Bacteroides ovatus and Escherichia coli have a negative effect on the total
production of butyrate by Lachnospiraceae bacterium.
Abstract
Major depressive disorder (MDD) is the leading cause of mental disability in the world. The pathophysiology of depression however remains unknown. Given the multiple disadvantages of the current pharmacological therapy for depression, it is important to look into another direction of treatment. Recently, the neuroinflammatory response and oxido-nitrosative stress are being attracted more and more attention due to their roles in depression pathogenesis. Previous animal studies showed that butyrate could regulate proinflammatory responses and oxido-nitrosative stress in different models of disease. Butyrate could therefore be of therapeutical relevance for the treatment of depression. Butyrate is produced endogenously by certain bacteria in the human gut. This research focused on the production of butyrate of human gut bacteria and can be considered as a pilot study for the interaction of human gut bacteria and the effect on butyrate production. A representative minimal human microbiome model was used, consisting of Lachnospiraceae bacterium (LB), Bacteroides ovatus (BO) and Escherichia coli (EC) in different combinations of cocultures. From the different cultures the growth was measured, and metabolite production was analyzed using HPLC. Co-culturing LB, BO and EC led to an increase of growth of the bacterial consortium. BO and EC however had a negative effect on the butyrate production of LB. The highest concentration of butyrate was measured in the monoculture of LB. The results of this study give us more information on which ratio of BO, EC and LB in the human gut could lead to the highest production of butyrate. Restoration of this balance between the human gut bacteria could lead to a higher endogenous production of butyrate and could play a key role in the treatment of depression.
Lilly Engelen
Bachelorthesis BMW
Supervisors: Tineke Banda & Pim van Leeuwen
26-03-2021
1. Introduction
Major depressive disorder (MDD) is the leading cause of mental disability in the world. With a yearly global point prevalence of 4.7% and a pooled annual incidence of 3.0%, it holds the eleventh place of leading causes of global disease burden.1 Characteristic symptoms of
depression include low mood, anhedonia, fatigue, concentration problems, hyper- or insomnia and recurrent thoughts of death.2 There is a large body of evidence linking depression to other comorbidities.3 This marks the importance of depression as a significant clinical and global health issue.4
Despite the impact of depression, the pathophysiology remains to be unknown.5 The majority of the pharmacological therapies for depression are used to target monoamine dysfunction in the brain, such as the selective monoamine oxidase inhibitors, serotonin reuptake inhibitors and tricyclic antidepressants. However, these antidepressants are effective in no more than one third of patients.6 In the patients who do benefit from these monoamine agents, there is a delay in therapeutic effect of several weeks.7 In addition, these
antidepressants can cause numerous side effects, such as headaches, dizziness, weight changes, sleep disruption and sexual dysfunction.8 These side effects are linked to poor adherence to the medication and may interfere with the treatment outcome.
Given the multiple disadvantages of the current pharmacological therapy for
depression, it is important to look into another direction. Over the last decade, studies have proven that the neuroinflammatory response and oxido-nitrosative stress seem to play a role in the pathogenesis of depression, especially in the prefrontal cortex and hippocampus area of the brain9. Earlier research has shown that butyrate has a significant therapeutical effect on MDD by regulation of neuroinflammatory response and oxido-nitrosative stress.10 Butyrate is a short-chain fatty acid (SCFA) and is produced endogenously in the colon by the
fermentation of non-digestible fibers by certain bacteria genera from the Firmicutes phylum. Butyrate is mostly known for the beneficial effects on both cellular energy metabolism and intestinal homeostasis, but numerous studies also point to the effect of butyrate on the brain via the gut-brain axis (GBA).11 This GBA consists of bidirectional communication between the central and the enteric nervous system. Recent studies have shown that there is also bidirectional communication between gut microbiota and the brain by means of neural, endocrine, immune and humoral links.12 Research has described that alterations in butyrate-producing bacteria can influence the peripheral and central nervous systems and brain functions, giving evidence for the theory of the microbiota-gut-brain axis.13
The microbe-derived metabolite butyrate can enter the circulation after absorption in the gut and is able to cross the blood-brain barrier.14 Here it can affect the brain through its anti-inflammatory properties including the reduction of pro-inflammatory cytokine expression interferon gamma [IFN-gamma], tumor necrosis factor-alfa [TNF-alfa] interleukin-1B [IL-1B], IL-6, IL-8). It can also induce IL-10, transforming growth factor-B (TGF-B) and nitric oxide synthase and reducing lymphocyte proliferation and activation.15
Often, studies make use of the sodium form of butyrate, since endogenously produced butyrate is difficult to use in practice. This is due to its offensive odor and absorption in the upper gut. It is to be assumed that sodium butyrate (SB) does not differ in function in comparison to butyrate.15 There is evidence that SB functions as a histone deacetylase (HDAC) inhibitor. By acting on the chromatin structure of DNA, SB can increase gene
expression of proteins related to neuronal synaptic plasticity.16 Other preclinical rodent studies have shown that SB has a positive effect on neurodegenerative diseases, such as spinal
muscular atrophy, ischemia-induced neuronal damage and Huntington’s disease.17 SB could have beneficial effects for the treatment of neurological disorders ranging from
neurodegenerative diseases to depression.
