As bioindicators, freshwater bivalves are crucial for the assessment of the contamination impact on different levels of biological integration. Escherichia coli is used as a bioindicator of water fecal contamination, representing a critical global concern, especially with the rise of multidrug-resistant (MDR) strains. Phylogenetic diversity, pathotypic characterization, and antibiotic resistance profiles of E. coli isolated from freshwater bivalves (Anodonta anatina) were assessed. Samples were collected from the Tua River in Northern Portugal, from two different sites, Chelas and Barcel, representing different degrees of contamination. Antimicrobial susceptibility testing was performed by the disk diffusion method, and characterizations of the phylogenetic groups and pathotypes were assessed by PCR-multiplex and real-time PCR-multiplex, respectively. Results showed that 60% of isolates were characterized as MDR, including resistance to carbapenems, considered the last resort against multidrug-resistant bacteria. Within this study, it was also possible to verify the antimicrobial resistance (AMR) profile differences between the two sampling sites, with bivalve isolates from the Chelas site showing a higher percentage of antibiotic resistance. Among the E. coli isolates, the highest prevalence (55%) was observed in group B1, followed by group D or E (15%), group A (10%), and group E or Clade I (10%). None of the isolates were classified as diarrheagenic E. coli (DEC). This work highlights the potential transmission of antimicrobial-resistant bacteria through bivalves in the food chain. The ‘One Health’ approach is crucial for combating antimicrobial resistance, namely in edible freshwater species, emphasizing active surveillance to protect human, animal, and environmental health against the spread of antibiotic-resistant bacteria in aquatic environments.

Coastal environments have been recognized as key reservoirs for antibiotic-resistant bacteria. The present study evaluated marine gastropods, Phorcus lineatus, as potential bioindicators to assess the spread of antibiotic-resistant bacteria. P. lineatus was sampled in four sites, with different anthropogenic pressures, along the northwest Portuguese coastal area. From these specimens, bacteria were isolated and tested for their antimicrobial susceptibility, followed by their phylogenetic and pathotypic determination. All the Escherichia coli isolates showed resistance to at least one antimicrobial agent. The highest levels of multidrug resistance (25%) were observed in E. coli isolates obtained from SITE 2, which is impacted by the city of Porto and industrial settlements, while nearly 17% of these isolates showed a multiple antibiotic resistance (MAR) index higher than 0.2. Among the isolates, phylogroups A and B2 were the most prevalent, followed by phylogroup B1. The isolates of phylogroup A showed a higher prevalence of antimicrobial resistance. This study offers valuable insights into the antibiotic resistance risks posed to marine ecosystems and underscores the need for microbiological monitoring and the development of effective management strategies. The findings suggest P. lineatus as a potential bioindicator of antibiotic-resistant bacteria in marine environments.

Background: Domestic refrigeration and egg handling are key factors in ensuring household food safety. Inadequate temperature control and poor hygiene in refrigerators can promote the survival and growth of foodborne pathogens. This study aimed to (i) characterize refrigerator temperature profiles and surface microbial contamination and (ii) screen eggs and egg-storage areas for the presence of Salmonella spp. and Campylobacter spp. Methods: Fifty domestic refrigerators were monitored twice in 2024 and 2025 in Porto, Portugal. The temperatures were continuously logged on the lowest shelf, which was swabbed for microbiological analysis. Surface hygiene was evaluated using total viable counts (TVC), Enterobacteriaceae, and Escherichia coli enumerated following ISO methods. Detection of pathogens Listeria monocytogenes, Salmonella spp., and Campylobacter spp. was performed using real-time PCR. Eggs (n = 92 in 2024; n = 88 in 2025), and domestic egg storage areas (total n = 76) were screened for Salmonella and Campylobacter. Results: The mean refrigerator temperatures were 6.0 ± 0.5 °C in 2024 and 6.1 ± 0.5 °C in 2025; 44% and 50% of the units, respectively, exceeded the recommended 6 °C threshold. In 2025, 31 (62%) and 33 (66%) refrigerators showed higher TVC and Enterobacteriaceae counts compared to 2024, whereas E. coli was only detected sporadically. L. monocytogenes, Salmonella spp., or Campylobacter spp. were not recovered from the refrigerator surfaces. Likewise, Salmonella and Campylobacter were not detected in any of the eggs or egg-storage sites. Indicator microorganism’s counts were not associated with the mean temperature. Conclusions: The absence of correlation between ΔT and Δ microbial counts suggests that behaviour-driven hygiene factors, rather than the relatively small year-to-year temperature differences observed, are more influential in determining household bioburden. Maintaining refrigerator temperatures ≤ 6 °C together with simple hygiene practices remains essential for reducing household food safety risks.