Diagnostic Microbiology and Infectious Disease 88 (2017) 287–289 Contents lists available at ScienceDirect Diagnostic Microbiology and Infectious Disease j ourna l homepage: www.e lsev ie r .com/ locate /d iagmicrob io Transfer of KPC-2 carbapenemase from Klebsiella pneumoniae to Enterobacter cloacae in a patient receiving meropenem therapy Evelin Rodrigues Martins a, Cássia Fernanda Estofolete a,b, Andressa Batista Zequini b, Louise Cerdeira c, Doroti de Oliveira Garcia d, Maria Fernanda Campagnari Bueno d, Gabriela Rodrigues Francisco d, Leonardo Neves de Andrade e, Ana Lúcia da Costa Darini e, Fernanda Modesto Tolentino g,f, Tiago Casella a,f, Nilton Lincopan c,h, Mara Corrêa Lelles Nogueira a,b,⁎ a Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, Brazil b Hospital de Base de São José do Rio Preto, São José do Rio Preto, Brazil c Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brazil d Instituto Adolfo Lutz, São Paulo, Brazil e Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Ribeirão Preto, Brazil f Universidade Estadual Paulista “Júlio de Mesquita Filho”, São José do Rio Preto, Brazil g Instituto Adolfo Lutz, São José do Rio Preto, Brazil h Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil a b s t r a c ta r t i c l e i n f o ⁎ Corresponding author. Tel.: +55-17-3201-5909; fax: E-mail addresses: evelin.rod.martins@gmail.com (E.R. cassiafestofolete@gmail.com (C.F. Estofolete), andressa.ze (A.B. Zequini), lcerdeira@gmail.com (L. Cerdeira), dogarci (D. de Oliveira Garcia), mf.campagnari@gmail.com (M.F.C gabis.francisco@gmail.com (G.R. Francisco), leoandrade02 (L.N. de Andrade), aldarini@fcfrp.usp.br (A.L. da Costa Dar fernandaTollentino@hotmail.com (F.M. Tolentino), tiago_ (T. Casella), lincopan@usp.br (N. Lincopan), ml.nogueira@ http://dx.doi.org/10.1016/j.diagmicrobio.2017.04.004 0732-8893/© 2017 Elsevier Inc. All rights reserved. Article history: Received 31 January 2017 Received in revised form 5 April 2017 Accepted 7 April 2017 Available online 12 April 2017 Keywords: Klebsiella pneumoniae Enterobacter cloacae blaKPC-2 Horizontal gene transfer Plasmid The horizontal transfer of a plasmid bearing the blaKPC-2 gene from K. pneumoniae to E. cloacae infecting the respiratory tract of a patient during meropenem therapy was elucidated. This finding is particularly worrisome, since these drugs are of last resort for multidrug-resistant Gram-negative pathogens. +55-17-3229-1777. Martins), quini@yahoo.com.br a@yahoo.com . Bueno), es@gmail.com ini), casella@yahoo.com.br famerp.br (M.C.L. Nogueira). © 2017 Elsevier Inc. All rights reserved. Carbapenems are the most effective antibiotics for the treatment of infections caused bymultidrug-resistant Gram-negative bacteria, main- ly extended-spectrumbeta-lactamase-producing Enterobacteriaceae. In this regard, the wide dissemination of the Klebsiella pneumoniae carbapenemase (KPC) among members of the Enterobacteriaceae fam- ily has become a global issue, since KPC can hydrolyze all β-lactam an- tibiotics (Doi and Paterson 2015). Moreover, most KPC producers often exhibit co-resistance to aminoglycosides and fluoroquinolones, dramatically limiting treatment options (Morrill et al. 2015). Pandemic KPC has occurred primarily by the dissemination of KPC-producing K. pneumoniae isolates belonging to international clones. On the other hand, the blaKPC gene, which is frequently located in transposon Tn4401, and carried on conjugative plasmids that vary in size and struc- ture, can spread among clinically significant Gram-negative species by horizontal gene transfer (HGT) (Mathers et al. 2015). Evidences for HGT of the blaKPC gene is supported on the isolation of several KPC- producing Enterobacteriaceae species in the same patient (Goren et al. 2010; Kocsis et al. 2014). On January 16, 2014, a 57-year-oldmanwas transferred from a local medical