Antibiotic resistance mediated by site‐specific mutations in the multidrug efflux transporter CmeB of zoonotic pathogen Campylobacter
Xiaolong Lin, Mengyu Zhao, Jianhong Gan, Haozheng Li, Min He, Fang Yang, Renqiao Wen, Tiejun Zhang, Quan Zhou, Ke Wu, Jinpeng Li, Chengyao Hou, Yang Cao, Hongning Wang, Yizhi Tang
TL;DR
The study identifies mutations in the CmeB protein of Campylobacter that enhance antibiotic resistance by improving the bacteria's ability to expel drugs.
Contribution
A new mutation-based mechanism of multidrug resistance in Campylobacter via CmeB efflux transporter is identified.
Findings
T136A and M292I mutations in CmeB are required for resistance to florfenicol and other antibiotics.
The mutations enhance hydrophobic interactions and stabilize antibiotic binding in the drug-binding pocket of CmeB.
CmeB-harboring mutations are widely distributed across 35 countries and multiple host species.
Abstract
The resistance‒nodulation‒division (RND) family of multidrug efflux transporters is widely distributed in Gram‐negative bacteria. Although their roles in mediating antibiotic resistance have been well known, our understanding of how they are altered to augment bacterial adaptation to antibiotic selection remains at an infancy stage. Here, we report the identification of a mutation‐based mechanism that empowers the function of the CmeB efflux protein, an RND‐type transporter in the zoonotic pathogen Campylobacter. During our surveillance study, we identified Campylobacter isolates that were phenotypically resistant to florfenicol but lacked known florfenicol resistance mechanisms. Using natural transformation and whole genome sequencing, we first linked the phenotype to sequence polymorphisms in the cmeB and subsequently demonstrated that both the T136A and M292I mutations in CmeB are…
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Figure 7| MIC (mg/l) | |||||||
|---|---|---|---|---|---|---|---|
| Strain | Description | FFC | CHL | CIP | ERY | TET | NAD |
| A31 |
| 32 | 64 | 128 | 32 | >128 | 128 |
| SH89 |
| 64 | 128 | 64 | 32 | >128 | 128 |
| TF31 | Transformant of NCTC 11168 carrying CmeABC from A31 | 16 | 16 | 2 | 2 | 1 | 16 |
| TFSH89 | Transformant of NCTC 11168 carrying CmeABC from SH89 | 16 | 16 | 2 | 2 | 1 | 16 |
| 11168‐A31‐ | Transformant of NCTC 11168 carrying CmeRAB from A31 | 16 | 16 | 2 | 2 | 1 | 16 |
| 11168‐A31‐ | Transformant of NCTC 11168 carrying CmeB from A31 and an A‐G mutation in the promoter region | 16 | 16 | 2 | 2 | 1 | 16 |
| 11168‐SH89‐ | Transformant of NCTC 11168 carrying CmeRAB from SH89 | 16 | 16 | 2 | 2 | 1 | 16 |
| 11168‐SH89‐ | Transformant of NCTC 11168 carrying CmeB from SH89 | 8 | 8 | 1 | 1 | 0.5 | 8 |
| 11168‐M7A | NCTC 11168 with T136A, A140G, N257D, V267I, M292I, N294D, and H303N mutations in CmeB and insertion of an A base in the promoter region | 16 | 16 | 1 | 2 | 1 | 16 |
| 11168‐M7 | NCTC 11168 with T136A, A140G, N257D, V267I, M292I, N294D, and H303N mutations in CmeB | 8 | 8 | 0.5 | 1 | 0.25 | 8 |
| 11168‐M4 | NCTC 11168 with T136A, N257D, V267I, and M292I mutations in CmeB | 8 | 8 | 0.5 | 1 | 0.25 | 8 |
| 11168‐M2A | NCTC 11168 with T136A and M292I mutations in CmeB and insertion of an A base in the promoter region | 16 | 16 | 1 | 2 | 1 | 16 |
| 11168‐M2 | NCTC 11168 with T136A and M292I mutations in CmeB | 8 | 8 | 0.5 | 1 | 0.25 | 8 |
| 11168‐ | NCTC 11168 with | 1 | 2 | 16 | 0.25 | 0.125 | 4 |
| 11168M2‐ | NCTC 11168 with T136A and M292I mutations in CmeB and | 8 | 8 | 64 | 1 | 0.25 | 8 |
| NCTC 11168 |
| 1 | 2 | 0.125 | 0.25 | 0.125 | 4 |
- —National Natural Science Foundation of China10.13039/501100001809
- —Sichuan Science and Technology Programs
- —National Key Research and Development Program of China10.13039/501100012166
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Taxonomy
TopicsSalmonella and Campylobacter epidemiology · Antibiotic Resistance in Bacteria · Bacterial Genetics and Biotechnology
INTRODUCTION
The resistance‒nodulation‒division (RND) family of multidrug efflux transporters is commonly present in Gram‐negative bacterial species1, 2. These efflux systems typically include an inner membrane transporter, a periplasmic fusion protein, and an outer membrane channel protein, which function together to actively expel a broad range of antimicrobial agents, such as antibiotics and other toxic compounds, from the cellular interior to the exterior, thereby reducing the concentration of antibiotics inside bacterial cells3, 4, 5. Overexpression of an RND efflux system or mutation in the inner membrane transporter proteins has been reported to increase the function of these efflux systems, contributing to the acquired resistance to antimicrobials6, 7.
Campylobacter spp., a genus of Gram‐negative bacteria, are well known for their roles in causing animal and human diseases8, 9. Particularly, Campylobacter jejuni and Campylobacter coli are major foodborne pathogens and are frequently associated with bacterial gastroenteritis in humans10, 11. Due to the widespread use of antibiotics in both human medicine and animal husbandry, antibiotic resistance in Campylobacter species has significantly increased, posing a major threat to public health12, 13. Hence, antibiotic‐resistant Campylobacter has been listed as a priority for control by the Centers for Disease Control and Prevention (CDC) (https://www.cdc.gov/antimicrobial-resistance/data-research/threats/index.html). Among the various mechanisms contributing to antibiotic resistance, multidrug efflux systems are important players in both intrinsic and acquired antimicrobial resistance. In Campylobacter, the best characterized and functionally the most important efflux system is the Campylobacter multidrug efflux (Cme) pump named CmeABC14, 15, which is a member of the RND‐type family of transporters.
The CmeABC efflux pump in Campylobacter is composed of three proteins, including CmeA (periplasmic fusion protein), CmeB (inner membrane transporter), and CmeC (outer membrane channel protein)14. Despite the presence of other antibiotic efflux transporters in Campylobacter, the CmeABC system is the major and predominant antibiotic efflux machinery in Campylobacter species16. By actively extruding substrates out of bacterial cells, CmeABC significantly reduces the susceptibility of Campylobacter to a range of antimicrobials including bile acids that are present in animal intestinal tract.17, 18, 19. Particularly, the synergistic role of CmeABC with other mechanisms in conferring acquired resistance to various antibiotics has been well documented in previous studies20, 21, 22, 23, but phenotypic resistance conferred by CmeABC alone has rarely been reported. The cmeABC operon is controlled by the CmeR transcriptional regulator, and overexpression of cmeABC has been linked to mutations in the promoter region of the efflux operon24, 25, 26. A naturally occurring CmeABC variant, named RE‐CmeABC, was identified in C. jejuni 26. This is a unique variant, as RE‐CmeB only shares 81% amino acid sequence identity with the CmeB in C. jejuni NCTC 11168 and forms a clade that is distinct from the CmeB sequences of C. jejuni and C. coli. Notably, RE‐CmeABC confers enhanced resistance to multiple antibiotics, such as chloramphenicol, florfenicol, tetracycline, erythromycin, and ciprofloxacin26, 27, 28. The molecular and structural basis of RE‐CmeB‐mediated antibiotic efflux has been elucidated recently, revealing key amino acids involved in binding to antibiotics29.
