Comparative transcriptomic analysis of Staphylococcus epidermidis associated with periprosthetic joint infection under in vivo and in vitro conditions.

Staphylococcus epidermidis is part of the commensal microbiota of the skin and mucous membranes, though it can also act as a pathogen in certain scenarios, causing a range of infections, including periprosthetic joint infection (PJI). Transcriptomic profiling may provide insights into mechanisms by which S. epidermidis adapts while in a pathogenic compared to a commensal state. Here, a total RNA-sequencing approach was used to profile and compare the transcriptomes of 19 paired PJI-associated S. epidermidis samples from an in vivo clinical source and grown in in vitro laboratory culture. Genomic comparison of PJI-associated and publicly available commensal-state isolates were also compared. Of the 1919 total transcripts found, 145 were from differentially expressed genes (DEGs) when comparing in vivo or in vitro samples. Forty-two transcripts were upregulated and 103 downregulated in in vivo samples. Of note, metal sequestration-associated genes, specifically those related to staphylopine activity (cntA, cntK, cntL, and cntM), were upregulated in a subset of clinical in vivo compared to laboratory grown in vitro samples. About 70% of the total transcripts and almost 50% of the DEGs identified have not yet been annotated. There were no significant genomic differences between known commensal and PJI-associated S. epidermidis isolates, suggesting that differential genomics may not play a role in S. epidermidis pathogenicity. In conclusion, this study provides insights into phenotypic alterations employed by S epidermidis to adapt to infective and non-infected microenvironments, potentially informing future therapeutic targets for related infections.

Human transcriptomic response to periprosthetic joint infection.

Periprosthetic joint infection (PJI), a devastating complication of total joint replacement, is of incompletely understood pathogenesis and may sometimes be challenging to clinically distinguish from other causes of arthroplasty failure. We characterized human gene expression in 93 specimens derived from surfaces of resected arthroplasties, comparing transcriptomes of subjects with infection- versus non-infection-associated arthroplasty failure. Differential gene expression analysis confirmed 28 previously reported potential biomarkers of PJI, including bactericidal/permeability increasing protein (BPI), cathelicidin antimicrobial peptide (CAMP), C-C-motif chemokine ligand 3 (CCL3), 4(CCL4) and C-X-C-motif chemokine ligand 2 (CXCL2), colony stimulating factor 2 receptor beta (CSF2RB), colony stimulating factor 3 (CSF3), alpha-defensin (DEFA4), Fc fragment of IgG receptor 1B (CD64B), intercellular adhesion molecule 1 (ICAM1), interferon gamma (IFNG), interleukin 13 receptor subunit alpha 2 (IL13RA2), interleukin 17D (IL17D), interleukin 1 (IL1A, IL1B, IL1RN), interleukin 2 receptors (IL2RA, IL2RG), interleukin 5 receptor (IL5RA), interleukin 6 (IL6), interleukin 8 (IL8), lipopolysaccharide binding protein (LBP), lipocalin (LCN2), lactate dehydrogenase C (LDHC), lactotransferrin (LTF), matrix metallopeptidase 3 (MMP3), peptidase inhibitor 3 (PI3), and vascular endothelial growth factor A (VEGFA), and identified three novel molecules of potential diagnostic use for detection of PJI, namely C-C-motif chemokine ligand CCL20, coagulation factor VII (F7), and B cell receptor FCRL4. Comparative analysis of infections caused by staphylococci versus bacteria other than staphylococci and Staphylococcus aureus versus Staphylococcus epidermidis showed elevated expression of interleukin 13 (IL13), IL17D, and MMP3 in staphylococcal infections, and of IL1B, IL8, and platelet factor PF4V1 in S. aureus compared to S. epidermidis infections. Pathway analysis of over-represented genes suggested activation of host immune response and cellular maintenance and repair functions in response to invasion of infectious agents. The data presented provides new potential targets for diagnosis of PJI and for differentiation of PJI caused by different infectious agents.

Cobalt and Chromium Ion Release in Metal-on-Polyethylene and Ceramic-on-Polyethylene THA: A Simulator Study With Cellular and Microbiological Correlations.

