969 research outputs found

    Impact of precursor-derived peracetic acid on post-weaning diarrhea , intestinal microbiota and predicted microbial functional genes in weaned pigs

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    Post-weaning diarrhea affects piglets in the nursery phase of production, leading to a substantial impact both at the farm and financial levels. The multifactorial etiology of this disease includes housing conditions, pig genetics, microbial composition, and metagenomic assets. Among the common therapeutic approaches, the widely used zinc oxide underwent a European Union ban in 2022 due to its negative environmental impact and correlation to increased antimicrobial resistance. During this study, we have tested two levels of inclusion of the potential antimicrobial alternative peracetic acid, delivered in water via the hydrolysis of the precursors sodium percarbonate and tetraacetylethylenediamine, in comparison to zinc oxide and an untreated control during a 2-week animal study. We assessed the microbial composition and predicted the metagenome, together with performance and physiological parameters, in order to describe the microbial functional role in etiopathology. Both zinc oxide and peracetic acid resulted in amelioration of the diarrheal status by the end of the trial period, with noticeable zinc oxide effects visible from the first week. This was accompanied by improved performance when compared to the first-week figures and a decreased stomach pH in both peracetic acid levels. A significant reduction in both stomach and caecal Proteobacteria was recorded in the zinc oxide group, and a significant reduction of Campylobacter in the stomach was reported for both zinc oxide and one of the peracetic acid concentrations. Among other functional differences, we found that the predicted ortholog for the zonula occludens toxin, a virulence factor present in pathogens like Escherichia coli and Campylobacter jejuni, was less abundant in the stomach of treated pigs compared to the control group. In water, peracetic acid delivered via precursor hydrolysis has the potential to be a valid intervention, an alternative to antimicrobial, to assist the weaning of piglets. Our findings support the view that post-weaning diarrhea is a complex multifactorial disease with an important metagenomic component characterized by the differential abundance of specific predicted orthologs and microbial genera in the stomach and caecum of pigs

    Impact of precursor-derived peracetic acid on post-weaning diarrhea , intestinal microbiota and predicted microbial functional genes in weaned pigs

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    Post-weaning diarrhea affects piglets in the nursery phase of production, leading to a substantial impact both at the farm and financial levels. The multifactorial etiology of this disease includes housing conditions, pig genetics, microbial composition, and metagenomic assets. Among the common therapeutic approaches, the widely used zinc oxide underwent a European Union ban in 2022 due to its negative environmental impact and correlation to increased antimicrobial resistance. During this study, we have tested two levels of inclusion of the potential antimicrobial alternative peracetic acid, delivered in water via the hydrolysis of the precursors sodium percarbonate and tetraacetylethylenediamine, in comparison to zinc oxide and an untreated control during a 2-week animal study. We assessed the microbial composition and predicted the metagenome, together with performance and physiological parameters, in order to describe the microbial functional role in etiopathology. Both zinc oxide and peracetic acid resulted in amelioration of the diarrheal status by the end of the trial period, with noticeable zinc oxide effects visible from the first week. This was accompanied by improved performance when compared to the first-week figures and a decreased stomach pH in both peracetic acid levels. A significant reduction in both stomach and caecal Proteobacteria was recorded in the zinc oxide group, and a significant reduction of Campylobacter in the stomach was reported for both zinc oxide and one of the peracetic acid concentrations. Among other functional differences, we found that the predicted ortholog for the zonula occludens toxin, a virulence factor present in pathogens like Escherichia coli and Campylobacter jejuni, was less abundant in the stomach of treated pigs compared to the control group. In water, peracetic acid delivered via precursor hydrolysis has the potential to be a valid intervention, an alternative to antimicrobial, to assist the weaning of piglets. Our findings support the view that post-weaning diarrhea is a complex multifactorial disease with an important metagenomic component characterized by the differential abundance of specific predicted orthologs and microbial genera in the stomach and caecum of pigs

    Virotyping and genetic antimicrobial susceptibility testing of porcine ETEC/STEC strains and associated plasmid types

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    IntroductionEnterotoxigenic Escherichia coli (ETEC) infections are the most common cause of secretory diarrhea in suckling and post-weaning piglets. For the latter, Shiga toxin-producing Escherichia coli (STEC) also cause edema disease. This pathogen leads to significant economic losses. ETEC/STEC strains can be distinguished from general E. coli by the presence of different host colonization factors (e.g., F4 and F18 fimbriae) and various toxins (e.g., LT, Stx2e, STa, STb, EAST-1). Increased resistance against a wide variety of antimicrobial drugs, such as paromomycin, trimethoprim, and tetracyclines, has been observed. Nowadays, diagnosing an ETEC/STEC infection requires culture-dependent antimicrobial susceptibility testing (AST) and multiplex PCRs, which are costly and time-consuming.MethodsHere, nanopore sequencing was used on 94 field isolates to assess the predictive power, using the meta R package to determine sensitivity and specificity and associated credibility intervals of genotypes associated with virulence and AMR.ResultsGenetic markers associated with resistance for amoxicillin (plasmid-encoded TEM genes), cephalosporins (ampC promoter mutations), colistin (mcr genes), aminoglycosides (aac(3) and aph(3) genes), florfenicol (floR), tetracyclines (tet genes), and trimethoprim-sulfa (dfrA genes) could explain most acquired resistance phenotypes. Most of the genes were plasmid-encoded, of which some collocated on a multi-resistance plasmid (12 genes against 4 antimicrobial classes). For fluoroquinolones, AMR was addressed by point mutations within the ParC and GyrA proteins and the qnrS1 gene. In addition, long-read data allowed to study the genetic landscape of virulence- and AMR-carrying plasmids, highlighting a complex interplay of multi-replicon plasmids with varying host ranges.ConclusionOur results showed promising sensitivity and specificity for the detection of all common virulence factors and most resistance genotypes. The use of the identified genetic hallmarks will contribute to the simultaneous identification, pathotyping, and genetic AST within a single diagnostic test. This will revolutionize future quicker and more cost-efficient (meta)genomics-driven diagnostics in veterinary medicine and contribute to epidemiological studies, monitoring, tailored vaccination, and management

