Structure and Function of ArnD. A Deformylase Essential for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance.

Covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N) mediates resistance to cationic antimicrobial peptides and polymyxin antibiotics in Gram-negative bacteria. The proteins required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4-amino-l-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC, requiring a deformylase activity downstream in the pathway to generate the final C55P-Ara4N donor. Here, we show that deletion of the arnD gene in an Escherichia coli mutant that constitutively expresses the arnBCADTEF operon leads to accumulation of the formylated ArnC product undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), suggesting that ArnD is the downstream deformylase. Purification of Salmonella typhimurium ArnD (stArnD) shows that it is membrane-associated. We present the crystal structure of stArnD revealing a NodB homology domain structure characteristic of the metal-dependent carbohydrate esterase family 4 (CE4). However, ArnD displays several distinct features: a 44 amino acid insertion, a C-terminal extension in the NodB fold, and sequence divergence in the five motifs that define the CE4 family, suggesting that ArnD represents a new family of carbohydrate esterases. The insertion is responsible for membrane association as its deletion results in a soluble ArnD variant. The active site retains a metal coordination H-H-D triad, and in the presence of Co2+ or Mn2+, purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid-base catalytic mechanism.

Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity.

Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.

Implication of Mn-cofactored superoxide dismutase in the tolerance of swarmer Pseudomonas aeruginosa to polymixin, ciprofloxacin and meropenem antibiotics.

The protective role of superoxide dismutase (Sod) against oxidative stress, resulting from the common antibiotic pathway of action, has been studied in the wild type and mutant strains of swarmer Pseudomonas aeruginosa, lacking Cytosolic Mn-Sod (sodM), Fe-Sod (sodB) or both Sods (sodMB).Our results showed that inactivation of sodB genes leads to significant motility defects and tolerance to meropenem. This resistance is correlated with a greater membrane unsaturation as well as an effective intervention of Mn-Sod isoform, in antibiotic tolerance.Moreover, loss of Mn-Sod in sodM mutant, leads to polymixin intolerance and is correlated with membrane unsaturation. Effectivelty, sodM mutant showed an enhanced swarming motility and a conserved rhamnolipid production. Whereas, in the double mutant sodMB, ciprofloxacin tolerance would be linked to an increase in the percentage of saturated fatty acids in the membrane, even in the absence of superoxide dismutase activity.The overall results showed that Mn-Sod has a protective role in the tolerance to antibiotics, in swarmer P.aeruginosa strain. It has been further shown that Sod intervention in antibiotic tolerance is through change in membrane fatty acid composition.

Polymyxins induce lipid scrambling and disrupt the homeostasis of Gram-negative bacteria membrane.

Polymyxins are increasingly used as the last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, efforts to address the resistance in superbugs are compromised by a poor understanding of the bactericidal modes because high-resolution detection of the cell structure is still lacking. By performing molecular dynamics simulations at a coarse-grained level, here we show that polymyxin B (PmB) disrupts Gram-negative bacterial membranes by altering lipid homeostasis and asymmetry. We found that the binding of PmBs onto the asymmetric outer membrane (OM) loosens the packing of lipopolysaccharides (LPS) and induces unbalanced bending torque between the inner and outer leaflets, which in turn triggers phospholipids to flip from the inner leaflet to the outer leaflet to compensate for the stress deformation. Meanwhile, some LPSs may be detained on the inner membrane (IM). Then, the lipid-scrambled OM undergoes phase separation. Defects are created at the boundaries between LPS-rich domains and phospholipid-rich domains, which consequently facilitate the uptake of PmB across the OM. Finally, PmBs target LPSs detained on the IM and similarly perturb the IM. This lipid Scramble, membrane phase Separation, and peptide Translocation model depicts a novel mechanism by which polymyxins kill bacteria and sheds light on developing a new generation of polymyxins or antibiotic adjuvants with improved killing activities and higher therapeutic indices.

CrrAB regulates PagP-mediated glycerophosphoglycerol palmitoylation in the outer membrane of Klebsiella pneumoniae.

The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.

Integrated metabolomic and transcriptomic analyses of the synergistic effect of polymyxin-rifampicin combination against Pseudomonas aeruginosa.

