Klebsiella pneumoniae is a Gram-negative bacterium commonly associated with bloodstream infections (BSIs), which can lead to severe clinical outcomes, especially in immunocompromised individuals or patients with underlying health conditions. The increasing prevalence of K. pneumoniae that produces extended-spectrum β-lactamases (ESBL) poses a significant challenge for treatment and infection control, necessitating a swift diagnostic approach and tailored antimicrobial therapy to improve patient outcomes. A total of 32 K. pneumoniae isolates were recovered from BSIs from December 2021 to August 2022. Whole-genome sequencing (WGS) was performed on the 14 ESBL-producing isolates. All ESBL isolates carried the bla_CTX-M-15 gene, together with other β-lactamase-encoding genes (bla_TEM-1, bla_SHV-28, bla_SHV-26, or bla_OXA-1). Three of the isolates also carried the bla_KPC-3 gene. Resistance genes to quinolones, sulfonamides, tetracycline, aminoglycosides, and chloramphenicol were also detected. We can conclude that the presence of ESBL-producing isolates among K. pneumoniae of BSIs raises concerns, since these enzymes limit the available treatment options, and future research must include studies on alternative therapies for dealing with resistant bacterial infections and developing new approaches to disease treatment.

One of the significant challenges facing modern medicine is the rising rate of antibiotic resistance, which impacts public health, animal health, and environmental preservation. Evaluating antibiotic resistance in wildlife and their environments is crucial, as it offers essential insights into the dynamics of resistance patterns and promotes strategies for monitoring, prevention, and intervention. Klebsiella and Raoultella genera isolates were recovered from fecal samples of wild animals and environmental samples using media without antibiotic supplementation. Antibiograms were performed for 15 antibiotics to determine the phenotypic resistance profile in these isolates. Extended-spectrum β-lactamase (ESBL) production was tested by the double-disc synergy test, and one ESBL-producing K. pneumoniae isolate was screened by PCR and whole-genome sequencing. Biofilm production was analyzed using the microtiter plate method. A total of 23 Klebsiella spp. and 3 Raoultella spp. isolates were obtained from 312 fecal samples from wild animals, 9 Klebsiella spp. and 4 Raoultella spp. isolates were obtained from 18 river and stream water samples, and 4 Klebsiella spp. and 3 Raoultella spp. isolates from 48 soil samples. Regarding antibiotic resistance, only one isolate of K. pneumoniae from soil samples was an ESBL-producer and showed resistance to six antibiotics. This isolate harbored multiple β-lactams genes (bla_CTX-M-15, bla_TEM-1, bla_SHV-28, and bla_OXA-1), as well as genes of resistance to quinolones, sulfonamides, tetracycline, aminoglycosides, and chloramphenicol, and belonged to the lineage ST307. Most of the Klebsiella spp. and Raoultella spp. isolates were biofilm producers (except for one Klebsiella isolate), and 45.6% were weak biofilm producers, with the remaining being moderate to strong biofilm producers. We can conclude that antibiotic resistance is not widespread in these environment-associated isolates, which is a positive epidemiological outcome. However, identifying a single ESBL-K. pneumoniae isolate should serve as a warning of potential hotspots of resistance emergence.

Bats are increasingly recognized as reservoirs for antimicrobial-resistant bacteria, playing a potential role in the dissemination of resistance genes across species and regions. In this study, 105 bats from 19 species in Portugal were sampled to investigate the presence, antimicrobial resistance, and genetic characteristics of Mammaliicoccus and Staphylococcus isolates. Thirteen Mammaliicoccus lentus and Staphylococcus epidermidis were recovered. Antimicrobial susceptibility testing revealed multidrug resistance in three isolates, with S. epidermidis carrying mph(C), msr(A), and dfrC genes, and M. lentus harboring salB, tet(K), and str. Notably, qacA was detected in S. epidermidis, highlighting its plasmid-associated potential for horizontal gene transfer to more pathogenic bacteria. Heavy metal resistance genes (arsB and cadD) were also identified, suggesting the role of environmental factors in co-selecting antimicrobial resistance. Molecular typing revealed the S. epidermidis strain as ST297, a clone associated with both healthy humans and invasive infections. These findings emphasize the need for monitoring bats as reservoirs of resistance determinants, particularly in the context of zoonotic and environmental health. The presence of mobile genetic elements and plasmids further underscores the potential for the dissemination of resistance. This study reinforces the importance of adopting a One Health approach to mitigate the risks associated with antimicrobial resistance.

