Zooming in on the intracellular microbiome composition of bacterivorous Acanthamoeba isolates.

Acanthamoeba, a free-living amoeba in water and soil, is an emerging pathogen causing severe eye infection known as Acanthamoeba keratitis. In its natural environment, Acanthamoeba performs a dual function as an environmental heterotrophic predator and host for a range of microorganisms that resist digestion. Our objective was to characterize the intracellular microorganisms of phylogenetically distinct Acanthamoeba spp. isolated in Australia and India through directly sequencing 16S rRNA amplicons from the amoebae. The presence of intracellular bacteria was further confirmed by in situ hybridization and electron microscopy. Among the 51 isolates assessed, 41% harboured intracellular bacteria which were clustered into four major phyla: Pseudomonadota (previously known as Proteobacteria), Bacteroidota (previously known as Bacteroidetes), Actinomycetota (previously known as Actinobacteria), and Bacillota (previously known as Firmicutes). The linear discriminate analysis effect size analysis identified distinct microbial abundance patterns among the sample types; Pseudomonas species was abundant in Australian corneal isolates (P < 0.007), Enterobacteriales showed higher abundance in Indian corneal isolates (P < 0.017), and Bacteroidota was abundant in Australian water isolates (P < 0.019). The bacterial beta diversity of Acanthamoeba isolates from keratitis patients in India and Australia significantly differed (P < 0.05), while alpha diversity did not vary based on the country of origin or source of isolation (P > 0.05). More diverse intracellular bacteria were identified in water isolates as compared with clinical isolates. Confocal and electron microscopy confirmed the bacterial cells undergoing binary fission within the amoebal host, indicating the presence of viable bacteria. This study sheds light on the possibility of a sympatric lifestyle within Acanthamoeba, thereby emphasizing its crucial role as a bunker and carrier of potential human pathogens.

Prevalence and seasonal variation of Acanthamoeba in domestic tap water in greater Sydney, Australia.

BACKGROUND: This study examined the prevalence of free-living Acanthamoeba in domestic tap water in the greater Sydney region, Australia, and determined any seasonal variation in prevalence. METHODS: Fifty-four participants were included in this study following approval from an institutional human research ethics committee. Each participant self-collected two samples (one in summer and another in winter) from the surface of the drain of the bathroom sink using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli and incubation at 32°C for two weeks. The plates were microscopically examined for the presence of free-living amoeba. DNA was isolated from 20 samples and a polymerase chain reaction (PCR) assay was performed for amplification of the partial sequence of the 18S ribosomal RNA gene. The PCR amplified products were sequenced using Sanger sequencing and genotyping was performed based on the variation in nucleotide sequences. RESULTS: A total of 97 samples were collected over the two collection periods, with 28.6 per cent of samples morphologically classified as Acanthamoeba. The summer period yielded 16 of 54 (29.6 per cent) samples classified as Acanthamoeba, while the winter period yielded 12 of 43 (27.9 per cent) samples classified as Acanthamoeba. There was no statistically significant difference (p = 0.85) between the prevalence of free-living Acanthamoeba in summer compared to winter. Phylogenetic analysis showed that 15 of 20 (75 per cent) isolates belonged to genotype T4, the most frequent genotype isolated in Acanthamoeba keratitis. CONCLUSION: The prevalence of free-living Acanthamoeba characterised morphologically in domestic tap water of the greater Sydney region was higher than expected, especially considering the low incidence of Acanthamoeba keratitis in Australia. However, this study did not find variation between seasons. As the T4 genotype was most common, Sydney-based practitioners must always consider Acanthamoeba as a possible causative organism in cases of microbial keratitis, regardless of the season.

Corynebacterium propinquum Keratitis Identified Using MALDI-TOF.

PURPOSE: To report a corneal infection due to Corynebacterium propinquum identified using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method. METHODS: A 94-year-old woman presented with suppurative keratitis. Her ocular history included Fuchs corneal dystrophy, corneal transplantation, and glaucoma. A gram-stained smear revealed coryneforms. Colonies on bacterial culture media were assayed by MALDI-TOF. RESULTS: Identification of C. propinquum was confirmed by RNA polymerase ß subunit (rpoB) gene sequencing. The patient was treated with topical cefazolin, gentamicin, and fluorometholone, and her vision improved to 20/160 unaided when last seen. CONCLUSIONS: MALDI-TOF can correctly identify isolates that are both uncommon and difficult to distinguish from related species. The ease and low running costs of this method will improve the laboratory diagnosis of ocular infections.

Acanthamoeba encephalitis: isolation of genotype T1 in mycobacterial liquid culture medium.

We report a case of Acanthamoeba encephalitis diagnosed from an antemortem brain biopsy specimen, where the organism was first isolated in mycobacterial liquid medium and first identified by using a sequence generated by a commercial panfungal sequencing assay. We correlate susceptibility results with clinical outcome.

Pythium insidiosum keratitis in an Australian child.

A 3-year-old girl from the Northern Territory developed suppurative keratitis after swimming in pools. A mycelial organism suspected to be Pythium insidiosum was cultured. Treatment with polyhexamethylene biguanide and voriconazole for 5 days was unsuccessful, and a corneal graft was performed. The infection was cleared and when last seen 14 months after surgery the patient had a stable graft and useful vision. The identification of the organism was confirmed by rRNA gene sequencing. P. insidiosum is an oomycete, an organism more closely related to kelp than to fungi. Masses of hyphae were seen in sections and, for the first time, the ultrastructure of P. insidiosum in human tissue is described. The staining characteristics of cultured hyphae were assessed; lactofuchsin and acridine orange were found to be the most useful methods. Although the diagnosis of P. insidiosum keratitis is not difficult, and the organism is susceptible in vitro to a number of antimicrobial agents, early corneal transplantation remains the treatment of choice.

Bacteria commonly isolated from keratitis specimens retain antibiotic susceptibility to fluoroquinolones and gentamicin plus cephalothin.

PURPOSE: Patients presenting with presumed infective keratitis were studied to determine predisposing factors, the current susceptibilities of the bacterial isolates to a range of relevant antibiotics, the success rate of topical antibiotic treatment of keratitis and predictors of failure of topical therapy. METHODS: Corneal scrapings taken from patients who presented between January 2002 and December 2003 to the Sydney Eye Hospital Emergency Department with keratitis were cultured. The minimum inhibitory concentration of selected antibiotics was determined for each bacterial isolate using an agar dilution technique. RESULTS: One hundred and twelve consecutive patients presented with corneal ulcers. Forty-seven of the 112 (42%) patients had a growth from the corneal scraping. Potential predisposing factors were identified in 64% of patients, most frequently contact lens wear (36% of patients). Coagulase-negative staphylococci were the most common species isolated. Other common organisms isolated include Pseudomonas aeruginosa, Corynebacterium spp., Staphylococcus aureus and Streptococcus spp. CONCLUSIONS: Most microorganisms isolated from patients with bacterial keratitis showed susceptibility to ciprofloxacin and aminoglycosides. Cephalothin plus aminoglycoside constituted an effective initial broad-spectrum antibiotic combination. The success rate of topical antibiotic treatment of corneal abscess is 89%. Predictors of failure include older age group, medium or large ulcer, culture-negative keratitis, hypopyon and poor visual acuity.

Antigens of selected Acanthamoeba species detected with monoclonal antibodies.

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.

Capnocytophaga canimorsus endophthalmitis following cataract surgery.

An elderly man developed endophthalmitis 1 week after cataract extraction and lens implantation. Intraocular samples were collected and the patient received intravitreal vanco-mycin and ceftazidime, and topical tobramycin. A Gram stain of vitreous humour revealed spindle-shaped Gram-negative bacilli. He was then given systemic clindamycin and topical ofloxacin. Capnocytophaga canimorsus, a member of the oral flora of dogs and cats, was cultured after 3 days. The infection resolved leaving the patient with a visual acuity of 6/60. An attempt was made to culture the organism from the mouth of the patient's pet dog. This was unsuccessful and the source of the infection remains unknown.