Multicentre MDR Elizabethkingia anophelis isolates: Novel random amplified polymorphic DNA with capillary electrophoresis systems to rapid molecular typing compared to genomic epidemiology analysis

Ming-Jr Jian,Hung-Sheng Shang,Yun-Hsiang Cheng,Jun-Ren Sun,Shih-Yi Lee,Yu-Hsuan Cheng,Shu-Chen Kuo,Hsing-Yi Chung,Cherng-Lih Perng

doi:10.1038/s41598-019-38819-w

Abstract

Elizabethkingia species are ubiquitous bacteria that uncommonly cause human infection. Elizabethkingia anophelis was first identified in 2011 from the mosquito Anopheles gambiae. The currently available bacterial typing systems vary greatly with respect to labour, cost, reliability, and ability to discriminate among bacterial strains. Polymerase chain reaction (PCR)-based fingerprinting using random amplified polymorphic DNA (RAPD) is commonly used to identify genetic markers. To our knowledge, no system coupling RAPD-PCR and capillary gel electrophoresis (CGE) has been utilized for the epidemiological typing of E. anophelis. Thus, the aim of the present study was to establish a reliable and reproducible molecular typing technique for E. anophelis isolates based on a multi-centre assessment of bacteraemia patients. Here, we used a rapid CGE-light-emitting diode-induced fluorescence (LEDIF)-based method in conjunction with RAPD-PCR to genotype E. anophelis with a high level of discrimination. All clinical isolates of E. anophelis were found to be typeable, and isolates from two hospitals formed two distinct clusters. The results demonstrated the potential of coupling RAPD and CGE as a rapid and efficient molecular typing tool, providing a reliable method for surveillance and epidemiological investigations of bacterial infections. The proposed method shows promise as a novel, cost-effective, high-throughput, first-pass typing method.

Highlights

The genus Elizabethkingia was named in honour of Elizabeth King, who first identified the bacterium in association with meningitis in infants
A wide variety of bacterial typing systems are currently available, which show substantial variations with respect to the effort required, cost, reliability, and ability to discriminate among bacterial strains
Random amplified polymorphic DNA (RAPD) analysis is based on the presence of primer-binding sites in the genome that are sufficiently similar to permit Polymerase chain reaction (PCR) amplification using a single primer with an arbitrary nucleotide sequence at a low annealing temperature

Summary

Introduction

The genus Elizabethkingia was named in honour of Elizabeth King, who first identified the bacterium in association with meningitis in infants. The confirmed number of cases in the United States 2015–2016 outbreak of E. anophelis was 65, in which 20 people died from the infection; all the cases were associated with a similar strain[6]. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) are powerful techniques for epidemiological typing, the random amplified polymorphic DNA (RAPD) PCR technique can rapidly process large amounts of strains in a relatively uncomplicated protocol[11], and is commonly applied to detect polymorphisms in taxonomic and phylogenetic studies[13]. We developed a rapid CGE-light-emitting diode-induced fluorescence (LEDIF)-based method in conjunction with RAPD-PCR amplification for genotyping. To our knowledge, this combination has never been utilized for the epidemiological typing of bacteria

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Results

Conclusion