A genomic island in Pseudomonas aeruginosa carries the determinants of flagellin glycosylation

Protein glycosylation has been long recognized as an important posttranslational modification process in eukaryotic cells. Glycoproteins, predominantly secreted or surface localized, have also been identified in bacteria. We have identified a cluster of 14 genes, encoding the determinants of the flagellin glycosylation machinery in Pseudomonas aeruginosa PAK, which we called the flagellin glycosylation island. Flagellin glycosylation can be detected only in bacteria expressing the a-type flagellin sequence variants, and the survey of 30 P. aeruginosa isolates revealed coinheritance of the a-type flagellin genes with at least one of the flagellin glycosylation island genes. Expression of the b-type flagellin in PAK, an a-type strain carrying the glycosylation island, did not lead to glycosylation of the b-type flagellin of PAO1, suggesting that flagellins expressed by b-type bacteria not only lack the glycosylation island, they cannot serve as substrates for glycosylation. Providing the entire glycosylation island of PAK, including its a-type flagellin in a flagellin mutant of a b-type strain, results in glycosylation of the heterologous flagellin. These results suggest that some or all of the 14 genes on the glycosylation island are the genes that are missing from strain PAO1 to allow glycosylation of an appropriate flagellin. Inactivation of either one of the two flanking genes present on this island abolished flagellin glycosylation. Based on the limited homologies of these gene products with enzymes involved in glycosylation, we propose that the island encodes similar proteins involved in synthesis, activation, or polymerization of sugars that are necessary for flagellin glycosylation.     

Interstrain transfer of the large pathogenicity island (PAPI-1) of Pseudomonas aeruginosa

The large Pseudomonas aeruginosa pathogenicity island PAPI-1 of strain PA14 is a cluster of 108 genes that encode a number of virulence features. We demonstrate that, in a subpopulation of cells, PAPI-1 can exist in an extrachromosomal circular form after precise excision from its integration site within the 3' terminus of the tRNA(Lys) gene. Circular PAPI-1 can reintegrate into either of the two tRNA(Lys) genes, including the one that was used for integration of small pathogenicity island PAPI-2 in strain PA14. The excision requires PAPI-1-encoded integrase, a member of the tyrosine recombinase family. PAPI-1 Soj contains the conserved domains of proteins that are related to chromosome and plasmid partition. soj plays a role in maintaining PAPI-1 and mutations in soj result in the loss of PAPI-1 from P. aeruginosa. We further demonstrate that, during coculture, the PAPI-1-containing strains are able to transfer it into P. aeruginosa recipient strains that do not harbor this island naturally. After transfer, PAPI-1 integrates into either of the two tRNA(Lys) genes. PAPI-1 encompasses many features of mobile elements, including mobilization and maintenance modules. Together with the virulence determinants, PAPI-1 plays an important role in the evolution of P. aeruginosa, by expanding its natural habitat from soil and water to animal and human infections.     

Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas

Genomic islands (GEI) comprise a recently recognized large family of potentially mobile DNA elements and play an important role in the rapid differentiation and adaptation of bacteria. Most importantly, GEIs have been implicated in the acquisition of virulence factors, antibiotic resistances or toxic compound metabolism. Despite detailed information on coding capacities of GEIs, little is known about the regulatory decisions in individual cells controlling GEI transfer. Here, we show how self-transfer of ICEclc, a GEI in Pseudomonas knackmussii B13 is controlled by a series of stochastic processes, the result of which is that only a few percent of cells in a population will excise ICEclc and launch transfer. Stochastic processes have been implicated before in producing bistable phenotypic transitions, such as sporulation and competence development, but never before in horizontal gene transfer (HGT). Bistability is instigated during stationary phase at the level of expression of an activator protein InrR that lays encoded on ICEclc, and then faithfully propagated to a bistable expression of the IntB13 integrase, the enzyme responsible for excision and integration of the ICEclc. Our results demonstrate how GEI of a very widespread family are likely to control their transfer rates. Furthermore, they help to explain why HGT is typically confined to few members within a population of cells. The finding that, despite apparent stochasticity, HGT rates can be modulated by external environmental conditions provides an explanation as to why selective conditions can promote DNA exchange.     

