Genomic Investigation of Two Acinetobacter baumannii Outbreaks in a Veterinary Intensive Care Unit in The Netherlands

Acinetobacter baumannii is a nosocomial pathogen that frequently causes healthcare-acquired infections. The global spread of multidrug-resistant (MDR) strains with its ability to survive in the environment for extended periods imposes a pressing public health threat. Two MDR A. baumannii outbreaks occurred in 2012 and 2014 in a companion animal intensive care unit (caICU) in the Netherlands. Whole-genome sequencing (WGS) was performed on dog clinical isolates (n = 6), environmental isolates (n = 5), and human reference strains (n = 3) to investigate if the isolates of the two outbreaks were related. All clinical isolates shared identical resistance phenotypes displaying multidrug resistance. Multi-locus Sequence Typing (MLST) revealed that all clinical isolates belonged to sequence type ST2. The core genome MLST (cgMLST) results confirmed that the isolates of the two outbreaks were not related. Comparative genome analysis showed that the outbreak isolates contained different gene contents, including mobile genetic elements associated with antimicrobial resistance genes (ARGs). The time-measured phylogenetic reconstruction revealed that the outbreak isolates diverged approximately 30 years before 2014. Our study shows the importance of WGS analyses combined with molecular clock investigations to reduce transmission of MDR A. baumannii infections in companion animal clinics

Cellular and humoral immune responsiveness to inactivated Leptospira interrogans in dogs vaccinated with a tetravalent Leptospira vaccine

Vaccination is commonly used to protect dogs against leptospirosis, however, memory immune responses induced by canine Leptospira vaccines have not been studied. In the present study, antibody and T cell mediated responses were assessed in dogs before and 2 weeks after annual revaccination with a commercial tetravalent Leptospira vaccine containing serogroups Canicola and Australis. Vaccination significantly increased average log 2 IgG titers from 6.50 to 8.41 in year 1, from 5.99 to 7.32 in year 2, from 5.32 to 8.32 in year 3 and from 5.32 to 7.82 in year 4. The CXCL-10 levels, induced by in vitro stimulation of PBMC with Canicola and Australis, respectively, significantly increased from 1039.05 pg/ml and 1037.38 pg/ml before vaccination to 2547.73 pg/ml and 2730.38 pg/ml after vaccination. IFN-γ levels increased from 85.60 pg/ml and 178.13 pg/ml before vaccination to 538.62 pg/ml and 210.97 pg/ml after vaccination. The percentage of proliferating CD4 + T cells in response to respective Leptospira strains significantly increased from 1.43 % and 1.25 % before vaccination to 24.11 % and 14.64 % after vaccination. Similar responses were also found in the CD8 + T cell subset. Vaccination also significantly enhanced the percentages of central memory CD4 + T cells from 12 % to 26.97 % and 27.65 %, central memory CD8 + T cells from 3 % to 9.47 % and 7.55 %, and effector CD8 + T cells from 3 % to 7.6 % and 6.42 %, as defined by the expression of CD45RA and CD62L, following stimulation with Canicola and Australis, respectively. Lastly, enhanced expression of the activation marker CD25 on T cells after vaccination was found. Together, our results show that next to IgG responses, also T cell responses are induced in dogs upon annual revaccination with a tetravalent Leptospira vaccine, potentially contributing to protection

Quantifying topical antimicrobial use before and during participation in an antimicrobial stewardship programme in Dutch companion animal clinics

The emergence of bacterial strains resistant to topical antimicrobials in both human and veterinary medicine has raised concerns over retaining the efficacy of these preparations. Yet, little information is available regarding the use of topical antimicrobials in either sector for planning targeted interventions. This study aims to quantify the use of topical antimicrobials in 44 Dutch companion animal clinics before and during their participation in an antimicrobial stewardship programme (ASP), to explore the effect of the intervention on topical antimicrobial use (AMU). Hence, prescription and clinic animal population data, collected from July 2012 until June 2018 were used. Specifically, the period from July 2012 until June 2015 was defined as pre-intervention period, whereas clinics started to participate in the ASP from March 2016 onwards. As quantification metric, the Defined Daily Dose for Animals (DDDA) was used and a mixed effect times series model with auto-regression was applied to monthly topical AMU data. The intervention effect was modelled using a step function with a change in (linear) time trend and clinic characteristics, as potential determinants of topical AMU, were assessed using a multivariable regression model. A seasonal pattern was identified, in the pre-intervention period, where topical AMU was highest in July-August and lowest in February-March. In addition, total topical AMU appeared to significantly decrease over time in the pre-intervention period and the proportion of dogs in the clinic was positively associated with topical AMU. The intervention effect was significant only for second line and for skin product AMU. This study demonstrates that during participation in an ASP, second line and skin product AMU decreased in Dutch companion animal clinics. Additionally, this study demonstrates the existence of a seasonal effect and a decrease in topical AMU over time already before introduction of a targeted intervention

