Vladimir Ferrafiat
email: vferrafiat@gmail.com

MD, MsC Elise Riquin

MD Elena Freri

MD Tiziana Granata

MD Nardo Nardocci

MD François Medjkane

PhD Claire Corfiotti

MD Alessandra Tozzo

Huges

MD Huges Pellerin

Xavier Benarous

PhD Julien Haroche

PhD Zahir Amoura

PhD Philippe Duverger

PhD Renaud Jardri

PhD Priscille Gerardin

PhD David Cohen

PhD Angèle Consoli

PhD Marie Raffin

Editing Julien Haroche

Editing Priscille Gerardin

Editing Renaud Jardri

Editing Philippe Duverger

Editing Zahir Amoura

Editing Angele Consoli

Psychiatric autoimmune conditions in children and adolescents: Is catatonia a severity marker?

Keywords: autism spectrum disorders, AE: autoimmune encephalitis, CAUS: causality assessment score, CNV: copy number variation, CSF: cerebral spinal fluid, CNS: central nervous system, ECT: electro-convulsive therapy, EEG: electroencephalogram, EOS: early onset schizophrenia, FDG PET: 18F-Flurodeoxyglucose positron emission tomography, HE: Hashimoto encephalopathy, TPO: thyroperoxidase, TG: thyroglobulin, ID: intellectual disability, IgG: immunoglobulin G, MRI: magnetic Resonance Imaging, OCD: obsessive compulsive disorder, PANS: Pediatric Acute-onset Neuropsychiatric Syndrome, PANDAS: pediatric autoimmune neuropsychiatric disorders associated with streptococcus infections, PCRS: Pediatric Catatonia Rating Scale, PE: plasma exchange, SLE: systemic lupus erythematosus Conceptualization, Methodology, Investigation, Data curation, Writing -Original Draft. Visualization. Elise Riquin: Methodology, Investigation, Data curation, Writing -Original Draft Methodology, Investigation, data curation, Writing -Original Conceptualization, Methodology, Investigation, Data curation, Writing, Writing -Review & Editing, Visualization. Marie Raffin Conceptualization, Methodology, Investigation, Data curation, Writing

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

Introduction

Pediatric catatonia is a rare and severe psychomotor syndrome associated with a large variety of psychiatric (early onset schizophrenia (EOS), autism spectrum disorder (ASD) and intellectual disability (ID)) [START_REF] Cohen | Clinical relevance of chronic catatonic schizophrenia in children and adolescents: evidence from a prospective naturalistic study[END_REF][START_REF] Cohen | Towards a valid nosography and psychopathology of catatonia in children and adolescents[END_REF][START_REF] Dhossche | Decalogue of catatonia in autism spectrum disorders[END_REF][START_REF] Withane | Electroconvulsive Treatment for Catatonia in Autism Spectrum Disorders[END_REF][START_REF] Ghaziuddin | Catatonia in Down syndrome; a treatable cause of regression[END_REF][START_REF] Fink | Catatonia in autistic spectrum disorders: a medical treatment algorithm[END_REF] and non-psychiatric conditions and with high rates of mortality and morbidity [START_REF] Cornic | Association of adolescent catatonia with increased mortality and morbidity: evidence from a prospective follow-up study[END_REF] . The prevalence of pediatric catatonia in the hospital setting is estimated at between 0.6% and 17.7%. Symptomatic treatment consists initially of high dosage benzodiazepines (e.g., lorazepam) [START_REF] Raffin | Treatment use in a prospective naturalistic cohort of children and adolescents with catatonia[END_REF] . In the case of resistance or life-threatening conditions, electro-convulsive therapy (ECT) is effective and safe in youth [START_REF] Dhossche | Decalogue of catatonia in autism spectrum disorders[END_REF][START_REF] Raffin | Treatment use in a prospective naturalistic cohort of children and adolescents with catatonia[END_REF][START_REF] Puffer | A 20 Year Practice Review of Electroconvulsive Therapy for Adolescents[END_REF][START_REF] Consoli | Electroconvulsive therapy in adolescents with the catatonia syndrome: efficacy and ethics[END_REF] . Approximately 20% of catatonias are secondary to an underlying medical condition, including genetic, neurological, infectious and autoimmune disorders [START_REF] Consoli | Medical and developmental risk factors of catatonia in children and adolescents: a prospective case-control study[END_REF][START_REF] Raffin | Catatonia in Children and Adolescents: A High Rate of Genetic Conditions[END_REF] . Therefore, specific etiological treatments can lead to catatonia improvement [START_REF] Consoli | Medical and developmental risk factors of catatonia in children and adolescents: a prospective case-control study[END_REF][START_REF] Ferrafiat | Catatonia and Autoimmune Conditions in Children and Adolescents: Should We Consider a Therapeutic Challenge?[END_REF][START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF][START_REF] Lahutte | Multidisciplinary approach of organic catatonia in children and adolescents may improve treatment decision making[END_REF][START_REF] Marra | Plasma exchange in patients with stuporous catatonia and systemic lupus erythematosus[END_REF] and have crucial impact on the prognosis [START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF][START_REF] Byrne | Earlier treatment of NMDAR antibody encephalitis in children results in a better outcome[END_REF][START_REF] Finke | Cognitive deficits following anti-NMDA receptor encephalitis[END_REF][START_REF] Florance | Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in children and adolescents[END_REF][START_REF] Dale | Autoimmune encephalitis in children: clinical phenomenology, therapeutics, and emerging challenges[END_REF][START_REF] Hacohen | Paediatric autoimmune encephalopathies: clinical features, laboratory investigations and outcomes in patients with or without antibodies to known central nervous system autoantigens[END_REF] . Autoimmune disorders, such as anti-NMDA receptor encephalitis [START_REF] Florance | Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in children and adolescents[END_REF][START_REF] Titulaer | Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study[END_REF] , Systemic Lupus Erythematosus (SLE) [START_REF] Tucker | Adolescent onset of lupus results in more aggressive disease and worse outcomes: results of a nested matched case-control study within LUMINA, a multiethnic US cohort (LUMINA LVII)[END_REF][START_REF] Hoffman | Juvenile-onset systemic lupus erythematosus: different clinical and serological pattern than adult-onset systemic lupus erythematosus[END_REF][START_REF] Livingston | Differences in clinical manifestations between childhood-onset lupus and adult-onset lupus: a meta-analysis[END_REF] , Hashimoto Encephalopathy (HE) [START_REF] Montagna | Hashimoto's encephalopathy: A rare proteiform disorder[END_REF][START_REF] Mahmud | Steroid-responsive encephalopathy associated with Hashimoto's thyroiditis in an adolescent with chronic hallucinations and depression: case report and review[END_REF][START_REF] Carlone | Acute psychiatric presentation of steroidresponsive encephalopathy: the under recognized side of autoimmune thyroiditis[END_REF] and Pediatric Autoimmune Disorders Associated with Streptococcus infections (PANDAS)/Pediatric Acute-onset Neuropsychiatric Syndrome (PANS) [START_REF] Swedo | Clinical presentation of pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections in research and community settings[END_REF][START_REF] Elia | PANDAS with catatonia: a case report. Therapeutic response to lorazepam and plasmapheresis[END_REF][START_REF] Schlansky | Pediatric Acute-Onset Neuropsychiatric Syndrome and Catatonia: A Case Report[END_REF] , are likely to exhibit an acute onset of severe psychiatric features at an early stage, including catatonia. The association between catatonia and severe psychiatric features (hallucinations, delusions, mood disorders and cognitive regression) remains rare and atypical in youths and is to be considered as a red flag for possible underlying autoimmune disorders. The identification of well characterized antibodies in plasma or cerebral spinal fluid (CSF) can be challenging, as a substantial proportion of youth with suspected autoimmune encephalitis is seronegative [START_REF] Dale | Autoimmune encephalitis in children: clinical phenomenology, therapeutics, and emerging challenges[END_REF][START_REF] Hacohen | Paediatric autoimmune encephalopathies: clinical features, laboratory investigations and outcomes in patients with or without antibodies to known central nervous system autoantigens[END_REF][START_REF] Lee | Tocilizumab in Autoimmune Encephalitis Refractory to Rituximab: An Institutional Cohort Study[END_REF] . Those conditions require the early introduction of aggressive immunosuppressive treatment to limit neurological and cognitive sequelae [START_REF] Byrne | Earlier treatment of NMDAR antibody encephalitis in children results in a better outcome[END_REF][START_REF] Florance | Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in children and adolescents[END_REF][START_REF] Titulaer | Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study[END_REF] . Moreover, autoimmune catatonia seems to respond well to the early introduction of plasma exchange [START_REF] Ferrafiat | Catatonia and Autoimmune Conditions in Children and Adolescents: Should We Consider a Therapeutic Challenge?[END_REF][START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF][START_REF] Marra | Plasma exchange in patients with stuporous catatonia and systemic lupus erythematosus[END_REF] . Despite potential adverse effects (e.g., infections, malignancies, infertility) [START_REF] Titulaer | Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study[END_REF][START_REF] Kashyape | Successful treatment of two paediatric cases of anti-NMDA receptor encephalitis with cyclophosphamide: the need for early aggressive immunotherapy in tumour negative paediatric patients[END_REF] , the risk-benefit ratio for using immunosuppressive treatment in youth remains favorable due to high mortality and morbidity rates [START_REF] Byrne | Earlier treatment of NMDAR antibody encephalitis in children results in a better outcome[END_REF][START_REF] Titulaer | Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study[END_REF][START_REF] Zekeridou | Treatment and outcome of children and adolescents with N-methyl-D-aspartate receptor encephalitis[END_REF] .

