Interleukin-1ß Polymorphisms Are Genetic Markers of Susceptibility to Periprosthetic Joint Infection in Total Hip and Knee Arthroplasty.

Periprosthetic joint infections (PJIs) are serious complications of prosthetic surgery. The criteria for the diagnosis of PJI integrate clinical and laboratory findings in a complex and sometimes inconclusive workflow. Host immune factors hold potential as diagnostic biomarkers in bone and joint infections. We reported that the humoral pattern-recognition molecule long pentraxin 3 (PTX3) predicts PJI in total hip and knee arthroplasty (THA and TKA, respectively). If and how genetic variation in PTX3 and inflammatory genes that affect its expression (IL-1ß, IL-6, IL-10, and IL-17A) contributes to the risk of PJI is unknown. We conducted a case-control study on a Caucasian historic cohort of THA and TKA patients who had prosthesis explant due to PJI (cases) or aseptic complications (controls). Saliva was collected from 93 subjects and used to extract DNA and genotype PTX3, IL-1ß, IL-6, IL-10, and IL-17A single-nucleotide polymorphisms (SNPs). Moreover, the concentration of IL-1ß, IL-10, and IL-6 was measured in synovial fluid and plasma. No association was found between PTX3 polymorphisms and PJI; however, the AGG haplotype, encompassing rs2853550, rs1143634, and rs1143627 in IL-1ß, was linked to the infection (p = 0.017). Also, synovial levels of all inflammatory markers were higher in cases than in controls, and a correlation emerged between synovial concentration of PTX3 and that of IL-1ß in cases only (Spearman r = 0.67, p = 0.004). We identified a relationship between rs2853550 and the synovial concentration of IL-1ß and PTX3. Our findings suggest that IL-1ß SNPs could be used for the early identification of THA and TKA patients with a high risk of infection.

Gut permeability may be associated with periprosthetic joint infection after total hip and knee arthroplasty.

A growing number of recent investigations on the human genome, gut microbiome, and proteomics suggests that the loss of mucosal barrier function, particularly in the gastrointestinal tract, may substantially affect antigen trafficking, ultimately influencing the close bidirectional interaction between the gut microbiome and the immune system. This cross-talk is highly influential in shaping the host immune system function and ultimately affecting the outcome of interventions. We hypothesized that the loss of mucosal barrier in the gut may be associatedto acute and chronic periprosthetic joint infections (PJI). Zonulin, soluble CD14 (sCD14), and lipopolysaccharide (LPS) were tested in plasma as part of a prospective cohort study of patients undergoing primary arthroplasty or revision arthroplasty because of an aseptic failure or PJI (as defined by the 2018 criteria). All blood samples were collected before antibiotic administration. Samples were tested using commercially available enzyme-linked immunosorbent assays as markers for gut permeability. A total of 134 patients were included in the study of which 44 patients had PJI (30 chronic and 14 acute), and the remaining 90 patients were categorized as non-infected that included 64 patients revised for aseptic failure, and 26 patients undergoing primary total joint arthroplasty. Both Zonulin (7.642 ± 6.077 ng/mL vs 4.560 ± 3.833 ng/mL; p < 0.001) and sCD14 levels (555.721 ± 216.659 ng/mL vs 396.872 ± 247.920 ng/mL; p = 0.003) were significantly elevated in the PJI group compared to non-infected cases. Higher levels of Zonulin were found in acute infections compared to chronic PJI (11.595 ± 6.722 ng/mL vs. 5.798 ± 4.841 ng/mL; p = 0.005). This prospective study reveals a possible link between gut permeability and the 'gut-immune-joint axis' in PJI. If this association continues to be borne out with a larger cohort and more in-depth analysis, it will have a clinically significant implication in managing patients with PJI. It may be that in addition to the administration of antimicrobials, patients with PJI and other orthopaedic infections may benefit from administration of gastrointestinal modulators such as pro and prebiotics.

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.

Better choice of the type of specimen used for untargeted metagenomic sequencing in the diagnosis of periprosthetic joint infections.

