Clostridium neonatale antimicrobial susceptibility, genetic resistance determinants, and genotyping: a multicentre spatiotemporal retrospective analysis.

BACKGROUND: Clostridium neonatale was isolated during an outbreak of neonatal necrotizing enterocolitis (NEC) in 2002. C. neonatale was validated as a new species within the genus Clostridium sensu stricto in 2018. In the present study, we evaluated the antimicrobial susceptibility, genetic determinants of resistance, and phylogenetic relationships of a collection of clinical isolates of C. neonatale. METHODS: C. neonatale strains (n = 68) were isolated from the stools of preterm neonates who either developed NEC or were asymptomatic carriers of C. neonatale in different periods and in different hospitals. Antimicrobial susceptibility was determined by the disc diffusion method. The MICs of clindamycin, cefotaxime and tetracycline were determined. Genetic determinants of resistance were screened by PCR (n = 68) and WGS (n = 35). Genotyping of the isolates was performed by MLST. RESULTS: Antimicrobial resistance was found to clindamycin (n = 24; 35%), cefotaxime (n = 7; 10%) and tetracycline (n = 1; 1%). One clindamycin-resistant isolate carried erm(B) by PCR. In addition, one isolate carrying tet(M) was tetracycline resistant (MIC = 16 mg/L) and 44 isolates carrying either tet(O), tet(32) or tet(M) were tetracycline susceptible (MICs < 16 mg/L). MLST showed that ST2 and ST15 were significantly associated with tet(32) (P < 0.0001) and tet(O) (P < 0.0001), respectively. From WGS, we identified aph(3')-IIa and blaTEM-116 genes and a blaCBP-1-like gene. CONCLUSIONS: C. neonatale is susceptible to anti-anaerobic molecules but resistant to clindamycin, cefotaxime and tetracycline. Genes encoding tetracycline ribosomal protection, macrolide-lincosamide-streptogramin B rRNA methyltransferase, aminoglycoside 3'-phosphotransferase and ß-lactamases have been identified in genomic regions flanked by mobile genetic elements.

Association between gut microbiota at 3.5 years of age and body mass index at 5 years: results from two French nationwide birth cohorts.

BACKGROUND/OBJECTIVES: The relationship between gut microbiota and changes in body mass index (BMI) or pediatric overweight in early life remains unclear, and information regarding the preterm population is scarce. This study aimed to investigate how the gut microbiota at 3.5 years of age is associated with (1) later BMI at 5 years, and (2) BMI z-score variations between 2 and 5 years in children from two French nationwide birth cohorts. SUBJECTS/METHODS: Bacterial 16S rRNA gene sequencing was performed to profile the gut microbiota at 3.5 years of age in preterm children (n = 143, EPIPAGE 2 cohort) and late preterm/full-term children (n = 369, ELFE cohort). The predicted abundances of metabolic functions were computed using PICRUSt2. Anthropometric measurements were collected at 2 and 5 years of age during medical examinations or retrieved from children's health records. Statistical analyses included multivariable linear and logistic regressions, random forest variable selection, and MiRKAT. RESULTS: The Firmicutes to Bacteroidetes (F/B) ratio at 3.5 years was positively associated with the BMI z-score at 5 years. Several genera were positively ([Eubacterium] hallii group, Fusicatenibacter, and [Eubacterium] ventriosum group) or negatively (Eggerthella, Colidextribacter, and Ruminococcaceae CAG-352) associated with the BMI z-scores at 5 years. Some genera were also associated with variations in the BMI z-scores between 2 and 5 years of age. Predicted metabolic functions, including steroid hormone biosynthesis, biotin metabolism, glycosaminoglycan degradation, and amino sugar and nucleotide sugar metabolism, were associated with lower BMI z-scores at 5 years. The unsaturated fatty acids biosynthesis pathway was associated with higher BMI z-scores. CONCLUSIONS: These findings indicate that the gut microbiota at 3.5 years is associated with later BMI during childhood, independent of preterm or term birth, suggesting that changes in the gut microbiota that may predispose to adult obesity begin in early childhood.

Core-genome multilocus sequence typing and core-SNP analysis of Clostridium neonatale strains isolated in different spatio-temporal settings.

