Isolation and Genomic Characterization of a Heat-Labile Enterotoxin 1-Producing Escherichia fergusonii Strain from a Human

ABSTRACT Escherichia fergusonii strains have been isolated from patients with diarrhea, but their virulence determinant has not been well elucidated. Here, we report the first isolation of a heat-labile enterotoxin 1 (LT1)-producing E. fergusonii strain (strain 30038) from a patient in Japan. The complete genome sequence of strain 30038 was determined and subjected to comparative genomics and phylogenetic analyses with 195 publicly available genomes of E. fergusonii. In addition to strain 30038, the elt1 gene was also identified in an E. fergusonii strain that is phylogenetically distinct and which was isolated from poultry in the United Kingdom. Fine genomic comparison revealed that these two strains share comparable elt1-bearing plasmids. However, an intriguing distinction arises in strain 30038, wherein the plasmid has integrated into the chromosome via a recombination process mediated by an insertion sequence. The production of active LT1 toxin by strain 30038 was verified through an in vitro assay using cultured cells. A large plasmid carrying 11 antimicrobial resistance genes was also identified in strain 30038. Our results indicate that extensive surveillance of elt1-positive E. fergusonii strains as diarrheagenic pathogens is needed. IMPORTANCE Escherichia fergusonii, a species closely related to Escherichia coli, is known to cause sporadic conditions in humans, including diarrhea. However, the critical virulence factors in E. fergusonii clinical isolates remain to be identified. This study shows the first isolation of an E. fergusonii strain carrying the elt1 gene, which encodes heat-labile enterotoxin 1, from a patient with diarrhea. Our analysis of public databases also revealed the presence of elt1-positive E. fergusonii strains isolated from poultry in the United Kingdom. Interestingly, while the elt1 gene in the poultry isolate was present on a large plasmid, in the human isolate it was integrated into the chromosome, which may confer stability on the elt1-carrying genetic element. Our findings highlight the need for extensive surveillance of elt1-positive E. fergusonii strains in livestock animals.

However, the isolation of LT-producing E. fergusonii strains from humans has not yet been reported. Moreover, there is no information on the virulence factors in the above-mentioned E. fergusonii strains from human patients (1). Here, we report the isolation of an LT1-positive E. fergusonii strain from an adult patient and describe its genomic features revealed by the determination of its complete genome sequence and genomic comparison with publicly available E. fergusonii genomes. The LT1-mediated cytotoxicity of the strain on Chinese hamster ovary (CHO) cells is also shown.
A bacterial strain was isolated using DHL agar (Eiken Kagaku, Japan) from a stool specimen from a 38-year-old male patient in a hospital in Miyazaki, Japan, in 2014. Through routine microbiological tests, in this strain (named strain 30038), the elt1 gene was identified by PCR using elt1 primers (6) and LT1 production was confirmed by a latex agglutination test (VET-RPLA; Denka Seiken, Tokyo, Japan). Strain 30038 was initially thought to be ETEC but was ultimately identified as E. fergusonii based on its biochemical features (see Table S1 in the supplemental material). Although the detailed clinical records of the patient were not available, the strain was most likely a causative agent of the patient's intestinal infection.
For genomic characterization, we determined the complete genome sequence of strain 30038 by hybrid assembly of Illumina short-read sequences and Oxford Nanopore Technologies (ONT) long-read sequences using Unicycler (7) (see the methods in the supplemental material for more details). The genome comprised a 4,687,599-bp chromosome and four plasmids (234,318 bp, 109,686 bp, 3,240 bp, and 1,506 bp in length) ( Fig.  S1 and S2). Unexpectedly, although known elt1 genes are exclusively carried on large plasmids (5), the elt1 gene was found on the chromosome in strain 30038.
To analyze the phylogenetic position of strain 30038 in E. fergusonii, we collected 195 E. fergusonii genomes available in the NCBI and EnteroBase (8) databases and constructed a core gene-based phylogenomic tree with strain 30038 (Fig. 1a and Table S2; see also methods in the supplemental material for more details). Among the 196 strains analyzed, 37 (18.8%) were isolated from livestock, 21 (10.7%) were from poultry, and 14 (7.1%, including strain 30038) were from humans; most were isolated in Europe (52.6% [n 5 103]) and North America (20.9% [n 5 41]) (Fig. 1b). No apparent bias in geographic distribution or isolation source was observed (Fig. 1a).
A search of the 196 genomes for virulence genes using an in-house virulence gene database (supplemental material), which included genes for the major E. coli virulence factors and known ETEC colonization factors (CFs) (see the methods in the supplemental material for more details), revealed that among the genes analyzed, strain 30038 contained only elt1 (Fig. 1a). In the other strains, elt1 was detected only in a strain distantly related to strain 30038 (strain RHB43-C11), which was isolated from a pooled fecal sample from a poultry farm in the United Kingdom (9). Although various CFs have been identified in human and animal ETEC strains (10), no known CFs were detected in either elt1-positive strain. However, the two elt1-positive E. fergusonii strains may possess previously unidentified CFs because 20% to 40% of ETEC clinical isolates contain no detectable CFs, and the presence of previously unidentified CFs in these isolates has been suspected (10). Although several E. fergusonii strains contained cdt-IA (cytolethal distending toxin), hes (hemagglutinin), or tia (invasion) from livestock (n 5 3) or unknown samples (n 5 8), the contribution of these potential virulence factors to human diseases remains unclear (11)(12)(13).
A search for acquired antimicrobial resistance (AMR) genes using the ARG-ANNOT database (14) revealed that strain 30038 contained 11 genes that confer resistance to six drug classes. Thus, this strain is genetically regarded as a multidrug-resistant (MDR) strain. These genes were all carried on plasmid 1 ( Fig. 1a and Fig. S2), which also contained the tellurium-resistant and mercury-resistant operons. Many other E. fergusonii strains (.70%) also carried at least one AMR gene, and 17 strains carried more than 10 genes (up to 25 genes) (Fig. 1a). In previous studies, E. fergusonii has been observed to be prevalent among livestock animals with a substantial degree of resistance to various antimicrobials, including tigecycline, colistin, and carbapenem, within China (15)(16)(17)(18)(19). Consequently, this bacterium could be regarded as a significant reservoir of AMR genes. As both Illumina read and ONT long-read data were available for strain RHB43-C11, we determined the complete genome sequence of strain RHB43-C11 by hybrid assembly to compare its elt1-bearing genomic region with that of strain 30038. The genome of RHB43-C11 comprised a 4,534,336-bp chromosome and six plasmids (227,114 bp, 133,429 bp, 95,369 bp, 94,932 bp, 45,971 bp, and 4,593 bp in length), and elt1 was found on a plasmid (plasmid 2) (Fig. 1c), as seen in most ETEC strains (5). However, this plasmid did not show homology to any genomes of ETEC virulence plasmids available in the NCBI database. Instead, it showed high sequence similarity to the LT1-bearing chromosomal region of strain 30038, with several small structural variations (Fig. 1c  and d). Moreover, the chromosome region of strain 30038 adjacent to the LT1-encoding region showed a high similarity to plasmid 4 of strain RHB43-C11 (Fig. 1c and d). These results and the comparison of this chromosome region of strain 30038 with the analogous region of strain RHB43-C11 indicate that the LT1-encoding region of strain 30038 is a composite plasmid that was generated by the fusion of plasmids very similar to plasmids 2 and 4 of RHB43-C11 and inserted into the iolS gene of strain 30038. The presence of IS15DI in the plasmid-plasmid and chromosome-plasmid junctions suggested that insertion sequence (IS)-mediated recombination involved both genetic events (Fig. 1c to e). The integrations of these two plasmid-like sequences were confirmed by PCR using primers located on the flanking regions of the integration sites (Fig. S3). The predominant observed state was the integrated form, as opposed to the circular form. Integrations of virulence plasmids into the chromosome by homologous recombination have also previously been reported for enteroinvasive E. coli and Shigella flexneri, and the plasmid integrations into chromosome have been proposed to be one of the strategies to ensure plasmid maintenance (20). It is hypothesized that the integration of the elt1-carrying plasmid into the chromosome in strain 30038 also confers stability. Accordingly, the two strains share not only elt1 but also genes for an iron utilization system and type IV-b pili homologous to those on plasmid R64. Type IVb pili may be involved in the horizontal transfer of LT1-encoding elements in these E. fergusonii strains.
We finally analyzed the production of LT1 toxin by strain 30038 using the CHO cell elongation assay (21). CHO cells (CHO-K1 cell line from Riken Cell Bank) treated with the culture supernatant of strain 30038 showed clear elongation, comparable to the cells treated with purified cholera toxin and the culture supernatant of a typical ETEC strain (Oita16058), confirming that strain 30038 produces active LT1 toxin (Fig. 1f).
In conclusion, we isolated an LT1-producing E. fergusonii strain named strain 30038 from the stool of an adult patient in Japan. In strain 30038, the elt1 gene was determined to be located on a plasmid that has been integrated into the chromosome. Additionally, a multidrug-resistant plasmid carrying 11 AMR genes was also identified in strain 30038. Given our findings, it is crucial to closely monitor the emergence and dissemination of multidrug-resistant LT1-producing strains of E. fergusonii within both human and animal populations. Such strains have the potential to contribute to the prevalence of diarrheal diseases. Moreover, further analyses of virulence mechanisms and the clinical impact of LT1-producing E. fergusonii may provide insights into their potential threat to public health.
Data availability. All sequence data generated in this study are available in the DDBJ/EMBL/GenBank BioProject under accession number PRJDB13909.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 3.1 MB.