Effect of Broad and Narrow Spectrum Antibiotics on Gram-Positive S.aureus and GramNegative E.

coli Natalia Prieto September 27 2013

Introduction: With the many different types of antibiotics available to the public, information on what an antibiotic is and how it actually affects an organism is important to understand. An antibiotic is a chemical made by a microorganism to inhibit or retard the growth of other microorganisms (CDC 2010). Antibiotics can be classified into two categories, broad and narrow spectrum. Broad spectrum antibiotics work in both gram-positive and gram-negative bacteria while narrow spectrum antibiotics only work on certain types of bacteria (Spectrum of Activity 2011). By using the method of Gram staining, bacteria can also be classified into two categories; gram-positive and gram-negative (Sadava et. al 2011). Gram-positive bacteria can be identified by the confirmation of the cell wall. This type of bacteria is characterized as having a thick peptidoglycan layer and will stain purple/blue. Gram-negative bacteria have a more complex cell wall involving two phospholipid bilayers and one peptidoglycan layer. Gram-negative bacteria stain red or pink (Sadava et. al 2011). This lab was focused on four antibiotics, ampicillin, erythromycin, penicillin, and tetracycline as well as two types of bacteria, gram-negative E.coli, and gram-positive S.aureus. Penicillin is considered to be a narrow spectrum antibiotic (Spectrum of Activity 2011) that works by disrupting the peptidoglycan cell wall of bacteria (Todar 2008). Erythromycin is a narrow-spectrum antibiotic that works by stopping protein synthesis (SRS Pharmaceuticals 2010). Ampicillin is considered to be a broad spectrum antibiotic that works by inhibiting the synthesis of the cell wall (Acred et al. 1962). Tetracycline is also a broad spectrum antibiotic (SRS Pharmaceuticals 2010) that works by stopping enzymatic reactions within the bacterial cells (Klajn).

It is predicted that ampicillin and tetracycline will be effective on both E.coli and S.aureus because they are broad spectrum antibiotics. It is also predicted that penicillin will be effective against S.aureus because S.aureus is a gram-positive bacteria with peptidoglycan that penicillin would affect. However E.coli, being gram-negative, will not be affected by penicillin since E.coli only has one thin peptidoglycan layer. It is also predicted that erythromycin will affect either E.coli or S.aureus because erythromycin is a narrow spectrum antibiotic that affects only certain types of bacteria.

Methods: Two agar plates were obtained. Using a sterile swab two bacterial lawns were prepared; one of S.aureus and E.coli. Four antibiotic discs were placed opposite each other on the surface of the lawn. A disk containing no antibiotics was placed in the middle of the lawn to be used as a control. The four antibiotics used were ampicillin, erythromycin, penicillin, and tetracycline. A Kirby Bauer test will be used to determine antibiotic susceptibility of E.coli and S.aureus. Standardized data on the sensitivity of the bacteria can be found on Table 1.5 in the lab manual (Seagull and Aronson, 2013).

Results: The S.aureus lawn of bacteria was not as clearly visible as was the E.coli lawn. Because of this the edges of the zones of inhibition in S.aureus were hard to identify and were much clearer in E.coli. The control disc, containing no antibiotic, did not yield a zone of inhibition for either E.coli or S.aureus (Figure 1).

Mean Diameter Zone of Inhibition (mm)

60 50 40 30 20 10 0 Ampicillin (Am) Erythromycin Penicillin Tetracycline (E) (P) (T) Antibitic (Disc Code) Control (C) S.aureus E.coli

Figure 1: Class mean diameter zone of inhibition (mm) of antibiotics Ampicillin, Erythromycin, Tetracycline, and the control on S.aureus and E.coli cultures (+/- One std dev).

All of the antibiotics presented to S.aureus affected the bacteria similarly on average. Penicillin affected S.aureus the least having the smallest zone of inhibition. For all of the antibiotics the standard deviation was the very high. The highest deviation was for penicillin while the smallest was for ampicillin (Figure 1). S.aureus was determined to be susceptible to all of the antibiotics based on the Kirby Bauer test (Figure 1). Ampicillin, erythromycin, and tetracycline had similar effects on E.coli (Figure 1). Erythromycin had the smallest zone of inhibition, tetracycline had the largest zone of inhibition. The standard deviation for these antibiotics was relatively small when compared to the zone of inhibition of S.aureus. E.coli did not yield a zone of inhibition when exposed to penicillin (Figure 1). By comparing experimental data with Table 1.5 in lab manual (Seagull and Aronson, 2013), E.coli was determined to be susceptible to ampicillin, erythromycin, and tetracycline and resistant to penicillin based on the Kirby Bauer test.

Ampicillin, erythromycin, penicillin, and tetracycline inhibited the S.aureus and yielded larger zones of inhibition than E.coli. The largest difference in the zones of inhibition was created by penicillin on S.aureus and E.coli. The smallest difference in the zones of inhibition was created by tetracycline. For the individual data, the zones of inhibition of S.aureus were variable. There was also a large difference between the zones of inhibition of S.aureus and E.coli. However, the data had the same trends as the average class data (Table 1). Table 1: Group Diameter zone of inhibition (mm) of antibiotics Ampicillin, Erythromycin, Tetracycline, and the control on S.aureus and E.coli Diameter of Diameter of Antibiotic (Disc Zone of Zone of Code) Inhibition in Inhibition in S.aureus (mm) E.coli (mm) Ampicillin (Am) 40 28 Erythromycin (E) 30 28 Penicillin (P) 27 0 Tetracycline (Te) 32 26 Control 0 0

For S.aureus ampicillin created the largest zone of inhibition. The smallest zone of inhibition was made by penicillin. The zones of inhibition created by the antibiotics on S.aureus varied from very big to very small. The zones of inhibition were larger S.aureus than in E.coli (Table 1). By comparing experimental data with Table 1.5 in lab manual (Seagull and Aronson, 2013), S.aureus was determined to be susceptible to all of the antibiotics based on the Kirby Bauer test. Except for penicillin, E.coli exhibited similar susceptibility to the antibiotics used (Figure 1). There was not much variability on the zones of inhibition of E.coli created by the antibiotics. Penicillin did not yield a zone of inhibition when presented to the E.coli (Table 1). By comparing experimental data with Table 1.5 in lab manual (Seagull and Aronson, 2013),

E.coli was determined to be susceptible to ampicillin, erythromycin, and tetracycline and resistant to penicillin based on the Kirby Bauer test.

Discussion: S.aureus was most sensitive to erythromycin but by a small margin. From the antibiotics we tested, S.aureus was not resistant to any of the antibiotics. E.coli was most sensitive to tetracycline. E.coli was resistant to penicillin. Erythromycin would be recommended for treatment of S.aureus as it had the most effect. Tetracycline would be recommended for treatment of E.coli. The difference in the results when considering gram-positive or gramnegative bacteria is that be that E.coli had smaller zones of inhibition than did S.aureus. E.coli was the most resistant to the antibiotics, apart from having smaller zones of inhibition, it was resistant to penicillin were S.aureus wasnt (Figure 1). The more complex cell wall of gramnegative-bacteria (Sadava, 2009), could be the reason of why E.coli was more resistant to the antibiotics. The results supported the prediction that ampicillin and tetracycline would be effective on both E.coli and S.aureus. The two antibiotics affected the two bacterial specimens. The results also supported that penicillin would be effective against S.aureus but not E.coli. E.coli was completely resistant to penicillin. A possible explanation for the penicillin-resistant strain of E.coli could be due to mutations that made E.coli resistant. A study conducted by Kristin Eriksson-Grennberg et.al (1965), found that E.coli resistant to penicillins carried a gene mutation that made it resistant to ampicillin. Another explanation is that resistance can be acquired through conjugation (Courvalin, 1994). A study performed by Patrice Courvalin (1994) found

that resistance of the microorganisms was obtained through conjugation of gram-positive and gram-negative bacteria. The results did not support the prediction that erythromycin would affect either E.coli or S.aureus Erythromycin affected E.coli and S.aureus. All of the antibiotics made a larger zone of inhibition in the S.aureus lawns. A possible reason for the larger diameters is that the complexity of gram-negative cell wall allows for permeability to be controlled (Nikaido, 1989). The lower the permeability of the cell wall the higher the chance for resistance (Nikiado, 1989). The zones of inhibition for S.aureus were similar to each other as were the zones of inhibition for E.coli.