To Örebro University

The inflammasome is a multiprotein complex that mediates caspase-1 activation with subsequent maturation of the pro-inflammatory cytokines IL-1β and IL-18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. The present study therefore aimed to investigate NLRP3 inflammasome activity in 20 S. aureus bacteremia patients, by repeated measurement during the first week of bacteremia, compared with controls. Caspase-1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (Fluorescent Labelled Inhibitor of Caspase-1), while IL-1β and IL-18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, mRNA expression of NLRP3, CASP1 (pro-caspase-1) and IL1B (pro-IL-1β) was analyzed by qPCR. We found induced caspase-1 activity in innate immune cells with subsequent release of IL-18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial inter-individual variation in caspase-1 activity between S. aureus bacteremia patients. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in S. aureus bacteremia could provide support in the effort to optimize management and treatment of each individual patient.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of infections in humans. Photodynamic therapy using blue light (450 nm) could possibly be used to reduce MRSA on different human tissue surfaces without killing the human cells. It could be less harmful than 300–400 nm light or common disinfectants. We applied blue light ± riboflavin (RF) to MRSA and keratinocytes, in an in vitro liquid layer model, and compared the effect to elimination using common disinfection fluids. MRSA dilutions (8 × 10⁵/mL) in wells were exposed to blue light (450 nm) ± RF at four separate doses (15, 30, 56, and 84 J/cm²). Treated samples were cultivated on blood agar plates and the colony forming units (CFU) determined. Adherent human cells were cultivated (1 × 10⁴/mL) and treated in the same way. The cell activity was then measured by Cell Titer Blue assay after 24- and 48-h growth. The tested disinfectants were chlorhexidine and hydrogen peroxide. Blue light alone (84 J/cm²) eliminated 70% of MRSA. This dose and riboflavin eradicated 99–100% of MRSA. Keratinocytes were not affected by blue light alone at any dose. A dose of 30 J/cm² in riboflavin solution inactivated keratinocytes completely. Disinfectants inactivated all cells. Blue light alone at 450 nm can eliminate MRSA without inactivation of human keratinocytes. Hence, a high dose of blue light could perhaps be used to treat bacterial infections without loss of human skin cells. Photodynamic therapy using riboflavin and blue light should be explored further as it may perhaps be possible to exploit in treatment of skin diseases associated with keratinocyte hyperproliferation.

Introduction: mRNA expressions of the major histocompatibility complex class II-related genes HLA-DRA and CD74 have been found to be promising markers for sepsis-induced immunosuppression. In the present study we aimed to study how expression of HLA-DRA and CD74 on intensive care unit (ICU) admission were related to death and/or secondary infections in patients with sepsis.

Methods: During a full year adult patients admitted to the ICU of Karolinska University Hospital Huddinge were consecutively subjected to blood sampling within 1 hour from ICU admission. Patients treated with antibiotic therapy were eligible for inclusion. The plausibility of infection (definite, probable, possible, none) was determined based on the Centers for Diseases Control (CDC) criteria. Patients with sepsis (definite/probable/possible infection and a SOFA score increase of >=2) were screened for death within 60 days and secondary infections 48 h to 60 days after ICU admission, using the CDC criteria. HLA-DRA and CD74 mRNA expressions were determined by reverse transcription quantitative PCR.

Results: Among 579 ICU admissions, a blood sample for RNA analysis was collected in 551 cases. Two hundred fifty-seven patients met the inclusion criteria and provided written informed consent. Sepsis was noted in 134 patients. The sepsis patients experienced death in 36 cases (27%), secondary infection in 32 cases (24%), and death and/or secondary infection in 60 cases (45%). Table 1 shows the results of HLA-DRA and CD74 expression related to death and secondary infections.

Conclusions: The mRNA expression of HLA-DRA on ICU admission was significantly decreased in patients with sepsis who died or contracted secondary infections within 60 days. CD74 expression was not significantly decreased in patients with negative outcome.

Background: Bacteria in the conjunctiva present a potential risk of vitreous cavity infection during 23-gauge pars plana vitrectomy (PPV). Current preoperative procedures used in Sweden include irrigation with chlorhexidine solution (CHX) 0.05% only and no iodine solutions. We evaluated the bacterial diversity and load before and after this single antibacterial measure.

Methods: In a prospective, consecutive cohort we investigated bacterial growth in samples from 40 eyes in 39 consecutive individuals subjected to vitrectomy. A conjunctival specimen was collected from each preoperative patient before and after irrigating of eye with CHX, 0.05% solution. Iodine was not used during any part of the surgery. One drop of chloramphenicol was administered prior to surgery. Samples from vitreous cavity were collected at the beginning and end of vitrectomy. All conjunctival specimens were cultured for different species and quantified using colony forming units (CFU).

