REVIEW

published: 13 June 2016

doi: 10.3389/fmicb.2016.00895

Global Dissemination of
Carbapenemase-Producing
Klebsiella pneumoniae:
Epidemiology, Genetic Context,
Treatment Options, and Detection
Methods
Chang-Ro Lee 1† , Jung Hun Lee 1† , Kwang Seung Park 1 , Young Bae Kim 2 ,
Byeong Chul Jeong 1 and Sang Hee Lee 1*
1
National Leading Research Laboratory of Drug Resistance Proteomics, Department of Biological Sciences, Myongji
University, Yongin, South Korea, 2 Division of STEM, North Shore Community College, Danvers, MA, USA
Edited by:
Miklos Fuzi,
Semmelweis University, Hungary
The emergence of carbapenem-resistant Gram-negative pathogens poses a
Reviewed by:
serious threat to public health worldwide. In particular, the increasing prevalence
Andrea Endimiani, of carbapenem-resistant Klebsiella pneumoniae is a major source of concern.
University of Bern, Switzerland K. pneumoniae carbapenemases (KPCs) and carbapenemases of the oxacillinase-
Sara Richter,
University of Padua, Italy 48 (OXA-48) type have been reported worldwide. New Delhi metallo-β-lactamase
*Correspondence: (NDM) carbapenemases were originally identified in Sweden in 2008 and have spread
Sang Hee Lee worldwide rapidly. In this review, we summarize the epidemiology of K. pneumoniae
sangheelee@[Link]
producing three carbapenemases (KPCs, NDMs, and OXA-48-like). Although the
† These authors have contributed
equally to this work.
prevalence of each resistant strain varies geographically, K. pneumoniae producing
KPCs, NDMs, and OXA-48-like carbapenemases have become rapidly disseminated.
Specialty section: In addition, we used recently published molecular and genetic studies to analyze the
This article was submitted to
Antimicrobials, Resistance
mechanisms by which these three carbapenemases, and major K. pneumoniae clones,
and Chemotherapy, such as ST258 and ST11, have become globally prevalent. Because carbapenemase-
a section of the journal
producing K. pneumoniae are often resistant to most β-lactam antibiotics and many
Frontiers in Microbiology
other non-β-lactam molecules, the therapeutic options available to treat infection
Received: 07 January 2016
Accepted: 26 May 2016 with these strains are limited to colistin, polymyxin B, fosfomycin, tigecycline, and
Published: 13 June 2016 selected aminoglycosides. Although, combination therapy has been recommended
Citation: for the treatment of severe carbapenemase-producing K. pneumoniae infections, the
Lee C-R, Lee JH, Park KS, Kim YB,
Jeong BC and Lee SH (2016) Global
clinical evidence for this strategy is currently limited, and more accurate randomized
Dissemination controlled trials will be required to establish the most effective treatment regimen.
of Carbapenemase-Producing
Moreover, because rapid and accurate identification of the carbapenemase type found
Klebsiella pneumoniae: Epidemiology,
Genetic Context, Treatment Options, in K. pneumoniae may be difficult to achieve through phenotypic antibiotic susceptibility
and Detection Methods. tests, novel molecular detection techniques are currently being developed.
Front. Microbiol. 7:895.
doi: 10.3389/fmicb.2016.00895 Keywords: carbapenemase, Klebsiella pneumoniae, epidemiology, KPC, NDM, OXA-48-like

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

INTRODUCTION enzymes are susceptible to only a few antibiotics such as colistin,

aminoglycosides, and tigecycline. Therefore, the mortality of the
The increasing prevalence of antibiotic resistance and the lack patient’s bloodstream infections caused by these bacteria is very
of new antibiotic drug development has gradually reduced the high (Munoz-Price et al., 2013).
treatment options for bacterial infections (Lee et al., 2013a; The epidemiology of K. pneumoniae producing KPCs varies
Nathan and Cars, 2014). In 2013, the Centers for Disease geographically. The endemic spread of these bacteria has been
Control and Prevention (CDC) named three microorganisms reported in the USA, China, Italy, Poland, Greece, Israel, Brazil,
that pose an urgent threat to public health: carbapenem- Argentina, Colombia, and Taiwan (Munoz-Price et al., 2013;
resistant (CR) Enterobacteriaceae (CRE), drug-resistant Neisseria Figure 1). Sporadic spread of KPC-producing K. pneumoniae has
gonorrhoeae, and Clostridium difficile (Zowawi et al., 2015). also been observed in many European countries including Spain,
Carbapenems (imipenem, meropenem, biapenem, ertapenem, France, Germany, the Netherlands, the UK, Ireland, Belgium,
and doripenem) are antibiotics used for the treatment of severe Sweden, and Finland, and in several countries in the Asia-Pacific
infections caused by multi-resistant Enterobacteriaceae, such as region, including India, South Korea, and Australia (Munoz-
Klebsiella pneumoniae and Escherichia coli (Nordmann et al., Price et al., 2013; Nordmann and Poirel, 2014). In the USA,
2009). However, over the past 10 years, CRE have increasingly the transmission of CR K. pneumoniae is primarily driven by
been reported worldwide (Nordmann et al., 2011a). In particular, the spread of organisms carrying KPC enzymes (Kaiser et al.,
K. pneumoniae have acquired carbapenemases, which are 2013), but other carbapenemase enzymes, such as the New-Delhi
enzymes capable of breaking down most β-lactams including metallo-β-lactamase (NDM), have also emerged (Lascols et al.,
carbapenems, and thus conferring resistance to these drugs (Jeon 2013). Within the USA, the prevalence of KPC-positive isolates
et al., 2015). High mortality rates have been reported in patients was relatively stable between 2007 and 2009 (5.9% in 2007, 4.9%
with bloodstream infections caused by CR K. pneumoniae in 2008, and 5.7% in 2009; Kaiser et al., 2013), and KPC-2 and
(Munoz-Price et al., 2013). Carbapenemases can be divided KPC-3 were the most frequently identified carbapenemases in
according to their dependency on divalent cations for enzyme K. pneumoniae (Deshpande et al., 2006).
activation into metallo-carbapenemases (zinc-dependent class The outbreaks caused by KPC-producing K. pneumoniae
B) and non-metallo-carbapenemases (zinc-independent classes have been reported in the USA (Woodford et al., 2004) and
A, C, and D; Jeon et al., 2015). The class A carbapenemases, Israel (Leavitt et al., 2007), but recently, similar outbreaks
such as the K. pneumoniae carbapenemase (KPC) enzymes, associated with patients traveling to endemic areas have also
have been identified worldwide in K. pneumoniae (Tangden been reported in many European counties. Since KPC-producing
and Giske, 2015). Various class B and D carbapenemases K. pneumoniae was identified in France, Italy, and Greece (Naas
have also been detected in hospital-acquired multi-resistant et al., 2005; Tsakris et al., 2008; Giani et al., 2009), the sporadic
K. pneumoniae (Nordmann et al., 2011a), whereas class C spread of KPC-producing K. pneumoniae has been observed in
carbapenemases have rarely been reported. In this review, many European countries including Spain (Robustillo Rodela
we summarize the epidemiology of the major four classes of et al., 2012), France (Carbonne et al., 2010), Germany (Wendt
carbapenemases and discuss their molecular genetics, methods et al., 2010; Steinmann et al., 2011), the Netherlands (Weterings
used for their detection, and the therapeutic options available for et al., 2015), the UK (Woodford et al., 2008; Virgincar et al.,
their treatment. 2011), Ireland (Roche et al., 2009; Morris et al., 2012), Belgium
(Bogaerts et al., 2010), Sweden (Samuelsen et al., 2009), and
Finland (Osterblad et al., 2012; Kanerva et al., 2015). KPC-
THE EPIDEMIOLOGY, GENETIC producing K. pneumoniae were also recently detected in eastern
CONTEXT, TREATMENT OPTIONS, AND European countries including the Czech Republic (Hrabak et al.,
DETECTION METHODS OF 2013b), Hungary (Toth et al., 2010), and Croatia (Bedenic et al.,
2012).
CARBAPENEM-RESISTANT In Greece, KPC-producing K. pneumoniae was first isolated in
K. pneumoniae August 2007 (Tsakris et al., 2008), and the prevalence of KPC-
producers among carbapenemase-producing K. pneumoniae
Class A Carbapenemases isolates collected at a tertiary Greek hospital increased from 0%
Epidemiology in 2003 to 38.3% in 2010 (Zagorianou et al., 2012). Most of the
Various class A carbapenemases forming six distantly related genotyped KPC-producing K. pneumoniae in Greece harbored
branches have been identified (Jeon et al., 2015). While some KPC-2 (Zagorianou et al., 2012). While many carbapenemase-
carbapenemases are chromosome-encoded (IMI-1, NMC-A, producing K. pneumoniae in the USA and Greece had KPC
SME enzymes, SHV-38, and SFC-1), others are plasmid-encoded enzymes (Nordmann et al., 2009; Zagorianou et al., 2012), several
(KPC enzymes, GES enzymes, and IMI-2). KPCs have been the studies in Spain showed that most carbapenemase-producing
most frequently observed class A carbapenemases since their first K. pneumoniae harbored OXA-48-like or class B carbapenemases,
description in the eastern the USA in 1996 (Yigit et al., 2001). Of and the rate of KPC- producing K. pneumoniae was very low
the many different KPC family variants (KPC-1 to KPC-22), the (2–3%; Oteo et al., 2013b; Palacios-Baena et al., 2016). These
most well-characterized variants are KPC-2 and KPC-3. KPCs are results indicate that the prevalent genotype of carbapenemase-
mostly plasmid-encoded enzymes and bacteria producing these producing K. pneumoniae varies geographically. For example, in

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

FIGURE 1 | Epidemiological features of KPC-producing Klebsiella pneumoniae. (1) USA; (2) Colombia; (3) Brazil; (4) Argentina; (5) Italy; (6) Greece; (7)
Poland; (8) Israel; (9) China; (10) Taiwan; (11) Canada; (12) Spain; (13) France; (14) Belgium; (15) Netherlands; (16) Germany; (17) UK; (18) Ireland; (19) Sweden; (20)
Finland; (21) Hungary; (22) India; (23) South Korea; (24) Australia; (25) Mexico; (26) Cuba; (27) Puerto Rico; (28) Uruguay; (29) Portugal; (30) Switzerland; (31) Austria;
(32) Czech Republic; (33) Denmark; (34) Norway; (35) Croatia; (36) Turkey; (37) Algeria; (38) Egypt; (39) South Africa; (40) Iran; (41) United Arab Emirates; (42)
Pakistan; (43) Russia; (44) Japan.

Italy which is a representative southern European country where In the Asia-Pacific region, the endemic dissemination of
KPC is becoming endemic, 89.5% of carbapenemase producers KPC-producing K. pneumoniae has been reported in China (Li
have been reported to have KPC-type enzymes, followed by et al., 2014) and Taiwan (Tseng et al., 2015), and the sporadic
VIM-1 (9.2%) and OXA-48 (1.3%; Giani et al., 2013). spread has been reported in India (Shanmugam et al., 2013),
In America, the endemic spread of KPCs has been reported in South Korea (Yoo et al., 2013), and Australia (Partridge et al.,
Colombia (Villegas et al., 2006; Rojas et al., 2013), Brazil (Peirano 2015). A novel KPC-15 variant which is closely homologous
et al., 2009; Fehlberg et al., 2012), and Argentina (Pasteran with KPC-4 was discovered in China (Wang et al., 2014b) and
et al., 2008; Gomez et al., 2011). In Canada, KPC-producing its enzymatic activity and phenotype was characterized (Wang
K. pneumoniae has sporadically been reported (Goldfarb et al., et al., 2014a). In China, the frequency of KPC-type enzymes
2009; Lefebvre et al., 2015), and since plasmid-mediated KPC- among carbapenemase producers was high (63%; Li et al., 2014).
producing K. pneumoniae was first detected in Ottawa in While ST258 is the predominant clone observed in European
Goldfarb et al. (2009), a laboratory surveillance program countries and the USA (Giani et al., 2013; Chen et al., 2014e;
found a high frequency (89.3%) of KPC-type enzymes among Bowers et al., 2015), ST11, which is closely related to ST258,
carbapenemase producers between 2010 and 2012 (Lefebvre et al., is the prevalent clone associated with the spread of KPC-
2015). The emergence of KPCs in Argentina was characterized producing K. pneumoniae in Asia (particularly in China and
by two patterns of dispersion: the first was the irregular Taiwan; Qi et al., 2011; Yang et al., 2013; Tseng et al., 2015). KPC-
occurrence of diverse Enterobacteriaceae harboring blaKPC−2 producing ST11 strain has also been reported in Latin America
in the IncL/M transferable plasmid in distant regions and the (Munoz-Price et al., 2013). Although it is unknown why ST11
second was the sudden clonal spread of K. pneumoniae ST258 is prevalent, a recent report showed that the KPC-producing
harboring blaKPC−2 in Tn4401a (Gomez et al., 2011). KPC- K. pneumoniae ST11 clone was resistant to serum killing (Chiang
producing K. pneumoniae was recently also detected in Cuba et al., 2016). In a Chinese hospital, another nosocomial outbreak
(Quinones et al., 2014), Mexico (Garza-Ramos et al., 2014), of KPC-2-producing K. pneumoniae was caused by multiple
Uruguay (Marquez et al., 2014), and Puerto Rico (Gregory et al., K. pneumoniae strains including ST37, ST392, ST395, and ST11,
2010). implying the horizontal transfer of blaKPC−2 gene between

