Jean-Baptiste Bahers
email: jeanbaptiste.bahers@univ-nantes.fr

Simron Singh
email: simron.singh@uwaterloo.ca

Mathieu Durand
email: mathieu.durand@univ-lemans.fr

Analyzing socio-metabolic vulnerability: Evidence from the Comoros archipelago

Keywords: socio-metabolic vulnerability, island metabolism, Socio-metabolic research, Small Islands Developing States (SIDS), Ndzuwani Island, Comoros archipelago, Material and Energy Flow Analysis

Small Island Developing States (SIDS) are often characterized as vulnerable owing to their unique geographies of smallness and remoteness, resource insecurity, and more recently from the impacts of climate change. These vulnerabilities are often manifested in resource insecurity, significant imports, poor waste management, and inability to develop economies of scale. In effect, sustaining small islands in an era of global environmental change is a task both scholars and policy makers are increasingly grappling with. Can small islands be sustainable? This research examines the social metabolism of an island system, and introduces the concept of "socio-metabolic vulnerability". As such, this research provides novel insights into the linkages between patterns of resource-use, systemic risks and vulnerability.

Results from a local material and energy flow analysis (local-MEFA) for the island of Ndzuwani (Comoros) suggests a very low level of resource-use, but at the same time heavy reliance on critical imports that cover vast distances, that are vulnerable to price and climate shocks.

Informal activities in resource extraction play an important role in lending both vulnerability and resilience to Ndzuwani. This study adds to the scarce body of literature that argues that small island economies would need to leverage resource-use patterns to build system resilience, along with bold policies and institutions that support material circularity, engages communities and fosters frugal innovation.

Introduction

Vulnerabilities associated with island economies

Islands are sites of compound events and multiple vulnerabilities [START_REF] Pelling | Small island developing states: natural disaster vulnerability and global change[END_REF]Uitto 2001, UNEP 2019). They suffer disproportionally from the effects of climate change such as sealevel rise, frequent and intense hurricanes, increased flooding, droughts, and water-stress [START_REF] Kelman | Hearing local voices from Small Island Developing States for climate change[END_REF]. Limited resources and waste absorption capacity, isolation from markets, and heavy reliance on imports to meet basic needs such as food and energy characterize island systems [START_REF] Baldacchino | Islands, island studies, island studies journal[END_REF]). Shocks such as hurricanes or pandemics result in abrupt breakdown of supplies and reveal the vulnerability of island economies [START_REF] Baldacchino | The Beak of the Finch: Insights into the Economic Development of Small Economies[END_REF].

According to the new Universal Vulnerability Index (UVI) of the Commonwealth (2021)1 , more than 20 years after the research of Briguglio on the economic vulnerabilities of islands [START_REF] Briguglio | Small island developing states and their economic vulnerabilities[END_REF], the Small Islands Developing States (SIDS) still have the lead as the most vulnerable territories. Building on the Economic and Environmental Vulnerability Index (EVI), the Multidimensional Vulnerability Index (MVI) highlights the acute and multidimensional vulnerability of SIDS by drawing on lessons learned from the COVID-19 pandemic. This MVI has been applied for 144 nations with a focus on six sectors (ecosystem services, food, human habitat, health, infrastructure and water), demonstrating the Caribbean countries are among the most vulnerable nations in the world [START_REF] Edmonds | A new composite climate change vulnerability index[END_REF]).

This research investigates the social metabolism of Comoros archipelago situated in the Mozambique Channel between Madagascar and the Swahili Coast in East Africa. It makes a theoretical contribution by introducing the concept of "socio-metabolic vulnerability", where "vulnerability" is defined as "the propensity or predisposition to be adversely affected" (IPCC 2014). The vulnerability analysis is a function of exposure, environmental effects, and recovery (also termed resilience or adaptive capacity) [START_REF] Turvey | Vulnerability Assessment of Developing Countries: The Case of Small-island Developing States[END_REF][START_REF] Lange | Ecological vulnerability in risk assessment -A review and perspectives[END_REF]. Several frameworks for vulnerability assessment exist [START_REF] Birkmann | Measuring vulnerability to promote disaster-resilient societies: Conceptual frameworks and definitions[END_REF], such as the school of the double structure of vulnerability [START_REF] Bohle | Vulnerability and criticality: perspectives from social geography[END_REF], the conceptual frameworks of the disaster risk community [START_REF] Bollin | Natural Disaster Network; Disaster Risk Management by Communities and Local Governments[END_REF] or the analytical framework for vulnerability assessment in the global environmental change community [START_REF] Turner | A framework for vulnerability analysis in sustainability science[END_REF].

Of these frameworks and indexes, none really consider resource-use dynamics as part of vulnerability analysis. Materials and energy flows are critical for the survival and wellbeing of any socio-economic system. By introducing the notion of socio-metabolic vulnerability, we examine the extraction, production, and circulation of resources within the system, and by way of trade, and how socio-spatial inequality is reproduced at both the local and global scale [START_REF] Schaffartzik | Global appropriation of resources causes high international material inequality -Growth is not the solution[END_REF]). Analysis of these vulnerabilities "includes several dimensions: impacts, exposure, sensitivity, capacity to adapt, and actual responses" (Romero Lankao and Qin 2011). A territory may thus be considered "vulnerable from a socio-metabolic perspective" due to the potential threats to its resource supplies, socioeconomic ways of functioning, and waste and emissions management [START_REF] Singh | Introduction: The Metabolism of Islands[END_REF].

Scholars are beginning to study islands from a socio-metabolic perspective, a field of research that systematically analyzes material stocks and flows associated with societal production and consumption. Socio-metabolic research (SMR) "links the study of socioeconomic processes with biophysical processes and thus plays a pivotal role in understanding society-nature interactions. It includes a broad range of systems science approaches for measuring, analysing and modelling of biophysical stocks and flows as well as the services they provide to society" [START_REF] Haberl | Contributions of sociometabolic research to sustainability science[END_REF]. Therefore, SMR offers crucial insights to develop strategies to reconfigure and reduce societies' use of natural resources that is compatible with ecological boundaries, while also providing essential services to reach social thresholds [START_REF] Singh | Introduction: The Metabolism of Islands[END_REF]). Understanding "island metabolism" [START_REF] Singh | Introduction: The Metabolism of Islands[END_REF]) and associated vulnerabilities is crucial for island economies to move towards more sustainable resource-use configurations, increase self-reliance, and resilience to impacts of climate change.

The objective of this research is to examine socio-metabolic vulnerability, defined as the propensity of the socio-economic system to be negatively affected by trends and patterns of social metabolism. We hypothesize that reconfiguring and restructuring of resource use patterns can be an effective adaptation strategy and build system resilience. In doing so, we focus on critical flows that lend Comoros its socio-metabolic vulnerability, mainly construction materials, biomass, wood, and waste, and identify key drivers and actors that influence the social metabolism of the archipelago. We ask: What are the trends and patterns of materials and energy flows in a given socio-economic system? What is driving those flows? What are the impacts of resource-use across geographical areas? What types of risks and vulnerabilities are associated with these flows?

Island metabolism: a scientific archipelago

Islands have "methodological utility" [START_REF] Chertow | The Hawaiian Islands: Conceptualizing an Industrial Ecology Holarchic System[END_REF]. With relatively well-defined boundaries, naturally and socially, they can be "real-world laboratories" [START_REF] Schäpke | Labs in the Real World: Advancing Transdisciplinary Research and Sustainability Transformation: Mapping the Field and Emerging Lines of Inquiry[END_REF] -an approach that is gaining momentum in addressing complex societal problems. In this respect, islands with clearly delineated boundaries are excellent geographies for sociometabolic (SMR) research, that is, tracking flows of matter and energy in and out of an economic system. An early MFA was for Trinket Island (India) [START_REF] Singh | Social Metabolism and Labour in a Local Context: Changing Environmental Relations on Trinket Island[END_REF][START_REF] Singh | Environmental relations and biophysical transition: the case of trinket island[END_REF], the same case later compared to the rise in material and energy consumption caused by excessive aid following the 2004 Asian tsunami (Singh andHaas 2016, Fetzel et al. 2018). Other notable "flow" studies on islands are Cuba (Eisenhut 2009), Iceland, Trinidad andTobago (Krausmann et al. 2014), Malta [START_REF] Conrad | Decoupling Economic Growth and Environmental Degradation: Reviewing Progress to Date in the Small Island State of Malta[END_REF], the Philippines (Martinico- [START_REF] Perez | Quel potentiel la connaissance du métabolisme territorial de l'île d'Anjouan offre-t-il en termes de mise en oeuvre de l'économie circulaire ? Mémoire de recherche[END_REF], Hawaii [START_REF] Chertow | The Hawaiian Islands: Conceptualizing an Industrial Ecology Holarchic System[END_REF]), New Caledonia [START_REF] Bahers | The "Metal-Energy-Construction Mineral" Nexus in the Island Metabolism: The Case of the Extractive Economy of New Caledonia[END_REF], and more recently, an investigation of long-term biomass flows in the Caribbean [START_REF] Rahman | Can the Caribbean localize its food system?: Evidence from biomass flow accounting[END_REF]. Some island SMR have focused only on problematic flows, in particular waste and energy.

