Characterisation of Colistin -Resistant Enterobacterales and Acinetobacter Strains Carrying mcr Genes from Asian Aquaculture Products

Aquaculture systems are widely recognised as hotspots for horizontal gene transfer, and the need for screening for bacteria carrying antimicrobial resistance genes in aquaculture systems is becoming more important. In this study, we characterised seventeen bacterial strains (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and A. nosocomialis) resistant to colistin originating from retailed aquaculture products imported from Vietnam to the Czech Republic. The mcr-1.1 gene was found located on plasmid types IncHI2, IncI2, and IncX4, as well as on the rarely described plasmid types IncFIB-FIC and IncFIB(K), phage-like plasmid p0111, and on the chromosome of E. coli. One E. coli strain carried the mcr-3.5 gene on IncFII(pCoo) plasmid in addition to the mcr-1.1 gene located on IncHI2 plasmid. K. pneumoniae was found to carry the mcr-1.1 and mcr-8.2 genes on IncFIA(HI1) plasmid. The mcr-4.3 gene was found on similar untypeable plasmids of A. baumannii and A. nosocomialis strains, pointing to the possible interspecies transfer of plasmids carrying the mcr-4 gene. Our results highlight that some aquaculture products of Asian origin can represent an important source of variable plasmids carrying mcr genes. The results showed an involvement of phages in the incorporation of the mcr-1 gene into plasmids or the chromosome in E. coli strains from aquaculture. The detection of E. coli with the mcr-1 gene in the chromosome points to the risks associated with the stabilisation of the mcr genes in the bacterial chromosome.


Introduction
Large amounts of antibiotics have been reported to be used in Asia, not only in public health but also as feed additives for the prevention or treatment of bacterial diseases in animal production, including aquaculture [1]. Antibiotic residues entering rivers and water used for aquaculture may then pose serious environmental risks to food production [2] because residues can persist there for a long time [3]. This fact is one of the reasons why antimicrobial resistance surveillance should be implemented in aquaculture farm products.
Colistin (CT) is a last-resort antibiotic used mainly for the treatment of infections caused by multidrug-resistant Gram-negative bacteria [4]. Resistance to colistin was long thought to be only chromosomally encoded, but this perspective changed in 2015 when Liu et al. described plasmid-mediated colistin resistance encoded by the mcr-1 gene [5]. Since the first report, the mcr-1 gene has been found in bacteria from various sources worldwide [6]. Subsequently described mcr-2 to mcr-10 genes [7][8][9][10][11][12][13][14][15] have emerged. The mcr genes have been found localised on various plasmid types as well as integrated in the chromosome [6], and the dissemination of mcr-mediated resistance represents a significant threat in the spreading of colistin resistance in clinically significant pathogenic bacteriae.g., Escherichia coli [16], Klebsiella pneumoniae [17], Salmonella enterica [18], and Acinetobacter baumannii [19].

Colistin Susceptibility
All the tested isolates were resistant to colistin, with minimum inhibitory concentrations (MICs) > 2 mg/L ( Table 1). The MIC of E. coli strains ranged from 4 to 8 mg/L, and only one E. coli strain CT226 carrying two copies of the mcr-1 gene had an MIC > 16 mg/L. On the other hand, another E. coli strain CT262 with both mcr-1 and mcr-3 genes had an MIC = 4 mg/L. The MIC of K. pneumoniae CT251 with mcr-1 and mcr-8 genes and Acinetobacter spp. strains CT237 and CT263 carrying the mcr-4 gene was >16 mg/L.

Multi-Locus Sequence Typing (MLST)
Whole-genome sequencing was applied and the 7 locus MLST showed a high variability between strains of E. coli. Only ST48 was identified in more than one strain (n = 4) originating from two samples of frog legs. Nevertheless, the strains varied in terms of their contents of ARGs and plasmids (Table S1). A. nosocomialis belonged to ST279, A. baumannii to ST490, and ST11 to the K. pneumoniae strain (Table 1).

