Antimicrobial resistance in the Spanish swine production: Impact of the production system and the antimicrobial use = Resistencias antimicrobianas en la producción porcina española: impacto del sistema de producción y del uso de antibióticos

207[ES] La resistencia a los antimicrobianos es una amenaza global para la salud pública y el medioambiente derivada del uso prolongado y frecuentemente inadecuado de antibióticos en medicina humana y veterinaria. En producción animal se han descrito diversos factores que pueden repercutir en el uso de antibióticos y, en consecuencia, en las resistencias, entre los que destacan el sistema de producción y las medidas de bioseguridad implementadas en la granja. En este sentido, las producciones de cerdo ibérico ecológica y extensiva se basan en sistemas de cría sostenibles y respetuosos con el entorno, constituyendo una excelente oportunidad para evaluar cómo las diferencias sostenidas de las distintas formas de uso de antibióticos repercuten en las resistencias antimicrobianas, no solo en los animales, sino también en el ambiente de la granja. El objetivo general de la presente tesis doctoral es ofrecer una descripción exhaustiva de las resistencias antimicrobianas en la producción porcina española, tanto en granjas intensivas como en las de producción ecológica-extensiva. Los estudios realizados se desarrollan en cuatro capítulos que abordan la evaluación del potencial de los microorganismos centinela en la monitorización de resistencias a los antimicrobianos y la caracterización genómica y metagenómica del resistoma y del mobiloma de las explotaciones porcinas. Las bacterias centinela recuperadas de granjas de producción ecológica-extensiva presentaron de forma sistemática menores resistencias antimicrobianas que aquellas procedentes de explotaciones porcinas intensivas, siendo el uso de antibióticos el factor más influyente. Además, aunque no se observó una relación directa entre el uso de antibióticos y la bioseguridad en las granjas, ciertas medidas, como la aplicación de protocolos de limpieza y desinfección adecuados, tuvieron un impacto aparente en la reducción de resistencias en Escherichia coli y Enterococcus spp. de granjas intensivas. La combinación de la caracterización fenotípica y genotípica de las resistencias antimicrobianas demostró el potencial de Escherichia coli, Enterococcus spp. y Staphylococcus spp. en la monitorización de resistencias en porcino, ya que estas bacterias resultaron ser importantes reservorios de determinantes de resistencia potencialmente movilizables. La vigilancia de las resistencias en Campylobacter coli también se demostró útil, pese a portar un menor número de determinantes de resistencia en su genoma. Por el contrario, los resultados de Salmonella enterica indicaron que no es un buen indicador de resistencia en estas granjas debido a su presencia esporádica en porcino y que su resistencia a los antimicrobianos está condicionada por el serotipo. El estudio genómico de las bacterias centinela demostró que la estructura tanto del resistoma como del mobiloma dependía del taxón, con mayores similitudes entre aquellas bacterias más relacionadas filogenéticamente. Este hecho fue corroborado en el estudio metagenómico, donde se observó que la composición del resistoma estaba primariamente asociada al tipo de muestra debido a cambios en las poblaciones bacterianas predominantes en cada nicho biológico. Por último, tanto el abordaje genómico como el metagenómico revelaron una clara interacción del resistoma y el mobiloma, la cual era más compleja en las granjas intensivas. En conjunto, esta tesis doctoral demuestra que el desarrollo de resistencias a los antimicrobianos está fuertemente ligado a los elementos genéticos móviles y al uso de antimicrobianos. [EN] Antimicrobial resistance is a global threat to public health and the environment derived from the prolonged and often inappropriate use of antimicrobials in human and veterinary medicine. In food-producing animals, several factors have been described that may have an impact on the use of antimicrobials and, consequently, on antimicrobial resistance, among which we can highlight the production system and the biosecurity measures implemented on the farm. In this sense, organic and extensive Iberian pig productions are based on sustainable and eco-friendly farming systems, providing an excellent opportunity to evaluate how sustained differences in the various forms of antimicrobial use impact antimicrobial resistance, not only in the animals, but also in the farm environment. The general objective of this doctoral thesis is to provide an in-depth characterization of antimicrobial resistance in Spanish swine production, both on intensive and organic-extensive farms. The studies carried out are developed in four chapters that address the evaluation of the potential of sentinel microorganisms in the monitoring of antimicrobial resistance and the genomic and metagenomic characterization of the resistome and mobilome of pig farms. Sentinel bacteria recovered from organic-extensive farms consistently showed lower antimicrobial resistance than those from intensive pig herds, with antimicrobial use as the most influential factor. In addition, although no direct relationship was observed between antimicrobial use and on-farm biosecurity, certain measures, such as the application of standardized cleaning and disinfection protocols, had an apparent impact on the reduction of antimicrobial resistance in Escherichia coli and Enterococcus spp. recovered from intensive pig herds. The combination of phenotypic and genotypic characterization of antimicrobial resistance demonstrated the potential of Escherichia coli, Enterococcus spp. and Staphylococcus spp. as sentinel microorganisms for antimicrobial resistance surveillance in swine, as these bacteria emerged as important reservoirs of potentially mobilizable antimicrobial resistance determinants. Likewise, antimicrobial resistance monitoring in Campylobacter coli proved to be useful even though this species harbored a reduced number of antimicrobial resistance determinants in its genome. In contrast, Salmonella enterica was not a good bioindicator due to its sporadic presence in swine production and the serotype-biased antimicrobial resistance. The genomic study of sentinel bacteria evidenced that the structure of both the resistome and mobilome was taxon-dependent, with greater similarities among those bacteria more phylogenetically related. This fact was corroborated in the metagenomic study, in which it was observed that the composition of the resistome was primarily determined by the type of sample due to changes in the predominant bacterial populations in each microecosystem. Finally, both genomic and metagenomic approaches revealed a clear interaction of the resistome and the mobilome, which was more complex on intensive farms. Overall, this doctoral thesis demonstrates that the development of antimicrobial resistance is strongly associated with mobile genetic elements and antimicrobial use

