Upregulation of AcrEF in Quinolone Resistance Development in Escherichia coli When AcrAB-TolC Function Is Impaired

Abstract

We studied mechanisms of drug resistance development in Escherichia coli strains lacking efflux pump components. E. coli K12 deletion mutants were subjected to increasing concentrations of ciprofloxacin (CIP) to determine the frequency of target gene mutations. We generated a series of mutants that were selected based on their minimum inhibitory concentrations (MICs) to CIP, as well as their corresponding point mutations in target genes. The mutants displayed a number of target modifications and, in particular, gyrA mutations altering codons Ser83Leu, Asp87Gly, and Asp87His as well as a change in parC at 78 (substitution of Gly for Asp). All these mutations were related to drug resistance. When exposed to CIP, mutants lacking efflux pump genes acrA and acrB demonstrated a low level of resistance that was because of point mutations in the target genes. High-level resistance was achieved with a 100- to 500-fold increase in expression of efflux pump genes acrE and acrF that compensated for the loss of AcrA and AcrB, and thus resulted in an obvious increase of CIP MIC. We demonstrate that an intact AcrAB-TolC efflux pump is crucial to the development of bacterial resistance. Its activity is complemented by expression of the alternative AcrEF efflux pump.

Introduction

Fluoroquinolones¹ and cephalosporins² are the most widely used antimicrobial classes for treatment of bacterial infections. Their extensive use in animal production and aquaculture has led to numerous resistance developments that present an increasing challenge to their effective treatment. Two primary resistance mechanisms against fluoroquinolones are point mutations in target enzyme genes and activated efflux pumps. The point mutations in key positions of DNA gyrases (i.e., gyrA and gyrB) and topoisomerase IV (i.e., parC and parE) lead to a decreased affinity between drug and target enzymes, resulting in a decrease in the inhibition of bacterial DNA replication.³ High level of resistance can occur because of point mutations in gyrA at the serine-83, asparagine-87 codons, and an additional mutation in serine-80 or glutamate-84.⁴ Mutations of gyrB or parE are rare and are often associated with low-level resistance.⁵

In Escherichia coli, activated efflux pumps are efficient drug exporters, and fluoroquinolones such as ciprofloxacin (CIP) are substrates of many efflux pumps, such as resistance-nodulation-division (RND) efflux pump family members AcrAB-TolC and AcrEF-TolC and the multidrug and toxic compound extrusion family members NorE.^6,7 When activated, these pumps are the primary mechanisms of multiple drug resistance.

The AcrAB-TolC system in E. coli responsible for multidrug resistance has been widely studied. This complex can export a variety of structurally unrelated drugs, dyes, and detergents.⁸ Other than AcrAB efflux pump, the acrEF operon also encodes an efflux pump. AcrE and AcrF are highly homologous to AcrA and AcrB,⁹ respectively, whereas AcrE shares 65% amino acid identity with AcrA, and AcrF shares 77% identity with AcrB.¹⁰

AcrAB gene expression is regulated by several factors, including AcrR, MarA, MppA, Rob, and SoxS.¹¹ AcrR protein is a negative regulator, binds as a dimer upstream of acrA and acrB genes, and inhibits efflux pump expression. Conversely, MarA is a positive regulator under the control of MarR and increases acrAB transcription efficiency. MarR also regulates and increases TolC expression by binding to a site nearby the tolC gene. In addition, SoxS is a small 107-amino acid protein that can trigger AcrAB and TolC expression, resulting in a reduced susceptibility to antimicrobials.¹² MarR can also enhance the function of the AcrAB-TolC pumps in collaboration with SoxS.

Bacteria resistance may be because of a decrease of outer membrane permeability.¹³ E. coli outer membrane proteins OmpC and OmpF are nonspecific hydrophilic protein channels that allow entry of nutrients and β-lactam antimicrobials.¹⁴ In E. coli, CIP preferentially enters cells through OmpF, and a lack of OmpF in Serratia spp. leads to increased resistance.¹⁵

When bacteria develop drug resistance, it is usually through multiple mechanisms that often work in concert to produce greater clinical resistance. This study focuses on the role of the AcrAB-TolC efflux pump in E. coli K12 using AcrAB-TolC knockout mutants. We tested its association with other efflux pumps under the selective pressure of CIP exposure.

