Detection and discrimination of two Brucella species by multiplex real-time PCR and high-resolution melt analysis curve from human blood and comparison of results using RFLP.

OBJECTIVES: Rapid and accurate detection of Brucella abortus and Brucella melitensis from clinical samples is so important because antibiotic treatment has major side effects. This study reveals a new method in detection of clinical samples of brucellosis using real-time PCR and high-resolution melt (HRM) curve analysis. MATERIALS AND METHODS: 160 brucellosis suspicious samples with more than 1/80 serum antibody titers were collected and the results were compared with the RFLP method. In order to amplify the sequences for HRM analysis, vdcc, int-hyp and glk and for RFLP, omp2a and omp2b with PstI and Hinf1 restriction endonuclease were used. At last, the accuracy and specificity of the two methods were compared with each other. RESULTS: Out of these 160 samples, multiplex real time PCR showed 108 positive samples (67.5%), including 56% B. melitensis and 44% B. abortus; whereas in PCR-RFLP 52 out of 160 samples were positive, where recognition of two species were accordant with HRM analysis, separation was based on the size of the amplified fragment. Using the designed primers and performing the assay, we confirmed this method to be much faster and have lower cost with more than 99% accuracy compared to methods such as RFLP. CONCLUSION: The present study showed that this technique, which scans gene segments and creates an analysis pattern for detection of clinical samples, is useful and more dominant compared with PCR-RFLP. Thus, this method can be used for brucellosis detection, and clinical and epidemiological research.

Antimicrobial Effect of Imipenem-Functionalized Fe2O3 Nanoparticles on Pseudomonas aeruginosa Producing Metallo ß-lactamases.

BACKGROUND: Resistant strains of Pseudomonas aeruginosa to imipenem was medical treatment problem, especially in burnt units of hospitals. OBJECTIVES: This study was conducted to evaluate the antimicrobial effect of Fe2O3 nanoparticles alone and functionalized with imipenem on P. aeruginosa starins producing metallo ß-lactamases (MBL). MATERIALS AND METHODS: A disk diffusion method was used to isolate a clinical P. aeruginosa producing Metallo ß-lactamases with imipenem resistance. The minimum inhibitory concentration (MIC) of Fe2O3 nanoparticles and imipenem were calculated against the bacteria. The antimicrobial effect of nanoparticles functionalized with the antibiotic was determined. Standard strain of P. aeruginosa ATCC: 27853 was used as control. RESULTS: The clinical sample was resistant to imipenem (up to 28 µg.mL-1). Similarly, MIC of the nanoparticles against the isolate was 160 µg.mL-1. Subsequently, the combination of 16 pg.mL-1 of antibiotic with 80 µg.mL-1 of Fe2O3 nanoparticles were able to inhibit the growth of the isolate. CONCLUSIONS: Fe2O3 nanoparticles functionalized with imipenem can impair antibiotic resistance mechanisms of bacteria as it can make the imipenem resistant the aforementioned bacterium more susceptible to weaker concentrations of antibiotic. It also has its own antibacterial effect in certain concentrations.