S Schönherr-Hellec

J Aires

Clostridia and necrotizing enterocolitis in preterm neonates

Keywords: necrotizing enterocolitis, preterm neonates, anaerobes, Clostridia, NEC

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

Introduction

Prematurity incidence is increasing worldwide. Survival of very preterm neonates (PN) has improved over the last decades and with it the incidence of major morbidities remains a serious concern. Necrotizing enterocolitis (NEC) is the most severe and life threatening gastrointestinal disease among PN that continues to account for substantial morbidity and mortality in neonatal intensive care units [START_REF] Battersby | Incidence of neonatal necrotising enterocolitis in high-income countries: a systematic review, Archives of Disease in Childhood -Fetal and Neonatal Edition[END_REF][START_REF] Gordon | Mapping the new world of necrotizing enterocolitis (NEC): review and opinion[END_REF]. In the US, NEC has been and is still in 2016 among the 10 leading causes of neonatal and postnatal death [START_REF] Heron | Deaths: leading causes for 2016[END_REF]. Although NEC was first recognized 50 years ago, its incidence remains unchanged in high-income countries ranging from 2% to 7% among babies born <32 weeks' gestation and from 5% to 22%, among those with a birth weight <1000 g (Figure 1) [START_REF] Battersby | Incidence of neonatal necrotising enterocolitis in high-income countries: a systematic review, Archives of Disease in Childhood -Fetal and Neonatal Edition[END_REF].

NEC usually occurs during the first weeks of life. PN with low to very low gestational age, or low to very low birth weight, have a higher risk for NEC [START_REF] Gordon | Mapping the new world of necrotizing enterocolitis (NEC): review and opinion[END_REF]. NEC clinical signs and biological markers are non-specific making the differential diagnosis of the disease with other enteropathies delicate and fulminant cases can occur [START_REF] Garg | Biomarkers of necrotizing enterocolitis: a review of literature[END_REF]. NEC pathophysiology still remains unclear, impairing the development of novel and effective strategies for disease prevention and treatment. NEC survivors are plagued with a number of short and long term complications, particularly those who had to undergo surgery, the most common complication being short bowel syndrome [START_REF] Neu | Necrotizing Enterocolitis[END_REF]. Consequently, surgically treated neonates may suffer from intestinal malabsorption leading to abnormal growth, neurodevelopmental impairment, or both [START_REF] Neu | Necrotizing Enterocolitis[END_REF].

Medically and surgically managed NEC is associated with significantly higher hospital costs. Estimated hospital charges were $96 902 for infants with medical NEC and $153 855 for those with surgical NEC [START_REF] Johnson | The cost of morbidities in very low birth weight infants[END_REF].

NEC is recognized as a multifactorial disease. Three factors are recognized to coalesce, i.e. immaturity of the PN, enteral feeding and bacterial colonization [START_REF] Neu | Necrotizing Enterocolitis[END_REF]. Although NEC pathophysiology remains incompletely understood, it is proposed that NEC originates from a defective interaction between intestinal microbiota and the host's intestinal inflammatory response [START_REF] Cho | The immunological landscape in necrotising enterocolitis[END_REF]. Since NEC initial descriptions, clinicians have believed that bacteria play a major role in the disease process. This belief has been strengthened by data on outbreaks, animal models, and more recently by next-generation sequence analysis of the gut microbiota.

Among the proposed etiological agents of NEC are some anaerobic bacteria and particularly clostridia. The present review gives an update on the role of clostridia in NEC.

Anaerobic bacteria & NEC

When considering strict anaerobic bacteria that have been associated with NEC, most belonged to the Clostridium genus, and particularly to the cluster I sensu stricto [START_REF] Zhou | Longitudinal analysis of the premature infant intestinal microbiome prior to necrotizing enterocolitis: a case-control study[END_REF][START_REF] Rozé | Nutritional strategies and gut microbiota composition as risk factors for necrotizing enterocolitis in very-preterm infants[END_REF]. Only few studies suggested that other anaerobic bacteria, e.g. Bacteroides, Prevotella, Sporacetigenium, may be involved in NEC development [START_REF] Brower-Sinning | Mucosa-associated bacterial diversity in necrotizing enterocolitis[END_REF]. To our knowledge only one study reported a significant increase in the presence and the abundance of the anaerobic bacteria Bacteroides dorei in the meconium of neonates who developed NEC compared to controls [START_REF] Heida | A necrotizing enterocolitis-associated gut microbiota is already present in the meconium: results of a prospective study[END_REF]. Therefore, for this review we will further detail the association between clostridia and NEC. Table 1 summarizes anaerobic bacteria identified in biological samples of PN with NEC.

Clostridia and NEC

Clostridia are among the most common anaerobes identified from the commensal fecal microbiota of PN [START_REF] Ferraris | Clostridia in premature neonates' gut: incidence, antibiotic susceptibility, and perinatal determinants influencing colonization[END_REF]. By contrast, Bacteroides and Bifidobacterium -this latter genus being known for its potential health benefits -seldom colonize PN [START_REF] Butel | Conditions of bifidobacterial colonization in preterm infants: a prospective analysis[END_REF]. Clostridia have also often been involved throughout NEC epidemiological studies. The existence of differences between studies involving clostridia in NEC and using bacterial culture may be due to real differences in etiologies, but also to methodology. First, correct anaerobic bacteria screening and identification was not systematically achieved [START_REF] Guner | Association of Escherichia coli O157:H7 with necrotizing enterocolitis in a full-term infant[END_REF][START_REF] Faustini | An epidemic of gastroenteritis and mild necrotizing enterocolitis in two neonatal units of a University Hospital in Rome, Italy[END_REF] or elimination of the non-spore-forming organisms was performed [START_REF] Sim | Dysbiosis anticipating necrotizing enterocolitis in very premature infants[END_REF].

Second, application of molecular techniques to the gut microbiota analysis showed that a large part of the microbiota cannot be cultivated under standard laboratory conditions. Studies using cultureindependent techniques may also underestimate clostridia. Indeed, methodologies are based on PCR techniques using universal primers or primers designed to amplify the variable 16S rRNA gene regions. Yet, design of primers is challenging as clostridia belong to a complex phylogenetic heterogeneous group with some cluster having high strain genetic diversity [START_REF] Kalia | Analysis of the unexplored features of rrs (16S rDNA) of the genus Clostridium[END_REF]. Therefore, clostridia may have not been systematically detected, all the more as they can be in a subdominant status. Indeed, metagenomics sequencing techniques targeting the bacterial 16S rDNA gene mostly enable the detection of dominant species and not those present at low titer.

