Gut microbiome and resistome characterization of pigs treated with commonly used post-weaning diarrhea treatments.

BACKGROUND: The global burden of antimicrobial resistance demands additional measures to ensure the sustainable and conscious use of antimicrobials. For the swine industry, the post-weaning period is critical and for many years, antimicrobials have been the most effective strategy to control and treat post-weaning related infections. Among them, post-weaning diarrhea causes vast economic losses, as it severely compromises piglets' health and growth performance. In this study, 210 piglets were transferred from a farm with recurrent cases of post-weaning diarrhea to an experimental farm and divided into six different treatment groups to determine the effect of the different treatments on the growth performance and survival, the microbiome, and the resistome in a cross-sectional and longitudinal study. The different treatments included antimicrobials trimethoprim/sulfamethoxazole, colistin, and gentamicin, an oral commercial vaccine, a control with water acidification, and an untreated control. An extra group remained at the farm of origin following the implemented amoxicillin routine treatment. A total of 280 fecal samples from pigs at four different sampling times were selected for metagenomics: before weaning-treatment at the farm of origin, and three days, two weeks, and four weeks post-treatment. RESULTS: The control group with water acidification showed a reduced death risk in the survival analyses and non-significant differences in average daily weight gain in comparison to the antibiotic-treated groups. However, the growth-promoting effect among antibiotic-treated groups was demonstrated when comparing against the untreated control group at the experimental farm. After four weeks of treatment, diversity indexes revealed significantly decreased diversity for the untreated control and the group that remained at the farm of origin treated with amoxicillin. For this last group, impaired microbial diversity could be related to the continuous amoxicillin treatment carried out at the farm. Analysis of the resistome showed that both gentamicin and amoxicillin treatments significantly contributed to the emergence of resistance, while trimethoprim/sulphonamide and colistin did not, suggesting that different treatments contribute differently to the emergence of resistance. CONCLUSIONS: Overall, this shotgun longitudinal metagenomics analysis demonstrates that non-antibiotic alternatives, such as water acidification, can contribute to reducing the emergence of antimicrobial resistance without compromising pig growth performance and gut microbiome.

The Susceptibility Trends of Respiratory and Enteric Porcine Pathogens to Last-Resource Antimicrobials.

Monitoring the antimicrobial susceptibility of last-resource antimicrobials for veterinary pathogens is urgently needed from a one-health perspective. The objective of this study was to analyze the antimicrobial susceptibility trends of Spanish porcine bacteria to quinolones, cephalosporins, and polymyxins. Isolates of Actinobacillus pleuropneumoniae, Pasteurella multocida, and Escherichia coli were isolated from sick pigs from 2019 to 2022. An antimicrobial susceptibility test was determined based on the minimal inhibitory concentration (MIC) following an internationally accepted methodology. The MIC categorization was based on distributing the range of MIC values in four categories, with category one being the most susceptible (lowest MIC value) and category four the least susceptible (highest MIC value). Moreover, clinical susceptibility (susceptible/non-susceptible) was also determined according to the CLSI and EUCAST clinical breakpoints. A logistic and multinomial logistic regression model was used to analyze the susceptibility data for dichotomized and categorized MIC data, respectively, for any pair of antimicrobial/microorganism. In general terms, the antimicrobial susceptibility of pig bacteria to these antimicrobials remained stable or increased in the last four years in Spain. In the case of A. pleuropneumoniae and quinolones, a significant temporal trend was observed where isolates from 2020 had significantly increased odds of being more susceptible than isolates from 2019. In the case of E. coli and polymyxins, a significant temporal trend was observed where isolates from 2020 and 2021 had significantly increased odds of being more susceptible than isolates from 2019 and 2020, respectively. Finally, significant odds of being less susceptible were only observed for cephalosporins and E. coli for 2020 versus 2019, stagnating for the rest of study period. These results provide sound data on critically important antimicrobials in swine medicine.

HEALTH ASSESSMENT OF CAPTIVE BEARDED VULTURES (GYPAETUS BARBATUS) IN CATALONIA (SPAIN).

