Detection of Five mcr-9-Carrying Enterobacterales Isolates in Four Czech Hospitals

Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the “last-resort” antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published.

IMPORTANCE Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published. Here, we report the first detection of Enterobacterales carrying mcr-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored bla VIM-1 , while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included bla VIM-4 . Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. bla VIM-4 was found in the MDR regions of IncHI2 plasmids, while bla VIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms. A significant increase in infections caused by carbapenemase-producing bacteria (1), coupled with the lack of novel antibiotics (2), has led to the revival of polymyxins as the "last-resort" antibiotic (3). Consequently, higher prevalence of colistin resistance among carbapenemase-producing Klebsiella pneumoniae strains has been reported worldwide (4). In K. pneumoniae, resistance to colistin is mainly mediated via chromosomal mutations in genes involved in lipopolysaccharide synthesis (5). However, in 2016, the first plasmid-mediated colistin resistance gene, mcr-1, was identified among Chinese Escherichia coli isolates (6). Following the first description, several reports describing the presence of mcr-1 in different host species and geographic areas were published (7,8). Thus far, the mcr gene family comprises mcr-1 to mcr-10 (9). These genes encode phosphoethanolamine transferases that catalyze the addition of phosphoethanolamine to the phosphate group of lipid A, reducing the negative charge of the bacterial outer membrane and attenuating its affinity for colistin, resulting in antibiotic resistance.
Among the mcr-like genes, mcr-1 and mcr-9 are the most widely disseminated. The mcr-9 gene has been identified from 40 countries across six continents. However, half of mcr-9-positive isolates (1,035/1,682 strains) were recovered in the United States, among which Salmonella enterica was the most common host species, especially in turkeys and chickens (9).
Here, we report the first detection of mcr-9-positive members of the Enterobacterales isolated from Czech hospitals.
In 2019, 4 isolates belonging to Enterobacter cloacae complex and one isolate belonging to Citrobacter freundii species were recovered from five patients admitted to Czech hospitals (see Table S1 in the supplemental material). In all isolates, which exhibited a meropenem MIC of .0.125 mg/ml (10), carbapenemase production was detected by a positive result in the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay (11). Screening for carbapenemase-encoding genes by PCR showed that all isolates carried bla VIM -like genes (12,13). Additionally, bacteria were positive for the presence of plasmid-mediated colistin resistance genes by PCR, as described previously (14). All VIM-producing isolates exhibited resistance to piperacillin, piperacillin-tazobactam, and cephalosporins, while the variations in the MICs of carbapenems that were observed (Table S2) might reflect the presence of additional resistance mechanisms in some of the isolates. Variations were also observed in the MICs of non-b-lactam antibiotics. However, all isolates were susceptible to colistin, according to data obtained by the broth dilution method (15) and interpreted according to EUCAST criteria (https://www.eucast.org/ clinical_breakpoints/).
To define the genetic units carrying mcr genes, the genomic DNAs of mcr-carrying clinical isolates were extracted using a NucleoSpin microbial DNA kit (Macherey-Nagel, Düren, Germany) and were sequenced using long-read sequencing technology on the PacBio Sequel I platform (Pacific Biosciences, Menlo Park, CA, USA). Library preparation was done following the manufacturer's recommendation for microbial multiplexing for the Express kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). DNA was sheared using Hydropore-long on a Megaruptor 2 device (Diagenode), and no size selection was performed during library preparation. The microbial assembly pipeline offered by SMRT Link v8.0 (Pacific Biosciences) was used to perform genome assembly with a minimum seed coverage of 30. For sequence analysis and annotation, BLAST (www.ncbi.nlm.nih.gov/ BLAST), the ISfinder database, and the open reading frame (ORF) finder tool (www .bioinformatics.org/sms/) were used. Comparative genome alignment was performed using Mauve v.2.3.1 (16). Figures were generated from sequence data using BRIG v.0.95 (17).
Analysis of whole-genome sequencing (WGS) data by PubMLST databases (https:// pubmlst.org/) revealed that the C. freundii isolate belonged to sequence type 95 (ST95). Additionally, 3 of 4 isolates belonging to E. cloacae complex were ST106, while the remaining isolate was assigned to ST764. In silico hsp60 typing of the genome sequences showed that four Enterobacter isolates belonged to the species Enterobacter hormaechei (18).
Analysis of WGS data using ResFinder 3.2 revealed that all isolates carried plasmidmediated colistin resistance mcr-9-like alleles. Furthermore, the three ST106 E. hormaechei isolates harbored the carbapenemase-encoding gene, bla VIM-1 , while the ST764 E. hormaechei and ST95 C. freundii isolates included the bla VIM-4 allele. Also, all isolates included additional genes for resistance to aminoglycosides, tetracyclines, trimethoprim, chloramphenicol, sulfonamides, quinolones, and/or macrolides ( Table 1). The presence of the resistance genes was confirmed by the antimicrobial resistance phenotypes (Table S1) of the isolates harboring those genes.