BACKGROUND: Understanding the mechanism of antimicrobial action is critical for improving antibiotic therapy. For the first time, we integrated correlative metabolomics and transcriptomics of Pseudomonas aeruginosa to elucidate the mechanism of synergistic killing of polymyxin-rifampicin combination. METHODS: Liquid chromatography-mass spectrometry and RNA-seq analyses were conducted to identify the significant changes in the metabolome and transcriptome of P. aeruginosa PAO1 after exposure to polymyxin B (1 mg/L) and rifampicin (2 mg/L) alone, or in combination over 24 h. A genome-scale metabolic network was employed for integrative analysis. RESULTS: In the first 4-h treatment, polymyxin B monotherapy induced significant lipid perturbations, predominantly to fatty acids and glycerophospholipids, indicating a substantial disorganization of the bacterial outer membrane. Expression of ParRS, a two-component regulatory system involved in polymyxin resistance, was increased by polymyxin B alone. Rifampicin alone caused marginal metabolic perturbations but significantly affected gene expression at 24 h. The combination decreased the gene expression of quorum sensing regulated virulence factors at 1 h (e.g. key genes involved in phenazine biosynthesis, secretion system and biofilm formation); and increased the expression of peptidoglycan biosynthesis genes at 4 h. Notably, the combination caused substantial accumulation of nucleotides and amino acids that last at least 4 h, indicating that bacterial cells were in a state of metabolic arrest. CONCLUSION: This study underscores the substantial potential of integrative systems pharmacology to determine mechanisms of synergistic bacterial killing by antibiotic combinations, which will help optimize their use in patients.

A Simple and Robust Liquid Chromatography With Tandem Mass Spectrometry Analytical Method for Therapeutic Drug Monitoring of Plasma and Cerebrospinal Fluid Polymyxin B1 and B2.

BACKGROUND: Polymyxin B is used as the last treatment resort for multidrug-resistant gram-negative bacterial infections. This study aimed to develop and validate a simple and robust liquid chromatography with tandem mass spectrometry analytical method for therapeutic drug monitoring of plasma and cerebrospinal fluid (CSF) polymyxin B1 and B2. METHODS: Plasma and CSF polymyxin B1 and B2 were chromatographically separated on a Thermo Hypersil GOLD aQ C18 column and detected using electrospray ionization mode coupled with multiple reaction monitoring. Blood and CSF samples for pharmacokinetic analysis were collected from 15 polymyxin B-treated patients. RESULTS: The calibration curve showed acceptable linearity over 0.2-10 mcg/mL for polymyxin B1 and 0.05-2.5 mcg/mL for B2 in the plasma and CSF, respectively. After validation, according to the Food and Drug Administration (FDA) method validation guideline, this method was applied for polymyxin B1 and B2 quantification in over 100 samples in a clinical study. CONCLUSIONS: A simple and robust method to measure polymyxin B1 and B2 in human CSF was first exploited and validated with good sensitivity and specificity, and successfully applied in polymyxin B pharmacokinetic analysis and therapeutic monitoring in Chinese patients.

Cryo-electron microscopy structures of ArnA, a key enzyme for polymyxin resistance, revealed unexpected oligomerizations and domain movements.

Gram-negative bacteria evade the attack of cationic antimicrobial peptides through modifying their lipid A structure in their outer membranes with 4-amino-4-deoxy-L-arabinose (Ara4N). ArnA is a crucial enzyme in the lipid A modification pathway and its deletion abolishes the polymyxin resistance of gram-negative bacteria. Previous studies by X-ray crystallography have shown that full-length ArnA forms a three-bladed propeller-shaped hexamer. Here, the structures of ArnA determined by cryo-electron microscopy (cryo-EM) reveal that ArnA exists in two 3D architectures, hexamer and tetramer. This is the first observation of a tetrameric ArnA. The hexameric cryo-EM structure is similar to previous crystal structures but shows differences in domain movements and conformational changes. We propose that ArnA oligomeric states are in a dynamic equilibrium, where the hexamer state is energetically more favorable, and its domain movements are important for cooperating with downstream enzymes in the lipid A-Ara4N modification pathway. The results provide us with new possibilities to explore inhibitors targeting ArnA.

Characterization of the Polymyxin D Synthetase Biosynthetic Cluster and Product Profile of Paenibacillus polymyxa ATCC 10401.

The increasing prevalence of polymyxin-resistant bacteria has stimulated the search for improved polymyxin lipopeptides. Here we describe the sequence and product profile for polymyxin D nonribosomal peptide synthetase from Paenibacillus polymyxa ATCC 10401. The polymyxin D synthase gene cluster comprised five genes that encoded ABC transporters (pmxC and pmxD) and enzymes responsible for the biosynthesis of polymyxin D (pmxA, pmxB, and pmxE). Unlike polymyxins B and E, polymyxin D contains d-Ser at position 3 as opposed to l-α,γ-diaminobutyric acid and has an l-Thr at position 7 rather than l-Leu. Module 3 of pmxE harbored an auxiliary epimerization domain that catalyzes the conversion of l-Ser to the d-form. Structural modeling suggested that the adenylation domains of module 3 in PmxE and modules 6 and 7 in PmxA could bind amino acids with larger side chains than their preferred substrate. Feeding individual amino acids into the culture media not only affected production of polymyxins D1 and D2 but also led to the incorporation of different amino acids at positions 3, 6, and 7 of polymyxin D. Interestingly, the unnatural polymyxin analogues did not show antibiotic activity against a panel of Gram-negative clinical isolates, while the natural polymyxins D1 and D2 exhibited excellent in vitro antibacterial activity and were efficacious against Klebsiella pneumoniae and Acinetobacter baumannii in a mouse blood infection model. The results demonstrate the excellent antibacterial activity of these unusual d-Ser3 polymxyins and underscore the possibility of incorporating alternate amino acids at positions 3, 6, and 7 of polymyxin D via manipulation of the polymyxin nonribosomal biosynthetic machinery.

Interaction of the antimicrobial peptide polymyxin B1 with both membranes of E. coli: a molecular dynamics study.

Antimicrobial peptides are small, cationic proteins that can induce lysis of bacterial cells through interaction with their membranes. Different mechanisms for cell lysis have been proposed, but these models tend to neglect the role of the chemical composition of the membrane, which differs between bacterial species and can be heterogeneous even within a single cell. Moreover, the cell envelope of Gram-negative bacteria such as E. coli contains two membranes with differing compositions. To this end, we report the first molecular dynamics simulation study of the interaction of the antimicrobial peptide, polymyxin B1 with complex models of both the inner and outer membranes of E. coli. The results of >16 microseconds of simulation predict that polymyxin B1 is likely to interact with the membranes via distinct mechanisms. The lipopeptides aggregate in the lipopolysaccharide headgroup region of the outer membrane with limited tendency for insertion within the lipid A tails. In contrast, the lipopeptides readily insert into the inner membrane core, and the concomitant increased hydration may be responsible for bilayer destabilization and antimicrobial function. Given the urgent need to develop novel, potent antibiotics, the results presented here reveal key mechanistic details that may be exploited for future rational drug development.

Use of a novel medium, the Polymyxin Ceftazidime Oxford Medium, for isolation of Listeria monocytogenes from raw or non-pasteurized foods.

Polymyxin Ceftazidime Oxford Medium (PCOM), a novel selective and differential plating medium for Listeria monocytogenes was compared with Modified Oxford Agar (MOX) for efficacy to isolate L. monocytogenes and other Listeria spp. naturally present in non-pasteurized Mexican-style cheese (n = 50), non-pasteurized fresh squeezed orange juice (n = 50), raw beef chunks (n = 36), and fresh cabbage (n = 125). Samples were collected from retail markets and farms in Mexico and tested following the US Department of Agriculture enrichment technique. Listeria spp. were isolated from 23.4% of analyzed samples, and from those, 75.0% corresponded to raw beef chunks, 38.0% to non-pasteurized Mexican-style cheese, and 30.0% to fresh squeezed orange juice. No Listeria spp. were isolated from fresh cabbage samples. L. monocytogenes was recovered from 15.3% of food samples analyzed. Non-pasteurized Mexican-style cheese showed the highest proportion of L. monocytogenes positive samples (36.0%), followed by orange juice (26.0%) and raw beef (25.0%). The frequency of isolation of Listeria spp. and L. monocytogenes was not different (P > 0.05) between PCOM and MOX. The advantages of using PCOM when comparing to MOX, include the easier way to identify Listeria species, the lower cost per plate and the availability of its ingredients for Latin-American countries.

Analytical and functional determination of polymyxin B protein binding in serum.

To enhance our understanding of the pharmacological properties of polymyxin B, serum protein binding for polymyxin B1, B2, and B3 and for isoleucine-polymyxin B1 was evaluated. Using equilibrium dialysis and ultrafiltration, comparable protein binding was found in all 4 components of polymyxin B (92% to 99%). Protein binding in human serum was further assessed using a functional assay, the results of which were in general agreement with previous findings (approximately 90%).

Significant accumulation of polymyxin in single renal tubular cells: a medicinal chemistry and triple correlative microscopy approach.

Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our approach represents a significant methodological advancement in determination of drug uptake for single-cell pharmacology.

Studies on tridecaptin B(1), a lipopeptide with activity against multidrug resistant Gram-negative bacteria.

Previously other groups had reported that Paenibacillus polymyxa NRRL B-30507 produces SRCAM 37, a type IIA bacteriocin with antimicrobial activity against Campylobacter jejuni. Genome sequencing and isolation of antimicrobial compounds from this P. polymyxa strain show that the antimicrobial activity is due to polymyxins and tridecaptin B1. The complete structural assignment, synthesis, and antimicrobial profile of tridecaptin B1 is reported, as well as the putative gene cluster responsible for its biosynthesis. This peptide displays strong activity against multidrug resistant Gram-negative bacteria, a finding that is timely to the current problem of antibiotic resistance.

Molecular Crowding Alters the Interactions of Polymyxin Lipopeptides within the Periplasm of E. coli: Insights from Molecular Dynamics.

The cell envelope of Gram-negative bacteria is a crowded tripartite architecture that separates the cell interior from the external environment. Two membranes encapsulate the aqueous periplasm, which contains the cell wall. Little is known about the mechanisms via which antimicrobial peptides move through the periplasm from the outer membrane to their site of action, the inner membrane. We utilize all-atom molecular dynamics to study two antimicrobial peptides, polymyxins B1 and E, within models of the E. coli periplasm crowded to different extents. In a simple chemical environment, both PMB1 and PME bind irreversibly to the cell wall. The presence of specific macromolecules leads to competition with the polymyxins for cell wall interaction sites, resulting in polymyxin dissociation from the cell wall. Chemical complexity also impacts interactions between polymyxins and Braun's lipoprotein; thus, the interaction modes of lipoprotein antibiotics within the periplasm are dependent upon the nature of the other species present.

Polymyxins and analogues bind to ribosomal RNA and interfere with eukaryotic translation in vitro.

Looking for targets: while the bactericidal activity of polymyxins is attributed to changes in membrane permeation, we show that these antibiotics can bind prokaryotic and eukaryotic A-sites, domains responsible for translational decoding. Polymyxin B, colistin and analogues also hinder eukaryotic translation in vitro. These new targets and effects might be partially responsible for the plethora of adverse effects by these potent bactericidal agents.

Lipopolysaccharide as an antibiotic target.

Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii are amongst the highest priority drug-resistant pathogens, for which new antibiotics are urgently needed. Whilst antibiotic drug development is inherently challenging, this is particularly true for Gram-negative bacteria due to the presence of the outer membrane, a highly selective permeability barrier that prevents the ingress of several classes of antibiotic. This selectivity is largely due to an outer leaflet composed of the glycolipid lipopolysaccharide (LPS), which is essential for the viability of almost all Gram-negative bacteria. This essentiality, coupled with the conservation of the synthetic pathway across species and recent breakthroughs in our understanding of transport and membrane homeostasis has made LPS an attractive target for novel antibiotic drug development. Several different targets have been explored and small molecules developed that show promising activity in vitro. However, these endeavours have met limited success in clinical testing and the polymyxins, discovered more than 70 years ago, remain the only LPS-targeting drugs to enter the clinic thus far. In this review, we will discuss efforts to develop therapeutic inhibitors of LPS synthesis and transport and the reasons for limited success, and explore new developments in understanding polymyxin mode of action and the identification of new analogues with reduced toxicity and enhanced activity.

Beyond the identifiable proteome: Delving into the proteomics of polymyxin-resistant and non-resistant Acinetobacter baumannii from Brazilian hospitals.

This work discloses a unique, comprehensive proteomic dataset of Acinetobacter baumannii strains, both resistant and non-resistant to polymyxin B, isolated in Brazil generated using Orbitrap Fusion Lumos. From nearly 4 million tandem mass spectra, the software DiagnoMass produced 240,685 quality-filtered mass spectral clusters, of which PatternLab for proteomics identified 44,553 peptides mapping to 3479 proteins. Crucially, DiagnoMass shortlisted 3550 and 1408 unique mass spectral clusters for the resistant and non-resistant strains, respectively, with only about a third with sequences (and PTMs) identified by PatternLab. Further open-search attempts via FragPipe yielded an additional ∼20% identifications, suggesting the remaining unidentified spectra likely arise from complex combinations of post-translational modifications and amino-acid substitutions. This highlights the untapped potential of the dataset for future discoveries, particularly given the importance of PTMs, which remain elusive to nucleotide sequencing approaches but are crucial for understanding biological mechanisms. Our innovative approach extends beyond the identifications that are typically subjected to the bias of a search engine; we discern which spectral clusters are differential and subject them to increased scrutiny, akin to spectral library matching by comparing captured spectra to themselves. Our analysis reveals adaptations in the resistant strain, including enhanced detoxification, altered protein synthesis, and metabolic adjustments. SIGNIFICANCE: We present comprehensive proteomic profiles of non-resistant and resistant Acinetobacter baumannii from Brazilian Hospitals strains, and highlight the presence of discriminative and yet unidentified mass spectral clusters. Our work emphasizes the importance of exploring this overlooked data, as it could hold the key to understanding the complex dynamics of antibiotic resistance. This approach not only informs antimicrobial stewardship efforts but also paves the way for the development of innovative diagnostic tools. Thus, our findings have profound implications for the field, as far as methods for providing a new perspective on diagnosing antibiotic resistance as well as classifying proteomes in general.

Discovery and Characterization of Polymyxin-Resistance Genes pmrE and pmrF from Sediment and Seawater Microbiome.

Polymyxins are the last-line antibiotics used to treat Gram-negative pathogens. Thus, the discovery and biochemical characterization of the resistance genes against polymyxins are urgently needed for diagnosis, treatment, and novel antibiotic design. Herein, we report novel polymyxin-resistance genes identified from sediment and seawater microbiome. Despite their low sequence identity against the known pmrE and pmrF, they show in vitro activities in UDP-glucose oxidation and l-Ara4N transfer to undecaprenyl phosphate, respectively, which occur as the part of lipid A modification that leads to polymyxin resistance. The expression of pmrE and pmrF also showed substantially high MICs in the presence of vanadate ions, indicating that they constitute polymyxin resistomes. IMPORTANCE Polymyxins are one of the last-resort antibiotics. Polymyxin resistance is a severe threat to combat multidrug-resistant pathogens. Thus, up-to-date identification and understanding of the related genes are crucial. Herein, we performed structure-guided sequence and activity analysis of five putative polymyxin-resistant metagenomes. Despite relatively low sequence identity to the previously reported polymyxin-resistance genes, at least four out of five discovered genes show reactivity essential for lipid A modification and polymyxin resistance, constituting antibiotic resistomes.

Genomic variations in polymyxin-resistant Pseudomonas aeruginosa clinical isolates and their effects on polymyxin resistance.

Infection with P. aeruginosa, one of the most relevant opportunistic pathogens in hospital-acquired infections, can lead to high mortality due to its low antibiotic susceptibility to limited choices of antibiotics. Polymyxin as last-resort antibiotics is used in the treatment of systemic infections caused by multidrug-resistant P. aeruginosa strains, so studying the emergence of polymyxin-resistant was a must. The present study was designed to define genomic differences between paired polymyxin-susceptible and polymyxin-resistant P. aeruginosa strains and established polymyxin resistance mechanisms, and common chromosomal mutations that may confer polymyxin resistance were characterized. A total of 116 CRPA clinical isolates from patients were collected from three tertiary care hospitals in China during 2017-2021. Our study found that polymyxin B resistance represented 3.45% of the isolated carbapenem-resistant P. aeruginosa (CRPA). No polymyxin-resistant isolates were positive for mcr (1-8 and 10) gene and efflux mechanisms. Key genetic variations identified in polymyxin-resistant isolates involved missense mutations in parR, parS, pmrB, pmrA, and phoP. The waaL and PA5005 substitutions related to LPS synthesis were detected in the highest levels of resistant strain (R1). The missense mutations H398R in ParS (4/4), Y345H in PmrB (4/4), and L71R in PmrA (3/4) were the predominant. Results of the PCR further confirmed that mutation of pmrA, pmrB, and phoP individually or simultaneously did affect the expression level of resistant populations and can directly increase the expression of arnBCADTEF operon to contribute to polymyxin resistance. In addition, we reported 3 novel mutations in PA1945 (2129872_A < G, 2130270_A < C, 2130272_T < G) that may confer polymyxin resistance in P. aeruginosa. Our findings enriched the spectrum of chromosomal mutations, highlighted the complexity at the molecular level, and multifaceted interplay mechanisms underlying polymyxin resistance in P. aeruginosa.