Abstract
Among 39 carbapenem-resistant Enterobacteriaceae (2.7% overall; Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae strains) isolated in 2006 and 2007 in India, 15 strains carried blaNDM-1 and 10 harbored a gene encoding a variant of the carbapenemase OXA-48, named blaOXA-181. One E. cloacae strain harbored blaVIM-6, and one K. pneumoniae strain carrying blaOXA-181 also possessed blaVIM-5. Multiple pulsed-field gel electrophoresis patterns and clonal dissemination within and among sites were observed. Isolates producing NDM-1 were disseminated in Indian health care facilities as early as 2006.
NDM-1 (New Delhi metallo-β-lactamase 1), which was initially reported for Klebsiella pneumoniae and Escherichia coli strains from India, now has been detected in several enterobacterial strains and in different countries (3, 6, 8, 10-13, 15). In this study, we report occurrences and characterizations of carbapenemase-producing Enterobacteriaceae collected from Indian hospitals during 2006 and 2007, including isolates producing NDM-1, VIM enzymes, and a variant of the OXA-48 carbapenemase OXA-181.
A total of 1,443 isolates of Enterobacteriaceae were collected from 14 hospitals in India as part of the SENTRY Antimicrobial Surveillance Program, 2006-2007. Isolates were susceptibility tested using the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method (4). Extended MIC ranges were determined using Etest (AB bioMérieux, Marcy l'Étoile, France). Categorical interpretations for all antimicrobials were those found in CLSI document M100-S20-U (5), and quality control results for E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were within specified ranges as published by CLSI (5).
All enterobacterial isolates with reduced susceptibility to imipenem, meropenem, or ertapenem (MIC, ≥2 μg/ml) were screened for production of carbapenemases. The modified Hodge test (MHT) was performed using imipenem and meropenem as substrates. Carbapenem-nonsusceptible isolates were PCR screened using custom primers targeting blaNDM-1, blaIMP, blaVIM, blaGES, blaKPC, and blaOXA-48. Isolates were also screened for genes encoding plasmid-mediated AmpC enzymes (9) and extended-spectrum β-lactamases (ESBLs) by using custom primers targeting blaCTX-M, blaOXA-2, blaOXA-10, blaOXA-1/-30, blaPER, blaPSE, blaBEL, and blaVEB. PCR amplicons were sequenced for both strands, and sequences were analyzed.
Twenty-six carbapenemase-producing strains (MHT and/or PCR positive) were identified among 39 enterobacterial isolates (2.7%; 1,443 overall) resistant to at least one of the carbapenems tested (imipenem, meropenem, or ertapenem) (3). These 26 isolates were recovered from patients hospitalized in New Delhi (two medical centers), Mumbai, and Pune in 2006 or 2007. Carbapenemase producers were highly resistant to β-lactams, including cefepime (MIC range, 4 to >256 μg/ml) (Tables 1 and 2), ceftazidime (64 to >256 μg/ml), aztreonam (4 to >256 μg/ml), and piperacillin-tazobactam (>64 μg/ml) and were generally resistant to amikacin (MICs, 8 to >32 μg/ml). Only one strain was susceptible to ciprofloxacin (MIC, ≤0.03 μg/ml) (Table 1), and all but two isolates were susceptible to polymyxin B (Tables 1 and 2). Tigecycline was the only antimicrobial agent active against all isolates (MICs, 0.12 to 2 μg/ml).
TABLE 1.
