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Diagnostic Microbiology and Infectious Disease 72 (2012) 363 – 366 www.elsevier.com/locate/diagmicrobio
Case Report
Fecal colonization of VIM-1–producing Klebsiella pneumoniae and in vivo transfer of multidrug-resistant IncN plasmid in a renal transplant patient☆ Umaer Naseer a , Bjørn Odvar Eriksen b , Arnfinn Sundsfjord a, c , Ørjan Samuelsen c,⁎ a
Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, 9038 Tromsø, Norway b Section of Nephrology, University Hospital of North Norway, 9038 Tromsø, Norway c Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, 9038 Tromsø, Norway Received 20 October 2011; accepted 19 December 2011
Abstract We report a case of long-term colonization of a carbapenemase (VIM)-producing Klebsiella pneumoniae clone in a renal transplant patient and demonstrate the in vivo transmission of a broad-host-range multidrug-resistant IncN plasmid containing blaVIM, blaSHV-12, and qnrS to Escherichia coli. © 2012 Elsevier Inc. All rights reserved. Keywords: VIM-1; Colonization; Plasmid; Resistance; IncN
1. Introduction Carbapenemases, including Klebsiella pneumoniae carbapenemase (KPC), metallo-β-lactamases (e.g., VIM and NDM-1), and OXA-48, are rapidly emerging in clinical relevant Gram-negative bacteria (Walsh, 2010). They mediate resistance to virtually all β-lactam antibiotics and disseminate through clonal spread and on mobile genetic elements often linked to other resistance determinants (Walsh, 2010). K. pneumoniae is an important nosocomial pathogen, and the increasing prevalence of multidrugresistant carbapenemase-producing K. pneumoniae worldwide is of particular concern as it limits therapeutic options and requires firm infection control measures. VIM-producing K. pneumoniae has been associated with hospital outbreaks in several countries particularly in the Mediterranean region (Walsh, 2010). Community-onset infections by VIM-producing K. pneumoniae are now also being reported ☆ This work was supported by a grant from the Northern Norway Regional Health Authority. ⁎ Corresponding author. Tel.: +47-77627043. E-mail address:
[email protected] (Ø. Samuelsen).
0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2011.12.010
(Poulou et al., 2010). However, there are limited scientific knowledge on the gastrointestinal colonization of carbapenemase-producing Enterobacteriaceae and unresolved issues with regard to adequate fecal screening systems (Carmeli et al., 2010).
2. Case report In August 2007, a blaVIM-1–positive K. pneumoniae isolate (K45-67) was recovered from a urethral stent used for draining a lymphocele in a patient undergoing a kidney transplant at Rikshospitalet University Hospital in Oslo, Norway (Samuelsen et al., 2011). Upon inquiry, a foreign link was determined, as the patient had undergone haemodialysis in Turkey 1 year prior to the transplant. Subsequently, the patient agreed to be sampled regularly at hospital admissions/polyclinic controls at the University Hospital of North Norway in order to monitor the gastrointestinal colonization of the strain K45-67 to determine whether increased infection control measures were required (a written informed consent was obtained from the patient for the use of clinical data). Twelve fecal samples were taken
− − + − − −
a AMC = Amoxicillin clavulanic acid; TZP = piperacillin tazobactam; CAZ = ceftazidime; FEP = cefepime; ATM = aztreonam; MEM = meropenem; IPM = imipenem; ETP = ertapenem; GEN = gentamicin; TOB = tobramycin; AMK = amikacin; CIP = ciprofloxacin; TIG = tigecycline; COL = colistin; SXT = trimethoprim–sulfamethoxazole.
