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Identification of Methicillin-resistant Staphylococcus aureus Isolated from Dairy Cow’s Milk in Tulungagung District, Indonesia

Aswin Rafif Khairullah1, Sri Agus Sudjarwo2, Mustofa Helmi Effendi3,*, Shendy Canadya Kurniawan4, Agus Widodo5, Otto Sahat Martua Silaen6, Sancaka Chasyer Ramandinianto7
1Division of Animal Husbandry, Faculty of Veterinary Medicine, Universitas Airlangga, Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
2Division of Basic Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
3Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
4Master Program of Animal Sciences, Department of Animal Sciences, Specialisation in Molecule, Cell and Organ Functioning, Wageningen University and Research, Wageningen 6708 PB, Netherlands.
5Department of Health, Faculty of Vocational Studies, Universitas Airlangga, Jl. Dharmawangsa Dalam Selatan No. 28-30, Kampus B Airlangga, Surabaya 60115, East Java, Indonesia.
6Doctoral Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6 Senen, Jakarta 10430, Indonesia.
7Lingkar Satwa Animal Care Clinic, Jl. Sumatera No. 31L, Gubeng, Surabaya 60281, East Java, Indonesia.

ABSTRACT

Background: Mastitis is one of the factors contributing to the health-related decreased milk production and quality for dairy cows. Mastitis in ruminants is a serious bacterial disease caused by Staphylococcus aureus. Staphylococcal bacteria are becoming increasingly resistant to many classes of antibiotics, particularly β-lactam families like the MRSA strain. Laboratory tests are required to determine the level of bacterial resistance and to identify MRSA isolates sourced from dairy cows in Tulungagung District. 

Methods: 110 milk samples were isolated on MSA media followed by Gram’s staining and biochemical tests. Kirby-bauer diffusion test-based assessment of antibiotic sensitivity. The S. aureus isolates that underwent the MRSA identification test were S. aureus isolates that had developed a resistance to β-lactam antibiotics.

Result: A total 81 samples of the 110 isolated milk samples were determined to be positive for S. aureus. Out of total isolates, 25 isolates of S. aureus had the highest level of oxacillin resistance. As many as 4 isolates were confirmed to be MultiDrug Resistance (MDR) and 17 MRSA isolates were discovered from 100 samples of dairy cows. Early diagnosis of MRSA infection is crucial since it can be challenging to treat because this type of bacteria is known to be resistant to several drugs and spreads readily.

INTRODUCTION

Milk is a nutrient dense (sources of nutrients mainly the protein). Even milk is regarded as a nutritious complement for the process of human growth and development. The reason for the significant requirement and desire for milk is its complete nutritious value (Smith et al., 2022). The production of nutritious and high-quality milk should be increased in order to boost revenue for everyone, especially dairy farmers and to promote public health by encouraging healthy milk consumption (Britt et al., 2018). However, Indonesia still has a high need and demand for milk that is inversely correlated with a low supply of milk, both in terms of quantity and quality (Khairullah et al., 2022). Mastitis is one of the factors contributing to the health-related decreased milk production and quality for dairy cows (Khairullah et al., 2020).
       
Dairy cows owned by businesses and small farmers alike frequently get mastitis, or udder inflammation, which results in significant losses (in milk production). In Indonesia, mastitis affects dairy cattle at a rate of 85% and because the majority of these cases are subclinical infections, prompt treatment or control is not always possible (Putra et al., 2023). This mastitis incidence may result in significant economic losses, particularly because of the decreased milk output, which may account for up to 25% of total production. Numerous pathogenic bacteria that enter the udder through the teat canal are one of the many causes that contribute to mastitis (Hughes and Watson, 2018). The incidence of mammary gland infection is caused by a number of predisposing factors, such as unsanitary milking, improper milking management, sores on the teats and the presence of pathogenic microorganisms in the cage environment (Widodo et al., 2022).
       
During manual milking, transmission of mastitis-causing pathogenic bacteria can take place between cows or from one udder’s teat to another. This could happen because of unhygiene in milker’s hands, the udder-washing water, the cloth used to dry the udder prior and after milking, or other tools used during milking (Goulart and Mellata, 2022).
       
