Prevalence of Staphylococcus aureus in udder milk, bulk milk, and milk products
The present study showed an overall prevalence of S. aureus of 15.64% (94/601) in raw milk and milk products in Ambo and Bako towns, Ethiopia. The highest prevalence was detected in bulk tank milk (17.50%), followed by ‘Ayib’ (16.67%) and individual cow level milk (15.29%). The higher S. aureus contamination of bulk milk than the individual animal level in the current study is in agreement with the finding of Addis et al. [23], and it could be due to milk cross-contamination during bulking and inadequate handling during transportation from farms to collection centers and at milk collection facilities.
The individual cow level prevalence of S. aureus in the present study was almost in agreement with the 15.5% prevalence reported by Asrat et al. [24] in Addis Ababa, Ethiopia. However, the current finding is higher than the 11.4% prevalence in Afar, Ethiopia by Beyene [25], 13.9% in Ambo and Guder town, Ethiopia by Megersa [26], and 8% in Bishoftu, Ethiopia by Addis et al. [23]. On the other hand, the current value is lower than the previous prevalence of 19.6% by Ayele et al. [27] in Sebeta, 19.3% by Abunna et al. [28] in Asella and 21.2% by Tessema and Tsegaye [29] in Alage ATVET College Dairy Farm, Ethiopia. Much higher prevalence was 39.1% at Asella by Bedada and Hiko [30], 46.5% in Assela, Ethiopia by Seyoum et al. [31], and 56.6% in Kombolcha by Asmelash et al. [32]. The variation in the magnitude of the prevalence might be due to differences in the management practices at farms, the sample collection procedure, hygienic milking and handling practices, and the use of towels for udder drying. Moreover, observations during sample collection showed unhygienic milking and milk handling, and poor farm management practices were observed during sample collection at the farm level, which might have contributed to the high prevalence rates of S. aureus in the current study.
The prevalence of S. aureus (17.50%) in bulk tank milk in the present study was much higher than the previous study by Wodajo et al. [33] who reported 3.2% from Asella Dairy Union Ada Primary Dairy Cooperative, Ethiopia, and lower than the 35.3% prevalence in Alage ATVET College Dairy Farm, Ethiopia reported by Tessema and Tsegaye [29], and 63.15% in Arsi Negelle town, Ethiopia by Tsige [34]. S. aureus is a contagious pathogen, which can spread from one animal to another or personnel by contact with cows during unhygienic milking procedures [32]. Accordingly, the differences in the prevalence of S. aureus from bulk tank milk from various studies could be due to milk cross-contamination during bulking and unhygienic handling during transportation from farms to collection centers and at milk collection facilities [23].
In the current study, the prevalence of S. aureus in raw bulk tank milk (17.50%) is higher than that of ‘Ayib’ (18.03%) and ‘Ergo’ (13.11%). Higher contamination with S. aureus in raw milk than in milk products, which is in agreement with the current study was reported by Lemma et al. [35]. The overall prevalence in the milk production of the current study 15.57% (19/122) is almost in agreement with the previous 11.3% prevalence of S. aureus by Lemma et al. [35] in Addis Ababa and 14.3% prevalence by Argaw et al. [10] from Jimma. However, the S. aureus contamination in ‘Ayib’ in the current study is much higher than that in ‘Ayib’ (5%) and lower than that in ‘Ergo’ (17.5%) reported by Argaw et al. [10] from Jimma, Ethiopia. This difference might be attributed to the difference in hygienic practices of dairy farmers, and milk product processors and sellers/vendors in the different regions of the country,
Risk factors for prevalence of S. aureus at cow and farm level
The statistically significant association of the prevalence of S. aureus with the risk factors namely breeds and age of the animal, history of previous treatments, tick infestation, lactation stage, udder washing, using an individual towel for each cow, hygiene of milkers, and use of detergents in the current study are in agreement with the previous study by Pittet et al. [36], reported using detergents can decrease, Ayele et al. [27] reported no practice of udder washing increases the prevalence of S. aureus and Yilma et al. [37] identified pre-milking udder preparation like udder washing and employing good milk handling practices play an important role in decreasing S. aureus in dairy farms. Even though not statistically significant, the numerically higher prevalence of S.aureus observed in farms where milking containers were washed by war water and soap could be due to the luke warm water, which is not enough to kill the bacteria. In addition it was found that most of the time even if they wash the milking container with hot or warm water, finally they rinse and wash with their hand using cod water just before they use it for milking. Thus such malpractice of washing the utensil with un washed hands be the cause of re-contamination of the utensils.
The significant association of the prevalence of S. aureus with the absence of individual towels for each cow and udder washing with a common towel before milking cows was reported by Sori et al. [38], Lakew et al. [39], and Asmelash et al. [32]. This might be the fact that sharing same towels for udder washing and drying for all cows udder, could spread the pathogenic bacteria from affected cows to healthy ones [40]. On the other hand, similar to the current study, previous studies by Biffa et al. [41] and Abraham [42] identified that higher infection in cows occurred at an early stage of lactation followed by medium and late stages of lactation. This could be because the mammary gland is more susceptible to new infection during the early and mid-lactation which may be due to the absence of udder washing and teat dipping, which in turn might have increased the presence of potential pathogens on the skin of the teat [43].
The current study also identified cow age as the risk factor for the prevalence of S. aureus, which is in line with the findings of Zenebe et al. [44], who stated that older cows were more affected than the younger ones. On the other hand, a high prevalence (16.25%) was detected in barns where there was infrequent cleaning of the barn than in those where daily cleaning was practiced (15.04%). The current finding also showed high prevalence recorded in Holstein Frisian cross (2.22%) and Jersey (3.47%) than locals (Borena and Horro) this is in agreement with Radostits et al. [40] and Elemo et al. [45] who reported that the position of the teat and udder anatomy of teat canal in these cows makes their injury.
In the current study floor and farm hygiene were significantly associated with the prevalence of S. aureus at farm levels. This finding was in agreement with the findings of Biffa et al. [41] in Southern Ethiopia, and Abera et al. [46] in Adama town, where high prevalence was detected in farms with the muddy floor as compared to those having concrete floor types. This might be due to the fact that dirty and muddy shared barns favor the proliferation and transition of pathogens like S. aureus in dairy farms. This is also consistent with the previous study, which identified that utensils on which food is displayed can get easily contaminated if left dirty or unprotected [47].
In the current study, the management system of the dairy farms and herd size were found to be the risk factors for the prevalence of S. aureus at the farm level. The prevalence of S. aureus in extensive (59.09%), semi-intensive (28.57%), and intensive (23.53%) in the present study is almost in agreement with Abebe et al., (2016), who reported that semi-intensive management type (63.2%) and intensive (36.8%) in Hawasa, but disagree with Shiferaw and Telila [48] where a significantly higher prevalence was reported in housed cows than pastured ones. On the other hand, the effect of tick infestation on udder/teat skin was significantly associated with the prevalence of S. aureus at the cow level. Similarly, Shiferaw and Telila [48] also reported a high incidence of S. aureus cow’s teat lesions due to tick infestation were frequently colonized by staphylococci.
Risk factors for Staphylococcus aureus prevalence in bulk tank milk
In the current study plastic containers were used for milking, storing, and transporting bulk milk to raw milk selling shops by the majority of the dairy farmers. The statistical analysis showed that the type of bulk milk containers was significantly associated with the prevalence of S. aureus. This finding is consistent with Omoe et al. [49] who reported that plastic containers have an inherent characteristic that makes them unsuitable for milk handling as they scratch easily, provide a hiding place for bacteria during cleaning, sanitation, and poor conductor heat, and thus will hinder effective sanitization by heat leading to bacterial contamination of milk. In addition, another factor like the practice of hand washing after touching money is significantly associated with the prevalence of S. aureus. The practice of fingering the nose and shaking hands for greeting, which could be the risk factors for the contamination of bulk raw milk was also observed in the study areas. In line with this current finding, Alemu [18], testified that paper currency notes in circulation, which are handled by a large number of people in a variety of personal and environmental settings, are contaminated with a variety of microbiological agents, the most of which are resistant to frequently used antibiotics, posing a risk to the community and those handling currency notes. Ayandele and Ayandele and Adeniyi [50] also reported high rates of microbial contamination arising from currency notes circulating in the world due to endemic bacterium families of Enterobacteriaceae, Mycobacterium tuberculosis, and S. aureus and arising from human normal skin flora were contaminants of money.
Risk factors for prevalence of Staphylococcus aureus in milk products
The present study showed that environmental sanitation (p = 0.004), type of product container (p < 0.001), hygiene product container (p < 0.019), and product handler (p < 0.001) were significantly associated with the prevalence of S. aureus in the milk products. These findings are in agreement with the report of Mamo et al. [51], who reported that milk products sold in an open market were contaminated with S. aureus in Hawasa, Ethiopia. Similar to the current finding, the type of product container as a potential risk factor was reported by Omoe et al. [49] and Soomro et al. [52], who suggested stainless steel for handling products than plastic containers, which are prone to bacterial contamination of milk products as they are easily scratched during cleaning and cannot be effectively sanitized by heat.
Contamination of S. aureus in bulk tank milk in this study was associated with poor hygiene of milk handler and container. This finding is in line with the finding of Fanta et al. [53], who reported that pre-milking udder preparation and employing good milk handling practices play an important role in minimizing S. aureus contamination at the farm. A study conducted in Sebeta, Ethiopia by Ayele et al. [27] also showed that many farmers did not effectively clean their hands before milking and utensils used for milking and storing milk. Such practices might facilitate the cross-contamination of microorganisms in the bulk milk supplied to different restaurants or direct consumers, who rent milk from dairy farms for home consumption.
Bacterial quality of bulk tank milk
According to the current study, the maximum bacterial load of 1.92x104 CFU/mL (6.28 log10) CFU/mL in farms and 1.86 x104 CFU/mL (6.27 log10) CFU/ml in restaurant, respectively. Based on international standards level ISO 6888 (ISO, 1999), such milk, having less than 105 CFU/mL count, is satisfactory and can be consumed without causing any risk to the health of the population. When the concentration of entero-toxigenic strain of S. aureus exceeds 105CFU/ml the strain is capable of producing toxin sufficient to cause enterotoxin [54]. The current study also showed a statistically significantly higher mean count of S. aureus when no hand washing was practiced (Mean1.75 ± 2.12 SD) and water was used as the only hand washing agent (Mean1.77 ± 1.2 SD) than when hand washing was done (Mean 0.68 ± 1.59 SD) and soap and water were used as hand washing agent (Mean 0.58 ± 2.0 SD). This finding is in line with Reta et al. [55], Yilma [56], and Welearegay et al. [57], who reported that the dairy farms, where water and detergent were used for cleaning had less contamination. This could be because cross-contamination can be avoided if the hand of milkers, utensils, or equipment is washed with detergents and water before use.
Antimicrobial susceptibility test
The prevalence and level of antimicrobial resistance in veterinary medicine were increasing worldwide. Particularly, the spread of antimicrobial-resistant staphylococci is presenting a challenge to both human and animal health professionals [58]. The antimicrobial susceptibility tests carried out in the current study showed the highest rate of resistance 65(98.48%) recorded against ampicillin, amoxicillin, and oxacillin followed by cefoxitin 61 (92.42%) and tetracycline 55 (83.33%). The high prevalence of resistance to oxacillin and cefoxitin in the current study is indicative of the existence of Methicillin-Resistant Staphylococcus aureus (MRSA), which is difficult to treat and becoming a very serious public health concern. Nearly in agreement with this finding, 100% (amoxicillin) and 64.7% (tetracycline) resistance was reported by Ayele et al. [27], respectively. However, the 83.33% resistance against tetracycline in the current study is higher compared to the 32.5% − 82.2% previously reported resistances from various sources [21, 24, 31, 45]. The existence of an alarming level of S. aureus resistance to tetracycline, ampicillin, and amoxicillin was also documented in earlier studies in other countries by El-Jakee et al. [59] and Daka et al. [60] and Gentilini et al. [61] suggesting a possible development of resistance from prolonged and indiscriminate usage of the antimicrobials. Cefoxitin-resistant isolates detected in the milk and milk products in the current study are in line with the findings of Elemo et al. [45]. The reason for the occurrence of antimicrobial resistant S. aureus isolates could be due to the arbitrary use of antimicrobials, self-medication, and administration of sub-therapeutic doses of antimicrobials to livestock for prophylactic purposes and limited updating of the long-used drug groups Abraha et al. [62].
Analysis of multidrug resistance patterns in the current study indicated that 43.94% (29/66) of S. aureus isolates from milk and milk products showed multiple drug resistance. In contrast to this finding, a higher (62.5%) and lower percent (2.4%) of multidrug-resistant S. aureus isolated from milk and milk products were reported by Gebremedhin et al. [21] and Regasa et al. [63], respectively. The emergence of resistance to numerous drugs represents public health hazard because foodborne outbreaks might be difficult to treat and the group of multidrug resistance S. aureus in the food supply represents a reservoir for communicable resistant genes [62].