NDM-1: A Local Clone Emerges with Worldwide Aspirations

Andrea Marra

Abstract

Are bacteria always going to outsmart us? With the emergence of the metallo-β-lactamase blaNDM-1 gene, it certainly seems so. Whereas at one time bacterial clones resided in hospitals or long-term care facilities, it is now apparent that they have the capability of thriving in the community and quickly spreading across countries and continents with few impediments, thanks to accessible, rapid global travel. Thus, under conditions favoring the organism (promiscuous or inappropriate antibiotic use and poor infection control procedures), what was at one time a local problem can rapidly become a worldwide health crisis. Given that the discovery and development of a new antibiotic can take a decade or more, multiply resistant pathogens can have ample time to wreak havoc before a successful novel agent comes to market. At one time a single drug, penicillin, was enough to raise expectations that new antibiotics were unnecessary; we have since seen that bacteria can generate stable resistance to every antibiotic in rapid fashion, with no detrimental effects on their pathogenicity.

Introduction

The antibiotic resistance determinant bla NDM-1 is a prime example of the ability of bacteria to accumulate resistance determinants, maintain infectivity and fitness and spread rapidly around the world. NDM-1 (New Delhi Metallo-β-lactamase-1) is a novel plasmid-borne metallo-β-lactamase (MBL) that has so far been isolated only in Enterobacteriaceae.[1] The identification of this resistance determinant was first reported in 2008;[2] by 2010,[1] it had spread from its base in New Delhi, India across the Indian subcontinent to Pakistan and Bangladesh and also in Australia and throughout the US, the UK, France and Canada.[1–7]
The bla NDM-1 gene was isolated from Klebsiella pneumoniae and Escherichia coli cultures from the same patient suffering from a urinary tract infection (UTI); the organisms were found to be resistant to all antibiotic classes with the exception of colistin.[2] Whereas NDM-1 shares the ability to hydrolyze carbapenems with other MBLs, several features make it especially worrisome. First, that this gene was found in K. pneumoniae and E. coli isolated from the same patient raises the possibility of facile horizontal resistance gene transfer in vivo; second, that it was found in Enterobacteriaceae suggests the opportunity for broad community dissemination; third, elevated MICs to many antibiotic classes indicates potential for widespread treatment failure as these organisms spread; and finally, the gene's location on mobile genetic elements allows rapid spread between pathogens with no effective antibiotics to counteract it.[1–3,8]
The generations of β-lactam antibiotics came about due to the steady evolution of bacterial resistance mechanisms that disable them. β-lactam agents (cephalosporins, carbapenems, clavulanate-type β-lactamase inhibitors, monobactams and penems) bind penicillin-binding proteins, which are involved in bacterial cell wall peptidoglycan biosynthesis; resistance to them is achieved by their efflux out of the cell, alteration of the penicillin-binding protein target, decreased porin production to limit cell entry or hydrolysis of the β-lactam itself.[9–14] One count estimates the number of different β-lactamases produced by disparate organisms to be over 950,[11] with four major classes: penicillinases, cephalosporinases (known as AmpC-type), extended-spectrum β-lactamases (ESBLs) and carbapenemases, with the latter two moving to the forefront in recent years (Table 1).[10,11,15–17] Until approximately a decade ago, ESBL-producing isolates were confined to hospitals, with K. pneumoniae being the main culprit. At present, however, community-acquired infections are of major concern, particularly UTIs, caused by ESBL-positive K. pneumoniae as well as E. coli, with different so-called CTX-M ESBLs predominating in different countries.[17,18] The number, diversity and rate of occurrence of ESBLs targeting penicillins and cephalosporins has led to more frequent use of carbapenems, which in turn has resulted in increased carbapenem resistance due to both serine carbapenemases and MBLs. For example, in India, 70–90% of Enterobacteriaceae are ESBL-producers; as a result, carbapenem use to treat these infections has increased, and so has carbapenem resistance.[1,18] In addition, in the UK at least, greater than 80% of ESBL-producing E. coli from bloodstream infections are fluoroquinolone-resistant, and greater than 40% are gentamicin-resistant, thus presenting more challenges to clinicians.[8] The serine carbapenemases found in Enterobacteriaceae, particularly in K. pneumoniae, are now endemic worldwide, with the plasmid-borne K. pneumoniae carbapenemases being the most prevalent.[11] The rapidly emerging MBLs are of concern because of their broad β-lactam resistance coverage and their transmissibility: most of the MBLs within clinical Enterobacteriaceae isolates are contained within gene cassettes on integrons.[11,15] The integron itself encodes an integrase gene, a recombination site and a promoter for expression of the gene contained therein, and often resistance elements for antiseptics (qac) and sulfonamide (sul).[15] Structures such as integrons allow facile transmission within the same cell, either into the chromosome or onto plasmids – once these elements are located on plasmids, transfer between cells becomes a matter of opportunity and pressure as antibiotic use encourages selection of resistance.
The bla NDM-1 gene is contained within such structures and perhaps not surprisingly has spread around the world rapidly from its likely origins on the Indian subcontinent. Although Yong et al. [2] have demonstrated that NDM-1 is not as robust as other MBLs (such as VIM-2 and IMP-1) in terms of binding to and hydrolysis of cephalosporins and penicillins, its real threat is its plasmid-borne location and the ease with which it is able to spread.[1–3,7] The K. pneumoniae isolate bearing bla NDM-1 carries three antibiotic resistance regions:[2] one region encodes NDM-1, plus an efflux pump and the bla DHA AmpC gene; the second encodes a rifampicin resistance gene (arr2), a novel erythromycin esterase gene (ereC), the gene CM1A7 (encoding chloramphenicol resistance), and qac/sul; and the third region contains yet another AmpC gene, bla CMY4.[2]
Sequencing of the original patient isolate revealed that the bla NDM-1 gene encoded a 27.5 kDa protein and had a lower G+C content than surrounding DNA, suggesting a non-Klebsiella origin. Kumarasamy et al. [1] molecularly characterized and studied the epidemiology of the NDM-1 isolates. For their analysis, the authors examined carbapenem-resistant Enterobacteriaceae from different sites on the Indian subcontinent and in the UK, analyzing the isolates by MIC, pulsed-field gel electrophoresis and PCR. The clinical isolates from India were primarily from two cities, Chennai and Haryana, in addition to isolates collected from three other cities in India and eight cities in Pakistan; the movement of this gene via patient travel was also investigated as isolates in the UK were screened for its presence. The majority of infections from which the NDM-1 Enterobacteriaceae were isolated were community-acquired UTIs, pneumonia and bloodstream infections.
The antibiograms of the NDM-1-producing isolates from the disparate sites told a disturbing story. In addition to being carbapenem-resistant, all the isolates were found to be nonsusceptible to several antibiotic classes. Since only a small number of isolates (all from the UK) remained susceptible to aztreonam (which is not hydrolyzed by MBLs including NDM-1), it became clear that the rest contained genes encoding ESBLs and/or the β-lactamase AmpC; indeed, sequence analysis revealed that the majority of the isolates carried bla CTX-M-15 and bla CMY-4. The UK isolates were also resistant to tobramycin, minocycline and amikacin, and most were resistant to ciprofloxacin and gentamicin as well. A total of 89% of the UK isolates were susceptible to colistin, and 64% were susceptible to tigecycline.[1]
The isolates from Chennai and Haryana followed a similar pattern: nearly all were resistant to all β-lactams (including aztreonam), fluoroquinolones, aminoglycosides and minocycline, with greater than 50% susceptible to tigecycline and all susceptible to colistin. Interestingly, all of the Haryana isolates were clonal, perhaps suggesting an increased fitness of this particular isolate. Most worrisome is the single K. pneumoniae isolate from Chennai that manifested resistance to all of the antibiotic classes mentioned above, with a MIC of colistin of 32 µg/ml and of tigecycline of 8 µg/ml. Though panresistant isolates such as this are rare, they have been reported in Greece[19,20] and significant spread would mean a return to the preantibiotic era.
As noted above, the presence of bla NDM-1 does not in itself result in the broad β-lactam resistance that is observed in all of these isolates; the major concern with this resistance determinant is the piggy-backing of this gene onto plasmids or transposons or integrons, mobile genetic elements that allow easy transfer between organisms (in the case of plasmids) or between plasmids and chromosomes (transposons and integrons). Indeed, when all of the Chennai, Haryana and UK isolates were analyzed for the presence of plasmids and the location of bla NDM-1, they were all found to harbor multiple plasmids (50–500 kb in size, in some cases as many as eight) and the gene was plasmid-borne in all of them. In addition, the bla NDM-1 gene was also in the chromosomes of three of the UK isolates and on more than one plasmid in others, reinforcing the idea that this gene is capable of movement within the same cell.[1]
The analysis by Kumarasamy et al. included transfer of plasmids from isolates to an E. coli laboratory strain.[1] The results suggest high transmissibility of bla NDM-1-carrying plasmids between Enterobacteriaceae; surely selective pressure through inappropriate or nonprescription use of antibiotics (and especially carbapenems) in India[1,21] as well as Greece[22] could select for this resistance element and significantly increase the numbers of bla NDM-1 isolates from patients. As this antibiotic resistance element is not confined to hospitals or long-term care facilities, but was for the most part isolated from community-acquired infections, infection control measures would be extremely difficult to implement.

Conclusion & Future Perspective

The carbapenems were developed to combat the increasing prevalence of β-lactamase enzymes in Gram-negative organisms and have become first-line therapy against serious infections. The wide-ranging and rapid emergence of a resistance element such as bla NDM-1 is cause for concern and has justifiably elicited coverage in the scientific and medical communities. The MBLs present many challenges to clinicians as well as drug developers: they have an incredibly broad range of activity against β-lactams, they are resistant to β-lactamase inhibitors, and they are commonly linked to aminoglycoside resistance genes. The isolates studied so far appear to remain susceptible to tigecycline and colistin, but both drugs have clinical limitations – tigecycline achieves low serum and urine levels and thus is not routinely prescribed for bacteremia or UTI; colistin has good efficacy in general but is weak against respiratory tract infections.[8,9,15,23] Ironically, the NDM-1 isolates were predominantly from patients suffering from UTIs, respiratory tract infections and bloodstream infections. Since its emergence in the UK in 2008, carbapenem-resistant Enterobacteriaceae carrying the bla NDM-1 gene have become the predominant Enterobacteriaceae carbapenem resistance determinant – from approximately 5% in 2008 to 44% in 2009.[1] In that time period, 37 bla NDM-1- producing Enterobacteriaceae were isolated from 25 sites across England, Scotland and Northern Ireland. More than half of the 29 patients involved had traveled to India or Pakistan within the previous year, and nearly half had been admitted to a hospital while there. The isolates were determined to be K. pneumoniae, E. coli, Enterobacter spp., Citrobacter freundii, Morganella morganii, and Providencia spp..[23] The isolation of this gene in Enterobacteriaceae – common flora – on mobile genetic elements, from patients in varied clinical settings around the world should herald a worldwide call for the development of global guidelines involving testing, treatment and infection control measures to contain what could become a serious global health issue.

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Andrea Marra

Rib-X Pharmaceuticals, Inc., 300 George Street, Suite 301, New Haven, CT 06511, USA. Tel.: +1 203 848 3349 Fax: +1 203 624 5627 amarra@rib-x.com

Acknowledgements
The author would like to acknowledge the expert technical advice and guidance of Tom Gootz in the preparation of this manuscript.

Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.
Future Microbiology. 2011;6(2):137-141. © 2011 Future Medicine Ltd