Background/Objectives: Nerolidol (NER) is a sesquiterpene alcohol with recognized antimicrobial potential, whose applications as a pure substance are limited by hydrophobicity, instability, and cytotoxicity. Invasomes, i.e., liposomes with terpene ingredients, offer a strategy to improve their delivery; however, the NER loading limits compatible with vesicle integrity are still unclear. Here, Nerolidol-loaded invasomes were produced using a controlled simil-microfluidic coaxial injection process. Methods and Results: As a preliminary step, unloaded liposomes were fabricated to consolidate operating conditions and ensure their reproducible colloidal properties. Thereafter, formulations with progressively decreasing nominal NER loads were investigated to evaluate vesicle size, polydispersity, ζ-potential, encapsulation efficiency, effective loading, and stability. High nominal loads promoted turbidity, size increase (by agglomeration coalescence phenomena), and structural instability, whereas formulations containing approximately 1–2% NER achieved nearly complete encapsulation, Z-average ≈ 300 nm, |ζ| > 30 mV, and satisfactory physical stability. Antimicrobial and cytotoxic profiles of representative formulations, previously evaluated in an independent study are here reported only to contextualize the practical relevance of the optimized systems, while the present work primarily focuses on process–formulation aspects and loading/stability limitations. Conclusions: Overall, the present work identifies a realistic loading window for Nerolidol invasomes and highlights the suitability of the simil-microfluidic approach to obtain scalable, well-controlled formulations, providing a rational basis for their future biological assessment. Nerolidol invasome systems indeed can be considered a promising, versatile platform for antimicrobial applications, including prospective use in animal feed.

Nerolidol (NER) is a sesquiterpene alcohol with recognized antimicrobial potential, whose applications as pure substance are limited by hydrophobicity, instability, and cytotoxicity. Invasomes, i.e. liposomes with terpene ingredients, offer a strategy to improve its delivery; however, the NER loading limits compatible with vesicle integrity are still unclear. Here, Nerolidol-loaded invasomes were produced using a controlled simil-microfluidic coaxial injection process. As a preliminary step, unloaded liposomes were fabricated to consolidate operating conditions and ensure their reproducible colloidal properties. Thereafter, formulations with progressively decreasing nominal NER loads were investigated to evaluate vesicle size, polydispersity, ζ-potential, encapsulation efficiency, effective loading, and stability. High nominal loads promoted turbidity, size increase (by agglomeration coalescence phenomena), and structural instability, whereas formulations containing approximately 1–2% NER achieved nearly complete encapsulation, Z-average ≈ 300 nm, |ζ| > 30 mV, and satisfactory physical stability. Antimicrobial and cytotoxic profiles of representative formulations, previously evaluated in an independent study, confirmed biological activity. Overall, this work identifies a realistic loading window for Nerolidol invasomes and highlights the suitability of the simil-microfluidic approach to obtain scalable, well-controlled formulations, providing a rational basis for their future biological assessment. Nerolidol invasomes systems indeed can be considered a promising versatile platform for antimicrobial applications, including prospective use in animal feed.

Eucalyptus globulus leaf extract (EGL) is a phytocomplex associated with antimicrobial and anti-inflammatory effects, but a comprehensive characterization of its chemical composition and its relationship with biological activity remains limited. In this study, we combined untargeted metabolomic profiling with functional antibacterial and antibiofilm assays to characterize EGL and to investigate the relationship between its chemical composition and biological activity. Untargeted UHPLC-TWIMS-QTOF analysis revealed a complex profile dominated by polyphenols and pentacyclic triterpenoids. Targeted quantification identified asiatic acid and ursolic acid as major triterpenoid components, present at concentrations of 47.29 ± 0.13 µg/mL and 16.12 ± 0.32 µg/mL, respectively. The antibacterial and antibiofilm activities of EGL were evaluated against clinically relevant Gram-positive pathogens, including Streptococcus pyogenes, Staphylococcus aureus, methicillin-resistant S. aureus and Streptococcus mutans. The two major triterpenoids identified in the extract, asiatic acid and ursolic acid, were tested separately as reference compounds to explore their individual antibacterial and antibiofilm activity in comparison with the whole extract. EGL inhibited bacterial growth with MIC values ranging from 62.5 µg/mL to 437.5 µg/mL, while the triterpenoids showed stronger activity, with MIC values of 2 µg/mL for ursolic acid and 4 µg/mL for asiatic acid against Streptococcus pyogenes. In antibiofilm assays, asiatic acid and ursolic acid showed MBIC values between 2 and 32 µg/mL, whereas the whole extract displayed partial inhibitory activity. Pre-exposure of bacterial cultures to EGL or its major triterpenoids enhanced antibiofilm efficacy, reducing MBIC values by up to fourfold. Overall, these results indicate that EGL is a chemically characterized multicomponent system with measurable antibacterial and antibiofilm activity against Gram-positive pathogens. The study highlights the importance of integrating chemical profiling with functional assays to better understand the activity of complex phytocomplexes and supports further investigation of phytochemical-based strategies targeting early stages of biofilm formation.

