Source: https://hzi.openrepository.com/handle/10033/620726
Timestamp: 2019-04-23 06:43:23+00:00

Document:
The pseudomonas quinolone signal (PQS) balances life and death in Pseudomonas aeruginosa populations.
When environmental conditions deteriorate and become inhospitable, generic survival strategies for populations of bacteria may be to enter a dormant state that slows down metabolism, to develop a general tolerance to hostile parameters that characterize the habitat, and to impose a regime to eliminate damaged members. Here, we provide evidence that the pseudomonas quinolone signal (PQS) mediates induction of all of these phenotypes. For individual cells, PQS, an interbacterial signaling molecule of Pseudomonas aeruginosa, has both deleterious and beneficial activities: on the one hand, it acts as a pro-oxidant and sensitizes the bacteria towards oxidative and other stresses and, on the other, it efficiently induces a protective anti-oxidative stress response. We propose that this dual function fragments populations into less and more stress tolerant members which respond differentially to developing stresses in deteriorating habitats. This suggests that a little poison may be generically beneficial to populations, in promoting survival of the fittest, and in contributing to bacterial multi-cellular behavior. It further identifies PQS as an essential mediator of the shaping of the population structure of Pseudomonas and of its response to and survival in hostile environmental conditions.
Phenotypic and genome-wide analysis of an antibiotic-resistant small colony variant (SCV) of Pseudomonas aeruginosa.
Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.
Acquired type III secretion system determines environmental fitness of epidemic Vibrio parahaemolyticus in the interaction with bacterivorous protists.
Genome analyses of marine microbial communities have revealed the widespread occurrence of genomic islands (GIs), many of which encode for protein secretion machineries described in the context of bacteria-eukaryote interactions. Yet experimental support for the specific roles of such GIs in aquatic community interactions remains scarce. Here, we test for the contribution of type III secretion systems (T3SS) to the environmental fitness of epidemic Vibrio parahaemolyticus. Comparisons of V. parahaemolyticus wild types and T3SS-defective mutants demonstrate that the T3SS encoded on genome island VPaI-7 (T3SS-2) promotes survival of V. parahaemolyticus in the interaction with diverse protist taxa. Enhanced persistence was found to be due to T3SS-2 mediated cytotoxicity and facultative parasitism of V. parahaemolyticus on coexisting protists. Growth in the presence of bacterivorous protists and the T3SS-2 genotype showed a strong correlation across environmental and clinical isolates of V. parahaemolyticus. Short-term microcosm experiments provide evidence that protistan hosts facilitate the invasion of T3SS-2 positive V. parahaemolyticus into a coastal plankton community, and that water temperature and productivity further promote enhanced survival of T3SS-2 positive V. parahaemolyticus. This study is the first to describe the fitness advantage of GI-encoded functions in a microbial food web, which may provide a mechanistic explanation for the global spread and the seasonal dynamics of V. parahaemolyticus pathotypes, including the pandemic serotype cluster O3:K6, in aquatic environments.
The Pseudomonas aeruginosa Chemotaxis Methyltransferase CheR1 Impacts on Bacterial Surface Sampling.
The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa.
Global genotype-phenotype correlations in Pseudomonas aeruginosa.
Once the genome sequence of an organism is obtained, attention turns from identifying genes to understanding their function, their organization and control of metabolic pathways and networks that determine its physiology. Recent technical advances in acquiring genome-wide data have led to substantial progress in identifying gene functions. However, we still do not know the function of a large number of genes and, even when a gene product has been assigned to a functional class, we cannot normally predict its contribution to the phenotypic behaviour of the cell or organism--the phenome. In this study, we assessed bacterial growth parameters of 4030 non-redundant PA14 transposon mutants in the pathogenic bacterium Pseudomonas aeruginosa. The genome-wide simultaneous analysis of 119 distinct growth-related phenotypes uncovered a comprehensive phenome and provided evidence that most genotypes are not phenotypically isolated but rather define specific complex phenotypic clusters of genotypes. Since phenotypic overlap was demonstrated to reflect the relatedness of genotypes on a global scale, knowledge of an organism's phenome might significantly contribute to the advancement of functional genomics.
Genomewide identification of genetic determinants of antimicrobial drug resistance in Pseudomonas aeruginosa.
