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1
Sampson, Timothy R., Xiang Liu, Max R. Schroeder, Colleen S. Kraft, Eileen M. Burd, and David S. Weiss. "Rapid Killing of Acinetobacter baumannii by Polymyxins Is Mediated by a Hydroxyl Radical Death Pathway." Antimicrobial Agents and Chemotherapy 56, no. 11 (August 20, 2012): 5642–49. http://dx.doi.org/10.1128/aac.00756-12.
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ABSTRACTAcinetobacter baumanniiis an opportunistic pathogen that is a cause of clinically significant nosocomial infections. Increasingly, clinical isolates ofA. baumanniiare extensively resistant to numerous antibiotics, and the use of polymyxin antibiotics against these infections is often the final treatment option. Historically, the polymyxins have been thought to kill bacteria through membrane lysis. Here, we present an alternative mechanism based on data demonstrating that polymyxins induce rapid cell death through hydroxyl radical production. Supporting this notion, we found that inhibition of radical production delays the ability of polymyxins to killA. baumannii. Notably, we demonstrate that this mechanism of killing occurs in multidrug-resistant clinical isolates ofA. baumanniiand that this response is not induced in a polymyxin-resistant isolate. This study is the first to demonstrate that polymyxins induce rapid killing ofA. baumanniiand other Gram-negatives through hydroxyl radical production. This significantly augments our understanding of the mechanism of polymyxin action, which is critical knowledge toward the development of adjunctive therapies, particularly given the increasing necessity for treatment with these antibiotics in the clinical setting.
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Li, Mengyao, Mohammad A. K. Azad, Maizbha U. Ahmed, Yan Zhu, Jiangning Song, Fanfan Zhou, Hak-Kim Chan, Tony Velkov, Qi Tony Zhou, and Jian Li. "Polymyxin Induces Significant Transcriptomic Perturbations of Cellular Signalling Networks in Human Lung Epithelial Cells." Antibiotics 11, no. 3 (February 24, 2022): 307. http://dx.doi.org/10.3390/antibiotics11030307.
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Inhaled polymyxins are increasingly used to treat pulmonary infections caused by multidrug-resistant Gram-negative pathogens. We have previously shown that apoptotic pathways, autophagy and oxidative stress are involved in polymyxin-induced toxicity in human lung epithelial cells. In the present study, we employed human lung epithelial cells A549 treated with polymyxin B as a model to elucidate the complex interplay of multiple signalling networks underpinning cellular responses to polymyxin toxicity. Polymyxin B induced toxicity (1.0 mM, 24 h) in A549 cells was assessed by flow cytometry and transcriptomics was performed using microarray. Polymyxin B induced cell death was 19.0 ± 4.2% at 24 h. Differentially expressed genes (DEGs) between the control and polymyxin B treated cells were identified with Student’s t-test. Pathway analysis was conducted with KEGG and Reactome and key hub genes related to polymyxin B induced toxicity were examined using the STRING database. In total we identified 899 DEGs (FDR < 0.01), KEGG and Reactome pathway analyses revealed significantly up-regulated genes related to cell cycle, DNA repair and DNA replication. NF-κB and nucleotide-binding oligomerization domain-like receptor (NOD) signalling pathways were identified as markedly down-regulated genes. Network analysis revealed the top 5 hub genes (i.e., degree) affected by polymyxin B treatment were PLK1(48), CDK20 (46), CCNA2 (42), BUB1 (40) and BUB1B (37). Overall, perturbations of cell cycle, DNA damage and pro-inflammatory NF-κB and NOD-like receptor signalling pathways play key roles in polymyxin-induced toxicity in human lung epithelial cells. Noting that NOD-like receptor signalling represents a group of key sensors for microorganisms and damage in the lung, understanding the mechanism of polymyxin-induced pulmonary toxicity will facilitate the optimisation of polymyxin inhalation therapy in patients.
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Aye, Su Mon, Irene Galani, Mei-Ling Han, Ilias Karaiskos, Darren J. Creek, Yan Zhu, Yu-Wei Lin, Tony Velkov, Helen Giamarellou, and Jian Li. "Lipid A profiling and metabolomics analysis of paired polymyxin-susceptible and -resistant MDR Klebsiella pneumoniae clinical isolates from the same patients before and after colistin treatment." Journal of Antimicrobial Chemotherapy 75, no. 10 (July 22, 2020): 2852–63. http://dx.doi.org/10.1093/jac/dkaa245.
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Abstract Background The increased incidence of polymyxin-resistant MDR Klebsiella pneumoniae has become a major global health concern. Objectives To characterize the lipid A profiles and metabolome differences between paired polymyxin-susceptible and -resistant MDR K. pneumoniae clinical isolates. Methods Three pairs of K. pneumoniae clinical isolates from the same patients were examined [ATH 7 (polymyxin B MIC 0.25 mg/L) versus ATH 8 (64 mg/L); ATH 15 (0.5 mg/L) versus ATH 16 (32 mg/L); and ATH 17 (0.5 mg/L) versus ATH 18 (64 mg/L)]. Lipid A and metabolomes were analysed using LC-MS and bioinformatic analysis was conducted. Results The predominant species of lipid A in all three paired isolates were hexa-acylated and 4-amino-4-deoxy-l-arabinose-modified lipid A species were detected in the three polymyxin-resistant isolates. Significant metabolic differences were evident between the paired isolates. Compared with their corresponding polymyxin-susceptible isolates, the levels of metabolites in amino sugar metabolism (UDP-N-acetyl-α-d-glucosamine and UDP-N-α-acetyl-d-mannosaminuronate) and central carbon metabolism (e.g. pentose phosphate pathway and tricarboxylic acid cycle) were significantly reduced in all polymyxin-resistant isolates [fold change (FC) > 1.5, P < 0.05]. Similarly, nucleotides, amino acids and key metabolites in glycerophospholipid metabolism, namely sn-glycerol-3-phosphate and sn-glycero-3-phosphoethanolamine, were significantly reduced across all polymyxin-resistant isolates (FC > 1.5, P < 0.05) compared with polymyxin-susceptible isolates. However, higher glycerophospholipid levels were evident in polymyxin-resistant ATH 8 and ATH 16 (FC > 1.5, P < 0.05) compared with their corresponding susceptible isolates. Conclusions To our knowledge, this study is the first to reveal significant metabolic perturbations associated with polymyxin resistance in K. pneumoniae.
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Moffatt, Jennifer H., Marina Harper, Paul Harrison, John D. F. Hale, Evgeny Vinogradov, Torsten Seemann, Rebekah Henry, et al. "Colistin Resistance in Acinetobacter baumannii Is Mediated by Complete Loss of Lipopolysaccharide Production." Antimicrobial Agents and Chemotherapy 54, no. 12 (September 20, 2010): 4971–77. http://dx.doi.org/10.1128/aac.00834-10.
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ABSTRACT Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.
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Malott, Rebecca J., Chia-Hung Wu, Tracy D. Lee, Trevor J. Hird, Nathan F. Dalleska, James E. A. Zlosnik, Dianne K. Newman, and David P. Speert. "Fosmidomycin Decreases Membrane Hopanoids and Potentiates the Effects of Colistin on Burkholderia multivorans Clinical Isolates." Antimicrobial Agents and Chemotherapy 58, no. 9 (June 23, 2014): 5211–19. http://dx.doi.org/10.1128/aac.02705-14.
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ABSTRACTBurkholderia cepaciacomplex (Bcc) pulmonary infections in people living with cystic fibrosis (CF) are difficult to treat because of the extreme intrinsic resistance of most isolates to a broad range of antimicrobials. Fosmidomycin is an antibacterial and antiparasitic agent that disrupts the isoprenoid biosynthesis pathway, a precursor to hopanoid biosynthesis. Hopanoids are involved in membrane stability and contribute to polymyxin resistance in Bcc bacteria. Checkerboard MIC assays determined that although isolates of the Bcc speciesB. multivoranswere highly resistant to treatment with fosmidomycin or colistin (polymyxin E), antimicrobial synergy was observed in certain isolates when the antimicrobials were used in combination. Treatment with fosmidomycin decreased the MIC of colistin for isolates as much as 64-fold to as low as 8 μg/ml, a concentration achievable with colistin inhalation therapy. A liquid chromatography-tandem mass spectrometry technique was developed for the accurate quantitative determination of underivatized hopanoids in total lipid extracts, and bacteriohopanetetrol cyclitol ether (BHT-CE) was found to be the dominant hopanoid made byB. multivorans. The amount of BHT-CE made was significantly reduced upon fosmidomycin treatment of the bacteria. Uptake assays with 1-N-phenylnaphthylamine were used to determine that dual treatment with fosmidomycin and colistin increases membrane permeability, while binding assays with boron-dipyrromethene-conjugated polymyxin B illustrated that the addition of fosmidomycin had no impact on polymyxin binding. This work indicates that pharmacological suppression of membrane hopanoids with fosmidomycin treatment can increase the susceptibility of certain clinicalB. multivoransisolates to colistin, an agent currently in use to treat pulmonary infections in CF patients.
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McConville, Thomas, Marla Giddins, Nenad Macesic, and Anne-Catrin Uhlemann. "707. Clarifying the Role of CrrB in Polymxyin-resistant Klebsiella pneumoniae Clinical Isolates Utilizing a Novel CRISPR-Cas9 System." Open Forum Infectious Diseases 5, suppl_1 (November 2018): S254—S255. http://dx.doi.org/10.1093/ofid/ofy210.714.
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Abstract Background Polymyxin resistance (PR) threatens the mainstay of therapy for carbapenem-resistant Enterobacteriaceae (CRE) infections. While mgrB disruption accounts for most cases of PR, missense mutations in crrB have been proposed as an alternative pathway for PR through PmrA/B/C upregulation of the pmrHFIJKLM operon. It remains unknown if CrrB acts as a positive or negative regulator on its downstream targets. Methods We assembled a CRISPR-Cas9 system for gene knockouts (KO) in CRE K. pneumoniae (CRKP) using zeocin as a selectable marker. We chose a polymyxin susceptible (PS) and a PR isolate with a missense mutation in crrB (L87V) (NR5337 and NR5083, respectively) for KO. Isolates were transformed with a crrB KO plasmid, grown with zeocin selection, induced with arabinose, and plated on low-salt LB-zeocin/arabinose. KOs were confirmed via PCR and Sanger sequencing. Polymyxin susceptibility was performed with broth-microdilution. Gene expression was determined by qRT-PCR of cDNA extracts. Results Colistin MIC following crrB KO of NR5337 (PS) remained unchanged. In contrast, crrB KO of NR5083 (PR), decreased polymyxin MIC (MIC >128 to 1.0 μg/mL). qRT-PCR of NR5083 did not show increased expression of pmrA/C, nor pmrK. NR5083 ^crrB showed a small decrease in phoQ expression, compared with NR5083, but similar expression of phoP, pmrA/C and pmrK (Table 1). Conclusion Polymyxin MIC decreased >128 fold after crrB KO in a PR isolate, but colistin MIC remained unchanged after KO in a PS isolate. CrrB mutations in PR isolates may confer a gain of function with CrrB acting as a positive regulator on its downstream targets. Contrary to previous literature, no upregulation of pmrA/C and pmrHFIJKLM was detected. Differences in crrB mutations or clonal background may explain this finding. CRISPR-Cas9 may serve as a reliable system for genetic manipulation of CRKP. Further data on the impact of individual crrB missense mutations are needed. Disclosures A. C. Uhlemann, Merck: Investigator, Grant recipient.
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Lin, Quei Yen, Yi-Lin Tsai, Ming-Che Liu, Wei-Cheng Lin, Po-Ren Hsueh, and Shwu-Jen Liaw. "Serratia marcescensarn, a PhoP-Regulated Locus Necessary for Polymyxin B Resistance." Antimicrobial Agents and Chemotherapy 58, no. 9 (June 23, 2014): 5181–90. http://dx.doi.org/10.1128/aac.00013-14.
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ABSTRACTPolymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly againstSerratia marcescens. To investigate the underlying mechanisms, Tn5mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5inserted into thearnBandarnCgenes. In other bacteria,arnBandarnCbelong to the seven-genearnoperon, which is involved in lipopolysaccharide (LPS) modification. LPSs ofarnmutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility inS. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression ofphoPandarnin the wild-type strain but not in thephoPmutant. Complementation of thephoPmutant with the full-lengthphoPgene restored the PB MIC and induction by PB and low Mg2+levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to thearnpromoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+levels protectedS. marcescensfrom a PB challenge in the wild-type strain but not in thephoPmutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression ofugd, a gene required for LPS modification, inS. marcescensthrough a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression ofarnAupon exposure to PB than did susceptible isolates. This is the first report to describe the role ofS. marcescensarnin PB resistance and its modulation by PB and Mg2+through the PhoP protein.
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Hussein, Maytham, Rafah Allobawi, Irini Levou, Mark A. T. Blaskovich, Gauri G. Rao, Jian Li, and Tony Velkov. "Mechanisms Underlying Synergistic Killing of Polymyxin B in Combination with Cannabidiol against Acinetobacter baumannii: A Metabolomic Study." Pharmaceutics 14, no. 4 (April 3, 2022): 786. http://dx.doi.org/10.3390/pharmaceutics14040786.
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Polymyxins have resurged as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As reports of polymyxin resistance in A. baumannii with monotherapy have become increasingly common, combination therapy is usually the only remaining treatment option. A novel and effective strategy is to combine polymyxins with non-antibiotic drugs. This study aimed to investigate, using untargeted metabolomics, the mechanisms of antibacterial killing synergy of the combination of polymyxin B with a synthetic cannabidiol against A. baumannii ATCC 19606. The antibacterial synergy of the combination against a panel of Gram-negative pathogens (Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) was also explored using checkerboard and static time-kill assays. The polymyxin B–cannabidiol combination showed synergistic antibacterial activity in checkerboard and static time-kill assays against both polymyxin-susceptible and polymyxin-resistant isolates. The metabolomics study at 1 h demonstrated that polymyxin B monotherapy and the combination (to the greatest extent) significantly perturbed the complex interrelated metabolic pathways involved in the bacterial cell envelope biogenesis (amino sugar and nucleotide sugar metabolism, peptidoglycan, and lipopolysaccharide (LPS) biosynthesis), nucleotides (purine and pyrimidine metabolism) and peptide metabolism; notably, these pathways are key regulators of bacterial DNA and RNA biosynthesis. Intriguingly, the combination caused a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids) compared to very minimal changes induced by monotherapies. At 4 h, polymyxin B–cannabidiol induced more pronounced effects on the abovementioned pathways compared to the minimal impact of monotherapies. This metabolomics study for the first time showed that in disorganization of the bacterial envelope formation, the DNA and RNA biosynthetic pathways were the most likely molecular mechanisms for the synergy of the combination. The study suggests the possibility of cannabidiol repositioning, in combination with polymyxins, for treatment of MDR polymyxin-resistant Gram-negative infections.
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Wang, Won-Bo, I.-Chun Chen, Sin-Sien Jiang, Hui-Ru Chen, Chia-Yu Hsu, Po-Ren Hsueh, Wei-Bin Hsu, and Shwu-Jen Liaw. "Role of RppA in the Regulation of Polymyxin B Susceptibility, Swarming, and Virulence Factor Expression in Proteus mirabilis." Infection and Immunity 76, no. 5 (March 3, 2008): 2051–62. http://dx.doi.org/10.1128/iai.01557-07.
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ABSTRACT Proteus mirabilis, a human pathogen that frequently causes urinary tract infections, is intrinsically highly resistant to cationic antimicrobial peptides, such as polymyxin B (PB). To explore the mechanisms underlying P. mirabilis resistance to PB, a mutant which displayed increased (>160-fold) sensitivity to PB was identified by transposon mutagenesis. This mutant was found to have Tn5 inserted into a novel gene, rppA. Sequence analysis indicated that rppA may encode a response regulator of the two-component system and is located upstream of the rppB gene, which may encode a membrane sensor kinase. An rppA knockout mutant of P. mirabilis had an altered lipopolysaccharide (LPS) profile. The LPS purified from the rppA knockout mutant could bind more PB than the LPS purified from the wild type. These properties of the rppA knockout mutant may contribute to its PB-sensitive phenotype. The rppA knockout mutant exhibited greater swarming motility and cytotoxic activity and expressed higher levels of flagellin and hemolysin than the wild type, suggesting that RppA negatively regulates swarming, hemolysin expression, and cytotoxic activity in P. mirabilis. PB could modulate LPS synthesis and modification, swarming, hemolysin expression, and cytotoxic activity in P. mirabilis through an RppA-dependent pathway, suggesting that PB could serve as a signal to regulate RppA activity. Finally, we demonstrated that the expression of rppA was up-regulated by a low concentration of PB and down-regulated by a high concentration of Mg2+. Together, these data highlight the essential role of RppA in regulating PB susceptibility and virulence functions in P. mirabilis.
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Ayoub Moubareck, Carole. "Polymyxins and Bacterial Membranes: A Review of Antibacterial Activity and Mechanisms of Resistance." Membranes 10, no. 8 (August 8, 2020): 181. http://dx.doi.org/10.3390/membranes10080181.
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Following their initial discovery in the 1940s, polymyxin antibiotics fell into disfavor due to their potential clinical toxicity, especially nephrotoxicity. However, the dry antibiotic development pipeline, together with the rising global prevalence of infections caused by multidrug-resistant (MDR) Gram-negative bacteria have both rejuvenated clinical interest in these polypeptide antibiotics. Parallel to the revival of their use, investigations into the mechanisms of action and resistance to polymyxins have intensified. With an initial known effect on biological membranes, research has uncovered the detailed molecular and chemical interactions that polymyxins have with Gram-negative outer membranes and lipopolysaccharide structure. In addition, genetic and epidemiological studies have revealed the basis of resistance to these agents. Nowadays, resistance to polymyxins in MDR Gram-negative pathogens is well elucidated, with chromosomal as well as plasmid-encoded, transferrable pathways. The aims of the current review are to highlight the important chemical, microbiological, and pharmacological properties of polymyxins, to discuss their mechanistic effects on bacterial membranes, and to revise the current knowledge about Gram-negative acquired resistance to these agents. Finally, recent research, directed towards new perspectives for improving these old agents utilized in the 21st century, to combat drug-resistant pathogens, is summarized.
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Yang, G., F. D. Coffman, and E. F. Wheelock. "Characterization and purification of a macrophage-triggering factor produced in Mycoplasma arginini-infected L5178Y cell cultures." Journal of Immunology 153, no. 6 (September 15, 1994): 2579–91. http://dx.doi.org/10.4049/jimmunol.153.6.2579.
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Abstract The supernatant of Mycoplasma arginini-infected murine L5178Y T lymphoma cell cultures (SN-L51) synergizes with small concentrations of IFN-gamma to activate murine peritoneal, thioglycollate-elicited macrophages (M phi) to exhibit cytostatic activity against tumor cells. Treatment of M phi with IFN-gamma and SN-L51 sequentially, but not in the reverse order, activates M phi, which indicates that SN-L51 contains a M phi-triggering factor (MTF). MTF activity could be inhibited by small concentrations of prostaglandin E2, but not by polymyxin B. M phi activated by IFN-gamma plus MTF produce cytostatic effects on tumor cells through a nitric oxide-dependent pathway. MTF activity in SN-L51 is associated with infection of L5178Y cells by M. arginini. Mycoplasma-free L5178Y cells do not produce MTF activity, infection of these L5178Y cells with M. arginini generates the activity, and supernatants of pure M. arginini cultures contain MTF activity. MTF activity is thermostable and resistant to acid, dilute alkali, proteases, and nucleases. MTF was partially purified by ammonium sulfate precipitation, chromatography, electrophoresis, and electroelution. On 12.5% SDS-urea gels, MTF activity migrated with a molecular mass of 2.5 to 4 kDa. MTF activity and the silver staining of this band was resistant to proteinase K; however, Coomassie staining of this band was abolished by proteinase K. The combined data suggest that MTF is either a stable peptide or a peptide linked to lipid or carbohydrate.