    European regulations on the use of antibiotics in veterinary medicine

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    Antimicrobial resistance endangers the successful combat of bacterial infections in humans and animals. The common use of antibiotic classes including those of high clinical value in human as well as veterinary medicine is a critical factor contributing to or suspected to promote the emergence of antibiotic resistance. New legal provisions laid down in veterinary drug legislations and related guidelines and advice are in force in the European Union to safeguard the effectiveness, accessibility and availability of antibiotics. Categorisation of antibiotics in classes of importance for treatment of infections of humans by the WHO was one of the first steps. This task is also undertaken for antibiotics for treatment of animals by the EMA's Antimicrobial Advice Ad Hoc Expert Group. The new veterinary Regulation (EU) 2019/6 has extended restrictions for use of some antibiotics in animals to a full ban of certain antibiotics. While some (but not all) antibiotic compounds not being authorized in veterinary medicine may still be used in companion animals more strict provisions were already applicable for treatment of food producing animal species. Distinct regulations are in place for the treatment of animals kept in large numbers in flocks. Initial regulations focussed on the protection of consumers from residues of veterinary drugs in food commodities, new regulations address prudent (not routinely) and responsible selection, prescription and use of antibiotics, and have improved the practicality for cascade use outside the terms of marketing authorisation. Mandatory recording of use of veterinary medicinal products for food safety reasons is extended to rules for veterinarians and owners or holders of animals to regularly report the use of antibiotics for the purpose of official surveillance of consumption. National sales data of antibiotic veterinary medicinal products have been collected on a voluntary basis until 2022 by ESVAC, which has created awareness of major differences between EU member states. A significant decline in sales was reported for third and fourth generation cephalosporines, polymyxins (colistin), and (fluoro)quinolones since the initiation in 2011

    Changes in the saliva proteome of pigs with diarrhoea caused by Escherichia coli

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    Infection with the Gram-negative bacterium Escherichia coli is one of the main causes of diarrhoea in pigs and currently represents a significant problem for producers. Saliva is a fluid that can be collected by non-invasive, non-stressful methods and contains analytes that change in disease. These changes can provide information on the pathophysiology of the disease and can be used as an aid to diagnosis or monitoring of therapy. The objective of the present work aims to identify potential alterations in the salivary proteome of pigs with diarrhoea caused by E. coli. For that reason, we used two groups of Large White post-weaning pigs, one control group and other infected group (E. coli group). We took samples of saliva from both groups and after we used two techniques for protein separation by isoelectric point/molecular mass and molecular mass. These are 2-DE gel electrophoresis and SDS PAGE, respectively. In addition to these techniques, a more sophisticated technique was used for protein identification called mass spectrometry. The total concentration of proteins in the infected group was three times higher than the control group. In the SDS-PAGE analysis, we have higher levels of salivary lipocalins and IgA bands and, in contrast, lower levels of odorant-binding proteins, protease inhibitor from the submandibular origin and prolactin inducible protein. In the two-dimensional profile analysis, we have higher levels of salivary lipocalins, adenosine deaminase, IgA bands and albumin peptides and, in contrast, lower levels of alpha- amylase, carbonic anhydrase, carbonate dehydratase VI and whole albumin. After this, a validation test was made in which pigs with diarrhoea by Escherichia coli had considerably greater levels of salivary adenosine deaminase activity in comparison to the control group (healthy group). Regarding this study, some of these proteins play a important role in physiological processes and in physiological/pathological conditions, it has been observed that these proteins suffer alterations at salivary proteome level. For this reason, it is possible to say that these techniques are used to identify new biomarkers in saliva which contributes for the discovery of new alternative diagnoses of diseases in the future; - Resumo: Alterações no Proteoma da Saliva de Suínos com Diarreia Causada por Escherichia coli - A infeção pela bactéria Gram-negativa Escherichia coli é uma das principais causas de diarreia em suínos e representa atualmente um problema significativo para os produtores. A saliva é um fluido que pode ser recolhido por métodos não invasivos e não stressantes e que contém analitos que se alteram em caso de doença. Estas alterações podem fornecer informações sobre a fisiopatologia da doença e podem ser utilizadas como auxiliares de diagnóstico ou na monitorização da terapêutica. O objetivo do presente trabalho é identificar potenciais alterações no proteoma salivar de suínos com diarreia causada por E. coli. Para tal, utilizámos dois grupos de suínos Large White pós-desmame, um grupo de controlo e outro grupo infetado (grupo E. coli). Recolhemos amostras de saliva de ambos os grupos e depois utilizámos duas técnicas de separação de proteínas, sendo elas a SDS-PAGE e a eletroforese bidimensional. Para além destas técnicas, foi utilizada uma técnica para a identificação de proteínas, denominada espetrometria de massa. A concentração total de proteínas no grupo infetado foi três vezes superior à do grupo de controlo. Na análise de SDS-PAGE, temos níveis mais elevados de lipocalinas salivares e bandas de IgA e, em contrapartida, níveis mais baixos de proteínas ligadoras de odorantes, inibidor de protease de origem submandibular e proteína induzível por prolactina. Na análise do perfil bidimensional, temos níveis mais elevados de lipocalinas salivares, adenosina deaminase, bandas de IgA e péptidos de albumina e, em contrapartida, níveis mais baixos de alfa-amilase, anidrase carbónica, carbonato desidratase VI e albumina total. Em seguida, foi efetuado um teste de validação em que os suínos com diarreia por Escherichia coli apresentavam níveis consideravelmente mais elevados de atividade da adenosina deaminase salivar em comparação com o grupo de controlo (grupo saudável). Relativamente a este estudo, algumas destas proteínas desempenham um papel importante em processos fisiológicos e em condições fisiológicas/patológicas, foi observado que estas proteínas sofrem alterações ao nível do proteoma salivar. Por esta razão, é possível afirmar que estas técnicas são valiosas para a identificação de novos biomarcadores na saliva que contribuem para a descoberta de novos diagnósticos alternativos de doenças no futuro

    Genomic traits of multidrug resistant enterotoxigenic Escherichia coli isolates from diarrheic pigs