Contribution of cats and dogs to SARS-CoV-2 transmission in households

INTRODUCTION: SARS-CoV-2 is known to jump across species. The occurrence of transmission in households between humans and companion animals has been shown, but the contribution of companion animals to the overall transmission within a household is unknown. The basic reproduction number ( R 0) is an important indicator to quantify transmission. For a pathogen with multiple host species, such as SARS-CoV-2, the basic reproduction number needs to be calculated from the partial reproduction numbers for each combination of host species. METHOD: In this study, the basic and partial reproduction numbers for SARS-CoV-2 were estimated by reanalyzing a survey of Dutch households with dogs and cats and minimally one SARS-CoV-2-infected human. RESULTS: For households with cats, a clear correlation between the number of cats and the basic reproduction number (Spearman's correlation: p 0.40, p-value: 1.4 × 10 -5) was identified, while for dogs, the correlation was smaller and not significant (Spearman's correlation: p 0.12, p-value: 0.21). Partial reproduction numbers from cats or dogs to humans were 0.3 (0.0-2.0) and 0.3 (0.0-3.5) and from humans to cats or dogs were 0.6 (0.4-0.8) and 0.6 (0.4-0.9). DISCUSSION: Thus, the estimations of within-household transmission indicated the likelihood of transmission from these companion animals to humans and vice versa, but the observational nature of this study limited the ability to establish conclusive evidence. This study's findings support the advice provided during the pandemic to COVID-19 patients to maintain distance from companion animals as a precautionary measure and given the possibility of transmission, although there is an overall relatively limited impact on the pandemic when compared to human-to-human transmission

Evidence of cat-to-human transmission of Staphylococcus felis.

Introduction. Staphylococcus felis is a coagulase-negative staphylococcal species that is commonly isolated from healthy cats. Like other commensal staphylococci, S. felis can cause opportunistic infections, e.g. otitis externa, skin and urinary tract infections, in cats. Gap Statement. Several studies have reported within-household transmission between humans and pets and human infections caused by coagulase-positive staphylococci. However, human infections with coagulase-negative staphylococci of zoonotic origin are relatively rare. Methodology. Culture of a surgical site infection in a 58-year-old woman who underwent a laminectomy revealed dominant growth of S. felis. The three cats owned by the patient were sampled to investigate potential within-household transmission. S. felis isolates were sequenced to investigate the relatedness of the isolates and to look for virulence factors and host specific genes. Results. All cats were colonized with S. felis. Comparative genomics of the isolates showed that each cat was colonized with a distinct genotype. The patient's isolate clustered with isolates of one of the cats. Sequence analysis of the studied isolates together with 29 publicly available S. felis genomes detected putative virulence factors that can be crucial in potential interspecies transmission. Conclusion. The current case is the first reported human infection caused by S. felis and highlights the zoonotic potential of this bacterial species. Evidence of cat-to-human transmission was shown by comparative genomics of isolates from the patient with isolates of her cats

Driving laboratory standardisation of bacterial culture and antimicrobial susceptibility testing in veterinary clinical microbiology in Europe and beyond.

Globally, antimicrobial resistance is one of the most important public health challenges in which the clinical microbiology laboratory plays a critical role by providing guidance for antimicrobial treatment. Despite the recognition of its importance, there is still a real need for standardized training of clinical microbiologists and harmonisation of diagnostic procedures. This is particularly true for veterinary clinical microbiology where additional challenges exist when microbiologists are trying to fulfil a professional role very similar to their colleagues working in human microbiology laboratories. The specific points that need addressing to improve the outputs of veterinary microbiology laboratories discussed here include 1) harmonisation of methodologies used by veterinary laboratories for antimicrobial susceptibility testing (AST); 2) specific guidelines for interpretation and reporting of AST results for animal pathogens; 3) guidelines for detection of antimicrobial resistance mechanisms in animal isolates; 4) standardisation of diagnostic procedures for animal clinical specimens and 5) the need to train more veterinary clinical microbiology specialists. However, there is now a plan to address these issues led by the European Network for Optimisation of Veterinary Antimicrobial Treatment (ENOVAT) which is bringing together experts in veterinary microbiology, pharmacology, epidemiology and antimicrobial stewardship from Europe and wider afield. ENOVAT is aiming to work with project partners towards standardisation and harmonisation of laboratory methodologies and optimisation of veterinary antimicrobial treatment. Ultimately, the project may provide a mechanism for standardisation and harmonisation of veterinary clinical microbiology methodologies, which could then be used as a template for implementation at a wider international level

