Bacterial sample:
Six isolates of Escherichia coli, harboring blaNDM−4, isolated from hospital patients of Silchar Medical College and Hospital, India were selected for the study (Table 1). Samples were taken as a part of standard care and day to day routine sampling as suggested by clinicians. The carriage of blaNDM−4 was confirmed by PCR sequencing of whole gene. Plasmid incompatibility was determined by PCR assay (2). Presence of blaNDM was determined by PCR assay using primers (NDM-F 5-GGGCAGTCGCTTCCAACGGT-3 and NDM-R 5-GTAGTGCTCAGTGTCGGCAT-3) (6). The isolates were identified by Gram staining method, standard biochemical characterization tests including IMViC test, urease test, triple sugar iron test, sugar fermentation test and nitrate reduction test and finally by 16 s rDNA sequencing
Table 1
Escherichia coli isolates obtained for this study
1 | NH-19 | Female | 12.5 years | Stool | Medicine |
2 | NH-36 | Male | 32 years | Urine | Medicine |
3 | NH-28 | Male | 27 years | Surgical wound | Surgery |
4 | NH-31 | Male | 62 years | Stool | OPD |
5 | NH-56 | Female | 48 years | Urine | OPD |
6 | NH-39 | Female | 10 years | Urine | Genital ward |
Plasmid Preparation And Transmission Assay:
Plasmids encoding blaNDM−4 were extracted by QIAprep Spin Miniprep Kit (Qiagen, Germany) as per manufacturer’s instruction. Isolated plasmids were subjected to transformation assay. The recipient strain used was E.coli JM107, E.coli DH5α, Klebsiella pneumonia, proteus mirabilis, Pseudomonas aeruginosa and Acinetobacter baumannii of clinical origin. Transformation was carried out by heat shock method (7). Transformants were selected on LB Agar plates containing ampicillin (100 µg/ml). Conjugation experiment was performed using blaNDM−4 harbouring clinical strains as donors and azide resistant E.coli J53 as recipient and transconjugants were selected on medium containing either imipenem (0.5 µg/ml) or ampicillin (100 µg/ml) alongwith sodium azide (100 µg/ml).
Plasmid Stability Within Different Hosts:
Plasmid stability analysis of blaNDM−4 producers in parent strain (E.coli) and transformants in different hosts i.e, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Proteus mirabilis, E.coli DH5α) was performed by the serial passage method for consecutive 70 days at 1:1000 dilutions without any antibiotic pressure (8) After each passage, 1 ml of the culture was diluted into 103 dilution with normal saline, and 40 µl of the diluted sample was spread on to the LB agar plate. After overnight incubation, 50 colonies from plates were randomly picked and subjected to phenotypic detection of MBL and further confirmed genotypically by PCR assay for the presence of blaNDM−4.
Plasmid copy number alteration and transcriptional expression of bla NDM−4 within broad host range against concentration gradient imipenem stress:
Single colony of each host isolate was inoculated into LB broth with 1 µg/ml, 2 µg/ml, 4 µg/ml and 8 µg/ml of imipenem and also without any antibiotic (considered as a control for the reaction) and was incubated at 37◦C for 4–7 hour till the OD reaches 0.9 at A600. cDNA was extracted from each condition, the reaction was performed using 10 µl of SYBR® Green PCR Master Mix (Applied Biosystem, Warrington, UK), 4 ng plasmid DNA as template and 3 µl of each primer (Table 2) (10 pmol) in a 20 µl reaction and the relative fold change was measured by ∆∆CT method and was normalized against a housekeeping gene rpsl of E. coli (9). The each set of reaction was run in triplicate and the experiment was repeated thrice. Quantitative Real Time PCR was done to determine the level of alteration of the plasmid encoding blaNDM− 4 using Step One Plus real time detection system (Applied biosystem, Warrington).
Table 2
Oligonucleotides used as primer for the amplification of carbapenamase gene
Name of target genes | Primer Sequence(5’-3’) | Amplification size (bp) |
Inc X3 F | 5’-GTTTTCTCCACGCCCTTGTTCA-3’ | 351 |
Inc X3 R | 5’-CTTTGTGCTTGGCTATCATAA-3’ |
NDM F | GGGCAGTCGCTTCCAACGGT | 476 |
NDM R | CGACCGGCAGGTTGATCTCC |
Susceptibility Testing:
The antibiotic susceptibility was done by Kirby Bauer disc-diffusion method against antibiotics as piperacillin-tazobactam (100/10 µg), amikacin (30 µg), gentamicin (10 µg), ciprofloxacin (5 µg), polymixin B (300units) ampicillin (30 µg), cotrimoxazole (10 µg) and carbenicillin (100 µg) (Hi-Media, Mumbai, India). Minimum inhibitory concentration was performed by agar dilution method against imipenem, meropenem, cefepime & aztreonam, cotrimoxazole, ampicillin, ciprofloxacin and the results were compared with standard CLSI guidelines (10). The antibiotic susceptibility of the transformants was also determined