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Baum, Ellen Z., Steven M. Crespo-Carbone, Barbara D. Foleno, Lee D. Simon, Jerome Guillemont, Mark Macielag, and Karen Bush. "MurF Inhibitors with Antibacterial Activity: Effect on Muropeptide Levels." Antimicrobial Agents and Chemotherapy 53, no. 8 (August 2009): 3240–47. http://dx.doi.org/10.1128/aac.00166-09.
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ABSTRACT MurF catalyzes the last cytoplasmic step of bacterial cell wall synthesis and is essential for bacterial survival. Our previous studies used a pharmacophore model of a MurF inhibitor to identify additional inhibitors with improved properties. We now present the characterization of two such inhibitors, the diarylquinolines DQ1 and DQ2. DQ1 inhibited Escherichia coli MurF (50% inhibitory concentration, 24 μM) and had modest activity (MICs, 8 to 16 μg/ml) against lipopolysaccharide (LPS)-defective E. coli and wild-type E. coli rendered permeable with polymyxin B nonapeptide. DQ2 additionally displayed activity against gram-positive bacteria (MICs, 8 to 16 μg/ml), including methicillin (meticillin)-susceptible and -resistant Staphylococcus aureus isolates and vancomycin-susceptible and -resistant Enterococcus faecalis and Enterococcus faecium isolates. Treatment of LPS-defective E. coli cells with ≥2× MIC of DQ1 resulted in a 75-fold-greater accumulation of the MurF substrate compared to the control, a 70% decline in the amount of the MurF product, and eventual cell lysis, consistent with the inhibition of MurF within bacteria. DQ2 treatment of S. aureus resulted in similar effects on the MurF substrate and product quantities. At lower levels of DQ1 (≤1× MIC), the level of accumulation of the substrate was less pronounced (15-fold greater compared to the amount for the control). However, a 50% increase in the amount of the MurF product compared to the control was reproducibly observed, consistent with the possible upregulation of muropeptide biosynthesis upon partial inhibition of this pathway. The overexpression of cloned MurF appeared to partly alleviate the DQ1-mediated inhibition of muropeptide synthesis. The identification of MurF inhibitors such as DQ1 and DQ2 that disrupt cell wall biosynthesis suggests that MurF remains a viable target for an antibacterial agent.
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De Oliveira, David M. P., Bernhard Keller, Andrew J. Hayes, Cheryl-Lynn Y. Ong, Nichaela Harbison-Price, Ibrahim M. El-Deeb, Gen Li, et al. "Neurodegenerative Disease Treatment Drug PBT2 Breaks Intrinsic Polymyxin Resistance in Gram-Positive Bacteria." Antibiotics 11, no. 4 (March 25, 2022): 449. http://dx.doi.org/10.3390/antibiotics11040449.
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Gram-positive bacteria do not produce lipopolysaccharide as a cell wall component. As such, the polymyxin class of antibiotics, which exert bactericidal activity against Gram-negative pathogens, are ineffective against Gram-positive bacteria. The safe-for-human-use hydroxyquinoline analog ionophore PBT2 has been previously shown to break polymyxin resistance in Gram-negative bacteria, independent of the lipopolysaccharide modification pathways that confer polymyxin resistance. Here, in combination with zinc, PBT2 was shown to break intrinsic polymyxin resistance in Streptococcus pyogenes (Group A Streptococcus; GAS), Staphylococcus aureus (including methicillin-resistant S. aureus), and vancomycin-resistant Enterococcus faecium. Using the globally disseminated M1T1 GAS strain 5448 as a proof of principle model, colistin in the presence of PBT2 + zinc was shown to be bactericidal in activity. Any resistance that did arise imposed a substantial fitness cost. PBT2 + zinc dysregulated GAS metal ion homeostasis, notably decreasing the cellular manganese content. Using a murine model of wound infection, PBT2 in combination with zinc and colistin proved an efficacious treatment against streptococcal skin infection. These findings provide a foundation from which to investigate the utility of PBT2 and next-generation polymyxin antibiotics for the treatment of Gram-positive bacterial infections.
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Mitra, Mautusi, Kevin Manoap-Anh-Khoa Nguyen, Taylor Wayland Box, Jesse Scott Gilpin, Seth Ryan Hamby, Taylor Lynne Berry, and Erin Harper Duckett. "Isolation and characterization of a novel Sphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources." F1000Research 9 (July 24, 2020): 767. http://dx.doi.org/10.12688/f1000research.25284.1.
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Background: Green micro-alga, Chlamydomonas reinhardtii (a Chlorophyte), can be cultured in the laboratory heterotrophically or photo-heterotrophically in Tris-Phosphate-Acetate (TAP) medium, which contains acetate as the carbon source. Chlamydomonas can convert acetate in the TAP medium to glucose via the glyoxylate cycle, a pathway present in many microbes and higher plants. A novel bacterial strain, CC4533, was isolated from a contaminated TAP agar medium culture plate of a Chlamydomonas wild type strain. In this article, we present our research on the isolation, and biochemical and molecular characterizations of CC4533. Methods: We conducted several microbiological tests and spectrophotometric analyses to biochemically characterize CC4533. The 16S rRNA gene of CC4533 was partially sequenced for taxonomic identification. We monitored the growth of CC4533 on Tris-Phosphate (TP) agar medium (lacks a carbon source) containing different sugars, aromatic compounds and saturated hydrocarbons, to see if CC4533 can use these chemicals as the sole source of carbon. Results: CC4533 is a Gram-negative, non-enteric yellow pigmented, aerobic, mesophilic bacillus. It is alpha-hemolytic and oxidase-positive. CC4533 can ferment glucose, sucrose and lactose, is starch hydrolysis-negative, resistant to penicillin, polymyxin B and chloramphenicol. CC4533 is sensitive to neomycin. Preliminary spectrophotometric analyses indicate that CC4533 produces b-carotenes. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of CC4533 show 99.55% DNA sequence identity to that of Sphingobium yanoikuyae strain PR86 and S. yanoikuyae strain NRB095. CC4533 can use cyclo-chloroalkanes, saturated hydrocarbons present in car motor oil, polyhydroxyalkanoate, and mono- and poly-cyclic aromatic compounds, as sole carbon sources for growth. Conclusions: Taxonomically, CC4533 is very closely related to the alpha-proteobacterium S. yanoikuyae, whose genome has been sequenced. Future research is needed to probe the potential of CC4533 for environmental bioremediation. Whole genome sequencing of CC4533 will confirm if it is a novel strain of S. yanoikuyae or a new Sphingobium species.
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Audrain, Bianca, Lionel Ferrières, Amira Zairi, Guillaume Soubigou, Curtis Dobson, Jean-Yves Coppée, Christophe Beloin, and Jean-Marc Ghigo. "Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides." Applied and Environmental Microbiology 79, no. 24 (October 4, 2013): 7770–79. http://dx.doi.org/10.1128/aem.02593-13.
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ABSTRACTAntimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response ofEscherichia coliupon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σEenvelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S4dermaseptin, also activate severalE. colienvelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes toE. colitolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show thatE. colisenses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway toE. colitolerance to antimicrobial peptides.
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Rodríguez-Santiago, Jonathan, Nadia Rodríguez-Medina, Elsa María Tamayo-Legorreta, Jesús Silva-Sánchez, Juan Téllez-Sosa, Josefina Duran-Bedolla, Alejandro Aguilar-Vera, Alba Neri Lecona-Valera, Ulises Garza-Ramos, and Celia Alpuche-Aranda. "Molecular and Genomic Insights of mcr-1-Producing Escherichia coli Isolates from Piglets." Antibiotics 11, no. 2 (January 26, 2022): 157. http://dx.doi.org/10.3390/antibiotics11020157.
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The use of colistin in food-producing animals favors the emergence and spread of colistin-resistant strains. Here, we investigated the occurrence and molecular mechanisms of colistin resistance among E. coli isolates from a Mexican piglet farm. A collection of 175 cephalosporin-resistant colonies from swine fecal samples were recovered. The colistin resistance phenotype was identified by rapid polymyxin test and the mcr-type genes were screened by PCR. We assessed the colistin-resistant strains by antimicrobial susceptibility test, pulse-field gel electrophoresis, plasmid profile, and mating experiments. Whole-Genome Sequencing data was used to explore the resistome, virulome, and mobilome of colistin-resistant strains. A total of four colistin-resistant E. coli were identified from the cefotaxime-resistant colonies. All harbored the plasmid-borne mcr-1 gene, which was located on conjugative 170-kb IncHI-2 plasmid co-carrying ESBLs genes. Thus, high antimicrobial resistance rates were observed for several antibiotic families. In the RC2-007 strain, the mcr-1 gene was located as part of a prophage carried on non-conjugative 100-kb-plasmid, which upon being transformed into K. variicola strain increased the polymyxin resistance 2-fold. The genomic analysis showed a broad resistome and virulome. Our findings suggest that colistin resistance followed independent acquisition pathways as clonal and non-genetically related mcr-1-harboring strains were identified. These E. coli isolates represent a reservoir of antibiotic resistance and virulence genes in animals for human consumption which could be potentially propagated into other interfaces.
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Avedissian, Sean N., Jiajun Liu, Nathaniel J. Rhodes, Andrew Lee, Gwendolyn M. Pais, Alan R. Hauser, and Marc H. Scheetz. "A Review of the Clinical Pharmacokinetics of Polymyxin B." Antibiotics 8, no. 1 (March 22, 2019): 31. http://dx.doi.org/10.3390/antibiotics8010031.
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Polymyxin B remains an antibiotic of last resort because of its toxicities. Although newer therapies are becoming available, it is anticipated that resistance to these agents will continue to emerge, and understanding the safest and most efficacious manner to deliver polymyxin B will remain highly important. Recent data have demonstrated that polymyxin B may be less nephrotoxic than colistin. Pharmacokinetically, polymyxin B is primarily eliminated via non-renal pathways, and most do not recommend adjusting the dose for renal impairment. However, some recent studies suggest a weak relationship between polymyxin B clearance and patient creatinine clearance. This review article will describe the clinical pharmacokinetics of polymyxin B and address relevant issues in chemistry and assays available.