IMPORTANCE: Clostridium neonatale has been isolated from the fecal samples of asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Taking advantage of a large collection of independent strains isolated from different spatio-temporal settings, we developed and established a cgMLST scheme for the molecular typing of C. neonatale. Both the cgMLST and cgSNP methods demonstrate comparable discrimination power. Results indicate geographic- and temporal- independent clustering of C. neonatale NEC-associated strains. No specific cgMLST clade of C. neonatale was genetically associated with NEC.

Occurrence of Neonatal Necrotizing Enterocolitis in Premature Neonates and Gut Microbiota: A Case-Control Prospective Multicenter Study.

BACKGROUND: Necrotizing enterocolitis (NEC) is still one of the leading causes of neonatal death. The present study reports the data from a French case-control prospective multicenter study. METHODS: A total of 146 preterm neonates (PNs) with or without NEC were included. Bacterial 16S rRNA gene sequencing was performed on stool samples (n = 103). Specific culture media were used to isolate Escherichia coli, Clostridium butyricum, and Clostridium neonatale, and strains were phenotypically characterized. RESULTS: The gut microbiota of PNs was dominated by Firmicutes and Proteobacteria, and five enterotypes were identified. The microbiota composition was similar between NEC cases and PN controls. However, differences were observed in the relative abundance of Lactobacillus genus, which was significantly lower in the NEC group, whereas that of the Clostridium cluster III was significantly higher (p < 0.05). Within enterotypes, several phylotypes were significantly more abundant in NEC cases (p < 0.05). Regarding perinatal factors, a statistical association was found between the gut microbiota and cesarean delivery and antifungal therapy. In NEC cases and PN controls, the carriage rates and virulence genes of uropathogenic E. coli were equivalent based on culture. No correlation was found between E. coli, C. butyricum, and C. neonatale carriages, beta-lactam resistance, and antibiotic treatment. CONCLUSIONS: At disease onset, our data support a microbiota dysbiosis between NEC and control infants at the genus level. In addition, it provides valuable information on bacterial antimicrobial susceptibility.

Early Life Factors Influencing Children Gut Microbiota at 3.5 Years from Two French Birth Cohorts.

Early life gut microbiota-influencing factors may play an important role in programming individuals long-term health and substantial efforts have been devoted into studying the development of the gut microbiota in relation to early life events. This study aimed to examine in a single study, the persistence of associations between 20 factors occurring in the early life and the gut microbiota at 3.5 years of 798 children from two French nationwide birth cohorts, EPIPAGE 2 (very preterm children) and ELFE (late preterm and full-term children). Gut microbiota profiling was assessed using 16S rRNA gene sequencing-based method. Upon thorough adjustment of confounding factors, we demonstrated that gestational age was one of the factors most associated with gut microbiota differences with a noticeable imprint of prematurity at 3.5 years of age. Children born by cesarean section harbored lower richness and diversity and a different overall gut microbiota composition independently of preterm status. Children who had ever received human milk were associated with a Prevotella-driven enterotype (P_type) compared to those who had never received human milk. Living with a sibling was associated with higher diversity. Children with siblings and those attending daycare centers were associated with a P_type enterotype. Maternal factors including the country of birth and preconception maternal body mass index were associated with some microbiota characteristics: children born to overweight or obese mothers showed increased gut microbiota richness. This study reveals that multiple exposures operating from early life imprint the gut microbiota at 3.5 years that is a pivotal age when the gut microbiota acquires many of its adult characteristics.

Neonatal necrotizing enterocolitis: Clostridium butyricum and Clostridium neonatale fermentation metabolism and enteropathogenicity.