N32 N32 N32 N32 N32 N32 0.5 0.5 0.5 0.5 1 1
COL TIG
1 1 1 2 2 2 N32 N32 N32 N32 N32 N32
CIP AMK
2 2 4 4 4 4 4 8 8 8 8 8
TOB GEN
2 2 4 4 4 4 2 0.5 1 1 2 1
ETP IPM
8 1 8 2 4 2 2 0.5 2 1 2 2
MEM ATM
128 32 64 128 128 256 64 16 64 32 64 64
FEP CAZ
N256 N256 N256 N256 N256 N256 N256 128 256 N256 N256 N256
TZP TEM-3
+ + + + + +
− + − − − −
SHV-11
+ − − + + + + + + + + + 15/8/2007 17/10/2007 24/10/2007 22/12/2007 5/5/2008 13/6/2008
K. pneumoniae E. coli E. coli K. pneumoniae K. pneumoniae K. pneumoniae
SHV-12
TEM-1
QnrS
64 N256 32 N256 N256 N256
AMC VIM-1
+ + + + + +
Antimicrobial susceptibility, MIC (mg/L) a Resistance determinants Species
K45-67 K46-61 K46-72 K47-76 K49-6 K49-47
This study documents the long-term fecal colonization by a multidrug-resistant VIM-producing K. pneumoniae clone and the in vivo spread of blaVIM-1 by means of plasmid transfer from K. pneumoniae to E. coli in vivo. Fecal presence of the VIM-producing K. pneumoniae strain was documented 10 months after its initial isolation in August
Date of isolation
3. Discussion
Isolate
during August 2007–November 2009 (1 per month over the first 10 months, and 2 per year over 1 year) and screened for bacterial growth with reduced susceptibility towards ceftazidime both directly on agar media with discs and after enrichment in brain hearth infusion broth containing ceftazidime (10 mg/L) and vancomycin (6 mg/L). Five fecal samples tested positive for isolates with reduced susceptibility to ceftazidime; 3 K. pneumoniae and 2 Escherichia coli isolates, spanning over a period of 10 months (August 2007–June 2008). Etests (bioMérieux, Marcy l'Etoile, France) were performed on the index isolate as well as on the 5 recovered isolates and interpreted according to the clinical breakpoints set by the European Committee for Antimicrobial Susceptibility Testing (EUCAST). All isolates expressed high-level resistance to penicillins, cephalosporins, and aztreonam, while the carbapenem MICs were variable (Table 1). Furthermore, all isolates were resistant to ciprofloxacin, trimethoprim– sulfamethoxazole, nitrofurantoin, and intermediate susceptible or resistant to tobramycin. Isolates were susceptible to amikacin, tigecycline, and colistin. Isolates were further screened by polymerase chain reactions (PCRs) and subsequent sequencing for the detection of highly prevalent β-lactamase genes (blaTEM, blaSHV, blaCTX-M, and blaVIM) as well as plasmid-mediated quinolone-resistance genes (qnrA, qnrB, and qnrS) to explore the linkage of qnr genes to β-lactamase genes (Samuelsen et al., 2011). All 6 isolates were positive for blaVIM-1 (Table 1). In addition, all K. pneumoniae isolates were positive for blaSHV-11, blaSHV-12, and qnrS, and all E. coli isolates were positive for blaSHV-12 and qnrS. Furthermore, 1 E. coli isolate was positive for blaTEM-1 and the other for blaTEM-3, a TEM-ESBL. Genetic fingerprinting of the 6 isolates confirmed clonal relationships (Tenover et al., 1995) between the 4 K. pneumoniae (indistinguishable) and between the 2 E. coli isolates (2-band difference) by XbaIpulsed field gel electrophoresis (PFGE, data not shown) (Naseer et al., 2009). Plasmid profiling by S1 nuclease PFGE revealed the presence of a single plasmid of approximately 50 kb in all K. pneumoniae isolates, and 3 plasmids of approximately 50, 100, and 145 kb were detected in the E. coli isolates (Fig. 1A). Southern blot followed by hybridization with a specific blaVIM probe identified the 50-kb plasmid as the blaVIM-1 carrier in all isolates (Fig. 1B). PCRbased replicon typing and subsequent hybridization typed this plasmid to the IncN family (Fig. 1C) (Carattoli et al., 2005; Naseer et al., 2009).
SXT
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Table 1 Recovered VIM-producing isolates and relevant characteristics
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Fig. 1. A) S1-nuclease digested plasmid PFGE of blaVIM-1–positive K. pneumoniae (n = 4) and E. coli (n = 2) isolates. B) Hybridization with blaVIM-specific DNA probe. C) Hybridization with IncN-specific DNA probe. Content in lane: M, PFGE low-range marker; 1, K45-67 (K. pneumoniae initial isolate); 2, K46-61 (E. coli); 3, K46-72 (E. coli); 4, K47-76 (K. pneumoniae); 5, K49-6 (K. pneumoniae); 6, K49-47 (K. pneumoniae); 7, negative control (K. pneumoniae).
