The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica

The PmrA/PmrB regulatory system of Salmonella enterica controls the modification of lipid A with aminoarabinose and phosphoethanolamine. The aminoarabinose modification is required for resistance to the antibiotic polymyxin B, as mutations of the PmrA-activated pbg operon or ugd gene result in strains that lack aminoarabinose in their lipid A molecules and are more susceptible to polymyxin B. Additional PmrA-regulated genes appear to participate in polymyxin B resistance, as pbgP and ugd mutants are not as sensitive to polymyxin B as a pmrA mutant. Moreover, the role that the phosphoethanolamine modification of lipid A plays in the resistance to polymyxin B has remained unknown. Here we address both of these questions by establishing that the PmrA-activated pmrC gene encodes an inner membrane protein that is required for the incorporation of phosphoethanolamine into lipid A and for polymyxin B resistance. The PmrC protein consists of an N-terminal region with five transmembrane domains followed by a large periplasmic region harboring the putative enzymatic domain. A pbgP pmrC double mutant resembled a pmrA mutant both in its lipid A profile and in its susceptibility to polymyxin B, indicating that the PmrA-dependent modification of lipid A with aminoarabinose and phosphoethanolamine is responsible for PmrA-regulated polymyxin B resistance.     

Polymyxin B-induced phase separation and acyl chain interdigitation in phosphatidylcholine/phosphatidylglycerol mixtures

Monolayers, fluorescence polarization, differential scanning calorimetry and X-ray diffraction experiments have been carried out to examine the effect of the polypeptide antibiotic polymyxin B on the phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) either pure or mixed with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC). It is shown that in both phosphatidylglycerol alone and phosphatidylglycerol/phosphatidylcholine mixtures, polymyxin B can induce either phase separation between lipid domains of various compositions or interdigitation of the acyl chains in the solid state, without segregation of the two lipids. Phase separation was observed by fluorescence and differential scanning calorimetry after addition of the antibiotic to vesicles composed of mixtures of DMPC and DPPG in conditions where polymyxin B did not saturate phosphatidylglycerol (DPPG to polymyxin B molar ratio, Ri, higher than 15). Phase separation was also observed in mixed monolayers of DPPC and of the 5:1 DPPG/polymyxin B complex, at high surface pressure. Acyl chain interdigitation was observed by X-ray diffraction in both 5:1 DPPG/polymyxin B mixtures and preformed 5:5:1 DMPC/DPPG/polymyxin B mixture, in which the antibiotic saturates phosphatidylglycerol (Ri 5). In both cases, raising the temperature gave rise to a complex double-peaked phase transition by differential scanning calorimetry, from the interdigitating phase to a normal L alpha lamellar phase. As it is known that polymyxin B does not interact with phosphatidylcholine, the data presented show that, when phosphatidylcholine and phosphatidylglycerol are mixed together, a phase perturbation such as acyl chain interdigitation, which normally affects only phosphatidylglycerol, is also felt by phosphatidylcholine.     

Polymyxin B inhibits insulin-induced glucose transporter and IGF II receptor translocation in isolated adipocytes.

In isolated adipocytes, polymyxin B inhibited insulin-induced glucose incorporation into lipids in a dose-dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin-induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low-density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin-induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes. Since low-molecular-mass GTP-binding proteins have been implicated in the regulation of vesicular trafficking, we have used [alpha-32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP-binding proteins were revealed in plasma membrane and low-density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low-molecular-mass GTP-binding proteins in plasma membranes, but not in low-density microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor-containing vesicles, perhaps at the fusion step between vesicles and plasma membranes.     

Isolation and characterization of a gene, pmrD, from Salmonella typhimurium that confers resistance to polymyxin when expressed in multiple copies.