Canine otitis externa is a common multifactorial condition frequently complicated by bacterial and fungal infections. The increasing prevalence of antimicrobial resistance has reduced the effectiveness of conventional therapies, underscoring the need for alternative treatment strategies. Antimicrobial peptides represent a promising option due to their broad-spectrum activity and low propensity to induce resistance. Among these, the Naja Cardiotoxin Peptide-3 (NCP-3), derived from cardiotoxin 1 of the Chinese cobra (Naja atra subsp. atra), has demonstrated potent bactericidal and fungicidal activity with minimal cytotoxicity. In this study, the in vitro antimicrobial activity of NCP-3 was evaluated against clinical isolates obtained from dogs with otitis externa, including multidrug-resistant bacteria and Malassezia pachydermatis. NCP-3 was effective against all reference tested strains except Proteus mirabilis. Among field strains, the highest bactericidal activity was observed on Staphylococcus pseudintermedius (MBC₅₀=3.1 µg/mL; MBC₉₀=6.3 µg/mL), whereas higher concentrations were required to inhibit Pseudomonadaceae (MBC₅₀=12.5 µg/mL). Neither MBC₅₀ nor MBC₉₀ were achieved for Enterobacteriaceae or M. pachydermatis. Time-kill assays demonstrated rapid bactericidal activity of NCP-3 against both reference and clinical Gram-positive strains, with complete inhibition achieved within five minutes. In contrast, Gram-negative reference strains required longer exposure times, with a partial inhibition only at 120 min. Overall, these findings indicate that NCP-3 exhibits stronger antimicrobial activity against Gram-positive bacteria, including resistant strains, than on Gram-negative bacteria and M. pachydermatis field strains. NCP-3 may therefore represent a promising candidate as therapeutic agent for canine otitis externa caused by Gram-positive pathogens, supporting further investigation into its potential applications in veterinary medicine.

This study evaluated two alternative bedding materials (poplar pellet-PP and vine pellets-VP) in against conventional wood shavings (WS) on production performance, health status, hygiene, immune-related genes expression and meat quality of broilers reared in organic-like conditions. A total of 252 male Ross-308 chicks were assigned to 9 pens in a randomized blocked design with 3 replicates; 50 % were slaughtered at 42d while the remaining at 84d in accordance with organic farming regulations. Broilers raised on VP resulted in lower body and carcass weight than those on PP, which had the lowest feed conversion ratio at 63d. Compared to WS broilers on PP were heavier, cleaner and had lower water consumption, water consumption ratio, and water to feed ratio in organic rearing period. Pelleted beddings were drier until mid-trial; PP had a consistently higher fiber content (aNDF, ADF, lignin) than WS. Despite higher microbiological contamination pelleted beddings did not affect Lactobacillus spp. growth. Poplar pellet increased footpad and hock score (HS), while VP improved HS and plumage cleanliness (CS) at 28d but worsened HS at 84d. A blood protein reduction was observed in pellet beddings leading to increased creatinine levels. Birds reared on PP had the greatest thickness of tunica mucosa, villus-height (VH) and VH /crypt depth ratio at 42d. At 84d, VP increased IL-8 expression and reduced survival rate compared to PP. Overall, from a production perspective, pelleted beddings are suitable for fast-growing broiler hybrids under conventional and organic regimens. The PP is particularly recommended for organic production systems.

Cysteine biosynthetic pathway, unique to bacteria and absent in the human host, represents a relevant reservoir of potential antimicrobial targets. Cysteine, in fact, is involved in redox homeostasis and is prone to the production of reactive oxygen species, and altering its metabolic equilibrium provokes severe consequences on bacterial fitness and antibiotic response. Here, we developed a library of benzotriazole-derived compounds targeting O-acetylserine sulfhydrylase (OASS)-the enzyme responsible for cysteine synthesis in most pathogenic bacteria-which act as alternate OASS substrates that can impair cysteine production. The library was tested on three clinically relevant gram-negative species, and the three most promising candidates, upon the validation of their mechanism of action in vitro, were demonstrated to have adjuvant effect in combination with selected approved antibiotics. While further optimization is required before their application, these molecules might find application as adjuvants of the antibiotic therapy and help to preserve existing drugs.

Plant-derived compounds have emerged as potential alternatives to traditional antimicrobials in livestock; however, their application may be limited by degradation in the gastrointestinal tract. Nanoliposome encapsulation offers a strategy to overcome these limitations. In this study, we investigated the effects of nerolidol encapsulation, by evaluating the antimicrobial activity of free-nerolidol (NER), nerolidol-loaded nanoliposomes (LN), and unloaded nanoliposomes (UN) (Lipobox™) using a Time-Kill assay. The cytotoxicity of these formulations was assessed through MTT assay on swine and bovine cell lines. NER was effective against MRSA, Enterococcus faecium, and Lactobacillus acidophilus at all time points, at concentrations ≥62.5, ≥15.63 and ≥1,000 μg/ml, respectively, but was ineffective against Gram-negative bacteria Conversely, LN and UN were effective against all bacteria, showing the best activity at 2,500 μg/ml. LN showed the greatest activity against MRSA up to 6 h while UN on E. faecium up to 4 h (P < 0.05). No difference between LN and UN on Salmonella Typhimurium up to 24 h and on E. coli up to 6 h at this concentration (P > 0.05) was observed. For L. acidophilus, both LN and UN were effective up to 6 h even at the lowest concentration (9.77 μg/ml). NER showed high cytotoxicity on MDBK and IPEC-J2 cells at all doses; while LN and UN were low-toxic at concentrations ≤ 1,250 μg/ml or ≤ 625 μg/ml, respectively. These results suggest that nanoliposomes themselves exhibit dose-dependent antimicrobial and cytotoxicity activity; however, when NER is encapsulated its spectrum of activity its enhanced.

Hospital-acquired infections (HAIs) are a significant concern in human medicine and have increasingly been reported in veterinary settings as well. Veterinary Teaching Hospitals may present a heightened risk for HAIs, partly due to the lack of standardized infection control measures and screening protocols. In May 2022, a cat previously hospitalized at a Veterinary Teaching Hospital for urinary tract obstruction developed a catheter-associated urinary tract infection (CAUTI). A multidrug-resistant (MDR) strain of Klebsiella pneumoniae was isolated. To investigate the suspected HAI and assess the presence of other MDR Gram-negative bacteria, an environmental screening of different hospital surfaces was conducted using both phenotypic and genotypic analyses. A total of 70 swabs were collected from environmental surfaces, 51 before and 19 after cleaning procedures. Sterility was observed in 19.6

BACKGROUND:Malassezia pachydermatis (MP) is implicated in severe dermatitis and otitis externa (OE) of companion animals and recently gained attention for its increasing resistance to azole compounds. For this reason, developing novel therapeutic strategies is of great interest. In a previous work, we used reference yeast isolates to evaluate several compounds bearing acyl/selenoureido moieties and primary/secondary sulfonamide groups for antifungal activity through organic selenium and carbonic anhydrase inhibition. OBJECTIVES:This work aimed to evaluate the antifungal efficacy of eight selenoureido compounds on 36 clinical MP isolates from dogs, compared to selected azoles, notably ketoconazole (KCZ), miconazole (MCZ) and fluconazole (FCZ). MATERIALS AND METHODS:MIC assays of 5g, 7a, 7c, 7k, 8c, 10c, 11b, 11f, KCZ, MCZ and FCZ were performed on 36 MP field isolates isolated from dogs affected by dermatitis and/or OE in which yeast aetiology was suspected. Minimum 50% and 90% inhibitory concentrations (MIC50 and MIC90) were calculated. MP identification was confirmed with a nested PCR for the internal transcribed spacer region of the rRNA gene. RESULTS:Overall, the MIC50 of the tested compounds on MP field isolates was higher than the MICs obtained on reference MP DSM 6172. Although KCZ showed the lowest MIC50 value, compounds 5g, 7a and 7k showed lower MIC50s than MCZ and FCZ. Five clinical isolates showed a MIC on azoles >MIC90. Compounds 7a (four of five), 10c (three of five) and 8c (three of five) showed lower MIC values on these isolates compared to the tested azoles, suggesting good activity in phenotypically azole-resistant MP. CONCLUSIONS AND CLINICAL RELEVANCE:Considering the increasing azole resistance of the Malassezia genus, selenoureido compounds could represent a potential topical treatment for dog skin and ear mycotic infections.

