1. Junior Consultant Paediatrics, District Hospital, Pirojpur, Bangladesh.
2. Former Professor & HoD of Paediatric Nephrology, Sir Salimullah Medical College, Dhaka, Bangladesh.

* Correspondence:  shawon.sb3@gmail.com

Keywords: Extended-Spectrum Beta-Lactamases, Urinary Tract Infections, children, antibiotic resistance

1. INTRODUCTION

Urinary tract infections (UTI) occurs when microorganisms invade and multiply in the urinary tract. Anatomically, UTIs are upper or lower tract infections. Upper tract UTI causes kidney and ureter inflammation (pyelonephritis). This causes abdominal pain, loin tenderness, fever, anorexia, vomiting, lethargy, and malaise [1]. Lower tract UTI causes bladder (cystitis) and urethra infection, causing lower abdominal or suprapubic pain, dysuria, urine frequency, and urgency [2]. Older children may have infection-related symptoms, younger patients often lack these typical indications, making upper and lower UTI difficult to distinguish [3]. The third most prevalent illness in children is UTI, behind respiratory and gastrointestinal infections [4]. Feverish newborns, ill children, and older children with urinary symptoms account for 6%–8% of UTI cases. Peak prevalence occurs in newborns, toddlers, and adolescents [5]. UTI is more likely in female and uncircumcised male babies due to bacterial skin flora concentration under the nappy, shorter female urethral distance, and foreskin surface area [6]. Toddler toilet training can cause volitional holding and bladder stasis, causing UTIs. When sexual activity upsets bacteria at the urethral entrance, prevalence rises in adolescent girls [7].  Due to poor sanitation, living methods, undernourishment, and environmental conditions, this disease is more prevalent in underdeveloped nations. Enterobacterales (previously called Enterobacteriaceae) are Gram-negative, non-spore-forming, facultative anaerobic bacilli that cause > 80% of UTIs [8]. Escherichia coli is the most common, Klebsiella, Enterobacter, and Proteus spp as well [9]. Other Gram-negative microbes like Pseudomonas aeruginosa can cause UTIs rarely. Enterococcus spp. (patients with a urinary catheter, urinary tract instrumentation, or anatomical abnormality), Staphylococcus saprophyticus (sexually active adolescents), and Streptococcus Group B (neonates) are Gram-positive bacterial pathogens [10]. Children rarely get UTIs from viruses or fungi, save for Candida species in premature neonates. Patients with structural defects may have several organisms [11].  ESBLs, plasmid-mediated β-lactamases, hydrolyze extended-spectrum Cephalosporins (Cefotaxime, Ceftriaxone, and Ceftazidime) and Oxyimino-monobactam (Aztreonam), but not Cephamycins (Cefoxitin and Cefotetan) or Carbapenems (Meropenem and Imipenem). ESBLs transmit resistance to antibiotics and related Oxyimino-β lactams.  Clavulanic acid, Sulbactam, and Tazobactam are ‘classical’ β-lactamase inhibitors that inhibit these enzymes [12]. ESBLs provide Gram-negative bacteria resistance by preventing β-lactams from reaching penicillin binding proteins (PBPs), decreasing affinity for PBPs, and destroying the antibiotic through β-lactamases [13]. The most prevalent resistance mechanism against β-lactam antibiotics is beta-lactamase enzyme synthesis. Because bacteria share mobile genetic components, ESBL enzymes have spread. Clonal development of ESBL-producing bacteria, such as E. coli ST131, which is resistant to quinolones, has caused global epidemics [14]. E. coli and Klebsiella spp. produce ESBLs, but Enterobacter, Proteus, Citrobacter, Morganella, Providencia, Salmonella, and Serratia can also cause dozens of infections. ESBL-related infections are concerning for various reasons. First, multidrug resistance makes them hard to treat (Jacoby, 1997). Second, ESBL infections may delay treatment [15]. Third, ESBL infections cause longer hospital stays and higher expenses, fourth, existing identification approaches may underestimate these organisms’ prevalence [16]. Finally, ESBL infections increase the likelihood of clinical failure and death in adults and children. Poor medication regulatory and control systems in many countries have led to antibiotic misuse and overuse in humans and animals [17]. These activities promote the spread of resistant bacterial strains into the community and clinic, lowering treatment outcomes. When infection prevention and management are lacking, drug-resistant pathogenic bacterial strains can spread anywhere [18]. Inappropriate prescribing, patient noncompliance with duration, dose, and frequency of antibiotics, and antibiotic use in livestock and fish farms are known sources of antibiotic resistance. Antibiotic resistance increases in nations where antibiotics are available without a prescription and self-medication is frequent [19]. In places with heavy antibiotic usage regardless of history, antibiotic resistance is more likely [20]. Patients under 15 and over 45 are more susceptible to ESBL, alarmingly, 25% of newborns and young (0–15 year olds) have UTI. Previous antibiotic exposure, UTI, hospitalization, underlying disease, urinary tract abnormalities, and immunosuppressant drug use have been linked to ESBL-positive UTIs in children [21].

