First reported genome of an mcr-9-mediated colistin-resistant Salmonella Typhimurium isolate from Brazilian livestock

Objectives: To investigate the genetic context of colistin resistance in anmcr-9-harbouring Salmonella Typhimurium ST19 strain from swine in Brazil. Methods: Minimum inhibitory concentrations (MIC) to colistin were determined by broth microdilution. Whole-genome sequencing was performed on an Illumina MiSeq system, followed by de novo genome assembly using SPAdes 1.13.1. The draft genome sequence was annotated in Prokka using KBase online server. Downstream analyses for resistome and plasmid detection were performed using online tools available at the Center for Genomic Epidemiology. The strain was typed in silico using MLST 2.0. Phylogenetic analysis involving 24 other genomes of Salmonella Typhimurium ST19 and mcr-9-harbouring Salmonella Typhimurium isolated from humans, livestock and foodstuff in different regions was also performed. Results: Assembly of the draft genome resulted in 5245 protein-coding sequences, 14 rRNAs, 83 tRNAs and a GC content of 51.81%. The strain was identified as Salmonella Typhimurium ST19 harbouring a 265.5-kb pN1566-2 plasmid carrying genes encoding resistance to colistin (mcr-9.1), aminoglycosides (aadA1), tetracycline [tet(C)] and sulfonamides (sul1). Our findings indicate that the Salmonella Typhimurium ST19 strain in this study showed low genetic variability compared with Salmonella Typhimurium ST19 isolated from swine and poultry in Brazil, and was less related to those reported in other countries. Conclusions: This is the first reported genome of a phenotypically colistin-resistant Salmonella Typhimurium harbouring the mcr-9 variant in Brazilian livestock. This genome will aid global investigations on epidemiological and evolutionary aspects of plasmid-mediated colistin resistance and the role of colistin-resistant Salmonella Typhimurium ST19 lineage as a zoonotic pathogen

Phenotypic and genotypic characterization of Salmonella spp. isolates in Portugal

Salmonellosis remains one of the most common causes of gastrointestinal infection in the world, and represents a considerable burden in developing and developed countries. As such, Salmonella surveillance is essential to control infection and decrease the economic burden of the disease, and the Portuguese National Institute of Health has been working in this field roughly since 1950. Considering the importance of the surveillance of Salmonella enterica, the main goal of this work was to contribute to a better knowledge of relevant serovars circulating in Portugal, either the most common, or serovars exhibiting important fitness traits that confer ecological advantages over other strains. In order to achieve this objective, first we analysed decades of data and established the trends of Salmonella enterica serovars circulating in Portugal. Additionally, whole-genome sequencing (WGS) was implemented at the National Reference Laboratory to the surveillance of Salmonella enterica, and was evaluated as a substitute of traditional typing methods, such as serotyping and multiple-locus variable number tandem repeat analysis. WGS proved to be a highly discriminatory method for the detection of suspected outbreaks and even previously undetected clusters. We identified several isolates carrying multiple resistance markers and mobile genetic elements that confer the potential of persistence and spread of these bacteria. This PhD dissertation highlights the need for the adjustment of the existing regulations for foodstuffs monitoring and the investigation of safe control measures in animal production, as a means to control the spread of resistant and persistent clones that may contribute to the increase of severe infections and to the spread of those markers to other bacteria. This work stands as an important contribution for the insight on Salmonella enterica isolates circulating in Portugal, as well as a contribution for the reinforcement of the capacitation of the Portuguese National Reference Laboratory

First Report of Salmonella Serovar Typhimurium and Monophasic Typhimurium Clinical Isolates Harboring mcr-9 in Portugal

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Phenotypic and genotypic characterization of Salmonella spp. isolates in Portugal

Tese de doutoramento em Biologia, Especialidade de Genética Molecular, apresentado à Faculdade de Ciências e Tecnologia. Universidade NOVA de Lisboa, 2019.Trabalho desenvolvido no Laboratório de Infeções Gastrintestinais do Departamento de Doenças Infeciosas, INSA.Orientador: João Paulo dos Santos Gomes, Investigador Auxiliar com Habilitação, INSA; Coorientador: Jorge Cândido Pinheiro da Costa Machado, Coordenador do Departamento de Doenças Infeciosas, INSASalmonellosis remains one of the most common causes of gastrointestinal infection in the world, and represents a considerable burden in developing and developed countries. As such, Salmonella surveillance is essential to control infection and decrease the economic burden of the disease, and the Portuguese National Institute of Health has been working in this field roughly since 1950. Considering the importance of the surveillance of Salmonella enterica, the main goal of this work was to contribute to a better knowledge of relevant serovars circulating in Portugal, either the most common, or serovars exhibiting important fitness traits that confer ecological advantages over other strains. In order to achieve this objective, first we analysed decades of data and established the trends of Salmonella enterica serovars circulating in Portugal. Additionally, whole-genome sequencing (WGS) was implemented at the National Reference Laboratory to the surveillance of Salmonella enterica, and was evaluated as a substitute of traditional typing methods, such as serotyping and multiple-locus variable number tandem repeat analysis. WGS proved to be a highly discriminatory method for the detection of suspected outbreaks and even previously undetected clusters. We identified several isolates carrying multiple resistance markers and mobile genetic elements that confer the potential of persistence and spread of these bacteria. This PhD dissertation highlights the need for the adjustment of the existing regulations for foodstuffs monitoring and the investigation of safe control measures in animal production, as a means to control the spread of resistant and persistent clones that may contribute to the increase of severe infections and to the spread of those markers to other bacteria. This work stands as an important contribution for the insight on Salmonella enterica isolates circulating in Portugal, as well as a contribution for the reinforcement of the capacitation of the Portuguese National Reference Laboratory.Salmonelose permanece uma das causas mais frequentes de infeção gastrintestinal no mundo e representa um enorme impacto económico não só em países em desenvolvimento, mas também em países desenvolvidos. Assim, a vigilância epidemiológica e laboratorial de Salmonella é essencial para o controlo da infeção e para a diminuição dos custos associados à doença. Desde 1950 que o Instituto Nacional de Saúde Doutor Ricardo Jorge tem tido um papel relevante na vigilância laboratorial desta doença. O principal objetivo deste trabalho prendeu-se com a contribuição para o melhor conhecimento de alguns dos serotipos de Salmonella que circulam em Portugal, quer mais frequentes, quer serotipos com características vantajosas para a sua perseverança. De forma a cumprir este objetivo, primeiramente foram analisados os dados acumulados ao longo de décadas de vigilância laboratorial e foram determinadas as tendências dos serotipos circulantes no país. Foi também implementada, no Laboratório Nacional de Referência, a sequenciação genómica aplicada à vigilância de Salmonella enterica, e foi feita a avaliação da substituição das técnicas clássicas de tipagem. A sequenciação genómica, como método altamente discriminatório permitiu, não só a confirmação de surtos previamente identificados, como também a sinalização de potenciais surtos não sinalizados pelas autoridades de saúde. Neste trabalho, identificámos também vários isolados com múltiplos marcadores de resistência e diversos elementos genéticos móveis que conferem a capacidade de persistência e disseminação a estas bactérias. Esta dissertação destaca a necessidade de revisão da regulamentação associada a géneros alimentícios e de estabelecimento de novas medidas de controlo de infeção na produção animal, de maneira a evitar a disseminação de marcadores de multirresistência e persistência, que contribuem grandemente para o aumento da severidade das infeções causadas por Salmonella, e também da disseminação dessas características altamente transmissíveis. Este trabalho representa uma importante contribuição para o conhecimento dos isolados de Salmonella enterica circulantes em Portugal, e também para o reforço da capacitação do Laboratório Nacional de Referência.N/

Of Animal and Men: The Importance of Animal Environment to Antimicrobial Resistance: A One Health Approach

The contribution of the animal environments to the worsening of the global antimicrobial resistance framework is related to the use of antimicrobials in subtherapeutic doses and, for long periods, establishing ideal conditions for the circulation of resistance genes, which can be transmitted to pathogens adapted to the human microbiota. The study of the animal environment as conducive to the acceleration of resistance evolution is an emerging and critical area for understanding the development and dissemination of resistance genes among the circulating bacteria. The connection between people, animals, and the environment allows us to consider antimicrobial resistance in an approach within the “One Health” concept, which provides a global strategy for expanding collaboration and interdisciplinary communication. This chapter will highlight the emergence of colistin resistance, a great challenge in antimicrobial resistance field. Also, it will focus on some agents included in the priority list of superbugs of the World Health Organization (WHO) or correlated species already identified in veterinary medicine, such as the critical superbugs; priority level 1, Carbapenem-resistant Acinetobacter baumannii, Carbapenem-resistant Pseudomonas aeruginosa, and ESBL-producing Carbapenemic-resistant Enterobacteriaceae; and the high-priority, level 2, methicillin-resistant Staphylococcus aureus (MRSA)

Reducing the risk of transmission of critical antimicrobial resistance determinants from contaminated pork products to humans in South-East Asia

Antimicrobial resistance (AMR) is a critical challenge worldwide as it impacts public health, especially via contamination in the food chain and in healthcare-associated infections. In relation to farming, the systems used, waste management on farms, and the production line process are all determinants reflecting the risk of AMR emergence and rate of contamination of foodstuffs. This review focuses on South East Asia (SEA), which contains diverse regions covering 11 countries, each having different levels of development, customs, laws, and regulations. Routinely, here as elsewhere antimicrobials are still used for three indications: therapy, prevention, and growth promotion, and these are the fundamental drivers of AMR development and persistence. The accuracy of detection of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) depends on the laboratory standards applicable in the various institutes and countries, and this affects the consistency of regional data. Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the standard proxy species used for indicating AMR-associated nosocomial infections and healthcare-associated infections. Pig feces and wastewater have been suspected as one of the hotspots for spread and circulation of ARB and ARG. As part of AMR surveillance in a One Health approach, clonal typing is used to identify bacterial clonal transmission from the production process to consumers and patients – although to date there have been few published definitive studies about this in SEA. Various alternatives to antibiotics are available to reduce antibiotic use on farms. Certain of these alternatives together with improved disease prevention methods are essential tools to reduce antimicrobial usage in swine farms and to support global policy. This review highlights evidence for potential transfer of resistant bacteria from food animals to humans, and awareness and understanding of AMR through a description of the occurrence of AMR in pig farm food chains under SEA management systems. The latter includes a description of standard pig farming practices, detection of AMR and clonal analysis of bacteria, and AMR in the food chain and associated environments. Finally, the possibility of using alternatives to antibiotics and improving policies for future strategies in combating AMR in a SEA context are outlined

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

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

Molecular Characterization and Survive Abilities of Salmonella Heidelberg Strains of Poultry Origin in Brazil

The aim of the study was to evaluate the genotypic and phenotypic characteristics of 20 strains of S. Heidelberg (SH) isolated from broilers produced in southern Brazil. The similarity and presence of genetic determinants linked to virulence, antimicrobial resistance, biofilm formation, and in silico-predicted metabolic interactions revealed this serovar as a threat to public health. The presence of the ompC, invA, sodC, avrA, lpfA, and agfA genes was detected in 100% of the strains and the luxS gene in 70% of them. None of the strains carries the blaSHV, mcr-1, qnrA, qnrB, and qnrS genes. All strains showed a multidrug-resistant profile to at least three non-β-lactam drugs, which include colistin, sulfamethoxazole, and tetracycline. Resistance to penicillin, ceftriaxone (90%), meropenem (25%), and cefoxitin (25%) were associated with the presence of blaCTX–M and blaCMY–2 genes. Biofilm formation reached a mature stage at 25 and 37°C, especially with chicken juice (CJ) addition. The sodium hypochlorite 1% was the least efficient in controlling the sessile cells. Genomic analysis of two strains identified more than 100 virulence genes and the presence of resistance to 24 classes of antibiotics correlated to phenotypic tests. Protein-protein interaction (PPI) prediction shows two metabolic pathways correlation with biofilm formation. Virulence, resistance, and biofilm determinants must be constant monitoring in SH, due to the possibility of occurring infections extremely difficult to cure and due risk of the maintenance of the bacterium in production environments

Genomic microbial epidemiology is needed to comprehend the global problem of antibiotic resistance and to improve pathogen diagnosis

Contamination of waste effluent from hospitals and intensive food animal production with antimicrobial residues is an immense global problem. Antimicrobial residues exert selection pressures that influence the acquisition of antimicrobial resistance and virulence genes in diverse microbial populations. Despite these concerns there is only a limited understanding of how antimicrobial residues contribute to the global problem of antimicrobial resistance. Furthermore, rapid detection of emerging bacterial pathogens and strains with resistance to more than one antibiotic class remains a challenge. A comprehensive, sequence-based genomic epidemiological surveillance model that captures essential microbial metadata is needed, both to improve surveillance for antimicrobial resistance and to monitor pathogen evolution. Escherichia coli is an important pathogen causing both intestinal [intestinal pathogenic E. coli (IPEC)] and extraintestinal [extraintestinal pathogenic E. coli (ExPEC)] disease in humans and food animals. ExPEC are the most frequently isolated Gram negative pathogen affecting human health, linked to food production practices and are often resistant to multiple antibiotics. Cattle are a known reservoir of IPEC but they are not recognized as a source of ExPEC that impact human or animal health. In contrast, poultry are a recognized source of multiple antibiotic resistant ExPEC, while swine have received comparatively less attention in this regard. Here, we review what is known about ExPEC in swine and how pig production contributes to the problem of antibiotic resistance

