Mehdi Bonnet

Régis Catherine Eckert

Régis Tournebize

Decolonization of asymptomatic carriage of multi-drug resistant bacteria by bacteriophages ?

Keywords: decolonization, microbiota, bacteriophage, multi-drugs resistant bacteria, Enterobacterales

published or not. The documents may come

Decolonization of asymptomatic carriage of multi-drug

resistant bacteria by bacteriophages ?

Introduction

The unceasing increase of infections by multidrug resistant (MDR) bacteria and their global spread among community and health care settings are a major public health concern.

The panel of antibiotics available to treat infections by MDR bacteria can be quickly limited leading to situations of therapeutic « dead end » that are not rare nowadays. Indeed, antimicrobial resistance (AMR) is estimated to have been responsible of between 3.62-6.57 million deaths in 2019 with death rate/100 000 ranging from 27.3 in western sub-Saharian countries to 6.3 in Australasia (Antimicrobial Resistance Collaborators, 2022). According to a Review on Antimicrobial Resistance at the British government request, this figure is expected to double to reach 10 million by 2050 [START_REF] Resistance | Tackling Drug-resistant Infections Globally[END_REF]. Among the large number of bacteria of medical interest, the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae and Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter sp) represents a group of pathogens for which new therapeutics are urgently needed. Indeed in 2017, the World Health Organization (WHO) listed carbapenemresistant (CRE) and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae (mainly E. coli and K. pneumoniae), carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii as bacteria of critical priority for research and development of new therapeutic strategies [START_REF] Stoma | Decolonization of Intestinal Carriage of MDR/XDR Gram-Negative Bacteria with Oral Colistin in Patients with Hematological Malignancies: Results of a Randomized Controlled Trial[END_REF]. More than 600 000 deaths are associated to each E. coli, S. aureus, K. pneumoniae and Streptococcus pneumoniae infections because of AMR (Antimicrobial Resistance Collaborators, 2022).

Intestinal colonization by MDR bacteria is a risk factor of infections by these bacteria and also represents a reservoir of antimicrobial resistance genes (ARG), with a risk of spread to other patients and within the community and transfer to other bacterial species [START_REF] Crits-Christoph | Good microbes, bad genes? The dissemination of antimicrobial resistance in the human microbiome[END_REF]. Fighting antimicrobial resistance and its consequences is thus a priority. In this context, decolonization strategies aiming to reduce or eradicate gut carriage of MDR bacteria are proposed to be a way to decrease AMR burden. This approach would be useful for patients with the higher risk of infection, such as, for instance, those identified as MDR carriers in intensive care units or some colonized immunocompromised patients hospitalized for hematologic diseases. The use of antibiotics to target MDR bacteria is currently the main decolonization strategy that has been developed and reported in studies [START_REF] Bar-Yoseph | Natural history and decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae carriage: systematic review and meta-analysis[END_REF]. However, 3 systematic reviews conclude that there is a lack of convincing evidence to recommend the implementation of routine digestive decolonization with antibiotics [START_REF] Bar-Yoseph | Natural history and decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae carriage: systematic review and meta-analysis[END_REF]Catho and Huttner, 2019;[START_REF] Tacconelli | ESCMID-EUCIC clinical guidelines on decolonization of multidrug-resistant Gram-negative bacteria carriers[END_REF]. Moreover, their use is controversial because of the risk of selecting and consequently disseminating resistant mutants.

Another consequence of the antibiotics use is the gut microbiota disruption, which is a risk factor for colonization by Clostridioides difficile and subsequent infection [START_REF] Langdon | The effects of antibiotics on the microbiome throughout development and alternative approaches for therapeutic modulation[END_REF]. Therefore, other alternative strategies to decolonize MDR bacteria carriers are needed: among them, fecal microbiota transplantation (FMT), probiotics or the use of bacteriophages are promising and being more investigated. In this review, we aim to summarize the current alternative strategies considered to decolonize the digestive tract of MDR bacteria with a focus on 2 major Enterobacterale: E. coli and K. pneumoniae.

Colonization

Enterobacterales like E. coli or K. pneumoniae resistant to third generation cephalosporins (mainly by Extended Spectrum Beta-Lactamases or ESBL production) or carbapenems (for instance by carbapenemase production) are considered as MDR. The genes encoding for these resistances are often carried by mobile genetic elements such as plasmids, that are often carrying multiple antimicrobial resistant genes and that can spread between different species of bacteria. Indeed the important antibiotic usage in the hospital leads to high ESBLs concentration that favors increased conjugation and spread of resistance within the gut microbiota [START_REF] Sommer | Functional characterization of the antibiotic resistance reservoir in the human microflora[END_REF][START_REF] Tschudin-Sutter | Contact Precautions for Preventing Nosocomial Transmission of Extended-Spectrum β Lactamase-Producing Escherichia coli: A Point/Counterpoint Review[END_REF] .

The acquisition of these MDR bacteria and their persistence in the digestive tract are asymptomatic. However this colonization is a source of MDR strains transmission between patients, subsequently responsible for infection or epidemics within healthcare establishments, but also in the community [START_REF] Vink | Acquisition of MDR-GNB in hospital settings: a systematic review and meta-analysis focusing on ESBL-E[END_REF]. The epidemiology of intestinal MDR carriage has been recently described in few reviews to which the reader can refer to for details [START_REF] Arzilli | Screening for antimicrobial-resistant Gram-negative bacteria in hospitalised patients, and risk of progression from colonisation to infection: Systematic review[END_REF]Campos-Madueno et al., 2023). The risk of progression from colonization to infection during hospitalization is estimated at 11% for patients colonized with MDR Gram negative bacteria [START_REF] Arzilli | Screening for antimicrobial-resistant Gram-negative bacteria in hospitalised patients, and risk of progression from colonisation to infection: Systematic review[END_REF] and 16.5% for patients colonized with carbapenemase producing Enterobacteriaceae (CPE) [START_REF] Tischendorf | Risk of infection following colonization with carbapenem-resistant Enterobactericeae: A systematic review[END_REF]. Infections by MDR bacteria have been shown to be associated with high mortality rates, particularly for CPE [START_REF] Arzilli | Screening for antimicrobial-resistant Gram-negative bacteria in hospitalised patients, and risk of progression from colonisation to infection: Systematic review[END_REF][START_REF] Tischendorf | Risk of infection following colonization with carbapenem-resistant Enterobactericeae: A systematic review[END_REF]. The most frequently reported infections following colonization are pulmonary infections, followed by urinary tract infections, bacteremia and finally skin and soft tissue infections and surgical site infections [START_REF] Arzilli | Screening for antimicrobial-resistant Gram-negative bacteria in hospitalised patients, and risk of progression from colonisation to infection: Systematic review[END_REF][START_REF] Tischendorf | Risk of infection following colonization with carbapenem-resistant Enterobactericeae: A systematic review[END_REF].

In hospital setting the rates of MDR can vary between 12 and 65% (Campos-Madueno et al., 2023). For instance at the time of admission, in a Turkish study, screening of the patients showed that 39.8% of patients were positive for ESBL and 12.5% for carbapenem resistant E. coli or K. pneumoniae [START_REF] Kizilates | Risk factors for fecal carriage of extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli and Klebsiella pneumoniae strains among patients at hospital admission[END_REF]. In France in 2014, 5.3% of the patients were ESBL carriers in an ICU [START_REF] Jalalzaï | Cessation of screening for intestinal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in a low-endemicity intensive care unit with universal contact precautions[END_REF]. However antibiotic consumption, previous and length of hospital stay, type of ward, patients' conditions or the geographic location largely impact these data. A systematic review from Arzilli and colleagues showed that the risk of acquiring Gram-negative MDR bacteria during a hospital stay is significant (9.4%). This risk varies significantly according to pathogen and type of hospital units, with notably higher rates for the Klebsiella genus (26.4%) and in high-risk units such as intensive care units (10.8%) [START_REF] Arzilli | Screening for antimicrobial-resistant Gram-negative bacteria in hospitalised patients, and risk of progression from colonisation to infection: Systematic review[END_REF]. Recent hospitalization or treatment with a beta-lactamase inhibitor/betalactam combination or carbapenem are also important risk factors for Multi-Resistant Enterobacterales colonization [START_REF] Detsis | ICU Acquisition Rate, Risk Factors, and Clinical Significance of Digestive Tract Colonization With Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae: A Systematic Review and Meta-Analysis[END_REF].

Since the first description of ESBL-bacteria fecal carriage in 2001 [START_REF] Mirelis | Community transmission of extended-spectrum beta-lactamase[END_REF], numerous reports have documented the evolution of the fecal prevalence of community carriage rates of MDR bacteria [START_REF] Woerther | Trends in human fecal carriage of extended-spectrum β-lactamases in the community: toward the globalization of CTX-M[END_REF]. Several meta-analysis indicate a general increase in MDR carriage, with an average carriage rates of around 15% worldwide but with notable regional differences. The figures can be as low as 2% in America, 4% in Europe, and reaching up to 15% in the eastern Mediterranean, 22% in Southeast Asia and Africa, and 46% in West Pacific [START_REF] Karanika | Fecal Colonization With Extended-spectrum Beta-lactamase-Producing Enterobacteriaceae and Risk Factors Among Healthy Individuals: A Systematic Review and Metaanalysis[END_REF]. A recent meta analysis has estimate the pooled prevalence of ESBL E. coli carriage in the community to 16.5% with an 8-fold increase since the beginning of the century (Bezabih et al., 2021). For instance, in a genomic investigation of ESBL Enterobacterales strains circulating in Singapore, the rate of ESBL carrier was 26.2% in healthy Singaporeans and 7.4 % were carrying a mcr1 mutation conferring resistance to colistin, drug used as a last line antibiotic [START_REF] Ding | Extended-Spectrum β-Lactamase-Producing and mcr-1-Positive Escherichia coli from the Gut Microbiota of Healthy Singaporeans[END_REF]. In Taiwan, the colonization rate by E. coli or K pneumoniae resistant to 3 rd generation cephalosporin (3GC) or presenting an-ESBL phenotype were 41.4% and 27.4% respectively [START_REF] Huang | Colonization With Multidrug-Resistant Organisms Among Healthy Adults in the Community Setting: Prevalence, Risk Factors, and Composition of Gut Microbiome[END_REF]. A smaller study with 85 participants performed in Mexico among healthy blood donors revealed that 84.7% of the participants were carriers of ESBL bacteria, with ESBL E. coli being colonizer of 74.1% of the cohort [START_REF] Tamez-Torres | High prevalence of MDR gram-negative bacteria in feces of healthy blood donors in Mexico[END_REF]. Data for carbapenem resistant Enterobacterales carriage in the community are scarce. A scoping review published in 2017 showed large variability in CRE prevalence rates in various samples, ranging from 0% to 29.5% [START_REF] Karbalaei | Probiotics and intestinal decolonization of antibioticresistant microorganisms; A reality or fantasy?[END_REF]. For instance, a recent study showed that carbapenemase producing Enterobacterale carriage rate in Pakistani urban community was 14.4% (Amina Habib, 2022). In the community, international travel, particularly to countries endemic for MDR bacteria, appears to be a major risk factor for their acquisition. The rate of acquisition of MDR Enterobacterales after international travel varies according to the studies, from 21 to 60% depending on the countries visited. Antibiotic therapy, particularly with beta-lactam antibiotics, as well as the occurrence of gastroenteritis during the stay are shown to be additional risk factors [START_REF] Hassing | International travel and acquisition of multidrug-resistant Enterobacteriaceae: a systematic review[END_REF][START_REF] Schwartz | Travel and the Spread of Drug-Resistant Bacteria[END_REF].