穿心莲内酯对铜绿假单胞菌PAO1株MexAB-OprM外排泵mRNA表达的影响

目的建立实时荧光定量PCR法检测铜绿假单胞菌MexAB-OprMmRNA水平的表达，探讨中草药穿心莲内酯对铜绿假单胞菌外排泵MexAB-OprM表达情况的影响。方法克隆铜绿假单胞菌mexAB-oprM操纵元中merB基因和30SrRNA基因rpsL，分别构建相应质粒作为实时荧光定量PCRDNA标准品。以不同浓度穿心莲内酯作用铜绿假单胞菌PA01株，采用实时荧光定量PCR法检测PA01菌株中mexB和rpsl。基冈mRNA的表达水平。结果成功构建标准曲线质粒，50、100、150和200／μg／ml。穿心莲内酯作用后PA01ⅢPTB基因mRNA表达量分别为0．04±0．03、0．06±0．07、0．09±0．03和0．04±0．03，对照组为0．24±0．04，差异有统计学意义（P〈0．05），穿心莲内酯组间差异无统计学意义（P〉0．05）。结论穿心莲内酯可在mRNA水平抑制外排泵Mex—AB-OprM的表达，这可能是其抗感染机制之一。     

Emerging Phage Resistance in Pseudomonas aeruginosa PAO1 Is Accompanied by an Enhanced Heterogeneity and Reduced Virulence

Bacterial surface structures of a proteinic nature and glycoconjugates contribute to biofilm formation and provide shields to host defense mechanisms (e.g., the complement system and phagocytosis). A loss or alteration of these molecules, leading to phage resistance, could result in fewer virulent bacteria. In this study, we evaluate the biology and phenotype changes in Pseudomonas aeruginosa PAO1 phage-resistant clones, which emerge in phage-treated biofilms. We characterize these clones for phage-typing patterns, antibiotic resistance, biofilm formation, pathogenicity, and interactions with the innate immune system. Another important question that we address is whether phage-resistant mutants are also generated incidentally, despite the phage treatment-selective pressure, as the natural adaptation of the living biofilm population. It is found that the application of different phages targeting a particular receptor selects similar phage resistance patterns. Nevertheless, this results in a dramatic increase in the population heterogeneity, giving over a dozen phage-typing patterns, compared to one of the untreated PAO1 sessile forms. We also confirm the hypothesis that “phage-resistant bacteria are more susceptible to antibiotics and host-clearance mechanisms by the immune system”. These findings support phage application in therapy, although the overall statement that phage treatment selects the less virulent bacterial population should be further verified using a bigger collection of clinical strains.     

Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.     

Fluoroquinolone-resistant Pseudomonas aeruginosa: assessment of risk factors and clinical impact

Purpose

Pseudomonas aeruginosa infections have been associated with considerable morbidity and mortality. Fluoroquinolones (FQ) are the only oral therapy available for P. aeruginosa infections, but resistance is increasingly prevalent.

Methods

We examined annual trends in FQ-resistant P. aeruginosa (FQRPA) from 1991 to 2000. Subsequently, inpatients with a clinical culture positive for P. aeruginosa between January 1, 1999 and December 31, 2000 were included in a case control study to identify risk factors for FQ resistance and a cohort study to examine the impact of FQ resistance on outcomes in P. aeruginosa.

Results

Annual prevalence of FQRPA increased from 15% in 1991 to 41% in 2000 (P <0.001 trend). Between 1999 and 2000, 332 P. aeruginosa isolates were FQ resistant and 540 were FQ susceptible. Prior FQ use was the only independent risk factor for FQRPA (adjusted OR = 3.43; 95% confidence interval [CI] 2.37, 4.96). Subjects with FQRPA had greater median hospital charges ($62,325 vs $48,734) (P =.007) and higher mortality (47.5% vs 35.5%) (P =.004). However, in a multivariate model, only imipenem resistance of the isolate was significantly associated with mortality. FQ resistance was not an independent risk factor.

Conclusions

FQRPA has increased significantly and is associated with prior FQ use. Limiting FQ use may curb the emergence of resistance among P. aeruginosa. FQRPA is associated with increased hospital charges, but other resistance patterns may have a more significant impact on mortality.     

