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Research Detail

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Ariful Islam
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Bakhtiar Lijon


Md. Mahadee Hasan
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abu Syem Muhammad Arif
Reptiles Farm Limited, Mymensingh, Bangladesh

Mahbubul Pratik Siddique
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Muhammad Tofazzal Hossain
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Ariful Islam
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

An attempt was undertaken to characterize the bacterial flora of saltwater crocodiles reared on a commercial farm at Valuka, Mymensingh, Bangladesh. Swab samples (n=60) comprised of the oral swab (n=20), nasal swab (n=20), and cloacal swab (n=20) were collected from apparently healthy farm-raised crocodiles. Samples were cultured into various selective media to isolate bacteria. Characterization of the isolated bacteria was performed by studying the cultural, staining, biochemical properties followed by polymerase chain reaction. The antimicrobial susceptibility patterns of bacteria were investigated against eight commonly used antibiotics by the disc diffusion method. A total of 135 bacterial isolates belonged to 5 genera such as Escherichia (22%), Salmonella (22%), Staphylococcus (21%), Bacillus (16%), and Vibrio (19%) were identified. A genus-specific PCR targeting 16s rRNA gene successfully identified Escherichia coli, Salmonella spp., and Staphylococcus spp. In PCR assay, 11 of 30 (36.67%) E. coli isolates were found positive for stx1 and stx2 genes, and 4 of 30 (13.33%) isolates were found positive for and hly gene. Five isolates of Staphylococcus spp. were found positive with nuc gene, indicating these were pathogenic strains of Staphylococcus aureus. Multidrug resistance profile was observed in E. coli, Salmonella spp., and Vibrio spp. The results of this study indicate that captive crocodiles in the farm harbor multidrug-resistant pathogenic bacteria, which may produce infection in crocodiles and might cause health problems if transmitted to a human.

  Antibiogram, Bacterial flora, Bangladesh, Crocodiles, PCR.
  Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh.
  
  
  Animal Health and Management
  Bacteria, Crocodile, Crocodile

The study's objective was to isolate and identify the bacterial flora from oral, nasal, and cloacal cavities of captive crocodiles in Bangladesh and to characterize them through staining, cultural, biochemical features, and polymerase chain reaction (PCR), and antibiotic sensitivity test.

The research work was conducted at the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh. The samples were collected from Reptiles Farm Ltd. (RFL); Valuka, Mymensingh, Bangladesh. To collect the oral swabs, a piece of wood was used to keep the mouth of the crocodile open. A tight rubber band was used to avoid biting. Then sterile cotton buds were introduced inside its mouth cavity and the swab was collected. For nasal swabs, sterile cotton buds were introduced into the nasal cavity of the crocodile. The cloacal swab was also collected by using sterile cotton buds. Aseptic measures were undertaken during the collection of samples. After collection, the swabs were transferred into cryovials containing 0.1% peptone water. Immediately after transferring in cryovials, all samples were brought to the Bacteriology Laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh, through cool chain maintaining. Samples were inoculated into the Nutrient broth and incubated for 24 hours at 37°C for better nourishment of the desirable organisms. Isolation of bacteria is carried out based on the cultural characteristics of different selective media. For this one loopful of an enrichment culture of oral, nasal, and cloacal swabs was separately streaked in duplicate onto Mannitol salt agar (MSA, Hi-media, India), Thiosulfate Citrate Bile Salts Sucrose agar (TCBS, Hi-media, India), Salmonella-Shigella (SS) agar (Hi-media, India), Eosin Methyline Blue agar (EMB, Hi-media, India), Blood agar( BA, Hi-media, India) respectively and incubated for 24 hours at 37 ºC. Single colony grown onto selective media were further sub-cultured onto the particular media until pure bacterial cultures were obtained. To identify the bacteria Gram's staining method (Ashikuzzaman et al., 2015), motility test (Lijon et al., 2015), sugar fermentation test (Hemraj et al., 2013), and biochemical tests such as Catalase (Hemraj et al., 2013), Indole (Hemraj et al., 2013), MR-VP (Hemraj et al., 2013) and coagulase tests (Islam et al., 2016) were performed. Polymerase chain reaction (PCR) method was used to identify E. coli by amplifying 585bp, 606 bp, 374 bp, 889 bp, and 497 bp fragments of 16srRNAgene , stx1 and stx2 gene, hly gene, and rfb0157 gene, respectively (Schippa et al., 2010; Heuvelink et al., 1995; Wieler et al., 1996; Paton and Paton, 1998). For detection of Salmonella spp. 469 bp and 274 bp fragments of 16srRNA gene and typh gene were amplified respectively by following PCR method (Cohen et al., 1993). Polymerase chain reaction (PCR) assay was also performed to identify Staphylococcus spp. by amplifying 241 bp and 279 bp fragments of 16s rRNA gene and nuc gene, respectively (Stuhlmeier and Stuhlmeier, 2003; Kalorey et al., 2007). Antibiotic sensitivity test of isolated E. coli (n=30), Salmonella spp. (n=30), Bacillus spp. (n=10), Staphylococcus spp. (n=10) and 10 Vibrio spp. (n=10) were performed against eight commercially available antibiotic discs such as Ampicillin, Azithromycin, Cephalexin, Ciprofloxacin, Chloramphenicol, Gentamicin, Penicillin and Vancomycin (Hi-media, India). The disc diffusion method was used to detect antibiotic susceptibility of the isolated bacteria according to the guidelines of Cockerill and Clinical and Laboratory Standard Institute (CLSI), 2012.

  International Journal of Biosciences - Vol. 19, No. 2, p. 139-152, 2021
  http://dx.doi.org/10.12692/ijb/19.2.139-152
Funding Source:
1.   Budget:  
  

Crocodiles harbor hazardous and highly pathogenic strains of some bacteria, which may cause disease at the time of stress and results in significant economic loss due to morbidity and mortality. Bacterial genera isolated in this study may cause public health hazards that implicate people's health risk who come in contact with the crocodiles. Food-borne pathogenic bacteria might also contaminate crocodile's meat during slaughtering and processing if proper hygienic measures are not undertaken, which can result in loss of their acceptance for export in the international market. Multidrug-resistant bacteria found in crocodiles can make it hard for treatment if there is an infection.

  Journal
  


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