Scandinavian Journal of Infectious Diseases, 2014; 46: 320–324
SHORT COMMUNICATION
Emergence of OXA-162-producing Klebsiella pneumoniae in Hungary
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LAURA JÁNVÁRI1, IVELINA DAMJANOVA2, ANDREA LÁZÁR3, KATALIN RÁCZ4, BÉLA KOCSIS5, EDIT URBÁN3 & ÁKOS TÓTH1 From the 1Department of Bacteriology, National Centre for Epidemiology, Budapest, 2Department of Phage Typing and Molecular Epidemiology, National Centre for Epidemiology, Budapest, 3Department of Clinical Microbiology, Albert Szent-Györgyi Clinical Centre, University of Szeged, Szeged, 4Department of Paediatric Clinic, Albert Szent-Györgyi Clinical Centre, University of Szeged, Szeged, and 5Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary
Abstract In August 2012, 2 carbapenem-resistant Klebsiella pneumoniae isolates from the University of Szeged were submitted to the National Reference Laboratory at the National Centre for Epidemiology to confirm the carbapenem resistance mechanism. PCR assays and sequencing revealed that the isolates harboured the blaOXA-162 carbapenemase gene, a very recently described variant of OXA-48, and the blaCTX-M-15 extended-spectrum β-lactamase gene. The isolates had indistinguishable PFGE patterns and belonged to sequence type ST15. To the best of our knowledge, this is the first description of OXA-48-like carbapenemase-producing bacteria in Hungary and of an OXA-162-type carbapenemase gene in the K. pneumoniae ST15 clone.
Keywords: OXA-162, ST15, carbapenemase, Hungary, Klebsiella pneumoniae
Introduction Carbapenem resistance among Enterobacteriaceae due to the production of carbapenemase enzymes is a growing clinical problem [1]. Based on the mechanism, carbapenemases can be either Ambler class A β-lactamases (e.g. KPC), metallo-β-lactamases (MBLs; e.g. VIM, NDM), or class D β-lactamases (OXA-48-like) [2]. Since carbapenemase-producing strains usually show a multidrug-resistant phenotype, the antibiotic therapy of infections caused by such strains is greatly limited. In Hungary, the first carbapenemase-producing strains of Enterobacteriaceae appeared in 2008, when a KPC-2-producing Klebsiella pneumoniae ST258 clone disseminated in the north-eastern part of Hungary [3]. The following year, VIM-type MBL-producing Enterobacteriaceae strains also emerged [4], and since then this has become the main carbapenemase type in Hungary. During the y 2010–2011, rapid country-wide dissemination of the VIM-4-producing
ST15 K. pneumoniae Hungarian Epidemic Clone was observed in Hungary [5,6]. In August 2012, a 1-month-old infant with tetralogy of Fallot was admitted to the paediatric intensive care unit (ICU) of the University of Szeged from a Romanian hospital for an elective surgical intervention. Prophylactic antibiotic therapy with meropenem (3 ⫻ 100 mg per day) was applied. Four weeks later, a K. pneumoniae isolate was cultured from an endotracheal aspirate sample, which was resistant to almost all antibiotics tested, even the carbapenems, thus the antibiotic regimen was changed to colistin. Two months later the patient died due to his underlying disease. After the isolation of the carbapenem-resistant K. pneumoniae, all infants treated in the same ward, close to the first patient, were screened weekly for the carriage of multidrug-resistant Enterobacteriaceae using BrillianceTM ESBL agar (Oxoid). A further carbapenem-resistant K. pneumoniae was isolated
Correspondence: Á. Tóth, National Centre for Epidemiology, Albert Flórián út 2–6, 1097, Budapest, Hungary. Tel: ⫹ 36 1 476 1337. E-mail: toth.akos@ oek.antsz.hu (Received 29 August 2013 ; accepted 18 December 2013) ISSN 0036-5548 print/ISSN 1651-1980 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2013.879993
