Genomic Characteristics of an Extensive-Drug-Resistant Clinical Escherichia coli O99 H30 ST38 Recovered from Wound

Background Antibiotic-resistant Escherichia coli is one of the major opportunistic pathogens that cause hospital-acquired infections worldwide. These infections include catheter-associated urinary tract infections (UTIs), ventilator-associated pneumonia, surgical wound infections, and bacteraemia. Objectives To understand the mechanisms of resistance and prevent its spread, we studied E. coli C91 (ST38), a clinical outbreak strain that was extensively drug-resistant. The strain was isolated from an intensive care unit (ICU) in one of Kuwait's largest hospitals from a patient with UTI. Methods This study used whole-genome sequencing (Illumina, MiSeq) to identify the strain's multi-locus sequence type, resistance genes (ResFinder), and virulence factors. This study also measured the minimum inhibitory concentrations (MIC) of a panel of antibiotics against this isolate. Results The analysis showed that E. coli C-91 was identified as O99 H30 ST38 and was resistant to all antibiotics tested, including colistin (MIC > 32 mg/L). It also showed intermediate resistance to imipenem and meropenem (MIC = 8 mg/L). Genome analysis revealed various acquired resistance genes, including mcr-1, blaCTX-M-14, blaCTX-M-15, and blaOXA1. However, we did not detect blaNDM or blaVIM. There were also several point mutations resulting in amino acid changes in chromosomal genes: gyrA, parC, pmrB, and ampC promoter. Additionally, we detected several multidrug efflux pumps, including the multidrug efflux pump mdf(A). Eleven prophage regions were identified, and PHAGE_Entero_SfI_NC was detected to contain ISEc46 and ethidium multidrug resistance protein E (emrE), a small multidrug resistance (SMR) protein family. Finally, there was an abundance of virulence factors in this isolate, including fimbriae, biofilm, and capsule formation genes. Conclusions This isolate has a diverse portfolio of antimicrobial resistance and virulence genes and belongs to ST38 O99 H30, posing a serious challenge to treating infected patients in clinical settings.


Background
Multi-drug resistant Escherichia coli are opportunistic pathogens causing hospital-acquired infections worldwide.These infections include catheter-associated urinary tract infections, ventilator-associated pneumonia, surgical wound infections, and bacteraemia.They often carry resistance genes to antibiotics, such as β-lactams and fluroquinolone, that are commonly used for treatment.
Genes encoding extended-spectrum β-lactamases (ESBLs) are often found on mobile genetic elements (MGEs) and are harbored within transposons or insertion sequences, thereby facilitating their spread to other strains.The most prevalent and dominant ESBL gene found in Enterobacteriaceae isolated from humans and food-producing animals is bla CTX-M-15 (1).Recently, a major concern has been the resistance to colistin, a polymixin, one of the last antibiotics in use after others failed.Colistin resistance gene mcr, which currently has ten variants, is usually found on plasmids of various incompatibility groups (IncX4, IncI2, and IncHI2) and often coexists with ESBLs (2, 3).In addition to ESBL genes, macrolide, tetracycline, aminoglycoside, fluoroquinolone, and carbapenem resistance genes can also coexist in a colistin-resistant isolate, limiting treatment options for hospitalized patients.Plasmid (mcr)-and chromosomal-mediated colistin resistance involve mutations in genes encoding enzymes that are associated with outer membrane modification of LPS by encoding a phosphoethanolamine transferase that catalyzes the addition of a phosphoethanolamine moiety to lipid A (3,4), such as the pmrC and pmrE and the pmrHFIJKLM operon (4).Previous studies on E. coli have revealed that mutations in the sensor histidine kinase pmrB are an important mechanism of colistin resistance, leading to the constitutive production of the enzymes ArnT and EptA that add a positive charge (4-amino-4-deoxy-L-arabinose and phosphoethanolamine, respectively) to the phosphate groups of lipid A and reducing the affinity of colistin to bind to lipid A (3,4).
To plan effective treatment guidelines, it is crucial to understand the mechanisms of resistance and epidemiology of multidrug-resistant (MDR) E. coli in both the community and hospitals.Given the burden of diseases caused by E. coli and its significant public health concern, hospitals should continuously monitor their antimicrobial treatment efficacy.Whole-genome sequencing (WGS)-based in silico approaches are valuable tools in gene analysis of outbreak strains that offer detailed epidemiological investigation and tracing of pathogens (5).In this study, we used WGS to characterize E. coli C91 (ST38), an extensively drug-resistant clinical outbreak strain isolated from patient zero in the intensive care unit (ICU) of one of the largest hospitals in Kuwait, with the intention of successfully treating the patients and containing its spread.

Sample Collection
A clinical E. coli isolate C91 was isolated from a post-surgical wound of a 53-year-old male admitted to ward 8/ICU (26/11/2016) and was initially identified by VITEK 2 ID system (bioMérieux, Marcy-l'Etoile, France).This patient was named patient zero.

Antibiotic Sensitivity Testing
Antimicrobial sensitivity testing was carried out according to the Clinical and Laboratory Standards Institute (2020) (6).

Whole-Genome Sequencing Analyses
Genomic deoxyribonucleic acid (DNA) was extracted using QIAamp ® DNA Mini Kit (Qiagen, Hilden, Germany) and quantified by the NanoDrop-800 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA) according to the manufacturer's instructions.
The WGS was performed by MicrobesNG, University of Birmingham, UK (https://microbesng.uk) using the Illumina MiSeq ® sequencer platform.The reads were trimmed using Trimmomatic, and the quality was assessed by MicrobesNG's in-house scripts combined with the following software packages: SAMtools (Sequence, Alignment/Map), Bedtools, and bwa-mem (Burrows-Wheeler Aligner).All statistics are based on contigs of size ≥ 500 bp unless otherwise noted.The trimmed data were assembled using the SPAdes algorithm assembler (version: 3.7.1);this de novo assembly of the quality-controlled reads was assembled to create a draft genome sequence, and variant calling was performed using VarScan.An automated annotation was performed using Prokka (version 1.13.3).The WGS of the isolate was submitted to Genbank Accession: SAMN10105215, ID: 10105215 (sample name: Escherichia coli strain Kuwait C-91).

Detection of Phages from WGS
Phaster tool (21) was used to identify prophage sequences.This tool classifies the phages into three classes (intact, questionable, and incomplete) based on their completeness (phage score).Additionally, by using the Proksee server, phages were identified with the VirSorter2 2.2.4 tool and were screened for antimicrobial resistance genes using the basic local alignment search tool (BLAST).

Description of the Isolate and Mapping Summary
The bacterial strain C91 was identified as E. coli O99 H30 ST38 according to two different schemes, Warwick and Pasteur Institute (Appendix 1).The draft genome was annotated using RAST (Table 1) and revealed a linear chromosome consisting of 5 532 235 base pairs, with 4 964 coding sequences, 87 transfer RNA (tRNA) genes, and several proteins with functional assignments.The genome was assembled using the SPAdes assembler (version: 3.7.1)from trimmed data, producing an N50 quality value of 181 117 and a L50 of 11, with an N75 of 97 722 and L75 of 20.The sample had a mapping rate of 76.81% against the reference genome (without Ns), with an average depth of 76.96X and over 90.93% coverage of more than 1X, a result that falls within the normal range.The genome mapping of antimicrobial resistance and virulence factors is shown in Figure 1, and the comparison of E. coli C91 to E. coli K12 MG1655 (GenBank: U00096.2) using NCBI and Proksee software is presented in Figure 2.

Antibiotic Sensitivity Testing and Resistance Genes
E. coli C91 was resistant to all antibiotics tested, including aminoglycosides, chloramphenicol, tetracycline, β-lactams (both alone and in combination with β-lactam inhibitors), ciprofloxacin, erythromycin, trimethoprim, gentamycin, and colistin.The MIC for these antibiotics was greater than 32 mg/L.It also demonstrated intermediate resistance to imipenem and meropenem with an MIC of 4 mg/L.The analysis of the genome of E. coli C91 revealed the presence of 200 antibiotic-resistance genes, including efflux pump complexes and antibiotic target protection proteins, as confirmed by CARD annotations.The genome analysis also revealed the presence of mcr-1, bla CTX-M-14 , bla CTX-M-15 , and bla OXA-1 genes, but not bla NDM or bla VIM .The bacterium was observed to have acquired resistance genes, including aac(3)-IIa, aac(6')Ib-cr, aadA1, qnrS1, catB4, tetA, mphA, ermB, and dfrA1, as shown in Table 3 (and Appendix 2) and Figure 1.Point mutations were also detected in chromosomal resistance genes, including gyrA, parC, and pmrB, leading to changes in amino acids, as shown in Table 4.The results of some of these mutational modifications are not clear.

Virulence Factors
This isolate has an abundance of virulence factors shown in Table 5 and Figure 1, including fimbriae, biofilm, and capsule formation genes.

Phage Analysis
Eleven prophage regions were identified in E. coli C91, from contig 1 -45, using the Phaster tool (Table 6 Appendix 3).Out of these regions, three are intact, seven are incomplete, and one is questionable.However, when the VirSorter2 2.2.4 tool was used in Proksee software, phages were also picked up from nodes 46-183 (

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Escherichia coli C91 also contains several multidrug efflux pumps, including the multidrug efflux pump mdf (A), which confers resistance to antibiotics, such as chloramphenicol, erythromycin, and fluoroquinolones (26).The present study also detected mutations in chromosomally encoded gyrA, gyrB, parC, pmrB, ampC, and cya genes causing resistance to fluoroquinolones, polymyxins, and fosfomycin.We did not detect bla NDM , bla VIM , nor bla OXA-48 in this isolate, althrough the MIC for imipenem was just below the cutoff point (MIC = 4).However, others have reported the prevalence of carbapenem resistance among Enterobacteriaceae in hospitals in Kuwait (27).
Antimicrobial resistance plasmids present in E. coli C91 comprise epidemic resistance plasmids IncFIB and IncFIC(FII), which can acquire resistance determinants and disseminate readily among Enterobacteriaceae and broad-range IncY, IncI2 (pMCR-1) carrying the mcr-1 gene.IncI1-I plasmids have been shown to propagate the resistance genes between different species (28).Therefore, this isolate has the potential to tolerate and resist conventional antibiotic therapies.
The identification of E. coli clones in the fields of taxonomy and epidemiology is predicated on a combination of O-and H-antigens.These antigens are characterized by variations in the sugars present in the O unit and the linkages between O units (29).There are currently 185 O antigens, and the O99 antigen consists of four d-rhamnose moieties in the backbone and two d-glucose moieties in the side chain.The O-antigen is synthesized and transported by an ABC transporter-dependent process and is considered an important virulence factor, offering selective advantages in specific niches.Pathogenic clones are often found to have a higher incidence of certain O antigens (29,30).
H-antigens (flagellins) are encoded by fliC genes, with 53 different serotypes of H-antigen identified (31).The diversity of H-antigens arises from lateral gene transfer and recombination of foreign DNA, generating alleles and antigenic variation (32).FimH genes encode a type I fimbria that enables adherence and infects the epithelial urinary tract tissue expressed in uropathogenic E. coli (UPEC).FliC genes encode proteins that promote successful host colonization and are involved in interleukin-6 (IL-6) and interleukin-8 (IL-8) release.FumC genes encode a protein that catalyzes fumarate oxidation to malate during the oxidative TCA cycle under aerobic conditions.FumC is required for E. coli fitness in vivo, and a loss of FumC results in delayed growth during iron limitation (33)(34)(35).
The H30 subclone has been reported to be responsible for the clonal dissemination of ST131 E. coli (36).Therefore, it is proposed that H30 provides ST38 clones with the advantage of propagation.Since E. coli sequence type ST38 has become prominently associated with hospitaland community-acquired infections worldwide (37)(38)(39), it is crucial to identify the subclones to increase the chances of successful treatments.
In conclusion, E. coli C91 (ST38) O99 H30 is a high-risk and globally disseminated extraintestinal pathogenic (ExPEC) strain that can cause invasive infections and resist multiple antibiotic treatments.This study used WGS and in silico analysis to identify the molecular characteristics of this isolate.
The obtained results showed that it contains genes encoding ESBLs that confer resistance to cephalosporins and other β-lactam antibiotics.Additionally, E. coli C91 (ST38) is resistant to macrolides, tetracyclines, aminoglycosides, and fluoroquinolones, making it extensively drug-resistant (XDR).Furthermore, it carries mcr-1 gene, which severely limits the treatment options.This isolate also encodes several virulence factors facilitating biofilm formation and adherence to tissues.Infections caused by XDR E. coli C91 (ST38) O99 H30 in the ICU might be life-threatening and require urgent treatment.

Figure 1 .
Figure 1.The gene map of E. coli C91 with labels showing the resistance (red) and virulence (blue) genes.

Table 1 .
Summary of the Statistics of the Assembled Genome of E. coli C91

Table 2 .
Plasmids Identified in E. coli C91

Table 4 .
Chromosomal Point Mutations and Their Phenotypic Characteristics Identified in E. coli C-91

Table 6 .
Phage Analysis with Phaster Tool Indicative of the Regions Containing Phages a a Region: The number assigned to the region.Region length: The length of the sequence of that region (in bp).Completeness: A prediction of whether the region contains an intact or incomplete prophage.#Total proteins: The number of ORFs present in the region.Most common phage: The phage(s) with the highest number of proteins most similar to those in the region.GC %: The percentage of GC nucleotides of the region.Iran J Pharm Res.2024; 23(1):e143910.

Table 3 .
Antimicrobial Resistance Genes and Their Phenotypic Characteristics Identified in E. coli