Emma C. L. Marrs

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection

Aim:  To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS).

Development of the Preterm Gut Microbiome in Twins at Risk of Necrotising Enterocolitis and Sepsis

The preterm gut microbiome is a complex dynamic community influenced by genetic and environmental factors and is implicated in the pathogenesis of necrotising enterocolitis (NEC) and sepsis. We aimed to explore the longitudinal development of the gut microbiome in preterm twins to determine how shared environmental and genetic factors may influence temporal changes and compared this to the expressed breast milk (EBM) microbiome. Stool samples (n = 173) from 27 infants (12 twin pairs and 1 triplet set) and EBM (n = 18) from 4 mothers were collected longitudinally. All samples underwent PCR-DGGE (denaturing gradient gel electrophoresis) analysis and a selected subset underwent 454 pyrosequencing. Stool and EBM shared a core microbiome dominated by Enterobacteriaceae, Enterococcaceae, and Staphylococcaceae. The gut microbiome showed greater similarity between siblings compared to unrelated individuals. Pyrosequencing revealed a reduction in diversity and increasing dominance of Escherichia sp. preceding NEC that was not observed in the healthy twin. Antibiotic treatment had a substantial effect on the gut microbiome, reducing Escherichia sp. and increasing other Enterobacteriaceae. This study demonstrates related preterm twins share similar gut microbiome development, even within the complex environment of neonatal intensive care. This is likely a result of shared genetic and immunomodulatory factors as well as exposure to the same maternal microbiome during birth, skin contact and exposure to EBM. Environmental factors including antibiotic exposure and feeding are additional significant determinants of community structure, regardless of host genetics.

Bacterial and fungal viability in the preterm gut: NEC and sepsis

Background and aims Evidence suggests that microbial communities in the preterm gut may influence the development of necrotising enterocolitis (NEC) and sepsis. Existing data often neglect fungi and whether bacteria were metabolically active or not. We sought to characterise the bacterial and fungal stool flora of preterm neonates and organism viability and evaluate any associations with NEC and sepsis. Patients 136 stools from 32 patients (<32 weeks gestation) were collected between birth and day 95. Seven infants developed NEC and 13 sepsis. Methods Stools were analysed by PCR-DGGE for assessment of the total bacterial and fungal communities by analysis of 16S rRNA and 28S rRNA, respectively. In 65 samples (25 infants), the viable (RNA) bacterial and fungal communities were analysed. Analyses were performed to examine the possible effects of demographic or treatment related factors and the development of NEC or sepsis. Results 80 (66 viable) bacterial species were identified overall and 12 fungal (none viable). Total bacterial communities significantly differed between healthy infants and those with NEC or sepsis, with Sphingomonas spp. significantly associated with NEC. Significant drivers of community structure differed based on either total or viable analysis. Antifungal prophylaxis was associated with altered bacterial community and a reduction in bacterial richness was observed in week 4, correlating with high antibiotic exposure. Conclusions Total and viable communities differ in preterm infants, and non-viable fungal species are present in infants on fungal prophylaxis. Exploration of viability and non-bacterial contributors to the total community may increase understanding of NEC and sepsis.

Routine Use of Probiotics in Preterm Infants: Longitudinal Impact on the Microbiome and Metabolome.

