In vitro Susceptibility and Evaluation of Techniques for Understanding the Mode of Action of a Promising Non-antibiotic Citrus Fruit Extract Against Several Pathogens

Copyright © 2019 de Nova, Carvajal, Prieto and Rubio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.[EN] The screening for alternatives to antibiotics is an urgent need for the pharmaceutical industry. One of these alternatives seems to be the citrus fruit extracts, which are showing a significant antibacterial activity against Gram-negative and Gram-positive bacteria. One of these citrus extracts, named BIOCITRO®, is assessed in this study to elucidate its bacteriostatic and bactericidal effect and its mode of action on the important pathogens Campylobacter coli, C. jejuni, Escherichia coli, Salmonella enterica ssp. enterica, Clostridium difficile, C. perfringens, and Staphylococcus aureus. For most of the strains tested of these bacteria the product was bactericidal as well as bacteriostatic at the same concentration, and the minimum bactericidal concentrations ranged from 16 to 256 μg/mL. Regarding the mode of action, important changes in the permeability, structure, composition and morphology of the bacterial envelope were evidenced using flow cytometry, Fourier transform infrared spectroscopy and scanning electron microscopy. The main effect of the product was found over carbohydrates and polysaccharides, inducing the release of microvesicles by the cells in addition to other specific effects. During the study, the techniques used were evaluated to clarify their contribution to the knowledge of the mode of action of the product. The survival test elucidated whether the modifications displayed using other techniques affected the viability of the cells or on the contrary, the cells remained viable even with evident changes in their structure, composition or morphology. Flow cytometry showed that for some strains the proportion of cells detected with altered membrane permeability were higher than the number of non-viable cells, and therefore the damage did not affect the viability of some cells. On the contrary, some cells observed using scanning electron microscopy with no apparent damage, were demonstrated non-viable using the survival test, making this technique indispensable in studies of the mode of action of antimicrobials to make a correct interpretation of the data from other techniques.SIThe research was partly financed by PROBENA (2015/00119/001), the company commercializing BIOCITRO® in the EU, but without any influence over the experimental procedures neither results

Coronavirus

Las enfermedades causadas por coronavirus son conocidas y han sido estudiadas en veterinaria muchos años antes que en medicina humana por su importancia y por sus efectos en distintas especies de aves y de mamíferos. La primera vacuna contra una enfermedad por coronavirus en animales, la bronquitis infecciosa aviar, se desarrolló en 1950 mientras que el primer coronavirus humano se describióen 1965. En el presente trabajo se hace una revisión histórica de los aspectos que pueden ser más interesantes de las enfermedades causadas por coronavirus, aportando algunas experiencias propias con enfermedades causadas por coronavirus en el cerdo, algunas de ellas emergentes e ilustrativas de lo que son estas enfermedades. Se resaltan las características epidemiológicas de las zoonosis causadas por coronavirus, su origen y el carácter preocupantemente emergente de las mismas en medicina humana; es por esto que el conocimiento de los antecedentes de la pandemia actual causada por SARS-CoV-2 resultan muy elocuentes a la hora de interpretar la actual emergencia sanitaria, que aunque sorpresiva no resulta un hecho inesperado

Estudio comparativo entre fijación del complemento y ELISA indirecto para la detección de anticuerpos antirrotavirus en terneros

[ES] Estudio comparativo entre fijación del complemento y ELISA indirecto para la detección de anticuerpos antirrotavirus en ternero

Aislamiento de rotavirus bovino en cultivos celulares de riñón fetal bovino

[ES] Aislamiento de rotavirus bovino en cultivos celulares de riñón fetal bovin

Tipificación serológica, sensibilidad a los agentes antimicrobianos y patogenicidad para el ratón de cepas de Escherichia coli aisladas de cerdos

[ES] Tipificación serológica, sensibilidad a los agentes antimicrobianos y patogenicidad para el ratón de cepas de Escherichia coli aisladas de cerdo

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)