AIMS: As a proven and comprehensive molecular technique, metagenomic next-generation sequencing (mNGS) has shown its potential in the diagnosis of pathogens in patients with periprosthetic joint infection (PJI), using a single type of specimen. However, the optimal use of mNGS in the management of PJI has not been explored. In this study, we evaluated the diagnostic value of mNGS using three types of specimen with the aim of achieving a better choice of specimen for mNGS in these patients. METHODS: In this prospective study, 177 specimens were collected from 59 revision arthroplasties, including periprosthetic tissues, synovial fluid, and prosthetic sonicate fluid. Each specimen was divided into two, one for mNGS and one for culture. The criteria of the Musculoskeletal Infection Society were used to define PJI (40 cases) and aseptic failure (19 cases). RESULTS: The sensitivity and specificity of mNGS in the diagnosis of PJI were 95% and 94.7%, respectively, for all types of specimen. The sensitivity and specificity were 65% and 100%, respectively, for periprosthetic tissues, 87.5% and 94.7%, respectively, for synovial fluid, and 92.5% and 94.7%, respectively, for prosthetic sonicate fluid. The mNGS of prosthetic sonicate fluid outperformed that for other types of specimen in the rates of detection of pathogens (84.6%), sequencing reads (> ten-fold) and the rate of genome coverage (> five-fold). CONCLUSION: mNGS could serve as an accurate diagnostic tool in the detection of pathogens in patients with a PJI using three types of specimen. Due to its superior perfomance in identifying a pathogen, mNGS of prosthetic sonicate fluid provides the most value and may partly replace traditional tests such as bacteriological culture in these patients. Cite this article: Bone Joint J 2021;103-B(5):923-930.

Proinflammatory biomarkers' level and functional genetic polymorphisms in periprosthetic joint infection.

OBJECTIVE: The aims of this study were 1) to identify the level of inflammatory biomarkers interleukin (IL)-1α, IL-1ß, IL-6, IL-8, IL-17, C-reactive protein (CRP), granulocyte colony-stimulating factor (GCSF), ferritin, and tumor necrosis factor (TNF)-α in serum and synovial fluid samples of patients who underwent revision arthroplasty surgery; 2) to establish the relationship between serum and synovial fluid levels; 3) to determine if any of the 11 genetic polymorphisms of TNFα, IL-1, IL-6, IL-8, IL-17, and GCSF on the encoding genes was associated with periprosthetic joint infection (PJI). METHODS: Synovial fluid and serum was collected from 88 patients who underwent revision arthroplasty surgery. The Musculoskeletal Infection Society definition was used to classify these patients into 2 groups: 36 PJIs and 52 aseptic failures. Synovial fluid and serum samples were tested for 9 biomarkers using a micro enzyme-linked immunosorbent assay. Genetic polymorphisms were evaluated with polymerase chain reaction and restriction endonuclease analysis. RESULTS: Synovial fluid-derived IL-1α, IL-1ß, IL-8, IL-17, CRP, GCSF, TNFα, and serum-derived IL-6, IL-17, ferritin, CRP were found suitable to classify PJI and aseptic failure. In addition, IL-17 and CRP levels demonstrated a positive correlation between synovial fluid and serum. TNFα-238, IL6-174, GCSF3R, and IL1 RN-VNTR genetic polymorphisms occurred more frequently in individuals with septic failure. CONCLUSION: Significant differences between the two groups were observed in the functional polymorphisms of the genes encoding the cytokines investigated. These differences could be interpreted as indicating that there is an association between PJI and genetic polymorphisms. LEVEL OF EVIDENCE: Level III, diagnostic study.

