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Background and Aim: Pseudomonas aeruginosa is a relevant opportunistic and difficult to treat pathogen due to its widespread environmental diffusion, intrinsic resistance to many classes of antimicrobials, high ability to acquire additional resistance mechanisms, and wide range of pathogenic factors. The present study aimed to investigate the prevalence of P. aeruginosa in canine clinical samples, the antimicrobial susceptibility against antipseudomonal antibiotics, and the presence of extracellular pathogenic factors of the isolates, as well as their ability to produce biofilm. Materials and Methods: Overall, 300 clinical specimens from dogs with pyoderma or abscesses (n=58), otitis (n=59), and suspected bladder infection (n=183) were analyzed by standard bacteriological methods. P. aeruginosa isolates were tested for their antimicrobial susceptibility by disk and gradient diffusion methods to determine the minimum inhibitory concentrations. The ability of the isolates to produce biofilm was investigated by a microtiter plate assay, while virulence genes coding for elastase (lasB), exotoxin A (toxA), alkaline protease (aprA), hemolytic phospholipase C (plcH), and exoenzyme S (ExoS) were detected by polymerase chain reaction method. Results: A total of 24 isolates of P. aeruginosa were found in clinical specimens (urine n=3, skin/soft tissue n=6, and ear canal n=15). No resistance was found to ceftazidime, gentamicin, aztreonam, and imipenem (IMI), while low levels of resistance were found to enrofloxacin (ENR) (4.2%) and piperacillin-tazobactam (8.3%). However, 41.7% and 29.2% of the isolates showed intermediate susceptibility to ENR and IMI, respectively. Disk and gradient diffusion methods showed high concordance. The majority of the isolates revealed a weak (33.3%) or intermediate (45.8%) ability to form biofilm, while the strong biofilm producers (20.8%) derived exclusively from the ear canal samples. All isolates (100%) were positive for lasB, aprA, and plcH genes, while exoS and toxA were amplified in 21 (87.5%) and 22 (91.7%) isolates, respectively. Conclusion: In the present study, P. aeruginosa isolates from canine clinical samples were characterized by low levels of antimicrobial resistance against antipseudomonal drugs. However, the high presence of isolates with intermediate susceptibility for some categories of antibiotics, including carbapenems which are not authorized for veterinary use, could represent an early warning signal. Moreover, the presence of isolates with strong ability to produce biofilm represents a challenge for the interpretation of the antimicrobial susceptibility profile. In addition, the high prevalence of the extracellular pathogenic factors was indicative of the potential virulence of the isolates.

Streptococcus equi subsp. zooepidemicus (SEZ) is a major problem in equine veterinary medicine. Typically, a commensal in horses, SEZ can cause severe disease including respiratory infections, septicaemia and reproductive tract infections under certain conditions. Recent evidence suggests that humans can also develop severe disease infection through direct contact with infected animals or the consumption of contaminated unpasteurised milk and milk products. This study investigates SEZ strains isolated from nasal swabs of equidae in central Italy in 2023 to describe the epidemiology and genomic characteristics of circulating strains. A sampling plan was implemented to randomly collect nasal swabs from equid farms in the Abruzzo and Molise regions. In addition, a sampling form was designed to collect information on risk factors related to the presence of the bacterium and the potential development of respiratory disease. Relative risk was used to measure the association between the presence of SEZ in the samples and various variables. The swabs were analysed by real-time PCR and isolation. To confirm the identification and characterise the strains, the isolates were fully sequenced by next-generation sequencing (NGS) using the Illumina platform. A total of 478 animals from 99 farms were sampled and 30% of the animals tested positive for SEZ (79% horses, 20% donkeys, 1% mules). Forty-five percent of the farms tested positive for SEZ. Monitoring the clonal spread of SEZ is essential to understand the ecology of this emerging zoonotic pathogen, to assess the risk, and to implement effective control measures. In addition, genomic assessments are recommended to investigate the pathogenicity of circulating strains. This study provides a comprehensive understanding of the epidemiology and genomic diversity of SEZ strains isolated in central Italy.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

Brucella ceti infections have been increasingly reported in cetaceans, although a very limited characterization of Mediterranean Brucella spp. isolates has been previously reported and relatively few data exist about brucellosis among cetaceans in Italy. To address this gap, we studied 8 cases of B. ceti infection in striped dolphins (Stenella coeruleoalba) stranded along the Italian coastline from 2012 to 2018, investigated thanks to the Italian surveillance activity on stranded cetaceans. We focused on cases of stranding in eastern and western Italian seas, occurred along the Apulia (N = 6), Liguria (N = 1) and Calabria (N = 1) coastlines, through the analysis of gross and microscopic findings, the results of microbiological, biomolecular and serological investigations, as well as the detection of other relevant pathogens. The comparative genomic analysis used whole genome sequences of B. ceti from Italy paired with the publicly available complete genomes. Pathological changes consistent with B. ceti infection were detected in the central nervous system of 7 animals, showing non-suppurative meningoencephalitis. In 4 cases severe coinfections were detected, mostly involving Dolphin Morbillivirus (DMV). The severity of B. ceti-associated lesions supports the role of this microbial agent as a primary neurotropic pathogen for striped dolphins. We classified the 8 isolates into the common sequence type 26 (ST-26). Whole genome SNP analysis showed that the strains from Italy clustered into two genetically distinct clades. The first clade comprised exclusively the isolates from Ionian and Adriatic Seas, while the second one included the strain from the Ligurian Sea and those from the Catalonian coast. Plotting these clades onto the geographic map suggests a link between their phylogeny and topographical distribution. These results represent the first extensive characterization of B. ceti isolated from Italian waters reported to date and show the usefulness of WGS for understanding of the evolution of this emerging pathogen.

