Research Article

Efficiency of Lactoferrin to Eradicate Multidrug Resistant Staphylococcus aureus Isolated from some Dairy Products

Sayed H. Al Habty1, Dina N.Ali2*

1Bacteriology department, Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt; 2Certified Food Hygiene Lab., Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt.

Received | October 24, 2022; Accepted | November 15, 2022; Published | December 12, 2022 ,

*Correspondence | Dina N Ali, Certified Food Hygiene Lab., Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt; Email: [email protected]

Citation | Al Habty SH, Ali DN (2023). Efficiency of lactoferrin to eradicate multidrug resistant staphylococcus aureus isolated from some dairy products. Adv. Anim. Vet. Sci. 11(1): 35-44.

DOI | http://dx.doi.org/10.17582/journal.aavs/2023/11.1.35.44

ISSN (Online) | 2307-8316

 

 

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Staphylococcus aureus is a significant and costly public health concern since it may enter the human nourishment chain and usually causing foodborne illness (Liu et al., 2022). Milk and milk products are known to be a source of S. aureus contamination whether they are collected from dairy animals enduring mastitis or from food handlers carrying the organism because of poor individual cleanliness (Bingol et al., 2012).

Moreover, the huge extent of Multidrug Resistant (MDR) S. aureus isolates may represent an open wellbeing chance due to the spread of drug-resistant zoonotic S. aureus (Gebremedhin et al., 2022).

The development of such resistant strains plays a fundamental position in restorative disappointment in each human and animal disease. The uncontrolled utilization of antibiotics in human and animals, in conjugation with terrible demonstrative methods and improper endorsing by way of unfit doctors, exacerbates the problem and constitutes a top-notch mission for the avoidance and control of this pathogen (Kimang’a, 2012). So, the consumers wanted to take natural compounds, an effective and non-antibiotic antimicrobial agent. Lactoferrin (LF) proved to be the goal of this concept since, it is a multifunctional protein to inhibit the growth of planktonic MDR S. aureus form (Sinha et al., 2013, Reznikov et al., 2018) or its quorum form (biofilm formation) (Ammons and Copié, 2013, Quintieri et al., 2020) rather than modulating the host immune status. Thus, it is recommended to be as a food additive (Duran and Kahve, 2017) especially those dairy foods.

So, the reason for this study was to detect MDR S. aureus in some dairy products. As well as, to assess the antibacterial activity of lactoferrin and its sensorial properties as a bio preservative in yogurt.

Materials and methods

Collection of samples

150 samples of dairy products items (yogurt, ice cream and Damietta cheese) 50 of each were collected from general stores and dairy shops in Assiut city, Egypt in sterile partitioned tubes, named and carried on ice box to be exchanged with the least delay to the research laboratory for bacteriological examination.

Identification and Enumeration of Staph. aureus:

From each sample, 1 gm was inoculated into 9 ml saline, and shacked well to adopt 10-fold serial dilution process, then a loopful was streaked on Baird Parker (Oxoid) agar for enumeration (ISO 6888-1: 2021) and suspected colonies (golden yellow with hallow zone) were picked up and sub cultured onto both blood (Difco) and mannitol salt (HiMedia) agar. According to Quinn et al. (2011), the suspected colonies were subjected to the biochemical affirmation (catalase, hemolysin, and coagulase tests).

Antimicrobial susceptibility test

It was carried out by Kirby-Bauer disc diffusion method (CLSI, M100 2020) utilizing Muller Hinton agar where each strain was tried against 10 antimicrobial discs; penicillin (PEN) ampicillin (AMP), oxacillin (OXA), amoxicillin /clavulanate (AMC), tetracycline (TET), neomycin (N), streptomycin (S), marbofloxacin (MAR), cefotaxim (CTX)and vancomycin (VA) (Oxoid,). The breadth of the inhibitory zone was measured with a caliper, and the comes about were recorded and deciphered using CLSI criteria.

