Evaluation of a Rapid Method for the Detection and Differentiation of Colistin-resistance Mechanisms in Gram-negative Bacteria

  • Ifeoma Adaeze Ekwueme

Student thesis: Doctoral Thesis

Abstract

Background
There has been a global increase in carbapenemase-producing Gram-negative organisms which has resulted in increased use of polymyxin E (colistin) for the treatment of multidrug-resistant (MDR) Gram-negative infections. To reduce the infection and mortality rate caused by MDR organisms, colistin has been the last resort drug. Currently there has been a widespread dissemination of Gram- negative organisms harbouring mobile colistin-resistance (mcr) genes worldwide. This is concerning and necessitates more attention. This continuing rise of infection causing serious public health issues require rapid identification and differentiation to combat the spread. Hence, there is need to understand the colistin-resistance mechanism in Gran-negative organisms and evaluate rapid methods to detect colistin-resistance mechanisms in Gram-negative bacteria and differentiate all the mcr genes. Broth microdilution (BMD) assay is the current gold standard method for colistin susceptibility testing, but this method associated with technical challenges. Rapid and reliable identification of colistin-resistant Gram-negative bacteria and characterisation of resistance mechanisms notably of transferrable colistin-resistance mcr genes will assist with implementation of infection prevention and control measures to prevent further spread of resistance. This study evaluated MALDIxin assay using prototype MBT Lipid A kit on MALDI Biotyper Sirius and Multiplex real-time PCR on Quant Studio 7 method for detection and differentiation of mobile colistin-resistance (mcr-1 to mcr-10) genes.
Methods
The SYBR® Green-based real-time PCR method was used for the 163 isolates. Working solutions were prepared for each of the primer pairs for both screening and supplementary, by adding 10 mL of forward and reverse stock primers to 80 µL of molecular grade water making a total volume of 100 µL for each set of primers. Melt curve analysis was used for positive results confirmation. MALDIxin assay using prototype MBT Lipid A kit on MALDI Biotyper Sirius identify L-Ara4N and pETN modified Lipid A within 30 minutes and able to differentiates chromosome-encoded and mcr-related colistin-resistance mechanisms.
Results
For the real-time PCR, expected results were obtained for all strains. The design of the screening assay was able to detect mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, while the supplementary primer set was able to differentiate the 5 mcr variants (mcr-1, mcr-2and mcr-6, mcr-9 and mcr-10). Positive results were confirmed by melt curve analysis. Results expected were all detected for all the isolates tested.
The MALDIxin assay using MBT Lipid A Kit was able to detect and differentiate between changes in Lipid A. Only 21 samples were tested. Peak 1796.2m/z detected only 5 out of 10 E. coli isolates, while peak 1919.2m/z detected on 4 out of the 10 E. coli isolates. The peaks 1796.2m/z and peak 1919.2m/z were both detected on 3 E. coli with mcr-1.
Conclusion
The performance of Prototype MBT Lipid A Kit for the detection and differentiation of colistin- resistance mechanisms in Gram-negative organisms using MALDI-TOF MS in negative ion mode proved to be an effective and rapid method. This assay detected colistin-resistance and was able to differentiate between chromosomal and plasmid mediated genes. The Multiplex RT-PCR assay proved rapid and easy to perform and proved 100% specificity and sensitivity for all expected results.
Date of Award22 Mar 2024
Original languageEnglish
Awarding Institution
  • University of Portsmouth
SupervisorGraham Mills (Supervisor), John Simon Young (Supervisor) & Sarah Fouch (Supervisor)

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