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Oral supplementation with lactic acid-producing bacteria during intake of clindamycin

      Objective: To study the effect of administration of clindamycin with or without supplementation of the intestinal microflora with Bifidobacterium bifidum and Lactobacillus acidophilus.
      Methods: Twenty-three healthy subjects received clindamycin by mouth for 7 days. Eleven of the subjects also received capsules containing lyophilized L. acidophilus and B. bifidum for 14 days. The other 12 subjects received placebo.
      Results: There was a marked decrease in total numbers of anaerobic bacteria during the administration of clindamycin. In the lactic acid bacteria-supplemented group, a tendency towards delayed reduction and earlier increase in bifidobacteria was observed, and two of 11 subjects (18%) were colonized with Clostridium difficile, in comparison with five of 12 (41%) in the placebo group. The total number of microorganisms was significantly higher in the lactic acid bacteria-supplemented group than in the placebo group (p = 0.02) 4 days after the end of clindamycin administration. The difference was mainly due to higher counts of Escherichia coli and enterococci. Mean levels of other enterobacteria increased less in the lactic acid bacteria-supplemented group than in the placebo group between days 0 and 14.
      Conclusions: The recolonization with aerobic and anaerobic microorganisms was faster in the lactic acid bacteria-supplemented group than in the placebo group. This may be of importance in preventing colonization with C. difficile.

      Key words

      INTRODUCTION

      The human gastrointestinal microflora is, under normal circumstances, relatively constant during a lifetime. An ecological system is created by the indigenous microorganisms and the host. One of the most important effects of this ecosystem is maintenance of colonization resistance against potentially pathogenic microorganisms. Bacterial interference, both antagonistic and cooperative, plays a key role in the colonization resistance against pathogenic microorganisms. Production of volatile fatty acids and bacteriocins and competition for attachment sites and nutrients contribute to the defense against pathogens. The intestinal anaerobic bacteria, particularly the Gram-positive species, have been considered to be of importance for colonization resistance [
      • Savage DC
      Microbial ecology of the gastrointestinal tract.
      ,
      • van der Waaij D
      Colonization pattern of the digestive tract by potentially pathogenic microorganisms: colonization-controlling mechanisms and consequences for antibiotic treatment.
      ]. As bifidobacteria and lactobacilli constitute a major part of the intestinal microflora in humans throughout life [
      • Mitsuoka T
      • Kaneuchi C
      Ecology of the bifidobacteria.
      ], they are probably of importance in colonization resistance.
      The equilibrium, however, may be affected by certain factors such as administration of antimicrobial agents. This can lead to several unwanted effects, such as overgrowth of resistant microorganisms and development of new antimicrobial-resistant strains [
      • Nord CE
      • Edlund C
      Ecological effects of antimicrobial agents on the human intestinal microflora.
      ]. The use of clindamycin is focused on treatment of anaerobic infections, especially infections caused by Bacteroides fragilis and other penicillin-resistant anaerobic bacteria. Unfortunately, oral and parenteral administration of the agent results in high concentrations of the drug in the colon, leading to considerable impact on the intestinal microflora. Clindamycin has in earlier studies been shown to reduce the number of lactobacilli and bifidobacteria [
      • Heimdahl A
      • Nord CE
      Effect of phenoxymethylpenicillin and clindamycin on the oral, throat and faecal microflora of man.
      ,
      • Lidbeck A
      • Edlund C
      • Gustafsson J-Å
      • Kager L
      • Nord CE
      Impact of Lactobacillus acidophilus on the normal intestinal microflora after administration of two antimicrobial agents.
      ,
      • Orrhage K
      • Brismar B
      • Nord CE
      Effect of supplements with Bifidobacterium longum and Lactobacillus acidophilus on the intestinal microbiota during administration of clindamycin.
      ]. Clindamycin is also a potent agent inducing Clostridium difficile-associated diarrhea [
      • Golledge CL
      • McKenzie T
      • Riley T
      Extended spectrum cephalosporins and Clostridium difficile.
      ]. C. difficile infection is responsible for virtually all cases of antimicrobial agent-induced pseudomembraneous colitis and 20–30% of cases of antibiotic-associated non-specific colitis and diarrhea without colitis [
      • George WL
      • Rolfe RD
      • Finegold SM
      Clostridium difficile and its cytotoxin in feces of patients with antimicrobial agent-associated diarrhea and miscellaneous conditions.
      ]. These pathogenic conditions are conventionally treated with other antimicrobial agents, but the relapse frequency is as high as 15–20% [
      • George WL
      • Volpicelli NA
      • Stiner DB
      • et al.
      Relapse of pseudomembraneous colitis after vancomycin therapy.
      ].
      A report by Gorbach et al. [
      • Gorbach SL
      • Chang T-W
      • Goldin B
      Successful treatment of relapsing Clostridium difficile colitis with Lactobacillus GG.
      ] indicated that Lactobacillus casei strain GG was useful in the treatment of relapsing colitis secondary to C. difficile infection. Increasing interest in the use of ecological methods to restore balance in the intestinal microflora has developed. Supplements, so-called probiotics, have been used for this purpose [
      • Lidbeck A
      • Gustafsson J-Å
      • Nord CE
      Impact of Lactobacillus acidophilus supplements on the human oropharyngeal and intestinal microflora.
      ,
      • Orrhage K
      • Lidbeck A
      • Nord CE
      Effect of Bifidobacterium longum supplements on the human faecal microflora.
      ,
      • Isolauri E
      • Juntunen M
      • Rautanen T
      • Sillanaukee P
      • Koivula T
      A human lactobacillus strain (Lactobacillus casei sp. strain GG) promotes recovery from acute diarrhea in children.
      ]. The term ‘probiotic’ has been defined by Fuller as ‘a live microbial feed supplement which beneficially affects the host animal by improving its microbial balance’ [
      • Fuller R
      Probiotics in man and animals.
      ]. Probiotic preparations usually contain viable human lactic acid-producing bacteria, especially lactobacilli and bifidobacteria. Lactobacilli and bifidobacteria are non-motile, non-sporulating Gram-positive rods. Lactobacilli are facultative or anaerobic rods, whereas bifidobacteria are strictly anaerobic rods. The main end products of their glucose fermentation are lactic and acetic acids in variable proportions.
      Several studies have shown interesting results of the use of probiotic supplements in connection with antibiotic therapy [
      • Black F
      • Einarsson K
      • Lidbeck A
      • Orrhage K
      • Nord CE
      Effect of lactic acid producing bacteria on the human intestinal microflora during ampicillin treatment.
      ,
      • Siitonen S
      • Vapaatalo H
      • Salminen S
      • et al.
      Effect of Lactobacillus GG yoghurt in prevention of antibiotic associated diarrhea.
      ]. The purpose of the present investigation was to study the intestinal microflora and pH in feces before, during and after administration of clindamycin with or without supplements of lyophilized strains of B. bifidum and L. acidophilus and other bacteria.

