The threat of carbapenem-resistant Enterobacteriaceae in Lebanon: An update on the regional and local epidemiology

Journal of Infection and Public Health (2012) 5, 233—243

The threat of carbapenem-resistant Enterobacteriaceae in Lebanon: An update on the regional and local epidemiology Rima I. El-Herte a, Souha S. Kanj a, Ghassan M. Matar b, George F. Araj c,∗ a

Division of Infectious Diseases, Department of Internal Medicine, American University of Beirut Medical Center, Beirut, Lebanon b Department of Experimental Pathology, Immunology and Microbiology, American University of Beirut Medical Center, Beirut, Lebanon c Department of Pathology & Laboratory Medicine, American University of Beirut Medical Center, Beirut, Lebanon Received 18 May 2011 ; received in revised form 16 November 2011; accepted 24 February 2012

KEYWORDS Carbapenemases; Lebanon; Multi-drug resistant Enterobacteriaceae; Detection; NDM-1; Treatment

∗

Summary Bacterial resistance to antimicrobial agents is increasing. Complex resistant mechanisms have resulted in a wide spectrum of species and strains with multidrug-resistant patterns. In addition to the production of extended-spectrum␤-lactamases (ESBLs), Gram-negative rods have acquired the capacity to hydrolyze carbapenem antibiotics by means of carbapenemases. The enzyme that has gained the most publicity recently is the New Delhi metallo-␤-lactamase (NDM-1). This enzyme and others are now spreading from their homeland on the Indian subcontinent to other continents, primarily via medical tourists. This spread contributes to be a global threat in an era when no potent antibiotics are expected to be developed. Patients coming from countries where antimicrobial use is not restricted, such as Iraq, may harbor similar organisms. Reports from the Middle East and Arabian countries describing the occurrence of carbapenem-resistant Enterobacteriaceae are rare. In this communication, an update on the epidemiology, prevalence and mechanisms of carbapenem-resistant Enterobacteriaceae in Lebanon and the surrounding region will be addressed in addition to the detection methods and required infection control practices. © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Corresponding author at: American University of Beirut Medical Center, Department of Pathology & Laboratory Medicine; PO Box 11-0236, Riad El Solh, 1107 2020 Beirut, Lebanon. E-mail address: [email protected] (G.F. Araj).

Background The history of antimicrobial resistance dates back to antimicrobial discovery and parallels its use. The

1876-0341/$ — see front matter © 2012 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.jiph.2012.02.003

234 mechanisms of resistance among Gram-negative Enterobacteriaceae vary widely [1]. The emergence of extended-spectrum-␤-lactamases (ESBLs) compromised the effect of most ␤-lactam antibiotics [2]. In this situation, carbapenem antibiotics remain the drug of choice and have been increasingly utilized to treat infections with ESBLproducing organisms [3]. However, the increasing rates of ESBLs isolates are leading to the overuse of carbapenems, creating antibiotic pressure on carbapenems and triggering bacterial resistance against this class of antimicrobial agents. Subsequently, carbapenemases have been increasingly detected among Enterobacteriaceae, limiting the existing antimicrobial armamentarium for treating these resistant isolates [4]. Reports of carbapenem-resistant Enterobacteriaceae have been published from different regions, including Europe, North America and Latin America [5]. Reports from Lebanon are scarce [6,7]. In addition, no publications are available describing the emergence of such strains from neighboring countries aside from Turkey, Israel, Oman [8] and Kuwait [9]. Devastating new carbapenemases, particularly NDM-1, have recently been reported from the Indian subcontinent and subsequently spread to other countries. The above-mentioned complexities and alarming threats warranted this update to shed light on the carbapenemases challenging clinical and laboratory settings and the treatment of the bacterial infections producing these enzymes. In addition, a description of the global and local epidemiology is provided.

