Ship ballast water should be disinfected before being discharged into the ocean to avoid the dispersal of non-native species into the marine environment. This study presents the results of using pressurized carbon dioxide (CO2) at less than 1.0 MPa for inactivating Escherichia coli and Enterococcus sp. in artificial seawater (3.4% salinity). The bactericidal effects of pressurized CO2 were assessed using a liquid-film-forming apparatus under various conditions of pressure, temperature, and working volume ratio (WVR). Additionally, leakage of proteins and nucleic acids from cells was measured. Cell morphology of untreated cells and cells treated with pressurized CO2 was assessed using scanning electron microscopy (SEM). Pressurized CO2 treatment affected both strains; however, Enterococcus sp. exhibited higher resistance to pressurized CO2 treatment than did E. coli. Under identical treatment conditions (0.7 MPa, 20 °C, and 50% WVR), more than 5.0 log reduction in the load of E. coli and Enterococcus sp. was achieved after treatments for 5 min and 20 min, respectively. Release of intracellular contents occurred during the treatment process and SEM images of E. coli and Enterococcus sp. revealed that morphological changes had occurred after the treatment with pressurized CO2. Hence, pressurized CO2 has potential applications for inactivating pathogens in ballast water.

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Disease outbreaks attributed to monsoon flood-induced pathogen exposure are frequently reported, especially in developing cities with poor sanitation. Contamination levels have been monitored in past studies, yet the sources, routes, and extents of contamination are not always clear. We evaluated pollution from municipal wastewater (MWW) discharge and investigated fecal contamination by Escherichia coli (E. coli) in three agricultural fields on the outskirts of Hue City, Vietnam. After E. coli concentration was determined in irrigation water (IRW), MWW, soil, vegetables (VEG), and manure, its dispersion from MWW was tracked using multilocus sequence typing (MLST) and phylogenetic analyses during the wet and dry seasons. IRW was severely contaminated; 94% of the samples were positive with E. coli exceeding the stipulated standards, while VEG contamination was very low in both seasons. The confirmed total number of isolates was comparable between the seasons; however, results from MLST and phylogenetic clustering revealed more links between the sites and samples to MWW during the wet season. The wet season had four mixed clusters of E. coli isolates from multiple locations and samples linked to MWW, while only one mixed cluster also linking MWW to IRW was observed during the dry season. The most prevalent sequence type (ST) complex 10 and two others (40 and 155) have been associated with disease outbreaks, while other STs have links to major pathotypes. Irrigation canals are significant routes for E. coli dispersion through direct links to the urban drainage-infested river. This study clarified the genotype of E. coli in Hue city, and the numerous links between the samples and sites revealed MWW discharge as the source of E. coli contamination that was enhanced by flooding.