      MATERIALS AND METHODS

       Subjects

      Twenty-three healthy subjects with no history of gastrointestinal, hepatic or renal disease participated in the study. They were divided into two groups, consisting of eight women and three men, and eight women and four men, respectively. The mean age was 29.5 years (range 21–54 years). None of the volunteers had been treated with antibiotics during the last 3 months or consumed lactic acid bacteria-fermented products for 2 weeks prior to the study. No other medication except clindamycin and the capsules of lactic acid bacteria was allowed during the investigation period. The investigation was performed as a randomized double-blind parallel group study and it was approved by the Local Ethics Committee of Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden and by the Agency of Medical Products, Sweden.

       Clinical examination

      The subjects visited the hospital before (day 0) and at the 14th and 28th day of the investigation. A routine physical examination was performed and fasting serum samples were taken at the first two visits. Recording of adverse events was performed at every visit and possible adverse events were monitored during the entire study.

       Administration of clindamycin

      The subjects received by mouth 150 mg clindamycin capsules (Dalacin®, Upjohn, Kalamazoo, Michigan, USA) with meals, four times a day for 7 days.

       Administration of lactic acid-producing bacteria

      Eleven subjects received capsules (Trevis® ACO, Pharmacia, Sweden) containing 3×109 colony-forming units (CFU) of lyophilized microorganisms of L. acidophilus La-CH5, B. bifidum Bb-12, Lactobacillus delbrueckii subsp. bulgaricus Lb-Y27 and Streptococcus salivarius subsp. thermophilus St-Y31 (Christian Hansen Biosystems, Denmark). Three capsules were administered after meals twice a day for 14 days, starting at the same time as the clindamycin administration. Thus, the daily dose of lactic acid bacteria was 2×1010 CFU. The other 12 subjects received placebo capsules administered in the same manner.

       Collection of fecal specimens

      Fecal samples were collected before taking the supplement (day 0) and on the 2nd, 4th and 7th day during the clindamycin and lactic acid bacteria/placebo administration period and again 4, 7, 14 and 21 days after withdrawal of the antimicrobial agent. The specimens were collected into sterile plastic containers, placed on ice chests and immediately sent to the laboratory, where they were stored at –70°C until analyzed. The freezing procedure was validated to ensure that the results of the study were not influenced by this technique and all specimens were treated in the same way [
      • Hardey MG
      • Hudson MJ
      • Swarbrick ET
      • et al.
      The rectal mucosa-associated microflora in patients with ulcerative colitis.
      ].