Currently, there is limited understanding of the functionality of butyrate and how it might affect the pathogenesis of depression. This research will focus on the endogenous production of butyrate of human gut bacteria and can be considered as a pilot study for the interaction of human gut bacteria and the effect on butyrate production. Given the complexity of the entire human gut microbiome, a minimal human microbiome model is used. This model consists of representative species of three different phyla which are commonly found in the human gut: Lachnospiraceae bacterium (LB), Bacteroides ovatus (BO) and Escherichia coli (EC).
The vast majority of the human gut microbiota is composed of the Firmicutes and Bacteroides phyla. Most Firmicutes are butyrate-producing-bacteria (BPB). LB belongs to the Firmicutes phylum and the Clostridiales order. LB is easy to cultivate and genetically related to other unculturable members of the Lachnospiraceae.18 LB mainly produces butyrate and for this pathway acetate is used. LB also produces other SCFAs like acetate and propionate.19 20 BO belongs to the Bacteroidetes phylum and the Bacteroidales order. BO produces SCFAs like acetate, succinate, lactate and proprionate.21 EC is also a common bacterium found in the human gut. It is among the first bacterial species to colonize the intestine early after birth and become a habitant throughout the life of the host.22 Cultures of EC mainly produce acetate
when grown under anaerobic circumstances. They also produce ethanol, formate and succinate.23
To further investigate the microbial dynamics and metabolic interaction of butyrate in the human gut, this study researches the effect of BO and EC on LB and the total production of butyrate in vitro is assessed. These three bacteria will be grown in different cocultures and the metabolite profiles will be analyzed via high-performance liquid chromatography
(HPCL).
As of yet, little is known about the interaction between the bacteria BO, EC and LB. Based on their metabolite profile, the expectation for the metabolite products in the
monocultures of LB, EC and LB is shown in figure 1A. According to Larsen et al. (2019) BO was negatively correlated with cumulative production of butyrate in an in vitro colon model.24 Therefore we hypothesize that BO will have a negative effect on the production of butyrate in a coculture with LB. Because the production of acetate from EC is beneficial for the butyrate pathway of LB, we hypothesize that in cocultures with LB and EC the butyrate production will be higher than in a monoculture of LB. This cross-feeding of acetate between BO, EC and EC is shown in figure 1B. In a triculture of BO, EC and LB the beneficial relationship between LB and EC will not weigh up to the negative effect of BO on the butyrate pathway of LB. Because BO, EC and LB all use the same nutrients for their metabolite pathways, there will be a higher competition in the tricultures. We hypothesize that this will lead to an earlier stagnation of growth and a lower production of metabolites, including butyrate.
In this study the metabolites butyrate and acetate will be looked at specifically with
HPLC, in order to research the cross-feeding interaction of these metabolites between BO, EC and LB. The concentration level of glucose in the different cultures will also be analyzed to interpret the nutrient utilization.
Figure 1. Metabolic interaction of B. ovatus, E. coli and L. bacterium. (A) Different metabolite products of BO, EC and LB. (B)
Cross-feeding of acetate occurs between the three bacteria. For the production of butyrate L. bacterium. For the production of butyrate L. bacterium utilizes acetate, which is produced by LB itself and both BO and EC.
2. Material and Methods
Bacterial strains
In this study, Lachnospiraceae bacterium, Bacteroides ovatus and Escherichia coli K-12 MG1655 strains were used. 100 μL was taken from freezer-stocks 100 μL and transferred into a tube containing 5 mL ssGut-medium. This was incubated overnight under anaerobic
conditions at 37°C using a hypoxic chamber (Whitley DG250 anaerobic workstation, Don Whitley Scientific). Of each bacterial strain 100 μLwas pipetted, plated out on petri dishes and left for 16 hours under anaerobic conditions at 37°C. The solid medium that was used on the petri dishes for LB and BO was made of blood-agar, since they are fastidious micro-organisms. This means that they will only grow if special nutrients are present in their culture medium, such as growth factors, lipids and minerals. EC is a non-fastidious micro-organism and for EC the solid medium consisted of LB-agar. More information about the ingredients and preparation for these mediums can be found in the appendix. For the inoculation of a preculture of each bacterium, a single colony was taken with a sterile loop and transferred into a tube containing 0.5 ml BHI (S)-medium. After 16 hours the tubes were resuspended.A portion of this suspension was diluted in ssGut-medium to obtain a volume of 10 ml with an optical density (OD) of 0.1 at 600 nm using a spectrophotometer (Fisher 40 Cell Density meter). To calculate the amount of preculture needed to make the main culture, formula 1 was applied.
!"#$%& () *"+# ,-.$-/%
*%"0-/%& () 1/%,-.$-/% x volume of main culture = volume of preculture
Formula 1. For measuring the amount of preculture needed to inoculate 10 mL main culture with an OD600 of
0.1
After confirming an OD600 of 0.1 for the 10 mL of suspension by measuring it with the spectrophotometer, the main cultures were ready for growth-curve analysis. In the initial phase (lag phase) time periods of one hour were used to measure the OD600. When the bacteria started to show an exponential growth rate (log phase), samples were taken each 30 minutes to measure the OD600. When the growth curve stagnated at an OD600 of
OD-CFU correlation
This procedure was used to calibrate OD to colony forming unit (CFU) counts, in relationship to the cell concentration of the culture. The 1 mL samples were diluted with ssGut-medium to an OD600 0.1 in 1 mL as the starting sample for the serial dilution. The first three dilutions were 1:20 and the last two dilutions 1:10. Of dilution 4, 5 and 6 100 μL was pipetted and plated out on petri dishes with the appropriate medium (blood-agar or LB-agar) in triplicate. This led to a final dilution factor of 8 x 104, 8 x 105 and 8 x 106. These petri dishes where incubated overnight under anaerobic conditions at 37°C. After approximately 18-20 hours CFUs start to appear, where each CFU represents one bacterial cell, assuming that one bacterial cell will give rise to one colony. Plates with more than 300 colonies cannot be
counted and are designated too many to count (TMTC). Plates with fewer than 30 colonies are designated too few to count (TFTC). Only petri plates containing between 30 and 300
colonies were selected for the OD-CFU correlation. By multiplying the colony count by the final dilution factor on each plate, the number of CFUs per mL in a starting sample with an OD600 of 0.1 were measured per bacteria.
Figure 2. OD-CFU correlation graphic. The different steps are shown for the serial dilutions from the starting
samples from the main cultures. For each bacterial strain, there are 12 plated petri dishes: 6 for positive controls and 6 for negative controls. Two starting samples of OD600 0.1 are three times diluted 1:20 and then two times
diluted 1:10. From dilution 3, 4 and 5 100 μL is plated out in triplicate, leading to a final dilution factor of 8 x 104,
8 x 105 and 8 x 106. After 18-20 hours the colonies can be counted on each plate. After multiplying the colony
Co-culturing
The different combinations of monocultures and cocultures are shown in table 1. The aim was that all cultures start at the OD600 of 0.1 with a volume of 10 mL. To measure the needed amount of the precultures, formula 1 was applied. In a coculture of two bacterial strains, the ss Gut-Medium was inoculated with an OD600 of 0.05 per strain. For the tricultures an OD600 of 0.033 per strain was used. The time stamps for measuring the OD600 for the growth curve analysis were after 1, 2, 3, 4, 6, 8 and 24 hours.
Table 1. Different combinations of bacterial strains for co-culturing. Cocultures consist of monocultures,
cocultures, a triculture and a control group. BO, B. ovatus; LB, L. bacterium; EC, E. coli. Bacterial strains monocultures BO LB EC cocultures BO + EC BO + LB EC + LB triculture BO + EC + LB control -
HPLC-analysis
To determine metabolic products used and produced by the microorganisms, the conditioned medium was run through an HPLC system (LC-20AT, SIL-20AC ht, RID-20A, SPD-M30A; Shimadzu) with the Rezex ROA-Organic acid H+ column (Phenomex, S/No: H17-364678). Elution was performed with H2SO4 in 30 minutes at a flow rate of 0.5 mL/min. The
absorbance of the effluent was monitored at 214 nm.
Statistical analysis
For the OD-CFU correlation the mean values and standard deviations were calculated. In order to assess the effect of the metabolic interactions between the bacteria, the
concentrations of the metabolites were plotted against the incubation time. A two-sample t-test was performed to assess significant difference between the metabolite concentration of the cocultures and the mean values of concentration of the monocultures.26 Statistical significance was accepted at p < 0.05 level.
3. Results
Growth curve
In figure 3 the growth curve of all cultures is shown, where the dotted lines represent the monocultures: blue for BO, orange for EC and grey for LB. The solid lines represent the cocultures: blue for the culture of BO and LB, yellow for BO and EC and green for LB and EC. The dark blue striped line represents the triculture of LB, EC and BO.The brown line represents the control group.
0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 4 8 12 16 20 24 Gr ow th (OD 600 )
Incubation time (hours)
BO EC
LB BO + EC
BO + LB LB + EC
LB + EC + BO
Figure 3. Growth curve of all examined cultures. B. ovatus (BO), E. coli (EC) and L. bacterium (LB) were cultured in different bacterial.
consortia. The mono-, co- and tricultures were inoculated at an OD600 of 0.1 and incubated for 24 hours at 37°C under anaerobic conditions.
The OD600 was measured after 1, 2, 3, 4, 6, 8 and 24 hours. The dotted lines represent the monocultures (BO – blue, EC – orange, LB –
grey), the solid lines the cocultures (BO+LB – blue, BO+EC – yellow, LB+EC – green) and the striped line represents the triculture (LB+EC+BO – dark blue). The lowest growth is measured in LB (maximum OD600 of 0.32 after 8 hours) and the highest in the triculture
of BO, EC and LB (maximum OD600 of 0.48 after 8 hours). Monocultures of BO and EC and their coculture show similar growth (reaching
OD600 of 0.42 after 24 hours). BO and EC show a higher growth curve when co-cultured with LB. In the control group there was no growth
detected (OD600 remained at 0.00 for 24 hours; not visible in figure).
All cultures show signs of exponential growth in the first 4 hours. With regards to the monocultures, LB has the lowest increase in OD600 throughout the total 24 hours of incubation, showing a maximum OD600 of 0.32 after 8 hours. The OD600 decreases to 0.27 after 24 hours. In comparison to the other monocultures, EC has the highest increase in OD600 with a maximum of 0.43 after 8 hours. BO reaches a maximum OD600 of 0.40 after hours, having a similar course of growth as EC. BO and EC both reach an OD600 of 0.42 after 24 hours. Looking at the coculture of BO and EC, the growth curve is similar to the growth curves of the monocultures of BO and EC, having a maximum OD600 of 0.40 after 8 hours and also reaching an OD600 of 0.42 after 24 hours. The coculture of LB and BO shows an OD600 of 0.44 after 8 hours and reaches its maximum OD600 of 0.50 after 24 hours. The coculture of LB and EC reaches its maximum OD600 of 0.46 after 8 hours and stagnates at 0.45 after 24 hours. During the exponential growth phase (0 – 4 hours) the triculture of BO, EC and LB shows the highest increase of growth in comparison to the other cultures. It reaches a maximum OD600 of 0.48 after 8 hours and stagnates at 0.46 after 24 hours.
