# Molecular Basis of Macrolide Resistance inCampylobacter Strains Isolated from Poultry in South Korea

Authors: Bai Wei; Min Kang
Journal: BioMed Research International
 (2018)

---

## Abstract

We investigated the molecular mechanisms underlying macrolide resistance in 38 strains ofCampylobacter isolated from poultry. Twenty-seven strains were resistant to azithromycin and erythromycin, five showed intermediate azithromycin resistance and erythromycin susceptibility, and six showed azithromycin resistance and erythromycin susceptibility. FourCampylobacter jejuni and sixCampylobacter coli strains had azithromycin MICs which were 8–16 and 2–8-fold greater than those of erythromycin, respectively. The A2075G mutation in the 23S rRNA gene was detected in 11 resistant strains with MICs ranging from 64 to ≥ 512 μg/mL. Mutations including V137A, V137S, and a six-amino acid insertion (114-VAKKAP-115) in ribosomal protein L22 were detected in theC. jejunistrains. Erythromycin ribosome methylase B-erm(B) was not detected in any strain. All strains except three showed increased susceptibility to erythromycin with twofold to 256-fold MIC change in the presence of phenylalanine arginine ß-naphthylamide (PAßN); the effects of PAßN on azithromycin MICs were limited in comparison to those on erythromycin MICs, and 13 strains showed no azithromycin MIC change in the presence of PAßN. Differences between azithromycin and erythromycin resistance and macrolide resistance phenotypes and genotypes were observed even in highly resistant strains. Further studies are required to better understand macrolide resistance inCampylobacter.


---

## Body

## 1. Introduction

Infection withCampylobacter spp. is considered to be the most common cause of bacterial gastroenteritis in humans worldwide. Macrolides are considered the first drug of choice for treatingCampylobacter gastroenteritis. Resistance to macrolides has been reported in a few scattered clinical isolates ofCampylobacter across the world. High prevalence of macrolide-resistantCampylobacter spp., especiallyC. coli, in animal meat has been reported [1], and this finding is of concern because of the risk of transmission of such isolates to human.Modification of the antibiotic target genes via methylation or mutation, and efflux of antibiotics from bacterial cells could induce macrolide resistance [2]. The most important macrolide resistance mechanism inCampylobacter involves the modification of ribosomal target sites and weakening of the interaction between the tunnel wall of the ribosome and the macrocyclic ring of the macrolide [3]. Point mutations in domain V of the 23S rRNA at positions 2,074 and 2,075 are the most common mechanisms for high-level macrolide resistance inCampylobacter spp. [4]. Several modifications in the ribosomal proteins L4 and L22 are associated with low- to intermediate-level macrolide resistance inCampylobacter [4]. The chromosomally encoded multidrug resistance-nodulation-cell division (RND) efflux system is involved in intrinsic and acquired macrolide resistance inCampylobacter spp. [4]. A ribosomal methylase, encoded by the erythromycin ribosome methylase B-erm(B) gene, located in the chromosomal multidrug resistance genomic island (MDRGI) inC. coli from swine, was reported for the first time in China in 2014 [5]. Subsequently, several reports emerged oferm(B)-harboringC. jejuni in animal meat including that of swine and chicken and in human diarrheal samples [6, 7]. Outside China, anerm(B)-positiveC. coli strain was isolated from chicken in Spain in 2016 [8].Macrolides are a class of natural or semisynthetic products comprising a large macrocyclic lactone ring to which one or more deoxy sugars are attached [2]. The lactone rings can be either 14-membered (clarithromycin, dirithromycin, erythromycin, and roxithromycin), 15-membered (azithromycin), or 16-membered (josamycin, kitasamycin, spiramycin, and tylosin) [2]. In general, modification of ribosomal targets and drug efflux confer cross-resistance to macrolides. During antibiotic treatment in clinical settings, erythromycin and azithromycin are widely used because they have a broad spectrum of activity not only against Gram-positive bacteria, but also against Gram-negative bacteria [9]. Azithromycin is more potent than erythromycin against Gram-negative bacteria includingCampylobacter, with a lower MIC [9]. However, azithromycin shows a higher MIC than erythromycin against Gram-positive bacteria and rarely acts against Gram-negative bacteria [10, 11]. The diverse mechanisms underlying resistance to erythromycin and azithromycin continue to be unclear.It is well known that the handling and/or consumption of chicken meat are the main causes of human infection withCampylobacter. Other poultry sources also pose a similar threat to human health. Even though the vertical transmission ofCampylobacter is questionable, breeder chicken harboring antibiotic-resistant bacteria could be a public health threat as they can horizontally transmit antibiotic-resistantCampylobacter to broiler chicken in the production chain, indirectly leading to human infection [12]. With the increasing consumption of duck meat around the world and worldwide reports of duck-related product-induced human campylobacteriosis, researchers are focusing more on antibiotic-resistantCampylobacter found in duck [13, 14]. Even though the occurrence of highly macrolide-resistantCampylobacterin breeder chicken and macrolide-resistantCampylobacter in duck meat has been reported [15, 16], the causes of macrolide resistance in these species have rarely been reported. Therefore, we investigated the genetic basis of macrolide resistance inCampylobacter from poultry sources including breeder chicken and chicken and duck meat and identified the isolated strains asC. jejuni andC. coli; these strains showed different levels of resistance to azithromycin and erythromycin, evaluated using molecular methods. Additionally, we sought to investigate the diverse mechanisms underlying the higher resistance to azithromycin, but not to erythromycin, shown by theCampylobacter strains.