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    Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) infections poses a significant challenge in global pig farming. To address this issue, the study was conducted to identify and characterize 19 ETEC isolates from fecal samples of diarrheic pigs sourced from large-scale farms in Sichuan Province, China. Whole-genome sequencing and bioinformatic analysis were utilized for identification and characterization. The isolates exhibited substantial resistance to cefotaxime, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, ampicillin, tetracycline, florfenicol, and sulfadiazine, but were highly susceptible to amikacin, imipenem, and cefoxitin. Genetic diversity among the isolates was observed, with serotypes O22:H10, O163orOX21:H4, and O105:H8 being dominant. Further analysis revealed 53 resistance genes and 13 categories of 195 virulence factors. Of concern was the presence of tet(X4) in some isolates, indicating potential public health risks. The ETEC isolates demonstrated the ability to produce either heat-stable enterotoxin (ST) alone or both heat-labile enterotoxin (LT) and ST simultaneously, involving various virulence genes. Notably, STa were linked to human disease. Additionally, the presence of 4 hybrid ETEC/STEC isolates harboring Shiga-like toxin-related virulence factors, namely stx2a, stx2b, and stx2e-ONT-2771, was identified. IncF plasmids carrying multiple antimicrobial resistance genes were prevalent, and a hybrid ETEC/STEC plasmid was detected, highlighting the role of plasmids in hybrid pathotype emergence. These findings emphasized the multidrug resistance and pathogenicity of porcine-origin ETEC strains and the potential risk of epidemics through horizontal transmission of drug resistance, which is crucial for effective control strategies and interventions to mitigate the impact on animal and human health

    University of Nebraska-Lincoln Agricultural Research Division 121st Annual Report. July 1, 2006 to June 30, 2007.

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    Our Mission..... 4 Foreword..... 5 Research Highlights..... 6 Faculty Awards and Recognitions....14 Graduate Student Awards and Recognitions...17 Undergraduate Honors Student Research Program...22 Variety and Germplasm Releases....23 Patents.....24 Administration.....25 Administrative Personnel....25 Organizational Chart....26 Administrative Units....27 IANR Research Facilities....28 Faculty.....29 Agricultural/Natural Resources Units....30 Education and Human Sciences Departments...39 Off-Campus Research Centers....40 Interdisciplinary Activities....41 Visiting Scientists/Research Associates....42 Research Projects.....47 Agricultural/Natural Resources Units....47 Education and Human Sciences Departments...52 Off-Campus Research Centers....52 Interdisciplinary Activities....53 Publications.....55 Agricultural/Natural Resources Units....60 Education and Human Sciences Departments...82 Off-Campus Research Centers....85 Research Expenditures....8

