Prevalence of Extended Spectrum Beta-Lactamase Producing Escherichia coli and Klebsiella species from Urinary Specimens of Children attending Friendship International Children ’ s Hospital

Extended-spectrum β-lactamase producing E. coli and K. pneumoniae is a serious threat to the patients. These organisms are major extended spectrum beta lactamase (ESBL) producers. The objective of this study was to determine the prevalence of Extended spectrum βlactamase producing strains of Escherichia coli and Klebsiella spp isolates from the urine sample of children visiting International Friendship Children Hospital. During the seven months, between June 2016 to December 2016, 1018 midstream urine samples(MSU) were collected from patients suspected of having UTI. The samples were investigated by conventional semi-quantitative culture technique and identification of E. coli and Klebsiella spp. was done by microscopy and biochemical test. Antibiotic susceptibility test of isolates was performed by modified Kirby Bauer Disc diffusion test. ESBL screening test was done by using 3rd generation Cephalosporin and confirmation done by combination disc diffusion method. Out of total 1018 MSU samples investigated, 200(19.64%) isolates of E. coli and 28(2.7%) isolates of Klebsiella spp. making a total of 228(22.39%) were found to cause significant bacteriuria. 76(33.33%) isolates, from those causing significant bacteriuria, were Multi-drug resistant organisms. Out of 228 isolates, 54(23.68%) were ESBL producers, that includes 51(25.5%) Escherichia coli and 3(12.5%) Klebsiella pneumoniae. ESBL producers were more common in in-patient (36.17%) than out-patient (20.44%). Most of the ESBL producers were resistance to amoxicillin, followed by Cotrimoxazole and Ciprofloxacin respectively. They were highly sensitive to Imipenem, Tigecycline, Amikacin, Piperacillin-Tazobactam, and Nitrofurantoin. High prevalence of ESBL producing E. coli and Klebsiella pneumoniae was found among children. Regular and routine monitoring of ESBL producing isolates is essential.


Introduction
Urinary tract infection (UTI) is a term applied to a variety of clinical conditions ranging in severity from asymptomatic which is carrier status in the urine to symptomatic acute infection of the kidney with resultant sepsis [1].The clinical symptoms of UTI usually include frequency, dysuria, pyuria, suprapubic tenderness, back pain, fever and urgency [2].The most common uropathogenic Gram negative bacteria are Escherichia coli and Klebsiella pneumoniae [3].Extended-spectrum β lactamases (ESBLs) are a group of enzymes with the ability to hydrolyze and cause resistance to the oxyiminocephalosporins (i.e.cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefepime) monobactams (i.e.aztreonam) [4].Beta-lactamases are among the most heterogeneous group of resistance enzymes.Despite a significant amount of amino acid sequence variability, betalactamases share a common overall topology [5].There are several ESBLs genotypes.These are SHV, TEM, and CTX-M [6].Other clinically important genotypes include OXA, VEB, PER, BES-1, BEL-1, SFO-1, TLA, and IBC [7]."Classical" ESBLs are derived from TEM and SHV enzymes whereas "Non-Classical" ESBLs are derived from enzymes other than TEM or SHV.90% of ampicillin resistance in E. coli is due to the production of .Based on different combinations of changes, currently, 195 TEM-type enzymes have been described.TEM-2, the first variant described, differed from TEM-1 through the substitution of a lysine for a glutamine at position 39 [6].A shift in the distribution of different ESBLs has recently occurred in Europe, with a dramatic increase of CTX-M enzymes over TEM and SHV variants [9].Since 2000, E. coli producing CTX-M β-lactamases have emerged worldwide as an important cause of communityonset urinary tract infections (UTIs) and this has been called the CTX-M pandemic [10].These enzymes were named for their greater activity against cefotaxime than other oxyimino-betalactam substrates (e.g.ceftazidime, ceftriaxone, or cefepime) [7].Rather than arising from mutation, they represent examples of plasmid acquisition of beta-lactamase genes normally found on the chromosome of Kluyvera species, a group of rarely pathogenic commensal organisms [9].In France, a novel derivative of OXA-10 (numbered OXA-28) was found in a P. aeruginosa isolate [11].The rapid increasing rate of ESBL production among Enterobacteriaceae, mainly E. coli and Klebsiella pneumonia, have created a serious global public health problem [12].Availability of limited treatment options for the infections caused ESBL producing bacteria makes the treatment very difficult and often results into treatment failure [13].Scant number of studies had been reported from Nepal assessing the ESBL producers among E. coli and Klebsiella spp in children.Hence the study was carried out to determine the prevalence of ESBL producing Escherichia coli and Klebsiella spp isolates from the urine samples of children.

