Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract: An alternative approach for the treatment of staphylococcal bovine mastitis

Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract: An alternative approach for the treatment of staphylococcal bovine mastitis

    Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract: An alternative approach for the treatment of staphylococcal bovine mastitis Auemph...

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    Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract: An alternative approach for the treatment of staphylococcal bovine mastitis Auemphon Mordmuang, Supayang Piyawan Voravuthikunchai PII: DOI: Reference:

S0034-5288(15)30026-6 doi: 10.1016/j.rvsc.2015.07.018 YRVSC 2925

To appear in: Received date: Revised date: Accepted date:

23 May 2014 6 July 2015 26 July 2015

Please cite this article as: Mordmuang, Auemphon, Voravuthikunchai, Supayang Piyawan, Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract: An alternative approach for the treatment of staphylococcal bovine mastitis, (2015), doi: 10.1016/j.rvsc.2015.07.018

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ACCEPTED MANUSCRIPT Rhodomyrtus tomentosa (Aiton) Hassk. leaf extract: An alternative approach for the treatment of

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Auemphon Mordmuang, Supayang Piyawan Voravuthikunchai *

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staphylococcal bovine mastitis

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Department of Microbiology and Excellent Research Laboratory on Natural Products, Faculty of Science and Natural Product Research Center of Excellence, Prince of Songkla

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University, Hat Yai, Songkhla 90112, Thailand

*Corresponding author. Tel./fax.: +66−74−446661

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Email address: [email protected] (S.P. Voravuthikunchai)

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ACCEPTED MANUSCRIPT Abstract

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Antibiotic residues in dairy products as well as emergence of antimicrobial resistance

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in foodborne pathogens have been recognized as global public health concerns. The present

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work was aimed to study a potent antibacterial extract from natural product as an alternative treatment for staphylococcal bovine mastitis. Staphylococcal isolates (n=44) were isolated from milk samples freshly squeezed from individual cows. All staphylococcal isolates were

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resistant to ampicillin, ciprofloxacin, erythromycin, gentamicin, penicillin, except

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vancomycin. Rhodomyrtus tomentosa leaf ethanolic extract was accessed for its antibacterial activity and anti−inflammatory potential. The extract exhibited profound antibacterial activity against all of staphylococcal isolates with MIC and MBC values ranged from 16−64 µg/ml

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and 64−>128 µg/ml, respectively. Moreover, the extract also exerted anti−protein denaturation and human red blood cell membrane stabilizing activity. The results support the

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use of Rhodomyrtus tomentosa extract could be applied to cure bovine mastitis and to reduce

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inflammatory injury caused by the bacterial infections.

Keywords: Antibacterial agent, Anti−inflammatory, Bovine mastitis, Rhodomyrtus tomentosa, Staphylococcus aureus

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ACCEPTED MANUSCRIPT 1.

Introduction

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Bovine mastitis is a frequent cause of economic loss in dairy cows, which adversely

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affects animal health, impinges on quality of milk, and reduces milk production. The disease

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is defined as inflammation of mammary gland tissue. It is also characterized by abnormalities of udders and changes in milk composition including increase in somatic cell counts (Galal Abdel Hameed et al., 2006). Mastitis begins after organisms pass through the teat canal.

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Invasion of the teat usually occurs during milking. Major mastitis causing microorganisms

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are coagulase−negative staphylococci, Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Corynebacterium sp., and Klebsiella sp. (Sharma et al., 2012). Staphylococcus aureus is one of the most common mastitis pathogens causing

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contagious and environmental mastitis (Saidi et al., 2013). It is associated with sub−clinical and clinical bovine mastitis. Recently, S. aureus has been recognized as facultative

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intracellular pathogen by its abilities to adhere and internalization within bovine mammary epithelial cell and phagocytic cells (Atalla et al., 2010). It can produce adherence factors and

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virulence toxins involved in pathogenesis of bovine cells such as endotoxin, exotoxin, α−toxin, haemolysin, etc. Intramammary infection of S. aureus is often causing tissue damage and inflammatory process in host (Craven et al., 2009). Moreover, it is also known as one of the most important agents in dairy products by accidentally mixed into bulk milk and can be transmitted to human (Sasidharan et al., 2011). Emergence of antibacterial resistance in foodborne pathogens has increased mainly as a result from irrational antibiotics used in livestock. The problem of S. aureus bovine mastitis is becoming worse due to widespread of multidrug−resistant strains (Sampimon et al., 2011). There have been reports on staphylococcal isolates obtained from mastitis resistant to existing antibiotics (Unakal and kaliwal, 2010; Wang et al., 2013). Antibiotic residue and disinfectant