    한국 돼지농장 유래 ESBL 생성 및 콜리스틴 내성 대장균의 분자역학과 내성 기전 연구

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    학위논문(박사) -- 서울대학교대학원 : 수의과대학 수의학과, 2023. 2. 조성범.3세대 세팔로스포린 및 콜리스틴 항생제는 인간의 다제내성세균 감염 질환 치료에 있어 최후의 항생제로 언급된다. 그러나 양돈산업에서는 이러한 항생제들이 돼지의 질병 치료 및 예방을 위하여 지속적으로 처방되어 왔고, 이러한 추세로 인해 돼지농장에서 ESBL 및 AmpC β-lactamase을 생성하는 대장균 (ESBL/AmpC 생성 대장균) 과 콜리스틴 내성 유전자인 mobilized colistin resistance gene (mcr)을 보유하는 대장균 (mcr 보유 대장균)의 분리율이 급격히 증가하였다. 돼지의 사육단계는 일령에 따라 이유자돈, 육성돈, 비육돈으로 나뉘며, 일반적으로 다른 사육단계의 돼지들은 분만사, 자돈사, 육성사, 비육사로 구분되어 사육된다. 돼지의 사육단계에 따라 다발하는 질병이 다르기 때문에 처방되는 항생제의 종류 및 양은 돼지 사육단계 따라 차이가 있으며, 이는 돼지의 사육단계별 항생제 내성균의 분포 및 특성을 다르게 하는 주된 요인으로 작용할 수 있다. ESBL/AmpC 생성 및 mcr 보유 대장균의 중요한 보균원인 돼지농장에서 이들 균주의 돼지의 사육단계별 분포 및 특성을 이해하는 것은 항생제 내성균을 제어하고 관리를 위한 중요한 초석으로 작용할 수 있다. 본 연구는 분자역학 및 내성 전달기전 분석을 기반으로 ESBL/AmpC 생성 및 mcr 보유 대장균에 대하여 돼지의 사육단계별 유병률과 특성의 차이를 분석하였다. 또한 공개 데이터베이스를 활용하여 사람, 돼지고기 등 다양한 유래 균주들과의 유전적 근연관계를 분석함으로써, 돼지농장 유래 균주의 공중보건학적 위해를 분석하고자 하였다. 마지막으로, 돼지농장 유래 균주들과 더불어 공개데이터베이스를 활용하여, 두 가지 주요 콜리스틴 내성 유전자인 mcr-1.1 및 mcr-3.1의 전달 기전에 대하여 비교유전체분석을 기반으로 분석하였다. 본 연구를 위하여 2017년 5월부터 2020년 3월까지 국내 돼지 농가수가 가장 많은 지역인 경기, 경북, 충남, 전남, 전북에 위치해 있는 11개의 돼지 농장을 대상으로 다단계 계층화 무작위 샘플링 (이유자돈, 육성돈, 비육돈, 임신모돈)을 실시하였고, 분리된 ESBL/AmpC 생산 및 mcr 보유 대장균이 분석에 포함되었다. 국내 양돈장에서의 ESBL/AmpC 생성 대장균의 유병률은 55.1%로 확인되었으며, 돼지의 사육단계별 균주의 유병률 및 특성이 다른 것으로 확인되었다. 이유자돈에서의 ESBL/AmpC 생성 대장균의 유병률은 86.3%로, 다른 사육단계 (육성돈 58.3%, 비육돈 48.4%, 임신모돈 43.1%)에서의 유병률과 비교하여 통계적으로 유의한 수준으로 높았다. 돼지의 사육단계별 비교에서 ESBL 생성 대장균은 모든 돼지 생산 단계에 분포해 있었으나, AmpC 생성 대장균은 육성돈 및 비육돈에서만 확인되었다. K-평균 군집 분석 기반 ESBL/AmpC 생성 대장균의 클론 분포 유사성 분석에서는, 같은 양돈장내 다른 돼지 생산 단계 유래 균주 간 높은 클론분포 유사성이 확인되었으며, 이는 농장내에서 사육단계간 교차 감염 가능성이 높음을 시사한다. 공개 데이터베이스(National Center for Biotechnology Information, NCBI)에 등록되어 있는 한국의 다양한 유래 균주와 비교분석 결과, 본 연구에서 분리된 돼지농장 유래 균주들은 인체, 돈육 유래 균주와 ESBL/AmpC 유형 및 클론유형을 공유하는 것이 확인되었으며, 특히 돼지농장 유래 균주 중 특히 ST101-B1, ST648-F, 그리고 ST457-F 등 장외 병원성 대장균 클론 타입이 공유되는 것이 확인되었다. 이는 돼지농장 유래 다제내성 장외 병원성 대장균 균주가 도축장, 돈육 등의 식품유통경로를 통해 인간에게 전염될 수 있다는 간접적인 과학적 증거를 제시한다. 국내 돼지농장에서 mcr-1 보유 대장균의 가중 유병률은 8.4%였다. 다른 사육단계와 비교하여 이유기(13.0%)에서 가장 높은 유병률을 보였으며, 이유자돈 유래 균주는 다른 사육단계 유래 균주와 비교하여 다제내성률이 통계적으로 유의한 수준으로 높았다. 전장유전체기반 분석에서 다제내성 및 병원성 이점을 가진 mcr-1 보유 대장균이 농장 내 돼지 단계 간 공유되는 것이 확인되었다. 반면, NCBI에 등록되어 있는 한국의 사람, 돈육, 돼지농장에서 분리된 균주 간에는 클론 타입이 전혀 공유되지 않는 것으로 확인되었으며, 이는 mcr-1의 환경 간 전파에 있어서 클론전파가 상대적으로 낮은 영향력을 가지고 있음을 시사한다. 한편, mcr-1 보유 대장균은 장외 및 장내 대장균 병원성 유전자와 바이오필름 형성과 같은, 균주의 생존에 이점을 주는 병원성을 보유하고 있는 것으로 확인되었다. 이러한 병원성 이점은 food-chain 환경 등 생존에 불리한 환경에서 mcr-1 보유 대장균의 생존 가능성을 높일 수 있으며, 수평전이 등을 통해 mcr-1을 다른 병원성 박테리아 등에 전달하는 중요한 공급원 역할을 할 수 있도록 도울 수 있음을 암시한다. mcr-1.1은 다른 내성 유전자나 삽입유전자 (Insertion sequence)이 없는 단순한 유전적 카세트 "mcr-1.1-pap2"를 기반으로 전달되었으며, 높은 수평전이빈도 (6.30 logCFU/ml)를 보였다. 이는 콜리스틴 내성 전파에 있어 mcr-1.1의 수평 전이가 주된 역할을 할 수 있음을 시사한다. 반면, mcr-3.1은 mcr-1.1와 비교하여 낮은 수평전이빈도 (0.97 logCFU/ml)를 보였으나, 다양한 항생제 및 중금속 내성 유전자 및 삽입유전자로 구성된 유전자 카세트의 형태로 전달되는 것이 확인되었다. 이는 mcr-3.1의 전파가 콜리스틴 내성뿐만 아니라 다제내성을 같이 전파함으로써 공중보건학적인 위해를 가져올 수 있음을 시사한다. 이 연구에서는 세계최초로 mcr-3.1 플라즈미드가 IS26을 매개로 하여 박테리아의 염색체 (Chromosome)에 통합될 수 있는 가능성을 보고하였다. 이 결과는 mcr-3.1가 수평 및 수직 전이를 통해 전달될 수 있음을 암시하며, mcr-3.1이 전세계적으로 전파될 수 있었던 성공이유 중 하나로 제안될 수 있다. 본 연구는 배양기법 기반 및 비교유전체분석을 통해 mcr-1.1 및 mcr-3.1에 의한 서로 다른 전달특성을 제시하였으며, 콜리스틴 내성을 제어하기 위하여 이러한 차이점을 고려한 적절한 전략의 필요성을 시사한다. 결론적으로, 본 논문은 돼지농장이 ESBL/AmpC 생성 및 mcr 보유 대장균의 중요한 보균원이며, 이들 균주가 food-chain을 통해 사람에게 전달되어 공중보건학적 위해가 될 수 있는 가능성에 대한 간접적인 과학적인 증거를 제시하였다. 돼지의 사육 단계에 따라 ESBL/AmpC 생성 및 mcr 보유 대장균의 유병률과 특성이 다르다는 것을 제시하였으며, 이들 균주의 중요한 저장소인 돼지농장에서 돼지의 사육단계별 다른 균주 특성을 고려한 다단계 체계적 전략의 필요성을 강조하였다. 또한 돼지 농장내에서 사육단계 간 ESBL/AmpC 생성 및 mcr 보유 대장균의 교차오염 가능성이 높음을 제시하였으며, 이러한 다제내성균을 제어하기 위하여 농장 내 교차오염을 줄이기 위한 노력이 필수적임을 시사하였다. 