Bacterial Persistence in Aquatic Environments: A Study of Green Fluorescent Protein (GFP)-Tagged Escherichia coli in Laboratory Microcosms

Received 02 Apr, 2023

Accepted 30 Aug, 2023

Published 02 Sep, 2023

Background and Objective: In-depth knowledge of survival potential and survival patterns of wild-type and genetically engineered E. coli in aquatic systems is scarce. The study was designed to investigate the relative persistence and survival of transformed E. coli DH5α and environmental isolates. Materials and Methods: Three different types of microcosms were set up and inoculated with GFP-transformed E. coli. Samples were collected at regular intervals and the survival and growth of the organisms were monitored by viable counts using GFP as a cellular marker. Survival curves were prepared to visualize bacterial persistence trends over time. Results: The obtained results suggested different survival patterns of E. coli in different microcosms. The survival of E. coli in filter-sterilized water samples was found greater than that in untreated water. A sharp decrease in the count of E. coli cells in untreated water, compared to that of filter-treated and/or autoclaved water suggests that the survival of E. coli in natural water might be limited due to deprivation of nutrients or biological interactions, such as competition and predation. Conclusion: Transformation of GFP in E. coli did not show a significant effect on the persistence in the laboratory microcosm underscoring the potential utility of GFP-tagged E. coli as a model organism for studying bacterial survival and distribution dynamics.

INTRODUCTION

Survival has been defined as the maintenance of viability under adverse environmental circumstances^1,2. After being discharged from the host to the environment, E. coli encounters adverse nutritional and physicochemical shocks or stresses that influence significantly its survival³. Survival has been shown to be affected by numerous interacting factors, which include competition⁴, predation⁵, nutrient availability⁶, temperature⁷, osmotic conditions⁸, radiation⁹, pH¹⁰, presence of antibiotics, algal toxins, heavy metals and other physicochemical factors^11,12. Although E. coli is an enteric organism, previous research indicated its survival in aquatic environments¹³. Due to the difficulty of observing a particular species in mixed populations, the survival and persistence of intestinal bacteria like E. coli in the environment are frequently poorly known. On the other hand, due to the existence of starving or damaged cells or possibly “viable but nonculturable” cells, the use of culture-based, conventional enumeration techniques alone may dramatically underestimate the number of viable cells^14,15. A unique method of monitoring such cell types in the environment can be accomplished by incorporating of certain cellular markers into the test bacteria, an example being a bioluminescent marker. It is now possible to identify and gauge the activity of recombinant bacteria in the environment by creating a bacterium with the bioluminescence trait^16,17. In this context, research has been carried out to investigate the use of a plasmid-borne green fluorescent protein (GFP) gene as a marker for monitoring the survival of E. coli in natural environments. The GFP can function as a reporter protein and in situ cell marker in a wide variety of prokaryotic and eukaryotic cells^18,19.

The GFP is expressed two to three times faster in E. coli and exhibits eighteen-fold greater fluorescence intensity than native GFP. The main benefits of the GFP-tagged technique over the lux-based system, which needs an aldehyde substrate for bioluminescence¹⁷, include its species independence and absence of need for any substrates or cofactors. Thus, GFP is a useful marker system that may be quickly identified by epifluorescence microscopy²⁰, spectrofluorometry, direct fluorescence measurement and fluorescence colony counting. Additionally, all growth phases, including those with starvation, are recognized by the green fluorescent phenotypes, allowing for nondestructive detection²¹.

The survival capacity of the E. coli strains in the environmental water can be studied by correlating the persistence and recovery of E. coli cells in laboratory microcosm experiments. Suitable laboratory systems that model particular environments to monitor the fate of genetically engineered bacteria in such environments before initiating field experiments are commonly termed microcosms²². Microcosms can serve as standard test systems that can be adapted to a variety of organisms and environmental conditions²² and therefore, are very useful and straightforward in examining microbial ecological processes^23-26.

Due to the restrictions placed on the release of these engineered microorganisms into the environment, studies in microcosm model systems are vital for studying their survival. However, by using a range of different microcosms rather than a single design, the results may prove more meaningful for predicting how strains will survive in an open environment³.