To date, there are few data regarding catatonia in autoimmune conditions. Most of the studies about autoimmune systemic disorders and autoimmune encephalitis have focused on neurological aspects, such as epilepsy and movement disorders and autonomic dysfunction, even though catatonia is considered as a movement disorder by some authors [START_REF] Granata | Pediatric NMDAR encephalitis: A single center observation study with a closer look at movement disorders[END_REF][START_REF] Graus | A clinical approach to diagnosis of autoimmune encephalitis[END_REF] . Psychiatric features in autoimmune conditions that are common have been broadly assessed. Assessments have mainly detailed psychotic and mood symptoms in the adult population. However, catatonia has often been reported in patients with autoimmune conditions, in particular in anti-NMDA encephalitis and lupus. One study proposed a subtype of catatonic anti-NMDA encephalitis [START_REF] Desena | Three phenotypes of anti-N-methyl-Daspartate receptor antibody encephalitis in children: prevalence of symptoms and prognosis[END_REF] . Our previous study underlined the key role of catatonia as a red flag for different types of autoimmune conditions [START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF] .

Here, we aimed to explore whether pediatric patients with autoimmune encephalitis exhibiting catatonia differed from patients with autoimmune encephalitis that did not. To do so, we retrospectively compared the following factors among a large series of patients with autoimmune encephalitis: 1) psychiatric and non-psychiatric clinical features and paraclinical characteristics; and 2) the clinical outcomes in terms of response to treatment, relapses, sequelae and death between patients with and without catatonia. Based on the high prevalence of catatonia in autoimmune disorders, our clinical experience with autoimmune catatonia 11,13- 16 , and on a study suggesting a predominant catatonic subtype of anti-NMDA receptor encephalitis with poorer treatment response [START_REF] Desena | Three phenotypes of anti-N-methyl-Daspartate receptor antibody encephalitis in children: prevalence of symptoms and prognosis[END_REF] , we hypothesized that catatonia might be a marker of severity in terms of initial clinical presentation and outcomes, with a higher risk of resistance to treatment for autoimmune conditions.

Methods

Participants

Inclusion criteria

Patients had to fulfill two main criteria: with immunosuppressive or immunomodulatory treatments was positive; iv) reasonable exclusion of alternative causes. To help define when a medical condition could be causal in pediatric catatonia [START_REF] Consoli | Medical and developmental risk factors of catatonia in children and adolescents: a prospective case-control study[END_REF] , we also retrospectively used a causality assessment score (CAUS). In autoimmune and pediatric setting, CAUS helps diagnose autoimmune conditions even in the absence of formal identification of autoantibodies with a validated threshold score ≥5, allowing early and aggressive use of immunosuppressive treatment [START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF] .

2. Patients should present acute severe neuropsychiatric symptoms, including a systematic assessment of catatonia. In addition to catatonia, the main psychiatric presentations included: an acute psychotic episode; mood disorders, such as manic, mixed or depressive episodes with or without psychotic features; severe cognitive regression (global or specific)

and isolated hallucinations (visual, auditory). In the French network, the diagnosis of catatonia was made when patients presented at least two motor symptoms or one motor and one non-motor symptom (Mutism, Negativism, Echolalia, Verbigeration, Withdrawal, Incontinence, Schizophasia, Acrocyanosis, Autonomic abnormality) indicative of severe behavioral and emotional impairment. The catatonic symptom list was based on a validated modified version of the Bush and Francis scale: the Pediatric Catatonia Rating Scale (PCRS) [START_REF] Benarous | Validation of the Pediatric Catatonia Rating Scale (PCRS)[END_REF] . In the Italian center, catatonia was systematically searched using DSM5 criteria.

They included the presence of three symptoms from the following list of twelve signs: stupor, catalepsy, waxy flexibility, mutism, negativism, posturing, mannerisms, stereotypy, agitation, grimacing, echolalia, and echopraxia.

Patient assessment

For each patient, each center systematically and retrospectively assessed: i) sociodemographic data (sex, age); ii) autoimmune and psychiatric history; iii) the type of autoimmune condition: autoimmune systemic disorders, definite AE and suspected AE; iv) the presence and type of catatonia, the presence and type of psychotic features (hallucinations, delusions), the presence of mood disorders (manic, depressive symptoms), ASD features, obsessive compulsive disorder (OCD), anxiety features, sleep disorders, and cognitive regression. Regarding the neurological signs, we systematically reported any movement disorders and seizures. All possible systemic localizations, such as cutaneous, digestive, or rheumatologic expression, were also systematically searched.

To maximize the accuracy of medical diagnoses, we used previously proposed guidelines for clinical and paraclinical investigations to help determine the medical conditions associated with catatonia and/or acute psychotic episodes [START_REF] Cornic | Association of adolescent catatonia with increased mortality and morbidity: evidence from a prospective follow-up study[END_REF][START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF][START_REF] Lahutte | Multidisciplinary approach of organic catatonia in children and adolescents may improve treatment decision making[END_REF][START_REF] Sedel | Psychiatric manifestations revealing inborn errors of metabolism in adolescents and adults[END_REF][START_REF] Benarous | Catatonia in children and adolescents: New perspectives[END_REF] . Neurological and global examinations were performed to identify medical conditions. Paraclinical investigations were performed accordingly to our previous published extensive panel when AE is suspected [START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF] .

Additional cerebral PET scanning was performed when the clinical presentation included fever and/or neurological signs and/or resistance to standard treatment and/or negative MRI.

The use of PET scan in AE remains as a research topic and is not available in all pediatric settings [START_REF] Morbelli | Role of (18)F-FDG-PET imaging in the diagnosis of autoimmune encephalitis[END_REF] . Some authors argue that FDG-PET imaging has low specificity regarding the cause of the disorder [START_REF] Graus | Role of (18)F-FDG-PET imaging in the diagnosis of autoimmune encephalitis -Authors' reply[END_REF] [START_REF] Turpin | 18F-Flurodeoxyglucose positron emission tomography with computed tomography (FDG PET/CT) findings in children with encephalitis and comparison to conventional imaging[END_REF] and adults (61 patients with AE, 85% PET abnormalities vs 68% MRI abnormalities) [START_REF] Probasco | Abnormal brain metabolism on FDG-PET/CT is a common early finding in autoimmune encephalitis[END_REF] . In our study, PET was considered abnormal based on local expert's interpretation. From the most recent literature data in adult and pediatric population, they usually consider the presence or absence of metabolism abnormalities mainly in cerebral cortex [START_REF] Probasco | Abnormal brain metabolism on FDG-PET/CT is a common early finding in autoimmune encephalitis[END_REF][START_REF] Turpin | 18F-Flurodeoxyglucose positron emission tomography with computed tomography (FDG PET/CT) findings in children with encephalitis and comparison to conventional imaging[END_REF] and basal ganglia [START_REF] Turpin | 18F-Flurodeoxyglucose positron emission tomography with computed tomography (FDG PET/CT) findings in children with encephalitis and comparison to conventional imaging[END_REF] The response to treatment was clinically and retrospectively evaluated with a threepoint scale based on the psychiatric and neurological clinical improvement evaluation by senior clinicians of each center: 0 for "no improvement," when no improvement of either psychiatric or neurological symptoms was found; 1 for "partial improvement," if at least one category of symptoms was improved; and 2 for "major improvement," when both psychiatric and neurological symptoms were improved. In addition to treatment response, we also reported possible poor outcomes, such as relapse, sequelae and death. The outcome assessments were performed knowing if the patient was catatonic or not. Relapse was considered when the patient presented a similar or more severe clinical (psychiatric and neurological) presentation within the coming year following discharge. Sequelae had to occur after treatment response and included: i) persistent seizures and/or movement disorders; ii) cognitive impairment, such as memory loss, language deficit, attention deficit, or persistence of the initial cognitive regression; and iii) persistent chronic psychiatric features requiring the long-term use of standard psychiatric treatment (e.g., antipsychotic, mood stabilizer, and antidepressant). The follow up period available for all patients was at least one year after treatment introduction.

Statistical Analysis

This is a retrospective analysis of pooled data from 5 centers. We compared patients with catatonia versus patients without catatonia for categorical variables: sex; autoimmune conditions; subtypes of catatonia (stuporous and/or agitated); psychiatric features; treatment response to first and/or second line treatments with the categories of "partial" and "major" improvement combined as "positive response" for binary percentages of improvement ; relapses; sequelae and death. The quantitative variables compared between the two groups included age and CAUS score.

Quantitative variables were described using the means and standard deviation.

Categorical variables were described using the numbers and percentages of occurrences.

Quantitative variables were compared using either Welch's t-test or Wilcoxon rank-sum test depending on the graphically assessed normality assumption. Categorical variables were compared using either Chi-squared test or Fisher's exact test if at least one expected count under the null hypothesis was less than five. Analyses were run using R software 3.4.0. A pvalue less than 0.05 were considered significant. Given the sample size and lack of statistical correction, this study is only exploratory.

