Cytotoxic clinical isolates of Pseudomonas aeruginosa identified during the Steroids for Corneal Ulcers Trial show elevated resistance to fluoroquinolones

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Abstract

Background

To determine the relationship between type three secretion genotype and fluoroquinolone resistance for P. aeruginosa strains isolated from microbial keratitis during the Steroids for Corneal Ulcers Trial (SCUT) and for two laboratory strains, PA103 and PAO1.

Methods

Confirmed P. aeruginosa isolates from the SCUT were divided into exoU(+) or exoU(−). The exoU(+) strains contained the gene encoding ExoU, a powerful phospholipase toxin delivered into host cells by the type three secretion system. Isolates were then assessed for susceptibility to fluoroquinolone, cephalosporin, and aminoglycoside antibiotics using disk diffusion assays. Etest was used to determine the MIC of moxifloxacin and other fluoroquinolones. Laboratory isolates in which the exoU gene was added or deleted were also tested.

Results

A significantly higher proportion of exoU(+) strains were resistant to ciprofloxacin (p = 0.001), gatifloxacin (p = 0.003), and ofloxacin (p = 0.002) compared to exoU(−) isolates. There was no significant difference between exoU(+) or exoU(−) negative isolates with respect to susceptibility to other antibiotics except gentamicin. Infections involving resistant exoU(+) strains trended towards worse clinical outcome. Deletion or acquisition of exoU in laboratory isolates did not affect fluoroquinolone susceptibility.

Conclusions

Fluoroquinolone susceptibility of P. aeruginosa isolated from the SCUT is consistent with previous studies showing elevated resistance involving exoU encoding (cytotoxic) strains, and suggest worse clinical outcome from infections involving resistant isolates. Determination of exoU expression in clinical isolates of P. aeruginosa may be helpful in directing clinical management of patients with microbial keratitis.

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Most cited references 32

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Risk factors and causative organisms in microbial keratitis.

Matthew Green, Andrew Apel, Fiona Stapleton (2008)

To establish the risk factors, causative organisms, levels of antibiotic resistance, patient demographics, clinical presentations, and clinical outcomes of microbial keratitis at a tertiary hospital in Australia. Patients who had a corneal scraping for culture over a 5-year period were identified through the local microbiology database, and a retrospective audit of their medical records was carried out. Clinical information was gathered from medical records, and smear, culture, and antibiotic resistance results were from the microbiology database. An index of disease severity was calculated for each patient from scores for the magnitude of the epithelial defect and anterior-chamber reaction and the location of the lesion. Associations between risk factors for keratitis and variables such as patient demographics, causative organism and antibiotic resistance, disease severity, and outcome were analyzed by using analysis of variance and chi tests with appropriate correction for multiple comparisons. Two hundred fifty-three cases of microbial keratitis in 231 patients were included. Sixty percent of patients were men, and there was a bimodal distribution in the age of presentation. Common risk factors for keratitis were contact lens wear (53; 22%), ocular surface disease (45; 18%), ocular trauma (41; 16%), and prior ocular surgery (28; 11%). Gram stains were positive in 33%, with a sensitivity of 53% and specificity of 89%. Cultures of corneal scrapings were positive in 65% of cases, and Pseudomonas aeruginosa (44; 17%), coagulase-negative staphylococci (22; 9%), Staphylococcus aureus (19; 8%), and fungi (7; 3%) were commonly recovered. P. aeruginosa was more common than other culture results in contact lens-related cases (55% vs. 0%-23%; P < 0.001), and S. aureus was more common than other culture results in ocular surgery-related cases (29% vs. 0%-21%; P < 0.001). Patients with keratitis related to prior ocular surface disease had more severe keratitis at the time of scraping (P = 0.037). Cultures positive for Fusarium, P. aeruginosa, and other Gram-negative organisms had statistically significantly more severe keratitis at the time of scraping, whereas patients with negative cultures had milder keratitis (P = 0.030). Only 2% of all bacterial isolates were resistant to ciprofloxacin, 20% of Gram-positive isolates were resistant to cephalothin, and no Gram-negative isolates were resistant to gentamicin. In this series, the most common risk factor for keratitis was contact lens wear and the most commonly isolated organism was P. aeruginosa.

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Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa.