Bearded vulture (Gypaetus barbatus) populations are declining worldwide primarily due to anthropogenic factors. A captive breeding program has been established in Spain, a country with one of the largest free-living populations in Europe, to further enhance the conservation efforts of this emblematic species. However, captive vulture populations can be exposed to different risks through food, such as drug residues and antimicrobial-resistant (AMR) bacteria. Health surveillance of species involved in captive breeding programs is important to face introduction of healthy animals in situ and to obtain baseline clinical data. The objective of this study was to assess the general health status of bearded vultures held in captivity in Catalonia (northeastern Spain) by carrying out hematologic, biochemical, toxicologic, and bacteriologic analyses. A total of 16 bearded vultures were sampled; the data obtained from one vulture, with a chronic tibiotarsal fracture, were excluded from the statistical analysis. Hematologic and biochemical parameters of the bearded vultures were mostly within the range of standard values as stated in previous studies. Basal feather and serum corticosterone levels were analyzed and described for the first time in this species. A total of 15 Escherichia coli isolates were obtained that were resistant to fluoroquinolones (80%), tetracycline (60%), trimethoprim and ampicillin (40%), sulfamethoxazole (33%), and colistin (20%), with 40% of them being multidrug resistant. Three of 15 isolates were carriers of the mcr-1 gene. Only the injured bird previously treated with enrofloxacin was positive for fluoroquinolone residues. Periodic monitoring for the presence of AMR bacteria would be recommended in captive breeding programs as a preventive action to establish future therapies.

Diverse Populations of Staphylococcus pseudintermedius Colonize the Skin of Healthy Dogs.

Staphylococcus pseudintermedius is a commensal bacterium of the canine skin but is also a key opportunistic pathogen that is responsible for most cases of pyoderma in dogs. The current paradigm indicates that infection arises when predisposing factors alter the healthy skin barrier. Despite their importance, the characteristics of the S. pseudintermedius populations colonizing the skin of healthy dogs are yet largely unknown. Here, we retrieved 67 complete circular genomes and 19 associated plasmids from S. pseudintermedius isolated from the skin of 9 healthy dogs via long-reads Nanopore sequencing. Within the S. pseudintermedius populations isolated from healthy skin, multilocus sequence typing (MLST) detected 10 different STs, distributed mainly by the host. 39% of the 18 representative genomes isolated herein were methicillin-resistant S. pseudintermedius (MRSP), and they showed, on average, a higher number of antibiotic resistance genes and prophages than did the methicillin-sensitive (MSSP). In summary, our results revealed that the S. pseudintermedius populations inhabiting the skin of healthy dogs are relatively diverse and heterogeneous in terms of MLST and methicillin resistance. In this study, all of the 67 commensal S. pseudintermedius populations that were isolated from healthy dogs contained antibiotic resistance genes, indicating the extent and severity of the problem of antimicrobial resistance in staphylococci with zoonotic potential. IMPORTANCE Staphylococcus pseudintermedius is a commensal canine bacterium that can become an opportunistic pathogen and is responsible for most cases of canine pyoderma. It can also cause occasional zoonotic infections. Infections caused by antibiotic-resistant Staphylococcus are a global concern. Skin commensal Staphylococcus pseudintermedius is understudied. To provide insight into the commensal strains circulating in healthy dogs, we performed whole-genome sequencing of 67 S. pseudintermedius isolates from different skin sites in 9 healthy dogs. Through the bioinformatic analysis of these genomes, we identified a genomic diversity that is more complete than those afforded by traditional molecular typing strategies. We identified 7 new STs. All of the isolates harbored genes associated with antibiotic resistance, and 39% of the representative genomes were methicillin-resistant. Our data provide critical insights for future skin infection control and antibiotic surveillance within veterinary medicine.

The fecal bacterial microbiome of the Kuhl's pipistrelle bat (Pipistrellus kuhlii) reflects landscape anthropogenic pressure.

