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Clinical isolates of Pseudomonas aeruginosa can be characterized as cytotoxic or invasive, based on their differing effects on host cells. Previous studies have shown that strain type influences pathology in animal models. The aim of this study was to determine if invasive and cytotoxic strains differentially impact clinical presentation, outcome, or therapeutic response in bacterial keratitis.
P.aeruginosa isolates from the NEI-funded Steroids for Corneal Ulcers Trial (SCUT) were subtyped as invasive or cytotoxic strains. The main outcome measure compared between the two subtypes was change in visual acuity at three months using Huber robust regression, adjusting for topical corticosteroid treatment.
Of the 101 confirmed P.aeruginosa isolates from the SCUT, 74 had a classically invasive or cytotoxic genotype. While corneal ulcers caused by genotypically invasive P.aeruginosa strains presented with significantly better visual acuity than those caused by genotypically cytotoxic P.aeruginosa strains when adjusting for the effect of ulcer location (p=0.008), invasive ulcers improved significantly less than cytotoxic ulcers at three months (0.35 logMAR (three and a half line difference), 95% CI 0.04 to 0.66 p=0.03). When compared with topical moxifloxacin alone, adjunctive treatment with topical corticosteroids was associated with significantly more improvement in visual acuity in the invasive subgroup (p=0.04), but was associated with less improvement in vision in the cytotoxic subgroup (p=0.07).
The results of this study suggest that rational profiling of differentially expressed virulence determinants, e.g. cytotoxicity and invasiveness for P.aeruginosa, could be used as a tool for decision making in management of infections to optimize outcome.
Pseudomonas aeruginosa is a significant cause of bacterial keratitis in both the United States and South India, accounting for between 8–29% and 11–48% of cases, respectively.1–7 However, not all P. aeruginosa strains impact host cells in the same manner. Two important virulence determinants are invasiveness and cytotoxicity.8–12 Invasive strains encode exoS, and therefore can sequester themselves intracellularly, replicating and stimulating membrane bleb formation within host cells.13–15 Cytotoxic strains lack exoS, and instead encode the acute cytotoxin exoU, which can quickly kill cells.16 Both ExoS and ExoU are effectors of the P. aeruginosa type III secretion system (T3SS). Effector proteins are injected into eukaryotic cells via the T3SS apparatus, activated within the targeted cell, and then trafficked to specific organelles, where they mediate a variety of phenotypic changes that ultimately result in cell death.17
Studies in cultured cells and in mouse models have compared invasive and cytotoxic strains. Cell culture studies show an inverse relationship between capacity for cell killing and capacity for invasion, while mouse models show different effects on pathology, immune responses, and response to antibiotics.8–10, 12, 18, 19 Although there have been some studies showing differences in ulcer presentation between these two subtypes of P. aeruginosa, a larger study in humans investigating differences in functional outcomes and therapeutic response is lacking.11, 20–24
The Steroids for Corneal Ulcers Trial (SCUT) was an NIH-funded, randomized, placebo-controlled trial investigating the effect of topical corticosteroids as adjunctive treatment to antibiotics for bacterial keratitis.25 The use of topical corticosteroids for bacterial keratitis has been controversial, and prior to this trial, there was insufficient evidence to guide clinical practice. The SCUT found no overall benefit or harm with adjunctive topical corticosteroid treatment across all types of bacterial corneal ulcers but suggested a benefit for patients with the most severe ulcers. The large sample size from the SCUT allows for further investigation by subgroup, including by organism type. The purpose of this pre-specified study was to determine whether the invasive and cytotoxic subtypes of P. aeruginosa have different clinical signs at baseline and result in a different response to therapy, which may allow for a more tailored treatment approach.
