A relative comparison of antibacterial strength between platelet lysate and platelet factor 4 in vitro

doi:10.21203/rs.3.rs-1789529/v1

Background

Recently the role of platelets in the tissue regeneration, wound healing, and prevention and control of infections have been reported. The treatment of bacterial infections has become a global challenge due to the spread of multidrug-resistant bacteria; therefore, the development of new treatment options for the mentioned infections is essential. We assessed the antibacterial effect of human platelet lysate (PL) and platelet factor 4 (PF4) on bacteria commonly found in nosocomial infections.

Methods

In this study, the antibacterial activity of PL and PF4 was tested on Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Enterococcus faecalis, Acinetobacter baumannii by well diffusion and serial micro-dilution methods.

Results

In vitro results showed that PF4 inhibited the growth of S. aureus, E. faecalis A. baumannii, as a new antibacterial agent. While PL had no discernible impact on the growth parameters of any bacterium tested.

Conclusion

PF4 that is a biocompatible and safe product could be potentially useful in nosocomial infections. The use of PF4 is an attractive strategy for treating A. baumannii and S. aureus, E. faecalis nosocomial infections because it inhibits bacterial growth.

Platelet factor 4

platelet lysate

antibacterial effect

nosocomial infections

Platelets are disk-shaped cells originating from the megakaryocyte lineage (1–3). The average longevity of platelets in the peripheral bloodstream is 5–10 days (4). A mature platelet may be categorized into one of the following cytoplasmic granule types: dense granules, α-granules, and lysosomes. Platelets cause skin cell regeneration, angiogenesis, collagen, and fibroblastic production. Their use is, however, restricted due to immunologic reactions. Studies have suggested that platelets are the cellular source of antibacterial activities (5–8). Numerous researchers have separated a lot of antibacterial substances from human and animal platelets (9). CXCL4/PF 4 is the first discovered chemokine. CXCL4 is highly abundant in platelet α-granules and is released during platelet activation (10, 11). It plays an important role in the chemotaxis of neutrophils and monocytes through binding to the chemokine receptor CXCR3, CXCL4 with significant contribution to angiogenesis (12), tissue repair (13), and regulation of tissue damage in complex inflammatory diseases, such as systemic sclerosis (14), melanoma (15) and antimalarial models (16). Recent findings have shown that CXCL4 participates in inflammation and innate immunity (17), particularly, bacterial clearance (18, 19). Pooled human platelet PL contains high levels of growth-promoting factors such as platelet-derived growth factor (PDGF)-AB, PDGF-AB/BB, and transforming growth factor (TGF)-β₁. Moreover, PL contains high concentrations of chemokines (CCL5/RANTES [regulated on activation, normal T cell expressed and secreted] and CXCL1/2/3) and soluble adhesion molecules (sCD40L, sVCAM [vascular cell adhesion molecule], and sICAM [intercellular adhesion molecule]). A variety of other cytokines, including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-la, IL-7, IL-8, macrophage inflammatory protein (MIP)-1α, MIP-1β, TGF-β₁ and interferon (IFN)-γ can be also found in PL (20). Antibiotics have been used to treat or prevent some types of bacterial infections, but they can cause various adverse reactions. Bacterial infections and antimicrobial resistance (AMR) are some of the principal public health problems associated with antibiotics consumption. Therefore, the purpose of the present study is to evaluate the effect of pooled human PL and CXCL4/PF4 on three Gram-negative and Gram-positive bacteria mainly responsible for wound and nosocomial infections.

Before performing this study, we attained the Ethic Code IR.UMSHA.REC.1398.494 from the Research Ethics Committee of Hamadan University of Medical Sciences. All experiments in this study were performed correctly in accordance with relevant named guidelines and regulations. The current research investigated the antibacterial properties of pooled human PL and PF4 on several standard bacteria including E. coli (ATCC 25922), S aureus (ATCC25923), E. faecalis (ATCC 29212), P. aeruginosa (ATCC 27853), A. baumannii (ATCC 19606), and K. pneumoniae (ATCC 10031). The mentioned bacterial strains were supplied by Pasteur Institute (Tehran, Iran) which were then inoculated on the Muller Hinton Agar medium followed by culturing on the plates and incubating at 37°C for 24 h. Upon growth, staining was carried out to determine their Gram-positive and Gram-negative properties. Fresh bacterial suspensions were prepared in sterile distilled water at an optical density corresponding to 0.5 McFarland (1 × 10⁸ CFU/mL) (21).