BACKGROUND: Anthropogenic disturbance has the potential to negatively affect wildlife health by altering food availability and diet composition, increasing the exposure to agrochemicals, and intensifying the contact with humans, domestic animals, and their pathogens. However, the impact of these factors on the fecal microbiome composition of wildlife hosts and its link to host health modulation remains barely explored. Here we investigated the composition of the fecal bacterial microbiome of the insectivorous bat Kuhl's pipistrelle (Pipistrellus kuhlii) dwelling in four environmental contexts with different levels of anthropogenic pressure. We analyzed their microbiome composition, structure and diversity through full-length 16S rRNA metabarcoding using the nanopore long-read sequencer MinION™. We hypothesized that the bacterial community structure of fecal samples would vary across the different scenarios, showing a decreased diversity and richness in samples from disturbed ecosystems. RESULTS: The fecal microbiomes of 31 bats from 4 scenarios were sequenced. A total of 4,829,302 reads were obtained with a taxonomic assignment percentage of 99.9% at genus level. Most abundant genera across all scenarios were Enterococcus, Escherichia/Shigella, Bacillus and Enterobacter. Alpha diversity varied significantly between the four scenarios (p < 0.05), showing the lowest Shannon index in bats from urban and intensive agriculture landscapes, while the highest alpha diversity value was found in near pristine landscapes. Beta diversity obtained by Bray-Curtis distance showed weak statistical differentiation of bacterial taxonomic profiles among scenarios. Furthermore, core community analysis showed that 1,293 genera were shared among localities. Differential abundance analyses showed that the highest differentially abundant taxa were found in near pristine landscapes, with the exception of the family Alcaligenaceae, which was also overrepresented in urban and intensive agriculture landscapes. CONCLUSIONS: This study suggests that near pristine and undisturbed landscapes could promote a more resilient gut microbiome in wild populations of P. kuhlii. These results highlight the potential of the fecal microbiome as a non-invasive bioindicator to assess insectivorous bats' health and as a key element of landscape conservation strategies.

Concordance between Antimicrobial Resistance Phenotype and Genotype of Staphylococcus pseudintermedius from Healthy Dogs.

Staphylococcus pseudintermedius, a common commensal canine bacterium, is the main cause of skin infections in dogs and is a potential zoonotic pathogen. The emergence of methicillin-resistant S. pseudintermedius (MRSP) has compromised the treatment of infections caused by these bacteria. In this study, we compared the phenotypic results obtained by minimum inhibitory concentration (MICs) for 67 S. pseudintermedius isolates from the skin of nine healthy dogs versus the genotypic data obtained with Nanopore sequencing. A total of 17 antibiotic resistance genes (ARGs) were detected among the isolates. A good correlation between phenotype and genotype was observed for some antimicrobial classes, such as ciprofloxacin (fluoroquinolone), macrolides, or tetracycline. However, for oxacillin (beta-lactam) or aminoglycosides the correlation was low. Two antibiotic resistance genes were located on plasmids integrated in the chromosome, and a third one was in a circular plasmid. To our knowledge, this is the first study assessing the correlation between phenotype and genotype regarding antimicrobial resistance of S. pseudintermedius from healthy dogs using Nanopore sequencing technology.

European-wide antimicrobial resistance monitoring in commensal Escherichia coli isolated from healthy food animals between 2004 and 2018.

OBJECTIVES: To describe the susceptibility of Escherichia coli to medically important antibiotics, collected over four periods (2004-2006, 2008-2009, 2013-2014, 2017-2018), from food-producing animals at slaughter. METHODS: Intestinal contents from cattle, pigs and broilers were randomly sampled (5-6 countries/host; ≥4 abattoirs/country; one sample/animal/farm) for isolation of Escherichia coli; antimicrobial susceptibilities were centrally determined by CLSI agar dilution. Clinical breakpoints (CLSI) and epidemiological cut-off values (EUCAST) were applied for data interpretation. RESULTS: In total, 10 613 E. coli strains were recovered. In broilers, resistance percentages were the lowest (P ≤ 0.01) in the latest time period. A significant decrease in MDR over time was also observed for broilers and a tendency for a decrease for pigs. Resistance to meropenem and tigecycline was absent, and resistance to azithromycin was 0.2%-2.0%. Also, low resistance to third-generation cephalosporins (1.1%-7.4%) was detected in broilers. Resistance to colistin varied between 0.1%-4.8%. E. coli from broilers showed high resistance to ciprofloxacin (7.3%-23.3%), whereas for cattle and pigs this was 0.2%-2.5%. Low/moderate resistance to chloramphenicol (9.3%-21.3%) and gentamicin (0.9%-7.0%) was observed in pigs and broilers. The highest resistance was noted for ampicillin (32.7%-65.3%), tetracycline (41.3%-67.5%), trimethoprim (32.0%-35.7%) and trimethoprim/sulfamethoxazole (27.5%-49.7%) from pigs and broilers, with marked country differences. MDR peaked in pigs and broilers with 24 and 26 phenotypes, with 21.9%-26.2% and 18.7%-34.1% resistance, respectively. CONCLUSIONS: In this pan-EU survey antibiotic susceptibility of commensal E. coli varied largely between antibiotics, animal species and countries. Resistance to critically important antibiotics for human medicine was absent or low, except for ciprofloxacin in broilers and ampicillin in pigs and broilers.

Revealing Genomic Insights of the Unexplored Porcine Pathogen Actinobacillus pleuropneumoniae Using Whole Genome Sequencing.

Actinobacillus pleuropneumoniae (APP) is the causative agent of pleuropneumonia in pigs, one of the most relevant bacterial respiratory diseases in the swine industry. To date, 19 serotypes have been described based on capsular polysaccharide typing with significant virulence dissimilarities. In this study, 16 APP isolates from Spanish origin were selected to perform antimicrobial susceptibility tests and comparative genomic analysis using whole genome sequencing (WGS). To obtain a more comprehensive worldwide molecular epidemiologic analyses, all APP whole genome assemblies available at the National Center for Biotechnology Information (NCBI) at the time of the study were also included. An in-house in silico PCR approach enabled the correct serotyping of unserotyped or incorrectly serotyped isolates and allowed for the discrimination between serotypes 9 and 11. A pangenome analysis identified the presence or absence of gene clusters to be serotype specific, as well as virulence profile analyses targeting the apx operons. Antimicrobial resistance genes were correlated to the presence of specific plasmids. Altogether, this study provides new insights into the genetic variability within APP serotypes, correlates phenotypic tests with bioinformatic analyses and manifests the benefits of populated databases for a better assessment of diversity and variability of relatively unknown pathogens. Overall, genomic comparative analysis enhances the understanding of transmission and epidemiological patterns of this species and suggests vertical transmission of the pathogen, including the resistance genes, within the Spanish integrated systems. IMPORTANCE Pleuropneumonia is one of the most relevant respiratory infections in the swine industry. Despite Actinobacillus pleuropneumoniae (APP) being one of the most important pathogens in the pig production, this is the first comparative study including all available whole genome sequencing data from NCBI. Moreover, this study also includes 16 APP isolates of Spanish origin with known epidemiological relationships through vertical integrated systems. Genomic comparisons provided a deeper understanding of molecular and epidemiological knowledge between different APP serotypes. Furthermore, determination of resistance and toxin profiles allowed correlation with the presence of mobile genetic elements and specific serotype, respectively.

Antimicrobial Susceptibility Testing of Porcine Bacterial Pathogens: Investigating the Prospect of Testing a Representative Drug for Each Antimicrobial Family.

