Detection of mecA gene and methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and risk factors from farms in Probolinggo, Indonesia

Background: Staphylococcus aureus is commonly found in dairy cows and is a source of contamination in milk. S. aureus that are resistant to beta-lactam antibiotics (especially cefoxitin) are referred to as methicillin-resistant Staphylococcus aureus (MRSA). The spread of MRSA cannot be separated from sanitation management during milking; it can originate from milk collected from the udder or from the hands of farmers during the milking process. The purpose of this study was to examine the level of MRSA contamination in dairy cow's milk and farmer's hand. Methods: A total of 109 samples of dairy cow’s milk and 41 samples of farmer’s hand swabs were collected at a dairy farm in Probolinggo, East Java, Indonesia. Samples were cultured and purified using mannitol salt agar (MSA). The profile of S. aureus resistance was established by disk diffusion test using a disk of beta-lactam antibiotics, namely oxacillin and cefoxitin. Results: The S. aureus isolates that were resistant to oxacillin and cefoxitin antibiotics were then tested for oxacillin resistance screening agar base (ORSAB) as a confirmation test for MRSA identity. S. aureus isolates suspected to be MRSA were then tested genotypically by polymerase chain reaction (PCR) method to detect the presence of the mecA gene. The results of the isolation and identification found 80 isolates (53.33%) of S. aureus. The results of the resistance test found that 42 isolates (15%) of S. aureus were resistant to oxacillin and 10 isolates (12.5%) were resistant to cefoxitin. The ORSAB test found as many as 20 isolates (47.62%) were positive for MRSA. In PCR testing to detect the presence of the mecA gene, three isolates (30%) were positive for the mecA gene. Conclusions: This study shows that several S. aureus isolates were MRSA and had the gene encoding mecA in dairy farms.


Introduction
Staphylococcus aureus is a pathogenic bacteria that can cause public health problems, because these bacteria often contaminate products of animal origin, including milk or commonly known as milk-borne disease (MBD). 1 This opportunistic bacterial pathogen that can be found in animals and humans. This bacterium can cause various diseases ranging from mild to systemic skin infections such as pneumonia, arthritis, and meningitis. [2][3][4] In previous studies, S. aureus was mostly transmitted to humans through contaminated milk. 5 S. aureus is commonly found on the skin and mucosa of livestock, especially dairy cows with subclinical or clinical mastitis, which is a source of contamination in milk. 6 If these bacteria are resistant to beta-lactam antibiotics is referred to as methicillin-resistant S. aureus (MRSA). 7 It has been noted in earlier investigations that MRSA can result in new health issues for both people and animals. 8 The high rate of MRSA contamination in dairy farms due to excessive administration of antibiotics in the treatment of dairy cows and the spread of these bacteria cannot be separated from sanitation management during milking. 3 Contamination can happen from milk that is collected from the udder as well as from the hands of farmers during the milking process. 9 The Probolinggo Regency, specifically in Krucil District, is one of the largest milk-producing centers in Indonesia. 10 Antibiotics have been widely used as treatment in cases of infection in dairy cattle in Probolinggo, especially in cases of mastitis, so contamination by MRSA in dairy farms in Probolinggo 11 is possible.
S. aureus evolved into strain MRSA because it received the insertion of a large DNA element between 20-100 kb called staphylococcal cassette chromosome mec (SCC mec), that underlies the change in normal penicillin-binding protein (PBP), namely PBP2 to PBP2a. 12 PBP2a is expressed by the gene encoding mecA contained in SCC mec which has a very low affinity for beta-lactams, so that event cultured on media containing high concentrations of beta-lactams, MRSA survives. 13 Molecular detection of the mecA gene using polymerase chain reaction (PCR) is often carried out to confirm the presence of MRSA isolates, but cannot be done in all laboratories because of the ability and cost constraints. 14 Constraints in the use of PCR can be replaced by examining MRSA using the disk diffusion method with the antibiotics oxacillin and cefoxitin, which is then continued with an examination using oxacillin resistance screening agar base (ORSAB). 15 The purpose of this study was to examine the level of MRSA contamination in dairy cow's milk and farmer's hand in Probolinggo, Indonesia, as well as to compare phenotypic detection methods using screening with oxacillin and cefoxitine diffusion disks, ORSAB, and confirming genotypes using PCR to detect mecA-coding genes. The sensitivity and specificity of the test show the effectiveness and ease of application of the MRSA detection method.