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OXA-162-producing K. pneumoniae in Hungary from the peri-anal sample of a 4-month-old boy with aortic coarctation; he was being treated with antibiotics due to a previous urinary tract infection caused by an extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae. His antibiotic therapy was changed from imipenem to colistin. The two positive infants received cohort care. Three months later he recovered and was discharged from the hospital. These 2 carbapenem-resistant K. pneumoniae isolates recovered from the University of Szeged were submitted to the National Centre for Epidemiology (NCE) to confirm the carbapenem resistance mechanism. The isolates proved to be OXA-48-like carbapenemase producers. The aim of this study was to characterize the first OXA-48-like carbapenemase-producing K. pneumoniae isolates in Hungary. Materials and methods Identification of the isolates at the NCE was carried out using the Micronaut-E semi-automatic identification system (Genzyme Virotech GmbH, Ruesselsheim, Germany). Antibiotic susceptibility was determined by gradient minimum inhibitory concentration (MIC) test strips (Liofilchem, Roseto, Italy),
and the results were interpreted in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (http://www. eucast.org). The putative production of a carbapenemase was tested by Carba NP test [7] and by modified Hodge test performed on a Mueller–Hinton plate containing 20 mg/ml bile [8]. Aminophenylboronic acid, dipicolinic acid, and cloxacillin in a combined disk test with meropenem was used for further classification of the carbapenemase enzyme [9]. The presence of carbapenemase [10], ESBL [11], acquired AmpC-encoding genes [12], and plasmid-mediated quinolone resistance determinants [13–18] was verified using PCR followed by sequencing. The possible clonal relationships among isolates were investigated by pulsed-field gel electrophoresis (PFGE) [11] and multilocus sequence typing (MLST) [19]. Mating assays with Escherichia coli using the rifampicinresistant Escherichia coli J53 as recipient, plasmid profile analysis, and plasmid replicon typing were performed as described previously [11,20–22]. Results Both carbapenem-resistant K. pneumoniae isolates were positive by the Carba NP test and by the modified
Table I. Clinical and in vitro characterization of OXA-162-producing Klebsiella pneumoniae isolates and the OXA-162-producing Escherichia coli J53 transconjugant. Isolate
Sex/age Specimen Pulsotype Sequence type ESBL type PMQRb MIC (mg/l) CAZ CTX IMP MEM ETP CO TGC CIP AK CN TOB SXT
321
5825/12
5868/12
J53
T5868a
Male/0 y Lower respiratory tract KP046 ST15 blaCTX-M-15 Negative
Male/0 y Peri-anal swab KP046 ST15 blaCTX-M-15 Negative
NA NA ND ND ND ND
NA NA ND ND Negative Negative
⬎ 256 ⬎ 256 8 8 ⬎ 32 1 4 ⬎ 32 2 64 8 16
128 ⬎ 256 4 8 ⬎ 32 1 8 ⬎ 32 8 32 8 4
0.125 0.032 0.125 0.006 0.004 1 0.25 0.008 0.25 0.064 0.125 0.032
0.064 0.25 0.5 0.064 0.25 1 0.25 0.004 0.5 0.25 0.125 0.008
NA, not applicable; ND, not determined; ESBL, extended-spectrum β-lactamase; PMQR, plasmidmediated quinolone resistance determinants; MIC, minimum inhibitory concentration; CAZ, ceftazidime; CTX, cefotaxime; IMP, imipenem; MEM, meropenem; ETP, ertapenem; CO, colistin; TGC, tigecycline; CIP, ciprofloxacin; AK, amikacin; CN, gentamicin; TOB, tobramycin; SXT, trimethoprim/ sulfamethoxazole. aT5868: transconjugant of 5868/12. bPMQR: plasmid-mediated quinolone resistance determinants (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6)-Ibcr, qepA, oqxAB).
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Discussion
Figure 1. Plasmid profiles of OXA-162-producing Klebsiella pneumoniae isolate 5868/12 and its transconjugant. Lanes M1 and M2, molecular weight markers; lane 1, 5868/12; lane 2, T5868.
Hodge test for carbapenemase production. We observed no synergism with meropenem in the presence of any inhibitors. The isolates were resistant to extended-spectrum cephalosporins, ertapenem, ciprofloxacin, gentamicin, tobramycin, and tigecycline, and intermediately susceptible to imipenem and meropenem. They remained susceptible only to colistin (Table I). PCR assays and sequencing revealed that the isolates harboured the blaOXA-162 carbapenemase gene and the blaCTX-M-15 ESBL gene. The ~60 kb IncL/M type plasmid carrying blaOXA-162 was successfully transferred to the recipient strain. Only 1 plasmid was found to have been possessed by the transconjugant, as was revealed by plasmid profiling (Figure 1). The carbapenem MIC values of the transconjugant (T5868) were slightly increased (Table I), but they remained within the susceptible category. Both isolates investigated in this study belonged to a previously identified Hungarian pulsotype designated KP046 and to the sequence type ST15 (Figure 2).
To the best of our knowledge, this is the first description of OXA-48-like carbapenemase-producing bacteria in Hungary and of the OXA-162-type carbapenemase gene in the K. pneumoniae ST15 international clone. OXA-48-like carbapenemases were first described in K. pneumoniae isolates in Turkey in 2004, and have since been found in several European countries (e.g. France, Belgium, Germany, the Netherlands, Italy, the UK, Ireland, Switzerland, Slovenia, and Spain) and non-European countries (e.g. Lebanon, Tunisia, Morocco, Israel, and India) [23]. Generally such cases have been sporadic, however a few outbreaks have also been reported [24]. OXA-162, a novel variant of OXA-48, has recently been identified in K. pneumoniae in Turkey (2012) [25]. Very recently, this variant has also been reported from Germany, in 3 different species [23]. In 2009 the CTX-M-producing KP046 clone caused an outbreak in the maternity ward of a hospital in Budapest, and during the following year this clone appeared in 3 paediatric wards in different hospitals [26]. These isolates were ciprofloxacin-resistant and CTX-M-positive like our OXA-162-producers, however they proved to be carbapenem-susceptible and carbapenemase-non-producers. Although, these OXA-162-producing isolates and the Hungarian Epidemic Clone share the same sequence type, the similarity of their PFGE patterns is below 85%, making a common descent unlikely. The origin of the blaOXA-162 gene in Hungary is unknown, however 2 different explanations for its presence can be proposed: (1) as the K. pneumoniae KP046 clone is circulating among Hungarian paediatric wards, it might have acquired the OXA-162 gene-harbouring plasmid from another species [23], which might have been carried asymptomatically by the Romanian patient; (2) it can be suspected that the OXA-162-positive K. pneumoniae ST15 clone was imported from Romania via cross-border transfer. This latter hypothesis might be supported by the fact that the K. pneumoniae ST15 clone has been detected among OXA-48-like-producing isolates in Europe [27,28]. Based on a report by the European Centre for Disease Prevention and Control [2], the NCE released guidance on the management of carbapenemase-producing Enterobacteriaceae in 2011. This guidance recommends active surveillance of high-risk patients, especially those transferred from healthcare facilities abroad. In accordance with these recommendations, active screening has been performed in the paediatric department of the University of Szeged in an attempt
OXA-162-producing K. pneumoniae in Hungary
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Isolate ID 98
100
96
94
92
klebsiepi 90
88
86
82
80
78
76
74
72
70
68
66
64
62
60
klebsiepi
84
85%
Dice (Opt:1.00%) (Tol 1.0%-2.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
233/03 K167/05 369/05 K115/11 K135/12 K130/11 K131/11 K132/11 K14/12 K149/11 K140/11 K241/09 K242/09 K42/10 5825/12 5868/12 K224/09 K227/09 K232/09 K4/10 K84/06 K9/07 K81/06/1 K94/06/1 K82/12 K89/11 K83/12 K177/07 K294/05/1 381/08 K260/05 K110/12 K116/10 K105/10 K107/10 K119/10 K106/10 K273/05 K93/05 161/05 K183/11 K134/06 K138/06 K4/08/1
Year of isolation/ Center 2003/FJ 1 2005/FJ 2 2005/BP 2 2011/CS 2012/SM 2011/BAZ 3 2011/BAZ 2 2011/BAZ 1 2012/BAZ 2 2011/BAZ 1 2011/BAZ 3 2009/BP 1 2009/BP 1 2010/BP 3 2012/CS 2012/CS 2009/BP 1 2009/BP 1 2009/BP 1 2010/NG 2006/BP 4 2007/PT 1 2006/BP 4 2007/PT 1 2012/BAZ 1 2011/BAZ 4 2012/BAZ 1 2007/BP 5 2005/BP 6 2008/BAZ 4 2005/BP 7 2012/BK 1 2010/PT 2 2010/PT 2 2010/PT 2 2010/BP 8 2010/PT 2 2005/BP 7 2005/BP 7 2005/BP 7 2011/BK 2 2006/BP 3 2006/BP 3 2008/PT 1
Pulsotype/ Sequence type N/ST15 N/ST15 N/ST15 N/ST15 N N N N N N N KP046 KP046 KP046 KP046/ST15 KP046/ST15 KP046 KP046 KP046 KP046 Z/ST525 Z Z Z Z Z Z S/ST11 S/ST11 S/ST258 S/ST11 KP053 KP053 KP053 KP053 KP053/ST11 KP053 R/ST147 R/ST147 R/ST147 R/ST147 L/ST274 L L
323
Sample
blood blood urine blood bloodC trachea throath urine urine wound blood stool stool stool trachea perianal urine stool stool stool stool throat blood blood blood urine blood blood blood urine blood wound urine urine urine throath urine CVC blood urine blood CSF blood blood
N/ ST15 (Hungarian Epidemic Clone)
KP046/ ST15
Z/ ST525
S/ ST11 or ST258 KP053/ ST11 R/ ST147
L/ ST274
Figure 2. Cluster analysis of macrorestriction profiles of ESBL- and/or carbapenemase-producing Klebsiella pneumoniae epidemic clones in Hungary.
to halt the further dissemination of the OXA-162producing K. pneumoniae ST15 clone.