Clinical signs of NEC are consistent with a clostridial etiology of the pathology. Pneumatosis intestinalis and portal venous gas are pathognomonic radiographic signs of NEC [START_REF] Buonomo | The radiology of necrotizing enterocolitis[END_REF] and thought to be caused by anaerobic bacteria, specifically clostridia. Among neonates with NEC, infants colonized with clostridia showed a more rapid progression of the disease. Clostridia have been isolated from the blood, feces, and peritoneal fluids of neonates suffering from NEC (Table 1).

Based on the phenotypic and immune inflammatory properties of clostridia strains isolated from NEC and controls PN, the existence of a specific clostridial signature associated with NEC was suggested [START_REF] Schönherr-Hellec | Clostridial strain-specific characteristics associated with necrotizing enterocolitis[END_REF]. A correlation between the presence of clostridia in tissue specimens from neonates with NEC was reported [START_REF] Smith | Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis[END_REF]. The involvement of clostridia in NEC pathogenesis was strengthened using animal models. In gnotobiotic quail different clostridia species were shown to be responsible for the development of digestive NEC-like lesions [START_REF] Waligora-Dupriet | Short-chain fatty acids and polyamines in the pathogenesis of necrotizing enterocolitis: Kinetics aspects in gnotobiotic quails[END_REF][START_REF] Butel | Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria[END_REF][START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF][START_REF] Bousseboua | Experimental cecitis in gnotobiotic quails monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis and from healthy newborns[END_REF]. A significant role of clostridia has also been reported in the preterm piglet model: clostridia species were largely absent in piglets without NEC and significantly overrepresented among colonic mucosa samples from piglets with NEC [START_REF] Azcarate-Peril | Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria[END_REF].

Clostridium butyricum

C. butyricum was first isolated from pig intestines by Prazmowski in 1880. It belongs to the cluster I sensus stricto and is the type species of the Clostridium genus [START_REF] Vos | Bergey's Manual of Systematic Bacteriology[END_REF]. This species can be isolated from a variety of sources including feces of humans and animals [START_REF] Ferraris | Clostridia in premature neonates' gut: incidence, antibiotic susceptibility, and perinatal determinants influencing colonization[END_REF][START_REF] Vos | Bergey's Manual of Systematic Bacteriology[END_REF][START_REF] Ghoddusi | Preliminary study on the isolation of Clostridium butyricum strains from natural sources in the UK and screening the isolates for presence of the type E botulinal toxin gene[END_REF].

Although C. butyricum is a commensal bacteria of healthy children and adults, some strains have been implicated in infant botulism or NEC [START_REF] Abe | Infantile botulism caused by Clostridium butyricum type E toxin[END_REF][START_REF] Shelley | Infant botulism due to C. butyricum type E toxin: a novel environmental association with pet terrapins[END_REF][START_REF] Fenicia | Intestinal toxemia botulism in Italy, 1984-2005[END_REF]. C. butyricum was first associated with NEC during an outbreak in 1977, after that C. butyricum was identified from PN blood cultures [START_REF] Howard | Outbreak of necrotising enterocolitis caused by Clostridium butyricum[END_REF]. In 1980, a toxigenic strain of C. butyricum has been isolated from the peritoneal fluid and cerebrospinal fluid of a neonate with NEC [START_REF] Sturm | Neonatal necrotizing enterocolitis associated with penicillin-resistant, toxigenic Clostridium butyricum[END_REF]. In 2015, the analysis of 363 fecal samples from PN with and without NEC using culture, pyrosequencing and/or quantitative PCR, allowed to specifically associate C. butyricum with NEC [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF]. This association was confirmed by a multicentric case-control study that analyzed fecal samples from 16 NEC infants matched with 78 controls [START_REF] Rozé | Nutritional strategies and gut microbiota composition as risk factors for necrotizing enterocolitis in very-preterm infants[END_REF]. Fluorescent in situ hybridization (FISH) analysis of intestinal biopsies surgically removed from neonates diagnosed with NEC showed a correlation between the presence of C. butyricum and histological pneumatosis intestinalis [START_REF] Smith | Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis[END_REF].

C. butyricum strains isolated from NEC cases can cause intestinal lesions in animals with necrotic areas, gas cysts, villus atrophy and edema with infiltrate of mononuclear cells [START_REF] Waligora-Dupriet | Short-chain fatty acids and polyamines in the pathogenesis of necrotizing enterocolitis: Kinetics aspects in gnotobiotic quails[END_REF][START_REF] Butel | Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria[END_REF][START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF][START_REF] Bousseboua | Experimental cecitis in gnotobiotic quails monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis and from healthy newborns[END_REF][START_REF] Azcarate-Peril | Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria[END_REF][START_REF] Popoff | Experimental cecitis in gnotoxenic chickens monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis[END_REF][START_REF] Popoff | Lesions produced by Clostridium butyricum strain CB 1002 in ligated intestinal loops in guinea pigs[END_REF]. Lactose fermentation, and particularly the production of butyric acid, was proposed as a prerequisite [START_REF] Waligora-Dupriet | Short-chain fatty acids and polyamines in the pathogenesis of necrotizing enterocolitis: Kinetics aspects in gnotobiotic quails[END_REF][START_REF] Butel | Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria[END_REF][START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF][START_REF] Bousseboua | Experimental cecitis in gnotobiotic quails monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis and from healthy newborns[END_REF], and colonization with bifidobacteria was protective [START_REF] Butel | Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria[END_REF]. In cellular models, C. butyricum NEC strains supernatants may show or not cytotoxic effects [START_REF] Schönherr-Hellec | Clostridial strain-specific characteristics associated with necrotizing enterocolitis[END_REF][START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF][START_REF] Popoff | Cytotoxic activity of Clostridium butyricum supernatants induced by butyrate[END_REF].

In the preterm piglet model, using quantitative PCR and terminal restriction fragment length polymorphism (T-RFLP) analysis of ileal mucosa, C. butyricum has been associated with NEC in preterm, cesarian-derived, and formula-fed piglets [START_REF] Azcarate-Peril | Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria[END_REF].

When comparing the phenotypic and immune inflammatory properties of NEC and control C. butyricum strains, a higher stress tolerance and higher IL-8 Caco-2 cells stimulation was observed for NEC strains [START_REF] Schönherr-Hellec | Clostridial strain-specific characteristics associated with necrotizing enterocolitis[END_REF]. It suggested that NEC strains may have a specific ecological role in the environmental gastrointestinal tract of PN with NEC.

