Epidemiological investigation and antimicrobial sensitivity of Staphylococcus aureus in dairy farms of selected towns in Western oromia, Ethiopia

Abstract

The aim of this study is to estimate prevalence, investigate potential risk factors and determine the antimicrobial sensitivity of Staphylococcus aureus in dairy farms of purposively selected towns in western Oromia: Bako, Nekemte and Gimbi. A cross-sectional study was conducted from November 2020 to June 2021. A total of 347 samples consisting of 255 raw milk, 49 milkers’ hand swab and 43 milking bucket swabs were collected and examined for Staphylococcus aureus using bacteriological culture and biochemical tests. Dairy animals were selected by simple random sampling. Risk factors for the occurrence of Staphylococcus aureus in dairy farms were assessed through interviews and personal observation. The isolates were tested for drug sensitivity using disk diffusion techniques. The overall prevalence of S. aureus was 18.44% (95% CI: 14.69–22.90). The proportion of isolation was 17.25% (95% CI: 13.07–22.43), 30.61% (95% CI: 19.03–45.29) and 11.63% (95% CI: 4.74–25.79) from raw milk, swabs of milkers’ hand and milking bucket, respectively showing insignificant difference (P < 0.05). Prevalence of Staphylococcus aureus within the breed, parity, lactation stage, hand washing at the milking interval and hygiene of the barn were also significant. The Antimicrobial test showed the highest susceptibility to ciprofloxacin (100%), gentamicin (90%), vancomycin (85%) and chloramphenicol (85%) but the highest resistance to penicillin G (85%), ampicillin (75%) and tetracycline (50%). None of the isolates showed multidrug resistance. Such Staphylococcus aureus prevalence with also the antibiotic resistant isolates suggests a public health risk thus the need to address this issue to protect both human and animal health.

Introduction

Staphylococcus aureus is a ubiquitous, versatile and highly adaptive pathogen that colonizes the skin and mucous membrane of the anterior nostrils, the gastrointestinal tract, the perineum, the urogenital tract and the pharynx [1]. It is also found in the environment and in various foods of animal origin [2, 3]. Staphylococcus aureus is a facultative anaerobic, non-motile, fermentative and non-spore-forming bacteria [4]. They are often found in clusters that resemble a bunch of grapes when observed under a light microscope after Gram staining. The name ‘Staphylococcus’ was derived from the Greek words, Staphyle (bunch of grapes) and kokkos (berry) [5]. The term aureus is derived from Latin, which refers to the color of gold, as colonies (often) have a golden color when growing on solid media [6].

The main niche and the largest reservoir of Staphylococcus aureus is human nares. On average, every third person is colonized with this facultative pathogen [7]. Although the largest reservoir is human nares, the second largest may well be cows [8]. The skin, mucous membranes, teats and udder of milking animals are important reservoirs [9]. However, the main reservoirs in dairy herds are infected udders and teat skin [8]. Staphylococcus aureus infected purchased animals and chronically infected animals are a major source of new Staphylococcus infections within a farm [9].

Staphylococcus aureus causes a diverse array of diseases [10]. In animals, it is most commonly reported as the cause of mastitis in milk producing animals and bumble foot in chickens [11, 12]. In pigs, Staphylococcus aureus causes exudative dermatitis (greasy pig disease) [13]. In dairy cattle, Staphylococcus aureus causes one of the most common forms of chronic mastitis. Although some cows may flare up clinical mastitis, the infection is usually subclinical and causes increased somatic cell counts but no detectable changes in milk or udder [14]. The primary mode of transmission of Staphylococcus aureus is cow-to-cow during milking, when milk from an infected cow comes into contact with the teat end of an uninfected cow [15, 16].

In Ethiopia, several studies have reported the prevalence of Staphylococcus aureus in dairy farms in recent years. For instance [17], reported a prevalence of 24% for udder milk in Central Ethiopia, l [18] reported a prevalence of 20% for udder milk and 23.3% bucket swab in Jigiiga City [19], reported in and around Asella a prevalence of 11.9%, 0.00%, 11.1%, 33.3% for udder milk, bucket swab, milkers hand swab and milkers nasal swab respectively, In Addis Ababa [20] reported a prevalence of 20% from udder milk, 25% from bucket swab, 25% from milkers swab [21]. reported from Gondar the prevalence of 18.33% from udder milk 18.33% and 25% from bucket swab [22], reported from Mukaturi and Sululta the prevalence of 15.3% from udder milk, 20% from bucket swab and 25% from milk swab [23], reported from Mekelle the prevalence of 17% from udder milk.

Various potential risk factors are involved in the occurrence of Staphylococcus aureus in dairy farms. The host risk factors include age, parity, stage of lactation, somatic cell count, breed, anatomy of the mammary glands/morphology of the udder and teat, udder positioning, relative distance between teats, milk fat content and immunity [24, 25]. Environmental risk factors include the proper functioning of the milking machine, udder trauma, hygiene, climate, nutrition, management, season, and housing conditions. The warm, humid, and moist climate favors the growth of bacteria and increases the likelihood of intramammary infection [26]. Pathogen risk factors include the number of bacteria, virulence, frequency of exposure (dirty farm floor, dirty milking machine, and dirty towel for drying teats frequently exposed to pathogens), ability to resist flushing out of the glands by milk and resistance to antimicrobials [27].