2. MATERIALS AND METHODS

The study follow the cross-sectional analytical study design, study place was department of Pediatrics and Department of Microbiology, Sir Salimullah Medical College Mitford Hospital (SSMCMH), Dhaka. On the basis of inclusion & exclusion criteria, all the patients from 01 month to 12 years of age presented to the pediatric outpatient department or admitted to pediatric inpatient ward with clinical feature(s) of UTI (fever, abdominal pain, loin pain, dysuria, frequency, urgency etc.) within the defined period were included. Data has been collected with the help of purposive sampling technique. Sample size was calculated by following formula:

           

n = the required sample size

p₁ = anticipated probability of exposure among cases

p₂ = anticipated probability of exposure among control

p = (p₁ + p₂) ÷ 2

Z = z- value of SND at a given level of significance

Zβ = z- value of SND at a given power

Here,

                p₁ = 79

p₂ = 52.2

p = (79 + 52.2) ÷ 2

= 65.6

Z = 1.96

Z𝛽 = 1.28

So calculated sample size:

n = 64

 A number of 64 in each group so the total sample (64+64) = 128. Data were collected by interview of the parents or guardians, clinical examination and laboratory investigations using the research instrument. Sociodemographic data including age (years), sex and clinical history of participants such as previous use of antibiotic, previous hospitalization, prior UTI, use of immunosuppressant drug within the past 3 months and associated diseases were recorded. All patients presented to the pediatric outpatient department or admitted to pediatric inpatient ward, SSMCMH, fulfilling the inclusion criteria were enrolled for the study. Glans penis and foreskin in male, while perineum including labia minora and majora in female were cleaned using tap water at room temperature prior to obtaining the urine sample. Freshly voided early morning mid-stream urine (MSU) from children 2 years and older and clean catch or sterile, adhesive urine bag specimen from children younger than 2 years were collected into properly labelled, dry, clean, sterile, transparent, screw-capped, wide-mouth, leak-proof plastic containers. After labeling the container, samples were transported immediately to the Clinical pathology and Microbiology laboratory of SSMC with a requisition form for routine examination and culture & antibiotic susceptibility test. The samples were analyzed and processed according to the standard protocol within 2 hours of collection. Using a calibrated wire loop of loop diameter 4mm, l0 μl of uncentrifuged urine were inoculated into 5% Blood agar and MacConkey agar media (Oxoid Ltd. England). Semi quantitative streaking method was used for quantification of bacterial load in urine. The inoculated plates were incubated at 37⁰C aerobically, after overnight incubation, plates were examined for growth and colony forming units (cfu) per ml were calculated. A specimen was considered positive for UTI if a single organism was cultured at a concentration of >10⁵ cfu/ ml. Pure isolates of bacterial pathogen were preliminarily characterized by their colony morphology and Gram-staining. All positive urine cultures showing significant bacteriuria were further identified by their characteristic appearance on their respective media and confirmed by the pattern of biochemical reaction using the standard procedures. Isolated bacteria were E. coli, Pseudomonas, Klebsiella, Acinetobacter, Providencia, Morganella, Serratia, Citrobacter and Proteus. A single colony represents one organism, if an inoculum of 0.01 ml produces 20 colonies, the number of organisms represents in 0.01 ml of urine is 20. So one ml urine contains 2 x 10³ organisms. A count of 1 x 10⁵ or more bacteria per ml of urine were considered as clinically significant [22]. Susceptibility to antimicrobial agents of all isolates were done by Kirby-Bauer modified disk-diffusion technique. Three to five isolated colonies of the organisms to be tested were picked from the pure culture plates by a sterile wire loop and suspended in 5 ml of nutrient broth in a screw capped test tube and mixed gently until it forms a homogenous suspension. In a good light the turbidity of suspension was matched to 0.5 McFarland standards in order to standardize the inoculums density. A sterile cotton swab was dipped into the suspension and the excess were removed by gentle rotation of the swab against the surface of the tube. The swab was then used to distribute the bacteria suspension evenly over the entire surface of Mueller-Hinton agar (Oxoid Ltd. England). The inoculated plates were left on the flat surface at room temperature to dry for 10-15 minutes. Then the antibiotic discs were placed on the inoculated plates. The antibiotics for disk diffusion testing were obtained from Oxoid that include: Amoxicillin (20 µg) + Clavulanic acid (10 µg), Ceftriaxone (30 µg), Ceftazidime (30 µg), Cefotaxime (30 µg), Aztreonam (10 µg), Cefixime (30 µg), Doxycycline (30 µg), Imipenem (10 µg), Meropenem (10 µg), Co-trimoxazole (23.75/1.25 µg), Ciprofloxacin (30 µg), Levofloxacin (5 µg), Gentamicin (10 µg), Amikacin (10 µg), Nitrofurantoin (300μg), Chloramphenicol (30 µg), Tetracycline (30 µg), Nalidixic acid (30 µg) were used to see the sensitivity patterns of the organism. The plates were then incubated at 37⁰C for 24 hours and reading was taken using a ruler on the underside of the plate measured the diameter of each zone of inhibition in mm. Zone of inhibition produced by each was considered into susceptibility categories namely Susceptible (S), Intermediate (I), and Resistant (R) by CLSI (2015). All the gram-negative isolates were tested for detection of ESBL by Double-Disc Synergy Test (DDST). Antimicrobial discs (Oxoid Ltd. England) Ceftazidime (CAZ) 30 μg, Cefotaxime (CTX) 30 μg, Ceftriaxone (CRO) 30 μg were used. Mueller Hinton agar plates were prepared and inoculated with standardized inoculums of the organism with sterile cotton swab. Disc containing 20 μg Amoxicillin and 10 μg Clavulanic acid was placed in the center of the inoculated plate. Third generation Cephalosporin disc of Ceftazidime, Ceftriaxone and Cefotaxime were placed about 20 mm distant from Amoxicillin-Clavulanate disc. The plate was incubated overnight at 37ºC. Extension of the inhibition zone of Ceftazidime, Ceftriaxone and Cefotaxime disc on the side exposed to the disc containing Amoxicillin and Clavulanic acid was considered positive for ESBLs. Standard strain of Klebsiella pneumoniae ATCC 700603 was used as ESBL-positive control and E. coli ATCC 25922 was used as ESBL-negative control. All Statistical analyses were done using Statistical Package for Social Science (SPSS) software version 27.0.

