The minimum Se requirement for male turkey poults is 0.3 μg Se/g--three times higher than requirements found in rodents--based on liver and gizzard glutathione peroxidase-4 (GPX4) and GPX1 ...activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only "predicted." Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA) codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.
Abstract
Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance ...(AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436
Escherichia coli
were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype–phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.
Objectives: To investigate the distribution of, associations between and the transferability of antimicrobial resistance genes in resistant Escherichia coli strains isolated from Norwegian meat and ...meat products. Methods: The 241 strains investigated were collected within the frame of the Norwegian monitoring programme for antimicrobial resistance in bacteria from feed, food and animals (NORM-VET) during the years 2000–2003. PCR was carried out for detection of resistance genes. Conjugation experiments were carried out with the resistant isolates from meat as donor strains and E. coli DH5α as the recipient strain. Statistical analyses were performed with the SAS-PC-System version 9.1 for Windows. Results: Resistance genes common in pathogenic E. coli were frequently found among the isolates investigated. Strains harbouring several genes encoding resistance to the same antimicrobial agent were significantly (P < 0.0001) more frequently multiresistant than others. Strong positive associations were found between the tet(A) determinant and the genetic elements sul1, dfrA1 and aadA1. Negative associations were found between resistance genes encoding resistance to the same antimicrobial agent: tet(A)/tet(B), sul1/sul2 and strA-strB/aadA1. The resistance genes were successfully transferred from 38% of the isolates. The transfer was more frequent from resistant isolates harbouring class 1 integrons (P < 0.001). Conclusions: Acquired resistance played a major role in conferring resistance among the isolates investigated. The possibility of transferring resistance increases both by increased multiresistance and by the presence of class 1 integrons. The conjugation experiments suggest that tet(A) and class 1 integrons are often located on the same conjugative plasmid.
Background
The use of biological therapy (biologicals) is established in the treatment of Crohn's disease. This study aimed to determine whether preoperative treatment with biologicals is associated ...with an increased rate of complications following surgery for Crohn's disease with intestinal anastomosis.
Methods
All patients receiving biologicals and undergoing abdominal surgery with anastomosis or strictureplasty were identified at six tertiary referral centres. Demographic data, and preoperative, operative and postoperative details were registered. Patients who were treated with biologicals within 2 months before surgery were compared with a control group who were not. Postoperative complications were classified according to anastomotic, infectious or other complications, and graded according to the Clavien–Dindo classification.
Results
Some 111 patients treated with biologicals within 2 months before surgery were compared with 187 patients in the control group. The groups were well matched. There were no differences between the treatment and control groups in the rate of complications of any type (34·2 versus 28·9 per cent respectively; P = 0·402), anastomotic complications (7·2 versus 8·0 per cent; P = 0·976) and non‐anastomotic infectious complications (16·2 versus 13·9 per cent; P = 0·586). In univariable regression analysis, biologicals were not associated with an increased risk of any complication (odds ratio (OR) 1·33, 95 per cent confidence interval 0·81 to 2·20), anastomotic complication (OR 0·89, 0·37 to 2·17) or infectious complication (OR 1·09, 0·62 to 1·91).
Conclusion
Treatment with biologicals within 2 months of surgery for Crohn's disease with intestinal anastomosis was not associated with an increased risk of complications.
Did not increase complications
Class I hydrophobins are a unique family of fungal proteins that form a polymeric, water-repellent monolayer on the surface of structures such as spores and fruiting bodies. Similar monolayers are ...being discovered on an increasing range of important microorganisms. Hydrophobin monolayers are amphipathic and particularly robust, and they reverse the wettability of the surface on which they are formed. There are also significant similarities between these polymers and amyloid-like fibrils. However, structural information on these proteins and the rodlets they form has been elusive. Here, we describe the three-dimensional structure of the monomeric form of the class I hydrophobin EAS. EAS forms a β-barrel structure punctuated by several disordered regions and displays a complete segregation of charged and hydrophobic residues on its surface. This structure is consistent with its ability to form an amphipathic polymer. By using this structure, together with data from mutagenesis and previous biophysical studies, we have been able to propose a model for the polymeric rodlet structure adopted by these proteins. X-ray fiber diffraction data from EAS rodlets are consistent with our model. Our data provide molecular insight into the nature of hydrophobin rodlet films and extend our understanding of the fibrillar β-structures continue to be discovered in the protein world.
Objectives
The aim of this study was to investigate the clonal epidemiology of methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates from dogs in Norway and to evaluate the ...antimicrobial resistance patterns and determine the biofilm-forming abilities of the isolates.
Methods
All MRSP index isolates from each MRSP-positive dog detected in Norway until June 2011 were included (n = 23). The MICs of antimicrobial agents were determined by the VetMIC™ microdilution method. The genetic relationship between the isolates was investigated by multilocus sequence typing (MLST) and PFGE. The isolates' abilities to form biofilm on polystyrene were studied.
Results
The MRSP isolates investigated grouped into 11 different sequence types (STs); MRSP ST106 occurred most frequently. There were a relatively smaller number of isolates belonging to ST71, the largely predominant ST in Europe. Isolates belonging to ST71 had a significantly greater ability to produce biofilm compared with the other isolates, and especially compared with MRSP ST106.
