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Outbreak of carbapenem-resistant Klebsiella pneumoniae producing KPC-3 in a tertiary medical centre in Israel
International Journal of Antimicrobial Agents 30 (2007) 525–529
Outbreak of carbapenem-resistant Klebsiella pneumoniae producing KPC-3 in a tertiary medical centre in Israel Zmira Samra a , Orit Ofir a , Yinon Lishtzinsky b , Liora Madar-Shapiro c , Jihad Bishara b,∗ a
Laboratory of Clinical Microbiology, Rabin Medical Center, Beilinson Hospital, Petah Tiqwa 49100, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel b Infectious Diseases Unit, Rabin Medical Center, Beilinson Hospital, Petah Tiqwa, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel c Hy Laboratories Ltd., Rehovot, Israel Received 4 May 2007; accepted 31 July 2007
Abstract This report describes an outbreak of carbapenem-resistant KPC-3-producing Klebsiella pneumoniae outside the USA. Ninety patients from different departments of a tertiary medical centre were diagnosed with carbapenem-resistant, extended-spectrum -lactamase (ESBL)-negative Klebsiella pneumoniae infection by standard methods over a 10-month period in 2006. Fifteen randomly selected outbreak isolates were subjected to randomly amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) as well as PCR amplification and sequencing of the KPC genes, and the findings were compared with two carbapenem-susceptible K. pneumoniae isolates (one ESBL-positive and one ESBL-negative). All the outbreak isolates were resistant to all fluoroquinolones and -lactam antibiotics tested, including carbapenems, and were sensitive only to colistin, gentamicin and most of them also to tigecycline. On RAPD-PCR, all 15 outbreak isolates were identical to each other and clearly distinguishable from control strains, indicating clonality. The KPC-3 enzyme was identified by nucleotide sequencing analysis in all outbreak isolates but not in the control strains. These findings should alert government and medical authorities to institute stringent control measures and to initiate research into therapeutic and preventive strategies. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Klebsiella pneumoniae; Carbapenem; Resistance; KPC-3; Israel
1. Introduction Klebsiella pneumoniae is a frequent nosocomial pathogen. It is currently the fourth most common cause of pneumonia and the fifth most common cause of bacteraemia in intensive care patients [1–3]. Its principal nosocomial reservoirs are contaminated medical equipment, hands of hospital staff and the gastrointestinal tract of patients. During the 1990s, increased use of cephalosporins was accompanied by the emergence of Enterobacteriaceae possessing extended-spectrum -lactamases (ESBLs). Studies from developed countries have identified several genera that ∗
Corresponding author. Tel.: +972 3 937 7511; fax: +972 3 937 7513. E-mail addresses: [email protected], [email protected] (J. Bishara).
produce these enzymes, however Klebsiella spp. account for the majority of isolates. These isolates are particularly problematic because they are frequently resistant to most classes of antimicrobial agents [4–6]. At present, carbapenems are considered the drug of choice for the treatment of serious infections caused by ESBLproducing pathogens. In K. pneumoniae and Enterobacter spp., carbapenem resistance has been attributed to the combination of high-level production of AmpC -lactamase and loss of outer membrane proteins [3,7–11]. Furthermore, although the finding of efficient carbapenem-hydrolysing lactamases in Enterobacteriaceae remains unusual, it appears to be increasing. Three distinct classes of -lactamases have the ability to hydrolyse carbapenems: the class A and class B metallo--lactamases, which are usually associated with Pseudomonas aeruginosa and Acinetobacter baumannii but
0924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.07.024
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may also be found in K. pneumoniae, and the class D (OXA) -lactamases [12]. Among class A -lactamases with hydrolytic activity against carbapenems, SME-1 and SME-2 were identified in carbapenem-resistant isolates of Serratia marcescens [13], GES-2 in isolates of P. aeruginosa [14] and IMI-1 and NMC-A in isolates of Enterobacter cloacae [15,16]. Class A KPC-type -lactamases efficiently hydrolyse penicillins, cephalosporins and aztreonam in addition to carbapenems, and are inhibited by clavulanic acid and tazobactam [2,3]. The present report describes an infectious outbreak outside the USA caused by carbapenem-resistant K. pneumoniae strains producing KPC-3.
2. Materials and methods 2.1. Setting The outbreak took place at the Rabin Medical Centre, Beilinson Hospital (Israel), a 900-bed, university-affiliated, primary and tertiary care hospital serving an urban population of ca. 1 000 000. It is also a referral centre for several hospitals in the vicinity. In addition to a haemato-oncological institution, the centre runs the largest solid organ transplantation programme in the country. The hospital has four intensive care units (ICUs), a general ICU (20 beds), a cardiac ICU (9 beds), a neurosurgical ICU (6 beds) and a cardiothoracic ICU (12 beds). In addition, each of the six departments of internal medicine manages between four to five mechanically ventilated patients at any given time. 2.2. Klebsiella isolates From February to November 2006, a total of 2219 K. pneumoniae isolates were identified in the institution. Among these, 135 (6%) carbapenem-resistant K. pneumoniae strains were isolated from 90 patients from different departments of the Rabin Medical Center, Beilinson Hospital. The isolates were identified by standard laboratory methods. Antibiotic susceptibility was tested by the disk diffusion method and the minimal inhibitory concentration (MIC) was determined by Etest (AB Biodisk, Solna, Sweden). MIC breakpoints were defined according to Clinical and Laboratory Standards Institute criteria [17]. ESBL production was tested using disks containing cefotaxime (30 g) and cefotaxime/clavulanic acid (30/10 g) as well as ceftazidime (30 g) and ceftazidime/clavulanic acid (30/10 g) (Oxoid, Basingstoke, UK). Isolates were selected at random for the molecular investigation, as follows: • outbreak strains: 15 single-patient, carbapenem-resistant, ESBL-negative isolates (5 from blood, 7 from wounds and body fluids and 3 from sputum); and
• control strains: one carbapenem-sensitive, ESBL-positive isolate and one carbapenem-sensitive, ESBL-negative isolate. All isolates were patient unique and there were no repeated isolates from the same patient. 2.3. Randomly amplified polymorphic DNA (RAPD) Total bacterial DNA was extracted from K. pneumoniae colonies using AccuPrepTM Genomic DNA Extraction Kit (Bioneer, Daejon, South Korea). RAPD polymerase chain reaction (PCR) was performed in a 20 L AccuPowerTM PCR PreMix (Bioneer) with 100 pmol of each primer (1290, 1254, 1252 and 1247) [18,19] as follows: initial denaturation at 92 ◦ C for 2 min, followed by 40 cycles of 92 ◦ C for 30 s, 40 ◦ C for 1 min and 72 ◦ C for 1.5 min, and a final incubation at 72 ◦ C for 10 min. The experiments were repeated twice to assess reproducibility. The amplified DNA fragments were separated on 2% (w/v) agarose gels, stained with ethidium bromide and photographed under ultraviolet light. DNA fingerprints were compared by visual inspection. Similar isolates with the same banding pattern were assigned to the same RAPD type. 2.4. PCR amplification and sequencing of resistance genes PCR amplification was performed on bacterial DNA using the AccuPowerTM PCR PreMix (Bioneer) and sets of primers for the detection of -lactamase resistance genes blaKPC [20], blaTEM [20], blaSHV [21], blaIMP [22], blaCTX-M [23] and blaOXA [24]. Amplification products were purified and sequenced on an ABI PRISM 3700 Genetic Analyzer automated DNA sequencer (Applied Biosystems Inc., Foster City, CA). Obtained sequences were aligned and compared with archived National Center for Biotechnology Information (NCBI) sequences for gene identification.
3. Results Of the 90 patients from whom the outbreak strains were isolated, 34% were hospitalised in medical departments, 29% in the ICU, 17% in general surgical departments, 11% in the department of solid organ transplantation and 9% in the department of cardiothoracic surgery. Carbapenem-resistant organisms were predominantly isolated from wounds and body fluid (48% of cases), followed by the bloodstream (23%), respiratory tract (19%) and urine (10%). MIC results for the tested isolates are given in Table 1. All 15 outbreak isolates were resistant to carbapenems (MIC > 32 g/mL) and fluoroquinolones (ciprofloxacin and ofloxacin, MIC > 32 g/mL). Two of them were also resistant to tigecycline (MIC = 8 g/mL and 16 g/mL). All isolates
Z. Samra et al. / International Journal of Antimicrobial Agents 30 (2007) 525–529 Table 1 Minimal inhibitory concentrations (MICs) of 15 carbapenem-resistant Klebsiella pneumoniae outbreak isolates to the tested antibiotics Antibiotic
Carbapenemsa Piperacillin/tazobactam Ceftazidime Cefotaxime Trimethoprim/sulfamethoxazole Fluoroquinolonesb Tetracycline Tigecycline Amikacin Gentamicin Colistin
MIC (g/mL) MIC50
MIC90
Range
>32 >256 >256 >256 >256 >32 32 1 >256 2 0.25
>32 >256 >256 >256 >256 >32 64 3 >256 2 0.38
>32 >256 >256 >256 >256 >32 8–96 1–16 >256 0.25–2.0 0.19–0.0.38
MIC50/90 , MIC for 50% and 90% of the isolates, respectively. a Carbapenems include imipenem, meropenem and ertapenem. b Fluoroquinolones include ofloxacin and ciprofloxacin.
