- Genome Report
- Open Access
Genomic characterization of Escherichia coli LCT-EC001, an extremely multidrug-resistant strain with an amazing number of resistance genes
- Xuelin Zhang†1, 4,
- Saisong Xiao†2,
- Xuege Jiang†1,
- Yun Li3,
- Zhongyi Fan1,
- Yi Yu1,
- Peng Wang1,
- Diangeng Li1,
- Xian Zhao1 and
- Changting Liu1Email author
© The Author(s) 2019
- Received: 26 October 2018
- Accepted: 13 April 2019
- Published: 21 May 2019
Multidrug resistance is a growing global public health threat with far more serious consequences than generally anticipated. In this study, we investigated the antibiotic resistance and genomic traits of a clinical strain of Escherichia coli LCT-EC001.
LCT-EC001 was resistant to 16 kinds of widely used antibiotics, including fourth-generation cephalosporins and carbapenems. In total, up to 68 determinants associated with antibiotic resistance were identified, including 8 beta-lactamase genes (notably producing ESBLs and KPCs), 31 multidrug efflux system genes, 6 outer membrane transport system genes, 4 aminoglycoside-modifying enzyme genes, 10 two-component regulatory system genes, and 9 other enzyme or transcriptional regulator genes, covering nearly all known drug-resistance mechanisms in E. coli. More than half of the resistance genes were located close to mobile genetic elements, such as plasmids, transposons, genomics islands, and insertion sequences. Phylogenetic analysis revealed that this strain may have evolved from E. coli K-12 but is a completely new MLST type.
Antibiotic resistance was extremely severe in E. coli LCT-EC001, mainly due to mobile genetic elements that allowed the gain of a large quantity of resistance genes. The antibiotic resistance genes of E. coli LCT-EC001 can probably be transferred to other bacteria. To the best of our knowledge, this is the first report of a strain of E. coli which has such a large amount of antibiotic resistance genes. Apart from providing an E. coli reference genome with an extremely high multidrug-resistant background for future analyses, this work also offers a strategy for investigating the complement and characteristics of genes contributing to drug resistance at the whole-genome level.
- Escherichia coli
- Antibiotic resistance
- High-throughput sequencing
According to the World Health Organization (WHO) report ‘Antimicrobial resistance: global report on surveillance 2014’, multidrug resistance is a growing global public health threat with far more serious consequences than generally anticipated. Out of the WHO member states, 50% reported that E. coli isolated from within these states was resistant to third-generation cephalosporins and fluoroquinolones—the best antibiotics available for treating multidrug-resistant bacteria. In February 2017, the WHO published its first ever list of antibiotic-resistant “priority pathogens”—a catalogue of 12 families of bacteria that pose the greatest threat to human health. E. coli was defined as one of the most critical multidrug-resistant bacteria, which were considered to have built-in abilities to find new ways to resist treatment and pass along genetic material that allows other bacteria to become drug-resistant as well. It is widely accepted that infections caused by antibiotic-resistant bacteria burden healthcare resources and increase the risk of poor clinical outcomes for patients. Global estimates suggest that more than 700,000 people per year die from drug-resistant infections . It is predicted that antibiotic-resistant infections will kill ~ 10 million people per year by 2050, costing the global economy ~ $100 trillion . The seriousness of this situation was surmised in the WHO report: ‘A post antibiotic era, in which common infections and minor injuries can kill, is instead a very real possibility for the 21st century’.
Revealing the mechanisms underlying drug resistance in bacterial pathogens is crucial in infection disease control and management. With significant progress in high-throughput sequencing and bioinformatics analysis of pathogens, whole-genome sequencing has become more accessible for the identification and tracking of multidrug-resistance (MDR) microorganisms in hospitals and communities . In this study, we isolated E. coli strain LCT-EC001 from a 78-year-old male patient with several health issues, including diabetes, hypertension and chronic obstructive pulmonary disease, who had received long-term therapy with multiple drugs. The drug resistance of E. coli strain LCT-EC001 was tested, and whole-genome sequencing was conducted to understand the genetic elements contributing to antibiotic resistance. This work contributes a clinically isolated drug-resistant E. coli strain as a valuable reference for future studies and presents a strategy for the comprehensive analysis of drug resistance at the whole-genome level.
Bacterial isolation and culture conditions
An E. coli isolate (designated LCT-EC001) was obtained from the sputum of a 78-year-old male patient who had several health issues (diabetes, hypertension and chronic obstructive pulmonary disease) and had received multidrug therapy over a long time period. The bacterium was inoculated in Brain Heart Infusion (Oxoid, UK) medium at 37 °C.
Antibiotic susceptibility test
The antibiotic susceptibility profile was tested using a VITEK 2 Compact System (bioMerieux Inc., USA) according to the manufacturer’s instructions as previously reported . 17 kinds of antibiotics tested are as follows: ampicillin, cefazolin, ampicillin/sulbactam, cefotetan, ceftriaxone, cefepime, ceftazidime, aztreonam, ertapenem, imipenem, amikacin, gentamicin, tobramycin, levofloxacin, ciprofloxacin, trimethoprim/sulfa, and nitrofurantoin.
High-throughput sequencing and assembly
Isolation of genomic DNA was carried out using the cetyltrimethylammonium bromide (CTAB) method. Total DNA obtained was subjected to quality control by agarose gel electrophoresis and quantified by Qubit . The genome of E. coli strain LCT-EC001 was sequenced with MPS (massively parallel sequencing) Illumina technology. Two DNA libraries were constructed: a paired-end library with an insert size of 500 bp and a paired-end library with an insert size of 5 kb. The 500 bp library and the 5 kb library were sequenced using an Illumina HiSeq 2000 platform (Illumina, USA). Quality control of the two paired-end library reads was performed using readfq (version 10) program  with the following steps: (1) Eliminate reads once its low quality nucleotide bases (Q-value ≤ 38) exceeding the threshold (40 bp by default), (2) Eliminate the reads containing Ns in the reads greater than the threshold (10 bases by default), (3) Eliminate reads whose overlap with the adapter exceeding the threshold (15 bp by default), and (4) Filter duplicates to keep only one copy of the totally same reads. For a library of 500 bp, 6.19% of reads were filtered, while 8.48% of reads were filtered for a library of 5 kb. The filtered reads were assembled by SOAPdenovo  to generate scaffolds. The parameters used for assembly were as follows: SOAPdenovo all -F -K 107 -k 107. All reads were used for further gap closure by using GapCloser (version 1.12)  with default parameters.
Gene prediction, annotation and protein classification
Gene prediction was performed on the LCT-EC001 genome assembly by GeneMarkS  with an integrated model that combined the GeneMarkS generated (native) and heuristic model parameters. Gene annotation was performed with a BLASTp  search (E-value less than 1·e−5, minimal alignment length percentage larger than 40%) against 4 databases in a standalone environment. The databases are KEGG (Kyoto Encyclopedia of Genes and Genomes, v2016.4) , COG (Clusters of Orthologous Groups, v2015.12) , GO (Gene Ontology, v2014.10) , and ncRNA (noncoding RNA database, tRNA: v1.3.1, rRNA: v1.2, and sRNA: v2013.8) [14–16]. A genome overview was created with Circos  to show annotation information. In addition, genomic islands (GIs), prophages, repeat regions, transfer elements, plasmids, and insertion sequences elements (IS elements) in LCT-EC001 were analyzed. Repetitive sequences were predicted using RepeatMasker . Tandem repeats were analyzed using Tandem Repeat Finder (TRF) . PHAST  was used for prophage prediction. IslandPath-DIOMB  was used to predict genomic islands and horizontal gene transfer by examining features such as dinucleotide sequence composition bias and the presence of mobility genes.
Phylogenetic analysis and multilocus sequence typing (MLST)
The genome datasets of the other 62 E. coli strains were compared with the genome of LCT-EC001 for SNP detection by using MUMmer with default settings (version 3.22). Then, the repeat regions of LCT-EC001 were detected by self-blast (choosing BLASTn parameter with blastall, using BLAST v2.2.23), TRF and RepeatMasker. After that, SNPs located in the repeat region were filtered. Based on the location array of SNPs, a phylogenetic tree was generated using the neighbor-joining method with 1000 bootstraps via MEGA6. MLST was performed with the web tool at http://cge.cbs.dtu.dk/services/MLST/, using the assembled genome. By comparing the sequences of seven housekeeping genes (ADK,FUMC,GYRB,ICD,MDH,PURA,RECA) in LCT-EC001 with that in the database, the MLST type was analyzed.
Analysis of antibiotic resistance genes
A BLASTp  search (E-value less than 1·e−5, minimal alignment length percentage larger than 40%) was performed against 3 databases for drug resistance analysis. The databases are ARDB (Antibiotic Resistance Genes Database), CARD and ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation). Then, the identified sequences were all BLAST searched online (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to match genes in NCBI. The identified resistance genes were further verified by PCR and Sanger sequencing. Location relationships between these identified genes and genomic islands, prophages, repeat regions, transfer elements, plasmids, and IS elements were analyzed.
Strain LCT-EC001 is resistant to most clinical antibiotics
Antimicrobial susceptibility profile of E. coli strain LCT-EC001
Normally, E. coli colonizes the intestines of humans and other animals . However, it is a frequent cause of community and hospital-acquired infections, such as those of the urinary tract, bloodstream, abdomen, skin and soft tissues under certain circumstances . This bacterium also causes pneumonia, neonatal meningitis and food-borne infections on a global scale . It is well accepted that antimicrobial resistance is related to widespread antibiotic use, especially their inappropriate use in humans and other animals, as well as in the food industry . With the increasing incidence of multidrug-resistant organisms, antibiotic resistance has now become a serious global public health problem.
Genomic features of the strain LCT-EC001
The genome summary of E. coli strain LCT-EC001
Genome size (bp)
N50 length (bp)
GC content (%)
Gene average length (bp)
GC content of genes (%)
Phylogenetic tree and MLST analysis of LCT-EC001
Analysis of the complement of antibiotic resistance genes
The drug resistance genes in LCT-EC001 covered nearly all known drug-resistance mechanisms in E. coli. Of these genes, 34 genes were detected from the ARDB database, 61 genes were detected from the CARD database, and 19 genes were detected from the ARG-ANNOT database (Additional file 7: Table S7). In addition, 6 of these genes were located in genome islands, 11 genes were located in plasmids, 3 genes were near transposons, 14 genes were near insertion sequences, and no genes were related to prophages or repeat regions (Additional file 7: Table S7). A more concerning problem is that antibiotic resistance traits in bacteria can transfer between each other, regardless of their genus , via mobile genetic elements (MGEs) such as plasmids , insertion sequences , integrons/transposons , and chromosomal fragments (including resistance islands) . A plasmid is a kind of extrachromosomal DNA molecule with the ability to autonomously replicate. A plasmid can harbor genes encoding β-lactams, even carbapenemases or extended-spectrum β-lactamases, and aminoglycosides  and genes producing antibiotic-target protecting proteins, antibiotic-modifying enzymes or multidrug efflux pumps . Plasmids can also acquire mobile genetic elements by encoding endonucleases/methylase restriction systems . Furthermore, plasmids can move from one bacterial cell to another by conjugal transfer , playing a vital role in the spread of resistance determinants among bacteria. An insertion sequence (IS) is an important MGE that widely exists in bacterial genomes, usually with a length of 0.6–2.0 kb . IS elements can help resistance genes to transfer between and within bacteria  and can upregulate downstream resistance genes . Integrons are another MGE responsible for the emergence and spread of antibiotic resistance genes, including β-lactamases, aminoglycosides, and fluoroquinolones . Transposons, like plasmids, have the potential to transfer horizontally or vertically among pathogens, driving the development of antibiotic resistance . A genomic island (GI), usually with a size of 4.5–600 kb and generated by lateral gene transfer (LGT), is a large continuous genomic region. In addition, GIs can carry tens to hundreds of genes, often important for bacterial evolution, such as antibiotic resistance .
It is worth mentioning that our genome is a draft genome comprising 18 contigs, which means there are 17 gaps of sequence missed and other drug-resistant genes that may not have been identified.
XZ, SX, XJ and CL designed the study and wrote the manuscript; YL and ZF analyzed the data; XZ, YY, PW, DL and XZ carried out the experiments. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
This Whole Genome Shotgun project of E. coli strain LCT-EC001 has been deposited at DDBJ/ENA/GenBank under the accession JMIC00000000. The version described in this paper is version JMIC02000000.
Consent for publication
Ethics approval and consent to participate
This work was supported by the National Natural Science Foundation of China (No: 81600011) and the China Postdoctoral Science Foundation (Grant No: 2016M592928).
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