2007. VIM-producing K. pneumoniae is very rarely isolated in Norway (Samuelsen et al., 2011). The only identified risk factor for VIM-producing K. pneumoniae acquisition in this patient was the haemodialysis performed in Turkey in 2006, a country with a high prevalence of VIM-producing K. pneumoniae (Walsh, 2010). Thus, the patient had been colonized by VIM-producing K. pneumoniae for almost 2 years. During this period, no infection was known to be caused by the VIM-producing bacteria. However, the patient had been treated with several courses of antibiotics including trimethoprim–sulfamethoxazole for 6 months after the kidney transplant (standard prophylactic treatment), doxycycline, amoxicillin, clarithromycin, and azithromax for respiratory/lung infections. The patient was considered negative for fecal carriage in November 2009 (3 consecutive negative samples). During the colonization period, the strain maintained its multidrug resistance profile and apparently transferred the 50-kb IncN plasmid carrying blaVIM-1 to E. coli. The presence of blaSHV-12 and qnrS in all isolates suggests colocalization on the same plasmid. The broad-host-range properties of IncN plasmids have previously been shown to be important in the endemic spread of blaVIM-1 in various countries (Psichogiou et al., 2008; Walsh, 2010), in addition to its importance in the worldwide spread of CTX-M group 1 extended spectrum β-lactamases and KPC-2 (Naseer and Sundsfjord, 2011). In conclusion, this study presents evidence for import and long-term colonization of a VIM-producing K. pneumoniae clone and in vivo plasmid dissemination. These results also highlight the importance of policies for fecal screening of patients identified with, or at risk of, colonization with carbapenemase-producing Enterobacteriaceae and the implementation of targeted infection control measures upon readmission to hospitals. This is particularly relevant for patients undergoing dialysis or renal transplantation (Wong et al., 2007). Educating patients as well as health care personnel regarding hand hygiene and disinfection procedures when performing dialysis is also important. Several studies have
highlighted travel as an important risk factor for infections with virulent multidrug-resistant strains of Enterobacteriaceae (Pitout et al., 2009; Tangden et al., 2010). Thus, in countries with a low prevalence of carbapenemase-producing clinically relevant Gram-negative bacteria, screening should be advised on patients who have been hospitalized in countries with known high prevalence rates of these bacteria. Acknowledgment Bjørg Haldorsen, Bettina Aasnæs, and Kine Susann Waade Edvardsen are acknowledged for excellent technical assistance. References Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ (2005) Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63:219–228. Carmeli Y, Akova M, Cornaglia G, Daikos GL, Garau J, Harbarth S, Rossolini GM, Souli M, Giamarellou H (2010) Controlling the spread of carbapenemase-producing Gram-negatives: therapeutic approach and infection control. Clin Microbiol Infect 16:102–111. Naseer U, Sundsfjord A (2011) The CTX-M conundrum: dissemination of plasmids and Escherichia coli clones. Microb Drug Resist 17:83–97. Naseer U, Haldorsen B, Tofteland S, Hegstad K, Scheutz F, Simonsen GS, Sundsfjord A; Norwegian ESBL Study Group (2009) Molecular characterization of CTX-M-15-producing clinical isolates of Escherichia coli reveals the spread of multidrug-resistant ST131 (O25:H4) and ST964 (O102:H6) strains in Norway. APMIS 117:526–536. Pitout JD, Campbell L, Church DL, Gregson DB, Laupland KB (2009) Molecular characteristics of travel-related extended-spectrum-β-lactamase-producing Escherichia coli isolates from the Calgary Health Region. Antimicrob Agents Chemother 53:2539–2543. Poulou A, Spanakis N, Pournaras S, Pitiriga V, Ranellou K, Markou F, Tsakris A (2010) Recurrent healthcare-associated community-onset infections due to Klebsiella pneumoniae producing VIM-1 metallo-betalactamase. J Antimicrob Chemother 65:2538–2542. Psichogiou M, Tassios PT, Avlamis A, Stefanou I, Kosmidis C, Platsouka E, Paniara O, Xanthaki A, Toutouza M, Daikos GL, Tzouvelekis LS (2008) Ongoing epidemic of blaVIM-1-positive Klebsiella pneumoniae in Athens, Greece: a prospective survey. J Antimicrob Chemother 61:59–63.
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