Bacterial colonization in the gut plays a pivotal role in neonatal necrotizing enterocolitis (NEC) development, but the relationship between bacteria and NEC remains unclear. In this study, we aimed to elucidate whether bacterial butyrate end-fermentation metabolites participate in the development of NEC lesions and confirm the enteropathogenicity of Clostridium butyricum and Clostridium neonatale in NEC. First, we produced C.butyricum and C.neonatale strains impaired in butyrate production by genetically inactivating the hbd gene encoding ß-hydroxybutyryl-CoA dehydrogenase that produces end-fermentation metabolites. Second, we evaluated the enteropathogenicty of the hbd-knockout strains in a gnotobiotic quail model of NEC. The analyses showed that animals harboring these strains had significantly fewer and less intense intestinal lesions than those harboring the respective wild-type strains. In the absence of specific biological markers of NEC, the data provide original and new mechanistic insights into the disease pathophysiology, a necessary step for developing potential novel therapies.

Very Preterm Children Gut Microbiota Comparison at the Neonatal Period of 1 Month and 3.5 Years of Life.

Prematurity is a risk factor for dysbiosis of the gut microbiota due to particular birth conditions and frequent prolonged hospitalization of neonates. Although gut microbiota colonization after birth and its establishment during the hospitalization period have been studied in preterm infants, data on gut microbiota following discharge, particularly during early childhood, are scarce. The present study investigated the relationship between gut microbiota at 1 month after birth (hospitalization period) and 3.5 years of age in 159 preterm children belonging to the French EPIFLORE prospective observational cohort study. Analysis using bacterial 16S rRNA gene sequencing showed that the gut microbiota of preterm neonates at 1 month was highly variable and characterized by six distinct enterotypes. In contrast, the gut microbiota of the same children at 3.5 years of age showed less variability, with only two discrete enterotypes. An absence of association between enterotypes at 1 month and 3.5 years of age was observed. While the alpha diversity of gut microbiota significantly increased between 1 month and 3.5 years of age, for both alpha and beta diversities, there was no correlation between the 1-month and 3.5-years time points. Comparison at 3.5 years between children born either preterm (n = 159) or full-term (n = 200) showed no differences in terms of enterotypes, but preterm children harbored a lower Shannon diversity index and a different overall composition of microbiota than full-term children. This study suggests that the characteristics of the early gut microbiota of preterm children are not predictive of the microbial community composition at 3.5 years of age. However, the impact of gestational age is still noticeable on the gut microbiota up to 3.5 years of age.

Gut Microbiota Diversity of Preterm Neonates Is Associated With Clostridioides Difficile Colonization.

In adults, Clostridioides difficile infections are associated with alterations of the intestinal bacterial populations. Although preterm neonates (PN) are frequently colonized by C. difficile, limited data are available regarding the relationship between C. difficile and the intestinal microbiota of this specific population. Therefore, we studied the intestinal microbiota of PN from two multicenter cohorts using high-throughput sequencing of the bacterial 16S rRNA gene. Our results showed that alpha diversity was significantly higher in children colonized by C. difficile than those without colonization. Beta diversity significantly differed between the groups. In multivariate analysis, C. difficile colonization was significantly associated with the absence of postnatal antibiotherapy and higher gestational age. Taxa belonging to the Lachnospiraceae, Enterobacteriaceae, Oscillospiraceae families and Veillonella sp. were positively associated with C. difficile colonization, whereas Bacteroidales and Bifidobacterium breve were negatively associated with C. difficile colonization. After adjustment for covariables, Clostridioides, Rothia, Bifidobacterium, Veillonella, Eisenbergiella genera and Enterobacterales were more abundant in the gut microbiota of colonized children. There was no significant association between C. difficile colonization and necrotizing enterocolitis in PN. Our results suggest that C. difficile colonization in PN is related to the establishment of physiological microbiota.

Core-, pan- and accessory genome analyses of Clostridium neonatale: insights into genetic diversity.

