Achieving Antimicrobial Stewardship on the Global Scale: Challenges and Opportunities.

Antimicrobial resistance (AMR) has been clearly identified as a major global health challenge. It is a leading cause of human deaths and also has a toll on animals, plants, and the environment. Despite the considerable socio-economic impacts, the level of awareness of the problem remains woefully inadequate, and antimicrobials are not generally recognized as a global common good, one that everyone has a role and responsibility to conserve. It is imperative for antimicrobial stewardship to be more widely implemented to achieve better control of the AMR phenomenon. The Food and Agriculture Organization (FAO) of the United Nations plays an important role in promoting and facilitating antimicrobial stewardship. The specific needs to be addressed and barriers to be overcome, in particular, in low- and middle-income countries in order to implement antimicrobial stewardship practices in agrifood systems are being identified. As a global community, it is essential that we now move beyond discussing the AMR problem and focus on implementing solutions. Thus, FAO provides multi-pronged support for nations to improve antimicrobial stewardship through programs to strengthen governance, increase awareness, develop and enhance AMR surveillance, and implement best practices related to antimicrobial resistance in agrifood systems. For example, FAO is developing a platform to collect data on AMR in animals and antimicrobial use (AMU) in plants (InFARM), working on a campaign to reduce the need to use antimicrobials, studying the use of alternatives to the use of antimicrobials (especially those used for growth promotion) and actively promoting the implementation of the Codex Alimentarius AMR standards. Together, these will contribute to the control of AMR and also bring us closer to the achievement of multiple sustainable development goals.

Social-technical interventions to reduce antimicrobial resistance in agriculture: evidence from poultry Farmer Field Schools in Ghana and Kenya.

OBJECTIVES: To assess the feasibility of the Farmer Field School approach to address the complex problem of antimicrobial resistance in agriculture, specifically within small-to-medium-scale layer poultry systems in Ghana and Kenya. Impact was assessed across three domains relevant to the emergence and selection of antimicrobial resistance, including infection, prevention, and control practices, engagement with animal health professionals, and knowledge, attitudes, and practices on antimicrobial use and antimicrobial resistance. METHODS: Farmer Field Schools were held in Ghana (N = 2) and Kenya (N = 3) across an eight-month period with an average of 18 participants in each school. After completion, a quantitative evaluation survey was administered to participants and a sample of non-participants (Ghana; N = 97) (Kenya; N = 103). Logistic and ordinary least squares regression were used to assess differences between participants and non-participants on the three domains. RESULTS: Participation in a layer poultry Farmer Field School in Ghana and Kenya is associated with self-reported reductions in antibiotic use, particularly for prevention, an increased investment in farm infection, prevention, and control practices, including the use of footbaths and personal protective equipment, and enhanced engagement with animal health professionals. CONCLUSIONS: Antimicrobial resistance is a complex problem driven by a wide range of practices and multiple stakeholders. To holistically address these factors requires the use of complex intervention approaches. The Farmer Field School approach offers a complex intervention methodology that can reduce the emergence and spread of antimicrobial resistance in agricultural systems through targeting the variety of on-farm and off-farm factors that drive resistance.

Narratives of veterinary drug use in northern Tanzania and consequences for drug stewardship strategies in low-income and middle-income countries.

INTRODUCTION: Awareness-raising campaigns play a central role in efforts to combat drug resistance. These campaigns assume that knowledge deficits drive poor practices that increase resistance. Therefore, increasing awareness will promote prudent practices and reduce resistance. However, most awareness campaigns have been developed and evaluated in high-income and public health settings. Consequently, it is not clear whether these campaigns are effective in low-income and middle-income countries and/or within animal health settings. METHODS: Focus group discussions and in-depth interviews were used to collect narratives of veterinary drug use among Maasai pastoralists (n=70), animal health professionals (n=10) and veterinary drug sellers (n=5). Thematic analysis was used to identify recurring themes across narratives and groups. RESULTS: Narratives of Maasai and animal health professionals indicated that Maasai treated their livestock with limited input from the professional sector and that non-prudent treatment practices were observed (eg, using antimicrobials as 'energizers'). Professionals linked these practices to knowledge and attitudinal deficits among the Maasai, while Maasai narratives highlighted the importance of climatic uncertainties and cultural beliefs surrounding veterinary care. CONCLUSION: Narratives of veterinary drug use from animal health professionals are consistent with the knowledge deficit assumption guiding awareness-raising efforts. In contrast, Maasai narratives highlight how animal health practices are patterned by cultural norms interacting with factors largely outside of Maasai control, including a constrained professional veterinary sector. If these cultural and structural contexts remain unconsidered in awareness-raising strategies, current campaigns are unlikely to motivate practices necessary to limit drug resistance, especially within low-income and middle-income settings.

A systematic approach toward progressive improvement of national antimicrobial resistance surveillance systems in food and agriculture sectors.

The first Food and Agriculture Organization of the United Nations (FAO) Action Plan on antimicrobial resistance (AMR), published in 2016, identified the need to develop capacity for AMR surveillance and monitoring in food and agriculture sectors. As part of this effort, FAO has developed the "Assessment Tool for Laboratories and AMR Surveillance Systems" (FAO-ATLASS) to assist countries in systematically assessing their AMR surveillance system in food and agriculture. FAO-ATLASS includes two different modules for surveillance and laboratory assessment. Each module includes two questionnaires that collect either qualitative or semi-quantitative data to describe and score the performance of national AMR surveillance system data production network, data collection and analysis, governance, communication and overall sustainability in a standardized manner. Based on information captured in the questionnaire by trained assessors (1) tables and figures describing the outputs of the surveillance system are automatically generated (2) a Progressive Improvement Pathway (PIP) stage, ranging from "1-limited" to "5-sustainable", is assigned to each laboratory assessed in the country, each area of the surveillance system and also to the overarching national AMR surveillance system. FAO-ATLASS allows national authorities to implement a strategic stepwise approach to improving their AMR surveillance systems via the FAO-ATLASS PIP system and provides an evidence base for actions and advocacy. The implementation of FAO-ATLASS at regional and global levels can contribute to harmonize and better coordinate strategies aimed at implementing an integrated AMR surveillance system under the One Health approach.

Knowing Is Not Enough: A Mixed-Methods Study of Antimicrobial Resistance Knowledge, Attitudes, and Practises Among Maasai Pastoralists.

Global, national, and local efforts to limit antimicrobial resistance (AMR) often stress the importance of raising awareness among users, sellers, and prescribers of antimicrobial drugs. This emphasis is founded upon two assumptions. First, awareness is limited, particularly concerning the links between antimicrobial use (AMU) and AMR. Second, "filling the awareness gaps" will motivate practises that will limit AMR. The first assumption is supported by knowledge, attitudes, and practises (KAP) surveys but these same studies provide mixed support for the second, with several studies finding that knowledge and attitudes are not correlated with related practises. This disconnect may arise as these surveys typically do not collect data on the cultural or historical contexts that pattern AMU. To explore how these contexts impact KAP related to AMU and AMR, we use a mixed-methods approach to examine veterinary practises among Maasai pastoralists in Tanzania. We combine a quantitative KAP survey (N = 195 households) with extensive qualitative data from focus group discussions (N = 55 participants). Results document limited awareness of AMR but also find that knowledge and attitudes are not correlated with practise. Thematic analysis of qualitative data pointed to three reasons behind this disconnect, including (1) Maasai self-perceptions as veterinary experts, (2) the central role of livestock in Maasai culture, and (3) the use of ethnoveterinary knowledge in animal health treatment. We argue that mixed-method approaches will be critical to developing the targeted awareness campaigns needed to limit the emergence and transmission of AMR.

Understanding Antimicrobial Use Contexts in the Poultry Sector: Challenges for Small-Scale Layer Farms in Kenya.

The poultry sector contributes significantly to Kenya's food and economic security. This contribution is expected to rise dramatically with a growing population, urbanization, and preferences for animal-source foods. Antimicrobial resistance is putting the poultry sector in Kenya-and worldwide-at risk of production losses due to the failure of medicines for animal (and human) health. The emergence and spread of antimicrobial resistance has been linked to overuse and misuse of antimicrobials in poultry and other sectors. Previous studies have documented poultry farmer antimicrobial use but without systematic consideration of the contexts (i.e., drivers) as important targets for behavior change, particularly in low- and middle-income countries. To improve understanding of antimicrobial use patterns in poultry systems, we conducted a mixed-methods knowledge, attitudes, and practices study of 76 layer farms in Kiambu County; Kenya. We found that commonly used antibiotics were often labeled for prophylactic, growth promotion, and egg production improvement purposes. Antimicrobial use was also motivated by the presence of diseases/disease symptoms, most of which could instead be managed through infection prevention measures. The results suggest that improving vaccination and biosecurity practices on farms and engaging with drug-makers to ensure proper labeling and marketing of antimicrobial drugs may represent important areas of opportunity for social behavior change communication and/or behavioral science interventions (i.e., nudges) to reduce disease burdens and promote prudent antimicrobial use. We conclude our findings with suggestions for further research into the behavioral insights at play in these scenarios to fuel future intervention development.

"We are doctors": Drivers of animal health practices among Maasai pastoralists and implications for antimicrobial use and antimicrobial resistance.

Animal health service providers can play an important role in limiting drug resistance by promoting responsible and prudent use of veterinary drugs. Recognizing this potential, international agencies and governments have called for these providers to receive drug stewardship training, particularly providers in low- and middle-income countries where top-down regulations (e.g., national regulation of veterinary prescriptions) are largely unfeasible. The success of these stewardship trainings to promote responsible and prudent use will depend on many factors, including understanding how livestock-keeping communities currently interact with animal health service providers. Here, we use a mixed methods approach to identify and understand animal health seeking practices among Maasai pastoralists in Tanzania. Combining qualitative interviews (N = 31) and structured surveys (N = 195), we show the majority of Maasai respondents (≈80 %) do not frequently consult animal health service providers with most relying on advice from family and friends. Logistic regression models of health seeking practices find that increasing age, education, observance of treatment failure, and herd disease burdens are associated with greater odds of seeking out health services. Quantitative results were supported by data from focus group discussions and in-depth interviews that showed Maasai view animal health service providers as measures of last resort, whose input is largely sought after self-treatment with veterinary drugs fail. We argue patterns of animal health seeking among the Maasai are partially the consequence of their high confidence in their own abilities in livestock disease and treatment and generally low confidence in the skills of animal health service providers. We link this high sense of self-efficacy to the culturally engrained process by which Maasai develop mastery in animal health and how the roles and norms in Maasai culture surrounding animal health influence Maasai perceptions of animal health professionals. Our results highlight the need for more research to understand Maasai perceptions of animal health service providers as well as the knowledge, attitudes, and practices of these providers. Finally, our study emphasizes that the success of drug stewardship trainings will require efforts to first understand the cultural and historical contexts driving health seeking practices that impact perceptions of animal health service providers and animal health practices more generally.

The Impacts of Animal Health Service Providers on Antimicrobial Use Attitudes and Practices: An Examination of Poultry Layer Farmers in Ghana and Kenya.

International organizations and governments have argued that animal health service providers can play a vital role in limiting antimicrobial resistance by promoting the prudent use of antimicrobials. However, there is little research on the impact of these service providers on prudent use at the farm level, especially in low- and middle-income countries where enforcement of prudent-use regulations is limited. Here, we use a mixed-methods approach to assess how animal health-seeking practices on layer farms in Ghana (n = 110) and Kenya (n = 76) impact self-reported antimicrobial usage, engagement in prudent administration and withdrawal practices and perceptions of antimicrobial resistance. In general, our results show that the frequency of health-seeking across a range of service providers (veterinarians, agrovets, and feed distributors) does not significantly correlate with prudent or non-prudent use practices or the levels of antimicrobials used. Instead, we find that patterns of antimicrobial use are linked to how much farmers invest in biosecurity (e.g., footbaths) and the following vaccination protocols. Our results emphasize that more research is required to understand the interactions between animal health service providers and farmers regarding antimicrobial use and antimicrobial resistance. Addressing these gaps will be crucial to inform antimicrobial stewardship training, curriculums and, guidelines whose ultimate purpose is to limit the selection and transmission of antimicrobial resistance.

Antimicrobials Used in Backyard and Commercial Poultry and Swine Farms in the Philippines: A Qualitative Pilot Study.