Clostridium neonatale

In 2002, an outbreak of NEC occurred in six neonates within a two-month period in a Canadian neonatal intensive care unit [START_REF] Alfa | An outbreak of necrotizing enterocolitis associated with a novel Clostridium species in a neonatal intensive care unit[END_REF]. Blood cultures from three premature neonates grew the same strain identified as C. neonatale based on the16S rRNA gene sequencing. neonatale by demonstrating that it was a new species belonging to cluster I of the Clostridium genus sensu stricto [START_REF] Bouvet | Aires, 16S rRNA gene sequencing, multilocus sequence analysis, and mass spectrometry identification of the proposed new species "Clostridium neonatale[END_REF]. C. neonatale was isolated and identified in samples from healthy PN suggesting that its belongs to neonates commensal microbiota [START_REF] Ferraris | A onestep multiplex PCR for differentiating the proposed new species "Clostridium neonatale" from closely related species[END_REF]. In 2017, a link between C. neonatale and NEC development was shown in a multicentric study [START_REF] Rozé | Nutritional strategies and gut microbiota composition as risk factors for necrotizing enterocolitis in very-preterm infants[END_REF]. In 2018, the formal description of Clostridium neonatale sp. nov. has been published [START_REF] Bernard | Clostridium neonatale sp. nov. linked to necrotizing enterocolitis in neonates and a clarification of species assignable to the genus Clostridium (Prazmowski 1880) emend. Lawson and Rainey 2016[END_REF].

In the preterm piglet, as for C butyricum, C. neonatale was significantly overrepresented among samples of colonic mucosa from piglets with NEC [START_REF] Azcarate-Peril | Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria[END_REF].

Clostridium perfringens

C. perfringens can be found in diverse environments and is a member of the gut microbiota of humans and animals [START_REF] Vos | Bergey's Manual of Systematic Bacteriology[END_REF]. However, C. perfringens has been constantly associated with various significant systemic and enteric diseases, including gas gangrene, food poisoning and non-foodborne diarrhea [START_REF] Vos | Bergey's Manual of Systematic Bacteriology[END_REF]. Indeed, C. perfringens is known to produce a variety of virulence factors that are involved in its pathogenicity. C. perfringens has been identified in the peritoneal liquid, the blood and fecal samples from PN with NEC [START_REF] Heida | A necrotizing enterocolitis-associated gut microbiota is already present in the meconium: results of a prospective study[END_REF][START_REF] Sim | Dysbiosis anticipating necrotizing enterocolitis in very premature infants[END_REF][START_REF] Kosloske | Clostridial necrotizing enterocolitis[END_REF][START_REF] Blakey | Enteric colonization in sporadic neonatal necrotizing enterocolitis[END_REF][START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and role of alpha toxin[END_REF][START_REF] Kotsanas | Novel use of tryptose sulfite cycloserine egg yolk agar for isolation of Clostridium perfringens during an outbreak of necrotizing enterocolitis in a neonatal unit[END_REF][START_REF] Schlapbach | Clostridium perfringens and necrotizing enterocolitis[END_REF][START_REF] Warren | Necrotizing enterocolitis and hemolysis associated with Clostridium perfringens[END_REF][START_REF] De La Cochetière | Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants: the putative role of Clostridium[END_REF].

C. perfringens colonization has been associated with a higher mortality rate among NEC cases (78% ) when compared to NEC cases colonized with other clostridial species (14% ) [START_REF] Kosloske | Clostridial necrotizing enterocolitis[END_REF]. C. perfringens identification prior to NEC diagnosis established a significant temporal relationship between early colonization by this species and the later development of the disease [START_REF] De La Cochetière | Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants: the putative role of Clostridium[END_REF]. Compared to the control group, C. perfringens onset of disease was earlier in life, the clinical course more severe, patients had a larger extent of gangrene, and the mortality rate was more than twice as high [START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and role of alpha toxin[END_REF]. C. perfringens has the potential to trigger a fulminant and often lethal course [START_REF] Schlapbach | Clostridium perfringens and necrotizing enterocolitis[END_REF]. In 2016, metagenomic analysis linked the presence of C. perfringens in the meconium with the development of the disease [START_REF] Heida | A necrotizing enterocolitis-associated gut microbiota is already present in the meconium: results of a prospective study[END_REF]. Although some studies involved the presence of C. perfringens type A toxinogenic strains in NEC [START_REF] Sim | Dysbiosis anticipating necrotizing enterocolitis in very premature infants[END_REF][START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and role of alpha toxin[END_REF][START_REF] Kotsanas | Novel use of tryptose sulfite cycloserine egg yolk agar for isolation of Clostridium perfringens during an outbreak of necrotizing enterocolitis in a neonatal unit[END_REF][START_REF] Warren | Necrotizing enterocolitis and hemolysis associated with Clostridium perfringens[END_REF], the toxin concentration did not correlate systematically with the severity of the disease [START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and role of alpha toxin[END_REF].

As for C. butyricum, C. perfringens has also been associated with NEC in several animal models [START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF][START_REF] Siggers | Elective cesarean delivery affects gut maturation and delays microbial colonization but does not increase necrotizing enterocolitis in preterm pigs[END_REF][START_REF] Siggers | Early administration of probiotics alters bacterial colonization and limits dietinduced gut dysfunction and severity of necrotizing enterocolitis in preterm pigs[END_REF][START_REF] Cilieborg | Preterm birth and necrotizing enterocolitis alter gut colonization in pigs[END_REF][START_REF] Cilieborg | The incidence of necrotizing enterocolitis is increased following probiotic administration to preterm pigs[END_REF][START_REF] Støy | Necrotizing enterocolitis in preterm pigs is associated with increased density of intestinal mucosa-associated bacteria including Clostridium perfringens[END_REF]. In quail animal model, pathological findings were similar to those of C. butyricum [START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF]. In the preterm piglet model of NEC, T-RFLP analysis showed a colonization frequency by C. perfringens up to 32 times higher in sick animals [START_REF] Siggers | Elective cesarean delivery affects gut maturation and delays microbial colonization but does not increase necrotizing enterocolitis in preterm pigs[END_REF][START_REF] Siggers | Early administration of probiotics alters bacterial colonization and limits dietinduced gut dysfunction and severity of necrotizing enterocolitis in preterm pigs[END_REF]. A higher abundance of C. perfringens in piglets that developed the disease was confirmed by FISH and quantitative PCR analysis [START_REF] Cilieborg | Preterm birth and necrotizing enterocolitis alter gut colonization in pigs[END_REF][START_REF] Cilieborg | The incidence of necrotizing enterocolitis is increased following probiotic administration to preterm pigs[END_REF][START_REF] Støy | Necrotizing enterocolitis in preterm pigs is associated with increased density of intestinal mucosa-associated bacteria including Clostridium perfringens[END_REF]. Furthermore NEC severity was correlated with bacterial density [START_REF] Støy | Necrotizing enterocolitis in preterm pigs is associated with increased density of intestinal mucosa-associated bacteria including Clostridium perfringens[END_REF].