Results: There was a significant 82% reduction in the total number of CFUs for all bacteria in all eyes (P < 0.0001), and 90% reduction for coagulase negative staphylococci (CoNS) alone (P = 0.0002). The number of eyes with positive bacterial growth in conjunctival samples decreased from 33 to 18 after irrigation with CHX (P = 0.0023). The most common bacteria prior to surgery were CoNS (70%), Propionibacterium acnes (55%) and Corynebacterium species (36%). No case of post-vitrectomy endophthalmitis was reported during mean follow-up time, which was 4.6 +/- 2.3 (range; 1.5 to 9) months.

Conclusions: Patients undergoing PPV harbored bacteria in conjunctiva capable of causing post-vitrectomy endophthalmitis. Preoperative preparation with CHX significantly reduced the bacterial load in the conjunctival samples subsequently leading to very low inoculation rates in recovered vitreous samples. Thus, CHX used as a single disinfectant agent might be an effective preoperative procedure for eye surgery in Sweden. This is a relatively small study but the results could be a reference for other intraocular surgeries.

To improve management of Staphylococcus aureus bacteremia (SAB), better understanding of host-pathogen interactions is needed. In vitro studies have shown that S. aureus bacteria induce dose-dependent immunosuppression that is evidenced by reduced expression of major histocompatibility complex (MHC) class II on antigen presenting cells. Thus, the aim of this study was to determine whether expression of the MHC class II-related genes HLA-DRA and CD74 is more greatly reduced in complicated SAB, with its probable higher loads of S. aureus, than in uncomplicated SAB. Adult patients with SAB were prospectively included and blood samples taken on the day of confirmation of SAB (Day 1) and on Days 2, 3, 5 and 7. HLA-DRA and CD74 mRNA expression was determined by quantitative reverse transcription PCR. Sepsis was defined according to the Sepsis-3 classification and SAB was categorized as complicated in patients with deep-seated infection and/or hematogenous seeding. Twenty patients with SAB were enrolled and samples obtained on all assessment days. HLA-DRA and CD74 expression did not differ significantly between patients with SAB and sepsis (n=13) and those without sepsis (n=7) on any assessment day. However, patients with complicated SAB (n=14) had significantly weaker HLA-DRA expression on all five assessment days than patients with uncomplicated SAB (n=6). Additionally, they tended to have weaker CD74 expressions. Neutrophil, monocyte and leukocyte counts did not differ significantly between complicated and uncomplicated SAB. In conclusion, patients with complicated SAB show weaker HLA-DRA expression than those with uncomplicated SAB during the first week of bacteremia.

Purpose: To compare elimination of methicillin-resistant Staphylococcus aureus (MRSA) by exposure of blue light alone and with riboflavin.

Methods: A reference strain of MRSA was cultured and diluted in PBS with and without riboflavin (0.01%). Fifteen microlitre was added on a microscope slide, creating a fluid layer with a thickness of around 400 microns. Both of the bacterial suspensions were exposed to blue light, and the effect between exposure with and without riboflavin was compared. Evaluation involved two different wavelengths (412 and 450 nm) of blue light with a lower (5.4 J/cm(2) ) and higher dose (approximately 28.5 J/cm(2) ). The effect of 412 nm light was also evaluated for a thicker fluid layer (1.17 mm). After exposure, colony-forming units (CFUs) were determined for each solution. All measurements were repeated eight times.

Results: The reductions in bacteria were similar for both wavelengths. With riboflavin, a statistically significant elimination was observed for both 412 and 450 nm (p < 0.001). At both dosages, the mean reduction was more pronounced with the presence of riboflavin than without it. Using the higher dose, CFU reduction was 99% and 98%, respectively, for 412 and 450 nm light. The bactericidal efficacy was high also in the deeper fluid layer (93%, higher dose).

Conclusion: Riboflavin enhanced the antibacterial effect on the exposed MRSA strain of blue light for both 412 and 450 nm blue light. This indicates that blue light could be considered for possible implementation in deep corneal infections.

Background: To evaluate the bactericidal effect of clinical ultraviolet A (UVA) settings used in photoactivated chromophore for infectious keratitis (PACK)-collagen cross-linking (CXL) in antibiotic-resistant and non-resistant bacterial strains.

Methods: Well-characterized bacterial strains from clinical isolates, without and with antibiotic resistance, were studied in a pairwise comparison. The evaluated pathogens were Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis. Bacteria were dispersed in PBS and diluted to a concentration of approximately 4x10(5)/ml. Riboflavin was added to a concentration of 0.01%. By spreading the solution on a microscope slide, a fluid film layer, with a thickness of around 400mm, was formed and UVA exposure followed. Eight separate exposures were made for each strain (n=8). The degree of elimination in resistant and non-resistant pathogens was compared.

Results: The bactericidal efficacy of exposure differed between the tested microorganisms, and the mean elimination ranged between 60 and 92%, being most extensive in both of the evaluated Pseudomonas strains and least in the E. faecalis strains. Similar reductions were seen in antibiotic-resistant and non-resistant strains, with the exception of S. aureus, in which the resistant strain metchicillin-resistant Staphylococcus aureus (MRSA) was eradicated in a greater extent than the non-resistant strain (P=0.030).

