Bacterial Antagonism and Synergism

BIO120 General Microbiology (Laboratory) st 1 Semester SY 2008-2009

Synergistic and Antagonistic Relationships of Escherechia coli , Bacill us cereus, , cereus, Bacillus subt ili s St aphylococc aphy lococcus us aur eus and Penicillium enicillium notat um 1

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Santos, Herbert J. , David, Jefty Jr. , Garcera, Acielle Angeli and Zuniega, Ralph Rommualdo 1

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Instructor, Institute of Biology, College of Science, University of the Philippines DIliman, Student, BIO120 (General Microbiology), Institute of Biology, College of Science, University of the Philippines DIliman, Quez on City, 1101 Philippines

ABSTRACT cereus, In the experiment, synergistic and antagonistic relationships between microorganisms were examined. Bacillu s cereus, St aphylococcus aphylococcus aur eus and Escherechia coli were placed in carbohydrate broth tubes and observed for their catabolic activities. It was found out that with the presence of E. col i , B. cereus and S. aureus were able to utilize lactose as energy sources since E. col i hydrolyzed hydrolyzed the lactose into glucose which the two other microbes can utilize. This is proved further by their inability to digest lactose in the absence of E. col i . However, sucrose broth tubes exhibited positive reactions for all three microbes whether in combination or alone further implying the ability of all three to utilize sucrose as energy source. Antagonistic activities were observed when Bacillu Bacillu s subti lis and Penicillium enicillium not atum were streaked in nutrient agar plates containing E. col i and S. aur eus. It was observed that B. subtil is was antagonistic to E. . However, based on literature P. notat um should coli and S. aur eus while P. notat um was antagonistic only to E. col i have been antagonistic to S. aur eus since it has a very strong anti-bacterial anti-bacterial action which has been the basis of its wide use in antibiotics. The use of other methods apart from the seeded plate method used in this experiment may eliminate such errors observed.

Keywords: syngergistic and antagonistic microbial relationships, lactose and sucrose broth tubes, pour and streak plate method

INTRODUCTION In real situations, nutrients and other essential resources are limited. Over time, a number of adaptations have evolved during the course of microbial and fungal existence to address their resource limitations. Microbes may compete with another microbe for nutrients and these competitions often result to inhibition of one of the microbes. This is termed as antagonism which is described as the inhibition of a bacterium by the products of another (Talaro and Talaro, 2002). However, bacteria or fungi may also rely on other species for nutrient supply. At some instances, both bacteria and/or fungi may benefit from one another. This is termed synergism. An example of which is when one species produces a substance not synthesized by another species but beneficial to it. To illustrate, a population of Streptococcus faecalis produces ornithine which Escherichia coli cannot synthesize. E. coli may then take up ornithine (a

Experiment 12 │ Group 3│ October 2, 2008

product of S. faecalis ) in order to synthesize protein. E. coli, however, produces putresine which is not synthesized by S. faecalis, and this nutrient may be beneficial to S. faecalis . Therefore, in the end, both microorganisms are benefited. Synergism, however are not obligatory dependencies between two or more microorganisms and these microorganisms can generally live on their own (Harley and Prescott, 2002). Bacterial antagonism depends on many factors. Among others, for example are competitions for an essential nutrient, the production of toxic metabolites or the release of antibiotic substances. Environmental pressures drive these microorganisms to produce substances that may inhibit the growth of competing species (Talaro and Talaro, 2002). For example, some species may produce bacteriocins, a substance similar to antibiotics but are generally very

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narrow in spectrum and may only act on a single species. Microbial interactions are also found in diseases. In this case, infection of two or more microorganism can either be beneficial or detrimental to the host. In the human body, normal floras are usually antagonistic to pathogens due to nutrient competition (Updyke and Probisher, 1947). However, synergistic interactions between two pathogens in the human body can lead to much potent infections and diseases (Meleney, 1931). These are only some of the actual applications. Understanding of microbial interactions can be applied to other fields. In one example, bacterial antagonism is employed to control growth of spoilage bacteria. Non-pathogenic bacteria that produce bacteriocins are used to control bacterial species that may produce spoilage (Tsigarida et al., 2003).

and/or sucrose), 10g tryptone, 5g yeast extract, 2ml of the indicator solution and 1000ml of distilled water. Durham tubes were added to nine lactose broth tubes and nine sucrose broth tubes. Synerg yne rg ism Test

Several organisms were tested for antagonistic activity. These are Escherichia coli, Staphylococcus aureus, and Bacillus cereus . Each type of broth was inoculated with each microorganism (i.e. each organism was inoculated to one tube containing sucrose broth and the other tube with lactose broth). To check for synergism, combinations of two organisms were inoculated in one tube containing sucrose broth or lactose broth. The tubes were incubated for 48 hours at 37°C and observed for gas productions and color change. Ant agonism Tes Test

It is therefore crucial to study microbial or fungal synergism or antagonism. The purpose of this experiment is to give a simple model of bacterial interaction that may exhibit antagonism or synergism between two species.

Materials and Methods M edia preparation preparation

Bromthymol Blue as indicator for Carbohydrate Broths were prepared from stock solution using 8g Bromthymol Blue, 250ml of 95% ethyl alcohol, and 250ml Distilled Water. Lactose and sucrose based broths were prepared using 5g sugar base (lactose

To illustrate the model for antagonism between microorganisms, four representative species were tested. These are Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Penicillium notatum . Nutrient agar media were liquefied and cooled to 0 about 50 C and inoculated with the initial test organism. These were then poured into sterile Petri plates and allowed to cool at room temperature before streaking with the second test organism. There are four set-ups for this test as summarized in Table 1. Observations were made after the plates were incubated at 37°C for 48 hours.

Table 1. Summary of set-ups used to illustrate antagonism between two species of bacteria or against a fungal species.

Set – up No. 1 2 3 4

Test organism 1 (Inoculated in liquid agar media) S. aureus S.aureus E. coli E. coli


Test Organism 2 (streaked on agar media) B. subtilis P. notatum B. subtilis P. notatum

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Results Growth of the microbes in different culture media such as lactose broth and sucrose broth were investigated. This is to test whether each bacterium or combination of them will use lactose or sucrose as energy source or if they will use both. Color change from green to yellow indicates fermentation and production of acids as by-product. Also, formation of gas will indicate fermentation and confirm growth. Table 2 summarizes the results of synergism tests for individual organisms and combinations of them. Combination of two microorganisms in a single culture media may indicate synergism if both of them can grow in that media. It can be be noted from the results that S. aureus and B. cereus are not synergistic since their combination did not have positive growths when inoculated in the lactose Broth. However, both organisms were able to ferment sucrose either together or apart. It can also be noted that E. coli combined with B. cereus or S. aureus can exhibit synergism. Note that E. coli in combination with the two previously mentioned microbes elicited growth in lactose broth and sucrose broth.

Table 3 summarizes the results for antagonism tests between two microorganisms. In this test, one microorganism is inoculated to the liquid agar media before being poured on the sterile plate. The other microorganism is streaked on the solid agar media containing the previous organism. These microorganisms are identified based on the colony growth, pour plated bacteria will show up as distinct circular colonies growing randomly on the plate and the streaked bacteria will grow along the streaking patterns (see Figure 1). Antagonism can be illustrated by growth of a single species on the agar plates. It can be noted that B. subtilis is antagonistic to both E. coli and S. aureus while P. notatum is antagonistic only to E. coli .

Discussion Synergism occurs when two organisms grow better together than apart (mutualism) or when the byproducts of one organism enhances the survival of another without affecting the contributory organisms (commensalism). This type of interaction can be observed in animal guts particularly in those of ruminants where a lot of bacteria are involved in the digestion of cellulose. Some of the microbes present are able to utilize cellulose only in the

Table 2. Summary of results for the individual species grown in lactose broth and sucrose broth. Antagonism or synergisms between two species are tested by growing them in the same culture medium.

Individual Organism E. coli B. cereus S. aureus Combination E. coli and B. cereus E. coli and S. aureus S. aureus and B. cereus

Lactose Broth Acid Gas + + * + -/+ + -

+ + -

Sucrose Broth Acid Gas + + + + + + +

+ + -

* conflicting results from two replicates of the same set-up, however, a negative results for this test is theoretically the valid outcome. The first replicate had a yellow-green appearance which prompted the (+ -) conclusion.


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Table 3. Summary of results for the antagonism tests. Two microorganisms are inoculated on the same Nutrients agar plates. The growth of each species were indicated by circular colony growth due to the pour plating method or by growth along streaked patterns.

Combination S. aureus and B. subtilis

Observations Growth was mostly B. subtilis which was streaked on a plate containing S. aureus. S. aureus was observed to grow in Nutrient Agar plate without B. subtilis.

Conclusions B. subtilis is antagonistic to S. aureus

S. aureus and P. notatum

Both S. aureus and P. notatum growths were observed on the Nutrient Agar plates.

No antagonism

E. coli and B. subtilis

B. subtilis was obrserved to grow on the streaked area of the plate. E. coli did not grow in combination with B. subtilis .

E. coli and P. notatum

Pour plated E. coli was observed to grow on the Nutrient Agar plate. However, the streaked P. notatum on the plate containing E. coli did not show signs of growth.

B. subtilis is antagonistic to E. coli E. coli is antagonistic to P. notatum

presence of other microbial species while some utilize the by-products of the cellulolytic species in a mechanism known as cross-feeding (Dehority, 1998). Synergism is also similar to the concept of syntrophy wherein one or two organisms cooperate in the degradation of complex compounds. A particular example of syntrophy occurs with methanogens which rely on other microbes to supply them with the necessary substrates needed to degrade organic compounds into CH 4. There are only a few substrates which can be converted by methanogens directly into methane thus making the digestion of organic compounds by other microbes a necessity (Madigan et al., 2002). In the experiment it is clearly seen that E. coli is synergistic with both B. cereus and S. aureus since it allows both organisms to utilize lactose as a carbon


source. This happens because E. coli has galactosidase which hydrolyzes lactose into galactose and glucose. Glucose can then be utilized by B. cereus and S. aureus as energy sources. The utilization of these sugars makes the medium acidic thus turning the neutral green color of Bromothymol blue into yellow (Wikipedia 2008). Furthermore, the production of a gas by E. coli accounts for the gas formations in the Durham tubes – a phenomenom absent when either B. cereus or S. aureus alone utilizes a sugar (Harley and Prescott, 2002). Sucrose is easily used by any microbe as its energy source so it is expected that positive results be observed for sucrose tubes containing solely B. cereus, E. coli and S. aureus or a combination thereof. Furthermore, E. coli was the only one to exhibit gas formations in its Durham tubes since it was the only one capable of gas productions.

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A

B

C

Figure 1. Results for the antagonism tests between two microorganisms. (Fig 1A) B. subtilis was found to be antagonistic against S. aureus. Note the growth of the streaked colonies and the absence of scattered colonies along the streaked lines. (Fig 1B) B. subtilis was also found to be antagonistic against E. coli. In this plate, E. coli was inoculated on the liquid NA media before being streaked with B. subtilis. However, only the streaked colonies were present. (Fig 1C) Randomly distributed colonies of E.coli were present in this plate; note that there are no visible growths of P. notatum that was streaked on the plate.

Antagonism refers to the reduction in growth and activities of an organism as a result of the living of another organism. In a natural setting like soil and water which often have a mixed microbiological population, numerous relations of association and antagonism exist. All the inhabiting organisms in the medium are affected, directly or indirectly, by one or more of the other constituent members of this population. These relationships were at first defined as due basically to competition for nutrients. If two organisms are grown on the same substrate, one overcomes the other sooner or later, and even kills it. The limited nutrients in the medium were believed by many to be responsible for this, with the fast-growing organism being more favorable than the slow grower. Moreover, when two organisms are capable of utilizing the same nutrients, but are differently affected by environmental conditions, such as reaction, air supply and temperature, the one that finds conditions more favorable for its growth, will develop more rapidly and in time be able to suppress the other (Waksman, 1941).

example, the effects produced upon each other by fungi in mixed cultures may be due to the formation of substances which exert detrimental or beneficial effects to one or both. Antagonism is therefore regarded as a very complex phenomenon. It is a result of numerous and often little known activities. It influences the morphology of the organism, the capacity for pigment production, and various physiological processes (Waksman, 1941).

However, it soon became clearer that antagonism among microorganisms can be due to other phenomena other than mere competition for nutrients. Like when two or more organisms live in close proximity, they may exert mutually antagonistic, indifferent or favorable effects. For

Several various methods have been used for the study of bacterial antagonism. One is Garre's method which has been employed extensively by many, either in its original form or with modifications. Agar or gelatin is poured into Petri dishes and is allowed to harden. It is then inoculated on the surface with


As for the mechanism of antagonism, various theories were made which generally are the following: nutrient exhaustion in medium; physicochemical changes due to the organism g rowing in the medium, which include changes in osmotic pressure, surface tension, oxidation-reduction potential and reaction; enzyme production either by the antagonist itself or as a result of autolysis of the antagonized cells; substance production and liberation which can cause a selective bacteriostatic and bactericidal effect, or fungistatic and fungicidal action; and space antagonism (Waksman, 1941).