We have isolated from Salmonella typhimurium a gene, designated pmrD, that confers resistance to the membrane-damaging drug, polymyxin B when expressed from the medium-copy-number plasmid pHSG576. The gene maps to 46 min on the standard genetic map, near the menB gene, and is therefore distinct from the previously described pmrA locus. We have mapped the polymyxin resistance activity to a 1.3-kb ClaI-PvuII fragment which contains a small open reading frame that could encode an 85-amino-acid peptide. When an omega-Tet insertion was made into the putative pmrD open reading frame (pmrD2::omega-Tet), the resulting plasmid no longer conferred polymyxin resistance, whereas an omega-Tet insertion into vector sequences had no effect. Maxicell analysis confirmed that a protein of the expected size is made in vivo. The PmrD protein shows no significant homology to any known protein, but it does show limited homology across the active site of the p15 acid protease from Rous sarcoma virus, indicating that the protein may have proteolytic activity. However, changing the aspartic acid residue at the putative active site to alanine reduced but did not eliminate polymyxin resistance. When pmrD2::omega-Tet replaced the chromosomal copy of pmrD, the resulting strain showed wild-type sensitivity to polymyxin and could be complemented to resistance by a plasmid that carried pmrD. The pmrA505 allele confers resistance to polymyxin when present in single copy on the chromosome or when present on a plasmid in pmrA+ pmrD+ cells. In combination with the pmrD(2)::-Tet mutation, the effect o the pmrA505 allele on polymyxin resistance was reduced, whether pmrA505 was present in the chromosome or on a plasmid. Conversely, a strain carrying an insertion in pmrA could be complemented to polymyxin resistance by a plasmid carrying the pmrA505 allele but not by a plasmid carrying pmrD. On the basis of these results, we suggest that polymyxin resistance is mediated by an interaction between PmrA or a PmrA-regulated gene product and PmrD.     

多粘菌素耐药性的研究进展

多粘菌素因在多重耐药革兰氏阴性菌上的治疗效果良好,再度被应用于临床,其耐药水平在多种抗菌药中曾一度较低,但目前有研究表明多粘菌素的耐药率有增加趋势。作为抗击多重耐药革兰氏阴性菌的最后一道防线,如何抑制其耐药的发生就显得尤为重要。本文就多粘菌素的耐药性现状、产生机制及防控措施三个方面进行了综述,为指导临床科学合理使用多粘菌素及革兰氏阴性菌耐药菌株传播和蔓延的防控措施提供理论依据。     

Polymyxin B diminishes blood flow to brown adipose tissue and lactating mammary gland in the rat. Possible mechanism of its action to decrease the stimulation of lipogenesis on refeeding.

Polymyxin B, a cyclic decapeptide antibiotic, increased blood glucose and lactate, and inhibited the stimulation of lipogenesis in interscapular brown adipose tissue and lactating mammary gland of starved-refed virgin and lactating rats respectively. Lipogenesis was not inhibited in white adipose tissue or liver. The antibiotic increased the haematocrit. The relative blood flow to brown adipose tissue and lactating mammary gland was decreased by polymyxin B, and this was accompanied by a decrease in tissue ATP content. In vitro polymyxin B did not affect glucose utilization or conversion into lipid, nor the stimulation by insulin of these processes in brown-adipose-tissue slices. Treatment of rats in vivo with polymyxin B resulted in decreased utilization of glucose in vitro in brown-adipose-tissue slices. Similarly, acini from mammary glands of polymyxin B-treated lactating rats had decreased rates of conversion of [1-14C]glucose to lipid. It is concluded that the effects of polymyxin B may be brought about by decreases in tissue blood flow. The possibility that these effects are secondary to inhibition of glucose utilization cannot be ruled out.     

Specific Assay of Aminoglycosidic- or Polymyxin-Type Antibiotics Present in Human Sera in Combination with Cephalosporins

Monitoring of serum concentrations of aminoglycosidic or polymyxin antibiotics when administered concurrently with cephalosporins or penicillins requires a special assay technique. Selective enzymatic degradation of the beta-lactam antibiotic from the serum specimen allows subsequent assay of the antibiotic being monitored. This report gives details of a simple procedure for laboratory production of a crude enzyme capable of degrading cephalosporins or penicillins. An assay procedure for quantitating aminoglycosidic or polymyxin antibiotics after enzymatic degradation of a coexisting beta-lactam antibiotic is described.     