The emergence of antimicrobial drug resistance is of enormous public concern due to the increased risk of delayed treatment of infections, the increased length of hospital stays, the substantial increase in the cost of care, and the high risk of fatal outcomes. A prerequisite for the development of effective therapy alternatives is a detailed understanding of the diversity of bacterial mechanisms that underlie drug resistance, especially for problematic gram-negative bacteria such as Pseudomonas aeruginosa. This pathogen has impressive chromosomally encoded mechanisms of intrinsic resistance, as well as the potential to mutate, gaining resistance to current antibiotics. In this study we have screened the comprehensive nonredundant Harvard PA14 library for P. aeruginosa mutants that exhibited either increased or decreased resistance against 19 antibiotics commonly used in the clinic. This approach identified several genes whose inactivation sensitized the bacteria to a broad spectrum of different antimicrobials and uncovered novel genetic determinants of resistance to various classes of antibiotics. Knowledge of the enhancement of bacterial susceptibility to existing antibiotics and of novel resistance markers or modifiers of resistance expression may lay the foundation for effective therapy alternatives and will be the basis for the development of new strategies in the control of problematic multiresistant gram-negative bacteria.
Evaluation of a microarray-hybridization based method applicable for discovery of single nucleotide polymorphisms (SNPs) in the Pseudomonas aeruginosa genome.
BACKGROUND: Whole genome sequencing techniques have added a new dimension to studies on bacterial adaptation, evolution and diversity in chronic infections. By using this powerful approach it was demonstrated that Pseudomonas aeruginosa undergoes intense genetic adaptation processes, crucial in the development of persistent disease. The challenge ahead is to identify universal infection relevant adaptive bacterial traits as potential targets for the development of alternative treatment strategies. RESULTS: We developed a microarray-based method applicable for discovery of single nucleotide polymorphisms (SNPs) in P. aeruginosa as an easy and economical alternative to whole genome sequencing. About 50% of all SNPs theoretically covered by the array could be detected in a comparative hybridization of PAO1 and PA14 genomes at high specificity (> 0.996). Variations larger than SNPs were detected at much higher sensitivities, reaching nearly 100% for genetic differences affecting multiple consecutive probe oligonucleotides. The detailed comparison of the in silico alignment with experimental hybridization data lead to the identification of various factors influencing sensitivity and specificity in SNP detection and to the identification of strain specific features such as a large deletion within the PA4684 and PA4685 genes in the Washington Genome Center PAO1. CONCLUSION: The application of the genome array as a tool to identify adaptive mutations, to depict genome organizations, and to identify global regulons by the "ChIP-on-chip" technique will expand our knowledge on P. aeruginosa adaptation, evolution and regulatory mechanisms of persistence on a global scale and thus advance the development of effective therapies to overcome persistent disease.
Beta-lactam resistance response triggered by inactivation of a nonessential penicillin-binding protein.
It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for beta-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in beta-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) beta-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for beta-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex beta-lactam resistance response, triggering overproduction of the chromosomal beta-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of beta-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases.
Pseudomonas aeruginosa cupA-encoded fimbriae expression is regulated by a GGDEF and EAL domain-dependent modulation of the intracellular level of cyclic diguanylate.
Cyclic-diguanylate (c-di-GMP) is a widespread bacterial signal molecule that plays a major role in the modulation of cellular surface components, such as exopolysaccharides and fimbriae, and in the establishment of a sessile life style. Here, we report that intracellular c-di-GMP levels influence cupA-encoded fimbriae expression in Pseudomonas aeruginosa. In an autoaggregative P. aeruginosa small colony variant (SCV) CupA fimbriae and the intracellular c-di-GMP concentration were found to be enhanced as compared with the clonal wild-type. The SCV morphology and the expression of CupA fimbriae were dependent on a functional PA1120 and morA gene both encoding a GGDEF domain. Overexpression of the GGDEF domain protein PA1120 complemented the PA1120 and the morA mutant with respect to CupA fimbriae expression. In agreement with these findings, overexpression of the EAL domain containing phenotypic variance regulator (PvrR) in the SCV resulted in a decreased intracellular level of c-di-GMP, a reduced cupA fimbriae expression and a switch to wild-type colony morphology.
A German external quality survey of diagnostic microbiology of respiratory tract infections in patients with cystic fibrosis.