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

different K. pneumoniae clones in China (Yang et al., 2013). In 2009), indicating the worldwide prevalence of K. pneumoniae
Taiwan, two novel KPC variants were identified; KPC-16 and co-harboring two carbapenemases.
KPC-17 differed from KPC-2 by two (P202S and F207L) and a Aside from KPC-type carbapenemases, other class A
single (F207L) amino acid substitutions, respectively (Yu et al., carbapenemases, such as GES-2, GES-4, GES-5, GES-6, GES-11,
2015). A nationwide survey in Taiwan between 2011 and 2013 GES-14, GES-18, SFC-1, SHV-38, NMC-A, SME-1, and IMI-type
reported the national spread of KPC-2 and KPC-17 (Tseng et al., enzymes, were rarely found in K. pneumoniae (Table 1).
2015). KPC-producing K. pneumoniae was recently also detected
in Japan (Saito et al., 2014), Pakistan (Pesesky et al., 2015), Iran Molecular and Genetic Context
(Nobari et al., 2014), and United Arab Emirates (Sonnevend The blaKPC in K. pneumoniae has been reported on numerous
et al., 2015a). In the Arabian Peninsula, the prevalence of plasmid types, such as IncF, IncI2, IncX, IncA/C, IncR, and
KPC-producing K. pneumoniae was very low in comparison to ColE1 (Garcia-Fernandez et al., 2012; Chen et al., 2014e;
NDM-1 and OXA-48-like carbapenemases (Sonnevend et al., Pitout et al., 2015), but the predominant plasmid type is IncF
2015b). Sonnevend et al. (2015a), two K. pneumoniae ST14 with FIIK replicons (Pitout et al., 2015). IncF often contains
strains producing KPC-2 were first identified in the United Arab several additional genes responsible for resistance to other
Emirates of the Arabian Peninsula. In Africa, several countries antibiotics, including aminoglycosides, tetracyclines, quinolones,
such as South Africa (Brink et al., 2012), Algeria (Bakour et al., trimethoprim, and sulfonamides (Pitout et al., 2015). Many
2015b), and Egypt (Metwally et al., 2013), have also isolated blaKPC genes are associated with a promiscuous transposon-
KPC-producing K. pneumoniae. related structure Tn4401, which is approximately 10 kb in
The coexistence of KPCs and other carbapenemases in size and consists of a transposase gene, a resolvase gene, the
K. pneumoniae was frequently reported worldwide, including blaKPC gene, and two insertion sequences, ISKpn6 and ISKpn7
in Italy (KPC-3/VIM-2 and KPC-2/VIM-1; Richter et al., 2012; (Figure 2A; Naas et al., 2008). This transposon has jumped to
Perilli et al., 2013), Colombia (KPC-2/VIM-24; Rojas et al., numerous plasmids that are commonly conjugative (Chen et al.,
2013), Brazil (KPC-2/NDM-1; Pereira et al., 2015), China (KPC- 2014e). In China, a novel genetic environment was detected (Shen
2/NDM-1, KPC-2/CMY-2, and KPC-2/IMP-4; Hu et al., 2014; et al., 2009). It contains an integration structure consisting of
Dong et al., 2015; Liu et al., 2015), Canada (KPC-3/CMY-2; a Tn3-based transposon and partial Tn4401 segment, with the
Leung et al., 2012), and Greece (KPC-2/VIM-1; Giakkoupi et al., gene order Tn3-transposase, Tn3-resolvase, ISKpn8, the blaKPC−2

TABLE 1 | The epidemiology of various carbapenemases in Klebsiella pneumoniae.

Molecular class Carbapenemase Geographical distribution

A SME types Not found

IMI types Not found
GES types Greece (Vourli et al., 2004), Finland (Osterblad et al., 2012), Brazil (Picao et al.,
2010), and South Korea (Jeong et al., 2005; Bae et al., 2007)
SFC-1, SHV-38, and NMC-A France (Poirel et al., 2003) and Brazil (Tollentino et al., 2011)
B OXA-23, OXA-24/40, OXA-51, Not found
OXA-58, OXA-134, OXA-143,
OXA-211, OXA-213, OXA-214,
OXA-229, and OXA-235
C DHA-1 Taiwan (Lee et al., 2012b), South Korea (Park et al., 2013), and China (Hu et al.,
2014)
CMY-2 and CMY-10 China (Hu et al., 2014), Canada (Leung et al., 2012), and Greece (Pournaras
et al., 2010b)
ADC-68 Not found
D IMP types Malaysia (Hamzan et al., 2015), Taiwan (Tseng et al., 2015), China (Chen et al.,
2015), Thailand (Rimrang et al., 2012), Ireland (Morris et al., 2016), Greece
(Lascols et al., 2013), Spain (Lascols et al., 2013), Italy (Lascols et al., 2013),
Turkey (Lascols et al., 2013), Austria (Zarfel et al., 2011), the USA (Limbago
et al., 2011; Rojas et al., 2013), and Mexico (Gales et al., 2012)
VIM types Greece (Pournaras et al., 2010b), Ireland (Morris et al., 2016), Spain (Pena
et al., 2014), Australia (Lascols et al., 2013), Croatia (Zujic Atalic et al., 2014),
the Czech Republic (Hrabak et al., 2013b), Hungary (Melegh et al., 2014), Italy
(Giani et al., 2013), Norway (Naseer et al., 2012), Austria (Zarfel et al., 2011),
Finland (Osterblad et al., 2012), Germany (Steinmann et al., 2011), France
(Birgy et al., 2011), China (Liu et al., 2015), India (Castanheira et al., 2011),
Philippines (Lascols et al., 2013), Iran (Rajabnia et al., 2015), Taiwan (Tseng
et al., 2015), Colombia (Rojas et al., 2013), Mexico (Gales et al., 2012), and
Algeria (Rodriguez-Martinez et al., 2010)
GIM-1, KHM-1, and SPM-1 Not found

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

FIGURE 2 | Structural features of representative genetic environments of blaKPC−2 , blaNDM−1 , and blaOXA−48 genes. (A) The blaKPC−2 -containing Tn4401
transposon from the plasmid pNYC (GenBank accession no. EU176011) is shown in horizontal arrows. Two inverted repeat sequences (IRL and IRR) of Tn4401 are
depicted in triangles at either end. Tn4401 has five isoforms which differ by deletions (68–255 bp) just upstream of the blaKPC gene [(a) deletion of 99 bp; (b) no
deletion; (c) deletion of 215 bp; (d) deletion of 68 bp; (e) deletion of 255 bp]. (B) The blaNDM−1 genetic context of pNDM_MGR194 (GenBank accession no.
KF220657) is shown in horizontal arrows. (C) The blaOXA−48 -containing Tn1999 transposon from the plasmid pOXA-48 (GenBank accession no. JN626286) is
shown in horizontal arrows.

gene, and the ISKpn6-like element (Shen et al., 2009). This These genes may be important for the successful dissemination
genetic structure is the chimera form of several transposon- of blaKPC -harboring plasmids. The IncFII plasmids are one of
associated elements. This transposon was also identified in many predominant blaKPC -harboring plasmids. pKpQIL, which is an
other countries (Chen et al., 2014e), and several variants with IncFIIK2 plasmid harboring Tn4401a, was initially identified in
various fragment insertions between the ISKpn8 and blaKPC Israel in 2006 (Leavitt et al., 2010), and then this plasmid and
gene have been found among Enterobacteriaceae in China its variants are believed to have spread to Italy, Poland, the UK,
(Shen et al., 2009; Li et al., 2011; Qi et al., 2011). Tn4401 Colombia, the Czech Republic, the USA, and other countries
has five isoforms which differ by deletions (68–255 bp) just (Baraniak et al., 2011; Garcia-Fernandez et al., 2012; Hidalgo-
upstream of the blaKPC gene [(a) deletion of 99 bp; (b) no Grass et al., 2012; Warburg et al., 2012; Hrabak et al., 2013b;
deletion; (c) deletion of 215 bp; (d) deletion of 68 bp; (e) Chen et al., 2014d,e), suggesting the wide dissemination of this
deletion of 255 bp; Chen et al., 2014e]. Notably, in many plasmid. The blaKPC gene has also been identified in other
cases, different Tn4401 isoforms was associated with different non-Tn4401 mobile elements that mostly have partial ISKpn6
blaKPC -harboring plasmids. Tn4401a was frequently found in genes (Shen et al., 2009; Gomez et al., 2011). Based on the
the blaKPC−3 -harboring IncFIIK2 plasmids (Leavitt et al., 2010; insertion sequence upstream of the blaKPC gene, they can be
Garcia-Fernandez et al., 2012; Chen et al., 2014d), and Tn4401b divided into three groups: group I, no insertion (Shen et al.,
and Tn4401d were often associated with the IncN and IncFIA 2009; Liu et al., 2012; Chen et al., 2014g); group II, insertion of
plasmids, respectively (Chen et al., 2013a, 2014c,e). Up to truncated blaTEM (Gomez et al., 2011); group III, insertion of
now, more than 30 blaKPC -harboring plasmids obtained from Tn5563/IS6100 (Wolter et al., 2009). These non-Tn4401 genetic
K. pneumoniae have been sequenced (Gootz et al., 2009; Shen elements harboring blaKPC sometimes have an IS26 transposon
et al., 2009; Jiang et al., 2010; Leavitt et al., 2010; Almeida (Liu et al., 2012; Chen et al., 2014g).
et al., 2012; Chen et al., 2013a,b, 2014b,c,e). One of common Carbapenemase genes often spread worldwide through clonal
features shared by these sequenced plasmids is the presence of the expansion in several successful pathogenic strains (Chen et al.,
tra operon, which encodes the plasmid conjugation machinery 2014e). For example, the dissemination of KPC-producing
proteins that induce the spread of plasmids (Chen et al., 2014e). K. pneumoniae in most countries including the USA and

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

FIGURE 3 | Epidemiological features of NDM-producing K. pneumoniae. (1) India; (2) Pakistan; (3) Bangladesh; (4) Canada; (5) USA; (6) Colombia; (7) Spain;
(8) France; (9) UK; (10) Italy; (11) Switzerland; (12) Greece; (13) Turkey; (14) Saudi Arabia; (15) Oman; (16) United Arab Emirates; (17) Kuwait; (18) Morocco; (19)
South Africa; (20) China; (21) South Korea; (22) Japan; (23) Taiwan; (24) Singapore; (25) Australia; (26) Mexico; (27) Guatemala; (28) Brazil; (29) Ireland; (30) Germany;
(31) Netherlands; (32) Czech Republic; (33) Poland; (34) Hungary; (35) Romania; (36) Croatia; (37) Norway; (38) Sweden; (39) Finland; (40) Russia; (41) Algeria; (42)
Tunisia; (43) Libya; (44) Egypt; (45) Kenya; (46) Madagascar; (47) Mauritius; (48) Israel; (49) Iraq; (50) Iran; (51) Yemen; (52) Sri Lanka; (53) Nepal; (54) Thailand; (55)
Vietnam; (56) Malaysia, (57) New Zealand.