Pooled human PL and PF4 preparations

PL was prepared based on Schallmoser and Strunk method (22). First, human platelet concentrates (PCs) were collected from the blood bank of Farshchian Heart and Vascular Hospital as waste products and insuring that they were not clinically applicable. PC bags were stored at -20°C until the experiments. The PCs were then placed in a water bath at 37°C to melt. Afterwards, 50-mL of thawed PCs were poured into the conical tube followed by centrifugation at 7600 rpm for 30 min and 4°C temperature in a refrigerated centrifuge (Eppendorf, Model 5430 R, Hamburg, Germany) to eliminate cell particles and debris known as platelet bodies. The supernatants were transferred into a new conical tube and kept at -70°C until usage. Recombinant PF4 protein with 20 µg concentration was provided by Abnova (Taipei City 114, Taiwan). 20 µg lyophilized PF4 was reconstituted in 3 mL PBS (BioBench, Tehran, Iran) and aliquoted in 200-µL volumes. The aliquots were stored at -20°C until usage.

Measurement of total protein in concentration PL

The total protein released in PL was assayed by the Bradford method and was established in 1976 (23). In a typical procedure, nine serial dilutions were prepared from bovine serum albumin standard (BSA, Fraction V, Roche Diagnostics GmbH, Germany) in 1X PBS. In the presence of an improved Coomassie Brilliant Blue G-250 (DNAbiotech Co., Tehran, Iran), a blue dye complex was formed, and the absorption was evaluated at 595 nm using a 96-well ELISA reader (DANA-3200, GARNI Medical Engineering Co., Tehran, Iran). The total protein of the unknown PL samples was determined based on the standard curve linear equation.

Antibacterial activity assessment

Agar-well diffusion

The antibacterial properties of PF4 and PL were tested by agar-well diffusion method using Mueller-Hinton agar (QUELAB Laboratories Inc., Montreal, Quebec, Canada). Four mm diameter wells were created in Mueller-Hinton agar Petri dishes (QC LAB, China) in which 50-µL aliquots of each PF4 and PL solution were dispensed. The agar Petri dishes experienced 24 hours of incubation at 37°C (24).

Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration (MIC) of the samples was measured using the broth microdilution approach. Each 96-well microtiter plate (Biologix Europe GmbH, Köln, Germany) contained positive controls (bacteria without antibacterial agents), negative controls (Mueller-Hinton only), and twofold serial dilutions of each antibacterial agent. The bacteria were initially cultured in Mueller-Hinton broth (QUELAB Laboratories Inc., Montreal, Quebec, Canada) followed by incubation at 37°C. After 24 h, when the bacterial growth reached the logarithmic phase, their turbidity was read at 600 nm to calculate their concentrations. Mueller-Hinton broth was added to all 96 wells of the microplate. In the microdilution method, the first well is diluted by 10% while the last one is set as control, as it contains no product. Then, a given level of the bacterial culture was added to the wells followed by 24 hours of incubation at 37°C. These ATCC strains were employed as controls: E. coli (ATCC 25922), S. aureus (ATCC25923), E. faecalis (ATCC 29212), P. aeruginosa (ATCC 27853), A. baumannii (ATCC 19606), and K. pneumoniae (ATCC 10031). The findings were interpreted based on the breakpoints of the Clinical and Laboratory Standards Institute (CLSI, 2020) (25).

Minimum bactericidal concentration (MBC)

the MBC is defined as the lowest concentration of antimicrobial PF4 and PL needed to kill 99.9% of the final inoculum after incubation for 24 h under a standardized set of conditions described in the document M26-A and the result of its culture on agar medium is negative (26).

Total protein concentration of PLs

We finally collected twenty-eight pooled PL samples, each one included in a 50-mL conical tube. As represented in Fig. 1, the total protein in PL was calculated from a formula in the Bradford method. Six PL samples with the highest concentration of protein were applied correspondingly to treat the bacterial strains.

