Isolation of Ethanol Reduced-Susceptibility Staphylococcus aureus Mutants Ashley J. Simenson, Nathanial J. Torres, and John E. Gustafson Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078

0%

Background Staphylococcus aureus is a Gram-positive pathogen commonly found in the noses of up to 50% of healthy people (1). Studying S. aureus is of importance based on the microbe’s ability to elude the effects of antimicrobial agents, notably, Methicillinresistant Staphylococcus aureus or MRSA. MRSA is resistant to over 50% of the antimicrobials available for the treatment of infections caused by this pathogen. In 2011, MRSA led to 80,461 severe invasive infections and 11,285 deaths. Comparatively, 7,638 people died in the same year due to complications of HIV/AIDS (2). The utilization of alcohol-based hand sanitizers, specifically those containing ethanol, has been used to combat and prevent the spread of MRSA infections within the public and hospital setting (3). Ethanol works to kill microbes by making the cytoplasmic membrane porous leading to the leakage of intracellular content (4) (Fig. 1) Therefore, it is imperative to determine whether or not S. aureus has the capability to evolve in such a way as to evade the effects of ethanol. From previous studies, the utilization of the gradient plate method was successful in isolating S. aureus mutants that demonstrated a reduced susceptibility to a membrane active hydrocarbon mixture tea tree oil (5). Therefore, S. aureus strain SH1000 was used to isolate ethanol reduced susceptibility mutants using the gradient plate technique (5).

20% Ethanol

Table 2. Ethanol minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays data. Strain

Parent Strain

SH1000

Figure 2. SH1000 ethanol reduced susceptibility isolation utilizing the gradient plate technique. MHA plate containing 0 to 20% ethanol grown at 37˚C for 48 hrs.

EtOH MICa

EtOH MBCa

9.67±0.52

16.33±0.52

A

SH1000

9.33±0.58

15.33±0.58

B

SH1000

9.67±0.58

15.33±0.58

C

SH1000

10.33±0.58

15.00±1.00

D

SH1000

9.33±0.58

14.00±1.00

E

SH1000

9.33±0.58

14.67±2.08

F

SH1000

9.67±0.58

15.00±1.00

G

SH1000

8.67±0.58

14.67±2.08

H

SH1000

9.00±1.00

15.00±1.73

I

SH1000

9.00

13.67±1.15

J

SH1000

9.00±1.00

13.00

ethanol (EtOH) % v/v ± SD, n=3 b Two-tailed t-test – p ≤0.05, compared with respective parent strain SH1000 (no significant results) a

CH3 – CH2 – OH

Results Figure 1. Ethanol causes the cellular membrane to become porous. As a result, intracellular content leaks from the cell. Figure 3. SH1000 and proposed mutants grown on an MHA plate containing 0 to 18% ethanol at 37˚C for 72 hrs.

Methods Gradient plate isolation technique: Gradient plates were made by elevating one end of a square petri plate (100 X 15 mm square shaped, Becton Dickinson Labware, Franklin Lakes, NJ) to a height of 6 mm and then adding 40 mL of Mueller-Hinton Agar (MHA) (granulated agar, Fisher Scientific, Pittsburgh, PA). The plates were left to solidify overnight. To make the second layer, MHA was combined with filter sterilized (0.2 μm Nylon, Fisher Scientific, Pittsburgh, PA) ethanol (200 proof-absolute anhydrous, Pharmco-AAPER, Brookfield, CT) to achieve a concentration of 20% ethanol in 40 mL of MHA. After drying for 4 hrs, an overnight culture prepared by inoculating a single colony from SH1000 into 2 mL of Mueller-Hinton Broth (MHB) (Becton Dickinson, Sparks, MD) incubated at 37˚C, 200 rpm, for over 12 hr was then diluted to an OD580 = 1.00. A sterile cotton swab (Fisher Scientific, Pittsburgh, PA) was then used to swab the entire surface of the ethanol gradient plate. The plates were then incubated for 48 hr at 37˚C and the growth (Fig. 2) was measured and recorded. After the incubation period, an inoculating loop was then dragged across the highest ethanol concentration where colonies appeared (Fig. 2), which was then used to streak an MHA plate to isolate individual colonies. Ten individual colonies were then selected from this primary streak plate and struck onto MHA plates. These colonies were then considered putative ethanol reduced susceptibility mutants. Gradient Plate Assay: Overnight cultures, prepared as above made from the putative ethanol mutants, were diluted to an OD580 = 1.00. Each isolated mutant was then swabbed onto a single lane of an 18% ethanol gradient plate, prepared as above. Growth of each strain was measured after 48 and 72 hrs of incubation (Fig. 3). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays: Overnight cultures were made using single colonies of the mutants isolated above and the parent strain SH1000. These cultures were then diluted to an OD580 = 0.01 and added to test tubes containing 8-40% ethanol in MHB. The test tubes were statically incubated for 24 hours at 37˚C after which the MIC was determined. To determine the MBC, 100 μL of test tubes containing the ethanol MIC and higher were then plated onto MHA plates and grown at 37˚C for 24 hr.