European countries is largely caused by expansion of a single KPC-2, while the cps-2 clade is primarily associated with KPC-
dominant strain, ST258 (Giani et al., 2013; Chen et al., 2014e; 3 (Chen et al., 2014a). Because the capsule polysaccharide can
Bowers et al., 2015). This strain is a prototype of a high-risk clone help K. pneumoniae to evade phagocytosis, the global success of
of K. pneumoniae. Recent data from Israel showed that the KPC- this strain may involve the capsule polysaccharide biosynthesis
producing K. pneumoniae ST258 clone remains the predominant regions cps-1 and cps-2. A recent report revealed a relationship
clone, representing 80% of the KPC-producing K. pneumoniae between the integrative conjugative element ICEKp258.2 and the
population (Adler et al., 2015). ST258 may be a hybrid clone global success of the ST258 clone (Adler et al., 2014). ICEKp258.2
that was created by a large recombination event between ST11 contains two specific gene clusters, a type IV pilus gene cluster
and ST442 (Pitout et al., 2015). It is unknown why the ST258 (i.e., pilV) associated with the uptake and exchange of plasmids
lineage is the most prevalent clone of KPC-producing Klebsiella and adherence to living and non-living surfaces, and a gene
species. The ST258 clone is highly susceptible to serum killing cluster of a type III restriction-modification system determining
in animal models and lacks well-known K. pneumoniae virulence host specificity in the exchange of certain compatible plasmids
factors, such as aerobactin genes, K1, K2, and K5 capsular antigen or mobile elements (Adler et al., 2014). Because these genes
genes, and the regulator of the mucoid phenotype gene rmpA associated with the restriction of plasmids and specific mobile
(Tzouvelekis et al., 2013; Pitout et al., 2015). Two recent reports elements were present only in ST258 and genetically related
revealed that the ST258 strains consist of two distinct genetic sequence types, this difference may explain the divergence of
clades and genetic differentiation between the two clades (-1 and ST258 predominantly harboring KPC and ST11, another high-
cps-2) results from an approximately 215-kb region of divergence risk clone that lacks ICEKp258.2, harboring a broad range of
that includes cps genes involved in capsule polysaccharide plasmids and carbapenemases, including KPC, NDM, OXA-
synthesis (Chen et al., 2014e; Deleo et al., 2014). Multiplex PCR 48, VIM, and IMP (Chen et al., 2014f; Pitout et al., 2015).
for the identification of two capsular types in K. pneumoniae Although the ICEKp258.2 of ST258 strains may contribute to
ST258 strains revealed a significant association between the cps global success, the precise reason for the predominance of the
type and KPC variant: the cps-1 clade is largely associated with ST258 strain in KPC-producing K. pneumoniae is still not entirely

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

FIGURE 4 | Epidemiological features of OXA-48-like-producing K. pneumoniae. (1) Turkey; (2) Morocco; (3) Tunisia; (4) Libya; (5) Egypt; (6) India; (7)
Argentina; (8) Spain; (9) France; (10) Germany; (11) Switzerland; (12) Belgium; (13) Netherlands; (14) UK; (15) Italy; (16) Israel; (17) Saudi Arabia; (18) Kuwait; (19)
Lebanon; (20) Japan; (21) Canada; (22) USA; (23) Ireland; (24) Poland; (25) Finland; (26) Hungary; (27) Romania; (28) Bulgaria; (29) Greece; (30) Russia; (31) Algeria;
(32) Senegal; (33) South Africa; (34) United Arab Emirates; (35) Oman; (36) Iran; (37) Sri Lanka; (38) Thailand; (39) Singapore; (40) South Korea; (41) Taiwan; (42)
Australia; (43) New Zealand.

understood. Recently, an outbreak of non-ST258 KPC-producing colonizing the human intestine appears frequent, for example
K. pneumoniae clones has been reported in the USA and Europe from K. pneumoniae to E. coli (Richter et al., 2011; Gona et al.,
(Ruiz-Garbajosa et al., 2013; Bonura et al., 2015; Garbari et al., 2014). Therefore, reservoirs in healthcare workers, patients, or
2015). the hospital environment may be a principle mode of spread in
The habitat of K. pneumoniae is not limited to humans but nosocomial outbreaks.
extends to the ecological environment, such as soil, water, and
sewage, and K. pneumoniae can survive in extreme environments Treatment Options
for long periods of time (Pitout et al., 2015). Therefore, Carbapenemase-producing K. pneumoniae strains are currently
K. pneumoniae producing KPCs were detected in various one of the most important nosocomial pathogens. Hospital
nosocomial environments, such as gowns and gloves (Rock et al., outbreaks of KPC-producing K. pneumoniae mainly affect
2014) and wastewater (Chagas et al., 2011; Galler et al., 2014). severely ill patients and are associated with an increased risk of
The frequency of KPC-producing K. pneumoniae contamination death (Ducomble et al., 2015; Tumbarello et al., 2015). KPC-
of gowns and gloves of healthcare workers is similar to that of producing K. pneumoniae bloodstream infections in intensive
contamination with methicillin-resistant Staphylococcus aureus care unit (ICU) have also been associated with increased
and vancomycin-resistant Enterococcus (Rock et al., 2014), mortality (Chang et al., 2015).
indicating fast transmission of KPC-producing Klebsiella species Because carbapenemase-producing K. pneumoniae are mostly
in a nosocomial environment. A long-term observation in resistant to several important antibiotic classes (β-lactam drugs,
a hospital with low-frequency outbreaks of KPC-producing fluoroquinolones, and aminoglycosides), antibiotics, such as
K. pneumoniae suggested the possible role of a persisting polymyxin B, colistin (polymyxin E), fosfomycin, tigecycline,
environmental reservoir of resistant strains in the maintenance of and sometimes selected aminoglycosides, are the last-resort
this long-term outbreak (Tofteland et al., 2013). After discharge agents. KPC-producing K. pneumoniae are usually resistant to
from the hospital, long-term (>3 years) carriage of KPC- all β-lactam antibiotics, but temocillin can be active against
producing K. pneumoniae is also possible (Lübbert et al., 2014), some KPC-producing K. pneumoniae, particularly in the case
and lateral gene transfer of KPC among Enterobacteriaceae of lower urinary tract infections (Adams-Haduch et al., 2009).

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

To maximize bacterial killing and minimize bacterial resistance, was also reported elsewhere (Pena et al., 2014; Bonura et al.,
combined therapy is sometimes recommended. Combination 2015; Parisi et al., 2015). These results indicate that the strict
therapy including a carbapenem, such as a combination of rules for colistin use are required to diminish the spread of
tigecycline, colistin, and meropenem, was strongly effective in the colistin resistance in the endemic regions of KPC-producing
treatment of KPC-producing K. pneumoniae, including colistin- K. pneumoniae.
resistant isolates (Tumbarello et al., 2012, 2015; Giamarellou Molecular and biochemical studies have shown that
et al., 2013; Hong et al., 2013; Daikos et al., 2014). The insertional inactivation of the mgrB gene, encoding a negative-
synergistic combination of colistin and rifampin was also feedback regulator of the PhoQ–PhoP signaling system,
effective in the treatment of colistin-resistant KPC-producing can be responsible for colistin resistance in KPC-producing
K. pneumoniae by slowing the selection of heteroresistant K. pneumoniae, due to the resulting up-regulation of the Pmr
subpopulations during colistin therapy (Tascini et al., 2013). lipopolysaccharide modification system (Cannatelli et al., 2013,
However, several reports have shown that combination therapy 2014b). A recent study analyzing a series of colistin-resistant
was not superior to monotherapy (de Oliveira et al., 2015; Toledo K. pneumoniae isolates of worldwide origin identified a single
et al., 2015). Thus, extensive studies will be required to assess amino acid change (T157P) in the PmrB protein as being
the effectiveness of combination therapy. A triple combination responsible for the overexpression of pmrCAB and pmrHFIJKLM
of colistin-doripenem-ertapenem was effective only in isolates operons involved in lipopolysaccharide modification, leading
with high levels of OmpK35 and OmpK36 porin expression to colistin resistance (Jayol et al., 2014). The relationship
(Hong et al., 2013). The expression level of OmpK36 was between colistin resistance and inactivation of the mgrB gene
also involved in the rapid induction of high-level carbapenem was further supported by analysis of clinical colistin-resistant
resistance in heteroresistant KPC-producing K. pneumoniae K. pneumoniae isolates producing KPC (Bonura et al., 2015;
populations (Adams-Sapper et al., 2015). Therefore, molecular Giani et al., 2015). The emergence of colistin resistance was also
characterization of the KPC-producing K. pneumoniae strain, associated with low-dosage colistin treatment (Cannatelli et al.,
such as the determination of the expression level of OmpK35 2014a). A recent report showed that the plasmid carrying the
and OmpK36, can be used to identify effective combination mcr-1 gene, which encodes a phosphoethanolamine transferase
regimens. However, as a minor effect of OmpK35 and OmpK36 enzyme catalyzing the addition of phosphoethanolamine to lipid
on carbapenem resistance of K. pneumoniae was also reported A, is a major contributor to colistin resistance in Gram-negative
(Zhang et al., 2014), more extensive studies on the role of bacteria and is spread through horizontal gene transfer (Liu
these proteins on K. pneumoniae carbapenem resistance are also et al., 2016). This mcr-1-harboring plasmid was also detected in
required. E. coli isolates collected from 78 (15%) of 523 samples of raw
Colistin (polymyxin E), an agent discovered more than meat, 166 (21%) of 804 animals, and 16 (1%) of 1322 samples
60 years ago, is a key component of the combination of from inpatients with infection, indicating the emergence of
antimicrobial regimens used for the treatment of severe KPC- this plasmid-mediated colistin resistance mechanism (Liu et al.,
producing K. pneumoniae infections (Cannatelli et al., 2014b). 2016).
Since the global spread of KPC-producing K. pneumoniae, Fosfomycin is a broad-spectrum antibiotic that inhibits
the emergence of colistin resistance in KPC-producing bacterial cell wall biogenesis by inactivating the enzyme UDP-
K. pneumoniae have been reported in many countries, including N-acetylglucosamine-3-enolpyruvyltransferase, also known as
Italy (Cannatelli et al., 2014b; Giani et al., 2015), the USA MurA (Brown et al., 1995). Fosfomycin has been used to
(Bogdanovich et al., 2011), Greece (Kontopoulou et al., 2010), treat KPC-producing K. pneumoniae, but recently, a high
Hungary (Toth et al., 2010), and Turkey (Labarca et al., 2014). fosfomycin resistance rate was observed is in countries with
The increasing prevalence of colistin-resistant K. pneumoniae higher usage (Giske, 2015). Only 43.4% of KPC-producing
producing KPC poses a threat to public health because colistin K. pneumoniae strains retained susceptibility to fosfomycin in
resistance increases the mortality due to KPC-producing a Chinese university hospital (Li et al., 2012) and a similar
K. pneumoniae bloodstream infections and reduces therapeutic fosfomycin susceptibility rate (39.2%) was observed in KPC-
options. A multicenter case-control-control study in Italy producing K. pneumoniae collected from 12 hospitals in China
showed that the rate of colistin resistance among KPC-producing (Jiang et al., 2015). Like colistin, fosfomycin resistance seems
K. pneumoniae blood isolates increased more than threefold to be associated with the plasmid containing the fosA3 gene
during the 4.5-years study period, and the 30-days mortality due which encodes glutathione S-transferase to modify fosfomycin
to colistin-resistant KPC-producing K. pneumoniae bloodstream and was characterized first in CTX-M-producing E. coli in Japan
infections was approximately 51% (Giacobbe et al., 2015). Data (Wachino et al., 2010). In China, the fosA3-harboring plasmid
collected from 21 hospital laboratories in Italy between 2013 was attributed to 55.6% of fosfomycin-resistant KPC-producing
and 2014 also showed that 43% of carbapenemase-producing K. pneumoniae strains (Jiang et al., 2015). Although the fosA3
K. pneumoniae were resistant to colistin, 6% were resistant gene is mainly associated with the blaCTX−M gene, the fosA3
to tigecycline, 16% were resistant to gentamicin, 82% were gene has also been characterized in atypical blaKPC -harboring
resistant to trimethoprim-sulfamethoxazole, and 1% were plasmids (Jiang et al., 2015; Li et al., 2015). In pFOS18 (Jiang
resistant to all four antibiotics, and colistin-resistant isolates et al., 2015) and pKP1034 (Xiang et al., 2015), the fosA3 and
were detected in all participating hospital laboratories (Monaco blaKPC−2 genes were located on different transposon systems,
et al., 2014). The progressive increase of colistin resistance whereas in pHS102707 belonging to the IncP1 group (Li et al.,

