“Stormy waters ahead”: global emergence of carbapenemases

Carbapenems, once considered the last line of defense against of serious infections with Enterobacteriaceae, are threatened with extinction. The increasing isolation of carbapenem-resistant Gram-negative pathogens is forcing practitioners to rely on uncertain alternatives. As little as 5 years ago, reports of carbapenem resistance in Enterobacteriaceae, common causes of both community and healthcare-associated infections, were sporadic and primarily limited to case reports, tertiary care centers, intensive care units, and outbreak settings. Carbapenem resistance mediated by β-lactamases, or carbapenemases, has become widespread and with the paucity of reliable antimicrobials available or in development, international focus has shifted to early detection and infection control. However, as reports of Klebsiella pneumoniae carbapenemases, New Delhi metallo-β-lactamase-1, and more recently OXA-48 (oxacillinase-48) become more common and with the conveniences of travel, the assumption that infections with highly resistant Gram-negative pathogens are limited to the infirmed and the heavily antibiotic and healthcare exposed are quickly being dispelled. Herein, we provide a status report describing the increasing challenges clinicians are facing and forecast the “stormy waters” ahead.

The bla KPC gene has been mapped to a highly conserved Tn3-based transposon, Tn4401 (Figure 1A), and five isoforms of Tn4401 are described (Naas et al., 2008;Cuzon et al., 2010;Kitchel et al., 2010). Plasmids carrying bla KPC are of various sizes and many carry additional genes conferring resistance to fluoroquinolones and aminoglycosides thus limiting the antibiotics available to treat infections with KPC-producing pathogens Rice et al., 2008). bla KPC has rarely been mapped to a chromosomal location (Villegas et al., 2007;Castanheira et al., 2009).
A predominant strain of K. pneumoniae appears responsible for outbreaks and the international spread of KPC-producing K. pneumoniae Kitchel et al., 2009a;Samuelsen et al., 2009). Congruent pulsed-field gel electrophoresis (PFGE) patterns also suggest a clonal relationship between outbreakassociated strains of KPC-producing K. pneumoniae recovered from different areas that are endemic (Navon-Venezia et al., 2009;Woodford et al., 2011). The Centers for Disease Control and Prevention (CDC) performed PFGE and multilocus sequence typing (MLST) on isolates submitted to their reference laboratory from 1996 to 2008. A dominant PFGE pattern was observed and noted to be of a specific MLST type, ST 258 (Kitchel et al., 2009a). A second sequence type, ST 14, was common in institutions in the Midwest (Kitchel et al., 2009b). These findings implied that certain strains of K. pneumoniae may be more apt to obtain and retain the bla KPC gene. Another study, however, analyzing 16 KPC-2 producing K. pneumoniae isolates from different geographic regions demonstrated diverse PFGE patterns and MLST types. This included four FIGURE 1 | Basic genetic construct of select carbapenemase genes. (A) Schematic representation of Tn4401 type of transposon associated with bla KPC which includes a transposase gene (tnpA), a resolvase gene (tnpR), as well as insertion sequences, ISKpn6 and ISKpn7 . (B) The bla NDM−1 construct demonstrates ISAba125 insertion sequence(s) upstream of the bla NDM−1 and a novel bleomycin resistance gene, ble MBL , downstream (Dortet et al., 2012). (C) bla OXA−48 is often mapped to a Tn1999 composite transposon where it is bracketed between two copies of the same insertion sequence, IS1999. Downstream of bla OXA−48 lies a lysR gene which encodes for a regulatory protein (Poirel et al., 2012b). www.frontiersin.org different MLST types in Colombia (ST 14,ST 337,ST 338,and ST 339) and two in Israel (ST 227 and ST 340). Although this study analyzed a smaller number of isolates, these findings suggest that the global propagation of KPC-2 is more complicated than the successful expansion of a fixed number of clones Qi et al., 2011). More recently, a study evaluating the MLST types associated with widespread KPC-2 production in K. pneumoniae in Greece suggested that although ST 258 predominates at least 10 additional sequence types were found to carry bla KPC−2 . Of note three (i.e., ST 147,ST 323,and ST 383) carried both KPC-2 as well as genes encoding VIM-type MBLs (Giakkoupi et al., 2011;Woodford et al., 2011). A retrospective study in Cleveland documented the presence of ST 36 in a long-term care facility for children (Viau et al., 2012).