Materials and Methods

Reagents, bacterial strains, and growth conditions

E. coli K12 was obtained from commercial sources. Plasmids pKD3, pKD4, and pKD46 obtained from commercial sources were used for mutagenesis procedures listed hereunder following a standard procedure.^16,17 Strains were grown in Luria–Bertani (LB) broth at 37°C with shaking.

Nalidixic acid (NAL), ofloxacin (OFL), norfloxacin (NOR), cefotaxime (CTX), erythromycin (ERY), and tetracycline (TET) were purchased from Xiangbo Ltd. (Guangzhou, Guangdong). Culture media were commercially obtained from Huankai Microbiology & Technology Ltd. (Guangdong, China). RNAiso Plus, Prime Script TM RT reagent Kit with gDNA Erase, and SYBR^® Premix Ex Taq™ (Tli RNaseH Plus) were purchased from Bo Biotechnology Ltd. (Dalian, Liaoning). Agar was purchased from Difco (England). Quantitative PCR strip tubes were obtained from Amgen Ltd.

Construction of acrA, acrB, or tolC deletion strains

Construction of acrA, acrB, or tolC deletion mutants was performed as previously described with some modifications.^16,17 Primers with extensions homologous to acrA, acrB, or tolC were annealed to plasmids pKD3 and pKD4 that contained bacteriophage λ FLP recognition sites to generate chloramphenicol and kanamycin knockout cassettes, respectively. PCR products were introduced into pure clonal E. coli K-12 by transformation in the presence of pKD46, generating deletion mutants designated as K12ΔacrA::kan, K12ΔacrB::kan, and K12ΔtolC::kan. Mutants were stored in broth containing 30% glycerin at −80°C.

Growth kinetics of E. coli K12 and deletion mutants

Overnight cultures of the E. coli K12 and its single gene deletion mutants were diluted with fresh LB broth to OD₆₀₀ = 0.1 and 30 μl was used to inoculate into 15 ml of fresh LB broth. The mixture was incubated with shaking at 200 rpm at 37°C and 100 μl aliquots were taken every hour for 11 hr. Each sample was diluted in sterile saline and 20 μl was plated onto agar plates for colony counting.

In vitro selection of CIP-resistant mutants

Parental and the deletion mutants K12ΔacrA::kan, K12ΔacrB::kan, and K12ΔtolC::kan were grown in LB broth at 37°C. Induction of E. coli K12 and deletion mutants upon exposure of stepwise concentrations of CIP was performed as previously described with modifications,¹⁸ that is, starting concentration at 0.06 and 0.015 mg per liter separately and increased at each step until high-level resistant mutants were selected. Serial mutants were designated as M1, M2, M3, M4, and M5 and each mutant was passaged five times on agar plates without CIP. The mutation frequency was calculated for each step.¹⁹

Quinolone resistance-determining regions mutation and antimicrobial susceptibility testing

Mutations in gyrA, gyrB, parC, and parE were confirmed by DNA sequence analysis (Yingjun Biotechnology Ltd., Shanghai) from gel-purified PCR products for each isolate using primers listed in Supplementary Table S1. The BLAST algorithm software was used for gene identification and comparison (National Center Biotechnology Information; www.ncbi.nlm.nih.gov). Susceptibility of mutants to antimicrobials was determined using the agar dilution method.²⁰ Minimum inhibitory concentration (MIC) was defined as the lowest concentration of a drug that inhibits visible growth. The following antimicrobials were tested in triplicates: NAL, OFL, NOR, CTX, ERY, and TET. ATCC 25922 was used as a quality control strain for the susceptibilities testing.

Real time-PCR

Gene expression levels were measured using total RNA extracted from 1.5 ml of overnight culture (OD₆₀₀ = 0.6) using the RNAiso Plus Kit (TaKaRa, Shiga, Japan). RNA was reverse transcribed using Prime Script RT Reagent Kit with gDNA Erase according to instructions provided by the manufacturer (Takara, Japan). Complementary DNA was quantified using an iCycler IQ5 instrument (Bio-Rad, USA) and data manipulation was done by utilizing software included with the instrument. Quantitative PCR was performed using 2 μl (100 ng/μl) of template, 0.6 μl (10 μmol/ml) gene-specific primers, and 10 μl of 2 × SYBR green master mix following instructions from the manufacturer. Cycling conditions were as follows: 94°C for 3 min, 94°C for 15 sec, 58°C for 15 sec, and 72°C for 30 sec for 40 cycles. Gene expression was calculated using the 2^−ΔΔCT method and all samples were normalized using 16S ribosomal RNA as an internal control.²¹ Gene-specific primers are listed in Supplementary Table S2.

Results

Characteristics of CIP-resistant mutants derived from deletion mutants

There were no significant differences between the growth rates for the three deletion mutants (K12ΔacrA::kan, K12ΔacrB::kan, and K12ΔtolC::kan) and their parent strain (E. coli K12) (Supplementary Fig. S1), indicating that growth of mutants was not impaired when AcrAB-TolC was inactivated. Meanwhile, susceptibilities of deletion mutants to fluoroquinolones, ampicillin, CTX, ERY, and TET were increased (Table 1).

Table 1.

Antimicrobial Susceptibility, Mutations, and Mutation Frequency of All the Strains Used in This Study

		Antimicrobial MIC (μg/ml)								Target gene mutation
Strains	CIP pressure (μg/ml)	CIP R ≥ 4	NAL R ≥ 32	NOR R ≥ 16	OFL R ≥ 2	CTX R ≥ 4	AMP R ≥ 32	TET R ≥ 16	ERY —	gyrA	gyrB	parC	parE	Frequency
K12		0.008	4	0.03	0.03	1	8	2	32	—	—	—	—	—
K12-M1	0.06	0.125	16	0.5	1	1	8	2	32	—	R342I	—	—	1.3 × 10⁻⁸
K12-M2	0.5	0.5	64	1	1	2	16	2	64	—	R342I	—	—	—
K12-M3	2	1	256	4	2	4	16	2	64	—	R342I	—	—	—
K12-M4	4	2	256	4	4	8	16	4	256	—	R342I	—	—	6.44 × 10⁻⁸
K12-M5	8	1	>256	8	4	8	32	8	256	—	—	—	—	7.43 × 10⁻¹⁰
K12ΔacrA		0.004	1	<0.004	<0.004	0.015	2	<0.25	2	—	—	—	—	—
K12ΔacrA-M1	0.015	0.015	64	0.125	0.125	0.125	2	<0.25	2	D87G	—	—	—	9.72 × 10⁻¹⁰
K12ΔacrA-M2	0.06	0.125	64	0.5	0.25	0.5	2	0.5	2	S83L	—	—	—	1.02 × 10⁻⁶
										D87G
K12ΔacrA-M3	0.5	4	64	8	8	16	2	0.5	2	S83L	—	G78D	—	—
										D87G
K12ΔacrA-M4	8	16	64	32	16	64	2	0.5	2	S83L	—	G78D	—	2.04 × 10⁻⁷
										D87G
K12ΔacrA-M5	16	64	>256	64	64	256	2	1	64	S83L	—	G78D	—	1.13 × 10⁻⁸
										D87G
K12ΔacrB		0.004	4	<0.004	<0.004	0.015	2	<0.25	4	—	—	—	—	—
K12ΔacrB-M1	0.015	0.03	32	0.125	0.06	0.125	2	<0.25	64	D87G	—	—	—	4.84 × 10⁻¹⁰
K12ΔacrB-M2	0.5	0.25	256	1	1	1	4	0.5	128	D87G	—	—	—	2.7 × 10⁻⁷
K12ΔacrB-M3	4	4	>256	16	16	32	8	8	256	S83L	—	—	—	—
										D87G
K12ΔacrB-M4	8	16	>256	64	32	128	8	8	256	S83L	—	—	—	6.74 × 10⁻⁸
										D87G
K12ΔacrB-M5	32	32	>256	64	64	128	8	8	256	S83L	—	—	I464M	9.62 × 10⁻¹¹
										D87G
K12ΔtolC		0.004	1	<0.004	<0.004	0.015	2	<0.25	2	—	—	—	—	—
K12ΔtolC-M1	0.015	0.03	32	0.125	0.125	—	—	—	—	S83L	—	—	—	4 × 10⁻⁹
K12ΔtolC-M2	0.06	0.125	64	0.25	0.125	—	—	—	—	S83L	—	—	—	4.97 × 10⁻⁷

There are no MIC interpretive standards for ERY.

AMP, ampicillin; CIP, ciprofloxacin; CTX, cefotaxime; D, aspartic acid; ERY, erythromycin; G, glycine; H, histamine; I, isoleucine; L, leucine; M, methionine; MIC, minimum inhibitory concentration; NAL, nalidixic acid; NOR, norfloxacin; OFL, ofloxacin; R, resistant; S, serine; TET, tetracycline.