OD-CFU correlation
To determine the number of CFUs per mL in a starting sample with an OD600 of 0.1 per bacteria, only petri plates containing between 30 and 300 colonies were selected for the OD-CFU correlation. For BO, EC and LB this range in colony count responded to a dilution of the starting samples of the cultures of 10-6, 10-6 and 10-5, respectively. From each bacterial strain two biological replicates were grown, and from each biological replicate from two serial dilutions were made. Each dilution was plated out in triplicate, totaling 12 petri dishes that were plated out per bacterial strain.
In table 2 the counted colonies for BO are shown, with a total CFU average of 51.6 ± 8.69, leading to an OD600[0.1]-CFU correlation of 5.16 x 107 ± 8.69 x 106. In table 3 the counted colonies for EC are shown, with a total CFU average of 48.09 ± 7.22, leading to an OD600[0.1]-CFU correlation of 4.81 x 107 ± 7.22 x 106. In table 4 the counted colonies for LB are shown, with a total CFU average of 13.33 ± 8.04, leading to an OD600[0.1]-CFU
correlation of 1.333 x 108 ± 8.04 x 107. Biological replicate 1 2 Technical replicate 1.1 1.2 2.1 2.2 CFUs TMTC TMTC 44 50 58 65 38 48 62 43 52 56 Total average 51.6 ± 8.69 OD600[0.1]-CFU 5.16 x 107 ± 8.69 x 106
Table 2. OD600[0.1]-CFU correlation for B. ovatus Shown are the CFUs and the total average with the standard deviation
of the petri dishes cultures of BO. From each bacterial strain two biological replicates were grown, and from each biological replicate from two serial dilutions were made. They were plated out in triplicate and grown under anaerobic conditions for 48 hours at 37°C. After multiplying the total average CFUs by the final dilution factor, the number of CFUs/mL for 0.1 OD600
Table 3. OD600[0.1]-CFU correlation for E. coli. Shown are the CFUs and the total average with the standard
deviation of the petri dishes cultures with EC. From each bacterial strain two biological replicates were grown, and from each biological replicate from two serial dilutions were made. They were plated out in triplicate and grown under anaerobic conditions for 48 hours at 37°C. After multiplying the total average CFUs by the final dilution factor, the number of CFUs/mL for 0.1 OD600 was measured.
Biological replicate 1 2 Technical replicate 1.1 1.2 2.1 2.2 CFUs TMTC 42 40 43 57 59 36 54 50 51 48 49 Total average 48.09 ± 7.22 OD600[0.1]-CFU 4.81 x 107 ± 7.22 x 106
Table 4. OD600[0.1]-CFU correlation for L. bacterium. Shown are the CFUs and the total average with the
standard deviation of the petri dishes cultures with LB. From each bacterial strain two biological replicates were grown, and from each biological replicate from two serial dilutions were made. They were plated out in triplicate and grown under anaerobic conditions for 48 hours at 37°C. After multiplying the total average CFUs by the final dilution factor, the number of CFUs/mL for 0.1 OD600 was measured.
Biological replicate 1 2 Technical replicate 1.1 1.2 2.1 2.2 CFUs 25 3 20 4 10 13 9 5 25 23 11 12 Total average 13.33 ± 8.04 OD600[0.1]-CFU 1.333 x 108 ± 8.04 x 107
Metabolite production
The metabolites produced by the cultures during the 24 hours of incubation were analyzed via HLPC to assess the metabolic interaction between BO, EC and LB. These metabolites were butyrate, acetate and lactate. The difference between the metabolite concentration of the co-cultures and the mean values of concentration of the monoco-cultures were statistically analyzed using a two-sample t-test (p-value < 0.05). The concentration of glucose, the common nutrient for all metabolism pathways of the bacterial strains, was also examined. Some bacterial consortia had a too small amount of culture to be examined with HLPC. This called for the 4-hour time point of the monoculture with BO and the triculture of BO, EC and LB.
In figure 4 the concentration of glucose in all examined cultures is shown for the total incubation of 24 hours. Glucose is utilized by all bacterial strains for their metabolism
pathways. There is significant decrease of glucose concentration in the control group from 104.38 mM to 81.00 mM in the first 2 hours. The glucose level remains this low for the total duration of 24 hours. In all cultures there is a significant decrease in glucose level after 2 hours, all remaining at 63 – 68 mM for 24 hours expect for the monoculture of BO and the coculture of LB and EC. BO increases to a concentration of 80.54 mM after 8 hours and reaches its maximum concentration of 81.50 mM after 24 hours. The coculture of LB and EC
60 65 70 75 80 85 90 95 100 105 0 4 8 12 16 20 24 Co nc en tr at io n (m M )
Incubation time (hours)
Glucose
BO EC LB BO + EC BO + LB LB + EC LB + EC + BO controlFigure 4. Production levels of glucose of all examined cultures. B. ovatus (BO), E. coli (EC) and L. bacterium (LB) were
cultured in different bacterial consortia. The mono-, co- and tricultures were inoculated at an OD600 of 0.1 and incubated for
24 hours at 37°C under anaerobic conditions. The metabolite production was measured after 2, 4, 8 and 24 hours using HLPC. BO – blue, EC – orange, LB – grey, BO + EC – yellow, BO + LB – light blue, LB + EC – green, LB + EC + BO – dark blue, control – brown. All cultures show a similar glucose concentration level during the 24 hours, except for the coculture of LB and EC (outlier of 79.98 mM at 4 hours) and the monoculture of BO. BO increases to 80.54 mM at 8 hours and to 81.50 mM at 24 hours. The control group decreases to a concentration of 81.00 mM and remains at this level for 24 hours.
shows an outlier of 79.98 mM at the 4-hour time point. After 8 hours it decreases to a concentration of 66.79 mM and reaches a total concentration of 67.07 mM after 24 hours.
