3.1. Physico-chemical characteristics of the Kongsfjorden water
In summer, the water temperature was between 5.4 to 6.6°C in Svalbard (Fig. 4.) (Table 1.). The salinity of the water was 31 ppt and the pH was 8.1. The summer Arctic temperature is drastically increasing each year. Mainly the temperature is influenced by the Arctic amplification and influx of warmer Atlantic water to the fjord (Ding et al., 2018; Hatha et al., 2021). The warming temperature in the Arctic leads to the melting of ice and glaciers, adding a considerable load of freshwater to the fjord. During fall (October 2018) water temperatures were ranging from 1.6 to 3.3°C. The salinity of the Kongsfjorden water was 32ppt and the pH was 7.8.
3.2. E. coli survival in the Kongsfjorden water during Arctic summer
The self-purifying factors in the Kongsfjorden water caused a steady decline of E. coli cell count during the Arctic summer (Fig. 1a.). The result indicated rapid inactivation of the suspended test organism in raw Kongsfjorden water. The experiment started with approximately 108 cells/ml of the test organism. The biotic factors in the water are the primary factor that leads to the rapid decrease of cell count. Within the 10th day of the experiment, the cell count reached zero in the microcosm to study the biotic factors. T90 (time required to inactivate 90% of the population) was reached within 24 h. Solar radiation was the second major factor that caused an impact on cell count. In 14 days, the test organism cell count declined to zero in solar radiation microcosm and T90 of the cells was reached within 48 h. The chemical composition such as organic and inorganic components of the water did not exert any negative impact on the cells and retained approximately 106 cfu/ml until the end of the experiment.
Within 10 days, the cell count of E. coli isolated from reindeer reached zero in both biotic and solar microcosms. Here in summer, the E. coli isolated from Barnacle goose showed complete elimination on the 14th day of the experiment, with the action of biotic factors and solar radiation present in the Arctic environment. But T90 of both isolates from B. leucopsis and reindeer reached within 24 h (Hatha et al., 2021). The effect of Antarctic solar radiation on the viability of sewage bacteria has been studied previously (Hughes, 2003).
3.3. E. coli survival in the Kongsfjorden water during the Arctic fall
Fall season conditions in the Arctic changed the survival capabilities of the E. coli cells (Fig. 1b, Fig. 2b). All the self-purifying factors initially exerted a small extent of negative impact on the cell count till the 3rd day of the experiment. The cell count in the microcosms was maintained between ~ 104−106 cfu/ ml till the end of the experiment. There was a slight increase in the cell count in the control (0.85% saline) setup. The results showed that the enteric bacteria introduced into the Arctic can survive prolonged periods when the temperature is very low, and they keep up the viability of the cell for a long time. Previous studies reported the survival of a typical mesophilic organism such as E. coli at a temperature of zero degree centigrade in seawater (Halton and Nehlsen, 1968) and Antarctic waters have been reported previously (Statham and McMeekin, 1994). During fall, the water temperature was between 3.3°C and 1.6°C (Fig. 4.). During fall, the T90 of the cells reached biotic, light, and chemical microcosm within a third of the experiment.
3.3 Effect of biotic factors on the survival of E. coli
This study found that biological factors were the main reason for the decline of test organisms in the Kongsfjorden water during summer. Within 10 days the bacterial count reached zero and T90 reached in 24h. The relative survival curve of the effects of the biological factors in the two seasons is presented in Fig. 3b. During the fall, cell count was decreased for 2 days of the experiment. Then showed some increase in cell count and maintained the count till the end of the experiment. The previous study found E. coli can survive in Arctic waters (Hatha et al., 2021).
The fate of the allochthonous organism in the fjord water is determined by many biotic factors such as protozoan predation, antibiotics, cell lysis by a bacteriophage, and competition. Autochthonous organisms are more adapted to this environment than the invader microbes like E. coli. Previous study the total heterotrophic bacterial load in the Kongsfjorden water during summer was observed to be between 104 to 106 cfu/ml (Hatha et al., 2021). The major phyla of bacteria found in Kongsfjorden water were Actinobacteria (39%), γ-Proteobacteria (23%), Firmicutes (14%), α-Proteobacteria (12%), and Bacteroidetes (12%) (Table 2). The major competing bacterial flora was Bacillus, Stenotrophomonas, Enterobacter, Pseudomonas, and Staphylococcus. In the fall, the bacterial count was 104 to106 CFU/ml and γ-Proteobacteria (33%) and α-Proteobacteria (31%) were the major phyla, followed by Actinobacteria (21%) Bacteroidetes (12%) and Firmicutes (3%) (Sinha et al., 2017). These bacteria compete for resources and space with the invader microbes. And also Actinobacteria is a group of bacteria that has the ability to produce antibiotics. For example, they can produce antibiotics including actinomycin, streptomycin, and streptothricin (Madigan et al., 1997; Bérdy, 2015). During summer, the Actinobacteria count was high compared to other phyla in the Kongsfjorden water.