Klebsiella pneumoniae carbapenemases-production can confer variable levels of carbapenem resistance with reported minimum inhibitory concentrations (MICs) ranging from susceptible to ≥16 μg/mL. Analysis of isolates displaying high-level carbapenem resistance demonstrated that increased phenotypic resistance may be due to increased bla KPC gene copy number or the loss of an outer membrane porin, OmpK35 and/or OmpK36. The highest level of imipenem resistance was seen with isolates lacking both porins and with augmented KPC enzyme production (Kitchel et al., 2010).

AMBLER CLASS B CARBAPENEMASES: METALLO-β-LACTAMASES
Class B β-lactamases ( Table 2) are referred to as MBLs and require a metal ion, usually zinc, for β-lactam hydrolysis (Walsh et al., 2005). Due to the dependence on Zn 2+ , catalysis is inhibited in the presence of metal-chelating agents like ethylenediaminetetraacetic acid (EDTA). MBL expression in Gram-negative bacteria confers resistance to penicillins, cephalosporins, and carbapenems. MBLs are not inhibited by the presence of commercially available β-lactamase inhibitors and susceptibility to monobactams (e.g., aztreonam) appears to be preserved in the absence of concomitant expression of other resistance mechanisms (e.g., ESBL production). The more geographically widespread MBLs include IMP (imipenem-resistant), VIM, and New Delhi metallo-β-lacta mase (NDM).
Chromosomal MBLs were the first to be identified and are the cause of carbapenem resistance observed in Bacillus cereus, Aeromonas spp., and Stenotrophomonas maltophilia (Walsh et al., 2005). However, of growing concern are the "mobile" MBLs that have been reported since the mid-1990s. Although most frequently found in carbapenem-resistant isolates of P. aeruginosa and occasionally Acinetobacter spp., there is growing isolation of these enzymes in Enterobacteriaceae.
Prior to the description of NDM-1, frequently detected MBLs include IMP-type and VIM-type with VIM-2 being the most prevalent. These MBLs are embedded within a variety of genetic structures, most commonly integrons. When these integrons are associated with transposons or plasmids they can readily be transferred between species.
In 1991, IMP-1, a plasmid-mediated MBL, was identified in an isolates of S. marcescens from Japan (Ito et al., 1995). Since then plasmid-mediated carbapenem resistance secondary to IMP-1 spread widely in Japan, Europe, Brazil, and other parts of Asia and in several species of Gram-negative bacilli including Acinetobacter spp. and Enterobacteriaceae. At the present time, 42 variants of IMP have been identified with most cases of IMP-mediated carbapenem resistance being reported from Asia and among P. aeruginosa (Bush and Jacoby, 2010).  (Salabi et al., 2010).

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy
A more commonly recovered MBL is the VIM-type enzyme. VIM-1 was first described in Italy in 1997 in P. aeruginosa (Lauretti et al., 1999). VIM-2 was next discovered in southern France in P. aeruginosa cultured from a neutropenic patient in 1996 (Poirel et al., 2000b). Although originally thought to be limited to non-fermenting Gram-negative bacilli, VIM-type MBLs are being increasingly identified in Enterobacteriaceae as well (Giakkoupi et al., 2003;Kassis-Chikhani et al., 2006;Morfin-Otero et al., 2009;Canton et al., 2012). To date, 37 variants of VIM have been described with VIM-2 being the most common MBL recovered worldwide.
NDM-1 was first identified in 2008. Due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens, NDM is poised to become the most commonly isolated and distributed carbapenemase worldwide. Initial reports frequently demonstrated an epidemiologic link to the Indian subcontinent where these MBLs are endemic (Kumarasamy et al., 2010). Indeed, retrospective analyses of stored isolates suggest that NDM-1 may have been circulating in the subcontinent as early as 2006 (Castanheira et al., 2011). Despite initial controversy, the Balkans may be another area of endemicity for NDM-1 (Struelens et al., 2010;Jovcic et al., 2011;Livermore et al., 2011c;Halaby et al., 2012). Sporadic recovery of NDM-1 in the Middle East suggests that this region may be an additional reservoir (Poirel et al., 2010a(Poirel et al., , 2011dGhazawi et al., 2012).
