Abstract Background Antimicrobial resistance due to carbapenemase expression poses a worldwide threat in healthcare. Inter-genus exchange of genetic information is of utmost importance in this context. Objectives Here, to the best of our knowledge, we describe the first detection and characterization of a KPC-2-producing Pseudomonas aeruginosa in Germany. Methods Characterization of the isolate was performed using MALDI-TOF MS, automated microdilution and MLST. Carbapenemase detection was performed using phenotypic and genotypic assays. The blaKPC-2-carrying plasmid was transformed into Escherichia coli NEB® 10-beta. The purified plasmid DNA was sequenced using the Illumina technique. Results The isolate expressed ST235 and was resistant to carbapenems. Antimicrobial susceptibility testing revealed colistin to be the only antimicrobial agent active in vitro. The blaKPC-2 gene was located on a replicon type lncHI1 plasmid as part of Tn4401. Conclusions The first detection (to the best of our knowledge) of plasmid-encoded KPC-2 in P. aeruginosa in Germany may point to a currently underestimated spread of carbapenemases among clinically relevant Gram-negative bacteria. Here, to the best of our knowledge, we also provide the first report of blaKPC-2 associated with the IncHI1 plasmid. Introduction Klebsiella pneumoniae carbapenemase (KPC) is a serine protease of the molecular class A and was first described in North Carolina in 1996.1 It is encoded by the blaKPC gene and almost always located within the Tn3-type transposon Tn4401, which is able to insert into plasmids of a huge variety of Gram-negative bacteria. Even though it is still most prevalent in K. pneumoniae,2 KPC has also been described in other Enterobacteriaceae like Escherichia coli and in non-fermenters like Pseudomonas or Acinetobacter species.3 This is remarkable, as the latter ones more frequently harbour class B metallo-β-lactamases (e.g. VIM, NDM) and class D β-lactamases, also referred to as oxacillinases (OXAs). In 2007, the first clinical isolate of KPC-producing Pseudomonas aeruginosa was identified in a Colombian hospital4 and has continuously spread throughout other countries, including the USA,5 China6 and Brazil.7 Interestingly, there has been no report of KPC-producing P. aeruginosa in Europe, yet. As of now we report its arrival in the European Union. Patient and methods The clinical isolate was detected in a mid-void urine sample of a patient with chronic myeloid leukaemia on continuous ibrutinib therapy. Comorbidities of the elderly patient included obesity, hyperlipidaemia, hypertension, diabetes mellitus II and consequently chronic kidney disease with impaired renal function. She had received several regimens of antibiotic therapy including quinolones, cephalosporins and piperacillin/tazobactam for suspected pneumonia and urosepsis prior to transfer to our hospital. On admission, at the time of sampling, the patient received meropenem intravenously for externally diagnosed ESBL E. coli bacteraemia. Nasal, pharyngeal and rectal swabs on admission did not yield multiresistant Gram-negative bacilli. Identification of the P. aeruginosa urine isolate and antimicrobial susceptibility testing based on EUCAST criteria were performed using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany) and automated microdilution (VITEK2, bioMérieux, Marcy-l’Étoile, France), respectively. MLST was performed as previously described.8 Briefly, DNA was extracted using the NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany) and fragments of seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA and trpE) were amplified and sequenced. The ST of the isolate was identified using the sequence definition tool of the P. aeruginosa MLST website (http://pubmlst.org/paeruginosa/). Disc diffusion tests as well as gradient strips (Etest) served as confirmation of the VITEK2 results and additional MIC determination of ceftazidime/avibactam, ceftolozane/tazobactam and colistin (all from Liofilchem srl, Terremoto, Italy). Colistin MIC was confirmed by microdilution. Initial carbapenemase screening of the isolate was performed on the fully automated BD MAX™ PCR platform (Becton Dickinson, Québec, Canada), which amplifies four targets (blaKPC, blaVIM, blaOXA and blaNDM genes encoding KPC 1–17, VIM 1–6 and 8–38, OXA-48-like and NDM, respectively) and detects amplicons via hydrolysis probes (Check-Points Health BV, Wageningen, The Netherlands).9 Hereupon, the presence of a carbapenemase was further phenotypically analysed with a cloxacillin combined disc test to exclude an intrinsic AmpC cephalosporinase. The modified Hodge test using K. pneumoniae ATCC 700603 as the indicator strain served to confirm carbapenemase expression. Genotypic confirmation was carried out using PCRs for VIM-, IMP-, NDM-, GES- and KPC-encoding genes and Sanger sequencing of amplificates.10 The blaKPC-2-carrying plasmid was purified and electroporated into E. coli NEB® 10-beta (New England Biolabs, Frankfurt am Main, Germany). The presence of blaKPC-2 in transformants was confirmed by PCR; replicon typing was performed as previously described.11 Plasmid DNA from a blaKPC-2-positive transformant was sequenced using the Illumina technique. Results and discussion The clinical isolate expressed ST235, which has been frequently described in the context of MDR within P. aeruginosa. It has also been frequently reported as a carrier of acquired carbapenemases. KPC-2-producing P. aeruginosa isolates from China expressed ST463, and information on STs is missing in other reports of KPC-2 in this species. Susceptibility testing of the isolate revealed MDR, including resistance to the novel antipseudomonal β-lactam/β-lactamase inhibitor combinations ceftolozane/tazobactam and ceftazidime/avibactam (Table 1). Multiplex real-time PCR on BD MAX™ repeatedly detected a blaKPC gene. KPC-2-type carbapenemase production was confirmed using single-locus PCRs as described above. PCR-based replicon typing showed that the blaKPC-2-carrying plasmid belonged to the Inc type HI1. Plasmid sequence analysis showed the blaKPC-2 gene to be located on a 10 kb contig as part of Tn4401. To the best of our knowledge, this is the first report of blaKPC-2 associated with an IncHI1 plasmid, indicating a continuous spread of KPC-encoding genes into a large variety of transferable plasmids. As no information on the Inc type of other blaKPC-2-carrying plasmids can be found in the literature, it remains unknown whether the finding of such a strain in Germany is based upon clonal dissemination or international spread of a single plasmid. Table 1. Susceptibility testing of the clinical isolate using different testing methods Antimicrobial agent VITEK2 N190a (mg/L) Disc diffusion (mm) Etest (mg/L) Broth microdilution (mg/L) Piperacillin/tazobactam ≥128 0 Ceftazidime ≥64 0 Ceftazidime/avibactam 24.0 Ceftolozane/tazobactam ≥256 Meropenem ≥16 0 ≥32 Imipenem b 0 Ciprofloxacin ≥4 0 Levofloxacin b 0 Gentamicin ≥16 Amikacin 0 Colistin 0.75 1.0 Antimicrobial agent VITEK2 N190a (mg/L) Disc diffusion (mm) Etest (mg/L) Broth microdilution (mg/L) Piperacillin/tazobactam ≥128 0 Ceftazidime ≥64 0 Ceftazidime/avibactam 24.0 Ceftolozane/tazobactam ≥256 Meropenem ≥16 0 ≥32 Imipenem b 0 Ciprofloxacin ≥4 0 Levofloxacin b 0 Gentamicin ≥16 Amikacin 0 Colistin 0.75 1.0 Interpretation of susceptibility testing was in accordance with EUCAST breakpoints. a bioMérieux, card for automated antimicrobial susceptibility testing of Enterobacteriaceae and non-fermenting Gram-negative bacilli. b Derived from the above substance tested in the panel. Table 1. Susceptibility testing of the clinical isolate using different testing methods Antimicrobial agent VITEK2 N190a (mg/L) Disc diffusion (mm) Etest (mg/L) Broth microdilution (mg/L) Piperacillin/tazobactam ≥128 0 Ceftazidime ≥64 0 Ceftazidime/avibactam 24.0 Ceftolozane/tazobactam ≥256 Meropenem ≥16 0 ≥32 Imipenem b 0 Ciprofloxacin ≥4 0 Levofloxacin b 0 Gentamicin ≥16 Amikacin 0 Colistin 0.75 1.0 Antimicrobial agent VITEK2 N190a (mg/L) Disc diffusion (mm) Etest (mg/L) Broth microdilution (mg/L) Piperacillin/tazobactam ≥128 0 Ceftazidime ≥64 0 Ceftazidime/avibactam 24.0 Ceftolozane/tazobactam ≥256 Meropenem ≥16 0 ≥32 Imipenem b 0 Ciprofloxacin ≥4 0 Levofloxacin b 0 Gentamicin ≥16 Amikacin 0 Colistin 0.75 1.0 Interpretation of susceptibility testing was in accordance with EUCAST breakpoints. a bioMérieux, card for automated antimicrobial susceptibility testing of Enterobacteriaceae and non-fermenting Gram-negative bacilli. b Derived from the above substance tested in the panel. The clinical and therapeutic significance of KPC-2-producing P. aeruginosa during the current hospitalization of the patient remains unclear, since the patient showed no clinical signs and symptoms of urogenital tract infection. Furthermore, clinical improvement was achieved with a dose-adjusted intravenous meropenem treatment (2 × 1 g owing to impaired renal function with a creatinine clearance of 45.8 mL/min/1.73 m2 according to BIS1) despite a meropenem MIC ≥32.0 mg/L. Clinical improvement therefore likely resulted from adequate therapy for an E. coli bacteraemia rather than from treatment of a P. aeruginosa isolate considered a mere colonizer. Consistently, we were able to cultivate the same isolate in a rectal swab 8 months after its first detection, indicating long-term colonization. P. aeruginosa colonization remains a major risk factor for consequent