Antibiotic Resistance due to Horizontal Gene Transfer in Bacteria Lab Section: L10 By: Jeevika Goyal (1209306), Sophie

Brevard, Tessa Klettl Keller To: Steven Botts Date: October 25th, 2013

Lab 2: Antibiotics Resistance and Horizontal Gene Transfer Introduction:

October 25th, 2013

Antibiotics were first discovered in 1928, by Alexander Fleming, and have been a primal source for treating bacterial infections (History of Learning Site, 2000-2013). Antibiotics- resistance occurs naturally among bacteria. This resistance among bacteria is transferable between different genus or species of bacteria. This process of exchanging genetic material with a partner is called Horizontal gene transfer. The genes for resistance are carried by bacteria on their plasmids or on transposons; which are used as vectors to transfer the desired gene. This transfer can occur by three different mechanisms: transformation, transduction or conjugation. These genes have been used in a variety of fields; such as Agriculture and medicine to treat severe problems. For example, in Agriculture these genes have been studied to prevent pathogenicity and to enhance crop yield. On the other hand, it has been studied in medicine to continually improve antibiotic medicines. (Bio2EE3 Lab 2 Manual, 2013) The purpose of this lab is to explore the degree of resistance that certain bacteria exhibit against certain antibiotics; and to observe the rate of gene transfer by mating two strains of E.Coli that will mate to produce potential offsprings with Ampicillin and Chloramphenical resistance. The E.Coli culture at various times will be obtained and various dilutions of it will be plated on agar containing Ampicllin and Chloramphenical. The organisms that have both strains of resistant antibiotics will be selected for. (Bio2EE3 Lab 2 Manual, 2013)It is expected that longer the bacterial cultural is incubated the more growth will be observed to have the greatest growth, as it will have had the maximum amount of time for both stains to have matted and produce subsequent generations with both strains. The second part of this lab is to explore the degree of susceptibility that a certain bacteria has for a certain antibiotic. It is hypothesized that antibiotics that affect the cell wall will be more harmful to gram positive compare to gram negative; while antibiotics that affect the protein activity will affect both bacterial types equally.

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Lab 2: Antibiotics Resistance and Horizontal Gene Transfer Methods: 2.1 Conjugative Transfer of Antibiotic Resistance Genes between E.coli Strains Refer to Bio 2EE3 lab manual (2013) for a detailed procedure 2.4 Sensitivity of four microorganisms to eight antibiotics Refer to Bio 2EE3 lab manual (2013) for a detailed procedure

October 25th, 2013

The only change made in the procedure was that the observations were supposed to be made 2 days after the lab; however, due to the long weekend, they were made 3 days after. This affected the bacterial count and the zone of inhibition; as they both grew in number due to increased incubation period. Results: 2.1 Conjugative Transfer of Antibiotic Resistance Genes between E.coli Strains In this part of experiment, the rate of plasmid transfer between E.coli Strains was examined by monitoring the rate of growth of E.coli colonies. Table 1.1 shows that there was a greater growth in E.coli sample that were incubated for 40 minutes. While the least amount of growth was observed in sample that was incubated for 10 minutes. In terms of dilutions, 10-1 had the most growth while no growth was observed in sample with 10-4 dilutions. The sample that was incubated for 10 minutes showed growth in dilutions 10-1 and 10-2.However the number of colonies in 10-2 dilution sample were only 3, which is outside the range of 5-200; and are therefore, considered unreliable. The sample at 20 minutes produced viable results in 10-2 and 10-3 dilutions; while sample at 30 minutes showed viable growth in the first three dilutions. As stated above, most growth was observed in sample incubated for 40 minutes; however, viable results were only produced in 10-3 dilutions. The numbers in Table 1.1 were converted into cfu/mL (Table 1.2) to get a more accurate value of colonies forming over time. It was done using the formula: colonies/ (amount plated * dilution factor). The pattern observed is same as that of Table 1.1; indicating the Page 3 of 10

Lab 2: Antibiotics Resistance and Horizontal Gene Transfer

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maximum amount of growth in E.coli sample that was incubated for 40 minutes with least amount of growth in sample incubated for 10 minutes. The hyphen in the table indicates that the colonies calculated were outside the range; thereby, cfu/mL for those values was not calculated. The averages of the colonies in Table 1.2, for respective dilution, were calculated and were graphed against time. Graph 1.1 shows an exponential growth of E.coli bacteria. It shows that the bacterial growth for first 20 minutes was slow and linear; however, they were growing steadily at an exponential rate after 30 minutes. Graph 1.2 is a Semi Logarithmic Graph of the same data. It shows that the growth in bacterial culture was increasing steadily for first 20 minutes, then it was consistent for next 10 minutes; after which it was again growing linearly. 2.4 Sensitivity of four microorganisms to eight antibiotics In this part of the experiment 4 strains of bacteria: E.coli; M. Luteus; P.Fluorescens and S.cerevisiae were subjected to 8 different types of antibiotics to measure their resistance to each. These antibiotics were ampicillin, chloramphenicol, gentamicin, kanamycin, penicillin, streptomycin, tetracycline and neomycin. The resistance to each antibiotic was measured by measuring the zone of inhibition; which is diameter of the clearing area around the each strain bacteria, for their respective antibiotic. For E.coli, the inhibition circle was largest in Chloramphenicol; while there was no inhibition zone observed in Penicillin. M.Luteus showed least resistance to Ampicillin, as it had the largest zone of inhibition diameter (3.6cm) around this antibiotic; while it showed the most resistance to Neomycin with diameter of 1.4 cm. P.fluorescens had the largest zone of inhibition around Tetracycline; however, it showed most resistance to Chloramphenicol and Penicillin. S. cerevisiae, on the other hand, was only susceptible to Neomycin, as it was the only antibiotic around which zone of inhibition was observed. Discussion: 2.1 Conjugative Transfer of Antibiotic Resistance Genes between E.coli Strains

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Lab 2: Antibiotics Resistance and Horizontal Gene Transfer

October 25th, 2013

It was hypothesised that the greatest growth will be observed in sample obtained from E.coli incubated for more than 30 minutes. This is because by this time most of the mating is supposed to have occurred; therefore, majority of the offsprings produced at this point must contain both ampicillin resistance and chloramphenicol resistance gene. The samples obtained at various times are grown on agar containing ampicilin and chloramphenicol; therefore, only organisms with both antibiotic resistance gene will be able to grow and reproduce. This is the reason, the sample obtained after 40 minute incubation should show a greater colony count. Similarly, least amount of growth was expected on agar plate containing sample obtained at 10 minutes, as this sample of bacteria have had least amount of time to mate and reproduce; therefore, very few bacteria at this point will contain both ampicillin and chloramphenicol resistance genes (Bio2EE3 Lab 2 Manual, 2013). This expected pattern was observed in the results, as it can be seen in table 1.2, that sample at 40 minutes was a very concentrated sample with both genes; as bacterial colonies from that culture were too many to count. Also, the growth was observed in all the dilution, except for 10-4 dilution, at thirty minutes. This suggests that most of the bacteria at this point have mated and subsequent generation contain both the antibiotic resistant gene. On the contrary, sample obtained after 20 and 10 minutes only produced colonies in the first two dilutions; indicating that very few bacteria have both the resistant gene. The average of bacterial colonies at various dilutions was graphed on a linear and logarithmic scale. The reason both the graphs differ because linear scale goes up by the same amount each time; while Logarithmic graph goes up by 10x each time. In comparison, Logarithmic graph is a more realistic representation, as it shows steady growth; unlike linear growth which shows a drastic growth from 30 to 40 minutes. If only the linear graph is considered, it would be assumed that most of the growth occurred only occurred in the last 10 minutes, as there is an exponential rise in the last ten minutes. However, this assumption would bias, as the growth was steady because bacteria are reproducing rapidly at a consistent rate. Therefore, log graph gives a better representation because it shows a steady growth; rather than a dramatic increase; thereby giving a better assumption of rate of plasmid transfer. Page 5 of 10

Lab 2: Antibiotics Resistance and Horizontal Gene Transfer 2.4 Sensitivity of four microorganisms to eight antibiotics

October 25th, 2013

In this part, the inhibition zone, clearing in bacterial cultural, is measured to examine an organisms susceptibility to a particular antibiotic. The greater the diameter of the inhibition zone is, the more susceptible the organism is to that antibiotic. It was expected that gram negative bacteria will be more resistant to antibiotics affecting cell wall synthesis; while the antibiotics affecting the protein activity will not discriminate between the two types of bacteria. The pattern observed aligned with the proposed idea as, for example E.coli shows least resistance to Chloramphenical while it shows the most resistance to Penicillin because the diameter around Chloramphenical is 3.4 cm and 0 cm around Pencillin. The reason it is most susceptible to Chloramphenical is because it inhibits a lot of enzyme activity which is required by bacteria to perform various cellular function (Mathers, 2013). It shows most resistance to Penicillin; because it affects bacteria by affecting its ability to make cell wall; however, E.coli is gram negative, and has an outer membrane that protects its cell wall, making Penicillin ineffective against E.coli.(Cells Alive,2013) M.luteus shows most susceptibility to Ampicillin, which has a diameter of 3.6cm, and most resistant to Neomycin with diameter of inhibition zone being 1.4cm. This is because Ampicillin disrupts the cell wall in a bacterial cell; and since M.luteus is a gram positive bacterium, it is more susceptible to it than a gram negative bacteria (Cliff Notes, 2013s). Neomycin damages the cell membrane; however, in gram positive, it is protected by cell wall, making it more resistant (Health24.com, 2013). P.Fluorescens is most resistant to Penicillin, Ampicillin and Chloramphenicol with their diameter being 0cm; while it is least resistant to Tetracycline. This is because all three Penicillin, Ampicillin and Chloramphenicol disrupts cell walls of an organism and since P.Fluorescens is gram negative, its cell wall is protected by its outer membrane; making these three antibiotics ineffective against P.Fluorescens. However, it most susceptible to Tetracycline because this antibiotics inhibits a lot of enzymes involved in cellular functions of the bacterium. (Rafal Klajn, 2013) S.cerevisae only showed susceptibility to Neomycin. It is an error because S.cerevisae is a fungus while Neomycin affects bacterial cell wall; which is why this fungus should be resistant to it. S.cerevisae is a fungus while most of the antibiotics affect either gram positive or negative or both. Page 6 of 10