Demographic information, antimicrobial profile, and molecular results from 15 clinical Enterobacteriaceae isolates producing NDM-1 collected in India from 2006 to 2007
| Parameter | Characteristic of or result for indicated isolateg |
||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 246-49D | 246-34D | 246-17D | 252-26D | 257-25A | 258-03D | 246-14A | 246-05A | 258-18A | 257-36D | 257-42D | 246-61A | 252-38C | 252-41C | 258-14C | |
| Speciesa | EC | EC | EC | EC | EC | EC | ECL | ECL | ECL | KPN | KPN | KPN | KPN | KPN | KPN |
| City | New Delhi | New Delhi | New Delhi | Mumbai | New Delhi | Pune | New Delhi | New Delhi | Pune | New Delhi | New Delhi | New Delhi | Mumbai | Mumbai | Pune |
| Collection year | 2006 | 2006 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 |
| Sourceb | SSSI | SSSI | SSSI | SSSI | BSI | SSSI | BSI | BSI | BSI | SSSI | SSSI | BSI | RTI | RTI | RTI |
| MIC (μg/ml) | |||||||||||||||
| Imipenem | 4 | 4 | 8 | 8 | 16 | 64 | 2 | 2 | 8 | 1* | 2 | 16 | 8 | 8 | 8 |
| Meropenem | 8 | 8 | 32 | 32 | 64 | 128 | 2 | 2 | 32 | 2 | 8 | 64 | 16 | 64 | 32 |
| Ertapenem | 32 | 32 | >32 | >32 | 32 | >32 | 4 | 4 | 32 | 4 | 32 | 32 | 8 | 32 | 16 |
| Doripenem | 4 | 2 | 16 | 8 | >32 | >32 | 1* | 2 | 4 | 2 | 4 | 16 | 8 | 16 | >32 |
| AMX/CLAc | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 |
| PIP/TAZd | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 |
| Ceftriaxone | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 |
| Ceftazidime | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 |
| Cefepime | >256 | 128 | >256 | >256 | >256 | >256 | 16 | 32 | >256 | 64 | 64 | >256 | 128 | >256 | 128 |
| Aztreonam | 128 | 64 | >256 | >256 | >256 | >256 | 4 | 32 | >256 | >256 | >256 | >256 | 64 | >256 | >256 |
| Amikacin | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 | >32 |
| Gentamicin | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 | >8 |
| Tobramycin | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 |
| Ciprofloxacin | >4 | >4 | >4 | >4 | >4 | >4 | ≤0.03 | >4 | >4 | >4 | >4 | >4 | >4 | >4 | >4 |
| Tetracycline | >8 | >8 | >8 | >8 | >8 | >8 | ≤2 | ≤2 | >8 | >8 | 8 | >8 | >8 | 4 | 4 |
| Tigecycline | 0.12 | 0.25 | 0.25 | 0.25 | 0.25 | 0.5 | 0.5 | 0.5 | 2 | 1 | 1 | 0.5 | 1 | 0.5 | 0.5 |
| Polymyxin B | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | 4 | 4 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 |
| MHT result | Weak pos | Weak pos | Pos | Weak pos | Weak pos | Weak pos | Pos | Pos | Pos | Weak pos | Weak pos | Weak pos | Weak pos | Weak pos | Weak pos |
| PFGE group | EC-A | EC-A | EC-B | EC-B1 | EC-B2 | EC-B3 | ECL-A | ECL-A | ECL-B | KPN-A | KPN-B | KPN-C | KPN-D | KPN-E | KPN-E |
| Gene locatione | P | P | P | C | P | C | P | P | P | P | P | P | P | P | Pf |
| Gene | |||||||||||||||
| blaCTX-M-15 | Neg | Pos | Pos | Pos | Neg | Pos | Neg | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos |
| blaOXA-1/-30 | Pos | Pos | Neg | Pos | Neg | Pos | Neg | Pos | Pos | Pos | Pos | Neg | Pos | Pos | Pos |
| blaOXA-2 | Neg | Neg | Neg | Pos | Pos | Pos | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg |
| blaCMY | blaCMY-4 | blaCMY-4 | Neg | Neg | Neg | Neg | blaCMY-6 | blaCMY-6 | Neg | blaCMY-6 | blaCMY-4 | Neg | Neg | blaCMY-4 | Neg |
| blaDHA-like | Pos | Pos | Pos | Neg | Neg | Neg | Pos | Pos | Neg | Pos | Pos | Neg | Neg | Neg | Neg |
EC, Escherichia coli; ECL, Enterobacter cloacae; KPN, Klebsiella pneumoniae.
SSSI, skin and skin structure infection; BSI, bloodstream infection; RTI, respiratory tract infection.
AMX/CLA, amoxicillin-clavulanate.
PIP/TAZ, piperacillin-tazobactam.
P, plasmidic; C, chromosomal.
Hybridization signals were detected in two plasmid bands.
*, susceptible by CLSI M100-S20-U criteria; pos, positive; neg, negative.
TABLE 2.
Demographic information, antimicrobial profile, and molecular results from clinical Enterobacteriaceae isolates producing OXA-181 and/or VIM variants collected in India from 2006 to 2007
| Parameter | Characteristic of or result for indicated isolateg |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 243-08A | 243-09A | 243-16C | 243-18C | 243-21C | 257-35A | 257-41D | 252-36A | 252-39A | 243-50C | 258-04A | |
| Carbapenemase | OXA-181 | OXA-181 | OXA-181, VIM-5 | OXA-181 | OXA-181 | OXA-181 | OXA-181 | OXA-181 | OXA-181 | OXA-181 | VIM-6 |
| Speciesa | KPN | KPN | KPN | KPN | KPN | KPN | KPN | KPN | KPN | ECL | ECL |
| City | Kolkata | Kolkata | Kolkata | Kolkata | Kolkata | New Delhi | New Delhi | Mumbai | Mumbai | Kolkata | Pune |
| Collection year | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2007 | 2006 | 2007 |
| Sourceb | BSI | BSI | RTI | RTI | RTI | BSI | SSSI | BSI | BSI | RTI | BSI |
| MIC (μg/ml) | |||||||||||
| Imipenem | 64 | 64 | 4 | 2 | 2 | 4 | 64 | 2 | 1* | 32 | 2 |
| Meropenem | 64 | 64 | 4 | 2 | 4 | 16 | 64 | 2 | 2 | 64 | 2 |
| Ertapenem | >32 | >32 | 8 | 32 | 16 | >32 | >32 | 4 | 8 | >32 | 4 |
| Doripenem | >32 | >32 | 1* | 2 | 2 | 32 | >32 | 2 | 1* | >32 | 2 |
| AMX/CLAc | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 | >16 |
| PIP/TAZd | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 | >64 |
| Ceftriaxone | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 |
| Ceftazidime | 128 | 64 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 |
| Cefepime | >256 | >256 | 64 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | 128 |
| Aztreonam | >256 | >256 | 128 | >256 | >256 | >256 | >256 | >256 | >256 | >256 | >256 |
| Amikacin | 8 | 8 | >32 | 16 | >32 | >256 | >32 | >32 | >32 | 8 | >32 |
| Gentamicin | ≤2 | >8 | >8 | ≤2 | >8 | ≤2 | >8 | >8 | >8 | >8 | >8 |
| Tobramycin | 16 | >16 | >16 | 16 | >16 | 16 | >16 | >16 | >16 | >16 | >16 |
| Ciprofloxacin | >4 | >4 | >4 | >4 | >4 | >4 | >4 | >4 | >4 | >4 | >4 |
| Tetracycline | >8 | >8 | 8 | 8 | >8 | 8 | >8 | ≤2 | ≤2 | >8 | >8 |
| Tigecycline | 1 | 1 | 1 | 0.5 | 1 | 0.5 | 1 | 0.25 | 0.5 | 2 | 1 |
| Polymyxin B | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 | ≤0.5 |
| MHT result | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos |
| PFGE group | KPN-F | KPN-F | KPN-G | KPN-H | KPN-H | KPN-E1 | KPN-I | KPN-I | KPN-I | ECL-C | ECL-B1 |
| Gene locatione | P | P | P | P | P | P | P | P | P | P | P |
| Gene | |||||||||||
| blaCTX-M-15 | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos | Pos |
| blaOXA-1/-30 | Pos | Pos | Pos | Neg | Neg | Pos | Pos | Pos | Pos | Pos | Pos |
| blaOXA-10 | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Posf |
| Plasmidic AmpC | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg | Neg |
KPN, Klebsiella pneumoniae; ECL, Enterobacter cloacae.
SSSI, skin and skin structure infection; BSI, bloodstream infection; RTI, respiratory tract infection.
AMX/CLA, amoxicillin-clavulanate.
PIP/TAZ, piperacillin-tazobactam.
P, plasmidic.
blaOXA-10 was located in the class 1 integron carrying blaVIM-6.
*, susceptible by CLSI M100-S20-U criteria; pos, positive; neg, negative.
