Fiona P. Brennan

Long-Term Persistence and Leaching of Escherichia coli in Temperate Maritime Soils

ABSTRACT Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 ± 0.77 CFU ml−1), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions.

Characterization of environmentally persistent Escherichia coli isolates leached from an Irish soil.

ABSTRACT Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.

Clay mineral type effect on bacterial enteropathogen survival in soil

Enteropathogens released into the environment can represent a serious risk to public health. Soil clay content has long been known to have an important effect on enteropathogen survival in soil, generally enhancing survival. However, clay mineral composition in soils varies, and different clay minerals have specific physiochemical properties that would be expected to impact differentially on survival. This work investigated the effect of clay materials, with a predominance of a particular mineral type (montmorillonite, kaolinite, or illite), on the survival in soil microcosms over 96 days of Listeria monocytogenes, Salmonella Dublin, and Escherichia coli O157. Clay mineral addition was found to alter a number of physicochemical parameters in soil, including cation exchange capacity and surface area, and this was specific to the mineral type. Clay mineral addition enhanced enteropathogen survival in soil. The type of clay mineral was found to differentially affect enteropathogen survival and the effect was enteropathogen-specific.

The nitrification inhibitor dicyandiamide increases mineralization–immobilization turnover in slurry-amended grassland soil

Nitrification inhibitors are used in agriculture for the purpose of decreasing nitrogen (N) losses, by limiting the microbially mediated oxidation of ammonium (NH 4 + ) to nitrate (NO 3 − ). Successful inhibition of nitrification has been shown in numerous studies, but the extent to which inhibitors affect other N transformations in soil is largely unknown. In the present study, cattle slurry was applied to microcosms of three different grassland soils, with or without the nitrification inhibitor dicyandiamide (DCD). A solution containing NH 4 + and NO 3 − , labelled with 15 N either on the NH 4 + or the NO 3 − part, was mixed with the slurry before application. Gross N transformation rates were estimated using a 15 N tracing model. In all three soils, DCD significantly inhibited gross autotrophic nitrification, by 79–90%. Gross mineralization of recalcitrant organic N increased significantly with DCD addition in two soils, whereas gross heterotrophic nitrification from the same pool decreased with DCD addition in two soils. Fungal to bacterial ratios were not significantly affected by DCD addition. Total gross mineralization and immobilization increased significantly across the three soils when DCD was used, which suggests that DCD can cause non-target effects on soil N mineralization–immobilization turnover.

Insights into the low-temperature adaptation and nutritional flexibility of a soil-persistent Escherichia coli

An understanding of the survival capacity of Escherichia coli in soil is critical for the evaluation of its role as a faecal indicator. Recent reports that E. coli can become long-term residents in maritime temperate soils have raised the question of how the organism survives and competes for niche space in the suboptimal soil environment. The ability of an environmental isolate to utilize 380 substrates was assessed together with that of a reference laboratory strain (E. coli K12) at both 15 and 37 °C. At 15 °C, the environmental strain could utilize 161 substrates, with only 67 utilizable by the reference strain, while at 37 °C, 239 and 223 substrates could be utilized by each strain respectively. An investigation into the cold response of the strains revealed that E. coli K12 was found to reduce the expression of biosynthetic proteins at 15 °C, while the environmental isolate seemed to switch on proteins involved in stress response, suggesting low-temperature adaptation in the latter. Taken together, the results indicate that the environmentally persistent E. coli strain is well adapted to use a wide range of nutrient sources at 15 °C and to direct its protein expression to maintain a relatively fast growth rate at low temperature.

Permeable reactive interceptors: blocking diffuse nutrient and greenhouse gases losses in key areas of the farming landscape

Engineered remediation technologies such as denitrifying bioreactors target single contaminants along a nutrient transfer continuum. However, mixed contaminant discharges to a water body are more common from agricultural systems. Indeed, evidence presented herein indicates that pollution swapping within denitrifying bioreactor systems adds to such deleterious discharges. The present paper proposes a more holistic approach to contaminant remediation on farms, moving from the use of ‘denitrifying bioreactors’ to the concept of a ‘permeable reactive interceptor’ (PRI). Besides management changes, a PRI should contain additional remediation cells for specific contaminants in the form of solutes, particles or gases. Balance equations and case studies representing different geographic areas are presented and used to create weighting factors. Results showed that national legislation with respect to water and gaseous emissions will inform the eventual PRI design. As it will be expensive to monitor a system continuously in a holistic manner, it is suggested that developments in the field of molecular microbial ecology are essential to provide further insight in terms of element dynamics and the environmental controls on biotransformation and retention processes within PRIs. In turn, microbial and molecular fingerprinting could be used as an in-situ cost-effective tool to assess nutrient and gas balances during the operational phases of a PRI.

