The Effect of Six Antimicrobial TNO Compounds on Germination and Outgrowth of Bacillus subtilis Spores.

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Germination and Outgrowth of Bacillus subtilis Spores.

Bachelor thesis Biomedical Sciences by Esther de Boer

Student number: 10324429 Supervisor: Soraya Omardien MSc Second assessor: Dr. S. A. J. Zaat

Research group: Molecular Biology and Microbial Food Safety Submitted on July 1, 2015

Abstract

Certain Gram-‐positive bacteria can form spores, which are metabolically dormant cell types that are resistant to harsh conditions. These characteristics also cause resistance of the spores to most of the currently used food treatments, which means spores can form a serious threat to food safety. Even though spores are non-‐pathogenic and incapable of causing food spoilage, they regain these possibilities when they become vegetative cells again. At this moment, there are no compounds available that can inhibit either germination or outgrowth; the two processes a spore goes through to become a vegetative cell again. In this research study, six antimicrobial compounds provided by TNO were tested for their effects on spore germination and outgrowth. Firstly, a MTT assay was performed to determine whether the TNO compounds had an effect on the oxidative metabolism. Subsequently, time-‐kill assays were performed to determine whether the compounds are bacteriostatic or bactericidal. To confirm whether the compounds inhibited germination or outgrowth, the spores were visualized using microscopy, after treatment with the compounds. Propidium iodide staining of the spores was also performed, to determine whether the compounds cause membrane damage, which was visualized using fluorescent microscopy. The MTT assay showed that all the TNO compounds inhibit the oxidative metabolism of the spores. The time-‐kill assay showed that the six TNO compounds are bactericidal. Lastly the results obtained from the microscopy showed that none of the TNO compounds inhibits germination, but that they all inhibit outgrowth. In TNO 1 and TNO6 this went along with a loss of membrane

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Introduction

Food safety in the Netherlands has improved in the past few decades, but foodborne infections remain a persistent issue, with an estimate of 700,000 illnesses a year in the Netherlands alone (van Kreijl, et al., 2006). In some industrialized countries, the amount of food related illnesses is even rising (Havelaar, Brul, de Jong, de Jonge, Zwietering, & ter Kuile, 2010), indicating the necessity of food safety research. Even though modern techniques have ensured the improvement of food safety, pathogens are sometimes able to survive these methods.

Some Gram-‐positive bacteria are capable of forming spores. A spore is a state of a bacterial cell in which it is resistant to harsh circumstances. Therefore, spores are harder to eliminate from food than other bacterial cell types and thus pose a novel threat to food safety (Tewari & Abdullah, 2014). Bacillus subtilis is an example of such a spore forming Gram-‐positive bacterium that is capable of sporulating and can cause food spoilage. However, unlike other Gram-‐positive spore forming bacteria such as Bacillus cereus, Bacillus anthracis or Clostridium difficile, B. subtilis is a non-‐pathogenic bacterium. It is therefore used as a model organism for spore forming Gram-‐positive bacteria (Stein, 2005) (Sietske & Diderichsen, 1991).

Spores are metabolically dormant, which suggests that there is no detectable metabolism and hardly any enzyme action (Setlow, 2006; Setlow, 2003; Cortezzo, Setlow, & Setlow, 2004). This means that a spore can survive long periods with little to no nutrients. An inner membrane, germ cell wall, cortex, outer membrane and spore coat surround the core of a Bacillus subtilis spore, which contains the DNA, RNA and ribosomes, but mostly consists of water and dipicolinic acid (DPA) (Figure 1). The (variable) water content of the core causes heat resistance in spores, while the spore coat makes them resistant to toxic chemicals. Spores are also resistant against radiation, though this mechanism is poorly understood. These resistant characteristics make spores a problem for the food industry, as the usual preservation methods, like heat treatment and food preservatives, do not manage to eliminate spores. The spores itself are never toxic or pathogenic, but in the right circumstances they can germinate and grow out, becoming vegetative cells that are able to produce toxins, such as cells of Bacillus cereus

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and Clostridium difficile.