       Microbiological procedures

      The samples were inoculated on non-selective and selective media and processed as described by Heimdahl and Nord [
      • Heimdahl A
      • Nord CE
      Effect of phenoxymethylpenicillin and clindamycin on the oral, throat and faecal microflora of man.
      ]. Bifidobacteria were cultured on a medium developed for isolation of bifidobacteria, BL agar, without addition of horse blood [
      • Teraguchi S
      • Uehara M
      • Ogasa K
      • Mitsuoka T
      Enumeration of bifidobacteria in dairy products.
      ] and lactobacilli were cultured on Rogosa SL agar (Difco, MI, USA). Aerobic and anaerobic microorganisms were identified by morphologic, serologic and biochemical tests and gas–liquid chromatography [
      • Heimdahl A
      • Nord CE
      Effect of phenoxymethylpenicillin and clindamycin on the oral, throat and faecal microflora of man.
      ]. The lower limit of detection was 102 microorganisms/g feces.

       Minimum inhibitory concentrations

      The minimum inhibitory concentrations (MICs) of clindamycin for L. acidophilus La-CH5 and B. bifidum Bb-12 were determined by an agar dilution method [
      • Heimdahl A
      • Nord CE
      Effect of phenoxymethylpenicillin and clindamycin on the oral, throat and faecal microflora of man.
      ].

       Measurement of pH in fecal samples

      The fecal samples were diluted 1:1 in sterile distilled water [
      • Marsh WW
      • Hentges DJ
      • Chavarria JF
      • et al.
      Influence of orally administered antibiotics on faecal pH and volatile fatty acid concentrations of infants and young children.
      ] and the pH of the mixture was measured with a pH meter (Radiometer PHM 92).

       Statistical analysis

      Statistical analysis was performed by testing the differences between the two treatment groups using the Wilcoxon rank sum test after logarithmic transformation of the microbiological variables. A p-value less than 0.05 was considered statistically significant.

      RESULTS

       Effect on total intestinal microflora

      Figure 1a shows the total numbers of aerobic and anaerobic microorganisms during the investigation period. The level of total microorganisms was significantly higher on day 11 in the group supplemented with lactic acid bacteria than in the placebo group (p = 0.02).
      Figure thumbnail gr1
      Fig. 1Effect of clindamycin and supplements containing lactic acid bacteria (LAB) or placebo on the (a) total intestinal microflora, (b) aerobic intestinal microflora, and (c) anaerobic intestinal microflora of 11 and 12 subjects, respectively. Mean values and standard errors (SE) are shown. Significant differences between the two groups. p = 0.02, *p < 0.05.

       Effect on aerobic intestinal microorganisms

      There were no significant differences in the aerobic microflora between the groups during the week of clindamycin administration (Figure 1b). During the second week, however, on day 11 in the group receiving the lactic acid bacterial supplement, the mean level of the total number of aerobic microorganisms was significantly increased compared to the placebo group (p < 0.05). A similar but later increase was seen in the placebo group on day 14. The difference was mainly due to higher counts of Escherichia coli and enterococci (Figure 2). Concerning the other Gram-negative aerobic rods, called enterobacteria in the Figure, there was an increase of 3–4 log10 in the lactic acid bacteria group and approximately 5 log10 in the placebo group between days 0 and 14.
      Figure thumbnail gr2
      Fig. 2Effect of clindamycin and supplements containing lactic acid bacteria or placebo on groups of the aerobic intestinal microflora in 11 and 12 subjects, respectively. The line represents interpolated geometric mean values. Note: ‘Enterobacteria’ comprise Gram-negative enterobacteria other than E. coli.

