.2) 6.2 (2.5–9.3) 14 <2.0 (<2.0–6.7) 15 <2.0 (<2.0–8.0) 3.8 (<2.0–6.6) 10 3.0 (<2.0–7.6) 10 
Yeasts 10 <2.0 3.9 (<2.0–5.4) <2.0 (<2.0–3.4) <2.0 (<2.0–3.5) 3.1 (<2.0–3.9) 3.0 (<2.0–4.0) 
Peptostreptococcus spp. <2.0 (<2.0–6.0) 5.5 (<2.0–7.0) <2.0 (<2.0–8.3) <2.0 2.5 (<2.0–7.7) 5.3 (<2.0–8.0) 
Lactobacillus spp. 13 4.9 (<2.0–6.7) 11 4.5 (<2.0–6.9) 11 5.8 (<2.0–8.0) 13 14 4.8 (<2.0–9.2) 13 3.2 (<2.0–7.8) 11 5.3 (<2.0–9.2) 13 
Bifidobacterium spp. 12 6.2 (4.8–7.8) 12 <2.0 (<2.0–8.7) 6.3 (<2.0–8.4) 11 13 7.1 (5.2–8.5) 12 <2.0 (<2.0–5.3) 3.9 (<2.0–8.3) 10 
Clostridium spp. 11 5.0 (<2.0–7.0) 11 <2.0 (<2.0–4.9) 5.7 (<2.0–8.0) 11 15 5.5 (<2.0–6.1) 13 <2.0 (<2.0–8.7) 4.8 (<2.0–7.9) 13 
Veillonella spp. 2.3 (<2.0–5.3) <2.0 3.6 (<2.0–7.1) 2.7 (<2.0–4.4) <2.0 3.0 (<2.0–4.2) 
Bacteroides spp. 14 7.3 (5.6–9.7) 14 6.0 (<2.0–9.5) 7.3 (3.0–9.9) 14 16 7.6 (<2.0–9.1) 14 2.6 (<2.0–8.1) 5.4 (<2.0–9.8) 11 

Table IV.

MIC50 and MIC90 values (mg/L) for microorganisms isolated in saliva in patients treated with (a) omeprazole–amoxycillin–metronidazole and (b) omeprazole–clarithromycin–metronidazole

 day 0 day 7 day 35 
  MIC50/90 range  MIC50/90 range P valuec  MIC50/90 range P valuec 
Microorganism nb (mg/L) (mg/L) nb (mg/L) (mg/L) day 0–7 nb (mg/L) (mg/L) day 0–35 
aMIC is tested for (a) amoxycillin and (b) clarithromycin. 
bNumber of strains tested. 
cAccording to Mann–Whitney U-test. 
NS, not significant; ND, not done. 
(a) Omeprazole–amoxycillin–metronidazole groupa            
Streptococcus spp. 58 0.032/0.125 0.032–0.25 44 0.25/1 0.032–1 <0.001 63 0.032/0.25 0.032–0.5 <0.05 
Staphylococcus spp. 0.064/0.125 0.064–0.125 0.125/0.125 0.032–0.25 NS 0.125/0.25 0.032–0.25 NS 
 Enterobacteriaceae 4/4 4–32 16/32 16–32 ND 2/2 2–64 ND 
Prevotella and Bacteroides spp. 16 0.25/1 0.064–2 – – ND 10 0.5/2 0.064–4 ND 
(b) Omeprazole–clarithromycin–metronidazole groupa            
Streptococcus spp. 65 0.032/0.032 0.032–2 53 1/16 0.032–64 <0.001 57 0.032/2 0.032–64 <0.001 
Staphylococcus spp. 12 0.125/0.25 0.125–32 0.125/32 0.125–32 NS 11 0.25/32 0.032–32 NS 
 Enterobacteriaceae 64 – 64/128 32–128 ND 128 – ND 
Prevotella and Bacteroides spp. 21 0.064/0.125 0.032–0.25 0.032/0.032 0.032–0.064 NS 22 0.064/0.125 0.032–0.25 NS 
 day 0 day 7 day 35 
  MIC50/90 range  MIC50/90 range P valuec  MIC50/90 range P valuec 
Microorganism nb (mg/L) (mg/L) nb (mg/L) (mg/L) day 0–7 nb (mg/L) (mg/L) day 0–35 
aMIC is tested for (a) amoxycillin and (b) clarithromycin. 
bNumber of strains tested. 
cAccording to Mann–Whitney U-test. 
NS, not significant; ND, not done. 
(a) Omeprazole–amoxycillin–metronidazole groupa            
Streptococcus spp. 58 0.032/0.125 0.032–0.25 44 0.25/1 0.032–1 <0.001 63 0.032/0.25 0.032–0.5 <0.05 
Staphylococcus spp. 0.064/0.125 0.064–0.125 0.125/0.125 0.032–0.25 NS 0.125/0.25 0.032–0.25 NS 
 Enterobacteriaceae 4/4 4–32 16/32 16–32 ND 2/2 2–64 ND 
Prevotella and Bacteroides spp. 16 0.25/1 0.064–2 – – ND 10 0.5/2 0.064–4 ND 
(b) Omeprazole–clarithromycin–metronidazole groupa            
Streptococcus spp. 65 0.032/0.032 0.032–2 53 1/16 0.032–64 <0.001 57 0.032/2 0.032–64 <0.001 
Staphylococcus spp. 12 0.125/0.25 0.125–32 0.125/32 0.125–32 NS 11 0.25/32 0.032–32 NS 
 Enterobacteriaceae 64 – 64/128 32–128 ND 128 – ND 
Prevotella and Bacteroides spp. 21 0.064/0.125 0.032–0.25 0.032/0.032 0.032–0.064 NS 22 0.064/0.125 0.032–0.25 NS 

Table V.

MIC50 and MIC90 values (mg/L) for microorganisms isolated in the gastric mucosa in patients treated with (a) omeprazole–amoxycillin– metronidazole, and (b) omeprazole–clarithromycin–metronidazole

 day 0 day 7  day 35  
  MIC50/90 range  MIC50/90 range P valuec  MIC50/90 range P valuec 
Microorganism nb (mg/L) (mg/L) nb (mg/L) (mg/L) day 0–7 nb (mg/L) (mg/L) day 0–35 
aMIC values are tested for (a) amoxycillin and (b) clarithromycin. 
bNumber of strains tested. 
cAccording to Mann–Whitney U-test 
NS, not significant; ND, not done. 
(a) Omeprazole–amoxycillin–metronidazole groupa            
Streptococcus spp. 49 0.032/0.5 0.032–4 39 0.5/2 0.032–4 <0.001 38 0.064/0.25 0.032–2 <0.05 
Staphylococcus spp. 10 0.032/0.5 0.032–1 11 0.5/1 0.125–1 <0.05 0.125/0.25 0.032–1 NS 
 Enterobacteriaceae – 16/64 4–64 ND – ND 
(b) Omeprazole–clarithromycin–metronidazole groupa            
Streptococcus spp. 34 0.032/0.25 0.032–128 52 1/16 0.032–>128 <0.001 22 0.032/4 0.032–16 NS 
Staphylococcus spp. 0.5/8 0.125–16 2/32 0.125–32 NS 12 0.125/0.25 0.125–0.25 NS 
 Enterobacteriaceae 64/64 64–64 64/64 64–64 ND 128/128 128–128 ND 
 day 0 day 7  day 35  
  MIC50/90 range  MIC50/90 range P valuec  MIC50/90 range P valuec 
Microorganism nb (mg/L) (mg/L) nb (mg/L) (mg/L) day 0–7 nb (mg/L) (mg/L) day 0–35 
aMIC values are tested for (a) amoxycillin and (b) clarithromycin. 
bNumber of strains tested. 
cAccording to Mann–Whitney U-test 
NS, not significant; ND, not done. 
(a) Omeprazole–amoxycillin–metronidazole groupa            
Streptococcus spp. 49 0.032/0.5 0.032–4 39 0.5/2 0.032–4 <0.001 38 0.064/0.25 0.032–2 <0.05 
Staphylococcus spp. 10 0.032/0.5 0.032–1 11 0.5/1 0.125–1 <0.05 0.125/0.25 0.032–1 NS 
 Enterobacteriaceae – 16/64 4–64 ND – ND 
(b) Omeprazole–clarithromycin–metronidazole groupa            
Streptococcus spp. 34 0.032/0.25 0.032–128 52 1/16 0.032–>128 <0.001 22 0.032/4 0.032–16 NS 
Staphylococcus spp. 0.5/8 0.125–16 2/32 0.125–32 NS 12 0.125/0.25 0.125–0.25 NS 
 Enterobacteriaceae 64/64 64–64 64/64 64–64 ND 128/128 128–128 ND 