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

2015), two genes were co-located in the same Tn1721-Tn3-like Detection Methods
transposon. Because a delay in the appropriate antibiotic therapy for
Tigecycline, a derivative of minocycline, is the first member severe infections of KPC-producing K. pneumoniae is strongly
of the glycylcycline class that acts as a protein synthesis inhibitor associated with unfavorable prognosis and increased mortality
by blocking the interaction of aminoacyl-tRNA with the A site rates (Karaiskos and Giamarellou, 2014), rapid detection of
of the ribosome (Rose and Rybak, 2006). Due to the increased CR strains is essential for the effective management of these
clinical use of tigecycline for treatment of KPC-producing infections (Lee et al., 2005b, 2006b, 2016). Various methods
K. pneumoniae, increased tigecycline resistance was reported for the identification of KPCs have been developed, including
(Zagorianou et al., 2012; Papadimitriou-Olivgeris et al., 2014; multiplex PCR assay (Spanu et al., 2012; Adler et al., 2014),
Weterings et al., 2015). In the Netherlands, all KPC-producing real-time PCR assay (Wang et al., 2012; Lee et al., 2015c),
K. pneumoniae isolates exhibited reduced susceptibility to DNA microarray (Peter et al., 2012; Braun et al., 2014), Raman
tigecycline (Weterings et al., 2015). Another report showed that spectroscopic analysis (Willemse-Erix et al., 2012), single-colony
during ICU stay, 17.9% (39/257) of patients became colonized whole-genome sequencing (Koser et al., 2014), matrix-assisted
by tigecycline-resistant KPC-producing K. pneumoniae during laser desorption ionization-time-of-flight mass spectrometry
their stay (Papadimitriou-Olivgeris et al., 2014). In a Greek (MALDI-TOF MS; Carvalhaes et al., 2014), loop-mediated
tertiary hospital during 2004 to 2010, 11.3% (34/301) of KPC- isothermal amplification (LAMP) method (Nakano et al., 2015),
producing isolates were resistant to tigecycline (Zagorianou et al., chromogenic medium (Vrioni et al., 2012), and a new phenotypic
2012). Overproduction of efflux pumps such as AcrAB and test, called the carbapenem inactivation method (CIM; van der
overexpression of RamA, a positive regulator of the AcrAB Zwaluw et al., 2015). False positive results can occur when
efflux system, seem to be major factors for decreased sensitivity the modified Hodge test is used to detect carbapenemases in
of K. pneumoniae strains to tigecycline (Ruzin et al., 2005; carbapenemase-negative K. pneumoniae clinical isolates (Wang
Rosenblum et al., 2011; Sun et al., 2013). A recent report in et al., 2011). Therefore, to improve the efficiency in the
China showed that the OqxAB efflux pump was also contributed phenotypic detection of KPC-producing K. pneumoniae isolates,
to tigecycline resistance in K. pneumoniae isolates (Zhong et al., the modified Hodge test can be combined with an EDTA
2014). disk test (Yan et al., 2014) or a disk test using boronic acid
Because KPC-producing K. pneumoniae sometimes remains compounds (Pournaras et al., 2010a). These methods enhanced
susceptible to several aminoglycosides such as gentamicin the sensitivity and specificity of KPC detection in K. pneumoniae
(Tzouvelekis et al., 2014), aminoglycosides can be used alone or isolates. In a new developed phenotypic test, called the CIM, a
in combination therapy to treat KPC-producing K. pneumoniae susceptibility-testing disk containing carbapenem was immersed
infections. Actually, gentamicin monotherapy or in combination in the suspension made by suspending an inoculation loop
with tigecycline was recently reported to reduce the mortality of bacterial culture (van der Zwaluw et al., 2015). After
from sepsis caused by K. pneumoniae ST512 clone producing incubation, the disk was placed on an agar plate inoculated
KPC-3, SHV-11, or TEM-1 (Gonzalez-Padilla et al., 2015). with a susceptible E. coli indicator strain. If the bacterial isolate
New weapons are always indispensable for combating KPC- produces carbapenemase, the susceptibility-testing disk will allow
producing K. pneumoniae infections (Lee et al., 2007, 2015a). the growth of the susceptible indicator strain. This method
The effectiveness of some antibiotics in development was showed high concordance with results obtained by PCR (van
also estimated against KPC-producing K. pneumoniae. Potent der Zwaluw et al., 2015). Nevertheless, these culture-based
inhibitors of serine β-lactamases, such as avibactam and phenotypic tests are time-consuming and cannot easily detect
MK7655, were effective against KPC-producing K. pneumoniae ESBLs and carbapenemases produced by Enterobacteriaceae,
infections (Temkin et al., 2014). Combination therapy with owing to varied levels of enzyme expression and the poor
avibactam and ceftazidime exhibited significant synergetic effects specificity of some antibiotic combinations (Okeke et al.,
against organisms with combinations of extended-spectrum 2011; Swayne et al., 2013). To overcome these limitations
β-lactamases (ESBLs), AmpCs, and KPC-2 (Wang et al., of phenotypic methods, various molecular-based diagnostic
2014e). Plazomicin (a novel aminoglycoside) also exhibited methods have been developed. Especially, the direct detection
significant activity against KPC-producing K. pneumoniae of the carbapenemase gene using multiplex PCR, real-time
(Temkin et al., 2014). The novel polymyxin derivatives with lower PCR, and DNA microarray method can increase the speed and
nephrotoxicity are under development (Vaara, 2010). A recent accuracy of detecting CR strains (Okeke et al., 2011; Solanki
report suggested that synthetic peptides with antimicrobial and et al., 2014). Because of the high genetic diversity of genes
antibiofilm activities are a promising strategy in the treatment coding for carbapenemase, the precise design of primers or
of infections caused by KPC-producing K. pneumoniae (Ribeiro probes is necessary for correctly amplifying or detecting only
et al., 2015). The in vitro activity of the next-generation expected carbapenemase genes. Therefore, already developed
aminoglycoside plazomicin alone and in combination with methods for bla gene detection are restricted to the detection
colistin, meropenem, fosfomycin or tigecycline was tested against of only limited types of bla genes (Lee et al., 2015b). However,
carbapenemase-producing Enterobacteriaceae (CPE) strains. the large-scale blaFinder was recently developed on the basis
When plazomicin was combined with meropenem, colistin of multiplex PCR, and this large-scale detection method can
or fosfomycin, synergy was observed against CPE isolates detect almost all bla genes, including KPCs, NDMs, OXA-
(Rodriguez-Avial et al., 2015). 48-likes, present in bacterial pathogens (Lee et al., 2015b).

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

Recently, mass spectrometry-based methods, such as MALDI- virtually all β-lactam antibiotics including carbapenems except
TOF MS and ultra-performance liquid chromatography–tandem for monobactams (Jeon et al., 2015). Class B carbapenemases
mass spectrometry (UPLC-MS/MS), have been shown to be were mostly identified in Enterobacteriaceae and include VIMs,
capable of characterizing carbapenemase-producing bacteria IMPs, and the emerging NDM group (Jeon et al., 2015). Among
(Carricajo et al., 2014; Carvalhaes et al., 2014). These methods them, NDM (New Delhi metallo-β-lactamase) is one of the most
are fast and accurate to routinely identify bacterial isolates clinically significant carbapenemases. NDM-1 was first detected
with great specificity and sensitivity (Hrabak et al., 2011; in 2008 in K. pneumoniae and E. coli in a patient returning
Patel, 2013; Carvalhaes et al., 2014; Lasserre et al., 2015), to Sweden from India and has since spread worldwide (Yong
but these systems do not accurately provide the carbapenem et al., 2009; Jeon et al., 2015). Thus far, 15 NDM variants have
minimum inhibitory concentrations (MICs) for carbapenemase- been assigned (Jeon et al., 2015), and most of them originated
producing K. pneumoniae (Patel, 2013). Several experiments from Asia (Nordmann and Poirel, 2014). NDMs shares very little
showed that this method is more rapid and accurate for identity with other metallo-β-lactamases (Nordmann and Poirel,
detection of carbapenemase activity in Gram-negative bacteria 2014).
than some methods including the modified Hodge test (Lee Since 2008, K. pneumoniae producing NDMs rapidly spread
et al., 2013b; Chong et al., 2015). The LAMP method has in many countries (Berrazeg et al., 2014; Figure 3). NDM-
emerged as a powerful gene amplification assay for the rapid producing K. pneumoniae are considered to be endemic in the
identification of microbial infections (Notomi et al., 2000). Indian subcontinent, including India, Pakistan, and Bangladesh
This method employs a DNA polymerase and a set of four (Nordmann et al., 2011b; Nordmann and Poirel, 2014). The
specially designed primers that recognize a total of six distinct sporadic spread has been reported in the USA (Rasheed et al.,
sequences on the target DNA. The assay amplifies the DNA 2013; Centers for Disease Control and Prevention, 2014; Doi
under isothermal conditions (63–65◦ C) with high degrees of et al., 2014), Canada (Mulvey et al., 2011; Borgia et al., 2012;
specificity, efficiency, and speed (Reddy et al., 2010). The cycling Lowe et al., 2013), Colombia (Escobar Perez et al., 2013; Ocampo
reaction continues with accumulation of 109 copies of target et al., 2015), Spain (Oteo et al., 2013b; Seara et al., 2015),
in less than an hour. The assay can be conducted in a water France (Arpin et al., 2012; Robert et al., 2014), Switzerland
bath or heating block instead of the thermal cycling using a (Poirel et al., 2011g; Spyropoulou et al., 2016), Italy (Gaibani
PCR machine (Notomi et al., 2000). The LAMP assay can be et al., 2011), the UK (Kumarasamy et al., 2010; Giske et al.,
applied for detection of KPC producers in the clinical laboratory 2012), Greece (Voulgari et al., 2014; Spyropoulou et al., 2016),
(Nakano et al., 2015) and has greater sensitivity, specificity, and Turkey (Poirel et al., 2014; Kilic and Baysallar, 2015), Morocco
rapidity compared to the phenotypic methods and PCR for (Poirel et al., 2011b; Barguigua et al., 2013), South Africa
the detection of KPC-producing K. pneumoniae (Solanki et al., (Brink et al., 2012; de Jager et al., 2015), Singapore (Chen
2013). et al., 2012; Balm et al., 2013; Ling et al., 2015), Saudi Arabia
Colistin has often been used as a therapeutic option for (Shibl et al., 2013; Zowawi et al., 2014), Oman (Poirel et al.,
the treatment of CR K. pneumoniae infections. However, 2011a; Zowawi et al., 2014), United Arab Emirates (Sonnevend
the imprudent use of colistin has caused rapid spread of et al., 2013; Dash et al., 2014), Kuwait (Jamal et al., 2012,
colistin resistance in K. pneumoniae producing carbapenemases, 2013; Sonnevend et al., 2015b), China (Qin et al., 2014; Jin
particularly the KPC-type carbapenemases (Monaco et al., 2014; et al., 2015; Liu et al., 2015), Japan (Yamamoto et al., 2013;
Giacobbe et al., 2015). This situation demonstrates the need Nakano et al., 2014), Taiwan (Chiu et al., 2013; Tseng et al.,
for the development of accurate and reliable methods for 2015), South Korea (Kim et al., 2012; Cho et al., 2015), and
detecting colistin resistance. Recently, several methods for the Australia (Shoma et al., 2014; Wailan et al., 2015). In India,
identification of colistin resistance were reported, including NDM-1 was the most common carbapenemase type detected
various routine colistin MIC testing methods, such as BMD, and accounted for 75.22% of the carbapenemase-producing
BMD-P80, AD, Etest, MTS, and Vitek2 (Dafopoulou et al., 2015; isolates (Kazi et al., 2015). In Singapore and the United Arab
Humphries, 2015); capillary electrophoresis method according to Emirates, NDM-1 also was the most common carbapenemase
characteristic surface properties of bacteria (Sautrey et al., 2015); type observed (44.4 and 100%, respectively; Dash et al., 2014;
and the micromax assay based on evaluation of the efficacy of Ling et al., 2015). The endemic spread of NDM-producing
antibiotics that affect cell wall integrity (Tamayo et al., 2013). K. pneumoniae has also been reported in the UK, which has
Because a recent report showed that the mcr-1 gene, involved close relationships with India and Pakistan (Nordmann and
in the modification of lipid A, is a major contributor to colistin Poirel, 2014). In China, NDM-1 has been found mostly in
resistance in Gram-negative bacteria (Liu et al., 2016), detection Acinetobacter spp., but data obtained from patients between
of this gene may be important in the detection of colistin June 2011 and July 2012 showed that 33.3% of the CRE
resistance. isolates, including K. pneumoniae, had NDM-1, suggesting the
possible transmission of blaNDM−1 -containing sequences from
Class B Carbapenemases Acinetobacter spp. to Enterobacteriaceae (Qin et al., 2014).
Epidemiology These findings reveal the emergence and active transmission
Class B β-lactamases are metallo-β-lactamases that require zinc of NDM-1-producing K. pneumoniae in China. Comparative
or another heavy metal for catalysis. Class B β-lactamases have analyses of the conserved NDM-1-encoding region among
a broad substrate spectrum and can catalyze the hydrolysis of different plasmids from K. pneumoniae and E. coli suggested that