Like KPCs, the conveniences of international travel and medical tourism have quickly propelled this relatively novel MBL into a formidable public health threat. Gram-negative bacilli harboring bla NDM have been identified worldwide with the exception of Central and South America.
NDM-1 was first identified in Sweden in a patient of Indian descent previously hospitalized in India (Yong et al., 2009). The patient was colonized with a K. pneumoniae and an E. coli carrying bla NDM−1 on transferable plasmids. In the UK, an increase in the number of clinical isolates of carbapenem-resistant Enterobacteriaceae was seen in both 2008 and 2009. A UK reference laboratory reported that at least 17 of 29 patients found to be harboring NDM-1 expressing Enterobacteriaceae had a history of recent travel to the Indian subcontinent with the majority having been hospitalized in those countries (Kumarasamy et al., 2010).
European reports suggest that horizontal transfer of bla NDM−1 exists within hospitals outside endemic areas. Of overwhelming concern are the reported cases without specific contact with the healthcare system locally or in endemic areas suggesting autochthonous acquisition (Kumarasamy et al., 2010;Kus et al., 2011;Arpin et al., 2012;Borgia et al., 2012;Nordmann et al., 2012b).
Surveillance of public water supplies in India indicates that exposure to NDM-1 may be environmental. Walsh et al. (2011) analyzed samples of public tap water and seepage water from sites around New Delhi. The results were disheartening in that bla NDM−1 was detected by PCR in 4% of drinking water samples and 30% of seepage samples. In this survey, carriage of bla NDM−1 was noted in 11 species of bacteria not previously described, including virulent ones like Shigella boydii and Vibrio cholerae.
The rapid spread of NDM-1 highlights the fluidity and rapidity of gene transfer between bacterial species. Although bla NDM−1 was initially and repeatedly mapped to plasmids isolated from carbapenem-resistant E. coli and K. pneumoniae, reports of both plasmid and chromosomal expression of bla NDM−1 has been noted in other species of Enterobacteriaceae as well as Acinetobacter spp. and P. aeruginosa (Moubareck et al., 2009;Bogaerts et al., 2010;Bonnin et al., 2011;Patel and Bonomo, 2011). Recently, bacteremia with a NDM-1 expressing V. cholerae has been described in a patient previously hospitalized in India colonized with a variety of Enterobacteriaceae previously known to be capable of carrying plasmids with bla NDM−1 (Darley et al., 2012).
In contrast to KPCs, the presence of a dominant clone among bla NDM−1 carrying isolates remains elusive (Poirel et al., 2011c). NDM-1 expression in E. coli has been noted among sequence types previously associated with the successful dissemination of other β-lactamases including ST 101 and ST 131 (Mushtaq et al., 2011). Mushtaq et al. (2011) analyzed a relatively large group of bla NDM−1 expressing E. coli from the UK, Pakistan, and India in order to potentially identify a predominant strain responsible for the rapid and successful spread of NDM-1. The most frequent sequence type identified was ST 101. Another study examining a collection of carbapenem-resistant Enterobacteriaceae from India demonstrates the diversity of strains capable of harboring bla NDM−1 . Carriage of bla NDM−1 was confirmed in 10 different sequence types of K. pneumoniae and 5 sequence types of E. coli (Lascols et al., 2011). This multiplicity was confirmed in a study looking at a collection of bla NDM−1 expressing Enterobacteriaceae from around the world (Poirel et al., 2011c). Of most concern is that NDM-1 has been identified in E. coli ST 131, the strain of E. coli credited with the global propagation of CTX-M-15 ESBLs (Mushtaq et al., 2011;Peirano et al., 2011;Pfeifer et al., 2011b;Woodford et al., 2011). Similar to KPCs, NDM-1 expression portends variable levels of carbapenem resistance and there is often concomitant carriage of a myriad of resistance determinants including other βlactamases and carbapenemases as well as genes associated with resistance to fluoroquinolones and aminoglycosides  www.frontiersin.org NDM-1 shares the most homology with VIM-1 and VIM-2. It is a 28-kDa monomeric protein that demonstrates tight binding to both penicillins and cephalosporins (Zhang and Hao, 2011). Binding to carbapenems does not appear to be as strong as other MBLs, but hydrolysis rates appear to be similar. Using ampicillin as a substrate, allowed for detailed characterization of the interactions between NDM's active site and β-lactams as well as improved evaluation of MBLs unique mechanism of β-lactam hydrolysis. More recent crystal structures of NDM-1 reveal the molecular details of how carbapenem antibiotics are recognized by dizinc-containing MBLs (King et al., 2012).
To date, NDM-1 remains the most common NDM variant isolated. Seven variants (NDM-1 to NDM-7) exist (Kaase et al., 2011;Nordmann et al., 2012a). It is currently held that bla NDM−1 is a chimeric gene that may have evolved from A. baumannii . Contributing to this theory is the presence of complete or variations of the insertion sequence, ISAba125, upstream to the bla NDM−1 gene in both Enterobacteriaceae and A. baumannii (Pfeifer et al., 2011a;Poirel et al., 2011a;Dortet et al., 2012;Toleman et al., 2012). This insertion sequence has primarily been found in A. baumannii.
A recent evaluation of the genetic construct associated with bla NDM−1 ( Figure 1B) has lead to the discovery of a new bleomycin resistance protein, BRP MBL . Evaluation of 23 isolates of bla NDM−1/2 harboring Enterobacteriaceae and A. baumannii noted that the overwhelming majority of them possessed a novel bleomycin resistance gene, ble MBL (Dortet et al., 2012). Co-expression of bla NDM−1 and ble MBL appear to be mediated by a common promoter (P NDM−1 ) which includes portions of ISAba125. It is postulated that BRP MBL expression may contribute some sort of selective advantage allowing NDM-1 to persist in the environment.
A contemporary evaluation of recently recovered NDM-1 producing A. baumannii isolates from Europe demonstrates that bla NDM−1 and bla NDM−2 genes are situated on the same chromosomally located transposon, Tn125 . Dissemination of bla NDM in A. baumannii seems be due to different strains carrying Tn125 or derivatives of Tn125 rather than plasmid-mediated or clonal (Bonnin et al., 2013;Poirel et al., 2012a).

CARBAPENEM-HYDROLYZING CLASS D β-LACTAMASES
Oxacillinases comprise a heterogeneous group of class D βlactamases which are able to hydrolyze amino-and carboxypenicillins (Poirel et al., 2010b). The majority of class D β-lactamases are not inhibited by commercially available β-lactamase inhibitors but are inhibited in vitro by NaCl. Over 250 types of oxacillinases are reported with a minority demonstrating low levels of carbapenem-hydrolyzing activity. This select group of enzymes is also referred to as the carbapenem-hydrolyzing class D βlactamases (CHDLs ; Table 3). CHDLs have been identified most frequently in Acinetobacter spp., however, there has been increasing isolation among Enterobacteriaceae, specifically OXA-48 (oxacillinase-48; Lascols et al., 2012;Mathers et al., 2012).
CHDLs can be intrinsic or acquired. A. baumannii does have naturally occurring but variably expressed chromosomal CHDLs, OXA-51, OXA-66, and OXA-69 (Brown et al., 2005;Héritier et al., 2005b). For the most part, in isolation the phenotypic carbapenem resistance associated with these oxacillinases is low. However, levels of carbapenem resistance appear to be increased in the presence of specific insertion sequences promoting gene expression (Figueiredo et al., 2009;Culebras et al., 2010). Additional resistance to extended-spectrum cephalosporins can be seen in the setting of co-expression of ESBLs and/or other carbapenemases (Castanheira et al., 2011;Mathers et al., 2012;Pfeifer et al., 2012;Voulgari et al., 2012;Potron et al., 2013).
The first reported "acquired" oxacillinase with appreciable carbapenem-hydrolyzing activity was OXA-23. OXA-23, or ARI-1, was identified from an A. baumannii isolate in Scotland in 1993 (the isolate was first recovered in 1985; Paton et al., 1993). Subsequently, OXA-23 expression has been reported worldwide (Mugnier et al., 2010) and both plasmid and chromosomal carriage of bla OXA−23 are described. The OXA-23 group includes OXA-27, found in a single A. baumannii isolate from Singapore (Afzal-Shah et al., 2001). With the exception of an isolate of Proteus mirabilis identified in France in 2002, this group of β-lactamases has been exclusively recovered from Acinetobacter species (Bonnet et al., 2002). Increased expression of OXA-23 has been associated with the presence of upstream insertion sequences (e.g., ISAba1 and ISAba4) acting as strong promoters (Corvec et al., 2007).
As civilian and military personnel began returning from Afghanistan and the Middle East, practitioners noted increasing Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy recovery of A. baumannii from skin and soft tissue infections. Drug resistance was associated with expression of both OXA-23 and OXA-58 (Hujer et al., 2006;Scott et al., 2007;Perez et al., 2010b). Many isolates carrying the bla OXA−58 gene concurrently carry insertion sequences (e.g., ISaba1, ISAba2, or ISAba3) associated with increased carbapenemase production and thus higher levels of carbapenem resistance. In one report increased gene copy number was also associated with a higher level of enzyme production and increased phenotypic carbapenem resistance (Bertini et al., 2007). Spread of OXA-type carbapenemases among A. baumannii appears to be clonal and in depth reviews of the molecular epidemiology and successful dissemination of these clones have been published (Woodford et al., 2011;Zarrilli et al., 2013). Two MLST schemes with three loci in common exist for A. baumannii -the PubMLST scheme (Bartual et al., 2005) and the Pasteur scheme (Diancourt et al., 2010). Both schemes assign different sequence types into clonal complexes (CC). Sequence types and CC from both schemes can be further categorized into the international (European) clones I, II, and III. It should be noted, however, that the molecular taxonomy of A. baumannii continues to evolve (Higgins et al., 2012a). OXA-23 producing A. baumannii predominantly belong to international clones I and II with a notable proportion being part of CC92 ( OXA-48 was originally identified in a carbapenem-resistant isolate of K. pneumoniae in Turkey (Poirel et al., 2004c). Early reports suggested that this enzyme was geographically restricted to Turkey. In the past few years, however, the enzyme has been recovered from variety of Enterobacteriaceae and has successfully circulated outside of Turkey with reports of isolation in the Middle East, North Africa, Europe (Carrer et al., 2010), and most recently the USA (Lascols et al., 2012;Mathers et al., 2012). The Middle East and North Africa may be secondary reservoirs for these CHDLs (Hays et al., 2012;Poirel et al., 2012c). Indeed, the introduction of OXA-48 expressing Enterobacteriaceae in some countries has been from patients from the Middle East or Northern Africa (Decre et al., 2010;Adler et al., 2011;Poirel et al., 2011e;Canton et al., 2012). In the USA, the first clinical cases were associated with ST 199 and ST 43 (Mathers et al., 2012).
Although much of the spread of OXA-48 is attributed to a specific plasmid, outbreak evaluations demonstrate that a variety of strains have contributed to dissemination of this emerging carbapenemase in K. pneumoniae. The same K. pneumoniae sequence type, ST 395, harboring bla OXA−48 was identified in Morocco, France, and the Netherlands (Cuzon et al., 2011;Potron et al., 2011a). ST 353 was associated with an outbreak of OXA-48 producing K. pneumoniae in London (Woodford et al., 2011) and ST 221 with an outbreak of OXA-48 in Ireland (Canton et al., 2012). OXA-48 production in K. pneumoniae, like KPC-expressing K. pneumoniae, has also been associated with ST 14 (Poirel et al., 2004c) and a recent outbreak in Greece was associated with ST 11 (Voulgari et al., 2012).
OXA-163, a single amino acid variant of OXA-48, was identified in isolates of K. pneumoniae and Enterobacter cloacae from Argentina and is unique in that it has activity against extendedspectrum cephalosporins . OXA-163 also has been identified in Egypt, which has a relatively prevalence of OXA-48, in patients without epidemiologic links to Argentina (Abdelaziz et al., 2012).
OXA-181 was initially identified among carbapenem-resistant Enterobacteriaceae collected from India (Castanheira et al., 2011). OXA-181 differs from OXA-48 by four amino acids, however, appears to be nestled in an entirely different genetic platform. The bla OXA−181 gene has been mapped to a different group of plasmids, the ColE family, and has been associated with an alternative insertion sequence, ISEcp1. The latter insertion sequence has been associated with the acquisition of other β-lactamases including CTX-M-like ESBLs. Like, OXA-48, it appears that OXA-181 may have evolved from a waterborne environmental species Shewanella xiamenensis (Potron et al., 2011b).
OXA-204 differs from OXA-48 by a two amino acid substitution. It was recently identified in a clinical K. pneumoniae isolate from Tunisia (Potron et al., 2013). Its genetic construct appears to be similar to that of OXA-181. OXA-232 was recently identified among K. pneumoniae isolates in France (Poirel et al., 2012c).

AVAILABLE AGENTS AND DRUGS IN DEVELOPMENT
Few antimicrobials are currently available to treat infections with carbapenemase-producing Gram-negative bacteria. Carriage of concurrent resistance determinants can result in decreased susceptibility non-β-lactams including the fluoroquinolones and aminoglycosides thus further compromising an already limited antimicrobial arsenal. What frequently remains available are the polymyxins (including colistin), tigecycline, and fosfomycin but susceptibilities to these agents are unpredictable (Falagas et al., 2011).
The reintroduction of polymyxins, both polymyxin B and colistin overlaps with the evolution of carbapenem resistance among Gram-negative bacilli. The clinical "resurgence" of these agents is well documented (Falagas and Kasiakou, 2005;Li et al., 2006a;Landman et al., 2008). Some experts advocate for the use of polymyxins in combination with other agents like rifampicin (Hirsch and Tam, 2010;Urban et al., 2010). In vitro evaluations of different combinations including carbapenems, rifamycins, and/or tigecycline demonstrate variable results (Bercot et al., 2011;Biswas et al., 2012;Deris et al., 2012;Jernigan et al., 2012). Most evaluations of the clinical outcomes or "effectiveness" of combination therapies have been retrospective (Qureshi et al., 2012;Tumbarello et al., 2012). Prospective clinical trials evaluating the superiority of colistin-based combination therapy over monotherapy are in their infancy. A real interest in combination therapy persists due to the concern of hetero-resistance (Li et al., 2006b;Poudyal et al., 2008;Lee et al., 2009;Yau et al., 2009;Meletis et al., 2011).
Early evaluations of the glycylcycline, tigecycline, demonstrated favorable in vitro activity against ESBL-producing Enterobacteriaceae and specific isolates of carbapenem-resistant A. baumannii and Enterobacteriaceae (Bratu et al., 2005;Fritsche et al., 2005;Noskin, 2005;Castanheira et al., 2008;Wang and Dowzicky, 2010). Tigecycline remains untested in prospective trials and reports of resistance are increasing (Navon-Venezia et al., 2007;Anthony et al., 2008;Wang and Dowzicky, 2010;Sun et al., 2012). The role of tigecycline in treating primary bloodstream infections or urinary tract infections remains undefined due less than therapeutic concentrations of drug achieved in the serum (Rodvold et al., 2006) and urine (Satlin et al., 2011). We also note that meta-analyses of pooled data from trials evaluating the use of tigecycline for a variety of indications suggest there is a excess mortality associated with the use of tigecycline over comparator regimens (Cai et al., 2011;Tasina et al., 2011;Yahav et al., 2011;Verde and Curcio, 2012). However, in the absence of other tested regimens tigecycline may be an appropriate or perhaps the only therapeutic option.
Growing resistance to both the polymyxins and tigecycline has resulted the revisiting of older drugs including chloramphenicol, nitrofurantoin, and temocillin (Livermore et al., 2011d). Fosfomycin is also one of these earlier antibiotics being reassessed (Falagas et al., 2008). In an in vitro evaluation of 68 KPCexpressing K. pneumoniae isolates, fosfomycin demonstrated in vitro activity against 87% of tigecycline and/or polymyxin nonsusceptible isolates and 83% of isolates that were resistant to both (Endimiani et al., 2010b). Fosfomycin may be a potential therapeutic option for patients infected with carbapenemase-producing Enterobacteriaceae if the infection is localized to the genitourinary tract. Unfortunately, fosfomycin does not demonstrate reliable activity against non-urinary pathogens. Fosfomycin demonstrated activity against only 30.2% of 1693 multidrug-resistant (MDR) P. aeruginosa isolates and 3.5% of 85 MDR A. baumannii isolates (Falagas et al., 2009). The individual studies included in this review did not employ uniform MDR definitions or consistent susceptibility breakpoints. Moreover, access to the parenteral fosfomycin is limited and the threshold for resistance is low (Rodriguez-Rojas et al., 2010;Karageorgopoulos et al., 2012). Concerns regarding the emergence of resistance have lead to an increasing interest in the utility of combination therapy (Michalopoulos et al., 2010;Bercot et al., 2011;Souli et al., 2011).