BACKGROUND: The goal of this pilot study was to design an external quality assessment (EQA) scheme for German cystic fibrosis (CF) clinical microbiology laboratories. Therefore, a multicentre study of 18 German CF laboratories was performed to evaluate their proficiency in analyzing CF respiratory secretions. METHODS: Simulated clinical specimens containing a set of four frequent CF pathogens, namely two Pseudomonas aeruginosa strains differing in morphotype (mucoid versus non-mucoid) and resistotype, one Staphylococcus aureus strain and one Burkholderia multivorans strain, were distributed to each laboratory. Isolation, identification and antimicrobial susceptibility testing (AST) of any bacterial pathogen present and completion of a questionnaire about applied microbiological protocols were requested. RESULTS: Three of four strains were isolated and identified correctly by almost all laboratories. B. multivorans was once misidentified as Burkholderia cenocepacia. Fourteen laboratories failed to detect the second multidrug resistant P. aeruginosa isolate. AST errors occurred most often for P. aeruginosa 2 followed by B. multivorans, P. aeruginosa 1 and S. aureus. Evaluation of the questionnaires revealed major differences in cultivation and identification techniques applied by the participating laboratories. CONCLUSIONS: A periodical EQA programme for German CF laboratories and standardized microbiological procedures seem to be necessary to advance diagnostic microbiology employed on CF respiratory tract specimens and may help to improve anti-infective treatment and infection control practices for CF patients.
Identification of Burkholderia cepacia complex pathogens by rapid-cycle PCR with fluorescent hybridization probes.
Members of the Burkholderia cepacia complex are important bacterial pathogens in cystic fibrosis (CF) patients. The B. cepacia complex currently consists of nine genetic subgroups (genomovars) of different epidemiological relevance and possibly of different pathogenic potential in humans. In this study, a new approach was developed for the rapid identification of B. cepacia genomovar I, Burkholderia multivorans (genomovar II), Burkholderia cenocepacia (lineage III-A and III-B), Burkholderia stabilis (genomovar IV) and Burkholderia vietnamiensis (genomovar V), which cause the large majority of infections in CF patients. The method was based on the detection of differences in the recA gene sequence by using rapid-cycle PCR and genomovar-specific fluorescence resonance energy transfer (FRET) probes. The genomovar status of all 39 B. cepacia complex strains tested (genomovars I-V) was identified by melting-curve analysis. Each FRET probe produced a specific fluorescence signal only with the respective genomovar, and not with other B. cepacia complex strains and Burkholderia spp. The identification system was easy to handle and revealed B. cepacia complex genomovar I-V status from culture isolates within about 1 h.
The Pseudomonas aeruginosa quinolone signal (PQS) has an iron-chelating activity.
Virulence factor production and the development of biofilms in Pseudomonas aeruginosa have been shown to be regulated by two hierarchically organized quorum-sensing systems activated by two types of small acyl-homoserine lactone signal molecules. Recently, a third type of bacterial signal molecule, the Pseudomonas quinolone signal (PQS), has been identified, which positively regulates a subset of genes dependent on the quorum-sensing systems. However, the molecular mechanism underlying PQS signalling has remained poorly understood. In this study the global transcriptional profile of P. aeruginosa in response to PQS revealed a marked upregulation of genes belonging to the tightly interdependent functional groups of the iron acquisition and the oxidative stress response. Remarkably, most of the differentially regulated genes, as well as the induction of rhlR, could be traced back to an iron-chelating effect of PQS. Our results amount to the elucidation of how PQS affects P. aeruginosa and have important implications for the understanding of the complex regulatory circuits involved in P. aeruginosa gene regulation.
Quorum-sensing antagonistic activities of azithromycin in Pseudomonas aeruginosa PAO1: a global approach.
The administration of macrolides such as azithromycin for chronic pulmonary infection of cystic fibrosis patients has been reported to be of benefit. Although the mechanisms of action remain obscure, anti-inflammatory effects as well as interference of the macrolide with Pseudomonas aeruginosa virulence factor production have been suggested to contribute to an improved clinical outcome. In this study we used a systematic approach and analyzed the impact of azithromycin on the global transcriptional pattern and the protein expression profile of P. aeruginosa PAO1 cultures versus those in untreated controls. The most remarkable result of this study is the finding that azithromycin exhibited extensive quorum-sensing antagonistic activities. In accordance with the inhibition of the quorum-sensing systems, virulence factor production was diminished and the oxidative stress response was impaired, whereas the type III secretion system was strongly induced. Moreover, P. aeruginosa motility was reduced, which probably accounts for the previously observed impaired biofilm formation capabilities of azithromycin-treated cultures. The interference of azithromycin with quorum-sensing-dependent virulence factor production, biofilm formation, and oxidative stress resistance in P. aeruginosa holds great promise for macrolide therapy in cystic fibrosis. Clearly quorum-sensing antagonist macrolides should be paid more attention in the management of chronic P. aeruginosa infections, and as quorum-sensing antagonists, macrolides might gain vital importance for more general application against chronic infections.

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