In addition to overexpression, which increases the contribution of RND efflux systems to antibiotic resistance, amino acid substitutions within the inner membrane transporters have also been reported to increase antibiotic efflux efficiency of the transporters, producing clinically relevant resistance to antibiotics. For example, a G288D substitution in AcrB of Salmonella was found to affect the function of the distal binding pocket and therefore resulted in resistance to ciprofloxacin6. Another two amino acid substitutions, M78I and P319L, in AcrB reduced the susceptibility of Salmonella to multiple antibiotic substrates30. Additionally, two mutations, K79 and G287, in MexY of Pseudomonas aeruginosa enhanced resistance to aminoglycoside31. In Campylobacter, overexpression of cmeABC and identification of the unique RE‐CmeB variant have been reported26, but gain‐of‐function point mutations within the CmeB have not been described. In this study, we identified two function‐enhancing amino acid mutations in the cmeB gene in natural Campylobacter isolates. By using a stepwise transformation strategy and whole‐genome sequencing, we discovered that the T136A and M292I substitutions in the drug‐binding pocket of CmeB were responsible for the antibiotic resistance phenotype. Structural modeling and molecular dynamics simulation indicated that the two mutations enhanced antibiotic binding and transport by CmeB. Finally, we determined the distribution of the two mutations in Campylobacter genomes deposited in the NCBI database and found that the T136A and M292I mutations occurred in both C. coli and C. jejuni that were derived from different countries and host species.
RESULTS
Association of CmeABC mutations with enhanced resistance to florfenicol and other antimicrobials
We conduct routine surveillance of antimicrobial resistance in Campylobacter strains isolated from food‐producing animals in China. From 2023 to 2024, a total of 495 Campylobacter strains (both C. jejuni and C. coli) were isolated from fresh feces and cecal contents of swine and chicken from Sichuan, Yunnan, and Guizhou provinces. Among the 495 isolates, 163 were selected for antimicrobial susceptibility testing, representing different animal hosts and different farms/slaughterhouses. Of the 163 isolates, all (100%) were resistant to cefotaxime, 150 (92.1%) resistant to ciprofloxacin (CIP), 65 (39.9%) resistant to gentamycin (GEN), 100 (61.3%) resistant to amikacin (AMK), 135 (82.8%) resistant to azithromycin (AZM), 157 (96.3%) resistant to doxycycline (DOX), and 0 resistant to tigecycline (TIG) and imipenem (IMP) (Figure 1A). Additionally, 77 (47.2%) of the tested strains were resistant to florfenicol, with the minimum inhibitory concentration (MIC) of florfenicol ranging from 8 to 128 mg/l. Florfenicol is a therapeutic antibiotic widely used for the treatment of respiratory diseases in food‐producing animals in China. Whole Genome Sequences (WGS) analysis of known florfenicol‐resistant determinants revealed that 67.5% (52/77) of the florfenicol‐resistant strains were positive for optrA, 18.2% (14/77) positive for fexA, 3.9% (3/77) positive for cfr(C), 10.4% (8/77) positive for RE‐cmeABC, and one strain positive for both optrA and cfr(C). The remaining one C. coli strain A31 was phenotypically resistant to florfenicol, but lacked all known florfenicol resistance mechanisms. A31 presented a florfenicol MIC of 32 mg/l and a chloramphenicol MIC of 64 mg/l (Table 1). Another C. coli strain, SH89, was isolated from a swine slaughterhouse in Sichuan Province in 2019 and presented a florfenicol MIC of 64 mg/l and a chloramphenicol MIC of 128 mg/l. Although this strain harbored a (C)‐carrying plasmid, we found that the florfenicol‐resistant phenotype was transferrable via natural transformation without the plasmid transfer, suggesting that there is a novel florfenicol resistance mechanism independent of cfr(C) in this isolate.
*Prevalence of antimicrobial resistance and relative expression levels of cmeABC. (A) Antimicrobial resistance rates of the 163 Campylobacter strains analyzed in this study. Data were based on minimum inhibitory concentration (MIC) assay results generated in this study. AMK, amikacin; AZM, azithromycin; CIP, ciprofloxacin; CTX, cefotaxime; DOX, doxycycline; FFC, florfenicol; GEN, gentamicin; IMP, imipenem; TIG, tigecycline. (B) Relative expression levels of cmeA, cmeB, and cmeC in NCTC 11168 and TF31, as determined by quantitative RT‐PCR. The expression level in NCTC 11168 is used as the baseline. *p < 0.05; **p < 0.01; **p < 0.001.
A31 and SH89 were chosen for studying potentially novel florfenicol resistance mechanisms. Their purified genomic DNA was used as the donor DNA to naturally transform C. jejuni NCTC 11168, and transformants were selected on plates containing 4 mg/l florfenicol. Transformants were obtained from both A31 and SH89 DNA, indicating that the florfenicol resistance phenotype was transferable between C. coli and C. jejuni. The control transformation with no donor DNA did not yield any transformants. A total of four transformants, including two A31‐derived transformants and two SH89‐derived transformants, were selected for WGS. Comparative analysis of their genome sequences with the whole genome of the recipient strain C. jejuni NCTC 11168 (GenBank accession number NC_002163) revealed that the transferred sequences in the transformants were all located in the cmeABC sequences of A31 and SH89. Two transformants named TF31 and TFSH89 carried the longest cmeABC fragments of A31 (4232 bp) and SH89 (4248 bp), respectively, covering partial cmeR, cmeA, and partial cmeB (Figure 2A,B). Compared with the recipient strain C. jejuni NCTC 11168, both TF31 and TFSH89 presented 16‐fold increases in florfenicol MIC and eightfold increases in chloramphenicol MIC (Table 1). Additionally, TF31 and TFSH89 presented increased resistance to other antimicrobial agents, including ciprofloxacin (16‐fold), tetracycline (8‐fold), erythromycin (8‐fold), and nalidixic acid (4‐fold) (Table 1). Sequence analysis of the cmeABC promoter regions of TF31 and TF89 revealed an A‐to‐G and a G‐to‐A mutation, respectively, in the inverted repeat (IR) of the CmeR binding site (Figure 2A,B). Since mutations in the IR are known to affect CmeR binding and consequently result in overexpression of cmeABC 24, we conducted real‐time RT‐PCR to compare expression levels of cmeABC in TF31 and C. jejuni NCTC 11168. Indeed, the expression of cmeA, cmeB, and cmeC in TF31 increased 21‐, 6‐ and 9‐fold, respectively, compared with C. jejuni NCTC 11168 (Figure 1B). Taken together, these results suggest that the cmeABC sequences in A31 and SH89 are associated with increased resistance to florfenicol and other antimicrobials.
