Effects of Efflux Pump Inhibitors on Erythromycin,Ciprofloxacin,and Tetracycline Resistance in Campylobacter spp. Isolates

Abstract

The aim was to assess the potency of the efflux pump inhibitors (EPIs) phenylalanine–arginine ß-naphthylamide (PAßN) and 1-(1-naphthylmethyl)-piperazine (NMP) and the putative natural EPI phenolic (−)-epigallocatechin gallate (EGCG) for the reversal of erythromycin, ciprofloxacin, and tetracycline resistance in Campylobacter jejuni and Campylobacter coli isolates. We investigated target mutations and resistant genes involved in erythromycin and tetracycline resistance and determined the roles of the bacterial drug efflux systems (cmeB, cmeF, and cmeR) in antimicrobial resistance. Our data show that most of the high-level erythromycin resistance and all of the tetracycline resistance can be explained through mutations in 23S rRNA and the presence of the tetO gene, respectively. The EPIs show the ability to partly reverse drug resistance in these Campylobacter isolates. Based on a fourfold or greater reduction in the erythromycin minimal inhibitory concentration (MIC), PAßN and NMP had clear effects in almost of all of the isolates tested. PAßN had a highly selective action on the ciprofloxacin and tetracycline MICs. Inactivation of cmeB increased susceptibility to all of the antimicrobials tested, whereas inactivation of cmeF and cmeR had no effects. A notable decrease in resistance to erythromycin and ciprofloxacin in the presence of subinhibitory concentrations of EGCG demonstrates the resistance-modifying activities of this natural EPI, and indicates its potential use in the control of Campylobacter spp. in the food chain.

Introduction

From the recent European Food Safety Authority annual report, it is evident that in 2009, Campylobacter spp. continued (since 2005) to be the most commonly reported gastrointestinal bacterial pathogen in humans, with a 4% overall increase compared to 2008, and with most member states reporting increases.⁸ In foodstuffs, Campylobacter spp. were mainly detected in fresh broiler meat (poultry), where an average of 31% of samples tested across the European Union (EU) were found to be positive. Similarly, 25% of the broiler flocks tested in the EU were found to be positive.^8,38

Although Campylobacter spp. infections usually result in self-limiting gastroenteritis, antimicrobial treatment is necessary for patients with severe and advance infections. Fluoroquinolones and erythromycin are the first-line agents for treatment of Campylobacter spp. infections, whereas tetracycline is sometimes listed as an alternative. However, the prevalence of resistant Campylobacter spp. is increasing worldwide. This is becoming a major concern for public health,²⁶ as seen more recently from several official reports of national and international surveillance programs, along with a number of other publications.⁷

Resistance to macrolides and quinolones in Campylobacter spp. has been mainly attributed to mutations in domain V of the 23S rRNA gene²⁸ and in the gyrA-encoded subunit of DNA gyrase, respectively.²⁹ Tetracycline resistance is primarily mediated by a plasmid-encoded tetO gene.³⁵ In addition to these target mutations, resistance to antimicrobials can be conferred via the efflux pumps, which limit the access of antimicrobials to their targets by actively pumping these molecules out of the bacterial cell, thereby preventing the intracellular accumulation necessary for lethality. Several putative and two well-known resistance–nodulation–cell division (RND) transporters, including the CmeABC and CmeDEF multidrug efflux systems, have been characterized in Campylobacter spp.^1,15,20 CmeABC contributes to resistance to a variety of antimicrobials, including erythromycin, ciprofloxacin, and tetracycline,^11,21 as well as to the multidrug resistant (MDR) phenotype.²³ CmeDEF has a more modest impact on antimicrobial resistance.^1,15 Lin et al.²¹ reported that cmeR encodes a transcriptional repressor, CmeR, which directly interacts with the cmeABC promoter and modulates the expression of cmeABC.

Pharmacological MDR efflux pump inhibition by an efflux pump inhibitor (EPI) has become an attractive area of research.³⁰ Several new putative bacterial EPIs have been described, and one of the first with known EPI activity was phenylalanine– arginine ß-naphthylamide (PAßN), as defined in Pseudomonas aeroginosa.²² Several studies have shown that PAßN can restore erythromycin susceptibility in Campylobacter spp. that show low-level resistance.^3,10,12,19 Another EPI, 1-(1-naphthylmethyl)-piperazine (NMP), has been shown to reverse MDR, as first shown in Escherichia coli,¹⁶ and later in Campylobacter jejuni and Campylobacter coli.¹³

With a reduction in the number of new agents and in antimicrobial development, there is a need for new resistance-modifying agents and EPIs that can restore the activities of the already-licensed antimicrobial agents. Piddock³¹ reported that natural and synthetic chemically simpler and safer compounds than PAβN, such as trimethoprim, can also act as EPIs in Gram-negative bacteria. Also, medicinal plant extracts can provide suitable lead compounds for future development and possible clinical use as EPIs for various Gram-negative bacteria.^9,34 Interestingly, (−)-epigallocatechin gallate (EGCG), the major polyphenol, or catechin, found in green tea (Camellia sinensis),¹⁴ shows resistance-modifying activities for norfloxacin in staphylococci and tetracycline.³² Low concentrations of catechins would result in the high-affinity binding sites of the efflux pumps being occupied preferentially, thereby enhancing efflux.³²

The aim here was to assess the potencies of the EPIs PAßN and NMP, and of the putative natural EPI EGCG, for the reversal of resistance in C. jejuni and C. coli isolated from food, animal, environmental water, and human sources. We also investigated target mutations and the resistant genes involved in erythromycin and tetracycline resistance. Moreover, we determined the role of bacterial drug efflux systems in antimicrobial resistance by comparing the sensitivities of wild-type C. jejuni 11168 and its mutants in terms of the specific efflux pump genes cmeB, cmeF, and cmeR.

