1. Introduction
Non-typhoidal Salmonella has been associated with many food-borne diseases all over the world [1]. Not only developing country, especially in the United States, it is the cause of an estimated 1.4 million illnesses annually [2]. Various foods, such as chicken, beef, and pork, have been implicated in outbreaks caused by Salmonella spp. [3] [4]. A characteristic of Salmonellae, which is a food poisoning-causing bacterium, is that it produces hydrogen sulfide (H2S) when cultured with using a medium containing a sulfur source. H2S is toxic to the human body [5].
So, it can be said that Salmonella is not a probiotic or beneficial bacterium. In other words, Salmonella is a bacterium rather harmful to humans.
H2S produced by Salmonella reduces the iron source contained in the medium, thereby forming iron sulfide, and a color change to black is observed. We have defined this as visualization of H2S production, and we have continued research that will lead to the development of new methods for detecting Salmonellae and testing the antimicrobial properties of materials [6].
First of all, when slices of lemon placed and cultured on which the Salmonella was densely inoculated, we discovered that black rings are formed around the lemon (Named as MY phenomenon) [7]. Next, experiments were continued to identify substances that significantly visualize the H2S production produced by Salmonella. As a result, it was revealed that H2S production of Salmonella is remarkably visualized in black under the influence of organic acids which are components of lemon, such as citric acid and ascorbic acid. Other citrus and acerola, kiwi fruit, strawberries, green peppers or paprika showed same [8] [9], Figure 1.
By the same phenomenon, a method for detecting Salmonella was devised by analyzing the expression state of iron sulfide (II) on the agar medium and/or the color change of the agar medium.
Next, it was also discovered that the H2S production of Salmonella is inhibited by the influence of salt content and antibacterial substances. Therefore, it became clear that visualization of H2S production may be an indicator of active growth of Salmonella [10] [11], Figure 2.
In addition, we discovered that H2S by Salmonella can be visualized by making the medium surface to anaerobic, and developed a new antibacterial test method. That is, when a substance that does not inhibit the growth of bacteria is
Figure 1. Effects of lemon slice on H2S production of Salmonella. [7].
placed on the medium and cultured for a predetermined time, then only the medium below the sample turns black. On the other hand, in the culture medium under the sample having the antimicrobial property, the phenomenon of black change is not observed [13], Figure 3. Thus, we have discovered a new antimicrobial test that will prove the antimicrobial properties of the material if Salmonella shows no visualization of H2S production [14].
The developed antibacterial test method has made it possible to test the antibacterial property of water-insoluble substances as well as water-soluble substances more quickly and more accurately than the current method [15] [16].
The above is the result of our research using the MY phenomenon of Salmonella. Furthermore, since it discovered that the organic acid which E. coli etc. produce also exhibits the MY phenomenon to on Salmonella, shows the knowledge below.
2. Materials and Methods
Media used: Deoxycholate-hydrogen sulfate-lactose (DHL) agar medium (Eiken Chemical Co., Ltd.). The characteristic of the medium is that of each of 1 L of solution contains 1 g of ammonium ferric citrate as a ferric source 2.3 g of sodium thiosulfate source as a sulfur source.
Figure 2. Effects of antimicrobial substances sodium chloride and copper ion on H2S production of Salmonella [11] [12].
Figure 3. After 24 hours of culture, observe from the back side of the medium in petri dish. Since aluminum is not antibacterial, H2S production is not observed beneath the medium. On the other hand, H2S is not produced because copper is antibacterial [14].
Used strain of E. coli: Growth at 44.5˚C in Escherichia coli Medium (EC Medium Eiken Kagaku Japan) from environmental isolates. The IMViC test with the characteristics of indole reaction (I), methyl red reaction (M), Voges-Proskauer reaction (Vi) and Simmons' citrate availability (C) shows a pattern of “++−−” [17] [18].
Salmonella strain: non typhoid, serotype Derby, isolated from the fresh market of Vientiane Lao P.D.R. and H2S producing strain.
Sterile Cotton swab (Eiken. Japan): used when smearing and inoculating Salmonella.
Sterile Toothpick (Daiso Japan): Use a pointed tip when inoculating E. coli.
The following three experiments were tried using the above materials.
The case not contacting E. coli and Salmonella directly:
Inoculate Salmonella and E. coli to two petri dishes of the same culture medium as follows.
A: The Salmonella was inoculated on half of the dish with a sterile cotton swab. Nothing was inoculated on the other half.
B: Similarly, Salmonella was smeared on half of the dish. Then E. coli was inoculated on the other half using a toothpick tip.
After incubation at 37˚C for 24 h in an incubator, observed the results.
The case Salmonella was contaminated with E. coli
The following procedure of C and D was performed on 2 sheets of DHL agar medium.
C: Using toothpick tip, inoculate E. coli into 5 spots on the medium of one dish at intervals like 5 in the dice.
D: The other dish was smeared with Salmonella and then inoculated E. coli 5 places as above.
Then, these were cultured at 37˚C for 24 hours.
From a petri dish in which E. coli was cultured with Salmonella, isolation of Salmonella was done as below E and F.
E: Portion of Salmonella only.
F: Coexistence of Salmonella and E. coli.