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

the transposable elements and two unknown inverted repeat- in Madagascar highlighted the potential for mother-to-child
associated elements flanking the NDM-1-encoding region aided transmission (Chereau et al., 2015). In India, analysis of
the spreading of this resistance determinant (Chen et al., 2012). Enterobacteriaceae, including K. pneumoniae, isolated from
Recently, in China, eight K. pneumoniae isolates producing the blood of septicaemic neonates, indicted that 14% of the
NDM-1 were identified in the neonatal ward of a teaching isolates possessed blaNDM−1 , and there was a significantly higher
hospital (Zhang et al., 2015), and four diverse types (NDM-1, incidence of sepsis caused by NDM-1-harboring isolates (Datta
KPC-2, VIM-2, and IMP-4) of carbapenemase of K. pneumoniae et al., 2014). In China, an outbreak of blaNDM−1 -producing
clones were identified in a single hospital in China (Liu et al., K. pneumoniae ST20 and ST17 isolates was identified in a
2015). neonatal unit (Jin et al., 2015). In Turkey, the spread of NDM-1-
The Balkan states (Livermore et al., 2011; Voulgari et al., producing K. pneumoniae in a neonatal ICU was reported (Poirel
2014), the Arabian Peninsula (Nordmann and Poirel, 2014), et al., 2014). In Colombia, NDM-1-producing K. pneumoniae
and North African countries (Dortet et al., 2014c), have also strains were identified from an outbreak that affected six neonatal
been recently considered as an additional reservoir of NDM patients (Escobar Perez et al., 2013).
producers. In the Arabian Peninsula, NDM-1 was the most As with KPC, coexistence of NDMs and other carbapenemases
frequently encountered carbapenemase (46.5%) followed by in K. pneumoniae has also been reported worldwide, in Brazil
OXA-48-like carbapenemases (32.5%; Sonnevend et al., 2015b). (NDM-1/KPC-2; Pereira et al., 2015), Malaysia (NDM-1/OXA-
In Greece, among 132 non-repetitive CRE isolates between 232; Al-Marzooq et al., 2015), South Korea (NDM-5/OXA-181;
2010 and 2013, 78 K. pneumoniae isolates with the blaNDM−1 Cho et al., 2015), China (NDM-1/IMP-4; Chen et al., 2015), India
gene were identified (Voulgari et al., 2014). In the USA, KPC- (NDM-1/OXA-232; Al-Marzooq et al., 2015), Turkey (NDM-
producing K. pneumoniae have been responsible for much of 1/OXA-48; Kilic and Baysallar, 2015), Pakistan (NDM-1/KPC-
the increase in carbapenemase-producing bacteria detection, but 2; Sattar et al., 2014), Switzerland (NDM-1/OXA-48; Seiffert
recent increases in NDM-producing K. pneumoniae have the et al., 2014), United Arab Emirates (NDM-1/OXA-48-like; Dash
potential to add to this burden (Rasheed et al., 2013; Centers for et al., 2014), Australia (NDM-1/OXA-48; Sidjabat et al., 2015),
Disease Control and Prevention, 2014). Morocco (NDM-1/OXA-48; Barguigua et al., 2013), Singapore
The movement of patients between countries may be a (NDM-1/OXA-181 and NDM-5/OXA-181; Balm et al., 2013),
trigger for the international spread of carbapenemase-producing and the USA (NDM-1/OXA-232; Doi et al., 2014).
K. pneumoniae (Berrazeg et al., 2014). Carbapenemase- Besides NDM-type carbapenemases, the IMP and VIM groups
producing Gram-negative bacteria including K. pneumoniae, have also been detected worldwide in K. pneumoniae, but
obtained from patients that had recently traveled outside Canada other carbapenemases, such as GIM-1, KHM-1, and SPM-1,
between 2010 and 2013, were found to be NDM-producing have been not found in K. pneumoniae (Table 1). Since IMP
K. pneumoniae, belonging to various sequence types (ST15, ST16, and VIP were first detected in Serratia marcescens in 1991
ST147, ST258, ST340, ST512, and ST972) with different plasmids and in Pseudomonas aeruginosa in 1996, respectively (Osano
(IncF, IncA/C, and IncL/M), and were imported from India to et al., 1994; Lauretti et al., 1999), IMP- and VIM-producing
Canada (Peirano et al., 2014). Therefore, more careful attention K. pneumoniae have spread in Europe and Asia, but were rarely
is required when treating patients with a recent history of foreign reported in other regions, such as America and Africa (Table 1).
hospitalization in countries where carbapenemase-producing Although in this review we have focused only on carbapenemase-
bacteria are prevalent. The international transportations of producing K. pneumoniae, the VIM group is one of the most
patients between countries recently resulted in the detection of commonly reported carbapenemases worldwide if we consider all
NDM-producing K. pneumoniae in Mexico (Barrios et al., 2014), bacteria species (the VIM groups have been mainly identified in
Guatemala (Pasteran et al., 2012), Brazil (Quiles et al., 2015), P. aeruginosa; Poirel et al., 2007).
the Netherlands (Bathoorn et al., 2015), Ireland (McDermott
et al., 2012), Poland (Baraniak et al., 2016), Czech Republic Molecular and Genetic Context
(Studentova et al., 2015), Croatia (Kocsis et al., 2016), Russia The blaNDM gene is frequently observed in the transposon
(Ageevets et al., 2014), Tunisia (Ben Nasr et al., 2013), Romania Tn125 (with two flanking ISAba125 elements) within NDM-
(Lixandru et al., 2015), Egypt (Bathoorn et al., 2013), Kenya producing species of the genus Acinetobacter (Partridge and
(Poirel et al., 2011f), Madagascar (Chereau et al., 2015), Iraq Iredell, 2012; Wailan et al., 2015). The blaNDM gene was
(Poirel et al., 2011e), Yemen (Gharout-Sait et al., 2014), Iran hypothesized to originate in the genus Acinetobacter (Toleman
(Shahcheraghi et al., 2013), Mauritius (Poirel et al., 2012b), Sri et al., 2012). In Enterobacteriaceae, the ISAba125 elements of
Lanka (Dortet et al., 2013), Thailand (Rimrang et al., 2012), the Tn125 structure carrying blaNDM are frequently truncated
Nepal (Tada et al., 2013), Vietnam (Hoang et al., 2013), Malaysia (Tn125) at various lengths and the Tn125 structure frequently
(Al-Marzooq et al., 2015), and New Zealand (Williamson et al., has different IS elements (Figure 2B; Partridge and Iredell,
2012). 2012; Wailan et al., 2015). The blaNDM genes in K. pneumoniae
The global dissemination of NDM-producing K. pneumoniae have been reported on numerous broad-host-range plasmid
also has a serious impact on neonatal mortality rates, particularly types, including IncA/C (Hudson et al., 2014), IncF (Hishinuma
in low-income countries where the burden of neonatal sepsis et al., 2013), IncR (Studentova et al., 2015), IncH (Villa
is high (Zaidi et al., 2005). Colonization of NDM-producing et al., 2012), IncN (Chen et al., 2014a), IncL/M (Peirano
K. pneumoniae isolates in pregnant women in the community et al., 2014), and IncX types (Wang et al., 2014d). The

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

predominant plasmid type responsible for spreading blaNDM−1 provide a reliable tool for the rapid detection of the ST258
is the IncA/C type plasmids (Poirel et al., 2011d; Pitout clone.
et al., 2015). Many IncA/C plasmids with blaNDM−1 also High-resolution genomic analysis of multidrug-resistant
carry various antibiotic resistance genes including 16S rRNA hospital outbreaks of K. pneumoniae through whole-genome
methylases (RmtA and RmtC), associated with aminoglycoside sequencing revealed the emergence of a capsule switching NDM-
resistance; CMY-type β-lactamases, associated with broad- 1 bearing K. pneumoniae ST15 strain, suggesting that further
spectrum cephalosporin resistance; and QnrA, associated with studies should concentrate on the diversity and spread of this
quinolone resistance (Pitout et al., 2015). Consequently, many specific clone (Chung The et al., 2015). ST15 harboring blaNDM−1
NDM-producing K. pneumoniae were susceptible only to has often been reported in many countries, including Spain
colistin, fosfomycin, and tigecycline (Nordmann and Poirel, (Ruiz-Garbajosa et al., 2013), Croatia (Kocsis et al., 2016),
2014). A novel NDM-1 variant (NDM-9) located on a novel Thailand (Netikul et al., 2014), Canada (Peirano et al., 2014),
IncH variant plasmid was recently identified in a clinical China (Hu et al., 2013), France (Arpin et al., 2012), and Morocco
K. pneumoniae isolate in China (Wang et al., 2014c). Because (Poirel et al., 2011b). In Bulgaria, this clone was responsible for
the blaNDM genes are located in numerous broad-host-range the clonal dissemination of KPC-2-producing K. pneumoniae
plasmids, the spread of NDM-1 is facilitated by horizontal (Markovska et al., 2015). Another whole-genome sequencing
gene transfer between bacteria. In China, CRE 21 strains analysis of CR K. pneumoniae strains, which were isolated
harboring the blaNDM−1 gene were found to consist of multiple from 26 individuals involved in infections in a Nepali neonatal
Enterobacteriaceae species including nine Enterobacter cloacae, unit, showed that three temporally separated cases were caused
three E. coli, three Citrobacter freundii, two K. pneumoniae, two by a single NDM-producing K. pneumoniae strain with four
K. oxytoca, and two Morganella morganii strains (Wang et al., conserved plasmids including a plasmid carrying blaNDM−1
2015). (Stoesser et al., 2014). The plasmids contained a large number
Unlike KPC, the blaNDM genes were detected in various of antimicrobial resistance and plasmid maintenance genes,
K. pneumoniae clones. ST11, a major high-risk sequence type of which may explain their persistence (Stoesser et al., 2014). These
KPC-producing K. pneumoniae in Asia, has also been associated reports suggest that whole-genome sequencing analysis play
with blaNDM−1 in K. pneumoniae identified in many countries, an important role in the elucidation of the factors that allow
such as the USA (Rasheed et al., 2013), Greece (Voulgari et al., emergence and persistence of resistance.
2014), Australia (Shoma et al., 2014), Switzerland (Seiffert et al.,
2014), the Czech Republic (Studentova et al., 2015), Spain Treatment Options
(Oteo et al., 2013b), and Thailand (Netikul et al., 2014). The New-Delhi metallo-β-lactamase-producing K. pneumoniae are
recent outbreak of NDM-producing K. pneumoniae ST11 in usually resistant to most β-lactam antibiotics but remain
Poland was caused by a clone similar to an isolate identified in susceptible to aztreonam (Nordmann et al., 2012a). As with
the Czech Republic in 2013 (Baraniak et al., 2016), indicating the case of KPC-producing K. pneumoniae, the effect of
the local spread of this clone. ST11 has also been associated combination therapy was tested in the treatment of NDM-
with OXA-48-like enzymes from isolates found in Argentina, producing K. pneumoniae infections. When double- and triple-
Turkey, and Spain (Lascols et al., 2013; Oteo et al., 2013b). antibiotic combinations of aztreonam, ciprofloxacin, colistin,
ST14, ST147, and ST340 have sometimes been associated with daptomycin, fosfomycin, meropenem, rifampin, telavancin,
blaNDM in K. pneumoniae in many countries (Peirano et al., tigecycline, and vancomycin were used in patients infected
2011, 2014; Poirel et al., 2011d; Giske et al., 2012; Osterblad with two NDM-producing K. pneumoniae strains susceptible
et al., 2012; Lascols et al., 2013; Gharout-Sait et al., 2014; to colistin, the combination of rifampin-meropenem-colistin
Lee et al., 2014; Shoma et al., 2014; Wang et al., 2014d; was the most effective regimen against these strains (Tangden
Izdebski et al., 2015; Sonnevend et al., 2015b). Over 50% of et al., 2014). The in vitro synergetic effect of the combination
NDM-producing K. pneumoniae isolates from India belonged therapy of colistin and fosfomycin against NDM-producing
to either ST11 or ST147 (Lascols et al., 2013). The analysis K. pneumoniae has also been reported (Bercot et al., 2011).
of clinical isolates of NDM-1-producing K. pneumoniae from The combination of polymyxin B and chloramphenicol used
India, the UK, and Sweden, showed that the most frequently against NDM-producing K. pneumoniae substantially enhanced
detected sequence types were ST14, ST11, ST149, ST231, bacterial killing and suppressed the emergence of polymyxin
and ST147 (Giske et al., 2012). Although ST258 is a high- resistance (Abdul Rahim et al., 2015). Combination therapy
risk KPC-producing K. pneumoniae clone, ST258 harboring including aztreonam and avibactam (a novel inhibitor of serine
blaNDM has never been reported, to the best of our knowledge. β-lactamases under development) was effective in the treatment
This phenomenon may result from the integrative conjugative of metallo-β-lactamase-producing bacterial infections (Wang
element ICEKp258.2, present only in ST258 and genetically et al., 2014e).
related sequence types (Adler et al., 2014). This genetic locus In the case of NDM-1-producing Enterobacteriaceae
contains a type IV pilus gene cluster and a type III restriction- infections, carbapenems have been suggested to still represent
modification system. Because these genes are associated with a viable treatment option (Wiskirchen et al., 2014b).
the restriction of plasmids and specific mobile elements, most Despite unfavorable in vitro MICs of NDM-producing
ST258 may predominantly harbor plasmids with the blaKPC K. pneumoniae, recent in vivo studies have demonstrated
gene. Therefore, PCR for this unique region (ICEKp258.2) can the efficacy of carbapenems against NDM-1-producing isolates