Carbapenemases and mechanisms of resistance The carbapenem antibiotic family includes biapenem, doripenem, ertapenem, faropenem (a closely related penem to carbapenems), imipenem, meropenem, panipenem, thienamycin and razupenem. These penems originally developed from thienamycin, a natural, fungal-derived product of Streptomyces cattleya [10]. The bactericidal activity of carbapenems results from the inhibition of cell wall synthesis. Because of their zwitterionic molecule charges, carbapenems readily penetrate the cell wall of most Gram-positive and Gramnegative bacteria to reach and act on targets pertaining to penicillin-binding proteins (PBPs 1A, 1B, 2, 3, 4, 5, 6). Several mechanisms account for the resistance to carbapenems; these include modifications in the target substrate PBP, outer membrane

R.I. El-Herte et al. permeability modifications, and the production of ESBLs and other hydrolyzing enzymes. The latter existed years before the license of carbapenem antibiotics for use in the USA and United Kingdom [4]. Hydrolyzing enzymes occur naturally in some Gram-negative bacteria, such as Stenotrophomonas maltophilia, Flavobacterium, Chryseobacterium spp. and Aeromonas hydrophila. Carbapenem-resistance among Enterobacteriaceae bacteria is primarily associated with the production of carbapenemases. Dual mechanisms, including an outer membrane permeability defect with the production of ␤-lactamases, such as AmpC cephalosporinases and ESBLs, particularly with the presence of the CTX-M variant, have also been described [11].

Classification There are various classifications of carbapenemases in use. The most commonly used is the Ambler classification, which classifies the carbapenemases into three classes, A, B, and D, depending on the amino acid sequence homology. Classes A and D are serine carbapenemases, while class B contains metallo-␤-lactamases (MBL) [12]. An update on the Ambler classification of carbapenemase classes is presented in Table 1. The carbapenemase genes are located on highly mobile genetic elements that contribute to their rapid spread and frequent transfer of multiple other antibiotic resistance genes. A brief description of these classes follows. Class A carbapenemases were first detected in 1982. They include the following enzymes: KPC (Klebsiella pneumoniae carbapenemase), SME (Serratia marsescens enzyme), NMC-A (not metalloenzyme carbapenemase class A), IMI (imipenemhydrolyzing) and GES (Guiana extended-spectrum), each of which have different family members. These enzymes hydrolyze all ␤-lactams including monobactams, have a high affinity for meropenem and hold less hydrolytic activity towards cephamycins and ceftazidime. This class can carry resistance to fluoroquinolones and aminoglycosides because the encoding genes for quinolone and aminoglycoside resistance are carried on the same plasmids. They are inhibited by clavulanic acid and tazobactam but not sulbactam. They are found in Enterobacteriaceae (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella enterica, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae and Serratia marcescens) and non-Enterobacteriaceae (e.g., Pseudomonas aeruginosa and Acinetobacte spp.). The KPC enzyme is the most clinically important in this group and was discovered in

The threat of carbapenem-resistant Enterobacteriaceae in Lebanon Table 1

235

Classes of carbapenemases according to Ambler classification.

Ambler class

Example of enzymes

Inhibition

Antimicrobial hydrolysis properties Yes

No Ceftazidime Cephamycin

A

KPC, IMI, SME, GES NMC-A

Clavulanic acid Tazobactam

All ␤-lactams Carbapenems Monobactam

B metallo-␤lactamses

IMP, VIM, GIM, SPM NDM-1, SIM

EDTA

All ␤-lactams Carbapenems

D oxacillinase

OXA-48

In vitro by NaCl

Penicillins Carbapenems Cephalosporins

C

AmpC CMY + mutation in OMP

Clavulanic acid Sulbactam Tazobactam

Penicillins Cephamycin Oxyiminocephalosporin Azteonam Carbapenem

1996 in North Carolina. The encoding gene blaKPC is plasmid mediated with nine different variants [4,13,14]. These enzymes have been reported in many countries, including Israel, India, France, Portugal, Holland, Greece, Japan, China, Korea, Brazil, Colombia, Argentina and the USA [4]. Class B carbapenemases are metallo-␤lactamases (MBL) and the most clinically significant carbapenemases. Enzymes in this class include IMP (active on imipenem), VIM