Quantifying Antimicrobial Use in Dutch Companion Animals

Antimicrobial resistance (AMR) is an increasing threat, both in human and in veterinary medicine. To reduce the selection and spread of AMR, antimicrobial use (AMU) should be optimized, also in companion animals. To be able to optimize AMU, a feasible method to quantify AMU and information on current AMU are needed. Therefore, a method to quantify AMU was developed, using the number of Defined Daily Doses Animal (DDDA). This method was used to explore applied antimicrobial classes and to identify differences in prescribing patterns in time and between veterinary clinics. Antimicrobial procurement data of the years 2012–2014 were collected retrospectively from 100 Dutch veterinary clinics providing care for companion animals. The mean number of DDDAs per clinic per year decreased significantly from 2012 to 2014. A shift in used classes of antimicrobials (AMs) was seen as well, with a significant decrease in use of third choice AMs (i.e., fluoroquinolones and third generation cephalosporins). Large differences in total AMU were seen between clinics ranging from 64-fold in 2012 to 20-fold in 2014. Despite the relative low and decreasing AMU in Dutch companion animal clinics during the study, the substantial differences in antimicrobial prescribing practices between clinics suggest that there is still room for quantitative and qualitative optimization of AMU

Molecular Characterization and Clinical Relevance of Taxonomic Reassignment of Staphylococcus schleiferi Subspecies into Two Separate Species, Staphylococcus schleiferi and Staphylococcus coagulans.

Staphylococcus schleiferi is an opportunistic pathogen in humans and dogs. Recent taxonomic reassignment of its subspecies (S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans) into two separate species (S. schleiferi and S. coagulans) lacks supporting data for diagnostic implications and clinical relevance. We aimed to confirm the reclassification of S. schleiferi by using genomic and matrix-Assisted laser desorption ionization-time of flight (MALDI-TOF) data for a large set of isolates from humans and animals to investigate their molecular epidemiology and clinical relevance. Routine MALDI-TOF analysis and Illumina sequencing were performed on 165 S. schleiferi isolates from the Netherlands. With 33 publicly available genomes, the study included 198 genomes from 149 dogs, 34 humans, and 15 other sources. The Type Strain Genome Server was used to identify species in the genomes, and the MALDI-TOF MS database was extended to improve species differentiation. MALDI-TOF did not discriminate between S. schleiferi and S. coagulans. Genome phylogeny distinguished the two species in two monophyletic clusters. S. schleiferi isolates originated from humans, while S. coagulans isolates were found in animals and three human isolates clustering with the animal isolates. The sialidase B gene (nanB) was a unique marker gene for S. schleiferi, whereas the chrA gene was exclusive for S. coagulans. The mecA gene was exclusively detected in S. coagulans, as were the lnu(A), blaZ, erm(B/C), tet(O/M), and aac(69)-Aph(299) genes. The MALDI-TOF database extension did not improve differentiation between the two species. Even though our whole-genome sequencing- based approach showed clear differentiation between these two species, it remains critical to identify S. schleiferi and S. coagulans correctly in routine diagnostics. IMPORTANCE This study clearly shows that S. schleiferi is a concern in human hospital settings, whereas S. coagulans predominantly causes infections in animals. S. coagulans is more resistant to antibiotics and can sometimes transmit to humans via exposure to infected dogs. Even though genome-based methods can clearly differentiate the two species, current diagnostic methods used routinely in clinical microbiology laboratories cannot distinguish the two bacterial species

Within-Household Transmission and Bacterial Diversity of Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius can be transmitted between dogs and their owners and can cause opportunistic infections in humans. Whole genome sequencing was applied to identify the relatedness between isolates from human infections and isolates from dogs in the same households. Genome SNP diversity and distribution of plasmids and antimicrobial resistance genes identified related and unrelated isolates in both households. Our study shows that within-host bacterial diversity is present in S. pseudintermedius, demonstrating that multiple isolates from each host should preferably be sequenced to study transmission dynamics

Defining the scope of the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet): a bottom-up and One Health approach

Background Building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) was proposed to strengthen the European One Health antimicrobial resistance (AMR) surveillance approach. Objectives To define the combinations of animal species/production types/age categories/bacterial species/specimens/antimicrobials to be monitored in EARS-Vet. Methods The EARS-Vet scope was defined by consensus between 26 European experts. Decisions were guided by a survey of the combinations that are relevant and feasible to monitor in diseased animals in 13 European countries (bottom-up approach). Experts also considered the One Health approach and the need for EARS-Vet to complement existing European AMR monitoring systems coordinated by the ECDC and the European Food Safety Authority (EFSA). Results EARS-Vet plans to monitor AMR in six animal species [cattle, swine, chickens (broilers and laying hens), turkeys, cats and dogs], for 11 bacterial species (Escherichia coli, Klebsiella pneumoniae, Mannheimia haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus hyicus, Streptococcus uberis, Streptococcus dysgalactiae and Streptococcus suis). Relevant antimicrobials for their treatment were selected (e.g. tetracyclines) and complemented with antimicrobials of more specific public health interest (e.g. carbapenems). Molecular data detecting the presence of ESBLs, AmpC cephalosporinases and methicillin resistance shall be collected too. Conclusions A preliminary EARS-Vet scope was defined, with the potential to fill important AMR monitoring gaps in the animal sector in Europe. It should be reviewed and expanded as the epidemiology of AMR changes, more countries participate and national monitoring capacities improve.Peer reviewe