Detected mcr Genes
The sequences of all strains were checked for the presence of the mcr genes. All tested E. coli (4 strains from one meat sample of pangasius fish, 2 strains from one meat sample of blue swimmer crab, and 8 strains from two samples of frog legs) and K. pneumoniae (1 strain from the sample of frog legs) carried the mcr-1.1 gene. In contrast to Enterobacterales strains, A. baumannii from the sample of frog legs and A. nosocomialis from the meat sample of pangasius fish carried the mcr-4.3 gene. One E. coli strain (CT262) originating from frog legs carried the mcr-3.5 gene in addition to mcr-1.1. The only K. pneumoniae strain tested carried mcr-8.2 together with mcr-1.1 (Table 1).

Genetic Environment of the mcr Genes on Plasmids
To determine the localisation of the mcr genes on plasmids or in the chromosome, long-read sequencing was performed. The IncHI2 plasmid type with the mcr-1.1 gene was the most prevalent (n = 7) and was present in E. coli originating from frog legs ( Table 1). The IncHI2 plasmids were approx. 215 to 292 kb in size and carried the mcr-1.1 gene in addition to multiple other ARGs ( Figure 1). Plasmids of E. coli strains CT249 and CT259 carried a replicon type IncN in addition to IncHI2. The ISApl1 transposase associated with the mcr-1 gene was found upstream of the mcr-1 gene in five out of seven of the IncHI2  (Figure 2c). The mcr-1.1 gene in the E. coli strain CT250 originating from frog legs was found on IncFIB(K) plasmid together with other ARGs (Table S1). A plasmid type IncFIB(K) of E. coli strain CT250 shared around 50% coverage with the IncHI2 plasmid type ( Figure 1). The main shared sequence included the mcr-1.1 gene. The mcr-1.1 gene in CT250 had one single-nucleotide polymorphism (SNP) in comparison with the reference gene mcr-1.1, but it did not lead to a change in amino acid. The ISApl1 transposase was found upstream of the mcr-1.1 gene in CT250 (Figure 2c).
To determine the localisation of the mcr genes on plasmids or in the chromosome, long-read sequencing was performed. The IncHI2 plasmid type with the mcr-1.1 gene was the most prevalent (n = 7) and was present in E. coli originating from frog legs ( Table 1). The IncHI2 plasmids were approx. 215 to 292 kb in size and carried the mcr-1.1 gene in addition to multiple other ARGs (Figure 1). Plasmids of E. coli strains CT249 and CT259 carried a replicon type IncN in addition to IncHI2. The ISApl1 transposase associated with the mcr-1 gene was found upstream of the mcr-1 gene in five out of seven of the IncHI2 plasmids ( Figure 2c). The mcr-1.1 gene in the E. coli strain CT250 originating from frog legs was found on IncFIB(K) plasmid together with other ARGs (Table S1). A plasmid type IncFIB(K) of E. coli strain CT250 shared around 50% coverage with the IncHI2 plasmid type ( Figure 1). The main shared sequence included the mcr-1.1 gene. The mcr-1.1 gene in CT250 had one single-nucleotide polymorphism (SNP) in comparison with the reference gene mcr-1.1, but it did not lead to a change in amino acid. The ISApl1 transposase was found upstream of the mcr-1.1 gene in CT250 (Figure 2c).  The E. coli strain CT262 with mcr-1.1 on IncHI2 plasmid also carried the mcr-3.5 gene found on IncFII(pCoo) plasmid ( Figure 3) containing other ARGs. Tn3 family transposase TnAs2 associated with the mcr-3 gene was found upstream, while dgkA diacylglycerol kinase and IS6 family transposase IS26 were found downstream of the mcr-3.5 gene.
The IncI2 plasmid type (n = 2) was found in E. coli from pangasius ( Table 1). The IncI2 plasmids were approx. 64 and 73 kb in size and carried only mcr-1.1 as ARG ( Figure 4). The ISApl1 transposase was found twice (once truncated) upstream of the mcr-1.1 gene located on IncI2 plasmid in strain CT228. No ISApl1 was found on the same plasmid type in strain CT226 (Figure 2e). The strain CT226 carried a second copy of the mcr-1.1 gene on a plasmid of IncX4 type, size approx. 33 kb, with no other ARGs ( Figure 5). The E. coli strain CT262 with mcr-1.1 on IncHI2 plasmid also carried the mcr-3.5 gene found on IncFII(pCoo) plasmid ( Figure 3) containing other ARGs. Tn3 family transposase TnAs2 associated with the mcr-3 gene was found upstream, while dgkA diacylglycerol kinase and IS6 family transposase IS26 were found downstream of the mcr-3.5 gene. The IncI2 plasmid type (n = 2) was found in E. coli from pangasius ( Table 1). The IncI2 plasmids were approx. 64 and 73 kb in size and carried only mcr-1.1 as ARG ( Figure 4). The ISApl1 transposase was found twice (once truncated) upstream of the mcr-1.1 gene The E. coli strain CT262 with mcr-1.1 on IncHI2 plasmid also carried the mcr-3.5 gene found on IncFII(pCoo) plasmid ( Figure 3) containing other ARGs. Tn3 family transposase TnAs2 associated with the mcr-3 gene was found upstream, while dgkA diacylglycerol kinase and IS6 family transposase IS26 were found downstream of the mcr-3.5 gene. The IncI2 plasmid type (n = 2) was found in E. coli from pangasius ( Table 1). The IncI2 plasmids were approx. 64 and 73 kb in size and carried only mcr-1.1 as ARG ( Figure 4). The ISApl1 transposase was found twice (once truncated) upstream of the mcr-1.1 gene   In the E. coli strain CT225, originating from pangasius, the mcr-1.1 gene was localised on a IncFIB(AP001918)-FIC(FII) plasmid carrying several other resistance genes ( Figure  6). The ISApl1 transposase was found upstream of the mcr-1.1 gene (Figure 2c).   In the E. coli strain CT225, originating from pangasius, the mcr-1.1 gene was localised on a IncFIB(AP001918)-FIC(FII) plasmid carrying several other resistance genes ( Figure  6). The ISApl1 transposase was found upstream of the mcr-1.1 gene (Figure 2c). In the E. coli strain CT225, originating from pangasius, the mcr-1.1 gene was localised on a IncFIB(AP001918)-FIC(FII) plasmid carrying several other resistance genes ( Figure 6). The ISApl1 transposase was found upstream of the mcr-1.1 gene (Figure 2c).
The E. coli strain CT229 originating from crab meat carried the mcr-1.1 gene on a p0111 plasmid type (Figure 7). When annotating the plasmid, many phage related proteins were found. Therefore, the plasmid sequence was analysed by Phaster [38] and a P1 phage was found in 98% of the plasmid sequence. The P1 phage was found to be intact, with a score of >90. The BLASTn results showed that it was 98% identical to phage P1 (accession number AF234172) at a 77% coverage. The E. coli strain CT229 originating from crab meat carried the mcr-1.1 gene on a p0111 plasmid type (Figure 7). When annotating the plasmid, many phage related proteins were found. Therefore, the plasmid sequence was analysed by Phaster [38] and a P1 phage was found in 98% of the plasmid sequence. The P1 phage was found to be intact, with a score of >90. The BLASTn results showed that it was 98% identical to phage P1 (accession number AF234172) at a 77% coverage. The plasmids of Acinetobacter spp. were not typed using PlasmidFinder [39] ,since the database focuses mainly on Enterobacteriaceae members and Gram-positive plasmid typing. The two Acinetobacter plasmids carried only the mcr-4.3 gene as ARG (Figure 8). The comparison showed a high identity in an approx. 17 kb segment of the plasmids of approx.  The E. coli strain CT229 originating from crab meat carried the mcr-1.1 gene on a p0111 plasmid type (Figure 7). When annotating the plasmid, many phage related proteins were found. Therefore, the plasmid sequence was analysed by Phaster [38] and a P1 phage was found in 98% of the plasmid sequence. The P1 phage was found to be intact, with a score of >90. The BLASTn results showed that it was 98% identical to phage P1 (accession number AF234172) at a 77% coverage. The plasmids of Acinetobacter spp. were not typed using PlasmidFinder [39] ,since the database focuses mainly on Enterobacteriaceae members and Gram-positive plasmid typing. The two Acinetobacter plasmids carried only the mcr-4.3 gene as ARG (Figure 8). The comparison showed a high identity in an approx. 17 kb segment of the plasmids of approx. The plasmids of Acinetobacter spp. were not typed using PlasmidFinder [39], since the database focuses mainly on Enterobacteriaceae members and Gram-positive plasmid typing. The two Acinetobacter plasmids carried only the mcr-4.3 gene as ARG (Figure 8). The comparison showed a high identity in an approx. 17 kb segment of the plasmids of approx. 24 and 25 kb sizes. Tn3 family transposase ISPsy42 was found upstream of mcr-4.3 in both plasmids.
The K. pneumoniae strain CT251 originating from frog legs carried both mcr-1.1 and mcr-8.2 on a IncFIA(HI1) plasmid of size approx. 37 kb (Figure 9). No other ARGs were found to be located on the plasmid. The transposon Tn6330 (ISApl1-mcr-1.1-orf -ISApl1) was found around the mcr-1.1 gene and the mcr-8.2 gene was found upstream of Tn6330 (Figure 2b).

Genetic Surroundings of the mcr-1 Gene in the Chromosome
The mcr-1.1 gene was found in the chromosome in two E. coli strains, CT227 and CT230, originating from meat samples of pangasius and crab. Both strains carried several other ARGs in their chromosomes (Table S1). When examining the genetic context of the mcr-1.1 gene in CT227, ISApl1 transposase was found downstream, along with several phagerelated sequences around the mcr-1.1 gene (Figure 2d). After submitting the sequence to Phaster [38], the results showed an Enterobacteria lambda phage (NC_001416) present around the mcr-1.1 gene with a questionable score of 70-90. BLASTn results showed a 98% Antibiotics 2021, 10, 838 9 of 16 identity at a 64% coverage with the phage sequence. On the contrary, no phage sequences were found around the mcr-1.1 gene in the CT230 strain. The context of the mcr-1.1 gene in the CT230 strain was ISApl1-IS5-orf -mcr-1.1-orf -ISApl1 (Figure 2a), and multiple copies of ISApl1 were present throughout the whole chromosome.

Co-Occurrence of Genes Encoding Resistance to Different Classes of Antimicrobials
The strains of E. coli were generally multiresistant, carrying genes encoding resistance to at least six antibiotic classes found by ResFinder [40]. All E. coli strains (n = 8) originating from frog legs carried genes encoding resistance to rifampicin and were also carrying genes encoding ESBL (bla CTX-M-55 , bla OXA-1 or bla VEB ) (Table S1). No genes encoding resistance to carbapenems were found. Genes encoding resistance to fluoroquinolones (qnrS1, aac(6')-Ibcr, or qepA1) were found in eleven strains of E. coli (Table S1) The complete resistance genes profiles with their localisation on the plasmid or chromosome of all tested bacterial strains are presented in Table S1.

Discussion
In this study, all tested isolates of Enterobacterales and Acinetobacter spp. originating from retailed aquaculture products with resistance to colistin were found to be positive for the presence of different variants of the mcr genes.
In this study, the MIC of E. coli strain CT226 with two copies of the mcr-1 gene (on IncX4 and IncI2 plasmids) was 16 mg/L. Interestingly, E. coli strain CT262 with two copies of the mcr genes (mcr-1 and mcr-3) had an MIC = 4 mg/L. The occurrence of multiple copies of mcr genes in one strain does not have to lead to increased resistance to colistin-e.g., in the case of the co-occurrence of mcr-1 and mcr-3 in E. coli from cattle in Spain (MIC = 4 mg/L) [41] or the co-occurrence of the mcr-1 gene on plasmid and in the chromosome of E. coli from swine in China (MIC = 4 mg/L) [42]. E. coli was predominantly associated with the mcr-1 gene, which is consistent with the worldwide prevalence of the mcr-1 gene in Enterobacterales of different origin [6]. In a study on retailed meat (poultry, beef, pork, and rabbit) from the Czech Republic, the mcr-1 gene was also found to be predominant in E. coli strains [43]. The MLST of E. coli varied and no correlation was observed.
The co-occurrence of mcr-1 and mcr-8 genes on one plasmid was observed in K. pneumoniae strain CT251 in this study. The co-occurrence of mcr-1 and mcr-8 genes in K. pneumoniae has been described before, but the genes were located on two different plasmids [44].
Colistin resistance in Acinetobacter species was long thought to be only chromosomally encoded [45]; however, recently several studies have reported the occurrence of plasmid mediated colistin resistance in Acinetobacter spp. The mcr-1 gene in Acinetobacter species has been found in clinical strains from China [46] and Pakistan [47]. Acinetobacter strains with mcr-1, mcr-2, and mcr-3 genes have been detected from clinical and environmental samples in Iraq [48]. In this study, the A. baumannii and A. nosocomialis strains carried the mcr-4.3 gene, which has already been found in A. baumannii strains from pig faeces in China [23], a meningitis case in Brazil [19], and human and food samples in the Czech Republic [24]. A. nosocomialis with mcr-4.3 has been described sporadically so far. Currently, this species has been associated with the mcr-4.3 gene only as NCBI database entry MG948623 (the sequence of the mcr-4.3 gene from A. nosocomialis from South Africa). The common backbone of mcr-4.3-carrying plasmids in Acinetobacter spp. found in this study was described by Bitar et al. [24], where he compared mcr-4-positive plasmids from the Czech Republic with the ones from China [23] and Brazil [19].
The most common plasmid types associated with the mcr-1 gene are IncX4, IncI2, and IncHI2 [49]. Of these, the IncI2 plasmid type is typical for Asia, whereas IncHI2 is typical for Europe [50,51]. Despite the Asian origin of the strains tested in this study, mcr-1 was predominantly found on IncHI2 (n = 7), followed by two IncI2 and one each of the IncX4, IncFIB-FIC, IncFIB(K), and p0111 plasmids. A previous study on Enterobacterales strains originating from Czech retailed meat samples focused on the characterisation of plasmids carrying the mcr-1 gene, and only the three most common plasmid types were described (IncX4, IncHI2, IncI2) [52]. Our results suggest that aquacultures and Asian countries can be a source of diversity among plasmids carrying the mcr-1 gene.
The IncHI2 plasmids are usually hundreds of kb in size and, apart from the mcr gene, they carry multiple other ARGs in the multidrug-resistant (MDR) area, which varies between the plasmids while the backbone is conserved [53]. This phenomenon was also observed in this study. Interestingly, most of the IncHI2 plasmids in E. coli isolated from the same sample varied among each other, suggesting that the evolution of this plasmid type is very fast. Only IncHI2 plasmids from E. coli strains CT258 and CT262 shared a 99.98% identity in 99% coverage, being approx. 273 kb in size. The strains CT258 and CT262 originated from the same sample of frog legs but belonged to different STs (Table 1) and carried different plasmid types (Table S1).
On the other hand, the IncX4 plasmids with mcr-1 are known to be very conserved [54], usually being approx. 33 kb in size and carrying no other resistance genes, which was also the case of IncX4 plasmid in the E. coli strain CT226.
The phage-like plasmid p0111 of E. coli strain CT229 shared a significant identity with the P1 phage (accession number AF234172). When undergoing lysogenic conversion, P1 phage does not incorporate into the chromosome but circularises as a plasmid. In this case, the transmission of the mcr-1 gene could have been achieved via transduction, subsequently leading to phage degradation. The occurrence of mcr-1 on phage-like plasmids has been reported before [55,56], and the mcr-1 gene has been found within metagenomic studies of phage populations in swine feedlot wastewater [57] or chicken faeces [58]. Additionally, the CT229 phage-like plasmid showed a >99.96% identity with a >98% coverage with plasmids (accession numbers MG288678 and MF455226) from K. pneumoniae and E. coli from China, suggesting the wider spread of this phage-like plasmid.
When comparing the plasmid IncFIB(AP001918)-FIC(FII) of E. coli CT225 with the public database, similar plasmids were found but none of them contained the mcr-1.1 gene (accession numbers, e.g., CP075063, AP023199, or CP055255). The mcr-1.1 has been found to be located on IncFIB plasmid types before [59,60]; however, to our best knowledge, even though the replicon type IncFIC has been found in strains containing the mcr-1 gene [61], the mcr-1 gene has not been localised on the IncFIC plasmid type. Similarly, when performing BLASTn search for the IncFIB(K) plasmid of E. coli CT250, the most similar plasmids found belonged to the IncHI2 type (accession numbers, e.g., MG385063, MN232189, or CP019214). This suggests a rare finding of the mcr-1 gene localised on the IncFIB-FIC and IncFIB(K) plasmid types.
Plasmid IncFIA(HI1) of K. pneumoniae CT251 showed a 99.61% identity in 73% coverage with MK262711.1, a larger plasmid p18-29mcr-8.2 (approx. 91 kb in size) of K. pneumoniae KP18-29 from a human urine sample from China carrying the mcr-8.2 gene. The transposon Tn6330 of plasmid CT251 was not present in p18-29mcr-8.2. The surroundings of the mcr-8.2 gene were found to be relatively conserved [62], and plasmid CT251 shared some previously described features: mcr-8.2 was flanked by IS903 and ISKpn26 and the genes dgkA and copR were found upstream of the mcr-8.2 gene. In K. pneumoniae strain CT251, Tn6330 was located between the mcr-8.2 gene and IS903. These findings suggest that Tn6330 with the mcr-1.1 gene was incorporated into the mcr-8.2-bearing plasmid in strain CT251.
The mobilisation of ARGs is often achieved using insertion sequences. In the case of the mcr-1 gene, ISApl1 has been found co-localised with mcr-1 forming a transposon Tn6330 when localised upstream and downstream of the gene [63]. In this study, different types of genetic arrangements around the mcr-1 gene were found (Figure 2), which is consistent with the previously described surroundings of the mcr-1 gene [63,64] and show the possibility of its transfer between plasmids and/or chromosomes.
The mcr-3.5-carrying plasmid IncFII(pCoo) in E. coli CT262 from frog legs imported to the Czech Republic from Vietnam was 99.9% identical in 87% coverage with AP018353-an mcr-3.2 gene carrying IncFII plasmid from pork meat from Vietnam [65], suggesting a possible dissemination of these plasmids carrying mcr-3 gene in the country. Another study in China [66] characterised E. coli strains positive for both mcr-1 and mcr-3 and localised these genes on different plasmids or chromosomes. The plasmid pCP55-IncFII with mcr-3.5 shared a 95.85% identity in a 60% coverage with the mcr-3.5 carrying plasmid of strain CT262 from this study.
The occurrence of mcr genes in the chromosome is not observed very often [64] but can represent a threat of stabilising the heritage of mcr-1 [67]. The chromosomal carriage of the mcr-1 gene has been detected in 36.8% of mcr-1-positive E. coli strains isolated from healthy residents in Vietnam [68] and found in two E. coli strains isolated in a medical setting in Vietnam [69]. In this study, the mcr-1 gene was found on the chromosome in two E. coli strains, CT227 and CT230, originating from pangasius and crab meat from Vietnam, respectively. As in the case of the phage-like plasmid p0111 of E. coli strain CT229, the strain CT227 with chromosomally located mcr-1 could have acquired the mcr-1 gene by lysogeny of phage. Similarly, it has been observed by Shen et al. that the most common phage-like region around the mcr-1 gene contains an incomplete phage Vibrio 12B8 (NC_021073), as found by Phaster [67]. In contrast to the tested E. coli strains with the mcr-1 gene on plasmids, strain CT227 carried only the IncY-type plasmid, along with some small replicons of a few kb in size. The strain CT230 did not carry any plasmids at all (Table S1). In a recent study on Czech travellers and expatriates living in the Czech Republic, one E. coli strain with mcr-1 in the chromosome was found and the strain carried only one additional plasmid with no ARGs [70]. Even though the occurrence of mcr genes in the chromosome is quite rare, it could represent a heritable repository and emerge again if new selective pressure appears.

Bacterial Isolates Collection with Colistin Resistance
In this study, seventeen colistin-resistant bacterial isolates originating from aquaculture products imported from Vietnam were analysed. The isolates were obtained from 53 retailed originally packed samples in the Czech Republic throughout the year 2019; out of these, four were positive for mcr-carrying bacteria (unpublished data). The mcr-positive isolates were detected in aquaculture products originating from Vietnam but from different producers. The samples of pangasius and crab meat originated from aquaculture products caught in freshwaters, whereas the samples of frog legs came from farmed frogs.

Genomic DNA Extraction, Whole-Genome Sequencing (WGS), and Genome Assembly
For short-read whole-genome sequencing, genomic DNA was extracted using the DNeasy Blood and Tissue kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). The preparation of DNA libraries and sequencing on the Illumina platform were carried out by LGC Genomics GmbH group (NextSeq, 2 × 150 bp).
To determine the localisation of mcr genes, Oxford Nanopore Technologies (ONT, Oxford, UK) long-read sequencing was applied. The genomic DNA was extracted using the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany). MinION libraries were prepared with a Ligation Sequencing Kit, #SQK-LSK109, (ONT, Oxford, UK) and sequenced in a #FLO-MIN106 R9.4 flow cell. Fast5 read files were base called and converted to fastq format using the software Guppy v 3.0.3+7e7b7d0 (ONT). The de novo hybrid assembly of long (ONT) and short (Illumina) reads was conducted using Unicycler v0.4.7 [71]. The contigs were checked for circularisation and size.

Conclusions
The mcr-1.1 gene was found located on mcr-1-associated plasmid types IncHI2, IncI2, and IncX4, as well as on the rarely described plasmid types IncFIB-FIC and IncFIB(K), phage-like plasmid p0111, and on the chromosome of E. coli from retailed aquaculture products imported to the Czech Republic from Vietnam. One E. coli strain carried the mcr-3.5 gene on IncFII(pCoo) plasmid in addition to the mcr-1.1 gene located on IncHI2 plasmid. The mcr-4.3 gene was found on similar plasmids of A. baumannii and A. nosocomialis strains, pointing to the possible interspecies transfer of plasmids carrying the mcr-4.3 gene. K. pneumoniae was found to carry the mcr-1.1 and mcr-8.2 genes on IncFIA(HI1) plasmid. This study highlights the risks involved in the spreading of bacteria resistant to colistin, being a last-resort antibiotic, as well as having other resistances, such as genes encoding resistance to beta-lactams or fluoroquinolones from aquaculture sources of Asian origin.