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 vitro evaluation of gentamicin activity against Spanish field isolates of Brachyspira hyodysenteriae

[EN]Background: The treatment of swine dysentery (SD) has become constrained in recent years due to the limited availability of effective drugs combined with a rise in antimicrobial resistance. Gentamicin, an aminoglycoside antibiotic, is authorised for the control of this disease in several European countries but has not been extensively used so far. In this study, the in vitro susceptibility of 56 Brachyspira hyodysenteriae field isolates was evaluated against gentamicin using a broth microdilution test. The molecular basis of decreased susceptibility to gentamicin was also investigated by sequencing the 16S rRNA gene and phylogenetic relatedness by multiple-locus variable number tandem-repeat analysis (MLVA). Results: Most B. hyodysenteriae isolates presented low minimum inhibitory concentration (MIC) values to gentamicin, with a mode of 2 µg/mL, a median or MIC50 of 4 µg/mL and percentile 90 or MIC90 of 16 µg/mL. The distribution of these values over the period studied (2011–2019) did not show a tendency towards the development of resistance to gentamicin. Differences in susceptibility among isolates could be explained by two point-mutations in the 16S rRNA gene, C990T and A1185G, which were only present in isolates with high MICs. These isolates were typed in three different MLVA clusters. Analyses of co-resistance between gentamicin and antimicrobials commonly used for the treatment of SD revealed that resistance to tiamulin and valnemulin was associated with low MICs for gentamicin. Conclusions: The results provide an accurate characterisation of antimicrobial sensitivity to gentamicin and possible mechanisms of resistance in Spanish B. hyodysenteriae isolates. These findings allow us to propose gentamicin as an alternative in the antibiotic management of SD, particularly in outbreaks caused by pleuromutilin resistant isolates.SIThe authors wish to thank Benjamín Rabanal for his contribution in some parts of the research and Diana Molina for her excellent technical assistance. Clara Vega (workplace ULE-02-B) and Manuel Gómez (LE131-18) are supported by grants from Junta de Castilla y León co-financed by the European Social Fund and Youth Employment Initiative. Héctor Argüello is supported by a grant from the Spanish Ministry of Education under the programme “Beatriz Galindo” (BEAGAL-18-106). Lucía Pérez-Pérez (PRE2020-093762) and Héctor Puente (FPU17/00466) are also supported by the Spanish Ministry of Education. We fnally want to thank the reviewer for the advice and ideas provided which clearly improved the quality of the manuscript.This work was supported by Fatro Ibérica S.L

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

Antimicrobial use and production system shape the fecal, environmental, and slurry resistomes of pig farms

P. 1-17Background: The global threat of antimicrobial resistance (AMR) is a One Health problem impacted by antimicrobial use (AMU) for human and livestock applications. Extensive Iberian swine production is based on a more sustainable and eco-friendly management system, providing an excellent opportunity to evaluate how sustained differences in AMU impact the resistome, not only in the animals but also on the farm environment. Here, we evaluate the resistome footprint of an extensive pig farming system, maintained for decades, as compared to that of industrialized intensive pig farming by analyzing 105 fecal, environmental and slurry metagenomes from 38 farms. Results: Our results evidence a significantly higher abundance of antimicrobial resistance genes (ARGs) on intensive farms and a link between AMU and AMR to certain antimicrobial classes. We observed differences in the resistome across sample types, with a higher richness and dispersion of ARGs within environmental samples than on those from feces or slurry. Indeed, a deeper analysis revealed that differences among the three sample types were defined by taxa-ARGs associations. Interestingly, mobilome analyses revealed that the observed AMR differences between intensive and extensive farms could be linked to differences in the abundance of mobile genetic elements (MGEs). Thus, while there were no differences in the abundance of chromosomal-associated ARGs between intensive and extensive herds, a significantly higher abundance of integrons in the environment and plasmids, regardless of the sample type, was detected on intensive farms. Conclusions: Overall, this study shows how AMU, production system, and sample type influence, mainly through MGEs, the profile and dispersion of ARGs in pig production.S