With limited land area, and high cost of exporting waste elsewhere has exacerbated sustainability challenges on islands [START_REF] Eckelman | Island Waste Management Systems[END_REF][START_REF] Mohee | Current status of solid waste management in small island developing states: A review[END_REF], UNEP 2019). Some archipelagos are known for their difficulties in managing waste, such as Samoa, whose open landfill has more than reached its capacity [START_REF] Brown | Garbage: How population, landmass, and development interact with culture in the production of waste[END_REF], Nauru, for its mining waste and phosphate-polluted water [START_REF] Gowdy | The Physical Destruction of Nauru: An Example of Weak Sustainability[END_REF], and Corfu, where 4,000 tonnes of waste which is left behind by tourists each year [START_REF] Skordilis | Modelling of integrated solid waste management systems in an island[END_REF]). Notable waste studies on islands using SMR include aluminum flows on Fiji [START_REF] Bahers | Ecologie industrielle, gestion des déchets et territoire insulaire. L'exemple de l'archipel des Fidji[END_REF]), e-waste in five Caribbean countries [START_REF] Mohammadi | Electronic waste in the Caribbean: An impending environmental disaster or an opportunity for a circular economy? Resources[END_REF], solid waste on Grenada [START_REF] Elgie | You can't manage what you can't measure: The potential for circularity in Grenada's waste management system[END_REF], plastic waste in Trinidad and Tobago [START_REF] Shah | Plastics Waste Metabolism in a Petro-Island State: Towards Solving a "Wicked Problem" in Trinidad and Tobago[END_REF], construction waste on Samothraki Island [START_REF] Noll | The expansion of the built environment, waste generation and EU recycling targets on Samothraki, Greece: An island's dilemma[END_REF], and disaster waste on St. Martin [START_REF] Popescu | The Impact of Hurricane Irma on the Metabolism of St. Martin's Island[END_REF]. Few studies focus only energy flows on islands, which is also a problematic issue. Cecchin [START_REF] Cecchin | Material flow analysis for a sustainable resource management in island ecosystems[END_REF] assesses local policies on the consumption of fossil fuels and on agricultural production on the Galapagos Islands. Lenzen [START_REF] Lenzen | Sustainable island businesses: a case study of Norfolk Island[END_REF][START_REF] Lenzen | Cultural and socio-economic determinants of energy consumption on small remote islands[END_REF] includes material flow in a case study of Norfolk Island businesses and an energy flow study determining the energy consumption for Cocos (Keeling) Islands. The social metabolism of the Balearics argues for a shift towards more resilient low-carbon islands due to the increase in the use and demand of imported fossil fuels (Ginard-Bosch and Ramos-Martín 2016).

Studies with a focus on material "stocks" are rare, and only recently scholars have started to pay attention to islands using Material Stock Analysis (MSA), a method to quantify materials that remain in use for longer than a year, such as buildings, infrastructure, machineries, electronics and all other durable goods [START_REF] Augiseau | Studying construction materials flows and stock: A review[END_REF]. The spatial distribution of stocks, such as type of materials, how much, and where they are located are of crucial importance for island sustainability. As stocks provide critical societal services to society, insights into stock dynamics can play an important role in building system resilience from impacts of climate change, such as sea-level rise and hurricanes. [START_REF] Tanikawa | Estimates of Lost Material Stock of Buildings and Roads Due to the Great East Japan Earthquake and Tsunami[END_REF] estimated the loss in material stocks following the earthquake and tsunami in Japan. [START_REF] Merschroth | Lost Material Stock in Buildings due to Sea Level Rise from Global Warming: The Case of Fiji Islands[END_REF]) estimated the loss in stocks in Fiji due to sea-level rise and global warming. Symmes et al. [START_REF] Symmes | The weight of islands: Leveraging Grenada's material stocks to adapt to climate change[END_REF]) conducted the first spatially explicit material stock-flow analysis in the Caribbean, focusing on Grenada's metabolism of construction materials, their distribution across the different sectors of the economy, and the potential impacts from sea-level rise. [START_REF] Bradshaw | GIS-Based Material Stock Analysis (MSA) of Climate Vulnerabilities to the Tourism Industry in Antigua and Barbuda[END_REF] explores the first material stockservice relationship for any island (Antigua and Barbuda) with consequences for island sustainability under sea-level rise scenarios.

Overall, however, since the seminal works by [START_REF] Singh | Social Metabolism and Labour in a Local Context: Changing Environmental Relations on Trinket Island[END_REF], [START_REF] Deschenes | An island approach to industrial ecology: towards sustainability in the island context[END_REF], [START_REF] Eckelman | Using Material Flow Analysis to Illuminate Long-Term Waste Management Solutions in Oahu, Hawaii[END_REF], research on islands from a SMR perspective has been inadequate. Recognizing the urgency of small islands to transition to more sustainable modes of production and consumption, the International Society for Industrial Ecology (ISIE) has recently founded a special section called "Island Industrial Ecology". Further impetus to this area of research has been given by a collection of papers in a special issue "The Metabolism of Islands" [START_REF] Singh | Introduction: The Metabolism of Islands[END_REF].

Methodology: The Material Flow Analysis (MFA) of Ndzuwani island

The study area of Ndzuwani island in the Comoros

The Union of the Comoros (or Comoros) is a Small Island Developing State (SIDS) facing a number of issues that drive vulnerability, and societal efforts to adapt to it [START_REF] Altschuler | Perceptions of climate change on the Island of Providencia[END_REF][START_REF] Magnan | Towards adaptation pathways for atoll islands. Insights from the Maldives[END_REF], Piggott-McKellar et al. 2020). With 846,281 inhabitants in 2020 and an average per capita GDP of 1,445 USD/cap/yr, it has one of the lowest nominal GDPs and is classified by the UN as a least developed country. Also, the Gini coefficient (the measurement of inequality) and HDI (Human development index) are very low, respectively the 141 st and the 160 th in the world according to the World Bank and the United Nations Development Program [START_REF] Belghith | Comoros Poverty Assessment[END_REF]. Comoros has numerous structural defects in the fields of health, education, infrastructure, transport, and tourism, partly due to a lack of public and private investment [START_REF] Lenzen | A vision of ecotourism in the Comoros Islands. The University of Sydney[END_REF]. As Taglioni (2008) notes, "history and geography have conspired to place this archipelago on the margins of political and economic development and global flows". This transpires in high levels of poverty and infant mortality, low literacy, and limited life expectancy. Despite this, there is little criminality and peace reigns in the streets of Mutsamudu, the capital city.

The volcanic archipelago of the Comoros lies in the Mozambique Channel between Madagascar and the Swahili Coast in East Africa. The archipelago is composed of four islands, Ndzuwani, Ngazidja, Mwali and, Mayotte (the latter is still a municipality of France). Ndzuwani is an island with very mountainous topography, limiting its habitability. Its capital, Mutsamudu, is the only cargo port on the island. In 2017 Ndzuwani had a population of 327,382 inhabitants (according to a UN estimate). It covers an area of only 424km 2 , making it the island with the highest population density in the Comoros (772 inhabitants per km 2 ). According to a recent research on deforestation [START_REF] Boussougou | Changements de la couverture forestière dans l'île d'Anjouan entre 1995 et[END_REF], the woodland represents 65% of the land use, the agricultural land 30% and the building zones 2%. It is also the poorest island in the archipelago, engendering migratory flows towards Mayotte, belonging to France.

The political situation in the Comoros seemed to have calmed down until reforms to the revolving presidency and to the length of periods of office seriously destabilized its internal and external politics in 2018, resulting in the army stepping in. Furthermore, relations with Mayotte continue to be very tense due to problems relating to migration and the close family ties between inhabitants of Mayotte and the Comoros. The Comoros also have a trade deficit with Mayotte and colonial ties with France remain strong. Research has also shown that the population of Mayotte is far less vulnerable to climate change (Wu-Tiu-Yen 2015) and to natural catastrophes [START_REF] Legoff | Les Comores et l'aléa cyclonique dans le contexte des changements climatiques : la vulnérabilité différenciée d'Anjouan et de Mayotte[END_REF] than that of Ndzuwani. Agriculture is the largest economic sector on the Comoros (accounting for about 40% of GDP), comprising both traditional subsistence agriculture [START_REF] Robineau | Société et économie d'Anjouan (Océan Indien)[END_REF]) and cash crops for export, such as vanilla, ylang-ylang, and cloves, controlled by local communities with a trading history [START_REF] Blanchy | Anjouan (Comores), un noeud dans les réseaux de l'océan Indien. Émergence et rôle d'une société urbaine lettrée et marchande (XVIIe-XXe siècle)[END_REF]. Thus, most of the population is rural (about 70%), but with a population shift towards the towns on the coast [START_REF] Gérard | Étalement urbain et transformation de la structure urbaine de deux capitales insulaires : Moroni et Mutsamudu, archipel des Comores (océan Indien)[END_REF].

Conducting a Material Flow Analysis (MFA): A case in "local studies"

This research follows the MFA research tradition (Baccini andH.Brunner 1991, Hendriks et al. 2000) with a local approach. It seeks to "understand the ways by which the 'local' is altered by global processes through interventions such as subsidies, markets, legal frameworks, creation of infrastructure" [START_REF] Singh | Complex Disasters on the Nicobar Islands[END_REF]. Local MFA becomes necessary in data-poor environments, and therefore secondary data collection is complemented by fieldwork that brings researchers in direct contact with actors and stakeholders. A local MFA also provides valuable insights into system dynamics through narratives provided by the stakeholders, thus allowing to interpret quantitative data within a certain socio-cultural context. Analyzing the social metabolism of a local system highlights problematic flows that may the propensity to negatively affect the system as a whole, and generate cascade effects throughout the system.

In this respect, an MFA is intended to provide a range of experiences of how local environments are shaped, contested and rendered vulnerable.

Data for Material Flow Analysis (MFA), secondary data on the physical flows entering and leaving the territory was compiled to the extent possible using national and international sources. Embedded in the System of Environmental Economic Accounting (SEEA) framework, MFA offers a consistent compilation of all resources entering the socioeconomic system, the changes in biophysical stocks within the system, and outflows to the environment (such as wastes and emissions), as well as exports to other socioeconomic systems (Eurostat 2018).

Standard headline indicators (Eurostat 2018) were used in this study, mainly Domestic Material Input (DMI) which is a sum of Imports and Domestic Extraction (DE), and Domestic Material Consumption (DMC), which is DMI -Exports. The flows accounted for, and derived indicators are consistent with the main categories of EUROSTAT (2018). These are construction materials (cement, lime or plaster), biomass (such as food and crops), fossils fuels (petroleum products, coal), metals (including manufactured appliances and metallurgical products, household equipment, cars, electronic products). On the output side, Domestic Processed Outputs (DPO) quantify solid waste, emissions and dissipative uses. Solid waste includes municipal, commercial and construction waste. The Net Addition to Stocks (NAS) corresponds to the accumulation of material in the economy per year, such as construction minerals in buildings and durable goods (Eurostat 2018).

Data collection was a combination of secondary sources research as well as two field visits, one in February 2018, and the more extended one between March and June 2018. Secondary sources research soon revealed one important challenge: the lack of a full and consolidated database. International studies of socio-economic metabolism generally use national and international statistics [START_REF] Krausmann | Growth in global materials use, GDP and population during the 20th century[END_REF], Fischer-Kowalski et al. 2011a). In our case, the available statistics did not cover all flows, especially local extraction (biomass and minerals production) and domestic processed outputs (or wastes). The main database used in MFA for mining data is the U.S. Geological Survey (USGS) [START_REF] Krausmann | Resource Use in Small Island States[END_REF][START_REF] Duro | Metabolic Inequality and Its Impact on Efficient Contraction and Convergence of International Material Resource Use[END_REF], Wiedenhofer et al. 2019), but the latest issue (USGS 2010) did not mention any local production of material in Comoros, what the field survey later invalidated.

To fill these significant data gaps that drive material flows (such as quarries), a brief field trip of three weeks was undertaken in February 2018. Field observation showed that there are two authorized quarries producing about 80% of the minerals mined on the island (sand of various grain size, gravel, and pebbles). Other data limitations were overcome by a workshop over two days with local actors to refine and reconstruct missing data for the MFA. Forty political, economic, and non-governmental organizations were invited to this workshop, who also participated in surveys to identify, assess and interpret the important flows. These were from, for example, the Comoros institutions (customs and port), Ndzuwani institutions (the economic and the environmental director for the Ndzuwani Governorate, and local ministers in charge of the environment, the economy, and urban development), the local actors (elected officials), local and environmental NGOs (Ouani vert, Commission Mutsamudu Propre, and Sac Marie), and international NGOs (Initiative Développement (ID), Naipenda Comores, and Gevalor).

During the workshop, the participants drew up a map of the material and energy flows that are imported, produced, exported and discharged on the island.

Statements by those local actors helped develop a better understanding of the visible and less visible flows circulating on the island. We also had the opportunity to meet and build a relationship with a customs official from Comoros. Data on imports and exports was gathered from the port of Mutsamudu in Ndzuwani, which is the only point of entry of goods to the island.

Customs data provided a rather accurate picture of flows entering or leaving the territory. This methodological stance is innovative in the field of territorial metabolism studies, which tend to be guided by national or international sources, as mentioned earlier. The map of flows, their characteristics, and their transformations, that was elaborated by local actors during the first field visit, and the workshop formed the basis of the 4-month field visit between March and June 2018 (Perez 2018).

In the first field visit, it became clear that informal activities generated a significant amount of material flows within the system. The objective of the second field visit was therefore to observe, quantify and assess the material flows induced by informal economic activities, such as sand extraction, waste, or the production of ylang-ylang essential oil. For that we conducted 20 interviews with key actors in the area of agriculture, the production of essential oil, forestry, fishing, the collection of waste on beaches, sand extraction, quarrying, and in the recovery of construction waste, scrap metal and other discarded materials from landfills. Interviews also involved understanding the end uses of recovered waste to make products such as plastic bags, tablecloths textiles, and plastic carpets. Interviews were semi-structured, open ended, and were conducted in Comorian and translated into French by a local collaborator. Opening questions were of general nature, for example, how they work, the goods (biomass, construction materials, manufactured products) and quantities they produce and from which source, or the waste they collect, and where they are sold. When possible, questions surrounding their working conditions, the social, political and environmental implications of their activities in the island economy were also asked. That enabled us to understand the political economy of metabolic links and emphasize the metabolic relationships and resultant antagonisms (Demaria andSchindler 2016, Martinez-Alier et al. 2016a). Practices and activities were also documented through photos (almost 250) during the field visits (cf. Figure 1). . An overview of data sources and steps of study taken is presented in Table 1. The total Domestic Material Consumption (DMC) -which is DMI minus Exportswas 480 kt or 1.5 t/cap in 2016. This DMC is very low, and actually Ndzuwani has a very low metabolic rate as compared to other researched islands so far (Table 2). Moreover, according to the International Resource Panel, the Union of Comoros appears to have one of the lowest metabolic rates (DMC: 1.7/cap) in the world (Fischer-Kowalski et al. 2011b), which makes it a very specific and relevant territory. The share of biomass is the most significant with 48% of total DMC, followed by construction minerals (28%) and fossil fuels (22%). Metals (such as manufactured appliances and metallurgical products) represent a very small share in consumption, which also explains the lack of durable goods equipment (household equipment, cars, electronic products) of inhabitants. In this, the annual net addition to stocks (NAS), that is to say the materials that remain in the territory, represents about 138 kt / year, so 0.42 t / cap. It consists mainly of construction minerals and few imported durable products.

Results

Indicators from Material Flow Analysis (MFA)

The DPO of 0.5 t / cap consists of solid waste, including construction materials (30%), dissipative uses (30%) from agricultural waste, and atmospheric emissions (40%) from the combustion of fossil fuels, wood, and from decomposition of organic matter. It should be noted that on Ndzuwani, unlike other resource-exporting countries, little waste results from the extraction and manufacture of exported products. However, the wastewater from the production of essential oils is significant [START_REF] Tongnuanchan | Essential Oils: Extraction, Bioactivities, and Their Uses for Food Preservation[END_REF]. Finally, recycling is not very significant compared to the production of island waste.

Comparing Ndzuwani's metabolism with other islands (Table 2), we find the former's use of materials is very low [START_REF] Singh | Social Metabolism and Labour in a Local Context: Changing Environmental Relations on Trinket Island[END_REF][START_REF] Krausmann | Resource Use in Small Island States[END_REF][START_REF] Martinico-Perez | The Socio-Economic Metabolism of an Emerging Economy: Monitoring Progress of Decoupling of Economic Growth and Environmental Pressures in the Philippines[END_REF][START_REF] Cerema | Comptabilisation des flux de matières à l'échelle de la Martinique[END_REF][START_REF] Bahers | The "Metal-Energy-Construction Mineral" Nexus in the Island Metabolism: The Case of the Extractive Economy of New Caledonia[END_REF]

Spatial distribution of resources imports

Figure 3 shows the spatial distribution of Ndzuwani imports, demonstrating a complex supply chain for sustaining a remote small island. The thickness of the arrows is proportional to the quantity of flow per country. Regional imports come from Mayotte, Madagascar, La Reunion, Tanzania, and we can also include South Africa that is 3000 km away. These regional imports account for only 7% of the total, but are very varied. For a low-income country, Ndzuwani is therefore well integrated into the circuits of global flow of goods. The biomass (in green) comes from thirty-six different countries, and many European countries. This mainly comprised of rice, sodas and meat products, especially a large amount of chicken wings from France. Indeed, these are products of very low quality, often discarded in France, and therefore cheap.

However, chicken wings are popular on the islands and thrive as part of the French colonial heritage. Construction minerals (in brown) are mainly composed of cement, sand, lime and plaster. Pakistan is the first partner in these materials, followed by the United Arab Emirates and China. Manufactured products (in black), which are imported in relatively small quantities (10 kg / cap / year), come from Asian countries such as China and India, and the Middle East (United Arab Emirates and Saudi Arabia).

Socio-metabolic vulnerabilities

This section highlights examples of socio-metabolic vulnerabilities as revealed by the MFA.

These socio-metabolic vulnerabilities are both related to inflows (imports and domestic extraction) and outflows (i.e. waste and emissions) of island metabolism.

Biomass metabolism and food insecurity

In 2017, the MFA shows that subsistence crops amount to 0.5t per capita, directly consumed by producers. Yet this is insufficient, and has to be supplemented by imported foodstuffs. A quarter of food comes from outside (cf. Figure 2), that is mostly low-quality protein as there are a lot of meat products. Moreover, the economic value of imported foodstuffs has quadrupled since 2005 (FAO 2018) It is therefore becoming very expensive to import food, which is one of the indicators of the declining food security, according to the FAO.

The food production has increased between 2006 and 2016 (up 65%) by expanding agricultural land for a population that has also increased by 30%. Yet, imports have grown to an even greater extent, by about 77% (FAO 2018). Thus, even with increased local production, local food makes up a decreasing share of domestic consumption, meaning the island is not on the path to food security. Indeed, the inhabitants also consume more food, and while malnutrition is decreasing, obesity is increasing due to consumption of cheap low quality proteins, fats, sweet beverages, and other processed foods. Obesity among adults has increased from 4.1% of the share of population in 2000 to 7.8% in 2016 (FAO 2018).

Extraction of sand and ecological consequences

The spatial distribution of MFA demonstrate that large physical and financial volumes of sand, cement, lime, and plaster are imported (mainly from Pakistan, amounting to 40 kt in 2016, and to a lesser extent from China and the United Arab Emirates -see the minerals brown arrows in Figure 2). This might seem surprising, but "sand is rarer than one thinks" [START_REF] Peduzzi | Sand, rarer than one thinks[END_REF] , so these bulk materials cross oceans despite their relatively low economic value per ton, making them very expensive to transport. This is due to the booming trade in construction material, created by increasing coastal urbanization. The size of urban areas doubled between 1995 and 2014 [START_REF] Boussougou | Changements de la couverture forestière dans l'île d'Anjouan entre 1995 et[END_REF]. In Ndzuwani, these imports result from the shortage of domestic resources.

In recent years quarries have been opened on Ndzuwani, leading to a drop in imports of sand and gravel (Customs data, 2016). Yet interviews with those involved showed that these domestically produced materials are too expensive for many households. At the same time, the informal extraction of sand on the coast and rivers continues to be popular when possible, and accounted for about 20% of building material extraction on the island in 2016. Furthermore, this practice is very labor intensive (cf. Figure 4) and poorly paid: "I extract a quantity of thirty buckets per day. When I reach about 4m³, about a month's work, I sell the sand for FRC50,000

[about €100]" (interview with worker extracting sand from the river at Ouani, 2018). It is also a disaster for the island's landscape, because the coasts are progressively disappearing and undergoing major erosion as a result of this practice [START_REF] Sinane | Fragilisation et modification des formations littorales meubles sur l'île d'Anjouan (Comores) : Quand l'érosion d'origine anthropique se conjugue au changement climatique[END_REF]). Furthermore, it endangers the road infrastructure along the coasts which sometimes collapse due to the receding beaches (cf. Figure 4). However, interviews indicate that the building sector generates very little waste, unlike in northern countries, for most of it is reused on site, due simply to lack of accessible raw materials: "the building sector is highly informal, and as the population cannot pay for new materials they reuse building waste and even make community contributions to fund the building of roads and mosques in their neighborhood or village" (interview with an inhabitant building his house, 2018). Secondary materials are thus fully reintegrated into local production cycles, giving rise to different forms of collective organization and informal activities. In this way, informal actors can be important levers for optimizing the management of flows on the island of Ndzuwani.

Deforestation for ylang-ylang oil production

The third vulnerability relates to large scale deforestation related to produce the ylang-ylang essential oil. The distillation of ylang-ylang used for perfumery is a flagship product of the island's economy, employing roughly 15% of the working population (interview with NGO official, 2018). There are about 270 distillation operations per year on the island, producing 3 liters of oil per distillation (cf. Figure 5). However, this process consumes large qualities of river water, ultimately drying them up, and similarly large quantities of timber, being one of the main causes of deforestation in Ndzuwani. About 1 ton of timber is consumed per distillation (cf.

Figure 5), and "most of the time they get a logger to select and cut the tree, but don't replant afterwards" (interview with NGO official, 2018). The wood is cut from natural forest and from agroforestry whose main species is Eucalyptus robusta, a colony plantation. The area of deteriorated forest is reported to have doubled between 1995 and 2014, from 8.3% to 15.3% of the land use [START_REF] Boussougou | Changements de la couverture forestière dans l'île d'Anjouan entre 1995 et[END_REF] with logging of timber being as high as 260 kg per capita in 2016. This weakens the economic viability of the essential oil production sector, which is based on deforestation and therefore about increasing loss of forest, that leads to less domestic wood resources over time.

Conflicts relating to "new" urban waste flows

As shown in the MFA in Figure 2, roughly 48 kt of household waste was produced in 2016, that is about 150 kg per capita. This is very low in comparison to high income countries such as France (530 kg/cap) or the United States (725 kg/cap) (OECD 2013). Urban waste is not externalized on Ndzuwani, unlike European or North American territories which export several categories of waste. Even if the quantity of waste is very low, these externalities are mainly processed under conditions that increase the island's fragility. It is in Ndzuwani a clearly visible negative externality, which could paradoxically induce more transparent and controlled management by these territories. Still, the lack of facilities to collect and process waste is stumbling blocks in its management. Indeed, some of the waste is thrown into rivers or on the seashore, sometimes after being burnt alongside beaches such as in the urbanized Mutsamudu bay, which is the main city of Ndzuwani (cf. Figure 3). The rest goes to landfill, or more accurately is placed in illegal dumps, some of which are very large, dotted around rural areas. Field observations show a significant part of this urban waste is comprised of plastic objects not previously found there. A very big issue is plastic flip-flops "bought very cheaply from China and lasting no more than two weeks" (interview with economic development officer, 2018). It is particularly difficult to combat the obsolescence of this object, which is another stigma of "cheap things" [START_REF] Patel | A History of the World in Seven Cheap Things: A Guide to Capitalism, Nature, and the Future of the Planet[END_REF]. This also represents a beginning shift from reuse and repair towards a throw-away society.

Since 2014, the need to "govern municipal waste" [START_REF] Bulkeley | Governing municipal waste: Towards a new analytical framework[END_REF] has led the local authorities to try to sort out this situation by selecting controlled landfill sites, even if the municipal waste is still not collected. However, this political decision soon sparked social conflict on the island (cf. Figure 6). These protests about dumps do not differ much from those in other countries, mainly involving residents who rightly fear the landfill may "overspill" (cf.

Figure 6) and worried about the systematic traffic of trucks transporting dangerous material.

Thus the first plan for a dump had to be abandoned in the face of resistance from the inhabitants of Ouani, the fifth main city of Ndzuwani very close to Mutsamudu, who organized protests against the project as soon as the government decree was issued in 2014. Protesters argued there were risks of increased numbers of mosquitoes and flies in the vicinity of the cities. Interviews indicated that for the inhabitants of Ouani it was also a matter of not becoming a "garbage town" (interview with economic development officer, 2018) receiving waste from all over the island.

The second project for an official landfill site closed to Domoni, the second city of the island started in 2014 with the support of European institutions and of a French NGO which conducted communication and awareness campaigns with inhabitants" (interview with economic development officer, 2018). However the site is not yet operational due to lack of oversight and the fact that it was soon "too full" (see Figure 6). Waste collection was halted in 2018 because waste could no longer be stored in the dump due to lack of space, and was spilling out across the access road and nearby stream. Waste collectors and residents protested, and managed to get the dump closed. It is currently abandoned, and no steps are envisaged to correct the situation or control the dump, for its waste cannot be burnt as a nearby village which might receive all the smoke. The first concerns the food security related to the biomass flow circulation. Thus, even with increased local production, local food makes up a decreasing share of domestic consumption, meaning the island is not on the path to self-sufficiency. Food security is one of the main issues for SIDS that determines their vulnerability and resilience [START_REF] Allen | Framing food security in the Pacific Islands: empirical evidence from an island in the Western Pacific[END_REF]. According to Lowitt et al. [START_REF] Lowitt | Environmental change and food security: the special case of small island developing states[END_REF]: "Global and regional economic and environmental change processes

[…] can have very serious implications for household food and nutrition security, potentially affecting the sustainability of fishing and farming systems and the health of the island populations that depend on them for food, nutrition and livelihoods" (p. 1296). The intersection of metabolic and economic aspects are therefore directly connected to the consequences for food security.

The second socio-metabolic vulnerability reveals the social and environmental consequences of sand extraction on island metabolism. The booming trade in construction material is created by increasing coastal urbanization in Ndzuwani. The size of urban areas doubled between 1995 and 2014 [START_REF] Boussougou | Changements de la couverture forestière dans l'île d'Anjouan entre 1995 et[END_REF], following a similar trajectory as in other African towns [START_REF] Crush | Rapid Urbanisation, Urban Food Deserts and Food Security in Africa[END_REF]. Despite the public authorities repeatedly legislating against the informal material extraction, this practice continues. Such activities result in unreliable buildings less able to resist climate hazards (particularly floods and cyclones) because of the poor quality of sand extracted. Critical analysis of the island metabolism brings out this sociometabolic vulnerability, in a picture combining high-impact environmental materiality, social constraints on labor, and defective infrastructure.

The third socio-metabolic vulnerability highlights the economic relationship between a resource (wood) and the export of goods (ylang-ylang essential oil). The circuits of various flows come together for ylang-ylang distillation, producing territorial vulnerability (threats to island forestry), material vulnerability (voluminous consumption of wood and river water), and social vulnerability (with a sector of precarious employment depending on these exports).

Furthermore, there is fierce global competition even in this niche capitalist market, leading to a race towards production and yields that are at odds with the island's capacity. Additionally, these products are exported unprocessed, limiting their added value. Exports continue to increase in volume (up about 30% from 2016 to 2017), accompanied by an increase in their sale price from which local producers benefit little.

Nevertheless, Ndzuwani remains dependent on global commodity markets, since the local industry has insufficient capacity to influence prices given the structure of the capitalist market, primarily driven by external demand. Analysis of the balance of forces between the island and its export territories goes to the heart of its socio-metabolic vulnerability. It highlights the tension between the economic need to export goods, the strong increase in demand from importing countries (particularly Chinese companies), and the need to commit large volumes of island resources, driving deforestation. In the long-term, these dependencies significantly add to the island's environmental, social, and economic fragilities. This situation is symptomatic of other island economies driven by export-oriented extractivism [START_REF] Krausmann | Resource Use in Small Island States[END_REF][START_REF] Schaffartzik | Global patterns of metal extractivism, 1950-2010: Providing the bones for the industrial society's skeleton[END_REF][START_REF] Bahers | The "Metal-Energy-Construction Mineral" Nexus in the Island Metabolism: The Case of the Extractive Economy of New Caledonia[END_REF].

The last socio-metabolic vulnerability that our study reveals regards with urban waste flows.

Waste generation and its management lends another vulnerability to the island, where environmental consequences (the illegal dumping of waste destroying coastal areas) are inextricably linked to material flows (induced by short-lived cheap products), socio-political resistance (conflicts and protests against waste infrastructure), and historical processes (with the shift towards a throw-away society). This situation of waste on Ndzuwani can be framed as an "environmental justice" issue [START_REF] Walker | Environmental Justice : Concepts, Evidence and Politics[END_REF][START_REF] Fragkou | Metabolisms of injustice: municipal solid-waste management and environmental equity in Barcelona's Metropolitan Region[END_REF], Martinez-Alier et al. 2016b[START_REF] Griffin | Environmental Justice and Resilience in the Urban Global South: An Emerging Agenda[END_REF]) that analyzes metabolic conflicts related to the unequal distribution of cost and benefits of environmental goods and services in space and time.

Finally, the concept of "socio-metabolic vulnerability" complements research in politicalindustrial ecology (Newell andCousins 2015, Pincetl and[START_REF] Pincetl | Why data for a political-industrial ecology of cities?[END_REF], which "provides the framework necessary to assess the quantities of resource flows, to track them across space and time, and to decipher intertwined social and environmental dynamics that both reveal internal urban inequities and link the city to the distant areas from which these flows originate" (Pincetl and Newell 2017, 382). A growing number of scholars within industrial ecology are increasingly focusing on the political dimensions of resource flows, an approach that is being referred to as "political-industrial ecology", "which entails an importing of method and an exporting of spatial sensitivity and critical political economy" (Newell and Cousins 2015, 271).

Its ambition is to combine methods from environmental assessment and political ecology to provide a social and economic critique of the flows of materials that travel between territories and produce environmental impacts at multiple scales (Cousins andNewell 2015, Goldstein and[START_REF] Goldstein | How to track corporations across space and time[END_REF]. Few empirical studies have been carried out so far. An important achievement of this research is precisely to engage a discussion with the PIE field to move towards a socio-metabolic analysis of island metabolism.

Within this context of Ndzuwani's metabolism, we also find numerous examples of adaptation and resilience building. Many local and international NGOs are implementing in situ solutions based on vernacular practice to close the material loop and reduce dependency on external flows. Such initiatives include recycled bags made from recuperated fabric and paper, ovens to optimize the combustion of wood, rugs made from the remains of plastic sandals (of which very large numbers are to be found in the illegal dumps along the coast), cooking pots made from aluminum cans, compressed cardboard pellets for burning as fuel, and tablecloths made from used textiles by groups of women. These solutions, though modest in terms of volume,

show that there are "socio-ecological alternatives" [START_REF] Ernstson | Urban Political Ecology in the Anthropo-obscene: Interruptions and Possibilities[END_REF] from "local voices" [START_REF] Kelman | Hearing local voices from Small Island Developing States for climate change[END_REF]. Certain communities also organize coastal cleanups every weekend, without waiting for the state to step in.

Furthermore, the government of the Union of the Comoros and the municipality of Ndzuwani are involved in a transition towards an integrated economy of resources (particularly forestry) and more sustainable waste management. New legislations are constantly being put in place, though implementation challenges remain, to make local authorities responsible for collecting and processing waste, introducing quotas to cap logging, and setting up a land registry to develop the territory and its irrigation infrastructure.

