Summary

Introduction: Although platelets are not part of the classical immune system, they have many features that indicate their role in the anti-infective host defense. They come into interactions with micro­organisms, which results in co-aggregation and co-adhesion or destruction of the microbes due to the action of antimicrobial peptides released from platelets.
The aim of this study was to evaluate the killing effect of platelets against planktonic and biofilm cultures of Staphylococcus aureus and to test their synergy with antibiotics.
Materials and Methods: S. aureus ATCC 29213; platelet rich plasma (1-3 days post shelf life). Evaluation of bactericidal activity of platelets or their lysates against planktonic cultures of S. aureus – CFU calculation after 4- and 24-hour co-incubation. Assessment of S. aureus biofilm viability under the influence of platelets – Live/Dead^® BacLight^TM Bacterial Viability Kit. Determination of minimum inhibi­tory concentrations (MICs) (oxacillin, vancomycin, linezolid) and estimation of the synergistic action of antibiotics and platelet lysates – a gradient-diffusion test strip.
Results: Microbicidal activity of „expired” platelets and their lysates has been shown as a significant reduction in the population of staphylococci in their planktonic cultures by 56-87% and a decrease in metabolic activity of biofilm formation by 7-38%. These activities were enhanced after activation with ADP. Platelet lysates showed a synergistic effect with β-lactam antibiotic (oxacillin) and glycopeptide (vancomycin) but not with oxazolidinone (linezolid).
Conclusions and Discussion: In summary, platelets even after the medical expiry date are still a good source of antimicro­bial low molecular weight proteins (PMPs). Testing of bacterial resistance to PMPs may be advisable as a predictive indicator of susceptibility to treatment of infections such as infective endocarditis and other local infections of biofilm nature.

Key words:blood platelets • platelet microbicidal peptides • S. aureus • biofilm • infective endocarditis

Introduction

Although blood platelets (thrombocytes) are not consi­dered as part of the classical immune system, they have many features indicating their important role in the anti­-infective defense of the host. These cells express recep­tors for the Fc fragment of immunoglobulin, complement component C3a/C5a, various chemokines and Toll-like receptors (TLRs). They have an ability to generate reac­tive oxygen (ROS) and nitrogen (NOS) species, and their intracellular granules are rich in peptides with microbici­dal activity [2,3,7,11,14,16,18]. The platelets undergo rapid stimulation by thrombin, ADP (adenosine diphosphate), collagen, and other mediators released into body fluids as a result of infection or exposed on the surface of the damaged tissues. Activated platelets interact mainly with the vascular endothelial cells; they stimulate the recruit­ment and facilitate the adhesion of neutrophils and other leukocytes to the affected tissue, acting as a specific con­nection between hemostasis, infection, inflammation and immunity processes [12,15,17]. They can also participa­te in direct contact (co-adhesion) with microorganisms, resulting in the creation of stable cell-to-cell aggregates (sequestration of bacteria) or pathogen destruction (pha­gocytosis/killing). Findings from several investigations aimed at elucidating this antimicrobial role of platelets revealed the presence of platelet microbicidal peptides (PMPs) and other released factors, which possess poten­tial antimicrobial properties. It has been agreed that PMP activity may have positive importance in the pathogene­sis of various types of infection and the resistance to their action is relevant to the outcome of the particular disease [2,4,5,7]. Special attention has been paid to the participa­tion of PMPs in pathogenesis of such diseases as sepsis, infective endocarditis (IE) and other chronic localized infections. It has been proved that these types of infec­tions are very often complicated by formation of biofilms / aggregates of bacteria or fungi. Besides IE and sepsis, examples of such pathologies are non-healing diabetic foot ulcers, cancer-related ulcers, burn-associated wo­unds, surgical site infections and many others [1,2,8,9,17]. It has been reported that bacteria recovered from patients with, for example, IE are consistently resistant to PMP­-induced killing, whereas similar species of bacteria iso­lated from other types of infections are susceptible [2,8]. For many years scientific interest of our team has been focused on the analysis of drug resistance and virulence factors of Staphylococcus aureus – bacteria taking part in the pathogenesis of the above-mentioned types of infec­tions (common endovascular pathogen). It is noteworthy that staphylococci are able to bind fibrinogen/fibrin, po­ssess procoagulant activity, easily aggregate and interact with platelets. Moreover, their activity leads to both clot formation (mainly based on direct surface protein inte­ractions and coagulase activity) and fibrinolysis (due to staphylokinase properties). These two processes could be relevant to the course of IE, making staphylococci one of its most important etiological agents [2,6,8,10,19]. A no­vel approach to alternative therapies of such infections, presented in this study, involves characterization of the antimicrobial role of platelets toward S. aureus. An addi­tional question which we want to ask is whether we can expect synergy between platelet-derived antimicrobial products and antibiotics routinely used for therapy of infective endocarditis and other infections. Knowledge regarding the role of platelets in the development and se­verity of various disorders besides thrombosis continues to emerge, particularly in the field of inflammation and the immune response to infection [5,7,13,16].