Clostridium neonatale is a potential opportunistic pathogen recovered from faecal samples in cases of necrotizing enterocolitis (NEC), a gastrointestinal disease affecting preterm neonates. Although the C. neonatale species description and name validation were published in 2018, comparative genomics are lacking. In the present study, we provide the closed genome assembly of the C. neonatale ATCC BAA-265T (=250.09) reference strain with a manually curated functional annotation of the coding sequences. Pan-, core- and accessory genome analyses were performed using the complete 250.09 genome (4.7 Mb), three new assemblies (4.6-5.6 Mb), and five publicly available draft genome assemblies (4.6-4.7 Mb). The C. neonatale pan-genome contains 6840 genes, while the core-genome has 3387 genes. Pan-genome analysis revealed an 'open' state and genomic diversity. The strain-specific gene families ranged from five to 742 genes. Multiple mobile genetic elements were predicted, including a total of 201 genomic islands, 13 insertion sequence families, one CRISPR-Cas type I-B system and 15 predicted intact prophage signatures. Primary virulence classes including offensive, defensive, regulation of virulence-associated genes and non-specific virulence factors were identified. The presence of a tet(W/N/W) gene encoding a tetracycline resistance ribosomal protection protein and a 23S rRNA methyltransferase ermQ gene were identified in two different strains. Together, our results revealed a genetic diversity and plasticity of C. neonatale genomes and provide a comprehensive view of this species genomic features, paving the way for the characterization of its biological capabilities.

Isolation and Characterization of Commensal Bifidobacteria Strains in Gut Microbiota of Neonates Born Preterm: A Prospective Longitudinal Study.

Bifidobacterial population dynamics were investigated using a longitudinal analysis of dominant species isolated from feces of neonates born preterm (singletons (n = 10), pairs of twins (n = 11)) from birth up to 16 months of age. We performed quantification, isolation, and identification of the dominant bifidobacteria strains. The genetic relationship of the isolates was investigated via pulsed field gel electrophoresis (PFGE) genotyping, and PCR was used to screen the specific genetic marker tet genes. Additionally, all of the isolated strains were phenotypically characterized by their response to gastro-intestinal stresses and the MIC determination of tetracycline. In the same individual, our results showed a turnover of the bifidobacteria dominant population not only at species but also at strain levels. In addition, we found clonally related strains between twins. A minority of strains were tolerant to gastric (6%) and intestinal (16%) stresses. Thirteen percent of the strains were resistant to tetracycline. This work is original as it provides insights at the strain level of the early life in vivo dynamics of gut microbiota bifidobacteria in preterm neonates. It highlights the need to take into consideration the fluctuation of bifidobacteria populations that may occur for one individual.

First 1000 Days of Life: Consequences of Antibiotics on Gut Microbiota.

The developmental origin of health and disease highlights the importance of the period of the first 1000 days (from conception to 2 years) of life. In particular, the process of gut microbiota establishment occurs within this time window. Therefore, determinants interfering with neonatal gut establishment may disrupt its physiological functions and potentially lead to negative health outcomes. Antibiotics are among perinatal determinants that can directly or indirectly affect the pattern of gut bacterial colonization, with a long-lasting impact on intestinal ecosystem functions. In this review, we will examine the impact of antibiotics on the intestinal microbiota during the perinatal period and first years of life, a key interval for development of an individual's health capital. Further, we will discuss the role of antibiotics during short- and long-term dysbiosis and their associated health consequences.

Characterization of Non-Toxigenic Clostridioides difficile Strains Isolated from Preterm Neonates and In Vivo Study of Their Protective Effect.

In a previous monocentric study in preterm neonates (PN), we described a high Clostridioides difficile colonization rate (74%) with two uncommon non-toxigenic strains (NTCD) belonging to PCR-ribotype (RT) (CE)847 and (CE)032. To determine the extent of carriage of both NTCD in other spatio-temporal settings, strains isolated in PN stools from two multicenter cohorts were characterized by PCR-ribotyping, MLVA and MLST. We also evaluated the protective role of two NTCD from these RT against C. difficile infection in a hamster caecitis model. Animals were administered either each NTCD alone (n = 7), or followed by a 027 strain (n = 9). A control group received only the 027 strain (n = 8). Clinical activity and colonization by C. difficile in stools were monitored daily until death or sacrifice at D20. We isolated 18 RT(CE)032 (ST-83) strains and 2 RT(CE)847 (ST-26) strains among 247 PN from both cohorts. Within each RT, strains were genetically related. The survival rate was significantly increased when animals received a RT(CE)847 or (CE)032 strain before the 027 strain (4/9 deaths, p = 0.029; 1/9 death, p = 0.0004, respectively). We describe two predominant uncommon NTCD strains, in a PN population from different healthcare facilities. Both NTCD provide a potential protection against C. difficile infection.