Acknowledgements We thank Wenna Pei for her excellent work reviewing and editing this article. Declaration of interest: All authors declare that they have no conflicts of interest. References [1] Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011;17:1791–8. [2] European Centre for Disease Prevention and Control. Risk assessment on the spread of carbapenemase-producing Enterobacteriaceae (CPE) through patient transfer between healthcare facilities, with special emphasis on cross-border transfer. Stockholm: ECDC; 2011.
[3] Tóth Á, Damjanova I, Puskás E, Jánvári L, Farkas M, Dobák A, et al. Emergence of a colistin-resistant KPC-2-producing Klebsiella pneumoniae ST258 clone in Hungary. Eur J Clin Microbiol Infect Dis 2010;29:765–9. [4] Kristóf K, Tóth Á, Damjanova I, Jánvári L, Konkoly-Thege M, Kocsis B, et al. Identification of a blaVIM-4 gene in the internationally successful Klebsiella pneumoniae ST11 clone and in a Klebsiella oxytoca strain in Hungary. J Antimicrob Chemother 2010;65:1303–5. [5] Damjanova I, Jánvári L, Kristóf K, Szabó D, Kenesei E, Szikra L, et al. Carbapenemase-producing Enterobacteriaceae strains in Hungary. Poster1683, 22nd European Congress of Clinical Microbiology and Infectious Diseases. 31 March- 3 April 2012, London, United Kingdom. [6] Melegh S, Kovács K, Gám T, Nyul A, Patkó B, Tóth A, et al. Emergence of VIM-4 metallo-β-lactamase-producing Klebsiella pneumoniae ST15 clone in the Clinical Centre University of Pécs, Hungary. Clin Microbiol Infect 2013 [Epub ahead of print]. doi: 10.1111/1469-0691.12293 [7] Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2012;18:1503–7. [8] Lee K, Kim CK, Yong D, Jeong SH, Yum JH, Seo YH, et al. Improved performance of the modified Hodge test with MacConkey agar for screening carbapenemase-producing
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L. Jánvári et al. Gram-negative bacilli. J Microbiol Methods 2010;83:149– 52. Giske CG, Gezelius L, Samuelsen Ø, Warner M, Sundsfjord A, Woodford N. A sensitive and specific phenotypic assay for detection of metallo-β-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect 2011;17:552–6. Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011;70:119–23. Damjanova I, Tóth Á, Pászti J, Hajbel-Vékony G, Jakab M, Berta J, et al. Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15type β-lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’? J Antimicrob Chemother 2008;62:978–85. Pérez-Pérez FJ, Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002,40:2153–62. Robicsek A, Strahilevitz J, Sahm DF, Jacoby GA, Hooper DC. Qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob Agents Chemother 2006;50:2872–4. Cavaco LM, Hasman H, Xia S, Aarestup FM. qnrD a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin. Antimicrob Agents Chemother 2009; 53:603–8. Wang M, Guo Q, Xu X, Wang X, Ye X, Wu S, et al. New plasmid-mediated quinolone resistance gene qnrC found in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother 2009;53:1892–7. Kim HB, Wang M, Park CH, Kim EC, Jacoby GA, Hooper DC. oqxAB encoding a multidrug efflux pump in human clinical isolates of Enterobacteriaceae. Antimicrob Agents Chemother 2009;53:3582–4. Yamane K, Wachino J, Suzuki S, Arakawa Y. Plasmid mediated qepA gene among Escherichia coli clinical isolates from Japan. Antimicrob Agents Chemother 2008;52: 1564–6.