3. RESULTS AND DISCUSSION

Data were expressed as frequency and percentage and mean ±SD unpaired student t-test and Chi-square test was done ns = not significant Majority of the patients were age ranges between 1-5 years in both group (p=0.854). There was male predominance in ESBL-UTI group (p=0.596).

Table 01: Age and sex distribution of the patients with ESBL-UTI and non-ESBL-UTI (N=128)

Variables	ESBL (n=64) No. (%)	Non-ESBL (n=64) No. (%)	p-value
Age groups
1 month-12 months 1 year-5 years 6 years-12 years	15(23.4%) 32(50.0%) 17(26.6%)	13(20.3%) 33(51.6%) 18(28.1%)	0.854ns
Mean±SD Range	3.76±3.41 (0.20-12) years	3.87±3.25 (0.10-12.0) years
Sex
Male	34(53.1%)	31(48.4%)	0.596ns
Female	30(46.9%)	33(51.6%)
Male: Female ratio	1.1:1	1:1.1

Figure 01: |/strong>Bar diagram shows distribution of the bacteria in ESBL-UTI and non-ESBL-UTI group.

E.coli was the commonest organism identified in both ESBL-UTI and non-ESBL-UTI group followed by Pseudomonas, Providencia and others.

Table 02: Antibiotic susceptibility pattern of the isolated bacteria in ESBL-UTI group (n=64)

