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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2024; 52(4): 513-516

https://doi.org/10.48022/mbl.2406.06014

Received: July 1, 2024; Revised: October 10, 2024; Accepted: October 14, 2024

Whole-Genome Sequences of Two Chloramphenicol-Florfenicol Resistance Gene-Carrying Enterococcus faecalis Isolates ES-3-1 and ES-11 from Pig Stools in South Korea

Jun Bong Lee, Nguyen Thi Mai Tho, Se Kye Kim, and Jang Won Yoon*

College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea

Correspondence to :
Jang Won Yoon,        jwy706@kangwon.ac.kr

Abstract

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We report the complete genome sequences of cfr(D)-positive Enterococcus faecalis strains, ES-3-1 and ES-11, which were isolated from pig stools in 2021. The assembled genomes revealed that cfr(D) was located in the Inc18 type plasmids: a 21,899 bp plasmid of ES-3-1 and a 15,026 bp plasmid of ES-11. The IS1216 mobile elements were found to frank the cfr(D) locus on these two plasmids, and fexA and poxtA2 genes co-existed with cfr(D). Both ES-3-1 and ES-11 strains belonged to sequence type 16, which has animal and human origins, suggesting the high possibility of cfr(D) transmission between animals and humans.

Keywords: Whole-genome sequence, cfr(D), Enterococcus faecalis, pig stool, sequence type 16

Body

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The emergence of transferable chloramphenicol-florfenicol resistance gene (cfr) in bacterial isolates from livestock poses a significant challenge to animal health as it confers resistance to florfenicol, which is often used in the veterinary field [1]. In addition, cfr-mediated linezolid resistance in bacteria of human origin cause a serious problem to treat multidrug-resistant Gram-positive bacterial infection [2]. The cfr encodes a methyltransferase that modifies the A-residue at position 2503 in 23S ribosomal RNA (rRNA) and thereby confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A [3]. This multidrug resistance gene was first identified in a Staphylococcus sciuri isolate in 1997, and its variants named as cfr(B)(detected in Staphylococcus, Enterococcus, and Clostridium), cfr(C) (detected in Campylobacter and Clostridium), cfr(D) (detected in Enterococcus, Vagococcus, and Streptococcus), and cfr(E) (detected only in Clostridium) were also described in different bacterial species [4]. Among these species, Enterococcus spp. are not only commensal bacteria in the gastrointestinal tract of human and animals but also opportunistic pathogens that cause the human diseases such as urinary infection, sepsis, and endocarditis [5]. Previous studies suggested that animal reservoirs were important sources of enterococci infections in humans, and two of the predominant species isolated from clinical sources are Enterococcus faecium and E. faecalis. For example, Larsen et al. demonstrated that a porcine-originated E. faecalis isolate belonging to sequence type 16 (ST16) causes endocarditis in humans [6]. Recently, cfr(D) was first identified in an E. faecium clinical isolate from France in 2015 [7], then in an E. faecalis clinical isolate from Spain in 2018 [8]. However, there have been few reports of cfr(D) in Enterococcus spp. of animal origin.

In this study, two cfr(D)-positive E. faecalis isolates (ES-3-1 and ES-11) were isolated from pig stools in 2021 and analyzed by whole genome sequencing (WGS). For this purpose, a single colony of each isolate was cultured in tryptic soy broth (BD Difco, Sparks, MD, USA) at 37℃with shaking (220 rpm) for 18 h. The bacterial genomic DNA was extracted using HiYield genomic DNA mini kit (Real Biotech Co., Taiwan). DNA samples were sent to a commercially available company (Sanigen, Republic of Korea) for WGS analysis.

Library preparation and DNA sequencing were performed as previously described [9]. Briefly, two separate genomic DNA libraries were constructed using Illumina TruSeq Nano DNA Sample Prep kit (Illumina, USA) and Nanopore Ligation Sequencing Kit (Oxford Nanopore, UK), respectively. WGS data of ES-3-1 and ES-11 were generated using a combination of Illumina short-read and Nanopore long-read. Illumina sequencing was performed on a 300 bp paired-end Illumina Miseq platform. Raw data from Illumina sequencing were processed to remove low quality bases and adapter sequences using Trimmomatic v0.39 with the optimized settings (LEADING: 10, TRAILING: 10, SLIDING WINDOW: 4:20, MINLEN: 200). Subsequently, high quality-trimmed Illumina reads were mapped to the phiX genome using bowtie2 v2.3.5.1 and filtered out by samtools v.1.9. Nanopore sequencing was performed on a MinION flow cell (MIN106 R9.4.1) for a 24 h run using MinKNOW with the default settings (MinKNOW core 5.0.0, Guppy 6.06). Nanopore reads were base-called with guppy base-caller v3.1.5, and reads with average Phred quality score lower than 7 and lengths lower than 1,000 bp were omitted using NanoFilt v2.8.0. To construct the complete genome sequences, Illumina and Nanopore sequencing data were used in a hybrid assembly performed in Unicycler v0.4.8. The chromosome and plasmids obtained were annotated using Prokka v1.14.6 and their genetic features were identified.

Bacterial species of ES-3-1 and ES-11 were determined by calculating the average nucleotide identity values of their assembled sequences (GCA_033811895.1 and GCA_033814155.1, respectively) in National Center for Biotechnology Information (NCBI) GeneBank database. The closest match was E. faecalis (GCA_022869705.1), with average nucleotide identity values of 99.07% and 98.9%, respectively. The acquired genes and chromosomal mutations related to antimicrobial resistance (AMR) were identified using NCBI’s AMRFinderPlus tool (v.3.11.4). Multilocus sequence typing (MLST) and mobile genetic element analysis were performed by using the MLST (v.2.0.9) and Mobile Element Finder (v.1.03), which are publicly available gene prediction programs from the web site (https://www.genomicepide-miology.org/services/). To determine the antimicrobial susceptibility phenotype of each E. faecalis isolate, the microbroth dilution method was used according to the National Antimicrobial Resistance Monitoring System (NARMS) guidelines. Sensititre panel KRVP2F (TREK Diagnostic Systems, USA) was utilized for the microbroth dilution method. Finally, high-quality complete genome sequences of ES-3-1 and ES-11 were deposited at the NCBI GeneBank data base under the accession numbers CP138637.1-CP138640.1 (ES-3-1) and CP138648.1-CP138652.1 (ES-11).

The assembled genome of ES-3-1 strain yielded one full circular chromosome and three plasmids. Sizes of the chromosome, a large plasmid, and two small plasmids were 2,933,922, 21,899, 5,165, and 4,847 bp, respectively (Table 1). The chromosome (Contig 1) contained 2,762 coding sequences (CDSs), 37.41% G+C content, 63 transfer RNA (tRNA), 12 rRNA, and multiple AMR genes (Table 1 and Fig. 1). Notably, a 21,899 bp plasmid (Contig 2) was the Inc18 type (rep1) plasmid harboring 35.24% G+C content and 22 CDSs that include cfr(D), fexA, and poxtA2 flanked by the IS6-like element IS1216 family transposase genes. The cfr(D) gene encoded a 349 amino acid protein, which shared 62% amino acid identity with Cfr.

Table 1 . Genetic and phenotypic features of Enterococcus faecalis ES-3-1 and ES-11.

FeaturesES-3-1ES-11
No. of contigs45
Genome size (bp)2,933,922 (Contig 1), 21,899 (Contig 2), 5,165 (Contig 3), 4,847 (Contig 4)3,137,925 (Contig 1), 74,882 (Contig 2), 71,645 (Contig 3), 56,79 (Contig 4), 15,026 (Contig 5)
G+C content (%)37.41 (Contig 1), 35.24 (Contig 2), 36.77 (Contig 3), 37.78 (Contig 4)37.25 (Contig 1), 35.45 (Contig 2), 36.03 (Contig 3), 34.39 (Contig 4), 35.66 (Contig 5)
No. of CDSs2,762 (Contig 1), 22 (Contig 2), 5 (Contig 3), 5 (Contig 4)3,068 (Contig 1), 73 (Contig 2), 91 (Contig 3), 73 (Contig 4), 16 (Contig 5)
No. of tRNA and rRNA63 (Contig 1) and 12 (Contig 1)68 (Contig 1) and 12 (Contig 1)
MLSTST16ST16
AMR genesContig 1: tet(M), abc-f, parC(S80I)a, erm(B), ant(6)-Ia, Inu(B), Isa(E), spw, aph(3’)-IIIa, sat4, aac(6’)-Ie/aph(2’’)-Ia, catAb, Isa(A)
Contig 2: poxtA2, cfr(D), fexAContig 5: poxtA2, cfr(D), fexA
AMR profileChloramphenicol, Gentamicin, Kanamycin, Streptomycin, Quinupristin/dalfopristin, Erythromycin, Tylosin, Tetracycline, Ciprofloxacinc
NCBI accession numberCP138637.1 (Contig 1), CP138638.1 (Contig 2), CP138639.1 (Contig 3), CP138640.1 (Contig 4)CP138648.1 (Contig 1), CP138649.1 (Contig 2), CP138650.1 (Contig 3), CP138651.1 (Contig 4), CP138652.1 (Contig 5)

aES-11 has a chromosomal mutation parC(S80I) solely.

bES-3-1 has a catA gene solely.

cES-11 is resistant solely to ciprofloxacin.

Figure 1.The chromosomes and cfr(D)-carrying plasmids of Enterococcus faecalis strains ES-3-1 and ES-11. Circular genome maps of ES-3-1 contig 1 (A) ES-3-1 contig 2 (B) ES-11 contig 1 (C) and ES-11 contig 5 (D). Marked features are represented from outside to the center. Rings 1 and 2 show the coding sequence (blue), transfer RNA (brown), and ribosomal RNA (purple) on the forward and reverse strands, respectively. Ring 3 represents the G+C content plot and ring 4 shows the positive and negative GC skew in green and purple, respectively.

On the other hand, ES-11 strain contained one circular chromosome (3,137,925 bp) and four plasmids (74,882, 71,645, 56,791, and 15,026 bp) (Table 1). The chromosome (Contig 1) harbored 3,068 CDSs, 37.25%G+C content, 68 tRNA, 12 rRNA, multiple AMR genes, and a single point mutation in parC (S80I) (Table 1 and Fig. 1). Interestingly, a 15,026 bp Inc18 plasmid (Contig 5) contained 35.66% G+C content and 16 CDSs including cfr(D), fexA, and poxtA2 flanked by the IS1216 family transposase genes. The cfr(D) nucleotide sequence of ES-11 was identical with that of ES-3-1.

Inc18 plasmids are widespread conjugative plasmids with a broad host-range. A previous study revealed transferable cfr-carrying Inc18 plasmid in E. faecalis from a healthy piglet in Brazil. Researchers found that the genetic environment of cfr gene in porcine E. faecalis isolate was similar to that from human Staphylococcus aureus isolate in the USA [10]. This indicates that two cfr(D)-carrying Inc18 plasmids from ES-3-1 and ES-11 can be transmitted to Gram-positive bacteria of human origin, subsequently posing a threat to public health.

Both ES-3-1 and ES-11 strains were identified as E. faecalis belonging to ST16 (Table 1). Previously, a ST16 E. faecalis isolate harboring cfr(D) and poxtA2 genes were isolated from healthy human in Switzerland, and the two resistance genes were located on the same Inc18 type plasmid [11]. Corresponding to AMR genotypes, they displayed multiple resistance to tetracycline, phenicols (chloramphenicol and florfenicol), macrolides (erythromycin and tylosin), aminoglycosides (gentamicin, kanamycin, and streptomycin), and quinupristin/dalfopristin. However, ES-3-1 and ES-11 exhibited the intermediate phenotype to linezolid (MIC=4 µg/ml), which is consistent with previous reports to non-linezolid resistant and cfr-positive E. faecalis isolates from swine farms in China [12].

Abbreviations

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CDSs, coding sequences; MLST, multilocus sequence typing; ST, sequence type; AMR, antimicrobial resistance.

Acknowledgments

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This work was supported by a grant from Research of Korea Centers for Disease Control and Prevention (2021ER220100), Republic of Korea.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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