Cutibacterium acnes contamination does not enhance the proinflammatory profile of platelet concentrates.

BACKGROUND: Cutibacterium acnes, a common anaerobic platelet concentrate (PC) contaminant, has been associated with rare mild adverse transfusion reactions and is often considered a harmless commensal. Notably, C. acnes can cause chronic infections and has been shown to induce the release of proinflammatory cytokines by immune cells. Since elevated concentrations of proinflammatory factors in PCs have been linked to noninfectious adverse reactions, this study aimed to assess whether C. acnes could elicit the release and accumulation of proinflammatory factors during PC storage, thereby enhancing the risk of such reactions. STUDY DESIGN/METHODS: Four ABO-matched buffy coat PCs were pooled and split into six units, each were inoculated with either saline (negative control), a Staphylococcus aureus isolate (positive control, 30 colony forming units [CFU]/unit), or four C. acnes PC isolates (10 CFU/mL) and stored at 20-24°C with agitation. Bacterial counts, platelet activation, and concentration of proinflammatory factors were assessed on days 0, 3, and 5. N = 3. RESULTS: C. acnes counts remained stable, while S. aureus proliferated reaching 108CFU/mL by the end of PC storage. By day 5, no significant differences in platelet activation or proinflammatory cytokine profiles were observed in C. acnes-contaminated PCs compared to the negative control (p > .05), while there was a significant increase (p ≤ .05) in sCD40L concentration (day 3), and platelet activation and IL-8 concentration (day 5) in S. aureus-contaminated units. DISCUSSION: C. acnes contamination does not promote the accumulation of proinflammatory factors in the absence of proliferation during storage and may not enhance the risk of inflammatory reactions when transfused to patients.

Proliferation of psychrotrophic bacteria in cold-stored platelet concentrates.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PC) are stored at 20-24°C to maintain platelet functionality, which may promote growth of contaminant bacteria. Alternatively, cold storage of PC limits bacterial growth; however, data related to proliferation of psychotrophic species in cold-stored PC (CSP) are scarce, which is addressed in this study. MATERIALS AND METHODS: Eight laboratories participated in this study with a pool/split approach. Two split PC units were spiked with ~25 colony forming units (CFU)/PC of Staphylococcus aureus, Klebsiella pneumoniae, Serratia liquefaciens, Pseudomonas fluorescens and Listeria monocytogenes. One unit was stored under agitation at 20-24°C/7 days while the second was stored at 1-6°C/no agitation for 21 days. PC were sampled periodically to determine bacterial loads. Five laboratories repeated the study with PC inoculated with lyophilized inocula (~30 CFU/mL) of S. aureus and K. pneumoniae. RESULTS: All species proliferated in PC stored at 20-24°C, reaching concentrations of ≤109 CFU/mL by day 7. Psychrotrophic P. fluorescens and S. liquefaciens proliferated in CSP to ~106 CFU/mL and ~105 CFU/mL on days 10 and 17 of storage, respectively, followed by L. monocytogenes, which reached ~102 CFU/mL on day 21. S. aureus and K. pneumoniae did not grow in CSP. CONCLUSION: Psychrotrophic bacteria, which are relatively rare contaminants in PC, proliferated in CSP, with P. fluorescens reaching clinically significant levels (≥105 CFU/mL) before day 14 of storage. Cold storage reduces bacterial risk of PC to levels comparable with RBC units. Safety of CSP could be further improved by implementing bacterial detection systems or pathogen reduction technologies if storage is beyond 10 days.

Comparable bacterial growth in platelet concentrates suspended in plasma and platelet additive solution and improved detection of bacterial contamination using a new generation automated culture system.