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ACCEPTED MANUSCRIPT agents accumulate in milk due extensive use in the treatment and control of the disease. It adversely affects environment, animal, human health, economy, and international food trade

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(Galal Abdel Hameed et al., 2006). Several non−traditional antimicrobial agents or medicinal

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plants have been tested in the recent years, in order to reduce the settlement of antibiotic

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residue in milk. Rhodomyrtus tomentosa (Aiton) Hassk. is a plant species belonging to the family Myrtaceae. Many recent studies have reported that ethanolic extract of Rhodomyrtus tomentosa leaves has pronounced effect against Gram−positive bacteria such as S. aureus, S.

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epidermidis, methicillin−resistant S. aureus (MRSA) (Saising et al., 2008), S. pyogenes, S.

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pneumoniae, S. mutans, Enterococcus faecalis, Bacillus subtili, and B. cereus (Limsuwan et al., 2009; Voravuthikunchai et al., 2010). However, anti−inflammatory potential of the

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ethanolic extract has not been studied.

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Therefore, the present work was aimed to further investigate the ability of Rhodomyrtus tomentosa extract to eradicate staphylococci in bovine mastitis as well as its

Materials and methods

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anti−inflammatory potential.

2.1. Bacterial isolation and culture condition

Staphylococcal isolates (n=44) were isolated from milk samples freshly squeezed from individual cows in dairy farms located in Phatthalung province, Thailand. Identification of isolates was based on colony characteristics on mannital salt agar (MSA, Merck, Germany), Gram staining, and catalase reaction. Coagulase test was used to differentiate between coagulase−positive and coagulase−negative staphylococcal isolates. Out of 44 isolates, 18 and 26 isolates were identified as coagulase−positive and coagulase−negative

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ACCEPTED MANUSCRIPT staphylococci, respectively. In addition, S. aureus ATCC 29213 was included as a reference strain. All bacterial isolates were cultured on tryptic soy agar (TSA, Becton Dickinson, Difco,

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France) at 37 °C for 18−24 h.

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2.2. Antibiotic susceptibility testing

All staphylococcal isolates were tested for susceptibility to antibiotic drugs by agar

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disk diffusion method according to Clinical and Laboratory Standards Institute guideline

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(CLSI, 2011). Six antibiotics were included in this study, which are most prescribed and used for treatment of bovine mastitis in dairy farms. Aminoglycosides were represented by

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gentamicin (10 µg), fluoroquinolones by ciprofloxacin (5 µg), while beta−lactamic drugs

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were represented by penicillin (10 units) and ampicillin (10 µg). Macrolides were represented by erythromycin (15 µg) and glycopeptide by vancomycin (30 µg). All of the drugs were

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purchased from Oxoid, UK. After measuring the growth inhibition zone, the isolates were categorized as sensitive or resistant by following the recommendations of CLSI (2011).

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Statistically significant difference between the two groups of staphylococci was analyzed using one−way analysis of variance (ANOVA). A probability P−values less than 0.05 was considered different significantly.

2.3. Preparation of Rhodomyrtus tomentosa ethanolic extract

Rhodomyrtus tomentosa leaves were dried in oven at 60 °C for 48 h and ground in an electric blender. Dried leaf powder was extracted with 95% ethanol at room temperature for 7 days. The extract was evaporated using a rotary evaporator (BUCHI Rotavapor R−114, Büchai Labortechnik AG, Swizerland) until it was completely dried and kept at 4 °C until

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ACCEPTED MANUSCRIPT use. The ethanol extract was dissolved in 10% dimethyl sulphoxide (DMSO, Sigma,

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Germany) and used for further study.

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2.4. Antibacterial activity of Rhodomyrtus tomentosa extract

Antibacterial activity of Rhodomyrtus tomentosa ethanolic extract was determined by agar disk diffusion method (CLSI, 2011). Fresh 3−5 colonies of staphylococci on TSA were

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inoculated in Mueller−Hinton broth (MHB, Difco) and incubated at 37 °C for 3−5 h. The

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bacterial suspensions were adjusted to McFarland turbidity standard No. 0.5 and spreaded on Mueller−Hinton agar (MHA, Difco) plates. The ethanol extract was dissolved in DMSO. Ten

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microliter of the extract from the stock solution (250 mg/ml) was loaded onto sterile filter

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paper disk (diameter 6 mm). The disks containing the ethanol extract were placed on seeded plates and incubated at 37 °C for 18 h. The diameters of inhibition zones (mm) were

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measured and the mean was calculated. The disk containing DMSO was used as a negative

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control. The assay was performed in triplicate.

2.5.Determination of minimum inhibitory concentration (MIC)

Antimicrobial activity was measured by determining the minimum inhibitory concentration of the extract needed to inhibit the growth of bacteria, a value called MIC, was evaluated by broth microdilution method according to CLSI (2011). In 96−well microtitre plate, 20 µl of the ethanolic extract was added and diluted by two−fold serial dilution to obtain final concentration ranging from 4−256 µg/ml. Total volume was adjusted to 100 µl by adding 80 µl of MHB in each wells, followed by 100 µl of the bacterial suspensions (106

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ACCEPTED MANUSCRIPT CFU/ml). The plates were incubated at 37 °C for 18 h. Vancomycin was used as a standard

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2.6. Determination of minimum bactericidal concentration (MBC)

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drug. The experiment was performed in triplicate.

Minimum bactericidal concentration (MBC) was determined subsequently to the MIC assays. MBC is the lowest concentration of the extract necessary for the complete

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suppression of bacterial growth. An aliquot of 0.1 ml from the wells with MIC was seeded on

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TSA and incubation at 37 °C for 18 h. The appearance of bacterial growth on the agar plate

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was evaluated for the MBC determination. The experiment was carried out in triplicate.

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2.7. Human red blood cell (HRBC) membrane stabilizing activity

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Membrane stabilizing activity assay was carried out as reported previously (Debnath et al., 2013). Alsever’s solution was prepared by dissolving 2% dextrose, 0.8% sodium

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citrate, 0.5% of 1M citric acid, and 0.42% sodium chloride in distilled water followed by sterilization. Blood was collected from healthy volunteers and mixed with equal volumes of Alsever’s solution. The blood was centrifuged (Centrifugen 5804, Eppendorf, Hamberg, Germany) at 3000 rpm for 20 min. Debris of packed cells was washed with 0.9% sodium chloride and suspended in normal saline solution (0.85% sodium chloride) to attain a suspension density of 6x1012 red blood cells/ml. Rhodomyrtus tomentosa ethanolic extract was tested with concentration ranging from 8−256 μg/ml. The assay mixture contained 1 ml of the extract, 1 ml phosphate buffer, 2 ml hyposaline (0.25% sodium chloride), and 0.5 ml HRBC suspension. Diclofenac sodium was used as a standard drug and 2 ml of distilled water was used as a control which produces 100% of haemolysis. All the assay mixtures were

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ACCEPTED MANUSCRIPT incubated at 37 °C for 30 min and centrifuged at 3000 rpm for 20 min. The hemoglobin content in the supernatant solution was estimated using UV−Visible spectrophotometer

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(GENESYS 10S UV-Vis) at 560 nm. Percentage inhibition of haemolysis was calculated

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using the following equation: (OD of control − OD of treated sample / OD of control) x 100.

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Statistical analysis was performed using t−test for dependent samples. P−values less than 0.05 were considered significant difference between the standard drug and the ethanolic

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2.8. Anti−protein denaturation activity

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extract effect.