Staphylococcus aureus is a highly adaptive pathogen that is constantly developing resistance to most of the antibiotics available [28]. Over the past few decades, antibiotic resistance of Staphylococcus aureus has gradually increased due to the evolution of bacteria and the abuse of antibiotics [29]. In the past, beta-lactam antibiotics have shown potent activity against Staphylococcus aureus, which, along with good safety profiles, makes them the antibiotics of choice for the treatment of Staphylococcal infections [30]. However, penicillin-resistant strains of Staphylococcus aureus emerged shortly after the antibiotic was introduced in the early 1940s [31]. Vancomycin resistance in S. aureus strains is a global issue and the resistance is linked to van genes which code for different resistance phenotypes and have the same name as the corresponding genes [32, 33]. Resistance rates of S. aureus to vancomycin are extremely high as noted by previous reports [32, 34]. The mechanisms involved in S. aureus resistance to tetracyclines are ribosomal protection by elongation of proteins, and activation of the efflux pump [33]. Acquisition of resistance to Macrolides and Lincosamides in S. aureus strains that includes modification of the target site by mutation or methylation of 23s rRNA subunit encoded, and activation of an efflux pump [35, 36].

To the best of the researchers’ knowledge, historically, Staphylococcus aureus has not been extensively studied in western Ethiopia and no such studies have been conducted to investigate its epidemiology in dairy farms of Bako, Nekemte and Gimbi towns. So, the objective of this study is to estimate the prevalence of Staphylococcus aureus in raw milk of lactating cows, swabs of milkers’ hands and milking bucket, to investigate the potential risk factors associated with its occurrence and to determine antimicrobial sensitivity of the isolated Staphylococcus aureus isolates in dairy farms of Bako, Nekemte and Gimbi towns.

Materials and methods

Study area description

The study was carried out in three purposively selected towns (Bako, Nekemte and Gimbi) based on their proximity to Ambo University Zoonoses and Food Safety Research Laboratory, the presence of large number of dairy farms and infrastructure accessibility. The towns are located in western part of Oromia Regional State, Ethiopia. Bako town is located in the west Shoa Zone of Oromia Regional State. The town is located at latitude and longitude of 09°08^’N and 37^o03^’E, and at an altitude of 1743 m above sea level [37]. Nekemte town is located in the east Wollega Zone of Oromia Regional State. The town has latitude and longitude of 9°5′N and 36°33′E. It is located at an elevation of 2088 m above sea level [38]. Gimbi town is located in the west Wollega Zone of Oromia Regional State. The town has a latitude and longitude of 9°10’N and 35°50’E and is located at an altitude between 1845 and 1930 m above sea level [39]. The map of Ethiopia depicting the location of the study areas is shown in Fig. 1.

Fig. 1.

Map of the study area. The study was conducted in the West Wollega zone in Gimbi town, East Wollega zone in Nekemte town and West Shoa zone in Bako town

Study population

The study population was lactating local Zebu and crossbred cows (Holstein Friesian cross indigenous Zebu and Jersey cross indigenous Zebu) in Bako, Nekemte and Gimbi town dairy farms which are kept under intensive, semi-intensive and extensive management systems. In addition to the cows, the milkers’ hand and milking buckets were part of the study.

Study design

A cross-sectional study design was conducted from November 2020 to June 2021. A structured questionnaire and personal observation were also used to assess the milking hygienic practices and risk factors associated with the occurrence of Staphylococcus aureus.

Sampling technique

The dairy farms found in each town were selected using a simple random sampling technique. The lactating cows in the selected farm were sampled by simple random sampling technique. The proportionality of sampling dairy cows was applied according to the number of dairy farms in the town and the population size of each herd. Before the formal study, the researcher and Artificial Inseminators of the respective Woredas carried out preliminary visits to obtain the consent of the farmers, to localize the farms and to give each respondent a brief description of the study.

Sample size determination

The sample size was calculated according to the formula by [40]. Using 95% confidence interval, 5% desired level of precision and with expected prevalence of Staphylococcus aureus 16.6% in dairy farms of Mukaturi and Sululta towns [22].

d.

Were,

n = sample size of the study population.

p = expected prevalence in the study area.

d = desired precision.

Accordingly, the calculated sample size for the current study was 214 animals; however, to increase precision the sample size was inflated to 255. Thus 255 animals were selected for milk sampling. In addition, 49 hand swabs from the milkers and 43 swabs from the milking buckets were purposively sampled. Therefore, a total of 347 samples were considered for the present study.

Data collection and laboratory investigation

Active observational survey

During the farm visit, personal observation was made to look for various hypothesized risk factors (body condition scores, presence of teat lesions, tick infestation, age, floor type and barn hygiene). The animal’s body condition score was categorized as poor, medium and good based on the criteria of [41]. The hygienic condition of the barn was classified as poor if there were bad smells, the feed trough and channel (for the waste drainage) were dirty, and when the flanks, udder and belly of the animals were soiled and rated good if none of the above defects were found [42].

Collection of samples

Milk samples were collected according to the National Mastitis Council procedure [43]. First the quarters were washed with clean water and wiped dry. The end of the teat was wiped with cotton wool soaked in 70% ethyl alcohol for disinfection. Approximately, 10 ml of raw milk was collected in a sterile universal bottle. The milk from all four quarters was collected in the same tube. To reduce the contamination of the teat ends during sampling, the close teats were sampled first and then followed by the far ones. The sample bottles were capped, labeled and stored in an ice-packed cool box and transported within 24 h to the Zoonoses and Food Safety Research Laboratory of Ambo University. Any presence of teat lesions and tick infestations on the udder of lactating cows were recorded during the process.

Swabs from the hands of milkers’ and milking buckets were collected with sterile, cotton-tipped swabs. Swab samples were taken from the hand of the milkers by rotating the swab 360° between the finger bases of both hands and the base of the milking bucket. The swab samples were transported in a sterilized universal bottle with 9 ml of peptone water. Upon arrival at the laboratory, all samples were stored in a refrigerator at 4 °C until processing.

Isolation and identification of Staphylococcus aureus

The collected samples were cultured directly on a blood agar base enriched with 7% defibrinated sheep blood. The inoculated plates were incubated aerobically at 37 °C and examined after incubation for 24 h. Incubation was extended to 48 h if no growth was observed within 24 h. Growth of the colonies (smooth and circular), the morphology (creamy, white/yellow) and the hemolytic properties were observed and recorded. Suspected colonies were then sub-cultured on nutrient agar plates and incubated for 24–48 h at 37 °C to obtain a pure culture. Gram’s staining was then used to identify the Gram reaction, cell shape and cell arrangement. Staphylococcus aureus is a gram-positive bacteria (staining purple by Gram staining) that is cocci-shaped and tends to cluster in what is described as grape-like. Suspected pure colonies from the nutrient agar plate were inoculated into nutrient slants and incubated at 37 °C for 24–48 h under aerobic culture conditions. The pure isolates in the culture medium were preserved and kept at 4^oC for further biochemical tests [4].

The final identification of Staphylococcus aureus was based on biochemical tests (catalase test, mannitol fermentation, 1% maltose fermentation and coagulase test). Pure cultures of the isolates to be tested for catalase were picked up from the nutrient agar plate using a bacteriological loop and mixed with a drop of 3% hydrogen peroxide on a clean slide. When mixed with 3% H₂O₂, catalase-positive organisms create oxygen bubbles that are visible to the naked eye, while catalase-negative organisms do not. The catalase test is important to distinguish Streptococci (catalase-negative) from Staphylococci, which are catalase-positive. Catalase positive colonies were selected and streaked on mannitol salt agar plates incubated at 37^oC and examined after 24 h. Mannitol Salt Agar is used as a selective and differential medium for the isolation and identification of Staphylococcus aureus from clinical and non-clinical specimens. Mannitol, the fermentable carbohydrate, the fermentation of which leads to acid production, is detected by the phenol red indicator, helps in the differentiation of Staphylococcal species. Staphylococcus aureus produces yellow colonies and a surrounding yellow medium [4].

To differentiate Staphylococcus aureus, a purple agar base with the addition of 1% maltose was used. The suspected colonies were inoculated with 1% maltose on a purple agar base media plate and incubated at 37 °C for 24 h. Staphylococcus aureus rapidly ferments maltose, changing the medium and colonies to yellow. Finally, a tube coagulase test is performed which is the standard test for the routine identification of Staphylococcus aureus. The tube coagulase test was performed in sterile tubes by adding 0.1 ml of selected isolates of Staphylococcus aureus grown on tryptone soya broth for 24 h at 37 °C to 0.5 ml of fresh rabbit plasma. The mixture within the tube was evaluated at 30 min intervals for the first 4 h of the test and then after 24 h of incubation, if any degree of clotting was visible when tilted, as Staphylococcus aureus produces clots [4].

Antimicrobial sensitivity test

A total of 20 randomly selected Staphylococcus aureus isolates were subjected to the antimicrobial sensitivity test using the Kirby-Bauer disc diffusion method according to the protocol of the Clinical and Laboratory Standards Institute [44]. The lists of antimicrobials tested were as follows; vancomycin (30 µg), penicillin G (10IU), tetracycline (30 µg), ciprofloxacin (5 µg), nalidixic acid (30 µg), ampicillin (10 µg), chloramphenicol (30 µg), gentamicin (10 µg), nitrofurantoin (30 µg) and ceftriaxone (30 µg). An inhibition zone diameter of each antimicrobial was then measured and interpreted as resistant, intermediate and susceptible by comparing it with recorded diameters of a control organism. Multiple antimicrobial resistance was recorded for isolates showing resistance to three or more antimicrobial classes [45].

Questionary survey

A structured questionnaire was used to collect data about milkers’ demography, milking practices and potential risk factors through interviewing farm owners or workers. Different risk factors thought to influence the occurrence of Staphylococcus aureus in the dairy herds (type of farm, management system, lactation stage, parity, average milk production, barn cleaning frequency and milking hygiene) were collected.

The variables were categorized into various categories. According to [46], the dairy farms were classified into large scale (> 30 dairy cows), medium scale (6–30 dairy cows) and small scale (≤ 5 dairy cows). However, no large-scale dairy farm was observed in the study area. The age of the cows examined was determined based on recorded birth dates, and owners’ information and by using the age determination described by [47]. The study cows were then categorized into young (3–6 years), adult (7–9 years) and old (> 9 years) [48]. The parity was categorized as cows that gave birth to one calf, two calves, three calves and ≥ four calves according to [49]. The lactation stage was classified as early (≤ 3 months), medium (4–6 months), and late (≥ 7 months) and the average daily milk yield was categorized as ≤ 7 l (low), between 8 and 15 l (medium) and > 15 l (high) [50].

Data management and analysis

All collected data was entered into a computer using Microsoft Excel and transferred to STATA version 14.0 (Stata Corp. College Station, TX, USA) for analysis. Descriptive statistics were used to calculate the prevalence of Staphylococcus aureus and to describe the antimicrobial sensitivity. Logistic regression was used to test the significance of the effect of various risk factors on the prevalence of Staphylococcus aureus. Univariable logistic regression was used in which the level of the risk factors for the occurrence of Staphylococcus aureus was compared using an odds ratio (OR). Variables that have P < 0.25 were investigated using a correlation matrix to assess collinearity and all non-collinear variables were offered to multivariable logistic regression. After checking for confounders, variables with a significant association (P < 0.05) with the dependent variable were retained in the final model. The model validity and predictive ability were assessed using the Hosmer-Lemeshow goodness-of-fit test and Receiver Operating Characteristic curve (ROC curve), respectively.

Results

Prevalence of Staphylococcus aureus

From the total of 347 samples collected and processed, the overall prevalence of Staphylococcus aureus was 18.44% (95% CI: 14.69–22.90). The percentage of isolation from raw milk of lactating cows, milkers’ hand and milking bucket swabs are listed in Table 1.

Table 1.

Percentage of Staphylococcus aureus isolates from Raw milk and swab samples

Sample type	Total samples examined	Number of positive samples (%)	(95% CI)
Raw milk	255	44 (17.25)	13.07–22.43
Milkers’ hand swab	49	15 (30.61)	19.03–45.29
Milking bucket swab	43	5 (11.63)	4.74–25.79

Risk factors associated with Staphylococcus aureus

Intrinsic risk factors

In the present study, the univariable logistic regression analysis showed that breed, age, parity, lactation stage and daily average milk yield of the cows were statistically significantly associated (P < 0.05) with the isolation rate of Staphylococcus aureus. Accordingly, the likelihood of isolation of Staphylococcus aureus from crossbred cows (OR = 2.27, CI:1.09–4.74, P = 0.028) was statistically significantly higher than that of local breeds. Similarly, older cows (OR = 3.34, CI: 1.39–7.97, P = 0.007) and adult cows (OR = 2.13, CI:1.00-4.51, P = 0.048) had statistically significantly higher odds of contracting Staphylococcus aureus than young cows. Cows that gave birth to one calf were statistically significantly less likely than cows that gave birth to three calves (OR = 4.47, CI:1.46–13.68, P = 0.009) and ≥ 4 calves (OR = 4.89, CI:1.69–14.15, P = 0.003) to be affected by Staphylococcus aureus. No statistically significant differences were observed between cows that gave birth to one calf and two calves.

The odds of Staphylococcus aureus in cows in the late lactation stage (OR = 3.48 CI: 1.44–8.43, P = 0.006) were statistically significantly higher than in cows in mid-lactation stage. Cows that produce medium milk (8–15 l/day) had significantly higher odds of Staphylococcus aureus (OR = 2.76, CI: 1.29–5.91, P = 0.009) than those that produce low milk (≤ 7 l/day). No statistically significant differences were seen between other milk production levels. The result of the univariable logistic regression analysis of Staphylococcus aureus with intrinsic risk factors is described below (Table 2).

Table 2.

Univariable logistic regression analysis of S. aureus with intrinsic risk factors

Risk Factors	No of examined	No of positive (%)	OR (95% CI)	P-value
Breed Local Cross	102 153	11(10.78) 33(21.57)	Ref 2.27 (1.09–4.74)	0.028**
Body condition score Poor Medium Good	99 119 37	19(19.19) 20(16.81) 5(13.51)	1.52 (0.52- 4.42) 1.29 (0.49- 3.72) Ref	0.442 0.634
Age Young Adult Old	146 73 36	17(11.64) 16(21.92) 11(30.56)	Ref 2.13(1.00-4.51) 3.34(1.39–7.97)	0.048** 0.007**
Parity 1 2 3 ≥4	74 67 49 65	5(6.76) 10(14.93) 12(24.49) 17(26.15)	Ref 2.42(0.78- 7.49) 4.47 (1.46–13.68) 4.89 (1.69–14.15)	0.125 0.009** 0.003**
Lactation stage Early Mid Late	89 87 79	13(14.61) 10(11.49) 21(26.58)	1.89 (0.76- 4.74) Ref 3.48(1.44–8.43)	0.169 0.006**
Daily average milk yield Low Medium High	102 108 45	11(10.78) 27(25.69) 6(13.33)	Ref 2.76 (1.29–5.91) 1.27 (0.44-3.69)	0.009** 0.657

