Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients

Chronic bronchopulmonary bacterial infections remain the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Recent community sequencing work has now shown that the bacterial community in the CF lung is polymicrobial. Identifying bacteria in the CF lung through sequencing can be costly and is not practical for many laboratories. Molecular techniques such as terminal restriction fragment length polymorphism or amplicon length heterogeneity-polymerase chain reaction (LH-PCR) can provide many laboratories with the ability to study CF bacterial communities without costly sequencing. The aim of this study was to determine if the use of LH-PCR with multiple hypervariable regions of the 16S rRNA gene could be used to identify organisms found in sputum DNA. This work also determined if LH-PCR could be used to observe the dynamics of lung infections over a period of time. Nineteen samples were analysed with the V1 and the V1_V2 region of the 16S rRNA gene. Based on the amplicon size present in the V1_V2 region, Pseudomonas aeruginosa was confirmed to be in all 19 samples obtained from the patients. The V1 region provided a higher power of discrimination between bacterial profiles of patients. Both regions were able to identify trends in the bacterial population over a period of time. LH profiles showed that the CF lung community is dynamic and that changes in the community may in part be driven by the patient's antibiotic treatment. LH-PCR is a tool that is well suited for studying bacterial communities and their dynamics.

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. ^