Emergence of colistin-resistant Enterobacter cloacae and Raoultella ornithinolytica carrying the phosphoethanolamine transferase gene, mcr-9, derived from vegetables in Japan

ABSTRACT Colistin, a last-line antimicrobial agent, is recommended for the treatment of severe infections caused by multidrug-resistant (MDR) microorganisms. However, reports on plasmid-mediated mobilized colistin resistance (mcr) genes have prompted the importance of routine colistin resistance surveillance. Herein, we report the emergence of Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1 carrying an mcr-9.1 gene in vegetables from Japan. A total of 308 colistin-resistant isolates were retrieved from 200 fresh vegetables in Hiroshima Prefecture, Japan. PCR detection of mcr-1 to mcr-9 was conducted. While none of the isolates detected positive for mcr-1 to mcr-8 genes, we found two (0.65%) positive strains, E. cloacae CST17-2 and R. ornithinolytica CST129-1, that harbored mcr-9.1 allele. These isolates were subjected to phenotypic susceptibility testing, whole-genome sequencing (WGS), PCR-based replicon typing, and conjugation experiment. We found that both isolates had high colistin resistance [minimum inhibitory concentration (MIC) 16 or >64 µg/mL] and showed MDR phenotypes. WGS of both isolates revealed mcr-9 on a plasmid of the IncHI2/HI2A backbone. The mcr-9-bearing plasmid, pCST17-2_1, was self-transferable, although the pCST129-1_1 plasmid was not. Despite being colistin-resistant, the so-called two-component regulatory operon, qseBC, which induces polymyxin resistance, was absent from the genetic arrangements downstream of mcr-9 in R. ornithinolytica CST129-1. Nonetheless, a conjugation experiment demonstrated that mcr-9 in a Raoultella-type background is capable of mediating colistin resistance. In silico genomic analysis and comparison revealed distinct genetic structures surrounding mcr-9, especially in the downstream vicinities. The E. cloacae CST17-2 strain is of sequence-type ST738, a sequence type that has emerged in mcr-9.1-containing E. cloacae. Remarkably, we report the first mcr-9-carrying colistin-resistant Enterobacteriaceae isolated from Japanese vegetables, which is a grave public health concern. Our findings highlight the importance of strict epidemiological monitoring to track and/or prevent further dissemination of mcr homologs across the vegetable industry. IMPORTANCE Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two mcr-9.1-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica. This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of mcr alleles.

IMPORTANCE Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice.Here, we described the genetic features of two mcr-9.1-carryingGram-nega tive bacteria with a colistin-resistant phenotype derived from vegetables in Japan.The colistin-resistant mcr-9.1, which has never been detected in vegetables, was located on a large plasmid in Enterobacter cloacae CST17-2 and Raoultella ornithinolytica CST129-1, suggesting a high chance of horizontal gene transfer.To the best of our knowledge, this is the first report of mcr-9 in R. ornithinolytica.This study indicates that fresh vegetables might be a potential source for the transmission of mcr-9 genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials.The study also provides study has identified mcr-9-carrying Enterobacter cloacae and Raoultella ornithinolytica with a colistin-resistant phenotype in vegetables.It was previously found that the two-component regulatory operon, qseBC, induces mcr-9 expression, leading to colistin resistance (32).Other studies tested 30 mcr-9-carrying Enterobacteriaceae and found that those isolates were susceptible to colistin and lacked the qseBC regulatory system (33).Further studies reported colistin resistance in isolates lacking such regulatory operon (34,35), indicating uncertainties as to what phenotype mcr-9 gene may carry.Hence, genomic epidemiological surveillance investigations will be useful in providing updated information on mcr-9 and the emergence of new resistance traits within the food chain and vegetable production continuum.
Considering that vegetables are part of a healthy diet and the increasing prevalence and wide dissemination of mcr genes have been reported, it is of the utmost urgency to investigate the occurrence of mcr-positive bacteria in vegetables in Japan.In a routine, prefectural surveillance to track the changes and the prevalence of antimicro bial resistance determinants among different local ecosystems (river water, sewage, meat, soil, and vegetables), our research group retrieved colistin-resistant GNB bacte rial isolates (MIC >2 µg/mL) from several vegetable samples collected in Hiroshima Prefecture, Japan.Although the use of colistin as a feed additive for food-producing animals has been abolished in Japan since 2018, its therapeutic administration to cattle and pigs has been allowed (36).Given that the current reports on mcr genes in Japan are mostly linked to E. coli and Salmonella enterica derived from livestock, we specula ted that the discharge of wastewater and sewage irrigation from animal farms might transfer colistin-resistant bacteria to vegetables.We then screened our collection of colistin-resistant isolates for the presence of mcr-1 to mcr-9.Moreover, it is worth noting that there are no reports describing the emergence of mcr-9-carrying Enterobacteriaceae in vegetables, apart from the recently reported E. hormaechei in the USA (31).There fore, in this study, we characterized the genetic features of vegetable-derived plasmidborne mcr-9.1 in colistin-resistant E. cloacae and R. ornithinolytica during a prefectural epidemiological or antimicrobial surveillance in Japan by whole-genome sequencing (WGS).

