Resistance to Bile Salts and Sodium Deoxycholate in Macrolide- and Fluoroquinolone-Susceptible and Resistant Campylobacter jejuni and Campylobacter coli Strains

Abstract

Campylobacter are the most commonly reported bacterial causes of human gastroenteritis, and they are becoming increasingly resistant to antibiotics, including macrolides and fluoroquinolones, those most frequently used for the treatment of campylobacteriosis. Active efflux mechanisms are involved in resistance of Campylobacter to a broad spectrum of antimicrobials, and are also essential for Campylobacter colonization in the animal intestine, through mediation of bile resistance. Acquisition of antibiotic resistance through resistance-conferring mutations can impose a fitness cost of Campylobacter. The aim of the present study was to determine whether macrolide and fluoroquinolone resistance in Campylobacter affects their tolerance to bile salts and sodium deoxycholate through the most frequent resistance-conferring mutations. Antimicrobial efflux was studied on the basis of restored sensitivity in the presence of the efflux-pump inhibitors (EPIs) phenylalanine–arginine beta-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine. In the 15 Campylobacter jejuni and 23 Campylobacter coli strains examined here, both of these EPIs partially reversed the resistance to bile salts and sodium deoxycholate. Erythromycin-sensitive C. coli strains were more resistant to bile salts and sodium deoxycholate than erythromycin-resistant strains. PAβN had greater effects on bile salt and sodium deoxycholate resistance in these erythromycin-resistant strains compared to erythromycin-sensitive strains. However, no differences were seen between the ciprofloxacin-sensitive and ciprofloxacin-resistant strains.

Introduction

C ampylobacter spp., and especially Campylobacter jejuni and Campylobacter coli, are the most commonly reported bacterial causes of food-borne gastroenteritis in humans worldwide. These bacteria are usually transmitted by contaminated food and drinking water.²² As well as their widespread occurrence, Campylobacter have become increasingly resistant to antibiotics, including the macrolides and fluoroquinolones (e.g., erythromycin and ciprofloxacin, respectively), the major drugs of choice for treatment of clinical campylobacteriosis, thus greatly compromising the effectiveness of these antibiotic treatments.^7,25

The chromosomal target mutations of the 23S rRNA gene (e.g., A2074C, A2074G, and A2075G) have been attributed to the greater part of macrolide resistance in Campylobacter.^9,11 In addition, the modifications in the rplD and rplV genes, which encode the ribosomal proteins L4 and L22, respectively, can also confer macrolide resistance.⁴ A single-point mutation in the quinolone-resistance-determining region (QRDR) of the gyrA gene, which encodes GyrA, a subunit of DNA gyrase, can lead to fluoroquinolone resistance in Campylobacter. However, there are many known mutations of gyrA, such as Thr86Ala, Ala70Thr, Thr86Lys, Asp90Asn, and Pro104Ser.^2,8 Double-point mutations of gyrA that combine Thr86Ile and Asp85Tyr or Asp90Asn or Pro104Ser have also been reported.⁸ However, the majority of fluoroquinolone-resistant C. jejuni and C. coli isolates have the C86T mutation on gyrA, which results in the Thr86Ile substitution.^8,24,29,30

Resistance acquired through chromosomal mutations has been shown to impose a fitness cost on the bacteria in the absence of antibiotic selection pressure, which can result in reductions in growth, virulence, colonization, and transmission.²⁶ Nevertheless, some resistant isolates have developed compensatory mutations that reduce the fitness cost associated with the acquisition of antibiotic resistance. In addition, some resistance-conferring mutations lead to no fitness cost, or even to enhanced fitness.^26,28 It has been shown that fluoroquinolone-resistant Campylobacter that carry the Thr86Ile mutation in gyrA can colonize chickens persistently, without losing the resistance phenotype and this resistance-associated mutation. Indeed, fluoroquinolone-resistant mutants can outcompete fluoroquinolone-susceptible strains when coinoculated in chickens, which suggests that fluoroquinolone-resistant mutants have enhanced fitness, which does not appear to be due to a compensatory mutation.¹⁹ Conversely, it was shown recently by Zeitouni and Kempf (2011)²⁷ that fluoroquinolone resistance can indeed impose a fitness cost in Campylobacter. In contrast to fluoroquinolone-resistant Campylobacter, erythromycin-resistant mutants show a fitness disadvantage when compared with erythromycin-sensitive wild-type strains, which has been linked to the resistance-conferring mutation in 23S rRNA.^18,28 The spontaneous compensatory mutation in the vicinity of the L16 ribosomal protein that evolved in vivo has resulted a colonization capacity of these mutants that is similar to the erythromycin-susceptible strains.²⁸ However, contrary evidence appears in the literature. In the study of Zeitouni et al. (2012),²⁸ macrolide-susceptible and resistant C. coli strains displayed similar levels of colonization, when inoculated in vivo in chickens, which suggest that macrolide resistance does not always impose a fitness cost in vivo.