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

in immunocompetent-mouse and neutropenic-mouse thigh and 100%, respectively, directly from spiked blood cultures
infection models (Wiskirchen et al., 2013, 2014b; MacVane (Dortet et al., 2014a). Comparative evaluation of the Carba
et al., 2014). Although high-dose, prolonged infusions of NP test with other detection methods was tested in many
ertapenem or doripenem induced reduction in bacterial density, reports (Vasoo et al., 2013; Huang et al., 2014; Yusuf et al.,
bacterial density was also reduced in standard infusions of 2014; Gallagher et al., 2015; Lifshitz et al., 2016). When the
ertapenem at 1 g every 24 h or of doripenem at 500 mg every Carba NP test and the modified Hodge test were compared,
8 h (Wiskirchen et al., 2013, 2014b). Notably, these efficacies the Carba NP test was more specific (100% versus 80%) and
were observed only against NDM-1-producing K. pneumoniae faster (Vasoo et al., 2013; Huang et al., 2014; Yusuf et al.,
(Wiskirchen et al., 2013, 2014b). In addition to carbapenems, 2014). When it was compared the performance of the Carba
this discordance between in vitro and in vivo activities against NP test and the commercially available imipenem hydrolysis-
NDM-1-producing K. pneumoniae was also observed in based rapid test (the Rosco Rapid CARB screen kit) for detecting
human simulated regimens of ceftazidime at 2 g every 8 h or CPE and P. aeruginosa, the Carba NP test showed superior
ceftazidime/avibactam at 2,000/500 mg every 8 h (MacVane specificity and sensitivity (Huang et al., 2014; Yusuf et al.,
et al., 2014). Despite experiments in immunocompetent-mouse 2014; Gallagher et al., 2015). A novel simplified protocol of
and neutropenic-mouse thigh infection models, these results the Carba NP test designed for carbapenemase detection direct
show that standard infusions of ertapenem and doripenem from bacterial cultures (instead of bacterial extracts) showed
could reduce bacterial density. Therefore, further experiments enhanced detection of carbapenemase producers (Pasteran et al.,
in human are required to determine whether carbapenems are 2015).
sometimes a viable treatment option for NDM-1-producing However, several reports showed that false-negative results
K. pneumoniae infections. in the Carba NP test were associated with mucoid strains or
The copy number of blaNDM−1 was assessed using Southern linked to enzymes with low carbapenemase activity, particularly
blotting and quantitative PCR under different conditions. The OXA-48-like (Tijet et al., 2013; Osterblad et al., 2014). To
blaNDM−1 sequence was maintained under antibiotic selection; overcome these problems, several derivatives of the Carba
however, removal of the antibiotic selection led to the emergence NP test were developed, such as the Rapidec Carba NP test
of susceptible bacterial populations with a reduced copy number (bioMérieux; Poirel and Nordmann, 2015), the CarbAcineto
or even the complete loss of the blaNDM−1 gene (Huang NP test for rapid detection of carbapenemase-producing
et al., 2013). The dynamic nature of the copy number of Acinetobacter spp. (Dortet et al., 2014b), the Rapid CARB
blaNDM−1 provides a strong argument for the prudent use of Screen (Rosco Diagnostica; Dortet et al., 2015), the Blue-
clinically important antibiotics to reduce the development and Carba test using bromothymol blue as a pH indicator
dissemination of antibiotic resistance among pathogenic bacteria solution (Garcia-Fernandez et al., 2016), a modified Carba
(Huang et al., 2013). NP test (Bakour et al., 2015a), and the BYG Carba test
based on an electro-active polymer biosensing technology
Detection Methods (Bogaerts et al., 2016). Many studies evaluated the performance
Because NDM-producing K. pneumoniae infections are also of the Rapidec Carba NP test (bioMérieux), which was
associated with significant in-hospital mortality (de Jager et al., introduced into the market for the detection of carbapenemase
2015), the rapid and accurate detection of NDM-producing production (Dortet et al., 2015; Hombach et al., 2015; Kabir
K. pneumoniae is becoming a major issue in limiting the spread et al., 2016; Lifshitz et al., 2016). These report showed
of CR bacteria. Several methods recently developed to detect that this method was user-friendly and had a high overall
NDM-producing K. pneumoniae include Xpert Carba-R based
R
performance, making it an attractive option for clinical
on real time PCR (Anandan et al., 2015), the Carba NP test laboratories (Kabir et al., 2016). Recently, performance evaluation
based on the color change of a pH indicator (Nordmann et al., of two biochemical rapid tests commercialized (the Rapidec
2012b), and its derivatives (Pires et al., 2013; Dortet et al., Carba NP test and the Rapid CARB Screen) was reported
2014b), and a method based on MALDI-TOF (Hrabak et al., and compared with the home-made Carba NP test (Dortet
2013a). Xpert Carba-R based on real time PCR effectively
R
et al., 2015). The Rapidec CARBA NP test possesses the
identified various carbapenemases of KPC, NDM, IMP, and best performance for rapid and efficient detection of CPE
VIM, with 100% sensitivity and 77% specificity (Anandan et al., (Dortet et al., 2015). The BYG Carba test based on a new
2015). However, this method failed to detect OXA-48-like and original electrochemical method detects the variations of
carbapenemases, in contrast to multiplex PCR (Anandan et al., conductivity of a polyaniline (an electro-sensing polymer)-
2015). coated electrode which is highly sensitive to the modifications
The Carba NP test using chromogenic medium is based on of pH and of redox activity occurring during the imipenem
the color change of a pH indicator (Nordmann et al., 2012b). enzymatic hydrolysis reaction (Bogaerts et al., 2016). In
The enzymatic hydrolysis of the β-lactam ring of a carbapenem comparison with PCR results, the BYG Carba test displayed
(usually imipenem) causes the acidification of an indicator sensitivity of 95% and specificity of 100% versus 89% and
solution (phenol red for the Carba NP test) that changes its 100%, respectively, for the Carba NP test (Bogaerts et al.,
color due to pH modification (Garcia-Fernandez et al., 2016). 2016). The development of these detection methods based on
This method could rapidly detect KPC, IMP, VIM, NDM, and inexpensive and affordable techniques can limit the spread of CR
OXA-48-like producers with sensitivity and specificity of 97.9 bacteria.

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

Class D Carbapenemases Similarly, a large outbreak of OXA-48 carbapenemase-producing

Epidemiology K. pneumoniae in a French university hospital was recently
Class D β-lactamases were referred to as oxacillinases (OXAs) attributed to the late implementation of successive cohort units
because they commonly hydrolyze isoxazolylpenicillins and a high level of staff movement between the infectious
(oxacillin, cloxacillin, and dicloxacillin) much faster than diseases and internal medicine ward (Semin-Pelletier et al.,
benzylpenicillin (Jeon et al., 2015). Of over 400 Class D 2015). These results suggest that practical guidelines are required
β-lactamases, only some variants actually possess carbapenemase to help hospitals confronting uncontrolled outbreaks. Because
activity. Based on their amino acid sequence, class D the gut of colonized patients is the main source of CPE,
carbapenemases were recently reclassified into 12 subgroups: accurate and stringent hygiene of endoscopic instruments is
OXA-23, OXA-24/40, OXA-48, OXA-51, OXA-58, OXA-134a, also important. A recent report partially attributed an outbreak
OXA-143, OXA-211, OXA-213, OXA-214, OXA-229, and OXA- of OXA-48-producing K. pneumoniae in a German University
235 (Jeon et al., 2015). Among them, only several subgroups hospital to complex instruments such as the duodenoscope (Kola
such as OXA-23, OXA-48, OXA-51, and OXA-58 are reported et al., 2015). Strict hygiene regulations for various nosocomial
in K. pneumoniae (Evans and Amyes, 2014). OXA-48 is the most environments, including endoscopic instruments as well as
efficient class D carbapenemase for imipenem and is one of the gowns and gloves, are required.
most prevalent class D carbapenemases (Jeon et al., 2015). The OXA-48-producing K. pneumoniae was recently also detected
OXA-48 was first identified in K. pneumoniae in Turkey in 2003 in Canada (Ellis et al., 2013), the USA (Mathers et al., 2013),
(Poirel et al., 2004), and thus far, 10 variants of the blaOXA−48 Ireland (Wrenn et al., 2014), Poland (Izdebski et al., 2015),
gene have been identified (Jeon et al., 2015). Turkey may be one Hungary (Janvari et al., 2014), Greece (Voulgari et al., 2013),
of the main reservoirs of OXA-48-producing K. pneumoniae Romania (Lixandru et al., 2015), Bulgaria (Markovska et al.,
(Nordmann and Poirel, 2014). Since 2003, the endemic spread 2015), Finland (Osterblad et al., 2012), Russia (Fursova et al.,
of these bacteria has been reported in countries such as Turkey, 2015), Algeria (Cuzon et al., 2015), United Arab Emirates
Morocco, Libya, Egypt, Tunisia, and India (Nordmann and (Ahn et al., 2015), Iran (Azimi et al., 2014), South Africa
Poirel, 2014; Figure 4). The sporadic spread has been reported (Brink et al., 2013), Senegal (Moquet et al., 2011), Taiwan
in France (Liapis et al., 2014; Semin-Pelletier et al., 2015), Spain (Ma et al., 2015), Singapore (Ling et al., 2015), South Korea
(Oteo et al., 2013b; Pena et al., 2014), Italy (Giani et al., 2013, (Jeong et al., 2015), and Australia (Espedido et al., 2013;
2014), Belgium (Cuzon et al., 2008; Huang et al., 2013), the Figure 4). In North America, the frequency of OXA-48-
Netherlands (Kalpoe et al., 2011; Dautzenberg et al., 2014), the like enzymes among carbapenemase-producing K. pneumoniae
UK (Dimou et al., 2012; Thomas et al., 2013), Germany (Pfeifer isolates was very low (11%; Lascols et al., 2013). A recent report
et al., 2012; Kola et al., 2015), Switzerland (Potron et al., 2012; in Romania showed that among 65 carbapenemase-producing
Seiffert et al., 2014), Argentina (Poirel et al., 2011c; Lascols et al., K. pneumoniae, the most frequently identified gene was the
2013), Lebanon (Beyrouthy et al., 2014), Israel (Adler et al., blaOXA−48 gene (78%), 12% were positive for blaNDM−1 gene, 6%
2013, 2015), Kuwait (Poirel et al., 2012a; Zowawi et al., 2014), had the blaKPC−2 gene (Lixandru et al., 2015). The recent spread
Saudi Arabia (Shibl et al., 2013; Liu et al., 2015), and Japan of OXA-48 and OXA-244 carbapenemase genes in Russia was
(Nagano et al., 2013; Hashimoto et al., 2014). The prevalence reported among Proteus mirabilis, E. aerogenes, and E. cloacae
of OXA-48 carbapenemases among carbapenemase-producing as well as K. pneumoniae (Fursova et al., 2015). In South
K. pneumoniae in Spain and France was particularly high (74 Africa, the emergence of a colistin-resistant OXA-181-producing
and 78%, respectively; Robert et al., 2014; Palacios-Baena et al., K. pneumoniae isolate was also reported (Brink et al., 2013),
2016). In Africa, OXA-48-producing K. pneumoniae have been and in the Netherlands, an OXA-48-producing K. pneumoniae
mainly reported in the northern countries, such as Morocco, was reported to infect two patients (Kalpoe et al., 2011), and a
Libya, Egypt, Tunisia, and Algeria (Figure 4). In the Arabian hospital-wide outbreak was successfully controlled (Dautzenberg
Peninsula, the prevalence of OXA-48-like carbapenemases et al., 2014).
among carbapenemase-producing K. pneumoniae was also high OXA-181, a derivative of OXA48 with the substitution of
(32.5–56%; Zowawi et al., 2014; Sonnevend et al., 2015b). Among a single amino acid, was first identified in India in Potron
CRE isolates in Lebanon, 88% produced OXA-48 carbapenemase et al. (2011), and then has been spread to many different
(Beyrouthy et al., 2014). In Saudi Arabia, 78% of carbapenemase- countries, such as the UK (Dimou et al., 2012), Romania
producing K. pneumoniae isolates harbored blaOXA−48 , and (Székely et al., 2013), Canada (Peirano et al., 2014), Oman
three strains of 47 blaOXA−48 -positive K. pneumoniae isolates (Potron et al., 2011), Singapore (Balm et al., 2013), Sri Lanka
were resistant to colistin, suggesting that colistin resistance is (Hall et al., 2014), South Korea (Cho et al., 2015), Australia
emerging in Saudi Arabia (Shibl et al., 2013). (Sidjabat et al., 2015), Japan (Kayama et al., 2015), and New
In France, examination of the epidemiologic features of Zealand (Williamson et al., 2011). However, in many cases,
an outbreak of OXA-48-producing K. pneumoniae in an the infections have been associated with India (Potron et al.,
ICU revealed that the outbreak was caused by environmental 2011; Williamson et al., 2011; Dimou et al., 2012; Hall et al.,
persistence of OXA-48-producing K. pneumoniae over 20 months 2014; Peirano et al., 2014). Other OXA-48-derivatives, such as
(Pantel et al., 2016). This report emphasizes the importance of OXA-204 (Potron et al., 2013), OXA-232 (Potron et al., 2013),
early environmental screening to interrupt the transmission of and OXA-163 (Poirel et al., 2011b), were recently identified in
carbapenemase-producing K. pneumoniae (Pantel et al., 2016). Tunisia, France, and Argentina, respectively, and OXA-244 and