Clostridium paraputrificum

One study has associated C. paraputrificum with NEC development [START_REF] Smith | Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis[END_REF]. In this study, FISH analysis of the inflamed intestinal tissue surgically removed from neonates diagnosed with NEC showed a correlation between the presence of C. paraputrificum and intestinal pneumatosis [START_REF] Smith | Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis[END_REF]. In quails animal model of NEC mono-associated with C. paraputrificum, hemorrhages predominated, and less thickening, pneumatosis and necrotic areas were observed as compared to C. perfringens and C. butyricum [START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF]. Furthermore, lesions were associated with the presence of high concentrations of iso-butyric acid instead of butyric acid.

Clostridium difficile

Clostridium difficile was first isolated as a commensal bacterium in healthy neonates [START_REF] Hall | Intestinal flora in new-born infants: with a description of a new pathogenic anaerobe, Bacillus difficilis[END_REF].

Nowadays, in developed countries, C. difficile is recognized as the major cause of antibioticassociated diarrhea and colitis in adults and is associated with a substantial morbidity and mortality [START_REF] Smits | Clostridium difficile infection[END_REF]. Its major virulence factors include toxins A (TcdA), B (TcdB) and potentially a binary toxin (Cdt) [START_REF] Smits | Clostridium difficile infection[END_REF]. Worldwide hyper-virulent strains have become a significant cause of nosocomial infections and outbreaks of C. difficile severe infections [START_REF] He | Emergence and global spread of epidemic healthcare-associated Clostridium difficile[END_REF]. Children less than 1 year old have been shown to be asymptomatic carriers of toxigenic and/or non-toxigenic C. difficile strains [START_REF] Lees | The role of Clostridium difficile in the paediatric and neonatal gut -a narrative review[END_REF]. The absence of C. difficile infection in children colonized by toxigenic strains is still a matter of debate. It could be due to the absence of toxin receptors, or to their inability to bind to their receptors [START_REF] Lees | The role of Clostridium difficile in the paediatric and neonatal gut -a narrative review[END_REF]. Gut microbiota has also been proposed to display a protective barrier effect against C. difficile overgrowth in neonates and infants [START_REF] Lees | The role of Clostridium difficile in the paediatric and neonatal gut -a narrative review[END_REF]. In the 1980's, two NEC outbreaks involved C. difficile toxigenic strains in fecal samples of PN with NEC [START_REF] Cashore | Clostridia colonization and clostridial toxin in neonatal necrotizing enterocolitis[END_REF][START_REF] Han | An outbreak of Clostridium difficile necrotizing enterocolitis: a case for oral vancomycin therapy?[END_REF]. But C. difficile involvement in NEC has been questioned [START_REF] Lishman | Clostridium difficile isolation in neonates in a special care unit. Lack of correlation with necrotizing enterocolitis[END_REF]. Of note, in quail model of NEC, a toxigenic C. difficile strain did not cause caecal NEC-like lesions [START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF].

Clostridia toxigenic/metabolic bacterial factors implicated in NEC

C. perfringens [START_REF] Sim | Dysbiosis anticipating necrotizing enterocolitis in very premature infants[END_REF][START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and role of alpha toxin[END_REF][START_REF] Kotsanas | Novel use of tryptose sulfite cycloserine egg yolk agar for isolation of Clostridium perfringens during an outbreak of necrotizing enterocolitis in a neonatal unit[END_REF][START_REF] Warren | Necrotizing enterocolitis and hemolysis associated with Clostridium perfringens[END_REF], C. difficile [START_REF] Cashore | Clostridia colonization and clostridial toxin in neonatal necrotizing enterocolitis[END_REF][START_REF] Han | An outbreak of Clostridium difficile necrotizing enterocolitis: a case for oral vancomycin therapy?[END_REF] and C. butyricum [START_REF] Sturm | Neonatal necrotizing enterocolitis associated with penicillin-resistant, toxigenic Clostridium butyricum[END_REF][START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF] toxigenic strains have been associated with NEC. However, either the presence of the toxin or its concentration did not correlate systematically with the severity of the disease [START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and role of alpha toxin[END_REF]. Some reports have suggested a potential role for clostridial toxigenic factors in NEC pathogenesis [START_REF] Sturm | Neonatal necrotizing enterocolitis associated with penicillin-resistant, toxigenic Clostridium butyricum[END_REF][START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF][START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and role of alpha toxin[END_REF].

Live bacteria, bacterial debris, cytoplasmic content and culture supernatants of C. butyricum and C. neonatale strains isolated from fecal samples of PN with and without NEC showed no or low-titer cytotoxic effect in vitro [START_REF] Schönherr-Hellec | Clostridial strain-specific characteristics associated with necrotizing enterocolitis[END_REF]. Additionally, cytotoxicity activity of PN fecal filtrates showed the absence of specific association between the observed cytotoxic effects between NEC and control samples. Therefore, evidence is still limited concerning the role of clostridial toxigenic factors in NEC development, and further investigations are needed.

Bacterial implication in the development of NEC has been proposed to be due to colonic fermentation of non-hydrolyzed lactose, a consequence of the immaturity of the intestinal lactasic equipment in PN [START_REF] Kien | Digestion, absorption, and fermentation of carbohydrates in the newborn[END_REF]. Overproduction of metabolites due to this undigested lactose may be triggering the inflammatory cascade, as shown in the preterm pig model of NEC [START_REF] Thymann | Carbohydrate maldigestion induces necrotizing enterocolitis in preterm pigs[END_REF].

Hence, either excessive or abnormal or both luminal production of short chain fatty acids (SCFAs) through the colonic bacterial fermentation can exceed the capacity of the immature intestinal mucosa of the PN [START_REF] Lin | Too much short chain fatty acids cause neonatal necrotizing enterocolitis[END_REF]. Fecal SCFAs concentrations in very PN without digestive disease (less than 33 weeks of GA) were reported to be lower than in late PN [START_REF] Favre | Diet, length of gestation, and fecal short chain fatty acids in healthy premature neonates[END_REF]. High SCFAs concentrations and butyrate present in C. butyricum culture supernatants displayed cytotoxic effects in vitro in fibroblast and VERO cells [START_REF] Popoff | Cytotoxic activity of Clostridium butyricum supernatants induced by butyrate[END_REF]. In animal models of NEC, SCFAs lead to the development of NEC-like lesions [START_REF] Waligora-Dupriet | Short-chain fatty acids and polyamines in the pathogenesis of necrotizing enterocolitis: Kinetics aspects in gnotobiotic quails[END_REF][START_REF] Waligora-Dupriet | Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model[END_REF][START_REF] Lin | Variable effects of short chain fatty acids and lactic acid in inducing intestinal mucosal injury in newborn rats[END_REF][START_REF] Nafday | Short-chain fatty acids induce colonic mucosal injury in rats with various postnatal ages[END_REF].

