Antimicrobial Activity of Plasma Rich in Platelets (PRP) on the Ocular Microbiota of Healthy Horses from Minas Gerais: In Vitro Study

In equine ophthalmology, ulcerative keratitis is among the most common conditions and, in general, arises as a consequence of some trauma suffered. Secondarily, subsequent contamination by pathogenic or resident bacteria of the horse's ocular microbiota may have undesirable consequences. Under physiological conditions, the normal microbiota coexists with the immune status of the host, serving as a barrier, ensuring the health of the ocular surface, and inhibiting the proliferation of pathogens. However, in the imbalance of immune barriers, the normal microbiota can become pathogenic and lead to infection, acting as an opportunistic agent. The present study aims to demonstrate the antimicrobial effect of platelet-rich plasma (PRP), its time of action, and its correlation with the concentration of its same components in vitro on Staphylococcus sciuri, a bacterium with high prevalence in the normal ocular microbiota of horses in the municipality of Minas Gerais. For the preparation of the PRP, eight adult Quarter Horse (QH) horses were used. The individual PRP was prepared by the double centrifugation protocol, and then, the PRPs were added to a pool, followed by testing their interaction in culture with Brain Heart Infusion (BHI) broth at different dilutions against five strains collected from different animals. After 3, 6, 12, and 18 hours, the colony formation units (CFU) count on a 5% horse blood agar plate was evaluated for each time point. Our study showed that Staphylococcus sciuri, the resident microorganism of the ocular conjunctival microbiota of horses, is more susceptible when compared to the standard strain “American Type Culture Collection” (ATCC-29213) Staphylococcus aureus, a pathogenic microorganism, which was used for the validation of our study. The antibacterial effect shown in this study was bacteriostatic for up to 6 hours. The most concentrated PRP dilutions, 1 : 1 and 1 : 2, were also most effective, suggesting that the antibacterial effect is volume dependent.


Introduction
Regenerative medicine and tissue engineering are rapidly growing felds that aim to restore or replace damaged or diseased tissues using a variety of approaches. One promising approach is the use of platelet-rich plasma (PRP), a concentrate of platelets obtained from the patient's own blood. PRP contains various growth factors and cytokines that can enhance the healing and regeneration of tissues.
Several studies have investigated the use of PRP in regenerative medicine and tissue engineering in both humans and animals. Te PRP has a positive impact on tissue regeneration particularly in bone and soft tissue healing [1]. PRP is found to stimulate cellular growth and diferentiation, increase collagen synthesis, and enhance angiogenesis [2]. It can also help reduce infammation and pain associated with tissue damage [3].
Te use of platelet concentrates has gained signifcant attention in recent years as a potential treatment for wound healing. Clinical studies show that these platelet concentrates are efective treatments for a variety of wounds, both in humans and animals, including chronic ulcers, burns, and surgical incisions [1,4,5].
Platelet concentrates have been used in a variety of clinical settings, including orthopedics [6,7], dentistry [8,9], and ophthalmology [10] to enhance tissue repair and regeneration. In veterinary medicine, PRP has been used to treat various conditions in animals. Te authors in [5] investigated the use of PRP in dogs with chronic skin wounds. Te study found that PRP treatment can improve wound healing and reduce the risk of infection. Tis shows that in addition to its regenerative properties, PRP has antibacterial properties, which have already been proven in various experimental models with animals [11][12][13].
Ulcerative keratitis is among the most common conditions in equine ophthalmology [14]. Although trauma is the primary cause, secondary resident or pathogenic bacterial contamination might result in severe sequelae.
Te bacterial isolates from the normal conjunctiva may vary depending on the geographic location, climate, age, sex, and habitat of the animals. Most commonly, aerobic Gram-positive bacteria predominate [19][20][21]. Te Staphylococcus genus is found in several studies, always being the 1st or 2nd most frequent genus among studies and with occurrences above 18% [17,[19][20][21]. Tus, it is possible that Staphylococcus spp. is the only truly resident microorganism and that the others are transient microorganisms, subject to environmental variations, but more studies are needed to confrm this relationship. In addition, the Staphylococcus genus is also the most prevalent microorganism in eye lesions in horses [22].
Treatments for ulcerative keratitis often have limited results. Terefore, there is a need to fnd new therapeutic strategies that are of multiple actions, are less artifcial, and are without potential allergens, such as preservatives or other products that, in the short or long term, can induce toxicity on the ocular surface, which is very vulnerable [23]. In this context, platelet-rich plasma (PRP), which is a blood-derived biological product, is used to promote better tissue repair, through the growth factors (GFs) contained in its composition, and fulflls most therapeutic goals for this type of eye pathology. Bezerra et al. [24] demonstrated the efectiveness of PRP against ulcerative keratitis in a horse, in which there was a reduction in consolidation time and a better healing quality. In addition to the therapeutic advantages already mentioned, its antimicrobial potential has been receiving attention. In humans, they reported that platelet concentrate has antimicrobial activity against bacteria such as Staphylococcus aureus (methicillinresistant and sensitive), Escherichia coli, and others [25,26]. In horses, the efect of PRP was also confrmed in an in vitro model against methicillin-resistant and sensitive Staphylococcus aureus [27,28]. Tese studies concluded that the combination of PRP components such as platelets and their alpha granules that release some microbicidal proteins and growth factors, such as leukocytes and plasma that contain complement and complement-binding proteins, produces a substance with antimicrobial efects [27,28].
Tis study aims to demonstrate the antimicrobial efect of platelet-rich plasma (PRP), its time of action, and its correlation with the concentration of its same components in vitro on Staphylococcus sciuri, a bacterium with high prevalence in the normal ocular microbiota of horse healthy in the municipality of Minas Gerais. To date, no studies have reported the antimicrobial action of PRP on bacteria isolated from the normal equine ocular microbiota, either in vitro or in vivo. For this study, several antimicrobial tests were applied, where one of the objectives of this fact was the search for techniques that could reduce the instability of the CFU variable, a gold standard test to evaluate bacterial growth, for which it was decided to also use spectrophotometry to evaluate the bacterial growth pattern of the diferent dilutions tested.
If the antimicrobial efect in this situation is confrmed, the use of antimicrobials for eye treatments in horses can be reduced, using an easily obtained biological therapy, with no reported side efects. In addition, this therapy will likely also have advantages in modulating infammation and tissue repair, as has been observed for other tissues and diseases.

Materials and Methods
Tis study was approved by the Ethics Committee on Animal Experimentation of the Federal University of Minas Gerais (CEUA/UFMG) under protocol number 231/2020.

Horses.
By means of a complete physical examination and a hematological examination, eight clinically healthy horses (4 males and 4 females) with a mean age of 7 years (range: 4-13) were selected. Te animals came from a farm where the environmental and management conditions were the same for everyone, respecting all animal welfare parameters. Horse owners were informed of the nature of the investigation and signed the appropriate authorization.
During the experiment, the animals were not removed from their environment of origin or their routine. Exclusion criteria in the study were changes in initial physical examination, recent use of anti-infammatory drugs and antibiotics (in the previous 7 days), recent systemic illness, or travel in the last month.

Blood Collection and Preparation of Equine PRP.
For blood extraction and PRP processing, a protocol adapted from [29] was used. Aseptic blood collection was performed by venipuncture of the external jugular vein, through the vacuum tube system, with a 22-G vacutainer needle (Agulha, BD Vacutainer, SP, Brazil) and vacuum tubes (Biocon ® ). In each collection, a total of 37 tubes per animal were extracted, extracting a volume of 130.6 ml of blood per animal. A sample was collected in a 1.0 ml tube of ethylenediaminetetraacetic acid (EDTA) to perform the initial blood count. Ten, 36 tubes of 3.6 ml with 3.2% sodium citrate (CIT) were collected per animal, to later make the PRP pool. Te samples were homogenized by slow manual inversion of the tubes.
Blood collected in tubes with CIT was processed using a double centrifugation protocol. In the frst step, the blood was transferred to sterile 15 ml falcon tubes to optimize processing time and immediately centrifuged (CENTRIBIO 80-2B ® , Biovera, Brazil), using 120 g for 5 min with a subsequent rest of 10 min in a vertical position. Te frst fraction of supernatant plasma (50%) was discarded. Te other 50% of plasma was aspirated at 4 mm above the bufy coat with the aid of a graduated pipette of up to 1 ml and a micropipette of 200 μL when it was close to the bufy coat. Tis fraction was transferred to other sterile 15 ml falcon tubes, and all processing was carried out under aseptic conditions, under a laminar fow hood. After the second centrifugation, performed at 240 g for 5 min with subsequent rest for 10 min in an upright position, 75% of the frst fraction was discarded. Te remaining 25% was pipetted to dissolve the pellet formed in the fnal portion of the tube. Tis last fraction was considered as PRP after the fnal platelet count above 300,000/μL, as described by [30][31][32]. Red blood cell and leukocyte counts were also performed using an impedance hemocytometer (Icounter Vet ® , DIAGNO, Brazil).
Finally, the PRP of each individual was transferred to a 100 ml polypropylene beaker to obtain a single pool of all animals, which yielded a fnal amount of 66 ml of PRP. From this pool, another aliquot was separated to obtain platelet concentration, erythrocyte, and leukocyte counts on an impedance hemocytometer.

Study Design and In Vitro Antibacterial Assay.
A pool of PRP without prior activation was analyzed to determine its antibacterial efect against six isolated strains of Staphylococcus sciuri and a standard strain of Staphylococcus aureus "American Type Culture Collection (ATCC-29213, OxoidCulti-Loops, KS, USA)." Due to the possible individual variability of the strains, six strains isolated from diferent animals of Staphylococcus sciuri (S1-S6) were used, the most prevalent microorganism in the ocular microbiota of healthy horses from the metropolitan area of Belo Horizonte-MG (data not yet published) from the bacterial bank of the Veterinary School of UFMG, Laboratory of Bacteriology. In addition to these strains, a standard strain (SS) of Staphylococcus aureus ATCC-29213 was included to ensure the reliability and reproducibility of the results.
Te minimum inhibitory concentration (MIC) was evaluated by the macrodilution method in tubes with Brain Heart Infusion Broth (BHI) (Brain Heart Infusion Broth ® , KASVI, Brazil) at all times, and samples were visually inspected, according to the standards established by the "National Committee for Clinical Laboratory Standard" (NCCLS).
Te experimental groups were confrmed for all bacterial strains tested by fve dilutions of PRP (1 : 1, 1 : 2, 1 : 4, 1 : 8, 1 : 16) together with BHI broth and 400 μL of bacteria to obtain a concentration end of inoculum of 1 × 10 5 CFU/ml. For each strain, four control groups were established, the BHI control consisted of only BHI broth, the PRP control consisted of BHI broth and pure PRP, the positive control group (PCG) consisted of BHI broth with bacteria, and a control group negative (NCP) formed by BHI broth with bacteria and 0.5 μL/ml of purifed erythromycin for antibiotic sensitivity test (Drogavet ® , Brazil), minimum inhibitory dose for the two bacteria tested according to NCCLS. In Figure 1, we can see the composition of each experimental group; all groups had a fnal volume of 4 ml.
All samples were incubated at 37°C for 24 hours. During this incubation time in the broth, aliquots were extracted for each dilution of PRP (1 : 1, 1 : 2, 1 : 4, 1 : 8, 1 : 16) and the PCG of 100 μL after 3, 6, 12, and 18 hours of incubation for all samples. Tese aliquots were diluted in phosphate bufer saline solution (PBS 90%) to 10 − ³. Immediately afterward, 100 μL of the sample was seeded on 5% horse blood agar plates (Base Blood Agar ® , KASVI, Brazil). All processing was performed under aseptic conditions in a laminar fow hood. Tese plates were labeled and incubated at 37°C for 24 hours for subsequent manual counting of CFUs in each plate. Te highest growth observed for any sample was limited to 300 CFU/plate for all dilutions. Te number of CFU/mL will be determined by the following formula: CFU/ mL � CFU/plate × 1/0.1 ml (aliquot factor) × 10³ (dilution factor) [28].
After completion of the 24 hours of culture in the broth for the NCG, a visual reading was carried out frst, looking for the formation of bacterial mass or turbidity in the samples, and then soon after, sowing in a Petri dish to evaluate bacterial growth and the efectiveness of the antibiotic in these microorganisms.
At the time of 0 hours, aliquots of the BHI control and PRP control groups were taken for seeding and cultivation in a Petri dish, to validate the sterility of the products.
To test a technique that could reduce the instability of the variable CFU, it was decided to also use spectrophotometry to evaluate the bacterial growth pattern of the diferent dilutions tested. For the reading of the optical density on the spectrophotometer, during the 24 hours of incubation of the samples, aliquots of 400 μL were extracted at times 0, 3, 6, 12, and 18 hours for each tested dilution of the product. Tese aliquots were read in a quartz microcuvette, using the spectrophotometer method (Spectrophotometer 35 D ® , Coleman, Brazil) in absorbance, with a wavelength of 620 nm, using BHI broth to zero the instrument and then reading the samples.

Statistical Analysis.
Statistical analysis was performed using the R software version 3.6.1 (R Core Team, 2019).
In this study, the horse does not constitute the experimental unit, as the individual character will not be considered, mainly because a pool of diferent PRPs will be carried out. Te number of animals used for blood collection was defned based only on the amount of blood needed for all analyses, using a volume much smaller than the maximum allowed per animal (8% of the live weight).
To test the diferences between strains, times, and dilutions, linear regression models were ftted. Te diferent strains, times, and dilutions were used as predictor variables.

Veterinary Medicine International
It was necessary to apply the logarithmic transformation to the response variables to control the variance and reduce the impact of outliers. After adjusting the model, the mean values were calculated, and their respective 95% confdence intervals and illustrative graphs were constructed. Multiple comparison tests (pairwise) were performed by applying the Sidak correction. For all tests, a signifcance level of 5% was used.

Results
One of the strains (S2) was removed from the CFU/ml and optical density data analysis because it showed bacterial inactivity during the experiment.

Blood Count and Assessment of Whole Blood Compared to PRP.
Te results of the hemograms of the individuals were within the reference values for the equine species. Te mean blood counts were 6.57 × 10 6 ± 0.38 red blood cells/μL, 11.02 × 10³ ± 2.5 leukocytes/μl, and 187 × 10³ ± 47.4 platelets/ μL.
After measuring the individual PRP values, they were grouped into a pool that was defned as the fnal product to be tested. Te values of this pool were 0.01 × 10 6 red blood cells/μL, 2.06 × 10 3 leukocytes/μL, and 382 × 10³ platelets/μL, with a platelet concentration of 2.05 times higher than the baseline concentration.

