Emergence of the cfr Gene in Vibrio diabolicus of Seafood Origin

ABSTRACT

The emergence and transmission of multidrug resistance (MDR) gene cfr have incurred great public health concerns worldwide. Recently, Gram-negative pathogens were found to carry cfr by various mobile elements. Here, we investigated a cfr-positive Vibrio diabolicus isolate by phenotyping and genomic analysis and found cfr in a translocatable structure (IS26-hp-cfr-IS26) among the MDR region in pNV27-cfr-208K, an emerging MDR plasmid in Vibrio species. This study highlights the necessity of surveillance of cfr in bacteria of diverse origins.

INTRODUCTION

The Cfr protein encoded by the cfr gene is a 23S rRNA methylase, facilitating bacterial resistance to several antibiotics, including phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A compounds (1), which are commonly used in clinical practice or the livestock industry. After the first identification of cfr in Staphylococcus sciuri in 2000, the cfr gene has been detected in both Gram-positive bacteria (Staphylococcus, Enterococcus, Streptococcus, Bacillus, etc.) and Gram-negative bacteria (Escherichia coli, Proteus, Campylobacter, Providencia, etc.) (2, 3). Although clinical threats caused by antibiotic resistance empowered by cfr in Gram-positive bacteria exist, emergence and transmission of cfr among Gram-negative bacteria are expanding the host range and further deteriorating the control measures. Thus, monitoring and surveillance in novel bacterial species are important for understanding its evolution in diverse bacteria. Vibrio diabolicus, a marine bacterium isolated from a deep-sea hydrothermal field, can produce exopolysaccharide (EPS), which has importance in the biotechnological industry and human health (4, 5). This bacterium is closely related to members of the Harveyi clade (e.g., Vibrio alginolyticus and Vibrio parahaemolyticus) that are commonly isolated from aquatic products and characterized as opportunistic pathogens in humans and marine animals (6). The emergence of important resistance genes in such marine pathogens constitutes a potential public health concern. Here, we first report the emergence of cfr in Vibrio diabolicus of seafood origin and identified a novel plasmid encoding cfr flanked by two IS26 copies.

During the surveillance of resistance genes in aquatic bacteria, a clam (Mactra veneriformis) was purchased from a supermarket in Nanjing, China, in January 2021. The sample was rinsed in 10 mL marine broth, incubated at 37°C for 4 h, and subsequently spread on thiosulfate citrate bile salts sucrose (TCBS) agar plates with florfenicol (25 μg/mL) to obtain florfenicol-resistant strains. After incubation at 37°C overnight, a strain, NV27, with a yellow colony was purified and selected for further investigation to decipher the underlying resistance mechanisms. The species of NV27 was identified by 16s rRNA sequencing and further confirmed by PubMLST based on whole-genome sequence data, and finally NV27 was confirmed as Vibrio diabolicus. Antimicrobial susceptibility testing was performed using the microdilution method according to Clinical and Laboratory Standards Institute (CLSI) guidelines (7). The data showed that strain NV27 is resistant to ampicillin, amoxicillin-clavulanic acid, ticarcillin, kanamycin, tetracycline, chloramphenicol, florfenicol, and colistin and is still susceptible to tigecycline, enrofloxacin, meropenem, imipenem, ceftriaxone, and ciprofloxacin (see Table S1 in the supplemental material). To learn the genomic basis of the resistance phenotype, the complete genome sequence of strain NV27 was obtained, successfully combining Illumina short-read and MinION Nanopore long-read sequencing platforms with hybrid de novo assembly with Unicycler as previously described (8–10). Different bioinformatics tools such as RAST, ResFinder, and BRIG were used to annotate and visualize the genomic comparison (11–13).

Genomic analysis revealed that strain NV27 harbors two chromosomes and two plasmids designated as pNV27-261K and pNV27-cfr-208K. Chromosome I is 3,238,151 bp in length with 45.00% GC content, and chromosome II is 1,794,367 bp with 44.90% GC content. Two tetracycline resistance genes, tet(34) and tet(35), were observed in chromosome I. In chromosome II, bla_CARB-57 was detected, and an online BLASTn result demonstrated bla_CARB-57 or its variants existed in few V. diabolicus and V. alginolyticus strains. Cloning and functional assays confirmed that bla_CARB-57 could mediate resistance to amoxicillin-clavulanic acid, ampicillin, and ticarcillin in E. coli BL21(DE3) (see Table S2 in the supplemental material). Notably, two untypeable plasmids from strain NV27 were 261,992 bp (pNV27-261K; 39.00% GC content) and 208,057 bp (pNV27-cfr-208K; 41.60% GC content) in length. No resistance genes were found in pNV27-261K, but a few resistance genes were identified in pNV27-cfr-208K (see Table S3 in the supplemental material), including cfr. However, most open reading frames (ORFs) predicted in pNV27-cfr-208K were hypothetical proteins without known functions. BLASTn analysis showed that pNV27-cfr-208K is 100% identical to pLMB157 at 80% coverage (hosted by Vibrio campbellii) and 100% identical to pVCGX2 at 95% coverage (hosted by V. campbellii). It is also 100% identical at 95% coverage to pSLV18-213K, encoding a novel carbapenemase-encoding gene, bla_VMB-2, hosted by V. diabolicus. Apart from cfr, pNV27-cfr-208K also carries other drug resistance genes, including tet(A), strA, strB, sul2, and floR. These antibiotic resistance genes are located in a multidrug resistance (MDR) region flanked by two ISShfr9 copies, forming a complex mosaic region ISShfr9-sul2-strA-strB-tet(A)-strB-lysR-floR-virD2-IS26-hp-cfr-IS26-ISCR2-ΔISShfr9 (Fig. 1A). Such an MDR genetic structure is different from those on pVCGX2 from V. campbellii and pSLV18-213K from V. diabolicus (Fig. 1B). The structure IS26-hp-cfr-IS26 inserted between virD2 and ISCR2 and ISShfr9-mexE-mexF-ISShfr9 were also found in pSLV18-213K but absent in pVCGX2. Reverse PCR results showed that IS26-hp-cfr-IS26 can form a circular intermediate containing cfr, one IS26 copy, and the hp with 2,699 bp in length. Through online BLASTn analysis, we found that IS26-hp-cfr-IS26 shared 99% identity at 100% query coverage with a PmSC1111 chromosome segment (Fig. 2A and B) hosted by Proteus mirabilis, and it also shows a similar genetic environment with plasmid pEC-01 from E. coli (Fig. 2C). Previous study revealed the different genetic environments of cfr (14), in which cfr was in the same orientation with IS26 (Fig. 2D to F). Similarly, different sizes of cfr-carrying segments could also be looped out with one IS26 copy via IS26-mediated recombination (15). Similar structures also appeared in plasmids or chromosomes hosted by Proteus vulgaris (Fig. 2G and H). Thus, it is implied that this cfr-bearing structure had the potential to be transferred to other Gram-negative bacteria by preferentially targeting another copy of IS26 (16), which is a universal mobile element in different Gram-negative bacteria. Moreover, Tn3-like element ISShfr9, encoding transposase with 52% identity to IS3000, was observed in MDR regions of different species, including Vibrio spp., Aeromonas spp., Proteus spp., etc., which indicated its potential role of facilitating antimicrobial resistance (AMR) transmission among different bacterial species (17).

FIG 1.

(A) Circular comparison of the cfr-bearing plasmid pNV27-cfr-208K and other similar plasmids pVCGX2, pSLV18-213K, and pLMB157 in the NCBI database. The outermost circle denotes the reference plasmid pNV27-cfr-208K with arrows for coding genes. (B) Linear comparison of the MDR regions distributed in pNV27-cfr-208K, pVCGX2, and pSLV18-213K. Red arrows represent the resistance genes.

FIG 2.

Linear comparison of the cfr genetic environments with other cfr harboring Gram-negative bacteria. Red arrows represent the resistance genes.

Conjugation assays of pNV27-cfr-208K to different recipients, such as E. coli C600, V. alginolyticus, and V. parahaemolyticus, were performed but failed. Through oriTfinder analysis (18), we found that there was no putative oriT region, relaxase, type IV secretion system, or type IV coupling protein predicted in pNV27-cfr-208K. The conjugation assay and plasmid analysis indicated that pNV27-cfr-208K was a nonconjugative and narrow-host-range MDR plasmid. In addition, the cfr gene in this study exhibits 99.81% identity with the first cfr gene reported in 2000 in Staphylococcus sciuri (3), with only two substitutions (C493A and C893A). Furthermore, cfr in this study was cloned into pET23a by the ClonExpress Ultra one-step cloning kit (Vazyme, China) with designed primers (see Table S4 in the supplemental material). Compared to the empty vector pET28a conferring MICs of 1 μg/mL to both antibiotics, expression of the cfr gene in E. coli BL21(DE3) increased MICs of chloramphenicol and florfenicol to 4 μg/mL (4-fold) and 8 μg/mL (8-fold), respectively. Recently, cooccurrence of cfr and other critical resistance genes such as tet(X) and mcr-1 in the same bacteria of animal origin was reported (19, 20), and cfr-positive E. coli strains were identified from vegetables (21), both of which imply that a One Health approach should be implemented to perform the surveillance.

In conclusion, we first described the emergence of cfr encoded in an MDR plasmid of Vibrio species origin, highlighting the role of mobile element IS26 in the transmission of cfr among Gram-negative bacteria. Monitoring the emergence and dissemination of cfr in different bacterial species of diverse origins should be performed as a routine work.

Data availability.

The complete sequences of strain NV27 were submitted to the NCBI database with the following accession numbers: CP085843 to CP085846.