In addition, AMR gut carriage following travel has been show to increase the spread of those bacteria within the household (reviewed in [START_REF] Schwartz | Travel and the Spread of Drug-Resistant Bacteria[END_REF].

Decolonization

The concept of decolonization, generally referred to in the literature as "decolonization", "loss of carriage" or "eradication" refers to the eradication of carriage of MDR strains. Yet, to date, there is no agreed definition of digestive decolonization of any MDR bacteria. In particular, it has been shown that the number of negative rectal swabs chosen to affirm loss of carriage influences the decolonization rate found in different studies. Although not statistically significant, higher decolonization rates were reported when a single negative sample is used to define decolonization compared with studies that require more than one. Higher rates of decolonization are also reported in studies in which resistant strains are identified only by phenotypic method, compared with those in which strains are also identified by genotypic method [START_REF] Bar-Yoseph | Natural history and decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae carriage: systematic review and meta-analysis[END_REF]. It is also important to note that it is very difficult to routinely distinguish persistent carriage from reacquisition. While the main reservoir of Gram negative bacteria in humans is the digestive tract, several other locations have already been described in the literature as sites of carriage, notably the urinary tract and inguinal folds [START_REF] Weintrob | Natural history of colonization with gram-negative multidrug-resistant organisms among hospitalized patients[END_REF]. These other sites could potentially be sources of reacquisition of the MDR strain in the same patient, perhaps highlighting the value of using decolonization strategies that are also active extra-intestinally.

One has to keep in mind that colonization by MDR bacteria can be dynamic and fluctuate in time. In the course of the natural history of digestive MDR bacteria carriage, spontaneous decolonization without intervention can occur. This is highlighted in a recent meta-analysis where 50% of ESBL carriers spontaneously decolonize in 2 months, while about 20% remain colonized up to 18 months after initial colonization. This occurs in patients discharged from hospitals, travelers or in the community [START_REF] Ling | Carriage Duration and Household Transmission of Enterobacterales Producing Extended-Spectrum Beta-Lactamase in the Community: A Systematic Review and Meta-Analysis[END_REF]. In a literature review by Bar-Yoseph et al., the proportion of patients colonized with ESBL Enterobacterales in healthcare facilities declines over time, but still persists at 1 year (around 80% at 1-month follow-up, 56% at 6 months and 36% at 1 year) [START_REF] Bar-Yoseph | Natural history and decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae carriage: systematic review and meta-analysis[END_REF]. These observations show that still a significant proportion of the population is unable to clear its microbiota from ESBL bacteria, and hence remain a vector for AMR transmission and colonization within the community. A study by Riccio at al has actually observed a household ESBL acquisition rate of ESBL from patient discharged from the hospital. This rate is 41% upon one week returning one housing, and 29% after two months. In this study, only one third of the index cases turned ESBL-negative after 3 months [START_REF] Riccio | Household acquisition and transmission of extended-spectrum β-lactamase (ESBL) -producing Enterobacteriaceae after hospital discharge of ESBL-positive index patients[END_REF]. Spontaneous natural decolonization of ESBL bacteria is thus not an option. In the hospital setting, colonization by MDR bacteria is a risk factor for developing infections or cross-transmission to other patients and hygiene and stewardships measures have proved to be effective in preventing cross-transmission.

Among the strategies considered to achieve digestive decolonization of MDR bacteria, the use of antibiotics accounts for the vast majority of data available to date. Given the limitations of this approach, other alternative strategies have been discussed and studied: the use of probiotics, FMT and bacteriophages.

Antibiotherapy

The vast majority of published data on digestive decolonization of MDR bacteria concerns the use of antibiotics to eradicate carriage of MDR Enterobacterales. Colistin and aminoglycosides (gentamicin, streptomycin, neomycin in particular) are the most widely used antibiotics for this purpose, alone or in combination, since they are not absorbed when administered orally and have no activity against the anaerobic bacteria of the microbiota. Out of 13 included studies on the decolonization of ESBL enterobacteria by antibiotics (mainly aminoglycosides and/or colistin), the rate of colonized patients is 37% at the end of antibiotic therapy for decolonization, and rises to 58% 1 month later [START_REF] Bar-Yoseph | Natural history and decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae carriage: systematic review and meta-analysis[END_REF]. Similar results are observed in a randomized controlled trial of 58 patients showing a significantly lower rate of rectal carriage at the end of treatment in those receiving antibiotic therapy (colistin or neomycin for 10 days) aimed at decolonization compared with a group receiving placebo (32% vs. 77%), with however a disappearance of the effect at day 7 and day 28 post-treatment [START_REF] Huttner | Decolonization of intestinal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae with oral colistin and neomycin: a randomized, double-blind, placebocontrolled trial[END_REF].

However, the use of antibiotics for decolonization exposes patients to the risk of emerging resistance to these antibiotics. This is particularly important for colistin, which is a treatment of last resort for MDR Gram-negative bacterial infections [START_REF] Yang | Agents of Last Resort: An Update on Polymyxin Resistance[END_REF]. The few randomized trials that have looked at this as a secondary endpoint show no increase in colistin resistance in the treated group [START_REF] Huttner | Decolonization of intestinal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae with oral colistin and neomycin: a randomized, double-blind, placebocontrolled trial[END_REF][START_REF] Saidel-Odes | A randomized, double-blind, placebo-controlled trial of selective digestive decontamination using oral gentamicin and oral polymyxin E for eradication of carbapenem-resistant Klebsiella pneumoniae carriage[END_REF][START_REF] Stoma | Decolonization of Intestinal Carriage of MDR/XDR Gram-Negative Bacteria with Oral Colistin in Patients with Hematological Malignancies: Results of a Randomized Controlled Trial[END_REF]. However, a prospective controlled trial from 2013 showed the emergence of secondary resistance to the antibiotics used for decolonization in 14% of patients treated, with notably 1 case of colistin resistance in 16 patients and 6 cases of gentamicin resistance in 26 patients [START_REF] Oren | Eradication of carbapenem-resistant Enterobacteriaceae gastrointestinal colonization with nonabsorbable oral antibiotic treatment: A prospective controlled trial[END_REF]. Hence, the current literature of clinical trials does not provide sufficient data to assess the emergence of resistance to antibiotics used for the eradication of digestive carriage of MDR bacteria. In addition, a growing body of literature demonstrate the emergence of colistin resistance, for instance, in Enterobacterales such as E. coli and K. pneumoniae [START_REF] Bastidas-Caldes | Worldwide Prevalence of mcr-mediated Colistin-Resistance Escherichia coli in Isolates of Clinical Samples, Healthy Humans, and Livestock-A Systematic Review and Meta-Analysis[END_REF][START_REF] Uzairue | Global Prevalence of Colistin Resistance in Klebsiella pneumoniae from Bloodstream Infection: A Systematic Review and Meta-Analysis[END_REF]. It therefore seems crucial to carefully take into account this phenomenon before considering wider clinical application

Unlike to what has been described for nasal decolonization of methicillin resistant S. aureus [START_REF] Sharara | Decolonization of Staphylococcus aureus[END_REF] [START_REF] Tacconelli | ESCMID-EUCIC clinical guidelines on decolonization of multidrug-resistant Gram-negative bacteria carriers[END_REF]. However, antibiotic decolonization of MDR Enterobacterales may be considered in specific patient populations, such as neutropenic patients, but with limited evidence (Tacconelli CMI 2019). Randomized controlled trials are needed to assess the potential efficacy of antibiotic decolonization, specifically targeting high-risk patients, such as those with hematological pathologies or solid organ transplants colonized during hospitalization.

Probiotics

An alternative strategy to antibiotics for the digestive decolonization of MDR bacteria is the use of probiotics that are defined, by the WHO as living micro-organisms that confer a health benefit on the host when administered in adequate quantities. Probiotics are already widely used for example in prevention of antibiotic associated diarrhea. Lactobacillus sp, Bifidobacterium sp, Saccharomyces boulardii are some examples of the large variety of bacteria that can be used. There is growing public and scientific interest in these micro-organisms with the aim of preventing or treating pathologies [START_REF] Seale | Probiotics: a new frontier for infection control[END_REF]. However analysis of 14 clinical studies failed to show a benefit of probiotics in decolonization of MDR or in prevention of acquisition of MDR, supporting the ESCMID guidelines [START_REF] Karbalaei | Probiotics and intestinal decolonization of antibioticresistant microorganisms; A reality or fantasy?[END_REF][START_REF] Tacconelli | ESCMID-EUCIC clinical guidelines on decolonization of multidrug-resistant Gram-negative bacteria carriers[END_REF]. Hence, there is currently no evidence demonstrating the efficacy of probiotics on the decolonization of MDR bacteria, and consideration must be given to the possible impact on the gut microbiota and its consequences, as well as the risks of infection associated with these microorganisms, particularly in immunocompromised patients [START_REF] Didari | A systematic review of the safety of probiotics[END_REF]. However, several parameters such as the type of probiotic strains used, dosage, frequency of administration could be more precisely defined in order to try to improve probiotics utilization for MDR decolonization. Indeed, few clinical and fundamental observations suggest that probiotics could be useful. For instance in a preliminary study conducted in Italy, 36 patients colonized by a carbapenemase producing K. pneumoniae, received either usual care or usual care + probiotic treatment for 14 days [START_REF] Nouvenne | Carbapenemase-producing Klebsiella pneumoniae in elderly frail patients admitted to medical wards[END_REF]. Loss of the CRE K. pneumoniae of stool samples was observed in 10 patients receiving probiotics versus 2 patients in the control group. Although these results seems promising, no data are available regarding the composition of the commercial symbiotic treatment used, nor about the kinetic of decolonization of the samples. In a more recent fundamental study, Klebsiella oxytoca was shown to outcompete carbapenemase producing K. pneumoniae strains in a murine model (Osbelt et al., 2021). In this work, mice were co-colonized or pre-colonized with K. oxytoca before colonization with a carbapenem resistant K. pneumoniae strain.

