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. 2022 Jun 8;11(6):661.
doi: 10.3390/pathogens11060661.

Surveillance of Amoebic Keratitis-Causing Acanthamoebae for Potential Bacterial Endosymbionts in Ontario, Canada

Nessika Karsenti  1 , Andrew Purssell  2 , Rachel Lau  3 , Filip Ralevski  3 , Shveta Bhasker  4 , Hira Raheel  1 , Andrea K Boggild  5   6   7
Affiliations

Surveillance of Amoebic Keratitis-Causing Acanthamoebae for Potential Bacterial Endosymbionts in Ontario, Canada

Nessika Karsenti et al. Pathogens. .

Abstract

Acanthamoeba spp. are the causative pathogens of several infections, including amoebic keratitis (AK), a vision-threatening infection. Acanthamoebae from corneal specimens of patients with AK harbor bacterial endosymbionts, which may increase virulence. We sought to understand the spectrum of bacterial endosymbionts present in clinical isolates of Acanthamoeba spp. identified in our reference parasitology laboratory. Isolates of Acanthamoeba spp. obtained from our biobank of anonymized corneal scrapings were screened for potential endosymbionts by PCR using primer pairs detecting bacteria belonging to orders Chlamydiales, Rickettsiales, or Legionellales and pan16S primers. Three primer pairs specific to the 18s rRNA gene of Acanthamoeba spp. were used for the amplification of Acanthamoeba DNA used for sequencing. Sanger sequencing of all PCR products was performed, followed by BLAST analysis for species identification. We screened 26 clinical isolates of Acanthamoeba spp. for potential endosymbionts. Five isolates (19%) were found to contain bacterial DNA belonging to Legionellales. Three (11%) contained members of the Rickettsiales and Pseudomonas genticulata was detected in a Rickettsia-positive sample. One strain (4%) contained Neochlamydia hartmannellae, a member of the Chlamydiales order. Bacterial endosymbionts are prevalent in clinical strains of Acanthamoeba causing AK isolated from corneal scrapings. The demonstration of these organisms in clinical Acanthamoeba isolates supports a potential exploration of anti-endosymbiont therapeutics as an adjuvant therapy in the treatment of AK.

Keywords: Acanthamoeba; amoebic keratitis; endosymbionts; surveillance.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Proportion of samples sent for testing that were diagnosed as positive for Acanthamoeba per year from June 2009 to June 2016. Numbers plotted correspond to those found in Table 2. A linear curve was fitted to the graph (equation and R squared provided).
Figure 2
Figure 2
Proportion of samples sent for testing that were diagnosed as positive for Acanthamoeba per month from June 2009 to June 2016.
Figure 3
Figure 3
Agarose gel electrophoresis showing positive detection of various endosymbionts in 27 clinical Acanthamoeba samples. (A) Chlamydiales PCR. (B) Legionellales PCR. (C) Rickettsiales PCR. L—DNA ladder with base pairs indicated; 1 to 28—clinical samples; “+”—positive control of the respective PCR; “L+”—Legionellales-positive control; “C+”—Coxiella-positive control; B—negative control. Endosymbiont positives in clinical samples are indicated by red boxes. There was insufficient DNA in clinical sample # 17 to be analyzed, and sample 4 was subsequently removed from the study.
Figure 3
Figure 3
Agarose gel electrophoresis showing positive detection of various endosymbionts in 27 clinical Acanthamoeba samples. (A) Chlamydiales PCR. (B) Legionellales PCR. (C) Rickettsiales PCR. L—DNA ladder with base pairs indicated; 1 to 28—clinical samples; “+”—positive control of the respective PCR; “L+”—Legionellales-positive control; “C+”—Coxiella-positive control; B—negative control. Endosymbiont positives in clinical samples are indicated by red boxes. There was insufficient DNA in clinical sample # 17 to be analyzed, and sample 4 was subsequently removed from the study.
Figure 4
Figure 4
Gel showing DNA detected outside of cells, when sample DNA was extracted without a freeze–thaw step, to determine whether bacteria detected were present within the collected sample but outside of Acanthamoeba cells. Lanes as follows: L—DNA ladder; 5—sample #A5 (no DNA detected); L+—Legionella-positive control (strong band detected); -—negative control (no DNA detected); B—water (no DNA detected); 22—A22 (DNA of interest highlighted in red box); 23—A23 (no DNA of interest detected); R+—Rickettsia control (band detected).
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