MDL

Dear Editor,
New Delhi metallo-beta-lactamases (NDM) is a nomenclature that Indians cannot be

proud of, NDM-1 is the designation for carbapenemases found in enterobacteriaceae
isolated from patients in the United Kingdom and elsewhere who have had healthcare
contact in India or Pakistan. [1],[2]
Carbapenems (imipenem, ertapenem, meropenem, doripenem) are a class of beta-lactam

antibiotics with a broad spectrum of activity against gram-positive, gram-negative, and
anaerobic bacteria.
Carbapenemase enzymes belonging to Ambler molecular classes A to D have been

detected in various clinical isolates. Of these the class B enzymes are clinically the most
significant. They are the metallo-beta-lactamase (MBL) enzymes of the IMP or VIM
series that have been reported worldwide. MBL enzymes, whose genes are plasmid and
integron located, hydrolyze virtually all beta-lactams except aztreonam. [3] Many of the
carbapenemase producers are frequently resistant to fluroquinolones and
aminoglycosides.
NDM-1 was first detected in a Klebsiella pneumoniae isolate from a Swedish patient of
Indian origin in 2008. The gene coding for this unique enzyme blaNDM-1 was found in
one of the three resistance-carrying regions of an integron. NDM-1 shares very little
identity with other MBLs. As well as possessing unique residues near the active site,
NDM-1 also has an additional insert between positions 162 and 166, which is not present
in other MBLs. NDM-1 has a molecular mass of 28 kD and is monomeric. [4]
NDM-1 have been isolated from K pneumoniae, Escherichia More Details coli, Citrobacter
freundii, Enterobacter cloacae, and Morganella morganii. [2] Other classes of
carbapenemases have already been found in K pneumoniae, E cloacae, Pseudomonas
aeruginosa, and Acinetobacter baumannii. [5]
The first clue to the presence of a carbapenemase comes from the increased minimum
inhibitory concentration (MIC) values or frank resistance of the enterobacteriaceae to
ertapenem, imipenem, or meropenem. NDM-1 is inhibited by EDTA like other MBL
enzymes; this has been demonstrated by the EDTA-disc synergy test. The carbapenemase
activity can be screened for by the modified Hodge test. [6] Further characterization and
identification of the enzyme can be done only by molecular methods.
Treatment of infections caused by pathogens producing carbapenemases, including

NDM-1, poses a serious challenge as these infections are resistant to all commonly used
antibiotics. [5] Treatment of patients should be guided by the susceptibilities of the
individual pathogens, and clinical laboratories must test for a wide range of antibiotics,
including tigecycline, colistin, polymyxin, and aztreonam. The use of antibiotic
combinations may have to be considered in desperate cases.
Carbapenems are the only reliably active antibiotics against many multiresistant gram-
negative pathogens, particularly those with extended-spectrum beta-lactamases (ESBLs)
and AmpC enzymes. [7] The emergence and diversity of carbapenemase-producing strains
is therefore a major concern and one that Indian microbiologists cannot afford to ignore.
The virtual nonexistence of antibiotic policies and guidelines in India to help doctors
make rational choices with regard to antibiotic treatment is a major driver of the
emergence and spread of multidrug resistance in India. This is augmented by the
unethical and irresponsible marketing practices of the pharmaceutical industry, and
encouraged by the silence and apathy of the regulating authorities. Poor microbiology
services in most parts of the country add to the problem.
Microbiologists in India have a very important role in the prevention of spread of these
dreaded multiresistant pathogens across the world. They should actively participate in the
clinical decision making with regard to the treatment of infections, influence the policies
and approach to infections and antimicrobials by the government, develop guidelines for
antibiotic therapy in their local hospitals, become infection-control doctors, set up
surveillance systems for drug-resistant organisms, and educate healthcare workers and
the general public about the dangers of multidrug resistant organisms, including hospital-
acquired infections.
~ References
1. Yong D, Giske CG, Toleman M, Walsh TR. A novel subgroup metallo-beta-

lactamase (MBL), NDM-1 emerges in Klebsiella pneumoniae (KPN) from India.
48th Annual ICAAC/IDSA 46th Annual Meeting, Washington DC, October 25-28,
2008. 2009;C1-105:87.
2. Health Protection Agency. National Resistance Alert: Carbapenemases in
Enterobacteriaceae. Health Protection Report 2009;3:news.
3. Nordmann P, Poirel L. Emerging carbapenemases in Gram-negative aerobes. Clin
Microbiol Infect 2002;8:321-31. [PUBMED] [FULLTEXT]
4. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al.
Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel
erythromycin esterase gene carried on a unique genetic structure in Klebsiella
pneumoniae sequence type 14 from India. Antimirob Agents Chemother
2009;53:5046-54.
5. Walsh TR. Clinically significant carbapenemases: An update. Curr Opin Infect Dis
2008;21:367-71. [PUBMED] [FULLTEXT]
6. Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH. Modified Hodge and EDTA-
disk synergy tests to screen metallo-beta-lactamase-producing strains of
Pseudomonas and Acinetobacter species. Clin Microbiol Infect 2001;7:88-
91. [PUBMED] [FULLTEXT]
7. Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing
Enterobacteriaceae: An emerging public-health concern. Lancet Infect Dis 2008;8
:159-66. [PUBMED] [FULLTEXT]
Characterization of a new metallo-beta-lactamase gene, bla(NDM-1),
and a novel erythromycin esterase gene carried on a unique genetic
structure in Klebsiella pneumoniae sequence type 14 from India.
Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR.
Yonsei University College of Medicine, Research Institute of Antimicrobial Resistance, Seoul, Republic of
Korea.
Abstract
A Swedish patient of Indian origin traveled to New Delhi, India, and acquired a urinary tract infection caused
by a carbapenem-resistant Klebsiella pneumoniae strain that typed to the sequence type 14 complex. The
isolate, Klebsiella pneumoniae 05-506, was shown to possess a metallo-beta-lactamase (MBL) but was
negative for previously known MBL genes. Gene libraries and amplification of class 1 integrons revealed
three resistance-conferring regions; the first contained bla(CMY-4) flanked by ISEcP1 and blc. The second
region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a new erythromycin
esterase gene; ereC; aadA1; and cmlA7. An intact ISCR1 element was shown to be downstream from the
qac/sul genes. The third region consisted of a new MBL gene, designated bla(NDM-1), flanked on one side
by K. pneumoniae DNA and a truncated IS26 element on its other side. The last two regions lie adjacent to
one another, and all three regions are found on a 180-kb region that is easily transferable to recipient strains
and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1 shares very little
identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2, with which it has only 32.4%
identity. As well as possessing unique residues near the active site, NDM-1 also has an additional insert
between positions 162 and 166 not present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is
monomeric, and can hydrolyze all beta-lactams except aztreonam. Compared to VIM-2, NDM-1 displays
tighter binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin (cefalotin), and
also to the penicillins. NDM-1 does not bind to the carbapenems as tightly as IMP-1 or VIM-2 and turns over
the carbapenems at a rate similar to that of VIM-2. In addition to K. pneumoniae 05-506, bla(NDM-1) was
found on a 140-kb plasmid in an Escherichia coli strain isolated from the patient's feces, inferring the
possibility of in vivo conjugation. The broad resistance carried on these plasmids is a further worrying
development for India, which already has high levels of antibiotic resistance
New Delhi metallo-beta-lactamase
From Wikipedia, the free encyclopedia
Jump to: navigation, search
This article documents a current event. Information may change rapidly as the event
progresses.
ND Metallo-beta-lactamase (NDM-1)[1] is a gene that makes bacteria resistant to

antibiotics of the carbapenem family. It encodes a type of beta-lactamase enzyme called a
carbapenemase. Bacteria that carry this gene are often referred to by news reporters as
"superbugs."[2] There are currently no new drugs in the research pipelines that aim to stop
NDM-1.[3] To date, some strains of E.coli and Klebsiella pneumoniae are known carriers
of the gene, but the gene can be transmitted from one strain of bacteria to another through
horizontal gene transfer.
Contents
[hide]
• 1 Function
• 2 Origin and spread
• 3 Indian response
• 4 References
• 5 External links
Function
The gene produces a metallo-beta-lactamase, an enzyme that hydrolyzes and inactivates

antibiotics in the beta-lactam family. Those antibiotics were, until recently, capable of
killing most bacteria by inhibiting the synthesis of one of their cell wall layers. The
resistance conferred by this gene therefore aids the expansion of bacteria that carry it
throughout a human host, since they will face less opposition/competition from
populations of antibiotic-sensitive bacteria, which will be diminished by the original
antibacterial treatment.
The following antibiotics are inactivated by the enzyme:
• cephalosporins
• penicillins
• carbapenem
Origin and spread
The gene was named after New Delhi, the capital city of India, as it was first described
by Yong et al. in 2009 in a Swedish national who fell ill with an antibiotic-resistant
bacterial infection that he acquired in India.[4] The infection was unsuccessfully treated in
a New Delhi hospital and after the patient's repatriation to Sweden, a carbapenem-
resistant Klebsiella pneumoniae strain bearing the novel gene was identified. The authors
concluded that the new resistance mechanism "clearly arose in India, but there are few
data arising from India to suggest how widespread it is."[4]
It has reportedly been found in Pakistan, India and most other Asian countries and has
been brought from that region to Europe by people undergoing hospitalization in those
countries. In several cases people went there to undergo cosmetic surgery at a lower cost,
getting infected during the procedure and bringing the resistant bacteria back to their
country of origin.
As of June 2010, there were three reported cases of Enterobacteriaceae isolates bearing
this newly described resistance mechanism in the US, the CDC stated that "All three U.S.
isolates were from patients who received recent medical care in India."[5] However, US
experts have stated that it is unclear if this strain is any more dangerous than existing
antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus, which
are already common in the USA.[6]
A study by a multi-national team was published in the August 2010 issue of the journal
The Lancet Infectious Diseases. This examined the emergence and spread of bacteria
carrying the NDM-1 gene. This reported on 37 cases in the United Kingdom, 44 isolates
with NDM-1 in Chennai, 26 in Haryana and 73 in various other sites in Pakistan and
India.[1] The authors' analysis of the strains showed that many carried NDM-1 on
plasmids, which will allow the gene to be readily transferred between different strains of
bacteria by horizontal gene transfer. All the isolates were resistant to multiple different
classes of antibiotics, including beta-lactam antibiotics, fluoroquinolones, and
aminoglycosides, but most were still susceptible to the polymyxin antibiotic colistin.
In early August a chemical compound, GSK-299423, was found to significantly fight

against antibiotic-resistant bacteria by making such bacteria unable to reproduce, citing a
likely treatment to the NDM-1 strain.[7]
Indian response
The Indian health ministry has disputed the conclusion that the gene originated in India or
Pakistan, describing this conclusion as "unfair" and stating that Indian hospitals are
perfectly safe for treatment.[8] Indian politicians have described linking this new drug
resistance gene to India as “malicious propaganda” and blamed multinational
corporations for what they describe as selective malignancy.[8][9] A Bharatiya Janata Party
politician has instead argued that the journal article is bogus and represented an attempt
to scare medical tourists away from India.[10] The Indian Ministry of Health released a
statement "strongly refut[ing]" naming the enzyme "New Delhi".[11]
In contrast, an editorial in the March 2010 issue of the Journal of Association of

Physicians of India blamed the emergence of this gene on the widespread misuse of
antibiotics in the Indian healthcare system, stating that Indian doctors have "not yet taken
the issue of antibiotic resistance seriously" and noting little control over the prescription
of antibiotics by doctors and even pharmacists.[12] The Times of India states that there is
general agreement among experts that India needs both an improved policy to control the
use of antibiotics and a central registry of antibiotic-resistant infections.[13]
The primary author of the 2010 Lancet study, who is based in the University of Madras,
has stated that he does not agree with the part of the article that advises people to avoid
elective surgeries in India.[13]
References
1. ^ a b Kumarasamy et. al. (2010). "Emergence of a new antibiotic resistance mechanism in

India, Pakistan, and the UK: a molecular, biological, and epidemiological study". The
Lancet Infectious Diseases. doi:10.1016/S1473-3099(10)70143-2.
2. ^ Jordan, Carol (11 August 2010). "World update: More aid planned for Pakistan". CNN.
http://news.blogs.cnn.com/2010/08/11/world-update-more-aid-planned-for-pakistan/.
Retrieved 13 August 2010.
3. ^ Globe and Mail: Scientists find new superbug spreading from India
4. ^ a b Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR. (December
2009). "Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a
novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella
pneumoniae sequence type 14 from India". Antimicrob Agents Chemother. 53 (12):
5046-54. doi:10.1128/AAC.00774-09. PMID 19770275. PMC 2786356.
http://aac.asm.org/cgi/content/full/53/12/5046?view=long&pmid=19770275.
5. ^ Detection of Enterobacteriaceae Isolates Carrying Metallo-Beta-Lactamase --- United
States, 2010
6. ^ McNeil Jr., Donald G. (11 August 2010). "Antibiotic-Resistant Bacteria Moving From
South Asia to U.S.". The New York Times.
http://www.nytimes.com/2010/08/12/world/asia/12bug.html?_r=1&hpw. Retrieved 13
August 2010.
7. ^ Alazraki, Melly (6 August 2010). "GlaxoSmithKline Finds Compound That Could Help
Fight 'Superbugs'". dailyfinance.com.
http://www.dailyfinance.com/story/glaxosmithkline-finds-compound-fight-
superbugs/19582888/. Retrieved 13 August 2010.
8. ^ a b Pandey, Geeta (12 August 2010). "India rejects UK scientists' 'superbug' claim". BBC
News. http://www.bbc.co.uk/news/world-south-asia-10954890. Retrieved 13 August
2010.
9. ^ "Linking India to superbug unfair and wrong, says India". Hindustan Times. 12 August
2010. http://www.hindustantimes.com/Linking-India-to-superbug-unfair-and-wrong-
says-India/Article1-585840.aspx. Retrieved 13 August 2010.
10. ^ http://expressbuzz.com/nation/superbug-an-mnc-conspiracy-bjp-leader/197607.html
11. ^ Sharma, Sanchita (13 August 2010). "‘Don’t blame superbug on India, it’s
everywhere’". Hindustan Times. http://www.hindustantimes.com/Don-t-blame-
superbug-on-India-it-s-everywhere/Article1-585926.aspx. Retrieved 13 August 2010.
12. ^ Abdul Ghafur K (March 2010). "An obituary- On the Death of antibiotics!". Journal of
Association of Physicians of India 58. http://www.japi.org/march_2010/article_01.html.
13. ^ a b Narayan, Pushpa (13 August 2010). "Indian author says superbug report is fudged".
The Times of India. http://timesofindia.indiatimes.com/city/chennai/Indian-author-says-
superbug-report-is-fudged/articleshow/6302479.cms. Retrieved 13 August 201
Indian author says superbug report is fudged
CHENNAI: A day after the Lancet report on a drug-resistant superbug NDM-1

