ABSTRACT

Introduction: New Delhi metallo-β-lactamase-1 (NDM-1) is a recently reported novel

plasmid-borne metallo-β-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-β-lactamase-1, enterobactericea

CHAPTER 01

INTRODUCTION

1

1.1 Carbapenem:

Carbapenems are a class of β-lactam antibiotics with a broad spectrum of

antibacterial activity. They have a structure that renders them highly resistant to most β-

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 multi‐drug resistant Gram‐negative 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.

2

Figure 1a. Primary mechanisms of b-lactam resistance in Enterobacteriaceae.Hydrophilic channels formed by porins allow the uptake of b-lactams throughthe outer membrane of the bacterium. In the periplasmic interspace, b-lactammolecules bind irreversibly to the penicillin-binding proteins (PBPs), leading to theinhibition of the peptidoglycan synthesis. Primary mechanisms of b-lactamresistance in Enterobacteriaceae include the following: (i) enzymatic inactivationof the antibiotic by chromosome- and/or plasmid-encoded enzymes possessinghydrolytic activity against b-lactam molecules; (ii) decreased outer membranepermeability through production of modified porins, loss of porin expression, or ashift in the types of porins found in the outer membrane; and (iii) efflux of theantibiotic 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 β-lactams. This subset of BLs belongs to Ambler classes A, B, and D.

Each class has specific enzyme types based on molecular differences

3

(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 β-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 β-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

4

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 β-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).

5

Table 1:Classification of the various carbapenem hydrolyzing beta‐lactamases:

1.5 New Delhi metallo-β-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),

6

plasmid-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 integron–encoded metallo-β-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-β-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

7

coli and Klebsiella pneumoniae which were resistant to all antibiotics except tigecycline and

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

8

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.

9

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

10

Figure 4. Geographic distribution of New Delhi metallo-β-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 ofunknown origin.

11

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

12

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-β-

lactamases, Verona imipenemase (VIM), German imipenemase (GIM), Săo 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 Gram-

negative 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

13

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. Chughtai Lahore Laboratory 50

2. Post Graduate Medical Institute laboratory 30

3. Jinnah Hospital laboratory 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:

15

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 µl TE 10:1

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

in TE 10:1.

Added 250 µl of supernatant and 750 µl of Tris-reagent in tube.

Mixed with pipette and vortexed.

16

Placed at room temperature for 05 mins.

Added 200 µl 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 µl 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 µl 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 µl

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 µg / µl.

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.

F -(5′-GGG CAG TCG CTT CCA ACG GT-3′)

17

And

R -(5′-GTA GTG CTC AGT GTC GGC AT-3′)

MASTER MIX:

The PCR master mix (25 µl) consisted of:

Buffer 2.5 µl

dNTP’s 2.5 µl

Taq Polymerase 0.4µl

MgCl2 2.0µl

Forward Primer 0.75 µl

Reverse Primer 0.75 µl

De-ionized water 15.1 µl

Template DNA 1 µl

Total (Master Mix) 25 µl

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 94°C, 60°C and 72°C,

respectively, for 30 s each. The final extension step was performed for 3 min at 72°C.

18

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

19

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

21

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.

22

CHAPTER 06

Reference

23

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