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the two species placed alternately, either parallel or radiating from the center. This method affords the advantage of permitting either simultaneous inoculations or a period of time for the growth of the inhibitor (Lewis, 1928). A more useful plate method of growing two species in mixed association is the seeded plate with surface inoculation. This was the method done in this experiment. The agar, seeded with one of the pair, is poured into a Petri dish and inoculated on the surface with the other. The extent of the inhibited zone is dependent on many several factors such as composition and consistency of the agar, its depth in the dish, amount of inoculum, susceptibility of the inhibited species and the normal growth rate of the two species. The seeded plate method is much useful as preliminary analysis, however, its application to the study of bacterial antagonism is limited and the results, when negative, are very often misleading (Lewis, 1928). As for this experiment, the obtained results somehow seem to be in-line with that of the literature, but with some discrepancies. In the test including S. aureus and P. notatum, results show that no antagonism occured, but in literature, S. aureus should have became transparent indicating lysis due to P. notatum's strong anti-bacterial action. P. notatum is antagonistic to S. aureus. Another one is that of E. coli and P. notatum again, where in the results, E. coli was shown to be antagonistic to P. notatum. However according to literature, P. notatum should have been antagonistic to E. coli though at low level since gram-negative bacteria are least sensitive to P. notatum's anti-bacterial action. In the next pairs, they show similar results to that of the literature. In that of E. coli and B. subtilis , literature tells that B. subtilis is antagonistic to various bacteria, meaning it is not that selective and so, it does not necessarily contradict results obtained where B. subtilis was found to be antagonistic to E. coli . The recent case is just similar to that of S. aureus and B. subtilis (Waksman, 1941). The reason behind the errors could have been from the experimenter's judgement, since the interpretation of results is somewhat subjective.


Furthermore, this can be accounted also to the disadvantages and limitations of the seeded plate method done. The experimenters would like to recommend the use of other methods such as collodion sack method which according to literature, gives more reliable results but is difficult and time consuming when many tests are to be made (Lewis, 1928). The knowledge of microbial interrelationships is useful in various industries such as the medical industry wherein the antagonistic activities of one microbe to another has led to the discoveries of a variety of antibiotics. Microbial interactions also play a role in the understanding of the mechanisms of food spoilage and the hope to preserve food longer (Tsigarida, et al. 2003). It is therefore seen that just as humans and other macro organisms rely or fight with each other for survival so thus microorganisms depend or compete with each other. A knowledge of such interactions can help us better understand the microbial world and its implications to human life.

References: Dehority, B. A. (1998) Microbial Interactions in the Rumen. Rev. Fac. Agron. (LUZ) Vol. 15 pp. 69-86 Harley J. and Prescott L. (2002) Laboratory Exercises in Microbiology 5th Ed. McGraw−Hill Companies Inc., USA (eBook) Lewis I. 1928. Bacterial Antagonism with Special Reference to the Effect of Pseudomonas fluorescens on Spore Forming Bacteria of Departments of Botany and Soils. Bacteriology, the University of Texas. pp 9193. Madigan M.T., Martinko, J.M. and Parker, J. Brock th Biology of Microorganisms 9 Ed. Prentice Hall Intl. Inc., USA pp. 677-678 Meleney, F. L. (1931) Bacterial Synergism In Disease Processes. Annals of Surgey Vol. 94, No. 6, pp. 961-981

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Talaro, K. P. and A. Talaro (2002) Foundations in Microbiology 4th Edition. McGraw Hill Publishing, USA (eBook) Tsigarida, E., I. S. Boziaris, and G. E. Nychas (2003) Bacterial Synergism or Antagonism in a Gel Cassette System. Applied And Environmental Microbiology, Vol. 69, No. 12, p. 7204–7209 Updyke, E. L. and M. Frobisher, Jr. (1947) A Study Of Bacterial Synergism With Reference to the Etiology Of Malignant Diphtheria. J. Bact. Path. Vol. 54, pp. 619-632 Waksman S. 1941. Antagonistic Relations to Microorganisms. New Jersey Agricultural Experiment Station, New Brunswick, New Jersey. pp 235-254. http://en.wikipedia.org Bromothymol Accessed: October 1, 2008


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Bacterial Antagonism and Synergism

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