Introduction

Colistin has become one of the last antibacterials to retain activity against Carbapenem-Resistant Gram-Negative Bacilli (CR-GNB) [1]. As a consequence, increased use of colistin has led to colistin resistance in GNB by modifications of the lipopolysaccharide [2]. These modifications consist in addition(s) of cationic groups such as 4-Amino-L-arabinose (L-Ara4N) and/or Phosphoethanolamine (pETN), to the lipid A of the outer-membrane of the GNB, which results in electrostatic repulsion between the added cationic group and the polymyxin. These modifications of the lipid A may be due to mutations in chromosome-encoded genes of the two-component systems PhoP/PhoQ or PmrA/PmrB, or of their regulator MgrB, which results in high MICs of colistin (8 to >32 mg/L) associated to a high-fitness cost for the bacteria [2,3].

The first plasmid-encoded resistance to polymyxin, named MCR-1, has been described late 2015 [4]. MCR-1 catalyzes the addition of a pETN to lipid A resulting in MICs of colistin ranging from 2 to 8 mg/L [2]. Mcr-1 genes have now been reported worldwide in Enterobacteriaceae (mostly Escherichia coli), recovered from human and animal samples [2,5]. On top of its ability to be transferred between entero bacterial species, recent reports indicate its very low (or lack) fitness cost for the bacteria fearing a rapid dissemination of this mechanism [6]. Currently, nine families of mcr genes have been assigned and seven reported in Enterobacteriaceae [7,8]. They form a very heterogeneous family of enzymes sharing only 29-82% amino-acid sequence identity with MCR-1. Mcr-1 gene and its variants (mcr-1.2 to mcr-1.7) are the most prevalent mcr-genes found in human enterobacterial isolates and are located on plasmids (pMCRs) belonging to replicon types IncI2, IncHI2 and IncX4 [2,5,9]. MCR-1-producing E. coli isolates have rarely been reported from the Americas [10,11]. Very recently mcr-1.5 variant was reported in Argentina and in Japan [10,12]. The aim of our study was to characterize the molecular determinant of colistin-resistance in nine E. coli isolates recovered from a University Hospital in Buenos Aires, Argentina. Six of them were MCR-1- or MCR-1.5-producing E. coli isolates. We also characterized their plasmidic location and compaired them with the previously described IncL2 mcr-harboring plasmid from Argentina [10].

Materials and Methods

Bacterial Strains

E. coli 6383, 2336, 1724, 1670, 979, 789, 4070, 94427 and 4222 clinical isolates were identified using Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry (MALDI Biotyper CA system, Bruker Daltonics, Billerica, MA, USA). Azide-resistant E. coli J53 was used for conjugation assays.

Antimicrobial Agents, Susceptibility Testing and Microbiological Techniques

The susceptibility of the nine E. coli isolates was determined by BD Phoenix^TM 100 ID/AST system (Becton Dickinson, Sparks Glencoe, MD, USA) using the Phoenix NMC-406 panel according to the manufacturer’s recommendations. At the same time, the nine isolates were evaluated for colistin resistance by the In-house colorimetric Andrade Screening Antimicrobial Test (ASAT) [13]. Colistin Minimal Inhibitory Concentration (MIC) values were determined using Sensititre^® broth microdilution method (Thermo Scientific, Villebon sur Yvette, France) and interpreted according to the EUCAST breakpoints, updated in 2016 (http://www.eucast.org). MALDI-TOF based technology, MALDIxin test (Bruker Daltonics, Billerica, MA, USA) was performed following the published recommendations, as previously reported [14].

PCR

Whole-cell DNAs of all the different E. coli clinical isolates were used as a template for PCR using mcr-1 specific primers, CLR5F 5’CGGTCAGTCCGTTTGTTC 3’ and CLR5R 5’ CTTGGTCGGTCTGTAGGG 3’ [4].

