Tigecycline was considered as one of the last-resort antibiotics to treat severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, especially carbapenem-resistant Enterobacteriaceae. The emergence of mobile high-level tigecycline resistance genes, tet(X3) and tet(X4) in Enterobacteriaceae and Acinetobacter species isolates from animals, food, and humans in China, raises concerns regarding antibiotic resistance globally.1,2 Subsequently, a few plasmid-mediated [for example, tet(X3.2), tet(X5), tet(X6), and tet(X7)] and chromosome-encoded [for example, tet(X6), tet(X14), and tet(X15)] tet(X)-like genes have been discovered.3–13 Although the tet(X4) gene has been identified in more than ten different Gram-negative species, with Escherichia coli being the most common,2,14–21tet(X4)-positive Salmonella enterica isolates of human origin remain unexplored. Here, we describe the first global report of the identification of tet(X4) in an MDR S. enterica serovar Llandoff strain isolated from a man's fecal sample in China. Salmonella species are important zoonotic pathogens worldwide, and they are frequently found to carry clinically relevant antibiotic resistance genes (ARGs; eg, blaNDM-1 and mcr-1) in plasmids.22,23 The emergence and spread of mobile tigecycline resistance genes in Salmonella isolates should be taken seriously, and continuous monitoring is urgently needed. Despite several efforts to study tet(X)-like genes, little is known about their extensive species distribution and spread. Therefore, we screened the sequences of tet(X)-like genes in 276,592 bacterial genomes (66,217 Salmonella genomes were included) to shed light on these fundamental issues.
Characteristics of tigecycline-resistant S. enterica strains
Through comparative genomic analysis of 2628 Salmonella draft genome sequences, we found that the tet(X4) gene was present in the isolate SH16G3606. The result indicated that the prevalence of tet(X4) in Salmonella in China is very low, accounting for 0.038% of the total number. SH16G3606 was recovered from a stool sample of a 63-year-old male (asymptomatic carrier) in Guangxi Municipality in China in 2016. Analysis of whole-genome sequencing (WGS) data and the subsequent serum agglutination assay (Statens Serum Institute, Denmark) confirmed it was S. enterica serovar Llandoff (1,3,19:z29:-). Multilocus sequence typing revealed a novel sequence type ST8300 for the S. Llandoff isolate.
Antimicrobial susceptibility testing (AST) results showed that the tet(X4)-positive S. Llandoff isolate was resistant to tigecycline, tetracycline, florfenicol, ampicillin, streptomycin, and trimethoprim/sulfamethoxazole with reduced susceptibility to ciprofloxacin, but it remained susceptible to gentamicin and colistin (Table 1). As SH16G3606 is resistant to >3 classes of antimicrobials, it can be considered as an MDR strain. Conjugation experiments showed that tigecycline resistance [tet(X4)-mediated resistance phenotype] can be successfully transferred from S. Llandoff into the recipient, E. coli J53, with a high transfer frequency of 1.2 × 10−3 (transconjugants/donor). The tet(X4) gene in transconjugants was confirmed by PCR. Susceptibility testing results showed that the transconjugants had a 32-fold increase in the MIC of tigecycline (8 mg/L) compared with the plasmid-less recipients (0.25 mg/L). In addition to tigecycline, the transconjugants were also resistant to tetracycline, florfenicol, ampicillin, streptomycin, and trimethoprim/sulfamethoxazole (Table 1).
Table 1 -
Results of antimicrobial susceptibility tests and genetic characterization of SH16G3606
||MIC (mg/L) of SH16G3606
||MIC (mg/L) of transconjugants-E. coli J53
||Mechanism of resistance
tet(A), tet(M), and tetR(A)
dfrA12, sul2, and sul3
I: intermediate; MIC: minimal inhibitory concentration; R: resistant; S: susceptible; –: none or not applicable.
Genetic characterization of the plasmid carrying the tet(X4) gene
The combined Illumina HiSeq and Nanopore sequencing results showed that SH16G3606 contained one chromosome of 4,688,541 bp (GC content: 52.2%) and a plasmid of 256,017 bp (named pSal21GXH-tetX4). Three different replicon types, IncHI1A, IncFIA(HI1), and IncHI1B(R27), were identified in pSal21GXH-tetX4. Moreover, the plasmid harbored additional ARGs encoding resistance to beta-lactam antibiotics (blaTEM-1), chloramphenicol (cmlA1), aminoglycosides (aadA2, aadA1), trimethoprim/sulfamethoxazole (dfrA12, sul2, and sul3), fluoroquinolones (qnrS1), florfenicol (floR) and tetracycline [tet(A), tet(M), and tetR(A)]. The ARG content was consistent with the resistance phenotypes we found for S. Llandoff isolate SH16G3606. Such a plasmid represents a high threat to public health since it confers MDR, including resistance to the last-resort antibiotic tigecycline.