Materials and Methods

Bacterial isolates and growth conditions

Fifty-four food, animal, water, and human isolates were used in this study (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr). They were isolated and identified phenotypically by multiplex polymerase chain reaction (mPCR), as described previously.³⁹ The cultures were stored at −80°C in a brain–heart infusion broth (Merck, Darmstadt, Germany) with defibrinated horse blood (Oxoid, Hampshire, UK) and glycerol (Kemika, Zagreb, Croatia). The isolates were subcultured on Columbia agar (Oxoid), supplemented with 5% horse blood (Oxoid) at 42°C under microaerophilic conditions in gas-tight containers (5% O₂, 10% CO₂, and 85% N₂). C. coli ATCC 33559, C. jejuni ATCC 33560, and C. jejuni NCTC 11168 were used as the reference strains. Knockout cmeB, cmeF, and cmeR mutants of C. jejuni NCTC 11168 were also used (the genomic DNA prepared from the corresponding mutant strains was obtained from Prof. Qijing Zhang, Iowa State University, USA). Purified DNA from the cmeB,²⁰ cmeF,¹ and cmeR²¹ mutants was used to transform C. jejuni NCTC 11168 using the standard biphasic method for natural transformation and construction of cmeB, cmeF, and cmeR mutants.³⁷ Our transformants of cmeB, cmeF, and cmeR were confirmed by PCR (A. Klančnik, unpublished results), using the following specific primers: cmeB BF1 (59-GCTGGATCCATA GGTCTTACAAAT-39) and cmeB CR (59-TTTTTAAAGCTT TAAGGTAATTTTCTT-39) (Lin et al.²⁰); cmeF FF1 (59 AAGT ACAACTCTCATTGCTTGCAT-39) and cmeF FR1 (59-TGGC TATTGCCATAGGAGAA-39) (Akiba et al.¹); and cmeR F (59-TAGAAAAGTATATTTGTATACCCT-39) and cmeR GSR4 (59-GAAATTTTTGGCTAATTATAT-39) (Lin et al.²¹).

Antimicrobial susceptibility testing

The Campylobacter spp. isolates were selected from different sources, primarily on the basis of high and intermediate erythromycin resistance. This was determined first by disk-diffusion assays and Epsilometer tests,¹⁸ and for 36 isolates, this was confirmed by the broth microdilution method.¹⁹ The minimal inhibitory concentrations (MICs), as the lowest concentrations of erythromycin, ciprofloxacin, and tetracycline where no growth was seen, were determined on the basis of the fluorescent signals measured using a microplate reader (Tecan, Männedorf/Zurich, Switzerland) after adding the CellTiter-Blue^® reagent (Promega Corporation, Madison, WI) to the culture medium.¹⁹ As this reagent can react with the natural antimicrobials, the MICs when EGCG was used were determined on the basis of the luminescent signal measured after adding the CellTiter-Glo^® reagent (Promega Corporation) to the culture medium.¹⁷ The breakpoints used were those defined by the Clinical and Laboratory Standards Institute: ≥32 mg/L for erythromycin (Sigma-Aldrich, Saint Louis, MO), ≥4 mg/L for ciprofloxacin (Fluka, Biochemika), and ≥16 mg/L for tetracycline (Fluka, Biochemika).⁴ MDR is defined as resistance to all three of these antimicrobials. For erythromycin resistance, isolates with MICs<8 mg/L are considered to be susceptible; with MICs between 8 and 32 mg/L, they are termed intermediate, and with MIC>256 mg/L, they show high-level resistance (HLR).

To demonstrate the contributions of the CmeABC and CmeDEF efflux pumps in antimicrobial resistance, the cmeB and cmeF mutants were tested with erythromycin, ciprofloxacin, and tetracycline, both in the absence and presence of the EPIs.

Effects of the chemical EPIs PAßN and NMP

To investigate the contributions of efflux pump activity to antimicrobial resistance, the MICs of the three antimicrobials tested (erythromycin, ciprofloxacin, and tetracycline) were determined both in the absence and presence of the EPIs PAßN and NMP, in all of the selected isolates, using the broth microdilution method.¹⁹ For this purpose, the Müller Hinton (MH) broth (Oxoid) was supplemented with PAßN (Sigma-Aldrich) or NMP (Chess, Mannheim, Germany). To initially determine the effects of various concentrations of NMP (for dose-dependent responses) on the susceptibility of C. coli VC 110722 to erythromycin and ciprofloxacin, the MICs were determined with 10, 20, 40, 60, 80, and 100 mg/L NMP, in the MH broth over 24 hours. This isolate was selected, because it did not have the mutation in the 23S rRNA gene, and it showed intermediate erythromycin resistance. On the basis of these susceptibilities (taking into account the lowest concentration with the highest effects on susceptibility), NMP (80 mg/L) was used for further testing. In the case of PAßN, the sublethal concentration of 20 mg/L was used, as confirmed in a previous study.¹⁹ Two independent experiments were conducted to confirm the reproducibility of the MIC data. The C. coli ATCC 33559, C. jejuni ATCC 33560, and C. jejuni NCTC 11168 strains were included as the reference strains.