Whole Genome Sequencing (WGS)

Colistin-resistant E. coli isolates were sequenced using Illumina technology as previously described [15], in order to determine their resistome and Multi Locus Sequence Type (MLST), by uploading assembled genomes using CLC Genomics Workbench software (CLC bio, Qiagen, Les Ulis, France) to the Resfinder v3.1 (https://cge.cbs.dtu.dk/services/ResFinder/) and MLST v2.0 (https://cge.cbs.dtu.dk/services/MLST/), respectively [16,17].

For the reconstruction of mcr-1- and mcr-1.5-carrying plasmids the obtained contigs were mapped against KY471308 (pMCR-M15049) [10], KY471309 (pMCR-M15224) [10] using CLC Genomics Workbench software (CLC bio, Qiagen). Gaps were filled by PCR amplification and Sanger sequencing. Open reading frames (ORFs) were annotated using the RAST server (rast.nmpdr.org) followed by manual comparative curation and determination of sequence similarity using the BLAST web server. Alignments with other IncI2 pMCRs were performed by using the BRIG tool [18].

Plasmid Characterization and Mating-Out Assay

Plasmid DNA from the different clinical isolates were extracted using the Kieser method [19]. To perform the filter mating-out assay, E. coli 6383, 1724, 1670, 979, 4070 and 4222 clinical isolates used as donors and recipient E. coli J53 were each grown overnight in BHI (brain heart infusion) broth supplemented with colistin (2 mg/L) for plasmid maintenance in donor cells. 0.25-ml donor culture was mixed with 4.75 ml BHI broth and incubated at 37°C for 5 h without shaking. Recipient cultures of E. coli J53 grown overnight were diluted 1:50 in BHI broth and incubated at 37°C for 5 h without shaking. After incubation, 200 μl of the donor culture was gently mixed with 800 μl of the recipient culture, and 200 μl of this mating mix was filtered through a 0.45-μm filter (Millipore). Filters were incubated on prewarmed plates at 37°C for 3 h. Mating assays were ended by placing the filters into 4ml of an ice-cold 0.9% NaCl solution, followed by vigorous agitation for 30s. Transconjugants were selected on Mueller-Hinton agar supplemented with colistin (2 mg/L) and azide (100 mg/ L). From the transconjugants harboring mcr-1 and mcr-1.5 genes, plasmid DNA was extracted using Kieser method and subsequently analyzed on a 0.7% agarose gel stained with ethidium bromide.

Nucleotide Sequence Accession Number

The nucleotide sequence of mcr-1.5 gene has been submitted to the EMBL/Genbank nucleotide sequence database under the accession number KY271416.1. The sequences of the plasmids reported here have been deposited in GenBank under accession MG594798 (p6383), MG594799 (p4222), MG594800 (p1724), MG598814 (p1670), MG598815 (p4070) and MG598816 (p979). The Whole Genome Shotguns have been deposited at DDBJ/ENA/GenBank under the accession PIJR00000000 (6383), PIIZ00000000 (1670), PIJA00000000 (4070), PIJB00000000 (979), PIJY00000000 (4222) and PIJS00000000 (1724).

Results and Discussion

Escherichia coli Isolates

9 colistin resistant E. coli isolates were recovered at a University Hospital in Buenos Aires, Argentina, between 2014 to 2016 from urine samples of 6 hospitalized- and 3 external patients. All of them were resistant according to Phoenix system and were positive for the ASAT test [13], indicating that this 9 isolates could growth in the presence of colistin. They displayed MIC values for colistin ranging from 4 mg/L to 16 mg/L (Table 1). PCR using mcr-1-like gene specific primers, revealed that six isolates were positive for mcr-1-like genes. Moreover, all the strains were analyzed by MALDIxin test giving a positive result for colistin resistance. Six of the isolates were positive for plasmid encoded resistance mechanism [14].

