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The spread of carbapenem-resistant bacteria is a global threat to public health and the role of pets in the rise in antimicrobial resistance is gaining attention worldwide. This study aimed to determine the fecal carriage of extended-spectrum β-lactamase (ESBL)-, AmpC- and carbapenemase (CP)-producing E. coli and associated risk factors in healthy and unhealthy stray cats admitted to the Veterinary Teaching Hospital of Lodi, University of Milan, Italy. Fecal samples collected in, 2020-2022 were microbiologically and molecularly analyzed. Overall, ESBL-/AmpC-/CP-producing E. coli were detected in 18/94 (19.1 %) stray cats. Twelve (12.8 %), 4 (4.3 %) and 7 (7.4 %) stray cats carried ESBL-, AmpC- and CP-producing E. coli phenotypes respectively, supported by the detection of bla CTX-M in all ESBL-producing E. coli, bla CMY-2 in all AmpC-producing E. coli and bla NDM (4/7; 57.1 %) or bla OXA-48 (3/7; 42.9 %) genes in CP-producing E. coli. Multiple combination of resistance genes and phenotypes were detected. MIC results showed that all E. coli were multidrug resistant isolates. Risk factors associated with ESBL-, AmpC- and/or CP-producing E. coli fecal carriage were hospitalization (P < 0.0001), antibiotic treatment during hospitalization (P < 0.0001) and unhealthy status (P < 0.0001). Fecal carriage of CP-producing E. coli is of concern and highlights the need of specific surveillance programs for CP-producing Enterobacteriaceae and antimicrobial stewardship in stray cats to reduce the emergence and spread of resistant bacteria. Limitations of this study suggest a One Health approach to characterize the whole genome of the isolates and the epidemiology of AMR bacteria among stray cats, including additional bacterial species and the environment., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest associated with this study., (Copyright © 2024. Published by Elsevier Ltd.)

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.

MDR bacteria are an emerging global threat to public health, and the role of dogs in the rise of antimicrobial resistance is under investigation. This study investigated the fecal shedding of extended-spectrum β-lactamase (ESBL)-, AmpC- and carbapenemase (CP)-producing Escherichia coli and associated risk factors in dogs admitted to the Veterinary Teaching Hospital of Lodi, University of Milan, or other veterinary clinics and kennels in Northen Italy. Feces collected in 2020-2022 were microbiologically and molecularly analyzed. ESBL-/AmpC-/CP-producing E. coli was detected in 14/100 (14%) dogs. Eleven (11%), five (5%) and one (1%) dogs carried ESBL-, AmpC- and CP-producing E. coli phenotypes, respectively, supported by the PCR detection of bla CTX-M and/or bla TEM in ESBL-producing E. coli ; bla CMY-2 and the presence of putative low-level AmpC production in AmpC-producing E. coli ; and bla OXA-48 in CP-producing E. coli . Different combinations of resistance genes and genetic features were observed. Multidrug resistance was observed in 13/14 (92.9%) E. coli isolates. Binary logistic regression analysis showed that ESBL-/AmpC-/CP-producing E. coli fecal shedding tended to be associated with antibiotic treatment (p = 0.058; OR = 3.87). The detection of ESBL-/AmpC-producing E. coli , along with the presence of a carbapenemase-resistant E. coli isolate from domestic dogs, although still limited, emphasizes the need for antimicrobial stewardship and specific surveillance programs, particularly for CP-producing bacteria in companion animals.

Background/Objectives : Waste milk harbors many bacteria and antibiotic residues. Calves fed with untreated waste milk have a higher incidence of scours and an increased risk of developing antimicrobial-resistant bacteria. This study aimed to evaluate the efficacy of treatment with copper ions on bacteria and antibiotics contained in bovine waste milk. Methods : Waste milk samples were collected from a dairy farm for seven weeks and were subjected to treatment with copper ions. Total bacterial counts, coliforms, Streptococcus , and Staphylococcus were assessed before and after treatment. Additionally, metagenomic analysis was performed to determine microbial diversity. Results : Before treatment, the total bacterial count average was 4.0 × 10 6 CFU/mL, 1.7 × 10 4 CFU/mL for coliforms, 2.6 × 10 6 CFU/mL for Streptococcus , and 5.4 × 10 2 CFU/mL for Staphylococcus Copper treatment significantly reduced bacterial counts within 15 min. Total bacteria decreased from 4.0 × 10 6 CFU/mL to 1.1 × 10 2 CFU/mL after 30 min; meanwhile, other groups were not detected. The most abundant groups were Lactococcus (29.94%), Pseudomonas (28.89%), and Enterobacteriaceae (21.19%). Beta-lactams were detected in five-sevenths samples, and in one sample they were detected before and at 15 min of treatment but not after 30 min. Conclusions : The effect of treatment with copper ions on the different bacterial groups was significantly effective but showed limited effect on the detection of antibiotics.

Non-aureus staphylococci and mammaliicocci (NASM) are the microorganisms most frequently isolated from milk. Given their numerosity and complexity, MALDI-TOF MS is one of the preferred species identification approaches. Nevertheless, reference mass spectra for the novel species Staphylococcus borealis were included only recently in the Bruker Biotyper System (MBT) library, and other species of veterinary interest such as S. rostri are still absent. This work provides an updated picture of the NASM species found in milk, gained by retrospectively analyzing the data relating to 21,864 milk samples, of which 6,278 from clinical mastitis (CM), 4,039 from subclinical mastitis (SCM), and 11,547 from herd survey (HS), with a spectrum library including both species. As a result, S. borealis was the second most frequently isolated NASM (17.07%) after S. chromogenes (39.38%) in all sample types, with a slightly higher percentage in CM (21.84%), followed by SCM (17.65%), and HS (14.38%). S. rostri was also present in all sample types (3.34%), reaching 8.43% of all NASM in SCM and showing a significant association (p < 0.01) with this condition. Based on our findings, the presence of S. borealis and S. rostri in milk and their potential association with mastitis might have been overlooked, possibly due to the difficulties in differentiating these species from other closely related NASM. Our results indicate that S. borealis could be a more frequent contributor to bovine udder infections than previously thought and that S. rostri should also not be underestimated considering its significant association with SCM., (© 2024. The Author(s).)

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.

Non-aureus staphylococci and mammaliicocci (NASM) are associated with bovine mastitis and increased milk somatic cell count (SCC) but their relationships with mammary gland health at the species level are not clearly defined. Regional differences have also been reported in their specific prevalence. The implementation of MALDI-TOF MS in milk microbiology is generating large and dependable datasets with the potential of providing useful epidemiological information. We present the retrospective analysis of 17,213 milk samples sent to our laboratory in 2021-2022, including 13,146 quarter samples from cows with subclinical (SCM) or clinical mastitis (CM) from 104 farms, and 4,067 composite herd survey (HS) samples from 21 farms. NASM were isolated from 21.12% of SCM, 11.49% of CM, and 15.59% of HS milk samples. The three most frequently identified NASM in SCM milk were Staphylococcus chromogenes (33.33%), S. haemolyticus (26.07%), and S. epidermidis (10.65%); together with S. microti and S. hyicus, these species were significantly more prevalent in quarters with SCM (p < 0.05). The three most frequently identified NASM in CM milk were S. chromogenes (31.69%), S. haemolyticus (21.42%), and Mammaliicoccus sciuri (18.38%), although no significant associations were found between these NASM species and CM. The three most frequently identified NASM in HS milk were S. chromogenes (44.49%), S. epidermidis (17.84%), and S. haemolyticus (17.23%), with S. chromogenes being isolated in all the farms sending HS milk (100%). In conclusion, this retrospective study provides the first information on the NASM species isolated from cow milk in Italy, expanding our knowledge on the epidemiology of NASM at the species level and providing further insights into their relationships with mammary gland health in modern dairy farms., (© 2023. The Author(s).)

