Polymyxin Resistance Research Articles

The role of oxidoreductases in determining the function of the neisserial lipid A phosphoethanolamine transferase required for resistance to polymyxin.

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.

Fosmidomycin decreases membrane hopanoids and potentiates the effects of colistin on Burkholderia multivorans clinical isolates.

Burkholderia cepacia complex (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 species B. multivorans were 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 by B. 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 clinical B. multivorans isolates to colistin, an agent currently in use to treat pulmonary infections in CF patients.

Probing the Penetration of Antimicrobial Polymyxin Lipopeptides into Gram-Negative Bacteria

The dry antibiotic development pipeline coupled with the emergence of multidrug resistant Gram-negative ‘superbugs’ has driven the revival of the polymyxin lipopeptide antibiotics. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections. The lack of molecular imaging probes that possess native polymyxin-like antibacterial activity is a barrier to understanding the resistance mechanisms and the development of a new generation of polymyxin lipopeptides. Here we report the regioselective modification of the polymyxin B core scaffold at the N-terminus with the dansyl fluorophore to generate an active probe that mimics polymyxin B pharmacologically. Time-lapse laser scanning confocal microscopy imaging of the penetration of probe (1) into Gram-negative bacterial cells revealed that the probe initially accumulates in the outer membrane and subsequently penetrates into the inner membrane and finally the cytoplasm. The implementation of this polymyxin-mimetic probe will advance the development of platforms for the discovery of novel polymyxin lipopeptides with efficacy against polymyxin-resistant strains.

Prevalence and Genetic Characterization of Carbapenem- and Polymyxin-Resistant Acinetobacter baumannii Isolated from a Tertiary Hospital in Terengganu, Malaysia.

Nosocomial infection caused by Acinetobacter baumannii is of great concern due to its increasing resistance to most antimicrobials. In this study, 54 nonrepeat isolates of A. baumannii from the main tertiary hospital in Terengganu, Malaysia, were analyzed for their antibiograms and genotypes. Out of the 54 isolates, 39 (72.2%) were multidrug resistant (MDR) and resistant to carbapenems whereas 14 (25.9%) were categorized as extensive drug resistant (XDR) with additional resistance to polymyxin B, the drug of “last resort.” Pulsed-field gel electrophoresis analyses showed that the polymyxin-resistant isolates were genetically diverse while the carbapenem-resistant isolates were clonally related. The 14 XDR isolates were further investigated for mutations in genes known to mediate polymyxin resistance, namely, pmrCAB, and the lipopolysaccharide biosynthesis genes, lpxA, lpxC, lpxD, and lpsB. All 14 isolates had a P102H mutation in pmrA with no mutation detected in pmrC and pmrB. No mutation was detected in lpxA but each polymyxin-resistant isolate had 2–4 amino acid substitutions in lpxD and 1-2 substitutions in lpxC. Eight resistant isolates also displayed a unique H181Y mutation in lpsB. The extent of polymyxin resistance is of concern and the novel mutations discovered here warrant further investigations.

Teaching ‘Old’ Polymyxins New Tricks: New-Generation Lipopeptides Targeting Gram-Negative ‘Superbugs’

The antimicrobial lipopeptides polymyxin B and E (colistin) are being used as a ‘last-line’ therapy for infections caused by multidrug-resistant Gram-negative pathogens. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections caused by the Gram-negative ‘superbugs’. This report details the structure–activity relationships (SAR) based design, in toto synthesis, and preclinical evaluation of a series of novel polymyxin lipopeptides with better antibacterial activity against polymyxin-resistant Gram-negative bacteria.

Structural Basis for Substrate Specificity in ArnB. A Key Enzyme in the Polymyxin Resistance Pathway of Gram-Negative Bacteria

Cationic Antimicrobial Peptides (CAMPs) represent a first line of defense against bacterial colonization. When fighting Gram-negative bacteria, CAMPs initially interact electrostatically with the negatively charged phosphate groups in lipid A and are thought to kill bacteria by disrupting their membrane integrity. However, many human pathogens, including Salmonella and Pseudomonas, have evolved lipid A modification mechanisms that result in resistance to CAMPs and related antibiotics such as Colistin. The addition of 4-amino-4-deoxy-l-Arabinose (Ara4N) to a phosphate group in lipid A is one such modification, frequently found in Pseudomonas isolated from cystic fibrosis patients. The pathway for biosynthesis of Ara4N-lipid A requires conversion of UDP-Glucuronic acid into UDP-Ara4N and subsequent transfer of the amino-sugar to lipid A. ArnB is a pyridoxal-phosphate (PLP) dependent transaminase that catalyzes a crucial step in the pathway: synthesis of UDP-Ara4N from UDP-4-keto-pentose. Here we present the 2.3 Å resolution crystal structure of an active site mutant of ArnB (K188A) in complex with the reaction intermediate aldimine formed by UDP-Ara4N and PLP. The sugar–nucleotide binding site is in a cleft between the subunits of the ArnB dimer with the uracil buried at the interface and the UDP ribose and phosphate groups exposed to the solvent. The Ara4N moiety is found in the 4C1 conformation and its positioning, stabilized by interactions with both the protein and cofactor, is compatible with catalysis. The structure suggests strategies for the development of specific inhibitors that may prove useful in the treatment of resistant bacteria such as Pseudomonas found in cystic fibrosis patients.