With the increase of CIP pressure, serial multidrug-resistant mutants were obtained from strains with acrA or acrB deletion, but not from the strain with tolC deletion, from which only susceptible mutants (MIC_CIP = 0.125 μg/ml) were obtained, despite great efforts being made (Table 1). For the K12ΔacrA::kan strain, with increasing CIP concentrations, the first mutation was detected in gyrA D87G at a frequency of 9.72 × 10⁻¹⁰ and MIC_CIP was elevated to 0.015 μg/ml. Additional mutation was found in gyrA S83L at a frequency of 1.02 × 10⁻⁶, the mutant was susceptible to CIP (MIC_CIP = 0.125 μg/ml). In mutants (K12ΔacrA-M3) presenting resistance to CIP (MIC_CIP = 4 μg/ml), a third mutation was detected in parC G78D, and no other mutation was found. For the K12ΔacrB::kan strain, the first mutation detected in gyrA D87G, and MIC_CIP was 0.03 μg/ml and the second mutation was found in gyrA S83L, and the mutant was still susceptible to CIP (MIC_CIP = 0.25 μg/ml), meanwhile, the first and the second mutations in the K12ΔacrB::kan strain were detected at a frequency similar to the K12ΔacrA::kan strain. The third mutation was found in parE I464M in the mutant (K12ΔacrB-M5) with high-level resistance to CIP (MIC_CIP = 32 μg/ml) at a frequency of 9.62 × 10⁻¹¹.

For K12ΔtolC::kan, induction by CIP exposure only yielded point mutations that changed codon 83 of gyrA with a corresponding MIC to CIP at 0.125 μg/ml (Table 1).

Relative expression of efflux pump genes and their regulators by real time-PCR

During the CIP exposure of parental strain K12, expression of the efflux pump genes acrA, acrB, and tolC increased. The expression levels of acrA and acrB were significantly higher in two of the three strains tested. The acrD, acrE, and acrF genes remained unchanged. In contrast, expression of ompF gene decreased without an accompanying change in ompC (Fig. 1A). Interestingly, there was no significant change in gene encoding the marR regulator, MarA. However, the regulator of the superoxide response regulon soxS increased between 8- and 18-fold among the three strains (Fig. 1A).

FIG. 1.

Relative expression levels of efflux pump genes and their regulators by RT-PCR. (A) Comparison of expression levels among series of mutants upon ciprofloxacin induction of Escherichia coli K12. (B) Comparison of gene expression levels of K12ΔacrA::kan-derived mutants. (C) Comparison of gene expression levels of K12ΔacrB::kan-derived mutants. (D) Comparison of gene expression levels of K12ΔtolC::kan-derived mutants. *p < 0.05; **p < 0.01. RT, real time.

For acrA deletion mutant (K12ΔacrA::kan), expression levels of tolC and other regulators were not significantly changed when concentration was increased in CIP exposure to 8 μg/ml, although the mutant displayed resistance to CIP (K12ΔacrA-M4, MIC = 16 μg/ml). However, K12ΔacrA-M5 mutants (CIP exposure 16 μg/ml, MIC = 64 μg/ml) showed significant increase in the expression of acrE and acrF. There was no change in acrD. Exposure of the strain (K12ΔacrA-M4 and K12ΔacrA-M5) to higher CIP concentrations resulted in a decreasing trend for ompF (Fig. 1B).

For acrB deletion strain (K12ΔacrB::kan), expression levels of tolC and regulators were not altered with the increasing CIP concentration (Fig. 1C). When the mutant displayed reduced susceptibility to CIP (K12ΔacrB-M2, MIC = 0.25 μg/ml), the expression of acrE and acrF elevated 100- and 500-fold. Furthermore, these two genes were constantly expressed in the CIP-resistant mutants. However, expression of transmembrane porin genes ompC and ompF did not change significantly.

For tolC deletion strain (K12ΔtolC::kan), the expression of acrB was found significantly increased when the mutant (K12ΔtolC-M2, MIC = 0.25 μg/ml) demonstrated a reduced susceptibility to CIP. There was no change in expression of regulatory genes marA and soxS, but there was significantly decreased expression in ompF (Fig. 1D).