Background: Probiotics are live microbial supplements that colonize the gut and potentially exert health benefit to the host. Objectives: We aimed to determine the impact of a probiotic (Infloran®: Lactobacillus acidophilus-NCIMB701748 and Bifidobacterium bifidum-ATCC15696) on the bacterial and metabolic function of the preterm gut while in the neonatal intensive care unit (NICU) and following discharge. Methods: Stool samples (n = 88) were collected before, during, and after probiotic intake from 7 patients, along with time-matched controls from 3 patients. Samples were also collected following discharge home from the NICU. Samples underwent bacterial profiling analysis by 16S rRNA gene sequencing and quantitative PCR (qPCR), as well as metabolomic profiling using liquid chromatography mass spectrometry. Results: Bacterial profiling showed greater Bifidobacterium (15.1%) and Lactobacillus (4.2%) during supplementation compared to the control group (4.0% and 0%, respectively). While Lactobacillus became reduced after the probiotic had been stopped, Bifidobacterium remained high following discharge, suggestive of successful colonisation. qPCR analysis showed a significant increase (p ≤ 0.01) in B. bifidum in infants who received probiotic treatment compared to controls, but no significant increase was observed for L. acidophilus (p = 0.153). Metabolite profiling showed clustering based on receiving probiotic or matched controls, with distinct metabolites associated with probiotic administration. Conclusions: Probiotic species successfully colonise the preterm gut, reducing the relative abundance of potentially pathogenic bacteria, and effecting gut functioning. Bifidobacterium (but not Lactobacillus) colonised the gut in the long term, suggesting the possibility that therapeutically administered probiotics may continue to exert important functional effects on gut microbial communities in early infancy.

Preterm gut microbiota and metabolome following discharge from intensive care

The development of the preterm gut microbiome is important for immediate and longer-term health following birth. We aimed to determine if modifications to the preterm gut on the neonatal intensive care unit (NICU) impacted the gut microbiota and metabolome long-term. Stool samples were collected from 29 infants ages 1–3 years post discharge (PD) from a single NICU. Additional NICU samples were included from 14/29 infants. Being diagnosed with disease or receiving increased antibiotics while on the NICU did not significantly impact the microbiome PD. Significant decreases in common NICU organisms including K. oxytoca and E. faecalis and increases in common adult organisms including Akkermansia sp., Blautia sp., and Bacteroides sp. and significantly different Shannon diversity was shown between NICU and PD samples. The metabolome increased in complexity, but while PD samples had unique bacterial profiles we observed comparable metabolomic profiles. The preterm gut microbiome is able to develop complexity comparable to healthy term infants despite limited environmental exposures, high levels of antibiotic administration, and of the presence of serious disease. Further work is needed to establish the direct effect of weaning as a key event in promoting future gut health.

Versatile routes to marine sponge metabolites through benzylidene rhodanines.

The first total synthesis of the marine natural products Psammaplin C and Tokaradine A is described. Benzylidene rhodanines were utilized as versatile intermediates toward the synthesis of seven brominated marine sponge metabolites through the optimization of protection group strategies. Spermatinamine demonstrated good inhibition of all cancer cell lines tested, in particular the leukemia K562 and colon cancer HT29 cell lines.

Synthesis and biological evaluation of purpurealidin E-derived marine sponge metabolites: aplysamine-2, aplyzanzine A, and suberedamines A and B.

Five purpurealidin-derived marine secondary sponge metabolies have been synthesized through the carbodiimide coupling of an appropriate bromotyrosine unit. The structure elucidations have been confirmed through direct comparison with spectroscopic data of isolated natural products. Aplyzanzine A has been shown to be the most active product against a broad bacterial and fungal screen, demonstrating MIC values 2 to 4 times lower than the other metabolites in this study. Compounds 2, 3, 4a, and 5-7 exhibit a modest inhibition against slow growing mycobacteria (MIC 25-50 μg/mL), including Mycobacterium tuberculosis. iso-Anomoian A and suberedamine B showed antitumor activity in the NCI-DTP60 cell line screen at single-digit micromolar concentrations, with iso-anomoian A inhibiting 53 cell lines. These molecules present novel scaffolds for further optimization.

