The effect of goal-directed crystalloid versus colloid administration on postoperative spirometry parameters: a substudy of a randomized controlled clinical trial.

BACKGROUND: Pulmonary function is impaired after major abdominal surgery and might be less impaired by restrictive fluid administration. Under the assumption of a fluid-sparing effect of colloids, we tested the hypothesis that an intraoperative colloid-based goal-directed fluid management strategy impairs postoperative pulmonary function parameters less compared to goal-directed crystalloid administration. METHODS: We performed a preplanned, single-center substudy within a recently published trial evaluating the effect of goal-directed crystalloids versus colloids on a composite of major complications. Sixty patients undergoing major open abdominal surgery were randomized to Doppler-guided intraoperative fluid replacement therapy with lactated Ringer's solution (n = 31) or unbalanced 6% hydroxyethyl starch 130/0.4 (n = 29). A blinded investigator performed bedside spirometry (Spirobank-G, Medical International Research, Rome, Italy) preoperatively as well as 6, 24, and 48 h postoperatively. RESULTS: Median total intraoperative fluid requirements were significantly higher during crystalloid administration compared to patients receiving colloids (4567 ml vs. 3044 ml, p = 0.01). Six hours after surgery, pulmonary function parameters did not differ significantly between the crystalloid - and the colloid group: forced vital capacity (FVC): 1.6 l (1.2-2 l) vs. 1.9 l (1.5-2.4 l), p = 0.15; forced expiratory volume in 1 second (FEV1): 1.1 l (0.9-1.6 l) vs. 1.4 l (1.2-1.7 l), p = 0.18; and peak expiratory flow (PEF): 2 l.sec-1 (1.5 - 3.6 l.sec -1) vs. 2.3 l.sec -1 (1.8 - 3.4 l.sec -1), p = 0.23. Moreover, postoperative longitudinal time × group interactions of FVC, FEV1, and PEF between 6 and 48 postoperative hours did not differ significantly. CONCLUSION: Postoperative pulmonary function parameters were similarly impaired in patients receiving goal-directed crystalloid administration as compared to goal-directed colloid administration during open abdominal surgery. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT00517127 , registered on August 16, 2007) and EudraCT (2005-004602-86).

Sex differences in pediatric caudal epidural anesthesia under sedation without primary airway instrumentation.

STUDY OBJECTIVE: To identify sex differences associated with caudal epidurals, the most commonly used technique of pediatric regional anesthesia, based on individually validated data of ultrasound-guided blocks performed between 04/2014 and 12/2020. METHODS: Prospectively collected and individually validated data of a cohort of children aged between 0-15 years was analyzed in a retrospective observational study. We included pediatric surgeries involving a primary plan of caudal epidural anesthesia under sedation (without airway instrumentation) and a contingency plan of general anesthesia. Sex-specific rates were analyzed for overall failure of the primary anesthesia plan, for residual pain, for block-related technical complications and for critical respiratory events. We used Fisher´s exact tests and multivariable logistic regressions were used to evaluate sex-specific associations. RESULTS: Data from 487 girls and 2060 boys ≤15 years old (ASA status 1 to 4) were analyzed. The primary-anesthesia-plan failure rate was 5.5% (95%CI 3.8%-7.8%) (N = 27/487) among girls and 4.7% (95%CI 3.9%-5.7%) (N = 97/2060) among boys (p = 0.41). Residual pain was the main cause of failure, with rates of 4.5% (95%CI 2.9-6.6%) (N = 22/487) among girls and 3.0% (95%CI 2.3-3.8%) (N = 61/2060) among boys (p = 0.089). Block-related technical complications were seen at rates of 0.8% (95%CI 0.3%-1.9%) (N = 4/487) among girls vs 2.5% (95%CI 0.5-2.7%) (N = 51/2060) among boys and, hence, significantly more often among male patients (p = 0.023). Male sex was significantly associated with higher odds (adjusted OR: 3.18; 95% CI: 1.12-9; p = 0.029) for such technical complications regardless of age, ASA status, gestational week at birth or puncture attempts. Critical respiratory events occurred at a 1.7% (95%CI 1.2%-2.3%) rate (N = 35/2060) twice as high among boys as 0.8% (95%CI 0.3%-1.9%) (N = 4/487) among girls (p = 0.21). CONCLUSIONS: While the the primary-anesthesia-plan failure rate was equal for girls and boys, technical complications and respiratory events are more likely to occur in boys.

Isolation, Characterization, Genome Analysis and Host Resistance Development of Two Novel Lastavirus Phages Active against Pandrug-Resistant Klebsiella pneumoniae.