The intestinal tract of the chicken is a natural host and a major reservoir for Campylobacter. Resistance to bile salts is thus essential for Campylobacter to survive in this intestinal environment. Active efflux is one of the resistance mechanisms used by Campylobacter to resist the bactericidal effects of bile.^12,16 It has been shown that the CmeABC major resistance-nodulation-cell division (RND) efflux system is essential for Campylobacter colonization in the animal intestine, through mediation of bile salt resistance.¹² The expression of this efflux pump is under the control of the transcriptional repressor CmeR that binds directly to the cmeABC promoter¹⁴ and is induced by the natural substrates of this efflux pump, the bile salts. Thus, the bile salts interact directly with CmeR, leading to its reduced binding to the cmeABC promoter and increased transcription of the efflux genes.¹⁵ Moreover, bile salts also induce the CmeR-dependent gene Cj0561c, which is strongly suppressed by the CmeR repressor and is essential for efficient poultry colonization.^6,13

A second RND efflux system, CmeDEF, has been identified in Campylobacter, which is functionally distinct from CmeABC. Despite the difference in their expression and contributions to the intrinsic antimicrobial resistance, both efflux pumps appear to function interactively, and the activities of both are required for optimal cell viability.¹

The aim of this study was to determine whether the macrolide and fluoroquinolone resistance through the most frequent mutations (A2075G and Thr86Ile, respectively) can affect the resistance to bile salts and the efflux mechanisms that are essential for bile salt resistance. The efflux was studied on the basis of restored sensitivity in the presence of the efflux-pump inhibitors (EPIs) phenylalanine–arginine beta-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP).

Materials and Methods

Bacterial strains and growth conditions

Thirty-eight C. jejuni and C. coli strains isolated from animals (poultry and pig), humans, and environmental water were used in this study, which were selected on the basis of the different antimicrobial resistances determined in our previous study.²¹ The cultures were stored at −80°C in a brain–heart infusion broth (Biolife) with 20% horse blood (Oxoid) and 20% glycerol (Kemika). The isolates were cultivated at 42°C under microaerophilic conditions (3% O₂, 10% CO₂, and 87% N₂) in gas-tight containers on Karmali or Columbia agar supplemented with 5% horse blood (Oxoid). The antibiotic resistance breakpoints were defined according to the recommendations of the Clinical and Laboratory Standards Institute.⁵ Strains with minimal inhibitory concentrations (MICs) of erythromycin >32 mg/L and MICs of ciprofloxacin >4 mg/L were defined as erythromycin resistant and ciprofloxacin resistant, respectively. The reference strains were used as the antibiotic susceptibility test controls: C. jejuni NCTC11168, C. jejuni ATCC33560, and C. coli ATCC33559.

Detection of mutations in the 23S rRNA gene by restriction fragment length polymorphism analysis

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol was used to detect the mutations at position 2075 of the 23S rRNA gene, as described by previously.^11,17 The DNA was extracted using QIAamp DNA mini kits (Qiagen). The primers used to amplify the fragments of the 23S rRNA gene are listed in Table 1. The 23S rRNA amplicons were digested with the BsmAI restriction enzyme (Fermentas). The fragments were separated on 2.5% agarose gels. The molecular sizes of the digests were determined by comparison with the molecular marker PCR 20 bp Low Ladder (Sigma) using Quantity One 1-D Analysis software, version 4.0.3.

Table 1.

