SEARCHOTHERophthalmology, cornea

other·ophthalmology, cornea, micronutrients, microbiology, infectious disease·PMC10795995

The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model

PLOS Neglected Tropical Diseases · 8 authors, 5 centres

AI SUMMARY

FIDELITY 76%

POPULATIONWistar rats aged 8–10 weeks

INTERVENTIONIntrastromal injection of Acanthamoeba containing naturally acquired intracellular Pseudomonas aeruginosa (strain AK1, ~10^4 trophozoites, ~4×10^4 bacteria)

COMPARISONIntrastromal injection of Acanthamoeba without intracellular bacteria (strain AK11), or injection of Acanthamoeba re-isolated after infection (AK10), or injection of intracellular P. aeruginosa alone (4×10^4 cells)

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TL;DR

This study shows that Acanthamoeba containing live intracellular Pseudomonas aeruginosa causes severe keratitis in rat corneas, whereas Acanthamoeba alone induces only mild infection. The findings suggest that intracellular bacteria in Acanthamoeba can act as a prerequisite for acute keratitis and may contribute to polymicrobial infections in patients.

Full summary

4,110 CHARS

**Background:** Acanthamoeba species are environmental hosts for various microorganisms and can cause Acanthamoeba keratitis (AK), a progressive corneal infection often associated with coinfections involving bacteria. The presence of bacterial endosymbionts has been linked to increased virulence, but the role of naturally acquired intracellular bacteria in AK severity in vivo remained unclear. This study investigated whether naturally occurring intracellular Pseudomonas aeruginosa within Acanthamoeba enhances keratitis in a rat model.

**Methods:** Acanthamoeba isolates were recovered from domestic tap water (AK1) and from a corneal swab of an AK patient (AK11), both confirmed as genotype T4F by sequencing. The AK1 strain was found to harbor intracellular P. aeruginosa (AK1-PA) by 16S rRNA PCR, MALDI-TOF MS, and FISH. TEM showed bacteria within phagolysosome-like structures and undergoing binary fission. Metabolic activity of intracellular bacteria was confirmed using alkDala labeling. The motility of AK1-PA (swimming, swarming, twitching) was compared to wild-type PA01, and the exoU and exoS genes were detected by PCR. Survival of AK1-PA versus PA01 was assessed in human monocyte-derived macrophages (hMDMs) by CFU counting at 3, 6, 12, and 24 h post-infection. In two phases, rats received intrastromal injections of: (Phase I) Acanthamoeba without intracellular bacteria (group A, n=4) or Acanthamoeba with intracellular P. aeruginosa (group B, n=4); (Phase II) Acanthamoeba re-isolated from group B corneas (AK10, group C, n=3) or P. aeruginosa alone re-isolated from group B (group D, n=3, ~4×10^4 cells). Clinical scores were assessed daily for 5 days using slit lamp. Histopathology (H&E, calcofluor white), MPO assay, and re-isolation of organisms were performed.

**Key Results:** The Acanthamoeba AK1 strain contained an average of 4±2.2 intracellular P. aeruginosa per trophozoite. AlkDala labeling confirmed the bacteria were metabolically active. AK1-PA possessed the exoU gene but not exoS; motility did not differ significantly from PA01. In hMDMs, AK1-PA showed significantly higher intracellular survival: at 3 h, >4-fold greater CFU than PA01 (p<0.05); at 24 h, ~3-fold greater (p<0.05). Confocal microscopy at 6 and 12 h showed significantly higher bacterial load per macrophage with AK1-PA (p<0.05). In vivo, group B (Acanthamoeba with intracellular P. aeruginosa) developed severe keratitis with ring infiltrate, ulceration, and corneal opacity within 48 h, with mean clinical scores significantly higher than group A at all time points (p≤0.05 to p≤0.0001). Group A (Acanthamoeba alone) showed only mild focal infiltrates. Histology of group B corneas revealed complete epithelial/endothelial collapse, hemorrhagic necrosis, and inflammatory infiltrates, while group A showed only focal necrosis. CFW staining and laser microdissection confirmed Acanthamoeba cysts in group B corneas. Re-isolated trophozoites (AK10) from group B had no intracellular bacteria. In phase II, group D (P. aeruginosa alone) developed moderate keratitis but significantly lower clinical scores than group B (p<0.05). Group C (AK10) showed mild keratitis. MPO levels were highest in group B (1312.3 ng/mL), compared to group D (791.3 ng/mL), group A (367.5 ng/mL), and group C (191.6 ng/mL); differences between group B and others were significant (p<0.05). The mean clinical scores of group C were significantly higher than group A (p<0.05), suggesting increased virulence after corneal passage.

**Clinical Implications:** The study demonstrates that Acanthamoeba harboring live intracellular P. aeruginosa induces significantly more severe keratitis in rats than Acanthamoeba alone or bacteria alone. The ability of these bacteria to survive and replicate within human macrophages suggests a potential for enhanced pathogenicity. The release of bacteria from amoebae in the cornea may drive polymicrobial keratitis. Routine assessment for intracellular bacteria in corneal isolates of Acanthamoeba may improve diagnosis and treatment, potentially warranting adjunctive antibiotic therapy.

PICO

PPOPULATION

Wistar rats aged 8–10 weeks

IINTERVENTION

Intrastromal injection of Acanthamoeba containing naturally acquired intracellular Pseudomonas aeruginosa (strain AK1, ~10^4 trophozoites, ~4×10^4 bacteria)

OOUTCOME

Severity of keratitis (clinical score, histopathology, myeloperoxidase activity), survival of P. aeruginosa in macrophages

STUDY TYPE

other

SPECIALTY

ophthalmology

SUMMARISED BY

AI pipeline

FIDELITY CHECK

76% · B

ORIGINAL PAPER

PMC10795995

The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model

PLOS Neglected Tropical Diseases

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ABSTRACT

Abstract Background Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba . This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. Methodology/Principal findings Household water and corneal swabs were collected from AK patients. Acanthamoeba strains and genotypes were confirmed by sequencing. Acanthamoeba isolates were assessed for the presence of intracellular bacteria using sequencing, fluorescence in situ hybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne–functionalized D–alanine (alkDala). Primary human macrophages were used to compare the intracellular survival and replication of the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats were challenged intrastromally with Acanthamoeba containing or devoid of P . aeruginosa and evaluated for the clinical response. Domestic water and corneal swabs were positive for Acanthamoeba . Both strains belonged to genotype T4F. One of the Acanthamoeba isolates harboured P . aeruginosa which was seen throughout the Acanthamoeba ’s cytoplasm. It was metabolically active and could be seen undergoing binary fission. This motile strain was able to replicate in macrophage to a greater degree than strain PAO1 ( p <0.05). Inoculation of Acanthamoeba containing the intracellular P . aeruginosa in rats eyes resulted in a severe keratitis with increased neutrophil response. Acanthamoeba alone induced milder keratitis. Conclusions/Significance Our findings indicate the presence of live intracellular bacteria in Acanthamoeba can increase the severity of acute keratitis in vivo . As P . aeruginosa is a common cause of keratitis, this may indicate the potential for these intracellular bacteria in Acanthamoeba to lead to severe polymicrobial keratitis.

CITATION EXPORT

Binod Rayamajhee, Mark Willcox, Fiona L Henriquez et al.. The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model. PLOS Neglected Tropical Diseases. (2024). https://doi.org/10.1371/journal.pntd.0011878

AUTHORS

8 · 5 CENTRES

Binod Rayamajhee, Mark Willcox, Fiona L Henriquez, Ajay Kumar Vijay, Constantinos Petsoglou, Gauri Shankar Shrestha, Hari Kumar Peguda, Nicole Carnt

1School of Optometry and Vision Science, Faculty of Medicine and Health, UNSW, Sydney, Australia
2School of Health and Life Sciences, University of the West of Scotland, Blantyre, Scotland, United Kingdom
3Sydney and Sydney Eye Hospital, Southeastern Sydney Local Health District, Sydney, Australia
4Save Sight Institute, University of Sydney, Sydney, Australia
5Universidade Federal de Sergipe, BRAZIL