Klebsiella pneumoniae is a global health threat and bacteriophages are a potential solution in combating pandrug-resistant K. pneumoniae infections. Two lytic phages, LASTA and SJM3, active against several pandrug-resistant, nosocomial strains of K. pneumoniae were isolated and characterized. Their host range is narrow and latent period is particularly long; however, their lysogenic nature was refuted using both bioinformatic and experimental approaches. Genome sequence analysis clustered them with only two other phages into the new genus Lastavirus. Genomes of LASTA and SJM3 differ in only 13 base pairs, mainly located in tail fiber genes. Individual phages, as well as their cocktail, demonstrated significant bacterial reduction capacity in a time-dependent manner, yielding up to 4 log reduction against planktonic, and up to 2.59 log on biofilm-embedded, cells. Bacteria emerging from the contact with the phages developed resistance and achieved numbers comparable to the growth control after 24 h. The resistance to the phage seems to be of a transient nature and varies significantly between the two phages, as resistance to LASTA remained constant while resensitization to SJM3 was more prominent. Albeit with very few differences, SJM3 performed better than LASTA overall; however, more investigation is needed in order to consider them for therapeutic application.

Redesigned pMAL expression vector for easy and fast purification of active native antimicrobial peptides.

AIMS: The aim of this study was to construct the improved pMAL expression vector to increase the efficacy of purification of small native peptides and their clear-cut separation from MBP tag. The modifications we introduced can be applied to many expression vectors. METHODS AND RESULTS: To improve the pMAL expression vector, we introduced the His6 tag and the enterokinase cleavage site (Ek) downstream from the MBP tag and Xa cleavage site on the original vector. For cloning of a desired peptide DNA, the enterokinase site contains a unique BsaBI restriction site adjacent to the original multi-cloning site. This redesigned pMAL vector was optimized for the purification of cytoplasmic (pMALc5HisEk) and periplasmic (pMALp5HisEk) peptides. The purification of native and active peptide (P) was obtained following two-step affinity chromatography. In the first step, the entire MBP-His6 -Ek-P fusion protein is purified using the Ni-NTA agarose column. This fusion protein was cleaved with active His6 tagged enterokinase. In the second step, the further purification was performed by column containing the mixture of amylose and Ni-NTA agarose resins. This removes both the MBP-His6 and His6 -enterokinase leaving pure native protein in solution. These new vectors and the two-step purification protocol were successfully applied in purification of active native small antimicrobial peptides (AMPs), lactococcin A and human ß-defensin. CONCLUSIONS: We constructed the improved pMAL expression vectors and established the pipeline and optimal conditions for their use in efficient purification of large amounts of active native small peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of expression vector impacts on the efficiency of expression and purification of desired proteins. The idea of redesigning pMAL vector was driven by the need for rapid purification of larger amounts of active native AMPs. This newly improved pMAL vector, the cloning strategy, expression conditions and two-step purification protocol represent a unique simple approach which can be applied in every laboratory.

C-protein α-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae.

Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein α-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein α-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.

Isolation, Characterization and Draft Genome Analysis of Bacteriophages Infecting Acidovorax citrulli.

Bacterial fruit blotch and seedling blight, caused by Acidovorax citrulli, is one of the most destructive diseases of melon and watermelon in many countries. Pathogen-free seed and cultural practices are major pillars of the disease control. However, use of bacteriophages as natural biocontrol agents might also contribute to the disease management. Therefore, we isolated 12 bacteriophages specific to A. citrulli, from phyllosphere and rhizosphere of diseased watermelon plants. The phage strains were characterized based on their host range, plaque and virion morphology, thermal inactivation point, adsorption rate, one step growth curve, restriction fragment length polymorphism (RFLP), and genomic analysis. Transmission electron microscopy of three phage strains indicated that they belong to the order Caudovirales, family Siphoviridae. All phages lysed 30 out of 32 tested A. citrulli strains isolated in Serbia, and did not lyse other less related bacterial species. They produced clear plaques, 2 mm in diameter, on bacterial lawns of different A. citrulli strains after 24 h of incubation. The thermal inactivation point was 66 or 67°C. They were stable at pH 5-9, but were sensitive to chloroform and inactivated in either 5 or 10 min exposure to ultraviolet (UV) light. RFLP analysis using EcoRI, BsmI and BamHI enzymes did not show genetic differences among the tested phages. Adsorption rate and one step growth curve were determined for the Acidovorax phage ACF1. Draft genome sequence of the ACF1 phage was 59.377 bp in size, with guanine-cytosine (GC) content 64.5%, including 89 open reading frames. This phage shared a very high genomic identity with Acidovorax phage ACPWH, isolated in South Korea. Evaluation of systemic nature of ACF1 strain showed that it can be absorbed by roots and translocated to upper parts of watermelon plants where it survived up to 10 days.