Elimination of carbapenem resistant K. pneumoniae was successfully completed in 80% of the mice after 42 days, versus 20% of the control group. To confirm these results in a more complex model, the authors used humanized microbiota mice that had received microbiota from either children or adults human donor. In these mice, K. oxytoca can impair MDR K. pneumoniae outgrowth by competiting for carbon sources, with different clearance kinetics depending of the human microbiota used. Overall, despite current absence of recommendation for the use of probiotics for MDR decolonization, this approach should still be considered as current research on the microbiota provides a wealth of new information about the role, function and impact of some commensals bacteria and their potential use as probiotics, as seen for instance with the development of Faecalibacterium prausnitii as a new generation probiotic for inflammatory bowel diseases [START_REF] Martín | Faecalibacterium: a bacterial genus with promising human health applications[END_REF]

Fecal Microbiota Transplantation

The gut represent a huge reservoir of antimicrobial resistant genes (Transferable drug resistance., 1969). So far, modify or replace the intestinal microbiota can represent an interesting strategy to decolonize the digestive tract from MDR bacteria. Fecal microbiota transplantation (FMT) consists of transferring the fecal microbiota of a healthy donor, from a stool sample, to the patient's gastrointestinal tract. This therapeutic strategy is notably recognized as safe and effective for the treatment of patients with recurrent C. difficile infections and is now the subject of international recommendations in this context [START_REF] Debast | European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection[END_REF][START_REF] Mcdonald | Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA)[END_REF]. FMT can also be considered as a strategy for eradicating digestive carriage of MDR bacteria. A 2019 systematic review conducted by Yoon et al. mentions the potential benefit of using FMT in the setting of digestive decolonization of MDR bacteria, but most of the studies included are not randomized controlled trials and thus sufficient evidence is still lacking to confirm this hypothesis [START_REF] Yoon | Efficacy and safety of fecal microbiota transplantation for decolonization of intestinal multidrug-resistant microorganism carriage: beyond Clostridioides difficile infection[END_REF]. A more recent systematic review, including 7 studies and 5 cases reports came to similar conclusions (Bilsen et al., 2022). Decolonization rates after FMT varied from 20 to 90% but the studies were heterogenous (pre-or post-treatment with antibiotic or not, type of FMT procedure, types of MDR bacteria, number of patients included, duration of follow-up). For instance one of the largest study including 35 patients showed promising results with 68.5 % of decolonization rate at one year [START_REF] Seong | Fecal Microbiota Transplantation for multidrug-resistant organism: Efficacy and Response prediction[END_REF]. In this study only 4 patients were colonized with carbapenemase strain (mostly K. pneumoniae carbapenemase producing strain) and 12 were colonized with both carbapenemase strain and vancomycin resistant Enterococcus. Among the 4 patients colonized with carbapenemase producing strain, 3 were successfully decolonized. A first multi-centric randomized clinical trial including 39 patients failed to obtain statistically significant results due to early termination of the study despite a slightly decreased of ESBL and/or carbapenemase producing Enterobacterales colonization compared to the control group: 41% of the patients colonized were negative at day 35-48 compared to 29% of the patients of the control group [START_REF] Huttner | A 5-day course of oral antibiotics followed by faecal transplantation to eradicate carriage of multidrug-resistant Enterobacteriaceae: a randomized clinical trial[END_REF]. A second randomized double blind trial study against placebo (KAPEDIS) regarding the efficacy of decolonization of KPC producing K. pneumoniae is currently ongoing [START_REF] Perez-Nadales | Randomised, double-blind, placebo-controlled, phase 2, superiority trial to demonstrate the effectiveness of faecal microbiota transplantation for selective intestinal decolonisation of patients colonised by carbapenemase-producing Klebsiella pneumoniae (KAPEDIS)[END_REF]. So far, according to Merrick et al.,13 randomized studies are actually ongoing or have been recently completed [START_REF] Merrick | Modulation of the Gut Microbiota to Control Antimicrobial Resistance (AMR)-A Narrative Review with a Focus on Faecal Microbiota Transplantation (FMT)[END_REF]. Number of participants recruited, inclusion criteria and primary and secondary outcomes of those studies show significant differences between the studies. 7/13 of those studies are randomized, double-blind, controlled trials; 5/13 are randomized, open-label, controlled trials, and 1/13 trial is randomized, participant-blinded, controlled. The number of estimated included participants ranges between 9 and 437 with 6/13 studies including more than 100 participants. The recruited participants have different origin, including adults inpatients colonized with MDR, to specific population having had renal transplantation. Colonizing bacteria are mainly ESBL or CRE bacteria identified either by culture or PCR. The fecal material is administrated mainly by capsule (7/13), retention enema (4/13) colonoscopy (1/13) and nosoduodenal tube (1/13). The primary outcome is decolonization (evaluated by culture or PCR) but at varying times post treatment with assessment ranging from 3 days to 6 months, with 9/13 studies with an evaluation set at 1 month. Overall, despite their difference in the studies design, those ongoing randomized clinical trial highlight the growing interest of this low risk therapeutic to decolonize MDR bacteria. Their results, when published, will hopefully provide a clearer view of FMT decolonization efficacy and potential future utilization.

Bacteriophages

Bacteriophages (or phages) are bacterial viruses discovered over 100 years ago by Frederick Twort and Félix d'Hérelle, who observed the presence of lysis halos on bacterial cultures [START_REF] D'herelle | Sur un microbe invisible antagoniste des bacilles dysentériques[END_REF][START_REF] Twort | An investigation on the nature of ultra-microscopic viruses[END_REF]. D'Hérelle was the first to call these viruses "bacteriophages", literally "bacteria eaters" and was also the first to successfully conduct phage therapy to treat typhoid in chickens: this was the birth of phagotherapy. The development of phage therapy took place mainly in the first half of the 20th century, before the era of antibiotics, and phages were used to treat a wide range of infections, with significant therapeutic success, particularly in the absence of alternatives at the time [START_REF] Marongiu | Reassessment of Historical Clinical Trials Supports the Effectiveness of Phage Therapy[END_REF][START_REF] Stacey | The Safety and Efficacy of Phage Therapy: A Systematic Review of Clinical and Safety Trials[END_REF].

Bacteriophages are ubiquitous and found in all ecosystems; they represent the largest biomass on Earth with an estimated number of 10 32 , for 10 8 different genomes (Chibani-Chennoufi et al., 2004;[START_REF] Rohwer | Global phage diversity[END_REF] and over 6,000 species listed by the International Committee on Taxonomy of Virus [START_REF] Krupovic | Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021[END_REF]. They can carry different type of genetic materials ((ssRNA, dsRNA, ssDNA, dsDNA), and have various structures and shapes.

There are two types of bacteriophage cycle: the "lytic" (or virulent/productive) cycle and the "lysogenic" (or temperate/dormant) cycle. The lysogenic cycle involves the integration of the phage's genetic material into the chromosome of the infected bacterium, forming a dormant prophage, without destruction of the bacterial cell or production of new bacteriophages. The phage genetic material is then transmitted vertically to daughter cells and also sometimes by horizontal transfer to neighboring bacteria. Prophages can be re-activated under the influence of abnormal environmental conditions or other external stress, restart replication and lytic infection cycle. The lytic cycle takes place in several phases, chronologically involving adsorption, injection of the genetic material contained in the capsid, replication, assembly of new viral particles and then lysis of the infected bacteria, enabling the release of new virions.

Bacteriophages characterized by a lytic cycle are those of clinical interest, since they induce a rapid and highly specific bactericidal effect. For this reason, they are seen as an alternative to tackle MDR in bacteria.

Numerous parameters and characteristics specific to each bacteriophage-host bacterium pair need to be characterized in vitro to guarantee successful use for clinical purposes, in particular burst size and multiplicity of infection (MOI). The burst size corresponds to the number of phages released by a lysed bacterium, while the MOI corresponds to the bacteriophage/bacterium ratio and is used to define the initial phage inoculum to be administered in in vitro and in vivo studies [START_REF] Glonti | In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success[END_REF].

Bacteriophages offer a number of advantages for their use as therapeutic agents, particularly in comparison with antibiotics. Firstly, phages are highly specific for their target bacteria, enabling them to be active only on the pathogen to be eliminated while preserving the microbiota: their host spectrum is very narrow [START_REF] Wittebole | A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens[END_REF]. Moreover, their replication at the site of infection can take place from the very first application and, unlike antibiotics, allows their concentration at the site of infection to increase over time. Moreover, several clinical studies on efficacy and/or safety of phagotherapy indicate that this approach is safe. Side effects that have been reported are generally accounted for by inflammatory bacterial contamination [START_REF] Stacey | The Safety and Efficacy of Phage Therapy: A Systematic Review of Clinical and Safety Trials[END_REF]. Moreover, reassessment of the old clinical trials that have been performed indicate that phagotherapy is efficient [START_REF] Marongiu | Reassessment of Historical Clinical Trials Supports the Effectiveness of Phage Therapy[END_REF], even though, efficiency of some recent clinical trials has been observed only in 2 out 7 clinical trials. Therapeutic efficiency requires the bacteriophages to be delivered to the right infectious sites and that the infectious sites harbors enough bacteriophage-sensitive bacteria [START_REF] Stacey | The Safety and Efficacy of Phage Therapy: A Systematic Review of Clinical and Safety Trials[END_REF]. Overall, the clinical trials that have been performed have allowed to better define the conditions more suited for an efficient therapy that will permit in the future to better assess phagotherapy efficacy.

