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Nosocomial Bacteria Inhibition with Polymyxin B: In Silico Gene Mining and In Vitro Analysis

by Jayendra Chunduru, Nicholas LaRoe, Jeremy Garza, Abdul N. Hamood and Paul W. Paré

Antibiotics 2024, 13(8), 745; https://doi.org/10.3390/antibiotics13080745 - 8 Aug 2024

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Multidrug-resistant bacteria present a significant public health challenge; such pathogens exhibit reduced susceptibility to conventional antibiotics, limiting current treatment options. Cationic non-ribosomal peptides (CNRPs) such as brevicidine and polymyxins have emerged as promising candidates to block Gram-negative bacteria. To investigate the capability of bacteria to biosynthesize CNRPs, and specifically polymyxins, over 11,000 bacterial genomes were mined in silico. Paenibacillus polymyxa was identified as having a robust biosynthetic capacity, based on multiple polymyxin gene clusters. P. polymyxa biosynthetic competence was confirmed by metabolite characterization via HPLC purification and MALDI TOF/TOF analysis. When grown in a selected medium, the metabolite yield was 4 mg/L with a 20-fold specific activity increase. Polymyxin B (PMB) was assayed with select nosocomial pathogens, including Pseudomonas aeruginosa, Klebsiella pneumonia, and Acinetobacter baumaii, which exhibited minimum inhibitory concentrations of 4, 1, and 1 µg/mL, respectively. Full article

(This article belongs to the Section Antimicrobial Peptides)

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Graphical abstract
Full article ">Figure 1
(A). Estimated non-ribosomal peptide length (average) in select genera (purple) and average cationic residues per peptide (blue): Paenibacillus n = 602 (range of peptide length = 1–30), Brevibacillus n = 249 (range of peptide length = 1–33), Streptomyces n = 2592 (range of peptide length = 1–56), Bacillus n = 4840 (range of peptide length = 1–26), Pseudomonas n = 1758 (range of peptide length = 1–48), and Burkholderia n = 1915 (range of peptide length = 1–28). (B). Fraction of bacteria that contain essential predicted residues for PPPB. legend for circle graph (C). Total number of organisms with the biological potential of producing polymyxin (* commercially available). Range of peptide length (n) (P. lentus DSM 25539 (1–15), P. sp. IHB B 3084, P. polymyxa CR1 (2–14), P. polymyxa ZF129 (3–13) P. polymyxa J (3–14), P. polymyxa Sb3-1 (4–12), P. polymyxa SQR-21, ATCC 15970, HY96-2 (4–13), P. polymyxa E681, YC0136, P. peoriae HS311 (4–14), P. sp. lzh-N1 (4–16), and P. sp. M-152 (4–18). Full article ">Figure 1 Cont.
(A). Estimated non-ribosomal peptide length (average) in select genera (purple) and average cationic residues per peptide (blue): Paenibacillus n = 602 (range of peptide length = 1–30), Brevibacillus n = 249 (range of peptide length = 1–33), Streptomyces n = 2592 (range of peptide length = 1–56), Bacillus n = 4840 (range of peptide length = 1–26), Pseudomonas n = 1758 (range of peptide length = 1–48), and Burkholderia n = 1915 (range of peptide length = 1–28). (B). Fraction of bacteria that contain essential predicted residues for PPPB. legend for circle graph (C). Total number of organisms with the biological potential of producing polymyxin (* commercially available). Range of peptide length (n) (P. lentus DSM 25539 (1–15), P. sp. IHB B 3084, P. polymyxa CR1 (2–14), P. polymyxa ZF129 (3–13) P. polymyxa J (3–14), P. polymyxa Sb3-1 (4–12), P. polymyxa SQR-21, ATCC 15970, HY96-2 (4–13), P. polymyxa E681, YC0136, P. peoriae HS311 (4–14), P. sp. lzh-N1 (4–16), and P. sp. M-152 (4–18). Full article ">Figure 2
Media effect on P. polymyxa growth [ATCC-recommended media (M178, blue), tryptic soy broth (TSB, red), tryptic soy broth with starch (20 g/L) (TSB S20, green), tryptic soy broth with starch (40 g/L) (TSB S40, purple), Luria–Bertani broth (LB, orange), and yeast extract peptone dextrose (YPD, dark green)] (three trials in triplicate and error bars are SDs). Full article ">Figure 3
Fragmented MS/MS peaks using TOF of 1203.3698 Da peak; y-axis on right shows absolute intensity. Full article ">Figure 4
PMB inhibits the growth of several bacterial pathogens. The MBIC of PMB to each strain was determined as described in the Materials and Methods section. (A) The effect of PMB on three P. aeruginosa multidrug-resistant strains (MRSN 17849, MRSN 18560, and MRSN 2108. (B) The effect of PMB on the K. pneumoniae strain KP-UTI-2 and the A. baumannii strain AB-10. Bars indicate the means of three independent experiments. *, p < 0.05; ****, p < 0.0001; ns, not significant. Statistical significance (****) was determined using a two-way ANOVA with Tukey’s multiple comparison test. The growth of P. aeruginosa strains MRSN-17849 and MRSN-2108 was inhibited by 4 mg/mL (no CFU was recovered). Similarly, the K. pneumoniae strain KP-UTI-2 and the A. baumannii strain AB-10 were inhibited by 2 mg/mL. In the graphs, we included 4–5 CFUs for each point to conduct the statistical analysis. Full article ">

Abstract

Coelibactin is a putative non-ribosomally synthesized peptide with predicted zincophore activity and which has been implicated in antibiotic regulation in Streptomyces coelicolor A3(2). The coelibactin biosynthetic pathway contains a stereo- and regio-specific monooxygenation step catalyzed by a cytochrome P450 enzyme (CYP105N1). We have determined the X-ray crystal structure of CYP105N1 at 2.9 Å and analyzed it in the context of the bacterial CYP105 family as a whole. The crystal structure reveals a channel between the α-helical domain and the β-sheet domain exposing the heme pocket and the long helix I to the solvent. This wide-open conformation of CYP105N1 may be related to the bulky substrate coelibactin. The ligand-free CYP105N1 structure has enough room in the substrate access channel to allow the coelibactin to enter into the active site. Analysis of typical siderophore ligands suggests that CYP105N1 may produce derivatives of coelibactin, which would then be able to chelate the zinc divalent cation. Full article

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