       Effect on the anaerobic intestinal microorganisms

      There was a marked decrease in total numbers of anaerobic bacteria during the administration of clindamycin in both groups. The lowest levels of total anaerobic microorganisms were reached on day 7 (Figure 1c). There was no significant difference between the groups. The numbers of members of the genera Lactobacillus, Bifidobacterium, Clostridium and Bacteroides are shown in Figure 3. In the lactic acid bacteria-supplemented group, the reduction in bifidobacteria during clindamycin administration tended to be delayed, as nine subjects (82%) still harbored detectable numbers of bifidobacteria on day 2, compared to four subjects (33%) in the placebo group. Also on day 14, during recolonization of the microflora, detectable levels of bifidobacteria were seen in 5/11 subjects (48%) in the lactic acid bacteria-supplemented group, compared to 1/12 (8%) in the placebo group. No difference between the groups in the number of lactobacilli was detected. In the lactic acid bacteria-supplemented group, the mean levels of total bacteroides had returned to initial values on day 14 as 9/11 (82%) were recolonized, while the increase in the placebo group tended to be delayed, with only 6/12 (50%) colonized subjects. Although the levels of total Clostridia were almost similar between the groups, as can be seen in Figure 3, only 2/11 subjects (18%) in the lactic acid bacteria-supplemented group were colonized with C. difficile as compared to 5/12 subjects (41%) in the placebo group. None of the subjects with bifidobacteria on day 14 was colonized by C. difficile, and of 17 subjects in total without bifidobacteria, seven were colonized by C. difficile.
      Figure thumbnail gr3
      Fig. 3Effect of clindamycin and supplements containing lactic acid bacteria or placebo on groups of the anaerobic intestinal microflora in 11 and 12 subjects, respectively. The line represents interpolated geometric mean values.

       Minimum inhibitory concentrations

      The MIC of clindamycin was 0.064 μg/L for L. acidophilus La-CH5 and 0.032 μg/L for B. bifidum Bb-12.

       pH of fecal samples

      There was no significant difference observed in fecal mean pH between the groups (Figure 4). However, there was a tendency towards lower values in the lactic acid bacteria-supplemented group than in the placebo group during the week of clindamycin administration. An increase in mean pH was seen in both groups 1 week after withdrawal of the antimicrobial agent.
      Figure thumbnail gr4
      Fig. 4Fecal mean pH of the two groups given clindamycin and lactic acid bacteria (LAB) or placebo.

       Clinical findings

      All subjects completed the trial without any serious adverse events. The most commonly reported side effects were nausea, diarrhea, flatulence and abdominal discomfort. On day 14, six subjects in the lactic acid bacteria-supplemented group and eight persons in the placebo group reported side effects of mild severity. During the last 2 weeks of the study, three subjects in the lactic acid bacteria-supplemented group had mild adverse events as against one mild and one moderate event in the placebo group.