Results

Demographic and clinical characteristics

The French series included 23 patients: 14 patients had catatonia, and 9 did not. The Italian series comprised 35 patients: 11 patients had catatonia, and 24 did not. Figure 1 summarizes the diagram flow of the study. In total, the sample included 58

Clinical Outcomes

First line therapies were efficient (major or partial improvement) for 46 (79%) patients and not efficient without any improvement for 12 (21%) patients. Second line therapies were efficient, with major or partial improvement for 26 (87%), and 4 (13%) patients had no improvement. Seven patients (12%) relapsed. Fifteen (26%) patients exhibited sequelae within the one-year follow up, including: persistent seizures (N=4), movement disorders (N=0), cognitive impairment (N=5, memory loss and language deficit), and chronic psychiatric features (N=6, anxiety and mood disorders). Outcome data were not available for two patients. Only one catatonic patient (2%) died from complications of severe autonomic dysfunction due to anti-NMDA encephalitis.

Interestingly most patients with suspected AE exhibited catatonia (N=10, 67%). Most of them fully responded to first lines including plasma exchange (N=13, 81%). Only 3 of them (19%) required both first and second line. Half of them (N=8) exhibited sequelae including cognitive impairment (N=3) and chronic psychiatric features (N=5, anxiety and mood disorders). Only one (6%) patient relapsed.

Patients with and without catatonia

Comparisons are given in Table 1. There were no differences between the two groups regarding sociodemographic data. A few differences emerged regarding clinical characteristics. Catatonic patients showed significantly more psychotic features than patients without catatonia (18 (72%) vs 9 (27.3%), p<0.001), including more delusional ideas and hallucinations. In addition, they had significantly more movement disorders (dystonia, choreiform or other non-specific abnormal movements): 25 (100%) vs 20 (60.6%), respectively, p<0.001. In contrast, patients without catatonia had significantly more anxiety manifestations than catatonic ones, with 33 (100%) vs 5 (20%), respectively, p= 0.012.

Regarding the response to immunomodulatory or immunosuppressive treatment, first line (high dosage corticoids and/or PE and/or IgG IV) and second line (including cyclophosphamide, mycophenolate mofetil, and azathioprine, rituximab) therapies were found to be more efficient for catatonic patients, with 24 (96%) versus 22 (66.7%) (p=0.006) and 17

(68%) versus 9 (27.3%) (p= 0.002), respectively, showing a major or partial clinical response.

However, they had more relapses than patients without catatonia, at 7 (29.2%) versus 0 (0%) (p=0.001), respectively. No significant differences regarding sequelae or death were found between the two groups. Catatonic patients received all high dose benzodiazepines, and none received ECT.

--Insert Fig 2--

Discussion

Few data exist regarding pediatric catatonia related to autoimmune conditions [START_REF] Ferrafiat | Catatonia and Autoimmune Conditions in Children and Adolescents: Should We Consider a Therapeutic Challenge?[END_REF][START_REF] Ferrafiat | Catatonia and Autoimmune Conditions in Children and Adolescents: Should We Consider a Therapeutic Challenge?[END_REF][START_REF] Benarous | Catatonia in children and adolescents: New perspectives[END_REF][START_REF] Mooneyham | Evaluation and Management of Autoimmune Encephalitis: A Clinical Overview for the Practicing Child Psychiatrist[END_REF][START_REF] Tanguturi | Anti-N-Methyl d-Aspartate Receptor Encephalitis and Electroconvulsive Therapy: Literature Review and Future Directions[END_REF] . This study gathers one of the largest samples over several university hospitals, allowing a multi-disciplinary diagnosis and therapeutic approach through various pediatric departments. First, our study underscores that autoimmune catatonia is significantly more associated with severe psychotic features compared to autoimmune conditions without catatonia, suggesting a more severe initial psychiatric presentation. Second, our results stress that patients with catatonia experienced more relapses that patients without catatonia. These results emphasize that autoimmune catatonia could imply a more severe form of autoimmune condition, supporting our hypothesis. However, we did not find a higher risk of poorer outcomes in terms of treatment resistance, sequelae or death among catatonic patients. Given our results, we discuss four main points: i) catatonia in immune-related conditions; ii) treatment; iii) anti-NMDA receptor encephalitis, as we observed 27 cases; and iv) the limitations of our study.

Our results support the idea that catatonia is associated with more severe forms of autoimmune disorders, but we can generalize this finding outside the context of anti-NMDA receptor encephalitis, as half of our cases were secondary to another causal encephalitis.

Catatonia was associated with more psychotic features, emphasizing the fact that autoimmune catatonia is likely to be associated with other severe psychiatric features, such as hallucinations, psychosis, cognitive regression, and fluctuation of symptoms, leading to an atypical psychiatric presentation in youths [START_REF] Consoli | Medical and developmental risk factors of catatonia in children and adolescents: a prospective case-control study[END_REF][START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF][START_REF] Bonnot | Secondary psychosis induced by metabolic disorders[END_REF] . Regarding the significant difference in anxiety features between the two groups, the non-catatonic patients were more likely to express their anxiety and to be diagnosed, whereas catatonic patients presented difficulties in identifying and therefore expressing their inner subjective feelings and experiences during the acute phase 2 . Furthermore, catatonic patients had more relapses. Even if the prognosis of neurocognitive sequelae is related to the timing of the introduction of immunosuppressive treatment [START_REF] Byrne | Earlier treatment of NMDAR antibody encephalitis in children results in a better outcome[END_REF][START_REF] Florance | Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in children and adolescents[END_REF][START_REF] Titulaer | Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study[END_REF][START_REF] Armangue | Autoimmune encephalitis in children[END_REF][START_REF] Luca | Anti-N-methyl-D-aspartate receptor encephalitis: a newly recognized inflammatory brain disease in children[END_REF][START_REF] Chapman | Anti-NMDA receptor encephalitis: diagnosis, psychiatric presentation, and treatment[END_REF][START_REF] Nosadini | Immune therapy in autoimmune encephalitis: a systematic review[END_REF] , it is notable that catatonic patients were more severe with more relapses regardless of their initial positive response to first or second line therapies. Hypothetically, catatonia as a clinical marker of risk of relapse would require earlier (even presumptively) and more aggressive immunosuppressive treatment to better prevent relapses and neurocognitive sequelae. In contrast to DeSena et al., who hypothesized that anti-NMDA encephalitis with catatonia showed a poor response to immunosuppressive treatments [START_REF] Desena | Three phenotypes of anti-N-methyl-Daspartate receptor antibody encephalitis in children: prevalence of symptoms and prognosis[END_REF] , our catatonic patients had a better response to either first or second line treatment than patients without catatonia.

These results might be explained by two factors: i) our catatonic patients were clinically more severe and therefore had faster access to immunotherapy and more combinations of first and second line treatments (only 4 catatonic patients did not receive second line treatments compared to 24 without catatonia); ii) the lack of standardized international treatment guidelines makes it difficult to interpret our treatment data, as patients' treatment plans and access to immunosuppressive treatment were heterogeneous and widely differed from one center to another. To illustrate this heterogeneity, we found that immunoglobulin was often used in our Italian sample. Plasma exchanges were only available in 3 centers due to limited access to this procedure. Cyclophosphamide was mainly used in one center according to a published multidisciplinary treatment algorithm [START_REF] Ferrafiat | A causality algorithm to guide diagnosis and treatment of catatonia due to autoimmune conditions in children and adolescents[END_REF] . The different treatments used in each center are detailed in Figure 2.

Regarding published data on treatment options, available studies suggest that the use of high dosage corticoids via pulses initially gives good response rates in pediatric populations [START_REF] Armangue | Autoimmune encephalitis in children[END_REF][START_REF] Luca | Anti-N-methyl-D-aspartate receptor encephalitis: a newly recognized inflammatory brain disease in children[END_REF][START_REF] Brunner | Difference in disease features between childhood-onset and adult-onset systemic lupus erythematosus[END_REF][START_REF] Levy | Systemic lupus erythematosus in children and adolescents[END_REF] . However, plasma exchanges and their peripheral action represent an interesting option in SLE and anti-NMDA receptor encephalitis [START_REF] Boers | Hashimoto's encephalopathy responding to plasmapheresis[END_REF][START_REF] Desena | Intravenous methylprednisolone versus therapeutic plasma exchange for treatment of anti-N-methyl-D-aspartate receptor antibody encephalitis: A retrospective review[END_REF][START_REF] Hussain | Effects of prednisone and plasma exchange on cognitive impairment in Hashimoto encephalopathy[END_REF][START_REF] Prytuła | Therapeutic plasma exchange in children with acute autoimmune central nervous system disorders[END_REF][START_REF] Suppiej | Plasma exchange in pediatric anti-NMDAR encephalitis: A systematic review[END_REF] and provide an efficient treatment for catatonia [START_REF] Ferrafiat | Catatonia and Autoimmune Conditions in Children and Adolescents: Should We Consider a Therapeutic Challenge?[END_REF][START_REF] Marra | Plasma exchange in patients with stuporous catatonia and systemic lupus erythematosus[END_REF][START_REF] Elia | PANDAS with catatonia: a case report. Therapeutic response to lorazepam and plasmapheresis[END_REF] . In the case of resistance to first line treatments [START_REF] Armangue | Autoimmune encephalitis in children[END_REF] or relapses, second line therapies are to be considered [START_REF] Byrne | Earlier treatment of NMDAR antibody encephalitis in children results in a better outcome[END_REF][START_REF] Florance | Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis in children and adolescents[END_REF][START_REF] Dale | Autoimmune encephalitis in children: clinical phenomenology, therapeutics, and emerging challenges[END_REF][START_REF] Titulaer | Treatment and prognostic factors for long-term outcome in patients with anti-NMDA receptor encephalitis: an observational cohort study[END_REF][START_REF] Stingl | An Update on the Treatment of Pediatric Autoimmune Encephalitis[END_REF] . The early introduction of second lines in youths ensure better outcomes [START_REF] Luca | Anti-N-methyl-D-aspartate receptor encephalitis: a newly recognized inflammatory brain disease in children[END_REF][START_REF] Stingl | An Update on the Treatment of Pediatric Autoimmune Encephalitis[END_REF][START_REF] Ishiura | Response of anti-NMDA receptor encephalitis without tumor to immunotherapy including rituximab[END_REF] . For example, despite rituximab's significant risk of infectious complications, a recent study supports its early off-label use in youths with significant morbidity secondary to autoimmune disorders [START_REF] Dale | Utility and safety of rituximab in pediatric autoimmune and inflammatory CNS disease[END_REF] .