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Campos, Miguel A., Miguel A. Vargas, Verónica Regueiro, Catalina M. Llompart, Sebastián Albertí, and José A. Bengoechea. "Capsule Polysaccharide Mediates Bacterial Resistance to Antimicrobial Peptides." Infection and Immunity 72, no. 12 (December 2004): 7107–14. http://dx.doi.org/10.1128/iai.72.12.7107-7114.2004.
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ABSTRACT The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, β-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.
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Lewis, Lisa A., Biswa Choudhury, Jacqueline T. Balthazar, Larry E. Martin, Sanjay Ram, Peter A. Rice, David S. Stephens, Russell Carlson, and William M. Shafer. "Phosphoethanolamine Substitution of Lipid A and Resistance of Neisseria gonorrhoeae to Cationic Antimicrobial Peptides and Complement-Mediated Killing by Normal Human Serum." Infection and Immunity 77, no. 3 (December 29, 2008): 1112–20. http://dx.doi.org/10.1128/iai.01280-08.
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ABSTRACT The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide β-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.
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Harshaw, Nathaniel S., Nicholas A. Stella, Kara M. Lehner, Eric G. Romanowski, Regis P. Kowalski, and Robert M. Q. Shanks. "Antibiotics Used in Empiric Treatment of Ocular Infections Trigger the Bacterial Rcs Stress Response System Independent of Antibiotic Susceptibility." Antibiotics 10, no. 9 (August 25, 2021): 1033. http://dx.doi.org/10.3390/antibiotics10091033.
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The Rcs phosphorelay is a bacterial stress response system that responds to envelope stresses and in turn controls several virulence-associated pathways, including capsule, flagella, and toxin biosynthesis, of numerous bacterial species. The Rcs system also affects antibiotic tolerance, biofilm formation, and horizontal gene transfer. The Rcs system of the ocular bacterial pathogen Serratia marcescens was recently demonstrated to influence ocular pathogenesis in a rabbit model of keratitis, with Rcs-defective mutants causing greater pathology and Rcs-activated strains demonstrating reduced inflammation. The Rcs system is activated by a variety of insults, including β-lactam antibiotics and polymyxin B. In this study, we developed three luminescence-based transcriptional reporters for Rcs system activity and used them to test whether antibiotics used for empiric treatment of ocular infections influence Rcs system activity in a keratitis isolate of S. marcescens. These included antibiotics to which the bacteria were susceptible and resistant. Results indicate that cefazolin, ceftazidime, polymyxin B, and vancomycin activate the Rcs system to varying degrees in an RcsB-dependent manner, whereas ciprofloxacin and tobramycin activated the promoter fusions, but in an Rcs-independent manner. Although minimum inhibitory concentration (MIC) analysis demonstrated resistance of the test bacteria to polymyxin B and vancomycin, the Rcs system was activated by sub-inhibitory concentrations of these antibiotics. Together, these data indicate that a bacterial stress system that influences numerous pathogenic phenotypes and drug-tolerance is influenced by different classes of antibiotics despite the susceptibility status of the bacterium.
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Pilonieta, M. Carolina, Kimberly D. Erickson, Robert K. Ernst, and Corrella S. Detweiler. "A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC." Journal of Bacteriology 191, no. 23 (September 18, 2009): 7243–52. http://dx.doi.org/10.1128/jb.00688-09.
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ABSTRACT Antimicrobial peptides (AMPs) kill or prevent the growth of microbes. AMPs are made by virtually all single and multicellular organisms and are encountered by bacteria in diverse environments, including within a host. Bacteria use sensor-kinase systems to respond to AMPs or damage caused by AMPs. Salmonella enterica deploys at least three different sensor-kinase systems to modify gene expression in the presence of AMPs: PhoP-PhoQ, PmrA-PmrB, and RcsB-RcsC-RcsD. The ydeI gene is regulated by the RcsB-RcsC-RcsD pathway and encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in broth and also for virulence in mice. We report here that ydeI is additionally regulated by the PhoP-PhoQ and PmrA-PmrB sensor-kinase systems, which confer resistance to cationic AMPs by modifying lipopolysaccharide (LPS). ydeI, however, is not important for known LPS modifications. Two independent biochemical methods found that YdeI copurifies with OmpD/NmpC, a member of the trimeric β-barrel outer membrane general porin family. Genetic analysis indicates that ompD contributes to polymyxin B resistance, and both ydeI and ompD are important for resistance to cathelicidin antimicrobial peptide, a mouse AMP produced by multiple cell types and expressed in the gut. YdeI localizes to the periplasm, where it could interact with OmpD. A second predicted periplasmic OB-fold protein, YgiW, and OmpF, another general porin, also contribute to polymyxin B resistance. Collectively, the data suggest that periplasmic OB-fold proteins can interact with porins to increase bacterial resistance to AMPs.
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Huang, Can, Wenqian Li, and Jingyu Chen. "Stringent Response Factor DksA Contributes to Fatty Acid Degradation Function to Influence Cell Membrane Stability and Polymyxin B Resistance of Yersinia enterocolitica." International Journal of Molecular Sciences 24, no. 15 (July 26, 2023): 11951. http://dx.doi.org/10.3390/ijms241511951.
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DksA is a proteobacterial regulator that binds directly to the secondary channel of RNA polymerase with (p)ppGpp and is responsible for various bacterial physiological activities. While (p)ppGpp is known to be involved in the regulation and response of fatty acid metabolism pathways in many foodborne pathogens, the role of DksA in this process has yet to be clarified. This study aimed to characterize the function of DksA on fatty acid metabolism and cell membrane structure in Yersinia enterocolitica. Therefore, comparison analysis of gene expression, growth conditions, and membrane permeabilization among the wide-type (WT), DksA-deficient mutant (YEND), and the complemented strain was carried out. It confirmed that deletion of DksA led to a more than four-fold decrease in the expression of fatty acid degradation genes, including fadADEIJ. Additionally, YEND exhibited a smaller growth gap compared to the WT strain at low temperatures, indicating that DksA is not required for the growth of Y. enterocolitica in cold environments. Given that polymyxin B is a cationic antimicrobial peptide that targets the cell membrane, the roles of DksA under polymyxin B exposure were also characterized. It was found that DksA positively regulates the integrity of the inner and outer membranes of Y. enterocolitica under polymyxin B, preventing the leakage of intracellular nucleic acids and proteins and ultimately reducing the sensitivity of Y. enterocolitica to polymyxin B. Taken together, this study provides insights into the functions of DksA and paves the way for novel fungicide development.
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Kumar, Nitin, Chih-Chia Su, Tsung-Han Chou, Abhijith Radhakrishnan, Jared A. Delmar, Kanagalaghatta R. Rajashankar, and Edward W. Yu. "Crystal structures of the Burkholderia multivorans hopanoid transporter HpnN." Proceedings of the National Academy of Sciences 114, no. 25 (June 5, 2017): 6557–62. http://dx.doi.org/10.1073/pnas.1619660114.
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Strains of the Burkholderia cepacia complex (Bcc) are Gram-negative opportunisitic bacteria that are capable of causing serious diseases, mainly in immunocompromised individuals. Bcc pathogens are intrinsically resistant to multiple antibiotics, including β-lactams, aminoglycosides, fluoroquinolones, and polymyxins. They are major pathogens in patients with cystic fibrosis (CF) and can cause severe necrotizing pneumonia, which is often fatal. Hopanoid biosynthesis is one of the major mechanisms involved in multiple antimicrobial resistance of Bcc pathogens. The hpnN gene of B. multivorans encodes an integral membrane protein of the HpnN family of transporters, which is responsible for shuttling hopanoids to the outer membrane. Here, we report crystal structures of B. multivorans HpnN, revealing a dimeric molecule with an overall butterfly shape. Each subunit of the transporter contains 12 transmembrane helices and two periplasmic loops that suggest a plausible pathway for substrate transport. Further analyses indicate that HpnN is capable of shuttling hopanoid virulence factors from the outer leaflet of the inner membrane to the periplasm. Taken together, our data suggest that the HpnN transporter is critical for multidrug resistance and cell wall remodeling in Burkholderia.
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Swaminathan, Subramanian, and Prithwijit Kundu. "Tigecycline: Role in the Management of cIAI and cSSTI in the Indian Context." Indian Journal of Clinical Medicine 10, no. 1-2 (June 2020): 24–30. http://dx.doi.org/10.1177/26339447211067579.
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The current millennium has witnessed an increased antimicrobial resistance which poses a mammoth challenge for public health management. This has resulted in an increase in morbidity and mortality, resulting in an increase in financial burden to the patients. A recent analysis from 10 hospitals in India reported that mortality rate increases by 1.57 times in patients suffering from multidrug resistance (MDR) bacterial infections as compared to patients infected with similar but susceptible infections. Due to the emergence of MDR and extensively drug-resistant (XDR) bacteria, most of the broad-spectrum antibiotics have been rendered ineffective. The mortality rate with Gram-negative strains is higher than with Gram-positive strains. Tigecycline is the first in class glycylcycline antibiotic with an expanded broad-spectrum activity. Tigecycline enters bacterial cells through energy-dependent pathways or via passive diffusion, to reversibly bind to the 30S ribosomal subunit. It has potent in vitro activity against Gram-negative carbapenemase producers, except Pseudomonas aeruginosa and Proteus spp. It also has good in vitro activity against Carbapenem-resistant Klebsiella pneumoniae strains. Hence, it is considered as a therapeutic option in XDR isolates. Recent meta-analyses have shown tigecycline to be as effective as its comparators with reducing mortality rates. Due to increased resistance reported in carbapenem-resistant isolates in Indian health-care settings, a colistin/polymyxin B-based combination therapy as a treatment option is being sought. A lower mortality rate has been reported with colistin-based combination therapy in Carbapenem-resistant Enterobacteriaceae-associated infections. Combinations with tigecycline, Fosfomycin, and chloramphenicol have shown to improve treatment outcomes. Tigecycline can be a good alternative in MDR and XDR complicated intra-abdominal and complicated skin and soft tissue infections. Appropriately designed clinical trials in Indian health-care setups will reinforce clinician’s confidence in using tigecycline in complex clinical situations.