      DISCUSSION

      The total numbers of microorganisms in the intestinal microflora increased earlier after clindamycin administration in the lactic acid bacteria-supplemented group than in the placebo group. This was mainly due to the aerobic component of the microflora, which showed an increase of E. coli and other Gram-negative rods and enterococci during the second week of the study. The significantly higher level on day 11 in the lactic acid bacterial-supplemented group may be considered as an accelerated recolonization of the aerobic microflora. The increase in the aerobic microflora may partly have compensated for the strongly suppressed anaerobic microflora; however, the same pattern was followed in the placebo group on day 14. Several recent studies [
      • Barza M
      • Giuliano M
      • Jacobus NV
      • Gorbach SL
      Effect of broad-spectrum parenteral antibiotics on ‘colonization resistance’ of intestinal microflora of humans.
      ,
      • Gorbach SL
      • Barza M
      • Giuliano M
      • Jacobus NV
      Colonization resistance of the human intestinal microflora: testing the hypothesis in normal volunteers.
      ] have failed to support the earlier hypothesis that only the anaerobic microflora is responsible for colonization resistance. It is possible that the importance of the aerobic and facultatively anaerobic part of the microflora in colonization resistance has been underestimated.
      The tendency towards better preserved levels of bifidobacteria on days 2 and 14 in the lactic acid bacteria-supplemented group in the present study were in accordance with observations made by Orrhage et al. [
      • Orrhage K
      • Brismar B
      • Nord CE
      Effect of supplements with Bifidobacterium longum and Lactobacillus acidophilus on the intestinal microbiota during administration of clindamycin.
      ], where subjects receiving a fermented milk preparation containing Bifidobacterium longum and L. acidophilus during and after administration of clindamycin showed a marked tendency towards higher counts of bifidobacteria in comparison with controls receiving placebo milk. The differences between the groups in that study were more pronouncedly, in favor of the probiotic-supplemented group. No effects on lactobacilli were detected in that investigation or in our own study, a finding in contrast to that of Lidbeck et al. [
      • Lidbeck A
      • Edlund C
      • Gustafsson J-Å
      • Kager L
      • Nord CE
      Impact of Lactobacillus acidophilus on the normal intestinal microflora after administration of two antimicrobial agents.
      ], who observed an increase of lactobacilli after administration of clindamycin in a group receiving L. acidophilus supplements given as a fermented milk product.
      The strains of lactobacilli and bifidobacteria used by us were very sensitive to clindamycin. Clindamycin is a drug that is well absorbed soon after ingestion and then excreted again, mainly through bile and feces, resulting in high concentrations in the colon. Although there was no significant difference in numbers of bifidobacteria or lactobacilli in the fecal samples, the possibility cannot be excluded that the ingested lactic acid bacteria may have been viable during most of their passage through the intestinal tract but then destroyed by the bactericidal effect of high concentrations of clindamycin in the colon.
      In the present study there was no significant effect on pH caused by intake of the lactic acid bacteria, although the lactic acid bacteria-supplemented group showed a trend towards a lower mean pH during the period of administration of clindamycin capsules. This tendency reflected the more pronounced and permanent effects on fecal pH, observed after administration of a fermented milk product in a similar study by Orrhage et al. [
      • Orrhage K
      • Brismar B
      • Nord CE
      Effect of supplements with Bifidobacterium longum and Lactobacillus acidophilus on the intestinal microbiota during administration of clindamycin.
      ].
      Fewer subjects in the lactic acid bacteria-supplemented group were colonized with C. difficile than in the placebo group. The risk of developing C. difficile diarrheal disease associated with the use of clindamycin is well established [
      • Aronsson B
      • Möllby R
      • Nord CE
      Antimicrobial agents and Clostridium difficile in acute enteric disease: epidemiological data from Sweden 1980–1982.
      ]. Simultaneous intake of yogurt containing B. longum with erythromycin was demonstrated to decrease the level of fecal clostridial spores and reduce the frequency of gastrointestinal disorders in healthy volunteers [
      • Colombel JF
      • Cortot A
      • Neut C
      • Romond C
      Yoghurt with Bifidobacterium longum reduces erythromycin-induced gastrointestinal effects.
      ]. Some strains of Lactobacillus and Bifidobacterium spp. have also been shown to inhibit the multiplication of C. difficile in vitro [
      • Rolfe R
      • Helebian S
      • Finegold S
      Bacterial interference between Clostridium difficile and normal fecal flora.
      ].
      Studies similar to the present investigation have shown more pronounced effects on preservation of the intestinal microflora. Possible reasons for the weaker effects of oral supplementation with this lyophilized preparation of lactic acid bacteria during intake of clindamycin may be suggested. One important factor may be the numbers of ingested microorganisms. In the present investigation, the number of microorganisms given in capsules was approximately 2×1010 CFU daily. In two earlier studies, showing more marked effects on preservation of the microflora in connection with clindamycin, the numbers of daily ingested microorganisms were 10–100 times higher [
      • Lidbeck A
      • Edlund C
      • Gustafsson J-Å
      • Kager L
      • Nord CE
      Impact of Lactobacillus acidophilus on the normal intestinal microflora after administration of two antimicrobial agents.
      ,
      • Orrhage K
      • Brismar B
      • Nord CE
      Effect of supplements with Bifidobacterium longum and Lactobacillus acidophilus on the intestinal microbiota during administration of clindamycin.
      ]. In addition, the probiotic product in those studies consisted of fermented milk. The vehicle for the probiotic has been reported to be of importance for the survival of ingested microorganisms [
      • Conway PL
      • Gorbach SL
      • Goldin BR
      Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells.
      ]. Microorganisms in a milk product may be better protected from the surrounding environment and better prepared for survival on entrance to the intestine than under lyophilized conditions. Another important factor in the survival, and possibly also the colonization, may be the strains of bacteria chosen. The strains administered in the present investigation have been used in a study concerning the prophylactic use of probiotic supplements to prevent traveller's diarrhea [
      • Black FT
      • Andersen PL
      • Ørskov J
      • Ørskov F
      • Gaarslev K
      • Laulund S
      Prophylactic efficacy of lactobacilli on traveller's diarrhea.
      ]. The study group comprised Danish tourists visiting Egypt, randomized into two groups, 40 subjects in the lactic acid bacteria-supplemented group and 41 subjects in the placebo group. The preparation significantly reduced the frequency of diarrhea from 71% to 43% in the lactic acid bacteria-supplemented group. The same preparation was also administered to eight healthy volunteers in a study by Nielsen et al. [
      • Nielsen OH
      • Jørgensen S
      • Pedersen K
      • Justesen T
      Microbial evaluation of jejunal aspirates and faecal samples after oral administration of bifidobacteria and lactic acid bacteria.
      ], investigating effects on the jejunal and fecal microfloras. No changes in the numbers of microorganisms or ratios between anaerobic and aerobic microorganisms were observed.
      The faster recolonization with a mixture of aerobic and anaerobic microorganisms in the lactic acid bacteria-supplemented group than in the placebo group may possibly be of importance in preventing colonization with C. difficile. Larger studies, however, are needed to confirm this hypothesis.

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