본 연구는 축산업계의 다양한 항생제 내성에 대한 심층적 접근을 위한 과학적 근거와 역학 모델을 제시함으로써 항생제 내성 관리 전략의 개선에 기여할 것으로 기대된다.The third-generation cephalosporins and colistin have been regarded as the critically important antibiotics (CIA) for treatment of multi-drug resistant (MDR) bacterial infection diseases in human. These antimicrobial agents have been continuously prescribed to prevent and control diseases in the swine industry. This trend made swine farms one of the most important reservoirs of extended-spectrum β-lactamase (ESBL)-/AmpC β-lactamase (AmpC)-producing and mobilized colistin resistance gene (mcr)-carrying Escherichia coli (ESBL/AmpC/MCR-EC). Pig production stages are divided into four stages, including weaning piglets, growing pigs, finishing pigs, and pregnant sows, and pigs of different stages are raised in separated barns. Since different diseases occur according to swine stages, the type and volume of antimicrobial treatment are different for each pig stage. Therefore, the distribution and characteristics of CIA-resistant bacteria could differ at each swine stage. In that point, understanding the distribution and characteristics of ESBL/AmpC/MCR-EC by swine stages could be an important cornerstone for control and management of CIA-resistant bacteria in swine farms. The present study aimed to investigate the risks of ESBL/AmpC/MCR-EC strains from swine farms according to swine production stages and to evaluate the potential threat of swine farm-derived strains to humans by understanding molecular epidemiological dynamics and resistance transfer mechanisms. For this study, multi-stage stratified sampling of swine feces was conducted for eleven swine farms located in South Korea between May 2017-March 2020, and whole genome sequence (WGS) of strains which uploaded in public database was utilized for comparative analysis. The ESBL/AmpC-EC strains were distributed throughout all swine stages (total prevalence: 55.1%). Prevalence and characteristics of ESBL/AmpC-EC strains were significantly different according to stages. Weaning piglets exhibited significantly higher prevalence (86.3%) relative to finishing pigs (48.4%). The CTX-M β-lactamase was the dominant ESBL type for all swine production stages, with the dominant type of CTX-M-55. Whereas, CMY β-lactamase was identified only in growing and finishing stages with the dominant type of CMY-2. The K-means similarity analysis showed clonal similarity between ESBL/AmpC-EC strains from different swine production stages within farms. This result suggests there is a high potential of cross-infection between stages, which enabling spread, persistence and reintroduction of ESBL/AmpC-EC clones within swine farms. In the comparative analysis using public database, the ESBL/AmpC types and clone types were shared between strains isolated from swine farms, pork meats and humans in South Korea. In particular, high-risk clones of swine farm-derived strains (ST101-B1, ST457-F, and ST648-F) were shared with strains from pork meats and humans. This result provides an indirect scientific evidence that swine farm-derived ESBL/AmpC-producing potentially high-risk clones could be transmitted to humans through food-chains. Total prevalence of mcr-1-carrying E. coli (MCR1-EC) was 8.4% in swine farms, with the highest prevalence from weaning piglets (13.0%). Weaning piglet-derived strains exhibited significantly higher multi-drug resistance (MDR) rate (quinolone, aminoglycoside, and chloramphenicol, etc.) compared to other stage-derived strains. WGS-based analysis showed that mcr-carrying intestinal pathogenic E. coli, with MDR and pathogenic advantages, were highly shared between swine stages. Whereas, between strains from different pig farms and sources (humans, pigs, and pork meats), highly heterogeneous clone types were identified. It suggests the lower contribution of clonal spread to colistin resistance spreading between environments. MCR1-EC with virulence advantages (e.g., intestinal/extraintestinal pathogenic E. coli or robust biofilm formation) accounted for nearly half of all strains. These results imply that MCR1-EC may act as an important source of mcr-1 horizontal transfer to other pathogenic bacteria in a harsh environment (e.g., food chain) based on its increased survivability. The mcr-1.1 showed high horizontal transfer frequency (6.30 logCFU/ml) and transferred with simple gene cassette without MDR and insertion sequences, mcr-1.1-pap2. This result suggests that mcr-1.1-mediated horizontal genetic transfer may provide a high contribution for colistin resistance spreading. Whereas, the mcr-3.1-cassette was bracketed by multiple insertion sequences (e.g., IS26, IS4321, etc.) and mainly transferred with MDR. This result implies that the transfer of mcr-3.1 would pose a significant challenge on public health by spreading with MDR. From this study, it was first reported that mcr-3.1-cassette may be integrated into bacterial chromosome via IS26-mediated transfer. This result implies that mcr-3.1 had dual pathways mediated by plasmid transfer (horizontal transmission) and chromosomal insertion (vertical transmission), enabling it to proliferate stably despite of its relatively lower horizontal transfer frequency (0.97 logCFU/ml). This study highlights the need for suitable strategies based on the different characteristics between mcr variants to control colistin resistance. In conclusion, swine farms may act a melting pot of high-risk pathogenic E. coli clones and CIA-associated resistance genes. This study provided an indirect scientific evidence swine farm-derived potentially high-risk zoonotic ESBL/AmpC/MCR-EC clones may be transferred to humans through food-chains using clonal spread and horizontal genetic transfer. Prevalence and characteristics of ESBL/AmpC/MCR-EC strains were significantly different according to stages in swine farms, an important reservoir of CIA-resistant bacteria, highlighting the importance of a multi-stage systemic policy to monitor and control CIA-resistant bacteria. WGS-based genetic relatedness analysis suggested the high possibility of cross-infection within swine farms, emphasizing the need for reduction of cross-infection in farms. This study is expected to contribute to the improvement of antimicrobial resistance management strategies by presenting scientific evidence and epidemiological models for an in-depth approach to different antibiotic resistance in the livestock industry.General introduction 1 Literature review 6 1. Antimicrobial use and resistance in swine industry 6 2. Antimicrobial resistance: from swine farms to humans 9 3. Mechanisms of antimicrobial resistance 10 3.1. Intrinsic antimicrobial resistance 10 3.2. Acquired antimicrobial resistance 11 4. Extended-spectrum β-lactamase (ESBL) 13 4.1. Resistance mechanisms of β-lactam drugs 13 4.2. β-lactamase types 13 4.3. ESBL β-lactamase types 14 4.4. AmpC β-lactamase types 14 4.5. Global occurrence of ESBL/AmpC β-lactamase 16 5. Mobilized colistrin resistance gene (mcr) 18 5.1. Colistin 18 5.2. Mechanisms of colistin resistance 18 5.3. mcr variants 19 5.4. Global occurrence of mcr 20 6. Comparative genomic analysis for resistance mechanisms 22 6.1. Single nucleotide polymorphism (SNP) 22 6.2. Horizontal transfer of antimicrobial resistance 23 6.3. Clonal spread of multi-drug resistant bacteria 24 Chapter 1. Prevalence, characteristics and clonal distribution of extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli following the swine production stages, and potential risks to humans 25 Abstract 26 1.1 Introduction 28 1.2 Material and Methods 30 1.3 Results 38 1.4 Discussion and Conclusion 45 Chapter 2. Prevalence, characteristics, and clonal distribution of Escherichia coli carrying mobilized colistin resistance gene mcr-1.1 in swine farms and their differences according to swine production stages 74 Abstract 75 2.1 Introduction 77 2.2 Material and Methods 79 2.3 Results 88 2.4 Discussion and Conclusion 95 Chapter 3. Different threats posed by two major mobilized colistin resistance genes –mcr-1.1 and mcr-3.1– revealed through comparative genomic analysis 125 Abstract 126 3.1 Introduction 127 3.2 Material and Methods 128 3.3 Results 131 3.4 Discussion and Conclusion 137 General Discussion and Conclusion 162 Reference 166 국문초록 201박