Detoxifying effect of antibiotics and their effectiveness in experimental plague at the stage of explicit intoxication]

The effect of antibiotics such as amikacin, rifampicin, doxycycline, polymyxin B and cefotaxime on the toxins of the plague microbe (lipopolysaccharide + fraction II according to Beiker) was studied in vitro and in vivo. The study on the antibiotic neutralization of plague toxins revealed that only polymyxin had toxin neutralizing capacity which depended on the dose. Investigation of the polymyxin effect at various stages of plague infection showed that when polymyxin in a dose of 1250 units and a mixture of plague toxins in lethal doses were administered simultaneously to albino mice, the positive effect amounted to 100 per cent. When the antibiotic was administered 30 or 60 minutes later, the antibiotic efficacy proved to be lower by 90 or 76.6 per cent, respectively. The intoxication in later periods (in 90-120 minutes) resulted in a decrease in animal survival up to 40-15 per cent. It was demonstrated on the model of the plague infection in albino mice that the use of amikacin, cefotaxime, rifampicin or doxycycline during polymyxin therapy at the stage of marked generalization of the infection provided a significant increase in the animal survival (60 to 80 per cent) as compared to that after the use of the same drugs alone (0 to 20 per cent).     

Protective effect of polymyxin B sulfate in experimental meningococcal infection in mice.

The mouse model of intraperitoneal meningococcal sepsis was used to evaluate the antiendotoxic activity of polymyxin B sulfate independent of its antibiotic effects. Administered either before or after the infective challenge therapeutic doses of polymyxin B sulfate produced small but significant increases in survival over unprotected animals. These results also suggest that endotoxin contributes to the outcome in this variety of Gram-negative infection.     

Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]¹polymyxin B₃. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]¹polymyxin B₃ for LPS was comparable to polymyxin B and colistin, and considerably greater (K_d < 1 μM) than for whole cells (K_d ∼ 6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]¹polymyxin B₃ to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]¹polymyxin B₃–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]¹polymyxin B₃–LPS complex due to steric hindrance from the dansyl[Lys]¹ fluorophore; this corresponded with diminished antibacterial activity (MIC ?16 μg/mL). Dansyl[Lys]¹polymyxin B₃ may prove useful as a screening tool for drug development.     

Mechanism of Polymyxin B Resistance in Proteus mirabilis

The lipids from three types of organisms-a Proteus mirabilis wild type highly resistant to polymyxin B, a polymyxin B-sensitive mutant derived from the wild type, and the wild type grown in the presence of sulfadiazine resulting in phenotypic conversion to polymyxin B sensitivity-were examined to determine the nature of polymyxin B resistance. The phospholipid compositions were nearly identical; each organism contained similar small amounts of N-methyl phosphatidylethanolamine in addition to comparable quantities of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. the fatty acid compositions were similar in the exponential phase of growth; in the stationary phase, sulfadiazine markedly inhibited the synthesis of cyclopropane fatty acids. Liposomes prepared from the dried lipids of the three types of organisms were extensively and similarly disrupted by the polymyxin. These findings suggest that polymyxin B resistance in P. mirabilis is determined by the cell envelope which prevents access of the antibiotic to the susceptible lipid target sites.     

Polymyxin B binding sites in Escherichia coli as revealed by polymyxin B-gold labeling

A complex of polymyxin B, bovine serum albumin, and colloidal gold was prepared and used for the ultrastructural localization of polymyxin B binding sites on thin sections of Epon-embedded Escherichia coli cells. Gold particles were found on the outer membrane of E. coli, which is consistent with reported biochemical findings. We concluded that gold labeling with polymyxin B is useful in localizing the binding sites of polymyxin.     

Novel polymyxin derivatives are less cytotoxic than polymyxin B to renal proximal tubular cells

The emergence of very multiresistant Gram-negative bacterial strains has reinstated polymyxins (polymyxin B, colistin), pentacationic lipopeptides, in the therapy, in spite of their nephrotoxicity. Extensive tubular reabsorption concentrates polymyxin in proximal tubular cells. The novel polymyxin derivatives NAB739, NAB7061 and NAB741 have their cyclic part identical to that of polymyxin B, but their side chain consists of uncharged octanoyl-threonyl-d-serinyl, octanoyl-threonyl-aminobutyryl, and acetyl-threonyl-D-serinyl respectively. In this study, we compared the toxicities of NAB739, NAB7061 and NAB741 with that of polymyxin B by using the porcine renal proximal tubular cell line LLC-PK1 electroporated or incubated with the selected compound. Both the ability to cause cell necrosis (quantified as the leakage of lactate dehydrogenase) and the ability to cause apoptosis (as quantified by counting apoptotic nuclei) were assessed. In electroporated cells, polymyxin B induced total (>85%) necrosis of the cells at 0.016 mM, whereas an approx. 8-fold concentration of NAB739 and NAB7961 and an approx. 32-fold concentration of NAB741 was required for the same effect. In cells treated without electroporation (incubated), polymyxin B elicited a marked degree (approx. 50%) of necrosis at 0.5mM, whereas the NAB compounds were inert even at 1mM. Neither polymyxin B nor the NAB compounds induced apoptosis.     

