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

1The increasing prevalence of antibiotic resistance and the lack of new antibiotic drug development has gradually reduced the treatment options for bacterial infections. In 2013, the Centers for Disease Control and Prevention (CDC) named three microorganisms that pose an urgent threat to public health: carbapenem-resistant (CR) Enterobacteriaceae (CRE), drug-resistant Neisseria gonorrhoeae, and Clostridium difficile. 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. 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. High mortality rates have been reported in patients with bloodstream infections caused by CR K. pneumoniae. 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; ). The class A carbapenemases, such as the K. pneumoniae carbapenemase (KPC) enzymes, have been identified worldwide in K. pneumoniae. 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.

2Various class A carbapenemases forming six distantly related branches have been identified. 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. Of the many different KPC family variants (KPC-1 to KPC-22), 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.

3The 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 (; 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. In the USA, the transmission of CR K. pneumoniae is primarily driven by the spread of organisms carrying KPC enzymes, but other carbapenemase enzymes, such as the New-Delhi metallo-β-lactamase (NDM), have also emerged. Within the USA, the prevalence of KPC-positive isolates was relatively stable between 2007 and 2009 (5.9% in 2007, 4.9% in 2008, and 5.7% in 2009; ), and KPC-2 and KPC-3 were the most frequently identified carbapenemases in K. pneumoniae.

4The outbreaks caused by KPC-producing K. pneumoniae have been reported in the USA and Israel, but recently, similar outbreaks associated with patients traveling to endemic areas have also been reported in many European counties. Since KPC-producing K. pneumoniae was identified in France, Italy, and Greece, the sporadic spread of KPC-producing K. pneumoniae has been observed in many European countries including Spain, France, Germany, the Netherlands, the UK, Ireland, Belgium, Sweden, and Finland. KPC-producing K. pneumoniae were also recently detected in eastern European countries including the Czech Republic, Hungary, and Croatia.

5In Greece, KPC-producing K. pneumoniae was first isolated in August 2007, and the prevalence of KPC-producers among carbapenemase-producing K. pneumoniae isolates collected at a tertiary Greek hospital increased from 0% in 2003 to 38.3% in 2010. Most of the genotyped KPC-producing K. pneumoniae in Greece harbored KPC-2. While many carbapenemase-producing K. pneumoniae in the USA and Greece had KPC enzymes, 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%;; ). These results indicate that the prevalent genotype of carbapenemase-producing 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%; ).

6In America, the endemic spread of KPCs has been reported in Colombia, Brazil, and Argentina. In Canada, KPC-producing K. pneumoniae has sporadically been reported, and since plasmid-mediated KPC-producing K. pneumoniae was first detected in Ottawa in, a laboratory surveillance program found a high frequency (89.3%) of KPC-type enzymes among carbapenemase producers between 2010 and 2012. The emergence of KPCs in Argentina was characterized by two patterns of dispersion: the first was the irregular occurrence of diverse Enterobacteriaceae harboring blaKPC-2 in the IncL/M transferable plasmid in distant regions and the second was the sudden clonal spread of K. pneumoniae ST258 harboring blaKPC-2 in Tn4401a. KPC-producing K. pneumoniae was recently also detected in Cuba, Mexico, Uruguay, and Puerto Rico.

7In the Asia-Pacific region, the endemic dissemination of KPC-producing K. pneumoniae has been reported in China and Taiwan, and the sporadic spread has been reported in India, South Korea, and Australia. A novel KPC-15 variant which is closely homologous with KPC-4 was discovered in China and its enzymatic activity and phenotype was characterized. In China, the frequency of KPC-type enzymes among carbapenemase producers was high (63%; ). While ST258 is the predominant clone observed in European countries and the USA, ST11, which is closely related to ST258, is the prevalent clone associated with the spread of KPC-producing K. pneumoniae in Asia (particularly in China and Taiwan;;; ). KPC-producing ST11 strain has also been reported in Latin America. 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. 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 blaKPC-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. KPC-producing K. pneumoniae was recently also detected in Japan, Pakistan, Iran, and United Arab Emirates. In the Arabian Peninsula, the prevalence of KPC-producing K. pneumoniae was very low in comparison to NDM-1 and OXA-48-like carbapenemases., 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, Algeria, and Egypt, have also isolated KPC-producing K. pneumoniae.