BACKGROUND: Microbial screening of platelet concentrates (PC) with automated culture methods is widely implemented to reduce septic transfusion reactions. Herein, detection of bacterial contamination in PC was compared between units prepared in plasma and a mix of plasma and platelet additive solution (PAS) and between the BACT/ALERT 3D and next generation BACT/ALERT VIRTUO systems. STUDY DESIGN/METHODS: Double apheresis units were split into single units, diluted in either PAS (PAS-PC) or plasma (plasma-PC), and tested for in vitro quality and sterility prior to spiking with ~30 CFU/unit of Staphylococcus epidermidis, Staphylococcus aureus, Serratia marcescens, and Klebsiella pneumoniae or ~10 CFU/mL of Cutibacterium acnes. Spiked PC were sampled for BACT/ALERT testing (36 and 48 h post-spiking) and colony counts (24, 36, and 48 h post-spiking). Times to detection (TtoD) and bacterial loads were compared between PC products and BACT/ALERT systems (N = 3). RESULTS: Bacterial growth was similar in plasma-PC and PAS-PC. No significant differences in TtoD were observed between plasma-PC and PAS-PC at the 36-h sampling time except for S. epidermidis which grew faster in plasma-PC and C. acnes which was detected earlier in PAS-PC (p < .05). Detection of facultative bacteria was 1.3-2.2 h sooner in VIRTUO compared with 3D (p < .05) while TtoD for C. acnes was not significantly different between the two systems. DISCUSSION: Comparable bacterial detection was observed in plasma-PC and PAS-PC with PC sampling performed at 36-h post blood collection. PC sampling at ≤36 h could result in faster detection of facultative pathogenic organisms with the VIRTUO system and improved PC safety.

Sebum Components Dampen the Efficacy of Skin Disinfectants against Cutibacterium acnes Biofilms.

At Canadian Blood Services, despite the use of 2% chlorhexidine and 70% isopropyl alcohol (standard disinfectant, SD) prior to venipuncture, Cutibacterium acnes evades eradication and is a major contaminant of platelet concentrates (PCs). Since C. acnes forms bacterial aggregates known as biofilms in the sebaceous niches of the skin, this study aimed to assess whether sebum-like components impact disinfectant efficacy against C. acnes leading to its dominance as a PC contaminant. C. acnes mono-species and dual-species biofilms (C. acness and a transfusion-relevant Staphylococcus aureus isolate) were formed in the presence and absence of sebum-like components and exposed to SD, a hypochlorous acid-based disinfectant (Clinisept+, CP), or a combination of both disinfectants to assess disinfectant efficacy. Our data indicate that sebum-like components significantly reduce the disinfectant efficacy of all disinfectant strategies tested against C. acnes in both biofilm models. Furthermore, though none of the disinfectants led to bacterial eradication, the susceptibility of C. acnes to disinfectants was heightened in an isolate-dependent manner when grown in the presence of S. aureus. The reduction of skin disinfection efficacy in the presence of sebum may contribute to the overrepresentation of C. acnes as a PC contaminant and highlights the need for improved disinfection strategies.

Nutrient supplementation of culture media improves the detection of Cutibacterium acnes in platelet components by an automated culture system.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PCs) contaminated with Cutibacterium acnes are often transfused prior to detection by the BACT/ALERT system. Though C. acnes is implicated in mild transfusion reactions, delayed clinical effects are unknown. This study assessed the ability to enhance C. acnes detection by supplementing culture media with Tween 80 (T80, an oleic acid source) and a commercial nutrient supplement. MATERIALS AND METHODS: Anaerobic culture bottles (BPN) were supplemented with T80 or oleic acid. T80-supplemented BPN bottles were inoculated with four C. acnes isolates (10 or 100 colony-forming units [CFU]/bottle) or other transfusion-relevant bacteria (10 CFU/bottle). Samples of plasma containing SSP+ (platelet additive solution [PAS]) (PAS-plasma) at different concentrations, plasma-PCs and PAS-PCs, spiked with two C. acnes isolates (10 CFU/bottle), were inoculated into T80-supplemented BPN bottles. Furthermore, plasma-PCs were spiked with C. acnes and tested in BPN bottles supplemented with the BD Difco Supplement VX (BDVx). Bottles were incubated in the BACT/ALERT system and times to detection (TtoD) were compared (N = 3). RESULTS: A reduction in TtoD of ≤3.5 days was observed for C. acnes in T80-supplemented BPN, while other species did not show the same effect. However, false positives were observed when T80-supplemented BPN was inoculated with PAS-plasma (except for 70% PAS:30% plasma), plasma-PCs or PAS-PCs. Oleic acid supplementation also resulted in false positives. Interestingly, BDVx-supplemented BPN reduced the TtoD of C. acnes in PCs by ≤1.2 days (p < 0.05), with no false-positive results. CONCLUSION: BDVx supplementation for detection of C. acnes from PCs could result in timely unit retrieval, preventing the transfusion of contaminated products. In clinical settings, T80 supplementation could significantly enhance C. acnes detection from non-blood-derived samples.

