Prevalence of Staphylococci in Milk Sold in Bharatpur

Milk is a fundamental dietary component for many people globally. However, it can pose microbiological risks to consumers if hygiene and sanitation standards are not properly upheld [1]. It serves as a significant source of carbohydrates, proteins, immunoglobulins, fatty acids, and various micronutrients. Ensuring the nutritional quality and safety of milk requires rigorous monitoring and evaluation. The nutrient profile of milk supports human health in multiple ways, offering beneficial components such as healthy fats, calcium, vitamins, and minerals. Unfortunately, milk can become contaminated from various sources, including pathogens associated with diseases like tuberculosis, mastitis, and brucellosis, as well as infections carried by milk handlers, such as dysentery and typhoid [2]. Dairy farms and their environments may harbor pathogens like Listeria, Salmonella, and pathogenic E. coli [3]. Over 90% of reported dairy-related illnesses are due to bacterial contamination, with at least 21 recognized milk-borne diseases. S. aureus is particularly significant in foodborne illnesses, causing an estimated 241,000 cases annually in the United States [4]. According to regulations by the US FDA and ICMSF-1996, the permissible limit for S. aureus in raw milk and dairy products is 104CFU/gm. 

Dairyborne pathogens such as Mycobacterium bovis, Brucella spp and Coxiella burnetii are also significant concerns. Mycobacterium bovis, responsible for bovine tuberculosis,can be transmitted through unpasteurized milk and cause respiratory and systemic symptoms. Brucella species lead to brucellosis, transmitted via raw milk and presenting with fever, joint pain, and fatigue [5]. Coxiella burnetii, the causative agent of Q fever, can also be present in raw milk and cause flulike symptoms or severe respiratory illness. Pasteurization is an essential preventive measure that effectively kills these pathogens and ensures the safety of dairy products, highlighting the importance of consuming pasteurized milk to reduce the risk of these diseases [5]. S. aureus is a significant foodborne pathogen responsible for causing Staphylococcal Food Poisoning (SFP). This bacterium is commonly found on human skin and in nasal passages, which makes it a frequent contaminant in food when handled improperly. S. aureus can enter food through direct contact with human carriers, such as food handlers who have not properly washed their hands or who have open wounds (Hennekinne, De Buyser, & Dragacci).This includes ensuring good hygiene among food handlers, such as frequent hand washing, and properly cooking and refrigerating foods.

In total, 30 milk samples were analyzed, consisting of 15 raw and 15 pasteurized samples sourced from different areas within Bharatpur. 30ml of raw milk was aseptically collected from the site immediately after milking into sterile screw-capped test tubes. For the pasteurized milk, 500ml packages available in the market were purchased and transported in plastic bags. 

For sample preparation, 1 ml of the sample was added to a sterile test tube that contained 9ml of sterile normal saline, applicable to both raw and pasteurized milk. To assess the total staphylococcal count, the pour plate method was utilized. A 1ml sample from each dilution was transferred to sterile petri dishes, mixed with Mannitol Salt Agar (MSA), and incubated at 37°C for 24 hours. After incubation, the plates were examined for distinct colonies, particularly golden yellow colonies, and the quantity of Staphylococcus species was calculated as colony-forming units per milliliter (CFU/ml), following the method described by Cheesbrough [6].

Total Staphylococcal Count 

Among 30 milk samples, Total Staphylococcus counts varied significantly, with raw milk showing higher contamination and positivity rates compared to pasteurized milk (Table 1). 

Table 1: Total staphylococcal count. 

Prevalence of S. Aureus and CoNS 

From the 30 milk samples tested, 17 were found to contain Staphylococcus species. Of these, 15 isolates were identified as S. aureus, while 2 were classified as other Coagulase-Negative Staphylococci (CoNS) (Figures (1-3)).

Figure 1: Prevalence of Staphylococcus aureus and CoNS.  

Figure 2: MDR among isolates. 

Figure 3: MRSA among isolates.

Antibiotic Susceptibility Pattern of Staphylococcus Species 

Among the 17 Staphylococcus spp. isolates, all demonstrated complete sensitivity to Cotrimoxazole, Amikacin, and Vancomycin. In contrast, resistance was noted for Clindamycin (29.41%), Penicillin G (100%), Ampicillin (76.47%), Tetracycline (17.64%), Cefotaxime (52.94%), Cefoxitin (23.53%) and Ciprofloxacin (17.64%) (Table 2).

Table 2: Antibiotic susceptibility pattern of staphylococci species isolated from milk.

Distribution of MDR Isolates 

Out of 17 Staphylococci isolates, 5(29.47%) showed Multi-Drug Resistance (MDR). None of the CoNS were MDR.

MRSA Among Isolates 

4(23.5%) of the S.aureus isolates shows the MRSA based on resistance to cefoxitin.

In a study conducted in Bharatpur, a total of 30 milk samples were collected, comprising 15 raw and 15 pasteurized samples. The analysis revealed that the Total Staphylococcus Count (TSC) varied between 2.07×10³ and 45×10³CFU/ml, with an overall mean TSC of 1.0648×10⁴CFU/ml. For the raw milk samples, the TSC ranged from 3.0×10³CFU/ml to 45×10³CFU/ml, resulting in a mean TSC of 1.3108×10⁴CFU/ml. Notably, 86.67% of these raw samples tested positive for Staphylococcus species. In contrast, the pasteurized milk samples showed a TSC range of 2.07×10³CFU/ml to 3.0×10³CFU/ml, with a mean TSC of 2.655×10³CFU/ml; only 26.67% of these samples were positive for Staphylococcus spp. The majority of milk samples fell within the acceptable limits as shown in table 1. However, these findings differ from those of Acharya et al. [7], who noted that the total bacterial count in milk samples exceeded established guidelines. Additionally, when comparing Staphylococcal counts, the observed range of 2.07×10³ to 45×10³ CFU/ml is significantly lower than the 14×10⁵ to 8×10⁶CFU/ml reported. 

In this investigation, out of 30 analyzed milk samples, 17 tested positive for Staphylococcus species. Specifically, 15 were identified as S. aureus, while the remaining 2 were categorized as other coagulase-negative Staphylococci (CoNS). Among pasteurized milk samples, 13.33% were confirmed to contain S. aureus, whereas a significant 86.67% of raw milk samples were found to be positive for this bacterium. These results contrast with those reported, who found 45% of raw milk samples tested positive for S. aureus. Additionally, the total Staphylococcus count differed significantly between raw and pasteurized milk (p<0.05). For pasteurized samples, the 13.33% positivity rate is consistent with findings from Achary et al. [7] and Arjyal et al. [8], who reported 12.5% positivity in pasteurized milk. Notably, detected a higher contamination rate of 20% in pasteurized milk, suggesting that unsatisfactory processing, improper cleaning, and contamination during packaging may play a role. 

While pasteurization effectively reduces S. aureus levels, it does not eliminate the enterotoxins produced by this bacterium, which can pose risks to consumers. 

The Antimicrobial Susceptibility Testing (AST) of S. aureus from milk samples revealed complete sensitivity to Cotrimoxazole, Amikacin, and Vancomycin. However, there was significant resistance observed against Penicillin G (100%), Ampicillin (76.47%), Tetracycline (17.64%), Cefotaxime (52.94%), Cefoxitin (23.53%), Ciprofloxacin (17.64%), and Clindamycin (29.41%). In a study by milk samples exhibited resistance rates of 86.04% for Penicillin G, 74.42% for Ampicillin, and lower rates of 4.65% for both Cefoxitin and Cotrimoxazole, which aligns with our findings. Conversely reported that S. aureus isolates showed high sensitivity to Ciprofloxacin (97.5%), Ceftriaxone (91.6%), and Tetracycline (89.9%), with Cefoxitin showing the least sensitivity at 62.2%. Found that S. aureus had 49.7% resistance to Penicillin G, 5.3% to Tetracycline, and 2.1% to Cefoxitin, indicating lower resistance compared to our results, which showed 100% resistance to Penicillin G, 20% to Tetracycline, and 26.67% to Cefoxitin. Additionally, while reported 100% sensitivity to Chloramphenicol, our findings differ. In research conducted in the Kathmandu valley by Parajuli et al. [9], S. aureus was found to be fully susceptible to Amikacin and Levofloxacin, similar to our results. However, their findings of 100% sensitivity to Chloramphenicol and 33.33% resistance to Penicillin, along with 67.7% resistance to Cefoxitin, contrast with our data. 

The variations in findings from different studies suggest that the antibiotic resistance patterns of Staphylococcus species are evolving. These bacteria are increasingly developing resistance to various antibiotics through multiple mechanisms. The isolation of antibiotic-resistant Staphylococcus species from milk poses significant challenges for human medicine, as these antibiotics are commonly used for treating both humans and animals [10]. 

In this study, we found multi-drug resistant S. aureus in both raw and pasteurized milk samples. Specifically, 5 out of 17 Staphylococcus isolates (29.47%) were classified as multi-drug resistant, with 2 (11.79%) coming from pasteurized milk and 3 (17.68%) from raw milk. This contrasts with the findings of Marjan et al. [11], who reported that nearly all (100%) pathogen isolates, including S. aureus from both types of milk, demonstrated multi-drug resistance. Our results align more closely with those of Rai et al. [12], who reported that 29.63% and 20-25% of S. aureus, respectively, were isolated from raw milk. 

Additionally, 23.58% (4 isolates) of the S. aureus identified from raw milk were confirmed as Methicillin-Resistant S. Aureus (MRSA), with those exhibiting a zone of inhibition of 21mm or less for cefoxitin disks meeting the CLSI [13]. Parajuli et al. [9], reported a slightly higher prevalence of MRSA at 33.33% in milk samples, while Acharya et al. [7], found that S. aureus from both raw and pasteurized milk in Bharatpur showed cefoxitin resistance at an average of 33.30%. The high morbidity and mortality associated with MRSA underline its clinical significance. Furthermore, our findings differ from those of Rai et al. [12], who detected MRSA in 66.67% of pasteurized and 37.70% of raw milk samples. In conclusion, conducting antibiotic susceptibility tests is essential for identifying effective treatments for diseases affecting both humans and animals [14,15].

This study focused on assessing the prevalence of staphylococci in milk samples collected from various locations in Bharatpur. Conducted in the microbiology laboratory at Birendra Multiple Campus, the results indicated that 86.67% (13 out of 15) of raw milk samples tested positive for Staphylococcus spp., while 26.67% (4 out of 15) of pasteurized milk samples were also positive. Among the 30 milk samples analyzed, 15 were identified as S. aureus, along with 2 coagulase-negative staphylococci (CoNS). Of the 17 Staphylococcus isolates examined, all demonstrated 100% sensitivity to Cotrimoxazole, Amikacin, and Vancomycin. Multi-drug resistance (MDR) was observed in 5 isolates (29.47%), while 4 isolates (23.53%) were identified as Methicillin-Resistant S. Aureus (MRSA) based on their resistance to Cefoxitin. The overall quality of milk sold in various areas of Bharatpur raises concerns, particularly given the detection of resistant S. aureus, which can lead to milk-borne illnesses and food poisoning. Therefore, it is essential to implement stringent monitoring of bacterial contamination in all milk products reaching consumers.

It is a pleasure to pay tribute to all the farmers and my grateful thanks also goes to all milk societies, dairys of Bharatpur, Chitwan for supporting during sample collection.

The authors declare that there is no conflict of interest regarding the publication of this article.

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