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Lymphoma ranks among the most prevalent neoplasms in veterinary oncology, frequently diagnosed in dogs, particularly in its multicentric form. While histopathology plays a crucial role in lymphoma diagnosis, prognosis and prediction of biological behavior, minimally invasive diagnostic methods are increasingly emerging as viable alternatives. This study aims to assess and compare various minimally invasive diagnostic techniques for multicentric lymphomas in dogs. A total of 38 dogs, encompassing various sexes, ages, and breeds, with clinical suspicion of multicentric lymphoma, was included in the study. Fine needle aspiration was employed to collect samples from lymph nodes, which were subsequently used for cytology, cell block preparation, PCR for antigen receptor rearrangement (PARR), and immunocytochemistry. Among the animals evaluated, 31 dogs received a cytological diagnosis of lymphoma, while 7 showed findings suggestive of lymphoma or lymphadenitis. Immunocytochemistry on cytological smears yielded inconclusive results in 50 % of cases, with 44.74 % diagnosed with B-cell lymphoma and 5.26 % with T-cell lymphoma. Cell block analysis identified lymphoma in 30 dogs and suggested lymphoma or a round cell neoplasm in 8 cases. Cell block immunocytochemistry confirmed lymphoma in 35 dogs, comprising 80 % B-cell and 20 % T-cell lymphomas. PARR revealed monoclonal rearrangement/clonality in 33 cases, with 84.85 % of these being B-cell and 15.15 % T-cell lymphomas. This study underscores the precision of minimally invasive techniques in diagnosing and characterizing multicentric lymphoma in dogs, reaffirming their significance in veterinary clinical practice., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Antimicrobial resistance mediated by extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales, as well as carbapenemase-producing Enterobacterales have globally increased among companion animals, posing a potential health risk to humans in contact with them. This prospective longitudinal study investigates the transfer of ESBL/AmpC- and carbapenemase-producing Enterobacterales between companion animals and their cohabitant humans in Portugal (PT) and the United Kingdom (UK) during animal infection. Fecal samples and nasal swabs collected from dogs and cats with urinary tract infection (UTI) or skin and soft tissue infection (SSTI), and their cohabitant humans were screened for resistant strains. Relatedness between animal and human strains was established by whole-genome sequencing (WGS). ESBL/AmpC-producing Enterobacterales were detected in companion animals (PT = 55.8%; UK = 36.4%) and humans (PT = 35.9%; UK = 12.5%). Carbapenemase-producing Enterobacterales carriage was observed in one dog from Portugal (2.6%) and another dog from the UK (4.5%). Transmission of index clinical ESBL-producing Escherichia coli and Klebsiella pneumoniae strains to cohabitant humans was observed in three Portuguese households (6.9%, n = 43), with repeated isolation of the index strains on fecal samples from the animals and their cohabiting humans. In addition, longitudinal sharing of E. coli strains carried by companion animals and their owners was observed in other two Portuguese households and two households from the UK. Furthermore, a multidrug-resistant ACT-24-producing Enterobacter hormaechei subsp. hoffmannii strains were also shared within another Portuguese household. These results highlight the importance of the household as an epidemiological unit in the efforts to mitigate the spread of antimicrobial resistance, further emphasizing the need for antimicrobial surveillance in this context, capable of producing data that can inform and evaluate public health actions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Menezes, Frosini, Weese, Perreten, Schwarz, Amaral, Loeffler and Pomba.)