It should be noted that legislation concerning bacteriophages does not facilitate their use for clinical purposes: health authorities of western countries generally supervises and authorizes their availability and use only within the framework of clinical trials or in a compassionate setting, notably for cases of infections by MDR bacteria escaping all lines of antibiotic therapy.

There are, of course, limits to the clinical use of bacteriophages. Their highly specific nature with regard to their host bacteria means that they have a very narrow spectrum of activity, sometimes limited only to particular strains or clones. In addition, bacteriophages can degrade rapidly under certain environmental conditions, and their stability and long term storage condition should be tested and frequently checked before being used in humans. Furthermore, only bacteriophages with a lytic cycle can be used for clinical purposes, while those with a lysogenic cycle may be responsible for horizontal transfer of antibiotic resistance genes [START_REF] Górski | Bacteriophage therapy for the treatment of infections[END_REF]. Finally, bacterial resistance to bacteriophages is also a well-documented mechanism that could hinder the utilization of bacteriophages. Bacteria possess a large panel of defense mechanisms ranging from mutating the bacteriophage receptor (LPS, capsule, outer membrane proteins, other transporters…), degrading the viral genetic material to expressing CRISPR_Cas or restriction-modification systems for instance that negatively affect the infectivity of the bacteriophages [START_REF] Egido | Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice[END_REF][START_REF] Roach | Phage therapy: awakening a sleeping giant[END_REF]. One approach to limit the emergence of such clones is to use a cocktail of different bacteriophages that are using different receptors for entry into bacteria. As emergence of a resistant clone is the selection of a stochastic mutant within a large population, the likelihood to select for bacteria that are mutated in several receptors is extremely low. Moreover, in the context of infection, the selection of bacteriophage-resistant bacteria can be detrimental to the bacteria as mutations of several types of phage receptors such as capsule, LPS or other receptor can lead to reduced bacterial virulence and increased sensitivity to the host immune system leading to clearance of the pathogen from the organism [START_REF] Egido | Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice[END_REF].

Use of phage as therapeutics have proven efficacy in some case reports and in compassional use but data for decolonization are lacking and need more studies to be implemented in clinic.

In the following section, we detail studies addressing decolonization of MDR bacteria by bacteriophages in in vitro, animal or human models, with an emphasis on E. coli and K. pneumoniae as models of the main MDR Enterobacterales. Studies concerning the treatment of gastrointestinal infections have already been discussed elsewhere (Javaudin et al., 2021b) and will not be considered here, as most studies involving non-MDR bacteria that are beyond of the scope of this review focusing on decolonization of MDR strains.

In vitro models

E. coli

Recapitulating partly the gut environment in vitro allows to quickly test and evaluate decolonization strategies. Bernasconi et al. used a simplified in vitro model of digestive tract colonization consisting of a fermenter inoculated with two pools of stools from healthy donors free of ESBL-or carbapenemase-producing Enterobacterales [START_REF] Bernasconi | Eradication of a Multidrug-Resistant, Carbapenemase-Producing Klebsiella pneumoniae Isolate Following Oral and Intra-rectal Therapy With a Custom Made, Lytic Bacteriophage Preparation[END_REF].

Colonization was achieved after inoculating these stools with a CTX-M-15 ESBL-producing strain of E. coli. After 3 consecutive introduction of a sterile filtrate of bacteriophages lysate (10 5 to 10 6 PFU/mL) of several pathogenic E. coli, Shigella spp., Salmonella spp., Proteus vulgaris/mirabilis, Pseudomonas aeruginosa, Staphylococcus spp. and Enterococcus spp., a significant decrease of the MDR strain population and the absence of restoration of this population after cessation of treatment were observed for one of the two stool pools during the 48-hour observation period of the experiment. The specificity of the bacteriophages towards the MDR E. coli was observed as the introduction of those phages had no effect on the total E. coli population of the microbiota while acting on the specific MDR strain. However, in one of the fermenter batch, the inhibition of the MDR strain was only temporal and the strain regrew.

Resistant mutants were isolated and found to have incorporated 2 additional plasmids and presented few Single Nucleotides Polymorphisms. One of them, in a glycosyl transferase family 2 protein domain is hypothesized to be responsible of the bacterial resistance to the phage by -14 -increasing the sugar content on the membrane thereby potentially blocking one or more phage receptors. A major limitation of this study is that it approximates the environment represented by the human digestive tract, thus neglecting possible interactions between the host's digestive tract cells, its microbiota, the bacterium of interest and the bacteriophages. Moreover, this in vitro model was built under aerobic conditions, preventing the growth of strictly anaerobic bacteria physiologically present in the human digestive tract.

Another in vitro work tried to assess the combined benefit of using phage to reduce the bacterial load of an MDR with the competitive action of a probiotic preventing the outgrowth of phageresistant MDR bacteria [START_REF] Laird | In Vitro Demonstration of Targeted Phage Therapy and Competitive Exclusion as a Novel Strategy for Decolonization of Extended-Spectrum-Cephalosporin-Resistant Escherichia coli[END_REF]. A bacteriophage specific of a CTX-M-1 ESBLproducing MDR E. coli was used in combination with a probiotic corresponding to a commensal wild type E. coli strain producing colicin bacteriocin. On the one hand, the bacteriophage alone incubated with the ESBL E. coli strain lead to a sharp decrease in the number of bacteria, followed by regrowth due to the selection of a bacterial mutant. On the other hand, when the commensal and the ESBL strains were co-cultured together, the growth of the ESBL strain was transiently inhibited by up to 3 log depending on the commensal strain used, indicating that the commensal strain can reduce, but not abolish the growth of the ESBL bacteria by exerting a competitive exclusion mechanism. However, when the bacteriophage and the commensal were used in combination, the bacteriophage quickly killed the majority of the ESBL bacteria, and the commensal outcompeted the ESBL strain, completely abolishing its regrowth. Hence, in vitro, a combined action of a bacteriophage and a probiotic was shown to be efficient at decolonizing ESBL bacteria. These experiments need to be confirmed in more complex in vitro system mimicking the gut and evaluated in animal models.

In vivo animal models

E. coli

A murine model where mice first received an oral treatment with streptomycin and then were colonized with a multi-resistant E. coli, was used to assess the efficacy of a cocktail of bacteriophages in decolonizing multi-resistant enterobacteria colonizing the digestive tract, and its impact on microbiota diversity [START_REF] Galtier | Bacteriophages to reduce gut carriage of antibiotic resistant uropathogens with low impact on microbiota composition[END_REF]. While the mice were under constant antibiotic pressure, when the three bacteriophages were administered individually or in cocktail, only a transient decrease was observed. But when the antibiotic pressure was released after colonization, and upon addition of a cocktail of the three phages, a strong reduction in the bacterial load was observed a week after phage injection, indicating a positive action of the bacteriophage cocktail. Furthemore, by assessing the abundance of different bacterial genera present in the stools through 16S rRNA sequencing, the authors were able to show a significantly lower impact of bacteriophages on the digestive microbiota of mice as compared to antibiotics usage.

The study by Javaudin and colleagues presents less optimistic data than the above-mentioned studies. In their murine model, they were unable to demonstrate the long-term efficacy of using a cocktail of bacteriophages specific to an ESBL E. coli (type not specified in the article) producing carbapenemase OXA-48, for digestive decolonization (Javaudin et al., 2021a). They were only able to demonstrate a transient 1-log decrease in the fecal concentration of the multiresistant strain following oral, oral and rectal administration or microencapsulation of bacteriophages. Similarly, another study showed only a 0.5-log transient decrease in the carriage of a CTX-M-15 ESBL-producing multi-resistant E. coli in a mouse colonization model [START_REF] Porter | Bacteriophage Cocktail and Microcin-Producing Probiotic Escherichia coli Protect Mice Against Gut Colonization With Multidrug-Resistant Escherichia coli Sequence Type 131[END_REF]. However, they observed a significant 3-log decrease in the strain of interest in the stools of the mice studied when the bacteriophage cocktail was combined with a probiotic (Microcine-C7 bacteriocin-producing E. coli), suggesting a potential synergy. It should be noted, however, that the effect remains transient (1 week maximum) and declines despite continued treatment.

Klebsiella pneumoniae

In a first study by Fang et al., one first phage (P24) targeting a K. pneumoniae strain producing carbapenemase type KPC-2 and a phage targeting a capsule-deficient mutant of that strain were isolated, characterized and tested for their capacity to modify the gut colonization by this strain (Fang et al., 2022). Several bacterial mutants resistant to the P24 phage were isolated and shown to be mutated for capsule production. In a murine colonization model, a moderate reduction in the K. pneumoniae MDR fecal population (2 log) was observed following rectal administration of a single bacteriophage, but resistant mutants emerged in all mice in the week time-frame of the experiment. When a cocktail of 2 bacteriophages was administered, a greater reduction in K. pneumoniae MDR was observed (from 4 to 6 log); the emergence of mutants resistant to both bacteriophages was nevertheless observed in one mouse. Some of the strains resistant to the phages had altered production of capsule or mutation in a cyclic di-GMP phosphodiesterase. These mutants were also shown to be less virulent in a Galleria mellonella larvae model.

Two other studies investigated decolonization by bacteriophages of germ-free mice colonized with human microbiota containing a carbapenem-resistant K. pneumoniae. A first study from Taiwan used colonized mice with human stool carrying a K64 K. pneumoniae producing carbapenemase of the K64 serotype, the main MDR clone circulating in Taiwan [START_REF] Liu | Decolonization of carbapenem-resistant Klebsiella pneumoniae from the intestinal microbiota of model mice by phages targeting two surface structures[END_REF]. Here also, two different phages were used alone or in combination. When administered orally, one phage was unable to decolonize the microbiota of the K64 CRE bacteria. The other was able to decolonize mice for 2 weeks before recolonization was observed. When the 2 phages were used in combination, most of the mice were also decolonized for 2 weeks. A stronger decolonization for 2 months was observed in half of the animal when the phages were administrated both intragastrically and intraperitonally.