8The coexistence of KPCs and other carbapenemases in K. pneumoniae was frequently reported worldwide, including in Italy (KPC-3/VIM-2 and KPC-2/VIM-1;; ), Colombia (KPC-2/VIM-24; ), Brazil (KPC-2/NDM-1; ), China (KPC-2/NDM-1, KPC-2/CMY-2, and KPC-2/IMP-4;;; ), Canada (KPC-3/CMY-2; ), and Greece (KPC-2/VIM-1; ), indicating the worldwide prevalence of K. pneumoniae co-harboring two carbapenemases.

9Aside from KPC-type carbapenemases, other class A carbapenemases, such as GES-2, GES-4, GES-5, GES-6, GES-11, GES-14, GES-18, SFC-1, SHV-38, NMC-A, SME-1, and IMI-type enzymes, were rarely found in K. pneumoniae (Table 1).

10The blaKPC in K. pneumoniae has been reported on numerous plasmid types, such as IncF, IncI2, IncX, IncA/C, IncR, and ColE1, but the predominant plasmid type is IncF with FIIK replicons. IncF often contains several additional genes responsible for resistance to other antibiotics, including aminoglycosides, tetracyclines, quinolones, trimethoprim, and sulfonamides. Many blaKPC genes are associated with a promiscuous transposon-related structure Tn4401, which is approximately 10 kb in size and consists of a transposase gene, a resolvase gene, the blaKPC gene, and two insertion sequences, ISKpn6 and ISKpn7 (Figure 2A; ). This transposon has jumped to numerous plasmids that are commonly conjugative. In China, a novel genetic environment was detected. It contains an integration structure consisting of a Tn3-based transposon and partial Tn4401 segment, with the gene order Tn3-transposase, Tn3-resolvase, ISKpn8, the blaKPC-2 gene, and the ISKpn6-like element. This genetic structure is the chimera form of several transposon-associated elements. This transposon was also identified in many other countries, and several variants with various fragment insertions between the ISKpn8 and blaKPC gene have been found among Enterobacteriaceae in China. 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; ]. Notably, in many cases, different Tn4401 isoforms was associated with different blaKPC-harboring plasmids. Tn4401a was frequently found in the blaKPC-3-harboring IncFIIK2 plasmids, and Tn4401b and Tn4401d were often associated with the IncN and IncFIA plasmids, respectively. Up to now, more than 30 blaKPC-harboring plasmids obtained from K. pneumoniae have been sequenced. One of common features shared by these sequenced plasmids is the presence of the tra operon, which encodes the plasmid conjugation machinery proteins that induce the spread of plasmids. These genes may be important for the successful dissemination of blaKPC-harboring plasmids. The IncFII plasmids are one of predominant blaKPC-harboring plasmids. pKpQIL, which is an IncFIIK2 plasmid harboring Tn4401a, was initially identified in Israel in 2006, 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, suggesting the wide dissemination of this plasmid. The blaKPC gene has also been identified in other non-Tn4401 mobile elements that mostly have partial ISKpn6 genes. Based on the insertion sequence upstream of the blaKPC gene, they can be divided into three groups: group I, no insertion; group II, insertion of truncated blaTEM; group III, insertion of Tn5563/IS6100. These non-Tn4401 genetic elements harboring blaKPC sometimes have an IS26 transposon.

11Carbapenemase genes often spread worldwide through clonal expansion in several successful pathogenic strains. 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. This strain is a prototype of a high-risk clone of K. pneumoniae. Recent data from Israel showed that the KPC-producing K. pneumoniae ST258 clone remains the predominant clone, representing 80% of the KPC-producing K. pneumoniae population. ST258 may be a hybrid clone that was created by a large recombination event between ST11 and ST442. 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. 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. 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 high-risk clone that lacks ICEKp258.2, harboring a broad range of plasmids and carbapenemases, including KPC, NDM, OXA-48, VIM, and IMP. 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.

