Losing My Resistance: Unexpected restoration of sensitivity of CRE following exposure to meropenem in combination with the new beta-lactamase inhibitor RPX7009
EMBARGOED UNTIL: Sunday, September 7, 2014, 3:00 P.M. EDT
(Session 126, Paper C-1193)
The Med.s Company, San Diego, CA, United States
The recent world-wide spread of β-lactamases that are capable of degrading meropenem and other carbapenems, the so called carbapenemases, threatens the usefulness of these exceptional drugs and is becoming the major clinical issue. In the United States and Europe, the most rapidly spreading carbapenemases are represented by the KPC (Klebsiella pneumonia carbapenemase) enzymes. Carbapenems are a sub-class of β-lactam antibiotics that combines the excellent safety profile of β-lactams with notable stability to the majority of β-lactamases, the main pathway of β-lactam resistance. Consequently, carbapenems are frequently antibiotics of “last defense” for the most resistant pathogens in serious infections.
Genes that encode KPC β-lactamase are located on various self-transferable plasmids (Gootz et al., 2009 AAC 53: 1998–2004); these plasmids can freely move between cells of either the same or different bacterial species. In all cases, the KPC gene is found as a part of the same transposon, Tn4401 located on the previously mentioned plasmids. A recent study indicates that Tn4401is a very active transposon capable of jumping from plasmid to plasmid at high frequency (Guzon et al., 2011, AAC 55: 5370-5373). This creates the perfect KPC breeding environment explaining the rapid worldwide dissemination of KPC.
Carbavance, a β-lactamase inhibitor/carbapenem combination product
Carbavance™ (meropenem/RPX7009) is a combination of the approved carbapenem antibiotic meropenem and an investigational β-lactamase inhibitor (BLI) RPX7009 being developed by The Medicines Company. This drug combination is being developed for intravenous (IV) administration for the treatment of patients with severe gram-negative infections, including those caused by bacteria resistant to currently available drugs.
RPX7009 is a greater than100-fold more potent inhibitor of KPC-2 than tazobactam and clavulanic acid (Tsivkovski et al., ICAAC 2012); it significantly (in many cases >256-fold) enhances the antimicrobial potency of meropenem (and other carbapenems), against strains that produce KPC and other (less frequently encountered) class A serine carbapenemases (Castanheira et al., ICAAC 2014); it improves the efficacy of meropenem against carbapenem-resistant, KPC-producing strains in various mouse infection models (Sabet et al., ICAAC 2014) and it is very safe as was demonstrated in the recently completed Phase 1 studies evaluating the safety, tolerability, and pharmacokinetics of RPX7009 alone, meropenem alone, and both in combination (Griffith et al., ICAAC 2014). Carbavance has been advanced into patient studies.
In preparation for initiation of clinical studies of Carbavance in infected patients, studies were conducted in an in vitro hollow fiber pharmacodynamic model of infection due to KPC-producing Enterobacteriaceae. The hollow fiber (HF) bioreactor system allows one to model human pharmacokinetics of drugs (determined in Phase 1 clinical trials) and enables exploration of various dosing regimens and their effect on bacterial growth, killing and resistance development in advance of patient studies. Using several KPC-producing strains of Klebsiella pneumonia with varying MICs to meropenem alone (32-512 mg/L) and in the presence of RPX7009 (≤0.06 – 8 mg/L) we found that the simulated human exposures of meropenem 2 g /RPX7009 2 g dosed as 3h infusions every 8 h was capable of highly efficient killing: the number of viable cells after the treatment was generally reduced by more than 10,000-fold (Tarazi et al., ICAAC 2014). Rare survivors, generally, 50-100 colonies of each strain, were collected to answer the question whether or not resistant mutants have been selected.
Analysis of the isolates demonstrated no selection of resistance. Moreover, it was discovered that 5-10 of survivors had become sensitive to carbapenems, similar to wild-type strains that do not produce any carbapenemase at all (MER MIC≤1 µg/ml). We used PCR to examine the isolates that lost carbapenem resistance for the presence of the KPC gene, the presence of transposon Tn4401, and the presence of plasmids that carried KPC in our strains. Since PCR primers failed to amplify either the KPC gene or Tn4401, we concluded that both of them were lost from cells that became sensitive to carbapenems. As for plasmids, PCR establishing that 1) most of the strains that were used in the study contained multiple plasmids, 2) that only one plasmid per strain carried the KPC gene, and 3) that the entire KPC-plasmid was lost in cells that became sensitive to meropenem after the exposure to carbavance.
Next, we tried to re-introduce the lost KPC plasmid back into these meropenem sensitive strains through bacterial mating (i.e., conjugation) using as plasmid donors, the original KPC-producing meropenem resistant strains that were not exposed to Carbavance. Multiple attempts to do this failed despite the fact that the same donor strains were perfectly capable to donate their plasmids to other mating partners. Thus these strains not only had lost the gene producing the β-lactamase, but had also the capability to resist introduction of new genes.
In conclusion, our preliminary results indicate that 5-10% of cells of KPC-producing strains of K. pneumonia that survived exposure to Carbavance in vitro became sensitive to carbapenems and other β-lactam antibiotics due to the elimination of the entire KPC plasmid. The loss of the KPC plasmids did not appear to be easily restored by re-introducing plasmids from other survivors still holding to their KPCs. The implications of these observations on clinical outcomes and epidemiology are not known but follow up analyses in patients with infection due to KPC-producing Enterobacteriaceae treated with Carbavance are planned.
This project has been funded in whole or in part with Federal funds from the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority, under Contract No. HHSO100201400002C.