ANALYSIS OF THE EFFICACY OF STREPTOMYCIN AND ERYTHROMYCIN AGAINST Escherichia coli THROUGH DISK DIFFUSION ASSAY

Delos Santos, D, Jagonob, C, Javillonar, P, Sandoval, B, and Valdez, C, Group 4, BIO1133, Far Eastern University, Manila, Philippines. ABSTRACT
There are many pathogenic organisms and these organism can be dealt if given the right bacteriostatic organism or drug. Disk diffusion assay is used to analyze the susceptibility of an organism which help in identifying the proper drug. In this experiment this is done by placing the antimicrobial disks (streptomycin and erythromycin) on the NA plate swabbed with Escherichia coli. After twenty-four hours of incubation, there were areas around the antimicrobial disks with no bacterial growth, the antimicrobial disk with larger zone of inhibition is streptomycin, proving it to be a stronger antibiotic for E. coli than erythromycin. Keywords: Disk Diffusion Assay, Kirb-Baurer Method, Susceptibility Test, Streptomycin, Erythromycin, Escherichia coli.

I. INTRODUCTION
Different antimicrobial agents have different effects on different organism. Some organisms may be completely resistant to a specific antimicrobial while others are highly susceptible (Perez, et al. 2007). Susceptibility as defined is the tendency of an organism to be to be affected by another organism (Microsoft Encarta. 2009). Organisms are categorized to be susceptible, intermediate, or resistant on a certain organism or antimicrobial drug based on the results of particular susceptibility test. Susceptibility test in microbiology implies different methods that are used by medical laboratories which broth dilution test, antimicrobial gradient method, and disk diffusion test (Reppun, MD, et al. n.d; Reller, et al. 2009). Disk diffusion method is also known as the Kirby-Bauer disk susceptibility test. Disk diffusion method is one of the oldest approaches to antimicrobial susceptibility testing methods in routine medical laboratories (Hudzicki, 2013). It is suitable for testing the majority of bacterial pathogens, including the more common fastidious bacteria, it is flexible in the range of antimicrobial agents that can tested. And also it includes no special equipment. The principle of this method is dependent upon the inhibition of

reproduction of a microorganism on the surface of a solid medium by an antimicrobial agent which diffuses into the medium from a filter paper disc. The performance of antimicrobial susceptibility test is important in confirming susceptibility that are chosen to detect resistance in individual bacterial isolates The objective of this paper is to test the effectiveness of the antimicrobial drugs, streptomycin -- a water-soluble aminoglycoside derived from Streptomyces griseus and erythromycin -- produced by a strain of Saccharopolyspora erythraea (formerly Streptomyces erythraeus) and belongs to the macrolide group of antibiotics (Rxlist Inc. 2013), in inhibiting the growth of the gram negative bacteria E. coli and to check which drug is more effective. As the experiment progresses the students will also learn about the processes and principles involved in disk diffusion method of susceptibility testing and to practice aseptic technique throughout the experiment.

II. MATERIALS AND METHODS

From a pure culture of E. coli a sterile inoculating loop was dipped to inoculate the bacteria on another sterile test tube with distilled water. The inoculated test tube was shaken carefully by

rolling the tube against the palms until the solution appears to be colloidal. This dilution will be used as the organisms in the experiment. The E. coli dilution is cultivated in a nutrient agar plate using a sterile swab through the swab plate method. Three control variables were added to the E. coli plate. The first was the negative control. A pair of forceps was dipped in an ethyl alcohol and flamed sterilized, it was used to grab a sterile paper disk with a size of 8 millimeters and was dipped in a test tube with distilled water, the paper disk was air dried, and was placed in the E. coli plate. The two positive controls are the antimicrobial disk, streptomycin and erythromycin, having the same size that of the negative control, the antimicrobial disks were carefully grabbed with a sterile forceps and was placed in the E. coli plate. The paper disks were placed as shown in Fig 1. The test was done in duplicate. The E. coli plates were then incubated for 24 hours.

Figure 2.0: E. coli plate 1 after incubation. Paper disk S (streptomycin), Paper disk E (erythromycin), and Paper disk P (water).

Figure 1.0: Placement of the paper disks.

After the incubation period a clear area around the positive controls were observed. These areas were measured using a foot rule in millimeters. All observations were recorded.