Effects of the putative EPI EGCG

To investigate the activity of the plant phenolic EGCG as a putative EPI and resistance-modifying agent, the MICs of erythromycin, ciprofloxacin, and tetracycline were determined in the absence and presence of EGCG (Sigma-Aldrich), using the broth microdilution method.¹⁷ The MH broth was supplemented with EGCG at the subinhibitory concentrations of 0.25 MIC for each of the Campylobacter spp. tested. Three independent experiments were conducted to confirm the reproducibility of these MIC data.

Detection of mutation in the 23S rRNA gene by PCR–restriction fragment length polymorphism

A PCR–restriction fragment length polymorphism (RFLP) protocol was used to detect the mutation at position 2075, as described previously.¹⁹ The mutation at position 2075 leads to five fragments after BsmAI digestion (311, 226, 102, 57, and 18 bp). The fragments were separated on 1.5% agarose gels.

Sequence analysis of the 23S rRNA gene

Sequence analysis was used to confirm the data obtained by PCR-RFLP and to detect potential mutations in the 23S rRNA gene. The sequencing of the amplified fragments of 23S rRNA was carried out as described previously.¹⁹

PCR amplification of the tetO gene

The primers used for amplification of the tetO gene were the same as described previously.² The amplification reactions were carried out in a 25-μL volume containing (final concentrations) 1.0 μL crude cell lysate, 2.5 μL 10×PCR buffer II, 1.5 μL MgCl₂ (25 mM), 1.25 μL dNTP (2 mM), 0.25 μL of each primer (1.0 μg/μL), and 0.2 μL AmpliTaq^® DNA Polymerase (5 U/μL). The PCR reagents were from Applied Biosystems. The PCR cycle included an initial denaturation at 95°C for 1 min, 30 cycles of denaturation for 15 sec at 95°C, primer annealing for 15 sec at 58°C, and an extension of denaturation for 30 sec at 72°C. The amplified PCR fragment was 559 bp and was revealed on 1.5% agarose gels.

Results

Antimicrobial susceptibility

A total of 54 food, animal, water and human isolates were identified as C. coli, C. jejuni, or Campylobacter spp. (Supplementary Table S1). Along with three reference strains, these were examined for erythromycin, ciprofloxacin, and tetracycline resistance using the broth microdilution method. Table 1 gives the distributions of the MICs (%) for these three antimicrobials.

Table 1.

Distribution of the Minimal Inhibitory Concentrations (%) of Erythromycin, Ciprofloxacin, and Tetracycline Across the 54 Campylobacter spp. Isolated from Food/Animals and Surface Water, and from Human Clinical Samples

	Distribution of MIC (%)
Antimicrobial	≤0.5	1	2	4	8	16	32	64	128	256	≥512	MIC₅₀ [mg/L]	MIC₉₀ [mg/L]
Erythromycin	24.1	14.8	13.0	7.4	5.6	1.9	1.9	9.3	3.7	1.9	16.7	2	512
Ciprofloxacin	42.6	1.9	5.6	11.1	9.3	9.3	9.3	3.7	5.6	0	1.9	2	64
Tetracycline	48.1	18.5	5.6	0	0	3.7	5.6	5.6	13.0	0	0	0.5	128

MIC, minimal inhibitory concentration.

The results of the antimicrobial susceptibilities among the full 57 Campylobacter spp. tested showed 18 (31%) as resistant to erythromycin, 26 (45%) resistant to ciprofloxacin, and 15 (26%) resistant to tetracycline (see Supplementary Table S1). Resistance to all three of these antimicrobials was more frequent among the C. coli isolates. For MDR, six isolates of C. coli (10%) showed resistance to all three of these antimicrobials (see Supplementary Table S1).

Target modifications in the 23S rRNA gene and the presence of the tetO gene

The PCR-RFLP procedure was used to test for the presence of the A2075G mutation in the 23S rRNA gene. Seven (78%) of the erythromycin HLR C. coli had the A2075G mutation (Table 2). Conversely, the A2075G mutation was not identified in any of the intermediate resistance and susceptible isolates. Additionally, the sequence analysis of the 714-bp amplicon of the 23S rRNA gene confirmed the data obtained by PCR-RFLP in these seven mutated HLR C. coli isolates. Finally, none of our isolates contained a 2074 mutation.

Table 2.

Detection of Point Mutations in the 23S rRNA Gene and tetO Gene in Erythromycin- and Tetracycline-Resistant Campylobacter Isolates, and in the Reference and Mutant Strains

	Target modifications of 23S rRNA
Strain/isolate	PCR-RFLP	Sequencing	tetO gene (PCR)
Reference strains
ATCC 33559	−	−	−
ATCC 33560	−	−	−
NCTC 11168	−	−	−
Collected isolates
VC 110722	−	−	+
VC 110725	−	−	+
10522	−	−	+
119/06	−	−	−
573/03	−	−	−
1846	−	−	−
1847	−	−	−
413/06	−	−	−
375/06	−	−	−
575	−	−	−
944	−	−	−
652/1	−	−	−
M 37	A2075G	A2075G	−
137	A2075G	A2075G	+
140	A2075G	A2075G	+
VC 7114	−	−	+
VC 11076	A2075G	A2075G	+
2235	A2075G	A2075G	−
171	A2075G	A2075G	+
809	A2075G	A2075G	+
803	−	−	−
FC 10	−	−	+
VC 81	−	−	+
FC 8	−	−	+
21F	−	−	+
17015	−	−	+
10522	−	−	+
804	−	−	+
Mutant strains of Campylobacter jejuni NCTC 11168
cmeB	−	−	−
cmeF	−	−	−
cmeR	−	−	−

+, presence of the A2075G mutation and/or tetO gene; −, absence of the A2075G mutation and/or tetO gene; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

The PCR procedure confirmed the presence of the tetO gene in all of the tetracycline-resistant isolates. None of the isolates that were susceptible to tetracycline had the tetO gene.

Effects of NMP on susceptibility of C. coli VC 110722 to erythromycin and ciprofloxacin are dose dependent

To determine the effects of various concentrations of NMP on C. coli VC 110722 resistance to erythromycin and ciprofloxacin, the MICs of these antimicrobials were determined in the presence of NMP. For measurement of the dose-dependency of this response, the VC 110722 isolate was grown for 24 hours in the MH broth containing different concentrations of the antimicrobials and of NMP. The sublethal concentrations of 10 and 20 mg/L NMP led to fourfold increases in erythromycin susceptibility. The higher concentration of 40 mg/L NMP promoted an eightfold increase in erythromycin susceptibility, whereas 80 and 100 mg/L NMP had the greatest effects, showing 64-fold increases in erythromycin susceptibility. For ciprofloxacin, 10, 20, and 40 mg/L NMP promoted twofold increases in susceptibility, and the higher concentrations of 80 and 100 mg/L NMP showed fourfold increases in ciprofloxacin susceptibility. On this basis, for the lowest concentration with the highest effects on susceptibility, the sublethal concentration of 80 mg/L NMP was used for further testing.

Effects of the EPIs PAßN and NMP

With the aim to study the efflux mechanisms involved in these isolates of 57 Campylobacter spp., 20 mg/L PAßN and 80 mg/L NMP were tested on the resistance to erythromycin, ciprofloxacin, and tetracycline. The effects of these EPIs on the MICs of these antimicrobials in the resistant Campylobacter spp. are given in Table 3.

Table 3.

Susceptibilities of the Campylobacter spp. Isolates; Reference Strains and Mutants to Erythromycin, Ciprofloxacin, and Tetracycline; and the Effects of Sublethal Concentrations of the Efflux Pump Inhibitors Phenylalanine–Arginine ß-Naphthylamide (20 mg/L) and 1-(1-Naphthylmethyl)-Piperazine (80 mg/L)

	MIC [mg/L] (fold reduction)
	Erythromycin			Ciprofloxacin			Tetracycline
Strain sample code	−EPI	+PAßN	+NMP	−EPI	+PAßN	+NMP	−EPI	+PAßN	+NMP
Reference strains
ATCC 33559	2	0.06 (32)	0.06 (32)	0.06	<0.06 (>1)	<0.06 (>1)	1	0.5 (2)	ND
ATCC 33560	1	0.25 (4)	1 (1)	0.25	0.25 (1)	0.13 (2)	0.5	0.13 (4)	ND
NCTC 11168	0.13	0.03 (4)	0.03 (4)	0.25	0.13 (2)	0.25 (1)	0.25	0.25 (1)	0.25 (1)
Collected isolates
10162	8	0.06 (128)	0.13 (64)	0.03	0.03 (1)	0.03 (1)	0.13	0.06 (2)	ND
VC 110722	8	0.13 (64)	0.25 (32)	128	<2 (≥64)	<2 (≥64)	128	64 (2)	128 (1)
VC 110725	8	0.06 (128)	8 (1)	0.25	0.25 (1)	0.125 (2)	64	4 (16)	64 (1)
10522	16	4 (4)	8 (2)	32	<0.06 (≥512)	<0.06 (≥512)	16	8 (2)	16 (1)
119/06	32	4 (8)	8 (4)	0.5	0.25 (2)	0.25 (2)	0.5	0.13 (4)	0.5 (1)
573/03	64	8 (8)	8 (8)	0.25	0.25 (1)	0.13 (2)	2	1 (2)	ND
1846	64	16 (4)	8 (8)	4	4 (1)	4 (1)	1	0.5 (2)	ND
1847	64	16 (4)	0.5 (128)	4	4 (1)	0.5 (8)	2	1 (2)	2 (1)
413/06	64	16 (4)	16 (4)	1	1 (1)	1 (1)	0.13	0.06 (2)	ND
375/06	64	2 (32)	4 (16)	0.5	0.5 (1)	0.25 (2)	0.25	0.06 (4)	ND
575	128	16 (8)	32 (4)	2	2 (1)	2 (1)	1	0.5 (2)	1 (1)
944	128	32 (4)	16 (8)	0.06	0.06 (1)	0.06 (1)	0.125	0.06 (2)	ND
652/1	256	32 (8)	32 (8)	32	16 (2)	16 (2)	0.06	0.06 (1)	ND
M 37	512	16 (32)	128 (4)	8	<0.06 (≥128)	0.25 (32)	1	0.25 (4)	1 (1)
137	>512	32 (≥32)	128 (≥8)	8	<0.06 (≥128)	8 (1)	32	8 (4)	ND
140	>512	32 (≥32)	128 (≥8)	32	32 (1)	8 (4)	64	16 (4)	64 (1)
VC 7114	>512	32 (≥32)	<2 (≥512)	8	8 (1)	4 (2)	128	32 (4)	ND
VC 11076	>512	32 (≥32)	32 (≥32)	64	<1 (≥64)	<1 (≥64)	128	<2 (≥64)	128 (1)
2235	512	16 (32)	16 (32)	0.25	0.25 (1)	<0.06 (4)	0.25	0.25 (1)	ND
171	512	32 (16)	128 (4)	16	16 (1)	8 (2)	32	8 (4)	32 (1)
809	512	8 (64)	32 (16)	4	4 (1)	4 (1)	32	16 (2)	32 (1)
803	512	32 (32)	<2 (≥256)	0.06	0.06 (1)	<0.06 (≥1)	0.13	0.13 (1)	ND
28264	0.5	<0.03 (>16)	<0.03 (>16)	4	4 (1)	4 (1)	0.5	0.13 (4)	ND
28233	1	0.13 (8)	0.06 (16)	4	4 (1)	2 (2)	0.5	0.13 (4)	ND
K51/1	1	0.06 (16)	0.5 (2)	4	4 (1)	4 (1)	0.25	0.25 (1)	0.25 (1)
FC 41	1	0.06 (16)	0.25 (4)	8	8 (1)	4 (2)	0.25	0.06 (4)	0.25 (1)
FC 10	4	0.06 (64)	0.5 (8)	16	16 (1)	8 (2)	128	64 (2)	128 (1)
GC 182	0.5	0.13 (4)	0.5 (1)	16	16 (1)	8 (2)	1	0.5 (2)	1 (1)
VC 81	2	2 (1)	0.5 (4)	16	8 (2)	8 (2)	128	32 (4)	128 (1)
GC 154	4	0.06 (64)	1 (4)	16	16 (1)	8 (2)	0.06	0.06 (1)	ND
3755	1	0.13 (8)	0.25 (4)	32	32 (1)	16 (2)	1	0.5 (2)	1 (1)
652/1	2	0.06 (32)	0.13 (16)	32	<0.06 (≥512)	<0.06 (≥512)	0.06	0.06 (1)	ND
573/03	1	0.03 (32)	0.5 (2)	32	32 (1)	8 (4)	0.25	0.13 (2)	ND
FC 8	4	0.13 (32)	0.5 (8)	64	32 (2)	16 (4)	64	16 (8)	64 (1)
3552	2	0.03 (64)	0.13 (16)	128	128 (1)	32 (4)	1	1 (1)	1 (1)
21F	1	0.13 (8)	0.06 (16)	128	64 (2)	128 (1)	128	8 (16)	128 (1)
17015	0.5	0.06 (8)	0.03 (16)	512	512 (1)	256 (2)	128	16 (8)	ND
10522	16	4 (4)	8 (2)	32	0.06 (512)	0.06 (512)	16	8 (2)	16 (1)
804	0.13	0.06 (2)	0.06 (2)	0.,06	0.06 (1)	<0.06 (>1)	16	8 (2)	ND
Mutant strains of C. jejuni NCTC 11168
cmeB	0.016	0.016 (1)	0.016 (1)	0.06	0.02 (4)	0.02 (4)	0.03	0.03 (1)	ND
cmeF	0.5	0.03 (16)	0.13 (16)	0.25	0.06 (4)	0.06 (4)	ND	ND	ND
cmeR	0.5	ND	0.06 (8)	0.125	0.03 (4)	0.03 (4)	ND	ND	ND

Fourfold or greater differences in the MICs were considered significant and are indicated by bold. The numbers in parenthesis indicate the fold reductions in the MICs of the antimicrobials tested.

−EPI, without efflux pump inhibitor; ND, not determined; NMP, 1-(1-naphthylmethyl)-piperazine; PAßN, phenylalanine–arginine ß-naphthylamide.

PAßN restored the susceptibilities to the level of the MICs of the susceptible isolates in all of the intermediate and two of the erythromycin-resistant isolates (Table 3). These isolates did not carry any mutation in the 23S rRNA gene. Very similar effects were seen with NMP for erythromycin susceptibility. Based on a fourfold or greater MIC reduction for erythromycin, the effects of PAßN and NMP were clear in almost all of the isolates tested. Additionally, in the erythromycin HLR isolates, PAßN and NMP increased the susceptibilities by at least 16-fold to 64-fold, and 4-fold to 256-fold, respectively (Table 3).

The effects of PAßN and NMP on ciprofloxacin resistance in the Campylobacter spp. tested were much more limited in comparison to erythromycin (Table 3). PAßN reduced the MICs in nine ciprofloxacin-resistant isolates, and in four of these, PAßN increased the susceptibility by twofold. Moreover, PAßN restored ciprofloxacin susceptibility in five of the ciprofloxacin-resistant isolates. In addition, clear effects of NMP on the ciprofloxacin MICs were seen for 20 of these ciprofloxacin-resistant isolates. NMP reduced the MICs by at least twofold to eightfold in 16 of these ciprofloxacin-resistant isolates, whereas it restored susceptibility in six of these ciprofloxacin-resistant isolates (Table 3). On the other hand, both of these EPIs had only small (twofold) effects, or no effects, on the ciprofloxacin MICs of the ciprofloxacin-susceptible isolates.

Clear effects on the tetracycline MICs were only seen for PAßN (Table 3), but not for NMP. Effects of PAßN were seen for all of the tetracycline-resistant isolates, with the tetracycline MICs reduced by at least 2-fold to 64-fold. In addition, in 7 of the 15 tetracycline-resistant isolates, PAßN restored the susceptibility (Table 3).

Effects of the putative EPI EGCG

The effects of EGCG were tested for selected Campylobacter isolates. The data show a high efficiency for EGCG, with MICs against the selected isolates of 39 to 625 mg/L (Table 4). In the present study, we investigated the resistance of Campylobacter spp. to erythromycin and ciprofloxacin in the absence and presence of 0.25 MIC of EGCG, for each isolate tested. This activity of EGCG as a putative EPI and a resistance-modifying agent in the selected Campylobacter spp. isolates is presented in Table 4.

Table 4.