Primers Used in This Study

Primer	Sequence	Amplification	Reference
Ery23SFwd	5′-GTAAACGGCCGTAACTA-3′	714-bp-long fragment of the 23S rRNA in Campylobacter spp.	Leser et al. (1997)¹⁷
Ery23SRev	5′-GACCGAACTGTCTCA CGACG-3′
GZgyrA4	5′-CAGTATAACGCATCGCAGCG-3′	368-bp-long fragment of gyrA in C. jejuni—positive control of MAMA-PCR	Zirnstein et al. (1999)²⁹
CampyMAMAgyrA1	5′-TTTTTAGCAAAGATTCTGAT-3′
CampyMAMAgyrA1	5′-TTTTTAGCAAAGATTCTGAT-3′	265-bp-long fragment of gyrA with C86T mutation in C. jejuni	Zirnstein et al. (1999)²⁹
CampyMAMAgyrA5	5′-CAAAGCATCATAAACTGCAA-3′
CampyMAMAgyrA1	5′-TTTTTAGCAAAGATTCTGAT-3′	265-bp-long fragment of gyrA in C. jejuni—negative control of MAMA-PCR (absence of C86T mutation in gyrA)	Zorman (2004)³¹
CampygyrJEJwild	5′-CAAAGCATCATAAACTGCAC-3′
GZgyrCcoli3F	5′-TATGAGCGTTATTATCGGTC-3′	505-bp-long fragment of gyrA in C. coli—positive control of MAMA-PCR	Zirnstein et al. (2000)³⁰
GZgyrCcoli4R	5′-GTCCATCTACAAGCTCGTTA-3′
GZgyrCcoli3F	5′-TATGAGCGTTATTATCGGTC-3′	192-bp-long fragment of gyrA with C86T mutation in C. coli	Zirnstein et al. (2000)³⁰
CampyMAMAgyrA8	5′-TAAGGCATCGTAAACAGCCA-3′
GZgyrCcoli3F	5′-TATGAGCGTTATTATCGGTC-3′	192-bp-long fragment of gyrA in C. coli—negative control of MAMA-PCR (absence of C86T mutation in gyrA)	Zorman (2004)³¹
CampygyrCOLIwild	5′-TAAGGCATCGTAAACAGCCG-3′

MAMA-PCR, mismatch amplification mutation assay PCR.

Detection of mutations in the gyrA gene by mismatch amplification mutation assay PCR

The resistance to ciprofloxacin was confirmed by mismatch amplification mutation assay (MAMA) PCR detection of mutations in the QRDR of the gyrA gene, using the primers as described previously.^29,30,31 The primers used to amplify the QRDR of the gyrA sequences in C. jejuni and C. coli are listed in Table 1.

Antimicrobial susceptibility testing

MICs of bile salts and sodium deoxycholate (Sigma-Aldrich) were determined using the broth microdilution method. This was carried out in the Mueller Hinton broth (Oxoid) with inocula of 10⁶ bacteria/ml using 96-well microtiter plates. Twofold serial dilutions of bile salts and sodium deoxycholate were used at concentrations 0.5–64 mg/ml. The microtiter plates were incubated for 24 hr at 42°C under microaerophilic conditions. The MICs were defined as the lowest concentration where no viable cells were present, and they were determined on the basis of fluorescent signals measured using a microplate reader (Tecan; excitation/emission wavelength 550/595 nm) after the addition of 20 μl CellTiter-Blue Reagent (Promega) to the culture medium, following the manufacturer's instructions. The assays were repeated twice in duplicate, to confirm the reproducibility of the MIC data.

Effects of the EPIs

The MICs of the bile salts and sodium deoxycholate were determined with the broth microdilution method, as described above, in the presence of two EPIs: PAβN (Sigma-Aldrich) and NMP (Chess). For this purpose, the Mueller Hinton broth was supplemented with PAβN or NMP to the final concentrations of 10 and 60 mg/L, respectively. Microdilution tests were also performed in preliminary independent experiments to determine the MICs of PAβN and NMP for all of the strains tested here. The concentrations of 10 mg/L PAβN and 60 mg/L NMP alone had no inhibitory effects on bacterial growth for any of these strains.

Statistical analysis

IBM PASW Statistic software, version 18.0., was used for statistical analyses. The MICs of the bile salts and sodium deoxycholate and the effects of the EPIs were compared with the independent-sample T-test to determine the significance of the differences in resistances between the erythromycin- or ciprofloxacin-sensitive and erythromycin- or ciprofloxacin-resistant strains, as well as between C. jejuni and C. coli. The correlations of the antimicrobial MIC distributions and the effects of the EPIs were compared by Pearson χ² tests. A Pearson coefficient was calculated for the correlation matrix between the bile salt or sodium deoxycholate MICs and the antibiotic MICs, and between the effects of the EPIs and the antibiotic MICs. The data were considered significant when p≤0.05.