Antimicrobial resistance (AMR) is a global health concern that has been linked to humans, animals, and the environment. The One Health approach highlights the connection between humans, animals, and the environment and suggests that a multidisciplinary approached be used in studies investigating AMR. The present study was carried out to identify and characterize the antimicrobial resistance profiles of bacteria isolated from wildlife and livestock feces as well as from surface water samples in Maiella National Park, Italy. Ecological and georeferenced data were used to select two sampling locations, one where wildlife was caught within livestock grazing areas (sympatric group) and one where wildlife was caught outside of livestock grazing areas (non-sympatric group). Ninety-nine bacterial isolates from 132 feces samples and seven isolates from five water samples were collected between October and December 2019. The specimens were examined for species identification, antibiotic susceptibility and molecular detection of antibiotic resistance. Forty isolates were identified as Escherichia coli, forty-eight as Enterococcus spp., eight as Streptococcus spp. and ten as other gram-negative bacteria. Phenotypic antibiotic resistance to at least one antimicrobial agent, including some antibiotics that play a critical role in human medicine, was detected in 36/106 (33.9%, 95% CI: 25–43) isolates and multidrug resistance was detected in 9/106 isolates (8.49%, 95% CI: 3.9–15.5). In addition, genes associated with antibiotic resistance were identified in 61/106 (57.55%, 95% CI: 47.5–67) isolates. The samples from sympatric areas were 2.11 (95% CI: 1.2–3.5) times more likely to contain resistant bacterial isolates than the samples from non-sympatric areas. These data suggest that drug resistant bacteria may be transmitted in areas where wildlife and livestock cohabitate. This emphasizes the need for further investigations focusing on the interactions between humans, wildlife, and the environment, the results of which can aid in the early detection of emerging AMR profiles and possible transmission routes.

The available data on antimicrobial resistance in pets are limited compared to those collected for food-producing animals. Bacterial urinary tract infections are some of the most important indications for antimicrobial use in pets, and empiric antimicrobial treatments are often administered in the presence of clinical signs. In this study, the results obtained from the laboratory investigations carried out on dogs and cats with urinary tract infections coming from veterinary clinics and practices in Central Italy were evaluated to provide additional data concerning the bacterial urinary pathogens and their antimicrobial resistance patterns in pets. A total of 635 isolates were collected from urine samples. Escherichia coli was the most common species recovered in dogs and cats, followed by Proteus mirabilis and Enterococcus spp. Furthermore, it was possible to isolate bacteria not usually described in other studies concerning pets such as Pantoea dispersa, Raoultella ornithinolytica, and Pasteurella pneumotropica (also known as Rodentibacter pneumotropicus). Based on the antimicrobial susceptibility results, 472/635 (74.3%) isolates were resistant to at least one antibiotic and 285/635 (44.8%) isolates were classified as multidrug-resistant. Monitoring the antibiotic resistance profiles in pet infections is important not only for the public health implications, but also to collect data useful for the treatment of diseases in pets.

Background Bacterial antimicrobial resistance is a significant global threat to public health, closely linked to the misuse of antimicrobials in human and veterinary medicine, aquaculture, and agriculture. The consequences of antimicrobial resistance overcome species boundaries and require a holistic approach for mitigation actions. The study of antimicrobial resistance in wildlife is thus increasingly relevant to understand the spread of antimicrobial resistance in the environment and the animal community, as well as to investigate the role of wildlife either as a carrier, reservoir, spillover, or indicator of antimicrobial resistance. The aim of this study is to describe the prevalence and type of antimicrobial resistance in bacterial isolates from wild mammals through systematic review and meta-analysis of the available literature, following the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) guidelines. Results Out of 5052 collected documents, 3795 were screened, and finally 139 studies on antimicrobial resistance in free-ranging wild mammals were included in the meta-analysis. The studies covered 37 countries, mostly European. The Enterobacterales Escherichia coli and Salmonella spp., as well as Campylobacter spp., were the most frequently targeted bacterial species, mainly in the Artiodactyla order and specifically in the Suidae and Cervidae families. Low to moderate prevalences of antimicrobial resistance were found in all the continents, countries, bacteria, host taxa, and antimicrobials included in the meta-analysis, even for critically important antimicrobials as defined by the World Health Organisation, with higher values in Africa and Asia, in carnivores, and in animal species with high adaptability to diverse habitats. Conclusion This meta-analysis showed that antimicrobial resistance in wild mammals is widespread and variable according to taxonomy, trophic source, and geographic location. The meta-analysis highlighted methodological gaps that need to be addressed to improve the interpretation and conclusions obtained from the data. Genetic analyses on antimicrobial resistance and population ecological data should be included in future analysis to achieve a standardised methodology and overcome current limitations. To date, wildlife appears to be an environmental indicator of antimicrobial resistance and should be included in antimicrobial resistance surveillance plans not only because this sentinel role but also to monitor potential spill-back to livestock and/or humans.

In this study, a culture-independent approach was applied to compare the microbiome composition and the abundance of the antimicrobial resistance genes (ARGs) aadA2 for aminoglycosides, tet(A), tet(B), tet(K), and tet(M) for tetracyclines, and mcr-1 for colistin in broiler litter samples collected from conventional and antibiotic-free flocks located in Central Italy. A total of 13 flocks and 26 litter samples, collected at the beginning and at the end of each rearing cycle, were submitted to 16s rRNA sequence analysis and quantitative PCR for targeted ARGs. Firmicutes resulted in the dominant phylum in both groups of flocks, and within it, the Clostridia and Bacilli classes showed a similar distribution. Conversely, in antibiotic-free flocks, a higher frequency of Actinobacteria class and Clostridiaceae, Lactobacillaceae, Corynebacteriaceae families were reported, while in the conventional group, routinely treated with antibiotics for therapeutic purposes, the Bacteroidia class and the Enterobacteriaceae and Bacillaceae families were predominant. All investigated samples were found to be positive for at least one ARG, with the mean values of aadA2 and tet(A) the highest in conventional flocks by a significant margin. The results suggest that antibiotic use can influence the frequency of resistance determinants and the microbial community in poultry flocks, even though other environmental factors should also be investigated more deeply in order to identify additional drivers of antimicrobial resistance.

Rose Bengal antigen and smooth lipopolysaccharide (s-LPS) were produced from a field strain of Brucella ceti (“homologous” antigens) and from the reference strain B. abortus S99 (“heterologous” antigens); they are currently used for the diagnosis of brucellosis in cattle, water buffaloes, sheep, goats, and pigs, as recommended in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (WOAH). “Homologous” and “heterologous” antigens were used in a rapid serum agglutination test (Rose Bengal test, RBT) and a competitive ELISA assay (c-ELISA) to test a panel of sera, blood, and other body fluids (cerebrospinal fluid, pericardial fluid, tracheal fluid, and aqueous humor) collected from 71 individuals belonging to five cetacean species (Stenella coeruleoalba; Tursiops truncatus; Grampus griseus; Globicephala melas; and Ziphius cavirostris), which were found stranded on the Italian coastline. Six animals were positive for Brucella spp. for bacterial isolation and/or PCR, and 55 animals were negative; for the remaining 10 animals, no PCR/isolation data were available. A total of 90 samples were tested. Results obtained from the two tests were compared in order to identify the most suitable antigen for the serological diagnosis of Brucella infection in cetaceans. The RBT performed with the “homologous” antigen showed the best results in comparison with the “heterologous” antigen: diagnostic sensitivity, specificity, and accuracy were 80.0%, 44.1%, and 46.9% for the “homologous” antigen and 80.0%, 17.0%, and 21.9% for the “heterologous” antigen. For the c-ELISA, “homologous” and “heterologous” s-LPS showed similar results (diagnostic sensitivity 66.7%, diagnostic specificity 97.3%, and diagnostic accuracy 95.0%). Therefore, the RBT using the “homologous” antigen and c-ELISA with “homologous” or “heterologous” s-LPS could be used in parallel for the detection of antibodies against Brucella spp. in cetaceans.

Between 2019 and 2021, 21 bottlenose dolphins were found stranded dead along the Ligurian Pelagos Sanctuary coast (Italy). For 11 animals, out of a total of 14 recovered, the cause of death was classified as natural, anthropic, or not determined based on gross and histological pathology and ancillary testing. Hexachlorobenzene (HCB), dichlorodiphenyltrichloroethanes (DDTs), and polychlorinated biphenyls (PCBs) were examined in their blubber, and results were discussed according to their toxicological properties. All specimens showed the following trend: PCBs > DDTs >> HCB, and the levels of cancerogenic, mutagenic, and teratogenic organochlorine compounds (T-OCs) were >50% of total OCs. Immunosuppressant organochlorine compound (IS-OC) levels in 10 out of 11 animals were above 50% of the total OCs. PCB levels always exceeded the threshold of 17 mg/kg lipid weight for PCB-induced adverse health effects. The results suggest that bottlenose dolphins living in the Pelagos Sanctuary undergo a high level of exposure to pathogens and OCs, betraying the designation of the Cetacean Sanctuary and, consequently, of a region created for their conservation. Immune dysfunction and infectious disease susceptibility appear to be highly connected with high levels of OC xenobiotics. These data are useful to understand health and mortality trends in cetacean populations, as well as for developing policies for cetacean conservation and management in this important protected area of the Mediterranean.