Research Article: Restoration of susceptibility to amikacin by 8-hydroxyquinoline analogs complexed to zinc

Date Published: May 29, 2019

Publisher: Public Library of Science

Author(s): Jesus Magallon, Kevin Chiem, Tung Tran, Maria S. Ramirez, Veronica Jimenez, Marcelo E. Tolmasky, Amal Al-Bakri.


Gram-negative pathogens resistant to amikacin and other aminoglycosides of clinical relevance usually harbor the 6’-N-acetyltransferase type Ib [AAC(6′)-Ib], an enzyme that catalyzes inactivation of the antibiotic by acetylation using acetyl-CoA as donor substrate. Inhibition of the acetylating reaction could be a way to induce phenotypic conversion to susceptibility in these bacteria. We have previously observed that Zn2+ acts as an inhibitor of the enzymatic acetylation of aminoglycosides by AAC(6′)-Ib, and in complex with ionophores it effectively reduced the levels of resistance in cellulo. We compared the activity of 8-hydroxyquinoline, three halogenated derivatives, and 5-[N-Methyl-N-Propargylaminomethyl]-8-Hydroxyquinoline in complex with Zn2+ to inhibit growth of amikacin-resistant Acinetobacter baumannii in the presence of the antibiotic. Two of the compounds, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and 5,7-diiodo-8-hydroxyquinoline, showed robust inhibition of growth of the two A. baumannii clinical isolates that produce AAC(6′)-Ib. However, none of the combinations had any activity on another amikacin-resistant A. baumannii strain that possesses a different, still unknown mechanism of resistance. Time-kill assays showed that the combination of clioquinol or 5,7-diiodo-8-hydroxyquinoline with Zn2+ and amikacin was bactericidal. Addition of 8-hydroxyquinoline, clioquinol, or 5,7-diiodo-8-hydroxyquinoline, alone or in combination with Zn2+, and amikacin to HEK293 cells did not result in significant toxicity. These results indicate that ionophores in complex with Zn2+ could be developed into potent adjuvants to be used in combination with aminoglycosides to treat Gram-negative pathogens in which resistance is mediated by AAC(6′)-Ib and most probably other related aminoglycoside modifying enzymes.

Partial Text

Among many mechanisms bacteria have evolved to resist antibiotics, enzymatic modification is one of the most efficient [1]. In the case of aminoglycosides, bactericidal antibiotics used to treat a wide range of bacterial infections, the most relevant mechanisms of resistance in the clinics are enzymatic inactivation by acetylation, nucleotidylation, or phosphorylation [1–3]. Although more than hundred aminoglycoside modifying enzymes have been identified in bacterial pathogens, the acetyltransferase AAC(6′)-Ib, which mediates resistance to amikacin and other aminoglycosides, is the most widespread among Gram-negative clinical isolates [4–6]. The progressive acquisition of this gene is eroding the usefulness of amikacin as well as other aminoglycosides. One way to overcome this problem is the design of new antimicrobials such as the recent introduction of plazomicin [7]. However, since this is a slow and expensive process and resistance will inevitably develop against the new antibiotics, these efforts must be complemented by other strategies to prolong the useful life of existing drugs [1, 2, 8–11]. In the case of aminoglycosides, in addition to the design of new molecules [7, 12–14], there is active research to find inhibitors of expression of aminoglycoside modifying enzymes [15–18] and to design enzymatic inhibitors [1, 2, 9, 10, 19–22]. A recent breakthrough in the search for inhibitors of enzymatic inactivation of aminoglycoside was the finding that Zn2+ and other metal ions inhibit the acetylation of aminoglycosides mediated by AAC(6′)-Ib in vitro [23]. Although concentrations beyond toxic levels were needed to interfere with resistance in growing bacteria, further research showed that the action of the metal was enhanced when complexed to ionophores, in which case low concentrations were sufficient to overcome resistance in several aminoglycoside-resistant bacteria [23–26]. We recently showed that two classes of ionophores, clioquinol (5-chloro-7-iodo-8-hydroxyquinoline)(CI8HQ) and pyrithione (N-hydroxypyridine-2-thione), when complexed to Zn2+ or Cu2+, significantly reduce the levels of resistance to amikacin in Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii strains harboring the aac(6′)-Ib gene [24–26]. CI8HQ and other substituted 8-hydroxyquinolines are being tested as treatment for cancer, neurodegenerative conditions such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, and lead poisoning [27–30]. The ongoing studies and uses of these compounds indicate that human toxicity is not a serious impediment in their development as drugs for diverse diseases [29, 31]. These facts make CI8HQ and other substituted 8-hydroxyquinolines excellent candidates to be used in combination with aminoglycosides in the treatment of resistant infections. In this work, we compared the effect of commercially available substituted 8-hydroxyquinolines complexed to Zn2+ on growth of amikacin-resistant A. baumannii clinical isolates.

Combination therapies consisting of an antibiotic and an inhibitor of resistance can be an invaluable tool in the search for solutions to the multidrug resistance problem [10]. While this strategy has already been reduced to practice in the case of pathogens resistant to β-lactams [42], efforts to develop inhibitors of resistance to aminoglycosides are still in experimental stages. We have recently found that ionophores complexed to Zn2+ or Cu2+ could be potentiators that decrease the levels of resistance to amikacin in K. pneumoniae and A. baumannii clinical isolates [23–25]. Since one of the ionophores that in complex with Zn2+ demonstrated activity as an inhibitor of the resistance to amikacin was CI8HQ, a substituted 8-hydroxyquinoline (8HQ), we expanded our studies to other compounds with these characteristics. Fig 1 shows the compounds tested in this work. The tests were carried out using as models three A. baumannii clinical isolates, two of them, A155 and A144, harboring the aac(6′)-Ib gene [34, 43]. The third strain, Ab33405 does not carry this gene and exhibits resistance to amikacin by a different mechanism. Although this mechanism remains to be elucidated, it most probably consists of phosphorylation mediated by the aphA6 gene found in its genome [34, 44].

Numerous approaches are being pursued to combat the current crisis of antibiotic resistance [10, 12]. In addition to the efforts to find or design new classes of antibiotics, researchers are looking for new scaffolds or attempting to modify existing antimicrobial families or designing compounds that act as adjuvant of these antibiotics by interfering with resistance [12, 46–50]. We have previously found that Zn2+, when complexed to ionophores such as pyrithione or CI8HQ, significantly reduces the levels of resistance to amikacin mediated by the AAC(6′)-Ib enzyme [23–25]. Since this enzyme is the most prevalent in amikacin resistant infections in the clinics [5], this finding represented a significant advance in the search for compounds that in combination with the antibiotic can help extend its useful life. The obvious potential these compounds have to be part of formulations composed of amikacin and the inhibitor warrant further research to find the best ionophores. Since CI8HQ is a derivative of 8HQ, in this work we tested combinations of Zn2+ with 8HQ and other commercially available derivatives. While CI8HQ and II8HQ show similar capacity to reverse resistance to amikacin, 8HQ and MP8HQ did not show any of the desired inhibitory activity, and B8HQ exhibited antimicrobial activity in the absence of the antibiotic. The disparity of effects found among these chemically related compounds shows the importance of assessing the activity of ionophores with similar structures. Since one of the most crucial problems exhibited by numerous compounds that are otherwise good drug or adjuvant candidates is their toxicity, it was interesting that the ionophores tested in this work did not show cytotoxicity in our assays. Furthermore, as substituted 8HQ derivatives are being researched as treatments of other human conditions, their low toxicity has also been established by other laboratories [29, 31]. Taken together, the results described in this work indicate that Zn2+ or other cations complexed to ionophores are firm candidates to be developed as potentiators to aminoglycosides to overcome resistance. In particular, CI8HQ and II8HQ are excellent candidates as adjuvants to overcome AAC(6′)-Ib -mediated resistance to amikacin.