Detection of metallo-beta-lactamase enzymes in Pseudomonas aeruginosa isolates by gold nanoparticles
3 الي 6 اسفند 1395، تهران - ايران
Presentation Type: Poster
Abstract: Background and Aim : Pseudomonas aeruginosa is a Gram negative opportunistic pathogen responsible for moderate to severe nosocomial infections. This organism has high intrinsic resistance to many antimicrobials including the extensively used carbapenems. An important mechanism of carbapenem inactivation in P. aeruginosa is production of metallo-β-lactamases (MBLs). The aims of this research were to design a novel biosensor for detection of MBL producing P. aeruginosa isolates using gold nanoparticles.
Methods : Imipenem susceptibility of 101 clinical P. aeruginosa isolates (51 from burn and 50 cystic fibrosis isolates) were determined by disc diffusion and measuring minimum inhibitory concentrations (MIC) according to the Clinical and Laboratory Standards Institute guidelines (CLSI). Production of MBLs was detected by the double disc synergy test (DDST) and presence of blaIMP, blaVIM, blaSPM genes by and PCR analysis. MBL production was also determined by designing a novel biosensor using gold nanoparticles linked with meropenem based on the iodometric method. Starch-stabilized gold nanoparticles (AuNPs) were synthesized by the reduction of HAuCl4 with sodium borohydride (NaBH4) as shown by a color change from yellow to dark red. Meropenem was linked to the surface of AuNPs by mixing 1.09 ×10-4 M concentration with AuNPs and stirring for 48 h before adding the starch-iodine solution. The resulting blue mixture was used as the biosensor for detection of MBL. P. aeruginosa fresh bacterial cultures (40 μl) were then mixed with the biosensor (10 μl, 2.5 ×10-4 M) and incubated at 37oC for 15 minutes. Decolourisation of the starch-iodine complex was interpreted as MBL production.
Results : Of the 51 burn P. aeruginosa isolates, 50 (98%) were resistant to imipenem (MIC of 16- 256 µg/ml) and among the 50 CF isolates, 5 (10%) showed resistance to imipenem (MIC of 16-32 µg/ml). MBL production was observed in 19 burn isolates (37.3%) using the double disc synergy test and PCR results showed that all MBL producing isolates harbored their related genes and one CF isolate harbored blaMBL. The same results were obtained using the biosensor designed in this research. The TEM micrograph of AuNPs showed an average diameter of 6.5 nm, with a relatively narrow size distribution and uniform shape. AuNPs were stable for at least 3 months at 4°C in a dark glass. The novel biosensor detected MBL production with 97% specificity and 100% sensitivity in as low as 10-15 CFU/ml.
Conclusion : High sensitivity (100%) of the designed novel biosensor suggests that nanoparticle technology could be a sensitive, fast and inexpensive means for detecting β- lactamase production in antibiotic resistant clinical isolates.