    Efectos de los Antibióticos y Óxido de Zinc y su retirada en el microbioma de los cerdos al destete

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    Post-weaning diarrhoea (PWD) is an infectious disease that causes significant productive and economic losses in pig production and often requires antimicrobial use. Antibiotic prophylaxis and metaphylaxis in animals are subject to more and more restrictions, especially in the EU, due to the risk of antimicrobial resistance. Zinc Oxide (ZnO) used in a range of 1500-3000ppm (dose referred as therapeutic or pharmacological) is also an effective treatment to prevent PWD. However, its therapeutic use was banned in the EU on the 28th of July 2022 due to environmental risk of soil pollution associated to its use. Finding alternative strategies to the use of antibiotics and ZnO to control PWD is crucial to ensure optimum levels of animal health and welfare and the economic viability of pig farms, ultimately resulting in high quality food production. A key step to find alternative strategies to the use of antibiotics and ZnO is to understand in detail their effects in the microbiome and the animal. This thesis focuses on the effects on the microbiome. The main causative agent of PWD is enterotoxigenic Escherichia coli. Antibiotics and ZnO are effective controlling E. coli overgrowth during the post-weaning period, although the exact mechanism of action of ZnO is not completely understood. On the other hand, microbiome dysbiosis occurring immediately post-weaning is described as both a possible risk factor and consequence of PWD. ZnO stabilizes the gut microbiome avoiding this dysbiosis, yet the exact taxonomic and functional changes triggered by ZnO in the microbiome are not completely characterized. In this thesis, we used shotgun whole metagenome sequencing to explore the effects of ZnO and antibiotics both, at species and functional level, in pigs gut microbiome in the first weeks post-weaning. In chapter 2, we studied the effects of ZnO and apramycin on the gut microbiome of the piglet a week post-weaning. Both, ZnO and apramycin had marked effects in gut microbiome taxonomy and functionality. Pigs fed the control diet with no ZnO or apramycin (Ct) exhibited high abundance of E. coli harbouring several virulence factors in animals not showing clinical signs of diarrhoea. This study was carried out in a low disease, high hygiene and biosecurity experimental farm where effects of different cleaning procedures were assessed as well. Treatment was the factor with strongest effect on the microbiome, whereas cleaning procedures had no remarkable effects. Given the results observed in the first study, the following studies described in chapters 3 and 4 were conducted in commercial farms to explore the effects of ZnO and antibiotics in commercial environments and between-farms variability in gut microbiome composition. Chapter 3 studied the impact of antibiotics and ZnO in pig microbiome comparing farms that had successfully removed ZnO and antibiotics to farms frequently using ZnO and antibiotic prophylaxis and metaphylaxis at weaning. Pig microbiome of farms using ZnO and antibiotics exhibited changes at days 7 and 14 post-weaning both at taxonomic and functional levels; these changes being more apparent in diarrhoea samples of 7 days post-weaning. Analysis of the environmental microbiome revealed a weak contribution of the environment to the gut microbiome of piglets, which shared few species early after weaning and within the 2 weeks post-weaning period studied. Chapter 4 studied the effects of removing antibiotics and ZnO on the microbiome of farms regularly using ZnO and antibiotics as prophylaxis and metaphylaxis. Results showed that antibiotics, and especially ZnO, maintain a stable microbiome composition (taxonomical and functional), inhibiting E. coli overgrowth both in normal and diarrhoeic conditions. Removal of ZnO and antibiotics generated an increase of E. coli abundance, as well as virulence related genes associated to the higher abundance of E. coli. Lastly, in chapter 5, e discussed the utility of shotgun sequencing in the study of microbiome changes caused by ZnO and diarrhoea, that could be triggered by antimicrobial and notantimicrobial ZnO-associated effects (both taxonomical and functional), and the effects of ZnO maintaining gut microbiome stability during the most critical period of post-weaning stage. From the results obtained in this thesis, the author concludes that weaning induces a sudden transition from a suckling pig microbiome to an adult like microbiome. The use of antibiotics and ZnO had a strong influence in this microbiome transition after weaning preventing piglet’s gut microbiome dysbiosis by inhibiting E. coli overgrowth and hence the presence of its associated virulence factors related genes as well as promoting a stable transition to an adult-like microbiome. Finally, environmental microbiota (i.e., weaning room environment) exerts minor effects on the composition of the microbiome of the piglet.La diarrea pos-destete (PWD, del inglés post-weaning diarrhoea) es una enfermedad infecciosa que causa pérdidas productivas y económicas en producción porcina y que a menudo requiere el uso de antimicrobianos. El uso profiláctico y metafiláctico de estos antimicrobianos para el tratamiento de la PWD está sujeto a cada vez más restricciones, especialmente en la UE, debido al riesgo de resistencias antimicrobianas. El óxido de zinc (ZnO) usado en concentraciones de 1500 a 3000ppm (referidas como concentraciones terapéuticas o farmacológicas) también se usa como un tratamiento eficaz para prevenir la PWD. Su uso se prohibió el 28 de julio de 2022 en la UE debido al riesgo ambiental de contaminación del suelo asociado a su uso. Encontrar estrategias alternativas al uso de los antibióticos y del ZnO es crucial para mantener los niveles óptimos de salud y bienestar animal, así como la rentabilidad de las granjas, asegurando la producción de alimentos de alta calidad. Un primer paso clave para encontrar estas estrategias alternativas a los antibióticos y el ZnO es entender en detalle sus efectos de en el microbioma y en el animal. Esta tesis se centrar en los efectos en el microbioma. El principal agente causal de la PWD es Escherichia coli enterotoxigénica. El ZnO y los antibióticos son efectivos para controlar el crecimiento excesivo de E. coli durante este período, aunque el mecanismo de acción exacto del ZnO no está totalmente claro. Por otro lado, la disbiosis del microbioma que ocurre en los días posteriores al destete uno de los nuevos posibles factores de riesgo y a su vez consecuencias descritas de la PWD. Se cree que el ZnO estabiliza el microbioma intestinal, pero hasta el momento, los cambios taxonómicos y funcionales exactos que provoca no están bien caracterizados. En esta tesis, utilizamos la secuenciación del metagenoma completo para caracterizar el efecto que tanto el ZnO como los antibióticos tienen en el microbioma intestinal del cerdo tanto a nivel taxonómico como funcional, en las primeras semanas posteriores al destete. En el capítulo 2, estudiamos el efecto del ZnO y de la apramicina en la respuesta del microbioma intestinal del cerdo al destete una semana pos-destete. Ambos tuvieron efectos marcados en la taxonomía y funcionalidad del microbioma intestinal. Los cerdos alimentados con dieta control sin ZnO ni antibióticos (Ct) exhibieron una gran abundancia de E. coli, que portaba varios factores de virulencia en animales que no mostraban signos clínicos de diarrea. Este estudio se realizó en una granja experimental de baja patología con altos niveles de higiene y bioseguridad en la que también se evaluaron los efectos de diferentes procedimientos de limpieza. El tratamiento fue el factor con mayor efecto en el microbioma, mientras que los procedimientos de limpieza no tuvieron efectos notables. Dados los resultados observados en el primer estudio, los siguientes estudios descritos en los capítulos 3 y capítulo 4 se realizaron en granjas comerciales para explorar los efectos de los antibióticos y el ZnO en entornos comerciales y la variabilidad entre granjas en la composición del microbioma intestinal. En el capítulo 3, se compararon granjas que usaban antibióticos y ZnO con granjas que los habían retirado. El microbioma de las granjas que utilizaban ZnO y antibióticos exhibió diferencias en los días 7 y 14 posteriores al destete, tanto a nivel de taxonómico como funcional; diferencias más evidentes en muestras de diarrea de 7 días post destete. El análisis del microbioma ambiental reveló una contribución débil al microbioma de los lechones, que compartían algunas especies consideradas como “core” que permanecían en el ambiente limpio de la sala de destete y en muestras iniciales y recogidas a las 2 semanas posdestete. En el capítulo 4 se estudió el impacto de la retirada de los antibióticos y ZnO en el microbioma porcino en granjas que utilizaban habitualmente antimicrobianos de forma profiláctica y metafiláctica al destete. Los resultados mostraron que los antibióticos, y sobretodo el ZnO, mantienen la composición del microbioma estable (taxonómica y funcionalmente), inhibiendo el crecimiento excesivo de E. coli tanto en condiciones normales como en diarrea. La retirada de ZnO y antibióticos en estas granjas generó un aumento en la abundancia de E. coli, así como genes relacionados con la virulencia asociados a la mayor abundancia de E. coli. Por último, en el capítulo 5, discutimos la utilidad de la secuenciación por medio de “Shotgun” en el estudio de los cambios en el microbioma causados por el ZnO y la diarrea, que podrían desencadenarse por efectos antimicrobianos y no antimicrobianos asociados al ZnO (tanto taxonómica como funcionalmente), y los efectos de ZnO manteniendo la estabilidad del microbioma intestinal durante el período más crítico de la etapa posterior al destete. De los resultados obtenidos en esta tesis, el autor concluye que el destete induce una transición brusca de un microbioma de lechón lactante a un microbioma de cerdo adulto. El uso de antibióticos y ZnO tiene una fuerte influencia en la transición del microbioma después del destete, previniendo la disbiosis intestinal en el microbioma del lechón al inhibir el crecimiento excesivo de E. coli y, por lo tanto, la presencia de sus genes relacionados con factores de virulencia, así como promover una transición estable hacia un microbioma similar al de un animal adulto. Finalmente, la microbiota ambiental (la presente en la sala de destete) ejerció efectos menores en la composición del microbioma de los lechones

    Avances en el estudio de biomarcadores de inflamación en saliva de cerdo

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    La sepsis es una condición grave y potencialmente mortal caracterizada por una respuesta inflamatoria sistémica desencadenada por un agente infeccioso que puede llevar finalmente a la disfunción orgánica e incluso a la muerte. Los biomarcadores pueden permitir una intervención más temprana y un mejor tratamiento de la sepsis, mejorando los resultados de salud y el bienestar de los pacientes, y reduciendo el desarrollo de resistencia a los antibióticos y las pérdidas económicas. En medicina humana, varios biomarcadores se utilizan rutinariamente para detectar infecciones bacterianas y guiar en el uso responsable de antibióticos, como la procalcitonina. En medicina veterinaria, a pesar de que las enfermedades inflamatorias e infecciosas generan muchos problemas de salud y pérdidas económicas en las granjas, este campo está poco explorado. Esta tesis de doctorado busca contribuir al campo del diagnóstico y la caracterización de la sepsis en cerdos profundizando y ampliando nuestra comprensión de los biomarcadores inflamatorios, su comportamiento y su relevancia en la sepsis en cerdos. Los objetivos específicos son los siguientes: • Objetivo 1: Investigación bibliográfica sobre la sepsis y los biomarcadores más comunes utilizados para diagnosticar y monitorearla en la medicina veterinaria. • Objetivo 2: Identificación de nuevos biomarcadores potenciales utilizando técnicas de proteómica. • Objetivo 3: Validación y medición de varios biomarcadores de inflamación, estrés oxidativo, bienestar y daño muscular, con posibles aplicaciones en la inflamación séptica y el estudio de sus posibles cambios en la sepsis y otras condiciones. • Objetivo 4: Desarrollo y validación de nuevos ensayos para diagnosticar sepsis: procalcitonina y presepsina. Para alcanzar estos objetivos, se utilizó la siguiente metodología: • Las muestras de cerdo se recolectaron principalmente de saliva, con esponjas de polipropileno; también se recolectó sangre mediante punción de la vena yugular. • Se realizaron análisis proteómicos de las muestras de cerdo utilizando técnicas basadas en gel y sin gel, incluyendo SDS-page y espectrometría de masas. • Se emplearon varios métodos como kits comerciales automatizados, ELISAs y ensayos quimioluminiscentes para analizar los biomarcadores estudiados, abarcando estrés, inflamación, estado redox y daño muscular. • Se desarrollaron anticuerpos policlonales y monoclonales para la detección de dos biomarcadores de sepsis, procalcitonina y presepsina. Los anticuerpos policlonales se generaron mediante la inmunización de un conejo y una cabra, mientras que los monoclonales se produjeron utilizando ratones como la especie elegida para la inmunización, con la posterior producción de hibridomas. • La tecnología utilizada para el desarrollo de los ensayos con los anticuerpos previamente mencionados fue AlphaLISA, que tiene diversas ventajas, como el uso mínimo de muestra y la ausencia de pasos de lavado. En resumen, esta investigación contribuyó al desarrollo de herramientas valiosas para el diagnóstico de infecciones y condiciones inflamatorias, con un enfoque integral que involucró la producción de anticuerpos, la recolección de muestras, el desarrollo de ensayos y el análisis de biomarcadores. Las conclusiones específicas de esta tesis de doctorado son: 1. Los biomarcadores con potencial en el diagnóstico y monitorización de la sepsis pueden clasificarse en tres categorías principales: (1) proteínas de fase aguda y citocinas, que han sido tradicionalmente utilizadas en medicina veterinaria para evaluar la inflamación; (2) PCT, PSE y otras proteínas más recientes que son más específicas de infecciones bacterianas; y (3) otros marcadores que pueden proporcionar información complementaria. 2. En los estudios proteómicos, la saliva y el suero mostraron patrones diferentes en respuesta a la inflamación séptica en un modelo inducido por LPS y la meningitis debida a Streptococcus suis. Además, se encontraron cambios en las proteínas de la saliva en otras enfermedades sépticas, como la diarrea causada por Escherichia coli. 3. La sepsis produce cambios en los analitos salivares relacionados con estrés, estado redox, inflamación y daño muscular, lo que abre la posibilidad de utilizarlos como posibles biomarcadores para este proceso en el cerdo. En esta línea, se requieren estudios adicionales para definir la capacidad y las aplicaciones de estos biomarcadores en el diagnóstico y monitorización de la sepsis, así como establecer valores de corte precisos. 4. La procalcitonina se midió por primera vez en la saliva porcina y mostró aumentos más elevados en cerdos con diferentes patologías sépticas, como en un modelo inducido por LPS, en meningitis por Streptococcus suis y en un brote de mordedura de cola; que en procesos inflamatorios no sépticos como un modelo inducido por trementina.Sepsis is a severe and life-threatening condition characterized by a systemic inflammatory response triggered by an infectious agent that can finally lead to organ dysfunction and even death. Biomarkers can allow an earlier intervention and better treatment of sepsis, improving the health outcomes and welfare of patients, and reducing development of antibiotic resistances and economic losses. In human medicine, several biomarkers are routinely used to early detect bacterial infections and guide in antibiotic stewardship, like procalcitonin. In veterinary medicine, even though inflammatory and infectious diseases lead to many health problems and economic losses in farms, this field is poorly explored. This PhD thesis seeks to contribute to the field of sepsis diagnosis and characterization in pigs by deepen and widen our understanding of inflammatory biomarkers, their behavior, and their relevance in sepsis in pigs. The specific objectives are: • Objective 1. Bibliographic research about sepsis and the current most common biomarkers used to diagnose and monitor it in veterinary medicine. • Objective 2. Identification of new potential biomarkers using proteomics techniques. • Objective 3. Validation and measurement of various biomarkers of inflammation, oxidative stress, welfare, or muscle damage, with potential application in septic inflammation and study of their possible changes in sepsis and other conditions. • Objective 4. Development and validation of new assays to diagnose sepsis: procalcitonin and presepsin. To this end, the methods that were used were: • Samples from pigs were collected mainly in saliva, using polypropylene sponges. Saliva is considered a non-invasive sample that ensures welfare in pigs and allows for serial sample collections, even in the same day, and by non-trained personnel. Blood was collected with comparative purposes, through jugular vein puncture. • Proteomic gel-based and gel-free analyses of pig samples were conducted to identify new biomarkers, including SDS-page and mass spectrometry. • Various methods, including automated commercial kits, ELISA, and chemiluminescent immunoassays, were employed to analyze the studied biomarkers, covering stress, inflammation, redox status, and muscle damage. • Polyclonal and monoclonal antibodies for the detection of two sepsis biomarkers, procalcitonin and presepsin, were developed: polyclonal antibodies through immunization of a rabbit and a goat; and monoclonal antibodies using mice as the chosen species for immunization, with the subsequent production of hybridomas. • The technology used for assays development with the previous antibodies was AlphaLISA, that has several advantages such as minimal sample usage and no need for washing steps.Overall, this research contributed to the development of valuable tools for diagnosing infections and inflammatory conditions, with a comprehensive approach that involved antibody production, sample collection, assay development, and biomarker analysis. The specific conclusions of this PhD these are: 1. The biomarkers with a potential in the diagnosis and monitoring of sepsis can be classified into three main categories: (1) acute phase proteins and cytokines, which have been traditionally used in veterinary medicine for evaluation of inflammation; (2) PCT, PSE, and other more recent proteins that are more specific of bacterial infections; and (3) other markers that can provide complementary information. 2. In the proteomic studies, saliva and serum showed different patterns in response to septic inflammation in a model induced by LPS and meningitis due to Streptococcus suis. In addition, changes in proteins in saliva were found in other septic diseases, such as diarrhoea caused by Escherichia coli. . Sepsis produces changes in salivary analytes related to stress, redox status, inflammation, and muscle damage, which opens the possibility of using them as potential biomarkers for this process in the pig. In this line, further studies are necessary to define the ability and applications of these biomarkers to diagnose and monitor sepsis, as well as establish accurate cut-off values. 4. Procalcitonin was measured for the first time in porcine saliva and showed higher increases in pigs in different septic conditions, such as in an LPS-induced model, Streptococcus suis meningitis, and a tail-biting outbreak, than in non-septic inflammatory processes like a turpentine-induced model
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