The Effect of Antibiotic Treatments on the Growth of Pseudomonas Stutzeri

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GSU Research Day Research Day 2017

Apr 7th, 4:00 PM - 6:00 PM The effect of antibiotic treatments on the growth of Pseudomonas stutzeri and Enterobacter gergoviae and the fermentation of several carbohydrates by Enterobacter gergoviae, Enterobacter aerogenes, and Pseudomonas aeruginosa Diana Acosta Biology Program, CAS, Governors State University, [email protected]

Timothy Gsell Governors State University, [email protected]

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Acosta, Diana and Gsell, Timothy, "The effect of antibiotic treatments on the growth of Pseudomonas stutzeri and Enterobacter gergoviae and the fermentation of several carbohydrates by Enterobacter gergoviae, Enterobacter aerogenes, and Pseudomonas aeruginosa" (2017). GSU Research Day. 48. https://opus.govst.edu/research_day/2017/Poster_sessions/48

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Introduction Abstract Conclusions • Several products manufactured by an agricultural Products produced by an agricultural chemical company, bloated in their product containers due to microbial fermentation. Products • In almost every sample, the AC colony counts were company experienced bloating after packaging or in and their components were tested for the presence of microbes, with Pseudomonas spp. and Enterbacter spp. the most prevalent. The higher than the EB counts. Enterobacter likely grew storage containers. use of preservatives, disinfectants, or antibiotics can be utilized to kill microbes to potentially prevent bloating. Hydrogen peroxide, six antibiotics (Vancomycin, Ampicillin, Doxycline, Ciprofloxacin, Streptomycin, and Gentamicin) and two preservatives (Proxel BN and on both plates. The difference between the CFU/ml • An unknown gas built up inside the containers causing Proxel GXL) at various concentrations, were examined to determine their disinfection efficacy against Pseudomonas stutzeri and of aerobic and Enterobacteriacea plates may show distortion of the containers. Enterobacter gergoviae. The results suggest that hydrogen peroxide, and the two preservatives were effective versus the control at the true CFU/ml count of Pseudomonas. • The gas is likely a product of bacterial fermentation inhibiting microbial growth. The bacteria were susceptible to all but two antibiotics, Vancomycin and Ampicillin. Enterobacter • Pseudomonas spp. was the most prevalent bacteria in (H2) or aerobic respiration (CO2). gergoviae, Enterobacter aerogenes, and Pseudomonas aeruginosa, were also examined to access whether they could ferment four the BiOLOG identifications followed by Enterobacter separate carbohydrates (xanthan gum, glycerol, propylene glycol, and a red pigment solution) commonly used in products. Each spp. • The products were tested and found to contain high bacterium was tested separately, and each Enterobacter spp. was tested in combination with Pseudomonas aeruginosa. The results levels of bacteria (10^6 -10^7). suggest that both Enterobacter spp., separately and in combination with Pseudomonas, can ferment glycerol (producing acid and gas), • Since Pseudomonas can degrade many organic • The samples primarily contained Pseudomonas spp. and xanthan (acid only). Enterobacter aerogenes had a higher rate of glycerol fermentation, although not significantly, than compounds, it is possible that it is able to degrade and Enterobacter spp. Enterobacter gergoviae, and both Enterobacter spp. produced more gas in the presence of Pseudomonas. None of the bacteria could the seed treatment products. ferment propylene glycol or the red pigment solution. Pseudomonas did not ferment glycerol but may weakly ferment xanthan. • The use of disinfectants, antibiotics, and preservatives . • High significance values (p<0.001) in the peroxide can potentially be used to prevent bloating from and preservative treatments against the control occurring. suggest that the treatments all had a marked effect • Carbohydrates used in products may be more easily Results on inhibiting the bacterial growth. fermented than others. These components may be • Bacterial isolation: Many species of Pseudomonas and two species of Enterobacter were found. • There was no significance between the exposure time replaced to reduce or prevent fermentation. • Colony counting: Figure 1 - 3. High numbers, 10^6 – 10^7, were present in many of the products. of the bacteria to the peroxide which suggests that . • Preservative trials: Figures 4 - 5. Both preservatives were significant against the control (P<0.001) with ~99% reductions in bacterial growth. There was little to no significance between the different preservatives or at different concentrations. there is no real difference in growth inhibition • Hydrogen peroxide trials: Figures 6 - 7. All treatments were significant against the control, P. stutzeri trials (P<0.01), E. gergoviae (P<0.001), with ~90% reductions in bacterial growth. There was no between long or short exposure times. significance between time treatments. Methods & Materials • Antibiotic trials: Figures 8 - 9. • Many the preservatives were not significant against • Both bacteria, had no measurable zone of inhibition for Ampicillin (AM10) or Vancomycin (VA30) indicating resistance. Ciprofloxacin (CIP), Doxycline (D30), Streptomycin (S10), and Gentamicin each other at different concentration or vs the other • Bacterial isolation: 15 products were streaked onto nutrient agar plates and (GM10) were all within susceptible ranges for both. preservative. This suggests that overall, the different incubated at 37°C to obtain isolated colonies (4). Isolated colonies were • For both bacteria, antibiotics were significant vs each other with P<0.001, except for AM10 vs VA30 (P>0.05), D30 vs S10 (P<0.05) in P. stutzeri trials, and D30 vs GM10 (P<0.05) in E. gergoviae grown on BUG media and then identified with BiOLOG GEN III (2). trials concentrations are not significantly different in terms • Colony counting: 15 products and 3 water samples were plated onto • Carbohydrate fermentation trials: Figure 10. of disinfectant ability and that the two preservatives Aerobic (AC) and Enterobacteriacea (EB) petrifilm, then incubated at 37°C • Glycerol: Gas and acid was produced when Enterobacter spp. were present. (3, 4). The number of countable colonies and dilution factor were used to have relatively similar biocide efficacy. • Propylene glycol and red pigment solution: There was no gas and no acid produced. calculate cfu/ml. N=3. • • Antibiotic resistance trials: Spread plates containing isolated bacteria were • Xanthan gum: All tubes showed some pH change resulting in a change from the initial red broth to orange or yellow. Enterobacter containing tubes had the most color change while In the fermentation trials, the gas and acid prepared on Mueller Hinton agar with six different antibiotic discs, then Pseudomonas by itself had the least color change. production by Enterobacter spp. suggests that incubated at 37°C (4). Zones of inhibition were measured and compared to • All bacteria combinations were significant (P< 0.05) against P. aeruginosa on days 2-4, except for E. gergoviae on days 1-4, E. gergoviae + P. aeruginosa on day 1, and P. aeruginosa on days 2-4. glycerol is easily fermented. There appeared to be an interpretation chart to determine the susceptibility or resistance to each • E. aerogenes and E. aerogenes + P. aeruginosa increased significantly from day 1 to day 2 (P<0.05). E. gergoviae + P. aeruginosa had a significant (p <0.01) increase in gas from day 1 to days 3-4. E. antibiotic (4). N=5. gergoviae did not have any significant day to day increase in gas production. large differences in gas production in glycerol between Enterobacter spp. On average, E. aerogenes

Figure 1: Mean aerobic colony forming units of Figure 2: Mean Enterobacteriacea colony forming Figure 3: Mean difference between Aerobic and produced more gas than E. gergoviae, and both seed treatment products units of seed treatment products Enterobacteriacea colony forming units of seed

4.00E+07 3.50E+07 treatment products species produced more gas when present with P.

3.00E+07 3.00E+07 aeruginosa. However, there was no real significance 3.00E+07

2.50E+07 2.00E+07

in gas produced compared to each other. Since there

2.00E+07 2.00E+07 1.50E+07 1.00E+07 was no gas and no acid production produced by any

CFU/ml CFU/ml 1.00E+07 1.00E+07 CFU/ml 0.00E+00 bacteria, red pigment solution and propylene glycol 5.00E+06 0.00E+00 -1.00E+07 0.00E+00 are likely not fermented. The color change in xanthan

-1.00E+07 -5.00E+06 -2.00E+07 Sample Sample Sample gum tubes was likely a result of weak acid production Left to right: Photo 1: Nutrient agar plates, Photo 2: AC and EB petrifilm, Photo 3: Antibiotic plates. H2O 1 H2O 2 1 2 3 4 H2O 1 H2O 2 1 2 3 4 H2O 1 H2O 2 1 2 3 4 from fermentation. P. aeruginosa had the least color • Preservative trials: Two preservatives (Proxel BN and Proxel GXL) were 5 6 7 8 9 10 5 6 7 8 9 10 5 6 7 8 9 10 change in xanthan. It is not known to undergo diluted to 0.1% - 0.5% concentration in tryptic soy broth. Isolated bacteria 11 12 13 14 15 16 11 12 13 14 15 16 11 12 13 14 15 16 were then added to test tubes, then incubated at 37°C (4). The density of fermentation reactions since it is an aerobic cell growth was determined by measuring absorbance values at 600 nm Figure 4: Comparison of CFU/ml for Enterobacter Figure 6: Mean CFU/ml of Enterobacter gergoviae Figure 8: Mean zone of inhibition (mm) of bacterium. This may indicate that it can ferment for a using a standard curve to convert to cfu/ml (5, 6). N=5. gergoviae treated with Proxel BN and Proxel GXL after 3% peroxide treatment Enterobacter gergoviae

• Hydrogen peroxide trials: Isolated bacteria were added to test tubes 1.E+10 1.E+10 40 short time in anaerobic conditions before dying out.

containing 3% hydrogen peroxide. At different time intervals, the peroxide 30 samples were transferred to test tubes containing tryptic soy broth (5). The 1.E+09 1.E+09

test tubes were then incubated at 37°C. The density of cell growth was 1.E+08 20 CFU/ml determined by measuring absorbance values at 600 nm using a standard CFU/ml 1.E+07 10 curve to convert to cfu/ml (5, 6). N=5 Preservative (% by volume) 1.E+08 Trial

• Carbohydrate fermentation trials: Four separate broths were prepared using (mm) inhibition of Zone 0 Control 0.1 BN 0.1 GXL 0.2 BN Antibiotic References the standard concentrations of four different carbohydrates in a phenol 0.2 GXL 0.3 BN 0.3 GXL 0.4 BN CONTROL 1 min 2 min D30 CIP S10 GM10 VA30 AM10 1. 2016. InStat, on GraphPad Software, Inc. http://www.graphpad.com/scientific- broth base. The broth was dispensed into separate test tubes containing 0.4 GXL 0.5 BN 0.5 GXL 5 min 10 min 15 min inverted Durham tubes, then autoclaved (4). Isolated bacteria were added software/instat/. Accessed Figure 9: Mean zone of inhibition (mm) of to test tubes, then incubated at 37°C. Color changes (indicating pH change) Figure 5: Comparison of CFU/ml for Pseudomonas Figure 7: Mean CFU/ml of Pseudomonas stutzeri 2. 2016. Microbial Identification and Microbial Community Analysis, on Biolog. Pseudomonas stutzeri were recorded every 24 hours for 96 hours. Gas bubbles inside the Durham stutzeri treated with Proxel BN and Proxel GXL after 3% peroxide treatment http://www.biolog.com/. Accessed July. tube were measured in mm with a ruler. N=5. 1.E+09 1.E+09 50 3. 2016. Quality Indicator Testing - 3M™ Petrifilm™ Plates, on 3M. • Statiscal analysis was performed using Instat Graphpad (1). 40 http://solutions.3m.com/wps/portal/3M/en_US/Microbiology/FoodSafety/prod

1.E+08 1.E+08 30 ucts/petrifilm-plates/. Accessed 1.E+07 CFU/ml CFU/ml 20 4. Harley JP, Prescott LM, Klein DA. 2002. Laboratory Exercises in Microbiology. McGraw-Hill, Boston, MA. 1.E+06 10 Preservative (% by volume) 1.E+07 5. Kim D-j, Chung S-g, Lee S-h, Choi J-w. 2012. Relation of microbial biomass to Trial 0 Control 0.1 BN 0.1 GXL 0.2 BN counting units for Pseudomonas aeruginosa. African Journal of Microbiology

Zone of inhibition (mm) inhibition of Zone Antibiotic 0.2 GXL 0.3 BN 0.3 GXL 0.4 BN CONTROL 1 min 2 min Research 6:4620-4622. D30 CIP S10 GM10 VA30 AM10 0.4 GXL 0.5 BN 0.5 GXL 5 min 10 min 15 min 6. 16. Pavia Uo. 2011. Identification of bacterial growth parameters.

Figure 10: AVERAGE GAS PRODUCTION IN 5% GLYCEROL VS TIME

15 Acknowledgements 10 Research Mentor: Timothy Gsell Ph.D. and fellow 5 undergraduate researchers, Caitlin Johnson and Samantha Roderick. Thanks to Governors State University and Dr. Mark

GAS BUBBLE SIZE (mm) SIZE BUBBLE GAS 0 Zhen et. al. for the ideas, supplies ,and equipment to preform 0 1 2 3 4 this experiment. A special thanks to Dr. Erin Grey-Avis, GSU LS- -5 TIME (days) AMP advisor and mentor, and GSU President Dr. Elaine Maimon From left to right: Photo 4: Glycerol fermentation (gas and acid produced), EA EA + PA EG EG + PA PA and Provost Dr. Deborah Bordelon for their support. Photo 5: Xanthan gum fermentation (acid produced).