Strains Behavior and Dilutions Over Time Related to Colony Forming Units (CFU) Counts. Te 5 strains of
Staphylococcus sciuri and the standard strain of Staphylococcus aureus selected for the study were analyzed according to their behavior at diferent times, considering the means of the PRP dilutions (1 : 1, 1 : 2, 1 : 4, 1 : 8, and 1 : 16) for CFU counts. Table 1 shows the median values and their respective quartiles. Te values recorded in the study as >300 × 10 4 were transformed to 300 × 10 4 .
At time 0, descriptive means were calculated for the samples taken from the BHI control, PRP control, and positive control groups for both Staphylococcus sciuri and Staphylococcus aureus. For the two microorganisms, the mean values obtained in the BHI control groups and the PRP control groups were 0.0 × 10 4 CFU/ml and 0.0 × 10 4 CFU/ml, respectively. Tese fndings refect sterility in the preparation of the BHI broth. Also, with regards to the PRP control group, it suggests that the product to be tested was not contaminated in its preparation. Te positive control group (PCG) presented a value of 8 × 10 4 CFU/ml, which demonstrates that bacteria were viable and able to replicate. Figure 2 shows the linear graph that represents the behavior of the CFU variable of each strain over time.
Comparing the diferences between strains for the same time, it was observed that at time 3, SS had a higher bacterial growth (P < 0.05) than all other strains of Staphylococcus sciuri (Figure 2). Within the other times (6, 12, and 18), there was no diference in bacterial growth between strains, regardless of the dilutions.
Comparing the diferences between times for the same strain in (Figure 2), it was observed that strains S1, S3, and S6 showed signifcant bacterial growth (P < 0.05) from time 12 onwards. Strains S4 and S5 started to grow signifcantly (P < 0.05) earlier, at time 6. Te S6 strain continued to grow signifcantly (P < 0.05) even after time 6. Te SS showed, over time, an unstable growth behavior.
Te 5 dilutions tested 1 : 1, 1 : 2, 1 : 4, 1 : 8, and 1 : 16 of our product (PRP) and PCG were also analyzed and compared with each other over time considering all strain means for the CFU scores. In Table 2, we fnd the median values and their respective quartiles.
In Figure 3, the diferences in bacterial growth (CFU/ml) were compared between the tested PRP dilutions and PCG over time. Tere was less growth of CFUs (P < 0.05) and therefore a greater antimicrobial efect in the 1 : 1 and 1 : 2 dilutions when compared to the 1 : 4 dilution onwards and PCG, at all times. Tis was also observed in Figure 4, considering all unifed strain means and time. In turn, when comparing the diferences between times for the same dilution (Figure 3), it is observed that all dilutions are observed to show bacterial growth (P < 0.05) at times 3, 6, and 12.

Strain Behavior and Dilutions Over Time Related to
Optical Density Measured by Absorbance. Te 5 strains of Staphylococcus sciuri and the standard strain of Staphylococcus aureus selected for the study were analyzed according to their behavior at diferent times. PRP dilutions were tested for absorbance using spectrophotometry at a wave of 620 nm, after which the means were used. Table 3 presents the descriptive values of the medians and their respective quartiles.
In Figure 5, the graph represents the absorbance behavior of the strains over time.
Comparing the diferences between strains for the same time in Figure 5, we found that S1 showed higher absorbance (P < 0.05) than strains S3, S4, S5, and SS at time 0, in any of the dilutions. Within the others, there was no diference between the optical density readings over time.
Comparing the diferences between times for the same strain in (Figure 5), regardless of the dilutions, we observed that S1 started reading at time 0 with a higher absorbance,  Veterinary Medicine International which was reduced (P < 0.05) soon after at time 3, staying that way until the end. Te increase in absorbance of S3, S6, and SS only became signifcant (P < 0.05) at time 18. At S5, this happened at time 12.
In Table 4, we fnd the descriptive values of the medians and their respective quartiles of each dilution over time, considering the means of the strains.
In Figure 6, when all strains were grouped together, the diferences in absorbance were compared between the tested PRP dilutions over time. Figure 7, both strains and times were grouped.