A recent study identified phages targeting either Wild Type or capsule mutants of K. pneumoniae [START_REF] Lourenço | Phages against Noncapsulated Klebsiella pneumoniae: Broader Host range, Slower Resistance[END_REF]. The authors showed that phages targeting capsule mutants had a broader host range and provide increased efficient at killing K. pneumoniae when used in combination with capsule-targeting bacteriophage. When used in vivo in OM12 mice, that have a defined model microbiota of 12 species, and colonized with a hypervirulent K2 K. pneumoniae, only a temporary 10-100x decrease in the bacteria load was observed when both phages were used alone or in combination. K. pneumoniae is known to strongly express a capsule, but the fact that the capsule-independent phage showed some activity indicate that within the gut microbiota, the bacteria is able to modulate its capsule expression. Although the K. pneunomiae was not a MDR strain, this study is interesting for phagotherapy approaches, as being able to target with phages both capsulated and acapsulated bacteria could increase potential therapeutic approaches based on phages.

In vivo human model

In 2020, Corbellino et al reported a clinical case of decolonization with a cocktail of bacteriophages in a patient colonized with carbapenemase-producing KPC-3 K. pneumoniae (Corbellino et al., 2020). The patient had a history of Crohn's disease in remission, and had a complex clinical history with a right nephrectomy and total cystectomy with left ureterostomy and ureteral stenting following recurrent episodes of lithiasis and urinary tract infection complicated by extensive cystic fibrosis. In addition, he presented with multi-site colonization (gastrointestinal tract, urinary tract and on a permanent external invasive device) by a MDR strain of K. pneumoniae, resistant to all beta-lactams (with the exception of Ceftazidime-Avibactam) because of the production of the carbapenemase. Several episodes of urinary tract infection and sepsis have been documented for this patient with this strain of K. pneumoniae, isolated in culture from urine, ureteral stent and blood cultures, and successfully treated empirically with Ceftazidime-Avibactam. However, after each infectious episode, the MDR strain reappeared and persisted in culture at all the sites tested. A cocktail of MDR strain-specific lytic bacteriophages was therefore used to decolonize the patient, administered orally and rectally for 3 weeks. No adverse effects were reported, and the treatment was well tolerated.

At the end of the bacteriophage treatment and until publication of the study, the MDR strain was never again isolated in culture on selective media from the sites previously colonized, i.e. from stools, rectal swabs, urine and the ureteral stent. In addition, molecular biology tests for carbapenemase genes on rectal swabs, carried out 5 days after the start of treatment with the phage cocktail and every 7 to 14 days during patient follow-up, were always negative. This is all the more encouraging given the technique's greater sensitivity than culture on selective media. Interestingly, only 2 other non MDR K. pneumoniae have since been isolated from the patient's stool or rectal swab, but these were sensitive to most antibiotics and belonged to two other clones. This confirms the close specificity of bacteriophages to their host bacteria, and therefore the possibility of highly selective decolonization directed almost exclusively against the strain of interest.

Conclusion

Decolonization of the gut microbiota from MDR bacteria hasn't yet been reproducibly achieved. Antibiotics have the drawback of not being selective enough. Bacteriophages however have the advantage of being selective and not alter the microbiota. But they haven't yet consistently shown to be effective. We believe that a reason for this is our yet limited knowledge of the interactions between phages and bacteria in the gut.

A parameter that has been overlooked when considering the use of bacteriophages for decolonization is the gut geography and differential localization of bacteria and phages in the digestive tract. The gut epithelium is indeed separated from the luminal microbiota by a mucus layer that protects it from bacteria and bacteria components. However, this barrier is not fully impenetrable and bacteria such as Acinetobacter, Delftia and Stenotrophomonas have been shown to establish niches in the colon crypts of mouse and humans [START_REF] Pedron | A crypt-specific core microbiota resides in the mouse colon[END_REF]Saffarian et al., 2019). Other bacteria such as Akkermensia muciniphila, Bacteroides spp or Bifidobacterium spp, but also K. pneumoniae [START_REF] Glover | Characterizing the mucin-degrading capacity of the human gut microbiota[END_REF][START_REF] Hudson | Klebsiella pneumoniae l-Fucose Metabolism Promotes Gastrointestinal Colonization and Modulates Its Virulence Determinants[END_REF], are known to be able to reside in the mucus layer and use it as a carbon source. Also, in mice gut colonization experiments with E. coli or K. pneumoniae, even though the bacteria are mainly observed in the lumen, some bacteria can be detected in the mucus (Caballero et al., 2015;[START_REF] Lourenço | The Spatial Heterogeneity of the Gut Limits Predation and Fosters Coexistence of Bacteria and Bacteriophages[END_REF] where they are not accessible to bacteriophages present in the lumen.

Similarly, several phages have been shown to interact with mucin [START_REF] Almeida | Bacteriophage Adherence to Mucus Mediates Preventive Protection against Pathogenic Bacteria[END_REF][START_REF] Barr | Bacteriophage adhering to mucus provide a non-host-derived immunity[END_REF]Chin et al., 2022;[START_REF] Green | Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract[END_REF] and the presence of phages in the mucus proposed to provide a non-host immunity against invasion or colonization of the gut mucosa by bacteria [START_REF] Barr | Bacteriophage adhering to mucus provide a non-host-derived immunity[END_REF]), (reviewed extensively in Rothschild-Rodriguez 2023). This means that bacteria localized in the mucus are, in the absence of mucus associatedbacteriophages targeting them, not visible from bacteriophages that cannot interact with and penetrate in the mucus and are luminal. These bacteria are thus a constant source of bacterium that, by being shed, can recolonize the lumen [START_REF] Lourenço | The Spatial Heterogeneity of the Gut Limits Predation and Fosters Coexistence of Bacteria and Bacteriophages[END_REF], and thus represent an inaccessible hidden reservoir of the bacteria to eradicate. However, they are target of phages that can interact with mucin, and be activated by mucin. Green and colleagues, using a E. coli non-MDR ExPEC strain were convincingly able to eradicate the colonizing bacterium with a mucin-targeting bacteriophage [START_REF] Green | Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract[END_REF]. When considering decolonization one should thus consider these different environments and isolate bacteriophages that also have a tropism for mucin. A cocktail of phages that can act in these different compartments might be more efficient at decolonizing the gut from a MDR strain. This capacity to interact with mucin is a parameter that has rarely been considered or discussed so far in the publications addressing gut decolonization by bacteriophages and should be more taken into account. Also, whenever an application of bacteriophages in in vivo models or in in humans is foreseen, applying a set of standardized protocols to characterize thoroughly the bacteriophages will be useful to anticipate or predict the efficiency of the bacteriophages in clinical applications [START_REF] Glonti | In Vitro Techniques and Measurements of Phage Characteristics That Are Important for Phage Therapy Success[END_REF].

Emergence of phage resistance is a common phenomenon and can be a limitation to the use of bacteriophage. In addition to the classic mutations in the bacterial phage receptors, several new mechanisms of the bacterial defense systems underlying it have been uncovered in the recent years [START_REF] Tal | SnapShot: Bacterial immunity[END_REF]Bernheim and Sorek, 2020). The acquisition of phage resistance can come with a fitness cost for the bacterium, which may in return lead to a reduced virulence and increased sensitivity to the host immune response (Fang 2022 Commun Biol) or decreased antibiotic resistance profile be modified with the recovery of susceptibility to certain antibiotics (Majkowska-Skrobek 2021 Environ. Microbiol). In order to better understand and predict better efficacy the mechanisms of phage resistance should therefore be investigated both in in vitro studies and in the host context (Gaboriaux and Debarbieux 2023). To counter this emergence of resistant mutants, 2 approaches are envisaged. One relies on the use of cocktails of phages targeting different bacterial phage receptors, in order to minimize phage-resistance evolution.

The second approach makes use of bacteriophages that select a bacterial resistance mechanism inducing a reduced virulence or increased sensitivity to antibiotics [START_REF] Torres-Barceló | Mitigation of evolved bacterial resistance to phage therapy[END_REF] 3677-3686. doi:10.1128/JB.186.12.3677-3686.2004. Chin, W. H., Kett, C., Cooper, O., Müseler, D., Zhang, Y., Bamert, R. S., et al. (2022).

Bacteriophages evolve enhanced persistence to a mucosal surface. Proc Natl Acad Sci USA 119, e2116197119. doi:10.1073/pnas.2116197119.

  , antibiotherapy has not shown a clear decolonization efficacy of the digestive tract (Tacconelli CMI 2019). To date, the review by Taconelli et al. constitutes the data reference on which the European recommendations of the European Society of Clinical Microbiology and Infection Diseases -European Committee on Infection Control (ESCMID-EUCIC) are based and do not recommend routine decolonization of ESBL and CRE bacteria carriage

  . :10.1016/j.jgar.2020.05.018.Bernheim, A. and Sorek, R. (2020) The pan-immune system of bacteria: antiviral defence as a community resource. Nat RevMicrobiol, 18,113-119. doi:10.1038/s41579-019-0278-2 Bezabih, Y. M., Sabiiti, W., Alamneh, E., Bezabih, A., Peterson, G. M., Bezabhe, W. M., et al. (2021). The global prevalence and trend of human intestinal carriage of ESBL-producing Escherichia coli in the community. Journal of Antimicrobial Chemotherapy 76, 22-29. doi:10.1093/jac/dkaa399. Bilsen, M. P., Lambregts, M. M. C., van Prehn, J., and Kuijper, E. J. (2022). Faecal microbiota replacement to eradicate antimicrobial resistant bacteria in the intestinal tract -a systematic review. Curr Opin Gastroenterol 38, 15-25. doi:10.1097/MOG.0000000000000792. Caballero, S., Carter, R., Ke, X., Sušac, B., Leiner, I. M., Kim, G. J., et al. (2015). Distinct but Spatially Overlapping Intestinal Niches for Vancomycin-Resistant Enterococcus faecium and Carbapenem-Resistant Klebsiella pneumoniae. PLoS Pathog 11, e1005132. doi:10.1371/journal.ppat.1005132. Campos-Madueno, E. I., Moradi, M., Eddoubaji, Y., Shahi, F., Moradi, S., Bernasconi, O. J., et al. (2023). Intestinal colonization with multidrug-resistant Enterobacterales: screening, epidemiology, clinical impact, and strategies to decolonize carriers. Eur J Clin Microbiol Infect Dis. doi:10.1007/s10096-023-04548-2.Catho, G., and Huttner, B. D. (2019). Strategies for the eradication of extended-spectrum betalactamase or carbapenemase-producing Enterobacteriaceae intestinal carriage. Expert Rev Anti InfectTher 17, 557-569. doi:10.1080/14787210.2019.1645007. Chibani-Chennoufi, S., Bruttin, A., Dillmann, M.-L., and Brüssow, H. (2004). Phage-host

	culture	system	model.	J	Glob	Antimicrob	Resist	22,	664-671.
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doi

It is still possible that, depending on the context (host, bacteria, bacteriophages), bacteriophages alone will not be sufficient to efficiently eradicate the MDR colonizing bacteria.