Antibiotics		Organisms
		E.coli (n=53)	Pseudomonas (n=4)	Acinetobacter (n=1)	Providencia (n=2)	Citrobacter (n=2)	Morganella (n=1)	Serratia (n=1)
Amoxyclav	S	3(5.7)	0(0.0)	0(0.0)	0(0.0)	1(50.0)	0(0.0)	0(0.0)
	R	50(94.3)	4(100.0)	1(100.0)	2(100.0)	1(50.0)	1(100.0)	1(100.0)
Amikacin	S	36(67.9)	4(100.0)	1(100.0)	1(50.0)	0(0.0)	0(0.0)	0(0.0)
	R	17(32.1)	0(0.0)	0(0.0)	1(50.0)	2(100.0)	1(100.0)	1(100.0)
Aztreonam	S	9(17.0)	1(25.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)
	R	44(83.0)	3(75.0)	1(100.0)	2(100.0)	2(100.0)	1(100.0)	1(100.0)
Ceftriaxone	S	0(0.0)	0(0.0)	1(100.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)
	R	53(100.0)	4(100.0)	0(0.0)	2(100.0)	2(100.0)	1(100.0)	1(100.0)
Cefixime	S	0(0.0)	0(0.0)	1(100.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)
	R	53(100.0)	4(100.0)	0(0.0)	2(100.0)	2(100.0)	1(100.0)	1(100.0)
Ceftazidime	S	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)
	R	53(100.0)	4(100.0)	1(100.0)	2(100.0)	2(100.0)	1(100.0)	1(100.0)
Cefotaxime	S	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)
	R	53(100.0)	4(100.0)	1(100.0)	2(100.0)	2(100.0)	1(100.0)	1(100.0)
Cephradine	S	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)
	R	53(100.0)	4(100.0)	1(100.0)	2(100.0)	2(100.0)	1(100.0)	1(100.0)
Ciprofloxacin	S	13(24.5)	3(75.0)	1(100.0)	2(100.0)	1(50.0)	0(0.0)	1(100.0)
	R	40(75.5)	1(25.0)	0(0.0)	0(0.0)	1(50.0)	1(100.0)	0(0.0)
Chloramphenicol	S	32(60.4)	3(75.0)	0(0.0)	0(0.0)	1(50.0)	1(100.0)	0(0.0)
	R	21(39.6)	1(25.0)	1(100.0)	2(100.0)	1(50.0)	0(0.0)	1(100.0)
Co-trimoxazole	S	7(13.2)	1(25.0)	0(0.0)	1(50.0)	0(0.0)	0(0.0)	0(0.0)
	R	46(86.8)	3(75.0)	1(100.0)	1(50.0)	2(100.0)	1(100.0)	1(100.0)
Cefuroxime	S	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)	0(0.0)
	R	53(100.0)	4(100.0)	1(100.0)	2(100.0)	2(100.0)	1(100.0)	1(100.0)
Colistin	S	52(98.1)	4(100.0)	1(100.0)	1(50.0)	2(100.0)	1(100.0)	1(100.0)
	R	1(1.9)	0(0.0)	0(0.0)	1(50.0)	0(0.0)	0(0.0)	0(0.0)
Doxycycline	S	29(54.7)	1(25.0)	1(100.0)	0(0.0)	1(50.0)	1(100.0)	1(100.0)
	R	24(45.3)	3(75.0)	0(0.0)	2(100.0)	1(50.0)	0(0.0)	0(0.0)
Gentamicin	S	28(52.8)	3(75.0)	1(100.0)	1(50.0)	0(0.0)	0(0.0)	0(0.0)
	R	25(47.2)	1(25.0)	0(0.0)	1(50.0)	2(100.0)	1(100.0)	1(100.0)
Imipenem	S	45(84.9)	4(100.0)	0(0.0)	2(100.0)	0(0.0)	1(100.0)	1(100.0)
	R	8(15.1)	0(0.0)	1(100.0)	0(0.0)	2(100.0)	0(0.0)	0(0.0)
Levofloxacin	S	17(32.1)	3(75.0)	1(100.0)	2(100.0)	1(50.0)	0(0.0)	1(100.0)
	R	36(67.9)	1(25.0)	0(0.0)	0(0.0)	1(50.0)	1(100.0)	0(0.0)

Table-2 continued

Table 02: Antibiotic susceptibility pattern of the isolated bacteria in ESBL-UTI group (n=64)

Antibiotic		Organisms
		E.coli (n=53)	Pseudomonas (n=4)	Acinetobacter (n=1)	Providencia (n=2)	Citrobacter (n=2)	Morganella (n=1)	Serratia (n=1)
Meropenem	S	41(77.4)	4(100.0)	0(0.0)	2(100.0)	0(0.0)	1(100.0)	1(100.0)
	R	12(22.6)	0(0.0)	1(100.0)	0(0.0)	2(100.0)	0(0.0)	0(0.0)
Nalidixic acid	S	10(18.9)	2(50.0)	0(0.0)	1(50.0)	0(0.0)	0(0.0)	0(0.0)
	R	43(81.1)	2(50.0)	1(100.0)	1(50.0)	2(100.0)	1(100.0)	1(100.0)
Nitrofurantoin	S	31(58.5)	1(25.0)	1(100.0)	0(0.0)	1(50.0)	0(0.0)	1(100.0)
	R	22(41.5)	3(75.0)	0(0.0)	2(100.0)	1(50.0)	1(100.0)	0(0.0)
Pefloxacin	S	21(39.6)	2(50.0)	1(100.0)	2(100.0)	1(50.0)	0(0.0)	1(100.0)
	R	32(60.4)	2(50.0)	0(0.0)	0(0.0)	1(50.0)	1(100.0)	0(0.0)
Tetracycline	S	40(75.5)	3(75.0)	1(100.0)	0(0.0)	2(100.0)	1(100.0)	0(0.0)
	R	13(24.5)	1(25.0)	0(0.0)	2(100.0)	0(0.0)	0(0.0)	1(100.0)

Figures in the parentheses indicate corresponding percentage;

S=sensitive, R=Resistant, Intermediate was counted as sensitive.

