Staphylococcus epidermidis undergoes global changes in gene expression during biofilm maturation in platelet concentrates.

BACKGROUND: Staphylococcus epidermidis forms surface-attached aggregates (biofilms) when grown in platelet concentrates (PCs). Comparative transcriptome analyses were undertaken to investigate differential gene expression of S. epidermidis biofilms grown in PCs. STUDY DESIGN AND METHODS: Two S. epidermidis strains isolated from human skin (AZ22 and AZ39) and one strain isolated from contaminated PCs (ST02) were grown in glucose-supplemented Trypticase Soy Broth (TSBg) and PCs. RNA was extracted and sequenced using Illumina HiSeq. Differential expression analysis was done using DESeq, and significantly differentially expressed genes (DEGs) were selected. DEGs were subjected to Kyoto encyclopedia of genes and genomes and Gene Ontology analyses. Differential gene expression was validated with quantitative reverse transcription-PCR. RESULTS: A total of 436, 442, and 384 genes were expressed in AZ22, AZ39, and ST02, respectively. DEG analysis showed that 170, 172, and 117 genes were upregulated in PCs in comparison to TSBg, whereas 120, 135, and 89 genes were downregulated (p < .05) in mature biofilms of AZ22, AZ39, and ST02, respectively. Twenty-seven DEGs were shared by all three strains. While 76 DEGs were shared by AZ22 and AZ39, only 34 and 21 DEGs were common between ST02, and AZ22 and AZ39, respectively. Significant transcriptional expression changes were observed in genes involved in platelet-bacteria interaction, biofilm formation, production of virulence factors, and resistance to antimicrobial peptides and antibiotics. CONCLUSION: Differential gene expression in S. epidermidis is triggered by the stressful PC storage environment. Upregulation of virulence and antimicrobial resistance genes could have clinical implications for transfusion patients.

The peptidoglycan and biofilm matrix of Staphylococcus epidermidis undergo structural changes when exposed to human platelets.

Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using biochemical and microscopy techniques, we investigated the structural changes of the peptidoglycan (PG) and the biofilm matrix of S. epidermidis biofilms formed in whole-blood derived PCs compared to biofilms grown in glucose-supplemented trypticase soy broth (TSBg). Both, the PG and the biofilm matrix are primary mechanisms of defense against environmental stress. Here we show that in PCs, the S. epidermidis biofilm matrix is mainly of a proteinaceous nature with extracellular DNA, in contrast to the predominant polysaccharide nature of the biofilm matrix formed in TSBg cultures. PG profile studies demonstrated that the PG of biofilm cells remodels during PC storage displaying fewer muropeptides variants than those observed in TSBg. The PG muropeptides contain two chemical modifications (amidation and O-acetylation) previously associated with resistance to antimicrobial agents by other staphylococci. Our study highlights two key structural features of S. epidermidis that are remodeled when exposed to human platelets and could be used as targets to reduce septic transfusions events.

Septic transfusion case caused by a platelet pool with visible clotting due to contamination with Staphylococcus aureus.

BACKGROUND: Contamination of platelet concentrates (PCs) with Staphylococcus aureus is one of the most significant ongoing transfusion safety risks in developed countries. CASE REPORT: This report describes a transfusion reaction in an elderly patient diagnosed with acute myeloid leukemia, transfused with a 4-day-old buffy coat PC through a central venous catheter. The transfusion was interrupted when a large fibrous clot in the PC obstructed infusion pump flow. Shortly afterward, a red blood cell (RBC) unit transfusion started. After septic symptoms were developed, the RBC transfusion was also interrupted. While the RBC unit tested negative for bacterial contamination, the PC and the patient samples were found to be contaminated with a S. aureus strain that exhibited the same phenotypic and genome sequencing profiles. The isolated S. aureus forms biofilms and produces the superantigen enterotoxin-like U, which was detected in a sample of the transfused PCs. The patient received posttransfusion antibiotic treatment and had her original central line removed and replaced. DISCUSSION: As the implicated PC had been tested for bacterial contamination during routine screening yielding negative results, this is a false-negative transfusion sepsis case. Using a point-of-care test could have prevented the transfusion reaction. This report highlights the increasing incidence of S. aureus as a major PC contaminant with grave clinical implications. Importantly, S. aureus is able to interact with platelet components resulting in visible changes in PCs. CONCLUSION: Visual inspection of blood components before transfusion is an essential safety practice to interdict the transfusion of bacterially contaminated units.

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.

The Legionella pneumophila kai operon is implicated in stress response and confers fitness in competitive environments.

Legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two-hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. Indeed, mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not circadian KaiB1C1. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments.

Circadian clock proteins in prokaryotes: hidden rhythms?

Circadian clock genes are vital features of eukaryotes that have evolved such that organisms can adapt to our planet's rotation in order to anticipate the coming day or night as well as unfavorable seasons. This circadian clock uses oscillation as a timekeeping element. However, circadian clock mechanisms exist also in prokaryotes. The circadian clock of Cyanobacteria is well studied. It is regulated by a cluster of three genes: kaiA, kaiB, and kaiC. In this review, we will discuss the circadian system in cyanobacteria, and provide an overview and updated phylogenetic analysis of prokaryotic organisms that contain the main circadian genes. It is evident that the evolution of the kai genes has been influenced by lateral transfers but further and deeper studies are needed to get an in depth understanding of the exact evolutionary history of these genes. Interestingly, Legionella pneumophila an environmental bacterium and opportunistic human pathogen that parasitizes protozoa in fresh water environments also contains kaiB and kaiC, but their functions are not known. All of the residues described for the biochemical functions of the main pacemaker KaiC in Synechococcus elongatus are also conserved in the L. pneumophila KaiC protein.