Next Article in Journal
Silver Nanoparticles Enhance Antimicrobial Efficacy of Antibiotics and Restore That Efficacy against the Melioidosis Pathogen
Next Article in Special Issue
Characterisation of Early Positive mcr-1 Resistance Gene and Plasmidome in Escherichia coli Pathogenic Strains Associated with Variable Phylogroups under Colistin Selection
Previous Article in Journal
Exudative Epidermitis in Combination with Staphylococcal Pyoderma in Suckling Piglets
Previous Article in Special Issue
The Intestinal Carriage of Plasmid-Mediated Colistin-Resistant Enterobacteriaceae in Tertiary Care Settings
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antibiotics
Volume 10
Issue 7
10.3390/antibiotics10070838
antibiotics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Alžběta Kalová
1,2,*,
Tereza Gelbíčová
1,
Søren Overballe-Petersen
3,
Eva Litrup
3 and
Renáta Karpíšková
1
1
Department of Microbiology and Antimicrobial Resistance, Veterinary Research Institute, 621 00 Brno, Czech Republic
2
Department of Experimental Biology, Faculty of Science, Masaryk University, 602 00 Brno, Czech Republic
3
Statens Serum Institut, 2300 Copenhagen, Denmark
*
Author to whom correspondence should be addressed.
Antibiotics 2021, 10(7), 838; https://doi.org/10.3390/antibiotics10070838
Submission received: 26 May 2021 / Revised: 29 June 2021 / Accepted: 7 July 2021 / Published: 9 July 2021
(This article belongs to the Special Issue Colistin Resistance: The Need for a One Health Approach)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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 bacteria—e.g., Escherichia coli [16], Klebsiella pneumoniae [17], Salmonella enterica [18], and Acinetobacter baumannii [19].
Plasmids can be characterised by many ways, and the most common is to divide them by their incompatibility (Inc) groups. Currently, there are 28 Inc groups of plasmids in Enterobacteriaceae [20]. The host range can be limited only to Enterobacteriaceae (e.g., IncF or IncX) or can be broader (e.g., IncA/C, IncH, or IncP) [20]. According to Carattoli [21], the major plasmid families associated with antimicrobial resistance genes (ARGs) in Enterobacteriaceae are IncF, IncA/C, IncL/M, IncI1, IncHI2, and IncN. Some of these plasmid groups can be linked to specific resistance genes—e.g., IncF plasmids are frequently described carrying genes encoding resistance to extended-spectrum beta-lactams, carbapenems, aminoglycosides, or fluoroquinolones; IncI2, IncX4, and IncP plasmids are associated with resistance to colistin encoded by the mcr-1 gene; IncHI1 and IncHI2 plasmid are reported to be associated with multidrug resistance (including colistin resistance mcr-1 and mcr-3 genes); and ColE plasmids are reported to carry colistin resistance genes mcr-4 and mcr-5 [20]. The plasmids of Acinetobacter baumannii belong to a limited number of plasmid lineages and only around one third of them carry any ARGs (the most frequent are genes encoding resistance to aminoglycosides, beta-lactams, or sulphonamides) [22]. Plasmids associated with genes encoding resistance to colistin often carry the mcr-4.3 gene [19,23,24]. The ARGs are often found located close to mobile elements such as insertion sequences (IS), which help them to spread between different plasmids and chromosomes [25]. Insertion sequences have been described as the most abundant and ubiquitous genes in nature [26], and some specific IS can be linked to particular ARG—e.g., ISApl1 is associated with the mcr-1 gene [5].
Although bacteria carrying mcr genes in poultry, pork, or beef meat have been described extensively [6,27], little is known about the detailed characteristics of plasmids carrying colistin resistance genes from aquaculture products [28].
In China, several mcr-1-positive bacterial strains have been reported in aquaculture products: E. coli from grass carp carrying the mcr-1 gene on IncI2, IncP, and IncX4 plasmids or in the chromosome [29]; E. coli and K. pneumoniae from duck-fish integrated fisheries, slaughterhouses, and fish markets with mcr-1 on IncHI2, IncI2, IncX4, and IncP plasmids [30]; and a first detected Vibrio parahaemolyticus bearing the mcr-1 gene on a transferable IncX4 plasmid originating from shrimps [31]. In Vietnam, extended-spectrum beta-lactamase (ESBL) producing E. coli harbouring the mcr-1 gene isolated from fish gut was detected by PCR in the Mekong delta [32].
In Europe, mcr-1-positive E. coli has been found in pangasius fillets and prawns imported from Vietnam to Denmark [33]. Similarly, in Norway, a scampi imported from Bangladesh was found to be positive for E. coli carrying the mcr-1 gene on the IncHI2 plasmid type [34].
The detection of the mcr-3 gene in aquaculture has been reported mostly as mcr-3-like gene in the bacterial species Aeromonas isolated from fish [35,36]. These aquatic bacteria are believed to be “the source” of the mcr genes and their phosphoethanolamine transferases show a significant identity with the mcr-3 gene found in E. coli [37]. None of the rest of the currently described mcr genes have been detected in bacterial isolates from aquaculture products as of yet.
This study aims to provide a detailed characterisation of mcr-positive strains isolated from retailed aquaculture products imported from Vietnam to the Czech Republic, with a special emphasis on the localisation of mcr genes along with genes encoding resistance to other antimicrobials.

2. Results

2.1. Bacterial Isolates

Seventeen bacterial isolates resistant to colistin were acquired from aquaculture products (frog legs, crab meat, and pangasius meat) originating from Vietnam and retailed in the Czech Republic in 2019. The tested collection consisted of fourteen E. coli isolates, one K. pneumoniae, one A. baumannii, and one A. nosocomialis (Table 1).

2.2. 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.

2.3. 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).

2.4. 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).

2.5. 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 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).
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 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).

2.6. 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 phage-related 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% 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.

