Reprinted from

www.antimicrobe.org

NDM-1—The Newest Recruit to the Army of β-lactamases

Sarah Turner, Ja’Nean Ray and Timothy Mietzner

In the recent popular press much discussion has been made of a new group of ‘super
bugs’ that are emerging from India and Pakistan. These organisms have a specific
resistance gene designated as NDM-1 (New Delhi metallo-β-lactamase-1). The gene
encoding this β-lactamases associated with organisms that demonstrate multiple
antimicrobial resistance profiles, limiting the treatment options for these infections.

NDM-1 was first described by Yong et al in December 2009 in a Swedish national

who fell ill with an antibiotic-resistant urinary tract infection that he acquired in New
Delhi, India. The infection was unsuccessfully treated in a New Delhi hospital which led
to the patient's repatriation to Sweden. Klebsiella pneumoniae which expressed a novel
metallo-β-lactamase (MBL) was identified as the source of the infection. This was the
first report identifying the NDM-1 gene and this organism was susceptible only to
fluoroquinolones and colistin. 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” (1).

In March 2010 the spread of NDM-1 was studied in a tertiary care unit in Mumbai,
India. In 3 months, 22 different species of Enterobacteria were identified as NDM-1
producers. NDM-1 was so pervasive that only two Klebsiella pneumoniae carbapenem
resistant strains did not produce the NDM-1 enzyme (2).
Much of the concern surrounding NDM-1 in the medical community is due to the fact
that the gene has not been confined to a specific species of bacteria, which has largely
been the case for methicillin-resistant Staphylococcus aureus (MRSA). Rather, this gene
can be transferred to many different types of Enterobacteriaceae. Thus, this broad host
range capacity gives it the potential to spread at an unprecedented rate (3).

The Indian response by members of Parliament disputed the claim made in the August
2010 publication of the Lancet that NDM-1 originated in India. The health ministry in
India asserted that the alarm was spurred by Western doctors fearing loss of business to
India by way of medical tourism. Prof. K.N. Prasad, a microbiologist at the Sanjay
Gandhi Post Graduate Institute of Medical Science argued that the sensationalization over
NDM-1 originating in India is unnecessary, saying “Already 40 types of bugs similar to
NDM-1 exist globally, that includes the German imipenem-resistant metallurgic strain.

The Indian variant is just the 41st strain. These things

happen.”[http://www.hindustantimes.com/Linking-India-to-superbug-unfair-and-wrong-
says-India/Article1-585840.aspx ]. Professor Kumarasamy (University of Madras), the
primary author of the 2010 Lancet study maintained that he does not agree with the part
of the article that advises people to avoid elective surgeries in India. He said that many of
the interpretations were added later without his permission or knowledge
[http://timesofindia.indiatimes.com/city/chennai/Indian-author-says-superbug-report-is-
fudged/articleshow/6302479.cms ]. NDM-1 isn't unique in attracting controversy over its
name. From the colloquial term “Swine Flu” for H1N1 in 2009 to Syphilis being known
as the “French disease” in 1530, denoting specific geographic locales as a name for a
pathogen can be contentious.

β -Lactamases and NDM-1

β-lactamases are the primary causes of bacterial resistance to β-lactam antibiotics.

These enzymes hydrolyze the β-lactam ring common to all penicillins and carbapenems
(Figure 1). These enzymes are quite diverse in nature and can be organized into classes.
In 1903, international efforts to construct the Panama Canal were being threatened by the
twin biological hazards of yellow fever and malaria. The French decided to leave the
endeavor in the hands of the Americans after enduring more than 12,000 deaths and an
estimated $40 million loss. Tropical diseases were a deadly enemy, and if American
efforts were to succeed), these diseases needed to be overcome.

Figure 1. Action of Beta Lactamase Cleaving the β-lactam Ring.