Lastly, informal economic activity is not the cause of vulnerability. On the contrary, it is sometimes the informal sector which can reduce certain aspects of socio-metabolic vulnerability (Kaviti [START_REF] Musango | Urban metabolism of the informal city: Probing and measuring the 'unmeasurable' to monitor Sustainable Development Goal 11 indicators[END_REF], Kovacic et al. 2020). The presence of informal actors may be one of the main levers for optimizing the management of flows on the island of Ndzuwani. We have observed, for example, that the reuse of building waste by informal actors is important and effective.

Figure1:

  Figure1: Photos of a) extracting sand in a river (Source: Perez, 2018) and b) depositing of burnt waste along Mutsamudu beach next to a sign forbidding this placed there by local inhabitants (Source: Bahers, 2018)

Figure 2 :

 2 Figure 2: Material flows (in kilotons) for the island of Ndzuwani in 2016

Figure 3 :

 3 Figure 3: Import flows from foreign countries (Note: The thickness of arrows corresponds to the volumes imported by foreign countries). Source: Customs data, 2016; realization: Bahers)

Figure4:

  Figure4: Photos of a) informal worker collecting sand on the coast (Source: Perez, 2018) and a collapsed road at Mutsamudu (Source: Bahers, 2018)

Figure 5 :

 5 Figure 5: Photos of a) Ylang-ylang still (Source: Bahers, 2018) and b) Distillation site with store of cut wood (Source: Durand, 2018)

Figure 6 :

 6 Figure 6: Photos of a) Lack of supervision of the "controlled" Domoni landfill (Source: Bahers, 2018) and b) protests to the siting of a dump in Ouani (Source: Bahers, 2018)

Table 1 .

 1 Source and steps of study for the material flow analysis.

	Data	Source	Steps of study
	DOMESTIC EXTRACTION
	-Biomass (and food)	FAO for Comoros	Secondary sources
	-Construction minerals	Interviews with local stakeholders, Interviews with	First field trip and
		informal actors, field observation	second field trip
	IMPORTS
	-Biomass (and food)	Customs data / interviews with port authority	First field trip
	-Construction minerals	Customs data / interviews with port authority	First field trip
	-Fossil fuels	Customs data / interviews with port authority	First field trip
	-Metals (and	Customs data / interviews with port authority	First field trip
	manufactured products)
	EXPORTS
	-Biomass	Customs data / interviews with port authority	First field trip
	DPO

  Fossilfuel imports were very small with 19 kt (or 0.06 t / cap). Metal imports have the smallest share with 8 kt. It has to be noted that 61% of metals imports are manufactured products. Domestic

	extraction had the larger share in DMI with 353 kt or 1.1 t /cap, with biomass, especially food
	from subsistence farming (cereals and vegetables) being 182 kt (or 0.5 t / cap) and wood 87
	kt (or 0.26 t / cap).

Figure 2 summarizes the results of the Material Flow Analysis (MFA) obtained for the island of Ndzuwani. In 2016, the Domestic Material Input (DMI) for the island was 483 kt (which is the sum of imports and domestic extraction). Imports in relation to DMI was only 27% or 130 kt (or 0.4 t / cap), contrary to what one might have thought intuitively. Imports were mostly of construction minerals (cement, lime or plaster) and biomass (such as food and crops), which represented 51 kt and 52 kt respectively in 2016 (or 80% of all imports (cf. Figure 2 &3).

  . For example, the DMC of Ndzuwani is 1.5 t/cap/yr, quite

	low as compared to the Philippines (6 t / cap), Trinidad Tobago (17 t / cap), and New Caledonia
	(29 t / cap). All three are extractive economies where industry is heavily slanted towards
	exporting local resources (metal ores for the Philippines, oil for Trinidad and Nickel for New
	Caledonia), with resultant high levels of energy use and production of waste. Equally, the
	amount of goods imported to Ndzuwani, at 0.4t per capita, is far lower than the 5.5t per capita
	in Martinique, the 8.8t per capita in Trinidad and Tobago or the 15.1 t per capita in Iceland.
	The Domestic Process Output (DPO) is also very low in comparison to other islands. Ndzuwani
	is close to the metabolic profile of Trinket, even if the Domestic Extraction (DE) is much more
	important in Trinket (5.8 t / cap), in particular the minerals.

Table 2 : Comparison of MFA indicators with others island metabolisms (Note: t/cap/yr = tons per capita per year mentioned closed to the countries; DE = Domestic extraction; DPO = Domestic Processed Output; DMC = Domestic Material Consumption; see section 4 for explanations of these indicators).

 2

	t/cap/yr	DE	Imports Exports DPO DMC	Source
	Ndzuwani (2018)	1.1	0.4	0.01	0.5	1.5
	Martinique (2015)	7.8	5.5	2.4	7	10.9	CEREMA 2019
	New Caledonia (2016)	36.3	14.3	21.7	13.9	29.0	Bahers et al. 2020
	Trinidad and Tobago	34.7	8.8	26.2	NA	17.4	Krausmann et al. 2014
	(2008)
	Iceland (2008)	14.0	15.1	6.1	NA	23.0	Krausmann et al. 2014
	Philippines (2014)	6.0	1.2	1.5	NA	5.9	Martinico-Perez et al.
							2018
	Trinket (2000)	5.8	0.4	2.4	NA	3.8	Singh et al. 2001

Available on < https://thecommonwealth.org/sites/default/files/inline/Universal%20Vulnerability%20Index %20Report.pdf>

Data availability statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Conclusion: Resisting island vulnerabilities

Materials and energy do not always flow on their own. They are deliberately organized by society in culturally and historically prescribed ways that shape a society's socio-metabolic vulnerability. Given the links between a material's life-cycle, its spatial trajectory, and its socioeconomic consequences, approaches need to be combined. By linking the materiality of social metabolism with political ecology relations [START_REF] Ernstson | Urban Political Ecology in the Anthropo-obscene: Interruptions and Possibilities[END_REF], as in the case of Ndzuwani, we find greater explanatory power to unpack the complex relationships between the drivers of material flows, decision-making institutions, and the resultant metabolic traps and vulnerabilities: this is the interest of the concept of socio-metabolic vulnerability. This study adds to the scarce body of literature that argues that small island economies would need to leverage resource-use patterns to build system resilience, along with bold policies and institutions that support material circularity, engages communities and fosters frugal innovation.

The case of Ndzuwani is crucial for understanding island sustainability in general. It suggests that island vulnerability is not a given because of their smallness or remoteness, a position held by Briguglio and colleagues [START_REF] Briguglio | Small island developing states and their economic vulnerabilities[END_REF][START_REF] Briguglio | Small island developing states and their economic vulnerabilities[END_REF]. Such a fatalist view of islands is challenged by a number of island scholars, among them Armstrong and Read (Armstrong andRead 2002, 2006), Baldacchino [START_REF] Baldacchino | Islands, island studies, island studies journal[END_REF][START_REF] Baldacchino | Studying islands: on whose terms? Some epistemological and methodological challenges to the pursuit of island studies[END_REF], [START_REF] Baldacchino | The Beak of the Finch: Insights into the Economic Development of Small Economies[END_REF]. This research supports the latter position, but with some caution. While islands have unique characteristics owing to their geographies, but when combined with a global historical political economy, vulnerabilities can be reproduced, as is evident from the Ndzuwani case, but also in the cases of Hawaii, Puerto Rico and Singapore [START_REF] Chertow | The Intimacy of Human-Nature Interactions on Islands[END_REF]. As such, a territorial island metabolism approach combined with historical and political context, as well as the analysis of formal and informal actors can lend power to understand island vulnerability. One can argue that the informal economy on Ndzuwani plays a significant role in maintaining lower levels of resource-use. As such, "local studies" allow to identify efforts and opportunities for adaptation and resilience building across space and time, efforts that are likely to be overlooked at high resolution. The research provides novel insights into the linkages between patterns of resource-use, systemic risks and vulnerability. Moving away from a linear metabolism (characterized by a one-way flow of materials) that does not benefit marginalized communities, to an equitable circular metabolism (e.g., by localizing resource supply, as well as closing material cycles) can be an important sustainability strategy for small island economies. To this end, while appropriate policies and institutions are relevant, the role of communities and frugal innovation equally need to be recognized in building resilient island systems, and therefore resist vulnerability.

Conflict of interest

The authors declare no conflict of interest.


domain: chim spi

text:
Djamel Drider

Rabah Boukherroub

Laetitia Le Devendec

Yanath Belguesmia

Noura Hazime

Gwenaelle Mourand

Frédéric Paboeuf

Isabelle Kempf
email: isabelle.kempf@anses.fr

Hazime Ab

Impact of colistin and colistin-loaded on alginate nanoparticles on pigs infected with a colistin

Keywords: Post-weaning diarrhea, pig, colistin, Enterotoxigenic Escherichia coli, alginate, 40 nanoparticles 41

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction 43

Post-weaning diarrhea (PWD) is one of the most frequent diseases encountered in pig produc-44 tion, leading to important economic losses. PWD is caused by Enterotoxigenic Escherichia coli 45 (ETEC) strains, belonging to different serogroups (e.g. O138, O139, and O141), and possessing 46 various adhesive factors (e.g. F4 (K88), F8, AIDA, and Eae) and virulence genes (e.g. entero-

Materials and Methods

92

Preparation of strains, media and inocula

93

The previously described E. coli strain 12-269M (Le [START_REF] Devendec | Development of a pig infection model with colistin-resistant Escherichia coli. 466[END_REF] was used. Briefly, 94 this mcr-1-positive E. coli strain has been isolated from PWD-suffering piglets. It was made 95 resistant to rifampicin. It is hemolytic, belongs to the phylogenetic group A, and contains the 96 virulence markers O149-F4, astA, capU, gad, hlyA, iha, ltcA, sta1, stb, terE, and ureD. It is 97 resistant to colistin, with a colistin MIC of 16 mg/L, and to sulfamethoxazole, trimethoprim, 98 tetracycline, ampicillin, gentamicin, chloramphenicol, and rifampicin. In addition to the mcr-1 99 gene, and mutations in the PhoP and PhoQ genes of unknown impact on colistin resistance, we 100 could detect the following resistance genes: sul1,sul2,sul3,dfrA1,tetA,cat,cmlA1,101 aac(3)-IIa, aac(3)-IVa, aadA1, aadA3, aph(3')-Ia, aph(4)-Ia, strA, and strB.

102

The inoculum was prepared by culture in Mueller Hinton (MH) broth containing colistin (2 103 mg/L) for 18 h at 37°C, under gentle agitation. The bacterial pellet obtained after centrifugation 104 (5,180 g, 5 min) was re-suspended in peptone buffered solution in order to obtain a titer of 105 approximately10 8 colony-forming units (CFU)/mL. The titer of the suspension was checked by 106 plating serial dilutions on MH media.

Preparation and characterization of colistin-loaded on alginate nanoparticles 108

The alginate nanoparticles (Alg NPs) were obtained using a top-down process with the BALL 109 MILL RETSCH (PM100S10, Eragny, France). To this end, 2 g of sodium alginate powder 110 (Sigma Aldrich, St Louis, MA USA) and 112 g of zirconium oxide beads were placed in a 111 grinding pot of 1.050 kg, for 10 h with a stirring at 440 rpm, and a pause every 5 min, followed 112 by a reverse rotation every 10 min. The Alg NPs were collected and dispersed in Milli-Q water 113 (500 µg/mL) under ultrasonication (BRANSON 2800, frequency: 40 KHz) for 1 h at 25°C.

114

Then, a solution containing colistin (60 µg) was adjusted at pH 6.5 with 1 M sodium hydroxide 115 prior to addition of 500 μg/mL of Alg NPs. The solution was introduced in an ultrasound bath 116 (FS100B, Decon Laboratories Ltd, Hove, UK) for 45 min at 50/60 Hz. The temperature of the 117 bath was maintained below 30 o C, in order to limit the aggregation of Alg NPs.

118

The amount of colistin adsorbed on the surface of the Alg NPs was determined by HPLC. To confirm that it belonged to the E. coli species [START_REF] Bej | Detection of Escherichia coli and 430 Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. 431[END_REF], to detect the presence of the 188 mcr-1 gene [START_REF] Liu | Emergence of plasmid-mediated colistin resistance 477 mechanism MCR-1 in animals and human beings in China: a microbiological and 478 molecular biological study[END_REF], to check that a positive result was obtained with a F4 antiserum 189 (Labocea, Ploufragan, France). For isolates that did not exhibit the expected results (presence 190 of mcr-1, and positive F4 agglutination test), we determined the phylogenetic group (Clermont 191 et al., 2013), and studied ERIC-PCR profiles (Rivera et al., 1995). The susceptibility of some both components makes it difficult to discriminate the peaks due to colistin (Figure S2).

239

However, the shift of the main peak of Alg NPs from 1617 to 1612 cm -1 suggests strong 240 interactions between colistin and the nanoparticles' surface.

241

Inoculum titer, pathology and bodyweight gains 242

The aim of this experimental study was to evaluate the impact of colistin, either its classical shown in Table S1. The mean body weight gains of the treated groups were lower, but there 259 was no significant difference between groups.

260 Thus, as previously reported, severe diarrhea was observed during the week after inoculation.

261

Similarly, Rhouma et al. [START_REF] Rhouma | In vivo therapeutic efficacy[END_REF]

Evolution of the titers of the dominant E. coli population and of the inoculated strain

274 Mean E. coli titers obtained on MC for fecal samples were globally stable, with a mean of 7.0 275 and ranging from 5.8 to 7.9 (

294

The mean titers of PM samples obtained on MC are shown in Table S2. The lowest titers were 295 observed for the jejunum. No significant difference between groups was observed between the 296 titers of the different organs.

297

The titers obtained on MCRif and the numbers of positive PM samples are summarized in Table 298 5. Concerning jejunum samples, only two jejunum samples, one from the ICC and one from the 299 ICNP groups were found positive on MCRif. The numbers of positive pigs did not differ 300 significantly. There was no significant differences between the mean titers of the different 301 organs of the groups.

302

These different results concerning the impact of the colistin treatments on the E. coli population 303 can be compared to those of previous trials of our group or others. We previously studied the 304 impact of a colistin treatment on the fecal E. coli population of non-inoculated pigs (Fleury et 305 al., 2016). We noticed a significant but transitory decrease in the total E. coli population during

306

treatment, but all the E. coli isolates remained susceptible to colistin. In the study of Viel et al. [START_REF] Rhouma | In vivo therapeutic efficacy[END_REF] on ETEC:F4 challenged piglets, administered or not colistin at the doses of 100 000 or 50 000 IU/kg, both doses significantly reduced the fecal E. coli total 315 population and also, contrary to our results, the excretion of the inoculated ETEC:F4 during the 316 treatment period. In our conditions, using a limited number of animals placed in strict 317 biosecurity conditions, and with individual administration of the medications, we observed a 318 non-significant impact of colistin at the 100,000 IU /kg on the excretion of the 12-269M strain.

319

This result is probably in relation with its colistin resistance (MIC of 16 mg/L), whereas the

327

To overcome colistin resistance, many authors have studied in vitro synergistic effect of com-328 bination of colistin with other antibiotics [START_REF] Macnair | Overcoming mcr-1 mediated colistin resistance with colistin 487 in combination with other antibiotics[END_REF]Yahav et al., 2012) or antimi-329 crobials [START_REF] Cannatelli | 436 Synergistic Activity of Colistin in Combination With Resveratrol Against Colistin-437 Resistant Gram-Negative Pathogens[END_REF][START_REF] Naghmouchi | 492 Synergistic effect between colistin and bacteriocins in controlling Gram-negative 493 pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial 494 cells[END_REF][START_REF] Madi-Moussa | Lacticaseicin 482 30 and Colistin as a Promising Antibiotic Formulation against Gram-Negative 483 β-Lactamase-Producing Strains and Colistin-Resistant Strains[END_REF]), but in 330 vivo studies are still rare. In Europe, the marketing authorizations for veterinary oral products 331 containing colistin in combination with other antimicrobial substances was withdrawn 332 (EMA/CVMP/CHMP, 2016). Thus, other strategies to enhance the antimicrobial activity of 333 colistin or other compounds are needed. An option is to optimize the delivery of the drug and 334 increase its in vivo half-life by using nanoparticles. Thus, colistin-loaded lipid nanoparticles 335 were found to be much more efficient for eradication of Pseudomonas aeruginosa biofilms 336 compared to free colistin, although they had the same activity against planktonic bacteria (Sans-337 Serramitjana et al., 2016). Similarly, colistin nanoparticles prepared via coacervate complexa-338 tion had equal or better in vitro antimicrobial activity compared to free colistin and could be 339 used in animal models of bacteremia or pneumoniae: the results revealed that they had lower 340 liver toxicity and their antimicrobial efficacy was comparable to free colistin [START_REF] Liu | Colistin nanoparticle assembly by coacervate complexation with polyanionic 472 peptides for treating drug-resistant gram-negative bacteria[END_REF].

341

Our results showed that the use of colistin/Alg NPs yielded globally similar clinical and bio-342 logical results as the commercial colistin formulation. The two detected differences were a sig-

  107

119

  this end, a solution of colistin loaded onto Alg NPs was previously dialyzed on an 8 kDa 120 membrane in order to remove non-associated colistin with the nanoparticles. The sample was 121 filtered through a 0.45 µm regenerated cellulose membrane before loading the column (injected 122 volume 40 μL). Equipment used during this experiment was the Shimadzu LC2010-HT 123 (Shimadzu, Tokyo, Japan) with a column C4 of 5 μm QS Uptisphere® 300 Å, 250 × 4.6 mm 124 (Interchim, Montluçon, France). The eluent composition was a mixture of two solvents A and 125 B (A: 0.1% trifluoroacetic acid in deionized water, and for B: 0.1% trifluoroacetic acid in 126 acetonitrile) with a flow rate of 1 mL/min. The column was heated to 40°C, and the elution of 127 the compounds was carried out by a linear gradient of 0 to 80% during 30 min. Samples 128 detection was performed at 215 and 254 nm. The concentration of colistin was determined using 129 quantification of obtained peaks area comparatively to samples of colistin with known 130 concentrations analyzed under the same conditions. The amount of colistin loaded on Alg NPs 131 was calculated as the difference between the initial amount of colistin used for the formulation 132 and the amount of colistin remaining in the supernatant. The percent of loading efficiency was 133 calculated by the following formula: zeta potential were determined by dynamic light scattering (DLS)

  , all the samples were diluted 1/10 in peptone buffered solution containing 178 20% glycerol. The 1/10 diluted aliquots were stored at -70°C. After thawing, decimal dilutions 179 were prepared. One hundred microliters of decimal dilutions was inoculated using EasySpiral® 180 Dilute (Interscience, Saint-Nom-la-Bretèche, France) onto MacConkey media (MC) to 181 determine the titers of the total E. coli population and onto MC supplemented with rifampicin 182 (250 mg/L)(MCRif) to assess the titers of the inoculated strain. The plates were incubated 183 overnight at 37°C. Moreover, for samples negative on MCRif, one mL of the 1/10 thawed 184 dilutions was enriched overnight in 9 mL of colistin-supplemented Brain Heart Infusion broth 185 (colistin 2 mg/L) before plating 100 µL on MCRif plates. The limit of detection was 10 CFU/g. 186 Whenever possible, one colony obtained on MCRif per sample was stored. PCR was used to 187

192

  of these isolates was compared to the susceptibility of the inoculated strain by determining the 193 MIC of colistin using a broth microdilution method on EUVSEC plates(Sensititre, 194 ThermoFisher Scientific, Dardilly, France).195In addition, fecal samples collected on Days 4, 10 and 17 were inoculated, after thawing, onto 196 the Chromagar Orientation TM agar medium (i2a, Montpellier, France) supplemented with 197 colistin (8 mg/L) and vancomycin (8 mg/L) (CAO-CV)[START_REF] Mourand | 489 Dissemination of the mcr-1 colistin resistance gene among pigs: an experimental 490 study[END_REF]. When E. coli 198 colonies developed, two colonies per sample were stored and further analyzed. Their identity 199 and the presence of the mcr-1 gene were checked by PCR, their resistance to rifampicin was 200 tested by culture on rifampicin-supplemented MH media (250 mg/L) and they were tested by 201 agglutination with the F4 antiserum.