center to the emergency department of the Hospital de Base de São José do Rio Preto, in São Paulo, Brazil. During admission, the patient presented pallor, cold sweats and dyspnea, and reported epigastric pain of high intensity, irradiating to the back. Previous medical history enclosed diabetes mellitus, hypertension, congestive heart failure by trypanosomiasis and an ischemic stroke. Chest computerized tomogra- phy showed a type B aortic aneurysm without rupture with partially thrombosed false lumen andbilateral consolidation in lung parenchyma plus pleural effusion. He was transferred to cardiac intensive care unit (C-ICU), presenting mental confusion, tachypnea and hypertension, evolving with worsening of symptoms and consciousness impairment, http://crossmark.crossref.org/dialog/?doi=10.1016/j.diagmicrobio.2017.04.004&domain=pdf http://dx.doi.org/10.1016/j.diagmicrobio.2017.04.004 mailto:evelin.rod.martins@gmail.com mailto:cassiafestofolete@gmail.com mailto:andressa.zequini@yahoo.com.br mailto:lcerdeira@gmail.com mailto:dogarcia@yahoo.com mailto:mf.campagnari@gmail.com mailto:gabis.francisco@gmail.com mailto:leoandrade02es@gmail.com mailto:aldarini@fcfrp.usp.br mailto:fernandaTollentino@hotmail.com mailto:tiago_casella@yahoo.com.br mailto:lincopan@usp.br mailto:ml.nogueira@famerp.br http://dx.doi.org/10.1016/j.diagmicrobio.2017.04.004 http://www.sciencedirect.com/science/journal/07328893 Fig. 1. PFGE profile and antimicrobial susceptibility profiles of E. cloacae strains EcTA01 and EcTA02. Black squares (resistant), gray squares (intermediate resistance), white squares (susceptible). SAM (ampicillin/sulbactam), TZP (piperacillin-tazobactam), FOX (cefoxitin), CAZ (ceftazidime), CTX (cefotaxime), FEP (cefepime), ETP (ertapenem), IPM (imipenem), MER (meropenem), AMK (amikacin), GEN (gentamicin), CIP (ciprofloxacin), TIG (tigecicline), COL (colistin). • Fig. 1 is a 2-column fitting image. 288 E.R. Martins et al. / Diagnostic Microbiology and Infectious Disease 88 (2017) 287–289 and requiring intubation andmechanical ventilation (MV). Amoxicillin/ clavulanic acid (500 mg tid) was empirically prescribed, due to the pneumonia hypothesis, and an endotracheal aspirate (EA) sample was collected. Three days after, the treatment was changed to piperacillin- tazobactam (4.5 g qid). Quantitative EA culture showed the presence of E. cloacae (b106 CFU/mL), but the antimicrobial susceptibility test was not performed. Patient's condition worsened, and on January 23, 2014, a new EA sample was collected and an E. cloacae (≥106 CFU/mL) susceptible to carbapenems (CS), amikacin and colistin (designated EcTA01) was isolated. Meropenen (500mg tid) was used for treatment and the patient evolved with clinical improvement. On February 6, 2014, hewas submitted to placement of an aortic endoprosthesis.With- out MV and invasive devices, he was discharged from C-ICU to hospital ward. On February 16, 2014, due to new septic condition, the patient was readmitted to C-ICU, being submitted to MV and placement of central venous and bladder catheters. Blood cultures were negative, but a carbapenem-resistant (CR) K. pneumoniae strain (designated KpU01) was isolated from a urine culture. One day later, another CR K. pneumoniae (designated KpTA01) and a CR E. cloacae (designated EcTA02)were isolated fromEA cultures. Treatmentwith sodiumcolistin methanesulfonate (3.000.000UI tid) and ciprofloxacin (500 mg bid) was initiated. However, on the fourth day of this treatment his clinical conditions deteriorated, leading to death by septic shock, after forty- one days of admission. E. cloacae strains EcTA01 (CS) and EcTA02 (CR) were isolated in different times from endotracheal aspirate cultures, and XbaI-PFGE clonal relatedness (Pereira et al. 2013) elucidated that both strains belong to the same clone (Fig. 1). The carriage of blaKPC