The effects of antibiotic use on the dynamics of the microbiome and resistome in pigs.

Antibiotics are widely used in pig farming across the world which has led to concerns about the potential impact on human health through the selection of antibiotic resistant pathogenic bacteria. This worry has resulted in the development of a production scheme known as pigs Raised Without Antibiotics (RWA), in which pigs are produced in commercial farms, but are ear-tagged as RWA until slaughter unless they receive treatment, thus allowing the farmer to sell the pigs either as premium priced RWA or as conventional meat. Development of antibiotic resistance in pig farming has been studied in national surveys of antibiotic usage and resistance, as well as in experimental studies of groups of pigs, but not in individual pigs followed longitudinally in a commercial pig farm. In this study, a cohort of RWA designated pigs were sampled at 10 time points from birth until slaughter along with pen-mates treated with antibiotics at the same farm. From these samples, the microbiome, determined using 16S sequencing, and the resistome, as determined using qPCR for 82 resistance genes, was investigated, allowing us to examine the difference between RWA pigs and antibiotic treated pigs. We furthermore included 176 additional pigs from six different RWA farms which were sampled at the slaughterhouse as an endpoint to substantiate the cohort as well as for evaluation of intra-farm variability. The results showed a clear effect of age in both the microbiome and resistome composition from early life up until slaughter. As a function of antibiotic treatment, however, we observed a small but significant divergence between treated and untreated animals in their microbiome composition immediately following treatment, which disappeared before 8 weeks of age. The effect on the resistome was evident and an effect of treatment could still be detected at week 8. In animals sampled at the slaughterhouse, we observed no difference in the microbiome or the resistome as a result of treatment status but did see a strong effect of farm origin. Network analysis of co-occurrence of microbiome and resistome data suggested that some resistance genes may be transferred through mobile genetic elements, so we used Hi-C metagenomics on a subset of samples to investigate this. We conclude that antibiotic treatment has a differential effect on the microbiome vs. the resistome and that although resistance gene load is increased by antibiotic treatment load, this effect disappears before slaughter. More studies are needed to elucidate the optimal way to rear pigs without antibiotics.

Pilot testing the EARS-Vet surveillance network for antibiotic resistance in bacterial pathogens from animals in the EU/EEA.

Introduction: As part of the EU Joint Action on Antimicrobial Resistance (AMR) and Healthcare-Associated Infections, an initiative has been launched to build the European AMR Surveillance network in veterinary medicine (EARS-Vet). So far, activities included mapping national systems for AMR surveillance in animal bacterial pathogens, and defining the EARS-Vet objectives, scope, and standards. Drawing on these milestones, this study aimed to pilot test EARS-Vet surveillance, namely to (i) assess available data, (ii) perform cross-country analyses, and (iii) identify potential challenges and develop recommendations to improve future data collection and analysis. Methods: Eleven partners from nine EU/EEA countries participated and shared available data for the period 2016-2020, representing a total of 140,110 bacterial isolates and 1,302,389 entries (isolate-antibiotic agent combinations). Results: Collected data were highly diverse and fragmented. Using a standardized approach and interpretation with epidemiological cut-offs, we were able to jointly analyze AMR trends of 53 combinations of animal host-bacteria-antibiotic categories of interest to EARS-Vet. This work demonstrated substantial variations of resistance levels, both among and within countries (e.g., between animal host species). Discussion: Key issues at this stage include the lack of harmonization of antimicrobial susceptibility testing methods used in European surveillance systems and veterinary diagnostic laboratories, the absence of interpretation criteria for many bacteria-antibiotic combinations of interest, and the lack of data from a lot of EU/EEA countries where little or even surveillance currently exists. Still, this pilot study provides a proof-of-concept of what EARS-Vet can achieve. Results form an important basis to shape future systematic data collection and analysis.

Survival of livestock-associated methicillin-resistant Staphylococcus aureus CC398 on different surface materials.