Characterization, Antibiofilm, and Depolymerizing Activity of Two Phages Active on Carbapenem-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a leading cause of healthcare-associated infections worldwide. Its various intrinsic and acquired mechanisms of antibiotic resistance make the therapeutic challenge even more serious. One of the promising alternative treatments that is increasingly highlighted is phage therapy, the therapeutic use of bacteriophages to treat bacterial infections. Two phages active against nosocomial carbapenem-resistant A. baumannii strain 6077/12, vB_AbaM_ISTD, and vB_AbaM_NOVI, were isolated from Belgrade wastewaters, purified, and concentrated using CsCl gradient ultracentrifugation. The phages were screened against 103 clinical isolates of A. baumannii from a laboratory collection and characterized based on plaque and virion morphology, host range, adsorption rate, and one-step growth curve. Given that phage ISTD showed a broader host range, better adsorption rate, shorter latent period, and larger burst size, its ability to lyse planktonic and biofilm-embedded cells was tested in detail. Phage ISTD yielded a 3.5- and 2-log reduction in planktonic and biofilm-associated viable bacterial cell count, respectively, but the effect was time-dependent. Both phages produced growing turbid halos around plaques indicating the synthesis of depolymerases, enzymes capable of degrading bacterial exopolysaccharides. Halos tested positive for presence of phages in the proximity of the plaque, but not further from the plaque, which indicates that the observed halo enlargement is a consequence of enzyme diffusion through the agar, independently of the phages. This notion was also supported by the growing halos induced by phage preparations applied on pregrown bacterial lawns, indicating that depolymerizing effect was achieved also on non-dividing sensitive cells. Overall, good rates of growth, fast adsorption rate, broad host range, and high depolymerizing activity, as well as antibacterial effectiveness against planktonic and biofilm-associated bacteria, make these phages good candidates for potential application in combating A. baumannii infections.

The effect of intraoperative goal-directed crystalloid versus colloid administration on perioperative inflammatory markers - a substudy of a randomized controlled trial.

BACKGROUND: Excessive perioperative fluid administration may result in iatrogenic endothelial dysfunction and tissue edema, transducing inflammatory markers into the bloodstream. Colloids remain longer in the circulation, requiring less volume to reach similar hemodynamic endpoints compared to crystalloids. Thus, we tested the hypothesis that a goal-directed colloid regimen attenuates the inflammatory response compared to a goal-directed crystalloid regime. METHODS: Patients undergoing moderate- to high-risk open abdominal surgery were randomly assigned to goal-directed lactated Ringer's solution (n = 58) or a hydroxyethyl starch 6% 130/0.4 (n = 62) fluid regimen. Our primary outcome was perioperative levels of pro- and anti-inflammatory cytokines. Secondary outcome was perioperative levels of white blood cell count (WBC), C-reactive protein (CRP), procalcitonin (PCT) and lipopolysaccharide-binding protein (LBP). Measurements were performed preoperatively, immediate postoperatively, on postoperative day one, two and four. RESULTS: The areas under the curve of Interleukin (IL) 6 (p = 0.60), IL 8 (p = 0.46), IL 10 (p = 0.68) and tumor necrosis factor α (p = 0.47) levels did not differ significantly between the groups. WBC, CRP and PCT values were also comparable. LBP, although significantly higher in the crystalloid group, remained in the normal range. Patients assigned to crystalloids received a median (IQR) amount of 3905 mL (2880-5288) of crystalloid. Patients assigned to colloids received 1557 mL (1207-2116) of crystalloid and 1250 mL (750-1938) of colloid. CONCLUSION: Cytokine and inflammatory marker levels did not differ between goal-directed crystalloid and colloid administration after moderate to high-risk abdominal surgery. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT00517127 ). Registered 16th August 2007.

Effect of goal-directed crystalloid- versus colloid-based fluid strategy on tissue oxygen tension: a randomised controlled trial.

