Development of Loop-Mediated Isothermal Amplification Assay for Detection of Clinically Significant Members of Acinetobacter calcoaceticus-baumannii Complex and Associated Carbapenem Resistance.

Amit Sharma,Rajni Gaind

doi:10.3389/fmolb.2021.659256

Abstract

Background: Acinetobacter calcoaceticus–baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by bla OXA-23. Clinically significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Early diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by bla OXA-23. Methodology: Universal LAMP primers were designed for the detection of significant members of the ACB complex and carbapenem resistance targeting the ITS 16S–23S rRNA and bla OXA-23 gene respectively. The optimal conditions for the LAMP assay were standardized for each primer set using standard ATCC strains. The sensitivity of the LAMP assay was assessed based on the limit of detection (LOD) using different DNA concentrations and colony counts. The specificity of LAMP was determined using the non-ACB complex and non-Acinetobacter species. The results of the LAMP assay were compared with those of polymerase chain reaction (PCR). Results: The optimal temperature for the LAMP assay was 65°C, and the detection time varied with various primers designed. Using the ITS Ab1 primer, LODs of LAMP and PCR assays were 100 pg/μl and 1 ng/μl of DNA concentration and 104 cfu/ml and 108 cfu/ml of colony count, respectively. The LAMP assay was 10- and 104-fold more sensitive than PCR using DNA concentration and colony count, respectively. The LAMP assay was found to be specific for clinically important ACB complex species. Significance of the study: The LAMP assay can be applied for early detection of significant species of the ACB complex from clinical samples and their carbapenem-resistant variants. Depending on the emerging pathogen and locally prevalent resistance genes, the LAMP assay can be modified for detection of colonization or infection by various resistant bugs.

Highlights

Acinetobacter species are a group of aerobic, non-fermentative, oxidase-negative, non-motile, gram-negative coccobacilli, and they have emerged as important opportunistic pathogens in healthcare facilities (Peleg et al, 2008; Howard et al, 2012; Akrami and Namvar, 2019)
The loop-mediated isothermal amplification (LAMP) assay was standardized for detection of clinically significant members of the Acinetobacter calcoaceticus–baumannii complex (A. baumannii, A. pittii, and A. nosocomialis) targeting the internal transcribing spacer (ITS) region between 16S and 23S rRNA genes and associated carbapenem resistance mediated by blaOXA-23
The amplified products from positive control (A. baumannii ATCC 19606 for ITS Ab1 and Ab2 and carbapenem-resistant Acinetobacter baumannii (CRAB)/05/ RICU/VMMC-SJH for the OXA-23 primer) were observed on 1% gel corresponding to a band size of 221, 223, and 200 bp, respectively (Figures 4A,B), while no bands were observed for negative (E. coli ATCC 25922 for ITS Ab1 and Ab2 and A. baumannii ATCC 19606 for OXA-23) and reagent controls

Summary

Introduction

Acinetobacter species are a group of aerobic, non-fermentative, oxidase-negative, non-motile, gram-negative coccobacilli, and they have emerged as important opportunistic pathogens in healthcare facilities (Peleg et al, 2008; Howard et al, 2012; Akrami and Namvar, 2019). Within the ACB complex, A. baumannii has emerged as an important pathogen associated with a variety of nosocomial infections, including bacteremia, pneumonia, meningitis, and urinary tract and surgical site infections (Manchanda et al, 2010; Chen et al, 2014). Acinetobacter calcoaceticus–baumannii (ACB) complex has emerged as an important nosocomial pathogen and is associated with life-threatening infections, especially among ICU patients, including neonates. Carbapenem resistance in Acinetobacter baumannii has emerged globally and is commonly mediated by blaOXA-23. Significant infections with carbapenem-resistant Acinetobacter baumannii (CRAB) are a major concern since therapeutic options are limited and associated mortality is high. Diagnosis of both the pathogen and resistance is important to initiate the optimal therapy and prevent selection of resistance. A loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of the ACB complex and carbapenem resistance mediated by blaOXA-23

Methods

Results

Conclusion