Complementing MDR decolonization by phages with the action of a probiotic preventing its outgrowth as observed by Laird and colleagues in vitro [START_REF] Laird | In Vitro Demonstration of Targeted Phage Therapy and Competitive Exclusion as a Novel Strategy for Decolonization of Extended-Spectrum-Cephalosporin-Resistant Escherichia coli[END_REF] could represent an interesting alternative approach that would need to be further investigated.


Yalini Senathirajah

Sylvia Pelayo

Human Factors and Organizational Issues

Keywords: Human factors, human-computer interaction, organizational issues, patient safety, health information technology

Objective: To select the best papers that made original and high impact contributions in the area of human factors and organizational issues in biomedical informatics in 2019. Methods: A rigorous extraction process based on queries from Web of Science® and PubMed/Medline was conducted to identify the scientific contributions published in 2019 that address human factors and organizational issues in biomedical informatics. The screening of papers on titles and abstracts independently by the two editors led to a total of 30 papers. These papers were discussed for a selection of 15 finalist papers, which were then reviewed by the two editors and by three external reviewers from internationally renowned research teams. Results: The query process resulted in 626 papers that reveal interesting and rigorous methods and important studies in human factors that move the field forward, particularly in clinical informatics and emerging technologies such as brain-computer interfaces. This year three papers were clearly outstanding and help advance the field. They provide examples of applying existing frameworks together in novel and highly illuminating ways, showing the value of theory development in human factors.

Conclusion:

The selected papers make important contributions to human factors and organizational issues, expanding and deepening our knowledge of how to apply theory and applications of new technologies in health.

Introduction

Papers published in 2019 on human factors and organizational issues (HFOI) show the emergence of new technologies, such as the multifaceted app suite for chronic disease self-management, brain-computer interface evaluation, and new combinations or improvement of existing methods and theories, particularly in analyzing workflows in clinical care and the cognitive burdens involved. None of the papers is explicitly focused on ethics, though much of the work has ethical implications in improving both provider and system performance and understanding diagnostic delays and errors. Papers show a certain maturity in the development of theory and applications in highly specific practical contexts.

The selection of papers on HFOI intends to illuminate current progress of HFOI research in biomedical informatics. The selected papers offer a glimpse of the quality and breadth of the work being conducted in the HFOI community.

Methods

We collected papers from 2019 by a standard search in PubMed and Web of Science ® . Searches were performed in October and December 2019 to identify peer-reviewed journal articles published in 2019, in the English language, related to HFOI research in biomedical informatics. In addition to the search of electronic databases, manual searches of key themes were performed in major biomedical journals (e.g., Journal of the American Medical Informatics Association, Methods of Information in Medicine, Journal of Medical Internet Research, etc.).

We used free-text and coded keywords.

Free-text keywords were listed in with respect to the questions addressed by the section. Relevant MeSH terms were also identified, and PubMed test queries were made iteratively. Two final queries were built, one based on MeSH terms for major topics and one on free-text keywords searched in titles or abstracts via PubMed and Web of science ® . This process led to an initial set of 626 papers that the two section editors independently evaluated using papers' titles and abstracts. Each editor chose the 15 papers considered as the best list of candidate best papers. The two selections overlapped, and the set was finally reduced to 15 papers by discussion. These were evaluated by both section editors and three other external reviewers, with scoring based on the topic's importance to medical informatics, scientific and/or practical impact, quality of scientific work, originality and innovativeness, coverage of related literature, organization and clarity of thought, and whether or not to include the paper as a best paper. Three papers were considered as being 'best papers' on the basis of their substantially higher scores by all reviewers and the agreement that they were excellent examples of HFOI studies that break new ground and add to our tools to advance the field (Table 1). A content summary of the selected best papers can be found in the appendix of this synopsis.

Conclusions

Noteworthy papers published in 2019 provide examples of applying existing frameworks together in novel and highly illuminating ways, showing the value of theory development in human factors. Wang et al., [START_REF] Wang | Understanding Health Information Technology Induced Medication Safety Events by Two Conceptual Frameworks[END_REF] applied the Sittig and Singh sociotechnical basis of errors framework [START_REF] Sittig | A New Socio-technical Model for Studying Health Information Technology in Complex Adaptive Healthcare Systems[END_REF] and Coeira's information value framework [START_REF] Coiera | A new informatics geography[END_REF] together to analyze US FDA (Food and Drug Administration) MAUDE (Manufacturer and User Facility Device Experience) data on health-IT related errors and near-misses. The dual analysis strategy permitted to understand and classify the main causes of error in this area, then to apply data-related causes to analyze how missing, partial, null, or false data caused downstream errors, calculate the probabilities and help identify the major impacts on outcomes. This analysis provides an excellent method to prioritize which types of errors should be dealt with early, and helps understand overall causation. It also paves the way for a formal reporting template and develops a knowledge base supported by case-based reasoning.

Lee et al., [START_REF] Lee | Development and User Evaluation of a Rare Disease Gene Prioritization Workflow Based on Cognitive Ergonomics[END_REF] aimed to understand and leverage a combination of cognitive prototype approach with computational approaches, using the best of each. Prototypical thinking is common in clinical diagnosis and genetic counseling. The authors showed that a tool which elicits prototypes before asking the geneticist to identify disease phenotypes had the same accuracy but took less time. Combining tools with both strategies would be optimal, leveraging the cognitive characteristics of experts. The paper is a nice illustration of the understanding and formalization of cognitive processes and how to draw lessons to design computer-assisted gene prioritization to augment clinical reasoning and cognitive characteristics of experts.

The aim of Patterson et al., [START_REF] Patterson | Reducing Delays to Diagnosis in Ambulatory Care Settings: A Macrocognition Perspective[END_REF] was to increase system resilience by using macrocognition concepts to analyze ambulatory care work in a partnership between clinicians and human factors experts. This helped identify a set of resilience strategies and ways in which information systems can be modified to increase safety. It augments the existing Systems Engineering Initiative for Patient Safety (SEIPS) theoretical framework [START_REF] Carayon | Work system design for patient safety: the SEIPS model[END_REF], bridges disciplines, and suggests design solutions.

Although not selected as best papers, the remaining candidate best papers were just as interesting. They also bring a real added value for the HFOI community.

Sumak et al., [START_REF] Šumak | An empirical evaluation of a handsfree computer interaction for users with motor disabilities[END_REF] present a very interesting study that contributes to the body of research and design for hands-free computer interaction, in particular for motor-impaired users who need innovative solutions for interacting with Information and Communication Technology (ICT). The authors aimed at investigating the benefits and drawbacks of a solution for touchless interaction with a computer using the EPOC+ brain-computer interface at home which allowed to measure and combine EEG and EMG signals with data from facial expressions. The authors studied user perceptions in terms of acceptance and use of the solution with a 5-step protocol that compared the user's current human computer interaction approach with the EPOC+, with 18 participants (10 non-disabled and 8 disabled). An interesting initial finding is that non-disabled users experienced significantly more difficulty using the EPOC+, although both groups required more time to use EPOC+ than their usual solution.

Chen et al., [START_REF] Chen | A Multi-Faceted Approach to Characterizing User Behavior and Experience in a Digital Mental Health Intervention[END_REF] made use of a multi-function suite of 13 clinical apps to address depression and other mental health issues, by measuring experience and user engagement via usage patterns, mechanisms of change, and usability. They used log data collected during treatment delivery, integrating text and visual analytics in a novel approach aiding analysis of a rich complex dataset. They found four clusters of different types of users based on app usage patterns, and compared them in terms of engagement and associated factors, highlighting mechanisms of change and usability perceptions. The paper has both practical and scientific value: it contributes to richer methods for understanding user engagement more holistically and it contains a detailed description of a log analysis work which may be helpful.

Giordanengo et al., [START_REF] Giordanengo | Design and Prestudy Assessment of a Dashboard for Presenting Self-Collected Health Data of Patients With Diabetes to Clinicians: Iterative Approach and Qualitative Case Study[END_REF] evaluated the design of a diabetes dashboard displaying patient-collected data to clinicians, using iterative participatory design with 14 clinicians. The study permits the assessment of the reliability of self-collected data, calculations, and highlighted conditions requiring investigation. 100% of participants deemed it useful for daily use. The study relies on an iterative participatory process involving workshops with patients, clinicians, and researchers to define which information should be available and how it should be displayed. Several methods are used to address main barriers to the use of self-collected data, such as visualization and scenarios, and the identification of medical events causes. The studied system does not limit the integration of data to specific companies or types of sensors, and includes personal goals. For all these reasons, this paper contributes greatly to HF tools for addressing chronic disease.

Yera et al., [START_REF] Year | Modelling the interactive behaviour of users with a medication safety dashboard in a primary care setting[END_REF] characterized medication dashboard use by 35 users over 10 months. They were able to distinguish pharmacist from non-pharmacist use, using features tied to exploration and dwell time via user interface events, with 80% accuracy using multilayer perception giving the highest precision and recall values. Primary users showed distinctive behavior with lower time between clicks on overview screens, and less visual search activity on patient risk screens and visualizations. This is a contri-Human Factors and Organizational Issues bution showing that using machine learning techniques to distinguish between classes of users both helps understand the differences in their behavior and model useful user interface improvements or customizations. The study includes new workflows to group population health monitoring activities and nudges to increase engagement.

Businger et al., [START_REF] Businger | Lessons Learned Implementing a Complex and Innovative Patient Safety Learning Laboratory Project in a Large Academic Medical Center[END_REF] described the Patient Safety Learning Lab approach, implemented in 12 units of a large academic medical center over 18 months, impacting 12,000 patient admissions. The paper describes implementation challenges and the approach in designing and implementing a tool suite integrated with a major commercial EHR to improve safety. It contributes to our knowledge of how to change health system culture and provide recommendations for each part of institutional structures. It provides a description of how one center implemented novel tools in a commercial EHR, using APIs and real-time data extraction, and thinking outside current workflows. Not all institutions have the same resources but the paper provides examples of execution in a certain type of system.

