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

Abstract

Citrobacter spp. 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 in Citrobacter spp., which leads to the postulation that Citrobacter spp. could potentially be a reservoir for spreading of antimicrobial resistant genes. In this study, we characterized two colistin-resistant Citrobacter spp. isolated from the feces of a healthy individual in Thailand. Based on MALDI-TOF and ribosomal multilocus sequence typing, both strains were identified as Citrobacter sedlakii and Citrobacter amalonaticus. Genomic analysis and S1-nuclease pulsed field gel electrophoresis/DNA hybridization revealed that Citrobacter sedlakii and Citrobacter amalonaticus harbored mcr-3.5 gene 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^-4 CFU/recipient cell Escherichia coli J53. Colistin-MICs of transconjugants increased ≥ 16-fold suggesting that mcr-3.5 on these plasmids can be expressed in other species. However, beside mcr, other major antimicrobial resistant determinants in multidrug resistant Enterobacterales were not found in these two isolates. These findings indicate that mcr gene continued to evolve in the absence of antibiotics selective pressure. Our results also support the hypothesis that Citrobacter could be a reservoir for spreading of antimicrobial resistant genes. To the best of our knowledge, this is the first report that discovered human-derived Citrobacter spp. that harbored mcr but no other major antimicrobial resistant determinants. Also, this is the first report that described the presence of mcr gene in C. sedlakii and mcr-3 in C. amalonaticus.

Keywords: citrobacter amalonaticus; citrobacter sedlakii; citrobacter spp.; colistin resistance; mcr gene; mcr-3.

Figure 1

Identification of Citrobacter spp. (A) Mass fingerprinting of C. sedlakii and C. amalonaticus from Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF-MS). (B) Sequence alignment of C. sedlakii strain SY-CS04 with C. sedlakii strain 3347689II (accession no. CP071070). (C) Sequence alignment of C. amalonaticus strain SY-CA35 with C. amalonaticus strain FDAARGOS_165 (accession no. CP014070).

Figure 2

Overview of genomic structure of Citrobacter isolates. (A) Citrobacter sedlakii SY-CS04. (B) Citrobacter amalonaticus strain SY-CA35. The inner circle and outer circle represent GC skew and GC content, respectively. The protein-coding gene on forward strand and reverse strand represent in blue and red, respectively.

Figure 3

Antimicrobial resistance and virulence-associated profiles of the 2 Citrobacter isolates. Blue squares indicate the presence of genes while white squares represent the absence of genes.

Figure 4

Roary matrix-based gene sequence analysis of 86 Citrobacter isolates. The source of the isolates is shown in the inner ring. The location of the isolates is depicted in the middle ring and the year of the isolates is indicated by the outer ring. Isolates in this study including SY-CS04 and SY-CA35 were colored in red.

Figure 5

Plasmid profile analysis of Citrobacter isolates harboring mcr-3.5 gene by S1-PFGE and DNA hybridization. (A) The profile of total DNA treated with S1 nuclease and (B) relative hybridization of mcr-3 probe. Lane M, molecular standard, which is Salmonella braenderup H9812 digested with XbaI. Lane 1, Citrobacter amalonaticus strain SY-CA35. Lane 2, Citrobacter sedlakii strain SY-CS04. Arrows indicate the locations of plasmid harboring mcr-3 gene.

Figure 6

Structure of the IncFII(pCoo) harboring mcr-3.5, including pSY_CS01 (A) and pSY_CA01 (B). The inner circle and outer circle represent GC skew and GC content, respectively. The arrows indicate the directions of gene transcription. The red arrows represent antimicrobial resistance genes, the green arrows show other functional genes, the blue arrows show mobile element-encoding genes, the yellow arrows show IncF plasmid conjugative element and grey arrows for hypothetical protein-encoding genes.

Figure 7

Circular comparison of IncFII(pCoo) harboring mcr-3.5, pSY_CS01 and pSY_CA01 with eight homologous plasmids with considerable query coverage.

Figure 8

Linear comparison of surrounding regions of mcr-3. The arrows indicate directions of gene transcription. mcr-3 is labeled in red arrow, while other antimicrobial resistance genes are labeled in orange. Mobile genetic elements are indicated in blue and other functional gene are in green.

Figure 9

Comparison of 14 mcr-3.5 regions from 16 plasmids. The arrows indicate directions of gene transcription. Shading in light blue denotes regions of homology (nucleotide identity 95%).