Well diffusion

The inhibition zone of bacterial growth was measured for each well on the culture medium. The mean values for the zone of inhibition produced by PL and PF4, positive control, and negative control against six bacteria are shown in Table 1. We observed that there was robust bacterial growth around the wells containing PF4 for S. aureus, E. faecalis, and A. baumannii on all plates. However, no growth was observed around the wells containing PL. We did not detect any activity against K. pneumoniae, P. aeruginosa, and E. coli for PF4 (Table 1 and Fig. 2).

Table 1

Well diffusion of two agents plus control antibiotics. The inhibitory zone of each agent is expressed as mm unit.
Bacteria Spp. Agent	Staphylococcus aureus	Enterococcus faecalis	Escherichia coli	Klebsiella pneumonia	Pseudomonas aeruginosa	Acinetobacter baumannii
PL	4	4	4	4	4	4
PF4	21	19	4	4	4	22
Chloramphenicol disk	-	-	22	18	-	-
Piperacillin disk	-	-	-	-	25	21
Vancomycin disk	19	17	-	-	-	-

MIC

In our study, the MIC was defined as the lowest PL and PF4 that could inhibit bacterial growth. After 24 hours of incubation, bacterial standard strains growth were observed in all wells that contained PL. Based on the results, PL had no remarkable antibacterial activity against all bacteria. PF4 at 0.02 µg/mL concentration was observed to inhibit the growth of S. aureus, and E. faecalis. PF4 at 0.04 µg/mL concentration was observed to inhibit the growth of A. baumannii. In addition, we did not observe such effects for K. pneumoniae, P. aeruginosa, and E. coli. (Table 2 and Fig. 3).

Table 2

Antibacterial effect of PF4 measured by MIC and MBC.
Bacteria Spp. Measurement	Staphylococcus aureus	enterococcus faecalis	Escherichia coli	Klebsiella pneumonia	Pseudomonas aeruginosa	Acinetobacter baumannii
MIC	0.206	0.206	-	-	-	0.412
MBC	0.412	0.412	-	-	-	0.825

MBC

In the current study, MBC was defined as the minimum product concentration required for killing 99.9% of all bacteria. Dilutions of 0.04, 0.04, and 0.08 µg/mL of the PF4 were considered for MBCs against S. aureus, E. faecalis, and A. baumannii, respectively. PL was unable to cause bacterial death in any well.

Antibiotics are used for treating of infections. Antibiotic resistance is continually increasing and leads to a public health threat, which is worsened by the overuse of antibiotics. Due to an increase in the use of synthetic antibiotics, the incidence of bacterial resistance to antibiotics have been increased and there is a need for new antibacterial agents. Bacterial infection as a major therapeutic challenge leads to morbidity and mortality worldwide (27). Use of antibiotics for the treatment of infections is limited because of a rise in the prevalence of multidrug resistance (MDR) bacteria. Also, the discovery of novel agents along with elevated antibacterial resistance in bacteria is increasing (28). In this research, we evaluated the impact of the platelet components on the inhibition of bacterial growth. The PL and PF4 results indicated that PF4 had a significant effect on bacterial growth inhibition. According to the findings of the agar-well diffusion and MIC assays, PL was found with no demonstrable activity against bacterial growth and could not completely inhibit the growth of the considered bacteria. PF4 and PL were employed to inhibit S aureus (ATCC25923), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), E. faecalis (ATCC 29212), A. baumannii (ATCC 19606), and K. pneumoniae (ATCC 10031). In vitro findings have indicated that PF4 could inhibit the growth of S. aureus (ATCC25923), E. faecalis (ATCC 29212) A. baumannii (ATCC 19606). Compared to Gram-negative bacteria, Gram-positive bacteria are more sensitive to PF4. Yue et al. demonstrated that CXCL4 is a critical chemokine playing a critical role in bacterial clearance in P. aeruginosa infection by using neutrophils to the lungs as well as intracellular bacterial killing (29).

Tang et al. evaluated the possible antibacterial activity of platelet proteins. They stimulated normal human platelets using human thrombin in vitro. Using reversed-phase high-performance liquid chromatography, components of the stimulated-platelet supernatants were purified to homogeneity. Purified peptides, which showed an inhibitory effect on Escherichia coli ML35 in the agar diffusion assay were characterized using amino acid analysis, mass spectrometry, and sequence determination, through which seven thrombin-releasable antimicrobial peptides were identified from human platelets: connective tissue activating peptide 3 (CTAP-3), PF4, RANTES, fibrinopeptide A (FP-A) platelet basic protein, thymosin β-4 (Tβ-4), and fibrinopeptide B (FP-B). Except for FP-A and FP-B, purification of all peptides from acid extracts of non-stimulated platelets were done. Then, in vitro antibacterial effect of seven released peptides was evaluated against E. coli and S. aureus and Candida albicans, and Cryptococcus neoformans. Each peptide was found with activity against a minimum of two organisms. Generally, they were stronger against bacteria than against fungi (30). This interaction can markedly affect the balance between infection and immunity. Jafarzadeh evaluated the antibacterial activity of human PC in Staphylococcus epidermidis, Pseudomonas aeruginosa, S. aureus, Micrococcus luteus, Escherichia coli, and Proteus vulgaris. Human PC exhibited antibacterial effect against S. aureus and S. epidermidis, while no effect was found against M. luteus, E. coli, Pseudomonas aeruginosa, and P. vulgaris (31).

In a study by Shariati et al., the potential antibacterial and wound healing properties of two different platelet-derived biomaterial (PdB) (CaCl₂-PdB and F-PdB) analyzed against methicillin-resistant S. aureus and P. aeruginosa. The results of disk diffusion (DD) and broth microdilution (BMD) showed that both PdB performed very well on MRSA, whereas P. aeruginosa was inhibited only by F-PdB and was less susceptible to MRSA than PdBs. The time-kill assay also showed that F-PdB has an antibacterial effect in four hours for two strains. Histopathological studies showed that the treated groups had fewer inflammatory cells and necrotic tissues (32). The antibacterial and wound healing effects of PL on K. pneumoniae and A. baumannii burn wound infections were investigated. PdB could inhibit the growth of A. baumannii at the highest concentration (0.5), whereas no inhibitory effects were found against K. pneumoniae. In contrast, PdB could significantly inhibit the bacterial growth in treated animal wounds than the control groups (P value < 0.05) (33).

The inhibitory effects of platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were determined on the growth of S. aureus, E. coli, Streptococcus agalactiae, K. pneumonia, P. aeruginosa, Staphylococcus epidermidis, Shigella sp., and Serratia sp .PRP–1 was generated using the one-step blood centrifugation method; whereas, for PRP–2 and PPP the two-step centrifugation protocol was used. The whole blood (WB) and platelet-poor plasma were found with no discernible impact on the growth parameters of the evaluated bacteria in the present study, PRP–1 could reduce the growth rate of some selected strains. Likewise, although PRP–2 could inhibit the growth of Shigella sp., E. coli, S. aureus, S. agalactiae, and S. epidermidis, it was not effective in the growth of P. aeruginosa, K. pneumoniae, and Serratia sp. (24).

Gilbertie’s study indicated that a PRP lysate (PRP-L) had antibiofilm effects and restored the antibacterial effect on synovial fluid biofilms in vitro and reported that PRP-L can augment current antibacterial treatment regimens which in turn reducing morbidity and mortality associated with infectious arthritis. The current assessed the inhibitory effects of PL and PF4 on the growth of common pathogenic bacteria, including E. coli, K. pneumonia, P. aeruginosa, and Staphylococcus spp. Their findings indicated in vitro inhibitory effects of PL and PF4 on the growth of these pathogenic bacteria (34).

The effect of platelet gel (PG) obtained from umbilical cord blood (UCB) on diabetic foot ulcers was assessed and no significant difference was found in the wound size between PG, PPP, and placebo groups (35). The antibacterial effect of PPP, PRP, PG, and solvent/detergent-treated PL biomaterials was evaluated on wound bacteria. It has been reported that plasma complements cause the antibacterial effect of plasma and platelet biomaterials against K. pneumoniae, E. coli, P. aeruginosa, and S. aureus (36). According to Palankar’s study, platelets kill bacteria through the bridge between innate and adaptive immunity using PF4 and FcγRIIA. Accordingly, through the connection between innate and adaptive immune mechanisms, platelets and anti-PF4/polyanion antibodies cause an antibacterial host reaction (37). The medication for the combination of delivery of PL and vancomycin (VCM) hydrochloride was developed by Rossi and colleagues as an anti-infective model drug for chronic skin ulcers. They found the mechanical strength to withstand packaging and handling stress capable of absorbing a huge amount of wound exudate for forming a protective gel on the lesion site (38).

In Gordon’s study on the antimicrobial effect of equine PL, findings demonstrated the potential value of PL as a broad-spectrum antimicrobial (39). Rostami investigated the in vitro evaluation of the antibacterial activity of platelet-rich plasma against selected oral and periodontal pathogens, PRP had strong in vitro antibacterial activity against Streptococcus Mitis, Streptococcus Mutans, and Neisseria Lactamica with the mean zone of inhibition diameters of 6.73 ± 0.52, 5.8 ± 0.43 and 6.67 ± 0.43 mm, respectively. PRP is an effective antibacterial agent along with conventional antibiotic treatments against oral and periodontal infections (40).The current research results indicated the in vitro inhibitory effects of PF4 on the growth of S. aureus, E. faecalis, and A. baumannii. More studies, both in vitro and in vivo, must examine the antibacterial effects of PF4 and PL.

The current study provides evidence that there is the antibacterial effect of PF4 on S. aureus and Enterococcus faecalis, A. baumannii, but PL had no similar qualification. Further researches on PF4 should be employed to determine the wide antibacterial spectrum, its antibacterial capacity along with antibiotics, and their efficacy in vivo. Subsequently, PF4 can be considered as an alternative for the infection control, preferably in the antibiotic-resistance challenges.

PL: platelet lysate

PF4: platelet factor 4

PDGF: platelet-derived growth factor

TGF-β₁: transforming growth factor beat-1

RANTES: regulated on activation, normal T cell expressed and secreted

VCAM: vascular cell adhesion molecule

ICAM: intercellular adhesion molecule

G-CSF: granulocyte colony-stimulating factor

GM-CSF: granulocyte-macrophage colony-stimulating factor

IL: interleukin

MIP: macrophage inflammatory protein

IFN: interferon

AMR: antimicrobial resistance

PC: platelet concentrate

MIC: minimum inhibitory concentration

MBC: minimum bactericidal concentration

MDR: multidrug resistance

CTAP-3: connective tissue activating peptide 3

FP-A: fibrinopeptide A

Tβ-4: thymosin β-4

FP-B: fibrinopeptide B

PdB: platelet-derived biomaterial

DD: disk diffusion

BMD: broth microdilution

PRP: platelet-rich plasma

PPP: platelet-poor plasma

WB: whole blood

PRP-L: PRP lysate

VCM: vancomycin

a. Funding

This project was completed with the financial support of the Vice-chancellor for Research and Technology, Hamadan University of Medical Sciences (Grant No. 9807165319).

b. Conflicts of interest/Competing interests

There is neither conflict of interest nor competing interest between everyone who is determined as an author of this manuscript.

c. Availability of data and material (data transparency)

Not applicable.

d. Code availability

Not applicable.

e. Ethics approval

Approval was gained from the Research Ethics Committee of Hamadan University of Medical Sciences (Ethics Committee Identifier: IR.UMSHA.REC.1398.494).

f. Authors' contributions

Material procurement, data processing, and analysis were done by Mahdane Roshani and Alireza Goodarzi equally. The first draft of the manuscript was commented by Mohammadreza Arabestani. All authors read and confirmed the final version of manuscript.

g. Consent to participate

All human platelet concentrates were used in this study after the expiry date of product ensuring without any clinical application in the future.

h. Consent for publication

Not applicable.

Acknowledgements

We should be friendly thankful for the kind collaborations of Mr. Vahid Delavari (conscientious technician of Hematology and Blood Bank Laboratory, Department of Medical Laboratory Sciences in Hamadan University of Medical Sciences) to organize some laboratory devices and instruments which this experiment was labor-intensive void of his assistance.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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No competing interests reported.

SupplementarymaterialScientificReports.pdf

A relative comparison of antibacterial strength between platelet lysate and platelet factor 4 in vitro

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Pooled human PL and PF4 preparations

Measurement of total protein in concentration PL

Antibacterial activity assessment

Agar-well diffusion

Minimum inhibitory concentration (MIC)

Minimum bactericidal concentration (MBC)

Results

Total protein concentration of PLs

Well diffusion

MIC

MBC

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1