Enterobacteriaceae producing OXA-48-like carbapenemases in Poland, 2013–January 2017

Enterobacteriaceae producing OXA-48-like carbapenemases in Poland, 2013–January 2017 Abstract Objectives To analyse OXA-48 (OXA-48/181)-type carbapenemase-producing Enterobacteriaceae reported in Poland from 2013 until January 2017. Methods Bacterial isolates were typed by PFGE and MLST. Genes coding for OXA-48/181 types and other β-lactamases were amplified and sequenced. Mobile elements with blaOXA-48/181-like genes were PCR mapped. blaOXA-48/181-carrying plasmids were characterized by nuclease S1-hybridization profiling, transfer assays and PCR-based replicon typing, while the chromosomal location of the genes was confirmed by the I-CeuI analysis. Results Fifty-four isolates from 52 patients in 20 hospitals (14 cities) were included, in 14 cases having probable foreign origins indicated. The organisms were genetically diverse and represented numerous pandemic clones, including Klebsiella pneumoniae ST395 (n = 23), ST11, ST15 and ST101, Escherichia coli ST38, ST410 and ST648, and Enterobacter cloacae complex ST78. These produced OXA-48 (n = 49), OXA-181 (n = 4) or OXA-232 (n = 1). One of five K. pneumoniae ST395 pulsotypes caused a multicentre outbreak with 18 cases, which significantly contributed to the total number of patients. Depending on the variant, the blaOXA-48/181-like genes were parts of the Tn1999-, Tn2013- or Tn2016-like transposons, with blaOXA-48 found in an ISEcp1-associated module (Tn2016-like) for the first time. Three genotypes, including E. coli ST38, had chromosomal blaOXA-48 genes, while others carried transmissible IncL (∼60 kb; blaOXA-48; n = 30), IncM (∼80–95 kb; blaOXA-48; n = 4), IncX3 (∼50 kb; blaOXA-181; n = 4) or non-typeable (∼90–160 kb; blaOXA-48 or blaOXA-232) plasmids. Conclusions Even though OXA-48/181 producers seem to occur infrequently in Poland, their epidemiology has been marked by various phenomena, namely multiple imports, several limited transmissions plus one larger clonal outbreak, and possible plasmid transfers. Introduction Carbapenemase-producing Enterobacteriaceae (CPE), including those with OXA-48-type enzymes, contribute greatly to the global crisis in antimicrobial chemotherapy.1 OXA-48 was identified first in 2001 in Turkey,2 and soon most of the south-east Mediterranean region became endemic for producers of this enzyme.1,2 Another large reservoir is the Indian subcontinent where organisms with the related OXA-181 have spread.1,3 Widespread travel and migration are at the origin of the increasing presence of OXA-48/181 types in Europe.1,2,4 The enzymes confer resistance to penicillins (with temocillin) and penicillin/inhibitor combinations, and low-level resistance to carbapenems, augmented by, e.g. permeability defects. The common resistance to oxyimino-β-lactams results from co-expression of ESBLs and/or AmpC-type cephalosporinases.2,blaOXA-48/181-like genes originate from Shewanella spp. and disseminate with mobile elements, such as blaOXA-48-carrying Tn1999-like transposons and IncL-type plasmids, or a blaOXA-181-carrying ISEcp1-associated transposon, Tn2013, and IncX3 plasmids.2,3,5 Clonal spread of some enterobacterial lineages, such as Klebsiella pneumoniae ST11, ST101, ST395 or Escherichia coli ST38, has also been significant in OXA-48/181 proliferation.4 The first OXA-48 producers reported in Poland in 2012 were Enterobacter cloacae ST89 from two patients from Białystok; one was described by Majewski et al.,6 whereas the second one, confirmed by the National Reference Centre for Susceptibility Testing (NRCST), was not preserved. In this study, all unique OXA-48/181-like isolates identified by the NRCST surveillance from 2013 to January 2017 were analysed. Methods Clinical isolates Fifty-four non-duplicate isolates (37 K. pneumoniae, 14 E. coli, and one of each Citrobacter freundii, Enterobacter aerogenes and E. cloacae complex) were collected from 52 patients in 20 hospitals in 14 Polish cities (Table 1). These isolates were submitted to the NRCST owing to carbapenem non-susceptibility and resistance to temocillin and/or positive Carba NP test (http://eucast.org). Species were re-identified with Vitek2 (bioMérieux, Marcy l’Etoile, France), and carbapenemase-associated phenotypes were analysed as described.7,8,blaOXA-48/181-like genes were detected by PCR and sequenced.7 Table 1. OXA-48/181-producing isolates: species, basic clinical and epidemiological data, clones, pulsotypes, transposons and plasmids with blaOXA-48/181-like genes, and other acquired β-lactamases Species and isolatea  Year of isolation  Hospital  Specimenb  Origin, including hospitalization history (H)c  STd  PFGE types  blaOXA-48-like genes  Types of transposons with blaOXA-48/181-like genes  Location of blaOXA-48/181e  ESBLs, AmpCs and other acquired β-lactamases  K. pneumoniae 2844/13  2013  Poznań  urine  Russia (H)  ST395  KpnA  blaOXA-48  Tn1999.1  ∼260 kbj  TEM-1  K. pneumoniae 1786/14  2014  Cracow I  BAL  Georgia  ST395  KpnB  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 655/15  2015  Cracow II  wound  Ukraine  ST395  KpnH  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4969/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 5083/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 3606/16  2016  Cracow III  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 3676/16  2016  Cracow I  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4378/16  2016  Cracow I  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5063/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5404/16  2016  Cracow III  urine  —  ST395  KpnN2  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5485/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5486/16  2016  Cracow III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6151/16  2016  Cracow I  blood  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6325/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6384/16  2016  Warsaw III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6713/16  2016  Cracow III  BAL  —  ST395  KpnN3  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6965/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7243/16  2016  Cracow III  wound  —  ST395  KpnN4  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7426/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 92/17  2017  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 94/17  2017  Cracow III  BAL  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 165/17  2017  Bochnia  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 283/17  2017  Cracow III  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4166/14  2014  Warsaw III  urine  Romania (H)g  ST15  KpnC  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 2673/15  2015  Ełk  urine  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼90; ∼105; NT  CTX-M-15; TEM-1  K. pneumoniae 3695/16  2016  Białystok  stool  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼160 kb; NT  CTX-M-15; TEM-1  K. pneumoniae 4245/14  2014  Bydgoszcz  BAL  Turkey  ST101  KpnD  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 1202/15f  2015  Warsaw IV  rectal swab  Tunisia (H)  ST101  KpnF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-3; TEM-1  K. pneumoniae 3651/16  2016  Siemianowice Śl.  wound  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4492/16  2016  Siemianowice Śl.  