The aim of this study was to evaluate the microbicidal ac­tivity of platelets and their products against S. aureus in suspension (planktonic) and sessile (biofilm) cultures. Expi­red platelet concentrates (platelet-rich plasma product in the bag) were used in this study. We asked the question if 6-8-day-old platelets themselves or their lysates prepa­red by freezing can still be microbicidally effective and may become a source of alternative or auxiliary products to antibiotics.

Material and Methods

The test organisms.

The reference strain of Staphylococcus aureus ATCC 29213 grown for 24 h at 37°C on Müller-Hinton agar (MHA) or tryptic-soy broth (TSB) supplemented with 0.25% D-(+)­-glucose (TSBGlc) was used.

Platelets, platelet-derived products.

The expired platelet concentrates were obtained from the Regional Blood Center in Lodz. It was platelet-rich plasma (PRP) from 5 donors, within 1-3 days after the expiry date. Total platelet count in the bag was equal to 3 x 10¹¹ and contained <1% of leukocytes. Cells taken from the bag were centrifuged (10 min, 2000 g), then washed twice in Tyrode buffer (Sigma, USA) and suspended in the culture medium RPMI-1640 (CytoGen, Poland) with a density of 108/ml. Platelets were counted by the photometric me­thod according to Walkowiak et al. [15]. The platelet su­spension was left without stimulation or was stimulated with ADP (adenosine diphosphate, Sigma, USA) at a final concentration of 20 mM, 15 min, 37°C. Thus, in the expe­riments platelets were used as: a) a suspension of unsti­mulated cells, b) a suspension of platelets after ADP sti­mulation, c) and d) as cell lysates prepared, respectively, from these two types of platelets (in a density equivalent to those used in point a/b), then they were frozen and thawed three times. Prepared lysates were kept frozen (-80°C) until testing, but no longer than 1 month.

Microbicidal activity of platelet-derived products against S. aureus planktonic cultures

In time-kill studies platelets or their lysates were used against S. aureus ATCC 29213 strain at the inoculum of 3 × 10⁷ CFU/ml in RPMI-1640. Platelets (or quantitatively equ­ivalent lysate) were mixed with bacteria in the proportion of 3:1 in a 24-well culture microplate. At predetermined time points (0, 4, and 24 h of incubation at 37°C), 100 µl samples were serially diluted in sterile normal saline. Then, a 100 µl aliquot of each dilution was plated onto an MHA plate for CFU counting, after further incubation at 37°C for 24 h. The results were reported as the mean CFU ± S.D. of all four replicates and the percentage of growth inhibition was calculated (compared to the control).

