FQ resistance mechanisms

The quinolones are a class of broad spectrum antimicrobials that are potent against both gram-negative and gram-positive bacteria (Andersson and MacGowan, Reference Andersson and MacGowan2003). According to their spectrum of activity, quinolones have been classified into four generations. The majority of quinolones currently used for clinical therapies are FQs, which are derived from the quinolones by a fluorine substitution at the C-6 or C-7 position, thereby increasing their activity against gram-negative bacteria (Andersson and MacGowan, Reference Andersson and MacGowan2003). Once inside bacterial cells, FQ antimicrobials exert their antibacterial effect by interacting with DNA gyrase and topoisomerase IV, resulting in double-stand DNA breaks and cell death (Jacoby, Reference Jacoby2005). Two main mechanisms of resistance to FQs are currently recognized in Campylobacter bacteria, including mutations that change the antibiotic's target and that reduce antibiotic intracellular accumulation. In other gram-negative bacteria, target protection mediated by the Qnr protein was also involved in FQ resistance (Martin-Gutierrez et al., Reference Martin-Gutierrez, Rodriguez-Martinez, Pascual, Rodriguez-Beltran and Blazquez2017), but this mechanism has not been reported in Campylobacter.

In gram-negative bacteria, gyrase is the main target of FQ antibiotics, whereas, in gram-positive bacteria, topoisomerase IV is more susceptible to the action of FQ (Jacoby, Reference Jacoby2005). Both enzymes consisting of two pairs of subunits, named GyrA and GyrB (DNA gyrase), and ParC and ParE (topoisomerase IV) (Payot et al., Reference Payot, Bolla, Corcoran, Fanning, Megraud and Zhang2006). Although most bacteria have both enzymes, Campylobacter lacks the parC and parE genes and thus they are not the targets of FQ antimicrobials in Campylobacter (Bachoual et al., Reference Bachoual, Ouabdesselam, Mory, Lascols, Soussy and Tankovic2001; Payot et al., Reference Payot, Cloeckaert and Chaslus-Dancla2002; Piddock et al., Reference Piddock, Ricci, Pumbwe, Everett and Griggs2003). Additionally, no mutations in gyrB have been associated with FQ resistance in Campylobacter (Bachoual et al., Reference Bachoual, Ouabdesselam, Mory, Lascols, Soussy and Tankovic2001). Therefore, mutations linked to FQ resistance in C. jejuni and C. coli mainly occur in GyrA. Specifically, resistance to FQs involves amino acid substitutions in a region of the GyrA termed the ‘quinolone-resistance-determining region’. This region is located within the DNA-binding domain on the surface of DNA gyrase and corresponding amino acids spans from position 51 to position 106 (E. coli numbering), with common mutations at amino acid positions 83 and 87 (position 86 and 90 in Campylobacter) (Friedman et al., Reference Friedman, Lu and Drlica2001). The most frequent mutation observed in FQ-resistant Campylobacter isolates is Thr-86-Ile, followed by Asp-90-Asn, Thr-86-Lys, Thr-86-Ala, Thr-86-Val, Asp-90-Tyr, and Ala-70-Thr (Wang et al., Reference Wang, Huang and Taylor1993; Engberg et al., Reference Engberg, Aarestrup, Taylor, Gerner-Smidt and Nachamkin2001; Luo et al., Reference Luo, Sahin, Lin, Michel and Zhang2003). The Thr-86-Ile mutation confers a high level of FQ resistance [ciprofloxacin minimum inhibitory concentration (MIC) ≥ 16 µg ml⁻¹] in Campylobacter, while other mutations are associated with a low to medium level of resistance (MIC = 1–8 µg ml⁻¹) (Luo et al., Reference Luo, Sahin, Lin, Michel and Zhang2003; Payot et al., Reference Payot, Bolla, Corcoran, Fanning, Megraud and Zhang2006; Yan et al., Reference Yan, Sahin, Lin and Zhang2006). Double mutations including Thr-86-Ile/Pro-104-Ser and Thr-86-Ile/Asp-90-Asn have also been linked to FQ resistance in Campylobacter (Payot et al., Reference Payot, Bolla, Corcoran, Fanning, Megraud and Zhang2006). Additionally, acquisition of high-level FQ resistance in Campylobacter does not require stepwise accumulation of point mutations in gyrA. Instead, a single point mutation in gyrA can lead to clinically relevant levels of resistance to FQ antimicrobials (Gootz and Martin, Reference Gootz and Martin1991; Wang et al., Reference Wang, Huang and Taylor1993; Ruiz et al., Reference Ruiz, Goni, Marco, Gallardo, Mirelis, Jimenez De Anta and Vila1998; Luo et al., Reference Luo, Sahin, Lin, Michel and Zhang2003; Yan et al., Reference Yan, Sahin, Lin and Zhang2006).

