Gut microbiota of Type 1 diabetes patients with good glycaemic control and high physical fitness is similar to people without diabetes: an observational study.

AIM: Type 1 diabetes is the product of a complex interplay between genetic susceptibility and exposure to environmental factors. Existing bacterial profiling studies focus on people who are most at risk at the time of diagnosis; there are limited data on the gut microbiota of people with long-standing Type 1 diabetes. This study compared the gut microbiota of patients with Type 1 diabetes and good glycaemic control and high levels of physical-fitness with that of matched controls without diabetes. METHODS: Ten males with Type 1 diabetes and ten matched controls without diabetes were recruited; groups were matched for gender, age, BMI, peak oxygen uptake (VO2max ), and exercise habits. Stool samples were analysed using next-generation sequencing of the 16S rRNA gene to obtain bacterial profiles from each individual. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was implemented to predict the functional content of the bacterial operational taxonomic units. RESULTS: Faecalibacterium sp., Roseburia sp. and Bacteroides sp. were typically the most abundant members of the community in both patients with Type 1 diabetes and controls, and were present in every sample in the cohort. Each bacterial profile was relatively individual and no significant difference was reported between the bacterial profiles or the Shannon diversity indices of Type 1 diabetes compared with controls. The functional profiles were more conserved and the Type 1 diabetes group were comparable with the control group. CONCLUSIONS: We show that both gut microbiota and resulting functional bacterial profiles from patients with long-standing Type 1 diabetes in good glycaemic control and high physical fitness levels are comparable with those of matched people without diabetes.

Microbiological profiles of sputum and gastric juice aspirates in Cystic Fibrosis patients.

Gastro-Oesophageal Reflux (GOR) is a key problem in Cystic Fibrosis (CF), but the relationship between lung and gastric microbiomes is not well understood. We hypothesised that CF gastric and lung microbiomes are related. Gastric and sputum cultures were obtained from fifteen CF patients receiving percutaneous endoscopic gastrostomy feeding. Non-CF gastric juice data was obtained through endoscopy from 14 patients without lung disease. Bacterial and fungal isolates were identified by culture. Molecular bacterial profiling used next generation sequencing (NGS) of the 16S rRNA gene. Cultures grew bacteria and/or fungi in all CF gastric juice and sputa and in 9/14 non-CF gastric juices. Pseudomonas aeruginosa(Pa) was present in CF sputum in 11 patients, 4 had identical Pa strains in the stomach. NGS data from non-CF gastric juice samples were significantly more diverse compared to CF samples. NGS showed CF gastric juice had markedly lower abundance of normal gut bacteria; Bacteroides and Faecalibacterium, but increased Pseudomonas compared with non-CF. Multivariate partial least squares discriminant analysis demonstrated similar bacterial profiles of CF sputum and gastric juice samples, which were distinct from non-CF gastric juice. We provide novel evidence suggesting the existence of an aerodigestive microbiome in CF, which may have clinical relevance.

Effect of crop management and sample year on abundance of soil bacterial communities in organic and conventional cropping systems.

AIMS: To identify changes in the bacterial community, at the phylum level brought about by varied crop management. METHODS AND RESULTS: Next-generation sequencing methods were used to compare the taxonomic structure of the bacterial community within 24 agricultural soils managed with either organic or conventional methods, over a 3-year period. Relative abundance of the proportionately larger phyla (e.g. Acidobacteria and Actinobacteria) was primarily affected by sample year rather than crop management. Changes of abundance in these phyla were correlated with changes in pH, organic nitrogen and soil basal respiration. Crop management affected some of the less dominant phyla (Chloroflexi, Nitrospirae, Gemmatimonadetes) which also correlated with pH and organic N. CONCLUSION: Soil diversity can vary with changing environmental variables and soil chemistry. If these factors remain constant, soil diversity can also remain constant even under changing land use. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of crop management on environmental variables must be considered when interpreting bacterial diversity studies in agricultural soils. Impact of land use change should always be monitored across different sampling time points. Further studies at the functional group level are necessary to assess whether management-induced changes in bacterial community structure are of biological and agronomic relevance.

Evaluation of a new chromogenic medium, chromID OXA-48, for recovery of carbapenemase-producing Enterobacteriaceae from patients at a university hospital in Turkey.

The purpose of this study was to evaluate a new chromogenic medium, chromID OXA-48, for the isolation of carbapenemase-producing Enterobacteriaceae (CPE) directly from rectal swabs. chromID CARBA and chromID OXA-48 are two chromogenic media that have been commercialized for the isolation of CPE directly from clinical samples. Both media were evaluated alongside a broth enrichment method recommended by the CDC for isolation of CPE, with rectal swabs from 302 unique hospitalized patients at the Hacettepe University Hospital, Ankara, Turkey. A total of 33 patients (11 %) were found to be colonized with CPE using a combination of all methods, and all CPE produced OXA-48 carbapenemase. Klebsiella pneumoniae was by far the most dominant species of CPE and was isolated from 31 patients. Culture on chromID OXA-48 offered the highest sensitivity (75.8 %) for detection of CPE compared with the other two methods (sensitivity for both other methods was 57.6 %) and also offered the highest specificity (99.3 %). However, a combination of methods (either chromID OXA-48 plus CDC method or chromID OXA-48 plus chromID CARBA) was necessary to achieve an acceptable sensitivity (90.9 %). For isolation of CPE, in a setting where OXA-48 carbapenemase is the dominant type of carbapenemase, chromID OXA-48 is a highly useful medium but using a combination of methods is optimal for adequate detection. The combined use of two chromogenic media offered acceptable sensitivity (90.9 %) and the highest specificity (98.5 %) and also allowed for isolation of CPE within 18-20 h.

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

AIMS: 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. METHODS AND RESULTS: One hundred and fifty-two stool samples from patients with diarrhoea were referred to the Microbiology Department and were investigated for the presence of CPE using two chromogenic culture media, Brilliance CRE and chromID CARBA. Thirteen patients (8·6%) were found to be colonized with CPE and all produced NDM-1 carbapenemase. Twelve of these patients (92%) were found to be colonized by culture on chromID CARBA compared with seven (54%) using Brilliance CRE. CONCLUSIONS: If only coloured colonies were considered as presumptive CPE, the sensitivity, specificity and positive predictive value were 54, 23 and 6% for Brilliance CRE and 85, 85 and 36% for chromID CARBA, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: We conclude that Enterobacteriaceae that produce NDM-1 carbapenemase can be found in patients from all major provinces of Pakistan and that chromID CARBA was the most effective of the two chromogenic media in this setting.

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). METHODS: Ninety-nine stools from 38 infants of median 27-week gestation were cultured; 44 stools from 27 infants had their microbial profiles determined by 16S. Ordination analyses explored effects of patient variables on gut communities. RESULTS: Standard microbiological culture identified a mean of two organisms (range 0-7), DGGE 12 (range 3-18) per patient. Enterococcus faecalis and coagulase negative staphylococci (CONS) were most common by culture (40% and 39% of specimens). Meconium was not sterile. No fungi were cultured. Bacterial community structures in infants with NEC and LOS differed from healthy infants. Infants who developed NEC carried more CONS (45% vs 30%) and less Enterococcus faecalis (31% vs 57%). 16S identified Enterobacter and Staphylococcus presence associated with NEC/LOS, respectively. CONCLUSIONS: Important differences were found in the gut microbiota of preterm infants who develop NEC/LOS. The relationship of these changes to current practices in neonatal intensive care requires further exploration.

Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis.