3. Results
The case not contacting E. coli and Salmonella directly: (Figure 4)
· Result of A: After cultivation, In the case of smear inoculation only with Salmonellae on one side of the petri dish, no MY phenomenon was shown that Salmonella produces H2S. Because no blackened is observed. This means iron sulfide is not formed (Figure 4). Repeat this experiment 10 times or more and get the same result.
· Result of B: However, in the petri dishes in which E. coli are inoculated on the other side which had not been coated with Salmonellae, a black line, which is a visualization of H2S production, is produced at the boundary where the Salmonella bacteria are cultured (Figure 4).
Repeat this experiment 10 times or more and get the same result.
The case E. coli is contaminated on smear Salmonella (Figure 5)
· Result of C: Where only E. coli is inoculated into the medium, all five locations turn red at the E. coli growth point (Figure 5 Left Panel). Repeat this experiment 10 times or more and get the same result.
· Result of D: The case E. coli contaminate on Salmonella (The fact is shown in Figure 6), black spots are observed in all five places where E. coli and Salmonella is coexist (Figure 5). Repeat this experiment 10 times or more and get the same result.
· Result of E: Only, Salmonella is isolated (Figure 6). Repeat this experiment 10 times or more and get the same result.
Figure 4. Left: Result of A. Salmonella cultured on one side. No M.Y. Phenomenon occurs. Right: Result of B. When culturing E. coli 1 cm away from the border of Salmonella, Black line appears. The M.Y. Phenomenon occurs.
Figure 5. Left: Result of C. Colonies of E. coli on DHL agar show red color. Right: Result of D. E. coli grown on inoculated Salmonella turned black. E. coli was identified by isolation from black part (see next Figure 6).
Figure 6. Left: Result of E. Salmonella colonies isolated from the part without E. coli. Right: Result of F. Salmonella colonies (black color) isolate from the part contaminated with E. coli (red color).
· Result of F: Both Salmonella and E. coli are isolated. The black color of Salmonella colonies is clearer than case E (Figure 6). Repeat this experiment 10 times or more and get the same result.
4. Discussion
The phenomenon of visualization of H2S production by Salmonella in a medium containing a sulfur source and an iron source has existed as a previously known fact [19] before we have discovered the MY phenomenon of forming Mido Ring around a sliced lemon.
In the case of using the DHL medium, the colony isolated alone is black in the center, but when Salmonella grows densely, H2S produced by bacteria is not visualized. This fact is already understood as a matter of course for those who have been testing for Salmonella. However, we are the first to document this fact.
The fact that we discovered next is that even under conditions where Salmonella is growing densely, if the surface is covered with a device that contains citric acid, ascorbic acid, etc. as well as sliced lemons, can visualize H2S production by Salmonella. For example, other sliced citrus fruits and other fruits showed same results [7].
These are the fact that author has made clear about this as MY Phenomenon. In this present study, it has been confirmed from the following three points that citrus fruit and other organic acids as well as E. coli promote the H2S production of Salmonella.
1) Compare the results of A and B in Figure 4. Only when E. coli is cultured on the other side, M.Y phenomenon, this time as a black line appearance, occurs at the place where Salmonella is densely grown and the borderline where the E. coli is cultured. The black line is considered to appear as a result of H2S produced by metabolism of sulfur source contained in the culture medium reacting with the iron source to form iron sulfide. It is suggested that Salmonella is active in H2S production near cultured E. coli. It can say that E. coli acts positively on Salmonella growth.
2) As the result of C, E. coli ferments lactose to produce acid and gas, when inoculated alone and cultured, it turns red. This is because in the DHL medium, the pH indicator turns red due to the organic acid produced by the E. coli. This fact is known.
However, in the result D, when E. coli is inoculated on densely inoculated Salmonellae, the M.Y phenomenon of black visualization occurs at the places where E. coli was inoculated. That is, the active H2S production of Salmonella is observed in the part inoculated with E. coli. Previous our studies have shown that Salmonella active H2S production shows that it has activated the growth of Salmonella itself [11]. Therefore, it is suggested that the contaminating E. coli has an effect of promoting the growth activity of Salmonella itself.
3) Similarly, black part from which both Salmonella and E. coli are isolated, it can be determined that the black part is a coexistence of Salmonella and E. coli. In the case result F from where E. coli and Salmonella co-exist, the black color is obviously remarkable as compared with the case where it is isolated from the part Salmonella alone sown in Result E. Therefore, for Salmonella, existence of E. coli is symbiosis. So, by what mechanism does E. coli activate Salmonella H2S and growth? As one of the reasons, it can be mentioned that the organic acid which is a metabolite produced from E. coli, may also be an energy source of Salmonella like the citrus component. Of course, in addition to E. coli, bacteria having similar effects on Salmonella are also considered to exist. However, this is a future research topic. In addition, it would be further research that whether the same phenomenon can be find as this research or not even in the intestines of humans or animals.
5. Conclusion
The following facts show that E. coli is assisting Salmonella growth.
· When E. coli is cultured near Salmonellae, visualization of hydrogen sulfide (MY phenomenon) production of Salmonella occurs near E. coli.
· When E. coli is cultured in DHL medium, it turns red due to the organic acid produced.
· When E. coli contaminates on densely cultured Salmonella, the MY phenomenon appears and therefore blackens.
· When Salmonella coexists with Escherichia coli, hydrogen sulfide production becomes significant rather than isolating Salmonella alone.
The relations suggest that bacterial symbiosis relation exist in bacterial flora.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number 16H05634 and 15K00894.
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