Emergence of plasmid-mediated colistin resistance mcr-3.5 gene in Citrobacter amalonaticus and Citrobacter sedlakii isolated from healthy individual in Thailand

Abstract

Citrobacterspp. are Gram-negative bacteria commonly found in environments and intestinal tracts of humans and animals. They are generally susceptible to third-generation cephalosporins, carbapenems and colistin. However, several antibiotic resistant genes have been increasingly reported inCitrobacterspp., which leads to the postulation thatCitrobacterspp. could potentially be a reservoir for spreading of antimicrobial resistant genes. In this study, we characterized two colistin-resistantCitrobacterspp. isolated from the feces of a healthy individual in Thailand. Based on MALDI-TOF and ribosomal multilocus sequence typing, both strains were identified asCitrobacter sedlakiiandCitrobacter amalonaticus. Genomic analysis and S1-nuclease pulsed field gel electrophoresis/DNA hybridization revealed thatCitrobacter sedlakiiandCitrobacter amalonaticusharboredmcr-3.5gene on pSY_CS01 and pSY_CA01 plasmids, respectively. Both plasmids belonged to IncFII(pCoo) replicon type, contained the same genetic context (Tn3-IS1-ΔTnAs2-mcr-3.5-dgkA-IS91) and exhibited high transferring frequencies ranging from 1.03×10-4- 4.6×10-4CFU/recipient cellEscherichia coliJ53. Colistin-MICs of transconjugants increased ≥ 16-fold suggesting thatmcr-3.5on these plasmids can be expressed in other species. However, besidemcr, other major antimicrobial resistant determinants in multidrug resistant Enterobacterales were not found in these two isolates. These findings indicate thatmcrgene continued to evolve in the absence of antibiotics selective pressure. Our results also support the hypothesis thatCitrobactercould be a reservoir for spreading of antimicrobial resistant genes. To the best of our knowledge, this is the first report that discovered human-derivedCitrobacterspp. that harboredmcrbut no other major antimicrobial resistant determinants. Also, this is the first report that described the presence ofmcrgene inC. sedlakiiandmcr-3inC. amalonaticus.