Pathogenic microorganisms released onto the soil from point or diffuse sources represent a public health concern. They can be transported by rainwater that infiltrates into subsoil and reach the groundwater where they can survive for a long time and contaminate drinking water sources. As part of the SCA.Re.S. (Evaluation of Health Risk Related to the Discharge of Wastewater on the Soil) project, we reviewed a selection of field-scale studies that investigated the factors that influenced the fate of microorganisms that were transported from the ground surface to the groundwater. A total of 24 studies published between 2003 and 2022 were included in the review. These studies were selected from the PubMed and Web of Science databases. Microbial contamination of groundwater depends on complex interactions between human activities responsible for the release of contaminants onto the soil, and a range of environmental and biological factors, including the geological, hydraulic, and moisture characteristics of the media traversed by the water, and the characteristics and the viability of the microorganisms, which in turn depend on the environmental conditions and presence of predatory species. Enterococci appeared to be more resistant in the underground environment than thermotolerant coliforms and were suggested as a better indicator for detecting microbial contamination of groundwater.

According to Italian Ministerial Decree No. 185 of 12 June 2003, water is considered suitable for irrigation if levels of fecal bacteria (i.e., Escherichia coli and Salmonella) are within certain parameters. The detection of other microorganisms is not required. The aim of this study is to determine the bacteriological quality of groundwater used for irrigation and the occurrence of enteric viruses (Norovirus, Enterovirus, Rotavirus, Hepatovirus A), and to compare the presence of viruses with the fecal bacteria indicators. A total of 182 wells was analyzed. Widespread fecal contamination of Apulian aquifers was detected (141 wells; 77.5%) by the presence of fecal bacteria (i.e., E. coli, Salmonella, total coliforms, and enterococci). Considering bacteria included in Ministerial Decree No. 185, the water from 35 (19.2%) wells was unsuitable for irrigation purposes. Among 147 wells with water considered suitable, Norovirus, Rotavirus, and Enterovirus were detected in 23 (15.6%) wells. No Hepatovirus A was isolated. Consequently, 58 wells (31.9%) posed a potential infectious risk for irrigation use. This study revealed the inadequacy of fecal bacteria indicators to predict the occurrence of viruses in groundwater and it is the first in Italy to describe the presence of human rotaviruses in well water used for irrigation.

Legionella is the causative agent of Legionnaires' disease, a flu-like illness normally acquired following inhalation or aspiration of contaminated water aerosols. Our recent studies revealed that climatic parameters can increase the number of reported cases of community-acquired Legionnaires' disease. Here, we evaluated the presence of Legionella in water networks and the distribution of Legionnaires' disease cases associated with touristic-recreational facilities in the Apulia region (southern Italy) during the period 2001-2017 using geostatistical and climatic analyses. Geostatistical analysis data revealed that the area with the highest concentration of Legionella in water systems also had the greatest number of cases of Legionnaires' disease associated with touristic-recreational facilities. Climatic analysis showed that higher daily temperature excursion (difference between maximum and minimum temperature) on the day of sampling was more often associated with Legionella-positive samples than Legionella-negative samples. In addition, our data highlighted an increased risk of Legionnaires' disease with increases in precipitation and average temperature and with decreases in daily temperature excursion (difference between maximum and minimum temperature over the course of 24 h in the days of incubation period of disease) and minimum temperature. Healthcare professionals should be aware of this phenomenon and be particularly vigilant for cases of community-acquired pneumonia during such climatic conditions and among the tourist population. The innovative geo-statistical approach used in this study could be applied in other contexts when evaluating the effects of climatic conditions on the incidence of Legionella infections.