In the SCUT, patients with culture-confirmed bacterial keratitis were randomized to receive adjunctive treatment with either topical 1% prednisolone phosphate or placebo after 48 hours of topical 0.5% moxifloxacin treatment. Specific methods of the trial, including inclusion and exclusion criteria as well as examination and treatment protocol, are described in detail elsewhere.26
Patients were evaluated at enrollment, three weeks, and three months by certified refractionists and ophthalmologists who performed visual acuity and slit-lamp examinations, respectively. Best spectacle-corrected visual acuity (BSCVA) was measured in logMAR units using a tumbling “E” chart at four meters, with 0.1 logMAR being approximately one line of acuity. Slit lamp examination was used to measure infiltrate/scar size, epithelial defect size, depth of the ulcer, and size of hypopyon, if present, as well as to assess for ocular adverse events. Infiltrate/scar size and epithelial defect size were evaluated to the nearest 0.1 millimeter by taking the geometric mean of the longest diameter and the longest perpendicular to the first measurement. Re-epithelialization was defined as the absence of an epithelial defect with administration of fluorescein. Depth of the ulcer was measured in thirds (>0–33%, >33–67%, or >67–100%). Size of hypopyon was measured to the nearest 0.5 mm. Ulcer location was assessed using photographs with an artificial 4mm pupil superimposed by cornea-specific software. Location was graded as completely central, partially central, or peripheral. Further details of how these assessments were done have been previously described.26
All corneal isolates from the SCUT with growth morphology and Gram stain characteristics consistent with P. aeruginosa were strain typed after confirming speciation by growth on centrimide agar. Lab personnel were masked to clinical data, both baseline data and treatment outcomes, until strain typing was completed. Bacteria were grown on Trypticase soy agar overnight, and rabbit corneal epithelial cells were exposed to bacterial strain suspensions. Corneal epithelial cells were washed and treated with gentamicin to kill extracellular bacteria. Isolates which were resistant to gentamicin were also treated with amikacin. Rabbit corneal epithelial cells were washed and lysed. Lysate was plated on MacConkey agar and incubated. Percent invasiveness was determined by comparing growth from the intracellular lysate of each isolate to growth from P. aeruginosa clinical isolate 6294, which was used as a positive control. Since the assay for invasion depends on sensitivity to either gentamicin or amikacin, percent invasiveness was not measured for gentamicin and amikacin resistant strains. However, P. aeruginosa speciation was confirmed for these strains with an Analytical Profile Index kit.
Percent cytotoxicity for each corneal isolate was determined by measuring the amount of lactate dehydrogenase released into the media by dead or dying host cells after bacterial exposure using a cytotoxicity detection kit (Roche Diagnostics, Indianapolis, IN). Values were compared to a positive control, P. aeruginosa clinical isolate 6206. In some experiments, invasive control 6294 had slightly lower LDH values than the media (baseline) control. Negative values for percent cytotoxicity corresponded to strains that were not cytotoxic and had negative LDH values after the media control had been included in the calculation. Values greater than one corresponded to invasiveness or cytotoxicity greater than the respective positive control.
Each bacterial isolate was genotyped using polymerase chain reaction (PCR). PCR was specifically performed on target loci for the four effectors, exoU, S, T, and Y, of the type III secretion system for P. aeruginosa. Strains 6206 and 6294 were used as positive controls for exoU and exoS amplification, respectively. Both strains were used as positive controls for exoY and exoT amplification. Negative controls were also used for amplification of each effector protein sequence. Strains which were positive for either exoU or exoS, but not both, were considered typical strains. Within this group, exoU+/exoS− strains were classified as classically cytotoxic while exoU−/exoS+ strains were classified classically invasive on the basis of genotype. Strains which were either positive or negative for both exoU and exoS were classified as atypical strains. More detailed methodology for the invasion and cytotoxicity assays as well as the PCR protocol with specific primers are described elsewhere.9, 24, 27
Univariate analyses for genotype were performed using either Fisher’s exact test or a two- mean-comparison t-test for categorical and continuous variables, respectively. Multivariate analyses were performed using Huber robust regression to assess the relationship between strain type, either genotype or phenotype, and clinical outcome. In these models, strain genotype was used as a dichotomous predictor variable, either classically invasive or classically cytotoxic. Strains with atypical genotypes were not used. Phenotype was used as a continuous predictor variable, measured as either percent invasiveness or percent cytotoxicity. All confirmed isolates were used in phenotype analyses except when percent invasiveness could not be measured. Baseline characteristics used were BSCVA enrollment, location of ulcer, epithelial defect size at presentation, infiltrate/scar size at enrollment, depth of ulcer at enrollment, size of hypopyon, age, and contact lens wear. Clinical outcome was measured using change in BSCVA at 3 months. Ulcer location was added as a covariate in analyses involving BSCVA to control for possible confounding. Additionally, treatment arm was added as a covariate to control for the possible effect of steroids on change in visual acuity at 3 months. An interaction term (treatment arm×strain genotype) was added to the model assessing genotype and clinical outcome to test if a differential effect of steroids on vision was present for cytotoxic versus invasive P. aeruginosa ulcers. Analyses were performed using STATA 11.0 (StataCorp, College Station, TX). P-values reported for all analyses are nominal values and have not been adjusted for multiple comparisons.
Informed consent was obtained for all subjects enrolled in the SCUT. Institutional Review Board approval was granted by the Aravind Eye Care System Institutional Review Board, the University of California, San Francisco Committee on Human Research, and the Dartmouth-Hitchcock Medical Center Committee for Protection of Human Subjects.
Of the 500 patients in the SCUT enrolled between September 1, 2006 and February 22, 2010, 111 corneal bacterial isolates were strain typed based on growth morphology and Gram stain characteristics consistent with P. aeruginosa. One hundred one of these isolates were confirmed P. aeruginosa based on the methodology described above. Of the 101 confirmed P. aeruginosa isolates, 27 were determined to have atypical genotypes defined as exoU+/exoS+ or exoU−/exoS−. The remaining 74 isolates had typical genotypes; 56 were classically invasive and 18 were classically cytotoxic. Percent invasiveness, expressed as a decimal, ranged from 0.03 to 3.32. Percent cytotoxicity ranged from −0.72 to 2.94. A significant difference was present between the genotypically cytotoxic and invasive strains for both percent invasiveness and percent cytotoxicity (Figure 1).
Baseline demographic features and clinical exam findings were compared between the two genotype groups (Table 1). The mean infiltrate/scar sizes of 4.66 and 3.61 mm for invasive and cytotoxic isolates, respectively, were significantly different (p=0.049, Table 1). The difference in enrollment BSCVA between the two genotype groups was not statistically significant (p=0.80, Table 1). Phenotypically, there was no significant association between percent cytotoxicity and enrollment BSCVA (0.10 logMAR, 95% CI −0.18 to 0.38 p=0.47). However, there was a statistically significant correlation between percent invasiveness and enrollment BSCVA; regression analysis showed that one hundred percent invasiveness was associated with an approximately two and a half line better visual acuity at enrollment compared zero percent invasiveness (−0.26 logMAR, 95%CI −0.51 to −0.01 p=0.04).
When controlling for location, P. aeruginosa ulcers caused by genotypically invasive strains had significantly better visual acuity at enrollment, approximately three and a half lines, than those caused by genotypically cytotoxic strains (−0.36 logMAR, 95%CI −0.63 to −0.10 p=0.008, Table 2). Phenotypically, increasing percent invasiveness was associated with a significantly better visual acuity at enrollment when controlling for location. One hundred percent invasiveness was associated with an approximately three line better visual acuity at enrollment than zero percent invasiveness (−0.32 logMAR, 95%CI −0.49 to −0.15 p<0.001, Table 2). Increasing percent cytotoxicity was associated with worse visual acuity at enrollment; however, this relationship was not significant (0.19 logMAR, 95%CI −0.002 to 0.39 p=0.052, Table 2).