OR = odds ratio, CI = confidence interval, ** significant

Extrinsic risk factors

In the current study, the occurrence of Staphylococcus aureus in raw milk showed a statistically significant association (P < 0.05) with regard to hand washing at milking interval, the type of floor, the frequency of floor cleaning and the barn hygiene. Accordingly, the odds of Staphylococcus aureus in cows that were milked without hand washing at milking intervals (OR = 3.01, CI:1.33–6.80, P = 0.005) was statistically significantly higher than those milked after washing hand at milking intervals. The likelihood of occurrence Staphylococcus aureus were statistically significantly higher in cows kept on the earthen floor (OR = 2.32, CI:1.11–4.84, P = 0.025) than those kept on concrete floor. In addition, there were statistically significantly higher odds of Staphylococcus aureus in cows from herds where the floor of the barn was cleaned more than once per week (OR = 3.80, CI:1.86–7.79, P = 0.001), once per week (OR = 4.44, CI:1.01–19.38, P = 0.047) and less than once per week (OR = 5.33, CI:1.17–24.29, P = 0.031) than those barns where floor was cleaned daily, as well there was also statistically significantly higher odds in cows kept in poor barn hygiene (OR = 2.77, CI:1.39–5.53. P = 0.004) than those kept in good barn hygienic condition. The result of the univariable logistic regression analysis of Staphylococcus aureus with extrinsic risk factors is described below (Table 3).

Table 3.

Univariable logistic regression analysis of S. aureus with extrinsic risk factors

Risk factors	No of examined	No of positive (%)	OR (95% CI)	P-value
Farm location Nekemte Bako Gimbi	101 82 72	21(20.79) 13(15.85) 10(13.89)	1.63 (0.72 − 3.70) 1.19 (0.49-2.85) Ref.	0.733 0.256
Type of farm Small scale Medium Scale	70 185	10(14.29) 34(18.38)	Ref. 1.35(0.63 − 2.90)	0.441
Management system Intensive Semi-intensive Extensive	28 146 81	2(7.14) 29(19.86) 13(16.05)	Ref. 2.48(0.76-11.78) 3.22(0.72-14.36)	0.265 0.251
Presence of teat lesions Yes No	53 202	13(24.53) 31(15.35)	1.79(0.86-3.73) Ref.	0.119
Tick infestation Yes No	84 171	17(20.24) 27(15.79)	1.35(0.69-2.65) Ref.	0.378
Pre-milking hand washing Yes No	237 18	40(16.88) 4(22.22)	1.4(0.44-4.49) Ref.	0.565
Washing hands at milking interval Yes No	94 161	8(8.51) 35(21.75)	Ref. 3.01(1.33–6.80)	0.005**
Pre-milking udder washing Yes No	76 179	11(14.47) 33(18.44)	Ref. 1.34(0.64 − 2.80)	0.445
Post-milking udder washing Yes No	53 202	5(9.43) 39(19.31)	Ref. 2.27 (0.86-6.15)	0.098
Towel use for udder drying Yes No	76 179	9(11.84) 35(19.55)	Ref. 1.80 (0.82-3.98)	0.140
Post-milking teat dipping Yes No	37 218	2(5.41) 42(19.27)	Ref. 4.18(0.96-18.06)	0.056
Type of floor Earthen Concrete	152 103	33(21.71) 11(10.68)	2.32(1.1–4.84) Ref.	0.025**
Frequency of floor cleaning Daily More than once/week Once/week Less than once/week	168 70 9 8	25(14.9) 13(18.6) 3(33.4) 3(37.5)	Ref. 3.80(1.86–7.79) 4.44(1.01–19.38) 5.33(1.17–24.29)	0.001** 0.047** 0.031**
Barn hygiene Poor Good	122 133	30(24.59) 14(10.53)	2.77(1.39–5.53) Ref.	0.004**

OR = odds ratio, CI = confidence interval, ** significant

Risk factors that were P < 0.25 in the univariable analyses (breed, age, parity, lactation stage, daily average milk yield, presence of teat lesions, hand washing at milking interval, post-milking udder washing, use of towel for udder drying, post-milking teat dipping, type of floor, frequency of floor cleaning and barn hygiene) were investigated using correlation matrix in order to assess collinearity. The independent variables were considered collinear when r > 0.5. Thus, the variables collinear with each other were parity and age (r = 0.8), average daily milk yield and breed (r = 0.9), post milking udder washing and post milking teat dipping (r = 0.8), post milking teat dipping and type of floor (r = 0.7) and floor cleaning frequency and barn hygiene (r = 0.7). Use of towel for udder drying and post milking teat dipping were removed from the model due to confounding. Finally, breed, parity, lactation stage, washing hands at the milking interval, presence of teat lesions and barn hygiene were subjected to multivariable logistic regression analysis.

The result of the multivariable logistic regression analysis showed that breed, parity, stage of lactation, hand washing at milking interval and barn hygiene were statistically significantly associated (P < 0.05) with the occurrence of Staphylococcus aureus. Accordingly, crossbred cows had statistically significantly (OR = 3.05, CI:1.47–18.07, P = 0.006) higher odds to contract Staphylococcus aureus compared to local breeds. Cows that gave birth to three calves (OR = 4.71 CI:1.47–15.07, P = 0.009) and ≥ 4 calves (OR = 4.34, CI:1.44–13.14, P = 0.009) were statistically significantly more likely to have Staphylococcus aureus positive result than cows that gave birth to one calf. The odds of Staphylococcus aureus in cows in late lactation stage (OR = 3.05, CI:1.19–7.82, P = 0.037) were statistically significantly higher than those in mid lactation stage. The odds of Staphylococcus aureus in dairy cows that were milked without washing hands at milking intervals (OR = 3.71, CI:1.94–11.46, P = 0.001) were statistically significantly higher than those cows that are milked after washing hands at the milking intervals. Similarly, the occurrence of Staphylococcus aureus in dairy cows that were kept in poor hygienic condition barns (OR = 2.53, CI:1.04–6.14, P = 0.040) were statistically significantly higher than those cows that have been kept in a good hygienic condition.

Hosmer-Lemeshow goodness-of-fit test result showed that the model fits the data reasonably well (P = 0.7998, χ² = 43.29). ROC curve (0.7658) indicated that the model had good predictive ability. The result of multivariable logistic regression analysis of Staphylococcus aureus with various risk factors is depicted in Table 4.

Table 4.

Multivariable logistic regression analysis of S. aureus with various risk factors

Risk factors		No of examined			No of positive (%)	OR (95% CI)			P-value
Breed Local Cross		102 153			11(10.78) 33(21.57)	Ref 3.05(1.47–18.07)			0.006**
Lactation stage Early Mid Late		89 87 79			15(14.61) 8(11.49) 21(26.58)	1.69 (0.63-4.59) Ref 3.05(1.19–7.82)			0.297 0.037**
Parity 1 2 3 ≥4		74 67 49 65			5(6.76) 10(14.93) 12(24.49) 17(26.15)	Ref 2.33(0.72 − 7.5) 4.71(1.47–15.07) 4.34(1.44–13.14)			0.157 0.009** 0.009**
Washing hands at milking interval Yes No		94 161			8(8.51) 35(21.75)	Ref 3.71 (1.94–11.46)			0.001**
Barn hygiene Poor Good		122 133			30(24.59) 14(10.53)	2.53(1.04–6.14) Ref			0.040**
Presence of teat lesions Yes No		53 202	13(24.53) 31(15.35)			2.23(0.91-5.04) Ref.			0.09

OR = odds ratio, CI = confidence interval, ** significant

Antimicrobial Sensitivity Pattern of Staphylococcus aureus

In the current study, 20 randomly selected Staphylococcus aureus isolates were subjected to 10 types of antimicrobials for an antimicrobial sensitivity test. Accordingly, the susceptibility pattern of the tested Staphylococcus aureus isolates was; ciprofloxacin (100%), followed by, gentamicin (90%), vancomycin (85%), chloramphenicol (85%), nitrofurantoin (75%), nalidixic acid (70%), ceftriaxone (60%), tetracycline (50%), ampicillin (20%) and penicillin (15%). Antimicrobial resistance was observed most frequently to penicillin G (85%), followed by ampicillin (75%), tetracycline (50%), ceftriaxone (30%), nalidixic acid (25%), nitrofurantoin (10%), vancomycin (10%) and chloramphenicol (5%). From the total isolates tested, 65.5% were susceptible, 5.5% were intermediate and 29% were resistant to the antimicrobials. The result is shown in Fig. 2.

Fig. 2.

Antibiotic resistance profile of the Staphylococcus aureus in dairy farms of Bako, Nekemte and Gimbi towns

Multidrug Resistance Pattern of Staphylococcus aureus

Of the 20 Staphylococcus aureus isolates examined, none of them were resistant to three or more antimicrobial classes.

Discussion

Considering the overall prevalence of Staphylococcus aureus in the selected dairy farms of Bako, Nekemte and Gimbi towns; the finding is comparable with reports of [51] around Addis Ababa (17.5%) [52], in Sebata (19.9%) and [53] in Mekelle (17.3%) dairy farms in Ethiopia. The present finding is lower when compared with a study conducted by [17] in central Ethiopia (25%) and [54] in Arsi Negele town (25.47%) dairy farms. However, it is higher than reports of [55] in Bishoftu town (8.25%) and [56] in Addis Ababa (5.2%) dairy farms. These disparities in Staphylococcus aureus prevalence could be related to differences in management systems, sample size, the environment of the sampled area, and farm owners’ and milkers’ awareness.