BACKGROUND: Zoonotic livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is widely spread in pig herds in many countries. However, the knowledge regarding the survival of LA-MRSA in the pig farm environment is currently limited. The aim of this study was to assess the survival of LA-MRSA on different surface materials found in the farm environment. The study investigated the survival of two different LA-MRSA strains belonging to the clonal complex (CC) 398 on four different surfaces: stainless steel, polypropylene plastic, K30 concrete and commercial concrete disk coupons. The survival of the bacteria over time was determined by the viable count method and, where possible, fitting a model to the observed data by using nonlinear least squares method to calculate the half-life ([Formula: see text]) for different strain and material combinations. RESULTS: The study showed that the half-life of the bacteria was longer on polypropylene plastic ([Formula: see text]=11.08-15.78 days) than on stainless steel ([Formula: see text]=2.45-7.83 days). On these materials, both LA-MRSA strains survived through the 14 week observation period. The bacterial decay was fastest on the concrete surfaces, where LA-MRSA became undetectable after 3-9 weeks. CONCLUSIONS: The survival of LA-MRSA in the pig farm environment may be affected by different surface materials. A more frequent sampling protocol (< 7 days) is needed to determine the half-life on concrete surfaces.

Intranasal delivery of a synthetic Entamoeba histolytica vaccine containing adjuvant (LecA + GLA-3 M-052 liposomes): In vitro characterization.

Amebiasis, a disease caused by the parasite Entamoeba histolytica, is estimated to cause millions of infections and at least 55,000 deaths globally each year. With no vaccine currently available, there is an urgent need for an accessible means of stimulating protective mucosal immunity. The objective of this study was to characterize the nasal spray of a novel amebiasis vaccine candidate from a syringe-based liquid atomization device, the Teleflex MAD Nasal™, in both adult and infant nasal airways. Human ergonomic testing was completed to determine realistic actuation parameters. Spray pattern, plume geometry, and droplet size distribution were measured to evaluate reproducibility of free plume characteristics. The Alberta Idealized Nasal Inlet (AINI) and three realistic infant nasal airways were used to determine the in vitro deposition profile in adult and infant airways, respectively. Collectively, in vitro results demonstrated the feasibility of delivering the vaccine candidate to target sites within the nasal airways. Penetration through the nasal airways that could lead to deposition in the lungs was below the limit of quantification for both adult and infant geometries, indicating a low likelihood of adverse events due to lung exposure. These results support continued investigation of intranasal delivery of the synthetic Entamoeba histolytica vaccine.

Association between Antibiotic Consumption and Resistance in Mink Production.

Antibiotic consumption is considered to be a main driver of antibiotic resistant bacteria. Mink breeding follows a distinctive seasonal reproduction cycle, and all of the mink produced in the northern hemisphere are bred, born, and pelted around the same time of year. Some of the diseases are age-related, which is reflected in the seasonal variation of antibiotic consumption. The seasonality makes mink a good model for the investigation of the association between antibiotic consumption and resistance. The objectives of this study were (1) to monitor the farm level of antibiotic resistance during one production cycle and (2) to assess the potential associations between antibiotic consumption and resistance. Twenty-four farms were included in this study (Denmark n = 20, Iceland n = 2, and The Netherlands n = 2), following a cohort of animals born in 2018. Staphylococcus delphini and Escherichia coli were isolated from samples of the carcasses and faeces and were collected randomly. The isolates were susceptibility tested and subsequently divided into the sensitive wildtype (WT) and the resistant non-wildtype (NWT) populations. The antibiotic consumption relative to the sampling periods was assessed as having a short-term or a long-term impact, i.e., in two explanatory factors. For both S. delphini and E. coli, a large between-farm variation of NWT profiles was detected. In the final multivariable, generalized linear mixed models, significant associations between NWT isolates and the consumption of specific antibiotics were found: the short-term use of tetracyclines in the growth period was associated with the occurrence of tetracycline NWT E. coli in the growth period (OR: 11.94 [1.78; 89.28]), and the long-term use of macrolide and tetracyclines was associated with the occurrence of erythromycin NWT S. delphini in the weaning period (OR: 18.2 [2.26; 321.36]) and tetracycline NWT S. delphini in the growth period (OR: 8.2 [1.27; 63.31]), respectively. Farms with zero consumption in the study years prior to sampling also had a substantial proportion of NWT isolates, indicating that NWT isolates are persistent and/or widely spread in the environment. Generally, a high occurrence of tetracycline NWTs was observed. NWT isolates with resistance against the most commonly used antibiotics were found on all the farms, stressing the need for routine surveillance and the prudent use of antibiotics. The results offer a preview of the complex relationship between consumption and resistance, demonstrating some significant associations between use and resistance. Moreover, antibiotic-resistant bacteria are present even on farms with no antibiotic consumption over extended periods, and theoretical explanations supported by the data are offered.

Review and Analysis of National Monitoring Systems for Antimicrobial Resistance in Animal Bacterial Pathogens in Europe: A Basis for the Development of the European Antimicrobial Resistance Surveillance Network in Veterinary Medicine (EARS-Vet).

