Pseudomonas aeruginosa Population Structure Revisited

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS ⁺/exoU ⁻ genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set.     

Developmental Stage Age Groups and African Population Structure: The Kusasi of the West African Savanna

African population structures based on censuses exhibit a distinctive pattern of distortion. It is often assumed that the cause for this distortion is systematic biases in age estimates by census enumerators and respondents influenced by perceptions of social and biological development. African developmental stage age groups are the cultural codification of such perceptions. I describe developmental stage age groups among the Kusasi of Bawku District in northeast Ghana, and analyze their age and sex structure for a sample of 1,132 individuals from the village of Zorse. I show that differences between men and women reflect differences in biological and social development, and that cultural concepts of developmental stages could influence age estimates to produce the pattern of distortions typical of those found in African population structures based on censuses. This is supported by a comparison of Bawku District population structure based on the Ghana census and an ethnographic sample census in Zorse which eliminated most age estimate biases.     

Incidence of Mucoid Pseudomonas aeruginosa from Clinical Sources

The high frequency of pulmonary infection by mucoid strains of Pseudomonas aeruginosa in patients with cystic fibrosis can be interpreted to represent an apparent mutation resulting from environmental factors within the host.     

The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD⁺ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 _Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60°C and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD⁺) were determined. The crystal structure represents that the subunit has more compact structure than homologs’ structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.     

Carbenicillin Resistance in Pseudomonas aeruginosa from Clinical Material

Strains of Pseudomonas aeruginosa resistant to 256 μg. per ml. or more of carbenicillin (Pyopen) were isolated from 17 patients over a period of three months. The infections were not solely due to cross-infection. Low dosage and attempted eradication of the organism from inaccessible sites, such as the bronchi or skin surfaces, by using systemic treatment are two possible causes. Restraint in treating infections of doubtful importance and the use of local applications to appropriate sites with or without systemic treatment is suggested.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa

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Structure of an extracellular cross-reactive polysaccharide from Pseudomonas aeruginosa immunotype 4

A neutral small molecular mass (approximately 6.5 kDa) polysaccharide comprising a pentasaccharide repeat unit was isolated from culture supernatants of Pseudomonas aeruginosa immunotype 4. The polysaccharide had a pentasaccharide repeating unit as follows (formula; see text) where Rha is rhamnose. The structure was determined using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride, methylation analysis, and 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments. The polysaccharide bound antibody raised to the lipopolysaccharide of the seven P. aeruginosa Fisher-Devlin immunotype strains. Inhibition assays demonstrated the presence of a serologically similar polysaccharide in supernatants of these strains. Affinity-purified antibody to the polysaccharide bound to lipopolysaccharide and whole cells of the immunotype strains of P. aeruginosa in a Western immunoblot and colony blot assay, respectively. This polysaccharide seems to contain an antigenic determinant present in the core of the P. aeruginosa lipopolysaccharide or may represent another minor polysaccharide substituent on the lipopolysaccharide in addition to the O side chain.     

Population Structure of Clinical Vibrio parahaemolyticus from 17 Coastal Countries,Determined through Multilocus Sequence Analysis

Vibrio parahaemolyticus is a leading cause of food-borne gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of clinical strains from worldwide collections remains largely undescribed, and the recorded outbreaks of V. parahaemolyticus gastroenteritis highlight the need for the subtyping of this species. We present a broad phylogenetic analysis of 490 clinical V. parahaemolyticus isolates from 17 coastal countries through multilocus sequence analysis (MLST). The 490 tested isolates fell into 161 sequence types (STs). The eBURST algorithm revealed that the 161 clinically relevant STs belonged to 8 clonal complexes, 11 doublets, and 94 singletons, showing a high level of genetic diversity. CC3 was found to be a global epidemic clone of V. parahaemolyticus, and ST-3 was the only ST with an international distribution. recA was observed to be evolving more rapidly, exhibiting the highest degree of nucleotide diversity (0.028) and the largest number of polymorphic nucleotide sites (177). We also found that the high variability of recA was an important cause of differences between the results of the eBURST and ME tree analyses, suggesting that recA has a much greater influence on the apparent evolutionary classification of V. parahaemolyticus based on the current MLST scheme. In conclusion, it is evident that a high degree of genetic diversity within the V. parahaemolyticus population and multiple sequence types are contributing to the burden of disease around the world. MLST, with a fully extractable database, is a powerful system for analysis of the clonal relationships of strains at a global scale. With the addition of more strains, the pubMLST database will provide more detailed and accurate information, which will be conducive to our future research on the population structure of V. parahaemolyticus.     

Structure of the Pseudomonas aeruginosa acyl-homoserinelactone synthase LasI

The LasI/LasR quorum-sensing system plays a pivotal role in virulence gene regulation of the opportunistic human pathogen, Pseudomonas aeruginosa. Here we report the crystal structure of the acyl-homoserine lactone (AHL) synthase LasI that produces 3-oxo-C12-AHL from the substrates 3-oxo-C12-acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine. The LasI six-stranded beta sheet platform, buttressed by three alpha helices, forms a V-shaped substrate-binding cleft that leads to a tunnel passing through the enzyme that can accommodate the acyl-chain of acyl-ACP. This tunnel places no apparent restriction on acyl-chain length, in contrast to a restrictive hydrophobic pocket seen in the AHL-synthase EsaI. Interactions of essential conserved N-terminal residues, Arg23, Phe27 and Trp33, suggest that the N-terminus forms an enclosed substrate-binding pocket for S-adenosyl-L-methionine. Analysis of AHL-synthase surface residues identified a binding site for acyl-ACP, a role that was supported by in vivo reporter assay analysis of the mutated residues, including Arg154 and Lys150. This structure and the novel explanation of AHL-synthase acyl-chain-length selectivity promise to guide the design of Pseudomonas aeruginosa-specific quorum-sensing inhibitors as antibacterial agents.     

