Ostriches (Struthio camelus) have fascinated humanity for centuries. Ostrich farming has grown globally, offering an inexpensive trade of feathers, meat, prized skin, and eggs, making them essential replacement animals in the multiple nations (Cooper et al., 2008). Additionally, ostriches are exhibited in the zoos and estates. There were numerous ostriches in the Sahara desert, which served as an area for hunting. Palestine, Iran, and the Arabian Desert have ostriches. In the late 19th century, Africa sent many ostriches to Australia, New Zealand, Europe, and North and South America (Boum, 2015). Ostrich farming is now an obscure part of South African agriculture, but it once dominated certain regions economies. Egypts 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 Africas 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 food-borne 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 Pseudo-monas 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.

Selection of study site and period  

This study was complemented at the bacteriology research laboratory of the Department of Microbiology at Hajee Mohammad Danesh Science and Technology University [HSTU] Bangladesh, during the period from July 2019 to December 2019. The research was carried out in Hajee Mohammad Danesh Science and Technology University [HSTU] ostrich farms in Dinajpur district. 

Sample collection and processing

A total of 32 samples including oropharyngeal swabs (Adult=2, Young=6), cloacal swabs (Adult=2, Young =6), environmental sand (Adult=2, Young=6) and feces (Adult=2, Young=6) were collected from ostrich farm and transferred with ice-containing bags in the bacteriology laboratory for microbiological analysis (Fig. 1). 

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 bacterio-logical and fungal agar media were derived from Hi-Media Laboratories Private Ltd. India. Primary identification of bacteria was made by using gram-staining 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 Scienti-fic-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).

Table 1: PCR primers and DNA extraction techniques.

Fig. 1: Collection of samples from faces (left) and cloacal swab (right)

Antibiotic sensitivity tests

According to Clinical and Laboratory Standards Insitute (CLSI), agar disc diffusion techniques were used to determine the antibiotic sensitivity patterns of isolates on Muller-Hinton agar plates (CLSI, 2014).  A total of 15 commercially available antibiotics, inclu-ding Kanamycin (30 µg), Amoxicillin (30 µg), Pipera-cillin (10 µg), Bacitracin (10 µg), Tetracyclin (15 µg), Erythromycin (15 µg), Azithromycin (15 µg), Cloxa-cillin (5 µg), Chloramphenicol (30 µg), Gentamicin (10 µg), Novobiocin (30 µg), Methicillin (5 µg), Cefi-xime (5 µg), Vancomycin (30 µg), 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 measure the zone of inhibition on a millimeter scale according to company guidelines.

Prevalence of isolates

Table 2: Prevalence of E. coli and Salmonella spp. from young ostrichs. 

Among thirty-two (32) samples, E. coli 8 (53.33%) and Salmonella spp. 7 (46.67%) were isolated from young ostrich, and in adult ostrich, E. coli 2 (40%) and Salmonella spp. 3 (60%) were identified. In young ostrich, E. coli 3 (75%) and Salmonella spp. 1 (25%) from oropharyngeal swab, and E. coli 5(45.45%) and Salmonella spp. 6 (54.55%) from feces were found (Table 2 and Fig. 

Fig. 2: Prevalence of bacterial isolates from young ostrich.

In adult ostrich, E. coli 1(50%) and Salmonella spp. 1 (50%) from cloacal swab, E. coli zero and Salmonella spp. 1 (100%) from oropharyngeal swab and E. coli 1(50%) and Salmonella spp. 1 (50%) from orophary-ngeal swab samples were found, respectively (Table 3 and Fig. 3).

Isolation and Identification of Bacteria

Table 3: Prevalence of E. coli and Salmonella spp. from adult ostrichs.

 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).

Fig. 3: Prevalence of bacterial isolates from adult ostrich.

Results of Antibiotic Sensitivity Test

Table 4: Result of biochemical test for the representative isolates of E. coli and Salmonella spp.

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, Tetra-cycline, Cloxacillin, Novobiocin, Cefixime (Fig. 6A). Salmonella spp. was 100% sensitive to Azithromycin and 100% resistant to Tetracycline, Piperacillin, Baci-tracin, Chloramphenicol and Methicillin (Fig. 6B).

Fig. 4: E. coli produce metallic sheen (greenish black) colony on EMB agar (A), Salmonella spp. on Brilliant Green Agar (B).

Results of PCR, gene sequencing and phylogenic tree analysis

16S rRNA gene region of E. coli was amplified with the universal primers, Forward primer 27F (5-AGAGTTTGATCCTEGGCTCAG3) and Reverse primer 1492 R (5-TACCTTGTTACGACTT3), and found at 1465 bp. For Salmonella spp. DNA was amplified with specific S139- F and S141- R primers, and a 284 bp band was found, which are shown in Fig. 7A and the phylogenic tree in Fig. 7B. Whole genome sequencing was experimented by the National Institute of Biotechnology at Savar, Dhaka, Bangladesh.

Table 5: Antibiotic sensitivity tests results of E. coli and Salmonella spp.

Fig. 5: E. coli show positive results in A: Methyl red, B: Voges-Proskauer, C: TSI, D: SC and E: SF.

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, Baci-tracin, Chloramphenicol and Methicillin (Fig. 6B).

Fig. 6: Antibiotic sensitivity test for E. coli (A) and Salmonella spp. (B)

Results of PCR, gene sequencing and phylogenic tree analysis 16S rRNA gene region of E. coli was amplified with the universal primers, Forward primer 27F (5-AGAGTTTGATCCTEGGCTCAG3) and Reverse primer 1492 R (5-TACCTTGTTACGACTT3), and found at 1465 bp. For Salmonella spp. DNA was amplified with specific S139- F and S141- R primers, and a 284 bp band was found, which are shown in Fig. 7A and the phylogenic tree in Fig. 7B. Whole genome sequencing was experimented by the National Institute of Biotechnology at Savar, Dhaka, Bangladesh.

Fig. 7: 284 bp of Salmonella enterica; L=500kb ladder; S1, S 2, S3: sample (A); Phylogenic tree analysis of Salmonella enterica (B).

In Bangladesh, very few ostrich farms are available. Different food items such as vegetables, green leaves, poultry feeds, rice, wheat and water are needed to feed ostrich. All these foods are related to microbial contamination during handling. Our research mainly focused on identifying microorganisms directly or indirectly related to the ostrich, the farm environment and persons working on ostrich farms. Different zoonotic bacteria cause different diseases and transmit to human through animals. This represents the latest research in Bangladesh to assess the microbial community in the oropharyngeal swabs, cloacal swabs, feces and environmental sand samples from ostrich farms, along with determining their antibiogram study. A previous study reported that there was very little information about the zoonotic potential of Salmonella spp. and E. coli strains (Youssef AI et al., 2017). Earlier researchers reported that ostriches are potential reservoirs of Salmonella spp. and E. coli as well as transmit important zoonotic bacteria from animals to humans (Jahan et al., 2017). In our study, most pre-valence E. coli (53.33%) were identified from different ages, which was lower than the observation of Asmaa et al. (2016), who found 58.4% E. coli in Ostrich Farms in Egypt. In our study, the overall cultural prevalence of Salmonella spp. from different ages ostrich was (46.67%), which was higher than the value (20.8%) observed by Asmaa et al. (2016).

The current study revealed that the molecular characterization of E. coli and Salmonella enterica that had been done by PCR amplification and phylogenic tree analysis. To determine the genetic diversity and evolutionary relationship between Salmonella spp. and other similar species, we applied phylogenic tree analysis with BLAST search tools. Overall on BLAST analysis, Salmonella_ enterica subsp_ salamae_strain_ DSM_9220 were identified in our sample and it was closely linked with Salmonella_ enterica_ subsp_ houtenae_strain_DSM_9221 and Salmonella_ enterica _subsp_ entarica_strain_Ty2. The present study repor-ted that E. coli can resist strong antibiotics such as Amoxicillin and Azithromycin (100%), and Chloramphenicol, Kanamycin and Gentamicin (75%), which are often used to treat pathogenic bacteria. A previous study by Jahan et al. (2017) showed that amoxicillin and azithromycin have strong antimicrobial activity against E. coli. And the result is similar to our current research. A previous study by Yadav et al. (2017) revealed that the rate of Salmonella spp. resistant to amoxicillin, ampicillin, tetracycline, erythromycin, and gentamycin was the highest (100%), followed by colistin sulfate (83.33%), pefloxacin (38.88%) enrofloxacin (38.88%), gentamycin (11.1%) and Ceftria-xone (0%). The present study reported that Salmonella spp. could be inhibited with strong antibiotics such as azithromycin (100%), followed by gentamycin (75%), which are often used to treat pathogenic bacteria, whereas they were more resistant to amoxicillin, piperacillin, bacitracin, tetracycline, cloxacillin (100%). Our study provides a similar result to (Janan et al., 2017).

The current study revealed that the molecular characterization of E. coli and Salmonella enterica that had been done by PCR amplification and phylogenic tree analysis. To determine the genetic diversity and evolutionary relationship between Salmonella spp. and other similar species, we applied phylogenic tree analysis with BLAST search tools. Overall on BLAST analysis, Salmonella_ enterica subsp_ salamae_strain_ DSM_9220 were identified in our sample and it was closely linked with Salmonella_ enterica_ subsp_ houtenae_strain_DSM_9221 and Salmonella_ enterica _subsp_ entarica_strain_Ty2. The present study reported that E. coli can resist strong antibiotics such as Amoxicillin and Azithromycin (100%), and Chloramphenicol, Kanamycin and Gentamicin (75%), which are often used to treat pathogenic bacteria. A previous study by Jahan et al. (2017) showed that amoxicillin and azithromycin have strong antimicrobial activity against E. coli. And the result is similar to our current research. A previous study by Yadav et al. (2017) revealed that the rate of Salmonella spp. resistant to amoxicillin, ampicillin, tetracycline, erythromycin, and gentamycin was the highest (100%), followed by colistin sulfate (83.33%), pefloxacin (38.88%) enrofloxacin (38.88%), gentamycin (11.1%) and Ceftriaxone (0%). The present study reported that Salmonella spp. could be inhibited with strong antibiotics such as azithromycin (100%), followed by gentamycin (75%), which are often used to treat pathogenic bacteria, whereas they were more resistant to amoxicillin, pipe-racillin, bacitracin, tetracycline, cloxacillin (100%). Our study provides a similar result to (Janan et al., 2017).

The current study revealed that the molecular characterization of E. coli and Salmonella enterica that had been done by PCR amplification and phylogenic tree analysis. To determine the genetic diversity and evolutionary relationship between Salmonella spp. and other similar species, we applied phylogenic tree analysis with BLAST search tools. Overall on BLAST analysis, Salmonella_ enterica subsp_ salamae_strain_ DSM_9220 were identified in our sample and it was closely linked with Salmonella_ enterica_ subsp_ houtenae_strain_DSM_9221 and Salmonella_ enterica _subsp_ entarica_strain_Ty2. The present study reported that E. coli can resist strong antibiotics such as Amoxicillin and Azithromycin (100%), and Chloramphenicol, Kanamycin and Gentamicin (75%), which are often used to treat pathogenic bacteria. A previous study by Jahan et al. (2017) showed that amoxicillin and azithromycin have strong antimicrobial activity against E. coli. And the result is similar to our current research. A previous study by Yadav et al. (2017) revealed that the rate of Salmonella spp. resistant to amoxicillin, ampicillin, tetracycline, erythromycin, and gentamycin was the highest (100%), followed by colistin sulfate (83.33%), pefloxacin (38.88%) enrofloxacin (38.88%), gentamycin (11.1%) and Ceftriaxone (0%). The present study reported that Salmonella spp. could be inhibited with strong antibiotics such as azithromycin (100%), followed by gentamycin (75%), which are often used to treat pathogenic bacteria, whereas they were more resistant to amoxicillin, piperacillin, bacitracin, tetracycline, cloxacillin (100%). Our study provides a similar result to (Janan et al., 2017). 

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 developing diseases caused by zoonotic bacteria.

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 inves-tors, prescribers, and the ostrich owners. Additionally, Bangladeshi ostrich farms must utilize antibiotics rationally to prevent multi-drug-resistant micro-organisms. Finally, precautions must be taken to prevent the spread of zoonotic diseases among the ostrich farming workers.

Ethical approval

The ethical committee of Hajee Mohammad Danesh Science and Technology University in Basherhat, Dinajpur, Bangladesh [HSTU] approved the researchs methodology [approval number: HSTU/IRT/94]. 

The Microbiology department at Hajee Mohammad Danesh Science and Technology University [HSTU] in Basherhat, Dinajpur, Bangladesh, supported laboratory facilities (bacteriological analysis) for this research. The IRT project gave financial support during research conduct.

The authors have no conflict of interest.