Sample collection and identification of bacteria
The cross-sectional study was carried out in Pathology Department of International Friendship Children Hospital, Maharajgunj, Kathmandu from June 2016 to December 2016.Patients under the age of 13 years or their guardians visiting the Pathology Department were directly interviewed for his/her clinical history during the sample collection.During the study period, 1018 midstream urine samples were collected and processed according to the standard laboratory methods by Forbes et al [14].Semi-quantitative culture technique was used to detect the presence of significant bacteriuria.The bacterial culture was done on MacConkey Agar (MA) and Blood Agar (BA) and incubated overnight at 37 o C for isolation of pure culture.Diagnosis of UTI was made when there were colony count exceeding 10 5 cfu/ml of urine specimen.The isolates were identified based on morphology, gram's staining, motility, and standard biochemical tests as described in Forbes et al [14].Modified Kirby-Bauer's disc diffusion method was employed for the antibiotic susceptibility test of potential pathogenic isolates as per standard technique on Muller Hilton Agar [15].Nitrofurantoin(300mcg), Amoxicillin (30mcg), Cotrimoxazole (25 mcg), Ciprofloxacin (5mcg), Ceftriaxone (30 mcg), Ceftazidime (30 mcg), Gentamicin (10 mcg), Imipenem (10 mcg) and Meropenem (10 mcg) were used for antimicrobial susceptibility testing.

Screening of ESBL producing strains of E. coli and K.pneumoniae
The screening test for the production of ESBL was performed using both Ceftazidime (CAZ) (30μg) and Ceftriaxone (CTR) (30μg) disks.If the zone of inhibition was less than or equal to 22mm for CAZ and/or less than or equal to 25mm for CTR, the isolate was considered a potential ESBL-producer as recommended by CLSI [16].

Phenotypic confirmation of ESBL production
Susceptible screened ESBL producers were subjected to combined disk test as recommended by the CLSI [16].Combination disk method used for the confirmation of ESBL-producing strains in which CAZ (30μg), alone and in combination with Clavulanic acid (CA) (10μg) were used.After incubating overnight at 37°C, ≥ 5 mm increase in the zone diameter for either antimicrobial agent which was tested in combination with Clavulanic acid (CAC) versus its zone when tested alone, was interpreted as positive for ESBL production.1).Most of the patient were of age group 1-5 (60.96%) followed by age <1 (18.42%) (Table 2).Out of 75 isolates from male, 12(16%) were ESBL producer and out of 153 isolates from female 42(27.54%)were ESBL producer.More female was infected as compared to male, also chi-square test suggests a significant association in between sex of a patient and ESBL producers(p-value<0.05) (Table 7).9).All of the 3 ESBL producing K. pneumoniae isolates were sensitive to Imipenem, Meropenem, Piperacillin-tazobactam, tigecycline, and Amikacin.All of the isolates were resistant to Cefepime and Amoxicillin.[20].The rate of infection found in female patients was 163/684 (23.83%) and in male, the rate of infections was found to be 80/354 (22.59%).There wasn't any huge difference between rate of infection in male and female children patient.This result is in contrast to the earlier studies by Thakur et al (2013) where 56.64% female and 43.36% male patient were infected [21].The growth positivity with 33.5% among female patients and 23.7% in male patients was observed in a similar study by Baral et al (2012)[22].The predominant isolate was E. coli (82.30%) followed by Klebsiella species (11.53%) and Staphylococcus aureus (3.29%).E. coli is a predominant isolate, because E. coli can bind to the glycol-conjugate receptor of the uroepithelial cells of human urinary tract so it can initiate infection itself.E. coli is isolated in 90% of infection and strains are characterized by presence of unique virulence determinant the pilus (Gal-Gal) receptor [23].Similar result was obtained by other studies [24,25].E. coli was found to be resistant towards amoxicillin (81.5%),Co-trimoxazole (35%), and Ciprofloxacin (38%).In our study, 25% of K. pneumoniae were resistant to both Ceftriaxone and Ceftazidime.Other studies from Nepal reported that the resistance rates of K. pneumoniae to thirdgeneration Cephalosporin were between 20% to 75% [26,27].Likewise, 25% and 8.3% of K. pneumoniae were found resistant to Nitrofurantoin and Gentamicin, respectively.All K. pneumoniae strains were resistant (100%) to Amoxicillin, while resistance rate for E. coli was 81.5%.Similar result was observed in a study carried out at Madagascar, where 80% of the E. coli isolates were resistant to amoxicillin [28].In our study, E. coli showed a high resistance rate 68% to Co-trimoxazole whereas the K. pneumoniae showed resistance rate 30% to Co-trimoxazole.A comparable resistance rate of 80% and 45% to Cotrimoxazole was shown among ESBL producing E. coli and K. pneumoniae isolates in a study conducted in Iran [29].Bazzaz et al (2009) reported ESBL-producing isolates of K. pneumoniae and E. coli as 59.2% [30].In this study, 17% isolates of K. pneumoniae were resistant to Ciprofloxacin.Resistant to fluoroquinolones is due to the result of alterations in target enzyme (DNA gyrase and topoisomerase IV) and because of change in drug entry and efflux [31].Klebsiella pneumoniae sensitivity towards Gentamicin was found in 79.2% which was significantly higher than that was reported in Karanchi, Pakistan where a resistance rate for gentamicin was 46.7% [32].However, the rate was only slightly higher than to the findings from previous study done in Nepal where the sensitivity was 72.9% [22].These significant variations may be attributed to selective pressures ESBL occurrence among E. coli and Klebsiella is of great concern since infections caused by these bacteria are very common and resistance of the organism may be due to the presence of capsule that gives some level of protection to the cells, presence of multidrug resistance efflux pump, they also spread easily, are pathogenic and efficient at acquiring and disseminating resistance plasmid [42].More female (27.45%) were infected as compared to male (16%).Similar result was obtained by Bhandari (2013) [17].

Results
Though some of our results were very much contrast with other studies, the prevalence rate of ESBL producing isolates in our study was considered significant and high.The higher number of ESBL producers might be due to more reliance on third generation Cephalosporins to treat Gram negative infections and unscrupulous hospital antibiotic policy.Moreover, high prevalence of ESBL isolates among children might be due to immaturity of the immune system in these infection in these age group.Further studies are required to know the current burden in these population.

Conclusion
High prevalence of ESBL producing E. coli and K. pneumoniae was observed in our study.E. coli is predominant ESBL producers than K. pneumoniae.Children under age five were found to be highly infected with urinary tract infection.Escherichia coli and Klebsiella spp are emerging highly as a multi-drug resistant.Imipenem, Tigecycline, Amikacin, Piperacillin-Tazobactam were found to be the most effective drug against the ESBL producing isolates.Nitrofurantoin and Gentamycin can be used as a drug of choice against non-ESBL producing isolates.

DECLARATION Ethics approval and consent to participate
Ethical approval was taken from the Ethical Review committee of International Friendship Children's Hospital and Tri-Chandra Multiple Campus.Informed consent form was obtained from parents of the participants before their participation.

Competing interests
We have read Nepal journal of biotechnology policy on declaration of competing interest and declare that we have no competing interests.

Table 2 :
Demographic distribution of E. coli and

Table 4 :
Antibiotic susceptibility pattern of K. pneumoniae isolates.

Table 5 :
Distribution of MDR isolates

Table 6 :
Detection of ESBL by combination disk method.

Table 7 :
Gender wise distribution of ESBL producing isolates.

Table 8 :
Department wise distribution of ESBL producers.
In our study, 25.5% of E. coli isolates and 12.5% of Klebsiella pneumoniae isolates were ESBL producer.These findings were comparable to the study done byChander and Shrestha (2013)in Nepal tertiary hospital in which 13.51% of E. coli and 16.55% of Klebsiella pneumoniae were ESBL producers [41].

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