Iron reductases from Pseudomonas aeruginosa

Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen.     

Population genetic analysis of Pseudomonas aeruginosa using multilocus sequence typing

To study the population genetic structure of Pseudomonas aeruginosa, we developed a multilocus sequence typing scheme. The sequences of internal fragments of seven housekeeping genes were obtained for 34 P. aeruginosa isolates from patients hospitalized in five different European cities. Twenty-six different allelic profiles were identified. The mean allelic diversity was 0.854 (range: 0.606-0.978), which was about six times greater than the results obtained with the multilocus enzyme electrophoresis method. Linkage disequilibrium was measured with the index of association. An index of 1.95+/-0.24 was calculated when all the strains were considered. This index was 1.76+/-0.27 when only one strain per sequence type was considered. Both results were different from 0, indicating linkage among loci, which means that the population structure of our set of P. aeruginosa isolates is clonal. The clonal structure of the population was also suggested by the congruence of the topology of the different trees obtained from the seven housekeeping genes. These results are in contrast to previous studies, finding a non clonal population structure. Since a small number of isolates was analyzed in this study, there might be a bias of selection which includes the possibility that they belong to widely disseminated epidemic clones. Another possibility is that recombination did not occurred homogeneously throughout the genome of P. aeruginosa, so that part of it has a clonal structure, while the remaining part of the genome is more frequently subject to recombination.     

Surface Structure of Intact Cells and Spheroplasts of Pseudomonas aeruginosa

This report describes the ultrastructural features of Pseudomonas aeruginosa after freeze-etching of intact cells and enzymatically prepared spheroplasts. Freeze-etching of intact cells revealed two convex layers of the cell wall and particles within the hydrophobic interior of the cell membrane. Areas of the membrane free of particles were sometimes elevated in the form of rather large dome-shaped structures. Spheroplasts were formed from intact cells by the addition of trypsin to a reaction mixture of lysozyme and ethylenediaminetetraacetic acid. Spheroplasts contained the outer lipoid layer of the cell wall. It was possible to observe this cell wall layer in freeze-etch preparations of spheroplasts. The spheroplast membrane like that of intact cells was cleaved along a central plane to expose particles and particle-free areas.     

铜绿假单胞菌的临床分布和耐药性变迁

目的调查温州医学院附属第一医院铜绿假单胞菌的临床分布和耐药性及其变迁情况,指导临床合理用药和控制耐药菌的产生。方法对该院2003年至2005年437株铜绿假单胞菌进行回顾性分析,用17种抗菌药物进行体外药敏试验。结果铜绿假单胞菌分布以呼吸道感染和创口分泌物感染为主。细菌对第3代头孢菌素耐药率高,对头孢他啶、亚胺培南、头孢哌酮/舒巴坦和氟喹诺酮类药物的耐药率均有逐年增加的趋势。耐药率增加与临床广谱抗菌药物用药量增加有关。结论铜绿假单胞菌的耐药性十分突出,应在药敏指导下合理选用抗菌药物,应该适当控制碳青霉烯类和第3代头孢菌素的使用,减缓耐药菌株的产生。     

2006-2009年铜绿假单胞菌医院感染临床分布及耐药性变迁

目的:调查铜绿假单胞菌(PA)4年来临床分布,了解临床分离PA时多种常用抗菌药物耐药谱的动态变迁,比较分析不同感染部位PA的耐药状况,为临床合理选用抗菌药物提供依据.方法:应用回顾性调查方法,对临床2006～2009年送检标本中分离的PA药敏试验进行统计分析.结果:队感染以呼吸道为主,主要分布在呼吸科和神经外科等科室;PA对临床常用的抗菌药物中左氧氟沙星和环丙沙星的耐药率相对上升较快,分别由2006年的25.8%、16.5%上升至2009年的52.8%、40.1%;对阿米卡星、亚胺培南、美洛培南、哌拉西林/他唑巴坦的耐药率相对较稳定,且耐药率较低;对部分抗生素的耐药率在2009年有所回落.结论:PA对阿米卡星、亚胺培南、美洛培南、哌拉西林/他唑巴坦的敏感性较高,而对其它10种抗菌药物耐药现象严重,临床应根据药敏结果选择单一或联用抗菌药物,可有效控制和减缓细菌耐药性的增长;2009年PA对部分抗生素耐药率下降进一步提示合理用药的重要性.     

Structure,Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.     

Pyocine Typing of Clinical Strains of Pseudomonas aeruginosa

A total of 954 clinical isolates of Pseudomonas aeruginosa were typed by their ability to produce pyocines. The strains of Pseudomonas were isolated from urines, bloods, sputa, stools, and miscellaneous infectious exudates or tissue of patients of the Mayo Clinic and four associated hospitals. About 80% of the typable strains could be grouped into three major pyocine types: A (30.9%), B (34.8%), and D (14.1%). These large groups could be divided into subtypes by using additional indicator strains. There was no significant difference in the distribution of types by either institutional or specimen source, except that urine specimens yielded the highest percentage of one type. By this procedure, 93% of all isolates could be typed. Repeated typing of serially transferred strains indicated that the procedure has a high degree of reliability. Several strains exhibited extreme fluctuation in inhibition pattern. The procedure is a simple and reliable method to monitor the patterns of nosocomial infections due to P. aeruginosa.