Screening for mcr-1 to mcr-9 and strain identification
During a One Health antimicrobial surveillance from June 2020 to December 2021, our research group recovered 308 colistin-resistant (MIC >2 µg/mL) GNB isolates from 200 fresh vegetable samples randomly collected in Hiroshima Prefecture, Japan.We have screened the isolates for the presence of mcr-1 to mcr-9.While none of the isolates detected positive for mcr-1 to mcr-8 genes, we found two (0.65%) positive strains, E. cloacae CST17-2 and R. ornithinolytica CST129-1 that carried the recently reported putative phosphoethanolamine transferase gene, mcr-9 (18).E. cloacae CST17-2 was isolated from a bean sprout (a common ingredient in many Asian cuisines), while R. ornithinolytica CST129-1 was an isolate from myoga (Japanese ginger) (Table 1).They were identified by amplifying and sequencing the 16S rRNA fragments and confirmed by WGS.

Genome characterization of mcr-9-carrying Enterobacteriaceae
A hybrid assembly of both Illumina MiSeq short reads and Oxford Nanopore long reads revealed the complete circular chromosome and plasmids contained in each strain.The chromosome of the E. cloacae CST17-2 strain was 4,819,105 bp in size, with an average guanine-cytosine (GC) content of 55% and harbored fosA (a gene encoding fosfomy cin resistance) and bla ACT (a chromosomal AmpC gene in Enterobacter that renders resistance to penicillins and cephalosporins) genotype (Table 3).Species identification based on average nucleotide identity (ANI, a metric for comparing genetic relatedness or to verify taxonomic identities) analysis and the k-mer algorithm identified the strain as E. cloacae, as it shared 99.95% sequence identity with the reference strain for E. cloacae A1137 (accession number NZ_CP021851.1), with a query coverage of 91%; given this, it was designated as E. cloacae CST17-2.Further analysis of the WGS data indicated that the strain was of sequence type ST738, a sequence type that has emerged in mcr-9.1-carryingE. cloacae.
The R. ornithinolytica CST129-1 strain was confirmed as R. ornithinolytica because it had ANI value of 99.62% with the reference strain for R. ornithinolytica Yangling I2 (83% query coverage; GenBank accession number NZ_CP013338.1)identified in China.The chromosome size was 5,451,172 bp, with 56% GC content, and carried two known antimicrobial resistance genes, bla PLA-1a and fosA (Table 3).