geneswas investigated by PCR and sequencing (Doyle et al. 2012). Plasmids from CR strains were extracted and trans- formed into Escherichia coli DH10B. Next, plasmid DNAs were extracted from transformants to construct a Nextera XT DNA library, which was sequenced using the MiSeq platform (Illumina, San Diego, CA), using paired-end reads (300 bp), which produced reads of 3.632,240, 2.814,910 and 148.966 base pair lengths for plasmids pKP4365 (KpU01), pKP4368 (KpTA01) and pEC4365 (EcTA02), respectively. De novo assembly was performed using A5-Miseq pipeline (Coil et al. 2015) and this assembly was optimized using Geneious v.R9 Fig. 2. Comparison of plasmids harboring the blaKPC-2 gene from K. pneumoniae KPU01 (A: pK accession number: KX783439) and K. pneumoniae KPTA01 (C: pKP4365, GenBank accession nu (Biomatters Ltd., New Zealand). The three final plasmid sequences were annotated using BLASTp and ISFinder (https://www-is.biotoul.fr/ blast.php); the annotation was curated using Artemis software version 16.0.11 (Carver et al. 2012). TheDDBJ/ENA/GenBank accession numbers are KX783440 for pKP4365, KX783441 for plasmid pKP4368, and KX783439 for pEC4365. During meropenem therapy, E. cloacae EcTA02 (CR) and K. pneumoniae KpTA01 (CR) were isolated from the same endotracheal sample, and curiously, both strains presented an identical ~15.7-kb plas- mid harboring blaKPC-2 (pEC4365 and pKP4365). Otherwise, the K. pneumoniae KpU01 (CR), isolated from the urine sample, carried a ~ 67.1-kb plasmid (pKP4368), which also harbored the blaKPC-2 gene. In all CR strains, the presence of the blaKPC-2 gene was associated with transposon Tn4401b (Fig. 2), whereas none of the studied plasmids pre- sented additional antibiotic resistance genes. Clinical and experimental studies on antibiotic therapy have showed that the choice of dose and treatment duration can influence the selec- tion of antibiotic-resistant bacteria (Olofsson and Cars 2007). Specifical- ly, to broad-spectrum beta-lactams, some studies have demonstrated that Enterobacteriaceae can acquire carbapenem resistance during anti- microbial therapy through horizontal transfer of an insertion sequence or plasmid (Ding et al. 2016). In this study, we report for the first time (to our knowledge) the transfer of KPC-2 from K. pneumoniae to E. cloacae in the same patient, during meropenem treatment, since DNA sequences of KPC-2-positive plasmids from the two species were identical (Richter et al. 2011). So, both selection of the KPC-2- producing E. cloacae and coexistence of K. pneumoniae producing KPC- 2, in the respiratory tract of the patient, were triggered by therapy with meropenem. On the other hand, horizontal transfer of identical blaKPC-2- or blaNDM-7-containing plasmids among several different En- terobacteriaceae genera has been previously documented (Cai et al. 2008; Chen et al. 2015), supporting the obtained result. In conclusion, clonal dissemination and horizontal plasmid transfer can play a significant role in the acquisition of carbapenem resistance in vivo, where acquisition of carbapenemases by Enterobacteriaceae can occur during antibiotic therapy. In this regard, treatment of infec- tion caused by KPC-positive bacteria is particularly worrisome, since P4368, GenBank accession number KX783441), E. cloacae ECTA02 (B: pEC4365, GenBank mber KX783440). • Fig. 2 is a 2-column fitting image. https://www.google.com.br/search?rlz=1C1PRFC_enBR548BR559&espv=2&biw=1440&bih=785&q=amoxicillin+clavulanic+acid&spell=1&sa=X&ved=0ahUKEwij3cm2ifTPAhVBlJAKHXpjD40QvwUIGSgA https://www.google.com.br/search?rlz=1C1PRFC_enBR548BR559&espv=2&biw=1440&bih=785&q=amoxicillin+clavulanic+acid&spell=1&sa=X&ved=0ahUKEwij3cm2ifTPAhVBlJAKHXpjD40QvwUIGSgA https://www-is.biotoul.fr/blast.php https://www-is.biotoul.fr/blast.php 289E.R. 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