Table 1. Distance of putative ethanol reduced-susceptibility mutants grown on18% ethanol gradient plates Strain Parent Strain Distance of Growth (mm)a SH1000

24.83±2.79

All results of the MIC and MBC assays of the suspected ethanol reduced-susceptibility mutants were not significantly different from that of the parent strain SH1000 (Table 1). This was an unexpected result. Previously, S. aureus mutants demonstrating reducedsusceptibility to the complex membrane active substances tea tree oil and household disinfectants were reported on. These mutants also demonstrated reduced susceptibility to alcohols. Therefore, we expected to be able to isolate ethanol reduced-susceptibility mutants with ease. However, results presented indicated that the method used for selection of ethanol reduced-susceptibility mutants of SH1000 proved unsuccessful. It is possible that by increasing inoculum size or by using liquid media, ethanol reduced-susceptibility mutants might be isolated from this organism. It is also possible that the MIC and MBC method employed cannot detect a difference in ethanol susceptibility between the mutants and parent strains. Additional work in the laboratory that is not shown demonstrated that mutants selected with this procedure grow the same distance on ethanol gradients as the parent strain, but the growth of the mutants is more pronounced and appears more dense. This dense growth appears to be a distinct phenotype unique to the ethanol reduced-susceptibility mutants. Statistically, the proposed mutants were no different in their susceptibility to ethanol than the parent strain SH1000. Since another method used to inoculate gradient plates yielded more promising mutants, that method will likely be used for future projects. Using methods other than MIC and MBC assays to classify mutants, such as growth curves and Kirby Bauer disk diffusion tests, may be more successful.

A B

SH1000 SH1000

24.67±3.51 21.33±4.62

C

SH1000

20.67±4.16

D

SH1000

22.0±7.21

E

SH1000

24.0±4.36

F

SH1000

22.33±3.06

G

SH1000

19.33±4.73

H

SH1000

22.67±1.53

Future Work

I

SH1000

22.33±2.31

J

SH1000

24.67±1.53

Determination of the genes responsible for the ethanol-reduced susceptibility phenotype and the mechanisms the cell uses to elude the effects of ethanol.

Distance of growth (mm) ± SD, n=3 b Two-tailed t-test – p ≤0.05, compared with respective parent strain SH1000 (no significant results) a

Acknowledgements The authors would like to acknowledge funding provided by the Oklahoma State University Freshman Research Program and the Oklahoma Agricultural Experimental Station.

References 1. Warnke P, Harnack T, Ottl P, Kundt G, Podbielski A. 2014. Nasal screening for Staphylococcus aureus - daily routine with improvementpotentials. PLOS one 9:1-7. 2. Hoyert DL, Xu J. 2012. Deaths: preliminary data for 2011. National Vital Statistics Reports 61:1-52. 3. Kampf G, Kramer A. 2004. Epidemiological background of hand hygiene and evaluation of the most important agent for scrubs and rubs. Clinical Microbiology Review 14:863-893. 4. Goldstein DB. 1986. Effect of alcohol on cellular membranes. Annals of Emergency Medicine 15:1013-1018. 5. Cuaron JA, Dulal S, Song Y, Singh AK, Montelongo CE, Yu W, Nagarajan V, Jayaswal RK, Wilkinson BJ, Gustafson JE. 2012. Tea tree oil-induced transcriptional alteration in Staphylococcus aureus. Phytotherapy Research 27:390-396.