Sampling
Milk samples were taken from the udders of female cows who were in lactation period, while the samples of farmer's hand swabs were taken from farmers who were milking. The sample size in this study refers to the formula used by Regasa et al. 16 in the study of the milk safety assessment of Staphylococcus aureus as follows:

REVISED Amendments from Version 2
Based on reviewer comments, we have removed some unnecessary information, we have added some information to the isolation and identification results, we have added the research sample calculation formula to the method, and we have added some research pictures. We have improved the discussion section accordingly. We have corrected the minor typos, and grammar and we want to thanks the reviewers for their valuable comments which improved the quality of manuscript.
Any further responses from the reviewers can be found at the end of the article P = Expected prevalence is 4.8% 17 d = Desired absolute precision (4%) Based on these calculations, 109 milk samples was obtained with the selection of dairy cooperatives purposively based on the amount of milk production in an area and the willingness of dairy cooperatives to participate in the study. Meanwhile, the number of farmer hand swab samples was adjusted to the number of dairy cows owned by each farmer in the dairy cooperative area, of which 41 cattle were obtained from 109 cows.
A total of 109 samples of dairy cow's milk and 41 samples of farmer's hand swabs were collected at a dairy farm in the Probolinggo region, East Java, Indonesia from July to September 2021. Dairy cow's milk samples were taken from each cow in the third press as much as 30 ml which was then stored in a 60 ml sample bottle; the farmer's hand swab samples were taken from each farmer after the milking process using a sterile cotton swab which was then stored on Amies medium.

Bacteria isolation and identification
As much as 1 ml of each milk sample was put into a 20 ml test tube filled with 9 ml of Mannitol Salt Broth (MSB) medium while for hand swab samples, the Amies medium was vortexed until it became liquid and then 1 ml was added into a 20 ml test tube which has been filled with 9 ml of MSB media. The test tube containing MSB which had been mixed with the sample was incubated in an incubator (Isuzu Model 2-2195, Jica) at 37°C for 24 hours. The samples were cultured and purified using Mannitol Salt Agar (MSA) (Oxoid CM0085) and then incubated at 37°C for 24 hours.
Microscopic examination of bacteria was done through Gram staining to visualise Gram-positive bacteria in the form of cocci and clusters. 18 The biochemical examination was carried out using a catalase test and a coagulase test. The catalase test was carried out by dripping 3% hydrogen peroxide (H 2 O 2 ) on bacterial colonies that had been placed on the surface of the glass. 19 The coagulase test was carried out by dripping 200 μl of rabbit plasma into a coagulase test tube containing bacterial colonies, which was then incubated at 37°C for 24 hours. 20

Oxacillin and cefoxitin disk diffusion methods
The test was carried out following the Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines: S. aureus was tested for susceptibility to the antibiotics oxacillin 1 μg and cefoxitin 30 μg (Oxoid) on Muller Hinton Agar (MHA) plates (Oxoid, CM0337). The identified isolates were purified on mannitol salt agar (HiMedia Pvt. Ltd., M118) and incubated at 37°C for 24 hours. Using a sterile cotton swab (AKD 10903610549), standardized isolates (0.5 McFarland standard) were evenly streaked on the surface of the MHA medium (Oxoid, CM0337). The oxacillin (1 μg) and cefoxitin (30 μg) antibiotic disks were placed side by side with a distance of 50 mm on MHA that had been inoculated with isolates, and then incubated at 37°C for 24 hours to measure the inhibition zone.
Oxacillin resistance screen agar test S. aureus isolates resistant to oxacillin 1 μg and cefoxitin 30 μg (Oxoid) were confirmed by ORSAB (HiMedia M1415) using S. aureus isolates from the MHA media; plus Oxacillin Resistance Selective Supplement (Supplement, HiMedia Pvt. Ltd., FD191). 21 Detection of the mecA gene All S. aureus isolates that were resistant to cefoxitin 30 μg and positive on ORSAB examination were then subjected to a PCR test to detect the presence of the mecA gene. 22 The DNA extraction process was carried out according to the QIAamp DNA Mini Kit protocol (51304 & 51306), where previously the isolates were purified on MSA (HiMedia Pvt. Ltd, M118) and inoculated on MHA (Oxoid, CM0337). The primer used was mecA F: 5 0 -AAA ATC GAT GGT AAA GGT TGG C-3 0 and mecA R: 5 0 -AGT TCT GCA GTA CCG GAT TTG C-3 0 . 23 The PCR master mix used GoTaq Green Master Mix (Promega, 9PIM712) which is a ready-to-use solution mixture containing Taq DNA polymerase, dNTPs, MgCl 2 , and a reaction buffer. DNA was amplified using a Thermal Cycler T100 machine (Bio-Rad, 186-1096) for 40 cycles in 25 μl of the reaction mixture with the following steps: denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 min with a final extension at 72°C for 5 min. A total of 10 μl of PCR product were analyzed by 2% agarose gel electrophoresis, and the gel was visualized under ultraviolet light. 24 A positive test indicated a PCR product in the 533-base pair (bp) band.

Result
The results of the isolation and identification tests yielded 80 (53.33%) S. aureus isolates from 150 samples taken at a dairy farm in Probolinggo, East Java, Indonesia. The 80 isolates that were positive for S. aureus consisted of 54 isolates from dairy cow's milk samples and 26 isolates from farmer's hand swab samples as shown in Table 1. S. aureus had phenotypic colony characteristics on MSA medium, namely a change in color in the medium from red to golden-yellow indicating mannitol fermentation, while the colonies had various pigments including white, golden, and yellow as shown in Figure 1. The Gram staining test showed the Gram-positive colonies in the form of cocci and clusters as shown in Figure 2, which were then confirmed by the catalase test and coagulase test as shown in Figures 3 and 4. 19 The disk diffusion method on MHA medium showed that 42 isolates exhibited resistance to oxacillin preparations, with a percentage of 52.5% (28 isolates came from dairy cow's milk samples and 14 isolates came from farmer's hand swab sample); on the other hand, 10 isolates showed resistance to cefoxitin, with a percentage of 12.5% (five isolates came from dairy cow's milk samples and five isolates came from farmer's hand swab samples) as shown in Table 2 and Figure 5.   No S. aureus isolate was found to simply be resistant to cefoxitin, according to the disc diffusion test results, and all isolates that were found to be resistant to cefoxitin were also found to be resistant to oxacillin, as shown in Table 3.
Confirmation of the phenotype test that for resistance to oxacillin and cefoxitin was followed by ORSAB test, with a blue culture coloration indicating positive results while a white coloration indicated negative results. The ORSAB test showed that of the 42 isolates of S. aureus that were resistant to oxacillin, 20 isolates (47.62%) were confirmed MRSA by the disk diffusion method, as shown in Table 4.
S. aureus isolates suspected to be MRSA (Phenotypically resistant to cefoxitin and positive for ORSAB) were then tested genotypically using PCR to detect the presence of the gene encoding mecA. A total of 10 isolates suspected to be MRSA were tested, from which three isolates (30% of the total isolates tested by PCR) were detected positive for the mecA gene, as shown in Figure 6. The results of the PCR test showed that isolates suspected to be MRSA were found to have the mecA gene, which is resistant to the antibiotics cefoxitin and oxacillin, as shown in Table 3.

Discussion
MBD is quite a common public health problem, because it not only has an impact on human health, also has an impact on the health of dairy cows, especially in the milk production and quality sector. 25 Several previous studies have reported that the incidence of contaminated milk by S. aureus resistant to antibiotics is found in both developed and developing countries. 26 Improper and unhygienic handling of milk, especially during the milking process, plays an important role in the occurrence of milk contamination. 27 33 showed that the difference in the number of isolates found could be influenced by differences in study design such as population and geographic distribution of the sample, infection control practices, and the type of antibiotic used, as seen in Figure 6.
The problem of the incidence of S. aureus infection continues to grow with the emergence of MRSA, which is resistant to all beta-lactam antibiotics, including monobactams and cephalosporins, which are a group of antibiotics often used to treat Staphylococcus infections. 34 MRSA infection causes treatment problems and facilitates its spread, so prompt and early diagnosis is needed to identify MRSA accurately. 35 In this study, 42 samples (52.5%) of S. aureus were found to be resistant to oxacillin disks, and 10 samples (12.5%) to cefoxitin disks. Miragaia 36 stated that the phenotypic detection of MRSA using disk diffusion still has not shown accurate results, and mecA genotyping using PCR is still the main recommendation even though it cannot be done routinely. However, even so, identification of MRSA with disk diffusion is still widely used because it can be done quickly and at a lower cost. 37 Diffusion disks using oxacillin and cefoxitin have the same sensitivity level of 100%, and specificities of 74.07% for oxacillin and 92.59% for cefoxitin. 38 However, several previous studies reported that the use of the cefoxitin disk diffusion method had a better sensitivity level than that of oxacillin in detecting MRSA, because the oxacillin disk diffusion method still has a high false positive rate. 39 Vyas et al. 38 stated that false positives could be influenced by beta-lactamase hyperproduction, resulting in the phenotypic expression of oxacillin resistance but without a genotypic resistance mechanism.
In this study, all isolates detected were resistant to the cefoxitin and oxacillin disks. All isolates detected to be resistant to oxacillin and cefoxitin were confirmed by ORSAB assay, in line with a report by Pourmand et al. 40 which stated that the ORSAB test has a specificity of 100%. In this study, 20 of the 42 isolates (47.62%) were found to be positive for MRSA. The sensitivity level confirmed the resistance strain being tested while the specificity was to the minimum inhibitory concentration (MIC). 41 Cefoxitin-resistant and ORSAB-positive S. aureus isolates were tested genotypically using PCR to detect the presence of the gene encoding mecA; these isolates also had positive results in all phenotypic methods (resistance to cefoxitin and oxacillin in the disk diffusion method and positive results in the ORSAB test This project contains the following extended data: • Sentence 2: "The purpose of this study was to examine the level of MRSA contamination in dairy cow's milk and farmer's hand swabs." Comment 2: Authors should delete the word "swabs" in the sentence because what is being actually assessed are the hands of the farmers. The swab is just a tool used to collect the sample.

Comment 3:
The keyword "Swab's hand" should be changed to "hand swabs" in the list of keywords.

Introduction:
The introduction was generally very good. I will suggest that the authors make a change in the last paragraph of this section: Last paragraph of introduction: The purpose of this study was to examine the level of MRSA contamination in dairy cow's milk and farmer's hand swab in Probolinggo, Indonesia, as well as to compare phenotypic detection methods using screening with oxacillin and cefoxitine diffusion disks, ORSAB, and confirming genotypes using PCR to detect mecAcoding genes.
Comment: I think the authors should remove the word "swab" as what is being actually assessed are the farmers' hands, just like I mentioned in my earlier suggestion in the abstract section.

Methods:
The methodology was well-detailed except for some important technical corrections which I have suggested:

Oxacillin and cefoxitin disk diffusion methods
The test was carried out following the Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines: S. aureus was tested for susceptibility to the antibiotics oxacillin 30 μg and cefoxitin 30 μg (Oxoid) on Muller Hinton Agar (MHA) plates (Oxoid, CM0337). The identified isolates were purified on mannitol salt agar (HiMedia Pvt. Ltd., M118), incubated at 37°C for 24 hours as a 0.5 McFarland suspension, and then taken using a sterile cotton swab of size S (AKD 10903610549). They were then wiped evenly on the surface of the MHA medium (Oxoid, CM0337). Disk. The oxacillin 30 μg and cefoxitin 30 μg antibiotic disks were placed side by side with a distance of 5 cm on MHA that had been inoculated with isolates, and then incubated at 37°C for 24 hours to measure the inhibition zone.

Comment 1:
Authors should correct the concentration of oxacillin antibiotic disc to 1 μg because oxacillin disc concentration from Oxoid, UK is 1 μg while that of cefoxitin is correct at the 30 μg indicated. I think this might have been an oversight during the writing of the manuscript.
○ Comment 2: Authors should take note of the bolded sections in the sentence and make corrections as I indicated below for the sentence to be more comprehensive and understandable. Also, 5cm is the same as 50mm, so it is preferable to indicate that the distance between the oxacillin and cefoxitin antibiotics was 50 mm instead of 5 cm since distance units in the CLSI charts are in mm. As I mentioned earlier, the sentence in the last section should be written as: "The identified isolates were purified on mannitol salt agar (HiMedia Pvt. Ltd., M118) and incubated at 37°C for 24 hours. Using a sterile cotton swab (AKD 10903610549), standardized isolates (0.5 McFarland standard) were evenly streaked on the surface of the MHA medium (Oxoid, CM0337). The oxacillin (1 μg) and cefoxitin (30 μg) antibiotic disks were placed side by side with a distance of 50 mm on MHA that had been inoculated with isolates, and then incubated at 37°C for 24 hours to measure the inhibition zone."

Comment 3:
The concentration of all the oxacillin discs in the manuscript should be changed to 1 μg.

Results:
The results are very clear and understandable. Data were properly interpreted and comprehensive. However, I suggested some important changes and corrections: Comment 1: The colour of S. aureus on mannitol salt agar (MSA) is golden-yellow. I will suggest authors use this all through the manuscript.
○ Sentence: "Based on the results of the disk diffusion test, no S. aureus isolate was to only be resistant to cefoxitin: all S. aureus isolates that were detected to be resistant to cefoxitin were also identified as resistant to oxacillin as shown in Table 3." Comment 2: I suggest that authors should rephrase this sentence to be more understandable.
Comment 3: I suggest that the authors delete the column "mecA detection using PCR" in Table 3 as it is empty and serves no function since the last column is already indicating the total isolates that harboured the mecA gene.

Discussion:
The discussion is good but needs some critical changes in some confusing sentences which I have suggested below: Sentence: ..contamination; this percentage is higher than the research conducted by Wang et al. 27 which isolated 195 milk samples, of which 90 samples (46.15%) were contaminated with S. aureus, and from another study conducted by Jahan et al. 28 who isolated 47 milk samples, of which 12 (25.53%) were contaminated with S. aureus.

Comment 1:
There is a mix-up in the sentence above. The sentence is stating that milk samples were isolated while what was actually isolated was the S. aureus from the milk samples. I will suggest that authors should re-write this section as ": ..contamination; this percentage is higher than the research conducted by Wang et al. "The Gram staining test showed the Gram-positive colonies in the form of cocci and clusters, which were then confirmed by the catalase test and coagulase test" -Change the line and add both biochemical test results (+/-).