South As. J. Biol. Sci. 2(Supp.1):140-149 ISSN 2249-6599 Phenotypic Characterization of Urinary Tract Infection Causing Escherichia coli in Paediatric age group along with Prevalence of Extended Spectrum Beta Lactamases in a Tertiary Care Centre Nalini K 1*, Janani P 1 and P. Sumathi 2 1 Department of Microbiology, Ayya Nadar Janakiammal College,Sivakasi-626123. * Part time research scholar, Bharathiar university, coimbatore. 2 Department of Biochemistry, Government Arts college,krishnagiri To cite this article: Nalini K, Janani P and P. Sumathi, 2012. Phenotypic Characterization of Urinary Tract Infection Causing Escherichia coli in Paediatric age group along with Prevalence of Extended Spectrum Beta Lactamases in a Tertiary Care Centre. South As. J. Biol.Sci., 2(Supp.1): 140 149. Abstract Antibiotic resistance as a phenomenon is, in itself, not surprising nor is it new. It is, however, newly worrying because it is accumulating and accelerating, while the world's tools for combating it decrease in power and number: The unnecessary use and misuse of antibiotics has led to a significant healthcare problem antibiotic resistance. CAUTIs comprise the largest institutional reservoir of antibiotic-resistant pathogens. In the present investigation, the urine sample were collected from 447 children who had the symptom of UTI.Among them, 280 urine samples were positive i.e., showed the presence of bacteria in the urine in which 119 samples were from hospitalized patients and 161 samples from outpatients. The study was conducted at Kanchi Kamakoti child trust hospital during the period of Nov 2009 to March 2010. Among the organisms isolated E.coli was found to be the predominant form. Antibiotic sensitivity pattern of the isolates were studied using different antibiotics like Amikacin, Gentamycin, Ciprofloxacin, Ofloxacin, Amoxicillin/ Clavulanic acid, Pipracillin/ Tazobactum, Meropenem, Ampicillin, Ceftazidime, Cefotaxime, Cotrimaxazole, Ceftriaxome, Cefuroxime, Cephalexin, Nitrofurantoin, Nalidixic acid. Antibiotic resistance is observed at differential rates for different antibiotics. No resistance is observed for Meropenam and more resistance is observed for Cephalexin, Cefotaxime, Ceftriaxome. Phenotypic detection of ESBL is confirmed in majority of E.coli. Key words: Beta lactam antibiotics, Antimicrobial resistance, urinary tract infections. Corresponding author: Nalini K. Email:nalininandhini@yahoo.com 140 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
Introduction Urinary tract infection is a serious health problem affecting millions of people. Infections of the urinary tract are the second most common type of infection in the body and account for about 8.3 million doctor visits every year. Urinary tract infections in men are not as common as in women but can be very serious when they do occur 1.Women have more UTIs than men because women s urethra is short, allowing bacteria for quick access to the bladder An infection occurs when tiny organisms usually bacteria from the digestive tract, cling to the opening of the urethra and begin to multiply. Escherichia coli is the most common organism isolated in the newborn period, accounting for up to 80 % of infections in most large series. This pathogen also is the most frequent cause of UTI at all ages. Other Enterobacteriacea, which can generate UTI, include: Klebsiella sps, Enterobacter sps, Citrobacter sps, Proteus sps, Providencia sps, Morgenella sps, Serratia sps, and Salmonella species Other bacteria associated with complicated or severe infection include Pseudomonas aeruginosa., and gram-positive organisms including Entercoccus species and S. saprophyticus. UTI infection is most commonly occurring disease in worldwide. The following countries also have high risk of UTI like south America (48%),USA (34%), Europe (9.5%),China (34-38%), Russia (40-90%).In Africa the percentage of UTI is >20%. Taiwan, Philipines and Singapore have >20% UTI respectively. In India the UTI infected persons are at elevated range 30-80 %. 2.In normal newborns, the incidence of bacteriuria determined by prospective bladder urine collection (suprapubic aspiration) is 0.1 to 1%. The risk of significant bacteriuria may be higher in high-risk newborns (2 to 6 %). UTI occurs 1.5 to 5 times in as many males as females in the neonatal period and is higher in uncircumcised than circumcised males. The incidence decreases in boys and increases in girls during the first six months after birth. Midstream clean catch specimen is the preferred type of specimen for culture and sensitivity testing because of the reduced incidence of cellular and microbial contamination. ESBL producing E.coli is antibiotic resistant strains of E.coli which secretes an enzyme called extended spectrum beta lactamases. Extended spectrum beta lactamases (ESBL) hydrolyse oxyimino beta lactams like Ceftazidime, Cephotaxime and Monobactam but have no effect on Cephamycins carbapenem and related compounds These enzymes are plasmid mediated enzymes and hydrolyse variety of beta lactams, including penicillin,second and third generation cephalosporin s, and aztreonam.esbl production carry resistance to many antibiotics like Amino glycosides, Fluoroquinolones, Tetracycline, Cotrimoxazole. ESBL producing organisms are reported in intensifying and diversified numbers worldwide. E.coli and Klebsiella are the major ESBL producing organisms. Other organisms reported to produce ESBL are Proteus species, Pseudomonas sps. The present study focuses on the antibiotic resistance 141 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
pattern of E.coli isolated from urine sample of different paediatric age groups and observing the prevalence of ESBL in these isolates. Materials and Methods Urine sample collection The clean voided midstream urine sample collected from the children in a sterile dry, wide necked, leak proof universal container.the container was labeled with date, name, and age of the patient and also labeled with proper registration number (lab number) of the patient. In some cases the urine also collected by suprapubic aspiration and catheterization method by using syringe and sterile catheter tube.urine sample were inoculated in the culture media as soon as possible after collection. The samples were otherwise refrigerated at 4 C to 6 C. It was then cultured within 24 hours. Detection of bacteriuria Bacteriuria was detected by quantitative urine culture by calibrated loop method. The microbial count in the urine sample was detected. In calibrated loop method, loops were made to deliver 0.01ml of urine. A loopful of uncentrifuged urine was inoculated on to the medium like blood agar and macconkey agar medium. These plates were incubated at 37 C for 24 hours. The number of colonies were counted and multiplied by 100 to calculate the number of organisms per ml of the urine 3 Cultural characterization Organisms were identified based on standard protocol. Different organisms form different types of colonies in the nutrient agar. From the colony morphology, the organism can be determined. Formation of mucous colonies indicates the presence of Klebsiella sps. Formation of swarming growth colonies indicates the presence of Proteus sps. Dry, rough colonies indicate the presence of E.coli. Macconkey agar and blood agar were also used to determine the type of the organism. The isolates were morphologically identified by gram stain and motility done by hanging drop method. Further biochemical detection was based on catalase and Oxidase tests, Fermentation and gas production in triple sugar iron medium, mannitol utlisation, indole production, Urease production, citrate utilization in the respective media. In addition to the above test, phenyl pyruvic acid test was also employed in case of non-lactose fermenting gram negative bacilli. Antibiotic sensitivity testing Antimicrobial susceptibility testing was done by Kirby Bauer s disc diffusion method as described by National committee for clinical laboratory standards 4 Muller Hinton agar plates were prepared by sterilization of the medium at 121 C for 15 minutes at 15 lbs pressure. The isolate was inoculated in nutrient broth and 0.5Mac Farland standard isolate in nutrient broth was soaked in sterile 142 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
cotton swab and spread on the surface of the plate. Antibiotic discs like Amikacin, Gentamycin, Ciprofloxacin, Ofloxacin, Amoxicillin/ Clavulanic acid, Pipracillin/ Tazobactum, Meropenem, Ampicillin, Ceftazidime, Cefotaxime, Cotrimaxazole, Ceftriaxome, Cefuroxime, Cephalexin Nitrofurantoin, Nalidixic acid were placed on the surface with the sterile forceps and incubated at 37 C for 24 hours. The diameter of the zone was measured and the sensitivity of the microorganisms was determined by comparing the measurement with the standard chart. Phenotypic detection of ESBL Double disk synergy test was used to detect the ESBL. The following antibiotics were used : Ampicillin (10µg), co-trimoxazole (25µg), Gentamycin (10µg), Nalidixic acid (30µg), Nitrofurantoin (300µg), Cephotaxime (30µg), and Amikacin (10µg).Gram negative bacteria with resistance or with decreased susceptibility to third generation cephalosporin s were tested for ESBL production.the Cephotaxime and a disc of amoxicillin/clavulinic acid (Augmentin)arranged in pairs. In the Muller Hinton agar plate the discs were arranged so that the distance between them was approximately twice the radius of the inhibition zone produced by Cephotaxime tested on its own. The test isolate was considered to produce ESBL, if the zone size around the antibiotic disc increased towards the Augmentin disc. 5 Results In the present investigation, the urine samples were collected from 447 children children in Kanchi Kamakoti Child Trust Hospital, Chennai, Tamil Nadu, who had the symptom of UTI. Information regarding the positive samples (>10 5 cells/ml) were reported in Table 1. The relationship between the sex and incidence and bacteriuria is illustrated in Table-2. Among the 215 children 110 patients were male (51%) and 105 patients were female (49%). By applying t test, it was found that there is no significant difference between the sex and incidence of UTI.Most of the UTI positive cases reported are community acquired. Table 1: Details of Urinary tract infected patients Types of patients Number of patients Positive samples Hospitalized patients 119 280 Out patients 161 143 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
Table 2: Data collection Based on Sex Patients detail No. of positive patients Male Female In Patients 92 43 49 Out Patients 123 67 56 Total 215 110 105 The relationship between the age distribution and incidence of bacteriuria is illustrated in figure 1. Age group was distributed as neonates (0-1 month), early infants, infants (1m-3m), preschools (3m-1 yr), toddler(1yr-5yr), adolescents(5yrs-10yrs )and 10yrs-teenage. Maximum infection occurs between the age group of 1 yr to 5 years (37.5%), which is followed by 3m 1 yr (30.3%) and 5 to 10 yrs children (15.3%) respectively. Neonates less than one month and early infants are less affected by UTI. Figure- 1. Age distribution and incidence of asymptomatic Bacteriuria Figure 2 showed various urine collection methods that are also included in the investigation. Out of 215 urine samples, most of the infected samples were from clean catch midstream urine (97.7%). The risk of UTI was decreased in both direct catheter (0.5%) and suprapubic sample (0.5%) collection methods. The causative organisms of UTI were represented in the Figure-3. Enterobacteriacea family (95.5%) caused the majority of the infection. Among the Enterobacteriacea, Escherichia coli was the common pathogen which caused ¾ th of the infection, which is followed by Klebsiella sps (7.5%), Proteus sps (7.1%) Morgeneela sps,enterobacter sps were isolated from 2 patients (0.7% o). Providanceae, Citrobacter were also isolated in minimum number of patients (0.35%). The non- fermentors like Pseudomonas (2.5%) and Acinetobacter (35%) also caused Urinary tract infection. Among gram positive 144 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
cocci, Enterococcus (3.5%) were seen in few case. Staphylococcus sps infection was not reported in this study. Fig- 2. Various urine collection methods Figure-3. Causative organisms of symptomatic Bacteriuria Figure- 4. Prevalence of Escherichia coli infecting Urinary tract 145 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
Among 119 hospitalized patients, Escherichia coli was found to be the major organism and caused UTI in 92 patients (77%). Extended spectrum beta lactamase (ESBL) producing strains were found in 44.6% of isolated E.coli. In community acquired urinary tract infection, Escherichia coli caused infection in 123 patients, among them 48.7% seemed to be ESBL producer.approximately 50% of E.coli produced ESBL. Figure 4 represents the prevalence of Escherichia coli infecting Urinary tract infection in different areas of hospital. 72.8% of Escherichia coli infection was isolated from general ward patients, which is followed by isolation in emergency ward (9.8%), new born ICU 5.4 % and 1% in pediatric ICU. Figure- 5. Antibiotic profile of UTI causing Escherichia coli strain Photo- 1. Antibiogram of E.Coli Photo -2. ESBL producing E.coli Figure 5 represented the antibiotic profile of UTI causing Escherichia coli strain. The sensitivity of Escherichia coli towards various antibiotics was determined based on the diameter of the zone formed in 146 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
muller Hinton agar. The Escherichia coli was more sensitive to the antibiotics viz Meropenem, Nitrofurantoin, Amikacin, Pipracillin/ tazobactum, Gentamycin, Cefaperazone/sulbactums. The organism was resistant to Ampicillin, Amoxicillin / Clavulinic acid, Cephalexin, Cefotaxime, Ceftriaxome, Ciprofloxacin, Ofloxacin, Nalidixic acid and Cotrimaxazole(Photo 1). Escherichia coli including ESBL showed 100% sensitive to Carbapenams. Though beta lactamase inhibitor were claimed to be sensitive to PIP/TAZ, 14 % of ESBL strains were resistant to this antibiotic. The ESBL strains showed high resistant to Ampicillin (94.5%) and Nalidixic acid (94%). The ESBL strains showed equal amount of resistant to third generation cephalosporin s like Cefotaxime and Ceftriaxome and it showed elevated resistant to first generation cephalosporin (89.8%). It also showed an equal amount of resistant to quinolones groups of antibiotics like ciprofloxacin (68.8%) and Ofloxacin (69.8%) (Photo 2). Discussion Urinary tract infection is the most common bacterial infection. Due to rising antibiotic resistance among uropathogens, it is important to have local hospital based knowledge of the organisms causing urinary tract infection and their antibiotics sensitivity pattern. Out of 447 urine samples received in this present study, bacteriuria was found in 280 samples (63%), while 167 (37%) samples were culture negative. Out of 4800 urine samples received for culture during the six month period significant bacteria was found in 1500 (31.25 %) samples, while 2610(54.38%) samples were culture negative 6. Among the 280 positive samples 119 were from hospitalized patients and 161 samples were from community acquired patients. Earlier work investigated that the common causative agent of Urinary tract infection was Escherichia coli i.e, 50% 7 and 83 % 8.. It may be due to the reason that the reservoir from which the Escherichia coli causing urinary tract infections were derived from patients own fecal flora.higher incidence of 49.8 % E.coli is followed by 37.8 % for Klebsiella species 9. Similar results were obtained in my study that of the 280 culture positive samples, the common isolates was Escherichia coli (76.7%) followed by Klebsiella species (7.5%) and Proteus species (7.1 %). Among the uropathogen isolated, gram negative bacteriuria was confirmed in 87 % cases, where as Gram positive bacteriuria was confirmed in 13 % of cases 10. In the present investigation the gram negative bacteriuria was confirmed in 96.4 %, where as gram positive bacteriuria was confirmed in 3.6% of cases. ESBL (extended spectrum β lactamases) is a common problem in hospitalized patients throughout the world. The prevalence of ESBL among clinical isolates varies greatly in different geographical areas and is rapidly changing over time. In my study, the extended spectrum β lactamases (ESBL) producing Escherichia coli was found to be only in a percentage 147 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
of 47 % (approximately 50%). ESBL production was observed in 41 % of Escherichia coli (143/ 353) and 40% in Klebsiella pneumoniae 11. In the present investigation it is revealed that out of 215 positive E.coli culture, male children were very susceptible to E. coli infection (51.8 %) rather than female(48.2%). The prevalence of E.coli infection was more predominant in the age group between 1 year to 5 years (37.7 %).Antimicrobial susceptibility of E.coli to Meropenem, Nitrofurantoin and Amikacin were found to be 100%, 89 % and 86 % respectively 10.Similar results were found in my investigation, where the E. coli was more susceptible to Meropenem 100%, Nitrofurantoin (93.5%) and Amikacin (93%) and E.coli was more resistance to Ampicillin (94.4 %) and Nalidixic acid (94%) respectively. Nitrofurantoin, constitute the reasonable option for treatment of UTIs.Globally, E.coli is the commonest cause of UTI in both community and hospital settings. A national survey of nosocomial UTI in the United States found that E. coli sps, Pseudomonas sps, Enterococcussps sps, Klebsiella species and Enterobacter are among the top 5 uropathogens 12 Conclusion Antibiotic resistance is of global concern and steps should be taken to reduce its prevalence. Susceptibility studies should be carried now and then to map out the resistance pattern in different geographical locations. This makes the clinicians to understand the pattern of resistance and design novel drugs to fight against antibiotic resistance. On phenotypic level there are two ways of fighting development and spread of drug resistant bacteria. The first is to reduce the use of antimicrobial agents to decrease the selection of resistant bacteria and second is to improve the hygienic measures to prevent the spread of resistant bacteria. References 1. Hyattsville K., 2004. Urinary tract infection in adults. National centre for health statistics, centers for disease control and prevention. 41:222-9. 2. Brenner MB. The Kidney. 2 nd ed.wb: Saunders company; 2000. p. 1475-76. 3. Raphael SS. Lynch s Medical Laboratory Technology. WB: Saundeers company; 1976. p. 667. 4. National committee for clinical laboratory standards. Methods for Disc susceptibility Tests for Bacteria that grow aerobically. NCCLS Documant M2 -A7.Wayne, National committee for clinical laboratory standards & 7 th edition.2000. 5. National committee for clinical laboratory standards. Performance standards for antimicrobial susceptibility testing. International supplement. NCCLS committee for clinical laboratory standards Wayne, National committee for clinical laboratory standards & 11 th edition.2001. 148 South As. J. Biol. Sci. 2(Suppl 1): 140 149 Nalini et al.
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