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Pitt, Miranda E., Minh Duc Cao, Mark S. Butler, Soumya Ramu, Devika Ganesamoorthy, Mark A. T. Blaskovich, Lachlan J. M. Coin, and Matthew A. Cooper. "Octapeptin C4 and polymyxin resistance occur via distinct pathways in an epidemic XDRKlebsiella pneumoniaeST258 isolate." Journal of Antimicrobial Chemotherapy 74, no. 3 (November 14, 2018): 582–93. http://dx.doi.org/10.1093/jac/dky458.
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Bina, J. E., R. A. Alm, M. Uria-Nickelsen, S. R. Thomas, T. J. Trust, and R. E. W. Hancock. "Helicobacter pylori Uptake and Efflux: Basis for Intrinsic Susceptibility to Antibiotics In Vitro." Antimicrobial Agents and Chemotherapy 44, no. 2 (February 1, 2000): 248–54. http://dx.doi.org/10.1128/aac.44.2.248-254.2000.
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ABSTRACT We previously demonstrated (M. M. Exner, P. Doig, T. J. Trust, and R. E. W. Hancock, Infect. Immun. 63:1567–1572, 1995) that Helicobacter pylori has at least one nonspecific porin, HopE, which has a low abundance in the outer membrane but forms large channels. H. pylori is relatively susceptible to most antimicrobial agents but less susceptible to the polycationic antibiotic polymyxin B. We demonstrate here that H. pylori is able to take up higher basal levels of the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN) thanPseudomonas aeruginosa or Escherichia coli, consistent with its enhanced susceptibility to hydrophobic agents. Addition of polymyxin B led to a further increase in NPN uptake, indicative of a self-promoted uptake pathway, but it required a much higher amount of polymyxin B to yield a 50% increase in NPN uptake inH. pylori (6 to 8 μg/ml) than in P. aeruginosa or E. coli (0.3 to 0.5 μg/ml), suggesting that H. pylori has a less efficient self-promoted uptake pathway. Since intrinsic resistance involves the collaboration of restricted outer membrane permeability and secondary defense mechanisms, such as periplasmic β-lactamase (which H. pylori lacks) or efflux, we examined the possible role of efflux in antibiotic susceptibility. We had previously identified in H. pylori 11637 the presence of portions of three genes with homology to potential restriction-nodulation-division (RND) efflux systems. It was confirmed that H. pylori contained only these three putative RND efflux systems, named here hefABC, hefDEF, andhefGHI, and that the hefGHI system was expressed only in vivo while the two other RND systems were expressed both in vivo and in vitro. In uptake studies, there was no observable energy-dependent tetracycline, chloramphenicol, or NPN efflux activity in H. pylori. Independent mutagenesis of the three putative RND efflux operons in the chromosome of H. pylori had no effect on the in vitro susceptibility of H. pylori to 19 antibiotics. These results, in contrast to what is observed inE. coli, P. aeruginosa, and other clinically important gram-negative bacteria, suggest that active efflux does not play a role in the intrinsic resistance of H. pylori to antibiotics.
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Chen, Bin, Hailiang Han, Junfeng Hou, Fei Bao, Heping Tan, Xiaocheng Lou, Guiyue Wang, and Fucheng Zhao. "Control of Maize Sheath Blight and Elicit Induced Systemic Resistance Using Paenibacillus polymyxa Strain SF05." Microorganisms 10, no. 7 (June 29, 2022): 1318. http://dx.doi.org/10.3390/microorganisms10071318.
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Maize (Zea mays L.) is an important crop in the world and maize sheath blight damages the yield and quality greatly. In this study, an antagonist strain, which exhibited antagonism against pathogenic fungi of maize and controlled maize banded leaf sheath blight in the field, was effectively isolated and named Paenibacillus polymyxa strain SF05. High cellulase and chitinase activity of the strain were detected in this study, which might contribute to degrading the cell wall of fungi. Furthermore, different resistant genes such as ZmPR1a, OPR1 and OPR7 were elicited differently by the strain in the leaves and stems of maize. In order to explain the biocontrol mechanism of P. polymyxa strain SF05, the genome was sequenced and then the genes involving the biocontrol mechanism including biofilm formation pathways genes, cell wall degradation enzymes, secondary metabolite biosynthesis gene clusters and volatile organic compounds biosynthesis genes were predicted. The study revealed the biocontrol mechanism of P. polymyxa strain SF05 preliminary and laid a foundation for further research of biocontrol mechanism of P. polymyxa.
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Cheng, Hsin-Yao, Yi-Fong Chen, and Hwei-Ling Peng. "Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43." Journal of Biomedical Science 17, no. 1 (2010): 60. http://dx.doi.org/10.1186/1423-0127-17-60.
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Semenzato, Giulia, Tania Alonso-Vásquez, Sara Del Duca, Alberto Vassallo, Christopher Riccardi, Marco Zaccaroni, Nadia Mucci, et al. "Genomic Analysis of Endophytic Bacillus-Related Strains Isolated from the Medicinal Plant Origanum vulgare L. Revealed the Presence of Metabolic Pathways Involved in the Biosynthesis of Bioactive Compounds." Microorganisms 10, no. 5 (April 27, 2022): 919. http://dx.doi.org/10.3390/microorganisms10050919.
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Multidrug-resistant pathogens represent a serious threat to human health. The inefficacy of traditional antibiotic drugs could be surmounted through the exploitation of natural bioactive compounds of which medicinal plants are a great reservoir. The finding that bacteria living inside plant tissues, (i.e., the endophytic bacterial microbiome) can influence the synthesis of the aforementioned compounds leads to the necessity of unraveling the mechanisms involved in the determination of this symbiotic relationship. Here, we report the genome sequence of four endophytic bacterial strains isolated from the medicinal plant Origanum vulgare L. and able to antagonize the growth of opportunistic pathogens of cystic fibrosis patients. The in silico analysis revealed the presence of gene clusters involved in the production of antimicrobial compounds, such as paeninodin, paenilarvins, polymyxin, and paenicidin A. Endophytes’ adaptation to the plant microenvironment was evaluated through the analysis of the presence of antibiotic resistance genes in the four genomes. The diesel fuel degrading potential was also tested. Strains grew in minimum media supplemented with diesel fuel, but no n-alkanes degradation genes were found in their genomes, suggesting that diesel fuel degradation might occur through other steps involving enzymes catalyzing the oxidation of aromatic compounds.
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Lee, Myeongseon, and Marcelo C. Sousa. "Structural Basis for Substrate Specificity in ArnB. A Key Enzyme in the Polymyxin Resistance Pathway of Gram-Negative Bacteria." Biochemistry 53, no. 4 (January 24, 2014): 796–805. http://dx.doi.org/10.1021/bi4015677.
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Adnan, Mohd, Arif Jamal Siddiqui, Emira Noumi, Sami Hannachi, Syed Amir Ashraf, Amir Mahgoub Awadelkareem, Mejdi Snoussi, et al. "Integrating Network Pharmacology Approaches to Decipher the Multi-Target Pharmacological Mechanism of Microbial Biosurfactants as Novel Green Antimicrobials Against Listeriosis." Antibiotics 12, no. 1 (December 20, 2022): 5. http://dx.doi.org/10.3390/antibiotics12010005.
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Listeria monocytogenes (L. monocytogenes) is a serious food-borne pathogen that can cause listeriosis, an illness caused by eating food contaminated with this pathogen. Currently, the treatment or prevention of listeriosis is a global challenge due to the resistance of bacteria against multiple commonly used antibiotics, thus necessitating the development of novel green antimicrobials. Scientists are increasingly interested in microbial surfactants, commonly known as “biosurfactants”, due to their antimicrobial properties and eco-friendly nature, which make them an ideal candidate to combat a variety of bacterial infections. Therefore, the present study was designed to use a network pharmacology approach to uncover the active biosurfactants and their potential targets, as well as the signaling pathway(s) involved in listeriosis treatment. In the framework of this study, 15 biosurfactants were screened out for subsequent studies. Among 546 putative targets of biosurfactants and 244 targets of disease, 37 targets were identified as potential targets for treatment of L. monocytogenes infection, and these 37 targets were significantly enriched in a Gene Ontology (GO) analysis, which aims to identify those biological processes, cellular locations, and molecular functions that are impacted in the condition studied. The obtained results revealed several important biological processes, such as positive regulation of MAP kinase activity, protein kinase B signaling, ERK1 and ERK2 cascade, ERBB signaling pathway, positive regulation of protein serine/threonine kinase activity, and regulation of caveolin-mediated endocytosis. Several important KEGG pathways, such as the ERBBB signaling pathway, TH17 cell differentiation, HIF-1 signaling pathway, Yersinia infection, Shigellosis, and C-type lectin receptor signaling pathways, were identified. The protein–protein interaction analysis yielded 10 core targets (IL2, MAPK1, EGFR, PTPRC, TNF, ITGB1, IL1B, ERBB2, SRC, and mTOR). Molecular docking was used in the latter part of the study to verify the effectiveness of the active biosurfactants against the potential targets. Lastly, we found that a few highly active biosurfactants, namely lichenysin, iturin, surfactin, rhamnolipid, subtilisin, and polymyxin, had high binding affinities towards IL2, MAPK1, EGFR, PTPRC, TNF, ITGB1, IL1B, ERBB2, SRC, and mTOR, which may act as potential therapeutic targets for listeriosis. Overall, based on the integrated network pharmacology and docking analysis, we found that biosurfactants possess promising anti-listeriosis properties and explored the pharmacological mechanisms behind their effect, laying the groundwork for further research and development.
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Timmusk, Salme, and E. Gerhart H. Wagner. "The Plant-Growth-Promoting Rhizobacterium Paenibacillus polymyxa Induces Changes in Arabidopsis thaliana Gene Expression: A Possible Connection Between Biotic and Abiotic Stress Responses." Molecular Plant-Microbe Interactions® 12, no. 11 (November 1999): 951–59. http://dx.doi.org/10.1094/mpmi.1999.12.11.951.
