Molecular Characterization of Multi Drug Resistance Escherichia coli isolated among Diabetes Mellitus Patients in Dongla State, Sudan

Background: Urinary tract infections (UTIs) caused by Escherichia coli have become a significant worldwide public health concern and is a common infectious disease in which level of antimicrobial resistance are alarming worldwide. Methods: Urine samples were collected form Diabetic patients clinically diagnosed by having UTI, during the period from November 2019 to April 2020 at diabetic center in Dongla, Antibiotic susceptibility tests were performed by Kirby-Bauer disc diffusion and B-lactamases genes were detected used PCR. Results: A total of 120 E. coli were Isolated from DM with UTI. All the isolated were shown to be resistant to Cefpodoxime (100%). The most efficient antibiotics were Colistin and imipenem (99.2% and 88.3 respectively as susceptibility rate) followed by Gentamycin (70%). High resistance rates were observed with ofloxacin (90.8%), Ciprofloxacin (77.5%), Amikacin (60.8%), ceftriaxone (58.3%) and Cefepime (51.7%). The most B-lactamase genes isolates were blaCTX-M -1 gene (64%) followed by blaCMY-G2 (55.8%), blaSHV gene (34.2%), blaNDM gene (10.8%), blaOXA-48 gene (5.8%), blaVIM gene (3.3%), blaKPC gene (2.5%) and blaIMP were not detected in any of the tested isolates. Conclusions: Urinary tract infection due to E. coli may be difficult to treat empirically due to high resistance to commonly used antibiotics. Continuous surveillance of multidrug resistant organisms and patterns of drug resistance are needed to prevent treatment failure and reduce selective pressure.

in both DM and non-DM patients [11]. Uropathogenic E. coli (UPEC) possesses a variety of pathogenicity determinants that make colonization of the urinary tract possible. These determinants include fimbrial (P, S/F1C and type 1 fimbriae) and non-fimbrial adhesins that mediate bacterial adherence to epithelial cells, siderophores (iron-acquisition systems), secreted toxins (haemolysin and cytotoxic necrotizing factor 1) and capsule forming polysaccharides for immune evasion [13][14][15]. Phylogenetic analysis classifies E. coli strains into four main groups (A, B1, B2 and D). Groups B2 and D are mainly associated with E. coli strains causing extraintestinal infections, whilst groups A and B1 are associated with commensal strains [16]. Worldwide, there is great concern due to the high rates of resistance to antimicrobials used in the treatment of infections caused by E. coli, particularly in developing countries. Frequent prescription of antibiotics, including the ones with broad-spectrum, may result in development of antibiotic-resistant urinary pathogens. Since patients with DM are more prone to have resistant pathogens, they inevitably require longer and more potent antimicrobial treatment [17]. Therefore, improved control of glycaemia in diabetics may help in controlling the UTIs. Accurate screening for UTI in diabetic patients is also critical to enable the appropriate treatment, avoiding related complications. In this study we aimed to assess the prevalence of E. coli and pattern of the antimicrobial drugs susceptibility by phenotyping and genotyping method.

Study design
A cross-sectional study was carrying out during the period from August 2019 to April 2020 at diabetic center in Dongla and Neelain university. The study includes patients clinically diagnosed by having one or more of the following symptoms: dysuria, frequency, urgency, suprapubic discomfort, or flank pain. Non-diabetic and pregnancy were excluded from the study. A total of 120 E. coli isolated form Diabetic patients (45 males and 75 females) with age group ranged from 10 to 80 years old. All patients were informed of the purpose of the study and their consent or that of their care provider was obtained before urine samples were collected.

Sample collection and Processing
Each patient was asked to collect approximately 10-20 ml of midstream urine into a sterile urine container. after giving proper instructions to avoid contamination and samples were processed in the laboratory within 2 hours of collection. None of the patients admitted to consuming antibiotics during the 2 weeks prior to urine sample collection.

Data collection
A structured questionnaire and referring to the patient clinical sheet were being used; demographic data and other Data (clinical symptoms, previous antibiotic, duration of antibiotic used). verbal consent was obtained from each patient enrolled in this study.