The monitoring of antimicrobial resistance (AMR) in bacterial pathogens of animals is not currently coordinated at European level. To fill this gap, experts of the European Union Joint Action on Antimicrobial Resistance and Healthcare Associated Infections (EU-JAMRAI) recommended building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet). In this study, we (i) identified national monitoring systems for AMR in bacterial pathogens of animals (both companion and food-producing) among 27 countries affiliated to EU-JAMRAI, (ii) described their structures and operations, and (iii) analyzed their respective strengths, weaknesses, opportunities and threats (SWOT). Twelve countries reported having at least one national monitoring system in place, representing an opportunity to launch EARS-Vet, but highlighting important gaps in AMR data generation in Europe. In total, 15 national monitoring systems from 11 countries were described and analyzed. They displayed diverse structures and operations, but most of them shared common weaknesses (e.g., data management and representativeness) and common threats (e.g., economic vulnerability and data access), which could be addressed collectively under EARS-Vet. This work generated useful information to countries planning to build or improve their system, by learning from others' experience. It also enabled to advance on a pragmatic harmonization strategy: EARS-Vet shall follow the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards, collect quantitative data and interpret AMR data using epidemiological cut-off values.

Microbiological Diagnoses on Clinical Mastitis-Comparison between Diagnoses Made in Veterinary Clinics versus in Laboratory Applying MALDI-TOF MS.

The present study compares the diagnoses on clinical bovine mastitis made in veterinary clinics using conventional diagnostic methods with diagnoses on the same samples made by a veterinary reference laboratory using MALDI-TOF MS as diagnostics. The study enables targeted and evidence-based consulting on prudent mastitis diagnostics and related antibiotic usage. In total, 492 samples from clinical mastitis were included. When applying MALDI-TOF MS as gold standard, only 90 out of 492 diagnoses made in veterinary clinics, equal to 18%, were correct. Four main findings were important: (1) the veterinary clinics overlooked contamination in mastitis samples; (2) the veterinary clinics only assigned 2 fully correct diagnoses out of 119 samples with mixed growth cultures; (3) the veterinary clinics made close to half of their diagnoses on pure culture erroneously; (4) the veterinary clinics applied a limited number of the relevant pathogen identifications on pure culture samples. Altogether, the present study shows that a large part of Danish clinical mastitis cases are misdiagnosed. Lack of correct diagnoses and diagnostic quality control may lead to the choice of wrong treatment and thus hamper prudent use of antibiotics. Hence, the present study warns a risk of overuse of antibiotics in Denmark. Consequently, the present study calls for training of veterinary clinics in diagnostics of mastitis pathogens and national guidelines on quality assurance of mastitis diagnostics.

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.

Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity.

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15-0.6 µM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 µM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 µg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

Microbial Aetiology, Antibiotic Susceptibility and Pathogen-Specific Risk Factors for Udder Pathogens from Clinical Mastitis in Dairy Cows.

Mastitis is one of the most important infectious diseases and one of the diseases that causes the greatest use of antibiotics in dairy cows. Therefore, updated information on the bacteria that cause mastitis and their antibiotic susceptibility properties is important. Here, for the first time in over 10 years, we updated the bacterial findings in clinical mastitis in Swedish dairy cows together with their antibiotic resistance patterns and risk factors for each bacterial species. During the period 2013-2018, samples from clinical mastitis were collected, together with information on the cows and herds of origin. The samples were cultured, and a total of 664 recovered bacterial isolates were subjected to susceptibility testing. Staphylococcus aureus (S. aureus) was the most common pathogen and accounted for 27.8% of diagnoses, followed by Streptococcus dysgalactiae (S. dysgalactiae) (15.8%), Escherichia coli (E. coli) (15.1%), Streptococcus uberis (S. uberis) (11.4%), Trueperella pyogenes (T. pyogenes) (7.7%), non-aureus staphylococci (NAS) (2.8%), Klebsiella spp. (2.7%), Enterococcus spp. (1.3%), and Streptococcus agalactiae (S. agalactiae) (1.2%). Various other bacteria accounted for 2.6%. Staphylococci were, in general, susceptible to most antibiotics, but 2.6% of S. aureus and 30.4% of NAS were resistant to penicillin. No methicillin-resistant staphylococci were found. All S. agalactiae were susceptible to penicillin. Bimodal and trimodal MIC distributions for penicillin in S. dysgalactiae and S. uberis, respectively, indicate acquired reduced susceptibility in some isolates. The mostly unimodal MIC distributions of T. pyogenes indicate that acquired resistance does usually not occur in this species. Among E. coli, 14.7% were resistant to at least one antibiotic, most often ampicillin (8.7%), streptomycin (7.8%), or sulphamethoxazole (6.9%). Klebsiella spp. had low resistance to tetracycline (9.1%) but is considered intrinsically resistant to ampicillin. Pathogen-specific risk factors were investigated using multivariable models. Staphylococcus aureus, S. dysgalactiae, and T. pyogenes were more common, while E. coli was less common in quarters with more than one pathogen. S. aureus and T. pyogenes were mostly seen in early lactation, while E. coli was more common in peak to mid lactation and S. dysgalactiae in early to peak lactation. Trueperella pyogenes and Klebsiella spp. were associated with a previous case of clinical mastitis in the current lactation. Staphylococcus aureus was associated with tie stalls and T. pyogenes with loose housing. All pathogens except E. coli and S. dysgalactiae had a seasonal distribution. In conclusion, the aetiological agents for clinical bovine mastitis have remained relatively stable over the last 10-15 years, S. aureus, S. dysgalactiae, E. coli and S. uberis being the most important. Resistance to penicillin among Gram-positive agents was low, and in general, antibiotic resistance to other compounds was low among both Gram-positive and Gram-negative agents.