Table VI.

MIC50 and MIC90 values (mg/L) for intestinal microorganisms isolated in patients treated with (a) omeprazole–amoxycillin–metronidazole and (b) omeprazole–clarithromycin–metronidazole

 day 0 day 7  day 35  
  MIC50/90 range  MIC50/90 range P valuec  MIC50/90 range P valuec 
Microorganism nb (mg/L) (mg/L) nb (mg/L) (mg/L) day 0–7 nb (mg/L) (mg/L) day 0–35 
aAntibiotic tested. 
bNumber of strains tested. 
cAccording to Mann–Whitney U-test 
NS, non significant. 
(a) Omeprazole–amoxycillin–metronidazole groupa            
Enterococcus spp. amoxycillina 37 0.125/0.5 0.064–2 43 0.25/0.5 0.064–1 <0.001 43 0.5/0.5 0.1251 <0.001 
 Enterobacteriaceae amoxycillin 42 4/>256 1–>256 42 64/>256 1–>256 <0.001 42 4/>256 0.5–>256 NS 
Bacteroides spp. amoxycillin 42 16/>256 8–>256 21 32/256 16–>256 NS 44 32/>256 0.064–>256 NS 
Bacteroides spp. metronidazole 42 0.5/1 0.064–1 21 0.5/1 0.25–1 NS 44 0.5/0.5 0.125–1 NS 
(b) Omeprazole–clarithromycin–metronidazole groupa            
Enterococcus spp. clarithromycina 43 0.5/1 0.032-8 37 >128/>128 4–>128 <0.001 44 0.5/>128 0.032–>128 NS 
 Enterobacteriaceae clarithromycin 45 16/64 16–>128 31 64/>128 32–>128 <0.001 46 32/64 0.064–>128 NS 
Bacteroides spp. clarithromycin 43 0.5/4 0.25–>128 25 >128/>128 0.25–>128 <0.001 32 8/>128 0.25–>128 <0.001 
Bacteroides spp. metronidazole 43 0.5/0.5 0.25–0.5 22 0.5/1 0.064–1 NS 34 0.5/1 0.25–2 NS 
 day 0 day 7  day 35  
  MIC50/90 range  MIC50/90 range P valuec  MIC50/90 range P valuec 
Microorganism nb (mg/L) (mg/L) nb (mg/L) (mg/L) day 0–7 nb (mg/L) (mg/L) day 0–35 
aAntibiotic tested. 
bNumber of strains tested. 
cAccording to Mann–Whitney U-test 
NS, non significant. 
(a) Omeprazole–amoxycillin–metronidazole groupa            
Enterococcus spp. amoxycillina 37 0.125/0.5 0.064–2 43 0.25/0.5 0.064–1 <0.001 43 0.5/0.5 0.1251 <0.001 
 Enterobacteriaceae amoxycillin 42 4/>256 1–>256 42 64/>256 1–>256 <0.001 42 4/>256 0.5–>256 NS 
Bacteroides spp. amoxycillin 42 16/>256 8–>256 21 32/256 16–>256 NS 44 32/>256 0.064–>256 NS 
Bacteroides spp. metronidazole 42 0.5/1 0.064–1 21 0.5/1 0.25–1 NS 44 0.5/0.5 0.125–1 NS 
(b) Omeprazole–clarithromycin–metronidazole groupa            
Enterococcus spp. clarithromycina 43 0.5/1 0.032-8 37 >128/>128 4–>128 <0.001 44 0.5/>128 0.032–>128 NS 
 Enterobacteriaceae clarithromycin 45 16/64 16–>128 31 64/>128 32–>128 <0.001 46 32/64 0.064–>128 NS 
Bacteroides spp. clarithromycin 43 0.5/4 0.25–>128 25 >128/>128 0.25–>128 <0.001 32 8/>128 0.25–>128 <0.001 
Bacteroides spp. metronidazole 43 0.5/0.5 0.25–0.5 22 0.5/1 0.064–1 NS 34 0.5/1 0.25–2 NS 

Table VII.

Frequency of resistant strains (R) isolated from saliva, gastric mucosa and faecal samples according to the NCCLS (1997)14

 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Sample day 0 day 7 day 35 day 0 day 7 day 35 
aNumber of resistant strains according to the NCCLS/number of strains tested. 
Percentages are shown in parentheses. 
Saliva             
Streptococcus spp. 0/58a (0) 0/44a (0) 0/63a (0) 3/65a (5) 39/53a (74) 15/57a (26) 
Staphylococcus spp. 0/6 (0) 0/5 (0) 0/8 (0) 1/12 (8) 1/3 (33) 4/11 (36) 
 Enterobacteriaceae 1/2 (50) 4/8 (50) 1/2 (50) 1/1 (100) 4/4 (100) 1/1 (100) 
Prevotella and Bacteroides spp. 0/16 (0) 0/0 (0) 0/10 (0) 0/21 (0) 0/2 (0) 0/22 (0) 
Gastric mucosa             
Streptococcus spp. 0/49 (0) 0/39 (0) 0/38 (0) 2/34 (6) 36/52 (70) 5/22 (23) 
Staphylococcus spp. 3/10 (30) 7/11 (64) 1/7 (14) 2/5 (40) 2/6 (33) 0/12 (0) 
 Enterobacteriaceae 0/1 (0) 3/7 (43) 0/1 (0) 3/3 (100) 3/3 (100) 2/2 (100) 
Faecal samples             
Enterococcus spp. 0/37 (0) 0/43 (0) 0/43 (0) 1/43 (2) 34/37 (92) 13/44 (29) 
Enterobacteriaceae 5/42 (12) 32/42 (76) 16/42 (38) 45/45 (100) 31/31 (100) 46/46 (100) 
Bacteroides spp. 42/42 (100) 21/21 (100) 44/44 (100) 1/43 (2) 19/25 (76) 19/32 (59) 
 Omeprazole–amoxycillin–metronidazole group Omeprazole–clarithromycin–metronidazole group 
Sample day 0 day 7 day 35 day 0 day 7 day 35 
aNumber of resistant strains according to the NCCLS/number of strains tested. 
Percentages are shown in parentheses. 
Saliva             
Streptococcus spp. 0/58a (0) 0/44a (0) 0/63a (0) 3/65a (5) 39/53a (74) 15/57a (26) 
Staphylococcus spp. 0/6 (0) 0/5 (0) 0/8 (0) 1/12 (8) 1/3 (33) 4/11 (36) 
 Enterobacteriaceae 1/2 (50) 4/8 (50) 1/2 (50) 1/1 (100) 4/4 (100) 1/1 (100) 
Prevotella and Bacteroides spp. 0/16 (0) 0/0 (0) 0/10 (0) 0/21 (0) 0/2 (0) 0/22 (0) 
Gastric mucosa             
Streptococcus spp. 0/49 (0) 0/39 (0) 0/38 (0) 2/34 (6) 36/52 (70) 5/22 (23) 
Staphylococcus spp. 3/10 (30) 7/11 (64) 1/7 (14) 2/5 (40) 2/6 (33) 0/12 (0) 
 Enterobacteriaceae 0/1 (0) 3/7 (43) 0/1 (0) 3/3 (100) 3/3 (100) 2/2 (100) 
Faecal samples             
Enterococcus spp. 0/37 (0) 0/43 (0) 0/43 (0) 1/43 (2) 34/37 (92) 13/44 (29) 
Enterobacteriaceae 5/42 (12) 32/42 (76) 16/42 (38) 45/45 (100) 31/31 (100) 46/46 (100) 
Bacteroides spp. 42/42 (100) 21/21 (100) 44/44 (100) 1/43 (2) 19/25 (76) 19/32 (59) 
*
Correspondence address. F82, Huddinge University Hospital, S-131 86 Huddinge, Sweden. Tel:+46-8-585-878-38; Fax: +46-8-711-3918; E-mail: carl.erik.nord@impi.ki.se

References

1.
Bell, G. D. & Powell, K. U. (
1993
). Eradication of Helicobacter pylori and its effect in peptic ulcer disease.
Scandinavian Journal of Gastroenterology
 
28, Suppl. 196,
7
– 11.
2.
Labenz, J., Gyenes E., Rühl G. H. & Börsch, G. (
1993
). Omeprazole plus amoxycillin: efficacy of various treatment regimens to eradicate Helicobacter pylori.
American Journal of Gastroenterology
 
88
,
491
–5.
3.
Lind, T., van Zanten, S. V., Unge, P., Spiller, R. C., Bayerdorffer, E., O'Morain, C. et al. (
1996
). Eradication of Helicobacter pylori using one-week triple therapies combining omeprazole with two antimicrobials: MACH I Study.
Helicobacter
 
1
,
138
–44.
4.
Hunt, R. H. (
1997
). Peptic ulcer disease: defining the treatment strategies in the era of Helicobacter pylori.
American Journal of Gastroenterology
 
92, Suppl. 4,
36S
–40S.
5.
The European Helicobacter pylori Study Group. (
1997
). Current European concepts in the management of Helicobacter pylori infection. The Maastricht Consensus Report.
Gut
 
41
,
8
– 13.
6.
Edlund, C. & Nord, C. E. (
1993
). Ecological impact of antimicrobial agents on human intestinal microflora.
Alpe Adria Microbiology Journal
 
2
,
137
–64.
7.
Stark, C. A., Adamsson, I., Edlund, C., Sjöstedt, S., Seensalu, R., Wikström, B. et al. (
1996
). Effects of omeprazole and amoxycillin on the human oral and gastrointestinal microflora in patients with Helicobacter pylori infection .
Journal of Antimicrobial Chemotherapy
 
38
,
927
–39.
8.
Möller, Å. J. R. (
1966
). Microbiological examination of root canals and periapical tissues of human teeth.
Scandinavian Dental Journal
 
74
,
5
–6.
9.
Oksanen, A., Bergström, M., Sjöstedt, S., Gad, A., Hammarlund, B. & Seensalu, R. (
1997
). Accurate detection of Helicobacter pylori infection with a simplified C13-urea breath test.
Scandinavian Journal of Clinical and Laboratory Investigation
 
57
,
689
–94.
10.
Heimdahl, A. & Nord, C. E. (
1979
). Effect of phenoxymethylpenicillin and clindamycin on the oral, throat and faecal microflora of man .
Scandinavian Journal of Infectious Diseases
 
11
,
233
–42.
11.
Adamsson, I., Seensalu, R., Sjöstedt, S., Wikström, B. & Nord, C. E. (
1998
). The value of different detection methods of Helicobacter pylori treatment .
Journal of Clinical Gastroenterology
 