The complete sequence of pSal21GXH-tetX4 was similar (99.9% nucleotide sequence identity) to that of pSa4-CIP (MG874042.1) derived from S. enterica serovar Typhimurium from pork meat in Hong Kong24 and pSESen370925 (AP020333.1) from S. enterica serovar Senftenberg from human in Japan (Figure 1A), and the three plasmids shared the same replicon type. However, the region containing tet(X4) and abh in pSal21GXH-tetX4 was not present in pSa4-CIP and pSESen3709 (Figure 1A).
A further BLASTn search for the tet(X4) gene against the National Center for Biotechnology Information (NCBI)-nr database identified a series of Enterobacteriaceae carrying the same sequence from humans and animals (Table S1, https://links.lww.com/IMD/A13). Genetic context analysis revealed that the tet(X4) gene in SH16G3606 was located between two copies of ISCR2 (Figure 1B), which was identical to the tet(X4)-harboring structure in E. coli described previously.2,21 The previous reports showed that ISCR2 could mediate the generation of a circular form, thereby facilitating transmission of the tet(X4) gene between different genetic environments.14,17,21 Moreover, ISCR2 was previously reported to be associated with various ARGs, such as floR, also identified in pSal21GXH-tetX4. The genetic contexts of tet(X4) in S. enterica and animal commensal E. coli isolates were similar, which suggests the possibility for it to spread to humans from animals. Therefore, we assumed that tet(X4) in the plasmid of pSal21GXH-tetX4 might be derived from E. coli due to horizontal gene transfer.
Global genomic characterization of S. enterica serovar Llandoff
To understand the global resistance patterns, plasmid profiles, and virulence profiles of S. Llandoff strains, an additional 20 strains were retrieved from the Enterobase and NCBI (Table S2, https://links.lww.com/IMD/A13). The WGS analysis revealed that the global strains contain resistance genes against two antibiotic families: aminoglycosides [aac(6’)-Iaa] and fosfomycins (including fosA7). Moreover, one chromosomal mutation ParC (T57S) in the quinolone resistance-determining region was found in twelve isolates (Table S3, https://links.lww.com/IMD/A13). A total of 33.33% of the isolates harbored plasmids, mainly Col(pHAD28), Col440I, IncY, ColpVC, and Col156 (Table S3, https://links.lww.com/IMD/A13).
The virulence gene profile for the 21 strains was identified using the virulence factor database (VFDB).26 A total of 51.38% of the genes (56/109) were conserved among all 21 strains (Table S4, https://links.lww.com/IMD/A13). Notably, the strains harbored the cytolethal distending toxin (cdtB) gene, which is considered as one of the typhoid toxins. Multiple genes associated with fimbrial adherence and the type III secretion system were detected in all isolates. Salmonella Pathogenic Islands (SPIs) were also explored in the isolates using SPIFinder.27 The SPI-1, SPI-2, SPI-3, and SPI-5 were identified in all strains (Table S5, https://links.lww.com/IMD/A13). CS54 island and C63PI were detected in four and sixteen isolates, respectively.
To further investigate the diversity and ecological features of S. Llandoff isolates worldwide, we collected genome sequences from NCBI GenBank and Enterobase (http://enterobase.warwick.ac.uk/) and found 20 additional S. Llandoff isolates (Figure S1, https://links.lww.com/IMD/A14). Genetic analysis of these S. Llandoff isolates revealed a geographical clustering pattern and suggested potential transmission from Guatemala to Mexico, the USA, and China (Figure S2, https://links.lww.com/IMD/A14). In short, we reported here for the first time the identification of an S. Llandoff strain isolated from a human in China.
Distribution of tet(X)-like genes in Gram-negative bacterial genomes
We subsequently searched for tet(X)-like genes in 276,592 bacterial genomes to fully understand the prevalence of these genes in bacterial species. Interestingly, six tet(X)-like genes, tet(X3) (n = 11), tet(X4) (n = 46), tet(X6) (n = 10), tet(X7) (n = 7), tet(X10) (n = 95), and tet(X12) (n = 2), were identified in various bacterial genomes (>49 species, Figure 2 and Table S6, https://links.lww.com/IMD/A13), including a broad spectrum of Gram-negative bacteria. In total, 171 isolates carrying tet(X)-like genes were distributed in >21 countries or areas across 6 continents. No isolates were positive for the genes tet(X5), tet(X8), tet(X9), tet(X11), or tet(X13). Similar to previous reports,19,28 the distribution of tet(X)-like genes varied in different countries; furthermore, 50, 44, 13, and 12 tet(X)-positive isolates were present in the USA, China, the United Kingdom, and Thailand, respectively.
