ABSTRACT

Introduction: New Delhi metallo--lactamase-1 (NDM-1) is a recently reported novel plasmid-borne metallo--lactamase that represents an emerging public health threat. Initially identified as a significant healthcare risk on the Indian sub-continent, it has rapidly become a global problem, posing significant diagnostic and management challenges. Aim: To determine the prevalence of blaNDM-1 gene in Carbapenem resistant Enterobacteriaceae isolated from clinical samples by polymerase chain reaction. Materials & Methods: The study was performed on routine samples subjected to culture and antibiotics susceptibility.100 Bacterial cultures of enterobactericea isolated from different types of clinical samples were randomly selected. Of which, 14 were reported resistant to imipenem on primary antibiotic susceptibility testing. These 14 isolates were further studied for the presence of gene blaNDM-1 by PCR. Results: Carbapenem-resistance was reported in 14% (14/100) isolates based on antibiotics susceptibility testing. Of these 14 isolates, Carbapenem-resistance gene, blaNDM-1, was detected in only (02 /14) isolates. Conclusion: Prevalence of blaNDM-1 containing enterobacteriaceae is not very high in this area. Key words: Carbapenem resistance, New Delhi metallo--lactamase-1, enterobactericea

CHAPTER 01 INTRODUCTION
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1.1 Carbapenem:
Carbapenems are a class of -lactam antibiotics with a broad spectrum of antibacterial activity. They have a structure that renders them highly resistant to most lactamases.i Carbapenem antibiotics were originally developed from thienamycin, a naturally derived product of Streptomyces cattleya.ii They have traditionally been reserved for therapy of suspected or confirmed infection in patients who are critically ill and infection in patients known or suspected to be colonized with multidrug resistant Gramnegative bacilli.

1.2 Carbapenem-resistant Enterobacteriaceae (CRE):

Carbapenem-resistant enterobacteriaceae is a family of gram-negative bacterium that are nearly immune to the carbapenem class of antibiotics, considered the "drug of last resort" for such infections.CREs are resistant to most classes of antimicrobials; frequently, the only therapeutic options available are polymixin, tigecycline, and sometimes aminoglycosides. Since therapeutic options are scarce, infections due to CRE are associated with severe and adverse clinical outcomes. These poor outcomes are due primarily to a delay in the initiation of effective antimicrobial therapy.iii

1.3 Mechanism of resistence:

There are a number of mechanisms of Carbapenem Resistance in Enterobacteriaceae. These include (1) Hyperproduction of ampC beta-lactamase with an outer membrane porin mutation (2) CTX-M extended-spectrum beta-lactamase with a porin mutation or drug efflux (3) Carbapenemase production.

Figure 1a. Primary mechanisms of b-lactam resistance in Enterobacteriaceae.

Hydrophilic channels formed by porins allow the uptake of b-lactams through the outer membrane of the bacterium. In the periplasmic interspace, b-lactam molecules bind irreversibly to the penicillin-binding proteins (PBPs), leading to the inhibition of the peptidoglycan synthesis. Primary mechanisms of b-lactam resistance in Enterobacteriaceae include the following: (i) enzymatic inactivation of the antibiotic by chromosome- and/or plasmid-encoded enzymes possessing hydrolytic activity against b-lactam molecules; (ii) decreased outer membrane permeability through production of modified porins, loss of porin expression, or a shift in the types of porins found in the outer membrane; and (iii) efflux of the antibiotic to the outside of the bacterium through production of an efflux pump.

1.4 Carbapenemases: Carbapenemases are enzymes that inactivate carbapenems and sometimes other classes of -lactams. This subset of BLs belongs to Ambler classes A, B, and D. Each class has specific enzyme types based on molecular differences
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(Table1).Carbapenemases are found in Enterobacteriaceae, Pseudomonas spp., and Acinetobacter spp. The majority of genes controlling carbapenemase production are transferable by plasmids. For the most part, BL-inhibitors, such as clavulanic acid, tazobactam, and sulbactam, are inactive against carbapenemases. Classification:iv I. Ambler Class A Carbapenemases:

Serine carbapenemases include many subtypes (Table 1). Gene(s) that control their production can be located on the bacterial chromosome or a plasmid. Serine carbapenemases can decrease bacterial susceptibility to all -lactams. Imipenem and cefoxitin induce chromosomal-based serine carbapenemase production, but not plasmid-borne production. This induction confers resistance to carbapenems, penicillin, and aztreonam, but not to extended spectrum -lactams. KPC is the most common carbapenemase in the United States. Although first recognized in K. pneumoniae, KPCs are now distributed in other Enterobacteriaceae.KPC-producing isolates are generally resistant to quinolones and aminoglycosides but usually susceptible to colistin and tigecycline.

II.

Ambler Class B Carbapenemases:

Class B carbapenemases are referred to as MBLs because they require the presence of zinc to function. Due to this zinc dependency, chelators such as EDTA inhibit MBL activity. Until 2009, the MBL carbapenemase subtype Verona integron-borne metallo betalactamase was the most widely disseminated. In 2009, a novel MBL subtype was recognized in a K. pneumoniae isolate from a Swedish patient originally treated in
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New Delhi, India. The enzyme was named NDM-1 (New Delhi MBL-1). NDM-1 is now disseminated worldwide in Enterobacteriaceae and other GNB. The term superbug is used to describe bacteria carrying the NDM-1 because these bacteria are resistant to most antibacterials with perhaps the exceptions of colistin, fosfomycin, and tigecycline. Although infections with bacteria possessing the NDM-1 enzyme can be fatal, NDM-1 bacterial colonization also occurs without disease.

III.

Ambler Class D Carbapenemases

This group of carbapenemases is of the oxacillinase (OXA) enzyme type. They have weak activity against carbapenems and are found primarily in Pseudomonas aeruginosa, Acinetobacter baumannii, and rarely in isolates of Enterobacteriaceae from the United States. OXA CPM genes can be located on bacterial chromosomes or plasmids. Activity of OXA carbapenemases can be increased by upstream elements that control gene expression.

IV.

AmpC -lactamase:

AmpC BLs belong to Ambler Class C. Genes for the production of AmpC-BL can exist on bacterial chromosomes or plasmids in P. aeruginosa and many Enterobacteriaceae. AmpC genes on the bacterial chromosome produce low levels of BLs (repressed) but can become de-repressed by induction by antibacterials such as cefoxitin. When de-repressed, the BL is hyperproduced. AmpC genes located on plasmids constitutively produce the BL. AmpC-BL has minimal activity against carbapenems and monobactam (eg, aztreonam).

Table 1:Classification of the various carbapenem hydrolyzing betalactamases:

1.5 New Delhi metallo--lactamase-1 (NDM-1):

NDM-1 was first detected in a Klebsiella pneumoniae isolate from a Swedish patient of Indian origin in 2008. It was later detected in bacteria in India, Pakistan, the United Kingdom, the United States,v Canada,vi Japanvii and Brazil.viii The most common bacteria that make this enzyme are Gram-negative such as Escherichia coli and Klebsiella pneumoniae, but the gene for NDM-1 can spread from one strain of bacteria to another by horizontal gene transfer. In contrast to several other carbapenemase genes, the blaNDM-1 gene is not associated with a single clone but rather with nonclonally related isolates and species. It has been identified mostly in E. coli and K. pneumoniae and to a lesser extent in other enterobacterial species .The level of resistance to carbapenems of NDM-1 producers may vary. Plasmids carrying the blaNDM-1 gene are diverse and can harbor a high number of resistance genes associated with other carbapenemase genes (oxacillinase-48 [OXA-48] types, VIM types), plasmid6

mediated cephalosporinase genes, ESBL genes, aminoglycoside resistance genes (16S RNA methylases), macrolide resistance genes (esterase), rifampin (rifampin modifying enzymes) and sulfamethoxazole resistance genes as a source of multidrug resistance and pandrug resistance . The association of such a high number of resistance genes in single isolates has been rarely observed, even among the other carbapenemase producers. Many NDM-1 producers remain susceptible only to tigecycline, colistin and to a lesser extent fosfomycin ixx.