Assessment of Neonatal Intensive Care Unit Practices and Preterm Newborn Gut Microbiota and 2-Year Neurodevelopmental Outcomes.

Importance: In very preterm newborns, gut microbiota is highly variable with major dysbiosis. Its association with short-term health is widely studied, but the association with long-term outcomes remains unknown. Objective: To investigate in preterm newborns the associations among practice strategies in neonatal intensive care units (NICUs), gut microbiota, and outcomes at 2 years. Design, Setting, and Participants: EPIFLORE is a prospective observational cohort study that includes a stool sample collection during the fourth week after birth. Preterm newborns of less than 32 weeks of gestational age (GA) born in 2011 were included from 24 NICUs as part of the French nationwide population-based cohort, EPIPAGE 2. Data were collected from May 2011 to December 2011 and analyzed from September 2016 to December 2018. Exposures: Eight NICU strategies concerning sedation, ventilation, skin-to-skin practice, antibiotherapy, ductus arteriosus, and breastfeeding were assessed. A NICU was considered favorable to a practice if the percentage of that practice in the NICU was more than the expected percentage. Main Outcomes and Measures: Gut microbiota was analyzed by 16S ribosomal RNA gene sequencing and characterized by a clustering-based method. The 2-year outcome was defined by death or neurodevelopmental delay using a Global Ages and Stages questionnaire score. Results: Of 577 newborns included in the study, the mean (SD) GA was 28.3 (2.0) weeks, and 303 (52.5%) were male. Collected gut microbiota was grouped into 5 discrete clusters. A sixth cluster included nonamplifiable samples owing to low bacterial load. Cluster 4 (driven by Enterococcus [n = 63]), cluster 5 (driven by Staphylococcus [n = 52]), and cluster 6 (n = 93) were significantly associated with lower mean (SD) GA (26.7 [1.8] weeks and 26.8 [1.9] weeks, respectively) and cluster 3 (driven by Escherichia/Shigella [n = 61]) with higher mean (SD) GA (29.4 [1.6] weeks; P = .001). Cluster 3 was considered the reference. After adjustment for confounders, no assisted ventilation at day 1 was associated with a decreased risk of belonging to cluster 5 or cluster 6 (adjusted odds ratio [AOR], 0.21 [95% CI, 0.06-0.78] and 0.19 [95% CI, 0.06-0.62], respectively) when sedation (AOR, 10.55 [95% CI, 2.28-48.87] and 4.62 [1.32-16.18], respectively) and low volume of enteral nutrition (AOR, 10.48 [95% CI, 2.48-44.29] and 7.28 [95% CI, 2.03-26.18], respectively) was associated with an increased risk. Skin-to-skin practice was associated with a decreased risk of being in cluster 5 (AOR, 0.14 [95% CI, 0.04-0.48]). Moreover, clusters 4, 5, 6 were significantly associated with 2-year nonoptimal outcome (AOR, 6.17 [95% CI, 1.46-26.0]; AOR, 4.53 [95% CI, 1.02-20.1]; and AOR, 5.42 [95% CI, 1.36-21.6], respectively). Conclusions and Relevance: Gut microbiota of very preterm newborns at week 4 is associated with NICU practices and 2-year outcomes. Microbiota could be a noninvasive biomarker of immaturity.

Carriage and colonization of C. difficile in preterm neonates: A longitudinal prospective study.

BACKGROUND: Premature neonates (PN) present multiple risk factors for high frequencies and high levels of colonization by C. difficile, yet data is missing about this specific pediatric population. Here, we investigated PN C. difficile carriage and colonization dynamics, analyzed the impact of perinatal determinants on colonization, and characterized the isolates. METHODS: A one year longitudinal monocentric prospective cohort study was performed on 121 PN. C. difficile strains isolated from fecal samples on selective medium were identified and characterized by PCR (tpi housekeeping gene; tcdA and tcdB, and binary toxin genes), capillary gel-based electrophoresis PCR-ribotyping, and Multi-Locus Variable-number tandem-repeat Analysis (MLVA). RESULTS: Of the 379 samples analyzed, 199 (52%) were C. difficile culture positive with the mean levels of C. difficile colonization decreasing significantly (P = .027) over time. During hospitalization, C. difficile colonization frequency increased up to 61% with 95% of the strains belonging to both non-toxigenic PCR-ribotypes (RTs) FR082 (35%) and 032 (60%). After hospital discharge, if a higher diversity in RTs was observed, RTs FR082 and 032 remained predominant (respectively 40% and 28%). MLVA showed clonal relationship within each FR082 and 032 RTs. Ten toxigenic strains (5%) were isolated, all tcdA+/tcdB+ except for one tcdA-/tcdB+, and all being acquired after hospitalization. At 1 week, the only factors found to be linked with a higher frequency of C. difficile colonization were a higher gestational age (P = 0.006) and a higher birth weight (P = 0.016). CONCLUSION: The dynamics of C. difficile colonization in PN followed a specific pattern. C. difficile colonization rapidly occurred after birth with a low diversity of non-toxigenic RTs. After hospitalization, non-toxigenic RTs diversity increased. Sporadic carriage of toxigenic strains was observed after hospitalization.

Clostridial Strain-Specific Characteristics Associated with Necrotizing Enterocolitis.

We aimed at identifying potential bacterial factors linking clostridia with necrotizing enterocolitis (NEC). We compared the phenotypic traits, stress responses, cellular cytotoxicity, and inflammatory capabilities of the largest collection of Clostridium butyricum and Clostridium neonatale strains isolated from fecal samples of NEC preterm neonates (PN) and control PNs. When strain characteristics were used as explanatory variables, a statistical discriminant analysis allowed the separation of NEC and control strains into separate groups. Strains isolated from NEC PN were characterized by a higher viability at 30°C (P = 0.03) and higher aerotolerance (P = 0.01), suggesting that NEC strains may have a competitive and/or survival advantage in the environmental gastrointestinal tract conditions of NEC PN. Heat-treated NEC bacteria induced higher production of interleukin-8 in Caco-2 cells (P = 0.03), suggesting proinflammatory activity. In vitro, bacteria, bacterial components, and fecal filtrates showed variable cytotoxic effects affecting the cellular network and/or cell viability, without specific association with NEC or control samples. Altogether, our data support the existence of a specific clostridial strain signature associated with NEC.IMPORTANCE Clostridia are part of the commensal microbiota in preterm neonates (PN). However, microbiota analyses by culture and metagenomics have linked necrotizing enterocolitis (NEC) and intestinal colonization with clostridial species. Nevertheless, little is known about the specific characteristics that may be shared by clostridia associated with NEC compared to commensal clostridia. Therefore, our goal was to identify specific bacterial factors linking clostridial strains with NEC. We report the existence of a specific bacterial signature associated with NEC and propose that activation of the innate immune response may be a unifying causative mechanism for the development of NEC independent of a specific pathogenic organism. The present study provides new insights into NEC pathophysiology that are needed for better diagnostics and strategies for implementing prevention of the disease.

Nutritional strategies and gut microbiota composition as risk factors for necrotizing enterocolitis in very-preterm infants.

