Global Dissemination of Carbapenemase-Producing Klebsiella pneumoniae: Epidemiology, Genetic Context, Treatment Options, and Detection Methods

The emergence of carbapenem-resistant Gram-negative pathogens poses a serious threat to public health worldwide. In particular, the increasing prevalence of carbapenem-resistant Klebsiella pneumoniae is a major source of concern. K. pneumoniae carbapenemases (KPCs) and carbapenemases of the oxacillinase-48 (OXA-48) type have been reported worldwide. New Delhi metallo-β-lactamase (NDM) carbapenemases were originally identified in Sweden in 2008 and have spread worldwide rapidly. In this review, we summarize the epidemiology of K. pneumoniae producing three carbapenemases (KPCs, NDMs, and OXA-48-like). Although the prevalence of each resistant strain varies geographically, K. pneumoniae producing KPCs, NDMs, and OXA-48-like carbapenemases have become rapidly disseminated. In addition, we used recently published molecular and genetic studies to analyze the mechanisms by which these three carbapenemases, and major K. pneumoniae clones, such as ST258 and ST11, have become globally prevalent. Because carbapenemase-producing K. pneumoniae are often resistant to most β-lactam antibiotics and many other non-β-lactam molecules, the therapeutic options available to treat infection with these strains are limited to colistin, polymyxin B, fosfomycin, tigecycline, and selected aminoglycosides. Although, combination therapy has been recommended for the treatment of severe carbapenemase-producing K. pneumoniae infections, the clinical evidence for this strategy is currently limited, and more accurate randomized controlled trials will be required to establish the most effective treatment regimen. Moreover, because rapid and accurate identification of the carbapenemase type found in K. pneumoniae may be difficult to achieve through phenotypic antibiotic susceptibility tests, novel molecular detection techniques are currently being developed.


INTRODUCTION
The increasing prevalence of antibiotic resistance and the lack of new antibiotic drug development has gradually reduced the treatment options for bacterial infections (Lee et al., 2013a;Nathan and Cars, 2014). In 2013, the Centers for Disease Control and Prevention (CDC) named three microorganisms that pose an urgent threat to public health: carbapenemresistant (CR) Enterobacteriaceae (CRE), drug-resistant Neisseria gonorrhoeae, and Clostridium difficile (Zowawi et al., 2015). Carbapenems (imipenem, meropenem, biapenem, ertapenem, and doripenem) are antibiotics used for the treatment of severe infections caused by multi-resistant Enterobacteriaceae, such as Klebsiella pneumoniae and Escherichia coli (Nordmann et al., 2009). However, over the past 10 years, CRE have increasingly been reported worldwide . In particular, K. pneumoniae have acquired carbapenemases, which are enzymes capable of breaking down most β-lactams including carbapenems, and thus conferring resistance to these drugs (Jeon et al., 2015). High mortality rates have been reported in patients with bloodstream infections caused by CR K. pneumoniae (Munoz-Price et al., 2013). Carbapenemases can be divided according to their dependency on divalent cations for enzyme activation into metallo-carbapenemases (zinc-dependent class B) and non-metallo-carbapenemases (zinc-independent classes A, C, and D; Jeon et al., 2015). The class A carbapenemases, such as the K. pneumoniae carbapenemase (KPC) enzymes, have been identified worldwide in K. pneumoniae (Tangden and Giske, 2015). Various class B and D carbapenemases have also been detected in hospital-acquired multi-resistant K. pneumoniae , whereas class C carbapenemases have rarely been reported. In this review, we summarize the epidemiology of the major four classes of carbapenemases and discuss their molecular genetics, methods used for their detection, and the therapeutic options available for their treatment.
THE EPIDEMIOLOGY, GENETIC CONTEXT, TREATMENT OPTIONS, AND DETECTION METHODS OF CARBAPENEM-RESISTANT K. pneumoniae