AIM: The aim of this study was to quantitatively and qualitatively assess the effect of sample storage on the metabolically active microbial community found in sputum samples from patients with cystic fibrosis (CF). METHODS: Sputum samples were collected and split in two equal aliquots one of which was immersed in RNAlater and refrigerated immediately, the second stored at room temperature for 24 h and RNAlater was subsequently added. mRNA was extracted, and RT-PCR-DGGE and qPCR analysis of the bacterial and fungal communities was carried out. RESULTS: Significant differences in the bacterial communities between the two protocols were observed but there were no significant difference seen in the fungal community analyses. Analysis by qPCR demonstrated that room temperature storage gave statistically significant increases in eubacteria and Pseudomonas spp. and a statistically significant decrease in those of Haemophilus influenzae. CONCLUSIONS: The analysis of metabolically active microbial communities from CF sputum using molecular techniques indicated that samples should be stored at 4 degrees C upon addition of RNAlater to obtain an accurate depiction of the CF lung microbiota. Also, storing respiratory samples at room temperature may cause an over representation of Pseudomonas aeruginosa and mask the presence of other clinically significant organisms.

The impact of zinc and lead concentrations and seasonal variation on bacterial and actinobacterial community structure in a metallophytic grassland soil.

The diversity and structure of bacterial and actinobacteral diversity and communities were determined in a metallophytic grassland soil from an upland site in northern England. The community profiles were subjected to multivariate analyses using correspondence and cluster analyses. The total bacterial community diversities and structures were not significantly affected by Pb and Zn concentration in the soil. However, the community structure did show changes between winter and summer samples. Raup and Crick analysis indicated that deterministic selection lead to winter profiles exhibiting significant similarity. The actinobacterial community was also unaffected by Pb and Zn concentration. However, seasonal changes were apparent as diversity were significantly lower in winter compared to summer profiles. Moreover, the community structure showed evidence of changes of structure based on the seasonal samples with winter samples showing significant similarity to each other.

Phenotypic variation in Streptomyces sp. DSM 40537, a lipoteichoic acid producing actinomycete.

AIMS: To reinvestigate the production of lipoteichoic acid (LTA) by the actinomycete strain Streptomyces sp. DSM 40537 (=ATCC 3351). METHODS AND RESULTS: LTA was extracted and purified from strain Streptomyces sp. DSM 40537. The identification of the LTA was confirmed by Western blotting with a monoclonal antibody. During these studies, two stable phenotypic variants of DSM 40537 were obtained, one of which released a distinctive orange pigment. 16S rRNA gene sequencing of each variant yielded identical sequences and allowed phylogenetic analysis to be performed. CONCLUSIONS: Streptomyces sp. DSM 40537 was shown to exhibit stable morphological variation. The strain was confirmed to be a LTA-producing actinomycete and to belong to the Streptomyces albidoflavus cluster within the genus Streptomyces. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide important support for the hypothesis that the distribution of LTA is linked to that of wall teichoic acids and emphasizes the need to reinvestigate LTA distribution in actinomycetes.

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. METHODS AND RESULTS: Bromoxynil degradation was monitored in soil microcosms using HPLC. Its impact on the bacterial community was determined using denaturing gradient gel electrophoresis (DGGE) and quantitative PCR of five bacterial taxa (Pseudomonads, Actinobacteria, alpha-Proteobacteria, Acidobacteria and nitrifying bacteria). Three applications of 10 mg kg(-1) of bromoxynil at 28-day intervals resulted in rapid degradation, the time for removal of 50% of the compound decreasing from 6.4 days on the first application to 4.9 days by the third. Bacterial population profiles showed significant similarity throughout the experiment. With the addition of 50 mg kg(-1) bromoxynil to soil, the degradation was preceded by a lag phase and the time for 50% of the compound to be degraded increased from 7 days to 28 days by the third application. The bacterial population showed significant differences 7 days after the final application of bromoxynil that correlated with an inhibition of degradation during the same period. CONCLUSIONS: These analyses highlighted that the addition of bromoxynil gave rise to significant shifts in the community diversity and its structure as measured by four abundant taxa, when compared with the control microcosm. These changes persisted even after bromoxynil had been degraded. SIGNIFICANCE AND IMPACT OF THE STUDY: Here we show that bromoxynil can exert an inhibitory effect on the bacterial population that results in decreased rates of degradation and increased persistence of the compound. In addition, we demonstrate that molecular approaches can identify statistically significant changes in microbial communities that occur in conjunction with changes in the rate of degradation of the compound in the soil.

The application of the herbicide bromoxynil to a model soil-derived bacterial community: impact on degradation and community structure.

AIMS: Bromoxynil degradation by soil micro-organisms has been shown to be co-oxidative in character. In this study, we investigate both the impact of the application of increasing bromoxynil concentrations on soil-derived bacterial communities and how these changes are reflected in the degradation of the compound. Our aim was to test the hypothesis that the addition of bromoxynil to a soil-derived bacterial community, and the availability of a readily utilizable carbon source would have an impact on bromoxynil degradation, and that would be reflected in the bacteria present in the soil community. METHODS AND RESULTS: Degradation of bromoxynil was observed in soil-derived communities containing 15 mg l(-1), but not 50 mg l(-1) of the compound, unless glucose was added. This suggests that the addition of carbon stimulates co-oxidative bromoxynil degradation by the members of the bacterial community. Measurable changes in the bacterial community indicated that the addition of bromoxynil led to deterministic selection on the bacterial population, i.e. the communities observed arise through the selection of specific micro-organisms that are best adapted to the conditions in the soil. The addition of bromoxynil was also shown to have a negative impact on the presence of alpha and gamma-proteobacteria in the soil community. CONCLUSION: Bromoxynil degradation is significantly inhibited in bacterial soil communities in the absence of readily accessible carbon. The application of bromoxynil appears to exert deterministic selection on the bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the effects of increasing bromoxynil concentrations on a model bacterial population derived from soil. Soil communities show qualitative and quantitative differences to bromoxynil application depending on the availability of organic carbon. These findings might have implications for the persistence of bromoxynil in agricultural soils.

The impact of bioaugmentation on metal cyanide degradation and soil bacteria community structure.

Metal cyanides are significant contaminants of many soils found at the site of former industrial activity. In this study we isolated bacteria capable of degrading ferric ferrocyanide and K2Ni(CN)4. One of these bacteria a Rhodococcus spp. was subsequently used to bioaugment a minimal medium broth, spiked with K2Ni(CN)4, containing 1 g of either an uncontaminated topsoil or a former coke works site soil. Degradation of the K2Ni(CN)4 was observed in both soils, however, bioaugmentation did not significantly impact the rate or degree of K2Ni(CN)4 removal. Statistical analysis of denaturing gradient gel electrophoresis profiles showed that the topsoil bacterial community had a higher biodiversity, and its structure was not significantly affected by either K2Ni(CN)4 or bioaugmentation. In contrast, profiles from the coke works site indicated significant changes in the bacterial community in response to these additions. Moreover, in both soils although bioaugmentation did not affect rates of biodegradation the Rhodococcus spp. did become established in the communities in broths containing both top and coke works soil. We conclude that bacterial communities from contaminated soils with low biodiversity are much more readily perturbed through interventions such as contamination events or bioaugmentation treatments and discuss the implications of these findings for bioremediation studies.

The impact of acrylonitrile and bioaugmentation on the biodegradation activity and bacterial community structure of a topsoil.

The analysis of the bacterial community within the soil using DGGE showed acrylonitrile (ACN) could lead to the selection of significantly similar communities. Moreover, Rhodococcus sp. AJ270 was successfully established in the soil community. High GC G+-bacteria also responded positively to ACN addition. Bioaugmentation or carbon addition had no impact on the rate or degree of ACN degradation. ACN could be readily degraded by the soil bacteria, however, the community structure was significantly affected by its addition as well as by the addition of carbon or Rhodococcus sp. AJ270. The bioaugmentation of the soil with this strain was successful, in that the organism became established within the community. ACN addition to a soil produces statistically significant changes in the bacterial community.

Potassium uptake and retention by Oceanomonas baumannii at low water activity in the presence of phenol.

Oceanomonas baumannii(T) (ATCC 700832) is a halotolerant bacterium capable of degrading phenol, which requires potassium in order for turgor growth to occur in minimal medium containing 5% NaCl (w/v). However, at this salinity growth can be inhibited by reduced potassium concentrations. The affinity for potassium (K(S)) was determined to be 219 microM and 408 microM for cultures utilising phenol and succinate respectively as the sole carbon source for growth. Rubidium but not caesium could substitute for potassium in alleviating growth inhibition due to potassium limitation. The effect of elevated phenol on potassium retention was studied, and it was shown that contrary to expectations, as external phenol concentration was increased the levels of intracellular potassium were significantly elevated. This observation correlated with changes in the cytoplasmic membrane, particularly the increase in the saturated:unsaturated fatty acid ratio from 0.47 to 1.44, and the decrease in the zwitterionic:anionic phospholipid ratio from 2.23 to 1.22. Both these changes promote membrane bilayer configurations and increase lipid ordering of the membrane reducing its permeability and inhibiting cation efflux.

Flavobacterium frigidarium sp. nov., an aerobic, psychrophilic, xylanolytic and laminarinolytic bacterium from Antarctica.

A psychrophilic, aerobic bacterium designated A2iT was isolated from marine sediment recovered from shallow waters surrounding Adelaide Island, Antarctica (67 degrees 34' S, 68 degrees 07' W). The organism exhibited xylanolytic and laminarinolytic activity and was halotolerant. Basic characterization showed that it was gram-negative, non-motile, yellow-pigmented (beta,beta-carotene-3,3'-diol) and positive for oxidase and catalase synthesis. Analysis of the 16S rDNA sequence suggests that the organism belongs to the Flexibacter-Cytophaga-Bacteroides phylum. On the basis of its 16S rDNA sequence, the bacterium is 96.8% similar to Flavobacterium columnare ATCC 43622--its closest relation. The genomic DNA G+C content was 35 mol%. Growth on xylan occurs optimally at 15 degrees C, though growth also occurs at 0 degrees C, and the doubling times are 9.6 and 34.8 h, respectively. The maximum growth temperature on xylan is at 24 degrees C. The bacterium is a neutrophile, growing across the pH range 5.6-8.4 and having an optimum at pH 7.5. Analysis of the 16S rDNA sequence, together with phenotypic characterization, suggests that the organism is a member of the genus Flavobacterium. DNA-DNA hybridization experiments have shown that it is a novel species; it is proposed, therefore, that the organism be designated as the type strain of Flavobacterium frigidarium sp. nov. (= ATCC 700810T = NCIMB 13737T).

Reclassification of [Pseudomonas] doudoroffii (Baumann et al. 1983) into the genus Oceanomonas gen. nov. as Oceanomonas doudoroffii comb. nov., and description of a phenol-degrading bacterium from estuarine water as Oceanomonas baumannii sp. nov.

A bacterium, isolate GB6T, capable of degrading phenol in the presence of elevated salinity was isolated from the estuary of the River Wear, UK. The bacterium was subjected to biochemical and molecular analysis to determine its taxonomic status. These studies indicated that the bacterium was a distinct species closely related to [Pseudomonas] doudoroffii. However, the phylogenetic analysis indicated that [Pseudomonas] doudoroffii was misclassified, as noted previously. Analysis of the characteristics of isolate GB6T and the type strain of [Pseudomonas] doudoroffii confirmed that these bacteria belonged to the same novel genus, which we have named Oceanomonas gen. nov. The type strain of Oceanomonas doudoroffii (Baumann et al. 1983) comb. nov. is ATCC 27123T (= DSM 7028T. The DNA-DNA homology between isolate GB6T and [Pseudomonas] doudoroffii is low and phenotypic differences between the two organisms are evident. Isolate GB6T (= ATCC 700832T = NCIMB 13685T) is therefore proposed as the type strain of a new species, Oceanomonas baumannii sp. nov.

The modification of the membrane of Oceanomonas baumannii when subjected to both osmotic and organic solvent stress.

Oceanomonas baumanniioff a novel halotolerant bacterium which was isolated from the estuary of the river Wear (Sunderland, UK). When grown in tryptone soya broth it can tolerate high levels of phenol, which is not utilised as a carbon source in this medium. However, the level of tolerance was reduced from 35 mM to 3 mM phenol as salinity increased from 1% to 12% NaCl (w/v). Increasing salinity up to 12% NaCl also decreased the growth rate 8-fold and caused modifications to the cytoplasmic membrane particularly anionic phosphatidylglycerol levels, which doubled at the expense of zwitterionic phosphatidylethanolamine. In addition, changes in the phospholipid fatty acid composition were noted, cis-vaccenic acid decreased significantly at higher salinities. Intracellular solute levels also increased with increasing salinity and there was an accumulation of the compatible solutes ectoine, glycine betaine and glutamate. The addition of phenol to osmotically compromised cultures led to a further modification of the cytoplasmic membrane phospholipid composition, in particular, that the decrease in zwitterionic phosphatidylethanolamine and the increase of anionic phospholipid species was much less pronounced. A further decrease in unsaturation, particularly in the proportion of cis-vaccenic acid, and the mean chain length of the fatty acids suggested that this response was important in maintaining membrane integrity in the presence of phenol.

Polymer hydrolysis in a cold climate.

In this review we discuss the activity of an ecologically significant group of psychrophilic bacteria, which are involved in the hydrolysis of plant cell wall polymers. Until now these organisms have been largely overlooked, despite the key role they play in releasing organic carbon fixed by primary producers in permanently cold environments such as Antarctica. This review details a specific group of plant cell wall polymer-degrading enzymes known as beta-glycanases. Studies on "cold" enzymes in general are in their infancy, but it has been shown that many exhibit structural and functional modifications that enable them to function at low temperature. beta-Glycanases in particular are intriguing because their substrates (cellulose and xylan) are very refractile, which may indicate that their "cold" modifications are pronounced. In addition, mesophilic beta-glycanases have been extensively studied and the current state of our knowledge is reviewed. This body of information can be exploited to enable meaningful comparative studies between mesophilic and psychrophilic beta-glycanases. The aim of such investigations is to obtain a deeper insight into those structural and functional modifications that enable these enzymes to function at low temperature and to examine the evolutionary relationship between mesophilic and psychrophilic beta-glycanases.

Osmoregulatory responses of bacteria isolated from fresh or composted, olive-mill waste-waters.

Halotolerant bacteria isolated from raw, olive-mill waste-waters (alpechin) and from composted alpechin could grow on solid medium containing up to 10% (w/v) NaCl. Most (70%) of these halotolerant isolates could also grow in liquid minimal medium with the same NaCl concentration and three isolates from this group were chosen for further study. When grown in tryptic soy broth (TSB), two isolates responded to lowering of water activity (a w) by addition of NaCl or sucrose in the expected manner: by increasing the proportion of membrane anionic lipids diphosphatidylglycerol or phosphatidylglycerol. Some solute-specific differences were observed. In contrast, the third isolate did not alter its membrane phospholipid composition significantly in response to growth in NaCl, whereas in sucrose there was an increase in phosphatidylethanolamine. This response is contrary to the accepted interpretation of the function of such a w-dependent changes, as being a mechanism for preserving the membrane lipid-bilayer phase. When all three isolates were grown in the presence of alpechin, there was a decrease in the proportion of phosphatidylglycerol and a rise in the level of phosphatidylethanolamine. Quantitative and qualitative differences in compatible solute composition were observed when the three isolates were grown in TSB with NaCl or sucrose added to lower a w. The major compatible solutes in two of the isolates were proline and betaine, whereas in the third they were proline, betaine and ectoine; one isolate also contained some trehalose when NaCl but not sucrose was the osmolyte.

Successful treatment of postoperative chylothorax using an external pleuroperitoneal shunt.

We report 3 patients with chylothorax who were successfully managed as outpatients using external pleuroperitoneal shunts. This external shunt has the advantage over subcutaneously placed shunts of pumping large volumes of fluid with each compression of the pumping chamber, of not causing the discomfort associated with pumping a subcutaneous chamber, of not becoming difficult to find in the subcutaneous space, and of being constructed of larger components which do not kink or become easily clogged with fibrinous debris.