Multi Drug Resistant Pathogens Causing Urinary Tract Infections in Children at Kathmandu Model Hospital

Introduction: Antibiotic resistance of urinary tract pathogens has increased globally. Updated knowledge of the antibiotic resistance patterns of uropathogens in the health institutes is important for the selection of an appropriate empirical antimicrobial therapy. The aim of this study was to evaluate the multi drug resistant urinary isolates in the children from 1 to15 years and evaluate the options for empiric antibiotic therapy. Materials and Methods: The study was conducted from December 2011 to May 2012 in the Bacteriology laboratory, Kathmandu Model Hospital. Urine samples received in the laboratory were processed for routine, culture and its sensitivity. The antimicrobial susceptibility of bacterial isolates was determined following Clinical and Laboratory Standard Institute (CLSI) recommended Kirby-Bauer Disc Diffusion method. Results: Of the total 372 urine samples received in the laboratory, 60 (16.13%) showed significant growth; of which 55.0 % (33/60) were MDR isolates. Escherichia coli were the predominant isolate from urine sample. Out of 49 Escherichia coli isolates, 27 (45.0%) were Multi drug resistant. Enterococcus faecalis (N=3) was the most predominant Gram positive isolate and 66.67 % (2/3) of this organism were multi drug resistant. Among the first line drugs used against gram negative isolates, nitrofurantoin was the most effective drug followed by quinolones, while among the second line drugs; meropenem was the most effective drug followed by chloramphenicol and amikacin, whereas; nitrofurantoin (100%) was the most effective drug for Gram positive isolates followed by norfloxacin and cefotaxime. Conclusion: High percentages of multi drug resistant uropathogens were revealed in children. Nitrofurantoin was found to be the most effective drug for gram positive, gram negative and multi drug resistant isolates.


Introduction
U rinary tract infection (UTI) is defi ned as bac teriuria along with urinary symptoms and is identifi ed by growth of a signifi cant number of organisms of single species in the urine.Urinary tract infections are common medical problems in children and are important cause of morbidity.UTI during childhood varies by age and gender.UTI commonly aff ects boys during the fi rst year of life 1 , but thereafter 3-5% of girls are aff ec ted1, increasing to 10% by the teenage years 2 .Although several diff erent microorganisms can cause UTI, including fungi and viruses, bacteria are the major causative organisms and responsible for more than 95% of UTI cases 3 .Treatment of urinary tract infections is compromised worldwide by the emergence of bacteria that are resistant to multiple antibiotics 4 .Overuse and use of incomplete course of antibiotics as well as empirical antimicrobial therapy has been the major contributing factor in the development of Multi Drug Resistant(MDR) bacteria 5 .
Multidrug resistance is defi ned as resistance to two or more diff erent structural classes of antimicrobial agents 6 .There is growing concern regarding the resistance to uropathogens to antibiotics.The clinical impact of drug resistance may be great or insignifi cant, depending on the level of resistance, the site of infection, and the availability of eff ective, nontoxic therapeutic alternatives 7 .This prospective study was conducted to identify UTI due to multidrug resistant uropathogens among children and to evaluate empiric antibiotic therapy.