Class A Carbapenemases
Epidemiology Various class A carbapenemases forming six distantly related branches have been identified (Jeon et al., 2015). While some carbapenemases are chromosome-encoded (IMI-1, NMC-A, SME enzymes, SHV-38, and SFC-1), others are plasmid-encoded (KPC enzymes, GES enzymes, and IMI-2). KPCs have been the most frequently observed class A carbapenemases since their first description in the eastern the USA in 1996 (Yigit et al., 2001). Of the many different KPC family variants (KPC-1 to , the most well-characterized variants are KPC-2 and KPC-3. KPCs are mostly plasmid-encoded enzymes and bacteria producing these enzymes are susceptible to only a few antibiotics such as colistin, aminoglycosides, and tigecycline. Therefore, the mortality of the patient's bloodstream infections caused by these bacteria is very high (Munoz-Price et al., 2013).
The epidemiology of K. pneumoniae producing KPCs varies geographically. The endemic spread of these bacteria has been reported in the USA, China, Italy, Poland, Greece, Israel, Brazil, Argentina, Colombia, and Taiwan (Munoz-Price et al., 2013; Figure 1). Sporadic spread of KPC-producing K. pneumoniae has also been observed in many European countries including Spain, France, Germany, the Netherlands, the UK, Ireland, Belgium, Sweden, and Finland, and in several countries in the Asia-Pacific region, including India, South Korea, and Australia (Munoz-Price et al., 2013;Nordmann and Poirel, 2014). In the USA, the transmission of CR K. pneumoniae is primarily driven by the spread of organisms carrying KPC enzymes (Kaiser et al., 2013), but other carbapenemase enzymes, such as the New-Delhi metallo-β-lactamase (NDM), have also emerged (Lascols et al., 2013). Within the USA, the prevalence of KPC-positive isolates was relatively stable between (5.9% in 2007, 4.9% in 2008, and 5.7% in 2009Kaiser et al., 2013), and KPC-2 and KPC-3 were the most frequently identified carbapenemases in K. pneumoniae (Deshpande et al., 2006).
In Greece, KPC-producing K. pneumoniae was first isolated in August 2007 (Tsakris et al., 2008), and the prevalence of KPCproducers among carbapenemase-producing K. pneumoniae isolates collected at a tertiary Greek hospital increased from 0% in 2003 to 38.3% in 2010 (Zagorianou et al., 2012). Most of the genotyped KPC-producing K. pneumoniae in Greece harbored KPC-2 (Zagorianou et al., 2012). While many carbapenemaseproducing K. pneumoniae in the USA and Greece had KPC enzymes (Nordmann et al., 2009;Zagorianou et al., 2012), several studies in Spain showed that most carbapenemase-producing K. pneumoniae harbored OXA-48-like or class B carbapenemases, and the rate of KPC-producing K. pneumoniae was very low (2-3%; Oteo et al., 2013b;Palacios-Baena et al., 2016). These results indicate that the prevalent genotype of carbapenemaseproducing K. pneumoniae varies geographically. For example, in Italy which is a representative southern European country where KPC is becoming endemic, 89.5% of carbapenemase producers have been reported to have KPC-type enzymes, followed by VIM-1 (9.2%) and OXA-48 (1.3%; Giani et al., 2013).
In the Asia-Pacific region, the endemic dissemination of KPC-producing K. pneumoniae has been reported in China  and Taiwan (Tseng et al., 2015), and the sporadic spread has been reported in India (Shanmugam et al., 2013), South Korea (Yoo et al., 2013), and Australia (Partridge et al., 2015). A novel KPC-15 variant which is closely homologous with KPC-4 was discovered in China (Wang et al., 2014b) and its enzymatic activity and phenotype was characterized (Wang et al., 2014a). In China, the frequency of KPC-type enzymes among carbapenemase producers was high (63%; Li et al., 2014). While ST258 is the predominant clone observed in European countries and the USA Chen et al., 2014e;Bowers et al., 2015), ST11, which is closely related to ST258, is the prevalent clone associated with the spread of KPCproducing K. pneumoniae in Asia (particularly in China and Taiwan; Qi et al., 2011;Yang et al., 2013;Tseng et al., 2015). KPCproducing ST11 strain has also been reported in Latin America (Munoz-Price et al., 2013). Although it is unknown why ST11 is prevalent, a recent report showed that the KPC-producing K. pneumoniae ST11 clone was resistant to serum killing (Chiang et al., 2016). In a Chinese hospital, another nosocomial outbreak of KPC-2-producing K. pneumoniae was caused by multiple K. pneumoniae strains including ST37, ST392, ST395, and ST11, implying the horizontal transfer of bla KPC−2 gene between different K. pneumoniae clones in China . In Taiwan, two novel KPC variants were identified; KPC-16 and KPC-17 differed from KPC-2 by two (P202S and F207L) and a single (F207L) amino acid substitutions, respectively . A nationwide survey in Taiwan between 2011 and 2013 reported the national spread of KPC-2 and KPC-17 (Tseng et al., 2015). KPC-producing K. pneumoniae was recently also detected in Japan (Saito et al., 2014), Pakistan (Pesesky et al., 2015), Iran (Nobari et al., 2014), and United Arab Emirates (Sonnevend et al., 2015a). In the Arabian Peninsula, the prevalence of KPC-producing K. pneumoniae was very low in comparison to NDM-1 and OXA-48-like carbapenemases (Sonnevend et al., 2015b). Sonnevend et al. (2015a), two K. pneumoniae ST14 strains producing KPC-2 were first identified in the United Arab Emirates of the Arabian Peninsula. In Africa, several countries such as South Africa (Brink et al., 2012), Algeria (Bakour et al., 2015b), and Egypt (Metwally et al., 2013), have also isolated KPC-producing K. pneumoniae.
These genes may be important for the successful dissemination of bla KPC -harboring plasmids. The IncFII plasmids are one of predominant bla KPC -harboring plasmids. pKpQIL, which is an IncFII K2 plasmid harboring Tn4401a, was initially identified in Israel in 2006 (Leavitt et al., 2010), and then this plasmid and its variants are believed to have spread to Italy, Poland, the UK, Colombia, the Czech Republic, the USA, and other countries (Baraniak et al., 2011;Garcia-Fernandez et al., 2012;Hidalgo-Grass et al., 2012;Warburg et al., 2012;Hrabak et al., 2013b;Chen et al., 2014d,e), suggesting the wide dissemination of this plasmid. The bla KPC gene has also been identified in other non-Tn4401 mobile elements that mostly have partial ISKpn6 genes (Shen et al., 2009;Gomez et al., 2011). Based on the insertion sequence upstream of the bla KPC gene, they can be divided into three groups: group I, no insertion (Shen et al., 2009;Liu et al., 2012;Chen et al., 2014g); group II, insertion of truncated bla TEM (Gomez et al., 2011); group III, insertion of Tn5563/IS6100 (Wolter et al., 2009). These non-Tn4401 genetic elements harboring bla KPC sometimes have an IS26 transposon (Liu et al., 2012;Chen et al., 2014g). Carbapenemase genes often spread worldwide through clonal expansion in several successful pathogenic strains (Chen et al., 2014e). For example, the dissemination of KPC-producing K. pneumoniae in most countries including the USA and European countries is largely caused by expansion of a single dominant strain, ST258 Chen et al., 2014e;Bowers et al., 2015). This strain is a prototype of a high-risk clone of K. pneumoniae. Recent data from Israel showed that the KPCproducing K. pneumoniae ST258 clone remains the predominant clone, representing 80% of the KPC-producing K. pneumoniae population (Adler et al., 2015). ST258 may be a hybrid clone that was created by a large recombination event between ST11 and ST442 (Pitout et al., 2015). It is unknown why the ST258 lineage is the most prevalent clone of KPC-producing Klebsiella species. The ST258 clone is highly susceptible to serum killing in animal models and lacks well-known K. pneumoniae virulence factors, such as aerobactin genes, K1, K2, and K5 capsular antigen genes, and the regulator of the mucoid phenotype gene rmpA (Tzouvelekis et al., 2013;Pitout et al., 2015). Two recent reports revealed that the ST258 strains consist of two distinct genetic clades and genetic differentiation between the two clades (-1 and cps-2) results from an approximately 215-kb region of divergence that includes cps genes involved in capsule polysaccharide synthesis (Chen et al., 2014e;Deleo et al., 2014). Multiplex PCR for the identification of two capsular types in K. pneumoniae ST258 strains revealed a significant association between the cps type and KPC variant: the cps-1 clade is largely associated with KPC-2, while the cps-2 clade is primarily associated with KPC-3 . Because the capsule polysaccharide can help K. pneumoniae to evade phagocytosis, the global success of this strain may involve the capsule polysaccharide biosynthesis regions cps-1 and cps-2. A recent report revealed a relationship between the integrative conjugative element ICEKp258.2 and the global success of the ST258 clone . ICEKp258.2 contains two specific gene clusters, a type IV pilus gene cluster (i.e., pilV) associated with the uptake and exchange of plasmids and adherence to living and non-living surfaces, and a gene cluster of a type III restriction-modification system determining host specificity in the exchange of certain compatible plasmids or mobile elements . Because these genes associated with the restriction of plasmids and specific mobile elements were present only in ST258 and genetically related sequence types, this difference may explain the divergence of ST258 predominantly harboring KPC and ST11, another highrisk clone that lacks ICEKp258.2, harboring a broad range of plasmids and carbapenemases, including KPC, NDM, OXA-48, VIM, and IMP (Chen et al., 2014f;Pitout et al., 2015). Although the ICEKp258.2 of ST258 strains may contribute to global success, the precise reason for the predominance of the ST258 strain in KPC-producing K. pneumoniae is still not entirely understood. Recently, an outbreak of non-ST258 KPC-producing K. pneumoniae clones has been reported in the USA and Europe (Ruiz-Garbajosa et al., 2013;Bonura et al., 2015;Garbari et al., 2015).
The habitat of K. pneumoniae is not limited to humans but extends to the ecological environment, such as soil, water, and sewage, and K. pneumoniae can survive in extreme environments for long periods of time (Pitout et al., 2015). Therefore, K. pneumoniae producing KPCs were detected in various nosocomial environments, such as gowns and gloves (Rock et al., 2014) and wastewater (Chagas et al., 2011;Galler et al., 2014). The frequency of KPC-producing K. pneumoniae contamination of gowns and gloves of healthcare workers is similar to that of contamination with methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus (Rock et al., 2014), indicating fast transmission of KPC-producing Klebsiella species in a nosocomial environment. A long-term observation in a hospital with low-frequency outbreaks of KPC-producing K. pneumoniae suggested the possible role of a persisting environmental reservoir of resistant strains in the maintenance of this long-term outbreak (Tofteland et al., 2013). After discharge from the hospital, long-term (>3 years) carriage of KPCproducing K. pneumoniae is also possible (Lübbert et al., 2014), and lateral gene transfer of KPC among Enterobacteriaceae colonizing the human intestine appears frequent, for example from K. pneumoniae to E. coli Gona et al., 2014). Therefore, reservoirs in healthcare workers, patients, or the hospital environment may be a principle mode of spread in nosocomial outbreaks.