The two major classification schemes are the functional scheme proposed by Bush
and Jacoby (4) and the molecular classification scheme proposed by Ambler (5). This
Ambler classification is cited in the majority of publications concerning NDM-1. β-
lactamases according to the Ambler system are classified by their amino acid sequence
(Figure 2). Class A, C and D are different serine-β-lactamases. Class B refers to all the
metallo-β-lactamases. There are similarities between the serine-β-lactamases that suggest
they may have descended from a common ancestor. However, metallo-β-lactamases are
very different from each other and probably are not related (6). Class A is the most
diverse. The resistance can be encoded on plasmids, integrons, or on the bacterial
chromosome (7).

Class A were the first β-lactamases described and hydrolyzed only simple penicillins.
Later, resistance against both narrow and broad spectrum cephalosporins emerged and
are now described as extended-spectrum β-lactamases (ESBLs) (8). A well known
example of Class A ESBLs is found in the well described MRSA strains. This class also
includes serine carbapenemases. Bacteria belonging to this group of β-lactamases are
resistant to carbapenems, cephalosporins, penicillins, and aztreonam. Their multi-drug
resistance is especially difficult to treat (7).
 Class B contains all the metallo-β-lactamases including NDM-1. This class requires
a bivalent metal ion, usually zinc, for the enzyme’s active site (6). Like Class A, the
resistance in class B can be due to plasmids, integrons or chromosomes. Class B is
usually resistant to everything except aztreonam. The plasmid carrying NDM-1 allows
the bacteria to hydrolyze all β-lactams except for aztreonam (7). Because this plasmid is
often transferred to bacteria that already has non-plasmid mediated resistance, it is able to
hydrolyze all antibiotics except for tigecycline or colistin. In some cases, the bacteria is
impervious even to these (3).

Figure 2. Relationship of β-lactamases as Described by Ambler.

Class C β-lactamases were initially identified as being resistant to cephalosporins.

The resistance is chromosomally mediated and is encoded by ampC (9). The transfer of
ampC to bacteria through plasmids is becoming more prevalent. These bacteria are
usually resistant to penicillins and cephalosporins. These bacteria poorly hydrolyze
cefepime and are sensitive to cloxacillin, oxacillin, and aztreonam (7).

Class D are named oxacillinases because they were identified to hydrolyze isoxazolyl
β -lactams such as oxacillin and methicillin. The resistance in this class is commonly
conferred by plasmids and integrons (9). This class can usually hydrolyze penicillins,
cephalosporins, extended-spectrum cephalosporins, and carbapenems. Currently, they
are inhibited by known β-lactamase inhibitors clavulanate, sulbactam, and tazobactam
with some exceptions (7).

Integrons and The Spread of Multiple Antibiotic Resistance

In 1986, the DNA sequence of several seemingly unrelated antibiotic resistance genes
heralded the first hints regarding integrons (10). Common regions were noted upstream
and downstream of various antibiotic resistance genes. These regions were found to be in
different places on various plasmids, suggesting that, like transposons, these elements
were mobile. However, the element differed from transposons in two important
characteristics: (i) transposons have direct or indirect repeat sequences at their ends, but
the regions surrounding the antibiotic resistance genes in the new elements were not
repeats, and (ii) the elements contained a site-specific integrase gene of the same family
as those found in phage but lacked many gene products associated with transposition.
Due to these differences, the elements were not grouped with transposons and were
named integrons.

Integrons are mobile DNA elements with the ability to capture genes, notably those
encoding antibiotic resistance, by site-specific recombination. Integrons have an integrase
gene (int), a nearby recombination site (attI), and a promoter. There are at least three
classes of integrons based upon the type of integrase gene they possess. Class 1 integrons
have been examined the most extensively. They consist of a variable region bordered by
5' and 3' conserved regions. The 5' region is made up of the int gene, attI, and the
promoter which drives transcription of genes within the variable region. The 3' region
consists of an ethidium bromide resistance locus (qacED1), a sulfonamide resistance gene
(sulI), and an open reading frame containing a gene of unknown function. The integrase
of Class 2 integrons is located within the 3' conserved region. Class 3 integrons have yet
to be thoroughly studied. Examples of Class 1 integrons are shown in Figure 3.

Figure 3. Examples of Class 1 Integrons (taken from

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/transposons/integrons/int
egrons.html )

The antibiotic resistance genes that integrons capture are located on gene cassettes.
The cassettes consist of a promoterless gene and a recombination site (attC). The
cassettes can exist as free, circular DNA but cannot be replicated or transcribed in this
form. A recombination event occurs between attI and attC, integrating the cassette into
the integron. The gene on the cassette is then bound by the attI site on the 5' side and by
attC on the 3' side. The evolution of an integron is illustrated in Figure 4.

Figure 4. Evolution of An Integron (taken from

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/transposons/integrons/int
egrons.html)
 In the original description of the NDM-1 gene, Yong et al (1) prepared a genomic
DNA library from the resistant K. pneumoniae clinical isolate. This library was
transformed into a -lactam sensitive E. coli background and selected for growth on β-
lactam-containing medium. From this three distinct antibiotic resistance conferring
regions were identified (Figure 5). These included genes for resistance to rifampin,
erythromycin, gentamicin, chloramphenicol, and three different β-lactamases (one of
which is NDM-1). The novel NDM-1 gene product displays tight binding to the
penicillins and cephalosporins. It is also active against the carbapenems. The only
antibiotics active in vitro against K. pneumoniae clinical isolates were tigecycline and
colistin. Perhaps more disturbing was the finding of the NDM-1 gene on a 140-kb
plasmid from a fecal E. coli isolate. This indicates that this phenotype is highly
transmissible and can be mobilized to a broad host range.

Figure 5. 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 encoding the efflux pump and lactate dehydrogenase
(gray diagonal lines) are of 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).
Epidemiology of NDM-1

As of November 2010 reports of NDM-1 producers were identified in India,

Pakistan, the UK, the Netherlands, Japan, Australia, France, Spain, Sweden, Finland,
Denmark, Austria, Belgium, Norway, Italy, Oman, Kenya, and the Balkan region. The
majority of these patients had recently traveled to the Indian subcontinent.

In November 2010 seventy-seven patient cases were reviewed in European countries.

Among these cases, four patients developed a blood-stream infection. Seven died in the
hospital and one was directly related to NDM-1 positive E. coli which caused septic
shock in a leg wound. Most of these patients had recently (within the last year) traveled
to the Indian subcontinent or the Balkan region, but other areas included Dubai and
Spain. The majority of these patients had been admitted to the hospital because of illness
or an accident, and a small minority of hospitalizations were due to medical tourism (6).

Two patient cases in Italy had no travel history, but were in a hospital unit where a
patient returning from India had stayed. Another two patients in the UK had no travel
history and could not be linked to another travel-associated case. In some isolates in this
study patients tested positive for the NDM-1 enzyme in K. pneumoniae from urine and E.
coli from feces suggesting in vivo transfer. At this moment evidence of secondary
nosocomial transmission of the NDM-1 enzyme in Europe is limited.

The patients who are most susceptible to a severe illness due to bacteria expressing
NDM-1 are those with comorbidities and/or needed invasive care procedures (6). In the
Indian subcontinent a pertinent concern is the NDM-1 is commonly found in E. coli
which is the number one cause of diarrhea in children. Antibiotic resistance in this
population can have potentially devastating consequences (3). Indirect fecal-oral
transmission and exposure to contaminated people, food and water are the most likely
ways these pathogens are spread. Countries that lack an adequate sanitation
infrastructure are in the greatest danger of having NDM-1 community based infections.
India is identified as a reservoir for NDM-1 caused by an enormous population of people
living in these conditions (11). It is possible that other areas lacking this infrastructure
are at risk of becoming NDM-1 endemic. Patients hospitalized in the Balkan region were
positive for NDM-1 producers. Between 2007- 2009 NDM-1 positive K. pneumoniae
was found in patients in a tertiary care center in Nairobi, Kenya. These patients had no
travel history to the Indian subcontinent or the UK, but there is a large population of
Indian diaspora to Kenya (12, 13).
 Clinicians and scientists are advocating preventative measures of NDM-1 infections
since pharmaceutical options are severely limited. Overuse of the few effective
antibiotics poses a risk of antibiotic resistance to those medications.

Treatment of NDM-1

NDM-1, like other MBL’s (metallo-β-lactamases), hydrolyzes all β-lactam antibiotics

except for aztreonam but it is usually inactivated by co-produced extended-spectrum β-
lactamases. Most of the bacteria producing the NDM-1 enzyme belong to the
Enterobacteriacea family and are usually only susceptible to colistin and occasionally
tigecycline (14).

The CDC recommends using the guidance outlined for carbapenem-resistant

Enterobacteriaceae for treating possible cases of NDM-1. This guideline includes
identifying the pathogen as carbapenem-resistant and placing patient into contact
precautions. Some circumstances may call for point-prevalence surveys or surveillance
testing among other high-risk patients.

The CDC is asking that carbapenem-resistant isolates from patients who have
received medical care in India or Pakistan for the last six months be forwarded through
the state public health laboratories to the CDC to determine whether or not the bacteria is
NDM-1 producing. Infection control by preventing transmission is of the utmost
importance when managing an NDM-1 positive patient
(http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5924a5.htm).

References

1. Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, Walsh TR.
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, 2009;53:5046-
54. [PubMed]

2. Deshpande P, Rodrigues C, Shetty A, Kapadia F, Hedge A, Soman R. New Delhi

Metallo-beta lactamase (NDM-1) in Enterobacteriaceae: treatment options with
carbapenems compromised. J Assoc Physicians India, 2010;58:147-9. [PubMed]

3. Nordmann P, Poirel L, Carrër A, Toleman MA, Walsh TR. How to detect NDM-1
producers. J Clin Microbiol. 2011;49(2):718-21 [PubMed]

4. Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-
lactamases and its correlation with molecular structure. Antimicrob Agents Chemother,
1995;39(6):1211-33. [PubMed]

5. Ambler RP. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci,
1980;289:321-31. [PubMed]
6. Garau G, Di Guilmi AM, Hall BG. Structure-based phylogeny of the metallo-beta-
lactamases. Antimicrob Agents Chemother, 2005;49:2778-84. [PubMed]

7. Drawz SM, Bonomo RA. Three decades of beta-lactamase inhibitors. Clin Microbiol
Rev 2010;23:160-201. [PubMed]

8. Coque TM, Baquero F, Canton R. Increasing prevalence of ESBL-producing

Enterobacteriaceae in Europe. Euro Surveill 2008;3:9. [PubMed]

9. Kong KF, Aguila A, Schneper L, Mathee K. Pseudomonas aeruginosa beta-lactamase

induction requires two permeases, AmpG and AmpP. BMC Microbiol 2010;10: 328.
[PubMed]

10. Cambray G, Guerout AM, Mazel D. Integrons. Annu Rev Genet, 2010;44:141-66.
[PubMed]

11. Webster PC. Alarm grows over management of antibiotic resistance file. CMAJ
2010;182(2):E141-2. [PubMed]

12. Poirel L, Al Maskari Z, Al Rashdi F, Bernabeu S, Nordmann P. NDM-1-producing

Klebsiella pneumoniae isolated in the Sultanate of Oman. J Antimicrob Chemother
2011;66:304-6. [PubMed]

13. Poirel L, Revathi G, Bernabeu S, Nordmann P. Detection of NDM-1-Producing

Klebsiella pneumoniae in Kenya. Antimicrob Agents Chemother, 2011;55(2):934-6.
[PubMed]

14. Struelens MJ, Monnet DL, Magiorakos AP, Santos O'Connor F, Giesecke J;
European NDM-1 Survey Participants. New Delhi metallo-beta-lactamase 1-producing
Enterobacteriaceae: emergence and response in Europe. Euro Surveill, 2010;15(46),pii:
19716. [PubMed]