12-Methyltetradecanoic acid, a branched-chain fatty acid, represses the extracellular production of surfactants required for swarming motility in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is known to produce surfactants that are involved in its swarming motility behavior, such as rhamnolipids and their precursors-3-(3-hydroxyalkanoyloxy)-alkanoic acids (HAAs). In P. aeruginosa PAO1, swarming motility is inhibited by some fatty acids, including branched-chain fatty acids and unsaturated fatty acids. In the present study, addition of 12-methyltetradecanoic acid (12-MTA, anteiso-C15:0) to an agar medium markedly repressed surfactant activity in the extracellular fraction of a P. aeruginosa culture in a drop collapse assay. Further, an extracellular fraction of a culture of rhlA mutant P. aeruginosa, which did not produce both rhamnolipids and HAAs, showed a complete loss of surfactant activity and markedly reduced swarming activity. In contrast, an extracellular fraction of a culture of rhlB mutant P. aeruginosa, which produced HAAs but not rhamnolipids, showed moderate swarming activity and weak extracellular surfactant activity that was lost on the addition of 12-MTA to the agar medium. Expression of the rhlAB operon from the plasmid pMR2 restored normal swarming motility on 12-MTA-containing agar medium. Taken together, these findings indicate that 12-MTA reduced extracellular surfactant activity, thus resulting in a swarming defect in P. aeruginosa PAO1.     

野生型铜绿假单胞菌PAO1和粘液型铜绿假单胞菌PA17播种型扩散的研究

目的:探讨PA17和PAO1生物膜形成过程中播种型扩散发生的相关机制.方法:SYTO9/PI双染色后,在激光共聚焦显微镜下观察1、3、5、7、9、t1d时PAO1及PA17生物膜形成的情况,real-time RT-PCR检测不同时间点PAO1及PA17的algR的表达.结果:PAO13 d时形成生物膜,但PA17此时...     

Deuterium isotope effect on the intramolecular electron transfer in Pseudomonas aeruginosa azurin

Intramolecular electron transfer in azurin in water and deuterium oxide has been studied over a broad temperature range. The kinetic deuterium isotope effect, k(H)/k(D), is smaller than unity (0.7 at 298 K), primarily caused by the different activation entropies in water (-56.5 J K(-1) mol(-1)) and in deuterium oxide (-35.7 J K(-1) mol(-1)). This difference suggests a role for distinct protein solvation in the two media, which is supported by the results of voltammetric measurements: the reduction potential (E(0')) of Cu(2+/+) at 298 K is 10 mV more positive in D(2)O than in H(2)O. The temperature dependence of E(0') is also different, yielding entropy changes of -57 J K(-1) mol(-1) in water and -84 J K(-1) mol(-1) in deuterium oxide. The driving force difference of 10 mV is in keeping with the kinetic isotope effect, but the contribution to DeltaS from the temperature dependence of E(0') is positive rather than negative. Isotope effects are, however, also inherent in the nuclear reorganization Gibbs free energy and in the tunneling factor for the electron transfer process. A slightly larger thermal protein expansion in H(2)O than in D(2)O (0.001 nm K(-1)) is sufficient both to account for the activation entropy difference and to compensate for the different temperature dependencies of E(0'). Thus, differences in driving force and thermal expansion appear as the most straightforward rationale for the observed isotope effect.     

Crystal structure of the electron transfer complex rubredoxin rubredoxin reductase of Pseudomonas aeruginosa

Crude oil spills represent a major ecological threat because of the chemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the few bacterial species able to metabolize such compounds. Three chromosomal genes, rubB, rubA1, and rubA2 coding for an NAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs) are indispensable for this ability. They constitute an electron transport (ET) pathway that shuttles reducing equivalents from carbon metabolism to the membrane-bound alkane hydroxylases AlkB1 and AlkB2. The RdxR-Rdx system also is crucial as part of the oxidative stress response in archaea or anaerobic bacteria. The redox couple has been analyzed in detail as a model system for ET processes. We have solved the structure of RdxR of P. aeruginosa both alone and in complex with Rdx, without the need for cross-linking, and both structures were refined at 2.40- and 2.45-A resolution, respectively. RdxR consists of two cofactor-binding domains and a C-terminal domain essential for the specific recognition of Rdx. Only a small number of direct interactions govern mutual recognition of RdxR and Rdx, corroborating the transient nature of the complex. The shortest distance between the redox centers is observed to be 6.2 A.     

