Carbapenemase-producing Enterobacterales strains causing infections in companion animals-Portugal.

An increase in Klebsiella pneumoniae carbapenem-resistant human nosocomial strains is occurring in Europe, namely with the blaOXA-48-like and blaKPC-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 blaKPC-3 gene was located on transposon Tn4401d on IncFIA type plasmid for the K. pneumoniae ST147 strains and on a IncN-type plasmid for the K. pneumoniae ST392 strain, while blaOXA-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.

Multiple myeloma in dogs: Use of the cell block technique as a new diagnostic tool.

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.

Dissemination of blaNDM-5-carrying IncX3-type plasmid among non-clonal Escherichia coli strains colonising a dog with a skin infection caused by a carbapenem-resistant Klebsiella pneumoniae, United Kingdom.

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 blaNDM-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.

Longitudinal study of ESBL/AmpC-producing Enterobacterales strains sharing between cohabiting healthy companion animals and humans in Portugal and in the United Kingdom.

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.

Genetic diversity and antimicrobial resistance profiles of Staphylococcus pseudintermedius associated with skin and soft-tissue infections in companion animals in Lisbon, Portugal.

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 (COWT) 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 SmaI 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.

The use of aminopenicillins in animals within the EU, emergence of resistance in bacteria of animal and human origin and its possible impact on animal and human health.

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 ß-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.

Exploring Efflux as a Mechanism of Reduced Susceptibility towards Biocides and Fluoroquinolones in Staphylococcus pseudintermedius.

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 (COWT) 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.

Co-Expression of T- and B-Cell Markers in a Canine Intestinal Lymphoma: A Case Report.

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.

mcr-1 colistin resistance gene sharing between Escherichia coli from cohabiting dogs and humans, Lisbon, Portugal, 2018 to 2020.

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.

Virulence Potential of Biofilm-Producing Staphylococcus pseudintermedius, Staphylococcus aureus and Staphylococcus coagulans Causing Skin Infections in Companion Animals.

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.

Occurrence and Biological Cost of mcr-1-Carrying Plasmids Co-harbouring Beta-Lactamase Resistance Genes in Zoonotic Pathogens from Intensive Animal Production.

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.

Longitudinal Study Detects the Co-Carriage of ESBL and mcr-1 and -4 Genes in Escherichia coli Strains in a Portuguese Farrow-to-Finish Swine Herd.

Cephalosporins and polymyxins are employed in antimicrobial protocols to control and treat neonatal infections and post-weaning diarrhoea in swine operations. We conducted a longitudinal study to evaluate the colonization and transmission of antibiotic-resistant Escherichia coli in sows and their piglets in a farrow-to-finish operation, focusing on characterization of Extended Spectrum Beta-Lactamase (ESBL) and mcr genes, virulence traits and genetic relatedness. A total of 293 E. coli isolates were obtained from faecal samples collected in five time points. At birth blaCTX-M-1group cluster was detected in E. coli isolates from 9 sows and 49 piglets (73.41%), while in the following four' piglets sampling moments it was detected in 91.8%, 57.6%, 71.4% and 97.4%. The gene mcr-1 was detected in E. coli from one sow and from three piglets from different litters at birth and increased in the first weeks of piglet life (68.85%, 100%, 90% and 8.1%). A new mcr-4 allele, mcr-4.7, was identified in 3.28%, 28.57%, 7.5% of E. coli isolates. Most mcr-positive E. coli isolates (96,7%) carried blaCTX-M-1Group genes and 93,33% carried both mcr-4 and mcr-1. CTX-M-1 and CTX-M-32 were the most predominant ESBLs. Plasmids belonged to IncI1, IncF and IncN groups. Most isolates belong to phylogenetic group B1; PAI IV536 marker was detected in nine isolates. The strains were kept in the different stages of the piglets' life. The use of ceftiofur and colistin may explain the high prevalence and co-selection of blaCTX-M-1Group and mcr-1 and/or -4 genes, contributing to the maintenance of resistant and virulent isolates throughout the pig life cycle that may reach the food chain.

Genomic epidemiological analysis of Klebsiella pneumoniae from Portuguese hospitals reveals insights into circulating antimicrobial resistance.

Klebsiella pneumoniae (Kp) bacteria are an increasing threat to public health and represent one of the most concerning pathogens involved in life-threatening infections and antimicrobial resistance (AMR). To understand the epidemiology of AMR of Kp in Portugal, we analysed whole genome sequencing, susceptibility testing and other meta data on 509 isolates collected nationwide from 16 hospitals and environmental settings between years 1980 and 2019. Predominant sequence types (STs) included ST15 (n = 161, 32%), ST147 (n = 36, 7%), ST14 (n = 26, 5%) or ST13 (n = 26, 5%), while 31% of isolates belonged to STs with fewer than 10 isolates. AMR testing revealed widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems. The most common carbapenemase gene was blaKPC-3. Whilst the distribution of AMR linked plasmids appears uncorrelated with ST, their frequency has changed over time. Before year 2010, the dominant plasmid group was associated with the extended spectrum beta-lactamase gene blaCTX-M-15, but this group appears to have been displaced by another carrying the blaKPC-3 gene. Co-carriage of blaCTX-M and blaKPC-3 was uncommon. Our results from the largest genomics study of Kp in Portugal highlight the active transmission of strains with AMR genes and provide a baseline set of variants for future resistance monitoring and epidemiological studies.

ESBL/pAmpC-Producing Escherichia coli Causing Urinary Tract Infections in Non-Related Companion Animals and Humans.

Urinary tract infections (UTI) caused by Escherichia coli are frequently diagnosed in humans and companion animals. Extended-spectrum beta-lactamase (ESBL)- and cephalosporinase (pAmpC)-producing Escherichia coli are worldwide-disseminated and frequently multidrug-resistant, hence leading to treatment failure and public health concerns. This study aimed to characterize and compare ESBL/pAmpC-producing E. coli strains causing community-acquired UTI in companion animals and non-related humans. Third-generation cephalosporin (3GC)-resistant E. coli (companion animals n = 35; humans n = 85) isolated from patients with UTI were tested against 14 antimicrobials following CLSI guidelines. PCR-based assays were used to detect the major E. coli phylogenetic groups, pathogenicity associated-islands (PAIs), virulence genes, and ESBLs/pAmpC resistance genes. ESBL/pAmpC-producing E. coli isolates were typed by multi-locus sequence typing (MLST) and PCR. E. coli strains from companion animals and humans shared two MDR high-risk clonal lineages: ST131 and ST648. To the best of our knowledge, this study reports the first description of E. coli ST131 clade C1-M27 and the clonal lineage ST131 clade A in humans with community-acquired UTI in Portugal. Considering that companion animals with UTI are generally treated at home by the owners, measures should be implemented to avoid the spread of multidrug-resistant high-risk clones to humans and their household environment.

Staphylococcus aureus Causing Skin and Soft Tissue Infections in Companion Animals: Antimicrobial Resistance Profiles and Clonal Lineages.

Staphylococcus aureus is a relevant agent of skin and soft tissue infections (SSTIs) in animals. Fifty-five S. aureus comprising all SSTI-related isolates in companion animals, collected between 1999 and 2018 (Lab 1) or 2017 and 2018 (Lab 2), were characterized regarding susceptibility to antibiotics and heavy metals and carriage of antimicrobial resistance determinants. Clonal lineages were established by PFGE, MLST and agr typing. Over half of the isolates (56.4%, 31/55) were methicillin-resistant S. aureus (MRSA), and 14.5% showed a multidrug resistance (MDR) phenotype. Resistance was most frequently observed for beta-lactams (81.8%, related to blaZ and/or mecA), fluoroquinolones (56.4%) and macrolides/lincosamides (14.5%, related to erm(A) or erm(C)). The distributions of heavy-metal MICs allowed the detection of non-wild-type populations associated with several resistance genes. The collection showed genetic diversity, with prevalence of clonal lineage ST22-agrI (45.5%, 25/55), comprising only MRSA isolates, and several less frequently detected clones, including ST5-agrII (14.6%, 8/55), ST398-agrI (9.1%, 5/55) and ST72-agrI (7.3%, 4/55). This work highlights the high frequency of SSTI-related MRSA strains that reflect the clonal lineages circulating both in companion animals and humans in Portugal, reinforcing the need for a One Health approach when studying staphylococci causing infections in companion animals.

Companion Animals-An Overlooked and Misdiagnosed Reservoir of Carbapenem Resistance.

The dissemination of antimicrobial-resistance is a major global threat affecting both human and animal health. Carbapenems are human use ß-lactams of last resort; thus. the dissemination of carbapenemase-producing (CP) bacteria creates severe limitations for the treatment of multidrug-resistant bacteria in hospitalized patients. Even though carbapenems are not routinely used in veterinary medicine, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals are being reported. NDM-5 and OXA-48-like carbapenemases are among the most frequently reported in companion animals. Like in humans, Escherichia coli and Klebsiella pneumoniae are the most represented CP Enterobacterales found in companion animals, alongside with Acinetobacter baumannii. Considering that the detection of carbapenemase-producing Enterobacterales presents several difficulties, misdiagnosis of CP bacteria in companion animals may lead to important animal and public-health consequences. It is of the upmost importance to ensure an adequate monitoring and detection of CP bacteria in veterinary microbiology in order to safeguard animal health and minimise its dissemination to humans and the environment. This review encompasses an overview of the carbapenemase detection methods currently available, aiming to guide veterinary microbiologists on the best practices to improve its detection for clinical or research purposes.

Molecular epidemiology of Clostridioides difficile in companion animals: Genetic overlap with human strains and public health concerns.

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.

Phenotypic and Molecular Traits of Staphylococcus coagulans Associated with Canine Skin Infections in Portugal.

Staphylococcus coagulans is among the three most frequent pathogens of canine pyoderma. Yet, studies on this species are scarce. Twenty-seven S. coagulans and one S. schleiferi, corresponding to all pyoderma-related isolations from these two species at two veterinary laboratories in Lisbon, Portugal, between 1999 and 2018 (Lab 1) or 2018 (Lab 2), were analyzed. Isolates were identified by the analysis of the nuc gene and urease production. Antibiotic susceptibility towards 27 antibiotics was evaluated by disk diffusion. Fourteen antibiotic resistance genes were screened by PCR. Isolates were typed by SmaI-PFGE. Two S. coagulans isolates (2/27, 7.4%) were methicillin-resistant (MRSC, mecA+) and four (4/27, 14.8%) displayed a multidrug-resistant (MDR) phenotype. We observed resistance to penicillin (17/27, 63.0%), fluoroquinolones (11/27, 40.7%), erythromycin and clindamycin (3/27, 11.1%), fusidic acid (3/27, 11.1%) and tetracycline (1/27, 3.7%). The blaZ and erm(B) genes were carried by 16 and 1 isolates resistant to penicillin and erythromycin/clindamycin, respectively. Only three S. coagulans carried plasmids. The single S. schleiferi isolate presented an MDR phenotype. SmaI-PFGE revealed a limited genetic diversity of S. coagulans, with a predominant lineage present from 2001 to 2018. This study describes the first MRSC causing canine infection in Portugal and reveals a high burden of antimicrobial resistance, with the emergence of MDR phenotypes within the main lineages.

Proposal of Epidemiological Cutoff Values for Apramycin 15 µg and Florfenicol 30 µg Disks Applicable to Staphylococcus aureus.

Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and florfenicol. The susceptibility to apramycin and florfenicol was evaluated by disk diffusion of five S. aureus collections, comprising 352 isolates of animal (n = 265) and human (n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ≥15 mm (ECOFF = 15 mm) for apramycin and ≥21 mm for florfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas five NWT isolates were identified for florfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and florfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.