  Wallis non-parametric test was used to compare weight gains or titers. The 204 distributions of pigs or isolates were analyzed using the Chi2-test or the Fisher exact test.205Analyses of variance were performed to evaluate the impact of the treatments on the bacterial 206 titers in fecal and PM samples.

  and Dynamic Light Scattering of alginate nanoparticles and colistin-loaded on 210 alginate nanoparticles (colistin/Alg NPs) 211 Scanning electron microscopy (SEM) imaging revealed nanostructures of different shapes with 212 an average size of 87 nm (Figure S1). Dynamic light scattering (DLS) permitted to determine 213 the hydrodynamic size of the Alg NPs before and after colistin loading acquired at pH 6.5. The 214 prepared Alg NPs (500 μg/mL) displayed a hydrodynamic size of 111 nm . The difference 215 observed in size of Alg NPs using the two techniques was ascribed to the presence a hydration 216 shell in the liquid phase (DLS), as compared to SEM recorded in a dry state. After loading of 217 colistin (60 μg/mL) on Alg NPs (500 μg/mL), the hydrodynamic size remained almost 218 unchanged (112 nm) while the zeta potential value changed from negative (-32.0 mV) for the 219 naked nanoparticles to slightly positive (+0.7 mV). With a pKa of ~10, colistin behaves as a 220 polycation under our experimental conditions. Therefore, loading of colistin on Alg NPs 221 neutralizes the negative charge and turns the surface charge to slightly positive. The result 222 clearly indicates the successful adsorption of colistin on the Alg NPs surface through 223 electrostatic interactions. However, other types of interactions such as hydrogen bonding and 224 dipole interactions, owing to the presence of many polar groups in colistin and Alg NPs cannot 225 be discarded. 226 The amount of colistin loaded on Alg NPs was about 60 μg, as determined using HPLC 227 analyses. Finally, the successful loading of colistin on the surface of Alg NPs was confirmed 228 by Fourier transform infrared (FTIR) spectroscopy (Figure S2). The FTIR spectrum of Alg NPs 229 comprises a broad band at around 3413 cm -1 ascribed to surface stretching vibrations of -OH 230 and adsorbed water along with bands at 2921 cm -1 (CH2 stretching), 1617 cm -1 (asymmetric 231 stretching vibration of COO groups), 1414 cm -1 (symmetric stretching vibration of COO 232 groups), and 1032 cm -1 (elongation of C-O groups). . The FTIR spectrum of pure colistin is 233 displayed in Figure S2 and consists of a broad stretching vibration band at ~3387 cm -1 assigned 234 to -OH and -NH stretching vibrations, and the characteristic amide I and II bands at respectively 235 1655 and 1533 cm -1 . The band at ~1114 cm -1 is attributed to antisymmetric stretching of the 236 sulphate counter ion. The results are in full agreement with the literature data (Freudenthal et 237 al., 2016). After colistin loading on Alg NPs, the overlap between the stretching vibrations of 238

  243 formulation (free form) or loaded on Alg NPs, on the E. coli fecal population, and the efficacy 244 on PWD. The experimental model was based on the results previously obtained with the 12-245 269M strain (Le Devendec et al., 2018). The inoculum titer was 1.68 × 10 8 CFU/mL, thus 246 1.68*10 9 CFU per pig instead of 7.68*10 7 CFU, in an attempt to obtain a more severe model. 247 No clinical signs were observed in the NINT group. Diarrhea and signs of discomfort were 248 detected in the other groups a few hours after inoculation, and were present on four out of eight 249 different pigs in the INT group (p=0.02). The distributions of affected/non affected pigs in the 250 two other groups (3/8 affected pigs in the ICC group and 0/8 affected pigs in the ICNP group) 251 were not significantly different between them and in comparison with the NINT and INT groups 252 (p>0.05). The numbers of individual diarrheic samples collected during the twelve days 253 following inoculation varied from 0 out of 120 in the NINT, and 0 out of 96 in the ICNP group, 254 to 3 out of 96 in the ICC group and 9 out of 96 in the INT group. Thus the numbers of diarrheic 255 samples were significantly higher in the INT group compared to the NINT (p=0.0005), and 256 ICNP groups (p=0.003). 257 The mean bodyweight gains for the different periods (Day -3 to Day 4, Day 11 or Day 18) are 258

307(

  Viel et al., 2018), the pigs were orally inoculated with a non-pathogenic mcr-1-positive E. coli, 308 with a MIC of 8 mg/L. The colistin treatment given at the recommended dose (100 000 309 IU/kg/day for five days) led to a non-significant decrease of the E. coli population, which 310 returned to its initial level within a few days after the last colistin administration. The mcr-1-311 positive E. coli population remained relatively constant, at a low level during and after the 312 treatment, and no selection of this resistant population was observed. According to Rhouma et 313 al.

320

  strain used by Rhouma et al. had a colistin MIC of <0.06 mg/L. We could also observe that the 321 dominant E. coli population on Day 5 had a colistin wild-type phenotype in INT pigs, whereas 322 a non-wild-type phenotype, with mcr-1 positive results, was obtained for the dominant E. coli 323 population excreted by ICC pigs, and to a lower extent by ICNP ones. Nevertheless, on Day 17, 324 the dominant E. coli population was of the wild-type phenotype, and mcr-1 negative in all 325 groups, suggesting once again that the modifications of the E. coli populations caused by 326 colistin are transitory.

.3. Animal experimental design 147

	165	of the inoculum. For all treated groups, treatment began on the evening of Day 0 and ended on
	166	the morning of Day 5. Pigs from the ICC group received colistin (Colivet solution, 2 000 000
	142 167	IU/mL, CEVA, Libourne, France) by oral gavage at the recommended dosage (50 000 IU/kg
	143 168	The colistin, Alg NPs and colistin/Alg NPs were analyzed, separately, in a pellet form, which of body weight twice a day). Pigs from groups ICNP were also treated twice a day by oral
	144 169	is made by mixing 1 mg of the sample with 175 mg of potassium bromide (KBr). After that, gavage (40,415 IU/kg per day, 50 mL per pig each time).
	145 170	the mixture was grounded properly to ensure uniform distribution in the KBr base. Finally, the The weight of each animal was recorded once a week. During the week, daily clinical
		grounded mixture was pressed in a hydraulic press by applying a pressure of ∼7 to 9 tons. examinations consisted of looking for general clinical signs and taking rectal temperatures.
	148	The experiment was performed in accordance with French animal welfare regulations and the
	149	protocol was approved by the Ethics Committee authorization (APAFiS no.
	150	2019010215289917). The experiment was conducted in the ANSES Ploufragan animal
	151	facilities (ANSES registration No. C-22-754-1). Strict biosecurity measures were implemented
	152	to avoid contamination of the pigs, including the use of an air filtration system and airlocks for
	153	each unit, unit-specific clothes, and compulsory showering after visiting the pigs. The trial was
	154	conducted with 34 specific-pathogen-free (SPF) Large White piglets, males and females,
	155	selected after genotyping the SPF available piglets for the polymorphism linked to the intestinal
	156	receptors of E. coli F4 (Le Devendec et al., 2018). Then, at six weeks of age, the piglets were
	157	randomized, on sex and weight, into four experimental groups, the F4-susceptible piglets being
	158	in priority placed into the inoculated groups (Table 1). The experimental groups were as follows:
	159	NINT (non-infected, non-treated group), INT (infected, non-treated group), ICC (infected,
	160	commercial colistin group), and ICNP (infected, colistin/Alg NPs). The animals did not receive
	137
	161	any antibiotic treatment prior to the trial and were given the same non-supplemented feed. Each
		measurements using a Malvern Zeta sizer Nano ZS instrument (Malvern Instruments Inc.,
	162	group was housed in a separate room, containing two pens of four or five animals.
	163	On the morning of Day 0, the ten NINT piglets were orally inoculated with 10 mL of sterile
		broth. On the same day, the 24 piglets of the infected groups were orally inoculated with 10 mL

138

Worcestershire, UK), equipped with a 4 mV He-Ne laser emitting at 633 nm. The capillary cell 139 was pre-rinsed with ethanol and purified water prior to loading samples. Samples were 140 dissolved in Milli-Q water at a concentration of 500 μg/mL.

141

FTIR analysis was performed on a Thermo Fisher Scientific, Nicolet 380 (Waltham, MA, USA). 146 2171 Individual fecal samples were collected from pigs on Days -3, 1, 2, 3, 4, 5, 7, 10, 14 and 17. 172 The pigs were sacrificed on Day 21 (groups ICC and ICNP), Day 22 (two pigs from group INT), 173 Day 23 (group NINT) or Day 24 (six pigs from group INT). The sacrificed animals were 174 autopsied and samples of the jejunum, ileum, cecum, colon, and rectum were collected. The 175 samples collected post-mortem (PM) contained both fecal material and scraped mucosa.

  inoculated four-week-old piglets, previously their model may be linked to the lower age of the animals at inoculation, or to other 265 experimental conditions. Concerning the clinical efficacy of the treatments, according to our

	266
	267	results, pigs of the ICNP group had a lower number of diarrheic samples, the other criteria
	268	(number of affected pigs and weight gains) being not significantly different. In the experiment
	269	described by Rhouma et al. (Rhouma et al., 2016) on ETEC:F4 challenged piglets,
	270	administration of colistin, at the doses of 100 000 or 50 000 IU/kg, resulted in lower diarrhea
		scores in challenged treated pigs compared to challenged untreated ones, but only during the

262

selected on the presence of the F4 receptor gene, with a colistin-susceptible ETEC:F4 strain, 263 and observed severe diarrhea scores, hypothermia and occasional deaths. The apparent higher 264 severity of 271 treatment period, and colistin administration did not affect the decreased body growth rate, 272 resulting from the ETEC:F4 strain inoculation 273

Table 2

 2 , either ICC or ICNP, were resistant to colistin, contrary to isolates from the INT 290 group (p<0.001 for each comparison). On the final sampling, all isolates obtained on non-

	284
	285	MCRif media from thawed fecal samples collected just at the end of the treatment period (Day
	286	5) and at the end of the trial (Day 17), from INT, ICC and ICNP pigs were determined (Table
		4). All isolates positive for the mcr-1 gene had a MIC of 8 or 16 mg/L, as the inoculated strain,

). In comparison with the INT group, the titer of the ICC 276 group was significantly lower on Days 2, 4 and 5 but higher on Day 10, the titer of the ICNP 277 was significantly lower on Day 2 and higher on Day 17.

278

The total numbers of samples positive on MCRif without enrichment ranged from 62 out of 72 279 (INT group) to 66 out of 72 (ICNP group) (p>0.05), whereas after enrichment, positive samples 280 were observed in 67 out of 72 (INT group) to 69 out of 72 (ICC group) (p>0.05) (Table

3

). No 281 statistically significant difference between the fecal sample titers of the groups on each 282 sampling day was observed.