Enesha Cobb, D Mariscal, R. Hauser … (2002)

Pseudomonas aeruginosa is a frequent cause of ventilator-associated pneumonia. Recent evidence suggests that production of type III secretion proteins is correlated with increased pathogenicity in both cellular and animal models of infection. The objective of this study was to determine whether this system contributes to disease severity in humans with ventilator-associated pneumonia. Retrospective pilot cohort study. University hospital. Thirty-five mechanically ventilated patients with bronchoscopically confirmed ventilator-associated pneumonia caused by P. aeruginosa. Ventilator-associated pneumonia was categorized as severe (patients died or had a recurrence of their pneumonia despite appropriate antibiotic therapy) or mild (patients uneventfully recovered from their pneumonia). The type III secretion genotypes and phenotypes of isolates cultured from the patients with ventilator-associated pneumonia were determined. Whereas every examined isolate harbored type III secretion genes, only 27 (77%) were capable of secreting detectable amounts of type III proteins in vitro. Twenty-two (81%) of the patients infected with these 27 isolates had severe disease. Of the eight isolates that did not secrete type III proteins, only three (38%) were cultured from patients with severe disease. Thus, infection with a type-III-secreting isolate correlated with severe disease (p < .05). In vitro assays indicated that ExoU, the type III effector protein most closely linked to mortality in animal models, was secreted in detectable amounts in vitro by 10 (29%) of the 35 examined isolates. Nine (90%) of these 10 isolates were cultured from patients with severe disease (p < .05 when compared with the nonsecreting isolates). In contrast, ExoS was secreted by 16 (46%) of the 35 examined isolates. Twelve (75%) of these 16 isolates were cultured from patients with severe disease (p = .14 when compared with the nonsecreting isolates). In patients with ventilator-associated pneumonia, type-III-secreting isolates were associated with worse clinical outcomes, suggesting that this secretion system plays an important role in human disease. Our findings support the hypothesis that antibodies targeted against these proteins may be useful as adjunctive therapy in intubated patients with P. aeruginosa colonization or infection.

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Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung.

R. Hauser, Ciara M. Shaver (2004)

Pseudomonas aeruginosa uses a type III secretion system to promote development of severe disease, particularly in patients with impaired immune defenses. While the biochemical and enzymatic functions of ExoU, ExoS, and ExoT, three effector proteins secreted by this system, are well defined, the relative roles of each protein in the pathogenesis of acute infections is not clearly understood. Since ExoU and ExoS are usually not secreted by the same strain, it has been difficult to directly compare the effects of these proteins during infection. In the work described here, several isogenic mutants of a bacterial strain that naturally secretes ExoU, ExoS, and ExoT were generated to carefully evaluate the relative contribution of each effector protein to pathogenesis in a mouse model of acute pneumonia. Measurements of mortality, bacterial persistence in the lung, and dissemination indicated that secretion of ExoU had the greatest impact on virulence while secretion of ExoS had an intermediate effect and ExoT had a minor effect. It is of note that these results conclusively show for the first time that ExoS is a virulence factor. Infection with isogenic mutants secreting wild-type ExoS, ExoS defective in GTPase-activating protein (GAP) activity, or ExoS defective in ADP-ribosyltransferase activity demonstrated that the virulence of ExoS was largely dependent on its ADP-ribosyltransferase activity. The GAP activity of this protein had only a minor effect in vivo. The relative virulence associated with each of these type III effector proteins may have important prognostic implications for patients infected with P. aeruginosa.

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Author and article information

Contributors

Durga S Borkar

Nisha R Acharya

Chelsia Leong

Prajna Lalitha

Muthiah Srinivasan

Catherine E Oldenburg

Vicky Cevallos

Thomas M Lietman

David J Evans

Suzanne M J Fleiszig

Journal

Journal ID (nlm-ta): BMC Ophthalmol

Journal ID (iso-abbrev): BMC Ophthalmol

Title: BMC Ophthalmology

Publisher: BioMed Central

ISSN (Electronic): 1471-2415

Publication date Collection: 2014

Publication date (Electronic): 24 April 2014

Volume: 14

Page: 54

Affiliations

[1 ]Department of Ophthalmology, University of California, 94143 San Francisco, CA, USA

[2 ]Francis I. Proctor Foundation, University of California, 94143 San Francisco, CA, USA

[3 ]School of Optometry, University of California, 688 Minor Hall, 94720 Berkeley, CA, USA

[4 ]Aravind Eye Care System, Madurai, Tamil Nadu, India

[5 ]Department of Epidemiology and Biostatistics, University of California, 94143 San Francisco, CA, USA

[6 ]College of Pharmacy, Touro University California, 94592 Vallejo, CA, USA

[7 ]Programs in Vision Science, Infectious Diseases and Immunity, and Microbiology, University of California, 94720 Berkeley, CA, USA

Article

Publisher ID: 1471-2415-14-54

DOI: 10.1186/1471-2415-14-54

PMC ID: 4008435

PubMed ID: 24761794

SO-VID: 577eb5f4-10dd-4ec2-96ad-8ce9b30879ad

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Cytotoxic clinical isolates of Pseudomonas aeruginosa identified during the Steroids for Corneal Ulcers Trial show elevated resistance to fluoroquinolones

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Abstract

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Clinical Virtual Research Center Journal of Ophthalmology

Most cited references 32

Risk factors and causative organisms in microbial keratitis.

Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa.

Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung.

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