Stephen P. Cummings

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).

The current and future applications of microorganism in the bioremediation of cyanide contamination

Inorganic cyanide and nitrile compounds are distributed widely in the environment, chiefly as a result of anthropogenic activity but also through cyanide synthesis by a range of organisms including higher plants, fungi and bacteria. The major source of cyanide in soil and water is through the discharge of effluents containing a variety of inorganic cyanide and nitriles. Here the fate of cyanide compounds in soil and water is reviewed, identifying those factors that affect their persistence and which determine whether they are amenable to biological degradation. The exploitation of cyanides by a variety of taxa, as a mechanism to avoid predation or to inhibit competitors has led to the evolution in many organisms of enzymes that catalyse degradation of a range of cyanide compounds. Microorganisms expressing pathways involved in cyanide degradation are briefly reviewed and the current applications of bacteria and fungi in the biodegradation of cyanide contamination in the field are discussed. Finally, recent advances that offer an insight into the potential of microbial systems for the bioremediation of cyanide compounds under a range of environmental conditions are identified, and the future potential of these technologies for the treatment of cyanide pollution is discussed.

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.

Diversity and activity of free-living nitrogen-fixing bacteria and total bacteria in organic and conventionally managed soils.

ABSTRACT Agricultural soils are heterogeneous environments in which conditions affecting microbial growth and diversity fluctuate widely in space and time. In this study, the molecular ecology of the total bacterial and free-living nitrogen-fixing communities in soils from the Nafferton Factorial Systems Comparison (NFSC) study in northeast England were examined. The field experiment was factorial in design, with organic versus conventional crop rotation, crop protection, and fertility management factors. Soils were sampled on three dates (March, June, and September) in 2007. Total RNA was extracted from all soil samples and reverse transcribed. Denaturing gradient gel electrophoresis (DGGE) and quantitative PCR (qPCR) were used to analyze nifH and 16S rRNA genes in order to study free-living diazotrophs and the total bacterial community, respectively. Crop rotation was shown to have a significant effect on total bacterial diversity (and that of free-living N fixers) (P ≤ 0.001). On all three dates, nifH activity was higher in the conventional crop rotation. In contrast, qPCR analysis of free-living N fixers indicated significantly higher levels of activity in conventionally fertilized plots in June (P = 0.0324) and in plots with organic crop protection in September (P = 0.0143). To our knowledge, the effects of organic and conventional farming systems on free-living diazotrophs have never been studied. An increased understanding of the impacts of management practices on free-living N fixers could allow modifications in soil management practices to optimize the activity of these organisms.

Nodulation of Sesbania species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia.

Summary Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a bacterial strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridization with R. radiobacter, R. rubi, R. vitis and R. huautlense gave only 44%, 5%, 8% and 8% similarity respectively, suggesting that IRBG74 is potentially a new species. Additionally, it contained no vir genes and lacked tumour‐forming ability, but harboured a sym‐plasmid containing nifH and nodA genes similar to those in other Sesbania symbionts. Indeed, IRBG74 effectively nodulated S. cannabina and seven other Sesbania spp. that nodulate with Ensifer (Sinorhizobium)/Rhizobium strains with similar nodA genes to IRBG74, but not species that nodulate with Azorhizobium or Mesorhizobium. Light and electron microscopy revealed that IRBG74 infected Sesbania spp. via lateral root junctions under flooded conditions, but via root hairs under non‐flooded conditions. Thus, IRBG74 is the first confirmed legume‐nodulating symbiont from the Rhizobium (Agrobacterium) clade. Cross‐inoculation studies with various Sesbania symbionts showed that S. cannabina could form fully effective symbioses with strains in the genera Rhizobium and Ensifer, only ineffective ones with Azorhizobium strains, and either partially effective (Mesorhizobium huakii) or ineffective (Mesorhizobium plurifarium) symbioses with Mesorhizobium. These data are discussed in terms of the molecular phylogeny of Sesbania and its symbionts.

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.

Gut microbiota in preterm infants: assessment and relevance to health and disease

In adults the microbial community of the gut (microbiota) influences a diverse range of health outcomes from obesity, diabetes, asthma and allergy to seemingly ‘remote’ diseases like Parkinsons disease.1 In preterm infants, establishment of the gut microbiota is also of importance for key morbidities like late onset sepsis (LOS) and necrotising enterocolitis (NEC), both significant causes of mortality.2 Many episodes of LOS are with gut derived organisms3 and changes in the intestinal barrier contribute to both LOS and NEC. The gut microbiota are key to developing barrier function, integrity, and mucosal and systemic immune function. They also ‘educate’ the gut associated lymphoid tissue, allowing the establishment of a ‘tolerant’ state between microbiota and the immune system, affecting intestinal function including tight junction structure and immune function.4–6 Patterns of initial colonisation affect host metabolic function: fat deposition, circulating leptin levels, and insulin resistance.6 In the preterm gut structural and immunological immaturity contribute to inflammatory necrosis and abnormal bacterial colonisation (dysbioses). This may result in decreased microbial diversity and an increased inflammatory response exacerbated by an immature innate immune response that increases the risk of diseases like NEC or LOS. An improved understanding of the microbiota of infants cared for in neonatal intensive care, and how this is affected by current practices may allow clinicians to promote more ‘healthy’ gut microbiota patterns, and may be associated with reductions in mortality and improvements in long term outcomes.7 ### What factors associated with preterm birth affect the gut microbiota? Early differences in delivery mode and care alter the development of the gut microbiota. Vaginally delivered term infants acquire their gut microbiota from maternal exposures (genital, stool, skin and breast milk flora) and the wider environment. In contrast, preterm infants are more commonly delivered by caesarean section, and have additional ‘risks and exposures’: antenatal and postnatal antibiotics, infection control measures …

The degradation of the herbicide bromoxynil and its impact on bacterial diversity in a top soil

Aims:  To study how repeated applications of an herbicide bromoxynil to a soil, mimicking the regime used in the field, affected the degradation of the compound and whether such affects were reflected by changes in the indigenous bacterial community present.

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.