Whole-Genome Sequencing of an Escherichia coli ST69 Strain Harboring bla_CTX-M-27 on a Hybrid Plasmid

Introduction

Both Gram-negative and Gram-positive bacteria are responsible for urogenital tract infections. However, among Gram-negative bacterial agents, Escherichia coli is the most common causative agent.^1,2 Based on data from the China Antimicrobial Surveillance Network (CHINET), in 2022, E. coli had the highest detection rate, comprising 18.97% of clinical isolates. Uropathogenic E. coli strains were recognized as typical bacterial agents for urinary tract infections, these groups of infectious diseases are more common among women rather than men, as well as female reproductive tract.^3,4 In 2021, a meta-analysis of 82 studies, with a sample size of 33,118, indicated that the prevalence of E. coli in vaginal samples from pregnant women is 32% and that of extended-spectrum-lactamase (ESBL)-producing E. coli in pregnant women is 34%.⁵ Perinatal infections caused by E. coli can result in premature birth, a low birth weight, and an increased risk of neonatal infection and death.^6,7 Notably, due to the misuse or overuse of antibiotics, antimicrobial resistance (AMR) is considered to feature among the top 10 threats to global health.⁸ The emergence and global spread of antibiotic-resistant E. coli have caused difficulties in clinical treatment and have become a public health threat.⁹ Third-generation cephalosporins are the most commonly used class of antibiotics in gynecology/obstetrics and pediatrics departments to treat infections caused by E. coli;^10,11 however, the prevalence of E. coli resistant to third-generation cephalosporins is increasing.¹²

Enterobacteriales bear different antibiotic resistance genes encoding Extended-Spectrum-ß-Lactamases (ESBLs) and Metallo-ß-Lactamases (MBLs) on their chromosomes and mobile genetic elements such as plasmids and transposons.¹³ In E. coli (typical representative of Enterobacteriales), ESBLs mainly confer third-generation cephalosporin resistance.¹⁴ TEM- and SHV-type ESBLs were initially described in the 1980s.^15,16 The first CTX-M-type ESBL (CTX-M-1), with higher hydrolytic activity against cefotaxime than against ceftazidime, emerged in 1995.¹⁷ Since then, several variants of CTX-M-1 have been found,¹⁸ and these are now the most prevalent ESBLs all over the world, with E. coli as their major bacterial host.¹⁹

Mobile genetic elements (eg plasmid, insertion sequence (IS), transposon, and integron) play a major role in facilitating the spread of resistance genes.²⁰ ESBL genes are mainly located on plasmids, and among the ESBL-carrying plasmids from E. coli, the most common replicon types are IncF, IncI1, IncN, IncHI1, and IncHI2.²¹ ESBL genes are always associated with many ISs (such as IS26, ISCR1, ISEcp1, and IS10), transposons (such as Tn2), and integrons.^22–24 Here, we report a multidrug resistant (MDR) E. coli ST69 strain carrying an IncF plasmid harboring bla_CTX-M-27 and other antibiotic resistance genes (ARGs) and present a comparative analysis of the MDR region of the IncF plasmid.

Materials and Methods

For this study, the data collection and analysis are shown in Figure S1.

Bacterial Strains and Identification

The strain EC6868 was isolated from the vaginal secretions of a 39-year-old pregnant woman at the Zhuhai People’s Hospital in December 2021. Species identification and antimicrobial susceptibility testing were conducted using a fully automatic VITEK 2 COMPACT system (BioMérieux, France) according to the manufacturer’s instructions. The results of antimicrobial sensitivity were interpreted based on the Clinical and Laboratory Standards Institute guidelines (M100-S24). The identity of EC6868 was further confirmed through 16S rRNA gene sequencing by using bacterial universal primers (27F/1492R).

Whole-Genome Sequencing, Assembly, and Annotation

Whole-genome sequencing of strain EC6868 was conducted by Genewiz Biotechnology Co. Ltd. (Suzhou, China) by using the Illumina paired-end sequencing and PacBio long reads sequencing. PacBio reads were assembled using HGAP4.0/Falcon 0.3 of WGS-Assembler 8.2.²⁵ The genome assembly was then polished with the software Pilon 1.22²⁶ using Illumina reads. The assembled genome (both chromosome and plasmids) of strain EC6868 was submitted to the GenBank database²⁷ and annotated using the NCBI Prokaryotic Annotation Pipeline (PGAP).²⁸

Bioinformatics Analysis of the Genome of Strain EC6868

Acquired ARGs in the genome of strain EC6868 were identified using the websever ResFinder 4.1,²⁹ with a minimum coverage of 60% and a minimum identity of 90%. Multilocus sequence typing (MLST) of strain EC6868 was performed using MLST 2.0,³⁰ selecting the database as “Escherichia coli #1” and using seven housekeeping genes, including adk, fumC, gyrB, icd, mdh, purA, and recA. Replicon types of the plasmids contained by strain EC6868 were determined using PlasmidFinder 2.1,³¹ with the database “Enterobacteriales”, minimum coverage of 60%, and minimum identity of 95%. The ISs adjacent to the ARGs in the EC6868 genome were identified using ISfinder.³² Sequence similarity search was performed using MegaBLAST³³ scans against the GenBank nr database. BLAST Ring Image Generator (BRIG) 0.95³⁴ and Easyfig2.2.5³⁵ were used for plasmid comparisons.

Nucleotide Sequence Accession Numbers

The genome sequence of strain EC6868, which contained a chromosome and two plasmids, was submitted to GenBank under the accession numbers CP095083–CP095085.

Results

Identification and Antimicrobial Susceptibility Testing of Strain EC6868

Strain EC6868 isolated from vaginal secretions of a pregnant woman, was identified as E. coli by the automatic VITEK 2 COMPACT system, which was then confirmed via 16SrRNA gene sequencing. E. coli strain EC6868 showed resistance to cephalosporins, including cefuroxime, cefuroxime axetil, and ceftriaxone, but conferred intermediate-level ceftazidime resistance (Table S1). In addition, it showed resistance to trimethoprim/sulfamethoxazole and intermediate-level resistance to levofloxacin (Table S1).

Genomic Features of the E. coli Strain EC6868

Genomic analysis revealed that the genome of E. coli strain EC6868 comprised a 5.25-Mb chromosome and two plasmids with sizes of 144,934 bp (pEC6868-1) and 7919 bp (pEC6868-2). MLST analysis indicated that E. coli strain EC6868 belonged to sequence type (ST) 69. PlasmidFinder results indicated that plasmid pEC6868-1 contained three replicons (Col156, IncFIB_AP001918, and IncFII), whereas plasmid pEC6868-2 was an untypeable plasmid.

ResFinder results indicated that E. coli EC6868 carried multiple ARGs located on both chromosomes and the plasmid pEC6868-1. The chromosome of E. coli EC6868 was found to harbor disinfectant-resistance genes (sitABCD). Further, the multidrug-resistance plasmid pEC6868-1 harbored ARGs conferring resistance to extended-spectrum cephalosporins (bla_CTX-M-27), aminoglycosides (aph(6)-Id, aph(3”)-Ib, and aadA5), macrolides (erm(B) and mph(A)-mrx(A)-mphR(A)), sulphonamides (sul1 and sul2), tetracycline (tet(A)-tetR(A)), and trimethoprim (dfrA17). Plasmid pEC6868-1 also harbored the antiseptic-resistance gene ΔqacE.

Comparative Analysis of the MDR Region of Plasmid pEC6868-1

The ARGs, as well as the adjacent IS elements carried by plasmid pEC6868-1, were located on the ~31.2-kb fragment of the plasmid, forming a large MDR region. The MDR region of pEC6868-1, flanked by ∆IS1R and ∆IS1R-∆IS26, which was nearly identical to part of the E. coli plasmid pB16EC0698-3 (100.00% coverage and 99.78% identity; Figure 1). In the MDR region of pEC6868-1, IS26 was found to be located upstream of the macrolide-resistance gene ermB and its leading peptide gene ermB(L) (Figure 1). The ESBL gene bla_CTX-M-27 was located on an ISEcp1-mediated transposition unit, with a truncated ISEcp1 (∆ISEcp1) located upstream of bla_CTX-M-27 and IS903B located downstream of bla_CTX-M-27, forming the genetic structure ∆ISEcp1-bla_CTX-M-27-IS903B (Figure 1). Based on the region ∆ISEcp1-bla_CTX-M-27-IS903B, the BLAST search hit from the database of GenBank (on November 16th, 2023), with the minimum coverage of 100% and minimum identity of 99%, showed that 1109 strains contain the structure (Figure 2). The structure was widely present in the species of Enterobacteriales, including Klebsiella pneumoniae (549), E. coli (347), Salmonella enterica (106), Proteus mirabilis (44) and the other 23 species (63) (Figures 2 and S2).

Figure 1 Comparative analysis of multidrug-resistance (MDR) region of plasmid pEC6868-1 and that of Escherichia coli plasmid pB16EC0698-3 generated using BRIG. Resistance, transposase, and integrase genes are shown in red, blue, and purple, respectively.

Figure 2 An overview of the species of Enterobacteriales harboring the structure “∆ISEcp1-blaCTX-M-27-IS903B.”.

