Detection of Methicillin-Resistant Staphylococcus aureus in Clinical and Subclinical Mastitis in Ruminants and Studying the Effect of Novel Green Synthetized Nanoparticles as One of the Alternative Treatments

Mastitis is an important disease in dairy animals worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of clinical and subclinical intramammary infections. In the current study, we isolated bacteria from 150 mastitic milk samples. Multiplex PCR was used to detect the methicillin resistance genes in S. aureus to detect the occurrence of MRSA isolates. Green synthesized titanium dioxide nanoparticles (TiO2 NPs) using aqueous leaf extracts of Artemisia herb Alba (A. herb Alba TiO2 NPs). The antibacterial efficacy of these nanoparticles was evaluated (in vitro and in vivo) against collected MRSA isolates using the disc diffusion method and SPF rats. Out of 150 mastitic milk samples, the frequency of S. aureus was 38 (25.3%), that of E. coli was 45 (30%), that of Klebsiella spp. Was 7 (4.7%), and that of Streptococcus spp. Was 11 (7.3%). Among 38 positive isolates of S. aureus, MRSA was 16 (42.1%) by antimicrobial sensitivity testing (AST) and 14 (38.8%) by multiplex PCR. The MRSA isolates were shown to have 100% resistance to penicillin and methicillin, 87.5% resistance to gentamicin, 50% resistance to cefoxitin and amoxicillin, and 75% resistance to ampicillin and ampicillin/sublactam with low resistance against erythromycin, ciprofloxacin, tetracycline, and levofloxacin by AST, respectively. A. herb Alba TiO2 NP formation was observed by changing the colour from white to dark green. The UV spectrum revealed absorbance peaks at 240–250 nm, and their sizes ranged from 42–66 nm and 11 to 45 nm by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). ‏A. herb Alba TiO2 NP suspensions were evaluated against MRSA, with the highest zone of inhibition (43 ± 0.45 mm) at a concentration of 40 μg/ml. Hematological parameters and histological examination after oral administration of 20 mg/kg of A. herb Alba TiO2 NPs indicated that A. herb Alba TiO2 NPs can be used as a new antimicrobial against resistant bacteria (MRSA) with consideration of the dose and methods of synthesis of plant-based compounds.


Introduction
Mastitis is a global challenge for dairy animals. It is associated with economic losses due to the reduction in milk production and quality because it afects animal health [1,2].
increased, so we are trying to fnd a new antimicrobial therapy for clinical mastitis (CM) and subclinical mastitis (SCM) caused by MDR S. aureus using green synthesis of metal oxide nanoparticles [18,19]. Based on natural plant extracts, TiO2 NPs have broad-spectrum activity against Gram-negative and Gram-positive bacteria and fungi [20].
In this study, the isolation of bacteria in mastitic milk samples from dairy cows, bufaloes, sheep, and goats was determined, and resistance genes in positive isolates of S. aureus causing mastitis were detected using multiplex PCR to evaluate their resistant state (MRSA). We evaluated the antibacterial efcacy of these nanoparticles against isolated MRSA in vitro and in vivo, in order to develop new strategies for reducing the spread of these resistant pathogens using new green synthesized NPs. Tis is the frst work to describe the preparation of A. herb Alba TiO2 NPs and investigate their antimicrobial efect against MRSA.

Ethics Statement.
Te experimental protocols and handling of experimental animals in this study were approved by Ethics Review Committee of National Research Centre, Egypt, under approval number: 19-149.

Collection of Samples and Study Localities.
A total of 150 milk samples were collected from animals sufering from clinical and subclinical mastitis (cows: 55; bufaloes: 32) from Monofa, Giza, Beni Suef, Siwa, Marsa Matruh, and Alexandria in Egypt, from December 2019 to April 2020, as shown in Table 1. Te milk samples were used for the isolation and identifcation of diferent bacteria causing mastitis. Subclinical mastitis cases were detected using the California mastitis test (CMT) by mixing 3 mL milk samples from each quarter and 3 mL CMT reagent using a partitioned plastic paddle, as previously described [21]; then, the selection of animals with clinical mastitis (CM) was based on swelling of the mammary glands and abnormalities in the milk (pus and blood streaks), positive CMT, and decreased milk yield [22]. Te udders of animals were cleaned with water and dried using cotton with 75% ethanol before samples were collected.

