Maria Aparecida Scatamburlo Moreira

Conventional milk indicators such as somatic cell count (SCC) and pH have limited specificity for monitoring caprine mastitis, particularly under non-antibiotic treatment protocols. This exploratory study evaluated the temporal behavior of milk N-acetyl-β-D-glucosaminidase (NAGase), SCC, total bacterial count (TBC), pH, and selected electrolyte indicators (Na, K, and Na/K ratio) across sequential non-antibiotic phases, including milk microbiota transplantation (MMT), Resilience (washout), and 7-epiclusianone, in an experimental Staphylococcus warneri mastitis model in goats. Using a paired-udder longitudinal design in six goats, treated udders showed sustained sodium elevation, potassium depletion, and increased Na/K ratio, indicating persistent blood–milk barrier disturbance across phases, whereas pH and TBC showed more variable responses. NAGase activity correlated positively with SCC and sodium and negatively with potassium, consistent with its association with mammary epithelial injury and inflammation. These findings suggest that NAGase, together with Na, K, and the Na/K ratio, may help characterize biomarker dynamics during non-antibiotic treatment. Given the exploratory design and small sample size, these findings require validation in larger studies before routine application.

Mastitis remains a major cause of antimicrobial use in dairy herds, contributing to the spread of antimicrobial resistance (AMR). Staphylococcus warneri has emerged as a recurrent pathogen associated with persistent and resistant infections in goats. To explore potential alternative agents, this study integrated in vitro assays with in silico modeling to evaluate the antimicrobial activity of usnic acid (UA) and potassium usnate (KU) against S. warneri isolates from clinical mastitis. Two strains with documented enrofloxacin persistence were characterized in vitro and analyzed using protein–protein interaction (PPI) network modeling and molecular docking (MD). KU displayed higher predicted affinity to persistence- and resistance-associated protein targets than UA. Prominent protein families included ABC transporters (ABC_tran and ATP-Binding_Cassette) related to resistance, and MenD_MenH_VitK_biosynth, Metallo-β-lactamase_sf, and HipB-associated domains linked to persistence. Functional PPI networks highlighted biologically relevant clusters centered on NorB, VraR, SrrB, ArlR, CodY, SarA, BcrA, and MazF. KU binds with higher affinity than reference antibiotics to key resistance (BcrA, TcaR) and persistence (GTP pyrophosphokinase, glycerol kinase) targets. These findings suggest that KU may interfere with essential bacterial survival pathways, supporting its potential as a potent natural antibacterial agent for combating resistant and persistent infections and supporting its continued investigation as a natural product–derived candidate for combating AMR in veterinary infections.

Multidrug efflux systems (MESs) are major contributors to antimicrobial resistance (AMR) in Staphylococcus aureus , yet their role in primary healthcare settings is poorly understood. Under a One Health framework, we investigated MES‐mediated resistance in 38 S. aureus isolates (27 from humans, 11 from dogs) from three Basic Health Units (BHUs) in Viçosa, Brazil. Isolates were characterized by antimicrobial susceptibility testing, PCR for six key efflux genes, and (GTG) ₅ ‐PCR fingerprinting. Phenotypic efflux activity was evaluated using ethidium bromide fluorescence assays. Thirty‐seven isolates were resistant to at least one antimicrobial, most commonly penicillin (57.9%) and erythromycin (55.3%), while all remained susceptible to chloramphenicol, trimethoprim, and linezolid. While the msrA gene was rare (10.5%), other efflux genes like norA/B/C , lmrS , and tet38 were nearly ubiquitous (> 94%). This high genetic prevalence contrasted with low phenotypic resistance, indicating that most MES genes were not expressed. Fingerprinting revealed seven genetic clusters, demonstrating the circulation of closely related strains between human and animal hosts across different health units. Eight isolates showed clear genotype–phenotype concordance, with MES activity confirmed phenotypically. The four msrA ‐positive, erythromycin‐intermediate isolates formed two clonal groups (100% similarity): one shared between two users from different BHUs and another shared between a healthcare worker and a dog from different BHUs, providing direct evidence of interhost and cross‐geographic AMR dissemination. Moreover, co‐colonization of a single individual with two genetically distinct tetracycline‐resistant strains (60% similarity) suggests possible horizontal gene transfer. Although phenotypic MES‐mediated resistance was limited (21%), we demonstrate the potential AMR spread across hosts and geographic boundaries, as primary healthcare settings harbor a significant reservoir of MES genes in S. aureus even if they are silent. These results highlight the critical need for integrated One Health surveillance in community settings to mitigate AMR dissemination.

Species of the genus Staphylococcus are commensals and opportunistic pathogens of relevance to One Health, occurring in humans, animals, and healthcare-associated environments. This study aimed to characterize the diversity and distribution of Staphylococcus spp. in primary healthcare units in Recife, Brazil, using samples from humans, companion animals living in the surrounding area, and environmental surfaces and healthcare equipment within the units. Nasal and hand swabs were collected from humans (n = 83), oropharyngeal swabs from dogs (n = 27) and cats (n = 19), and swabs from environmental surfaces and healthcare equipment (n = 28) between July 2022 and January 2023. Isolates were obtained by culture and identified by MALDI-TOF MS. In humans, S. epidermidis was the most frequent species (33.7%), with a higher proportion among healthcare workers. In animals, S. felis (26.1%) and S. pseudintermedius (13.0%) predominated. In environmental surface and healthcare equipment samples, S. aureus was the most frequent species (23.8%). Species occurrence was associated with sample groups (χ2 = 46.05; p < 0.0001), indicating distinct distribution patterns and overlap in species occurrence across human, animal, and environmental sources. MALDI-TOF MS enabled rapid identification of diverse species, including less commonly reported taxa, highlighting its potential for microbiological surveillance in primary healthcare from a One Health perspective.

Mastitis remains a major challenge in dairy goat farming, and the increasing prevalence of antibiotic-resistant pathogens highlights the urgent need for alternative management strategies. This pilot study evaluated the feasibility of a sequential protocol combining intramammary milk-derived microbial transfer (MMT) with 7-epiclusianone in an experimental Staphylococcus warneri-induced caprine mastitis model. Six healthy primiparous Parda Alpina goats were infected in the right udder with S. warneri and received MMT for nine consecutive days from a screened healthy donor. Two days after MMT, intramammary 7-epiclusianone was administered for 7 days. Left udders served as uninfected controls. Pathogen load was assessed using a singleplex S. warneri-specific Polymerase Chain Reaction (PCR) and a multiplex real-time PCR panel targeting common mastitis-associated pathogens. Sequential treatment was well tolerated, and preliminary trends suggested reduced detection intensity of S. warneri, particularly following 7-epiclusianone administration. Changes in detection patterns were also observed for other pathogens. This study demonstrates the feasibility and safety of integrating MMT with a phytocompound-based intervention for managing intramammary infection in an experimental goat mastitis model. While preliminary results suggest potential for pathogen modulation, these findings require confirmation in larger, independently controlled trials to determine individual and combined effects and to rule out contributions from natural immune resolution.

Antimicrobial resistance is a challenge for public health, exacerbated by the indiscriminate use of these agents in humans and animals. Staphylococcus aureus, frequently found in fresh meat, poses a considerable risk due to its ability to transfer resistance genes. This study assessed the phenotypic and genotypic resistance profiles of Staphylococcus aureus isolated from 120 samples of beef, pork, and chicken sold in Recife, Pernambuco, Brazil. Samples were collected from markets, butcher shops, and street markets. Phenotypic identification was conducted using catalase tests and Gram staining, with confirmation through polymerase chain reaction (PCR) by amplifying the nuc gene. The resistance profile was evaluated using the disk diffusion method for 21 antimicrobials, following Clinical and Laboratory Standards Institute guidelines. In contrast, the resistance genes blaZ, mecA, mecC, norA, norC, tet(38), and msrA were analyzed by PCR. Among the 152 Staphylococcus spp. colonies isolated, 6.67% (10/152) were confirmed as S. aureus. Of these, 70% (7/10) were isolated from pork and 30% (3/10) from chicken, with no detection in beef samples. All isolates exhibited multidrug resistance, showing universal resistance to penicillin, rifampin, tetracycline, doxycycline, erythromycin, clindamycin, and linezolid. Genotypically, all isolates carried the norC gene, while 90% (9/10) harbored tet(38), and 60% (6/10) tested positive for norA and blaZ. No detection of mecA, mecC, or msrA was observed. These results underscore the urgent need for health education promoting the rational use of antimicrobials and coordinated actions between human and animal health sectors to mitigate antimicrobial resistance, which poses a serious threat to public health.

This study aimed to identify extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in chicken, beef, and pork meats sold in Recife, Pernambuco, Brazil. A total of 120 meat samples (40 of each type) were collected from supermarkets, butcher shops, and open-air markets across the city’s eight health districts, using convenience sampling. The samples were processed in a microbiology laboratory, and E. coli was identified through selective isolation on eosin-methylene blue agar and biochemical tests. Phenotypic resistance was assessed using disk diffusion tests on Müller–Hinton agar with cefotaxime, ceftazidime, and ceftriaxone disks, followed by the double-disk synergy test to confirm ESBL production. Genotypic analysis was conducted by polymerase chain reaction to detect the blaTEM and blaSHV genes. Of the 40 E. coli isolates obtained, 34 (85%) exhibited phenotypic resistance, while 21 (52.5%) and 23 (57.5%) tested positive for the blaSHV and blaTEM genes, respectively. A higher prevalence of blaSHV was observed in pork samples (73.3%, 11/15), whereas blaTEM was more prevalent in beef (70%, 7/10). The presence of resistant bacteria in commercial meats highlights contamination risks in the production chain and underscores the need for surveillance and public awareness to protect human health.

Mastitis significantly impacts dairy goat herds, causing substantial economic losses. The escalating challenge of multidrug-resistant bacteria has prompted research into alternative therapeutic strategies, including milk microbiota transfer and phytochemical treatments. This study evaluates milk chemical elements as potential biomarkers during alternative treatments for Staphylococcus warneri-induced goat mastitis. Seven female Parda Alpina goats were studied, with six receiving sequential treatments: milk microbiota transplantation (MMT) from the seventh goat (donor), two days later, intra-mammary 7-epiclusianone administration followed. The right udder of the six goats received the treatments and different time points while the left udder served as the control. Milk samples (n = 120) were analyzed using X-ray Energy Dispersive Spectroscopy (XEDS) to monitor trace and macro elements. Milk elemental level variations were observed across both treatments. Notable changes included increased iron levels from MMT initiation, reduced copper and zinc levels during MMT, and decreased sodium levels following 7-epiclusianone treatment. These results suggest zinc, copper, iron, and sodium could serve as potential biomarkers for monitoring mastitis treatment effectiveness. Additional research with broader and more diverse sample populations would help validate these findings and explore the method's broader applicability.