Richardson et al., [START_REF] Richardson | Live Usability Testing of Two Complex Clinical Decision Support Tools: Observational Study[END_REF] conducted a study of live usability testing which showed how even a small study may reveal more different problems in 'live' in situ testing than in usual laboratory think-aloud testing, providing additional areas in which system redesign was needed, particularly for clinical decision support. This calls attention to the need for flexible and widely applicable clinical decision support tools to achieve the fastest way to perform tasks.

Want et al., [START_REF] Wang | Design and Evaluation of an Integrated, Patient-Focused Electronic Health Record Display for Emergency Medicine[END_REF] study concerned work-centered usability methods to evaluate integrated patient-focused status displays for Emergency Department (ED) communication and situational awareness. The study uses multiple design methods to show ED data in a prioritized and highly cognitively supporting way, using focus groups, surveying, and scenario-based study. There are some limitations with the static tests, small numbers, and incomplete functions of the prototype, but the paper provides an interesting insight.

Setiawan et al., [START_REF] Setiawan | An Adaptive Mobile Health System to Support Self-Management for Persons With Chronic Conditions and Disabilities: Usability and Feasibility Studies[END_REF] provided an example of testing for a very interesting and specialized app architecture, in this case aimed at patients with chronic conditions and disabilities (PwCCDs). This allows studying a highly customizable app suite with clinician-and patient-facing components in a large study via survey, and performing feasibility studies with smaller numbers, in real deployment. It shows means of usability testing of more complex multi-stakeholder applications with shifting components and stakeholder levels of participation, and showed clear relationship of usability with use.

Woolridge et al., [START_REF] Wooldridge | Complexity of the pediatric trauma care process: implications for multi-level awareness[END_REF] provided a fascinating paper with a detailed and groundbreaking large study of pediatric trauma care processes, highlighting and providing techniques to understand the true complexity of processes across the multiple levels found in healthcare. The authors argued for shifting from eliminating to coping with complexity, tools for awareness, reliance and adaptation across individuals, team and organizational levels, with sociotechnical solutions. The paper greatly contributes to the understanding of how to realistically improve care and safety in complex settings.

Cho et al., [START_REF] Cho | Eye-tracking retrospective thinkaloud as a novel approach for a usability evaluation[END_REF] demonstrated an improvement on usual eye-tracking protocols by devising a retrospective think-aloud, and related findings from eye tracking measures to success of failure in task completion. The new method asked participants to share their inner experience using the app and explain their experience and gaze patterns during errors making it possible to catch instances in which the user had more confidence than warranted, i.e., problems that would be undetected in traditional usability testing.

Kutney-Lee et al., [START_REF] Lee | Electronic Health Record Adoption and Nurse Reports of Usability and Quality of Care: The Role of Work Environment[END_REF] presented a large (~12,000 person) study of nurses in 353 hospitals, using regression models to assess EHR adoption level, work environment, and usability outcomes. The paper illuminates our understanding of how EHR implementation and the work environment affect usability in practice, helping evaluate their effects on care quality. The study found that nurses are still often not included in selection, modification, and other aspects of EHR deployment, with high rates of dissatisfaction, and that advanced functionality of EHRs is needed to achieve desired effects, with satisfaction increasing with the EHR level of sophistication. Relying on the two conceptual frameworks and the National Coordinating Council for Medication Errors Reporting and Prevention (NCC MERP) taxonomy, a retrospective analysis of 9-year safety reports from the US FDA (Food and Drug Administration) MAUDE (Manufacturer and User Facility Device Experience) database (45,624 MAUDE reports were extracted; 3,521 reports were identified as HIT-related reports) was conducted. A hundred and fifty-two unique reports met the inclusion criteria.

Results show a variety of contributing factors to risks which are mostly associated with clinical content followed by human-computer interface, and people. While Organizational and system-related contributing factors are critical underlying factors, they were not directly highlighted. Results also provide an overview of how likely certain effect may be brought on one step and how it affects the other adjacent steps (e.g., receiving wrong information causes making a wrong decision with a probability of 0.62, and the wrong informa-tion may lead to a wrong (probability: 0.38) or delayed (probability: 0.56) care process, with a high chance of resulting in patient harm (probability: 0.94)).

The application of the two conceptual frameworks to the MAUDE database may be a general approach to analyze any event reports in the context of HIT. It may enhance the understanding of HIT-induced problems, along with their causes and the identification of the most significant risks to patient safety. This may have implication on (i) how to formalize risk-related reporting template for HIT, and (ii) the development of a knowledge base supported by case-based reasoning.

Lee JY, van Karnebeek CDM, Wasserman WW Development and user evaluation of a rare disease gene prioritization workflow based on cognitive ergonomics J Am Med Inform Assoc 2019;26(2):124-33

This paper is interested in understanding human cognitive capabilities and applying this understanding to support human cognition via the design and evaluation of a human-system interaction for optimized performance in gene prioritization activities. The authors designed a novel workflow in which clinicians recalled known genetic diseases with similarity to patient phenotypes to inform data interpretation. This prototype-based workflow was evaluated against the common computational approach based on physician-specified sets of individual patient phenotypes. An evaluation was conducted as a web-based user study, in which 18 clinicians analyzed two simulated patient scenarios using a randomly assigned workflow. Data analysis compared the two workflows with respect to accuracy and efficiency in diagnostic interpretation, efficacy in collecting detailed phenotypic information, and user satisfaction.

Results demonstrated that gene interpretation could be accelerated using the prototype-based workflow by facilitating prototypical thinking. It was likely that participants employed some level of proto-typical thinking in both workflows, while the reasoning process was more efficiently facilitated by the prototype-based workflow.

Patterson ES, Su G, Sarkar U Reducing delays to diagnosis in ambulatory care settings: A macrocognition perspective Appl Ergon 2020 Jan;82:102965

In this paper, the authors want to characterize contributors to diagnostic delays by physicians that can be mitigated by work system redesign. To do so, they overlay concepts from the theoretical framework of macrocognition upon a foundation of human factors concepts, including a perspective on how to advance patient safety known as Safety-II. In the outpatient care setting, complex tasks, conducted by a primary care provider, are provided for five macrocognition functions: sensemaking, re-planning, detecting problems, deciding, and coordinating. Relying on an interdisciplinary team, the authors held a series of meetings to redesign systems in order to reduce delays to diagnosis by helping users to avoid missed symptoms, forgotten follow-up activities, and delayed actions.

For each critical task, relying on the five macrocognition functions, the authors identified vulnerabilities in care provision in the outpatient setting that can contribute to diagnostic delays. Resilience strategies were proposed that can mitigate these vulnerabilities with work system redesign. The primary contribution of this paper is a set of resilience strategies that could be supported by innovations in health information technology (HIT) in future research.

Results augment an existing theoretical framework providing examples of how HIT could be redesigned to make complex tasks easier. This effort represents a preliminary step in a line of potentially useful research for reducing patient harm by providing ideas for interventions that go beyond training or education. The paper is a nice contribution of constructing a bridge between how human factors engineers conceptualize cognitive work in a way that can suggest innovative design solutions.

Human

  Factors and Organizational Issues Appendix: Summary of Best Papers Selected for the 2020 Edition of the IMIA Yearbook, Section Human Factors and Organizational Issues Wang J, Liang H, Kang H, Gong Y Understanding health information technology induced medication safety events by two conceptual frameworks Appl Clin Inform 2019;10:158-67 This paper reveals a new perspective to Health Information Technology (HIT) induced medication errors through the application of two conceptual frameworks: the information value chain (Coiera, 2016) used in conjunction with the sociotechnical model for HIT-related errors (Sittig & Singh, 2010) can enhance measurement and facilitate identification of the most significant risks to patient safety.

Table 1

 1 Best paper selection of articles for the IMIA Yearbook of Medical Informatics 2020 in the section 'Human Factors and Organizational Issues'. The articles are listed in alphabetical order of the first author's surname.

	Section
	HumanFactors and Organizational Issues
	 Lee JY, van Karnebeek CDM, Wasserman WW. Development and user evaluation of a rare disease gene prioritization workflow
	based on cognitive ergonomics. J Am Med Inform Assoc 2019;26(2):124-33.
	 Wang J, Liang H, Kang H, Gong Y. Understanding health information technology induced medication safety events by two
	conceptual frameworks. Appl Clin Inform 2019;10:158-67.
	 Patterson ES, Su G, Sarkar U. Reducing delays to diagnosis in ambulatory care settings: A macrocognition perspective. Appl
	Ergon2020;82:102965.

Published online: 2020-08-21

Acknowledgement

We would like to acknowledge the support of Adrien Ugon, Martina Hutter, Kate Fultz Hollis, Lina Soualmia, Brigitte Séroussi, and the whole Yearbook Editorial Committee as well as the reviewers for their contribution to the selection process of the Human Factor and Organizational Issues section for the IMIA Yearbook 2020.


HORTICULTURAL AGROFORESTRY SYSTEMS: A MODELLING FRAMEWORK TO COMBINE DIVERSIFICATION AND ASSOCIATION EFFECTS

Keywords: intercropping, fruits, vegetables, Land Equivalent Ratio, Modern Portfolio Theory

While much research has been devoted to analyze the benefits of intercropping and agroforestry systems on yield, through the concept of Land Equivalent Ratio, little literature is available on the benefits of such systems for reducing yield variability. In the present study, we intend to introduce the notion of yield variability in the Land Equivalent Ratio and to combine both concepts in a new framework. Through an application of this new framework on cases selected from literature, we show how intercropping or agroforestry systems may result in both an increase in yield and a decrease in its variability. This exploratory work may nevertheless be completed with further studies to confirm and evaluate the situations under which such benefits can be expected.

Introduction

In a context of increasing environmental awareness and evolving relationships between farmers and consumers, new farming organizations have emerged in the last decades thus creating innovative agro-ecological systems [START_REF] Wezel | Agroecological practices for sustainable agriculture. A review[END_REF]. One of these systems, known as mixed horticultural system, which corresponds to the intercropping of fruit trees and vegetables, is attracting a growing interest in Europe, especially among new entrants into farming [START_REF] Warlop | The smart project: a focus on fruit trees and vegetables agroforestry systems in France[END_REF].

