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Imipenem resistance in clinical Escherichia coli from Qom, Iran
BMC Research Notes volume 11, Article number: 314 (2018)
The emergence of metallo-β-lactamase-producing Enterobacteriaceae is a worldwide health concern. In this study, the first evaluation of MBL genes, bla IMP and bla VIM , in Escherichia coli resistant to imipenem isolated from urine and blood specimens in Qom, Iran is described. Three hundred urine and blood specimens were analysed to detect the presence of E. coli. Resistance to imipenem and other antimicrobials was determined by disk diffusion and MIC. MBL production was screened using CDDT. PCR was also carried out to determine the presence of bla IMP and bla VIM genes in imipenem-resistant isolates.
In total, 160 E. coli isolates were collected from March to May 2016. According to disk diffusion, high-level of resistance (20%) to cefotaxime was observed, whereas the lowest (1%) was detected for tetracycline. In addition, five isolates showed resistance to imipenem with a MIC ≥ 4 µg/mL. CDDT test confirmed that five isolates were MBL-producing strains, but no bla IMP and bla VIM genes were detected. Results of this study show a very low level of resistance to imipenem in our geographical area.
In recent years, the emergence of antimicrobial resistance in bacteria, mainly Enterobacteriaceae and other Gram-negative bacteria, has become a major concern for health systems worldwide . The impact of resistance on cost and length of hospitalization and increase in morbidity and mortality of patients is now obvious. Resistance to carbapenems is considered at high frequency among Gram-negative bacteria, e.g. Pseudomonas aeruginosa, Acinetobacter spp., and Enterobacteriaceae [2,3,4]. Bacterial β-lactamases can be categorized into four molecular classes based on the amino acid sequence . Metallo-β-lactamases (MBLs) or B class β-lactamases are distinct from the serine β-lactamases (classes A, C, and D) due to a zinc ion(s) in their structure. Except monobactams, these enzymes are able to hydrolyse all β-lactam antibiotics, such as penicillins, cephalosporins and carbapenems. Different types of MBL genes have been described, such as blaIMP, blaVIM, blaSPM-1, blaGIM-1, blaSIM-1, blaKMH-1, blaDIM-1 and blaNDM-1 genes [6, 7]. Among these, IMP (imipenemase) and VIM (Verona integron-encoded MBL) types are the most common MBLs that have been recently recognized in Enterobacteriaceae . MBLs are encoded both by chromosomal and acquired genes, called resident and acquired MBLs, respectively. Acquired MBLs can spread horizontally via mobile genetic elements among Enterobacteriaceae and other Gram-negative bacteria of clinical importance. In addition, studies showed evidence of a broad distribution of MBLs across different geographical areas, and that is considered a serious threat [9, 10]. Thus, although different studies on MBL-producing Escherichia coli from some Iranian provinces have been performed, this is the first study carried out in Qom city.
Bacterial isolation and identification
This cross-sectional study was conducted on patients admitted to the Ali Ebne Abitaleb Center, Qom, Iran and the Microbiology Department of Qom University of Medical Sciences. During a 3-month period (March to May 2016), 300 blood and urine samples were collected from 300 patients, mean age 40 years, with some common symptoms (e.g., fever, leukocytosis, dysuria, pyuria, and bladder pain), following their consent. Patients with a previous antimicrobial consumption were excluded from the study. Isolation and identification of E. coli was performed by a standard procedure. Briefly, blood and urine samples were cultured onto Eosin Methylene Blue agar (Merck, Germany) and MacConkey agar (Merck, Germany) at 37 °C for 24 h. Colonies were confirmed by Gram staining and biochemical testing (e.g., catalase, oxidase, Triple Sugar Iron Agar, Sulfide Indole Motility (SIM), Methyl Red (MR)/Voges-Proskauer (VP), citrate and urease).
Antimicrobial susceptibility testing (AST)
Antimicrobial susceptibility of isolates was determined by standard disk diffusion method as recommended by the CLSI . The antimicrobial disks used were imipenem (IMP, 10 µg), ceftazidime (CAZ, 30 µg), amikacin (AK, 30 µg), tobramycin (TOB, 10 µg), gentamicin (GM, 10 µg), tetracycline (TE, 30 µg), ceftriaxone (CRO, 30 μg), norfloxacin (NOR, 10 µg) ciprofloxacin (CP, 5 µg), cefexime (CFM, 5 µg) nalidixic acid (NA, 30 µg), trimethoprim/sulfamethoxazole (SXT, 2.5 µg), amoxicillin (AMX, 25 μg) and cefotaxime (CTX, 30 µg) (MAST, UK). E. coli ATCC 25922 was used as a quality control strain.