were sensitive only to colistin (MIC = 0.19–0.38 g/mL) and gentamicin (MIC = 0.25–2 g/mL) and 13 were also sensitive to tigecycline (MIC = 1–2 g/mL). 3.1. RAPD analysis All 15 carbapenem-resistant, ESBL-negative K. pneumoniae outbreak isolates were indistinguishable from each other, indicating clonality (Fig. 1, lanes 1, 4–17), and were different from the control isolates (Fig. 1, lanes 2 and 3). 3.2. PCR amplification and sequencing of resistance genes All the outbreak isolates were positive for TEM-1, SHV11 and OXA genes and negative for the IMP and CTX-M -lactamase genes.
Fig. 1. Randomly amplified polymorphic DNA (RAPD) analysis of carbapenem-resistant, extended-spectrum -lactamase (ESBL)-negative Klebsiella pneumoniae isolates (lanes 1, 4–17), a carbapenem-sensitive, ESBL-positive isolate (lane 2) and a carbapenem-sensitive, ESBL-negative isolate (lane 3).
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PCR analysis of the blaKPC gene showed that all the 15 outbreak K. pneumoniae isolates were KPC-positive, whilst the control isolates were KPC-negative. Genotyping of the blaKPC PCR products from all 15 outbreak isolates was performed by nucleotide sequencing analysis. The results showed 100% identity with the KPC-3 compared with the gene bank sequences.
4. Discussion Here we describe an infectious outbreak caused by K. pneumoniae producing KPC-3 isolated in our hospital over a 10-month period. RAPD analysis showed that these isolates were genetically identical, indicating single clonality. Furthermore, PCR analysis of additional -lactamase genes such as blaTEM , blaSHV , blaIMP , blaOXA and blaCTX-M revealed an identical gene profile, supporting our hypothesis for a clonal KPC-3-related infectious outbreak. Nosocomially acquired carbapenem-resistant K. pneumoniae isolates were first seen at the Tisch Hospital in New York in April 2000 [3]. All isolates were recovered from respiratory secretions of patients in the ICU. In 2001, Yigit et al. reported the first recovery of KPC-1 -lactamase from a carbapenemresistant strain of K. pneumoniae [25]. The enzyme was found to reside on a transferable plasmid. Soon afterwards, reports of KPC-2 expression by isolates of K. pneumoniae [3,26,27], Klebsiella oxytoca [28], Salmonella enterica [29] and E. cloacae [30] surfaced throughout the northeastern USA. Later, studies from New Jersey [31] and New York [2] identified carbapenem-resistant isolates of Escherichia coli and K. pneumoniae, respectively, carrying the blaKPC-3 gene. The blaKPC-3 gene is known to confer reduced susceptibility to carbapenem compared with blaKPC-2 , which differs from it by only a single nucleotide. In some reports, carbapenem resistance was also apparently associated with diminished production of outer membrane porins [2,25]. Recently, E. coli isolates producing KPC-2 were reported from another institution in Israel. The enzyme was found in four genetically unrelated carbapenem-resistant E. coli isolates from four epidemiologically unrelated patients [32]. In Israel in general, and in our institution in particular, carbapenems are used to treat serious Gram-negative infections. Between 1997 and 2002, the incidence of multidrug-resistant A. baumannii bloodstream infections (BSIs) increased twoto four-fold in three Israeli hospitals, accounting for 3.5–18% of all hospital-acquired BSIs. This was associated with an increase in carbapenem resistance, reaching 35–54%, and a dramatic increase in carbapenem consumption [33]. Indeed, until recently carbapenem resistance was largely confined to nosocomially acquired P. aeruginosa and A. baumannii species. A recent report by Leavitt et al. [34] describes a similar increase in carbapenem-resistant K. pneumoniae strains possessing KPC-2 and KPC-3 from 2004 to 2006 in Tel Aviv Medical Center, which is in close vicinity to our hospital. In
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their outbreak, KPC-3-producing K. pneumoniae belonged to more than one clone. The present study, besides being the second documentation of the appearance of a KPC-3-producing K. pneumoniae strain in Israel, raises major concerns because of the rapid spread of these isolates to almost all the hospital’s departments within a few months. Outbreaks can be controlled if they are detected early and if strict measures are implemented. However, this strain has a particular epidemic potential and interhospital transmission is likely to occur during patient transfer. Therefore, active surveillance for this multidrug-resistant microorganism at a national level should be implemented. Indeed, recommendations for the surveillance, prevention and control of KPC-producing K. pneumoniae were disseminated to all hospitals in Israel in February 2007. In addition, a national laboratory network consisting of a National Reference Centre was set up to characterise isolated strains. Current efforts are focused on reinforcing the recommendations in all hospitals, detecting and controlling clusters early and limiting the spread of the KPC-producing K. pneumoniae. Our findings should promote government and health authorities in Israel to conduct further studies in order to clarify the impact of resistance on the clinical course and outcome of the disease, to delineate the risk factors and to develop therapeutic and preventive strategies. Funding: None. Competing interests: None declared. Ethical approval: Not required. References [1] Centers for Disease Control and Prevention. National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2003, issued August 2003. Am J Infect Control 2003;31:481–98. [2] Woodford N, Tierno Jr PM, Young K, et al. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A -lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother 2004;48:4793–9. [3] Bradford PA, Bratu S, Urban C, et al. Emergence of carbapenemresistant Klebsiella species possessing the class A carbapenemhydrolyzing KPC-2 and inhibitor-resistant TEM-30 -lactamases in New York City. Clin Infect Dis 2004;39:55–60. [4] Bishara J, Livne G, Ashkenazi S, et al. Antibacterial susceptibility of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. Isr Med Assoc J 2005;7:298–301. [5] Livermore DM. -Lactamase-mediated resistance and opportunities for its control. J Antimicrob Chemother 1998;41:25–41. [6] Garau J. Beta-lactamases: current situation and clinical importance. Intensive Care Med 1994;20(Suppl. 3):S5–9. [7] Bradford PA, Urban C, Mariano N, et al. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC -lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother 1997;41: 563–9. [8] Martinez-Martinez L, Pascual A, Hernandez-Alles S, et al. Roles of lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrob Agents Chemother 1999; 43:1669–73.
[9] Cao VTB, Arlet G, Ericsson BM, et al. Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmidmediated CMY-4 and permeability alteration. J Antimicrob Chemother 2000;46:895–900. [10] Chow JW, Shlaes DM. Imipenem resistance associated with the loss of a 40 kDa outer membrane protein in Enterobacter aerogenes. J Antimicrob Chemother 1991;28:499–504. [11] Raimondi A, Traverso A, Nikaido H. Imipenem- and meropenemresistant mutants of Enterobacter cloacae and Proteus rettgeri lack porins. Antimicrob Agents Chemother 1991;35:1174–80. [12] Yan JJ, Ko WC, Tsai SH, et al. Outbreak of infection with multidrugresistant Klebsiella pneumoniae carrying blaIMP-8 in a university medical center in Taiwan. J Clin Microbiol 2001;39:4433–9. [13] Queenan AM, Torres-Viera C, Gold HS, et al. SME-type carbapenem-hydrolyzing class A beta-lactamases from geographically diverse Serratia marcescens strains. Antimicrob Agents Chemother 2000;44:3035–9. [14] Poirel L, Weldhagen GF, Naas T, et al. GES-2, a class A -lactamase from Pseudomonas aeruginosa with increased hydrolysis of imipenem. Antimicrob Agents Chemother 2001;45:2598–603. [15] Rasmussen BA, Bush K, Keeney D, et al. Characterization of IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae. Antimicrob Agents Chemother 1996;40:2080–6. [16] Nordmann P, Mariotte S, Naas T, et al. Biochemical properties of a carbapenem-hydrolyzing -lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli. Antimicrob Agents Chemother 1994;38:1262–70. [17] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Fifteenth informational supplement. Approved standard MS100-S16. Wayne, PA: CLSI; 2006. [18] Hopkins KL, Hilton AC. Optimization of random amplification of polymorphic DNA analysis for molecular subtyping of Escherichia coli O157. Lett Appl Microbiol 2001;32:126–30. [19] Cusick SM, O’Sullivan DJ. Use of a single, triplicate arbitrarily primedPCR procedure for molecular fingerprinting of lactic acid bacteria. Appl Environ Microbiol 2000;66:2227–31. [20] Lomaestro BM, Tobin EH, Shang W, et al. The spread of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae to upstate New York. Clin Infect Dis 2006;43:e26–8. [21] N¨uesch-Inderbinen MT, H¨achler H, Kayser FH. Detection of genes coding for extended-spectrum SHV beta-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test. Eur J Clin Microbiol Infect Dis 1996;15:398–402. [22] Senda K, Arakawa Y, Ichiyama S, et al. PCR detection of metallobeta-lactamase gene (blaIMP ) in gram-negative rods resistant to broadspectrum beta-lactams. J Clin Microbiol 1996;34:2909–13. [23] Bauernfeind A, Stemplinger I, Jungwirth R, Ernst S, Casellas JM. Sequences of -lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other -lactamases. Antimicrob Agents Chemother 1996;40:509– 13. [24] Dale JW, Godwin D, Mossakowska D, Stephenson P, Wall S. Sequence of the OXA2 beta-lactamase: comparison with other penicillin-reactive enzymes. FEBS Lett 1985;191:39–44. [25] Yigit H, Queenan AM, Anderson GJ, et al. Novel carbapenemhydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 2001;45:1151–61. [26] Moland ES, Black JA, Ourada J, et al. Occurrence of newer lactamases in Klebsiella pneumoniae isolates from 24 US hospitals. Antimicrob Agents Chemother 2002;46:3837–42. [27] Moland ES, Hanson ND, Herrera VL, et al. Plasmid-mediated, carbapenem-hydrolysing -lactamase, KPC-2, in Klebsiella pneumoniae isolates. J Antimicrob Chemother 2003;51:711–4. [28] Yigit H, Queenan AM, Rasheed JK, et al. Carbapenem-resistant strain of Klebsiella oxytoca harboring carbapenem-hydrolyzing -lactamase KPC-2. Antimicrob Agents Chemother 2003;47:3881–9.
