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- Exploring flagellar contributions to motility and virulence in Arcobacter butzleriPublication . Santos, Raquel; Mateus, Cristiana; Oleastro, Mónica; Ferreira, SusanaFlagella is a well-known bacterial structure crucial for motility, which also plays pivotal roles in pathogenesis. Arcobacter butzleri, an enteropathogen, possesses a distinctive polar flagellum whose functional aspects remain largely unexplored. Upon investigating the factors influencing A. butzleri motility, we uncovered that environmental conditions like temperature, oxygen levels, and nutrient availability play a significant role. Furthermore, compounds that are found in human gut, such as short-chain fatty acids, mucins and bile salts, have a role in modulating the motility, and in turn, the pathogenicity of A. butzleri. Further investigation demonstrated that A. butzleri ΔflaA mutant showed a reduction in motility with a close to null average velocity, as well as a reduction on biofilm formation. In addition, compared with the wild-type, the ΔflaA mutant showed a decreased ability to invade Caco-2 cells and to adhere to mucins. Taken together, our findings support the role of environmental conditions and gut host associated compounds influencing key physiological aspects of the gastrointestinal pathogen A. butzleri, such as motility, and support the role of the flagellum on bacterial virulence.
- Genomic Analysis of Antibiotic Resistance and Virulence Profiles in Escherichia coli Linked to Sternal Bursitis in Chickens: A One Health PerspectivePublication . Ribeiro, Jessica; Silva, Vanessa; Freitas, Catarina; Pinto, Pedro; Vieira-Pinto, Madalena; Batista, Rita; Nunes, Alexandra; Gomes, João Paulo; Pereira, José Eduardo; Igrejas, Gilberto; Barros, Lillian; Heleno, Sandrina A.; Reis, Filipa S.; Poeta, PatríciaAbstract: Sternal bursitis is an underexplored lesion in poultry, often overlooked in microbiological diagnostics. In this study, we characterized 36 Escherichia coli isolates recovered from sternal bursitis in broiler chickens, combining phenotypic antimicrobial susceptibility testing, PCRbased screening, and whole genome sequencing (WGS). The genetic analysis revealed a diverse population spanning 15 sequence types, including ST155, ST201, and ST58. Resistance to tetracycline and ciprofloxacin was common, and several isolates carried genes encoding β-lactamases, including blaTEM-1B. Chromosomal mutations associated with quinolone and fosfomycin resistance (e.g., gyrA p.S83L, glpT_E448K) were also identified. WGS revealed a high number of virulence-associated genes per isolate (58–96), notably those linked to adhesion (fim, ecp clusters), secretion systems (T6SS), and iron acquisition (ent, fep, fes), suggesting strong pathogenic potential. Many isolates harbored virulence markers typical of ExPEC/APEC, such as iss, ompT, and traT, even in the absence of multidrug resistance. Our findings suggest that E. coli from sternal bursitis may act as reservoirs of resistance and virulence traits relevant to animal and public health. This highlights the need for including such lesions in genomic surveillance programs and reinforces the importance of integrated One Health approaches.
- Screening and in silico characterization of prophages in Helicobacter pylori clinical strainsPublication . Ferreira, Rute; Pinto, Graça; Presa, Eva; Oleastro, Mónica; Silva, Catarina; Vieira, Luís; Sousa, Claúdia; Pires, Diana; Figueiredo, Ceu; Melo, LuísThe increase of antibiotic resistance calls for alternatives to control Helicobacter pylori, a Gram-negative bacterium associated with various gastric diseases. Bacteriophages (phages) can be highly effective in the treatment of pathogenic bacteria. Here, we developed a method to identify prophages in H. pylori genomes aiming at their future use in therapy. A polymerase chain reaction (PCR)-based technique tested five primer pairs on 74 clinical H. pylori strains. After the PCR screening, 14 strains most likely to carry prophages were fully sequenced. After that, a more holistic approach was taken by studying the complete genome of the strains. This study allowed us to identify 12 intact prophage sequences, which were then characterized concerning their morphology, virulence, and antibiotic-resistance genes. To understand the variability of prophages, a phylogenetic analysis using the sequences of all H. pylori phages reported to date was performed. Overall, we increased the efficiency of identifying complete prophages to 54.1 %. Genes with homology to potential virulence factors were identified in some new prophages. Phylogenetic analysis revealed a close relationship among H. pylori-phages, although there are phages with different geographical origins. This study provides a deeper understanding of H. pylori-phages, providing valuable insights into their potential use in therapy.
- A single amino acid substitution in CspA increases germination sensitivity and broadens bile salt germinant specificity in clostridioides difficile sporesPublication . Roseiro, Isabel; Nunes, Alexandra; Martins, Diogo; Alves, Frederico; Persson, Søren; Henriques, Adriano O.; Oleastro, Mónica; Serrano, MónicaOnce regarded primarily as a healthcare-associated pathogen, Clostridioides difficile has increasingly been reported as a cause of community-acquired infection, raising questions about the contribution of animal and environmental reservoirs to persistence and transmission. Ribotype 033 (RT033) is predominantly associated with animal and environmental reservoirs, yet key traits underlying its ecology are poorly defined. Here we show that RT033 strains produce spores with enhanced germination sensitivity and expanded germinant specificity, enabling germination at low concentrations of bile-salts and in response to bile-salts that are typically inhibitory, including those more typical of animal hosts. Genetic analysis identified a single amino acid substitution (R1036I) in CspA, a component of the CspBAC germination apparatus, as the determinant of this phenotype. Expression of the RT033 cspBAC operon in a laboratory strain was sufficient to confer increased sensitivity and expanded bile-salt responsiveness. The R1036I substitution disrupts a conserved salt bridge at the CspA:CspC interface while not causing major destabilisation of the complex in the absence or presence of germinants and co-germinants. Together, our findings reveal a naturally occurring rewiring of bile-salt sensing in spores of RT033 strains that likely reflects adaptation to animal-associated bile-acid environments and may influence persistence, transmission, and zoonotic potential of this lineage.
- When disinfection fails: Biocide tolerance as a driver of campylobacter persistence and resistancePublication . Fonseca, Inês M.; Martins, Inês; Oleastro, Mónica; Ferreira, SusanaCampylobacter spp. constitutes a significant global public health hazard as it is a leading cause of reported foodborne diseases. Human infection is predominantly acquired through the ingestion of contaminated food, unpasteurized milk and untreated water, prompting the widespread implementation of chemical disinfection across several sectors, from healthcare, domestic environments, and food-processing to animal husbandry. While these biocidal agents encompass multiples classes with different modes of action and efficacy, growing evidence suggests that their extensive and repeated use may unintentionally promote bacterial persistence, tolerance and adaptive responses. Although biocide resistance has been documented in several foodborne pathogens, data on biocide tolerance in Campylobacter spp. remain limited. Available studies report variable degrees of reduced susceptibility to commonly used biocides among isolates originating from poultry production, food-processing environments, and water systems. Importantly, while biocide-induced adaptive responses in Campylobacter spp. may potentially overlap with antimicrobial resistance mechanisms, the extent to which these agents drive co-selection, persistence, or dissemination requires further elucidation. Evidence remains limited on the effects of long-term and repeated exposure under realistic processing conditions, the interplay between stress-induced gene regulation and stable genetic changes, and the contribution of mobile genetic elements, biofilm formation, and microbial communities in shaping antimicrobial resistance evolution. In light of the global health burden imposed by campylobacteriosis and the rising challenge of antimicrobial-resistant Campylobacter, this review brings together current evidence on the role of biocides in shaping bacterial survival, adaptation, and resistance mechanisms.