Isolation and Identification of Escherichia Species from

Int.J.Curr.Microbiol.App.Sci (2020) 9(11): 2084-2090
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 9 Number 11 (2020) Journal homepage: http://www.ijcmas.com

Original Research Article

https://doi.org/10.20546/ijcmas.2020.911.248

Isolation and Identification of Escherichia Species from Faecal Sample of an Elephant and Antibiogram Assay
S. D. Audarya1*, R. Gangil1, D. Chhabra1, R. Sharda1, M. Haque2, N. Shrivastava3, S. Shukla3 and U. K. Garg3

1Department of Veterinary Microbiology ,2Department of Veterinary Parasitology, 3Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry,
Nanaji Deshmukh Veterinary Science University, Harnyakhedi, Dr. Ambedkar Nagar, Mhow-453446, Indore, Madhya Pradesh, India
*Corresponding author

Keywords
Elephant, Escherichia species, Antibiogram assay, Biochemical characterization, Carbohydrate utilization
Article Info
Accepted: 15 October 2020 Available Online: 10 November 2020

ABSTRACT
In the present study, faecal and blood samples were collected, aseptically, from an elephant arrived to the Veterinary Clinics of the College. Immediately, samples were used for bacterial isolation. A bacterial isolate, isolated from the faecal sample of an elephant was identified at the genus level to be of Escherichia species by adopting Enterobacteriaceae identification kit used for studying biochemical characteristics and carbohydrate utilization tests (Gram negative bacilli showing mainly ONPG positive, oxidase negative and nitrate reduction positive). Mueller Hinton’s agar (MHA) was used to conduct antibiogram assay by using the faecal culture. A total of 14 number of antibiotics from 7 different groups were utilized in the investigation namely Chloramphenicol, Ciprofloxacin, Ofloxacin, Levofloxacin, Nitrofurantoin, Norfloxacin, Tetracycline, Streptomycin, Amoxyclov, Amikacin, Ampicillin, Doxycycline hydrochloride, Penicillin G and Erythromycin. Only two antibiotics, Levofloxacin (Quinolone group) and Nitrofurantoin (Nitrofuran antibacterial) were found to be sensitive in the test.

Introduction
Elephants are one of the heaviest mammals on the earth. It belongs to the family Elephantidae. The family is further divided into two genera; Loxodonta and Elephas. African elephant (Loxodonta africana) and Asian elephant (Elephas maximus) both originated in sub-Saharan Africa. Further, the

African elephant is divided into two subspecies, the savanna elephant Loxodonta africana africana and Loxodonta africana cyclotis (Poole, 1996). Asian elephants are different than their African counterparts in several aspects (smaller size, small ears, only some male Asian elephants have tusks). Elephants help maintain biodiversity of the ecosystems. However, African elephants are

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mainly poached for ivory and Asian elephants face threats due to habitat loss from humananimal conflict (https://www.worldwildlife .org/species/elephant#:~:text=Elephants%20h elp%20maintain%20forest%20and,allow%20 passage%20for%20other%20animals). In 2017, there were around 27,000 elephants (Elephas maximus) in India of which 2500 kept in captivity. The majority (around 1700) of the captive elephants belonged to private individuals). The rest of the captive elephants (800) were with zoos, circuses and temples. Though, a wildlife protection act 1972 is in force, still animals like elephants face inhumane conditions at some places. (https://scroll.in/article/954150/india-ispushing-a-plan-to-protect-elephants-in-thewild-but-is-ignoring-animals-incaptivity#:~:text=Despite%20the%20odds%2 C%20India%20 has, in % 202017% 20 was %20 around % 2027% 2C000). Asian elephants also suffer from infectious diseases including those that cause diarrhoea due to Escherichia coli (Senthilkumar et al., 2020).
In the present study, clinical samples were collected from a she elephant (suffering due to unknown ailments) brought to the Veterinary Clinics for clinical investigation. Isolation and identification of bacteria in pure culture is important for further characterization of the isolate and its antibiogram studies.
Such studies will help in understanding the antibacterial susceptibility profile of the bacterial isolate that can be of help in future for treatment purposes. Hence, the present investigation, reports isolation of a bacteria in pure culture from a faecal sample of an elephant. The study also report, results of antibiogram assay from faecal culture and results of biochemical and carbohydrate utilization tests on the bacterial isolate (the isolate closely resembled to Escherichia species).

Materials and Methods
Elephant
A female elephant (close to 35 years old) named Laxmi was brought at Teaching Veterinary Clinical Complex on 12/05/2014 located in the premises of the college by lifting with a Crain from Indore. It was lying in the recumbent condition and the cause of suffering was not known (Fig. 1).
Collection of clinical samples
Blood sample was collected aseptically from the ear vein in the sterile container. Faecal sample was collected per rectal in the sterile container. Both the samples were collected for bacteriological examination in the morning hours.
Transportation of the samples
The collected samples were immediately brought to the Department of Veterinary Microbiology laboratory on ice.
Inoculation of the samples in liquid culture media and growth
In the laboratory broth, the samples were immediately inoculated into the separate tubes with sterile Brain Heart Infusion (BHI) broth, a liquid culture media. After 4-5 hours of inoculation, there was no turbidity in the tube inoculated with blood sample.
Inoculation of the samples on solid culture medias, nutrient agar, MacConkey’s agar and Eosin Methylene Blue (EMB) agar
Blood and faecal samples were further inoculated onto nutrient agar and MacConkey’s agar (Fig. 2). On overnight incubation at 37οC, there was no growth in the plate inoculated with the blood sample but

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plates inoculated with faecal sample indicated growth in nutrient agar as well as pink growth due to lactose fermentation on MacConkey’s agar. Very next day the bacterial growth on MacConkey’s agar was streaked on both EMB agar plates (Fig. 3).

Enterobacteriaceae identification kit

Hi25TM Enterobacteriaceae identification kit

employing standardized colorimetric

identification system (biochemical and

carbohydrate utilization tests), KB003

(HiMedia, India) was used to identify

Enterobacteriaceae species (Fig. 4). The kit

employs

standardized

colorimetric

identification system comprised of 25 tests

(biochemical and carbohydrate utilization

tests) and used in the study. 50 µl of freshly

grown bacterial culture was added in each

well of both the strips provided by the manufacturer and incubated at 37 οC at least

for 18 hours before addition of reagents for

required tests.

Antibiogram assay

Antimicrobial susceptibility test or

antibiogram assay was conducted using

Mueller Hinton’s agar, MHA (Bauer et al.,

1966). Faecal sample inoculated BHI broth

(approx. 3 ml) showing turbidity after 4 hours

was inoculated on MHA plates by pouring.

Excess broth was decanted and MHA plates

were kept for drying inside the laminar air

flow cabinet for 5 minutes. Thereafter,

antibiotic sensitivity discs were placed at their

respective position on MHA. The plates were incubated overnight at 37 οC. On the next day,

after 15 hours of incubation results were

recorded. A total of 14 no of antimicrobials

were employed in the test based on disc

diffusion technique. Antimicrobials/

antibiotics used in the study; namely from

groups,

Chloramphenicol

group-

Chloramphenicol (30 mcg), Quinolone group-

Ciprofloxacin (5 mcg), Norfloxacin (10 mcg), Ofloxacin (5 mcg), Levofloxacin (5 mcg), Tetracycline group- Tetracycline (30 mcg), Doxycycline hydrochloride (30 mcg), Nitrofuran antibacterial- Nitrofurantoin (300 mcg), Aminoglycoside group-Amikacin (30 mcg), Streptomycin (10 mcg), Penicillins group- Ampicillin (10 mcg), Penicillin G (10 units), Amoxyclov (30 mcg), and MacrolidesErythromycin (15 mcg). The results were interpreted as sensitive or resistant as per the manufacturer’s instructions.
Results and Discussion
In the present study, liquid culture, BHI broth inoculated with faecal sample exhibited turbidity on incubation at 37 οC for 4-5 hours duration indicating bacterial growth. On Gram’s staining of bacterial smear prepared from broth, coco-bacillary to bacillary organisms were visualized under oil immersion microscopy. Faecal sample inoculated onto nutrient agar and MacConkey’s agar and incubated overnight at 37 οC indicated bacterial growth. Pink coloured colony grown on MacConkey’s agar was due to lactose fermentation. The next day, growth on nutrient agar and MacConkey’s agar, both, were streaked on EMB agar. Bacterial growth on EMB agar proved to be of Gram negative, rods, however there was no specific metallic sheen observed. On Gram’s staining, growth on MacConkey’s agar and EMB agar proved to be of gramnegative bacilli.
Hi25TM Enterobacteriaceae identification kit, KB003 (HiMedia, India) was employed to identify Enterobacteriaceae spp. as per manufacturer’s instructions. The results of biochemical and carbohydrate utilization tests were presented in the figure 2 and compared with the Enterobacteriaceae spp. identification chart given by the manufacturer (HiMedia, India). After following the

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identification chart, it was concluded that the isolate in the study fall close to the genus Escherichia but species level identification was difficult. Gram negative bacteria possessing tests results of ONPG positive, oxidase negative and nitrate reduction positive are placed in the genus Escherichia (Willey et al., 2008). Similar results are observed in the present study. The species level identification for Escherichia species identified was not possible in the study,

however, Escherichia coli infection was reported in dung samples of diarrheic captive Asian elephants of Tamil Nadu state in India (Senthilkumar et al., 2020). Escherichia coli bacteria were also isolated from different biological and environmental sources (Zinnah et al., 2007). DNA isolation from faecal samples was reported (Zhang et al., 2006). Faecal genotyping for elephant species was also described, recently (Bourgeois et al., 2019).

Fig.1 She elephant

Fig.2 Bacterial culture from faecal and blood samples on agar

Nutrient agar

McConkey’s agar

Fig.3 Pure culture on EMB agar. (Bacteria reveals no metallic sheen; cultures derived from Nutrient agar (A) and MacConkey’s agar (B) respectively streaked on EMB agar, Gram’s
staining indicated Gram negativity (C)

A

B

C

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Fig.4 Results of biochemical and carbohydrate utilization tests for a bacterial isolate from faecal sample of elephant
Plate.1 ONPG (+), Lysine utilisation (+), Ornithine utilisation (+), Urease (-), Phenylalanine deamination (-), Nitrate reduction (+), H2S production (-), Citrate utilisation (+), VP (-), MR (+),
Indole (-), Malonate (-)

Plate.2 Esculin hydrolysis (-/+ after 24 hrs), Arabinose (+), Xylose(+), Adonitol (+), Rhamnose (+), Cellobiose (-), Melibiose (+), Saccharose (-/+ after 24 hrs), Raffinose (-/+ after 24 hrs), Trehalose (+), Glucose (+), Lactose (+)

Oxidase test (-): Not shown

In the antibiogram assay, out of 7 different groups of antibiotics, only two groups Nitrofuran antibacterial (Nitrofurantoin) and Quinolone group (Levofloxacin) were found to be sensitive. However, except Levofloxacin other members in the Quinolone group of antibiotics used in the investigation (Ciprofloxacin, Norfloxacin, Ofloxacin) were ineffective. Levofloxacin is the synthetic broad-spectrum antibacterial agent. Levofloxacin is the L-isomer of the racemate Ofloxacin, a Quinolone. It inhibits enzymes required for DNA replication, transcription, repair and recombination (bacterial topoisomerase IV and DNA gyrase, type II topoisomerases). It is effective against aerobic gram-negative microorganisms. Some bacteria resistant to other quinolones may be susceptible to Levofloxacin (https://www. accessdata.fda.gov/drugsatfda_docs/label/2006

/020634s040,020635s043,021721s007lbl.pdf).

The present study supports this above

statement. Nitrofurantoins reduced by

bacterial flavoproteins to reactive

intermediates. These reactive intermediates

inactivate or alter bacterial ribosomal proteins

or other macromolecules (inhibition of

processes related to protein synthesis, aerobic

energy metabolism, DNA synthesis, RNA

synthesis and cell wall synthesis).

Nitrofurantoin is shown to be effective

against gram positive and negative aerobes

(Staphylococcus

saprophyticus

and

Escherichia coli, in-vitro as well as in clinical

infections)

(https://www.accessdata.fda.gov/drugsatfda_d

ocs/label/2009/020064s019lbl.pdf). Our study

also indicated, the in-vitro effectiveness of

Nitrofurantoin against isolated Escherichia

species.

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Reports from all over the world showed increase in emergence of antibiotic resistant Escherichia coli strains from various clinical sources like uterine infections, mastitic milk, urinary infections, diarrheic cases and sepsis (Iredell et al., 2016), samples from animals (Marshall and Levy, 2011; Onen et al., 2015; Armanullah et al., 2018) and interestingly from environmental samples (Stephanie, 2015). The use of antibiotics in the treatment of diseases and other factors have been linked to the development of resistant microorganisms (Adzitey, 2015). Some of the main anthropogenic source of the antimicrobial resistance bacteria in wildlife already suggested were landfills, insufficiently treated wastewater draining into rivers and lakes and waste from intensively managed livestock farm (Schroeder, et al.,2002; Aarestrup, et al., 2008; Ahlstrom et al., 2019).
Faecal-bacterial transmission between banded mongoose (Mungos mungo) and humans in northern Botswana using Escherichiacoli as a model organism was evaluated (Pesapane et al., 2013). There are very few reports available on the studies of bacterial isolates from elephants. Hence, the present investigation, reports bacterial isolation in pure culture from a faecal sample of an elephant. The study also reports results of antibiogram assay from culture of faecal sample and results of biochemical characteristics and carbohydrate utilization tests on the bacterial isolate from elephant.
Acknowledgements
The authors are thankful to the supporting staff at the Department of Veterinary Microbiology.
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How to cite this article:

Audarya, S. D., R. Gangil, D. Chhabra, R. Sharda, M. Haque, N. Shrivastava, S. Shukla and Garg, U. K. 2020. Isolation and Identification of Escherichia Species from Faecal Sample of an Elephant and Antibiogram Assay. Int.J.Curr.Microbiol.App.Sci. 9(11): 2084-2090. doi: https://doi.org/10.20546/ijcmas.2020.911.248

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