Campylobacter species, particularly C. jejuni and C. coli, are leading zoonotic foodborne pathogen that can cause gastrointestinal infections (such as diarrhea) and immune diseases (such as Guillain-Barré syndrome) in humans (Silva et al., 2011). Unlike in humans, Campylobacter causes mostly asymptomatic infections in food-animals, including poultry, cattle, and pigs, and is considered a commensal bacterium (Burnham and Hendrixson, 2018). According to the World Health Organization (WHO), the major route of human campylobacteriosis is via the food-chain of animal products (such as meat and milk) which are contaminated with Campylobacter, present in animal feces, during the slaughter process (Jaakkonen et al., 2020).

Since 2013, the US Centers for Disease Control and Prevention has classified drug-resistant Campylobacter as a serious antibiotic resistance threat (CDC, 2013). In recent years, Campylobacter has become increasingly resistant to clinically important antimicrobials and has developed multiple mechanisms of antimicrobial resistance. These include mutations in target genes, such as 23S rRNA mutations that confer resistance to macrolides and, gyrA mutations associated with fluoroquinolone resistance; multidrug efflux pump CmeABC/RE-CmeABC extruding structurally diverse compounds and antimicrobials; and horizontally acquired antimicrobial resistance genes, such as tet(O), erm(B), fexA, and fosXCC (Aarestrup et al., 2008). Among the two major species of Campylobacter, C. jejuni and C. coli, which are mainly responsible for human infection, C. coli is known to be more resistant to antimicrobials (Liu et al., 2019b).

The cfr gene, which encodes 23S rRNA methyltransferase, confers resistance to five antimicrobial classes, namely phenicols, lincosamides, oxazolidionones, pleuromutilins, and streptogramin A, known as PhLOPS_A phenotype (Schwarz et al., 2000; Long et al., 2006). WHO classified lincosamides, pleuromutilins, and streptogramin A as “critically important antimicrobials: highly important” and warned that resistance to these antimicrobial agents would make it difficult to treat bacterial infection (WHO, 2019). Since the first report of cfr gene in Staphylococcus sciuri in 2006, five types of the cfr gene family have been reported: cfr, cfr(B), cfr(C), cfr(D), and cfr(E) (Schwarz et al., 2000; Deshpande et al., 2015; Hansen and Vester, 2015; Candela et al., 2017; Tang et al., 2017; Sassi et al., 2019; Stojković et al., 2019). Tang et al. (2017) first identified plasmid-mediated multi-drug resistance gene cfr(C) in C. coli isolates of feedlot cattle origin (Tang et al., 2017). Since the first report of cfr(C) in Campylobacter, three additional studies reported the presence of cfr(C) in Campylobacter species (Zhao et al., 2019; Liu et al., 2019a; Tang et al., 2020). However, the horizontal transferability of Campylobacter-derived cfr(C) has been reported in only one study (Tang et al., 2017), and the transfer mechanism of cfr(C) is still unclear and requires further research.

An understanding of genetic variation of cfr(C) and their transmission mechanisms can help control the propagation of antimicrobial multi-resistance, a serious problem when it is transferred to humans. Thus, in this study we analyzed genetic background and horizontal transfer unit of cfr(C) in C. coli strains through comparative genomic analysis to elucidate the genetic variation and horizontal transfer mechanism of the Campylobacter-derived cfr(C) gene from swine sources.

The rRNA methyltransferase N, encoded by cfr gene, causes m⁸-modification of A2503 of 23S rRNA, which is known to confer resistance to two antimicrobial agents, florfenicol and clindamycin (Long et al., 2006). Even though cfr(C) is known to induce antimicrobial resistance to critically important antimicrobials to humans, its transfer mechanism is still unclear and requires further research. In this study, nine novel cfr(C)-positive C. coli strains were isolated from swine sources (swine farms and slaughterhouses). To elucidate the horizontal transfer mechanism of the Campylobacter-derived cfr(C) gene, we analyzed the genetic background and horizontal transfer unit of cfr(C) in C. coli strains through comparative genomic analysis.