283

The colistin minimum inhibitory concentration (MIC) of 186 isolates obtained on MC and 287 whereas those negative for the mcr-1 gene were susceptible, with a MIC of 0.5-2 mg/L. Just at 288 the end of the treatment, many isolates obtained on non-supplemented media from colistin-289 treated pigs291 supplemented media, thus belonging to the dominant E. coli population were mcr-negative.

292

Most isolates obtained on MCRif contained the mcr-1 gene and were resistant to colistin, except 293 for the ICNP group on Day 17, but only five isolates were available.

4. Loss and transfer of colistin resistance 353

  343nificantly lower number of diarrheic samples compared to INT pigs, which was not observed 344 in the ICC group, and the reduction of the total E. coli population was observed only on Day 2, 345 whereas it was observed on three sampling times in the ICC group. The efficacy of colistin/Alg 346 NPs should now be studied in various experimental and field conditions, including infections 347 caused by E. coli strains with a large range of antimicrobial susceptibility, as it could decrease 348 the total amount of colistin that is used, in line with the national (EcoAntibio plans) and inter-349 national recommendations (EMA/CVMP/CHMP, 2016). The decrease of the use of colistin in 350 animals would lead to a decrease of the amount of colistin spread via manures in the environ-Our experiment was also an opportunity to evaluate the loss of colistin resistance between E.All fecal samples collected on Day 4, except one sample from the ICC group, gave E. coli

	389
	390	colonies on Chromagar Orientation TM agar medium; supplemented with colistin and
	391	vancomycin (CAO-CV), presenting the characteristics of the inoculated strain (presence of
	392	mcr-1, F4 agglutination and resistance to rifampicin). This result confirms our previous in vitro
	393	and in vivo observations of the non-transferability of the mcr-1 gene of E. coli 12-269M (Le
	394	Devendec et al., 2018). None of the samples collected on Days 10 and 17 enabled the detection
		of the inoculated strain, nor of mcr-1-positive E. coli.
	351
		ment, as colistin is not or only poorly absorbed in the intestinal tract (Rhouma et al., 2016).
	354
	355	coli strains. Analysis of 196 fecal isolates obtained on MCRif without enrichment showed that
	356 405	190 (97%) shared the characteristics of the inoculated strain 12-269M (presence of the mcr-1
	357 406	gene and positive reaction with the F4 antiserum). Four isolates, one from the INT group, one Supporting information
	358 407	from the ICC group and two from the ICNP group) were F4-positive and mcr-1-negative. They Figure S1: Characterization of alginate nanoparticles by scanning electron microscopy (SEM)
	359 408	belonged to the phylogenetic group A like the inoculated strain, and shared the same ERIC-Figure S2: FTIR spectra of (a) alginate nanoparticles, (b) colistin, (c) alginate nanoparticles
	360	PCR profile, one band excepted; the colistin MICs of these isolates were 0.25 or 0.5 mg/L. Two loaded with colistin (colistin/Alg NPs).
	361	isolates were F4-negative, and mcr-1-negative; one of these shared the phylogenetic group and
	362	the ERIC-PCR profile of the inoculated strain, one band excepted, but the other one had another
		ERIC-PCR profile.

352

3.

395

4. Conclusion 396

In conclusion, our study revealed that, under our experimental conditions, colistin/Alg NPs had 397 a slightly better therapeutic effect than free colistin, on the diarrhea induced by the colistin-398 resistant ETEC 12-269M. Both treatments led to a transitory decrease of the total E. coli fecal 399 population, with a majority of resistant E. coli isolates during treatment, but the dominant E. 400 coli population was found susceptible at the end of the trial. The loss of the mcr-1 gene of the 401 inoculated strain was detected more often in PM samples, and may indicate an in vivo biological 402 cost of this resistance for this strain. Further studies are needed to evaluate, in experimental or 403 field conditions, the therapeutic efficacy of colistin/Alg NPs against infections caused by vari-404 ous E. coli strains, as such an approach could lead to a decrease of colistin use.

409

Table S1 :

 S1 Growth: mean bodyweight gains (kg) in the different groups 410

Table S2 .

 S2 Titers obtained on MacConkey media from the post-mortem samples

	411
	412
	413

Table 2 .

 2 Mean E. coli titers obtained on MacConkey media

	533
							Days
	Group (n) D-3	D1	D2	D3	D4	D5	D7	D10	D14	D17
	NINT (10)* 6.43±0.61 a 6.77±0.77 b 6.79±0.76 ab 6.79±0.88 a 6.66±0.66 ab 6.83±0.46 a 6.38±0.42 bc 6.37±0.59 b 6.45±0.33 b 6.48±0.38 b
	INT (8)	7.25±0.66 a 7.94±1.06 a 7.67±0.90 a 7.66±1.13 a 7.75±1.24 a 7.06±0.82 a 6.71±0.52 abc 6.90±0.39 b 6.94±0.57 ab 6.57±0.28 b
	ICC (8)	6.99±0.73 a 7.41±0.45 ab 6.06±1.09 b 6.56±0.89 a 6.11±1.40 b 5.77±1.07 b 6.20±0.58 c 7.73±0.55 a 7.03±0.51 ab 6.99±0.41 ab
	ICNP (8)	6.44±0.35 a 7.36±0.49 ab 6.45±0.85 b 7.20±0.75 a 7.40±0.66 ab 6.43±0.62 ab 6.63±0.51 abc 6.66±0.26 b 7.60±0.91 a 7.40.37± a
	536
	537

*The NINT group contained ten piglets up to Day 14, and then two piglets were removed from this group, to be included into another assay For 534 each day, titers sharing a common superscript are not significantly different. NINT: non-infected, non-treated group; INT: infected, non-treated 535 group; ICC: infected, treated with commercial colistin; ICNP: infected, treated with colistin-loaded alginate nanoparticles

Table 3 .

 3 Culture on MCRif: numbers of positive samples and mean E. coli titers Number of samples positive without enrichment out of eight analyzed samples; b Number of samples positive with or without enrichment out of eight analyzed samples; c Mean titer on MCRif; d Total number of positive samples without enrichment out of 72 tested samples; e Total number of positive samples with or without enrichment out of 72 tested samples; NINT: non-infected, non-treated group; INT: infected, non-treated group;

	Days

a ICC: infected, treated with commercial colistin; ICNP: infected, treated with colistin-loaded alginate nanoparticles; MCRif: MacConkey media

Table 5 .

 5 Titers and numbers of positive results obtained on MCRif media from post-mortem samples Mean of eight samples for each group and each organ; b Number of positive samples out of eight analyzed samples; c Total of positive samples out of 40 analyzed samples for each group;

						Total of	Total of
	Groups Jejunum Ileon	Caecum	Colon	Rectum	positive	positive
						organs c	pigs d
	0±0.00 a 0.75±0.71 2.32±0.67 2.58±0.47 2.36±0.72
	INT
	0 b	5	8	8	8	29	8
	0.25±0.71 1.76±1.00 2.13±1.11 1.96±1.31 1.45±1.01
	ICC
	1	7	7	6	7	28	8
	0.125±0.35 1.69±1.42 1.81±1.31 1.34±1.37 1.24±1.38
	ICNP
	1	6	6	5	4	22	6

a d Total of pigs positive for at least one organ, out of eight analyzed pigs. NINT: non-infected, non-treated group; INT: infected, non-treated group; ICC: infected, treated with commercial colistin; ICNP: infected, treated with colistin-loaded alginate nanoparticles; MCRif: MacConkey media supplemented with rifampicin; all the samples from the NINT group were negative on MCRif

Acknowledgments

416

The authors thank Jean-Marie GUIONNET (ANSES) for technical assistance. 417

Funding 418 This work was funded by from the French National Research Agency (ANR) Sincolistin-15-419 CE21-0015. The funders had no role in the design of the study, in the collection, analyses, or 420 interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

421 422

Among the 75 studied PM isolates obtained on MCRif without enrichment, 56 were mcr-1-364 positive and F4-positive, 17 were mcr-1-negative, and F4-positive and 2 were mcr-1-negative 365 and F4-negative. Thus the ratio of mcr-negative isolates was higher for PM samples (19 out of 366 75) compared to fecal ones (4 out of 196, p<0.001). These 19 mcr-1-negative isolates were 367 obtained from pigs from the three inoculated groups; they had a colistin MIC of 0.25 or 0.5 368 mg/L and shared an ERIC profile similar, one band excepted, to the one of the inoculated strain.

369

The detection of isolates rifampicin-resistant, mcr-1 negative, F4-positive, with an ERIC-PCR 370 profile similar to E. coli 12-269M, and with a colistin MIC of 0.25-0.5 mg/L suggested that the 371 loss of the mcr-1 gene from the strain 12-269M occurred on several occasions, as it was 372 observed for isolates from the three inoculated groups. In comparison with the MIC determined 373 for the 12-269M strain, the mcr-1-negative isolates had decreased MICs, which are comparable 374 to those of wild-type E. coli strains (https://mic.eucast.org/search), suggesting that the colistin 375 resistance in E. coli 12-269M was associated only with the mcr-1 gene and not with the 376 mutations in the PhoP and PhoQ genes. This resistance gene loss had already been observed 377 during our previous experiments (Le [START_REF] Devendec | Development of a pig infection model with colistin-resistant Escherichia coli. 466[END_REF], and we did not further analyse the 378 mcr-1-negative isolates. The ratio of isolates missing the mcr-1 gene was higher for PM samples 379 compared to fecal samples; this difference may be due to a later sampling time for PM samples, 380 or to the differences between fecal and PM samples which were collected at different levels of 381 the digestive tract, and contained both digesta and scrapped mucosa. Further trials with sacrifice 382 of animals at different times and separate analysis of feces, digesta and mucosa could help us 383 to understand this phenomenon. As up to a quarter of PM 12-269M isolates had lost the mcr-1 384 gene after three weeks, it would be interesting to evaluate whether this ratio would further 385 increase with an extended observation period. This would indicate that, in vivo, for this studied 386 strain, the mcr-1 resistance, or the plasmid bearing this gene, is associated with a substantial 387 biological cost [START_REF] Choi | 439 Comparison of Fitness Cost and Virulence in Chromosome-and Plasmid-Mediated 440 Colistin-Resistant Escherichia coli[END_REF].

Conflict of interest 414

The authors declare no conflict of interest. and pharmacokinetics of colistin sulfate in an experimental model of Enterotoxigenic