Amikacin in combination with zinc pyrithione prevents growth of a carbapenem-resistant/multidrug-resistant Klebsiella pneumoniae isolate

Sir,

The morbidity and mortality of Klebsiella pneumoniae infections increased in recent years due to the acquisition of multidrug resistance and the emergence of hypervirulent variants. This bacterium is currently the third most prevalent bloodstream pathogen. In particular, acquisition of resistance to carbapenems (carbapenem-resistant K. pneumoniae, CRKP) complicates treatment and increases mortality [1]. Further compounding this grim picture, the COVID-19 pandemic may be accelerating evolution and dissemination of resistant pathogenic bacteria. A recent study on COVID-19 and non-COVID-19 hospitalized patients found that 34.1% and 28.2% tested positive for CRKP, respectively [2]. Strategies to control this problem must be rapidly developed to reduce the dangerous increase and dissemination of multidrug-resistant bacterial pathogens. K. pneumoniae VA360, a recent CRKP isolate from the Cleveland Clinic, OH [3], is resistant to aminoglycosides, including amikacin, and carbapenems. The minimal inhibitory concentration (MIC) values (expressed in μg/ml) of the carbapenems meropenem, cefepime, doripenem, imipenem, and ertapenem were 64, >256, >32, 12, and >32, respectively. The MIC values (expressed in μg/ml) of the aminoglycosides amikacin, netilmicin, kanamycin, tobramycin, and gentamicin were 128, >256, >256, 64, and 3, respectively. The aminoglycoside resistance profile is characteristic bacteria producing AAC(6’)-I enzymes [3]. K. pneumoniae VA360 belongs to the multilocus sequence type 258 (ST258), the group most frequently isolated from CRKP-caused infections. The resistance to β-lactams is due to bla_TEM-1, bla_SHV-11, bla_SHV-12, bla_KPC-2 genes, and resistance to amikacin is mediated by aac(6’)-Ib [3]. The rapid reduction of alternative antimicrobial options can be dealt with with the introduction of new antibiotics. However, the slow and expensive nature of this option requires other alternative strategies. One of them is the design of inhibitors of the enzymatic inactivation of antibiotics, which is the primary mechanism of resistance to β-lactams and aminoglycosides [4]. In the case of amikacin, the most prevalent resistance mechanism is acetylation catalyzed by the 6’-N-acetyltransferase type Ib [AAC(6’)-Ib]. Based on our recent findings that Zn²⁺ complexed to ionophores such as pyrithione can restore susceptibility to aminoglycosides mediated by AAC(6’)-Ib [4], we tested the action of this combination on the CRKP strain VA360.

Bacteria were cultured in Lennox Luria or Mueller-Hinton (MH) broth. The effect of the zinc pyrithione complex (ZnPT) on resistance to amikacin was evaluated culturing K. pneumoniae VA360 in 100-μl MH broth containing 0.5% dimethyl sulfoxide with the required additions. Assays were carried in microtiter plates using the BioTek Synergy 5 microplate reader. The cells were incubated for 20 h at 37°C with shaking and the OD₆₀₀ was measured every 20 min. Time-kill assays were performed adding 10⁶ CFU/ml to MH broth with the additions indicated, incubating at 37°C with shaking, and determining CFU/ml after 4, 8, 20, and 32 h. Checkerboard assays were carried out as described before [5] using variable concentrations of meropenem and amikacin in the presence of 5 μM ZnPT. MIC values were determined using commercial Etest strips (Liofilchem S.r.l., Italy) following the procedures recommended by the supplier.

Fig. 1A shows that in the presence of 5 μM ZnPT, a concentration of 32 μg/ml amikacin blocked the growth of K. pneumoniae VA360. Addition of any of these components without the other did not prevent bacterial growth (Fig. 1A). Combinations of 5 μM ZnPT with lower than 32 μg/ml amikacin fail to inhibit growth. The extended lag time before growing at normal rates observed in the presence of ZnPT has been described previously [4]. The bactericidal nature of the action of the combination amikacin/ZnPT was confirmed using time-kill assays. Amikacin at 32 μg/ml exerted bactericidal activity if ZnPT was also present in the culture medium (Fig. 1B). Checkerboard assays designed to assess whether the presence of a carbapenem (meropenem) could potentiate the combination amikacin/ZnPT showed no improvement, underscoring the robust resistance to carbapenem exhibited by this strain.

Fig. 1.

Effect of ZnPT on resistance to amikacin. A, Bacterial growth. Cells were cultured at 37°C with shaking in 100 μl Mueller-Hinton broth with the additions shown in the figure. Growth was assessed by periodic measuring of the OD₆₀₀. B, Time-kill assay. K. pneumoniae VA360 cells (10⁶ CFU/ml) were inoculated into 100 μl Mueller-Hinton broth and incubated at 37°C with shaking. CFU/ml were determined after 4, 8, 20, and 32 h incubation. All assays were carried out in duplicate.

In conclusion, the results described here indicate that the combination amikacin/ZnPT overrides the resistance to amikacin in a multidrug-resistant CRKP. Furthermore, the significant reduction in amikacin inhibitory concentration produced by the combination of the aminoglycoside with ZnPT is very encouraging and could be an alternative for hard-to-treat multidrug-resistant K. pneumoniae.