2.7. 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 (blaCTX-M-55, blaOXA-1 or blaVEB) (Table S1). No genes encoding resistance to carbapenems were found. Genes encoding resistance to fluoroquinolones (qnrS1, aac(6’)-Ib-cr, or qepA1) were found in eleven strains of E. coli (Table S1).
K. pneumoniae strain CT251 with both mcr-1.1 and mcr-8.2 genes carried both the blaCTX-M-65 and blaSHV-182 genes encoding ESBL. Both Acinetobacter spp. strains carried the mcr-4.3 gene. A. nosocomialis strain CT237 carried blaADC-68 gene encoding ESBL. On the other hand, A. baumannii strain CT263 carried blaADC-25, encoding a cephalosporinase.
The complete resistance genes profiles with their localisation on the plasmid or chromosome of all tested bacterial strains are presented in Table S1.

3. 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.

4. Materials and Methods

4.1. 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.
The minimum inhibitory concentration (MIC) of colistin was determined by the microdilution method (Erba Lachema, Brno, Czech Republic) and evaluated according to EUCAST (European Committee on Antimicrobial Susceptibility Testing, 2019, https://www.eucast.org/clinical_breakpoints/, accessed on 7 April 2020).

4.2. 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.

4.3. Multilocus Sequence Typing (MLST)

E. coli sequence type was determined by the Achtman MLST scheme (www.enterobase.warwick.ac.uk/species/e.coli, accessed on 17 June 2020), whereas the Pasteur MLST scheme was used for the Klebsiella pneumoniae isolate (https://bigsdb.pasteur.fr/klebsiella/klebsiella.html, accessed on 17 June 2020) and Acinetobacter spp. isolates (https://pubmlst.org/abaumannii/, accessed on 17 June 2020).

4.4. Genetic Analysis of Plasmids and Antibiotic Resistance Genes

Plasmid types and resistance genes in Enterobacterales isolates were evaluated using PlasmidFinder [39] and ResFinder [40,72], available at https://cge.cbs.dtu.dk/services/ (accessed on 22 September 2020). For Acinetobacter spp. isolates, the ARGs were analysed by CARD [73] (https://card.mcmaster.ca/analyze/rgi, accessed on 22 September 2020). The annotation of genes was carried out using the Prokka v1.13.7 software [74] and RAST software [75] (https://rast.theseed.org/FIG/rast.cgi, accessed on 9 December 2020). The plasmids of identical type in this study were compared between each other using BRIG [76] v0.95 (Blast Ring Image Generator, http://brig.sourceforge.net/, accessed on 10 December 2020). BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 10 December 2020), with default parameters, was used on unique mcr-carrying plasmid sequences from this study to search for similar plasmids available in the NCBI database. PHASTER [38] (PHAge Search Tool Enhanced Release, https://phaster.ca/, accessed on 14 December 2020) was used to identify and annotate prophage sequences possibly surrounding the mcr genes.

5. 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.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/antibiotics10070838/s1: Table S1: Detailed localisation of antimicrobial resistance genes in tested strains.

Author Contributions

Conceptualisation, R.K. and A.K.; Methodology, A.K., T.G. and S.O.-P.; Investigation, A.K., T.G. and S.O.-P.; Resources, R.K. and E.L.; Data Curation, A.K. and S.O.-P.; Writing—Original Draft Preparation, A.K.; Writing—Review and Editing, R.K. and T.G.; Visualisation, A.K.; Supervision, R.K. and E.L.; Funding Acquisition, R.K. and E.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Ministry of Health of the Czech Republic, grant no: NV 18-09-00254. All rights reserved.

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

The sequencing data after hybrid assembly are available at Mendeley data, doi:10.17632/gj4pg2rbtp.1.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Henriksson, P.J.G.; Rico, A.; Troell, M.; Klinger, D.H.; Buschmann, A.H.; Saksida, S.; Chadag, M.V.; Zhang, W. Unpacking Factors Influencing Antimicrobial Use in Global Aquaculture and Their Implication for Management: A Review from a Systems Perspective. Sustain. Sci. 2018, 13, 1105–1120. [Google Scholar] [CrossRef] [Green Version]
  2. Han, Q.F.; Zhao, S.; Zhang, X.R.; Wang, X.L.; Song, C.; Wang, S.G. Distribution, Combined Pollution and Risk Assessment of Antibiotics in Typical Marine Aquaculture Farms Surrounding the Yellow Sea, North China. Environ. Int. 2020, 138, 105551. [Google Scholar] [CrossRef] [PubMed]
  3. Jia, J.; Guan, Y.; Cheng, M.; Chen, H.; He, J.; Wang, S.; Wang, Z. Occurrence and Distribution of Antibiotics and Antibiotic Resistance Genes in Ba River, China. Sci. Total Environ. 2018, 642, 1136–1144. [Google Scholar] [CrossRef] [PubMed]
  4. Catry, B. Use of Colistin-Containing Products within the European Union and European Economic Area (EU/EEA): Development of Resistance in Animals and Possible Impact on Human and Animal Health. Int. J. Antimicrob. Agents 2015, 10, 297–306. [Google Scholar] [CrossRef] [PubMed]
  5. Liu, Y.-Y.; Wang, Y.; Walsh, T.R.; Yi, L.-X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; et al. 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. 2016, 16, 161–168. [Google Scholar] [CrossRef]
  6. Luo, Q.; Wang, Y.; Xiao, Y. Prevalence and Transmission of Mobilized Colistin Resistance (Mcr) Gene in Bacteria Common to Animals and Humans. Biosaf. Health 2020, 8, 71–78. [Google Scholar] [CrossRef]
  7. Xavier, B.B.; Lammens, C.; Ruhal, R.; Kumar-Singh, S.; Butaye, P.; Goossens, H.; Malhotra-Kumar, S. Identification of a Novel Plasmid-Mediated Colistin-Resistance Gene, Mcr-2, in Escherichia Coli, Belgium, June 2016. Euro. Surveill. 2016, 21, 30280. [Google Scholar] [CrossRef]
  8. Yin, W.; Li, H.; Shen, Y.; Liu, Z.; Wang, S.; Shen, Z.; Zhang, R.; Walsh, T.R.; Shen, J.; Wang, Y. Novel Plasmid-Mediated Colistin Resistance Gene Mcr-3 in Escherichia Coli. mBio 2017, 8, e00543-17. [Google Scholar] [CrossRef] [Green Version]
  9. Carattoli, A.; Villa, L.; Feudi, C.; Curcio, L.; Orsini, S.; Luppi, A.; Pezzotti, G.; Magistrali, C.F. Novel Plasmid-Mediated Colistin Resistance Mcr-4 Gene in Salmonella and Escherichia Coli, Italy 2013, Spain and Belgium, 2015 to 2016. Eurosurveillance 2017, 22, 30589. [Google Scholar] [CrossRef] [Green Version]
  10. Borowiak, M.; Fischer, J.; Hammerl, J.A.; Hendriksen, R.S.; Szabo, I.; Malorny, B. Identification of a Novel Transposon-Associated Phosphoethanolamine Transferase Gene, Mcr-5, Conferring Colistin Resistance in d-Tartrate Fermenting Salmonella Enterica Subsp. Enterica Serovar Paratyphi B. J. Antimicrob. Chemother. 2017, 72, 3317–3324. [Google Scholar] [CrossRef] [Green Version]
  11. AbuOun, M.; Stubberfield, E.J.; Duggett, N.A.; Kirchner, M.; Dormer, L.; Nunez-Garcia, J.; Randall, L.P.; Lemma, F.; Crook, D.W.; Teale, C.; et al. Mcr-1 and Mcr-2 (Mcr-6.1) Variant Genes Identified in Moraxella Species Isolated from Pigs in Great Britain from 2014 to 2015. J. Antimicrob. Chemother. 2017, 72, 2745–2749. [Google Scholar] [CrossRef] [Green Version]
  12. Yang, Y.-Q.; Li, Y.-X.; Lei, C.-W.; Zhang, A.-Y.; Wang, H.-N. Novel Plasmid-Mediated Colistin Resistance Gene Mcr-7.1 in Klebsiella Pneumoniae. J. Antimicrob. Chemother. 2018, 73, 1791–1795. [Google Scholar] [CrossRef] [Green Version]
  13. Wang, X.; Wang, Y.; Zhou, Y.; Li, J.; Yin, W.; Wang, S.; Zhang, S.; Shen, J.; Shen, Z.; Wang, Y. Emergence of a Novel Mobile Colistin Resistance Gene, Mcr-8, in NDM-Producing Klebsiella Pneumoniae. Emerg. Microbes Infect. 2018, 7, 1–9. [Google Scholar] [CrossRef] [Green Version]
  14. Carroll, L.M.; Gaballa, A.; Guldimann, C.; Sullivan, G.; Henderson, L.O.; Wiedmann, M. Identification of Novel Mobilized Colistin Resistance Gene Mcr-9 in a Multidrug-Resistant, Colistin-Susceptible Salmonella Enterica Serotype Typhimurium Isolate. mBio 2019, 10, e00853-19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Wang, C.; Feng, Y.; Liu, L.; Wei, L.; Kang, M.; Zong, Z. Identification of Novel Mobile Colistin Resistance Gene Mcr-10. Emerg. Microbes Infect. 2020, 9, 508–516. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Tkadlec, J.; Kalova, A.; Brajerova, M.; Gelbicova, T.; Karpiskova, R.; Smelikova, E.; Nyc, O.; Drevinek, P.; Krutova, M. The Intestinal Carriage of Plasmid-Mediated Colistin-Resistant Enterobacteriaceae in Tertiary Care Settings. Antibiotics 2021, 10, 258. [Google Scholar] [CrossRef] [PubMed]