Detection and Genetic Diversity of Porcine Coronavirus Involved in Diarrhea Outbreaks in Spain

[EN]Porcine enteric coronaviruses include some of the most relevant viral pathogens to the swine industry such as porcine epidemic diarrhea virus (PEDV) or porcine transmissible gastroenteritis virus (TGEV) as well as several recently identified virus such as swine enteric coronavirus (SeCoV), porcine deltacoronavirus (PDCoV) or swine enteric alphacoronavirus (SeACoV). The aim of this study is the identification and characterization of enteric coronaviruses on Spanish pig farms between 2017 and 2019. The study was carried out on 106 swine farms with diarrhea outbreaks where a viral etiology was suspected by using two duplex RT-PCRs developed for the detection of porcine enteric coronaviruses. PEDV was the only coronavirus detected in our research (38.7% positive outbreaks, 41 out of 106) and neither TGEV, SeCoV, PDCoV nor SeACoV were detected in any of the samples. The complete S-gene of all the PEDV isolates recovered were obtained and compared to PEDV and SeCoV sequences available in GenBank. The phylogenetic tree showed that only PEDV of the INDEL 2 or G1b genogroup has circulated in Spain between 2017 and 2019. Three different variants were detected, the recombinant PEDV-SeCoV being the most widespread. These results show that PEDV is a relevant cause of enteric disorders in pigs in Spain while new emerging coronavirus have not been detected so far. However, the monitoring of these virus is advisable to curtail their emergence and spread.SIThis work was supported by the program from the National Institute of Agricultural and Food Research and Technology (INIA project E-RTA2015-0003-C02-02) of Spanish Government. HP, ÓM-A, and HA were supported by Spanish Government (FPU17/00466, FPU16/03485, and BEAGAL-18-106, respectively) and MG-G by Junta de Castilla y León (LE131-18)

In vitro activity of essential oils against microbial isolates from otitis externa cases in dogs

[EN] Despite the number of studies focused on the potential use of essential oils (EOs) as an alternative to conventional treatments of canine external otitis, there is controversy about their antimicrobial activity which could be explained by differences in technical or biological aspects. This study focuses on the antimicrobial activity of three single EO compounds (thymol, cinnamaldehyde and carvacrol) and two EOs (clove and oregano) against clinical isolates recovered from canine otitis externa cases (14 bacterial isolates belonging to five different genera and six Malassezia pachydermatis isolates). All compounds showed activity and cinnamaldehyde exhibited the highest bactericidal and fungicidal activity. The susceptibility was lower among bacterial isolates than fungal isolates, being this difference more evident for Gram-positive bacteria. No relationship between antibiotic multi-resistant profile and susceptibility to compounds was observed. To sum up, our results provide appropriate information about appropriate concentrations of promising candidates for the topic treatment of canine otitis.SIManuel Gómez-García was supported by grant from Junta de Castilla y León co-financed by the European Social Fund (LE131-18). Héctor Puente (FPU17/00466) and Óscar Mencía-Ares (FPU16/03485) were supported by the Spanish Ministry of Education. Héctor Argüello was supported by the Spanish Ministry of Education under the programme Beatriz Galindo (BEAGAL-18-106)

