Detection of Zoonotic Potential of Salmonella and Escherichia coli Isolated from Ostriches and Determine Their Antibiogram Study

The present research was conducted for molecular characterization of important zoonotic bacteria isolated from different samples in ostrich and also determined their antimicrobial activity. For this current research, 32 samples were randomly collected from 8 ostriches at different ages, of which 8 were oropharyngeal, 8 were cloacal swabs, 8 were environmental sand samples, and 8 were feces samples. In addition, the bacteria were isolated and identified by using standard microbiological methods, including cultural, biochemical and molecular techniques. 16S rRNA gene was used to detect Escherichia coli and Salmonella spp. molecularly. The Kirby-Bauer disc diffusion method was used to determine the antibiotic sensitivity test. Out of 32 samples, E. coli 8 (53.33%) and Salmonella spp. 7 (46.67%) were identified in young ostrich, while in adult ostrich, E. coli 2 (40%) and Salmonella spp. 3 (60%) were detected. According to our study, E. coli was the most predominant isolate found in cloacal swabs and ostrich feces. Escherichia coli were most sensitive to Amoxicillin and Azithromycin (100%), followed by Kanamycin, Chloramphenicol and Gentamicin (75%), while 100% resistant to Piperacillin, Bacitracin, Tetracycline, Cloxacillin, Novobiocin, Cefixime. Salmonella spp. was 100% sensitive to Azithromycin and also 100% resistant to Tetracycline, Piperacillin, Bacitracin, Chloramphenicol and Methicillin. Our research concluded that E. coli and Salmonella spp. are multi-drug resistant bacteria, and appropriate antibiotics should be used in ostrich farms to protect the multi-drug resistant bacteria. We suggest farm owners increase public awareness about zoonotic diseases and those working on ostrich farms.

certain regions' economies. Egypt's ostrich industry continues to expand, as are its farms (Cooper et al., 2008). Egyptian ostrich farms produced 7.27 eggs/ hen/month, compared to South Africa's 5.99 (Youssef and Afifi, 2017;Rahman et al., 2019). Cooper et al. (2009) reported that ostrich eggs are highly nutritious. Additionally, ostrich eggs may contain different pathogenic bacteria. A previous study investigated that 19.3% of several bacterial isolates were found in ostrich eggs (Youssef et al., 2017). In Bangladesh, many ostrich farm owners breed ostrich on their farms for a high quantity of meat production. During the handling of foods, farm owners attach ostrich and ostrich eggs and meat, which could be a potential risk of much zoonotic disease transmission. Recently, ostrich farming and ostrich exhibition have been increased, and as a result, people are more conscious about the zoonotic disease risk associated with this bird, its products and by-products (Youssef et al., 2017). Adult ostriches have a high level of resistance to several diseases. Nevertheless, young birds, especially when being moved from their nests to the farm area, are more susceptible to the dangers posed by parasites and bacteria such as hemolytic E. coli, Campylobacter spp., and the Salmonella spp. (Cooper, 2005). Escherichia coli is a part of the intestinal microbiota of poultry, including ostriches, yet pathogenic strains cause colibacillosis, and poultry deaths often start with it (Scerbova et al., 2016). Salmonella is found in clinically healthy ostrich and diseased ostriches (de Freitas Neto et al., 2009). Enterotoxigenic E. coli strains cause watery diarrhoea in animals and birds worldwide (Marzouk et al., 2004). Escherichia coli in ostrich products may impede meat and other product trade. As a widespread human foodborne infection, it threatens public health (Foley et al., 2008;Smith et al., 2008). Ostriches are widely farmed, although little is known about infections in their eggs. Bacterial infections inhibit extensively ostrich breeding. In ostriches, E. coli, Salmonella, and Pseudomonas infections are most important (Wieliczko et al., 2000).
Antimicrobial resistance in microorganisms from animals, including food-producing animals, pets, fish, and wild animals, has generated interest significantly (Schwarz et al., 2010). Only a few specific studies have been conducted on the antimicrobial resistance of organisms isolated from ostriches in Bangladesh. Therefore, this research aimed to: isolate and identify the zoonotic potential of Salmonella and E. coli strains from ostriches, molecular characterization of isolated strains by PCR, DNA sequencing and phylogenetic tree analysis and to determine antimicrobial resistance.

Isolation and identification of bacteria
Samples were suspended in a sterile saline solution. The suspension was inoculated into nutrient agar and nutrient broth for the primary isolation of bacteria (Parvez et al., 2016). For sub-culturing of the suspected bacteria, we have used different bacteriological agar media like MacConkey agar, Eosin Methylene Blue agar, Mannitol Salt Agar, Cetrimide agar and Salmonella-Shigella agar. All bacterial culture Petri dishes were incubated at 37˚C overnight for the confirmation of bacterial growth. Pure culturing of bacteria was then done by following the methods described earlier (Kundu et al., 2021). All bacteriological and fungal agar media were derived from Hi-Media Laboratories Private Ltd. India. Primary identification of bacteria was made by using gramstaining methods, which showed morphological and staining characteristics under microscopy (Merchant and Packer, 1967). Using standard methods, bacteria were identified by different biochemical tests, inclu-ding catalase, oxidase, indole, MR-VP, Simon citrate, and motility urease (Cheesbrough, 2003).
DNA extraction of E. coli and Salmonella spp. and phylogenic tree analysis E. coli and Salmonella spp. were identified by biochemical tests. DNA was extracted from E. coli and Salmonella with a robotic DNA extractor (Maxwell-16, source: Promega-USA) as per manufacturer instructions. Genomic DNA purity and concentration of E.
coli and Salmonella were measured with a Nano-drop spectrophotometer (ND-200, source: Thermo Scientific-USA). The final PCR band was found in agar gel electrophoresis and visualized and photo-graphed by a UV transilluminator. The PCR primer marks gene and PCR cycling procedure are demonstrated in Table 1. By applying the neighbor-joining method of 1000 replicates, a phylogenic tree was measured with the MEGA6 program (Tamura et al., 2013).  , and Cefradin (25 µg) were used. All antibiotic discs were purchased from (Oxoid Limited, UK). After biochemical identification, colonies of the pure isolates were spread on Muller-Hinton agar, and selected antibiotic discs were placed using sterile forceps. Finally, incubate the plates at 37°C for 24 hours, and then observe and the measure the zone of inhibition on a millimeter scale according to company guidelines.

Isolation and Identification of Bacteria
In this research, a total of 20 isolates were isolated and identified by cultural and biochemical tests. E. coli produces metallic sheen (greenish black) colonies on EMB agar (Fig. 4A), and Salmonella spp. produce white colony on Brilliant Green agar (Fig. 4B). E. coli demonstrated positive results for MR-VP, TSI, SC and SF in young ostrich, while negative results showed in TSI and OXI in adult ostrich below (Fig. 5 and Table  4). Salmonella spp. gives positive results for MR-VP, SC and SF in young ostrich, whereas negative results showed in TSI and OXI in adults (Table 4).

Results of Antibiotic Sensitivity Test
A total of 15 commercially available antibiotics were used to determine the antibiotic sensitivity tests of this research. Escherichia coli were most sensitive to Amoxicillin and Azithromycin (100%), followed by Kanamycin, Chloramphenicol and Gentamicin (75%), while 100% resistant to Piperacillin, Bacitracin, Tetracycline, Cloxacillin, Novobiocin, Cefixime (Fig. 6A). Salmonella spp. was 100% sensitive to Azithromycin and 100% resistant to Tetracycline, Piperacillin, Bacitracin, Chloramphenicol and Methicillin (Fig. 6B).   The obtained results of our study mentioned that E. coli and Salmonella are the most predominant pathogens, which are mainly found in ostrich feces. Spreads of zoonotic bacteria and causes of diseases in animals and humans are common in ostrich farms due to a lack of proper management. The persons directly or indirectly involved in an ostrich farm as well as an immune-compromised person and pregnant women, have a high potential risk of the developing diseases caused by zoonotic bacteria.

CONCLUSION:
In the present study, the most prevalent bacteria were found to be E. coli and Salmonella spp., which were isolated from the most significant sources, such as cloacal swabs and ostrich faces, and were responsible for the transmission of zoonotic pathogens from animals to humans. Due to wide use of antibiotics without proper prescription microorganisms exhibit their resistance character in ostrich farm in Bangladesh as well as other countries in the world. Usually, hygiene levels in ostrich farms determine the presence of these microorganisms, and contamination may result from domestic ostrich sanitation and handling. Thus, ostrich farms need proper antimicrobials and biosecurity. Ostrich farming is a developing sector in Bangladesh. Therefore, this study will benefit investors, prescribers, and the ostrich owners. Additionally, Bangladeshi ostrich farms must utilize antibiotics rationally to prevent the multi-drug-resistant micro-organisms. Finally, the precautions must be taken to prevent the spread of zoonotic diseases among the ostrich farming workers.

CONFLICTS OF INTEREST:
The authors have no conflict of interest.