Compared to the previously reported sequences of cfr(C), two strains (CC021 and CC024) carried incomplete amino acid coding sequences of the cfr(C) gene. Insertion or deletion of one or two adenines at the polyadenine site, such as 19delA (amino acid: M7fs) identified in CC021, generates premature stop codons by downstream frameshifting, leading to premature chain termination (Park et al., 2000). Tang et al. (2020) reported loss of resistance to florfenicol and clindamycin of cfr(C) due to other nucleotide deletion mutations in the C. coli JP10 strain, in which a frameshift mutation occurred due to nucleotide deletion at position 738–756 of cfr(C). The cfr(C) amino acid sequence of CC024 (366 amino acids) exhibited 95.51% identity to that of C. coli Tx40 (379 amino acids). Notably, the original CC024 strain, as well as its transformant strain, showed resistance to florfenicol. According to a study of cfr protein structure and function, the function of the cfr protein analog was ensured by a highly conserved amino acid sequence around the C-terminus (G.DIdAACGQL) (Atkinson et al., 2013). In this study, although the amino acid sequence of the cfr(C) gene was truncated in the CC024 strain, the conserved C-terminal region of cfr(C) was preserved, which may ensure the function of cfr(C).

Remarkably, the transformants of CC027 and CC032 with three missense non-synonymous (ns) SNPs (281A > C, 532A > C, and 952A > G) showed weak resistance to florfenicol (4–8 mg/L). These three missense nsSNPs were also identified in the florfenicol resistant cfr(C)-positive strains, CC135 and CC159. The amino acid substitutions (E94A, K178Q, and I318V), due to three SNP sites, have also been identified in the florfenicol and clindamycin-resistant C. coli JZ_1_79 and SH96 strains reported by Tang et al. (2020). The cfr(C)-encoded rRNA methyltransferase has functional amino acid sites including three substrate entrance sites and eight substrate modulation sites (Atkinson et al., 2013). Considering that the mutation sites of CC027 and CC032 do not overlap with the mutation sites of the previous report, it can be inferred that the three SNPs of these two strains are not associated with the phenotype for florfenicol resistance. Therefore, in relation to decreased resistance to phenicols found in strains CC027 and CC032, further research on the genetic background and expression of cfr(C) is needed.

Except for CC159, all eight cfr(C)-positive C. coli strains showed resistance to clindamycin, although the resistance level was different between strains depending on the carriage of 23S rRNA A2075G substitution: four strains with 23S rRNA A2075G substitutions showed high-level clindamycin resistance, whereas strains without the 23S rRNA A2075G substitution showed low-level resistance. The 23S rRNA A2075G substitution is known to be the most common mechanism underlying high-level resistance to the macrolide and lincosamide classes of antimicrobial agents in Campylobacter (Perez-Boto et al., 2010). Considering that the resistance of bacteria to lincosamide is generally mediated by the combination of three mechanisms (ribosomal target methylation or mutation, efflux of antimicrobials, and drug inactivation) (Leclercq, 2002), strains with simultaneous cfr(C)-induced A2503 methylation and A2075G substitution could exhibit high resistance to the lincosamide class. Our result suggests that cfr(C)-induced A2503 methylation may lead to low-level resistance to clindamycin, which could be enhanced by the acquisition of A2075G substitution.

cfr(C) is encoded on transferable plasmids (Tang et al., 2017;Liu et al., 2019a; Tang et al., 2020) or the chromosome of cfr(C)-positive C. coli strains (Liu et al., 2019a; Tang et al., 2020). In this study, the cfr(C) gene was encoded on the bacterial chromosome in the form of gene cassettes in eight cfr(C)-positive C. coli strains, except the gene cassette of strain CC024, and a total of six different gene cassettes were identified. Consistent with previous studies, all six cfr(C) gene cassettes carried the aminoglycoside resistance gene, aphA3, which was encoded upstream of cfr(C) in the same direction (Tang et al., 2017; Zhao et al., 2019; Liu et al., 2019a; Tang et al., 2020). Three cassette types, namely GC-1 (CC021), GC-2 (CC041, 042, and 046), and GC-5 (CC024), carried the aminoglycoside-streptothricin resistance gene cluster “aphA3-sat4-aadE” in the upstream of the cfr(C)-cassette gene. The “aphA3-sat4-aadE” cluster was also reported to be encoded in the upstream of the cfr(C)-cassette in the C. coli SHP40 strain, where the cfr(C) gene was first reported on the chromosome (Liu et al., 2019a). In contrast, the aminoglycoside resistance gene cluster “aphA3-aadE” without “sat4” was encoded in the upstream of the cfr(C)-cassette in the plasmids of 2 C. coli strains, Tx40 and N61925F (Tang et al., 2017; Zhao et al., 2019). The aphA3 gene is an aminoglycoside-resistance determinant, encoding aminoglycoside 3′-phosphotransferase, and the sat4 gene is a streptothricin-resistance determinant, encoding streptothricin N-acetyltransferase (Lysnyansky and Borovok, 2021b). The “aphA3-sat4-aadE” cluster is one of the highly transmissible and disseminated clusters, mainly found in the plasmids of gram-positive (Staphylococcus and Enterococcus) and gram-negative bacteria (Campylobacter) (Boerlin et al., 2001; Lysnyansky and Borovok, 2021a; Guirado et al., 2022). The aphA3-sat4-aadE cluster is transmitted with various antimicrobial-resistance genes, such as ermB, optrA, and tet (O) (Werner et al., 2001, 2003; Palmieri et al., 2012). Although the aphA3-sat4-aadE cluster has been reported to be mediated by mobile genetic elements, such as Tn5405 (Werner et al., 2001), recent studies have also shown that in Campylobacter spp., this resistance gene cluster may be mediated by a process of homologous recombination (Qin et al., 2012). Homologous recombination may enable horizontal transfer and chromosomal integration of gene clusters without mobile genetic elements (de Vries and Wackernagel, 2002). In the case of GC-4, another resistance gene cluster, “ant1-pnp-lnu(G),” was encoded in the upstream of the cfr(C)-cassette. The lincosamide resistance gene, lnu (E), is encoded in the upstream of the ISEnfa5-flanked cfr gene cassette in the Streptococcus suis RN4220 strain (Zhao et al., 2014).