1.6 Global Spread of Carbapenemase producing Enterobacteriaceae:

Carbapenemases increasingly have been reported in Enterobacteriaceae in the past 10 years. Klebsiella pneumoniae carbapenemases have been reported in the United States and then worldwide, with a marked endemicity at least in the United States and Greece. Metalloenzymes (Verona integronencoded metallo--lactamase, IMP) also have been reported worldwide, with a higher prevalence in southern Europe and Asia. Carbapenemases of the oxacillinase-48 type have been identifi ed mostly in Mediterranean and European countries and in India. Recent identify caution of New Delhi metallo--lactamase-1 producers, originally in the United Kingdom, India, and Pakistan and now worldwide, is worrisome.xi

1.8 Prevalence in Pakistan:

Gram-negative bacteria from the Enterobacteriaceae family with resistance to carbapenems conferred by New Delhi metallo-b-lactamase 1 (NDM-1) were identified in isolates from India (Chennai, Haryana and other sites), the UK and Pakistan. Isolates from Mumbai (32 isolates), Varanasi (13), and Guwahati (three) in India, and 25 isolates from eight cities in Pakistan (Charsadda, Faisalabad, Gujrat, Hafizabad, Karachi, Lahore, Rahim Yar Khan, and Sheikhupura) were NDM-1-positive bacteria.NDM-1 was found mostly among Escherichia coli and Klebsiella pneumoniae which were resistant to all antibiotics except tigecycline and
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colistin. Many of the UK NDM-1 positive patients had travelled to India or Pakistan in the past year or had been admitted to a hospital in these countries for procedures ranging from bone marrow transplant to cosmetic surgery.ix

The colonization rate of NDM-1 producers in one Pakistani city has been estimated at more than 18% by Perry, J.D. et al. (2011).xii To date there is only one reported death attributed to an infection with a bacterium carrying NDM-1 occurring in a Belgian being repatriated in Belgium after a car accident during a trip in Pakistan.xiii

Figure 2.A) Worldwide geographic distribution of Klebsiella pneumoniae carbapenemase (KPC) producers. Gray shading indicates regions shown separately: B) distribution in the United States; C) distribution in Europe; D) distribution in China.

Figure 3. Worldwide (A) and European (B) geographic distribution of Verona integron encoded metallo--lactamase (VIM) and IMP enterobacterial producers.

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Figure 4. Geographic distribution of New Delhi metallo--lactamase-1 producers, July 15, 2011. Star size indicates number of cases reported. Red stars indicate infections traced back to India, Pakistan, or Bangladesh; green stars indicate infections traced back to the Balkan states or the Middle East; and black stars indicate contaminations of unknown origin.

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AIMS & OBJECTIVES

To determine the prevalence of blaNDM-1 gene in Carbapenem Resistant Enterobacteriaceae isolated from clinical samples by Polymerase chain reaction (PCR).

CHAPTER 02
REVIEW OF LITERATURE

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Yong D et al, in 2009 stated that, the most recently emerged carbapenemase is the New Delhi metallo-beta-lactamase (NDM-1). NDM-1 was named after the place (New Delhi, India) where it was assumed to have originated, similar to other nomenclature for metallo-lactamases, Verona imipenemase (VIM), German imipenemase (GIM), So Paulo MBL (SPM) and Seoul imipenemase (SIM). The first report of NDM-1 producing strain (Klebsiella pneumoniae) was from Sweden in December 2009 (the patient had received medical care in New Delhi). The strain was found to be resistant to all antibiotics except colistin.xiv According to Kumarasamy KK et al (2010), since its first description, NDM-1-producing organisms have been reported in hospitalized patients in Europe, Australia, and North America, in the studies that focused primarily on hospitalized patients returning from South Asian countries, India and Pakistan, in particular.ix Munir A et al also stated that International travel and the use of multiple healthcare systems contribute to the rapid spread of blaNDM-1, with potentially serious consequences.xv M. A. Islam et al (2012), conducted a study for NDM-1 in Bangladesh, a country that, along with India and Pakistan, forms the Indian subcontinent and found that 3.5% of the Gramnegative organisms produced NDM-1.xvi In the Kumarasamy et al study, it was noted that blaNDM -1 also coexisted with amino glycoside resistant genes like blaOXA- 23 and armAxvii. A similar study by Poirel et al, observed that the NDM-1 gene in a strain of Citrobacter freundii was accompanied by 9 different types of beta lactamase
xviii