Regarding anti-NMDA receptor encephalitis, most studies are recent, and it is the most frequent form of AE. This was also the case in our sample, at nearly half of the cases, and 49% of those cases had catatonia. Movement disorders, such as stereotyped movements, dystonia, chorea and catatonia, are frequent and are considered to be key symptoms exhibited by patients [START_REF] Granata | Pediatric NMDAR encephalitis: A single center observation study with a closer look at movement disorders[END_REF][START_REF] Dash | Proportion and spectrum of movement disorders in adolescent and adult patients of autoimmune encephalitis of non-neoplastic aetiology[END_REF][START_REF] Duan | Variations of movement disorders in anti-Nmethyl-D-aspartate receptor encephalitis: A nationwide study in Taiwan[END_REF][START_REF] Dalmau | Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies[END_REF] . Graus et al. included rigidity and abnormal postures (that belong to catatonic features) among the movement disorders in new diagnosis criteria for anti-NMDA receptor encephalitis [START_REF] Graus | A clinical approach to diagnosis of autoimmune encephalitis[END_REF] . Interestingly, two studies focusing on catatonia found that patients who had hypokinetic disorders, such as catatonia, took significantly longer to improve or did not fully recover compared to other forms of movement disorders [START_REF] Granata | Pediatric NMDAR encephalitis: A single center observation study with a closer look at movement disorders[END_REF][START_REF] Dash | Proportion and spectrum of movement disorders in adolescent and adult patients of autoimmune encephalitis of non-neoplastic aetiology[END_REF] . DeSena et al. proposed distinguishing 3 phenotypes of anti-NMDA receptor antibody encephalitis in children [START_REF] Desena | Three phenotypes of anti-N-methyl-Daspartate receptor antibody encephalitis in children: prevalence of symptoms and prognosis[END_REF] : type

1 "classic anti-NMDA receptor antibody encephalitis with predominant movement disorder and epilepsy"; type 2 "psychiatric predominant NMDA receptor encephalitis subtype"; and type 3 "catatonia/stupor predominant anti-NMDA receptor encephalitis sub phenotype." Type 3 patients were often very severe and showed the poorest response even to aggressive immunotherapies [START_REF] Desena | Three phenotypes of anti-N-methyl-Daspartate receptor antibody encephalitis in children: prevalence of symptoms and prognosis[END_REF] . Anti-NMDA encephalitis emphasizes that our results cross paths with those previous available data in terms of severity, whether the severity is defined by the initial clinical presentation, treatment response, frequency of relapses or sequelae. Hence, our results extend the concept of catatonia as a marker of severity to autoimmune conditions in general.

However, the results should be interpreted in the context of several limitations. First, the number of subjects recruited was low, despite the multicentric recruitment. This could be explained by the very low prevalence of catatonia in youths compared to other psychiatric disorders [START_REF] Cohen | Clinical relevance of chronic catatonic schizophrenia in children and adolescents: evidence from a prospective naturalistic study[END_REF] and the low prevalence of autoimmune conditions among the pediatric population [START_REF] Dale | Autoimmune encephalitis in children: clinical phenomenology, therapeutics, and emerging challenges[END_REF][START_REF] Stingl | An Update on the Treatment of Pediatric Autoimmune Encephalitis[END_REF] . The statistical analysis should be regarded as exploratory. Second, to increase the number of patients with probable, suspected and definite AE, we grouped different samples that had recruitment biases, leading to heterogeneity. Indeed, 60% of the cases came from the Italian autoimmune encephalitis specialized center, with a lower proportion of catatonia compared to the French network specialized in pediatric catatonia. Additionally, the two centers had a different first-step inclusion criterion, highlighting center-based biases. As a consequence, generalization may not be valid. Third, we used a multicenter clinical sample of acutely ill patients recruited in university teaching hospitals that may have been particularly

enriched for subjects with more severe forms of neuropsychiatric autoimmune conditions.

Conclusion

Despite its exploratory nature, this study enhances the need to consider catatonia as a marker of possible underlying autoimmune conditions, as many children and adolescents with suspected autoimmune encephalitis are seronegative [START_REF] Dale | Autoimmune encephalitis in children: clinical phenomenology, therapeutics, and emerging challenges[END_REF] . It also underscores the idea that catatonia can be a marker of severity and morbidity in terms of associated psychiatric symptoms and outcomes. Finally, the response of autoimmune catatonia to treatment, along with a higher relapse rate further supports the existing literature for both early and aggressive immunosuppressive treatments.

  This publication involves current clinical practice and is based on a clinical study in compliance with the Ethics of the University Centers. The study was conducted and approved according to the hospital ethics committee's regulations. The French sample included every child and adolescent inpatient admitted for a severe acute psychiatric presentation (catatonia, acute psychosis episode, cognitive regression, hallucinations, and mood disorders) and a diagnosis of autoimmune conditions from 4 departments of Child and Adolescent Psychiatry belonging to a National network for rare psychiatric diseases: i) University Hospital La Pitie -Salpêtrie re, Paris, France between 1993 and 2017; ii) University Hospital Charles Nicolle, Rouen, France between 2013 and 2017; iii) University Hospital of Lille, France between 2015 and 2017; iv) University Hospital of Angers, France between 2015 and 2017. The Italian sample included every child and adolescent inpatient admitted for suspicion of acute encephalopathy to the Department of Pediatric Neuroscience at the Foundation IRCCS Neurological Institute "Carlo Besta", Milan, Italy between 2010 and 2017.

  including: i) hypometabolisms in frontal and/or occipital lobes; ii) hypermetabolisms involving every five lobes; and iii) basal ganglia hypermetabolisms. All paraclinical data were retrospectively assessed by each center.Due to the absence of treatment guidelines, each center had its own treatment algorithm. Etiological treatments were reviewed and systematically retrospectively classified into three groups: i) tumor removal; ii) first line treatments, including corticoids, intravenous immunoglobulins (IgG IV) and plasma exchanges (PE); and iii) second line treatments, including cyclophosphamide, mycophenolate mofetil, azathioprine, and rituximab. Despite differences in treatment algorithm, all centers ensured an early and timely introduction of both lines.

  neurological symptoms with movement disorders and seizures; systemic localization; plasma biological arguments; CSF biological arguments; abnormal EEG; abnormal MRI; abnormal PET scan; type of treatment used with tumor removal, first line and second line treatments;

Figure 2 .

 2 Figure 2. Participants' first and second line treatments according to catatonia IgG IV: intravenous immunoglobulins; PE: plasma exchange; CYC: cyclophosphamide; MYP: mycophenolate mofetil; AZP: azathioprine; RTX: rituximab

Table 1 . Socio-demographics and clinical characteristics of the patients with neuropsychiatric autoimmune conditions with and without catatonia AE

 1 : autoimmune encephalitis, ASD: autism spectrum disorder, OCD: obsessive-compulsive disorder, CSF: cerebro-spinal fluid; *N=56; In most cases we used Chi2 tests for comparisons except when indicated

	Total	P
	(N=58)
	Socio-demographics

This study was performed without any specific support.


Soufian Ider

Yanath Belguesmia
email: yanath.belguesmia@univ-lille.fr

Guillaume Cazals

Rabah Boukherroub

Françoise Coucheney

Mabrouk Kihal

Christine Enjalbal

Djamel Drider

Rhizophila Soufian Ider

The antimicrobial peptide oranicin P16 isolated from Trichosporon asahii ICVY021, found in camel milk's, inhibits Kocuria rhizophila

Keywords: Trichosporon asahii, yeast antimicrobial peptide, Kocuria rhizophila, camel milk

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

Introduction

Isolation and characterization of non-conventional yeasts from foods and their applications as biopreservative agents has received limited investigation. Camel milk was recently targeted as a potential source of autochthonous non-conventional yeasts and was shown to have Trichosporon asahii strains [START_REF] Ider | Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria[END_REF]. Trichosporon spp. are opportunistic organisms that are distributed throughout nature [START_REF] Colombo | Current knowledge of Trichosporon spp. and Trichosporonosis[END_REF], but their presence in the human microbiome seems to be limited [START_REF] Mariné | On and under the skin: Emerging basidiomycetous yeast infections caused by Trichosporon species[END_REF]. These basidiomycetes yeast-like anamorphic organisms can be found in different ecological niches including the skin and gut of healthy individuals [START_REF] Cho | Molecular evidence that the opportunistic fungal pathogen Trichosporon asahii is part of the normal fungal microbiota of the human gut based on rRNA genotyping[END_REF][START_REF] Zhang | The opportunistic yeast pathogen Trichosporon asahii colonizes the skin of healthy individuals: Analysis of 380 healthy individuals by age and gender using a nested polymerase chain reaction assay[END_REF], dairy products as spoilage yeasts (Garnier et al., 2017), soft churupi soup, Indian fermented foods, and camel milk [START_REF] Ider | Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria[END_REF]Rai et al.2016), indicating their capabilities of adaptation and thriving in various host and environmental niches.

Fungal spoilage is a major concern for foods and causes between 5 and 10% of the world's food production losses (Garnier et al., 2017). The presence of such microorganisms in dairy and derivative products could lead to a visible growth of fungi or to the production of unwanted metabolites causing organoleptic deterioration, and possibly a safety hazard [START_REF] Hymery | Filamentous fungi and mycotoxins in cheese: A review[END_REF][START_REF] Ledenbach | Microbiological spoilage of dairy products[END_REF]. The use of biopreservation has become another way to handle/control the development of spoilage microorganisms as well as that of food-borne pathogens (Garnier et al., 2017).

T. asahii is also emerging as a biotechnological tool for its ability to decontaminate polluted environments by accumulating large amounts of oils [START_REF] Ageitos | Oily yeasts as oleaginous cell factories[END_REF], and also converting isoeugenol into vanillin [START_REF] Ashengroph | Bioconversion of isoeugenol to vanillin and vanillic acid using the resting cells of Trichosporon asahii[END_REF]. Strains of T. asahii were reported to produce lipase [START_REF] Singh | Novel S-enantioselective lipase TALipB from Trichosporon asahii MSR54: Heterologous expression, characterization, conformational stability and homology modeling[END_REF], aspartic-type peptidase (Valle et al., 2017), and antioxidant molecules [START_REF] Zhang | Study on antioxidant enzymatic activities of Trichosporon asahii[END_REF]. Furthermore, the T. asahii GSY10 strain was able to produce lipid from molasses, and was suggested as a feed supplement in dairy cattle (Paserakung et al., 2015). [START_REF] Chen | Screening and identification of yeasts antagonistic to pathogenic fungi show a narrow optimal pH range for antagonistic activity[END_REF] reported the antagonistic effects of T. asahii strains against fungi through production of extracellular pH-dependent compounds.

In this study, the inhibitory properties of 6 T. asahii strains isolated from camel milk that had been collected from arid and semi-arid regions of Algeria [START_REF] Ider | Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria[END_REF], were tested against a range of target organisms including fungi and bacteria. Only T. asahii ICVY021 showed an inhibition against Kocuria rhizophila CIP 53.45 through production of an extracellular, heat-stable and proteinaceous peptide. K. rhizophila, which belongs to the Micrococcaceae family, was described as a contaminant of camel milk [START_REF] Elhaj | Aerobic bacteria and fungi associated with raw camel's milk[END_REF][START_REF] Takarada | Complete genome sequence of the soil actinomycete Kocuria rhizophila[END_REF] with enzymatic activities able to induce spoilage of dairy product [START_REF] Pukančíková | Natural microflora of raw cow milk and their enzymatic spoilage potential[END_REF]. Moreover K. rhizophila is responsible for human infection [START_REF] Moissenet | Persistent bloodstream infection with Kocuria rhizophila related to a damaged central catheter[END_REF], and for diseases in rainbow trout or brown trout (Pękalaa et al., 2018). Therefore this study aimed at characterizing the antibacterial peptide, designated oranicin P16, produced by T. asahii strain ICVY021.

Materials and methods

Growth conditions and media

T. asahii strains ICVY021, ICVY022, ICVY023, ICVY024, ICVY025 and ICVY026 were isolated from camel milk in arid and semi-arid regions of Algeria (i.e.,Béchar,Biskra,Djelfa,, respectively) [START_REF] Ider | Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria[END_REF]. T. asahii ICVY021 was chosen because of its antimicrobial activity, while the other 5 strains were randomly isolated among many other strains without antimicrobial activity. Strains were plated onto YEG agar (5.0 g.L - 1 yeast extract; 20.0 g.L -1 glucose; 15.0 g.L -1 agar-agar, Sigma Aldrich Co., St. Louis, MO, USA), supplemented with 0.1% (w/v) chloramphenicol (Sigma Aldrich). The plates were incubated for 3 days at 25 °C. Yeast colonies were purified by streaking onto YEG agar and stored at -80 °C in YEG broth supplemented with 30% (v/v) glycerol for a maximum of three months.

Genomic DNA extraction

Total DNA was extracted using the Bust n'Grab method [START_REF] Harju | Rapid isolation of yeast genomic DNA: Bust n'Grab[END_REF]. Cells from 1.5 mL of the overnight yeasts cultures in YEG broth were pelleted in a microcentrifuge tube at 8,000 g (8680 rpm in a FA-45-30-11 rotor, Model 5804R centrifuge, Eppendorf, Hamburg, Germany) for 10 min at 4°C. The cell pellets were suspended in a lysis buffer (2% Triton X-100; 1% SDS; 100 mM NaCl; 100 mM Tris-HCl at pH 8.0; 0.1 mM EDTA, Sigma Aldrich), and disrupted by two repetitive heat shock cycles at -80 °C (for two min) and 95 °C (for one min). DNA was extracted using the chloroform procedure as follows: 200 μL of chloroform (Sigma Aldrich) was added to the tubes and Vortexed (Vortex Genie 2, Dutscher, Brumath, France) for two min and centrifuged at 20,000 g, three min at room temperature (20-25 °C).

The aqueous layer was transferred to a tube containing 400 μL of ice-cold 100% ethanol (Sigma Aldrich). The samples were precipitated for 5 min at room temperature, and centrifuged at 20,000 g for 5 min at room temperature. Supernatants were pipetted and the DNA pellets were washed with 0.5 mL of 70% ethanol followed by drying at 37 °C. DNA was re-suspended in 10 mM Tris-EDTA buffer (pH 8.0, Sigma Aldrich) and its concentration determined with a Nanodrop Biowave II (Biochrom, Harvard, UK), by measuring their absorbance at 260nm UV wavelength.

Yeast identification using an API 20C AUX strip and molecular methods

The API 20C AUX identification procedure was done in accordance with the manufacturer's instructions (bioMérieux, Marcy-l'Étoile, France). Cultures of ~10 6 colony forming units (CFU) mL -1 were prepared in YEG broth, and 5 drops were dispersed in an API C medium vial provided by the manufacturer and homogenized by Vortexing. After homogenizing, the inoculum was deposited on the wells of the test strip, and incubated at 25°C for 48 h. The wells with growth were allocated a plus sign, whereas those with the absence of growth were allocated a minus sign. The results were transformed into numerical codes using a correspondence table provided by the manufacturer. The isolates were identified using the 20C analytical profile index (API 20C) available online (https://apiweb.biomerieux.com/).

The sequences of the 26S rDNA and ITS1-5.8-ITS2 were determined using the polymerase chain reaction (PCR) products gathered from the genomic DNA of the yeast cells, extracted using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA). The sequencing of the 26S rDNA and ITS1-5.8-ITS2 were done using primers and programs shown in Table I. The PCR products were separated on a 1% agarose gel (Bio-Rad, Hercules, CA, USA) labelled with 0.05% GelRed (Thermo Fisher Scientific Co., Waltham, MA, USA)

for 2 h at 100 V. The gels were analysed using PyElph 1.4 software available online (https://pyelph.software.informer.com/1.4/).

The PCR products were sequenced at Eurofins (Hamburg, Germany). A blast search of the nucleotide sequences was done through the nucleotide Blastn software (https://blast.ncbi.nlm.nih.gov/Blast.cgi) of the US National Center for Biotechnology Information (NCBI). The nucleotide sequences obtained for these samples were deposited at the NCBI database with the accession numbers given in Table II.

Repetitive-element-palindromic-PCR (Rep-PCR) fingerprints

The Rep-PCR was done with the unique primer 5'-GTG GTG GTG GTG GTG-3' (GTG5, Eurofins) [START_REF] Ceugniez | Yeast diversity in a traditional French cheese "Tomme d'orchies" reveals infrequent and frequent species with associated benefits[END_REF][START_REF] Ouoba | Genotypic diversity of lactic acid bacteria isolated from African traditional alkaline-fermented foods[END_REF][START_REF] Ouoba | The microbiology of Bandji, palm wine of Borassus akeassii from Burkina Faso: Identification and genotypic diversity of yeasts, lactic acid and acetic acid bacteria[END_REF]. The PCR program used included an initial denaturation at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 1 min, hybridation at 40 °C for 1 min and elongation at 72 °C for 8 min. The final elongation was done for 16 min at 72 °C. For each reaction 150 ng of DNA, master mix PCR 2X (Thermo Fisher Scientific), ultra-pure water (Thermo Fisher Scientific) and 2 µM primer were used. The PCR products and 5 µL of DNA marker (Thermo Scientific, Vilnius, Lithunia), at 0.1 µg µL -1 , were deposited and separated on a 1% agarose gel labelled with 0.05% GelRed for 2 h at 100 V. The gels were visualized and photographed with a Gel-Doc 2000® (Bio-Rad), and the DNA profiles (size and number of amplicons) were established with GelCompar II (v6.6.11) software (Biosystematica, Ceredigion, Wales, UK).

Assessment of antagonistic properties of T. asahii strains

The inhibitory properties of culture supernatants of T. asahii ICVY021, ICVY022, ICVY023, ICVY024, ICVY026 and ICVY026 strains were assessed against different fungi and Gram- rhizophila strains isolated from a meat grinder in a Danish Slaughterhouse, and kindly provided by Professor J. Herschend (Copenhagen, Denmark) were also tested. T. asahii strains were grown in YEG broth for 48 h at 25 °C with shaking (160 rpm). Culture supernatants (CS) were obtained by centrifugation at 6,000 g (6,690 rpm in a FA-45-6-30 rotor), for 10 min at 4 °C, and stored at this same temperature for a maximum of 2 days before use. One mL of target strain suspension in Mueller Hinton (MH) broth (Sigma Aldrich), previously adjusted at OD600nm to 0.0025, was flooded on MH agar plates. After drying the plates at room temperature in a laminar flow hood (ADS Laminaire, Aulnay-sousbois, France) for <20 min, wells of ~6 mm were made with sterile pipette tips (VWR, Radnor, PA, USA) in each plate, and filled with 50 µL of CS. After 2 h of incubation at 4 °C, allowing diffusion of CS, plates were incubated at 37 °C for 48 h. In all these experiments, the presence or absence of inhibition zones was visually determined.

Purification of the inhibitory compound from T. asahii ICVY021

The active CS obtained after the culture of T. asahii ICVY021 was purified using Amberlite ® XAD ® -2 polymeric adsorbent resin (Sigma Aldrich). CS (50 mL) were mixed with the XAD resin (2 g) and incubated overnight at 20 °C with orbital agitation at 100 rpm. The mixture was vacuum filtrated through a 0.2 µm filter (VWR) and the filtrate removed. The XAD resin was resuspended and washed twice with 20 mL of ultrapure water (Thermo Fisher Scientific), and incubated at 20 °C with agitation at 100 rpm for 20 min. The XAD resin containing the active compound(s) was separated again by vacuum filtration as described above and resuspended in a mix of 10 mL of ultrapure water + 10 mL methanol, and incubated at 100 rpm, 20 °C for 20 min. The water/methanol phase containing the active compound, designated oranicin P16, was recovered using a syringe after the resin settled, and the solvent was removed using a speed vacuum concentrator (SpeedVac, Thermo Fisher Scientific).

Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the purified inhibitory compound oranicin P16 produced by T. asahii ICVY021

Following the growth of K. rhizophila CIP 53.45 in MH medium at 37 °C, 1 colony was randomly chosen and added to 10 mL of MH broth. Samples of 10 μL of this bacterial suspension were added to the 96 wells of bioassay microplates containing serial dilutions of active compound prepared as indicated above. The MIC was defined as the lowest concentration of this active compound that inhibited the visible growth of the target strain after overnight incubation at 37 °C.

The MBC was determined by subculturing the inoculated samples, used for the MIC assay, in MH agar without oranicin P16. The MBC was defined as the concentration of active compound that reduced the viability of the initial inoculum of K. rhizophila CIP 53.45 by ≥99.9%, as determined by counting the cells on MH plates, compared to the untreated inoculum tested using the same conditions.

In situ activity of oranicin P16 in skim milk

To assess the in situ activity of the purified active compound, a time-killing experiment was done. Tubes containing a commercial sterilized skim milk (Carrefour ® , Carrefour group, France, Boulogne-Billancourt, France) were supplemented with 10% (v/v) of PBS, or nisin (Sigma Aldrich) at a final concentration of 1 µg mL -1 , or with the active compound oranicin P16 at the MIC value. A volume of 20 µL of a K. rhizophila CIP 53.45 suspension of ~2×10 9 CFU mL -1 in PBS, as determined by cell-counting, was introduced in the prepared tubes containing 2 mL of the supplemented skim milk with PBS (control), nisin or oranicin P16, and incubated at 37 °C for 6 h. The killing kinetics of the nisin and the oranicin P16 were monitored using viable bacterial counts on agar plates compared to the control. Aliquots were taken at 0, 2, 4 and 6 h, and serially diluted in saline solution for determination of viable counts. Diluted samples (100 μL) were plated on the MH agar plates and colonies were counted after overnight incubation at 37 °C.

Molecular mass and amino acids sequence of the purified inhibitory compound

Liquid chromatography -mass spectrometry (LC/MS) analyses, using positive mode with electrospray ionisation (ESI) and high resolution time of flight (TOF) mass analyzer, were done using an Acquity H-Class (Waters Corp., Milford, MA, USA) system equipped with a Kinetex EVO C18 column (1.9 mm particle size, 150 × 2.1 mm), (Phenomenex, Torrance, CA, USA). The mobile phase A consisted of water while mobile phase B was acetonitrile both containing 0.1% formic acid. The gradient started at 5% mobile phase B and increased to 100% mobile phase B over 40 min. The flow rate was 0.5 mL min -1 and the eluent was directed to the atmospheric pressure ionisation source of the Synapt G2-S (Waters) tandem hybrid Quadrupole-TOF mass spectrometer operating in positive electrospray ionization mode. Capillary and cone voltages were 3000 and 30 V, respectively. Source and desolvation temperatures were 120 and 450 °C, respectively. The molecular weight of the purified peptide was determined by the deconvolution of the resulting signal. MS/MS analyses were done using collision induced dissociations (CID) fragmentation at 40 eV. All data were acquired and processed using Masslynx 4.2 software (Waters). Then the sequences were blasted using the NCBI protein database using BlastP online software (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The matching peptides, with highest sequences homology, were then compared manually.

2.10. Transmission electron microscopy (TEM) of K. rhizophila CIP53.45 treated with the inhibitory compound K. rhizophila CIP 53.45 was grown in 2 mL MH medium overnight and divided equally in two different tubes after Vortexing. After centrifugation at 8,000 g at 4 °C for 10 min, the supernatants were discarded and the cells pellets were washed twice with PBS. In the first tube, the pellets were resuspended and treated with the oranicin P16 at MBC, determined as described previously, and resuspended in sterile ultrapure water. In the second tube (control tube), the pellets were resuspended in sterile ultrapure water, and the samples were incubated overnight at 37 °C.

For TEM, samples were fixed in 2.5% glutaraldehyde (Merck Co., Kenilworth, NJ, USA) buffered with 0.1 M sodium cacodylate (Merck), post-fixed in 1% osmium tetroxide (EMS, Hatfield, PA, USA) in the same buffer and "en bloc" stained with 2% uranyl acetate (VWR).

After washing with acetonitrile and air-drying, samples were embedded in epon-like resin (EMbed-812, EMS). Ultrathin sections of ~90 nm were cut using a Leica UC7 ultramicrotome (Wetzlar, Germany) and collected on 150 mesh hexagonal barred copper grids. The samples were observed at 200 kV using a Jeol JEM-2100 microscope (Jeol Ltd., Tokyo, Japan) coupled with a Gatan Orius SC200D camera (Gatan Inc., Oxford, UK) with magnification of ~20,000 to 40,000x

2.11. Hemolysis and resistance to antifungal drugs of T. asahii ICVY021 antagonistic strain

The hemolytic activity of antagonistic strain was determined by streaking fresh colonies on blood agar plate (VWR). The plate was incubated at 37 °C for 24-48 h. The development of a clear zone around the colonies was considered as a positive result. The in vitro studies of sensitivity to amphotericin B, 5-fluorocytosine, fluconalzol, voriconazol, caspofungin, posaconazol and anidulafungin (bioMérieux) were done using the E-test method (bioMérieux) according to CLSI (Clinical Laboratory Standards Institute) guidelines. Briefly, an inoculum of T. asahii ICVY021 was prepared and adjusted to ~10 6 cells mL -1 . A sample of 0.5 mL of this suspension was inoculated onto plates containing RPMI 1640 agar (1.5%) (Sigma Aldrich) with 2% glucose (Sigma Aldrich) using a sterile cotton swab. After a period of 15 min, the E-test strips (5.7×0.5 cm) containing the antifungal drugs, with concentrations ranging from 0.002 to 256 µg mL -1 , were applied on the plates (9 cm diameter). The MIC which is the lowest concentration allowing the complete inhibition of yeast growth was obtained after 48 h of incubation at 25° C.

The interpretation of MIC values was done using the data given for T. asahii by [START_REF] Kanafani | Resistance to antifungal agents: Mechanisms and clinical impact[END_REF], in accordance with the recommendations approved by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2017) and the CLSI (CLSI, 2018) using Candida albicans as a reference strain [START_REF] Cuenca-Estrella | ESCMID guideline for the diagnosis and management of Candida diseases 2012: Diagnostic procedures[END_REF]. According to these recommendations, the T. asahii was considered resistant to the antifungal agents tested when their MIC were equal or higher to the following cut off concentrations: 2 mg L -1 for amphotericin B, 50 mg L -1 for 5-fluorocytosine, 8 mg L -1 for fluconalzol, 1 mg L -1 for voriconazol, 1 mg L -1 for caspofungin, 0.06 mg L -1 for posaconazol and 1 mg L -1 for anidulafungin.

Cytotoxicity assay

Intestinal human colorectal tumour Caco-2 cells (Sigma Aldrich) were grown in 96-well cell culture plates and pre-incubated for 7 days at 37 °C, 5% CO2 atmosphere in Dulbecco modified Eagle medium (DMEM, Pan-Biotech GmbH, Aidenbach, Germany) supplemented with 10% of fetal bovine serum (FBS, Thermo Fisher Scientific), 2 mM glutamine (Pan-Biotech GmbH), 100 µg mL -1 of penicillin and 100 µg mL -1 of streptomycin (Pan-Biotech GmbH). A 24 h old culture of T. asahii ICVY021 were diluted to 2×10 8 CFU mL -1 in antibiotic and serum free DMEM. A suspension of 100 µL was added to each well containing ~2.10 5 Caco-2 cells. Furthermore, the cytotoxicity on the Caco-2 cells was measured after 24 h of contact by determining the dehydrogenase activity of mitochondria with the CCK-8

(Counting Kit-8) assay (Dojindo, Kumamoto, Japan). Briefly, after incubation cells were washed with DMEM without any supplementation to remove non-adherent yeasts. To eliminate adherent yeasts, 50 μL of amphotericin B (Sigma Aldrich), at a final concentration of 5 mg L -1 in PBS, were added to each well. After 2 h of incubation at 37 °C, the solution of amphotericin B was removed and the wells were filled with 100 µL of DMEM supplemented with 5 µL of CCK-8 (Dojindo) reagent and incubated for an additional 2 h at 37 °C. Wells with Caco-2 cells treated with Triton X100 solution (Sigma Aldrich), diluted to a final concentration of 0.1% in DMEM were used as a positive control of cytotoxicity whereas wells with non-treated Caco-2 cells served as a negative control. An additional negative control using wells with Caco-2 cells treated with amphotericin B, with the same conditions as described before, but not exposed to yeast was also used.

formazan from the water-soluble tetrazolium salt: WST-8(2-(2-methoxy-4nitrophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)2H tetrazolium, monosodium salt)). The formation of formazan was measured at 450 nm using a microplate reader (Xenius SAFAS, Monaco), and compared with the absorbance of non-treated cells considered as the control.

Adhesion assay

The adhesion abilities of T. asahii ICVY021 on Caco-2 cells were determined as reported by

Ait [START_REF] Seddik | Anti-pathogenic and probiotic attributes of Lactobacillus salivarius and Lactobacillus plantarum strains isolated from feces of Algerian infants and adults[END_REF]. Caco-2 cells/well (4×10 4 ) were seeded in 24-well tissue culture plate, incubated for 7 days at 37 °C (5% CO2 and 95% air). Cells of T. asahii ICVY021 grown in YEG broth for 48 h at 25 °C were harvested by centrifugation at 6,000 g at 4 °C for 10 min, and resuspended in DMEM without FBS and antibiotics to a final concentration of 10 5 CFU/well. To determine the effect of proteins or carbohydrates on the adhesion process of T.

asahii ICVY021 with confluent Caco-2 monolayers, the yeast cells were treated with trypsin from porcine pancreas (Sigma Aldrich) at a final concentration of 2.5 mg mL -1 for 1 h at 37°C, or with 50 mM of periodic acid (Sigma Aldrich) in 10 mM PBS (pH 7.4) for 30 min at 37 °C (Ait [START_REF] Seddik | Anti-pathogenic and probiotic attributes of Lactobacillus salivarius and Lactobacillus plantarum strains isolated from feces of Algerian infants and adults[END_REF]. Both samples were washed twice with PBS and resuspended in DMEM without FBS and antibiotics. Untreated yeast-cells at ~10 5 CFU/well or treated ones with trypsin or periodic acid were applied to confluent Caco-2 cells and incubated for 2 h at 37 °C (5% CO2 and 95% air). The Caco-2 monolayers were washed twice with 500 μL of PBS to remove non-adherent yeast cells. Lysates obtained after incubation with Triton X100 (Sigma Aldrich) at 0.1% (v/v) for 15 min, were serially diluted and plated on YEG agar to determine the number of adherent yeast cells, untreated and treated with trypsin or periodic acid.

Statistical analysis

Data of cytotoxicity and adhesion assays were expressed as a mean ± standard error calculated over three independent experiments. Analysis of statistical significance was done using one way ANOVA and the post-hoc Tukey Test (P < 0.05) using XL-STAT (Addinsoft, Bordeaux, France).

Results

API 20C AUX strips and molecular methods are reliable methods for identification of T. asahii strains

After 72 h at 25 °C colonies of T. asahii ICVY021, ICVY022, ICVY023, ICVY024, ICVY025 and ICVY026 on YEG-agar plates were white, dried and wrinkled with irregular margins (Fig. 1). All the strains were identified as T. adahii using API 20C AUX strips and molecular methods. The scores obtained using the API 20C AUX system ranged from 90 to 100% (Table II), while those obtained after blasting 26 rDNA and ITS1-5.8-ITS2 regions sequences ranged between 99 to 100% (Table II).

T. asahii ICVY021 showed a slightly different DNA pattern

The analysis of rep-PCR DNA fingerprints and their related dendrogram delineated two main groups of yeasts. The first is composed of ICVY022, ICVY023, ICVY024, ICVY024, ICVY025 strains, and the second contains only T. asahii ICVY021 (Fig. 2).

T. asahii ICVY021 is the only strain with inhibitory properties

The inhibitory properties of T. asahii strains were tested against different organisms including fungi, and both Gram positive and Gram negative bacteria. CS were tested as sources of inhibitory compound(s). Only T. asahii ICVY021 showed an inhibition zone against K.

rhizophila CIP 53.45 (Fig. 3). The CS was heat-treated for 10 min at 60, 80 and 100 °C, and tested against K. rhizophila CIP 53.45. The antibacterial activity had slightly decreased with treatment at 100 °C and 80 °C but not at 60 °C (data not shown). Protease treatments abolished the anti-K. rhizophila CIP 53.45 activity, suggesting a proteinaceous active compound.

For the in situ assessment of this activity, killing curves were obtained in a commercial skim milk using K. rhizophila CIP 53.45 as a target organism, treated or not with nisin at 1 µg mL - 1 , and the purified oranicin P16, used at the MIC value (215 µg mL -1 ). Microbial counts were measured as CFU mL -1 and the resulting averaged data were transformed to Log CFU mL -1 .

The cell number in the treated skim milk with oranicin P16 decreased by ~1 Log CFU mL -1 , whereas the untreated sample was unaffected showing a growing population, which reached 6.61 Log CFU mL -1 (Fig. 4). Nevertheless, nisin showed the strongest inhibitory activity with a rapid decreasing number of the viable and cultivable cells, by 3 Logs after 6 h, therefore killing >99.9% of the initial bacterial populations.

Resistance to antifungal drugs, hemolytic and cytotoxic effects of T. asahii ICVY021

T. asahii ICVY021 was tested against 6 antifungal drugs routinely used in treatment of yeast infections. The MIC values obtained were 0.125 mg L -1 for amphotericin B, 200 mg L -1 for 5fluorocytosine, 0.380 mg L -1 for fluconazole, 0.02 mg L -1 for voriconazole, 0.047 mg L -1 for posaconazole and 32 mg L -1 for anidulafungin. T. asahii ICVY021 do not show hemolytic activity, as no blood hemolysis was observed around the grown colonies after 24-48 h of growth at 25 °C. Likewise, no cytotoxic effect of T. asahii ICVY021 was observed with Caco-2 cells after 24 h of contact with T. asahii (Fig. 5A). Taken together, these results began to establish the safety of T. asahii ICVY021.

The adhesion ability was also investigated using the human cell line Caco-2. As shown in Fig. 5B, the results showed than 10% of T. asahii ICVY021 cells were adhesive. This score is weak compared to those for dairy yeasts (Živković et al., 2015), but remains higher than that observed for Candida species [START_REF] Al-Seraih | Characterization of Candida famata isolated from poultry feces for possible probiotic applications[END_REF]. Moreover, the treatment with trypsin or periodic acid led to a decrease of the percentage of adhesive T. asahii ICVY021 to <1%.

The inhibitory proteinaceous sample contains a peptide with a molecular weight of 4,607

Da.

To identify the compound(s) responsible for the antimicrobial activity, the inhibitory proteinaceous sample was analysed using LC/MS/MS with a reversed-phase gradient designed to separate a wide range of organic compounds. Among all the chromatographic peaks detected, one of them was found to show the features of a peptide sequence. Indeed, the corresponding mass spectrum obtained in the positive mode with ESI and high resolution TOF mass analyser showed multiple charged molecular ions ranging from +3 up to +6 protonations, as seen in Fig. 6 (top). These MS signals were deconvoluted to obtain the molecular weight of 4,607 Da as shown in Fig. 6 (bottom). The sequencing of such a long peptide was directly attempted using LC/MS/MS with CID without any proteolytic step.

