The antimicrobial activity of vitamin D 3 and omega-3 combination against pathogenic microorganisms

doi:10.21203/rs.3.rs-2762979/v1

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The antimicrobial activity of vitamin D 3 and omega-3 combination against pathogenic microorganisms

https://doi.org/10.21203/rs.3.rs-2762979/v1

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Food supplemnts such as vitamin D₃ and omega-3 have a significant impact on controlling pathogens. This study aims to evaluate the antimicrobial activities of combined vitamin D₃ and omega-3 against selected pathogens. Minimum inhibitory activities of different serial dilutions of vitamin D₃ (1.8 µM-216.6 µM) or omega-3 (0.8 mM-110 mM) or combined (vitamin D₃: 0.8 µM -108.2 µM and omega-3: 0.3 mM -55 mM) have been investigated on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. All the tested concentrations of vitamin D₃ and omega-3 as a separate treatement were significantly different from the positive control in terms of microbial log-reduction. The highest concentration of vitamin D₃ demonstrated a complete eradication of the tested microorganisms. Conversely, omega-3 had a lower effect on them. Combining 27 µM vitamin D₃ and 13.5mM omega-3 resulted in ~ 0.4 to 0.6 log reduction of S. aureus, E.coli, P. aeuregnosa and C. albicans. On the other hand, C. albicans had 1.1 log reduction at a combination of 108.4 µM vitamin D₃ and 55 mM of omega-3. Proposed mechanisms of the lowered antimicrobial activity when using the combination are discussed. These findings showed decreased antimicrobial effect of the combination and suggest a similar in vivo study to evaluate wheather taking the combination together or not.

antimicrobial agents

food suppliments

vitamin D3

Omeg

Dysbiosis due to antibiotic treatment and surgery may lead to the infection by the opportunistic colonization of toxin-producing Clostridium difficile, S.aureus, P.aeurignosa as well as sepsis of invasive Escherichia coli, causing pseudomembranous enterocolitis (Larcombe et al. 2016; Dong et al. 2018; Santana et al. 2022). An imbalance in gut bacteria composition has been noticed to be associated with intestinal symptoms, such as bloating, abdominal pain, and diarrhea (Brunkwall et al. 2021). This may be aggravated into more serious diseases like autoimmune diseases and cognitive function disturbances (Critchfield et al. 2011; Opazo et al. 2018).

The concept of adjunctive therapy in research has been extensively considered recently (Bellerba et al. 2021; Lebiecka et al. 2021; Rizzo and Brandi 2021). Adunctive therapy allows the use of lower doses of original treatment and/or less time required to expose the body to a drug and, therefore, fewer side effects encountered. Using adjunctive therapy in infection cases is being increasingly invistigated (Hilpert 2021; Inchingolo et al. 2021; Wong et al. 2021). One of the proposed options as adjunctive therapy in cases of gut microbiome dysbiosis is vitamin D₃ and omega-3 supplements (Sarris 2017; Hilpert 2021; Wellington et al. 2021). These supplements have been widely tested on human and animal models as immune-modulatory agents and gut dysbiosis regulators (Cantorna et al. 2004; Husson et al. 2016; Bellerba et al. 2021; Wellington et al. 2021; Hahn et al. 2022).

There is an established link between health state and vitamin D₃ intake in gut microbiota diversity (Singh et al. 2020). Vitamin D₃ has also been shown to potentially reduce gut opportunistic pathogens, particularly the class of Gamma-proteobacteria Pseudomonas spp. and Escherichia or Shigella spp (Wellington et al. 2021). Singh et al., 2020 investigated the effect of vitamin D₃ supplementation on gut microbiota, where gut microbiota showed a significant change in Firmicutes abundance level by a decrease of 5% and an increase of 3% in Beacteroidetes abundance with a general increase in the abundance of Bifidobacterium and Akkermansia (Singh et al. 2020).

Omega-3 modulates the gut microbiome in three aspects, by controlling the type and abundance of gut microbes, by altering levels of pro-inflammatory mediators like lipopolysaccharides, and by regulating the levels of Short Chain Fatty Acids (SCFA) and SCFA producing bacteria(Gentile and Weir 2018; Healy-Stoffel and Levant 2018; Labrousse et al. 2018). Omega-3 may also have beneficial effects on the gut microbiota by decreasing the growth of Enterobacteria, increasing the growth of Bifidobacteria, hence, inhibiting the inflammatory response associated with metabolic toxicity (Rizos et al. 2021). Such supplementation leads to omega-3 increasing the abundance of Bifidobacteria in the gut of male Sprague-Dawley rats (Guinane and Cotter 2013). It also could prevent gut microbiota dysregulation by increasing the number of beneficial lactic acid-producing bacteria in the mice fed a high-fat diet (Mujico et al. 2013; Robertson et al. 2017).

Many studies showed that there is an antimicrobial activity of vitamin D₃ (Tintino et al. 2016; Saputo et al. 2018; Almoudi et al. 2021) and omega-3 (Dhouioui et al. 2016; Sun et al. 2016; Sun et al. 2017). However, the impact of the combination has not been studied yet. Therefore, this study aims to evaluate the antimicrobial effects of the commercially available vitamin D₃ and omega-3 as separate and as combined adjunctive therapy against gut pathogenic microorganisms Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans.

Preparation of vitamin D₃ and omega-3

A commercially available product of vitamin D₃ (cholecalciferol) Hi Dee™ was used in this study with a concentration 250 µg/mL purchased from C-town Pharmacy - Amman, Jordan and stored in 2–8\(℃ \text{f}\text{r}\text{i}\text{d}\text{g}\text{e}\). Also, a commercially available omega-3 source as Wild Salmon & Fish Oils Jamison^® with 1000 mg/mLas a total content (containing 300 mg/mL of omega-3 specified as EPA and DHA) purchased from C-town community Pharmacy - Amman, Jordan and stored at room temperature.

Vitamin D₃ and omega-3 separate treatments were prepared for this assay in the microdilution method using a 96-well plate. The starting concentration of vitamin D₃ or omega-3 in the first well of the 96 well plate was diluted three times as the total volume of well is 300µl (100µl of vitamin D₃ stock equivelant to 650 µM, 100µl of broth media and 100µl of bacterial/yeast inoculum) or (100µl of omega-3 stock (329.9 mM), 100µl of broth media and 100µl of bacterial/yeast inoculum), this gives a starting concentration of 216.6 µM, continued with 1:2 ratio sequence of serial dilutions down to 1.8 µM for vitamin D₃ and a starting concentration of 110 mM, continued with 1:2 ratio sequence of serial dilutions down to 0.9 mM for omega-3.

Combination treatment of both vitamin D₃ and omega-3 was made by mixing 1mL of vitamin D₃ (10,000UI, 650 µM) and 1mL of omega-3 (1099.6 mM) with 0.5% Dimethyl Sulfoxide (DMSO; AZ Chem for chemicals, Canada). The overall mixture (vitamin D₃ concentration of 5000UI (325 µM ), 165 Mm of omega-3 and DMSO) had been mixed using a vortex before it was serially diluted in a 96-well plate. Starting concentration in the first well in the 96 well plate was diluted 3 times as the total volume of the well was 300µl (100µl of combination stock solution (325 µM of vitamin D₃ and 329.9 mM of omega-3), 100µl of broth media and 100µl of bacterial/yeast inoculum), this gave a starting concentration of 108.2 µM of vitamin D₃ and 55 mM of omega-3 continued with serial dilutions down to 0.8 µM of vitamin D₃ and 0.3 mM of omega-3.

Bacterial strains and growth conditions

The three bacterial strains, P. aeruginosa (ATCC 27853), S. aureus (ATCC 25923), and E. coli (ATCC 14169) were sub-cultured in nutrient broth (NB) (Oxoid, UK), while C. albican (ATCC 90028) was grown on Sabouraud-dextrose broth (SDB) (Oxoid, UK) and incubated with each bacterial inoculum overnight (16 ± 2 hrs) at 37 ± 2 ᴼC for bacteria and 32ᴼC for yeast. Microorganisms were resuspended in NB to an OD600 nm = 0.08 measured using an absorbance microplate reader (EPOCH, USA). According to 0.5 McFarland (equivalent to 1.5 × 10⁸ CFU/mL). For C. albicans, it was resuspended in SDB to 0.5 McFarland standard OD530 = 0.12–0.15 was used, corresponding to 1 ~ 5 × 10⁶ CFU/mL.

Minimum inhibitory concentration (MIC)

This study determined the minimum inhibitory concentration (MIC) using the broth microdilution method against E. coli, S. aureus, P. aeruginosa and C. albicans. Microbial inoculum of an approximate concentration of 1.5×10⁸ CFU/mL for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and for C. albicans, the microbial inoculum of an approximate concentration of 1 ~ 5 × 10⁶ CFU/mL. This assay was tested in triplicates along with positive and negative controls. Negative or positive controls were bacterial inoculum in NB/alone or treated with 50mg/mL Gentamicin Sulphate (Cytiva, USA), respectively. For yeast, 250µg/mL of amphotericin B (cytiva, HyClone™ Utah) was used a s a positive control. Then the plates were incubated aerobically at 37 ± 2°C for 24 hours. The growth of bacteria was determined at OD600 nm and OD530 nm for Candida using a microplate reader (Bio-Tek, Germany). The microbial growth was assessed by converting the mean absorbance OD600 and OD530 to CFU/mL according to linear curve equations for negative control growth in each type of bacteria and subtracting the log CFU/mL of the tested trial from the log negative control to calculate log reductions.

Log reductions = log₁₀(A + 1)-Log₁₀(B + 1) (Breed and Dotterrer 1916; Goldman and Green 2015)

Where, A = number of viable organisms without treatment, and B = number of viable organisms after treatment.

Statistical analysis

All results are expressed as mean ± SD (n = 3) using GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA), performing multiple unpaired t-test per treatment. Statistical significance was determined using the Holm-Sidak method(Dinno 2015), with alpha value set at 0.05. Our p-value had < 0.001 significance level throughout the experimental trials.

Effect of vitamin D ₃ and omega-3 separately and in combination on E. coli bacterial growth

Gentamicin positive control had 5.5 log reductions onto E.coli growth, while vitamin D₃ separate treatment showed a higher growth inhibition effect on E. coli at 83.3µg/mL concentration having 7 log reductions (Fig. 1A). There was a decrease in log reductions as the concentration of vitamin D₃ decreased. Showing that 41.7µg/mL concentration of vitamin D₃ had 2 log reduction only. (Fig. 1A). On the other hand, the log reduction of E. coli reached only 0.4 when using omega-3 as a separate treatment at a concentration of 110 mM (Fig. 1A). There was also a decrease in log reductions as the concentration of omega-3 decreased showing that 50mg/mL concentration of omega-3 had only 0.3 log reduction. (Fig. 1A). All conentrations of vitamin D₃ and omega-3 separate treatment had a significant difference than the positive control with a p-value < 0.001, but the most effective one compared to positive genatmicin control was 216.6 µM concentration of vitamin D₃ separate treatment.

The effect of the combination treatment of both vitamin D₃ and omega-3 on E. coli (Fig. 1B) demonstrated a range of inhibition of log reductions reaching maximally to 0.6 log for the combination of 27 µM vitamin D₃ and 13.5 mM of omega-3 together. Any concentration less than or equal to 3.4 µM + 1.8 mM did not show any pronounced effect. Furthermore, the highest starting concertation (108.2 µM of vitamin D₃ with 55 mM of omega-3) also weakly affected bacterial growth (Fig. 1B).

Effect of vitamin D ₃ and omega-3 separately and in combination on S. aureus

Gentamicin positive control had 5.5 log reductions onto S. aureus growth, while vitamin D₃ separate treatment showed a higher inhibition of S. aureus growth reaching 7 log reduction at a concentration of 216.6 µM (Fig. 2A).A similar pattern to that described for E. coli growth was also detected here; as vitamin D₃ concentration decreased, the log reductions also decreased. The 108.2 µM concentration of vitamin D₃ had only 1 log reduction (Fig. 2A). Meanwhile, when using omega-3 alone against S. aureus, 110 mM of omega-3 decreased the growth of S. aureus by only 0.2 log reduction. There was a decrease in log reductions as the concentration of omega-3 decreased. Showing that 55 mM concentration of omega-3 had less than 0.2 log reductions. (Fig. 2A). All conentrations of vitamin D₃ and omega-3 separate treatment had a significant difference than the positive gentamicin control with a p-value < 0.001, but the most closer one to positive genatmicin control was 216.6 µM concentration of vitamin D₃ sepearte treatment (Fig. 2A).

The effect of combination treatment of both vitamin D₃ and omega-3 together against S. aureus (Fig. 2.B) showed a range of inhibition of log reductions reaching maximally to 0.4 log for the combination of 54.1 µM vitamin D₃ and 27.5 mM of omega-3 together. Lower dilutions had stable log reduction values leveling maximally to 0.3 log reductions only (Fig. 2B).

Effect of vitamin D ₃ and omega-3 separately and in combination on P. aeruginosa

Gentamicin positive control had 5 log reductions onto P. aeruginosa growth, while vitamin D₃ separate treatment showed a higher inhibition of P. aeruginosa growth reaching 6.5 log reduction at a concentration of 216.6 µM (Fig. 3A). There was a decrease in log reductions as the concentration of vitamin D₃ decreased. The 108.2 µM concentration of vitamin D₃ had only a 1.5 log reduction (Fig. 3A). On the other hand, omega-3 separate treatment had only 0.3 log reduction onto P. aeruginosa growth when using 110 mM of omega-3 alone (Fig. 3A). There was a slight decrease in log reductions as the concentration of omega-3 decreased. As 55 mM concentration of omega-3 had only 0.2 log reduction. (Fig. 3A). All conentrations of vitamin D₃ and omega-3 separate treatment had a significant difference than the positive gentamicin control with a p-value < 0.001, but the most closer one to positive genatmicin control was 216.6 µM concentration of vitamin D₃ sepearte treatment (Fig. 3A).

The effect of the combination treatment of vitamin D₃ and omega-3 together was also tested against P. aeruginosa (Fig. 3B) showing a range of inhibition of log reductions reaching maximally to 0.5 log for the combination of 27 µM vitamin D₃ and 13.5 mM of omega-3. Any concentration less than or equal to 13.5 µM of vitamin D₃ combined with 6.9 mM of omega-3 did not show any pronounced effect. Furthermore, the highest starting concertation (216.6 µM of vitamin D₃ mixed with 55 mM of omega-3) also weakly affected bacterial growth (Fig. 3B).

Effect of vitamin D ₃ and omega-3 separately and in combination on C. albicans yeast

Amphotericin B positive control had 6 log reductions onto C. albicans growth, while vitamin D₃ separate treatment showed a higher inhibition of C. albicans growth reaching 7 log reduction at a concentration of 216.6 µM (Fig. 4A). A similar pattern to that described for E. coli, S. aureus, P. aeruginosa growth was also detected with C. albicans, as vitamin D₃ concentration decreased, the log reductions also decreased. Showing that 108.2 µM concentration of vitamin D₃ had only 0.2 log reduction. (Fig. 4A). On the other hand, when using omega-3 alone against C. albicans, 110 mM and less had no clear growth inhibition on C. albicans. (Fig. 4A). All conentrations of vitamin D₃ and omega-3 separate treatment had a significant difference than the positive amphotericin B control with a p-value < 0.001, but the most closer one to positive amphotericin B control was 216.6 µM concentration of vitamin D₃ sepearte treatment (Fig. 4A).

The effect of combination treatment of both vitamin D₃ and omega-3 together was also tested against C. albicans shown in Fig. 4B, showing a range of inhibition of log reductions reaching maximally to 1.1 log for the combination of 108.2 µM vitamin D₃ and 50mg/mL of omega-3 together. Any concentrations equal to or less than 6.8 µM of vitamine D3 with 3.4 mM of omega-3 had lower log reduction values reaching to 0.6 log reduction or less (Fig. 4B).

Vitamin D₃ has been studied as an effective supplement to human health in many areas (Cantorna et al. 2004; Stoffels et al. 2006; Cannell and Hollis 2008; Huang et al. 2013; Singh et al. 2020). In our study a full log reduction was noticed on Gram positive, Gram negative pathogenic bacteria, and C. albicans after treatment with 216.6 µM (3300IU) of vitamin D₃. At lower concentaration of vitamin D₃, the log reduction decreased (1–2 log reduction) for the bacteria, while it showed no significant effect on C. albicans. These results are in agreement with a previous in vivo study that showed that vitamin D₃ is effective to general human health and in limiting the growth of pathogenic gut bacteria and infection in the recommended daily doses used in ranges of 50µg/mL to 125µg/mL (130–325 µM) (Stroud et al. 2008; Mocanu et al. 2009; Pludowski et al. 2018; Waterhouse et al. 2019; Charoenngam et al. 2020). Moreover, in vitro studies suggested that vitamin D₃ has inhibitory activity on Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli (Cannell and Hollis 2008; Kempker et al. 2012; Bekele et al. 2018). In the presence of high doses ranging from 50,000 to 90,000IU/mL (1250µg/mL-2250µg/mL) of vitamin D₃, the microorganisms showed a significant growth inhibition (Kempker et al. 2012). In another in vivo study, Gram-positive bacteria, invasive pneumococcal disease, meningococcal disease, and group A streptococcal disease were more common when vitamin D₃ levels were low, raising the possibility that pharmacological doses of vitamin D₃ could be an effective adjuvant therapy (Cannell and Hollis 2008; Bekele et al. 2018).

Omega − 3 is a long chain-polyunsaturated fatty acid (LC-PUFA), an essential type of nutrient to human body, that can exist in many forms of fatty acid chains like docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA), and docosapentaenoic acid (DPA) (Saini and Keum 2018). Omega-3 has proven its efficacy as adjunctive therapy in autism and ADHD alongside cardiovascular diseases and gut microbiota dysbiosis (He 2009; Manson et al. 2012; Costantini et al. 2017; Robertson et al. 2017; Infante et al. 2020). In the current study, omega-3 had a minimum effect on both Gram positive and Gram negative bacterial log reductions. While E. coli and P. aeruginosa had low log reduction (0.4 and 0.3, respectively) when treated with 100mg/mL of omega-3 treatment, Gram positive bacteria S. aureus had insignificant effect. Meanwhile, C. albicans did not show any significant effect even when treated with the highest concentration of omega-3 (Fig. 4A). In a recent study, it was noted that the pure Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) separate preparation obtained for investigation use, promoted the complete killing of S. epidermidis and S. aureus in planktonic form by using the highest concertation 5 mg/mL. On the other hand, the fatty acids in the study mentioned above showed low activity against P. aeruginosa (Coraça-Huber et al. 2021). Furthermore, a study made by using the bio-converted EPA and DHA omega-3 components displaying antibacterial activities against Gram-positive bacteria (Bacillus subtilis, Listeria monocytogenes, Staphylococcus aureus) and Gram-negative bacteria (Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, Salmonella enteritidis, S. typhimurium) (Shin et al. 2007; Kannan et al. 2021). The growth inhibition by both bio-converted EPA and DHA was similar against Gram-positive bacteria, but the bio-converted extract of DHA was more effective than EPA against Gram-negative bacteria (Shin et al. 2007). Another study investigated the antimicrobial activity of the short, medium and long-chain fatty acids for oral microorganisms especially C. albicans. Long chain fatty acids (LCFAs), including n-3(omega-3), n-6, n-7, and n-9 fatty acids have been reported to have anti-inflammatory and antibacterial activities. The SCFAs and LCFAs showed limited bio-activity against the oral fungal species (Huang et al. 2011). To explain this different outcome than our study, it is suggested in a recent investigation made on components of omega-3 (Pal et al. 2020), stating that the intake of EPA and DHA together may cause competition of both molecules against each other through interposed mechanisms. First, EPA and DHA may compete for residency in membrane phospholipids of biomolecules surfaces like cell membranes. Moreover, EPA and DHA induce different effects onto plasma membrane structures, building an additional layer of competition between the fatty acids in successive signaling pathways. Third, DHA regulates membrane EPA levels by lowering its rate of conversion to EPA’s longer product n-3 docosapentaenoic acid DHA(Pal et al. 2020).