The random insertion of a gene cassette into cfr(C) has the potential to significantly affect the fitness and survival of Campylobacter, as the insertion of the cfr(C) gene cluster may interfere with crucial cellular processes of the bacterium. The insertion sites of GC-1 and -5, harboring genomic islands, were different from those of the potD gene reported by Tang et al. (2020). GC-1 was inserted into the pxpC gene, which encodes 5-oxoprolinase. 5-Oxoproline, a metabolite from the breakdown of glutamine, has been reported to inhibit bacterial growth when it accumulates without proper disposal (Niehaus et al., 2017). GC-5 was inserted into the bacteriohemerythrin-encoding gene. Hemerythrin helps maintain bacterial homeostasis through intracellular oxygen- and redox-sensing functions. Cells cannot rapidly adapt to environmental changes without hemerythrin (Kitanishi, 2022). Our results suggest that the cleavage of these two genes, caused by the insertion of cfr(C)-carrying GCs, could have detrimental effects on the survivability of C. coli strains. The GC-3, -4, and -6 types were inserted between genes without gene cleavage. The insertion site of GC-3 downstream of the rpoBC gene has been previously reported (Tang et al., 2020). However, the other two types of insertion sites, pnp to sbnD and ATPase-encoding gene to yfhM, are novel positions. In contrast, GC-2 (CC024) was inserted into a circular genetic element without a self-replication module, such as the replication initiation protein gene repA. This genetic element of CC024 was similar in genetic composition to the previously reported plasmids of C. jejuni and C. coli. In addition, differences in GC contents between insertion sites and other coding regions indicated that the cfr(C)-carrying cluster was not included in ancestral genetic elements. TraG, truncated by GC-2, plays major roles in conjugation, and is associated with the invasion of C. jejuni into epithelial cells (Poly et al., 2005). Thus, truncation of the traG gene could attenuate the pathogenicity and survivability of Campylobacter.

Of the six types of gene cassette, GC-1, -4, and -5 formed circular intermediates and were horizontally transferred to recipient C. coli strains. The transferred circular intermediate form was 3,678 bp in length and consisted of the four coding sequences “pcp-hp-cfr(C)-aphA3.” Although the present study found differences in the length and composition of cfr(C)-positive strains, the pcp gene-mediated circular intermediate “pcp-hp-ppo-cfr(C)-aphA3-sat4-aadE” has also been reported in the C. coli SHP40 strain (Liu et al., 2019a). Similar to our study, the cfr(C)-cassette was also encoded in the bacterial chromosome of the C. coli SHP40 strain. The pcp gene encodes an enzyme that is conserved in a variety of organisms, from prokaryotes (archaea and bacteria) to eukaryotes (Barrett et al., 2012). The pcp gene is found to be inserted at multiple sites in the bacterial genome, including chromosomes and plasmids, and this multiple insertion of pcp could mediate the intermolecular transfer of gene clusters by forming the circular intermediate: the circular intermediates released from chromosome/plasmids could be integrated into other chromosome/plasmids containing a homologous sequence, such as the pcp gene (Liu et al., 2019a). These characteristics of the pcp gene can facilitate the transmission of cfr(C), which forms a cluster with pcp. Similarly, tetO-mediated circular intermediates of ermB, escaping from original plasmids/chromosome, could be integrated into other bacterial genomes by targeting the multiple inserted tetO homologous sequence (Liu et al., 2019b). In particular, the CC041, CC042, and CC046 strains that form GC-4 have been reported as hyper-aerotolerant strains, along with ST830, which is related to clinical isolates, according to our previous study (Guk et al., 2021). Since these strains not only transfer cfr(C) to other strains but also have a higher probability for transmission to humans than other C. coli strains, hyper-aerotolerant cfr(C)-positive C. coli potentially carry higher risk than other cfr(C)-positive C. coli strains.

In GC-3, 4, 5, and 6, cfr(C) was flanked by the IS607 family member ISChh1. The IS607 family is one of the major insertion sequences (ISs) that mediate transfer of antimicrobial resistance in Campylobacter spp. (Chen et al., 2018). For example, IS607 has been reported to mediate the transfer of the optrA gene, which induces resistance to oxazolidinones and phenicols, as well as of fexA, a florfenicol–chloramphenicol resistance gene (Tang et al., 2021). The IS607 family encoded in the upstream of the cfr(C)-gene cassette was also identified in the plasmids of the JZ_1_79, SH89, and JZ_1_74 strains reported by Tang et al. (2020). Among the three cfr(C)-cassette types flanked by IS607, GC-3 and GC-6 were bracketed by two copies of ISChh1 in the same orientation at both sides of the gene cassette and formed the ISChh1-mediated circular intermediates “ISChh1-aphA3-cfr(C)-lnu(G)-pnp-antl1-hp-ATPase” and “ISChh1-aphA3-cfr(C)-hp,” respectively. Even though the circular intermediates of GC-3 and -6, namely CIR-2 and -3, were not horizontally transferred in our study, two copies of IS elements in the same orientation can form a circular intermediate and can be transferred using the mechanism “copy-out-paste-in,” since the IS607 family is a highly active IS element (He et al., 2019). Tang et al. (2020) confirmed the existence of IS607 only upstream of the cfr(C)-cassette but could not confirm the formation of circular intermediates by the IS607 family. GC-4 and -5, bracketed by the ISChh1 segment and pcp, formed a circular intermediate mediated by pcp regardless of the position of the ISChh1 segment. These results suggest that two copies of IS607 in the same orientation play an important role in the formation of a circular intermediate form of gene cassettes.

The CIR-1s of five strains (CC021, 041, 042, 046, and 135) were horizontally transferred from the chromosomes into the plasmid of the recipient strain CC166 in the conjugation assay. For all five strains, the pcp and aphA3 genes were present at the insertion sites, suggesting that insertion of CIR-1 may have been mediated by homologous recombination with pcp and aphA3. The conjugation of gene cassettes targeting homologous gene regions has been reported in several studies. Qin et al. (2012) suggested that the aminoglycoside resistance gene island of C. coli strain SX81 may be acquired from the C. coli strain RM2228 and mediated by homologous recombination of the cadF and CCO1582 genes (Qin et al., 2012). Taken together with our results, this suggests that the use of pcp, which is a commonly encoded gene in Campylobacter, as a vector in the horizontal transfer of cfr(C)-carrying gene cassettes may be a possible reason for the global success of cfr(C) propagation.

This is the first study to elucidate the horizontal transferability of cfr(C) and random insertion site of the chromosome-encoded cfr(C) cassette in Campylobacter spp. The results reveal novel cfr(C) variants along with their associated genetic environments in C. coli isolates and indicate the flexibility of C. coli in acquiring new antimicrobial resistance genes. The transferability of chromosomal cfr(C) to humans may be attributed to the global spread of multidrug resistance against clinically important antimicrobials. Thus, enhanced surveillance is needed to monitor the emergence and spread of cfr(C) in Campylobacter from swine sources as well as other pathogens.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, PRJNA1000181.

J-UA: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. SL: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – original draft, Writing – review & editing. J-HG: Conceptualization, Formal analysis, Methodology, Writing – original draft. JW: Conceptualization, Formal analysis, Methodology, Writing – original draft. HS: Writing – review & editing. SC: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing.