This study aimed to update the Streptococcus suis serotype distribution in Spain by analysing 302 clinical isolates recovered from diseased pigs between 2020 and 2022. The main objectives were to identify prevalent serotypes, differentiate specific serotypes 1, 14, 2, and 1/2, investigate specific genotypic and phenotypic antimicrobial resistance features, and explore associations between resistance genes and phenotypic resistances. Serotypes 9 (21.2%), 1 (16.2%), 2 (15.6%), 3 (6%), and 7 (5.6%) were the most prevalent, whereas serotypes 14 and 1/2 corresponded with 4.3% and 0.7% of all isolates. Antimicrobial resistance genes, including tet(O), erm(B), lnu(B), lsa(E), tet(M), and mef(A/E), were analysed, which were present in 85.8%, 65.2%, 7%, 7%, 6.3%, and 1% of the samples, respectively. Susceptibility testing for 18 antimicrobials revealed high resistance levels, particularly for clindamycin (88.4%), chlortetracycline (89.4%), and sulfadimethoxine (94.4%). Notably, seven significant associations (p < 0.0001) were detected, correlating specific antimicrobial resistance genes to the observed phenotypic resistance. These findings contribute to understanding the S. suis serotype distribution and its antibiotic resistance profiles in Spain, offering valuable insights for veterinary and public health efforts in managing S. suis-associated infections.

Staphylococcus hyicus is a significant pathogen in swine, primarily causing exudative epidermitis. Addressing S. hyicus infections requires both the characterization of virulence and antimicrobial resistance (AMR) in farm-recovered isolates. This study aimed to characterize the virulence, AMR, and biofilm formation of S. hyicus isolates from Spanish swine farms. A total of 49 isolates were analyzed, originating from animals with cutaneous, reproductive, and systemic clinical signs. Half of the isolates (49.0%) were positive for at least one virulence factor (VF) gene, with SHETA being the most frequent (28.6%). A high frequency of multidrug resistant (MDR) isolates was observed (83.7%), with significant resistance to commonly used antimicrobials, including lincosamides (83.7%), pleuromutilins (81.6%), penicillins (75.5%), and tetracyclines (73.5%). All isolates exhibited robust in vitro biofilm formation capacity (DC = 15.6 ± 7.0). Significant associations were found between VFs, biofilm formation, and AMR patterns, highlighting the link between the resistance to lincosamides and pleuromutilins (p < 0.001; Φ = 0.57) and macrolides (p < 0.001; Φ = 0.48), and the association of AMR with the ExhC and ExhD VF genes. These findings underscore the need for targeted diagnostics to improve management and therapeutic strategies to mitigate the impact of S. hyicus on swine production.

Glaesserella parasuis (Gp) is the etiological agent of Glässer's disease (GD), which causes important economic losses for the pig intensive production worldwide. This organism uses a smart protein-based receptor to acquire specifically iron from the porcine transferrin. This surface receptor consists of transferrin-binding protein A (TbpA) and transferrin-binding protein B (TbpB). TbpB has been considered the most promising antigen to formulate a based-protein vaccine with broad-spectrum of protection against GD. The purpose of our study was to determine the capsular diversity of Gp clinical isolates collected in different Spanish regions between 2018 and 2021. A total of 68 Gp isolates were recovered from porcine respiratory or systemic samples. A species-specific PCR based on tbpA gene, followed by multiplex PCR for typing Gp isolates were performed. Serovars 5, 10, 2, 4 and 1 were the most prevalent and involved almost 84% of isolates. TbpB amino acid sequences from 59 of these isolates were analyzed, and a total of ten clades could be established. All of them showed a wide diversity with respect to capsular type, anatomical isolation site and geographical origin, with minor exceptions. Regardless of the serovars, the in silico analysis of TbpB sequences revealed that a vaccine based on a TbpB recombinant protein could potentially prevent Glässer's disease outbreaks in Spain.

Glaesserella (Haemophilus) parasuis, the causative agent of Glässer’s disease, is present in most pig farms as an early colonizer of the upper respiratory tract. It exhibits remarkable variability in virulence and antimicrobial resistance (AMR), with virulent strains capable of inducing respiratory or systemic disease. This study aimed to characterize the virulence and the AMR profiles in 65 G. parasuis isolates recovered from Spanish swine farms. Virulence was assessed using multiplex leader sequence (LS)-PCR targeting vtaA genes, with all isolates identified as clinical (presumed virulent). Pathotyping based on ten pangenome genes revealed the virulent HPS_22970 as the most frequent (83.1%). Diverse pathotype profiles were observed, with 29 unique gene combinations and two isolates carrying only potentially non-virulent pangenome genes. AMR phenotyping showed widespread resistance, with 63.3% classified as multidrug resistant, and high resistance to clindamycin (98.3%) and tylosin (93.3%). A very strong association was found between certain pathotype genes and AMR phenotypes, notably between the virulent HPS_22970 and tetracycline resistance (p < 0.001; Φ = 0.58). This study reveals the wide diversity and complexity of G. parasuis pathogenicity and AMR phenotype, emphasizing the need for the targeted characterization of clinical isolates to ensure appropriate antimicrobial treatments and the implementation of prophylactic measures against virulent strains.

Introduction: Streptococcus suis is a zoonotic pathogen of great relevance to the swine industry, characterized by high genetic diversity and multiple serovars (SVs) with varying clinical prevalence. Biofilm formation represents a key factor in its virulence, antimicrobial resistance and infection persistence. Methods: We integrated gene expression profiling of biofilm-associated genes by RT-qPCR and antimicrobial susceptibility in planktonic and mature biofilm against five antibiotics in S. suis field isolates belonging to SV1, SV2, SV7 and SV9. Results: Expression of quorum sensing and adhesion genes (luxS, fbps, sadP and srtA) was significantly higher in SV2, the poorest biofilm formers, and inversely correlated with biofilm biomass, suggesting these factors act during early biofilm establishment. Correlation analysis indicated coordinated regulation among genes involved in quorum sensing, adhesion and capsule synthesis. Antimicrobial susceptibility testing revealed a high frequency of non-wild type phenotypes in planktonic cells for tetracycline, erythromycin and clindamycin (>80%), while ampicillin and ciprofloxacin were less frequent. Mature biofilms exhibited a significant increase in antimicrobial tolerance for all antibiotics tested, with SV2 showing the greatest susceptibility. Conclusions: These data highlight serovar-specific biofilm regulation patterns and enhanced drug tolerance in established S. suis biofilms.

Glaesserella parasuis (G. parasuis) is a key pathogen responsible for swine respiratory disease, and the development of broadly protective vaccines is hampered by its high antigenic diversity. The iron-acquisition protein TbpB is a conserved vaccine candidate, but the cellular immune responses it elicits, particularly T-cell subset dynamics during immunization and challenge, remain insufficiently defined. This study characterized these responses after oral immunization of colostrum-deprived piglets with the TbpB^Y167A mutant. Ten colostrum-deprived piglets were allocated to immunized and non-immunized (PBS) groups, immunized at days 15 and 30 of life and subsequently challenged with G. parasuis (45 days old); peripheral blood mononuclear cells were collected at baseline, after each immunization, and at 1 and 3 days post-infection. Multiparametric flow cytometry was used to quantify major leukocyte subsets and T-cell phenotypes defined by sIgM, CD172a, CD3, TCRγδ, CD8α/β, CD4 and CD27 expression. Booster immunization induced significant expansion of B cells (p < 0.01), TCRγδ T cells (p < 0.01), CD8⁺ αβ T cells (p < 0.001) and CD4⁺ memory T cells (p < 0.01) in immunized piglets compared with controls. After challenge, CD8⁺ cytotoxic T cells in immunized animals rapidly shifted from naïve to memory phenotypes, peaking at 48–72 h (p < 0.01). These biphasic T-cell dynamics are consistent with the protective efficacy previously demonstrated for this vaccine in colostrum-deprived piglets, and support a key contribution of TCRγδ, CD8⁺ cytotoxic and CD4⁺ memory T cells to immunity against G. parasuis and to the design of next-generation vaccines.