Caracterización y detección genética de Enterobacterias resistentes a colistina en muestras de origen animal
Marta Valero Lorenzo defended this Degree Final Project
October 11st, 2018
Justification. Colistin resistance poses a major Public Health concern. The first description of a plasmid mediated gene codifying for colistin resistance took place in 2015, since this year eight genes have been described. Those genes codify for colistin resistance by modifications in Lipid A, target of this antimicrobial in the bacterial cell wall. As a plasmid mediated genes, could imply horizontal transmission of resistance. Besides, colistin classification as “critically important antibiotic for human medicine” by the WHO, and the growth of colistin resistance, stablish the need to develop and validate detection methods.
The objectives of this study include an assesment of recently described culture medium CHROMID® Colistin R agar (BioMérieux, Marcy-L’Etoile, France), and the evaluation of the performance of molecular techniques (PCR and real-time PCR), as well as to stablish a comparison between molecular and culture methods.
Methodology. Using fattening pigs samples collected in 2012, 2013 and 2015, a culture of the faeces was performed in the mentioned medium according to the specifications of the manufacturer just with some modifications. Identification techniques were performed in the colonies obtained (MALDI-TOF, API 20E). qPCR for detection of mcr-1 was carried out after optimization of a standard curve. Conventional PCR for the detection of multiple resistance genes (mcr-1-mcr-4) was also conducted in this study.
Conclusions. The results show an excellent correlation between pink-coloured colonies and identification as Escherichia coli (100%, CI 95: 88,88-100). Besides, several species not included in the manufacturer specifications were cultured as well. Regarding qPCR, results were positive in 20/20 and 19/20 samples in 2012 and 2015, proving its great sensitivity. Conventional PCR turned out less sensitive due to the samples used (cultured isolates). Mcr-1 was found in a higher proportion of samples, also noting the presence of mcr-4 in isolates identified as Citrobacter freundii.