Antimicrobial susceptibility testing
Antimicrobial sensitivity testing of all isolates was performed on diagnostic sensitivity test plates according to the Kirby-Bauer method [20] following the definition of the Committee of Clinical Laboratory International Standards [21]. Bacterial inoculums were prepared by suspending the freshly grown bacteria in 5mL sterile saline. A sterile cotton swab was used to streak the surface of Mueller Hinton agar plates. Filter paper disks containing a designated concentration of the antimicrobial drugs were obtained from Hi-Media Laboratories in the following concentrations: Amikacin Meropenem (10μg) ciprofloxacin (5μg), ofloxacin (5μg), colistin (10μg), Cefepime (30μg). The diameters of zone of inhibition were interpreted according to CLSI standards. Media and disks were tested for quality control with E. coli standard strain.

Molecular detection
DNA for molecular detection was extracted after bacterial lysis according to the extraction protocol prepared by the Community Reference Laboratory for Antimicrobial Resistance (CRL, 2009).
Briefly, a few colonies taken from fresh culture medium and transferred to phosphate buffered saline (pH 7.3). The suspension was then heated at 100°C for 15 minutes. Boiled suspension was transferred directly on ice, this was followed by vortexing and the suspension was then centrifuged at 12000 rpm for 5 minutes to sediment the debris, the clear supernatant was used as template DNA in PCR method.
The universal primers were designed for b-lactamase genes, including ESBL genes ( bla SHV and bla CTX-M-1), AmpC genes ( bla C-MY-G2), carbapenemases genes ( bla KPC, bla IMP, bla VIM, bla NDM and bla-OXA-48). Details for the primers were shown in (Table 1). PCR was performed and the test was carried out in total volume of 25 µl, con- 5 µl of the PCR product was analyzed using 1% or 1.5% Agarose gel electrophoresis and stained with 0.15% Ethidium bromide and the product was visualized using UV gel documentation [22,23].

Results
A total of 120 isolates were identified as E. coli from DM with UTI (45 males and 75 females, 6.7% had type 1 and 93.3% had type II DM with age group ranged from 10 to 80 years old), by routine phenotypic methods including Gram's staining, colony morphology and standard conventional biochemical tests. About 25 % and 35% of the UTI positive patients were in age between 40-50 and 50-60 years, respectively (Table 2). This result indicated that the emergence of UTI raised with the increase in age of the patient.
E. coli strains showed differences in susceptibility and resistance patterns to the antimicrobials tested. All of the isolated uropathogenic E. coli were shown to be resistant to Cefpodoxime (100%).

Discussion
Urinary tract infections (UTIs) are the most common human infectious disease affecting the bladder, kidneys and urinary tracts [24]. Kidney stones, diabetes, weak immune system can increase the risk of UTIs [25]. Patients with significant bacteriuria have at least two symptoms referable to the urinary tract infection (dysuria, urgency, frequency, incontinence, suprapubic pain, flank pain or costovertebral angle tenderness, fever (temp ≥ 38°C) and chills are said to be symptomatic. Complications of UTI include urosepsis, renal impairment, and renal abscess [26,27]. In the present study all urine sample (100%) revealed significant growth. the most isolated agents from urinary tract infections vary, almost all of them are caused by single microorganism type. In this study the most frequently isolated microorganism was E. coli with a rate of (75%).
Uropathogenic Escherichia coli (UPEC), is one of the main causes of community (80-90%) and nosocomial acquired UTIs (30-50%) [34][35][36][37]. Also, our results reveal higher prevalence of urinary tract infections in female patients than in male [38][39][40]. this mainly due to short urethra, absence of prostatic secretion, pregnancy, and easy contamination of the urinary tract with fecal flora [41]. The emergence of high rates of antibiotic resistance and MDR-pheno-type from urinary tract infections related bacteria becomes a public health concern worldwide. Our results findings that E. coli isolated were showed the following resistance rates; Cefpodoxime (100%), In recent years, multidrug resistant caused by ESBLs are reported to be associated with higher morbidity and mortality rates [48].
The proportion of the ESBL positive cases was highest, followed by AmpC-producing stains, and carbapenemases-producing stains.
ESBLs are mainly mediated by plasmid, while AmpCs are mainly mediated by chromosome. CTX-M types are the major phenotypes of domestic ESBLs, Widespread dissemination of these genes has been described in Africa and elsewhere, followed by SHV type [49].

Conclusions
In this study, a high prevalence of resistance to β-lactams as well as to other antimicrobials were observed in E. coli isolates from DM patients with UTI. The study showed the importance of continuous monitoring programming of multidrug resistance in our hospitals.
It also showed the need for developing attempts to decrease the