Antimicrobial susceptibility testing is necessary to carry out antimicrobial stewardship but a limited number of drugs belonging to each antimicrobial family has to be tested for technical limitations and economic resources. In this study, we have determined the minimal inhibitory concentration, using microdilution following international standards (CLSI), for 490 Actinobacillus pleuropneumoniae, 285 Pasteurella multocida, 73 Bordetella bronchiseptica, 398 Streptococcus suis and 1571 Escherichia coli strains from clinical cases collected in Spain between 2018 and 2020. The antimicrobial susceptibility pattern was deciphered using a principal component analysis for each bacterium and a matrix correlation (high > 0.8, medium 0.5−0.8 and low < 0.5) was obtained for each pair of antimicrobials. No significant associations were observed between MIC patterns for different antimicrobial families, suggesting that co-selection mechanisms are not generally present in these porcine pathogens. However, a high correlation was observed between the fluroquinolones (marbofloxacin and enrofloxacin) for all mentioned pathogens and for ceftiofur and cefquinome for E. coli and S. suis. Moreover, a significant association was also observed for tetracyclines (doxycycline and oxytetracycline) and B. bronchiseptica and tildipirosin/tulathromycin for P. multocida. These results suggest that generally, a representative drug per antimicrobial class cannot be selected, however, for some drug−bug combinations, MIC values from one representative drug could be extrapolated to the whole antimicrobial family.

Whole-Genome Sequencing and De Novo Assembly of 67 Staphylococcus pseudintermedius Strains Isolated from the Skin of Healthy Dogs.

We have de novo assembled 67 Staphylococcus pseudintermedius genomes, with median values of 2.6 Mbp size and 99.43% completeness, 2,386 coding sequences, 19 complete rRNAs, 59 tRNAs, and 4 noncoding RNAs. We released 51 single-contig complete genomes and 16 genomes with a circular main contig using Nanopore sequencing.

Whole genome sequencing and de novo assembly of Staphylococcus pseudintermedius: a pangenome approach to unravelling pathogenesis of canine pyoderma.

BACKGROUND: Staphylococcus pseudintermedius is the main aetiological agent of canine pyoderma. Whole genome sequencing is the most comprehensive way of obtaining relevant genomic information about micro-organisms. HYPOTHESIS/OBJECTIVES: Oxford Nanopore technology enables quality sequencing and de novo assembly of the whole genome of S. pseudintermedius. Whole genome analysis of S. pseudintermedius may help to better understand the pathogenesis of canine pyodermas. METHODS AND MATERIALS: Twenty-two strains of S. pseudintermedius isolated from the skin of five healthy dogs and 33 strains isolated from skin of 33 dogs with pyoderma were analysed. DNA was extracted and sequenced using Oxford Nanopore MinION, a new technology that delivers longer reads in a hand-held device. The pangenome was analysed and visualised with Anvi'o 6.1. RESULTS: Nanopore technology allowed the sequencing and de novo assembly of the genomes of 55 S. pseudintermedius strains isolated from healthy dogs and from dogs with pyoderma. The average genome size of S. pseudintermedius was 2.62 Mbp, with 48% being core genome. Pyoderma isolates contained a higher number of antimicrobial resistance genes, yet the total number of virulence factors genes did not change between isolates from healthy dogs and from dogs with pyoderma. Genomes of meticillin-resistant S. pseudintermedius (MRSP) strains were larger than those of meticillin-susceptible (MSSP) strains (2.80 Mbp versus 2.59 Mbp), as a consequence of a greater presence of antimicrobial resistance genes, phages and prophages. CONCLUSIONS AND CLINICAL IMPORTANCE: This technique allows much more precise and easier characterisation of canine S. pseudintermedius populations and may lead to a better understanding of the pathogenesis of canine pyodermas.

Stochastic Assessment of the Economic Impact of Streptococcus suis-Associated Disease in German, Dutch and Spanish Swine Farms.

The economic assessment of animal diseases is essential for decision-making, including the allocation of resources for disease control. However, that assessment is usually hampered by the lack of reliable data on disease incidence, or treatment and control measures, and that is particularly true for swine production diseases, such as infections caused by Streptococcus suis. Therefore, we deployed a questionnaire survey of clinical swine veterinarians to obtain the input data needed for a stochastic model to calculate the costs caused by S. suis, which was implemented in three of the main swine producing countries in Europe: Germany, the Netherlands and Spain. S. suis-associated disease is endemic in those countries in all production phases, though nursery was the phase most severely impacted. In affected nursery units, between 3.3 and 4.0% of pigs had S. suis-associated disease and the mortalities ranged from 0.5 to 0.9%. In Germany, the average cost of S. suis per pig (summed across all production phases) was 1.30 euros (90% CI: 0.53-2.28), in the Netherlands 0.96 euros (90% CI: 0.27-1.54), and in Spain 0.60 euros (90% CI: 0.29-0.96). In Germany, that cost was essentially influenced by the expenditure in early metaphylaxis in nursery and in autogenous vaccines in sows and nursery pigs; in the Netherlands, by expenditure on autogenous vaccines in sows and nursery pigs; and in Spain, by the expenditures in early metaphylaxis and to a lesser extent by the mortality in nursery pigs. Therefore, the differences in costs between countries can be explained to a great extent by the measures to control S. suis implemented in each country. In Spain and in Germany, use of antimicrobials, predominantly beta-lactams, is still crucial for the control of the disease.

Prevalence of Salmonella in Free-Range Pigs: Risk Factors and Intestinal Microbiota Composition.

Extensive pig systems are gaining importance as quality production systems and as the standard for sustainable rural development and animal welfare. However, the effects of natural foods on Salmonella epidemiology remain unknown. Herein, we assessed the presence of Salmonella and the composition of the gut microbiota in pigs from both Salmonella-free and high Salmonella prevalence farms. In addition, risk factors associated with the presence of Salmonella were investigated. The pathogen was found in 32.2% of animals and 83.3% of farms, showing large differences in prevalence between farms. Most isolates were serovars Typhimurium monophasic (79.3%) and Bovismorbificans (10.3%), and exhibited a multi-drug resistance profile (58.6%). Risk factor analysis identified feed composition, type/variety of vegetation available, and silos' cleaning/disinfection as the main factors associated with Salmonella prevalence. Clear differences in the intestinal microbiota were found between Salmonella-positive and Salmonella-negative populations, showing the former with increasing Proteobacteria and decreasing Bacteroides populations. Butyrate and propionate producers including Clostridium, Turicibacter, Bacteroidaceae_uc, and Lactobacillus were more abundant in the Salmonella-negative group, whereas acetate producers like Sporobacter, Escherichia or Enterobacter were more abundant in the Salmonella-positive group. Overall, our results suggest that the presence of Salmonella in free-range pigs is directly related to the natural vegetation accessible, determining the composition of the intestinal microbiota.

Antimicrobial Resistance in Pasteurellaceae Isolates from Pyrenean Chamois (Rupicapra pyrenaica) and Domestic Sheep in an Alpine Ecosystem.

Antimicrobial resistance (AMR) has spread worldwide due to the inappropriate use of antimicrobial drugs in human and veterinary medicine, becoming a public health problem. However, little is known about its occurrence and maintenance in wild animals, and very few studies have been carried out in ecosystems subjected to low human pressure. In our study, nasal and lung swabs were collected from hunted Pyrenean chamois (Rupicapra pyrenaica), and nasal swabs from sympatric domestic sheep were also collected. The swabs were cultured in agar plates to obtain bacterial isolates from the Pasteurellaceae family. The presence of AMR was assessed in a total of 28 Pasteurellaceae isolates from 45 Pyrenean chamois, and 9 isolates from sympatric domestic sheep found in the National Hunting Reserve of Freser-Setcases (Northeastern Pyrenees, Spain). The isolates belonged to one of the following three species: Pasteurella multocida, Mannheimia haemolytica and Bibersteinia trehalosi. Some P. multocida and M. haemolytica isolates tested positive for AMR. The statistical analysis revealed no differences between the AMR levels from chamois and domestic sheep isolates. However, one P. multocida of chamois origin presented resistance to cephalosporins and fluoroquinolones, which are antibiotics of critical importance for human health. Further studies are required to elucidate potential routes of dissemination of AMR genes in natural environments and assess any significant persistence in wildlife to design risk mitigation actions.