invasive disease in critically ill patients with increased morbidity and mortality.12 Although only a minority of colonized patients will suffer invasive infection, they may still serve as sources of nosocomial transmission. Huge clonal outbreaks with carbapenem-resistant, particularly KPC-2-producing, P. aeruginosa in hospitalized patients have been repeatedly reported.13 Since similar outbreaks with increased patient morbidity and mortality caused by carbapenem-resistant Enterobacteriaceae (CRE) have been recognized for some time,14 the CDC regularly provides up-to-date infection control recommendations for CRE involving close cooperation between clinical microbiologists, hospital hygienists and clinical practitioners.15 As with CRE-colonized patients, intensified infection control measures are mandatory for hospitalized patients colonized with MDR Gram-negative non-fermenters since MDR accounts for increased morbidity and mortality.16 Proof of KPC-2-producing P. aeruginosa will thus result in consequences for patients’ further healthcare. To date, roughly 25% of carbapenem-resistant P. aeruginosa isolates in Germany express carbapenemases.17 Systematic screening for carbapenemases in P. aeruginosa may therefore not seem worthwhile at first glance. Yet many of the currently recognized carbapenemases rapidly spread even across genus borders, as is the case for KPC-2 located on Tn4401. Colonized patients may serve as a neglected source for the extensive transmission of antimicrobial resistance between a multitude of Gram-negative bacteria. The impressive travel route around the world of KPC-2-producing P. aeruginosa, beginning far away in Columbia, proceeding to the USA, China and Brazil, and finally reaching Europe, highlights the threat of rapid carbapenemase emergence as a worldwide challenge. The most worrisome impact of such spread has recently been further exemplified for GIM-1.18 Since risk factors for MDR bacteria, such as malignancy19 or treatment with multiple antimicrobial substances,20 are regularly present in high-risk populations, we suggest coordinated screening procedures in these patients, as has been recommended for CRE. Conclusions Here, to the best of our knowledge, we report the first case of KPC-2-producing ST235 P. aeruginosa in Europe and also for the first time, to the best of our knowledge, the association of blaKPC-2 with the IncHI1 plasmid. These findings underline the importance of molecular methods for carbapenemase identification, plasmid sequencing and MLST for our epidemiological understanding. Since colonization of the gastrointestinal tract along with Enterobacteriaceae may allow horizontal gene transfer and thus inter-genus spread of MDR, it seems justified to screen Gram-negative non-fermenting bacilli for carbapenemases in high-risk patients based on specific phenotypic data obtained with routine antimicrobial susceptibility testing. To date, P. aeruginosa and possibly other non-fermenters might still be a neglected source of unperceived carbapenemase allocation. Acknowledgements We thank Beate Wirths, Martin Streicher and Susanne Friedrich for excellent technical assistance. We also very much appreciate the support provided by Felix Lange, Jennifer Schauer and Martina Cremanns regarding molecular biological methods. Funding The work of the German National Reference Laboratory for Multidrug-Resistant Gram-negative Bacteria is supported by the Robert Koch Institute with funds provided by the German Ministry of Health (grant no. 1369–402). Transparency declarations N. P. has received speaker fees from bioMérieux. S. G. G. has received speaker fees from Becton Dickinson, bioMérieux and Bio-Rad. All other authors: none to declare. References 1 Yigit H , Queenan AM , Anderson GJ et al. Novel carbapenem-hydrolyzing β-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae . Antimicrob Agents Chemother 2001 ; 45 : 1151 – 61 . Google Scholar CrossRef Search ADS PubMed 2 Kitchel B , Rasheed JK , Patel JB et al. Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolates in the United States: clonal expansion of multilocus sequence type 258 . Antimicrob Agents Chemother 2009 ; 53 : 3365 – 70 . Google Scholar CrossRef Search ADS PubMed 3 Arnold RS , Thom KA , Sharma S et al. Emergence of Klebsiella pneumoniae carbapenemase-producing bacteria . South Med J 2011 ; 104 : 40 – 5 . Google Scholar CrossRef Search ADS PubMed 4 Villegas MV , Lolans K , Correa A et al. 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Antimicrob Agents Chemother 2007 ; 51 : 1967 – 71 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: email@example.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Mar 24, 2018
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