EFFLUX AS AN ARISING CAUSE OF DRUG RESISTANCE IN E. COLI

Rakesh kumar * Savita Jandaik and Pooja Patial. Department of Microbiology Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Multidrug resistant strains of bacteria which are characterized as serious public health problem may arise due to over expression of efflux pumps. Efflux pumps in bacteria can be detected by using specialized instruments. In the present study simple instrument free agar cartwheel method was used to detect efflux pumps in E.coli with some modification. Four isolates of E.coli were collected and processed. Biochemical analysis of all the strains was done using standard protocol. Further cartwheel assay was performed. MIC method was used to confirm the observations of agar cartwheel assay. In Cartwheel assay, a total of 4 strains of E.coli were analyzed for the presence of efflux pumps, active efflux pump was detected in 1 isolate while it was not observed in 3 strains. Bacterial isolate which contains efflux pump also showed high MIC value for the antibiotics used. But in the presence of an EPI, a considerable decrease in the MIC of antibiotics was observed among isolate containing efflux pump, while there was no decrease in MIC of antibiotics for strains without efflux pumps. Present study revealed that cartwheel assay to detect efflux pumps is a reliable, fast and sensitive technique, which may reduce time and efforts to detect efflux pumps.

…………………………………………………………………………………………………….... Introduction:-
Escherichia coli is a principal facultative organism present in the human gastrointestinal tract. Pathogenic strains of E. coli have the capability to cause different types of diseases in humans and animals because of the presence of definite colonisation factors, virulence factors and pathogenically associated genes. E. coli major cause of nosocomial infections in humans is a Gram-negative, flagellated, rod-shaped, Oxidase negative, motile, facultative anaerobe and classified under the family Enterobacteriaceae 1 . E. coli is genetically classified as the most versatile bacteria and is major source of many plasmid and phage mediated genes. It causes septicemia and diarrhoea in overall hosts including man, avian and animals such as cattle, piglets, kids, foals, lambs and buffaloes. The pathogenic strains of E. coli which are isolated from hospitalized patients were more resistant to amoxicillin, ciprofloxacin, cephalexin and Gentamycin compared to those from outpatients 2 . Some strains of E. coli develop resistance to third-generation cephalosporin's and monobactams. Most members of gram negative bacteria family are less susceptible to most classes of the antibiotics, mainly lipophilic and amphiphilic ones; this is due to the presence of outer membrane in gram negative bacteria. ). Constant increase in drug resistant strains of bacteria becomes a big threat to human health 3 . Multidrug efflux pumps unfavorably affect the clinical effectiveness of existing antibiotics and also affect the process of discovery of new antibiotics 4 . Active efflux is established as ISSN: 2320-5407 Int. J. Adv. Res. 5 (1), 1999-2007 2000 important factor of bacterial resistance in case of most classes of antibiotics. The mechanism of active efflux is mediated by efflux pumps [5][6][7] . Efflux pumps are known as membrane associated active transporters which help in expelling out toxic compounds, including antibiotics from the cell [5][6][7][8] .
Antibiotic efflux was first reported in 1980, in case of E. coli bacteria as a mechanism for tetracycline resistance. Recently the role of efflux mechanism is reported in bacterial resistance in case of almost all major groups of antibiotics 5,7,[9][10][11] . Bacterial resistance to most of the classes of antibiotics is provided by membrane transporter proteins which are known as drug efflux pumps 12 . Efflux pumps are present as single component system or multicomponent systems. Increasing resistance of bacteria against most classes of antibiotics in recent days becomes a big problem 13 . Resistance in antibiotics develops very rapidly because of the changes in the expression of efflux pumps. So it is important to characterize new antibiotics, efflux pump inhibitors and the agents which are resistance modifying 14 . Resistance of bacterial isolates to most classes of antibiotics is widespread mostly in developing countries. Schedule monitoring of antibiotic resistance gives data for the antibiotic therapy and control of resistance, and evaluates the effectiveness of both of these 15 . In spite of the fact that antibiotics are originally emerged for the treatment of infectious disease caused by bacteria in humans, multidrug resistance to most classes of antibiotics becomes a worldwide problem. Efflux pumps are reported in both gram positive and gram negative bacteria and as well as in eukaryotic organisms. Some of the bacterial efflux pumps are selective for only one substrate while some of them are not. The non-selective group of efflux pumps are involved in shifting of wide range of compounds and different classes of antibiotics which confer a multiple drug resistance phenotype 5,6-11 . The AcrAB efflux system present in E. coli is multidrug efflux pump which is a major efflux system present in E. coli and responsible for intrinsic resistance of the bacteria against antibiotics, dyes and detergents [16][17] . The AcrAB efflux pump existing in E. coli belongs to resistance-nodulation-division (RND) family of efflux pumps which is a more usual in Gramnegative bacteria. AcrAB -TolC is specified as one of the best efflux pump in case of E. coli [18][19][20][21][22][23][24][25][26][27] . AcrAB efflux pump is the only single efflux pump of RND family in case of E. coli which plays an important role in antibiotic resistance against different antibiotics because of its prenominal expression. The Ethidium bromide (Et Br) agar cartwheel assay is a newly investigated simple, instrument free method used for the detection of efflux pump activity in bacteria 28 . The present study was aimed to detect efflux pumps in Gram negative bacteria E.coli with the help of EtBr agar Cartwheel assay.

