Rosa Araujo

Infectious agents shape fish populations by inducing lethal and sublethal changes that alter nutrient metabolism and metal bioaccumulation. These shifts can manifest as changes in the ionome—the specific combination of essential and non-essential chemical elements defining the whole-body composition of an individual. Understanding how pathogens shape the fish ionome is critical for developing advanced monitoring tools and clarifying the ecological roles of hosts and their pathogens. This study reports the first documented outbreak of Citrobacter freundii, Pseudomonas aeruginosa, Pseudomonas mosselii, and Shewanella xiamenensis bacterial infections in wild-caught eastern mosquitofish (Gambusia holbrooki) from three populations in Extremadura, southwestern Spain. Under laboratory-controlled conditions, we established associations between these bacterial outbreaks and the whole-fish body ionome of G. holbrooki. We compared 19 chemical elements and seven elemental ratios among diseased fish, healthy fish at the outbreak, and individuals fully recovered 100 days post-infection following antibiotic treatment. The fish ionome clearly discriminated between diseased and healthy states, and the response was consistent across all three populations. Our findings support the utility of whole-fish body elemental composition in G. holbrooki as a biomarker for environmental monitoring. Furthermore, as the bacterial infections were associated with the capture and transport-induced stress of wild individuals, this study provides critical data on the opportunistic pathogens that may be co-introduced into recipient ecosystems through the release of this widely distributed invasive fish species.

The use of lagooning as a complementary natural method of treating secondary effluents of wastewater treatment plants has been employed as an affordable and easy means of producing reclaimed water. However, using reclaimed water for some purposes, for example, for food irrigation, presents some risks if the effluents contain microbial pathogens. Classical bacterial indicators that are used to assess faecal contamination in water do not always properly indicate the presence of bacterial or viral pathogens. In the current study, the presence of faecal indicator bacteria (FIB), heterotrophic bacterial counts (HBC), pathogens and opportunistic pathogens, such as Legionella spp., Aeromonas spp., Arcobacter spp., free-living amoeba (FLA), several viral indicators (human adenovirus and polyomavirus JC) and viral pathogens (noroviruses and hepatitis E virus) were analysed for 1 year in inlet and outlet water to assess the removal efficiency of a lagooning system. We observed 2.58 (1.17–4.59) and 1.65 (0.15–3.14) log reductions in Escherichia coli (EC) and intestinal enterococci (IE), respectively, between the inlet and outlet samples. Genomic copies of the viruses were log reduced by 1.18 (0.24–2.93), 0.64 (0.12–1.97), 0.45 (0.04–2.54) and 0.72 (0.22–2.50) for human adenovirus (HAdV), JC polyomavirus (JCPyV) and human noroviruses (NoV GI and GII), respectively. No regrowth of opportunistic pathogens was observed within the system. FLA, detected in all samples, did not show a clear trend. The reduction of faecal pathogens was irregular with 6 out of 12 samples and 4 out of 12 samples exceeding the EC and IE values, specified in the Spanish legislation for reclaimed water (RD 1620/2007). This data evidences that there is a need for more studies to evaluate the removal mechanisms of lagooning systems in order to optimize pathogen reduction. Moreover, surveillance of water used to irrigate raw edible vegetables should be conducted to ensure the fulfilment of the microbial requirements for the production of safe reclaimed water.

Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L^-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed.

The extended aeration activated sludge (EAAS) process is one of the most applied biological processes in small towns. Here, we study the abundance and viability of total bacterial cells in two wastewater treatment plants (WWTPs) operating with an EAAS process. We use flow cytometry (FCM) combined with SYTO13 and propidium iodide (PI) dyes as a rapid, easy, reliable and accurate microbial monitoring tool. A disaggregation procedure with an ultrasonic bath was designed to detach total bacterial cells from activated sludge flocs for subsequent FCM analysis. This procedure permitted the recovery of total bacterial cells from sludge flocs without affecting bacterial viability, as indicated by bacterial strain controls. Since FCM is a multi-parameter technique, it was possible to determine total bacterial abundance and their viability in the activated sludge. As a comparative method, epifluorescence microscopy was also used to quantify total bacterial cells; both methods produced similar results. The FCM analysis revealed relative microbial stability in both the WWTPs. The total bacterial abundance quantified by FCM in the two plants studied was 1.02–6.23 × 10¹¹ cells L⁻¹ with 70–72 % viability, one logarithm less than that reported in the literature for WWTPs using the conventional activated sludge process. This can be explained by the difference in the operational parameters between the conventional plant and EAAS, mainly the organic loading rate.