Role of AmpR and alginate production in Pseudomonas aeruginosa biofilm antibiotic resistance associated with cystic fibrosis

Pseudomonas aeruginosa is an ubiquitous Gram-negative opportunistic pathogen that is commonly found in nosocomial infections, immunocompromised patients and burn victims. In addition, P. aeruginosa colonizes the lungs of cystic fibrosis patients, leading to chronic infection, which inevitably leads to their demise. In this research, I analyzed the factors contributing to P. aeruginosa antibiotic resistance, such as the biofilm mode of growth, alginate production, and 13-lactamase synthesis. Using the biofilm eradication assay (MBEC™ assay), I exposed P. aeruginosa to B-lactams (piperacillin, ceftazidime, and cefotaxime ), aminoglycosides ( amikacin, tobramycin and gentamicin), and a fluoroquinolone ( ciprofloxacin) at various concentrations. I analyzed the effects of biofilm on P. aeruginosa antibiotic resistance, and confirmed that the parent strain PAO 1 biofilms cells were > 100 times more resistant than planktonic (freefloating) cells. The constitutively alginate-producing strain PDO300 exhibited an altered resistance pattern as compared to the parent strain P AO 1. Finally, the role of AmpR, the regulator of ampC-encoded 13-lactamase expression was analyzed by determining the resistance of the strain carrying a mutation in the ampR gene and compared to the parent strain PAOl. It was confirmed that the loss of ampR contributes to increased antibiotic resistance.

Characterization of the amp genes involved in the regulation of beta-lactamase expression in Pseudomonas aeruginosa

Antibiotic resistance, production of alginate and virulence factors, and altered host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection. Failure of antibiotic therapy has been attributed to the emergence of P. aeruginosa strains that produce β-lactamase constitutively. In Enterobacteriaceae, β-lactamase induction involves four genes with known functions: ampC, ampR, ampD, and ampG, encoding the enzyme, transcriptional regulator, amidase and permease, respectively. In addition to all these amp genes, P. aeruginosa possesses two ampG paralogs, designated ampG and ampP. In this study, P. aeruginosa ampC, ampR, ampG and ampP were analyzed. Inactivation of ampC in the prototypic PAO1 failed to abolish the β-lactamase activity leading to the discovery of P. aeruginosa oxacillinase PoxB. Cloning and expression of poxB in Escherichia coli confers β-lactam resistance. Both AmpC and PoxB contribute to P. aeruginosa resistance against a wide spectrum of β-lactam antibiotics. The expression of PoxB and AmpC is regulated by a LysR-type transcriptional regulator AmpR that up-regulates AmpC but down-regulates PoxB activities. Analyses of P. aeruginosa ampR mutant demonstrate that AmpR is a global regulator that modulates the expressions of Las and Rhl quorum sensing (QS) systems, and the production of pyocyanin, LasA protease and LasB elastase. Introduction of the ampR mutation into an alginate-producing strain reveals the presence of a complex co-regulatory network between antibiotic resistance, QS alginate and other virulence factor production. Using phoA and lacZ protein fusion analyses, AmpR, AmpG and AmpP were localized to the inner membrane with one, 16 and 10 transmembrane helices, respectively. AmpR has a cytoplasmic DNA-binding and a periplasmic substrate binding domains. AmpG and AmpP are essential for the maximal expression of β-lactamase. Analysis of the murein breakdown products suggests that AmpG exports UDP-N-acetylmuramyl-L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D-alanine-D-alanine (UDP-MurNAc-pentapeptide), the corepressor of AmpR, whereas AmpP imports N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid-Ala-γ-D-Glu-meso-diaminopimelic acid (GlcNAc-anhMurNAc-tripeptide) and GlcNAc-anhMurNAc-pentapeptide, the co-inducers of AmpR. This study reveals a complex interaction between the Amp proteins and murein breakdown products involved in P. aeruginosa β-lactamase induction. In summary, this dissertation takes us a little closer to understanding the P. aeruginosa complex co-regulatory mechanism in the development of β-lactam resistance and establishment of chronic infection. ^

Potential Application of Phage Therapy Against Pseudomonas aeruginosa Biofilm Infection in Cystic Fibrosis Patients

Majority of the microbial activity in humans is in the form of biofilms i.e. an Exopolysaccharide-enclosed bacterial mass. Unlike planktonic cells and the cells on the surface of the biofilm, the biofilm-embedded cells are more resistant to the effects of the antibiotics and the host cellular defense mechanisms. A combination of biofilm growth and inherent resistance prevents effective antibiotics treatment of Pseudomonas aeruginosa infections including those in patients with cystic fibrosis. This has lead to an increasing interest in alternative modalities of treatment. Thus, phages that multiply in situ, only in the presence of susceptible hosts can be used as natural, self-limiting, and deeply penetrating antibacterial agents. The objective of this study is to identify effective phages against a collection of P. aeruginosa isolates (PCOR strains) including the prototype PAOl and the isogenic constitutively alginate-producing PD0300 strains.These PCOR strains were tested against six phages (P105, P134, P140, P168, P175B and P182). Analysis shows 69 % of the PCOR isolates are sensitive and the rest are resistant to all six phages. These phages were then tested for their ability to inhibit biofilm formation using a modified biofilm assay. The analysis demonstrated that the sensitive strains showed increased resistance but none of the sensitive strains from the initial screening were resistant. Using the minimum biofilm eradication concentration (MBEC) assay for biofilm formation, the biofilm eradication ability of the phages was tested. The data showed that a higher volume of phage was required to eradicate preformed biofilms than the volume required to prevent colonization of planktonic cells. This data supports the idea of phage therapy more as a prophylactic treatment.

Molecular Mechanisms Involved in Regulating the Mucoid-to-Nonmucoid Conversion of Pseudomonas aeruginosa Associated with Cystic Fibrosis Patients

Pseudomonas aeruginosa is an opportunistic pathogen that has received attention because of its close association with cystic fibrosis (CF). Chronic pulmonary infection with the mucoid P. aeruginosa is the leading cause of mortality in CF patients. This bacterium has the ability to sense and adapt to the harsh environment in the CF lung by converting from a nonmucoid to a mucoid state. The mucoid phenotype is caused by overproduction of a polysaccharide called alginate. Alginate production is regulated by the algT/U operon containing five genes, algT/U-mucA-mucB-mucC-mucD. Alginate overproduction in CF isolates has been partially attributed to a loss-of-function mutation in mucA that results in the overexpression of algT. This mucoid phenotype is unstable, reverting to the nonmucoid form when the isolates are cultured outside of the CF lung. This study was undertaken to determine the mechanisms involved in the conversion from the mucoid to the nonmucoid form. Thirty-six spontaneous nonmucoid variants of a known mucoid isolate with a mucA mutation were analyzed. Ten of these isolates were complemented in trans by plasmids containing the algT operon and the algT gene. Chromosomal DNA was extracted and the mucA and algT genes were amplified by the polymerase chain reaction. Sequence analysis of the genes showed that these mutants retained the original mucA mutation but acquired secondary mutations in the algT gene.

Comparative transcriptome analyses of Pseudomonas aeruginosa

One of the hallmarks of bacterial survival is their ability to adapt rapidly to changing environmental conditions. Niche adaptation is a response to the signals received that are relayed, often to regulators that modulate gene expression. In the post-genomic era, DNA microarrays are used to study the dynamics of gene expression on a global scale. Numerous studies have used Pseudomonas aeruginosa--a Gram-negative environmental and opportunistic human pathogenic bacterium--as the model organism in whole-genome transcriptome analysis. This paper reviews the transcriptome studies that have led to immense advances in our understanding of the biology of this intractable human pathogen. Comparative analysis of 23 P. aeruginosa transcriptome studies has led to the identification of a unique set of genes that are signal specific and a core set that is differentially regulated. The 303 genes in the core set are involved in bacterial homeostasis, making them attractive therapeutic targets.