Question

Procedure

Four different colors of beads, representing four species of bacteria, all possible residents of the microbiome, will be utilized. The species differ naturally in their resistance to a particular antibiotic. Three different antibiotic treatment regimens will be modeled:

• following doctor’s orders
• ending antibiotic treatment early
• skipping Day 3 of treatment

Your initial population of bacteria is represented as follows:

• 40 beads that represent Species 1, the most susceptible to the antibiotic (susceptible on Day 1 of treatment)
• 30 beads that represent Species 2 (susceptible on Day 2), slightly more resistant than Species 1
• 20 beads that represent Species 3 (susceptible on Day 3), slightly more resistant than Species 2
• 10 beads that represent Species 4 (susceptible on Day 4), the most resistant species of all.

Thus the species vary in the concentration of antibiotic and/or the duration of exposure necessary to kill them. At the beginning of each “day:”

• The antibiotic is taken (except where noted below).
• Ten percent of the total number of bacteria is removed through the action of host immune system defenses (species are assumed to be equally susceptible to these responses). The total number of bacteria to be removed is calculated, and then taken randomly from among the four species. Do this by having one group member close their eyes, touch one of the four cups holding the beads, then remove one from the cup. Continue until the calculated number of bacteria has been removed from the population. On Day 0 and each subsequent day, 10% of the bacteria is removed.
• If the species is susceptible on that particular day, then half of those bacteria are removed. For instance, on Day 1 of the experiment, Species 1 is susceptible and half of those microbes are removed from the population. By Day 4, all the species would be susceptible and half the bacteria from each cup would be removed.
• If a species is not susceptible on a particular day, then the number of bacteria is actually doubled, simulating microbial division. For instance, on Day 1 of the experiment, Species 2, 3, and 4 are not susceptible, so

the number in their respective cups would be doubled. On Day 4, only Species 4 is not susceptible, so that population would be the only one to be doubled.

• Each group should make their calculations and use the data collection sheet to enter the number of each species of bacteria present on each day until 0 bacteria are counted in each species. The number of days necessary to reach 0 will vary according to the antibiotic regimen, and for some regimens will never occur.

Regimens

• Following doctor’s orders: Follow the directions above exactly.
• Ending treatment early: Discontinue antibiotic use on Day 5. At this point, a patient on antibiotics would begin to feel better and might stop taking their prescribed medication. Once antibiotic use is discontinued, 10%

of the total number of microbes is removed randomly at the start of each day. Following that, all species will double in number as antibiotic is no longer present.

• Skip Day 3 of treatment: Often, patients think it’s no big deal if they skip a day or two of a prescribed antibiotic regimen. Assume that when a day of taking the antibiotic is skipped, the antibiotic concentration will fall to 0 and numbers of all species will double on that day. Following that, the “susceptibility clock” is reset to Day 1. In other words, after the skipped day, only Species 1 is susceptible. Two days after the skipped day, Species 1 and 2 are susceptible, etc.

Questions

1. Assume Species 1, 2, and 3 are normal gut inhabitants and are not pathogenic. Species 4 represents a pathogen such as C. difficile. Explain, using data from this simulation, how C. diff can be caused by overuse and misuse of antibiotics.
2. How do the number and distribution of bacteria differ when the prescribed treatment regimen is followed and when antibiotic treatment is stopped prematurely?
3. What are the effects of skipping a day of treatment?
4. Dr. Lita Proctor, Program Director for the Human Microbiome Project, has said this about antibiotic treatment: “We can often cause more problems than we cure in many cases when we take antibiotics, especially when we don’t take the full regimen.” How would you respond to this statement?
5. Explain why the following statement is incorrect: “Antibiotics have created resistant bacteria.”
6. How does this simulation provide support for treatments such as FMT for infections such as C. diff ?
7. Does this simulation change any thoughts you have on antibiotic use? How?

Answer 1

When we use the normal course of antibiotics and follow the doctor's order exactly, on the eigth day of tge antibiotic course, all the species of bacteria along with the pathogenic species 4 would be removed completely from the body. But as in regimen 1 when the antibiotic is discontinued in day 5, species 1,2,3 and also 4 would double in number increasing in population every single day with species 4 causing pathogenecity. If however according to the regiman 2 when day 3 of the antibiotic course has been skipped, the pathogenic load would increase. Even if the antibiotic course is now started again, it would take a total of 12 days to remove the species 4 completely from the body.

The data can be summarised as follows. Table 1 represent the number of species 1,2,3 and 4 if normal course of antibiotics if followed according to the doctor's orders and all the given criteria are met.

Day	Species 1	Species 2	Species 3	Species 4
0	40-3=37	30-3=27	20-2=18	10-2=8
1(10% population removed by body's natural immunity. Also only Species 1 is susceptible and the rest are not susceptible. Those who are susceptible, their number is reduced to half but those non susceptible will be doubled.)	17	50	32	12
2(10% population removed by body's immune immunity. Also Species 1 and 2 are susceptible and the rest are non susceptible)	7	24	58	20
3( 10% of the population are removed by body's natural immunity. Also Species 1, 2 and 3 are susceptible while 4 is not susceptible)	2	11	28	36
4 (10% of the population is removed by body's natural immunity. Also all the Species are susceptible)	0	5	13	17
5(10% removal by body's natural immunity and half the number of each Species removed by antibiotic susceptibility)	0	2	6	8
6( the above condition prevails)	0	0	3	4
7	0	0	0	2
8	0	0	0	0

Now if antibiotic course is skipped on the day 3, the whole scenario of the population load of the bacteria would change and the antibiotics have to be continued for much longer time to remove the pathogenic species 4 completely from the body. The following table represents the condition if antibiotic is skipped on day 3 the number of each Species would vary from the original table

Day	Species 1	Species 2	Species 3	Species 4
0	37	27	18	8
1	17	50	32	12
2	7	24	58	20
3( when antibiotic is skipped which would reboot the whole system doubling the population of each species after 10% removal by the body's natural immunity)	8	42	110	36
4( thw whole population of different species of bacteria would follpw the day 1 of the antibiotic course when only Species 1 is susceptible with its population being reduced to half while the rest of the Species would double because of non susceptible nature after 10% removal by body's natural immunity.)	0	68	206	58
5. (Species. 2 is susceptible whilethe rest are not)	0	29	390	94
6(Species 2 and 3 are susceptible while 4 is not)	0	6	187	154
7(Species 4 also become susceptible)	0	0	86	72
8	0	0	39	32
9	0	0	8	9
10	0	0	4	4
11	0	0	1	2
12	0	0	0	0

Thus, the table can be studied to understand how skipping the antibiotic for a single day can increase the bacterial load in the body causing pathogenecity of the Species 4 which would require intake of antibiotics for a much longer duration.

If however the antibiotic course is discontinued on day 5 of the organinal antibiotic course, the number of bacteria of all Species 2, 3 and 4 would increase to 8,24 and 32 and would increase to double their number every single day if antibiotics are not started again.