The General Stress Response Is Conserved in Long-Term Soil-Persistent Strains of Escherichia coli

ABSTRACT Although Escherichia coli is generally considered to be predominantly a commensal of the gastrointestinal tract, a number of recent studies suggest that it is also capable of long-term survival and growth in environments outside the host. As the extraintestinal physical and chemical conditions are often different from those within the host, it is possible that distinct genetic adaptations may be required to enable this transition. Several studies have shown a trade-off between growth and stress resistance in nutrient-poor environments, with lesions in the rpoS locus, which encodes the stress sigma factor RpoS (σS). In this study, we investigated a unique collection of long-term soil-persistent E. coli isolates to determine whether the RpoS-controlled general stress response is altered during adaptation to a nutrient-poor extraintestinal environment. The sequence of the rpoS locus was found to be highly conserved in these isolates, and no nonsense or frameshift mutations were detected. Known RpoS-dependent phenotypes, including glycogen synthesis and γ-aminobutyrate production, were found to be conserved in all strains. All strains expressed the full-length RpoS protein, which was fully functional using the RpoS-dependent promoter reporter fusion PgadX::gfp. RpoS was shown to be essential for long-term soil survival of E. coli, since mutants lacking rpoS lost viability rapidly in soil survival assays. Thus, despite some phenotypic heterogeneity, the soil-persistent strains all retained a fully functional RpoS-regulated general stress response, which we interpret to indicate that the stresses encountered in soil provide a strong selective pressure for maintaining stress resistance, despite limited nutrient availability. IMPORTANCE Escherichia coli has been, and continues to be, used as an important indicator species reflecting potential fecal contamination events in the environment. However, recent studies have questioned the validity of this, since E. coli has been found to be capable of long-term colonization of soils. This study investigated whether long-term soil-persistent E. coli strains have evolved altered stress resistance characteristics. In particular, the study investigated whether the main regulator of genes involved in stress protection, the sigma factor RpoS, has been altered in the soil-persistent strains. The results show that RpoS stress protection is fully conserved in soil-persistent strains of E. coli. They also show that loss of the rpoS gene dramatically reduces the ability of this organism to survive in a soil environment. Overall, the results indicate that soil represents a stressful environment for E. coli, and their survival in it requires that they deploy a full stress protection response.

Impact of Soil Type, Biology and Temperature on the Survival of Non-Toxigenic Escherichia Coli O157

The occurrence of microbial enteropathogens in the environment can represent a serious risk to human health. The fate of enteropathogens introduced into the soil environment is dependent on a wide range of complex interacting environmental factors. While the effect of abiotic factors on enteropathogen survival has been widely examined, the interaction of enteropathogens with the soil microbial community is poorly understood. This study investigated the effect of soil biology and soil type on the survival of a non-toxigenic strain of Escherichia coli O157 under different temperature regimes. Soil microcosms of two soil types, with and without an intact microbial community, were inoculated with the enteropathogen surrogate, and survival was determined over a 64-day period, encompassing a shift from cold to ambient temperatures. In both soil types bacterial numbers decreased in soil with an intact microfl ora, while in the absence of an intact community E. coli populations increased. This effect was temperature specifi c, with E. coli populations remaining stable at low temperature, regardless of treatment. Soil type was of importance in survival at both cold and ambient temperatures. This work highlights the signifi cance of the soil microbial community in suppressing enteropathogens in soil, and of investigating die-off in a multi-factorial manner.

Roles for RpoS in survival of Escherichia coli during protozoan predation and in reduced moisture conditions highlight its importance in soil environments

The soil is a complex ecosystem where interactions between biotic and abiotic factors determine the survival and fate of microbial inhabitants of the system. Having previously shown that Escherichia coli requires the general stress response regulator, RpoS, to survive long term in soil, it was important to determine what specific conditions in this environment necessitate a functional RpoS. This study investigated the susceptibility of soil-persistent E. coli to predation by the single-celled eukaryotes Acanthamoeba polyphaga and Tetrahymena pyriformis, and the role RpoS plays in resisting this predation. Strain-specific differences were observed in the predation of E. coli strains, with soil-persistent strain COB583 being the most resistant to predation by both protozoans. RpoS and curli, proteinaceous fibres used for attachment to biotic and abiotic surfaces, increased the ability of E. coli to resist predation by A. polyphaga and T. pyriformis. Furthermore, soil moisture content impacted the survival of E. coli BW25113 but wild-type COB583 had similar survival irrespective of soil moisture content. Overall, this study confirmed that RpoS contributes to the resistance of E. coli to protozoan predation and that RpoS is crucial for the increased fitness of soil-persistent E. coli against predation and reduced moisture in soil.

Absence of Curli in Soil-Persistent Escherichia coli Is Mediated by a C-di-GMP Signaling Defect and Suggests Evidence of Biofilm-Independent Niche Specialization

Escherichia coli is commonly viewed as a gastrointestinal commensal or pathogen although an increasing body of evidence suggests that it can persist in non-host environments as well. Curli are a major component of biofilm in many enteric bacteria including E. coli and are important for adherence to different biotic and abiotic surfaces. In this study we investigated curli production in a unique collection of soil-persistent E. coli isolates and examined the role of curli formation in environmental persistence. Although most soil-persistent E. coli were curli-positive, 10% of isolates were curli-negative (17 out of 170). Curli-producing E. coli (COB583, COB585, and BW25113) displayed significantly more attachment to quartz sand than the curli-negative strains. Long-term soil survival experiments indicated that curli production was not required for long-term survival in live soil (over 110 days), as a curli-negative mutant BW25113ΔcsgB had similar survival compared to wild type BW25113. Mutations in two genes associated with c-di-GMP metabolism, dgcE and pdeR, correlated with loss of curli in eight soil-persistent strains, although this did not significantly impair their survival in soil compared to curli-positive strains. Overall, the data indicate that curli-deficient and biofilm-defective strains, that also have a defect in attachment to quartz sand, are able to reside in soil for long periods of time thus pointing to the possibility that niches may exist in the soil that can support long-term survival independently of biofilm formation.