Susceptibilities of the Campylobacter Spp. Isolates; Reference Strains and Mutants to Erythromycin, Ciprofloxacin, and Tetracycline; and the Effects of (−)-Epigallocatechin Gallate

		MIC [mg/L] (fold reduction)
	MIC [mg/L]	Erythromycin		Ciprofloxacin		Tetracycline
Strain sample code	EGCG	−EPI	+EGCG ^a	−EPI	+EGCG ^a	−EPI	+EGCG ^a
Reference strains
ATCC 33559	156	2	1 (2)	0.06	0.06 (1)	0.5	0.5 (1)
ATCC 33560	156	1	1 (1)	1	1 (1)	1	1 (1)
NCTC 11168	313	0.13	0.02 (8)	0.25	0.25 (1)	0.25	0.032 (8)
Collected isolates
804		0.125	0.125 (1)	0.06	0.06 (1)	16	4 (4)
GC 182	625	0.5	0.5 (1)	16	8 (2)	1	0.5 (2)
3755	313	1	0.5 (2)	32	32 (1)	1	1 (1)
573/03	39	1	0.5 (2)	32	16 (2)	2	2 (1)
K 51/1	625	1	1 (1)	4	4 (1)	0.25	0.25 (1)
FC 41	313	1	1 (1)	8	8 (1)	0.25	0.25 (1)
VC 81	625	2	1 (2)	16	16 (1)	128	128 (1)
3552	313	2	2 (1)	128	64 (2)	1	1 (1)
GC 154	156	4	1 (4)	16	8 (2)	0.06	0.06 (1)
FC 8	625	4	4 (1)	64	8 (8)	64	2 (32)
FC 10	39	4	2 (2)	16	16 (1)	128	128 (1)
10162	39	8	0.5 (16)	0.03	0.03 (1)	0.125	0.125 (1)
VC 110722	78	8	2 (4)	128	4 (32)	128	128 (1)
VC 110725	78	8	2 (4)	0.25	0.25 (1)	64	64 (1)
10522	39	16	1 (16)	32	8 (4)	16	16 (1)
413/06	156	64	2 (32)	1	0.25 (4)	0.125	0.125 (1)
375/06	78	64	64 (1)	0.5	0.5 (1)	0.25	0.5 (0.5)
M 37	39	512	256 (2)	8	4 (2)	1	1 (1)
137	313	>512	128 (≥4)	8	1 (8)	32	8 (4)
140	313	>512	64 (≥8)	32	8 (4)	64	8 (4)
VC 7114	313	>512	512 (≥1)	8	8 (1)	128	16 (8)
VC 11076	156	>512	256 (≥2)	64	8 (8)	128	128 (1)
2235	78	512	128 (4)	0.25	0.25 (1)	0.25	0.25 (1)
171	78	512	128 (4)	16	32 (0.5)	32	32 (1)
809	156	512	32 (16)	4	4 (1)	32	32 (1)
803	39	512	64 (8)	0.06	0.06 (1)	0.125	0.125 (1)
Mutant strains of C. jejuni NCTC 11168
cmeB	78	0.02	0.02 (1)	0.06	0.06 (1)	0.03	0.03 (1)
cmeF	78	0.5	0.25 (2)	0.25	0.5 (0.5)	ND	ND
cmeR	313	0.5	1 (0.5)	0.125	0.5 (0.25)	ND	ND

0.25 MIC of EGCG.

Fourfold or greater differences in the MICs were considered significant and are indicated by bold. The numbers in parenthesis indicate the fold reductions in the MIC of the antimicrobials tested.

EGCG, (−)-epigallocatechin gallate.

EGCG resulted in a notable decrease in resistance to erythromycin, where it reduced the MICs by at least 2-fold to 32-fold in 19 of the 22 Campylobacter spp. tested (11 erythromycin resistant, 4 intermediate, and 4 erythromycin sensitive). Clear effects of EGCG on these erythromycin MICs were seen in particular for seven erythromycin-resistant and four intermediate isolates, where EGCG reduced the erythromycin resistance by at least 4-fold to 32-fold. Moreover, EGCG restored susceptibility to erythromycin in all of the intermediate resistance isolates (Table 4) and in one of the resistant isolates (Table 4; isolate 413/06).

The effects of EGCG on ciprofloxacin resistance in the selected Campylobacter spp. were much smaller in comparison to erythromycin. It reduced the ciprofloxacin MICs by at least 2-fold to 32-fold in 12 of 23 isolates (11 ciprofloxacin resistant and one ciprofloxacin sensitive). Additionally, EGCG restored ciprofloxacin susceptibility in one ciprofloxacin-resistant isolate (Table 4; isolate 137).

EGCG showed a modulatory activity also in combination with tetracycline, where in four of 15 (26%) tetracycline-resistant isolates, it decreased the MICs by fourfold to eightfold (Table 4).

Contributions of the CmeABC and CmeDEF efflux pumps to antimicrobial resistance

In studying the efflux mechanisms involved in these antimicrobial resistances of Campylobacter spp., the reference strain C. jejuni NCTC 11168 was assessed both with the EPIs PAßN and NMP, and also with its cmeB, cmeF, and cmeR mutants (Table 3). Both of these EPIs increased the susceptibility of this wild-type strain to erythromycin by fourfold. Additionally, PAßN reduced the ciprofloxacin MIC by twofold, although with tetracycline, no effects of the EPIs were seen. Moreover, EGCG reduced the erythromycin MIC by eightfold in this C. jejuni NCTC 11168 reference strain, whereas it had no effects on the susceptibility to ciprofloxacin (Table 4). On the other hand, insertional inactivation of the cmeB gene increased the susceptibility of this C. jejuni NCTC 11168 reference strain to the antimicrobials erythromycin (eightfold), ciprofloxacin (fourfold), and tetracycline (eightfold). In contrast, the inactivation of the cmeF and cmeR genes had no effects on the MICs of these antimicrobials.