Minimum inhibitory concentrations (MICs) of imipenem were also determined using the broth microdilution method . Eight different dilutions (256, 128, 64, 32, 16, 8, 4 and 2 μg/mL) were prepared in Mueller–Hinton Broth (Merck, Germany) followed by the transfer of 100 μL into 96-well microtiter plates. Twenty-four hours cultures of E. coli colonies were suspended in 0.9% saline at 0.5 McFarland and then diluted (1:20). Finally, 10 μL of the bacterial suspension was added to the wells and microplates were incubated aerobically at 37 °C for 18–24 h. E. coli strains with a MIC ≥ 4 µg/mL were defined as resistant strains .
Phenotypic identification of MBLs
Phenotypic detection of MBLs was evaluated by the combination disk diffusion test (CDDT) using EDTA/imipenem and imipenem disks (MAST, UK) as previously described by Fallah et al. .
DNA extraction and detection of MBL genes
DNA was extracted from resistant isolates using the boiling method . PCR was performed by using the primers listed in Table 1 and in a total volume of 25 μL composed by 12.5 µL of PCR Master Mix (SinaClon, Iran), 5 µL of extracted DNA, 10 p.m. of each primer synthesized by Bioneer (Korea), and 5.5 µL of DNase/RNase-free sterile water.
PCR reaction was carried out in a thermocycler (Eppendorf, Hamburg, Germany) as follows: denaturation at 94 °C for 3 min (1 cycle), followed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min and a single, final, elongation step at 72 °C for 10 min. Finally, amplified products were analysed by electrophoresis in a 2% agarose gel run at 95 V for 100 min in 1X TBE containing DNA Safe Stain (SinaClon, Iran) and gels were visualized under UV light.
Out of 300 clinical specimens, 160 (53%) were found positive to E. coli and all were urine specimen. Sixty percent of strains were isolated from female and 40% from male patients. Disk diffusion test showed that the highest resistance was against cefotaxime (31 isolates, 20%) and ceftazidime (27 isolates, 17%) and the lowest to tetracycline (1 case, 1%). No resistance to amikacin and tobramycin was observed among isolates. Only five isolates (3%) were found resistant to imipenem (Fig. 1). In CDDT assay, all five imipenem-resistant strains were confirmed positive for MBL enzymes (Fig. 2) and they had a MIC ≥ 4 µg/mL. PCR did not detect any blaIMP and blaVIM genes in MBL-producing strains.
Over the years, the widespread use of antimicrobials has forced the emergence of resistance in pathogenic bacteria, and this has seriously challenged their clinical effectiveness. In the past, carbapenems, mainly imipenem, were considered drugs of the first line in empirical treatment of severe bacterial infections. However, a dramatic increase of resistance to these antimicrobials has being observed in the last years and this can reduce treatment options in the near future . MBLs are effective enzymes in hydrolysing all β-lactams, except for aztreonam. IMP-1 and VIM-1 have been the first enzymes to be discovered in Japan (1990) and in Italy (1999), respectively. Afterward, many variants have been described in other countries. Currently, more than 20 different IMP and VIM types are known [9, 15, 16]. MBL-producing bacteria, e.g. E. coli, Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii, have been reported from different parts of Iran [3, 17,18,19] and this has become a major threat to public health . E. coli is considered an opportunist pathogen and cause of urinary infections, bacteraemia, neonatal meningitis, but the emergence of MBL-producing strains is rapidly growing and threating successful therapy . Therefore, detection of resistance in E. coli from different areas is crucial to avoid its spread among bacteria.
In this study, all E. coli positive cultures were obtained from urine samples. This finding is in agreement with Ntirenganya et al.  who found 55.2% of positive cases related to urine specimens. In our E. coli isolates, the highest level of resistance was detected against cefotaxime (20%), ceftazidime (17%), ciprofloxacin (14%) and trimethoprim/sulfamethoxazole (14%). The lowest level of resistance was identified against imipenem (3%), gentamycin and tetracycline (1%), while no resistance to tobramycin and amikacin (0%) was detected. In other Iranian regions, different resistance levels have been reported. Mansouri  reported very high resistance rates of E. coli to trimethoprim/sulfamethoxazole (93.4%) and amoxicillin (91.4%) in Kerman city, Iran. Unlike our study, the Authors did not report resistance to imipenem. Pouladfar et al.  reported two imipenem-resistant urinary E. coli (1.9%) in Shiraz, Iran. The full susceptibility of our isolates to tobramycin and amikacin is similar to that obtained in other countries, such as Spain . However, resistance to these antimicrobials has been observed in other studies. For example, Soleimani et al.  detected resistance to aminoglycosides among uropathogenic E. coli isolated from Tehran, Iran. In addition, they showed that 24.6 and 3.62% of isolates were resistant to tobramycin and amikacin, respectively. Another report from Punjab, Pakistan, showed 59% and 91% of resistance to tobramycin and amikacin, respectively [26, 27].
MIC results were comparable with those obtained at disk diffusion, which showed 3% of isolates resistant to imipenem. A very low level of resistance was observed by Moayednia et al.  in Isfahan city, Iran. The Authors detected 0.3% (2/720) of hospital E. coli isolates positive for MBL, while no MBL-producing isolates were detected in non-hospital E. coli. In contrast to our study, Zeighami et al.  did not detect any MBL-positive E. coli in Zanjan City, Iran. Besides Iran, the presence of MBL-producing E. coli has been reported from other parts of the world. Bora et al.  in Nepal indicated a high prevalence of MBLs with 18.98 and 21.08% for E. coli and K. pneumonia, respectively. Other studies carried out in Korea and India also detected an increasing frequency of MBL positive strains within the Enterobacteriaceae family [31, 32].
In this study, no bla IMP and bla VIM genes were found, but we cannot exclude that other MBL genes might have been involved in resistance. It is cleared that the resistance to carbapenems may be attributed to outer membrane protein deletion and significantly a decrease in cell permeability can be seen . In study of the Ranjan and co-worker , bla KPC gene could not be detected among isolated E. coli, while they were positive for bla NDM and blaOXA-48. Additionally, although phenotypic tests are easy and have an excellent sensitivity, the use of EDTA can give false positive results due to the instability of the bacterial membrane [35, 36]. In agreement with our study, Lee et al.  found only one out of three imipenem-resistant E. coli isolates as MBL producer and harbouring MBL genes in southern Taiwan. In another study carried out by Peirano et al. , the majority of VIM- and IMP-producing Enterobacteriaceae were K. pneumoniae and only one E. coli strain was found positive. A high prevalence of MBL-producing E. coli was detected in the study of Nahid et al.  in Pakistan. The Authors showed that, out of 145 E. coli, 50 (34.48%) were MBL producers and VIM and IMP genes were present in eight and two strains, respectively.
In conclusion, our study is the first description of MBL-producing E. coli in Qom, Iran. Overall, strains showed a low resistance level to imipenem and none was found positive for bla IMP and bla VIM genes. Therefore, phenotyping assays are recommended for detecting resistance in E. coli, as well as a guideline on the prudent use of antimicrobials should be developed for preventing the emergence and spread of resistance.
Due to financial constraints, we could not focus on other genes only.
minimum inhibitory concentration
combination disk diffusion test
- E. coli :
Verona integron-encoded MBL
polymerase chain reaction
Clinical & Laboratory Standards Institute
Nepal K, Pant ND, Neupane B, Belbase A, Baidhya R, Shrestha RK, et al. Extended spectrum beta-lactamase and metallo beta-lactamase production among Escherichia coli and Klebsiella pneumoniae isolated from different clinical samples in a tertiary care hospital in Kathmandu, Nepal. Ann Clin Microbiol Antimicrob. 2017;16:62.
Robledo IE, Aquino EE, Vazquez GJ. Detection of the KPC gene in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii during a PCR-based nosocomial surveillance study in Puerto Rico. Antimicrob Agents Chemother. 2011;55:2968–70.
Limbago BM, Rasheed JK, Anderson KF, Zhu W, Kitchel B, Watz N, et al. IMP-producing carbapenem-resistant Klebsiella pneumoniae in the United States. J Clin Microbiol. 2011;49:4239–45.
Cornaglia G, Mazzariol A, Lauretti L, Rossolini GM, Fontana R. Hospital outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-1, a novel transferable metallo-beta-lactamase. Clin Infect Dis. 2000;31:1119–25.
Bush K, Jacoby GA. Updated functional classification of β-lactamases. Antimicrob Agents Chemother. 2010;54:969–76.
Moloughney JG, Thomas JD, Toney JH. Novel IMP-1 metallo-beta-lactamase inhibitors can reverse meropenem resistance in Escherichia coli expressing IMP-1. FEMS Microbiol Lett. 2005;243:65–71.
Queenan AM, Bush K. Carbapenemases: the versatile beta-lactamases. Clin Microbiol Rev. 2007;20:440–58 (table of contents).