The objectives of this study were to investigate the survivality of GFP-tagged E. coli in aquatic microcosms in the laboratory setting and to assess the potential utility of GFP-tagged E. coli as a model organism for studying bacterial survival and distribution in aquatic ecosystems.

MATERIALS AND METHODS

Study area: This work has been conducted in the Environmental Biotechnology Laboratory of the Department of Microbiology, University of Dhaka. This study takes 12 months (March, 2008 to February, 2009) from inception to completion.

Preparation of competent cells for transformation: A new overnight culture of 100 mL of the chosen strain of E. coli DH5a collected from the institutional repository was added to 100 mL of LB broth and it was then cultured at 37°C to an OD₆₀₀ of 0.5 to 0.6. Cells were extracted by centrifugation (750-1000 x g, 12-15 min, 4°C) after being frozen on ice for 10-15 min. After being pelleted, the cells were resuspended in 0.25 culture volume of ice-cold 0.1 M CaCl₂ solution and held on ice for 30 min before being harvested as usual. Cells were kept on ice for at least 2 hrs (ideally overnight, as the cells are most competent the day after storage). Glycerol was added to the mixture to reach a final concentration of 20% for storage²⁰.

Transformation of 2 L of DNA solution (pGFP) and one hundred microliters of competent cells was combined and incubated on ice for 20-30 min. The cells were promptly cooled on ice for 2 min after being heat shocked at 42°C for 1 min. With the addition of 1 mL of LB broth and an hour of incubation at 37°C, they were given time to recover. The converted cells were plated onto a selective LB agar plate containing 0.5% arabinose and ampicillin (100 g L^–1) antibiotic.

Determination of transformation efficiency: The transformation efficiency was calculated by dividing the number of colonies by the amount (μg) of DNA added.

Laboratory microcosm set-up: Seven 250 mL conical flasks were taken for microcosm study. Each flask contained 100 mL of pond water (collected from the pond in front of Dhaka University Shahidullah Residential Hall). Inoculum consisted of 200 μL of actively growing cells of about 10⁷ cells/mL concentration resuspended in normal saline. The following three different types of microcosms were used for this study:

	•	Microcosm with non-sterile pond water containing either transformed DH5α or transformed environmental strains
	•	Microcosm with filter-sterilized (using 0.22 μm millipore filter) pond water containing either transformed or untransformed parent strains of DH5α (without pGFP)
	•	Microcosm with filter-sterilized (using 0.22 μm millipore filter) pond water containing either transformed or untransformed parent environmental strains of E. coli (without pGFP)

Two additional microcosm experiments were set up using transformed E. coli DH5α in autoclaved distilled and autoclaved pond waters.

One flask of non-sterile pond water was taken as a control where no inoculum was added to check for the presence of any non-GFP conferred green fluorescent colonies. Treatments were performed in triplicate and microcosm flasks were regularly kept in a shaking incubator to provide proper aeration and agitation.

Counting interval: Water samples were collected from microcosm flasks on days 0, 1, 2, 3, 4, 7, 10, 15, 30 and 50. Samples were diluted appropriately and GFP- fluorescent cell counts (in case of transformed inocula) and total viable cell (in case of parent inoculum) counts were performed²⁷ on LB agar medium supplemented with ampicillin and arabinose as well as non-supplemented LB agar respectively. Finally, a plot of time intervals in days versus log of CFU mL^–1 of water sample was prepared for each type of microcosm.

Statistical analysis: For the growth study, the viable counts were expressed as Log₁₀ CFU mL^–1 and plotted against incubation time (hr) to obtain the growth curves for each sample. Data were analyzed by the Gompertz equation to produce fitted growth curves. All statistical tests were considered significant at a confidence level of 95% (p<0.05).

RESULTS

Transfer of green fluorescent protein containing plasmid (pGFP) to E. coli strains: The control strain E. coli DH5α and one avian strain of E. coli were transformed with GFP. The transformation efficiency of E. coli DH5α was found to be 5.0×10⁴ CFU μg^–1 and the transformation efficiency of the environmental E. coli strain was 1.0×10⁴ CFU μg^–1.

