Lactose fermenting enteroinvasive Escherichia coli from diarrhoeal cases confers enhanced virulence

Abstract

Enteroinvasive Escherichia coli (EIEC), known for causing bacillary dysentery akin to Shigella species, comprises both lactose-fermenting (LF) and non-lactose-fermenting (NLF) isolates. While NLF-EIEC is a well-established pathogen associated with acute dysentery and harbours classical Shigella-like virulence factors, the role of LF-EIEC in human disease remains underexplored. In this study, we sought to characterize LF-EIEC clinical isolates and assessed their pathogenic potential in comparison to NLF-EIEC. Among 13,682 diarrhoeal stool specimens, six LF and nine NLF-EIEC were isolated, predominantly belonging to serogroups O28ac, O125, O136, and O152. Unlike other E. coli, all the EIEC isolates were non-motile. Both the types of EIEC had multiple plasmids harbouring several virulence encoding genes (ipaBCD, ial, virF, sig, sepA and ipaH). Resistance to recent generation antibiotics were mostly confined to NLF-EIEC but some of the LF-EIEC were resistant only to ceftriaxone. Higher invasion ability and significant increase in the expression of virulence encoding genes by the LF-EIEC (p < 0.05) were noted during infection to Int407 cell-line. Additionally, LF-EIEC exhibited extensive colonization of the mouse intestine and expressed severe keratoconjunctivitis in guinea pigs. Together, our findings highlight LF-EIEC as an emerging pathogenic variant warranting heightened surveillance and comprehensive investigation to better understand its epidemiological and clinical significance.

Keywords: Antibiotic resistance; Bacterial infections; Diarrhoea; Enteroinvasive Escherichia coli; Keratoconjunctivitis; Pathogenesis.

Fig. 1

Restriction fragment length polymorphism (RFLP) pattern of XbaI digested clinical LF and NLF-EIEC isolates. Dendrogram showed about 60% similarity between the LF and NLF-EIEC isolates indicating the clonal relationship among the isolates.

Fig. 2

Antimicrobial resistance of LF and NLF-EIEC isolated from Kolkata, India. (a) Percentage of LF and NLF isolates showing resistance to different antibiotics. NLF isolates showed greater resistance than the LF to commonly used antibiotics. (b) EIEC isolates were categorized into 3 different antimicrobial classes to determine MDR phenotypes. MDR-3, MDR-4 and MDR-5 represent isolates that are resistant to at least three, four and five antimicrobial classes, respectively. Very few of the isolates were found show the MDR phenotype and mostly confined to the NLF. (c) Presence of different antibiotic resistance conferring genes. β-lactam genes such as bla_CTXM3, bla_OXA1 and bla_TEM9 were prevalent in both LF and NLF isolates whereas, fluoroquinolone resistance gene qnrS was only found in NLF isolates. AMG aminoglycosides, SFM sulfonamides, TRM trimethoprim, FLQ fluoroquinolones.

Fig. 3

Invasive property of LF and NLF-EIEC isolates was estimated infecting Int407 cell line. S. flexneri 2a from our lab collection was included as a reference in this assay. With the CFU counts, LF isolates were found to be more invasive than the NLF and Shigella. Statistical significance was calculated using Two-way ANOVA between LF and NLF groups. Error bars represent standard deviations from the mean. ****p < 0.0001.

Fig. 4

Transcription profiles of common virulence associated genes of LF and NLF-EIEC expressed during infection of Int407 cell monolayer after 2 h. Real-time PCR was performed, and C_t values were normalized to recA expression using the 2^−ΔΔCt method, with the expression of a S. flexneri 2a set to 1.0. The graphs represent the mean expression levels from three independent experiments performed in duplicate. One-way analysis of variance (ANOVA) and Dunnett’s multiple-comparison tests were used to determine statistical significance. The error bars represent standard deviations from the mean. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5

Intestinal gut colonization in BALB/c mice was determined with representative LF and NLF-EIEC during different time points from 6 h to 5-days. Higher colonization was observed with the LF isolates and gradually decreased over time. Systemic spread to the liver and spleen was found for the LF isolates. Both the types have similar shedding patterns in the stool. Specific variation in the colonization pattern was observed among LF isolates. A non-pathogenic commensal E. coli HK2082022 has also been included as a reference. All the time points with the isolates were performed at least in triplicate. Error bars represent mean ± standard deviation.

Fig. 6

Hematoxylin and Eosin-stained tissue sections of different parts of mice orally infected with LF and NLF-EIEC and challenged for 24 h. Damages in liver and spleen were almost equally observed in both the EIEC isolates. Shapes with different colors represent: Inflammatory cell infiltrates within mucosa (arrows) and submucosa (arrowheads); goblet cell loss (green arrowhead); crypt loss (White arrowhead); hemorrhage (red arrowhead); Inflammation (brown arrow); Extensive granulomas (black arrow); Portal inflammation (green arrow); Lobular inflammation (yellow arrow); Acidophil bodies (white arrow); Infiltrating leukocytes (red arrow).