Antimicrobial Peptides Epinecidin-1 and Beta-Defesin-3 Are Effective against a Broad Spectrum of Antibiotic-Resistant Bacterial Isolates and Increase Survival Rate in Experimental Sepsis

The antimicrobial peptides human Beta-defensin-3 (hBD-3) and Epinecidin-1 (Epi-1; by Epinephelus coioides) could be a promising tool to develop novel antibacterials to combat antibiotic resistance. The antibacterial activity of Epi-1 + vancomycin against methicillin-resistant Staphylococcus aureus (22 isolates) and Epi-1 + hBD-3 against carbapenem-resistant isolates of Klebsiella pneumoniae (n = 23), Klebsiella aerogenes (n = 17), Acinetobacter baumannii (n = 9), and Pseudomonas aeruginosa (n = 13) was studied in vitro. To evaluate the in vivo efficacy of hBD-3 and Epi-1, ICR (CD-1) mice were injected intraperitoneally with a lethal dose of K. pneumoniae or P. aeruginosa. The animals received a single injection of either sterile saline, hBD-3 monotherapy, meropenem monotherapy, hBD-3 + meropenem, or hBD-3 + Epi-1. Studied peptides showed antibacterial activity in vitro against all studied clinical isolates in a concentration of 2 to 32 mg/L. In both experimental models of murine sepsis, an increase in survival rate was seen with hBD-3 monotherapy, hBD-3 + meropenem, and hBD-3 + Epi-1. For K. pneumoniae-sepsis, hBD-3 was shown to be a promising option in overcoming the resistance of Klebsiella spp. to carbapenems, though more research is needed. In the P. aeruginosa-sepsis model, the addition of Epi-1 to hBD-3 was found to have a slightly reduced mortality rate compared to hBD-3 monotherapy.

Novel Antimicrobial Peptides Designed Using Recurrent Neural Network Reduce Mortality in Experimental Sepsis

The amino acid sequences of 198 novel peptides were obtained using a generative long short-term memory recurrent neural network (LTSM RNN). To assess their antimicrobial effect, I synthesized 5 out of 198 generated peptides. The PEP-38 and PEP-137 peptides were active in vitro against carbapenem-resistant isolates of Klebsiella aerogenes (n=12) and K. pneumoniae (n=18). PEP-137 was also active against Pseudomonas aeruginosa (n=17). The remaining three peptides (PEP-36, PEP-136 and PEP-174) showed no antibacterial effect. Then I investigated the effect of PEP-38 and PEP-137 (a single intraperitoneal administration of a 100 µg dose 30 min after infection) on animal survival in an experimental murine model of K. pneumoniae-induced sepsis. As a control, I used two groups of mice: one received sterile saline, and the other received inactive in vitro PEP-36 (a single 100 µg dose). The PEP-36 peptide was shown to provide the highest survival rate (66.7%). PEP-137 showed a survival rate of 50%. PEP-38 was found to be ineffective. The data obtained can be used to develop new antibacterial peptide drugs to combat antibiotic resistance.

Immunohistochemical analysis of GFAP expression in the experimental sepsis-associated encephalopathy

Pathophysiology of sepsis-associated encephalopathy (SAE) is linked to blood-brain barrier breakdown, neuroinflammation and neurotransmitter imbalance in the brain. Astroglia, the most abundant cell population within the brain, plays the critical role in control of all kinds of homeostatic processes, thereby regulating the adaptive reactions of the brain to various challenges. Astroglia are highly heterogenous across the brain regions, therefore, damaging factors stimulate heterogenous astroglial reactivity and response in different brain regions. The aim of this study was determining immunohistochemical features of GFAP expression in various brain regions in the model of rodent experimental sepsis. Materials and methods. The experiment was performed in Wistar rats: control group of 5 sham-operated rats and the main group of 20 rats subjected to cecum ligation and puncture (CLP) procedure. The immunohistochemical study of GFAP expression in the sensorimotor cortex, subcortical white matter, hippocampal, thalamic and caudate nucleus/putamen regions was performed from 20 to 48 hours of the postoperative period. Results. Starting from the 12th hour after CLP, animals began display progressive increase in signs of periorbital exudation, piloerection, fever-/hypothermia, diarrhea, social isolation, lethargy, and respiratory impairment. In the period of 20–38 hours, 9 animals showed expressed previously listed symptoms and were euthanized (CLP-B – lethal group), 11 rats survived until 48 hours of the experiment (CLP-A – survived group). In the lethal group, starting from 20 to 38 hours after the CLP procedure, a significant (relative to control) regionally-specific dynamic increase in the level of GFAP expression was observed in the brain: in the cortex – by 465 %, in the subcortical white matter – by 198 %, in the hippocampus – by 250 %, from the 23rd hour – in the caudate nucleus/putamen by 18 %. In the thalamus, no significant changes in the level of GFAP expression were observed. In the cortex and hippocampus of survived animals, 48 h after CLP, higher values of GFAP expression were observed comparing to the group of non-survived animals. Conclusions. Under conditions of the experimental SAE, an early dynamic increase in the astroglial reactivity was observed in the cortex, hippocampus, white matter, and caudate nucleus/putamen of the brain with the most significant increase of indicators in the cortex and hippocampus, which potentially indicates relatively more vulnerable areas of the brain to damaging factors, as well as places of the most active intercellular interaction in the condition of systemic inflammation. Higher values of GFAP expression in the cortex and hippocampus of survived animals at 48 hours of the experiment, compared with indicators of non-survived group, indicate increased astroglial reactivity in these brain regions at the noted time period, accompanied by relatively more favorable clinical course of the disease.

