Lab 5: Determination of Antimicrobial Effects of Microbial Extracts

Introduction   

An anti-microbial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbiocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body. Antibiotics are the thing that inhibit the growth of pathogenic and spoilage microorganism. Organic acids and bacteriocins are included among this antimicrobial compound.

Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are typically considered to be narrow spectrum antibiotics, though this has been debated.^[1] They are phenomenological analogous to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse.

The lactic acid bacteria (LAB) comprise a clade of Gram-positive, low-GC, acid-tolerant, generally non-sporulation, non-respiring rod or cocci that are associated by their common metabolic and physiological characteristics.  Proteinaceous bacteriocins are produced by several LAB strains and provide an additional hurdle for spoilage and pathogenic microorganisms. The range of inhibitory activity by these bacteriocins of lactic acid bacteria can be either narrows, inhibiting only those strains that are closely related to the producer organism or wide, inhibiting a diverse group of Gram-positive microorganisms. Thebacteriocins have been reported to inhibit the growth of bacteria; in this experiment we use them to inhibit growing of Staphylococcus aureus and Escherichia coli.

Staphylococcus aureus is a facultative anaerobic, Gram-positive coccus, and is the most common cause of staph infections. It is frequently part of the skin flora found in the nose and on skin. S. aureus can cause a range of illnesses from minor skin infections, such as pimple, impetigo, boils (furuncles).  It is still one of the five most common causes of nosocomial infections, often causing postsurgical wound infections.

Escherichia coli is a Gram-negative rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some can cause serious food poisoning in humans. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K₂ and by preventing the establishment of pathogenic bacteria within the intestine.

Part I: Determination of bacteriocin activity via agar diffusion test

The agar diffusion test, or the Kirby-Bauer disk-diffusion method, is a means of measuring the effect of an antimicrobial agent against bacteria grown in culture.

The bacteria in question are swabbed uniformly across a culture plate. A filter-paper disk, impregnated with the compound to be tested, is then placed on the surface of the agar. The compound diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is thezone of inhibition. Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.

Part II: Determination of bacteriocin activity via optical density

Optical density, measured in a spectrophotometer, can be used as a measure of the concentration of bacteria in a suspension. As visible light passes through a cell suspension the light is scattered. Greater scatter indicates that more bacteria or other material is present. The amount of light scatter can be measured in a spectrophotometer. Typically, when working with a particular type of cell, you would determine the optical density at a particular wavelength that correlates with the different phases of bacterial growth. Generally we will want to use cells that are in their mid-log phase of growth. Typically the OD₆₀₀ is measured.

Objective

Refer to the lab manual

Material and reagents

Refer to the lab manual

Procedure (Part I: Determination of bacteriocin activity via agar diffusion test)

Refer to the lab manual

Material and reagents

Refer to the lab manual

Procedure (Part I: Determination of bacteriocin activity via agar diffusion test)

Refer to the lab manual

Result (part 1)

Calculations 

Inhibition zone :

Serial dilutions of extracellular extract

Y axis : Abs600  or OD600                                                                         X axis : Serial dilutions of extracellular extract

m and c : Constants

One arbitrary unit (AU) is defined as the dilution factor of the extracellular extract that inhibited 50% of the spoilage / pathogenic bacteria growth and expressed as AU/ml

Control : Abs600 = Z. Thus, 50% of Z = Z/2

Y = mx + c . Therefore , x = ( Y – c)/m

When Y = Z/2 , thus, x= (Z /2-c)/m

Data sheet

Part I. Determination of bacteriocin activity via agar diffusion test.

Strains of LAB	Strains of spoilage/pathogenic bacteria	Inhibition zone (cm )
LAB species	Staphylococcus aureus	(0.85+0.80)/2=0.825
	Escherichia coli	(0.70+0.70)/2=0.70

Discussion (part 1)

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms.

The extracts of the lactic acid bacteria gave zones of inhibition onto the indicator pathogen strains tested. In the agar well diffusion assay a linear relationship existed between response (diameter or area of the zone of inhibition). The strains inhibited were Escherichia coli and Staphylococcus aureus. The diameter of inhibition zone obtained from Escherichia coliwas0.700cm while the diameter of inhibition zone obtained from Staphylococcus aureuswas 0.825cm.Staphylococcus aureus was a gram positive bacteria while Escherichia coli were gram negative bacteria. The result showed us that the diameter of inhibited zone in Staphylococcus aureus was larger than Escherichia coli. This was because a gram positive indicator bacterium was much more sensitive to bacteriocin of lactic acid bacteria strains than gram negative indicator bacteria. The resistance of gram negative bacteria was attributed to the particular nature of their cellular envelope, the mechanisms of action described for bacteriocin bringing in a phenomenon of adsorption.

According to Bhunia et al. (1991) the pediocin(bacteriocin produced by  Pediococcusacidilactici) interacts with lipoteichoic acids absent in gram negative bacteria. These molecules play the role of site of necessary not specific reception to produce the bactericidal effect. Some bacteriocins produced by gram positive bacteria had a broad spectrum of activity. These variations of sensibility were due to the characteristic of indicators strains and thus in level of hurt caused by the inhibitive factor.

Procedure (Part II: Determination of bacteriocin activity via optical density)

Refer to the lab manual

Result (part2)

Part II. Determination of bacteriocin activity via optical density

Serial dilution of extracellular extract

Dilutions	OD600 of spoilage/pathogenic bacteria
	Staphylococcus aureus	Escherichia coli
0X	0.304	0.623
2X	1.087	2.044
10X	1.207	2.283
50X	1.022	2.525
100X	1.794	2.012
Equation	y = 0.3259x + 0.9197	y = 0.2915x + 0.2083
OD600  of control	0.322	0.322
50% of OD600	0.161	0.161
AU/ml	-2.33	-0.162

Staphylococcus aureus

 

Escherichia coli

Discussion (Part II)

            The activity of bacteriocins was difficult to quantify and was dependent on the determination method. The most widely used techniques were based on the evaluation of growth inhibition caused on a sensitive bacterial strain, either in solution or in agar plates. A standardized bioassay does not exist because bacteriocinswere significantly different among them, making difficult the use of a common standard, and results depend on experimental conditions. The advantages of using this method were the elimination of diffusion related problems, its quickness, commodity and low cost. Major experiment errors came from the cell sedimentation and interference of sample colour. In addition, time of reaction was generally critical and the relationship between the bacteriocin concentration and the inhibitory response usually follow a sigmoidal curve making use of complex regression models of limited practical application.The logarithm of the dose while a non-linear equation was used to model the sigmoidal dose/response curve in photometric assays (PA). The dose/response curves were used to define titters of the standard solutions in arbitrary units and to develop quantitative assays for all the bacteriocins.

Generally, the OD value is higher from the 100X dilution. It indicated that the LAB has stronger antimicrobial effect on the pathogenic bacteria compare to a more diluted solution. The OD₆₀₀ values in 100X dilutions had the highest value in both pathogenic bacteria where Staphylococcosaurens is 1.794 and Escherichia coli is 2.012.So, the more diluted the extracellular extract, the greater the value of OD_600. Both of the graphs of Staphylococcus aureusand Escherichia coli are obtained by plotting the optical density of spoilage/pathogenic bacteria Staphylococcus aurensor Escherichia coli (OD₆₀₀) against different serial dilutions of extracellular extract. The graph demonstrated a less linear relationship between serial dilutions of extracellular extract and OD₆₀₀, with the regression analysis giving an R² value of 0.4825 for Staphylococcus aureus.While the graph of Escherichia colidemonstrated a more linear relationship between serial dilutions of extracellular extract and OD₆₀₀, with the regression analysis giving an R² value of 0.751.

Conclusion

In summary, the present results clearly suggest the potential usefulness of the bacteriocins produced by Lactic acid bacteria (LAB) as bio preservatives against both Escherichia coli and Staphylococcus aureus. Both acidification and the production of hydrogen peroxide by LAB were ruled out as the source of the inhibition. LAB competed with spoilage microorganisms, such as certain Gram-negative bacteria for nutrients or space with. Moreover, the shelf-life of food products could extend the production of organic acids, hydrogen peroxide, low molecular weight metabolites (such as diacetyl and bacteriocins) due to their inhibiting effect on the growth of spoilage and pathogenic bacteria. For these reasons, antagonistic effects against pathogenic bacteria exhibited by harmless LAB applied interest as bio preservatives – with the subsequent reduction in the use of antibiotics – in future aquaculture activities.

Bacteriocins have been defined as proteinaceous substances exhibiting bactericidal activity against closely related species. Currently they are receiving increased attention because of their inhibitory activity against food spoilage and food-borne pathogenic bacteria such as Listeria monocytogenes. Commercial nisin preparations have been evaluate in food systems. It was now widely used as bio preservatives in the food industry due to their antibacterial properties. This allowed a more strict microbial control of a variety of commercial food products.

Determination of bacteriocin activity by the agar diffusion assay in which inhibition zones are produced in plates in a procedure similar to that of antibiograms, is undoubtedly the most commonly used despite the inconveniences and limitations of its application. The performance of the method, which is laborious and time-consuming, depends largely on human ability and judgment and precision can’t be achieved when inhibition zones are unclear or not perfectly circular. Diffusion-related difficulties of the active substance represent another important limitation of agar diffusion assays. The need to eliminate diffusion-related problems associated with the agar techniques, introduced liquid medium methods, which make use of indicator organisms and quantify the bacteriocin concentration from the percentage of growth inhibition in the indicator organism. Since then, applications of turbidometric assays by spectrophotometer can be found in a number of reports in which, as with the agar diffusion assay, various indicator microorganisms were used, in procedures that show large variability regarding bacteriocin extraction, general experimental conditions and definition of the bacteriocin unit. Sensitivity limits and linearity of responses to various bacteriocin levels vary significantly among different test-microorganisms in both bioassays, the lower sensitivity limits depending on both the test-microorganism and the applied method. Very low nisin concentrations, e.g. 1 IU/ml, were more safely determined in the turbidometric assay (spectrophotometer) through determination of the percentage of inhibition of growth of the indicator microorganism. This method proved to be more suitable for determination of nisin in processed food samples.

Although the agar diffusion assay is the most widely used method in routine measurements of bacteriocin activity, turbidometry (spectrophotometer) offers a simpler, faster and more reliable alternative since diffusion related problems are eliminated, the degree of human intervention and judgment is low, and very low bacteriocin concentrations can be quantified.

Reference

http://en.wikipedia.org/wiki/Antimicrobial

http://en.wikipedia.org/wiki/Lactic_acid_bacteria

http://en.wikipedia.org/wiki/Bacteriocin

http://en.wikipedia.org/wiki/Staphylococcus_aureus

http://en.wikipedia.org/wiki/E_coli

http://en.wikipedia.org/wiki/Agar_diffusion_test

http://people.hofstra.edu/beverly_clendening/Adv_Molecular_Biology/Protocols/Measuring_Optical_Dens.html

http://www.pjbs.org/pjnonline/fin202.pdf

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-3YCMHFW-W&_user=4187955&_coverDate=04%2F30%2F1995&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_searchStrId=1706486002&_rerunOrigin=scholar.google&_acct=C000012438&_version=1&_urlVersion=0&_userid=4187955&md5=61b54cfa49439f60490f5e41f689a1fa&searchtype=a

http://www.academicjournals.org/ajfs/PDF/Pdf2009/Jan/Khalil%20et%20al.pdf