Genetic-relatedness of peri-implants and buccal Candida albicans isolates determined by RAPD-PCR / Relación genética de aislamentos de candida albicans por rapd-pcr en surcos en surcos peri-implantarios de cavidad bucal

Molecular techniques have been used in recent studies toidentify a wide range of potential bacterial pathogens inperi­implant pockets of the oral cavity. However, the prevalence and molecular epidemiology of yeasts and species distribution related to peri­implantitis are as yet unknown. The aim of thisstudy was to determine the prevalence and distribution of yeasts in peri­-implant biofilm and to study genetic relatedness of Candida albicans.Yeasts recovered from peri­implant biofilm samples (n=89) andbuccal samples (n=120) were studied in 40 immunocompetent non­-smoking patients who visited the dental clinic of the Asociación Implantodontológica Argentina, Buenos Aires, Argentina, and had received oral rehabilitation with implants for more than five years. Yeasts recovered from samples were studied by typing assays using RAPD­PCR. The prevalence of yeasts in the peri­implant sulcus was 73% (n=29). C. albicans was the most prevalent species identified in this study population. The RAPD analysis showed identical genotypes inmost C. albicans spp. from the two different sampling sites: buccal and peri­implant. These findings suggest that periimplant biofilm is an ecological niche that favors the growth of yeast species. Most C. albicans found in peri­implant biofilmoriginate from the endogenous infection caused by commensalstrains.

Las técnicas moleculares se han utilizado en estudios recientespara identificar una gran diversidad de patógenos bacterianosde surcos periimplantarios de cavidad bucal. Sin embargo, laprevalencia y epidemiología molecular de especies de levadurasen relación con la periimplantitis son aún desconocidas. Elobjetivo de este estudio fue determinar la prevalencia ydistribución de las levaduras en la biopelícula periimplantaria yestudiar la relación genética de Candida albicans. Se estudiaron40 pacientes inmunocompetentes no fumadores que se asistieronen la clínica dental de la Asociación ImplantodontológicaArgentina, Buenos Aires, Argentina, y que habían recibidorehabilitación oral con implantes durante más de cinco años.Las levaduras aisladas de las muestras de biopelículaperiimplantaria (n = 89) y bucales (n = 120), fueron identificadaspor métodos micológicos tradicionales y moleculares. Se obtuvoel ADN de C. albicans y se realizaron estudios moleculares porRAPD ­PCR. La prevalencia de levaduras en el surco alrededordel implante fue de 73 % (n = 29). C. albicans fue la especie másfrecuente identificada en esta población de estudio. El análisisRAPD permitió identificar idénticos genotipos de C. albicans enambos nichos ecológicos estudiados, periimplantar y bucal.Según los resultados obtenidos, el surco periiplantario es unnicho ecológico que favorece el crecimiento de especies delevaduras del género Candida. La mayoría de los aislamientosde C. albicans periimplantarios se originan a partir de lainfección endógena causada por cepas comensales.

IL-12 promotes myeloid-derived suppressor cell recruitment and bacterial persistence during Staphylococcus aureus orthopedic implant infection.

Staphylococcus aureus is a leading cause of human prosthetic joint infections (PJIs) typified by biofilm formation. We recently identified a critical role for myeloid-derived suppressor cells (MDSCs) in S. aureus biofilm persistence. Proinflammatory signals induce MDSC recruitment and activation in tumor models; however, the mechanisms responsible for MDSC homing to sites of biofilm infection are unknown. In this study, we report that several cytokines (IL-12p40, IL-1ß, TNF-α, and G-CSF) and chemokines (CXCL2, CCL5) were significantly elevated in a mouse model of S. aureus PJI. This coincided with significantly increased MDSC infiltrates concomitant with reduced monocyte, macrophage, and T cell influx compared with uninfected animals. Of the cytokines detected, IL-12 was of particular interest based on its ability to possess either pro- or anti-inflammatory effects mediated through p35-p40 heterodimers or p40 homodimers, respectively. MDSC recruitment was significantly reduced in both p40 and p35 knockout mice, which resulted in enhanced monocyte and neutrophil influx and bacterial clearance. Adoptive transfer of wild-type MDSCs into infected p40 knockout animals worsened disease outcome, as evidenced by the return of S. aureus burdens to levels typical of wild-type mice. Tissues obtained from patients undergoing revision surgery for PJI revealed similar patterns of immune cell influx, with increased MDSC-like cells and significantly fewer T cells compared with aseptic revisions. These findings reveal a critical role for IL-12 in shaping the anti-inflammatory biofilm milieu by promoting MDSC recruitment.