In vitro activity of mecillinam against Enterobacteriaceae with NDM-1 carbapenemase

exhibited a similar pulsotype. These isolates were obtained from two patients treated at different branches. Thus the results indicated than an interhospital dissemination of clones had occurred. In Taiwan the first case of ST11 KPC-2-producing K. pneumoniae was identified in a patient who returned from mainland China in 2011. 3 Chiu et al. 2 reported that rapid intra-and interhospital dissemination of ST11 KPC-2-producing K. pneumoniae continued in Taiwan during 2012, observing that ST11 KPC-2-producing K. pneumoniae became an endemic problem and appeared to be homogeneous in Taiwan. This was the first time that ST11 KPC-2-producing K. pneumoniae isolates had been reported in our hospitals. A total of 47 isolates (36 carbapenem-resistant K. pneumoniae, 7 carbapenem-resistant E. coli and 4 carbapenem-resistant E. cloacae) showed co-resistance to all carbapenems but without any carbapenemase identified. The porin or efflux pump mutation needs to be investigated further in these isolates. In this study the data showed a decrease in AmpC and ESBL producers within the study period; in contrast, an increase in carbapenemase producers was found (Table S2, available as Supplementary data at JAC Online). Further nationwide surveillance of KPC-K. pneumoniae and IMP-E. cloacae has to be performed. The antibiotic stewardship of carbapenems and strict infection control measures should be enforced. 2 Chiu SK, Wu TL, Chuang YC et al. National surveillance study on carbape-nem non-susceptible Klebsiella pneumoniae in Taiwan: the emergence and rapid dissemination of KPC-2 carbapenemase. 4 Huang SR, Liu MF, Lin CF et al. Molecular surveillance and clinical outcomes of carbapenem-resistant Escherichia coli and Klebsiella pneu-moniae infections. 6 Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. 7 Yu WL, Cheng KC, Chi CJ et al. Characterisation and molecular epidemiology of extended-spectrum b-lactamase-producing Enterobacter cloacae isolated from a district teaching hospital in Taiwan. et al. Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii. Sir, We read with interest the recent paper by Janså ker et al., 1 who described 39 patients with urinary tract infection (UTI) caused by Enterobacteriaceae with extended-spectrum b-lactamases (ESBLs) who were treated with pivmecillinam. The authors reported the bacteriological cure rate to be 79% with a clinical cure reported in 16 of 19 evaluable patients (84%). A number of studies have reported in vitro susceptibility of ESBL-producing Enterobacteriaceae to mecillinam despite the fact that mecillinam is labile to …

Stool bacterial load in preterm infants with necrotising enterocolitis

Resected gut tissue in necrotising enterocolitis (NEC) has a higher bacterial load than controls. Quantitative PCR was performed on longitudinal NEC and control stool samples (n=72). No significant difference in the total bacterial load was found between samples at diagnosis compared to controls or temporally within NEC.

A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive Bacteria by Targeting FtsZ.

Inhibition of FtsZ assembly has been found to stall bacterial cell division. Here, we report the identification of a potent carbocyclic curcumin analogue (2d) that inhibits Bacillus subtilis 168 cell proliferation by targeting the assembly of FtsZ. 2d also showed potent inhibitory activity (minimum inhibitory concentrations of 2-4 mg/L) against several clinically important species of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In addition, 2d displayed a significantly reduced inhibitory effect on human cervical cancer cells in comparison to its effect on bacterial cells. Using live cell imaging of GFP-FtsZ by confocal microscopy, 2d was found to rapidly perturb the cytokinetic FtsZ rings in Bacillus subtilis cells. The immunofluorescence imaging of FtsZ also showed that 2d destroyed the Z-ring in bacteria within 5 min. Prolonged treatment with 2d produced filamentous bacteria, but 2d had no detectable effect either on the nucleoids or on the membrane potential of bacteria. 2d inhibited FtsZ assembly in vitro, whereas it had minimal effects on tubulin assembly. Interestingly, 2d strongly enhanced the GTPase activity of FtsZ and reduced the GTPase activity of tubulin. Furthermore, 2d bound to purified FtsZ with a dissociation constant of 4.0 ± 1.1 μM, and the binding of 2d altered the secondary structures of FtsZ. The results together suggested that the non-natural curcumin analogue 2d possesses powerful antibacterial activity against important pathogenic bacteria, and the evidence indicates that 2d inhibits bacterial proliferation by targeting FtsZ.