DECRYPTING THE DIVERSITY OF MICROBIOME IN AQUACULTURE

Aquaculture can play an important role in reducing the overexploitation of natural re- sources and feeding the world’s growing population. However, the use of e.g., antibiotics in aquaculture can favor the development of resistant bacteria and jeopardize the safety of its products. Thus, this Ph.D. thesis aimed to contribute to the deciphering of aquaculture’s mi- crobiome and resistome, as well as to the understanding of the role of mobile genetic elements (MGE) in the dissemination of resistance genes in these environments. Several approaches were used, to obtain the results that most reflect the microbiome and resistome of seabream and bivalve mollusks from aquaculture. All microbiomes studied were very diverse, encompassing commensal and pathogenic bacteria from seabream and bivalve mollusks (e.g., Aeromonas,Kocuria, Pseudomonas and Vibrio genera), as well as bacteria important in human medicine (e.g., Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae). Twenty-one new se- quence types were described in Aeromonas spp., Citrobacter sp., Enterobacter spp., Shewanella spp., Staphylococcus sp. and Vibrio spp. Decreased susceptibilities to phenicols, oxytetracy- cline, β-lactams (namely carbapenems), quinolones, glycopeptides, mupirocin, erythromycin, and colistin were found. The resistome also revealed a great diversity of genes in all samples studied associated with antibiotics (e.g., blaTEM-1B, mecA, sul2, mcr-9.1), disinfectants (e.g.,formA-type), and heavy metals (e.g., sil) resistance. Twenty-five different genes related with increased virulence were also detected. Thirteen new β-lactams resistance genes were identi- fied (e.g., blaCTX-M-246, blaFOX-18, and blaOXA-958) and 35 other resistance genes, namely for antibi- otics (e.g., mcr-9 and qnrD2), heavy metals (e.g., emrA and mdtE) and disinfectants ( sitABCD- type), and virulence factors (e.g., astA and hlyF) were here described for the first time associated with aquaculture. Our results suggest that some of these resistance genes (e.g., erm(T)-type,qnrB19, catA1-type, tet(A), dfrA-type, aph(6)-Id, qacE∆ 1 and merA) are being disseminated by MGE such as plasmids, class 1 integrons, and Tn As1. These findings not only expand our knowledge about aquaculture’s microbiome and resistome, but also provide the necessary xiv information to implement the most suitable measures to control antibiotic resistance in aqua- culture environments.A aquacultura pode desempenhar um papel importante na redução da sobre-explora- ção dos recursos naturais e no fornecimento de alimentos para a crescente população mundial. No entanto, o uso de, por exemplo, antibióticos em aquacultura pode favorecer o desenvolvi- mento de bactérias resistentes e comprometer a segurança dos seus produtos. Assim, esta tese de Doutoramento teve como objetivo contribuir para a decifração do microbioma e resistoma em aquacultura, bem como compreender o papel dos elementos genéticos móveis na disse- minação de genes de resistência nestes ambientes. Foram usadas diversas abordagens com o intuito de obter os resultados que melhor refletem o microbioma e o resistoma das douradas e moluscos bivalves de aquacultura. Os microbiomas estudados apresentaram uma grande diversidade, englobando bactérias comensais e patogénicas das douradas e moluscos bivalves (ex.: os géneros Aeromonas, Kocuria, Pseudomonas e Vibrio), assim como bactérias importan- tes em medicina humana (ex.: Staphylococcus aureus, Escherichia coli e Klebsiella pneumo- niae). Foram descritos 21 novos sequence type em Aeromonas spp., Citrobacter sp., Entero- bacter spp., Shewanella spp., Staphylococcus sp. e Vibrio spp. Foram encontradas suscetibili- dades diminuídas aos fenicois, oxitetraciclina, β-lactâmicos (como carbapenemes), quinolonas, glicopéptidos, mupirocina, eritromicina e colistina. Também os resistomas revelaram uma grande diversidade de genes associados a resistências aos antibióticos (ex.: blaTEM-1B, mecA,sul2, mcr-9.1), desinfetantes (ex.: formA-type) e metais pesados (ex.: sil). Foram também dete- tados 25 genes diferentes, associados a uma maior virulência. Encontram-se aqui descritos 13 novos genes de resistência aos β-lactâmicos (ex.: blaCTX-M-246, blaFOX-18, blaOXA-958) e outros 35 genes de resistência, nomeadamente aos antibióticos (ex.: mcr-9 e qnrD2), metais pesados (ex.:emrA e mdtE) e desinfetantes ( sitABCD-type), e fatores de virulência (ex.: astA e hlyF) são aqui identificados pela primeira vez associados à aquacultura. Os nossos resultados sugerem que alguns destes genes de resistência (como erm(T)-type, qnrB19, catA1-type, tet(A), dfrA-type, aph(6)-Id, qacE∆ 1 e merA) estarão a ser disseminados por elementos genéticos móveis, como plasmídeos, integrões de classe 1 e Tn As1. Estes estudos não só ampliam o nosso conheci- mento sobre o microbioma e o resistoma em aquacultura, mas também providenciam a infor- mação necessária para a implementação das medidas mais adequadas ao controlo da resis- tência aos antibióticos em aquacultura