Detection of extended-spectrum beta-lactamase-producing E. coli and Klebsiella spp. in effluents of different hospitals sewage in Biratnagar, Nepal

Objective This study was aimed to determine prevalence and resistance pattern like multidrug resistant (MDR) or ESBL nature of E. coli and Klebsiella spp. from various sewage drain samples with an idea to deliver baseline information that could be utilized for defining guidelines for the treatment of hospital sewages. Results Of 10 sewage samples analyzed, 7 (70%) contained E. coli while 6 (60%) contained Klebsiella. Except one sample, all positive samples contained both E. coli and Klebsiella spp. E. coli isolates were resistant to ampicillin, amoxicillin, cefoxitin, cefuroxime, and cefpodoxime; while 85.7% were resistant to amoxicillin/clavulanate, ceftazidime, cefotaxime and ceftriaxone. 71.4%, 57.1%, 42.9%, and 28.6% were resistant to aztreonam, trimethoprim/sulfamethoxazole, nitrofurantoin, and gentamicin. Most were sensitive to chloramphenicol, ofloxacin, ciprofloxacin, and azithromycin. 85.7% and 57.1% of E. coli were MDR and ESBL isolates, respectively. Klebsiella were resistant to ampicillin, amoxicillin, and amoxicillin/clavulanate. 83.4% of Klebsiella were resistant to cefoxitin. 66.7% of strains were resistant to cefuroxime, ceftazidime, cefotaxime, ceftriaxone, and cefpodoxime. Klebsiella showed 50% resistant to aztreonam and trimethoprim/sulfamethoxazole, while 33.3% were resistant to chloramphenicol, nitrofurantoin, ofloxacin, and ciprofloxacin. Klebsiella were sensitive to azithromycin and gentamicin. 66.7% and 33.3% of Klebsiella were MDR and ESBL isolates, respectively.


Introduction
Sewage designates raw sewage, sewage sludge or septic tank waste containing about 95.5% water and 0.1% to 0.5% organic and inorganic materials. Hospital wastewaters are generated in different sections of hospital like surgery units, ICU, laboratories, patient wards, clinical wards, laundries and possess a quite variable compositions depending on the activities involved [1]. A variety of microorganisms are present in water for examples bacteria, fungi, protozoa etc. Bacteria like Shigella, E. coli, Klebsiella, Vibrio, Salmonella, etc. are found in sewage drain water [2]. Coliforms are mainly from family Enterobacteriaceae that are aerobic or facultative aerobic, gram negative, non-spore forming enteric bacilli and basically found in human colon which are introduced into environment by human feces [3,4].
Antibiotics are fractionally metabolized by patients and are then ejected into the hospital sewage. Along with excreta, they pass through sewage system and end up in the environment, mainly in the water area [5]. Hospital sewage discharge a variety of multi resistant bacteria and substances like antimicrobial, pharmaceutical, disinfectants, heavy metals, radioisotopes, and drugs not metabolized by patients [6].
Multiple drug resistance (MDR) for Gram negative and Gram-positive bacteria means "resistant to three or more antimicrobial classes. " Extended-spectrum β-lactamases Open Access BMC Research Notes *Correspondence: Mahato.sanjay@gmail.com (ESBL) are enzymes that impart resistant to extendedspectrum (third generation) cephalosporins (e.g. ceftazidime, cefotaxime and ceftriaxone) and monobactams (e.g. aztreonam). The most common ESBL producing bacteria are few strains of E. coli and Klebsiella pneumoniae [7,8].
The main aim of this study was to determine prevalence and resistance pattern like MDR or ESBL nature of E. coli and Klebsiella pneumoniae from various sewage drain samples since they can cause serious public health problem. This study could deliver baseline information that could be utilized for defining guidelines for the treatment of hospital sewages.

Sample collection method and characteristics
During March to October 2018, a total of 10 sewages samples were collected aseptically from different hospitals of Biratnagar city (Table 1). For this, the sample was collected nearby the center of the flow channel, at approximately 10-15 cm depth from the water surface, where the turbulence was at maximum and the possibility of settling was minimized. Skimming the water surface or dragging the bottle was avoided. The sewage water was first mixed and then 500 ml sample was taken in the sterile high-density polyethylene (HDPE) bottle aseptically. Each sample bottle was properly labelled with date, code number and time with the help of the marker.

Sample transportation and processing
Samples collected were placed on 4 °C ice box to inhibit the growth of microorganisms and were immediately transported (within 2 h) to microbiology research lab for the analysis. Distilled water was used as control during analysis.

Isolation and identification of E. coli and Klebsiella spp.
The samples collected from the hospital wastewater were serially diluted in 0.85% saline water and dilution 10 −2 and 10 −3 were inoculated onto Eosin Methylene Blue (EMB) Agar and MacConkey Agar for Escherichia coli and Klebsiella spp. by spread plate method and were incubated aerobically at 37 °C for 24-48 h. After incubation, colonies were picked on their colony morphology like colonial appearance, size, elevation, color, margin, and opacity. All the selected colonies were, then, subcultured on nutrient agar plate to obtain pure culture for the microscopic and biochemical identification. TSI (triple sugar iron), SIM (sulfate/indole/motility), Methyl Red test, Voges-Proskauer test, citrate agar, catalase test, oxidase test, and urea hydrolysis test were performed to identify the organisms [9].

Antimicrobial susceptibility tests
The identified isolates of Escherichia coli and Klebsiella spp. were submitted to antimicrobial susceptibility testing according to the guidelines of the Clinical and Laboratory Standards Institute

Criterion for multidrug resistance
Isolates which demonstrated the resistance to at least one agent in three or more classes of the drug were defined as multidrug resistant (MDR) [10,11].

ESBL detection
Isolates exhibiting a zone of inhibition of growth for ceftazidime and ceftriaxone ≤ 22 mm and ≤ 25 mm, respectively, were submitted to the combined disc test to check for ESBL-producing strains [12]. The combined disc methodology used to detect ESBL-producing E. coli and Klebsiella spp. was performed as per CLSI [10]. The antimicrobials used were ceftazidime (30 μg) and ceftazidime/clavulanic acid (30/10 μg), and cefotaxime (30 μg) and cefotaxime/clavulanic acid (30/10 μg). Results were interpreted as per the criteria established by the CLSI [10]. An increase of 5 mm in a zone of inhibition of growth for combined drugs to ceftazidime or cefotaxime were confirmatory for ESBL-producing strains [10,12].

Multiple antibiotic resistance (MAR) index
MAR index is the number of antibiotics to which test isolate displayed resistance divided by the total number of antibiotics to which the test organism has been evaluated for sensitivity. MAR index for each isolate was calculated as per the guidelines of Krumperman [13].

Data analysis
The data were statistically analyzed using Statistical Package for Social Sciences (SPSS v21) software package. Chi square test at p-value < 0.05 was considered statistically significant.
There is no significant relationship between the type of bacterial strains (like E. coli and Klebsiella) and their response to the antibiotics at df = 1, p = 0.05.

Discussion
The main aim of this study was to determine prevalence and resistance pattern like MDR or ESBL nature of E. coli and Klebsiella pneumoniae from various sewage drain samples. Presence of 70% of E. coli and 60% of Klebsiella in sewage may have direct link with the human feces in many cases [14]. Sewage mass is liquid mass containing excessive amount of organic matter which acts as a nutrient medium for all the bacteria [15]. Excessive number of MDR and ESBL E. coli and Klebsiella show that drainage system of Biratnagar hospitals is highly infectious and life threatening if contaminated with water and food [16].
The result of E. coli showing 100% resistance to ampicillin, amoxicillin, cefoxitin, cefuroxime, and cefpodoxime was higher than Belachew et al. [17] showing 91.3% resistance to ampicillin, 70% resistance to cefuroxime and ceftriaxone, Cefpodoxime (74%), amoxicillin/clavulanate (52%), cefoxitin (43%), ceftazidime (65%). In this study, 85.7% of E. coli were resistant to amoxicillin/ clavulanate, ceftazidime, cefotaxime and ceftriaxone. Resistance to nitrofurantoin was similar in Belachew et al. [17]. Resistance to aztreonam and chloramphenicol were higher than the findings of Florica et al. [18]. On the contrary, resistance to trimethoprim/sulfamethoxazole (57.1%), gentamicin (28.6%) and ciprofloxacin (14.3%) in our study was lower than Belachew et al. [17] showing 67%, 43%, and 52%, respectively. It has been observed that none of the hospitals in Biratnagar have waste treatment system as a result, 85.7% of E. coli species had multi-drug resistance, which is, higher compared to previously reported results in Ethiopia (78%) [17] and Romania (60.34%) [18]. Such a high resistance rate may be a Klebsiella pneumoniae showed 100% resistance to ampicillin and amoxicillin/clavulanate which was higher than the study of Ethiopia (94%) [17] and Romania (70.7%) [18]. Resistance to penicillin antibiotics like ampicillin has become very common in the world and our finding is in line with this evidence. Resistance of 66.7% for ceftazidime, cefotaxime, and ceftriaxone was higher than the findings of Romania [18] as 8.6%, 17.2% and 13.8%, respectively. Resistance shown by Trimethoprim/sulfamethoxazole (50%), chloramphenicol (33.3%) and ciprofloxacin (33.3%) were much higher than found in Romania as 22.4%, 5.2% and 6.9%. Similarly, the resistance to cefoxitin, cefuroxime, cefpodoxime, and nitrofurantoin in this study was found to be much higher than other studies [17]. A high rate of MDR (66.7%) was observed for Klebsiella spp. which was higher than results reported in Ethiopia (40.5%) [17], Romania (33%) [18] and Mexico (50%) [19]. However, MDR rate of the current finding was lower than previously reported results in Brazil (77.5%) [20]. Such variation may be due to the difference in antimicrobial use and availability of waste treatment system in hospital sewage [17].

Conclusion
This study builds the importance to enquire the involvement of hospital liquid waste discharge in the development and distribution of antibiotics resistance in the environment. There is rise in resistant bacteria like E. coli and Klebsiella in hospital wastewater. The government must implement some rules and laws for proper treatment of hospital wastewater before entry to main municipal wastewater. Sewage treatment plant must be established in hospital for their effluents and sludge coming from the hospital's units.

Limitations
The standard strain E. coli (ATCC 25922) and K. pneumoniae (ATCC 13883) could not be used.