Z. Samra et al. / International Journal of Antimicrobial Agents 30 (2007) 525–529 [29] Miriagou V, Tzouvelekis LS, Rossiter S, et al. Imipenem resistance in a Salmonella clinical strain due to plasmid-mediated class A carbapenemase KPC-2. Antimicrob Agents Chemother 2003;47:1297–300. [30] Hossain A, Ferraro MJ, Pino RM, et al. First report of plasmid-encoded carbapenem hydrolyzing enzyme KPC-2 in Enterobacter cloacae. In: Abstracts of the 43rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). Washington, DC: American Society for Microbiology; 2003. p. 75 [Abstract C1-264]. [31] Hong T, Moland ES, Abdalhamid B, et al. Escherichia coli: development of carbapenem resistance during therapy. Clin Infect Dis 2005;40:e84–6.
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[32] Navon-Venezia S, Chmelnitsky I, Leavitt A, et al. Plasmid-mediated imipenem-hydrolyzing enzyme KPC-2 among multiple carbapenemresistant Escherichia coli clones in Israel. Antimicrob Agents Chemother 2006;50:3098–101. [33] Paul M, Weinberger M, Sigeman-Igra Y, et al. Acinetobacter baumannii: emergence and spread in Israeli hospitals 1997–2002. J Hosp Infect 2005;60:256–60. [34] Leavitt A, Navon-Venezia S, Chmelnitsky I, Schwaber MJ, Carmeli Y. Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae strains in an Israeli Hospital. Antimicrob Agents Chemother 2007;51:3026–9.
Outbreak of carbapenem-resistant Klebsiella pneumoniae producing KPC-3 in a tertiary medical centre in Israel Zmira Samra a , Orit Ofir a , Yinon Lishtzinsky b , Liora Madar-Shapiro c , Jihad Bishara b,∗ a
Laboratory of Clinical Microbiology, Rabin Medical Center, Beilinson Hospital, Petah Tiqwa 49100, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel b Infectious Diseases Unit, Rabin Medical Center, Beilinson Hospital, Petah Tiqwa, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel c Hy Laboratories Ltd., Rehovot, Israel Received 4 May 2007; accepted 31 July 2007
Abstract This report describes an outbreak of carbapenem-resistant KPC-3-producing Klebsiella pneumoniae outside the USA. Ninety patients from different departments of a tertiary medical centre were diagnosed with carbapenem-resistant, extended-spectrum -lactamase (ESBL)-negative Klebsiella pneumoniae infection by standard methods over a 10-month period in 2006. Fifteen randomly selected outbreak isolates were subjected to randomly amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) as well as PCR amplification and sequencing of the KPC genes, and the findings were compared with two carbapenem-susceptible K. pneumoniae isolates (one ESBL-positive and one ESBL-negative). All the outbreak isolates were resistant to all fluoroquinolones and -lactam antibiotics tested, including carbapenems, and were sensitive only to colistin, gentamicin and most of them also to tigecycline. On RAPD-PCR, all 15 outbreak isolates were identical to each other and clearly distinguishable from control strains, indicating clonality. The KPC-3 enzyme was identified by nucleotide sequencing analysis in all outbreak isolates but not in the control strains. These findings should alert government and medical authorities to institute stringent control measures and to initiate research into therapeutic and preventive strategies. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Klebsiella pneumoniae; Carbapenem; Resistance; KPC-3; Israel
1. Introduction Klebsiella pneumoniae is a frequent nosocomial pathogen. It is currently the fourth most common cause of pneumonia and the fifth most common cause of bacteraemia in intensive care patients [1–3]. Its principal nosocomial reservoirs are contaminated medical equipment, hands of hospital staff and the gastrointestinal tract of patients. During the 1990s, increased use of cephalosporins was accompanied by the emergence of Enterobacteriaceae possessing extended-spectrum -lactamases (ESBLs). Studies from developed countries have identified several genera that ∗
Corresponding author. Tel.: +972 3 937 7511; fax: +972 3 937 7513. E-mail addresses: [email protected], [email protected] (J. Bishara).
produce these enzymes, however Klebsiella spp. account for the majority of isolates. These isolates are particularly problematic because they are frequently resistant to most classes of antimicrobial agents [4–6]. At present, carbapenems are considered the drug of choice for the treatment of serious infections caused by ESBLproducing pathogens. In K. pneumoniae and Enterobacter spp., carbapenem resistance has been attributed to the combination of high-level production of AmpC -lactamase and loss of outer membrane proteins [3,7–11]. Furthermore, although the finding of efficient carbapenem-hydrolysing lactamases in Enterobacteriaceae remains unusual, it appears to be increasing. Three distinct classes of -lactamases have the ability to hydrolyse carbapenems: the class A and class B metallo--lactamases, which are usually associated with Pseudomonas aeruginosa and Acinetobacter baumannii but
0924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2007.07.024
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Z. Samra et al. / International Journal of Antimicrobial Agents 30 (2007) 525–529
may also be found in K. pneumoniae, and the class D (OXA) -lactamases [12]. Among class A -lactamases with hydrolytic activity against carbapenems, SME-1 and SME-2 were identified in carbapenem-resistant isolates of Serratia marcescens [13], GES-2 in isolates of P. aeruginosa [14] and IMI-1 and NMC-A in isolates of Enterobacter cloacae [15,16]. Class A KPC-type -lactamases efficiently hydrolyse penicillins, cephalosporins and aztreonam in addition to carbapenems, and are inhibited by clavulanic acid and tazobactam [2,3]. The present report describes an infectious outbreak outside the USA caused by carbapenem-resistant K. pneumoniae strains producing KPC-3.
2. Materials and methods 2.1. Setting The outbreak took place at the Rabin Medical Centre, Beilinson Hospital (Israel), a 900-bed, university-affiliated, primary and tertiary care hospital serving an urban population of ca. 1 000 000. It is also a referral centre for several hospitals in the vicinity. In addition to a haemato-oncological institution, the centre runs the largest solid organ transplantation programme in the country. The hospital has four intensive care units (ICUs), a general ICU (20 beds), a cardiac ICU (9 beds), a neurosurgical ICU (6 beds) and a cardiothoracic ICU (12 beds). In addition, each of the six departments of internal medicine manages between four to five mechanically ventilated patients at any given time. 2.2. Klebsiella isolates From February to November 2006, a total of 2219 K. pneumoniae isolates were identified in the institution. Among these, 135 (6%) carbapenem-resistant K. pneumoniae strains were isolated from 90 patients from different departments of the Rabin Medical Center, Beilinson Hospital. The isolates were identified by standard laboratory methods. Antibiotic susceptibility was tested by the disk diffusion method and the minimal inhibitory concentration (MIC) was determined by Etest (AB Biodisk, Solna, Sweden). MIC breakpoints were defined according to Clinical and Laboratory Standards Institute criteria [17]. ESBL production was tested using disks containing cefotaxime (30 g) and cefotaxime/clavulanic acid (30/10 g) as well as ceftazidime (30 g) and ceftazidime/clavulanic acid (30/10 g) (Oxoid, Basingstoke, UK). Isolates were selected at random for the molecular investigation, as follows: • outbreak strains: 15 single-patient, carbapenem-resistant, ESBL-negative isolates (5 from blood, 7 from wounds and body fluids and 3 from sputum); and
• control strains: one carbapenem-sensitive, ESBL-positive isolate and one carbapenem-sensitive, ESBL-negative isolate. All isolates were patient unique and there were no repeated isolates from the same patient. 2.3. Randomly amplified polymorphic DNA (RAPD) Total bacterial DNA was extracted from K. pneumoniae colonies using AccuPrepTM Genomic DNA Extraction Kit (Bioneer, Daejon, South Korea). RAPD polymerase chain reaction (PCR) was performed in a 20 L AccuPowerTM PCR PreMix (Bioneer) with 100 pmol of each primer (1290, 1254, 1252 and 1247) [18,19] as follows: initial denaturation at 92 ◦ C for 2 min, followed by 40 cycles of 92 ◦ C for 30 s, 40 ◦ C for 1 min and 72 ◦ C for 1.5 min, and a final incubation at 72 ◦ C for 10 min. The experiments were repeated twice to assess reproducibility. The amplified DNA fragments were separated on 2% (w/v) agarose gels, stained with ethidium bromide and photographed under ultraviolet light. DNA fingerprints were compared by visual inspection. Similar isolates with the same banding pattern were assigned to the same RAPD type. 2.4. PCR amplification and sequencing of resistance genes PCR amplification was performed on bacterial DNA using the AccuPowerTM PCR PreMix (Bioneer) and sets of primers for the detection of -lactamase resistance genes blaKPC [20], blaTEM [20], blaSHV [21], blaIMP [22], blaCTX-M [23] and blaOXA [24]. Amplification products were purified and sequenced on an ABI PRISM 3700 Genetic Analyzer automated DNA sequencer (Applied Biosystems Inc., Foster City, CA). Obtained sequences were aligned and compared with archived National Center for Biotechnology Information (NCBI) sequences for gene identification.
