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Absence of bactericidal activity of orally administered wood creosote on human intestinal bacterial flora

Norio Ogata

R & D Center, Taiko Pharmaceutical Co., Ltd., Kyoto, Japan

E-mail : aa

Takanori Miura

R & D Center, Taiko Pharmaceutical Co., Ltd., Kyoto, Japan

DOI: 10.15761/IMM.1000321

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Abstract

Wood creosote is a mixture of simple phenolic compounds that has long been used for over a century as an antidiarrheal medicine. While there are many pharmacological studies that explain its antidiarrheal effects, it has long been a matter of controversy whether the antidiarrheal activity of wood creosote is attributable to its putative bactericidal effect on the human intestinal bacterial flora. The objective of our study was to investigate the putative bactericidal effect of wood creosote in the human intestine when given in an ordinary therapeutic dose. To this end, we used an in vitro test to measure the minimal inhibitory concentration of wood creosote for various bacteria from the human gut. In addition, we also quantified the copy numbers of bacterial 16S ribosomal DNA in the feces of healthy human participants after administration of a therapeutic dose of wood creosote.We determined the minimal inhibitory concentration of wood creosote to be >128 µg/ml, a level far greater than thatof commonly used antibacterial agents. Copy numbers of bacterial 16S ribosomal DNA in human feces after theadministration of a therapeutic dose of wood creosote did not changesignificantly ( p > 0.05) from that before dosing. Taken together, we conclude that wood creosote given at anordinarytherapeutic dose has no significant antibacterial effect in the human lower intestine where most of the intestinal bacterial flora resides. Thus, the antidiarrheal properties of wood creosote are not attributable to its effect on the intestinal bacterial flora, but rather to its other effects on the intestine.

Keywords

wood creosote, intestine; bacterial flora, minimal inhibitory concentration, ribosomal DNA

Introduction

Wood creosote (WC) is a mixture of phenolic compounds, such as phenol, guaiacol, p -cresol, creosol, 4-ethylguaiacol, obtained by the fractional distillation of wood tar [1]. In some countries WC, under the trade name of Seirogan, has long been used as an antidiarrheal medicine [2]. WC and its constituent compounds are known to suppress enterotoxin-induced intestinal fluid secretion via chloride channels[3-5] and to suppress smooth muscle contraction [6,7]. Indeed, one of the major constituents of WC, 4-ethylguaiacol, is known to possess the latter activity [8]. According to the findings of Morino et al . the antimotility effect of WC is attributable toits inhibition of Ca2+ mobilization in cells of the gastrointestinal tract [9].Ataka et al. suggested from the results of experimentsconducted on rats that stress-induced diarrhea might be inhibited by blocking serotonin receptors in the colon [10].

These reported findings could well explain the antidiarrheal effect of WC. However, it has long been a matter of controversy whether the antidiarrheal effect of WC is attributable to its putative antibacterial activity or not. In fact, WC contains well-known aseptic agents, such as, phenol and p -cresol [1], whichwere previously used as antiseptic agents in hospitals. Medicinal WC taken orally at an ordinary therapeutic doseis immediately absorbed from the gastrointestinal tract;a peak blood concentration of its metabolites being 30 min after administration [11,12]. These findingssuggest that WC is absorbed from the upper gastrointestinal tract, namely stomach and duodenum [12]. Moreover, these observations further suggest that the concentration of WC in the lower ileum and colon, where most of the intestinal bacterial florasin human reside, is extremely low. Consequently, the potential antibacterial effect of WC would not be discernible in the lower gastrointestinal tract.If this interpretation is the case, the antidiarrheal effect of WC is unrelated to its potential bactericidal activity. The aim of this study was to investigate whether or not WC taken orally at its ordinary therapeutic dose displays antibacterial activity in the human gastrointestinal tract.

Materials and methods

In vitrobacterial study

Minimal inhibitory concentration (MIC) of WC (lot TA-03; Taiko Pharmaceutical, Osaka, Japan) on the growth of various human gut isolates (Don Whitley Scientific, Shipley, UK) was testedaccording to the reportedMIC test guidelines [13]. Agar plates containing culture medium suitable for each bacterium were used. WC diluted to several concentrations in distilled water was added in the agar platesjust before they solidified. Special care was exercised to make a homogeneous stock solution of WC in water (2664 µg/ml) by vigorous agitation. After spreading bacteriaon the surface of each plate at two bacterial densities, the plates were incubated at 35 –42ºC for 24 h in aerobic or anaerobic conditions appropriate for each bacterium under investigation. Following incubation,the number of colonies on the plate was counted and the MIC determined. MIC was defined as the concentration of WC at which no bacterial colony appeared on the plates.