BAL  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4396/16  2016  Białystok  rectal swab  —  ST11  KpnP  blaOXA-48  Tn2016-like  ∼90 kbj  CTX-M-15; DHA-1; TEM-1  K. pneumoniae 2805/16  2016  Grodzisk Maz.  urine  —  ST13  KpnL  blaOXA-48  Tn1999.2  ∼60 kb; IncL  –  K. pneumoniae 1504/16  2016  Cracow I  urine  —  ST14  KpnK  blaOXA-48  Tn1999.1  ∼95 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 432/15  2015  Cracow II  wound  Ukraine  ST23  KpnG  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-55; TEM-1  K. pneumoniae 3086/16  2016  Warsaw V  throat swab  —  ST39  KpnM  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5774/16  2016  Szczecin  rectal swab  Oman (H)  ST231  KpnQ  blaOXA-232  ΔTn2013-like  ∼110 kb; NT  CTX-M-15; SHV; TEM-1  K. pneumoniae 4999/14  2014  Siemianowice Śl.  rectal swab  Egypth,i  ST336  KpnE  blaOXA-48  Tn1999.2  chr  CTX-M-15; TEM-1  E. coli 5240/13  2013  Warsaw II  stool  Egypt  ST38  EcoB  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 1414/14  2014  Słupsk  vaginal swab  Egypti  ST38  EcoC  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2365/14  2014  Warsaw III  stool  —  ST38  EcoE  blaOXA-48  Tn1999.2  chr  CTX-M-24; TEM-1  E. coli 4616/14  2014  Siemianowice Śl.  wound  Egypt  ST38  EcoG  blaOXA-48  Tn1999.2  chr  TEM-1  E. coli 4674/14  2014  Warsaw III  stool  —  ST38*  EcoK  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 972/15  2015  Sosnowiec  urine  —  ST38  EcoH  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2636/15  2015  Grodzisk Maz.  rectal swab  —  ST38  EcoI  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 1524/16  2016  Warsaw III  rectal swab  —  ST38  EcoJ  blaOXA-48  Tn1999.1  chr  CTX-M-15; TEM-1  E. coli 1987/14  2014  Kielce  wound  —  ST410  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 1988/14  2014  Kielce  BAL  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 2251/14  2014  Kielce  rectal swab  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 4008/13  2013  Warsaw I  stool  Cambodia (H)  ST648  EcoA  blaOXA-48  —  chr  CTX-M-15; TEM-1  E. coli 1704/16  2016  Warsaw I  stool  multiple travels  ST648  EcoL  blaOXA-48  —  chr  CTX-M-15  E. coli 4157/14  2014  Siemianowice Śl.  wound  Egypth,i  ST744  EcoF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  C. freundii 5976/16  2016  Kielce  urine  —  ST124  —  blaOXA-181  Tn2013  ∼50 kb; IncX3  —  E. aerogenes 4188/14  2014  Warsaw III  blood  Romania (H)g  NA  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  E. cloacae 150/17  2017  Biała Podlaska  rectal swab  —  ST78  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  Species and isolatea  Year of isolation  Hospital  Specimenb  Origin, including hospitalization history (H)c  STd  PFGE types  blaOXA-48-like genes  Types of transposons with blaOXA-48/181-like genes  Location of blaOXA-48/181e  ESBLs, AmpCs and other acquired β-lactamases  K. pneumoniae 2844/13  2013  Poznań  urine  Russia (H)  ST395  KpnA  blaOXA-48  Tn1999.1  ∼260 kbj  TEM-1  K. pneumoniae 1786/14  2014  Cracow I  BAL  Georgia  ST395  KpnB  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 655/15  2015  Cracow II  wound  Ukraine  ST395  KpnH  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4969/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 5083/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 3606/16  2016  Cracow III  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 3676/16  2016  Cracow I  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4378/16  2016  Cracow I  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5063/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5404/16  2016  Cracow III  urine  —  ST395  KpnN2  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5485/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5486/16  2016  Cracow III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6151/16  2016  Cracow I  blood  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6325/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6384/16  2016  Warsaw III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6713/16  2016  Cracow III  BAL  —  ST395  KpnN3  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6965/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7243/16  2016  Cracow III  wound  —  ST395  KpnN4  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7426/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 92/17  2017  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 94/17  2017  Cracow III  BAL  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 165/17  2017  Bochnia  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 283/17  2017  Cracow III  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4166/14  2014  Warsaw III  urine  Romania (H)g  ST15  KpnC  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 2673/15  2015  Ełk  urine  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼90; ∼105; NT  CTX-M-15; TEM-1  K. pneumoniae 3695/16  2016  Białystok  stool  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼160 kb; NT  CTX-M-15; TEM-1  K. pneumoniae 4245/14  2014  Bydgoszcz  BAL  Turkey  ST101  KpnD  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 1202/15f  2015  Warsaw IV  rectal swab  Tunisia (H)  ST101  KpnF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-3; TEM-1  K. pneumoniae 3651/16  2016  Siemianowice Śl.  wound  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4492/16  2016  Siemianowice Śl.  BAL  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4396/16  2016  Białystok  rectal swab  —  ST11  KpnP  blaOXA-48  Tn2016-like  ∼90 kbj  CTX-M-15; DHA-1; TEM-1  K. pneumoniae 2805/16  2016  Grodzisk Maz.  urine  —  ST13  KpnL  blaOXA-48  Tn1999.2  ∼60 kb; IncL  –  K. pneumoniae 1504/16  2016  Cracow I  urine  —  ST14  KpnK  blaOXA-48  Tn1999.1  ∼95 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 432/15  2015  Cracow II  wound  Ukraine  ST23  KpnG  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-55; TEM-1  K. pneumoniae 3086/16  2016  Warsaw V  throat swab  —  ST39  KpnM  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5774/16  2016  Szczecin  rectal swab  Oman (H)  ST231  KpnQ  blaOXA-232  ΔTn2013-like  ∼110 kb; NT  CTX-M-15; SHV; TEM-1  K. pneumoniae 4999/14  2014  Siemianowice Śl.  rectal swab  Egypth,i  ST336  KpnE  blaOXA-48  Tn1999.2  chr  CTX-M-15; TEM-1  E. coli 5240/13  2013  Warsaw II  stool  Egypt  ST38  EcoB  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 1414/14  2014  Słupsk  vaginal swab  Egypti  ST38  EcoC  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2365/14  2014  Warsaw III  stool  —  ST38  EcoE  blaOXA-48  Tn1999.2  chr  CTX-M-24; TEM-1  E. coli 4616/14  2014  Siemianowice Śl.  wound  Egypt  ST38  EcoG  blaOXA-48  Tn1999.2  chr  TEM-1  E. coli 4674/14  2014  Warsaw III  stool  —  ST38*  EcoK  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 972/15  2015  Sosnowiec  urine  —  ST38  EcoH  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2636/15  2015  Grodzisk Maz.  rectal swab  —  ST38  EcoI  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 1524/16  2016  Warsaw III  rectal swab  —  ST38  EcoJ  blaOXA-48  Tn1999.1  chr  CTX-M-15; TEM-1  E. coli 1987/14  2014  Kielce  wound  —  ST410  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 1988/14  2014  Kielce  BAL  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 2251/14  2014  Kielce  rectal swab  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 4008/13  2013  Warsaw I  stool  Cambodia (H)  ST648  EcoA  blaOXA-48  —  chr  CTX-M-15; TEM-1  E. coli 1704/16  2016  Warsaw I  stool  multiple travels  ST648  EcoL  blaOXA-48  —  chr  CTX-M-15  E. coli 4157/14  2014  Siemianowice Śl.  wound  Egypth,i  ST744  EcoF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  C. freundii 5976/16  2016  Kielce  urine  —  ST124  —  blaOXA-181  Tn2013  ∼50 kb; IncX3  —  E. aerogenes 4188/14  2014  Warsaw III  blood  Romania (H)g  NA  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  E. cloacae 150/17  2017  Biała Podlaska  rectal swab  —  ST78  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  a Isolates are ordered according to the species, ST and pulsotype; STs are ordered according to numbers of isolates classified; in clusters of the same ST and pulsotype the isolates are ordered chronologically. b Rectal (and throat) swabs and stool samples were collected during microbiological screening at admission or during regular CPE surveillance procedures in some centres. c Documented stay, including hospitalization (H) for a 1 year period before the OXA-48/181 CPE identification. d ST, sequence type; a new ST is marked in bold; NA, not applicable; asterisks indicate the STs that were adopted from fully analysed isolates of the same pulsotypes. e Bold font refers to blaOXA-48/181 plasmid replicon types detected in E. coli recombinants obtained for isolates representing different genotypes (pulsotype plus blaOXA-48/181 plasmid size); italics represent replicons confirmed by PCR for other clinical isolates of the individual genotypes; chr, chromosomal location; NT, not-typeable. f Sequence of the blaOXA-48 gene, type of transposon and ST of the isolate were reported previously.7 g Isolates from the same patient. h Isolates from the same patient. i Travel of a family member. j These plasmids could not be transferred to E. coli despite repeated mating and electroporation attempts. Molecular analysis E. coli and K. pneumoniae isolates were typed by PFGE,8 and along with C. freundii and E. cloacae complex by MLST (http://pubmlst.org/cfreundii/, http://pubmlst.org/ecloacae, http://mlst.warwick.ac.uk/mlst/dbs/Ecoli and http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). The location of the blaOXA-48/181-like genes in transposons was checked by PCR mapping and confirmed by sequencing for selected isolates.3,9,10 Identification of blaOXA-48/181-carrying plasmids was performed by the nuclease S1 analysis (TaKaRa, Otsu, Japan)8 and hybridization, using the ECL Direct Nucleic Acid Labelling System (GE Healthcare, Little Chalfont, UK). Twenty-three isolates of all species/pulsotypes and blaOXA-48/181 plasmid sizes were subjected to plasmid transfer experiments with E. coli A15RifR (mating) or E. coli TOP10 (Invitrogen, Carlsbad, CA, USA; electroporation), respectively.8 Recombinants were selected with 32 mg/L temocillin, plus 128 mg/L rifampicin in case of transconjugants. Plasmid DNAs from the recombinants were used in the PCR-based replicon typing (PBRT),11–13 and the replicons detected were subsequently checked in all isolates of individual genotypes. Chromosomal location of blaOXA-48 genes was confirmed by the I-CeuI analysis.8 Other selected β-lactamase genes (blaKPC, blaIMP, blaNDM, blaVIM, blaCTX-M, blaSHV-5/-12, blaTEM, blaCMY-2 and blaDHA types) were identified in clinical isolates by PCRs and sequencing.8 Susceptibility testing Susceptibility of the isolates and E. coli recombinants was tested by broth microdilution or agar dilution for fosfomycin, as recommended by EUCAST (http://eucast.org). Results Clinical and epidemiological data The NRCST runs the CPE surveillance in Poland, collecting suspected isolates with basic clinical and epidemiological data. The 54 study isolates represented all 52 OXA-48/181-type cases confirmed from 2013 to January 2017, from hospitals in 11/16 major regions (Table 1). In 14 cases there was information on the patient’s arrival from or stay in another country prior to admission (a family member in two cases), sometimes with hospitalization. The history of one patient, a victim of terrorism in Tunisia in 2015, was published previously (K. pneumoniae 1202/15).7 Thirty-four patients developed infections, while 18 patients were colonized only. Clonality data The 37 K. pneumoniae isolates were grouped into 17 pulsotypes and 11 STs, of which ST395 prevailed (n = 23; six hospitals) and varied with five pulsotypes (Table 1). A single pulsotype (ST395/KpnN) comprised 18 isolates from four hospitals, mainly in the Cracow area, including one centre with 13 cases. Only three other K. pneumoniae pulsotypes of various STs had two isolates each. The 14 E. coli isolates were of 12 pulsotypes and four STs. E. coli ST38 was prevalent (n = 8; six hospitals) but each isolate represented another pulsotype. E. coli ST410 comprised three homogeneous isolates from a hospital in Kielce. blaOXA-48/181-like genes and their genetic context OXA-48/181 types were the only carbapenemases in the study isolates. blaOXA-48 was confirmed in 49 isolates, while blaOXA-181 and blaOXA-232 were confirmed in four and one isolates, respectively (Table 1). The blaOXA-48 genes were located mainly in Tn1999.1 (GenBank accession number AY236073) (n = 29) and Tn1999.2 (JN714122) (n = 15) transposons.2 However, in three K. pneumoniae isolates (ST15/KpnI and ST11) the ISEcp1 element was placed 46 bp upstream of blaOXA-48, a configuration described for blaOXA-204 as Tn2016 (JQ809466).14 The blaOXA-181 and blaOXA-232 genes were in Tn2013-like structures with ISEcp1 128 bp upstream of the blaOXAs (JN205800 and JX423831, respectively).3,10 PCR mapping failed to identify the blaOXA-48 context in two E. coli ST648 isolates. In PFGE clusters with more than one isolate, all isolates had the same blaOXA gene in the same mobile element. Plasmid versus chromosomal location of blaOXA-48/181-like genes Forty-three isolates of all species and multiple clones contained blaOXA-48/181-carrying plasmids of ∼50–260 kb, whereas 11 isolates of E. coli ST38 and ST648, and K. pneumoniae ST336 had blaOXA-48 in the chromosome (Table 1). Of 23 selected isolates with blaOXA-48/181 plasmids, 20 isolates produced transconjugants, and one plasmid was electroporated. The PBRT revealed mainly IncL (plasmids of ∼60 kb with blaOXA-48), followed by IncM (∼80–95 kb; blaOXA-48), IncX3 (∼50 kb; blaOXA-181) and non-typeable (∼90–160 kb; blaOXA-48/-232) replicons. The data correlated with typing, except for two K. pneumoniae ST15/KpnI isolates with non-typeable blaOXA-48 plasmids of varying size. Other acquired β-lactamases All but five isolates had additional β-lactamases, the profiles of which corresponded to their genotypes (Table 1). CTX-M-type ESBLs dominated, especially CTX-M-15 (n = 39). Only one transconjugant was resistant to oxyimino-β-lactams and had CTX-M-15 (Table S1, available as Supplementary data at JAC Online), indicating that ESBLs/AmpCs were mostly not co-encoded with OXA-48/181 types by the same plasmids (Table S1). Susceptibility The isolates were resistant (n = 51) or intermediate (n = 3) to at least one carbapenem (Table S1), with relatively high MICs (imipenem MIC50, 16 mg/L; ertapenem MIC50, >32 mg/L), indicating other, possibly permeability-based mechanisms.2 All ESBL producers were resistant to at least cefotaxime of the oxyimino-β-lactams. Major groups were resistant to ciprofloxacin (n = 45), gentamicin (n = 43) or fosfomycin (n = 35), but not to colistin (K. pneumoniae, n = 15; E. coli, n = 1; mcr-1 gene not detected), amikacin or tigecycline (n = 12 each). Twenty K. pneumoniae isolates, including multiple ST395/KpnN outbreak isolates, were susceptible to agents of one or two therapeutic groups only (if at all). Discussion This is, to our knowledge, the first country-wide analysis of OXA-48/181-type CPE in Poland. With 52 cases reported, one non-preserved and two from other studies,6,15 the organisms have been much less frequent than in other European countries,1,2,4 and in Poland these have been far behind CPE with NDM- or KPC-like carbapenemases.8,16 It is possible that due to diagnostic difficulties,2 the NRCST OXA-48/181 records have been underestimated (of note was the relatively high-level carbapenem resistance of the isolates collected). The analysis revealed genetic diversity of the organisms in general, likely arising from multiple foreign imports, as exemplified by 14 cases. The countries of possible origin included those with only few original or indirect OXA-48/181 reports (Russia and Georgia)17–19 or, as far as we are aware, no internationally available data (Ukraine and Cambodia). However, five clusters of isolates resulting from on-site transmission were also discerned. The K. pneumoniae ST395/KpnN OXA-48 outbreak observed in Cracow from mid-2016 has contributed 18 cases to the overall situation. Locally, plasmid dissemination has occurred, like in Kielce, where after the E. coli ST410 OXA-181 outbreak, C. freundii ST124 OXA-181 with an IncX3-like plasmid of identical fingerprint (result not shown) was recovered. Most of the STs observed represent international lineages, identified with various resistance determinants, including OXA-48/181 types.1,4,20,21 OXA-48-producing K. pneumoniae ST11, ST101, ST395 and E. coli ST38 of similar characteristics have spread in endemic regions and in Europe,4,17,22–24 with K. pneumoniae ST395 outbreaks, for example, in France or Hungary.25,26 Apart from the outbreak organism, four other K. pneumoniae ST395 and eight E. coli ST38 genotypes were identified with different origins, repeatedly Egypt for E. coli ST38 as elsewhere,4,22 underlining the significance of these in OXA-48/181 epidemiology. The study reaffirmed the broad spread and resistance of the emerging E. cloacae complex ST78.20,21 The blaOXA-48/181-like genes were located in Tn1999-like transposons and two elements formed by ISEcp1 placed upstream, Tn2013 and Tn2016. These structures mark multiple mobilizations of blaOXA-48/181-like genes from their natural hosts.2,4,10 Interestingly, K. pneumoniae ST15/KpnI and ST11 from the same hospital/region had blaOXA-48 in the Tn2016-like element. To our knowledge, Tn2016 has been observed with the blaOXA-204 gene,14 while blaOXA-48 only in Tn1999-like transposons so far.2 This indicates repeated mobilizations of blaOXA-48 and suggests evolution of blaOXA-204 from the Tn2016-located blaOXA-48 gene. Conjugative pOXA-48-like plasmids of ∼62 kb,27 widely observed in diverse organisms, are major factors in the dissemination of blaOXA-48-like genes.2,4 These were classified into the IncL/M group, recently updated as the IncL type.11 The prevailing ∼60 kb IncL-like plasmids in this study were most probably pOXA-48 variants. However, three K. pneumoniae genotypes had IncM-type plasmids of ∼80–95 kb. To our knowledge, this is the first report on IncM blaOXA-48 plasmids, but their presence among the previous ‘IncL/M’ molecules is likely. The blaOXA-181 genes correlated with IncX3-like plasmids as in other reports.5,24 In summary, OXA-48/181-type CPE have been relatively rare in Poland so far. Their epidemiology has comprised multiple imports, several localized transmissions, one larger clonal outbreak and plasmid transfers. The study revealed some possibly new characteristics of these organisms, including the presence of the blaOXA-48 gene in the ISEcp1 context. Acknowledgements Parts of this study were shown at the 12th β-Lactamase Meeting, Gran Canaria, Spain, 28 June–1 July 2014 (Poster No. 29.11); and the 11th International Meeting on Microbial Epidemiological Markers, Estoril, Portugal, 9–12 March, 2016 (Poster No. PO36).  We thank M. Herda for her assistance, and other colleagues for the study material: A. Byś, K. Dzierżanowska-Fangrat, T. Gadomski, A. Guzek, L. Horoń, B. Jaglarz, M. Jarczyńska, A. Jurczak, E. Mik, E. Podsiadły, A. Sierzputowska, M. Skarżycka and B. Witkowska. The authors are very thankful to curator teams of the Medical University in Białystok, Poland, the National Center for Global Health and Medicine in Tokyo, Japan, the University of Warwick, UK, and the Institut Pasteur in Paris, France, for curating the MLST data of C. freundii, E. cloacae, E. coli and K. pneumoniae, respectively. Members of the OXA-48-PL Study Group P. Chrystyniuk: Voivodship Hospital, Słupsk; B. Durnaś: Świętokrzyskie Oncology Centre, Kielce; J. Kędzierska: University Hospital, Cracow; A. Mól: University Hospital of Lord’s Transfiguration, Poznań; E. Swoboda-Kopeć, M. Wróblewska: Public Central Teaching Hospital, Medical University of Warsaw, Warsaw; E. Tomanek: Center for Burns, Siemianowice Śląskie; B. Wcisło-Wach: Rydygier Specialist Hospital, Cracow. Funding This work was partially financed by grant UMO-2012/07/B/NZ6/03528 from the Polish National Science Centre, SPUB MIKROBANK from the Polish Ministry of Science and Higher Education, and grant DS-5.26/2016 from the National Medicines Institute. Transparency declarations None to declare. Supplementary data Table S1 appears as Supplementary data at JAC Online. References 1 Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin Microbiol Infect  2014; 20: 821– 30. Google Scholar CrossRef Search ADS PubMed  2 Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother  2012; 67: 1597– 606. Google Scholar CrossRef Search ADS PubMed  3 Potron A, Nordmann P, Lafeuille E et al.   Characterization of OXA-181, a carbapenem-hydrolyzing class D β-lactamase from Klebsiella pneumoniae. Antimicrob Agents Chemother  2011; 55: 4896– 9. Google Scholar CrossRef Search ADS PubMed  4 Potron A, Poirel L, Rondinaud E et al.   Intercontinental spread of OXA-48 β-lactamase-producing Enterobacteriaceae over a 11-year period, 2001 to 2011. Euro Surveill  2013; 18: pii=20549. 5 Liu Y, Feng Y, Wu W et al.   First report of OXA-181-producing Escherichia coli in China and characterization of the isolate using whole-genome sequencing. Antimicrob Agents Chemother  2015; 59: 5022– 5. Google Scholar CrossRef Search ADS PubMed  6 Majewski P, Wieczorek P, Sacha PT et al.   Emergence of OXA-48 carbapenemase-producing Enterobacter cloacae ST89 infection in Poland. Int J Infect Dis  2014; 25: 107– 9. Google Scholar CrossRef Search ADS PubMed  7 Izdebski R, Bojarska K, Baraniak A et al.   NDM-1- or OXA-48-producing Enterobacteriaceae colonising Polish tourists following a terrorist attack in Tunis, March 2015. Euro Surveill  2015; 20: pii=21150. 8 Baraniak A, Izdebski R, Fiett J et al.   NDM-producing Enterobacteriaceae in Poland, 2012-14: inter-regional outbreak of Klebsiella pneumoniae ST11 and sporadic cases. J Antimicrob Chemother  2016; 71: 85– 91. Google Scholar CrossRef Search ADS PubMed  9 Carrer A, Poirel L, Eraksoy H et al.   Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in Istanbul, Turkey. Antimicrob Agents Chemother  2008; 52: 2950– 4. Google Scholar CrossRef Search ADS PubMed  10 Potron A, Rondinaud E, Poirel L et al.   Genetic and biochemical characterisation of OXA-232, a carbapenem-hydrolysing class D β-lactamase from Enterobacteriaceae. Int J Antimicrob Agents  2013; 41: 325– 9. Google Scholar CrossRef Search ADS PubMed  11 Carattoli A, Seiffert SN, Schwendener S et al.   Differentiation of IncL and IncM plasmids associated with the spread of clinically relevant antimicrobial resistance. PLoS One  2015; 10: e0123063. Google Scholar CrossRef Search ADS PubMed  12 Johnson TJ, Bielak EM, Fortini D et al.   Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae. Plasmid  2012; 68: 43– 50. Google Scholar CrossRef Search ADS PubMed  13 Carattoli A, Bertini A, Villa L et al.   Identification of plasmids by PCR-based replicon typing. J Microbiol Methods  2005; 63: 219– 28. Google Scholar CrossRef Search ADS PubMed  14 Potron A, Nordmann P, Poirel L. Characterization of OXA-204, a carbapenem-hydrolyzing class D β-lactamase from Klebsiella pneumoniae. Antimicrob Agents Chemother  2013; 57: 633– 6. Google Scholar CrossRef Search ADS PubMed  15 Majewski P, Wieczorek P, Ojdana D et al.   Altered outer membrane transcriptome balance with AmpC overexpression in carbapenem-resistant Enterobacter cloacae. Front Microbiol  2016; 7: 2054. Google Scholar CrossRef Search ADS PubMed  16 Baraniak A, Izdebski R, Zabicka D et al.   Multiregional dissemination of KPC-producing Klebsiella pneumoniae ST258/ST512 genotypes in Poland, 2010-14. J Antimicrob Chemother  2017; 72: 1610– 6. Google Scholar CrossRef Search ADS PubMed  17 Ageevets VA, Partina IV, Lisitsyna ES et al.   Emergence of carbapenemase-producing Gram-negative bacteria in Saint Petersburg, Russia. Int J Antimicrob Agents  2014; 44: 152– 5. Google Scholar CrossRef Search ADS PubMed  18 Adler A, Shklyar M, Schwaber MJ et al.   Introduction of OXA-48-producing Enterobacteriaceae to Israeli hospitals by medical tourism. J Antimicrob Chemother  2011; 66: 2763– 6. Google Scholar CrossRef Search ADS PubMed  19 Fursova NK, Astashkin EI, Knyazeva AI et al.   The spread of blaOXA-48 and blaOXA-244 carbapenemase genes among Klebsiella pneumoniae, Proteus mirabilis and Enterobacter spp. isolated in Moscow, Russia. Ann Clin Microbiol Antimicrob  2015; 14: 46. Google Scholar CrossRef Search ADS PubMed  20 Izdebski R, Baraniak A, Herda M et al.   MLST reveals potentially high-risk international clones of Enterobacter cloacae. J Antimicrob Chemother  2015; 70: 48– 56. Google Scholar CrossRef Search ADS PubMed  21 Fernandez J, Montero I, Martinez O et al.   Dissemination of multiresistant Enterobacter cloacae isolates producing OXA-48 and CTX-M-15 in a Spanish hospital. Int J Antimicrob Agents  2015; 46: 469– 74. Google Scholar CrossRef Search ADS PubMed  22 Poirel L, Bernabeu S, Fortineau N et al.   Emergence of OXA-48-producing Escherichia coli clone ST38 in France. Antimicrob Agents Chemother  2011; 55: 4937– 8. Google Scholar CrossRef Search ADS PubMed  23 Jayol A, Poirel L, Dortet L et al.   National survey of colistin resistance among carbapenemase-producing Enterobacteriaceae and outbreak caused by colistin-resistant OXA-48-producing Klebsiella pneumoniae, France, 2014. Euro Surveill  2016; 21: doi:10.2807/1560-7917.ES.2016.21.37.30339. 24 Skalova A, Chudejova K, Rotova V et al.   Molecular characterization of OXA-48-like-producing Enterobacteriaceae in the Czech Republic and evidence for horizontal transfer of pOXA-48-like plasmids. Antimicrob Agents Chemother  2017; 61: pii=e01889-16. 25 Cuzon G, Ouanich J, Gondret R et al.   Outbreak of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in France. Antimicrob Agents Chemother  2011; 55: 2420– 3. Google Scholar CrossRef Search ADS PubMed  26 Kovacs K, Nyul A, Mestyan G et al.   Emergence and interhospital spread of OXA-48-producing Klebsiella pneumoniae ST395 clone in Western Hungary. Infect Dis (Lond)  2017; 49: 231– 3. Google Scholar CrossRef Search ADS PubMed  27 Poirel L, Bonnin RA, Nordmann P. Genetic features of the widespread plasmid coding for the carbapenemase OXA-48. Antimicrob Agents Chemother  2012; 56: 559– 62. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Abstract

Abstract Objectives To analyse OXA-48 (OXA-48/181)-type carbapenemase-producing Enterobacteriaceae reported in Poland from 2013 until January 2017. Methods Bacterial isolates were typed by PFGE and MLST. Genes coding for OXA-48/181 types and other β-lactamases were amplified and sequenced. Mobile elements with blaOXA-48/181-like genes were PCR mapped. blaOXA-48/181-carrying plasmids were characterized by nuclease S1-hybridization profiling, transfer assays and PCR-based replicon typing, while the chromosomal location of the genes was confirmed by the I-CeuI analysis. Results Fifty-four isolates from 52 patients in 20 hospitals (14 cities) were included, in 14 cases having probable foreign origins indicated. The organisms were genetically diverse and represented numerous pandemic clones, including Klebsiella pneumoniae ST395 (n = 23), ST11, ST15 and ST101, Escherichia coli ST38, ST410 and ST648, and Enterobacter cloacae complex ST78. These produced OXA-48 (n = 49), OXA-181 (n = 4) or OXA-232 (n = 1). One of five K. pneumoniae ST395 pulsotypes caused a multicentre outbreak with 18 cases, which significantly contributed to the total number of patients. Depending on the variant, the blaOXA-48/181-like genes were parts of the Tn1999-, Tn2013- or Tn2016-like transposons, with blaOXA-48 found in an ISEcp1-associated module (Tn2016-like) for the first time. Three genotypes, including E. coli ST38, had chromosomal blaOXA-48 genes, while others carried transmissible IncL (∼60 kb; blaOXA-48; n = 30), IncM (∼80–95 kb; blaOXA-48; n = 4), IncX3 (∼50 kb; blaOXA-181; n = 4) or non-typeable (∼90–160 kb; blaOXA-48 or blaOXA-232) plasmids. Conclusions Even though OXA-48/181 producers seem to occur infrequently in Poland, their epidemiology has been marked by various phenomena, namely multiple imports, several limited transmissions plus one larger clonal outbreak, and possible plasmid transfers. Introduction Carbapenemase-producing Enterobacteriaceae (CPE), including those with OXA-48-type enzymes, contribute greatly to the global crisis in antimicrobial chemotherapy.1 OXA-48 was identified first in 2001 in Turkey,2 and soon most of the south-east Mediterranean region became endemic for producers of this enzyme.1,2 Another large reservoir is the Indian subcontinent where organisms with the related OXA-181 have spread.1,3 Widespread travel and migration are at the origin of the increasing presence of OXA-48/181 types in Europe.1,2,4 The enzymes confer resistance to penicillins (with temocillin) and penicillin/inhibitor combinations, and low-level resistance to carbapenems, augmented by, e.g. permeability defects. The common resistance to oxyimino-β-lactams results from co-expression of ESBLs and/or AmpC-type cephalosporinases.2,blaOXA-48/181-like genes originate from Shewanella spp. and disseminate with mobile elements, such as blaOXA-48-carrying Tn1999-like transposons and IncL-type plasmids, or a blaOXA-181-carrying ISEcp1-associated transposon, Tn2013, and IncX3 plasmids.2,3,5 Clonal spread of some enterobacterial lineages, such as Klebsiella pneumoniae ST11, ST101, ST395 or Escherichia coli ST38, has also been significant in OXA-48/181 proliferation.4 The first OXA-48 producers reported in Poland in 2012 were Enterobacter cloacae ST89 from two patients from Białystok; one was described by Majewski et al.,6 whereas the second one, confirmed by the National Reference Centre for Susceptibility Testing (NRCST), was not preserved. In this study, all unique OXA-48/181-like isolates identified by the NRCST surveillance from 2013 to January 2017 were analysed. Methods Clinical isolates Fifty-four non-duplicate isolates (37 K. pneumoniae, 14 E. coli, and one of each Citrobacter freundii, Enterobacter aerogenes and E. cloacae complex) were collected from 52 patients in 20 hospitals in 14 Polish cities (Table 1). These isolates were submitted to the NRCST owing to carbapenem non-susceptibility and resistance to temocillin and/or positive Carba NP test (http://eucast.org). Species were re-identified with Vitek2 (bioMérieux, Marcy l’Etoile, France), and carbapenemase-associated phenotypes were analysed as described.7,8,blaOXA-48/181-like genes were detected by PCR and sequenced.7 Table 1. OXA-48/181-producing isolates: species, basic clinical and epidemiological data, clones, pulsotypes, transposons and plasmids with blaOXA-48/181-like genes, and other acquired β-lactamases Species and isolatea  Year of isolation  Hospital  Specimenb  Origin, including hospitalization history (H)c  STd  PFGE types  blaOXA-48-like genes  Types of transposons with blaOXA-48/181-like genes  Location of blaOXA-48/181e  ESBLs, AmpCs and other acquired β-lactamases  K. pneumoniae 2844/13  2013  Poznań  urine  Russia (H)  ST395  KpnA  blaOXA-48  Tn1999.1  ∼260 kbj  TEM-1  K. pneumoniae 1786/14  2014  Cracow I  BAL  Georgia  ST395  KpnB  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 655/15  2015  Cracow II  wound  Ukraine  ST395  KpnH  