Genetic susceptibility to prosthetic joint infection following total joint arthroplasty: A systematic review.

BACKGROUND: Prosthetic joint infection (PJI) is the most common cause of total joint arthroplasty failure and revision surgery. Genetic polymorphisms could be determinant factors for PJI. METHODS: We performed a systematic research of Medline, Pubmed, Embase, Cochrane Library, and Google Scholar, and identified 11 studies with 34 kinds of gene polymorphisms, were included in the synthesis. RESULTS: Our data suggest that the C allele and genotype C/C for MBL-550 SNP, genotype A/A for MBL-54 SNP and G allele for MBL-221 SNP increase the risk of PJI, while G allele and genotype G/G for MBL-550 SNP decrease the risk of PJI in Caucasian populations. Several other genes reported by single-center studies also contribute to the genetic susceptibility to septic PJI. No definitive conclusions could be achieved due to the small amount of data in the included studies. CONCLUSION: Several genes contribute to the genetic susceptibility to PJI following total joint arthroplasty. Further studies will enhance the understanding of PJI, and may inform and direct early interventions.

Osteoprotegerin gene polymorphism is not associated with prosthetic joint infection after total joint arthroplasty in the Czech population.

BACKGROUND AND AIMS: Osteoprotegerin (OPG; official gene symbol: TNFRSF11B) is considered a negative regulator of bone resorption via inhibition of osteoclast differentiation. Further, OPG expression has been detected in Prosthetic Joint Infection (PJI) a serious complication limiting the overall outcome of total joint arthroplasty (TJA). As OPG may be a candidate molecule for PJI pathogenesis, we investigated whether genetic variation in the OPG promoter, namely the SNP at position -163 was associated with PJI. METHODS: OPG -163 T/C SNP (rs3102735) was genotyped by polymerase chain reaction with sequence specific primers (PCR-SSP) in 98 Czech patients with PJI and two Czech control groups: 1) aseptic TJA control [251 patients with TJA who did not develop PJI at least 6 yrs. after the surgery] and 2) population control (185 healthy control subjects without TJA). RESULTS: The distribution of OPG -163 SNP genotypes complied with the Hardy-Weinberg equilibrium in all three groups. The allele frequencies of OPG -163 SNP were similar in patients with PJI (minor allele frequency: 0.14), those with aseptic TJA (0.13) and population controls (0.14, P>0.05). Further, there was no significant difference in genotype or phenotype frequency (carriage rate) between patients with PJI and both control groups (P>0.05). CONCLUSIONS: In a Czech population, the OPG -163 T/C SNP has not been found to be associated with PJI.

Variation in the IL1B, TNF and IL6 genes and individual susceptibility to prosthetic joint infection.

BACKGROUND: Prosthetic joint infection (PJI) is an important failure mechanism of total joint arthroplasty (TJA). Here we examine whether the particular genetic variants can lead to increased susceptibility to PJI development. RESULTS: We conducted a genetic-association study to determine whether PJI could be associated with functional cytokine gene polymorphisms (CGP) influencing on innate immunity response. A case-control design was utilized and previously published criteria for PJI were included to distinguish between cases and control subjects with/without TJA. Six single nucleotide polymorphisms (SNPs) located in the genes for interleukin-1beta (SNP: IL1B-511, +3962), tumour necrosis factor alpha (TNF-308, -238) and interleukin-6 (IL6-174, nt565) were genotyped in 303 Caucasian (Czech) patients with TJA (89 with PJI / 214 without PJI), and 168 unrelated healthy Czech individuals without TJA. The results showed that carriers of the less common IL1B-511*T allele were overrepresented in the group of TJA patients with PJI (69%) in comparison with those that did not develop PJI (51%, p = 0.006, p(corr) = 0.037) and with healthy controls (55%, p = 0.04, p(corr) = N.S.). There was no significant difference in the distribution of the remaining five investigated CGPs and their haplotypes between groups. CONCLUSION: A functional variant of the gene encoding for IL-1beta was preliminarily nominated as a genetic factor contributing to the susceptibility to PJI. Our results should be independently replicated; studies on the functional relevance of IL1B gene variants in PJI are also needed.