From the agronomic perspective, this type of system presents two major advantages: (i) firstly, it reduces the overall risk on production through a diversification effect [START_REF] Letourneau | Does plant diversity benefit agroecosystems? A synthetic review[END_REF][START_REF] Isbell | Benefits of increasing plant diversity in sustainable agroecosystems[END_REF]) (ii) secondly, it benefits from positive interactions between crops through an association effect [START_REF] Vandermeer | The Ecology of Intercropping[END_REF]). The risk reduction arising from diversification which is a well-studied mechanism in the field of economy [START_REF] Markowitz | Portfolio Selection[END_REF]) has been formalized in the Modern Portfolio Theory (MPT). Yet, this theory, primarily established to design a portfolio of assets in finance, does not take in account for assets that interact together such as crops in associations. On the other hand, the effect of crop association on production is covered in agricultural sciences by the concept of Land Equivalent Ratio (LER) that provides a standardized basis to assess intercropping system performance. However, this approach is still limited since it does not account for the effect of diversification on risk reduction. Our objective was therefore to combine these two theoretical approaches in a unified framework to formalize the effect of intercropping on both production and risk. After presenting the model linking diversification and association effects, we apply this unified framework on examples based on a literature review.

Material and methods

• Formalizing the effect of association: the Land Equivalent Ratio

When two crops are cultivated simultaneously on the same surface, interactions between the two crops may lead to an overall production different from the weighted sum of the production of each crop cultivated in sole crop. This association effect is classically assessed in the literature by the so-called Land Equivalent Ratio [START_REF] Mead | The Concept of a "Land Equivalent Ratio" and Advantages in Yields from Intercropping[END_REF][START_REF] Vandermeer | The Ecology of Intercropping[END_REF]. For a given proportion (k) of crop A in the association with crop B, the value of the LERk corresponds to the area that would be needed to produce the same amount of crops in two separate sole crops. LERk is formalized as follows:

𝐿𝐸𝑅 𝑘 = 𝐿𝐸𝑅 𝑘 𝐴 + 𝐿𝐸𝑅 1-𝑘 𝐵 = 𝑌 𝑘 𝐴 𝑆 𝐴 + 𝑌 1-𝑘 𝐵 𝑆 𝐵 ( 1
)
where Yki is the yield of crop i cultivated in intercropping, and Si represents the yield of a sole crop i.

An LER greater than 1 means that the intercropping system mixing crop A and crop B produces more than their respective sole crop for the same cultivated area.

• Formalizing the effect of diversification: the Modern Portfolio Theory

The Modern Portfolio Theory (MPT) formalizes how risk can be reduced in a context of assets diversification [START_REF] Markowitz | Portfolio Selection[END_REF]. The general idea behind this theory is that when assets are combined in a portfolio and when asset returns are not perfectly correlated, the portfolio risk is reduced compared to single assets portfolio. Applied to agriculture, a diversified portfolio becomes a combination of several crops within a farm.

According to this theory, the expected return of a portfolio P is the weighted sum of each individual assets in the portfolio:

𝐸(𝑅 𝑃 ) = ∑ 𝑤 𝑖 𝑖 𝐸(𝑅 𝑖 )
(2) with wi the weight of crop i (that is, the proportion of crop i in the portfolio) and E(Ri) the expected yield of crop i On the other hand, the risk of a portfolio is measured by the standard deviation, σP:

𝜎 𝑃 ² = ∑ ∑ 𝑤 𝑖 𝑤 𝑗 𝜎 𝑖𝑗 𝑗 𝑖 (3)
with σij the yield standard deviation of crop i when i=j, and the covariance of crop i and j when i≠j

The MPT has already been used in agriculture [START_REF] Knoke | Optimizing agricultural land-use portfolios with scarce data-A non-stochastic model[END_REF] but always considering farms as portfolios of non-interacting crops. When applying MPT to agroforestry systems, the challenge is to integrate the effects of interaction between associated crops on the yield of intercropped cultures.

• Combining MPT and LER

Combining both Modern Portfolio Theory and Land Equivalent Ratio requires improving Eq. ( 2) so as to account for interactions between the two crops:

𝐸 𝑘 (𝑅 𝑃 ) = 𝐸(𝑅 𝐴 )𝐿𝐸𝑅 𝑘 𝐴 + 𝐸(𝑅 𝐵 )𝐿𝐸𝑅 𝑘 𝐵 (

• Applying the new conceptual framework to cases selected from literature

A literature review was carried out for fruits and vegetables intercropping data in the Web of Science database, with the terms "agroforestry" OR "intercropping" OR "fruit" OR "orchard" OR "vegetable". The search allowed to identify 24 studies containing LER data of fruits and/or vegetables intercropping systems. Furthermore, the search identified 12 studies which had data fitting our model requirements: crop yields, yield variability and LERs. For illustrative purpose, the model was run on three studies for which sufficient data was available: [START_REF] Guvenc | Increasing Productivity with Intercropping Systems in Cabbage Production[END_REF][START_REF] Blazewicz-Wozniak | The effect of intercropping on yielding of root vegetables of Apiaceae family[END_REF][START_REF] Rao | Agronomic and economic benefits of coffee-banana intercropping in Uganda's smallholder farming systems[END_REF].

Results

• Characteristics of fruits and vegetables intercropping systems

The review of intercropping studies involving vegetables and/or resulted in 277 LER measures (often several experiments per study). On average, intercropping was more efficient than sole cropping, since 251 out of the 277 calculated LER values were larger than 1. The median LER was 1.28 and the mean 1.36. However, the standard deviation on LER was 0.36, suggesting a high variability in LERs depending on crop species, treatments and experimental designs (Figure 2). • Illustration with three cases from literature Among the reviewed papers, the risk-return combination curves took two possible functional shapes. The first functional form appears when a low yield -low risk crop is combined with a high yield -high risk crop (Figure 3a; 15 of the 24 studies). In the example given here (cabbage-bean intercropping), diversification led to a strong reduction in risk and association made it possible to reach the lowest level of risk for a yield similar to the one obtained with cabbage only. The second functional form is obtained when a high yield -low risk crop is combined with a low yield -high risk crop (Figure 3b; 9 of the 24 studies). Interestingly, in this functional form, adding a high-risk / low-yield crop to a low-risk / high-yield monoculture is still beneficial in terms of risk reduction. In the example given here (carrot-parsley intercropping), risk reduction was moderate and yield improvement was particularly high. The only agroforestry systems for which we found sufficient data in the literature is the coffee-banana system (Figure 3c). It corresponds to the first functional form. In this particular example, risk reduction is marginal but yield improvement can be important. The main difference with the cabbage-bean association is that in the coffee-banana system, no combination of the two crops makes it possible to reach at the same time both a lower risk and a higher yield than the sole crops.

Discussion

The two perspectives examined here were Modern Portfolio Theory, and Land Equivalent Ratio. The former quantifies the effect of diversification on risk, the latter measures the effect of association on production. This research has merged both approaches in a combined framework in order to assess horticultural-agroforestry system performances. The results show how some fruit and vegetable combinations can outperform monoculture in both the risk and production dimensions. In other words, that it is possible to reduce the overall risk related to production while maintaining or even increasing the global yield thanks to the association of fruits and vegetables. Even though application of this framework to real situations was limited in this study (due to the scarcity of literature data), our results indicate that a two dimensional analysis (risk and return) of agroforestry systems adds insight into the assessment of such systems. Further evaluations of yield and associated standard deviations in a wide variety of agroforestry systems are now needed to evaluate the conditions in which such benefits can be expected. It would be especially interesting to extend this approach to more diversified systems. From the theoretical point of view, extension of the framework to situations with more than two crops is straightforward and does not present any mathematical difficulty. The issue once again lies in the availability of data since (to our knowledge) no study have evaluated yield and standard deviations of more than two crops in associations compared to sole crops.

Besides the issue of data availability on production, a further limitation of our study is that we limited our analysis to crop production. However, crop productivity does not directly reflect economic profitability. To further enhance and improve our model, more information on production costs and returns (especially in intercropping systems) would also be necessary [START_REF] Betters | Planning optimal economic strategies for agroforestry systems[END_REF].

Finally, this study only accounts for the effect of association on production and does not consider the effect of crop association on risk. While many studies have focused on the effect of crop association on overall production, the particular issue of production variability in agroforestry systems remains unstudied [START_REF] Mead | The Concept of a "Land Equivalent Ratio" and Advantages in Yields from Intercropping[END_REF]. A consensus seems to emerge on the fact that crop association can decrease the risk associated with each crop [START_REF] Vandermeer | The Ecology of Intercropping[END_REF][START_REF] Rao | Agronomic and economic benefits of coffee-banana intercropping in Uganda's smallholder farming systems[END_REF] but this effect has rarely been quantified. Therefore, our hypothesis that the individual risk associated to each crop remains unchanged under intercropping can be considered as a conservative hypothesis; and integrating an effect of association on risk is likely to strengthen our conclusion that crop association benefits both production and risk reduction.

  4) with E(Ri) the expected yield of crop i ϵ{A;B} and LERki the partial LER of crop i at the proportion of k in the portfolio. Graphically, this new framework is represented in Figure 1. The solid curve displays the risk and yield of crops portfolio for different proportions of crop A and B in a situation where only the diversification effect is considered (Δσ formalized by MPT). The dotted curve represents the same combination of crops in a situation where both the diversification effect and the association effects are considered (ΔY formalized by our new framework combining MPT and LER). The dashed line represents the weighted average of risks and returns.

Figure 1 :

 1 Figure 1: Possible return and risk combinations for a theoretical intercropping system for different proportions of crop A and crop B. Solid curve: diversification effect (Δσ formalized by

Figure 2 :

 2 Figure 2: Cumulative frequencies (a) and frequency distribution (b) of land equivalent ratios (LER) for fruits and vegetables intercropping experiments.

Figure 3 :

 3 Figure 3: Risk-return combinations observed for different types of interactions. The interactions are detailed in the body of the text. Dots ( ) represent the performances of intercropping systems for different proportions of crop A and B in the case where only the diversification effect is considered, triangles ( ) represent the performances of the same intercropping systems in the case where both the diversification effect and the association effect are considered.

Acknowledgments: The present work was carried out with the support of the "Fondation de France".