Survival potential of transformed E. coli in different types of microcosms: In the case of non-sterilized pond water containing microcosms, the initial inoculum of both E. coli DH5α and the environmental strain was about 10⁶ CFU mL^–1. No significant change in the viable count was observed in the case of E. coli

DH5α until after 24 hrs (Fig. 1) whereas, a sharp decrease in the viable count of the environmental strain was observed from the very beginning. A gradual decrease in the cell count was observed in both types of microcosms with time intervals and after 10 days of inoculation, no pGFP-containing cells were observed.

The survival of E. coli in filtered water samples was found relatively better than that in untreated water. In filter-sterilized pond water, the reference DH5α strain and the environmental E. coli strain demonstrated a consistent survival pattern (Fig. 2 and 3). Initial inoculum of E. coli DH5α in filter-sterilized pond water was about 10⁶ CFU mL^–1 and initially there was no significant change in the viable count within 48 hrs. Then a slow, gradual decrease in the cell count was observed with time intervals, the untransformed strain showing somewhat better survival (Fig. 2). On day 20, 100 μL water was spread on arabinose arabinose-containing plate but no fluorescent colony was observed. Then on day 22, 10 mL of the water sample was filtered using a 0.22 μm millipore filter and the filter paper was placed on an arabinose-containing plate. However, no fluorescence was observed.

On the other hand, the initial inoculum of environmental isolate was about 10⁵ to 10⁷ CFU mL^–1 and it showed a gradual decrease in the cell count with time intervals from the very beginning (Fig. 3). Due to heavy contamination, further counting after 10 days became impossible.

Fig. 1: Graph showing the culturability of both DH5α and environmental strains of E. coli in non-sterilized pond water

Fig. 2: Graph showing the culturability of both transformed and untransformed DH5α strains of E. coli in filter-sterilized pond water

Fig. 3: Graph showing the culturability of both transformed and untransformed environmental strains of E. coli in filter-sterilized pond water

Fig. 4: Graph showing the culturability of transformed DH5α strain of E. coli in both autoclaved distilled water and autoclaved pond water

Microcosm with autoclaved pond water showed the maximum growth response of the E. coli DH5α strain. The initial inoculum was 10⁵ CFU mL^–1 and the setup was continued for 48 days, a gradual fall in cell count was observed with time. On the other hand, the growth response was minimal in autoclaved distilled water where the cultivability was observed for only 7 days (Fig. 4). After that, no green fluorescent colony was observed in the plates. The spread plate done on nutrient agar media did not reveal any colony.

The control flask was regularly checked by plating with arabinose and without arabinose-containing media. No green fluorescent colonies were observed during the period of investigation.

DISCUSSION

The microcosm model described in the present study is a simple but useful way to study the fate and survival of genetically modified E. coli in aquatic environments. In order to assess long-term bacterial viability in nutrient-limited aerobic settings and mixed microbial communities with diverse types of water, the study successfully transformed pGFP into E. coli DH5 and an environmental strain.

The maintenance of GFP fluorescence in cells under various settings and the connection between GFP fluorescence and cell viability were studied using microcosms containing sterilized water samples. GFP was highly stable under nonselective conditions and produced brightly fluorescence colonies which were easily detectable with a standard epifluorescent microscope^27,28.

The present study revealed that there was a significant difference between the survival of E. coli in sterilized and non-sterilized pond water. In this study, the survival of DH5α and the environmental strain of E. coli was observed for 10 days in non-sterile pond water. Growth of transformed E. coli cells was dramatically reduced in the non-sterilized microcosms compared to the sterilized ones, suggesting that indigenous microorganisms play an important role in the die-off of E. coli in the environment. After 10 days no single GFP colony was observed in the plates.

The obtained result revealed the plausible impact of indigenous predators and competitor microbes in the environment. Thus, the survival of E. coli in nature might be limited by biological interactions. Escherichia coli in untreated water quickly perished as a result of competition and predation^29,30. Other genetically modified strains used in soil experiments with similar outcomes have been reported^31-33. It has frequently been demonstrated that biotic variables reduce the lifespan of bacteria put into different aquatic environments. These factors include competition for nutrients³⁴ and the possibility of bacteriophage infection³⁵. Several studies have shown the importance of protozoans in controlling bacterial populations^36,37. In a study of the factors affecting the survival and growth of introduced bacteria in natural water bodies, it has been reported that the growth of the introduced species was limited due to the availability of carbon and sometimes nitrogen and phosphorus^38,39.

