Journal of Current Biomedical Reports jcbior.com Volume 1, Number 2, 2020 eISSN: 2717-1906 1 Brief report The occurrence of antibiotic resistance, ESBLs, MBL and NDM-1 in Uropathogenic Escherichia coli in central part of Iran Farshad Kakian1, Javad Fathi1, Fatemeh Alvandi2, Mahnaz Moumivand2, Abbas Rabei Gholami3, Abolfazl Gholipour4, Mohamad hossein Rezaei5, Behnam Zamanzad6,* 1Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran 2Department Microbiology, Hamadan University of Medical Sciences, Hamadan, Iran 3Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran 4Cellular and Molecular Research Center, Basic Health Sciences Institute, Shahrekord University of Medical Sciences, Shahrekord, Iran 5Cellular and Molecular Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran 6Department of Microbiology and Immunology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran Abstract Extended spectrum β-lactamases (ESBLs) are enzymes that capable of destroying the antibiotics of β- lactam, and cephalosporin, and Metallo-β-lactamase enzymes (MBL) can also deactivate all β-lactams and carbapenems. This study aimed to determine ESBLs and MBLs enzymes and the frequency of NDM-1 gene. In this study, 200 Escherichia coli isolates of women with urinary tract infection were collected (100 inpatients and 100 outpatients). Minimum inhibitory concentration (MIC) for ceftazidime and meropenem was determined by E-test. A phenotypic confirmation test was used to detect ESBL enzymes. MBLs production was performed with modified Hodge test (MHT) and EDTA disk synergy (EDS) test. PCR was used for detecting the presence of NDM-1 gene. From 200 isolates, 93 isolates produce ESBL enzymes. Overall, 97 isolates were resistant to ceftazidime, and 38 isolates resistant to meropenem. The results of the MHT and EDS positive tests were 41 and 16 isolates, respectively. NDM-1 was not found in any of the patients. The prevalence of E. coli isolates producing both ESBLs and MBLs enzymes, is a serious threat to clinical infections. Accordingly, for the control and treatment of these strains, rapid and accurate identification can be greatly helpful. Keywords: Escherichia coli, MIC, ESBL, NDM-1 1. Introduction One of the most important infections that affect parts of the urinary tract is urinary tract infection (UTI). Bacteria are the main reason for this infection. The most common bacterial cause for UTI is Escherichia coli. UTIs report about 150 million cases each year and are more common in women than men [1-3]. Up to 10% of women have a UTI in a given year, * Corresponding author: Dr. Behnam Zamanzad, MD, Ph.D Department of Microbiology and Immunology, School of Medicine, Shahrekord University of Medical Sciences, Shahrekord, Iran Tel/Fax: +98 38 33346732; +98 38 3334911 Email: bzamanzad22@gmail.com http://orcid.org/0000-0001-8348-7827 Received: November, 5, 2020 Accepted: November, 30, 2020 and half of them have at minimum one infection at some point in their lifetime [4]. There is a rising worry for multidrug-resistant bacteria that produce extended-spectrum β-lactamases (ESBLs). E. coli becomes progressively resistant to expanded- spectrum cephalosporins by the production of ESBLs [5,6]. Class D β-lactamases or oxacillinases (OXA) are commonly distributed between clinically relate Gram- https://jcbior.com/ https://en.wikipedia.org/wiki/Escherichia_coli Kakian et al. 2 negative bacteria [7,8]. Carbapenem antibiotics are now used to treat infections caused by β-lactam resistant bacteria [2,9]. This antibiotic is used to treat infections caused by penicillin or cephalosporin- resistant Gram-negative bacilli because this antibiotic, in addition to a broad spectrum of action, has acceptable stability against most β-lactamase [2,10,11]. Resistance to carbapenem is mainly created with Metallo-β-lactamases (MBL) such as NDM (New Delhi Metallo-β-lactamase), a class B type of β- lactamases that know bivalent metal ions [12,13]. Recently, a new MBL named NDM-1 appeared in E. coli [14,15]. This gene causes resistance to penicillins, cephalosporins, and carbapenems [16]. This study aimed to determine ESBL, and MBL production ability and determination of the frequency of NDM gene in E. coli strains isolated from UTI in women. 2. Materials and Methods 2.1 Bacterial strain collection and identification In this descriptive-analytical study, 200 E. coli isolates with urinary tract infection were collected from women with UTI in two groups of inpatients (n=100) and outpatients (n=100) in teaching hospitals from Shahrekord, Iran from 2016 to 2017. In this study, patients who were diagnosed with UTI 48-72 hours after hospitalization were considered as inpatient samples. Patients who have not been hospitalized in the past month were also used as outpatient samples. Isolates were detected using standard tests including Gram stain, microscopy analysis and culture in EMB media, MacConkey agar, and Blood agar (Merck, Germany), and other biochemical tests. 