## 2. Materials and Methods

### 2.1. Origin ofCampylobacter Strains and Minimal Inhibitory Concentrations (MIC) Determination

A total of 38 strains ofCampylobacter (15 strains ofC. jejuni and 23 strainsC. coli) showing either intermediate resistance or resistance to azithromycin and erythromycin were used in this study (Tables 1 and 2). The strains were isolated from poultry between 2013 and 2016 in a previous study [16]. The sample sources were divided into four types, and the number of strains collected from each source was as follows: (1) in 176 feces samples from breeder chicken farms, 88 isolates ofCampylobacter were collected and 17 strains (oneC. jejuni and 16C. coli) showed resistance to azithromycin; (2) in 1,003 samples (feces and environmental samples) from broiler chicken farms, 55 isolates were collected and none of them showed resistance to azithromycin or erythromycin; (3) in 249 chicken meat samples from retail markets, 104 isolates were collected and 15 strains (10C. jejuni and fiveC. coli) showed resistance to azithromycin; and (4) in 106 duck meat sample from retail markets, 102 isolates were collected and six strains (fourC. jejuni and twoC. coli) showed resistance to azithromycin. The MICs of azithromycin and erythromycin were determined using agar or broth dilution methods and the breakpoints were as defined by the National Antimicrobial Resistance Monitoring System (NARMS) for azithromycin: susceptible, ≤ 2 μg/mL; intermediate, 4 μg/mL; and resistant, ≥ 8 μg/mL. The MIC breakpoints for erythromycin were susceptible, ≤ 8 μg/mL; intermediate, 16 μg/mL; and resistant, ≥ 32 μg/mL [17]. The reference strainC. jejuni ATCC 33560 was used as the quality control strain.Table 1
Characteristics of macrolide resistance genes inCampylobacter jejuni strains showing varied resistance to azithromycin and erythromycina.
Strain Source MIC (μg/mL) c m e B c e r m B c Mutations L22insertions Azithromycin Erythromycin 23SrRNA L4 L22 -EPI +EPI F o l d b -EPI +EPI F o l d b 33560 ATCC 0.25 0.06 4 1 0.25 4 nt nt wt wt wt wt A16-CF-329-3 CM 4 0.5 8 0.5 0.5 1 + - wt V196A A103V, S109A wt CM13-HL-010-1 CM 4 0.125 32 8 0.25 32 + - wt V80I, V196A A103V, S109A, V137A wt CM13-HL-012-1 CM 4 0.5 8 8 0.125 64 + - wt V80I, V196A A103V, S109A, V137A wt CM13-MWC-BS-004 CM 8 0.125 64 0.5 0.025 20 + - wt V196A A103V, S109A, V137A wt A16-CF-127-1-S1 BC 8 0.25 32 1 0.25 4 + - wt wt wt wt A16-CF-318-3 CM 8 0.25 32 1 0.015 67 + - wt V121A, T177S, M192I, V196A V137S 114VAKKAP115 DM13-MP-WS-002 DM 8 0.5 16 64 0.25 256 + - wt V196A A103V, S109A, V137A wt CM13-OP-004-1 CM 8 0.25 32 64 0.5 128 + - wt wt wt wt A16-CF-129-1-S1 CM 8 0.125 64 128 0.5 256 + - wt wt wt wt CM13-HL-BS-021 CM 16 1 16 32 2 16 + - wt V196A wt wt DM13-JDW-WL-007 DM 16 0.25 64 64 0.5 128 - - wt V196A V137A 114VAKKAP115 DM13-JDW-WL-009 DM 16 16 1 64 64 1 + - wt V121A, T177S, M192I, V196A S109A, V137A 114VAKKAP115 CM13-MWC-WL-008 CM 16 1 16 64 1 64 + - wt V196A A103V, S109A, V137A wt CM13-DW-BL-005 CM 16 1 16 64 1 64 + - wt V196A I65V, A103V, S109A, V137A wt DM13-JW-SS-017 DM 32 0.5 64 64 0.5 128 + - wt V121A, V196A I65V, A103V, S109A, V137A wt a. Abbreviations: ATCC, American Type Culture Collection; CM, chicken meat; BC, breeder chicken; DM, duck meat; EPI: efflux pump inhibitor; nt: not test; A: Ala; I: Ile; K: Lys; M: Met; P: Pro; S: Ser; V: Val; T: Thr; wt: wild type. b. Fold values were calculated as MIC without EPI/MIC with EPI. c. +/-, indicates whether or not the target genes were detected. The MIC breakpoints for azithromycin: susceptible, ≤ 2μg/mL; intermediate, 4 μg/mL; and resistant, ≥ 8 μg/mL; for erythromycin: susceptible, ≤ 8 μg/mL; intermediate, 16 μg/mL; and resistant, ≥ 32 μg/mL.Table 2
Characteristics of macrolide-resistance genes inCampylobacter coli strains showing varied resistance to azithromycin and erythromycina.
Strain Source MIC (μg/mL) cmeB c ermB c Mutations Azithromycin Erythromycin 23S rRNA L4 L22 -EPI +EPI Foldb -EPI +EPI Foldb A16-CF-319-1 CM 4 1 4 1 0.5 2 - - wt wt wt A16-CF-330-1 CM 4 1 4 1 0.5 2 - - wt V196A I65V, A74G A16-CF-130-1-S1 BC 8 8 1 2 0.5 4 + - wt wt I65V, A74G A16-CF-128-1-S1 BC 8 8 1 128 64 2 - - wt V121A, V196A I65V, A74G CM13-HL-BS-020 CM 16 0.5 32 32 0.5 64 - - wt V196A I65V, A74G CM13-HL-BL-031 CM 16 1 16 64 0.25 256 - - wt V196A I65V, A74G CM13-MC-BS-006 CM 16 0.125 128 64 2 32 - - wt V196A I65V, A74G DM13-MWC-SS-013 DM 32 2 16 4 0.125 32 - - wt V196A I65V, A74G DM13-MP-WS-007 DM 32 0.5 64 8 0.5 16 - - wt V196A I65V, A74G A16-CF-130-2-S1 BC 64 64 1 32 32 1 - - wt V196A I65V, A74G A16-CF-107-2-S2 BC 64 64 1 64 32 2 - - A2075G wt I65V, A74G, S109A A16-CF-128-1-S4 BC 128 2 64 128 4 32 - - wt V196A I65V, A74G, S109A A16-CF-124-2-S3 BC 128 4 32 128 2 64 - - wt V196A I65V, A74G, S109A A16-CF-107-2-S4 BC ≥512 64 8 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-124-1-S1 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-124-1-S4 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-126-2-S1 BC ≥512 64 8 ≥512 32 16 - - A2075G wt I65V, A74G, S109A A16-CF-124-1-S5 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-126-2-S2 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G V196A I65V, A74G, S109A A16-CF-126-2-S4 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G V121A, V196A I65V, A74G, S109A A16-CF-118-2-S3 BC ≥512 ≥512 1 ≥512 64 8 + - A2075G V121A, V196A I65V, A74G, S109A A16-CF-126-1-S3 BC ≥512 ≥512 1 ≥512 32 16 + - A2075G V121A, V196A I65V, A74G, S109A A16-CF-126-2-S3 BC ≥512 ≥512 1 ≥512 32 16 + - A2075G V121A, V196A I65V, A74G, S109A a. Abbreviations: CM: chicken meat; BC: breeder chicken; DM: duck meat; EPI: efflux pump inhibitor; A, Ala; G: Gly; I: Ile; S: Ser; V: Val; wt: wild type. b. Fold values were calculated as (MIC without EPI)/(MIC with EPI). c. +/-, indicates whether or not the target genes were detected. The MIC breakpoints for azithromycin: susceptible, ≤ 2μg/mL; intermediate, 4 μg/mL; and resistant, ≥ 8 μg/mL; for erythromycin: susceptible, ≤ 8 μg/mL; intermediate, 16 μg/mL; and resistant, ≥ 32 μg/mL.