Germination is the first step spores take to become a vegetative cell again (Setlow, 2003). Germination is usually induced by the presence of certain nutrients, called germinants. The inner membrane of the B. subtilis spores contains three germinant receptors: GerA, responding to L-‐alanine and L-‐valine, and GerB and GerK, responding to L-‐asparagine, D-‐glucose, D-‐fructose and K+ _{(Luu,
Cruz-‐Mora,
Setlow,
Feeherry,
Doona,
&} Setlow, 2015). Presence of all these germinants is necessary to initiate germination, however the exact mechanism in which germination is activated is not completely understood. After activation, the spore goes through germination stage I, characterized by the release of Ca2+_{-‐DPA
and
core
hydration,
and
stage
II,
in
which
the
cortex
is} hydrolysed and resistance is lost, before proceeding to outgrowth. Outgrowth requires ATP and is the last stage of a spore turning into a vegetative cell: in this stage the spore gets rid of the spore coats and the (oxidative) metabolism is restored as well (Keijser, et al., 2007). Current food treatments, like heat treatments, seem efficient in killing spores or preventing them from germinating and growing out (Setlow, Loshon, Genest, Cowan, Setlow, & Setlow, 2002) (Setlow P. , 2000). In treated food, changing circumstances in food transport or storage, for example, can still lead to spore germination and outgrowth.

Figure 1: Different layers of the B. subtilis spore. From outside to inside: spore coats, outer membrane, cortex, germ cell wall, inner membrane and core.

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This indicates the need for new methods or antimicrobial compounds to prevent germination or outgrowth.

Figure 2: Spore outgrowth and germination stages. After activation by germinants binding to germinant receptors in the inner membrane of the spore, the spore enters germination stage I. In this stage, among other things, Ca2+_{-‐DPA
is
released
and
the
core
is
partially
hydrated.
In
stage
II
the
cortex
is
lost
and
the
core}

expanses gets hydrated further. After germination the spore enters outgrowth, where the metabolism is completely restored and

Previously, a TNO compound library, consisting of 512 compounds, was screened for antimicrobial activity against Bacillus subtilis spores. The effect of the TNO compounds on growth of the bacteria was determined, based on optical density (OD) measurements over time. Six compounds with antimicrobial activity showed potential and were selected for further analysis. The chemical structure and minimal inhibitory concentration (MIC) of these compounds is shown in Appendix D. The effect of the compounds on the oxidative metabolism was studied using a 3-‐(4,5-‐dimethylthiazol-‐2-‐ yl)-‐2,5-‐diphenyltetrazolium bromide (MTT) assay and to determine whether the TNO compounds are bacteriostatic or bactericidal, time-‐kill assays were performed. Fluorescence microscopy was performed to determine whether germination or

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outgrowth is inhibited and the effect of the TNO compounds on the membranes using propidium iodide. Based on the first screening of these compounds it is expected that they will have an inhibiting effect on either germination or outgrowth. Which of the two is affected can however not be said based on the findings of the previous group.

Taking the outcomes of the experiments into account, the effect of the TNO compounds on germination and outgrowth in Bacillus subtilis is described: whether they are bactericidal or bacteriostatic and how they achieve their effects on Bacillus subtilis spores.

Materials and methods

Preparation of Bacillus subtilis spores

For the preparation of spores, Bacillus subtilis strain 168 was used. The method was followed as described by Abhyankar et al. (2011) (Appendix A). In short, one B. subtilis colony was inoculated in 5 ml tryptic soy broth (TSB) and incubated at 37° C, 200 rpm until an OD600 of 0.3-‐0.4 was reached. This step ensured that the spores used were in the exponential phase. A serial dilution of the spores in MOPS medium (see Appendix B for composition) was made from 10-‐1_{to
10}-‐7 _{and
incubated
at
37°
C,
200
rpm,
to
condition} the cells to the medium used for spore production. A dilution with an OD600 of 0.3-‐0.4 was selected and 1 ml of this dilution was inoculated in 20 ml pre-‐warmed MOPS medium until an OD600 of 0.3-‐0.4 was reached again. From this culture, 2.5 ml was inoculated in 250 ml MOPS medium and incubated for 96 hours at 37° C, 200 rpm. The spores were washed once with 1% Tween to have a final concentration of 0.01% Tween, after which the spores were pelleted at 5000 rpm for 15 min at 4° C and the supernatant was discarded. Afterwards, four washes were done with sterile MilliQ water in the same manner to remove vegetative cells and debris. An additional purification step was performed to remove remaining vegetative cells, using Histodenz (Appendix C). The spores were resuspended in 750 µl 20% Histodenz and then added to 800 µl 50% Histodenz. The spores were pelleted at 15000 rpm for 1 hour at 4° C and the supernatant was discarded. The spores were stored at 4° C for four weeks.