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The method of Williams et al. (2008) was employed for anti−protein denaturation

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assay. A solution of 0.2% (w/v) bovine serum albumin (BSA) was prepared in Tris buffer saline and pH was adjusted to 6.8 using 1M glacial acetic acid. Solutions of 8−256 µg/ml of

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Rhodomyrtus tomentosa ethanolic extracts were prepared using DMSO as a solvent. Five hundred microliters of each concentration of the extract was added to tubes containing 4.5 ml

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of BSA solution. The control consists of 4.5 ml of BSA solution and 0.5 ml of 1% DMSO. The standard consists of 0.5 ml diclofenac sodium (8−256 µg/ml) in 4.5 ml of BSA solution. Test mixtures were heated at 72 °C for 5 min and then cooled for 10 min at room temperature. The absorbance of the mixtures was determined by UV−Visible spectrophotometer (GENESYS 10S UV-Vis) at 660 nm. The percentage inhibition of protein denaturation was determined on a % basis relative to the control using the following formula: % inhibition of protein denaturation = 100 − (OD of control − (OD of tested − OD of extract) / OD of control) x 100. The experiment was carried out in triplicate.

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Results

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3.1. Antibiotic susceptibility of staphylococcal isolates

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Antibiotic resistance patterns of staphylococci to representative drugs from different

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antibiotic groups were determined (Fig. 1). Antibiotics used for susceptibility testing include ampicillin, ciprofloxacin, erythromycin, gentamicin, penicillin, and vancomycin. All staphylococcal isolates were resistant to most antibiotics, except vancomycin. Moreover,

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resistance to more than one antibiotic occurred in both of coagulase−positive and

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coagulase−negative staphylococci. Among the coagulase−positive isolates, 8.71%, 6.21%, 3.46%, 13.93%, and 20.05% were resistant to ampicillin, ciprofloxacin, erythromycin, gentamicin, and penicillin, respectively. While 1.41%, 10.72%, 2.71%, 16.29%, and 19.15%

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of coagulase−negative staphylococci were resistant to these antibiotics. No significant

(p>0.05).

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differences were observed in drug resistance patterns between both groups of the bacteria

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3.2. Agar disk diffusion assay

Antibacterial activity of Rhodomyrtus tomentosa ethanolic extract was preliminary tested by agar disk diffusion assay (Table 1). The ethanolic extract at a concentration of 2.5 mg/disk demonstrated strong antibacterial activity against all staphylococci isolates. Coagulase−positive and coagulase−negative staphylococci produced inhibition zones ranging from 8.7−15.5 mm and 11.3−22.2 mm, respectively.

3.3. Determination of MIC and MBC

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ACCEPTED MANUSCRIPT The ethanolic extract possessed strong antibacterial activity against all tested staphylococcal isolates with MIC and MBC values ranged from 16−64 µg/ml and 64−>128

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µg/ml, respectively. Minimum inhibitory concentration of the ethanolic extract required to

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staphylococcal isolates (MIC90) was at 64 µg/ml (Table 2).

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against the growth of 90% of both coagulase−positive and coagulase−negative

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3.4. Human red blood cell (HRBC) membrane stabilizing assay

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Prevention of HRBC lysis was taken as evaluation of anti−inflammatory activity of Rhodomyrtus tomentosa ethanolic extract and standard diclofenac sodium. This study was observed that the hypotonicity induced HRBC lysis was inhibited by the agents in a

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concentration dependent manner (8−256 µg/ml). Significant differences (p>0.05) were observed in stabilization activity between the ethanolic extract and standard drug except the

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activity at a concentration of 32 µg/ml (Fig. 2). The maximum percentage inhibition of haemolysis of the ethanolic extract and the standard drug was found to be 97% at

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concentration 8 µg/ml and 256 µg/ml, respectively (Fig. 2).

3.5. Anti−protein denaturation assay

Anti−inflammatory activity of the ethanolic extract was further evaluated against heat–induced denaturation of bovine serum albumin. The result is given in Table 3. The inhibition of protein denaturation of the ethanolic extract and standard diclofenac sodium (8−256 µg/ml) was concentration dependent, since increasing concentration the activity was also increased. The inhibitory activity of the standard drug was found to be less activity compared with the activity of the ethanolic extract in each concentration. The percentage

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ACCEPTED MANUSCRIPT inhibition of protein denaturation of the ethanolic extract and the standard drug were ranged

Discussion and conclusions

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from 11.30−97.47% and 9.92−88.28%, respectively (Table 3).

In this study, out of 44 isolates of staphylococci obtained from the milk samples, eighteen isolates (40.9%) were identified as coagulase−positive and twenty−six isolates

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(59.1%) as coagulase−negative. This finding agrees with earlier studies which reported that

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coagulase−negative staphylococci was predominant over coagulase−positive staphylococci (Taponen and Pyörälä, 2009). Although, coagulase−negative staphylococci infection is

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relatively widespread and common in subclinical cases, the infection is transient in clinical

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and chronic cases (Waller et al., 2011).

In veterinary care, β−lactam antibiotics were usually recommended for the treatment

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of herds with udder health problems as well as bovine mastitis. However, extensive use of antibiotics may lead to resistance development in causative pathogens. This has become an

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important public health concern. There is increasing evidence suggesting that the development of antibiotic resistance in staphylococci causing bovine mastitis due to the presence of β−lactamase enzyme produced by the bacteria (Keserű et al., 2011). The results from the present study demonstrated that staphylococcal isolates were susceptible to most antibiotics tested. Coagulase−negative staphylococci were more susceptible to antimicrobial drugs than coagulase−positive staphylococci. However, approximately 20% of both coagulase−positive and coagulase−negative staphylococci were resistant to penicillin. Our results were similar to Swedish study which reported 7.1% of S. aureus and 12.5% of coagulase−negative staphylococci were resistant to penicillin due to β−lactamase production (Bengtsson et al., 2009). Interestingly, a study of phenotypic penicillin, oxacillin and

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ACCEPTED MANUSCRIPT macrolide−lincosamide compounds resistance of coagulase−negative staphylococci in Netherlands revealed that 40.6% of the isolates exhibited resistance to a single compound or

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a single class, and 10.6% of the isolates were resistant to multiple drug classes (Sampimon et

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al., 2011).

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Medicinal plants remain sources of natural compounds that have been studied extensively as alternative antimicrobial therapeutic agents employed to reduce the problems associated with antibiotic−resistant bacteria and drug residue in milk. Previous studies have

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shown the efficacy of plant extracts against many pathogens. Methanol extract of Cenchrus

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ciliaris and Coccinia grandis showed antibacterial activity against S. aureus causing mastitis with MIC values of 125 µg/ml (Mubarack et al., 2011). Moreover, natural compounds from

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certain plants including carvacrol (Nostro et al., 2007), salvipisone (Kuźma et al., 2007), and

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berberine (Wang et al., 2009) have been demonstrated to exert effects on staphylococci with MIC at 0.015−0.031% v/v, 9.37−18.75 μg/ml, and 64−256 μg/ml, respectively.

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In comparison to other study, the present study demonstrated the potential antibacterial activity of Rhodomyrtus tomentosa ethanolic extract against staphylococci

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isolated from bovine mastitis with MIC values ranging from 16−64 µg/ml. Rhodomyrtone, a principal compound isolated from the plant extract has been suggested to play an important role in the prospective antibacterial effect (Saising et al., 2008; Sianglum et al., 2011). Pertaining to the effect of rhodomyrtone on the bacterial cells, earlier report with proteomic analysis suggested that this compound might have effects on proteins associated with cell wall biosynthesis and cell division, protein degradation, stress response and oxidative stress, cell−surface antigen and virulence factors, as well as various metabolic pathways (Sianglum et al., 2011). According to general knowledge, degeneration of protein is one of the important factors causing inflammations. Rhodomyrtus tomentosa ethanolic extract exhibited protection

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ACCEPTED MANUSCRIPT of protein denaturation activity in a concentration dependent manner, comparable to that of standard diclofenac sodium. Anti−inflammatory potential of the extract was further evaluated

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by human red blood cell (HRBC) membrane stabilization assay. In this study, the ethanolic

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extract demonstrated the maximum percentage inhibition of HRBC lysis of 97% at 8

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µg/ml.The red blood cell membrane structure is analogous to lysosomal membrane. Thus, the inhibition of haemolysis implies that the ethanolic extract may as well stabilize lysosomal membrane and prevent the release of lysosomal complements of activated host inflammatory

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response. Phenolic compound and flavonoids are important plant constituents that display

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beneficial effects on antioxidant and anti-inflammatory activities (Tsai et al., 2014). In addition to rhodomyrtone, other chemical constituents including phenolic compounds,

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flavonoids, triterpenoids, steroids, and tannins have been isolated from Rhodomyrtus

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tomentosa (Hui et al., 1975; Liu et al., 1998). The presence of these contents in this plant suggests that it may be responsible for anti−inflammatory property of the Rhodomyrtus

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tomentosa ethanolic extract.