Genomic structure surrounding the mcr-9 gene
Genome visualization of mcr-9-associated plasmids revealed different genetic orienta tions.Analysis of the genetic structure of pCST17-2_1 showed that mcr-9 was enclosed by two intact insertion sequences (ISs), IS903 (IS5 family) and IS5075 (IS110 family) located at the upstream and downstream boundaries, respectively.Within this region, a hypothetical protein, wbuC (encoding a functionless putative cupin metalloprotein), was located downstream, followed by a two-component regulatory system consisting of a sensor histidine kinase encoding qseC and a response regulator qseB genes (Fig. 3).The presence of similar downstream proteins induces colistin resistance in Enterobacteriaceae as previously reported (40,41).The IS5075-qseB-qseC-wbuC-mcr-9-IS903 operon was in an arrangement similar to that found within multiple genera of Enterobacteriaceae, including E. hormaechei (NZ_MK933279.1),E. coli (GCA_900500325), S. enterica (CP026661.1),E. cloacae (NZ_MF344583), and Leclercia adecarboxylata (MH909331.1).Unlike E. cloacae CST17-2, we observed a different genetic disposition for R. ornithinolytica CST129-1, which was characterized by a lack of downstream qseB-like and qseC-like regulatory operons (Fig. 3).The fact that mcr-9 conferred colistin resistance, despite the absence of the qseB/qseC system, brings new insights to the idea that mcr-9 may be activated by other proteins from R. ornithinolytica or that resistance is mediated by an unknown mechanism.

Transferability of mcr-9-harboring plasmid
To better understand whether the plasmids carrying mcr-9 could naturally restore colistin resistance in heterologous bacterial hosts, we conducted a series of conjugation experiments using multiple recipient backgrounds.Interestingly, E. cloacae CST17-2 successfully transferred its resistance plasmid to E. coli J53 and conferred colistin resistance, indicating the self-transferability of this plasmid (Table 2).Despite several attempts in conjugation between R. ornithinolytica CST129-1 and E. coli J53, transconjugants carrying mcr-9 were not obtained (Table 2).We repeated this process using two strains of R. ornithinolytica (MA-1-1-r and CAA-1-5-r) with different resistance phenotypes as recipients.Both strains were isolated from a sewage effluent (discharged from Asahi-cho Sewage Workstation, Hiroshima City) on 11 May 2021 in the same region and tested susceptible to colistin (MIC ≤ 1µg/mL).Plasmid transfer in this context was also unsuccessful even at varying conjugation temperatures of 25°C, 30°C, and 37°C (Table 4).We then speculated that mcr-9 may be activated by other proteins within Raoultella spp.In order to verify what function Raoultella background has on mcr-9 expression and considering the fact that Raoultella is an Enterobacteria ceae closely related to Klebsiella (42), we further included a colistin-susceptible (MIC, 0.125 µg/mL) Klebsiella pneumoniae LM22-1 (accession number DRA009528) (43) as a donor (Table 4).K. pneumoniae LM22-1 carries mcr-9 on a 281,252-bp plasmid of IncHI2/ HI2A backbone, which is in a cassette with a similar genetic context as R. ornithinolytica CST129-1.Interestingly, K. pneumoniae transferred its colistin-susceptible mcr-9-carrying plasmid to both R. ornithinolytica (MA-1-1-r and CAA-1-5-r) recipients.When determin ing colistin MIC for the Raoultella transconjugants (carrying the Klebsiella plasmid), we noticed a remarkable increase in MIC (4-32 µg/mL) (Table 5), suggesting that mcr-9 was highly expressed in a Raoultella-type background, which supported our hypothesis.This indicated that unknown proteins originating from Raoultella spp.might be potential players in mcr-9 activation, leading to colistin resistance.We then aligned the amino acid sequences of mcr-9 derived from E. cloacae CST17-2 and R. ornithinolytica CST129-1 with that of K. pneumoniae LM22-1.We found a mutational event characterized by a single amino acid deletion (539 vs. 540 aa) in the aligned sequences of mcr-9 compared to that found in K. pneumoniae LM22-1 (Fig. S1), further confirming mcr-9.1 identified in this study.It is important to note that mcr-9.1 is characterized by 539 aa.