Bacterial example

Klebsiella pneumoniae Escherichia coli Enterobacter cloacae Pseudomonas aeruginosa Serratia spp. Salmonella enterica Citrobacter freundii Aztreonam Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli Acinetobacter spp. Citrobacter freundii Serratia marcescens Morganella morganii Providencia spp. Acinetobacter Aztreonam spp. ExtendedProteus spectrumcephalosporins mirabilis Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli Cefepime Enterobacteriaceae

(Verona integron-encoded metallo-␤-lactamase), GIM (German imipenemase), SPM (Sao Paulo metallo-␤-lactamase) and SIM (Seoul imipenemase). IMP-1 was the first metallo-␤-lactamase identified in Japan in 1991 from a Serratia marcescens isolate. These enzymes hydrolyze all ␤-lactams and carbapenems but not aztreonam. They are inhibited by ethylenediaminetetraacetic (EDTA), a metal ion chelator, and resistant to all of the commercially available ␤-lactamase inhibitors.

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R.I. El-Herte et al.

Table 2 Summary of different carbapenemases recovered in different countries neighbouring Lebanon. + indicates present and the number next to each + indicates the subtypes of the different enzymes. OXA included subtypes 1,47,48; VIM included subtypes 1,2,4,5,12,19; KPC included subtypes 2 and 3. Country

Types of detected carbapenemase

Author/year

OXA

Lebanon Matar et al./2008 Matar et al./2010 El-Herte et al./2012

48 48 48

Turkey Poirel et al./2004 Gac ¸ar et al./2005 Aktas¸ et al./2008 Gülmez et al./2008 Carrer et al./2008 Carrer et al./2010 Egypt Cuzon et al./2009 Kuwait Jamal et al./2011 Oman Poirel et al./2010 Saudi Arabia Guerin et al./2005 Israel Navon-Venezia et al./2006 Leavitt et al./2007 Lopez et al./2010 Greece Giakkoupi et al./2003 Loli et al./2006 Ikonomidis et al./2007 Ikonomidis et al./2007

VIM

KPC

NDM-1

+

+1,47,48 5 48 48 48 48 48 + + 48

Psichogiou et al./2008 Tsakris et al./2008 Tokatlidou et al./2008 Giakkoupi et al./2009 Giakkoupi et al./209 Pournaras et al./2009 Hawser et al./2009 Pournaras et al./2010 Kontopoulou et al./2010 Zioga et al./2010

The genes responsible for this are chromosomal or plasmid-encoded and naturally present in Bacillus cereus, Aeromonas spp. and Stenotrophomonas maltophilia [13,15—24]. They have also been found in several Gram-negative bacteria, such as Enterobacteriaceae, Pseudomonas aeruginosa, Brevium diminuta, Pseudomonas fluorescens, Burkholderia cepacia, Achromobacter (Alcaligenes) xylosoxydans, Pseudomonas putida and Acinetobacter baumannii [13,14]. Reports on these enzymes

2 3 3 1 1 4 +1/+2 12 1 2 12 1

19 1

2 2 2 KPC not subtyped 2 2 2

have been noted from different countries around the world, including some from our region (e.g., Greece and Turkey) [13]. In 2008, a new metallo␤-lactamase, NDM-1, was identified in a Swedish patient previously hospitalized in India [25]. As of 2009, NDM-1 had spread from India and Pakistan via medical tourists to the UK, USA, France, Germany, Australia and other countries [26]. In the Gulf region, NDM-1 was first reported in Oman [8], Iraq [27] and, more recently, Kuwait [9] and Lebanon

The threat of carbapenem-resistant Enterobacteriaceae in Lebanon [28]. The bacteria carrying this enzyme are Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Providencia spp. and Morganella morganii. These bacteria were primarily recovered from community-acquired urinary tract infections, pneumonia, and blood stream infections [26]. Bacteria producing this enzyme hydrolyze all ␤-lactam antibiotics and are usually resistant to other classes of antibiotics; thus far, these bacteria only remain susceptible to colistin and tigecycline. The blaNDM-1 gene encoding this enzyme has been located on a plasmid and chromosomes in three UK isolates, suggesting in situ movement [29]. The isolates were from different clones, except for the ones recovered in Haryana, India, which were monoclonal [29]. The bacteria producing MBLs, especially NDM-1, represent a great burden because of their potential for rapid spread throughout the continents and because they often harbor genes encoding for resistance to other classes of antibiotics on the same plasmid (quinolones and aminoglycosides) in an era in which few new potent antimicrobial agents active against carbapenem-resistant organisms are on the horizon. Class D carbapenemases are also called oxacillinhydrolyzing ␤-lactamases (OXAs) [13]. The enzyme was first purified from a multidrug-resistant Acinetobacter baumannii strain isolated in 1985 from a patient in Scotland and first described in 1993 [30]. Its spectrum of hydrolysis covers penicillin, imipenem and meropenem but not the extendedspectrum cephalosporins and aztreonam. Their activity is poorly inhibited by clavulanic acid but is inhibited at variable levels by sodium chloride in vitro. In isolation, the enzyme was designated ARI-1 (for Acinetobacter resistant to imipenem) but was later labeled OXA-23. Most of the OXA-type carbapenemase genes appear to be chromosomally located in Acinetobacter spp., Pseudomonas aeruginosa, Shewanella oneidensis, Shewanella algae, Ralstonia pickettii, and other rare Enterobacteriaceae. Unfortunately, this enzyme was also recovered from some isolates of K. pneumoniae, E. coli and Proteus spp. Currently, 121 different variants of class D ␤-lactamases have been identified on the protein level, and 45 of these, namely the OXA-48 enzyme encoded by the blaOXA-48 gene, exhibit carbapenem-hydrolyzing activities. This enzyme was reported in different countries around the world, including those from our region (e.g., Turkey, Egypt, Saudi Arabia and Lebanon) [6,7,13,14,30,31]. The other mechanisms by which Enterobacteriaceae are resistant to carbapenems are the expression of AmpC and an altered outer membrane

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protein (OMP) that causes decreased permeability to carbapenems; these strains usually remain susceptible to cefepime and imipenem but may become ertapenem resistant. AmpC, which belongs to class C ␤-lactamases, was described in 2006, when it was isolated from a virulent strain of Enterobacter aerogenes. These strains are resistant to penicillins, cephalosporins, cephamycins, oxyiminocephalosporins, ertapenem and aztreonam but not to cefepime. The gene is plasmid mediated and often carries multiple other genes for resistance to aminoglycosides, chloramphenicol, quinolones, sulfonamide, tetracycline and trimethoprim, as well as genes for other ␤-lactamases. Benzylpenicillin, ampicillin, amoxicillin and cephalosporins (cefazolin and cephalothin) are strong inducers and good substrates for AmpC ␤-lactamase. Cefoxitin and imipenem are also strong inducers but are much more stable for hydrolysis. Cefotaxime, ceftriaxone, ceftazidime, cefepime, cefuroxime, piperacillin and aztreonam are weak inducers and weak substrates but can be hydrolyzed if enough enzymes are produced. Consequently, the MICs of weakly inducing oxyimino-␤-lactams are dramatically increased with AmpC hyperproduction. Clavulanic acid, sulbactam, and tazobactam have much less effect on AmpC ␤-lactamases, although some are inhibited by tazobactam or sulbactam. Thus, organisms producing enough AmpC ␤lactamase will typically have a positive ESBL screening test but fail the confirmatory test involving increased sensitivity with clavulanic acid. The culprit enzyme is CMY (cephamycin hydrolyzing) [32]. The development of resistance upon therapy is a major concern; an accompanying loss of outer membrane porins can further augment the resistance phenotype. The gold standard for confirming their presence is using polymerase chain reactions [33]. Table 1 summarizes the different classes of carbapenemases.