Discussion

Multidrug efflux pumps AcrAB-TolC in E. coli are composed of a trimer consisting of the outer membrane channel TolC, the periplasmic adaptor protein AcrA, and inner membrane protein AcrB.^22–24

The periplasmic adaptor protein AcrA is a key component of multidrug efflux pumps and connects TolC to AcrB.^22–25 AcrA is a functional subunit of intact multicomponent transporters and can also associate with other transporters belonging to RND, adenosine triphosphate-binding cassette, major facilitator families of proteins,^24,26 and the outer membrane channel TolC for drug efflux activity. The AcrB protein has recently been crystallized,²⁷ demonstrating that it is an inner membrane protein responsible for binding and transferring drug molecules. AcrB also determines specificity of substrates for the AcrAB-TolC efflux pump. Thus, AcrB is important for the stability and pumping activity of the AcrAB-TolC system,²⁸ and an intact AcrB in the trimer AcrAB-TolC is necessary for drug efflux activity.²⁹ The outer membrane channel protein TolC is not associated with substrate specificity or the direction of extrusion, and can associate with other efflux pumps of the RND family. TolC enters the periplasm membrane using a spiral structure and connects with AcrA. When the inner membrane AcrB protein senses and captures a substrate, TolC binds AcrAB to become a protein complex and opens the TolC pumping channel, resulting in drug export.³⁰

The integrity of the trimer system is crucial for the development of drug resistance. Disruption of any of these three components results in hypersusceptibility of bacteria to many antimicrobials, organic solvents, and bile salts.⁸

This study demonstrated that, once the AcrAB-TolC is impaired, the resistant mutants were even harder to select under the increasing CIP concentration compared with the parental strain, because the mutation frequency was 100 times lower in the former than in the latter (Table 1). Although acrA or acrB was deleted, the mutants were obtained and reached a high level of resistance to CIP. However, when tolC was deleted, it was difficult to select the mutant. During the development of resistance in the mutants with deletion of acrA or acrB, resistance-associated mutations in gyrase (gyrA S83L, D87N) and topoisomerase IV (parC G78D) played a role in the earlier stage. With the increase of MIC to CIP, no additional mutations were detected. Our findings suggested that other RND efflux transporters presumably exerted alternative roles when AcrAB is impaired in the development of higher CIP resistance. TolC plays an important role in the trimer structure of the AcrAB-TolC complex.^30,31 Our findings were consistent with Ricci et al., and confirmed that mutants lacking the tolC gene were rarely resistant to CIP.³²

In E. coli, acrD and acrEF also encode efflux pumps, AcrD pumps out aminoglycoside antimicrobials, and AcrE and AcrF share 65% and 77% of similarity to AcrA and AcrB, respectively.³³ AcrE and AcrF can also associate with TolC to form an AcrAB-TolC-like trimer efflux structure.³⁴ AcrEF and AcrAB have a similar substrate spectrum, and the two periplasmic adaptor proteins are interchangeable so that AcrA can also function with AcrF. Previous studies have shown that multidrug resistance phenotype in AcrAB-deficient cells can be conferred by the overproduction of AcrEF.³⁵ Our study showed that the overexpression of acrEF complemented AcrAB function^10,35 and contributed to the selection of resistant and high-level resistant mutants when acrA or acrB was inactivated. During the detection of resistance phenotypes, no additional resistance-associated mutation was detected though increased MIC to CIP was found (K12ΔacrA-M4, K12ΔacrA-M5, K12ΔacrB-M4, and K12ΔacrB-M5). The expression of acrE and acrF increased more than 100-fold. If tolC was impaired, drugs could not be extruded by acrEF alone. It was confirmed by our findings that mutants were hard to select under the increasing CIP concentration. In E. coli, OmpF is a major outer membrane protein and is associated with a reduced intracellular accumulation of fluoroquinolones, resulting in fluoroquinolone resistance.³⁶ The pore size of OmpF in E. coli is slightly larger than OmpC, and OmpF is the major channel in E. coli for CIP.^15,37 The reduction in expression of OmpF was only found in the tolC inactivated mutants, suggesting less penetration of CIP into the cell and contributing to the reduced susceptibility to CIP.

In summary, we demonstrate that an intact AcrAB-TolC efflux pump is crucial to development of bacterial resistance. Once AcrAB is impaired, AcrEF may play an important alternative function.

Footnotes

Acknowledgments

This work was supported, in part, by the National Natural Science Foundation of China (Grant No. 31272602) and Guangdong Provincial Department of Education project (Grant No. 2012KJCX0019).

Disclosure Statement

No competing financial interests exist.

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