27
,
138
–42.
12.
Nord, C. E. & Edlund, C. (
1990
). Impact of antimicrobial agents on human intestinal microflora .
Journal of Chemotherapy
 
2
,
218
–37.
13.
National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.
14.
National Committee for Clinical Laboratory Standards. (1997). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria—Fourth Edition: Approved Standard M11-A4. Vol. 17, No. 2. NCCLS, Villanova, PA.
15.
Blaser, M. J. (
1998
). Helicobacter pylori and gastric diseases.
British Medical Journal
 
316
,
1507
–10.
16.
NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease. (
1994
). Helicobacter pylori in peptic ulcer disease.
Journal of the American Medical Association
 
272
,
65
–9.
17.
Hunt, R. H. (
1993
). Hp and pH: implications for the eradication of Helicobacter pylori.
Scandinavian Journal of Gastroenterology
 
28, Suppl. 196
,
12
–6.
18.
Cederbrant, G., Kahlmeter, G., Schalen, C. & Kamme, C. (
1994
). Additive effect of clarithromycin combined with 14-hydroxy clarithromycin, erythromycin, amoxycillin, metronidazole, or omeprazole against Helicobacter pylori.
Journal of Antimicrobial Chemotherapy
 
34
,
1025
–9.
19.
Sjöstedt, S., Sagar, M., Lindberg, G., Wikström, B., Nord, C. E. & Seensalu, R. (
1998
). Prolonged and profound acid inhibition is crucial in Helicobacter pyloritreatment with a proton pump inhibitor combined with amoxycillin.
Scandinavian Journal of Gastroenterology
 
33
,
39
– 43.
20.
Peterson, W. L. (
1997
). The role of antisecretory drugs in the treatment of Helicobacter pylori infection.
Alimentary Pharmacology and Therapeutics
 
11, Suppl. 1
,
21
–5.
21.
Adamsson, I., Edlund, C., Seensalu, R., Sjöstedt, S.& Nord, C. E. (
1998
). The normal gastric microflora and Helicobacter pylori; before, during and after treatment with omeprazole and amoxycillin.
Clinical Microbiology and Infection
 
4
,
308
–15.
22.
Edlund, C., Stark, C. & Nord, C. E. (
1994
).The relationship between an increase in β-lactamase activity after oral administration of three new cephalosporins and protection against intestinal ecological disturbances .
Journal Antimicrobial Chemotherapy
 
34
,
127
–38.
23.
van der Waaij, D. (
1989
). The ecology of the human intestine and its consequences for overgrowth by pathogens such as Clostridium difficile.
Annual Review of Microbiology
 
43
,
69
–87.
24.
Brismar, B., Edlund, C. & Nord, C. E. (
1991
). Comparative effects of clarithromycin and erythromycin on the normal intestinal microflora .
Scandinavian Journal of Infectious Diseases
 
23
,
635
– 42.
25.
Kager, L., Ljungdahl, I., Malmborg, A. S. & Nord C. E. (
1981
). Effect of tinidazole prophylaxis on the normal microflora in patients undergoing colorectal sugery.
Scandinavian Journal of Infectious Diseases
 
26, Suppl.
,
84
–91.
26.
Bergan, T., Solhaug, J. H., Soreide, O. & Leinebo, O. (
1985
). Comparative pharmacokinetics of metronidazole and tinidazole and their tissue penetration.
Scandinavian Journal of Gastroenterology
 
20
,
945
–50.
27.
Finegold, S. (
1995
). Anaerobic infections in humans: an overview.
Anaerobe
 
1
,
3
–9.
28.
Goodwin, C. S. & Armstrong, J. A. (
1990
). Microbiological aspects of Helicobacter pylori (Campylobacter pylori).
European Journal of Clinical Microbiology and Infectious Diseases
 
9
,
1
–13.