Genomic Insights into the Mobilome and Resistome of Sentinel Microorganisms Originating from Farms of Two Different Swine Production Systems

[EN] Antimicrobial resistance (AMR) is a threat to public health due to long-term antimicrobial use (AMU), which promotes the bacterial acquisition of antimicrobial resistance determinants (ARDs). Within food-producing animals, organic and extensive Iberian swine production is based on sustainable and eco-friendly management systems, providing an excellent opportunity to evaluate how sustained differences in AMU impact the development and spread of AMR. Here, through a whole-genome sequencing approach, we provide an in-depth characterization of the resistome and mobilome and their interaction in 466 sentinel bacteria, namely, Escherichia coli, Enterococcus spp., Campylobacter coli, and Staphylococcus spp., recovered from 37 intensive and organic-extensive pig farms. Both ARDs and mobile genetic elements (MGEs) were primarily taxon-associated, with higher similarities among bacteria which were closely phylogenetically related. E. coli exhibited the most diverse resistome and mobilome, with 85.4% mobilizable ARDs, 50.3% of which were plasmid-associated. Staphylococcus spp. exhibited a broad repertoire of ARDs and MGEs, with 52.3% of its resistome being mobilizable. Although Enterococcus spp. carried the highest number of ARDs per isolate and its plasmidome was similar in size to that of E. coli, 43.7% of its resistome was mobilizable. A narrow spectrum of ARDs constituted the C. coli resistome, with point mutations as its main AMR driver. A constrained AMU, as observed in organic-extensive herds, determined a reduction in the quantitative composition of the resistome and the complexity of the resistome-mobilome interaction. These results demonstrate taxon-associated AMR-MGE interactions and evidence that responsible AMU can contribute to reducing AMR pressure in the food chainSIWe acknowledge the excellent technical assistance provided by Diana Molina, Lucía Pérez, and Clara Vega, the help with data management provided by Laura Uelze, and the help by Katharina Thomas and Beatrice Baumann in sequencing library preparation. We also thank the veterinary practitioners and farmers for their willingness to collaborate and, in particular, Álvaro Fernández-Blanco for his support in contacting the farms. O.M.-A. received a grant from the Spanish Government (Ministerio de Universidades), no. EST 19/00806, for a short stay at the German Federal Institute for Risk Assessment. O.M.-A. also holds a grant from the Spanish Government (Ministerio de Educación y Formación Profesional), no. FPU 16/03485. H.A. is financially supported by the “Beatriz Galindo” Program from the Spanish Government (Ministerio de Educación y Formación Profesional), no. BEAGAL-18-106. Study design was performed by A.C., H.A. and A.A.-O. Samples were prepared by O.M.-A. M.B. performed the whole-genome sequencing. C.D. performed the computational analyses, with contribution from J.F.C.-D. Statistical analyses were performed by O.M.-A. with contributions from C.D. and J.F.C.-D. B.M., H.A. and A.A.-O. provided technical and scientific support on the analysis. O.M.-A., H.A., A.C., A.A.-O., B.M., M.B. and C.D. participated in writing the manuscript or contributed to its revision. All authors revised the manuscript and approved the final versio

First identification and characterization of rotavirus H in swine in Spain

Rotaviruses (RVs) are classified into nine species or groups (RVA-RVD and RVF-RVJ). RVA, RVB and RVC are well-recognized as etiological agents of enteric disease on swine farms and have been identified in all countries with a relevant pork production. Contrarily, RVH has only been identified on swine farms from Japan and more recently from Brazil, USA, South Africa and Vietnam but not yet in Europe. The occurrence of RVH was investigated in 103 Spanish pig herds. Nine farms were positive and we achieved a complete nucleotide sequences in three isolates while another RVH isolate was partially sequenced. Mean nucleotide identities with the RVH sequences available in GenBank ranged between 69.4 and 93.7 %. Phylogenetically, all genomic segments of Spanish RVH isolates clustered closely with other porcine RVH strains but were distantly related to human RVH as well as bat RVH strain. To the best of our knowledge this is the first report of RVH on swine farms in Europe including its characterization by means of complete genome sequencing.info:eu-repo/semantics/acceptedVersio

Combined in-vitro and on-farm evaluation of commercial disinfectants used against Brachyspira hyodysenteriae

