Label‐free quantitative proteomics identifies unique proteomes of clinical isolates of the Liverpool Epidemic Strain of Pseudomonas aeruginosa and laboratory strain PAO1

2021 ◽  
pp. 2100062
Author(s):  
Mara C. Goodyear ◽  
Nicole Garnier ◽  
Jonathan R. Krieger ◽  
Jennifer Geddes‐McAlister ◽  
Cezar M. Khursigara
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quantitative Proteomics ◽  
Laboratory Strain ◽  
Clinical Isolates ◽  
Epidemic Strain ◽  
Label Free ◽  
Strain Pao1

scholarly journals A Rapid Phenotypic Whole-Cell Screening Approach for the Identification of Small-Molecule Inhibitors That Counter β-Lactamase Resistance in Pseudomonas aeruginosa

2017 ◽  
Vol 23 (1) ◽  
pp. 55-64 ◽  
Author(s):  
Deanna Collia ◽  
Thomas D. Bannister ◽  
Hao Tan ◽  
Shouguang Jin ◽  
Taimour Langaee ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Laboratory Strain ◽  
Multidrug Resistant ◽  
First Line ◽  
Strain Pao1 ◽  
Whole Cell ◽  
Bacterial Sensitivity ◽  
Opportunistic Human Pathogen

Pseudomonas aeruginosa is an opportunistic human pathogen that is prevalent in hospitals and continues to develop resistance to multiple classes of antibiotics. Historically, β-lactam antibiotics have been the first line of therapeutic defense. However, the emergence of multidrug-resistant (MDR) strains of P. aeruginosa, such as AmpC β-lactamase overproducing mutants, limits the effectiveness of current antibiotics. Among AmpC hyperproducing clinical isolates, inactivation of AmpG, which is essential for the expression of AmpC, increases bacterial sensitivity to β-lactam antibiotics. We hypothesize that inhibition of AmpG activity will enhance the efficacy of β-lactams against P. aeruginosa. Here, using a highly drug-resistant AmpC-inducible laboratory strain PAO1, we describe an ultra-high-throughput whole-cell turbidity assay designed to identify small-molecule inhibitors of the AmpG. We screened 645,000 compounds to identify compounds with the ability to inhibit bacterial growth in the presence of cefoxitin, an AmpC inducer, and identified 2663 inhibitors that were also tested in the absence of cefoxitin to determine AmpG specificity. The Z′ and signal-to-background ratio were robust at 0.87 ± 0.05 and 2.2 ± 0.2, respectively. Through a series of secondary and tertiary studies, including a novel luciferase-based counterscreen, we ultimately identified eight potential AmpG-specific inhibitors.

scholarly journals Contribution of the MexX-MexY-OprM Efflux System to Intrinsic Resistance in Pseudomonas aeruginosa

2000 ◽  
Vol 44 (9) ◽  
pp. 2242-2246 ◽  
Author(s):  
Nobuhisa Masuda ◽  
Eiko Sakagawa ◽  
Satoshi Ohya ◽  
Naomasa Gotoh ◽  
Hideto Tsujimoto ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laboratory Strain ◽  
Immunoblot Analysis ◽  
Rabbit Serum ◽  
Deficient Mutant ◽  
Efflux System ◽  
Intrinsic Resistance ◽  
Strain Pao1 ◽  
Isogenic Mutants ◽  
Increased Susceptibility

ABSTRACT To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/ormexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of themexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of amexAB-oprM- and mexXY-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.

scholarly journals Use of suppression subtractive hybridization to examine the accessory genome of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa

2006 ◽  
Vol 55 (6) ◽  
pp. 677-688 ◽  
Author(s):  
Catherine H. M. Smart ◽  
Martin J. Walshaw ◽  
C. Anthony Hart ◽  
Craig Winstanley
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Suppression Subtractive Hybridization ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Subtractive Hybridization ◽  
Epidemic Strain ◽  
Strain Pao1 ◽  
The Uk ◽  
Specific Sequences

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa has been highly successful at colonizing cystic fibrosis (CF) patients throughout the UK, has replaced previously established strains in CF patients, has caused infections of non-CF parents of CF patients, and can cause greater morbidity in CF than other strains of P. aeruginosa. Using suppression subtractive hybridization (SSH) to identify strain-specific sequences, a diagnostic test for the LES based on PCR amplification of SSH sequence PS21 had previously been developed. In this study, the SSH sequence database of LES was substantially increased, using both extension of previous sequences and new rounds of subtraction. Of 92 SSH sequences identified as present in the LES but absent from strain PAO1, 25 were assessed for prevalence amongst a strain panel consisting mainly of LES and non-LES CF isolates. Preliminary analysis of genome sequence data indicated that all SSH sequences that were LES specific or found only rarely in other strains of P. aeruginosa were present on one of three contigs. All of the SSH sequences screened were either unstable amongst LES isolates or were not completely LES specific. Rare false positives were found with the PS21 test. The authors suggest that a second PCR assay designed to detect SSH sequence LESF9 can be used to confirm the identity of the most prevalent CF epidemic lineage in the UK.

scholarly journals Priming with intranasal lactobacilli prevents Pseudomonas aeruginosa acute pneumonia in mice.

2020 ◽  
Author(s):  
Marie-Sarah FANGOUS ◽  
Philippe Gosset ◽  
Nicolas Galakhoff ◽  
Stéphanie Gouriou ◽  
Charles-Antoine Guilloux ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Virulence Factors ◽  
Saline Solution ◽  
Clinical Isolates ◽  
Control Groups ◽  
Strain Pao1 ◽  
Log Reduction ◽  
Animal Survival

Abstract Background : Increasing resistance to antibiotics of Pseudomonas aeruginosa leads to therapeutic deadlock and alternative therapies are needed. We aimed to evaluate the effects of Lactobacillus clinical isolates in vivo, through intranasal administration on a murine model of Pseudomonas aeruginosa pneumonia.Results : We screened in vitro 50 pulmonary clinical isolates of Lactobacillus for their ability to decrease the synthesis of two QS dependent-virulence factors (elastase and pyocyanin) produced by Pseudomonas aeruginosa strain PAO1.Two blends of three Lactobacillus isolates were then tested in vivo: one with highly effective anti-PAO1 virulence factors properties (blend named L.rff for L. rhamnosus, two L. fermentum strains), and the second with no properties (blend named L.psb, for L. paracasei, L. salivarius and L. brevis). Each blend was administered intranasally to mice 18h prior to PAO1 pulmonary infection. Animal survival, bacterial loads, cytological analysis, and cytokines secretion in the lungs were evaluated at 6 or 24h post infection with PAO1. Intranasal priming with both lactobacilli blends significantly improved 7-day mice survival from 12% for the control PAO1 group to 71% and 100% for the two groups receiving L.rff and L.psb respectively. No mortality was observed for both control groups receiving either L.rff or L.psb. Additionally, the PAO1 lung clearance was significantly enhanced at 24h. A 2-log and 4-log reduction was observed in the L.rff+PAO1 and L.psb+PAO1 groups respectively, compared to the control PAO1 group. Significant reductions in neutrophil recruitment and proinflammatory cytokine and chemokine secretion were observed after lactobacilli administration compared to saline solution, whereas IL-10 production was increased. Conclusions : These results demonstrate that intranasal priming with lactobacilli acts as a prophylaxis, and avoids fatal complications caused by Pseudomonas aeruginosa pneumonia in mice. These results were independent of in vitro anti-Pseudomonas aeruginosa activity on QS-dependent virulence factors. Further experiments are required to identify the immune mechanism before initiating clinical trials.

scholarly journals C-Terminal Region of Pseudomonas aeruginosa Outer Membrane Porin OprD Modulates Susceptibility to Meropenem

2001 ◽  
Vol 45 (6) ◽  
pp. 1780-1787 ◽  
Author(s):  
Simone F. Epp ◽  
Thilo Köhler ◽  
Patrick Plésiat ◽  
Mehri Michéa-Hamzehpour ◽  
Joachim Frey ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Basic Amino Acid ◽  
Clinical Isolates ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Strain Pao1 ◽  
External Loop ◽  
Outer Membrane Porin ◽  
Major Alteration