Clostridia and host factors implicated in NEC

Data suggest that the immature preterm or fetal gut is not prepared for the excessive stimulation of initial postnatal colonization and hence unable to down-regulate the inflammatory responses [START_REF] Neu | Necrotizing Enterocolitis[END_REF]. The inflammatory cascade may be triggered by a primary event or chain of events.

Particularly, NEC originates from a defective interaction between intestinal microorganisms and the host's immune response. Studies have focused on the role of the cellular Toll-like receptor (TLR) 4 that recognizes the lipopolysaccharides of Gram-negative bacteria [START_REF] Kawasaki | Toll-like receptor signaling pathways[END_REF] as well as some surface layer proteins of C. diffcile [START_REF] Ryan | A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins[END_REF]. TLR4 knockout mice were shown to be protected from the development of NEC [START_REF] Leaphart | A critical role for TLR4 in the pathogenesis of necrotizing enterocolitis by modulating intestinal injury and repair[END_REF]. Additionally, an up-regulation of ileal TLR4 and TLR2 was found prior to histological evidence of mucosal injury in the rat model of NEC, suggesting a causative role in NEC pathogenesis [START_REF] Le Mandat | Expression of TLR-2, TLR-4, NOD2 and pNF-κB in a neonatal rat model of necrotizing enterocolitis[END_REF][START_REF] Zhou | Glutamine downregulates TLR-2 and TLR-4 expression and protects intestinal tract in preterm neonatal rats with necrotizing enterocolitis[END_REF][START_REF] Liu | Changes in intestinal Toll-like receptors and cytokines precede histological injury in a rat model of necrotizing enterocolitis[END_REF]. However, data about anaerobic bacteria and particularly clostridia involvement in the inflammatory process during NEC are scarce. In 2018, C. butyricum strains isolated from fecal samples of PN with NEC were shown to induce higher in vitro IL-8 secretion in human intestinal epithelial cells as compared to control ones [START_REF] Schönherr-Hellec | Clostridial strain-specific characteristics associated with necrotizing enterocolitis[END_REF].

Conclusion

Necrotizing enterocolitis (NEC) is a devastating enigmatic disease which incidence did not decrease for several decades. New strategies of treatment or prevention are limited by the lack of knowledge of the exact pathophysiology of the disease. Among anaerobic bacteria, clostridia

  Although C. neonatale was proposed to form a novel species of the Clostridium genus, since 2002, its formal classification as a new species was not validated. The confusing status of this proposed species explains the absence of data about its isolation, identification, or clinical significance. As a consequence, misidentification or/and underrepresentation of "C.neonatale" populations during previous NEC studies may have occurred. In 2014, a ployphasic study based on a genetic and phenotypic analysis clarified the status of C.

FiguresFig. 1 .

 1 Figures

Conflict of interest

The authors declare that they have no conflict of interest.


Anca-Elena Dascalu

Alina Ghinet

Muriel Billamboz

Dr Emmanuelle Lipka
email: emmanuelle.lipka@univ-lille.fr

Performance comparison of chlorinated chiral stationary phases in supercritical fluid chromatography for separation of selected pyrrolidone derivatives

Keywords: antibacterial compounds, chirality, enantiomers, flow-rate, particle size

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

Introduction

Supercritical fluid chromatography (SFC) is an important technique in drug discovery related analysis due to its many advantages comparing to the commonly used high-performance liquid chromatography (HPLC) technique. The environmental-friendly CO2 based mobile phase, the increased efficiency together with the short analysis time (due to high flow-rate) and the lower operational costs give SFC benefits over many analysis techniques related to drug development [START_REF] Fekete | Evolution and current trends in liquid and supercritical fluid chromatography[END_REF][START_REF] Desfontaine | Supercritical fluid chromatography in pharmaceutical analysis; a review[END_REF], particularly in chiral separation where preparative scale remains its main application run by industrial actors more than academic ones [START_REF] Arnaud | Supercritical fluid chromatography seeks users[END_REF]. Halogenated polysaccharide based CSPs originally developed by Chankvetadze, et al. for HPLC [START_REF] Chankvetadze | Chloromethylphenylcarbamate derivatives of cellulose as chiral stationary phases for high-performance liquid chromatography[END_REF][START_REF] Chankvetadze | Dimethyl-, dichloro-and chloromethylphenylcarbamates of amylose as chiral stationary phases for high-performance liquid chromatography[END_REF][START_REF] Chankvetadze | 3-Fluoro-, 3-chloro-and 3bromo-5-methylphenylcarbamates of cellulose and amylose as chiral stationary phases for highperformance liquid chromatographic enantioseparation[END_REF] have reached the market in 2005. The coated chlorinated carbamate phases were deeply characterized [START_REF] Khater | Insights into chiral recognition mechanism in supercritical fluid chromatography IV. Chlorinated polysaccharide stationary phases[END_REF] and have found applications [START_REF] West | Effects of mobile phase composition and temperature on supercritical fluid chromatography enantioseparation of chiral fluoro-oxoindoletype compounds with chlorinated polysaccharide stationary phases[END_REF][START_REF] Zehani | Exploring chiral separation of 3-carboxamido-5aryl isoxazole derivatives in supercritical fluid chromatography on amylose and cellulose tris dimethyl-and chloromethyl phenylcarbamate polysaccharide based stationary phases[END_REF][START_REF] Wu | From analytical methods to large scale chiral SFC using chlorinated chiral stationary phases[END_REF][START_REF] West | Enantioseparation of novel chiral sulfoxides on chlorinated polysaccharide stationary phases in supercritical fluid chromatography[END_REF][START_REF] Albals | Chiral separations of cathinone and amphetamine-derivatives: Comparative study between capillary electrochromatography, supercritical fluid chromatography and three liquid chromatographic modes[END_REF] as their immobilized versions [START_REF] Klerck | Pharmaceutical enantiomers resolution using immobilized polysaccharide-based chiral stationary phases in supercritical fluid chromatography[END_REF][START_REF] Dasilva | Evaluation of non-conventional polar modifiers on immobilized chiral stationary phases for improved resolution of enantiomers by supercritical fluid chromatography[END_REF][START_REF] Zhang | Complementary enantiorecognition patterns and specific method optimization aspects on immobilized polysaccharide-derived chiral stationary phases[END_REF][START_REF] Lee | On the method development of immobilized polysaccharide chiral stationary phases in supercritical fluid chromatography using an extended range of modifiers[END_REF]. In 2018, there were only two studies concerning the use of cellulose tris(3-chloro-4-methylphenylcarbamate) chiral column [START_REF] Alvarenga | Enantioselective separation of (±)-β-hydroxy-1,2,3-triazoles by supercritical fluid chromatography and high-performance liquid chromatography[END_REF][START_REF] Zhao | Enantioseparation of napropamide by supercritical fluid chromatography: Effects of the chromatographic conditions and separation mechanism[END_REF] and articles dealing with cellulose tris (3,5-dichlorophenylcarbamate) [START_REF] Lipka | Separation of enantiomers of native amino acids with polysaccharide-based chiral columns in supercritical fluid chromatography[END_REF][START_REF] Hegade | Chiral stationary phase optimized selectivity supercritical fluid chromatography: a strategy for the separation of mixtures of chiral isomers[END_REF][START_REF] Pirrone | Supercritical fluid chromatographyphotodiode array detection-electrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances[END_REF] in SFC. To get compare the separation ability of these two columns, a pyrrolidin-2-one family, new class of antibacterial compounds under evaluation, was chosen (Fig. 1). In the context of green chemistry, a lot of efforts have been made for the valorification of natural resources and industrial waste. The pyroglutamic acid is a raw material, derived from sugar beet molasses. The pyroglutamic acid (1) and its derivatives, present a chiral center and like a great number of pharmaceutical molecules, each enantiomer has its own pharmacological properties thus underlining the importance of their separation. The pyrrolidin-2-one scaffold is well known as a privileged synthon for a broad range of heterocyclic compounds as well as for high potential to afford small compounds with interesting biological activities. Therefore, simple pyrrolidones are often fundamental parts of the structure of antimicrobial [START_REF] Matviiuk | Synthesis of 3-heteryl substituted pyrrolidine-2,5diones via catalytic Michael reaction and evaluation of their inhibitory activity against InhA and Mycobacterium tuberculosis[END_REF], antiviral [START_REF] Ghosh | Substituent effects on P2cyclopentyltetrahydrofuranyl urethanes: Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors[END_REF] and antitumor compounds [START_REF] Cramer | Synthesis and biological properties of cylindramide derivatives: evidence for calcium-dependent cytotoxicity of tetramic acid lactams[END_REF], as well as products that target the central nervous system [START_REF] Kaminski | Design, synthesis and anticonvulsant properties of new N-Mannich bases derived from 3-phenylpyrrolidine-2,5-diones[END_REF], or other biological systems. Considering the fact that the manufacturer presented baseline separation of thalidomide on the Lux Cellulose-2, and chlortalidone on the Lux i-Cellulose-5, chemical compounds which show structural similarities with the pterolactame derivatives used in this study, we expected similar enantioseparation ability for our analytes. Therefore, the aim of this work was to compare the separation performances of each chiral selector, the influence of the flow-rate and percentage of methanol on retention and resolution.

Material and methods

Chemicals

The molecules used are 5-methoxy-2-pyrrolidone 2 derivatives (racemic compound 3: 5-anilinopyrrolidin-2-one, racemic compound 4: 5-(benzylamino)pyrrolidin-2-one, racemic compound 5: The used methanol, ethanol, isopropanol or acetonitrile were HPLC grade and was purchased from VWR (Strasbourg, France). Carbon dioxide (CO2) with purity of 99.995% was purchased from Linde (Saint-Priest, France).

Sample solutions

For analytical screening, solutions of samples were prepared in methanol at 1 mg.mL -1 . The solutions were always degassed by an ultrasonic bath and filtered on a 0.45 m PTFE syringe-filter (15 mm diameter) prior to be used. Two chiral analytical columns were used for this study. Lux Cellulose-2 and Lux i-Cellulose-5, purchased from Phenomenex ® (Le Pecq, France) having dimensions 250 mm × 4.6 mm i.d. with 5 µm or 3 µm particle size (Fig. 2). The silica gel of the Lux Cellulose-2 column is coated with cellulose tris(3-chloro-4-methylphenylcarbamate) chiral selector, and the silica gel of the Lux i-Cellulose-5 column is immobilized with cellulose tris(3,5-dichlorophenylcarbamate) chiral selector.

Chromatographic system and conditions

The chromatographic system used was an SFC-PICLAB hybrid 10-20 apparatus (PIC Solution, Avignon, France) equipped with an autosampler comprised a 48-vials plate (model Alias, Emmen, Netherlands), three model 40 P pumps: two for CO2 and a third for the modifier (Knauer, Berlin, Germany), a column oven with a Valco ten-position column selection valve, and a Valco six-position solvent switching valve. The pump head used for pumping the CO2 was cooled to -8 • C by a cryostat (model Minichiller, Huber, Offenburg, Germany. The system was also composed of a Smartline 2600 diode array detector (DAD) (Knauer, Berlin, Germany). After the detector, the outlet pressure was controlled by a back-pressure regulator (BPR). The outlet regulator tube was heated to 55 °C to avoid ice formation during the CO2 depressurization. The system was controlled and the data were acquired with the SFC PicLab Analytic Online v.3.1.2 software and the data were processed with the Analytic Offline v.3.2.0 software (PIC Solution, Avignon, France). During the separation screening, the mobile phase was always CO2-modifier mixtures with 20% of either methanol, ethanol, isopropanol or acetonitrile and during the separation optimization, the mobile phase was always CO2-modifier mixtures with the proportion of methanol ranging from 7.5 to 15%, the flow rate was ranging between 2 and 4 mL/min. All analyses were run in isocratic mode. The column oven temperature was 40 • C and the outlet pressure was 150 bar. The wavelength was 210 nm and the injected volume was 20 µL.

Chromatographic parameters

The resolution factor from our study, was calculated using: Rs = 2(tR2-tR1)/(ω1+ω2), where tR1 and tR2 are the retention times of the peaks of enantiomers and ω1 and ω2 are the peak widths measured at the baseline between tangents drawn to the peak sides. The retention (or capacity) factor (k) is a mean of measuring the retention of an analyte on the chromatographic column for each enantiomer, k1 and k2 are calculated by: k = (tR-t0)/t0 where t0 was measured using 1, 3, 5-tritert-butylbenzene (TTBB).

Results and discussion

Selection of stationary and mobile phases

Two columns packed with two types of chiral selectors Lux Cellulose-2 and Lux i-Cellulose-5, were examined for their separative performances towards six antibacterial compounds. In SFC and in HPLC as well, polysaccharide stationary phases play a crucial role in chiral separation.

Thus the two columns were firstly tested at 4 mL/min with 10% of either acetonitrile, isopropanol, ethanol and methanol. As previously reported, aprotic solvent as acetonitrile leads to the highest retention times, followed by isopropanol and then ethanol for which almost all compounds were not separated. In terms of resolution and analysis times, best results were observed with methanol and are summarized for the six derivatives in Tables 1 and2.

From these results it can be seen that compounds 7 and 8 are not separated on Lux cellulose-5 CSP. In addition, on this column, three derivatives i.e 3, 4 and 5 were only partially separated (R<1.5). This behavior is also observed on Lux cellulose-2 for compounds 3, 5, 7 and 8. The effect of a decrease of the flow-rate on the resolution was further explored.

