Efficacy of Bifidobacterium animalis subsp. lactis (BB-12), B. infantis and Lactobacillus acidophilus (La-5) probiotics to prevent gut dysbiosis in preterm infants of 28+0–32+6 weeks of gestation: a randomised, placebocontrolled, double-blind, multicentre trial: the PRIMAL Clinical Study Protocol

Introduction The healthy ‘eubiosis’ microbiome in infancy is regarded as the microbiome derived from term, vaginally delivered, antibiotic free, breastfed infants at 4–6 months. Dysbiosis is regarded as a deviation from a healthy state with reduced microbial diversity and deficient capacity to control drug-resistant organisms. Preterm infants are highly sensitive to early gut dysbiosis. Latter has been associated with sepsis and necrotising enterocolitis, but may also contribute to long-term health problems. Probiotics hold promise to reduce the risk for adverse short-term outcomes but the evidence from clinical trials remains inconclusive and none has directly assessed the effects of probiotics on the microbiome at high resolution. Methods and analysis A randomised, double blind, placebo-controlled study has been designed to assess the safety and efficacy of the probiotic mix of Bifidobacterium animalis subsp. lactis, B. infantis and Lactobacillus acidophilus in the prevention of gut dysbiosis in preterm infants between 28+0 and 32+6 weeks of gestation. The study is conducted in 18 German neonatal intensive care units. Between April 2018 and March 2020, 654 preterm infants of 28+0–32+6 weeks of gestation will be randomised in the first 48 hours of life to 28 days of once daily treatment with either probiotics or placebo. The efficacy endpoint is the prevention of gut dysbiosis at day 30 of life. A compound definition of gut dysbosis is used: (1) colonisation with multidrug-resistant organisms or gram-negative bacteria with high epidemic potential or (2) a significant deviation of the gut microbiota composition as compared with healthy term infants. Dysbiosis is determined by (1) conventional microbiological culture and (2) phylogenetic microbiome analysis by high-throughput 16S rRNA and metagenome sequencing. Persistence of dysbiosis will be assessed at 12-month follow-up visits. Side effects and adverse events related to the intervention will be recorded. Key secondary endpoint(s) are putative consequences of dysbiosis. A subgroup of infants will be thoroughly phenotyped for immune parameters using chipcytometry. Ethics and dissemination Ethics approval was obtained in all participating sites. Results of the trial will be published in peer-review journals, at scientific meetings, on the website (www.primal-study.de) and via social media of parent organisations. Trial registration number DRKS00013197; Pre-results

Design rules for environmental biodegradability of phenylalanine alkyl ester linked ionic liquids

Ionic liquids (ILs) are perceived as a promising group of chemicals within the realm of green chemistry. They are increasingly of interest for open environmental applications. Rules for the design of biodegradable and preferably completely mineralizing ILs after their introduction into the environment are highly desirable. In this study, the impact of the length of the alkyl chain and of the cationic head group on the environmental biodegradability ofl-phenylalanine ester (PheC(n)) derived ILs using the pyridinium (PyPheC(n)), imidazolium (ImPheC(n)) and cholinium (CholPheC(n)) head groups was systematically studied. This included high-resolution mass spectrometry monitoring of formed products of incomplete mineralization. Completely biodegradable ILs were only identified in the PyPheC(n)series. PyPheC(2)and PyPheC(4)were fully mineralizable within 42 days with no detectable transformation products (TPs), whereas mineralization of longer chain length PyPheC(n)ILs required more time. Our results show biodegradation by microorganisms was affected by an alkyl chain length of at least 10 carbon atoms. Biodegradation of the ILs started with biotic ester hydrolysis independent of the alkyl chain length or core scaffold. In contrast, the second step of microbial degradation was an amide bond cleavage which was only shown for ILs with a pyridinium or imidazolium core, even after a test prolongation up to 42 days. Hence, the nature of the cationic head group and the chain length impacted on the mineralization and biodegradability of the ILs. For ILs with an imidazolium core, amide bond cleavage resulted in the formation of recalcitrant 1-carboxymethylimidazolium (ImAc). Whereas for most ILs with a pyridinium core, amide bond cleavage resulted in 1-carboxymethylpyridinium (PyAc) which was mineralizable, although not readily biodegradable according to the test guideline 301D. In the case of cholinium ILs, no further degradation of cholinium phenylalanine amide TP was observed after the hydrolysis of the ester to the alkyl alcohol. The biodegradability of ILs decreased in the order PyPheC(n)> ImPheC(n)> CholPheC(n), but a key finding is that ImPhe is degraded to ImAc but not further (cf.favourable PyAc mineralisation). These results can be used to design ILs with the property to be effectively biodegraded and mineralized in the environment