New Delhi metallo-β-lactamase-1 among Escherichia coli strains isolated from leukemia patients in Iran: two case reports

Background Escherichia coli has appeared as an important opportunistic pathogen responsible for nosocomial infections in patients with immunodeficiency, particularly in leukemia patients. New Delhi metallo-beta-lactamase is an enzyme originally found in Enterobacteriaceae. Case presentation In this study, 80 isolates of Escherichia coli and Klebsiella pneumoniae were collected over the course of 2 years from two medical centers in Tehran, Iran. Production of carbapenemase was detected in the isolates using modified Hodge test. New Delhi metallo-beta-lactamase-1 genes were detected by polymerase chain reaction amplification with specific primers. Two New Delhi metallo-beta-lactamase-1-producing Escherichia coli strains were isolated from two Iranian patients with leukemia. These two patients were 6 and 15 years old, one female and the other male, from two oncology centers in Iran. The isolates were resistant to carbapenems (imipenem, meropenem), and two isolates were positive for carbapenemase production by modified Hodge test. Conclusions The emergence of New Delhi metallo-beta-lactamase-1-producing Escherichia coli is a threat for leukemia patients in oncology and hematology departments. We conclude that the incidence of multidrug resistant pathogens has increased among patients with leukemia and is life threatening.


Introduction
The globally increasing prevalence of New Delhi metalloβ-lactamase-1 (NDM-1)-producing Enterobacteriaceae is a concerning phenomenon in immunocompromised patients. Previous results indicated that bla NDM-1 gene can be carried on incompatibility group N (IncN) plasmids of different sizes along with other resistance factors. bla NDM-1 can confer resistance to almost all the β-lactams. Thus, bacteria carrying NDM-1 gene are considered to be resistant to all antibiotic classes except colistin and ciprofloxacin. The gene was scarcely integrated into the chromosome. The sequencing of this gene suggests a new enzyme, unrelated to hitherto known metallo β-lactamases (MBLs). The most similar known type is Verona integron-encoded metallo-β-lactamase (VIM-1/VIM-2), sharing 32.4% resemblance [1]. Immunosuppressive, sepsis, and radiation therapy can be the differential diagnosis of exotic infection [2][3][4][5].
In a study conducted in China, the presence of this gene was first reported in a patient with leukemia, but it has mainly been reported in patients with neutropenia. Our study is the first report from Iran. The purpose of this study was to investigate the existence of NDM-1 gene as a risk factor for life-threatening infection in patients with leukemia.

Case presentation
In this study, 80 isolates of Escherichia coli and Klebsiella pneumoniae were collected from two medical centers in Tehran, Iran: the Hematology-Oncology Research Center, Dr. Shariati Hospital, and the Mahak Pediatric Oncology Center, between 2014 and 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 confirmatory test. Isolates were identified using standard microbiological and biochemical procedures [6]. In our previous study, antibiotic susceptibility of the isolates was tested by Kirby-Bauer disk diffusion method for imipenem, ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, levofloxacin, 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 [7]. The carbapenem-resistant strains were investigated for carbapenemase production by modified Hodge test (MHT) according to the CLSI guidelines, in which E. coli ATCC25922 was used as the positive control [8]. Furthermore, the combined disk diffusion method was applied for bla MBL detection using two IPM (10 µg) disks and ethylenediaminetetraacetic acid (EDTA) 0.5 M solution [9]. For diagnosis of the NDM1, polymerase chain reaction (PCR) was performed on DNA extracted by boiling, using NDM-F-specific primers: CAA CTG GAT CAA GCA GGA GA, NDM-R TCG ATC CCA ACG GTG ATA TT (Bioneer Company, Korea) [10].
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 confirmed using the National Center for Biotechnology Information (NCBI) website (http:// www. ncbi. nlm. nih. gov/ BLAST). A total of 56 E. coli and 24 K. pneumoniae isolates were obtained 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, eight (10%) of E. coli isolates were imipenem-resistant. Out of 80 isolates, 8 (10%) isolates were found to be carbapenemresistant. The major MBL and carbapenem-resistant species were E. coli. The first NDM-1-containing 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-1-positive E. coli isolates (labeled as E1-E2) were recovered from the urine and blood samples of two different hospitalized patients. Both isolates were positive for the modified Hodge test and MBL producers (Tables 1 and 2).
Eyvazi et al. recently reported bla NDM-1 -producing E. coli in Iran for the first time [11]. Shahcheraghi et al. detected the first bla NDM-1 -producing K. pneumonia in Iran as well [12].
In October 2011, Laurent Poirel et al. reported bla NDM-1 -producing K. pneumoniae and E. coli. in a 16-year-old male patient admitted to the hematology unit of a hospital in Istanbul, Turkey [13].
In a study in 2010 on a patient transferred from Iraq to France, NDM-1-producing K. pneumoniae was also reported [14].
Reports have also declared the existence of bla NDM-1 -producing Enterobacteriaceae in Pakistan and Afghanistan [15,16]. The bla NDM-1 -producing bacteria could also be found in environmental samples and drinking water [17]. In our investigation, PCR confirmed that the NDM-1-producing E. coli harbored quinolone resistance gene B (qnrB) and bla CTXM-9 . This result was similar to the other reports on strains carrying bla NDM-1 that also harbored other β-lactamase genes [18]. 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 first report about metallo-β-lactamase-6 (NDM-6) among K. pneumoniae in New Delhi. ST147 strains were also isolated from dialysis patients in Iran [19]. Firoozeh et al. reported 20 (11.1%) K. pneumoniae isolates harboring bla NDM-1 gene (20).
We inferred that it is important to evaluate the health condition of immunocompromised patients, especially those with leukemia. Moreover, patients receiving chemotherapy may develop bloodstream infections. Bacterial infections can result in significant morbidity and mortality due to the development of febrile neutropenia and bacteremia.

Conclusion
This is the first report on the bla NDM-1 -producing E. coli strains isolated from leukemia patients in Iran. NDM-1-producing E. coli also harbored genes encoding cefotaxime-resistant (CTX-M) group, TEM, quinolone resistance (QNR), and insertion sequence of resistance enzymes. The coincidence of NDM-1 with other antibiotic-resistance genes may further limit the treatment options and makes infection control procedures more challenging among leukemia patients. Our results show the emergence of bla NDM-1 as an alarm to our health services, particularly among immunocompromised patients.