Flow-rate selection

Six different flow-rates were tested between 2 and 4 mL/min with step of 0.5 mL/min, on the two columns. All the results are summarized in Tables 1 and2. On Lux cellulose-2 CSP, all the compounds benefited from this decrease of flow-rate leading to higher resolution values. For example, for pyrrolidone derivative 3, the resolution value goes from 1.15 to 1.47 at 4 and 2 mL/min respectively. Following the Purnell's equation resolution mainly depends on the efficiency value and for all the compounds, on the two CSPs, this parameter increases with a decreasing flow-rate (Tables 1 and2). However this improvement was not sufficient for compounds 7 and 8 for which resolution values were equal to 1.19 and 1.00 respectively at this lower flow-rate. It must be noticed that except for pyrrolidone derivative 6, the resolution values obtained on Lux cellulose-2 are higher than on Lux cellulose-5. In addition, one can notice the unusual high values of retention factors for derivative 6 in comparison to other compounds, whatever the CSP was. This behavior can be explained by the presence of a supplementary carbonyl moiety on this structure inducing supplementary dipole-dipole interactions and hydrogen bonding with the CSP.

It is noteworthy to say that lower flow-rates (i.e 1.0 and 1.5 mL/min) were also tested but led to large peaks and long retention times (data not shown). In order to improve further the resolution, particularly for derivatives 7 and 8, the influence of the percentage of methanol with a flow-rate conserved at 2 mL/min was explored on the Lux cellulose-2.

Percentage of methanol in the mobile phase

In a general manner, in SFC the modifier plays a key role and its effects are numerous: i) the organic solvent changes the polarity of the mobile phase, ii) it changes the density of the mobile phase, particularly when pressure and temperature conditions are such that fluid compressibility is high that is to say close to the critical point and when the fluid is more gas-like, iii) the solvent changes the polarity and possibly the three dimensional structure of the CSP through its extensive adsorption on the CSP surface [START_REF] West | Enantioselective separation with supercritical fluids[END_REF]. In SFC, the percentage of co-solvent affects very strongly the retention time and then the resolution. The percentage of methanol was varied between 7.5 and 15% by step of 2.5%. All results are summarized in Table 3. An increase in co-solvent proportion generally leads to a decrease of the retention factors. This behavior is observed in Table 3. In addition one can see that the decrease in retention factor is more and more diminished when the co-solvent percentage is increased: large variation in retention is observed at small percentages of modifier, for instance k1 is going from 34.38 to 15.54 when the percentage ranging from 7.5 to 10%, whereas further increase in modifier proportions causes less modification (k1 varying from 8.42 to 5.49 when the percentage ranging from 12.5 to 15%) for compound 4, this being a classical behavior in SFC. Best Rs/tR2 ratio depends on each compound. For derivatives 3, 4 and 6, the best resolutions in shorter analysis time were achieved with 15% of methanol and for 5 and 8 under 7.5% of co-solvent. All the five optimized chromatograms are represented on the Figure 3. Considering the separation of compound 7 on the Lux cellulose-2 packed with the 5 µm particles this latter is not fully baseline resolved.

Reduction of the particle size effect on resolution

Since 2010, there were some studies concerning the use of chiral columns packed with smaller particles [START_REF] Hamman | A high throughput approach to purifying chiral molecules using 3 μm analytical chiral stationary phases via supercritical fluid chromatography[END_REF][START_REF] Hegstad | Enantiomeric separation and quantification of citalopram in serum by ultrahigh performance supercritical fluid chromatography-tandem mass spectrometry[END_REF][START_REF] Regalado | Chromatographic resolution of closely related species: separation of warfarin and hydroxylated isomers[END_REF][START_REF] Regalado | Chromatographic separation and assignment of absolute configuration of hydroxywarfarin isomers[END_REF][START_REF] Yang | Development and validation of an enantioselective SFC-MS/MS method for simultaneous separation and quantification of oxcarbazepine and its chiral metabolites in beagle dog plasma[END_REF][START_REF] Vera | Comparing the selectivity and chiral separation of d-and l-fluorenylmethyloxycarbonyl chloride protected amino acids in analytical high performance liquid chromatography and supercritical fluid chromatography; evaluating throughput, economic and environmental impact[END_REF][START_REF] Regalado | Pushing the speed limit in enantioselective supercritical fluid chromatography[END_REF]. The mean diameter or particle size (dp), of the spherical supports used for the stationary phase of a column, is a physical dimension that has a significant impact on the performance of the column. Cellulose tris(3-chloro-4-methylphenylcarbamate) columns with 3µm particle are now commercially available from different suppliers. Thus this particle size was tested to improve the separation of derivative 7. It can be seen from Figure 3 that the 3µm stationary phase provides slightly faster analyses times and improved resolution equal to 1.90.

Conclusive remarks

The performance of Lux i-Cellulose-5 (5µm) and Lux Cellulose-2 (3 and 5 µm), 250 × 4.6 mm columns with respectively an immobilized and a coated chlorinated polysaccharide stationary phases were evaluated towards six pyrrolidone derivatives, with respect to flow-rate and methanol concentration using supercritical fluid chromatography. The optimum linear velocity corresponded to a low flow-rate equal to 2 mL/min. The Lux cellulose-2 (5µm) CSP was found to separate the enantiomers of five derivatives out of six thanks to various percentages of methanol as a co-solvent. Concerning the last derivative 7, the benefit of small particle (3 µm) leading to improved efficiency resulting in better chiral separation, was highlighted in this experimental study. This study result is of practical significance for future separation of similar analytes.

Figure captions

5-( 2 -

 2 phenylhydrazino)pyrrolidin-2-one, racemic compound 6: N'-(5-oxopyrrolidin-2yl)benzohydrazide, racemic compound 7: 5-(benzyloxy)pyrrolidin-2-one, racemic compound 8: 5-(1-phenylethoxy)pyrrolidin-2-one), synthesized in the frame of this project. Unlike compounds 1 to 7 bearing a single asymmetric center, compound 8 has two asymmetrical carbons and the complete separation of the four expected stereoisomers was also attempted on CSPs.

2. 3 .

 3 Chiral supercritical fluid chromatography apparatus 2.3.1. Stationary phases

Figure 1

 1 Figure 1 Chemical structures of pyroglutamic acid 1, pyrrolidin-2-one 2 and antibacterial

Figure 2

 2 Figure 2 Chemical structures of studied chiral stationary phases.

Figure 3

 3 Figure 3 Detailed chromatograms showing best chromatographic performance for the six

Figure 1

 1 Figure 1

  Figure 2

Table 1 .