Legionellae are opportunistic bacteria that cause various conditions after exposure to contaminated aerosols, ranging from a serious type of pneumonia to a mild case of an influenza-like illness. Despite the risks of exposure, little is known about the occurrence of Legionella in natural environments and, even though studies have shown that there is a potential risk of transmission via inhalation, it does not have to be detected in groundwater that is used for irrigation. The culture methods traditionally used to detect Legionella have several limits that can be partly solved by applying molecular techniques. Samples from 177 wells in Apulia, Southern Italy, were collected twice, in winter and in summer, and analyzed. When compared with the guidelines, 145 (81.9%) of the sampled wells were suitable for irrigation use. The culture-based method highlighted the presence of different species and serogroups of Legionella in 31 (21.2%) of the 145 wells that were shown to be suitable for irrigation use. A greater number of wells returned positive results for Legionella in summer than in winter (p = 0.023), and the median concentrations were mostly higher in summer (500 CFU/L) than in winter (300 CFU/L). The median temperature in the Legionella positive well waters was significantly higher than that in the negative ones, both in winter and in summer (p < 0.001). Using molecular techniques, Legionella non-pneumophila was found in 37 of the 114 wells earlier detected as suitable for irrigation use but negative for Legionella by the culture-based methods. The distribution of Legionella differ significantly in porous aquifers compared to the karst-fissured ones both with quantitative polymerase chain reaction (qPCR) (p = 0.0004) and viable cells by propidium monoazide (PMA-qPCR) (p = 0.0000). Legionella concentrations were weakly correlated with temperature of water both with qPCR (ρ = 0.47, p = 0.0033) and PMA-qPCR (ρ = 0.41, p = 0.0126). Our data suggest that water that aerosolizes when sprinkled on plants represents a potential source of Legionellosis, with a higher risk from exposure in summer. On a practical level, this finding is important for workers (farmers and gardeners) who are in contact with waters used for irrigation.

This study investigated the environmental contamination of groundwater as a consequence of the discharge of treated wastewater into the soil. The investigation focused on a wastewater treatment plant located in an area fractured by karst in the Salento peninsula (Apulia, Italy). Water samples were collected at four sites (raw wastewater, treated wastewater, infiltration trench, and monitoring well), monthly from May to December 2019 (with the exception of August), and were tested for (1) panel of bacteria; (2) enteric viruses; and (3) chemical substances. A gradual reduction in the concentration of bacteria, viruses and contaminants of emerging concern was observed across the profile of soil fissured by karst. All monitored bacteria were absent from the monitoring well, except for Pseudomonas aeruginosa. Pepper mild mottle virus and adenovirus were detected at all sampling sites. Personal care products and X-ray contrast media showed the greatest decrease in concentration from infiltration trench to the monitoring well, while the highest residual concentrations in the monitoring well were found for anticonvulsants (78.5%), antimicrobials (41.3%), and antipsychotic drugs (38.6%). Our results show that parameters provided by current law may not always be sufficient to evaluate the sanitary risk relating to the discharge of treated wastewater to the soil.

As a complement to clinical disease surveillance, the monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in wastewater can be used as an early warning system for impending epidemics. This study investigated the dynamics of SARS-CoV-2 in untreated wastewater with respect to the trend of coronavirus disease 2019 (COVID-19) prevalence in Southern Italy. A total of 210 wastewater samples were collected between May and November 2020 from 15 Apulian wastewater treatment plants (WWTP). The samples were concentrated in accordance with the standard of World Health Organization (WHO, Geneva, Switzerland) procedure for Poliovirus sewage surveillance, and molecular analysis was undertaken with real-time reverse-transcription quantitative PCR (RT-(q) PCR). Viral ribonucleic acid (RNA) was found in 12.4% (26/210) of the samples. The virus concentration in the positive samples ranged from 8.8 × 10² to 6.5 × 10⁴ genome copies/L. The receiver operating characteristic (ROC) curve modeling showed that at least 11 cases/100,000 inhabitants would occur after a wastewater sample was found to be positive for SARS-CoV-2 (sensitivity = 80%, specificity = 80.9%). To our knowledge, this is the first study in Italy that has applied wastewater-based epidemiology to predict COVID-19 prevalence. Further studies regarding methods that include all variables (meteorological phenomena, characteristics of the WWTP, etc.) affecting this type of wastewater surveillance data would be useful to improve data interpretation.