3.2. Survival of E. coli and S. Typhimurium
The results of the survival study indicate that both E. coli
Typhimurium are able to survive for up to 21 days in all three configurations that received moisture (ED, PI, and CI). Both microorganisms withstood the first 24 h of the experiment after sediment inoculation, which was evident from the culture counts of the two tested configurations (PI and ED; Figure 3
). Notably, even though the atmospheric temperature reached 43 °C within a few hours of inoculation, E. coli
counts increased marginally, and S.
Typhimurium counts increased nearly 10 times of the initial number. This initial growth could reflect the fact that fecal microorganisms are adapted to animal gut environments (37 °C for mammals and 42 °C for birds). Sediment properties that are widely known to influence bacterial survival include available nutrients [28
], organic matter [29
], particle size [30
], and clay content [31
]. Among these, estuarine bank sediment is generally known to have finer particles [32
], as well as higher organic matter content [34
], which can offer several advantages, such as protection from UV light [35
] and nutritional support. Therefore, it is perhaps not surprising to see extended survival of fecal microorganisms, including the potential pathogen, in estuarine banks even in unfavorable weather conditions. This, in turn, suggests that in the absence of other stressors, S.
Typhimurium can probably grow in estuarine bank sediments in high-temperature conditions when moisture is available and potentially serve as a source of this pathogen to the water column if re-suspended.
The initial phase of growth in the first 24 h was followed by a sharp die-off, consistent across organisms and configurations (Figure 3
). The CD system had the highest die-off rates for both types of bacteria during the experiment; 1.54/day for E. coli
and 1.72/day for S.
Typhimurium. Since significant losses in soil moisture took place during the early phase of the experiment (moisture content decreased from 40% to <1%), these very fast die-off rates were consistent with the literature [11
]. In addition, high die-off rates might have been facilitated by very high air temperatures [38
] coupled with high radiation levels [39
The two test organisms in the ED systems behaved similarly to the CD systems in the first week of the experiment (Figure 3
). The die-off rate for E. coli
was calculated to be 1.51/day and for S.
Typhimurium it was 1.64/day. Interestingly, when inundation was restored to these systems after Day 7 (although the ED system received a shorter duration of inundation compared to PI counterparts), an increase in moisture content coincided with the detection of E. coli
colonies on both Days 14 and 21 (in two of the three replicates on both days). S.
Typhimurium was also detected on Day 21 in one of the replicate boxes while the NI controls remained negative. An increase in moisture level due to inundation restoration might have influenced this regrowth/recurrence of both test organisms.
The detection of culturable bacteria after a period of drying indicates that both bacteria may survive either in dormant form or at least survive in very low densities. It is important to note that although above the limit of detection, the colony counts were below 30 cfu/plate, and therefore may have higher associated uncertainties. However, colonies were reproducibly isolated within the replicate, suggesting a level of survival was possible. Dormancy and subsequent regrowth of S.
Typhimurium upon increasing moisture has previously been reported [40
]. Also, desiccation has been shown for both E. coli
Typhimurium to significantly enhance resistance towards environmental stressors, including high temperature [41
]. This could perhaps partly explain the recurrence of these two organisms in the ED systems. Therefore, it is possible that bank sediments, which only receive moisture very intermittently (i.e., bank sediments above the usual high-tide mark, which are inundated only during king tides or high flows) could support the growth of viable fecal pathogens that, if washed, could get resuspended back into the water column [43
]. Resuspension could also occur due to other natural and anthropogenic activities, like storms, floods, recreational activities, and commercial dredging, which have all been known to cause resuspension of sediment borne bacteria and causing elevated levels of fecal organisms [44
]. Therefore, extended survival of potential fecal pathogens, like S.
Typhimurium, in estuarine bank sediment could comprise a potential human health risk.
PI systems had higher levels of survival for both E. coli
Typhimurium compared to the desiccated CD and ED systems (Figure 3
). For PI, the decay rate of E. coli
was 0.14/day, while a slightly higher decay rate was observed for S.