Diagrams showing the cmeR‐cmeABC region of various transformants generated in this study. (A) Campylobacter jejuni NCTC 11168 transformants generated using the genomic DNA or PCR‐amplified DNA of A31 as the donor DNA. (B) C. jejuni NCTC 11168 transformants generated using the genomic DNA or PCR‐amplified DNA of SH89 as the donor DNA. (C) Transformants generated by using synthesized cmeB gene fragments containing the four amino acid substitutions (T136A, N257D, V267I, and M292I), two amino acid substitutions (T136A and M292I) plus an A‐to‐G mutation in the inverted repeat (IR) of the promoter region, or two amino acid substitutions (T136A and M292I) without a promoter mutation, respectively. Bold letters indicated the left and right arms of the IR. Nucleotide changes in the IR region are colored red. Each red bar in panel C indicates a single amino acid substitution.
Identification of CmeB mutations associated with enhanced resistance to antimicrobials
When the sequences of the transformants were compared with that of C. jejuni NCTC 11168, we noted that none of the transformants, whether A31‐derived or SH89‐derived, acquired the entire cmeABC operon from the donor DNA, and all retained the native cmeC of NCTC 11168. To further determine which gene mutations in cmeABC play a major role in enhancing florfenicol resistance, the cmeRAB, cmeRA, and cmeB gene sequences were PCR amplified from A31 and SH89, respectively, and the purified PCR products were used as donor DNAs for natural transformation. The transformants were selected on Mueller Hinton (MH) agar plates containing 4 mg/l florfenicol. The PCR products containing the cmeRA sequences did not yield any transformants, while the PCR products containing cmeRAB or cmeB produced transformants on plates with 4 mg/l florfenicol. Notably, all the transformants contained a partial or complete cmeB sequence from A31 or SH89, and no transformants contained only cmeR or cmeA from the donor DNA, suggesting that cmeB mutations played an important role in florfenicol resistance. Importantly, the PCR products contained only cmeB sequences from A31 and SH89 also successfully generated transformants, and DNA sequence analysis confirmed the transfer of the cmeB sequences of A31 and SH89 into C. jejuni NCTC 11168. The resulting transformants were named 11168‐A31‐cmeB and 11168‐SH89‐cmeB, respectively. In the IR of the promoter region of 11168‐A31‐cmeB, which is located upstream of cmeA, there was a spontaneous A‐to‐G mutation (Figure 2A), which is known to result in overexpression of the cmeABC operon25. Phenotypically, 11168‐A31‐cmeB presented the same antibiotic resistance profile as that of TF31 (Table 1), suggesting that the mutations in cmeR and cmeA were not necessary for the enhanced function. Similarly, 11168‐SH89‐cmeB, despite the absence of the A‐to‐G mutation in the promoter region (Figure 2B), presented increased resistance to florfenicol (8‐fold), chloramphenicol (4‐fold), ciprofloxacin (8‐fold), erythromycin (4‐fold), tetracycline (4‐fold), and nalidixic acid (2‐fold) (Table 1). Together, these results indicated that the transferred mutations in cmeB, along with a mutation in the IR of the promoter region, produced MICs comparable with those of the transformants that carried the entire cmeABC operon from A31 or SH89, suggesting that cmeB mutations played a major role in enhancing antibiotic resistance.
Localization of the specific CmeB mutations responsible for the enhanced antibiotic resistance function
Among the transformants derived from the cmeB sequences of A31 or SH89, one generated from SH89 cmeB contained only seven amino acid substitutions (T136A, A140G, N257D, V267I, M292I, N294D, and H303N) in the N‐terminal region (Figure S1) of CmeB but presented the same antibiotic MICs as the transformants with the entire cmeB sequence of A31 or SH89. Additionally, sequencing of the cmeR‐cmeA intergenic region revealed a nucleotide insertion of base A between the left and right arms of the IR in the promoter region (Figure 2B). This transformant was named 11168‐M7A, which presented increased resistance to florfenicol (16‐fold), chloramphenicol (8‐fold), ciprofloxacin (8‐fold), erythromycin (8‐fold), tetracycline (8‐fold), and nalidixic acid (4‐fold) (Table 1). To further confirm the function of the 7 amino acid mutations in the enhanced resistance to multiple antibiotics, the 5' sequence (853 bp, from nucleotide 57 to 909) of cmeB containing the 7 amino acid mutations was amplified from SH89 and naturally transformed into C. jejuni NCTC11168. The transformants were selected on MH plates containing 2 µg/ml of florfenicol. The transformant was designated 11168‐M7 and presented increased resistance to the tested antibiotics, including florfenicol (8‐fold), chloramphenicol (4‐fold), ciprofloxacin (4‐fold), erythromycin (4‐fold), tetracycline (2‐fold), and nalidixic acid (2‐fold) (Table 1). Sequence analysis did not reveal any mutation in the IR of the promoter region of 11168‐M7. Together, the data suggest that the seven amino acid mutations are likely involved in the increased antibiotic resistance.
The CmeB amino acid sequences of A31 and SH89 are 97.5% homologous, and four (T136A, N257D, V267I, and M292I) out of the seven amino acid mutations in SH89 were also conserved in A31 (Figure S1). Comparison of the cmeB sequences of florfenicol‐resistant transformants revealed that the four mutations were present in all A31 cmeB‐derived and SH89 cmeB‐derived transformants, suggesting that they might be functionally important. To confirm this, we synthesized a C. jejuni NCTC 11168 cmeB gene fragment carrying the four amino acid substitutions (T136A, N257D, V267I, and M292I), which was then used as the donor DNA for natural transformation of 11168. The transformant, designated 11168‐M4, was selected on MH agar plates containing 2 mg/l of florfenicol. Sanger sequencing confirmed that the four amino acid mutations were successfully introduced into transformant 11168‐M4, and no mutations in the IR of the promoter region were detected (Figure 2C). As shown in Table 1, the MICs of florfenicol, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, and nalidixic acid were 8‐, 4‐, 4‐, 4‐, 2‐, and 2‐fold greater, respectively, in 11168‐M4 than in the recipient strain NCTC 11168. These data suggest that the four amino acid mutations are involved in the increase of antibiotic MICs.
To further determine whether all of the four amino acid mutations in CmeB are required for the increased resistance, four DNA fragments, each of which is a 631 bp sequence of the 5' region of cmeB containing a combination of three amino acid mutations, were synthesized and naturally transformed into C. jejuni NCTC 11168. Interestingly, only the constructs containing both the T136A and M292I changes in CmeB yielded transformants on MH agar plates containing 2 mg/l florfenicol, suggesting that the T136A and M292I mutations play an essential role in florfenicol resistance. The other donor DNA containing one of the two mutations did not yield transformants. To verify the role of the T136A and M292I changes in antibiotic resistance, a cmeB sequence with both T136A and M292I mutations was synthesized and naturally transformed into 11168, and the transformant designated 11168‐M2 was also selected on MH agar plates containing 2 mg/l florfenicol. Sanger sequencing confirmed that the two amino acid mutations were successfully introduced into the transformant 11168‐M2 (Figure 2C), in which no other nucleotide changes were observed. Compared with 11168‐M4, 11168‐M2 had the same MICs of florfenicol, chloramphenicol, ciprofloxacin, erythromycin, and tetracycline (Table 1). To verify the cooperative requirement between T136A and M292I changes, we also synthesized C. jejuni 111168 cmeB sequences that contained either T136A or M292I, and used them for natural transformation, but no florfenicol‐resistant transformants were obtained on MH plates containing 2 µg/ml of florfenicol. Additionally, we synthesized a fragment of C. jejuni NCTC 11168 cmeB sequence with the T136I and M292I mutations, and naturally transformed it into 11168, but no transformant was obtained. Together, these results strongly indicate that the T136A and M292I mutations in CmeB function together for the enhanced resistance to florfenicol and other antimicrobials, but the T136A or M292I mutation alone is not sufficient to mediate increased drug resistance. To further confirm the role of these two mutations in enhancing drug resistance in comparison with A31 and SH89, we introduced an A‐to‐G mutation in the IR region of the cmeABC promoter in 11168‐M2, and the resulting construct was named 11168‐M2A (Figure 2C), which presented florfenicol and chloramphenicol MICs comparable to those of TF31 and TF89 (Table 1), indicating that the two amino acid changes plus the mutation in the IR were sufficient to elevate the MICs to the level conferred by the cmeABC from A31 and SH89.
