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Molecular Detection of Methicillin-resistant Staphylococcus Aureus (mrsa) in Ornamental Birds Having Public Health Significance

Sumaiya Binte Zaman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Abdus Sobur
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Jannat Hossain
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Amrita Pondit
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Mst. Minara Khatun
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md Abu Choudhury
The University of Queensland, UQ Centre for Clinical Research UQCCR, Herston, Brisbane, Australia.

Md. Tawyabur
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Md. Tanvir Rahman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

There has been an increase in the demand for ornamental birds as pets in Bangladesh. However, issues of Antimicrobial Resistance (AMR) in ornamental birds remain poorly understood. Methicillin-resistant Staphylococcus aureus (MRSA) are major human health problem. Present study was designed to investigate the potentiality of ornamental birds as a source for MRSA that could transmit to human. A total of 70 samples were collected randomly from various ornamental birds and bird handlers working in the shops. A semi-structured questionnaire-based interview was also conducted with bird shop attendants to suspect the possible AMR origin and transmission. Isolation and identification of MRSA were based on culture, disk diffusion method followed by PCR. Hemolytic activities were tested on blood agar plates Among the 70 samples, 40 (57.14%) were found positive for S. aureus. Phenotypically 77.50% S. aureus were found resistant to oxacillin (methicillin), while by PCR, only ten (25%) isolates were found positive for mecA gene. Both the ornamental birds and bird handlers carried MRSA. Among the MRSA isolates, phenotypically six isolates were found resistant to vancomycin e.g.,VRSA (vancomycin-resistant Staphylococcus aureus).As far we know, this is the first report in Bangladesh describing molecular detection of MRSA from ornamental birds and bird handlers. From this study it is evident that ornamental bird carries MRSA that could transmit to human.

Keywords: Ornamental birds, Zoonoses, Antibiotic resistance, MRSA, VRSA, mecA

Location: Mymensingh city, Bangladesh

Start Date: 00-04-2018

End Date: 00-07-2018

Program Area: Animal Health and Management

Commodity: Bird

Objectives:

To determine the Molecular detection of methicillin-resistant Staphylococcus aureus (MRSA) in ornamental birds having public health significance

Methodology:

Collection of samples and data: The study was conducted during April to July, 2018 in Mymensingh city, Bangladesh. All the samples were collected on random basis from ornamental bird shops located in Mymensingh city, and bird handlers working there. A total of 64 cloacal and tracheal swab samples of different ornamental birds and 6 samples from hand washing of bird handlers (shop keepers) were collected aseptically and transferred immediately to the Bacteriology laboratory, Department of Microbiology and Hygiene, Bangladesh Agricultural University for bacteriological analysis. The summary of samples collected with their number and source are presented. During the sample collection, a semi-structured questionnaire-based interview were also conducted with bird shop owners, and bird handlers working in the shop (n=30) focusing their KAP (knowledge, attitude and practices) on disease occurrence, antibiotics used, hygiene of the shop, types of birds available etc. to have some idea regarding AMR origin and transmission among birds and bird handlers. Isolation and identification of Staphylococcus aureus: Initially Staphylococcus spp. was isolated by culturing on Mannitol Salt agar (MSA). Yellow colored colonies on MSA were Gram’s stained and the Gram-positive cocci bacteria having clustered arrangement was considered as S. aureus (Habibullah et al., 2017). The genomic DNA from S. aureus was extracted by boiling method (Begum et al., 2016). Final confirmation of detection of S. aureus was done by PCR targeting nuc gene as previously described (Bakeet and Darwish, 2014). All the PCR were done in a final 25 μl reaction with 12.5 μl master mixture 2X (Promega, USA), 2 μl genomic DNA (around 30 ng), 1 μl (100 pmol) each primer and 8.5 μl nuclease free water. Amplified products were analyzed by electrophoresis in 1.5% agarose gel. Ethidium bromide was used for staining the product and visualized under ultraviolet trans-illuminator (Biometra, Germany). A 100bp DNA ladder (Promega, USA) was used as molecular weight marker. The hemolytic activity of the isolated bacteria was detected following growth on blood agar as described by other (Habibullah et al., 2017). Antibiotic sensitivity test: Antibiotic sensitivity test was performed by disk diffusion test on Mueller-Hinton agar (Hodeida, India) plates having a concentration of bacteria equivalent to 0.5 McFarland standards (Bauer et al. 1966). The plates were incubated at 37°C aerobically for 18-24 hours to observe the results. Results of the antibiotic sensitivity tests were recorded as sensitive, intermediately sensitive, or resistant, and the zone of growth inhibition was compared with the zone size interpretative tables provided by the Clinical and Laboratory Standards Institute (CLSI, 2011). The antibiotics disc used to determine the antibiogram were methicillin (oxacillin) at the dose of 5μg/disc. In addition, gentamicin (10 μg), ciprofloxacin (10 μg), chloramphenicol (10 μg), oxytetracycline (30 μg) and vancomycin (30 μg) were also used in the antibiogram study. Any isolate showing resistance against oxacillin was considered as MRSA and resistance against vancomycin was considered as VRSA. Molecular detection of methicillin-resistant S. aureus (MRSA): Molecular detection of methicillin-resistant S. aureus (MRSA) was done by PCR using the standard primers and protocol (Habibullah et al., 2017) targeting mecA gene. All the PCR were done in final 25 μl volume as described earlier. Statistical analysis: All the data generated were incorporated into the excel sheet (MS-2013) and performed descriptive statistics using SPSS software (SPSS-22.0, IBM, USA) to compute the frequencies of MRSA.

Source of Information: J Bangladesh Agril Univ 18(2): 415–420, 2020

Article Link (Online Journal): https://doi.org/10.5455/JBAU.80165

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

MRSA are important zoonotic pathogens. In this study MRSA were successful detected from ornamental birds for the first time in Bangladesh. Some of the isolates were found resistant to vancomycin (VRSA). Present findings demonstrate that ornamental birds are potential reservoir of MRSA which poses a threat to bird handlers. Further research work is now required to identify the possible origin of these MRSA and VRSA in ornamental birds and their interspecies transmission. In addition, these birds need to be under surveillance for early the detection of MRSA so that risk of their transmission to human could be minimized.

Knowledge Management: Journal