A multiphasic approach to solve misidentification of Cutibacterium acnes as Atopobium vaginae during routine bacterial screening of platelet concentrates using the VITEK 2 system.

Skin flora bacteria, such as Cutibacterium acnes , are the predominant contaminants of blood products used for transfusion. Platelet concentrates (PCs), a therapeutic product used to treat patients with platelet deficiencies, are stored at ambient temperature under agitation, providing ideal conditions for bacterial proliferation. At Canadian Blood Services, PCs are screened for microbial contamination using the automated BACT/ALERT culture system. Positive cultures are processed and contaminating organisms are identified using the VITEK 2 system. Over a period of approximately 2 years, several PC isolates were identified as Atopobium vaginae to a high level of confidence. However, since A. vaginae is associated with bacterial vaginosis and is not a common PC contaminant, a retrospective investigation revealed that in all cases C. acnes was misidentified as A. vaginae . Our investigation demonstrated that the media type used to grow PC bacterial isolates can have a significant impact on the results obtained on the VITEK 2 system. Furthermore, other identification methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD-TOF MS) and PCR amplification of the 16S RNA gene were only partially successful in the identification of C. acnes . Therefore, our findings support a multiphasic approach when PC isolates are identified as A. vaginae by the VITEK 2 system for proper identification of C. acnes using macroscopic, microscopic and other biochemical analyses.

Assessment of bacterial growth in leukoreduced cold-stored whole blood supports overnight hold at room temperature prior to filtration: A pilot study.

BACKGROUND AND OBJECTIVES: Whole blood (WB) transfusion has regained attention to treat trauma patients. We reported no significant changes in in vitro quality through 21 days of cold storage for leukoreduced WB (LCWB) when time to filtration was extended from 8 to 24 h from collection. This study evaluated the impact of extended WB-hold at room temperature (RT) prior to leukoreduction on proliferation of transfusion-relevant bacteria. MATERIALS AND METHODS: WB units were spiked with suspensions of Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Listeria monocytogenes prepared in saline solution (SS) or trypticase soy broth (TSB) to a concentration of ~0.2 CFU/ml (N = 6). Spiked units were held at RT for 18-24 h before leukoreduction and cold-stored for 21 days. Bacterial growth was determined on days 2, 7, 14 and 21. In vitro quality of WB inoculated with unspiked diluents was assessed. RESULTS: K. pneumoniae and S. pyogenes proliferated in WB prior to leukoreduction reaching concentrations ≤102 CFU/ml. These bacteria, however, did not proliferate during the subsequent cold storage. S. aureus did not survive in WB while L. monocytogenes reached a concentration of ~102 CFU/ml by day 21. LCWB in vitro quality was not affected by SS or TSB. CONCLUSION: Extended WB-hold prior to leukoreduction allowed proliferation of bacteria able to resist immune clearance, although they did not grow to clinically significant levels. While L. monocytogenes proliferated in LCWB, clinically relevant concentrations were not reached by day 21. These data suggest that transfusing LCWB may not pose a significant bacterial contamination safety risk to transfusion patients.

Bacterial survival in whole blood depends on plasma sensitivity and resistance to neutrophil killing.

BACKGROUND: Whole blood (WB) is held at room temperature for not more than 24 hours before blood component manufacturing. The ability of several culture collection, skin-derived, and transfusion-related bacteria to survive in WB stored at 22 ± 2°C for 24 hours was investigated in this study. STUDY DESIGN AND METHODS: Twenty-one bacteria of the species Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus capitis, Streptococcus agalactiae, Serratia liquefaciens, Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, and Yersinia enterocolitica were inoculated into 7-mL aliquots of WB at a concentration of 500 colony-forming units (CFU)/mL. Spiked WB was stored aerobically at 22 ± 2°C, and bacterial viability and growth were monitored at 3, 8, and 24 hours during WB storage. Bacteria that showed decreased viability during WB incubation were further characterized for their sensitivity to plasma factors and neutrophil killing. RESULTS: There were three different scenarios for bacterial behavior during the hold of WB at 22 ± 2°C. Five bacteria proliferated (p < 0.03), 11 remained viable or showed low proliferation, and a third group of five bacteria had decreased or lost viability (p < 0.01). Three of the latter five bacteria were plasma-sensitive while the other two were plasma-resistant but susceptible to neutrophil killing (p = 0.01). CONCLUSIONS: The bactericidal activity of WB can be the result of plasma sensitivity or neutrophil killing. Bacteria with a starting inoculum of 500 CFU/mL, and able to resist WB immune factors, can proliferate to clinically significant levels posing a potential safety risk to transfusion patients. Results of this pilot study should be validated under standard WB collection and storage conditions.

Does Treatment Order Matter? Investigating the Ability of Bacteriophage to Augment Antibiotic Activity against Staphylococcus aureus Biofilms.

The inability to effectively treat biofilm-related infections is a major clinical challenge. This has been attributed to the heightened antibiotic tolerance conferred to bacterial cells embedded within biofilms. Lytic bacteriophages (phages) have evolved to effectively infect and eradicate biofilm-associated cells. The current study was designed to investigate the ability of phage treatment to enhance the activity of antibiotics against biofilm-forming Staphylococcus aureus. The biofilm positive S. aureus strain ATCC 35556, the lytic S. aureus phage SATA-8505, and five antibiotics (cefazolin, vancomycin, dicloxacillin, tetracycline, and linezolid), used to treat S. aureus infections, were tested in this study. The ability of the SATA-8505 phage to augment the effect of these antibiotics against biofilm-associated S. aureus cells was assessed by exposing them to one of the five following treatment strategies: (i) antibiotics alone, (ii) phage alone, (iii) a combination of the two treatments simultaneously, (iv) staggered exposure to the phage followed by antibiotics, and (v) staggered exposure to antibiotics followed by exposure to phage. The effect of each treatment strategy on biofilm cells was assessed by enumerating viable bacterial cells. The results demonstrate that the treatment of biofilms with either SATA-8505, antibiotics, or both simultaneously resulted in minimal reduction of viable biofilm-associated cells. However, a significant reduction [up to 3 log colony forming unit (CFU)/mL] was observed when the phage treatment preceded antibiotics. This effect was most pronounced with vancomycin and cefazolin which exhibited synergistic interactions with SATA-8505, particularly at lower antibiotic concentrations. This in vitro study provides proof of principle for the ability of phages to augment the activity of antibiotics against S. aureus biofilms. Our results also demonstrate that therapeutic outcomes can be influenced by the sequence in which these therapeutic agents are administered, and the nature of their interactions. Further investigation into the interactions between lytic phages and antibiotics against various biofilm-forming organisms is important to direct future clinical translation of efficacious antibiotic-phage combination therapeutic strategies.

Enhancing blood donor skin disinfection using natural oils.

BACKGROUND: Effective donor skin disinfection is essential in preventing bacterial contamination of blood components with skin flora bacteria like Staphylococcus epidermidis. Cell aggregates of S. epidermidis (biofilms) are found on the skin and are resistant to the commonly used donor skin disinfectants chlorhexidine-gluconate and isopropyl alcohol. It has been demonstrated that essential oils synergistically enhance the antibacterial activity of chlorhexidine-gluconate. The objective of this study was to test plant-extracted essential oils in combination with chlorhexidine-gluconate or chlorhexidine-gluconate plus isopropyl alcohol for their ability to eliminate S. epidermidis biofilms. STUDY DESIGN AND METHODS: The composition of oils extracted from Artemisia herba-alba, Lavandula multifida, Origanum marjoram, Rosmarinus officinalis, and Thymus capitatus was analyzed using gas chromatography-mass spectrometry. A rabbit model was used to assess skin irritation caused by the oils. In addition, the anti-biofilm activity of the oils used alone or in combination with chlorhexidine-gluconate or chlorhexidine-gluconate plus isopropyl alcohol was tested against S. epidermidis biofilms. RESULTS: Essential oil concentrations 10%, 20%, and 30% were chosen for anti-biofilm assays, because skin irritation was observed at concentrations greater than 30%. All oils except for O. marjoram had anti-biofilm activity at these three concentrations. L. multifida synergistically enhanced the anti-biofilm activity of chlorhexidine-gluconate and resulted in the highest anti-biofilm activity observed when combined with chlorhexidine-gluconate plus isopropyl alcohol. Gas chromatography-mass spectrometry revealed that the main component contributing to the activity of L. multifida oil was a natural terpene alcohol called linalool. CONCLUSION: The anti-biofilm activity of chlorhexidine-gluconate plus isopropyl alcohol can be greatly enhanced by L. multifida oil or linalool. Therefore, these components could potentially be used to improve blood donor skin disinfection.

Noninvasive pH monitoring for bacterial detection in platelet concentrates.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) remains the prevalent posttransfusion infectious risk. The pH SAFE system, a noninvasive method used to measure pH of PC for quality control, was evaluated herein as a rapid method to detect bacterial contamination in PCs. STUDY DESIGN AND METHODS: Pairs of ABO-D-matched apheresis and buffy coat PCs were pooled and split into two pH SAFE platelet bags. One of the bags served as the control unit, while the other was inoculated with one of nine clinically relevant bacteria (target concentration approx. 1 colony-forming units [CFUs]/mL). The pH of both PCs was measured over 7 days of storage at approximately 4-hour intervals during daytime. One-milliliter samples were taken at the testing points to determine bacterial concentration. RESULTS: PCs with pH values of less than 6.6 or with a pH change over time (ΔpH/Δtime) greater or equal than 0.046 pH units/hr are suspected of being contaminated. pH decreased significantly during storage in all bacterially inoculated PC at concentrations of more than 10(7) CFUs/mL (p < 0.0001). A significant decrease in pH (p < 0.0001) was noticed as early as 28 hours in units with Bacillus cereus and as late as 125 hours in units containing Staphylococcus epidermidis. Interestingly, PCs containing Gram-negative species showed a decline in pH followed by a rebound. CONCLUSIONS: The pH SAFE system allows for repeated, noninvasive pH screening during PC storage. A significant decrease in pH could serve as an indicator of clinically significant levels of bacterial contamination. Since differences in pH decline were observed among bacterial species, continuous pH monitoring in PCs is recommended.

Biofilm formation by Staphylococcus capitis strains isolated from contaminated platelet concentrates.

Bacterial contamination of platelet concentrates (PCs) poses the greatest infectious risk in modern transfusion medicine despite the implementation of measures such as improved skin disinfection and first aliquot diversion. The majority of PC contaminants are commensal skin flora introduced by venipuncture at the time of blood collection. The predominant organisms are Gram-positive coagulase-negative staphylococci such as Staphylococcus capitis. This bacterium has been implicated in numerous instances of infection and sepsis, likely for its ability to form surface-associated communities of micro-organisms encased in extracellular materials, known as biofilms. In the present study, five strains of S. capitis isolated from contaminated PCs were assessed for their ability to produce extracellular polysaccharide (slime), a canonical indicator of biofilm-formation ability, on Congo red agar plates. Biofilm formation was evaluated in both glucose-enriched trypticase soy broth (TSBg) and in PCs by using a crystal violet staining assay. The chemical nature of the biofilms was evaluated by disruption assays using sodium metaperiodate and proteinase K. In addition, biofilm architecture was observed by scanning electron microscopy. The presence of the biofilm-associated icaR and icaADBC genes was also examined by PCR. While only two out of the five S. capitis strains formed biofilms in TSBg, all strains formed biofilms in PCs. The ability of strains to produce extracellular polysaccharide and their possession of wild-type ica genes were not exclusive predictors of biofilm formation in TSBg or PCs; different profiles of biofilm markers were observed among isolates. This is likely due to the proteinaceous composition of the S. capitis biofilm matrix. Interestingly, an ica-negative, non-slime-producing isolate was capable of biofilm formation in PCs. Together, these data indicate that the platelet storage environment stimulates biofilm formation in S. capitis in the absence of extracellular polysaccharide production and that multiple bacterial factors and regulatory elements are likely involved in biofilm formation in this milieu.