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.

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.

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.

Staphylococci and streptococci are common causes of intramammary infection in small ruminants, and reliable species identification is crucial for understanding epidemiology and impact on animal health and welfare. We applied MALDI-TOF MS and gap PCR-RFLP to 204 non-aureus staphylococci (NAS) and mammaliicocci (NASM) and to 57 streptococci isolated from the milk of sheep and goats with mastitis. The top identified NAS was Staphylococcus epidermidis (28.9%) followed by Staph. chromogenes (27.9%), haemolyticus (15.7%), caprae, and simulans (6.4% each), according to both methods (agreement rate, AR, 100%). By MALDI-TOF MS, 13.2% were Staph. microti (2.9%), xylosus (2.0%), equorum, petrasii and warneri (1.5% each), Staph. sciuri (now Mammaliicoccus sciuri, 1.0%), arlettae, capitis, cohnii, lentus (now M. lentus), pseudintermedius, succinus (0.5% each), and 3 isolates (1.5%) were not identified. PCR-RFLP showed 100% AR for Staph. equorum, warneri, arlettae, capitis, and pseudintermedius, 50% for Staph. xylosus, and 0% for the remaining NASM. The top identified streptococcus was Streptococcus uberis (89.5%), followed by Strep. dysgalactiae and parauberis (3.5% each) and by Strep. gallolyticus (1.8%) according to both methods (AR 100%). Only one isolate was identified as a different species by MALDI-TOF MS and PCR-RFLP. In conclusion, MALDI-TOF MS and PCR-RFLP showed a high level of agreement in the identification of the most prevalent NAS and streptococci causing small ruminant mastitis. Therefore, gap PCR-RFLP can represent a good identification alternative when MALDI-TOF MS is not available. Nevertheless, some issues remain for Staph. haemolyticus, minor NAS species including Staph. microti, and species of the novel genus Mammaliicoccus., (© 2022. The Author(s).)

Mastitis is one of the most impacting diseases in dairy farming, and its sensitive and specific detection is therefore of the greatest importance. The clinical evaluation of udder and mammary secretions is typically combined with the milk Somatic Cell Count (SCC) and often accompanied by its bacteriological culture to identify the causative microorganism. In a constant search for improvement, several non-enzymatic milk proteins, including milk amyloid A (M-SAA), haptoglobin (HP), cathelicidin (CATH), and lactoferrin (LF), have been investigated as alternative biomarkers of mastitis for their relationship with mammary gland inflammation, and immunoassay techniques have been developed for detection with varying degrees of success. To provide a general overview of their implementation in the different dairy species, we carried out a systematic review of the scientific literature using the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines. Our review question falls within the type "Diagnostic test accuracy questions" and aims at answering the diagnostic question: "Which are the diagnostic performances of mastitis protein biomarkers investigated by immunoassays in ruminant milk?". Based on 13 keywords combined into 42 searches, 523 manuscripts were extracted from three scientific databases. Of these, 33 passed the duplicate removal, title, abstract, and full-text screening for conformity to the review question and document type: 78.8% investigated cows, 12.1% sheep, 9.1% goats, and 6.1% buffaloes (some included more than one dairy species). The most frequently mentioned protein was M-SAA (48.5%), followed by HP (27.3%), CATH (24.2%) and LF (21.2%). However, the large amount of heterogeneity among studies in terms of animal selection criteria (45.5%), index test (87.9%), and standard reference test (27.3%) resulted in a collection of data not amenable to meta-analysis, a common finding illustrating how important it is for case definitions and other criteria to be standardized between studies. Therefore, results are presented according to the SWiM (Synthesis Without Meta-analysis) guidelines. We summarize the main findings reported in the 33 selected articles for the different markers and report their results in form of comparative tables including sample selection criteria, marker values, and diagnostic performances, where available. Finally, we report the study limitations and bias assessment findings., (© 2022. The Author(s).)

Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant., (© 2022. The Author(s).)

The milk somatic cell count (SCC) is a standard parameter for monitoring intramammary infections (IMI) in dairy ruminants. In goats, however, the physiological increase in SCC occurring in late lactation heavily compromises its reliability. To identify and understand milk protein changes specifically related to IMI, we carried out a shotgun proteomics study comparing high SCC late lactation milk from goats with subclinical Staphylococcus aureus IMI and from healthy goats to low SCC mid-lactation milk from healthy goats. As a result, we detected 52 and 19 differential proteins (DPs) in S. aureus-infected and uninfected late lactation milk, respectively. Unexpectedly, one of the proteins higher in uninfected milk was serum amyloid A. On the other hand, 38 DPs were increased only in S. aureus-infected milk and included haptoglobin and numerous cytoskeletal proteins. Based on STRING analysis, the DPs unique to S. aureus infected milk were mainly involved in defense response, cytoskeleton organization, cell-to-cell, and cell-to-matrix interactions. Being tightly and specifically related to infectious/inflammatory processes, these proteins may hold promise as more reliable markers of IMI than SCC in late lactation goats. SIGNIFICANCE: The biological relevance of our results lies in the increased understanding of the changes specifically related to bacterial infection of the goat udder in late lactation. The DPs present only in S. aureus infected milk may find application as markers for improving the specificity of subclinical mastitis monitoring and detection in dairy goats in late lactation, when other widespread tools such as the SCC lose diagnostic value., Competing Interests: Conflict of interest The authors declare no conflict of interest. Supplementary data Supplementary data to this article can be found online., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We present a proteomic dataset generated from half-udder Alpine goat milk. The milk samples belonged to 3 groups: i) mid-lactation, low somatic cell count, uninfected milk (MLU, n=3); ii) late lactation, high somatic cell count, uninfected milk (LHU, n=3); and late lactation, high somatic cell count, Staphylococcus aureus subclinically infected milk (LHS, n=3). The detailed description of results is reported in the research article entitled "Impact of Staphylococcus aureus infection on the late lactation goat milk proteome: new perspectives for monitoring and understanding mastitis in dairy goats". After milk defatting, high speed centrifugation and trypsin digestion of milk with the FASP protocol, peptide mixtures were analyzed by LC-MS/MS on a Q-Exactive. Peptide identification was carried out using Sequest-HT in Proteome Discoverer. Then, the Normalized Abundance Spectrum Factor (NSAF) value was calculated by label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation by Uniprot was carried out by reporting biological process, molecular function and cellular component. The MS data have been deposited to the ProteomeXchange via the PRIDE with the dataset identifier PXD017243., (© 2020 The Author(s).)

Acute gastroenteritis is one of the most common diseases in humans worldwide. Viral gastroenteritis is a global problem in infants and young children. In this study the incidence of diarrhea was assessed in 877 hospitalized children under five years old, over a period of 24 months and distributed in 470 cases of diarrhea and 407 age-matched group with other pathologies, as control group. Two antigen detection techniques based on enzyme immunoassay (EIA) and latex particles were used for detection of rotavirus and adenovirus. Rotavirus A was a major cause of gastroenteritis with 23.6% of cases, being 90% of these cases in young children. Adenovirus infections was detected by EIA with frequency of 6.4%. Rotavirus and adenovirus were detected in 10.1 and 1.7% of stools from control group, respectively. Interestingly, the frequency of the youngest children in the control group excreting Rotavirus A was comparable to that detected in stools from diarrheic children. We cannot rule out the existence of other enteric viruses because the etiology of 171 cases of diarrhea was not determined and active search for astrovirus and calicivirus was not done. This is the first study that shows the presence of enteric viruses in the infantile population from Western Brazilian Amazonia and it was important to help physicians in the treatment of viral gastroenteritis.