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Infection, Genetics and Evolution

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Short communication

Plasmid carrying mcr-9 from an extensively drug-resistant NDM-1-producingKlebsiella quasipneumoniae subsp. quasipneumoniae clinical isolate

Diego Facconea,b,1, Florencia Martinoa,1, Ezequiel Albornoza, Sonia Gomeza,b, Alejandra Corsoa,Alejandro Petronia,⁎

a Servicio Antimicrobianos, Departamento de Bacteriología, Instituto Nacional de Enfermedades Infecciosas-ANLIS “Dr. Carlos G. Malbrán”, Av. Vélez Sarsfield 563(C1282AFF), Ciudad Autónoma de Buenos Aires, Argentinab Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290 (C1425FQB), Ciudad Autónoma de Buenos Aires, Argentina

A R T I C L E I N F O

Keywords:Klebsiella quasipneumoniaeColistinmcrIncHI2QuinoloneqnrB4

A B S T R A C T

The extensively drug-resistant NDM-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae M17277(Kqsq-M17277) was only susceptible to colistin and fosfomycin. Whole genome sequencing of Kqsq-M17277 wasperformed through Illumina (short reads) and Oxford Nanopore Technologies (long reads). Hybrid assembly ofshort and long reads rendered circular contigs of the Kqsq-M17277 chromosome and three plasmids of 477,340(p17277A_477), 138,998 (p17277C_138, which harbored blaNDM-1) and 123,307 bp (p17277B_123). In silicoanalysis showed that p17277A_477 was not previously described, belonged to the IncHI2 incompatibility groupand contained 51 complete or partial (≥300 bp) insertion sequences and Tn3 family transposons, which en-compassed, as a whole, 14% of the p17277A_477 sequence. In silico search for antimicrobial resistance genesrevealed that p17277A_477 harbored the recently described plasmid-encoded mcr-9 gene (colistin resistance),besides other resistance genes. This is the first worldwide report of a K. quasipneumoniae harboring mcr-9 andblaNDM-1 and the first report of mcr-9 in Latin America. This gene was embedded in a genetic structure, i.e.,IS903B-like/mcr-9/wbuC/IS26, that was found in 71% of the mcr-9-harboring sequences of the NCBI NucleotideCollection database. The qseC and qseB genes involved in mcr-9 expression were not found in Kqsq-M17277,which could explain its colistin susceptibility. Fifteen additional resistance genes were detected in p17277A_477.Ten of them [aac(6′)-IIc, aadA2, aph(3′)-Ia, blaDHA-1, dfrA12, ereA2, mphA, qnrB4, sul1 and tet(D)] were foundclustered within a 44,907-bp-long array of IS26-bounded resistance genes. The plasmid-mediated quinoloneresistance gene qnrB4 was located in a complex class 1 integron within this IS26-bounded resistance gene cluster.p17277A_477 was self-transferable by conjugation to Escherichia coli J53, azide-resistant. The Tra1 and Tra2conjugative transfer regions of the IncHI2 plasmid R478 were found by in silico search. p17277A_477 is acomplex plasmid that provides a large baggage of both resistance genes and genetic resources for plasmid re-arrangements and additional resistance gene acquisition.

Nine mobile colistin resistance (mcr) genes have been described(Carroll et al., 2019). These genes encode for phosphoethanolaminetransferases that modify the lipid A moiety of lipopolysaccharide de-creasing polymixin activity (Nang et al., 2019). These genes have beenmainly described in Enterobacteriaceae and more commonly in Escher-ichia coli recovered from animal, food, human and environment (Nanget al., 2019). mcr-9 was recently described in E. coli, Klebsiella pneu-moniae, Enterobacter spp. and Salmonella enterica serotype Typhimuriumclinical isolates (Carroll et al., 2019; Chavda et al., 2019; Kieffer et al.,2019; Wang et al., 2019; Yuan et al., 2019). mcr-9 encodes for aphosphoethanolamine transferase, which could confer colistin

resistance. However, this gene does not confer colistin resistance innormal conditions in an E. coli wild-type background and its expressionto reach a colistin-resistant phenotype seems to be dependent on bothcolistin induction and the presence of two genes, qseC and qseB, adja-cently located to mcr-9 (Carroll et al., 2019; Kieffer et al., 2019).

Recently, we described the NDM-1-producing K. quasipneumoniaesubsp. quasipneumoniae M17277 (Kqsq-M17277) isolate recovered froma rectal swab sample from a 4-year-old patient suffering erythrodermicpsoriasis (Martino et al., 2019). MICs were determined according to theCLSI guidelines (CLSI, 2019), using the agar dilution method for all theantibiotic tested except colistin and tigecycline, for which the broth

https://doi.org/10.1016/j.meegid.2020.104273Received 6 October 2019; Received in revised form 26 February 2020; Accepted 3 March 2020

⁎ Corresponding author.E-mail address: apetroni@anlis.gob.ar (A. Petroni).

1 D.F. and F.M. contributed equally to this work.

Infection, Genetics and Evolution 81 (2020) 104273

Available online 04 March 20201567-1348/ © 2020 Elsevier B.V. All rights reserved.

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microdilution method was used. Kqsq-M17277 showed an extensivelydrug-resistance profile (MICs, μg/ml), which included resistance toimipenem (16), meropenem (64), ceftazidime (> 256), cefotaxime(> 256), cefepime (256), aztreonam (256), ciprofloxacin (32), ami-kacin (> 64), gentamicin (> 64), tigecycline (16), minocycline (> 64),trimethoprim-sulfamethoxazole (> 2/38) and chloramphenicol(> 256). Kqsq-M17277 was only susceptible to fosfomycin (16) andcolistin (0.25).

Whole genome sequencing of Kqsq-M17277 was performed through

short reads (MiSeq, Illumina) and long reads (MinION, OxfordNanopore Technologies). Hybrid assembly of short and long reads wasdone with Unicycler v0.4.7 (Wick et al., 2017) and rendered circular(closed) contigs of the Kqsq-M17277 chromosome (5.16 Mb) and threeplasmids of 477,340 bp (p17277A_477), 138,998 bp [p17277C_138;formerly named as pKQN17277 (Martino et al., 2019)] and 123,307 bp(p17277B_123). p17277C_138 was previously described and harboredblaNDM-1 and rmtC among other resistance genes (Martino et al., 2019).

Analysis of p17277A_477 (average GC content of 48.7%) with

Fig. 1. BRIG comparison of p17277A_477 with the plasmids of the NCBI nt database with maximal similarity. The 10 complete plasmid sequences that showed thehighest total scores when p17277A_477 was queried against the NCBI nt database were compared with the sequence of this plasmid, used as the BRIG reference (redoutermost ring). The innermost ring shows nucleotide coordinates (base 1 is in the first codon of the repHIA gene). The arrows indicate deduced open reading framesand their orientations. The mcr-9 structure (see text) and the conjugative transfer regions, Tra1 and Tra2, are indicated by thick arched red lines. The partitioning Parmodules, Par1 and Par2, which split Tra2, are indicated by a filled arched black box. Only the transfer genes that bordered Tra1 and both portions of Tra2 are shown.Included plasmids, from inner to outer rings and from top to bottom in the legend, are (accession number): pGMI14-002_1 (CP028197.1); pGW1 (CP028975.1);pSA20094620.1 (CP030186.1); pIMP26 (MH399264.1); pAUSMDU8141-1 (CP022696.1); pIMP-4-EC62 (MH829594.1); unnamed1 (CP020529.1); pIMP4-SEM1(KX810825.1); pEC-IMPQ (EU855788.1); and pEC-IMP (EU855787.1). (For interpretation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

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ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/, accessed 27June 2019) revealed the presence of a gene that was 100% identical tothe recently described mcr-9 (NCBI accession no. NG_064792.1), be-sides other resistance genes (see below). This is the first report of mcr-9in Latin America. PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/, accessed 10 August 2019) characterizedp17277A_477 as an IncHI2 plasmid, as found for the previously re-ported mcr-9-harboring plasmids (Chavda et al., 2019; Kieffer et al.,2019; Yuan et al., 2019).

