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Introduction: Groundwater is considered the best candidate for drinking water supply in the karst area. The groundwater water resources, however, are vulnerable to pathogenic microorganism contamination because of the typically thin soil layers overlying aquifers and the high permeability of the aquifer host rock, resulting in short residence times and low natural attenuation capacities. Until now, little attention has been paid to the critical environmental factors affecting the pathogenic microorganism contamination in soil-groundwater systems in the karst area. Methods: In the study, orthogonality column experiments with controlling ambient temperatures, pH values of inlet water, and soil porosities were carried out to investigate the transport and lifespan of pathogenic microorganisms in the leachate of agricultural soils in the karst area of Yunnan province, China. The pathogenic indicators, i.e., total bacteria count (TBC) and total coliforms count (TCC), and hydrochemical parameters, i.e., pH and permanganate index (CODMn) in the leaching water, were systematically monitored. Results and Discussion: The results showed that bacteria including coliforms can survive for prolonged periods of time in karst soils. The soils overlying the karst rocks were unable to impede the bacteria from seeping into the groundwater. The soils, in turn, likely served as both reservoirs and incubators for pathogenic bacteria. The ambient temperature was the most predominant influential factor affecting both TBC and TCC. The bacteria concentrations were proportional to the temperature in the leachate. Therefore, more attention should be paid to temperature variations in protecting the water supply, particularly in the high-temperature period, such as during the summer months. [ABSTRACT FROM AUTHOR]

Agricultural soil contaminated by manure is becoming an important source for the transmission of foodborne pathogens. There is an urgent need for a rapid and accurate method for viable pathogen detection in agricultural soil samples. Propidium monoazide (PMA) is a DNA-binding dye that can inhibit the amplification of DNA from dead cells through subsequent quantitative polymerase chain reaction (qPCR), thus allowing for viable cells detection and quantification. The objective of this study was to detect viable Escherichia coli O157:H7 in the agricultural soils by PMA-qPCR. In this study, cell extraction and gradient density centrifugation were incorporated before PMA-qPCR to reduce the interference of soil particle including turbidity and a high ratio of dead cells. The optimized treatment conditions were determined as follows, the maximum removal of DNA from dead cells was achieved by 1.067 g/mL Percoll of centrifugation and 50 μM PMA treatment. Under these conditions, the turbidity of paddy soil suspensions decreased from 3500 to 28.4 nephelometric turbidity units (NTU), and the ratio of viable cells to dead cells increased from 0.001 to 1.025%. For typical agricultural soils collected in China, as low as 102colony-forming units (CFU)/g of viable cells could be accurately detected in the presence of a large number of dead cells (107 CFU/g) by the optimized PMA-qPCR. Significantly, with comparable accuracy, the optimized PMA-qPCR assay was more sensitive, accessible and rapid than conventional culture methods. In addition, the viable but non-culturable (VBNC) state of E. coli O157:H7 cells in paddy soils, which often escaped the detection by conventional culture methods, could be quantitatively characterized by the optimized PMA-qPCR method. Potentially, the optimized PMA-qPCR can be further applied for viable pathogens detection and give insight into the prevalence of VBNC E. coli O157:H7 in agricultural soil.

Agricultural soil contaminated by manure is becoming an important source for the transmission of foodborne pathogens. There is an urgent need for a rapid and accurate method for viable pathogen detection in agricultural soil samples. Propidium monoazide (PMA) is a DNA-binding dye that can inhibit the amplification of DNA from dead cells through subsequent quantitative polymerase chain reaction (qPCR), thus allowing for viable cells detection and quantification. The objective of this study was to detect viable Escherichia coli O157:H7 in the agricultural soils by PMA-qPCR. In this study, cell extraction and gradient density centrifugation were incorporated before PMA-qPCR to reduce the interference of soil particle including turbidity and a high ratio of dead cells. The optimized treatment conditions were determined as follows, the maximum removal of DNA from dead cells was achieved by 1.067 g/mL Percoll of centrifugation and 50 μM PMA treatment. Under these conditions, the turbidity of paddy soil suspensions decreased from 3500 to 28.4 nephelometric turbidity units (NTU), and the ratio of viable cells to dead cells increased from 0.001 to 1.025%. For typical agricultural soils collected in China, as low as 102colony-forming units (CFU)/g of viable cells could be accurately detected in the presence of a large number of dead cells (107 CFU/g) by the optimized PMA-qPCR. Significantly, with comparable accuracy, the optimized PMA-qPCR assay was more sensitive, accessible and rapid than conventional culture methods. In addition, the viable but non-culturable (VBNC) state of E. coli O157:H7 cells in paddy soils, which often escaped the detection by conventional culture methods, could be quantitatively characterized by the optimized PMA-qPCR method. Potentially, the optimized PMA-qPCR can be further applied for viable pathogens detection and give insight into the prevalence of VBNC E. coli O157:H7 in agricultural soil. [ABSTRACT FROM AUTHOR]

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Introduction: Groundwater is considered the best candidate for drinking water supply in the karst area. The groundwater water resources, however, are vulnerable to pathogenic microorganism contamination because of the typically thin soil layers overlying aquifers and the high permeability of the aquifer host rock, resulting in short residence times and low natural attenuation capacities. Until now, little attention has been paid to the critical environmental factors affecting the pathogenic microorganism contamination in soil-groundwater systems in the karst area., Methods: In the study, orthogonality column experiments with controlling ambient temperatures, pH values of inlet water, and soil porosities were carried out to investigate the transport and lifespan of pathogenic microorganisms in the leachate of agricultural soils in the karst area of Yunnan province, China. The pathogenic indicators, i.e., total bacteria count (TBC) and total coliforms count (TCC), and hydrochemical parameters, i.e., pH and permanganate index (COD Mn ) in the leaching water, were systematically monitored., Results and Discussion: The results showed that bacteria including coliforms can survive for prolonged periods of time in karst soils. The soils overlying the karst rocks were unable to impede the bacteria from seeping into the groundwater. The soils, in turn, likely served as both reservoirs and incubators for pathogenic bacteria. The ambient temperature was the most predominant influential factor affecting both TBC and TCC. The bacteria concentrations were proportional to the temperature in the leachate. Therefore, more attention should be paid to temperature variations in protecting the water supply, particularly in the high-temperature period, such as during the summer months., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor QL declared a shared affiliation with the authors at the time of review., (Copyright © 2023 Ning, Wang, Guo and Zhang.)

The community of ammonia-oxidizing prokaryotes was examined in an agricultural soil treated for 6 seasons with contrasting nitrogen (N) sources. Molecular tools based on the gene encoding ammonia monooxygenase were used to characterize the ammonia oxidizer communities and their abundance. Soil DNA was extracted from soils sampled from silage corn plots that received no additional N (control), dairy waste compost (DC), liquid dairy waste (LW), and ammonium sulfate (AS) treatments at approximately 100 and 200 kg available N ha-1 over 6 years. The N treatment affected the quantity of ammonia oxidizers based on estimates of amoA by real-time PCR. Ammonia oxidizing bacteria (AOB) were higher in soils from the AS200, AS100, and LW200 treatments (2.5 x107, 2.5x107, and 2.1 x107 copies g-1 soil, respectively) than in the control (8.1x106copies/g) while the abundance of amoA encoding archaea (AOA) was not significantly affected by treatment (3.8x107copies g-1 soil, average). The ratio of AOA/AOB was higher in the control and compost treated soils, both treatments have the majority of their ammonium supplied through mineralization of organic nitrogen. Clone libraries of partial amoA sequences indicated AOB related to Nitrosospira multiformis and AOA related to uncultured Nitrososphaera similar to those described by soil fosmid 54d9 were prevalent. Profiles of the amoC-amoA intergenic region indicated that both Nitrosospira- and Nitrosomonas-type AOB were present in all soils examined. In contrast to the intergenic amoC-amoA profile results, Nitrosomonas-like clones were recovered only in the LW200 treated soil-DNA. The impact of six years of contrasting nitrogen sources applications caused changes in ammonia oxidizer abundance while the community composition remained relatively stable for both AOB and AOA.

The community of ammonia-oxidizing prokaryotes was examined in an agricultural soil treated for six seasons with contrasting nitrogen (N) sources. Molecular tools based on the genes encoding ammonia monooxygenase were used to characterize the ammonia oxidizer (AO) communities and their abundance. Soil DNA was extracted from soils sampled from silage corn plots that received no additional N (control), dairy waste compost, liquid dairy waste (LW), and ammonium sulfate (AS) treatments at approximately 100 and 200 kg available N ha(-1) over 6 years. The N treatment affected the quantity of AO based on estimates of amoA by real-time PCR. Ammonia oxidizing bacteria (AOB) were higher in soils from the AS200, AS100, and LW200 treatments (2.5 × 10(7), 2.5 × 10(7), and 2.1 × 10(7)copies g(-1) soil, respectively) than in the control (8.1 × 10(6) copies g(-1) soil) while the abundance of amoA encoding archaea [ammonia oxidizing archaea (AOA)] was not significantly affected by treatment (3.8 × 10(7) copies g(-1) soil, average). The ratio of AOA/AOB was higher in the control and compost treated soils, both treatments have the majority of their ammonium supplied through mineralization of organic nitrogen. Clone libraries of partial amoA sequences indicated AOB related to Nitrosospira multiformis and AOA related to uncultured Nitrososphaera similar to those described by soil fosmid 54d9 were prevalent. Profiles of the amoC-amoA intergenic region indicated that both Nitrosospira- and Nitrosomonas-type AOB were present in all soils examined. In contrast to the intergenic amoC-amoA profile results, Nitrosomonas-like clones were recovered only in the LW200 treated soil-DNA. The impact of 6 years of contrasting nitrogen sources applications caused changes in AO abundance while the community composition remained relatively stable for both AOB and AOA.