  17. Lin, Y.-C.; Kuroda, M.; Suzuki, S.; Mu, J.-J. Emergence of the Mcr-1 Colistin Resistance Gene in Extended-Spectrum β-Lactamase-Producing Klebsiella Pneumoniae in Taiwan. J. Global Antimicrob. Resist. 2021, 24, 278–284. [Google Scholar] [CrossRef]
  18. Li, Y.; Zhang, Y.; Chen, M.; Hu, J.; Zhang, H.; Xiang, Y.; Yang, H.; Qiu, S.; Song, H. Plasmid-Borne Colistin Resistance Gene Mcr-1 in a Multidrug Resistant Salmonella Enterica Serovar Typhimurium Isolate from an Infant with Acute Diarrhea in China. Int. J. Infect. Dis. 2021, 103, 13–18. [Google Scholar] [CrossRef]
  19. Martins-Sorenson, N.; Snesrud, E.; Xavier, D.E.; Cacci, L.C.; Iavarone, A.T.; McGann, P.; Riley, L.W.; Moreira, B.M. A Novel Plasmid-Encoded Mcr-4.3 Gene in a Colistin-Resistant Acinetobacter Baumannii Clinical Strain. J. Antimicrob. Chemother. 2020, 75, 60–64. [Google Scholar] [CrossRef]
  20. Rozwandowicz, M.; Brouwer, M.S.M.; Fischer, J.; Wagenaar, J.A.; Gonzalez-Zorn, B.; Guerra, B.; Mevius, D.J.; Hordijk, J. Plasmids Carrying Antimicrobial Resistance Genes in Enterobacteriaceae. J. Antimicrob. Chemother. 2018, 73, 1121–1137. [Google Scholar] [CrossRef] [Green Version]
  21. Carattoli, A. Resistance Plasmid Families in Enterobacteriaceae. Antimicrob. Agents Chemother. 2009, 53, 2227–2238. [Google Scholar] [CrossRef] [Green Version]
  22. Salgado-Camargo, A.D.; Castro-Jaimes, S.; Gutierrez-Rios, R.-M.; Lozano, L.F.; Altamirano-Pacheco, L.; Silva-Sanchez, J.; Pérez-Oseguera, Á.; Volkow, P.; Castillo-Ramírez, S.; Cevallos, M.A. Structure and Evolution of Acinetobacter Baumannii Plasmids. Front. Microbiol. 2020, 11, 1283. [Google Scholar] [CrossRef]
  23. Ma, F.; Shen, C.; Zheng, X.; Liu, Y.; Chen, H.; Zhong, L.; Liang, Y.; Liao, K.; Xia, Y.; Tian, G.-B.; et al. Identification of a Novel Plasmid Carrying Mcr-4.3 in an Acinetobacter Baumannii Strain in China. Antimicrob. Agents Chemother. 2019, 63, e00133-19. [Google Scholar] [CrossRef] [Green Version]
  24. Bitar, I.; Medvecky, M.; Gelbicova, T.; Jakubu, V.; Hrabak, J.; Zemlickova, H.; Karpiskova, R.; Dolejska, M. Complete Nucleotide Sequences of Mcr-4.3 -Carrying Plasmids in Acinetobacter Baumannii Sequence Type 345 of Human and Food Origin from the Czech Republic, the First Case in Europe. Antimicrob. Agents Chemother. 2019, 63, e01166-19. [Google Scholar] [CrossRef] [PubMed]
  25. Siguier, P.; Gourbeyre, E.; Chandler, M. Bacterial Insertion Sequences: Their Genomic Impact and Diversity. FEMS Microbiol. Rev. 2014, 38, 865–891. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Aziz, R.K.; Breitbart, M.; Edwards, R.A. Transposases Are the Most Abundant, Most Ubiquitous Genes in Nature. Nucleic Acids Res. 2010, 38, 4207–4217. [Google Scholar] [CrossRef] [Green Version]
  27. Schwarz, S.; Johnson, A.P. Transferable Resistance to Colistin: A New but Old Threat: Table 1. J. Antimicrob. Chemother. 2016, 71, 2066–2070. [Google Scholar] [CrossRef] [PubMed]
  28. Shen, Y.; Zhang, R.; Schwarz, S.; Wu, C.; Shen, J.; Walsh, T.R.; Wang, Y. Farm Animals and Aquaculture: Significant Reservoirs of Mobile Colistin Resistance Genes. Environ. Microbiol. 2020, 22, 2469–2484. [Google Scholar] [CrossRef] [Green Version]
  29. Lv, L.; Cao, Y.; Yu, P.; Huang, R.; Wang, J.; Wen, Q.; Zhi, C.; Zhang, Q.; Liu, J.-H. Detection of Mcr-1 Gene among Escherichia Coli Isolates from Farmed Fish and Characterization of Mcr-1 -Bearing IncP Plasmids. Antimicrob. Agents Chemother. 2018, 62, e02378-17. [Google Scholar] [CrossRef] [Green Version]
  30. Shen, Y.; Lv, Z.; Yang, L.; Liu, D.; Ou, Y.; Xu, C.; Liu, W.; Yuan, D.; Hao, Y.; He, J.; et al. Integrated Aquaculture Contributes to the Transfer of Mcr-1 between Animals and Humans via the Aquaculture Supply Chain. Environ. Int. 2019, 130, 104708. [Google Scholar] [CrossRef]
  31. Lei, T.; Zhang, J.; Jiang, F.; He, M.; Zeng, H.; Chen, M.; Wu, S.; Wang, J.; Ding, Y.; Wu, Q. First Detection of the Plasmid-Mediated Colistin Resistance Gene Mcr-1 in Virulent Vibrio Parahaemolyticus. Int. J. Food Microbiol. 2019, 308, 108290. [Google Scholar] [CrossRef]
  32. Hoa, T.T.T.; Nakayama, T.; Huyen, H.M.; Harada, K.; Hinenoya, A.; Phuong, N.T.; Yamamoto, Y. Extended-spectrum Beta-lactamase-producing Escherichia Coli Harbouring Sul and Mcr–1 Genes Isolates from Fish Gut Contents in the Mekong Delta, Vietnam. Lett. Appl. Microbiol. 2020, 71, 78–85. [Google Scholar] [CrossRef] [PubMed]
  33. Ellis-Iversen, J.; Seyfarth, A.M.; Korsgaard, H.; Bortolaia, V.; Munck, N.; Dalsgaard, A. Antimicrobial Resistant E. Coli and Enterococci in Pangasius Fillets and Prawns in Danish Retail Imported from Asia. Food Control 2020, 114, 106958. [Google Scholar] [CrossRef]
  34. Slettemeås, J.S.; Urdahl, A.-M.; Mo, S.S.; Johannessen, G.S.; Grave, K.; Norström, M.; Steinbakk, M.; Sunde, M. Imported Food and Feed as Contributors to the Introduction of Plasmid-Mediated Colistin-Resistant Enterobacteriaceae to a ‘Low Prevalence’ Country. J. Antimicrob. Chemother. 2017, 72, 2675–2677. [Google Scholar] [CrossRef]