As for the combination treatment effect of vitamin D₃ and omega-3 on bacterial and fungal growth, our study showed that for Gram-negative bacteria, E. coli and P. aeruginosa, 27 µM of vitamin D₃ and 13.5 mM of omega-3 had a maximum effect of 0.5–0.6 log reductions. On the other hand, the same combination only had an approximately 0.4 log reduction for Gram positive bacteria, S. aureus, with no obvious effect on the highest concentration. While C. albicans had a different outcome, where 1.1 log reduction was noticed at a combination of 108.2 µM vitamin D₃ and 55 mM of omega-3 and 0.6–0.7 log reduction was seen for concentrations from 54.1 µM and 27.5 mM till 0.8 µM and 0.3 mM of vitamin D₃ and omega-3 combined. The “weaker” effect of the combination could be understood by a possible chemical interaction between vitamin D₃ and omega- 3. There is alcohol groups in the chemical structure of vitamin D₃ and carboxylic acid moieties in the chemical structure of omega-3 (Sebrell 1971; Valenzuela and Valenzuela 2013). Such interaction might result in the esterification of both structures producing a bulkier compound (Neises and Steglich 1978; Hughes et al. 1988), offering an obstacle in crossing through bacterial membranes. Considering that the esterification reaction needs special conditions like acidic media and heat (Neises and Steglich 1978). These conditions are present in the in vitro experiments as the heat source is supplied through incubation conditions and acidic moieties are mainly embedded in the Gram-positive bacteria cell wall (Gupta et al. 2021). Combination treatment had the lowest effect on log reductions of S. aureus (Gram-positive bacteria). S. aureus bacteria have many acid components on the surface of its cell wall, e.g., teichoic and lipoteichoic acids, in addition to multiple layers of peptidoglycan containing N-acetylmuramic acid (Gupta et al. 2021). Whereas combination treatment had a greater effect on the log reductions of Gram-negative bacteria, E. coli and P. aeruginosa, within recommended daily doses. This could possibly be due to Gram-negative bacteria having only a thin layer of peptidoglycan and limited acidic moieties on its cell wall surface (Beveridge 1999).

C. albicans demonstrated a different behavior in combination treatment since it prefers an acidic environment, thus, it can adjust the pH of its surroundings (Mayer et al. 2013). This way C. albicans encourages the formation of ester bonds, producing the bulky structure of the supplements used. Nevertheless, C. albicans can possibly degrade ester bonds through the secretion of enzymes and polymorphism to benefit from the carbon skeleton of supplements (Sorgo et al. 2013). Considering that C. albicans might secret enzymes and proteins as a form of nutritional acquisition (Gil-Bona et al. 2015), allowing the penetration of separated supplement structures and producing the log reduction effect on its growth curve.

Furhtermore, in our study, the high concentrations of vitamin D₃ 108.2 µM and omega-3 55 mM combination treatment was noticed to have lower inhibitory effects than combination with lower concentrations on both Gram positive and negative bacteria. It is proposed that both Gram positive and negative bacteria showed Eagle effect (Goldstein and Rosdahl 1981; Grandière-Pérez et al. 2005; Jarrad et al. 2018; Prasetyoputri et al. 2019), where bacteria exposed to drug concentrations higher than an optimal bactericidal concentration have paradoxically increased levels of survival than at the optimal bactericidal concentration due to a decreased rate of cell death (Goldstein and Rosdahl 1981; Prasetyoputri et al. 2019). This phenomenon has been explained by few mechanisms. The first mechanism is the aggregation of molecules in aqueous media. It is possible that the aggregation of both omega-3 and vitamin D₃ in vitro occur at the surface of bacteria leading to a larger agglomerated bulky structure, as mentioned previously. The agglomerated form might be detected as bacterial colonies leading to higher growth readings(Jarrad et al. 2018). The second mechanism suggested is the reduced expression of binding receptors on the cell wall of bacteria, these receptors are used by drug treatment to enter and eradicate bacteria. The third mechanism might be bacteria self-antagonizing its own receptors as a form of resistance to high concentrations of drug exposure, halting down the effect of antimicrobial effect. Hence, detecting higher bacterial growth readings will be observed in such case too (Goldstein and Rosdahl 1981; Jarrad et al. 2018).

It is suggested based on our in vitro observation that taking vitamin D₃ alone as adjunctive therapy in the recommended daily dose is advantageous in cases of pathogenic gut infections. It does not only potentiate immunity and gut microbiota abundance but also it has a log reduction effect onto the growth of Gram positive, negative, and fungal microorganism. On the other hand, it is not advised to consider omega-3 supplement as adjunctive therapy, depending on our observations, omega-3 had minimum log reduction effect on pathogenic bacteria.

On the other hand, in case of combination treatment of both vitamin D₃ and omega-3, log reductions were lower than that of separated therapy and thus vitamin D₃ lost its strong log reduction effect on pathogenic gut bacteria. The argument here is how much combination adjunctive treatment can help in reducing pathogenicity and slowing down the growth rate of gut pathogens. As mentioned previously that 1 log reduction is indicating that 90% of microorganism load is reduced (De Vries and Hamilton 1999), and so 0.5 log reduction indicate 66.6% of microorganism load is reduced. This may reduce the pathogenic microbial infectious dose and reduce the load on immune system to defeat the infection. By this, we highlight that the goal here is not to fully eradicate the infection rather than offering an adjunctive therapy for the immune system to attain hemostasis over pathogenic state again.

In conclusion, the combination of both supplements (vitamin D3 and Omega-3) resulted in decreased antimicrobial effect of vitamin D₃ and omega-3 individually. Therefore, depending on our results, we suggest a similar in vivo study to evaluate if the combination should be taken together or not. Subsequently, a recommendation about taking both suplplements together or leaving a time-separation interval can be made. Further studies are required to elucidate any proposed chemical interaction between both supplement chemical structures, to understand the mechanism of bacterial eradication of separate and combined treatments, and to further orchestrate the best use either separate or combined treatment as antimicrobial supplement adjunctive option.

Acknowledgement

The authors highly acknowledge the support from the Applied Science Private University in using the lab facilities to conduct this research.

Funding

This work was supported partially by the Applied Science Private University fund # DRGS/2017-2018-6

Competing interests: The authors declare there are no competing interests

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The antimicrobial activity of vitamin D 3 and omega-3 combination against pathogenic microorganisms

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Preparation of vitamin D₃ and omega-3

Bacterial strains and growth conditions

Minimum inhibitory concentration (MIC)

Statistical analysis

Results

Discussion

Declarations

Acknowledgement

Funding

References

Additional Declarations

Status:

Version 1

The antimicrobial activity of vitamin D 3 and omega-3 combination against pathogenic microorganisms

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Preparation of vitamin D3 and omega-3

Bacterial strains and growth conditions

Minimum inhibitory concentration (MIC)

Statistical analysis

Results

Discussion

Declarations

Acknowledgement

Funding

References

Additional Declarations

Status:

Version 1

Preparation of vitamin D₃ and omega-3