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

OXA-245 were first reported in Spain (Oteo et al., 2013a). All composite transposon was very low (<1.0 × 10−7 ; Aubert et al.,
of these countries are regions with a high prevalence of OXA- 2006).
48-producing K. pneumoniae (Figure 4). In addition, OXA-232- OXA-48 has most often been found in K. pneumoniae, but
producing K. pneumoniae has been reported in various countries, OXA-48 was also found in various Enterobacteriaceae, because
such as the USA (Doi et al., 2014), Singapore (Teo et al., 2013), of the high conjugation rate of the pOXA-48a (Potron et al.,
India (Al-Marzooq et al., 2015), and South Korea (Jeong et al., 2014). Molecular and epidemiological analyses in a German
2015). These results indicate that besides OXA-48, its many hospital showed the horizontal gene transfer of the OXA-48-
derivatives also spread worldwide. OXA-163, which differs from containing plasmid from K. pneumoniae to E. coli (Gottig et al.,
OXA-48 by a four amino acid deletion and a single amino acid 2015). Besides E. coli, OXA-48 has also been identified in
substitution, has lower carbapenemase activity than OXA-48, K. oxytoca, Enterobacter spp., Providencia rettgeri, C. freundii,
but this enzyme exhibits extended activity against expanded- and S. marcescens (Poirel et al., 2012c; Berger et al., 2013). The
spectrum cephalosporins, and its activity is partially inhibited blaOXA−48 gene was identified in all Enterobacteriaceae isolates
by clavulanic acid, a β-lactamase inhibitor (Poirel et al., 2011c). from the index case in Spain, indicating the capacity of OXA-
OXA-247, a derivative of OXA-163 with a single amino acid 48 carbapenemase to spread among Enterobacteriaceae by the
substitution, was recently reported in Argentina (Gomez et al., horizontal gene transfer (Arana et al., 2015).
2013), where OXA-163-producing K. pneumoniae were often Similar to NDM, the blaOXA−48−like genes were detected in
reported (Lascols et al., 2013). various K. pneumoniae clones. ST11 has often been associated
The coexistence of OXA-48-like and other carbapenemases in with blaOXA−48−like in K. pneumoniae isolated in many countries,
K. pneumoniae was also frequently reported worldwide, such as in such as Spain (Oteo et al., 2013a,b, 2015; Ruiz-Garbajosa et al.,
Turkey (OXA-48/NDM-1; Kilic and Baysallar, 2015), Switzerland 2013; Branas et al., 2015), Taiwan (Ma et al., 2015), Libya
(OXA-48/NDM-1; Seiffert et al., 2014), United Arab Emirates (Lafeuille et al., 2013), Turkey (Lascols et al., 2013), Argentina
(OXA-48-like/NDM-1; Dash et al., 2014), Australia (OXA- (Lascols et al., 2013), and Greece (Voulgari et al., 2013). In Spain,
48/ NDM-1; Sidjabat et al., 2015), Morocco (OXA-48/NDM-1; a large outbreak was initiated in 2013 by a OXA-48-producing
Barguigua et al., 2013), India (OXA-181/VIM-5; Castanheira K. pneumoniae ST11 clone, and this strain was detected in 44
et al., 2011), Singapore (OXA-181/NDM-1 and OXA-181/NDM- patients (Branas et al., 2015). The ST11 isolates carried various
5; Balm et al., 2013), the USA (OXA-232/NDM-1; Doi et al., carbapenemases, including NDM-1, VIM-1, OXA-48, KPC-2,
2014), and India (OXA-232/NDM-1; Al-Marzooq et al., 2015). and OXA-245 (Lascols et al., 2013; Oteo et al., 2013b, 2015).
Other class D carbapenemases, such as OXA-23, OXA-24/40, In addition, ST14, ST15, ST101, ST147, and ST405 harboring
OXA-51, OXA-58, OXA-134a, OXA-143, OXA-211, OXA-213, blaOXA−48−like have often been reported in many countries, such
OXA-214, OXA-229, and OXA-235, were mainly identified as the USA (Lascols et al., 2013), Spain (Oteo et al., 2013b, 2015;
in Acinetobacter species such as A. baumannii but not in Ruiz-Garbajosa et al., 2013; Arana et al., 2015; Cubero et al.,
K. pneumoniae (Table 1; Evans and Amyes, 2014). 2015), the Czech Republic (Hrabak et al., 2015), Libya (Lafeuille
et al., 2013), India (Lascols et al., 2013), Germany (Gottig et al.,
Molecular and Genetic Context 2015), Finland (Osterblad et al., 2012), France (Liapis et al.,
Unlike KPCs and NDMs, one highly transferable IncL group 2014), and Japan (Hashimoto et al., 2014). Recent results from
plasmid (pOXA-48a) was mainly responsible for the spread of 83 hospitals in Spain showed that OXA-48 (71.5%) and VIM-
the blaOXA−48 gene in K. pneumoniae (Figure 2C; Pitout et al., 1 (25.3%) were the most frequently detected carbapenemases,
2015). The molecular epidemiology of OXA-48 in European and and the most prevalent sequence types were ST11 and ST405
North African countries showed that in 92.5% of the isolates, for K. pneumoniae (Oteo et al., 2015). However, the molecular
the blaOXA−48 gene was located on this self-conjugative IncL/M epidemiology of OXA-48-producing enterobacterial isolates
type plasmid (Potron et al., 2013). The blaOXA−48 gene was collected from European and north-African countries between
found only in the IncL group of IncL/M type plasmids (Carattoli 2001 and 2011 indicated that ST101 was the most commonly
et al., 2015). In contrast to the IncA/C plasmids of NDM-1, observed sequence type in K. pneumoniae isolates, accounting for
the pOXA-48a plasmid contains blaOXA−48 , a unique antibiotic 17 out of 67 isolates (25.4%), followed by ST395 and ST15 (each
resistance gene (Pitout et al., 2015). The conjugation rate of the seven isolates, 10.5%; Potron et al., 2013). Two outbreaks of OXA-
pOXA-48a plasmid was very high (1 × 10−1 ); therefore, this self- 48-producing K. pneumoniae ST101 clones were reported in
conjugative plasmid can conjugate at a very high frequency to Spain (Pitart et al., 2011; Cubero et al., 2015). OXA-48-producing
Enterobacteriaceae (Potron et al., 2014). Inactivation of the tir K. pneumoniae appear to vary geographically. As with NDM, to
gene, which is known to encode a transfer inhibition protein, the best of our knowledge, ST258 harboring blaOXA−48−like has
was recently reported to be responsible for a 50- to 100-fold never been reported.
increase in the efficiency of transfer of the pOXA-48a plasmid
(Potron et al., 2014), explaining the spread of the pOXA-48a Treatment Options
plasmid with blaOXA−48 . Recently, the blaOXA−48−like gene has OXA-48-like-producing K. pneumoniae are usually resistant
also been reported on other plasmids and genetic elements, such to most β-lactam antibiotics, but OXA-48-producing
as IncA/C types (Ma et al., 2015), IncH types (Wang et al., K. pneumoniae without ESBLs remain susceptible to the
2014c), and Tn1999 (Poirel et al., 2012a). In comparison to the expanded-spectrum cephalosporins (Nordmann et al., 2012a;
pOXA-48a plasmid, the transmission frequency of the Tn1999 Munoz-Price et al., 2013). In addition, OXA-48-like-producing