The microbial composition of the initial insult can predict the prognosis of experimental sepsis

We hypothesized that the composition of sepsis-inducing bacterial flora influences the course of fecal peritonitis in rodents. Saline or fecal suspensions with a standardized dose range of bacterial colony-forming units (CFUs) were injected intraperitoneally into Sprague–Dawley rats. The qualitative composition of the initial inoculum and the ascites was analyzed separately by MALDI-TOF mass spectrometry. Invasive monitoring was conducted in separate anesthetized groups (n = 12–13/group) after 12, 24, 48 and 72 h to determine rat-specific organ failure assessment (ROFA) scores. Death and ROFA scores peaked at 24 h. At this time, 20% mortality occurred in animals receiving a monomicrobial E. coli suspension, and ROFA scores were significantly higher in the monomicrobial subgroup than in the polymicrobial one (median 6.5; 5.0–7.0 and 5.0; 4.75–5.0, respectively). ROFA scores dropped after 48 h, accompanied by a steady decrease in ascites CFUs and a shift towards intra-abdominal monomicrobial E. coli cultures. Furthermore, we found a relationship between ascites CFUs and the evolving change in ROFA scores throughout the study. Hence, quantitatively identical bacterial loads with mono- or polymicrobial dominance lead to a different degree of sepsis severity and divergent outcomes. Initial and intraperitoneal microbiological testing should be used to improve translational research success.

Predicting Experimental Sepsis Survival with a Mathematical Model of Acute Inflammation

Sepsis is characterized by an overactive, dysregulated inflammatory response that drives organ dysfunction and often results in death. Mathematical modeling has emerged as an essential tool for understanding the underlying complex biological processes. A system of four ordinary differential equations (ODEs) was developed to simulate the dynamics of bacteria, the pro- and anti-inflammatory responses, and tissue damage (whose molecular correlate is damage-associated molecular pattern [DAMP] molecules and which integrates inputs from the other variables, feeds back to drive further inflammation, and serves as a proxy for whole-organism health status). The ODE model was calibrated to experimental data from E. coli infection in genetically identical rats and was validated with mortality data for these animals. The model demonstrated recovery, aseptic death, or septic death outcomes for a simulated infection while varying the initial inoculum, pathogen growth rate, strength of the local immune response, and activation of the pro-inflammatory response in the system. In general, more septic outcomes were encountered when the initial inoculum of bacteria was increased, the pathogen growth rate was increased, or the host immune response was decreased. The model demonstrated that small changes in parameter values, such as those governing the pathogen or the immune response, could explain the experimentally observed variability in mortality rates among septic rats. A local sensitivity analysis was conducted to understand the magnitude of such parameter effects on system dynamics. Despite successful predictions of mortality, simulated trajectories of bacteria, inflammatory responses, and damage were closely clustered during the initial stages of infection, suggesting that uncertainty in initial conditions could lead to difficulty in predicting outcomes of sepsis by using inflammation biomarker levels.

Plasma hyaluronan, hyaluronidase activity and endogenous hyaluronidase inhibition in sepsis: an experimental and clinical cohort study

Background Plasma hyaluronan concentrations are increased during sepsis but underlying mechanisms leading to high plasma hyaluronan concentration are poorly understood. In this study we evaluate the roles of plasma hyaluronan, effective plasma hyaluronidase (HYAL) activity and its endogenous plasma inhibition in clinical and experimental sepsis. We specifically hypothesized that plasma HYAL acts as endothelial glycocalyx shedding enzyme, sheddase. Methods Plasma hyaluronan, effective HYAL activity and HYAL inhibition were measured in healthy volunteers (n = 20), in patients with septic shock (n = 17, day 1 and day 4), in patients with acute pancreatitis (n = 7, day 1 and day 4) and in anesthetized and mechanically ventilated pigs (n = 16). Sixteen pigs were allocated (unblinded, open label) into three groups: Sepsis-1 with infusion of live Escherichia coli (E. coli) 1 × 108 CFU/h of 12 h (n = 5), Sepsis-2 with infusion of E. coli 1 × 108 CFU/h of 6 h followed by 1 × 109 CFU/h of the remaining 6 h (n = 5) or Control with no E. coli infusion (n = 6). Results In experimental E. coli porcine sepsis and in time controls, plasma hyaluronan increases with concomitant decrease in effective plasma HYAL activity and increase of endogenous HYAL inhibition. Plasma hyaluronan increased in patients with septic shock but not in acute pancreatitis. Effective plasma HYAL was lower in septic shock and acute pancreatitis as compared to healthy volunteers, while plasma HYAL inhibition was only increased in septic shock. Conclusion Elevated plasma hyaluronan levels coincided with a concomitant decrease in effective plasma HYAL activity and increase of endogenous plasma HYAL inhibition both in experimental and clinical sepsis. In acute pancreatitis, effective plasma HYAL activity was decreased which was not associated with increased plasma hyaluronan concentrations or endogenous HYAL inhibition. The results suggest that plasma HYAL does not act as sheddase in sepsis or pancreatitis.