Fecal carriage of Extended Spectrum β-Lactamases ( ESBL ) Producing Escherichia coli and Klebsiella spp . among School Children in Pokhara , Nepal

Extended-spectrum β-lactamases (ESBL) producing microbes in recent years have been a major problem in developing countries like Nepal, with limited treatment options. This study aimed to determine the prevalence of ESBL producing E. coli and Klebsiella spp. in school children in Pokhara, Nepal. The study was conducted from June to October, 2015 at the microbiology laboratory of Manipal Teaching Hospital, Pokhara, Nepal. Antibiotic Susceptibility Test (AST) was done after isolation and identification of bacterial isolates. Then, presence of ESBL enzymes in E. coli and Klebsiella spp. were tested by combination disc diffusion test using cefotaxime and ceftazidime alone and with clavulanic acid. Out of total 309 school children, 211 (68%) bacterial isolates were detected from stool samples. Among them, E. coli and Klebsiella spp. were detected in 97 (46%) and 39 (19%) stool samples respectively. Bacteria isolated from 14 (5%) stool samples were multi-drug resistant (MDR) positive. After applying combined disk method, 88 (29%) isolates were found to be ESBL producer. Emerging prevalence rate of ESBL producing E. coli and Klebsiella spp. are major problem in medical history. Therefore, rapid need of surveillance for effective management of such MDR-strain is required.

Patients suffering from ESBL producers have high mortality, longer hospital stay, high health care cost, and longer antibiotic therapy as compared to those patients suffering from non-ESBL producers [9].Furthermore, they pose significant therapeutic challenges in the daily management of infectious diseases due to their resistance to additional classes of antibiotics reducing the effectiveness of alternative antimicrobial regimens [10].Worldwide, several studies [1,5,6,8] have been conducted but only a few data are available concerning the genetic characterization of clinical isolates from Nepal [7,11].The acquisition and expression of ESBL enzymes among E. coli and Klebsiella spp.have posed a serious public health problem.This study was conducted to determine the ESBL producing bacteria in school going children without any clinical symptoms to observe the fecal carriage.

Study designing and setting
The study was conducted in Pokhara Lekhanath Municipality-26, where samples were collected

Sample collection and Processing
Around one gram of fresh stool sample was collected in a clean, well labeled, and sterile screw-capped plastic container.Collected samples were kept in an icebox, transported within 1 hour, and processed immediately following Standard Operating Procedures (SOPs) of Microbiology to the laboratory of Manipal Teaching Hospital (MTH) in Pokhara, Nepal.If there was a delay in processing the specimens were kept in a freezer at 2-8°C.The general procedure of sample processing were explained in the flowchart (Figure 1).

Isolation and identification of E. coli and Klebsiella spp.by culture method
Stool samples were inoculated onto MacConkey agar plates, which were incubated at 37°C for 24 hours.After proper incubation, plates were observed for the growth of the bacteria.Isolated colonies were sub-cultured in nutrient agar and then plates were incubated at 37°C for 24 hours.Gram-positive and negative bacteria were initially confirmed by gram staining and thereafter, identified by conventional biochemical tests such as (MRVP, Indole, motility, protease, citrate, & O/F).

Screening and Confirmation of ESBL producing Strains
All the E. coli isolates were subjected to the screening test for ESBL detection.Screening test for ESBL detection was done according to the CLSI guidelines.Isolates showing inhibition zone size of ≤ 22 mm with ceftazidime (30 μg), ≤ 25 mm with ceftriaxone (30 μg), and ≤ 27 mm with cefotaxime (30 μg) were interpreted as screening test positive for ESBL production [18].For the confirmatory test for ESBL, two or three colonies of organisms were suspended in 0.5 ml of sterile broth and the turbidity matched to 0.5 McFarland.Using a sterile cotton swab the broth culture was uniformly swabbed on MHA.All the E. coli isolates which were resistant to at least ceftazidime, ceftriaxone and/or cefotaxime were subjected to the ESBL confirmatory test using ceftazidime (30 μg) and ceftazidime-clavulanic acid (30 μg & 10 μg) and the cefotaxime (30 μg) and cefotaxime-clavulanic acid (30 μg & 10 μg) combination disks.The tests were interpreted and a difference of 5 mm between zone of inhibition of a single disk and in combination with clavulanic acid (inhibitor) was confirmed to be produced by an ESBL positive isolate [18].Klebsiella pneumoniae ATCC 700603 was used as a control strain.

Statistical analysis
Pearson's Chi-square test was used to determine the significant association of dependable variables.WinPepi software (version 11.65) was used for quantitative data analysis.A p-value < 0.05 was considered to be statistically significant at 95 % CI.

Discussion
ESBL producing gram-negative bacilli, particularly K. pneumoniae and E.coli, recently arose worldwide in a hospital and Community-Acquired Infections (CAIs) as serious pathogens, and has been progressively rising.This upsurge introduces additional difficulties in treating patients affected by EBSLs since it puts at risk the activity of numerous wide-spectrum antibiotics [7].The present study exhibits that out of the 211 clinical isolates, 88 (42%) were found to be ESBL producers, and these results were in accordance with a previously conducted study [11,18].Additionally, the ratio of positive cases for the male to female children in this study was 1.2:1, which contrasted another study where the ratio for male to female patients was 1:2.2 [7].However, this difference in the ratio may be due to a gender bias of male and female samples initially in both the experiments.Thus, gender may not play a role in ESBL susceptibility of a person.Furthermore, E. coli positive cases were found in 97 (31%) clinical isolates, which was the predominant organism in this case.Conversely, another study found E. coli to be positive in 81% of their samples, which is remarkably higher than that of our study [7].The high occurrence of positive result may suggest that there is a possibility of normal flora of human E. coli.Anti-microbial Resistance (AMR) is a major public health problem in patient care in both developed and developing countries.Therefore, in this study, we analyzed eight different antibiotics.Among eight antibiotics, Piperacillin-Tazobactam resistant was predominant in both E. coli (62%) and K. pneumoniae (67%), which differed from previous studies [13-15].Piperacillin-Tazobactam is commonly used to treat intraabdominal, lower respiratory, urinary tract, and gynecological and skin/soft tissue infections, as well as for fever in patients with neutropenia [16].The predominance of the resistant bacteria for these antibiotics could imply that illnesses associated to E. coli & K. spp.are fairly prevalent in the Pokhara and have been treated just as normally.In addition, the rates of resistance to cefotaxime, ceftazidime, gentamycin, and amikacin for E. coli presented in this study are 50%, 60%, 10%, and 11% respectively.However, this contrasts another study, where the resistance of E. coli were found 99%, 78%, 29% and 4% to cefotaxime, ceftazidime, gentamycin, and amikacin respectively [17].This may suggest that the E. coli collected from Nepal has not been exposed to the four antibiotics quite as much as in other more developed places, which could probably mean that it is still possible to more easily manage the ESBLs in Nepal effectively

E. coli (%)
compared to places with higher antibiotic exposure and resistance.From the eight antibiotics that were analyzed, Imipenem was the most effective drug; however, it should not be administered as the empirical drug unless the infection is life-threatening, since carbapenems are considered the drug of last resort.If this situation is not taken seriously and misuse the carbapenem drugs then carbapenem resistant bacteria may evolve.In this study, 4.5% MDR strains were detected, which was very low compared with other studies 64.0%, and 64.9% [19].This may be the case due to the overexposure of the antibiotics to the bacteria in external environments and animals, whereas in the related district it may not be the case.Furthermore, 84% E. coli was confirmed as ESBL producer followed by K. pneumoniae, (9%) and K. oxytoca (7%).A similar result found in the study where the researchers reported 91% E. coli and 8% K. pneumonia [7].Conversely, the findings of another study [20] showed the variable result with E. coli isolates, 33(9%) were ESBL producers.The identification of ESBL producing organisms is a major challenge in the clinical world and, due to the selective pressure caused by heavy use of expanded-spectrum cephalosporins, lapses in effective infection control measures and affinity of these enzymes for different substrates, outbreaks are increasing [21].An antibacterial choice is often complicated by MDR leading to over-prescription and misuse of antibiotics.As indicated by the present and previous findings, it appears to be mandatory to include ESBL detection in routine laboratory practice so as to limit the rapid spread of ESBL producing organisms.AMR can be tackled by following continuously the 5 steps viz.awareness; surveillance; stewardship; research & innovation; infection prevention & control accordance with the "One Health" concept in agriculture, veterinary, environment and human.

Limitation of study:
We

Figure 1 :
Figure 1: Flowchart of isolation and identification of E. coli and Klebsiella spp.from collected stool samples

in nosocomial Escherichia coli from urinary tract infections in Pakistan. Afr J Biotechnol. 2009, 8(16): 3921-3926]
couldn't perform genotyping test and MBL test in bacterial isolates due to a limited resource and time limitation.available as per reasonable request to the corresponding author.