Polymyxins with Potent Antibacterial Activity against Colistin-Resistant Pathogens: Fine-Tuning Hydrophobicity with Unnatural Amino Acids.

In view of the increased prevalence of antimicrobial resistance among human pathogens, antibiotics against multidrug-resistant (MDR) bacteria are in urgent demand. In particular, the rapidly emerging resistance to last-resort antibiotic colistin, used for severe Gram-negative MDR infections, is critical. Here, a series of polymyxins containing unnatural amino acids were explored, and some analogues exhibited excellent antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Hydrophobicity of the compounds within this series (as measured by retention in reversed-phase analytical HPLC) exhibited a discernible correlation with their antimicrobial activity. This trend was particularly pronounced for colistin-resistant pathogens. The most active compounds demonstrated competitive activity against a panel of Gram-negative pathogens, while exhibiting low in vitro cytotoxicity. Importantly, most of these hits also retained (or even had increased) potency against colistin-susceptible strains. These findings infer that fine-tuning hydrophobicity may enable the design of polymyxin analogues with favorable activity profiles.

Antisense Peptide Nucleic Acid-Diaminobutanoic Acid Dendron Conjugates with SbmA-Independent Antimicrobial Activity against Gram-Negative Bacteria.

Precision antisense antibacterial agents may be developed into novel antibiotics in the fight against multidrug-resistant Gram-negative bacteria. In this study, a series of diaminobutanoic acid (DAB) dendrons are presented as novel carriers for the delivery of antisense antibacterial peptide nucleic acids (PNAs). The dendron-PNA conjugates targeting the essential acpP gene exhibit specific antisense antimicrobial bactericidal activity against Escherichia coli and Klebsiella pneumoniae at one-digit micromolar concentrations, while showing low toxicity to human cells. One compound selected from a structure-activity relationship series showed high stability in mouse and human serum (t1/2 ≫ 24 h) as well as in vivo activity against a multidrug-resistant, extended spectrum beta-lactamase-producing E. coli in a murine peritonitis model. The compound was also well tolerated in mice upon i.v. administration up to a dose of 20 mg/kg, and in vivo fluorescence imaging indicated clearance via renal excretion with slight accumulation in the kidneys and liver. Thus, DAB-based dendrons constitute a promising new chemistry platform for development of effective delivery agents for antibacterial drugs with possible in vivo use.

Evaluation of accessible regions of Escherichia coli fimH mRNA through computational prediction and experimental investigation.

BACKGROUND AND OBJECTIVES: This study aimed to investigate the accessible regions of the fimH mRNA using computational prediction and dot-blot hybridization to increase the effectiveness of antisense anti-virulence therapeutics against Uropathogenic Escherichia coli. MATERIALS AND METHODS: We predicted the secondary structure of the E. coli fimH mRNA using the Sfold and Mfold Web servers and RNA structure 5.5 program. Considering the predicted secondary structure, accessible regions in mRNA of fimH were determined and oligonucleotides complementary to these regions were synthesized and hybridization activity of those oligonucleotides to the fimH Digoxigenin (DIG) labeled mRNA was assessed with dot-blot hybridization. RESULTS: When searching the fimH gene in the GenBank database, two lengths for this gene was discovered in different strains of E. coli. The difference was related to the nine bases in the first part of the gene utilizing either of two translation initiation sites. Based on the bioinformatics analyses, five regions lacking obvious stable secondary structures were selected in mRNA of fimH. The result of dot-blot hybridization exhibited strongest hybridization signal between the antisense oligonucleotide number one and fimH labeled mRNA, whereas hybridization signals were not seen for the negative control. CONCLUSION: The results obtained here demonstrate that the region contains start codon of fimH mRNA could act as the potential mRNA target site for anti-fimH antisense therapeutics. It is recommended in the future both of utilizing translation initiation sites be targeted with antisense oligomers compounds.

Antibiotic Potentiation in Multidrug-Resistant Gram-Negative Pathogenic Bacteria by a Synthetic Peptidomimetic.

The peptidomimetic H-[NLys-tBuAla]6-NH2 (CEP-136), which exhibits low inherent antimicrobial activity against Gram-negative bacteria (MIC = 16-64 µM), was shown to significantly potentiate the antibacterial activity of several clinically important antibiotics against the human pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Thus, the antibacterial spectrum of rifampicin, clarithromycin, and azithromycin could be extended to include also these Gram-negative bacteria. Additionally, the potentiation effect was demonstrated in a panel of clinically relevant multidrug-resistant isolates including extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing as well as colistin-resistant strains. For some peptidomimetic-antibiotic combinations, the strong synergy corresponded to a more than 50-fold reduction of the minimal inhibitory concentration of the antibiotic. Mechanistic studies indicate that the potentiation arises from a permeabilization effect exerted on the outer membrane lipopolysaccharide layer of the Gram-negative bacteria without significant disruption of the inner membrane. Furthermore, the peptidomimetic enhancer exhibited only a marginal effect on the viability of mammalian HepG2 cells even at concentrations 100-fold higher than that enabling the antibiotic enhancement. Also, a low hemolytic activity combined with limited in vivo acute toxicity of CEP-136 in healthy mice allowed in vivo validation of the potentiation effect on both rifampicin and azithromycin treatment in a murine peritonitis model. Thus, CEP-136 is an interesting hit compound for further development of effective adjuvants for repurposing antibiotics for use against infections by multidrug-resistant Gram-negative bacteria.

Identification and characterization of the type II toxin-antitoxin systems in the carbapenem-resistant Acinetobacterbaumannii.

Carbapenem -resistant A. baumannii (CRAB) is a major cause of both community-associated and nosocomial infections that are difficult to control and treat worldwide. Among different mediators of pathogenesis, toxin-antitoxin (TA) systems are emerging as the most prominent. The functional diversity and ubiquitous distribution in bacterial genomes are causing significant attention toward TA systems in bacteria. However, there is no enough information on the prevalence and identity of TA systems in CRAB clinical isolates. This study aimed to identify type II toxin-antitoxin systems in carbapenem-resistant A. baumannii (CRAB) isolates. A total of 80 A. baumannii isolates were collected from different clinical samples. Antibiotic resistance patterns of A. baumannii isolates were evaluated phenotypically and genetically. The frequency of type II TA genes was evaluated in CRAB isolates using PCR. Moreover, the expression level of the most prevalent TA encoding genes in some clinical isolates were evaluated by RT-qPCR. To determine whether the SplT and SplA are functional, the growth of E. coli BL21 cells (DE3/pLysS) harboring pET28a, pET28a-splTA, and pET28a-splT were analyzed by kill-rescue assay. All of the isolates were resistant to third generation of cephalosporins, ciprofloxacin and levofloxacin, whereas, 72%, 81% and 87% were resistant to amikacin, carbapenems and tetracycline, respectively. The cheTA in 47 isolates (72.5%) and splTA in 39 isolates (60%) of 65 isolates were the most common genes encoding type II TA among CRAB isolates. RT-qPCR demonstrated that cheTA and splTA transcripts are produced in the clinical isolates. There was a significant correlation between the presence of splTA genes and blaOXA-24 in CRAB isolates. Over-expression of the splT gene in E. coli results in inhibition of bacterial growth, whereas co-expression of splTA effectively restores the growth. This study presents the first identification of the type II TA systems among the carbapenem -resistant A. baumannii isolates, in Iran.