Lab 2: Antibiotics Resistance and Horizontal Gene Transfer

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(Health24.com, 2013)The most versatile antibiotic is tetracycline because it affects the protein activity rather than the cell walls; which is why it can be used against both gram positive and gram negative bacteria (Rafal Klajn, 2013)

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Lab 2: Antibiotics Resistance and Horizontal Gene Transfer Appendix:

Time (min) Dilutions 10-1 10-2 10-3 10-4 10 37 3 0 0 20 98 56 3 0 30 148 18 10 0 40 TNTC TNTC 23 0

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Table 1.1: Table depicting the growth of colonies over time; thus, indicating the increase in Plasmid transfer over time. * disregard values outside the range 5-200
Colony Forming Units/mL (cfu/mL) Time (min) Dilutions 10-1 10 20 30 40 -

10-2 10-3 10-4

=3700cfu/ mL -

=9800cfu/mL

=14800cfu/mL -

= 56000cfu/mL ((9800+56000)/2)=32 900cfu/mL

=18000cfu/mL =100000cfu/mL ((14800+18000+100000)/3)= 44266 cfu/mL =230000cfu/mL 230000cfu/mL

Avg 3700 cfu/mL

Table 1.2: Conversion of values in Table 1.1 into cfu/mL

Inhibition Diameter (cm) in various Bacterial Cultures Treatment/ Antibiotic Penicillin (P2) Kanamycin(K30) Gentamicin(GM10) Ampicillin(AM2) Tetracycline(TE5) Neomycin(N30) Streptomycin(S10) Chloramphenicol (C30) Escherichia coli 0cm 1.2cm 0.8cm 1.6cm 1.8cm 0.8cm 1cm 2.4cm Bacteria Type Micrococcs Pseudomonas luteus Fluorescens 2.3cm 0 cm 2 cm 1.6 cm 1.5 cm 1 cm 3.6 cm 0 cm 2.4 cm 1.8 cm 1.4 cm 1.1 cm 2.2 cm 1.6 cm 3.4 cm 0 cm Saccharomyces cerevisiae 0 cm 0 cm 0 cm 0 cm 0 cm 1 cm 0 cm 0 cm

Table 1.3: Table summarizes the degree of resistance exhibited by E.coli, M.luteus, P.Fluorescens and S.cerevisiae towards P2, k30, Gm10, AM2, TE5, N30, S10, C30. Page 8 of 10

Lab 2: Antibiotics Resistance and Horizontal Gene Transfer

October 25th, 2013

Coorelation between exconjugants of E.coli of different diloutions and time

250000

200000 exconjugnts (cfu/mL)

150000

100000

50000

0 0 5 10 15 20 25 30 35 40 45 Time (min)

Graph 1.1: Graph depicting an exponential growth of E.coli colonies over time

Coorelation between exconjugants of E.coli of different diloutions and time

1000000 100000 exconjugants (cfu/mL) 10000 1000 100 10 1 0 5 10 15 20 25 30 35 40 45 TIme (min)

Graph 1.2: Logarithmic graph depicting a linear relationship between growth of E.coli colonies and time Page 9 of 10

Lab 2: Antibiotics Resistance and Horizontal Gene Transfer Work Cited:

October 25th, 2013

Bio2EE3 Lab 2 Manual (2013). Lab 2: Production of Gene Transfer and Antibiotic Resistance. McMaster University. Cells Alive (2013). How Penicillin kills Bacteria. [ONLINE] Available at: http://www.cellsalive.com/pen.htm. [Last Accessed October 22, 2013]. CliffNotes (2013). Antibiotics. [ONLINE] Available at: http://www.cliffsnotes.com/sciences/biology/microbiology/control-of-microbial-growth/antibiotics. [Last Accessed October 21, 2013]. Drugs.com (2013). Ampicilin. [ONLINE] Available at: http://www.drugs.com/cdi/ampicillin.html. [Last Accessed October 21, 2013]. health24.com (2013). Neomycin. [ONLINE] Available at: http://www.health24.com/Medical/Meds-andyou/Medication/Neomycin-Client-20120721. [Last Accessed October 22, 2013]. History of Learning Site (2000-2013). Antibiotics. [ONLINE] Available at: http://www.historylearningsite.co.uk/antibiotics.htm. [Last Accessed October 21, 2013]. Mathers, C. (2013). Effect of chloramphenicol on bacteria. [ONLINE] Retrieved from http://www.ehow.com/about_6514623_effect-chloramphenicol-bacteria.html . [Last Accessed October 21, 2013]. Rafal Klajn (2013). Antimicrobial Properties. [ONLINE] Available at: http://www.chm.bris.ac.uk/motm/tetracycline/refer.htm. [Last Accessed October 21, 2013]. Wessner, (2013). Microbiology: Applications and Connections. 1st ed. Canada: John Wiley & Sons Canada Ltd..

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