Conclusions
A heterogeneous clonal distribution was observed among MRSP from dogs in Norway. As opposed to previous findings in Europe, MRSP clones other than ST71 have spread in Norway, such as MRSP ST106. The results also show that MRSP ST71 is possibly a good biofilm producer, and this may in turn be a contributing factor to the nosocomial character of MRSP ST71.
In 2012 and 2014 the Norwegian monitoring programme for antimicrobial resistance in the veterinary and food production sectors (NORM-VET) showed that 124 of a total of 406 samples (31%) of Norwegian ...retail chicken meat were contaminated with extended-spectrum cephalosporin-resistant Escherichia coli. The aim of this study was to compare selected cephalosporin-resistant E. coli from humans and poultry to determine their genetic relatedness based on whole genome sequencing (WGS).
Escherichia coli representing three prevalent cephalosporin-resistant multi-locus sequence types (STs) isolated from poultry (n=17) were selected from the NORM-VET strain collections. All strains carried an IncK plasmid with a blaCMY-2 gene. Clinical E. coli isolates (n=284) with AmpC-mediated resistance were collected at Norwegian microbiology laboratories from 2010 to 2014. PCR screening showed that 29 of the clinical isolates harboured both IncK and blaCMY-2. All IncK/blaCMY-2-positive isolates were analysed with WGS-based bioinformatics tools.
Analysis of single nucleotide polymorphisms (SNP) in 2.5 Mbp of shared genome sequences showed close relationship, with fewer than 15 SNP differences between five clinical isolates from urinary tract infections (UTIs) and the ST38 isolates from poultry. Furthermore, all of the 29 clinical isolates harboured IncK/blaCMY-2 plasmid variants highly similar to the IncK/blaCMY-2 plasmid present in the poultry isolates.
Our results provide support for the hypothesis that clonal transfer of cephalosporin-resistant E. coli from chicken meat to humans may occur, and may cause difficult-to-treat infections. Furthermore, these E. coli can be a source of AmpC-resistance plasmids for opportunistic pathogens in the human microbiota.
Purpose
Variability in functional outcome after ileal pouch-anal anastomosis (IPAA) is to a large extent unexplained. The aim of this study was to use MRI to evaluate the morphology, emptying pattern ...and other pathology that may explain differences in functional outcome between well-functioning and poorly functioning pouch patients. A secondary aim was to establish a reference of normal MRI findings in pelvic pouch patients.
Methods
From a previous study, the best and worst functioning patients undergoing IPAA surgery between 2000 and 2013 had been identified and examined with manovolumetric tests (
N
= 47). The patients were invited to do a pelvic MRI investigating pouch morphology and emptying patterns, followed by a pouch endoscopy.
Results
Forty-three patients underwent MRI examination. We found no significant morphological or dynamic differences between the well-functioning and poorly functioning pouch patients. There was no correlation between urge volume and the volume of the bony pelvis, and no correlation between emptying difficulties or leakage and dynamic MRI findings. Morphological MRI signs of inflammation were present in the majority of patients and were not correlated to histological signs of inflammation. Of the radiological signs of inflammation, only pouch wall thickness correlated to endoscopic pouchitis disease activity index scores.
Conclusion
It seems MRI does not increase the understanding of factors contributing to functional outcome after ileal pouch-anal anastomosis. Unless there is a clinical suspicion of perianal/peripouch disease or pelvic sepsis, MRI does not add value as a diagnostic tool for pelvic pouch patients. Endoscopy remains the golden standard for diagnosing pouch inflammation.
Until recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated β-lactam resistance in Staphylococcus ...pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius.
We included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-μg cefoxitin and 1-μg oxacillin discs from three manufacturers and Mueller–Hinton agar from two manufacturers.
Cefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6–20 mm and 19–30 mm. For cefoxitin 16% (95% CI 14.8–18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6–2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively.
This investigation confirms that the 1-μg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-μg cefoxitin disc. For a 1-μg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all β-lactam antibiotics except those with activity against methicillin-resistant staphylococci.
To develop a comprehensive mutation analysis system with a high rate of detection, to develop a tool to predict the chance of detecting a mutation in the L1CAM gene, and to look for ...genotype-phenotype correlations in the X-linked recessive disorder, L1 syndrome.
DNA from 367 referred patients was analysed for mutations in the coding sequences of the gene. A subgroup of 100 patients was also investigated for mutations in regulatory sequences and for large duplications. Clinical data for 106 patients were collected and used for statistical analysis.
68 different mutations were detected in 73 patients. In patients with three or more clinical characteristics of L1 syndrome, the mutation detection rate was 66% compared with 16% in patients with fewer characteristics. The detection rate was 51% in families with more than one affected relative, and 18% in families with one affected male. A combination of these two factors resulted in an 85% detection rate (OR 10.4, 95% CI 3.6 to 30.1). The type of mutation affects the severity of L1 syndrome. Children with a truncating mutation were more likely to die before the age of 3 than those with a missense mutation (52% vs 8%; p=0.02).
We developed a comprehensive mutation detection system with a detection rate of almost 20% in unselected patients and up to 85% in a selected group. Using the patients' clinical characteristics and family history, clinicians can accurately predict the chance of finding a mutation. A genotype-phenotype correlation was confirmed. The occurrence of (maternal) germline mosaicism was proven.
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