3. Results Of the 90 patients from whom the outbreak strains were isolated, 34% were hospitalised in medical departments, 29% in the ICU, 17% in general surgical departments, 11% in the department of solid organ transplantation and 9% in the department of cardiothoracic surgery. Carbapenem-resistant organisms were predominantly isolated from wounds and body fluid (48% of cases), followed by the bloodstream (23%), respiratory tract (19%) and urine (10%). MIC results for the tested isolates are given in Table 1. All 15 outbreak isolates were resistant to carbapenems (MIC > 32 g/mL) and fluoroquinolones (ciprofloxacin and ofloxacin, MIC > 32 g/mL). Two of them were also resistant to tigecycline (MIC = 8 g/mL and 16 g/mL). All isolates
Z. Samra et al. / International Journal of Antimicrobial Agents 30 (2007) 525–529 Table 1 Minimal inhibitory concentrations (MICs) of 15 carbapenem-resistant Klebsiella pneumoniae outbreak isolates to the tested antibiotics Antibiotic
Carbapenemsa Piperacillin/tazobactam Ceftazidime Cefotaxime Trimethoprim/sulfamethoxazole Fluoroquinolonesb Tetracycline Tigecycline Amikacin Gentamicin Colistin
MIC (g/mL) MIC50
MIC90
Range
>32 >256 >256 >256 >256 >32 32 1 >256 2 0.25
>32 >256 >256 >256 >256 >32 64 3 >256 2 0.38
>32 >256 >256 >256 >256 >32 8–96 1–16 >256 0.25–2.0 0.19–0.0.38
MIC50/90 , MIC for 50% and 90% of the isolates, respectively. a Carbapenems include imipenem, meropenem and ertapenem. b Fluoroquinolones include ofloxacin and ciprofloxacin.
were sensitive only to colistin (MIC = 0.19–0.38 g/mL) and gentamicin (MIC = 0.25–2 g/mL) and 13 were also sensitive to tigecycline (MIC = 1–2 g/mL). 3.1. RAPD analysis All 15 carbapenem-resistant, ESBL-negative K. pneumoniae outbreak isolates were indistinguishable from each other, indicating clonality (Fig. 1, lanes 1, 4–17), and were different from the control isolates (Fig. 1, lanes 2 and 3). 3.2. PCR amplification and sequencing of resistance genes All the outbreak isolates were positive for TEM-1, SHV11 and OXA genes and negative for the IMP and CTX-M -lactamase genes.
Fig. 1. Randomly amplified polymorphic DNA (RAPD) analysis of carbapenem-resistant, extended-spectrum -lactamase (ESBL)-negative Klebsiella pneumoniae isolates (lanes 1, 4–17), a carbapenem-sensitive, ESBL-positive isolate (lane 2) and a carbapenem-sensitive, ESBL-negative isolate (lane 3).
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PCR analysis of the blaKPC gene showed that all the 15 outbreak K. pneumoniae isolates were KPC-positive, whilst the control isolates were KPC-negative. Genotyping of the blaKPC PCR products from all 15 outbreak isolates was performed by nucleotide sequencing analysis. The results showed 100% identity with the KPC-3 compared with the gene bank sequences.
4. Discussion Here we describe an infectious outbreak caused by K. pneumoniae producing KPC-3 isolated in our hospital over a 10-month period. RAPD analysis showed that these isolates were genetically identical, indicating single clonality. Furthermore, PCR analysis of additional -lactamase genes such as blaTEM , blaSHV , blaIMP , blaOXA and blaCTX-M revealed an identical gene profile, supporting our hypothesis for a clonal KPC-3-related infectious outbreak. Nosocomially acquired carbapenem-resistant K. pneumoniae isolates were first seen at the Tisch Hospital in New York in April 2000 [3]. All isolates were recovered from respiratory secretions of patients in the ICU. In 2001, Yigit et al. reported the first recovery of KPC-1 -lactamase from a carbapenemresistant strain of K. pneumoniae [25]. The enzyme was found to reside on a transferable plasmid. Soon afterwards, reports of KPC-2 expression by isolates of K. pneumoniae [3,26,27], Klebsiella oxytoca [28], Salmonella enterica [29] and E. cloacae [30] surfaced throughout the northeastern USA. Later, studies from New Jersey [31] and New York [2] identified carbapenem-resistant isolates of Escherichia coli and K. pneumoniae, respectively, carrying the blaKPC-3 gene. The blaKPC-3 gene is known to confer reduced susceptibility to carbapenem compared with blaKPC-2 , which differs from it by only a single nucleotide. In some reports, carbapenem resistance was also apparently associated with diminished production of outer membrane porins [2,25]. Recently, E. coli isolates producing KPC-2 were reported from another institution in Israel. The enzyme was found in four genetically unrelated carbapenem-resistant E. coli isolates from four epidemiologically unrelated patients [32]. In Israel in general, and in our institution in particular, carbapenems are used to treat serious Gram-negative infections. Between 1997 and 2002, the incidence of multidrug-resistant A. baumannii bloodstream infections (BSIs) increased twoto four-fold in three Israeli hospitals, accounting for 3.5–18% of all hospital-acquired BSIs. This was associated with an increase in carbapenem resistance, reaching 35–54%, and a dramatic increase in carbapenem consumption [33]. Indeed, until recently carbapenem resistance was largely confined to nosocomially acquired P. aeruginosa and A. baumannii species. A recent report by Leavitt et al. [34] describes a similar increase in carbapenem-resistant K. pneumoniae strains possessing KPC-2 and KPC-3 from 2004 to 2006 in Tel Aviv Medical Center, which is in close vicinity to our hospital. In
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their outbreak, KPC-3-producing K. pneumoniae belonged to more than one clone. The present study, besides being the second documentation of the appearance of a KPC-3-producing K. pneumoniae strain in Israel, raises major concerns because of the rapid spread of these isolates to almost all the hospital’s departments within a few months. Outbreaks can be controlled if they are detected early and if strict measures are implemented. However, this strain has a particular epidemic potential and interhospital transmission is likely to occur during patient transfer. Therefore, active surveillance for this multidrug-resistant microorganism at a national level should be implemented. Indeed, recommendations for the surveillance, prevention and control of KPC-producing K. pneumoniae were disseminated to all hospitals in Israel in February 2007. In addition, a national laboratory network consisting of a National Reference Centre was set up to characterise isolated strains. Current efforts are focused on reinforcing the recommendations in all hospitals, detecting and controlling clusters early and limiting the spread of the KPC-producing K. pneumoniae. Our findings should promote government and health authorities in Israel to conduct further studies in order to clarify the impact of resistance on the clinical course and outcome of the disease, to delineate the risk factors and to develop therapeutic and preventive strategies. Funding: None. Competing interests: None declared. Ethical approval: Not required. References [1] Centers for Disease Control and Prevention. National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2003, issued August 2003. Am J Infect Control 2003;31:481–98. [2] Woodford N, Tierno Jr PM, Young K, et al. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A -lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother 2004;48:4793–9. [3] Bradford PA, Bratu S, Urban C, et al. Emergence of carbapenemresistant Klebsiella species possessing the class A carbapenemhydrolyzing KPC-2 and inhibitor-resistant TEM-30 -lactamases in New York City. Clin Infect Dis 2004;39:55–60. [4] Bishara J, Livne G, Ashkenazi S, et al. Antibacterial susceptibility of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. Isr Med Assoc J 2005;7:298–301. [5] Livermore DM. -Lactamase-mediated resistance and opportunities for its control. J Antimicrob Chemother 1998;41:25–41. [6] Garau J. Beta-lactamases: current situation and clinical importance. Intensive Care Med 1994;20(Suppl. 3):S5–9. [7] Bradford PA, Urban C, Mariano N, et al. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC -lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother 1997;41: 563–9. [8] Martinez-Martinez L, Pascual A, Hernandez-Alles S, et al. Roles of lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrob Agents Chemother 1999; 43:1669–73.
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