Truncated glucagon-like peptide I, an insulin-releasing hormone from the distal gut

AbstractBy hydrophobic gel permeation and high pressure liquid chromatography we isolated from pig intestinal mucosa a peptide which corresponds to proglucagon 78–107 as suggested by chromatography and determination of its N-terminal sequence. Natural and synthetic proglucagon 78–107 dose dependently and potently increased insulin secretion from the isolated perfused pig pancreas. Proglucagon 78–107 also secreted by the small intestine may participate in the hormonal control of insulin secretion

The molecular characterisation of Escherichia coli K1 isolated from neonatal nasogastric feeding tubes

Background: The most common cause of Gram-negative bacterial neonatal meningitis is E. coli K1. It has a mortality rate of 10–15%, and neurological sequelae in 30– 50% of cases. Infections can be attributable to nosocomial sources, however the pre-colonisation of enteral feeding tubes has not been considered as a specific risk factor. Methods: Thirty E. coli strains, which had been isolated in an earlier study, from the residual lumen liquid and biofilms of neonatal nasogastric feeding tubes were genotyped using pulsed-field gel electrophoresis, and 7-loci multilocus sequence typing. Potential pathogenicity and biofilm associated traits were determined using specific PCR probes, genome analysis, and in vitro tissue culture assays. Results: The E. coli strains clustered into five pulsotypes, which were genotyped as sequence types (ST) 95, 73, 127, 394 and 2076 (Achman scheme). The extra-intestinal pathogenic E. coli (ExPEC) phylogenetic group B2 ST95 serotype O1:K1:NM strains had been isolated over a 2 week period from 11 neonates who were on different feeding regimes. The E. coli K1 ST95 strains encoded for various virulence traits associated with neonatal meningitis and extracellular matrix formation. These strains attached and invaded intestinal, and both human and rat brain cell lines, and persisted for 48 h in U937 macrophages. E. coli STs 73, 394 and 2076 also persisted in macrophages and invaded Caco-2 and human brain cells, but only ST394 invaded rat brain cells. E. coli ST127 was notable as it did not invade any cell lines. Conclusions: Routes by which E. coli K1 can be disseminated within a neonatal intensive care unit are uncertain, however the colonisation of neonatal enteral feeding tubes may be one reservoir source which could constitute a serious health risk to neonates following ingestion

PCR-Based Identification of Klebsiella pneumoniae subsp. rhinoscleromatis, the Agent of Rhinoscleroma

Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma

Assessing the accuracy of current near infra-red reflectance spectroscopy analysis for fresh grass-clover mixture silages and development of new equations for this purpose

The purpose of this study was to ascertain whether Near Infra-Red Reflectance Spectroscopy (NIRS) prediction equations calibrated on grass silage samples, could accurately predict the chemical composition of mixed grass-clover silage samples, and furthermore, to develop and calibrate new grass-clover equations should the grass-based equations be insufficiently accurate for these silages. A set of 94 silage samples from mixed grass-clover swards (clover concentration (CC) ranging from 4 to 1000 g/kg as fed; determined manually) were analysed for chemical composition using reference laboratory techniques, in vivo digestible organic matter in the dry matter (DOMD, in sheep), and in situ degradability of dry matter and crude protein (in cows). The same samples were scanned fresh (undried and unmilled, as is standard practice for silage analysis within UK laboratories) using NIRS (at AFBI, Northern Ireland) and grass-based prediction equations applied. Predicted and observed results were compared. Of 15 chemical components that were tested for prediction accuracy, only volatile-corrected dry matter and nitrogen were well predicted (RPD values of 4.9 and 2.4 respectively, with low root mean square errors of prediction (RMSEP)). Neutral detergent fibre and DOMD showed low RPD values, however the predicted and observed datasets had no significant bias between them and were therefore also considered as fit for purpose. Variables with significant bias between predicted and observed datasets that were not considered suitably accurate included crude protein, acid detergent fibre, microbial dry matter yield and the effective degradability of protein. For many components, bias could be attributed at least in part to CC and changes in the fractionation of nutrients present. For some variables such as crude protein, grass-based equations were sufficiently accurate at low CCs but became inaccurate as CC increased, as expected. In response to inadequate prediction accuracy of certain nutrients, new grass-clover equations were calibrated using the obtained spectra. These were validated and results indicated that the grass-clover-based equations outperformed their grass-based counterparts. The adoption of new grass-clover equations, or alternatively, with further development, the use of a CC correction factor to the existing grass-based equations, is recommended for commercial laboratories offering undried and unmilled silage analysis on samples containing clover

Heat Resistance Mediated by a New Plasmid Encoded Clp ATPase, ClpK, as a Possible Novel Mechanism for Nosocomial Persistence of Klebsiella pneumoniae

Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably thermotolerant providing a conceivable explanation of its persistence in the hospital environment. This marked phenotype is mediated by a novel type of Clp ATPase, designated ClpK. The clpK gene is encoded by a conjugative plasmid and we find that the clpK gene alone renders an otherwise sensitive E. coli strain resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections

Occurrence of L-iduronic acid and putative D-glucuronyl C5-epimerases in prokaryotes

Glycosaminoglycans (GAGs) are polysaccharides that are typically present in a wide diversity of animal tissue. Most common GAGs are well-characterized and pharmaceutical applications exist for many of these compounds, e.g. heparin and hyaluronan. In addition, also bacterial glycosaminoglycan-like structures exist. Some of these bacterial GAGs have been characterized, but until now no bacterial GAG has been found that possesses the modifications that are characteristic for many of the animal GAGs such as sulfation and C5-epimerization. Nevertheless, the latter conversion may also occur in bacterial and archaeal GAGs, as some prokaryotic polysaccharides have been demonstrated to contain L-iduronic acid. However, experimental evidence for the enzymatic synthesis of L-iduronic acid in prokaryotes is as yet lacking. We therefore performed an in silico screen for D-glucuronyl C5-epimerases in prokaryotes. Multiple candidate C5-epimerases were found, suggesting that many more microorganisms are likely to exist possessing an L-iduronic acid residue as constituent of their cell wall polysaccharides

Molecular Characterization of a 21.4 Kilobase Antibiotic Resistance Plasmid from an α-Hemolytic Escherichia coli O108:H- Human Clinical Isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes