From Chennai, 75 E coli, 60 Klebsiella spp, and six other Enterobacteriaceae resistant to carbapenems were isolated from 3521 (4%) Enterobacteriaceae analysed throughout 2009. Of these 141 carbapenem-resistant Enterobacteriaceae, 44 (19 E coli, 14 K pneumoniae, seven Enterobacter cloacae, two Proteus spp, one Citrobacter freundii, and one Klebsiella oxytoca) were NDM-1 positive (about 1% of all resistant isolates). During that same period, 47 carbapenem-resistant isolates (24%) of 198 from Haryana were identified; from these, 26 (13%) were positive for NDM-1, and all were K pneumoniae. The Indian isolates from Chennai and Haryana were primarily from community acquired urinary tract infections, pneumonia, and blood-stream infections. The age range was 4–66 years with a mean of 36 years (SD 20) and a female to male ratio of about two to one.
In the UK resistant isolates increased in both 2008 and 2009 (). Isolates with the NDM-1 enzyme, which was first detected in the UK in 2008, became the predominant carbapenemase-producing Enterobacteriaceae in 2009, accounting for 32 (44%) of 73 carbapenemase producers. During 2008–09, 37 Enterobacteriaceae isolates with the NDM-1 enzyme were referred from 25 laboratories across England with single representatives also from Scotland and Northern Ireland. These were identified as K pneumoniae (21 isolates), E coli (seven), Enterobacter spp (five), Citrobacter freundii (two), Morganella morganii (one), and Providencia spp (one). They were from 29 patients and had been isolated from urine (15 patients), blood (three), burn or wound swab (four), sputum (two), central line tip (one), throat swab (one), or unknown specimens (three). The mean age of the patients was 60 years (SD 24; range 1–87), with 17 male patients and 12 female patients. At least 17 patients had a history of travelling to India or Pakistan within 1 year, and 14 of them had been admitted to a hospital in these countries. Reasons for these admissions included renal or bone marrow transplantation, dialysis, cerebral infarction, chronic obstructive pulmonary disease, pregnancy, burns, road traffic accidents, and cosmetic surgery.
Numbers of carbapenemase-producing Enterobacteriaceae referred from UK laboratories to the UK Health Protection Agency's national reference laboratory from 2003 to 2009
Isolates, NDM-1-positive bacteria from Mumbai (32 isolates), Varanasi (13), and Guwahati (three) in India, and 25 isolates from eight cities in Pakistan (Charsadda, Faisalabad, Gujrat, Hafizabad, Karachi, Lahore, Rahim Yar Khan, and Sheikhupura) were also analysed in exactly the same manner but in laboratories in India and Pakistan. These isolates were from a range of infections including bacteraemia, ventilator-associated pneumonia, and community-acquired urinary tract infections.
All the isolates producing the NDM-1 enzyme were resistant to several antibiotic classes (). The 37 UK isolates were all resistant to imipenem and ertapenem, although a single M morganii isolate remained susceptible, at least in vitro, to meropenem (MIC 2 mg/L). Only four UK isolates remained susceptible to the monobactam aztreonam (MICs ≤1 mg/L), which is unaffected by metallo-carbapenemases including NDM-1; the other UK isolates were all resistant to all β-lactams, including aztreonam, suggesting the concurrent presence of additional β-lactamases including ESBLs and AmpC enzymes—identified by sequencing as mainly blaCTX-M-15 and blaCMY-4. All 37 isolates were resistant to amikacin and tobramycin, although one isolate was susceptible to gentamicin and three to ciprofloxacin. MICs of minocycline were consistently 2 mg/L or greater, interpreted as non-susceptible with the BSAC and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for doxycycline, but most (33 of 37) were susceptible to colistin (MICs ≤4 mg/L) and 26 were suseptible to tigecycline (MICs ≤1 mg/L; ).
Antibiotic susceptibilities for NDM-1-positive Enterobacteriaceae isolated in the UK and north (Chennai) and south India (Haryana)
90% minimum inhibitory concentration (MIC90) for Enterobacteriaceae from Chennai and Haryana, India, and the UK
The 44 isolates from Chennai were similarly resistant to all β-lactam antibiotics, fluoroquinolones, and aminoglycosides, apart from two that were sensitive to gentamicin. 39 were resistant to minocycline with MICs >2 mg/L, 19 to tigecycline, and three to colistin ( and ). Two of the three isolates resistant to colistin were Proteus
spp, which are intrinsically resistant, and the third was a K pneumoniae
strain (colistin MIC >32 mg/L; tigecycline MIC 8 mg/L). Although several reports from Greece have noted K pneumoniae
isolates as colistin resistant, we believe our isolate is truly pan-resistant.24,25
Most of the 26 Haryana isolates were resistant to all β-lactam and non-β-lactam antibiotics, although three were susceptible to aztreonam and one to ciprofloxacin and amikacin. Minocycline MICs for the Haryana isolates were 8–16 mg/L and ten isolates had intermediate resistance (2 mg/L) to tigecycline by EUCAST criteria. None were resistant to colistin ( and ).
The 21 Klebsiella isolates from the UK had different PFGE profiles and were typed to 19 distinct groups with only two related pairs, both of which included isolates from epidemiologically linked patients, probably representing cases of cross-infection. All the UK E coli isolates were different. The Chennai isolates were also very different, with none similar to each other. By contrast, the 26 NDM-positive K pneumoniae isolates from Haryana belonged to a single PFGE profile suggesting clonal spread. We could not prove statistically significant strain relatedness between the Indian and UK isolates.
Isolates from Chennai, Haryana, and the UK's Antibiotic Resistance Monitoring and Reference Laboratory were analysed for the location of the blaNDM-1 gene by S1 digestion of DNA, and then PFGE and direct probing of the gels with a radiolabelled blaNDM-1 gene. Each of the three groups of isolates typically carried several plasmids, with some isolates having up to eight plasmids ().
The difference in plasmid numbers from a selection of Indian isolates
Indian isolates had blaNDM-1 exclusively on plasmids. Plasmids from the non-clonal Chennai isolates ranged from 50 kb to 350 kb, whereas those from the clonal K pneumoniae from Haryana were predominately either 118 kb (54%) or 50 kb (36%). The UK isolates had a more diverse range of plasmid sizes, 80 kb to greater than 500 kb. Three UK isolates also carried blaNDM-1 on their chromosome, suggesting in-situ movement of blaNDM-1. There were many plasmids of identical size in isolates collected from India and the UK (data not shown), suggesting plasmid movement between bacterial isolates. In some isolates, blaNDM-1 was carried on more than one plasmid ().
Hybridisation results of UK isolates with blaNDM-1
47 isolates from Chennai (33) and Haryana (14) were randomly chosen for further investigation with PCR and DNA probing to verify the origin of replication (incompatibility type) for plasmids carrying blaNDM-1
Plasmids carrying blaNDM-1
from the 14 isolates from Haryana could not be typed. 13 of the 33 isolates from Chennai carried blaNDM-1
on A/C-type plasmids and one blaNDM-1
positive plasmid was incompatibility type FI/FII. Similarly, when the 32 randomly selected UK isolates were assessed with the same methods, 22 carried A/C type plasmids. The other blaNDM-1
positive plasmids from India and the UK that were A/C and FI/FII negative could not be typed.
Transconjugants were created in E coli
J53 from the 33 Chennai and 32 UK isolates; however, the isolates from Haryana did not produce transconjugates.22
All transconjugants were shown by PCR to contain blaNDM-1
. We compared the sizes of the plasmids in the clinical strains with those of the transconjugants and, in about 10% of cases, the plasmid had altered in size during transfer. In most cases the plasmid had lost DNA but two of 102 had gained DNA during transfer.
In addition to the collections of isolates from Chennai and Haryana detailed above, we have confirmed by PCR alone the presence of genes encoding NDM-1 in carbapenem-resistant Enterobacteriaceae isolated from Guwahati, Mumbai, Varanasi, Bangalore, Pune, Kolkata, Hyderabad, Port Blair, and Delhi in India, eight cities (Charsadda, Faisalabad, Gujrat, Hafizabad, Karachi, Lahore, Rahim Yar Khan, and Sheikhupura) in Pakistan, and Dhaka in Bangladesh () suggesting widespread dissemination.
Distribution of NDM-1-producing Enterobacteriaceae strains in Bangladesh, Indian, Pakistan, and the UK