The CmeABC efflux pump contributes significantly to both intrinsic and acquired resistance of C. jejuni to FQ antimicrobials by reducing the accumulation of FQs in Campylobacter cells (Ge et al., Reference Ge, McDermott, White and Meng2005). In wild type 81–176, inactivation of CmeB led to a 8-fold reduction in the MIC of ciprofloxacin, suggesting that CmeABC contributes to the intrinsic resistance of Campylobacter to FQs (Lin et al., Reference Lin, Michel and Zhang2002). Even in the presence of resistance-conferring GyrA mutations, insertional mutagenesis of CmeABC led to drastic reduction in ciprofloxacin MIC in FQ^R isolates, indicating the importance of CmeABC in FQ resistance (Luo et al., Reference Luo, Sahin, Lin, Michel and Zhang2003). Overexpression of CmeABC, either by inactivating its repressor CmeR or mutating the promoter region of cmeABC, increased the resistance to FQs in Campylobacter (Lin et al., Reference Lin, Akiba, Sahin and Zhang2005a; Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). The recently identified CmeABC variant (RE-CmeABC) showed a much higher efficiency in the efflux function and conferred an exceedingly high-level resistance (ciprofloxacin MIC ≥ 256 µg ml⁻¹) to FQs in the presence of GyrA mutations (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). The RE-CmeABC appears to be increasingly prevalent in China, where FQs have been widely used for animal production practices (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). By reducing the intracellular concentration of antibiotics, CmeABC facilitates and promotes the emergence of FQ^R Campylobacter under selection pressure because GyrA mutations alone are not sufficient to survive the killing effect of ciprofloxacin (Yan et al., Reference Yan, Sahin, Lin and Zhang2006). In the absence of a functional CmeABC, many spontaneous gyrA mutants would not be able to emerge under antibiotic selection (Yan et al., Reference Yan, Sahin, Lin and Zhang2006).

Macrolide resistance mechanisms

Macrolide antibiotics, such as erythromycin, azithromycin, clarithromycin, and relithromycin, are a class of natural products that consist of a large macrocyclic lactone ring, which are usually 14-, 15-, or 16-membered (Tenson et al., Reference Tenson, Lovmar and Ehrenberg2003). Macrolides inhibit protein synthesis by binding to the ribosome that includes 23S rRNA and ribosomal proteins. Macrolides are usually used for the treatment of gram-positive cocci (mainly staphylococci and streptococci), gram-positive bacilli, gram-negative cocci, and some gram-negative bacilli, such as Campylobacter and helicobacter (Leclercq, Reference Leclercq2002). For clinical therapy of campylobacteriosis, macrolides such as erythromycin are often considered the drug of choice. Three mechanisms have been reported for macrolide resistance in bacteria, which include (i) modification of target sites by mutation or methylation, (ii) active efflux of antibiotics from bacterial cells, and (iii) antibiotic inactivation. In Campylobacter, the first two mechanisms have been documented, but macrolide inactivation by the action of esterases or phosphotransferases has not been reported.

In Campylobacter, positions 2074 and 2075 of the 23S rRNA correspond to positions 2058 and 2059 in E. coli, respectively. These two nucleotides interact directly with macrolide antibiotics and mutations in the two sites impair the binding of macrolides to 23S rRNA (Tenson et al., Reference Tenson, Lovmar and Ehrenberg2003). To date, four types of point mutations at 23S rRNA have been linked to macrolide resistance in Campylboacter, including A2074C, A2074G, A2074T, and A2075G. Among these point mutations, A2075G has been observed most frequently (Jensen and Aarestrup, Reference Jensen and Aarestrup2001; Vacher et al., Reference Vacher, Menard, Bernard and Megraud2003, Reference Vacher, Menard, Bernard, Santos and Megraud2005). C. jejuni and C. coli have three copies of 23S rRNA (rrn operon). In most clinical strains that are highly resistant to erythromycin (MIC > 128 µg ml⁻¹), all three copies of the rrn operons were mutated (Jensen and Aarestrup, Reference Jensen and Aarestrup2001; Niwa et al., Reference Niwa, Chuma, Okamoto and Itoh2001; Gibreel et al., Reference Gibreel, Kos, Keelan, Trieber, Levesque, Michaud and Taylor2005). When the A2074T mutation occurred only in some of the rrn operons, it only conferred a low level resistance to macrolide (Vacher et al., Reference Vacher, Menard, Bernard, Santos and Megraud2005). However, when the A2074T mutations happened in all three copies of 23S rRNA genes, the mutant strains were highly resistant to macrolide (Ohno et al., Reference Ohno, Wachino, Saito, Jin, Yamada, Kimura and Arakawa2016).

Modification of the ribosomal protein L4 and L22 has also been found conferring macrolide resistance in Campylobacter. L4 and L22 were encoded by the rplD and rplV genes, respectively, and both were considered as a portion of the peptide exit tunnel of the 50S ribosome. Amino acids spanning positions 63–74 are reported to be the most important target regions of the L4 protein (Corcoran et al., Reference Corcoran, Quinn, Cotter and Fanning2006). Mutation in this region had been reported in several bacteria with high levels of erythromycin resistance (Chittum and Champney, Reference Chittum and Champney1994; Tait-Kamradt et al., Reference Tait-Kamradt, Davies, Appelbaum, Depardieu, Courvalin, Petitpas, Wondrack, Walker, Jacobs and Sutcliffe2000; Malbruny et al., Reference Malbruny, Nagai, Coquemont, Bozdogan, Andrasevic, Hupkova, Leclercq and Appelbaum2002). In Campylobacter, the G74D modification alone was found to confer low to medium resistance to macrolides (Cagliero et al., Reference Cagliero, Mouline, Cloeckaert and Payot2006). Outside the 63–74 amino acid region of L4, several other amino acid substitutions were associated with macrolide resistance in both Campylobacter and Streptococcus (Doktor et al., Reference Doktor, Shortridge, Beyer and Flamm2004; Corcoran et al., Reference Corcoran, Quinn, Cotter and Fanning2006). The L22 modifications, including insertion, mutation, and deletion are also involved in macrolide resistance in Campylobacter. Corcoran et al. (Reference Corcoran, Quinn, Cotter and Fanning2006) identified a unique A103V substitution in the L22 protein, which was linked to high level erythromycin resistance in both C. jejuni and C. coli. Three to four amino acid insertions at position 86 or 98 of the L22 protein were also observed in macrolide-resistant isolates (Caldwell et al., Reference Caldwell, Wang and Lin2008; Lehtopolku et al., Reference Lehtopolku, Kotilainen, Haanpera-Heikkinen, Nakari, Hanninen, Huovinen, Siitonen, Eerola, Jalava and Hakanen2011).

Recently, a new mechanism of macrolide resistance in Campylobacter has emerged (Qin et al., Reference Qin, Wang, Zhang, Zhang, Deng, Shen, Wu, Wang, Zhang and Shen2014), which is mediated by the erm(B) gene that encodes a rRNA methyltransferase. This enzyme adds a methyl group to the A2058 (E. coli numbering) position located within a conserved region of domain V of the 23S rRNA. Methylation at this site gives rise to cross-resistance to macrolide, lincosamide, and streptogramin B (MLS_B phenotype). To date, 43 erm (erythromycin ribosome methylase) genes have been reported (http://faculty.washington.edu/marilynr/), but only erm(B) has been detected in Campylobacter, including C. jejuni and C. coli in China and Europe (Qin et al., Reference Qin, Wang, Zhang, Zhang, Deng, Shen, Wu, Wang, Zhang and Shen2014; Deng et al., Reference Deng, Wang, Zhang and Shen2015; Florez-Cuadrado et al., Reference Florez-Cuadrado, Ugarte-Ruiz, Quesada, Palomo, Dominguez and Porrero2016). In the first report of erm(B) in C. coli, it was identified in a 12,035 bp genomic segment on the chromosome and was found to confer a high-level resistance to erythromycin (MIC 512 µg ml⁻¹). This segment contained 17 open reading frames (ORFs), 8 of which were antibiotic resistance determinants, including erm(B), tet(O), and 6 genes encoding aminoglycoside-modifying enzymes (Qin et al., Reference Qin, Wang, Zhang, Zhang, Deng, Shen, Wu, Wang, Zhang and Shen2014). Thus, the genomic segment was named as the MDR genomic island (MDRGI). This MDRGI can be transferred between C. jejuni and C. coli via natural transformation (Qin et al., Reference Qin, Wang, Zhang, Zhang, Deng, Shen, Wu, Wang, Zhang and Shen2014). The erm(B) gene was also later identified in C. jejuni, where it was associated with several antimicrobial resistance genes [tet(O), aadE and aad9] in a MDRGI that was inserted in the chromosome at a different location when compared with that in C. coli (Deng et al., Reference Deng, Wang, Zhang and Shen2015). In Europe, the identified erm(B) in C. coli was also located in a MDRGI, but the MDGRI contents were different from those found in China (Florez-Cuadrado et al., Reference Florez-Cuadrado, Ugarte-Ruiz, Quesada, Palomo, Dominguez and Porrero2016). Interestingly, the erm(B)-carrying MDRGIs have different G + C contents from the rest of the chromosome, which suggests that Campylobacter acquired erm(B) from other bacterial organisms via horizontal gene transfer (Florez-Cuadrado et al., Reference Florez-Cuadrado, Ugarte-Ruiz, Quesada, Palomo, Dominguez and Porrero2016). The emergence of erm(B) in Campylobacter is alarming because it alone confers a high-level resistance to macrolide antibiotics and is horizontally transferable. Thus, the spread of erm(B)-positive Campylobacter should be continuously monitored.

The multidrug efflux pump CmeABC also contributes significantly to macrolide resistance in Campylobacter. This was first demonstrated by an insertional mutation of the cmeB gene in wild-type 81–176, which resulted in a 4-fold decrease in the MIC of erythromycin (Lin et al., Reference Lin, Michel and Zhang2002). Additionally, overexpression of CmeABC by mutating the CmeR repressor led to 4-fold increase in the resistance to erythromycin (Lin et al., Reference Lin, Akiba, Sahin and Zhang2005a). Even in the highly resistant strains (harboring the resistance-conferring mutations in the 23S rRNA), inactivating the cmeB gene led to a drastic reduction in the MIC of erythromycin (Cagliero et al., Reference Cagliero, Mouline, Payot and Cloeckaert2005; Lin et al., Reference Lin, Yan, Sahin, Pereira, Chang and Zhang2007) suggesting that the point mutations in 23S rRNA and CmeABC function synergistically in conferring high-level macrolide resistance.

Florfenicol resistance mechanisms

Florfenicol is a fluorinated derivative of thiamphenicol and has only been used in veterinary medicine since its introduction in the mid-1990s (Syriopoulou et al., Reference Syriopoulou, Harding, Goldmann and Smith1981). In the USA, florfenicol is currently indicated for the treatment of bovine respiratory disease and bovine interdigital phlegmon. Florfenicol has a broad antibacterial spectrum against both gram-positive and gram-negative organisms, and shows a good tissue penetration property due to its lipophilicity (Schwarz et al., Reference Schwarz, Kehrenberg, Doublet and Cloeckaert2004). Once in a bacterial cell, florfenicol binds to the peptidyltransferase center to prevent the peptide chain elongation, resulting in inhibition of protein synthesis and bacterial death. Over the years, bacteria have developed mechanisms to counteract the selection pressure from florfenicol, including (i) modification or protection of the antibiotic targets and (ii) decrease of intracellular concentration by reducing the permeability and increasing efflux.

Functioning as an rRNA methyltransferase, Cfr adds a methyl group at position A2503 of 23S rRNA and plays an important role in bacterial resistance to florfenicol (Kehrenberg et al., Reference Kehrenberg, Schwarz, Jacobsen, Hansen and Vester2005). Given that position A2503 of 23S rRNA is located at the peptidyl transferase center, which is the target of a number of antimicrobial agents, modification of this position affects binding of multiple classes of antibiotics. Indeed, antimicrobial susceptibility testing revealed that Staphylococcus aureus and E. coli strains expressing the cfr gene showed resistance to five chemically distinct classes of antimicrobials, including phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (known as the PhLOPS_A phenotype) (Long et al., Reference Long, Poehlsgaard, Kehrenberg, Schwarz and Vester2006). The Cfr-mediated resistance to oxazolidinones is especially alarming as this class of antibiotics is considered the last resort for the treatment of MDR gram positives (French, Reference French2001). The cfr gene was first discovered on a 16.5-kb plasmid from S. sciuri isolate of bovine origin in 2000 (Schwarz et al., Reference Schwarz, Werckenthin and Kehrenberg2000). Since its first discovery, cfr has been detected in a number of Gram-positive and Gram-negative bacteria (Schwarz et al., Reference Schwarz, Werckenthin and Kehrenberg2000; Dai et al., Reference Dai, Wu, Wang, Wang, Wang, Huang, Xia, Li and Shen2010; Wang et al., Reference Wang, He, Schwarz, Zhou, Shen, Wu, Wang, Ma, Zhang and Shen2012a, Reference Wang, Wang, Schwarz, Shen, Zhou, Lin, Wu and Shenb; Liu et al., Reference Liu, Wang, Schwarz, Li, Shen, Zhang, Wu and Shen2013). The cfr gene is often carried by transferable plasmids with additional antibiotic resistance genes, facilitating its dissemination and emergence in different bacterial species and under various selective conditions (Wang et al., Reference Wang, Li, Song, Liu, He, Liu, Wu, Schwarz and Shen2013; Liu et al., Reference Liu, Wang, Dai, Wu and Shen2014; Li et al., Reference Li, Wu, Wang, Fan, Schwarz and Zhang2015; Zhang et al., Reference Zhang, Sun, Wang, Lei, Schwarz and Wu2015). Later, a cfr-like variant, named cfr(B), was discovered in a mobile genetic element in both Peptoclostridium difficile and Enterococcus faecium of human origin (Deshpande et al., Reference Deshpande, Ashcraft, Kahn, Pankey, Jones, Farrell and Mendes2015; Hansen and Vester, Reference Hansen and Vester2015). Cfr(B) shares 74.9% amino acid (aa) sequence identity with the original Cfr and confers the same MDR phenotype (Deshpande et al., Reference Deshpande, Ashcraft, Kahn, Pankey, Jones, Farrell and Mendes2015).

Although florfenicol is not used for treating Campylobacter infection, its use in animal production imposes a selection pressure on Campylobacter. Recently, a novel cfr-like gene, named cfr(C), was identified in Campylobacter coli and Clostridium difficile (Candela et al., Reference Candela, Marvaud, Nguyen and Lambert2017; Tang et al., Reference Tang, Dai, Sahin, Wu, Liu and Zhang2017a). In Campylobacter, cfr(C) was located on a conjugative plasmid of ~48 kb (Tang et al., Reference Tang, Dai, Sahin, Wu, Liu and Zhang2017b) and encodes a 379 aa protein that only shows 55.1 and 54.9% aa identity to the original Cfr from Staphylococcus sciuri (Schwarz et al., Reference Schwarz, Werckenthin and Kehrenberg2000) and the recently reported Cfr(B) from E. faecium, respectively (Deshpande et al., Reference Deshpande, Ashcraft, Kahn, Pankey, Jones, Farrell and Mendes2015). Cloning of cfr(C) into C. jejuni NCTC11168 and conjugative transfer of the cfr(C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins, and oxazolidinones, which resulted in 8- to 256-fold increase in their MICs in both C. jejuni and C. coli. These findings established cfr(C) as a novel MDR gene and represent the first report of a cfr-like gene in a foodborne pathogen. In Clostridium difficile, cfr(C) is located on a putative 24 kb-transposon and also confers resistance to PhLOPS_A (Candela et al., Reference Candela, Marvaud, Nguyen and Lambert2017). In addition to Cfr, mutation in the antibiotic target also confers resistance to florfenicol. For example, a G2073A mutation in all three copies of the 23S rRNA was shown to mediate resistance to chloramphenicol and florfenicol in C. jejuni (Ma et al., Reference Ma, Shen, Naren, Li, Xia, Wu, Shen, Zhang and Wang2014a).

The typical CmeABC in C. jejuni NCTC 11168 had limited effect on florfenicol resistance (Tang et al., Reference Tang, Dai, Sahin, Wu, Liu and Zhang2017b). However, the recently identified ‘super’ efflux pump variant, RE-CmeABC, is much more potent in conferring resistance to florfenicol and other antibiotics (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). The Re-CmeABC was discovered from MDR C. jejuni isolates, and transfer of this efflux mechanism to different C. jejuni strains resulted in a >32-fold increase in the MIC of florfenicol, suggesting its powerful role in the extrusion of florfenicol.

The floR gene, encoding a MDR efflux pump, mediates resistance to chloramphenicol and florfenicol (Arcangioli et al., Reference Arcangioli, Leroy-Setrin, Martel and Chaslus-Dancla1999). It was first been discovered in Salmonella typhimurium DT104 (Arcangioli et al., Reference Arcangioli, Leroy-Setrin, Martel and Chaslus-Dancla1999) and had also been detected in C. coli (Frye et al., Reference Frye, Lindsey, Meinersmann, Berrang, Jackson, Englen, Turpin and Fedorka-Cray2011). floR encodes a protein of 404 amino acids, which functions as efflux transporter. Interestingly, pp-flo, florSt, flo, and floR, are closely related even though they were assigned different names in the literature (Kim and Aoki, Reference Kim and Aoki1996; Arcangioli et al., Reference Arcangioli, Leroy-Setrin, Martel and Chaslus-Dancla1999; Bolton et al., Reference Bolton, Kelley, Lee, Fedorka-Cray and Maurer1999). Functionally, they all confer resistance to both chloramphenicol and florfenicol. Sequence alignment showed 96–100% identity in their nucleotide sequences and 88–100% identity in the amino acid sequences. The fexA and fexB genes, coding for phenicol specific efflux pumps, also confer resistance to florfenicol. They have been found in Staphylococcus, Bacillus, and Enterococcus, but not in Campylobacter (Dai et al., Reference Dai, Wu, Wang, Wang, Wang, Huang, Xia, Li and Shen2010; Liu et al., Reference Liu, Wang, Wu, Schwarz, Shen, Jeon, Ding, Zhang and Shen2012; Gomez-Sanz et al., Reference Gomez-Sanz, Kadlec, Fessler, Zarazaga, Torres and Schwarz2013).

Beta-lactam resistance mechanisms

Beta-lactam antibiotics, such as penicillin, inhibit the growth of bacteria by disrupting peptidoglycan cross-linking during bacterial cell wall biosynthesis. Although beta-lactam antibiotics are not commonly prescribed for the treatment of Campylobacter infection, recent studies have proposed that oral beta-lactam, such as co-amoxiclav, could be an appropriate choice when Campylobacter is resistant to both FQ and macrolides. In Campylobacter, two mechanisms of beta-lactam resistance have been documented. One is the production of beta-lactamase OXA-61 and the other one is the multidrug efflux pump.

Several studies have reported that the majority of Campylobacter isolates were ampicillin resistant, and resistance was more common among C. coli isolates than among C. jejuni isolates (Li et al., Reference Li, Mehrotra, Ghimire and Adewoye2007; Griggs et al., Reference Griggs, Peake, Johnson, Ghori, Mott and Piddock2009; Komba et al., Reference Komba, Mdegela, Msoffe, Nielsen and Ingmer2015). The genome sequence of C. jejuni NCTC 11168 revealed the presence of a putative chromosomally encoded class D beta-lactamase (Cj0299) (Parkhill et al., Reference Parkhill, Wren, Mungall, Ketley, Churcher, Basham, Chillingworth, Davies, Feltwell, Holroyd, Jagels, Karlyshev, Moule, Pallen, Penn, Quail, Rajandream, Rutherford, van Vliet, Whitehead and Barrell2000). The corresponding gene in a clinical human isolate GC015 has been functionally characterized and was shown to confer a ≥32-fold increase in the MICs of ampicillin, piperacillin, and carbenicillin in C. jejuni (Alfredson and Korolik, Reference Alfredson and Korolik2005). The expression level of the gene can also modulate the susceptibility of Campylobacter to beta-lactams. For example, a single nucleotide mutation (G–T transversion) in the promoter region of bla _OXA-61 led to overexpression of bla _OXA−61 and consequently ≥256-fold increase in beta-lactam resistance in C. jejuni (Zeng et al., Reference Zeng, Brown, Gillespie and Lin2014). A mutator phenotype resulting from a single amino acid change (G199W) in MutY increased the mutation frequency of the G–T transversion in the bla _OXA−61 promoter region and consequently elevated the spontaneous ampicillin resistance mutation frequency in C. jejuni (Dai et al., Reference Dai, Muraoka, Wu, Sahin and Zhang2015). In addition to OXA-61, other uncharacterized beta-lactamase genes may exist in Campylobacter (Griggs et al., Reference Griggs, Peake, Johnson, Ghori, Mott and Piddock2009). CmeABC also plays an important role in intrinsic resistance to beta-lactam antibiotics as mutation of CmeB resulted in 32-fold reduction in ampicillin MIC (Lin et al., Reference Lin, Michel and Zhang2002).

Tetracycline resistance mechanisms

Tetracyclines are an important class of antibiotics widely used in both human and animal medicine (Chopra, Reference Chopra2001). This class of antibiotics prevent bacterial growth by inhibiting protein synthesis with interaction of the antibiotics to the ribosomal 30S subunit (Chopra, Reference Chopra2001). The most important mechanism of resistance to tetracyclines results from acquisition of genetically mobile tetracycline resistance (tet) genes, which encode proteins that either extrude tetracyclines, or confer ribosomal protection (Chopra and Roberts, Reference Chopra and Roberts2001). In Campylobacter spp., two mechanisms of tetracycline resistance were reported, including (i) ribosomal protection protein tet(O) and (ii) endogenous efflux mediated by CmeABC. tet(O) is the only teracycline-resistance gene identified in Campylobacter so far. The Tet(O) protein binds to the tetracycline molecule and promote its release from the target site on the ribosome (Connell et al., Reference Connell, Trieber, Dinos, Einfeldt, Taylor and Nierhaus2003). The Tet(O) gene may be located on plasmids or the chromosome. The G + C content (40%) of tet(O) is higher than that of Campylobacter genomes (~30%), suggesting that Campylobacter might have obtained the gene from other bacteria by horizontal gene transfer. The multidrug efflux pump, CmeABC, has been shown to contribute to both intrinsic and acquired resistance to tetracycline (Lin et al., Reference Lin, Michel and Zhang2002; Gibreel et al., Reference Gibreel, Wetsch and Taylor2007). In the CmeB mutant stain of 81–176 (harboring tet(O)), the MIC of tetracycline was decreased by 8-fold (Lin et al., Reference Lin, Michel and Zhang2002).

Aminoglycoside resistance mechanisms

The aminoglycoside antibiotics is a class of broad spectrum antibacterial agents used for the treatment of both Gram-positive and Gram-negative organisms. Aminoglycoside antibiotics exert their antibacterial activity by binding the 30S ribosomal subunit, thus disturbing the biosynthesis of proteins (Mingeot-Leclercq et al., Reference Mingeot-Leclercq, Glupczynski and Tulkens1999). Gentamicin is an important aminoglycoside and is used in human beings for treatment of severe infection, including the systemic infection caused by Campylobacter. Gentamicin is also approved for the prevention of bacterial infection-associated death in young food animals, including day-old chicks and 1- to 3- day-old turkey poults. Due to the nephro- and ototoxicity, the consumption of gentamicin has significantly decreased. However, the increasing antimicrobial resistance to newer agents has prompted physicians to reevaluate the use of these old antibiotic compounds (Falagas et al., Reference Falagas, Grammatikos and Michalopoulos2008).

