Review. Confronting carbapenemaseproducing. Future Microbiology

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1 Confronting carbapenemaseproducing Klebsiella pneumoniae Antonis Markogiannakis 1, Leonidas S Tzouvelekis 2, Mina Psichogiou 3, Efi Petinaki 4 & George L Daikos* 3 1 Department of Pharmacy, Laiko General Hospital, Athens, Greece 2 Department of Microbiology, Medical School, University of Athens, Greece 3 First Department of Propaedeutic Medicine, Medical School, University of Athens, Greece 4 Department of Microbiology, Medical School, University of Thessaly, Larissa, Greece *Author for correspondence: Tel.: Fax: gdaikos@med.uoa.gr The ongoing spread of carbapenemase-producing (CP) multidrug-resistant enterobacteria, primarily Klebsiella pneumoniae, has undoubtedly caused a public health crisis of unprecedented dimensions. The scientific community has been struggling with these highly problematic nosocomial pathogens for more than a decade. Faced with the current situation, one cannot help but wish we could have done better, earlier. However, significant steps have been and are currently being made towards a better understanding of transmission routes of CP microorganisms and in designing strategies that could effectively curb this devastating epidemic. Most importantly, the systematic evaluation of accumulating experimental and clinical data has paved the way to a more rational management of CP-infected patients. In addition, systematic efforts of the industry have led to the development of novel antibacterial agents that are active against CP strains and expected to be introduced to clinical practice in the immediate future. Future Microbiology During the last two decades we have witnessed the emergence and global dissemination of Gram-negative bacterial strains, including enterobacteria, producing the so-called carbapenemases. The term refers to a variety of structurally and functionally diverse -lactamases, the most common being the Klebsiella pneumoniae carbapenemase (KPC) enzymes (molecular class A), the metallo- -lactamases (M Ls) VIM, IMP and ew Delhi M L (DM; molecular class B) and the XA-48 and its derivatives (molecular class D). These carbapenemases exhibit extensive hydrolysis spectra, including many of the clinically available -lactams. Additionally, they exhibit decreased susceptibility (KPC) or resistance to mechanism-based inhibitors (M Ls and XA-48). In addition to the variety it shields, the term carbapenemase also does not accurately reflect these enzymes substrate preference. Unlike the tailor-made penicillinases, such as the TEMs and SHVs, acquired carbapenemases do not have carbapenems as their preferred substrate (TABLE 1). Indeed, the collective name of these particular -lactamases reflects their clinical impact more than their hydrolytic properties. From the epidemiological view, the situation with the carbapenemase-producing (CP) enterobacterial species, especially CP K. pneumoniae (CP-Kp), is complex and evolving [1]. CP enterobacteria continue to spread worldwide, currently affecting acute as well as long-term care facilities in countries where, until recently, they occurred in a sporadic fashion if at all [1 3]. A systematic evaluation of the accumulated clinical data has already called for the revision of therapeutic approaches against severe infections caused by CP-Kp strains. For example, monotherapy using either colistin (considered as the first-choice antimicrobial for these infections) or tigecycline, has been questioned [1,4 6]. Furthermore, a discussion on the benefits of using carbapenems a possibility that would have been out of the question not so long ago has been initiated [7]; in fact, in some settings, the therapeutic potential of carbapenem-based combinations against CP-Kp hospital-acquired infections has already been tested with positive results [8 10]. evertheless, systematic efforts (e.g., large-scale controlled trials) to define the optimal therapeutic regimens have not yet been undertaken. This is partly due to the fact that CP-Kp strains mostly affect severely ill, immunocompromised patients treated in intensive care units (ICUs), in whom numerous confounding factors hamper a clear-cut evaluation of any antimicrobial regimens. There are also additional open issues regarding CPs, including their modes of transmission, detection methods and infection control policies. In the present review, we have attempted a critical appraisal of the existing epidemiological, experimental and clinical data, focusing on issues that remain debatable. ovel anti-cp Keywords antibiotic combinations antibiotic resistance antimicrobial therapy carbapenemases epidemiology infection control Klebsiella pneumoniae novel antimicrobials part of /FMB Future Medicine Ltd Future Microbiol. (2013) 8(9), ISS

2 Markogiannakis, Tzouvelekis, Psichogiou, Petinaki & Daikos Table 1. Hydrolytic efficiency of representative variants of the carbapenemase types encountered in Klebsiella pneumoniae clinical isolates against selected -lactam antibiotics. -lactam Carbapenemase (hydrolytic efficiency [k cat /K m (s -1 M -1 )]) KPC-2 VIM-1 IMP-1 DM-1 XA-48 Imipenem Meropenem <0.001 Cefotaxime Ceftazidime D Cephalothin Penicillin-G Data for KPC-2, VIM-1, IMP-1, DM-1 and XA-48 were derived from [11], [ ] and [17], respectively. k cat : Turnover number; K m : Michaelis constant; D: ot determined. compounds that are in an advanced stage of development are also presented. Antibiotic resistance patterns of CP-Kps Resistance to -lactams KPCs (KPC-2 KPC-15) are typical class A -lactamases possessing the characteristic amino acid motifs of this family of enzymes. The arrangement of the active site residues allows the accommodation and efficient acylation of virtually all -lactam antibiotics, including carbapenems and cephamycins [11 13]. Consequently, KPC producers commonly exhibit high-level resistance to penicillins (with the exception of temocillin, a derivative of ticarcillin with limited clinical usefulness), older cephalosporins, as well as newer oximino derivatives, cefamycins and aztreonam. The frequent coproduction of extended-spectrum -lactamases (ESBLs), such as SHV and CTX-M, as well as acquired AmpCs (mostly CMYs), contribute to the high MICs observed for the latter drugs. MICs of carbapenems may vary considerably among CP-Kps, though according to the breakpoints of the Clinical and Laboratory Standards Institute (CLSI) most of them are classified as resistant [14]. The class B M Ls of VIM (VIM-1 VIM-38), IMP (IMP-1 IMP-44) and DM (DM-1 DM-8) types, though phylogenetically distant, exhibit similarities in their hydrolysis spectra being active against all -lactams except aztreonam [15,16]. However, the frequent coproduction of ESBLs active against aztreonam, especially among VIM producers, precludes the use of this drug. Similarly to KPC producers, M L-positive K. pneumoniae strains display high-level resistance to penicillins and cephalosporins, while the range of carbapenem MICs is wide. f the acquired carbapenemases encountered in K. pneumoniae, XA-48 and its point mutants XA-162, -163, -181, -204 and -232 exhibit the weakest activity against carbapenems as well as the least extensive substrate spectrum, sparing oximino-cephalosporins. Yet, the vast majority of XA-48-positive K. pneumoniae isolates coproduce -lactamases such as CTX-M, which are capable of hydrolyzing the latter drugs [17,18]. Resistance to non- -lactams Virtually all CP-Kps exhibit extensive resistance phenotypes that include most clinically available drug classes. This is due to the fact that the vast majority of CP-Kps, apart from carbapenemaseencoding genes, also possess a variety of other resistant determinants mainly genes encoding aminoglycoside-modifying enzymes located either on the plasmid carrier of the carbapenemase gene or on coresident resistance plasmids. Moreover, several chromosomal mutations frequently encountered among clinical K. pneumoniae strains contribute to overall antibiotic resistance (e.g., by decreasing outer membrane permeability, upregulating efflux pumps and lowering the affinity between topoisomerases and quinolones, among others). Thus, with the exception of gentamicin (which retains some good activity, mainly against KPC producers), CP-Kps commonly exhibit in vitro resistance to most of the clinically important non- -lactam antimicrobial agents, such as aminoglycosides and fluorinated quinolones [19]. The good in vitro activity of colistin and tigecycline against CP enterobacteria has been shown in numerous studies. In vivo, however, these drugs are inherently problematic. Both exhibit complex pharmacokinetics that cannot be considered optimal for the treatment of bloodstream, lower respiratory and urinary tract infections caused by CP-Kps (as discussed below). evertheless, due to the dearth of drugs 1148 Future Microbiol. (2013) 8(9)

3 Confronting carbapenemase-producing Klebsiella pneumoniae to which CP-Kps are susceptible in vitro, colistin and, to a lesser extent, tigecycline, readily become the choice antibiotics. ot surprisingly, the consequence of their systematic use, especially in endemic settings, has been the increasing isolation frequency of colistin- and/or tigecycline-resistant CP-Kps [20 23]. Global spread of CP-Kp strains KPC producers: a single clone pandemic? The most widespread CP-Kp strains are those producing KPC-type -lactamases (predominant variants KPC-2 and KPC-3). KPCpositive strains have caused extensive hospital outbreaks in the northeast regions of the USA during the last decade (mainly in the states of Y and J). However, sporadic isolations had already been noticed during the late 1990s in the south-eastern (C) and the mid-atlantic regions (MD), indicating that these pathogens had in fact been circulating unnoticed for some years [24,25]. According to a recent report from the CDC, the prevalence of CP-Kp in the USA has increased from 1.6% in 2001 to 10.4% in 2012 [26]. An increasing proportion of KPCpositive K. pneumoniae has also been reported in Latin America, though a clear link with the US epidemic has not been established [22,27]. It has been suggested that transfer of patients carrying KPC-producing K. pneumoniae from the USA to Israel [28] and then to northern and western European countries (where isolation frequency remains low [1]), supposedly through Greece, was one of the main transmission routes leading to these microorganisms becoming widespread [29]. This hypothesis appears to be in line with the common clonal origin (sequence type 258 [ST258]) of the majority of the respective isolates. evertheless, it should be emphasized that sequence typing delineates phylogenetic origins rather than epidemiological associations. Also, index cases supporting the proposed transmission paths have not always been identified with certainty in the limited outbreaks described in Germany and The etherlands [30,31]. f note, in Chinese hospitals (also an endemic setting) KPC-producing K. pneumoniae isolates belong mainly to the likely founder of the clonal complex 258, ST11, differing from ST258 in a single locus [32]. Although we have sporadically identified ST11 CP-Kps in Greece, we are not aware of medical tourism cases from the latter country to China. We believe that the issue regarding the rapid international spread of the ST258 clone should remain open and some of the explanations discussed above should better serve as working hypotheses. Although isolates of clonal complex 258 remain predominant, KPC-Kps belonging to other lineages have emerged in many geographic areas due to the acquisition of bla KPC -encoding plasmids. The latter have been classified into several incompatibility groups such as FII, and L/M, a fact reflecting the mobilization capacity of the bla KPC genes that are invariably associated with isoforms of Tn4401 as well as the important role played by transmissible plasmids in the further dispersion of these genes [33 35]. M L-producers: a continuous threat In stark contrast to the KPC picture just described, the three acquired M L types, namely VIM, IMP, and DM, have been encountered in a variety of phylogenetically distinct K. pneumoniae strains causing polyclonal outbreaks. Moreover, the respective bla genes have been identified in a wide range of different plasmids [1,34,36]. Producers of IMP- and VIM-type enzymes were frequently isolated during the last decade in Far East and the Mediterranean countries, respectively, where they are still endemic [1,3,37]. The most affected country was Greece. VIM-positive K. pneumoniae predominated among multidrugresistant strains isolated in Greek tertiary care hospitals until 2008 [38]. Their decline during coincided with the rapid dissemination of KPC producers in that setting [39]. otwithstanding the repeated occurrence of IMP and VIM producers in various countries, these strains have largely remained confined in their original foci. DM is the most recent M L type detected in K. pneumoniae. The epicenter of the DM producers is the Indian subcontinent where, apart from their prevalence in the hospital setting, they have also been detected in various environmental niches. The recent emergence of these microorganisms in Europe, orth America, the Far East and Australia (apparently associated with the transfer of patients from India, Pakistan and Bangladesh, as well as the high immigration rates from these areas), led some investigators to hypothesize that DM producers may be capable of achieving a global spread similar to that presently seen with KPC-producing enterobacteria [40 42]. Hopefully, efforts to contain these microorganisms will be successful. XA-48 producers: the newcomers XA-48-producing K. pneumoniae caused extensive hospital outbreaks in Turkey during the end of the last decade, though it had been isolated from clinical samples in this country since

4 Markogiannakis, Tzouvelekis, Psichogiou, Petinaki & Daikos About the same time, such strains were also being isolated in other Middle Eastern and orth African countries underscoring the lag time between emergence and detection of multidrug-resistant strains [43]. The Middle East and orth Africa are still regarded as the most important foci of XA-48 producers. There have been various studies suggesting that transfer of patients carrying XA-48-positive K. pneumoniae from these areas to Europe has led to occasional outbreaks [18]. Moreover, the increasing number of studies reporting spread of XA-48 producers in diverse geographic areas (e.g., Latin America, South Africa and the USA) clearly suggests that these pathogens have reached a global epidemic status. Concluding remarks The data summarized above show that the transfer of patients from endemic to nonendemic areas is of undeniable importance for the international spread of CP-Kps. Consequently, policies to curb cross-border diffusion have been proposed [44]. otwithstanding the ethical and political issues (readers are reminded of the discussion regarding the correctness of the designation DM) arising, these approaches may need revision as indicated by the current dispersion patterns of CP-Kps. We do not argue against such measures. n the contrary, we believe that they should be redesigned and intensified based on international collaborative efforts that would include a drastic reallocation of the available resources: containing CP-Kps immediately and directly in their main foci is infinitely more efficient not to mention beneficial to international public health than trying to limit their subsequent spread towards other areas. What sounds like an ambitious project is bound to be extremely rewarding and effective in the mid-to-long term. It is also worth mentioning studies reporting the isolation of apparently autochthonous carbapenemase producers around the world, including western Europe and the Far East [45,46]. Assuming that the de novo emergence of such strains in diverse regions during the same time period is rather unlikely, these findings may indicate that the methods currently applied to trace transmission routes must be improved. CP-Kp strains in the acute care health setting Acquisition risks, transmission & control of CP-Kp strains Although CP-Kps can affect any hospitalized patient in endemic setting, these organisms cause life-threatening infections such as bacteremia and pneumonia mostly in critically ill patients with severe underlying diseases and comorbidity conditions. Consequently, several variables commonly linked with this subset of hospitalized patients have been recognized as independent risk factors for CP-Kp acquisition, those most frequently reported being prolonged hospitalization, stay in an ICU, poor functional status, previous use of antibiotics, malignancies, solid organ or stem cell transplantation, use of multiple invasive devices among others. However, the independence of each of the above factors may be rather an overstatement, since risks for colonization/infection by CP-Kps have mainly been assessed in settings where these microorganisms had been established and, in most cases, had already reached epidemic, if not indeed endemic, status. Therefore, a straightforward answer to the question: Who is prone to acquire a CP-Kp? could well be the inevitable tautology: The seriously ill patient treated in a facility where transmission of CP-Kps is largely uncontrolled. A fraction of colonized patients will develop infection. The colonization/infection ratio may vary from 10 to 30% depending mainly on patient characteristics [47,48] [Daikos GL, Unpublished Data]. The modes of CP-Kp transmission do not differ from those of other multiresistant enterobacteria. Person-to-person transmission via the hands of nursing and medical staff is most probably the main route of dissemination in healthcare facilities (FIGURE 1). Involvement of environmental sources has also been proposed, but its contribution is probably less important. In the majority of cases, the first step is ingestion of the microorganism followed by its establishment in the patient s gut flora. The median carriage time has been estimated to be at least several months, and in patients with invasive devices, poor functional status and comorbidity conditions appear to be longer [49,50]. This relatively long colonization time, when coupled with serious infection control shortfalls in a facility, will raise the number of colonized patients. In a study using the classic Ross Macdonald model for vector-borne diseases, it was estimated that, in a setting with low compliance with standard hygiene practices, an average of two secondary cases may occur per primary case of CP-Kp colonization [51]. In a worst case scenario, the gradually increasing colonization pressure would eventually lead to the predominance of CP-Kps. Until recently, the policies to control the spread of CP-Kps and other CP enterobacterial 1150 Future Microbiol. (2013) 8(9)

5 Confronting carbapenemase-producing Klebsiella pneumoniae species were those applied for other MDR Gram- negatives. However, experience from successful control attempts undertaken at various levels (from single wards to national health systems) has now allowed the formulation of more focused and detailed guidelines [44,52,53]. Some differences do exist, but there is a general agreement on the main actions that must be taken. Briefly, in order to be timely and effective, an intervention must include, at least, active surveillance cultures, isolation or cohorting of colonized/infected patients together with contact precautions and the appointment of dedicated nursing staff. Results must be evaluated on a regular basis, and, in case of failure, root-cause ana lysis must be performed. The potential effectiveness of the aforementioned measures has recently been supported by a mathematical model predicting that, in a high prevalence setting, a reduction of 60 90% in colonized patients on admission, through active surveillance cultures, contact precautions and isolation/cohorting, in combination with 60% compliance in hand hygiene, would result in a drastic decline in CP-Kp prevalence in a few months (FIGURE 2) [43]. Surveillance & detection: current trends Methods aiming to detect carbapenemase production appeared in the literature almost simultaneously with the emergence of CP Gramnegatives. There has been a wide variety of detection approaches that can be grossly classified as phenotypic, biochemical and molecular. Based on published data, we support that the straightforward recognition of carbapenemase-positive isolates by interpreting reading of phenotypes, including those obtained from automated systems, should be avoided [54 56]. f the phenotypic methods, the modified Hodge test still the only screening method recommended by the CLSI [14] is based on the hydrolysis of ertapenem or meropenem by whole cells of carbapenemase producers. The major shortcoming of the modified Hodge test is its relatively low specificity (frequent false positives among CTX-M and/or AmpC producers) [57]. Synergy between carbapenems and inhibitors (boronates for KPCs and chelating agents for M Ls) in a combined disk assay is a reliable approach despite some limitations, such as the requirement for additional time and the inability to detect XA-48-type carbapenemases [1,57]. These problems can be overcome by the available molecular methods, which are mainly PCR-based. umerous inhouse simplex, multiplex, and real-time PCR Uncolonized patients Uncontaminated HCW assays have been employed in the identification of carbapenemase genes. The industry has also developed various reliable detection methods based on PCR hybridization and real-time PCR as well as microarray technology [58 60]. A biochemical approach to detect carbapenemase production is the photometric monitoring of imipenem hydrolysis by crude enzyme preparations derived from the suspected isolate. The method is considered as the gold standard but can usually only be applied in reference laboratories [57]. Also, matrix-assisted laser desorption ionization time of flight mass spectrometry, a method capable of identifying carbapenem hydrolysis products, has been used to confirm carbapenemase activity in Gram-negative isolates [61]. At this point, it is worth mentioning the CarbaP test, a recently developed rapid, reliable and lowcost chromogenic reaction based on imipenem hydrolysis by whole cell suspensions that can be readily integrated in the work flow of a clinical laboratory [62]. The routine application of carbapenemase detection techniques in the clinical laboratory is no longer recommended by the CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [14,201]. However, confirmation of the presence of carbapenemases should be performed for epidemiological purposes, for example, identification of positive isolates derived after CP carrier screenings. The latter rely increasingly on the use of commercially available selective culture media [63]. It is possible that these media that are being continuously refined could be routinely used in surveillance studies in the near future. Colonized patients Contaminated HCW Figure 1. Clonal transmission of carbapenemase-producing Klebsiella pneumonia strains from patient to patient through healthcare workers in a bed ward during a relatively short time period (6 8 months). Boxes depict four population groups: uncolonized patients, colonized patients, uncontaminated HCWs and contaminated HCWs. Solid arrows depict movement between population groups and dashed arrows depict transmission of carbapenemase-producing Klebsiella pneumonia between patients and HCWs. HCW: Healthcare worker. Adapted from [51]

6 Markogiannakis, Tzouvelekis, Psichogiou, Petinaki & Daikos CP-Kp colonization (%) Time (days) Figure 2. Impact of infection control measures on the prevalence of carbapenemaseproducing Klebsiella pneumonia colonization in an hyperendemic setting. The evaluated scenarios include: (A) hand hygiene compliance of 60% (line with triangles); (B) hand hygiene compliance of 60% in combination with 60% reduction of colonized admissions by active surveillance cultures, isolation/cohorting and contact precautions (line with circles); and (C) hand hygiene compliance of 60% in combination with 90% reduction of colonized admissions by active surveillance cultures, isolation/cohorting and contact precautions (line with squares). CP-Kp: Carbapenemase-producing Klebsiella pneumonia. Adapted from [51]. Antibiotic therapy of CP-Kp infections Although numerous clinical studies have dealt with the antimicrobial treatment of CP-Kpcaused infections, controlled comparative trials are missing. Consequently, a systematic appraisal of the efficacy of the various treatment schemes used so far is quite difficult. Indeed, compilation of data from different types of studies, some of which include a fairly small number of patients, cannot lead to solid conclusions. The lack of a universally accepted definition for combination therapy is also problematic. Some investigators consider administration of at least two antibiotics as combination therapy, regardless of in vitro susceptibility, while, for others, combination therapy is defined as the use of at least two in vitro-active drugs. Further complications arise from the use of different carbapenem breakpoints (CLSI or EUCAST; TABLE 2) in clinical studies evaluating therapeutic efficacy of antibiotic schemes that include carbapenems. Apart from the difficulties in comparing clinical outcome data, applying different breakpoints may hamper the settingup of large scale international efforts aiming to optimize therapeutic practices. In defining carbapenem susceptibility status, EUCAST, based primarily on pharmacokinetic/pharmacodynamic (PK/PD) and clinical data, recommended higher values than the CLSI. However, lowering the breakpoints and reporting the CP-Kp isolates exhibiting, for instance, meropenem MICs of 4 or 8 µg/ml as resistant, decreases the opportunity Table 2. Carbapenem clinical breakpoints for Enterobacteriaceae according to European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute. Carbapenem EUCAST CLSI S R Screening cutoff S I R Imipenem 2 >8 > Ertapenem 0.5 >1 > Meropenem 2 >8 > Doripenem 1 >4 D Values (mg/l) are presented as in [14, 209]. CLSI: Clinical and Laboratory Standards Institute; EUCAST: European Committee on Antimicrobial Susceptibility Testing; I: Intermediate; D: ot defined; R: Resistant; S: Susceptible Future Microbiol. (2013) 8(9)

7 Confronting carbapenemase-producing Klebsiella pneumoniae for some patients to obtain the potential therapeutic benefit of carbapenem-based combination schemes. It should be also emphasized that, in certain clinical studies, carbapenem MICs have been determined by automated systems that have been repeatedly blamed for serious inconsistencies [54 56]. otwithstanding these limitations, there have been ample data that justify a current shift to combination therapy. As we have discussed previously, use of a single agent in treating patients with CP-Kp bloodstream infection (BSI) or pneumonia was associated with unacceptably high mortality rates, while combination therapy performed significantly better, though still suboptimally [1,64]. Further support for the superiority of the combination regimens that may include up to three drugs was provided by two recent studies conducted in the USA and Italy that included cohorts of patients infected with KPC-producing K. pneumoniae [9,10]. otably, in the Italian study, carbapenem-containing combinations appeared to be the most efficacious even when used against CP-Kps resistant to these drugs according to the current criteria of the CLSI. Therefore, using antibiotic combinations should clearly be regarded as a step towards a more efficacious treatment of CP-Kp infections. n the other hand, it cannot be denied that the relevant data are largely observational and lacking in experimental support. Superiority of treatment with two or more drugs apparently results from synergy or additive effect. The potential synergistic activity of a wide variety of antibiotic combinations against CP-Kps, producing either KPCs or M Ls, has been examined in many in vitro studies, using a variety of time-kill methodologies. These studies may provide some useful preliminary information. However, results are sometimes inconsistent and also depend heavily on the concentrations of the drugs tested. More valid data regarding synergy through drug combinations can be obtained by using animal infection models and controlled clinical trials, but the scarcity of such studies is remarkable. Lately, Hirsch and colleagues have documented a clear synergistic effect of doripenem amikacin combination against CP-Kps fully resistant to both drugs in the neutropenic murine pneumonia model [65]. In addition, two multicenter prospective studies aiming to evaluate the therapeutic potential of imipenem colistin [202] and meropenem colistin [203] combinations are underway. In the meantime, the available experimental and clinical data can and should be revisited in order to optimize the use of commonly used antimicrobial agents. Although there is a clear trend towards increasing resistance to colistin among CP-Kps, this drug remains one of the most in vitro-active ones against these pathogens. It can be reasonably expected that its use, mostly in combination schemes, will continue. However, the low efficacy of colistin monotherapy suggests that the currently employed schemes, administered in two or three divided doses across the day, are suboptimal and should be reconsidered [1]. Indeed, the complex pharmacokinetics of colistin have not yet been fully elucidated. Consequently, the fine balance between efficacy and toxicity of colistin remains to be defined. As things stand, the design of dosing schemes in subsets of patients, such as the critically ill, transplant patients and those with impaired renal function, rests on what could, not unfairly, be described as guesswork. The commonly used schemes have been based primarily on data from animal infection models, indicating a correlation between exposure time and antibacterial activity [66]. Kinetic studies in humans have shown that it takes at least 2 days for the drug to achieve steady state [67]. An initial loading dose of 9 ml IU can overcome this disadvantage; this practice is now widely accepted by clinicians. More to the point, colistin concentrations attained in serum are most probably insufficient for isolates with MICs >0.5 mg/l [68]. Also, its concentrations in bronchial secretions are significantly lower, even below detection levels [69,70]. Thus, testing colistin at higher dosages given at longer intervals may be worth trying. The long half-life of colistin, as well as its concentration-dependent killing and adaptive resistance phenomena [68,71,72], favor these suggested changes. However, the issues of nephro- and neuro-toxicity should be priorities, if any effort to enhance colistin efficacy by changing the dosing schemes is to be undertaken. The shortage of active antimicrobials and the generally low MICs of tigecycline against CP- Kps have led to the use of this drug, occasionally as a single agent, in the treatment of CP-Kp BSIs and pneumonia, in spite of these not being included in the US FDA-approved indications of the drug (skin and skin structure infections, complicated intra-abdominal infections and community-acquired pneumonia). Efficacy of tigecycline monotherapy in the former infections, however, seems to be poor, as expected from its PK/PD features [1]. Even so, the drug can be useful against various CP-Kp infections

8 Markogiannakis, Tzouvelekis, Psichogiou, Petinaki & Daikos including BSIs as part of combination regimens including a carbapenem and/or colistin but the relevant data are still limited [9,10]. ccasionally, tigecycline has been used at doses higher than those approved (e.g., 100 mg every 12 h) [73]. otwithstanding its potential toxicity, the large volume of distribution of tigecycline [74] makes it unlikely that a meaningful increase in plasma and epithelial lining fluid concentration can be attained by doubling its daily dose. ne of the most debatable issues regarding therapy of infections caused by CP-Kps and other CP enterobacteria is the use of carbapenems [7,75]. We have previously supported the use of these antibiotics based on clinical observations as well as data from animal experimental infections [1]. Lately, we have introduced (in a limited number of ICUs and hematology wards in Greek hospitals) an algorithm in which a carbapenem (preferentially meropenem) combined with a suitable aminoglycoside or colistin is the first choice for primary and secondary BSIs caused by CP-Kps, provided that the meropenem MIC of the infecting strain is 4 mg/l [64]. Results so far appear to be promising. thers, however, oppose the use of carbapenems against CPs irrespective of the MIC value, with the argument that such practice is not justified by the available clinical data [76]. Irrespective of this as-yet unresolved debate, for a carbapenem to be useful in a combination scheme it must be given at high doses and by prolonged infusion in order to exploit its PK/PD features and maximize its in vivo activity [77,78]. ther drug classes, mostly aminoglycosides that occasionally exhibit good in vitro activity against CP-Kps, have also been used in combination regimens. Evidently, any active aminoglycosides should be among the first-choice antibiotics for the treatment of urinary tract infections caused by CP-Kps. There have also been indications from a limited number of clinical studies that carbapenem aminoglycoside combinations may exhibit satisfactory therapeutic efficacy against CP-Kp bloodstream and lower respiratory tract infections [1,64]. Parenteral fosfomycin has been proposed as an adjunct to other active agents against serious CP-Kp infections but clinical experience is limited to a few small studies producing conflicting results [79,80]. This drug may deserve more attention since it exhibits good in vitro activity against CP-Kps and seems to be well tolerated at relatively high doses. It should be stressed, however, that the selection rate of fosfomycin-resistant variants among enterobacterial species is high [81]. Moreover, in the case of K. pneumoniae ST258, cross-resistance to -lactams is also possible [80]. Readers should bear in mind that the above discussion is based on studies mainly reporting on infections caused by KPC- and VIM-producing isolates. Similar data regarding infections due to XA-48 and DM producers are still limited. As with the other CP-Kps, combinations including colistin and/or tigecycline are frequently employed in treating the respective infections [18,40]. However, the relatively low carbapenem MICs in a significant portion of XA-48-producing K. pneumoniae should also allow treatment with carbapenem-based combinations [82,83]. Furthermore, oximino-cephalosporins may be an option for XA-48-positive K. pneumoniae not coproducing an ESBL [84]. ovel antimicrobials Antibiotics Plazomicin A derivative of sisomicin, plazomicin (FIGURE 3) is a new-generation aminoglycoside active against Gram-negative pathogens, mainly Enterobacteriaceae. Most importantly, the in vitro activity spectrum of the drug also includes bacterial strains producing any of the clinically important aminoglycoside-modifying enzymes. Plazomicin, however, is inactive against strains producing ribosomal methyltransferases such as most of the DM-positive Enterobacteriaceae [85]. The kinetic profile of plazomicin resembles that of other aminoglycosides, although at the currently tested dosing scheme in healthy individuals (intravenous infusion of 15 mg/kg over 10 min), the C max of the drug is much higher and the T1/2 more prolonged compared with the clinically available aminoglycosides [86]. Also, animal and Phase I studies have indicated no oto- or nephro-toxicity [87]. Results from a Phase II study in patients with complicated urinary tract infection including cases with bacteremia are expected to be published soon [204]. In a press release, the manufacturer announced that all objectives met in Phase II plazomicin complicated urinary tract infections study [205]. BAL30072 This monocyclic -lactam (a siderophore monosulfactam; FIGURE 3) exhibits potent in vitro activity against a wide range of Gram-negative species by inhibiting multiple penicillin binding proteins. BAL30072 overcomes decreased permeability and resists hydrolysis by several clinically relevant -lactamases, including M Ls, XA-48 and KPCs, though it is susceptible to 1154 Future Microbiol. (2013) 8(9)

9 Confronting carbapenemase-producing Klebsiella pneumoniae common ESBLs such as the SHV-type enzymes [88,89]. BAL30072, either alone or combined with meropenem, has shown promising activity against various Gram-negative species including M L-producing enterobacteria both in vitro and in experimental animal infections [90,91]. Plazomicin H H 2 H 2 H H H Carbapenemase inhibitors Avibactam This non- -lactam compound (a bridged diazabicyclo-octanone; FIGURE 3) is expected to be introduced soon in the clinic combined with ceftazidime, aztreonam or ceftaroline. Avibactam is capable of inhibiting virtually all serine -lactamases, including KPCs, at low concentrations [92], thus restoring even in KPC producers the activity of various -lactams, including oximino-cephalosporins. The therapeutic efficacy of the ceftazidime avibactam combination against KPC-positive K. pneumoniae has been documented in murine infection models [93]. The results of a Phase II study involving hospitalized patients with complicated urinary tract infections showed that its efficacy and safety were comparable to those of imipenem [94]. Additional clinical trials to evaluate ceftazidime avibactam combinations in a variety of nosocomial infections have been designed [206]. MK-7655 The second member of the diazabicyclooctanone family of -lactamase inhibitors, MK-7655 (FIGURE 3), is a potent inhibitor of class A and C -lactamases [95]. A pronounced in vitro synergy between MK-7655 and imipenem against KPC-producing K. pneumoniae has been shown [96]. A Phase II study of the safety, tolerability and efficacy of imipenem plus MK-7655 versus imipenem alone to treat complicated intra-abdominal infections is underway [207]. RPX7009 This is a boron-containing compound with activity against class A -lactamases including KPC types, as well as most AmpC enzymes [97]. RPX7009 is being developed in combination with biapenem, a carbapenem that is less vulnerable to M Ls and XA-48 enzymes as compared with other carbapenems. A biapenem/rpx7009 combination produced significant bacterial killing in experimental infections caused by KPC-producing strains and a Phase I study evaluating the safety, tolerability, and pharmaco kinetics of RPX7009 in healthy adults is in progress [208]. H H 3 C H 2 H 3 C H H H Avibactam Conclusion The grave public health consequences of the ongoing worldwide dissemination of CP enterobacteria, especially K. pneumoniae, have been exhaustively discussed [1,2,36,98]. Genes encoding distinct -lactamases, such as KPC and M Ls of the VIM, IMP and DM types, capable of hydrolyzing virtually all -lactams, including carbapenems, have spread via mobile units to a variety of K. pneumoniae clones, some of which have achieved global dissemination. Although infections by these pathogens have been plaguing the healthcare sector for over a decade, too many critical issues of epidemiology, detection, control measures and, most importantly, therapeutic approach remain open, and the need for these to be resolved, or at least adequately tackled, is urgent. At present, the most widespread CP-Kps are the bla KPC -carrying strains, many of which belong to ST258. Additionally, in the last few years, producers of XA-48 carbapenemase, which probably emerged in the Middle East, are increasingly being isolated in various regions. n the other hand, producers of the M L types VIM, IMP and DM, though also detected worldwide, are, so far, being isolated at high frequencies mostly H H 2 H 2 S 3 a H S H H RPX7009 S B H H H MK-7655 S 3 H S 3 a BAL30072 Figure 3. ovel agents active against carbapenemase producers expected to be introduced to clinical practice soon

10 Markogiannakis, Tzouvelekis, Psichogiou, Petinaki & Daikos in their original foci (the Mediterranean region, the Far East and the Indian subcontinent, respectively). Regardless of this epidemiological variability, curbing the further spread of carbapenemase producers is a clear international priority. Special measures aiming to limit cross-border diffusion from high- to low-prevalence countries have been implemented. The current epidemiological patterns of CP-Kps, however, suggest that these are not yet satisfactory, calling for a better elucidation of transmission routes. Introduction of these pathogens in a particular setting does not necessarily lead to dominance and endemicity. evertheless, propagation of CP-Kps in a healthcare institution is a clear indicator of the inadequacy of currently applied infection control measures. Fortunately, sound studies have shown that successful containment of carbapenemase producers is feasible. European expert committees and the CDC have recently issued detailed recommendations that include, among others, active surveillance cultures, isolation/cohorting of colonized/infected patients, contact precautions and assignment of dedicated nursing staff. CP-Kp strains mainly affect hospitalized patients with severe underlying conditions, but are also spreading in long-term care facilities. Among the wide spectrum of infections they cause, BSI and pneumonia are the most lifethreatening. Virtually all CP-Kps exhibit multidrug resistance phenotypes. Thus, the dearth of treatment options has led to the extensive use of colistin and tigecycline, since these are the most active drugs in vitro. However, systematic assessment of clinical data accumulated over a decade has demonstrated beyond any doubt that the therapeutic potential of the aforementioned drugs, especially in monotherapy schemes, is suboptimal. It has been suggested that the commonly used colistin dosage regimens are inappropriate and hence associated with possible clinical failure. Indeed, while this drug s toxicity is notorious, its complex pharmacokinetics have not yet been fully elucidated. Consequently, the balance between colistin s efficacy and toxicity remains to be fine-tuned. Regarding tigecycline, its bacteriostatic activity and, most importantly, its unfavorable kinetic properties (low concentrations in serum and epithelial lining fluid) may explain the poor efficacy of monotherapy in serious CP-Kp infections, including primary and secondary BSIs. Adding to these quandaries, there is a notable trend towards increasing MICs for both colistin and tigecycline, evidently associated with the systematic use of these agents. Therapy with carbapenems remains debatable. evertheless, it is reasonable to hypothesize that treating patients with prolonged infusion of maximum doses thus exploiting the PK/PD features of carbapenems may be effective against strains with relatively low MICs for these drugs. The overall safer current trend in the therapy of CP-Kp infections is the use of combination schemes including up to three antibiotics. So far, carbapenem-based combinations (e.g., a carbapenem plus either colistin or an aminoglycoside and/or tigecycline) seem to be the most efficacious. Antibiotic combinations are clearly promising as a more efficacious treatment of CP-Kp infections, though the available data are still limited. Large-scale controlled trials along with experimental studies are needed to validate and refine the combination schemes. ovel antimicrobials active against CP enterobacteria as well as inhibitors active against the most common carbapenemases have been developed by the industry. Some of them, such as plazomicin (an aminoglycoside resistant to all clinically important modifying enzymes), the sulfactam BAL30072 (a monocyclic -lactam active against M L producers) and the non- -lactam inhibitors avibactam and MK-7655 (both highly active against KPC -lactamases), are under advanced clinical testing and may prove valuable in the near future. Future perspective We may reasonably expect that application of the comprehensive infection control measures proposed by the USA and EU public health authorities could drastically limit the spread of CP enterobacterial pathogens, and possibly even eradicate them in low-prevalence settings in a relatively short time period. An utterly successful outcome, however, is far from certain. The recommended policies are demanding in both expertise and, more importantly, resources that are not available in many of the affected areas. Consequently, it would be safer to predict that the problem will persist, at least in the developing countries. ne may be more optimistic regarding the future of antibiotic therapy for CP enterobacteria infections. So far, attempts to assess clinical data, though fragmentary, have facilitated the rationalization of patient management. Reaching a consensus regarding the minimum requirements for the presentation and evaluation of clinical data should become a priority. This is a prerequisite for the setting up of large-scale international trials aiming to define optimal antibiotic treatments for specific patient groups Future Microbiol. (2013) 8(9)

11 Confronting carbapenemase-producing Klebsiella pneumoniae The introduction of novel antibacterial agents will also provide additional therapeutic options. Considering the recent development of several promising compounds that are highly active against CPs as well as potent carbapenemase inhibitors (some of which are to be used in clinical practice quite soon), it is expected that the renewed interest of the industry in producing novel antimicrobials will continue and, probably, be intensified. Technological progress in various relevant areas such as organic synthesis, modeling and high-throughput screening are most likely to facilitate this process. It can safely be surmised that the conventional, though still successful, strategy to design new improved molecules belonging to already established drug classes (recent examples being plazomicin and BAL30072) will remain prevalent in the near future [99]. onetheless, there have been systematic efforts to produce innovative compounds capable of interfering with metabolic pathways of the Gram-negative cell that have not yet been exploited. otable among others are various hydroxamic acid derivatives that can arrest the growth of a wide variety of Gramnegative species including K. pneumoniae, by impeding the biosynthesis of lipid A [100]. In this respect, development of -lactamase inhibitors is a step ahead: the diazabicyclooctanes avibactam and MK-7655 may indeed be the precursors of a brand new family of non- -lactam broad-spectrum inhibitors. Executive summary Antibiotic resistance patterns of carbapenemase-producing Klebsiella pneumoniae strain The global spread of carbapenemase-producing (CP) and extensively drug-resistant enterobacteria of clinical importance, especially Klebsiella pneumoniae (CP-Kp), is currently one of the most pressing public health problems. Global spread of CP-Kp strain High incidence of CP-Kps is commonly seen in developing countries, though increasing isolation frequencies are also being observed in industrialized countries. Cross-border transfer has been recognized as an important contributor to the ongoing evolution of this epidemic. CP-Kp strain in the acute care setting CP-Kp colonizations and infections occur mainly in acute healthcare settings and, to a lesser extent, in long-term care facilities. Claims of community-acquired CP-Kp infections have not been effectively documented. umerous independent risk factors for CP-Kp colonization/infection have been reported. Most relevant studies, however, have been conducted in endemic settings, suggesting that the key factor is the inadequacy of infection-control measures. CP-Kps preferentially affect hospitalized patients with severe underlying conditions causing a variety of life-threatening infections, such as bacteremia and pneumonia, with high mortality rates. Antibiotic therapy of CP-Kp infections The dearth of therapeutic options has led to the extensive use of colistin and tigecycline owing to their in vitro activity against CP-Kps. Yet, the efficacy of these drugs, especially when used in monotherapy, is disappointing. Moreover, colistin- and tigecycline-resistant CP-Kps are being increasingly reported. Currently, there is a shift towards therapy with antibiotic combination regimens. The hitherto available clinical data clearly indicate the superiority of this approach, where carbapenem-based combinations are the most efficacious, presumably because of carbapenems favorable pharmacokinetic/pharmacodynamic characteristics, especially when bloodstream infection and pneumonia are being treated. ovel antimicrobials ovel antibiotics active against CP enterobacteria as well as compounds with potent inhibitory activity against carbapenemases are expected to be available soon. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. o writing assistance was utilized in the production of this manuscript. References Papers of special note have been highlighted as: of interest of considerable interest 1. Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin. Microbiol. Rev. 25(4), (2012). Extensive review on microbiology, epidemiology, infection control and treatment for carbapenemase-producing Enterobacteriaceae, with emphasis on Klebsiella pneumoniae. 2. ordmann P, Dortet L, Poirel L. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol. Med. 18(5), (2012). 3. Cantón R, Akova M, Carmeli Y et al. Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe. Clin. Microbiol. Infect. 18(5), (2012). 4. Hirsch EB, Tam VH. Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause