/prof_nk_ganguly_-_ndm1

Special Session: NDM-1 and other Emerging Pathogens: A Microcosm of the Resistance Problem

NDM-1: The Indian Perspective N.K. Ganguly Former Director General – Indian Council of Medical Research (ICMR) President – Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Distinguished Biotechnology Research Professor National Institute of Immunology, New Delhi

Origin and Spread of NDM-1 

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NDM-I belongs to the Metallo-β-lactamase (MBL) family of enzymes that can split a large number of Carbapenems at this current point of time.

First reported in UK in 2009 from a Swedish patient with a hospital exposure in India.1 Report from SENTRY Antimicrobial Surveillance Program from Indian Hospitals (2006-2007).2 ◦ 39 Carbapenem resistant Enterobacteriaceae (overall 2.7 % prevalence) ◦ Out of these 39 isolates,15 harbored blaNDM-1 ◦ NDM-1 was isolated from E. coli, K. pneumoniae, E. cloacae.

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The blaCTX-M-15 gene jumped from the chromosome of its natural hosts, Kluyvera spp, to plasmids that have subsequently spread widely.3 1. Yong et al. An(microb Agents Chemother 2009; 53: 5046–54 2. Castanheira et al. An(microb Agents Chemother 2011; 55: 1274-­‐8 3. Lar>que et al. An(microb Agents Chemother 2006; 50: 1282-­‐6.

Genetic Characterization of NDM-1  NDM-1

shares very little identity with other Metallo-β-lactamases (MBLs).  Most similar MBLs being VIM-1/VIM-2, with which it has only 32.4% identity. 1  First characterized MBL from India was also VIM-2. 2

1. Yong et al. An(microb Agents Chemother 2009; 53:5046–54.

2. Toleman et al. An(microb Agents Chemother 2007; 51: 2636–8.

Structure of NDM-1 and its sequence alignment with its homologue proteins VIM-4, FEZ-1 and CphA

Liang et al. PLoS One, August 2011; Vol 6, Issue 8

Structure-Function Activity of NDM-1 on Antibiotics Molecular models of NDM-1 and its complex with antibiotics Imipenem (orange sticks) and Carbapenem (yellow sticks) in the active site of NDM-1.

Hydrolysis Activity of NDM-1 by Enzymatic Assays

Liang et al. PLoS One, August 2011; Vol 6, Issue 8

Crystal Structure of NDM-1 NDM-1 adopts the canonical MBL fold that facilitates a two zinc-catalyzed mechanism of Ă&#x;-lactam antibiotic hydrolysis. However, NDM-1 displays a larger active site and an electrostatic profile that can accommodate a wide variety of substrate molecules.

Zinc coordination: Zinc ions and ligands are in green and gray for apo-NDM-1 and hAMP-bound NDM-1. Coordination interactions are blue dashes, and hAMP is in cpk colored stick representation (carbons are magenta).

The apo-NDM-1 crystal structure helps provide a molecular basis for broad-spectrum antibiotic resistance and represents a valuable step in the informed design of novel MBL inhibitors.

King et al. Protein Science 2011; 20: 1484-1491

NDM-1: Diagnostics 

Direct detection of blaNDM-1 by PCR with specific primers targeting the gene.

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PCR and sequencing used to identify other resistant genes blaCMY4 and blaCTX-M-15 carried by the bacterial isolates.

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NDMs detected by a simple PCR, costs ~ INR 200.1

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TaqMan Real Time PCR 2 for detection carbapenem resistance in E.coli, K. pneumoniae and Acinetobacter baumannii. ◦ Advantages: High precision.

1. Deshpande et al. J Assoc Physicians India 2010; 58: 147-9.

2. Manchanda et al. Indian J Med Microbiol 2011; 29 (3): 249-53.

NDM-1: Therapeutics 

Organisms with this resistance determinant will be difficult to treat as therapeutic options are limited.

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Colistin and Tigecycline are the only available agents for treatment. 1 ◦ Both have limitations.

1. Dizbay et al. J Antibiotics 2010; 63: 51-3.

Studies at Sir Ganga Ram Hospital (SGRH), New Delhi

Prevalence of NDM-1 in Various Clinical Specimens at SGRH

The prevalence of NDM-­‐1 ranged from 6.7% in E.coli to 38% K. pneumoniae Project funded by Department of Research, Sir Ganga Ram Hospital

Association between Carbapenem Consumption and Resistance in P. aeruginosa and A. baumannii

p value for A. baumannii< 0.5

No carbapenem resistant Enterobacteriaceae isolated J Applied Therapeu>c Research 7(2)2009; WHO/EMP/MAR/2009.2 ; Published on 03 May 2009 (124 pages); Under Review: IJMR 2011

NDM-1 at a Sick Newborn Care Unit, West Bengal, India

Sulagna Basu, Division of Bacteriology National Institute of Cholera and Enteric Diseases, Kolkata, India

Sick Newborn Care Unit (SNCU)

Level II SNCU in the District Hospital § An overwhelming proportion of neonatal deaths occur in developing countries. § The development of sick newborn care units (SNCUs) at rural hospitals in India has made a difference in infant survival rates, but infection control still remains a concern. § The point prevalence survey was carried out on a single day in a 20-bed level II SNCU in Birbhum District Hospital, West Bengal, India. § The Department of Neonatology, IPGMER & SSKM Hospital at Kolkata, West Bengal, acts as a nodal centre for the rural SNCUs.

Sequence of Events In the event of a high prevalence of multidrug resistance, it was decided to conduct a point prevalence survey of the unit in November 2009

The environment of the SNCU and the four body-sites were sampled (umbilicus, groin, mouth and rectum)

Shortly after this survey, 4 fresh neonates admitted to the SNCU after the survey developed septicemia with Escherichia coli ----------- all four died within 72 hours even before the antibiogram report was received.

National Institute of Cholera and Enteric Diseases, Kolkata, India

1 2 3 4

5

6 7 8 9

10 M

1500bp 1000bp 500bp 300bp

100bp

Lane M: 100 bp DNA ladder; Lane 1-10: NDM-1 harboring representative E. coli isolates

National Institute of Cholera and Enteric Diseases, Kolkata, India

Description of the Isolates P. aeruginosa-2 K. pneumoniae -3 A. junii-4 During the survey

E. coli-16 A. baumannii-7 S. maltophilia-3

After survey

4 E. coli were isolated from blood of 4 fresh neonates

P. aeruginosa-2 E. cloacae-2 K. pneumoniae-1

National Institute of Cholera and Enteric Diseases, Kolkata, India

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E. coli isolated from 4 neonates

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All isolates were imipenem-resistant

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Resistant genes identified: blaNDM-1,

2 Imipenem-sensitive E. coli were isolated

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Resistant Genes identified:blaCTX-M-15, blaTEM-1, blaOXA-1, blaCMY-59, rmtB

blaCTX-M-15, blaTEM-1, blaOXA-1, blaCMY-59,

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Phylo group - D

rmtB

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Virulence genes – PapC

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Phylo group – D

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Virulence genes – papC

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Integron- Integron 1

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14 Imipenem-resistant E. coli were isolated

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Resistant Genes identified : blaNDM-1, blaCTX-M-15, blaTEM-1, blaCMY-42, rmtB

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Phylo group - A

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Integron- Integron 1

Genetic Diversity of the Isolates Blood

Colonized

PFGE analysis of E. coli isolates revealed that the isolates belonged to two different clones

Imipenem resistant Imipenemresistant

Imipenem sensitive

4 Imipenem-resistant blood isolates and 2 Imipenem-sensitive bodysite isolates were indistinguishable: possessed the same virulence factor (papC) and belonged to Phylo Group D. All 14 body-site isolates were clonal: Imipenem- resistant and belonged to Phylo Group A.

Detection of NDM-1 in other Gram Negative Bacteria Ø No E. coli was isolated from environmental specimens. Ø A search for NDM-1 possessing gram negative bacteria in the environment and the body sites showed Other NDM-1 harboring GNBs (apart from E. coli) wereq A. baumannii (body-site)-blaNDM-1, blaOXA-23, blaOXA-51, armA, Integron-1 q S. maltophilia (body-site)-blaNDM-1, blaCTX-M-15, blaTEM-1, blaCMY-42, rmtB, Integron-1 q K. pneumoniae (wash basin)-blaNDM-1, blaCTX-M-15, rmtC, Integron-1 q K. pneumoniae (wash basin)- blaNDM-1, blaCTX-M-15, blaTEM-1, blaSHV-1, blaOXA- 1, blaCMY-2, armA, Integron-1 GNB: Gram Negative Bacteria

L.6:Transconjugant of E. coli (groin) L.8: Transconjugant of E. coli (Blood)

Lane 1: Acinetobacter baumannii (mouth); Lane 2 & 3: Klebsiella pneumoniae (wash basins); Lane 4: transconjugant of Stenotrophomonas maltophilia (umbilicus); Lane 5: Stenotrophomonas maltophilia (umbilicus); Lane 6: transconjugant of Escherichia coli (groin); Lane 7: Escherichia coli (groin); Lane 8: transconjugant of Escherichia coli (blood); Lane 9: Escherichia coli (blood); Lane 10 & 11: Escherichia coli K12 V517 and Shigella flexneri YSH 6000 were used as a guide to molecular size respectively.

ImR E. coli (blood and body-site) and S. maltophilia (body-site) isolates possessed two mega plasmids. Transconjugants possessed only a single mega plasmid of 83 Kb.

Imipenem resistance was found to be transferable from E. coli (body-sites and blood) and S. maltophilia (body-site).

National Institute of Cholera and Enteric Diseases, Kolkata, India

Schematic Representation of Possible Transfer of blaNDM-1 in SNCU The point prevalence survey in November 2009 Imipenem-resistant body-site isolates

Imipenem-resistant environmental isolates

E. coli, Imipenem-resistant, NDM-1, CMY-42, phylo group A, Integron-1 S. maltophilia, Imipenem-resistant, NDM-1, CMY-42, Integron-1

K. pneumoniae, Imipenem-resistant, NDM-1, CMY-2, Integron-1

A. baumannii, imipenem-resistant, NDM-1, Integron-1 Transfer of NDM-1

Imipenem-sensitive body-site isolates E. coli, Imipenem-sensitive, CMY-59, phylo group D, papC

Imipenem-resistant blood isolates E. coli, Imipenem-resistant, NDM-1, CMY-59, phylo group D, papC, Integron-1

Indicated that the imipenem resistance must have been acquired by the imipenem-sensitive isolates, probably during the period between the survey and when the neonates were infected with E. coli.

National Institute of Cholera and Enteric Diseases, Kolkata, India

v The neonates in the SNCU colonized with NDM-1 possessing isolates did not have sepsis and were subsequently discharged. v Though these 4 neonates were admitted to the SNCU after the survey, it is evident from the dates of admission and discharge that there was an overlap in hospital stay between the neonates whose body-sites yielded E. coli and those who developed septicemia. v After the cases of septicemia with Imipenem E. coli were detected, standard precautions were reinforced. Strict attention was paid to hand-washing with liquid soap and water. In addition, hand hygiene with chlorhexidine/alcohol was introduced after this outbreak.

The time-frame of isolation and clonal identity indicated a possible transfer of blaNDM-1 from Imipenem-resistant GNBs to the Imipenem-sensitive E. coli which subsequently caused septicemia.

This establishes the promiscuous nature of blaNDM-1 and emphasizes the need for early recognition of similar isolates.

GNB: Gram Negative Bacteria

NDM-1: Key Questions

Nomenclature  Is

the name NDM-1 apt?

◦ Nomenclature needs to be in sync with other drug-resistant strains of bacteria – e.g.

MDR-TB, XDR-TB, MRSA etc.  Were

the standard taxonomic criteria adhered to?

 Is

there a need to create new taxonomic guidelines?

Transmission Dynamics 

What is the geographical circulation pattern?

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What are the major sites of infection?

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Who are the major vulnerable groups?

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What are the newer emerging strains?

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What are its survival strategies?

Tackling the NDM-1 Problem  What

are the vulnerable targets in the genome?

 Can

a drug be developed in time?

 What

strategies can be adopted for management?

The Way Forward ???  Stringent

infection control in hospitals.

◦ Need for policies for infection control that are practicable to their circumstances.  Good

sanitation in the community to restrict the spread of infectious clones.  Judicious use of antibiotics.  Co-ordinated International Surveillance.  What else???