 1 Chromatographic parameters obtained for all compounds under different flow-rates with 10% of MeOH on Lux Cellulose-2, 5 µm

	Compound	Flow-rate (mL/min)	tr1 (min) tr2 (min)	k1	k2	α	Rs	N
		2	28.42	31.5	17.90	19.94	1.11	1.47
		2.5	24.25	28.41	20.29	23.94	1.18	1.97
	3	3	19.93	23.44	19.76	23.42	1.19	1.88
		3.5	16.85	19.79	19.06	22.57	1.18	1.67
		4	15.32	17.22	19.27	21.79	1.13	1.15
		2	24.88	29.79	15.54	18.81	1.21	2.83
		2.5	19.36	23.26	16.00	19.42	1.21	2.72
	4	3	16.05 19.329 15.72	19.13	1.22	2.44
		3.5	13.58	16.35	15.17	18.47	1.22	2.16
		4	11.80	14.19	14.62	17.78	1.22	1.87
		2	11.04	12.53	6.34	7.33	1.16	1.71
		2.5	8.69	9.84	6.63	7.64	1.15	1.51
	5	3	7.21	8.19	6.51	7.53	1.16	1.42
		3.5	6.03	6.89	6.19	7.20	1.16	1.34
		4	5.39	6.09	6.14	7.07	1.15	1.15
		2	50.31	68.64	32.46	44.64	1.38	3.59
		2.5	38.42	52.24	32.73	44.86	1.37	3.47
	6	3	31.50	43.22	31.82	44.02	1.38	3.32
		3.5	26.53	36.31	30.58	42.23	1.38	3.05
		4	22.73	31.34	29.08	40.46	1.39	2.88
		2	8.15	8.92	4.42	4.93	1.12	1.19
		2.5	6.39	7.01	4.61	5.15	1.12	1.16
	7	3	5.33	5.84	4.55	5.08	1.12	1.04
		3.5	4.54	4.97	4.40	4.91	1.12	0.89
		4	3.93	4.29	4.20	4.68	1.11	0.79
		2	11.36	12.5	6.55	7.31	1.12	1.00
		2.5	8.92	9.92	6.84	7.71	1.13	0.95
	8	3	7.44	8.26	6.75	7.60	1.13	1.01
		3.5	6.31	6.99	6.52	7.33	1.12	1.08
		4	5.49	6.05	6.26	7.01	1.12	0.97

Table 2 .

 2 Chromatographic parameters obtained for all compounds under different flow-rates with 10% of MeOH on i-Lux Cellulose-5, 5 µm

	Compound	Flow-rate (mL/min)	tr1 (min) tr2 (min)	k1	k2	α	Rs	N
		2	29.06	32.19	18.76	20.90	1.11	1.50
	3	2.5 3	22.62 18.49	25.23 20.56	17.85 17.07	20.03 19.09	1.12 1.12	1.30 1.14
		3.5	15.57	17.15	16.49	18.27	1.11	0.97
		4	13.34	14.80	20.52	22.87	1.11	0.96
		2	24.34	27.89	15.61	18.04	1.16	1.80
		2.5	19.15	21.92	14.96	17.27	1.15	1.46
	4	3	15.61	17.98	14.26	16.58	1.16	1.31
		3.5	13.04	14.86	13.66	15.70	1.15	1.10
		4	11.21	12.83	17.08	19.69	1.15	1.05
		2	11.93	13.81	7.14	8.42	1.18	1.84
		2.5	9.38	10.82	6.81	8.02	1.18	1.68
	5	3	7.65	8.85	6.48	7.65	1.18	1.59
		3.5	6.56	7.56	6.37	7.49	1.18	1.41
		4	5.64	6.49	8.09	9.46	1.17	1.24
		2	59.53	84.85	39.63	56.92	1.44	4.79
		2.5	45.82	67.78	37.18	55.48	1.49	4.68
	6	3	37.06	54.68	35.22	52.45	1.49	4.12
		3.5	30.53	45.21	33.29	49.80	1.50	3.76
		4	26.28	39.15	41.38	62.14	1.50	3.43
		2	7.42	-	4.07	-	-	-	-
		2.5	5.93	-	3.94	-	-	-	-
	7	3 3.5	4.88 4.09	--	3.76 3.59	--	--	--	--
		4	3.56	-	4.74	-	-	-	-
		2	11.15	-	6.61	-	-	-	-
		2.5	8.79	-	6.32	-	-	-	-
	8	3	7.21	-	6.04	-	-	-	-
		3.5	6.05	-	5.79	-	-	-	-
		4	5.28	-	7.51	-	-	-	-

Table 3 .

 3 Chromatographic parameters obtained for all compounds at 2 mL/min under different modifier percentages on Lux Cellulose-2, 5 µm

	Compound	% MeOH	tr1 (min)	tr2 (min)	k1	k2	α	Rs
		7.5%	50.44	59.51	34.77	41.21	1.18	1.89
	3	10%	28.421	31.5	19.30	21.50	1.11	1.47
		12.5%	21.67	25.42	14.70	17.42	1.18	1.91
		15%	16.26	18.81	10.87	12.73	1.17	2.83
		7.5%	41.75	50.77	28.61	35.01	1.22	1.87
	4	10%	24.88	29.79	16.77	20.28	1.21	2.83
		12.5%	17.53	20.96	11.70	14.19	1.21	1.90
		15%	13.30	15.684	8.71	10.45	1.20	1.69
		7.5%	16.03	18.7	10.37	12.26	1.18	1.72
	5	10%	11.04	12.53	6.89	7.95	1.15	1.61
		12.5%	8.26	9.36	4.99	5.78	1.16	1.10
		15%	6.67	7.54	3.87	4.50	1.16	1.09
		7.5%	-	-	-	-	-	-
	6	10%	50.317	68.64	34.94	48.03	1.37	3.59
		12.5%	31.44	42.71	21.78	29.95	1.37	2.97
		15%	21.42	28.81	14.64	20.03	1.37	2.50
		7.5%	17.01	19.12	11.06	12.56	1.14	1.02
	7	10%	11.36	12.5	7.11	7.93	1.11	1.00
		12.5%	8.66	9.48	5.28	5.87	1.11	0.98
		15%	6.96	7.55	4.08	4.51	1.11	1.06
		7.5%	11.74	13.2	7.33	8.36	1.14	1.79
	8	10%	8.20	8.96	4.86	5.40	1.11	1.17
		12.5%	6.38	7.03	3.62	4.09	1.13	1.10
		15%	5.20	5.64	2.80	1.75	0.63	0.90

Acknowledgments

A.E.D. is grateful to the ''Fondation de la Catho de Lille'' for her PhD-scholarship. This project is supported by the State as part of the "Programme d'Investissement d'Avenir", under reference ANR-10-IEED-0004-01.