Microbial strains:-
The strains of Escherichia coli used in the presentstudy were procured from Sabine Schuster and Winfried V. Kern, Center for Infectious Diseases and Travel Medicine, University Hospital, and Department of Medicine, Albert-Ludwig's-University, Freiburg, Germany and from MTCC and NCTC. The E. coli strains used were one knock out strain 1-DC14, wild type strain K-12 AG100 and two standard strains with NCTC number 12923 and MTCC number 1302. Gram's staining:-Gram's staining was done following the methodology of Aneja 29 .The smear on a glass slide was covered with few drops of the primary stain (Crystal violet). After a minute of exposure to the staining solution, the slide was washed with water. The smear was treated with few drop of Gram's Iodine and allowed to stand for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant. The slide was again washed with water and then decolorized with absolute ethyl alcohol or acetone. After the smear was decolorized, it was washed with water without any delay. The smear was finally treated with few drops of counter stain (Safranin).The slide was washed in water; excess water was removed using a blotting paper, dried in air and heat fixed before observing under microscope.

Carbohydrate fermentation test:-
Four test tubes were taken containing four different kinds of broth namely glucose, lactose, sucrose, Mannitol. Each tube consist of 1% sugar, 1% peptone water and 1% Andrade's reagent. Inverted Durham tube were kept in each tube. Tubes were autoclaved at 15 lb. (121°C) for 30 minutes. After cooling tubes were inoculated with loopful culture of E.coli and incubated at 37°C for 24 hours. The broth was examined for acid and gas formation.
Positive test result: Acid production was indicated by the colour change from red to yellow. Negative test result: No colour change.
Motility test:-Semi solid motility medium was prepared in a test tube and inoculated with E.coli culture with a straight wire by making a single stab down the center of the tube about 8-10 mm deep into the medium and incubated at 37°C for 24hours. Examined after one day.
Positive result:-Diffuse, hazy growths that spread throughout the medium rendering it slightly opaque. Negative result:-Generally give growths that are confined to the stab-line, have sharply defined margins and the surrounding material completely transparent.

Oxidase test:-
Oxidase test is used to identify bacteria that produces oxidase enzyme of bacterial electron transport chain. A filter paper was soaked in substrate tetra methyl-p-phenylenediamine dihydrochloride. A loop full colony of E.coli culture was smeared onto the filter paper strip.

Positive test result:
Dark blue-purple colour change within 10-30sec. Negative test result: No colour change or colour change after more than 30 sec.

Agar cartwheel assay for the detection of efflux pumps:-
The Etbr-agar cartwheel assay method was used to assess the presence of efflux activity in E.coli strains following the methodology of with slight modifications [28][29][30] . All the strains of E.coli were grown in nutrient broth and incubated at 37°C for 24 hours. The TSA plates are then divided into four sectors, forming a cartwheel pattern. E. coli culture was inoculated by swabbing on tryptic soya agar plates starting from the center of the plate to the margin, these plates containing different concentrations (0.5µg/ml, 1µg/ml and 2.0µg/ml) of Etbr. Plates were incubated at 37°C for 16 hours and then observed under UV light (Gel Doc-it2 310 imager).The minimum concentration of Etbr that produces fluorescence of the bacterial mass was recorded. (fig.1)

MIC Determination:-
The MIC was determined in all strains of E.coli used for Etbr agar cartwheel assay to confirm the presence of efflux pumps. Pure cultures of E.coli were diluted in nutrient broth to a concentration between 1x10 5 and 1x10 6 cfu/mL. A stock dilution of the antibiotics i.e., Ciprofloxacin, Tetracycline and Erythromycin were made at approximately 100x the level of the expected MIC for E.coli. The concentrations used for each of the antibiotics were (0.003-128) μg/mL and Further 1:1 dilutions of antibiotics were made with nutrient broth in 96 well microtiter plates. Then the bacterial culture was inoculated from lower concentration to higher concentration of antibiotic in equal amount (30µl). Positive and negative control was also included. Then microtiter plates were incubated at 37°C for 24 hrs. And then plates were observed in ELISA plate reader (BioTek) at 490 nm. MIC was recorded as the lowest concentration where no growth was visually observed. Then MIC of antibiotics was again observed in the presence of an EPI (CCCP).     figure 3. Agar cartwheel assay was performed for all 4 strains of E. coli, out of these four strains efflux pump was detected only in K12-AG100 strain and the other three strains (knockout strain 1-DC14, MTCC strain 1302 and NCTC strain 12923) have shown florescence hence confirms the absence of efflux pump.

Minimum Inhibitory Concentration:-
The observations of Etbr agar cartwheel assay was further confirmed by the determination of MIC values for selected antibiotics, known to be efflux pump substrates viz., Tetracycline, Erythromycin and ciprofloxacin.  (Table 2 and Fig 4). The observations of MIC assay were found similar to those of EtBr agar cartwheel assay.

Discussion:-
Escherichia coli has recognized as an emerging opportunistic pathogen of clinical relevance. The intrinsic resistance of E.coli has been attributed due to the outer membrane barrier. From many years, role of efflux mechanisms in bacterial resistance to most classes of antibiotics 5,7,9,11 . Ethidium bromide is a substrate for all efflux pumps and is not allowed to accumulate inside the bacterial cell 32 (2013) reported that Gram negative bacteria Acinetobacter has shown significant increase in the fluorescence after the incubation at 4°C while less fluorescence was observed at 37°C. The observations of the present study suggested that the drug efflux mechanism is an energy mediated mechanism and direct perpetuate to the temperature when the temperature decreased the energy of membrane decreased and the other factor may be responsible for less efflux is low metabolic rate at low temperature, which is not able to produce sufficient energy, hence the available energy is not sufficient to efflux out the accumulated compounds. Therefore, more fluorescence occurs at low temperature. In addition to this, the isolates having potentially less active efflux systems showed higher fluorescence and isolates having more active efflux systems showed less fluorescence 32 . In the present study, the MIC value was determined for the selected antibiotics, known as efflux pump substrates viz., Tetracycline, Erythromycin and Ciprofloxacin. K12-AG100 strain with known AcrAB-TolC efflux pump have shown very high MIC values ranging from 0.06 μg/mL to 128 μg/mL confirm the presence of AcrAB-TolC efflux pump. While the other three strains have shown very low MIC value. Further the assay was performed with an Efflux pump inhibitor (EPI) CCCP. A significant decrease was observed in MIC of antibiotics for the resistant strain and these values were ranging from 0.003 μg/mL to 64μg/mL. While the MIC of control sensitive strains (1 MTCC-1302 and 1NCTC-12923) without efflux pump was not change and remain constant even in the presence of an EPI (CCCP). Similarly, Abdi-Ali et al., (2006) has reported significant decrease in MIC of resistant antibiotics used in combination with EPI (CCCP). The observations of the study again suggested that the drug resistance in MDR isolates is mediated by an active efflux pump which effluxed out the accumulated drug.

Conclusion:-
EtBr agar cartwheel assay is a simple trustworthy method and can be used for the detection of efflux pumps in bacteria and on the basis of present study it can be concluded that efflux pump over expression is an leading cause of drug resistance in E.coli therefore proper measures should be used to solve this problem.