E ciency of lactoferrin to eradicate multidrug resistant Staphylococcus aureus isolated from some dairy products


 Back ground: This study aimed to investigate Staphylococcus aureus (S. aureus) in some dairy products, Genotypically and phenotypically antibiotic resistance pattern as well as to evaluate the effectiveness of lactoferrin (LF) as a bio preservative for yoghurt. Methods: A total of 150 samples from yoghurt, ice cream and Damietta cheese (50 for each) in Assiut city, Egypt. Antimicrobial susceptibility against antibiotics commonly used in human and animals was tested using the disc diffusion method, S. aureus isolates, PCR was applied on multidrug resistant (MDR) S. aureus isolates for detection of blaz, mecA and VanA genesResults: The highest prevalence S. aureus was detected in yoghurt (38%) followed by Damietta cheese (30%) and ice cream (14%). Antimicrobial susceptibility against antibiotics S. aureus isolates showed high resistance to tetracycline, penicillin, oxacillin, ampicillin, streptomycin, amoxicillin/clavulanate and neomycin, in different percentages. blaz, mecA and VanA genes were detected at 60% for blaz gene, 40% for mecA gene and 20% for vanA gene. Lactoferrin has a satisfactory antibacterial activity MIC at 20mg/ml and MLC at 40mg/ml. The results revealed that 40mg/ml LF in yoghurt could inhibit MDR S. aureus at 2nd day while, 20mg/ml at the fourth day.Conclusion: The study concluded that LF can be used as a bio preservative in yoghurt due to its highest antimicrobial activity and acceptable sensorial properties.


Background
Staphylococcus aureus is one of the world's ubiquitous, yet sophisticated and captivating microorganisms due to its special world epidemiology [1]. S.aureus has robust pathogenicity due to its wide distribution, excessive pollution rate and fast transmission. It reasons a variety of medical manifestations, from mild-local, super cial pores and skin lesions to serious invasive diseases, and may additionally even threaten life, [2].
Milk and milk products are known to be a source of S. aureus contamination whether they are collected from cows suffering from mastitis or from food handlers carrying the microbe because of poor personal hygiene [3].
Over the past decade, the hassle of antimicrobial resistance in the African continent has gained one-of-a-kind interest. However, little is regarded about the actual extent of the problem due to the fact surveillance for antimicrobial resistance is carried out in solely a few countries [4].
Bacterial resistance to most conventional antibiotics has become a clinical and public health problem. Infections due to multidrug resistant microorganisms, such as methicillin resistant S. aureus (MRSA) considered a challenge, which ought to be controlled due to its high treatment coasts, therapeutic failure and death. Vancomycin is the mainstay treatment of MRSA invasive infection [5]. However, emerging isolates with reduced vancomycin susceptibility have also been detected [6]. Furthermore, MRSA and vancomycin resistant S. aureus (VRSA) isolates have been identi ed as an emerging pathogen in livestock animals that is readily transferable to humans in contact with livestock [1,7]. However, occurrence of vancomycin-resistant MRSA strains is scary recorded in animals because the drug not used in veterinary practice in many countries.
The emergence of such resistant strains plays a necessary position in therapeutic failure in each human and animal infections. The uncontrolled use of antibiotics in human and animals, together with bad diagnostic techniques and inappropriate prescribing by way of unquali ed physicians, exacerbates the problem [8] and constitutes a top-notch mission for the prevention and control of this pathogen.
Because of the accelerated issues of antibiotics as antibiotic resistance, direct toxicity, hyper sensitivity and fantastic infection, the consumers wanted to take natural compounds so, lactoferrin has attracted more attention because Food and Drug Administration (FDA) licensed the Lactoferrin (LF) as " Generally Recognized as Safe (GRAS)" [9].
Lactoferrin has been the center of attention of extra excessive research. Due to its unique antimicrobial, immune modulatory, and even antineoplastic properties, it looks to have terri c practicable in practical medicine. Nevertheless, a whole lot research and many experiments nonetheless need to be carried out in order to acquire a better understanding of its pastime and interactions and to allow the full and secure utilization of this glycoprotein [10]. Also, lactoferrin proved its ability for immunity enhancement in Egypt, [11].
Forti cation of yoghurt with lactoferrin can add more health bene ts, yogurt is one of the most popular fermented dairy products that have been traditionally consumed for their numerous potential health bene ts [12].
So, the purpose of this study was to detect MDR S. aureus in some milk products. As well as, to assess the antibacterial activity of lactoferrin and its sensorial properties as a bio preservative in yoghurt.

Collection of samples:
A total of 150 samples of dairy products include (yoghurt, ice cream and Damietta cheese (50 each) were collected from supermarkets and dairy shops in Assiut city, Egypt. The samples were collected in sterile separate tubes, labeled and carried on ice tank to be transferred with a minimum delay to the laboratory for bacteriological examination.
2.2 Enumeration and identi cation of Staph. aureus: Enumeration of S. aureus using paired barker agar according to [13]. A loopful from each colony was streaked onto blood agar and mannitol salt agar (MSA) (HiMedia) and incubated aerobically overnight at 37 °C for growth. The typical Staphylococcus spp. of golden yellow with hemolysis and yellow colonies on blood agar and MSA, respectively were picked for further biochemical investigations. The biochemical investigations included microscopic appearance with Gram stain, catalase test, hemolysin test, and coagulase test [14].

Antimicrobial susceptibility test :
The Clinical Laboratory Standards Institute's Kirby-Bauer disc diffusion method was used to determine antimicrobial susceptibility in vitro [15]. Each S. aureus isolate was cultured for 4-5 hours at 37 degrees Celsius in Mueller Hinton broth, and the turbidity was compared to a 0.5 McFarland reference solution. Each isolate's pure broth culture was placed on Mueller Hinton agar with sterile cotton swabs and allowed to dry.
The antimicrobial discs were put and incubated for 24 hours at 37 degrees Celsius. The diameter of the inhibitory zone was measured with a caliper, and the results were documented and interpreted using CLSI criteria [15]. The isolates were tested against 16 antibiotic discs that corresponded to eight different classes of Oxoid-supplied chemotherapeutic drugs. The used antibiotic discs were Penicillin (PEN) Ampicillin

Multiple Antibiotic resistant Index (MARI)
Resistance was judged by the inhibition zone diameter to determine the MAR index that was de ned as a/b, where (a) represents the number of antibiotics to which the isolated strain was resistant and (b) represents the number of all tested antibiotics. [16]. Isolate showing resistance to one agent in at three or more different classes of antimicrobials were considered multi drug resistant (MDR) [17]. Isolates with MARI values of more than 0.2 or 20% were considered highly resistant.
2.5 Molecular con rmation of penicillin resistant S.aureus, MRSA and VRSA isolates: The isolates were sent to the Reference Laboratory for Veterinary Quality Control on Poultry Production in Animal Health Research Institute, Dokki, Giza, Egypt for detection of 23S rRNA gene , blaz gene, mecA gene and vanA gene. Characteristic to S. aureus, penicillin resistance, methicillin resistance and vancomycin resistance, respectively. DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modi cations from the manufacturer's recommendations. Primers used were supplied from Metabion (Germany) are listed in table (A) The MIC and minimum lethal concentration (MLC) of lactoferrin were carried out using broth dilution method, as described by [22]. Lactoferrin was purchased from Canada, lactovegetarian, EN: 131947. Item number AOR 04110. The following concentrations of lactoferrin solution were prepared, 0.65, 1.25, 2.5, 5, 10, 20 and 40mg/ml in distilled water and sterilized by 0.45 mm lter and freshly used. Genotyped strains of MDR S. aureus were sub-cultured onto 5% sheep blood agar plates and incubated aerobically at 37 °C for 24h. Selected 3-4 colonies and inoculated in tryptic soy broth then incubated at 37 °C for 2-6 h. Suspensions turbidity was adjusted to match of 0.5 McFarland standard then diluted to obtain a nal concentration of 10 5 CFU /ml approximately. Two-fold serial of lactoferrin (0.65, 1.25, 2.5, 5, 10, 20 and 40 mg/ ml (w/v) were prepared separately using sterile Muller Hinton broth. Each tube was inoculated with a suspension of the 100 µL from CFU/ml.
The inoculated tubes together with the control tube (tubes contained broth only) were incubated aerobically at 37 °C for 24h. The MIC of lacoferrin was detected by lowest concentration of lacoferrin that inhibits growth of the organism with lack of visible turbidity. To determine the MLC, 100 µL from each clear tube (no visible growth) was spread onto sterile Muller Hinton agar (Oxoid, UK) for 24 hours incubation. MLC was detected as the lowest concentration of LF that killed the tested MDR Staphylococcus organisms (No growth on the plate). The mean MIC and MLC was recorded from triple readings in each test.

Agar well diffusion test:
The antibacterial test was conducted using the agar diffusion method summarized as the following. A concentration of 10 5 CFU /ml bacterial strain culture of MDR S. aureus was prepared and spread uniformly on the dried surface of a MHA plate by using sterile cotton swab. Multiple wells of 6 mm were made in the agar plate by using sterile cork pourer 50 µL of LF were added to each of the wells containing concentrations of 0.65, 1.25, 2.5, 5, 10, 20 and 40mg/ml The plates were incubated for 24 h at 37ºC ± 1ºC, under aerobic conditions. After incubation Inhibition of the bacterial growth was measured in mm. The tests were made in triplicate [23].
2.8 Anti-bacterial properties of lactoferrin on MDR S. aureus in yoghurt:

Bacterial suspension inoculation:
A fresh culture of MDR S. aureus isolate was adjusted to give an initial count of approximately a MacFarland 0.5 standard. The growth density was adjusted to match (4.5 log). [24].

Yoghurt preparation:
Raw milk was once boiled for 10 min. then unexpectedly cold and inoculated with 2% yoghurt way of life at 45ºC. One ml of MDR S.aureus pressure suspension (which organized as before) blended with 100 ml of organized milk and divided into suitable sterile jars, lactoferrin was once added at concentrations 10, 20 and 40 mg/ ml for every respectively, and every other tremendous control jars except lactoferrin incubated at 40 ºC until curdling. Control jar (free from pressure suspension, lactoferrin as a terrible control) used to be additionally stored. The jars have been saved at refrigerator temperature (5±2 ºC). The inoculated jars have been examined bacteriologically for the remember of S.aureus using Baird-Parker media (37ºC for 24-48h) at time zero, after curdling and, every 2 days until the cease of the experiment.

Sensory evaluation:
Control yoghurt jars (free from the preceding microorganism however inoculated with lactoferrin concentrations of 10, 20 and 40mg/ml respectively) had been prepared as beforehand mentioned and every one was once subjected to the preceding treatments. Thirty panelist had been selected in groups of one-of-a-kind ages, intercourse (18 females and 12 males), and education to style the trials. The grasp of shoppers toward samples with a range of conc. of lactoferrin was once studied with recognize to three one-of-a-kind attributes (odor, taste and over all acceptability (OAA) [25]. The stage of settlement used to be scored as percentages.

Statistical Analysis
The statistical analysis was performed using programs GraphPadPrism 5.04 (GraphPad, Inc., San Diego, USA) and Statistical 12.0 (Dell, Inc., Tulsa, USA). The bacterial count represented by mean ±SE. The data represented by using the Microsoft Excel Spreadsheet.

Discussion
Staphylococcus aureus is a versatile bacterium that causes a wide range of diseases in humans and animals. It causes mild skin infections to life-threatening disorders including pneumonia and meningitis [26]. Milk and milk products are known to be a source of S. aureus contamination, whether gathered from mastitis-affected cows or food workers who have the organism due to poor personal hygiene. Therefore, this study investigated the presence of S. aureus in some dairy products in Assiut city, Egypt. S. aureus could be detected in Yoghurt, Ice cream and Damietta Cheese samples at percentages of 38, 14 and 30% respectively. The staphylococcus count ranged from 2.6 to 4.3 with a mean value of 3.5±2 log 10 cfu/ g in yoghurt samples while in ice cream samples ranged from 1.6 to 3.3 with a mean value 2.9±1.5 log 10 cfu/ g and in Damietta Cheese samples it ranged from 1 to 4.3 with a mean value 3.2±2.1 log 10 cfu/ g as shown in Table (1). These results were agreed with those reported by [27,28,29]. But lower than [30] and higher than [31,32]. The high incidence in raw milk could be attributed to environmental pollution, cross contamination between the milk and each other and poor handling during transportation, besides, shedding of S. aureus from infected animals is another cause of contamination of milk and dairy food [33,34].
The massive consumption of antibiotics by human and animals resulted in increased number of bacterial populations' resistance thus creating critical public health problems. For the present study S. aureus isolates showed resistance to most antibiotics tested as shown in Figure ( 1 ).The resistance rate was 100%, 95.1%, 92.7%, 87.8%, 85.4%, 80.5% and 73.2% for tetracycline, penicillin, oxacillin, ampicillin, streptomycin, amoxicillin/clavulanate and neomycin, respectively. For tetracycline our results were agreed with those of [35,36] who their results were 100% and 80%, respectively. For penicillin the present results agreed with results of [37,38] who their results were 94.4%, and 96%, respectively. Moreover, our results for Oxacillin and Ampicillin were nearly similar to with that recorded by [36, 38] (86%) and (93%), respectively. The resistance against large number of antibiotics may be related to long term of sub dose applications and even misuse usage.
The differences in the results of antibiotic resistances among studies may be attributed to antibiotic resistances not only from country to another but also from area to other according to the lows of drug handling and abuse. It is interesting to note that resistance rates were observed against marbo oxacin, ceftiofur and vancomycin was (0.00%, 2.4% and 7.3%, respectively. These results were in agreement with that obtained by [39,40,41,42]. The highly e cacy of these antibiotics may be attributed to their recent use and expensive costs in veterinary medicine. Determination of the multiple antibiotic resistance index (MARI) of the isolates shows that most of isolates were resistant to three or more antibiotics with over all mean value about 0.54. This is nearly agreed with previous work of [43,44] who cited that MARI was 0.61 and 0.48, respectively. On the other hand lesser index was reported by [45,42] as 0.23 and 0.3, respectively. The high MARI con rming that there were high antibiotic misuses in such area or country.
The use of PCR considers an excellent method for diagnosing of suspected pathogen due to its rapid and sensitive results [46]. Suspected isolates which show multi drug resistance phynotyically were examined by PCR for genotypically assessments of 23S rRNA gene, blaz gene, mecA gene and vanA gene. Our results clari ed that all tested isolates were harbored 23S rRNA as in Photo (1).
Penicillin used to be notably very positive in opposition to most staphylococcal infections, but S. aureus started out producing βlactamase enzyme in the mid1940s, which destroys the penicillin β-lactam ring. Later, more than 90% of S. aureus strains had been penicillin resistant [47]. So, to detect the resistance to penicillin through the production of β-lactamase due to the presence of blaz gene that coded for an alteration of penicillin-binding protein 2a which reduced the a nity for β-lactam antibiotics [48].
Results obtained in this study represent resistance to penicillin by using blaz gene in (60%) of examined MDR isolates, Bands with approximate size of 833 bp were detected for blaz gene. Photo 2. In agreement with our study the blaz gene was detected in 59.2%, 60, 65 and 97% [49,50,51,52] Christine et al.,2021 . High blaz genes might indicate an increased use, and possibly misuse of β lactams in the study farms [53,5,54] encoding for the beta-lactamases blaz gene in staphylococci at 42.9% and 28.8% of S.aureus isolates, respectively.
Antimicrobial resistance in methicillin-resistant strains of S.aureus (MRSA) is related with the acquisition of a cell genetic element referred to as the staphylococcal cassette chromosome mec, which carries the mecA gene, encoding the low-a nity penicillin-binding protein 2a and confers resistance to the β-lactam antibiotics [55]. So, our outcomes revealed the presence of mecA gene in (40%) of the examined samples. Bands with approximate size of 310 bp had been detected for mecA gene, Photo (3). These were lower than [29,51] who revealed that the presence of mecA gene was 66.7 and 75% of MDR S. aureus, respectively. Also [49,50] who found a very high ratio of MRSA reach at 77% to 100%.
Glycopeptides such as vancomycin has become the cornerstone for treating MRSA infections over the ultimate twenty years [56]. Conceivably, the excessive utilization of vancomycin outcomes in the alarming signi cant of its resistance and the existence of two kinds of glycopeptide resistant S. aureus. The rst type, vancomycin intermediate resistant S. aureus (VISA) [57]. The 2nd one, vancomycin-resistant S. aureus (VRSA) is related with the acquirement of vanA operon from Enterococcus faecalis by a horizontal gene transfer [58].
Vancomycin has been an effective agent in opposition to the MRSA infections for decades [7]. But in July 2002, the situation changed when the Centers for Disease Control and Prevention (CDC) in the USA documented the rst sample of S.aureus that used to be resistant to both vancomycin and methicillin [59]. In the present work the examined isolates showed 20% vancomycin resistant by using vanA gene detection. Bands with approximate dimension of 885 bp had been detected for vanA gene as in Photo (4 The presence of resistance to many antimicrobials, emphasizing the need for new herbal anti-microbial retailers to deal with S. aureus infection, Lactoferrin is a protein that takes place naturally in milk and nowadays is increasingly supplemented in foods for its a couple of features and its application into meals upkeep is gaining superb interest due to consumers' vogue [ The antibacterial activity of Lactoferrin was performed using agar well diffusion method as in Table (2). The results revealed that the concentration of 40 mg / ml gave the highest observed zone of inhibition (29± 1.7 mm diameter) followed by 20 mg and 10 mg as 22± 2.1mm and 13.4±2.7 mm, respectively. Our nding (22± 2.1mm) was similar to that recorded by [66,67]. The latter authors cited that inhibitory effect of camel LF against E. coli and S. aureus was observed to be the very potent. According to zone diameter interpretation charts of [15] Marbo oxacin (>24 mm), Ceftiofur, (>26mm) and Vancomycin (>18mm) which consider the highly susceptible antibiotics against MDR Staphylococci as in Figure (1). In Table (2) 40 mg/ml LF come superior than the highly susceptible previously mentioned antibiotics against MDR Staphylococci in regarding to zone of inhibition diameter. The good results of LF may be due to it exhibits a broad spectrum antibacterial activity through main mechanisms, its iron chelating capacity that reduce the iron by bacteria, and binding to the bacterial membrane, changing its stability and consequently, the viability of bacteria [69].
As shown in Figure 2 we used three concentrations of lactoferrin 40, 20 and 10mg/ml. 40 mg/ml reduced the count of S.aureus at 1 st day and completely inhibit its growth at 2 nd day but 20 and 10mg/ml inhibit S.aureus growth at 4 th and 6 th day, respectively. While in positive control S.aureus could survive in yoghurt till the 6 th day with a mean count 2.4 log 10 CFU/gm, and that may be due to increase the acidity of yoghurt, S.aureus organisms are the most sensitive bacterial species to acidity [70]. Moreover, [71] cited that increased degree of LF, the bacterial load reduced dramatically in contrast to the control, thereby growing the shelf life of some dairy product. The results of sensory evaluation of the present study revealed that the samples treated with different concentrations of lactoferrin were generally acceptable; moreover the samples treated with 10, 20 and 40mg/ml concentrations of lactoferrin were given the highest score during the whole period of experiment as shown in Figure (3).Addition of bovine LF to yoghurt did not substantially affect the physicochemical properties of the product and increased the growth of lactic acid bacteria [72]. Also, [11] found that forti cation of milk with lactoferrin did not interfere with yoghurt manufacture and the sensory properties of produced yoghurt was acceptable.

Conclusion
The present study concluded that most of the isolated S. aureus in dairy products are MDR against several antibiotic groups. A high prevalence of S. aureus in milk and some milk products is considered a public health hazard and might be involved as a cause of high prevalence of human food poisoning. Moreover, LF considers a suitable food preservative in yoghurt due to powerful antibacterial activity and its good sensorial properties.