Background: The pathophysiology of necrotizing enterocolitis (NEC) remains poorly understood.Objective: We assessed the relation between feeding strategies, intestinal microbiota composition, and the development of NEC.Design: We performed a prospective nationwide population-based study, EPIPAGE 2 (Etude Epidémiologique sur les Petits Ages Gestationnels), including preterm infants born at <32 wk of gestation in France in 2011. From individual characteristics observed during the first week of life, we calculated a propensity score for the risk of NEC (Bell's stage 2 or 3) after day 7 of life. We analyzed the relation between neonatal intensive care unit (NICU) strategies concerning the rate of progression of enteral feeding, the direct-breastfeeding policy, and the onset of NEC using general linear mixed models to account for clustering by the NICU. An ancillary propensity-matched case-control study, EPIFLORE (Etude Epidémiologique de la flore), in 20 of the 64 NICUs, analyzed the intestinal microbiota by culture and 16S ribosomal RNA gene sequencing.Results: Among the 3161 enrolled preterm infants, 106 (3.4%; 95% CI: 2.8%, 4.0%) developed NEC. Individual characteristics were significantly associated with NEC. Slower and intermediate rates of progression of enteral feeding strategies were associated with a higher risk of NEC, with an adjusted OR of 2.3 (95% CI: 1.2, 4.5; P = 0.01) and 2.0 (95% CI: 1.1, 3.5; P = 0.02), respectively. Less favorable and intermediate direct-breastfeeding policies were associated with higher NEC risk as well, with an adjusted OR of 2.5 (95% CI: 1.1, 5.8; P = 0.03) and 2.3 (95% CI: 1.1, 4.8; P = 0.02), respectively. Microbiota analysis performed in 16 cases and 78 controls showed an association between Clostridium neonatale and Staphylococcus aureus with NEC (P = 0.001 and P = 0.002).Conclusions: A slow rate of progression of enteral feeding and a less favorable direct-breastfeeding policy are associated with an increased risk of developing NEC. For a given level of risk assessed by propensity score, colonization by C. neonatale and/or S. aureus is significantly associated with NEC. This trial (EPIFLORE study) was registered at clinicaltrials.gov as NCT01127698.

One-Step Multiplex PCR Assay for Differentiating Proposed New Species "Clostridium neonatale" from Closely Related Species.

"Clostridium neonatale" sp. nov., previously involved in an outbreak of neonatal necrotizing enterocolitis, was recently proposed as a new species of the Clostridium genus sensu stricto. We developed a one-step multiplex colony PCR for C. neonatale identification and investigated C. neonatale intestinal colonization frequency in healthy preterm neonates.

16S rRNA gene sequencing, multilocus sequence analysis, and mass spectrometry identification of the proposed new species "Clostridium neonatale".

In 2002, an outbreak of necrotizing enterocolitis in a Canadian neonatal intensive care unit was associated with a proposed novel species of Clostridium, "Clostridium neonatale." To date, there are no data about the isolation, identification, or clinical significance of this species. Additionally, C. neonatale has not been formally classified as a new species, rendering its identification challenging. Indeed, the C. neonatale 16S rRNA gene sequence shows high similarity to another Clostridium species involved in neonatal necrotizing enterocolitis, Clostridium butyricum. By performing a polyphasic study combining phylogenetic analysis (16S rRNA gene sequencing and multilocus sequence analysis) and phenotypic characterization with mass spectrometry, we demonstrated that C. neonatale is a new species within the Clostridium genus sensu stricto, for which we propose the name Clostridium neonatale sp. nov. Now that the status of C. neonatale has been clarified, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can be used for better differential identification of C. neonatale and C. butyricum clinical isolates. This is necessary to precisely define the role and clinical significance of C. neonatale, a species that may have been misidentified and underrepresented during previous neonatal necrotizing enterocolitis studies.

Bifidobacterium longum and Bifidobacterium breve isolates from preterm and full term neonates: comparison of cell surface properties.

We compared autoaggregation, surface hydrophobicity and Caco-2 cells adhesion capabilities of independent Bifidobacterium breve (n = 22) and Bifidobacterium longum (n = 25) strains isolated from preterm (n = 20) and full term neonates (n = 27). Concerning strains properties, a correlation between autoaggregation and surface hydrophobicity was found for B. longum (r = 0.40, p = 0.048), B. breve (r = 0.57, p = 0.005), and all strains independently of the species consideration (r = 0.46, p = 0.001). The absence of difference in adhesion capabilities between preterm and full term neonate strains suggests a strain-dependent property. However, B. longum strains from preterm neonates (n = 10) showed higher autoaggregation ability (p = 0.044). Additionally, independently of species consideration, preterm neonates strains showed lower surface hydrophobicity (p = 0.027). As far as species are considered, preterm neonate B. breve strains (n = 10) showed significantly lower surface hydrophobicity percentages (p = 0.043). Our results suggest the existence of variations in bifidobacteria membrane structure and/or composition that may reflect adaptation of these bacteria to the intestinal environment of either preterm or full term neonates. Such information is of interest when considering the use of bifidobacteria probiotic strains for modulation of preterm neonates gut microbiota.