The certification of organic dairy farms entails the verification of livestock products that are obtained without the use of antibiotics, synthetic antibacterial agents, growth promoters, and hormones in livestock feeds. Further, this certification is granted to farms wherein the livestock are raised in accordance with specific certification standards outlined by the National Agriculture Products Quality Management Service, South Korea (Guidelines for Non-Antibiotic Products; No. 2021-02). This certification is a mechanism aimed at supplying safe dairy products to consumers; however, the restrictions imposed on antimicrobial use may pose challenges in managing various diseases (11). In particular, the management of mastitis may be more difficult under these conditions (11), resulting in an increased probability of occurrence of clinical and subclinical mastitis in organic dairy farms.

Bovine mastitis is the most prevalent and economically burdensome disease affecting dairy herds worldwide (10), and the most frequently isolated Gram-positive microorganisms are staphylococci and streptococci, while coliform bacteria are the most prevalent Gram-negative microorganisms (9,10). Especially, Staphylococcus aureus is the most predominant mastitis-causing bacterial strain in Canada (6), Denmark (9), and Sweden (10). Previously, microbiological examination was performed according to the standards described in the Guidelines of National Mastitis Council (7) as follows: inoculation of ten microliters of the quarter sample onto a blood agar plate (BAP) and MacConkey agar plates, identification of bacterial colonies based on their gross morphology, classification of streptococci and staphylococci using gram staining and catalase test, differentiation between S. aureus and coagulase negative staphylococci (CNS) via a coagulase reaction utilizing rabbit plasma, and subsequent classification.

However, recently, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is being commonly employed for bovine mastitis diagnosis because it is a fast and reliable method for identifying mastitis-causing bacteria (5,8,9). Furthermore, MALDI-TOF mass spectrometry technique provides qualitative and quantitative results identical to those of multiplex quantitative PCR assays (8) and a more concrete differentiation resolution than biochemical identification (1) for diagnosing subclinical mastitis. In addition, the VITEK^® MS system is extensively utilized for the assessment of minimum inhibitory concentration (MIC) in commercial antimicrobial MIC determination kits owing to its expediency and ability to perform multiple antimicrobial MIC tests. These technologies have been widely used for the diagnosis of bovine mastitis-causing pathogens because of their fast and easy applicability. However, there are few reports of these technologies being used to establish management strategies for bovine mastitis in organic dairy farms.

Therefore, the objective of the present study was to identify mastitis-causing pathogens using MALDI-TOF mass spectrometry in an organic dairy farm and to evaluate the effects of antimicrobial restriction on bacterial antimicrobial susceptibility.

The experimental protocol devised for this study was approved by the Animal Care and Use Committee of the Kangwon National University Laboratory (KW-231106-1; Chuncheon, Korea).

Sample collection, milk composition, and culture conditions

The dairy farm used in this study was certified as an organic dairy farm in December 2021 and was operated in accordance with the organic dairy farm certification guidelines (National Agriculture Products Quality Management Service) a year before certification. A total of 43 Holstein cows (17 primiparous and 24 multiparous; 2.00 ± 0.17 parity) were used in this study, and 172 quarter milk samples were aseptically collected by skilled veterinarians from an organic dairy farm located in Gangwon-do in November 2022. The farm has loose stalls and milking parlors. On the day of the visit, the farmers wore gloves for udder washing and used post-milking teat dipping; however, they shared towels for udder washing among multiple cows.

Production information regarding day in milk; milk yield; somatic cell count (SCC); composition of milk fat, protein, and solid not fat; and milk urea nitrogen concentration were downloaded from the monthly test results of the Dairy Cattle Improvement Center (http://www.dcic.co.kr). Ten microliters of the quarter samples were inoculated on to BAPs, and all plates were incubated aerobically for 24 hours at 37°C. Then, one pure colony of culture positive plates was sub-cultured aerobically for 20 to 24 hours at 37°C on BAP for MALDI-TOF mass spectrometry assay.

MALDI-TOF mass spectrometry

Species identification of the bacterial colonies was performed using POSTBIO (https://www.pobanilab.com; Korea). Mass spectra were obtained using VITEK^® MS PRIME (BIOMERIEUX, France). One pure colony was selected and added to 10 μL of 70% formic acid; 1 μL of solution was then mixed with 0.5 μL of matrix solution, dried, and evaluated using MALDI-TOF MS on a Vitek MS platform. Spectral data were analyzed by comparison with typical spectra.