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

K. pneumoniae sometimes remains susceptible to several 96.3% sensitivity and 97.7% specificity (Tsakris et al., 2015).
aminoglycosides such as gentamicin (Tzouvelekis et al., 2014). ChromID OXA-48, based on chromogenic media, have been
Unlike for NDMs (Wiskirchen et al., 2013, 2014b; MacVane commercialized for the direct isolation of CPE from clinical
et al., 2014), carbapenems may not be a reliable treatment samples (Girlich et al., 2013), with an estimated 91% sensitivity
option for OXA-48 producer infection (Wiskirchen et al., 2014a). and 100% specificity (Girlich et al., 2013).
Combination therapy with sulbactam, meropenem, and colistin
was more effective in isolates producing NDM carbapenemase Emerging Class C Carbapenemases
than those producing OXA-48-like carbapenemases, suggesting Class C β-lactamases confer resistance to penicillins,
that the identification of the carbapenemase type helps determine cephalosporins, and cephamycins (cefoxitin and cefotetan) and
the combination most likely to clear the infection (Laishram are not significantly inhibited by clinically applied β-lactamase
et al., 2015). The combination of fosfomycin with imipenem, inhibitors such as clavulanic acid (Jeon et al., 2015). Although
meropenem, and tigecycline was also synergistic against OXA 48- four class C carbapenemases (ACT-1, CMY-2, CMY-10, and
positive K. pneumoniae strains in vitro with the ratios of 42, 33, ADC-68) have been reported, ACT-1 and CMY-2 exhibit
and 33%, respectively (Evren et al., 2013). Similarly, the in vitro reduced susceptibility to carbapenems, particularly ertapenem,
assays indicate that imipenem-containing combinations were only when combined with permeability defects, due to their
effective against serine-β-lactamase producers (KPC, OXA-48), low catalytic efficiencies (Kcat /Km ) for imipenem (0.007 and
while no synergy was observed for all NDM-1 producers (Poirel 0.04 M−1 ·S−1 , respectively; Mammeri et al., 2010). CMY-10
et al., 2016). However, because carbapenems were effective with the catalytic efficiency of 0.14 M−1 ·S−1 for imipenem
against NDM-1-producing isolates in vivo (Wiskirchen et al., was the first reported carbapenemase among plasmidic class
2013, 2014a; MacVane et al., 2014), the effect of carbapenem C β-lactamases (Kim et al., 2006), and this enzyme was also a
combination therapy on carbapenemase-producing isolates class C extended-spectrum β-lactamase with extended substrate
should be determined in vivo, particularly in the case of specificity for extended-spectrum cephalosporins (Lee et al.,
NDM-1-producing bacteria. 2009, 2012a; Jeon et al., 2015). Among the chromosomal class C
Despite the effectiveness of combination therapies, the β-lactamases, ADC-68 identified in A. baumannii was the first
prognosis for bloodstream infections caused by OXA-48- reported enzyme possessing both class C extended-spectrum
producing Enterobacteriaceae remains poor, and the 30- β-lactamase and carbapenemase activities (Jeon et al., 2014,
days mortality reached 50% (Navarro-San Francisco et al., 2015), and its catalytic efficiency for imipenem is 0.17 M−1
2013). A similar result was reported in OXA-48-producing S−1 . CMY-10-producing K. pneumoniae was identified only in
K. pneumoniae infections in a tertiary hospital in Spain (Pano- South Korea (Lee et al., 2005a, 2006a) and ADC-68-producing
Pardo et al., 2013). Although OXA-48-producing K. pneumoniae K. pneumoniae was never reported.
were susceptible to several antibiotics, including amikacin (97.2% Many reports showed that carbapenem resistance can be
susceptible), colistin (90.1%), tigecycline (73%), and fosfomycin triggered by the loss of two major porins, OmpK35 and
(66.2%), in-hospital mortality among patients with OXA-48- OmpK36, in combination with ESBLs or Ambler class C AmpC
producing K. pneumoniae infections was 43.5% (Pano-Pardo cephalosporinases, and the production of carbapenemase (Wang
et al., 2013). Therefore, to prevent delay in diagnosis and et al., 2009; Shin et al., 2012; Tsai et al., 2013). A recently
initiation of optimal antimicrobial therapy, rapid identification genetically engineered mutant of K. pneumoniae showed that
of OXA-48-producing isolates is required. several carbapenems (imipenem, meropenem, and doripenem)
remain effective against these carbapenemase-independent CR
Detection Methods strains (Tsai et al., 2013). Therefore, laboratory testing for
As with KPCs and NDMs, various detection methods were susceptibility to imipenem, meropenem, and doripenem can
developed to identify OXA-48-like carbapenemases (Tsakris improve the accuracy of identification of these isolates (Tsai et al.,
et al., 2010; Giske et al., 2011; Girlich et al., 2013; Naas 2013).
et al., 2013; Lee et al., 2015c). Accurate differentiation of the
various carbapenemase types, such as KPC-type, NDM-type, and
OXA-48-type enzymes, is crucial for controlling the spread of CONCLUSION
carbapenem resistance among Enterobacteriaceae (Nordmann
et al., 2012a). Many phenotypic detection methods to allow We analyzed the epidemiology of K. pneumoniae producing
differentiation between class A and class B carbapenemases true carbapenemases (Ambler molecular class A, B, D, and
were developed using boronic acid derivatives and EDTA or several carbapenemases of class C) responsible for non-
dipicolinic acid (Tsakris et al., 2010; Giske et al., 2011). Recently, susceptibility to carbapenems without additional permeability
a specific phenotypic method to differentiate a single OXA-48 defects. Many types of CR K. pneumoniae have been identified
producer from those producing other carbapenemase types (e.g., worldwide (Figures 1, 3, and 4). During the past 3 years,
KPC-types, NDM-types) was also developed (Tsakris et al., 2015). many countries have reported the arrival of carbapenemases
This method was based on an imipenem disk and two blank disks previously unreported in those countries. For example, although
adjacent to the imipenem disk, loaded with the tested strain and K. pneumoniae producing KPC- and NDM-type carbapenemases
impregnated with EDTA and EDTA plus phenyl boronic acid, have been extensively reported in the USA, a K. pneumoniae
respectively (Tsakris et al., 2015). This novel method exhibited producing OXA-48-type carbapenemases was recently detected

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Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

in the USA (Mathers et al., 2013). Since a first report associated with quinolone resistance. These features may
of NDM-1 in 2008, this carbapenemase has rapidly spread be linked to the current global success of NDM-producing
worldwide, and NDM-producing K. pneumoniae still continues K. pneumoniae.
to be found in new countries, implying that NDM-producing The current spread of OXA-48-producing bacteria is
K. pneumoniae is still spreading quickly. Despite the global attributed to the pOX-48a plasmid, which belongs to the IncL
dissemination of KPC, NDM, and OXA-48, the prevalence of group of IncL/M type plasmids. Although the pOXA-48a
carbapenemases varies geographically. The frequency of KPC- plasmid contains blaOXA−48 , a unique antibiotic resistance gene,
and NDM-producing strains was significantly higher in the the conjugation rate of the pOXA-48a plasmid was very high,
USA, Canada, Greece, Taiwan, Colombia, and China, whereas which may be responsible for its global spread in K. pneumoniae.
OXA-48-producing strains were rarely found in those countries The high pOXA-48a conjugation rate was recently attributed to
(Figures 1, 3, and 4). In Argentina, despite the extensive spread mutations in the tir gene known to encode a transfer inhibition
of KPC- and OXA-48-producing strains, no NDM-producing protein, which may lead to a 50- to 100-fold increase in the
K. pneumoniae has been reported. Although NDM- and OXA- efficiency of transfer of the pOXA-48a plasmid. Furthermore,
48-producing K. pneumoniae significantly spread in Turkey, the pOXA-48a plasmid is self-conjugative. Therefore, these
KPC-producing strains have rarely been reported there. In Brazil, specific features of the pOXA-48a plasmid may explain the global
KPC-producing K. pneumoniae has been mainly reported. In the dissemination of OXA-48-type carbapenemases.
Arabian Peninsula, OXA-48 and NDM producers are common, ST11, ST14, ST101, ST147, and ST258 are major
whereas KPC-type, VIM-type, or IMP-type producers are rare. carbapenemase-producing K. pneumoniae clones. ST258
In India, Spain, France, Italy, and the UK, all three types of was mainly found in KPC-producing K. pneumoniae, whereas
carbapenemases have been frequently reported (Figures 1, 3, other clones were found in various carbapenemase-producing
and 4). K. pneumoniae regardless of carbapenemase types. Well-
ST258 is an important strain responsible for the extensive designed epidemiological and molecular studies will be required
global spread of KPC-producing K. pneumoniae. Although the to understand the dynamics of transmission, risk factors, and
precise reason for the predominance of the ST258 strain in reservoirs of these K. pneumoniae clones. This will provide
KPC-producing K. pneumoniae is not fully understood, recent information essential for preventing infections and the spread of
molecular studies unveiled the genetic characteristics of this these risky sequence types.
strain. The ST258 strains consists of two distinct genetic clades Most currently available antibiotics may be not sufficiently
(cps-1 and cps-2) derived from genetic differentiation in genes effective for the treatment of all types of carbapenemase
involved in capsule polysaccharide biosynthesis. In addition, producers in monotherapy. Combination therapy of
the integrative conjugative element ICEKp258.2, present only in carbapenems with polymyxin B, colistin, rifampin, fosfomycin, or
ST258 and genetically related sequence types, may be linked to tigecycline has been reported to effectively treat carbapenemase-
the global success of the ST258 clone. ICEKp258.2 contains a producing K. pneumoniae. Despite these data supporting
type IV pilus gene cluster and a type III restriction-modification the use of combination therapy for treatment of severe
system. The type IV pilus gene cluster ICEKp258.2, particularly carbapenemase-producing K. pneumoniae infections, current
pilV, may contribute to the global success of ST258 clone. clinical evidence for treatment guidelines are limited and more
The type III restriction-modification system associated with accurate randomized controlled in vivo studies are required.
the restriction of plasmids and specific mobile elements may Moreover, considerable caution is required when applying these
explain the differences observed between ST258 predominantly therapies. For example, temocillin can actively treat against
harboring KPC and ST11, another high-risk clone that lacks some KPC-producing K. pneumoniae, particularly lower urinary
ICEKp258.2, harboring various carbapenemases, such as NDM- tract infections, and NDM-producing K. pneumoniae is often
1, OXA-48, KPC-2, VIM-1, and OXA-245. IncF with FIIK susceptible to aztreonam. OXA-48-producing K. pneumoniae
replicons, a plasmid most commonly identified in ST258 with remain susceptible to the expanded-spectrum cephalosporins
blaKPC , often contains several genes associated with resistance in approximately 20% of cases without ESBLs. In the case of
to other antibiotics, such as aminoglycosides, tetracyclines, NDM-1-producing K. pneumoniae, carbapenems were recently
quinolones, trimethoprim, and sulfonamides. The features of this reported to represent a viable treatment option for infections
plasmid may also play an important role in the current global caused by these bacteria, despite unfavorable in vitro MICs.
success of ST258. Because these results imply that carbapenems can sometimes
The rapid global spread of NDM-type carbapenemases may be a viable treatment option for infection with carbapenemase
be partly attributed to the dissemination of various epidemic producers, more extensive studies on the effect of carbapenem
broad-host-range plasmids bearing the blaNDM genes. NDM- monotherapy will be required in the case of NDM-producing
type carbapenemases were found in various plasmids such K. pneumoniae infections.
as IncA/C, IncF, IncR, IncH, IncN, IncL/M, and IncX types. The accurate and rapid detection of the genotype of
The IncA/C type plasmids, most common plasmids associated carbapenemases can minimize the delay to appropriate
with spread of the blaNDM genes, often have various antibiotic prescription of antibiotics. Many detection kits based on
resistance genes, such as 16S rRNA methylases associated with various phenotypic or molecular techniques, such as multiplex
aminoglycoside resistance, CMY-type β-lactamases associated PCR assay, real-time PCR assay, DNA microarray, Raman
with broad-spectrum cephalosporin resistance, and QnrA spectroscopic analysis, single-colony whole-genome sequencing,

Frontiers in Microbiology | [Link] 17 June 2016 | Volume 7 | Article 895

Lee et al. Carbapenem-Resistant Klebsiella pneumoniae

MALDI-TOF MS, loop-mediated isothermal amplification AUTHOR CONTRIBUTIONS

method, chromogenic medium, and new phenotypic test
methods, have been developed. Through the imprudent use C-RL, JL, and SL contributed to the conception and the design of
of colistin which is a key component used for the treatment the review and C-RL, JL, KP, YK, BJ, and SL researched and wrote
of severe carbapenemase-producing K. pneumoniae infections, the review.
the rapid spread of colistin resistance was recently reported in
K. pneumoniae producing carbapenemases, particularly KPC-
type carbapenemases. This situation strongly demonstrates the FUNDING
need for the development of novel accurate and reliable methods
for detecting resistance to clinically important antimicrobial This review was supported by the National Research Lab Program
agents, such as colistin. Hospital interventions can effectively through the National Research Foundation of Korea (NRF)
reduce the spread of carbapenemase-producing K. pneumoniae. funded by the Ministry of Science, ICT & Future Planning
Standard infection control guidelines should be implemented (No. 2011-0027928); the Cooperative Research Program
upon the detection of carbapenemase-producing K. pneumoniae, for Agriculture Science & Technology Development (No.
and carbapenemase-producing K. pneumoniae positive patients PJ01103103) of Rural Development Administration in South
should be individually isolated and treated according to strict Korea; and the National Research Foundation of the Ministry
standard guidelines. of Education, South Korea (NRF-2015R1C1A1A02037470).

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restructuring of New Delhi metallo-β-lactamase-1 (NDM-1)-producing ST42 conducted in the absence of any commercial or financial relationships that could
Klebsiella pneumoniae emerging in Japan. J. Infect. Chemother. 19, 118–127. doi: be construed as a potential conflict of interest.
10.1007/s10156-012-0470-z
Yan, Y., Yang, H., Pan, L., Sun, K., Fan, H., Lu, Y., et al. (2014). Improving the Copyright © 2016 Lee, Lee, Park, Kim, Jeong and Lee. This is an open-access article
efficiency of the modified Hodge test in KPC-producing Klebsiella pneumoniae distributed under the terms of the Creative Commons Attribution License (CC BY).
isolates by incorporating an EDTA disk. Curr. Microbiol. 69, 47–52. doi: The use, distribution or reproduction in other forums is permitted, provided the
10.1007/s00284-014-0552-5 original author(s) or licensor are credited and that the original publication in this
Yang, J., Ye, L., Guo, L., Zhao, Q., Chen, R., Luo, Y., et al. (2013). A nosocomial journal is cited, in accordance with accepted academic practice. No use, distribution
outbreak of KPC-2-producing Klebsiella pneumoniae in a Chinese hospital: or reproduction is permitted which does not comply with these terms.

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