First Report of New Delhi Metallo-β-Lactamase-1 (NDM-1) Among Escherichia Coli Strains Isolated from Leukemia Patients in Iran

Background: Escherichia coli has appeared as an important opportunistic pathogen responsible for nosocomial infections in Patients with immunodeciency particularly in leukemia patients. New Delhi metallo-β-lactamase (NDM-1) is an enzyme that class of beta-lactam antibiotics and is used in treatment of multi-drug resistant (MDR) infections. Case Presentation: In this study, 80 isolates of Escherichia coli and Klebsiella pneumoniae collected over the course of two years from two medical centers of Tehran, Iran. Production of carbapenemase was detected of the isolates using MHT. New Delhi metallo-beta-lactamase 1 (bla NDM-1 ) genes were detected by polymerase chain reaction (PCR) ampli ﬁ cation with speci ﬁ c primers 2 bla NDM-1 producing E.coli strains were isolated from 2 leukemia of patients, The isolates were resistant to carbapenems (imipenem, meropenem),two isolates were positive for carbapenemase production by Modied Hodge test. Conclusions: The emerging of NDM-1 producing E. coli is a threat for leukemia patients in Oncology and hematology departments. We concluded that the incidence of MDR pathogens increased amang patients with leukemia and is life threatening. with leukemia, especially in patients with neutropenia (2) .our study is also the rst report in Iran. The purpose of this study was to investigate the existence of NDM1 gens as a risk factor for life-threatening infection in patients with leukemia patients. The bacteria were isolated and stored in University of Medical Sciences. All the clinical specimens were quickly sent to the laboratory and analyzed for conrmatory test. Isolates were identied using standard microbiological and biochemical procedures (3). In our previous study, antibiotic susceptibility of the isolates was tested by Kirby-Bauer disk diffusion method for imipenem, ceftazidime, ceftriaxone, cefotaxime, ciprooxacin, levooxacin, amikacin, ampicillin, and gentamicin (all from Mast, UK). Moreover, the combination disk method was employed to detect extended-spectrum β-lactamase (ESBL) producing isolates; while the minimum inhibitory concentrations (MICs) of selected antimicrobials were determined by the broth microdilution method. The results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines (4). The carbapenem-resistant strains were investigated for carbapenemase production by modied hodge test (MHT) according to the CLSI guidelines in which, E. coli ATCC25922 was used as the positive control (5). Furthermore, the combined disk diffusion method was applied for bla MBL detection using 2 IPM (10 µg) disks and


Background
The global increasing prevalence of NDM-1-producing Enterobacteriaceae is a concerning phenomenon in immunocompromised patients. Previous results indicated that bla NDM−1 gene can be carried on the IncN plasmids of different sizes along with other resistance factors. bla NDM−1 can confer resistance to almost all the β-lactams. Thus the bacteria carrying NDM-1 gene are considered resistant to all antibiotic classes except colistin and cipro oxacin. The gene was scarcely integrated into the chromosome. The sequencing of this gene suggests a new enzyme unrelated to hitherto known as MBLs. The most similar known type is Verona integron-encoded metallo-β-lactamase (VIM-1/VIM-2) sharing a 32.4% resemblance (1).
In a study conducted in China, the presence of this gene was rst reported in a patient with leukemia, especially in patients with neutropenia (2) .our study is also the rst report in Iran. The purpose of this study was to investigate the existence of NDM1 gens as a risk factor for life-threatening infection in patients with leukemia patients.

Case Presentation
In this study, 80 isolates of Escherichia coli and Klebsiella pneumoniae were collected from two medical centers of Tehran, Iran: The Hematology-Oncology Research Center, Dr. Shariati Hospital, and the Mahak Pediatric Oncology Center between 2014-2015.
The bacteria were isolated and stored in Hamadan University of Medical Sciences. All the clinical specimens were quickly sent to the laboratory and analyzed for con rmatory test. Isolates were identi ed using standard microbiological and biochemical procedures (3). In our previous study, antibiotic susceptibility of the isolates was tested by Kirby-Bauer disk diffusion method for imipenem, ceftazidime, ceftriaxone, cefotaxime, cipro oxacin, levo oxacin, amikacin, ampicillin, and gentamicin (all from Mast, UK). Moreover, the combination disk method was employed to detect extended-spectrum βlactamase (ESBL) producing isolates; while the minimum inhibitory concentrations (MICs) of selected antimicrobials were determined by the broth microdilution method. The results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines (4). The carbapenem-resistant strains were investigated for carbapenemase production by modi ed hodge test (MHT) according to the CLSI guidelines in which, E. coli ATCC25922 was used as the positive control (5). Furthermore, the combined disk diffusion method was applied for bla MBL detection using 2 IPM (10 µg) disks and EDTA 0.5 M solution (6). For diagnosis of the NDM1, PCR was performed on DNA extracted by boiling, using NDM-Fspeci c primers: CAACTGGATCAAGCAGGAGA, NDM-R TCGATCCCAACGGTGATATT (Bioneer Company, Korea) (7).
The sequencing of amplicons in both directions was carried out by the Bioneer Company (Daejeon, South Korea). The data were analyzed using FinchTV software (Geospiza, USA), and the sequences were then con rmed using the NCBI web site (http://www.ncbi.nlm.nih.gov/ BLAST). A total of 56 E. coli and 24 K. pneumoniae isolates were achieved from urine, blood, sputum, wound, and vagina. Among the 80 isolates, 52 (63%) strains were ESBL producers, followed by 5 (6.25%) metallo β-lactamase (MBL) producers. According to our antimicrobial susceptibility test on E. coli and K. pneumoniae isolates, 8 (10%) of E. coli isolates were imipenem-resistant. Out of 80 isolates, 8(10%) isolates were found to be carbapenems-resistant. The major MBL and carbapenems-resistant species were Escherichia coli. The rst NDM1containing isolate was obtained from a 26-year-old subject diagnosed with acute leukemia. The second was obtained from a 2-year-old child with acute leukemia. These two NDM-1positive E. coli isolates (labeled as E1-E2) were recovered from the urine and blood samples of two different hospitalized patients. All two isolates were positive for the modi ed Hodge test and (MBL) producers (

Discussion
The present study reports NDM-1-producing E.coli strain from the bloodstream and infected urinary tract of two patients. PCR results con rmed that the NDM-1producing E.coli harbored qnrA, qnrB, qnrS and bla CTXM−1 , CTX−M2 , CTX−M8 , CTX−M9 , CTX−M25 and the insertion sequence of ISECP1,IS26,IS903. The two NDM1-producing E.coli isolates did not contain qnrS genes but they carried bla TEM gene. In a study in 2010 on a patient transferred from Iraq to France, NDM-1-producing K. pneumoniae was also been reported (11).
Reports have also declared the existence of bla NDM−1 producing Enterobacteriaceae in Pakistan and Afghanistan (12,13).
The bla NDM−1 producing bacteria could be also found in environmental samples and drinking-water (14). In our investigation, PCR con rmed that the NDM1-producing E.coli harbored quinolone resistance gene B (qnrB) and bla CTXM−9 .
This result was similar to the other reports on strains carrying blaNDM-1 which also harbored other β-lactamase genes (2).
An NDM-1-producing E.coli strain was detected in the bloodstream of a patient in this study that exhibited high resistance to all tested β-lactam antibiotics, which can be attributed to the production of bla NDM−1 and other resistant genes.
Bahramian et al. published the rst report about metallo-β-lactamase-6 (NDM-6) among K. pneumoniae in New Delhi. ST147 strains were also isolated from dialysis patients in Iran (15) all the authors agree to publish the manuscript in the European journal of medical science.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests Author's contribution: MR, FA and SD performed microbiological and molecular tests and write the manuscript. AG, A H and HG play role in Project Administration. MRA supervised all of the stages of designing the study, conducting the research and writing the manuscript.