created a global scare, India hit out at the study, which it said was funded by
pharma companies that make antibiotics to treat such cases. While the Union
health ministry issued a statement on Thursday, which took offence to the naming
of the bug after the national capital, the report's Chennai-based lead
authorKarthikeyan Kumarasamy dissociated himself from parts of it.
"The study was funded by the European Union and two pharmaceutical
companies, Wellcome Trust and Wyeth, which produce antibiotics for treatment
of such cases. It also needs to be highlighted that several of the authors have
declared conflict of interest in the publication," the health ministry said.
Kumarasamy said he had not written many of the interpretations in the report;
they were added later without his permission or knowledge. "I do not agree with
the last paragraph which advises people to avoid elective surgeries in India. While
I did the scientific work, correspondence author Timothy R Walsh of Cardiff
University was assigned to edit the report," he told TOI. According to the study,
based on a survey of patients in Indian cities, a multi-drug resistant strain of
bacteria was spreading from Indian hospitals.
The report implied that India is the prime source of the superbug which is
resistant to even the most powerful antibiotics, and advised patients to avoid
elective surgeries in the country.
The National Centre for Disease Control (NCDC) which met on Thursday to
discuss the Lancet report termed it "severely biased" against India. "It's not been
decided whether we should write to the editor of the journal, but we have
condemned and disagreed with the conclusions and inferences the report has
drawn. Since this is a scientific report, we are looking at scientific ways to contest
it. So far, the UK government has not issued any alarm. If they do so, we will take
it up with the UK government," said Indian Council of Medical Researchdirector
Dr V M Katoch.
"There are several such (multi-drug resistant) strains found in the UK and the US.
In fact, some of them are deadlier than the present superbug. But it makes news
only when it comes from India. They have made a mountain out of a mole hill,"
said PM Bhargava, former director of Centre for Cellular and Molecular Biology,
Hyderabad.
"When a deadly hospital infection like MRSA was cited in the USA more than a
decade ago, India had no cases. At least 15,000 people died of hospital-acquired
infections in the US last year. It is no different in the UK," he said.
The name of the superbug, New Delhi metallo-beta-lactamase, has also come in
for severe criticism. "Should AIDS be named after the US? Or should MRSA
(another superbug) be named after the UK? Why only India's name is tagged to
the new bug?" says Delhi-based cardiothoracic surgeon Dr Naresh Trehan.
What has surprised doctors even more is that the report came soon after a report
by International Society of Aesthetic Plastic Surgery ranked India among the top
five nations for cosmetic surgical and non-surgical procedures.
The Union health ministry in a statement on Thursday said: "It should have been
highlighted that getting infection by such drug resistant bacteria is a matter of
chance, is a global phenomenon and is preventable by sound infection prevention
strategies which are followed in any good hospital. While such organisms may be
circulating more commonly in the world due to international travel but to link this
with the safety of surgery hospitals in India and citing isolated examples to show
that due to the presence of such organism in Indian environment, India is not a
safe place is wrong.
Meanwhile, experts agree on the urgent need to have a registry for such infections
and also an antibiotics policy. "We are working towards having both. Without
such records, we are often not equipped to scientifically challenge such reports,"
said Katoch
Read more: Indian author says superbug report is fudged - Chennai - City - The
Times of India http://timesofindia.indiatimes.com/city/chennai/Indian-author-
says-superbug-report-is-fudged/articleshow/6302479.cms#ixzz0wU2jHjSu
An obituary- On the Death of antibiotics!
Abdul Ghafur K
Consultant in Infectious Diseases and Clinical Mycology, Apollo Hospital, Chennai.

Homosapiens is an alien species on earth. This planet belongs to bacteria. There are more
bacteria on earth than all other living organisms. The human body contains more number of
bacteria than human cells themselves. We lived with arrogant optimism that we had conquered
infections, at least the bacterial infections, if not the viruses. How wrong we were! Bacteria have
finally reclaimed their premier status and superiority and won the war against humans. They are
literally mocking our intellect, knowledge and antibiotic weaponry.
Indian Health care professionals involved in the treatment of patients with severe infections
especially healthcare associated infections will agree that it is commonplace to come across pan-
resistant Gram negative bacterial infections where we do not have a single effective antibiotic
option. We are, therefore, forced to use a cocktail of antibiotics to which the bacteria is resistant
with the infinitesimally small hope of a synergistic effect. Immunocompromised patients especially
transplant and cancer chemotherapy patients, who develop infections, are known to have high
mortality rates. Until a few years ago we could at least try our powerful antibiotics against these
infections. With increasing pan resistant bacteria, we will be forced to stop organ transplantation,
bone marrow transplantation and cancer chemotherapy. We are going to face this catastrophic
situation in the near future - not in a decade or so but within a few years time. May I encourage
the readers to examine the excellent article “Antibiotic-Resistant Bugs in the 21st Century —A
Clinical Super-Challenge” by Cesar A Arias, in one of the recent issues of NEJM.l
The easiest way of tackling the superbug problem is to use the notorious ostrich strategy which
denies the existence of the problem: stop looking for these bugs, stop looking for the hidden
resistance mechanisms and closing your eyes even if you find them. A National Resistance Alert
from UK, issued in January 2009,2 warned of an increasing number of carbapenem-resistant
strains of Enterobacteriaceae being identified in UK hospital patients, a significant proportion of
whom had received medical treatment in India and Pakistan. This new resistance mechanism is
named as “New Delhi Metallo-1” (NDM-1). At the time of the publication of the UK HPA warning,
the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL), UK identified a total 22
isolates of NDM-1.
We have now data from one of our own hospitals. Deshpande P and team from Hinduja National
hospital, Mumbai have isolated 22 NDM-1 producing Enterobacteriaceae, from span of just 3
months and a single hospital.3 This is the first Indian study on NDM-1 and an eye opener on how
deep a trouble we are in. If a single hospital can isolate such a significant number of bacteria with
a new resistance gene in a short period of time; the data from all the Indian hospitals, if available
would potentially be more interesting and shocking than the human genome project data, which is
considered as a discovery more important than the moon landing itself. I congratulate the authors
and their departments for doing such an important study, especially considering the fact that
majority of Indian hospitals are struggling to hide their resistance statistics.
We come across MDR or even pan resistant Gram negative bugs quite often and such bugs are
reported in almost all major centers in India and most of international centers though to a lesser
extent than India. We Indians are the leaders in antibiotic resistance. Many of MDR superbugs
are from bacterial cultures taken at the time of admission to the hospital. By the time a patient is
being admitted to a tertiary care centre, that patient has already visited many other hospitals and
doctors and has received multiple courses of different antibiotics. These patients are literally
walking culture plates of superbugs and you don’t have to be Nostradamus to predict their clinical
outcome
When we are called to manage patients with severe infections due to pan resistant bugs, we do
really wonder whether we are living in pre-Alexander Fleming years without antibiotics and then
with a shock, but no surprise, realise that we have reached the end of antibiotic era. Still, the
Indian medical community remains in a state of denial. We have not yet taken the issue of
antibiotic resistance seriously. We believe that Dr. Fleming has discovered penicillin only early
this morning and consider antibiotic resistance a problem of next century where in fact antibiotics
are dead and the foul smell of decay is already around us. You may call me a pessimist, but I
sincerely believe that it is too late to save antibiotics; unless you have divine powers to bring the
dead back to the life.
There is no restriction on the usage of higher end antibiotics in India. Indian doctors need not
justify their prescription. Any doctor can prescribe and in some cases even pharmacists can
dispense without prescription meropenem in a situation where ampicillin would have been
adequate and at the same time prescribing ampicillin in a case where meropenem would have
been the right choice. Under usage of antibiotics is as dangerous as overusing them. Choosing
the right antibiotic is critical and requires adequate knowledge on the spectrum of antibiotics.
Unfortunately a good percentage of our medical community lack that knowledge. India is a
country where infectious diseases are rampant and we manage plenty of these cases every day.
This experience gave us a false sense of confidence on antibiotic usage. We consider ourselves
masters of the encyclopedic knowledge of antibiotics where in fact some of us do not even think
about the spectrum of an antibiotic and others do not even consider the need of an antibiotic
before giving a prescription. The Indian medical curriculum lacks importance on the teaching of
infectious diseases to undergraduate and post graduate students. An MD General Medicine
candidate can clear his or her examination without reading the chapter on infectious diseases and
antibiotic usage. Our curriculum is still revolving around the colonial concepts. When are we
going to teach our students the difference between cefotaxime and meropenem rather than
spoon feeding them with the dynamics of opening snap, release reflexes and tidal percussion?
The pharmaceutical industry, both international and Indian generic, has made its own contribution
to this resistance saga. The lack of restriction on the usage of newer antibiotics with specialist
spectrum has given fertile ground for companies to exert their excessive pressure on doctors to
increase prescription of antibiotics. The aim of any industry is to make profit. So let us not blame
the industry, let us blame ourselves. We are the individuals making prescriptions, not the medical
representatives. Indian doctors should update their awareness on antibiotics and antibiotic
resistance. That should not be through the pamphlets issued by companies, but through unbiased
medical information. The pharma companies should realise that indiscriminate usage of
antibiotics in a situation where there is no good new antibiotics in the pipeline, is like killing a duck
laying golden eggs.
Most of the Government hospitals in India are not worried about resistance. They are still
struggling to get hold of life saving antibiotics. The private practitioners, private and corporate
hospitals are the breeding grounds for resistance. There are very few hospitals in India with
infectious diseases and infection control specialists. Only in a minority of Indian private hospitals
are antibiotic policy and antibiotic stewardship implemented. The majority of private and corporate
hospitals are in denial, either purposefully or due to ignorance. I have come across many hospital
administrators in India claiming zero infection in their hospitals. It is sad to say that many of these
hospitals do not have the necessary microbiology laboratory support or trained infection control
specialists to look for resistance. The claim of zero infection is in fact an innocent advertisement
of the lack of necessary infection control infrastructure in that hospital.
‘Bad bugs, No drugs: No ESKAPE!’ is an excellent article by the infectious disease society of
America on the superbug problem.4 There is a dramatic increase in the prevalence of superbugs
and there is an equally dramatic drop in the number of new antibiotics available. We need new
antibiotics to treat difficult Gram negative infections. But for the time being we can only dream
about these new molecules. The pipeline of antibiotic research and development is nearly dry.
There are some good gram positive antibiotics in the pipeline, not even a single promising
antibiotic in the advanced stage of development which is useful against resistant gram negative
bacteria. Research and development of any antibiotic is a huge investment for any
pharmaceutical company. Unlike anti-diabetics or anti- hypertensives, which do not become
resistant with usage, antibiotics frequently lose their utility within a few years of coming to market.
So antibiotic development is not a profitable option for pharmaceutical companies and that is one
of the reasons why the pipeline is getting drier. It is the responsibility of the medical community
especially the Indian medical community to save the antibiotics which remain, by prudent and
sensible use of these drugs.
Carbapenems are the most powerful tools against Gram negative bacteria. Due to the extensive
misuse of these antibiotics, a good proportion of Pseudomonas and Acinetobacters species in
Indian hospitals have developed carbapenem resistant mutations. Now even our
Enterobacteriaceae are becoming resistant to carbapenem. I congratulate the authors for their
excellent study and the result of this study is the final nail on the antibiotic coffin. It may be
overconfidence however to believe that this study will open the eyes of Indian medical
community.
The art of war is deception; that is deceiving the enemy. But in the war against microbes we have
deceived ourselves by misusing, under using and overusing antibiotics. Our country, India, is the
world leader in antibiotic resistance, in no other country antibiotics been misused to such an
extent. Microbes are the ultimate warriors. They have sophisticated weapons and use ingenious
methods of attacks. They have always been many steps ahead of us. Even in the twenty first
century with all the developments in the modern medicine, when we face microbes, we feel
helpless. Whatever weapons we had in the form of antibiotics, we ourselves have ruined them.
Indian medical community has to be ashamed of the NDM-1 (“New Delhi Metallo-1”) gene. Even
though we have not contributed to carbopenem development, we have contributed a resistance
gene with a glamorous name. The overuse of antibiotics is embedded in our Indian gene. It is an
Indian tradition. Why should we Indians worry? We can always depend on honey, yoghurt and
cow’s urine. At any rate within a few years,- these products may be more useful than antibiotics!
References
1. Cesar A. Arias. Antibiotic-Resistant Bugs in the 21st Century —A Clinical Super-