BACKGROUND: The aims of this study were to determine the levels of cobalt (Co) and chromium (Cr) ions generated in simulators from metal-on-polyethylene (MoP) and ceramic-on-polyethylene (CoP) constructs. Furthermore, we aimed to investigate the cytotoxic effect of these ion levels on native tissues and their potential to modify periprosthetic joint infection risk. METHODS: We used in vitro culture of human adipose-derived mesenchymal stem cells (AMSCs) and Staphylococcus epidermidis cultures, respectively. Ten hip simulator constructs (5 MoP and 5 CoP) were assembled and run for 1,000,000 cycles in bovine serum and evaluated for CoCr concentration. Cytotoxicity and growth impact on AMSCs and S. epidermidis was compared between CoCr and inert silicon dioxide. RESULTS: After 1,000,000 cycles, mean MoP and CoP Co concentration was 2264 and 0.6 ng/mL, respectively (P < .001). Mean MoP and CoP Cr concentration was 217 and 4.3 ng/mL, respectively (P < .001). Mean MoP Co:Cr ratio was 10:1. Co ions were significantly more toxic to human AMSCs than control silicon dioxide in a dose-response manner (P < .001). S. epidermidis growth was not significantly impacted by Co concentrations observed in the simulators. CONCLUSION: MoP constructs built in ideal conditions generated substantial CoCr debris, highlighting a baseline risk with these implants that may be exacerbated by host factors or imperfect surgical technique. Evaluation of impact on AMSCs suggests that debris levels produced under simulator conditions can be cytotoxic. In addition, these concentrations did not potentiate or inhibit S. epidermidis growth, suggesting that elevated periprosthetic joint infection rates with adverse local tissue reaction are related to other factors potentially associated with tissue necrosis.

Loop structures in the 5' untranslated region and antisense RNA mediate pilE gene expression in Neisseria gonorrhoeae.

Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.

Gonorrhea - an evolving disease of the new millennium.

Etiology, transmission and protection: Neisseria gonorrhoeae (the gonococcus) is the etiological agent for the strictly human sexually transmitted disease gonorrhea. Infections lead to limited immunity, therefore individuals can become repeatedly infected. Pathology/symptomatology: Gonorrhea is generally a non-complicated mucosal infection with a pustular discharge. More severe sequellae include salpingitis and pelvic inflammatory disease which may lead to sterility and/or ectopic pregnancy. Occasionally, the organism can disseminate as a bloodstream infection. Epidemiology, incidence and prevalence: Gonorrhea is a global disease infecting approximately 60 million people annually. In the United States there are approximately 300, 000 cases each year, with an incidence of approximately 100 cases per 100,000 population. Treatment and curability: Gonorrhea is susceptible to an array of antibiotics. Antibiotic resistance is becoming a major problem and there are fears that the gonococcus will become the next "superbug" as the antibiotic arsenal diminishes. Currently, third generation extended-spectrum cephalosporins are being prescribed. Molecular mechanisms of infection: Gonococci elaborate numerous strategies to thwart the immune system. The organism engages in extensive phase (on/off switching) and antigenic variation of several surface antigens. The organism expresses IgA protease which cleaves mucosal antibody. The organism can become serum resistant due to its ability to sialylate lipooligosaccharide in conjunction with its ability to subvert complement activation. The gonococcus can survive within neutrophils as well as in several other lymphocytic cells. The organism manipulates the immune response such that no immune memory is generated which leads to a lack of protective immunity.

H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/ -10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.

pilS loci in Neisseria gonorrhoeae are transcriptionally active.

Piliation is an important virulence determinant for Neisseria gonorrhoeae. PilE polypeptide is the major protein subunit in the pilus organelle and engages in extensive antigenic variation due to recombination between pilE and a pilS locus. pilS were so-named as they are believed to be transcriptionally silent, in contrast to the pilE locus. In this study, we demonstrate the presence of a small, pil-specific RNA species. Through using a series of pilE deletion mutants, we show by Northern blotting and quantitative reverse transcriptase PCR analysis (qRT-PCR), that these smaller RNA species are not derived from the primary pilE transcript following some processing events, but rather, arose through transcription of the pilS loci. Small transcriptome analysis, in conjunction with analysis of pilS recombinants, identified both sense and anti-sense RNAs originating from most, but not all, of the pilS gene copies. Focusing on the MS11 pilS6 locus, we identified by site-directed mutagenesis a sense promoter located immediately upstream of pilS6 copy 2, as well as an anti-sense promoter immediately downstream of pilS6 copy 1. Whole transcriptome analysis also revealed the presence of pil-specific sRNA in both gonococci and meningococci. Overall, this study reveals an added layer of complexity to the pilE/pilS recombination scheme by demonstrating pil-specific transcription within genes that were previously thought to be transcriptionally silent.