Biochemical characterization and antibiotic susceptibility pattern of Escherichia coli causing infections in chickens of Chhattisgarh plains

Introduction

Huge economic losses occurs as a result of mortality and decreased productivity of affected chickens, mainly during peak egg production Pathogenic E. coli strains including intestinal pathogenic E. coli (InPEC) and extra-intestinal pathogenic E. coli (ExPEC) depending on the location of the infection (Panth, 2019) [23] . APEC belongs to the ExPEC group is a major pathogen responsible for morbidity and mortality in chickens and causing high economic losses in poultry industries (Dhama et al., 2013; Mellata, 2013 and Tawab et al., 2014) [7,18,32] . APEC strains can affect all age groups of chickens, most commonly found in young chicks of up to 3 weeks of age. Many conventional bacteriological techniques, serological tests and molecular techniques are helpful for the identification of pathogenic E. coli and for the detection of certain virulence genes. Pathogenic E. coli possess one or more virulence properties that may help in establishment of the infection. Virulence properties of E. coli can be detected by Congo red binding and haemolytic activity. The association between the binding of Congo red dye and the pathogenicity of E. coli was confirmed by Berkhoff and Vinal, (1986) [5] . Conventional culture technique is commonly used for isolation of E. coli. This includes non-selective pre-enrichment (nutrient agar) followed by selective enrichment (MacConkey agar) and plating on selective agar (EMB). E. coli produce pink lactose fermenting colonies on MacConkey agar (MCA) after 24hrs. of incubation at 37°C, then performing subculture on EMB agar produce green metallic sheen. Suspected colonies are confirmed by various biochemical tests. According to Ewers et al., (2005), a variety of virulence factors have been implicated in promoting these extraintestinal diseases in avian species, including adhesins (F1-, P-, AC/I-, and F17-fimbriae, curli fibers and fimbrial adhesins), hemolysins (hemolysin E and tsh), antibactericidal factors (outer membrane protein A, iss, lipopolysaccharide, K1-capsule) and toxins (heat stable toxin, cyto-/verotoxin, flagella toxin, and vat toxin). All these virulence genes that contribute to the pathogenesis of Colibacillosis in chickens were also reported by (Johnson et al., 2008; Zenda et al., 2013 and Ibrahim et al., 2019) [12] . Keeping the above facts into consideration, the present study was undertaken with the following objectives:  Isolation of Escherichia coli on pre-enrichment and selective enrichment media.  Determination of biochemical characteristics and biofilm formation phenomenon of E. coli

Materials and Methods

Gram's staining of E. coli Single pink lactose fermenting colony was taken aseptically from MCA plate using a sterile loop and made smear on cleaned glass slide and then stained by Gram's method of staining (Himedia, K001-1KT). Following staining, slide was examined under microscope to determine the size and shape of the organism (Merchant and Packer, 1967).

Motility test

The motility test was performed according to the method described by (Cowan, 1985) [6] to differentiate the motile bacteria from the non-motile one by hanging drop method. A drop of 24 hrs. old pure broth culture of E. coli was taken on the clean glass slide and observed the movement of bacteria under microscope.

Biochemical tests

Biochemical tests such as catalase test, indole production, methyl red (MR), Voges-Proskauer (VP), citrate utilization, oxidase test and carbohydrate fermentation test were carried out as per the standard procedures described by Khaton et al., (2008) and Ievy et al., (2020) [14,11] .

Catalase test

A loop full of pure colony from each isolate was picked up and transferred on a glass slide. One-two drops of 3% Hydrogen Peroxide (H2O2) were placed over culture to observe effervescence of bubbles for confirmation of catalase.

Indole production test

Peptone broth (supplemented with NAD) tubes were prepared freshly and inoculated with each isolate and incubated at 37ºC for 48 hrs. After incubation, 0.5 ml Kovac's reagent was added into tubes and observed for appearance of red colour ring at the junction of medium and reagent to confirm Indole production.

Methyl Red test

Methyl red-Voges-Proskauer (MR-VP) broth was prepared in a tube and inoculated freshly pure culture and incubate at 37°C for 48 hrs. following incubation, 5 drop of a (0.05%) methyl red solution was added and observed the colour change in the tubes. Development of red or pink colour was interpreted as positive test.

Voges-Proskauer test MR-VP broth was prepared in a tube and inoculated freshly pure single colony of the each isolate of E. coli and incubated at 37°C for 48 hrs. After incubation, 0.6ml of VP reagent-1 and 0.2 ml of VP reagent-2 was added for each ml of culture, the ingredients were mixed thoroughly and allowed to stand. The appearance of pink colour indicated to positive test.

Citrate utilization test

Simmons citrate agar HiMedia, (M099S) slant was prepared in 10 ml tube by autoclaving at 15 lbs and 121°C. Small amount of 24 hrs old pure culture of E. coli inoculated into the slant by means of streaking in a zig-zag pattern. The tubes were incubated at 37°C for 48 hrs. Following incubation, colour change at the surface of slant from green to blue was interpreted as positive

Carbohydrate fermentation test

Carbohydrate fermentation tests was performed to detect the ability of the organism to ferment different sugars. Test was carried out by inoculating loop full fresh pure culture of E. coli into the 5ml Andrade peptone water (HiMedia, M885) then single carbohydrate disc was added in each tube and inverted Durham tube was placed inside the each tube and incubate at 37°C for 72 hrs. After incubation, colour changed and gas production was observed. Acid production was indicated by the colour change from red to yellow of the medium and the gas production was noted by the appearance of gas bubbles in the inverted Durham's tube (Edwards and Ewing, 1972 and Tonu et al., 2011) [33] .

Biofilm formation of E. coli

A simple qualitative approach for detecting biofilm production using Congo red agar (CRA) media, described by Freeman et al., (1989) was followed. Brain heart infusion agar 37 g/l, sucrose 50 g/l, and Congo red indicator 0.8 g/l were used to make CRA media. The first Congo red stain was made as a concentrated aqueous solution and autoclaved separately from the other medium constituents (121 o C for 15 min.). Then, at 55 0 C, it was put to autoclaved brain heart infusion agar with sucrose. CRA plates were inoculated with test organisms and aerobically incubated for 24 hours at 37°C. Biofilm was identified by black colonies with a dry crystalline appearance (Katongole et al., 2020) [13] .

Antibiogram of E. coli isolates

Antibiogram of E. coli isolates was studied by disc diffusion method and interpretation was made as per Clinical and Laboratory Standards Institute (CLSI, 2019). Twelve numbers of antibiotic discs (Himedia laboratories, Mumbai) are used as enlisted in Table . 4. The overnight grown broth culture was spread on Muller Hinton Agar Base (HiMedia, M173) and the antibiotic discs were impregnated and incubated at 37ºC for 24-48 hrs in BOD incubator (Sahoo et al., 2012) [25] . After incubation, result was interpreted as per CLSI guidelines as given in the Table . 1. MacConkey agar (MCA) (Fig. 1). Following 24 hours of incubation at 37°C, 24 of these isolates produced a characteristic green metallic sheen on Eosin Methylene Blue (EMB) agar (Fig. 2). These findings align with those reported by previous authors ( [33,25,26,30,17,1,16] . The E. coli isolation rate observed in this study (68.08%) is comparable to the isolation rates reported by Kilic et al. (2007) [15] , Ramasamy et al. (2008) [24] , and Sarkar et al. (2013) [26] , who documented rates of 48%, 51.9%, and 67.3%, respectively.

Gram's staining and Motility test

All 32 isolates showed Gram-negative, pink coloured, rodshaped, short chain in Gram's stain (Fig. 3). previous workers described similar staining characteristics of E. coli on Gram's stain ( [14,21,33] All the E. coli isolates were found to be motile with hanging drop's preparation. The results of the motility test are akin to the findings of (Khaton et al., 2008 and Dey et al., 2013) [14] .

Biochemical Characterization of E. coli isolates

All the isolates that have morphological characters of E. coli were subjected to biochemical test for identification. All the 32 isolates showed biochemical reactions pertaining to E. coli. The isolates were found to be positive for indole production, Methyl red (MR), catalase test and negative for Voges-Proskauer (VP) and citrate utilization test (Fig. 4). All the 32 isolates revealed similar IMViC pattern (+ + --). These findings in our study were in agreement with earlier reports by (Khaton et [14,33,26,19,20,29,22] . All the isolates fermented 7 sugars such as glucose, lactose, maltose, galactose, mannitol, sucrose and dextrose with the production of acid and gas. Acid production was indicated by the color change from reddish to yellow and gas production was noted by the presence of gas bubbles in the inverted Durham's tubes and all the isolates were failed to fermented inositol and adonitol (Fig. 5; [8,19,29] reported all this isolates fermented glucose, lactose and mannitol but, only 3% of isolates fermenting adonitol. DRG -isolates of Durg, RAI-isolates of Raipur, RJN-isolates of Rajnandgaon, SJP-isolates of Surajpur, NT-Not tested

Biofilm formation

For this test pure colony of the E. coli isolates were picked and streaked on CRA plates and incubated at 37°C for 24 hours aerobically. Out of 24 EMB positive E. coli isolates, 17 isolates produced dark coloured colonies with dry crystalline consistency on Congo red agar plate were considered positive biofilm formation (Fig. 6) whereas, red colonies negative biofilm forming E. coli (Fig. 7). These findings were in accordance with the findings of Katongole et al., (2020) [13] who showed the dark crystalline colonies of pathogenic E. coli on Congo red agar.

In contrast to the present findings observed by Berkhoff and Vinal (1986) [5] , Ahmed et al., (2009) and Seifi et al., (2015) [27] who reported that biofilm positive E. coli isolates produce grayish white colonies on CRA.

Antibiogram of APEC isolates

In the present study, antibiotic sensitivity pattern of all the PCR positive E. coli isolates were determined using disc diffusion method. The E. coli isolates were tested against 12 commonly used antibiotic discs viz. chloramphenicol, amikacin, streptomycin, tetracycline, gentamicin, ciprofloxacin, cefixime, cefotaxime, amoxycillin+clavulanic acid (amoxyclav), trimethoprim, levofloxacin and Azithromycin (Fig. 8). The antibiotic susceptibility of each isolates was shown in the form of percentage of sensitivity, intermediate and resistant (Table 3 andGraph 1). E. coli isolates were highly sensitive to antibiotic chloramphenicol (94.11%) followed by amikacin (88.23%), streptomycin (76.47%), gentamicin (58.82% and ciprofloxacin (41.17%), whereas highest resistant was observed to antibiotic levofloxacin (100%) fallowed trimethoprim (94.11%), amoxiclav (70.58%), cefotaxime (52.94%), tetracycline (47.05%), azithromycin (41.17%) and cefixime (35.29%). Intermediate resistance was observed to antibiotics azithromycin, cefixime (47.17%) followed by tetracycline, cefotaxime (35.29%), gentamicin (29.41%), amoxyclav (23.52%) and trimethoprim (5.88%). The highest susceptibility of E. coli to chloramphenicol was also reported by previous authors Akond et al., (2009) [2] , Sahoo et al., (2012) [25] and Shecho et al., (2017) [28] . In contrast, Omer et al., (2010) [21] detected almost all the E. coli isolates resistant to chloramphenicol isolated from chicken affected with Colibacillosis. Similarly, sensitivity to amikacin (88.23%) observed during the present study was also well supported by Subedi et al., (2018) [31] and Choudhari et al., (2020) [4] with sensitivity percentage of E. coli isolates observed were (84%) and ( 83 In the present study the APEC isolates clearly demonstrated higher resistance to most of the antibiotics (Graph 2) studied might be due to increased use of antibiotics as feed additives, growth promotion and therapeutic purpose for the prevention of diseases in poultry. For this reason, these antibiotics are ineffective or less effective against pathogenic E coli strains in the present time (Omer et al., 2010) [21] .

Conclusion

Based on cultural characteristics and morphological observation, a total of 24 E. coli isolates were successfully recovered and identified from EMB agar plates. The comprehensive antibiogram profiling of these E. coli isolates revealed that the highest level of sensitivity was exhibited toward chloramphenicol, which was followed in decreasing order of susceptibility by amikacin, streptomycin, gentamycin, and ciprofloxacin. Conversely, a significant patterns of antimicrobial resistance were observed, with the highest resistance rates recorded for levofloxacin, followed by trimethoprim, cefotaxime, cefixime, amoxyclav, tetracycline, and azithromycin. These findings strongly emphasized the urgent necessity for the judicious and evidence-based selection of antibiotics to ensure effective clinical treatment outcomes. Furthermore, the phenotypic evaluation of virulence factors showed that a majority of the E. coli isolates (70.83%) were identified as biofilm formers when tested on Congo Red Agar (CRA).

Acknowledgement

Acknowledgements:The authors wish to express their gratitude to the Dean, College of Veterinary Sciences, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, for providing the necessary facilities to conduct this research.

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