487Infections Caused by Enterobacteriaceae Species following Cardiac Surgery: Impact of Polymyxin Resistance on Treatment Outcomes

487Infections Caused by Enterobacteriaceae Species following Cardiac Surgery: Impact of Polymyxin Resistance on Treatment Outcomes

Strong cross-system interactions drive the activation of the QseB response regulator in the absence of its cognate sensor

Bacterial two-component systems (TCSs) mediate specific responses to distinct conditions and/or stresses. TCS interactions are highly specific between cognate partners to avoid unintended cross-talk. Although cross-talk between a sensor kinase and a noncognate response regulator has been previously demonstrated, the majority of reported interactions have not been robust. Here, we report that in the case of the quorum-sensing Escherichia coli (Qse)BC TCS, absence of the cognate sensor QseC leads to robust, constitutive activation of the QseB response regulator by the noncognate polymyxin resistance (Pmr) sensor kinase PmrB. Remarkably, the noncognate PmrB exhibits a kinetic preference for QseB that is similar to QseC. However, although PmrB readily phosphorylates QseB in vitro, it is significantly less efficient at dephosphorylating QseB, compared with QseC, thereby explaining the increased levels of active QseB in the qseC mutant. In addition to PmrB activating QseB on the protein level, we found that the PmrA response regulator contributes to qseB transcription in the absence of QseC and PmrA specifically binds the qseBC promoter, indicative of a direct regulation of qseBC gene transcription by PmrAB under physiological conditions. Addition of ferric iron in the growth medium of wild-type uropathogenic E. coli induced the expression of qseBC in a PmrB-dependent manner. Taken together, our findings suggest that (i) robust cross-talk between noncognate partners is possible and (ii) this interaction can be manipulated for the development of antivirulence strategies aimed at targeting uropathogenic Escherichia coli and potentially other QseBC-PmrAB-bearing pathogens.

Structural Basis of a Physical Blockage Mechanism for the Interaction of Response Regulator PmrA with Connector Protein PmrD from Klebsiella pneumoniae

In bacteria, the two-component system is the most prevalent for sensing and transducing environmental signals into the cell. The PmrA-PmrB two-component system, responsible for sensing external stimuli of high Fe(3+) and mild acidic conditions, can control the genes involved in lipopolysaccharide modification and polymyxin resistance in pathogens. In Klebsiella pneumoniae, the small basic connector protein PmrD protects phospho-PmrA and prolongs the expression of PmrA-activated genes. We previously determined the phospho-PmrA recognition mode of PmrD. However, how PmrA interacts with PmrD and prevents its dephosphorylation remains unknown. To address this question, we solved the x-ray crystal structure of the N-terminal receiver domain of BeF3(-)-activated PmrA (PmrA(N)) at 1.70 Å. With this structure, we applied the data-driven docking method based on NMR chemical shift perturbation to generate the complex model of PmrD-PmrA(N), which was further validated by site-directed spin labeling experiments. In the complex model, PmrD may act as a blockade to prevent phosphatase from contacting with the phosphorylation site on PmrA.

Extracellular DNA-induced antimicrobial peptide resistance in Salmonella enterica serovar Typhimurium