Isolation and Phenotypic Identifcation of Bacteria from
Mastitic Milk Samples. Te collected milk sample was kept in a sterile tube and transported to the laboratory within 4 h. Milk samples were cultured on nutrient agar, blood agar, mannitol salt agar, and MacConkey's agar (Oxoid, Hampshire, UK), and then the cultured plates were incubated for 24-48 h at 37°C. Te pure colonies were frst identifed according to their Gram staining, morphological characteristics, hemolysis, and biochemical characteristics as described in [23]. Positive isolates of S. aureus were examined by multiplex PCR to determine their MRSA status [24]. Isolates of S. aureus were kept at −20°C in tryptic soy broth (TSB; Becton Dickinson, Wokingham, UK), containing 20% volume of glycerol. In brief, a pure colony was collected from freshly grown culture into an Eppendorf tube containing molecular-grade water (100 ul), followed by mixing gently. Ten, the mixture was boiled for 10 minutes, cooled for 10 minutes, and centrifuged for 10 minutes at 2400 rpm. Finally, the supernatant was collected as the source for the genomic DNA; it was stored at −20°C until used for multiplex PCR to determine the MRSA status of S. aureus [26].

Preparation of Green Synthesis of A. herb Alba TiO2
NPs. Te green synthesis of TiO2 NPs was prepared according to the methods described in [28]. In brief, for the synthesis of A. herb Alba TiO2 NPs, 25 mL of aqueous extract of A. herb Alba was mixed with 225 mL of a 5 mM (TiO2) solution and incubated for 24 h under light conditions at room temperature. A greenish colour was developed, which confrms the generation of TiO2 NPs. Te prepared TiO2 NPs were separated by centrifugation (15,000×g, 20 min), washed thrice with distilled water, and dried overnight at 120°C to obtain a porous fne powder that was characterized to confrm the success of NP preparation.

Characterization of Artemisia herb Alba-Based TiO2 NPs
(1) UV Visible Spectrophotometer. Te formation and bioreduction of A. herb Alba TiO2 NPs were monitored by a UVvisible spectrophotometer (UV-vis spectra) of synthesized NPs [29] at the National Research Centre (NRC), Egypt.
(2) Electron Microscopy. Te size of TiO2 NPs particles was evaluated using transmission electron microscopy (TEM). Te grid was prepared by placing an aqueous suspension of A. herb Alba TiO2 NPs on a TEM grid and drying in air overnight before imaging. Separate images were taken at magnifcations of 20,000xs to 100,000xs at NRC using a TEM unit; SigmaScan Pro software was used to measure the average size of A. herb Alba TiO2 NPs [30]. In scanning electron microscopy (SEM) analysis, a fne powder of A. herb Alba TiO2 NPs was used. Te images were recorded using SEM at NRC, Egypt, to observe the shape and size of the green synthesized NPs [31].

Antibacterial Activity of A. herb Alba TiO2 NPs.
Te antibacterial activity of synthesized A. herb Alba TiO2 NPs was evaluated against isolated MRSA by using the disc Tey were cultivated on Mueller-Hinton medium and impregnated with sterile discs [32][33][34]. Te discs were flled with A. herb Alba TiO2 NPs with various concentrations (5, 10, 20, 30, and 40 μg/ml), and an empty disc with sterile distilled water was used as a negative control. Inhibition zones were measured after incubation with the inoculated plate at 37°C for 24 h.

In Vivo Experiments to Study the Efect of Artemisia herb
Alba-Based Titanium Dioxide Nanoparticles 2.7.1. Experimental Animals. Twenty adult male and female healthy SPF Webster rats weighing between 150 and 190 g were used in this research. Tey were obtained from the animal house of NRC, Cairo, Egypt, and randomly divided into four groups for the experiment. All rats were transported to randomly chosen polypropylene cages containing sawdust sheets with net wire tops in a fltered-air room. Tey were kept under daily observation at a normal room temperature (20-25°C) and a relative humidity of 60-70% and received food and water [35]. SPF Webster rats were randomly divided into four groups, with fve animals in each group. Group 1 (G1): healthy control rats were treated with saline (negative control); group 2 (G2): rats (S/C) were injected twice in the back with 0.2 ml of (3.0 × 107 CFU/l) cell suspension of MRSA in saline/rat for two days (positive control); group 3 (G3): MRSA infected group rats were orally administered ciprofoxacin (20 mg/kg BW); group 4 (G4): on the 21st day, all rats were weighed and blood was collected after being anaesthetized with chloroform by the ocular sinus puncture method. Ten, the blood samples were stored at room temperature for hematological analysis.

