colistin and Hemolysis

Multidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller-Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

Topics: Animals; Cell Membrane; Colistin; Drug Resistance, Multiple, Bacterial; Erythrocytes; Escherichia coli; Hemolysis; Membrane Potentials; Microbial Sensitivity Tests; Pore Forming Cytotoxic Proteins; Sheep

The last three decades have seen a dwindling number of novel antibiotic classes approved for clinical use and a concurrent increase in levels of antibiotic resistance, necessitating alternative methods to combat the rise of multi-drug resistant bacteria. A promising strategy employs antibiotic adjuvants, non-toxic molecules that disarm antibiotic resistance. When co-dosed with antibiotics, these compounds restore antibiotic efficacy in drug-resistant strains. Herein we identify derivatives of tryptamine, a ubiquitous biochemical scaffold containing an indole ring system, capable of disarming colistin resistance in the Gram-negative bacterial pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli while having no inherent bacterial toxicity. Resistance was overcome in strains carrying endogenous chromosomally-encoded colistin resistance machinery, as well as resistance conferred by the mobile colistin resistance-1 (mcr-1) plasmid-borne gene. These compounds restore a colistin minimum inhibitory concentration (MIC) below the Clinical & Laboratory Sciences Institute (CLSI) breakpoint in all resistant strains.

Topics: Animals; Anti-Bacterial Agents; Cattle; Colistin; Drug Resistance, Multiple, Bacterial; Erythrocytes; Gram-Negative Bacteria; Hemolysis; Humans; Microbial Sensitivity Tests; Polymyxins; Tryptamines

The aim of this study was to investigate the presence of plasmid-mediated colistin resistance genes in Escherichia coli from pigs affected by post-weaning diarrhoea (PWD).. DNA samples collected from 51 E. coli isolates from Italian pigs affected by PWD in 2015-2016 were studied. Isolates were classified as presumptively resistant to colistin by routine susceptibility testing and were investigated for the presence of the mcr-1 gene of plasmid origin by PCR. E. coli isolates testing negative for mcr-1 were analysed for the presence of a novel plasmid-mediated gene, mcr-2. Isolates were characterised for fimbrial [F4 (k88), F5 (k99), F6 (987P), F18 and F41] and toxin (LT, STa, STb and Stx2e) determinants by PCR as well as for the occurrence of haemolysis by phenotypic observation. Susceptibility to apramycin, cefquinome, enrofloxacin, florfenicol, gentamicin, tetracycline and trimethoprim/sulfamethoxazole (SXT) was also determined by disk diffusion.. Most of the isolates showed the presence of at least one virulence factor, confirming their pathogenic potential. The presence of mcr-1 was shown in 37 (72.5%) of the 51 isolates. All of the mcr-1-negative isolates tested negative for the mcr-2 gene. Moreover, 80.4% of the isolates were resistant to apramycin, 9.8% to cefquinome, 54.9% to enrofloxacin, 52.9% to florfenicol, 76.5% to gentamicin, 96.1% to tetracycline and 78.4% to SXT.. This is the first report documenting the presence of the mcr-1 gene in pathogenic E. coli isolated from pigs affected by PWD in Italy.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Bacterial Toxins; Colistin; Diarrhea; Disk Diffusion Antimicrobial Tests; DNA, Bacterial; Drug Resistance, Bacterial; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae, Bacterial; Genes, Bacterial; Hemolysis; Italy; Swine; Swine Diseases; Virulence Factors; Weaning

Multiple strains of Acinetobacter baumannii have developed multidrug resistance (MDR), leaving colistin as the only effective treatment. The cecropin-α-melittin hybrid BP100 (KKLFKKILKYL-NH2) and its analogs have previously shown activity against a wide array of plant and human pathogens. In this study, we investigated the in vitro antibacterial activities of 18 BP100 analogs (four known and 14 new) against the MDR A. baumannii strain ATCC BAA-1605, as well as against a number of other clinically relevant human pathogens. Selected peptides were further evaluated against strains of A. baumannii that acquired resistance to colistin due to mutations of the lpxC, lpxD, pmrA, and pmrB genes. The novel analogue BP214 showed antimicrobial activity at 1 to 2 μM and a hemolytic 50% effective concentration (EC50) of >150 μM. The lower activity of its enantiomer suggests a dual, specific and nonspecific mode of action. Interestingly, colistin behaved antagonistically to BP214 when pmrAB and lpxC mutants were challenged.

Topics: Acinetobacter baumannii; Acyltransferases; Amidohydrolases; Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Colistin; Drug Resistance, Bacterial; Erythrocytes; Gene Expression; Hemolysis; Humans; Microbial Sensitivity Tests; Molecular Sequence Data; Mutation; Oligopeptides; Solid-Phase Synthesis Techniques; Structure-Activity Relationship; Transcription Factors

Dextrin-colistin conjugates have been developed with the aim of achieving reduced clinical toxicity associated with colistin, also known as polymyxin E, and improved targeting to sites of bacterial infection. This study investigated the in vitro ability of such dextrin-colistin conjugates to bind and modulate bacterial lipopolysaccharide (LPS), and how this binding affects its biological activity. These results showed that colistin and amylase-activated dextrin-colistin conjugate to a lesser extent induced aggregation of LPS to form a stacked bilayer structure with characteristic dimensions, although this did not cause any substantial change in its secondary structure. In biological studies, both colistin and dextrin-colistin conjugate effectively inhibited LPS-induced hemolysis and tumor necrosis factor α (TNFα) secretion in a concentration-dependent manner, but only dextrin-colistin conjugate showed no additive toxicity at higher concentrations. This study provides the first direct structural experimental evidence for the binding of dextrin-colistin conjugates and LPS and gives insight into the mode of action of dextrin-colistin conjugates.

Topics: Amylases; Animals; Anti-Bacterial Agents; Bacteria; Cell Line; Colistin; Dextrins; Endotoxins; Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Limulus Test; Lipopolysaccharides; Rats; Tumor Necrosis Factor-alpha

Pathogens resistant to most conventional antibiotics are a harbinger of the need to discover novel antimicrobials and anti-infective agents and develop innovative strategies to combat them. The aim of this study was to assess the in vitro activity of colistin alone or in combination with two bacteriocins, nisin A and pediocin PA-1/AcH, against Salmonella choleraesuis ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Yersinia enterocolitica ATCC 9610, and Escherichia coli ATCC 35150 (O157:H7). The strain most sensitive to colistin was enterohemorrhagic E. coli O157:H7, which was inhibited at a concentration of about 0.12 μg/ml. When nisin A (1.70 μg/ml) or pediocin PA-1/AcH (1.56 μg/ml) was combined with colistin, the concentrations required to inhibit E. coli O157:H7 were 0.01 and 0.03 μg/ml, respectively. The in vitro antigenotoxic effect of colistin was determined by using the comet assay method to measure the level of DNA damage in freshly isolated human peripheral blood leukocytes (PBLs) incubated with colistin for 1 h at 37°C. Changes in the tail extents of PBLs of about 69.29 ± 0.08 μm were observed at a final colistin concentration of about 550 ng/ml. Besides the synergistic effect, the combination of colistin (1 mg/ml) and nisin (2 mg/ml) permitted us to re-evaluate the toxic effect of colistin on Vero (monkey kidney epithelial) cells.

Topics: Animals; Bacteriocins; Chlorocebus aethiops; Colistin; Drug Resistance, Multiple, Bacterial; Drug Synergism; Epithelial Cells; Escherichia coli O157; Gram-Negative Bacteria; Hemolysis; Humans; Kidney; Leukocytes; Nisin; Pediocins; Vero Cells

The emergence of Acinetobacter sp. strains resistant to all antibacterial agents including colistin necessitates the development of new types of antimicrobial agents. Six cationic α-helical frog skin-derived peptides (CPF-AM1, PGLa-AM1, B2RP-ERa, [E4K]alyteserin-1c, [D4K]B2RP and [G4K]XT-7) were selected for this study on the basis of potent growth-inhibitory activity against Gram-negative bacteria and low haemolytic activity against human erythrocytes. All peptides were active against a range of colistin-susceptible [minimum inhibitory concentration (MIC)≤2 μg/mL] and colistin-resistant (MIC≥64 μg/mL) clinical isolates of multidrug-resistant strains of Acinetobacter baumannii and Acinetobacter nosocomialis. The most potent peptides against the colistin-resistant strains were [D4K]B2RP and [E4K]alyteserin-1c (MIC=4-16 μg/mL for both). The MIC values of these peptides against the colistin-susceptible strains were in the same range. The frog peptides show potential for development into drugs to treat infections caused by pandrug-resistant Gram-negative pathogens.

Topics: Acinetobacter baumannii; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Anura; Colistin; Drug Evaluation, Preclinical; Drug Resistance, Bacterial; Erythrocytes; Hemolysis; Humans; Microbial Sensitivity Tests; Skin

Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

Topics: Animals; Colistin; Complement Activating Enzymes; Complement Activation; Complement C3; Complement C3-C5 Convertases; Complement Pathway, Alternative; Complement Pathway, Classical; Guinea Pigs; Hemolysis; Humans; In Vitro Techniques; Rabbits; Sheep

The cellular intermediate EAC14 was successfully prepared by incubating optimally sensitized sheep erythrocytes (EA) with normal serum diluted in complement diluent buffer containing suitable amounts of inhibitors of EAC142 formation from EAC14 and C2, namely polymyxin E and polymyxin B. EAC14 cells could also be prepared by incubating EA with normal serum diluted in Mg2+ free complement diluent buffer in the presence or absence of sucrose. The cellular intermediates prepared by these methods exhibited characteristic EAC142 formation and decay profiles and were found to be suitable for C2 estimations.

Topics: Animals; Colistin; Complement C1; Complement C2; Complement C4; Complement Pathway, Classical; Erythrocytes; Guinea Pigs; Hemolysis; Humans; Polymyxin B; Reference Values; Sheep

Topics: Chromatography, DEAE-Cellulose; Colistin; Hemolysin Proteins; Hemolysis; Streptococcus

Topics: Anti-Bacterial Agents; Colistin; Cysteine; Erythromycin; Hemolysin Proteins; Hemolysis; Puromycin; Ribonucleases; RNA; Streptococcus pyogenes

Topics: Agar; Animals; Anti-Bacterial Agents; Chloramphenicol; Colistin; Diffusion; Dihydrostreptomycin Sulfate; Drug Resistance, Microbial; Escherichia coli; Escherichia coli Infections; Furazolidone; Hemolysis; Kanamycin; Microbial Sensitivity Tests; Neomycin; Paper; Serotyping; Swine; Swine Diseases; Tetracycline