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(See editorial commentary by Bronomo, on pages 485–487.)
Carbapenem resistance in Klebsiella pneumoniae is most notably due to the K. pneumoniae carbapenemase (KPC) β-lactamase. In this report, we describe the occurrence of a newly described mechanism of carbapenem resistance, the NDM-1 β-lactamase, in a patient who received medical attention (but was not hospitalized) in India.
The emergence of carbapenem resistance in K. pneumoniae has become a substantial clinical problem, most typically attributed to production of KPC . KPC-producing organisms are most frequently found in the United States, but sizeable outbreaks have also occurred in Israel and Greece [2, 3]. Numerous other countries have now also been affected by KPC-producing organisms. KPC-producing K. pneumoniae cause considerable clinical problems because they are multidrug resistant, lacking susceptibility to β-lactam antibiotics, fluoroquinolones, and aminoglycosides . Thus, therapy for clinically significant isolates rests on the use of tigecycline or polymyxins, both of which have been associated with development of resistance during treatment . In addition, a dominant strain of KPC-producing K. pneumoniae (sequence type 258, as determined by multilocus sequence typing [MLST]) accounted for 70% of isolates in one study , suggesting some particular adaptiveness of this very resistant strain for the health care setting.
Carbapenem resistance in K. pneumoniae may be due to other causes; these include combinations of outer-membrane permeability loss and β-lactamase production  and the production of metallo-β-lactamases, such as those of the IMP or VIM groups . With the exception of Greece , most countries have been spared the widespread occurrence of IMP- or VIM-producing K. pneumoniae. In this Brief Report, we review the latest cause of carbapenem resistance in K. pneumoniae to be described—the New Delhi metallo-β-lactamase enzyme, NDM-1. There is emerging evidence that NDM-1–producing K. pneumoniae is destined to create clinical issues at least as substantial as those caused by KPC-producing strains. By virtue of its epicenter in the huge population of India, the number of individuals affected by NDM-1–producing K. pneumoniae may already exceed that of KPC-producing K. pneumoniae.
An 87–year-old woman was brought to a hospital in Australia directly from the airport, immediately after arriving from India. The patient was an Australian resident of Indian origin who had visited Khanna, in the state of Punjab, from November 2009 to January 2010. While in India, she developed a chronic draining foot ulcer. She was treated in India with an unknown intravenous antibiotic, which she administered at home. She was never hospitalized in India.
At the time of arrival, she developed fever (temperature, 38.9 degrees), dysuria, and suprapubic pain. Urine culture grew K. pneumoniae and Escherichia coli resistant to multiple antibiotics. The K. pneumoniae isolate was resistant to ertapenem, imipenem, meropenem, ceftazidime, cefotaxime, cefoxitin, piperacillin-tazobactam, ticarcillin-clavulanate, nalidixic acid, ciprofloxacin, amikacin, gentamicin, and trimethoprim-sulphamethoxazole, as determined on the basis of CLSI standards . The organism was susceptible to aztreonam, chloramphenicol, colistin (minimum inhibitory concentration [MIC], 0.25 μg/mL), and tigecycline (MIC, 1 μg/mL). The MICs of meropenem and doripenem were >32 μg/mL. Empirical treatment was given with intravenous ticarcillin-clavulanate. Despite the lack of susceptibility to this combination treatment, the patient's symptoms resolved, and she was discharged from hospital. Both the K. pneumoniae and E. coli isolates were also grown from a rectal swab specimen, which was plated on MacConkey agar that contained 8 μg/mL gentamicin.
Phenotypic detection of a metallo-β-lactamase was made in the K. pneumoniae isolate by using inhibition of the enzyme by EDTA . Polymerase chain reaction (PCR) and sequencing for antibiotic-resistance genes was positive for blaNDM-1, blaCMY-6, blaSHV, and blaDHA. The isolate was also positive for aac-6′-1b and rmtC [11–13]. PCR for the detection of blaNDM-1 was performed using forward primer (5′-GGGCCGTATGAGTGA-3′) and reverse primer (5′-GAAGCTGAGCACCGCATTAG-3′), which amplifiy a 758-bp fragment.
Transferability of blaNDM-carrying plasmids to laboratory strains of E. coli was conducted by transformation of extracted plasmids [12, 13] into Top10 E. coli (Invitrogen) and by conjugation with rifampin-resistant E. coli K - 12. Transformants and transconjugants were selected on Luria-Bertani agar supplemented with ceftazidime, 2 μg/mL. blaNDM-1 together with blaCMY-6, aac-6’-1b, and rmtC were successfully transferred by transformation and conjugation, which were confirmed by PCR and sequencing. To differentiate the successful transconjugants from the donor (NDM-1 producing K. pneumoniae), E. coli species-specific PCR was performed for the transconjugants . The size of the plasmid was ~70kb. The transformants and transconjugants were resistant to ertapenem, meropenem, imipenem, ceftazidime, cefotaxime, cefoxitin, amikacin, and gentamicin. The MICs in the transformants to meropenem and doripenem were 32 and 24 μg/mL, respectively.
MLST was performed as described on the MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). Allelic numbers were obtained on the basis of sequences of 7 housekeeping genes . According to this typing scheme, the K. pneumoniae isolate was ST147.
Antibiotic-resistant K. pneumoniae has been a notable hospital pathogen for ≥4 decades. Sequentially, aminoglycoside resistance in K. pneumoniae in the 1970s, third-generation cephalosporin resistance by way of extended-spectrum β-lactamases in the 1980s and 1990s, and then carbapenem resistance in this century have been major problems. KPC-producing K. pneumoniae has become a substantial international issue . The presence of KPC producers increases reliance on polymyxins or tigecycline as “workhorse” therapy. Increased use of any antibiotic hastens development of resistance to that class. Numerous reports of polymyxin- or tigecycline-resistant K. pneumoniae now exist [15–24]. Given the lack of new antibiotics active against multidrug-resistant gram-negative bacilli, a potential now exists for K. pneumoniae resistant to all commercially available antibiotics. The emergence of NDM-producing K. pneumoniae heightens this risk.
The first isolate from India known to produce NDM was retrospectively found in a survey of isolates from 2006 . A number of reports now document the widespread occurrence of NDM-producing K. pneumoniae in India and Pakistan. NDM producers have been detected in ≥10 major population centers in India, traversing both the north and south of the country [25, 26]. Of consecutive carbapenem-resistant Enterobacteriaceae collected in 3 months in late 2009 in a single hospital in Mumbai, 91.7% were NDM producers . Although surveillance studies are lacking, it is known that at least 10% of K. pneumoniae in some hospitals in India are carbapenem resistant (T.R.W.; unpublished data). Similar problems exist in Pakistan, where NDM producers have been documented in at least 8 cities . Given the populations of India (1184 million) and Pakistan (170 million), it can be appreciated that NDM producers may already be creating a massive problem in this region. Enterobacteriaceae other than K. pneumoniae, such as E. coli and Enterobacter cloacae, are also affected .
Since the first report of NDM-producing K. pneumoniae from Sweden in December 2009 (the patient had received medical care in New Delhi) , NDM producers have been detected in the United Kingdom [26, 28], the United States , Kenya , Japan , Canada , Belgium , the Netherlands , and Australia [33, 34]. In the United Kingdom, 25 different laboratories have reported NDM-producing organisms . In the United States, the Centers for Disease Control and Prevention have reported three NDM-producing isolates, from patients in 3 different states . The vast majority of the patients in these countries had received prior medical care in India [26–29, 33, 34].
Medical care in India and Pakistan is often sought by individuals visiting relatives in their country of origin. The Indian diaspora (a term used to describe people of Indian origin living permanently outside of India) are estimated to number > 24 million: 11 nations (including the United States, Saudi Arabia, United Kingdom, and Canada) have nonresident Indian populations exceeding 1 million (http://indiandiaspora.nic.in). The Pakistani diaspora is also considerable, estimated to number ≥7 million individuals (http://www.opf.org.pk). In addition, “medical tourism” to India is increasingly popular. A recent case of NDM-producing Providencia rettgeri in an Australian who received elective plastic surgery in India is illustrative of this phenomenon . Current data would suggest that recent hospitalization in India or Pakistan greatly increases the risk that an individual is colonized with an NDM-producing strain. Strong consideration should be given to screening patients who have undergone recent hospitalizations in India or Pakistan for carbapenem-resistant organisms and for preemptively using contact isolation precautions.
The isolate recovered from the patient who we describe was K. pneumoniae ST147. The only previous report using MLST showed an NDM-producer that was ST14 . The vast majority of KPC-producing K. pneumoniae isolates are ST258, although occasional KPC-producers are ST14 . In a recently published evaluation, 26 NDM-producers from a single institution in Haryana, India, belonged to a single pulsed-field gel electrophoresis profile implying clonal spread . However, isolates from another Indian institution showed no similarity with each other . British isolates have also been quite diverse . There is clear evidence from the Haryana molecular epidemiology that institutional outbreaks can occur. However, global spread appears to be due to a wide diversity of strains, indicating that a wide variety of NDM producers are circulating in India. Thus far, 2 NDM-producing E. coli isolates (1 from Canada and 1 from Australia) were ST101 [34, 35].
Although the isolate we characterized was susceptible to colistin, the full threat of NDM producers was illustrated by a recent report in which 1 NDM-producing K. pneumoniae isolate had a colistin MIC > 32 mg/L . Without doubt, NDM producers are destined to provide problems at least as great as KPC producers (Table 1). That the full extent of this impending catastrophe may be played out in developing nations should not reduce the need for urgent intervention.
Potential conflicts of interest.RL.N. has received grants from the National Institutes of Health and Australian National Health and Medical Research Council, and also received. Travel/ accommodations/meeting expense reimbursement from ICAAC. DL.P. is a consultant for Leo Pharmaceuticals, Novartis, Johnson&Johnson, Merck, and AstraZeneca. T.R.W. has received institutional grant support from Wyeth, and payment for lectures/speaker's bureau from Pfizer.