Hematological Analysis.
Whole blood samples were immediately collected in an ethylene diamine tetra acetic acid (EDTA)-coated vacutainer for hematological analysis.

Histological Examination.
Tissue samples of the lungs, liver, and kidney of each rat were collected and immersed directly in 10% neutral bufered formalin for histopathological investigations. After 72 hours, the samples were processed, embedded in parafn wax, and sectioned (5 ml) for hematoxylin and eosin (H&E) staining according to [37]. Histological slides were examined and photographed by a light microscope camera.

Statistical
Analysis. Te data were analyzed using Student's t-test and the data were expressed as mean ± division (SD) Te value of p < 0.05 was considered as a signifcant value against the control.  Table 3. Staphylococcus aureus was isolated from mastitic milk samples of dairy animals based on staining, golden yellowish colonies on MSA plates. Te isolates showed complete (β) hemolysis on blood agar and

Frequency of Bacteria in
Veterinary Medicine International coagulase tests, in addition to biochemical tests, which were all confrmed as positive isolates of S. aureus. Te patterns of antimicrobial sensitivity in Table 4 exhibited that out of 38 S. aureus isolates, 16 isolates (MRSA) were resistant to common available antibiotics. All 16 isolates were 100% resistant to penicillin G and methicillin, 87.5% resistant to gentamicin, 75% resistant to amoxicillin, ampicillin, and ampicillin/sublactam, 50% resistant to cefoxitin, 37.5% resistant to erythromycin, 31% resistant to ciprofoxacin, and 25% resistant to tetracycline and levofoxacin.

UV-Vis Spectra Analysis.
Te formation of TiO2 NPs in this study was analyzed by UV-visible spectrophotometer ( Figure 2). Te surface plasmon resonance (SPR) of the synthesized NPs appears as peaks due to the characteristic SPR of TiO2 NPs. Te absorption spectra of A. herb Alba TiO2 NPs formed in the solution had absorbance peaks at around 240-250 nm for aqueous plant extract exposed to TiO2.

Electron Microscopy of A. herb Alba TiO2
NPs. Te shape and size of nanoparticles were investigated. Te SEM and TEM examinations were carried out to understand the morphology and size distribution of titanium dioxide nanoparticles. Te SEM micrograph of the TiO2 NPs synthesized showed that there was a dense agglomeration of particles and they were irregular in shape. TEM refection and size allocation of TiO2 NPs, which were a mixture of diverse sizes and shapes, the composition of particles as shown in Figure 3.

Antibacterial Efect of A. herb Alba TiO2
NPs. Te antibacterial activity of various concentrations of synthesized A. herb Alba TiO2 NPs was evaluated against MRSA using disc difusion assay. Te mean inhibition zone (in millimetres) around each disc was estimated (Figures 4(a)-4(c)). Te ability of A. herb Alba TiO2 NPs, leaf extract of A. herb Alba, and antibiotics to inhibit bacterial growth of MRSA is listed in Table 6. Te highest zone of inhibition (43 ± 0.45 mm) was observed at the highest concentration of A. herb Alba TiO2 NPs (40 μg/ml).

Results of Oral Administration of A. herb Alba TiO2 NPs on Experimental Rats.
No death or toxic symptoms were observed due to administration of A. herb Alba TiO2 NPs during the experiment period; the use of A. herb Alba TiO2 NPs for 20 days did not cause any harmful efects on body weight or growth.

Hematological Analysis.
Hematological parameters were analyzed to diferentiate between rats that were treated with A. herb Alba TiO2 NP antibiotics after experimentally infected with MRSA and those that were not treated, as shown in Table 7. Rats infected with MRSA without treatment (G2) have shown a decrease in Hb when compared to control group rats (G1) and treated rats (G3 and G4). Tere was an increase in WBC in the A. herb Alba TiO2 NP treated group (G4). However, the total number of lymphocytes was    Table 7 showed signifcant change (p > 0.05) in (monocytes, eosinophils, and basophils) between infected group (G2) and control negative group (G1). Te results showed that the number of NEUT in (G2) was 0.52 (10 3 /cm), while in the control group (G1), it was 0.87 (10 3 /cm) which recorded signifcant decrease. Te number of LYM showed a signifcant increase in (G2) after infection reaching 13.00 (10 3 /cm) when compared to the control negative (G1) which reached 7.3 (10 3 /cm).