Multiple Antibiotic resistant Index (MARI)

Resistance was calculated to decide the MARI that was defined as a/b, where (a) spoken to the number of antibiotics to which the isolated strain was resistant and (b) spoken to the number of all tested antibiotics. (Kumar et al., 2012). The isolate that appeared resistant to three or more distinctive classes of antimicrobials was considered multi drug resistant (MDR) (Magiorakos et al., 2012). Isolates with MARI values of more than 0.2 were considered exceedingly resistant.

Molecular genotyping study

Was done on isolated MDR S. aureus strains for the detection of 23S rRNA, blaz, mecA and vanA genes in the Reference Research Laboratory for veterinary quality control on poultry production in Animal Health Research Institute, Dokki, Giza, Egypt. DNA extraction from samples was performed utilizing the QIAamp DNA scaled-down unit (Qiagen, Germany, GmbH) with adjustments from the manufacturer’s suggestions. Primers used were supplied from Metabion (Germany) and are recorded in Table (A).

Detection of minimum inhibitory concentration (MIC) and minimum lethal (MLC) concentration of LF

Against the different isolated strains were carried out using the broth dilution method (Stephen, 2005). Pure Lactoferrin was purchased from Canada, lactovegetarian, EN: 131947, item number AOR 04110. The following concentrations of lactoferrin solution were prepared, (40, 20, 10, 5, 2.5, 1.25 & 0.65 mg/ ml w/v) in distilled water and sterilized by 0.45 mm filter and freshly used. Genotyped strains of MDR S.aureus were sub cultured onto 5% sheep blood agar plates and brooded aerobically at 37 °C for 24h. Chosen 3-4 colonies and inoculated in tryptic soy broth then incubated at 37 °C for 2-6 h. Suspensions turbidity was balanced to coordinate with 0.5 McFarland standards and then diluted to obtain a last concentration of 105 CFU /ml approximately. Bi-fold serial dilution of lactoferrin was prepared separately using sterile Muller Hinton broth. Each tube was injected with a suspension of 100 µL from CFU/ml. The inoculated tubes together with the control positive tube (tubes contained broth only) and negative control (non-inoculated either MDR S. aureus or LF) were brooded aerobically at 37 °C for 24h. The MIC of LF was identified as the most reduced concentration of LF that inhibits the growth of the organism with a 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 of incubation. MLC was detected as the lowest concentration of LF that killed the tested MDR S. aureus organisms (no growth on the plate). The mean MIC and MLC were recorded from triple readings in each test.

In vitro agar well diffusion testing of LF anti MDR S. aureus activity

MDR S. aureus was spread uniformly on the dried surface of a Muller Hinton Agar plate by utilizing a sterile cotton swab. Multiple wells of 6 mm were made within the agar plate by using sterile cork pourer 50 µL of LF were inoculated in each of the wells containing distinctive concentrations. The plates were incubated for 24 h at 37ºC ± 1ºC,

 

Table A: Primers arrangements, target genes, amplicon sizes and cycling conditions.

Target gene	Primers sequences	Amplified segment (bp)	Primary denatu ration	Amplification (35 cycles)			Final extension	Ref.
				Secon dary denatur ation	Ann ealing	Ext ension
S. aureus 23S rRNA	ACGGAGTTAC AAAGGACGAC     AGCTCAGCCT TAACGAGTAC	1250	94˚C 5 min.	94˚C 30 sec.	55˚C 1 min	72˚C 1.2 min.	72˚C 12 min.	Bhati et al., 2016
blaZ	TACAACTGTAA TATCGGAGGG     CATTACACT CTTGGCGGTTTC	833	94˚C 5 min.	94˚C 30 sec.	50˚C 40 sec.	72˚C 50 sec.	72˚C 10 min.	Bagcigil et al. 2012
mecA	GTA GAA ATG ACT GAA CGT CCG ATA A   CCA ATT CCA CAT TGT TTC GGT CTA A	310	94˚C 5 min.	94˚C 30 sec.	50˚C 30 sec.	72˚C 30 sec.	72˚C 7 min.	McClure et al., 2006
vanA	CATGACGTATC GGTAAAATC   ACCGGGCAG RGTATTGAC	885	94˚C 5 min.	94˚C 30 sec.	50˚C 40 sec.	72˚C 50 sec.	72˚C 10 min.	Patel et al., 1997

 

under aerobic conditions. After incubation inhibition of the bacterial growth was measured in mm. The tests were made in triplicate (Elsherif and Ali, 2019).

In vivo testing of LF anti MDR S. aureus activity:

Bacterial suspension inoculation: A fresh culture of MDR S. aureus isolate was adjusted to MacFarland 0.5 standard, where the growth density was adjusted to match (4.5 log) to be standard inoculum strain (Ruparelia et al., 2008).

Yogurt preparation: Yogurt was prepared according to (Zakaria et al., 2020) with slight modification, Raw buffalo’s milk used for yogurt manufacturing was purchased from local markets in Egypt. Lyophilized starter cultures containing rise to blend of Streptococcus thermophilus and Lactobacillus delbrueckii sub spp bulguricus (YoFlex® Express 2.0 Chr-Hansen, Denmark), which in customary utilize in dairy plants, were used. Crude buffalo’s milk was pasteurized at 85 °C for 5 min in a stainless steel two fold coat holder some time recently being cooled to inoculation temperature (42°C). After cooling, one ml of arranged standard inoculum of MDR S. aureus was mixed well and divided into suitable jugs. One of them was cleared out to be as control positive and LF was added to the others with 10, 20 and 40 mg/ml. Then the starter culture was inoculated at a concentration of 1:1000 and the blends were poured into Polyethylene yogurt cartons (200 gm capacity) and kept at 42 °C in an incubator. Then kept at the refrigerator at 4o C. A negative control made up of buffalo milk samples without the addition of LF was made in parallel. The inoculated jars have been examined bacteriologically for the existence of viable inoculated MDR S.aureus by  streaked a loopful onto Baird-Parker plates at 37ºC for 24-48h as time zero experiment, after curdling and every 2 days until the cease of the experiment (6th day).

Sensory evaluation: Control yogurt jars (free from the preceding microorganism however inoculated with lactoferrin concentrations of 10, 20 and 40mg/ml respectively) had been prepared as already said and each one was once subjected to the going before medications. Thirty panelists had been chosen in different ages and instruction to style the trials. Different concentrations of lactoferrin were once studied with recognize to three one-of-a-kind attributes (odor, taste and over all acceptability (OAA) (Fernandes et al., 2008). The arrangement of settlement utilized to be scored as percentages.

Statistical Analysis

The factual examination was performed utilizing programs GraphPadPrism 5.04 (GraphPad, Inc., San Diego, USA) and measurable 12.0 (Dell, Inc., Tulsa, USA). The bacterial count was represented by mean ±SE. The data was represented by utilizing the Microsoft Excel Spreadsheet.

Results

As shown in Table (1) S. aureus could be detected in Yogurt, Ice cream and Damietta Cheese samples at percentages of 38, 14 and 30% respectively. The Staphylococcus aureus count ranged from 2.6 to 4.3 with a mean value of 3.5±2 log 10cfu/ g in yogurt samples while in ice cream samples ranged from 1.6 to 3.3 with a mean value 2.9±1.5 log 10cfu/ g and in Damietta Cheese samples it extended from 1 to 4.3 with a mean value 3.2±2.1 log 10cfu/ g.

 

Table 1: Statistical analytical results of S. aureus examination of examined dairy products.

Samples types	Results of S. aureus counts (log 10cfu/ g)
	Positive Samples		Min.	Max.	Mean ±SE
	No./50	%
Yoghurt	19	38	2.6	4.3	3.5±2
Ice Cream	7	14	1.6	3.3	2.9±1.5
Damietta Cheese	15	30	1	4.3	3.2±2.1
Total	41	27.3	5.2	11.9	9.6±5.6

 

Table (2) declared that the resistance rate was 100%, 95.1%, 92.7%, 87.8%, 85.4%, 80.5% and 73.2% for tetracycline, penicillin, ampicillin, amoxicillin/clavulanate, streptomycin, oxacillin and neomycin, respectively. Resistance rates were observed against marbofloxacin, ceftiofur and vancomycin was (0.00%, 2.4% and 7.3%), respectively. Determination of the multiple antibiotic resistance index (MARI) of the isolates shows that most of isolates were extremely resistant to three or more antibiotics with over all mean value about 0.54.

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 clarified that all tested isolates were harbored 23S rRNA ,blaz, mecA and VanA genes at 100, 60, 40 and 20% as in Photo 1, 2, 3 and 4.

At Table 3, Lactoferrin proved to have antibacterial activity against MDR S. aureus at 10 mg/ml for MIC and 40 mg for MLC. Zone of inhibition for 40 mg was (29± 1.7 mm diameter) followed by 20 mg and 10 mg as 22± 2.1mm and 13.4±2.7 mm, respectively.

 

Table 2: Drug resistance of S. aureus strains isolated from dairy products (n = 41).

Antibiotic	Resistant		Sensitive
	No.	%	N0.	%
Tetracycline TE 30 mcg	41	100	0	0
Penicillin P 10 mcg	39	95.1	2	4.9
Ampicillin AMP 10 mcg	38	92.7	3	7.3
Amoxicillin/clavulanate AMC 20/10 mcg	36	87.8	5	12.2
Streptomycin S 10 mcg	35	85.4	6	14.6
Oxacillin OXA 1mcg	33	80.5	8	19.5
Neomycin N 30 mcg	30	73.2	11	26.8
Vancomycin VAN 30 mcg	3	7.3	38	92.7
Ceftiofur XNL	1	2.4	40	97.6
Marbofloxacin MAR	0	0	41	100
MDRI	0.54

As shown in Figure 1 we used three concentrations of lactoferrin 40, 20 and 10mg/ml. 40 mg/ml reduced the count of S.aureus at 1st day and completely inhibit its growth at 2nd day but 20 and 10mg/ml inhibit S.aureus growth at 4th and 6th day, respectively.

Figure 2 represented that fortification of milk with lactoferrin did not interfere with yogurt manufacture and the sensory properties of produced yogurt were acceptable.

 

Table 3: The inhibitory effect of different concentrations of lactoferrin on MDR S. aureus isolates:

Concentrations	Zone of inhibition (mm)
40mg/ml	29± 1.7
20mg/ml	22± 2.1
10mg/ml	13.4±2.7
5mg/ml	0
2.5mg/ml	0
1.25mg/ml	0
0.65mg/ml	0

 

 

Discussion

S. aureus is one of the foremost important food-borne pathogens universally because it produces different heat stable toxins (Ahmed et al., 2019) and invasive enzymes (Dai et al., 2019). Indeed post pasteurization, this microorganism or its enterotoxins might still stay in pasteurized milk (Rall et al., 2008, Dai et al., 2019) threatening their human consumers. The present work showed that S. aureus was recognized in yogurt, Damietta cheese and ice cream (Table 1). Our comes about were concurred with those detailed by Ibrahim et al. (2015), Ahmed et al. (2019) and Zeinhom and Abed, (2021). But lower than Hanaa and Jehan (2015) and higher than Sasidharan et al., (2011), Mashael and Ashwag (2021).The undesired existence of S. aureus in Egyptian dairy products was recognized by a few thinks about (Hanaa and Jehan 2015, Ibrahim et al., 2015, Ahmed et al., 2019, Zeinhom and Abed, 2021) declaring environmental pollution, cross contamination between the milk and each other or poor handling during transportation.

The human potential hazard will be maximized when the contaminant is MDR S. aureus, so the display investigation was outlined to think about the MDR design of the isolated S. aureus strains. The study showed in (Table 2) the distinctive resistance percentage for tetracycline, penicillin, ampicillin, amoxicillin/clavulanate, streptomycin, oxacillin and neomycin and MDRI were with an overall mean of 0.54. Since, usually nearly agreed with past work of Bakheet et al. (2017) and Sineke et al. (2021) who cited that MARI was 0.61 and 0.48 , respectively. On the other hand lesser file was detailed by Amoako et al. (2019) and Aliyu et al. (2020) as 0.23 and 0.3, respectively. MDRI value just ≥ 0.2 is considered high and might be originated from environments with abuse of antibiotics where resistance developed and spread (Subramani and Vignesh 2012). It is curiously to note that resistance rates were watched against marbofloxacin, ceftiofur and vancomycin was low. These comes about were similar with that gotten by Firouzi et al. (2010), Kroemer et al. (2012), Idriss et al. (2014) and Aliyu et al. (2020). The high efficacy of these antibiotics may be attributed to their recent use and expensive costs in veterinary medicine. The isolated MDR S.aureus were public health hazardous pathogens.

The 23S rRNA sequencing was a powerful instrument for the recognition of S. aureus isolated from the examined samples which clarified that all isolates harbored both 23S rRNA and nuc genes. Bands with approximate size of 1250 bp were detected for 23S rRNA gene (Photo 1).

S. aureus produces an extracellular thermo stable nuclease that encoded by nuc gene; one of the foremost effective recognizing characteristics for S. aureus from other Staphylococcus spp. Therefore, nuc gene was cautioned as a unique marker gene (Sahebnasagh et al., 2014).

Penicillin used to be notably very positive in opposition to most staphylococcal infections, but S. aureus started out producing β-lactamase enzyme in the mid1940s, which destroys the penicillin β-lactam ring. Later, more than 90% of S. aureus strains had been penicillin resistant. (Khan et.al., 2013). So, to distinguish the resistance to penicillin through the production of β-lactamase due to the presence of blaz gene, Bands with approximate size of 833 bp were detected (Photo 2).

In agreement with our study the blaz gene was identified in 59.2% to 65 and 97% (Shukla et al., 2004; Naas et al., 2005; Christine et al., 2021; Ahmed et al., 2018). High blaz genes might demonstrate the expand utilization, and conceivably misuse, of β lactams in the study farms (Yang et al., 2016). Shifeng et al. (2021) and Schmidt et al. (2017) encoding for the beta-lactamases blaz gene in staphylococci at 42.9% and 28.8% of S.aureus isolates.

Antimicrobial resistance in methicillin-resistant strains of S.aureus (MRSA) is related with the securing of a cell genetic element alluded to as the staphylococcal cassette chromosome mec, which carries the mecA gene, encoding the low-affinity penicillin-binding protein 2a and confers resistance to the β-lactam antibiotics (Katayama et al., 2000). 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).

Our results were lower than Zeinhom and Abed, (2021) and Christine et al. (2021) who confirmed the presence of mecA gene in 66.7 and 75% of MDR S. aureus isolates, also (Shukla et al., 2004; Naas et al., 2005) found a really tall extent of methicillin resistant S. aureus (MRSA) strains with rates of 77% to 100%. The presence of mecA in MDR S.aureus have been detailed around the world in numerous previous studies (Kreausukon et al., 2012; Awad et al., 2017; Abed et al., 2018).

Vancomycin has been a viable operator in a restriction to MRSA infections for decades (Yan guang et al., 2020). But in July 2002, the circumstances changed when the Centers for Disease Control and Prevention (CDC) in the USA archived the first sample of S.aureus that used to be resistant to both vancomycin and methicillin (CDC, 2002) So, we examined resistance of vancomycin by using vanA gene and  (20%) of isolates were resist to vancomycin. Bands with approximate dimension of 885 bp had been detected for vanA gene (Photo 4).

Antibiotic have been utilized in animal production during the last decades worldwide resulted in emergence and increment resistance bacteria to antibiotics (Abadi et al., 2019), so, getting rid of food MDR S. aureus is of extraordinary concern. As antimicrobial peptides (AMPs) are considered as promising approaches leading to novel potential antimicrobial drugs (León-Buitimea et al., 2020), LF which is an iron protein found in human and bovine milk is considered as an AMP and multifunctional glycoprotein of the innate immune system (Zarzosa-Moreno et al., 2020) owing to binding to bacteria and their cell wall products (Kell et al., 2020) that called heparan sulfate proteoglycans (HSPGs), which are cell-surface and extracellular matrix macromolecules (Lang et al., 2011). Within the present study, the in vitro broth dilution of LF revealed MIC & MLC values against MDR S. aureus as 10 & 40 mg/ml respectively (Table 3). With agar diffusion method different concentrations of lactoferrin (0.65, 1.25, 2.5, 5, 10, 20 and 40mg/ml) against S.aureus to decide the suitable concentration to be applied in yogurt manufacturing. The results showed that 40mg/ml was the best one while 10mg/ml was the minimum inhibitory concentration.