An increase in Klebsiella pneumoniae carbapenem-resistant human nosocomial strains is occurring in Europe, namely with the bla OXA-48-like and bla KPC-like genes. We determined the prevalence of carbapenemase-producing Enterobacterales clinical strains in companion animals in Portugal and characterized their mobile genetic elements. Susceptibility data of a consecutive collection of 977 Enterobacterales clinical strains from a Portuguese private veterinary diagnostic laboratory were evaluated (January-December 2020). Additional phenotypical and genotypical assays were performed in a subset of 261 strains with a resistant phenotype. Whole-genome sequencing was performed for carbapenemase-producing strains. The frequency of carbapenemase-producing Enterobacterales clinical strains in companion animals in Portugal was 0.51% ( n = 5/977). Thus, five strains were characterized: (i) one OXA-181-producing K. pneumoniae ST273, (ii) two KPC-3-producing K. pneumoniae ST147; (iii) one KPC-3-producing K. pneumoniae ST392; and (iv) one OXA-48-producing E. coli ST127. The bla KPC-3 gene was located on transposon Tn 4401d on IncFIA type plasmid for the K. pneumoniae ST147 strains and on a IncN-type plasmid for the K. pneumoniae ST392 strain, while bla OXA-181 gene was located on an IncX3 plasmid. All de novo assembled plasmids and plasmid-encoded transposons harboring carbapenemase genes were homologous to those previously described in the human healthcare. No plasmid replicons were detected on the OXA-48-producing E. coli ST127. The dissemination of carbapenem resistance is occurring horizontally via plasmid spreading from the human high burden carbapenem resistance setting to the companion animal sector. Furthermore, companion animals may act as reservoirs of carbapenem resistance. Implementation of carbapenemase detection methods in routine clinical veterinary microbiology is urgently needed., Importance: This is the first study on the prevalence of carbapenemase-producing Enterobacterales (CPE) clinical strains from companion animals in Portugal. Despite the generally low prevalence of CPE in companion animals, it is imperative for veterinary diagnostic laboratories to employ diagnostic methods for carbapenemase detection. The resemblance found in the mobile genetic elements transporting carbapenemase genes between veterinary medicine and human medicine implies a potential circulation within a One Health framework., Competing Interests: The authors declare no conflict of interest.

Background: The diagnosis of multiple myeloma (MM) in dogs may be challenging and complex. The cell blocks are a diagnostic technique that allows the characterization of neoplastic cells and, therefore, might help in the diagnosis of atypical MM., Objective: The objective of the present work is to describe three clinical cases in which the cell blocks and immunohistochemistry contributed to the definitive diagnosis of canine MM., Methods: Three dogs, one female and two males, with different clinical signs, were presented for consultation with anemia, hyperproteinemia with monoclonal gammopathy, and the presence of plasmacytosis in the bone marrow. Cytologic analysis of the spleen was performed in two dogs and was suggestive of the presence of lymphocytes or plasma cells of a neoplastic nature in one of the cases and plasma cell hyperplasia associated with extramedullary hematopoiesis in the other. Given the hypotheses of lymphoid neoplasms with a plasma cell phenotype, cell blocks from aspiration punctures were performed for immunohistochemical analysis with anti-CD3, CD20, CD79αcy, PAX5, and MUM1 antibodies., Results: The results revealed positive staining for MUM1 in 80% of the cells in the spleen cell block and for CD20 and MUM1 in 70% of the cells in the bone marrow cell blocks, with negative staining for the other antibodies. The immunophenotyping results allowed the diagnosis of MM in the three cases and excluded other lymphoid neoplasms., Conclusions: This work reinforces the importance of using cell blocks in the diagnosis of neoplasms by demonstrating their potential to aid the diagnosis of MM., (© 2024 American Society for Veterinary Clinical Pathology.)

