739. Multidrug Resistant Pseudomonas aeruginosa (MDR-Pa) causing prosthetic valve endocarditis (PVE): a link between resistance and virulence
Session: Poster Abstract Session: Antimicrobials: Resistance Mechanisms
Friday, October 4, 2013
Room: The Moscone Center: Poster Hall C
Background: Successful treatment of infections caused by MDR-Pa is thwarted by the emergence of antibiotic resistance and biofilm formation. We analyzed isolates of MDR-Pa that were recovered from a patient with PVE who required surgery. All isolates were resistant to carbapenems and ciprofloxacin, but we noted a change in cephalosporin susceptibility over time. Our goal was to decipher the molecular basis of resistance and to understand the link with virulence in the Pa isolates.

Methods: Five sequential Pa blood isolates collected over 7 months were analyzed. Multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) were conducted. PCR amplification and sequencing was performed for the following genes: ampD, ampR, blaPDC, gyrA, gyrB, parC, parE, and oprD. Acquired carbapenemase genes were also screened: blaVIM, -KPC, -IMP. Expression levels of the AmpC of Pa, Pseudomonas derived cephalosporinase (PDC), was assessed by western blot.  

Results: All isolates belonged to sequence type 298, were identical by rep-PCR, and displayed resistance to ciprofloxacin (MIC >2 µg/mL), meropenem and imipenem (MICs >8 µg/mL) by Microscan. All contained mutations in gyrA (T83I), parC (S87L) and a premature stop codon at amino acid 79 in oprD, but no carbapenemase genes. While the initial isolate was susceptible to aztreonam, cefepime (MIC ≤ 8 µg/mL) and ceftazidime (MIC = 4 µg/mL), subsequent isolates were resistant (MIC >16 µg/mL). PDC-16 was found in all isolates. Sequencing of ampR genes revealed a D135N mutation in cephalosporin-resistant isolates only, which correlated with high-level (derepressed) PDC production by western blot.

Conclusion: Stepwise and/or point mutations in ampR, quinolone resistance determining regions (QRDRs), and porin channels contributed to the observed MDR phenotype. The D135N mutation, previously described only once in Pa, causes a conformational change in the AmpR regulator and results in increased PDC production, thus explaining the emergence of cephalosporin resistance. Notably, AmpR is a global transcriptional regulator associated with alginate production and biofilms, thus crucially linking resistance and virulence in this persistent PVE infection.  Prolonged antibiotic therapy in PVE may select for Pa that are both more resistant and virulent.

T. Nicholas Domitrovic, BA1, Andrea M. Hujer, BS2, Federico Perez, MD1, Steve Marshall, MS1, Kristine M. Hujer, BS2, Laila Woc-Coburn, M.D., D.T.M.&H.3, Mark Parta, MD4 and Robert A. Bonomo, MD1, (1)Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH, (2)Case Western Reserve University, Cleveland, OH, (3)Baylor College of Medicine, Houston, TX, (4)NIH, Bethesda, MD


T. N. Domitrovic, None

A. M. Hujer, None

F. Perez, None

S. Marshall, None

K. M. Hujer, None

L. Woc-Coburn, None

M. Parta, None

R. A. Bonomo, NIH: Grant Investigator, Research grant
VAMC: Grant Investigator, Research grant
Rib-X: Scientific Advisor, Consulting fee
Merck: Grant Investigator, Research grant
Astra-Zeneca: Grant Investigator, Grant recipient
Steris: Grant Investigator, Grant recipient

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