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

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.

Keywords: Acinetobacter calcoaceticus–baumannii (ACB) complex; carbapenem-resistant Acinetobacter baumannii (CRAB); internal transcribing spacer (ITS) 16S–23S rRNA; limit of detection (LOD); loop-mediated isothermal amplification (LAMP) assay; polymerase chain reaction (PCR).

FIGURE 1

Summary of the study workflow.

FIGURE 2

(A) shows the alignment of sequence of diverse isolates of A. baumannii (N=4), A. pittii (N = 4), and A. nosocomialis (N=2) and demonstrates that the sequences are conserved across diverse isolates of three species except few locations. (B) shows the alignment of our consensus ITS 16S–23S rRNA sequence with the sequence of other Acinetobacter species (A. calcoaceticus, A. lwoffii, A. radioresistens, A. haemolyticus, and A. junii). The presence of the non-conserved region at locations from 140 to 210 (where our two to three primers are aligned) demonstrates that our LAMP primers were not specific for other Acinetobacter species.

FIGURE 3

A universal consensus sequence of the ITS 16S–23S rRNA gene that was used to design the LAMP primers for detection of significant members of the Acinetobacter calcoaceticus–baumannii (ACB) complex. Red color bases signify the mixed bases used in place of different bases present in A. baumannii, A. nosocomialis, and A. pittii. The sequences sites for ITS Ab1 and Ab2 LAMP primers are designated by upper and lower horizontal arrows, respectively. Right and left arrows indicate sense and reverse complementary sequences that were used.

FIGURE 4

Comparison of the limit of detection (LOD) of LAMP and PCR assays from direct culture for identification of significant members of the ACB complex using ITS Ab1 and Ab2 primers and detection of carbapenem resistance using the OXA-23 primer. (A) shows the LOD using different DNA concentrations. Lane for LAMP and PCR: PC, positive control; NC, negative control; RC, reagent control; DNA concentration (/μl) from Lanes 1 to 10; 100, 10, 1 ng, 100, 10, 1 pg, 100 fg, 10 fg, 1 fg, and 0.1 fg, respectively. (B) shows the LOD using different colony counts. Lane for LAMP and PCR: PC, positive control; NC, negative control; RC, reagent control; colony count (cfu/ml) from Lanes 1 to 7: 1.0 × 108, 107, 106, 105, 104, 103, and 102, respectively. In (A,B), in the LAMP assay, green and orange colors are indicative of positive and negative results, respectively, and in the PCR assay, the presence and absence of bands are indicative of positive and negative results, respectively.