### 2.2. Characterization of Macrolide Resistance inCampylobacterStrains

Genomic DNA templates for PCR were prepared using freshCampylobacter colonies on 5 % sheep blood agar plates (Komed, Seongnam, South Korea) by adding 100 μl sterile distilled water and boiling in a heater block at 100°C for 15 min. Mutations at positions 2,074 and 2,075 of the domain V of 23S rRNA gene were analyzed by sequencing all three copies of 23S rRNA. Three separate reactions were employed to amplify the three copies of the 23S rRNA gene in allC. jejuni andC. coli strains [3]. Subsequently, potential macrolide resistance-associated mutations were identified by sequencing a 308-bp fragment from each copy of the target gene [3].In addition, to assess the contribution of mutations within the ribosomal genesrplD andrplV encoding L4 and L22, respectively, to macrolide resistance, sequence analysis of these genes was performed for all 38 strains. L4- and L22-encoding genes were amplified as previously described [18]. The presence of the recently reported macrolide resistance-related ribosomal RNA methylase gene,erm(B), was confirmed using a method described by Zhang et al. [6]. To investigate the role of drug efflux in macrolide resistance,cmeB,an efflux pump gene (1,070 bp), was amplified using the method described by Pumbwe et al. [19].PCR products were purified using JET-SORB gel extraction kit (Genomed, Kampenhout, Belgium) following the manufacturer’s instructions. Subsequently, they were sequenced using an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences were analyzed and compared with the reference sequence using the software MEGA (version 5.0). A macrolide-susceptible strain of NCTC 11168 (GenBank: AL111168.1) was used as a reference strain to analyze the mutations in genes encoding 23S rRNA, L4, and L22.


### 2.3. Effects of an Efflux Pump Inhibitor (EPI) on Macrolide Resistance

To investigate the contributions of efflux pump activity to macrolide resistance, the MICs of azithromycin and erythromycin were determined in presence of the EPI phenylalanine arginine ß-naphthylamide (PAßN, Sigma, St. Louis, Missouri). The broth microdilution method was used to determine the MICs in the presence of 20μg/mL PAßN in Mueller-Hinton broth (Oxoid, Basingstoke, England). TheC. jejuni ATCC 33560 was used as the reference strain.


## 2.1. Origin ofCampylobacter Strains and Minimal Inhibitory Concentrations (MIC) Determination

A total of 38 strains ofCampylobacter (15 strains ofC. jejuni and 23 strainsC. coli) showing either intermediate resistance or resistance to azithromycin and erythromycin were used in this study (Tables 1 and 2). The strains were isolated from poultry between 2013 and 2016 in a previous study [16]. The sample sources were divided into four types, and the number of strains collected from each source was as follows: (1) in 176 feces samples from breeder chicken farms, 88 isolates ofCampylobacter were collected and 17 strains (oneC. jejuni and 16C. coli) showed resistance to azithromycin; (2) in 1,003 samples (feces and environmental samples) from broiler chicken farms, 55 isolates were collected and none of them showed resistance to azithromycin or erythromycin; (3) in 249 chicken meat samples from retail markets, 104 isolates were collected and 15 strains (10C. jejuni and fiveC. coli) showed resistance to azithromycin; and (4) in 106 duck meat sample from retail markets, 102 isolates were collected and six strains (fourC. jejuni and twoC. coli) showed resistance to azithromycin. The MICs of azithromycin and erythromycin were determined using agar or broth dilution methods and the breakpoints were as defined by the National Antimicrobial Resistance Monitoring System (NARMS) for azithromycin: susceptible, ≤ 2 μg/mL; intermediate, 4 μg/mL; and resistant, ≥ 8 μg/mL. The MIC breakpoints for erythromycin were susceptible, ≤ 8 μg/mL; intermediate, 16 μg/mL; and resistant, ≥ 32 μg/mL [17]. The reference strainC. jejuni ATCC 33560 was used as the quality control strain.Table 1
Characteristics of macrolide resistance genes inCampylobacter jejuni strains showing varied resistance to azithromycin and erythromycina.
Strain Source MIC (μg/mL) c m e B c e r m B c Mutations L22insertions Azithromycin Erythromycin 23SrRNA L4 L22 -EPI +EPI F o l d b -EPI +EPI F o l d b 33560 ATCC 0.25 0.06 4 1 0.25 4 nt nt wt wt wt wt A16-CF-329-3 CM 4 0.5 8 0.5 0.5 1 + - wt V196A A103V, S109A wt CM13-HL-010-1 CM 4 0.125 32 8 0.25 32 + - wt V80I, V196A A103V, S109A, V137A wt CM13-HL-012-1 CM 4 0.5 8 8 0.125 64 + - wt V80I, V196A A103V, S109A, V137A wt CM13-MWC-BS-004 CM 8 0.125 64 0.5 0.025 20 + - wt V196A A103V, S109A, V137A wt A16-CF-127-1-S1 BC 8 0.25 32 1 0.25 4 + - wt wt wt wt A16-CF-318-3 CM 8 0.25 32 1 0.015 67 + - wt V121A, T177S, M192I, V196A V137S 114VAKKAP115 DM13-MP-WS-002 DM 8 0.5 16 64 0.25 256 + - wt V196A A103V, S109A, V137A wt CM13-OP-004-1 CM 8 0.25 32 64 0.5 128 + - wt wt wt wt A16-CF-129-1-S1 CM 8 0.125 64 128 0.5 256 + - wt wt wt wt CM13-HL-BS-021 CM 16 1 16 32 2 16 + - wt V196A wt wt DM13-JDW-WL-007 DM 16 0.25 64 64 0.5 128 - - wt V196A V137A 114VAKKAP115 DM13-JDW-WL-009 DM 16 16 1 64 64 1 + - wt V121A, T177S, M192I, V196A S109A, V137A 114VAKKAP115 CM13-MWC-WL-008 CM 16 1 16 64 1 64 + - wt V196A A103V, S109A, V137A wt CM13-DW-BL-005 CM 16 1 16 64 1 64 + - wt V196A I65V, A103V, S109A, V137A wt DM13-JW-SS-017 DM 32 0.5 64 64 0.5 128 + - wt V121A, V196A I65V, A103V, S109A, V137A wt a. Abbreviations: ATCC, American Type Culture Collection; CM, chicken meat; BC, breeder chicken; DM, duck meat; EPI: efflux pump inhibitor; nt: not test; A: Ala; I: Ile; K: Lys; M: Met; P: Pro; S: Ser; V: Val; T: Thr; wt: wild type. b. Fold values were calculated as MIC without EPI/MIC with EPI. c. +/-, indicates whether or not the target genes were detected. The MIC breakpoints for azithromycin: susceptible, ≤ 2μg/mL; intermediate, 4 μg/mL; and resistant, ≥ 8 μg/mL; for erythromycin: susceptible, ≤ 8 μg/mL; intermediate, 16 μg/mL; and resistant, ≥ 32 μg/mL.Table 2
Characteristics of macrolide-resistance genes inCampylobacter coli strains showing varied resistance to azithromycin and erythromycina.
Strain Source MIC (μg/mL) cmeB c ermB c Mutations Azithromycin Erythromycin 23S rRNA L4 L22 -EPI +EPI Foldb -EPI +EPI Foldb A16-CF-319-1 CM 4 1 4 1 0.5 2 - - wt wt wt A16-CF-330-1 CM 4 1 4 1 0.5 2 - - wt V196A I65V, A74G A16-CF-130-1-S1 BC 8 8 1 2 0.5 4 + - wt wt I65V, A74G A16-CF-128-1-S1 BC 8 8 1 128 64 2 - - wt V121A, V196A I65V, A74G CM13-HL-BS-020 CM 16 0.5 32 32 0.5 64 - - wt V196A I65V, A74G CM13-HL-BL-031 CM 16 1 16 64 0.25 256 - - wt V196A I65V, A74G CM13-MC-BS-006 CM 16 0.125 128 64 2 32 - - wt V196A I65V, A74G DM13-MWC-SS-013 DM 32 2 16 4 0.125 32 - - wt V196A I65V, A74G DM13-MP-WS-007 DM 32 0.5 64 8 0.5 16 - - wt V196A I65V, A74G A16-CF-130-2-S1 BC 64 64 1 32 32 1 - - wt V196A I65V, A74G A16-CF-107-2-S2 BC 64 64 1 64 32 2 - - A2075G wt I65V, A74G, S109A A16-CF-128-1-S4 BC 128 2 64 128 4 32 - - wt V196A I65V, A74G, S109A A16-CF-124-2-S3 BC 128 4 32 128 2 64 - - wt V196A I65V, A74G, S109A A16-CF-107-2-S4 BC ≥512 64 8 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-124-1-S1 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-124-1-S4 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-126-2-S1 BC ≥512 64 8 ≥512 32 16 - - A2075G wt I65V, A74G, S109A A16-CF-124-1-S5 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G wt I65V, A74G, S109A A16-CF-126-2-S2 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G V196A I65V, A74G, S109A A16-CF-126-2-S4 BC ≥512 ≥512 1 ≥512 64 8 - - A2075G V121A, V196A I65V, A74G, S109A A16-CF-118-2-S3 BC ≥512 ≥512 1 ≥512 64 8 + - A2075G V121A, V196A I65V, A74G, S109A A16-CF-126-1-S3 BC ≥512 ≥512 1 ≥512 32 16 + - A2075G V121A, V196A I65V, A74G, S109A A16-CF-126-2-S3 BC ≥512 ≥512 1 ≥512 32 16 + - A2075G V121A, V196A I65V, A74G, S109A a. Abbreviations: CM: chicken meat; BC: breeder chicken; DM: duck meat; EPI: efflux pump inhibitor; A, Ala; G: Gly; I: Ile; S: Ser; V: Val; wt: wild type. b. Fold values were calculated as (MIC without EPI)/(MIC with EPI). c. +/-, indicates whether or not the target genes were detected. The MIC breakpoints for azithromycin: susceptible, ≤ 2μg/mL; intermediate, 4 μg/mL; and resistant, ≥ 8 μg/mL; for erythromycin: susceptible, ≤ 8 μg/mL; intermediate, 16 μg/mL; and resistant, ≥ 32 μg/mL.


## 2.2. Characterization of Macrolide Resistance inCampylobacterStrains

Genomic DNA templates for PCR were prepared using freshCampylobacter colonies on 5 % sheep blood agar plates (Komed, Seongnam, South Korea) by adding 100 μl sterile distilled water and boiling in a heater block at 100°C for 15 min. Mutations at positions 2,074 and 2,075 of the domain V of 23S rRNA gene were analyzed by sequencing all three copies of 23S rRNA. Three separate reactions were employed to amplify the three copies of the 23S rRNA gene in allC. jejuni andC. coli strains [3]. Subsequently, potential macrolide resistance-associated mutations were identified by sequencing a 308-bp fragment from each copy of the target gene [3].In addition, to assess the contribution of mutations within the ribosomal genesrplD andrplV encoding L4 and L22, respectively, to macrolide resistance, sequence analysis of these genes was performed for all 38 strains. L4- and L22-encoding genes were amplified as previously described [18]. The presence of the recently reported macrolide resistance-related ribosomal RNA methylase gene,erm(B), was confirmed using a method described by Zhang et al. [6]. To investigate the role of drug efflux in macrolide resistance,cmeB,an efflux pump gene (1,070 bp), was amplified using the method described by Pumbwe et al. [19].PCR products were purified using JET-SORB gel extraction kit (Genomed, Kampenhout, Belgium) following the manufacturer’s instructions. Subsequently, they were sequenced using an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences were analyzed and compared with the reference sequence using the software MEGA (version 5.0). A macrolide-susceptible strain of NCTC 11168 (GenBank: AL111168.1) was used as a reference strain to analyze the mutations in genes encoding 23S rRNA, L4, and L22.


## 2.3. Effects of an Efflux Pump Inhibitor (EPI) on Macrolide Resistance

To investigate the contributions of efflux pump activity to macrolide resistance, the MICs of azithromycin and erythromycin were determined in presence of the EPI phenylalanine arginine ß-naphthylamide (PAßN, Sigma, St. Louis, Missouri). The broth microdilution method was used to determine the MICs in the presence of 20μg/mL PAßN in Mueller-Hinton broth (Oxoid, Basingstoke, England). TheC. jejuni ATCC 33560 was used as the reference strain.


## 3. Results

### 3.1. Variation in Resistance to Azithromycin and Erythromycin

Among the 38 testedCampylobacter strains, 27 strains, of which nine wereC. jejuni and 18 wereC. coli, were resistant to azithromycin and erythromycin (Tables 1 and 2). A total of five strains (threeC. jejuni and twoC. coli) showed intermediate resistance to azithromycin with an MIC of 4 μg/mL and were susceptible to erythromycin with MICs ranging from 0.5 to 8 μg/mL. A total of six strains (threeC. jejuni and threeC. coli) were resistant to azithromycin and susceptible to erythromycin with MICs ranging from 8–32 μg/mL and 0.5–8 μg/mL, respectively. MICs of azithromycin against four strains ofC. jejuni (A16-CF-329-3, CM13-MWC-BS-004, A16-CF-127-1-S1, and A16-CF-318-3) and six strains ofC. coli (A16-CF-319-1, A16-CF-330-1, A16-CF-130-1-S1, DM13-MWC-SS-013, DM13-MP-WS-007, and A16-CF-130-2-S1) were 8–16 and 2–8-fold higher, respectively, than those of erythromycin.


### 3.2. Sequence Analysis of the 23S rRNA

All of the 15C. jejuni strains, including one strain (A16-CF-129-1-S1) showing a high-level resistance to erythromycin with an MIC of 128 μg/mL, harbored a wild-type 23S rRNA sequence (Table 1).The point mutation A2075G was detected in all three copies of the 23S rRNA gene from 11 strains ofC. coli, which were resistant to azithromycin and erythromycin with MICs ranging from 64 to ≥ 512 μg/mL (Table 2). Two strains ofC. coli(A16-CF-128-1-S4 and A16-CF-124-2-S3) showed high-level resistance to azithromycin and erythromycin (MIC = 128 μg/mL) and did not harbor any mutations in the 23S rRNA gene, and one strain (A16-CF-128-1-S1) showed high-level resistance to erythromycin (MIC = 128 μg/mL) and did not harbor any mutations in the 23S rRNA gene. The other five strains ofC. coli were resistant to both azithromycin and erythromycin, with no mutations in the 23S rRNA gene.


### 3.3. Investigation of the Ribosomal Proteins L4 and L22

Analysis of amino acid sequences of the ribosomal proteins L22 and L4 from theC. jejuni andC. coli strains revealed the presence of different combinations of amino acid substitutions (Tables 1 and 2). The following amino acid substitutions were identified in L4 fromC. jejuni; V80I, T177S, and M192I in two isolates, V121A in three isolates, and V196A in 12 isolates. The following amino acid substitutions were identified in L22 fromC. jejuni; I65V in two isolates, A103V in eight isolates, S109A in nine isolates, V137S in one isolate, and V137A in eight isolates. In addition to the amino acid substitutions observed in L22 sequences, a six-amino acid sequence (VAKKAP) present between positions 114 and 115 was also identified in three azithromycin-resistant strains ofC. jejuni.Minimal genetic diversity in L4 and L22 amino acid substitutions was observed in 23 strains ofC. coli from poultry. The substitution V121A was identified in L4 from fiveC. coli strains and the substitution V196A was identified in L4 from 15C. coli strains; the substitutions I65V and A74G were identified in L22 from nineC. coli strains and the substitutions I65V, A74G, and S109A were identified in L22 from 13C. coli strains.


### 3.4. PCR Detection of thecmeB anderm(B) Gene

The presence ofcmeB in 14 (93.3 %)C. jejuni strains and 4 (17.4 %)C. coli strains was confirmed using PCR. However, none of the investigated strains ofC. jejuni andC. coli harborederm(B), as observed in the PCR results obtained.


### 3.5. Efficacy of an EPI

The effects of PAßN on the MICs of macrolide antibiotics in theC. jejuni andC. colistrains are shown in Tables 1 and 2. The presence of PAßN greatly decreased the MICs of azithromycin and erythromycin against most ofC. jejuni andC. colistrains. In theCampylobacter strains with no mutations in the 23S rRNA gene, all the azithromycin intermediate/resistant strains were restored to susceptibility except for four strains (DM13-JDW-WL-009, A16-CF-128-1-S1, A16-CF-130-2-S1, and A16-CF-130-1-S1), and all erythromycin-resistant strains were restored to susceptibility except three strains (DM13-JDW-WL-009, A16-CF-128-1-S1, and A16-CF-130-2-S1).All the strains showed increased susceptibility to erythromycin in the presence of PAßN with at least two-fold to 256-fold MIC change, except twoC. jejuni strains and oneC. coli strain which showed no MIC change. The effect of PAßN on azithromycin MIC was lesser than that on erythromycin MIC, with 13 strains (oneC. jejuni and 12C. coli) of the 38 strains showing no MIC change in the presence of PAßN. In addition, of the 11 strains carrying mutations in the 23S rRNA gene, nine showed no change in azithromycin MIC while all 11 strains showed a 2–16-fold decrease in erythromycin MIC.


## 3.1. Variation in Resistance to Azithromycin and Erythromycin

Among the 38 testedCampylobacter strains, 27 strains, of which nine wereC. jejuni and 18 wereC. coli, were resistant to azithromycin and erythromycin (Tables 1 and 2). A total of five strains (threeC. jejuni and twoC. coli) showed intermediate resistance to azithromycin with an MIC of 4 μg/mL and were susceptible to erythromycin with MICs ranging from 0.5 to 8 μg/mL. A total of six strains (threeC. jejuni and threeC. coli) were resistant to azithromycin and susceptible to erythromycin with MICs ranging from 8–32 μg/mL and 0.5–8 μg/mL, respectively. MICs of azithromycin against four strains ofC. jejuni (A16-CF-329-3, CM13-MWC-BS-004, A16-CF-127-1-S1, and A16-CF-318-3) and six strains ofC. coli (A16-CF-319-1, A16-CF-330-1, A16-CF-130-1-S1, DM13-MWC-SS-013, DM13-MP-WS-007, and A16-CF-130-2-S1) were 8–16 and 2–8-fold higher, respectively, than those of erythromycin.


## 3.2. Sequence Analysis of the 23S rRNA

All of the 15C. jejuni strains, including one strain (A16-CF-129-1-S1) showing a high-level resistance to erythromycin with an MIC of 128 μg/mL, harbored a wild-type 23S rRNA sequence (Table 1).The point mutation A2075G was detected in all three copies of the 23S rRNA gene from 11 strains ofC. coli, which were resistant to azithromycin and erythromycin with MICs ranging from 64 to ≥ 512 μg/mL (Table 2). Two strains ofC. coli(A16-CF-128-1-S4 and A16-CF-124-2-S3) showed high-level resistance to azithromycin and erythromycin (MIC = 128 μg/mL) and did not harbor any mutations in the 23S rRNA gene, and one strain (A16-CF-128-1-S1) showed high-level resistance to erythromycin (MIC = 128 μg/mL) and did not harbor any mutations in the 23S rRNA gene. The other five strains ofC. coli were resistant to both azithromycin and erythromycin, with no mutations in the 23S rRNA gene.


## 3.3. Investigation of the Ribosomal Proteins L4 and L22

Analysis of amino acid sequences of the ribosomal proteins L22 and L4 from theC. jejuni andC. coli strains revealed the presence of different combinations of amino acid substitutions (Tables 1 and 2). The following amino acid substitutions were identified in L4 fromC. jejuni; V80I, T177S, and M192I in two isolates, V121A in three isolates, and V196A in 12 isolates. The following amino acid substitutions were identified in L22 fromC. jejuni; I65V in two isolates, A103V in eight isolates, S109A in nine isolates, V137S in one isolate, and V137A in eight isolates. In addition to the amino acid substitutions observed in L22 sequences, a six-amino acid sequence (VAKKAP) present between positions 114 and 115 was also identified in three azithromycin-resistant strains ofC. jejuni.Minimal genetic diversity in L4 and L22 amino acid substitutions was observed in 23 strains ofC. coli from poultry. The substitution V121A was identified in L4 from fiveC. coli strains and the substitution V196A was identified in L4 from 15C. coli strains; the substitutions I65V and A74G were identified in L22 from nineC. coli strains and the substitutions I65V, A74G, and S109A were identified in L22 from 13C. coli strains.


## 3.4. PCR Detection of thecmeB anderm(B) Gene

The presence ofcmeB in 14 (93.3 %)C. jejuni strains and 4 (17.4 %)C. coli strains was confirmed using PCR. However, none of the investigated strains ofC. jejuni andC. coli harborederm(B), as observed in the PCR results obtained.


## 3.5. Efficacy of an EPI

The effects of PAßN on the MICs of macrolide antibiotics in theC. jejuni andC. colistrains are shown in Tables 1 and 2. The presence of PAßN greatly decreased the MICs of azithromycin and erythromycin against most ofC. jejuni andC. colistrains. In theCampylobacter strains with no mutations in the 23S rRNA gene, all the azithromycin intermediate/resistant strains were restored to susceptibility except for four strains (DM13-JDW-WL-009, A16-CF-128-1-S1, A16-CF-130-2-S1, and A16-CF-130-1-S1), and all erythromycin-resistant strains were restored to susceptibility except three strains (DM13-JDW-WL-009, A16-CF-128-1-S1, and A16-CF-130-2-S1).All the strains showed increased susceptibility to erythromycin in the presence of PAßN with at least two-fold to 256-fold MIC change, except twoC. jejuni strains and oneC. coli strain which showed no MIC change. The effect of PAßN on azithromycin MIC was lesser than that on erythromycin MIC, with 13 strains (oneC. jejuni and 12C. coli) of the 38 strains showing no MIC change in the presence of PAßN. In addition, of the 11 strains carrying mutations in the 23S rRNA gene, nine showed no change in azithromycin MIC while all 11 strains showed a 2–16-fold decrease in erythromycin MIC.


## 4. Discussion

In this study,Campylobacterstrains isolated from breeder chicken and chicken and duck meat between 2013 and 2016 were used to investigate the molecular mechanisms underlying macrolide resistance inC. jejuni andC. coli. Our data revealed that point mutations at positions 2,075 in domain V of the 23S rRNA gene contributed to high-level azithromycin and erythromycin resistance in 11Campylobacter strains. These mutations were not present inC. jejuni andC. coli strains with a low-level or intermediate resistance to azithromycin and erythromycin. This finding supports previously published reports that suggested a predominant role for this mutation in macrolide resistance [4, 20]. The binding site substitutions of A2075G, A2074G, and A2074C in the 23S rRNA gene inC. jejuni andC. coli have been implicated in high-level resistance to azithromycin and erythromycin in the field and in the laboratory [4]. The substitution A2075G was the most prevalent genetic mutation conferring high macrolide resistance in the field, suggesting A2075G may provide specific biological or survival advantages compared to A2074G and A2074C [3, 20–22]. Our results were also consistent with those of previous studies in Korea that suggested that the A2075G mutation in the 23S rRNA gene appeared to be the main contributor to high macrolide resistance [23, 24].As observed in several other species of Gram-negative bacteria, RND efflux pumps confer resistance to macrolides inCampylobacter spp. Inhibition of the efflux pumps using EPIs increases the susceptibility ofCampylobacter to macrolides [20]. Our results showed that an EPI promoted a marked decrease in resistance to both azithromycin and erythromycin in most ofCampylobacter strains (Tables 1 and 2). In highly erythromycin-resistant strains, the presence of the A2075G 23S rRNA gene mutation and efflux pump activity indicated synergism between these two resistance mechanisms inCampylobacter [20]. However, this may not apply to azithromycin because nine out of 11 strains with the A2075G 23S rRNA mutation showed no azithromycin MIC change in the presence of PAßN. Our result was consistent with that of a previous study which found that high azithromycin resistance inCampylobacter was mainly due to the A2075G 23S rRNA mutation [25]. These results suggest that the A2075G 23S rRNA mutation inCampylobacter was sufficient to confer high-level resistance to azithromycin, while the mutation synergizes with the drug efflux pump system to confer high erythromycin resistance. Further studies are required to assess the contribution of different mechanisms to erythromycin and azithromycin resistance inCampylobacter.A number of previous studies have reported that modifications in the ribosomal proteins L4 and L22 were associated with a lower level of macrolide resistance [26]. Numerous substitutions and insertions in the ribosomal protein sequences in macrolide-resistant strains have been documented. Amino acids at the positions 63–74 are a part of the most important target region in L4; no variation was found in this region of L4 in the present study. In contrast, the most frequent changes, V121A and V196A, were located outside the important target region. The mutations at positions 121 and 196 were identified in susceptible and resistant isolates in previous studies. This suggests that these substitutions are unlikely to contribute directly to macrolide resistance [27]. Other substitutions such as V80I, T177S, and M192I were also identified in erythromycin-susceptible and -resistant isolates previously [23, 27]. In L22, substitutions including I65V, A74G, A103V, and S109A were identified in erythromycin-susceptible and -resistant isolates previously [27, 28]. The mutations V137A and V137S, located in the β-hairpin region close to the C-terminus, were identified inC. jejunistrains. L22 consists of a small α plus β domain, with the β-hairpin contributing to the formation of the polypeptide tunnel exit at the surface of the ribosome. The mutation in this domain might change the surface properties and block macrolide binding [29]. Nevertheless, isolation ofCampylobacter strains harboring such substitutions and showing reduced susceptibility to erythromycin did not lead to the elucidation of the mechanistic significance of these amino acid substitutions in the present study.In addition to single amino acid substitutions, a six-amino acid insertion (114-VAKKAP-115) within theβ-hairpin region of L22 was found in threeC. jejunistrains showing azithromycin resistance and reduced susceptibility to erythromycin (Table 1). Amino acid insertions in L22 have been reported in a number of bacterial species, both Gram-negative and Gram-positive [30, 31]. For example, insertions at position 86 or 98 in L22 reportedly conferred macrolide resistance inC. jejuniandC. coli [26], and a six-amino acid insertion between T108 and V109 in L22 ofStreptococcus conferred resistance to azithromycin and erythromycin [32]. The significance of this insertion in L22 ofCampylobacter in relation to macrolide resistance needs to be investigated. In the present study, all strains which showed reduced susceptibility to azithromycin and erythromycin, except oneC. coli strain, carried the mutations I65V and A74G. These substitutions were not observed inC. jejunistrains. The significance of the coexistence of these amino acid substitutions is unknown. The occurrence of such mutations may be associated with local environmental conditions and other selective pressures; most of the previously reportedC. coli strains, carrying both the amino acid substitutions, were isolated from chicken and swine in Korea [23]. Further studies are required to assess whether the coexistence of these amino acid substitutions contributes toCampylobactermacrolide resistance.Erythromycin, the first 14-membered macrolide, is active against Gram-positive and some Gram-negative microorganisms. To improve acid stability and oral bioavailability of erythromycin, the first 15-membered macrolide, azithromycin, was developed by inserting a basic nitrogen atom into the macrocyclic ring [33]. Azithromycin exhibited enhancedin vitro andin vivo potency against Gram-positive and Gram-negative bacteria compared to erythromycin [34]. Bacteriostatic and bactericidal activity of azithromycin againstCampylobacter was up to four times more potent than that of erythromycin [9]. In this study, we found fourC. jejuni strains and sixC. coli strains showing higher resistance to azithromycin than to erythromycin; and the MIC of azithromycin was 8–16 and 2–8-fold higher against theC. jejuni strains and theC. coli strain, respectively, compared to that of erythromycin. Additionally, even in the presence of PAßN, twoC. jejuni strains (CM13-MWC-BS-004 and A16-CF-318-3) and fiveC. coli (A16-CF-319-1, A16-CF-330-1, A16-CF-130-1-S1, DM13-MWC-SS-013, and A16-CF-130-2-S1) showed higher MICs of azithromycin than of erythromycin. These results were in agreement with those of previous studies in which erythromycin had a lower MIC than azithromycin against bothCampylobacter and other Gram-positive bacteria [10, 23, 35, 36]. Ribosomal protein polymorphisms might affect the MICs of different macrolides, and an amino acid substitution (A86E) was identified in L22 from azithromycin-resistant and erythromycin-susceptibleCampylobacter in a previous study [37]. Further, azithromycin was found to be less affected by drug efflux compared with erythromycin, and inactivation of thecmeB gene led to greater MIC change of erythromycin than of azithromycin [25]. In the present study, the substitution A86E was not observed in L22 fromC. jejuni andC. colistrains, and higher MICs of azithromycin than of erythromycin were observed even in the presence of an EPI. This may indicate different contributing factors for macrolide resistance, apart from efflux pump-mediated mechanisms. Further studies are required to verify the diverse mechanisms underlying erythromycin and azithromycin resistance and to facilitate the development of strategies to control macrolide-resistantCampylobacter. In addition, considering that poultry meat is a common source of human pathogens, careful thought must be given to select effective antibiotics againstCampylobacter strains from poultry meat showing higher resistance to azithromycin than to erythromycin.A relatively low prevalence ofcmeB inC. coli strains compared with that inC. jejuni strains was found in this study. This was consistent with findings of a recent study on turkeys reported by Olah et al. [38]. The primers used were unable to detectcmeB due to the high sequence variation ofcmeB inC. coli isolates [39]. Differences betweenC. jejuni andC. coli strains should be further investigated.The geneerm(B) is involved in a major mechanism underlying macrolide resistance in other bacteria, especially Gram-positive bacteria [2]. The transferableerm(B) gene located in MDRGI on the chromosome and plasmids with high prevalence in Gram-positive bacteria has been described in highly erythromycin-resistantCampylobacter strains [5]. An increase in the incidence oferm(B)-carryingCampylobacter strains was reported in China, and their prevalence increased from 0.7 % during 2007–2009 to 6.4 % during 2011–2012 [40]. It is noteworthy that a recent study showed a particularly high prevalence (15.1 %) oferm(B) in strains in a province of southern China in 2017 [41]. The isolation of anerm(B)-carrying clinicalC. jejuni strain in 1994 suggests thaterm(B) has a property of diffusional spread along time and space [7]. Following the characterization oferm(B)-positiveC. coli isolated from chicken in Europe, researchers focused on the transferableerm(B), located in MDRGI on the chromosome as well as on plasmids. Theerm(B) gene has not been detected in macrolide-resistantC. jejuni andC. coli from various sources including human clinical specimens, retail meat, and fecal samples from food animals in the USA or in macrolide-resistantC. coli from colon contents of swine in France [37, 42]; in this study,erm(B) was not detected inC. jejuni andC. coli strains from poultry including breeder chicken or chicken and duck meat between 2013 and 2016. Continuous monitoring oferm(B) inCampylobacter is required, due to its highly transmittable nature.Recent studies have shown that whole-genome sequencing (WGS) analysis can potentially be a rapid approach to define resistance genotypes and predict resistance phenotypes of bacteria with great sensitivity and specificity, and numerous proof-of-principle studies have also highlighted the value of WGS as a primary diagnostic tool to detect antibiotic resistance [43]. InCampylobacter, although a recent study showed that the correlation between resistance phenotypes and genotypes was 100 % in terms of resistance to tetracycline, fluoroquinolones, and erythromycin [37], numerous studies have reported differences between macrolide resistance phenotypes and genotypes ofCampylobacter from various sources. These studies revealed thatC. jejuni andC. coli strains were resistant to macrolides but did not harbor the corresponding genes or mutations required for resistance [3, 27, 35, 44]. In this study, 27Campylobacter strains with both azithromycin and erythromycin resistance did not harbor any mutations within the corresponding genes. Further, three strains were did not harbor any mutation even in the presence of EPI. In such a situation, even after WGS, phenotypic testing would be necessary to confirm macrolide susceptibility of strains given that only deep WGS can detect resistance.In the present study, we investigated the macrolide resistance ofC. jejuni andC. coli from breeder chicken and chicken and duck meat. Chicken meat is a well-known and a major source for campylobacteriosis in humans; macrolide-resistantCampylobacter could be transmitted from duck and breeder chicken to humans. In our study,Campylobacter with high resistance to azithromycin and erythromycin was found in breeder chicken. Vertical transmission ofCampylobacter is questionable and it has been reported that antibiotic-resistantCampylobacter from breeder chicken showed clonal homology to that found in humans [1]. In addition, the increased consumption of ducks, especially in Asia, also increases the risk of transmission of antibiotic-resistantCampylobacter to humans [13, 14]. Therefore, monitoring populations of macrolide-resistantCampylobacter in poultry, including breeder chicken and duck, is required.It is noteworthy that allCampylobacter strains in the present study showing high resistance to azithromycin and erythromycin belonged to a singleC. coli population isolated from breeder chicken. Similar to a previous study, macrolide resistance has been usually observed inC. coli, and it is believed that the high resistance inCampylobacter is due to extensive exposure to macrolide derivatives [45]. AllCampylobacterstrains with low or high macrolide resistance were found in breeder chicken from a single integrated company with no difference in management practices or antibiotic usage. This may suggest that polymorphisms, which cause macrolide resistance, could develop even under similar environment pressure. This is in agreement with a previous study in which it was shown thatCampylobacter utilizes complex and different mechanisms to develop macrolide resistance in the field [46]. Therefore, further studies are required to elucidate mechanisms underlying the development of macrolide resistance inCampylobacterduring chicken growth.


## 5. Conclusions

In conclusion, the data presented here confirmed previous findings, revealing that a mutation in the 23S rRNA gene at position 2075 showed high-level azithromycin resistance inCampylobacter and that the 23S rRNA gene mutation acts synergistically with drug efflux to causes erythromycin resistance. Studies using a larger number ofC. jejuni andC. coli strains showing high resistance to azithromycin but not erythromycin are required to investigate the diverse mechanisms underlying azithromycin and erythromycin resistance inCampylobacter spp. Further investigations are required to elucidate the significance of the observed amino acid substitutions V137A and V137S and the six-amino acid insertion identified in L22 fromC. jejunistrains. None of the investigated strains ofC. jejuni andC. coli from chicken and duck harborederm(B); further surveillance might be required to confirm this. Because differences between macrolide resistance phenotypes and genotypes ofCampylobacter showing high resistance were found, further studies are needed to improve our understanding of macrolide resistance inCampylobacter. To prevent and control macrolide resistance inCampylobacter, mechanisms underlying resistance development during chicken growth need to be elucidated. In addition, populations of macrolide-resistantCampylobacter in poultry including breeder chicken and duck need to be monitored.



---

*Source: 4526576-2018-07-05.xml*