Figure 5 shows the highest production of butyrate in the monoculture of LB, with a total concentration of 59.51 mM in 24 hours. After 4 hours, the cocultures of LB and EC (26.16 mM) and LB and BO (22.88 mM) show a higher butyrate concentration than the monocultures of EC and BO, but between 8 to 24 hours the cocultures of LB and EC and LB and BO show the same butyrate production as the monocultures. After 24 hours the cocultures with LB (LB + EC, LB +BO) show a lower production of butyrate (24.20 mM and 24.78 mM) than the monoculture of LB (59.51 mM). The concentration of butyrate remains low in the triculture of BO, EC and LB, reaching a maximum concentration of 21.48 mM after 24 hours.
There was a significant decrease of butyrate concentration in the coculture of BO and LB in comparison to the mean concentration level of the monocultures of BO and LB (t-test, p < 0.05) (fig. 6). There was no significant difference in butyrate concentration in the other cultures compared to the butyrate concentration of their monocultures (t-test, p > 0.05).
0 10 20 30 40 50 60 70 0 4 8 12 16 20 24 Co nc en tr at io n (m M )
Incubation time (hours)
Butyrate
BO EC LB BO + EC BO + LB LB + EC LB + EC + BO controlFigure 5. Production levels of butyrate of all examined cultures. B. ovatus (BO), E. coli (EC) and L. bacterium (LB) were
cultured in different bacterial consortia. The mono-, co- and tricultures were inoculated at an OD600 of 0.1 and incubated for
24 hours at 37°C under anaerobic conditions. The metabolite production was measured after 2, 4, 8 and 24 hours using HLPC. BO – blue, EC – orange, LB – grey, BO + EC – yellow, BO + LB – light blue, LB + EC – green, LB + EC + BO – dark blue, control – brown. All cultures show a similar butyrate concentration level during the 24 hours, except for the monoculture of LB. LB reaches a maximum concentration of 59.51 mM after 24 hours. The cocultures with LB (LB + EC, BO + LB) show a
As shown in figure 7 the highest yield of concentration of acetate can be seen in the monoculture of BO, reaching a maximum concentration of 384.67 mM after 24 hours of incubation. The monoculture of EC and the coculture of BO and EC show a slightly lower production rate of acetate, reaching a maximum after 24 hours of 314.96 mM and 319.09 mM, respectively. The monoculture of LB shows the lowest production of acetate, stagnating around 200 mM for the entire incubation time (2 – 24 hours). The coculture of LB and EC shows an outlier of 305.57 mM at the 4-hour time-point and decreases to 275.35 mM at the 8-hour timepoint. The coculture of LB and EC reaches a maximum acetate concentration of 301.21 mM after 24 hours of incubation. The coculture of LB and BO reaches a maximum concentration of 307.36 mM after 24 hours. The triculture of BO, EC and LB has a similar growth curve as the coculture of LB and BO and reaches a maximum concentration of 306.81 mM after 24 hours. The control group decreases to a concentration of 213.54 mM at the 2-hour time-point and stagnates around this level for 24 hours.
There was a significant decrease of acetate concentration in the coculture of EC and LB in comparison to the mean concentration level of the monocultures of EC and LB (t-test, p Figure 6. Box-plot distribution of butyrate concentration. A two-sample t-test (p = 0.05) was performed to analyze the
difference in butyrate concentration between the cocultures of B. ovatus (BO), E. coli (EC) and L. bacterium (LB) (grey) and the mean butyrate concentration of the monocultures (white). There is a significant difference between the coculture of BO + LB and the monocultures of BO and LB (p = 0.026). There is no significant difference between the coculture of EC + LB and the monocultures of EC and LB (p > 0.05). There is no significant difference between the coculture of BO + EC and the monocultures of BO and EC (p > 0.05). There is no significant difference between the coculture of BO + EC + LB and the monocultures of BO, EC and LB (p > 0.05).
< 0.05) (fig. 8). There was no significant difference in butyrate concentration in the other cultures compared to the butyrate concentration of their monocultures (t-test, p > 0.05).
170 220 270 320 370 0 4 8 12 16 20 24 Co nc en tr at io n (m M )
Incubation time (hours)
Acetate
BO EC LB BO + EC BO + LB LB + EC LB + EC + BO controlFigure 7. Production levels of acetate of all examined cultures. B. ovatus (BO), E. coli (EC) and L. bacterium (LB) were
cultured in different bacterial consortia. The mono-, co- and tricultures were inoculated at an OD600 of 0.1 and incubated for
24 hours at 37°C under anaerobic conditions. The metabolite production was measured after 2, 4, 8 and 24 hours using HLPC. BO – blue, EC – orange, LB – grey, BO + EC – yellow, BO + LB – light blue, LB + EC – green, LB + EC + BO – dark blue, control – brown. The monoculture of BO shows the highest acetate concentration during the 24 hours, reaching a maximum concentration of 384.67 after 24 hours. The monoculture of LB shows the lowest acetate concentration, stagnating around 200 mM during the 24 hours. All cocultures of LB show a lower concentration than BO, EC and BO + EC, except for the coculture of LB and EC (outlier of 305.57 mM at 4 hours). The concentration of the control group decreases to 213.54 mM after 2 hours and remains at this level during the 24 hours.