Genomic Analysis

The genome of the 9 E. coli isolates was analyzed by searching for acquired resistance genes and for point mutations involved in resistance. Only contigs bigger than 300-bp were retained for further analysis. All the E. coli isolates presented with the exception of E. coli 1724, resistance genes to β-lactams (blaTEM-1 or blaCTX-M-2) and aminoglycosides (Table 1). Four of the six isolates mcr-1 positive presented genes that conferred resistance to tetracyclines and sulphonamides. In E. coli 1724 only qnrB19, a plasmid-encoded quinolone resistance gene was found. An mcr-1 allele was identified in E. coli isolates 4070, 979, 4222 and 1724 (Table 1). In isolates 6383 and 1670 a novel mcr-1-variant was identified that presents a single nucleotide substitution (C1354T) resulting in an amino acid change of H452Y. Upon submission to GenBank nucleotide sequence database under the accession number KY271416.1 it was assigned as mcr-1.5 variant.

In the isolates negative for any known mcr-gene, point mutations in chromosomal genes involved in colistin resistance mechanisms were found. Isolate 2336 harbored a mutated pmrA gene that led to a G53W substitution, which has not yet been reported in pmrA gene of E. coli. However, mutations at position 53 of PmrA in K. pneumoniae (G53C and G53S) and in Salmonella enterica (G53E, G53R) have been shown to be responsible for colistin resistance [20,21]. The isolate 789 harbored a mutated pmrB gene that led to a P94L substitution, which has not been described in E. coli but a substitution P94Q has already been identified in S. enterica [21]. The isolate 94427 harbored a mutation in pmrB gene that resulted in a substitution V88E in PmrB that has never been described so far in colistin resistance. However, mutations close by (L82R, S85R) have already been incriminated in colistin resistance, thus, suggesting the importance of this region in the functionality of PmrB [20,22].

The MLST analysis revealed that the clinical E. coli isolates belonged to different sequence types (ST), except for 6383 and 1670 isolates that belonged to the same ST (ST-602) (Table 1). According to E. coli MLST database (http://mlst.ucc.ie/mlst/dbs/Ecoli), colistin-resistant E. coli of ST-744 and ST-101, have been reported in South America (Colombia and Brazil), from human, poultry or livestock samples, but not from Argentina. ST-602, was reported once from a companion animal in Brazil, and ST-2722 from reptiles in Australia and from poultry in Denmark, but never from human samples or from South America. E. coli 979 belongs to ST-410, a hyper epidemic clone and founder of the widely disseminated clonal complex 23 (CC23) [23]. There is evidence that E. coli ST-410 has been successful for interspecies transmission between wild animals, food-producing animals, companion animals, humans, and the environment, increasing the risk of becoming a successful pandemic clone [24]. ST-410 carrying mcr-1 gene and bla_CTX-M genes have been recovered from turkey meat in Germany [25] and from a human blood culture in Brazil [26].

Plasmid Analysis

Whole genome sequence analysis and mating-out assay results indicated that mcr-1 gene or its variant were inserted onto a IncI2 type plasmid of c.a 60 kb (Figure 1) and were closely related to pMCR-M15049 (KY471308) [10]. The transconjugants showed the same MIC values for colistin (4 mg/L) than the clinical strains, revealing the possibility of dissemination through the conjugative mechanism.

Comparative sequence analysis between the previously described pMCR-M15049 and the plasmids harbored by the six colistin resistant E. coli, revealed a conserved backbone length, responsible for its replication, maintenance, and transfer, and similar overall genetic arrangements (Figure 2). The comparison of the immediate genetic environment of mcr-1.5 genes showed that mcr-1.5 and pap2 genes, were bracketed by two copies of ISApI1, likely forming a composite transposon, Tn6330, in plasmids p6383 and p1670 as in pMCR-M15049 [10], while in mcr-1 gene environment ISApI1 were absent (Figure 2). It has been proposed that the presence or absence of this IS correlates with the adaptation of mcr-1 to a new host. A composite transposon indicates a recent acquisition of this marker whereas the absence of ISApl1 or the presence of a single copy suggests that it has been already adapted [27]. Nevertheless, a recent report demonstrated that the composite transposon Tn6330, can translocate to other plasmids [28], which notoriously increases, the possibility of dissemination to plasmids from other incompatibility groups. Mcr genes are commonly located in plasmid of the incompatibility groups IncI2, HI1, HI2, P, X1/X2, X3/X4, X4, FI, FIP and FII, which are self-transferable plasmids responsible for inter-species dissemination worldwide [27] In south America only a few reports identified the mcr-1 carried plasmids. In these, plasmids belong to the incompatibility groups X4 and I2 carry and an initial analysis of the plasmid backbone suggests that these elements are highly similar to those reported in China, Europe or North America [27]. The plasmid sequence comparison of the p6383, p1670 and pMTY17668-MCR1.5 (AP018110) which harbored the only reported mcr-1.5 in Japan [12], showed a conserved backbone, with only mcr-1.5 as resistance gene, indicating that this plasmid is disseminated also in Japan.

