Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

Successful High-Dosage Monotherapy of Tigecycline in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Background: Recent scientific reports on the use of high dose tigecycline monotherapy as a "drug of last resort" warrant further research into the use of this regimen for the treatment of severe multidrug-resistant, Gram-negative bacterial infections. In the current study, the therapeutic efficacy of tigecycline monotherapy was investigated and compared to meropenem monotherapy in a newly developed rat model of fatal lobar pneumonia-septicemia. Methods: A Klebsiella pneumoniae producing extended-spectrum ß-lactamase (ESBL) and an isogenic variant producing K. pneumoniae carbapenemase (KPC) were used in the study. Both strains were tested for their in vitro antibiotic susceptibility and used to induce pneumonia-septicemia in rats, which was characterized using disease progression parameters. Therapy with tigecycline or meropenem was initiated at the moment that rats suffered from progressive infection and was administered 12-hourly over 10 days. The pharmacokinetics of meropenem were determined in infected rats. Results: In rats with ESBL pneumonia-septicemia, the minimum dosage of meropenem achieving survival of all rats was 25 mg/kg/day. However, in rats with KPC pneumonia-septicemia, this meropenem dosage was unsuccessful. In contrast, all rats with KPC pneumonia-septicemia were successfully cured by administration of high-dose tigecycline monotherapy of 25 mg/kg/day (i.e., the minimum tigecycline dosage achieving 100% survival of rats with ESBL pneumonia-septicemia in a previous study). Conclusions: The current study supports recent literature recommending high-dose tigecycline as a last resort regimen for the treatment of severe multidrug-resistant bacterial infections. The use of ESBL- and KPC-producing K. pneumoniae strains in the current rat model of pneumonia-septicemia enables further investigation, helping provide supporting data for follow-up clinical trials in patients suffering from severe multidrug-resistant bacterial respiratory infections.

Antimicrobial activity of two novel antimicrobial peptides AA139 and SET-M33 against clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles.

Colistin is an antimicrobial peptide (AMP) used as a drug of last resort, although plasmid-mediated colistin resistance (MCR) has been reported. AA139 and SET-M33 are novel AMPs currently in development for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. As many AMPs have a similar mode of action to colistin, potentially leading to cross-resistance, the antimicrobial activity of AA139 and SET-M33 was investigated against a collection of 50 clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles, including colistin-resistant strains. The collection was genotypically characterised and susceptibility to clinically relevant antibiotics was determined. Susceptibility to AA139 and SET-M33 did not differ among the collection despite differences in underlying mechanisms of resistance or susceptibility to colistin. For three colistin-susceptible and three colistin-resistant strains with distinct MDR profiles as well as an additional MCR-producing strain, the bactericidal activity of AA139, SET-M33 and colistin during 24 h of exposure was examined. Following 24 h of exposure to AA139, SET-M33 or colistin, the seven strains were tested for changes in susceptibility to the respective AMPs. AA139 and SET-M33 showed a concentration-dependent bactericidal effect irrespective of bacterial susceptibility to colistin. Exposure to low colistin concentrations resulted in the development of colistin resistance in colistin-susceptible strains, whereas susceptibility to AA139 and SET-M33 following exposure to the respective AMPs was maintained. The two novel AMPs remained effective against colistin-resistant strains and may be promising novel drugs for the treatment of clinically and genotypically diverse MDR K. pneumoniae infections, including infections associated with colistin-resistant bacteria.

Differences in Systemic IgA Reactivity and Circulating Th Subsets in Healthy Volunteers With Specific Microbiota Enterotypes.

Changes in the intestinal microbiota have been associated with the development of immune-mediated diseases in humans. Additionally, the introduction of defined bacterial species into the mouse intestinal microbiota has been shown to impact on the adaptive immune response. However, how much impact the intestinal microbiota composition actually has on regulating adaptive immunity remains poorly understood. Therefore, we studied aspects of the adaptive immunity in healthy adults possessing distinct intestinal microbiota profiles. The intestinal microbiota composition was determined via Illumina sequencing of bacterial 16S rRNA genes extracted from the feces of 35 individuals. Blood B-cell and T-cell subsets from the same individuals were studied using flow cytometry. Finally, the binding of fecal and plasma Immunoglobulin A (IgA) to intestinal bacteria (associated with health and disease) Bacteroides fragilis, Prevotella copri, Bifidobacterium longum, Clostridium difficile, and Escherichia coli was analyzed using ELISA. Unsupervised clustering of microbiota composition revealed the presence of three clusters within the cohort. Cluster 1 and 2 were similar to previously-described enterotypes with a predominance of Bacteroides in Cluster 1 and Prevotella in Cluster 2. The bacterial diversity (Shannon index) and bacterial richness of Cluster 3 was significantly higher than observed in Clusters 1 and 2, with the Ruminococacceae tending to predominate. Within circulating B- and T-cell subsets, only Th subsets were significantly different between groups of distinct intestinal microbiota. Individuals of Cluster 3 have significantly fewer Th17 and Th22 circulating cells, while Th17.1 cell numbers were increased in individuals of Cluster 1. IgA reactivity to intestinal bacteria was higher in plasma than feces, and individuals of Cluster 1 had significant higher plasma IgA reactivity against B. longum than individuals of Cluster 2. In conclusion, we identified three distinct fecal microbiota clusters, of which two clusters resembled previously-described "enterotypes". Global T-cell and B-cell immunity seemed unaffected, however, circulating Th subsets and plasma IgA reactivity were significantly different between Clusters. Hence, the impact of intestinal bacteria composition on human B cells, T cells and IgA reactivity appears limited in genetically-diverse and environmentally-exposed humans, but can skew antibody reactivity and Th cell subsets.

Investigations into the killing activity of an antimicrobial peptide active against extensively antibiotic-resistant K. pneumon iae and P. aeruginosa.

SET-M33 is a multimeric antimicrobial peptide active against Gram-negative bacteria in vitro and in vivo. Insights into its killing mechanism could elucidate correlations with selectivity. SET-M33 showed concentration-dependent bactericidal activity against colistin-susceptible and resistant isolates of P. aeruginosa and K. pneumoniae. Scanning and transmission microscopy studies showed that SET-M33 generated cell blisters, blebs, membrane stacks and deep craters in K. pneumoniae and P. aeruginosa cells. NMR analysis and CD spectra in the presence of sodium dodecyl sulfate micelles showed a transition from an unstructured state to a stable α-helix, driving the peptide to arrange itself on the surface of micelles. SET-M33 kills Gram-negative bacteria after an initial interaction with bacterial LPS. The molecule becomes then embedded in the outer membrane surface, thereby impairing cell function. This activity of SET-M33, in contrast to other similar antimicrobial peptides such as colistin, does not generate resistant mutants after 24h of exposure, non-specific interactions or toxicity against eukaryotic cell membranes, suggesting that SET-M33 is a promising new option for the treatment of Gram-negative antibiotic-resistant infections.