Detection of extended spectrum beta lactamase from multidrug resistance Escherichia coli from various clinical sample in District Peshawar, Pakistan

Gram negative bacteria’s such as Escherichia coli is one of the most common community-acquired as well as the nosocomial pathogen responsible for a large number of infections. Extendedspectrum beta-lactamase producing strains of Escherichia coli has become a great therapeutic challenge to the clinicians in managing such infections. This study was carried out in order to find out multidrug-resistant Escherichia coli from various clinical samples. A total of 70 E. coli isolates were obtained from various clinical samples. These already identified samples were obtained from Lady Reading Hospital. Antibiotic susceptibility test was done by disc diffusion method. The present study shows that (55.71%) samples were ESBL positive. The percentage of isolates sensitive to current antibiotics were highest for Polymyxin b (98.57%) and highest for Ceftriaxone (12.85%). While the percentage of isolates resistant to current antibiotics were highest for that Cotrimoxazole (84.28%), and lowest for Polymyxin b (1.42%). ESBL producing strains of E. coli causes therapeutic failure and also contribute to multidrug resistance. Therefore monitoring of antimicrobial resistance in developing countries is necessary to optimize empiric treatment and the prudent use of antimicrobials.


Introduction
Escherichia coli is one of the most common community-acquired pathogens responsible for a large number of nosocomial infections [1]. E. coli is a gram-negative, anaerobic, rod-shaped, coliform bacterium of the genus Escherichia, commonly occur as normal flora of small intestine of warm-blooded organisms [2]. The majority of E. coli strains are harmless. These harmless strains are part of the normal flora of the gut [3-5], which are benefited by producing vitamin K in their hosts [6] and prevent the colonization of the intestine with other pathogenic bacteria [7]. The E. coli related disease for the first time is diagnosed in 1982 from undercooked cow meat and unpasteurized milk [8]. The transmission of E.coli occurs through contaminated water (water which comes in contact with the feces of animals), while using it for drinking, for washing vegetables and fruits, and swimming in contaminated pools and lakes. It is important to note that the age and immune system of a person affected by a disease play a major role in its progress. Recent studies show's that infants aged younger than 5 years are more susceptible to this bacterium. Eating undercooked meat and burgers, eating on surfaces at service restaurants, using the immune-suppressing medication can attract E. coli related disease [9]. Most of the E. coli strains do not cause any disease [10], but virulent strains are responsible for gastroenteritis, urinary tract infections, and neonatal meningitis. It can also cause severe abdominal cramps, diarrhea that normally turns bloody within 24 hours, and sometimes fever. In rarer cases, virulent strains are also responsible for bowel necrosis (tissue death) and perforation without progressing to the hemolytic-uremic syndrome, peritonitis, mastitis, septicemia, and Gram-negative pneumonia Although bacterial resistance to broadspectrum βlactam antibiotics are major concerns and the primary focus for clinicians and researcher, until recently not many studies have conducted in Peshawar, Pakistan to detect ESBL's producing E coli strains. The present study was conducted with an aim to determine the ESBL enzyme in multidrugresistant E. coli isolated from various clinical samples.

Materials and methods Study design
Prospective study was designed to determine the ESBL enzyme in multidrug-resistant E. coli isolated from various clinical samples.

Sampling
The study was centered on 70 samples of E. coli, which were collected from Lady Reading Hospital (LRH) in Peshawar, during the period of (February 2018 to August 2018). The Samples were obtained using sterile techniques to circumvent contamination Antibiotic sensitivity testing Antibiotic susceptibility test was done by applying the Kirby-Bauer disk diffusion method, according to the Clinical and Laboratory Standard Institute (CLSI). Commercially available antibiotic discs were used. For example Amikacin (AK), Cefepime (FEP), Ciprofloxacin (CIP), Ceftriaxone (CRO), Amoxicillin (AMC), Tazobactam (TZP), Imipenem (IPM), Co-trimoxazole (SXT) and Polymyxin b (PB). Phenotypic detection of ESBL ESBL production was confirmed phenotypically by Double-disc synergy test (DDST) according to the Clinical and Laboratory Standards Institute (CLSI) criteria for ESBL screening. According to the CLSI protocol, DDST was done by using amoxicillin (30 µg)/clavulanic acid (10 µg) and cefepime (30 µg). The discs were placed 25 mm apart from each other on Muller-Hinton agar (MHA) plate inoculated with 0.5 McFarland suspension of the tested isolates. The plate was incubated overnight at 37ºC. AMC may or may not be sensitive, and FEP will be resistant but will be sensitive upto some extent towards AMC disc, which is due synergistic effect of clavulanic acid. The window formation indicated the presence of ESBL

Statistical analysis
All the data were presented in graphs and figures and was expressed in percentages.

Results
A total of 70 identified samples of E. coli were collected from the patients with the age 1day-90years, out of which 32 (45.71%) males and 38 (54.28%) females. Different specimens e.g urine, pus, wound swab were received from the indoor or admitted patients in Lady reading hospital Peshawar KPK (Fig.  1) depicts the sample wise distribution of clinical isolates of E. coli. Antibiotic susceptibility testing in our isolates, we have found increased percentage (98.57%) of isolates showed sensitivity to polymyxin b followed by imipenem, which showed sensitivity of (90%). 84.28% of E .coli isolates showed resistance to co.trimoxazole. However, we have observed an elevated level of resistance to other routinely used antibiotics. The Growth of Resistant E. coli strain has been shown ( Fig.  2), while cumulative susceptibility pattern of E.coli isolates were shown (Table 1).
The resistant antibiotic strains were tested for their ability to produce ESBL, the percentage of which has been shown (Fig. 3), while ESBL positive strain is shown (Fig. 4).   Goudarzi et al. [20] in Saudi Arabia reported it is 30.6% respectively. The inconsistency in reported results can be attributed to the frequent use of antibiotics in that particular region which will make an organism more resistant to extendedspectrum beta-lactam antibiotics.

Conclusion
The present study shows the prevalence of ESBL in E. coli was found to be 55.71% in Lady reading hospital Peshawar. PB (Polymyxin b) (98.57%) followed by IMP (Imipenem) (90%) are the best choice for "E. coli" with the highest sensitivity. Among the resistivity of antibiotics, the highest resistivity is that of SXT (Co-trimoxazole) (84.28%), which is the lowest acting antibiotic on these bacteria. It is concluded that the Multidrug resistance (MDR) is one of the major problems in various infections from all over the world. Several Hospitals belong to developing countries do not perform routine culture test procedure, because of limited resources, however, the present study supports the culture sensitivity test for every kind of infection, and also take some steps for reducing the resistivity, by minimizing the use of antibiotics, use of synergistic combinations, improving the hygienic measures and conduct further research to find the antibiotic pattern of resistance in E. coli.