Discussion
Te use of platelet-rich plasma (PRP) represents a simple form of treatment in equine ophthalmology, which can stimulate an increase in the proliferative and migratory capacity of corneal cells, reducing the time for consolidation and providing better healing in ocular surface injuries [23,33]. In equine veterinary medicine, few studies have evaluated the antimicrobial efect of blood components. Tis study is the frst to evaluate the antimicrobial efect of PRP against the Staphylococcus sciuri microorganism of the ocular microbiota of healthy horses. Until now, equine blood products have shown an antimicrobial efect against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) [27,28] and Escherichia coli [21].
Our PRP presented a diference, however not high, between the mean platelet values of the individual horses compared to the values found in the pool. Tis happened due to the fact that the individual factor afected the concentration of the pool, since some animals concentrated more platelets than others.
Although there is no standardization of protocols for the acquisition of ideal cellular components in equine PRP, the product used in this study was classifed as a PRP with adequate platelet concentration [28,[30][31][32] and considered as a pure-platelet-rich plasma (P-PRP) [34] or as leukoreduced platelet concentrate [35], making it a safer product for use in equine ophthalmology, considering that PRP with high concentrations of leukocytes promotes catabolic processes in fabrics [30].
In this study, it was observed that SS (S. aureus) had greater growth at 3 hours, which is logical considering that it is a pathogenic agent [36]. Te other strains (S1-S6) are strains of the ocular microbiota of healthy animals; therefore, they possibly do not have the same replication and colonization efciency. It was clear that PRP was efective in inhibiting these bacteria as early as 3 hours. Te inhibition of PRP on SS only occurred after 6 hours, which may mean that the inhibitory factors present in PRP needed a longer time to have an efect. From a clinical point of view, this may be particularly important when trying to identify S. aureus as the causative agent of ulcerative keratitis in horses [22]. Diferent results were observed by other researchers, who  cultivated PRP and other blood products, with MSSA and MRSA directly on the blood agar plate, and read the plates at 6 and 24 hours, comparing their treatments and the control between them. On reading after 6 hours of cultivation in the case of PRP, it totally inhibited bacterial growth compared to its control group. At 24 hours, the CFU/ml values of PRP were lower than those obtained in the control group [28]. Another interesting and occasional fnding in this study was the coagulation of SS samples from all subgroups since time 3. Tis fact is supported by the literature, since Staphylococcus aureus has a protein called coagulase that allows the conversion of fbrinogen to fbrin, interacting with von Willebrand factor binding protein, which binds to prothrombin (factor II of the clotting cascade), forming a complex called staphylothrombin [37] which then uses fbrinogen binding proteins such as aggregation factor (A) to adhere to fbrin clots and form fbrin-containing bacterial aggregates [38]. Tis fbrin membrane makes the bacteria resistant to phagocytosis through this mechanism [24,27]. Tis mechanism clearly demonstrates that there was an interaction of PRP with the bacteria, which could not have been demonstrated in MIC curves with antibiotics. Possibly, this mechanism infuenced the greater bacterial growth of SS at time 3 when compared to Staphylococcus sciuri strains. It Table 3: Medians of absorbance (A 620 ) with their respective quartiles of strains over time, considering all means of dilutions. Time 0  Time 3  Time 6  Time 12  Time 18  1°q  ua  Median  3°q  ua   1°q  ua  Median  3°q  ua   1°q  ua  Median  3°q  ua   1°q  ua  Median  3°q  ua   1°q  ua  Median  3°q  ua  S1 0     Although the present study also proposed to analyze and defne a MIC for PRP using the macrodilution method in BHI broth, this did not prove to be an efective technique for evaluating the efects of PRP in the methodology used. Furthermore, our study aimed to make observations at shorter time intervals and not just after 24 hours of cultivation. Another important factor was the fact that the density of PRP made it difcult to visually evaluate the samples, especially the SS ATCC subgroups of S. aureus, due to the coagulation of the samples. No studies were found that used the visual evaluation methodology to defne MIC in equine PRP. Tus, it was decided to interpret the results of the dilutions in the CFU counts, which is the gold standard test to evaluate bacterial growth.

Strains
In this study, PRP was not activated, but the results showed that there was indeed an inhibitory efect, a fnding that generates controversy compared to [39] who stated in their study that blood products without prior activation did not present an inhibitory efect. On the other hand, the authors in [28,40] showed that activated PRP preparations had lower antimicrobial activity against some microorganisms tested compared to inactivated preparations. Tese fndings are believed to be due to complement consumption during activation of coagulation [41]. Platelet activation was evident in SS since it promoted the clotting of samples after contact with PRP. Tis event is supported by the literature since platelets have several mechanisms of interaction with bacteria, which causes them to activate [42,43]. In addition, although Staphylococcus sciuri strains 1-6 do not have some virulence factors to generate PRP coagulation, the interaction between product and bacteria was evident from the results obtained, showing inhibition of bacterial growth of these strains when compared to the group positive control.
Te PRP in this study showed an inhibition of bacterial growth of all strains tested during the frst hours of incubation, but without reaching the complete breakdown of the microbial load, showing a recovery of bacterial growth. With this, it can be indicated that PRP presented a bacteriostatic inhibitory activity instead of a microbicidal one, and these results are compatible with what some authors have said, who also evaluated the potential of PRP and other blood products [26,28,[44][45][46][47].
Te time that the PRP tested in this study showed an inhibitory efect on bacterial growth was up to 6 hours for both Staphylococcus sciuri and Staphylococcus aureus. Other authors reported a time of inhibitory efect of PRP and other blood products against diferent microorganisms between a range of 4-8 hours, which is compatible with those  obtained in this study [26,28,[44][45][46][47]. Tis inhibitory time can be variable and discrepant due to several factors, including the intrinsic characteristics of the bacterial strains used, which may have diferent susceptibility to diferent platelet concentrates and their composition, or to the different sensitivity of the test used to assess antibacterial activity.
Taking into account the results obtained in this study, we can say that the use of PRP can be used either pure 1 : 1 or diluted up to 1 : 2, presenting a bacteriostatic efect of up to 6 hours, against all strains of Staphylococcus sciuri, a resident microorganism of the ocular conjunctival microbiota of healthy horses and against Staphylococcus aureus (SS), a multidrug-resistant pathogenic microorganism. Terefore, it can also be inferred that the antimicrobial properties of PRP are concentration dependent, which is consistent with the results of [48]. Although this study did not assess which of the cellular components of PRP is responsible for antimicrobial activity, our results support the hypothesis that the in vitro antimicrobial efect can probably be attributed to platelets [49,50] and plasma complement [28,39], since our PRP was a leukoreduced blood product. However, it is also possible that the antimicrobial efect of PRP under in vivo conditions could be mediated by other biological mechanisms that include leukocytes or the combination of all these components makes PRP a biological therapy with antimicrobial efects [28,41,44].
Although this study has proposed using absorbance as a measure of optical density by spectrophotometry in the search for a faster, more sensitive method with more stable data compared to CFUs. Tis search was restricted by the stable behavior of the spectrophotometry results, therefore, without following any pattern either positive or negative in bacterial growth and by the fact that PRP, being a blood derivative that contains a mixture of platelets, leukocytes, growth factors, serum, and plasma proteins [27,39], directly afects the absorbance result, reading all this PRP cellularity and, in addition, the bacteria are alive or dead. In addition, PRP presented in its 1 : 1 and 1 : 2 dilutions greater (P < 0.05) absorbance than the other dilutions, a fact that supports that the same concentration of PRP interfered with the results of this test. An interesting fnding in this test was the fact that the 1 : 16 dilution, with the lowest concentration of PRP, showed a signifcant diference in bacterial growth at time 3, time in which it had the lowest absorbance, compared to time 18, when it had its highest absorbance value. Tis fact demonstrates a tendency for this strain to increase its absorbance due to the same fact that lower concentrations of PRP do not lead to an efective antibacterial efect as in higher concentrations.

Conclusions
Under the conditions of the present experiment, evaluating the antimicrobial efect of PRP against Staphylococcus sciuri, a resident microorganism of the normal conjunctival ocular microbiota of healthy horses in the city of Minas Gerais, the following conclusions can be obtained: (i) Strains isolated from the normal conjunctival microbiota of Staphylococcus sciuri were more susceptible to the antimicrobial efect of PRP than the ATCC strain of Staphylococcus aureus (ii) PRP showed a bacteriostatic antimicrobial efect in vitro for up to 6 hours, at 1 : 1 and 1 : 2 dilutions (iii) Te antimicrobial efect of PRP is concentrationdependent (iv) Spectrophotometry is not an applicable method to assess the interaction and behavior of bacteria against PRP

Data Availability
Te data used to support the fndings of this study may be released upon application to the Escola de Veterinaria-Universidade Federal de Minas Gerais, who can be contacted at luisdsb94@gmail.com.

Conflicts of Interest
Te authors declare that there are no conficts of interest.