Table 03: Antibiotic susceptibility pattern of the isolated bacteria in non-ESBL-UTI group (n=64)

Antibiotics		Organisms
		E.coli (n=59) No. (%)	Klebsiella (n=1) No. (%)	Proteus (n=1) No. (%)	Pseudomonas (n=3) No. (%)
Amoxyclav	S	38(64.4%)	0(0.0%)	0(0.0%)	0(0.0%)
	R	21(35.6%)	1(100.0%)	1(100.0%)	3(100.0%)
Amikacin	S	58(98.3%)	1(100.0%)	1(100.0%)	3(100.0%)
	R	1(1.7%)	0(0.0%)	0(0.0%)	0(0.0%)
Aztreonam	S	59(100.0%)	1(100.0%)	1(100.0%)	1(33.3%)
	R	0(0.0%)	0(0.0%)	0(0.0%)	2(66.7%)
Ceftriaxone	S	59(100.0%)	1(100.0%)	0(0.0%)	2(66.7%)
	R	0(0.0%)	0(0.0%)	1(100.0%)	1(33.3%)
Cefixime	S	53(89.8%)	1(100.0%)	0(0.0%)	0(0.0%)
	R	6(10.2%)	0(0.0%)	1(100.0%)	3(100.0%)
Ceftazidime	S	59(100.0%)	1(100.0%)	1(100.0%)	3(100.0%)
	R	0(0.0%)	0(0.0%)	0(0.0%)	0(0.0%)
Cefotaxime	S	59(100.0%)	1(100.0%)	1(100.0%)	2(66.7%)
	R	0(0.0%)	0(0.0%)	0(0.0%)	1(33.3%)
Cephradine	S	38(64.4%)	0(0.0%)	0(0.0%)	0(0.0%)
	R	21(35.6%)	1(100.0%)	1(100.0%)	3(100.0%)
Ciprofloxacin	S	49(83.1%)	0(0.0%)	0(0.0%)	1(33.3%)
	R	10(16.9%)	1(100.0%)	1(100.0%)	2(66.7%)
Chloramphenicol	S	52(88.1%)	1(100.0%)	1(100.0%)	1(33.3%)
	R	7(11.9%)	0(0.0%)	0(0.0%)	2(66.7%)
Co-trimoxazole	S	27(45.8%)	1(100.0%)	0(0.0%)	0(0.0%)
	R	32(54.2%)	0(0.0%)	1(100.0%)	3(100.0%)
Cefuroxime	S	50(84.7%)	1(100.0%)	1(100.0%)	0(0.0%)
	R	9(15.3%)	0(0.0%)	0(0.0%)	3(100.0%)
Colistin	S	59(100.0%)	1(100.0%)	1(100.0%)	3(100.0%)
	R	0(0.0%)	0(0.0%)	0(0.0%)	0(0.0%)
Doxycycline	S	42(71.2%)	1(100.0%)	1(100.0%)	1(33.3%)
	R	17(28.8%)	0(0.0%)	0(0.0%)	2(66.7%)

Table-3 continued

Table 03: Antibiotic susceptibility pattern of the isolated bacteria in non-ESBL-UTI group (n=64)

Antibiotics		Organisms
		E.coli (n=59) No. (%)	Klebsiella (n=1) No. (%)	Proteus (n=1) No. (%)	Pseudomonas (n=3) No. (%)
Gentamicin	S	46(78.0%)	1(100.0%)	0(0.0%)	2(66.7%)
	R	13(22.0%)	0(0.0%)	1(100.0%)	1(33.3%)
Imipenem	S	59(100.0%)	1(100.0%)	1(100.0%)	1(33.3%)
	R	0(0.0%)	0(0.0%)	0(0.0%)	2(66.7%)
Levofloxacin	S	49(83.1%)	1(100.0%)	0(0.0%)	3(100.0%)
	R	10(16.9%)	0(0.0%)	1(100.0%)	0(0.0%)
Meropenem	S	59(100.0%)	1(100.0%)	1(100.0%)	1(33.3%)
	R	0(0.0%)	0(0.0%)	0(0.0%)	2(66.7%)
Nalidixic acid	S	12(20.3%)	1(100.0%)	1(100.0%)	0(0.0%)
	R	47(79.7%)	0(0.0%)	0(0.0%)	3(100.0%)
Nitrofurantoin	S	46(78.0%)	1(100.0%)	0(0.0%)	0(0.0%)
	R	13(22.0%)	0(0.0%)	1(100.0%)	3(100.0%)
Pefloxacin	S	49(83.1%)	1(100.0%)	0(0.0%)	2(66.7%)
	R	10(16.9%)	0(0.0%)	1(100.0%)	1(33.3%)
Tetracycline	S	49(83.1%)	1(100.0%)	1(100.0%)	3(100.0%)
	R	10(16.9%)	0(0.0%)	0(0.0%)	0(0.0%)

S=sensitive, R=Resistant, Intermediate was counted as sensitive.

Table 04: Pattern of antibiotic resistance between ESBL-UTI and non-ESBL-UTI group (N=128)

Antibiotic	ESBL (n=64) No. (%)	Non-ESBL (n=64) No. (%)	p-value
Amoxiclav	60(93.8%)	26(40.8%)	<0.001s
Amikacin	22(34.4%)	1(1.6%)	<0.001s
Aztreonam	54(84.4%)	2(3.1%)	<0.001s
Ceftriaxone	63(98.4%)	2(3.1%)	<0.001s
Cefixime	63(98.4%)	10(15.6%)	<0.001s
Ceftazidime	64(100.0%)	0(0.0%)	<0.001s
Cefotaxime	64(100.0%)	1(1.6%)	<0.001s
Cephradine	64(100.0%)	26(40.6%)	<0.001s
Ciprofloxacin	43(67.2%)	14(21.9%)	<0.001s
Chloramphenicol	27(42.2%)	9(14.1%)	0.001s
Co-trimoxazole	55(85.9%)	36(56.3%)	<0.001s
Cefuroxime	64(100.0%)	12(18.8%)	<0.001s
Colistin	2(3.1%)	0(0.0%)	0.043s
Doxycycline	30(46.9%)	19(29.7%)	0.100ns
Gentamicin	31(48.4%)	15(23.4%)	0.002s
Imipenem	11(17.2%)	2(3.1%)	0.020s
Levofloxacin	39(60.9%)	11(17.2%)	<0.001s
Meropenem	15(23.4%)	2(3.1%)	0.002s
Nalidixic acid	51(79.7%)	50(78.1%)	0.473ns
Nitrofurantoin	29(45.3%)	17(26.6%)	0.007s
Pefloxacin	36(56.3%)	12(18.8%)	<0.001s
Tetracycline	17(26.6%)	10(15.6%)	0.270ns

 Chi-square test (c²) was done to analyze the data. s= significant, ns = not significant, ESBL-producing bacteria were significantly resistant to commonly used antibiotics.

Table 05: Distribution of the study subjects by Urine examination (N=128)

Urine examination	ESBL (n=64) No. (%)	Non-ESBL (n=64) No. (%)	p-value
Pus cell (per HPF)
<5	10(15.6%)	8(12.5%)	0.299ns
5-10	30(46.8%)	28(43.8%)
>10	20(31.3%)	18(28.1%)
Plenty	4(6.3%)	10(15.6%)
RBC (per HPF)
<5	54(84.4%)	57(89.1%)	0.927 ns
5-10	6(9.4%)	4(6.2%)
>10	4(6.2%)	3(4.7%)
Ep cell (per HPF)
<5	59(92.2%)	60(93.8%)	0.193 ns
5-10	5(7.8%)	2(3.1%)
>10	0(0.0%)	2(3.1%)
Albumin
Nil	25(39.1%)	24(37.5%)	0.555 ns
Trace	8(12.5%)	14(21.9%)
1+	8(12.5%)	5(7.8%)
2+	5(7.8%)	4(6.2%)
3+	18(28.1%)	17(26.6%)
pH
<5	3(4.7%)	0(0.0%)	0.080 ns
>5	61(95.3%)	64(100.0%)
Specific gravity
1.0-1.015	8(12.5%)	3(4.7%)	0.115 ns
1.020-1.030	56(87.5%)	61(95.3%)
Nephrotic range proteinuria	21(32.8%)	15(23.4%)	0.555 ns

Chi-square test (c²) was done to analyze the data.,  s= significant, ns = not significant

None of the urinary findings were statistically significant between two groups.

