133. High throughput sequencing unveils unexpected roles for the OprD protein and the type IVa pili of Pseudomonas  aeruginosa affecting colonization and dissemination in mice
Session: Oral Abstract Session: Bacterial Pathogenesis and Virulence
Friday, October 21, 2011: 9:00 AM
Room: 151AB
Background: P. aeruginosa causes significant morbidity and mortality in immunocompromised hosts and ventilated patients. High throughput sequencing can identify genes necessary for colonization and dissemination in animal models and is a formidable tool to analyze full genomes to obtain a complete view of genes involved in pathogenesis.

Methods: A bank of 3x105 random transposon (Tn) mutants of P. aeruginosa PA14 was used to compare DNA needed for growth in LB, survival in water, colonization of the murine GI tract and dissemination to the spleen following induction of neutropenia. Genomic DNA was purified from P. aeruginosa recovered from each source, digested with MmeI, an enzyme cleaving 16 bp outside the inserted Tn, fragments sequenced on an Illumina Genome Analyzer and over 14 million sequences used to quantify changes in levels of genomic fragments carrying the Tn. Sequence comparisons revealed the genes necessary for colonization and dissemination.

Results: Of 5977 genes in PA14, 121, 391, 2583 and 5484 genes were found essential for survival in LB, water, gut colonization and spleen dissemination, respectively.  No mutant with a Tn inserted into previously described virulence factor genes was able to disseminate to the spleen. Interestingly, there was a strong positive selection for colonization of the cecum by interrupting the oprD gene and the genes involved in type IVa pilus production. Notably, more than 90% of the mutants disseminating to the spleen were oprD deficient. These results were confirmed in an in vivo colonization competition assay between the PA14 wild-type strain and mutants in the oprD and pilE genes. Induced or natural deletion of the oprD gene in P. aeruginosa strains from laboratory and clinical sources resulted in more resistance to carbapenems, better survival at pH4.5 and greater resistance to the action of the complement and phagocytes.

Conclusion: Mutations in the oprD gene are the main mechanism of resistance of P. aeruginosa to the carbapenems. These mutants appear to have a dramatic advantage in GI colonization and dissemination to the spleen, in part through a high resistance to the gastric acidic pH and to the action of the immune system antibacterial effectors. Potentially, carbapenem resistance may promote virulence.


Subject Category: B. Bacterial pathogenesis, studies in animal models, molecular pathogenicity

Skurnik David, M.D., Ph.D.1,2, Damien Roux, M.D., Ph.D.1,2, Yoder-Himes Deborah, Ph.D.3, Cattoir Vincent3, Xi Lu1,2, Roland Leclercq4, Stephen Lory, Ph.D3 and Gerald Pier, Ph.D1,2, (1)Channing Laboratory, Brigham and Women's Hospital, Boston, MA, (2)Medicine, Harvard Medical School, Boston, MA, (3)Microbiology, Harvard Medical School, Boston, MA, (4)Côte de Nacre Hosp, Caen, France

Disclosures:

S. David, None

D. Roux, None

Y. H. Deborah, None

C. Vincent, None

X. Lu, None

R. Leclercq, None

S. Lory, None

G. Pier, None

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