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Determining the oxidative metabolism of spores after treatment with the TNO compounds

The effect of the TNO compounds on the oxidative metabolism of these spores was tested using 3-‐(4,5-‐dimethylthiazol-‐2-‐yl)-‐2,5-‐diphenyltetrazolium bromide (MTT) as a reagent (Appendix E). Two ml spores were heat-‐activated at 70° C for 30 minutes. The spores (final OD600 of 0.2) were added to TSB, buffered with MOPS (pH 7.4), containing the compounds at the minimal inhibitory concentration (MIC) (Appendix D). Spores (final OD600 of 0.2) inoculated into MOPS buffer (pH 7.4) were used as a negative control for germination. Spores inoculated in TSB (final OD600 of 0.2) without TNO compounds were used as a positive control for germination and outgrowth. Each experiment was performed with four experimental repeats and with one reagent blank.

At 0 min, 60 min and 120 min, four aliquots of 100 µl were taken for each TNO compound, the reagent blanks and the controls and pipetted on a 96-‐wells plate. These aliquots were diluted 2x and 4x in inhibition mixture, containing D-‐alanine, D-‐histidine, MOPS buffer and MilliQ (Appendix E). As L-‐alanine is a germinant for B. subtilis spores, D-‐ alanine is supposed to inhibit germination. To each well 50 µl 1 mg/ml MTT was added. The 96-‐wells plates were incubated for 60 min at 37° C, 200 rpm. The dilution in inhibition mixture was done to prevent further germination after the treatment period. After the incubation with MTT, 100 µl DMSO was added to each well to dissolve the formazan crystals. The plates were incubated for 5 min at 37° C, 200 rpm. The OD570 was measured using a microtiter plate reader.

Time-‐kill assay of B. subtilis spores after exposure to the TNO compounds

Time-‐kill assays were performed for B. subtilis after treating the spores with the different TNO compounds to determine whether the compounds are bacteriostatic or bactericidal. Spores were heat-‐activated for 30 min at 70° C and inoculated into MOPS buffered TSB (pH 7.4) to have a final OD600 of 0.2. The TNO compounds were added to the medium at their MICs. Spores inoculated in buffered TSB without TNO compounds were used as a control. The mixtures were incubated at 37° C, 200 rpm and 50 µl aliquots were taken at

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0 minutes (before addition of the compounds) and at 30, 60 and 120 minutes after addition of the compounds. The samples were diluted in inhibition mixture from 10-‐1_to 10-‐7 _{(Appendix
E).

From
each
dilution
an
aliquot
of
10
µl
was
taken
twice
and
spotted
on} a TSB plate. These plates were incubated at 37° C overnight. The next day a CFU count was performed. From these data killing curves were drawn. The counts were repeated at day 2 and 3 to validate the data.

Determination of the effect of the compounds on germination, outgrowth and membrane integrity