Results

Target mutation in the 23S rRNA

The PCR-RFLP procedure was used to detect the A2075G mutation in the 23S rRNA. After BsmAI digestion, this mutation at position 2075 led to five fragments of 311, 226, 102, 57, and 18 bp. Eight of the 38 Campylobacter strains tested (21.1%) belonged to the erythromycin-resistant group and showed the A2075G mutation (Table 2). The A2075G mutation was not identified in any of the erythromycin-sensitive strains.

Table 2.

Minimal Inhibitory Concentrations of Bile Salts and Sodium Deoxycholate in the Absence and Presence of Phenylalanine–Arginine Beta-Naphthylamide and 1-(1-Naphthylmethyl)-Piperazine

	MICs
	Bile salts (mg/ml)			Sodium deoxycholate (mg/ml)
Strain (source)	−EPI	+PAβN	+NMP	−EPI	+PAβN	+NMP	ERY (mg/L)	CIP (mg/L)	Target mutation of 23S rRNA	Target mutation of gyrA
C. jejuni (poultry)
158	16	16	16	32	16	16	0.5	8	–	C86T
K49/4	64	64	64	128	128	128	0.5	1	–	–
K34/1	16	16	16	8	8	8	0.5	8	–	C86T
C. coli (poultry)
137	32	32	32	64	64	32	1024	16	A2075G	C86T
171	32	0.5	8	16	16	8	512	16	A2075G	C86T
140	32	0.5	8	16	16	16	1024	32	A2075G	C86T
K27/4	64	64	64	32	32	1	2	16	–	C86T
K30/2	32	4	4	8	4	4	0.5	4	–	–
K32/2	16	4	8	16	16	16	2	16	–	C86T
K33/1	64	64	64	128	128	128	0.5	4	–	–
M37	32	16	32	32	16	0.5	1024	0.25	A2075G	–
C. jejuni (human)
6272-05	64	64	64	64	64	64	0.5	8	–	C86T
375-06	16	0.5	8	8	8	1	512	16	A2075G	C86T
481	128	128	128	128	64	64	0.5	16	–	C86T
3552	64	64	64	32	32	16	1	128	–	C86T
413-06	64	64	64	32	32	16	0.25	0.25	–	–
C. coli (human)
557	64	64	64	32	16	16	0.25	4	–	–
6553-05	16	0.5	4	2	0.5	0.5	1024	32	A2075G	C86T
2235	32	0.5	0.5	32	0.5	1	512	0.25	A2075G	–
3341-05	16	16	8	16	0.5	1	0.25	0.125	–	–
10162	64	32	32	64	32	64	0.125	32	–	–
10522	32	32	32	64	32	64	0.125	32	–	–
C. coli (pig)
VC7114	16	0.5	8	1	1	0.5	1	8	–	C86T
VC11076	32	32	32	32	16	16	1024	64	A2075G	C86T
VC110722	64	64	64	32	32	32	4	128	–	C86T
VC110725	64	32	64	16	8	2	2	0.25	–	–
C. jejuni (water)
653	32	32	32	4	4	4	0.5	0.125	–	–
807	64	64	64	8	1	4	0.5	2	–	–
850	32	32	16	64	64	64	0.25	0.125	–	–
2782	64	64	64	128	128	128	1	0.125	–	–
1845	32	32	32	8	8	4	0.5	4	–	–
C. coli (water)
803	16	8	8	32	32	16	0.25	0.125	–	–
652/1	32	16	16	32	16	16	0.125	32	–	–
944	16	8	8	128	128	128	0.5	0.125	–	–
655	64	64	32	64	64	64	1	16	–	C86T
Reference strains
NCTC 11168 C. jejuni	64	64	64	32	32	32	0.125	0.25	–	–
ATCC 33560 C. jejuni	64	64	64	64	64	32	1	1	–	–
ATCC 33559 C. coli	64	64	64	64	64	32	2	0.063	–	–

MICs determined with the broth microdilution method. MIC changes of at least fourfold are indicated in bold.

+PAβN, 10 mg/L; +NMP, 60 mg/L.

–, absence of target mutation; MIC, minimal inhibitory concentration; EPI, efflux-pump inhibitor, PAβN, phenylalanine–arginine beta-naphthylamide; NMP, 1-(1-naphthylmethyl)-piperazine; ERY, erythromycin; CIP, ciprofloxacin.

Target mutation in the gyrA gene

The MAMA-PCR assay was used to detect the C86T mutation in the gyrA gene. Fifteen ciprofloxacin-resistant strains out of the 38 strains tested (39.5%) showed the C86T mutation (Table 2). The C86T mutation was not present in four further ciprofloxacin-resistant strains and in any of the ciprofloxacin-sensitive strains.