Challenge.NEJM 2009, 360:439-443
2. HPA.National Resistance Alert: carbapenemases in Enterobacteriaceae. Health
Protection Report [serial online] 2009; 3 (4) : news. Available at:
http://www.hpa.org.Uk/hpr/archives / 2009 / news0409.htm#enterora.
3. Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hegde A, Soman R. New Delhi
Metallo-b lactamase (NDM-1) in Enterobacteriaceae: Treatment options with
Carbapenems Compromised. JAPI 2010, 58:146-148.
4. Helen W. Boucher, et al. Bad Bugs, No Drugs: No ESKAPE! An Update from the
Infectious Diseases Society of America -IDSA Report on Development Pipeline. Clin
Infcet Dis 2009;48:1-12
Emergence of a new antibiotic resistance

mechanism in India, Pakistan, and the UK: a
molecular, biological, and epidemiological study
Karthikeyan K Kumarasamy MPhil a, Mark A Toleman PhD b, Prof Timothy R Walsh PhD b ,
Jay Bagaria MD c, Fafhana Butt MD d, Ravikumar Balakrishnan MD c, Uma Chaudhary MD e,
Michel Doumith PhD c, Christian G Giske MD f, Seema Irfan MD g, Padma Krishnan PhD a, Anil V
Kumar MD h, Sunil Maharjan MD c, Shazad Mushtaq MD c, Tabassum Noorie MD c, David L
Paterson MD i, Andrew Pearson PhD c, Claire Perry PhD c, Rachel Pike PhD c, Bhargavi Rao MD c,
Ujjwayini Ray MD j, Jayanta B Sarma MD k, Madhu Sharma MD e, Elizabeth Sheridan PhD c,
Mandayam A Thirunarayan MD l, Jane Turton PhD c, Supriya Upadhyay PhD m, Marina Warner
PhD c, William Welfare PhD c, David M Livermore PhD c, Neil Woodford PhD c
Summary
Background
Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi

metallo-β-lactamase 1 (NDM-1) are potentially a major global health problem. We investigated
the prevalence of NDM-1, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the
UK.
Methods
Enterobacteriaceae isolates were studied from two major centres in India—Chennai (south
India), Haryana (north India)—and those referred to the UK's national reference laboratory.
Antibiotic susceptibilities were assessed, and the presence of the carbapenem resistance gene
blaNDM-1 was established by PCR. Isolates were typed by pulsed-field gel electrophoresis of
XbaI-restricted genomic DNA. Plasmids were analysed by S1 nuclease digestion and PCR typing.
Case data for UK patients were reviewed for evidence of travel and recent admission to
hospitals in India or Pakistan.
Findings
We identified 44 isolates with NDM-1 in Chennai, 26 in Haryana, 37 in the UK, and 73 in other
sites in India and Pakistan. NDM-1 was mostly found among Escherichia coli (36) and Klebsiella
pneumoniae (111), which were highly resistant to all antibiotics except to tigecycline and
colistin. K pneumoniae isolates from Haryana were clonal but NDM-1 producers from the UK
and Chennai were clonally diverse. Most isolates carried the NDM-1 gene on plasmids: those
from UK and Chennai were readily transferable whereas those from Haryana were not
conjugative. Many of the UK NDM-1 positive patients had travelled to India or Pakistan within
the past year, or had links with these countries.
Interpretation
The potential of NDM-1 to be a worldwide public health problem is great, and co-ordinated
international surveillance is needed.
Funding
European Union, Wellcome Trust, and Wyeth
Characterization of a New Metallo-β-Lactamase Gene, blaNDM-1, and a

Novel Erythromycin Esterase Gene Carried on a Unique Genetic
Structure in Klebsiella pneumoniae Sequence Type 14 from India
Dongeun Yong,1,2 Mark A. Toleman,2 Christian G. Giske,3 Hyun S. Cho,4 Kristina
Sundman,5 Kyungwon Lee,1 and Timothy R. Walsh2*
Yonsei University College of Medicine, Research Institute of Antimicrobial Resistance,

Seoul, Republic of Korea,1 Department of Medical Microbiology, Cardiff University, Cardiff,
United Kingdom,2 Clinical Microbiology, MTC—Karolinska Institutet, Karolinska University
Hospital, Stockholm, Sweden,3 Yonsei University College of Life Science and
Biotechnology, Seoul, Republic of Korea,4 Department of Clinical Microbiology, Örebro
University Hospital, Örebro, Sweden5
Received 10 June 2009/ Returned for modification 7 August 2009/ Accepted 10

