a Department of Immunology, Microbiology, Pathology and Infectious Diseases b Centre of Gastroenterology, Huddinge University Hospital, Karolinska Institute, Stockholm c University College of South Stockholm, Stockholm, Sweden
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Abstract |
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Introduction |
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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.
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Materials and methods |
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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 3182 years). The OCM group consisted of 10 women and six men, with a mean age of 58.8 years (range 3375 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, 0.258 mg/L for amoxycillin and 0.516 mg/L for clarithromycin.
Microbial cultivation
The saliva and faecal samples were suspended in a prereduced peptone yeast glucose medium, diluted 10-fold to 107, 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 104 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 ß-lactamase activity
Faecal strains belonging to the Bacteroides spp. group were assayed for ß-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 MannWhitney U-test.
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Results |
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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 0.101.70 mg/L). In the gastric biopsies, five patients had detectable levels of amoxycillin, median value 5.03 mg/kg (range 1.18116 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.
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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.
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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.
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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 MannWhitney 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.
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The frequency of ß-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 ß-lactamase assay. Among patients not treated with ß-lactam agents (OCM), the proportion of ß-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).
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Discussion |
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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 ß-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 ß-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.
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Notes |
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References |
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Received 22 January 1999; returned 8 April 1999; revised 28 May 1999; accepted 22 June 1999