Our S. aureus prevalence in raw milk is relatively similar to the findings of [57] in the Hawassa area (17.9%) [37], around Addis Ababa (16.2%) and [58] in and around Addis Ababa (17.2%) dairy farms. This finding is lower than reports of [59] in Mekelle (46.4%) [60], in Alage TVET College dairy farm (28.2%) and [23] in Mekelle (26.3) dairy farms. However, this finding is higher than that of [61] in Holeta (13.8%) and [20] in and around Asella (11.9%) dairy farms. Although the prevalence of Staphylococcus aureus has been reported to vary with various factors, the high presence of Staphylococcus aureus in milk in the present study areas might be due to poor hygienic and sanitary conditions during milking.

Our S. aureus prevalence in swabs of milker’s hand is relatively in line with [52] in Sebata (32%) and [53] in Mekelle (31%) dairy farms. This finding is higher when compared with a study previously conducted by [19] in and around Asella (11.1%) [20], in Addis Ababa (25%) and [62] in Bishoftu (26.7%) dairy farms. However, this finding is slightly lower than the report of [54] who reported 34.28% at Arsi Negele town dairy farms. This high prevalence of Staphylococcus aureus could be related to the fact that at least 30% of people carry the bacterium in their nasal passages, and throats, and contaminate their hands by coughing or sneezing, implying that milkers could be possible sources. The variation in the isolation rate could be attributable to differences in hand-washing practices in the study areas.

Considering the prevalence of S. aureus in swabs of milking buckets; our finding is in agreement with reports of [63] in Ambo and Guder (9%) and [52] in Sebata (11.1%) dairy farms. However, the present finding is lower than that of [18] in Jigjiga (23.3%) and [20] in Addis Ababa (25%) dairy farms. In contrast to this finding [19], reported 0% in and around Asella town dairy farms. This variation might be due to variations in washing practices of milking buckets and mixing of milk from Staphylococcus aureus positive cows with others.

In the present study, the presence of Staphylococcus aureus was statistically significantly influenced by breed. The odds of finding a cross breed cow with a positive Staphylococcus aureus result were 3.05 times higher than in the local breed. This is in agreement with the findings of previous works by [64] in Wolaita Sodo and [65] in and around Asella town dairy farms. This variation of Staphylococcus aureus occurrence in breeds’ level could be due to the influence of some inheritable characteristics such as the capacity of milk production, teat characteristic and udder conformation [66]. However, this finding is in disagreement with [51] in and around Addis Ababa and [67] in Holeta area dairy farms who reported that there was no significant association between breed and occurrence of Staphylococcus aureus.

The current study revealed that the prevalence of Staphylococcus aureus significantly varied with the stage of lactation which was highest in the late stage of lactation. Evidence on the trend of prevalence increment at the late lactation stage was also produced in previous studies by [68] in Addis Ababa and [58] in and around Asella town dairy farms. The increased risk of Staphylococcus aureus in the late lactation stage can be due to the accumulated exposure to the pathogen that cows undergo throughout lactation. However, this finding is in disagreement with [51] in and around Addis Ababa and [22] in Mukaturi and Sululta dairy farms who reported no significant association between lactation stage and occurrence of Staphylococcus aureus.

The prevalence of Staphylococcus aureus was statistically significantly lower in primiparous. The odds of Staphylococcus aureus occurrence were 4.71 times higher in cows that gave birth to three calves and 4.34 higher in cows that gave birth to more than four calves than the primiparous cows. The current finding conforms to reports of [69] in Dire-Dawa and [70] in and around Asella town dairy farms. The increasing prevalence of Staphylococcus aureus along with parity could be due to multiparous cows in the studied herds might have had cumulatively several exposures to Staphylococcus aureus from poor hygiene during milking. Poor integrity of the teat canal due to aging, decreased immunity, or a more pendulous udder prone to injury in older cows might all have increased the susceptibility of the older animals [71, 72]. This result is disagreeable with a report of [48] in Adama smallholder dairy farms in which there was no statistically significant variation among levels of parity and prevalence of Staphylococcus aureus.

This study revealed that there is a statistically significant association between the occurrence of Staphylococcus aureus and hand washing at the milking interval. The odds of Staphylococcus aureus occurrence in cows that are milked without washing hands at the milking interval were 3.71 times higher than those that are milked after washing hands at the milking interval. This finding is in line with the reports of [73] in Bahir Dar and [74] in and around Gonder dairy farms. This implies that Staphylococcus aureus could be transmitted easily from infected to uninfected udder quarters or cows through the milkers’ hands as infected udder is the primary reservoir [75]. asserted that Staphylococcus aureus is well adapted to survive in the udder and usually establishes a mild subclinical infection of long duration, from which it is shed in milk, facilitating transmission to healthy animals, mainly during the milking process.

This study also revealed the odds of Staphylococcus aureus occurrence were statistically significantly 2.53 times higher in cows kept in poor hygiene barns than those kept in good hygienic condition barn. This finding is in agreement with [73] in Bahir Dar and [66] in Holeta area dairy farms. Keeping cows under unhygienic conditions increases the bacterial load and potential transmission of mastitis-causing organisms, to enter the udder through the teat orifice [50]. In the studied areas, the majority of the dairy farms do not wash the udder before milking and this might be one factor for the spread of Staphylococcus aureus. Furthermore, poor hygiene will make control methods such as teat dipping and sanitization ineffective [76].

The antimicrobial sensitivity test carried out in this study indicated that Staphylococcus aureus isolates displayed differences in the antimicrobial susceptibility patterns. The highest susceptibility of Staphylococcus aureus observed in our study is slightly in agreement with other studies carried out by [54] who reported, vancomycin (92.7%), gentamicin (95.3%) [77], reported, vancomycin (100%), gentamicin (100%) and chloramphenicol (81.8%) and [78] reported ciprofloxacin (100%), gentamicin (100%) and vancomycin (100%). The high susceptibility observed in these antimicrobials might be due to these antibiotics being the least frequently used in the study area in veterinary services and also might imply that such antibiotics can be used to treat infections caused by Staphylococcus aureus.

The highest resistance of Staphylococcus aureus towards penicillin G and ampicillin observed in the current study is comparable with the findings of [19, 22] who reported 93.1%, 97.6% and 95.5% resistance to penicillin G, respectively and [64, 66] who reported 55.1% and 66.7% resistance to ampicillin respectively. This high level of resistance to penicillin might be due to penicillin are the most commonly used antibiotic in the study areas for veterinary use and Staphylococcus aureus produces a penicillinase enzyme (a type of β-lactamase) that hydrolyses the beta-lactam ring of penicillin. It is believed that around 50% of mastitis causing Staphylococcus aureus strains produce β-lactamase [29].

The highest resistance of Staphylococcus aureus towards tetracycline observed in this study is relative to reports by [64] (50%) and [58] (82.2%) resistance to tetracycline. High tetracycline resistance could be associated with the fact this antibiotic is commonly used in the treatment of infections in the current study area. Lack of stringent regulation and monitoring in the dispensing and use of antimicrobials in the country also might contribute to the occurrence of high antimicrobial resistance to these drugs. In this study, none of Staphylococcus aureus isolates showed multi antimicrobial resistance to different classes of antimicrobials, this finding is similar with the report of [78].

Conclusion and recommendations

The present study, conducted in dairy farms of Bako, Nekemte and Gimbi towns, revealed a considerable presence of Staphylococcus aureus, posing potential public health and animal health concerns. The bacterium was isolated from raw milk, milkers’ hands, and milking buckets, highlighting the dairy production environment’s numerous sources of contamination. Breed type, parity, lactation stage, hand hygiene while milking, and barn cleanliness were risk factors that were strongly linked to the occurrence of Staphylococcus aureus, indicating both extrinsic and intrinsic factors that contribute to its presence. The antimicrobial susceptibility profile demonstrated that while Staphylococcus aureus isolates were highly sensitive to ciprofloxacin, gentamicin, vancomycin, and chloramphenicol, they showed high resistance to commonly used antibiotics such as penicillin G and ampicillin. Encouragingly, no multidrug-resistant strains were identified. These findings emphasize the urgent need for improved farm-level hygiene practices, responsible antibiotic use, and further molecular-level investigations to prevent the spread of antibiotic-resistant Staphylococcus aureus strains in the region. Overall, the study contributes valuable insights into the epidemiology of Staphylococcus aureus and calls for integrated efforts in training, surveillance, and intervention strategies to ensure safe milk production and safeguard public health.

Based on the above conclusion, the following recommendations were forwarded:

• Training on proper milking hygiene, hygiene of the cow and barn area should be given to all actors involved in milk production by concerned body.

• Close monitoring and rational use of antibiotics must be practiced to prevent antibiotic resistance from the pathogen.

• Further work must address the factors influencing the occurrence of Staphylococcus aureus in dairy farms of the study area.

• Further study on the molecular characterization of Staphylococcus aureus and detection of genes responsible for antimicrobial resistance needs to be carried out in the study area.

Limitation

The limitation of this study was the lack of molecular tests using Polymerase Chain Reaction (PCR) to confirm the presence of Staphylococcus aureus in dairy farms. The absence of PCR in this study might have underestimated the true prevalence of Staphylococcus aureus in dairy farms. In future studies, the use of molecular tests may increase the reliability and validity of the findings.

Author contributions

Authors’ Contribution: (A) Kirubel Paulos Gutama: conceptualization; methodology; data curation; formal analysis; visualization; investigation; writing- original draft, writing–review and editing. (B) Manyazewal Anberber: supervision; visualization; data curation; formal analysis; writing original draft; writing–review and editing. All authors reviewed the manuscript.

Funding Declaration

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Not applicable.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information files.

Declarations

Ethics approval and consent to participate

Informed consent was obtained from each participant. All the information obtained from the study participants was kept confidential.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.