Conclusion: UVA-riboflavin settings used in PACK-CXL are effective in reducing both antibiotic-resistant and non-resistant bacteria. Antibiotic resistance does not appear to be protective against the photooxidative exposure.

Introduction: A decrease in the expression of monocyte surface protein HLA-DR (mHLA-DR), measured by flow cytometry (FCM), has been suggested as a marker of immunosuppression and negative outcome in severe sepsis. However, FCM is not always available due to sample preparation that limits its use to laboratory operational hours. In this prospective study we evaluated dynamic changes in mHLA-DR expression during sepsis in relation to changes in HLA-DRA gene expression and Class II transactivator (CIITA), measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).

Aims: The aims of this study were: 1. to validate the robustness of qRT-PCR measurement of HLA-DRA- and CIITA-mRNA expression, in terms of reproducibility; and 2. to see if changes in expression of these genes reflect changes in mHLA-DR expression during the course of severe and non-severe bacteraemic sepsis.

Methods and Findings: Blood samples were collected from 60 patients with bacteraemic sepsis on up to five occasions during Days 1-28 after hospital admission. We found the reproducibility of the qRT-PCR method to be high by demonstrating low threshold variations (<0.11 standard deviation (SD)) of the qRT-PCR system, low intra-assay variation of Ct-values within triplicates (≤0.15 SD) and low inter-assay variations (12%) of the calculated target gene ratios. Our results also revealed dynamic HLA-DRA expression patterns during the course of sepsis that reflected those of mHLA-DR measured by FCM. Furthermore, HLA-DRA and mHLA-DR recovery slopes in patients with non-severe sepsis differed from those in patients with severe sepsis, shown by mixed model for repeated measurements (p<0.05). However, during the first seven days of sepsis, PCR-measurements showed a higher magnitude of difference between the two sepsis groups. Mean differences (95% CI) between severe sepsis (n = 20) and non-severe sepsis (n = 40) were; on day 1-2, HLA-DRA 0.40 (0.28-0.59) p<0.001, CIITA 0.48 (0.32-0.72) p = 0.005, mHLA-DR 0.63 (0.45-1.00) p = 0.04, day 7 HLA-DRA 0.59 (0.46-0.77) p<0.001, CIITA 0.56 (0.41-0.76) p<0.001, mHLA-DR 0.81 (0.66-1.00) p = 0.28.

Conclusion: We conclude that qRT-PCR measurement of HLA-DRA expression is robust, and that this method appears to be preferable to FCM in identifying patients with severe sepsis that may benefit from immunostimulation.

The aim was to investigate whether the detection and quantification of Staphylococcus epidermidis DNA in blood could distinguish S. epidermidis blood stream infections (BSIs) from blood culture contaminations in patients with hematological malignancies. The hld gene was chosen to identify S. epidermidis DNA and DNA in blood samples was detected by real-time PCR. Blood samples were obtained simultaneously with blood cultures positive for S. epidermidis (n = 30), during blood culture-negative episodes (n = 10) and episodes of bacteremia with other bacteria than S. epidermidis (n = 4) and from healthy blood donors (n = 10). In addition, DNA from S. epidermidis and a selection of other bacterial species were analyzed. Three different sets of criteria were used to classify episodes with positive blood cultures with S. epidermidis as BSIs or contaminations. All DNA preparations from S. epidermidis (n = 48) were hld-positive, but other bacterial species (n = 13) were negative. Sixteen (53%) of 30 blood samples from patients with blood cultures positive for S. epidermidis were hld-positive, but none of the controls. There was no clear association between a positive hld PCR and episodes interpreted as BSIs. In conclusion, hld PCR failed to distinguish S. epidermidis BSIs from blood culture contaminations in patients with hematological malignancies.

Purpose To investigate whether ultraviolet light (UVA) at 365 nm can inhibit/eliminate Acanthamoeba growth and if riboflavin would potentiate such an association.

Method: Acanthamoeba castellanii in a fluid medium with a concentration of approximately 1.7 x 10(4) protozoa/ml were prepared with (0.01 %) and without riboflavin. Exposure of UVA (dose 5.475 J/cm(2)) took place twice, with each illumination period followed by culturing of 10 mu l in peptone yeast-extract glucose (PYG) medium for 7 days. Every suspension prepared had a non-exposed control solution. Determination of Acanthamoeba was conducted daily, by count in Burker chamber days 4 through 7 after exposure. Statistical analysis was done by repeated-measurement ANOVA and post-hoc analysis for unpaired samples.

Results: The exposure of ultraviolet light resulted in an inhibited growth of Acanthamoeba compared to the non-exposed solutions, with a statistically significant reduction over time (p = 0.0003). The addition of riboflavin did not amplify the effect, and there were no tendencies for an interaction effect between UVA and riboflavin. The antiprotozoal effect of the UVA wavelength, utilized in CXL, is solely mediated by ultraviolet light, and riboflavin does not seem to amplify the antimicrobial efficacy.