Several mechanisms of gentamicin resistance in Campylobacter have been reported. aacA4 encodes an aminoglycoside 6′-N-acytyltransfearse, confer resistance to aminoglycosides containing purpurosamine ring including gentamicin, and was the first gentamicin resistant gene found in C. jejuni isolates (Lee et al., Reference Lee, Sanchez, Zimmer, Idris, Berrang and McDermott2002). The gene, aph(2 ″ )-If was identified on a MDR conjugative plasmid from a clinical strain of C. jejuni, which was isolated from a US soldier deployed to Thailand (Nirdnoy et al., Reference Nirdnoy, Mason and Guerry2005). Although this gene was initially considered as a bifunctional enzyme and annotated as aac(6 ′ -Ie)/aph(2 ″ -Ia) (also named aacA/aphD), later it was confirmed as a monofunctional aminoglycoside kinase and named as aph(2 ″ )-If (Toth et al., Reference Toth, Frase, Antunes and Vakulenko2013). A recent study from China found that aph(2 ″ )-If was chromosomally encoded and has become the predominant gentamicin resistance determinant in Campylobacter isolates of chicken and swine origin (Yao et al., Reference Yao, Liu, Wang, Zhang and Shen2017). A genomic island containing multiple genes encoding aminoglycoside inactivating enzymes has been detected on transmissible plasmids in C. jejuni as well as in the chromosome of C. coli (Nirdnoy et al., Reference Nirdnoy, Mason and Guerry2005; Qin et al., Reference Qin, Wang, Zhang, Chen, Shen, Deng, Wu and Shen2012). Another gentamicin resistant gene, aph(2 ″ )-Ig, which share 28% amino acid identity with aph(2 ″ )-If , was detected on a 55 kb conjugative MDR plasmid that shared 95% nucleotide sequence identity with a pTet plasmid in Campylobacter (Chen et al., Reference Chen, Mukherjee, Hoffmann, Kotewicz, Young, Abbott, Luo, Davidson, Allard, McDermott and Zhao2013). A recent study identified nine variants of gentamicin resistance genes in Campylobacter isolates from the NARMS program, including aph(2 ″ )-Ib, -Ic, -Ig, -If, -If1, -If3 and -Ih, aac(6 ′ )Ie/aph(2 ″ )-Ia and aac(6 ′ )-Ie/aph(2 ″ )-If2 (Zhao et al., Reference Zhao, Mukherjee, Chen, Li, Young, Warren, Abbott, Friedman, Kabera, Karlsson and McDermott2015). These recent findings clearly indicate a rising trend of aminoglycoside resistance and the continuous emergence of new gentamicin resistance mechanisms in Campylobacter.

MDR mechanisms

Different from specific resistance mechanisms conferred by target or antibiotic modification, the multidrug efflux pump confers a broad spectrum of resistance to structurally unrelated antimicrobials. In Campylobacter, two MDR mechanisms have been described including Cfr (described above) and multidrug efflux transporters, among which the RND type of transporters are the most significant for antibiotic resistance. In Campylobacter, CmeABC and CmeDEF are the functionally characterized RND-type of efflux systems. However, CmeDEF only contribute moderately to intrinsic resistance, while CmeABC plays a significant role in both intrinsic and acquired resistance of Campylobacter to different antibiotics (Lin et al., Reference Lin, Michel and Zhang2002; Akiba et al., Reference Akiba, Lin, Barton and Zhang2006; Gibreel et al., Reference Gibreel, Wetsch and Taylor2007). CmeABC is a tripartite multidrug efflux pumps and consists a periplasmic fusion protein (CmeA), an inner membrane efflux transporter (CmeB) and an outer membrane protein (CmeC) (Lin et al., Reference Lin, Michel and Zhang2002). The three proteins function together to form an intact efflux system that extrudes antibiotics and toxic compounds. CmeB forms a trimeric structure in the bacterial membrane. A recent study using X-ray crystallography and single-molecule fluorescence resonance energy transfer imaging revealed that the CmeB transporter undergoes conformational transitions uncoordinated and independent of each other, suggesting a novel transport mechanism where CmeB protomers function independently within the trimer (Su et al., Reference Su, Yin, Kumar, Dai, Radhakrishnan, Bolla, Lei, Chou, Delmar, Rajashankar, Zhang, Shin and Yu2017). The function of CmeABC in antibiotic resistance has been demonstrated in many published studies (Lin et al., Reference Lin, Michel and Zhang2002; Hannula and Hanninen, Reference Hannula and Hanninen2008; Guo et al., Reference Guo, Lin, Reynolds and Zhang2010; Mavri and Smole Mozina, Reference Mavri and Smole Mozina2013). In addition to conferring resistance to antibiotics, CmeABC also plays a significant role in bile resistance and thus is essential for Campylobacter colonization in the intestinal tract (Lin et al., Reference Lin, Sahin, Michel and Zhang2003).

The recent discovery of RE-CmeABC further demonstrates the key role of CmeABC in conferring MDR (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). The CmeB of RE-CmeABC is unique and shares only ~80% amino acid identity with the CmeB in NCTC 11168 and other strains. This efflux variant is much more powerful than the typical CmeABC in the extrusion of antibiotics. For example, transforming C. jejuni NCTC 11168 with RE-CmeABC showed 32-, 16-, 8-, 4-, and 4-fold increases in the MICs of florfenicol, chloramphenicol, ciprofloxacin, erythromycin, and tetracycline, respectively, compared with the recipient strain NCTC 11168 that has a typical CmeABC (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). Notably, Re-CmeABC confers exceedingly high-level resistance to FQs, resulting in a ciprofloxacin MIC ≥ 256 µg ml⁻¹ in FQ-resistant C. jejuni isolates. Re-CmeABC also contributes to enhanced emergence of FQ-resistant mutants under antibiotic selection, and drug accumulation assays confirmed the enhanced efflux function of Re-CmeABC (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). Interestingly, RE-CmeABC was found to be much more prevalent in C. jejuni (~35%) than in C. coli (~3%), and the proportion of C. jejuni harboring Re-CmeABC appeared to be on the rise in China (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016). This trend is probably driven by the extensive use of antibiotics for animal production in China and suggests a fitness advantage for C. jejuni strains carrying RE-CmeABC. The findings on Re-CmeABC also explains why florfenicol resistance is highly prevalent, and more so in C. jejuni than in C. coli in China (described above). Additionally, homologs of Re-CmeABC are found in the GenBank database and are deposited by investigators from different countries, suggesting that RE-CmeABC is not unique to China. The exact mechanism for the enhanced function of Re-CmeABC is unknown, but structural modeling suggested that sequence variations in the drug-binding pocket of CmeB may enhance its interaction with antibiotics and consequently increase its efflux function (Yao et al., Reference Yao, Shen, Wang, Deng, Liu, Naren, Dai, Su, Wang, Wang, Wu, Yu, Zhang and Shen2016).

The expression of cmeABC is subject to regulation. CmeR, a transcriptional repressor of cmeABC, directly interacts with the cmeABC promotor region and represses the transcription of this operon (Guo et al., Reference Guo, Wang, Shi, Barton, Plummer, Reynolds, Nettleton, Grinnage-Pulley, Lin and Zhang2008; Routh et al., Reference Routh, Su, Zhang and Yu2009). Insertional mutagenesis of cmeR or point mutation in the binding sites of CmeR abolish the binding of CmeR to the promoter, releasing the repression and enhancing the expression of cmeABC (Cagliero et al., Reference Cagliero, Maurel, Cloeckaert and Payot2007; Guo et al., Reference Guo, Wang, Shi, Barton, Plummer, Reynolds, Nettleton, Grinnage-Pulley, Lin and Zhang2008). CosR, a response regulator in C. jejuni, modulates the oxidative stress response and also plays a role in the repression of cmeABC expression (Hwang et al., Reference Hwang, Zhang, Ryu and Jeon2012; Grinnage-Pulley et al., Reference Grinnage-Pulley, Mu, Dai and Zhang2016). cosR is an essential gene in Campylobacter, but knockdown of cosR expression by use of antisense peptide nucleic acid increased the transcriptional levels of cmeABC (Hwang et al., Reference Hwang, Zhang, Ryu and Jeon2012). CosR directly binds to the promoter region of cmeABC, but the binding site is independent of the one bound by CmeR (Grinnage-Pulley and Zhang, Reference Grinnage-Pulley and Zhang2015). The fact that CmeABC is regulated by multiple mechanisms indicates that it may respond to multiple signals in the host or environment. For example, bile acids, which are present in the intestinal tract of animals, strongly induce the expression of cmeABC by inhibiting the function of CmeR (Lin et al., Reference Lin, Cagliero, Guo, Barton, Maurel, Payot and Zhang2005b; Gu et al., Reference Gu, Su, Shi, Li, McDermott, Zhang and Yu2007). This induced expression of CmeABC facilitates Campylobacter adaptation to the intestinal environment as it plays a key role in Campylobacter resistance to bile (Lin et al., Reference Lin, Sahin, Michel and Zhang2003). Additionally, Salicylate, a nonsteroidal anti-inflammatory compound, is also shown to induce cmeABC expression by inhibiting binding of CmeR to the promoter of cmeABC (Shen et al., Reference Shen, Pu and Zhang2011). These examples illustrate the essential functions of CmeABC beyond antibiotic resistance.