2.2 Determination of MIC and ESBL production The minimum inhibitory concentration (MIC) test was done by the Epsilon test (E-test) of Liofilchem Company (Italy) with a concentration range of 0.002- 32 μg/mL to meropenem and ceftazidime. Evaluation and determination of inhibition zones were performed after culturing and incubating the plates at 37 °C overnight, using the CLSI guidelines. The phenotypic combined disc method was also used to determine ESBL enzymes [8]. E. coli ATCC 25922 and Klebsiella pneumonia ATCC 1705 were considered as negative and positive controls. 2.3 MHT and EDS test In this study modified Hodge test (MHT) and EDTA disk synergy test (EDS) were performed to detect MBLs enzymes. Further, E. coli ATCC 25922 and Klebsiella pneumonia ATCC1705 were considered as negative and positive controls for running MHT, respectively [17,18]. 2.4 DNA extraction and PCR At this stage, after culturing samples on media, DNA was extracted using the boiling method [8]. The Single-PCR technique was also used to detect NDM-1 and 16s rRNA genes. Primers for this study are shown in Table 1 [19-21]. The presence of the 16srRNA gene (internal gene for E. coli) was investigated to approve the phenotypic tests for E. coli detection. 2.5 Statistical analysis The statistical analysis was performed using SPSS, version 23.0 (SPSS IBM, NY, USA). The results are presented as descriptive statistics in terms of relative frequency 3. Results In this study, the age range of patients was 2-88 years. The maximum and minimum prevalence of isolates were allocated to the internal wards and intensive care units (ICU) respectively. In this study, the result of resistance to ceftazidime was 73% for inpatients and 25% for outpatients. Resistance to meropenem was 28% for inpatients and 10% for outpatients. From 100 isolates from inpatients, 70 isolates (70%), and from 100 isolates from outpatients, 23 (23%) isolates produce ESBLs. The results of the MHT and EDS tests were positive in 21% and 20% of inpatient samples and 8% and 8% of outpatient samples, respectively. After the electrophoresis of PCR products, NDM-1 gene was not found in any of the PCR. 4. Discussion Table 1. Primers used in this study Target Primer sequence (5' to 3') Size (bp) NDM-F GGTTTGGCGATCTGGTTTTC 624 bp NDM-R CGGAATGGCTCATCACGATC 16s rRNA-F AGGCCTTCGGGTTGTAAAGT 420 bp 16s rRNA-R ACCTCCAAGTCGACATCGTT Kakian et al. 3 Resistant E. coli is considered a health- threatening pathogen and causes many diseases in humans, especially those with underlying diseases and immune deficiencies. The infections owing to E. coli are treated with broad-spectrum antibiotics [22-24]. Antibiotic resistance in these bacteria is increasing and is spreading all over the world [2]. Carbapenem and β- lactam antibiotics are drugs of choice for E. coli infections. It is very valuable to early detection of ESBL and MBL in bacteria to control and prevent the spread of resistance strain in society and hospitals. These results and information can lead to a proper prognosis and selection of the appropriate medication by the physician [25,26]. We observed that 93 isolates produce ESBL enzymes. Also, 97 isolates resistance to ceftazidime and 38 isolates resistance to meropenem. NDM gene was not funded in the patients. In a similar study, in Pakistan, it was observed that among 116 E. coli isolates from UTIs, 98 isolates were resistant to meropenem and imipenem and a total of 66 isolates were found to be ESBL producers [27]. A study in India showed that imipenem and ceftazidime resistance in clinical isolates of E. coli were 9%, and 62.1%, respectively. They also showed 61.1% of E. coli isolates were ESBLs [28]. Jamil et al. from Pakistan reported that among 25 carbapenem-resistant E. coli, 16% contained MBL by phenotypic test [29]. In another study by Qamar et al. of 100 E. coli isolates, 81% were MBL producers and 18.4% were NDM-1 positive [30]. In a similar study by Koraei et al. from southwest of Iran, in 376 E. coli isolates, NDM-1gene was not found in any isolates [31]. In this study, we had some limitations. The main limitations were the study of two medical centers and the small sample size. The emergence and spread of antibiotic resistance by ESBL and MBL producer isolates have been a global problem that indicates the excessive use of antibiotics. These types of antibiotic resistance are a serious problem in clinical therapeutic selections in the hospital as well as in the community. So, it is necessary to report ESBL/MBL production along with the daily sensitivity reporting, which will help the clinicians in determining correct antibiotics. Acknowledgments We thanks all the people and Staffs who assisted us in conducting this work. Author Contributions FK, AG, BZ: design of study; FK, AG, BZ, MHR: acquisition of data; FA, AR, MM and JF: evaluation of data, preparation of the manuscript; FK, BZ, AG and JF: assessment of data. All authors have read and approved the manuscript. Conflict of Interests We have not competing interests. Ethical declarations The study protocol was obtained by the ethics code of IR.SKUMS.REC.1395.215 from Shahrekord University of Medical Sciences. However, we only used leftovers from clinical specimens. Financial Support This research was supported by the Shahrekord University of Medical Sciences, Shahrekord, Iran (Grant No. 2540). References 1. Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ. 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