Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa.

Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxin-sensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.     

Antibody inhibition of ferripyochelin binding to Pseudomonas aeruginosa cell envelopes

A 14K molecular weight protein which has been shown to bind ferripyochelin has been purified from cell envelopes of Pseudomonas aeruginosa low iron grown cells. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to be free of contamination by lipopolysaccharide or carbohydrate. Antiserum to this protein was made in rabbits and was shown to react with the purified protein by immunoblot assay. The immunoglobulin G fraction of this antiserum blocked binding of [59Fe]pyochelin to isolated cell envelopes of P. aeruginosa in a dose-dependent fashion.     

铜绿假单胞菌耐消毒剂基因检测

目的了解深圳市人民医院临床分离的铜绿假单胞菌耐季胺盐类消毒剂基因(qac)的检出率及基因型。方法收集深圳市人民医院近几年铜绿假单胞菌临床分离菌株63株,应用PCR法检测菌株的qacA/B、qacC、qacG、qacJ和qacE△1基因。结果63株铜绿假单胞菌中,qacE△1基因阳性51株(80.9%),qacA/B基因阳性5株(7.9%),其余的基因型未检出。结论我院临床分离的铜绿假单胞菌中耐季铵盐类消毒剂基因检出率较高,主要为qacE△1型。     

Antibiotic susceptibility of clinical isolates of Pseudomonas aeruginosa

Three hundred and twenty two clinical isolates of Pseudomonas aeruginosa collected in Morelia, México, were analyzed for in vitro susceptibility to five antibiotics by agar dilution tests. Antibiotic resistance was shown by 50% of total isolates. Frequencies of resistance were: streptomycin, 47%; gentamicin, 13%; tobramycin, 8%; and carbenicillin, 7%; no amikacin resistance was found. The more common resistance patterns were streptomycin, gentamicin-streptomycin, and tobramycin-gentamicin-streptomycin. Resistance to either tobramycin, gentamicin or carbenicillin was found mainly in pyocin type 10 isolates. The proportion of antibiotic resistant isolates ranged from 37 to 75% in four hospitals, and amounted 24% in three clinical laboratories.     

Comparison of virulence between clinical and environmental Pseudomonas aeruginosa isolates.

New strains of Pseudomonas aeruginosa were isolated from clinical and environmental settings in order to characterize the virulence properties of this opportunistic pathogen. P. aeruginosa was frequently recovered from oil-contaminated samples but not from non-oil-contaminated soils. The virulence of five environmental and five clinical strains of P. aeruginosa was tested using two different models, Drosophila melanogaster and Lactuca sativa var. capitata L. There was no difference in the virulence between the two groups of isolates in either of the models. Since environmental P. aeruginosa strains are used for bioaugmentation in bioremediation programs, the results presented here should be taken into account in the future design of degradative consortia and/or in establishing containment measures.     

Two unusual pilin sequences from different isolates of Pseudomonas aeruginosa.

The pilin genes of two Pseudomonas aeruginosa strains isolated from two different patients with cystic fibrosis were cloned and sequenced. The predicted protein sequences of these two pilins had several unusual features compared with other published P. aeruginosa pilin sequences.     

Protein alterations in isolated outer membranes of polymyxin-resistantPseudomonas aeruginosa isolates

Isolated outer membranes fromPseudomonas aeruginosa strains resistant to polymyxin were compared with isolated outer membranes from polymyxin-sensitive strains as to their protein composition as determined by slab polyacrylamide gel electrophoresis. Both the porin protein and the H1 protein were decreased greatly in concentration in the outer membranes from the polymyxin-resistant strains. This confirms previous findings reduced concentrations of these proteins in cell envelopes of these strains. No evidence was found for these decreases being the result of an artifactual loss of these proteins from the outer membrane due to conditions used for growth or for preparation of cell envelopes. Nevertheless, the role of these outer membrane proteins in mediating polymyxin resistance is uncertain.     

临床分离铜绿假单胞菌对氟喹诺酮类抗菌药物的耐药性检测

目的了解临床分离铜绿假单胞菌对喹诺酮类等抗菌药物的耐药性。方法琼脂稀释法测定86株铜绿假单胞菌对5种氟喹诺酮类抗菌药物以及头孢吡肟、美罗培南的耐药性。结果铜绿假单胞菌对诺氟沙星、氧氟沙星、左氧氟沙星、环丙沙星、加替沙星的耐药率分别为50%、61.6%、51.2%、48.8%、51.2%;对头孢吡肟和美罗培南的耐药率分别为30.2%、23.2%。结论铜绿假单胞菌对氟喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Metabolic flux analyses of Pseudomonas aeruginosa cystic fibrosis isolates

Pseudomonas aeruginosa is a metabolically versatile wide-ranging opportunistic pathogen. In humans P. aeruginosa causes infections of the skin, urinary tract, blood, and the lungs of Cystic Fibrosis patients. In addition, P. aeruginosa's broad environmental distribution, relatedness to biotechnologically useful species, and ability to form biofilms have made it the focus of considerable interest. We used ¹³C metabolic flux analysis (MFA) and flux balance analysis to understand energy and redox production and consumption and to explore the metabolic phenotypes of one reference strain and five strains isolated from the lungs of cystic fibrosis patients. Our results highlight the importance of the oxidative pentose phosphate and Entner-Doudoroff pathways in P. aeruginosa growth. Among clinical strains we report two divergent metabolic strategies and identify changes between genetically related strains that have emerged during a chronic infection of the same patient. MFA revealed that the magnitude of fluxes through the glyoxylate cycle correlates with growth rates.     

Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa

Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.     

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.     

Early cell envelope alterations by tobramycin associated with its lethal action on Pseudomonas aeruginosa

The immediate activities of the aminoglycoside antibiotic tobramycin were investigated in Pseudomonas aeruginosa PAO1. The lethal action of a low concentration of tobramycin (8 micrograms ml-1) occurred rapidly (1-3 min) and was associated with leakage of certain cellular components into the supernatant. The presence of magnesium at the time of initial exposure protected cells by preventing uptake of tobramycin; however, magnesium addition following a brief exposure did not restore viability. Analyses of supernatant material revealed a rapid 2-fold increase in protein released following tobramycin treatment. A prominent 29 kDa protein, observed by SDS-PAGE in the released material was identified as the periplasmic beta-lactamase. Brief exposure to tobramycin did not result in major morphological damage or cell lysis as observed by transmission electron microscopy, and release of LPS was not a primary event. Although activity at the ribosomal level was observed by 2-3 min, leakage was detected after only 1 min. These data indicate that leakage of cellular components, particularly beta-lactamase, occurs simultaneously, if not prior to inhibition of protein synthesis by tobramycin.     

The cell walls of Pseudomonas aeruginosa. General composition

1. Cell walls of Pseudomonas aeruginosa were prepared and analysed. 2. Separate preparations were found to be reproducible, e.g. the phosphorus contents of all batches lay in the range 2.0-2.1%. 3. Ninhydrin-positive compounds in hydrolysates accounted for 43-46% of the cell wall and 79-87% of the nitrogen of the cell wall. Examination of the results for individual ninhydrin-positive compounds showed that 5-15% of the cell wall was murein and about 30% protein. 4. Trypsin treatment of crude cell-wall preparations preferentially liberated arginine, lysine and leucine, but it was not clear whether these arose from cell-wall or cytoplasmic proteins.     

Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.

The exopolysaccharide alginate of Pseudomonas aeruginosa was shown to be important in determining the degree of cell detachment from an agar surface. Nonmucoid strain 8822 gave rise to 50-fold more sloughed cells than mucoid strains 8821 and 8830. Alginate anchors the bacteria to the agar surface, thereby influencing the extent of detachment. The role of the P. aeruginosa alginate lyase in the process of cell sloughing was investigated. Increased expression of the alginate lyase in mucoid strain 8830 led to alginate degradation and increased cell detachment. Similar effects were seen both when the alginate lyase was induced at the initial stage of cell inoculation and when it was induced at a later stage of growth. It appears that high-molecular-weight alginate polymers are required to efficiently retain the bacteria within the growth film. When expressed from a regulated promoter, the alginate lyase can induce enhanced sloughing of cells because of degradation of the alginate. This suggests a possible role for the lyase in the development of bacterial growth films.     

Chemotaxis in Pseudomonas aeruginosa.

A chemotaxis system for Pseudomonas aeruginosa was defined by using the method of Adler. Cells were attracted to compounds in the order ammonium chloride greater than amino acids greater than organic acids. Two sugars were assayed and elicited no response. Comparisons with other model systems are discussed.