  35. Liu, D.; Song, H.; Ke, Y.; Xia, J.; Shen, Y.; Ou, Y.; Hao, Y.; He, J.; Li, X.; Zhou, Y.; et al. Co-Existence of Two Novel Phosphoethanolamine Transferase Gene Variants in Aeromonas Jandaei from Retail Fish. Int. J. Antimicrob. Agents 2020, 55, 105856. [Google Scholar] [CrossRef] [PubMed]
  36. Eichhorn, I.; Feudi, C.; Wang, Y.; Kaspar, H.; Feßler, A.T.; Lübke-Becker, A.; Michael, G.B.; Shen, J.; Schwarz, S. Identification of Novel Variants of the Colistin Resistance Gene Mcr-3 in Aeromonas Spp. from the National Resistance Monitoring Programme GERM-Vet and from Diagnostic Submissions. J. Antimicrob. Chemother. 2018, 73, 1217–1221. [Google Scholar] [CrossRef]
  37. Cabello, F.C.; Tomova, A.; Ivanova, L.; Godfrey, H.P. Aquaculture and Mcr Colistin Resistance Determinants. mBio 2017, 8, e01229-17. [Google Scholar] [CrossRef] [Green Version]
  38. Arndt, D.; Grant, J.R.; Marcu, A.; Sajed, T.; Pon, A.; Liang, Y.; Wishart, D.S. PHASTER: A Better, Faster Version of the PHAST Phage Search Tool. Nucleic Acids Res. 2016, 44, W16–W21. [Google Scholar] [CrossRef] [Green Version]
  39. Carattoli, A.; Zankari, E.; García-Fernández, A.; Voldby Larsen, M.; Lund, O.; Villa, L.; Møller Aarestrup, F.; Hasman, H. In Silico Detection and Typing of Plasmids Using PlasmidFinder and Plasmid Multilocus Sequence Typing. Antimicrob. Agents Chemother. 2014, 58, 3895–3903. [Google Scholar] [CrossRef] [Green Version]
  40. Bortolaia, V.; Kaas, R.S.; Ruppe, E.; Roberts, M.C.; Schwarz, S.; Cattoir, V.; Philippon, A.; Allesoe, R.L.; Rebelo, A.R.; Florensa, A.F.; et al. ResFinder 4.0 for Predictions of Phenotypes from Genotypes. J. Antimicrob. Chemother. 2020, 75, 3491–3500. [Google Scholar] [CrossRef] [PubMed]
  41. Hernández, M.; Iglesias, M.R.; Rodríguez-Lázaro, D.; Gallardo, A.; Quijada, N.; Miguela-Villoldo, P.; Campos, M.J.; Píriz, S.; López-Orozco, G.; de Frutos, C.; et al. Co-Occurrence of Colistin-Resistance Genes Mcr-1 and Mcr-3 among Multidrug-Resistant Escherichia Coli Isolated from Cattle, Spain, September 2015. Eurosurveillance 2017, 22, 30586. [Google Scholar] [CrossRef]
  42. Sun, J.; Li, X.-P.; Fang, L.-X.; Sun, R.-Y.; He, Y.-Z.; Lin, J.; Liao, X.-P.; Feng, Y.; Liu, Y.-H. Co-Occurrence of Mcr-1 in the Chromosome and on an IncHI2 Plasmid: Persistence of Colistin Resistance in Escherichia Coli. Int. J. Antimicrob. Agents 2018, 51, 842–847. [Google Scholar] [CrossRef] [PubMed]
  43. Gelbíčová, T.; Baráková, A.; Florianová, M.; Jamborová, I.; Zelendová, M.; Pospíšilová, L.; Koláčková, I.; Karpíšková, R. Dissemination and Comparison of Genetic Determinants of Mcr-Mediated Colistin Resistance in Enterobacteriaceae via Retailed Raw Meat Products. Front. Microbiol. 2019, 10, 2824. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Hala, S.; Antony, C.P.; Momin, A.A.; Alshehri, M.; Ben-Rached, F.; Al-Ahmadi, G.; Zakri, S.; Baadhaim, M.; Alsaedi, A.; Thaqafi, O.A.A.; et al. Co-Occurrence of Mcr-1 and Mcr-8 Genes in Multi-Drug-Resistant Klebsiella Pneumoniae from a 2015 Clinical Isolate. Int. J. Antimicrob. Agents 2021, 57, 106303. [Google Scholar] [CrossRef] [PubMed]
  45. Lima, W.G.; Alves, M.C.; Cruz, W.S.; Paiva, M.C. Chromosomally Encoded and Plasmid-Mediated Polymyxins Resistance in Acinetobacter Baumannii: A Huge Public Health Threat. Eur. J. Clin. Microbiol. Infect. Dis. 2018, 37, 1009–1019. [Google Scholar] [CrossRef] [PubMed]
  46. Li, S.; Duan, X.; Peng, Y.; Rui, Y. Molecular Characteristics of Carbapenem-Resistant Acinetobacter Spp. from Clinical Infection Samples and Fecal Survey Samples in Southern China. BMC Infect. Dis. 2019, 19, 900. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Hameed, F.; Khan, M.A.; Muhammad, H.; Sarwar, T.; Bilal, H.; Rehman, T.U. Plasmid-Mediated Mcr-1 Gene in Acinetobacter Baumannii and Pseudomonas Aeruginosa: First Report from Pakistan. Rev. Soc. Bras. Med. Trop. 2019, 52, e20190237. [Google Scholar] [CrossRef] [Green Version]
  48. Al-Kadmy, I.M.S.; Ibrahim, S.A.; Al-Saryi, N.; Aziz, S.N.; Besinis, A.; Hetta, H.F. Prevalence of Genes Involved in Colistin Resistance in Acinetobacter Baumannii: First Report from Iraq. Microb. Drug Resist. 2020, 26, 616–622. [Google Scholar] [CrossRef]
  49. Sun, J.; Zhang, H.; Liu, Y.-H.; Feng, Y. Towards Understanding MCR-like Colistin Resistance. Trends Microbiol. 2018, 26, 794–808. [Google Scholar] [CrossRef] [PubMed]
  50. Meinersmann, R.J. The Biology of IncI2 Plasmids Shown by Whole-Plasmid Multi-Locus Sequence Typing. Plasmid 2019, 106, 102444. [Google Scholar] [CrossRef]
  51. Matamoros, S.; van Hattem, J.M.; Arcilla, M.S.; Willemse, N.; Melles, D.C.; Penders, J.; Vinh, T.N.; Thi Hoa, N.; Bootsma, M.C.J.; van Genderen, P.J.; et al. Global Phylogenetic Analysis of Escherichia Coli and Plasmids Carrying the Mcr-1 Gene Indicates Bacterial Diversity but Plasmid Restriction. Sci. Rep. 2017, 7, 15364. [Google Scholar] [CrossRef] [Green Version]
  52. Zelendova, M.; Papagiannitsis, C.C.; Valcek, A.; Medvecky, M.; Bitar, I.; Hrabak, J.; Gelbicova, T.; Barakova, A.; Kutilova, I.; Karpiskova, R.; et al. Characterization of the Complete Nucleotide Sequences of Mcr-1-Encoding Plasmids From Enterobacterales Isolates in Retailed Raw Meat Products From the Czech Republic. Front. Microbiol. 2021, 11, 604067. [Google Scholar] [CrossRef]
  53. Madec, J.-Y.; Haenni, M. Antimicrobial Resistance Plasmid Reservoir in Food and Food-Producing Animals. Plasmid 2018, 99, 72–81. [Google Scholar] [CrossRef]
  54. Ageevets, V.; Lazareva, I.; Mrugova, T.; Gostev, V.; Lobzin, Y.; Sidorenko, S. IncX4 Plasmids Harbouring Mcr-1 Genes: Further Dissemination. J. Global Antimicrob. Resist. 2019, 18, 166–167. [Google Scholar] [CrossRef] [PubMed]
  55. Zhang, C.; Feng, Y.; Liu, F.; Jiang, H.; Qu, Z.; Lei, M.; Wang, J.; Zhang, B.; Hu, Y.; Ding, J.; et al. A Phage-Like IncY Plasmid Carrying the Mcr-1 Gene in Escherichia Coli from a Pig Farm in China. Antimicrob. Agents Chemother. 2017, 61, e02035-16. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  56. Li, R.; Xie, M.; Lv, J.; Wai-Chi Chan, E.; Chen, S. Complete Genetic Analysis of Plasmids Carrying Mcr-1 and Other Resistance Genes in an Escherichia Coli Isolate of Animal Origin. J. Antimicrob. Chemother. 2016, 72, 696–699. [Google Scholar] [CrossRef] [Green Version]
  57. Wang, M.; Xiong, W.; Liu, P.; Xie, X.; Zeng, J.; Sun, Y.; Zeng, Z. Metagenomic Insights Into the Contribution of Phages to Antibiotic Resistance in Water Samples Related to Swine Feedlot Wastewater Treatment. Front. Microbiol. 2018, 9, 2474. [Google Scholar] [CrossRef] [Green Version]
  58. Yang, Y.; Xie, X.; Tang, M.; Liu, J.; Tuo, H.; Gu, J.; Tang, Y.; Lei, C.; Wang, H.; Zhang, A. Exploring the Profile of Antimicrobial Resistance Genes Harboring by Bacteriophage in Chicken Feces. Sci. Total Environ. 2020, 700, 134446. [Google Scholar] [CrossRef]
  59. Shen, Y.; Wu, Z.; Wang, Y.; Zhang, R.; Zhou, H.-W.; Wang, S.; Lei, L.; Li, M.; Cai, J.; Tyrrell, J.; et al. Heterogeneous and Flexible Transmission of Mcr-1 in Hospital-Associated Escherichia Coli. mBio 2018, 9, e00943-18. [Google Scholar] [CrossRef] [Green Version]
  60. Nordmann, P.; Lienhard, R.; Kieffer, N.; Clerc, O.; Poirel, L. Plasmid-Mediated Colistin-Resistant Escherichia Coli in Bacteremia in Switzerland. Clin. Infect. Dis. 2016, 62, 1322–1323. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Zając, M.; Sztromwasser, P.; Bortolaia, V.; Leekitcharoenphon, P.; Cavaco, L.M.; Ziȩtek-Barszcz, A.; Hendriksen, R.S.; Wasyl, D. Occurrence and Characterization of Mcr-1-Positive Escherichia Coli Isolated From Food-Producing Animals in Poland, 2011–2016. Front. Microbiol. 2019, 10, 1753. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Chen, F.-J.; Lauderdale, T.-L.; Huang, W.-C.; Shiau, Y.-R.; Wang, H.-Y.; Kuo, S.-C. Emergence of Mcr-1, Mcr-3 and Mcr-8 in Clinical Klebsiella Pneumoniae Isolates in Taiwan. Clin. Microbiol. Infect. 2021, 27, 305–307. [Google Scholar] [CrossRef]
  63. Snesrud, E.; McGann, P.; Chandler, M. The Birth and Demise of the ISApl1-Mcr-1-ISApl1 Composite Transposon: The Vehicle for Transferable Colistin Resistance. mBio 2018, 9, 16. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  64. Li, R.; Yu, H.; Xie, M.; Chen, K.; Dong, N.; Lin, D.; Chan, E.W.-C.; Chen, S. Genetic Basis of Chromosomally-Encoded Mcr-1 Gene. Int. J. Antimicrob. Agents 2018, 51, 578–585. [Google Scholar] [CrossRef]
  65. Yamaguchi, T.; Kawahara, R.; Harada, K.; Teruya, S.; Nakayama, T.; Motooka, D.; Nakamura, S.; Nguyen, P.D.; Kumeda, Y.; Van Dang, C.; et al. The Presence of Colistin Resistance Gene Mcr-1 and -3 in ESBL Producing Escherichia Coli Isolated from Food in Ho Chi Minh City, Vietnam. FEMS Microbiol. Lett. 2018, 365, fny100. [Google Scholar] [CrossRef]
  66. Li, R.; Du, P.; Zhang, P.; Li, Y.; Yang, X.; Wang, Z.; Wang, J.; Bai, L. Comprehensive Genomic Investigation of Coevolution of Mcr Genes in Escherichia Coli Strains via Nanopore Sequencing. Global Chall. 2021, 5, 2000014. [Google Scholar] [CrossRef]
  67. Shen, C.; Zhong, L.-L.; Ma, F.; El-Sayed Ahmed, M.A.E.-G.; Doi, Y.; Zhang, G.; Liu, Y.; Huang, S.; Li, H.-Y.; Zhang, L.; et al. Genomic Patterns and Characterizations of Chromosomally-Encoded Mcr-1 in Escherichia Coli Populations. Gut. Pathog. 2020, 12, 55. [Google Scholar] [CrossRef]
  68. Yamaguchi, T.; Kawahara, R.; Hamamoto, K.; Hirai, I.; Khong, D.T.; Nguyen, T.N.; Tran, H.T.; Motooka, D.; Nakamura, S.; Yamamoto, Y. High Prevalence of Colistin-Resistant Escherichia Coli with Chromosomally Carried Mcr-1 in Healthy Residents in Vietnam. mSphere 2020, 5, e00117-20. [Google Scholar] [CrossRef] [Green Version]
  69. Tada, T.; Nhung, P.H.; Shimada, K.; Tsuchiya, M.; Phuong, D.M.; Anh, N.Q.; Ohmagari, N.; Kirikae, T. Emergence of Colistin-Resistant Escherichia Coli Clinical Isolates Harboring Mcr-1 in Vietnam. Int. J. Infect. Dis. 2017, 63, 72–73. [Google Scholar] [CrossRef] [Green Version]
  70. Krutova, M.; Kalova, A.; Nycova, E.; Gelbicova, T.; Karpiskova, R.; Smelikova, E.; Nyc, O.; Drevinek, P.; Tkadlec, J. The Colonisation of Czech Travellers and Expatriates Living in the Czech Republic by Colistin-Resistant Enterobacteriaceae and Whole Genome Characterisation of E. Coli Isolates Harbouring the Mcr-1 Genes on a Plasmid or Chromosome: A Cross-Sectional Study. Travel Med. Infect. Dis. 2021, 39, 101914. [Google Scholar] [CrossRef] [PubMed]
  71. Wick, R.R.; Judd, L.M.; Gorrie, C.L.; Holt, K.E. Unicycler: Resolving Bacterial Genome Assemblies from Short and Long Sequencing Reads. PLoS Comput. Biol. 2017, 13, 22. [Google Scholar] [CrossRef] [Green Version]
  72. Zankari, E.; Allesøe, R.; Joensen, K.G.; Cavaco, L.M.; Lund, O.; Aarestrup, F.M. PointFinder: A Novel Web Tool for WGS-Based Detection of Antimicrobial Resistance Associated with Chromosomal Point Mutations in Bacterial Pathogens. J. Antimicrob. Chemother. 2017, 72, 2764–2768. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Alcock, B.P.; Raphenya, A.R.; Lau, T.T.Y.; Tsang, K.K.; Bouchard, M.; Edalatmand, A.; Huynh, W.; Nguyen, A.-L.V.; Cheng, A.A.; Liu, S.; et al. CARD 2020: Antibiotic Resistome Surveillance with the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 2019, 48, D517–D525. [Google Scholar] [CrossRef] [PubMed]
  74. Seemann, T. Prokka: Rapid Prokaryotic Genome Annotation. Bioinformatics 2014, 30, 2068–2069. [Google Scholar] [CrossRef] [PubMed]
  75. Aziz, R.K.; Bartels, D.; Best, A.A.; DeJongh, M.; Disz, T.; Edwards, R.A.; Formsma, K.; Gerdes, S.; Glass, E.M.; Kubal, M.; et al. The RAST Server: Rapid Annotations Using Subsystems Technology. BMC Genom. 2008, 9, 75. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  76. Alikhan, N.-F.; Petty, N.K.; Ben Zakour, N.L.; Beatson, S.A. BLAST Ring Image Generator (BRIG): Simple Prokaryote Genome Comparisons. BMC Genom. 2011, 12, 402. [Google Scholar] [CrossRef] [Green Version]
Antibiotics 10 00838 g001 550
Figure 1. Genetic comparison of IncHI2 plasmids of E. coli strains CT249, CT248, CT258, CT262, CT267, CT259, CT260, and IncFIB(K) plasmids of E. coli strain CT250. The identity was calculated in comparison to plasmids of strain CT249 (red circle). The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the reference (red) plasmid.
Figure 1. Genetic comparison of IncHI2 plasmids of E. coli strains CT249, CT248, CT258, CT262, CT267, CT259, CT260, and IncFIB(K) plasmids of E. coli strain CT250. The identity was calculated in comparison to plasmids of strain CT249 (red circle). The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the reference (red) plasmid.
Antibiotics 10 00838 g001
Antibiotics 10 00838 g002 550
Figure 2. Types of genetic surroundings around the mcr-1.1 gene in tested strains of Enterobacterales. (a) The mcr-1.1 gene in the chromosome of E. coli CT230 in an atypical Tn6330 with IS5 and open reading frame (orf) inserted upstream of the mcr-1.1 gene. (b) The mcr-1.1 gene in the complete Tn6330 with mcr-8.2 located upstream of the transposon in K. pneumoniae CT251 plasmid. (c) ISApl1 upstream of the mcr-1.1 gene on plasmids in E. coli strains CT225, CT228, CT229, CT248, CT250, CT258, CT260, CT262, and CT267. (d) orf and ISApl1 downstream of the mcr-1.1 gene in E. coli CT227 (chromosome). (e) No ISApl1 sequence on plasmids in E. coli strains CT226, CT249, and CT259.
Figure 2. Types of genetic surroundings around the mcr-1.1 gene in tested strains of Enterobacterales. (a) The mcr-1.1 gene in the chromosome of E. coli CT230 in an atypical Tn6330 with IS5 and open reading frame (orf) inserted upstream of the mcr-1.1 gene. (b) The mcr-1.1 gene in the complete Tn6330 with mcr-8.2 located upstream of the transposon in K. pneumoniae CT251 plasmid. (c) ISApl1 upstream of the mcr-1.1 gene on plasmids in E. coli strains CT225, CT228, CT229, CT248, CT250, CT258, CT260, CT262, and CT267. (d) orf and ISApl1 downstream of the mcr-1.1 gene in E. coli CT227 (chromosome). (e) No ISApl1 sequence on plasmids in E. coli strains CT226, CT249, and CT259.
Antibiotics 10 00838 g002
Antibiotics 10 00838 g003 550
Figure 3. Visualisation of the mcr-3.5-carrying IncFII(pCoo) plasmid of E. coli strain CT262. The outer arrows show the ARGs, insertion sequences, and other genes and/or replication proteins present in the plasmid.
Figure 3. Visualisation of the mcr-3.5-carrying IncFII(pCoo) plasmid of E. coli strain CT262. The outer arrows show the ARGs, insertion sequences, and other genes and/or replication proteins present in the plasmid.
Antibiotics 10 00838 g003
Antibiotics 10 00838 g004 550
Figure 4. Genetic comparison of the IncI2 plasmids of E. coli strains CT228 and CT226. The identity was calculated in comparison to the plasmids of strain CT228 (red circle). The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the reference (red) plasmid.
Figure 4. Genetic comparison of the IncI2 plasmids of E. coli strains CT228 and CT226. The identity was calculated in comparison to the plasmids of strain CT228 (red circle). The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the reference (red) plasmid.
Antibiotics 10 00838 g004
Antibiotics 10 00838 g005 550
Figure 5. Visualisation of the mcr-1.1-carrying IncX4 plasmid of E. coli strain CT226. The outer arrow shows the ARG present in the plasmid.
Figure 5. Visualisation of the mcr-1.1-carrying IncX4 plasmid of E. coli strain CT226. The outer arrow shows the ARG present in the plasmid.
Antibiotics 10 00838 g005
Antibiotics 10 00838 g006 550
Figure 6. Visualisation of the mcr-1.1-carrying IncFIB-FIC plasmid of E. coli strain CT225. The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the plasmid.
Figure 6. Visualisation of the mcr-1.1-carrying IncFIB-FIC plasmid of E. coli strain CT225. The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the plasmid.
Antibiotics 10 00838 g006
Antibiotics 10 00838 g007 550
Figure 7. Visualisation of the mcr-1.1-carrying p0111 phage-like plasmid of E. coli strain CT229. The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the plasmid.
Figure 7. Visualisation of the mcr-1.1-carrying p0111 phage-like plasmid of E. coli strain CT229. The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the plasmid.
Antibiotics 10 00838 g007
Antibiotics 10 00838 g008 550
Figure 8. Genetic comparison of Acinetobacter spp. plasmids of strains CT263 and CT237. The identity was calculated in comparison to the plasmids of strain CT263 (red circle). The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the reference (red) plasmid.
Figure 8. Genetic comparison of Acinetobacter spp. plasmids of strains CT263 and CT237. The identity was calculated in comparison to the plasmids of strain CT263 (red circle). The outer arrows show the ARGs, insertion sequences, and/or replication proteins present in the reference (red) plasmid.
Antibiotics 10 00838 g008
Antibiotics 10 00838 g009 550
Figure 9. Visualisation of the mcr-1.1 and mcr-8.2 carrying IncFIA(HI1) plasmid of K. pneumoniae strain CT251. The outer arrows show the ARGs, insertion sequences, other genes, and/or replication proteins present in the plasmid.
Figure 9. Visualisation of the mcr-1.1 and mcr-8.2 carrying IncFIA(HI1) plasmid of K. pneumoniae strain CT251. The outer arrows show the ARGs, insertion sequences, other genes, and/or replication proteins present in the plasmid.
Antibiotics 10 00838 g009
Table
Table 1. Summary table of the tested bacterial strains with mcr-mediated colistin resistance originating from aquaculture products.
Table 1. Summary table of the tested bacterial strains with mcr-mediated colistin resistance originating from aquaculture products.
Strain IDSourceSpeciesColistin MIC (mg/L)MLSTmcr Genemcr Gene Localisation
(Plasmid Type/
Chromosome)
CT225pangasiusE. coli4ST155mcr-1.1IncFIB(AP001918)-FIC(FII)
CT226pangasiusE. coli>16ST2253mcr-1.1IncX4, IncI2
CT227pangasiusE. coli4ST206mcr-1.1chromosome
CT228pangasiusE. coli8ST156mcr-1.1IncI2
CT229crabE. coli4ST1011mcr-1.1p0111
CT230crabE. coli8ST6745mcr-1.1chromosome
CT248frog legsE. coli4ST4481mcr-1.1IncHI2
CT249frog legsE. coli4ST48mcr-1.1IncHI2-N
CT250frog legsE. coli4ST2179mcr-1.1IncFIB(K)
CT258frog legsE. coli4ST48mcr-1.1IncHI2
CT259frog legsE. coli8ST8680mcr-1.1IncHI2-N
CT260frog legsE. coli4ST48mcr-1.1IncHI2
CT262frog legsE. coli4ST609mcr-1.1, mcr-3.5mcr-1/IncHI2,
mcr-3/IncFII(pCoo)
CT267frog legsE. coli4ST48mcr-1.1IncHI2
CT251frog legsK. pneumoniae>16ST11mcr-1.1 + mcr-8.2IncFIA(HI1)
CT237pangasiusA. nosocomialis>16ST279mcr-4.3untypeable plasmid
CT263frog legsA. baumannii>16ST490mcr-4.3untypeable plasmid
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Kalová, A.; Gelbíčová, T.; Overballe-Petersen, S.; Litrup, E.; Karpíšková, R. Characterisation of Colistin -Resistant Enterobacterales and Acinetobacter Strains Carrying mcr Genes from Asian Aquaculture Products. Antibiotics 2021, 10, 838. https://doi.org/10.3390/antibiotics10070838

AMA Style

Kalová A, Gelbíčová T, Overballe-Petersen S, Litrup E, Karpíšková R. Characterisation of Colistin -Resistant Enterobacterales and Acinetobacter Strains Carrying mcr Genes from Asian Aquaculture Products. Antibiotics. 2021; 10(7):838. https://doi.org/10.3390/antibiotics10070838

Chicago/Turabian Style

Kalová, Alžběta, Tereza Gelbíčová, Søren Overballe-Petersen, Eva Litrup, and Renáta Karpíšková. 2021. "Characterisation of Colistin -Resistant Enterobacterales and Acinetobacter Strains Carrying mcr Genes from Asian Aquaculture Products" Antibiotics 10, no. 7: 838. https://doi.org/10.3390/antibiotics10070838

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop