Molecular Detection of Multidrug Resistant Staphylococcus aureus Isolated from Bovine Mastitis Milk in Bangladesh

The current study was conducted to isolate and identify multidrug-resistant Staphylococcus aureus (MDR-SA) from mastitis milk samples and to determine their antimicrobial susceptibility pattern. A total of 48 bovine mastitis (BM) milk samples were collected from different parts of the Rangpur division, Bangladesh. After the collection of milk samples, mastitis was confirmed using the California mastitis test. Isolation and identification of Staphylococcus aureus were performed using conventional cultural and biochemical tests as well as using molecular methods of PCR. Nucleotide sequence analysis of the 23S rRNA gene of Staphylococcus aureus was determined. The antibiogram of the isolated bacteria was conducted using the disc diffusion method. Phylogenetic analysis of 23S rRNA was done using MEGA 7, ClustalW multiple sequence alignment, and NCBI-BLAST tools, where the sequence of the isolate showed 98% to 99% identity. Antibiogram test using 15 antimicrobial agents showed that all of the Staphylococcus aureus isolates were classified as multidrug-resistant (MDR). It was found that the isolates were resistant to tetracycline, novobiocin, methicillin, vancomycin, and cephradine, and the isolates were sensitive to ciprofloxacin, azithromycin, norfloxacin, levofloxacin, gentamicin, and amoxicillin. The detection of MDR-SA in mastitis milk is alarming and represents a great public health concern. The findings of the present study help identify Staphylococcus aureus at the molecular level using 23S rRNA gene sequencing and will help select the appropriate and effective antimicrobial agent to control BM in the northern part of Bangladesh.


Introduction
Bovine mastitis (BM) is an extremely common and genuine risk to the dairy industry throughout the world. BM is difficult to control due to the multifactorial nature of the disease, of which Staphylococcus spp. is notoriously involved [1]. Staphylococcus aureus (S. aureus) is a potential pathogen that is responsible for intramammary infections and decreases the health status of cows, leading to huge economic losses [2]. It is the leading pathogen that frequently appears in BM, however, its identification is comparatively costly [3]. Yearly costs related to the treatment of mastitis dairy livestock are more than one million dollars in the USA [4][5][6]. Antibiotics are indiscriminately used to control BM. Antibiotic therapy is one of the most common practices for controlling BM, and antimicrobial drugs like chloramphenicol, ciprofloxacin, novobiocin, vancomycin, and tetracycline were reported to have poor effectiveness against S. aureus [7][8][9][10]. Several antibiotics have been used for BM treatment, however, nowadays, treatment failure is commonly reported [11]. The deliberate use of antimicrobial agents in veterinary practice is a major factor for spreading antibiotic-resistant bacterial pathogens to human populations, thus may potentially lead to public health issues [5,11]. Methicillin-resistant Staphylococcus aureus (MRSA) has been reported as a threat to both humans and animals [12][13][14]. Multidrug-resistant (MDR) S. aureus infections have been associated with high morbidly and economic losses. S. aureus is often found in dairy cow's milk and has been clinically proven to be a cause of mastitis [15]. It has been reported that humans sometimes consume raw milk [16]. Nowadays, livestock-associated and community-associated MRSA have been reported and are of great public health concern [17][18][19][20]. The consumption of contaminated milk and milk products was reported to be a source of infections by S. aureus [21]. S. aureus infections in humans may lead to septicemia, pneumonia, and dermatitis [22]. There has been limited research about the detection of S. aureus in Bangladesh [23][24][25]. In recent years, the prevalence of BM in the Rangpur division has been reported to be gradually increasing. In the present study, 23S rRNA gene analysis was used to identify in-depth gene-specific S. aureus in the Rangpur division from BM milk. In addition, the study was conducted to determine the antimicrobial resistance pattern of S. aureus and the phylogenetic relatedness of the isolated S. aureus from BM, which may help control the imprudent use of antibiotics in veterinary practices for the treatment of BM and to increase public awareness.

Ethics Statement
The experimental procedures and protocols used in this study were approved by the Animal Ethics and Welfare Committee of the Institute of Research and Training, Hajee Mohammad Danesh Science and Technology University (approval number HSTU/RIT/2556).

Sample Collection and Processing
A total of 48 milk samples (10 mL) from cattle (Bos indicus) suffering from mastitis were collected from nine selected dairy farms of the Rangpur division of Bangladesh in September 2017 to January 2018 (Table 1). To confirm the mastitis in the cattle, the California Mastitis Test (CMT) was performed by mixing milk sample (3 mL from each quarter) and commercial CMT reagent (3 mL) using a partitioned plastic paddle, as previously described [26]. After screening the presence of mastitis by CMT tests, all milk samples were transferred to the Microbiology Laboratory, Department of Microbiology, Hajee Mohammad Danesh Science and Technology University (HSTU), for further microbiological processing.  [27]. Samples were also cultured on blood agar medium (Merck, Darmstadt, Germany) enriched with defibrinated ox blood to determine the hemolytic activity of the isolates. The isolates were identified using conventional biochemical tests, as described previously [27][28][29].

DNA Extraction
A single pure colony of S. aureus strain BARC001 culture from SAN 110 was cultured overnight in Luria Bertani broth (HI-MEDIA ® , Mumbai, India) at 37 • C. Genomic DNA of Staphylococcus aureus (n = 48) was extracted using a Wizard ® Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Extracted DNA was quantified at 260/280 nm by Nanodrop (Thermo Fisher Scientific, Dreieich, Germany) and stored at −20 • C.

Sequencing and Phylogenetic Analysis
The nucleotide sequence of the 23S rRNA gene was obtained by Sanger sequencing method using the ABI 3130 Genetic Analyzer (Applied Biosystems ® , Thermo fisher scientific, Waltham, MA, USA) and sequence data were submitted to NCBI nucleotide sequence database for analysis using the BLAST tool, the ClustalW multiple sequence alignment, and neighbor-joining method [34]. The phylogenetic tree was constructed using MEGA software [35]. The evolutionary distances were computed using the maximum composite likelihood method [36].

Isolation and Identification of MDR-SA
A total of 48 clinical mastitis milk samples were collected from dairy cow farms from different areas of the Rangpur division (Table 1) (Table 1). It was found that S. aureus was isolated from all mastitis milk samples obtained from dairy cows. S. aureus showed gray-white colonies on SAN 110 and golden yellowish colonies on MSA plates. Isolates showed complete (β) hemolysis on ox blood agar. The isolates were confirmed as S. aureus using different conventional biochemical tests.
The genomic DNA was used to confirm identity using the 23S rRNA and species-specific PCR. Isolates showed a band at 1267 bp ( Figure 1). PCR products (10 pico-moles) were submitted to the National Institute of Biotechnology (NIB), Savar, Dhaka, Bangladesh for partial length sequencing of the 23S rRNA gene in both directions.

Sequencing and Phylogenetic Analysis
The nucleotide sequence of the 23S rRNA gene was obtained by Sanger sequencing method using the ABI 3130 Genetic Analyzer (Applied Biosystems®, Thermo fisher scientific, Waltham, MA, USA) and sequence data were submitted to NCBI nucleotide sequence database for analysis using the BLAST tool, the ClustalW multiple sequence alignment, and neighbor-joining method [34]. The phylogenetic tree was constructed using MEGA software [35]. The evolutionary distances were computed using the maximum composite likelihood method [36].

Isolation and Identification of MDR-SA
In total, 48 clinical mastitis milk samples were collected from dairy cow farms from different areas of the Rangpur division. Based on cultural examinations, four bacterial species, including Staphylococcus spp. (100%, n = 48/48), Escherichia coli (100%, n = 48/48), and Klebsiella sp., (62.5%, n = 30/48), were identified (Table 1). It was found that S. aureus was isolated from all mastitis milk samples obtained from dairy cows, while other bacteria were not detected. S. aureus showed graywhite colonies on SAN 110 and golden yellowish colonies on MSA plates. Isolates showed complete (β) hemolysis on ox blood agar. The isolates were confirmed as S. aureus using different conventional biochemical tests The genomic DNA was used to confirm identity using the 23S rRNA and species-specific PCR. Isolates showed a band at 1267 bp ( Figure 1). PCR products (10 pico-moles) were submitted to the National Institute of Biotechnology (NIB), Savar, Dhaka, Bangladesh for partial length sequencing of the 23S rRNA gene in both directions. BLAST analysis revealed 98% homology with NCBI GenBank data set. Phylogenetic analysis was done by ClustalW multiple sequence alignment and the neighbor-joining method (Figure 2). BLAST analysis revealed 98% homology with NCBI GenBank data set. Phylogenetic analysis was done by ClustalW multiple sequence alignment and the neighbor-joining method (Figure 2). The sequence analyses revealed that the S. aureus isolates were identical and were closely related to isolates previously reported from South Korea, France, the United Kingdom, and the USA.

Antibiogram of S. aureus Isolates
The results of the antibiogram test of S. aureus isolated from mastitis milk samples against 15 antimicrobial agents revealed that all isolates tested were resistant to methicillin (MET-5 µg), novobiocin (NV-30 µg), vancomycin (VA-30 µg), tetracycline (TE-30 µg), and cephradine (CH-30 µg). On the other hand, isolates showed intermediate resistance to chloramphenicol (C-30 µg), bacitracin (B-10 µg), erythromycin (E-15 µg), and nalidixic acid (NA-30 µg). The tested isolates were sensitive to six out of the 15 tested antimicrobial agents as shown in Table 2. The sequence analyses revealed that the S. aureus (n = 1) Strain BARS001 was closely related to isolates previously reported from South Korea, France, the United Kingdom, and the USA.

Discussion
Bovine mastitis (BM) is an inflammatory disease of dairy cows caused by various opportunistic bacteria that reduce milk yield and deteriorate the quality of milk composition [37,38]. BM is a threat to dairy farms and farmers [39]. The findings of the current study showed that the prevalence of S. aureus in mastitis milk samples was 100% (n = 48). Cultural, biochemical, as well as molecular tests using the 23S rRNA gene amplification were used to identify S. aureus.
The presence of S. aureus in milk in the present study was found to be higher than previous studies [16,[40][41][42][43][44]. The high prevalence rate of S. aureus in the present study may be explained by differences in geographical distribution, immunological status, as well as biosecurity practices of the study areas in comparison to other studies. The antibiogram showed that all the isolates were classified as MDR, similar to a previous study [45]. In the current study, levofloxacin (26 mm) and ciprofloxacin (29 mm) demonstrated the highest antibacterial activity based on the zones of inhibition, which may be recommended for use to treat BM in the Rangpur division of Bangladesh. Nevertheless, a study by Jahan [16] revealed that more than 83.33% S. aureus from raw milk were susceptible to ciprofloxacin. The results of the antibiogram from the current study were similar to those previously reported [16,46,47]. All isolates in the present study were classified as multidrug-resistant (MDR) [48]. The 23S rRNA sequencing was a powerful tool for the identification of Staphylococcus aureus isolated from bovine mastitis and its evolutionary features. In this study, 23S rRNA gene sequencing was used to identify coagulase-positive (CoPS) S. aureus isolates of BM at the species level. The detection of MDR-SA is alarming for human health, as it has zoonotic potential. Livestock may play an important role in transmitting the bacteria to humans through milk [49,50], which may represent a great threat to public health, farm-associated workers, and veterinarians. In recent years, the incidence of S. aureus has been increasing, and health care-associated MRSA and livestock-associated MRSA have commonly been reported, which has a major impact on public health [51,52]. The phylogenetic analysis showed that the MDR-SA isolate is closely related to those previously reported from other countries and regions [9,16,17].

Conclusions
Livestock associated CoPS -SA, which is MDR, is notorious for its appearance in the Rangpur division, causing BM. Current research suggests that it is essential to stop the deliberate use of antibiotics to prevent MDR-SA. Proper hygienic practices and the maintenance of biosecurity measures are two of the most important steps to reduce infections. The findings of the present study help determine the evolutionary feature of S. aureus in the Rangpur division, and the antibiogram results help select the appropriate antimicrobial agents for the treatment of infected animals. The isolated S. aureus may be used for the future development of a mastitis vaccine using local isolates as a prophylactic measure in livestock.