Killing Dynamics of Carbapenems against Pseudomonas aeruginosa Harboring Varied Determinants of Carbapenem Resistance

Background: Ideal dose of the antimicrobials should be decided by considering their killing dynamics since sufficient elimination of the causative microorganisms is critical for proper antimicrobial treatment. In this study, the bactericidal activities of carbapenems by resistance mechanisms were assessed for carbapenem-resistant Pseudomonas aeruginosa. Methods: Minimal inhibition concentrations (MICs) of carbapenems were determined by broth dilution method and the resistance mechanisms were identified by PCR and DNA sequencing. The expression levels of efflux pumps were determined by reverse transcriptase real-time PCR. Time-kill curves were plotted by time-course numeration of the viable cells grown under imipenem and meropenem at 1× and 4×MICs, respectively. Results: One P. aeruginosa strain was susceptible, whereas three were resistant to carbapenems by defective OprD, efflux pump overproduction, and/or IMP-6 production. The susceptible strain had imipenem and meropenem MICs of 2 and 1 mg/L, respectively. The MICs were elevated by eight-fold by defective OprD, 16and 32-fold by the pump overproduction, and fourand >64-fold by the combination of two determinants and the IMP-6 carbapenemase. While both the carbapenems showed time-dependent bactericidal activity to the susceptible isolate, either of the carbapenem-resistant determinants, such as decreased membrane permeability, carbapenemase production, or the defective OprD, presented concentration-dependent bacteriostatic activity. Conclusion: Different killing dynamics of the carbapenems were observed depending on the resistance determinants, and the results would guide a proper treatment strategy for the patients using these drugs.


INTRODUCTION
Pseudomonas aeruginosa is an opportunistic pathogen causative for healthcare-associated infections [1].
Carbapenems are a major treatment option for infections caused by drug-resistant P. aeruginosa. However, increasing proportion of the carbapenem resistance in the P. aeruginosa clinical strains is of concern [2]. The resistance mechanism of the drug includes i) membrane impermeability either by deficiency of the outermembrane porin OprD or by overexpression of the resistance-nodulation cell division (RND) efflux pumps such as MexAB-OprM and MexXY-OprM [3] and ii) acquisition of the carbapenemase gene, such as a class A Guiana extended-spectrum beta-lactamase (GES) of some subtypes and class B imipenemase (IMP) and Verona integron-encoded metallo-beta-lactamase (MBLs) (VIM) [4]. The level of resistance depends on the resistance determinants. The carbapenemase confers mid-to high-level resistance, while membrane impermeability leads only reduced susceptibility. The clinical strains often carry two or more resistance determinants and the interplay of multiple determinants has been demonstrated to confer higher level resistance to carbapenems [5].
Since elimination of the infection-causative microorganisms is critical to succeed the treatment, antimicrobial doses should be decided in consideration of the killing dynamics. Consequently, characterizing the killing effect of antimicrobials, including its bacteriostatic and bactericidal activity, is principal to optimize antimicrobial pharmacodynamics. While many efforts have been made to know the killing dynamics of drugs against drug-susceptible bacteria [6], that against the drug-resistant bacteria harboring single or multiple resistance determinants to carbapenems has been insufficiently established and the altered killing dynamics by common resistance determinant is needed. And in this study, we evaluated the killing dynamics of carbapenems against P. aeruginosa strains having varied common mechanisms of carbapenem resistance.

MATERIALS AND METHODS
Bacterial strains and Antimicrobial susceptibility testing were used for quality control [7].

DNA manipulation and polymerase chain reaction (PCR)
Total DNA was extracted using MG Genomic DNA Purification kit (MG MED Inc., Seoul, Korea). PCR and following Sanger sequencing was carried out for known acquired carbapenemases, such as bla IMP , bla VIM , bla KPC , bla NDM , and bla GES [8], and the oprD gene [9].

Determination of the efflux pump overexpression
Total RNA was extracted from exponentially growing bacterial cells by using RNeasy® plus mini (Qiagen, Hilden, Gemany). Levels of mRNA for the three RND efflux pumps, and the house-keeping rpoB gene was quantified by LightCycler® 480 instrument II (Roche Diagnostics, Rotkreuz, Switzerland) using the LightCycler® RNA amplification kit with SYBR green I (Roche Diagnostics) using gene-specific primer pairs 5ʹ-CAAGGGCGTCGGTGACTTCCA-3ʹ and 5ʹ-ACCTGGGAACCGTCGGGATTGA-3ʹ for mexB, 5ʹ-GGACCACGCCGAAACCGAACG-3ʹ and 5ʹ-CGCCGCAACTGACCCGCTACA-3ʹ for mexY, 5ʹ-GGACGGCTCGCTGGTCCGGCT-3ʹ and 5ʹ-CGACGAAGCGCGAGGTGTCGT-3ʹ for mexD, 5ʹ-CTTGGTACGACCGTTCACGT-3ʹ and 5ʹ-GCTGAAACTGAACCACCTGG-3ʹ for rpoB with the following cycles: predenaturtation at 95°C for 30 s, followed by 45 cycles at 95°C for 5 s, 58°C for 10 s, and 72°C for 20 s. The transcriptome levels of each pump were normalized to that of rpoB, and relative levels were calculated by dividing those of the P. aeruginosa A1701 DS . Each experiment was performed in duplicate twice independently.

Time-kill assay
Each strain was grown in MH broth till mid-exponential phase and were diluted to have 10 6 colony forming units (CFU)/mL. Drugs were then added at 1× (2 mg/L imipenem and 1 mg/L meropenem) and 4 × MICs (8 mg/L imipenem and 4 mg/L meropenem). CFUs were numerated by plating every two hours and plotted. The killing kinetics were determined twice independently, and verified the results being reproducible.
Bactericidal activity was defined as a ≥3.0 log CFU/mL decrease in bacterial count at any time point during the 24-hour exposure of the drug.

Carbapenem susceptibility and the relevant mechanism
The four P. aeruginosa strains studied included one carbapenem-susceptible and three carbapenem-resistant strains ( Table 1) Except the carbapenem-susceptible P. aeruginosa A1701 DS , the three P. aeruginosa strains had single or multiple mechanisms of carbapenem resistance (Table 1). Defective OprD was identified in P. aeruginosa B1707 ∆oprD and B1605 ∆oprD/ pump↑/IMP strains. Overexpression of the RND pump was observed in two P.  Finally, both the RND pump-overproducing strains, the F1709 pump↑ and the B1605 ∆oprD/ pump↑/IMP were not killed by meropenem at all, while concentration-dependent killing dynamics by imipenem were observed.

DISCUSSION
Adequate antimicrobial therapy is critical for the outcome of the patients with infectious diseases and the in vitro kinetics of bacterial killing is one of the key factors for guiding the proper antimicrobial choice as dead bugs don�t mutate [10]. Even though the definitions of �bacteriostatic� and �bactericidal� are clear, no antimicrobial agents could be definitively categorized into the two, since the killing kinetics of the drug is dependent on the targeting bacteria, inoculum size, and so on [11].
The carbapenems are one of the last-line drugs to treat P. aeruginosa infections. Since the drugs are used empirically for the critically ill patients, resistance to the drug is critical for the patient�s outcome [12]. The three resistance determinants, i.e. OprD deficiency, overproduction of the RND efflux pump, and MBL production, are common mechanisms of resistance to carbapenems globally in P. aeruginosa [13]. In addition, the IMP-6-producing P. aeruginosa used in the study is disseminated in South Korea since the end of the 2000s [9,14]. Thus, bacterial killing effects of carbapenems against the P. aeruginosa carrying the resistance determinants was needed to be evaluated.  Carbapenems showed a time-dependent bactericidal activity to the susceptible isolate as the most betalactams do. Certain regrowth of the bacteria by 24 hours under the 1×MIC imipenem was likely due to the collaborative effect of the poor chemical stability of the drug and the intrinsic cephalosporinase leaky in P. aeruginosa, which has a very weak carbapenemase-hydrolyzing activity. While deficiency of the OprD elevated the MICs of both carbapenems equally by eight folds, clear concentration-dependent killing kinetics of the meropenem was observed against the OprD-defective strain by eliminating 3.0 log CFU/mL in eight hours at the 4×MIC. Conceivably, the difference was led by imipenem preference of the porin [15].
Meropenem MICs were much more elevated by overproduction of the efflux pump, especially that of the MexAB-OprM having preference to meropenem [3,16]. The IMP-6 carbapenemase production resulted in additional elevation of the meropenem MIC on top of the RND efflux pump overexpression. Imipenem susceptibility was not affected by the IMP-6 making a good correlation with the previous report of the enzyme hydrolyzing efficiently the meropenem rather than the imipenem [17]. Further attention is needed to the carbapenemase producers, since they often harbor class 1 integron including multiple gene cassettes of drug resistance [18] resulting in multidrug-resistance elevating mortality of the victimized patients [19].
Notably, the mechanisms of carbapenem resistance threat the patients not only by limiting the drug choices for treatment, but also by putting the patients at risk through its alternative role as a virulence factor, and the point highlights the importance to identify the actual mechanism of carbapenem resistance. Recently, the OprD deficiency in P. aeruginosa was recognized as a risk factor for the patients with P. aeruginosa bloodstream infections by augmenting the 6-week mortality from 17.2% to 33.3% [20]. The RND efflux pump is also known to be associated with the bacterial virulence mostly by involving in the biofilm formation and quorum sensing [21,22].
This study has an obvious imperfection of limited number of strains. However, the killing kinetics of carbapenems would help to better optimize the antimicrobial pharmacodynamics against P. aeruginosa clinical strains harboring common mechanisms of carbapenem resistance. To conclude, both the timedependent killing kinetics and bactericidal activity of carbapenems were affected by any of the common resistance determinants. The defective OprD diminished the killing activity of imipenem rather than that of meropenem, while the RND pump overproduction and the meropenemase MBL IMP-6 weaken the activity of meropenem than that of imipenem.

CONFLICTS OF INTEREST
No potential conflicts of interest relevant to this article were reported.