Characterization of Proteobacterial Plasmid Integron-Encoded qac Efflux Pump Sequence Diversity and Quaternary Ammonium Compound Antiseptic Selection in Escherichia coli Grown Planktonically and as Biofilms

ABSTRACT

Qac efflux pumps from proteobacterial multidrug-resistant plasmids are integron encoded and confer resistance to quaternary ammonium compound (QAC) antiseptics; however, many are uncharacterized and misannotated. A survey of >2,000 plasmid-carried qac genes identified 37 unique qac sequences that correspond to one of five representative motifs: QacE, QacEΔ1, QacF/L, QacH/I, and QacG. Antimicrobial susceptibility testing of each cloned qac member in Escherichia coli highlighted distinctive antiseptic susceptibility patterns that were most prominent when cells grew as biofilms.

INTRODUCTION

Small multidrug resistance (SMR) family efflux pump genes, annotated as “qac,” are transmitted by proteobacterial integrons carried by multidrug-resistant plasmids (1, 2), making them distinct from other chromosomally inherited SMR members such as Gdx (SugE) and the archetypical member EmrE (3). SMR efflux pump proteins are small (100 to 120 amino acid residues), integral plasma membrane-spanning proteins that act as drug-H⁺ antiporters. These proteins are composed of only 4 transmembrane α-helices, which multimerize into functional homodimers (1, 4). Qac efflux pumps are named for their ability to confer resistance to quaternary ammonium compound (QAC) antiseptics and have become important genetic biomarkers to predict class 1 integron presence, bacterial antiseptic tolerance, and QAC environmental pollution (5, 6). In proteobacteria, there are numerous integron-associated qac members, annotated as qacE, qacEΔ1, qacF, qacG, qacH, qacI, and qacL in various sequence databases (2, 7, 8); however, only qacE, qacEΔ1, and qacF have been cloned and characterized in Escherichia coli to determine their antimicrobial susceptibility (9–13). qacEΔ1 is the most frequently detected member, because it forms part of the 3′ conserved class 1 integron region. QacEΔ1 is identical to QacE (WP_000679427.1) until its 95th residue, where it has an in-frame insertion element that extends its 4th transmembrane helix by 16 amino acids at the C terminus (Fig. 1), resulting in QacEΔ1 inactivation and reduced ethidium bromide (ET) efflux activity compared to QacE (9). QacE and QacF are predicted to expel a wide range of QACs based on antimicrobial susceptibility testing (AST) (10–13), but the antimicrobial selectivity conferred by other annotated qac sequences (qacG/H/I/L) is inferred from their homology to SMR members. This has resulted in many misannotated qac members and a lack of naming consensus, as well as little comparison of AST methods that involve planktonic, colony, or biofilm growth conditions, which are the main aims of this study.

FIG 1.

A multiple-protein sequence alignment of the 37 unique Qac sequences identified from this study and a summary of their amino acid sequence motifs. Amino acids are designated by single-letter abbreviations and color coded based on the Taylor scheme in Jalview software v.2.10.5 (18). The consensus motif below the main alignment shows each Qac motif. Colored amino acids in each motif indicate unique positions differentiating QacF/L, QacH/I, QacG, and QacEΔ1. Boldface residues in the consensus and Qac motifs indicate conserved motif positions in the small multidrug protein subclass of the SMR family. The cylinder diagram at the bottom of the alignment shows the start and ends of the four transmembrane α-helices (TMH) separated by loops (L1 and L2) and turns (T1) of the known SMR protein secondary structure.

To characterize proteobacterial plasmid qac sequence diversity and homology, we collected 2,953 qac sequences encoded by proteobacterial plasmids deposited in the GenBank, INTEGRALL (14), UniProt (https://www.uniprot.org), and Comprehensive Antimicrobial Resistance Database (CARD) (15) databases. Plasmid sequences were retrieved using QacE (WP_000679427.1) as a query sequence by tBLASTn (16) analysis. After performing a multiple-sequence alignment of translated Qac sequences with the online server Clustal Omega (17) in Jalview (18), we identified a total of 37 unique Qac protein sequences for final analysis, all with highly variable and inconsistent annotations (Fig. 1; see Tables S1 and S2 in the supplemental material). To accurately classify each Qac sequence, we performed a maximum likelihood phylogenetic analysis using PhyML v.3.0 (19), with archetypical SMR members EmrE (NP_415057.1) and Gdx/SugE (NP_418572.4) as SMR family comparators (Fig. 2). This analysis reconfirmed that all Qac members were closely related to the SMR family member EmrE, in agreement with previous findings (3, 20). It also revealed that Qac sequences grouped into one of three distinct clades: Qac annotated as (i) QacF/L/H/I, (ii) QacG or QacE, and (iii) QacE and QacEΔ1 (Fig. 2). The alignment of the 37 unique Qac sequences revealed 5 sequence motifs in all 4 transmembrane helices distinguishing QacE from QacEΔ1 and from QacG. The alignment also identified that QacF/L annotated sequences as well as QacH/I were in fact identical to each other (98 to 100% identity) (Fig. 1). Amino acid variations in each Qac motif occurred most often (61 to 82% frequency) at unconserved residue positions in the previously published SMR motif (3, 4) (Fig. 1). Our attempts to identify a Qac sequence progenitor from bacterial genomes using tBLASTn were unsuccessful, as many qac genes are also transmitted on chromosomally integrated integrons as well as phages/prophages. Based on the high pairwise sequence identities between each Qac to either QacE or EmrE (Fig. 1), we propose that qac sequences have likely originated from a single qac progenitor incorporated into an integron that is rapidly diverging over time into these qac variants.

FIG 2.

The phylogenetic relatedness and taxonomic distribution of the 37 unique Qac sequences identified in this study. Shown is the maximum likelihood dendrogram of the 37 unique Qac protein sequences translated from qac sequences identified in the survey of 2,953 proteobacterial plasmids. Branch node confidence values were determined by performing 1,000 bootstrap replicates using an approximate likelihood ratio testing method. Sequences of archetypical E. coli SMR members EmrE (NP_415075) and Gdx/SugE (NP_418572.4) are highlighted in bold. Other sequences shown in bold indicate qac genes that were selected for AST based on their detection frequency in various plasmids. The taxonomic origins (according to proteobacterial order) of each Qac sequence identified from various plasmids is shown as a bar chart on the right-hand panel of the dendrogram. Numbered bars in this panel indicate the total number of qac sequences identified from plasmids isolated in Enterobacterales (purple), Pseudomonadales (dark blue), Alteromondales (light blue), Vibrionales (green), and Burkholderiales (red) species, where gray bars indicate indeterminate species.

This sequence analysis reconfirmed that qacEΔ1 is the predominant qac representative (2,736 qacEΔ1 genes/2,953 total qac genes [92.6%]) (Fig. 2; see Fig. S1 and Table S1 in the supplemental material), given that qacEΔ1 is part of the 3′ conserved region of most class 1 integrons (9). The remaining qac genes were less frequently detected from plasmids (8% of 2,368 plasmids surveyed), where qacG was the second most predominant member (90/2,953 [3.0%]), followed by qacH/I (82/2,953 [2.8%]), qacF/L (29/2,953 [1.0%]), and qacE (16/2,953 [0.5%]) (Fig. 2; Fig. S1 and Table S1). The majority of all qac sequences we identified were from class 1 integrons (93.0 to 100% of all plasmids), with a few qac genes detected at very low frequency (<4%) from class 2 or 3 integrons (Fig. S1). This indicates that qac genes predominate in class 1 integrons, but caution should be taken when using these genes as genetic markers for class 1 integrons.

To determine the substrate selectivity of the five representative Qac sequence motifs, we gene synthesized and cloned qacE (NP_044260.1), qacEΔ1 (YP_003264406.1), qacF/L (YP_006961976.1), qacH/I (L0FU64), and qacG (YP_006965429.1) in the isopropyl-β-d-1-thiogalactopyranoside (IPTG)-inducible expression vector pMS119EH (21) (Tables 1 and 2), using the same cloning, plasmid expression, and AST methods described in a recent study of gdx/sugE (22). We chemically transformed each plasmid into Escherichia coli K-12 BW25113 (wild type) (23), as well as strain KAM32 (24), which lacks a competing dominant efflux pump gene, acrB, and an additional efflux pump gene, mdtK, improving qac substrate selectivity determination by AST. To determine differences in antiseptic resistance that may be attributed to different cell growth physiologies, as noted in our previous study (21), we performed three different AST culturing techniques in Luria-Bertani (LB) medium with 100 μg/ml ampicillin selection and 0.05 mM IPTG addition to determine the MICs for each cloned vector transformant. We measured planktonic growth using 96-well broth microdilution plating techniques and cell colony growth on agar spot plating as described by Slipski et al. (22). We also determined the minimal biofilm eradication concentration (MBEC) for transformants grown as biofilms using the MBEC device (Innovotech, Inc., Canada) as described in reference 22. All AST involved a library of 13 antimicrobials commonly tested in previous SMR studies (as reviewed in reference 4) (Tables 1 and 2). For AST, we applied a significance threshold of 4-fold or greater when determining differences in MIC and MBEC values to account for potential 2-fold-value errors.

TABLE 1.

Summary of antimicrobial MIC and MBEC values determined for E. coli BW25113 transformed with various qac gene vectors and grown as planktonic (broth), colony (agar), and biofilm (MBEC device) cultures for 24 h at 37°C

Condition and vectora	MIC or MBEC (μg/ml)b
	CPC	CET	BZK	DDAB	CDEB	CTAB	DOM	ET	AC	MV	CHX	ERY	TOB
MICc
Agar (n = 3)
pMS119EH	8	64	16	4	4	64	32	256	80	320	8	64	4
pEmrE	8	64	32	8	8	64	32	>1,024	320	640	8	32	4
pQacE	8	64	32	8	8	128	32	512	160	320	8	64	8
pQacEΔ1	8	64	32	4	4	64	32	256	160	320	8	64	4
pQacF	8	64	32	8	8	64	32	512	160	320	8	64	4
pQacG	8	64	32	8	8	64	32	256	80	320	8	64	8
pQacH	8	64	32	8	8	128	32	512	320	320	8	64	8
Broth (n = 3)
pMS119EH	8–16	8–16	8–16	1.5	8	2	8	NA	NA	512	1	38	4
pEmrE	8–16	8–16	8–16	2	8	4	8	NA	NA	>1,024	2	38	4
pQacE	8–16	8–16	8–16	2	8	4	8	NA	NA	256	2	38	4–8
pQacEΔ1	8–16	8–16	8–16	1.5	8	2	8	NA	NA	512	2	38	4
pQacF	8–16	16–32	8–16	2	8	4	8	NA	NA	256	1	38	4–8
pQacG	8–16	8–16	8–16	2	8	4	8	NA	NA	512	1	38	4–8
pQacH	8–16	8–16	8–16	2	8	2	8	NA	NA	512	1	38	4
MBEC (n = 6)d
pMS119EH	64	32	64	8-16	16	128	32	3,750	64	4,096	128	1,024	4
pEmrE	256	64	256	32	64–128	32	32–64	7,500	512	16,384	256	2,048	8
pQacE	32	64	256	8–16	32	32	32	7,500	256	16,384	512	2,048	32
pQacEΔ1	32	256	64	8–16	21	128	32	3,750	64	16,384	256–512	1,024	8
pQacF	128	128	64	16–32	26	128	32	7,500	149	16,384	128	2,048	16–32
pQacG	256	256	128	32	64	128	32–64	3,750	64	8,129	128	2,048	8
pQacH	256	128	256	32	128	128	32–64	7,500	512	16,384	128	2,048	32

^aAll genes were directionally cloned in the multiple-cloning site of pMS119EH at 5′ XbaI and 3′ HindIII restriction sites.

^bAntimicrobial abbreviations: CPC, cetylpyridinium chloride; CET, cetrimide bromide; BZK, benzalkonium chloride; DDAB, didecyldimethylammonium bromide; CDEB, cetyldimethylethylammonium bromide; CTAB, cetyltrimethylammonium bromide; DOM, domiphen bromide; ET, ethidium bromide; MV, methyl viologen dichloride; CHX, chlorhexidine dichloride; AC, acriflavine; ERY, erythromycin; TOB, tobramycin. Boldface numbers indicate ≤4- or ≥4-fold changes in MIC/MBEC AST values compared to pMS119EH transformants under the same AST growth conditions. NA, data not available because the drug concentration absorbance values exceeded detection thresholds.

^cAST involved 10⁻⁴ dilutions of plasmid-transformed cultures adjusted to an optical density at 600 nm of 1.0 as the starting inoculum in Luria-Bertani medium with 100 μg/ml ampicillin and 0.05 mM isopropyl β-d-1-thiogalactopyranoside (IPTG).

^dShown are results from 24-h biofilms on the MBEC device pegged lid followed by drug incubation for 24 h in LB broth prior to AST.

TABLE 2.

Summary of AST MIC and MBEC values determined for E. coli KAM32 transformed with various qac vectors and grown as planktonic (broth), colony (agar spot), and biofilm (MBEC) cultures for 24 h at 37°C

Condition and vectord	MIC or MBEC (μg/ml)a
	CPC	CET	BZK	DDAB	CDEB	CTAB	DOM	ET	AC	MV	CHX	ERY	TOB
MICb
Agar (n = 3)
pMS119EH	2	1	1	0.5	2	4	2	12	1	80	4	2	4
pEmrE	8	4	2	2	8	8	4	256	8	160	4	2	2
pQacE	8	8	4	1	8	8	4	32	4	80	4	2	2
pQacEΔ1	2	4	1	0.5	4	4	2	8	4	80	4	2	4
pQacF	8	8	4	2	8	8	4	8	4	80	4	2	8
pQacG	8	8	4	2	8	4	2	8	1	80	4	2	8
pQacH	8	4	4	2	8	8	8	32	4	80	4	2	4
Broth (n = 3)
pMS119EH	1.2	2	2	0.5	2	1	1	16	8	80	1	1–2	2
pEmrE	2.4	4	2	1	2	1	1–2	64	64	320	1	1	2–4
pQacE	1.2–2.4	4	2	1	2	1	1–2	64	16–32	80	1	1–2	2
pQacEΔ1	1.2–2.4	2	2	0.5	2	1	1	32	8	80	1	1	2
pQacF	1.2–2.4	8	4	2	2	2	1–2	16	8	80	1	2	2–4
pQacG	2.4	4	4	2	4	2	1–2	16	8	160	1	2	2–4
pQacH	1.2–2.4	4	4	2	4	1	1–2	32	16–32	160	1	1–2	2–4
MBEC (n = 6)c
pMS119EH	128	16	32	16	16	8	8	64	32	4,096	32	64	2–4
pEmrE	32	32	16	16	64	8	26	512	256	1,6384	64	128	8–16
pQacE	32	32	16	8	32–64	8	16	256	128–256	1,6384	64	128	8
pQacEΔ1	64	64	32	6	16	4	8	64	64	1,6384	64	64–128	8
pQacF	64	64	32	16	32–64	4	32	256	64	4,096	32	64	16
pQacG	256	128	32	16	32–64	16	8	32–64	16–32	1,6384	64	64	8
pQacH	32	16	16	16	32–64	16	3	341	256	4,096	64	128	16–32

^aAntimicrobial abbreviations: CPC, cetylpyridinium chloride; CET, cetrimide bromide; BZK, benzalkonium chloride; DDAB, didecyldimethylammonium bromide; CDEB, cetyldimethylethylammonium bromide; CTAB, cetyltrimethylammonium bromide; DOM, domiphen bromide; ET, ethidium bromide; MV, methyl viologen dichloride; CHX, chlorhexidine dichloride; AC, acriflavine; ERY, erythromycin; TOB, tobramycin. Boldface numbers indicate ≤4- or ≥4-fold changes in MIC/MBEC AST values compared to pMS119EH transformants under the same AST growth conditions.

^bAST involved 10⁻⁴ dilutions of plasmid-transformed cultures adjusted to an optical density at 600 nm of 1.0 as the starting inoculum in Luria-Bertani medium with 100 μg/ml ampicillin and 0.05 mM isopropyl β-d-1-thiogalactopyranoside (IPTG).

^cShown are results from 24-h biofilms grown on the MBEC device pegged lid incubated with drug for 24 h in LB broth prior to AST.

^dAll genes were directionally cloned in the multiple-cloning site of pMS119EH at 5' XbaI and 3' HindIII restriction sites.

Nearly all BW25113/pQac transformants we examined using broth or agar AST methods had MIC values that were statistically insignificant (within a 2-fold MIC difference or identical) to the vector control pMS119EH, with the exception of the pEmrE transformant (Table 1). BW25113/pEmrE transformant agar colony AST results showed higher QAC resistance (MIC values of ≥4-fold) to ethidium bromide (ET) and acriflavine (AC) than to the control vector pMS119EH (Table 1). This is in agreement with previous agar spot plate AST findings for emrE transformants exposed to these intercalating dyes (25). In broth, only BW25113/pEmrE conferred significant resistance (>4-fold MIC) to methyl viologen (MV) compared to all other transformants and controls (Table 1); MV is one of the original substrates initially identified for EmrE (26). All BW25113 qac gene transformants grown as biofilms demonstrated a significant increase in resistance (≥4-fold change) to at least one QAC, intercalating dye, and/or antibiotic for each SMR transformant compared to the parental vector control (Table 1). The biofilm AST results shown in Tables 1 and 2 were repeated in duplicate based on 6 transformed biological replicates (n = 6). The biofilm AST profile of recognized antimicrobial compounds was unique for each pQac transformant we tested, reflecting their sequence motif differences (Fig. 1). BW25113/pQacH transformant biofilms conferred resistance to the broadest range of antimicrobials (6 QACs plus tobramycin [TOB]), with pQacE, -F/L, or -G transformants resistant to 3 to 4 antimicrobials, and pQacEΔ1 expectedly conferring resistance to the fewest substrates (cetrimide bromide [CET] and MV in Table 1). Therefore, in the wild-type efflux pump BW25113 strain, pQacE, pQacF, pQacH, and pQacG transformants grown as biofilms confer unique antimicrobial resistance profiles to a limited range of QACs compared to pEmrE (Table 1).

To improve substrate selection identification conferred by each representative qac, we repeated AST with KAM32 ΔacrB ΔmdtK/pQac transformants (Table 2). As previously reported (22, 27), KAM32 has slower growth and higher drug susceptibility than BW25113, resulting in lower MIC and MBEC values for all antimicrobials we tested compared to BW25113/pMS119EH (Table 2). Broth and agar spot plate AST results for KAM32 transformed with pEmrE or pQac vectors (including pQacEΔ1) demonstrated a significant increase (≥4-fold) in MIC values for one or more QACs compared to pMS119EH (Table 2) or compared to the same AST results from BW25113 transformants (Table 1). These findings show that AST in strains lacking competing efflux pumps helped identify a broader range of QACs selected for by each qac gene when grown planktonically or as colonies. The KAM32 agar AST findings are in agreement with previous qac studies, as we identified increased resistance to similar QAC substrates (ET, cetyltrimethylammonium bromide [CTAB], and benzalkonium chloride [BZK]) (Tables 1 and 2), as reported for previous agar plate AST studies of qacE and qacEΔ1 (9, 12, 13), as well as QacF (10, 11). However, KAM32/pQac transformant biofilm AST unexpectedly identified fewer antimicrobials that significantly increased MBEC values compared to the control vector (Table 2) or compared to BW25113 biofilm results (Table 1). KAM32 transformant biofilm MBEC results identified enhanced antimicrobial susceptibility (≤4-fold reduction in MBEC values) for pQacEΔ1 and pQacF exposed to QACs, CET, BZK, didecyldimethylammonium bromide (DDAB), and CTAB (Table 2). Enhanced susceptibility was also observed for biofilm BW25113/pEmrE and -pQacE transformants for CTAB (Table 1). This suggests that overexpression of these qac efflux pumps works against the cell under these biofilm growth conditions, making cells more susceptible to the aforementioned QACs. The ability of SMR members to confer enhanced antimicrobial susceptibility in the presence of different antimicrobials has been reported in previous studies (28, 29) and may be due to amino acid variations that switch these pumps from exporters to importers for these particular drugs.

In conclusion, our findings reveal that many proteobacterial plasmid-carried qac genes are misannotated in sequencing databases, and the comprehensive Qac motif comparison herein can improve annotation of qac variants. We observed that qacH/I variants had the broadest antimicrobial recognition profile when grown as biofilms, whereas qacEΔ1 transformants conferred significant QAC resistance to the smallest number of QACs (CET and MV), indicating that even this relatively inactive qac variant can still confer limited QAC resistance. Our analysis also importantly shows that qac efflux pumps are most effective when E. coli cells grow as a biofilm and least effective when cells grow as planktonic cultures, which is concerning when considering that biofilm prevention and eradication strategies frequently rely on the use of QAC disinfectants (30). Altogether, this information provides more context to ongoing antimicrobial resistance genetic surveillance studies by providing qac-specific antimicrobial phenotypes to uncharacterized qac genes, clear and improved annotations, and identification of optimal growth physiologies influencing their conferred phenotypes.