1. Background
Bile is the exocrine secretion of the liver, which is a product of cholesterol metabolism (1-3). It is stored in the gallbladder and enters into the duodenum via the common bile duct (1, 2). Its major components include various amino acids such as glycine and taurine, as well as bile acids, cholesterol, phospholipids and bile pigments, biliverdin, and bilirubin (4, 5). Bile contains proteins, especially mucin glycoproteins, and a wide range of mineral salts ions, and anti-oxidants (4, 6). According to this structure, bile has a wide range of functions throughout the body (7).
Evidence suggest that anti-bacterial effects could be a potential effect of ox-bile (2, 5). In this regard, some studies have mentioned the bactericidal role of bile salts (8, 9), and its bacteriostatic function has been explained (10, 11). Bile salts provoke intestinal cell proliferation and prevent apoptosis, decrease mucosal damage, and improve survival after intestinal injury (1, 12). Bile acid may protect the intestine from injury or infection and help with compensation (12). It inhibits bacterial overgrowth and endotoxin absorption too (13). Concomitantly, bile has anti-inflammatory, immunomodulatory, and anti-oxidant effects. Indeed, it offers protection against oxidative stress and detoxifies a wide range of free radicals to control bacterial infections (14-16). The balance of bile acids and microbiota is also important for human health since dysbiosis and secondary bile acid deficiency can cause intestinal inflammation to progress (17).
Zootherapy is an important alternative therapy in some countries (18). This is because three million years ago animal bile’s was used in traditional Chinese medicine for the treatment of different infections and inflammations such as ocular infections and skin diseases, suppurative otitis media, sinusitis, rhinitis, tonsillitis, gingivitis, laryngitis as well as pharyngitis, bronchitis, pneumonia, cystitis, and hepatitis (19, 20).
In previous studies of traditional Persian medicine, several therapeutic indications have been mentioned for bile. For example, a study reported topical application of bile for the treatment of ear infections, purulent ulcer, and smallpox (21). In addition, some studies demonstrated that gram-positive and gram-negative bacteria have different rates of resistance or sensitivity to bile, gram-positive bacteria seem to be more sensitive to bile (2). To the best of our knowledge, although numerous uses of animal bile, especially ox-bile (bile of caw) were reported, there is no evidence of approved ox-bile related drugs in Iran.
2. Objectives
The aim of this study was to evaluate the anti-bacterial effects of ox-bile on some clinically important gram-positive and gram-negative bacteria.
3. Methods
3.1. Ox-bile Preparation
The bovine gallbladder was obtained from Marvdasht slaughterhouse (Marvdasht, Iran). Immediately after slaughtering and evisceration of the male cattle (1.5 - 2 years old), under aseptic conditions, their gallbladder was removed from the liver junction, the bile duct was ligated, and they were transported to the laboratory in plastic bags under cold conditions. In the laboratory, the surface of the gallbladders was sterilized with a hot spatula, their contents were sucked with a 50 mL syringe, collected in a sterile beaker, and then sterilized with a 0.22 µm syringe filter.
3.2. Sterility Status
Ox-bile samples were then examined for their sterility status through culture on different media, including EMB agar (MERCK, Germany), Blood agar (MERCK, Germany), Thioglycolate broth (MERCK, Germany), and TSB broth (MERCK, Germany) following incubation under both aerobic and anaerobic conditions. The aerobic cultures were checked for 48 hours and the anaerobic cultures for one week.
3.3. Minimum Inhibitory Concentration
The well diffusion and micro-broth dilution tests were performed to determine the minimum inhibitory concentration (MIC) of ox-bile against gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa (PAO1), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis, and Propionibacterium acnes (ATCC 6919).
Through the well diffusion method, bacterial suspension of each species (equal to 0.5 McFarland turbidity) was cultured on distinct Muller Hinton agar plate wells were made, four different concentrations of ox-bile (3, 1/5, 0/75, 0/375%) according to two folds serial dilution with primary concentration of 6% bile were prepared and 50 μL of each prepared concentration of ox-bile was inoculated in the wells. In each plate, one well was dedicated to penicillin as a positive control. This test was done twice for each examined bacterium.
For the MIC test, the procedure was done based on CLSI guidelines (22). This test was also done twice for each bacterium
Finally, the plates were incubated in the proper atmosphere and temperature given the bacterium they contained. P. aeruginosa, E. coli, S. aureus, and S. epidermidis were incubated aerobically, but P. acnes was incubated anaerobically.
3.4. Ethical Statement
The study was approved by the Research Ethics Committee of School of Medicine- Shiraz University of Medical Sciences (IR.SUMS.MED.REC.1400.117).
4. Results
After 24 - 48 hours of incubation, plates of well diffusion assay were examined for inhibition zone, no inhibition zone was detected in any of the plates. Microplates were examined for the well with no growth, but no inhibitory effects were detected for any concentrations of ox-bile against the examined bacteria.
5. Discussion
The results of our study revealed that ox-bile did not show any anti-bacterial effect against evaluated gram-positive and gram-negative bacteria, including P. aeruginosa, E. coli, S. aureus, S. epidermidis, and P. acnes in the in-vitro setting.
Not concurring with our results, previous studies have mentioned anti-bacterial effects of bile and several mechanisms, especially damaging the bacterial cell membranes, DNA and RNA via oxidative stress mechanisms, resulting in alteration of protein expression and cell wall disruption (23, 24). Also, it has been reported that it affects the lipid bilayer structure of bacterial membranes due to hydrophobicity and the amphipathic nature of the membrane (25, 26). Further, there are reports that indicate the detergent role of bile acids and potential antimicrobial activity (27, 28).
Some evidences showed that ox-bile has an anti-bacterial effect on gram-positive and gram-negative bacteria as Kandell and Bernstein reported that bile salts could induce damage to the DNA of E. coli (9, 29). Yan and Zou mentioned that bile powder has a significant inhibitory effect on S. aureus and E. coli (30). In another study, the zone of inhibition against S. aureus was 28 mm at 100 mg/mL and for E. coli it was 29 mm (31). Prieto et al. reported that the MICs of ox-bile for Salmonella typhimurium and Salmonella typhi were 18% and 12%, respectively (32). Amine et al. explored the mechanism of action of ox-bile on Salmonella typhimurium at the molecular level. They revealed that alteration of seq A and its protein changed the phospholipids and fatty acids and enhanced bacterial sensitivity to bile acid (25). In another study, both E. coli and Salmonella enterica were sensitive to bile due to the alteration of cellular functions (32, 33).
On the other hand, some investigations reported different information and mentioned that E. coli is considered to be bile-resistant or Listeria monocytogenes strains are inherently bile-resistant (2, 34). These differences in bile tolerance can be related to the concentration of bile, exposure to various pH and temperatures, or the type and structure of bile (35, 36). Other factors include alteration of the cell membrane structure and composition, especially different lipopolysaccharides of the outer membrane, as well as changes in membrane electric charge and hydrophobicity (6). Capacity of cell wall to maintain intracellular homeostasis and altered activity of critical enzymes (2, 37), differences in gene transcription and protein expression are important too (9, 38).
There were some limitations in our study. First, we selected some positive and negative micro-organisms to test their sensitivity against ox-bile in this study. Therefore, it is not possible to generalize these results to all gram positive and negative micro-organisms. In this regard, evaluation of the anti-bacterial efficacy of ox-bile against other micro-organism and fungal agents are necessary. Next, it is possible that the animal race is a factor that can affect the results, so we recommend that this factor will be considered in further studies. Our suggestion for other researchers is to work on different organisms of specific diseases and to evaluate its efficacy on bacteria apart from the bacterial strains examined in this study or detecting other mechanisms for ox-bile such as anti-fungal effects. Another suggestion is to combine ox-bile with herbal medication for evaluating the other possible effects of ox-bile such as enhancing the penetration of drugs to the bacterial structures or their synergistic anti-bacterial effects.
5.1. Conclusions
According to the results of this study, the use of ox-bile to inhibit the growth or kill of the evaluated organisms is not recommended, and the evaluation of the antibacterial utility of ox-bile warrants further research.
