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ANTIBIOTIC SUSCEPTIBILITY PATTERN OF ESCHERICHIA COLI ISOLATED FROM CHILDREN WITH URINARY TRACT INFECTION

Objective: This work aimed to detect the antibiotic susceptibility pattern of Escherichia coli isolated from children, as it is the most predominant pathogen of urinary tract infection (UTI). 
Methods: About 530 urine samples were collected and tested using the modified Kirby–Bauer disk diffusion method to find the susceptibility pattern of isolated bacteria. 
Results: Out of a total of 530 samples, 114 (21.50%) showed significant growth. A total of 8 different types of bacteria were isolated from the growth of positive samples. Among the isolates, E. coli 66 (57.8%) was found to be the most predominant organism followed by Klebsiella pneumoniae 18(15.8%), Proteus spp. 10 (8.8%), Staphylococcus aureus 8 (7.0%), Acinetobacter spp. 4 (3.5%), CoNS 4 (3.5%), Enterobacter spp. 2 (1.8%), and Pseudomonas aeruginosa 2 (1.8%). In the present study, out of 66 E. coli, 37 (56.1%) were multidrug-resistant strain. E. coli showed 94.0% resistance to ceftriaxone followed by ceftazidime 86.5% and cefotaxime 70.3%. Imipenem (91.9%) followed by amikacin (89.2%) seems to be the effective drug against UTI causing E. coli in children. 
Conclusion: Multidrug resistance may possess difficulties with the choice of therapeutic options for the treatment of severe infections.


INTRODUCTION
The urinary tract infection (UTI) is the colonization of a pathogen occurring anywhere in the urinary tract (kidney, ureter, bladder, and urethra). UTI is a common condition in children. Approximately 1 in 10 girls and 1 in 30 boys will have a UTI by the age of 16 years [1,2]. During UTI, multiplication of the organism takes place in the urinary tract and there is the presence of more than a hundred thousand organisms in a 1-mL urine sample [3]. The most common pathogen is Escherichia coli, accounting for approximately 85 % of UTIs in children. Renal parenchymal defects are present in 3-15 % of children within 1-2 years of their first diagnosed UTI [4]. Around 90% of UTIs were caused by E. coli [5].
The most common symptoms of UTI are dysuria, frequency, and urgency in urination, suprapubic pain, and possible hematuria. The urine may have an unpleasant odor and appear cloudy. When UTI will persist in children for more than a week with systemic symptoms of persistent fever, chills, nausea, and vomiting [6]. Antibiotics susceptibility test measures the ability of an antibiotic or another antimicrobial agent to inhibit the growth of microorganisms [7]. The choice of use of antibiotics and the duration of treatment depend on the history of the patient and the bacterial agent identified. For uncomplicated UTI disease is cured with 1 or 2 days of treatment and to ensure that the infection is cured antibiotics should be taken within a week or 2 weeks [8]. The objective of this research is to identify the incidence of E. coli causing UTI in children and determine the antibiotic susceptibility pattern of the isolates.

Sample collection and transport
The urine samples were collected by suprapubic bladder aspiration and midstream urine sample methods. In the suprapubic bladder aspiration method, urine was withdrawn directly into a syringe through a percutaneously inserted needle, thereby ensuring contamination-free specimen [9]. The midstream urine samples were collected from children who are toilet trained. Children are instructed to clean the periurethral area and were asked to pass urine at first by allowing and losing some urine and a midstream urine sample was collected in a sterile container. The collected urine sample was transported as soon as possible and 1.8% boric acid was added in case of delay.

Sample processing
The urine sample was processed by macroscopic and microscopic examination. The macroscopic examination observed the color and turbidity. In a microscopic examination, 5-10 mL of urine sample was centrifuged at 3000 rpm for 10 min and the sediment was observed under a microscope [10].

Culture of the urine sample
The culture of urine specimens was done on 5% blood agar (BA), MacConkey agar using a semiquantitative culture method [10]. About 0.001 mL of urine sample was streaked in culture media using a sterile inoculating loop and incubated in an inverted position at 37°C for 24 h. The photograph of a pure culture of E. coli in BA was shown in Fig. 1.

Identification of isolates
Identification of major isolates was done using standard microbiological techniques as described in the Bergey's manual that involves the morphological appearance of colonies, staining reactions, and biochemical properties such as Gram's staining, catalase, oxidase, coagulase, indole, methyl red, Voges-Proskauer, citrate utilization, triple sugar iron, urease, and sulfide indole motility tests.

Antibiotics susceptibility test
The antimicrobial susceptibility test of isolates was done by modified Kirby-Bauer disk diffusion method as recommended by the Clinical and Laboratory Standards Institute [11] Wayne, USA, using Muller-Hinton Agar (MHA). The isolated colonies were transferred to the nutrient broth and were incubated for 4 h until the turbidity of bacterial growth was similar to that of 0.5 McFarland standards. The sterile cotton swab was dipped into the tube containing culture and inoculated over dried MHA by carpet culture technique. About six antimicrobial disks each 6 mm in diameter were placed on the inoculated plates on a 90 mm diameter plate. The plates were left at room temperature for the diffusion of antibiotics from the disk. It was incubated at 37°C for 18 h and the plates were examined to ensure confluent growth and the diameter of each zone of inhibition in mm was measured [8].

Multidrug-resistant (MDR) analysis
The multidrug-resistant strain was evaluated using the Kirby-Bauer disk diffusion technique [10]. Those isolates which showed resistance to 3 or more than 3 groups of antibiotics were considered multidrugresistant [12]. Different types of drug in E. coli are shown in Fig. 2.

Quality control
The standard protocol was followed to maintain the quality of each test. All the agar plates were incubated at 37°C for 24 h before use to examine for any contamination that occurred during media preparation and storage. The antibiotic susceptibility test of the isolates was standardized using control strains preserved in the hospital.

Purity plate and statistical analysis
The purity plate was used to ensure the inoculation for the biochemical tests is pure culture and also to confirm whether the biochemical tests performed are in an aseptic condition or not. Before and after performing biochemical tests, the same inoculum was subcultured in nutrient agar media and incubated. It was observed for the appearance of pure growth of organisms. The measurements were statistically analyzed using Statistical Package for the Social Science version 20 software packages. Scheme 1 represents the flow sheet for the urine samples for the detection of UTI.

RESULTS AND DISCUSSION
Five hundred thirty urine samples were collected from the suspected UTI patients at International Friendship Children Hospital spanning between September 2015 and April 2016 to detect the incidence and antibiotic susceptibility pattern in E. coli.

Distribution of bacterial isolates in total processed samples
Out of the total 530 samples, 114 (21.50%) showed significant growth. A total of 8 different types of bacteria were isolated from positive samples.

Distribution frequency of E. coli in different hospital departments
The distribution of E. coli in different hospital departments is presented in Table 3. Among the total of 530 urine samples, 375 (70.8%) and 155 (29.2%) samples were from outdoor and indoor patients, respectively. Culture positive cases for E. coli for indoor and outdoor patients were 32 (48.5%) and 34 (51.5%), respectively.

Antibiotic susceptibility pattern of E. coli in first-and second-line drugs in children
Among the seven first-line drugs used in patients, E. coli was found to be resistant against ceftriaxone (94.0%) followed by gentamicin (66.7%), ampicillin (65.2%), and cotrimoxazole (63.6%), respectively. While nitrofurantoin was found more sensitive among first-line drugs with (77.3%) sensitivity followed by norfloxacin (68.2%) and ciprofloxacin (54.5%), respectively, as shown in Table 4.

MDR pattern in E. coli isolates
Most of the isolated E. coli pathogens showed resistance to more than 3 groups of antibiotics. It was found that 19.6% were sensitive to all antibiotics used in the study and 15.2% were resistant to 1 drug, 9.1%, 13.6%, and 42.4% isolates were resistant to 2, 3, and >3 drugs, respectively. Total MDR was found to be 37 (56.1%) out of 66 isolated samples which are shown in Table 5. Table 6 shows the distribution pattern of MDR E. coli in male and female children of different age groups. From observations, it was found that 37 (56.10%) were found to be MDR E. coli out of a total of 66 E. coli    Table 1. This result is also in harmony with the study done at international context [13] 84%, [14] 68.2%, [15] 80.0%, [16] 52.1%, [17] 76.7%, and [18] 49.3% [19], also found the predominance of Gram-negative in urine samples [20], states Gram-negative bacteria constituted the major group of isolates comprising 80.0%. The study revealed the colonic bacterial flora of the Enterobacteriaceae family is recognized to produce high UTIs, through the main pathway of canaliculi [21] in children.

Different age and gender-wise distribution of MDR in E. coli
Among the 114 (21.5%) growth positive samples in children, 33 (20.1%) were from males and 131 (79.9%) were from females. The male to female infection ratio of 1:4 was diagnosed to have UTI.
Research studies performed by Akram et al. [22], also found the male to female ratio of UTI as 1:2.9 and [23] as 1:2. Many research experiments found that UTI cases in male children are higher than that of female but still the UTI cases are found more severe in female children in Nepal.
In the present study, a total of 530 suspected urinary tract infected sample were processed, among them, 375 (70.8%) samples were from outdoor patients while 155 (29.2%) accounted for the indoor patient resulting high incidence of UTI among outpatient 51.5% than inpatient 48.5% as presented in Table 3. Similar findings were observed by the study done by Jesse et al. [24]. The proximity of the anus and the short urethral opening in females along with their hygienic habits are highly prone to UTIs [25].
Among the Gram-negative bacteria isolated, E. coli showed the highest percent of resistance toward first-line antibiotics, that is, ceftriaxone 94.0% and ceftazidime 86.5% as in Table 4. The highest percent of resistance toward first-line antibiotics was found for ceftriaxone (94.0%) followed by gentamicin (66.7%), and ampicillin (65.2%), respectively. According to Sharma et al. [26], E. coli is highly resistant to ampicillin (71.2%) in infants which are also similar to this study. Nitrofurantoin has shown an overall low resistance rate in both out-      patients and hospitalized, which can be considered as the first-line therapy.
The highest percent of resistance toward second-line antibiotics was found for ceftazidime (86.5%) followed by cefotaxime (70.3%) and ofloxacin (51.4%), respectively, shown in Table 5. This finding is in agreement with the study conducted by Acharya et al. [27]. Imipenem was found to be the most effective drug against E. coli with susceptibility of 91.9% followed by amikacin with susceptibility of 89.2%. This finding was similar to the study conducted by Noor et al. [28]. The antimicrobial sensitivity pattern differs in different studies as well as different times even in the same hospital, because of the emergency of resistance strain as a result of indiscriminate use of antibiotics [29].
In this study, out of 66 positive isolates of E. coli, 37 (56.1%) were found to be MDR for E. coli. The high level of MDR observed in E. coli is due mediated by β-lactamase, which hydrolyze the β-lactam ring inactivating the antibiotics. The classical Team-1, Team-2, and SHV-1 enzymes are the predominant plasmid-mediated β-lactamase of Gram-negative rods [30].

CONCLUSION
The high prevalence rate of UTI in our study concludes E. coli as the predominant pathogen in children. The study showed that E. coli 66 (57.8%) was the major organism in UTI followed by K. pneumoniae 18 (15.8%) while other organisms were less dominant. Imipenem and Amikacin can be used as drugs (second line) of choice for multi-drug resistant (MDR) for E. coli. However, nitrofurantoin (first-line drug) can be used to treat UTI caused by E. coli. The high prevalence rate of UTI in our study concludes E. coli as the predominant pathogen in children.
The finding of the study also demonstrated the fact that females were more susceptible to UTI than males may be due to anatomical proximity of the urinary system in females including sanitation and menstruation.