12The 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. Therefore, K. pneumoniae producing KPCs were detected in various nosocomial environments, such as gowns and gloves and wastewater. 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, 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. After discharge from the hospital, long-term (>3 years) carriage of KPC-producing K. pneumoniae is also possible, and lateral gene transfer of KPC among Enterobacteriaceae colonizing the human intestine appears frequent, for example from K. pneumoniae to E. coli. Therefore, reservoirs in healthcare workers, patients, or the hospital environment may be a principle mode of spread in nosocomial outbreaks.

13Carbapenemase-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. KPC-producing K. pneumoniae bloodstream infections in intensive care unit (ICU) have also been associated with increased mortality.

14Because 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. 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 colistin-resistant isolates. 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. However, several reports have shown that combination therapy was not superior to monotherapy. 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. The expression level of OmpK36 was also involved in the rapid induction of high-level carbapenem resistance in heteroresistant KPC-producing K. pneumoniae populations. 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.

15Colistin (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 KPC-producing K. pneumoniae infections. 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, the USA, Greece, Hungary, and Turkey. 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. The progressive increase of colistin resistance was also reported elsewhere. 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.

16Molecular and biochemical studies have shown that insertional inactivation of the mgrB gene, encoding a negative-feedback 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. 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. 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. The emergence of colistin resistance was also associated with low-dosage colistin treatment. 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. 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.

17Fosfomycin is a broad-spectrum antibiotic that inhibits bacterial cell wall biogenesis by inactivating the enzyme UDP-N-acetylglucosamine-3-enolpyruvyltransferase, also known as MurA. 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. 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 KPC-producing K. pneumoniae collected from 12 hospitals in China. 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. In China, the fosA3-harboring plasmid was attributed to 55.6% of fosfomycin-resistant KPC-producing K. pneumoniae strains. Although the fosA3 gene is mainly associated with the blaCTX-M gene, the fosA3 gene has also been characterized in atypical blaKPC-harboring plasmids. In pFOS18 and pKP1034, the fosA3 and blaKPC-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.

18Tigecycline, 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. Due to the increased clinical use of tigecycline for treatment of KPC-producing K. pneumoniae, increased tigecycline resistance was reported. In the Netherlands, all KPC-producing K. pneumoniae isolates exhibited reduced susceptibility to tigecycline. 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. In a Greek tertiary hospital during 2004 to 2010, 11.3% (34/301) of KPC-producing isolates were resistant to tigecycline. 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. A recent report in China showed that the OqxAB efflux pump was also contributed to tigecycline resistance in K. pneumoniae isolates.

19Because 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. New weapons are always indispensable for combating KPC-producing K. pneumoniae infections. 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. Combination therapy with avibactam and ceftazidime exhibited significant synergetic effects against organisms with combinations of extended-spectrum β-lactamases (ESBLs), AmpCs, and KPC-2. Plazomicin (a novel aminoglycoside) also exhibited significant activity against KPC-producing K. pneumoniae. The novel polymyxin derivatives with lower nephrotoxicity are under development. 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.

20Because 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, rapid detection of CR strains is essential for the effective management of these infections. Various methods for the identification of KPCs have been developed, including multiplex PCR assay, real-time PCR assay, DNA microarray, Raman spectroscopic analysis, single-colony whole-genome sequencing, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS; ), loop-mediated isothermal amplification (LAMP) method, chromogenic medium, and a new phenotypic test, called the carbapenem inactivation method (CIM; ). 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 or a disk test using boronic acid compounds. 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. 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. 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. 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. 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. However, the large-scaleblaFinder 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. 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. These methods are fast and accurate to routinely identify bacterial isolates with great specificity and sensitivity, but these systems do not accurately provide the carbapenem minimum inhibitory concentrations (MICs) for carbapenemase-producing K. pneumoniae. 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. The LAMP method has emerged as a powerful gene amplification assay for the rapid identification of microbial infections. 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. The cycling reaction continues with accumulation of 109 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. The LAMP assay can be applied for detection of KPC producers in the clinical laboratory and has greater sensitivity, specificity, and rapidity compared to the phenotypic methods and PCR for the detection of KPC-producing K. pneumoniae.

21Colistin 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. 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; capillary electrophoresis method according to characteristic surface properties of bacteria; and the micromax assay based on evaluation of the efficacy of antibiotics that affect cell wall integrity. 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, detection of this gene may be important in the detection of colistin resistance.

22Class 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. Class B carbapenemases were mostly identified in Enterobacteriaceae and include VIMs, IMPs, and the emerging NDM group. 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. Thus far, 15 NDM variants have been assigned, and most of them originated from Asia. NDMs shares very little identity with other metallo-β-lactamases.

23Since 2008, K. pneumoniae producing NDMs rapidly spread in many countries (; Figure 3). NDM-producing K. pneumoniae are considered to be endemic in the Indian subcontinent, including India, Pakistan, and Bangladesh. The sporadic spread has been reported in the USA, Canada, Colombia, Spain, France, Switzerland, Italy, the UK, Greece, Turkey, Morocco, South Africa, Singapore, Saudi Arabia, Oman, United Arab Emirates, Kuwait, China, Japan, Taiwan, South Korea, and Australia. In India, NDM-1 was the most common carbapenemase type detected and accounted for 75.22% of the carbapenemase-producing isolates. In Singapore and the United Arab Emirates, NDM-1 also was the most common carbapenemase type observed (44.4 and 100%, respectively;; ). The endemic spread of NDM-producing K. pneumoniae has also been reported in the UK, which has close relationships with India and Pakistan. In China, NDM-1 has been found mostly in Acinetobacter spp., but data obtained from patients between June 2011 and July 2012 showed that 33.3% of the CRE isolates, including K. pneumoniae, had NDM-1, suggesting the possible transmission of blaNDM-1-containing sequences from Acinetobacter spp. to Enterobacteriaceae. These findings reveal the emergence and active transmission of NDM-1-producing K. pneumoniae in China. Comparative analyses of the conserved NDM-1-encoding region among different plasmids from K. pneumoniae and E. coli suggested that the transposable elements and two unknown inverted repeat-associated elements flanking the NDM-1-encoding region aided the spreading of this resistance determinant. Recently, in China, eight K. pneumoniae isolates producing NDM-1 were identified in the neonatal ward of a teaching hospital, and four diverse types (NDM-1, KPC-2, VIM-2, and IMP-4) of carbapenemase of K. pneumoniae clones were identified in a single hospital in China.

24The Balkan states, the Arabian Peninsula, and North African countries, 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%; ). In Greece, among 132 non-repetitive CRE isolates between 2010 and 2013, 78 K. pneumoniae isolates with the blaNDM-1 gene were identified. In the USA, KPC-producing 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.

25The movement of patients between countries may be a trigger for the international spread of carbapenemase-producing K. pneumoniae. Carbapenemase-producing 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. 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, Brazil, the Netherlands, Ireland, Poland, Czech Republic, Croatia, Russia, Tunisia, Romania, Egypt, Kenya, Madagascar, Iraq, Yemen, Iran, Mauritius, Sri Lanka, Thailand, Nepal, Vietnam, Malaysia, and New Zealand.

26The 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. Colonization of NDM-producing K. pneumoniae isolates in pregnant women in the community in Madagascar highlighted the potential for mother-to-child transmission. In India, analysis of Enterobacteriaceae, including K. pneumoniae, isolated from the blood of septicaemic neonates, indicted that 14% of the isolates possessed blaNDM-1, and there was a significantly higher incidence of sepsis caused by NDM-1-harboring isolates. In China, an outbreak of blaNDM-1-producing K. pneumoniae ST20 and ST17 isolates was identified in a neonatal unit. In Turkey, the spread of NDM-1-producing 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.

27As with KPC, coexistence of NDMs and other carbapenemases in K. pneumoniae has also been reported worldwide, in Brazil (NDM-1/KPC-2; ), Malaysia (NDM-1/OXA-232; ), South Korea (NDM-5/OXA-181; ), China (NDM-1/IMP-4; ), India (NDM-1/OXA-232; ), Turkey (NDM-1/OXA-48; ), Pakistan (NDM-1/KPC-2; ), Switzerland (NDM-1/OXA-48; ), United Arab Emirates (NDM-1/OXA-48-like; ), Australia (NDM-1/OXA-48; ), Morocco (NDM-1/OXA-48; ), Singapore (NDM-1/OXA-181 and NDM-5/OXA-181; ), and the USA (NDM-1/OXA-232; ).

28Besides 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, 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 carbapenemase-producing 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; ).

29The blaNDM gene is frequently observed in the transposon Tn125 (with two flanking ISAba125 elements) within NDM-producing species of the genus Acinetobacter. The blaNDM gene was hypothesized to originate in the genus Acinetobacter. In Enterobacteriaceae, the ISAba125 elements of the Tn125 structure carrying blaNDM are frequently truncated (Tn125) at various lengths and the Tn125 structure frequently has different IS elements (Figure 2B;; ). The blaNDM genes in K. pneumoniae have been reported on numerous broad-host-range plasmid types, including IncA/C, IncF, IncR, IncH, IncN, IncL/M, and IncX types. The predominant plasmid type responsible for spreading blaNDM-1 is the IncA/C type plasmids. Many IncA/C plasmids with blaNDM-1 also carry various antibiotic resistance genes including 16S rRNA methylases (RmtA and RmtC), associated with aminoglycoside resistance; CMY-type β-lactamases, associated with broad-spectrum cephalosporin resistance; and QnrA, associated with quinolone resistance. 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. Because the blaNDM 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 blaNDM-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.

30Unlike KPC, the blaNDM 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 blaNDM-1 in K. pneumoniae identified in many countries, such as the USA, Greece, Australia, Switzerland, the Czech Republic, Spain, and Thailand. 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, 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. ST14, ST147, and ST340 have sometimes been associated with blaNDM in K. pneumoniae in many countries. Over 50% of NDM-producing K. pneumoniae isolates from India belonged to either ST11 or ST147. 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. Although ST258 is a high-risk KPC-producing K. pneumoniae clone, ST258 harboring blaNDM 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 restriction-modification system. Because these genes are associated with the restriction of plasmids and specific mobile elements, most ST258 may predominantly harbor plasmids with the blaKPC gene. Therefore, PCR for this unique region (ICEKp258.2) can provide a reliable tool for the rapid detection of the ST258 clone.

31High-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 blaNDM-1 has often been reported in many countries, including Spain, Croatia, Thailand, Canada, China, France, and Morocco. In Bulgaria, this clone was responsible for the clonal dissemination of KPC-2-producing K. pneumoniae. 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 blaNDM-1. The plasmids contained a large number of antimicrobial resistance and plasmid maintenance genes, which may explain their persistence. 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.

32New-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 NDM-producing K. pneumoniae infections. When double- and triple-antibiotic 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. The in vitro synergetic effect of the combination therapy of colistin and fosfomycin against NDM-producing K. pneumoniae has also been reported. The combination of polymyxin B and chloramphenicol used against NDM-producing K. pneumoniae substantially enhanced bacterial killing and suppressed the emergence of polymyxin resistance. 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.

33In the case of NDM-1-producing Enterobacteriaceae infections, carbapenems have been suggested to still represent a viable treatment option. 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. 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. Notably, these efficacies were observed only against NDM-1-producing K. pneumoniae. 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. 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.

34The copy number of blaNDM-1 was assessed using Southern blotting and quantitative PCR under different conditions. The blaNDM-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 blaNDM-1 gene. The dynamic nature of the copy number of blaNDM-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.

35Because NDM-producing K. pneumoniae infections are also associated with significant in-hospital mortality, 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® Carba-R based on real time PCR, the Carba NP test based on the color change of a pH indicator, and its derivatives, and a method based on MALDI-TOF. Xpert® 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.

36The 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. 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. Comparative evaluation of the Carba NP test with other detection methods was tested in many reports. When the Carba NP test and the modified Hodge test were compared, the Carba NP test was more specific (100% versus 80%) and faster. When it was compared the performance of the Carba NP test and the commercially available imipenem hydrolysis-based rapid test (the Rosco Rapid CARB screen kit) for detecting CPE and P. aeruginosa, the Carba NP test showed superior specificity and sensitivity. 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.

37However, 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. To overcome these problems, several derivatives of the Carba NP test were developed, such as the Rapidec Carba NP test (bioMérieux; ), the CarbAcineto NP test for rapid detection of carbapenemase-producing Acinetobacter spp., the Rapid CARB Screen (Rosco Diagnostica; ), the Blue-Carba test using bromothymol blue as a pH indicator solution, a modified Carba NP test, and the BYG Carba test based on an electro-active polymer biosensing technology. Many studies evaluated the performance of the Rapidec Carba NP test (bioMérieux), which was introduced into the market for the detection of carbapenemase production. These report showed that this method was user-friendly and had a high overall performance, making it an attractive option for clinical laboratories. 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. The Rapidec CARBA NP test possesses the best performance for rapid and efficient detection of CPE. 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. 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. The development of these detection methods based on inexpensive and affordable techniques can limit the spread of CR bacteria.

38Class D β-lactamases were referred to as oxacillinases (OXAs) because they commonly hydrolyze isoxazolylpenicillins (oxacillin, cloxacillin, and dicloxacillin) much faster than benzylpenicillin. Of over 400 Class D β-lactamases, only some variants actually possess carbapenemase activity. Based on their amino acid sequence, class D carbapenemases were recently reclassified into 12 subgroups: OXA-23, OXA-24/40, OXA-48, OXA-51, OXA-58, OXA-134a, OXA-143, OXA-211, OXA-213, OXA-214, OXA-229, and OXA-235. Among them, only several subgroups such as OXA-23, OXA-48, OXA-51, and OXA-58 are reported in K. pneumoniae. OXA-48 is the most efficient class D carbapenemase for imipenem and is one of the most prevalent class D carbapenemases. The OXA-48 was first identified in K. pneumoniae in Turkey in 2003, and thus far, 10 variants of the blaOXA-48 gene have been identified. Turkey may be one of the main reservoirs of OXA-48-producing K. pneumoniae. Since 2003, the endemic spread of these bacteria has been reported in countries such as Turkey, Morocco, Libya, Egypt, Tunisia, and India (; Figure 4). The sporadic spread has been reported in France, Spain, Italy, Belgium, the Netherlands, the UK, Germany, Switzerland, Argentina, Lebanon, Israel, Kuwait, Saudi Arabia, and Japan. The prevalence of OXA-48 carbapenemases among carbapenemase-producing K. pneumoniae in Spain and France was particularly high (74 and 78%, respectively;; ). In Africa, OXA-48-producing K. pneumoniae have been mainly reported in the northern countries, such as Morocco, Libya, Egypt, Tunisia, and Algeria (Figure 4). In the Arabian Peninsula, the prevalence of OXA-48-like carbapenemases among carbapenemase-producing K. pneumoniae was also high (32.5–56%;; ). Among CRE isolates in Lebanon, 88% produced OXA-48 carbapenemase. In Saudi Arabia, 78% of carbapenemase-producing K. pneumoniae isolates harbored blaOXA-48, and three strains of 47 blaOXA-48-positive K. pneumoniae isolates were resistant to colistin, suggesting that colistin resistance is emerging in Saudi Arabia.

39In 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. This report emphasizes the importance of early environmental screening to interrupt the transmission of carbapenemase-producing K. pneumoniae. 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. 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. Strict hygiene regulations for various nosocomial environments, including endoscopic instruments as well as gowns and gloves, are required.

40OXA-48-producing K. pneumoniae was recently also detected in Canada, the USA, Ireland, Poland, Hungary, Greece, Romania, Bulgaria, Finland, Russia, Algeria, United Arab Emirates, Iran, South Africa, Senegal, Taiwan, Singapore, South Korea, and Australia (; Figure 4). In North America, the frequency of OXA-48-like enzymes among carbapenemase-producing K. pneumoniae isolates was very low (11%; ). A recent report in Romania showed that among 65 carbapenemase-producing K. pneumoniae, the most frequently identified gene was the blaOXA-48 gene (78%), 12% were positive for blaNDM-1 gene, 6% had the blaKPC-2 gene. The recent spread of OXA-48 and OXA-244 carbapenemase genes in Russia was reported among Proteus mirabilis, E. aerogenes, and E. cloacae as well as K. pneumoniae. In South Africa, the emergence of a colistin-resistant OXA-181-producing K. pneumoniae isolate was also reported, and in the Netherlands, an OXA-48-producing K. pneumoniae was reported to infect two patients, and a hospital-wide outbreak was successfully controlled.

41OXA-181, a derivative of OXA48 with the substitution of a single amino acid, was first identified in India in, and then has been spread to many different countries, such as the UK, Romania, Canada, Oman, Singapore, Sri Lanka, South Korea, Australia, Japan, and New Zealand. However, in many cases, the infections have been associated with India. Other OXA-48-derivatives, such as OXA-204, OXA-232, and OXA-163, were recently identified in Tunisia, France, and Argentina, respectively, and OXA-244 and OXA-245 were first reported in Spain. All of these countries are regions with a high prevalence of OXA-48-producing K. pneumoniae (Figure 4). In addition, OXA-232-producing K. pneumoniae has been reported in various countries, such as the USA, Singapore, India, and South Korea. These results indicate that besides OXA-48, its many derivatives also spread worldwide. OXA-163, which differs from OXA-48 by a four amino acid deletion and a single amino acid substitution, has lower carbapenemase activity than OXA-48, but this enzyme exhibits extended activity against expanded-spectrum cephalosporins, and its activity is partially inhibited by clavulanic acid, a β-lactamase inhibitor. OXA-247, a derivative of OXA-163 with a single amino acid substitution, was recently reported in Argentina, where OXA-163-producing K. pneumoniae were often reported.

42The coexistence of OXA-48-like and other carbapenemases in K. pneumoniae was also frequently reported worldwide, such as in Turkey (OXA-48/NDM-1; ), Switzerland (OXA-48/NDM-1; ), United Arab Emirates (OXA-48-like/NDM-1; ), Australia (OXA-48/ NDM-1; ), Morocco (OXA-48/NDM-1; ), India (OXA-181/VIM-5; ), Singapore (OXA-181/NDM-1 and OXA-181/NDM-5; ), the USA (OXA-232/NDM-1; ), and India (OXA-232/NDM-1; ).

43Other class D carbapenemases, such as OXA-23, OXA-24/40, OXA-51, OXA-58, OXA-134a, OXA-143, OXA-211, OXA-213, OXA-214, OXA-229, and OXA-235, were mainly identified in Acinetobacter species such as A. baumannii but not in K. pneumoniae (Table 1; ).

44Unlike KPCs and NDMs, one highly transferable IncL group plasmid (pOXA-48a) was mainly responsible for the spread of the blaOXA-48 gene in K. pneumoniae (Figure 2C; ). The molecular epidemiology of OXA-48 in European and North African countries showed that in 92.5% of the isolates, the blaOXA-48 gene was located on this self-conjugative IncL/M type plasmid. The blaOXA-48 gene was found only in the IncL group of IncL/M type plasmids. In contrast to the IncA/C plasmids of NDM-1, the pOXA-48a plasmid contains blaOXA-48, a unique antibiotic resistance gene. The conjugation rate of the pOXA-48a plasmid was very high (1 × 10-1); therefore, this self-conjugative plasmid can conjugate at a very high frequency to Enterobacteriaceae. 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, explaining the spread of the pOXA-48a plasmid with blaOXA-48. Recently, the blaOXA-48-like gene has also been reported on other plasmids and genetic elements, such as IncA/C types, IncH types, and Tn1999. In comparison to the pOXA-48a plasmid, the transmission frequency of the Tn1999 composite transposon was very low (<1.0 × 10-7; ).

45OXA-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. Molecular and epidemiological analyses in a German hospital showed the horizontal gene transfer of the OXA-48-containing plasmid from K. pneumoniae to E. coli. Besides E. coli, OXA-48 has also been identified in K. oxytoca, Enterobacter spp., Providencia rettgeri, C. freundii, and S. marcescens. The blaOXA-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.