BLAST comparisons against the NCBI Nucleotide Collection (nt da-tabase; accessed 14 August 2019) revealed that p17277A_477 was notpreviously described given that the 10 complete plasmid sequences,with the highest total scores (99.4–99.8% overall identity withp17277A_477), showed only 58–66% coverage of the p17277A_477sequence. Moreover, a comparison through BLAST Ring ImageGenerator (BRIG) of those 10 highest-scoring plasmid NCBI entries withp17277A_477 showed that the latter had extensive regions that wereabsent in the former (Fig. 1). In addition, besides the mcr-9 immediategenetic surroundings, all of them shared an IncHI2 backbone thatmostly included regions essential for plasmid replication, maintenanceand conjugative transference (Fig. 1).

Analysis with ISFinder (https://isfinder.biotoul.fr/, accessed 10August 2019) showed that p17277A_477 contained 51 complete orpartial (≥300 bp) sequences of 25 different insertion sequences (IS)and Tn3 family transposons (Tn), several of which were found inmultiple copies (e.g., IS26 was present in 12 complete copies). These 51IS/Tn belonged to 16 IS families or subgroups within a given family andencompassed, as a whole, 14% (65,059 bp) of the p17277A_477 se-quence. In this plasmid, mcr-9 was embedded in a genetic structurerecently described, i.e., IS903B-like/mcr-9/wbuC/IS26 (Kieffer et al.,2019) (Fig. 2). Two reported variants of this structure harbored the qseCand qseB genes downstream of wbuC, which were proposed as a two-component system involved in mcr-9 expression (Kieffer et al., 2019).The absence of the qseC/qseB system in p17277A_477, the chromosomeand the other plasmids of Kqsq-M17277 could explain its colistin sus-ceptibility.

BLAST search for the IS903B-like/mcr-9/wbuC/IS26 structure in thent database (accessed 14 August 2019) showed that it was the mostcommon structure, since it was found, with 100% of query coverageand 99.7–100% identity, in 40 of the 56 (71%) hits that contained acomplete mcr-9. These 40 hits corresponded to complete sequences ofplasmids (n = 35) and chromosomes (n = 2), or partial plasmid se-quences (n = 3, with IS903B-like/mcr-9/wbuC/IS26-flanking regions≥94,809 bp) of Enterobacteriaceae isolates, mostly Enterobacter spp.,Klebsiella spp. and Salmonella spp.. These findings might represent athreat to public health due to the broad and silent dissemination of mcr-9 among Enterobacteriaceae isolates.

ResFinder also detected 15 additional resistance genes in

IS1B-likeIS903B-like IS26 IS26mcr-9

wbuC306,809 311,092 314,580

mcr-9 structure

catA2

orf1

Salmonella TyphimuriumR9_3274_R1, node 632,661 bp

99.96%identity

Fig. 2. Genetic environment of mcr-9. A 7772-bp region of p17277A_477 thatharbored mcr-9 is depicted at the top (relevant positions in the p17277A_477complete sequence are indicated by vertical arrows). IS are represented by greyboxes with white triangles that indicate the tnpA transcription sense. IS903B-like and IS1B-like showed 97.1% and 98.6% identity with the NCBI accessionno. X02527 and X17345, respectively. Genes/open reading frames (orf) aredisplayed by arrowed boxes (orf1, unknown function). The recently described4284 bp-long mcr-9 structure (Kieffer et al., 2019) is indicated. The mcr-9-harboring contig of the assembly of S. Typhimurium R9_3274_R1 (node 63,accession no. NZ_NAAN01000063.1), which was the first reported mcr-9-con-taining isolate (Carroll et al., 2019), is shown at the bottom and its locationrelative to p17277A_477 is indicated by dashed lines. The node 63 differed fromits corresponding p17277A_477 sequence in only one nucleotide.

IS1247-likeelement

ISCR1

tet(D)

pspoperon

Class 1 integron

aadA2

dfrA12

mphA

3’CS 5’CS

aph(3')-Ia

3’CS2 5’CS

blaDHA-1

sapoperon

qnrB4

3’CS1

ereA2

aac(6')-IIc

Complex class 1 integron

sul1 sul1 sul1

ampR

A B C TSDIS6100 DCBAF

oriIS

99.86-99.94% identityCP028537, CP040696, KY978628,LT994835, MH399264

A

B

Fig. 3. Resistance cluster of p17277A_477. A) The IS26-bounded resistance gene cluster of p17277A_477 (positions 390,913-435,819; 44,907 bp) is depicted. Forsimplicity, only relevant genes are shown, as arrowed grey (resistance genes) and white (other genes) boxes. All the IS found in this region are represented by black(IS26) and grey (other IS) boxes with white triangles that indicate the tnpA transcription sense. The genes commonly found in the surroundings of qnrB genes areshown (operon psp: genes pspD to eF encode for peripheral inner membrane phage-shock proteins; operon sap: genes sapA to C encode for antimicrobial peptide ABCtransporter periplasmic binding proteins). The oriIS of ISCR1 is indicated by an empty diamond. The transposition of the IS1247-like-based element, which containedthree additional orfs and split ereA2, was evidenced by the presence of a 4-bp target site duplication (TSD, black ovals). B) Comparison of the IS26-bounded resistancegene cluster with the NCBI nt database. The accession no. of the five entries that showed the longest hits with the resistance cluster of p17277A_477 are indicated atthe left. The sequences of these five hits (same length, 28,538 bp) are represented by the thick horizontal line and the range of similarity between these sequences andtheir counterpart in the cluster of IS26-bounded resistance genes of p17277A_477 is shown above the line.

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p17277A_477: aph(3″)-Ib, aph(6)-Id, blaSHV-12, catA2, sul2, aac(6′)-IIc,aadA2, aph(3′)-Ia, blaDHA-1, dfrA12, ereA2, mphA, qnrB4, sul1 and tet(D).The last 10 genes were found clustered within an array of IS26-boundedresistance genes located 85,641 bp downstream of mcr-9 (Fig. 3A). Itwas reported that the arrays of IS26-bounded resistance genes resultfrom IS26 transpositions, which constitute an IS26-mediated me-chanism of resistance gene clustering different from and independent ofintegron- or ISCR-mediated rearrangements (Harmer and Hall, 2016;He et al., 2015). In addition, we found herein that the IS26-boundedresistance gene cluster included a class 1 integron and a complex class 1integron that harbored qnrB4 and blaDHA-1 in its variable region 2

(Fig. 3A). Both integrons provide to the IS26-bounded resistance genecluster three additional loci (i.e., two attI and ISCR1) for resistance geneacquisition. In mcr-1-harboring IncHI2 plasmids, multi-drug resistance(MDR) regions that did not include the mcr-1 gene, as occurred inp17277A_477, were previously described (Li et al., 2017). A BRIGcomparison of IncHI2 plasmids that contained different mcr genesshowed that the complete IS26-bounded resistance gene cluster ofp17277A_477 was not found in any of the other mcr-harboring IncHI2plasmids (Fig. 4). Moreover, the search for this resistance gene clusterin the nt database (accessed 1 September 2019) revealed five entrieswith the longest hits (28,538 bp), which only comprised the IS26-

Fig. 4. BRIG comparison of mcr-harboring IncHI2 plasmids. IncHI2 plasmids that harbored mcr genes were selected from the NCBI nt database using BLAST andcompared with the sequence of p17277A_477, used as the BRIG reference (red outermost ring). The innermost ring shows nucleotide coordinates (base 1 is in the firstcodon of the repHIA gene). The arrows indicate deduced open reading frames and their orientations. All the antimicrobial resistance genes and the two intI1 genesdetected in p17277A_477 are shown. The IS26-bounded resistance gene cluster (MDR region) and the conjugative transfer regions, Tra1 and Tra2, are indicated bythick arched red lines. The partitioning Par modules, Par1 and Par2, which split Tra2, are indicated by a filled arched black box. Only the transfer genes that borderedTra1 and both portions of Tra2 are shown. Included plasmids, from inner to outer rings and from top to bottom in the legend, are (accession number): pOSUKPC4(CP024910); pK29 (EF382672); pD610-HI2 (MG288680); pSA20094620.1 (CP030186); pECJS-B60-267 (KX254341); pECJS-59-244 (KX084394); p17S-208(MH077952); pWJ1 (KY924928). The mcr gene harbored by each plasmid is indicated at the right of the legend. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)