During this experiments, the selected cations from the quadrupole (Q) collide with argon gas in the collision cell to induce fragmentation. Products ions derived from CID were then mass analysed by TOF analyser. Although partial sequence coverage was expected from fragmentation of a very large peptide with these low energy CID conditions [START_REF] Mouls | Low energy peptide fragmentations in an ESI-Q-Tof type mass spectrometer[END_REF], the aim of such identification study was to propose a sequence tag that can be used for further search of known peptides and proteins in databases. For that purposes, all protonated molecular ions were dissociated but only the penta-charged ion (M+5H) 5+ detected at m/z 922.6 provided many fragments that allowed the identification of the PPFP(K/Q) sequence tag. The first series of fragment ions without decarbonylation (Fig. In conclusion, two putative sequence tags were evidenced with PPFPK and CTHV(L/I)(K/Q) or TCHV(L/I)(K/Q). These sequences were compared to those found in the NCBI protein database, and showed a partial homology with proteins, with undefined function, from the plant Lupinus angustifolius and the bacterium Candidatus curtissbacteria. More information could be deduced from peptide proteolysis prior to MS/MS sequencing to dissociate shorter peptides more amenable to CID fragmentations.

TEM

The observation of the control sample by TEM showed the typical shape of the K. rhizophila cells, consisting of spherical cells of 1.0-1.5 µm diameter, occurring in pairs, tetrads and packets. When, these cells were treated with the inhibitory compound, oranicin P16 at the MBC = 860 µg mL -1 , from T. asahii ICVY021, they showed altered walls with apparent stoppage of the cell division process. The cells seem stuck at the same stage with widespread mortality with difficulties to distinguish the plasma membrane compared to the untreated cells (Fig. 8A,B).

Discussion

The presence of Trichosporon spp strains in food and food derivative products was recently reported for French foods [START_REF] Ceugniez | Fungal diversity of "Tomme d'Orchies" cheese during the ripening process as revealed by a metagenomic study[END_REF]Garnier et al., 2017), and also in Algerian camel milk [START_REF] Ider | Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria[END_REF]. T. asahii strains ICVY021, ICVY022, ICVY023, ICVY024, ICVY025 and ICVY026 from Algerian camel milk [START_REF] Ider | Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria[END_REF] were studied for their ability to inhibit other microorganisms and characterized for different aspects. No differences were observed in their morphologies aspects following their growth on the YEG agar medium. All the colonies from these different strains were white, dried and wrinkled with irregular margins (Fig. 1). Morphological changes have been reported for clinical strains [START_REF] Ichikawa | Switching of colony morphology and adhesion activity of Trichosporon asahii clinical isolates[END_REF], but not for food ones. These changes in the morphological aspects can be associated with different factors such as production of β-N-acetylhexosaminidase [START_REF] Ichikawa | Phenotypic switching and beta-N-acetylhexosaminidase activity of the pathogenic yeast Trichosporon asahii[END_REF], or exposure to oxidants [START_REF] Zong | Growth of Trichosporon asahii in presence of the oxidants, hydrogen peroxide, diamide and menadione[END_REF]. Additional studies are necessary to say whether these morphological changes constitute a general metabolic scheme, or are uniquely related to pathogenic phenotypes. The data and statistical analysis enabled us to categorize T. asahii strains ICVY021, ICVY022, ICVY023, ICVY024, ICVY025, and ICVY026 from camel milk origin into two main branches. The first contained only strain ICVY021, and the second contained the other ones (Fig. 2). T. asahii ICVY021 and ICVY022 isolated in the Ghardaïa region, have similar DNA patterns, unlike the other strains isolated in different locations.

T. asahii is an opportunistic basidiomycetous yeast-like anamorphic organism thriving in different ecological niches because of their metabolic adaptation and genome plasticity. The strains from food sources need further assessments to determine their safety and ability to produce antimicrobial substances. These are two important traits that can help promote these strains for industrial applications.

T. asahii ICVY021 produces an antimicrobial peptide, designated oranicin P16, in reference to Oran, an Algerian city, where the strain was isolated and characterized for its antibacterial function. Oranicin P16, of 4,607 Da, was produced uniquely after 48 h of growth, and consisted of two sequence tags of PPFPK and CTHV(L/I)(K/Q) or TCHV(L/I)(K/Q).

Moreover, TEM analysis of K. rhizophila CIP 53.45 strain treated with oranicin P16 indicated a possible action on its cell-division process. The size, stability to high temperatures or alteration by proteases of oranicin P16 are typical of bacteriocins from Gram-positive and Gram-negative bacteria [START_REF] Drider | Prokarytotic Antimicrobial Peptides: From Genes to Applications[END_REF]. T. asahii ICVY021 was not hemolytic, and was sensitive to clinical antifungal drugs. Mechanisms by which Trichosporon species resist antifungal drugs remain to be better understood, although similarities with Candida and Aspergillus species were reported [START_REF] Kushima | Antifungal susceptibility and drug-resistant mechanism of Trichosporon[END_REF]. Moreover, the genes ERG11 encoding the lanosterol 14-α-demethylase enzyme, which is the primary target of azoles [START_REF] Kushima | Antifungal susceptibility and drug-resistant mechanism of Trichosporon[END_REF], can be lost with a single amino acid substitution [START_REF] Kushima | A new amino acid substitution at G150S in lanosterol 14-α demethylase (Erg11 protein) in multi-azoleresistant Trichosporon asahii[END_REF]. T. asahii ICVY021 was slightly resistant to 5-fluorocytosine and anidulafungin but this may not be a critical point because the in vitro MIC values are not always systematically consistent with the therapeutical doses [START_REF] Rex | Has antifungal susceptibility testing come of age?[END_REF]. Therefore, more accurate methods such as the minimum effective concentration (MEC) are needed [START_REF] Kurtz | Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase[END_REF].

These data show the involvement of proteins and polysaccharides at the cell-surface of T.

asahii ICVY021 in the adhesion to eukaryotic cells.

Conclusion

The capability of a non-conventional yeast from camel milk, namely T. asahii strain ICVY021 to produce an extracellular antimicrobial peptide of 4,607 Da, with two putative sequence tags of PPFPK and CTHV(L/I)(K/Q) or TCHV(L/I)(K/Q) were studied. This peptide is active only against K. rhizophila CIP 53.45 and likely acts as an amitotic peptide.

From a biotechnology point of view, the purified peptide, named oranicin P16, may be a valid alternative to antibiotics against K. rhizophila human infections or outbreaks of the diseases in rainbow or brown trout. Therefore, the determination of the whole sequence of this antibacterial peptide as well as its expression in heterologous hosts need to be done.

  negative and Gram-positive bacteria, as target organisms. These included Candida albicans ATCC 10231, Fusarium oxysporum (Charles Viollette Institute collection, Lille University, Villeneuve d'Ascq, France), Escherichia coli ATCC 8739, Salmonella enterica subs. Enterica serotype Newport (Bioecoagro Lab collection, Lille University), Staphylococcus aureus ATCC 25923, Listeria monocytogenes 162 (Al Seraih et al., 2017). K. salsicia and 8 other K.

  7) were attributed to y ions starting at m/z 358.21 (noted yn) up to m/z 924.5 (yn+5). The last residue of this series with a mass of 128 can be attributed to either K or Q. Nevertheless, the detection of the m/z 84 ion related to the dehydrated immonium ion of K might favour the PPFPK sequence than PPFPQ.Two other ion series were related to the b ions due the loss of CO and formation of the corresponding a ions. The two sequences V(L/I)(K/Q) or (L/I)V(K/Q) can be envisaged since two possibilities may be obtained for the bn+1 ion at either m/z 441 or 455. The residue detected at a mass difference of 128 Da can be attributed as previously to isobaric K or Q. In the same way, a mass difference of 113 Da corresponds to isomeric L or I. Moreover, the immonium ion of H was detected at m/z 110 so this peptide contained this residue. Taking into account that three other ions at m/z 159, 170 and 187 were assigned as a/b ions and have experienced neutral losses (H2O, NH3 noted ° and *, respectively) are often observed in CID.They can be related to the bn ion (m/z 342) by the mass difference of H (expected bn-1 at m/z 205 and an-1 at m/z 177 but were not detected, but the bn-1° at m/z 187, bn-1°* at m/z 170 and an-1° at m/z 159 were detected). These result implied that the position of H can be proposed after V or after (L/I) giving the partial sequence HV(L/I)(K/Q) or H(L/I)V(K/Q). Finally, the residual mass corresponding to be the smallest b ion (m/z 205) can be the dipeptide CT or TC.Four high probable sequences can be suggested CTHV(L/I)(K/Q), CTH(L/I)V(K/Q), or TCHV(L/I)(K/Q), and TCH(L/I)V(K/Q). The detection of an internal ion at m/z 237 related to the VH or HV chain could favour the two possibilities CTHV(L/I)(K/Q) or TCHV(L/I)(K/Q).

Acknowledgements

SI received a short term Ph.D. grant from the Algerian Government. Experiments done at Lille University were supported by la région des Hauts-de-France through the Contrat-Plan-Etat-Région (CPER) Alibiotech [2016-2020]. The authors would like to thank Dr. Xavier Lepoutre from Hôpital Victor Provo de Roubaix for his kind assistance in assessing the antifungal drugs. Authors would like to thank Anne-Sophie Lacoste and Loïc Brunet of the UMS2014/US41 PLBS-BICeL Campus Cité Scientifique facility for access to instruments

Conflicts of interests

The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.