A successful outcome of a post-surgical wound infection management by a carbapenem-resistant Klebsiella pneumoniae is described in a dog. Four multidrug-resistant and carbapenem-resistant Escherichia coli strains belonging to ST410 (n = 1) and ST648 (n = 3) were isolated from faecal samples and nasal swabs of this dog at admission to a veterinary hospital in the United Kingdom, and one month after discharge. Whole-genome sequencing analysis suggests dissemination of a 46,161-bp IncX3 bla NDM-5 -carrying plasmid among E. coli strains from the different lineages. In this study, the E. coli ST648 strains were virtually identical to each other (5 SNPs difference) indicating dissemination and persistence of this clone over time and across different anatomical sites in the same dog maybe due to the prolonged antimicrobial therapy. The carbapenemase carrying plasmid also showed homology with other publicly available plasmid sequences from Asian countries. These results suggests that plasmids may be a major vehicle in mediating the dissemination of carbapenem-resistance. Further studies investigating the selection and flow of plasmids carrying important resistance genes amongst companion animals are needed as it may further contaminate other environments posing a threat to public health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aminopenicillins have been widely used for decades for the treatment of various infections in animals and humans in European countries. Following this extensive use, acquired resistance has emerged among human and animal pathogens and commensal bacteria. Aminopenicillins are important first-line treatment options in both humans and animals, but are also among limited therapies for infections with enterococci and Listeria spp. in humans in some settings. Therefore, there is a need to assess the impact of the use of these antimicrobials in animals on public and animal health. The most important mechanisms of resistance to aminopenicillins are the β-lactamase enzymes. Similar resistance genes have been detected in bacteria of human and animal origin, and molecular studies suggest that transmission of resistant bacteria or resistance genes occurs between animals and humans. Due to the complexity of epidemiology and the near ubiquity of many aminopenicillin resistance determinants, the direction of transfer is difficult to ascertain, except for major zoonotic pathogens. It is therefore challenging to estimate to what extent the use of aminopenicillins in animals could create negative health consequences to humans at the population level. Based on the extent of use of aminopenicillins in humans, it seems probable that the major resistance selection pressure in human pathogens in European countries is due to human consumption. It is evident that veterinary use of these antimicrobials increases the selection pressure towards resistance in animals and loss of efficacy will at minimum jeopardize animal health and welfare., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Extended-spectrum beta-lactamase (ESBL)- and plasmid-mediated cephalosporinase (AmpC)-producing Enterobacterales (ESBL/AmpC-E) are an increasing healthcare problem in both human and veterinary medicine. The aim of this study was to investigate the possible sharing of ESBL/AmpC-E strains between healthy companion animals and humans of the same household in Portugal (PT) and the United Kingdom (UK). In a prospective longitudinal study, between 2018 and 2020, faecal samples were collected from healthy dogs (n=90), cats (n=20) and their cohabiting humans (n=119) belonging to 41 PT and 44 UK households. Samples were screened for the presence of ESBL/AmpC-E and carbapenemase-producing bacteria. Clonal relatedness between animal and human strains was established by using REP-PCR fingerprinting method, followed by whole-genome sequencing (WGS) of selected strains. ESBL/AmpC-E strains were detected in companion animals (PT=12.7%, n=8/63; UK=8.5%, n=4/47) and humans (PT=20.7%, n=12/58; UK=6.6%, n=4/61) in at least one timepoint. REP-PCR identified paired multidrug-resistant ESBL/AmpC-producing Escherichia coli strains from companion animals and owners in two Portuguese households (4.8%) and one UK household (2.3%). WGS analysis of nine E. coli strains from these three households confirmed that interhost sharing occurred only between the two animal-human pairs from Portugal. Three shared strains were identified: one CTX-M-15-producing E. coli strain in a cat-human pair (O15-H33-ST93) and two CTX-M-15- and