Antimicrobial susceptibility

In total, 38 animal, human, and environmental water C. jejuni and C. coli isolates were tested for susceptibility to bile salts and sodium deoxycholate. The results from the MIC testing are presented in Table 2. The distributions of the MICs in C. jejuni and C. coli from this testing are presented in Figure 1A and B. No statistically significant differences in resistances to bile salts and sodium deoxycholate were found between C. jejuni and C. coli (T-test, p=0.103 and p=0.527, respectively).

FIG. 1.

(A, B) Distribution of the bile salt and sodium deoxycholate minimal inhibitory concentrations (MICs) in C. jejuni and C. coli. (C, D) Distribution of the bile salt and sodium deoxycholate MICs in Campylobacter strains with different antibiotic resistance levels.

In these tests, 30 (78.9%) erythromycin-sensitive and 8 (21.1%) erythromycin-resistant, and 19 (50%) ciprofloxacin-sensitive and 19 (50%) ciprofloxacin-resistant strains were included, as determined in our previous study.²¹ The MIC distributions from the MIC testing in those sensitive and resistant strains are presented in Figure 1C and D. The erythromycin-sensitive strains were more resistant to bile salts (T-test, p=0.001) and sodium deoxycholate (T-test, p=0.03) than the erythromycin-resistant strains. However, there were no statistically significant differences in the bile salt and sodium deoxycholate resistances between the ciprofloxacin-sensitive and ciprofloxacin-resistant strains, as well as between the strains with or without the Thr86Ile mutation (T-test, p>0.05).

A Pearson correlation matrix was calculated for the distributions of the MICs of the bile salts and sodium deoxycholate and the MICs of erythromycin and ciprofloxacin. A statistically significant negative correlation was seen for MICs of bile salts and erythromycin (p=0.049, r_xy=−0.322), which additionally confirmed the results of the independent-sample T-test. No statistically significant correlations were observed for sodium deoxycholate and erythromycin, as well as for ciprofloxacin (p>0.05).

Effects of NMP and PAβN on the bile salt and sodium deoxycholate resistance

The efficiencies of the EPIs PAβN and NMP were determined for each strain, in terms of the bacterial resistance to bile salts and sodium deoxycholate. The MICs from these experiments are presented in Table 2 and the effects of these EPIs in Table 3.

Table 3.

Effects of Efflux Pump Inhibitors Phenylalanine–Arginine Beta-Naphthylamide and 1-(1-Naphthylmethyl)-Piperazine on Bile Salt and Sodium Deoxycholate Resistance in Campylobacter jejuni and Campylobacter coli and in Strains with Different Antibiotic Resistance Levels

	PAβN (10 mg/L)			NMP (60 mg/L)
Species	MIC reduction	n/N	(%)	MIC reduction	n/N	(%)
Bile salts
C. jejuni	32	1/15	(6.7)	2	2/15	(13.3)
C. coli	2–64	13/23	(56.5)	2–64	13/23	(56.5)
Erythromycin-sensitive	2–32	8/30	(26.7)	2–8	11/30	(36.7)
Erythromycin-resistant	2–64	6/8	(75)	4–64	4/8	(50)
Ciprofloxacin-sensitive	2–64	6/19	(31.6)	2–64	6/19	(31.6)
Ciprofloxacin-resistant	2–64	8/19	(42.1)	2–4	9/19	(47.4)
Total	2–64	14/38	(36.8)	2–64	15/38	(39.5)
Sodium deoxycholate
C. jejuni	2–8	3/15	(20)	2–8	8/15	(53.3)
C. coli	2–64	11/23	(47.8)	2–64	15/23	(65.2)
Erythromycin-sensitive	2–32	8/30	(26.7)	2–32	16/30	(53.3)
Erythromycin-resistant	2–64	5/8	(62.6)	2–64	7/8	(87.5)
Ciprofloxacin-sensitive	2–64	6/19	(31.6)	2–64	12/19	(63.2)
Ciprofloxacin-resistant	2–4	7/19	(36.8)	2–32	11/19	(57.9)
Total	2–64	13/38	(34.2)	2–64	23/38	(60.5)

n/N, number of strains/number of total strains tested.