BackgroundThe Salmonella enterica serovar Typhimurium PhoPQ two component system (TCS) is activated by low Mg2+ levels, low pH and by antimicrobial peptides (AP). Under Mg2+ limitation, the PhoPQ system induces pmrD expression, which post-translationally activates the PmrAB TCS. PhoPQ and PmrAB control many genes required for intracellular survival and pathogenesis. These include the polymyxin resistance (pmr) operon, which is required for aminoarabinose modification of LPS and protecting the outer membrane from antimicrobial peptide disruption and killing. Extracellular DNA is a ubiquitous polymer in the matrix of biofilms and accumulates in some infection sites. Extracellular DNA chelates cations and thus activates the Pseudomonas aeruginosa PhoPQ/PmrAB systems, leading to expression of the orthologous arn (pmr) operon.ResultsHere we show that extracellular DNA induces expression of the S. Typhimurium pmr antimicrobial peptide resistance operon in a PhoPQ and PmrAB-dependent manner. Induction of the pmr genes by DNA was blocked when present with excess Mg2+. Exogenous DNA led to increased resistance of planktonic cultures to aminoglycosides, antimicrobial peptides (AP) and ciprofloxacin, but only AP resistance was PhoPQ/PmrAB-dependent. Extracellular DNA was shown to be a matrix component of S. Typhimurium biofilms cultivated in flow chambers and on glass surfaces. A pmrH-gfp fusion was highly expressed in flow chamber biofilms cultivated in medium with repressing levels of 10 mM Mg2+ and co-localized with eDNA. Expression of pmrH-lux was monitored in plastic peg biofilms and shown to require PhoPQ and PmrAB. Biofilms had higher levels of pmrH expression compared to planktonic cultures. We propose that DNA accumulation in biofilms contributes to the increased pmrH-lux expression in biofilms.ConclusionsThe Salmonella PhoPQ/PmrAB systems and antimicrobial peptide resistance are activated by the cation chelating properties of extracellular DNA. DNA-induced AP resistance may allow immune evasion and increased survival of S. Typhimurium biofilms formed during extracellular growth stages of an infection or outside the host.

Polymyxin Resistance of Pseudomonas aeruginosa phoQ Mutants Is Dependent on Additional Two-Component Regulatory Systems

Pseudomonas aeruginosa can develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistant phoQ mutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of the colRS genes, individually or in tandem, abrogated the polymyxin resistance of a ΔphoQ mutant, as did individual or tandem deletion of cprRS. Individual deletion of colR or colS in a ΔphoQ mutant also suppressed 4-amino-L-arabinose addition to lipid A, consistent with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion of colRS or cprRS in the ΔphoQ mutant or individual deletion of cprR or cprS failed to suppress 4-amino-L-arabinose addition to lipid A, indicating that this modification alone is not sufficient for PhoPQ-mediated polymyxin resistance in P. aeruginosa. Episomal expression of colRS or cprRS in tandem or of cprR individually complemented the Pm resistance phenotype in the ΔphoQ mutant, while episomal expression of colR, colS, or cprS individually did not. Highly polymyxin-resistant phoQ mutants of P. aeruginosa isolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of colRS and cprS; when expressed in a ΔphoQ background, these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance in P. aeruginosa, indicate that addition of 4-amino-L-arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate that colRS and cprS mutations can contribute to high-level clinical resistance.

Resistência às Polimixinas: velhos antibióticos, últimas opções terapêuticas

Polymyxins are polypeptide antimicrobials that act in the cell membranes and promote decrease of the cell wall integrity. These antimicrobials are used in the clinical practice for treatment of the multi-drug resistant Gram negative bacilli infections as the last therapeutic option. The polymyxin resistance involves lipopolysaccharide modifi cations that decrease the affi nity of the antimicrobial with the cell surface. These modifi cations are regulated by two component systems that are active by environmental infl uences as cation presence, pH or polymyxin exposure. The environmental infl uences initiate the action of the genes that develop the polymyxins resistant phenotype. The polymyxins viability maintenance is essential for the treatment for multi-drug resistant bacilli infections, while new therapeutic options are not available.KEYWORDS polimixins antimicrobial resistance

The Pseudomonas aeruginosa PhoP-PhoQ Two-Component Regulatory System Is Induced upon Interaction with Epithelial Cells and Controls Cytotoxicity and Inflammation

The adaptation of Pseudomonas aeruginosa to its environment, including the host, is tightly controlled by its network of regulatory systems. The two-component regulatory system PhoPQ has been shown to play a role in the virulence and polymyxin resistance of P. aeruginosa as well as several other Gram-negative species. Dysregulation of this system has been demonstrated in clinical isolates, yet how it affects virulence of P. aeruginosa is unknown. To investigate this, an assay was used whereby bacteria were cocultured with human bronchial epithelial cells. The interaction of wild-type (WT) bacteria that had adhered to epithelial cells led to a large upregulation of the expression of the oprH-phoP-phoQ operon and its target, the arn lipopolysaccharide (LPS) modification operon, in a PhoQ-dependent manner, compared to cells in the supernatant that had failed to adhere. Relative to the wild type, a phoQ mutant cocultured on epithelial cells produced less secreted protease and lipase and, like the phoQ mutant, piv, lipH, and lasB mutants demonstrated reduced cytotoxicity toward epithelial cells. Mutation in phoQ also resulted in alterations to lipid A and to increased inflammatory LPS. These data indicate that mutation of phoQ results in a phenotype that is similar to the less virulent but more inflammatory phenotype of clinical strains isolated from chronic-stage cystic fibrosis lung infections.

Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria

Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both gram-negative and gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the gram-positive system responsible for D-alanylation of teichoic acids. Such machinery was not thought to be used by gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between gram-positive and gram-negative organisms.

When it comes to drug discovery not all Gram‐negative bacterial biodefence pathogens are created equal: Burkholderia pseudomallei is different

When it comes to drug discovery not all Gram‐negative bacterial biodefence pathogens are created equal: <i>Burkholderia pseudomallei</i> is different

Repeated isolation of Pseudomonas aeruginosa isolates resistant to both polymyxins and carbapenems from 1 patient

Emergence of polymyxin resistance in carbapenem-resistant isolates is a great concern in clinical settings because it may mean the end of treatment options against Gram-negative bacterial infections. Polymyxin-nonsusceptible and -susceptible Pseudomonas aeruginosa isolates resistant to carbapenems and harboring blaIMP-6 were alternatively isolated from a patient. In vitro antimicrobial susceptibility testing, multilocus sequence typing, and pulsed-field gel electrophoresis were performed. Metallo-β-lactamase genes such as blaIMP, blaVIM, blaSPM, blaGIM, and blaSIM and blaOXA-50 were detected by polymerase chain reaction. Sequences of 2-component systems, PmrAB and PhoPQ, were also determined. All showed ST235 and the same pulsotype. Amino acid substitutions were identified in PmrB and PhoP from polymyxin-nonsusceptible isolates. Colistin exposure might be associated with the recovery of polymyxin-nonsusceptible isolates in this patient.

Differential Role of Two-Component Regulatory Systems (&amp;lt;i&amp;gt;phoPQ&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;pmrAB&amp;lt;/i&amp;gt;) in Polymyxin B Susceptibility of &amp;lt;i&amp;gt;Pseudomonas aeruginosa&amp;lt;/i&amp;gt;

Polymyxins are often considered as a last resort to treat multidrug resistant P. aeruginosa but polymyxin resistance has been increasingly reported worldwide in clinical isolates. Polymyxin resistance in P. aeruginosa is known to be associated with alterations in either PhoQ or PmrB. In this study, mutant strains of P. aeruginosa carrying amino acid substitution, a single and/or dual inactivation of PhoQ and PmrB were constructed to further understand the roles of PhoQ and PmrB in polymyxin susceptibility. Polymyxin B resistance was caused by both inactivation and/or amino acid substitutions in PhoQ but by only amino acid substitutions of PmrB. Alterations of both PhoQ and PmrB resulted in higher levels of polymyxin B resistance than alteration of either PhoQ or PmrB alone. These results were confirmed by time-killing assays suggesting that high-level polymyxin resistance in P. aeruginosa is caused by alterations of both PhoQ and PmrB.

Antimicrobial Resistance and Clones ofAcinetobacterSpecies andPseudomonas aeruginosa

Antimicrobial resistance in bacteria is problematic in clinical settings and is a growing threat to public health. Multidrug-resistant and pandrug-resistant non-fermenters such as Acinetobacter spp. and Pseudomonas aeruginosa have recently emerged as a great concern worldwide. Particularly, the prevalence of carbapenem resistance in Acinetobacter spp. and P. aeruginosa is problematic, and emergence of polymyxin resistance is ominous. In this review, we discuss carbapenem and polymyxin resistance in Acinetobacter spp. and P. aeruginosa isolates and their major clones.

Complete Genome Sequences of Three Pseudomonas aeruginosa Isolates with Phenotypes of Polymyxin B Adaptation and Inducible Resistance

Clinical "superbug" isolates of Pseudomonas aeruginosa were previously observed to be resistant to several antibiotics, including polymyxin B, and/or to have a distinct, reproducible adaptive polymyxin resistance phenotype, identified by observing "skipped" wells (appearance of extra turbid wells) during broth microdilution testing. Here we report the complete assembled draft genome sequences of three such polymyxin resistant P. aeruginosa strains (9BR, 19BR, and 213BR).

PmrB Mutations Promote Polymyxin Resistance of Pseudomonas aeruginosa Isolated from Colistin-Treated Cystic Fibrosis Patients

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance, P. aeruginosa isolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-function pmrB alleles that conferred polymyxin resistance to strains with a wild-type or pmrAB deletion background. Double mutant pmrB alleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutant pmrB alleles induced transcription from the promoter of the arnB operon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate that pmrB gain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.