Distribution of CmeB A136 and I292 mutations in Campylobacter isolates examined in this study
Among the 77 florfenicol‐resistant Campylobacter isolates examined in this study, LZ‐1‐95 was another isolate in addition to A31 and SH89 that also harbored the T136A and M292I substitutions in its CmeB protein (Figure 3). Additionally, all three strains contained mutations in the IR of their cmeABC promoter. Among the 86 florfenicol‐susceptible strains, with the exception of three C. coli strains carrying a nonfunctional cfr(C) gene (thus not resistant to florfenicol), all the remaining strains did not contain any other known florfenicol resistance genes (Figure 3). Although 22 strains (25.6%, 22/86) contained the T136A mutation and 15 strains (17.4%, 15/86) contained the M292I in their CmeB, none of the phenotypically susceptible strains contained both T136A and M292I mutations (Figure 3). This result further corroborates that both amino acid changes are required for the florfenicol resistance phenotype. On the phylogenetic tree constructed using core genome SNPs, the three C. coli strains harboring T136A and M292I in their CmeB were distributed in different branches (Figure 3), suggesting that they were genetically divergent.
Core genome SNP‐based phylogeny of the 163 Campylobacter isolates examined in this study. The maximum‐likelihood phylogenetic tree was constructed by using the core genomes of the strains with MEGA 11. The innermost ring to the outermost ring represents florfenicol‐resistant or florfenicol‐susceptible phenotypes, the presence or absence of the known florfenicol resistance gene, the presence or absence of the T136A mutation, and the absence or presence of the M292I mutation, respectively. The three C. coli strains harboring the T136A and M292I changes in their cmeB genes were labeled with a red star.
The synergistic role of the T136A and M292I mutations with the gyrA mutation in conferring high‐level resistance to ciprofloxacin
CmeABC is known to function synergistically with other mechanisms, such as gyrA mutations, in conferring clinically relevant antibiotic resistance20, 23, 32. As shown with 11168‐M2 (Table 1), the T136A and M292I mutations in CmeB resulted in 4‐fold increase in ciprofloxacin MIC. To determine whether the mutated CmeB works synergistically with a gyrA mutation in elevating ciprofloxacin MIC, we introduced the C257T mutation into the gyrA gene (ACA to ATA; Thr > Ile) in both NCTC 11168 and 11168‐M2, and the resistant mutants were designated 11168‐gyrA and 11168‐M2‐gyrA, respectively. Compared to wild‐type C. jejuni 11168, the MICs of ciprofloxacin for 11168‐gyrA and 11168‐M2‐gyrA increased 128‐fold and 512‐fold, respectively, reaching 16 and 64 mg/l (Table 1). This result indicates that, coupled with the T86I GyrA substitution, the T136A and M292I mutations in CmeB confer a high level of resistance to ciprofloxacin.
The T136A and M292I mutations enhance antibiotic efflux as determined by structural modeling and molecular dynamics simulation
Previously, amino acids involved in antibiotic binding of RE‐CmeB were elucidated by using Cryo‐electron microscopy (Cryo‐EM)29. To help explain the enhanced function of the CmeB containing the T136A and M292I mutations, we conducted protein structural modeling and molecular dynamics simulation of CmeB interaction with florfenicol and ciprofloxacin by referring to the previously determined structures of RE‐CmeB complexed with antibiotics29. The modeling results indicated that the two amino acids are in the drug binding pocket, interfacing with both ciprofloxacin and florfenicol (Figure 4A). The T136A and M292I mutations enhance hydrophobic interactions with the antibiotics while simultaneously reducing the size of the side chains, providing additional space in the drug‐binding pocket (Figure 4A). Molecular dynamics simulations revealed significant disparities in binding strengths with the antibiotics. Although the interactions between florfenicol and the CmeB in 11168‐M2 appeared to be stronger than that in strain NCTC 11168, there were no significant differences in the binding free energy between florfenicol and the CmeB proteins of the four strains (Figure 4C). However, root mean square deviation (RMSD) analysis indicates that the binding of CmeB to both florfenicol and ciprofloxacin in 11168‐M2, A31, and SH89 is more energetically favorable than that in 11168 (Figure 4B,C), suggesting that the two amino acid mutations in CmeB stabilize antibiotic binding. The binding free energies between ciprofloxacin and CmeB in strains NCTC 11168, 11168‐M2, A31, and SH89 were −78.858 ± 8.710, −88.506 ± 8.613, −283.199 ± 2.206, and −228.744 ± 5.834 kJ/mol, respectively, suggesting a substantially stronger interaction of ciprofloxacin with CmeB in 11168‐M2, A31, and SH89 than in NCTC 11168 (Figure 4C). Given that both the T136A and M292I changes are necessary for the antibiotic resistance function, we further performed mutual information analysis to examine their interaction in antibiotic binding, which revealed a strong correlation in motion between positions 136 and 292 (Figure 5), suggesting that the two amino acids are functional partners in antibiotic binding. These data strongly suggest that the T136A and M292I substitutions increase antibiotic binding affinity, create a large space in the drug‐binding pocket to accommodate antibiotics, and function interactively to increase the stability of CmeB‐antibiotic complexes in Campylobacter.
Structural modeling and molecular dynamics simulation of antibiotic efflux by CmeB. (A) Patterns of florfenicol (left) and ciprofloxacin (right) at positions 136 and 292 (green for 11168‐M2 and yellow for 11168). The CmeB structures were constructed with Modeller by using the Cryo‐electron microscopy structures of RE‐CmeB as the template (PDB code: 8GJL). For florfenicol, the binding energies for T136 and M292 are −0.126 and −3.505 kJ/mol, while for A136 and I292 are −0.246 and −5.902 kJ/mol. For ciprofloxacin, the binding energies for T136 and M292 are −3.998 and −3.014 kJ/mol, while for A136 and I292 are −3.606 and −6.091 kJ/mol. (B, C) Molecular dynamics simulation showing the changes in the RMSD of the CmeB with ciprofloxacin (B) and florfenicol (C) over the 0–30 ns time frame. The binding free energy between CmeB and the two antibiotics during the 20–30 ns period of the molecular dynamics simulation is labeled at the bottom of each panel. BE, binding energy; RMSD, root mean square deviation.