According to Pearson χ² tests, there were no statistically significant correlations (p>0.05) between the distributions of the effects of both the EPIs and the MICs of the bile salts and sodium deoxycholate. However, PAβN had greater effects on bile salt resistance in the erythromycin-resistant strains than in the erythromycin-sensitive strains (T-test, p=0.022). There were no other statistically significant differences in the effects of both of the EPIs between erythromycin-sensitive and erythromycin-resistant strains, between ciprofloxacin-sensitive and ciprofloxacin-resistant strains, and between C. jejuni and C. coli (T-tests, p>0.05).

A Pearson correlation matrix was also calculated for the distributions of the effects of both the EPIs and the MICs of erythromycin and ciprofloxacin. There were statistically significant correlations for the effects of PAβN for bile salt and sodium deoxycholate resistance and for the MICs of erythromycin (p=0.001, r_xy=0.508, and p=0.025, r_xy=0.364, respectively). No statistically significant correlations were observed for ciprofloxacin.

Discussion

The fitness cost of macrolide and fluoroquinolone resistance in Campylobacter has been well documented. Studies assessing the fitness of drug-resistant Campylobacter have been conducted in vitro in culture media and food matrices, and in vivo in the chicken digestive tract.^{8,18,19,27,28} Both in vitro culturing and chicken colonization studies have suggested that macrolide resistance incurs an obvious fitness cost in C. jejuni.^10,18,28 This fitness disadvantage was linked to resistance-conferring mutations in the 23S rRNA.¹⁸ Conversely, fluoroquinolone-resistant Campylobacter mutants did not carry such a fitness burden or even outcompete fluoroquinolone-susceptible strains when coinoculated in chickens.¹⁹ Still, contrary evidence has appeared in the literature. Zeitouni and Kempf (2011)²⁷ reported that acquisition of fluoroquinolone resistance through the mutation in the gyrA gene might or might not impose a fitness cost on C. jejuni and C. coli, depending on the conditions of the study, and the environment, and the bacterial species. However, the exact mechanisms involved in this competition are not known.

As bile salt resistance is essential for in vivo adaptation of Campylobacter in the intestinal tract,^12,16 in this study, we examined the resistance to bile salts and sodium deoxycholate of macrolide and fluoroquinolone-resistant and susceptible Campylobacter strains. As well as the efflux mechanisms, and particularly the RND efflux pumps, CmeABC and CmeDEF, involved in the bile salt resistance, we also determined the effects on these resistances of two EPIs, PAβN and NMP, which are considered to inhibit the RND type of efflux pumps.^20,23 The present study indicates that erythromycin-resistant Campylobacter strains show lower tolerance to bile salts and sodium deoxycholate. Moreover, correlations that were found for the effects of PAβN on bile salt and sodium deoxycholate resistance and on erythromycin resistance levels demonstrate that erythromycin resistance affects the efflux mechanisms involved in bile salt resistance. These results might explain the previous findings relating to the impaired fitness in macrolide-resistant Campylobacter isolates.^10,18,28 However, the majority of erythromycin-resistant strains, tested in our study, belong to C. coli species, for which the in vivo biological cost of macrolide resistance was not observed.²⁸ This indicates that macrolide resistance through the mutation in the 23S rRNA gene might or might not impose a fitness cost on Campylobacter, as it was found for fluoroquinolone resistance.²⁷ A more detailed picture of the influence of this resistance-conferring mutation on the bile salt resistance in Campylobacter might emerge when performing antimicrobial susceptibility testing in a larger amount of erythromycin-resistant C. jejuni isolates, covering also different environmental conditions (e.g., pH of environment), since these might significantly affect the activity of efflux systems in bacteria.³

Conversely, fluoroquinolone resistance in Campylobacter does not impact on the resistance to bile salts and sodium deoxycholate, nor on the efflux pump activity involved in this resistance. These findings are consistent with the previous report where fluoroquinolone-resistant and fluoroquinolone-sensitive Campylobacter isolates showed equal resistance levels to cholic acid and produced equal amounts of the CmeABC protein.¹⁹

The mechanisms underlying the adaptation of Campylobacter in the intestinal tract are still largely unknown and will be the subject of further investigations. Understanding precisely how resistance mutations affect the adaptation of Campylobacter in the animal host will help to predict and control the emergence and spread of these antibiotic-resistant Campylobacter.

Footnotes

Acknowledgment

The authors thank the Slovenian Research Agency for a Ph.D. grant to A.M. that enabled this study on Campylobacter spp.

Author Disclosure Statement

The authors declare that they have no competing financial interests.

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