Typhimurium (0.21/day). The decrease in cell counts after Day 1 may be associated with very high air temperatures and solar radiation, which also resulted in a very low moisture content (close to 5%; Figure 2
). It has previously been reported that Gram-negative bacteria require a moisture content of 93% or more for optimal growth [46
]. However, despite some variation among the three biological replicates for the PI configurations, it was evident that the scenario still supported the survival of both test organisms for up to three weeks. Therefore, under more favorable conditions (lower atmospheric temperature, lower solar radiation, and higher moisture), it is likely that survival of these fecal organisms (including potential pathogens) would be higher.
Survival rates of E. coli
Typhimurium in the PI configuration were found to be comparable. This contradicts the notion that fecal pathogens cannot survive in estuarine sediments, and suggests that E. coli
could be a reliable indicator of this pathogen in this scenario. Furthermore, the extended survival of a potential fecal pathogen in the tidally influenced zone of an estuarine bank could have human health implications. This is especially the case for bank sediments, where natural and anthropogenic activities (including tide, storm, flood, recreation, dredging, etc.) can readily cause resuspension into the water column [10
The CI systems showed slightly higher die-off rates when compared to PI (0.20/day for E. coli
and 0.24/day for S.
Typhimurium). It was interesting to see higher die-off rates compared to those of PI, especially since the organisms in the CI systems were exposed to higher moisture levels and reduced solar irradiation. However, the water used for inundating the experimental containers was stagnant in the CI configurations, which led to some interference due to the formation of algae after the first week, and this may have resulted in a faster die-off. The algal growth resulted in lower TP and TN concentrations in the inundating water in the CI systems compared to the other configurations (Table A2
In the estuarine context, better survival of E. coli in areas of coastal bank sediment with intermittent drying effect compared to continuously moist sediment has previously been reported by Solo-Gabriele et al. (2000). They argued that this better survival was due to having a better competitive advantage over the predators existing in natural sediment. Although our study attempted to exclude the biotic factors by sterilizing the soil beforehand, the CI systems with algal interference supports the idea that algal bloom (which could occur in estuarine systems) could potentially reduce the survival of fecal organisms in estuarine bank sediments. In this scenario, even under these algae-influenced conditions, the survival rates of both organisms were again comparable, reinforcing the ability of E. coli to represent S. Typhimurium in complex systems, including those with algae blooms. It is also important to note that apart from the CI systems, no other configurations showed any sign of algal interference.
This study was conducted under open atmospheric conditions, which led to some challenges, including changing TP and TN concentrations. However, other studies have demonstrated that increases in TP does not necessarily impact the survival of fecal organisms, like E. coli
]. Likewise, increased levels of organic or inorganic nitrogen do not necessarily impact the survival of fecal bacteria in ambient waters [48
]. Therefore, it can be assumed that the changes in TP and TN concentrations did not significantly influence the die-off rates of the test organisms. In fact, conducting the study in an ambient environment allowed a unique opportunity to explore the comparative survival of these organisms under highly unfavorable conditions.
In combination, the survival data suggests, for the first time, that both E. coli
Typhimurium were able to withstand severe environmental conditions in estuarine bank sediments. Overall, the die-off rates observed in this study ranged from 0.14/day to 1.54/day for E. coli
and 0.21/day to 1.72/day for S.
Typhimurium. In three of the four configurations (i.e., CD, PI, CI), die-off rates for the two organisms were similar. A wide range of die-off rates has been reported in soil sediment systems for these organisms [27
], and differences have been attributed to both biotic and abiotic factors [11
] that are also encountered in bank sediments.
This study investigated the influence of different bank sediment scenarios on the survival of two fecal microorganisms in potentially extreme weather conditions with respect to temperature, radiation, and desiccation. Our underlying hypothesis is that if the test organisms survive in this study’s extreme climatic conditions, then they can also survive in the less extreme conditions that exist in many systems year-round. With an absence of previous studies focusing on bank sediments under similar experimental conditions, the results of this study significantly enhance our knowledge of the survival of fecal organisms in estuarine bank sediments. Further, this study demonstrates that, without studying fecal pathogen survival in all environments (estuarine bank sediment in this case), perhaps it could be premature to conclude that E. coli is not a good indicator organism.