Antibiotics represent the first line therapy for bovine mastitis. However, the increasing prevalence of multidrug-resistant organisms (MDROs) highlights the need for alternative therapeutic approaches. This study evaluated the antimicrobial and antibiofilm activities of Eucalyptus globulus leaf extract (EGL-L), ursolic acid (UA) and asiatic acid (AA) against Staphylococcus aureus (SA), Streptococcus uberis (SU), Streptococcus agalactiae (SAG), and Enterococcus spp. (EN) isolated from bovine mastitis, 39.7% of which were MDROs. The minimal inhibitory concentration (MIC) assay demonstrated that all the compounds exhibited antimicrobial activity against the tested bacteria, including MDROs. However, EGL-L was less effective (p < 0.001) than UA or AA against field strains. UA was more effective against SAG and SU compared to SA (p < 0.001), whereas AA was more effective against SU than SA (p < 0.001). Conversely, EGL-L exhibited similar inhibitory effects on all bacteria. The biofilm-forming ability of the bacterial strains was also assessed, and the minimal biofilm inhibitory concentrations (MBICs) of the compounds were evaluated for moderate and strong biofilm producers. None of the compounds were able to completely inhibit biofilm formation. However, MBIC80 values within the tested concentration range were achieved for 15 out of 32 strains with EGL-L and for 27 out of 32 strains with UA and AA. These findings highlight a promising alternative to conventional antimicrobials for AA and UA, showing potential for topical intramammary use for the control and prevention of bovine mastitis, especially because of their efficacy against biofilm formation. Future research should focus on toxicity assessments and formulation development for potential topical administration.

Essential oils (EOs) and nature identical compounds (NICs) express different antimicrobial activity (AAc) which can be affected by variability in composition, stability over time, and the carrier employed for their inclusion in feedstuff and products for animal treatments. The aim of the present study was to evaluate the stability over time of the AAc of 8 EOs and 5 NICs, alone and in combination with Tween 20, on four major bacterial livestock pathogens (E. coli, S. Typhimurium, S. aureus and MRSA). For this purpose, minimal inhibitory concentration (MIC) and checkerboard assay (CkA) tests were performed on EOs and NICs at batch first use (T0 – only MIC), and after 12 (T1) and 24 (T2) months. Several EOs showed initial MICs lower than 2% (v/v) and, except for clove bud and cinnamon EOs, reduced their efficacy over time. The best NICs’ AAc was showed at T1 by cinnamic aldehyde (against MRSA), carvacrol (against E. coli), and thymol (except against MRSA), while at T2 the MIC values decreased for most of the NICs. The CkA between EOs and Tween 20 showed different interactions (Fractional Inhibitory Concentration -FIC- index from 0.06 to 32.04) based on bacteria and time. No interactions were found between NICs and Tween 20 (FIC index from 1.0 to 3.0). The AAc of EOs and NICs changes over time. Therefore, the preservation interval of these additives is critical for the AAc of feedstuff and products intended for livestock animals. Moreover, the NICs seem more suitable for the association with Tween 20.

Bovine neonatal enteritis is a major cause of losses in cattle production, involving microbial and extra-microbial causative factors. Failure of passive transfer (FPT) plays a critical role in the development of this disease, and colostrum management and quality have a direct impact on FPT. The aim of this study was to investigate the effects of breed, parity and calf gender on colostrum IgG content and newborn calf immunity. Understanding the influence of these factors could be useful in implementing effective strategies to improve calf health. Breed emerged as a major factor affecting colostrum quality and calf immunity. IgG levels were compared in colostrum and newborn calf serum across dairy (Italian Friesian, Reggiana and Bianca Modenese) and cow-calf type (Piemontese and Limousine) breeds. Italian Friesian cattle showed significantly lower IgG levels in both their colostrum and newborn calf serum compared to the other breeds. Parity did not significantly affect overall colostrum quality or FPT prevalence. However, first-calf heifers had a lower prevalence of inadequate colostrum compared to multiparous cows, suggesting their colostrum is suitable for colostrum banks. With regard to gender, the analysis of IgG levels and FPT prevalence in Italian Friesian newborn calves revealed a significant sex disparity. Females exhibited higher IgG levels and lower FPT prevalence compared to males, suggesting potential management practices influencing these outcomes. In conclusion, results revealed significant differences in IgG concentration between colostrum and newborn calf serum samples across breed, parity, and calf gender. Newborn calves are particularly vulnerable and prone to enteritis. Poor colostrum management and breeding practices that prioritize high milk production can lead to lower IgG levels, further compromising calf immunity. While colostrum banks and cow vaccination offer potential solutions, limitations exist. Moreover, effective control requires close cooperation between farmers and veterinarians, which is often lacking.

Azelaic Acid (AzA) is a 9-carbon atom dicarboxylic acid, with numerous pharmacological uses in dermatology. Its effectiveness in papulopustular rosacea and acne vulgaris, among other dermatological disorders such as keratinization and hyper-pigmentation, is thought to be related to its anti-inflammatory and antimicrobial properties. It is a by-product of Pityrosporum fungal mycelia metabolism but also it is found in different cereals such as barley, wheat, and rye. Diverse topical formulations of AzA exist in commerce, and it is mainly produced via chemical synthesis. In this study we describe the extraction of AzA from whole grains and whole-grain flour (Triticum durum Desf.) through green methods. Seventeen different extracts were prepared and analyzed for their AzA content by HPLC-MS methods and then screened for their antioxidant activity using spectrophotometric assays (ABTS, DPPH, and Folin–Ciocalteu). Minimum-inhibitory-concentration (MIC) assays against several bacterial and fungal pathogens were performed, to validate their antimicrobial activity. The obtained results indicate that whole grain extracts provide a wider spectrum of activity than the flour matrix; in particular, the Naviglio® extract showed higher AzA content, while the hydroalcoholic ultrasound-assisted extract provided better antimicrobial and antioxidant activity. The data analysis was performed using principal component analysis (PCA), as an unsupervised-pattern-recognition technique, to extract useful analytical and biological information.

The comprehensive identification of secondary metabolites represents a fundamental step for the assessment of bioactivities and pharmacological properties of traditional herbal drugs. Rumex usambarensis (Dammer) Dammer has been described as a multipurpose remedy in different African traditional pharmacopoeias, but its phytochemical profile has not been properly investigated. Herein we report a high throughput metabolomic screening, based on ultra-high performance liquid chromatography-travelling wave ion mobility spectrometry quadrupole time-of-flight (UHPLC-TWINS-QTOF), which was performed for the first time on different R. usambarensis plant parts. By applying high-resolution mass spectrometry-based metabolomics and chemometric analysis, a complete discrimination of different aerial parts was obtained, with the annotation of 153 significant metabolites in leaves, stems, and flowers, suggesting an easy authentication and discrimination route. Phytochemical data were correlated to antimicrobial and antioxidant properties. Flavonoids, benzopyranes, chromones, and xanthones derivatives, along with a richer phytocomplex, might be responsible for the stronger bioactivities obtained from flowers.

A short-term study was conducted to compare the effect of using poplar wood chips (PWC) instead of wheat straw (WS) litter in dairy cows. A total of 38 lactating Holstein cows (204 ± 119 days in milk, 26.9 ± 6.5 kg of milk yield [MY]) were housed in a tiestall farm for a 10-d trial including 5 d of adaptation followed by 5 sampling days (from d 5 to 10). Cows were divided into 2 homogeneous groups: one group was bedded with WS, and the second with PWC. Both litter materials were provided in the amount of 7 kg/stall per d. Each group was composed of 3 subgroups of 6 or 7 cows; the subgroups were physically separated along the feeding line by wooden boards. During the sampling days, fecal composition, used litter composition, and bacterial count (Clostridium spp., Salmonella spp., Escherichia coli, Lactobacillus, and total bacterial count) were analyzed by subgroup twice a day. On d 1 and from d 5 to 10, udder hygiene score and cow cleanliness score were also evaluated individually twice a day. Meanwhile MY, milk hygiene (total bacterial count [TBC], coliform bacterial count [CBC], and spore-forming units [SFU]) and quality were measured and analyzed from 9 animals per group. Moreover, individual animal behavior (body position and behavioral traits) and subgroup dry matter intake were measured on d 9 and 10. Fecal dry matter did not differ between groups, PWC had the lowest used litter moisture and N content favoring the highest clean cow frequency, but also gave rise to the greatest used litter microbial contamination. The MY, milk quality, TBC, SFU, and CBC were similar. The lying behavior frequency was similar between groups. However, the PWC group showed the lowest sleeping frequency, the highest frequency of other behaviors (including discomfort signs), and the lowest dry matter intake. However, despite this apparent reduction in cow comfort, no biologically important differences were observed in this short-term study between cows on PWC and WS in milk production or hygiene.

A dairy herd must be considered an integrated production unit. From newborn calves to milking cows, animal health status is pivotal to get satisfactory economic results. The neonatal phase of calves is a period of life that needs extra care due to their vulnerability. Diarrhea is considered the main pathology affecting newborn calves. Conversely, respiratory disease is the main cause of losses after sixty days of life. The present study aimed at assessing the impact of neonatal diarrhea on growth, milk productivity and health status in a selected population of dairy cattle. A case-control study involving 300 calves from 5 large dairy herds located in the Po Valley (Italy) was carried out. All animals received lived-modified or inactivated, monovalent or combined vaccines for immunization to Bovine Herpesvirus 1 (BoHV-1), Bovine Viral Diarrhea Virus (BVDV) and Bovine Respiratory Syncytial Virus (BRSV). Particularly, most calves were administered with IBR marker and BRSV vaccines by intranasal route as priming immunization treatment. The enrolled animals were divided in two groups, each consisting of 150 calves. In the group A (cases) were included the animals experiencing severe neonatal diarrhea. Conversely, the group B (controls) included calves showing no clinical signs of neonatal enteritis. The animals were monitored in order to measure the body weight at birth, 6 and 15 months of age and milk production. Moreover, mortality rate and incidence of respiratory disease episodes during life were recorded. Animals of Group A showed a lower body weight at 6 and 15 months of age and a significant loss of milk production compared to those of the Group B. Moreover, incidence of respiratory disease and relapses were significantly higher in Group A. Results support the thesis that neonatal enteritis has a negative impact on weight gain during the grow period, milk production and is related to severe respiratory disease onset.

This study is focused on resistance to carbapenems and third-generation cephalosporins in Gram-negative microorganisms isolated from swine, whose transmission to humans via pork consumption cannot be excluded. In addition, the common carriage of carbapenem-resistant (CR) bacteria between humans and pigs was evaluated. Sampling involved 300 faecal samples collected from slaughtered pigs and 300 urine samples collected from 187 hospitalised patients in Parma Province (Italy). In swine, MIC testing confirmed resistance to meropenem for isolates of Pseudomonas aeruginosa and Pseudomonas oryzihabitans and resistance to cefotaxime and ceftazidime for Escherichia coli, Ewingella americana, Enterobacter agglomerans, and Citrobacter freundii. For Acinetobacter lwoffii, Aeromonas hydrofila, Burkolderia cepacia, Corynebacterium indologenes, Flavobacterium odoratum, and Stenotrophomonas maltophilia, no EUCAST MIC breakpoints were available. However, ESBL genes (blaCTXM-1, blaCTX-M-2, blaTEM-1, and blaSHV) and AmpC genes (blaCIT, blaACC, and blaEBC) were found in 38 and 16 isolates, respectively. P. aeruginosa was the only CR species shared by pigs (4/300 pigs; 1.3%) and patients (2/187; 1.1%). P. aeruginosa ST938 carrying blaPAO and blaOXA396 was detected in one pig as well as an 83-year-old patient. Although no direct epidemiological link was demonstrable, SNP calling and cgMLST showed a genetic relationship of the isolates (86 SNPs and 661 allele difference), thus suggesting possible circulation of CR bacteria between swine and humans.

Klebsiella pneumoniae is the most common Klebsiella species infecting animals and is one of the causing agents of mastitis in cows. The rise of antimicrobial resistance in K. pneumoniae, particularly in strains producing extended-spectrum β-lactamases (ESBLs) and/or carbapenemases, is of concern worldwide. Recently (Regulation UE No 2022/1255), carbapenems and cephalosporins in combination with β-lactamase inhibitors have been reserved only to human treatments in the European Union. The aim of this study was to investigate the role of cattle as carrier of human pathogenic carbapenem-resistant (CR) and ESBL-producing K. pneumoniae. On this purpose, a study involving 150 dairy farms in Parma province (Northern Italy) and 14 non replicate K. pneumoniae isolates from patients admitted at Parma University-Hospital was planned. Four multidrug resistant (MDR) K. pneumoniae strains were detected from 258 milk filters collected between 2019 and 2021. One carbapenemase KPC-3-positive K. pneumoniae ST307 (0.4 %; 95 % CI - 0.07 - 2.2) was detected in milk filters. The isolate also harboured OXA-9, CTX-M-15 and SHV-106 determinants, together with genes conferring resistance to aminoglycosides (aac(3')-IIa, aph (3″)-Ib, aph (6)-Id), fluoroquinolones (oqxA, oqxB, qnrB1), phosphonic acids (fosA6), sulphonamides (sul2), tetracyclines (tet(A)6) and trimethoprim (dfrA14). One KPC-3-producing K. pneumoniae ST307 was identified also among the human isolates, thus suggesting a possible circulation of pathogens out of the clinical settings. The remaining three bovine isolates were MDR ESBL-producing K. pneumoniae characterized by different genomic profiles: CTX-M-15, TEM-1B and SHV-187 genes (ST513); CTX-M-15 and SHV-145 (ST307); SHV-187 and DHA-1 (ST307). Occurrence of ESBL-producing K. pneumoniae in milk filters was 1.2 % (95 % CI 0.4-3.4). All the isolates showed resistance to aminoglycosides, 3rd-generation cephalosporins, and fluoroquinolones. Among the human isolates, two multidrug resistant ESBL-producing K. pneumoniae ST307 were found, thus confirming the circulation of this high-risk lineage between humans and cattle. Our findings suggest that food-producing animals can carry human pathogenic microorganisms harboring resistance genes against carbapenems and 3rd-generation cephalosporins, even if not treated with such antimicrobials. Moreover, on the MDR K. pneumoniae farms, the antimicrobial use was much higher than the Italian median value, thus highlighting the importance of a more prudent use of antibiotics in animal productions.

Fungal promoted infections are becoming a severe health global emergency due to drug-resistant phenomena and zoonosis. This work investigated compounds bearing acyl-/selenoureido moieties and primary/secondary sulfonamide groups as novel antifungal agents acting through organism-directed selenium toxicity and inhibition of the newly emergent therapeutic target, the Carbonic Anhydrases (CAs; EC 4.2.1.1). Reported data clearly indicate that seleno-containing scaffolds with respect to the standard-of-care drugs showed appreciable antifungal activity, which was suppressed when the chalcogen was replaced with its cognate isosteric elements sulfur and oxygen. In addition, such compounds showed excellent selectivity against Malassezia pachydermatis over its related genus strains Malassezia furfur and Malassezia globosa. Safe cytotoxicity profiles on bovine kidney cells (MDBK) and human HaCat cells, as well as the shallow hemolytic activity on defibrinated sheep blood, allowed us to consider these compounds as up-and-coming novel antifungals.

Antibacterial adjuvants are of great significance, since they allow one to downscale the therapeutic dose of conventional antibiotics and reduce the insurgence of antibacterial resistance. Herein, we report that O-acetylserine sulfhydrylase (OASS) inhibitors could be used as colistin adjuvants to treat infections caused by critical pathogens spreading worldwide, Escherichia coli, Salmonella enterica serovar Typhimurium, and Klebsiella pneumoniae. Starting from a hit compound endowed with a nanomolar dissociation constant, we have rationally designed and synthesized a series of derivatives to be tested against S. Typhimurium OASS isoenzymes, StOASS-A and StOASS-B. All acidic derivatives have shown good activities in the nanomolar range against both OASS isoforms in vitro. Minimal Inhibitory Concentrations (MICs) were then evaluated, as well as compounds' toxicity. The compounds endowed with good activity in vitro and low cytotoxicity have been challenged as a potential colistin adjuvant against pathogenic bacteria in vitro and the fractional inhibitory concentration (FIC) index has been calculated to define additive or synergistic effects. Finally, the target engagement inside the S. Typhimurium cells was confirmed by using a mutant strain in which the OASS enzymes were inactivated. Our results provide a robust proof of principle supporting OASS as a potential nonessential antibacterial target to develop a new class of adjuvants.