Comparison of loop-mediated isothermal amplification, multiplex PCR, and REP- PCR techniques for identification of carbapenem-resistant Acinetobacter baumannii clinical isolates

Aysar Abbood Al jebur; Neda Soleimani; Seyed Masoud  Hosseini

doi:10.18502/ijm.v15i5.13871

Aysar Abbood Al jebur Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
Neda Soleimani Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
Seyed Masoud Hosseini Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran

DOI: https://doi.org/10.18502/ijm.v15i5.13871

Keywords: Acinetobacter baumannii; Antibiotic resistance; Loop-mediated isothermal amplification (LAMP); Multiplex polymerase chain reaction; Repetitive extragenic palindromic polymerase chain reaction

Abstract

Background and Objectives: Acinetobacter baumannii, an opportunistic pathogen, is related to hospital-acquired infections and increased mortality. This study aimed to develop the loop-mediated isothermal amplification (LAMP) test for the fast-de- tecting of A. baumannii isolates as well as determining genetic relatedness for these isolates via the REP-PCR technique.

Materials and Methods: LAMP primers and multiplex PCR primers were designed for recognizing A. baumannii isolates harboring the bla SHV-1 , bla PER-1 , bla , AMPC, qnr, and aac (6)-1 genes, were collected (October 2020 to February 2021) from Shahid Motahari Hospital, Tehran, Iran. Combination disc test (CDT) results were used to assess the phenotypic iden- tification of isolates from ESBL producers. The sensitivity of the LAMP method was evaluated using a range of serial dilu- tions of genomic DNA. Results were compared between the LAMP technique, and multiplex PCR. The genetic diversity of clinical isolates was determined by REP-PCR.

Results: Among one hundred A. baumannii samples and based on the combined disc test, 56% of isolates were ESBL pro- ducers. The sensitivity of the LAMP technique for the identification of A. baumannii was 4.06 ng/μl whilst the multiplex PCR was (16.2 ng/μl). Regarding multiplex PCR, (68%) of the isolates were bla SHV-1 positive, (40%) bla , (85%) aac (6́)-1, AMPC (67%), bla TEM-1 (63%), and (15%) qnr respectively. While in LAMP, (69%) of isolates were bla positive, (86%) aac (6')-1, and (20%) qnr. The results of AMPC, bla TEM-1 , and bla PER-1 genes showed 100% compatibility between multiplex PCR and LAMP assays. The results of REP-PCR indicated there were 17 clones, clone A at 14% was the most prevalent of the isolates.

Conclusion: Wherever equipment and financial constraints are crucial, the LAMP test offers a better and more potent detec- tion rate for the identification of A. baumannii isolates than multiplex PCR. Furthermore, the genetic diversity of A. bauman- nii in these clinical isolates showed frequent commonality of genotypes.