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Assessing the effects of variable LB nutrient concentration on Escherichia coli and Lactobacillus plantarum growth in solid agar A study in the co-culture growth dynamics of bacterial biofilms MINA KODSI Rensselaer Polytechnic Institute Department of Biology Troy, NY 12180 EPEC’s are a pathogenic strain of the common Escherichia coli bacterium naturally found in the lower intestine of mammalian species. This pathogenic strain is known for its ability to secrete toxins into the epithelial cells lining the intestine walls – altering host cell monolayer resistance, macromolecular per- meability, and morphology. The objective of this experiment was to determine the ideal nutrient con- centration that enables Lactobacillus plantarum to outgrow Escherichia coli when grown in co-culture biofilms. Co-cultures of E. coli and L. plantarum and single cultures of both bacterial species were cul- tivated under 3 LB nutrient concentrations (20g/L, 40g/L, and 60g/L) for 24 hours. Cellular growth dy- namics were analyzed using Coomassie Blue staining, and ImageJ analysis. Percent cover-age values were obtained for each culture and plotted to identify cross-trends between the bacteria growing in isolation and together in a co-culture. At an LB nutrient concentration of 40g/L, covering 71% of the plate it was cultivated in, the co-culture exhibited its highest plate coverage, and exhibited growth pat- terns similar to that of the single culture of L. plantarum. This concludes that L. plantarum’s capability in outgrowing E. coli is maximized at low to medium nutrient concentrations. This study also supports the idea that LB nutrient is greatly influential on the growth dynamics exhibited in L. plantarum and E. coli biofilms. INTRODUCTION Escherichia coli is a gram-negative rod shaped bacte- rium that is naturally found in the gut of most mammalian species. While most strains of E. coli are harmless and exhibit beneficial symbiotic effects in food digestion, pathogenic strains such as, Enteropathogenic E. coli (EPEC) are a highly invasive. EPEC bacterium secretes toxins, such as shiga toxins (also found in Shigella bacte- rium), that cause inflammation in epithelial host cells lin- ing the intestinal wall 3 . The diffusion of these toxins into the epithelial cells of the intestine is due to E. coli’s ability to form bacterial biofilms across epithelial host cells. Helpful bacterial strains, known as probiotics, prevent and aid in the digestion of pathogenic strains of bacteria, like EPEC. Lactobacillus plantarum is a highly effective probiotic agent, and recently the 299V strain of L. planta- rum was recognized as the most impactful probiotic strain available. Like E. coli, L. plantarum can form bac- terial biofilms across the epithelial cells lining the intes- tine 2 . L. plantarum’s ability to form large-surface area biofilms is why it effectively prevents pathogenic E. coli toxins from diffusing across the apical membrane of the intesti- nal wall and into the epithelial cells. Thus, these toxins are unable to alter monolayer resistance, macromolecu- lar permeability, and host cell morphology in the epithelial cells; changes that cause the symptoms associated with EPEC infection 3 . However, the efficacy of this probiotic is largely dependent upon L. plantarum’s capability to outgrow E. coli in the formation of bacterial biofilms. This capability can vary greatly due to several growth factors including; nutrient availability, moisture levels, medium permeability, availability of growth space, temperature, and waste production 1 . Our purpose therefore, was to analyze one of the most significant cross-species growth factors that impacts biofilm formation and cellular growth dynamics exhibited in both bacterial species: nutrient availability. We cultivated co-cultures of E. coli and L. plantarum and single cultures of both bacterial species under various LB nutrient concentrations for 24 hours. Percent of plate coverage was obtained by use of ImageJ analysis, and the values were plotted to identify cross-trends in the bacterial growth dynamics exhibited in all three cultures at the three different LB nutrient concentrations. Our results concluded that L. plantarum’s capability in out- growing E. coli is maximized in low to medium LB nutri- ent concentrations. PURPOSE OF RESEARCH The purpose of this study was to analyze the influence of LB nutrient concentration on the colony plate coverage exhibited in both pure cultures and co-cultures of L. plantarum and E. coli. We hope to identify trends that will maximize L. plantarum’s ability to outcompete E. coli in co-culture biofilms– possibly leading to more effective probiotic defense against pathogenic bacterial infections.

EPEC and Probiotic control

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Page 1: EPEC and Probiotic control

Assessing the effects of variable LB nutrient concentration on Escherichia coli and Lactobacillus plantarum growth in

