Additional modifications to the Blumgart pancreaticojejunostomy: Results of a propensity score-matched analysis versus Cattel-Warren pancreaticojejunostomy

Background: Postoperative pancreatic fistula continues to occur frequently after pancreatoduodenectomy. Methods: We have described a modification of the Blumgart pancreaticojejunostomy. The modification of the Blumgart pancreaticojejunostomy was compared to the Cattel-Warren pancreaticojejunostomy in cohorts of patients matched by propensity scores based on factors predictive of clinically relevant postoperative pancreatic fistula, which was the primary endpoint of this study. Based on a noninferiority study design, 95 open pancreatoduodenectomies per group were needed. Feasibility of the modification of the Blumgart pancreaticojejunostomy in robotic pancreatoduodenectomy was also shown. All pancreaticojejunostomies were performed by a single surgeon. Results: Between October 2011 and May 2019, there were 415 pancreatoduodenectomies with either a Cattel-Warren pancreaticojejunostomy (n = 225) or a modification of the Blumgart pancreaticojejunostomy (n = 190). There was 1 grade C postoperative pancreatic fistula in 190 consecutive modification of the Blumgart pancreaticojejunostomies (0.5%). Logistic regression analysis showed that the rate of clinically relevant postoperative pancreatic fistula was not affected by consecutive case number. After exclusion of robotic pancreatoduodenectomies (the Cattel-Warren pancreaticojejunostomy: 82; modification of the Blumgart pancreaticojejunostomy: 66), 267 open pancreatoduodenectomies were left, among which the matching process identified 109 pairs. The modification of the Blumgart pancreaticojejunostomy was shown to be noninferior to the Cattel-Warren pancreaticojejunostomy with respect to clinically relevant postoperative pancreatic fistula (11.9% vs 22.9%; odds ratio: 0.46 [0.21–0.93]; P = .03), grade B postoperative pancreatic fistula (11.9% vs 18.3%; P = .18), and grade C postoperative pancreatic fistula (0 vs 4.6%; P = .05) as well as to all secondary study endpoints. The modification of the Blumgart pancreaticojejunostomy was feasible in 66 robotic pancreatoduodenectomies. In this subgroup with 1 conversion to open surgery (1.5%), a clinically relevant postoperative pancreatic fistula occurred after 9 procedures (13.6%) with no case of grade C postoperative pancreatic fistula and a 90-day mortality of 3%. Conclusion: The modification of the Blumgart pancreaticojejunostomy described herein is noninferior to the Cattel-Warren pancreaticojejunostomy in open pancreatoduodenectomy. This technique is also feasible in robotic pancreatoduodenectomy

Sortase A Substrate Specificity in GBS Pilus 2a Cell Wall Anchoring

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtAΔN40) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtAΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus

Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis

The Streptococcus pneumoniae Pilus-1 Displays a Biphasic Expression Pattern

The Streptococcus pneumoniae pilus-1 is encoded by pilus islet 1 (PI-1), which has three clonal variants (clade I, II and III) and is present in about 30% of clinical pneumococcal isolates. In vitro and in vivo assays have demonstrated that pilus-1 is involved in attachment to epithelial cells and virulence, as well as protection in mouse models of infection. Several reports suggest that pilus-1 expression is tightly regulated and involves the interplay of numerous genetic regulators, including the PI-1 positive regulator RlrA. In this report we provide evidence that pilus expression, when analyzed at the single-cell level in PI-1 positive strains, is biphasic. In fact, the strains present two phenotypically different sub-populations of bacteria, one that expresses the pilus, while the other does not. The proportions of these two phenotypes are variable among the strains tested and are not influenced by genotype, serotype, growth conditions, colony morphology or by the presence of antibodies directed toward the pilus components. Two sub-populations, enriched in pilus expressing or not expressing bacteria were obtained by means of colony selection and immuno-detection methods for five strains. PI-1 sequencing in the two sub-populations revealed the absence of mutations, thus indicating that the biphasic expression observed is not due to a genetic modification within PI-1. Microarray expression profile and western blot analyses on whole bacterial lysates performed comparing the two enriched sub-populations, revealed that pilus expression is regulated at the transcriptional level (on/off regulation), and that there are no other genes, in addition to those encoded by PI-1, concurrently regulated across the strains tested. Finally, we provide evidence that the over-expression of the RrlA positive regulator is sufficient to induce pilus expression in pilus-1 negative bacteria. Overall, the data presented here suggest that the observed biphasic pilus expression phenotype could be an example of bistability in pneumococcus

Supramolecular Organization of the Repetitive Backbone Unit of the Streptococcus pneumoniae Pilus

Streptococcus pneumoniae, like many other Gram-positive bacteria, assembles long filamentous pili on their surface through which they adhere to host cells. Pneumococcal pili are formed by a backbone, consisting of the repetition of the major component RrgB, and two accessory proteins (RrgA and RrgC). Here we reconstruct by transmission electron microscopy and single particle image reconstruction method the three dimensional arrangement of two neighbouring RrgB molecules, which represent the minimal repetitive structural domain of the native pilus. The crystal structure of the D2-D4 domains of RrgB was solved at 1.6 Å resolution. Rigid-body fitting of the X-ray coordinates into the electron density map enabled us to define the arrangement of the backbone subunits into the S. pneumoniae native pilus. The quantitative fitting provide evidence that the pneumococcal pilus consists uniquely of RrgB monomers assembled in a head-to-tail organization. The presence of short intra-subunit linker regions connecting neighbouring domains provides the molecular basis for the intrinsic pilus flexibility

Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceabilit

Significance of ante mortem indicators on turkey meat inspection

The significance of parameters concerning the "transport" (loading, transport, unloading) was analysed as turkey meat inspection tools, in particular "biomass/cage". "birds/cage", "length of transport". The study, carried out on a considerable number of turkeys (81,449 males and 152,705 females) within the period of a whole year, has showed that stressing transport, as marked by some inspection indicators ("dead on arrival", "whole carcasses post mortem condemnation", "birds with traumatic pathology"), does not only affect product losses, but also both slaughtering hygiene and meat quality