Motion correlation determined by mutual information analysis. Correlations of amino acid position 136 with other amino acid sites in CmeB of Campylobacter jejuni NCTC 11168, 11168‐M2, A31, and SH89 are shown in the presence of ciprofloxacin (CIP; upper panels) or florfenicol (FFC; lower panels). The red triangle indicates position 292, and an arrow indicates the location of 136. The horizontal dashed line above zero in each panel indicates the correlation value between position 292 and 136.
Global distribution and phylogenetic relationship of A136‐ and I292‐containing CmeB in Campylobacter isolates
To determine the global distribution of the T136A and M292I substitutions of CmeB in Campylobacter, we analyzed the genomes of 121,814 Campylobacter isolates deposited in the NCBI GenBank database, from which 899 were found to carry the two mutations in their CmeB sequences, including 153 C. coli, 704 C. jejuni, and 32 other Campylobacter spp. (Figure 6A). Overall, the phylogenetic tree constructed using core genome sequences indicated that the 899 Campylobacter isolates belonged to multiple lineages, indicating they were genetically diverse (Figure 6A). These strains were derived from 1991 to 2024 from 35 countries (Figure 6B). Besides, these strains were isolated from various animal species (mule deer, dog, duck, alpaca, sheep, shellfish, swine, cow, bird, and poultry), humans, environment, foods, or unknown sources, among which poultry accounted for the largest proportion (162/899,18.3%) of the isolates (Figure 6C). Among the 899 strains, 246 (246/899, 27.7%) were untyped and the remaining 653 strains belonged to 222 known sequence types. The top three sequence types were ST‐45 (132/899, 14.9%), ST‐177 (45/899, 5.1%), and ST‐9432 (22/899, 2.5%) (Figure 6D).
Phylogenetic analysis and global distribution of Campylobacter isolates containing the T136A and M292I mutations in their cmeB genes. (A) The core genome SNP‐based phylogenetic tree of 889 Campylobacter isolates (153 Campylobacter coli, 704 Campylobacter jejuni, and 32 other Campylobacter spp.) containing the T136A and M292I mutations in their cmeB genes. The sequence data were derived from the NCBI database, and the maximum‐likelihood phylogenetic tree was constructed with MEGA11. The innermost circle to the outermost circle represents the species, the year, the location, and the host of the isolate. The red stars denote the strains analyzed in this study. (B) The distribution of Campylobacter isolates containing the T136A and M292I mutations in different years. (C) The percentage of host types of Campylobacter isolates with T136A and M292I mutations. (D) Percentage of sequence types of Campylobacter isolates containing the T136A and M292I mutations.
We further selected 37 C. coli strains whose cmeB sequences were closely related to the cmeB in A31 for detailed phylogenetic analysis, which showed three lineages (I, II, and III), and seven strains of chicken origin derived in 2020 and 2021 from China were indistinguishable based on their core genomes (Figure 7A). Similarly, two C. coli strains isolated from a cow in 2016 from Switzerland were identical, and three C. coli strains of chicken origin isolated in 2019 from China shared the same core genome (Figure 7A). These results suggest that some C. coli strains harboring A136 and I292 in their CmeB experienced localized clonal expansion. Detailed analysis of the 37 selected cmeB sequences revealed that 15 of them were identical or nearly identical, forming a single branch on the phylogenetic tree (Figure 7B). Interestingly, all of the 15 isolates were derived from China and were clustered in lineage III of the core genome tree (Figure 7A), suggesting that these strains were genetically related and might be originated from a single ancestor.
Phylogenetic analysis and distribution of selected Campylobacter isolates containing the T136A and M292I mutations in their cmeB genes. (A) Core genome SNP‐based phylogenetic tree of 37 selected Campylobacter coli strains whose cmeB sequences are closely related to the cmeB gene in A31. The dashed‐line boxes indicate the three lineages. (B) Phylogenetic tree of the cmeB sequences of the 37 C. coli strains selected for detailed analysis. C. coli 33559 and C. jejuni NCTC 11168 are included as references in the tree. In both panels, the red stars denote the strains used in this study.
DISCUSSION
In this study, we identified a novel antimicrobial resistance mechanism that involves two amino acid substitutions (T136A and M292I) in the RND‐type multidrug transporter CmeB and increases Campylobacter resistance to multiple classes of antibiotics (Table 1). Particularly, the two mutations alone were sufficient to elevate the MICs of ciprofloxacin and florfenicol to or above their epidemiological cutoff values in Campylobacter (https://www.eucast.org/mic_and_zone_distributions_and_ecoffs), indicating the clinical relevance of the resistance mechanism. This new antibiotic resistance mechanism is present in both C. coli and C. jejuni and distributed in multiple cmeB sequence types. Thus, this mechanism is distinct from the previously identified RE‐CmeB 26, which is a unique cmeB variant that is divergent from the cmeB sequences in both C. jejuni and C. coli and is predominantly detected in C. jejuni. As another evidence for the importance of this new mechanism, it was found in Campylobacter isolates from many different countries and various sources, including humans.
RND‐type efflux transporters play an important role in antibiotic resistance for Gram‐negative bacteria, and some well‐characterized efflux systems include E. coli AcrB, C. jejuni CmeB, Pseudomonas aeruginosa MexB, and Neisseria gonorrheae MtrD, for which high‐resolution structural data are available2, 33, 34, 35, 36, 37. Overexpression of these efflux transporters has been known to increase bacterial resistance to antibiotics, but mutation‐based functional enhancement in structural genes is being increasingly recognized as a new mechanism for antibiotic resistance. The mutations may occur in various regions of the inner membrane transporters, affecting the structural and functional properties of the proteins38. The transmembrane domains of RND‐type transporters are typically conserved, whereas the porter domain, where the antibiotic‐binding pocket is located, is flexible, facilitating adaptation to environmental challenges, including antibiotic selection. Most of the reported gain‐of‐function mutations in the RND‐type transporters occur in the porter domain, altering the substrate specificity or affinity to antibiotics. For example, the G288D substitution in AcrB of Salmonella Typhimurium altered the substrate specificity of the drug binding pocket and enhanced resistance to fluoroquinolone antibiotics1. Another study demonstrated that the M78I and P319L substitutions in AcrB confer high‐level ciprofloxacin resistance through dual mechanisms: enhanced substrate binding affinity at critical sites along the efflux pathway and increased flexibility that facilitates AcrB's functional rotation cycle30. Additionally, the R717L/R717Q mutations occurring in the drug binding pocket of AcrB were reported to reduce the susceptibility of Salmonella to macrolide antibiotics39, 40. Similarly, the R714 to H, L, or C changes in the MtrD efflux transporter decreased the susceptibility of N. gonorrheae to azithromycin7. Regarding the T136A and M292I mutations in CmeB identified in this study, they are also localized in the drug‐binding pocket (Figure 4A)29. Notably, the two mutations were identified from natural isolates that were resistant to florfenicol and other antibiotics, not from experimentally induced mutants. Therefore, the functional enhancement conferred by the T136A and M292I changes may have occurred in Campylobacter as a result of evolution in response to antibiotic selection pressure.
The CmeABC multidrug efflux pump is the predominant antibiotic efflux system in Campylobacter and plays an important role in reducing intracellular accumulation of antimicrobials. The efflux system is primarily controlled by CmeR, a transcriptional regulator modulating the expression of cmeABC 29, 41, 42. CmeR belongs to the TetR family of transcriptional regulators and negatively regulates cmeABC expression by binding to the IR sequence in the promoter region of the operon24. The cmeR gene is immediately upstream of the cmeABC operon, and mutations in either CmeR or the IR sequence disrupt this repression, leading to the overexpression of cmeABC 25, 43, 44, 45. Typically, CmeABC functions synergistically with other antibiotic resistance mechanisms (such as the C257T mutation in gyrA) to confer clinically relevant antibiotic resistance23, and overexpression of the efflux pump alone only resulted in a modest reduction in antibiotic susceptibility. Consistent with previous findings, we discovered that mutations in the IR sequence of the promoter region resulted in the overexpression of cmeABC (Figure 1B) and a modest increase in antibiotic MICs (Table 1). Thus, the contribution of the T136A and M292I mutations to antibiotic resistance is much larger than cmeABC overexpression, although the functions of the two mechanisms further elevated antibiotic MICs (Table 1). Furthermore, the synergy of the T136A and M292I mutations with other antibiotic resistance mechanisms was clearly shown with 11168‐M2, in which introduction of the C257T mutation increased ciprofloxacin MIC to 64 mg/l, 4‐fold above the MIC of the gyrA mutant in NCTC 11168 (Table 1). This finding suggests that the presence of the T136A and M292I mutations and other antibiotic resistance determinants may make Campylobacter exceedingly resistant to antibiotics, potentially compromising the efficacy of clinical therapies.
RE‐CmeABC is a functionally enhanced efflux pump of CmeABC in Campylobacter and has been identified in a number of clinical isolates from various host species, including humans in different regions28, 45, 46. RE‐CmeB is a distinct variant and is divergent from the CmeB sequences in both C. jejuni and C. coli. It only shares ~81% homology with the CmeB in C. jejuni NCTC 1116826, 45. Therefore, the RE‐CmeB sequences are clustered together, forming a unique branch on the phylogenetic tree. A recent study uncovered that the enhanced function RE‐CmeABC was due to a number of amino acid changes in RE‐CmeB, and the variant efflux system likely originated from C. coli 47. Different from RE‐CmeB, the cmeB genes containing T136A and M292I substitutions do not belong to a single variant and are distributed in different CmeB sequence types. Additionally, the two substitutions alone are sufficient to elevate the function of the efflux pump, which is in contrast to the large number of amino acid mutations involved in the functional gain of RE‐CmeB. Despite these differences, the functional consequences are similar for both RE‐CmeB and the newly identified mutations, as they increase the ability of Campylobacter to resist antibiotics. Thus, identification of the T136A and M292I mutations of CmeB in this study further enriches the mechanisms utilized by Campylobacter to counteract the action of antimicrobials.
Export of antibiotics by an RND‐type transporter involves a three‐step process including access, binding, and extrusion2, 5, 48. Each monomer of the trimeric membrane complex contains two distinct drug‐binding pockets, including a deep distal binding pocket (DBP) and a proximal binding pocket (PBP)37. The amino acid residue differences in the drug binding pocket influence the substrate specificity and binding affinity. Therefore, amino acid substitutions in the drug‐binding pocket may alter the activity of an efflux pump. Similar to the RND‐type transporters in other Gram‐negative bacteria, CmeB in Campylobacter also possesses a DBP and a PBP29. Notably, hydrophobic interaction was found to play an important role in drug binding. Based on the Cryo‐EM structures of RE‐CmeB antibiotic complexes, I291 (I292 in 1116‐M2) and I136 (T137 in 11168‐M2) are in the DBP and are directly involved in the interaction with antibiotics29. Structural modeling and molecular dynamics simulation performed in this study indicated both the T/A136 (T135 in RE‐CmeB) and M/I292 were at the binding interface of DBP of CmeB, and the T136A and M292I mutations increased the hydrophobic interaction with antibiotics and reduced the binding energy, stabilizing antibiotic binding by CmeB (Figures 4 and 5). Additionally, the two amino acid mutations reduced steric hindrance in the drug binding pocket, potentially creating a larger space to transport antibiotics. Thus, the triple effects of the mutations may have contributed to the potent function of CmeB containing the T136A and M292I substitutions in expelling antibiotics. Notably, the two mutations were not able to function independently, which suggests that they are interdependent in conferring antibiotic resistance. This is consistent with the result from mutual information analysis (Figure 5), which revealed functional interaction of the two residues in CmeB.
The 899 Campylobacter isolates harboring the A136 and I292 in their cmeB genes were distributed globally and present in various hosts. The fact that these isolates are genetically diverse (Figure 6) and the two mutations exist in divergent CmeB sequence types (Figure 7) suggests that the two mutations probably developed by evolution and adaptation in response to antibiotic selection pressure. Interestingly, many of the isolates identified in the database are C. jejuni, although the two gain‐of‐function mutations were originally identified from C. coli. Given that transfer of the two mutations to C. jejuni NCTC 11168 increased antibiotic MICs (Table 1), the C. jejuni isolates carrying the two mutations in their cmeB genes are expected to have an enhanced ability to resist antibiotics. Additionally, we observed that the two mutations were associated with Campylobacter isolates from humans (Figure 6), indicating the clinical relevance of the mutations and their potential role in facilitating Campylobacter adaptation to clinical treatments. Interestingly, 15 CmeB sequences harboring the two mutations were either identical or almost identical and are clustered together in the CmeB‐based phylogenetic tree (Figure 7B). This also correlates with the fact that they all belonged to one lineage (III) on the core genome SNP‐based phylogenetic tree (Figure 7A), suggesting that these isolates were genetically related, although they were derived from different hosts and countries (Figure 7A). Seven of the 15 isolates also showed identical core genomes (Figure 7A), and they were all derived from chickens in China, suggesting that clonal expansion was involved in their regional spread.
In conclusion, we identified the T136A and M292I mutations in the drug‐binding pocket of CmeB as a new mechanism for mediating resistance to florfenicol and other antimicrobial agents in Campylobacter. This mechanism is present in both C. jejuni and C. coli, and it alone is sufficient to confer clinically relevant resistance to ciprofloxacin and florfenicol. The finding enriches our understanding of the molecular mechanisms underlying mutation‐based functional enhancement of RND‐type transporters in Gram‐negative pathogens. It also enhances our knowledge about the evolution of CmeABC in facilitating Campylobacter adaptation to antibiotic selection pressure. Given that the two mutations are sufficient to elevate MICs of florfenicol and ciprofloxacin above their epidemiological cutoff values, they may be used as potential targets for surveillance of antibiotic‐resistant Campylobacter. The findings could also guide the design of next‐generation inhibitors with improved efficacy against the CmeB efflux pump. Additionally, the identified mutations could be used for predicting the risk associated with treatment failure clinically. From this perspective, continued monitoring of their occurrence in Campylobacter isolates on a global scale is warranted.
MATERIALS AND METHODS
Campylobacter strains and antimicrobial susceptibility testing
For surveillance of antimicrobial resistance in Campylobacter, we routinely collect fecal samples from food‐producing animals for isolation of Campylobacter. From 2023 to 2024, 495 Campylobacter strains were isolated from fresh feces and the cecal contents of swine and chickens collected from Sichuan, Yunnan, and Guizhou provinces, China. Among the Campylobacter isolates, C. coli A31, with a florfenicol resistance phenotype but lacking a known florfenicol resistance genotype, was isolated from slaughterhouses in Sichuan Province in 2023. C. coli SH89 was isolated from a swine slaughterhouse in Sichuan Province in 201949. Although it harbored a (C)‐carrying plasmid, we found that the florfenicol‐resistant phenotype was transferable via natural transformation even without transfer of the plasmid, indicating that there was a novel florfenicol resistance mechanism. All Campylobacter strains were grown in MH broth or agar (Sigma) at 42°C under microaerobic conditions (5% O_2_, 10% CO_2_, 85% N_2_). When needed, the culture media were supplemented with 2, 4, or 8 mg/l florfenicol.
Among the 495 Campylobacter isolates, 163 were randomly selected for antimicrobial susceptibility testing. Among the 163 Campylobacter strains, 35 were derived from chicken and 128 were from swine, 23 were C. jejuni, and 140 were C. coli. The MICs of 10 antibiotics were determined via broth microdilution as recommended by the Clinical and Laboratory Standards Institute (CLSI)50. The 10 antibiotics tested in this study included cefotaxime, imipenem, ciprofloxacin, gentamicin, amikacin, azithromycin, doxycycline, tigecycline, chloramphenicol, and florfenicol. For identification of specific amino acid mutations in CmeB that contributed to the enhanced multidrug resistance phenotype, C. coli isolates A31, SH89, and LZ‐1‐95, C. jejuni NCTC 11168, and NCTC 11168‐derived strains and transformants were analyzed for susceptibility to florfenicol, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, and nalidixic acid via the broth microdilution method recommended by the CLSI.
Detection of florfenicol resistance determinants by PCR
Using an MIC ≥ 8 mg/l as the breakpoint50, a total of 77 florfenicol‐resistant Campylobacter strains were selected for PCR detection of known florfenicol resistance genes in Campylobacter, including optrA, fexA, (C) and RE‐cmeABC. PCR detections were conducted using the primers listed in Table S1.
Natural transformation
Natural transformation was performed using protocols established in prior studies51. Briefly, the genomic DNA of C. coli A31 and SH89 was purified and served as the donor DNA. C. jejuni NCTC 11168, which is sensitive to florfenicol (MIC = 1 mg/l), served as the recipient strain. Selection of transformants was performed on MH agar plates supplemented with 4 mg/l florfenicol. A reaction free of donor DNA was used as the negative control to monitor the occurrence of spontaneous resistant mutants.
To determine which gene mutations in cmeABC play a major role in enhancing florfenicol resistance, the cmeRAB, cmeRA, and cmeB gene sequences were PCR amplified from A31 and SH89 using primers listed in Table S1, and the amplified PCR products were used as donor DNA to transform C. jejuni NCTC 11168. The transformants were selected on MH agar plates containing 4 mg/l florfenicol.
To locate the mutations responsible for the enhanced florfenicol resistance, the 5' sequence (853 bp; nt 57 to 909) of the cmeB gene in SH89 was amplified via PCR using primers 11168M7A‐F and 11168M7A‐R (Table S1). The purified PCR product was used as donor DNA to transform C. jejuni NCTC 11168, and the transformants were subsequently selected on MH agar plates containing 2 mg/l florfenicol. The resulting transformant was named 11168‐M7.
To identify the specific resistance‐conferring amino acid mutations in the cmeB genes of A31 and SH89, a 631 bp fragment (nt 296 to 926) of the C. jejuni NCTC 11168 cmeB gene with artificially introduced T136A, N257D, V267I, and M292I amino acid substitutions was synthesized by Tsingke Company (Tsingke Biotechnology, Co., Ltd.). This synthesized DNA fragment was introduced into NCTC 11168 via natural transformation. The transformants were selected on plates containing 2 mg/l florfenicol and designated 11168‐M4. Additionally, four DNA constructs, representing the four different combinations of any three of the four amino acid mutations, were synthesized and naturally transformed into C. jejuni NCTC 11168. Furthermore, the cmeB gene fragments with the two T136A and M292I mutations, and with one of the two mutations alone, were synthesized, and the synthesized DNAs were introduced into C. jejuni NCTC 11168 via natural transformation. The transformants 11168‐M3 (with three mutations in cmeB) and 11168‐M2 (with two mutations in cmeB) were selected on plates containing 2 mg/l florfenicol. To measure the additive effect of the promoter mutation on antibiotic MICs, we introduced an A‐to‐G mutations in the promoter region of 11168‐M2 by natural transformation using a synthesized 376 bp construct, covering the 3' region of cmeR (108 bp), the cmeABC promoter region with an A‐to‐G mutation in the IR (94 bp), and the 5' region of cmeA (174 bp), into NCTC 11168‐M2, the transformants were subsequently selected on MH agar plates containing 8 mg/l florfenicol. All the transformants mentioned above were analyzed via PCR and Sanger sequencing of the cmeB gene or promoter region to confirm that the desired mutations were introduced into NCTC 11168.
Induction of the T86I mutation in the gyrA gene
C. jejuni strains NCTC 11168 and 11168‐M2 were used for the induction of the C257T (T86I) mutation in the gyrA gene, which is known to confer fluoroquinolone resistance in Campylobacter 52. The two strains were initially cultured on MH agar plates at 42°C under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂) overnight. Colonies from the overnight culture were suspended in antibiotic‐free MH broth, and the cell density was adjusted to an OD_600_ of 0.2. Aliquots of 100 µl of the bacterial suspension were spread onto MH agar plates supplemented with 4 mg/l ciprofloxacin. The plates were incubated at 42°C under microaerophilic conditions for 48 h. Single colonies were picked from the ciprofloxacin‐containing plates. The gyrA gene was amplified via PCR using specific primers (Table S1), and Sanger sequencing was subsequently performed to verify the presence of the T86I (C257T) mutation in the gyrA gene.
WGS
The 163 Campylobacter isolates examined in this study for antimicrobial susceptibility and various transformants of C. jejuni NCTC 11168 were subjected to whole genome sequencing analysis. Bacterial DNA was isolated using the Tiangen Bacterial DNA Extraction Kit. WGS was performed on the Illumina HiSeq platform (Novogene). Raw sequencing reads underwent quality assessment with fastaQC v0.12.1 and adapter trimming with Cutadapt v4.253, 54. High‐quality reads were de novo assembled into contigs using SAPdes v3.15.255. To close the whole genome of A31, its genome was sequenced by both Oxford Nanopore GridION (Novogene) and Illumina platforms. Hybrid assembly of short‐read Illumina and long‐read Nanopore data was conducted with Unicycler v0.5.0 to generate complete genome sequences56.
Real‐time RT‒PCR analysis of cmeABC transcription
The expression levels of cmeABC were determined by using a previously described real‐time RT‒PCR method17. Briefly, C. jejuni NCTC 11168 and transformant TF31 were grown in MH overnight at 42°C under microaerobic conditions. Then, each culture was treated with two volumes of TaKaRa RNA/DNA Stabilization Reagent to preserve RNA integrity. Cells were harvested by centrifugation for RNA extraction. Total RNA was extracted via the TRIzol method. Total RNA was treated with gDNA Eraser (TaKaRa) to remove DNA contamination, which was confirmed by regular PCR. Four pairs of primers were designed to amplify the 16S rRNA, cmeA, cmeB, and cmeC genes, respectively (Table S2). Reactions were carried out using the TaKaRa One‐Step SYBR PrimeScript PLUS RT‐PCR Kit under the following cycling conditions: 2 min at 95°C and then 39 cycles of 5 s at 95°C and 30 s at 60°C. The 16S rRNA gene was used as a reference gene for normalization of samples. Three independent experiments were performed for each sample. Fold differences in cmeABC expression between TF31 and NCTC 11168 were determined using the 2−ΔΔCt method reported in a previous study57.
Analysis of the T136A and M292I mutations in CmeB of the 163 Campylobacter isolates
The whole‐genome sequences of the 163 Campylobacter isolates examined in this study were used to determine the presence of T136A and M292I mutations in CmeB, and the information was used to correlate with the antibiotic resistance profiles of these Campylobacter strains. The maximum‐likelihood phylogenetic tree was constructed by using the core genomes of the strains with MEGA1158.
Structural modeling
We constructed four CmeB models with the well‐established homology modeling software, Modeller59, by using the Cryo‐EM structures of RE‐CmeB (PDB code: 8GJL) as the template29. RE‐CmeB and the CmeB in C. jejuni NCTC 11168 share 81% amino acid identities. To explore the correlation between protein structure and antibiotic resistance, we constructed the binding conformation of CmeB with ciprofloxacin or florfenicol by referencing their respective Cyo‐EM structures. With the structures of 8GJL and 8GK4 as the templates29, the template‐based docking method, FitDock60, was used to build the CmeB‐ciprofloxacin complex and the CmeB‐florfenicol complex, respectively. The ligand for 8GJL is ciprofloxacin, and the ligand for 8GK4 is chloramphenicol, which shows a similarity score (PC‐score) of 0.96 to florfenicol.
Molecular dynamics simulation
We used Open Babel61 to protonate and hydrogenate the antibiotics at pH 7. Then, we prepared the topology and structure files of the antibiotics with the Antechamber tool62. For the CmeB protein, we constructed the topology and structure files using Gromacs63, selecting the AMBER99SB‐ILDN force field and the TIP3P water model. During this process, we reconstructed the hydrogen atoms of the protein according to the force field. We placed the protein and small molecules in a periodic simulation box, filled it with solvent water molecules using the SPC water model, and added Na^+^ and Cl^–^ ions to maintain electrical neutrality, achieving a NaCl concentration of 0.15 mol/l. Once the system was ready, we used Gromacs for energy minimization, followed by 100 ps of NVT equilibration, 100 ps of NPT equilibration, and a 30 ns molecular dynamics simulation. After the simulation, we calculated the interaction energy between the antibiotics (ciprofloxacin and florfenicol) and the protein (CmeB) using the g_mmpbsa tool64 in Gromacs. We converted the Gromacs trajectory files to DCD format using VMD65, and employed the bio3D package66 in R to perform normal mode analysis, calculating the motion correlation between mutation sites T136A and M292I.
Global prevalence analysis of A136‐ and I292‐contaning CmeB in Campylobacter isolates
To analyze the global prevalence of the T136A and M292I dual mutations in the CmeB efflux pump of Campylobacter, we screened 121,814 publicly available Campylobacter genomes from the NCBI database. Briefly, all Campylobacter CmeB protein sequences were retrieved from NCBI, and sequences harboring concurrent T136A and M292I substitutions were identified via multiple sequence alignment (MEGA software). Then, using NCBI's “identical protein groups” tool, genomic entries linked to dual‐mutated CmeB sequences were extracted. Python scripts automated the download of 889 C. jejuni genomes carrying these mutations (0.73% prevalence). Among the 899 isolates, 147 cmeB sequence types were identified and were aligned with ClustalW. The maximum‐likelihood phylogenetic trees based on core genome sequence and cmeB sequences were constructed with MEGA1158.
AUTHOR CONTRIBUTIONS
Xiaolong Lin: Conceptualization; formal analysis; investigation; methodology; software. Mengyu Zhao: Data curation; investigation; methodology. Jianhong Gan: Formal analysis; methodology; software. Haozheng Li: Investigation; methodology. Min He: Data curation; validation. Fang Yang: Data curation; investigation; methodology. Renqiao Wen: Data curation; investigation; methodology. Tiejun Zhang: Data curation; software. Quan Zhou: Investigation; software. Ke Wu: Data curation; software. Jinpeng Li: Methodology; software. Chengyao Hou: Data curation. Yang Cao: Software; writing—original draft. Hongning Wang: Data curation; Funding acquisition; resources. Yizhi Tang: Conceptualization; funding acquisition; resources; supervision; writing—original draft; writing—review and editing.
ETHICS STATEMENT
No animal or human research was involved in this study.
CONFLICT OF INTERESTS
The authors declare no conflict of interests.
Supporting information
Figure S1. Alignment of amino acid sequences of CmeB from Campylobacter jejuni NCTC 11168 and Campylobacter coli A31, SH89, and LZ‐1‐95. Different amino acids in the four proteins are highlighted in yellow. The red boxes highlight the four conserved substitutions in A31, SH89, and LZ‐1‐95.
Tables.
The reference list from the paper itself. Each links out to its DOI / PubMed record.
- 1Blair JM , Richmond GE , Piddock LJ . Multidrug efflux pumps in Gram‐negative bacteria and their role in antibiotic resistance. Future Microbiol. 2014;9:1165–1177.25405886 10.2217/fmb.14.66 · doi ↗ · pubmed ↗
- 2Du D , Wang‐Kan X , Neuberger A , van Veen HW , Pos KM , Piddock LJV , et al. Multidrug efflux pumps: structure, function and regulation. Nat Rev Microbiol. 2018;16:523–539.30002505 10.1038/s 41579-018-0048-6 · doi ↗ · pubmed ↗
- 3Zwama M , Yamaguchi A . Molecular mechanisms of Acr B‐mediated multidrug export. Res Microbiol. 2018;169:372–383.29807096 10.1016/j.resmic.2018.05.005 · doi ↗ · pubmed ↗
- 4Du D , Wang Z , James NR , Voss JE , Klimont E , Ohene‐Agyei T , et al. Structure of the Acr AB‐Tol C multidrug efflux pump. Nature. 2014;509:512–515.24747401 10.1038/nature 13205 PMC 4361902 · doi ↗ · pubmed ↗
- 5Li XZ , Plésiat P , Nikaido H . The challenge of efflux‐mediated antibiotic resistance in Gram‐negative bacteria. Clin Microbiol Rev. 2015;28:337–418.25788514 10.1128/CMR.00117-14PMC 4402952 · doi ↗ · pubmed ↗
- 6Blair JMA , Bavro VN , Ricci V , Modi N , Cacciotto P , Kleinekathӧfer U , et al. Acr B drug‐binding pocket substitution confers clinically relevant resistance and altered substrate specificity. Proc Natl Acad Sci USA. 2015;112:3511–3516.25737552 10.1073/pnas.1419939112 PMC 4371985 · doi ↗ · pubmed ↗
- 7Ma KC , Mortimer TD , Grad YH . Efflux pump antibiotic binding site mutations are associated with azithromycin nonsusceptibility in clinical Neisseria gonorrhoeae isolates. m Bio. 2020;11:e 01509‐20.32843551 10.1128/m Bio.01509-20PMC 7448274 · doi ↗ · pubmed ↗
- 8Mc Nulty CM , Wise R . Rapid diagnosis of Campylobacter‐associated gastritis. Lancet. 1985;325:1443–1444.10.1016/s 0140-6736(85)91865-32861379 · doi ↗ · pubmed ↗