Question

Part III – Restoring Susceptibility

Katelyn had been working for Dr. Johnson for a month, and while she had become quite good at measuring inhibition zones, she didn’t know why she was doing all this work. She had gotten very curious after she began doing all the measurements on a new set of antibiotics. This experiment involved

infecting mice with MRSA and tracking ho the MRSA grew over time.

Data was collected by counting the cells of MRSA taken from fluid samples from the nice. The cells were measured by taking one gram of the fluid and spreading it over plates, but now Katelyn counted the colonies that grew on the plate after 24 hours. Because there were so many, she actually measured the colonies as “log CFU/g.” A CFU is a colony forming unit, or essentially a cell that will divide into a colony that can be seen. Because there can be so many, Katelyn measured them on a logarithmic (log) scale. The raw data in her lab notebook looked like the following:

Table 1: Effect of Treatment on MRSA in Mice After 24 Hours of Drug Treatment as log CFU/g
		Treatment
Trial	Control	FtsZ inhibitor	Imipenem	FtsZ Inhibitor + Imipenem
1	9.11	7.55	6.98	2.21
2	8.25	8.12	8.12	4.55
3	9.05	9.27	9.01	7.98
4	9.37	8.02	8.33	5.64
5	8.80	7.65	7.64	1.25
6	9.25	8.30	7.77	9.98
7	9.41	7.99	8.21	6.78
8	9.11	7.71	7.98	3.45
9	8.61	8.22	7.68	2.45
10	9.12	8.11	8.21	1.01

Questions

5. Looking at the numbers in Table 1, do the FtsZ inhibitor and/or Imipenem have any effect on the

number of MRSA colonies, either alone or together? Keep in mind that a log value means each

integer increase is actually a ten-fold increase in the number of cells; please be as specific as possible

with your answer.

6. What do you think FtsZ Inhibitor and Imipenem are

Next, Katelyn further analyzed the data she collected by calculating the average and standard error:

Table 2: Average Effect of Treatment on MRSA in Mice After 24 Hours of Drug Treatment as log CFU/g
		Treatment
	Control	FtsZ inhibitor	Imipenem	FtsZ Inhibitor + Imipenem
Average	9.008	8.094	7.993	4.53
SE	0.114	0.153	0.169	0.954

Question

7. Does Table 2 change your interpretation of the experimental data from Question 6? Why or why not?

She then made the following graph (Figure 9):

Figure 9. Effects of treatments on MRSA numbers in mice. Samples were taken at 24 hours post-infection. (Figure modified from Tan et al. 2012).

Katelyn was very excited by the results, but she didn’t know what an FtsZ inhibitor was, or what

imipenem was. She decided you ask Dr. Johnson what his research was all about.

“Dr. Johnson, look at these results I got from the last round of plates,” Katelyn said as she handed him a

copy of the results above. “What exactly are we testing here?

Dr. Johnson looked at the results and smiled. “These are great! This could really change the way we deal

with antibiotic resistance.

“To answer your question, ß-lactam antibiotics are still the most heavily used antibiotics, though resistance is a big problem. Most treatments have changed to using multidrug resistance in the hopes of

allowing the antibiotic to still function while at least slowing down the resistance mechanism.

“Another approach involves looking for other proteins that could be inhibited, and looking for existing

inhibitors to make into drugs. Instead of looking just for new antibiotics, we’re looking for new targets.”

Dr. Johnson handed Katelyn a few papers to read. In them she learned that the protein, FtsZ, helps “pinch off” the new cells at the end of cell division. This involves interacting with the cell wall as it is synthesized, and if FtsZ is interfered with, cell wall synthesis stops too. This prevents cell division and

the microbe can no longer reproduce.

Dr. Johnson tested the new target idea by using a recently discovered inhibitor of FtsZ to see what effects that had on a MRSA infection. As part of the study, the inhibitor was tested by itself and in combination with imipenem, a ß-lactam antibiotic, resulting in the above data.

Questions

8. How effective was the FtsZ inhibitor alone in treating MRSA in mice?

9. How effective was the imipenem alone in treating MRSA in mice?

10. How effective was the combination of the inhibitor and the ß-lactam antibiotic in treating MRSA in

mice?

11. How would you explain the results illustrated in Figure 9?

Answer 1

5) By looking into the table we can clearly say that the FtsZ inhibitor and Imipenem combined together have effect on the number of MRSA colonies as the log CFU/g of both of the drug combined together has shown a decrease in the number of colonies, compared to the individual treatment with FtsZ inhibitor and Imipenem alone.

6) A highly conserved prokaryotic cell division protein FtsZ is considered as a promising target by inhibiting bacterial cytokinesis. Inhibition of FtsZ assembly restrains the cell-division complex known as divisome, which results in filamentation, leading to lysis of the cell. Imipenem is a beta-lactam antibiotic belongings to the subgroup of carbapenems. Imipenem acts as an antimicrobial through the inhibition of cell wall synthesis of various gram-positive and gram-negative bacteria. This inhibition of cell wall synthesis in gram-negative bateria is attained by binding to penicillin-binding proteins (PBPs). FtsZ inhibitor & Imipenem alone both are not highly effective, but together they can perform at a much higher effectiveness.

7) Yes. From this table we can see that the standard error of FtsZ inhibitor+Imipenem is large compared to FtsZ inhibitor and Imipenem alone, which means that it is not a correct representation of the population. So we cant accurately say from the data set that FtsZ inhibitor and Imipenem combined together has a great effect on MRSA. (Standard error refers to the standard deviation of the distribution of sample means taken from a population. The smaller the standard error, the more representative the sample will be of the overall population.The more data points involved in the calculations of the mean, the smaller the standard error tends to be. When the standard error is small, the data is said to be more representative of the true mean. In cases where the standard error is large, the data may have some notable irregularities.)

8) It was not as effective as the combination of Ftsz inhibitor and Imipenem. The filamentous temperature-sensitive protein Z (FtsZ) is an essential protein for bacterial cell division and viability. FtsZ self-assembles into polymers and then forms a Z-ring at the site of division, which serves as a scaffold to recruit other key protein constituents of the cell division machinery. Inhibiting ftsZ gene expression or disrupting FtsZ protein activity may prove to be a novel therapeutic strategy against bacterial infections, by disrupting bacterial growth patterns. Some studies indicated that a small-molecule inhibitor of FtsZ could treat infections caused by S. aureus. Recently, several other studies indicated that small-molecule inhibitors of FtsZ PC190723 also could treat infections caused by S. aureus. In one study, the researcher conjugated a locked nucleic acid (LNA) that targeted ftsZ mRNA with the peptide (KFF)3K, to generate peptide-LNA (PLNA). They found out that PLNA787 inhibited bacterial growth and resolved lethal Mu50 infections in epithelial cell cultures. PLNA787 also improved the survival rates of Mu50-infected mice and was associated with reductions of bacterial titers in several tissue types. The inhibitory effects on ftsZ mRNA and FtsZ protein expression and inhibition of the bacterial cell division process are considered to be the major mechanisms of PLNA. PLNA787 demonstrated activity against MRSA infections in vitro and in vivo.

9) It was not as effective as the combination of Ftsz inhibitor and Imipenem. Imipenem, a carbapenem, acts as an antimicrobial through the inhibition of cell wall synthesis of various gram-positive and gram-negative bacteria. It does not have bactericidal action against MRSA but it has a broad antibacterial spectrum covering both Gram-positive and Gram-negative bacteria, and has a stronger bactericidal activity than other β-lactams.