Analysis of plasmid sequences showed that, in all isolates, the mcr-9 allele was carried on IncHI2 plasmids (Table 1) (p48212_MCR, p48880_MCR_VIM, p48946_MCR, p49790_MCR, and p51929_MCR_VIM). Plasmids p48212_MCR, p48946_MCR, and p49790_MCR showed high degrees of similarity to each other (99% coverage and 99% identity), while lower diversity was observed in plasmids p48880_MCR_VIM (90% coverage and 99% identity) and p51929_MCR_VIM (77% coverage and 99% identity) compared to p48212_MCR. All plasmids exhibited sequences closely related to other mcr-9-carrying IncHI2 plasmids, like pC45-VIM4 from E. cloacae complex isolate C45 (GenBank accession no. LT991958) and pC45-001 from E. hormaechei strain C45, recovered from a clinical sample (GenBank accession no. CP042552) in Australia (Fig. 1), and typed as sequence type 1 (ST1) following the IncHI2 pDLST scheme (19). IncHI2 plasmid backbones were composed of regions for replication (reHI2), conjugative transfer (trh genes), and plasmid maintenance (par gene). Additionally, IncHI2 plasmids carried tellurium resistance genes (terZABCDEF), commonly associated with this plasmid family, in addition to terY1, terY2, and terW (20). Also, genes conferring arsenic resistance (arsCBRH) were found in IncHI2 plasmids. In all IncHI2 plasmids, the mcr-9 allele was inserted upstream the pcoS gene, as observed in other IncHI2 plasmids like pC45-001 (GenBank accession no. CP042552). In all IncHI2 plasmids except p48880_MCR, the mcr-9 gene was bounded by an IS903B element (upstream) and an ORF (downstream), encoding a cupin fold metalloprotein, followed by IS26. However, in plasmid p48880_MCR, carrying the mcr-9.2 allele, an IS1R insertion sequence was found downstream of the mcr-9.2 gene. In all isolates, qseC and qseB regulatory genes were not found in association with the mcr-9 gene. Based on previous studies (21,22), in the presence of subinhibitory concentrations of colistin, qseC and qseB genes can induce the expression of the mcr-9 gene, leading to increased MICs. The data mentioned above may explain the susceptibility to colistin. The mcr-9 genes from all clinical strains were transferred to the azide-resistant laboratory strain E. coli A15 by conjugation, confirming the ability of IncHI2 plasmids to further disseminate mcr-9 in other clones or species.
Moreover, at least one multidrug resistance (MDR) region was identified in each mcr-9carrying IncHI2 plasmids. Differences in MDR regions were observed among mcr-9-carrying IncHI2 plasmids. Interestingly, the carbapenemase-encoding gene bla VIM-4 was found in the MDR regions of IncHI2 plasmids p48880_MCR_VIM and p51929_MCR_VIM, as previously described for plasmid pME-1a, which was characterized from an Enterobacter hormaechei isolate harboring bla VIM-4 and mcr-9, recovered from a pediatric patient in a U.S. hospital (21). In plasmid p48880_MCR_VIM, the bla VIM-4 gene was part of the class 1 integron In416, comprising the bla VIM-4 , aacA7, dfrA1, DaadA1, and smr2 cassettes, while the class 1 integron In1174, which includes an array of aacA4 and bla VIM-4 gene cassettes, was identified in plasmid p51929_MCR_VIM.
On the other hand, in isolates ENCL48212, ENCL48946, and ENCL49790, the bla VIM-1 gene was localized on plasmids (p48212_VIM, p48946_VIM, and p59790_VIM) of approximately 55 kb. The bla VIM-1 -carrying plasmids shared extensive similarity with plasmid p16005813B from Leclercia adecarboxylata strain 16005813 (72% coverage and 99% identity; GenBank accession no. MK036884) (Fig. 2), encoding IMP-8 carbapenemase. The bla VIM-1 -carrying plasmids could not be typed by the PCR-based replicon typing (PBRT) method (23). However, in the plasmid sequences, repA-like sequences of 612 bp exhibiting 99% identity with the repA gene of pKPC-CAV1193 (GenBank accession no. CP013325) from Klebsiella pneumoniae strain CAV1193 were identified. Additionally, a complete transfer region was not found in pKPC-CAV1193-like plasmids, explaining the failure of bla VIM-1 -positive plasmids to be transferred, by conjugation experiments, to the azide-resistant laboratory strain E. coli A15, which was used as a recipient.
The MDR regions of VIM-1-encoding plasmids included the class 1 integron In110, whose variable region comprised bla VIM-1 , aacA4, and aadA1 (24). In all three VIM-1encoding plasmids, In110 was localized in a Tn1696-like sequence (nucleotides 13689 to 30156 in p48212_VIM). The IRi of In110 was located between the resI and resII sites of the Tn1696 module in precisely the same position as In4 in Tn1696. The 39 conserved segment of the integron was bounded with a Tn1696 fragment, consisting of IS6100, the resI site, and the mer operon. The Tn1696-like transposons were flanked by IRtnp and IRmer of Tn1696, with IRtnp being disrupted by IS4321 while IRmer remained intact. Target site duplications of 6 bp (CAGCAG) were identified at the boundaries of IRs of the Tn1696-like sequence, indicating its transposition within pKPC-CAV1193-like plasmids. Interestingly, resistance islands composed of the class 1 integron In110 associated with a Tn1696-like sequence have been previously identified in plasmids pKpn-431cz and pLec-476cz, characterized from VIM-1-producing Enterobacterales isolates of Czech origin (25). Additionally, in p48212_VIM, p48946_VIM, and p59790_VIM plasmids, the resistance genes bla TEM-1 , as part of the Tn3 transposon, and qnrS1 were found.
In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9positive bacteria of clinical origin identified in the Czech Republic (Fig. S1). Previous reports from the Czech Republic described the emergence of the mcr-1.1 allele in Enterobacterales recovered from retail meat and the mcr-4.3 allele in an Acinetobacter baumannii strain isolated from a clinical sample (26,27). Despite the fact that all 5 mcr-9carrying isolates were colistin susceptible, the identification of these isolates highlights the risk for the hidden spread of important resistance determinants such as plasmidmediated colistin resistance genes. Additionally, these 5 isolates cocarried the carbapenemase-encoding gene bla VIM and several other resistance genes that conferred resistance to aminoglycosides, tetracyclines, trimethoprim, chloramphenicol, sulfonamides, quinolones, and/or macrolides (Table 1), limiting therapeutic choices.
Based on epidemiological data, the 5 mcr-9-carrying isolates were recovered from three different hospitals, with two of them belonging to the same territory, suggesting three independent insertion events of MCR resistance mechanisms in Czech hospitals. In agreement with epidemiological data, the genomic data confirmed this suggestion. E. hormaechei isolates ENCL48212, ENCL48946, and ENCL49790 belonged to the same sequence type (ST106) and harbored similar IncHI2 plasmids carrying mcr-9.1 and similar pKPC-CAV1193-like plasmids carrying bla VIM-1 . On the other hand, the C. freundii CIFR51929 isolate included an IncHI2 plasmid cocarrying mcr-9.1 and bla VIM-4 resistance genes. In plasmid p51929_MCR_VIM, the bla VIM-4 gene was part of the class 1 integron In1174. Finally, the E. hormaechei isolate ENCL48880, which belonged to ST764, harbored the mcr-9.2 and bla VIM-4 genes localized on IncHI2 plasmid, p48880_MCR_VIM. In p48880_MCR_VIM, the mcr-9.2 allele was found in a slightly different genetic environment than the mcr-9.1 allele in p48212_MCR, p48946_MCR, p49790_MCR, and p51929_MCR_VIM. Unlike p51929_MCR_VIM, the bla VIM-4 gene was part of the class 1 integron In416 in p48880_MCR_VIM.
The association of the IncHI2 plasmid group with mcr-1 or mcr-9 genes has been frequently reported (21,28). However, the carriage of the bla VIM-1 gene on pKPC-CAV1193-like plasmids was described for the first time. Interestingly, MDR regions of the bla VIM-1 -carrying plasmids p48212_VIM, p48946_VIM, and p49790_VIM exhibited extensive similarity to the respective regions of plasmid pKpn-431cz (Fig. 2), previously described from Enterobacterales recovered from Czech hospitals (25). Thus, the acquisition of the bla VIM-1 -carrying MDR region from pKpn-431cz by a pKPC-CAV1193-like plasmid is a plausible hypothesis regarding the formation of p48212_VIM, p48946_VIM, and p49790_VIM plasmids. These findings highlight the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. FIG S1, PDF file, 0.2 MB.

ACKNOWLEDGMENTS
The study was supported by research project grant NU20J-05-00033, provided by the Czech Health Research Council, by the Charles University Research Fund PROGRES (project number Q39), and by project CZ.02.1.01/0.0/0.0/16_019/0000787 "Fighting Infectious Diseases," provided by the Ministry of Education Youth and Sports of the Czech Republic.
We have no conflicts to declare.