Carbapenemases producing enterobacteriaceae reported from Lebanon and neighboring countries Reports on the presence of carbapenem-resistant Enterobacteriaceae in the region are limited to a few countries: Lebanon, Turkey, Greece, Israel, Egypt, Kuwait and Saudi Arabia. Most of the reports are from Turkey, Greece and Israel. In Lebanon, OXA-48 (class D) was the first carbapenemase reported, in 2008, followed by another report in 2010; both were produced by E. coli and K. pneumoniae. Genetic studies did not reveal any

238 genomic relatedness between the Lebanese and Turkish strains harboring the plasmid encoding the OXA-48 gene, indicating that the dissemination of carbapenem resistance is not clonal but through horizontal plasmid transfer [6,7]. In 2009, a study from Lebanon revealed that 2.5% of E. coli and 7.84% of K. pneumoniae were ESBL producers and carbapenem resistant [antimicrobial brochure at the American University of Beirut Medical Center (AUB-MC)]. Most recently, E. coli and K. pneumoniae with NDM-1 were recovered from Iraqi patients referred for treatment in France [27] and in Lebanon [28]. A summary of the different carbapenemases recovered from patients in Lebanon and its neighboring countries is presented in Table 2. In Turkey, the earliest report on carbapenemases was published in 2001 and described class D OXA-48. Subsequently, many reports were published about VIM (VIM-5) and IMP recovered from different Enterobacteriaceae that were not genetically related to the strains recovered in Lebanon [15—18,34—36]. In Greece, several reports have been published about encountered carbapenemases. They were class A KPC-2 and class B VIM types 1, 2, 4, 12 and 19 and recovered from different Enterobacteriaceae [19—24,37—41]. Reports from Israel have described the detection of different Enterobacteriaceae producing class A KPC subtype-2 and -3 since 2004 [42—46]. The spread of a KPC-3 producing K. pneumonia from Israel to Colombia, the UK, Norway, Sweden and the USA via medical tourists has also recently been reported [46]. Although the various countries discussed share regional proximity, it is interesting to note the differences in the recovered carbapenemases. For example, similarities in the KPC enzymes were noted between Israel and Greece, and similarities between the OXA enzymes were noted between Turkey and Lebanon. These similarities may be attributed to interactions between different populations and communities in these countries; the exact factors that promote such differences remain to be identified. The recovery of these isolates from Egypt was noted in only one report, which described K. pneumoniae producing class D OXA-48 in an Egyptian patient hospitalized in France [47]. There is only one report from Saudi Arabia of Pseudomonas aeruginosa harboring the blaVIM-2 from a Saudi patient hospitalized in France [48]. Recently, Poirel et al. reported a transfer of NDM-1 from Iraq to France through an Iraqi medical tourist [26]. Moreover, infection with NDM1-producing K. pneumoniae has also been reported very recently in Kuwait [9].

R.I. El-Herte et al. Thus far, no reports of carbapenem-resistant Enterobacteriaceae have been published from Iran, Jordan, or Syria. Table 2 summarizes the enzymes reported from isolates recovered in different countries in the Middle East.

Detection The prompt detection of carbapenemase-producing bacteria is of paramount importance, as it has an impact on both appropriate therapeutic measures and infection control practices. Though carbapenemase production can be inferred from the resistance profile of the isolate, further phenotypic and genotypic tests should be done for confirmation. A carbapenem-resistant Enterobacteriaceae is usually suspected when resistance to ertapenem or increased MIC (see detail in the coming paragraph) to other carbapenems that remains within the susceptibility range is observed. For example, in one study, the positive predictive value and specificity of ertapenem resistance for KPC detection were 74% and 99.2%, respectively [49]. This is attributed to the fact that resistance to carbapenem can be due to the presence of CTX-M, ESBLs and porin mutations [49]. Definitive identification, however, was achieved with molecular testing for high sensitivity and specificity (100% and 96.4%, respectively) [50].

Screening: disk diffusion and MIC susceptibility testing Screening begins when a strain is suspected to have reduced susceptibility to carbapenems (decreased inhibition zone diameter or increased MIC). In disk diffusion susceptibility testing, a 10 ␮g ertapenem disk is used as the surrogate marker to screen for carbapenem resistance in the tested isolates. A zone of inhibition or mutant detection at the intermediate (16—18 mm) or resistant (≤15 mm) breakpoint zones should be analyzed further using MIC and Hodge testing. The strains typically indicate high MIC (presented below) regardless of the test used (broth dilution, E test, or automated system) and can vary according to different strains, but the MIC is usually greater than that of the correspondent wild type of the bacteria. A falsenegative result can be due to a low inoculum. The automated susceptibility testing instruments have variable sensitivity, ranging from 7% to 87% [51,52]. A strain is considered carbapenem resistant if the following breakpoints are found, according to the CLSI (updated January 2011) [53]: in disk

The threat of carbapenem-resistant Enterobacteriaceae in Lebanon

239

Table 3 Summary of most commonly used confirmatory methods for carbapenemases. DPA = dipicolinic acid; MBL = metallo-␤-lactamase; EDTA = ethylenediaminetetraacetic acid; KPC = Klebsiella pneumoniae carbapenemase; OXA = oxacillinase; BA = boronic acid; CTX-M = cefotaximase. Test

Sensitivity/specificity

Comments

Modified Hodge test

100%/78%

Carbapenemases inhibition

100%/98%

Amino-acid amplification

100%/97%

Chromogenic agar media

100%/98%

Cannot differentiate among the different types of carbapenemases DPA inhibits MBL EDTA inhibits MBL,KPC, OXA BA inhibits KPC, AmpC False positive in AmpC plus Omp, CTX-M Available only in referral centers allows differentiation of carbapenemases Cannot differentiate among the different classes of carbapenemases

diffusion testing against 10 ␮g meropenem, 10 ␮g doripenem, and 10 ␮g ertapenem; a zone diameter ≤19 mm (the imipenem disk performs poorly as a screen for carbapenemases); and in MIC testing with ≥4 mg/l against imipenem, meropenem and doripenem, and MIC ≥1 mg/l against ertapenem. The use of ertapenem as the surrogate test is due to it being the least active carbapenem against KPCs, and the use of this drug in both manual and automated susceptible testing methods has been found to be highly sensitive for detecting KPC [49].

Confirmation The most commonly used confirmatory tests for detecting carbapenemases are the modified Hodge test, carbapenemase inhibition tests and molecular methods (Table 3) [53,54]. Polymerase chain reaction (PCR) is the most reliable, sensitive and specific test for detecting carbapenemases. PCR followed by spectrophotometric measurements of carbapenem hydrolysis are considered to be the reference standard methods for detecting carbapenemase production [50,55].

Modified Hodge test This test, also known as the cloverleaf method, is recommended by the CLSI to phenotypically detect the presence of carbapenemases. The test is based on the growth of a susceptible strain of E. coli ATCC 25922 around the area of the resistant strain producing carbapenemases. This test is done by inoculating a 0.5 McFarland dilution of E. coli ATCC 25922 on a Mueller Hinton agar plate. A 10 ␮g meropenem or 10 ␮g ertapenem susceptibility disk is placed in the middle of the inoculated plate. A

wetted swab with the suspected test organism (0.5 McFarland) is then streaked from the edge of the disk to the edge of the plate. Up to four organisms can be tested on the same plate with one drug. After 16—24 h of incubation, the plate is examined for a cloverleaf-type indentation at the intersection of the test organism and E. coli within the zone of inhibition of the carbapenem susceptibility disk. The sensitivity of this test is excellent (95—100%), but it cannot differentiate between the different types of carbapenemases (specificity = 78%) [54]. The test might give false-positive results if the strain produces ESBLs, AmpC, CTX-M or a weak MBL and has an alteration in the outer membrane protein [50,55,56].

Carbapenemase inhibition test This test is based on the enhanced inhibition zone of the resistant strain when carbapenemase inhibitors are inoculated within the carbapenem disk. There are many carbapenemases inhibitors; the most important carbapenemases inhibitors are dipicolinic acid (DPA), EDTA, boronic acid (BA) and cloxacillin [55]. The DPA inhibits all of the MBL. The EDTA inhibits the MBL, KPC, CTX-M and the oxacillinases. The BA will inhibit class A KPC, class C AmpC and CTX-M ESBLs. Various studies have been performed regarding this method with various inhibitor concentrations. Optimal results are obtained if a Mueller—Hinton agar plate is streaked with a 0.5 McFarland inoculum of the tested organism followed by a 10 ␮g meropenem disk, 10 ␮g meropenem disk plus 1000 ␮g DPA [55], 10 ␮g meropenem disk plus 750 ␮g EDTA, 10 ␮g meropenem disk plus 600 ␮g APBA, and 10 ␮g meropenem disk plus 750 ␮g cloxacillin [57]. An increase ≥5 mm in the inhibitory zone diameter

240 around the carbapenem disk imbibed with the inhibitor confirms the presence of carbapenemases. A false-positive result might be obtained if the strain has an OMP modification and produces ESBL, AmpC or CTX-M. The sensitivity and specificity are 100% and 98—100%, respectively [50,55—57].

Molecular methods The definitive method for confirming the type of carbapenemase present is PCR amplification and sequence analysis of extracted DNA, which permits the identification and sequencing of the different encoding genes, e.g., pertaining to the class A enzymes blaKPC , blaIMI , blaNMC , and blaGES [12]; metallo-␤-lactamases (MBLs) blaVIM , blaIMP , blaGIM , blaSPM , blaSIM [12] and blaNDM ; and class D OXAtype carbapenemases blaOXA-23 and blaOXA-48 [12], with the sensitivity and specificity of these methods reaching 100% and 97%, respectively [58,59]. Though these tests are only available in research and referral centers, some clinical labs are turning to PCR amplification as a means of detection to overcome problems faced with phenotypic bench tests [50]. Other genes for antimicrobial resistance should be searched for (e.g., a quinolone resistance gene), as they often coexist on the same plasmid. Table 3 summarizes the detection and confirmatory tests. When treating with a patient with a highrisk probability of carrying a carbapenem-resistant organism, it is recommended to screen samples from rectal swabs, tracheal secretions and surgical sites. Organisms demonstrating decreased breakpoints should be confirmed by one of the above tests.

Treatment Limited options are currently available to treat carbapenem-resistant Enterobacteriaceae, as they are resistant to all ␤-lactam antibiotics and very often carry the same transposon genes responsible for resistance to TMP-SMX, aminoglycosides and fluoroquinolones [60]. Active agents include tigecycline, colistin and fosfomycin [61]. Tigecycline is a glycylcycline, which inhibit protein translation in bacteria by binding to the 30S ribosomal subunit and blocking the entry of aminoacyl tRNA molecules into the A site of the ribosome. This prevents the incorporation of amino acid residues into elongating peptide chains. This treatment is indicated for the skin, soft tissue and intraabdominal infections. Its high tissue distribution