solid agar A study in the co-culture growth dynamics of bacterial biofilms

MINA KODSI

Rensselaer Polytechnic Institute Department of Biology

Troy, NY 12180 EPEC’s are a pathogenic stra in of the common Escherichia col i bacter ium natural ly found in the lower intest ine of mammalian species. This pathogenic stra in is known for i ts abi l i ty to secrete toxins into the epithel ia l cel ls l in ing the intest ine wal ls – alter ing host cel l monolayer resistance, macromolecular per-meabi l i ty, and morphology. The object ive of th is experiment was to determine the ideal nutr ient con-centrat ion that enables Lactobaci l lus plantarum to outgrow Escherichia col i when grown in co-culture biof i lms. Co-cultures of E. col i and L. plantarum and single cultures of both bacter ia l species were cul-t ivated under 3 LB nutr ient concentrat ions (20g/L, 40g/L, and 60g/L) for 24 hours. Cel lu lar growth dy-namics were analyzed using Coomassie Blue stain ing, and ImageJ analysis. Percent cover-age values were obtained for each culture and plotted to ident i fy cross-trends between the bacter ia growing in isolat ion and together in a co-culture. At an LB nutr ient concentrat ion of 40g/L, cover ing 71% of the plate i t was cult ivated in, the co-culture exhibited i ts highest plate coverage, and exhibited growth pat-terns s imi lar to that of the s ingle culture of L. plantarum. This concludes that L. plantarum’s capabi l i ty in outgrowing E. col i is maximized at low to medium nutr ient concentrat ions. This study also supports the idea that LB nutr ient is great ly inf luent ia l on the growth dynamics exhibited in L. plantarum and E. col i b iof i lms.

INTRODUCTION Escherichia coli is a gram-negative rod shaped bacte-rium that is naturally found in the gut of most mammalian species. While most strains of E. coli are harmless and exhibit beneficial symbiotic effects in food digestion, pathogenic strains such as, Enteropathogenic E. coli (EPEC) are a highly invasive. EPEC bacterium secretes toxins, such as shiga toxins (also found in Shigella bacte-rium), that cause inflammation in epithelial host cells lin-ing the intestinal wall3. The diffusion of these toxins into the epithelial cells of the intestine is due to E. coli’s ability to form bacterial biofilms across epithelial host cells. Helpful bacterial strains, known as probiotics, prevent and aid in the digestion of pathogenic strains of bacteria, like EPEC. Lactobacillus plantarum is a highly effective probiotic agent, and recently the 299V strain of L. planta-rum was recognized as the most impactful probiotic strain available. Like E. coli, L. plantarum can form bac-terial biofilms across the epithelial cells lining the intes-tine2. L. plantarum’s ability to form large-surface area biofilms is why it effectively prevents pathogenic E. coli toxins from diffusing across the apical membrane of the intesti-nal wall and into the epithelial cells. Thus, these toxins are unable to alter monolayer resistance, macromolecu-lar permeability, and host cell morphology in the epithelial cells; changes that cause the symptoms associated with EPEC infection3. However, the efficacy of this probiotic is largely dependent upon L. plantarum’s capability to outgrow E. coli in the formation of bacterial biofilms. This capability can vary greatly due to several growth factors

including; nutrient availability, moisture levels, medium permeability, availability of growth space, temperature, and waste production1. Our purpose therefore, was to analyze one of the most significant cross-species growth factors that impacts biofilm formation and cellular growth dynamics exhibited in both bacterial species: nutrient availability. We cultivated co-cultures of E. coli and L. plantarum and single cultures of both bacterial species under various LB nutrient concentrations for 24 hours. Percent of plate coverage was obtained by use of ImageJ analysis, and the values were plotted to identify cross-trends in the bacterial growth dynamics exhibited in all three cultures at the three different LB nutrient concentrations. Our results concluded that L. plantarum’s capability in out-growing E. coli is maximized in low to medium LB nutri-ent concentrations. PURPOSE OF RESEARCH The purpose of this study was to analyze the influence of LB nutrient concentration on the colony plate coverage exhibited in both pure cultures and co-cultures of L. plantarum and E. coli. We hope to identify trends that will maximize L. plantarum’s ability to outcompete E. coli in co-culture biofilms– possibly leading to more effective probiotic defense against pathogenic bacterial infections.

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MATERIALS AND METHODS In order to examine the growth of these bacterial species – in isolation and together – we prepared 9 solid agar plates. The nutrient used in all cultures was Lysogeny Broth (LB) and all cultures were prepared in identical dishes to reduce extraneous variables. Cultures were all inoculated with both strains of bacterium from standard culture tubes of each. A total of 3 different cultures were prepared at each of the 3 nutrient concentrations. Preparation: 100mL of LB agar was prepared at each of the following concentrations: 20g/L, 40g/L, and 60g/L. After prepara-tion, they were placed in 3 separate flasks and auto-claved. Finally, each flask (concentration) was used to pour 3 identically sized petri dishes; one dish inoculated with E. coli, one dish inoculated with L. plantarum, and one dish inoculated with both bacterial cultures. These dishes were then left to grow for 24 hours in a 37 °C incubator. Staining & Imaging: To observe the general colony growth patterns of both bacteria strains, we stained the cultures with Coomassie blue stain without disrupting their growth in the agar me-dium. Each of the cultures were covered with Coomass-ie stain and left for 90 seconds. The dishes were then rinsed from the stain using distilled water and then im-aged using a stereomicroscope for colony growth analy-sis with ImageJ.