CTX-M-55/CMY-2-producing E. coli strains, in a dog-human pair (O8:H9-ST410 and O11:H25-ST457, respectively) at different timepoints. These E. coli clonal lineages are human pandemic, highlighting the role of companion animals living in close contact with humans in the dissemination and persistence of antimicrobial resistance in the household environment., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Staphylococcus pseudintermedius is the main bacterial pathogen of skin and soft-tissue infections (SSTIs) in companion animals. Antimicrobial resistance in this species is a growing public health concern. This study aims to characterize a collection of S. pseudintermedius causing SSTIs in companion animals, establishing the main clonal lineages and antimicrobial resistance traits. The collection corresponded to all S. pseudintermedius ( n  = 155) causing SSTIs in companion animals (dogs, cats and one rabbit) collected between 2014 and 2018 at two laboratories in Lisbon, Portugal. Susceptibility patterns were established by disk diffusion for 28 antimicrobials (15 classes). For antimicrobials without clinical breakpoints available, a cut-off value (CO WT ) was estimated, based on the distribution of the zones of inhibition. The blaZ and mecA genes were screened for the entire collection. Other resistance genes (e.g., erm , tet , aadD, vga (C) , dfrA (S1)) were searched only for those isolates showing an intermediate/resistance phenotype. For fluoroquinolone resistance, we determined the chromosomal mutations in the target genes grlA and gyrA . All the isolates were typed by PFGE following Sma I macrorestriction and isolates representative of each PFGE type were further typed by MLST. Forty-eight out of the 155   S. pseudintermedius isolates (31.0%) were methicillin-resistant ( mecA + , MRSP). Multidrug-resistant (MDR) phenotypes were detected for 95.8% of the MRSP and 22.4% of the methicillin-susceptible (MSSP) isolates. Of particular concern, only 19 isolates (12.3%) were susceptible to all antimicrobials tested. In total, 43 different antimicrobial resistance profiles were detected, mostly associated with the carriage of blaZ , mecA , erm (B), aph3-IIIa , aacA-aphD, cat pC221 , tet (M) and dfr (G) genes. The 155 isolates were distributed within 129 PFGE clusters, grouped by MLST in 42 clonal lineages, 25 of which correspond to new sequence types (STs). While ST71 remains the most frequent S. pseudintermedius lineage, other lineages that have been replacing ST71 in other countries were detected, including ST258, described for the first time in Portugal. This study revealed a high frequency of MRSP and MDR profiles among S. pseudintermedius associated with SSTIs in companion animals in our setting. Additionally, several clonal lineages with different resistance profiles were described, evidencing the importance of a correct diagnosis and selection of the therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Morais, Costa, Leal, Ramos, Andrade, Ferreira, Abrantes, Pomba and Couto.)

Staphylococcus pseudintermedius is the main bacterial cause of skin and soft tissue infections (SSTIs) in companion animals, particularly dogs. The emergence of methicillin-resistant S. pseudintermedius (MRSP) strains, frequently with multidrug resistance phenotypes is a public health concern. This study aimed to evaluate efflux, a resistance mechanism still poorly characterized in S. pseudintermedius , as a contributor to biocide and fluoroquinolone resistance. Susceptibility to the efflux pump substrates ethidium bromide (EtBr), tetraphenylphosphonium bromide (TPP) and ciprofloxacin (CIP) was evaluated by minimum inhibitory concentration (MIC) determination for 155 SSTIs-related S. pseudintermedius in companion animals. EtBr and TPP MIC distributions were analyzed to estimate cut-off (CO WT ) values. The effect of the efflux inhibitors (EIs) thioridazine and verapamil was assessed upon MICs and fluorometric EtBr accumulation assays, performed with/without glucose and/or EIs. This approach detected a non-wild type population towards TPP with increased efflux, showed to be strain-specific and glucose-dependent. Resistance to fluoroquinolones was mainly linked to target gene mutations, yet a contribution of efflux on CIP resistance levels could not be ruled out. In sum, this study highlights the relevance of efflux-mediated resistance in clinical S. pseudintermedius , particularly to biocides, and provides a methodological basis for further studies on the efflux activity on this important pathogen of companion animals.