[18] Park CH, Robicsek A, Jacoby GA, Sahm D, Hooper DC. Prevalence in the United States of aac(6’)-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother 2006;50:3953–5. [19] Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol 2005;43:4178–82. [20] Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005;63:219–28. [21] Poirel L, Bonnin RA, Nordmann P. Genetic features of the widespread plasmid coding for the carbapenemase OXA-48. Antimicrob Agents Chemother 2012;56:559–62. [22] Villa L, García-Fernández A, Fortini D, Carattoli A. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J Antimicrob Chemother 2010;65: 2518–29. [23] Pfeifer Y, Schlatterer K, Engelmann E, Schiller RA, Frangenberg HR, Stiewe D, et al. Emergence of OXA-48-type carbapenemase-producing Enterobacteriaceae in German hospitals. Antimicrob Agents Chemother 2012;56:2125–8. [24] Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother 2012;67:1597–606. [25] Kasap M, Torol S, Kolayli F, Dundar D, Vahaboglu H. OXA162, a novel variant of OXA-48 displays extended hydrolytic activity towards imipenem, meropenem and doripenem. J Enzyme Inhib Med Chem 2013;28:990–6. [26] Damjanova I. Molecular epidemiological investigation of SHV- and CTX-M-type extended-spectrum β-lactamaseproducing Klebsiella pneumoniae isolates in Hungary. PhD dissertation. Budapest: Semmelweis University; 2011. [27] Österblad M, Kirveskari J, Hakanen AJ, Tissari P, Vaara M, Jalava J. Carbapenemase-producing Enterobacteriaceae in Finland: the first years (2008–11). J Antimicrob Chemother 2012;67:2860–4. [28] Lascols C, Peirano G, Hackel M, Laupland KB, Pitout JD. Surveillance and molecular epidemiology of Klebsiella pneumoniae isolates that produce carbapenemases: first report of OXA-48-like enzymes in North America. Antimicrob Agents Chemother 2013;57:130–6.
SHORT COMMUNICATION
Emergence of OXA-162-producing Klebsiella pneumoniae in Hungary
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LAURA JÁNVÁRI1, IVELINA DAMJANOVA2, ANDREA LÁZÁR3, KATALIN RÁCZ4, BÉLA KOCSIS5, EDIT URBÁN3 & ÁKOS TÓTH1 From the 1Department of Bacteriology, National Centre for Epidemiology, Budapest, 2Department of Phage Typing and Molecular Epidemiology, National Centre for Epidemiology, Budapest, 3Department of Clinical Microbiology, Albert Szent-Györgyi Clinical Centre, University of Szeged, Szeged, 4Department of Paediatric Clinic, Albert Szent-Györgyi Clinical Centre, University of Szeged, Szeged, and 5Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary
Abstract In August 2012, 2 carbapenem-resistant Klebsiella pneumoniae isolates from the University of Szeged were submitted to the National Reference Laboratory at the National Centre for Epidemiology to confirm the carbapenem resistance mechanism. PCR assays and sequencing revealed that the isolates harboured the blaOXA-162 carbapenemase gene, a very recently described variant of OXA-48, and the blaCTX-M-15 extended-spectrum β-lactamase gene. The isolates had indistinguishable PFGE patterns and belonged to sequence type ST15. To the best of our knowledge, this is the first description of OXA-48-like carbapenemase-producing bacteria in Hungary and of an OXA-162-type carbapenemase gene in the K. pneumoniae ST15 clone.
Keywords: OXA-162, ST15, carbapenemase, Hungary, Klebsiella pneumoniae
Introduction Carbapenem resistance among Enterobacteriaceae due to the production of carbapenemase enzymes is a growing clinical problem [1]. Based on the mechanism, carbapenemases can be either Ambler class A β-lactamases (e.g. KPC), metallo-β-lactamases (MBLs; e.g. VIM, NDM), or class D β-lactamases (OXA-48-like) [2]. Since carbapenemase-producing strains usually show a multidrug-resistant phenotype, the antibiotic therapy of infections caused by such strains is greatly limited. In Hungary, the first carbapenemase-producing strains of Enterobacteriaceae appeared in 2008, when a KPC-2-producing Klebsiella pneumoniae ST258 clone disseminated in the north-eastern part of Hungary [3]. The following year, VIM-type MBL-producing Enterobacteriaceae strains also emerged [4], and since then this has become the main carbapenemase type in Hungary. During the y 2010–2011, rapid country-wide dissemination of the VIM-4-producing
ST15 K. pneumoniae Hungarian Epidemic Clone was observed in Hungary [5,6]. In August 2012, a 1-month-old infant with tetralogy of Fallot was admitted to the paediatric intensive care unit (ICU) of the University of Szeged from a Romanian hospital for an elective surgical intervention. Prophylactic antibiotic therapy with meropenem (3 ⫻ 100 mg per day) was applied. Four weeks later, a K. pneumoniae isolate was cultured from an endotracheal aspirate sample, which was resistant to almost all antibiotics tested, even the carbapenems, thus the antibiotic regimen was changed to colistin. Two months later the patient died due to his underlying disease. After the isolation of the carbapenem-resistant K. pneumoniae, all infants treated in the same ward, close to the first patient, were screened weekly for the carriage of multidrug-resistant Enterobacteriaceae using BrillianceTM ESBL agar (Oxoid). A further carbapenem-resistant K. pneumoniae was isolated
Correspondence: Á. Tóth, National Centre for Epidemiology, Albert Flórián út 2–6, 1097, Budapest, Hungary. Tel: ⫹ 36 1 476 1337. E-mail: toth.akos@ oek.antsz.hu (Received 29 August 2013 ; accepted 18 December 2013) ISSN 0036-5548 print/ISSN 1651-1980 online © 2014 Informa Healthcare DOI: 10.3109/00365548.2013.879993
Scand J Infect Dis Downloaded from informahealthcare.com by University of Utah on 11/25/14 For personal use only.
