Citrobacter braakii carrying plasmid-borne mcr-1 colistin resistance gene from ready-to-eat food from a market in the Chaco region of Bolivia

Sir, Polymyxins (colistin and polymyxin B) have recently regained a role as last-resort antibiotics for treatment of infections caused by XDR Gram-negative bacteria.1 Consequently, the emergence and diffusion of colistin resistance is of great concern. Acquisition of colistin resistance is mainly associated with chromosomal mutations.1 However, transferable resistance, mediated by the plasmid-borne mcr-type genes (encoding phosphoethanolamine transferases that modify lipid A), has recently been reported.1,mcr-1 was the first to be reported and, since its first identification in China,2 it has been detected worldwide in Enterobacteriaceae (mostly Escherichia coli) isolated from animals, food, environments and humans.1 The mcr-1 gene has been associated with different plasmid replicons such as IncI2, IncHI1, IncHI2, IncP, IncFII, IncFIB and IncX4,3 and was found only rarely to be chromosomally encoded.4 In Latin America, E. coli harbouring mcr-1 have been described in Brazil, Ecuador, Venezuela and Argentina from food-producing animals and human clinical samples.5–8 Moreover, a study reported intestinal colonization by mcr-1-positive E. coli in two unrelated travellers, who had visited Peru, Bolivia and Colombia, suggesting the presence of the gene in those countries.9 Here we report on the first detection of an mcr-1-positive Citrobacter braakii isolated in the Bolivian Chaco region from ready-to-eat food. In 2013, a pilot study was performed in the local markets of Camiri (Cordillera Province, Santa Cruz Department) and Villa Montes (Gran Chaco Province, Tarija Department), with the aim of investigating the presence of antibiotic-resistant Enterobacteriaceae in samples of ready-to-eat food. A total of 100 food samples were analysed as follows. A sliver of each sample was incubated at 35°C in tryptic soy broth plus 2 mg/L vancomycin for 24 h, and then the broth (10 μL) was plated onto MacConkey agar. Overall, 83 food specimens yielded bacterial colonies on MacConkey agar, and these were successively pooled and stored at −80°C pending further analysis. Screening for the presence of the mcr-1 gene2 identified one mcr-positive bacterial pool, obtained from a boiled potato. Plating the bacterial pool onto MacConkey agar plus 2 mg/L colistin allowed recovery of an isolate, named CA-26, identified as Citrobacter freundii by MALDI-TOF MS (bioMérieux Inc., Marcy-l’Étoile, France). Susceptibility testing performed by reference broth microdilution10 and interpreted according to the EUCAST breakpoints (http://www.eucast.org/clinical_breakpoints/) revealed that CA-26 was susceptible to all tested antibiotics (Table S1, available as Supplementary data at JAC Online) with the exception of colistin (MIC = 8 mg/L). A conjugation experiment,11 using E. coli J53 (F−met pro Azir) as recipient and selection with colistin (2 mg/L) and sodium azide (150 mg/L) yielded J53 transconjugants at a frequency of 7×10−5 ± 2×10−5 transconjugants per recipient. Transconjugants carried the mcr gene, as assessed by PCR, and were resistant to colistin (MIC = 4 mg/L). Genomic DNA of CA-26 was extracted using a QIAsymphony automated station (Qiagen, Hilden, Germany) and subjected to WGS using a HiSeq Illumina platform (Illumina, San Diego, CA, USA). The draft genome assembly included 94 contigs, with an estimated genome size of 5 072 546 bp and an average coverage of 80×. Analysis of 16S rRNA, leuS, recN, rpoB, pyrG and fusA gene sequences12 unambiguously identified CA-26 as C. braakii. In silico analysis of the antimicrobial resistome confirmed the presence of a plasmid-borne mcr-1 gene and of the chromosomal blaCMY-82 (accession no. KJ207203) AmpC-type β-lactamase gene. The sequence of the mcr-1-bearing plasmid, named pCA-26, was assembled in two contigs and the complete sequence was achieved using PCR and Sanger sequencing. Plasmid pCA-26 was 62 214 bp long, with an average GC content of 42.7%, and belonged to the IncI2 lineage. Its overall structure was similar to that of other IncI2 plasmids carrying mcr-1, previously described from other areas (99% nucleotide identity over a sequence coverage ranging from 85% to 96%; Table S2). The main differences consisted of the presence of additional IS elements or of an ISEcp1-blaCTX-M-55/64 resistance module in some of the other plasmids (Figure 1). The mcr-1-pap cassette in pCA-26 was inserted in a conserved position (i.e. downstream of the nikB locus) compared with other IncI2-type plasmids, and was not associated with any flanking mobile element (e.g. ISApl1). An SNP-based phylogenetic analysis identified pA31-12, an mcr-1- and blaCTX-M-55-carrying plasmid from an E. coli isolated from chicken in China in 2012, as the closest relative to pCA-26 (Table S2). The similarity of the mcr-1 genetic support to that from isolates from China suggests a possible epidemiological link between China and Bolivia, as also previously suggested.11 Nevertheless, a possible spread at the food, environmental and human level among Latin American countries could not be excluded. In fact, a recent report,5 showing the presence of the mcr-1 gene in Brazilian livestock, dated the presence of mcr-1 in Latin America back to 2012, suggesting that this resistance determinant has been circulating in Latin America for at least 5 years. Circular map of plasmid pCA-26. The circles, from the outermost to the innermost, show: (i) coding DNA sequences (CDSs) encoded on the plus and minus DNA strands (annotation is reported for known genes only); and (ii) the G + C content, shown as deviation from the average G + C content of the entire molecule. Main differences against other mcr-1-carrying IncI2 plasmids are indicated by dotted lines and bold and underlined text. CDSs are shown in the two outer circles and the GC content is shown in the circle inside these. To the best of our knowledge this is the first description of the mcr-1 gene in C. braakii, and also the first direct report of mcr-1 from Bolivia. This finding underlines that the mcr-1 determinant could be widespread throughout Latin America, and further underscores its ability to horizontally disseminate among different enterobacterial species. The draft genome of C. braakii CA-26 and the complete sequence of plasmid pCA-26 have been deposited under GenBank accession numbers MTJW00000000 and KY624633, respectively. The study was supported by grants from the Regione Toscana (Italy), Progetti di Iniziativa Regionale (PIR) 2012 (‘Supporto e assistenza tecnica all’implementazione del Sistema Unico de Salud dello Stato Plurinazionale di Bolivia: progetto pilota nella regione del Chaco’). None to declare. Tables S1 and S2 are available as Supplementary data at JAC Online.