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Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology.

Tayabali AF, Coleman G, Nguyen KC - PLoS ONE (2015)

Bottom Line: However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested.Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains.No relationship was observed between strains and levels of peripheral leukocytes.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Laboratory, Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Environmental Health Centre, Health Canada, Ottawa, Ontario, Canada.

ABSTRACT
Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.

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Related in: MedlinePlus

Cellular Viability During Pseudomonas Exposure.HT29 cells were exposed to Pseudomonas strains for 24 h at 37°C. Bacteria were rinsed from the wells, and one mg mL-1 MTT was added. MTT formazan generated after two h incubation was solubilized with DMSO and quantified by absorbance at 505 nm with a multiwell spectrophotometer. Data represent the mean bioreduction activities of three wells ± standard deviation compared to saline-treated wells. Asterisks indicate statistically different values compared to control exposures as determined using ANOVA and Dunnett’s Multiple Comparison Test (p < 0.05).
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pone.0143604.g001: Cellular Viability During Pseudomonas Exposure.HT29 cells were exposed to Pseudomonas strains for 24 h at 37°C. Bacteria were rinsed from the wells, and one mg mL-1 MTT was added. MTT formazan generated after two h incubation was solubilized with DMSO and quantified by absorbance at 505 nm with a multiwell spectrophotometer. Data represent the mean bioreduction activities of three wells ± standard deviation compared to saline-treated wells. Asterisks indicate statistically different values compared to control exposures as determined using ANOVA and Dunnett’s Multiple Comparison Test (p < 0.05).

Mentions: In order to determine whether the bacteria could cause damage to cultured cells in vitro, exposure assays were conducted with the colonic epithelial cell line, HT29. Twenty-four hour exposures were followed by a measurement of cellular metabolic activity (bioreduction activity) as measured with the MTT assay. As shown in Fig 1, the Pa strains (except Pa10752) demonstrated most toxicity (15 to 40% of control cell bioreduction). Observation of the exposure wells by microscopy revealed that the loss in MTT formazan was due to both the loss of cell adhesion and formazan production. Exposure to either Pa10752 or the other Pseudomonas strains resulted in non-statistically significant changes in bioreduction ranging from 77 to 98% of control.


Virulence Attributes and Host Response Assays for Determining Pathogenic Potential of Pseudomonas Strains Used in Biotechnology.

Tayabali AF, Coleman G, Nguyen KC - PLoS ONE (2015)

Cellular Viability During Pseudomonas Exposure.HT29 cells were exposed to Pseudomonas strains for 24 h at 37°C. Bacteria were rinsed from the wells, and one mg mL-1 MTT was added. MTT formazan generated after two h incubation was solubilized with DMSO and quantified by absorbance at 505 nm with a multiwell spectrophotometer. Data represent the mean bioreduction activities of three wells ± standard deviation compared to saline-treated wells. Asterisks indicate statistically different values compared to control exposures as determined using ANOVA and Dunnett’s Multiple Comparison Test (p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664251&req=5

pone.0143604.g001: Cellular Viability During Pseudomonas Exposure.HT29 cells were exposed to Pseudomonas strains for 24 h at 37°C. Bacteria were rinsed from the wells, and one mg mL-1 MTT was added. MTT formazan generated after two h incubation was solubilized with DMSO and quantified by absorbance at 505 nm with a multiwell spectrophotometer. Data represent the mean bioreduction activities of three wells ± standard deviation compared to saline-treated wells. Asterisks indicate statistically different values compared to control exposures as determined using ANOVA and Dunnett’s Multiple Comparison Test (p < 0.05).
Mentions: In order to determine whether the bacteria could cause damage to cultured cells in vitro, exposure assays were conducted with the colonic epithelial cell line, HT29. Twenty-four hour exposures were followed by a measurement of cellular metabolic activity (bioreduction activity) as measured with the MTT assay. As shown in Fig 1, the Pa strains (except Pa10752) demonstrated most toxicity (15 to 40% of control cell bioreduction). Observation of the exposure wells by microscopy revealed that the loss in MTT formazan was due to both the loss of cell adhesion and formazan production. Exposure to either Pa10752 or the other Pseudomonas strains resulted in non-statistically significant changes in bioreduction ranging from 77 to 98% of control.

Bottom Line: However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested.Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains.No relationship was observed between strains and levels of peripheral leukocytes.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Laboratory, Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Environmental Health Centre, Health Canada, Ottawa, Ontario, Canada.

ABSTRACT
Pseudomonas species are opportunistically pathogenic to humans, yet closely related species are used in biotechnology applications. In order to screen for the pathogenic potential of strains considered for biotechnology applications, several Pseudomonas strains (P.aeruginosa (Pa), P.fluorescens (Pf), P.putida (Pp), P.stutzeri (Ps)) were compared using functional virulence and toxicity assays. Most Pa strains and Ps grew at temperatures between 28°C and 42°C. However, Pf and Pp strains were the most antibiotic resistant, with ciprofloxacin and colistin being the most effective of those tested. No strain was haemolytic on sheep blood agar. Almost all Pa, but not other test strains, produced a pyocyanin-like chromophore, and caused cytotoxicity towards cultured human HT29 cells. Murine endotracheal exposures indicated that the laboratory reference strain, PAO1, was most persistent in the lungs. Only Pa strains induced pro-inflammatory and inflammatory responses, as measured by elevated cytokines and pulmonary Gr-1 -positive cells. Serum amyloid A was elevated at ≥ 48 h post-exposure by only some Pa strains. No relationship was observed between strains and levels of peripheral leukocytes. The species designation or isolation source may not accurately reflect pathogenic potential, since the clinical strain Pa10752 was relatively nonvirulent, but the industrial strain Pa31480 showed comparable virulence to PAO1. Functional assays involving microbial growth, cytotoxicity and murine immunological responses may be most useful for identifying problematic Pseudomonas strains being considered for biotechnology applications.

Show MeSH
Related in: MedlinePlus