46Similar to NDM, the blaOXA-48-like genes were detected in various K. pneumoniae clones. ST11 has often been associated with blaOXA-48-like in K. pneumoniae isolated in many countries, such as Spain, Taiwan, Libya, Turkey, Argentina, and Greece. In Spain, a large outbreak was initiated in 2013 by a OXA-48-producing K. pneumoniae ST11 clone, and this strain was detected in 44 patients. The ST11 isolates carried various carbapenemases, including NDM-1, VIM-1, OXA-48, KPC-2, and OXA-245. In addition, ST14, ST15, ST101, ST147, and ST405 harboring blaOXA-48-like have often been reported in many countries, such as the USA, Spain, the Czech Republic, Libya, India, Germany, Finland, France, and Japan. Recent results from 83 hospitals in Spain showed that OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequently detected carbapenemases, and the most prevalent sequence types were ST11 and ST405 for K. pneumoniae. However, the molecular epidemiology of OXA-48-producing enterobacterial isolates collected from European and north-African countries between 2001 and 2011 indicated that ST101 was the most commonly observed sequence type in K. pneumoniae isolates, accounting for 17 out of 67 isolates (25.4%), followed by ST395 and ST15 (each seven isolates, 10.5%; ). Two outbreaks of OXA-48-producing K. pneumoniae ST101 clones were reported in Spain. OXA-48-producing K. pneumoniae appear to vary geographically. As with NDM, to the best of our knowledge, ST258 harboring blaOXA-48-like has never been reported.

47OXA-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. In addition, OXA-48-like-producing K. pneumoniae sometimes remains susceptible to several aminoglycosides such as gentamicin. Unlike for NDMs, carbapenems may not be a reliable treatment option for OXA-48 producer infection. 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. The combination of fosfomycin with imipenem, meropenem, and tigecycline was also synergistic against OXA 48-positive K. pneumoniae strains in vitro with the ratios of 42, 33, and 33%, respectively. 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. However, because carbapenems were effective against NDM-1-producing isolates in vivo, the effect of carbapenem combination therapy on carbapenemase-producing isolates should be determined in vivo, particularly in the case of NDM-1-producing bacteria.

48Despite the effectiveness of combination therapies, the prognosis for bloodstream infections caused by OXA-48-producing Enterobacteriaceae remains poor, and the 30-days mortality reached 50%. A similar result was reported in OXA-48-producing K. pneumoniae infections in a tertiary hospital in Spain. Although OXA-48-producing K. pneumoniae were susceptible to several antibiotics, including amikacin (97.2% susceptible), colistin (90.1%), tigecycline (73%), and fosfomycin (66.2%), in-hospital mortality among patients with OXA-48-producing K. pneumoniae infections was 43.5%. Therefore, to prevent delay in diagnosis and initiation of optimal antimicrobial therapy, rapid identification of OXA-48-producing isolates is required.

49As with KPCs and NDMs, various detection methods were developed to identify OXA-48-like carbapenemases. 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. 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. 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. This novel method exhibited 96.3% sensitivity and 97.7% specificity. ChromID OXA-48, based on chromogenic media, have been commercialized for the direct isolation of CPE from clinical samples, with an estimated 91% sensitivity and 100% specificity.

50Class 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. 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 (Kcat/Km) for imipenem (0.007 and 0.04 M-1⋅S-1, respectively; ). 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, and this enzyme was also a class C extended-spectrum β-lactamase with extended substrate specificity for extended-spectrum cephalosporins. 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, and its catalytic efficiency for imipenem is 0.17 M-1 S-1. CMY-10-producing K. pneumoniae was identified only in South Korea and ADC-68-producing K. pneumoniae was never reported.

51Many 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. A recently genetically engineered mutant of K. pneumoniae showed that several carbapenems (imipenem, meropenem, and doripenem) remain effective against these carbapenemase-independent CR strains. Therefore, laboratory testing for susceptibility to imipenem, meropenem, and doripenem can improve the accuracy of identification of these isolates.

52We analyzed the epidemiology of K. pneumoniae producing true carbapenemases (Ambler molecular class A, B, D, and several carbapenemases of class C) responsible for non-susceptibility 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. 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 KPC- and 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).

53ST258 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 FIIK replicons, a plasmid most commonly identified in ST258 with blaKPC, 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.

54The rapid global spread of NDM-type carbapenemases may be partly attributed to the dissemination of various epidemic broad-host-range plasmids bearing the blaNDM genes. NDM-type 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 blaNDM 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.

55The 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 blaOXA-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.

56ST11, 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. Well-designed 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.

57Most 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 carbapenemase-producing 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.

58The 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 KPC-type 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.

59C-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.

60The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.