First finding of Streptococcus phocae infections in mink (Neovison vison).

Streptococcus phocae infection has been described in salmon, sea otters, and several families of pinnipeds. The pathology of the infected animals has mainly been located in the respiratory tract and reproductive system, and with indications of septicemia. In this study, we report the finding of S. phocae in diagnostic material from three unrelated cases of farmed mink. Since S. phocae initially has been described in pinnipeds, two isolates from wild harbor seals were included. All isolates originated from Denmark. To our knowledge, this is the first report of S. phocae infection in mink. The animals (three mink, two seals) were necropsied, and samples were collected for bacteriology, virology, and histopathology. Additionally, the S. phocae isolates were whole genome sequenced and compared to sequences of previously reported isolates from other host species. S. phocae was isolated from the lungs of one mink and one seal with bacteremia, and from one seal with pneumonia. The two remaining mink had dermal infections on the paws and S. phocae was isolated from the lesions. The analysis of the sequence data showed that the three mink isolates and one seal isolate were closely related. Further investigation is needed to conclude whether S. phocae is establishing as commensal in farmed mink and to uncover the infection related pathology in mink. Streptococcus phocae has been described as an emerging pathogen in other species, therefore future awareness and surveillance of this pathogen is crucial.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Occurrence and Survival of Livestock-Associated MRSA in Pig Manure and on Agriculture Fields.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is widespread in European pig production, and an increasing number of humans attract infections with this bacterium. Although most infections occur in humans with direct livestock contact, an increasing number of infections occur in humans without any established livestock contact. There have been speculations that at least some of these infections may be connected to the exposure of liquid pig manure for example spread as fertilizers. The present study therefore undertook to measure the presence of LA-MRSA in liquid pig manure and on the surface of soils fertilized with liquid manure and investigate the survival of the bacterium in manure. The results showed that LA-MRSA could be detected in 7 out of 20 liquid manure samples and in 12 out of 186 soil samples. However, the bacterium was not more frequently detected in samples collected after compared to before the spreading of liquid manure on the fields, thus suggesting that other sources of LA-MRSA on agriculture fields likely exist. The decimation time in liquid manure was >32 days at 5 °C in vitro but decreased with increasing temperature. Based on these results, liquid manure does not appear to be an important risk factor for human exposure to LA-MRSA.

Building the European Antimicrobial Resistance Surveillance network in veterinary medicine (EARS-Vet).

Antimicrobial resistance (AMR) should be tackled through a One Health approach, as stated in the World Health Organization Global Action Plan on AMR. We describe the landscape of AMR surveillance in the European Union/European Economic Area (EU/EEA) and underline a gap regarding veterinary medicine. Current AMR surveillance efforts are of limited help to veterinary practitioners and policymakers seeking to improve antimicrobial stewardship in animal health. We propose to establish the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) to report on the AMR situation, follow AMR trends and detect emerging AMR in selected bacterial pathogens of animals. This information could be useful to advise policymakers, explore efficacy of interventions, support antimicrobial stewardship initiatives, (re-)evaluate marketing authorisations of antimicrobials, generate epidemiological cut-off values, assess risk of zoonotic AMR transmission and evaluate the burden of AMR in animal health. EARS-Vet could be integrated with other AMR monitoring systems in the animal and medical sectors to ensure a One Health approach. Herein, we present a strategy to establish EARS-Vet as a network of national surveillance systems and highlight challenges of data harmonisation and bias. Strong political commitment at national and EU/EEA levels is required for the success of EARS-Vet.