Change in BSCVA at three months was compared between patients with ulcers caused by invasive and cytotoxic P. aeruginosa strains. Genotypically invasive strains were associated with an approximately three and a half line less improvement in visual acuity compared to genotypically cytotoxic strains (0.35 logMAR, 95%CI 0.04 to 0.66 p=0.027, Table 3). When controlling for the effect of location on change in BSCVA at three months, genotypically invasive strains were associated with an approximately four line less improvement in visual acuity compared to genotypically cytotoxic strains (0.40 logMAR, 95%CI 0.09 to 0.70 p=0.013, Table 4). Sensitivity analyses demonstrated that treatment arm, either corticosteroid or placebo, did not affect these results; the corticosteroid treatment difference, approximately one line, was not significant (−0.09 logMAR, 95%CI −0.36 to 0.17 p=0.49). However, an interaction term between treatment arm and strain type added to the model was significant (p=0.005, Table 5), suggesting a differential effect of corticosteroids on ulcers caused by invasive and cytotoxic P. aeruginosa strains. (Figure 2)
Analyses of phenotypic properties of each corneal isolate supported these findings. One hundred percent invasiveness was associated with an approximately two line smaller improvement in BSCVA at three months versus zero percent invasiveness (0.22 logMAR, 95%CI 0.03 to 0.41 p=0.023, Table 3). Similarly, one hundred percent cytotoxicity was associated with an approximately three greater improvement in BSCVA at three months compared to zero percent cytotoxicity(−0.29 logMAR, 95%CI −0.50 to −0.08 p=0.007, Table 3). Treatment arm was added to these models as a covariate, was not significant. However, an interaction term between percent invasiveness and treatment significant (−0.37 logMAR, 95% CI −0.74 to −0.004 p=0.047, Table 5), supporting a differential effect corticosteroids on change in visual acuity as percent invasiveness varied.
Several studies have compared the effect of cytotoxicity and invasiveness of P. aeruginosa the cornea in mouse models and have shown a difference in pathogenesis.8, 9, 19 The few studies done in humans with corneal infection have demonstrated an association between strain type and age, as contact lens wear, but the effect on clinical correlates, such as visual acuity, is unknown.20, 23 In this study, we found that ulcers caused by invasive strains presented with better visual acuity than caused by cytotoxic strains, but they showed less improvement at three months compared to cytotoxic strain ulcers. Additionally, ulcers in the corticosteroid treatment arm improved more than those placebo arm within the invasive subgroup but not within the cytotoxic subgroup.
At presentation, we found that patients with genotypically invasive strain ulcers had significantly better visual acuity compared to patients with genotypically cytotoxic ulcers when controlling for ulcer location. Similarly, analyses based on phenotypic rather than genotypic characteristics, i.e. percent invasiveness and percent cytotoxicity, supported these findings. In a mouse model, cytotoxic P. aeruginosa strains have been previously associated with more may be severe corneal edema, which may be expected to cause decreased visual acuity.19
The SCUT demonstrated no overall difference in visual acuity at three months with adjunctive topical corticosteroid treatment.25 Analysis of the data from all patients with confirmed P. aeruginosa infection demonstrated the same result.28 In the current study, we found a statistically significant difference between the two treatment arms in the invasive P. aeruginosa subgroup. On average, patients with invasive ulcers in the steroid treatment arm had a two and a half line greater improvement in BSCVA from enrollment to three month follow-up compared to the placebo treatment arm. Phenotypic analyses supported this finding; increasing percent invasiveness was associated with a greater difference in visual acuity improvement between the steroid and placebo arms.
Prior studies have showed that neutrophilic infiltration into the center of the cornea, not just damage caused by bacteria, is the hallmark of a pathologic response to invasive P. aeruginosa corneal infection in mouse models.9, 19, 29 In contrast, cytotoxic strains suppress neutrophil infiltration as a direct result of exoU expression.29 The improved outcomes we observed in the steroid treatment arm of the invasive subgroup may reflect the local inhibition of PMN infiltration due to topical corticosteroid exposure. However, modification of other host responses may also play a role in the effect of topical corticosteroids on P. aeruginosa corneal ulcers. We found that patients with cytotoxic ulcers in the corticosteroid arm had approximately five and a half lines less improvement in visual acuity at three month follow-up compared to those in the placebo arm; however, this result did not quite reach significance. One prior study showed delayed clearance of cytotoxic, but not invasive, P. aeruginosa strains from the ocular surface in mice deficient in surfactant protein D, an immunologic protein present in the tear film.30 Systemic literature has shown that inhaled corticosteroids decrease serum surfactant protein D levels.31 This raises the question of whether there is a negative effect of topical corticosteroids on immunologic proteins, such as surfactant protein D, that are important for clearance of cytotoxic P. aeruginosa at the level of the ocular surface.
We examined differences in several other clinical characteristics between these two P. aeruginosa subtypes. Specifically, we found that cytotoxic ulcers presented with an approximately one millimeter smaller infiltrate size on average compared to invasive ulcers in our sample. While previous studies have found that ulcers caused by cytotoxic strains are more common in patients younger than 50 years old, we did not find a significant difference in the age of patients with cytotoxic and invasive ulcers.20, 24 However, the relatively young age of patients in our sample may have made it hard to assess the association between strain type and older age. Additionally, one prior study found that corneal isolates from contact lens wearers were more likely to be cytotoxic.23 We were not able to find this relationship, but there were only five contact lens wearers included in the study.
There are a few potential limitations to consider. This study focused on two specific pathogen virulence determinants of P. aeruginosa without considering differences in host factors or other pathogen virulence determinants that might also impact corneal ulcer healing.29 For instance, factors such as tear film composition and presence of specific surfactant proteins at the ocular surface have been shown to affect the capacity to recover from P. aeruginosa keratitis.27, 32–34 Additionally, almost of the patients in the sample studied were enrolled at the Aravind Eye Care System in India. Differences in demographics, contact lens wear, and mechanism of injury may affect the generalizability of the findings to other populations. It is also possible that geographic location may have had an effect on pathogen genotype distribution. For example, there were 27 atypical isolates, which were neither genotypically classically invasive nor cytotoxic. In comparison, a study of 55 human corneal P. aeruginosa isolates from Australia reported only two atypical genotypes.23 Moreover, previous studies reported an approximately even distribution between the cytotoxic and invasive genotypes.20–22 In this study, only 18 of the 74 corneal isolates with typical genotypes were cytotoxic. This difference in distribution may be attributed to the fact that cytotoxic strains have been reported more commonly among infections associated with contact lens wear, which was rare among the SCUT patient population.23 Of interest is the finding that the proportion of cytotoxic strains isolated from the SCUT study participants resembled the distribution found among canines with P. aeruginosa keratitis.24 Additionally, a large sample of clinical nonocular P. aeruginosa isolates had a similar proportion of cytotoxic strains.35 While the small sample of cytotoxic strains could have affected the findings, particularly for subgroup analyses of the cytotoxic strains, the clinical data was collected prospectively a standardized manner at set time points, increasing the ability to detect relationships.
Currently, the microbiological methods used in this study have been performed in a basic science laboratory setting. However, the distinction between cytotoxic and invasive P. aeruginosa genotypes can be made using PCR, which is inexpensive and available in many clinical microbiology laboratory settings. Additionally, the time needed to obtain PCR results may be acceptable given that corticosteroid treatment could be added after 48 hours of antibiotic treatment, as was done in SCUT.
In summary, the results of this study showed that P. aeruginosa bacterial keratitis caused by genotypically invasive strains presented with significantly better visual acuity than genotypically cytotoxic ulcers but had less improvement in visual acuity at three months. They also revealed that adjunctive treatment with topical corticosteroids had a differential effect on invasive and cytotoxic ulcers. The results of this study suggest the potential to guide management decisions using existing therapies, such as corticosteroid treatment, based on these specific virulence determinants. Additionally, they illustrate the concept that not all infections caused by pathogens of a single species present or respond to treatment similarly. Further studies to elucidate differences in clinical presentation and therapeutic response based on specific virulence determinants for other pathogens may be warranted.
The Steroids for Corneal Ulcers Trial was funded by National Eye Institute grant U10 EY015114. Dr. Acharya is supported by National Eye Institute grant K23 EY017897 and a Research to Prevent Blindness Career Development Award. The UCSF Department of Ophthalmology is supported by National Eye Institute core grant, EY02162, an unrestricted grant from Research to Prevent Blindness, the South Asia Research Fund, and That Man May See, Inc. Dr. Fleiszig is supported by National Institutes of Health grants RO1 EY011221 and RO1 AI079192. Dr. Ghanekar, Dr. Li, and Chelsia Leong were supported by National Institutes of Health grant T35 EY07139-18. Alcon provided moxifloxacin (Vigamox) for the trial but did not play a role in the design and conduct of the study, in the collection, analysis, and interpretation of the data, or in the preparation, review, and approval of this manuscript. Dr. Acharya had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Conflicts of Interest: None of the authors have any financial or personal disclosures to report.