DISCUSSION
MDR organisms, including colistin-and carbapenem-resistant Enterobacterales, have been recognized as an unprecedented global concern (19).Currently, one of the most worrisome issues pertains to the frequent occurrence of plasmid-borne mcr alleles, resulting in the reduced effectiveness of colistin, one of the few therapeutic options for treating severe MDR infections.In fact, the increasing prevalence of the novel putative phosphoethanolamine transferase gene, mcr-9, among colistin-resistant Enterobacteria ceae is of growing concern and indicates a high propensity for poor clinical outcomes in the future because its mechanism of resistance is still unclear.
mcr-9 has been found in multiple bacterial species, including S. enterica (44,45), E. coli (32,45), E. hormaechei (40,46), Enterobacter kobei/Enterobacter asburiae (47), and K. pneumoniae (43), and is mostly hosted in the E. cloacae complex isolated from humans and animals (48).Understanding the transmission routes and mechanisms of resistance is crucial for making tailored decisions to controlling the spread of mcr alleles.To the best of our knowledge, the detection of mcr-9 in MDR Enterobacteriaceae isolated from vegetables has been reported only once (31).Therefore, vegetables might be a potential transmission route for mcr-9-producing MDR bacteria.
in Japan.Additionally, we reported the emergence of mcr-9 in R. ornithinolytica, an Enterobacteriaceae closely related to Klebsiella spp.R. ornithinolytica is often isolated from aquatic and soil environments and has been found to be associated with commun ity-acquired infection (8).Susceptibility testing indicated that both isolates exhibited MDR phenotypes and mediated colistin resistance (MIC 16 or >64 µg/mL), which were remarkably higher than that previously reported in mcr-9-carrying S. Typhimurium (18).The mcr-9 in this Salmonella strain was characterized by 64.5% amino acid identity to mcr-3 and was naturally susceptible to colistin (MIC 0.25-0.5 µg/mL).It has previ ously been shown that a two-component regulatory system, qseBC, induces mcr-9 expression, leading to colistin resistance (32).Genomic analysis of E. cloacae CST17-2 revealed a functional two-component regulatory system encoding qseBC genes located downstream of mcr-9, further supporting the role of these tandem genes in inducing colistin resistance (Fig. 3).Such genetic arrangements have previously been identified in mcr-9-carrying colistin-resistant S. enterica strains in Korea (44) and China (40).The fact that mcr-9 is not as concerning as other mcr homologs (37), from a public health perspective, should be reconsidered, as epidemiological investigations and susceptibility testing continue to reveal different emerging resistance phenomena related to this gene.It appears that the changing genetic disposition associated with mcr-9 continues to facilitate its effect on colistin, resulting in reduced susceptibility patterns among Enterobacterales.Surprisingly, contrary to other studies that reported colistin susceptibility among Enterobacteriaceae lacking chromosomal or plasmid qseBC from Egypt (49), Lebanon (46), and Japan (43), we have found a unique colistin-resistant phenotype (MIC 16 µg/mL) within R. ornithinolytica CST129-1, although the functional qseBC-like cassette responsi ble for inducing colistin resistance among Enterobacterales was absent from the genetic arrangements downstream of mcr-9.Since no functional mutations were detected on the R. ornithinolytica CST129-1 chromosome, we speculated that this resistance phenotype was suggestive of an unknown mechanism or that other proteins in Raoultella might have activated mcr-9 expression, leading to colistin resistance.Although conjugation failed to transfer pCST129-1_1 to E. coli J53 and the two R. ornithinolytica (MA-1-1-r and CAA1-5-r) recipients, we proved our assumption in another conjugation assay by using a colistin-susceptible mcr-9-carrying K. pneumoniae LM22-1 as a donor to R. ornithinolytica (MA-1-1-r and CAA1-5-r).In this context, we found that mcr-9 was highly expressed in the transconjugants, leading to a remarkable increase in colistin MIC levels, further supporting the role mcr-9-carrying Raoultella spp.may play in mediating colistin resistance among the Enterobacteriaceae.
Multiple Enterobacterial species, including Salmonella (45), E. coli (45), E. asburiae (50), Citrobacter telavivum (51), and E. hormaechei (52) with the so-called qseBC genes, have been reported to exhibit colistin-susceptible phenotypes.In contrast, some other studies also found that lack of such a tandem system in certain bacterial species was associated with colistin-resistant phenotypes, as with E. roggenkampii (34), E. cloacae complex (48), and S. Newport (35).These observations suggest that the mechanisms leading to a reduced susceptibility to polymyxin are more related to other factors (not mentioning chromosomal point mutations), in which a different strain background may be a contributing factor underlying the mystery behind mcr-9 expression.As a matter of growing concern, though mcr-9 expression is governed by a regulatory system, it may not necessarily be associated with colistin susceptibility in the absence of plasmid-borne qseBC genes as previously reported.
Evidence suggests that heavy metal ions impose a selective pressure favoring the co-selection of metal and antibiotic resistance genes (53)(54)(55).Two main mechanisms have been reported to govern this process.One is co-resistance, in which genes with specific resistance phenotypes co-exist on the same mobile genetic element (i.e., plasmid or transposons), and the second is cross-resistance, where bacteria can develop resistance to both antibiotics and heavy metals at the same time (54,56).It appears that co-selection is a mechanism of drug resistance because transcriptional and translational responses related to metal ions and antibiotics are genetically linked, in which bacteria exposed to heavy metals are simultaneously resistant to metals and antibiotics (54,57,58).Considering that the genetic context of mcr-9 consists of copper-binding proteins and metal efflux systems, we postulate that the presence of plasmid genes encoding metal ion resistance could be potential players in the induction of a positive feedback response leading to mcr-9 expression and consequently mediating colistin resistance.However, further studies are required to draw definitive conclusions regarding this speculation.Very recently, a different phenomenon of colistin resistance was reported in a Japanese research laboratory, in which E. asburiae strain carrying chromosomal mcr-9 coupled with qseBC showed susceptibility to colistin (MIC 0.5 µg/mL) in a static culture incubation using broth microdilution but did grow at 8 µg/mL in a shaking culture incubation (59).In contrast, a remarkable increase in colistin MIC was observed when the standardized cation-adjusted Muller-Hinton broth was amended with casein, peptone, and tryptone, further supporting the argument that several other factors within the immediate environment might be responsible for mcr-9 expression.It is also worth mentioning that the observed colistin MIC for this isolate was relatively higher (32-128 µg/mL) compared to those lacking the regulatory operon, which is supportive of the functional role of the qseBC tandem system.
Further analysis of the genetic context surrounding mcr-9 revealed genetic structures and arrangements distinct from those previously reported (34,40,49).However, a similar structure exists between the E. cloacae CST17-2 strain identified in this study and the E. hormaechei strain reported in China (40), in which mcr-9 was bracketed by two ISs (Fig. 3).These mobile elements facilitate the mobilization of resistance genes in different plasmids.Considering that mcr-9 and the two-component regulatory operons were identified within these flanking vicinities, they indicated the potential for being carried along during such mobilization and transposition events.We observed that the upstream regions of mcr-9 within the R. ornithinolytica CST129-1 strain were homologous to pAMS-38a (NZ_CP051133.1)and pN260-1 (NZ_AP023448.1)reported in Egypt (49) and Japan (34), respectively, but displayed different orientations downstream.Collectively, these insights suggest that clinical infections due to mcr-9-carrying strains would be problematic when treated with colistin since some bacterial species in this context seem to have elevated colistin MIC beyond the steady-state plasma concentration of 2 µg/mL (60).
WGS analysis showed that both isolates harbored mcr-9 on a large DLST HI2/HI2A plasmid (>300,000 bp).No other determinants of antibiotic resistance were detected on these plasmids.Comparative genome analysis revealed that these plasmids shared homologies with similar mcr-9-carrying HI2 plasmids downloaded from public database (Fig. 1 and 2).We then conducted conjugation assays to investigate the transferability of these epidemic plasmids and the expression of mcr-9 in heterologous bacterial hosts.Transconjugants with high colistin MIC were obtained when conjugating pCST17-2_1, indicating that mcr-9 was located on a self-transferable plasmid (Table 2).This obser vation is consistent with previous reports that the worldwide dissemination of mcr-9 is mainly driven by HI2/HI2A plasmids (18,48,49).Furthermore, similar conjugative mcr-9-carring plasmids have been found in food and clinical isolates in Japan (34,43,61), suggesting a One Health perspective on the transmission of such mcr homolog.Either the fact that R. ornithinolytica cannot conjugate was attributed to a lack of conjugative transfer mechanisms or the fact that the genetic backgrounds of the recipients were not suitable for this specific plasmid.Evidence suggests that most very large plasmids tend to be less mobilizable (62); hence, successful conjugation may not be easily achieved in certain bacterial species.
The E. cloacae CST17-2 strain belongs to the sequence type ST738.A search within NCBI database revealed 386 E. cloacae genomes; however, ST738 was not found in mcr-9-containing strains, suggesting that this ST type has emerged in mcr-9.1-carryingE. cloacae.It is worth mentioning that mcr-9 was previously reported to have originated from a yet-to-identify bacterial species that is closely related Buttiauxella spp.(19), given that this might relate to the uncertainties as to what function this mcr allele may carry.Further studies will be conducted to elucidate other mechanisms, if any, that may be associated with mcr-9 expression.
In conclusion, our findings indicate that fresh vegetables constitute a possible transmission route for the dissemination of colistin-resistant genes among Enterobacter ales.The detection of highly colistin-resistant MDR strains in food sources is frustrating and poses a food safety risk because these strains can be transmitted to humans.Here, we described the first complete genomic study of the characteristics of colistin-resistant mcr-9.1-carryingEnterobacteriaceae isolated from fresh vegetables in Japan.To the best of our knowledge, this is the first report of foodborne R. ornithinolytica carrying mcr-9.Further research, including extensive epidemiological investigations and continuous susceptibility testing, is required to assess the occurrence of novel resistance traits in all prefectures of Japan and other countries worldwide.It is equally important to investigate other unknown mechanisms that lead to reduced susceptibility to colistin both in vitro and in vivo.

Bacterial isolation and total genomic DNA extraction
During the period from June 2020 to December 2021, 200 fresh vegetables were purchased from popular supermarkets in Hiroshima Prefecture, Japan.Bacterial isolation was as follows: briefly, a 25-g portion of each vegetable sample was homogenized in 225 mL of buffered peptone water (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) using a stomacher blender for 1 min.The resulting suspensions were directly plated on MacConkey agar (Eiken Chemical Co., Ltd., Tochigi, Japan) containing 2 µg/mL of colistin (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the plates were incubated at 37°C for 24 h.After incubation, bacterial colonies were preferentially selected and re-cultured on similar antibiotic-containing agar plates.Pure cultures of 308 isolates were obtained from Luria-Bertani (LB) agar plates (Nacalai Tesque Inc., Kyoto, Japan) without antibiotics.Bacterial genomic DNA was extracted by using the boiled lysate method as previously described (63).DNA was stored at −20°C prior to molecular screening.

Antimicrobial susceptibility testing
The MICs of 12 antimicrobial agents were determined by using the standardized broth microdilution method according to the interpretative criteria described by the Clini cal and Laboratory Standards Institute (CLSI 2020) (67).The breakpoint for colistin was determined according to the European Committee on Antimicrobial Susceptibil ity Testing (EUCAST 2021) (68).Antimicrobial agents included ampicillin, cefotaxime, ceftazidime, cefoxitin, meropenem, kanamycin, gentamicin, chloramphenicol, ciprofloxa cin, tetracycline, colistin, and fosfomycin.A stock solution of each antimicrobial agent was prepared and diluted according to the CLSI recommendations.Quality control for MIC analysis was performed using E. coli American Type Culture Collection (ATCC) 25922.

Whole genome sequencing
Complete genome sequencing was performed by using next-generation sequencing (NGS) platforms.Total bacterial genomic DNA was prepared using a Qiagen Genomictip 20/G kit (Qiagen) in accordance with the manufacturer's guidelines.DNA sequenc ing libraries for Illumina MiSeq (short reads) were prepared using an Enzymatic 5× WGS fragmentation mix and WGS ligase reagents (Qiagen, Hilden, Germany) following the manufacturer's recommendations.For Oxford Nanopore sequencing, a library was prepared by using a Rapid Barcoding Sequencing Kit (SQK-RBK004) (Oxford Nanopore Technologies, Oxford, UK).Sequencing libraries were purified, loaded onto an FLO-MIN106 flow cell, and sequenced using a GridION device (Oxford Nanopore Technolo gies).

Conjugation assay
Transferability of mcr-9-bearing plasmids was determined by using filtermating conjugation assays, as previously described (63).Exponential-phase lysogeny broth cultures of the donor and azide-resistant E. coli J53 recipient strains were used.Both the donor and recipient strains were mixed in the ratio 1:9 (100-µL donor:900-µL recipient) and centrifuged for 3 min at 6,000 rpm.The supernatant was removed, and the pellets were resuspended in 200-µL LB broth.The resulting suspension was then plated on a conjugation filter on LB agar and incubated for 3-5 h at 37°C.Transconjugants were selected on LB agar containing 100-µg/mL sodium azide and 4-µg/mL colistin.Transconjugants were confirmed by PCR targeting the mcr-9 gene and antimicrobial susceptibility testing.Unlike E. cloacae CST17-2, the mcr-9-bearing plasmid within R. ornithinolytica CST129-1 was not successfully transferred to E. coli J53, after two-time trials.We repeated the experiment at 25°C and 30°C, but conjugation failed again at these temperatures.We further used two different strains of colistin-susceptible (MIC ≤ 1 µg/mL) R. ornithinolytica (MA-1-1-r and CAA-1-5-r) as recipients for repeat conjugation at varying temperatures, although this was unsuccessful.Furthermore, a colistin-suscep tible (MIC 0.125 µg/mL) K. pneumoniae strain LM-22-1 (accession number DRA009528) carrying mcr-9 on a 281,251-bp plasmid of IncHI2 backbone was used as a donor to R. ornithinolytica (MA-1-1-r and CAA-1-5-r) because we hypothesized that mcr-9 may be expressed by other proteins in Raoultella spp.In this context, conjugation was successful leading to mcr-9 activation.

PCR-based replicon typing
Plasmids were isolated from mcr-9-carrying isolates as well as the E. coli J53 transconju gant using the alkaline lysis method as previously described (70).PCR-based replicon typing was then performed targeting the IncHI2 plasmid incompatibility group as previously described (71).

Sequence alignment of mcr-9 proteins
From the plasmid sequences of E. cloacae CST17-2, R. ornithinolytica CST129-1, and K pneumoniae LM22-1, the nucleotide sequences of mcr-9 were retrieved using the SnapGene genome visualizer and translated into amino acid sequence using the EMBOSS Transeq tool (https://www.ebi.ac.uk/Tools/st/emboss_transeq/).The translated protein sequences were then imported into ClustalX v2.1 and saved into a FASTA file format.Then, multiple protein sequence alignment was performed using Clustal Omega method available in SnapGene Viewer software v7.0.

FIG 3
FIG 3 The genetic context of mcr-9.Comparative analysis between the genetic environments of mcr-9 identified in this study with other mcr-9-carrying HI2 plasmids is shown.Different genetic structures were identified, with intact IS903 located at the immediate upstream regions of all the mcr-9 genes.The downstream vicinities are different for each plasmid.mcr-9 genes are indicated with red arrows.Regions of homology (~99.90%sequence similarity as determined by pairwise alignment of those regions) are highlighted in gray rectangles.Arrows indicate the positions and directions of the transcriptional orientations of the genes.The colistin MICs of those isolates are indicated next to each plasmid where R is resistant and S means susceptible.NCBI GenBank accession numbers are as follows: pCST17-2_1 (this study), pMCR-SCNJ07 (NZ_MK933279.1),pCST129-1_1 (this study), pAMS-38a (NZ_CP051133.1),and pN260-1 (NZ_AP023448.1).

TABLE 3
Genomic features of chromosomes and plasmids identified in mcr-9-carrying colistin-resistant E. cloacae and R. ornithinolytica a

plasmid Length (bp) GC % MLST/pMLST Incompatibility group Resistance genes Tellurium ion resistance genes
a ND, not detected.

TABLE 5
MIC of antimicrobials for Raoultella transconjugants and K. pneumoniae LM22-1 b a MICs were adapted from the published article (refer to the published article on K. pneumoniae LM22-1, accession number DRA009528).The resistance results are shown in bold font.