Genomic characterization of ST38 NDM-5-producing Escherichia coli isolates from an outbreak in the Czech Republic

ABSTRACT A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-β-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

isolates were detected in the same region of the Czech Republic (Brno city and its surroundings), and the presence of the bla NDM-5 gene on the chromosome suggests a clonal outbreak.
The genomic relatedness of the Czech isolates was evaluated against all the ST38 E. coli genomes (n = 2,322) in the Enterobase database (https://enterobase.warwick.ac.uk/).Using Parsnp v1.2 from the Harvest suite (3), SNPs-based phylogeny was constructed on the 2,322 genomes along with the 26 genomes from this study using jch8249 as a reference.The unrooted phylogeny showed that all the Czech genomes (in this study) were grouped in one clade.All other clades were collapsed to have a more detailed view.The Czech genomes of this study clustered together in a subclade along with seven isolates from Australia (n = 4), Vietnam (n = 2), and Norway (n = 1) (Fig. 2).Interestingly, only the Czech isolates and the seven isolates mentioned above had the bla NDM-5 gene.
Furthermore, the clonality of the isolates in this outbreak was assessed (and the seven genomes detected in the same subclade) using Snippy v4.5 (4) as described before ( 5), using the genome of jch8249 as a reference (selected randomly).The results showed that the number of SNPs in both coding and non-coding regions varied from 2 to 16 SNPs between the isolates in this study and from 15 to 25 SNPs in the seven genomes found in the same subclade (Table S4), confirming the clonality of the isolates causing the outbreak in general and more specifically in Brno.The genomes from Vietnam had the least SNPs compared to the reference (15) followed by the genome from Norway (16).Remarkably, in some cases, it detected the presence of the same isolate in multiple patients.In SurGal Clinic Brno, the two strains (56235 and 57464) were isolated 1 month apart yet had the same two SNPs when compared against the reference.Also, 55393 and jch767, isolated from Syn Lab and University Hospital Brno, respectively, shared the same five SNPs and were isolated 6 months apart.Finally, 60272 and 604647, which were isolated from University Hospital Brno and Chronicare Mund Brno, respectively, on the same day, shared the same nine SNPs.These results showed the high clonality of the strains circulating within that region.
Moreover, the clonality of the strains was assessed by assessing the CRISPR array sequences.The spacers in CRISPR arrays were added in chronological order; therefore, it could be used to trace the clonality and the origin of the isolates and define them as a population of strains that were subjected to the same environmental condi tions including geographic location (region) and community/hospital settings (6).The genome assemblies of this study along with the seven genomes in the same subclade were uploaded to CRISPRCasFinder (https://crisprcas.i2bc.paris-saclay.fr/) which showed that all the strains had the CRISPR/Cas I-E type with two CRISPR arrays (7).The CRISPR arrays of all isolates were identical (100%).This implies the presence of a possible single case, which was the ancestor of this outbreak.
In all the Czech isolates, an ≈27-kb region carrying bla NDM-5 was found.This region also harbored other antibiotic resistance genes including aadA2, mphA, sul1, dfrA12, and ermB for resistance to aminoglycosides, macrolides, sulphonamides, and trimethoprim.BLASTn comparison showed that the region was identical in all the studied isolates and the seven related genomes mentioned earlier and exhibited high similarity with sequences from plasmids, like a 161,700-bp IncF (F90:A2:B33) plasmid (pNDM_p30_L1, accession number CP085061.1)isolated from human E. coli in the UK in 2021 and a 148,746-bp IncF (F90:A2:B20) plasmid (pIsolateD_A, accession number CP135489.1)isolated from human E. coli in Canada in 2023.The ≈27 kb region was flanked by two IS26 in the same orientation.No direct repeats were found in the insertion site suggesting a possible homologous recombination.The region was blasted on PHASTER (https://phaster.ca/)(8) but no phages were found.Long sequencing reads revealed that the insertion site of this region is in the same position in the chromosomes.Thus, the plasmid region seemed to be successfully integrated into the chromosome of NDM-5producing E. coli ST38 via homologs recombination or transposition and remained stable as well since the clone itself appeared to be successful.
Furthermore, the WGS data were analyzed with the virulence factor database (VFDB) (http://www.mgc.ac.cn/VFs/) (9).The 26 isolates from this study carried similar virulence genes (with some slight variations).The observed virulence genes were associated with adherence and fimbrial adherence determinants, autotransporters, iron uptake, invasion, T6SS secretion system, and hemolysin A toxin.Phenotypically, biofilm formation was assessed, as described previously (10).Results showed that 13 out of 26 (50%) isolates produced strong biofilm, 9 (35%) were moderate, and 4 (15%) were considered weak biofilm producers.These virulence profiles seem to be highly conserved among the ST38 genomes.Biofilm formation was achieved after 3 days of incubation and signifi cantly reached maximum adherence after 6 days of incubation.The one-way analysis of variance test showed a significant association (P value <0.05) between the incubation time and the biofilm formation (Fig. S1).
In conclusion, E. coli ST38 is a high-risk clone reported in many countries to carry different antibiotic resistance determinants (11)(12)(13).Here, we showed the acquisition of NDM-5 carbapenemase by this clone and its successful spread in one region of the Czech Republic.Recently, similar reports from different countries, especially Germany, have been published (11), indicating the dissemination of NDM-5 carbapenemase in various E. coli clonal lineages.Our results showed that this outbreak was clonally driven with minimal genomic variation between the isolates.The bla NDM-5 was chromosomally encoded, and the genetic environment around it was likely acquired from previously identified plasmids.The region seemed to be stable and harbored multiple insertion sequences and antibiotic resistance genes which implies the possibility of acquiring more resistance genes and integrating to other replicons and/or clones.Additionally, we showed that the presence of premature stop codons in the sequence of ompF contrib uted to the increased meropenem MICs.The isolates show high antibiotic resistance and virulence profiles confirming the successful dissemination and persistence in Brno for over a year.Further monitoring of this clone is needed since the situation infers a possible outbreak on a national scale.

FIG 1
FIG 1 The 2D map of Brno and adjacent regions showing the number of detected isolates identified as NDM-5-producing ST38 E. coli strains.Dark red points correspond to 26 isolates, light red to 9, dark pink to 3, and light pink to 1.

FIG 2
FIG 2The phylogenetic analysis of the clade harboring Czech isolates.The Czech isolates node is colored in red.The outer membrane ring corresponds to the isolate's reporting country.