Introduction: The changing epidemiology of Clostridioides difficile reflects a well-established and intricate community transmission network. With rising numbers of reported community-acquired infections, recent studies tried to identify the role played by non-human reservoirs in the pathogen's transmission chain. This study aimed at describing the C. difficile strains circulating in canine and feline populations, and to evaluate their genetic overlap with human strains to assess the possibility of interspecies transmission., Methods: Fecal samples from dogs ( n = 335) and cats ( n = 140) were collected from two populations (group A and group B) in Portugal. C. difficile isolates were characterized for toxigenic profile and PCR-ribotyping. The presence of genetic determinants of antimicrobial resistance was assessed in all phenotypically resistant isolates. To evaluate the genetic overlap between companion animals and human isolates from Portugal, RT106 ( n = 42) and RT014/020 ( n = 41) strains from both sources were subjected to whole genome sequencing and integrated with previously sequenced RT106 ( n = 43) and RT014/020 ( n = 142) genomes from different countries. The genetic overlap was assessed based on core-single nucleotide polymorphism (SNP) using a threshold of 2 SNP., Results: The overall positivity rate for C. difficile was 26% (76/292) in group A and 18.6% (34/183) in group B. Toxigenic strains accounted for 50% (38/76) and 52.9% (18/34) of animal carriage rates, respectively. The most prevalent ribotypes (RT) were the toxigenic RT106 and RT014/020, and the non-toxigenic RT010 and RT009. Antimicrobial resistance was found for clindamycin (27.9%), metronidazole (17.1%) and moxifloxacin (12.4%), associated with the presence of the ermB gene, the pCD-METRO plasmid and point mutations in the gyrA gene, respectively. Both RT106 and RT014/020 genetic analysis revealed several clusters integrating isolates from animal and human sources, supporting the possibility of clonal interspecies transmission or a shared environmental contamination source., Discussion: This study shows that companion animals may constitute a source of infection of toxigenic and antimicrobial resistant human associated C. difficile isolates. Additionally, it contributes with important data on the genetic proximity between C. difficile isolates from both sources, adding new information to guide future work on the role of animal reservoirs in the establishment of community associated transmission networks and alerting for potential public health risk., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alves, Castro, Pinto, Nunes, Pomba, Oliveira, Silveira, Gomes and Oleastro.)

An 8-year-old female neutered Labrador retriever was presented for a second opinion consultation due to vomiting and lethargy, having failed to respond to symptomatic therapy. Blood analysis revealed hyperbilirubinemia and hypoalbuminemia, associated with hypocobalaminemia. An abdominal ultrasound identified diffused bowel thickening and hypoechoic hepatomegaly. An ultrasound-guided liver fine-needle aspiration was performed for cytology and also for cell block immunocytochemistry. Gastric and duodenal biopsies were collected by gastroduodenoscopy. Liver cytology showed numerous lymphocytes, suggesting lymphoma at the hepatic infiltration stage, and immunocytochemistry in the cell block of the hepatic aspirate indicated co-expression of CD3 and CD20 in the lymphoid cells present. The histopathology of gastric and duodenal biopsies supported the hypothesis of gastrointestinal lymphoma due to heavy lymphoid infiltration of the gastric epithelium and intestinal mucosa, including the villi. Concurrent immunohistochemistry was performed using CD3, CD20, PAX5, and CD79αcy antibodies. Immunomarking was positive for CD3 and CD20, which overlapped populations of lymphoid cells, and was negative for all other antibodies. In the clonality test, lymphocyte co-expression of CD3 and CD20 was confirmed by monoclonal rearrangement of T-cell gamma receptors. The final diagnosis was type 2 enteropathy-associated T-cell lymphoma with hepatic infiltration. Co-expression was examined in conjunction with the PARR result in the presence of T-cell monoclonal rearrangement.