Table 06: Possible risk factors of study population (N=128)

Possible risk factors	ESBL (n=64) No. (%)	Non-ESBL (n=64) No. (%)	OR (95% CI)	p-value
Previous hospitalization	24(37.5%)	11(17.2%)	2.89 (1.3-6.6)	0.010s
H/O previous UTI	8(12.5%)	1(1.6%)	9.0 (1.1-74.2)	0.016 s
Previous use of antibiotics	46(71.9%)	15(23.4%)	8.3 (3.8-18.5)	<0.001 s
First generation cephalosporin	2(3.1%)	6(9.4%)	0.38 (0.07-2.04)	0.144ns
Second generation cephalosporin	2(3.1%)	1(1.6%)	2.03 (0.18-22.9)	0.559 ns
Third generation cephalosporin	43(67.2%)	10(15.6%)	11.1 (4.7-25.9)	<0.001s
Fourth generation cephalosporin	4(6.3%)	0(0.0%)	–	0.042s
Immunosuppressant drug	10(15.6%)	3(4.7%)	3.8 (0.98-14.4)	0.041s
Associated diseases	25(39.1%)	16(25%)	1.92(-2.2-30.4)	0.66 ns
Uncircumcised boys	30(88.2%)	26(83.9%)	1.44(-7.92-16.52)	0.611ns

Odds ratio (OR) with 95% CI and Chi-square test were done to analyze the data, s=significant, ns = not significant

Some patient had history of previous use of more than one generation of antibiotics. Previous hospitalization, H/O previous UTI and previous use of antibiotics (third generation Cephalosporin) were found as risk factors for ESBL-UTI.

Table 07: Independent risk factors for ESBL-positive UTI

		p-value	OR	95% C.I
				Lower	Upper
	Previous hospitalization	.750	.826	.255	2.675
	H/O previous UTI	.320	.308	.030	3.140
	Previous use of antibiotic	.596	.646	.129	3.242
	Third generation cephalosporin	.270	.156	.030	1.808
	Constant	.000	2.840

Multivariate binary logistic regression analysis was done, where none of them was found as an independent risk factor for ESBL-UTI. E. coli was the most isolated organism in both the ESBL-UTI (84.4%) and non-ESBL-UTI (92.2%) groups. E. coli was the most commonly isolated bacteria in UTI patients by Afroz, Albaramki, Alim, Awean, Islam, Kim, Yang, and Kim, Rahman, and Topaloglu. High E. coli ratios cause autoinfection since the bacteria are in the feces. E. coli can also create a biofilm on the bladder wall that resists the immune system. This study found that Pseudomonas caused 6.3% of ESBL-UTI and 4.7% of non-ESBL-UTI. Pseudomonas as the second main cause of UTI. Klebsiella was the second biggest cause and a major risk factor for ESBL development in other investigations. Different environmental circumstances, host characteristics, healthcare and education programs, socioeconomic levels, and cleanliness practices in each nation may affect uropathogen kind and distribution. The ESBL-UTI group had a considerably greater rate of AMR than the non-ESBL-UTI group. ALL ESBL isolates were resistant to Ceftazidime, Cefotaxime, and Cefuroxime, followed by Ceftriaxone, Cefixime, Amoxiclav, and Co-trimoxazole (p< Colistin, Imipenem, and Meropenem had the lowest resistance.