Host inflammatory responses to first isolation of Pseudomonas aeruginosa from sputum in cystic fibrosis

Pseudomonas aeruginosa infection of the respiratory tract in patients with cystic fibrosis is a major determinant of morbidity and mortality. However, it has been postulated that the earliest phase of colonization is not associated with injury. To test this hypothesis we determined the association of the first recorded isolation of P. aeruginosa from the sputum on circulating markers of the inflammatory response in 6 patients with cystic fibrosis. At this time circulating C-reactive protein was increased in all 6 and neutrophil elastase α₁-antitrypsin complex (elastasecomplex) was increased in 5 patients compared with healthy controls. This inflammatory response was associated with a reduction in the FEV, and FVC of all patients [FEV, 1.42 ± 0.87 L (mean ± SD) at first isolation vs. 2.08 ± 0.74 L before isolation; P < 0.05; FVC, 1.94 ± 0.93 L vs. 2.87 ±1.01 L, P < 0,05]. At a median interval of 10 months, 5 patients had raised titres of positive IgG antibody to P. aeruginosa, indicating significant exposure to this organism. At this time, lung function had returned to preinfection levels, whilst 3 patients showed continuing features of an inflammatory response, and the group mean value for elastase-complex was raised. Our findings demonstrate that at the time of first isolation of P. aeruginosa from the sputum of patients with cystic fibrosis, there is a concomitant systemic host response and an acute deterioration of pulmonary function. © 1993 Wiley-Liss, Inc.     

同时产PER-1型和TEM-1型β内酰胺酶铜绿假单胞菌的检出

目的　分析多重耐药铜绿假单胞菌临床株 (编号 0 10 7)对 β内酰胺类抗生素耐药情况及其原因。 方法　用改良三维试验分析 0 10 7株产生的 β内酰胺酶表型 ,以碱裂解法提取质粒 ,用聚合酶链反应 (PCR)方法扩增质粒的酶基因型别 ,并对阳性产物测序。结果　 0 10 7号铜绿假单胞菌临床株产生超广谱 β内酰胺酶 (ESBLs) ,携有blaPER - 1基因 ,同时还有blaTEM - 1型基因。结论　 0 10 7号菌株耐 β内酰胺类抗生素耐药的可能原因是产生PER - 1型的ESBLs和TEM - 1型的广谱酶。     

Detection and characterization of variant Salmonella genomic island 1s from Salmonella Derby isolates

The purpose of this study is to investigate the distribution and structure of Salmonella genomic island 1 (SGI1) among Salmonella enterica serovar Derby isolates from swine and their rearing environment. Three variants of SGI1s, specifically SGI1-A, C, and I, were identified by PCR mapping. The results of macro-restriction analysis and DNA sequencing of SGI1 flanking regions revealed that there are at least two genomic lineages of Derby strains bearing SGI1s.     

产金属β-内酰胺酶铜绿假单胞菌的基因检测及耐药性研究

目的　研究本地区铜绿假单胞菌临床分离株中产金属 β -内酰胺酶流行株的主要基因型和耐药性。 方法　用纸片扩散法进行抗生素协同敏感试验 ,检测产金属 β -内酰胺酶的铜绿假单胞菌 ,用琼脂稀释法检测其最低抑菌浓度 (MIC) ,分别用金属酶IMP - 1,IMP - 2 ,VIM - 1和VIM - 2引物进行聚合酶链反应 (PCR)并对扩增产物进行序列分析。结果　在 82株耐亚胺培南铜绿假单胞菌中 ,抗生素协同敏感试验阳性 7株 ,PCR检测金属 β -内酰胺酶阳性株也为这 7株 ,且基因型均为VIM - 2型。产酶株对亚胺培南、头孢噻肟、头孢他啶、哌拉西林 /他唑巴坦等常用抗铜绿假单胞菌 β -内酰胺类抗生素不敏感 ,对氨曲南较为敏感。结论　本地区铜绿假单胞菌所产金属 β-内酰胺酶为VIM - 2型 ,且产酶株耐药性强 ,应注意对其进行临床检测和监控。     

Pseudomonas aeruginosa and the airways disease of cystic fibrosis

The gram-negative organism Pseudomonas aeruginosa plays a major role in the morbidity and mortality of patients with cystic fibrosis (CF). Inherent properties of this organism, together with alterations in the CF airway, lead to colonization of the CF patient and, ultimately, contribute to the destructive lung lesion of CF. Innovative antibiotic therapies for the CF patient are discussed, including new potent oral and parenteral pseudomonicidal antibiotics, the re-emergence of nebulized delivery systems, and the validation of home intravenous therapy.