Conclusion

We have characterized 4 MCR-1 and 2 MCR-1.5 producing E. coli isolates recovered from a University hospital in Argentina. These isolates were not clonally-related and belonged to hospitalized or external patients. All the mcr-carrying plasmids belonged to the IncI2 incompatibility group, demonstrating the role of this plasmid-type in the dissemination of the mcr genes worldwide. Carbapenem-resistance rates among Enterobacteriaceae are very high in Argentina and is mainly due to KPC-2 dissemination [29]. Therefore, the use of colistin has drastically increased, being in many instances the last active therapeutic option. As a consequence, carbapenem-resistant bacteria that become colistin-resistant may raise, as exemplified by isolate 2336, that became colistin-resistant due to a chromosomal mutation in PmrA. The finding that plasmid-encoded colistin resistance is now also spreading in Argentinian hospitals and in the community, portrays a very scary scenario.

Funding

This work was supported by the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) by a grant from the French National Research Agency (ANR-10-LABX-33) and by DIM Malinf, Ile de France.

Transparency Declaration

None to declare

Figure 1: Plasmid extractions and analysis by Kieser method of the MCR-1-producing isolates [19].

WT: E. coli clinical isolates and; TC: the respective colistin resistant E. coli J53 transconjugants carrying pMCR plasmid. M: marker.

Figure 2: Sequence alignment of IncI2-type mcr-carrying plasmids. pMCR-M15049 (KY471308) was used as a reference to align and compare plasmids obtained from the clinical E. coli isolates of this study. The outer circle with red arrows indicates annotations from the reference sequence. Gaps in the inner circles are missing regions when compared with the reference. The sequences of the plasmids reported here have been deposited in GenBank under accession MG594798 (p6383), MG594799 (p4222), MG594800 (p1724), MG598814 (p1670), MG598815 (p4070) and MG598816 (p979).

References

1. Zavascki AP, Goldani LZ, Li J, Nation RL (2007) Polymyxin B for the treatment of multidrug-resistant pathogens: a critical review. J Antimicrob Chemother 60: 1206-1215.
2. Jeannot K, Bolard A, Plesiat P (2017) Resistance to polymyxins in Gram-negative organisms. Int J Antimicrob Agents 49: 526-535.
3. Choi MJ, Ko KS (2015) Loss of hypermucoviscosity and increased fitness cost in colistin-resistant Klebsiella pneumoniae sequence type 23 strains. Antimicrob Agents Chemother 59: 6763-6773.
4. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, et al. (2016) Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 16: 161-168.
5. Poirel L, Jayol A, Nordmann P (2017) Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes. Clin Microbiol Rev 30: 557-596.
6. Yang Q, Li M, Spiller OB, Andrey DO, Hinchliffe P, et al. (2017) Balancing mcr-1 expression and bacterial survival is a delicate equilibrium between essential cellular defence mechanisms. Nat Commun 8: 2054.
7. Partridge SR, Di Pilato V, Doi Y, Feldgarden M, Haft DH, et al. (2018) Proposal for assignment of allele numbers for mobile colistin resistance (mcr) genes. J Antimicrob Chemother 73: 2625-2630.
8. Carroll LM, Gaballa A, Guldimann C, Sullivan G, Henderson LO, et al. (2019) Identification of Novel Mobilized Colistin Resistance Gene mcr-9 in a Multidrug-Resistant, Colistin-Susceptible Salmonella enterica Serotype Typhimurium Isolate. MBio 10: e00853-19.
9. Sun J, Zhang H, Liu YH, Feng Y (2018) Towards Understanding MCR-like Colistin Resistance. Trends Microbiol 26: 794-808.
10. Rapoport M, Faccone D, Pasteran F, Ceriana P, Albornoz E, et al. (2016) First description of mcr-1-mediated colistin resistance in human infections caused by Escherichia coli in Latin America. Antimicrob Agents Chemother 60: 4412-13.
11. Mulvey MR, Mataseje LF, Robertson J, Nash JH, Boerlin P, et al. (2016) Dissemination of the mcr-1 colistin resistance gene. Lancet Infect Dis 16: 289-290.
12. Ishii Y, Aoki K, Endo S, Kiyota H, Aoyagi T, et al. (2017) Spread of mcr-1.5 in the community: an emerging threat. Int J Antimicrob Agents 51: 161-162.
13. Rodriguez CH, Maza J, Tamarin S, Nastro M, Vay C, et al. (2019) In-house rapid colorimetric method for detection of colistin resistance in Enterobacterales: A significant impact on resistance rates. J Chemother 17:1-4.
14. Dortet L, Bonnin RA, Pennisi I, Gauthier L, Jousset AB, et al. (2018) Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test. J Antimicrob Chemother 73: 3359-3367.
15. Jaidane N, Bonnin RA, Mansour W, Girlich D, Creton E, et al. (2018) Genomic Insights into Colistin-Resistant Klebsiella pneumoniae from a Tunisian Teaching Hospital. Antimicrob Agents Chemother 62: e01601-17.
16. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, et al. (2012) Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67: 2640-2644.
17. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, et al. (2012) Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 50: 1355-1361.
18. Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. (2011) BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12: 402.
19. Kieser T (1984) Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid. 12: 19-36.
20. Olaitan AO, Diene SM, Kempf M, Berrazeg M, Bakour S, et al. (2014) Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study. Int J Antimicrob Agents 44: 500-507.
21. Sun S, Negrea A, Rhen M, Andersson DI (2009) Genetic analysis of colistin resistance in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 53: 2298-2305. 
22. Cannatelli A, Di Pilato V, Giani T, Arena F, Ambretti S, et al. (2014) In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment. Antimicrob Agents Chemother 58: 4399-4403. 
23. Rocha IV, Andrade CADN, Campos TL, Rezende AM, Leal NC, et al. (2017) Ciprofloxacin-resistant and extended-spectrum β-lactamase-producing Escherichia coli ST410 strain carrying the mcr-1 gene associated with bloodstream infection. Int J Antimicrob Agents 49: 655-656.
24. Turrientes MC, González-Alba JM, del Campo R, Baquero MR, Cantón R, et al. (2014) Recombination blurs phylogenetic groups routine assignment in Escherichia coli: setting the record straight. PLoS ONE 9: e105395.
25. Schaufler K, Semmler T, Wieler LH, Wöhrmann M, Baddam R, et al. (2016) Clonal spread and interspecies transmission of clinically relevant ESBL-producing Escherichia coli of ST410 another successful pandemic clone? FEMS Microbiol Ecol 92: pii: fiv155.
26. Falgenhauer L, Waezsada SE, Gwozdzinski K, Ghosh H, Doijad S, et al. (2016) Chromosomal locations of mcr-1 and blaCTX-M-15 in fluoroquinolone-resistant Escherichia coli ST410. Emerg Infect Dis 22: 1689-1691.
27. Quiroga C, Nastro M, Di Conza J (2019) Current scenario of plasmid-mediated colistin resistance in Latin America. Rev Argent Microbiol. 51: 93-100.
28. Li R, Xie M, Zhang J, Yang Z, Liu L, et al. (2017) Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant. J Antimicrob ChemotheR 72: 393-401.
29. Gomez SA, Pasteran FG, Faccone D, Tijet N, Rapoport M, et al. (2011) Clonal dissemination of Klebsiella pneumoniae ST258 harboring KPC-2 in Argentina. Clin Microbiol Infect 17: 1520-1524.