We report the emergence of colistin resistance in Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae after 8 days of colistin-based therapy, resulting in relapse of bloodstream infection and death. Disruption of the mgrB gene by insertion of a mobile genetic element was found to be the mechanism, which was replicated in vitro after exposure to subinhibitory concentrations of colistin and meropenem

Bonomo R.A.

Chen L.

Domitrovic T.N.

Hujer A.M.

Jacobs M.R.

Kanwar A.

Kreiswirth B.N.

Marshall S.H.

Perez F.

Quinones-Mateu M.

Rojas L.J.

Rudin S.D.

Tomas M.

Van Duin D.

Carolina Digital Repository

BRIEF REPORT • OFID • 1Open Forum Infectious DiseasesB R I E F  R E P O R TEmergence of Resistance to Colistin During the Treatment of Bloodstream Infection Caused by Klebsiella pneumoniae Carbapenemase–Producing Klebsiella pneumoniaeAnubhav Kanwar,1,3 Steven H. Marshall,2 Federico Perez,1,2,3,4 Myreen Tomas,1 Michael R. Jacobs,9 Andrea M. Hujer,2,4 T. Nicholas Domitrovic,2,4 Susan D. Rudin,2,4 Laura J. Rojas,2,5 Barry N. Kreiswirth,10 Liang Chen,10 Miguel Quinones-Mateu,1,5  David van Duin,11 Robert A. Bonomo1–8; for the Antibacterial Resistance Leadership Group1Division of Infectious Diseases and HIV Medicine, Department of Medicine, University Hospitals Cleveland Medical Center, Cleveland, Ohio; 2Research Service and 3Geriatric Research Education and Clinical Center, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio; Departments of  4Medicine and  9Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio; Departments of 5Molecular Biology and Microbiology, 6Pharmacology, 7Biochemistry, and 8Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, Cleveland, Ohio; 10Public Health Research Institute Center New Jersey Medical School - Rutgers, The State University of New Jersey, Newark, New Jersey; 11Division of Infectious Diseases, University of North Carolina, Chapel Hill, North CarolinaWe report the emergence of colistin resistance in Klebsiella pneumoniae carbapenemase (KPC)–producing Klebsiella pneumoniae after 8 days of colistin-based therapy, resulting in relapse of bloodstream infection and death. Disruption of the mgrB gene by insertion of a mobile genetic element was found to be the mechanism, which was replicated in vitro after expos-ure to subinhibitory concentrations of colistin and meropenem.Keywords.  carbapenemase; colistin resistance; Klebsiella pneumoniae. Carbapenem-resistant Klebsiella pneumoniae, commonly medi-ated in the United States by production of Klebsiella pneumoniae carbapenemase (KPC), is a cause of difficult-to-treat infections associated with high mortality. Among the only currently available agents with reliable activity against KPC-producing K.  pneumo-niae are polymyxin B, colistin, and combinations containing novel inhibitors of KPC that restore the activity of β-lactams. Therefore, reports describing the decreased activity of ceftazidime-avibactam, one of the combinations, and the emergence of resistance to colis-tin in KPC-producing K. pneumoniae are of special concern [1, 2]. Although the plasmid-mediated mcr genes are increasingly reported as a cause of colistin resistance in Escherichia coli, the most common mechanism of colistin resistance in K. pneumoniae is insertional inactivation of the mgrB gene [1]. The temporal pace and factors leading to colistin resistance through this mechanism are unknown. We report a case of a 52-year-old man (Figure 1) with neutropenia and chronic myelogenous leukemia who devel-oped a central line–associated bloodstream infection with KPC-producing K. pneumoniae. The organism (isolate 1, Kpn918) had decreased ceftazidime-avibactam susceptibility in the absence of previous treatment with that agent. Despite removal of the line and 8 days of combination therapy with meropenem, tigecycline and colistin (loading dose of 5 mg/kg ideal body weight, followed by 1.75 mg/kg every 12 hours, adjusting for a creatinine clearance <50 mL/min), he relapsed with colistin-resistant KPC–K. pneumo-niae bloodstream infection (isolate 2, Kpn926) and died from the infection in the setting of persistent neutropenia.METHODSTo define the molecular mechanism of treatment-emergent colistin resistance and characterize the genetic background and evolution of colistin resistance, we performed the following microbiological tests. Antibiotic susceptibility testing was per-formed with disc diffusion assay and broth microdilution. In the case of colistin, broth macrodilution was performed in triplicate [3]. Results were interpreted according to Clinical and Laboratory Standards Institute guidelines, except for tigecycline and colistin minimum inhibitory concentrations (MICs), which were inter-preted according to guidelines from the European Committee on Antimicrobial Susceptibility Testing [4, 5]. Multilocus sequence typing (MLST), wzi sequencing, and repetitive sequence-based polymerase chain reaction (rep-PCR) assessed genetic relat-edness. Whole-genome sequencing of the isolates Kpn918 and Kpn926 was performed using the MiSeq platform (Illumina Inc., San Diego, CA) and analyzed using ResFinder, PlasmidFinder, and BLAST [6–8]. Additionally, blaKPC, blaSHV, blaNDM, blaVIM, blaIMP, blaTEM, blaOXA-48-like, ompK35/36, mcr-1/2, and mgrB genes were queried using established PCR primers, and plasmid rep-licon typing with PCR was performed [9]. To better understand the evolution of colistin resistance, isolate 1 (Kpn918, colis-tin-susceptible) was exposed to serial passages with 0.25 µg/mL of colistin and 2 µg/mL of meropenem.RESULTSAntibiotic susceptibility testing of both isolates revealed resist-ance to meropenem (MIC > 8  µg/mL), ceftazidime (MIC > 16 µg/mL; zone of inhibition, 6 mm), aztreonam (MIC > 16 µg/mL), and tigecycline (MIC, 2  µg/mL). Ceftazidime-avibactam displayed decreased susceptibility (MIC, 4  µg/mL; zone of Open Forum Infectious Diseases®© Published by Oxford University Press on behalf of Infectious Diseases Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.DOI: 10.1093/ofid/ofy054Received 22 December 2017; editorial decision 27 February 2018; accepted 18 April 2018.Correspondence: R. A. Bonomo, MD, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, 10701 East Boulevard, Cleveland, OH-44106 (robert.bonomo@va.gov).2 • OFID • BRIEF REPORTinhibition, 19 mm). In regards to colistin, isolate 1 demonstrated an MIC of 0.5 µg/mL (susceptible) while isolate 2 had an MIC of 32  µg/mL (resistant). Among antibiotic combinations tested using disc diffusion, only ceftazidime-avibactam combined with aztreonam resulted in an increased zone of inhibition (to 24 mm). Genetic analysis indicated that Kpn918 and Kpn926 belonged to ST258, contained wzi 29, and shared 98.4% similarity according to rep-PCR. Each harbored blaKPC-2, blaSHV-2, and blaTEM-1. Both isolates possessed FIIK plasmids harboring blaKPC-2 on a Tn4401a transposon. Neither contained additional carbapenemase genes or mcr-1/2. IS903, a 1057 base-pair-long sequence was detected within mgrB in isolate 2 (colistin-resistant). Insertional inactiva-tion of mgrB also occurred after 3 serial passages of isolate 1 in 0.25 µg/mL of colistin; however, a different element was found (IS4). In ompK35 and ompK36 from both isolates, we identified mutations that led to a stop codon at amino acid position 50 in ompK35, and insertions encoding glycine and aspartic acid at amino acid positions 134 and 135 in ompK36. Supplementary Figure 1 shows the results of draft whole-genome sequencing.DISCUSSIONThis report describes the occurrence of treatment-emer-gent colistin-resistant KPC–K.  pneumoniae after 8  days of colistin-based combination therapy and highlights important observations. First, colistin resistance emerged due to disrup-tion of mgrB by IS903. Similar emergence of colistin resistance was previously reported after 30 days of colistin therapy due to disruptive insertion of IS4-like insertion sequence into mgrB [1]. Insertion sequences are self-transmissible elements that can integrate into and excise from the chromosomes. The strain of K. pneumoniae infecting this patient belongs to ST258, the most common strain of KPC-producing K.  pneumoniae in the United States and other countries, and insertional events may represent the genetic basis of its success [10, 11]. We hypothesize that this strain is prone to random transposition of insertion sequences, explaining the variability in insertion sequences found in isolate 2 in vitro during exposure to sub-inhibitory concentrations of colistin. The cause of transposi-tion of insertion sequences is not known, although exposure to subinhibitory concentrations of antibiotics may be associ-ated with such events, as reproduced in vitro in the original isolate. Antibiotic-induced bacterial stress responses can lead to insertional mutations that contribute to methicillin resist-ance in Staphylococcus aureus and to deletions of regulatory genes in E. coli resistant to fluoroquinolones [12, 13]. Second, we found that the porin genes ompK35 and ompK36 in both Piperacillin-tazobactamCiprofloxacin Levofloxacin TigecyclineTobramycin ColistinMeropenemMeropenemCeftazidime-avibactamAdmitted with fever,dyspnea, and fatigue,Found to have WBC~154 000 cells/microliterDetected a fever of39.4ºC. Found to havecentral line-associatedKlebsiella oxytocabacteremia. Central lineremovedNegative blood culturesC: Celsius; COL-R: colistin-resistance; ID: infectious disease serviceSep. 1Sep. 4Sep. 5Sep. 9Sep. 10Sep. 12Sep. 15Sep. 18Sep. 19Sep. 20Sep. 25Sep. 26Negative blood cultures Patient diedFever of  38.6ºC followed bydevelopment of  septicshock. ID consulted. Foundto have carbapenem-resistant Klebsiellapneumoniae bacteremia.Central line removedFever of  39°C. Found tohave carbapenem-resistant Klebsiellapneumoniae with COL-R in 2/2 blood culturesMeropenemAntibiotics administeredHospital courseFigure 1.  Timeline of clinical events, laboratory findings, and antibiotics administered during the hospital course. Abbreviations: COL-R, colistin resistance; ID, infectious diseases; WBC, white blood cell.BRIEF REPORT • OFID • 3isolates contained mutations previously linked to reduced sus-ceptibility to ceftazidime-avibactam and carbapenems [2]. Of note, our patient had not been treated with ceftazidime-avibac-tam; therefore, reinfection with a new strain or horizontal gene transfer from a strain previously exposed to ceftazidime-avibac-tam cannot be dismissed. Approximately 5 cases of carbape-nem-resistant K.  pneumoniae occur at our hospital monthly. Although analyses of genetic relatedness suggest that both iso-lates from this case are clonally related, genome sequencing of KPC-producing K. pneumoniae from our hospital reveal that highly similar subpopulations can coexist over time, includ-ing isolates containing blaKPC-2 in IncFIIK plasmids, as in this patient [14]. In conclusion, clinicians should strongly suspect emergence of resistance to colistin during treatment with colis-tin-based combination therapies in patients with persistent or relapsing KPC–K.  pneumoniae bloodstream infection. These observations highlight the dynamic nature of resistance to colistin, often a “last-line agent” in critically ill patients.Supplementary DataSupplementary materials are available at Open Forum Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corre-sponding author.AcknowledgmentsFinancial support. This work was supported by funds from the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) under award numbers R01AI100560, R01AI063517, R21AI114508, and R01AI072219 to R.A.B. This study was also supported in part by funds and/or facilities provided by the Cleveland Department of Veterans Affairs, award number 1I01BX001974 to R.A.B., from the Biomedical Laboratory Research and Development Service of the Department of Veterans Affairs Office of Research and Development, and the Geriatric Research Education and Clinical Center VISN 10 to R.A.B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Department of Veterans Affairs. This work was also supported by the NAIAD of the NIH (UM1AI104681) and the Clinical and Translational Science Collaborative of Cleveland (UL1TR000439) from the National Center for Advancing Translational Sciences component of the NIH and NIH Roadmap for Medical Research.Potential conflicts of interest. All authors: no reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.References1. Cannatelli A, D’Andrea MM, Giani T, et al. In vivo emergence of colistin resist-ance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrob Agents Chemother 2013; 57:5521–6.2. Shields RK, Clancy CJ, Hao B, et al. Effects of Klebsiella pneumoniae carbapene-mase subtypes, extended-spectrum β-lactamases, and porin mutations on the in vitro activity of ceftazidime-avibactam against carbapenem-resistant K. pneumo-niae. Antimicrob Agents Chemother 2015; 59:5793–7.3. Rojas LJ, Salim M, Cober E, et  al; Antibacterial Resistance Leadership Group. Colistin resistance in carbapenem-resistant Klebsiella pneumoniae: laboratory detection and impact on mortality. Clin Infect Dis 2017; 64:711–8.4. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. CLSI supplement M100. 27th ed. Wayne, PA: Clinical Laboratory Standards Institute; 2017.5. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for Interpretation of MICs and Zone Diameters. Version 8.0. 2018. Basel, Switzerland: European Society of Clinical Microbiology and Infectious Diseases. Available at: http://www.eucast.org. Accessed 1 January 2018.6. 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Emergence of resistance to colistin during the treatment of bloodstream infection caused by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae

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Emergence of resistance to colistin during the treatment of bloodstream infection caused by Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae

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