Interestingly, tet(X3) and tet(X4) were mainly identified in Acinetobacter and E. coli, respectively, which have been identified from the human, animal, and environmental origins as well as in human and animal gut microbiomes.1,2,15,17,29–31 We found that the tet(X4) gene had been present in eight S. enterica isolates, including the serovars Agona, Typhimurium, Stanley, Livingstone, and Kentucky from England during 2014–2020, and one S. enterica isolate from the USA (Table S7, https://links.lww.com/IMD/A13), while the phenotype of tigecycline resistance has not been reported. Because contigs carrying tet(X4) were too short, we were not able to determine whether tet(X4) was located on the chromosome or plasmid in S. enterica strains in England (Figure S3, https://links.lww.com/IMD/A14). Tet(X6) was detected in various bacterial species, such as Acinetobacter johnsonii, Acinetobacter sp., Acinetobacter variabilis, Chryseobacterium sp., Proteus mirabilis, and E. coli. Tet(X7) was identified in E. coli, Pseudomonas aeruginosa, Proteus vulgaris, Enterobacter hormaechei, and S. Typhimurium. We also noted that the amino acid sequence of tet(X10) is highly similar to those of tet(X) and tet(X2). Most identified tet(X) genes belonged to Bacteroides, Acinetobacter, and E. coli. Bacteroides were found to be the main reservoir of tet(X) genes in humans, while tet(X) genes in Acinetobacter and E. coli were more often identified in animals. However, a previous report32 also indicated that Flavobacteriaceae were the potential ancestral source of tet(X). These findings expand our understanding of the bacterial species carrying tet(X)-like genes and illustrated that the newly discovered tet(X)-like genes have a wide distribution among different bacterial species from diverse sources worldwide. Therefore, further studies focusing on the epidemiology and transmission mechanism of tet(X)-like genes, especially tet(X)-like genes in foodborne and clinical pathogens, are warranted to better understand the public threat of the emergence of tigecycline resistance among human clinical pathogens.
The genetic context of the tet(X7) gene
Genetic analysis indicated that rteC (tetracycline regulation of excision) and sul1 were present in the upstream and downstream flanking region of the tet(X7) gene, respectively (Figure 1C). Notably, the tet(X7) gene and mcr-1 are co-existing in a single plasmid in E. coli, which is the same as previous reports.11,33,34 In addition, the tet(X7) gene in the downstream flanking region of rteC and the insertion element IS91 in a clinical P. aeruginosa isolate had sequences similar to those of contigs from Proteus vulgaris, and E. hormaechei, which indicates that IS91 may mediate the transmission of the tet(X7) gene between different genetic environments. We were not able to determine whether an insertion element is present downstream due to a lack of tet(X7)-bearing contigs available for comparison. Therefore, long-read sequencing is needed to fully assess the transferability of tet(X7) in foodborne and clinical isolates.
S. enterica is a major pathogen of humans and animals as well as an important source and reservoir of genes that encode antimicrobial resistance. For example, fluoroquinolone-resistant Salmonella spp. have been listed by the World Health Organization in 2017 as high priority pathogens posing a risk to human health and in need for the research and development of new antibiotics.35 In this study, we isolated and characterized an MDR and ciprofloxacin non-susceptible S. enterica serovar Llandoff strain from a human fecal sample in China. S. Llandoff is a rarely reported serovar. By using a comparative genomics analysis, we characterized not only the antimicrobial resistance and virulence gene profile, but also the phylogenetic relationships of the global Salmonella Llandoff genomes.
Through AST, conjugation, and WGS, we confirmed that the S. Llandoff strain carries the mobile tigecycline resistance gene tet(X4), which is responsible for the tigecycline resistance phenotype. It is noteworthy that this is the first report of the detection of plasmid-mediated tigecycline resistance gene tet(X4) in S. enterica strains isolated from humans in China. A retrospective study reported that the emergence of plasmid-mediated tigecycline resistance gene tet(X4) in farm animals in China is a recent event.16 However, it was present at low prevalence across China, but was highly endemic in northwestern China.17
Tigecycline is one of the last-resort treatments for serious infections. However, the clinical potential of tigecycline has been significantly compromised by the emergence and spread of plasmid-mediated tet(X)-like genes1,2,4,7,11,33 and resistance-nodulation-division efflux pump gene cluster,36,37 as well as chromosome-encoded tet(X)-like genes.5,6,8–10,12 Although Pan and colleagues screened the distribution of five variants of tet(X) genes [including tet(X), tet(X1), tet(X2), tet(X3), and tet(X4)], which identified a total of 155 isolates carrying tet(X) genes,28 systematic studies on the globally spread features of tet(X)-like genes are still limited. Because of limited genome data, especially the lack of genomes for tigecycline-resistant isolates, we are concerned that the worldwide distribution of tet(X)-like genes might be underestimated. Therefore, we retrieved these newly identified tet(X)-like gene sequences and traced these genes in available public bacterial datasets to understand the bacterial species and geographical distribution. We were surprised to find that a total of 171 strains carried tet(X)-like genes were distributed in >21 countries or areas across 6 continents. The emergence of tet(X)-like genes could be shared across many bacterial species, which has raised global attention.
In summary, we report for the first time, to our knowledge, the identification of a S. enterica serovar Llandoff strain isolated from a human in China. The plasmid-mediated tigecycline resistance mechanism tet(X4) was identified in a conjugative plasmid of MDR S. Llandoff isolate SH16G3606, which is a potential host for the spread of tigecycline resistance. The tet(X4) gene identified in this study was located between two copies of ISCR2, which was highly similar to that found in both pig and chicken commensal E. coli strains, indicating that it may be transmitted from food animals to humans. Moreover, we collected the largest dataset to date of sequenced bacterial genomes containing novel tet(X)-like genes through an extensive search of public sequences, which revealed that novel tet(X)-like genes are widespread in different bacterial species, including foodborne and clinical pathogens. Further studies are needed to understand the prevalence and dissemination of tigecycline-resistant S. enterica isolates and effective measures should be taken to control its spread.
Materials and methods
S. enterica genomes and bacterial strains
To explore the distribution of the recently reported tet(X)-like genes in Salmonella (Table S8, https://links.lww.com/IMD/A13), we analyzed 2628 S. enterica genomes from various sources (including human, animal, food, and environment) generated by our laboratory. A tet(X4)-carrying S. enterica serovar Llandoff strain was isolated from a human stool sample collected from Guangxi municipality in China in 2016. In addition, 20 S. Llandoff isolates (Table S2, https://links.lww.com/IMD/A13) with assembled contigs in FASTA format from the Enterobase (http://enterobase.warwick.ac.uk/, accessed September 5, 2020) and NCBI were collected and used for comparative analysis. The S. Llandoff isolates were used in this study to represent various geographical locations, including the UK (n = 4), France (n = 4), Mexico (n = 3), Guatemala (n = 2), the United States (n = 1), Netherlands (n = 1), Canada (n = 1), Germany (n = 1), Israel (n = 1), and Northern Ireland (n = 1). The location of the remaining isolate was not available (Figure S1, https://links.lww.com/IMD/A14).
Retrospective analysis of tet(X)-like genes in 276,592 public bacterial genomes
We screened the sequences of tet(X)-like genes (Table S8, https://links.lww.com/IMD/A13) in 276,592 bacterial draft genomes downloaded from the NCBI GeneBank (https://ftp.ncbi.nlm.nih.gov/genomes/refseq/bacteria, including 63,589 Salmonella genomes, accessed by September 2, 2020). Matches with 100% identity and 100% coverage were retrieved from GenBank. The retrieved sequences with a reference of each tet(X)-like gene were used to analyze gene environments.
Antimicrobial susceptibility testing
AST was conducted by agar dilution, with E. coli ATCC 25922 as the quality control strain, and interpreted according to the Clinical and Laboratory Standards Institute guideline. The tigecycline MIC value for the Salmonella isolate was 8 mg/L, meaning that the isolate was classified as resistant based on European Committee on Antimicrobial Susceptibility Testing and Food and Drug Administration tigecycline breakpoints.38
The transmission efficiency of the plasmid-mediated resistance genes was assessed by a conjugation experiment using the filter mating method. The conjugation assay was performed using tet(X4)-positive strains as the donors and E. coli J53 (AZIR) as the recipient, and Mueller-Hinton agar plates containing sodium azide (100 mg/L) and tigecycline (0.5 mg/L) were used for selection. Polymerase chain reaction (PCR) confirmed the tet(X4) gene in transconjugants according to a previous report.1
WGS and bioinformatic analysis
The total genomic DNA of the tigecycline-resistant isolate was extracted and sequenced using the Illumina HiSeq and Nanopore sequencing platform. Trimmomatic was used to remove adapters and low-quality sequences (https://github.com/usadellab/Trimmomatic), and only high-quality reads were selected for downstream analysis. Hybrid assembly was performed using Unicycler (https://github.com/rrwick/Unicycler).39 Annotation of genome sequences was performed using Prokka version 1.13.3 (https://github.com/tseemann/prokka).40 Acquired genes and chromosomal mutations mediating antimicrobial resistance, and plasmid replicon types were determined using ResFinder,41 PointFinder,42 and PlasmidFinder,43 respectively. Insertion sequences were investigated using online tools (https://cge.cbs.dtu.dk/services/). The BLAST ring image generator44 and Easyfig45 were used to visualize genetic comparisons. In addition, the virulence factors were identified using VFDB (http://www.mgc.ac.cn/VFs/).26 SPIs were identified using SPIFinder version 2.0.27
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