Several authors have reported antibacterial activity of lactoferrin in vitro against pathogens and reported that lactoferrin prevented the increase of S. aureus population (Murdock and Matthews 2002, Da Silva et al., 2012; Ombarak et al., 2019).

Also, Kutila et al. (2003), illustrated the antibacterial impact of lactoferrin was tried on isolates of (S. aureus) originally isolated from bovine mastitis but in lower concentrations 0.67 mg/ml, 1.67 mg/ml, and 2.67 mg/ml.

Subsequently, the in vivo study was designed to inoculate fresh prepared yogurt with 40 (MLC dose), 20 and 10 (MIC dose) mg/ml, where the MLC dose reduced the count of MDR S. aureus at the 1st day and completely inhibit its growth at the 2nd day, while the (MIC dose) inhibited MDR S. aureus development at the 6th day but 20 mg of LF killed MDR S. aureus growth at the 4th day, respectively. MDR S. aureus strain could survive in the positive control group (induced infected and non LF inoculated) up to the 6th day with a mean count 2.4 log 10 cfu/gm, it could be detected but in reduced count and that may be due to increasing the acidity of yogurt, S.aureus organisms are the most sensitive bacterial species to acidity (Bergdoll and Lee Wong, 2006) declaring that MLC dose was promising to fight the MDR S. aureus in dairy food manufacture and production.

It was once discovered that, as the lactoferrin degree in the item expanded, the bacterial boom reduced dramatically in contrast to the control, thereby growing the shelf life of some dairy products, (Shashikumar and Puranik, 2011).

The antibacterial activity of lactoferrin is mainly via 2 mechanisms, the first involves sequestration of iron in sites of infection, which deprives the bacteria from this nutrient and causes a bacteriostatic effect (Superti, 2020). The second mechanism is the direct interaction between lactoferrin molecule and the infectious microorganism (Latorre et al., 2010).

Lactoferrin interacts with lipopolysaccharide to destroy the outer membrane of Gram-negative bacteria (Leitch and Willcox, 1999). In some cases, positively charged amino acids in lactoferrin can interact with anionic molecules on certain bacterial, viral, fungal, and parasite surfaces, causing cell lysis (Gruden and Ulrih, 2021; Kell et al., 2020; Roseanu et al., 2010).

Through the sensory experiment, since the all groups (control negative as well as the three treated groups) did not alter in odor, texture and over all acceptability, LF is recommended to be added. The addition of bovine lactoferrin to yogurt did not considerably influence the physicochemical properties of this fermented product. However, the growth of Lactic Acid Bacteria (LAB) is upgraded with the addition of LF (Franco et.al., 2010). Also, Zakaria et al. (2020) found that fortifying of milk with lactoferrin did not interfere with yogurt manufacture and the sensory properties of produced yogurt was acceptable.

Conclusion

The study showed that S.aureus is widely prevalent especially in yogurt (38%) which may cause a public health risk due to its widespread consumption and concluded that most of the isolated S. aureus in dairy products are MDR against several antibiotic groups with high MAR index and different prevalence rates (yogurt, cheese and ice cream respectively). LF proved to be a good promising food preservative at a dose ≥ 40 mg/ml and it is considered a suitable food preservative in yogurt due to its powerful antibacterial activity and its good sensorial properties.

Abbreviations

LF: lactoferrin, MRSA: methicillin resistant Staphylococcus aureus, VARSA: vancomycin resistant Staphylococcus aureus, MDR: multi drug resistant.

Conflict of interests

The authors declare that they have no conflict of interests.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Ethics approval

This research did not involve experiments on humans or animals and received the ethical approval of the Animal Health Research Institute, Agriculture Research Center, Egypt.

Availability of data and material

All data generated or analyzed during this study were sent to the journal.

Authors’ contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Dina Nour- Eldin Ali and Sayed Al Habty. The first draft of the manuscript was written by Dina Nour- Eldin Ali and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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