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This paper addresses changes in plant gene expression induced by inoculation with plant-growth-promoting rhizobacteria (PGPR). A gnotobiotic system was established with Arabidopsis thaliana as model plant, and isolates of Paenibacillus polymyxa as PGPR. Subsequent challenge by either the pathogen Erwinia carotovora (biotic stress) or induction of drought (abiotic stress) indicated that inoculated plants were more resistant than control plants. With RNA differential display on parallel RNA preparations from P. polymyxa- treated or untreated plants, changes in gene expression were investigated. From a small number of candidate sequences obtained by this approach, one mRNA segment showed a strong inoculation-dependent increase in abundance. The corresponding gene was identified as ERD15, previously identified to be drought stress responsive. Quantification of mRNA levels of several stress-responsive genes indicated that P. polymyxa induced mild biotic stress. This suggests that genes and/or gene classes associated with plant defenses against abiotic and biotic stress may be co-regulated. Implications of the effects of PGPR on the induction of plant defense pathways are discussed.
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Fischer, Utz, Simon Hertlein, and Clemens Grimm. "The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity." Acta Crystallographica Section D Biological Crystallography 71, no. 3 (February 26, 2015): 687–96. http://dx.doi.org/10.1107/s1399004714026686.
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The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD+-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.
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Hussein, Maytham, Labell J. M. Wong, Jinxin Zhao, Vanessa E. Rees, Rafah Allobawi, Rajnikant Sharma, Gauri G. Rao, Mark Baker, Jian Li, and Tony Velkov. "Unique mechanistic insights into pathways associated with the synergistic activity of polymyxin B and caspofungin against multidrug-resistant Klebsiella pneumoniae." Computational and Structural Biotechnology Journal 20 (2022): 1077–87. http://dx.doi.org/10.1016/j.csbj.2022.02.021.
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Han, Mei-Ling, Sue C. Nang, Yu-Wei Lin, Yan Zhu, Heidi H. Yu, Hasini Wickremasinghe, Christopher K. Barlow, et al. "Comparative metabolomics revealed key pathways associated with the synergistic killing of multidrug-resistant Klebsiella pneumoniae by a bacteriophage-polymyxin combination." Computational and Structural Biotechnology Journal 20 (2022): 485–95. http://dx.doi.org/10.1016/j.csbj.2021.12.039.
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Zhao, Jinxin, Mei-Ling Han, Yan Zhu, Yu-Wei Lin, Yi-Wen Wang, Jing Lu, Yang Hu, Qi Tony Zhou, Tony Velkov, and Jian Li. "Comparative metabolomics reveals key pathways associated with the synergistic activity of polymyxin B and rifampicin combination against multidrug-resistant Acinetobacter baumannii." Biochemical Pharmacology 184 (February 2021): 114400. http://dx.doi.org/10.1016/j.bcp.2020.114400.
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Abdelkhalek, Ahmed, Abdulaziz A. Al-Askar, Toufic Elbeaino, Hassan Moawad, and Hamada El-Gendi. "Protective and Curative Activities of Paenibacillus polymyxa against Zucchini yellow mosaic virus Infestation in Squash Plants." Biology 11, no. 8 (July 30, 2022): 1150. http://dx.doi.org/10.3390/biology11081150.
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The use of microbial products as natural biocontrol agents to increase a plant’s systemic resistance to viral infections is a promising way to make agriculture more sustainable and less harmful to the environment. The rhizobacterium Paenibacillus polymyxa has been shown to have strong biocontrol action against plant diseases, but its antiviral activity has been little investigated. Here, the efficiency of the culture filtrate of the P. polymyxa strain SZYM (Acc# ON149452) to protect squash (Cucurbita pepo L.) plants against a Zucchini yellow mosaic virus (ZYMV, Acc# ON159933) infection was evaluated. Under greenhouse conditions, the foliar application of the culture filtrate of SZYM either in protective or curative treatment conditions enhanced squash growth, reduced disease severity, and decreased ZYMV accumulation levels in the treated plants when compared to the non-treated plants. The protective treatment group exhibited the highest inhibitory effect (80%), with significant increases in their total soluble carbohydrates, total soluble protein content, ascorbic acid content, and free radical scavenging activity. Furthermore, a considerable increase in the activities of reactive oxygen species scavenging enzymes (superoxide dismutase, polyphenol oxidase, and peroxidase) were also found. In addition, the induction of systemic resistance with a significant elevation in the transcriptional levels of polyphenolic pathway genes (CHS, PAL, and C3H) and pathogenesis-related genes (PR-1 and PR-3) was observed. Out of the 14 detected compounds in the GC–MS analysis, propanoic acid, benzenedicarboxylic acid, tetradecanoic acid, and their derivatives, as well as pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) were the primary ingredient compounds in the ethyl acetate extract of the SZYM-culture filtrate. Such compounds may act as elicitor molecules that induce systemic resistance against viral infection. Consequently, P. polymyxa can be considered a powerful plant growth-promoting bacterium (PGPB) in agricultural applications as well as a source of bioactive compounds for sustainable disease management. As far as we know, this is the first time that P. polymyxa has been shown to fight viruses in plants.
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Pi, Hongfei, Abiodun D. Ogunniyi, Bhumi Savaliya, Hang Thi Nguyen, Stephen W. Page, Ernest Lacey, Henrietta Venter, and Darren J. Trott. "Repurposing of the Fasciolicide Triclabendazole to Treat Infections Caused by Staphylococcus spp. and Vancomycin-Resistant Enterococci." Microorganisms 9, no. 8 (August 10, 2021): 1697. http://dx.doi.org/10.3390/microorganisms9081697.
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One approach to combat the increasing incidence of multidrug-resistant (MDR) bacterial pathogens involves repurposing existing compounds with known safety and development pathways as new antibacterial classes with potentially novel mechanisms of action. Here, triclabendazole (TCBZ), a drug originally developed to treat Fasciola hepatica (liver fluke) in sheep and cattle, and later in humans, was evaluated as an antibacterial alone or in combination with sub-inhibitory concentrations of polymyxin B (PMB) against clinical isolates and reference strains of key Gram-positive and Gram-negative bacteria. We show for the first time that in vitro, TCBZ selectively kills methicillin-sensitive and methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius at a minimum inhibitory concentration (MIC) range of 2–4 µg/mL, and vancomycin-resistant enterococci at a MIC range of 4–8 µg/mL. TCBZ also inhibited key Gram-negative bacteria in the presence of sub-inhibitory concentrations of PMB, returning MIC90 values of 1 µg/mL for Escherichia coli, 8 µg/mL for Klebsiella pneumoniae, 2 µg/mL for Acinetobacter baumannii and 4 µg/mL for Pseudomonasaeruginosa. Interestingly, TCBZ was found to be bacteriostatic against intracellular S. aureus but bactericidal against intracellular S. pseudintermedius. Additionally, TCBZ’s favourable pharmacokinetic (PK) and pharmacodynamic (PD) profile was further explored by in vivo safety and efficacy studies using a bioluminescent mouse model of S. aureus sepsis. We show that repeated four-hourly oral treatment of mice with 50 mg/kg TCBZ after systemic S. aureus challenge resulted in a significant reduction in S. aureus populations in the blood to 18 h post-infection (compared to untreated mice) but did not clear the bacterial infection from the bloodstream, consistent with in vivo bacteriostatic activity. These results indicate that additional pharmaceutical development of TCBZ may enhance its PK/PD, allowing it to be an appropriate candidate for the treatment of serious MDR bacterial pathogens.
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Kong, Qingke, David A. Six, Qing Liu, Lillian Gu, Shifeng Wang, Praveen Alamuri, Christian R. H. Raetz, and Roy Curtiss. "Phosphate Groups of Lipid A Are Essential for Salmonella enterica Serovar Typhimurium Virulence and Affect Innate and Adaptive Immunity." Infection and Immunity 80, no. 9 (July 2, 2012): 3215–24. http://dx.doi.org/10.1128/iai.00123-12.
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ABSTRACTLipid A is a key component of the outer membrane of Gram-negative bacteria and stimulates proinflammatory responses via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. Its endotoxic activity depends on the number and length of acyl chains and its phosphorylation state. InSalmonella entericaserovar Typhimurium, removal of the secondary laurate or myristate chain in lipid A results in bacterial attenuation and growth defectsin vitro. However, the roles of the two lipid A phosphate groups in bacterial virulence and immunogenicity remain unknown. Here, we used anS. TyphimuriummsbB pagL pagP lpxRmutant, carrying penta-acylated lipid A, as the parent strain to construct a series of mutants synthesizing 1-dephosphorylated, 4′-dephosphorylated, or nonphosphorylated penta-acylated lipid A. Dephosphorylated mutants exhibited increased sensitivity to deoxycholate and showed increased resistance to polymyxin B. Removal of both phosphate groups severely attenuated the mutants when administered orally to BALB/c mice, but the mutants colonized the lymphatic tissues and were sufficiently immunogenic to protect the host from challenge with wild-typeS. Typhimurium. Mice receivingS. Typhimurium with 1-dephosphorylated or nonphosphorylated penta-acylated lipid A exhibited reduced levels of cytokines. Attenuated and dephosphorylatedSalmonellavaccines were able to induce adaptive immunity against heterologous (PspA ofStreptococcus pneumoniae) and homologous antigens (lipopolysaccharide [LPS] and outer membrane proteins [OMPs]).
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Chacon, Ofelia, Luiz E. Bermudez, Denise K. Zinniel, Harpreet K. Chahal, Robert J. Fenton, Zhengyu Feng, Kathy Hanford, L. Garry Adams, and Raúl G. Barletta. "Impairment of d-alanine biosynthesis in Mycobacterium smegmatis determines decreased intracellular survival in human macrophages." Microbiology 155, no. 5 (May 1, 2009): 1440–50. http://dx.doi.org/10.1099/mic.0.024901-0.
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d-Alanine is a structural component of mycobacterial peptidoglycan. The primary route of d-alanine biosynthesis in eubacteria is the enantiomeric conversion from l-alanine, a reaction catalysed by d-alanine racemase (Alr). Mycobacterium smegmatis alr insertion mutants are not dependent on d-alanine for growth and display a metabolic pattern consistent with an alternative pathway for d-alanine biosynthesis. In this study, we demonstrate that the M. smegmatis alr insertion mutant TAM23 can synthesize d-alanine at lower levels than the parental strain. The insertional inactivation of the alr gene also decreases the intracellular survival of mutant strains within primary human monocyte-derived macrophages. By complementation studies, we confirmed that the impairment of alr gene function is responsible for this reduced survival. Inhibition of superoxide anion and nitric oxide formation in macrophages suppresses the differential survival. In contrast, for bacteria grown in broth, both strains had approximately the same susceptibility to hydrogen peroxide, acidified sodium nitrite, low pH and polymyxin B. In contrast, TAM23 exhibited increased resistance to lysozyme. d-Alanine supplementation considerably increased TAM23 viability in nutritionally deficient media and within macrophages. These results suggest that nutrient deprivation in phagocytic cells combined with killing mediated by reactive intermediates underlies the decreased survival of alr mutants. This knowledge may be valuable in the construction of mycobacterial auxotrophic vaccine candidates.
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McLeod, G. I., and M. P. Spector. "Starvation- and Stationary-phase-induced resistance to the antimicrobial peptide polymyxin B in Salmonella typhimurium is RpoS (sigma(S)) independent and occurs through both phoP-dependent and -independent pathways." Journal of bacteriology 178, no. 13 (1996): 3683–88. http://dx.doi.org/10.1128/jb.178.13.3683-3688.1996.
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Hussein, Maytham, Mei-Ling Han, Yan Zhu, Qi Zhou, Yu-Wei Lin, Robert E. W. Hancock, Daniel Hoyer, Darren J. Creek, Jian Li, and Tony Velkov. "Metabolomics Study of the Synergistic Killing of Polymyxin B in Combination with Amikacin against Polymyxin-Susceptible and -Resistant Pseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 64, no. 1 (October 14, 2019). http://dx.doi.org/10.1128/aac.01587-19.
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ABSTRACT In the present study, we employed untargeted metabolomics to investigate the synergistic killing mechanism of polymyxin B in combination with an aminoglycoside, amikacin, against a polymyxin-susceptible isolate of Pseudomonas aeruginosa, FADDI-PA111 (MIC = 2 mg/liter for both polymyxin B and amikacin), and a polymyxin-resistant Liverpool epidemic strain (LES), LESB58 (the corresponding MIC for both polymyxin B and amikacin is 16 mg/liter). The metabolites were extracted 15 min, 1 h, and 4 h following treatment with polymyxin B alone (2 mg/liter for FADDI-PA111; 4 mg/liter for LESB58), amikacin alone (2 mg/liter), and both in combination and analyzed using liquid chromatography-mass spectrometry (LC-MS). At 15 min and 1 h, polymyxin B alone induced significant perturbations in glycerophospholipid and fatty acid metabolism pathways in FADDI-PA111 and, to a lesser extent, in LESB58. Amikacin alone at 1 and 4 h induced significant perturbations in peptide and amino acid metabolism, which is in line with the mode of action of aminoglycosides. Pathway analysis of FADDI-PA111 revealed that the synergistic effect of the combination was largely due to the inhibition of cell envelope biogenesis, which was driven initially by polymyxin B via suppression of key metabolites involved in lipopolysaccharide, peptidoglycan, and membrane lipids (15 min and 1 h) and later by amikacin (4 h). Overall, these novel findings demonstrate that the disruption of cell envelope biogenesis and central carbohydrate metabolism, decreased levels of amino sugars, and a downregulated nucleotide pool are the metabolic pathways associated with the synergistic killing of the polymyxin-amikacin combination against P. aeruginosa. This mechanistic study might help optimize synergistic polymyxin B combinations in the clinical setting.
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Zhu, Yan, Jinxin Zhao, Mohd Hafidz Mahamad Maifiah, Tony Velkov, Falk Schreiber, and Jian Li. "Metabolic Responses to Polymyxin Treatment in Acinetobacter baumannii ATCC 19606: Integrating Transcriptomics and Metabolomics with Genome-Scale Metabolic Modeling." mSystems 4, no. 1 (February 5, 2019). http://dx.doi.org/10.1128/msystems.00157-18.
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ABSTRACT Multidrug-resistant (MDR) Acinetobacter baumannii has emerged as a very problematic pathogen over the past decades, with a high incidence in nosocomial infections. Discovered in the late 1940s but abandoned in the 1970s, polymyxins (i.e., polymyxin B and colistin) have been revived as the last-line therapy against Gram-negative “superbugs,” including MDR A. baumannii. Worryingly, resistance to polymyxins in A. baumannii has been increasingly reported, urging the development of novel antimicrobial therapies to rescue this last-line class of antibiotics. In the present study, we integrated genome-scale metabolic modeling with multiomics data to elucidate the mechanisms of cellular responses to colistin treatment in A. baumannii. A genome-scale metabolic model, iATCC19606, was constructed for strain ATCC 19606 based on the literature and genome annotation, containing 897 genes, 1,270 reactions, and 1,180 metabolites. After extensive curation, prediction of growth on 190 carbon sources using iATCC19606 achieved an overall accuracy of 84.3% compared to Biolog experimental results. Prediction of gene essentiality reached a high accuracy of 86.1% and 82.7% compared to two transposon mutant libraries of AB5075 and ATCC 17978, respectively. Further integrative modeling with our correlative transcriptomics and metabolomics data deciphered the complex regulation on metabolic responses to colistin treatment, including (i) upregulated fluxes through gluconeogenesis, the pentose phosphate pathway, and amino acid and nucleotide biosynthesis; (ii) downregulated TCA cycle and peptidoglycan and lipopolysaccharide biogenesis; and (iii) altered fluxes over respiratory chain. Our results elucidated the interplay of multiple metabolic pathways under colistin treatment in A. baumannii and provide key mechanistic insights into optimizing polymyxin combination therapy. IMPORTANCE Combating antimicrobial resistance has been highlighted as a critical global health priority. Due to the drying drug discovery pipeline, polymyxins have been employed as the last-line therapy against Gram-negative “superbugs”; however, the detailed mechanisms of antibacterial killing remain largely unclear, hampering the improvement of polymyxin therapy. Our integrative modeling using the constructed genome-scale metabolic model iATCC19606 and the correlative multiomics data provide the fundamental understanding of the complex metabolic responses to polymyxin treatment in A. baumannii at the systems level. The model iATCC19606 may have a significant potential in antimicrobial systems pharmacology research in A. baumannii.
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Zhao, Jinxin, Soon-Ee Cheah, Kade D. Roberts, Roger L. Nation, Philip E. Thompson, Tony Velkov, Zongjun Du, Matthew D. Johnson, and Jian Li. "Transcriptomic Analysis of the Activity of a Novel Polymyxin against Staphylococcus aureus." mSphere 1, no. 4 (July 27, 2016). http://dx.doi.org/10.1128/msphere.00119-16.
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ABSTRACT S. aureus is currently one of the most pervasive multidrug-resistant pathogens and commonly causes nosocomial infections. Clinicians are faced with a dwindling armamentarium to treat infections caused by S. aureus, as resistance develops to current antibiotics. This accentuates the urgent need for antimicrobial drug discovery. In the present study, we characterized the global gene expression profile of S. aureus treated with FADDI-019, a novel synthetic polymyxin analogue. In contrast to the concentration-dependent killing and rapid regrowth in Gram-negative bacteria treated with polymyxin B and colistin, FADDI-019 killed S. aureus progressively without regrowth at 24 h. Notably, FADDI-019 activated several vancomycin resistance genes and significantly downregulated the expression of a number of virulence determinants and enterotoxin genes. A synergistic combination with sulfamethoxazole was predicted by pathway analysis and demonstrated experimentally. This is the first study revealing the transcriptomics of S. aureus treated with a novel synthetic polymyxin analog. Polymyxin B and colistin are exclusively active against Gram-negative pathogens and have been used in the clinic as a last-line therapy. In this study, we investigated the antimicrobial activity of a novel polymyxin, FADDI-019, against Staphylococcus aureus. MIC and time-kill assays were employed to measure the activity of FADDI-019 against S. aureus ATCC 700699. Cell morphology was examined with scanning electron microscopy (SEM), and cell membrane polarity was measured using flow cytometry. Transcriptome changes caused by FADDI-019 treatment were investigated using transcriptome sequencing (RNA-Seq). Pathway analysis was conducted to examine the mechanism of the antibacterial activity of FADDI-019 and to rationally design a synergistic combination. Polymyxin B and colistin were not active against S. aureus strains with MICs of >128 mg/liter; however, FADDI-019 had a MIC of 16 mg/liter. Time-kill assays revealed that no S. aureus regrowth was observed after 24 h at 2× to 4× MIC of FADDI-019. Scanning electron microscopy (SEM) and flow cytometry results indicated that FADDI-019 treatment had no effect on cell morphology but caused membrane depolarization. The vancomycin resistance genes vraRS, as well as the VraRS regulon, were activated by FADDI-019. Virulence determinants controlled by SaeRS and the expression of enterotoxin genes yent2, sei, sem, and seo were significantly downregulated by FADDI-019. Pathway analysis of transcriptomic data was predictive of a synergistic combination comprising FADDI-019 and sulfamethoxazole. Our study is the first to examine the mechanism of the killing of a novel polymyxin against S. aureus. We also show the potential of transcriptomic and pathway analysis as tools to design synergistic antibiotic combinations. IMPORTANCE S. aureus is currently one of the most pervasive multidrug-resistant pathogens and commonly causes nosocomial infections. Clinicians are faced with a dwindling armamentarium to treat infections caused by S. aureus, as resistance develops to current antibiotics. This accentuates the urgent need for antimicrobial drug discovery. In the present study, we characterized the global gene expression profile of S. aureus treated with FADDI-019, a novel synthetic polymyxin analogue. In contrast to the concentration-dependent killing and rapid regrowth in Gram-negative bacteria treated with polymyxin B and colistin, FADDI-019 killed S. aureus progressively without regrowth at 24 h. Notably, FADDI-019 activated several vancomycin resistance genes and significantly downregulated the expression of a number of virulence determinants and enterotoxin genes. A synergistic combination with sulfamethoxazole was predicted by pathway analysis and demonstrated experimentally. This is the first study revealing the transcriptomics of S. aureus treated with a novel synthetic polymyxin analog.
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Han, Mei-Ling, Yan Zhu, Darren J. Creek, Yu-Wei Lin, Alina D. Gutu, Paul Hertzog, Tony Purcell, et al. "Comparative Metabolomics and Transcriptomics Reveal Multiple Pathways Associated with Polymyxin Killing in Pseudomonas aeruginosa." mSystems 4, no. 1 (January 8, 2019). http://dx.doi.org/10.1128/msystems.00149-18.
