The hydrophobic uptake pathway across the outer membrane of the antibiotic supersusceptible Pseudomonas aeruginosa mutant Z61

Abstract The Pseudomonas aeruginosa antibiotic supersusceptible mutant Z61 was 50–400-fold more susceptible than its wild-type parent K799 to 5 hydrophobic antibiotics. The strain Z61 outer membrane also demonstrated enhanced permeability towards a hydrophobic fluorescent probe. Strain Z61 cells had an altered cell surface, as revealed by phase-partitioning experiments, a lower amount of Lipid A phosphate, and a reduction in the number of Mg²⁺ binding sites in Lipid A, as demonstrated by dansyl polymyxin competition experiments. An antibiotic permation pathway directly through the outer membrane bilayer, rather than through porin proteins, is proposed for strain Z61.     

The Occurrence of Multidrug-Resistant Pseudomonas aeruginosa on Hydrocarbon-Contaminated Sites

The main aim of this paper was the comprehensive estimation of the occurrence rate and the antibiotic-resistance conditions of opportunistic pathogen Pseudomonas aeruginosa in hydrocarbon-contaminated environments. From 2002 to 2007, 26 hydrocarbon-contaminated sites of Hungary were screened for the detection of environmental isolates. Altogether, 156 samples were collected and examined for the determination of appearance, representative cell counts, and antibiotic-resistance features of P. aeruginosa. The detected levels of minimal inhibitory concentrations of ten different drugs against 36 environmental strains were compared to the results of a widely used reference strain ATCC 27853 and four other clinical isolates of P. aeruginosa. Based on our long-term experiment, it can be established that species P. aeruginosa was detectable in case of 61.5% of the investigated hydrocarbon-contaminated sites and 35.2% of the examined samples that shows its widespread occurrence in polluted soil–groundwater systems. In the course of the antibiotic-resistance assay, our results determined that 11 of the examined 36 environmental strains had multiple drug-resistance against several clinically effective antimicrobial classes: cephalosporins, wide spectrum penicillins, carbapenems, fluoroquinolones, and aminoglycosides. The fact that these multiresistant strains were isolated from 8 different hydrocarbon-contaminated sites, mainly from outskirts, confirms that multiple drug-resistance of P. aeruginosa is widespread not only in clinical, but also in natural surroundings as well.     

Protein-lipopolysaccharide interactions. 1. The reaction of lysozyme with Pseudomonas aeruginosa LPS.

Lysozyme (EC 3.2.1.17) complexes with extracted Pseudomonas aeruginosa LPS in two distinct stages. The initial stage does not produce turbidity detectable by nephelometry (measured as nephelos units (N) per time) but does permit low-speed sedimentation of the lysozyme-lipopolysaccharide (LPS) complex. This association is 100% disrupted by the action of 0.1 M Mg2+. Monovalent cations at equal ionic strength to the Mg2+ concentration used for these studies failed to alter significantly the lysozyme-LPS complex, indicating that the role of Mg2+ was not strictly an ionic one. The study of lysozyme-LPS complexes may provide a model system for investigating in vivo protein-LPS interactions.     

Mutational Analysis of Zinc Resistance in Pseudomonas putida Strain S4

The genes for resistance to any essential metal ion are generally tightly regulated. In Pseudomonas putida strain S4, a multiple metal-resistant strain, mutational analysis gave strong evidence to the presence of the same for the expression of Zn resistance. Zn-sensitive mutants showed a lower MTC of Zn and expressed the Zn resistance genes with a lower efficacy. Non-complementation between these mutants suggests that they are possibly involved in the same function. Altered response to Zn of these mutants assisted in predicting the involvement of a repressor protein regulating the expression of Zn resistance genes. Zn hypersensitive mutant, on the other hand, appears to have an unregulated Zn uptake. This seems to provide the sensor component in the regulation. Zn resistance in strain S4 consists of three steps, viz., uptake, efflux, and binding, which are shared by a Zn homeostasis mechanism as well. Received: 14 February 2001 / Accepted: 13 March 2001     

In Vitro Effects of Carbenicillin Combined with Gentamicin or Polymyxin B Against Pseudomonas aeruginosa

Disodium carbenicillin and gentamicin sulfate have both shown promise in the treatment of infections caused by Pseudomonas aeruginosa. This study was designed to explore possible synergistic relationships among the new as well as the established antimicrobial agents used to treat such infections. With an agar dilution technique, minimum inhibitory concentrations of 27 strains of P. aeruginosa were determined in two-dimensional tests. Graphs of equal biological activity (isobolograms) demonstrated moderate synergistic effects of the carbenicillin-gentamicin combination over therapeutically feasible concentration ranges. In contrast, the combination of carbenicillin and polymyxin B showed only additive or slightly antagonistic effects. Tests of bacterial killing confirmed the presence of carbenicillin-gentamicin synergy in 3 of 6 strains of P. aeruginosa, but did not show true antagonism between carbenicillin and polymyxin B. Clinical trials of both drug combinations are advisable to determine whether therapeutic results can be improved, and whether the dosages of gentamicin or polymyxin B can thereby be reduced to lessen their toxic hazards.     

Detection and Characterization of Plasmids in Pseudomonas aeruginosa Strain PAO

Using a variety of techniques, it has been established that the sex factor FP2 has a density of 1.717 g/cm(3) (corresponding to a guanine plus cytosine [G + C] content of 58%) and a mean circular contour length of 28.5 +/- 0.6 mum (corresponding to a molecular weight of 59 x 10(6)). Another sex factor FP39 has a density of 1.719 g/cm(3) (corresponding to a G + C content of 60%) and a mean circular contour length of 26.5 +/- 0.5 mum (corresponding to a molecular weight of 55 x 10(6)). It appears that all, or nearly all, of the FP sex factor deoxyribonucleic acid occurs as covalent circular molecules under the conditions employed. In addition, these procedures have been used to demonstrate that strain PAO, a naturally occurring female (i.e., FP(-)) strain of Pseudomonas aeruginosa, harbors a number of cryptic plasmids having similar densities to the bulk of the cell deoxyribonucleic acid (1.726 g/cm(3)) and occurring as covalent circular molecules.     

Mechanism of Copper Resistance in a Copper Mine Isolate Pseudomonas putida Strain S4

The mechanism of copper resistance in a multiple-metal-resistant natural isolate Pseudomonas putida strain S4 is based on inducible efflux. Active extrusion of copper ions occurs from the cytoplasm during the exponential phase of growth. Involvement of ATPase in the efflux of copper ions has been demonstrated by employing specific inhibitors. The effluxed copper is not thrown out of the cell, but remains in a bound form (to a protein) in the periplasm. Thus, a balance between the intracellular level, to fulfill the metabolic requirements, and the periplasmic sequestration, to evade toxicity, is maintained by this isolate. Received: 11 February 2002 / Accepted: 7 March 2002     

Occurrence of Staphylococcus aureus and Pseudomonas aeruginosa in Israeli coastal water

The occurrence of Pseudomonas aeruginosa and coagulase-positive Staphylococcus aureus in seawater from beaches of central Israel was investigated from June 1983 until June 1985. P. aeruginosa was monitored in 652 samples of seawater from 34 beaches, and S. aureus was monitored in 628 samples. P. aeruginosa was found in 44.8% of samples (6.5% with 1 bacterium per 100 ml of water), and S. aureus was recovered from 60.7% of samples (5.3% with 1 organism per 100 ml), compared with 91.6% of samples with total coliforms (TC) and 82.2% with fecal coliforms (FC). The correlation between the presence of P. aeruginosa to that of TC and FC was 99.1 and 98.3%, respectively, while S. aureus was found in 4.3 and 8% of samples where TC and FC, respectively, were absent. Monitoring of S. aureus as a supplementary indicator in populated beaches is recommended because it will add valuable information on the sanitary quality of the seawater.     

Interaction of Pseudomonas Bacteriophage 2 with the Slime Polysaccharide and Lipopolysaccharide of Pseudomonas aeruginosa Strain BI

Purified slime polysaccharide B and lipopolysaccharide of Pseudomonas aeruginosa strain BI were shown to possess receptor-like properties in inactivating Pseudomonas phage 2, whereas lipoprotein and glycopeptide fractions were devoid of activity. On a weight basis, slime polysaccharide B was more effective than lipopolysaccharide in inactivating phage. The specificity of the reaction with slime polysaccharide B was indicated by the fact that slime polysaccharide A of P. aeruginosa strain EI failed to inactivate phage 2. Electron micrographs showed phage 2 in typical, tail-first position of attachment on intact cells of strain BI, slime polysaccharide B, and lipopolysaccharide. Tail fibers were discernible during phage attachment.     

Prophage F116: Evidence for Extrachromosomal Location in Pseudomonas aeruginosa Strain PAO

F116 is a temperate-generalized transducing phage of Pseudomonas aeruginosa. Genetic evidence leads to the conclusion that F116 prophage DNA is maintained extrachromosomally as a plasmid. Preliminary physical evidence is presented to support this hypothesis.     

2010年宜昌市铜绿假单胞菌的耐药性分析

了解宜昌市铜绿假单胞菌（Pseudomonas aeruginosa）临床分离株的耐药现状。宜昌市城区5所医院临床分离的铜绿假单胞菌菌株,用K-B法作药敏试验,并根据统计其耐药情况及耐药表型（模式）分析可能存在的耐药机制。临床分离的铜绿假单胞菌共1 575株,耐药率依次为阿米卡星7.1%、美罗培南17.2%、头孢吡肟20.4%、头孢哌酮/舒巴坦21.0%、哌拉西林/他唑巴坦22.5%、环丙沙星23.1%、庆大霉素23.4%、头孢他啶25.0%、亚胺培南25.2%、哌拉西林30.4%、氨曲南34.5%、复方新诺明59.0%、米诺环素75.6%。多重耐药（MDR）和泛耐药（PDR）株分别占41.5%和0.17%。对各种抗假单胞菌药物分别耐药的菌株仍有13%～25.7%对阿米卡星敏感,提示在严重铜绿假单胞菌感染患者的治疗中,β内酰胺类抗假单胞菌药加氨基糖苷类仍是一个很好的联合用药组合。细菌耐药性仍呈增长趋势,临床上感染多重耐药和泛耐药的铜绿假单胞菌的治疗仍很棘手,应合理使用抗生素,尽量延缓耐药菌株的出现。     

Identification of the Biosynthetic Gene Cluster for the Pseudomonas aeruginosa Antimetabolite l-2-Amino-4-Methoxy-trans-3-Butenoic Acid

l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) (Fig. ​(Fig.1)1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a γ-substituted vinylglycine, a naturally occurring amino acid with a β,γ-C=C double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. ​(Fig.1).1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), γ-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3, 5, 10, 21, 22, 29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Open in a separate windowFIG. 1.Chemical structures of the γ-substituted vinylglycines AMB, aminoethoxyvinylglycine, and rhizobitoxine.     

Occurrence of Staphylococcus aureus and Pseudomonas aeruginosa in Israeli coastal water.

The occurrence of Pseudomonas aeruginosa and coagulase-positive Staphylococcus aureus in seawater from beaches of central Israel was investigated from June 1983 until June 1985. P. aeruginosa was monitored in 652 samples of seawater from 34 beaches, and S. aureus was monitored in 628 samples. P. aeruginosa was found in 44.8% of samples (6.5% with 1 bacterium per 100 ml of water), and S. aureus was recovered from 60.7% of samples (5.3% with 1 organism per 100 ml), compared with 91.6% of samples with total coliforms (TC) and 82.2% with fecal coliforms (FC). The correlation between the presence of P. aeruginosa to that of TC and FC was 99.1 and 98.3%, respectively, while S. aureus was found in 4.3 and 8% of samples where TC and FC, respectively, were absent. Monitoring of S. aureus as a supplementary indicator in populated beaches is recommended because it will add valuable information on the sanitary quality of the seawater.     

Occurrence of beta-lactamase type ESBL and IBL in Pseudomonas aeruginosa rods]

The aim of the study was to determine extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among Pseudomonas aeruginosa strains. A total of 43 strains isolated from humans (6), hospital sink (1), fish (15), cattle (5), swine (5), dog (1), redder (1) fur animals (9) were studied. ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al. (8). Clavulonate and tazobactam were used as the inhibitors of ESBL. Inducible beta-lactamases were determined using double disc method according to Sanders (15). Cefoxitin was the inductor of these beta-lactamases. The susceptibility study was carried out using the disc diffusion method according to NCCLS standards. A total of 8 ESBL (18.6% of all strains) and 31 (72%) IBL producing strains were detected. The obtained results indicate the necessity of monitoring of ESBL- and IBL-producing strains of Pseudomonas aeruginosa.     

First Report of OXA-4, an ESBL Isolated from Pseudomonas aeruginosa a South Indian Strain

The OXA-type β-lactamases are so named because of their oxacillin-hydrolyzing abilities. In this study we characterize an extended spectrum β-lactamase, designated OXA-4, produced by a clinical isolate of Pseudomonas aeruginosa. ESBL production was detected by double disk synergy test. The P. aeruginosa isolate was obtained from endotracheal suction tip of 84 years old male patient diagnosed with CVA and hypertension. ESBL producing OXA β-lactamases was detected by PCR with primers specific to the conserved regions of the coding genes. Iso electric focusing was done to confirm the significance, sequencing the amplified product was also done. In the phenotypic identification, the strain was highly resistant to third generation cephalosporins and also to imipenem. The PCR amplified product for OXA β-lactamase was viewed at 919 bp. The pI point for the same was identified at 7.2. With the help of sequencing the amplified OXA β-lactamase was identified as OXA-4 gene. Here we report P. aeruginosa producing OXA-4 ESBL for the first time in the Indian subcontinent.     

Identification of Genes Involved in Pseudomonas aeruginosa Biofilm-Specific Resistance to Antibiotics

Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic susceptibility, we previously identified 3 genes or operons of P. aeruginosa UCBPP-PA14 (ndvB, PA1875–1877 and tssC1) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents.     

4-Amino-4-deoxy-l-arabinose in LPS of enterobacterial R-mutants and its possible role for their polymyxin reactivity

Abstract The content of 4-amino-4-deoxy- l -arabinopyranose ( l -Arap4N) and the phosphate substitution pattern of the LPS of various strains from Salmonella minnesota, Yersinia enterocolitica and Proteus mirabilis was determined by GC/MS, HPLC and ³¹P-NMR. These data allowed us to examine the possible role of these components for the polymyxin B-binding capacity of LPS and for the minimal inhibiting concentration (MIC) and the minimal bactericidal concentration (MBC) of polymyxins B and E towards the respective R-mutants. Contrary to other investigated Re-, Rd- and Rc-mutants of S. minnesota , strain R595 (Re-mutant) showed about a 90% substitution of the ester-linked phosphate-group with l -Arap4N, whereas the l -Arap4N content of the other S. minnesota strains amounted to 17–25%. Neither the binding capacity of LPS to polymyxin B, determined by a bioassay, nor the MIC- and MBC-values of the R-mutants were significantly affected by this alteration. Similar results were obtained after using the temperature-dependent changes in the l -Ara p4N-content and phosphate substitution pattern of Y. enterocolitica 75R . In order to explore the relevant polymyxin B binding site, lipid A samples with or without substitution of their ester-linked phosphate group were prepared and subjected to the polymyxin-binding assay. The results obtained so far indicated that the inner core bound l -Arap4N, detected in all resistant strains investigated, may play a decisive role in the decreased binding of polymyxin B, responsible for the bacterial resistance towards polymyxin(s).     

The merG Gene Product Is Involved in Phenylmercury Resistance in Pseudomonas Strain K-62

The physiological function of a new gene, hereby designated merG, located between merA and merB on the broad-spectrum mer operon of Pseudomonas strain K-62 plasmid pMR26 was investigated. The 654-bp merG gene encodes a protein with a canonical leader sequence at its N terminus. The processing of the signal peptide of this protein was dose-dependently inhibited by sodium azide, a potent inhibitor of protein export. These results suggest that the mature MerG protein (ca. 20 kDa) may be located in the periplasm. Deletion of the merG gene from the broad-spectrum mer operon of pMR26 had no effect on the inorganic mercury resistance phenotype, but rendered the bacterium more sensitive to phenylmercury than its isogenic wild-type strain. Escherichia coli cells bearing pMU29, which carries a deletion of the merG gene, took up significantly more phenylmercury than the bacteria with the intact plasmid pMRA17. When the merG gene in a compatible plasmid was transformed into the E. coli strain carrying pMU29, the high uptake of and high sensitivity to phenylmercury were almost completely restored to their original levels. These results demonstrate that the merG gene is involved in phenylmercury resistance, presumably by reducing in-cell permeability to phenylmercury.