[EN] Background: Swine dysentery (SD) is a severe infectious disease with a relevant impact on pig production usually caused by Brachyspira hyodysenteriae, although B. hampsonii causes an identical clinical picture. SD control relies on antimicrobials, good management practices and strict biosecurity with cleaning and disinfection as crucial tools to avoid the pathogen transmission. This study evaluates the in-vitro efficacy of an array of commercial disinfectants against a collection of B. hyodysenteriae isolates using broth tests. The efficacy of cleaning and disinfection protocols was also evaluated on two farms with endemic SD using surface swabs collected in emptied pens before and after cleaning and disinfection procedures, using both real-time PCR and bacterial microbiological culture. Results: Most of the commercial disinfectants evaluated were effective against all B. hyodysenteriae isolates tested, with a reduction of more than 5.00 log10 CFU/mL (bactericidal efficacy of 99.999%). However, some isolates exhibited reduced susceptibility to Virkon-S and Limoseptic disinfectants. The evaluation of cleaning and disinfection protocols on farms with SD outbreaks showed that approximately half the pens tested (n = 25) were positive by real-time PCR after pigs removal (mean B. hyodysenteriae counts 5.72 ± 1.04 log10 CFU/mL) while almost 20% of the pens remained positive after cleaning (n = 7) and disinfection (n = 5) procedures although with significantly lower, mean estimates (4.31 ± 0.43 log10 CFU/mL and 4.01 ± 0.55 log10 CFU/mL, respectively). Conclusions: These results show the efficacy of disinfectants against B. hyodysenteriae but also stress the need to implement adequately the cleaning and disinfection protocols on pig farms and review and revise their efficiency periodically.SIThis study was supported by the Ministerio de Ciencia e Innovación (PID2019-110662RB-I00) and the Castilla y León Rural Development Pro‑ gramme co-fnanced by EAFRD (47/18/125/G0

In-depth in vitro Evaluation of the Activity and Mechanisms of Action of Organic Acids and Essential Oils Against Swine Enteropathogenic Bacteria

P. 1-13Alternative antimicrobials require a deep understanding of their action mechanisms by in vitro assays which support science-based field use. This study focuses on the characterization of bactericidal mechanisms of potential antimicrobial compounds, two organic acids and three single essential oil (EO) compounds against swine enteropathogenic bacteria Escherichia coli, Salmonella enterica subsp. enterica serovar Typhimurium, and Clostridium perfringens. Target concentrations of the compounds were evaluated using the inhibitory potential of the vapor phase and bacterial viability after short-term exposure, while cell targets were disclosed using flow cytometry (FC), Fourier-transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). All tested compounds exhibited vapor phase activity against the three bacterial species, except sodium salt of coconut fatty acid distillates against C. perfringens. Survival test results evidenced that effects on bacterial viability were concentration dependent and higher in single EO compounds than in organic acids. In detail, thymol and its isomer carvacrol were the most effective compounds. Further characterization of thymol and cinnamaldehyde activity revealed that thymol main target was the cell membrane, since it caused striking damages in the membrane permeability, integrity and composition evidenced by FC and FTIR in the three enteric pathogens. In contrast, cinnamaldehyde was more effective against enterobacteria than against C. perfringens and only caused slightly damages at the highest concentration tested. Its target at the molecular level differed between enterobacteria and C. perfringens isolates. The SEM micrographs allowed us to confirm the results previously obtained for both EO compounds by other techniques. Altogether, the study showed the straight effect of these antimicrobials, which could constitute relevant information to optimize their feed inclusion rates in field studies or field use.S

Detection and genetic characterization of enteric viruses in diarrhoea outbreaks from swine farms in Spain

[EN] Background The aim of this work was to study the prevalence and distribution of Porcine astrovirus (PAstV), Porcine kobuvirus (PKoV), Porcine torovirus (PToV), Mammalian orthoreovirus (MRV) and Porcine mastadenovirus (PAdV) as well as their association with widely recognized virus that cause diarrhoea in swine such as coronavirus (CoVs) and rotavirus (RVs) in diarrhoea outbreaks from Spanish swine farms. Furthermore, a selection of the viral strains was genetically characterized. Results PAstV, PKoV, PToV, MRV and PAdV were frequently detected. Particularly, PAstV and PKoV were detected in almost 50% and 30% of the investigated farms, respectively, with an age-dependent distribution; PAstV was mainly detected in postweaning and fattening pigs, while PKoV was more frequent in sucking piglets. Viral co-infections were detected in almost half of the outbreaks, combining CoVs, RVs and the viruses studied, with a maximum of 5 different viral species reported in three investigated farms. Using a next generation sequencing approach, we obtained a total of 24 ARN viral genomes (> 90% genome sequence), characterizing for first time the full genome of circulating strains of PAstV2, PAstV4, PAstV5 and PToV on Spanish farms. Phylogenetic analyses showed that PAstV, PKoV and PToV from Spanish swine farms clustered together with isolates of the same viral species from neighboring pig producing countries. Conclusions Although further studies to evaluate the role of these enteric viruses in diarrhoea outbreaks are required, their wide distribution and frequent association in co-infections cannot be disregard. Hence, their inclusion into routine diagnostic panels for diarrhoea in swine should be considered.SIThis work was supported by the program of the National Institute of Agricultural and Food Research and Technology (INIA project E-RTA2015-0003-C02-01 and E-RTA2015-0003-C02-02) of Spanish Government. H. Puente, O. Mencía-Ares, L. Pérez-Pérez, M. Cortey and H. Argüello were supported by Spanish Government (FPU17/00466, FPU16/03485, PRE2020-093762, RYC-2015-1715-4 and BEAGAL- 18-106, respectively) and M. Gómez-García by Junta de Castilla & León (LE131-18)

Abortos infecciosos en pequeños rumiantes domésticos de la Península Ibérica: Optimización de los procedimientos de muestreo para el diagnóstico molecular

[EN] Introduction: Infectious abortions have a major impact on small domestic ruminant farms, i.e., sheep and goats, both in terms of profitability and health status. Therefore, rapid and sensitive diagnosis is essential to minimize losses. Currently, molecular techniques, such as qPCR, are routinely used for their diagnosis, which imply the need to manipulate all abortive material, with consequent biosafety risks. Here, we evaluate the frequency of the main abortifacient pathogens in small domestic ruminants in the Iberian Peninsula and also assess an alternative approach for the optimization of sampling for molecular diagnosis. Results: A total of 392 clinical cases were analyzed from April 2020 to May 2021, evidencing that the main causative agents of abortion detected were Coxiella burnetii (49.0%), Chlamydia abortus (38.3%) and, to a lesser extent, Toxoplasma gondii (10.2%), Salmonella enterica (7.1%) and Campylobacter spp. (6.1%). An uneven distribution of these pathogens was observed between ruminant species, with a higher frequency (p < 0.05) of T. gondii, S. enterica and Campylobacter spp. in sheep than goat abortions, and among geographic areas, highlighting the higher frequency (p < 0.05) of T. gondii and Campylobacter spp. in the north compared to southeastern Spain. The alternative sampling method, consisting on the use of fetal tongues and placental swabs in replacement of the whole fetus and placental tissue, offered a very good agreement with the classical method for all pathogens, except for low concentrations of C. burnetii, which seems to have a doubtful role in abortion when its concentration in the abortifacient material is low. Conclusions: This study reveals a high frequency of infectious etiology in abortions of small domestic ruminants in the Iberian Peninsula and validates for the first time an alternative sampling method for molecular diagnosis that will help to provide rapid and accurate results while minimizing biosafety risks.S

Evaluación in vitro del efecto antiviral de compuestos naturales sobre el coronavirus de la diarrea epidémica porcina

[EN] Organic acid and essential oils (EOs), well-known antimicrobials, could also possess antiviral activity, a characteristic which has not been completely addressed up to now. In this study, the effect of two organic acids (formic acid and sodium salt of coconut fatty acid distillates) and two single EO compounds (thymol and cinnamaldehye) was evaluated against porcine epidemic diarrhea virus (PEDV). The concentration used for each compound was established by cytotoxicity assays in Vero cells. The antiviral activity was then evaluated at three multiplicities of infection (MOIs) through visual cytopathic effect (CPE) evaluation and an alamarBlue assay as well as real-time reverse-transcription PCR (RT-qPCR) and viral titration of cell supernatants. Formic acid at at a dose of 1,200 ppm was the only compound which showed antiviral activity, with a weak reduction of CPE caused by PEDV. Through the alamarBlue fluorescence assay, we showed a significant anti-CPE effect of formic acid which could not be observed by using an inverted optical microscope. RT-qPCR and infectivity analysis also showed that formic acid significantly reduced viral RNA and viral titers in a PEDV MOI-dependent manner. Our results suggest that the antiviral activity of formic acid could be associated to its inhibitory effect on viral replication. Further studies are required to explore the anti-PEDV activity of formic acid under field conditions alone or together with other antiviral agents.SIThis study was supported by the program of the National Institute of Agricultural and Food Research and Technology (INIA project E-RTA2015-0003-C02-02) of Spanish Government. MG-G was supported by grant from Junta de Castilla y León co-financed by the European Social Fund (LE131-18). HP (FPU17/00466) and ÓM-A (FPU16/03485) were supported by the Spanish Ministry of Education. HA was supported by the Spanish Ministry of Education under the programme Beatriz Galindo (BEAGAL-18-106)