Transformed cells did not survive in filter-sterilized water for more than 10 days. One reason behind this may be the toxins and bacteriophages that might not be removed by filtration and thus, might play an adverse role in the survival and growth of E. coli. According to reports, autoclaving has advantages over filtering that may be attributable to one or more of the following factors: (i) Destruction of bacteriophages, (ii) Inactivation of thermolabile toxic substances like antibiotics and (iii) Increase in nutrients that are readily available³⁰. Unfortunately, because of the contamination problem, counting of transformed environmental strains was not continued after 10 days.

In autoclaved distilled water, comparatively short survival of transformed E. coli DH5α strain indicated that the unavailability of nutrients in autoclaved distilled water and the probable release and accumulation of toxic materials in the cells might limit their survival. However, DAPI (4’,6-diamidino-2-phenylindole) staining of water samples would have explained whether the cells remained viable but nonculturable state.

On the other hand, the highest rate of survivability was observed in autoclaved pond water. The numbers of CFU mL^–1 in this water treatment declined steadily and were surviving until 48 days and onwards. The persistence of E. coli in autoclaved water suggests its survival pattern in nature that might be limited by biological interactions, such as competition with the natural microbial flora⁴⁰. Another significant finding from this experiment is that autoclaving improves the dissolved organic carbon's liability, potentially creating a better carbon supply for E. coli than regular pond water. Bacteriophages are destroyed by autoclaving and thermolabile harmful chemicals like antibiotics are rendered inactive^41,42. In this study, there was a consistently significant difference in the survival of E. coli in sterilized and non-sterilized water. However, research carried out by Banning et al.²⁷ obtained no consistent significant difference between the survival of E. coli in sterilized and non-sterilized water. The reason behind this might be they performed the investigation under anaerobic conditions. A similar result was also found in previous studies^41,42.

This study provides insights into the survivality of GFP-tagged E. coli in laboratory aquatic microcosms, which can help in understanding the fate and behavior of bacterial populations in natural aquatic environments. The implications of these findings extend beyond the laboratory setting, as they provide insights into the behavior and potential ecological consequences of GFP-tagged E. coli in natural aquatic environments. The results of this study can help in designing better strategies for managing bacterial contamination in aquatic environments, such as drinking water reservoirs, swimming pools and wastewater treatment plants. The study may stimulate further research into the molecular mechanisms underlying bacterial survival in aquatic environments, leading to a better understanding of microbial ecology and evolution.

CONCLUSION

Our findings revealed that GFP-tagged E. coli exhibited a persistent presence in the laboratory microcosms over time. In this experiment, no significant difference was observed among the survival patterns of the parent and transformed strains of E. coli. The uniformity of such response indicates that the presence of pGFP did not adversely affect the fitness of the host strains. Furthermore, our study highlights the importance of monitoring and managing bacterial populations in aquatic systems to mitigate potential risks associated with microbial contamination.

SIGNIFICANCE STATEMENT

Escherichia coli is a ubiquitous bacteria with potential aquatic transmission capability. In-depth knowledge of survival potential and survival patterns of wild-type and genetically engineered E. coli in aquatic systems is scarce. This study employed water microcosms to avoid issues related to the release of genetically engineered E. coli into the environment and to mimic real aquatic environmental conditions. Transformed E. coli showed comparable survival in microcosms compared to untransformed E. coli, indicating genetically modified pathogens can survive in aquatic environments. This study shed light on the possibility that enteropathogenic E. coli may survive in aquatic environments for a prolonged period.

REFERENCES

1. Roszak, D.B. and R.R. Colwell, 1987. Survival strategies of bacteria in the natural environment. Microbiol. Rev., 51: 365-379.
2. Petersen, F. and J.A. Hubbart, 2020. Physical factors impacting the survival and occurrence of Escherichia coli in secondary habitats. Water, 12: 1796.
3. Haruta, S. and N. Kanno, 2015. Survivability of microbes in natural environments and their ecological impacts. Microb. Environ., 30: 123-125.
4. Scanferlato, V.S., D.R. Orvos, G.H. Lacy and J. Cairns Jr., 1990. Enumerating low densities of genetically engineered Erwinia carotovora in soil. Lett. Appl. Microbiol., 10: 55-59.
5. Casida Jr., L.E., 1980. Bacterial predators of Micrococcus luteus in soil. Appl. Environ. Microbiol., 39: 1035-1041.
6. Cook, K.L. and C.H. Bolster, 2007. Survival of Campylobacter jejuni and Escherichia coli in groundwater during prolonged starvation at low temperatures. J. Appl. Microbiol., 103: 573-583.
7. Wiebe, W.J., W.M. Sheldon Jr. and L.R. Pomeroy, 1992. Bacterial growth in the cold: Evidence for an enhanced substrate requirement. Appl. Environ. Microbiol., 58: 359-364.
8. Ahl, T., K. Christoffersen, B. Riemann and O. Nybroe, 1995. A combined microcosm and mesocosm approach to examine factors affecting survival and mortality of Pseudomonas fluorescens Ag1 in seawater. FEMS Microbiol. Ecol., 17: 107-116.
9. Barcina, I., J.M. González, J. Iriberri and L. Egea, 1990. Survival strategy of Escherichia coli and Enterococcus faecalis in illuminated fresh and marine systems. J. Appl. Bacteriol., 68: 189-198.
10. Liu, Y., C.K. Lim, Z. Shen, P.K.H. Lee and T. Nah, 2023. Effects of pH and light exposure on the survival of bacteria and their ability to biodegrade organic compounds in clouds: Implications for microbial activity in acidic cloud water. Atmos. Chem. Phys., 23: 1731-1747.
11. Henis, Y., K.R. Gurijala and M. Alexander, 1989. Factors involved in multiplication and survival of Escherichia coli in lake water. Microb. Ecol., 17: 171-180.
12. Wang-Ngarm, S., P. Chareonsudjai and S. Chareonsudjai, 2014. Physicochemical factors affecting the growth of Burkholderia pseudomallei in soil microcosm. Am. J. Trop. Med. Hyg., 90: 480-485.
13. Chao, W.L. and R.L. Feng, 1990. Survival of genetically engineered Escherichia coli in natural soil and river water. J. Appl. Bacteriol., 68: 319-325.
14. Liang, L.N., J.L. Sinclair, L.M. Mallory and M. Alexander, 1982. Fate in model ecosystems of microbial species of potential use in genetic engineering. Appl. Environ. Microbiol., 44: 708-714.
15. Smith, J.J., J.P. Howington and G.A. McFeters, 1994. Survival, physiological response and recovery of enteric bacteria exposed to a polar marine environment. Appl. Environ. Microbiol., 60: 2977-2984.
16. Rattray, E.A., J.I. Prosser, K. Killham and L.A. Glover, 1990. Luminescence-based nonextractive technique for in situ detection of Escherichia coli in soil. Appl. Environ. Microbiol., 56: 2268-3374.
17. Shaw, J.J., F. Dane, D. Geiger and J.W. Kloepper, 1992. Use of bioluminescence for detection of genetically engineered microorganisms released into the environment. Appl. Environ. Microbiol., 58: 267-273.
18. Errampalli, D., K. Leung, M.B. Cassidy, M. Kostrzynska, M. Blears, H. Lee and J.T. Trevors, 1999. Applications of the green fluorescent protein as a molecular marker in environmental microorganisms. J. Microbiol. Methods, 35: 187-199.
19. Tsien, R.Y., 1998. The green fluorescent protein. Annu. Rev. Biochem., 67: 509-544.
20. Valdivia, R.H., A.E. Hromockyj, D. Monack, L. Ramakrishnan and S. Falkow, 1996. Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions. Gene, 173: 47-52.
21. Pérez-Arellano, I. and G. Pérez-Martı́nez, 2003. Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei. FEMS Microbiol. Lett., 222: 123-127.
22. Awong, J., G. Bitton and G.R. Chaudhry, 1990. Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments. Appl. Environ. Microbiol., 56: 977-983.