Another biotic factor that affects the survival of E.coli grazing by the Protozoans in the fjord water. Bhaskar et al. (2020 studied the abundance of ciliates and dinoflagellates in Kongsfjorden water during the summer and fall seasons. Athecate dominated dinoflagellates during both seasons in the Kongsfjorden water (Table 3). Their results describe that dinoflagellates' abundance ranged from 20.3 × 106 cells/m2 to 126 × 106 cells/m2 during summer and was relatively low in fall, which ranges from 2.34 × 106 cells/m2 to 19.1 × 106 cells/m2. Among ciliates, aloricate ciliates were more dominant than loricate. Ciliates at the surface ranged from as low as 0.069 × 103 cells/L (KF1) to 3.69 × 103 cells/L (KF4) during summer. Ciliate abundance increased with depth (up to 20 m). Strombidium spp. (55.28%) and Mesodinium rubrum (36.66%) were dominant during summer. Among the loricates and the aloricates, Strombidium spp. (85.72%) and Tintinnid spp. (92.15%) dominated in the fall.
3.5. Effect of Solar radiation on the survival of E.coli
Solar radiation is the second most impacted factor which determines the survival of cells in the Kongsforden waters in summer. During the summer, the 24h exposure to sunlight leads to the decline of E.coli cell count to zero within the 14th day of the experiment. T90 reached within 2 days. We observed E. coli cell count reached T90 within 8h of the experiment conducted in tropical estuary water (Chandran and Hatha, 2005). UV-B and Ozone reaching the Arctic during summer and fall were different, higher UV-B and Ozone were in the Arctic environment during summer. UV radiation is lethal to the microorganism; exposure leads to creating mutations in the DNA. During fall the UV-B and Ozone are comparatively very low. Relative survival curves of E. coli in summer and fall (Fig. 3a).
A study from the South Pole, Antarctica, found the rapid decline of faecal bacterial cells in the Antarctic during summer because of UV radiation (Statham and McMeekin, 1994). Within ~ 50 mins the E. coli was completely removed from the microcosm. And also found selective removal of UV-B and UV-A radiation could increase the survival of the cells in the experiment setup for up to 5h. They also found a negative effect of visible light on cell survival.
A previous study found E.coli isolates from Barnacle goose carriers of multidrug-resistant bacteria and genes. The study found that the E.coli isolates were resistant to four out of eleven antibiotics tested. Most importantly, the isolates were shown highest against colistin (100%), the “last resort” of antibiotics. Followed by ampicillin (39%) amoxicillin (12%), tetracycline (7%), and ceftazidime (2%) (Hatha et al., 2013) (Fig. 5.). This showed the possible role of Barnacle geese in the dissemination of antibiotic resistance genes to the pristine Arctic environment. The droppings of the bird find their way to the Kongsfjorden along with the meltwater from the tundra during the Arctic summer (Hatha et al., 2021). A study found that the reindeer species in the Ny-Ålesund region were feed Barnacle goose dropping to meet the daily energy requirements (van der Wal and Loonen, 1998). This is showing how the faecal dropping of the bird connected with the flora and fauna of the Arctic.
The prolonged survival of pathogenic and antibiotic-resistant E. coli strains in the Arctic may threaten the pristine environment ecosystem. Long survival will lead to the accumulation of antibiotic-resistant genes in the environment. The horizontal gene exchange mechanism within the microbes also leads to the exchange of ARGs with the environment microbe and intestine microbiome of the animals. Glady-Croue et al. (2018) showed that solar radiation may reshape the ARB community in the discharged sewage water and support the survival of certain ARBs. This may show the possibility of retaining ARB in the Arctic environment also during summer.