III. RESULTS AND DISCUSSION

The clear areas found in the E. coli plate are called zone of inhibition (Fig 2.0 and 2.1). These are the zones where the antibiotic in the positive control disks prevented the growth of E. coli (Madigan, et al. 2012) The paper disk (Fig 3.0) with water was expected to have no zone of inhibition for the reason that water is does not contain any antimicrobial agent against E. coli while on the other hand the presence of the zone of inhibition on the positive controls (Fig 3.1 and 3.2) proved that the

Figure 2.1: E. coli plate 2 after incubation. Paper disk S (streptomycin), Paper disk E (erythromycin), and Paper disk P (water).

Figure 3.0: Paper disk with water in (right) plate 1 and (left) plate 2.

antimicrobial disks used were able to impede the growth of the bacteria. To test which antimicrobial drug have greater efficacy against E. coli the researchers measured the diameter of the zone of inhibition to check for their bioactivity, which was assessed using the following rating system: (1) very active, > 19 mm zone of inhibition; (2) active, 13-19 mm zone of inhibition; (3) partially active, 10-12 mm zone of inhibition; and (4) inactive, < 10 mm zone of inhibition (Santiago, et al. 2010; Quinto and Santos 2005).

the criteria published by the Clinical and Laboratory Standards Institute (CLSI, formerly the National Committee for Clinical Laboratory Standards or NCCLS) (Reller, et al. 2009).
Table 1.1: Erythromycin ZOI and bioactivity.

Erythromycin

Plate 1 Plate 2

Zone of Inhibition (ZOI) in millimeters 16 18

Bioactivity Level

Active Active

As seen on the tables presented the streptomycin disks have larger zone of inhibition thus it can be deduced that it is a better antimicrobial drug against E. coli than erythromycin.

IV. CONCLUSION
Seeing the results after the incubation it can already be concluded that both of the antimicrobial disks did inhibit the growth of the E. coli and with further observation it can be seen that the streptomycin inhibited a larger area than erythromycin proving that streptomycin is a stronger antibiotic for E. coli. Disk diffusion method is really an easy way to check the susceptibility of organisms to other organisms and also the inhibiting effect of bacteriostatic organisms to certain pathogenic organisms. It might not be that accurate but still knowing that a certain organism can hinder microbial growth is a great start for a study. The more complex pathogens for which susceptibility testing is necessary are healthcare-associated pathogens or nosocomial pathogens, those acquired in hospitals and other healthcare settings. Hospital infection-control microbiologists generate and examine susceptibility data to generate periodic reports called antibiograms. These reports define the susceptibility of clinically isolated organisms to the antibiotics in current use. Antibiograms are used to monitor control of known pathogens, to track the emergence of new pathogens, and to identify the emergence of antibiotic resistance, all at the local level (Madigan, et al. 2012). May it be a simple susceptibility method or a complex one, all will show great results if done properly and carefully.

Figure 3.1: Streptomycin disk in (right) plate 1 and (left) plate 2.

Figure 3.2: Erythromycin disk in (right) plate 1 and (left) plate 2.

The measurements for the zones of inhibition were tabulated.

Table 1.0: Streptomycin ZOI and bioactivity.

Streptomycin

Plate 1 Plate 2

Zone of Inhibition (ZOI) in millimeters 22 24

Bioactivity Level

Very Active Very Active

The diameter of the zone is related to the susceptibility of the isolate and to the diffusion rate of the drug through the agar medium. The zone diameters of each drug are interpreted using

V. References
Madigan, M, Martinko, J, Stahl, D, and David, C. Brock Biology of Microorganisms 13th ed. Pearsons Education, Inc. 2012. Pages 888892. Reller, B, Weinstein, M, Jorgensen, J, and Ferraro, MJ. Clinical Infectious Diseases. Oxford Journals. July 2009. Vol. 49. Issue 11. Pp. 1749-1755. Perez, J, Beaman, C, Hinterlong, B, Hoekstra, B, and Leavell, S. Antimicrobial Susceptibility Test. Microbugz 2007. Retrieved Aug 2013. http://austincc.edu/microbugz/antimicrobial _susceptibility.php. Reppun, T, MD, and Watase, R, MT. Diagnostic Laboratory Services, Inc. Antimicrobial Susceptibility Testing. N.d. Santiago, K.A, Borricano, J.N, Canal, J, Marcelo, D.M, Perez, M.C, and dela Cruz, T.E. Antibacterial activities of fruticose lichens collected from selected sites in Luzon Island, Philippines. Philippine Science Letters. Vol.3. No.2. 2010. Hudzicki, J. Kirby-Baurer Disk Diffusion Susceptibility Test Protocol. ASM MicrobeLibrary. April 2013. http://www.microbelibrary.org/component/r esource/laboratory-test/3189-kirby-bauerdisk-diffusion-susceptibility-test-protocol.