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ABSTRACT Polymyxins are a last-line therapy against multidrug-resistant Pseudomonas aeruginosa; however, resistance to polymyxins has been increasingly reported. Therefore, understanding the mechanisms of polymyxin activity and resistance is crucial for preserving their clinical usefulness. This study employed comparative metabolomics and transcriptomics to investigate the responses of polymyxin-susceptible P. aeruginosa PAK (polymyxin B MIC, 1 mg/liter) and its polymyxin-resistant pmrB mutant PAKpmrB6 (MIC, 16 mg/liter) to polymyxin B (4, 8, and 128 mg/liter) at 1, 4, and 24 h, respectively. Our results revealed that polymyxin B at 4 mg/liter induced different metabolic and transcriptomic responses between polymyxin-susceptible and -resistant P. aeruginosa. In strain PAK, polymyxin B significantly activated PmrAB and the mediated arn operon, leading to increased 4-amino-4-deoxy-L-arabinose (L-Ara4N) synthesis and the addition to lipid A. In contrast, polymyxin B did not increase lipid A modification in strain PAKpmrB6. Moreover, the syntheses of lipopolysaccharide and peptidoglycan were significantly decreased in strain PAK but increased in strain PAKpmrB6 due to polymyxin B treatment. In addition, 4 mg/liter polymyxin B significantly perturbed phospholipid and fatty acid levels and induced oxidative stress in strain PAK, but not in PAKpmrB6. Notably, the increased trehalose-6-phosphate levels indicate that polymyxin B potentially caused osmotic imbalance in both strains. Furthermore, 8 and 128 mg/liter polymyxin B significantly elevated lipoamino acid levels and decreased phospholipid levels but without dramatic changes in lipid A modification in wild-type and mutant strains, respectively. Overall, this systems study is the first to elucidate the complex and dynamic interactions of multiple cellular pathways associated with the polymyxin mode of action against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has been highlighted by the recent WHO Global Priority Pathogen List due to multidrug resistance. Without new antibiotics, polymyxins remain a last-line therapeutic option for this difficult-to-treat pathogen. The emergence of polymyxin resistance highlights the growing threat to our already very limited antibiotic armamentarium and the urgency to understand the exact mechanisms of polymyxin activity and resistance. Integration of the correlative metabolomics and transcriptomics results in the present study discovered that polymyxin treatment caused significant perturbations in the biosynthesis of lipids, lipopolysaccharide, and peptidoglycan, central carbon metabolism, and oxidative stress. Importantly, lipid A modifications were surprisingly rapid in response to polymyxin treatment at clinically relevant concentrations. This is the first study to reveal the dynamics of polymyxin-induced cellular responses at the systems level, which highlights that combination therapy should be considered to minimize resistance to the last-line polymyxins. The results also provide much-needed mechanistic information which potentially benefits the discovery of new-generation polymyxins.
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Purcell, Alexandria B., Brent W. Simpson, and M. Stephen Trent. "Impact of the cAMP-cAMP Receptor Protein Regulatory Complex on Lipopolysaccharide Modifications and Polymyxin B Resistance in Escherichia coli." Journal of Bacteriology, April 18, 2023. http://dx.doi.org/10.1128/jb.00067-23.
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Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance.
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Yi, Wenshi, Chao Chen, and Xiuhai Gan. "Polymyxin B1 and E2 From Paenibacillus polymyxa Y-1 for Controlling Rice Bacterial Disease." Frontiers in Cellular and Infection Microbiology 12 (March 28, 2022). http://dx.doi.org/10.3389/fcimb.2022.866357.
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To discover novel microbial pesticide for controlling rice bacterial disease, polymyxin B1 and E2 were firstly isolated from the supernatant of fermentation broth of Paenibacillus polymyxa Y-1 by bioactivity tracking separation. It is shown that polymyxin B1 and E2 had remarkable in vitro inhibitory activities to Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) with the EC50 values of 0.19 μg/ml and 0.21 μg/ml against Xoo, and 0.32 μg/ml and 0.41 μg/ml against Xoc, respectively, which were better than those of Zhongshengmycin (0.31 μg/ml and 0.73 μg/ml) and Bismerthiazol (77.48 μg/ml and 85.30 μg/ml). Polymyxins B1 and E2 had good protection and curative activities against rice bacterial leaf blight (BLB) and rice bacterial leaf streak (BLS) in vivo. The protection and curative activities of polymyxins B1 (45.8 and 35.8%, respectively) and E2 (41.2 and 37.0%, respectively) to BLB were superior to those of Zhongshengmycin (34.8 and 29.8%, respectively) and Bismerthiazol (38.0 and 33.5%, respectively). Meanwhile, the protection and curative activities of polymyxins B1 (44.8 and 39.8%, respectively) and E2 (42.9 and 39.9%, respectively) to BLS were also superior to those of Zhongshengmycin (39.7 and 32.0%, respectively) and Bismerthiazol (41.5 and 34.3%, respectively). Polymyxin B1 exerted the anti-pesticide properties via destroying the cell integrity of Xoo, reducing its infectivity and enhancing rice resistance against pathogens through activating the phenylpropanoid biosynthesis pathway of rice. It is indicated that polymyxin B1 and E2 were potential microbial pesticides for controlling rice bacterial disease.
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Jones, Rebekah A., Holly Shropshire, Caimeng Zhao, Andrew Murphy, Ian Lidbury, Tao Wei, David J. Scanlan, and Yin Chen. "Phosphorus stress induces the synthesis of novel glycolipids in Pseudomonas aeruginosa that confer protection against a last-resort antibiotic." ISME Journal, May 24, 2021. http://dx.doi.org/10.1038/s41396-021-01008-7.
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AbstractPseudomonas aeruginosa is a nosocomial pathogen with a prevalence in immunocompromised individuals and is particularly abundant in the lung microbiome of cystic fibrosis patients. A clinically important adaptation for bacterial pathogens during infection is their ability to survive and proliferate under phosphorus-limited growth conditions. Here, we demonstrate that P. aeruginosa adapts to P-limitation by substituting membrane glycerophospholipids with sugar-containing glycolipids through a lipid renovation pathway involving a phospholipase and two glycosyltransferases. Combining bacterial genetics and multi-omics (proteomics, lipidomics and metatranscriptomic analyses), we show that the surrogate glycolipids monoglucosyldiacylglycerol and glucuronic acid-diacylglycerol are synthesised through the action of a new phospholipase (PA3219) and two glycosyltransferases (PA3218 and PA0842). Comparative genomic analyses revealed that this pathway is strictly conserved in all P. aeruginosa strains isolated from a range of clinical and environmental settings and actively expressed in the metatranscriptome of cystic fibrosis patients. Importantly, this phospholipid-to-glycolipid transition comes with significant ecophysiological consequence in terms of antibiotic sensitivity. Mutants defective in glycolipid synthesis survive poorly when challenged with polymyxin B, a last-resort antibiotic for treating multi-drug resistant P. aeruginosa. Thus, we demonstrate an intriguing link between adaptation to environmental stress (nutrient availability) and antibiotic resistance, mediated through membrane lipid renovation that is an important new facet in our understanding of the ecophysiology of this bacterium in the lung microbiome of cystic fibrosis patients.
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Han, Mei-Ling, Yan Zhu, Darren J. Creek, Yu-Wei Lin, Dovile Anderson, Hsin-Hui Shen, Brian Tsuji, et al. "Alterations of Metabolic and Lipid Profiles in Polymyxin-ResistantPseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 62, no. 6 (April 9, 2018). http://dx.doi.org/10.1128/aac.02656-17.
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ABSTRACTMultidrug-resistantPseudomonas aeruginosapresents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance inP. aeruginosahas been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistantP. aeruginosastrains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-typeP. aeruginosastrain K ([PAK] polymyxin B MIC, 1 mg/liter) and its pairedpmrBmutant strains, PAKpmrB6and PAKpmrB12(polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) inspeE(encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6compared to that in PAKpmrB12. Our results indicate that spermidine may play an important role in high-level polymyxin resistance inP. aeruginosa. Interestingly, bothpmrBmutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12mutant exhibited much lower levels of phospholipids than the PAKpmrB6mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance inP. aeruginosaand highlights its impacts on bacterial metabolism.
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Sun, Lang, Pernille Kronholm Rasmussen, Yinlei Bai, Xiulan Chen, Tanxi Cai, Jifeng Wang, Xiaojing Guo, et al. "Proteomic Changes of Klebsiella pneumoniae in Response to Colistin Treatment and crrB Mutation-Mediated Colistin Resistance." Antimicrobial Agents and Chemotherapy 64, no. 6 (March 30, 2020). http://dx.doi.org/10.1128/aac.02200-19.
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ABSTRACT Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually over the past few years. Although studies on polymyxin mechanisms are expanding, systemwide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how Klebsiella pneumoniae adapts to colistin (polymyxin E) pressure, we carried out proteomic analysis of a K. pneumoniae strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in K. pneumoniae involve several pathways, including (i) gluconeogenesis and the tricarboxylic acid (TCA) cycle, (ii) arginine biosynthesis, (iii) porphyrin and chlorophyll metabolism, and (iv) enterobactin biosynthesis. Interestingly, decreased abundances of class A β-lactamases, including TEM, SHV-11, and SHV-4, were observed in cells treated with colistin. Moreover, we present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant K. pneumoniae strains. The polymyxin-resistant strain Ci, a mutant of K. pneumoniae ATCC BAA 2146, showed a missense mutation in crrB. This crrB mutant, which displayed lipid A modification with 4-amino-4-deoxy-l-arabinose (l-Ara4N) and palmitoylation, showed striking increases in the expression of CrrAB, PmrAB, PhoPQ, ArnBCADT, and PagP. We hypothesize that crrB mutations induce elevated expression of the arnBCADTEF operon and pagP via PmrAB and PhoPQ. Moreover, the multidrug efflux pump KexD, which was induced by crrB mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediated colistin resistance, which may offer valuable information on the management of polymyxin resistance.