Staphylococcus aureus is a major pathogen causing intramammary infection and mastitis in dairy cows. S. aureus genotypes (GT) can differ significantly in their ability to diffuse and persist in the herd; while the association of virulence gene carriage with epidemiological behavior remains unclear, a role for secreted proteins has been postulated. We characterized the secretome of six S. aureus strains belonging to two genotypes with opposite within-herd prevalence, GTB (high) and GTS (low), corresponding to sequence types (ST) 8 and 398, by high-resolution tandem mass spectrometry and differential analysis with Proteome Discoverer. Data are available via ProteomeXchange with identifier PXD029571. Out of 720 identified proteins, 98 were unique or more abundant in GTB/ST8 and 68 in GTS/ST398. GTB/ST8 released more immunoglobulin-binding proteins, complement and antimicrobial peptide inhibitors, enterotoxins, and metabolic enzymes, while GTS/ST398 released more leukocidins, hemolysins, lipases, and peptidases. Furthermore, GTB/ST8 released the von Willebrand factor protein, staphylokinase, and clumping factor B, while GTS released the staphylococcal coagulase and clumping factor A. Hence, GTB/ST8 secretomes indicated a higher propensity for immune evasion and chronicity and GTS/ST398 secretomes for cellular damage and inflammation, consistent with their epidemiological characteristics. Accordingly, GTS/ST398 secretions were significantly more cytotoxic against bovine PBMCs in vitro. Our findings confirm the crucial role of extracellular virulence factors in S. aureus pathogenesis and highlight the need to investigate their differential release adding to gene carriage for a better understanding of the relationship of S. aureus genotypes with epidemiological behavior and, possibly, disease severity.

Dogs have been reported as potential carriers of antimicrobial-resistant bacteria, but the role of cats has been poorly studied. The aim of this study was to investigate the presence and the risk factors associated with the fecal carriage of extended-spectrum β-lactamase and AmpC (ESBL/AmpC)-producing Escherichia coli (E. coli) in pet and stray cats. Fecal samples were collected between 2020 and 2022 from healthy and unhealthy cats and screened for ESBL/AmpC-producing E. coli using selective media. The presence of ESBL/AmpC-producing E. coli was confirmed by phenotypic and molecular methods. The evaluation of minimum inhibitory concentrations (MICs) was performed on positive isolates. Host and hospitalization data were analyzed to identify risk factors. A total of 97 cats’ samples were collected, and ESBL/AmpC-producing E. coli were detected in 6/97 (6.2%), supported by the detection of bla_CTX-M (100%), bla_TEM (83.3%), and bla_SHV (16.7%) genes and the overexpression of chromosomal ampC (1%). All E. coli isolates were categorized as multidrug-resistant. Unhealthy status and previous antibiotic therapy were significantly associated with ESBL/AmpC-producing E. coli fecal carriage. Our results suggest that cats may be carriers of ESBL/AmpC-producing E. coli, highlighting the need for antimicrobial stewardship in veterinary medicine and an antimicrobial-resistance surveillance program focusing on companion animals, including stray cats.

This study investigated the presence, distribution, and antimicrobial resistance profiles of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in a dairy herd located in Northern Italy. The feces of clinically healthy calves, their mothers, and the cows treated for mastitis, as well as water, environmental samples, and waste milk were collected and subjected to bacteriological culture on CHROMagar^TM ESBL plates. A questionnaire was administered to identify risk factors. The isolates were identified as E. coli by MALDI-TOF MS and subjected to the double-disk synergy test (DDST) and minimal inhibitory concentration (MIC) assay. As a result, ESBL E. coli was isolated from the feces of 28 of 37 (75.67%) calves, the feces of 2 of 3 (66.67%) treated cows, 8 of 14 (57.15%) environmental samples, and waste milk. All ESBL isolates showed multiple resistances and were categorized as multidrug-resistant (MDR). Several risk factors for ESBL E. coli selection and diffusion were identified, including lack of routine cleaning of calf feeding and housing equipment, administration of waste milk to male calves, and blanket dry cow therapy. In conclusion, this study highlighted the presence of MDR, ESBL E. coli in the feces of most dairy calves, and their association with different sample sources. Accordingly, adding to the prudent use of antibiotics, the adoption of adequate farm hygiene and biosecurity measures might also help prevent the spread and transmission of ESBL E. coli within the herd.

In recent years, research on fish has seen remarkable advancements, especially in aquaculture, ornamental fish industry, and biomedical studies. Immunohistochemistry has become crucial in fish research, aiding in physiological and pathological investigations. However, the use of antibodies originally developed for mammals has raised concerns about their cross-reactivity and specificity in fish. This study systematically evaluated the reactivity of commonly used antibodies for diagnostic purposes, especially in fish pathology, including pan-cytokeratin, vimentin, S-100, glial fibrillary acidic protein, and desmin in the tissue of Sparus aurata, Dicentrarchus labrax, Oncorhynchus mykiss, and Carassius auratus. Western immunoblotting was employed to assess antibody specificity. The results revealed that the pan-cytokeratin and glial fibrillary acidic protein antibodies cross-react with all tested fish species, while S-100 demonstrated specific staining in sea bream, goldfish, and rainbow trout tissues. Conversely, vimentin and desmin antibodies displayed no reactivity. In conclusion, the anti-cytokeratin clone AE1/AE3 and the polyclonal rabbit anti-glial fibrillary acidic protein antibody, which are extensively used in mammals, were validated for fish immunohistochemical studies. Regrettably, D33 anti-desmin and V9 anti-vimentin clones are unsuitable for immunohistochemistry in the tested fish. These findings underscore the need for species-specific antibodies and proper validation for accurate immunohistochemistry analyses in fish research.

Background: In dogs, bacterial urinary tract infections are a frequent cause of antimicrobial prescription, increasing the risk of selecting antibiotic-resistant bacteria. This study analyzed resistance patterns, the presence of extended-spectrum β-lactamases (ESBLs) and biofilm-forming capacity in E. coli and K. pneumoniae previously isolated from urine samples collected from 133 selected dogs admitted to the Veterinary Teaching Hospital of Milan, Italy, in 2021 and 2023. Methods: The E. coli and K. pneumoniae isolates were bacteriologically and genetically analyzed. Results: Overall, 53/133 (39.8%) samples had a positive microbiological culture. Thirty-four E. coli/K. pneumoniae isolates were detected, accounting for 26.5% of the examined samples. The 34 isolates included 28 E. coli and 6 K. pneumoniae. Four (11.8%) were ESBL-producing bacteria, all supported by bla_CTX-M gene belonging to group 1. The K. pneumoniae isolates were significantly associated with ESBL production (p < 0.05). MIC analysis showed 11 (32.4%) multidrug-resistant isolates. Biofilm-forming capacity was observed in 23 (67.6%) isolates, regardless of bacterial species, including 20 weakly and 3 moderately adherent bacteria. All moderate biofilm producers were K. pneumoniae. Multidrug resistance (MDR) was significantly more present in strains with moderate biofilm-forming ability compared to strains with weak ability to form biofilm (p < 0.05). E. coli was confirmed as the most commonly identified urinary isolate in dogs. Conclusions: The high presence of ESBL producers and MDR in K. pneumoniae suggests mandatory in vitro susceptibility testing in the presence of this bacterium in dogs with UTI. The association of moderate biofilm production with MDR highlights the need for monitoring and surveillance of bacterial prevalence and resistance patterns of urinary isolates in dogs.