Employing MIC Data for Mink Pathogens to Propose Tentative Epidemiological Cut-Off Values: A Step Toward Rationalizing Antimicrobial Use in Mink.

Optimizing antimicrobial dosage regimens and development of breakpoints for antimicrobial susceptibility testing are important prerequisites for rational antimicrobial use. The objectives of the study were (1) to produce MIC data for four mink pathogens and (2) to employ these MIC data to support the development of tentative epidemiological cut-off values (TECOFFs), which may be used for future development of mink-specific antimicrobial dosages and breakpoints. Broth microdilution was used to establish MIC distributions for 322 mink bacterial isolates of clinical origin from six European mink-producing countries. The included species were E. coli (n = 162), S. delphini (n = 63), S. canis (n = 42), and P. aeruginosa (n = 55). Sixty-four E. coli isolates and 34 S. delphini isolates were whole-genome sequenced and analyzed for antimicrobial resistance genes. No EUCAST MIC data are available on S. delphini and S. canis, hence tentative ECOFFs were suggested for the majority of the tested antimicrobials. For E. coli and P. aeruginosa, the wildtype distributions were in accordance with EUCAST data. Overall, the genotypes of the sequenced isolates were in concordance with the phenotypes. These data constitute an important piece in the puzzle of developing antimicrobial dosages and clinical breakpoints for mink. Until pharmacokinetic and clinical data become available, the (tentative) ECOFFs can be used for monitoring resistance development and as surrogates for clinical breakpoints.

F4- and F18-Positive Enterotoxigenic Escherichia coli Isolates from Diarrhea of Postweaning Pigs: Genomic Characterization.

This study aimed to characterize in silico enterotoxigenic Escherichia coli F4- and F18-positive isolates (n = 90) causing swine postweaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance, prophage content, and metal tolerance rates. F4 strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 sequence types (STs). F18 strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23, and ST42. The highest rates of antimicrobial resistance were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and ampicillin. No resistance was found toward ciprofloxacin, cefotaxime, ceftiofur, and colistin. Genes conferring tolerance to copper (showing the highest diversity), cadmium, silver, and zinc were predicted in all genomes. Enterotoxin genes (ltcA, 100% F4, 62% F18; astA, 100% F4, 38.1% F18; sta, 18.8% F4, 38.1% F18; stb, 100% F4, 76.2% F18) and fimbria-encoding genes typed as F4ac and F18ac were detected in all strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. A highly diverse variety of plasmids was found; IncFII, IncFIB, and IncFIC were prevalent among F4 isolates, while IncI1 and IncX1 were dominant among F18 strains. Interestingly, F4 isolates from the early 1990s belonged to the same clonal group detected for most of the F4 strains from 2018 to 2019 (ONT:H10-A-ST100-CH27-0). The small number of single-nucleotide polymorphism differences between the oldest and recent F4 ST100 isolates suggests a relatively stable genome. Overall, the isolates analyzed in this study showed remarkably different genetic traits depending on the fimbria type.IMPORTANCE Diarrhea in the postweaning period due to enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by zinc oxide administration (to be discontinued by 2022). In addition, a breeding program has been implemented that aims to reduce the prevalence of this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among diarrhea-associated isolates by whole-genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC ST100 isolates from China, the United States, and Spain. High rates of AMR and high diversity of virulence genes were detected. The characterization of diarrhea-related ETEC is important for understanding the disease epidemiology and pathogenesis and for implementation of new strategies aiming to reduce the impact of the disease in pig production.

Evidence for action: a One Health learning platform on interventions to tackle antimicrobial resistance.

Improving evidence for action is crucial to tackle antimicrobial resistance. The number of interventions for antimicrobial resistance is increasing but current research has major limitations in terms of efforts, methods, scope, quality, and reporting. Moving the agenda forwards requires an improved understanding of the diversity of interventions, their feasibility and cost-benefit, the implementation factors that shape and underpin their effectiveness, and the ways in which individual interventions might interact synergistically or antagonistically to influence actions against antimicrobial resistance in different contexts. Within the efforts to strengthen the global governance of antimicrobial resistance, we advocate for the creation of an international One Health platform for online learning. The platform will synthesise the evidence for actions on antimicrobial resistance into a fully accessible database; generate new scientific insights into the design, implementation, evaluation, and reporting of the broad range of interventions relevant to addressing antimicrobial resistance; and ultimately contribute to the goal of building societal resilience to this central challenge of the 21st century.

Spread of LA-MRSA CC398 in Danish mink (Neovison vison) and mink farm workers.

More than 55 million mink skins were produced globally in 2017. As a consequence, a large number of people are employed in mink production worldwide. In Denmark, farmed mink were found to constitute a reservoir of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 and 6000 mink farm workers in Denmark are potentially exposed to LA-MRSA CC398. The study aim was to elucidate the source of LA-MRSA CC398 in mink farms and to investigate possible transmission to humans. In total, 161 LA-MRSA CC398 isolates from mink (n = 65), mink feed (n = 16) and humans (n = 80) with reported contact to mink, were whole-genome sequenced and compared to 183 LA-MRSA CC398 isolates from Danish pigs and an international collection of 89 S. aureus CC398 isolates. Most of the mink-associated isolates clustered within the predominant LA-MRSA CC398 lineages circulating in the Danish pig production, supporting that pigs are a source of LA-MRSA CC398 in mink feed, mink, and mink farmers.

Sequence Analysis of Plasmids in Vibrio anguillarum from Different Fish and Locations.

The genetic diversity of Vibrio anguillarum pJM1-like plasmids was investigated. Plasmids were isolated from 18 V. anguillarum serovar O1 strains collected from different geographic locations and fish species. The plasmids were sequenced and compared with the complete sequence of the published virulence plasmid pJM1. All 18 strains contained pJM1-like plasmids with approximately 65 kbp and all plasmids encoded the virulence genes responsible for the anguibactin iron sequestering system. The plasmids were highly conserved but minor differences were observed in some genes. A single nucleotide polymorphisms (SNPs) analysis showed 0-11 nucleotide variations between each of the 18 plasmids and the pJM1 plasmid. Compared with the sequence of pJM1, nonsynonymous SNPs were identified in fatC, angR, angL, pJM1_p19, and angE. In particular, a mutation found in 15 out of 18 sequenced plasmids in angR has previously been linked to hyperproduction of anguibactin and was found in all the European isolates. However, overall the pJM1-like plasmids isolated from V. anguillarum serovar O1 exhibited a high degree of conservation regardless of their geographical origin or fish species.

A link between the newly described colistin resistance gene mcr-9 and clinical Enterobacteriaceae isolates carrying blaSHV-12 from horses in Sweden.

OBJECTIVES: The aim of this study was to investigate the occurrence of the newly described transferable colistin resistance gene mcr-9 in extended-spectrum ß-lactamase (ESBL)-producing clinical Enterobacteriaceae isolates from horses in Sweden. METHODS: A total of 56 whole-genome sequenced ESBL-producing Enterobacteriaceae isolates from horses were subjected to in silico detection of antimicrobial resistance genes and identification of plasmid replicons types. The colistin minimum inhibitory concentration (MIC) for mcr-positive isolates was determined by broth microdilution. Relatedness between Enterobacteriaceae carrying mcr genes was determined by multilocus sequence typing (MLST) and core genome MLST. RESULTS: Thirty ESBL-producing Enterobacteriaceae isolates from horses were positive for the colistin resistance gene mcr-9. These isolates included Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca and Citrobacter freundii and belonged to diverse MLST sequence types within each species. Two of the mcr-9-containing isolates originated from the same horse. All mcr-9-positive isolates had colistin MICs below or equal to the EUCAST epidemiological cut-off value of 2 mg/L and were negative for the two potential regulatory genes qseB-like and qseC-like for mcr-9. Except for one isolate carrying only blaTEM-1B, all of the isolates carried blaSHV-12 and blaTEM-1B, and were all considered multidrug-resistant as they harboured genes encoding resistance to aminoglycosides, chloramphenicol, fosfomycin, macrolides, quinolones, sulfonamides, trimethoprim and tetracyclines. Plasmid replicon types IncHI2 and IncHI2A were detected in all mcr-9-positive isolates. CONCLUSION: The occurrence of mcr-9 was common among clinical ESBL-producing Enterobacteriaceae isolates from horses in Sweden and was linked to the ESBL-encoding gene blaSHV-12 and plasmid replicon types IncHI2 and IncHI2A.