September 2009
ABSTRACT
Top
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
A Swedish patient of Indian origin traveled to New Delhi, India, and acquired a urinary
tract infection caused by a carbapenem-resistant Klebsiella pneumoniae strain that typed
to the sequence type 14 complex. The isolate, Klebsiella pneumoniae 05-506, was shown
to possess a metallo-β-lactamase (MBL) but was negative for previously known MBL
genes. Gene libraries and amplification of class 1 integrons revealed three resistance-
conferring regions; the first contained blaCMY-4 flanked by ISEcP1 and blc. The second
region of 4.8 kb contained a complex class 1 integron with the gene cassettes arr-2, a
new erythromycin esterase gene; ereC; aadA1; and cmlA7. An intact ISCR1 element was
shown to be downstream from the qac/sul genes. The third region consisted of a new MBL
gene, designated blaNDM-1, flanked on one side by K. pneumoniae DNA and a truncated
IS26 element on its other side. The last two regions lie adjacent to one another, and all
three regions are found on a 180-kb region that is easily transferable to recipient strains
and that confers resistance to all antibiotics except fluoroquinolones and colistin. NDM-1
shares very little identity with other MBLs, with the most similar MBLs being VIM-1/VIM-2,
with which it has only 32.4% identity. As well as possessing unique residues near the
active site, NDM-1 also has an additional insert between positions 162 and 166 not
present in other MBLs. NDM-1 has a molecular mass of 28 kDa, is monomeric, and can
hydrolyze all β-lactams except aztreonam. Compared to VIM-2, NDM-1 displays tighter
binding to most cephalosporins, in particular, cefuroxime, cefotaxime, and cephalothin
(cefalotin), and also to the penicillins. NDM-1 does not bind to the carbapenems as tightly
as IMP-1 or VIM-2 and turns over the carbapenems at a rate similar to that of VIM-2. In
addition to K. pneumoniae 05-506, blaNDM-1 was found on a 140-kb plasmid in an
Escherichia coli strain isolated from the patient's feces, inferring the possibility of in vivo
conjugation. The broad resistance carried on these plasmids is a further worrying
development for India, which already has high levels of antibiotic resistance.
INTRODUCTION
Top
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
REFERENCES
The growing increase in the rates of antibiotic resistance is a major cause for concern in
both nonfermenting bacilli and isolates of the Enterobacteriaceae family. β-Lactams have
been the mainstay of treatment for serious infections, and the most active of these are
the carbapenems, which are advocated for use for the treatment of infections caused by
extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, particularly
Escherichia coli and Klebsiella pneumonia (21). However, carbapenemases are
increasingly being reported; and the most prevalent of these would appear to be KPC,
which has recently been characterized in the United States, Israel, Turkey, China, India,
the United Kingdom, and Nordic countries (20, 24, 26). KPC has invariably been found in
K. pneumoniae, although recent reports indicate that it can cross species boundaries (2).
OXA-48 is a class D carbapenemase that is mainly found in K. pneumoniae and that is
now reported from Turkey, China, India, and the United Kingdom (3, 5, 9). The mobile
class B enzymes or metallo-β-lactamases (MBLs) have been mainly found in
Pseudomonas aeruginosa, but they are increasingly being found in Enterobacteriaceae,
particularly isolates from Greece and Turkey (39). Those mainly found in K. pneumoniae
are the VIM-1/VIM-4 cluster (6).
The majority of resistance genes in Enterobacteriaceae are carried on class 1 integrons,

and this is also true for the carbapenemases, such as VIM-1/VIM-4 (24, 40). In such
cases, the genes are mobilized and evinced as gene cassettes and can theoretically move
as separate entities independent of the integrase gene. Indeed, the majority of the mobile
MBL genes are found as gene cassettes. These include blaIMP, blaVIM, blaGIM, blaSIM, and
blaKMH (41). The exception to these are genes encoding AIM-1 and SPM-1, which are
adjacent to and thought to be mobilized by ISCR elements via a transposition event called
rolling circle recombination (36).
Herein, we report on the genetic and biochemical characterization of a new subgroup of

MBL, designated NDM-1, originating from New Delhi, India. We also report on its novel
genetic context and describe a new erythromycin resistance gene, designated ereC.
Top
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and susceptibility testing. The identification of K. pneumoniae 05-

506 was performed with automated systems (the Phoenix and BD systems). Etest strips
(AB bioMerieux, Solna, Sweden) were used to determine susceptibility, and the
susceptibility results were interpreted according to the CLSI guidelines. E. coli J53 and E.
coli TOPO (Strategene, Amsterdam, The Netherlands) were used in the conjugation and
cloning experiments, respectively.
Phenotypic and molecular detection of MBL. Hodge and imipenem-EDTA double-disc
synergy tests as well as Etest were used to screen for MBL production. In addition, the
carbapenemase activities of cell sonicates from overnight broth cultures (Mueller-Hinton
broth; BD) were determined by spectrophotometric assays. These were undertaken by
using 150 µM imipenem as the substrate and 299 nm for the measurement of hydrolysis.
The assays were performed with or without EDTA (25 mM) to examine the inhibition of
carbapenemase activity. The strains were screened for the presence of known mobile MBL
genes (blaVIM, blaIMP, blaSPM-1, blaGIM-1, blaSIM-1, blaAIM-1) by PCR with the primers reported
previously (22). The strains were also screened for the presence of other β-lactamase
genes (blaCTX, blaCMY, etc.) as well as class 1, 2, and 3 integron structures (24). Strains
known to harbor MBL genes were used as positive controls for the PCR analysis.
Conjugation. E. coli J53 was used as the recipient in the conjugation studies, and mating
was carried out on blood agar medium without selection (25). After 18 h, the mixed
cultures were taken from the plates, suspended in saline, and plated onto MacConkey
medium containing sodium azide (100 µg/ml) and ceftazidime (8 µg/ml). Confirmation
that conjugation had taken place in E. coli J53 was carried out by indole testing and
testing for the presence of the MBL gene by PCR analysis.
DNA cloning. Cloning experiments were performed by using the cloning vector pK18, as
described previously (35). Restriction endonucleases BamHI, EcoRI, and HindIII
(Invitrogen, Carlsbad, CA) were used to cut both plasmid and K. pneumoniae 05-506
chromosomal DNA. Transformation was carried out by using electroporation and E. coli
TOP10 cells (Invitrogen Ltd, Paisley, United Kingdom). Transformants were selected on LB
agar plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (30 µg/ml),
ceftazidime (6 µg/ml), and kanamycin (25 µg/ml). Recombinant plasmids were recovered
with a QIAprep spin miniprep kit (Qiagen, West Sussex, United Kingdom). Restriction
endonuclease analysis of the plasmid was carried out with a variety of restriction enzymes
to determine the size of the ligated nucleotide segment.
The PCR-amplified open reading frame of NDM-1 was digested with the appropriate
enzymes and was subsequently ligated into the pVFT3S (six-His—trx fusion vector)
bacterial expression vector. The construct was transformed into E. coli BL21 groEL/groES
(pGro7/BL21; Takara Bio Inc., Japan).
DNA sequencing and computer analysis. Both strands of the clones from the libraries
that were produced were sequenced by using M13 forward and M13 reverse primers. The
cloned insets containing blaNDM-1, the complex class 1 integron, and blaCMY-4 were fully
sequenced. The nucleotide sequences, deduced amino acid sequences, and phylogenetic
relationships were analyzed by using the Lasergene software package (DNAStar, Madison,
WI). The sequences obtained were compared to the sequences available on the Internet
(http://www.ebi.ac.uk/fasta33/).
Pulsed-field gel electrophoresis (PFGE) and genomic DNA digestion with

endonucleases. Genomic DNA was prepared in agarose blocks and digested with the
restriction enzymes XbaI and SpeI (Roche Diagnostics, Mannheim, Germany), I-CeuI
(New England Biolabs, Beverly, MA), and S1 nuclease (Invitrogen, Abingdon, United
Kingdom). The DNA fragments were separated by use of a CHEF-DR III apparatus (Bio-
Rad, Hercules, CA) for 20 h at 6 V/cm and 14°C with initial and final pulse times of 0.5
and 30 s, respectively.
Southern blot analysis. Southern blotting was performed on agarose gels by in-gel
hybridization with the blaNDM-1 probe labeled with 32P (Strategene) and by a recently
described random primer method (22).
MLST. Multilocus sequence typing (MLST) of K. pneumoniae was performed as described

by Diancourt et al. (7). Experimentally determined DNA sequences were uploaded into
the MLST database
(http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html),
and allelic numbers as well as sequence types (STs) were obtained.
ß-Lactamase purification. Cell lysates were centrifuged at 12,000 rpm for 30 min at
4°C. The supernatant was obtained and loaded onto Ni-nitrilotriacetic acid agarose. The
NDM-1 protein was eluted by the use of elution buffer with 300 mM imidazole. The protein
was separated from the His-trx tag by tobacco etch virus protease and was finally purified
by gel filtration on a GS320 column.
Kinetic measurements. Purified ß-lactamase was used to determine the kinetic

parameters kcat and Km. Reactions were performed at 30°C in 1 ml of assay buffer (10 mM
HEPES, pH 7.5). The assays were performed in a UV-1601PC spectrophotometer
(Shimadzu Corp., Tokyo, Japan) by observing the changes in absorption resulting from
the opening of the ß-lactam ring at the specific wavelengths for each of the each
antimicrobial agents evaluated, as described previously (18).
Nucleotide sequence accession number. The nucleotide sequence data reported in

the present study have been assigned EMBL/GenBank nucleotide accession number
FN396876.
RESULTS
Top
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
REFERENCES
Clinical details for K. pneumoniae 05-506. A 59-year-old male patient who was
originally from India but who had lived in Sweden for many years and who often returned
to India had type 2 diabetes mellitus and had had multiple strokes. In November 2007,
he traveled to India and on 5 December was hospitalized in Ludhiana, Punjab, with a
large gluteal abscess. In December 2007, he was admitted to a hospital in New Dehli,
where he was again operated on and where he developed a decubital ulcer. On 8 January
2008 he was referred to Örebro, Sweden. During his stay in New Dehli he received
amoxicillin (amoxicilline)-clavulanic acid, metronidazole, amikacin, and gatifloxacin (all of
them parenterally).
Clinical isolate K. pneumoniae 05-506 was derived from a urinary culture on 9 January
2008. He had no clear symptoms of urinary tract infection at that time. The amount of
bacteria found in a culture of his urine was only 1,000 CFU/ml. Both ESBL-positive
Escherichia coli and a carbapenem-susceptible Acinetobacter sp. were isolated from his
deep wounds. An ESBL-positive E. coli strain was also found in a culture of fluid resulting
from external otitis.
On 6 March 2008, the patient was discharged to a nursing home. On 1 April a new urine
sample for culture was taken, and an ESBL-producing K. pneumoniae isolate was found.
The original carbapenem-resistant K. pneumoniae isolate has never been found in any
other cultures of samples from the patient. As K. pneumoniae 05-506 was carbapenem
resistant and positive by the MBL Etest (AB bioMerieux), it was investigated further.
Moreover, fecal samples were collected from the patient during his stay at the nursing
home to identify the source of 05-506; however, while 05-506 could not be recovered, an
MBL-positive E. coli isolate was recovered and was designated E. coli NF-NDM-1.
Typing and phenotypic characterization of K. pneumoniae 05-560, E. coli NF-

NDM-1, and transformants. ST14 is a single-locus variant of ST15, which has been
encountered with KPC- and CTX-M-producing K. pneumoniae strains (19, 30). We have
also found ST14 among U.S. KPC producers (19).
The susceptibility patterns of K. pneumoniae 05-560, E. coli NF-NDM-1, E. coli TOP10, E.

coli TOP10(pNDM-1), E. coli J53, and E. coli J53(pNDM-1) are listed in Table 1. K.
pneumoniae 05-560 was resistant to all β-lactams and was sensitive only to colistin (Table
1). The susceptibility profile of E. coli NF-NDM-1 largely reflects that of 05-560, apart
from susceptibility to aztreonam, cefepime, and ciprofloxacin, to which the level of
resistance of 05-560 was not quite as high. When the cloned MBL gene and its adjacent
DNA were expressed in an E. coli background, the strain was highly resistant to all
cephalosporins, apart from cefepime (MIC, 8 µg/ml), and was sensitive to aztreonam.
However, the aztreonam MICs differed significantly for TOP10 blaNDM-1 (MIC, 0.25 µg/ml)
and J53 blaNDM-1 (MIC, 24 µg/ml), even though the aztreonam MICs for TOP10 and J53
were very similar. Interestingly, the MICs of imipenem and meropenem were relatively
high for both E. coli TOP10 and J53 compared with those for strains with the other cloned
MBL genes (31, 41).
View this TABLE 1. Antimicrobial susceptibility patterns of K. pneumoniae
table: 05-560, E. coli NF-NDM-1, transformants, and conjugants
[in this
window]
[in a new
window]
Phenotypic and molecular screening for MBLs. The results of the MBL screening
tests were positive by the imipenem-EDTA double-disc synergy test, the MBL Etest (256
µg/ml with imipenem and 3 µg/ml with imipenem plus inhibitor), and the determination of
carbapenemase activities by spectrophotometry (data not shown). However, PCR analysis
failed to detect previously known MBL genes when the control genes blaVIM, blaIMP, blaSPM-1,
blaGIM-1, blaSIM-1, blaAIM-1 were used. PCR did detect a class 1 integron (intI and qacE 1/sul)
of 4.8 kb.
Nucleotide and deduced amino acid sequences of three resistance-conferring

regions. Total DNA from K. pneumoniae 05-560 was used to construct a genomic library
to isolate all genes conferring β-lactam resistance. After the plasmids of resistant colonies
were analyzed by restriction profiling, it was evident that there were two different types of
β-lactam resistance. One group carried a putative AmpC enzyme, on the basis of the fact
that it was resistant to the effects of EDTA but sensitive to inhibition with cloxacillin. The
other group possessed a putative MBL on the basis of the fact that it was resistant to the
effects of cloxacillin and clavulanic acid but sensitive to EDTA. Restriction of the
recombinant plasmids showed that the AmpC-type genes are carried on a 2.1-kb
fragment and that the MBL gene is carried on a 4.3-kb fragment. Additionally, the 4.8-kb
fragment containing the class 1 integron was also sequenced.
The class 1 integron contains a unique set of genes, some of which have been previously
reported from Asia. Immediately next to the intI gene is an arr-2 cassette that mediates
resistance to rifampin (rifampicin) and that has been found in isolates of Pseudomonas
aeruginosa and Acinetobacter baumannii from China and Hong Kong, respectively
(GenBank accession numbers EU886979 and AY038837, respectively) (1, 14, 27, 38). It
has also been identified on the chromosome of Streptomyces coelicolor (GenBank
accession number NC003888). Next to aar-2 is a novel erythromycin esterase gene
(ereC) which has 92% nucleotide sequence identity with ereA from E. coli (GenBank
accession number AY183453) (Fig. 1). The predicted protein sequence differs from the
EreA sequence at an additional 10 amino acids at the N terminus and also has 31 amino
acid substitutions throughout the sequence. At present, only two erythromycin esterases,
ereA and ereB, have been found (GenBank accession numbers DQ157752 and X03988,
respectively), and these share only 25% identity in their amino acid sequences (Fig. 1).
Adjacent to the esterase gene is aadA1, which mediates resistance to gentamicin, and the
cmlA7 gene cassette, which mediates resistance to chloramphenicol, is present between
aadA1 and qac/sul (Fig. 2).
FIG. 1. Alignment of novel erythromycin esterase

EreC (this study) with EreA and EreA2. Amino
acid changes between EreC and EreA are
indicated with asterisks. The single amino acid
View larger version (32K): difference between EreA and EreA2 is highlighted
[in this window] in gray.
[in a new window]
[Download PowerPoint slide]
FIG. 2. Three characterized antibiotic resistance-

conferring regions from K. pneumoniae 05-506.
(A) The 4.3-kb region is linked to the 4.8-kb
complex class 1 integron region. The genes
View larger version (18K):
encoding the efflux pump and lactate
[in this window]
dehydrogenase (gray diagonal lines) are of
[in a new window]
Klebsiella origin. blaNDM-1 (dark gray) is flanked
between the pathogenicity island (vertical black
lines) and IS26/Tn3 (black small squares). This
region lies downstream of the 4.8-kb complex
class 1 integron containing Int (checkered area),
arr-2, ere2A, aadA1, and cmlA7 as gene
cassettes and qac 1 (white boxes). Downstream
is an intact copy of ISCR1 (black and white
diagonal lines). Arrows, direction of
transcription; black ellipses, 59-base elements;
, genes that are truncated. (B) blaCMY-4 (gray) is
located between ISEcP1 (black) and blc (white), as
reported previously (16
16).
16
The AmpC fragment is shown in Fig. 2 and carries blaCMY-4, which is responsible for the
AmpC phenotype. Upstream of blaCMY-4 is an intact copy of ISEcP1, which is probably
responsible for the transposition and mobility of blaCMY-4. Downstream is blc, which is a
common feature in many blaCMY regions and which has been reported by Kang et al. (16).
The 4.3-kb fragment contained a novel MBL gene, designated blaNDM-1 (Fig. 2). This section
of DNA was a chimera of truncated and intact genes. The blaNDM-1 gene was found between
a section of K. pneumoniae chromosomal DNA and truncated sections of the IS26 and Tn3
transposase genes. The Klebsiella DNA consisted of a putative efflux protein gene
adjacent to another gene encoding L-lactate hydrogenase, and both were 99.5% identical
to a section of chromosomal DNA from K. pneumoniae MGH78578 (GenBank accession
number CP000647) and found in the same orientation in which they are present on the
Klebsiella chromosome. The lactate dehydrogenase gene was truncated and missing
approximately 750 bp of the 5' end of the gene. Immediately upstream of the lactate
dehydrogenase gene was a short internal part of the ampC gene, blaDHA-1 (base pair
coordinates 1268 to 1478), consisting of the DNA encoding amino acids 196 to 265. This
gene was truncated at both ends both by the lactate dehydrogenase gene and by a
section of DNA encoding the 5' end of a phosphoribosyl anthalinate isomerase gene. This
section of DNA (coordinates 1478 to 2100) has identities ranging from 55% to 68% to the
same gene from various members of the alphaproteobacteria, including Rhodobacter
spp., Rhodopseudomonas spp., Caulobacter spp., and Novosphingobium spp. The highest
degree of identity was found with Novosphingobium aromaticivor, with 68% nucleotide
sequence identity over 575 nucleotides. Again, this gene was truncated and missing the
DNA encoding the last 19 amino acids of the C terminus of the protein. The blaNDM-1 gene is
encoded within a 1,350-bp section of DNA with a lower G+C content (57%) than that of
the surrounding DNA, which has a G+C content of 62 to 65% and which consists of a
gene of 807 nucleotides and flanking DNA on either side. Upstream of the blaNDM-1 gene is
250 nucleotides of DNA that contains several sequences that show similarity to
conventional promoter sequences, and it is likely that the promoter driving the expression
of blaNDM-1 has been acquired along with the gene (Fig. 2). Farther upstream is a truncated
left-hand end of an IS26 insertion sequence, including 270 bp of the left-hand end of the
element with the left-hand inverted repeat and DNA encoding the first 74 amino acids of
the transposase. This element lies adjacent to a tnpA gene of a Tn3-like transposon
(coordinates 3748 to 4318), which is itself truncated and missing the 5' end of the gene
encoding the first 128 amino acids of the transposase (Fig. 2).
Structural properties of NDM-1. The blaNDM-1 open reading frame encodes a putative
protein of 269 amino acids with a molecular mass of approximately 27.5 kDa (Fig. 3). The
NDM-1 sequence and the behavior of NDM-1 during gel filtration and mass spectrometry
(data not shown) indicate that it is actively present as a monomer. Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis analysis showed an approximate molecular
mass of 28 kDa (data not shown). NDM-1 possesses a leader peptide, with the probable
cleavage site occurring at position 19 between two alanines which are in a very similar
position to the cleavage site for the VIM MBLs (Fig. 3). NDM-1 also shares the additional
loop of the VIM enzymes at positions 34 and 47 but also possesses a unique additional
sequence at positions 162 to 166 (blaNDM-1 and not BBL numbering) (Fig. 3) (4). NDM-1
shares very little identity with other MBLs and is the most closely related to VIM-1/VIM-2,
with which it has only 32.4% identity (Fig. 4). Curiously, NDM-1 also has a unique HXHXD
motif among the mobile MBLs, as it contains an alanine between the two histidines. NDM-
1 also possesses a tyrosine at position 222 instead of the universally conserved
tryptophan (Fig. 3). The mature peptide has a theoretical pI of 6.9.
FIG. 3. Alignment of the amino acid sequences of

NDM-1 with the amino acid sequences of IMP-1,
IMP-2, IMP-8, VIM-1, VIM-2, GIM-1, SPM-1,
SIM-1, and KHM-1. Conserved residues
coordinating zinc ions are denoted with
asterisks. Key amino acids are also numbered
according to the BBL scheme (12
12).
12 Additional
View larger version (61K): amino acids unique to NDM-1 are enclosed in a
[in this window] light gray box. Amino acid substitutions near the
[in a new window] active site are highlighted in plain boxes (13
13).
13
FIG. 4. Phylogenetic tree obtained for MBL

subgroup B1. The numbers for the substitutions
should be multiplied by 100. The alignment used
View larger version (4K):
was prepared with the CLUSTALW program. The
[in this window]
GenBank accession numbers for the sequences
[in a new window]
window]
are as follows: IMP-1, S71932; IMP-2, AJ243491;
IMP-8, AF322577; VIM-1, Y18050; VIM-2,
AF191564; GIM-1, AJ620678; SPM-1, AJ492820;
SIM-1, AY887066; KHM-1, AB364006.
Functional properties of NDM-1. Analysis of the purified NDM-1 enzyme by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a single band
corresponding to a size of 28 kDa (data not shown). Table 2 lists the kcat and Km values
and compares them with the kinetic values of IMP-1 and VIM-2. NDM-1 shows tight
binding (low Km values) to most cephalosporins and, in particular, to cefuroxime,
cefotaxime, and cephalothin (cefalotin). It also shows relatively tight binding to the
penicillins, which is unusual for an MBL, as both IMP-1 and VIM-2 possess higher Km
values (Table 2). Interestingly, NDM-1 does not bind to the carbapenems as tightly as
IMP-1 or VIM-2. The rate of turnover (kcat) of penicillins by NDM-1 is lower than that by
IMP-1 and VIM-2 and is generally unusual for MBLs. The kcat values for the cephalosporins
are more similar to those of IMP-1 than to those of VIM-2, yet the kcat values for the
carbapenems (20 and 12 s–1 for imipenem and meropenem, respectively) are more similar
to those for VIM-2 (10 and 1 s–1, respectively) than IMP-1 (46 and 50 s–1, respectively)
(Table 2). Due to the fact that most of the Km values are low, the kcat/Km ratios are
reasonably high and comparable to those of VIM-2. The exception is that of ceftazidime,
which has a Km of 181 µM and a kcat of 5 s–1 and which thus presents with a kcat/Km of 0.03.
View this table: TABLE 2. Steady-state kinetic constants of NDM-1, IMP-1,

[in this window] and VIM-2
[in a new
window]
Plasmid analysis and back probing with blaNDM-1. The plasmids carrying blaNDM-1 from
both K. pneumoniae 05-506 and E. coli NF-NDM-1 were successfully transferred to E. coli
J53 at a frequency of approximately 10–4 to 10–5. PCR was undertaken with primers
directed to ISCR1 and blaNDM-1, and both were shown to be carried by pNDM-1. To examine
the sizes of the plasmids from 05-506, E. coli J53(pNDM-1), and E. coli NF-NDM-1,
genomic DNA was isolated from each of the strains, restricted with S1 nuclease, and
examined by PFGE. The corresponding gel was blotted with blaNDM-1, and the data clearly
show that blaNDM-1 is on a 180-kb plasmid in 05-506 and E. coli J53(pNDM-1), but in E. coli
NF-NDM-1 it is on a smaller plasmid of 140 kb (Fig. 5). Inc typing was unsuccessful, and
plasmid pNDM-1 is currently being sequenced to identify the origin of replication and
other functions carried by pNDM-1.
FIG. 5. PFGE of plasmids from K. pneumoniae 05-506,
conjugant E. coli J53(pNDM-1), and E. coli NF-NDM-1 (from the
patient's normal flora). The bands with white arrows showed
positive signals by Southern blot hybridization with the NDM-1
probe (data nor shown). K. pneumoniae 05-506 carries blaNDM-1
on a 180-kb plasmid, whereas E. coli NF-NDM-1 carries blaNDM-
1 on a plasmid of approximately 140 kb.
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As the broader genetic context was not known and ISCR1 had been found in a large
antibiotic resistance-conferring region, K. pneumoniae 05-506 was also digested with
XbaI, SpeI, I-CeuI, and S1 nuclease and was then probed with radiolabeled blaNDM-1. The
probing results were compared to those of probing of ISCR1 to ascertain whether blaNDM-1
and ISCR1 are on the same genetic structure (Fig. 6). The probing data for ISCR1 and
blaNDM-1 are identical and clearly indicated probing to the same 25-kb region, suggesting
that the regions are adjacent to one another.
FIG. 6. PFGE findings (A and C) and Southern blot

hybridization (B and D) with blaNDM-1 (A and B)
and an ISCR1 probe (C and D). The genomic DNA
of the index strain (K. pneumoniae 05-506) was
undigested (lane 1) or was digested with S1
nuclease (lane 2), XbaI (lane 4), SpeI (lane 5), or
View larger version (85K): I-CeuI (lane 6). Lane 3, DNA size marker.
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DISCUSSION
Top
ABSTRACT
INTRODUCTION
MATERIALS AND
AND METHODS
RESULTS
DISCUSSION
REFERENCES
K. pneumoniae 05-506 types as ST14, a single-locus variant of type ST15. Recently, K.

pneumoniae ST15 has been branded as a new methicillin-resistant S. aureus type of
pathogen due to its wide distribution and its carriage of blaCTX-M15 and other resistance
markers (19, 30). Strain 05-506 clearly arose in India, but there are few data arising
from India to suggest how widespread it is. We are currently undertaking studies in
several Indian cities to examine these points. Interestingly, there appears to be the
possible transfer of blaNDM-1 in vivo either from K. pneumoniae to E. coli or vice versa, but
more interestingly, the plasmids carrying blaNDM-1 in the two species are of different sizes.
This evidence would suggest that there is rearrangement in vivo which could result from
either duplication and insertion, e.g., transposition or rolling circle replication from the
smaller plasmid, or deletion from the larger plasmid (33, 34). The plasmid carrying blaNDM-
1 also carries blaCMY-4 and the complex class 1 integron carrying several antibiotic
resistance-conferring genes (33), and it has also shown itself to naturally have a broad
host range. When the plasmid was transferred to E. coli J53, the E. coli strain containing
pNDM-1 was resistant to all antibiotics except colistin and ciprofloxacin and was shown by
blotting and PCR to carry blaCMY-4, the ISCR region, and blaNDM-1. Therefore, the rapid
dissemination of this plasmid among clinical bacteria would be a nightmare scenario.
This is the first report of an MBL gene not carried in a class 1 integron or adjacent to
ISCR1 elements. It appears to have an intact promoter sequence that is not dependent
on the IS26 element inserted upstream, even though IS26 is found adjacent to other
resistance genes and pathogenicity islands (8, 10, 11, 15). The plasmid clearly contains
Klebsiella housekeeping genes (Fig. 2) as part of this capturing DNA machinery, and
therefore, these have probably been purloined from the Klebsiella chromosome.
Furthermore, restriction digests and probing experiments indicate that blaNDM-1 and ISCR1
are on a DNA fragment of less than 25 kb (Fig. 6). While Tn3 and IS26 are capable of
movement, the amelioration of such a large region spanning from blaNDM-1 to ISCR1 could
have occurred only through a series of duplications and homologous recombination (34).
The known location of ISCR1 in all sequences analyzed so far is at the end of a complex
class 1 integron that is more often than not contained within a Tn3-like transposon, and
therefore, it is possible that the ISCR1 element is upstream of the blaNDM-1 gene and
farther downstream of the Tn3 transposase. Additionally, the chimeric structure of the
4.3-kb insert with its composition of fused truncated and nontruncated genes is a classic
signature of ISCR1 transposition activity (22, 34, 37).
NDM-1 not only is a new subclass of the B1 group of MBLs but also possesses novel
amino acids near the active site, suggesting that it has a novel structure. NDM-1
possesses only 32.4% identity with VIM-2, and therefore, molecular replacement or
threading cannot be undertaken to reasonably ascertain its structure. However, NDM-1
does show some similarities to the VIM group of MBLs; namely, it possesses the
additional sequences at positions 226 to 228 (BBL numbering) not present in other MBLs
(12). These residues contain R228 in VIM-2, which is thought to stabilize C221 and
possibly H263, yet in NDM-1 the substitute R228A is unlikely to play such a role. VIM-2
also possesses Y224, which is in close proximity to C221, whereas NDM-1 has a lysine,
which is likely to serve a similar role. Crystallographic insights into the mechanism of
action of VIM-2 by Garcia-Saez et al. suggest that Y67 pivots in toward the enzyme away
from bulk solvent and in so doing stabilizes H263; but NDM-1, like other non-VIM MBLs,
possesses the substitution Y67V, which is unlikely to function in the same manner (13).
However, NDM-1 does possess S69, which is in close proximity to Y67 and less so to
R121 and H263 (13). The role of these substitutions is not apparent, as NDM-1 possesses
kinetics similar to those of VIM-2 (23, 29). In general, NDM-1 binds more tightly (it has a
lower Km) to most β-lactam substrates and hydrolyzes them less well (it has a lower kcat),
with the exception being the less bulky carbapenems, for which NDM-1 possesses
relatively high Km and kcat values for both imipenem and meropenem (23, 29).
In addition to blaNDM-1, this study also characterized a new erythromycin esterase gene,
designated ereC (28). The esterase gene is part of the complex class 1 integron
containing the Asian rifampin resistance gene, arr-2, which is also contained as a gene
cassette (17). If this structure is stable, together with blaNDM-1, it will act as a unique
genetic marker for the prevalence and stability of plasmid pNDM-1 throughout Indian
Enterobacteriaceae strains. We are currently doing molecular epidemiological studies of
Indian Enterobacteriaceae carbapenem-resistant strains to examine the prevalence and
range of pNDM-1 among clinical and nonclinical isolates. In a country where there is little
control on antibiotic prescriptions, the rapid dissemination of such a plasmid is alarming.
ACKNOWLEDGMENTS
This work was funded by EU grant LSHM-CT-2005-018705 and Wellcome Trust grant
084627/Z/08.
D. Yong thanks Seok H. Jeong and Yunsop Chong for their valuable advice.
FOOTNOTES
* Corresponding author. Mailing address: Department of Medical Microbiology, School of

Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom. Phone: 44
(0)1453 811744. Fax: 44 (0)2920 742161. E-mail: WalshTR@Cardiff.ac.uk
Published ahead of print on 21 September 2009.
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Top
ABSTRACT
INTRODUCTION
RESULTS
DISCUSSION
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Dear Editor,

New Delhi metallo-beta-lactamases (NDM) is a nomenclature that Indians cannot be

Carbapenems (imipenem, ertapenem, meropenem, doripenem) are a class of beta-lactam

Carbapenemase enzymes belonging to Ambler molecular classes A to D have been

Treatment of infections caused by pathogens producing carbapenemases, including

1. Yong D, Giske CG, Toleman M, Walsh TR. A novel subgroup metallo-beta-

Jump to: navigation, search

ND Metallo-beta-lactamase (NDM-1)[1] is a gene that makes bacteria resistant to

The gene produces a metallo-beta-lactamase, an enzyme that hydrolyzes and inactivates

The following antibiotics are inactivated by the enzyme:

In early August a chemical compound, GSK-299423, was found to significantly fight

In contrast, an editorial in the March 2010 issue of the Journal of Association of

1. ^ a b Kumarasamy et. al. (2010). "Emergence of a new antibiotic resistance mechanism in

Indian author says superbug report is fudged

CHENNAI: A day after the Lancet report on a drug-resistant superbug NDM-1

An obituary- On the Death of antibiotics!

Consultant in Infectious Diseases and Clinical Mycology, Apollo Hospital, Chennai.

1. Cesar A. Arias. Antibiotic-Resistant Bugs in the 21st Century —A Clinical Super-

Emergence of a new antibiotic resistance

Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi

European Union, Wellcome Trust, and Wyeth

Characterization of a New Metallo-β-Lactamase Gene, blaNDM-1, and a

Yonsei University College of Medicine, Research Institute of Antimicrobial Resistance,

Received 10 June 2009/ Returned for modification 7 August 2009/ Accepted 10

The majority of resistance genes in Enterobacteriaceae are carried on class 1 integrons,

Herein, we report on the genetic and biochemical characterization of a new subgroup of

MATERIALS AND METHODS

Bacterial strains and susceptibility testing. The identification of K. pneumoniae 05-

Pulsed-field gel electrophoresis (PFGE) and genomic DNA digestion with

MLST. Multilocus sequence typing (MLST) of K. pneumoniae was performed as described

Kinetic measurements. Purified ß-lactamase was used to determine the kinetic

Nucleotide sequence accession number. The nucleotide sequence data reported in

Typing and phenotypic characterization of K. pneumoniae 05-560, E. coli NF-

The susceptibility patterns of K. pneumoniae 05-560, E. coli NF-NDM-1, E. coli TOP10, E.

Nucleotide and deduced amino acid sequences of three resistance-conferring

FIG. 1. Alignment of novel erythromycin esterase

FIG. 2. Three characterized antibiotic resistance-

FIG. 3. Alignment of the amino acid sequences of

FIG. 4. Phylogenetic tree obtained for MBL

View this table: TABLE 2. Steady-state kinetic constants of NDM-1, IMP-1,

FIG. 6. PFGE findings (A and C) and Southern blot

K. pneumoniae 05-506 types as ST14, a single-locus variant of type ST15. Recently, K.

* Corresponding author. Mailing address: Department of Medical Microbiology, School of

Published ahead of print on 21 September 2009.

1. Arlet, G., D. Nadjar, J. L. Herrmann, J. L. Donay, P. H. Lagrange, and A.

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