Unhygienic handler adds Staphylococcus aureus, a causative agent of Mastitis in ruminants. Mastitis is a serious bacterial disease which is the primary cause of subclinical or clinical mastitis in dairy cows, leading to enormous losses for the dairy sector (Tarazona-Manrique et al., 2019). Mastitis brought on by S. aureus manifests as subacute or persistent inflammation. Despite the fact that these bacteria can grow and develop well in milk. S. aureus contamination might happen because of the presence of causative bacterium in fresh milk either during milking or processing. The teats of infected cows are the primary reservoir of S. aureus (Exel et al., 2023).
       
Human affecting S. aureus can cause infection, particularly Methicillin-Resistant Staphylococcus aureus (MRSA) is known to be resistant to several medications (Yunita et al., 2020), it is challenging to treat this illness. There are several different types of Staphylococcal infections, including as pneumonia, bacteremia and postoperative wound contamination (Decline et al., 2020). S. aureus has a very high potential for producing a wide range of illnesses and food poisoning in both people and animals (Rahmaniar et al., 2020). It is known that S. aureus produces a variety of heat stable enterotoxins in milk. There have been reports of S. aureus enterotoxin food poisoning in milk and dairy products (Tyasningsih et al., 2022).
       
Treatment of S. aureus in cases of mastitis is known to be challenging, particularly due to the difficulty in selecting the appropriate type of antibiotic in the field and the ease with which resistance develops (Sharun et al., 2021). Staphylococcal bacteria are becoming increasingly resistant to many classes of antibiotics, particularly β-lactam families like the MRSA strain, its resistance to various types of antibiotics is developing rapidly (Alexander et al., 2023). The plasmid, which allows for quick transmission between Staphylococci, contains the gene for the penicillinase enzyme. Additionally, numerous hospitals have noted an increase in the prevalence of S. aureus and MRSA, which are often both types of these bacteria that are MDR (Waruwu et al., 2022).
       
Sendang (a sub district in Tulungagung district) is one of a major milk-producing regions of East Java (Khairullah et al., 2023) revealed that breeders poor care practices for dairy cows with subclinical mastitis eventually progresses to clinical mastitis. There have been numerous reports of subclinical mastitis cases in Tulungagung district, which is consistent with the high volume of milk production and the presence of dairy cattle (Ramandinianto et al., 2020). Mastitis instances are frequently caused by infections caused by the MRSA bacteria. Making the appropriate antibiotic choice can lower the number of mastitis cases (Wilm et al., 2021).
       
The incidence of MRSA infection can be a health problem for the community. Laboratory tests are required to determine the level of bacterial resistance and to identify MRSA isolates sourced from dairy cows in Tulungagung district, so that this research data can be useful for the prevention and treatment of mastitis incidents on cattle farms of dairy industry.

MATERIALS AND METHODS

Study area and sample collection
 
The study was carried out from March 2023 to May 2023. Dairy cow milk samples were collected from different dairy farms in the Sendang Subdistrict region of the Tulungagung district, while bacterial isolation and sensitivity testing were conducted at the Veterinary Microbiology Laboratory, Faculty of Veterinary Medicine, Airlangga University. A total of 110 milk samples were collected. A 60 ml sample bottle was used to hold each milk sample that was collected. The samples are then transported to the lab and were being kept under refrigerated conditions using a cooling box until analysis.
 
Isolation and identification of S. aureus
 
Milk samples obtained from enrichment media were purified and cultivated on Mannitol Salt Agar (MSA) medium before being incubated at 37°C for 24 hours (Dilnessa and Bitew, 2016). Identification was carried out by evaluation based on morphological cultural characteristics, followed by microscopic analysis using Gram’s staining technique, which reveals clusters of Gram-positive bacteria in the shape of coccus (Sadiq et al., 2020). The catalase test and the coagulase test were used in biochemical assays to identify the S. aureus species. Hydrogen peroxide (H2O2) 3% was used to clean glass objects to perform catalase tests and the solution was then mixed with one colony’s growth product (Lagos et al., 2016). For the coagulase test, S. aureus-related colonies were extracted from MSA media using ose (Check the ose), then these colonies were added to 3 ml of Nutrient Broth media and cultured at 37°C for 24 hours. A vortex was used to thoroughly mix 1 ml of rabbit plasma before incubation, which was followed by a 24-hour incubation period (Javid et al., 2018).
 
Antibiotic sensitivity test
 
Kirby-bauer diffusion test-based assessment of antibiotic sensitivity. This method was carried out by taking S. aureus bacterial isolates from MSA media using a sterile cotton swab in a suspension containing physiological saline with standard Mc Farland turbidity of 0.5 and then wiping it evenly on the surface of the Mueller Hinton Agar (MHA) media. The MHA media surface was covered with antibiotic discs, which were subsequently incubated for 24 hours at 37°C (Mutmainnah et al., 2020). There were 4 types of antibiotics used for the sensitivity test in this study, namely the aminoglycoside group (Gentamicin 10 μg), the macrolide group (Erythromycin 15 μg), the tetracycline group (Tetracycline 30 μg) and the β-lactam group (Cefoxitin 30 μg and oxacillin 30 μg). Each antibiotic disc was attached to the MHA media’s surface at a distance of 25 to 30 mm. The Clinical and Laboratory Standard Institute (CLSI, 2020) provides the basis for the interpretive standard for determining the diameter of the inhibitory zone.
 
MRSA identification test
 
The S. aureus isolates that underwent the MRSA identification test were S. aureus isolates that had developed a resistance to β-lactam antibiotics (Cefoxitin and oxacillin). Oxacillin Resistance Screening Agar Base (ORSAB) media that had been combined with Oxacillin Resistance Selective Supplement was used to inoculate the bacterial colonies from MHA medium in order to confirm the presence of MRSA. Following that, ORSAB medium was incubated for 24 hours at 37°C. When the bacterial colonies on ORSAB media turn blue, it is a positive indicator of MRSA (Ibrahim et al., 2017).

RESULTS AND DISCUSSION

Bacterial isolates
 
Based on the results of the sample examination and the results of the morphological culture, Gram staining and biochemical testing, 81 samples (73.64%) of the 110 isolated milk samples were determined to be positive for S. aureus (Table 1). The emergence of golden yellow bacterial colonies on MSA media suggested successful morphological culture of S. aureus (Fig 1). The presence of purple colonies and globular, clustered forms during Gram staining indicates a positive Gram result (Fig 2). The formation of gas bubbles in the catalase test (Fig 3) and plasma clots in the coagulase test are signs of positive biochemical tests for S. aureus (Fig 4).
 

Table 1: Isolation and identification of S. aureus.


 

Fig 1: S. aureus colonies in MSA.


 

Fig 2: Gram-stained S. aureus colonies under a microscope with a magnification of 1000x.


 

Fig 3: Catalase test results indicate S. aureus positivity.


 

Fig 4: Coagulase test results indicate S. aureus positivity.


       
MSA media was utilized for the isolation in this study because S. aureus can ferment mannitol employing streak media with multilevel patterns for early culture (Pumipuntu et al., 2017). Gram-stained S. aureus bacteria displayed purple coloration under a 1000x magnification microscope and a clustered, spherical morphology that is typical of Staphylococcus germs (Kobayashi et al., 2020). A positive catalase test result means that the bacteria are either aerobic or facultatively anaerobic, meaning that they use oxygen to breathe (Linzner et al., 2022). A positive coagulase test result means S. aureus can produce coagulase enzymes that can cause the activation of nonproteolytic pro-thrombin and fibrinogen cleavage (Trivedi et al., 2018).
 
Antibiotic resistance of S. aureus
 
According to this investigation, 25 isolates of S. aureus had the highest level of oxacillin resistance. While there are 18 isolates of tetracycline, 11 isolates of erythromycin, 9 isolates of cefoxitin and 6 isolates of gentamicin-resistant S. aureus (Table 2).
 

Table 2: Isolated S. aureus resistance profile by antibiotic group.


       
Numerous factors contribute to the development of bacteria that are resistant to antibiotics, including inadequate use of antibiotics, a populace that is reluctant to use such medications, treatment antibiotics that are the same but have been used repeatedly, inadequate research into new antibiotics and inadequate government oversight of the manufacture and distribution of antibiotics (Cook and Wright 2022). An opportunistic pathogenic bacteria called S.aureus is frequently found in both people and animals (Howden et al., 2023). This bacterium may cause financial losses by reducing the quantity and quality of milk produced by dairy cows (Tesfaye et al., 2021).
       
The profile of antibiotic resistance derived from the results of the S. aureus resistance test to antibiotics revealed that 27 S. aureus isolates (33.33%) were found to be resistant to the 1 class of antibiotics tested out of a total of 81 S. aureus isolates. Whereas 9 isolates (11.11%) were resistant to 2 classes of antibiotics and 4 isolates (4.99%) were confirmed to be MDR because it was resistant to three or more classes of antibiotics (Table 2 and Fig 5).
 

Fig 5: Analyze the susceptibility to antibiotics of a S. aureus isolate cultured on MHA.


       
As many as 4 out of every 81 S. aureus isolates were labeled as multidrug resistant because they exhibited resistance to at least 3 different antibiotic classes. A latent resistance gene’s expression, a gene with a resistance determinant, or genetic mutation are the main ways that bacterial colonies survive in a threatened state (Palma et al., 2020). The majority of bacterial information is encoded by chromosomes and in MDR bacteria, this results in a multistep mutation that gradually increases resistance (Gogry et al., 2021). Extrachromosomal genes, which can be found in plasmids or bacteriophages, are present in some bacteria (Deutsch et al., 2018).
       
On transposons and integrons, where the factor R plasmids, also known as infectious plasmids, can be transferred, resistance factors can be transferred from chromosomes to plasmids or vice versa (Partridge et al., 2018). The Resistance Transfer Factor (RTF) segment and the r-determinant (r-unit) make up the R factor itself, with the RTF segment allowing the transfer of the R factor and the r-units each carrying characteristics associated with antibiotic resistance (Helinski 2022).
       
Dairy cows from Tulungagung district produced milk that included four isolate of S. aureus that was MDR (Table 3). This may account for the fact that Tulungagung district still had a low number of MDR cases of S. aureus, with 4 isolates from 110 milk samples analyzed.
 

Table 3: S. aureus isolates with a profile MDR.


 
Confirmation of MRSA
 
The findings of the MRSA identification of S. aureus isolates that were known to be resistant to cefoxitin and oxacillin revealed that 17 out of 25 isolates were positive on the ORSAB test (Table 4 and Fig 6). This demonstrates that 17 MRSA isolates (68%) were discovered from 110 samples of dairy cows that were analyzed, demonstrating that MRSA infection rates in dairy farms in Tulungagung district are quite high.
 

Table 4: Total number confirmed MRSA by ORSAB.


 

Fig 6: ORSAB test for MRSA identification.


       
A significant DNA element called SCCmec, measuring 20-100 kb, is inserted into the S. aureus strain to transform it into an MRSA strain (Harkins et al., 2017). The Penicillin Binding Protein 2a (PBP 2a) genes, which are responsible for MRSA resistance, are encoded by the Staphylococcal Cassette Chromosome mec (SCCmec) proteins mecA and mecC (Miragaia 2018). Changes in the typical PBP, specifically PBP 2 to PBP 2a, cause MRSA resistance to all β-lactam class antibiotics (Da Costa et al., 2018). PBP 2a has a very low affinity for β-lactams, allowing the MRSA strain to survive and produce the bacterial cell wall even when it is cultivated in media containing high levels of β-lactams (Fergestad et al., 2020).
       
A dangerous bacterial strain called MRSA is frequently seen in people, but it can also colonize and infect other species, including livestock, wildlife, pets and poultry (Silva et al., 2023). MRSA in animals is significant not just from an economic and welfare point of view, but also because these strains have the potential to serve as a reservoir for zoonotic infections in humans (Correia et al., 2019). Early diagnosis of MRSA infection is crucial since it can be challenging to treat because this type of bacteria is known to be resistant to several drugs and spreads readily (Hassoun et al., 2017).

CONCLUSION

Milk from dairy cows in Tulungagung district, had up to 17 MRSA strains. A nearby veterinarian must assess the efficacy of using antibiotics to treat mastitis infection as a kind of therapy. Since no isolates of bacteria in this investigation were gentamicin-resistant, gentamicin antibiotics can still be utilized as an alternate treatment for mastitis infection.

ACKNOWLEDGEMENT

The authors would like to acknowledge the Penelitian Pascasarjana-Penelitian Disertasi Doktor funding from Penelitian Drtpm Kemendikbudristek Year 2023, with a grant from Universitas Airlangga,  Surabaya, Indonesia under No.: 741/UN3.LPPM/PT.01.03/2023.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.

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