Antimicrobial susceptibility test

Antimicrobial susceptibility test for determination of the MIC was performed using the VITEK^® MS system with VITEK2 AST-GP81 and VITEK2 AST-GN98 cards (BIOMERIEUX, France), and target antibiotics were amikacin, amoxicillin/clavulanic_acid, ampicillin, benzylpenicillin, cefalotin, cefazolin, cefixime, cefotaxime, cefovecin, cefpodoxime, cephalexin, chloramphenicol, ciprofloxacin, clindamycin, doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, meropenem, metronidazole, minocycline, nitrofurantoin, oxacillin, pradofloxacin, trimethoprim/sulfamethoxazole, and vancomycin. The antimicrobial resistance or susceptibility of the isolates was determined according to the guidelines of the Clinical and Laboratory Standards Institute (3,4).

The subclinical mastitis infection rates in the cows and quarters were 58.1% (25/43) and 25.6% (44/172), respectively. Colony patterns of bacterial isolates (n = 44) cultured on BAP were shown in Fig. 1. Species identification using MALDI-TOF mass revealed that S. aureus (40.9%) was the most prominent bacterial isolate, followed by S. chromogenes (22.7%), S. epidermis (18.2%), S. simulans (11.4%), S. haemolyticus (2.3%), S. muscae (2.3%), and S. xylosus (2.3%) (Table 1).

Table 1 . Identification of bacterial species using MALDI-TOF mass spectrometry.

Sample No.	Cattle No.	Quarter*	Species
1	2	B	S. chromogenes
2	4	C	S. chromogenes
3	7	D	S. epidermidis
4	9	B	S. aureus
5		C	S. epidermidis
6		D	S. epidermidis
7	17	B	S. simulans
8		D	S. simulans
9	18	A	S. haemolyticus
10		C	S. chromogenes
11	22	A	S. aureus
12	28	A	S. aureus
13	33	A	S. aureus
14		B	S. aureus
15		C	S. aureus
16		D	S. epidermidis
17	37	A	S. aureus
18		B	S. simulans
19		C	S. simulans
20		D	S. chromogenes
21	39	A	S. epidermidis
22		B	S. xylosus
23	41	C	S. epidermidis
24	42	D	S. epidermidis
25	45	B	S. chromogenes
26	46	C	S. aureus
27		D	S. aureus
28	48	A	S. aureus
29		B	S. aureus
30	49	D	S. chromogenes
31	50	D	S. muscae
32	52	A	S. aureus
33		C	S. simulans
34		D	S. epidermidis
35	59	A	S. aureus
36		C	S. aureus
37		D	S. aureus
38	60	C	S. aureus
39		D	S. aureus
40		D	S. aureus
41	62	A	S. chromogenes
42	65	B	S. chromogenes
43	68	D	S. chromogenes
44	78	C	S. chromogenes

*A, right front; B, right rear; C, left front; D, left rear quarter; S., Staphylococcus..

Figure 1. Colony patterns of bacterial isolates (n = 44) cultured on blood agar plate.

In the susceptibility test, all isolates were 100% susceptible to 24 of 28 antibiotics, except for benzylpenicillin, cefalotin, cefpodoxime, and trimethoprim/sulfamethoxazole. The resistance rates of S. aureus, S. chromogenes, and S. muscae isolates to trimethoprim/sulfamethoxazole were 27.8%, 10%, and 100%, respectively, and the resistance rates of S. epidermis and S. xylosus to benzylpenicillin were 50% and 100%, respectively. S. chromogenes, S. epidermis, S. simulans, S. haemolyticus, and S. xylosus were resistant to cefalotin and cefpodoxime (Table 2).

Table 2 . Antimicrobial susceptibility rate of Staphylococcus spp. isolates (n = 44) based on minimal inhibitory concentration.

Antimicrobials	Antimicrobial susceptibility* (%)
	S. aureus (n = 18)	S. chromogenes (n = 10)	S. epidermidis (n = 8)	S. simulans (n = 5)	S. haemolyticus (n = 1)	S. muscae (n = 1)	S. xylosus (n = 1)
Amikacin	100	100	100	100	100	100	100
Amoxicillin/clavulanic acid	100	100	100	100	100	100	100
Ampicillin	100	100	100	100	100	100	100
Benzylpenicillin	100	100	50.0	100	100	100	0
Cefalotin	100	0	0	0	0	100	0
Cefazolin	100	100	100	100	100	100	100
Cefixime	100	100	100	100	100	100	100
Cefotaxime	100	100	100	100	100	100	100
Cefovecin	100	100	100	100	100	100	100
Cefpodoxime	100	0	0	0	0	100	0
Cephalexin	100	100	100	100	100	100	100
Chloramphenicol	100	100	100	100	100	100	100
Ciprofloxacin	100	100	100	100	100	100	100
Clindamycin	100	100	100	100	100	100	100
Doxycycline	100	100	100	100	100	100	100
Enrofloxacin	100	100	100	100	100	100	100
Erythromycin	100	100	100	100	100	100	100
Florfenicol	100	100	100	100	100	100	100
Gentamicin	100	100	100	100	100	100	100
Marbofloxacin	100	100	100	100	100	100	100
Meropenem	100	100	100	100	100	100	100
Metronidazole	100	100	100	100	100	100	100
Minocycline	100	100	100	100	100	100	100
Nitrofurantoin	100	100	100	100	100	100	100
Oxacillin	100	100	100	100	100	100	100
Pradofloxacin	100	100	100	100	100	100	100
Trimethoprim/sulfamethoxazole	72.2	90.0	100	100	100	0	100
Vancomycin	100	100	100	100	100	100	100

The day in milk; milk yield; SCC; composition of milk fat, protein, and solid not fat; and milk urea nitrogen concentration of cows (n = 43) were 218.1 ± 22.6 days; 26.8 ± 1.31 kg/day; 265.5 ± 65.2 × 10³; 3.73 ± 0.10%, 3.42 ± 0.05%, and 8.91 ± 0.05%; and 15.6 ± 0.76 mg/dL, respectively.

Previously, identification of bacterial strains using MALDI-TOF mass spectrometry revealed that the most predominant Gram-positive bacterial isolate was S. aureus (79/294 isolates) in clinical or subclinical bovine mastitis in Denmark (9) and 13 out of 33 isolates from cows presenting with subclinical mastitis in Brazil (1). In Korea, S. aureus (9.0%), followed by S. chromogenes (6.8%), was the most predominant of the 1,142 Gram-positive isolates, according to the 2023 annual report of the Animal and Plant Quarantine Agency, South Korea (2023 Dairy cow mastitis management project; Publication registration No. 11-1543061-000093-10). In the present study, the 40.9% detection rate of S. aureus was higher than that reported in nationwide studies in Denmark (26.9%) and Korea (9.0%) but similar to that reported in Brazil (39.3%). We assumed that this was because nationwide studies included clinical and subclinical mastitis, while the present and previous Brazilian studies only focused on subclinical mastitis. In addition, S. aureus is more frequently identified in bulk tanks in organic farms in the United States than in conventional farms (2). The highest infection rate of S. aureus was the most common conclusion reported in various studies and annual reports in Korea. Therefore, the different drug restrictions implemented for organic farm certification did not have a significant impact on the ranking of bovine mastitis-causing bacterial infections; however, they did result in a notably higher rate of Staphylococcus spp. infections. Moreover, we suspect that this was owing to the insufficient use of disinfectants during the milking procedure and the worsening of Staphylococcus spp. infection under restricted antibiotic use in mastitis management.

Staphylococcus aureus is classified as the major mastitis-causing bacteria along with Streptococcus agalactiae, E. coli, Klebsiella spp., Mycoplasma spp., and environmental Streptococcus and has an impact on milk quality, production, and SCC owing to its ability to survive in the udder for extended periods of time (6). According to the 2023 annual report, the antibiotic resistance rate of S. aureus was relatively high for ampicillin (39.2%) and sulfadimethoxine (16.5%), which is inconsistent with our result that the resistance rates to trimethoprim/sulfamethoxazole and ampicillin were 27.8% and 100%, respectively. Moreover, 5 of 10 S. aureus infected cows showed multiple quarters of infection in the present study. Although our findings may be preliminary without comparison to other studies or data prior to the implementation of antibiotic restriction on the same farm, we hypothesize that antibiotic restriction may alter the antibiotic resistance properties of S. aureus and induce severe multiple quarter infections in an organic dairy farm.

In contrast, CNS is classified as a minor mastitis-causing bacterium, including various types of staphylococci (9,12). In the present study, S. chromogenes, S.epidermis, S. simulans, S. haemolyticus, S. muscae, and S. xylosus were detected using MALDI-TOF mass spectrometry, and the infection rate of CNS was higher than that of S. aureus (59.1% vs. 40.9%). In addition, CNS isolates, except for S. muscae, showed 100% antibiotic resistance to cefalotin and cefpodoxime in the present study, which is not consistent with the 2023 annual report showing relatively low antibiotic resistance of CNS isolates compared with S. aureus isolates. Therefore, these results are consistent with the antibiotic resistance of S. aureus, and antibiotic restriction may also have an impact on CNS antibiotic resistance.

In conclusion, the insufficient use of disinfectants during milking practices might deteriorate Staphylococcus spp. infection under restricted antibiotic use in mastitis management strategies, which should be improved by hygienic milking practices to produce high-quality milk. Furthermore, our findings suggest the need for further large-scale studies, especially for organic dairy farms that cannot use various antibiotics, synthetic antibacterial agents, growth promoters, or hormones for certification, thereby indicating the need for accurate subclinical mastitis management strategies operated by farmers together with veterinary practitioners, even if mastitis does not appear clinically in organic dairy farms.

This study was supported by 2022 Research Grant from Kangwon National University.

The authors have no conflicting interests.