DOI: 10.1002/cmdc.201600095
Communications
Structure–Resistance Relationships: Interrogating
Antiseptic Resistance in Bacteria with Multicationic
Quaternary Ammonium Dyes
Megan E. Forman,[a] Madison H. Fletcher,[b] Megan C. Jennings,[b] Stephanie M. Duggan,[a]
Kevin P. C. Minbiole,*[a] and William M. Wuest*[b]
Bacterial resistance toward commonly used biocides is a wide-
spread yet underappreciated problem, one which needs not
only a deeper understanding of the mechanisms by which re-
sistance proliferates, but also means for mitigation. To advance
our understanding of this issue, we recognized a polyaromatic
structural core analogous to activators of QacR, a negative
transcriptional regulator of the efflux pump QacA, and envi-
sioned a series of quaternary ammonium compounds (QACs)
based on this motif. Using commercially available dye scaf-
folds, we synthesized and evaluated the antimicrobial activity
of 52 novel QACs bearing 1–3 quaternary ammonium centers.
Striking differences in antimicrobial activity against bacteria
bearing QAC resistance genes have been observed, with up to
a 125-fold increase in minimum inhibitory concentration (MIC)
for select structures against bacteria known to bear efflux
pumps. Based on these findings, general trends in structure–
resistance relationships have been identified, laying the
groundwork for future mechanistic studies.
Gram-negative bacteria[4,5] and has presumably arisen through
overuse and prolonged sub-lethal exposure. These compounds
can, in fact, activate numerous resistance mechanisms, includ-
ing physiological changes to bacterial cell membranes, as well
as the production of transporter proteins, which efflux antibac-
terial agents.[6] More specifically, the qacAB/R system is one of
the primary methods by which Gram-positive bacteria, specifi-
cally S. aureus, minimizes exposure to QACs. An overview of
the resistance machinery is depicted in Figure 1A. Although
QACs are lytic to cell membranes, they are capable of entering
the cell at sub-MIC values by passive diffusion. The compounds
can then either be exported by the basal level of QacA (a
transmembrane efflux pump) activity that is present, or bind
with QacR, a negative transcriptional regulator of qacA. Follow-
ing the binding of QACs to the recognition site, QacR disasso-
ciates, allowing the transcription of the qacA gene. This leads
to the increased production of QacA and the rapid efflux of an-
timicrobial compounds from the cell. Other efflux proteins in
Gram-positive bacteria include NorA;[1,7] an analogous system
has also been observed in Gram-negative bacteria through the
efflux pumps AcrAB-TolC in E. coli (EC) and MexAB-OprM in
P. aeruginosa (PA).[4,5,8,9]
The proliferation of antibiotic-resistant bacteria is a significant
threat to human health, and accordingly, the scientific and
medical communities are making a concerted effort to stem
the tide of resistance. Less alarm has been sounded, however,
about a directly related problem: the decrease in efficacy of
antiseptics often used by the general public to decontaminate
surfaces and by hospitals to sterilize equipment. Over the past
thirty years the identification of bacterial isolates with quater-
nary ammonium compound (QAC) resistance genes has risen
dramatically,[1] and as a result, there have been efforts to better
understand the mechanisms by which antiseptics can lose effi-
cacy.[2,3] Resistance to traditional disinfectants such as benzal-
konium chloride (BAC) and didecyldimethylammonium chlo-
ride (DDAC) has been identified in both Gram-positive and
It has been posited that efflux pumps are, in fact, multidrug
transporters with alternate primary functions, having evolved
to recognize and export a wide range of antibacterial and bio-
cidal scaffolds.[2,6] The evolutionary origins of some of these re-
sistance mechanisms have been attributed to the recognition
of natural product QACs such as berberine, sanguinarine, and
chelerythrine produced by plants.[2,3] This is evidenced by the
crystal structure of berberine bound to QacR, which highlights
the key electrostatic (acidic amino acid residues) and p–p (aro-
matic residues) interactions as shown in Figure 1B.[2] Brennan
et al. demonstrated that commercially available dyes—crystal
violet and malachite green—fit neatly into the binding site for
berberine; they noted, however, that this recognition motif
was limited to mono- and biscationic QACs.[2]
Previous research from our group[3] and others confirm this
common QacR recognition motif, with ample evidence that
mono- and biscationic QACs and those with aryl moieties dis-
play significant increases in MIC values (up to 60-fold) for
qacA/B-bearing bacteria. In contrast, some of the most potent
antiseptics developed in our lab are multicationic; these often
exhibit low-micromolar concentrations against both Gram-pos-
itive and Gram-negative bacteria.[10–15] We thus sought to
expand on these earlier findings, exploring series of com-
pounds with varied cationic character as well as aromatic
[a] M. E. Forman, S. M. Duggan, Prof. K. P. C. Minbiole
Department of Chemistry, Villanova University,
800 East Lancaster Avenue, Villanova, PA 19085 (USA)
[b] M. H. Fletcher, M. C. Jennings, Prof. W. M. Wuest
Department of Chemistry, Temple University,
1901 North 13thStreet, Philadelphia, PA 19122 (USA)
E-mail: wwuest@temple.edu
Supporting information for this article can be found under http://
synthesis, characterization and spectral data, biological protocols, and
complete biological data.
ChemMedChem 2016, 11, 958 – 962
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