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bounded complex class 1 integron (Fig. 3B). These findings indicatethat the IS26-bounded resistance gene cluster of p17277A_477 was notpreviously described and also suggest that transpositions of IS26-basedelements could be involved in the dissemination of the qnrB4-harboringcomplex class 1 integron (Harmer and Hall, 2016; He et al., 2015).

p17277A_477 was successfully transferred to azide-resistant E. coliJ53 by conjugation at 30 °C, using sodium azide (200 μg/ml) pluschloramphenicol (64 μg/ml) and ciprofloxacin (0.06 μg/ml) for trans-conjugant selection. The presence of p17277A_477 in a transconjugantwas corroborated by S1 nuclease assay (Martino et al., 2019), PCR plusDNA sequencing of mcr-9 and analysis of antimicrobial susceptibility.The search for the conjugative machinery was performed with or-iTfinder (http://202.120.12.134/oriTfinder/oriTfinder.html) (Li et al.,2018), which revealed that the components of a predicted conjugativesystem were clustered in two distantly located regions (positions243,350–260,643 and 2025–36,540 in p17277A_477), as reported forthe well-conserved conjugative system of IncHI plasmids (Alvarez,2018) (Fig. 4). These regions were nearly identical (99.94% and99.97% identity) to the Tra1 and Tra2 regions, respectively, that makeup the conjugative system of the IncHI2 plasmid R478 (Gilmour et al.,2004). Interestingly, in p17277A_477, we found that parR, located inone of the two functionally partitioning modules inserted between thetransfer genes of Tra2 (Par2), was split by an IS903B-like element(97.4% identical to IS903, accession no. X02527) and its characteristic9-bp target site duplication. In the mcr-9-harboring IncHI2 plasmidpME-1a, an IS903B-like insertion was also found in the Par2 region(Chavda et al., 2019) but in a different location regardingp17277A_477. Though Par1 is the major determinant and may ac-complish partitioning by itself (Lawley and Taylor, 2003), the loss ofparR in a plasmid as large as p17277A_477 could increase the chancefor elimination after segregation (Gilmour et al., 2004).

This is the first report of a K. quasipneumoniae harboring mcr-9 andblaNDM-1. Despite its recent definition as a species, there is convincingevidence that K. quasipneumoniae is able to sustain in hospitalized pa-tients, to persist in the hospital environment for a long time as well as todisseminate between patients and sink drains (Mathers et al., 2019).Moreover, this species has been shown to take up plasmids from otherenterobacteria and harbor several resistance plasmids (Mathers et al.,2019; Yang et al., 2019). In addition to such epidemiologically relevantfeatures of K. quasipneumoniae, the blaNDM-1/rmtC-harboring plasmidp17277C_138 (Martino et al., 2019) and the plasmid p17277A_477described here provide Kqsq-M17277 with additional mechanisms ofdrug resistance, including fluoroquinolones, that may be useful underthe broad selective pressure of the hospital setting. Most importantly,p17277A_477 provides a large baggage of genetic resources for plasmidrearrangements and resistance gene acquisition.

Accession number. Sequence of p17277A_477 has been assignedGenBank accession number CP043927.

Declaration of competing interest

None.

Acknowledgments

This work was supported by the Agencia Nacional de PromociónCientífica y Tecnológica (ANPCYT) (grant number PICT 2015-01728 to

A.P.) and by the regular federal budget of the National Ministry ofHealth of Argentina. F.M. was supported by a fellowship from ANPCYT.

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