3.2 Discussion

A study found that all ESBL isolates were resistant to cefuroxime, ceftazidime, and cefotaxime and 99% to ampicillin and ceftriaxone. All isolates were sensitive to Meropenem, Imipenem, and Colistin, regardless of ESBL status [23]. ESBL E. coli in India exhibited high resistance to third-generation cephalosporins, quinolones, and co-trimoxazole, but less resistance to Gentamicin, Levofloxacin, Nitrofurantoin, Netilmicin, and Imipenem [24]. All ESBL isolates in a Finnish study were resistant to Cephalosporins and susceptible to Meropenem. ESBL-producing bacteria showed increased resistance to non-β-lactam antibiotics, such as Trimethoprim (76%), Norfloxacin (33%), and Sulphatrimethoprim (76%) [25]. The only clinically meaningful Trimethoprim resistance in ESBL-negative bacteria was 27%. In our country, oral third-generation antibiotics like Cefixime are overprescribed [26]. Through antibiotic selection pressure (ESBLs), this method may increase gram-negative bacteria drug resistance. In this study, 50% of ESBL-UTI children were 1–5 years old [27]. A recent study found more UTIs among 1-5-year-olds, and most instances are over 2 [28]. ESBL-UTI risk was independent for children under one year old, a CMH, Dhaka study found that UTIs were more common in children under 5 (62.5%) [29]. Possibly because younger children aren’t toilet-trained. Fecal flora ascending infection is more common in this age range [30]. In this study, ESBL-UTI members were mostly men (1.1:1), but not significantly so (p=0.596), a study found no gender correlation however several research have linked male sex to risk factors [31]. Other research indicated female predominance, as females are more likely to get UTIs [32]. Short urethras and other factors may make this condition more common in girls. In early infancy, boys are more prone to congenital deformity, but as they get older, the gender ratio reversesd [33]. Physician visit preferences may explain male predominance in UTI. Significant risk factors reported in this study by univariate analysis include prior antibiotic use (p<0.001), past UTI (p=0.016), and prior hospitalization (p=0.010). Children with antibiotic history had 8.3 times the risk of ESBL-UTI. Third-generation Cephalosporin was 11 times riskier. Different trials show different effects with prior antibiotic use, multiple studies indicated that antibiotic use was an independent risk factor for ESBL-UTI [34]. Penicillin, second- and third-generation Cephalosporin, Quinolones, Carbapenems, and Aminoglycosides, and suppression treatment (TMP/SMX, Nitrofurantoin) were risk factors for ESBL-UTI [35]. Previous antibiotic therapy did not increase the risk of ESBL-UTI in children [36]. In a meta-analysis of five investigations, uropathogens from children who had taken antibiotics were more than 10 times more likely to be resistant. It increased risk may last up to six months after antibiotic treatment, It also found that restricting third-generation Cephalosporins can suppress ESBL-producing bacteria in hospitals [37].  Children with a 3-month hospitalization history had a 3-fold higher risk of ESBL-UTI, findings showed that hospitalization within 3 months was an independent risk factor for ESBL-UTI in children [38]. Children may acquire ESBL-producing bacteria via healthcare processes and become reservoirs for them. Returning to the community may cause ESBL-UTI [39].  In this study, previous UTI in the recent 3 months increased ESBL-UTI risk by 9 times and [40] support this finding as well. However, [41] observed that children having a history of UTI within 12 months were less likely to contract ESBL-producing Enterobacteriacea. Multiple hospital stays and broad-spectrum antibiotics may generate ESBL-producing bacteria [42]. None were independent risk factors for ESBL-UTI in children on multivariate binary logistic regression, [44] supported this finding. A retrospective Korean investigation found no risk factors in up to 60% of babies with community-acquired ESBL-producing uropathogens [45].

4. CONCLUSIONS

The Pediatrics and Microbiology departments of Sir Salimullah Medical College Mitford Hospital in Dhaka conducted a cross-sectional study to investigate the risk factors for urinary tract infections caused by ESBL-producing bacteria in children ranging in age from one month to twelve years based on the results of the study. In both the ESBL-UTI (84.4%) and the non-ESBL-UTI (92.2%) groups, E. coli was the bacterium that became the most isolated. As far as Afroz, Albaramki, Alim, Awean, Islam, Kim, Yang, and Kim, Rahman, and Topaloglu were concerned, the bacteria that was most frequently isolated from UTI patients was E. coli. Due to the fact that the bacteria are present in the feces, high E. coli ratios might lead to autoinfection. E. coli has the ability to produce a biofilm on the bladder wall that is resistant to the immune system. According to the findings of this investigation, Pseudomonas was responsible for 6.3% of ESBL-UTI and 4.7% of non-ESBL-UTI. Pseudomonas was the second most common cause of urinary tract infections (UTIs). According to the findings of earlier investigations, Klebsiella was the second most significant cause and a significant risk factor for the development of ESBL. There are a variety of factors that can influence the type of uropathogen and its dissemination, including the environment, the features of the host, the healthcare and education programs, the socioeconomic levels, and the cleaning practices of each nation. The ESBL-UTI group had an antimicrobial resistance rate that was much higher than the non-ESBL-UTI group’s rate. There was a high level of resistance among all ESBL isolates to Ceftazidime, Cefotaxime, and Cefuroxime. The next most resistant isolates were Ceftriaxone, Cefixime, Amoxiclav, and Co-trimoxazole (p< Colistin, Imipenem, and Meropenem).  E. coli was the most prevalent of the uropathogens that were isolated, and this was true for both the ESBL and the non-ESBL-UTI groups. The Cephalosporin group of antibiotics was ineffective against almost all of the ESBL-producing bacteria, followed by Amoxiclav and Co-trimoxazole as the most effective antibiotics. Colistin emerged as the antibiotic with the least amount of resistance, followed by imipenem and meropenem. Prior hospitalization and a history of urinary tract infections were the two most important risk factors for urinary tract infections (UTIs) caused by ESBL-producing bacteria. The most prominent risk factor was a history of antibiotic use in the past. An independent risk factor for ESBL-positive urinary tract infections was not discovered in any of them. During the process of selecting a suitable empirical therapy, the risk factors and antibiotic susceptibility pattern that were discovered in this study will be of great assistance.It is recommended that ESBL testing be incorporated into the standard clinical practice. It is important for clinicians to use caution while administering antibiotics.

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