A systematic review on the association between genetic predisposition and dental implant biological complications.

OBJECTIVES: The aim of this systematic review was to evaluate the relationship between genetic polymorphisms and dental implant biological complications. MATERIAL AND METHODS: All prospective, cross-sectional and retrospective studies reporting on dental implant loss/peri-implantitis/peri-implant marginal bone loss after loading in association with genetic polymorphism were considered for inclusion. A thorough search of electronic databases, supplemented by checking bibliographies of review articles was performed by two independent reviewers. Quality assessment of the included studies was conducted independently and in duplicate by two reviewers as part of the data extraction process. RESULTS: The search provided 344 related articles. Twenty-two publications were identified for possible inclusion and finally, seven articles met the defined inclusion criteria. Four studies which investigated the potential relationship between early implant loss and IL-1, IL-2, IL-6, TNF-α or TGF-ß1 genotype revealed no evidence to support this association. In two of the three studies which evaluated peri-implantitis in relation to IL-1 genotype, the findings indicate that IL-1RN (intron 2), IL-1A (-899), IL-1B (+3954) gene polymorphisms were correlated to increased peri-implant tissue infection and destruction. CONCLUSIONS: Methodological and study design issues restricted the possibility to draw robust conclusions. Within the limits of this review, it might be concluded that there is no obvious association between specific genetic polymorphism and dental implant failure in terms of biological complications, although a tendency should be underlined showing the potential link between IL-1 genotype and peri-implantitis. Well designed and adequately powered prospective cohort studies are needed to provide further information.

Association of the composite IL-1 genotype with peri-implantitis: a systematic review.

OBJECTIVE: Cytokine gene polymorphisms may modulate the host response to the bacterial challenge and influence susceptibility to peri-implantitis. OBJECTIVE: To systematically review the evidence of an association between the interleukin-1 (IL-1) composite genotype, i.e. presence of the allele 2 in the gene clusters IL-1A (-889) and in IL-1B (+3953), and peri-implantitis. MATERIAL AND METHODS: An electronic search in the National Library of Medicine-computerized bibliographic database MEDLINE and a manual search were performed. The search was conducted for longitudinal clinical trials comparing progression of peri-implantitis in IL-1 genotype positive (carrying allele 2) with IL-1 genotype negative (not carrying allele 2) subjects. Selection of publications, extraction of data and validity assessment were made independently by two reviewers. RESULTS: The search provided 44 titles of which two longitudinal publications were included. CONCLUSION: Based on the findings from this study, there is not enough evidence to support or refute an association between the IL-1 genotype status and peri-implantitis. Systematic genetic testing for the assessment of the risk of peri-implantitis cannot be recommended as a standard of care at this time.

Genetic susceptibility to total hip arthroplasty failure: a preliminary study on the influence of matrix metalloproteinase 1, interleukin 6 polymorphisms and vitamin D receptor.

BACKGROUND: Matrix metalloproteinase (MMP)1, interleukin(IL)6 and vitamin D receptor (VDR) have been implicated in the biological cascade of events initiated by particulate wear debris and bacterial infection, resulting in periprosthetic bone loss around loosened total hip replacements (THRs). The individual responses to such stimuli may be dictated by genetic variation. OBJECTIVE: To study the effect of single-nucleotide polymorphisms (SNPs) within these candidate genes. METHODS: A case-control study of the MMP1, IL6 and VDR genes was performed for possible association with deep sepsis or aseptic loosening. All cases included in the study were Caucasian patients with osteoarthritis who had received a cemented Charnley total hip arthroplasty (THA) and polyethylene acetabular cup. Cases consisted of 91 patients with early aseptic loosening and 71 patients with microbiological evidence of deep infection on surgery. Controls consisted of 150 patients with THAs that were clinically asymptomatic for over 10 years and showed no radiographic features of aseptic loosening. DNA samples from all individuals were genotyped using Taqman allelic discrimination. RESULTS: The C allele (p = 0.001; OR = 3.27; 95% CI 2.21 to 4.83) and C/C genotype (p = 0.001) for the MMP1 SNP were highly associated with aseptic failure when compared with controls. No statistically significant relationships were found between aseptic loosening and the MMP2, MMP4, IL6 -174 or VDRL SNPs. The T allele (p = 0.007; OR = 1.76; 95% CI 1.16 to 2.66) and T/T genotype (p = 0.028) for VDR-T were statistically associated with osteolysis owing to deep infection as compared with controls. No statistically significant relationship was found between septic failure and any of the other SNPs examined in this study. CONCLUSIONS: Aseptic loosening and possibly deep infection of THR may be due to the genetic influence of candidate susceptibility genes. SNP markers may serve as predictors of implant survival and aid in pharmacogenomic prevention of THR failure.