Human study

The study was a single-hospital open trial without a control placebo. Thirty-four healthy adultparticipants of age 20 – 50 years were initially enrolled, from which five males and five femaleparticipants eligible for the study were selected. Written informed consent was obtained from each participant. Exclusion criteria were those undergoing treatment with adoctor or dentist, having a fever or diarrhea, pregnant or may be pregnant, allergic to WC, constipated, onmedication, or thosewho had taken other medicines within 7days of initiatingthe study.Standard blood chemistry analyses, urine analyses and physical analyses, includingan electrocardiogram examination, were performed for each participant just before the commencement of the study, and all the data were confirmed to be normal. The study protocol and consent form were approved by Miyawaki Orthopedic Clinic Clinical Study Ethics Committee (Hokkaido, Japan). The study was registered to University Hospital Medical Information Network (UMIN Clinical Trial Registry; UMIN000026926) before the commencement of the trial.

Feces were collected on day 1, day 2 and day 3 of the study from each participant. WC (capsule medicine, Seirogan Quick C, Taiko Pharmaceutical) was then given orally on day 4 at a dose of 3 capsules (each capsule containing 45 mg WC) within 30 min after a meal. This dosing was done for each meal (three times a day); a total of 9 capsules were administered to each participant on day 4. Feces were again collected on day 5, day 6 and day 7. When feces could not be obtained on a scheduled day, the collection of feces was done on the following day(s), and the entire study schedule was shifted tolater day(s). All feces were stored frozen until analysis. On the day of analysis DNA was extracted from the feces as described previously [14]. Next, thecopy number of 16Sribosomal DNA (rDNA) of target bacteria was quantified by real-time PCR using SYBR premix Ex Taq II (Takara, Otsu, Japan) and a Rotor-Gene Q machine (Qiagen, Hilden, Germany) for Bifidobacterium longum . In some experiments, digital PCR [15] was employed to quantifythe 16S rDNA copy number of DNA samples using Taqman Gene Expression Master Mix and aBiomark System (Fluidigm, South San Francisco, CA, USA) for 16S rDNA of Enterococcus faecalis , Bacteroides fragilis and Clostridium perfringens . The copy number of 16S rDNA was expressed pergram of feces.

Statistical analysis

The copy number of 16S rDNA of each bacterium was first expressed as a mean and standard deviation of pre-dose three days (day 1, 2 and 3) from each subject. Next, mean and standard deviation of post-dose three days (day 5, 6 and 7) was obtained from each participant. Then, the pre-dose and post-dose means were compared for each participant by a paired one-sided Student’s t -test. The difference was considered significant if p < 0.05.

Results

Initially,we attempted to use real-time PCR to quantifythe copy numbers of 16S rDNA of bacteria in feces. A time course of the change in copy number of 16S rDNA of Bifidobacterium longum in feces is shown in Table 1. Although there are some fluctuations in the copy numbers of 16S rDNA both in male and female, the difference was not significant ( p = 0.33 and 0.47, respectively)when compared before and after the dosing of WC for thetwo participants. Similar trends were found for other participants (data not shown). Similar results were obtained when investigating other bacteria (data not shown). Next, we compared means of five male participants and five female participants for pre-dose and post-dose copy numbers of rDNA from Bifidobacterium longum (Table 2), Bacteroides fragilis (Table 3), Clostridium perfringens (Table 4) and Enterococcus faecalis (Table 5). The copy number did not change significantly for all these four bacteria ( p = 0.48, 0.36, 0.13 and 0.14, respectively). Therefore, we concluded that WC given orally at a usual therapeutic dose is ineffective at killing intestinal bacterial flora. We next measured MIC of WC against bacteria of human isolates. As shown in Table 6 and 7, MIC of WC for all the bacteriatested was >128µg/ml, suggesting it has no significant antibacterial activity.

Table 1. Time course of the copy numbers of 16S rDNA of Bifidobacterium longum in the feces of two subjects

Subject name

Experiment day

Copies per gram feces

Male 5

1

9.0 x 1010

Male 5

2

1.5 x 1011

Male 5

3

2.1 x 1011

Mean  SD (day 1,2,3)

1.5 x 1011  6.0 x 1010

Male 5

5

2.3 x 1011

Male 5

6

6.6 x 1010

Male 5

7

6.2 x 1010

Mean  SD (day 5,6,7)

1.2 x 1011  9.6 x 1010

p value (day 1,2,3 vs 5,6,7)

0.33

Female 6

1

2.2 x 1010

Female 6

2

2.4 x 1010

Female 6

3

4.4 x 109

Mean  SD (day 1,2,3)