S. aureus biofilm eradication under the influence of platelet-derived products.

The suspension of S. aureus ATCC 29213 prepared from fresh overnight culture in TSBGlc was added to the wells of a 96-well polystyrene plate (Nunc, Denmark) and cul­tured for 24 h at 37°C. Biofilms were carefully washed and undiluted platelet lysates were applied (100 µl/well). Af­ter subsequent 24-h co-incubation, the degree of biofilm survival (%) was assessed using a LIVE/DEAD® BacLight™ Bacterial Viability Kit (Molecular Probes, USA), as recom­mended by the manufacturer. Results are presented as the percentage of the formed biofilm, calculated from mean fluorescence values (measured at 485/535 nm for green Syto9 and at 485/620 nm for red PI) ± S.D. in comparison to control biofilm considered as 100% grow rate. The as­say was done in quadruplicate to obtain the statistical average of the result.

Determination of antibiotics’ MICs and assessment of their synergistic activity with platelet lysates.

The prepared inocula of S. aureus ATCC 29213 (density of 0.5 according to McFarland scale) were lawned evenly by a sterile cotton swab on the surface of the A) control MHA or B) MHA containing blood platelet lysate (with the ratio 1:1). Antibiotic gradient strips – MIC Test Strip (LiofilChem, Italy) containing oxacillin, vancomycin or linezolid (the range of gradient concentrations for all antibiotics was 0.002-32 mg/l) – were used. MHA plates with overlayered strips were then incubated at 37°C for 24 h and the inhibition zones were measured. Differences in MIC values obtained on the control and test plates were recorded (end-points were determined according to the manufacturer’s instructions).

Results and Discussion

The aim of the present study was to provide answers to two important questions: whether blood platelets, when they are medically useless (shelf life – 5 days), possess an­tibacterial an­tibacterial activity and whether they could be a source of products which can enhance antibiotic activity. Indeed, our results confirm these two assumptions by showing that the biocidal activity of such platelets, as well as their products, is significant against S. aureus planktonic and biofilm cultures. The source of thrombocytes was plate­let rich plasma (PRP) obtained from the Regional Blood Center in Lodz. The cells were used at a density of 108/ ml, which corresponds to the average content of platelets in 1 ml of whole blood. It was shown that platelets, even after the expired time for transfusion (total time 6-8 days of life), maintain significant microbicidal activity. Direct contact with the platelet suspension limited the number of S. aureus, assessed after 4-hour co-incubation as CFU, by 85-87%. Quantitatively equivalent platelet lysates were also active, since those obtained from nonactivated cells were able to limit the multiplication of the tested bacteria by 56.70 ± 9.54%, and when platelets prior to lysis were activated with ADP, the effect increased to 72.70 ± 5.45% of inhibition. After extending the time of co-incubation of platelets or their products (lysates) with staphylococ­ci, the observed results were weaker (data not shown). It was due to the multiplication of bacteria in inoculum which were not destroyed during the first 4 hours of co­-incubation. As shown in Table 1, both platelets and their lysates were more effective after activation with ADP. It can be assumed that platelet activation with thrombin, instead of ADP used by us, could have given a much bet­ter response with respect to the release of antimicrobial products. However, due to the expected strong platelet aggregation and clot formation caused by thrombin, we decided to use ADP.

Table 1. The influence of platelets and released products on Staphylococ­cus aureus ATCC 29213 planktonic culture growth and biofilm eradication

Although ADP is regarded as a weak agonist of circula­ting blood platelets, it is an important mediator of pla­telet activation induced by other stimulators (thrombin, collagen), which promote ADP release from intraplatelet storage pools, such as dense granules, where it is present in high concentrations. Additionally, ADP acts synergisti­cally to all other platelet agonists, even faint ones, such as serotonin, adrenaline or chemokines [2,3,12,21]. In our hands, platelet activation with ADP, although not able to deliver a spectacular effect, has revealed that ADP is an equally potent agonist to the occurrence of microbicidal/ anti-biofilm action. In the present study the impact of cer­tain variables, such as pH, was not included, although we are aware that in this way we could have obtained a more reliable result. It has been reported that platelet-derived peptides exerted greater activities and border spectra under slightly acidic conditions [2,18,19]. Thus, it will be interesting to continue the research on the isolation of proteins with microbicidal activity, the assessment of their stability and the impact of physical and chemical conditions on their activity.

In the present study we made another important and unique observation concerning the activity of platelets and their products against S. aureus biofilm. Viability of the sessile biomass was measured by differential staining using a LIVE/DEAD^® BacLight^™ Bacterial Viability Kit, compared to the control biofilm (Table 1). ADP-activated platelets and their lysates applied onto already formed biofilm reduced its viability by approximately 32-38%, as­sessed after a further 24 h of incubation. Given the known extremely high resistance of biofilm, even partial destruc­tion of the biofilm by platelet-derived products should be regarded as encouraging. Many in vitro studies and clinical observations indicate that complete eradication of biofilm-embedded bacteria by chemotherapy is hard to achieve [1,8,10]. Therefore, it is important to search for compounds that could increase the susceptibility of biofilms to conventional therapeutic agents. It is hoped that antimicrobial peptides from blood platelets could be such prospective compounds as alternative or supple­mentary to classical pharmacological products to combat infections. We observed that platelet lysate used even in a sub-inhibitory concentration (diluted 1:1 in agar diffu­sion test), enhanced S. aureus susceptibility to β-lactam antibiotic (oxacillin) and to glycopeptide antibiotic (van­comycin), but not to oxazolidinone (linezolid). A typical view of the test in which a synergistic effect of platelet ly­sate and antibiotics has been examined is shown in Fig. 1.

Figure 1. Synergistic effect of vancomycin and ADP-activated platelet lysate against S. aureus ATCC 29213 evaluated by gradient-diffusion strip test as MIC value. A – control plate; B – medium with platelet lysate (1:1 dilution).

Both antibiotics which in our study showed synergism with platelet-derived lysates are known to have a bac­tericidal effect, by interfering with cell wall synthesis. It can be suggested that in the presence of oxacillin or vancomycin the uptake of platelet-derived peptides is facilitated, and in turn, due to a combined action, MICs of antibiotics were lowered. MIC of oxacillin assessed on a control plate was 0.25 mg/l but on agar containing platelet lysate it was 0.19 mg/l. Similarly, MIC of vanco­mycin dropped from 1.0 mg/l (control) to 0.5 mg/l (test plate). This is consistent with earlier reports concerning synergy of platelet-derived microbicidal proteins, iso­lated from fresh thrombocytes, with antibiotics such as nafcillin, ampicillin and vancomycin [2,8,9,10,20]. The similar activity of a product coming from outdated pla­telets, presented in this study, can be considered inte­resting. The observed effect of increased sensitivity of bacteria to antibiotics due to platelet proteins has ano­ther, more important effect in vivo: the possibility of re­duced adhesion of bacteria to platelet aggregates. Such an initial observation was reported many years ago by Yeaman et al. [19,20]. In summary, the group of results presented here suggests that platelets may enhance the effects of antibiotics used against S. aureus. Of course, it is possible that certain antimicrobial peptides also fa­cilitate the host defense functions of other endogenous mechanisms of innate or adaptive immunity.

In the light of our results it is clear that platelets signifi­cantly contribute to antimicrobial host defense and po­tentially enhance the antimicrobial mechanisms of di­stinct classes of conventional anti-staphylococcal agents. The activity of platelets/released antimicrobial products towards planktonic and biofilm S. aureus cultures obse­rved in this study are relevant, although the achieved ranges of inhibition were not significant. However, it can be suggested that after further processing, the expired platelet concentrate will have a potential medical appli­cation as a source of prophylactic or therapeutic modern biodrugs. In view of the rising prevalence of multi-resi­stant staphylococci such combined therapy may be very useful in future [8,9,21]. Here are a few data supporting the submission of the presented opportunities for discus­sion: in Poland there are 21 Regional Centers for blood donation, and 2 Departmental Centers under the super­vision of the National Blood Center. Taking into account the diversity of platelet-rich products prepared in each regional blood center and their short time of suitability for transfusion (platelets are discarded after five days of storage at 22°C), they seem to be a very rich and inexhau­stible source of natural biocidal peptides.

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The authors have no potential conflicts of interest to declare

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