Wendt C, Schutt S, Dalpke AH, Konrad M, Mieth M, Trierweiler-Hauke B, et al. First outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in Germany. Eur J Clin Microbiol Infect Dis. 2010;29:563–70.
Cornaglia G, Giamarellou H, Rossolini GM. Metallo-beta-lactamases: a last frontier for beta-lactams? Lancet Infect Dis. 2011;11:381–93.
Cornaglia G, Akova M, Amicosante G, Canton R, Cauda R, Docquier JD, et al. Metallo-beta-lactamases as emerging resistance determinants in Gram-negative pathogens: open issues. Int J Antimicrob Agents. 2007;29:380–8.
Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement. CLSI document M100-S25, vol. 35. Wayne: Clinical And Laboratory Standards Institute; 2015. p. 3.
Fallah F, Borhan RS, Hashemi A. Detection of bla(IMP) and bla(VIM) metallo-beta-lactamases genes among Pseudomonas aeruginosa strains. Int J Burns Trauma. 2013;3:122–4.
Shams S, Bakhshi B, Tohidi Moghadam T. In Silico analysis of the cadF gene and development of a duplex polymerase chain reaction for species-specific identification of Campylobacter jejuni and Campylobacter coli. Jundishapur J Microbiol. 2016;9:e29645.
Derevianko II, Nefedova LA, Lavrinova LN. Effectiveness of imipenem/cilastatin (Tienam, MSD) in treating complicated infections in urology. Urologiia. 2002;3:21–5.
Leiros HK, Borra PS, Brandsdal BO, Edvardsen KS, Spencer J, Walsh TR, et al. Crystal structure of the mobile metallo-beta-lactamase AIM-1 from Pseudomonas aeruginosa: insights into antibiotic binding and the role of Gln157. Antimicrob Agents Chemother. 2012;56:4341–53.
Lee K, Lee WG, Uh Y, Ha GY, Cho J, Chong Y. VIM- and IMP-type metallo-beta-lactamase-producing Pseudomonas spp. and Acinetobacter spp. in Korean hospitals. Emerg Infect Dis. 2003;9:868–71.
Shirani K, Ataei B, Roshandel F. Antibiotic resistance pattern and evaluation of metallo-beta lactamase genes (VIM and IMP) in Pseudomonas aeruginosa strains producing MBL enzyme, isolated from patients with secondary immunodeficiency. Adv Biomed Res. 2016;5:124.
Shigemoto N, Kayama S, Kuwahara R, Hisatsune J, Kato F, Nishio H, et al. A novel metallo-β-lactamase, IMP-34, in Klebsiella isolates with decreased resistance to imipenem. Diagn Microbiol Infect Dis. 2013;76:119–21.
Aksoy MD, Cavuslu S, Tugrul HM. Investigation of Metallo beta lactamases and oxacilinases in carbapenem resistant Acinetobacter baumannii strains isolated from inpatients. Balkan Med J. 2015;32:79–83.
Bahramian A, Eslami G, Hashemi A, Tabibi A, Heidary M. Emergence of fosfomycin resistance among isolates of Escherichia coli harboring extended-spectrum and AmpC β-lactamases. Acta Microbiol Immunol Hung. 2017. https://doi.org/10.1556/030.64.2017.030.
Jafri SA, Qasim M, Masoud MS, Rahman MU, Izhar M, Kazmi S. Antibiotic resistance of E. coli isolates from urine samples of urinary tract infection (UTI) patients in Pakistan. Bioinformation. 2014;10:419–22.
Ntirenganya C, Manzi O, Muvunyi CM, Ogbuagu O. High prevalence of antimicrobial resistance among common bacterial isolates in a tertiary healthcare facility in Rwanda. Am J Trop Med Hyg. 2015;92:865–70.
Mansouri S, Kalantar Neyestanaki D, Shokoohi M, Halimi S, Beigverdi R, Rezagholezadeh F, et al. Characterization of AmpC, CTX-M and MBLs types of beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli producing extended spectrum beta-lactamases in Kerman, Iran. Jundishapur J Microbiol. 2014;7:e8756.
Pouladfar G, Basiratnia M, Anvarinejad M, Abbasi P, Amirmoezi F, Zare S. The antibiotic susceptibility patterns of uropathogens among children with urinary tract infection in Shiraz. Medicine. 2017;96:37.
Sáenz Y, Zarazaga M, Briñas L, Lantero M, Ruiz-Larrea F, Torres C. Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. Int J Antimicrob Agents. 2001;18:353–8.
Soleimani N, Aganj M, Ali L, Shokoohizadeh L, Sakinc T. Frequency distribution of genes encoding aminoglycoside modifying enzymes in uropathogenic E. coli isolated from Iranian hospital. BMC Res Notes. 2014;7:842.
Sohail M, Khurshid M, Saleem HGM, Javed H, Khan AA. Characteristics and antibiotic resistance of urinary tract pathogens isolated from Punjab, Pakistan. Jundishapur J Microbiol. 2015. https://doi.org/10.5812/jjm.19272v2.
Moayednia R, Shokri D, Mobasherizadeh S, Baradaran A, Fatemi SM, Merrikhi A. Frequency assessment of beta-lactamase enzymes in Escherichia coli and Klebsiella isolates in patients with urinary tract infection. J Res Med Sci. 2014;19:S41–5.
Zeighami H, Haghi F, Hajiahmadi F. Molecular characterization of integrons in clinical isolates of betalactamase-producing Escherichia coli and Klebsiella pneumoniae in Iran. J Chemother. 2014. https://doi.org/10.1179/1973947814Y.0000000180.
Bora A, Sanjana R, Jha BK, Mahaseth SN, Pokharel K. Incidence of metallo-beta-lactamase producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in central Nepal. BMC Res Notes. 2014;7:557.
Yong D, Choi YS, Roh KH, Kim CK, Park YH, Yum JH, et al. Increasing prevalence and diversity of metallo-beta-lactamases in Pseudomonas spp., Acinetobacter spp., and Enterobacteriaceae from Korea. Antimicrob Agents Chemother. 2006;50:1884–6.
Datta S, Wattal C, Goel N, Oberoi JK, Raveendran R, Prasad KJ. A ten year analysis of multi-drug resistant blood stream infections caused by Escherichia coli & Klebsiella pneumoniae in a tertiary care hospital. Indian J Med Res. 2012;135:907–12.
Ye Y, Xu L, Han Y, Chen Z, Liu C, Ming L. Mechanism for carbapenem resistance of clinical Enterobacteriaceae isolates. Exp Ther Med. 2018;15:1143–9.
Ranjan A, Shaik S, Mondal A, Nandanwar N, Hussain A, Semmler T, et al. Molecular epidemiology and genome dynamics of New Delhi metallo-β-lactamase-producing extraintestinal pathogenic Escherichia coli strains from India. Antimicrob Agents Chemother. 2016;60:6795–805.
Yousefi S, Farajnia S, Nahaei MR, Akhi MT, Ghotaslou R, Soroush MH, et al. Detection of metallo-beta-lactamase-encoding genes among clinical isolates of Pseudomonas aeruginosa in northwest of Iran. Diagn Microbiol Infect Dis. 2010;68:322–5.
Giske CG, Gezelius L, Samuelsen O, Warner M, Sundsfjord A, Woodford N. A sensitive and specific phenotypic assay for detection of metallo-beta-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect. 2011;17:552–6.
Lee MF, Peng CF, Hsu HJ, Chen YH. Molecular characterisation of the metallo-beta-lactamase genes in imipenem-resistant Gram-negative bacteria from a university hospital in southern Taiwan. Int J Antimicrob Agents. 2008;32:475–80.
Peirano G, Lascols C, Hackel M, Hoban DJ, Pitout JD. Molecular epidemiology of Enterobacteriaceae that produce VIMs and IMPs from the SMART surveillance program. Diagn Microbiol Infect Dis. 2014;78:277–81.
Nahid F, Khan AA, Rehman S, Zahra R. Prevalence of metallo-beta-lactamase NDM-1-producing multi-drug resistant bacteria at two Pakistani hospitals and implications for public health. J Infect Public Health. 2013;6:487–93.
SS and AH involved in the management of the project and writing up the paper. ME, SK and ES involved in collecting of samples and performing of the study. AP involved in analysis of results. All authors read and approved the final manuscript.
We wish to thank the Research Council of Qom University of Medical Sciences for supporting the study.
The authors declare that they have no competing interests.
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The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Ethics approval and consent to participate
The clinical samples collected were in line with the patients’ diagnostic stages and no additional samples were taken. This project was submitted to and approved by 27th Ethical Committee of Qom University of Medical Sciences. The satisfaction of each patient was done before their participation. Firstly, the work was explained verbally for each patient and then the informed consent form was studied and signed by them.
The study was fully funded by Qom University of Medical Sciences.
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Shams, S., Hashemi, A., Esmkhani, M. et al. Imipenem resistance in clinical Escherichia coli from Qom, Iran. BMC Res Notes 11, 314 (2018) doi:10.1186/s13104-018-3406-6
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