OXA-162-producing K. pneumoniae in Hungary from the peri-anal sample of a 4-month-old boy with aortic coarctation; he was being treated with antibiotics due to a previous urinary tract infection caused by an extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae. His antibiotic therapy was changed from imipenem to colistin. The two positive infants received cohort care. Three months later he recovered and was discharged from the hospital. These 2 carbapenem-resistant K. pneumoniae isolates recovered from the University of Szeged were submitted to the National Centre for Epidemiology (NCE) to confirm the carbapenem resistance mechanism. The isolates proved to be OXA-48-like carbapenemase producers. The aim of this study was to characterize the first OXA-48-like carbapenemase-producing K. pneumoniae isolates in Hungary. Materials and methods Identification of the isolates at the NCE was carried out using the Micronaut-E semi-automatic identification system (Genzyme Virotech GmbH, Ruesselsheim, Germany). Antibiotic susceptibility was determined by gradient minimum inhibitory concentration (MIC) test strips (Liofilchem, Roseto, Italy),
and the results were interpreted in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (http://www. eucast.org). The putative production of a carbapenemase was tested by Carba NP test [7] and by modified Hodge test performed on a Mueller–Hinton plate containing 20 mg/ml bile [8]. Aminophenylboronic acid, dipicolinic acid, and cloxacillin in a combined disk test with meropenem was used for further classification of the carbapenemase enzyme [9]. The presence of carbapenemase [10], ESBL [11], acquired AmpC-encoding genes [12], and plasmid-mediated quinolone resistance determinants [13–18] was verified using PCR followed by sequencing. The possible clonal relationships among isolates were investigated by pulsed-field gel electrophoresis (PFGE) [11] and multilocus sequence typing (MLST) [19]. Mating assays with Escherichia coli using the rifampicinresistant Escherichia coli J53 as recipient, plasmid profile analysis, and plasmid replicon typing were performed as described previously [11,20–22]. Results Both carbapenem-resistant K. pneumoniae isolates were positive by the Carba NP test and by the modified
Table I. Clinical and in vitro characterization of OXA-162-producing Klebsiella pneumoniae isolates and the OXA-162-producing Escherichia coli J53 transconjugant. Isolate
Sex/age Specimen Pulsotype Sequence type ESBL type PMQRb MIC (mg/l) CAZ CTX IMP MEM ETP CO TGC CIP AK CN TOB SXT
321
5825/12
5868/12
J53
T5868a
Male/0 y Lower respiratory tract KP046 ST15 blaCTX-M-15 Negative
Male/0 y Peri-anal swab KP046 ST15 blaCTX-M-15 Negative
NA NA ND ND ND ND
NA NA ND ND Negative Negative
⬎ 256 ⬎ 256 8 8 ⬎ 32 1 4 ⬎ 32 2 64 8 16
128 ⬎ 256 4 8 ⬎ 32 1 8 ⬎ 32 8 32 8 4
0.125 0.032 0.125 0.006 0.004 1 0.25 0.008 0.25 0.064 0.125 0.032
0.064 0.25 0.5 0.064 0.25 1 0.25 0.004 0.5 0.25 0.125 0.008
NA, not applicable; ND, not determined; ESBL, extended-spectrum β-lactamase; PMQR, plasmidmediated quinolone resistance determinants; MIC, minimum inhibitory concentration; CAZ, ceftazidime; CTX, cefotaxime; IMP, imipenem; MEM, meropenem; ETP, ertapenem; CO, colistin; TGC, tigecycline; CIP, ciprofloxacin; AK, amikacin; CN, gentamicin; TOB, tobramycin; SXT, trimethoprim/ sulfamethoxazole. aT5868: transconjugant of 5868/12. bPMQR: plasmid-mediated quinolone resistance determinants (qnrA, qnrB, qnrS, qnrC, qnrD, aac(6)-Ibcr, qepA, oqxAB).
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Discussion
Figure 1. Plasmid profiles of OXA-162-producing Klebsiella pneumoniae isolate 5868/12 and its transconjugant. Lanes M1 and M2, molecular weight markers; lane 1, 5868/12; lane 2, T5868.
Hodge test for carbapenemase production. We observed no synergism with meropenem in the presence of any inhibitors. The isolates were resistant to extended-spectrum cephalosporins, ertapenem, ciprofloxacin, gentamicin, tobramycin, and tigecycline, and intermediately susceptible to imipenem and meropenem. They remained susceptible only to colistin (Table I). PCR assays and sequencing revealed that the isolates harboured the blaOXA-162 carbapenemase gene and the blaCTX-M-15 ESBL gene. The ~60 kb IncL/M type plasmid carrying blaOXA-162 was successfully transferred to the recipient strain. Only 1 plasmid was found to have been possessed by the transconjugant, as was revealed by plasmid profiling (Figure 1). The carbapenem MIC values of the transconjugant (T5868) were slightly increased (Table I), but they remained within the susceptible category. Both isolates investigated in this study belonged to a previously identified Hungarian pulsotype designated KP046 and to the sequence type ST15 (Figure 2).
To the best of our knowledge, this is the first description of OXA-48-like carbapenemase-producing bacteria in Hungary and of the OXA-162-type carbapenemase gene in the K. pneumoniae ST15 international clone. OXA-48-like carbapenemases were first described in K. pneumoniae isolates in Turkey in 2004, and have since been found in several European countries (e.g. France, Belgium, Germany, the Netherlands, Italy, the UK, Ireland, Switzerland, Slovenia, and Spain) and non-European countries (e.g. Lebanon, Tunisia, Morocco, Israel, and India) [23]. Generally such cases have been sporadic, however a few outbreaks have also been reported [24]. OXA-162, a novel variant of OXA-48, has recently been identified in K. pneumoniae in Turkey (2012) [25]. Very recently, this variant has also been reported from Germany, in 3 different species [23]. In 2009 the CTX-M-producing KP046 clone caused an outbreak in the maternity ward of a hospital in Budapest, and during the following year this clone appeared in 3 paediatric wards in different hospitals [26]. These isolates were ciprofloxacin-resistant and CTX-M-positive like our OXA-162-producers, however they proved to be carbapenem-susceptible and carbapenemase-non-producers. Although, these OXA-162-producing isolates and the Hungarian Epidemic Clone share the same sequence type, the similarity of their PFGE patterns is below 85%, making a common descent unlikely. The origin of the blaOXA-162 gene in Hungary is unknown, however 2 different explanations for its presence can be proposed: (1) as the K. pneumoniae KP046 clone is circulating among Hungarian paediatric wards, it might have acquired the OXA-162 gene-harbouring plasmid from another species [23], which might have been carried asymptomatically by the Romanian patient; (2) it can be suspected that the OXA-162-positive K. pneumoniae ST15 clone was imported from Romania via cross-border transfer. This latter hypothesis might be supported by the fact that the K. pneumoniae ST15 clone has been detected among OXA-48-like-producing isolates in Europe [27,28]. Based on a report by the European Centre for Disease Prevention and Control [2], the NCE released guidance on the management of carbapenemase-producing Enterobacteriaceae in 2011. This guidance recommends active surveillance of high-risk patients, especially those transferred from healthcare facilities abroad. In accordance with these recommendations, active screening has been performed in the paediatric department of the University of Szeged in an attempt
OXA-162-producing K. pneumoniae in Hungary
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Isolate ID 98