R.I. El-Herte et al. and the limited clinical experience for urinary and primary bloodstream infections make it a poor candidate for treating such infections, despite high in vitro susceptibility [62]. Reports describing the use of tigecycline for this indication have ranged from failure to favorable results when used either as a monotherapy or in combination with colistin [57,63]; the major side effects are nausea, vomiting, anaphylaxis/anaphylactoid reactions, acute pancreatitis, hepatic cholestasis, and jaundice. Colistin, or colistimethate sodium, is a surfaceactive agent that penetrates into and disrupts the bacterial cell membrane. It has been shown to have bactericidal activity against most strains. Though developed long ago, it is being reused in desperate situations in the era of antibiotic resistance to treat infections with these resistant strains [57,64]. It has a high rate of success when used in combination therapy but not as a monotherapy. However, Enterobacteriaceae resistant to colistin have been recently described [40]. The concern with colistin is its nephrotoxicity, decreased creatinine clearance, increased urea, respiratory distress and apnea. Fosfomycin inhibits bacterial cell wall biogenesis by inactivating the enzyme UDP-Nacetylglucosamine-3-enolpyruvyltransferase, also known as MurA. This enzyme catalyzes the committed step in peptidoglycan biosynthesis. In CLSI 2011 [53], it is indicated for use against E. coli UTI isolates only. However, a recent study reported excellent in vitro activity of fosfomycin against K. pneumoniae strains producing KPC resistant to either colistin and/or tigecycline. However, no clinical correlation was found; the susceptibility rates were 93% for the overall group, 87% for the tigecycline and/or colistin non-susceptible subgroup, and 83% for the extremely drug-resistant subgroup (tigecycline and colistin non-susceptible subgroup) [65]. The most common side effects are diarrhea, headache, vaginitis, nausea, rhinitis, back pain, dysmenorrhea, pharyngitis, dizziness, abdominal pain, anaphylaxis and hearing loss. Because of the above-noted limitations in therapeutic choices, several drugs have been under research and development. For example, the newly developed drug BAL30376, which is a combination of BAL19764 (a metallo-␤-lactamase stable monobactam), BAL 29880 (bridged monobactam to inhibit AmpC) and clavulanate (inhibits ESBLs), was active against strains producing MBL and OXA-48 but demonstrated poor activity against KPCproducing Enterobacteriaceae due to clavulanate’s poor inhibitory activity against these enzymes [66]. In a phase II trial study, NXL104 (a ␤-lactamase inhibitor) in combination with ceftazidime or aztreonam was tested against carbapenem-resistant

The threat of carbapenem-resistant Enterobacteriaceae in Lebanon Enterobacteriaceae. Ceftazidime + NXL104 was active against strains with the OXA-48 enzyme or combinations of impermeability and an ESBL or AmpC enzyme and against most Klebsiella spp. with the KPC enzyme, but it was not effective against metallo-␤-lactamase producers. Aztreonam + NXL104 was active against all strains, including those with metallo-␤-lactamases, e.g., the NDM-1 gene [67]. LK-157 is a novel tricyclic carbapenem that has been shown to have potent activity against class A and class C ␤-lactamases, indicating the need for further development [68]. The activity of BLI-489, a bicyclic penem molecule, was tested against a wide variety of enzymes but still must be assessed against KPCproducing organisms [69]. Lastly, ACHN-409, a new-generation aminoglycoside (neoglycoside), is currently under investigation and appears to have potent in vitro activity against KPC-producing isolates [70]. Due to the likely spread of these highly lifethreatening bacteria, physicians should remain highly suspicious of infections with these resistant organisms for any patient presenting from endemic areas. Thus, screening suspected patients (e.g., by sampling rectal, tracheal, and wound swabs) is warranted upon admission to the hospital. Moreover, prompt infection control measures with proper contact isolation should be implemented until the infection or colonization with these highly resistant bacteria has been ruled out.

Conclusions Similar to many other countries worldwide, Lebanon and its neighboring countries are now facing a dangerous threat with the emergence of carbapenem-resistant Enterobacteriaceae. The currently available antibiotics for treatment have limited efficacy. There are few potent antimicrobial agents with activity against these multidrugresistant bacteria; there are very few agents that have good prospects. A multidisciplinary approach to limit the spread of these organisms is essential. Acute attention and a high degree of alertness should be maintained when facing any patient who has been in areas endemic with these carbapenemases, such as the Indian subcontinent and recently, Iraq. Prompt detection, proper prevention, antimicrobial stewardship and adequate infection control measures should aid in limiting the spread of these organisms.

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Funding No funding sources.

Competing interests None declared.

Ethical approval Not required.

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