Materials and Methods
This is the prospective study conducted at Kathmandu Model Hospital on Pediatric patients (1-15 years) attending for the treatment of suspected UTI case.Three hundred and seventy two urine samples were examined from clinically suspected urinary tract infection during the research period of six months (December 2011-May 2012) using culture and sensitivity tests.All the patient or parents of the patient were instructed carefully for collection of morning midstream urine specimens.They were given a sterile, dry and clean collection bottles for urine collection.All the urine specimens were processed within 30 minutes of collection.
Culture of all urine specimens was done on 5% Blood Agar and Mac Conkey Agar plate utilizing semiquantitative culture method (Fig 1).Shaking with hand to ensure a uniform suspension of bacteria vigorously mixed the urine specimens.Then, an inoculating loop of standard dimension was used to take up approximately fi xed and known volume (0.001ml) of mixed urine and placed on the center of the plate.The drop was spread in a line and then over the entire surface of the agar plate.After inoculation, the culture plates were incubated in an inverted position at 37 o C for 24 hours. 8After 24 hours, the numbers of colonies were counted on each plate, which was multiplied by 1000 to calculate the number of organisms per ml in the specimen.Samples showing 10 5 or more organisms per ml of urine were taken as signifi cant.Colony counts less than this was considered as non-signifi cant 8 .
Gram's staining and various biochemical tests identifi ed pure culture of bacterial growth.Diff erent biochemical media used were Triple Sugar Iron Agar, Sulphite Indole motility Agar, Urease agar, Simmons's Citrate Agar, Methyl Red / Voges Proskauer Test and Oxidation Fermentation medium.Catalase, Coagulase and Oxidase Tests were also performed.The antibiotics used as fi rst line drugs for Gram negative bacteria were Amoxycillin (10 mcg), Cefotaxime (30 mcg), Ciprofl oxacin .The antibiotic impregnated discs were placed on the surface of the agar plate and then incubated at 37 o C for 18 hours 9 .The diff erent inhibition zones were measured and interpreted the results on the basis of zone size compared with standard interpretive table given by manufacturer.The organisms which showed resistant to all fi rst line antibiotics except Nitrofurantoin were tested for second line drugs.

Results
Of the 372 urine samples processed, 60 (16.13%) samples showed signifi cant growth where as majority of samples i.e. 284 (76.34%) showed no growth and 28 (7.53%) showed growth of no signifi cance (Table 1 UTI was signifi cantly high in female children (p=0.004) in comparison to male.There was no signifi cant diff erence seen in MDR infection between male and female (p=0.8).However, the signifi cant diff erence was seen in MDR infection between age group 1 to 5 and above 5 year's group (p=0.0001)Gram negative bacteria were predominant; constituting 56 (93.33%) of the total 60 isolates and 33(55.0%)were MDR.Among Gram negatives, Escherichia coli were the most frequently isolated species with 49 (81.67%).Gram positive organisms constituted only 4 (6.67%) of total isolates, and 2 (3.33%) of them were MDR.Both MDR isolates were Enterococcus faecalis (Table 3).Among the common antibiotics used as fi rst line against gram negative isolates, nitrofurantoin showed a susceptibility of 55/56 (98.21%).Quinolones (Ciprofl oxacin, Norfl oxacin and Ofl oxacin) followed Nitrofurantoin with susceptibility of 38/56 (67.86%).Among the second line antibiotics used, Meropenem was found to be most eff ective drug with susceptibility of 16/17 (94.12%) followed by chloramphenicol and Amikacin with a susceptibility of 15/17 (88.24%).Most of the Gram negative isolates i.e. 45 (80.36%) were resistant to Amoxycillin (Table 4, 5).
Among the gram positive isolates, Nitrofurantoin was the most eff ective drug with susceptibility of 4/4 (100%) which was followed by Cefotaxime and Norfl oxacin (Table 6).
Out of 60 isolates, 24 (40.0%)isolates were resistant to >3 drugs where as only 3 isolates of Escherichia coli and one isolate of Salmonella Paratyphi were sensitive to all antibiotics used.Among 56 gram negative isolates, 31(51.67%)isolates were MDR whereas of the 4 gram positive isolates, 2 (50.0%) isolates were MDR (Table 7).

Discussion
The emergence of multi drug resistance in uropathogens is of great public health concern.Prevalence of these organisms varies according to species, antibiotic use and geographical area.The increasing prevalence of infections caused by antibiotic resistant bacteria makes the empirical treatment of UTIs diffi cult.In the current study, we isolated 60 (16.13%) uropathogens among 372 urine culture samples.The study demonstrates that E. coli (81.67%) remains the leading uropathogen responsible for UTIs which was supported by several previous studies. 10,11The frequency of UTI is greater in female children as compared to male 10,11,12 and our results were similar to these reports showing 68.33% of patients were female.
Paediatric UTI causing bacteria are becoming increasingly resistant to commonly used antibiotics such as fl uroquinolones and third generation cephalosporins.Cotrimoxazole (Trimethoprim-sulphamethoxazole), fl uroquinolones, or nitrofurantoin are recommended for empirical treatment of uncomplicated UTI 13,14 .However, several reports from worldwide indicated the excessive increase in the emergence of trimethoprimsulphamethoxazole resistant E. coli 15,16 .Cotrimoxazole was replaced by fl uroquinolones and cephalosporins because of high level of resistance to this drug but unfortunately after sometime resistance to these drugs was also detected and published.Our study also showed the similar fi ndings with 28.5-30.35%resistance to quinolones, 39.2-50.0%resistance to cephalosporins and 48.21% resistance to cotrimoxazole for gram negative isolates 17 .The results showed a considerable increase in resistivity of gram negative isolates to amoxycillin (80.36%) which was supported by several studies 11,12,18 .Nitrofurantoin demonstrated better activity against gram negative (98.21% susceptible) as well as gram positive isolates (100.0%susceptible), in agreement with data published by others 17,18,19 .The high level susceptibility of uropathogens to nitrofurantoin may be the narrow spectrum of activity, narrow tissue distribution (low or undetectable serum concentration) and limited contact with bacteria outside the urinary tract 20 .According to our study, the fi rst line antibiotics to be used for the treatment of UTI is nitrofurantoin.In our study, we defi ned those organisms as MDR which were resistant to two or more diff erent structural classes of antibiotics 6 .According to this, 33 (55.0%)MDR isolates were detected.Of the 33 MDR isolates, 27(45.0%)were E. coli.This result was supported by previous other studies 21,22,23 .Our study demonstrated the highest resistance to Ceftriaxone (94.12%) among 17 MDR isolates.This may be due to the production of ESBL enzymes or other resistance mechanisms which could not be addressed because of limited resources.The resistance to cephalosporin is explained though the enzymatic mechanisms and effl ux pumps 24 .It has been reported that pathogenic E. coli isolates have relatively high potential for developing resistance. 25Among the antibiotics used in the second line, meropenem was the most active drug with susceptibility of 94.12% followed by amikacin and chloramphenicol with susceptibility of 88.24%.These fi ndings reveals stronger propensity of uropathogens towards multiple drugs resistance limiting few therapeutic options for the treatment.

Conclusion
The results of the present study suggest that prevalence of MDR E. coli is alarmingly high and the most appropriate fi rst line oral antibiotic for empiric treatment of uri nary tract infection at our hospital is nitrofu rantoin and meropenem, amikacin and chloramphenicol as second line agents.Antibacterial resistance patterns need to be up dated periodically to ensure proper empiric treatment of UTI.

Fig 1 :Fig 2 :
Fig 1: Signifi cant Growth of E. coli in Mac Conkey Agar ).The age and gender wise distribution of children with UTI is shown in Table 2. UTI was commonly found in young female children of age 1-5 years.MDR isolates were common in young children (46.67%) of the 1-5 years age group.Children of age group 6-10 years showed three (5.0%) and age group 11-15 years showed only two (3.34%)MDR isolates.Of the total MDR isolates, 31.67 occurred in young female children of age 1-5 years.

Table 3 :
Pattern of microbial isolates

Table 1 :
Growth profi le of urine sample

Table 2 :
Age and gender wise distribution of infected patients with MDR isolates

Table 4 :
Antibiotic Susceptibility Pattern of Gram-negative Isolates towards fi rst line antibiotics

Table 5 :
Antibiotic Susceptibility Pattern of gram negative isolates towards second line antibiotics

Table 6 :
Antibiotic Susceptibility Pattern of Gram-positive Isolates

Table 7 :
Status of antibiotic resistance among MDR isolatesMulti Drug Resistant Pathogens Causing Urinary Tract Infections in Children at Kathmandu Model Hospital