. The blaNDM -1 gene is located on plasmid, which can

be easily transferred to other bacteria by conjugation, and it is found to be located on plasmid of different sizes, which predicts its mobility and pathogenesis.xix Laboratory detection of NDM -1 involves: 1) Identification of the microorganism, 2) Detection of metello -beta- lactamase production and 3) Identification of bla NDM -1.xx,ix,xv

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In a study by Bores A et al, it was observed that crude mortality and attributable mortality in the patients of carbapenemase producing Kliebsella pneumoniae bacteremia was 71.9% and 50% respectively.xxi In a similar study by Patel G et al, it was noted that mortality among the cases with carbapenemase resistant Klebsiella pneumoniae was significantly more as compared to control (40% Vs 20%). Timely administration of in vitro sensitive antibiotics was not associated with survival.
xxii

Young D et al, described the various factors like over the counter use of antibiotics, irrational use of antibiotics, easy accessibility of higher antibiotics, poor sanitation, high prevalence of diarrhea and overcrowding are considered responsible for development and spread of these organisms.xv

CHAPTER 03
MATERIALS AND METHODS

This study was conducted in the Lahore. The practical work was done at Department of Microbiology, Institute of Public Health, Lahore, Pakistan. In this study 100 clinical isolates of enterobacteriaceae were selected, including E. coli, K. pneumonia, Pseudomons, Proteus. Of which 14 isolates, E. coli (06), K. pneumonia (05), Pseudomons (03), that was resistant to carbapenms on routine susceptibility testing analyzed for resistant blaNDM-1 gene by PCR. The nature of samples was urine, pus, respiratory secretions and blood. The specimens were collected from three different laboratories of Lahore.

1. 2. 3.

Chughtai Lahore Laboratory Post Graduate Medical Institute laboratory Jinnah Hospital laboratory

50 30 20

14

Total samples collected

100

STUDY DESIGN:
This was a cross sectional study design.

SAMPLE SETTING:
Department of Microbiology, IPH Lahore.

SAMPLE SIZE:
100 samples.

SAMPLING:
Convenient sampling INCLUSION CRITERIA All the enterobactericae cultur resistant to Carbapenems. EXCLUSION CRITERIA Culture of the enterobactericae sensitive to Carbapenems. DURATION OF STUDY:

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 October, 2011 to January, 2012.

DATA ANALYSIS:
Data is analyzed by using SPSS version 10.0 and get frequency of bla-NDM 1 positive bacteria.

METHODOLOGY:
Extraction of DNA: (PHENOL & CHLOROFORM METHOD): DNA extraction for carbapenem resistant bacterial strains was performed by using the phenol chloroform extraction method.

The loop full colony of bacteria from pure culture was picked from a plate using a sterilized wire loop and transferred to a 1.5ml eppendorf tube containing 1ml of PBS (Phosphate Buffer Saline).

Centrifuged at 13000 rpm for 5 mins. Supernatant was discarded and the pellet was re-suspended in 100 l TE 10:1

Boiled the suspension (or heat at 95C) for 05 mins. Dilute the lysed DNA to 10 fold in TE 10:1.

Added 250 l of supernatant and 750 l of Tris-reagent in tube. Mixed with pipette and vortexed.
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Placed at room temperature for 05 mins. Added 200 l of chloroform, mixed by inverting the tube 50-60 times and vortexed for 10 secs.

Placed at room temperature for 10 mins. Centrifuged for 12 mins. at 13000-15000 rpm. During this, labeled new 1.5 ml microtube & put 500 l isopropanol in tube. Transfered the upper transparent phase in isopropanol containing tube. Inverted the tube 50 times gently ant placed at room temp for 10 mins. Precipitated DNA from the interphase and organic phase with ethanol (add 0.3ml of 100% ethanol per 750 l of tri-reagent.

Sedimented the DNA by centrifugation at 12000-13000 rpm for 5 mins. Removed the Phenol-ethanol supernatant. Washed the DNA pellet twice in a solution containing 0.1 M sodium citrate in 10 % ethanol (used 01 ml of solution per 750 l of tri-reagent.

Repeated this DNA wash 2 times and in each wash, stored the DNA pellet in washing solution for 30 mins. at room temperature with periodic mixing and centrifuge at 12000-13000 rpm for 5 mins.

Removed the ethanol wash and briefly air dry the DNA pellet by keeping tubes open for 3-5 mins at room temp.

Dissolved the DNA pellet in 8 mM NaOH by slowly passing through the pipette. This gave a DNA conc. of 0.2-0.3 g / l.

POLYMERASE CHAIN REACTION (PCR):

Detection of bla NDM-1 gene was performed by the conventional PCR assay. PRIMER DESIGNING: A 475 bp region of NDM -1 gene was amplified through the conventional PCR assay using NDM-1-specific primers.

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F And R

-(5-GGG CAG TCG CTT CCA ACG GT-3)

-(5-GTA GTG CTC AGT GTC GGC AT-3)

MASTER MIX: The PCR master mix (25 l) consisted of: Buffer dNTPs Taq Polymerase MgCl2 Forward Primer Reverse Primer De-ionized water Template DNA Total (Master Mix) 2.5 l 2.5 l 0.4l 2.0l 0.75 l 0.75 l 15.1 l 1 l 25 l

PROCESS OF PCR:
All PCRs were carried out adhering to the standard precautions to avoid contamination. These included preparation of reaction mixtures and clinical specimens in two separate places (DNA-free PCR cabinet and DNA-Preparation PCR room) and the use of gloves, laboratory coats, facemasks. PCR primers and extracted DNA were added to a PCR master mix. The initial step of the reaction was denaturation of DNA at 94 o C for 3 min, followed by 30 cycles of denaturation, annealing and extension at 94C, 60C and 72C, respectively, for 30 s each. The final extension step was performed for 3 min at 72C.

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ELECTROPHORETIC DETECTION OF PCR PRODUCT:

Each PCR product was loaded on to an ethidium bromide stained 1 % agrose in Tris Borate EDTA (TBE), with 50 bp marker and run at 20 V for about one hour prior to viewing under UV to check for the presence of 475 bp PCR product.

RESULTS:
100 clinical isolates of enterobacteriacea were selectd for this study from 3 different labs. Of which 14 Carbapenem resistant strains are analyzed for blaNDM-1 gene by PCR. From these 14, blaNDM-1 gene was found only in 02. Figurur:05

Frequency of carbapenum resistance out of 100 sample are 14 Which is recorded in this research
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Figurur:06

Frequency of bla-NDM gene from resistance cases is 13% that was recoded

20

Figurur:07

Ratio of resistant groups

Is 50% for both groups

CHAPTER 05

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Discussion
NDM-1 producing bacteria are the most common cause of urinary infections. They can also cause bloodstream infections, pneumonia, and wound infections. Most patients will have fever and fatigue. Symptoms reflect the site of the infection and it does not differ between bacteria that express NDM-1 and those that do not. However, patients who have bacteria producing NDM-1 are difficult to treat and are at higher risk for complications.

These results show that there is not a high prevalence of bla-ndm gene in this area. But it is not considered that there is no risk at all and studies and research should be carried out to get more confirmed results about the prevalence of these almost untreatable creatures. Because we are already facing a high rate of other life threatening diseases like HCV (and not should forget Dengue), bla-ndm gene can be a great health problem in future, if we not take serious actions.

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CHAPTER 06 Reference

.Livermore DM, Woodford N (October 2000). "Carbapenemases: a problem in waiting?". Current Opinion in Microbiology
ii

Birnbaum J, Kahan FM, Kropp H, MacDonald JS (June 1985). "Carbapenems, a new class of beta-lactam antibiotics. Discovery and development of imipenem/cilastatin". American Journal of Medicine

iii

Paul M, Shani V, Muchtar E, Kariv G, Robenshtok E, Leibovici L. Systematic review and meta-analysis of the efficacy of appropriate empiric antibiotic therapy for sepsis. Antimicrob Agents Chemother 2010;54(11):48514863.
iv

Review of Carbapenemases and AmpC-beta Lactamases Frederic J. Marsik, PhD, ABMM, and Sumathi Nambiar, MD, MPH

Stephen Smith (13 September 2010). "New drug-resistant 'superbug' arrives in Mass." . The Boston Globe. Retrieved 18 September 2010.

vi

"Superbug detected in GTA" . Toronto Star. 22 August 2010.

vii

Shino Yuasa (8 September 2010). "Japan confirms first case of superbug gene". The Boston Globe. Associated Press.
viii

"Drug-resistant bacteria kills 18 in Brazil" . Inquirer. Agence France-Presse. 10 October 2010. Retrieved 21 October 2010.

ix

Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 2010;10:597 602. doi:10.1016/S1473-3099(10)70143-2

Nordmann P, Poirel L, Toleman MA, Walsh TR. Does broadspectrum -lactam resistance due to NDM-1 herald the end of the antibiotic era for treatment of infections caused by Gram-negative bacteria? J Antimicrob Chemother. 2011;66:689 92. doi:10.1093/jac/dkq520
x

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xi

Patrice Nordmann, Thierry Naas, and Laurent Poirel . Global Spread of Carbapenemase producing Enterobacteriaceae. Emerging Infectious Diseases , October 2011: Vol. 17, No. 10, pp. 17911798. study. Lancet Infect Dis 2010; 10 : 597-602.
xii

Perry, J.D. et al. (2011) Prevalence of faecal carriage of Enterobacteriaceae with NDM- 1 carbapenemase at military hospitals in Pakistan, and evaluation of two chromogenic media. J. Antimicrob. Chemother. 66, 22882294

xiii

Rolain MJ, Panola P, Cornaglia G. New Delhi metallo-beta-lactamase (NDM-1): towards a new pandemia? Clinical Microbiology and infection 2010;16:12.

Yong D, Toleman MA, Giske CG, et al. Characterization of a new metallo-lactamase gene, blaNDM-1, and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother. 2009;53:5046-54.
xiv

Muir A, Weinbren MJ (2010) New Delhi metallo--lactamase: a cautionary tale. J Hosp Infect 75:239240
xv

xvi

Islam, M. A., P. K. Talukdar, A. Hoque, M. Huq, A. Nabi, D. Ahmed, K. A. Talukder, M. A. Pietroni, J. P. Hays, A. Cravioto, and H. P. Endtz. 2012. Emergence of multidrug- resistant NDM-1-producing Gram-negative bacteria in Bangladesh. Eur.J.Clin.Microbiol.Infect.Dis. doi:10.1007/s10096-012-1601-2 [doi].

xvii

Coexistence of blaOXA-23 with blaNDM-1 and armA in clinical isolates of Acinetobacter baumannii from India. Karthikeyan K, Thirunarayan MA, Krishnan P J Antimicrob Chemother. 2010 Oct; 65(10):2253-4.

xviii

Extremely drug-resistant Citrobacter freundii isolate producing NDM-1 and other carbapenemases identified in a patient returning from India. Poirel L, Ros A, Carricajo A, Berthelot P, Pozzetto B, Bernabeu S, Nordmann P Antimicrob Agents Chemother. 2011 Jan; 55(1):447-8.

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New Delhi metallo--lactamase and multidrug resistance: a global SOS? Bonomo RA Clin Infect Dis. 2011 Feb 15; 52(4):485-7.
xix

xx

New Delhi Metallo-beta lactamase (NDM-1) in Enterobacteriaceae: treatment options with carbapenems compromised. Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hedge A, Soman R J Assoc Physicians India. 2010 Mar; 58():147-9.
xxi

Attributable mortality rate for carbapenem-resistant Klebsiella pneumoniae bacteremia. Borer A, Saidel-Odes L, Riesenberg K, Eskira S, Peled N, Nativ R, Schlaeffer F, Sherf M Infect Control Hosp Epidemiol. 2009 Oct; 30(10):972-6.

xxii

Outcomes of carbapenem-resistant Klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies. Patel G, Huprikar S, Factor SH, Jenkins SG, Calfee DP Infect Control Hosp Epidemiol. 2008 Dec; 29(12):1099-106.

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