In addition, both of the EPIs used here, PAßN and NMP, reduced the ciprofloxacin MICs by fourfold in the cmeB, cmeF, and cmeR mutants. PAßN and NMP also increased the susceptibility for erythromycin by 16-fold in the cmeF mutant, and NMP reduced the erythromycin MIC in the cmeR mutant. Unlike PAßN and NMP, EGCG did not reduce the ciprofloxacin MICs in these cmeB, cmeF, and cmeR mutants (Table 4), and it had only a small effect in the reduction of the erythromycin MIC in the cmeF mutant.

Discussion

Campylobacter is a leading food-borne pathogen, and it causes gastroenteritis in humans. This pathogenic organism is increasingly resistant to antimicrobials, especially to fluoroquinolones and macrolides, which are the most frequently used antimicrobials for treatment of campylobacteriosis when clinical treatment is warranted.²³ This problem of antimicrobial resistance induced us to select Campylobacter isolates from food, animal, water, and human clinical samples, and to assess these for resistance to erythromycin, ciprofloxacin, and tetracycline. We focused our investigation on mutations in region V of the 23S rRNA gene and on the presence of the tetO gene, to assess the involvement of the efflux mechanisms in antimicrobial resistance using EPIs. Furthermore, we also wanted to determine the roles of the bacterial drug efflux systems (CmeABC, CmeDEF, and CmeR) in resistance to erythromycin, ciprofloxacin, and tetracycline, so we compared the sensitivities of wild-type C. jejuni 11168 with C. jejuni 11168 with mutations in the specific efflux pump genes cmeB, cmeF, and cmeR.

In our study, only the A2075G mutation was detected in seven of the erythromycin HLR C. coli (erythromycin MIC>256 mg/L). As other studies have shown,^3,5,10,28 this mutation has been recognized as the most common mechanism for erythromycin resistance in C. jejuni and C. coli. Interestingly, no A2075G mutation was identified in two of the erythromycin HLR C. coli isolated from food and water, with a similar situation reported by Gibreel et al.¹⁰

All tetracycline-resistant Campylobacter isolates (MICs≥16 mg/L) carried the tetO gene, as amplified with PCR. The same has been reported from another study.⁶

Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents, as well as to antimicrobials. Efflux occurs due to the activity of membrane transporter proteins that are widely known as multidrug efflux systems. These are implicated in a variety of physiological processes other than drug efflux.^30,36 Today, it is still a challenging task to identify natural substrates and inhibitors, due mainly to the complexity of these multidrug efflux systems, and especially in Gram-negative bacteria. As observed in several other species of Gram-negative bacteria, RND efflux pumps confer resistance to various antimicrobials in Campylobacter spp. In this organism, the CmeABC and CmeDEF efflux systems are the primary RND efflux pumps that contribute to antimicrobial, dye, and detergent resistance, including for erythromycin, ciprofloxacin, and tetracycline resistance.^12,20,23 Inhibiting the efflux pumps using EPIs (e.g., PAßN and NMP) is a new approach to increasing the susceptibilities of campylobacters to different antimicrobials.

Our study provides more comprehensive and detailed information of the effects of PAßN and NMP on erythromycin, ciprofloxacin, and tetracycline resistance in Campylobacter isolates. The data obtained confirmed that both of these EPIs promote marked decreases in resistance to erythromycin. Similarly, Hannula and Hänninen¹³ reported about twofold to eightfold decreases in four erythromycin low-level resistant C. jejuni isolates in the presence of 100 mg/L NMP. Therefore, both of these EPIs indeed reduce the MICs of these susceptible isolates of Campylobacter spp. Our data indicate on the active role of efflux in erythromycin-sensitive and intermediate-resistance Campylobacter spp.

Additionally, PAßN and NMP increase the susceptibility of the erythromycin HLR isolates (MIC>256 mg/L). Of particular interest, two erythromycin HLR C. coli isolates from food (pig) and water (Table 2; MIC>512 mg/L; isolates VC 7114 and 803) were revealed to have no A2075G mutation, by PCR-RFLP and sequencing of the 23S rRNA gene fragment. Additional studies are needed here to confirm the suggestion that the main mechanism of erythromycin resistance in these cases will be through efflux. In erythromycin HLR isolates with the A2075G 23S rRNA mutation, the presence of efflux pump activities suggests synergism between these two drug-resistance mechanisms in Campylobacter spp.

The effects of PAßN and NMP on ciprofloxacin resistance in the Campylobacter spp. tested were much more limited in comparison to erythromycin, but were still remarkable. Significantly lower effects, or indeed no effects, of PAßN and NMP on ciprofloxacin resistance have been shown in other studies.^3,5,13 Additionally, noticeable effects of PAßN were clearly seen for all of the tetracycline-resistant isolates. Similar data have been reported for one tetracycline-resistant isolate of C. jejuni.^13,25 Another study, by Gibreel et al.,¹¹ also showed that inactivation of the cmeB gene resulted in complete restoration of tetracycline susceptibility, which suggested that the efflux mechanism here is mediated mainly by the CmeABC efflux pump, which would thus be the major contribution to acquired tetracycline resistance in C. jejuni.

Our data presented here show that both PAßN and NMP have EPI activities, and they confirm that there are differences in the actions of these inhibitors. This suggests differential competition with substrates for the binding sites on the efflux pumps in Campylobacter spp. PAßN is thought to have a dual mechanism of action, namely through substrate competition with potentiate antimicrobials for efflux pumps, and permeabilization of the outer membrane. However, NMP does not act through inhibition of the proton-motive force, but will also be active through competitive inhibition of the efflux pumps.³³ In the present study, the involvement of efflux mechanisms in erythromycin resistance was seen for all of the intermediate-resistance and for two HLR isolates through the use of PAßN and NMP. Conversely, in comparison to erythromycin, quite different susceptibility patterns were seen for ciprofloxacin and tetracycline. Efflux was involved in ciprofloxacin and tetracycline resistance in only some of the resistant isolates, which suggests that both of the EPIs, PAßN and NMP, are very effective in their competition with the antimicrobials tested. Irrespective of these interesting data, it appears that in the case of ciprofloxacin and tetracycline resistance, the effects of the EPIs are strain specific.

To date, only one EPI has been documented for use in the treatment of bacterial infections in human medicine.³⁶ The EPIs are usually of low specificity and are potentially toxic to the animal host,²⁴ which highlights the need for the exploration of novel approaches for inhibition of the efflux transporters. However, alternative compounds from plants with potential antimicrobial and/or resistance-modifying activities have a generally recognized as safe status, and as such, they can also be used to control the development of antimicrobial resistant bacteria in the food chain, and especially in meat.^17,27 In the present study, EGCG was thus evaluated for its antibacterial and efflux inhibitory activity against these Campylobacter spp. isolated from different sources and with determined resistances against erythromycin and ciprofloxacin.

EGCG resulted in a notable decrease in resistance to erythromycin. In comparison to PAßN and NMP, the effects of EGCG on erythromycin resistance were smaller, although they were still remarkable. As well as EGCG having high antimicrobial efficiencies against this Campylobacter spp., the subinhibitory EGCG concentrations used here are clearly demonstrated to also promote changes in erythromycin and ciprofloxacin activities. As seen here for the testing of the activities of this putative natural EPI in several sensitive and resistant isolates of Campylobacter spp., EGCG indeed shows effects on erythromycin- and ciprofloxacin-resistant and sensitive isolates, since EGCG appears to inhibit the efflux of both of these antimicrobials. This efficiency of EGCG shown in the present study represents the first evidence of modifying activities for this natural plant phenolic compound, as a resistance-modifying agent in Campylobacter spp. As a natural modulator of drug resistance, EGCG should extend the useful lifetime of conventional antimicrobials, such as erythromycin, ciprofloxacin, and tetracycline. Additionally, it should improve the treatment of MDR strains for which the majority of therapeutic antimicrobials have no further clinical use.

It has been suggested that use of EPIs is not an ideal approach for the assessment of the role of active efflux in conferring antimicrobial resistance in campylobacters, when compared to the insertional inactivation approaches, as also reported by Gibreel et al.¹¹ In the present study, we also used cmeB, cmeF, and cmeR knockout mutants of the C. jejuni NCTC 11168 reference strain. Here, this insertional inactivation of the cmeB gene resulted in a clear decrease in the MICs of erythromycin, ciprofloxacin, and tetracycline, thus confirming the involvement of efflux in these antimicrobial resistances. In contrast, inactivation of the cmeF and cmeR genes had no effects on the MICs of these antimicrobials. On the other hand, the availability of EPIs is useful to directly modulate the activities of the efflux pumps, thereby restoring a susceptible phenotype.³⁰ Additionally, our data show that both PAßN and NMP and the natural inhibitor EGCG can have an EPI activity in the wild-type strain NCTC 11168 as well as in the cmeB, cmeF, and cmeR mutants.

It is known that CmeDEF interacts with CmeABC in conferring antimicrobial resistance, and as shown by Akiba et al.,¹ we also confirm here that CmeABC is the predominant efflux pump in C. jejuni. Thus, CmeDEF has a secondary role in conferring intrinsic resistance to antimicrobials. In addition, both of the EPIs used here, PAßN and NMP, reduced the ciprofloxacin MICs in the cmeB and cmeF mutants, which suggests that other efflux pumps are involved in this resistance. However, as recently described by Jeon et al.,¹⁵ it is most likely that the CmeG efflux pump is at least mainly involved in ciprofloxacin resistance.

In conclusion, our study shows that erythromycin and tetracycline resistance can be largely explained through mutations in 23S rRNA and the presence of the tetO gene, respectively, the main described mechanisms among Campylobacter spp. However, in the case of erythromycin resistance, additional studies are required to confirm the synergy between efflux pumps and mutations in these ribosomal proteins, L4 and L22. Furthermore, we have confirmed that in erythromycin-resistant isolates, active efflux is provided by the predominant role of CmeABC along with a minor contribution from CmeDEF, which can act synergistically with previously mentioned mechanisms. The findings of the present study demonstrate the ability of two chemical EPIs, PAßN and NMP, and of the natural putative EPI EGCG, to partly reverse the drug resistance in Campylobacter, while also indicating the different mechanisms of action of these compounds. Collectively, our investigation has provided new information concerning the complexity of the efflux mechanisms, and some of the factors that affect the susceptibility to the antimicrobials. Furthermore, these data indicate new approaches for Campylobacter spp. risk management.

Footnotes

Acknowledgments

This work was supported by the Slovenian Research Agency (ARRS) through a Ph.D. grant to M.K., and partly through other projects, including postdoctoral grants to A.K. (Z1 2190), CRP V4-1079, and V4-1080.

Disclosure Statement

The authors declare that they have no competing financial interests.

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