1.7 x 1010  1.1 x 1010

Female 6

5

1.7 x 1010

Female 6

6

1.1 x 1010

Female 6

7

2.1 x 1010

Mean  SD (day 5,6,7)

1.6 x 1010  5.0 x 109

p value (day 1,2,3 vs 5,6,7)

0.47

Table 2. Copy numbers of 16S rDNA of Bifidobacterium longum per gram of feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

5.1 x 1010

3.1 x 1010

Male 2

3.3 x 106

1.1 x 107

Male 3

7.3 x 108

8.6 x 108

Male 4

2.9 x 1010

3.6 x 1010

Male 5

1.5 x 1011

1.2 x 1011

Female 6

1.7 x 1010

1.9 x 1010

Female 7

1.2 x 1011

1.4 x 1011

Female 8

6.7 x 106

6.4 x 106

Female 9

1.9 x 1011

1.8 x 1011

Female 10

9.0 x 109

4.6 x 1010

Mean

5.6 x 1010

5.7 x 1010

SD

7.0 x 1010

6.4 x 1010

p = 0.48 (pre- vs post-dose)

Table 3. Copy numbers of 16S rDNA of Bacteroides fragilis per gramof feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

3.3 x 108

2.8 x 108

Male 2

6.1 x 108

9.2 x 109

Male 3

3.1 x 1010

7.5 x 108

Male 4

1.4 x 1010

7,2 x 1010

Male 5

3.4 x 109

1.5 x 109

Female 6

4.9 x 108

5.7 x 108

Female 7

1.8 x 109

3.0 x 109

Female 8

4.4 x 1010

3.9 x 1010

Female 9

1.7 x 109

8.8 x 108

Female 10

6.3 x 109

2.7 x 109

Mean

1.0 x 1010

1.3 x 1010

SD

1.5 x 1010

2.4 x 1010

p = 0.36 (pre- vs post-dose)

Table 4. Copy numbers of 16S rDNA of Clostridium perfringens per gramof feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

2.9 x 1010

8.7 x 1010

Male 2

3.2 x 1010

6.6 x 1010

Male 3

5.0 x 1010

3.2 x 1010

Male 4

1.4 x 1010

4.5 x 1010

Male 5

1.5 x 1010

2.5 x 1010

Female 6

7.3 x 109

4.0 x 1010

Female 7

1.9 x 1010

1.0 x 1010

Female 8

4.6 x 1010

4.9 x 1010

Female 9

5.0 x 1010

4.8 x 1010

Female 10

5.7 x 1010

2.2 x 1010

Mean

3.2 x 1010

4.3 x 1010

SD

1.8 x 1010

2.2 x 1010

p = 0.13 (pre- vs post-dose)

Table 5. Copy numbers of 16S rDNA of Enterococcus faecalis per gramof feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

2.7 x 1010

9.5 x 1010

Male 2

3.4 x 1010

5.2 x 1010

Male 3

4.1 x 1010

2.6 x 1010

Male 4

9.5 x 109

3.5 x 1010

Male 5

1.7 x 1010

3.1 x 1010

Female 6

6.0 x 109

4.4 x 1010

Female 7

4.6 x 1010

3.4 x 1010

Female 8

4.3 x 1010

5.3 x 1010

Female 9

5.4 x 1010

4.7 x 1010

Female 10

5.6 x 1010

2.5 x 1010

Mean

3.3 x 1010

4.4 x 1010

SD

1.8 x 1010

2.1 x 1010

p = 0.14 (pre- vs post-dose)

Table 6. Minimal inhibitory concentration (MIC, ?g/ml) of wood creosote against indigenous bacterial strains.

Name of bacteria

Name of strain

Concentration of

original broth

(cfu/ml)

Dilution

1-fold

Dilution

100-fold

Bifidobacterium longum

DWC 2285

3.6 x 108

> 128

> 128

Bifidobacterium adolescentis

DWC 1480

4.1 x 108

> 128

> 128

Bacteroides fragilis

DWC 0310

1.2 x 109

> 128

> 128

Bacteroidesdistasonis

DWC 0308

1.7 x 109

> 128

> 128

Clostridium perfringens

DWC 2246

3.0 x 108

> 128

> 128

Clostridium difficile

DWC 2178

2.7 x 108

> 128

> 128

Enterococcus faecalis

DWC 1706

3.4 x 108

> 128

> 128

Enterococcus faecalis

DWC 1687

3.9 x 108

> 128

> 128

Table 7.Minimal inhibitory concentration (MIC, µg/ml) of wood creosote against pathological bacterial strains.