Treatment Options
Carbapenemase-producing K. pneumoniae strains are currently one of the most important nosocomial pathogens. Hospital outbreaks of KPC-producing K. pneumoniae mainly affect severely ill patients and are associated with an increased risk of death (Ducomble et al., 2015;Tumbarello et al., 2015). KPCproducing K. pneumoniae bloodstream infections in intensive care unit (ICU) have also been associated with increased mortality .
Because carbapenemase-producing K. pneumoniae are mostly resistant to several important antibiotic classes (β-lactam drugs, fluoroquinolones, and aminoglycosides), antibiotics, such as polymyxin B, colistin (polymyxin E), fosfomycin, tigecycline, and sometimes selected aminoglycosides, are the last-resort agents. KPC-producing K. pneumoniae are usually resistant to all β-lactam antibiotics, but temocillin can be active against some KPC-producing K. pneumoniae, particularly in the case of lower urinary tract infections (Adams-Haduch et al., 2009).
To maximize bacterial killing and minimize bacterial resistance, combined therapy is sometimes recommended. Combination therapy including a carbapenem, such as a combination of tigecycline, colistin, and meropenem, was strongly effective in the treatment of KPC-producing K. pneumoniae, including colistinresistant isolates (Tumbarello et al., 2012(Tumbarello et al., , 2015Giamarellou et al., 2013;Hong et al., 2013;Daikos et al., 2014). The synergistic combination of colistin and rifampin was also effective in the treatment of colistin-resistant KPC-producing K. pneumoniae by slowing the selection of heteroresistant subpopulations during colistin therapy (Tascini et al., 2013). However, several reports have shown that combination therapy was not superior to monotherapy (de Oliveira et al., 2015;Toledo et al., 2015). Thus, extensive studies will be required to assess the effectiveness of combination therapy. A triple combination of colistin-doripenem-ertapenem was effective only in isolates with high levels of OmpK35 and OmpK36 porin expression (Hong et al., 2013). The expression level of OmpK36 was also involved in the rapid induction of high-level carbapenem resistance in heteroresistant KPC-producing K. pneumoniae populations (Adams-Sapper et al., 2015). Therefore, molecular characterization of the KPC-producing K. pneumoniae strain, such as the determination of the expression level of OmpK35 and OmpK36, can be used to identify effective combination regimens. However, as a minor effect of OmpK35 and OmpK36 on carbapenem resistance of K. pneumoniae was also reported , more extensive studies on the role of these proteins on K. pneumoniae carbapenem resistance are also required.
Colistin (polymyxin E), an agent discovered more than 60 years ago, is a key component of the combination of antimicrobial regimens used for the treatment of severe KPCproducing K. pneumoniae infections (Cannatelli et al., 2014b). Since the global spread of KPC-producing K. pneumoniae, the emergence of colistin resistance in KPC-producing K. pneumoniae have been reported in many countries, including Italy (Cannatelli et al., 2014b;Giani et al., 2015), the USA (Bogdanovich et al., 2011), Greece (Kontopoulou et al., 2010), Hungary (Toth et al., 2010), and Turkey (Labarca et al., 2014). The increasing prevalence of colistin-resistant K. pneumoniae producing KPC poses a threat to public health because colistin resistance increases the mortality due to KPC-producing K. pneumoniae bloodstream infections and reduces therapeutic options. A multicenter case-control-control study in Italy showed that the rate of colistin resistance among KPC-producing K. pneumoniae blood isolates increased more than threefold during the 4.5-years study period, and the 30-days mortality due to colistin-resistant KPC-producing K. pneumoniae bloodstream infections was approximately 51% . Data collected from 21 hospital laboratories in Italy between 2013 and 2014 also showed that 43% of carbapenemase-producing K. pneumoniae were resistant to colistin, 6% were resistant to tigecycline, 16% were resistant to gentamicin, 82% were resistant to trimethoprim-sulfamethoxazole, and 1% were resistant to all four antibiotics, and colistin-resistant isolates were detected in all participating hospital laboratories (Monaco et al., 2014). The progressive increase of colistin resistance was also reported elsewhere (Pena et al., 2014;Bonura et al., 2015;Parisi et al., 2015). These results indicate that the strict rules for colistin use are required to diminish the spread of colistin resistance in the endemic regions of KPC-producing K. pneumoniae.
Molecular and biochemical studies have shown that insertional inactivation of the mgrB gene, encoding a negativefeedback regulator of the PhoQ-PhoP signaling system, can be responsible for colistin resistance in KPC-producing K. pneumoniae, due to the resulting up-regulation of the Pmr lipopolysaccharide modification system (Cannatelli et al., 2013(Cannatelli et al., , 2014b. A recent study analyzing a series of colistin-resistant K. pneumoniae isolates of worldwide origin identified a single amino acid change (T157P) in the PmrB protein as being responsible for the overexpression of pmrCAB and pmrHFIJKLM operons involved in lipopolysaccharide modification, leading to colistin resistance (Jayol et al., 2014). The relationship between colistin resistance and inactivation of the mgrB gene was further supported by analysis of clinical colistin-resistant K. pneumoniae isolates producing KPC (Bonura et al., 2015;Giani et al., 2015). The emergence of colistin resistance was also associated with low-dosage colistin treatment (Cannatelli et al., 2014a). A recent report showed that the plasmid carrying the mcr-1 gene, which encodes a phosphoethanolamine transferase enzyme catalyzing the addition of phosphoethanolamine to lipid A, is a major contributor to colistin resistance in Gram-negative bacteria and is spread through horizontal gene transfer (Liu et al., 2016). This mcr-1-harboring plasmid was also detected in E. coli isolates collected from 78 (15%) of 523 samples of raw meat, 166 (21%) of 804 animals, and 16 (1%) of 1322 samples from inpatients with infection, indicating the emergence of this plasmid-mediated colistin resistance mechanism (Liu et al., 2016).
Fosfomycin is a broad-spectrum antibiotic that inhibits bacterial cell wall biogenesis by inactivating the enzyme UDP-N-acetylglucosamine-3-enolpyruvyltransferase, also known as MurA (Brown et al., 1995). Fosfomycin has been used to treat KPC-producing K. pneumoniae, but recently, a high fosfomycin resistance rate was observed is in countries with higher usage (Giske, 2015). Only 43.4% of KPC-producing K. pneumoniae strains retained susceptibility to fosfomycin in a Chinese university hospital  and a similar fosfomycin susceptibility rate (39.2%) was observed in KPCproducing K. pneumoniae collected from 12 hospitals in China (Jiang et al., 2015). Like colistin, fosfomycin resistance seems to be associated with the plasmid containing the fosA3 gene which encodes glutathione S-transferase to modify fosfomycin and was characterized first in CTX-M-producing E. coli in Japan (Wachino et al., 2010). In China, the fosA3-harboring plasmid was attributed to 55.6% of fosfomycin-resistant KPC-producing K. pneumoniae strains (Jiang et al., 2015). Although the fosA3 gene is mainly associated with the bla CTX−M gene, the fosA3 gene has also been characterized in atypical bla KPC -harboring plasmids (Jiang et al., 2015;Li et al., 2015). In pFOS18 (Jiang et al., 2015) and pKP1034 (Xiang et al., 2015), the fosA3 and bla KPC−2 genes were located on different transposon systems, whereas in pHS102707 belonging to the IncP1 group , two genes were co-located in the same Tn1721-Tn3-like transposon.
Tigecycline, a derivative of minocycline, is the first member of the glycylcycline class that acts as a protein synthesis inhibitor by blocking the interaction of aminoacyl-tRNA with the A site of the ribosome (Rose and Rybak, 2006). Due to the increased clinical use of tigecycline for treatment of KPC-producing K. pneumoniae, increased tigecycline resistance was reported (Zagorianou et al., 2012;Papadimitriou-Olivgeris et al., 2014;Weterings et al., 2015). In the Netherlands, all KPC-producing K. pneumoniae isolates exhibited reduced susceptibility to tigecycline (Weterings et al., 2015). Another report showed that during ICU stay, 17.9% (39/257) of patients became colonized by tigecycline-resistant KPC-producing K. pneumoniae during their stay (Papadimitriou-Olivgeris et al., 2014). In a Greek tertiary hospital during 2004 to 2010, 11.3% (34/301) of KPCproducing isolates were resistant to tigecycline (Zagorianou et al., 2012). Overproduction of efflux pumps such as AcrAB and overexpression of RamA, a positive regulator of the AcrAB efflux system, seem to be major factors for decreased sensitivity of K. pneumoniae strains to tigecycline (Ruzin et al., 2005;Rosenblum et al., 2011;Sun et al., 2013). A recent report in China showed that the OqxAB efflux pump was also contributed to tigecycline resistance in K. pneumoniae isolates .
Because KPC-producing K. pneumoniae sometimes remains susceptible to several aminoglycosides such as gentamicin , aminoglycosides can be used alone or in combination therapy to treat KPC-producing K. pneumoniae infections. Actually, gentamicin monotherapy or in combination with tigecycline was recently reported to reduce the mortality from sepsis caused by K. pneumoniae ST512 clone producing KPC-3, SHV-11, or TEM-1 (Gonzalez-Padilla et al., 2015). New weapons are always indispensable for combating KPCproducing K. pneumoniae infections (Lee et al., , 2015a. The effectiveness of some antibiotics in development was also estimated against KPC-producing K. pneumoniae. Potent inhibitors of serine β-lactamases, such as avibactam and MK7655, were effective against KPC-producing K. pneumoniae infections (Temkin et al., 2014). Combination therapy with avibactam and ceftazidime exhibited significant synergetic effects against organisms with combinations of extended-spectrum β-lactamases (ESBLs), AmpCs, and KPC-2 (Wang et al., 2014e). Plazomicin (a novel aminoglycoside) also exhibited significant activity against KPC-producing K. pneumoniae (Temkin et al., 2014). The novel polymyxin derivatives with lower nephrotoxicity are under development (Vaara, 2010). A recent report suggested that synthetic peptides with antimicrobial and antibiofilm activities are a promising strategy in the treatment of infections caused by KPC-producing K. pneumoniae . The in vitro activity of the next-generation aminoglycoside plazomicin alone and in combination with colistin, meropenem, fosfomycin or tigecycline was tested against carbapenemase-producing Enterobacteriaceae (CPE) strains. When plazomicin was combined with meropenem, colistin or fosfomycin, synergy was observed against CPE isolates (Rodriguez-Avial et al., 2015).

Detection Methods
Because a delay in the appropriate antibiotic therapy for severe infections of KPC-producing K. pneumoniae is strongly associated with unfavorable prognosis and increased mortality rates (Karaiskos and Giamarellou, 2014), rapid detection of CR strains is essential for the effective management of these infections (Lee et al., 2005b(Lee et al., , 2006b(Lee et al., , 2016. Various methods for the identification of KPCs have been developed, including multiplex PCR assay (Spanu et al., 2012;Adler et al., 2014), real-time PCR assay (Wang et al., 2012;Lee et al., 2015c), DNA microarray (Peter et al., 2012;Braun et al., 2014), Raman spectroscopic analysis (Willemse-Erix et al., 2012), single-colony whole-genome sequencing (Koser et al., 2014), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS; Carvalhaes et al., 2014), loop-mediated isothermal amplification (LAMP) method (Nakano et al., 2015), chromogenic medium (Vrioni et al., 2012), and a new phenotypic test, called the carbapenem inactivation method (CIM; van der Zwaluw et al., 2015). False positive results can occur when the modified Hodge test is used to detect carbapenemases in carbapenemase-negative K. pneumoniae clinical isolates . Therefore, to improve the efficiency in the phenotypic detection of KPC-producing K. pneumoniae isolates, the modified Hodge test can be combined with an EDTA disk test (Yan et al., 2014) or a disk test using boronic acid compounds (Pournaras et al., 2010a). These methods enhanced the sensitivity and specificity of KPC detection in K. pneumoniae isolates. In a new developed phenotypic test, called the CIM, a susceptibility-testing disk containing carbapenem was immersed in the suspension made by suspending an inoculation loop of bacterial culture (van der Zwaluw et al., 2015). After incubation, the disk was placed on an agar plate inoculated with a susceptible E. coli indicator strain. If the bacterial isolate produces carbapenemase, the susceptibility-testing disk will allow the growth of the susceptible indicator strain. This method showed high concordance with results obtained by PCR (van der Zwaluw et al., 2015). Nevertheless, these culture-based phenotypic tests are time-consuming and cannot easily detect ESBLs and carbapenemases produced by Enterobacteriaceae, owing to varied levels of enzyme expression and the poor specificity of some antibiotic combinations (Okeke et al., 2011;Swayne et al., 2013). To overcome these limitations of phenotypic methods, various molecular-based diagnostic methods have been developed. Especially, the direct detection of the carbapenemase gene using multiplex PCR, real-time PCR, and DNA microarray method can increase the speed and accuracy of detecting CR strains (Okeke et al., 2011;Solanki et al., 2014). Because of the high genetic diversity of genes coding for carbapenemase, the precise design of primers or probes is necessary for correctly amplifying or detecting only expected carbapenemase genes. Therefore, already developed methods for bla gene detection are restricted to the detection of only limited types of bla genes (Lee et al., 2015b). However, the large-scale blaFinder was recently developed on the basis of multiplex PCR, and this large-scale detection method can detect almost all bla genes, including KPCs, NDMs, OXA-48-likes, present in bacterial pathogens (Lee et al., 2015b).
Recently, mass spectrometry-based methods, such as MALDI-TOF MS and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), have been shown to be capable of characterizing carbapenemase-producing bacteria (Carricajo et al., 2014;Carvalhaes et al., 2014). These methods are fast and accurate to routinely identify bacterial isolates with great specificity and sensitivity (Hrabak et al., 2011;Patel, 2013;Carvalhaes et al., 2014;Lasserre et al., 2015), but these systems do not accurately provide the carbapenem minimum inhibitory concentrations (MICs) for carbapenemaseproducing K. pneumoniae (Patel, 2013). Several experiments showed that this method is more rapid and accurate for detection of carbapenemase activity in Gram-negative bacteria than some methods including the modified Hodge test (Lee et al., 2013b;Chong et al., 2015). The LAMP method has emerged as a powerful gene amplification assay for the rapid identification of microbial infections (Notomi et al., 2000). This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. The assay amplifies the DNA under isothermal conditions (63-65 • C) with high degrees of specificity, efficiency, and speed (Reddy et al., 2010). The cycling reaction continues with accumulation of 10 9 copies of target in less than an hour. The assay can be conducted in a water bath or heating block instead of the thermal cycling using a PCR machine (Notomi et al., 2000). The LAMP assay can be applied for detection of KPC producers in the clinical laboratory (Nakano et al., 2015) and has greater sensitivity, specificity, and rapidity compared to the phenotypic methods and PCR for the detection of KPC-producing K. pneumoniae (Solanki et al., 2013).
Colistin has often been used as a therapeutic option for the treatment of CR K. pneumoniae infections. However, the imprudent use of colistin has caused rapid spread of colistin resistance in K. pneumoniae producing carbapenemases, particularly the KPC-type carbapenemases (Monaco et al., 2014;Giacobbe et al., 2015). This situation demonstrates the need for the development of accurate and reliable methods for detecting colistin resistance. Recently, several methods for the identification of colistin resistance were reported, including various routine colistin MIC testing methods, such as BMD, BMD-P80, AD, Etest, MTS, and Vitek2 (Dafopoulou et al., 2015;Humphries, 2015); capillary electrophoresis method according to characteristic surface properties of bacteria (Sautrey et al., 2015); and the micromax assay based on evaluation of the efficacy of antibiotics that affect cell wall integrity (Tamayo et al., 2013). Because a recent report showed that the mcr-1 gene, involved in the modification of lipid A, is a major contributor to colistin resistance in Gram-negative bacteria (Liu et al., 2016), detection of this gene may be important in the detection of colistin resistance.

Class B Carbapenemases
Epidemiology Class B β-lactamases are metallo-β-lactamases that require zinc or another heavy metal for catalysis. Class B β-lactamases have a broad substrate spectrum and can catalyze the hydrolysis of virtually all β-lactam antibiotics including carbapenems except for monobactams (Jeon et al., 2015). Class B carbapenemases were mostly identified in Enterobacteriaceae and include VIMs, IMPs, and the emerging NDM group (Jeon et al., 2015). Among them, NDM (New Delhi metallo-β-lactamase) is one of the most clinically significant carbapenemases. NDM-1 was first detected in 2008 in K. pneumoniae and E. coli in a patient returning to Sweden from India and has since spread worldwide (Yong et al., 2009;Jeon et al., 2015). Thus far, 15 NDM variants have been assigned (Jeon et al., 2015), and most of them originated from Asia . NDMs shares very little identity with other metallo-β-lactamases .
The Balkan states (Livermore et al., 2011;Voulgari et al., 2014), the Arabian Peninsula , and North African countries (Dortet et al., 2014c), have also been recently considered as an additional reservoir of NDM producers. In the Arabian Peninsula, NDM-1 was the most frequently encountered carbapenemase (46.5%) followed by OXA-48-like carbapenemases (32.5%; Sonnevend et al., 2015b). In Greece, among 132 non-repetitive CRE isolates between 2010 and 2013, 78 K. pneumoniae isolates with the bla NDM−1 gene were identified (Voulgari et al., 2014). In the USA, KPCproducing K. pneumoniae have been responsible for much of the increase in carbapenemase-producing bacteria detection, but recent increases in NDM-producing K. pneumoniae have the potential to add to this burden (Rasheed et al., 2013; Centers for Disease Control and Prevention, 2014).
The movement of patients between countries may be a trigger for the international spread of carbapenemase-producing K. pneumoniae (Berrazeg et al., 2014). Carbapenemaseproducing Gram-negative bacteria including K. pneumoniae, obtained from patients that had recently traveled outside Canada between 2010 and 2013, were found to be NDM-producing K. pneumoniae, belonging to various sequence types (ST15, ST16, ST147, ST258, ST340, ST512, and ST972) with different plasmids (IncF, IncA/C, and IncL/M), and were imported from India to Canada (Peirano et al., 2014). Therefore, more careful attention is required when treating patients with a recent history of foreign hospitalization in countries where carbapenemase-producing bacteria are prevalent. The international transportations of patients between countries recently resulted in the detection of NDM-producing K. pneumoniae in Mexico , Guatemala (Pasteran et al., 2012), Brazil (Quiles et al., 2015), the Netherlands ( The global dissemination of NDM-producing K. pneumoniae also has a serious impact on neonatal mortality rates, particularly in low-income countries where the burden of neonatal sepsis is high (Zaidi et al., 2005). Colonization of NDM-producing K. pneumoniae isolates in pregnant women in the community in Madagascar highlighted the potential for mother-to-child transmission (Chereau et al., 2015). In India, analysis of Enterobacteriaceae, including K. pneumoniae, isolated from the blood of septicaemic neonates, indicted that 14% of the isolates possessed bla NDM−1 , and there was a significantly higher incidence of sepsis caused by NDM-1-harboring isolates (Datta et al., 2014). In China, an outbreak of bla NDM−1 -producing K. pneumoniae ST20 and ST17 isolates was identified in a neonatal unit (Jin et al., 2015). In Turkey, the spread of NDM-1producing K. pneumoniae in a neonatal ICU was reported . In Colombia, NDM-1-producing K. pneumoniae strains were identified from an outbreak that affected six neonatal patients (Escobar Perez et al., 2013).
As with KPC, coexistence of NDMs and other carbapenemases in K. pneumoniae has also been reported worldwide, in Brazil Besides NDM-type carbapenemases, the IMP and VIM groups have also been detected worldwide in K. pneumoniae, but other carbapenemases, such as GIM-1, KHM-1, and SPM-1, have been not found in K. pneumoniae (Table 1). Since IMP and VIP were first detected in Serratia marcescens in 1991 and in Pseudomonas aeruginosa in 1996, respectively (Osano et al., 1994;Lauretti et al., 1999), IMP-and VIM-producing K. pneumoniae have spread in Europe and Asia, but were rarely reported in other regions, such as America and Africa (Table 1). Although in this review we have focused only on carbapenemaseproducing K. pneumoniae, the VIM group is one of the most commonly reported carbapenemases worldwide if we consider all bacteria species (the VIM groups have been mainly identified in P. aeruginosa; Poirel et al., 2007).

Molecular and Genetic Context
The bla NDM gene is frequently observed in the transposon Tn125 (with two flanking ISAba125 elements) within NDMproducing species of the genus Acinetobacter (Partridge and Iredell, 2012;Wailan et al., 2015). The bla NDM gene was hypothesized to originate in the genus Acinetobacter . In Enterobacteriaceae, the ISAba125 elements of the Tn125 structure carrying bla NDM are frequently truncated (Tn125) at various lengths and the Tn125 structure frequently has different IS elements (Figure 2B; Partridge and Iredell, 2012;Wailan et al., 2015). The bla NDM genes in K. pneumoniae have been reported on numerous broad-host-range plasmid types, including IncA/C (Hudson et al., 2014), IncF (Hishinuma et al., 2013), IncR , IncH , IncN , IncL/M (Peirano et al., 2014), and IncX types (Wang et al., 2014d). The predominant plasmid type responsible for spreading bla NDM−1 is the IncA/C type plasmids (Poirel et al., 2011d;Pitout et al., 2015). Many IncA/C plasmids with bla NDM−1 also carry various antibiotic resistance genes including 16S rRNA methylases (RmtA and RmtC), associated with aminoglycoside resistance; CMY-type β-lactamases, associated with broadspectrum cephalosporin resistance; and QnrA, associated with quinolone resistance (Pitout et al., 2015). Consequently, many NDM-producing K. pneumoniae were susceptible only to colistin, fosfomycin, and tigecycline . A novel NDM-1 variant (NDM-9) located on a novel IncH variant plasmid was recently identified in a clinical K. pneumoniae isolate in China (Wang et al., 2014c). Because the bla NDM genes are located in numerous broad-host-range plasmids, the spread of NDM-1 is facilitated by horizontal gene transfer between bacteria. In China, CRE 21 strains harboring the bla NDM−1 gene were found to consist of multiple Enterobacteriaceae species including nine Enterobacter cloacae, three E. coli, three Citrobacter freundii, two K. pneumoniae, two K. oxytoca, and two Morganella morganii strains .
Unlike KPC, the bla NDM genes were detected in various K. pneumoniae clones. ST11, a major high-risk sequence type of KPC-producing K. pneumoniae in Asia, has also been associated with bla NDM−1 in K. pneumoniae identified in many countries, such as the USA (Rasheed et al., 2013), Greece (Voulgari et al., 2014), Australia (Shoma et al., 2014), Switzerland (Seiffert et al., 2014), the Czech Republic , Spain (Oteo et al., 2013b), and Thailand (Netikul et al., 2014). The recent outbreak of NDM-producing K. pneumoniae ST11 in Poland was caused by a clone similar to an isolate identified in the Czech Republic in 2013 (Baraniak et al., 2016), indicating the local spread of this clone. ST11 has also been associated with OXA-48-like enzymes from isolates found in Argentina, Turkey, and Spain (Lascols et al., 2013;Oteo et al., 2013b). ST14, ST147, and ST340 have sometimes been associated with bla NDM in K. pneumoniae in many countries (Peirano et al., 2011(Peirano et al., , 2014Poirel et al., 2011d;Giske et al., 2012;Osterblad et al., 2012;Lascols et al., 2013;Gharout-Sait et al., 2014;Lee et al., 2014;Shoma et al., 2014;Wang et al., 2014d;Izdebski et al., 2015;Sonnevend et al., 2015b). Over 50% of NDM-producing K. pneumoniae isolates from India belonged to either ST11 or ST147 (Lascols et al., 2013). The analysis of clinical isolates of NDM-1-producing K. pneumoniae from India, the UK, and Sweden, showed that the most frequently detected sequence types were ST14, ST11, ST149, ST231, and ST147 (Giske et al., 2012). Although ST258 is a highrisk KPC-producing K. pneumoniae clone, ST258 harboring bla NDM has never been reported, to the best of our knowledge. This phenomenon may result from the integrative conjugative element ICEKp258.2, present only in ST258 and genetically related sequence types . This genetic locus contains a type IV pilus gene cluster and a type III restrictionmodification system. Because these genes are associated with the restriction of plasmids and specific mobile elements, most ST258 may predominantly harbor plasmids with the bla KPC gene. Therefore, PCR for this unique region (ICEKp258.2) can provide a reliable tool for the rapid detection of the ST258 clone.
High-resolution genomic analysis of multidrug-resistant hospital outbreaks of K. pneumoniae through whole-genome sequencing revealed the emergence of a capsule switching NDM-1 bearing K. pneumoniae ST15 strain, suggesting that further studies should concentrate on the diversity and spread of this specific clone . ST15 harboring bla NDM−1 has often been reported in many countries, including Spain (Ruiz-Garbajosa et al., 2013), Croatia (Kocsis et al., 2016), Thailand (Netikul et al., 2014), Canada (Peirano et al., 2014), China (Hu et al., 2013), France (Arpin et al., 2012), and Morocco . In Bulgaria, this clone was responsible for the clonal dissemination of KPC-2-producing K. pneumoniae (Markovska et al., 2015). Another whole-genome sequencing analysis of CR K. pneumoniae strains, which were isolated from 26 individuals involved in infections in a Nepali neonatal unit, showed that three temporally separated cases were caused by a single NDM-producing K. pneumoniae strain with four conserved plasmids including a plasmid carrying bla NDM−1 (Stoesser et al., 2014). The plasmids contained a large number of antimicrobial resistance and plasmid maintenance genes, which may explain their persistence (Stoesser et al., 2014). These reports suggest that whole-genome sequencing analysis play an important role in the elucidation of the factors that allow emergence and persistence of resistance.

Treatment Options
New-Delhi metallo-β-lactamase-producing K. pneumoniae are usually resistant to most β-lactam antibiotics but remain susceptible to aztreonam . As with the case of KPC-producing K. pneumoniae, the effect of combination therapy was tested in the treatment of NDMproducing K. pneumoniae infections. When double-and tripleantibiotic combinations of aztreonam, ciprofloxacin, colistin, daptomycin, fosfomycin, meropenem, rifampin, telavancin, tigecycline, and vancomycin were used in patients infected with two NDM-producing K. pneumoniae strains susceptible to colistin, the combination of rifampin-meropenem-colistin was the most effective regimen against these strains (Tangden et al., 2014). The in vitro synergetic effect of the combination therapy of colistin and fosfomycin against NDM-producing K. pneumoniae has also been reported (Bercot et al., 2011). The combination of polymyxin B and chloramphenicol used against NDM-producing K. pneumoniae substantially enhanced bacterial killing and suppressed the emergence of polymyxin resistance (Abdul Rahim et al., 2015). Combination therapy including aztreonam and avibactam (a novel inhibitor of serine β-lactamases under development) was effective in the treatment of metallo-β-lactamase-producing bacterial infections (Wang et al., 2014e).
In the case of NDM-1-producing Enterobacteriaceae infections, carbapenems have been suggested to still represent a viable treatment option (Wiskirchen et al., 2014b). Despite unfavorable in vitro MICs of NDM-producing K. pneumoniae, recent in vivo studies have demonstrated the efficacy of carbapenems against NDM-1-producing isolates in immunocompetent-mouse and neutropenic-mouse thigh infection models (Wiskirchen et al., 2013(Wiskirchen et al., , 2014bMacVane et al., 2014). Although high-dose, prolonged infusions of ertapenem or doripenem induced reduction in bacterial density, bacterial density was also reduced in standard infusions of ertapenem at 1 g every 24 h or of doripenem at 500 mg every 8 h (Wiskirchen et al., 2013(Wiskirchen et al., , 2014b. Notably, these efficacies were observed only against NDM-1-producing K. pneumoniae (Wiskirchen et al., 2013(Wiskirchen et al., , 2014b. In addition to carbapenems, this discordance between in vitro and in vivo activities against NDM-1-producing K. pneumoniae was also observed in human simulated regimens of ceftazidime at 2 g every 8 h or ceftazidime/avibactam at 2,000/500 mg every 8 h (MacVane et al., 2014). Despite experiments in immunocompetent-mouse and neutropenic-mouse thigh infection models, these results show that standard infusions of ertapenem and doripenem could reduce bacterial density. Therefore, further experiments in human are required to determine whether carbapenems are sometimes a viable treatment option for NDM-1-producing K. pneumoniae infections.
The copy number of bla NDM−1 was assessed using Southern blotting and quantitative PCR under different conditions. The bla NDM−1 sequence was maintained under antibiotic selection; however, removal of the antibiotic selection led to the emergence of susceptible bacterial populations with a reduced copy number or even the complete loss of the bla NDM−1 gene (Huang et al., 2013). The dynamic nature of the copy number of bla NDM−1 provides a strong argument for the prudent use of clinically important antibiotics to reduce the development and dissemination of antibiotic resistance among pathogenic bacteria (Huang et al., 2013).

Detection Methods
Because NDM-producing K. pneumoniae infections are also associated with significant in-hospital mortality (de Jager et al., 2015), the rapid and accurate detection of NDM-producing K. pneumoniae is becoming a major issue in limiting the spread of CR bacteria. Several methods recently developed to detect NDM-producing K. pneumoniae include Xpert R Carba-R based on real time PCR , the Carba NP test based on the color change of a pH indicator , and its derivatives (Pires et al., 2013;Dortet et al., 2014b), and a method based on MALDI-TOF (Hrabak et al., 2013a). Xpert R Carba-R based on real time PCR effectively identified various carbapenemases of KPC, NDM, IMP, and VIM, with 100% sensitivity and 77% specificity . However, this method failed to detect OXA-48-like carbapenemases, in contrast to multiplex PCR .
The Carba NP test using chromogenic medium is based on the color change of a pH indicator . The enzymatic hydrolysis of the β-lactam ring of a carbapenem (usually imipenem) causes the acidification of an indicator solution (phenol red for the Carba NP test) that changes its color due to pH modification (Garcia-Fernandez et al., 2016). This method could rapidly detect KPC, IMP, VIM, NDM, and OXA-48-like producers with sensitivity and specificity of 97.9 and 100%, respectively, directly from spiked blood cultures (Dortet et al., 2014a). Comparative evaluation of the Carba NP test with other detection methods was tested in many reports (Vasoo et al., 2013;Huang et al., 2014;Yusuf et al., 2014;Gallagher et al., 2015;Lifshitz et al., 2016). When the Carba NP test and the modified Hodge test were compared, the Carba NP test was more specific (100% versus 80%) and faster (Vasoo et al., 2013;Huang et al., 2014;Yusuf et al., 2014). When it was compared the performance of the Carba NP test and the commercially available imipenem hydrolysisbased rapid test (the Rosco Rapid CARB screen kit) for detecting CPE and P. aeruginosa, the Carba NP test showed superior specificity and sensitivity (Huang et al., 2014;Yusuf et al., 2014;Gallagher et al., 2015). A novel simplified protocol of the Carba NP test designed for carbapenemase detection direct from bacterial cultures (instead of bacterial extracts) showed enhanced detection of carbapenemase producers (Pasteran et al., 2015).
However, several reports showed that false-negative results in the Carba NP test were associated with mucoid strains or linked to enzymes with low carbapenemase activity, particularly OXA-48-like Osterblad et al., 2014). To overcome these problems, several derivatives of the Carba NP test were developed, such as the Rapidec Carba NP test (bioMérieux; Poirel and Nordmann, 2015), the CarbAcineto NP test for rapid detection of carbapenemase-producing Acinetobacter spp. (Dortet et al., 2014b), the Rapid CARB Screen (Rosco Diagnostica; Dortet et al., 2015), the Blue-Carba test using bromothymol blue as a pH indicator solution (Garcia-Fernandez et al., 2016), a modified Carba NP test (Bakour et al., 2015a), and the BYG Carba test based on an electro-active polymer biosensing technology (Bogaerts et al., 2016). Many studies evaluated the performance of the Rapidec Carba NP test (bioMérieux), which was introduced into the market for the detection of carbapenemase production (Dortet et al., 2015;Hombach et al., 2015;Kabir et al., 2016;Lifshitz et al., 2016). These report showed that this method was user-friendly and had a high overall performance, making it an attractive option for clinical laboratories (Kabir et al., 2016). Recently, performance evaluation of two biochemical rapid tests commercialized (the Rapidec Carba NP test and the Rapid CARB Screen) was reported and compared with the home-made Carba NP test (Dortet et al., 2015). The Rapidec CARBA NP test possesses the best performance for rapid and efficient detection of CPE (Dortet et al., 2015). The BYG Carba test based on a new and original electrochemical method detects the variations of conductivity of a polyaniline (an electro-sensing polymer)coated electrode which is highly sensitive to the modifications of pH and of redox activity occurring during the imipenem enzymatic hydrolysis reaction (Bogaerts et al., 2016). In comparison with PCR results, the BYG Carba test displayed sensitivity of 95% and specificity of 100% versus 89% and 100%, respectively, for the Carba NP test (Bogaerts et al., 2016). The development of these detection methods based on inexpensive and affordable techniques can limit the spread of CR bacteria.
In France, examination of the epidemiologic features of an outbreak of OXA-48-producing K. pneumoniae in an ICU revealed that the outbreak was caused by environmental persistence of OXA-48-producing K. pneumoniae over 20 months (Pantel et al., 2016). This report emphasizes the importance of early environmental screening to interrupt the transmission of carbapenemase-producing K. pneumoniae (Pantel et al., 2016).
Similarly, a large outbreak of OXA-48 carbapenemase-producing K. pneumoniae in a French university hospital was recently attributed to the late implementation of successive cohort units and a high level of staff movement between the infectious diseases and internal medicine ward (Semin-Pelletier et al., 2015). These results suggest that practical guidelines are required to help hospitals confronting uncontrolled outbreaks. Because the gut of colonized patients is the main source of CPE, accurate and stringent hygiene of endoscopic instruments is also important. A recent report partially attributed an outbreak of OXA-48-producing K. pneumoniae in a German University hospital to complex instruments such as the duodenoscope (Kola et al., 2015). Strict hygiene regulations for various nosocomial environments, including endoscopic instruments as well as gowns and gloves, are required.

Molecular and Genetic Context
Unlike KPCs and NDMs, one highly transferable IncL group plasmid (pOXA-48a) was mainly responsible for the spread of the bla OXA−48 gene in K. pneumoniae ( Figure 2C; Pitout et al., 2015). The molecular epidemiology of OXA-48 in European and North African countries showed that in 92.5% of the isolates, the bla OXA−48 gene was located on this self-conjugative IncL/M type plasmid (Potron et al., 2013). The bla OXA−48 gene was found only in the IncL group of IncL/M type plasmids (Carattoli et al., 2015). In contrast to the IncA/C plasmids of NDM-1, the pOXA-48a plasmid contains bla OXA−48 , a unique antibiotic resistance gene (Pitout et al., 2015). The conjugation rate of the pOXA-48a plasmid was very high (1 × 10 −1 ); therefore, this selfconjugative plasmid can conjugate at a very high frequency to Enterobacteriaceae (Potron et al., 2014). Inactivation of the tir gene, which is known to encode a transfer inhibition protein, was recently reported to be responsible for a 50-to 100-fold increase in the efficiency of transfer of the pOXA-48a plasmid (Potron et al., 2014), explaining the spread of the pOXA-48a plasmid with bla OXA−48 . Recently, the bla OXA−48−like gene has also been reported on other plasmids and genetic elements, such as IncA/C types (Ma et al., 2015), IncH types (Wang et al., 2014c), and Tn1999 . In comparison to the pOXA-48a plasmid, the transmission frequency of the Tn1999 composite transposon was very low (<1.0 × 10 −7 ; Aubert et al., 2006).
OXA-48 has most often been found in K. pneumoniae, but OXA-48 was also found in various Enterobacteriaceae, because of the high conjugation rate of the pOXA-48a (Potron et al., 2014). Molecular and epidemiological analyses in a German hospital showed the horizontal gene transfer of the OXA-48containing plasmid from K. pneumoniae to E. coli (Gottig et al., 2015). Besides E. coli, OXA-48 has also been identified in K. oxytoca, Enterobacter spp., Providencia rettgeri, C. freundii, and S. marcescens (Poirel et al., 2012c;Berger et al., 2013). The bla OXA−48 gene was identified in all Enterobacteriaceae isolates from the index case in Spain, indicating the capacity of OXA-48 carbapenemase to spread among Enterobacteriaceae by the horizontal gene transfer (Arana et al., 2015).

Treatment Options
OXA-48-like-producing K. pneumoniae are usually resistant to most β-lactam antibiotics, but OXA-48-producing K. pneumoniae without ESBLs remain susceptible to the expanded-spectrum cephalosporins Munoz-Price et al., 2013). In addition, OXA-48-like-producing K. pneumoniae sometimes remains susceptible to several aminoglycosides such as gentamicin . Unlike for NDMs (Wiskirchen et al., 2013(Wiskirchen et al., , 2014bMacVane et al., 2014), carbapenems may not be a reliable treatment option for OXA-48 producer infection (Wiskirchen et al., 2014a). Combination therapy with sulbactam, meropenem, and colistin was more effective in isolates producing NDM carbapenemase than those producing OXA-48-like carbapenemases, suggesting that the identification of the carbapenemase type helps determine the combination most likely to clear the infection (Laishram et al., 2015). The combination of fosfomycin with imipenem, meropenem, and tigecycline was also synergistic against OXA 48positive K. pneumoniae strains in vitro with the ratios of 42, 33, and 33%, respectively (Evren et al., 2013). Similarly, the in vitro assays indicate that imipenem-containing combinations were effective against serine-β-lactamase producers (KPC, OXA-48), while no synergy was observed for all NDM-1 producers (Poirel et al., 2016). However, because carbapenems were effective against NDM-1-producing isolates in vivo (Wiskirchen et al., 2013(Wiskirchen et al., , 2014aMacVane et al., 2014), the effect of carbapenem combination therapy on carbapenemase-producing isolates should be determined in vivo, particularly in the case of NDM-1-producing bacteria.

Detection Methods
As with KPCs and NDMs, various detection methods were developed to identify OXA-48-like carbapenemases (Tsakris et al., 2010;Giske et al., 2011;Girlich et al., 2013;Naas 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 carbapenem resistance among Enterobacteriaceae . Many phenotypic detection methods to allow differentiation between class A and class B carbapenemases were developed using boronic acid derivatives and EDTA or dipicolinic acid (Tsakris et al., 2010;Giske et al., 2011). Recently, a specific phenotypic method to differentiate a single OXA-48 producer from those producing other carbapenemase types (e.g., KPC-types, NDM-types) was also developed (Tsakris et al., 2015). This method was based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid, respectively (Tsakris et al., 2015). This novel method exhibited 96.3% sensitivity and 97.7% specificity (Tsakris et al., 2015). ChromID OXA-48, based on chromogenic media, have been commercialized for the direct isolation of CPE from clinical samples (Girlich et al., 2013), with an estimated 91% sensitivity and 100% specificity (Girlich et al., 2013).

Emerging Class C Carbapenemases
Class C β-lactamases confer resistance to penicillins, cephalosporins, and cephamycins (cefoxitin and cefotetan) and are not significantly inhibited by clinically applied β-lactamase inhibitors such as clavulanic acid (Jeon et al., 2015). Although four class C carbapenemases (ACT-1, CMY-2, CMY-10, and ADC-68) have been reported, ACT-1 and CMY-2 exhibit reduced susceptibility to carbapenems, particularly ertapenem, only when combined with permeability defects, due to their low catalytic efficiencies (K cat /K m ) for imipenem (0.007 and 0.04 M −1 ·S −1 , respectively; Mammeri et al., 2010). CMY-10 with the catalytic efficiency of 0.14 M −1 ·S −1 for imipenem was the first reported carbapenemase among plasmidic class C β-lactamases (Kim et al., 2006), and this enzyme was also a class C extended-spectrum β-lactamase with extended substrate specificity for extended-spectrum cephalosporins (Lee et al., , 2012aJeon et al., 2015). Among the chromosomal class C β-lactamases, ADC-68 identified in A. baumannii was the first reported enzyme possessing both class C extended-spectrum β-lactamase and carbapenemase activities (Jeon et al., 2014(Jeon et al., , 2015, and its catalytic efficiency for imipenem is 0.17 M −1 S −1 . CMY-10-producing K. pneumoniae was identified only in South Korea (Lee et al., 2005a(Lee et al., , 2006a and ADC-68-producing K. pneumoniae was never reported. Many reports showed that carbapenem resistance can be triggered by the loss of two major porins, OmpK35 and OmpK36, in combination with ESBLs or Ambler class C AmpC cephalosporinases, and the production of carbapenemase (Wang et al., 2009;Shin et al., 2012;Tsai et al., 2013). A recently genetically engineered mutant of K. pneumoniae showed that several carbapenems (imipenem, meropenem, and doripenem) remain effective against these carbapenemase-independent CR strains (Tsai et al., 2013). Therefore, laboratory testing for susceptibility to imipenem, meropenem, and doripenem can improve the accuracy of identification of these isolates (Tsai et al., 2013).

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

AUTHOR CONTRIBUTIONS
C-RL, JL, and SL contributed to the conception and the design of the review and C-RL, JL, KP, YK, BJ, and SL researched and wrote the review.