RESULTS Images were obtained using stereomicroscopy (Figure 1) and analyzed with ImageJ software to create a plot of nutrient concentration vs. plate coverage of the colony (Figure 2). Pure Cultures: We observed a similar trend in both bacterial strains that showed a higher rate of growth with greater plate cover-age as the nutrient concentration increased. As we doubled the concentration from 20 to 40g/L, the isolated culture of E. coli exhibited a minor increase in growth size. However, under the same increase of nutrient con-centration, the isolated culture of L. plantarum exhibited a major increase in growth, growing 53% larger than its initial size as the concentration doubled. Surprisingly, when the concentration was tripled from 20g/L to 60g/L, E. coli did exhibit a significant change in growth – in-creasing by more than 60% of its initial size at 20g/L. However, L. plantarum did not increase in size signifi-cantly, but rather exhibited a much more modest 10% increase in plate coverage. Co-cultures: Initially, at 20g/L, the co-culture exhibited similar growth patterns to both pure cultures. However the co-culture deviated from the expected trend in growth, and showed a peak growth in size at 40g/L. The size decreased, however, by 10% when the concentration was increased to 60g/L. Furthermore, while the co-culture seemed to grow very similarly to the isolated culture of L. plantarum from 20 to 40g/L, at concentrations higher than 40g/L, the growth pattern seemed to mimic that of the isolated culture of E. coli. Possible implications of this will be mentioned in the discussion.

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Figure 2: Plot of colony plate coverage vs. LB nutrient concentration for the 9 cultures

Culture area analysis using the ImageJ program was done by calculating area values for each dish and comparing this value to the area of the bacterial colony outgrowth on each dish. Percent coverage values for each culture were then calculated from these values and graphed (above). Values indicate how much of the dish each culture was able to spread out and cover within a 24 hour period – indicating the relative ease with which each bacteria strain was able to spread and, in vivo, to form biofilms. Notice a domination of the co-culture growth by L. plantarum at the 40g/L concentration and likewise for E. coli at 60g/L.

E. coli dominates

L. plantarum dominates

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DISCUSSION

Our study proved very successful in examining the cross-species growth dynamics of both E. coli and L. plantarum. We discovered that nutrient availability is one of the major factors affecting E. coli and L. plantarum competition.

Pure Culture Analysis:

From both our quantitative data obtained by ImageJ analysis (Figure 2) and qualitative data seen in the culture images (Figure 1), we were able to draw significant con-clusions regarding the growth patterns of E. coli and L. plantarum in regards to nutrient concentration. In re-gards to E. coli, we noticed that this strain of bacterium tends to favor high concentrations of nutrients. At higher concentration levels (60g/L), E. coli sees a major in-crease in growth (more than 60%) while lower concen-trations (20 and 40g/L) did not seem to have a significant impact on E. coli colony size. On the other hand, L. plantarum seems to be on the other side of the equation, favoring low to medium concentration levels (as seen in the 53% increase in size from 20 to 40g/L). This infor-mation allows us to draw to the conclusion that diets high in calories or carbohydrates may increase chances of infection, as E. coli gains the advantage in out com-peting L. plantarum within epithelial cells lining the lower intestines.

Co-Culture Analysis:

Even more astounding results were discovered when we compared the co-culture growth to both pure cultures. At the lower concentrations, 20 and 40g/L, the co-culture followed a growth pattern almost identical to L. plantarum. This could be further evidence of L. planta-rum’s maximum efficiency and dominance over E. coli and low to medium nutrient concentrations. However, as the concentration increased to 60g/L, the co-culture showed a stark change towards the growth pattern of E. coli. These results help to further our conclusion that E. coli gains an advantage over probiotics like L. plantarum at high nutrient concentrations.

CONCLUSION:

Thus far we can conclude that variable nutrient concen-trations can indeed affect the competition of E. coli with probiotic agents. At nutrient concentrations higher than 40g/L, there was a clear dominance in the co-culture by E. coli, while L. plantarum remained the dominant bacte-rium at low to medium nutrient concentrations. Further research can examine growth dynamics of co-cultures when grown within various LB broth concentrations. A study using SDS-PAGE to determine what protein sig-nals are altered in both L. plantarum and E. coli when grown in co-culture biofilms at various nutrient concen-trations may also be done.

Future studies may examine the environment of the in-testine in terms of nutrient availability in both: intestines inhabited by E. coli naturally found in the lower intestine and intestines with pathogenic E. coli strains. If compari-son between the two environments affirms E. coli achieves dominance in high nutrient environments, new dietary measures could be implemented as a means of treatment and prevention of EPEC infection in the intes-tine. REFRENCES:

1. MacWilliams, Maria P., and Min-Ken Liao. "Luria Broth (LB) and Luria Agar (LA) Media and Their Uses Protocol." Am. J Med 62 (2006): 293-30.

2. Mangell, Peter, et al. “Lactobacillus plantarum 299v inhibits Escherichia coli-induced intesti-nal permeability." Digestive diseases and sci-ences 47.3 (2002): 511-516.

3. Papoff, Paola, et al. "Gut microbial transloca-tion in critically ill children and effects of sup-plementation with pre- and pro-biotics.” In-ternational journal of microbiology, 2012.