Few agents are in the advanced stages of development with demonstrable in vitro activity against carbapenemase-producing organisms. These include β-lactamase inhibitors, aminoglycoside derivatives, polymyxin derivatives, and novel monobactams and monobactams-β-lactamase inhibitor combinations.
Avibactam, or NXL104, is a β-lactamase inhibitor which has been tested in combination with ceftazidime, ceftaroline, and aztreonam against several carbapenemase-producing Enterobacteriaceae with impressive decreases in MICs (Livermore et al., 2008(Livermore et al., , 2011bEndimiani et al., 2009a;Mushtaq et al., 2010c). Cephalosporin-avibactam combinations do not inhibit MBLs. Avibactam in combination with aztreonam, however, does seem to demonstrate activity against isolates harboring a variety of carbapenem resistance mechanisms including MBLs (Livermore et al., 2011b). Regrettably, the avibactam and aztreonam combination is not currently in clinical trials. The combination of ceftazidime-avibactam has been evaluated against collections of non-fermenting Gram-negative pathogens and its role remains undefined (Mushtaq et al., 2010b). In some evaluations of ceftazidime non-susceptible isolates of P. aeruginosa decrease MICs were noted with the addition of avibactam (Mushtaq et al., 2010b;Walkty et al., 2011;Crandon et al., 2012;Levasseur et al., 2012). The combinations of ceftarolineavibactam and ceftazidime-avibactam are currently in clinical trials.
Methylidene penems (penem-1 and penem-2) are β-lactamase inhibitors and appear to be potent inhibitors of KPC-2 (Papp-Wallace et al., 2010). The combination of cefepime with penem-1 demonstrated lower cefepime MICs in 88.1% of the 42 KPCproducing K. pneumoniae isolates evaluated . MK-7655 is a novel β-lactamase being evaluated in combination with imipenem against carbapenem-resistant Gramnegative bacilli (Hirsch et al., 2012). ME1071, formerly CP3242 (Bassetti et al., 2011, is a maleic acid derivative that competitively inhibits MBLs. Earlier studies demonstrated concentration-dependent decreases in carbapenem MICs in MBL-producing P. aeruginosa (Ishii et al., 2010), A. baumannii, and select Enterobacteriaceae (Shahid et al., 2009) A contemporary pre-clinical evaluation of ME1071 in combination with various type 2 carbapenems (i.e., biapenem, doripenem, meropenem, imipenem) confirms remarkable decreases in the carbapenem MICs for Enterobacteriaceae and A. baumannii harboring IMP, VIM, and NDM-type MBLs (Livermore et al., 2013). Irrespective of the candidate carbapenem, ME1071 activity against NDM MBLs was less than that of VIM-type and IMP-type MBLs. Of note, biapenem was the carbapenem with the lowest baseline MICs to the MBLs, but it is commercially unavailable in many countries including the USA. Other MBL-specific inhibitors are in pre-clinical development (Chen et al., 2012).
Plazomicin (ACHN-490) is an aminoglycoside derivative with potent activity against some carbapenem-resistant Gram-negative bacilli (Zhanel et al., 2012). Studies have noted that susceptibilities to aminoglycosides vary among KPC-producing K. pneumoniae. In one evaluation, 48% of 25 tested isolates were susceptible to amikacin, 44% to gentamicin, and 8% to tobramycin. Plazomicin demonstrated an MIC 90 significantly lower than that of amikacin (Endimiani et al., 2009c). In vitro studies also indicate that depending on the aminoglycoside resistance mechanisms present, Plazomicin may have activity against select isolates of P. aeruginosa and A. baumannii (Aggen et al., 2010;Landman et al., 2011). Susceptibility to plazomicin in the setting of resistance to other aminoglycosides appears to be dependent on the mechanism of aminoglycoside resistance (Livermore et al., 2011a).
NAB739 and NAB7061 are polymyxin derivatives that may be less nephrotoxic than commercially available polymyxins. In a small in vitro study, NAB739 displayed activity against nine carbapenemase-producing polymyxin-susceptible isolates of Enterobacteriaceae (Vaara et al., 2010). A contemporary evaluation of NAB739 demonstrated higher MICs compared to those of polymyxin B in a collection of polymyxin-susceptible and non-susceptible Enterobacteriaceae, P. aeruginosa, and A. baumannii . NAB7061 when used in combination with rifampicin or clarithromycin demonstrated synergistic activity against seven strains of carbapenemase-producing Gramnegative bacilli including one polymyxin-resistant strain (Vaara et al., 2010). It remains unclear what role these agents will play in the setting the increasing burden of infections with carbapenemase-producing Enterobacteriaceae.
The activity of the siderophore monosulfactam, BAL30072, has been against non-fermenting carbapenemase-producing Gramnegative bacilli (Page et al., 2010). In one study, susceptibility to BAL30072 was noted in 73% of 200 isolates of carbapenemaseproducing A. baumannii, the majority of which were of the same OXA-23 producing clone (Mushtaq et al., 2010a). In that same study, smaller percentages of susceptibility were noted in a selection of carbapenem-resistant Burkholderia cepacia and P. aeruginosa isolates. Recent evaluations of BAL30072 confirm that there may be a role for this agent in the treatment of resistant A. baumannii infections (Russo et al., 2011;Higgins et al., 2012b). BAL 30376 is a combination of a siderophore monobactam with clavulanic acid. In two studies, this combination demonstrated reasonable in vitro activity against CHDL, including OXA-48, and MBLs but not KPCs (Livermore et al., 2010;Page et al., 2011).

CONCLUDING REMARKS
In the last 5 years, we have witnessed the global spread of carbapenem resistance among Gram-negative organisms. The notion that multidrug resistance among these pathogens is limited to isolated outbreaks among the critically ill has met the ultimate challenge with NDM-1 (Kumarasamy et al., 2010). The conveniences of travel and medical tourism have introduced resistance mechanisms across states, countries, and even continents at an alarming rate (Rogers et al., 2011;van der Bij and Pitout, 2012). Rates of resistance in some countries may be underestimated due to the lack of organized reporting structures and limited resources. Long-term healthcare facilities are now recognized reservoirs for the continued propagation of MDR organisms (Urban et al., 2008;Aschbacher et al., 2010;Perez et al., 2010a;Ben-David et al., 2011;Prabaker et al., 2012;Viau et al., 2012).
Until the introduction of accurate, affordable, and readily accessible diagnostics and reliably effective antimicrobials a major focus remains containment and eradication of these organisms within the healthcare environment. Many cite a "bundle" type approach that includes administrative support, active surveillance, antimicrobial stewardship, and augmented infection control practices (Centers for Disease Control and Prevention, 2009;Schwaber et al., 2011;Snitkin et al., 2012). Just as with drug development (Tillotson, 2010), the future savings of investing in prevention is not as tangible as the immediate capital investment required to allot appropriate resources including advanced laboratory platforms, experienced laboratory personnel, dedicated nursing staff, and infection control personnel (Bilavsky et al., 2010). Expanding these efforts to non-acute healthcare settings is recommended to begin to stem the evolving pandemic of carbapenem resistance (Gupta et al., 2011).
The prudent use of antibiotics is essential in combating the continuing evolution of resistance (Marchaim et al., 2012). This may be even more crucial in areas where non-prescription antimicrobial use is common and continues to be unregulated. In an age where multidrug resistance is so widespread, even the appropriate use of broad-spectrum antibiotics has contributed to our current state.
Research funding and support for the description of resistance mechanisms, validation of current infection control practices, and antimicrobial development must be prioritized. Institutions supporting infection control, state of the art microbiology laboratories, and antimicrobial stewardship programs should receive recognition and incentives for their foresight. Despite these continuing challenges, considerable progress has been made to identify at-risk populations and to describe resistance determinants. Collaborative efforts (Kitchel et al., 2009a;Struelens et al., 2010;Canton et al., 2012) have led to a better understanding and awareness of the epidemiology and the contribution of antimicrobial use and the environment to the propagation of antimicrobial resistance. These joint efforts have proven crucial for the propagation of information about carbapenemases. Continuing to encourage these partnerships is imperative in the ongoing struggle against antimicrobial resistance and to prevent antimicrobials from essentially becoming obsolete.