The highest increase in lactate concentration can be seen in the monoculture of LB, reaching a maximum concentration of 125.43 mM after 24 hours (fig. 9). The rest of the cultures have a similar growth curve, all reaching a maximum lactate concentration between 40 – 50 mM. The coculture of LB and EC shows an outlier of 55.66 mM at the 4-hour time-point. The coculture of BO and LB shows an outlier of 48.77 mM at the 4-hour time-point. The lactate concentration in the control has an increase of 1.75 mM at the 4-hour time-point and stays at 0.00 mM for the remaining 24 hours.
There was a significant decrease of lactate concentration in the coculture of BO and LB in comparison to the mean concentration level of the monocultures of BO and LB (t-test, p < 0.05) (fig. 10). There was also a significant decrease in lactate concentration in the triculture of BO, EC and LB in comparison to the mean concentration level of the
monocultures of BO, EC and LB (t-test, p < 0.05). There was no significant difference in butyrate concentration in the other cultures compared to the butyrate concentration of their monocultures (t-test, p > 0.05).
Figure 8. Box-plot distribution of acetate concentration. A two-sample t-test (p = 0.05) was performed to analyze the
difference in butyrate concentration between the cocultures of B. ovatus (BO), E. coli (EC) and L. bacterium (LB) (grey) and the mean butyrate concentration of the monocultures (white). There is a significant difference between the coculture of EC + LB and the monocultures of EC and LB (p = 0.045). There is no significant difference between the coculture of BO + LB and the monocultures of BO and LB (p > 0.05). There is no significant difference between the coculture of BO + EC and the monocultures of BO and EC (p > 0.05). There is no significant difference between the coculture of BO + EC + LB and the monocultures of BO, EC and LB (p > 0.05).
0 20 40 60 80 100 120 140 0 4 8 12 16 20 24 Co nc en tr at io n (m M )
Incubation time (hours)
Lactate
BO EC LB BO + EC BO + LB LB + EC LB + EC + BO controlFigure 9. Production levels of lactate of all examined cultures. B. ovatus (BO), E. coli (EC) and L. bacterium (LB) were cultured
in different bacterial consortia. The mono-, co- and tricultures were inoculated at an OD600 of 0.1 and incubated for 24 hours at 37°C
under anaerobic conditions. The metabolite production was measured after 2, 4, 8 and 24 hours using HLPC. BO – blue, EC – orange, LB – grey, BO + EC – yellow, BO + LB – light blue, LB + EC – green, LB + EC + BO – dark blue, control – brown. The monoculture of LB shows the highest lactate concentration during the 24 hours, reaching a maximum concentration of 125.43 mM. In the rest of the cocultures there is a similar lactate production, with a maximum concentration range of 40 – 50 mM after 24 hours. There is an outlier at the 4-hour time-point in the coculture of BO and LB (48.77 mM) and in the control group (1.75 mM). The concentration of lactate remains at 0.00 mM for 24 hours.
Figure 10. Box-plot distribution of lactate concentration. A two-sample t-test (p = 0.05) was performed to analyze the difference
in butyrate concentration between the cocultures of B. ovatus (BO), E. coli (EC) and L. bacterium (LB) (grey) and the mean butyrate concentration of the monocultures (white). There is a significant difference between the coculture of BO + LB and the monocultures of BO and LB (p = 0.026). There is a significant difference between the triculture of BO + EC + LB and the monocultures of BO,
4. Discussion
All cultures of BO, EC and LB showed signs of exponential growth in the first 4 hours of incubation. With regards to the monocultures, the lowest increase of growth was measured in LB throughout the total 24 hours of incubation. EC had the highest increase of growth of the monocultures in the first 8 hours, but EC and BO stagnated around the same OD600 of 0.42 after 24 hours. The coculture of BO and EC had a similar course of growth as to the
monocultures of BO and EC. This means that BO and EC show similarity in growth and do not negatively affect each other’s growth. The growth in the cocultures of LB and BO is higher than that of the monocultures of LB and BO. This also accounts for the coculture of LB and EC in comparison to the monocultures of LB and EC. The coculture of LB and BO has a higher increase of growth in comparison to the coculture of LB and EC. The triculture of BO, EC and LB had the highest increase of growth during the first 16 hours in comparison to when the bacteria are grown in mono- and cocultures. This could mean that BO, EC and LB all benefit from being cocultured together, in a way that the total growth of the bacterial consortium increases. These results do not support the hypothesis that because BO, EC and LB all use the same products, there will be signs of competition and an earlier stagnation of growth. A possible explanation could be that because of cross-feeding these three bacterial strains can benefit from each other and utilize each other’s metabolites for their own bacterial cell growth. Since the spectrophotometer is not fit to determine the ratio between the different bacterial cells, it is not possible to draw a conclusion about which specific bacterium benefits the most from being cocultured with another bacterium. The OD-CFU correlation could be used to estimate the ratio between the bacterial cells of BO, EC and LB. The measured OD600[0.1]-CFU correlations for BO, EC and LB in this study were 5.16 x 107 ± 8.69 x 106, 4.81 x 107 ± 7.22 x 106 and 1.333 x 108 ± 8.04 x 107, respectively. However, the standard deviations for the OD-CFU correlations for BO, EC and LB were too high to make an accurate assumption about the ratio of bacterial cells in the examined cultures.