23. Sautour, M., P. Mary, N.E. Chihib and J.P. Hornez, 2003. The effects of temperature, water activity and pH on the growth of Aeromonas hydrophila and on its subsequent survival in microcosm water. J. Appl. Microbiol., 95: 807-813.
24. Konopka, A., M. Carrero-Colon and C.H. Nakatsu, 2007. Community dynamics and heterogeneities in mixed bacterial communities subjected to nutrient periodicities. Environ. Microbiol., 9: 1584-1590.
25. Randa, M.A., M.F. Polz and E. Lim, 2004. Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR. Appl. Environ. Microbiol., 70: 5469-5476.
26. Duguma, D., M.W. Hall, P. Rugman-Jones, R. Stouthamer, J.D. Neufeld and W.E. Walton, 2015. Microbial communities and nutrient dynamics in experimental microcosms are altered after the application of a high dose of Bti. J. Appl. Ecol., 52: 763-773.
27. Banning, N., S. Toze and B.J. Mee, 2002. Escherichia coli survival in groundwater and effluent measured using a combination of propidium iodide and the green fluorescent protein. J. Appl. Microbiol., 93: 69-76.
28. Blaustein, R.A., Y. Pachepsky, R.L. Hill, D.R. Shelton and G. Whelan, 2013. Escherichia coli survival in waters: Temperature dependence. Water Res., 47: 569-578.
29. Carlucci, A.F., P.V. Scarpino and D. Pramer, 1961. Evaluation of factors affecting survival of Escherichia coli in sea water: V. studies with heat- and filter-sterilized sea water. Appl. Microbiol., 9: 400-404.
30. Adebisi, O.O., I.D. Gbala, F.T. Akinsolu and A.B. Olayemi, 2019. Effect of physical stresses on survivability and post-exposure antibiotic susceptibility of coliforms in environmental waters and wastewaters. Afr. J. Clin. Exp. Microbiol., 20: 315-323.
31. Devanas, M.A., D. Rafaeli-Eshkol and G. Stotzky, 1986. Survival of plasmid-containing strains of Escherichia coli in soil: Effect of plasmid size and nutrients on survival of hosts and maintenance of plasmids. Curr. Microbiol., 13: 269-277.
32. Walter, M.V., K. Barbour, M. McDowell and R.J. Seidler, 1987. A method to evaluate survival of genetically engineered bacteria in soil extracts. Curr. Microbiol., 15: 193-197.
33. Becker, W., T.D. Nennich and S.F. Atkinson, 2016. Survivability of bovine derived Escherichia coli subjected to temperatures typical of summer in Texas. Texas J. Agric. Nat. Resour., 23: 19-25.
34. Fakruddin, M., K.S.B. Mannan and S. Andrews, 2013. Viable but nonculturable bacteria: Food safety and public health perspective. ISRN Microbiol., 2013.
35. Wommack, K.E. and R.R. Colwell, 2000. Virioplankton: Viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev., 64: 69-114.
36. Sherr, E.B. and B.F. Sherr, 1987. High rates of consumption of bacteria by pelagic ciliates. Nature, 325: 710-711.
37. van Elsas, J.D., A.V. Semenov, R. Costa and J.T. Trevors, 2011. Survival of Escherichia coli in the environment: Fundamental and public health aspects. ISME J., 5: 173-183.
38. Scheuerman, P.R., J.P. Schmidt and M. Alexander, 1998. Factors affecting the survival and growth of bacteria introduced into lake water. Arch. Microbiol., 150: 320-325.
39. Artz, R.R.E. and K. Killham, 2002. Survival of Escherichia coli O157:H7 in private drinking water wells: Influences of protozoan grazing and elevated copper concentrations. FEMS Microbiol. Lett., 216: 117-122.
40. Flint, K.P., 1987. The long-term survival of Escherichia coli in river water. J. Appl. Bacteriol., 63: 261-270.
41. Leff, L.G. and Leff A.A., 1996. Use of green fluorescent protein to monitor survival of genetically engineered bacteria in aquatic environments. Appl. Environ. Microbiol., 62: 3486-3488.
42. Ding, M., J. Li, X. Liu, H. Li, R. Zhang and J. Ma, 2018. Exploring links between water quality and E. coli O157:H7 survival potential in well waters from a rural area of Southern Changchun City, China. J. Water Health, 16: 300-310.