Complete Nucleotide Sequences of Two VIM-1-Encoding Plasmids from Klebsiella pneumoniae and Leclercia adecarboxylata Isolates of Czech Origin

Abstract

Two multidrug resistance (MDR) plasmids, carrying the VIM-1-encoding integron In110, were characterized. Plasmid pLec-476cz (311,758 bp), from a Leclercia adecarboxylata isolate, consisted of an IncHI1 backbone, a MDR region, and two accessory elements. Plasmid pKpn-431cz (142,876 bp), from a sequence type 323 (ST323) Klebsiella pneumoniae isolate, comprised IncFII Y -derived and pKPN3-like sequences and a mosaic region. A 40,400-bp sequence of pKpn-431cz was identical to the MDR region of pLec-476cz, indicating the en bloc acquisition of the VIM-1-encoding region from one plasmid by the other.KEYWORDS carbapenemases, metallo-␤-lactamases, IncHI1, IncFII Y , integrative conjugative elements V IM-producing Enterobacteriaceae have been observed since 2001 in Greece (1). For at least a decade, VIM producers were the main carbapenemase-producing Enterobacteriaceae (CPE) in Europe (2). In the Czech Republic, the first two cases of VIMproducing Enterobacteriaceae were identified in 2011. The first case was a sequence type 323 (ST323) Klebsiella pneumoniae (Kpn-431cz) isolate cultured in April 2011 from a bronchoalveolar lavage sample of a patient treated in a Czech hospital. The second case included a Leclercia adecarboxylata (Lec-476cz) isolate recovered (3) during a survey study focused on compliance with hand hygiene among the staff of a different Czech hospital in May 2011. Interestingly, the two isolates carried the VIM-1 carbapenemase-encoding integron In110 (bla VIM-1 -aacA4-aadA1) (4), localized on plasmids pKpn-431cz and pLec476cz. In the present study, we characterized the complete nucleotide sequences of pKpn-431cz and pLec-476cz in order to examine the nature of the genetic elements involved in the acquisition and spread of In110 in the Czech Republic.The bla VIM-1 -carrying plasmids were transferred from the clinical strains to rifampinresistant Escherichia coli A15 by conjugation in mixed broth cultures. Transconjugants were selected on MacConkey agar plates supplemented with rifampin (150 g/ml) and ampicillin (50 g/ml). Plasmid pKpn-431cz was transferred by conjugation at 37°C while pLec-476cz was capable of transferring at 30°C. Both bla VIM-1 -positive transconjugants exhibited similar resistance phenotypes (Table 1), showing resistance to piperacillin, piperacillin-tazobactam, and cephalosporins and decreased susceptibility to imipenem, while they remained susceptible to meropenem and ertapenem. Plasmid analysis revealed that the transconjugants harbored bla VIM-1 -positive plasmids of different sizes (ϳ150 kb [pKpn-431cz] and ϳ290 kb [pLec-476cz]) (5). The two plasmids were non-

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

“…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.…”

“…Target site duplications of 6 bp ( CAGCAG) were identified at the boundaries of IRs of the Tn 1696 -like sequence, indicating its transposition within pKPC-CAV1193-like plasmids. Interestingly, resistance islands composed of the class 1 integron In110 associated with a Tn 1696 -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 Tn 3 transposon, and qnrS1 were found.…”

Implication of different replicons in the spread of the VIM-1-encoding integron, In110, in Enterobacterales from Czech hospitals

“…Also, three (p52323cz_VIM, p53828cz_VIM and p60214cz_ VIM) out of four IncHI1 (pNDM-CIT) plasmids carried the In110. These plasmids were identical to VIM-1-encoding plasmid pLec-476cz (Supplementary Figure S2), which was previously characterized from a Leclercia adecarboxylata isolate (Papousek et al, 2017) recovered during a survey study focused on compliance with hand hygiene among the staff of a different Czech hospital in May 2011. On the other hand, the fourth IncHI1 (pNDM-CIT) plasmid (p56322_VIM), was carried by a ST674 C. freundii isolate.…”

“…In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported ( Papousek et al, 2017 ; Papagiannitsis et al, 2018 ), from 2011 till 2015. Interestingly, those isolates carried the VIM-1 carbapenemase-encoding integron In110 ( bla VIM-1 - aacA4 - aadA1 ; Lombardi et al, 2002 ).…”

First Report of Coexistence of Three Different MDR Plasmids, and That of Occurrence of IMP-Encoding Plasmid in Leclercia adecarboxylata

“…L. adecarboxylata is generally susceptible to cephalosporins, carbapenems, tetracyclines, aminoglycosides, quinolones, and chloramphenicol (Stock et al, 2004). Nevertheless, L. adecarboxylata isolates have evolved to acquire foreign antibiotic resistance genes, which encode extended-spectrum β-lactamases (ESBLs) SHV-12 (Mazzariol et al, 2003) and CTX-M-3 (Shin et al, 2012), and carbapenemases IMP-1 (GenBank accession number KJ531212), IMP-4 (Betteridge et al, 2013; Leung et al, 2013), KPC-2 (Geffen et al, 2013; Weingarten et al, 2018), NDM-1 (Sun et al, 2015; Hoyos-Mallecot et al, 2017; Riazzo et al, 2017) and VIM-1 (Papagiannitsis et al, 2013; Papousek et al, 2017); from these strains, one NDM-encoding plasmid pP10164-NDM (accession number KP900016) (Sun et al, 2015), one VIM-encoding pLec-476cz (accession number KY320277) (Papousek et al, 2017), and several KPC-encoding plasmids (Weingarten et al, 2018) have been fully sequenced.…”