In the monoculture of LB the highest concentration of butyrate was measured. All other cultures showed a similar production rate of butyrate during the 24 hours. There was no significant difference found in the butyrate concentration of the coculture of EC and LB in comparison to the mean concentration of the monocultures EC and LB. These results do not support the hypothesis that because LB utilizes acetate for its butyrate production, LB would benefit from co-culturing with EC and that there would be a higher production of butyrate. There was a significant decrease found in the butyrate concentration of the co-culture of BO and LB in comparison to the mean concentration of the monocultures of BO and LB.
Co-culturing BO and LB leads to a lower production of butyrate. These results are in line with the hypothesis that BO would negatively influence the butyrate production of LB. The production of butyrate remains low in the triculture of BO, EC and LB. The earlier given explanation for this was that because of the competitive relationship between BO, EC and LB, there would be a decrease in metabolism. This study however showed that this competitive relationship did not apply, and in fact showed that BO, EC and LB benefitted each other’s growth in the triculture. The highest concentrations of acetate were found in the monocultures of BO and EC and in the coculture of these bacteria. This is consistent with the hypothesis that acetate is mainly produced by EC and BO. The concentration of acetate was higher in the monoculture of BO than in the monoculture of EC. BO thus contributes to a higher acetate production than EC. This could explain the fact that the coculture of BO and LB has a higher increase of growth in comparison to the coculture of EC and LB, possibly because there is a higher concentration of acetate available for LB to utilize for the formation of butyrate. However, the significant decrease of butyrate in the coculture of BO and LB contradicts this theory. A possible explanation would be that BO utilizes less nutrients of the medium than EC, which makes a coculture of EC and LB more competitive than a coculture of BO and LB. The results of the HPLC-analysis of glucose support this. The highest concentration of glucose, next to the control group, is found in the monoculture of BO. This means that BO does not use a high amount of glucose for its own bacterial cell growth. The concentration of glucose in the monoculture of EC is similar to the monoculture of LB. This could explain why the production of butyrate remains low in the coculture of EC and LB, because there is a
competitive relationship between EC and LB. LB is therefore not able to maintain its butyrate production when cultured with EC. This and the fact that BO negatively influences the
butyrate production of LB, give an explanation for the low concentration of butyrate in the triculture of BO, EC and LB, despite the total increase in growth of the bacterial consortium. The lowest concentration of acetate was found in the monoculture of LB. This is due to the fact that LB utilizes acetate in its metabolic pathway to produce its main product butyrate. Because of this cross-feeding, the production of acetate is also lower in cocultures with LB. There was a significant difference found in acetate concentration of the coculture of EC and LB in comparison to the mean concentration of the monocultures of EC and LB. It is
important to take into account that there was an outlier detected in the acetate production of the coculture of LB and EC, which makes this significant difference unreliable. A similar outlier was detected in the coculture of LB and EC in the glucose concentration at the same
control group, assuming there are no bacteria present who could utilize the glucose for their metabolism. A possible explanation would be that the control group could be contaminated. This explanation is however unlikely, since there was no growth detected in the OD600-analysis in the control group for 24 hours. The HPLC-OD600-analysis did not measure metabolite production (butyrate, acetate, lactate) in the control group for the total incubation of 24 hours. Therefore, there was no active metabolism present in the control group. It can be argued that HPLC could be an unreliable method to detect metabolites in bacterial cultures, but a higher number of experimental runs is required to support this statement. Due to the fact that this HPLC-analysis has been run only once, no conclusions can be made about the reliability of the HPLC-method.
Contrary to the hypothesis that lactate is mainly produced by BO, there is a large increase in the lactate concentration in the monoculture of LB instead of the monoculture with BO. It was to be expected that the highest yield of lactate concentration would be measured in the
monoculture of BO, but the lactate concentration remained at a low level, similar to the other cultures. There was a significant difference in lactate concentration of the cocultures of BO and LB in comparison to the mean concentration of the monocultures BO and LB. This significant difference was also found between the triculture of BO, EC and LB and the mean concentration of the monocultures. When LB is cocultured with BO or with BO and EC in a triculture, there is a significant decrease in lactate production. A possible explanation for the high levels of lactate in the monoculture of LB could be that lactate is produced by BPB, especially when they undergo rapid cell division under anaerobic conditions.27 This was however not the case in the monoculture of LB, since the lowest increase of growth was measured in LB throughout the total 24 hours of incubation. BPB, like LB, are also able to utilize lactate for the formation of butyrate.27 One would expect that because of the
production and utilization of lactate in LB, the net concentration of lactate would remain at a low level. However, in the monoculture of LB there was a high yield of lactate concentration for the total duration of 24 hours. Therefore, the results from this study do not agree with the literature.
Summarizing, co-culturing BO, EC and LB had the most beneficial effect on the total growth of the bacterial consortium. The concentration of butyrate however remained low in the triculture. This study showed evidence of the utilization of acetate by LB for the formation of butyrate, as the concentration of butyrate was high and of acetate low in the monoculture of LB. Contrary to the hypothesis, EC did not have a positive effect on the butyrate production
of LB. According to the previous findings of Larsen et al. (2019), BO indeed had a negative effect on the butyrate production of LB.