Pyocin sensitivity of pseudomonas aeruginosa pretreated with antibiotics.

Strains of Pseudomonas aeruginosa exposed to subinhibitory concentrations of either polymyxin B, gentamicin, or carbenicillin were compared with untreated organisms for changes in sensitivity to pyocin. Pretreatment with polymyxin B or gentamicin resulted in a decreased sensitivity to pyocin, while carbenicllin pretreatment did not alter pyocin sensitivity.     

Adaptive resistance to polymyxin in Pseudomonas aeruginosa due to an outer membrane impermeability mechanism

The isolated outer membrane from cells of a Pseudomonas aeruginosa strain exhibiting adaptive resistance to polymyxin was not affected by polymyxin treatment, as monitored by electron microscopy of negatively stained preparations. This was in sharp contrast with extensive disruption by polymyxin of the outer membranes of the parent polymyxin-sensitive strain and the resistant strain following reversion to greater polymyxin sensitivity. The isolated cytoplasmic membrane of the polymyxin-resistant strain, on the other hand, remained sensitive to the disruptive effects of polymyxin treatment. The permeability characteristics of the resistant strains appear to be altered, as indicated by differences in minimal inhibitory concentrations for a variety of antibiotics between the polymyxin-sensitive and polymyxin-resistant strains. No evidence was found for a polymyxin-inactivating enzyme in osmotic shock fluid from the polymyxin-resistant strain. No evidence for a cytoplasmic membrane repair mechanism was found in the polymyxin-resistant strain. These observations suggest that the mechanism of adaptive polymyxin resistance in this model system is the alteration of the outer membrane so that it excludes polymyxin from reaching the still sensitive cytoplasmic membrane.     

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).     

The Influence of Calcium or Manganese on the Resistance to EDTA, Polymyxin B or Cold Shock, and the Composition of Pseudomonas aeruginosa Grown in Glucose- or Magnesium-depleted Batch Cultures

Cultures were batch grown in simple salts media in which growth was limited either by depletion of glucose and magnesium (C/Mg-dep) or by glucose alone (C-dep). Cultures were also grown in these media supplemented by calcium and/or manganese.
All cultures grown in the C-dep media were sensitive to ethylenediaminetetraacetic acid (EDTA), polymyxin and also to cold shock but were relatively resistant to ethyleneglycol-bis(2-aminoethyl ether)-N, N-tetraacetic acid (EGTA). Inclusion of calcium or manganese in the growth medium enhanced lysis by EDTA. Cultures grown in the basic C/Mg-dep media were resistant to EDTA, EGTA, polymyxin and to cold shock. Sensitivity to these agents was retained by cultures grown in C/Mg-dep media supplemented with Ca²⁺ and/or Mn²⁺. Cells grown in C/Mg-dep media with added Mn²⁺ were more sensitive to EDTA and polymyxin than those from the unsupplemented C/Mg-dep media but still resistant compared with C-dep cultures. All cultures from supplemented C/Mg-dep media were more sensitive to EGTA than those from any of the C-dep media.
Whole cells and cell walls from these various media had differing amounts of cell wall, phosphorus, amino sugar, carbohydrates, readily extractable lipid (REL), total phospholipid (PL), and especially differences in cell wall divalent metal cation content.
The differences in PL, REL and amino sugars and carbohydrate did not correlate with the response of C-dep and C/Mg-dep bacteria to EDTA, EGTA or polymyxin. The results are discussed in relation to the hypothesis that the sensitivity of Pseudomonas aeruginosa to polymyxin and EDTA is more dependent on outer membrane cation content rather than on other components, e.g. PL and lipopolysaccharide.     

Decreased particulate NADH oxidase activity in Bacillus subtilis spores after polymyxin B treatment

The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed.