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First report of OXA-48–producing Escherichia coli in Croatia and confirmed intergenic transfer of a plasmid-carrying blaOXA-48 from Klebsiella pneumoniae

The emergence of OXA-48-producing Escherichia coli (E. coli) in Denmark and of Klebsiella pneumonia in Hungary were recently reported in the present journal [1, 2]. In addition to this worrying threat in parts of Europe, we here describe our concomitant isolation of OXA-48-producing E. coli and K. pneumoniae from a patient admitted to the University Hospital Center of Zagreb. A carbapenem-resistant E. coli and K. pneumoniae were isolated from a screening sample (stool sample) of a female patient hospitalized at the haematology department and treated with vancomycin, meropenem and colistin. The screening policy of this high-risk department consisted of screening on admission and weekly screening at five sites: nose, groin, axillary area, urine and stool. Colonies with a typical morphology were identified by standard microbiological testing and confirmed by MALDI Biotyper (Bruker Germany). Antimicrobial susceptibility testing to a wide range of antibiotics (ampicillin, amoxicillin–clavulanate, ceftazidime, cefuroxime, ceftriaxone, cefepime, cefotaxime, amikacin, gentamicin, piperacillin–tazobactam, ciprofloxacin, imipenem, meropenem, ertapenem, trimetothoprim–sulphamethoxazole and colistin) was performed on Mueller Hinton agar by the standard disk diffusion method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [3]. The minimum inhibitory concentration (MIC) was determined for imipenem, meropenem and colistin. MICs were interpreted using the criteria established by EUCAST. The screening detection of carbapenemases was also performed according to EUCAST. Routinely, all isolates showing resistance to at least one of the tested carbapenems were screened with a Carba NP test (bioMerieux Ltd). The presence of genes encoding for ESBLs (blaSHV, blaTEM and blaCTX-M), group A carbapenemases (blaKPC), metallo-beta-lactamases (blaVIM, blaIMP and blaNDM) and carbapenem-hydrolyzing oxacillinases (blaOXA-48) was determined by PCR as described previously [4]. The two isolates, E. coli (22646-1) and K. pneumoniae (22646-2), were whole genome sequenced on an Illumina MiSeq using the MiSeq reagent kit v2 generating 250-bp paired-end reads as described previously [5]. De novo assembly was performed using CLC Genomics Workbench version 10.0.1 (Qiagen, Hilden, Germany) with optimal word sizes based on the maximum N50 (the largest scaffold length, N, such that 50% of the assembled genome size is contained in scaffolds with a length of at least N) value as described before [5]. The MLST type and the presence of resistance genes was determined by uploading assembled sequences to MLST v1.7 and ResFinder v2.1 (Center for Genomic Epidemiology, Technical University of Denmark, Lingby, Denmark) [6]. Assembled genomes of both isolates were queried against each other using webact online tool (http://www.webact.org/WebACT/home) and further analysed in ACT [6, 7]. Identified contigs that showed high DNA sequence similarity were analysed by blast for the identification of the reference plasmid which turned out to be pEC745_OXA48 (GenBank: CP015075.2). Further, E. coli and K. pneumoniae genomes were compared to the reference plasmid using webact online tool, visualized using dnaplotter [8] and further analysed in ACT. The isolates were identified as E. coli and K. pneumoniae by MALDI Biotyper. Both strains were resistant to ampicillin, amoxicillin–clavulanate, cefuroxime, Table 1. Results of MLST and WGS analysis. 22646-1 22646-2 Bacterial species Escherichia coli Klebsiella pneumoniae MLST ST205 ST552 Phenotype Resistance gene Identity Identity aac(3)-lla 100.00 – aac(3)-lld 99.88 99.88 aadA1 100.00 – aadA16 – 99.65 aadA5 100.00 – Aminoglycoside resistance strA 100.00 99.88 strB 100.00 99.88 blaCTX-M-3 100.00 blaOXA-48 100.00 100.00 blaSHV-2 – 100.00 Beta-lactam resistance blaTEM-135 99.88 – blaTEM-1B – 100.00 Fluoroquinolone and aminoglycoside resistance aac(6')lb-cr – 100.00 Fosfomycin resistance fosA 96.90 QnrB6 – 100.00 Quinolone resistance oqxA 98.98 oqxB – 99.11 Rifampicin resistance ARR-3 – 100.00 sul1 100.00 100.00 Sulphonamide resistance sul2 100.00 100.00 Tetracycline resistance tet(A) 100.00 – tet(D) 100.00 dfrA1 100.00 – Trimethoprim resistance dfrA17 100.00 dfrA27 – 100.00 Figure 1. DNAc comparison of identified plasmids of 22646-1 and 22646-2 isolates with the reference plasmid pEC745_OXA48 (GenBank: CP015075.2). Grey ring represents reference plasmid ; green ring shows identified sequence matches corresponding to the reference plasmid of E. coli 22646-1 and blue ring shows identified sequence matches corresponding to the reference plasmid of K. pneumoniae 22646-2. Open reading frames are indicated as arrows in light blue. Resistance gene blaOXA-48 is indicated in red (The colour figure is available in the online version). 2 Z. BO
SNJAK ET AL. Downloaded by [Linköping University Library] at 21:05 26 October 2017 ceftriaxone, cefepime, cefotaxime, gentamicin, piperacillin–tazobactam, ciprofloxacin, imipenem, meropenem and ertapenem and susceptible to amikacin and colistin. K. pneumoniae showed intermediate susceptibility to ceftazidime, while the E. coli isolate was resistant to ceftazidime. The MICs of imipenem, meropenem and colistin were, 128, 128 and 1.0 lg/ml, respectively. The Carba NP test was positive for both strains. PCR revealed the presence of the blaOXA-48 gene in both the E. coli and K. pneumoniae isolates. In addition, the E. coli isolate possessed the blactx-m and blaTEM genes. MLST analysis of sequenced isolates revealed that the E. coli isolate (22646-1) belongs to ST205 and that the K. pneumoniae isolate (22646-2) belongs to ST552. Both isolates contain various resistance genes of which aac(3)-IId, strA, strB, blaOXA-48, sul1, sul2 are present in both (Table 1). Plasmid analyses confirmed suspected interspecies transfer of a plasmid-carrying blaOXA-48 (Figure 1). This plasmid shares overall high genetic similarity to plasmid pEC745_OXA48, which was reported previously (GenBank: CP015075.2). Carbapenemases of the oxacillinase-48 type have been identified mostly in Mediterranean and southern European countries with rapid spread across Europe [9]. So far, dissemination of the OXA-48-type carbapenem resistance has been found mainly to occur through K. pneumoniae. Here, we report coexistence of OXA- 48-producing E. coli (ST205) and K. pneumoniae (ST552) isolates in a patient hospitalised at the haematology department. To our knowledge, this is the first report of an OXA-48- producing E. coli isolate in Croatia. Plasmid analyses confirm suspected interspecies transfer of a plasmid carrying the blaOXA-48 gene, an event that has been described before [10]. Identified plasmids were highly similar to reference plasmid pEC745_OXA48. This plasmid type had been identified in K. pneumonia, E. coli or Raoultella planticola isolated in various countries like Turkey, Lebanon, Serbia, France, Germany and Morocco [9]. Due to effective infection control measures, carbapenem-resistant E. coli and K. pneumoniae isolates identified in a single patient did not spread to other patients or other hospital units. As carbapenem-resistant isolates are associated with high morbidity and mortality, it is important to prevent further emergence of carbapenem resistance. Therefore, screening programmes focusing on early detection of carbapenemase genes are key for preventing the spread of resistance and may guide adequate empirical therapy for critically ill patients. As there is a relatively high rate of K. pneumoniae producing OXA-48 in our hospital, this active surveillance should not only to be performed in high-risk departments, In addition, further rationalisation of the use of antibiotics and infection control measures should be implemented. Such approaches will also contribute to the knowledge on the emergence of OXA-48-like- producing Enterobacteriaceae.

E. coli, transfer , blaOXA-48 , Klebsiella pneumoniae

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