BackgroundThe emergence of colistin resistance is a One Health antimicrobial resistance challenge worldwide. The close contact between companion animals and humans creates opportunities for transmission and dissemination of colistin-resistant bacteria.AimTo detect potential animal reservoirs of colistin-resistant Escherichia coli and investigate the possible sharing of these bacteria between dogs, cats and their cohabiting humans in the community in Lisbon, Portugal.MethodsA prospective longitudinal study was performed from 2018 to 2020. Faecal samples from dogs and cats either healthy or diagnosed with a skin and soft tissue or urinary tract infection, and their cohabiting humans were screened for the presence of colistin-resistant E . coli. All isolates were tested by broth microdilution against colistin and 12 other antimicrobials. Colistin-resistant isolates were screened for 30 resistance genes, including plasmid-mediated colistin resistance genes ( mcr-1 to mcr-9 ), and typed by multilocus sequence typing. Genetic relatedness between animal and human isolates was analysed by whole genome sequencing.ResultsColistin-resistant E. coli strains harbouring the mcr-1 gene were recovered from faecal samples of companion animals (8/102; 7.8%) and humans (4/125; 3.2%). No difference between control and infection group was detected. Indistinguishable multidrug-resistant E . coli ST744 strains harbouring the mcr-1 gene were found in humans and their dogs in two households.ConclusionsThe identification of identical E . coli strains containing the plasmid-mediated mcr-1 gene in companion animals and humans in daily close contact is of concern. These results demonstrate the importance of the animal-human unit as possible disseminators of clinically important resistance genes in the community setting.

Colistin is classified as a high-priority critical antimicrobial by the World Health Organization (WHO). A better understanding of the biological cost imposed by mcr-plasmids is paramount to comprehending their spread and may facilitate the decision about the ban of colistin in livestock. This study aimed to assess the prevalence of mcr and ESBL genes from 98 Escherichia coli and 142 Salmonella enterica isolates from food-producing animals and the impact of the mcr-1 acquisition on bacterial fitness. Only mcr-1 was identified by multiplex PCR (mcr-1 to mcr-10) in 15.3% of E. coli. Colistin MICs ranged between 8−32 mg/L. In four isolates, blaTEM-1, blaCTX-M-1, and blaCTX-M-15 co-existed with mcr-1. The IncH12, IncHI1, IncP, IncN, and IncI plasmids were transferred by conjugation to E. coli J53 at frequencies of 10−7 to 10−2 cells/recipient. Growth kinetics assays showed that transconjugants had a significantly lower growth rate than the recipient (p < 0.05), and transconjugants’ average growth rate was higher in the absence than in the presence of colistin (1.66 versus 1.32 (p = 0.0003)). Serial transfer assay during 10 days demonstrated that plasmid retention ranged from complete loss to full retention. Overall, mcr-1-bearing plasmids impose a fitness cost, but the loss of plasmids is highly variable, suggesting that other factors beyond colistin pressure regulate the plasmid maintenance in a bacterial population, and colistin withdrawal will not completely lead to a decrease of mcr-1 levels.

Coagulase-positive staphylococci (CoPS) account for most bacteria-related pyoderma in companion animals. Emergence of methicillin-resistant strains of Staphylococcus pseudintermedius (MRSP), Staphylococcus aureus (MRSA) or Staphylococcus coagulans (MRSC), often with multidrug-resistant (MDR) phenotypes, is a public health concern. The study collection comprised 237 staphylococci ( S. pseudintermedius ( n = 155), S. aureus ( n = 55) and S. coagulans ( n = 27)) collected from companion animals, previously characterized regarding resistance patterns and clonal lineages. Biofilm production was detected for 51.0% (79/155), 94.6% (52/55) and 88.9% (24/27) of the S. pseudintermedius , S. aureus and S. coagulans, respectively, and was a frequent trait of the predominant S. pseudintermedius and S. aureus clonal lineages. The production of biofilm varied with NaCl supplementation of the growth media. All S. pseudintermedius and S. aureus strains carried icaADB . Kaplan-Meier survival analysis of Galleria mellonella infected with different CoPS revealed a higher virulence potential of S. aureus when compared with other CoPS. Our study highlights a high frequency of biofilm production by prevalent antimicrobial-resistant clonal lineages of CoPS associated with animal pyoderma, potentially related with a higher virulence potential and persistent or recurrent infections.