Fifteen putative carbapenemase-producing strains carried blaNDM-1 (Table 1), and 10 strains harbored a novel blaOXA-48-like gene (Table 2). This new gene displayed 45 nucleotide substitutions leading to four amino acid differences compared to OXA-48 (T104A, N110D, E168Q and S171A) and was named the OXA-181 gene. Additionally, blaVIM gene variants (blaVIM-6 and blaVIM-5) were detected for two strains, one also carrying blaOXA-181 (Table 2). The gene encoding NDM-1 was detected in E. coli (six strains) (Table 1), K. pneumoniae (six strains), and Enterobacter cloacae (three strains), whereas blaOXA-181 was found in K. pneumoniae (10 strains, one also carrying the blaVIM-5 gene) (Table 2) and E. cloacae (1 strain). Another E. cloacae strain carried blaVIM-6 (Table 2).
Pulsed-field gel electrophoresis (PFGE) was used to evaluate clonality among carbapenemase-producing Enterobacteriaceae (14). NDM-1 producing E. coli isolates were additionally evaluated by multilocus sequence typing (MLST; http://mlst.ucc.ie/mlst/dbs/Ecoli). All six carbapenemase-producing E. coli strains carried blaNDM-1 and clustered within two PFGE groups (EC-A and EC-B) (Table 1). The two EC-A strains were recovered from New Delhi, while the four strains belonging to EC-B were from four sites, two in New Delhi and one each in Mumbai and Pune. MLST analyses showed that EC-A strains belong to ST-167 and that all EC-B subtypes were ST-101. Interestingly, a ST-101 E. coli strain carrying blaNDM-1 was recently described in Australia (11). In the present study, two E. coli isolates were collected in 2006, while all remaining NDM-1 strains were recovered in 2007. These are the earliest NDM-1-producing isolates reported to date, indicating that isolates producing this carbapenemase have been present in India earlier than previously appreciated.
Five pulsotypes were observed among the six K. pneumoniae strains carrying blaNDM-1. Two strains showing identical PFGE profiles were detected in Pune and Mumbai (KPN-E) (Table 1). The remaining nine K. pneumoniae strains harbored blaOXA-181, were from Kolkata (55.6%), and belonged to five clones. Clonality within medical sites was documented (Table 2). Furthermore, one OXA-181-producing K. pneumoniae strain from New Delhi was genetically related to an NDM-1-carrying K. pneumoniae type detected in Pune and Mumbai (KPN-E) (Tables 1 and 2). The strain carrying blaOXA-181 and blaVIM-5 had a unique genetic background.
A great variety of enzymes was detected for E. cloacae strains, and organisms of this bacterial species harbored NDM-1, OXA-181, or VIM-6. Two NDM-1 producers from New Delhi were identical, and isolates from Pune harboring blaNDM-1 or blaVIM-6 were also genetically related (Table 2).
A large proportion of carbapenemase-producing isolates also harbored blaCTX-M-15 (23 of 26 isolates; 88.5%) and/or blaOXA-30 (20 of 26; 76.9%), and these β-lactamases were detected in strains producing the NDM-1, OXA-181, or VIM enzyme. Three genetically related NDM-1-producing E. coli strains from the same medical center were also positive for the blaOXA-2 gene (Table 1). Furthermore, plasmid-mediated AmpC genes (blaCMY-4, blaCMY-6, and/or blaDHA-like genes) were detected among eight NDM-1-producing isolates (Table 1). None of the OXA-181- or VIM-producing isolates carried plasmid-mediated AmpC genes (Table 2).
Carbapenemase genes were predominantly plasmid located as demonstrated by S1 nuclease and ICeuI experiments followed by hybridizations with specific probes. blaNDM-1 was detected in plasmids ranging in size from 50 to 450 kb in 13 strains, and this metallo-β-lactamase (MβL) gene was embedded in the chromosome of two E. coli strains (from Mumbai and Pune). Hybridization signals were noted for two plasmid bands in one K. pneumoniae isolate (257-25A) (Table 1). The gene encoding OXA-181 was carried in plasmids of similar molecular sizes (200 to 250 kb) in all strains carrying this gene; however, a few organisms displayed hybridization signals in multiple extrachromosomal bands.
Oligonucleotides targeting blaNDM-1 and blaOXA-48 genetic surroundings reported earlier (11, 16) were designed and used against isolates producing these enzymes yielding negative results. Class 1 integron mapping was performed as described previously (1, 2). The gene encoding VIM-6 detected in E. cloacae was located in the first position of a 3.9-kb class 1 integron carrying also the aacA4 and blaOXA-10 genes. This genetic arrangement was previously described for P. aeruginosa isolates from hospitals located in several Asian nations (2), including the medical institution in Pune where this VIM-6-producing E. cloacae strain was recovered. The blaVIM-5 gene was also embedded in a class 1 integron lacking the common structures observed in the 3′ conserved sequence (qacEΔ1/sul1 or tniC gene) (7); however, genes encoding aminoglycoside resistance (aacA4 and aadA1) were detected downstream of the MβL gene.
Negative or weakly positive MHT results were observed for 11 of 15 NDM-1-producing strains (Table 1 and Fig. 1). All remaining carbapenemase-producing isolates yielded positive MHT results. It is noteworthy that all NDM-1-producing E. cloacae strains displayed the lowest carbapenem MIC values (1 to 4 μg/ml for imipenem and meropenem) among the species tested but were all MHT positive. Disk approximation tests using EDTA or mercaptopropionic acid as ion-chelating agents yielded positive results for all NDM-1-producing strains. These findings are particularly worrisome, because MHT has been broadly recommended to clinical microbiology laboratories for the detection and epidemiological evaluation of carbapenemase-producing bacteria (5). The newly published CLSI carbapenem susceptibility breakpoints (≤1 μg/ml) (5) will minimize these false-susceptible errors.
FIG. 1.
Modified Hodge test (MHT) for NDM-1-producing isolates showing weakly positive results (arrows). Positive and negative controls are included for comparison. MER, meropenem; IMI, imipenem.
The gene encoding NDM-1 was a prevalent mechanism of carbapenem resistance, being detected in 38.5% of the isolates surveyed and in at least 12 unique strains of three bacterial species. This gene was carried by distinct plasmids. Moreover, blaNDM-1-carrying K. pneumoniae strain displayed greater clonal diversity than that of the E. coli and E. cloacae strains harboring the same gene. This suggests greater genetic exchange among strains of this bacterial species.
NDM-1-producing strains appear to be an emerging worldwide problem (3, 6, 8, 10-13, 15), and by our findings, the detection of these strains by the MHT can be problematic (i.e., false-negative results can occur), delaying the implementation of infection control measures and allowing the continued dissemination of the strains. Further studies seem necessary to establish preventive strategies to control the dissemination of strains harboring blaNDM-1 and to elucidate the mechanism of dissemination of this β-lactamase gene.
Nucleotide sequence accession number.
The sequence of the OXA-181 gene was submitted to GenBank under accession number HM992946.
Acknowledgments
We thank the participants of the SENTRY Antimicrobial Surveillance Program in India: Padma Srikanth, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Chennai; Thomas Kuruvilla, Lisie Hospital, Cochin; Ratna Rao, Apollo Hospital, Hyderabad; D. S. Chitnis, Choitram Hospital, Indore; Anil Kumar and Kavitha Radhakrishnan Dinesh, Amrita Institute, Kochi; Sankar Sengupta, Ruby Hospital Institute of Child Health, Kolkata; Suganthi Rao, Kasturba Medical College, Manipal; Preeti Mehta, Seth Gordhandas Sunderdas Medical College and King Edward Memorial Hospital, Mumbai; Deepthi Nair, Safdarjung Hospital, New Delhi; Chand Wattal, Sir Ganga Ram Hospital, New Delhi; Neeta Munshi, Ruby Hall Clinic, Pune; Molly Madan, Pandit Deen Dayal Upadhaya Medical College, Rajkot; D. K. Mendiratta and Vijayashri Deotale, Kasturba Hospital, Sevagram; Bennett Abraham, Somerville Memorial CSI Mission Hospital, Trivandrum; and Anand Manoharan, Christian Medical College, Vellore.
Footnotes
Published ahead of print on 28 December 2010.
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