BACKGROUND: Sufficient tissue oxygen tension may reduce the risk of postoperative wound infections. Supplemental administration of crystalloids increases subcutaneous oxygen tension (Psqo2). Colloids remain longer in the intravascular system and might therefore increase Psqo2 even more than crystalloids. Therefore, we tested the hypothesis that goal-directed colloid administration increases the perioperative Psqo2 more compared with crystalloid administration. METHODS: We randomly assigned 80 patients undergoing elective open abdominal surgery to receive fluid boluses of hydroxyethyl starch (HES) or lactated Ringer's (LR) solution guided by oesophageal Doppler. Intraoperative Psqo2 was measured in the upper arm. After operation, we measured the Psqo2 in the upper arm and in the surgical wound. RESULTS: Forty patients were enrolled in each group. Patients in the colloid group received HES solution 750 ml (500; 1000) and LR solution 1500 ml (1000; 2000). Patients in the crystalloid group received LR solution 2825 ml (2000; 3960). The goal-directed administration of colloids did not improve intraoperative Psqo2 in the arm compared with crystalloid administration (11.4 kPa [9.0; 16.6] vs 11.2 kPa [8.6; 15.1], respectively; P=0.58). Postoperative arm Psqo2 was 8.1 kPa (6.5; 9.6) in the colloid group and 7.3 kPa (5.7; 9.1) in the crystalloid group (P=0.11). Postoperative surgical wound Psqo2 was 10.7 kPa (8.6; 13.4) in the colloid group and 10.1 kPa (8.1; 12.7) in the crystalloid group (P=0.68). CONCLUSIONS: Goal-directed colloid administration did not increase Psqo2 compared with goal-directed crystalloid administration in patients undergoing open abdominal surgery. CLINICAL TRIAL REGISTRATION: NCT00517127.

TSE clearance during plasma products separation process by Gradiflow(TM).

BACKGROUND AND OBJECTIVES: Recent experimental evidence from rodent models suggests a potential risk for transmissible spongiform encephalopathy (TSE) transmission by blood. The emergence of a new variant Creutzfeldt-Jakob disease (vCJD) has raised increased concerns about the safety of blood components and plasma products derived from vCJD-infected donors. Recent risk-minimisation strategies have included a ban on the use of UK-sourced plasma for the preparation of licensed blood products and leukodepletion of blood donations for fear of possible transmission of the human TSE via blood or blood components. The aim of this study was to investigate the capability and efficacy of a preparative electrophoresis system (Gradiflow) in the removal of TSE contaminants during the separation of plasma products. MATERIALS AND METHODS: Using hamster adapted scrapie 263 K as a model for TSE agent, albumin and IgG separation from human plasma by Gradiflow were performed separately by spiking a 263 K scrapie microsomal fraction to the feed material at each process step. Samples from pre- and post-Gradiflow separation process were titrated to the end-point for the detection of the disease-associated, proteinase K resistant form of the pathogenic prion protein (PrP(Sc)) by Western blot. RESULTS: Under all conditions tested, a greater than 3 log(10) reduction was achieved with no PrP(Sc) detected in any of the pooled products for either of the IgG or albumin separations. These data show that Gradiflow processing has clear advantages for concurrent purification of plasma products and in-process TSE removal. CONCLUSIONS: Our findings suggest that Gradiflow process is a viable alternative to remove causative TSE agents during plasma products separation, potentially eliminating the risk of TSE agents transmission.

Structural requirements for catalysis, expression, and dimerization in the CD26/DPIV gene family.

Dipeptidyl peptidase IV (DP-IV/CD26), fibroblast activation protein (FAP), DP-like 1 (DPL1), DP8, DP9, and DPL2 comprise the CD26 gene family. CD26/DP-IV has roles in liver disease, T cell costimulation, chemokine biology, type II diabetes, and tumor biology. DPIV substrates include the glucagonlike peptides, neuropeptide Y, and the chemokines CCL3, CCL5, CCL11, CCL22, and CXCL12. We have proposed that the extracellular region of CD26 is analogous to prolyl oligopeptidase in consisting of an alpha/beta hydrolase domain contributed by both N- and C-terminal portions of the polypeptide and a seven-blade beta-propeller domain. Replacing the C-terminal portion of the predicted alpha/beta hydrolase domain of CD26 (residues 501-766) with the homologous portion of DP8 or DP9 produced intact proteins. However, these chimeric proteins lacked dimerization and peptidase activity, suggesting that CD26 dimerization requires the C-terminal portion of the alpha/beta hydrolase domain. Deleting some N-terminal residues of the alpha/beta hydrolase domain of CD26 ablated peptidase activity and greatly diminished cell surface expression. Together with previous data that CD26 peptidase activity requires the C-terminal 20 residues, this suggests that peptidase activity requires the entire alpha/beta hydrolase domain. The catalytic triad of DP8 was shown to be Ser(739)-Asp (817)-His(849). Glu(259) of DP8, a residue distant from the catalytic triad yet greatly conserved in the CD26 gene family, was shown to be required for peptidase activity. These data concord with our predicted CD26 structure, indicate that biosynthesis of a functional fragment of CD26 is difficult, and confirm the functional homology of DP8 with CD26.