Chicken and pork are the most frequently consumed meat products in the Philippines. Swine and poultry are reared in either commercial farms (CMf) or backyard farms (BYf); the latter production system is relatively common and essential to food security in low- and middle-income countries (LMICs) such as the Philippines. Similar to resource-limited LMICs, antimicrobial use (AMU) surveillance has not yet been established; thus, AMU in food animals is a knowledge gap in understanding the emergence of antimicrobial resistance (AMR) in zoonotic foodborne bacteria in the country. This qualitative AMU pilot study aims to describe the antimicrobial active ingredients (AAIs) used and associated AMU practices (e.g., source of AAIs and informed AMU decisions) by poultry and swine CMf and BYf in the Philippines. Ninety-three farms across four regions in the Philippines voluntarily provided AMU information as part of a larger biosecurity and good practices study. The percentage of farms using AAI over the total number of farms was the metric used to describe AMU. In total, there were 30 AAIs used (CMf: n =27 and BYf: n = 13); per farm, the number of AAIs used ranged from 1 to 7. The spectrum of AAIs was more diverse in swine (n = 24) compared to poultry (n = 18). Enrofloxacin was the most frequently reported AAI in poultry (33%) and swine (36%) farms. Respiratory diseases were the most frequently reported reason for AMU in both species. Between production systems, significant differences were observed in the percentage of farms using amoxicillin (27% CMf vs. 3% BYf), colistin (17% CMf vs. 3% BYf), and oxytetracycline (12% CMf vs. 39% BYf). In terms of AMU practices, of important concern was the over-the-counter access of AAIs at retail outlets and the limited veterinary oversight in BYf. Our data indicated that antimicrobials critically important for human medicine are frequently used in poultry and swine farms in the Philippines. This study can inform the development of guidelines for curbing AMR through prudent AMU and serves as a reference point for AMU surveillance capacity development in the Philippines.

Description and determinants of the faecal resistome and microbiome of farmers and slaughterhouse workers: A metagenome-wide cross-sectional study.

BACKGROUND: By studying the entire human faecal resistome and associated microbiome, the diversity and abundance of faecal antimicrobial resistance genes (ARGs) can be comprehensively characterized. Prior culture-based studies have shown associations between occupational exposure to livestock and carriage of specific antimicrobial resistant bacteria. Using shotgun metagenomics, the present study investigated 194 faecal resistomes and bacteriomes from humans occupationally exposed to ARGs in livestock (i.e. pig and poultry farmers, employees and family members and pig slaughterhouse workers) and a control population (Lifelines cohort) in the Netherlands. In addition, we sought to identify determinants for the human resistome and bacteriome composition by applying a combination of multivariate (NMDS, PERMANOVA, SIMPER and DESeq2 analysis) and multivariable regression analysis techniques. RESULTS: Pig slaughterhouse workers and pig farmers carried higher total ARG abundances in their stools compared to broiler farmers and control subjects. Tetracycline, ß-lactam and macrolide resistance gene clusters dominated the resistome of all studied groups. No significant resistome alpha diversity differences were found among the four populations. However, the resistome beta diversity showed a separation of the mean resistome composition of pig and pork exposed workers from broiler farmers and controls, independent of their antimicrobial use. We demonstrated differences in resistome composition between slaughter line positions, pig versus poultry exposed workers, as well as differences between farmers and employees versus family members. In addition, we found a significant correlation between the bacteriome and resistome, and significant differences in the bacteriome composition between and within the studied subpopulations. Finally, an in-depth analysis of pig and poultry farms - of which also farm livestock resistomes were analysed - showed positive associations between the number of on-farm working hours and human faecal AMR loads. CONCLUSION: We found that the total normalized faecal ARG carriage was larger in persons working in the Dutch pork production chain compared to poultry farmers and controls. Additionally, we showed significant differences in resistome and bacteriome composition of pig and pork exposed workers compared to a control group, as well as within-population (farms, slaughterhouse) compositional differences. The number of on-farm working hours and the farm type (pig or broiler) that persons live or work on are determinants for the human faecal resistome. Overall, our results may suggest direct or indirect livestock contact as a determinant for human ARG carriage. Future studies should further focus on the connection between the human and livestock resistome (i.e. transmission routes) to substantiate the evidence for livestock-associated resistome acquisition.

Towards a bottom-up understanding of antimicrobial use and resistance on the farm: A knowledge, attitudes, and practices survey across livestock systems in five African countries.