ABSTRACT We investigated the unusual susceptibility to meropenem observed for seven imipenem-resistant clinical isolates of Pseudomonas aeruginosa. These strains were genetically closely related, expressed OprD, as determined by Western blot analyses, and were resistant to imipenem (>5 μg/ml) but susceptible to meropenem (<1 μg/ml). The oprD genes from two isolates were entirely sequenced, and their deduced protein sequences showed 93% identity with that of OprD of strain PAO1. The major alteration consisted of the replacement of a stretch of 12 amino acids, located in putative external loop L7 of OprD, by a divergent sequence of 10 amino acid residues. The oprD gene variants and the wild-typeoprD gene were cloned and expressed in a definedoprD mutant. The meropenem MICs for strains carrying theoprD genes from clinical isolates were four times lower than that for the strain carrying the wild-type oprDgene. Imipenem activities, however, were comparable for all strains. Furthermore, meropenem hypersusceptibility was obtained with a hybrid OprD porin that consisted of the PAO1 oprD gene containing loop L7 from a clinical isolate. These results show that the C-terminal portion of OprD, in particular, loop L7, was responsible for the unusual meropenem hypersusceptibility. Competition experiments suggested that the observed OprD modifications in the clinical isolates did not affect antagonism between imipenem and the basic amino acidl-lysine. We further propose that shortening of putative loop L7 of the OprD porin by 2 amino acid residues sufficiently opens the porin channel to allow optimal penetration of meropenem and increase its activity. In contrast, this alteration would not affect susceptibility to a smaller carbapenem molecule, such as imipenem.

scholarly journals Role of ampD Homologs in Overproduction of AmpC in Clinical Isolates of Pseudomonas aeruginosa

2008 ◽  
Vol 52 (11) ◽  
pp. 3922-3927 ◽  
Author(s):  
Amber J. Schmidtke ◽  
Nancy D. Hanson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Model System ◽  
Clinical Isolates ◽  
Rna Expression ◽  
Wild Type ◽  
Sequence Analyses ◽  
Strain Pao1 ◽  
Unknown Factor ◽  
Recycling Pathway

ABSTRACT AmpD indirectly regulates the production of AmpC β-lactamase via the cell wall recycling pathway. Recent publications have demonstrated the presence of multiple ampD genes in Pseudomonas aeruginosa and Escherichia coli. In the prototype P. aeruginosa strain, PAO1, the three ampD genes (ampD, ampDh2, and ampDh3) contribute to a stepwise regulation of ampC β-lactamase and help explain the partial versus full derepression of ampC. In the present study, the roles of the three ampD homologs in nine clinical P. aeruginosa isolates with either partial or full derepression of ampC were evaluated. In eight of nine isolates, decreased RNA expression of the ampD genes was not associated with an increase in ampC expression. Sequence analyses revealed that every derepressed isolate carried mutations in ampD, and in two fully derepressed strains, only ampD was mutated. Furthermore, every ampDh2 gene was of the wild type, and in some fully derepressed isolates, ampDh3 was also of the wild type. Mutations in ampD and ampDh3 were tested for their effect on function by using a plasmid model system, and the observed mutations resulted in nonfunctional AmpD proteins. Therefore, although the sequential deletion of the ampD homologs of P. aeruginosa can explain partial and full derepression in PAO1, the same model does not explain the overproduction of AmpC observed in these clinical isolates. Overall, the findings of the present study indicate that there is still an unknown factor(s) that contributes to ampC regulation in P. aeruginosa.

scholarly journals Analysis of the Phospholipid Profile of the Collection Strain PAO1 and Clinical Isolates of Pseudomonas aeruginosa in Relation to Their Attachment Capacity

2021 ◽  
Vol 22 (8) ◽  
pp. 4003
Author(s):  
Caroline Le Sénéchal ◽  
Mathilde Puges ◽  
Christophe Barthe ◽  
Patricia Costaglioli ◽  
Caroline Tokarski ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Clinical Isolates ◽  
Mass Spectrometry Analysis ◽  
Strain Pao1 ◽  
Spectrometry Analysis ◽  
Collection Strain ◽  
Strain Dependent ◽  
Very High ◽  
Phospholipid Profile

Bacteria form multicellular and resistant structures named biofilms. Biofilm formation starts with the attachment phase, and the molecular actors involved in this phase, except adhesins, are poorly characterized. There is growing evidence that phospholipids are more than simple structural bricks. They are involved in bacterial adaptive physiology, but little is known about their role in biofilm formation. Here, we report a mass spectrometry analysis of the phospholipid (PL) profile of several strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients. The aim of our study was to evaluate a possible link between the PL profile of a strain and its attachment phenotype. Our results showed that PL profile is strongly strain-dependent. The PL profile of P. aeruginosa PAO1, a collection strain, was different from those of 10 clinical isolates characterized either by a very low or a very high attachment capacity. We observed also that the clinical strain’s PL profiles varied even more importantly between isolates. By comparing groups of strains having similar attachment capacities, we identified one PL, PE 18:1-18:1, as a potential molecular actor involved in attachment, the first step in biofilm formation. This PL represents a possible target in the fight against biofilms.

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