Pathogenic Phenotype and Genotype ofPseudomonas aeruginosaIsolates from Spontaneous Canine Ocular Infections

Abstract

Purpose This study was designed to determine whether the ability to adversely affect corneal epithelial cell health is a factor common to Pseudomonas aeruginosa keratitis strains and to assess the prevalence of each pathogenic phenotype and genotype in a canine model of naturally-acquired P. aeruginosa ocular infection. Methods P. aeruginosa ocular isolates were collected by sampling 100 dogs without disease (six isolates collected) and by sampling dogs with conjunctivitis (two isolates), endophthalmitis (one isolate), active keratitis (12 isolates), and resolved P. aeruginosa keratitis (four isolates). Phenotype was determined in vitro by quantifying corneal epithelial cell invasion by gentamicin survival assays, and cytotoxic activity by Trypan blue exclusion assays. Genotyping was performed for genes encoding the type III secreted effectors. Results The ratio of invasive to cytotoxic strains with 95% confidence intervals (CI) was 0.83 (CI, 0.42– 0.99) for conjunctival microflora isolates, 0.80 (CI, 0.54 – 0.94) for ocular infection isolates, and 1.0 (CI, 0.45–1.0) for strains isolated post-resolution of keratitis. Among ocular infection isolates, invasive and cytotoxic strains were significantly (P ≤ 0.02) associated with older and younger dogs, respectively. Visible adverse effects on epithelial cells were significantly (P ≤ 0.03) more frequent for keratitis strains (6/12) than other strains (1/13), but only three of these keratitis strains and the single non-keratitis strain possessed ExoU. Conclusions Invasive strains predominated in the dogs of this study. Only keratitis strains had visible adverse effects on epithelial cells without overt cytotoxicity, suggesting virulence strategies affecting live corneal epithelial cell health are selected for among keratitis strains.

Characterisation of Pseudomonas aeruginosa related to bovine mastitis

“…The presence of four TTSS genes (exoS, T, Y and U) among the 122 P. aeruginosa isolates was examined by PCR using gene-specific primers, as described previously (Ledbetter et al, 2009). The chromosomal DNA was ex-tracted with a DNeasy ® blood and tissue kit (Qiagen Inc., USA) according to the manufacturer's instructions for Gram-negative bacteria.…”

Antimicrobial susceptibility and minimal inhibitory concentration of <i>Pseudomonas aeruginosa</i> isolated from septic ocular surface disease in different animal species

“…Discussion Animals, especially dogs, are well known naturallyoccurring P. aeruginosa ulcerative keratitis models. The infection is considered appropriate for comparative investigations since this bacterial agent in canine infections is congruent with the classic genotypephenotype pattern reported for human corneal isolates (Ledbetter et al, 2009). P. aeruginosa strains isolated from ocular infections have a particularly important role when prior ocular surface disease provides an opportunity for colonization and stabilization of an eye infection.…”

“…Considering, septic ocular surface disease typically requires an initial injury, such as mechanical or chemical trauma, before a microorganism can infect the cornea (Mayo et al, 1986). Specifically, bacterial infections of ocular surfaces are commonly the result of a predisposing factor or disease that allows the overgrowth of a bacterium that is or is not a part of the ocular surface microbiota (Quinn et al, 1994;Moore et al 1995;Ledbetter et al, 2009). Pseudomonas aeruginosa, a gram-negative bacillus that is widely distributed in nature, is commonly considered a transitional opportunistic agent and part of the normal ocular surface microbiota (Quinn et al, 1994).…”

“…Pseudomonas aeruginosa, a gram-negative bacillus that is widely distributed in nature, is commonly considered a transitional opportunistic agent and part of the normal ocular surface microbiota (Quinn et al, 1994). However, P. aeruginosa is also frequently isolated from ocular surface infections, especially from ocular septic keratitis (Ledbetter et al, 2009). This microorganism is considered one of the most destructive bacteria associated with eye infections in any species (Gutierrez, 1972), having a variety of mechanisms and virulence factors which allow efficient colonization and subsequently compromised ocular tissues and most corneal layers.…”

“…This is particularly true for ocular surface infections, where a drug can be delivered directly to the site of infection at a high concentration compared to that of systemic intravenous administration (Levison and Levison, 2009). Due to the aggressive nature of P. aeruginosa infections and the devastating consequences they can have on the eye, the lag time between diagnosis and antimicrobial susceptibility test results might enable significant and irreversible damage to be done to the eye prior to initiation of proper therapy.…”