Fluorescence microscopy was used to determine how the TNO compounds effect germination and outgrowth of the B. subtilis spores. Propidium iodide was used as a staining to determine if of the TNO compounds cause a loss of membrane integrity, preventing outgrowth. Propidium iodide can not pass membranes of living cells and shows a peak in fluorescence when bound to nucleic acids (Krishan, 1975). Therefore, a peak in fluorescence indicates a loss of membrane integrity or a damaged membrane. The spores were incubated with TSB buffered with MOPS (pH 7.4) with a final OD600 of 0.2, after which the TNO compounds were added at their MICs. The samples were incubated for 120 min at 37° C, 200 rpm. After this incubation 0.75 µl 20 mM propidium iodide was added to each 500 µl sample and incubated in the dark at room temperature for 15 min. The samples were petted using centrifugation at 14000 rpm for 5 min. One µl of the pellet was spotted onto a 2% agarose pad (containing 2% agarose in MilliQ) and examined under the BX microscope. Propidium iodide has an excitation spectrum of 500-‐ 550 nm and maximum emission at 639 nm (Wolff, Chien, & van Winkle, 2000). Two UV-‐ filters were used: UV1 = 1, UV2 = 0.068 to take images. The images were analysed using ImageJ and cells were counted using the Cell Counter plugin. The images were divided into the following categories: phase bright spores, phase dark spores, cells in outgrowth, vegetative cells and elongated vegetative cells. This categorisation was based on images provided by Pandey, Ter Beek, Vischer, Smelt, Brul, & Manders, 2013. The images were analysed for a second time, categorizing the cells in propidium iodide positive and propidium iodide negative cells.

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Results

Oxidative metabolism was measured using MTT as a reagent and the amount of formazan formed was interpreted as an indication of the presence of oxidative metabolism. In outgrowth spores restore the oxidative metabolism and ATP is required: absence of oxidative metabolism would suggest that outgrowth is inhibited. The standard deviation graph in Figure 3 expresses the results as OD570. The graph showed an increase in formazan production for the positive control, which were spores incubated in TSB with AGFK, indicating that in the absence of the TNO compounds oxidative metabolism is reached in these test conditions. The graph showed a stable OD570 for the negative control, which where the spores incubated with only MOPS, indicating that no oxidative metabolism is present. The results obtained for the six TNO compounds showed no increase in OD570 after 120 minutes, suggesting that formazan was not produced and that oxidative metabolism did not take place. The absence of oxidative metabolism suggests that outgrowth is inhibited. The spores were also diluted in inhibition mixture, to prevent further germination of spores and to enable the measurement of the effects directly after the treatment period. The results suggested that there was no difference between diluted and undiluted samples; even though the absolute OD570-‐values were lower for the diluted samples, there is no difference in the relative values (see Appendix F for the standard deviation graphs of the diluted samples).

Colony forming units (CFU) were counted after the treatment at various time points to determine whether the TNO compounds have a bacteriostatic or bactericidal effect. Figure 4 shows the results of the CFU counts for the six TNO compounds and a blank (without antimicrobial compounds). No effect of the compounds was observed in the first hour, but after 120 minutes a 90% or stronger decline in CFU/ml was found. As these results showed that all six TNO compounds caused a decline in CFU/ml, these data suggest that the TNO compounds have a bactericidal effect rather than a bacteriostatic effect. It also appears that TNO1 is the most effective compound. This bactericidal effect was found when the spores were incubated with each TNO compound for 120 minutes.

The oxidative metabolism results suggested that germination or outgrowth was inhibited; therefor microscopy was employed to confirm these findings. Phase contrast

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microscopy was employed to determine if germination or outgrowth was inhibited. Propidium iodide staining was then performed to see if the membrane damage was the cause of the inhibition of outgrowth. In Figure 6 to Figure 15 the overlay images of the microscopy are shown, in which the phase contrast images and the fluorescence images are combined. Phase bright spores are spores did not germinate, while phase dark spores are germinated spores. Also vegetative cells are found in the images, which are cells that have gone through both germination and outgrowth. Figure 18 and Figure 17 show graphs for the different stages of germination and outgrowth and the propidium iodide staining have been quantified from the images (for the absolute cell counts see Table 1 and Table 2 in Appendix G). The image taken after incubation in H2O and the negative control (Figure 5 & Figure 6) both show phase bright spores, indicating that the experimental conditions do not promote spore germination and outgrowth. Figure 7 shows the positive control, for which vegetative cells are found, confirming that the culturing conditions are sufficient for spore germination and outgrowth.

The results after treatment with TNO1 shows that 95 per cent of the cells are phase dark spores, the remaining cells are phase bright spores (Figure 8 & Figure 18). This indicates that germination does take place, but outgrowth is inhibited. 91 per cent of the cells show a propidium iodide positive staining, which indicates loss of membrane integrity (Figure 17). For TNO6 a similar effect is found, but with only 65 per cent of the cells showing a propidium iodide positive staining (Figure 15 & Figure 17). The results for TNO4 also show that 91 per cent of the cells is a phase dark spore, but after this treatment the remaining cells are also phase dark spores, in which outgrowth is initiated, but not continued (Figure 13 & Figure 18). This suggests that TNO4 and TNO6 also do not affect germination and inhibit outgrowth, although with less or without effect on the membrane integrity.

TNO2 treated spores were in phase dark in 96 per cent of the cells, which suggest spore germination is not affected. Two per cent of the cells are phase dark spores that have lost their original shape, but seem unable to grow out into vegetative cells (Figure 10, Figure 9 & Figure 18). Germinated spores treated with TNO2 showed propidium iodide positive staining in 35 per cent of the cells (Figure 17). TNO3 treated spores are

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phase dark spores for 36 per cent and phase dark spores attempting to grow out in 59 per cent. Two per cent of the cells consists of elongated vegetative cells (Figure 18). The spores are propidium iodide negative for 88 per cent (Figure 12, Figure 11 & Figure 17). Most notable is the image for TNO5, which shows spores that have lost their original shape for 74 per cent (Figure 14 & Figure 18). The cells are all phase dark and some of them have already shed the spore coat (Figure 14 & Figure 16). The shed coats can be seen in the images as a small appendix to the spores. The remaining cells are phase bright or phase dark spores that have not proceed to outgrowth. There are no vegetative cells, which suggests that complete outgrowth is inhibited. The cells are propidium iodide positive for 96 per cent. These results together indicate a homogenous effect treatment TNO1, TNO2, TNO4 and TNO6, with more than 91 per cent of the cells in the same stage.

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Figure 4: Standard deviation curves of the time-‐kill assay Bacillus subtilis. The CFU count at t=0 min is used as a reference to determine the relative growth of B. subtilis after treatment with the different TNO compounds, showing the relative CFU count in % at a log2-‐scale.

-‐0,2 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8

0 min 60 min 120 min

O p ti ca l d en si ty a t 5 7 0 n m Time (min)

OD

570

: oxidative metabolism

MOPS only TSB only TNO1 TNO2 TNO3 TNO4 TNO5 TNO6

Figure 3: Standard deviation graph depicting the oxidative metabolism assay results for the six TNO compounds, the positive control with TSB only and the negative control with MOPS only. OD570 was measured

over time (t=0, 60 & 120 min) for all samples: this graph shows the average value of experimental repeats for each time point.

0,015625 0,0625 0,25 1 4 16 64 256 1024

0 min 30 min 60 min 120 min

Rel at ive CF U co u nt ( %) Time (min)

Time-‐kill assay

Blank TNO1 TNO2 TNO3 TNO4 TNO5 TNO6

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Figure 6: Fluorescence image of B. subtilis spores in 1x MOPS, stained with propidium iodide. The image shows phase bright spores.

Figure 5: Fluorescence image of B. subtilis spores in H2O, stained with propidium iodide.

The image shows phase bright spores.

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Figure 7: Fluorescence image of B. subtilis spores in TSB, stained with propidium iodide. The image shows elongated vegetative cells that have not divided yet.

Figure 8: Fluorescence image B. subtilis spores incubated with TNO1, stained with propidium iodide. The image shows fluorescent, phase dark spores.

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Figure 10: Fluorescence image of B. subtilis spores incubated with TNO2, stained with propidium iodide. The image shows phase dark spores with some fluorescence and also some odd shaped phase dark spores. These might be germinated cells attempting to grow out.

Figure 9: Close-‐up, obtained from a fluorescence image stained with propidium iodide, of two spores that have lost their original shapes under influence of TNO2.

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Figure 12: Fluorescence image of B. subtilis spores incubated with TNO3, stained with propidium iodide. The image shows phase dark spores and an elongated, non-‐dividing vegetative cell.

Figure 11: Close-‐up of B. subtilis spores incubated with TNO3 that have lost their original shape and (partly) shed their coats, obtained from a fluorescence image stained with propidium iodide.

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Figure 14: Fluorescence image of B. subtilis spores incubated with TNO5, stained with propidium iodide. The image shows phase dark spores that have lost their original shape, probably due to too much water uptake.

Figure 13: Fluorescence image of B. subtilis spores incubated with TNO4, stained with propidium iodide. The image shows dark phase spores with some fluorescence.

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Figure 15: Fluorescence image of B. subitlis spores incubated with TNO6, stained with propidium iodide. The image shows phase dark spores and some fluorescence.

Figure 16: Close up of three B. subtilis spores, incubated with TNO5 and stained with propidium iodide. This image shows three spores that have already shed their coat, but got stuck in this stage of outgrowth.

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Figure 18: Cell count of all the images of the fluorescence microscopy, sorted by TNO compound. In all the images cells were counted and categorised in different stages: phase bright spores, phase dark spores, cells in outgrowth, vegetative cells and elongated vegetative cells.

Figure 17: Graph showing the relative amount of PI positive cells in all the samples, calculated after cell count on all images was performed using the Cell Counter plugin in ImageJ.

0 10 20 30 40 50 60 70 80 90 100

TNO1 TNO2 TNO3 TNO4 TNO5 TNO6 TSB MOPS

R el at iv e am ou n t o f _ lu or esc en t c el ls in % Samples

Relative _luorescence

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Discussion

Six TNO compounds were tested for their effect on B. subtilis spore germination and outgrowth using an oxidative metabolism assay, killing curves based on CFU counts and fluorescence microscopy using a propidium iodide staining. The hypothesis was that the TNO compounds would inhibit either germination or outgrowth, but which stage would be affected was unknown.

Initially to determine whether the TNO compounds affect germination or outgrowth, the oxidative metabolism of the spores after treatment with the compounds was determined, using MTT as a reagent. MTT can be converted into purple formazan by bacteria using NAD(P)H-‐dependent oxidoreductases. As this conversion requires the presence of NAD(P)H, it gives an indication of the presence and activity of an oxidative metabolism. Given that formazan is a purple molecule, an OD570-‐measurement can be used to determine the amount of formazan formed and therefore the amount of oxidative metabolism present (Berridge, Herst, & Tan, 2005) (Twentyman & Luscombe, 1987). The results obtained from the oxidative metabolism assay showed no increase in OD570 for each of the six TNO compounds. This means that no formazan was formed and thus no oxidative metabolism was present. However, it is not possible to conclude about the effects of the TNO compounds on germination, based on these outcomes. It can be concluded that the TNO compounds all inhibit the oxidative metabolism.

To determine whether the compounds are bactericidal or bacteriostatic, a killing curve was conducted by counting the colony forming units. This count was performed after different incubation times with the six TNO compounds. The results showed that all six TNO compounds caused a decline in colony forming units, suggesting that the compounds are bactericidal. This bactericidal effect was found after 120 minutes of incubation with the TNO compounds. TNO1 seems to be the most effective in it’s bactericidal effect. For shorter incubation times, no consistent results were found. This suggests that it takes the TNO compounds at their MIC 120 minutes to be active and have an bactericidal effect.

To find out whether the TNO compounds affect germination and outgrowth and to distinguish between the effects of the different TNO compounds, microscopy was used to

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take images of the spores after exposure to the TNO compounds for 120 minutes. The results show that most of the spores are in the dark phase: this shows that germination is not affected, but that outgrowth is. The results obtained after treatment with TNO1, TNO4 or TNO6 showed that these TNO compounds completely inhibit outgrowth. This prevention of outgrowth might be due to the inhibitory effect these compounds have on the oxidative metabolism, which is needed for outgrowth.

The results obtained for TNO2, TNO3 and TNO5 showed that outgrowth is initiated but not completed. The results for TNO2 showed phase dark spores that have lost their original shape. This might imply that the spores attempting to grow out, but can not complete outgrowth. It might also mean that the spores are continuing water uptake after germination has completed, which distorts their shape. Similar results were obtained in the results for TNO3 and TNO5. However, in these results the phase dark spores have shed their coat as well and thus have proceeded to outgrowth, but seem unable to develop into a vegetative cell. It can be assumed that these two compounds target a different process than TNO2. The process inhibited would have to take place after shedding of the spore coat. It can be concluded that after treatment with TNO3 and TNO5 outgrowth is initiated, but not completed. Lastly, the results after treatment with TNO3 also show some vegetative cells. This might indicate two situations: either TNO3 does not have an effect on outgrowth at all, or it is a slower acting compound. It seems unlikely that TNO3 does not have an effect on outgrowth, as the killing curves and oxidative metabolism assay show opposing results. This suggests more strongly that TNO3 is a slow acting compound, which would allow for some spores to germinate and grow out before the compound starts working. Interestingly, the vegetative cells found after incubation with TNO3 do not divide. Whether this is due to the general habit of B. subtilis spores to take some time over division or due to an effect of TNO3 is not known. This implies the need for repetition of this experiment for TNO3 with an incubation time of more than 120 min, to determine whether the elongated vegetative cells divide in the end. To research if TNO3 prevents the division of daughter cells, the use of a DNA and a cell membrane dye in a future experiment could be employed. The combination of two such dyes, for example DAPI and Nile Red, would be able to determine whether there is

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DNA replication or initiation of the division into daughter cells. This also indicates a general shortcoming of this experiment: after 120 minutes an effect of the TNO compounds is found, but it is unknown what happens when the incubation time is extended. These results might also explain why the TNO compounds only show their effect after 120 minutes in the oxidative metabolism assay and the time-‐kill assay: before the time point of 120 minutes, germination has not yet taken place. As the compounds show their effect in the outgrowth stage, it makes sense that no effects would be found after 60 min (Pandey R. , Ter Beek, Vischer, Smelt, Brul, & Manders, 2013). A hypothesis to explain the effect on outgrowth and not on germination might be that the TNO compounds can not pass the spore cortex. As the spore cortex is lost in germination, the spores might only be susceptible to the compounds after germination. In conclusion it can be said that all the TNO compounds inhibit outgrowth, but that they do not all have the same effect on this process.

To find out more about how the TNO compounds affect the spore, a propidium iodide staining was done. A peak in fluorescence indicates that the membrane is damaged. If the membrane is damaged the membrane potential will be lost, which means that the cells become less efficient in their metabolism, as the electron transport chain can not take place. This means that not all the available ATP can be harvested from nutrients. The results show fluorescence in most of the spores treated with TNO1 and TNO6. This indicates that the membrane integrity is compromised after treatment with these TNO compounds. Whether the membrane is the target of the compounds or that it gets compromised as a result of other processes being targeted, can not be said from these images. It can not be concluded therefor that the compounds target the membrane. However, the results show that with TNO2, TNO3, TNO4 and TNO5 the membrane is still intact in the majority of the cells. Therefore further research is needed to determine how these compounds affect the spores, as the results of the oxidative metabolism assay showed that the oxidative metabolism is affected. It would also be useful to do this experiment again, as in this research study only one biological repeat was done. Also more cells could be counted when this experiment would be repeated, leading to more

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representative results. It might be useful to do a repeat of this experiment using SYTOX Green as a staining, to validate the results obtained using propidium iodide as a staining. The results for the cell count show that treatment with TNO1, TNO2, TNO4 and TNO6 shows a homogenous effect. This makes these compounds especially interesting for the food industry, where homogenous effects are desired. Heterogeneous effects might lead to the presence of spores that are not affected and can germinate and grow out in a later stadium anyway. However, the images taken and counted might not be representative for the entire cell population, because prior to the taking of the images, areas with a large amount of cells were selected. A repeat of this experiment, focused on representative counting, would be useful to determine how homogenous the effects of all the compounds really are.

Future research

In further research, different stainings might be used to see how the TNO compounds are affecting the spores. Firstly a DiOC6 staining could be used to show membrane potentials (Laflamme, et al., 2005). All the compounds damage the spores’ oxidative metabolism, which suggests that the membrane potential might be lost. However, the PI staining shows that there is no loss of membrane integrity for some of the TNO compounds. A possible explanation could be that the membrane is still integer, but has lost its membrane potential. Laflamme et al. also suggest that one of the first events to happen in germination is the activation of the membrane potential: production of ATP was detected 3-‐4 minutes after the start of germination (2005). An advantage of using the DiOC6 staining is that it works quickly and is able to detect these quick effects. If the membrane potential is indeed damaged by some of the TNO compounds, this means that a process in germination is affected, but that its effects are only found in the outgrowth stage.

Secondly, it is important to find out what the toxic effects of the used TNO compounds are on humans. This was already proven to be important by Robert Koch in the 19th_{century:
after
discovering
that
mercuric
is
effective
in
killing
Bacillus
anthracis} spores, he treated animals infected with such spores by injecting mercuric chloride. This however caused the death of the treated animals because of mercury poisoning (Franklin

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& Snow, 2005). As this project focused on researching antimicrobial compounds for the food industry, it should be researched if and in which concentrations these compounds can safely be ingested.

Lastly, it would be interesting to research whether these TNO compounds affect germination and/or outgrowth in other spore forming bacteria as well. Especially their effect on pathogenic bacteria, like Clostridium difficile or Bacillus cereus, is of interest, as these kind of pathogenic bacteria pose the greatest threat to food safety. The experiments that should be done to investigate this would be similar to the experiments used in this research study.

Conclusion

In conclusion can be said that the TNO compounds show promising effects for the inhibition of outgrowth in B. subtilis spores. The effects of all the compounds are bactericidal. TNO1, TNO2, TNO4 and TNO6 show homogeneity in the inhibition of outgrowth. This is desirable for a treatment used in the food industry, as prevention of outgrowth hinders the formation of vegetative cells that could lead to food spoilage or produce toxins. The current results seem promising and future research might lead to the application of these antimicrobial compounds in the food industry.

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References

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Appendices

Appendix A: Sporulation protocol Day 1:

-‐ Streak out bacterial culture on a TSB solid medium and incubate overnight at 37° C.

Day 2:

-‐ Select a single colony and transfer it to 5 ml TSB (pH adjusted to 7.5) -‐ Incubate culture at 37° C, 200 rpm until the OD600 reaches 0.3-‐0.4.

-‐ Make a serial dilution of the culture ranging from 10-‐1_{to
10}-‐7_{in
MOPS
medium} (pH 7.5). Incubate the cultures at 37° C, 200 rpm overnight and pre-‐warm a 20 ml MOPS culturing medium in the 37° C incubater for the next day’s inoculation.

Day 3:

-‐ Select one of the dilutions, incubated the previous day, with an OD600 of 0.3-‐0.4. -‐ Inoculate 1 ml of this culture intho the pre-‐warmed 20 ml MOPS medium.

-‐ Incubate 20 ml of the culture at 37° C, 200 rpm until an OD600 of 0.3-‐0.4 was reached.

-‐ Inoculate 2.5/5 ml culture into 250/500 ml MOPS medium.

-‐ Incubate the final sporulation culture at 37° C, 200 rpm for 96 hours.

Day 7:

-‐ Determine the sporulation yield. A 99.9% spore yield is desired (spores/vegetative cells).

-‐ Pellet the spores at 5000 rpm for 15 min at 4° C. Discard the supernatant. -‐ Resuspend the pellet in 40 ml sterile MilliQ water. Add 1% (v/v) Tween. -‐ Pellet the spores at 5000 rpm for 15 min at 4° C. discard the supernatant.

-‐ Resuspend the pellet in 40 ml sterile MilliQ water. Determine the spore harvesting yield.

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-‐ Pellet the spores at 5000 rpm for 15 min at 4° C. Discar the supernatant. Repeat this wash 2 to 3 times.

-‐ Finally resuspend the spore pellet in 40 ml 1x MOPS and store at 4° C for 4 weeks. Aliquotes of the spores can be stored at -‐80° C for 8 weeks.