Interestingly, except for ermB-ermB(L) and bla_CTX-M-27, other ARGs carried by plasmid pEC6868-1 were located on the 18.9-kb IS26-composite transposon. Overall, the 18.9-kb IS26-composite transposon was composed of three parts as follows: (1) the mphA-mrx(A)-mphR(A) operon conferring macrolide resistance was flanked by IS26 and IS6100, forming the IS26-mphA-mrx(A)-mphR(A)-IS6100 transposable structure (Figure 1); (2) one fragment of pEC6868-1 containing tet(A)-tetR(A), aph(6)-Id, aph(3”)-Ib, and sul2 located adjacent to IS26-∆TnAs3-eamA and IS26-mphA-mrx(A)-mphR(A)-IS6100 (Figure 1); (3) notably, a complex class 1 integron, including intI1, dfrA17, aadA5, ∆qacE, and sul1, was also found in the MDR region of plasmid pEC6868-1, located adjacent to IS26-∆ISKpn72-∆IS1R-∆IS26 (Figure 1).

Based on the 18.9-kb IS26-composite transposon of plasmid pEC6868-1, the BLAST searches against the nr database of GenBank showed that the 18.9-kb region was widely present on plasmids of E. coli (77 plasmids), mainly distributed in ST131, ST38, and ST405 (Figure 3). Notably, pEC6868 in our study was the first report on a plasmid harboring the 18.9-kb IS26-composite transposon in E. coli ST69 in China. Another ST69 strain of E. coli containing the 18.9-kb structure was found in the United States, 2017 (Table S2). Surprisingly, 75 of the 77 plasmids harboring the 18.9-kb IS26-composite transposon were found to be the IncF plasmids, including IncFIA, IncFIB and IncFII subtypes, accounting for 97.4% of all the 18.9-kb structure-harboring plasmids of E. coli. In addition, four plasmids harboring the 18.9-kb IS26-composite transposon were also found in K. pneumoniae (Figures 3 and S2).

Figure 3 Details of replicon types and STs of host strains of the 81 plasmids harboring 18.9-kb IS26-composite transposon. Phylogenetic cladogram based on the presence/absence of orthologous gene families of the 81 plasmids harboring 18.9-kb IS26-composite transposon were constructed. Ec and Kp represent E. coli and K. pneumoniae, respectively.

Moreover, we found that the region within this 18.9 kb IS26-composite transposon, containing IS26-mphA-mrx(A)-mphR(A)-IS6100 and the complex class 1 integron, was not only present on the plasmids but also on the chromosomes of some pathogens, such as E. coli, K. pneumoniae, Klebsiella michiganensis, Proteus mirabilis, Shigella flexneri, and Morganella morganii (Figure 4).

Figure 4 Comparison of the gene cluster containing IS26-mphA-mrx(A)-mphR(A)-IS6100 and the complex class 1 integron carried by plasmid pEC6868-1with chromosomes of other pathogenic bacteria other than Escherichia coli. Resistance, transposase, and integrase genes are shown in red, blue, and yellow, respectively.

Discussion

In this study, we report a third-generation cephalosporin-resistant strain, E. coli ST69 (EC6868), isolated from the vaginal secretions of a 39-year-old pregnant woman. E. coli ST69 was determined to be one of the multidrug resistant E. coli clones of phylogenetic group D, which are widespread among different hosts, often causing urinary tract infections and exhibiting resistance to antibiotics.^36–38 ST69 E. coli strains have been reported to carry the bla_VIM-harboring IncA plasmid,³⁹ bla_NDM-1-harboring IncI1 plasmid,⁴⁰ and mcr-1-harboring IncHI2 plasmid.⁴¹ In this work, we report that the ST69 E. coli strain carries one hybrid plasmid, with three replicons (Col156, IncFIB_AP001918, and IncFII), harboring bla_CTX-M-27. IncF plasmids, comprising one of the most common incompatibility types, have been identified globally in Enterobacteriales. Moreover, they vary in size (50–200 kb) and replicon types, which harbor huge amount of ARGs and confer resistance to all major classes of antibiotics, such as beta-lactams, chloramphenicol, aminoglycosides, quinolones, and tetracyclines.^42–45 Notably, the IncF-bla_CTX-M association found in E. coli EC6868 in our study has been widely observed in E. coli isolates of both human and animal origins; for example, the IncF plasmid R100 is responsible for the spread of bla_CTX-M-14 in the UK and France.^46,47

Strain EC6868 in our study was found to be a CTX-M-type ESBL-producing E. coli isolate. CTX-M-14 and CTX-M-15 are the most common variants of CTX-M, and CTX-M-27 has increased rapidly in terms of its prevalence.⁴⁸ Notably, the detection of CTX-M-27 in E. coli isolates from patient has been rising and is particularly alarming due to its presence in clonal groups including ST10, ST69, and ST131.^49,50 The ∆ISEcp1 marker was located upstream of bla_CTX-M-27 and carried by pEC6868-1 (ΔISEcp1-bla_CTX-M-27-IS903B) was found in another study, which points that the conserved region responsible for transmission of bla_CTX-M-27 was ISEcp1-bla_CTX-M-27-IS903B.⁵¹ ISEcp1 belongs to the IS1380 family,⁵² and the ISEcp1-like element is associated with several bla_CTX-M gene types in Enterobacteriaceae, including bla_CTX-M-14, bla_CTX-M-24, bla_CTX-M-22, and bla_CTX-M-79.⁵³ IS903B, located downstream of bla_CTX-M-27 carried by pEC6868-1, is a small insertion element of 1057 bp that is transposed predominantly via a conservative “cut-and-paste” mechanism.⁵⁴

Five copies of intact IS26 and one ∆IS26 were found within the MDR region of plasmid pEC6868-1. The 820-bp IS26 was first described in 1983⁵⁵ and was determined to be present in numerous early antibiotic-resistant bacteria, playing a critical role in disseminating ARGs among Gram-negative bacteria.⁵⁶ IS26 is frequently associated with genes encoding antibiotic resistance factors,⁵⁷ and it can contribute to the expression of the ARGs by supplying a promoter −35 region which can be coupled with a −10 region in the adjacent ARG.⁵⁸

In this study, two translocatable units conferring macrolide resistance, IS26-ermB(L)-erm(B) and IS26-mphA-mrx(A)-mphR(A)-IS6100, were detected in plasmid pEC6868-1. In IS26-ermB(L)-erm(B), the ermB encodes a ribosomal methylase which can reduce the affinity of macrolides for the ribosome, resulting in high level of macrolide resistance.⁵⁹ Macrolide antibiotics promote ribosome stalling on the ErmB(L) (regulatory leader peptide), ultimately inducing the expression of ermB.⁶⁰ In the gene cluster mphA-mrx(A)-mphR(A) for high-level macrolide inactivation, mphA encodes a phosphotransferase that inactivates erythromycin and mrx(A) encodes a protein required for expression of MphA, whereas MphR(A) encoded by mphR(A) negatively regulates the expression of mphA at the transcriptional level.⁶¹ The IS26-mph(A)-mrx(A)-mph(R)(A)-IS6100 unit, which is linked to the global dissemination of macrolide-resistance genes,⁶² is the most common form in plasmids carrying mphA.⁶³

Class 1 integrons are prevalent genetic elements that play a key role in the spread of antibiotic resistance,⁶⁴ allowing bacteria to capture and exchange ARGs embedded in the gene cassettes. Moreover, acquisition of gene cassettes is catalyzed by integrase encoded by int1, which is a site-specific recombinase.⁶⁵ Class 1 integrons are structurally composed of three core elements, specifically an integrase gene (intI1), a primary recombination site (attI1), and a common promoter enabling the transcription of gene cassettes.⁶⁶ In all integrons, detection rate of class 1 integrons in clinical isolates is highest at 50–70%, which is linked to the production and spread of antibiotic-resistant bacteria.^67,68

Conclusion

Here, we describe the genomic characteristics of a 3GC-R and ESBL-producing E. coli strain EC6868 belonging to ST69 harboring bla_CTX-M-27 isolated from a pregnant patient in China. The strain carried multiple-resistance genes conferring resistance to aminoglycosides, macrolides, sulfonamides, tetracycline, and trimethoprim. The bla_CTX-M-27 gene located on a hybrid plasmid pEC6868-1 (IncFII/IncFIB/Col156). The ESBL gene bla_CTX-M-27 was flanked by the truncated ISEcp1 (∆ISEcp1) and the IS903B (∆ISEcp1-bla_CTX-M-27-IS903B), which was widely present in the species of Enterobacteriales, especially in K. pneumoniae, E. coli, and Salmonella enterica. Except for bla_CTX-M-27, other ARGs carried by plasmid pEC6868-1 were mainly located on the 18.9-kb IS26-composite transposon (five copies of intact IS26 and one copy of truncated IS26). The 18.9-kb structure was widely present on IncF plasmids of E. coli, mainly distributed in ST131, ST38, and ST405. Notably, pEC6868 in our study was the first report on a plasmid harboring the 18.9-kb structure in E. coli ST69 in China. In addition, Tn3, IS6100 and class 1 integron also play important role in the dissemination of acquired ARGs carried by the plasmid pEC6868-1 of E. coli strain EC6868.