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4969/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 5083/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 3606/16  2016  Cracow III  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 3676/16  2016  Cracow I  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4378/16  2016  Cracow I  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5063/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5404/16  2016  Cracow III  urine  —  ST395  KpnN2  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5485/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5486/16  2016  Cracow III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6151/16  2016  Cracow I  blood  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6325/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6384/16  2016  Warsaw III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6713/16  2016  Cracow III  BAL  —  ST395  KpnN3  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6965/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7243/16  2016  Cracow III  wound  —  ST395  KpnN4  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7426/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 92/17  2017  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 94/17  2017  Cracow III  BAL  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 165/17  2017  Bochnia  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 283/17  2017  Cracow III  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4166/14  2014  Warsaw III  urine  Romania (H)g  ST15  KpnC  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 2673/15  2015  Ełk  urine  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼90; ∼105; NT  CTX-M-15; TEM-1  K. pneumoniae 3695/16  2016  Białystok  stool  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼160 kb; NT  CTX-M-15; TEM-1  K. pneumoniae 4245/14  2014  Bydgoszcz  BAL  Turkey  ST101  KpnD  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 1202/15f  2015  Warsaw IV  rectal swab  Tunisia (H)  ST101  KpnF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-3; TEM-1  K. pneumoniae 3651/16  2016  Siemianowice Śl.  wound  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4492/16  2016  Siemianowice Śl.  BAL  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4396/16  2016  Białystok  rectal swab  —  ST11  KpnP  blaOXA-48  Tn2016-like  ∼90 kbj  CTX-M-15; DHA-1; TEM-1  K. pneumoniae 2805/16  2016  Grodzisk Maz.  urine  —  ST13  KpnL  blaOXA-48  Tn1999.2  ∼60 kb; IncL  –  K. pneumoniae 1504/16  2016  Cracow I  urine  —  ST14  KpnK  blaOXA-48  Tn1999.1  ∼95 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 432/15  2015  Cracow II  wound  Ukraine  ST23  KpnG  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-55; TEM-1  K. pneumoniae 3086/16  2016  Warsaw V  throat swab  —  ST39  KpnM  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5774/16  2016  Szczecin  rectal swab  Oman (H)  ST231  KpnQ  blaOXA-232  ΔTn2013-like  ∼110 kb; NT  CTX-M-15; SHV; TEM-1  K. pneumoniae 4999/14  2014  Siemianowice Śl.  rectal swab  Egypth,i  ST336  KpnE  blaOXA-48  Tn1999.2  chr  CTX-M-15; TEM-1  E. coli 5240/13  2013  Warsaw II  stool  Egypt  ST38  EcoB  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 1414/14  2014  Słupsk  vaginal swab  Egypti  ST38  EcoC  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2365/14  2014  Warsaw III  stool  —  ST38  EcoE  blaOXA-48  Tn1999.2  chr  CTX-M-24; TEM-1  E. coli 4616/14  2014  Siemianowice Śl.  wound  Egypt  ST38  EcoG  blaOXA-48  Tn1999.2  chr  TEM-1  E. coli 4674/14  2014  Warsaw III  stool  —  ST38*  EcoK  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 972/15  2015  Sosnowiec  urine  —  ST38  EcoH  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2636/15  2015  Grodzisk Maz.  rectal swab  —  ST38  EcoI  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 1524/16  2016  Warsaw III  rectal swab  —  ST38  EcoJ  blaOXA-48  Tn1999.1  chr  CTX-M-15; TEM-1  E. coli 1987/14  2014  Kielce  wound  —  ST410  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 1988/14  2014  Kielce  BAL  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 2251/14  2014  Kielce  rectal swab  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 4008/13  2013  Warsaw I  stool  Cambodia (H)  ST648  EcoA  blaOXA-48  —  chr  CTX-M-15; TEM-1  E. coli 1704/16  2016  Warsaw I  stool  multiple travels  ST648  EcoL  blaOXA-48  —  chr  CTX-M-15  E. coli 4157/14  2014  Siemianowice Śl.  wound  Egypth,i  ST744  EcoF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  C. freundii 5976/16  2016  Kielce  urine  —  ST124  —  blaOXA-181  Tn2013  ∼50 kb; IncX3  —  E. aerogenes 4188/14  2014  Warsaw III  blood  Romania (H)g  NA  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  E. cloacae 150/17  2017  Biała Podlaska  rectal swab  —  ST78  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  Species and isolatea  Year of isolation  Hospital  Specimenb  Origin, including hospitalization history (H)c  STd  PFGE types  blaOXA-48-like genes  Types of transposons with blaOXA-48/181-like genes  Location of blaOXA-48/181e  ESBLs, AmpCs and other acquired β-lactamases  K. pneumoniae 2844/13  2013  Poznań  urine  Russia (H)  ST395  KpnA  blaOXA-48  Tn1999.1  ∼260 kbj  TEM-1  K. pneumoniae 1786/14  2014  Cracow I  BAL  Georgia  ST395  KpnB  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 655/15  2015  Cracow II  wound  Ukraine  ST395  KpnH  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4969/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 5083/15  2015  Cracow II  urine  —  ST395  KpnJ  blaOXA-48  Tn1999.1  ∼80 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 3606/16  2016  Cracow III  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 3676/16  2016  Cracow I  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4378/16  2016  Cracow I  urine  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5063/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5404/16  2016  Cracow III  urine  —  ST395  KpnN2  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5485/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5486/16  2016  Cracow III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6151/16  2016  Cracow I  blood  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6325/16  2016  Cracow III  wound  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6384/16  2016  Warsaw III  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6713/16  2016  Cracow III  BAL  —  ST395  KpnN3  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 6965/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7243/16  2016  Cracow III  wound  —  ST395  KpnN4  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 7426/16  2016  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 92/17  2017  Cracow III  urine  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 94/17  2017  Cracow III  BAL  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 165/17  2017  Bochnia  rectal swab  —  ST395*  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 283/17  2017  Cracow III  BAL  —  ST395  KpnN1  blaOXA-48  Tn1999.1  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4166/14  2014  Warsaw III  urine  Romania (H)g  ST15  KpnC  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 2673/15  2015  Ełk  urine  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼90; ∼105; NT  CTX-M-15; TEM-1  K. pneumoniae 3695/16  2016  Białystok  stool  —  ST15  KpnI  blaOXA-48  Tn2016-like  ∼160 kb; NT  CTX-M-15; TEM-1  K. pneumoniae 4245/14  2014  Bydgoszcz  BAL  Turkey  ST101  KpnD  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 1202/15f  2015  Warsaw IV  rectal swab  Tunisia (H)  ST101  KpnF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-3; TEM-1  K. pneumoniae 3651/16  2016  Siemianowice Śl.  wound  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4492/16  2016  Siemianowice Śl.  BAL  —  ST152  KpnO  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 4396/16  2016  Białystok  rectal swab  —  ST11  KpnP  blaOXA-48  Tn2016-like  ∼90 kbj  CTX-M-15; DHA-1; TEM-1  K. pneumoniae 2805/16  2016  Grodzisk Maz.  urine  —  ST13  KpnL  blaOXA-48  Tn1999.2  ∼60 kb; IncL  –  K. pneumoniae 1504/16  2016  Cracow I  urine  —  ST14  KpnK  blaOXA-48  Tn1999.1  ∼95 kb; IncM  CTX-M-15; TEM-1  K. pneumoniae 432/15  2015  Cracow II  wound  Ukraine  ST23  KpnG  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-55; TEM-1  K. pneumoniae 3086/16  2016  Warsaw V  throat swab  —  ST39  KpnM  blaOXA-48  Tn1999.2  ∼60 kb; IncL  CTX-M-15; TEM-1  K. pneumoniae 5774/16  2016  Szczecin  rectal swab  Oman (H)  ST231  KpnQ  blaOXA-232  ΔTn2013-like  ∼110 kb; NT  CTX-M-15; SHV; TEM-1  K. pneumoniae 4999/14  2014  Siemianowice Śl.  rectal swab  Egypth,i  ST336  KpnE  blaOXA-48  Tn1999.2  chr  CTX-M-15; TEM-1  E. coli 5240/13  2013  Warsaw II  stool  Egypt  ST38  EcoB  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 1414/14  2014  Słupsk  vaginal swab  Egypti  ST38  EcoC  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2365/14  2014  Warsaw III  stool  —  ST38  EcoE  blaOXA-48  Tn1999.2  chr  CTX-M-24; TEM-1  E. coli 4616/14  2014  Siemianowice Śl.  wound  Egypt  ST38  EcoG  blaOXA-48  Tn1999.2  chr  TEM-1  E. coli 4674/14  2014  Warsaw III  stool  —  ST38*  EcoK  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 972/15  2015  Sosnowiec  urine  —  ST38  EcoH  blaOXA-48  Tn1999.1  chr  CTX-M-24; TEM-1  E. coli 2636/15  2015  Grodzisk Maz.  rectal swab  —  ST38  EcoI  blaOXA-48  Tn1999.1  chr  TEM-1  E. coli 1524/16  2016  Warsaw III  rectal swab  —  ST38  EcoJ  blaOXA-48  Tn1999.1  chr  CTX-M-15; TEM-1  E. coli 1987/14  2014  Kielce  wound  —  ST410  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 1988/14  2014  Kielce  BAL  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 2251/14  2014  Kielce  rectal swab  —  ST410*  EcoD  blaOXA-181  Tn2013  ∼50 kb; IncX3  CTX-M-15; CMY-2; TEM-1  E. coli 4008/13  2013  Warsaw I  stool  Cambodia (H)  ST648  EcoA  blaOXA-48  —  chr  CTX-M-15; TEM-1  E. coli 1704/16  2016  Warsaw I  stool  multiple travels  ST648  EcoL  blaOXA-48  —  chr  CTX-M-15  E. coli 4157/14  2014  Siemianowice Śl.  wound  Egypth,i  ST744  EcoF  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  C. freundii 5976/16  2016  Kielce  urine  —  ST124  —  blaOXA-181  Tn2013  ∼50 kb; IncX3  —  E. aerogenes 4188/14  2014  Warsaw III  blood  Romania (H)g  NA  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  E. cloacae 150/17  2017  Biała Podlaska  rectal swab  —  ST78  —  blaOXA-48  Tn1999.2  ∼60 kb; IncL  —  a Isolates are ordered according to the species, ST and pulsotype; STs are ordered according to numbers of isolates classified; in clusters of the same ST and pulsotype the isolates are ordered chronologically. b Rectal (and throat) swabs and stool samples were collected during microbiological screening at admission or during regular CPE surveillance procedures in some centres. c Documented stay, including hospitalization (H) for a 1 year period before the OXA-48/181 CPE identification. d ST, sequence type; a new ST is marked in bold; NA, not applicable; asterisks indicate the STs that were adopted from fully analysed isolates of the same pulsotypes. e Bold font refers to blaOXA-48/181 plasmid replicon types detected in E. coli recombinants obtained for isolates representing different genotypes (pulsotype plus blaOXA-48/181 plasmid size); italics represent replicons confirmed by PCR for other clinical isolates of the individual genotypes; chr, chromosomal location; NT, not-typeable. f Sequence of the blaOXA-48 gene, type of transposon and ST of the isolate were reported previously.7 g Isolates from the same patient. h Isolates from the same patient. i Travel of a family member. j These plasmids could not be transferred to E. coli despite repeated mating and electroporation attempts. Molecular analysis E. coli and K. pneumoniae isolates were typed by PFGE,8 and along with C. freundii and E. cloacae complex by MLST (http://pubmlst.org/cfreundii/, http://pubmlst.org/ecloacae, http://mlst.warwick.ac.uk/mlst/dbs/Ecoli and http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). The location of the blaOXA-48/181-like genes in transposons was checked by PCR mapping and confirmed by sequencing for selected isolates.3,9,10 Identification of blaOXA-48/181-carrying plasmids was performed by the nuclease S1 analysis (TaKaRa, Otsu, Japan)8 and hybridization, using the ECL Direct Nucleic Acid Labelling System (GE Healthcare, Little Chalfont, UK). Twenty-three isolates of all species/pulsotypes and blaOXA-48/181 plasmid sizes were subjected to plasmid transfer experiments with E. coli A15RifR (mating) or E. coli TOP10 (Invitrogen, Carlsbad, CA, USA; electroporation), respectively.8 Recombinants were selected with 32 mg/L temocillin, plus 128 mg/L rifampicin in case of transconjugants. Plasmid DNAs from the recombinants were used in the PCR-based replicon typing (PBRT),11–13 and the replicons detected were subsequently checked in all isolates of individual genotypes. Chromosomal location of blaOXA-48 genes was confirmed by the I-CeuI analysis.8 Other selected β-lactamase genes (blaKPC, blaIMP, blaNDM, blaVIM, blaCTX-M, blaSHV-5/-12, blaTEM, blaCMY-2 and blaDHA types) were identified in clinical isolates by PCRs and sequencing.8 Susceptibility testing Susceptibility of the isolates and E. coli recombinants was tested by broth microdilution or agar dilution for fosfomycin, as recommended by EUCAST (http://eucast.org). Results Clinical and epidemiological data The NRCST runs the CPE surveillance in Poland, collecting suspected isolates with basic clinical and epidemiological data. The 54 study isolates represented all 52 OXA-48/181-type cases confirmed from 2013 to January 2017, from hospitals in 11/16 major regions (Table 1). In 14 cases there was information on the patient’s arrival from or stay in another country prior to admission (a family member in two cases), sometimes with hospitalization. The history of one patient, a victim of terrorism in Tunisia in 2015, was published previously (K. pneumoniae 1202/15).7 Thirty-four patients developed infections, while 18 patients were colonized only. Clonality data The 37 K. pneumoniae isolates were grouped into 17 pulsotypes and 11 STs, of which ST395 prevailed (n = 23; six hospitals) and varied with five pulsotypes (Table 1). A single pulsotype (ST395/KpnN) comprised 18 isolates from four hospitals, mainly in the Cracow area, including one centre with 13 cases. Only three other K. pneumoniae pulsotypes of various STs had two isolates each. The 14 E. coli isolates were of 12 pulsotypes and four STs. E. coli ST38 was prevalent (n = 8; six hospitals) but each isolate represented another pulsotype. E. coli ST410 comprised three homogeneous isolates from a hospital in Kielce. blaOXA-48/181-like genes and their genetic context OXA-48/181 types were the only carbapenemases in the study isolates. blaOXA-48 was confirmed in 49 isolates, while blaOXA-181 and blaOXA-232 were confirmed in four and one isolates, respectively (Table 1). The blaOXA-48 genes were located mainly in Tn1999.1 (GenBank accession number AY236073) (n = 29) and Tn1999.2 (JN714122) (n = 15) transposons.2 However, in three K. pneumoniae isolates (ST15/KpnI and ST11) the ISEcp1 element was placed 46 bp upstream of blaOXA-48, a configuration described for blaOXA-204 as Tn2016 (JQ809466).14 The blaOXA-181 and blaOXA-232 genes were in Tn2013-like structures with ISEcp1 128 bp upstream of the blaOXAs (JN205800 and JX423831, respectively).3,10 PCR mapping failed to identify the blaOXA-48 context in two E. coli ST648 isolates. In PFGE clusters with more than one isolate, all isolates had the same blaOXA gene in the same mobile element. Plasmid versus chromosomal location of blaOXA-48/181-like genes Forty-three isolates of all species and multiple clones contained blaOXA-48/181-carrying plasmids of ∼50–260 kb, whereas 11 isolates of E. coli ST38 and ST648, and K. pneumoniae ST336 had blaOXA-48 in the chromosome (Table 1). Of 23 selected isolates with blaOXA-48/181 plasmids, 20 isolates produced transconjugants, and one plasmid was electroporated. The PBRT revealed mainly IncL (plasmids of ∼60 kb with blaOXA-48), followed by IncM (∼80–95 kb; blaOXA-48), IncX3 (∼50 kb; blaOXA-181) and non-typeable (∼90–160 kb; blaOXA-48/-232) replicons. The data correlated with typing, except for two K. pneumoniae ST15/KpnI isolates with non-typeable blaOXA-48 plasmids of varying size. Other acquired β-lactamases All but five isolates had additional β-lactamases, the profiles of which corresponded to their genotypes (Table 1). CTX-M-type ESBLs dominated, especially CTX-M-15 (n = 39). Only one transconjugant was resistant to oxyimino-β-lactams and had CTX-M-15 (Table S1, available as Supplementary data at JAC Online), indicating that ESBLs/AmpCs were mostly not co-encoded with OXA-48/181 types by the same plasmids (Table S1). Susceptibility The isolates were resistant (n = 51) or intermediate (n = 3) to at least one carbapenem (Table S1), with relatively high MICs (imipenem MIC50, 16 mg/L; ertapenem MIC50, >32 mg/L), indicating other, possibly permeability-based mechanisms.2 All ESBL producers were resistant to at least cefotaxime of the oxyimino-β-lactams. Major groups were resistant to ciprofloxacin (n = 45), gentamicin (n = 43) or fosfomycin (n = 35), but not to colistin (K. pneumoniae, n = 15; E. coli, n = 1; mcr-1 gene not detected), amikacin or tigecycline (n = 12 each). Twenty K. pneumoniae isolates, including multiple ST395/KpnN outbreak isolates, were susceptible to agents of one or two therapeutic groups only (if at all). Discussion This is, to our knowledge, the first country-wide analysis of OXA-48/181-type CPE in Poland. With 52 cases reported, one non-preserved and two from other studies,6,15 the organisms have been much less frequent than in other European countries,1,2,4 and in Poland these have been far behind CPE with NDM- or KPC-like carbapenemases.8,16 It is possible that due to diagnostic difficulties,2 the NRCST OXA-48/181 records have been underestimated (of note was the relatively high-level carbapenem resistance of the isolates collected). The analysis revealed genetic diversity of the organisms in general, likely arising from multiple foreign imports, as exemplified by 14 cases. The countries of possible origin included those with only few original or indirect OXA-48/181 reports (Russia and Georgia)17–19 or, as far as we are aware, no internationally available data (Ukraine and Cambodia). However, five clusters of isolates resulting from on-site transmission were also discerned. The K. pneumoniae ST395/KpnN OXA-48 outbreak observed in Cracow from mid-2016 has contributed 18 cases to the overall situation. Locally, plasmid dissemination has occurred, like in Kielce, where after the E. coli ST410 OXA-181 outbreak, C. freundii ST124 OXA-181 with an IncX3-like plasmid of identical fingerprint (result not shown) was recovered. Most of the STs observed represent international lineages, identified with various resistance determinants, including OXA-48/181 types.1,4,20,21 OXA-48-producing K. pneumoniae ST11, ST101, ST395 and E. coli ST38 of similar characteristics have spread in endemic regions and in Europe,4,17,22–24 with K. pneumoniae ST395 outbreaks, for example, in France or Hungary.25,26 Apart from the outbreak organism, four other K. pneumoniae ST395 and eight E. coli ST38 genotypes were identified with different origins, repeatedly Egypt for E. coli ST38 as elsewhere,4,22 underlining the significance of these in OXA-48/181 epidemiology. The study reaffirmed the broad spread and resistance of the emerging E. cloacae complex ST78.20,21 The blaOXA-48/181-like genes were located in Tn1999-like transposons and two elements formed by ISEcp1 placed upstream, Tn2013 and Tn2016. These structures mark multiple mobilizations of blaOXA-48/181-like genes from their natural hosts.2,4,10 Interestingly, K. pneumoniae ST15/KpnI and ST11 from the same hospital/region had blaOXA-48 in the Tn2016-like element. To our knowledge, Tn2016 has been observed with the blaOXA-204 gene,14 while blaOXA-48 only in Tn1999-like transposons so far.2 This indicates repeated mobilizations of blaOXA-48 and suggests evolution of blaOXA-204 from the Tn2016-located blaOXA-48 gene. Conjugative pOXA-48-like plasmids of ∼62 kb,27 widely observed in diverse organisms, are major factors in the dissemination of blaOXA-48-like genes.2,4 These were classified into the IncL/M group, recently updated as the IncL type.11 The prevailing ∼60 kb IncL-like plasmids in this study were most probably pOXA-48 variants. However, three K. pneumoniae genotypes had IncM-type plasmids of ∼80–95 kb. To our knowledge, this is the first report on IncM blaOXA-48 plasmids, but their presence among the previous ‘IncL/M’ molecules is likely. The blaOXA-181 genes correlated with IncX3-like plasmids as in other reports.5,24 In summary, OXA-48/181-type CPE have been relatively rare in Poland so far. Their epidemiology has comprised multiple imports, several localized transmissions, one larger clonal outbreak and plasmid transfers. The study revealed some possibly new characteristics of these organisms, including the presence of the blaOXA-48 gene in the ISEcp1 context. Acknowledgements Parts of this study were shown at the 12th β-Lactamase Meeting, Gran Canaria, Spain, 28 June–1 July 2014 (Poster No. 29.11); and the 11th International Meeting on Microbial Epidemiological Markers, Estoril, Portugal, 9–12 March, 2016 (Poster No. PO36).  We thank M. Herda for her assistance, and other colleagues for the study material: A. Byś, K. Dzierżanowska-Fangrat, T. Gadomski, A. Guzek, L. Horoń, B. Jaglarz, M. Jarczyńska, A. Jurczak, E. Mik, E. Podsiadły, A. Sierzputowska, M. Skarżycka and B. Witkowska. The authors are very thankful to curator teams of the Medical University in Białystok, Poland, the National Center for Global Health and Medicine in Tokyo, Japan, the University of Warwick, UK, and the Institut Pasteur in Paris, France, for curating the MLST data of C. freundii, E. cloacae, E. coli and K. pneumoniae, respectively. Members of the OXA-48-PL Study Group P. Chrystyniuk: Voivodship Hospital, Słupsk; B. Durnaś: Świętokrzyskie Oncology Centre, Kielce; J. Kędzierska: University Hospital, Cracow; A. Mól: University Hospital of Lord’s Transfiguration, Poznań; E. Swoboda-Kopeć, M. Wróblewska: Public Central Teaching Hospital, Medical University of Warsaw, Warsaw; E. Tomanek: Center for Burns, Siemianowice Śląskie; B. Wcisło-Wach: Rydygier Specialist Hospital, Cracow. Funding This work was partially financed by grant UMO-2012/07/B/NZ6/03528 from the Polish National Science Centre, SPUB MIKROBANK from the Polish Ministry of Science and Higher Education, and grant DS-5.26/2016 from the National Medicines Institute. Transparency declarations None to declare. Supplementary data Table S1 appears as Supplementary data at JAC Online. References 1 Nordmann P, Poirel L. The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide. Clin Microbiol Infect  2014; 20: 821– 30. Google Scholar CrossRef Search ADS PubMed  2 Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Mar 1, 2018

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