Detection of Beta-Lactam-Resistant Escherichia coli and Toxigenic Clostridioides difficile Strains in Wild Boars Foraging in an Anthropization Gradient.

Disease transmission among wild boars, domestic animals and humans is a public health concern, especially in areas with high wild boar densities. In this study, fecal samples of wild boars (n = 200) from different locations of the Metropolitan Area of Barcelona were analyzed by PCR to explore the frequency of ß-lactamases and extended cephalosporin and carbapenem resistance genes (ESBLs) in Escherichia coli strains and the presence of toxigenic Clostridioides difficile. The prevalence of genes conferring resistance to ß-lactam antimicrobials was 8.0% (16/200): blaCMY-2 (3.0%), blaTEM-1b (2.5%), blaCTX-M-14 (1.0%), blaSHV-28 (1.0%), blaCTX-M-15 (0.5%) and blaCMY-1 (0.5%). Clostridioides difficile TcdA+ was detected in two wild boars (1.0%), which is the first report of this pathogen in wild boars in Spain. Moreover, the wild boars foraging in urban and peri-urban locations were more exposed to AMRB sources than the wild boars dwelling in natural environments. In conclusion, the detection of E. coli carrying ESBL/AmpC genes and toxigenic C. difficile in wild boars foraging in urban areas reinforces the value of this game species as a sentinel of environmental AMRB sources. In addition, these wild boars can be a public and environmental health concern by disseminating AMRB and other zoonotic agents. Although this study provides the first hints of the potential anthropogenic sources of AMR, further efforts should be conducted to identify and control them.

Transmission of Similar Mcr-1 Carrying Plasmids among Different Escherichia coli Lineages Isolated from Livestock and the Farmer.

Colistin use has mostly been stopped in human medicine, due to its toxicity. However, nowadays, it still is used as a last-resort antibiotic to treat hospital infections caused by multi-drug resistant Enterobacteriaceae. On the contrary, colistin has been used in veterinary medicine until recently. In this study, 210 fecal samples from pigs (n = 57), calves (n = 152), and the farmer (n = 1) were collected from a farm where E. coli harboring mcr-1-mcr-3 was previously detected. Samples were plated, and mcr-genes presence was confirmed by multiplex-PCR. Hybrid sequencing which determined the presence and location of mcr-1, other antibiotic resistance genes, and virulence factors. Eighteen colistin resistant isolates (13 from calves, four from pigs, and one from the farmer) contained mcr-1 associated with plasmids (IncX4, IncI2, and IncHI2), except for two that yielded mcr-1 in the chromosome. Similar plasmids were distributed in different E. coli lineages. Transmission of mcr-1 to the farmer most likely occurred by horizontal gene transfer from E. coli of calf origin, since plasmids were highly similar (99% coverage, 99.97% identity). Moreover, 33 virulence factors, including stx2 for Shiga toxin E. coli (STEC) were detected, highlighting the role of livestock as a reservoir of pathotypes with zoonotic potential.

Feed additives for the control of post-weaning Streptococcus suis disease and the effect on the faecal and nasal microbiota.

Medicated feed is a common strategy to control the occurrence of Streptococcus suis disease in swine production, but feed additives may constitute an alternative to metaphylaxis. In a farm with post-weaning S. suis disease, the following additives were tested: lysozyme (Lys), medium chain fatty acids plus lysozyme (FA + Lys), FA plus a natural anti-inflammatory (FA + antiinf) and amoxicillin (Amox). During the course of the study, FA + antiinf and Amox groups showed lower prevalence of clinical signs compatible with S. suis disease than the rest of the groups. Piglets from the FA + antiinf group showed high diversity and richness in their nasal and faecal microbiota. Diet supplements did not have major effects on the faecal microbiota, where the genus Mitsuokella was the only differentially present in the FA + Lys group. In the nasal microbiota, piglets from FA + antiinf presented higher differential abundance of a sequence variant from Ruminococcaceae and lower abundance of an unclassified genus from Weeksellaceae. In general, we detected more significant changes in the nasal than in the feacal microbiota, and found that parity of the dams affected the microbiota composition of their offspring, with piglets born to gilts exhibiting lower richness and diversity. Our results suggest that additives could be useful to control post-weaning disease when removing antimicrobials in farms.