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klebsiepi 90
88
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klebsiepi
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85%
Dice (Opt:1.00%) (Tol 1.0%-2.0%) (H>0.0% S>0.0%) [0.0%-100.0%]
233/03 K167/05 369/05 K115/11 K135/12 K130/11 K131/11 K132/11 K14/12 K149/11 K140/11 K241/09 K242/09 K42/10 5825/12 5868/12 K224/09 K227/09 K232/09 K4/10 K84/06 K9/07 K81/06/1 K94/06/1 K82/12 K89/11 K83/12 K177/07 K294/05/1 381/08 K260/05 K110/12 K116/10 K105/10 K107/10 K119/10 K106/10 K273/05 K93/05 161/05 K183/11 K134/06 K138/06 K4/08/1
Year of isolation/ Center 2003/FJ 1 2005/FJ 2 2005/BP 2 2011/CS 2012/SM 2011/BAZ 3 2011/BAZ 2 2011/BAZ 1 2012/BAZ 2 2011/BAZ 1 2011/BAZ 3 2009/BP 1 2009/BP 1 2010/BP 3 2012/CS 2012/CS 2009/BP 1 2009/BP 1 2009/BP 1 2010/NG 2006/BP 4 2007/PT 1 2006/BP 4 2007/PT 1 2012/BAZ 1 2011/BAZ 4 2012/BAZ 1 2007/BP 5 2005/BP 6 2008/BAZ 4 2005/BP 7 2012/BK 1 2010/PT 2 2010/PT 2 2010/PT 2 2010/BP 8 2010/PT 2 2005/BP 7 2005/BP 7 2005/BP 7 2011/BK 2 2006/BP 3 2006/BP 3 2008/PT 1
Pulsotype/ Sequence type N/ST15 N/ST15 N/ST15 N/ST15 N N N N N N N KP046 KP046 KP046 KP046/ST15 KP046/ST15 KP046 KP046 KP046 KP046 Z/ST525 Z Z Z Z Z Z S/ST11 S/ST11 S/ST258 S/ST11 KP053 KP053 KP053 KP053 KP053/ST11 KP053 R/ST147 R/ST147 R/ST147 R/ST147 L/ST274 L L
323
Sample
blood blood urine blood bloodC trachea throath urine urine wound blood stool stool stool trachea perianal urine stool stool stool stool throat blood blood blood urine blood blood blood urine blood wound urine urine urine throath urine CVC blood urine blood CSF blood blood
N/ ST15 (Hungarian Epidemic Clone)
KP046/ ST15
Z/ ST525
S/ ST11 or ST258 KP053/ ST11 R/ ST147
L/ ST274
Figure 2. Cluster analysis of macrorestriction profiles of ESBL- and/or carbapenemase-producing Klebsiella pneumoniae epidemic clones in Hungary.
to halt the further dissemination of the OXA-162producing K. pneumoniae ST15 clone.
Acknowledgements We thank Wenna Pei for her excellent work reviewing and editing this article. Declaration of interest: All authors declare that they have no conflicts of interest. References [1] Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011;17:1791–8. [2] European Centre for Disease Prevention and Control. Risk assessment on the spread of carbapenemase-producing Enterobacteriaceae (CPE) through patient transfer between healthcare facilities, with special emphasis on cross-border transfer. Stockholm: ECDC; 2011.
[3] Tóth Á, Damjanova I, Puskás E, Jánvári L, Farkas M, Dobák A, et al. Emergence of a colistin-resistant KPC-2-producing Klebsiella pneumoniae ST258 clone in Hungary. Eur J Clin Microbiol Infect Dis 2010;29:765–9. [4] Kristóf K, Tóth Á, Damjanova I, Jánvári L, Konkoly-Thege M, Kocsis B, et al. Identification of a blaVIM-4 gene in the internationally successful Klebsiella pneumoniae ST11 clone and in a Klebsiella oxytoca strain in Hungary. J Antimicrob Chemother 2010;65:1303–5. [5] Damjanova I, Jánvári L, Kristóf K, Szabó D, Kenesei E, Szikra L, et al. Carbapenemase-producing Enterobacteriaceae strains in Hungary. Poster1683, 22nd European Congress of Clinical Microbiology and Infectious Diseases. 31 March- 3 April 2012, London, United Kingdom. [6] Melegh S, Kovács K, Gám T, Nyul A, Patkó B, Tóth A, et al. Emergence of VIM-4 metallo-β-lactamase-producing Klebsiella pneumoniae ST15 clone in the Clinical Centre University of Pécs, Hungary. Clin Microbiol Infect 2013 [Epub ahead of print]. doi: 10.1111/1469-0691.12293 [7] Nordmann P, Poirel L, Dortet L. Rapid detection of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2012;18:1503–7. [8] Lee K, Kim CK, Yong D, Jeong SH, Yum JH, Seo YH, et al. Improved performance of the modified Hodge test with MacConkey agar for screening carbapenemase-producing
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