The nutritional and economic potentials of livestock systems are compromised by the emergence and spread of antimicrobial resistance. A major driver of resistance is the misuse and abuse of antimicrobial drugs. The likelihood of misuse may be elevated in low- and middle-income countries where limited professional veterinary services and inadequately controlled access to drugs are assumed to promote non-prudent practices (e.g., self-administration of drugs). The extent of these practices, as well as the knowledge and attitudes motivating them, are largely unknown within most agricultural communities in low- and middle-income countries. The main objective of this study was to document dimensions of knowledge, attitudes and practices related to antimicrobial use and antimicrobial resistance in livestock systems and identify the livelihood factors associated with these dimensions. A mixed-methods ethnographic approach was used to survey households keeping layers in Ghana (N = 110) and Kenya (N = 76), pastoralists keeping cattle, sheep, and goats in Tanzania (N = 195), and broiler farmers in Zambia (N = 198), and Zimbabwe (N = 298). Across countries, we find that it is individuals who live or work at the farm who draw upon their knowledge and experiences to make decisions regarding antimicrobial use and related practices. Input from animal health professionals is rare and antimicrobials are sourced at local, privately owned agrovet drug shops. We also find that knowledge, attitudes, and particularly practices significantly varied across countries, with poultry farmers holding more knowledge, desirable attitudes, and prudent practices compared to pastoralist households. Multivariate models showed that variation in knowledge, attitudes and practices is related to several factors, including gender, disease dynamics on the farm, and source of animal health information. Study results emphasize that interventions to limit antimicrobial resistance should be founded upon a bottom-up understanding of antimicrobial use at the farm-level given limited input from animal health professionals and under-resourced regulatory capacities within most low- and middle-income countries. Establishing this bottom-up understanding across cultures and production systems will inform the development and implementation of the behavioral change interventions to combat antimicrobial resistance globally.

Occupational Exposure and Carriage of Antimicrobial Resistance Genes (tetW, ermB) in Pig Slaughterhouse Workers.

OBJECTIVES: Slaughterhouse staff is occupationally exposed to antimicrobial resistant bacteria. Studies reported high antimicrobial resistance gene (ARG) abundances in slaughter pigs. This cross-sectional study investigated occupational exposure to tetracycline (tetW) and macrolide (ermB) resistance genes and assessed determinants for faecal tetW and ermB carriage among pig slaughterhouse workers. METHODS: During 2015-2016, 483 faecal samples and personal questionnaires were collected from workers in a Dutch pig abattoir, together with 60 pig faecal samples. Human dermal and respiratory exposure was assessed by examining 198 carcass, 326 gloves, and 33 air samples along the line, next to 198 packed pork chops to indicate potential consumer exposure. Samples were analyzed by qPCR (tetW, ermB). A job exposure matrix was created by calculating the percentage of tetW and ermB positive carcasses or gloves for each job position. Multiple linear regression models were used to link exposure to tetW and ermB carriage. RESULTS: Workers are exposed to tetracycline and macrolide resistance genes along the slaughter line. Tetw and ermB gradients were found for carcasses, gloves, and air filters. One packed pork chop contained tetW, ermB was non-detectable. Human faecal tetW and ermB concentrations were lower than in pig faeces. Associations were found between occupational tetW exposure and human faecal tetW carriage, yet, not after model adjustments. Sampling round, nationality, and smoking were determinants for ARG carriage. CONCLUSION: We demonstrated clear environmental tetracycline and macrolide resistance gene exposure gradients along the slaughter line. No robust link was found between ARG exposure and human faecal ARG carriage.

Attributable sources of community-acquired carriage of Escherichia coli containing ß-lactam antibiotic resistance genes: a population-based modelling study.

BACKGROUND: Extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC), plasmid-mediated AmpC-producing E coli (pAmpC-EC), and other bacteria are resistant to important ß-lactam antibiotics. ESBL-EC and pAmpC-EC are increasingly reported in animals, food, the environment, and community-acquired and health-care-associated human infections. These infections are usually preceded by asymptomatic carriage, for which attributions to animal, food, environmental, and human sources remain unquantified. METHODS: In this population-based modelling study, we collected ESBL and pAmpC gene data on the Netherlands population for 2005-17 from published datasets of gene occurrences in E coli isolates from different sources, and from partners of the ESBL Attribution Consortium and the Dutch National Antimicrobial Surveillance System. Using these data, we applied an established source attribution model based on ESBL-EC and pAmpC-EC prevalence and gene data for humans, including high-risk populations (ie, returning travellers, clinical patients, farmers), farm and companion animals, food, surface freshwater, and wild birds, and human exposure data, to quantify the overall and gene-specific attributable sources of community-acquired ESBL-EC and pAmpC-EC intestinal carriage. We also used a simple transmission model to determine the basic reproduction number (R0) in the open community. FINDINGS: We identified 1220 occurrences of ESBL-EC and pAmpC-EC genes in humans, of which 478 were in clinical patients, 454 were from asymptomatic carriers in the open community, 103 were in poultry and pig farmers, and 185 were in people who had travelled out of the region. We also identified 6275 occurrences in non-human sources, including 479 in companion animals, 4026 in farm animals, 66 in wild birds, 1430 from food products, and 274 from surface freshwater. Most community-acquired ESBL-EC and pAmpC-EC carriage was attributed to human-to-human transmission within or between households in the open community (60·1%, 95% credible interval 40·0-73·5), and to secondary transmission from high-risk groups (6·9%, 4·1-9·2). Food accounted for 18·9% (7·0-38·3) of carriage, companion animals for 7·9% (1·4-19·9), farm animals (non-occupational contact) for 3·6% (0·6-9·9), and swimming in freshwater and wild birds (ie, environmental contact) for 2·6% (0·2-8·7). We derived an R0 of 0·63 (95% CI 0·42-0·77) for intracommunity transmission. INTERPRETATION: Although humans are the main source of community-acquired ESBL-EC and pAmpC-EC carriage, the attributable non-human sources underpin the need for longitudinal studies and continuous monitoring, because intracommunity ESBL-EC and pAmpC-EC spread alone is unlikely to be self-maintaining without transmission to and from non-human sources. FUNDING: 1Health4Food, Dutch Ministry of Economic Affairs, and the EU's Horizon-2020 through One-Health European Joint Programme.

Associations between antimicrobial use and the faecal resistome on broiler farms from nine European countries.

OBJECTIVES: To determine associations between farm- and flock-level antimicrobial usage (AMU), farm biosecurity status and the abundance of faecal antimicrobial resistance genes (ARGs) on broiler farms. METHODS: In the cross-sectional pan-European EFFORT study, conventional broiler farms were visited and faeces, AMU information and biosecurity records were collected. The resistomes of pooled faecal samples were determined by metagenomic analysis for 176 farms. A meta-analysis approach was used to relate total and class-specific ARGs (expressed as fragments per kb reference per million bacterial fragments, FPKM) to AMU (treatment incidence per DDD, TIDDDvet) per country and subsequently across all countries. In a similar way, the association between biosecurity status (Biocheck.UGent) and the resistome was explored. RESULTS: Sixty-six (38%) flocks did not report group treatments but showed a similar resistome composition and roughly similar ARG levels to antimicrobial-treated flocks. Nevertheless, we found significant positive associations between ß-lactam, tetracycline, macrolide and lincosamide, trimethoprim and aminoglycoside antimicrobial flock treatments and ARG clusters conferring resistance to the same class. Similar associations were found with purchased products. In gene-level analysis for ß-lactams and macrolides, lincosamides and streptogramins, a significant positive association was found with the most abundant gene clusters blaTEM and erm(B). Little evidence was found for associations with biosecurity. CONCLUSIONS: The faecal microbiome in European broilers contains a high diversity of ARGs, even in the absence of current antimicrobial selection pressure. Despite this, the relative abundance of genes and the composition of the resistome is positively related to AMU in European broiler farms for several antimicrobial classes.

The antimicrobial resistome in relation to antimicrobial use and biosecurity in pig farming, a metagenome-wide association study in nine European countries.

OBJECTIVES: Previous studies in food-producing animals have shown associations between antimicrobial use (AMU) and resistance (AMR) in specifically isolated bacterial species. Multi-country data are scarce and only describe between-country differences. Here we investigate associations between the pig faecal mobile resistome and characteristics at the farm-level across Europe. METHODS: A cross-sectional study was conducted among 176 conventional pig farms from nine European countries. Twenty-five faecal samples from fattening pigs were pooled per farm and acquired resistomes were determined using shotgun metagenomics and the Resfinder reference database, i.e. the full collection of horizontally acquired AMR genes (ARGs). Normalized fragments resistance genes per kilobase reference per million bacterial fragments (FPKM) were calculated. Specific farm-level data (AMU, biosecurity) were collected. Random-effects meta-analyses were performed by country, relating farm-level data to relative ARG abundances (FPKM). RESULTS: Total AMU during fattening was positively associated with total ARG (total FPKM). Positive associations were particularly observed between widely used macrolides and tetracyclines, and ARGs corresponding to the respective antimicrobial classes. Significant AMU-ARG associations were not found for ß-lactams and only few colistin ARGs were found, despite high use of these antimicrobial classes in younger pigs. Increased internal biosecurity was directly related to higher abundances of ARGs mainly encoding macrolide resistance. These effects of biosecurity were independent of AMU in mutually adjusted models. CONCLUSIONS: Using resistome data in association studies is unprecedented and adds accuracy and new insights to previously observed AMU-AMR associations. Major components of the pig resistome are positively and independently associated with on-farm AMU and biosecurity conditions.

Molecular relatedness of ESBL/AmpC-producing Escherichia coli from humans, animals, food and the environment: a pooled analysis.

Background: In recent years, ESBL/AmpC-producing Escherichia coli (ESBL/AmpC-EC) have been isolated with increasing frequency from animals, food, environmental sources and humans. With incomplete and scattered evidence, the contribution to the human carriage burden from these reservoirs remains unclear. Objectives: To quantify molecular similarities between different reservoirs as a first step towards risk attribution. Methods: Pooled data on ESBL/AmpC-EC isolates were recovered from 35 studies in the Netherlands comprising >27 000 samples, mostly obtained between 2005 and 2015. Frequency distributions of ESBL/AmpC genes from 5808 isolates and replicons of ESBL/AmpC-carrying plasmids from 812 isolates were compared across 22 reservoirs through proportional similarity indices (PSIs) and principal component analyses (PCAs). Results: Predominant ESBL/AmpC genes were identified in each reservoir. PCAs and PSIs revealed close human-animal ESBL/AmpC gene similarity between human farming communities and their animals (broilers and pigs) (PSIs from 0.8 to 0.9). Isolates from people in the general population had higher similarities to those from human clinical settings, surface and sewage water and wild birds (0.7-0.8), while similarities to livestock or food reservoirs were lower (0.3-0.6). Based on rarefaction curves, people in the general population had more diversity in ESBL/AmpC genes and plasmid replicon types than those in other reservoirs. Conclusions: Our 'One Health' approach provides an integrated evaluation of the molecular relatedness of ESBL/AmpC-EC from numerous sources. The analysis showed distinguishable ESBL/AmpC-EC transmission cycles in different hosts and failed to demonstrate a close epidemiological linkage of ESBL/AmpC genes and plasmid replicon types between livestock farms and people in the general population.

Farmers' knowledge and expectations of antimicrobial use and resistance are strongly related to usage in Dutch livestock sectors.

Comprehensive strategies to improve on-farm antimicrobial use (AMU) are needed to contain antimicrobial resistance (AMR). Little is known about farmers' motivating and enabling factors, and about their influence on AMU. In a cross-sectional online survey, Dutch dairy, veal and pig farmers (n=457) reported their on-farm AMU as "Defined Daily Dose Animal" per year (DDDAF) and completed a detailed questionnaire on their view, knowledge and behavior towards AMU and AMR. Exploratory factor analysis (EFA) on the questionnaire items identified four psychological factors labeled as 'referent beliefs', 'perceived risk', 'knowledge', and 'undesired attitude to regulations'. Linear regression was done to explore the relationship between the obtained factors and on-farm AMU across the three animal sectors. Dairy farmers showed the highest factor scores for 'knowledge' and the lowest for 'perceived risk'. 'Knowledge' scores were significantly and inversely related to AMU (P=0.0004). Borderline significant associations with AMU were found for 'perceived risk' and 'undesired attitude to regulations' (negative and positive relationships respectively). There were no apparent differences for these relationships between the three livestock sectors. Behavioral interventions in farmers such as educational campaigns or increased support by veterinarians could empower farmers with more prudent and rational practices, eventually reducing AMU in food animals.

Risk factors for ESBL-producing Escherichia coli on pig farms: A longitudinal study in the context of reduced use of antimicrobials.

The presence of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) in food animals is a public health concern. This study aimed to determine prevalence of ESBL-E. coli on pig farms and to assess the effect of reducing veterinary antimicrobial use (AMU) and farm management practices on ESBL-E. coli occurrence on pig farms. During 2011-2013, 36 Dutch conventional pig farms participated in a longitudinal study (4 sampling times in 18 months). Rectal swabs were taken from 60 pigs per farm and pooled per 6 pigs within the same age category. Presence of ESBL-E. coli was determined by selective plating and ESBL genes were characterized by microarray, PCR and gene sequencing. An extensive questionnaire on farm characteristics and AMU as Defined Daily Dosages per Animal Year (DDDA/Y) was available for the 6-month periods before each sampling moment. Associations between the presence of ESBL-E. coli-positive pigs and farm management practices were modelled with logistic regression. The number of farms with ESBL-E. coli carrying pigs decreased from 16 to 10 and the prevalence of ESBL-E. coli-positive pooled pig samples halved from 27% to 13%. Overall, the most detected ESBL genes were blaCTX-M-1, blaTEM-52 and blaCTX-M-14. The presence of ESBL-E. coli carrying pigs was not related to total AMU, but it was strongly determined by the presence or absence of cephalosporin use at the farm (OR = 46.4, p = 0.006). Other farm management factors, related with improved biosecurity, were also plausibly related to lower probabilities for ESBL-E. coli-positive farms (e.g. presence of a hygiene lock, pest control delivered by a professional). In conclusion, ESBL-E. coli prevalence decreased in pigs during 2011 and 2013 in the Netherlands. On pig farms, the use of cephalosporins was associated with the presence of ESBL-E. coli carrying pigs.

Quantitative assessment of antimicrobial resistance in livestock during the course of a nationwide antimicrobial use reduction in the Netherlands.

OBJECTIVES: To quantify associations between antimicrobial use and acquired resistance in indicator Escherichia coli over a period of time which involved sector-wide antimicrobial use reductions in broilers and pigs (years 2004-14), veal calves (2007-14) and dairy cattle (2005-14). Prevalence estimates of resistance were predicted for a hypothetical further decrease in antimicrobial use. METHODS: Data reported annually for the resistance surveillance programme in the Netherlands were retrieved. Two multivariate random-effects logistic models per animal sector were used to relate total and class-specific antimicrobial use (as defined daily dosages per animal per year, DDDA/Y) with the probability of E. coli resistance to a panel of 10 antimicrobial agents. RESULTS: Positive dose-response relationships (ORs) were obtained from all models. Specific resistance phenotypes were more often associated with total antimicrobial use than with class-specific use. The most robust associations were found in pigs and veal calves. Resistance to historically widely used antimicrobials (e.g. penicillins, tetracyclines) was, in relative terms, less influenced by drug use changes over time than resistance to newer or less prescribed antimicrobials (e.g. third-/fourth-generation cephalosporins, fluoroquinolones). In pigs and veal calves, prevalence estimates for the most common resistance phenotypes were projected to decline ∼5%-25% during 2014-16 if total antimicrobial use reduction reached 80%; projections for poultry and dairy cows were more modest. CONCLUSIONS: Epidemiological evidence indicated that drug use history and co-selection of resistance are key elements for perpetuation of resistance. Data suggest that recent Dutch policies aimed at reducing total use of antimicrobials have decreased E. coli resistance in the pig and veal calf production sectors while the impact on the dairy cattle and poultry sectors is less clear.

Transmission through air as a possible route of exposure for MRSA.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is highly prevalent in pigs and veal calves. The environment and air in pig and veal calf barns is often contaminated with LA-MRSA, and can act as a transmission source for humans. This study explores exposure-response relationships between sequence type 398 (ST398) MRSA air exposure level and nasal ST398 MRSA carriage in people working and/or living on farms. Samples and data were used from three longitudinal field studies in pig and veal calf farm populations. Samples consisted of nasal swabs from the human participants and electrostatic dust fall collectors capturing airborne settled dust in barns. In both multivariate and mutually adjusted analyses, a strong association was found between nasal ST398 MRSA carriage in people working in the barns for >20 h per week and MRSA air levels. In people working in the barns < 20 h per week there was a strong association between nasal carriage and number of working hours. Exposure to ST398 MRSA in barn air seems to be an important determinant for nasal carriage, especially in the highly exposed group of farmers, next to duration of contact with animals. Intervention measures should therefore probably also target reduction of ST398 MRSA air levels.