Name of bacteria

Name of strain

Concentration of

original broth

(cfu/ml)

Dilution

100-fold

Dilution

10000-fold

Salmonella enteritidis

DWC 2224

6.9 x 108

> 128

> 128

Salmonella virchow

DWC 2223

8.2 x 108

> 128

> 128

Shigella

sonnei

DWC 2216

3.5 x 108

> 128

> 128

Shigelladysenteriae

DWC 2201

2.9 x 108

> 128

> 128

Campylobacter jejuni

DWC 2030

3.7 x 108

> 128

> 128

Campylobacter coli

DWC 2032

> 5.0 x 108

> 128

> 128

Discussion

We quantitated mean values of the copy numbers of 16S rDNA of three-day pre-dose samples of feces and three-day post-dose samples of feces. Based on the relatively short doubling times of bacteria in the human gut ( e.g., 60 min for Staphylococcus aureus (16)), we reasoned that a three-day period was a suitable length of time for measuring the mean copy numbers of 16S rDNA in order to estimate numbers of bacteria in the intestine. In fact, as shown in Table 1, the time course of the 16S rDNA copy number did not change significantly during each 3-day period of sampling. Thus, we concluded that the three-day period of fecal sampling before and after the WC dosing is sufficient to estimate the numbers of bacteria in the intestine.

MIC of common antibiotics are of the order of 0.1 – 10µg/ml [17,18]. The MICs of WC found in bacteria tested in our experiments was greater than 128 µg/ml (Table 6, 7). Furthermore, WC administered orally is immediately absorbed from the upper gastrointestinal tract [12]. This suggests that the concentration of WC administered orally at the ordinary therapeutic dose would not attain aconcentration greater than 128 µg/ml in the lower gastrointestinal tract, such as the lower ileum and colon, where most of the intestinal bacterial floras reside. Thus, we speculate that the antidiarrhealeffect of WC is not attributable to its putative antimicrobial effect, but due primarily to its effect insuppressing intestinal motility and inhibition of intestinalfluid secretion via the chloride channel [3-8]. Furthermore, the absence of a noticeable antibacterial effect of orally administered WC suggests that it will not disturb the bacterial flora of the human intestine.

Competing interest

We declare no competing interest.

References

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Editorial Information

Editor-in-Chief

Ivan Gout
University College London
Ricardo H. Alvarez
Cancer Treatment Centers of America

Article Type

Research Article

Publication history

Received: February 05, 2018
Accepted: February 21, 2018
Published: February 24, 2018

Copyright

©2017 Ogata N. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Ogata N, Miura T (2017) Absence of bactericidal activity of orally administeredwood creosote on human intestinal bacterial flora. IntegrMol Med 4: DOI: 10.15761/IMM.1000321

Corresponding author

Norio Ogata, MD, PhD

R & D Center, Taiko Pharmaceutical Co., Ltd., 1-2-1 Hikaridai, Seikacho, Sorakugun, Kyoto 619-0237, Japan, Tel: 81-774-98-2716; Fax: 81-774-98-2737

Table 1. Time course of the copy numbers of 16S rDNA of Bifidobacterium longum in the feces of two subjects

Subject name

Experiment day

Copies per gram feces

Male 5

1

9.0 x 1010

Male 5

2

1.5 x 1011

Male 5

3

2.1 x 1011

Mean  SD (day 1,2,3)

1.5 x 1011  6.0 x 1010

Male 5

5

2.3 x 1011

Male 5

6

6.6 x 1010

Male 5

7

6.2 x 1010

Mean  SD (day 5,6,7)

1.2 x 1011  9.6 x 1010

p value (day 1,2,3 vs 5,6,7)

0.33

Female 6

1

2.2 x 1010

Female 6

2

2.4 x 1010

Female 6

3

4.4 x 109

Mean  SD (day 1,2,3)

1.7 x 1010  1.1 x 1010

Female 6

5

1.7 x 1010

Female 6

6

1.1 x 1010

Female 6

7

2.1 x 1010

Mean  SD (day 5,6,7)

1.6 x 1010  5.0 x 109

p value (day 1,2,3 vs 5,6,7)

0.47

Table 2. Copy numbers of 16S rDNA of Bifidobacterium longum per gram of feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