Klebsiella pneumoniae is an important pathogen associated with multidrug resistance and virulence in both human and animal populations. While its prevalence and resistance patterns are well documented in clinical settings, data on K. pneumoniae in food-producing animals remain scarce. This study aimed to isolate and characterize multidrug-resistant K. pneumoniae strains from healthy rabbits raised for human consumption, with a focus on antimicrobial resistance genes, plasmid content, and associated mobile genetic elements. A total of 295 fecal samples were collected from rabbits across 20 commercial farms in northern Portugal. Isolates were confirmed using MALDI-TOF MS, tested for hypermucoviscosity, and subjected to antimicrobial susceptibility testing (EUCAST). Whole-genome sequencing (WGS) was performed to determine sequence types (STs), resistance genes, plasmids, and resistance determinants for metals and biocides. Six K. pneumoniae isolates were recovered, showing extensive antimicrobial resistance profiles, including ESBL genes such as bla_CTX-M-15, bla_SHV-28, and bla_TEM-1. The most frequent ST was ST307. Multiple genes resistant to heavy metals were identified. Plasmid analysis revealed the presence of IncFII, IncN, and ColRNAI types. Network analysis showed clusters of genetically related isolates and highlighted shared resistance mechanisms. The presence of multidrug-resistant K. pneumoniae in healthy rabbits destined for human consumption underscores the zoonotic potential of this species and the need for surveillance in the animal–food–human interface. These findings contribute to a better understanding of resistance ecology in the context of One Health.

Background/Objectives: Enterobacterales are widely distributed in animals and environmental matrices and represent important reservoirs of antimicrobial resistance within a One Health framework. Gulls are particularly relevant because of their synanthropic behavior and frequent contact with anthropogenic waste, which may facilitate the acquisition and dissemination of resistant bacteria. Therefore, this study aimed to investigate the diversity, genomic backgrounds, and resistance determinants of Enterobacterales recovered from gulls and coastal environments in Portugal. Methods: A total of 265 samples, including seagull feces, saltwater, stagnant water, and sand, were collected from four coastal locations. Enterobacterales isolates were recovered and characterized by whole-genome sequencing, and comparative genomic analyses were performed for selected chromosomal beta-lactamase regions. Results:Enterobacterales were recovered from 41 samples and comprised 11 Klebsiella spp., 11 Leclercia spp., 9 Enterobacter spp., and 3 Citrobacter spp. isolates. Isolates were recovered mainly from seagull feces, followed by sand, and Berlenga Grande and Miramar showed the highest diversity. Klebsiella spp. and Enterobacter spp. concentrated most clinically relevant resistance determinants, including chromosomal bla_SHV-like, bla_OXY-like, and bla_ACT-like genes. By contrast, Leclercia pneumoniae was mainly associated with sand and showed a limited resistome, whereas Citrobacter braakii carried relevant genes such as bla_CMY and qnrB10. Comparative genomic analysis showed conserved chromosomal contexts surrounding bla_SHV-like, bla_OXY-like, and bla_ACT-like regions. Conclusions: These findings indicate that gull-associated fecal contamination may play an important role in the introduction and dissemination of resistant Enterobacterales in coastal ecosystems, with subsequent spread to sand and water. Coastal gulls and beach-associated matrices may therefore act as relevant reservoirs and indicators of antimicrobial resistance circulation at the human–environment interface.

The aquatic environment is a reservoir of antibiotics, pathogenic and native microorganisms, and antibiotic resistance genes (ARGs), constituting a key aspect of the One Health approach. Thus, the problem of antibiotic resistance is no longer restricted to bacterial pathogens but is a consequence of the interplay between distinct clinical and environmental drivers. In the project “ExplorAR—Exploring the Aquatic Resistome”, we investigated the putative role of cyanobacteria (CB) in the freshwater resistome, using a multidisciplinary approach based on phenotypic/genotypic/genomic tools. In this presentation, we disclose the main results of the project: (i) the development of a microdilution-based antibiotic susceptibility assay for CB; (ii) the establishment of an antibiotic susceptibility profile of CB genus/species; (iii) the identification of CB strains with reduced susceptibility to multiple antibiotic classes; (iv) the identification of ARGs in CB genomes; (v) the characterization of the antibiotic resistance profile of CB-associated bacteria; (vi) mapping the occurrence of CB and ARGs in surface freshwater reservoirs by high-throughput sequencing approaches. Overall, ExplorAR strongly supports the hypothesis that CB are environmental players in the emergence and dissemination of ARGs in water environments, contributing to the problem, and challenges, of antibiotic resistance.