Foraging at Solid Urban Waste Disposal Sites as Risk Factor for Cephalosporin and Colistin Resistant Escherichia coli Carriage in White Storks (Ciconia ciconia).

White stork (Ciconia ciconia) may act as a reservoir and vehicle of cephalosporin resistant (CR) Escherichia coli. Between 2011 and 2014, we sampled white storks from colonies exposed to different degrees of anthropic pressure across the major areas of natural distribution of white storks in Spain. Cloacal swab samples (n = 467) were obtained from individuals belonging to 12 different colonies from six different regions. Additionally, 70 samples were collected from recently deposited droppings at the base of nesting platforms. We phenotypically characterized E. coli isolates, confirmed presence of CR genes and classified plasmids. Risk factors for acquiring these genes were assessed. Overall, 8.8% (41 out of 467) storks carried CR E. coli in their cloaca and five (7.1%) were identified from recently deposited droppings; therefore, 46 isolates were further characterized. Of them, 20 contained bla CTX-M- 1, nine bla CMY- 2, six bla CTX-M- 14, four bla SHV- 12, three bla CTX-M- 15, two bla CTX-M- 32, one bla CTX-M- 1 together with bla CMY- 2, and one bla CTX-M- 1 together with bla SHV- 12. All were multidrug-resistant, and four harbored the plasmid-mediated colistin resistance mcr-1 gene. CR genes were associated with the presence of IncI1, IncFIB, and IncN replicon families. XbaI-macrorestriction analysis revealed a great diversity among most of the XbaI-PFGE types, but indistinguishable types were also seen with isolates obtained from different locations. Clonal complex 10 was the most common among CR E. coli and two bla CTX-M- 15 positive isolates were identified as B2-ST131. Carriage of CR E. coli was significantly higher in colonies located close to solid urban waste disposal sites in which foraging on human waste was more likely and in one case to cattle grazing. The co-occurrence of bla CMY- 2 and mcr-1 on plasmids of E. coli isolated from wild birds as early as 2011 is of note, as the earliest previous report of mcr-1 in wild birds is from 2016. Our study shows that foraging at landfills and in association with cattle grazing are important risk factors for the acquisition of CR E. coli in white storks.

Differential Viral-Host Immune Interactions Associated with Oseltamivir-Resistant H275Y and Wild-Type H1N1 A(pdm09) Influenza Virus Pathogenicity.

Oseltamivir is a common therapy against influenza A virus (IAV) infections. The acquisition of oseltamivir resistance (OR) mutations, such as H275Y, hampers viral fitness. However, OR H1N1 viruses have demonstrated the ability to spread throughout different populations. The objective of this work was to compare the fitness of two strains of OR (R6 and R7) containing the H275Y mutation, and a wild-type (F) pandemic influenza A (H1N1) 2009 (pdm09) virus both in vitro and in vivo in mice and to select one OR strain for a comparison with F in ferrets. R6 showed faster replication and pathogenicity than R7 in vitro and in mice. Subsequently, R6 was selected for the fitness comparison with the F strain in ferrets. Ferrets infected with the F virus showed more severe clinical signs, histopathological lung lesions, and viral quantification when compared to OR R6-infected animals. More importantly, differential viral kinetics correlated with differential pro-inflammatory host immune responses in the lungs of infected ferrets, where OR-infected animals developed a protective higher expression of type I IFN and Retinoid acid Inducible Gene I (RIG-I) genes early after infection, resulting in the development of milder disease. These results suggest the presence of early specific viral-host immune interactions relevant in the development of influenza-associated lung pathology.