Clare Lanyon

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.

Comparison of Four Chromogenic Culture Media for Carbapenemase-Producing Enterobacteriaceae

ABSTRACT Four chromogenic media for carbapenemase-producing Enterobacteriaceae (CPE) and two selective broths were challenged with a collection of Enterobacteriaceae with well-defined β-lactamases and 100 stool samples. With low inocula of 130 isolates of CPE, the sensitivities of the four chromogenic media were as follows: Brilliance CRE, 78%; chromID Carba, 91%; chromID ESBL, 96%; and Colorex KPC, 56%. The corresponding sensitivities of Trypticase soy broth plus ertapenem or meropenem were 78% and 47%, respectively.

Prevalence and molecular characterization of Enterobacteriaceae producing NDM-1 carbapenemase at a military hospital in Pakistan and evaluation of two chromogenic media

The aim of this study was to assess the frequency and genotypic diversity of carbapenemase-producing Enterobacteriaceae (CPE) in stool samples from patients attending a military hospital in Pakistan. Further aims included the identification of factors that might predispose to faecal carriage and evaluation of 2 chromogenic culture media: Brilliance CRE and chromID CARBA. Of 175 patients, 32 (18.3%) had faecal carriage of CPE and all produced NDM-1 carbapenemase. All of these 32 patients were detected using chromID CARBA compared with 20 patients (62.5%) detected using Brilliance CRE (P = 0.0015). Duration of hospitalization and treatment with co-amoxyclav were statistically associated with a higher likelihood of carriage of CPE (P ≤ 0.05). The majority of NDM-1-producing Enterobacteriaceae co-produced CTX-M-1 group extended spectrum β-lactamase, and one third produced armA-type methylase. NDM-1 carbapenemase was most commonly found amongst commensal types of Escherichia coli, especially phylogenetic group B1.

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.

Polymicrobial airway bacterial communities in adult bronchiectasis patients.

BackgroundChronic airway infection contributes to the underlying pathogenesis of non-cystic fibrosis bronchiectasis (NCFBr). In contrast to other chronic airway infections, associated with COPD and CF bronchiectasis, where polymicrobial communities have been implicated in lung damage due to the vicious circle of recurrent bacterial infections and inflammation, there is sparse information on the composition of bacterial communities in NCFBr. Seventy consecutive patients were recruited from an outpatient adult NCFBr clinic. Bacterial communities in sputum samples were analysed by culture and pyrosequencing approaches. Bacterial sequences were analysed using partial least square discrimination analyses to investigate trends in community composition and identify those taxa that contribute most to community variation.ResultsThe lower airway in NCFBr is dominated by three bacterial taxa Pasteurellaceae, Streptococcaceae and Pseudomonadaceae. Moreover, the bacterial community is much more diverse than indicated by culture and contains significant numbers of other genera including anaerobic Prevotellaceae, Veillonellaceae and Actinomycetaceae. We found particular taxa are correlated with different clinical states, 27 taxa were associated with acute exacerbations, whereas 11 taxa correlated with stable clinical states. We were unable to demonstrate a significant effect of antibiotic therapy, gender, or lung function on the diversity of the bacterial community. However, presence of clinically significant culturable taxa; particularly Pseudomonas aeruginosa and Haemophilus influenzae correlated with a significant change in the diversity of the bacterial community in the lung.ConclusionsWe have demonstrated that acute exacerbations, the frequency of exacerbation and episodes of clinical stability are correlated, in some patients, with a significantly different bacterial community structure, that are associated with a presence of particular taxa in the NCFBr lung. Moreover, there appears to be an inverse relationship between the abundance of P. aeruginosa and that of of H. influenzae within the NCFBr lung bacterial community. This interaction requires further exploration.

Use of Faropenem as an Indicator of Carbapenemase Activity in the Enterobacteriaceae

ABSTRACT The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10 μg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other β-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using the CLSI/EUCAST methodology with discs of faropenem (10 μg), temocillin (30 μg), and four carbapenems (each 10 μg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a United Kingdom reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-μg faropenem disc compared with only 6% of other β-lactamase producers (sensitivity, 99%; specificity, 94%). A “double zone” around 10-μg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of ≤23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. The presence of no zone of inhibition around a 30-μg temocillin disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a United Kingdom reference laboratory, growth up to a 10-μg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity), with a specificity of 87%. Disc susceptibility testing using faropenem (10 μg) is a simple, convenient, and highly predictive screening test for carbapenemase-producing Enterobacteriaceae.

Prevalence of NDM-1 carbapenemase in patients with diarrhoea in Pakistan and evaluation of two chromogenic culture media.

To evaluate two chromogenic media, Brilliance CRE and chromID CARBA, with stool samples referred to the Public Health Laboratories Division of the National Institute of Health in Islamabad, and assess the prevalence of carbapenemase‐producing Enterobacteriaceae (CPE) in this population.

The transmission of nosocomial pathogens in an intensive care unit: a space-time clustering and structural equation modelling approach.

We investigated the incidence of cases of nosocomial pathogens and risk factors in an intensive treatment unit ward to determine if the number of cases is dependent on location of patients and the colonization/infection history of the ward. A clustering approach method was developed to investigate the patterns of spread of cases through time for five microorganisms [methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter spp., Klebsiella spp., Candida spp., and Pseudomonas aeruginosa] using hospital microbiological monitoring data and ward records of patient-bed use. Cases of colonization/infection by MRSA, Candida and Pseudomonas were clustered in beds and through time while cases of Klebsiella and Acinetobacter were not. We used structural equation modelling to analyse interacting risk factors and the potential pathways of transmission in the ward. Prior nurse contact with colonized/infected patients, mediated by the number of patient-bed movements, were important predictors for all cases, except for those of Pseudomonas. General health and invasive surgery were significant predictors of cases of Candida and Klebsiella. We suggest that isolation and bed movement as a strategy to manage MRSA infections is likely to impact upon the incidence of cases of other opportunist pathogens.

A metagenomic approach to characterize temperate bacteriophage populations from Cystic Fibrosis and non-Cystic Fibrosis bronchiectasis patients

Pseudomonas aeruginosa (Pa), normally a soil commensal, is an important opportunistic pathogen in Cystic Fibrosis (CF) and non-Cystic Fibrosis Bronchiectasis (nCFBR). Persistent infection correlates with accelerated decline in lung function and early mortality. The horizontal transfer of DNA by temperate bacteriophages can add gene function and selective advantages to their bacterial host within the constrained environment of the lower lung. In this study, we chemically induce temperate bacteriophages from clonal cultures of Pa and identify their mixed viral communities employing metagenomic approaches. We compared 92 temperate phage metagenomes stratified from these clinical backgrounds (47 CF and 45 nCFBR Pa isolates) using MG-RAST and GeneWise2. KEGG analysis shows the complexity of temperate phage accessory gene carriage increases with duration and severity of the disease. Furthermore, we identify the presence of Ig-like motifs within phage structural genes linked to bacterial adhesion and carbohydrate binding including Big_2, He_Pig, and Fn3. This study provides the first clinical support to the proposed bacteriophage adherence to mucus (BAM) model and the evolution of phages interacting at these mucosal surfaces over time.