In conclusion, Rhodomyrtus tomentosa extract possesses remarkable antibacterial

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activity against staphylococci. Further study demonstrated that the ethanolic extract also exerts anti−inflammatory potential. The results support the use of Rhodomyrtus tomentosa extract as an alternative agent against staphylococcal bovine mastitis and could be applied to reduce inflammatory injuries and tissue damages caused by bacterial infections.

Acknowledgements

This work was supported by Research and Researcher for Industries, Ph.D. program, The Thailand Research Fund (Grant No. PHD 56I0006, Fiscal year 2013−2018), the Higher Education Research Promotion and National Research University Project of Thailand, Office

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ACCEPTED MANUSCRIPT of the Higher Education Commission, Ministry of Education, and TRF Senior Research

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Scholar, The Thailand Research Fund.

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Medical Journal 57, 327−331.

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ACCEPTED MANUSCRIPT Figure legends

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Fig. 1. Antibiotic susceptibility profiles of staphylococci isolates, black bars and white bars

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represent antibiotic susceptibility of coagulase−positive (n=18) and coagulase−negative

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staphylococci (n=26), respectively. The values obtained from three experiments, performed in triplicate (mean±SD).

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Fig. 2. Effects of Rhodomyrtus tomentosa ethanolic extract on hypotonicity−induced human

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red blood cell (HRBC) lysis. Black bars and gray bars represent the percentage HRBC membrane stabilization activity of the ethanolic extract and diclofenac sodium, respectively.

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Each symbol indicates the means ± SD for at least duplicates. * and ** values are

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significantly different between the standard drug and the ethanolic extract ( p<0.05).

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Fig. 1

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Fig. 2

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Table 1 Antibacterial activity of Rhodomyrtus tomentosa ethanolic extract at concentration 2.5 mg/disk against coagulase−positive and coagulase−negative staphylococci isolated from bovine mastitis

Number of isolates (%)

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Coagulase−positive (n=18)

11−15

88.89

16−>20

0.00

Coagulase−negative

0.00 88.46 11.54

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11.11

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(n=26) ≤10

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Inhibition zone (mm)

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ACCEPTED MANUSCRIPT Table 2 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Rhodomyrtus tomentosa ethanolic extract and vancomycin against coagulase−positive and coagulase−negative staphylococci isolated from bovine mastitis

Rhodomyrtus tomentosa ethanolic extract

vancomycin

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Tested isolates

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Antibacterial agent concentration (µg/ml)

MIC90

MBC

MIC

MIC90

MBC

coagulase−positive staphylococci (n=18)

32−64

64

>128

0.5−1

1

0.5−2

coagulase−negative staphylococci (n=26)

16−64

64

0.25−1

1

0.5−2

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MIC

64−>128

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S. aureus 64 − 128 0.5 − ATCC 29213 MIC90; Minimum inhibitory concentration required to inhibit the growth of 90% of staphylococci isolates

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ACCEPTED MANUSCRIPT Table 3 Effect of Rhodomyrtus tomentosa ethanolic extract and diclofenac sodium on inhibition of bovine serum albumin denaturation

11.30 ± 0.77 30.33 ± 1.65 65.12 ± 2.81 87.05 ± 0.29 94.31 ± 4.33 97.47 ± 1.72

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8 16 32 64 128 256

9.92 ± 1.00 16.68 ± 0.60 31.41 ± 0.23 61.51 ± 1.40 75.97 ± 0.49 88.28 ± 0.75

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The value expresses as mean ± SD.

Diclofenac sodium

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Rhodomyrtus tomentosa ethanolic extract

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% inhibition of protein denaturation

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Concentration (µg/ml)

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ACCEPTED MANUSCRIPT Highlights  This finding provided evidence for prevalence of staphylococcal bovine mastitis.  R. tomentosa extract possessed high antibacterial potency against staphylococci.  The extract showed anti−protein denaturation activity and HRBC stabilization.

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 The extract could be used to reduce inflammatory injury due to bacterial infection.

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 The extract could be applied as an alternative agent against bovine mastitis.

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