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Detection of Pseudomonas aeruginosa Serogroup G Using Real-Time PCR for Novel Target Genes Identified Through Comparative Genomics.

ABSTRACT: Accurate serotyping is essential for effective infection control. Pseudomonas aeruginosa serogroup G is one of the most common serogroups found in water. Conventional serotyping methods are not standardized and have several shortcomings. Therefore, a robust method for rapidly identifying P. aeruginosa serotypes is required. This study established a real-time PCR method for identifying P. aeruginosa serogroup G strains using novel target gene primers based on comparative genomic analysis. A total of 343 genome sequences, including 16 P. aeruginosa serogroups and 67 other species, were analyzed. Target genes identified were amplified using real-time PCR for detecting P. aeruginosa serogroup G strains. Eight serogroup G genes, PA59_01276, PA59_01887, PA59_01888, PA59_01891, PA59_01894, PA59_04268, PA59_01892, and PA59_01896, were analyzed to determine specific targets. A real-time fluorescence quantitative PCR method, based on the novel target PA59_01276, was established to detect and identify serogroup G strains. The specificity of this method was confirmed using P. aeruginosa serogroups and non-P. aeruginosa species. The sensitivity of this real-time PCR method was 4 × 10² CFU/mL, and it could differentiate and detect P. aeruginosa serogroup G in the range of 4.0 × 10³-4.0 × 10⁸ CFU/mL in artificially contaminated drinking water samples without enrichment. The sensitivity of these detection limits was higher by 1-3 folds compared to that of the previously reported PCR methods. In addition, the G serum group was accurately detected using this real-time PCR method without interference by high concentrations of artificially contaminated serum groups F and D. These results indicate that this method has high sensitivity and accuracy and is promising for identifying and rapidly detecting P. aeruginosa serogroup G in water samples. Moreover, this research will contribute to the development of effective vaccines and therapies for infections caused by multidrug-resistant P. aeruginosa.

SUBMITTER: Wang C

PROVIDER: S-EPMC9263582 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for both acute and chronic infections in humans. In particular, its ability to form biofilm, on biotic and abiotic surfaces, makes it particularly resistant to host's immune defenses and current antibiotic therapies as well. Innovative antimicrobial materials, like hydrogel, silver salts or nanoparticles have been used to cover new generation catheters with promising results. Nevertheless, biofilm remains a major health problem. For instance, biofilm produced onto endotracheal tubes (ETT) of ventilated patients plays a relevant role in the onset of ventilation-associated pneumonia. Most of our knowledge on Pseudomonas aeruginosa biofilm derives from in vitro studies carried out on abiotic surfaces, such as polystyrene microplates or plastic materials used for ETT manufacturing. However, these approaches often provide underestimated results since other parameters, in addition to bacterial features (i.e. shape and material composition of ETT) might strongly influence biofilm formation.We used an already established biofilm development assay on medically-relevant foreign devices (CVC catheters) by a stably transformed bioluminescent (BLI)-Pseudomonas aeruginosa strain, in order to follow up biofilm formation on ETT by bioluminescence detection. Our results demonstrated that it is possible: i) to monitor BLI-Pseudomonas aeruginosa biofilm development on ETT pieces in real-time, ii) to evaluate the three-dimensional structure of biofilm directly on ETT, iii) to assess metabolic behavior and the production of microbial virulence traits of bacteria embedded on ETT-biofilm.Overall, we were able to standardize a rapid and easy-to-perform in vitro model for real-time monitoring Pseudomonas aeruginosa biofilm formation directly onto ETT pieces, taking into account not only microbial factors, but also ETT shape and material. Our study provides a rapid method for future screening and validation of novel antimicrobial drugs as well as for the evaluation of novel biomaterials employed in the production of new classes of ETT.

Dataset Information

Detection of Pseudomonas aeruginosa Serogroup G Using Real-Time PCR for Novel Target Genes Identified Through Comparative Genomics.

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