Examination of Rats' Tissue Section of the Lung, Liver, and Kidney by Light Microscopy.
Specimens of lung sections from the control negative group (G1) using H&E staining magnifcation (x20) showed normal histological features of the lung. Specimens of the lung section of an infected group (G2) with MRSA showed thickening of the inter-alveolar   Veterinary Medicine International septum, catarrhal bronchitis, infltration with infammatory cells, lymphocytic aggregates, hemorrhage in the bronchi, and interstitial pneumonia. Specimens of lung sections of (G3) rats showed that the pulmonary blood vessels were dilated, thickened, congested, and about to rupture. Specimens of lung sections of rats treated with A. herb Alba TiO2 NPs (G4) showed infltration with infammatory cells, alveolar emphysema with characteristic giant alveoli and bronchitis, thick inter-alveolar septa, and narrowing of the alveolar sac. Te histological pattern of the lung tissue treated with A. herb Alba TiO2 NPs was the best, and the pattern of the lung tissue histologically was similar to the control negative group as shown in Figure 5. Specimens of liver sections from control negative (G1) rats' livers showed normal tissues of hepatic cells using H&E staining. Te infected group with MRSA (G2) rat liver showed focal areas of congestive necrosis infltrated by leucocytes and slight vacuolar degeneration of some hepatic cells. In rats treated   with antibiotics (G3), liver sections showed centro lobular congestion surrounded by slight degeneration of hepatocytes. In rats treated with A. herb Alba TiO2 NPs (G4), liver sections showed blood sinusoids with slight degeneration of hepatocytes as shown in Figure 5. Te kidney sections of the negative control group (G1) showed a normal picture of glomeruli, and rat kidneys infected with MRSA (G2) showed interstitial hemorrhage, necrosis in tubules, and interstitial infltration of infammatory cells; rats treated with antibiotics (G3) showed hemorrhage and degenerated tubules and glomeruli, and rats treated with A. herb Alba TiO2 NPs (G4) showed apparently normal renal tubules and glomeruli with slight hemorrhage and congestion as shown in Figure 6.

Discussion
Livestock is the major reservoir of MRSA infection, with specifc genotypic and phenotypic traits of 3% S. aureus infection [38][39][40]. Te prevalence of S. aureus associated with intramammary infections in CM and SCM mastitis in dairy animals was 25.3% as shown in Tables 3 and 4, while other studies in Egypt (35.9%) [41,42], and disagree with the lower prevalence (6.5%) reported by [40,43,44]. In contrast, the percent of S. aureus infections associated with CM and SCM mastitis compared to other previous studies may be due to bioflm formation, beside diferences in immunological status of the animals, isolation protocols, geographical distribution, and hygienic protocols of the study areas. Te frequency of MRSA infections increased steadily after the introduction of β-lactam antimicrobials, as shown in Table 4 [45]. Te prevalence of MRSA was determined by S. aureus isolates from cows, bufaloes, sheep, and goats, sufering from CM and SCM mastitis more than percentage previously recorded in Egypt, may be due to the fact that this study used molecular techniques for detection of MRSA as shown in Table 5 [46]. Te increase in the prevalence of MRSA may be due to the widespread usage of antimicrobials in recent years [47]. Confrmation of resistants' genes in the collected MRSA isolates, including mecA (331pb/314pb), blaZ, ermB, tetM, and tetK by multiplex PCR as shown in Figure 7.
On analysis of S. aureus sensitivity showed resistance to penicillin G and methicillin was 100% and this result is in agreement with [48][49][50]. S. aureus resistance to β-lactam antibiotics is due to mecA gene, which encodes a modifed penicillin binding protein that confers resistance to methicillin and other penicillin derivatives carried on a mobile gene element called the "staphylococcal cassette-  Veterinary Medicine International 9 chromosome-mec" (SCC-mec) [51][52][53][54]. Te 75% of MRSA isolates sensitive to tetracycline and levofoxacin by AST is in agreement with [55], resistant to penicillin up to 100%, and cefoxitin 42.7% [56][57][58] [63,64]. Te green TiO2 NPs had antibacterial activity by forming superoxide radicals (O2), which produce reactive oxygen species (ROS) in bacterial cells, leading to breaking of the bacterial cell membrane [65]. Another reason for the antibacterial activity of TiO2 NPs is their ability to inhibit the bioflm in multidrug-resistant bacteria by inducing cell lysis and destructing the cell wall of these bacteria, leading to cell death and eventually inhibiting bioflm formation [66,67].
Hematological parameters changes, A. herb Alba TiO2 NPs experimentally treatment of infected rats group retained hematological parameters to normal values as control negative group as they have the ability to control the infection in 20 days and the high oxidizing power of the free radicals, which can directly destroy bacteria cells and inhibit the activity of ATPase, which leads to the reduction of the essential ATP to reserve the bacterial cell life and signifcant inhibitory efect on bacteria lead to recovery infected group rates to normal values as in group (G4) treated with A. herb Alba TiO2 NPs leads to a signifcant improvement in Hb, RBC, HCT, MCV, and MCHC levels as a results with [68]. Te group treated with A. herb Alba TiO2 NPs showed a signifcant increase in the neutrophil numbers of 0.72 (10 3 / cm) compared to control infected groups (G2) without treatment and a reduction in the number of lymphocytes of 7.40 (10 3 /cm) in (G4) as shown in Table 7 [69,70]. Green synthesized TiO2 NPs have hydroxyl and superoxide anions that disturb the cell wall and reduce the growth of several microorganisms, such as E. coli and S. aureus.
Histopathological examination of infected groups with MRSA revealed an efect on lung tissue because it caused thickening of the inter-alveolar septum, infltration with infammatory cells, catarrhal bronchitis, lymphocytic aggregates, lymphocytic granuloma, hemorrhage in the bronchi, and interstitial pneumonia in the lung.  Furthermore, in the liver, it causes congestive necrosis and vacuolar degeneration of some hepatic cells, as shown in Figures 5, 6, and 8, and interstitial hemorrhage and infltration of infammatory cells in the kidney. Using A. herb Alba TiO2 NPs for the treatment of MRSA decreased the severity of damage in tissues but did not return to normal. Terefore, more studies are necessary to determine its toxicity. In this study, laboratory rats that had been experimentally infected with MRSA strains and treated with A. herb Alba TiO2 NPs showed an improvement in the values of hematological parameters, in addition to the signifcant restoration of the rates of WBCs, lymphocytes, and granules after treatment [71,72]. A. herb Alba TiO2 NPs had antimicrobial property for treatment of clinical and subclinical mastitis against multidrug-resistant bacteria especially MRSA.

Conclusion
Te current study was conducted to develop possible alternative economically therapeutic agents from green synthesis of A. herb Alba TiO2 NPs for curing clinical and subclinical mastitis in ruminants in Egypt, for which conventional antimicrobial therapy may fail to provide a response. Te highest rates of MRSA strains from positive S. aureus isolates were detected by AST and multiplex PCR assays. Phenotypically, resistance of positive isolates of S. aureus against 11 available antibiotics showed 100% resistance to penicillin and methicillin. Te most predominant resistance genes associated with MRSA strains in mastitic milk were mecA, blaZ, ermB, tetM, and tetK. Te green A. herb Alba TiO2 NPs at concentration of 40 μg/mL (average size of 11 to 45 nm) on MRSA-induced mastitis in vitro. While, in vivo study evaluated the efcacy of A. herb Alba TiO2 NPs in diferent hematological parameter, and histopathological examination in diferent organs of rats in diferent groups. Te study fndings support that A. herb Alba TiO2 NP green synthesized can be used as a novel antimicrobial therapeutic agent in MRSA causing clinical and subclinical mastitis. However, further studies are still needed to safety and toxicity at a level of genome and cells must be conducted in order to better comprehend the nano interactions with organisms and environment.

Data Availability
All data generated or analyzed during this study are available from the corresponding author upon request.

Ethical Approval
Tis study was approved ethically by the Medical Research Ethical Committee, National Research Centre, Egypt, under registration number: 19-149.

Conflicts of Interest
Te authors declare that they have no conficts of interest.