Distinct gene expression of receptor activator of nuclear factor-kappaB and rank ligand in the inflammatory response to variant morphologies of UHMWPE particles.

Recent studies have examined the role of wear debris-induced bone resorption in the aseptic loosening of orthopedic prostheses. Research has shown that inflammation depends not only on the amount of particulate debris, but also the shape and size of the accumulated wear particles. Our previous studies have demonstrated that variant shapes of ultra-high molecular weight polyethylene (UHMWPE) particles induce diverse cellular and apoptotic responses in a murine inflammation model. Since enhanced osteoclastogenesis is recognized as a hallmark of bone loss in prosthetic loosening, we have now investigated the gene expression of receptor activator of nuclear factor-kappaB (RANK) and receptor activator of nuclear factor-kappaB ligand (RANKL) during the inflammatory response to different shapes of UHMWPE particles. Two shapes of UHMWPE particles (globular or elongated) were implanted in established air pouches on BALB/c mice, and pouches harvested 7 days after stimulation with UHMWPE particles. Gene levels of RANK, RANKL, TNFalpha, IL-1beta, and cathepsin K (CK) were quantified by real time RT-PCR, and TRAP staining of pouch membrane was used to evaluate osteoclastogenesis. We found that (i) elongated particles generated significantly higher RANK and RANKL gene expression than globular particles in pouch tissue; (ii) elongated particles provoked significantly higher IL-1beta and TNFalpha gene expression; (iii) a positive association was found between tissue inflammation status and the gene level of RANK/RANKL; and (iv) elongated particles stimulated significantly higher CK gene expression in comparison with globular particles. Histology revealed that clusters of TRAP+ cells were located in regions in contact with elongated particles. Overall, these data suggest that the morphology of wear debris may be a critical factor in the pathogenesis of prosthetic loosening.

Impact of the regulatory loci agr, sarA and sae of Staphylococcus aureus on the induction of alpha-toxin during device-related infection resolved by direct quantitative transcript analysis.

The cytotoxic alpha-toxin (encoded by hla) of Staphylococcus aureus is regulated by three loci, agr, sarA and sae, in vitro. Here, we assess the regulation of hla in a guinea pig model of device-related infection by quantifying RNAIII (the effector molecule of agr) and hla directly in exudates accumulating in infected devices without subculturing of the bacteria. LightCycler reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the transcripts. Strains RN6390 and Newman expressed considerably smaller amounts of RNAIII in the guinea pig than during in vitro growth. The residual RNAIII expression decreased during the course of infection and was negatively correlated with bacterial densities. As with RNAIII, the highest hla expression was detected in both strains early in infection. Even in strain Newman, a weak hla producer in vitro, a pronounced expression of hla was observed during infection. Likewise, four S. aureus isolates from cystic fibrosis (CF) patients expressed Q1hla despite an inactive agr during device-related infection as in the CF lung. Mutation of agr and sarA in strain Newman and RN6390 had no consequence for hla expression in vivo. In contrast, the mutation in sae resulted in severe downregulation of hla in vitro as well as in vivo. In conclusion, S. aureus seems to be provided with regulatory circuits different from those characterized in vitro to ensure alpha-toxin synthesis during infections.