Analysis of causative agents of urinary tract infections in children and their antibiotic sensitivity pattern : A hospital based study *

Introduction: Urinary tract infections (UTIs) are an important cause of morbidity and mortality in children. Objective: To determine the common bacterial pathogens causing UTI in children less than 12 years old and their antimicrobial susceptibility patterns at the Colombo South Teaching Hospital, Sri Lanka. Method: A prospective study was done by analyzing records of urine samples received at the Department of Microbiology, Colombo South Teaching Hospital, from 1 May 2011 to 30 April 2012. Urine samples were inoculated on Cystine Lactose Electrolyte Deficient agar, according to standard operating procedures. The isolates were initially identified by Gram staining and colony characteristics. Gram positive organisms were further identified by catalase, coagulase and bile-aesculin tests. Lactose fermentation or non-lactose fermentation (NLF) of Gram negative organisms was recorded. The NLF isolates were further identified by oxidase, urease and Kleigler Iron Agar tests. Antibiotic susceptibility tests were carried out using the Stokes method. Results: A total of 2620 urine samples was analyzed. Of the 426 (16.3%) pure growths, Gram negative isolates of bacteriuria (>10 colony forming units/ml of urine) and Gram positive isolates of bacteriuria (>10 colony forming units/ml of urine) were found in 264 (62%) and 56 (13.1%) patients respectively. The female: male ratio of the total positive samples was 2.1:1. LF and NLF Coliforms (excluding Proteus, Providencia, and Morganella spp.) were isolated in 142 (33.3%) and 68 (16%) samples respectively. Proteus spp. were isolated in 43 (10.1%) samples 38 (88.4%) of which were identified in males. The sensitivity of LF Coliforms to ______________________________________________ Lecturer in Microbiology, Faculty of Medicine, General Sir John Kotelawala Defence University, Ratmalana, Consultant Microbiologist, Department of Microbiology, Colombo South Teaching Hospital, Kalubowila, Sri Lanka *Correspondence: nilanthisenanayake@yahoo.com Received on 30 September 2014: Accepted after revision on 21 November 2014 The authors declare that there are no conflicts of interest Open Access Article published under the Creative Commons CC-BY-NC-ND Licence. nitrofurantoin, cephalexin and ampicillin was 83.9%, 57.4% and 11.3% respectively. The sensitivity of NLF Coliforms to nitrofurantoin, cephalexin and ampicillin was 85.3%, 36.8% and 7.4% respectively. Conclusions: Enterobacteriaceae were the predominant pathogens causing UTI in our population and showed high resistance against ampicillin and cephalexin. Proteus infections were predominant in males. (


Introduction
Urinary tract infection (UTI) is a common health problem during childhood and an important cause of morbidity and mortality in the first 2 years of life 1 .Symptoms of UTI may be minimal and non-specific in infants and small children 2 .Therefore, diagnosis of UTI cannot be made on symptomatology alone and urine examination is advocated in children with minimal suspicion of UTI 3,4 .UTI may lead to life threatening complications, like septicaemia and renal scarring.Renal scarring is the most common cause of hypertension in later childhood leading to renal failure in adulthood 2,5 .Recognition of UTI in children should be made as early as possible to prevent these complications 2 .Therefore, early diagnosis of UTI is of utmost importance 5 .According to western publications at least 80% of UTI in children are caused by Escherichia coli followed by organisms like Proteus, Enterococcus, Pseudomonas, Klebsiella, Citrobacter and Staphylococcus species 5 .Effective antimicrobial therapy for UTI can reduce adverse events in this age group.Most recent studies, particularly in developing countries, on the antimicrobial susceptibility of bacterial pathogens causing UTI in children showed high levels of antibiotic resistance [6][7][8][9] .In addition, the prevalence of antimicrobial resistance in young children with UTI is increasing and varies among different geographical and regional locations and various socioeconomic strata 10 .Therefore, it is necessary to identify the bacterial agents responsible for UTI and to determine their sensitivity to antibiotics in our paediatric population.

Objective
To determine the common bacterial pathogens causing UTI in children less than 12 years old and their antimicrobial susceptibility patterns at the Colombo South Teaching Hospital (CSTH), Sri Lanka Method A prospective descriptive study was conducted to find the causative agents of UTI in children and their antibiotic sensitivity pattern by analyzing the records of urine samples received at the Department of Microbiology, CSTH from 1 st May 2011 to 30 th April 2012.Urine samples received at the Department of Microbiology, from children less than 12 years which have been collected by the midstream or clean catch methods were included in the study.These samples were well mixed and inoculated on Cystine Lactose Electrolyte Deficient (CLED) agar, using calibrated 0.001 ml (1µl) wire loop according to the standard operating procedures.The CLED plates were incubated aerobically at 35 0 C overnight.The isolates were initially identified by Gram staining and colony Pure bacterial colony counts of 10 5 or more [≥10 5 Colony Forming Units (CFU)/ml of urine] was considered as significant for Gram negative isolates and a pure bacterial colony count of 10 4 or more (≥10 4 CFU/ml of urine) was considered as significant for Gram positive isolates.Gram positive organisms were further identified by catalase, slide/tube coagulase and bile aesculin tests.Lactose fermentation (LF) or non-lactose fermentation (NLF) of Gram negative organisms were recorded.The NLF isolates were further identified by oxidase, urease and Kleigler Iron Agar (KIA) tests.

Results
A total of 2,620 urine samples was analyzed.Of the 426 (16.3%) pure growths Gram negative isolates of bacteriuria (>10 5 CFU/ml of urine) and Gram positive isolates of bacteriuria (>10 4 CFU/ml of urine) were found in 264 (62%) and 56 (13.1%) patients respectively.The female to male ratio of the pure growths was 2.1:1.0.
The distribution of Gram negative urinary tract causative agents is shown in Table 1.The percentages of the Gram negative isolates that were sensitive to the tested antibiotics are given in Table 3.

Discussion
UTIs are of major clinical importance due to high morbidity and mortality rates among children 11 .In the present study, of 2,620 urine samples received at the laboratory, 426 (16.3%) yielded pure growths.In previous studies Enterobacteriaceae was identified as the predominant pathogen in children with UTIs [12][13] .Similar to findings of other studies, the most frequently detected pathogen among children with UTIs in the present study was LF and NLF Coliforms, which belongs to the Enterobacteriaceae group (49.3%).Of the NLF Coliforms urease positivity is seen in Proteus, Providencia, and Morganella species.Of these organisms the commonest UTI causing organism is Proteus species.In present study, of the NLF Coliforms urease test positivity was seen in 43 (10.1%)urine isolates.Therefore the urease test positive isolates were most likely to have been Proteus species.Proteus species was isolated in 43 (10.1%)samples and of the Proteus species 38 (88.4%) were identified in males.Previous studies have shown that Proteus species causes more UTIs in males [14][15][16] .In one study from Turkey Proteus species were isolated only in male patients 14 .This has been explained on the relatively high motility of the Proteus species allowing it to swarm the long urethra of the males and ascend to cause the infection 15 .
In the present study, it was found that among the tested antibiotics, nitrofurantoin, gentamicin and cefotaxime were the most active agents, whereas ampicillin, cephalexin and cotrimoxazole were the least active agents against LF and NLF Coliforms (excluding Proteus spp.).Based on our results, Proteus spp.which mainly caused infections in males showed the highest sensitivity to nalidixic acid and gentamicin.Nitrofurantoin cannot be used in Proteus infections as it is active in acidic urine but due to the ability of Proteus species in splitting urea to release ammonia which in turn makes urine alkaline.When Proteus species were identified nitrofurantoin was reported as resistant irrespective of in vitro sensitivity to avoid treatment failure.According to our results if the laboratory failed to identify Proteus species approximately 40% of the positive cultures will have Nitrofurantoin reported as sensitive antibiotic leading to treatment failure.Staghorn calculus is more common in males (4:1) in Sri Lanka 17 .Staghorn calculi are associated with Proteus infections and childhood Proteus infections may contribute to the formation of staghorn calculi in adult life.The resistance to antibiotics varies in different societies and in different countries.In general, in poor and underdeveloped countries, the prevalence of antimicrobial resistance is particularly high, which reflects the irrational and excessive use of antimicrobial agents 18 .Therefore monitoring antimicrobial resistance of bacteria causing urinary tract infection is crucial to improve quality of healthcare.

Conclusions
The present study demonstrated that Enterobacteriaceae, the predominant pathogens causing UTIs in children, show significant resistance to ampicillin and cephalexin.Therefore this higher resistance status should be taken into consideration in the prescription of antibiotics for empirical use or for the development of antibiotic guidelines.This study also showed the importance of identifying Proteus species and reporting nitrofurantoin as resistant in the antibiotic sensitivity report irrespective of in vitro susceptibility status.

Table 1 Distribution of Gram negative causative agents
Of the 43 Proteus species isolated 38 (88.4%) were identified in males.All urea positive isolates which could belong to Proteus, Providencia, and Morganella species were reported as Proteus spp. as it is the commonest pathogen of these organisms causing UTIs.The distribution of Gram positive urinary tract causative agents is shown in Table2.

Table 3 : Percentage sensitivity of the antibiotics to Gram negative organisms
Proteus species showed the highest sensitivity to nalidixic acid and gentamicin and the lowest sensitivity to nitrofurantoin which cannot be used in treatment due to the urea splitting action of Proteus species.Therefore, nitrofurantoin was reported as resistant if the organism was identified as Proteus species irrespective of in vitro sensitivity results.Cefoxitin sensitivity of Staphylococcus aureus and coagulase negative staphylococci were 50% and 26.3% respectively.Of the Enterococci species.63% were sensitive to ampicillin.
Of the Gram negative isolates LF and NLF Coliforms (excluding Proteus, Providencia, and Morganella spp.) showed the highest sensitivity to nitrofurantoin, gentamicin, and cefotaxime and the lowest to ampicillin.