For a follow-up research a higher number of biological and technical replicates would be necessary to reduce the standard deviation in order to measure a more accurate OD-CFU correlation. The use of flow cytometric identification could also be a solution to distinguish between species of bacteria, in order to make an accurate estimation of the ratio between the different bacterial species in the cocultures.28 This could provide more data to give a more accurate conclusion on how BO, EC and LB interact each other’s bacterial cell growth.
The mini microbiome model of BO, EC and LB gives us more understanding about the complex metabolic interaction between bacteria which are representative of the human gut microbiome. It especially provides more knowledge about the effect of BO and EC on the butyrate production by the butyrate-producing LB. Previous studies have shown that butyrate can be of therapeutical relevance in the pathogenesis of depression. The results of this study give us more information on which ratio of BO, EC and LB in the human gut could lead to the highest production of butyrate. Restoration of this balance between the human gut bacteria could lead to a higher endogenous production of butyrate and could play a key role in the treatment of depression. Developing colon butyrate producing bacteria as probiotics, could facilitate this endogenous increase of butyrate production. However, according to Vacca et al. (2020) an increase in species of the Lachnospiraceae family is associated with aging.20
Butyrate producers from this family would not be suitable for the use of probiotic treatment. Other common butyrate producing bacteria in the colon belong to the Butyricicoccus genus from the Clostridiaceae family.29 In another study, the safety of Butyricicoccocus
pullicaecorum as a probiotic was assessed in healthy individuals. The study showed that the probiotic intervention did not cause disruptive alterations in the composition or metabolic activity of health-associated microbiota.30 This study however did not assess the effect of this probiotic on the long-term run in these individuals.
Not only could an increase of the endogenous concentration of butyrate in the human gut serve as a possible treatment, the pharmaceutical supplementation of butyrate could also be effective. This strategy is more efficient to increase the butyrate concentration, instead of indirectly targeting the butyrate production of bacteria in the gut. It is also advantageous that there would be less interference with the microbiota of the patients. Through oral
supplementation, butyrate could be absorbed via the gastro-intestinal route, enter the blood circulation and cross the blood-brain barrier. Butyrate could regulate neuroinflammatory
of depression, like the prefrontal cortex and the hippocampus. It could relieve depression-like symptoms and improve overall quality of life in patients who suffer from depression.
Future research should study the therapeutic effects of oral supplementation of butyrate in patients with MDD. Given the disadvantages of the current existing pharmaceutical therapies for MDD and the global impact of this disease, it is of social relevance to research potential new treatments of depression.
5. References
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6. Appendix
LB agar plates protocol
1. Weigh out the following components in a 500 mL bottle
reagent mass / volume Tryptone 5 gr
NaCl 5 gr Yeast Extract 2.5 gr Distilled water 500 mL
Agar 7.5 gr
2. Mix the reagents well
3. Autoclave for 20 minutes at 120 °C
4. Leave the solution to cool to approximately 55 °C
5. Prepare petri dishes in a sterile environment
6. Pour around 10-20 mL solution per plate
7. Leave the plates to set and cool
8. Stack the plates upside down and store in the fridge at 4 °C
Blood agar plates protocol
1. Weigh out the following components in a 1L bottle
reagent mass / volume BHI 37 gr
Distilled water 950 mL Agar 15 gr
2. Heat to boiling to dissolve the medium completely (use a stirring bean for this) 3. Autoclave for 15 minutes at 120 °C
4. Leave the solution to cool to approximately 55 °C
5. Add, in a sterile environment, 25 mL defibrinated sheep blood 6. Mix well using the mixing bean on a magnetic stirring plate 7. Prepare petri dishes in a sterile environment
8. Pour around 10-20 mL solution per plate
9. Leave the plates to set and cool
ssGut-medium
1. Add the following ingredients to a 1L flask reagent mass / volume Tryptone 2 gr Peptone 2 gr Yeast extract 1 gr Meat extract 5 gr MgSO4-7 H2O 0.0025 gr NaHCO3 0.4 gr NaCl2 0.08 gr K2HPO4 71.7 gr KH2PO4 28.3 gr CaCl2 0.008 gr FeSO4 0.75 mg Acetic acid 1.7 mL Propionic acid 2 mL Butyric acid 2 mL L-cysteine hydrochloride 2 gr
Trace element solution 10 mL 2. Add the following sugars
Soluble starch mass / volume
D-glucose 0.4 gr D-cellobiose 1 gr
Maltose 1 gr Fructose 1 gr
3. Autoclave for 20 minutes at 120 °C
4. Let the medium cool down to room temperature
5. Add the following chemicals to the medium
component mass / volume Vitamin solution 10 mL
Vitamin K solution 1 mL Tween80 0.7 mL Histidine-Hematin solution 1 mL
6. To ensure that all the dissolved oxygen is out of the medium, put the bottle with ssGut-medium in the anaerobic cabinet for at least 24 hours
BHI-(S) medium
1. Mix the following components in a 1L flask
reagent mass / volume Brain heart infusion (BHI) broth 37 gr
Distilled water 1 L Yeast Extract 2.5 gr Resazurin solution
(25 mg / 100 mL distilled water) 4 mL
2. Autoclave for 20 minutes at 120 °C
3. Let the medium cool down to room temperature
4. Add the following sterile components
component amount L-Cysteine 0.5 gr Hemin Solution 10 mL
Vitamin K1 0.2 mL
5. Store in the fridge at 4 °C