5.1 x 1010

3.1 x 1010

Male 2

3.3 x 106

1.1 x 107

Male 3

7.3 x 108

8.6 x 108

Male 4

2.9 x 1010

3.6 x 1010

Male 5

1.5 x 1011

1.2 x 1011

Female 6

1.7 x 1010

1.9 x 1010

Female 7

1.2 x 1011

1.4 x 1011

Female 8

6.7 x 106

6.4 x 106

Female 9

1.9 x 1011

1.8 x 1011

Female 10

9.0 x 109

4.6 x 1010

Mean

5.6 x 1010

5.7 x 1010

SD

7.0 x 1010

6.4 x 1010

p = 0.48 (pre- vs post-dose)

Table 3. Copy numbers of 16S rDNA of Bacteroides fragilis per gramof feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

3.3 x 108

2.8 x 108

Male 2

6.1 x 108

9.2 x 109

Male 3

3.1 x 1010

7.5 x 108

Male 4

1.4 x 1010

7,2 x 1010

Male 5

3.4 x 109

1.5 x 109

Female 6

4.9 x 108

5.7 x 108

Female 7

1.8 x 109

3.0 x 109

Female 8

4.4 x 1010

3.9 x 1010

Female 9

1.7 x 109

8.8 x 108

Female 10

6.3 x 109

2.7 x 109

Mean

1.0 x 1010

1.3 x 1010

SD

1.5 x 1010

2.4 x 1010

p = 0.36 (pre- vs post-dose)

Table 4. Copy numbers of 16S rDNA of Clostridium perfringens per gramof feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

2.9 x 1010

8.7 x 1010

Male 2

3.2 x 1010

6.6 x 1010

Male 3

5.0 x 1010

3.2 x 1010

Male 4

1.4 x 1010

4.5 x 1010

Male 5

1.5 x 1010

2.5 x 1010

Female 6

7.3 x 109

4.0 x 1010

Female 7

1.9 x 1010

1.0 x 1010

Female 8

4.6 x 1010

4.9 x 1010

Female 9

5.0 x 1010

4.8 x 1010

Female 10

5.7 x 1010

2.2 x 1010

Mean

3.2 x 1010

4.3 x 1010

SD

1.8 x 1010

2.2 x 1010

p = 0.13 (pre- vs post-dose)

Table 5. Copy numbers of 16S rDNA of Enterococcus faecalis per gramof feces.

Subject name

Pre-dose (mean of 3 days)

Post-dose (mean of 3 days)

Male 1

2.7 x 1010

9.5 x 1010

Male 2

3.4 x 1010

5.2 x 1010

Male 3

4.1 x 1010

2.6 x 1010

Male 4

9.5 x 109

3.5 x 1010

Male 5

1.7 x 1010

3.1 x 1010

Female 6

6.0 x 109

4.4 x 1010

Female 7

4.6 x 1010

3.4 x 1010

Female 8

4.3 x 1010

5.3 x 1010

Female 9

5.4 x 1010

4.7 x 1010

Female 10

5.6 x 1010

2.5 x 1010

Mean

3.3 x 1010

4.4 x 1010

SD

1.8 x 1010

2.1 x 1010

p = 0.14 (pre- vs post-dose)

Table 6. Minimal inhibitory concentration (MIC, ?g/ml) of wood creosote against indigenous bacterial strains.

Name of bacteria

Name of strain

Concentration of

original broth

(cfu/ml)

Dilution

1-fold

Dilution

100-fold

Bifidobacterium longum

DWC 2285

3.6 x 108

> 128

> 128

Bifidobacterium adolescentis

DWC 1480

4.1 x 108

> 128

> 128

Bacteroides fragilis

DWC 0310

1.2 x 109

> 128

> 128

Bacteroidesdistasonis

DWC 0308

1.7 x 109

> 128

> 128

Clostridium perfringens

DWC 2246

3.0 x 108

> 128

> 128

Clostridium difficile

DWC 2178

2.7 x 108

> 128

> 128

Enterococcus faecalis

DWC 1706

3.4 x 108

> 128

> 128

Enterococcus faecalis

DWC 1687

3.9 x 108

> 128

> 128

Table 7.Minimal inhibitory concentration (MIC, µg/ml) of wood creosote against pathological bacterial strains.

Name of bacteria

Name of strain

Concentration of

original broth

(cfu/ml)

Dilution

100-fold

Dilution

10000-fold

Salmonella enteritidis

DWC 2224

6.9 x 108

> 128

> 128

Salmonella virchow

DWC 2223

8.2 x 108

> 128

> 128

Shigella

sonnei

DWC 2216

3.5 x 108

> 128

> 128

Shigelladysenteriae

DWC 2201

2.9 x 108

> 128

> 128

Campylobacter jejuni

DWC 2030

3.7 x 108

> 128

> 128

Campylobacter coli

DWC 2032

> 5.0 x 108

> 128

> 128