Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

In orthopedic patients, foreign body-associated infections, especially periprosthetic joint infections (PJIs), are a devastating complication of ...

Paul-Ehrlich-Institute

Colonization of livestock with bacteria resistant to antibiotics is considered a risk for the entry of drug-resistant pathogens into the food chain. For this reason, there is a need for novel concepts to address the eradication of drug-resistant commensals on farms. In the present report, we evaluated the decontamination measures taken on a farm contaminated with methicillin-resistant Staphylococcus aureus (MRSA) and Enterobacteriaceae expressing extended-spectrum β-lactamases (ESBL-E). The decontamination process preceded the conversion from piglet breeding to gilt production. Microbiological surveillance showed that the decontamination measures eliminated the MRSA and ESBL-E strains that were detected on the farm before the complete removal of pigs, cleaning and disinfection of the stable, and construction of an additional stable meeting high-quality standards. After pig production was restarted, ESBL-E remained undetectable over 12 months, but MRSA was recovered from pigs and the environment within the first 2 days. However, spa (Staphylococcus aureus protein A gene) typing revealed acquisition of an MRSA strain (type t034) that had not been detected before decontamination. Interestingly, we observed that a farmworker who had been colonized with the prior MRSA strain (t2011) acquired the new strain (t034) after 2 months. In summary, this report demonstrates that decontamination protocols similar to those used here can lead to successful elimination of contaminating MRSA and ESBL-E in pigs and the stable environment. Nevertheless, decontamination protocols do not prevent the acquisition of new MRSA strains.

Eradication of methicillin-resistant Staphylococcus aureus and of Enterobacteriaceae expressing extended-spectrum beta-lactamases on a model pig farm.

Livestock-associated bacteria with resistance to two or more antibiotic drug classes have heightened our awareness for the consequences of antibiotic consumption and spread of resistant bacterial strains in the veterinary field. In this study we assessed the prevalence of concomitant colonization with livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) and enterobacteriaceae expressing extended-spectrum betalactamases (ESBL-E) in farms at the German-Dutch border region. Nasal colonization of pigs with MRSA (113/547 (20.7%)) was less frequent than rectal colonization with ESBL-E (163/540 (30.2%)). On the individual farm level MRSA correlated with ESBL-E recovery. The data further provide information on prevalence at different stages of pig production, including abattoirs, as well as in air samples and humans living and working on the farms. Notably, MRSA was detected in stable air samples of 34 out of 35 pig farms, highlighting air as an important MRSA transmission reservoir. The majority of MRSA isolates, including those from humans, displayed tetracycline resistance and spa types t011 and t034 characteristic for LA-MRSA, demonstrating transmission from pigs to humans. ESBL-E positive air samples were detected on 6 out of 35 farms but no pig-to-human transmission was found. Detection of ESBL-E, e.g. mostly Escherichia coli with CTX-M-type ESBL, was limited to these six farms. Molecular typing revealed transmission of ESBL-E within the pig compartments; however, related strains were also found on unrelated farms. Although our data suggest that acquisition of MRSA and ESBL-E might occur among pigs in the abattoirs, MRSA and ESBL-E were not detected on the carcasses. Altogether, our data define stable air (MRSA), pig compartments (ESBL-E) and abattoir waiting areas (MRSA and ESBL-E) as major hot spots for transmission of MRSA and/or ESBL-E along the pig production chain.

Analysis of Transmission of MRSA and ESBL-E among Pigs and Farm Personnel.

Total joint arthroplasty is a common operation worldwide with infection rates between 1% and 3%. In cases of suspected periprosthetic joint infection, it is very challenging to rule out the causative microorganisms. In this study, we compared the appearance of periprosthetic membranes with the microbiological results obtained from cultures of sonication fluid and the correlation between classical microbiological cultures and cultures of sonication fluid. The results confirmed a strong correlation of bacterial growth in sonication fluid cultures with bacterial growth in classical microbiological cultures. Most importantly, however, our study documented a highly significant correlation of periprosthetic membranes typical for periprosthetic joint infection (PJI) with bacterial growth in sonication fluid. Sonication fluid cultures yielded a better sensitivity than tissue cultures (72.34-60.87%). These 3 methods are useful tools in diagnosing PJIs, and even more, sonication fluid cultures should be included in the diagnostic path of PJI.

Comparison of bacterial growth in sonication fluid cultures with periprosthetic membranes and with cultures of biopsies for diagnosing periprosthetic joint infection.

The innate immune system harbors a multitude of different receptor systems and cells that are constantly prepared to sense and eliminate invading microbial pathogens. Staphylococcus aureus enters the body on its exposed epithelial surfaces, e.g., on skin and mucosa. The initial interaction with epithelial cells is governed by Toll-like receptor (TLR)-2-mediated local production of soluble mediators, including cytokines, chemokines, and antimicrobial peptides. The overall goal is to achieve a steady state of immune mediators and colonizing bacteria. Following cell and tissue invasion clearance of bacteria depends on intracellular microbial sensors and subsequent activation of the inflammasomes. Tissue-resident mast cells and macrophages recruit neutrophils, macrophages, and NK cells. This inflammatory response supports the generation of IL-17 producing NKT, γδ T cells, and T helper cells. Local dendritic cells migrate to the lymph nodes and fine-tune the adaptive immune response. The scope of this chapter is to provide an overview on the major cell types and receptors involved in innate immune defense against S. aureus. By segregating the different stages of infection from epithelial barrier to intracellular and systemic infection, this chapter highlights the different qualities of the innate immune response to S. aureus at different stages of invasiveness.

The Innate Immune Response Against Staphylococcus aureus.

Periprosthetic joint infection (PJI) is one of the most challenging complications in orthopedic surgery. In cases of suspected periprosthetic joint infection several diagnostic methods are available. In this study we investigated the performance of the newly available Unyvero i60 implant and tissue infection (ITI) multiplex PCR System. 62 specimens from 31 patients with suspected PJI or aseptic loosening of a painful joint arthoplasty were included in this study. Besides the established diagnostic procedures we included a commercial multiplex PCR detection system for diagnosis of PJI. The PCR results obtained from analysis of sonication and synovial fluids (62 specimens) showed a sensitivity of 66.7%, a specificity of 100%, a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 68.4% when compared to cultural methods. Notably, cultures from sonication fluid displayed a sensitivity of 88.9%, a specificity of 61.5%, a PPV of 76.2% and a NPV of 80.0% when compared to tissue cultures. In conclusion, multiplex PCR is an additional - rapid - method for diagnosing PJI. Positive results with the PCR assay used in this study were always confirmed by subsequent matching culture positivity. However, apart from the time saved the nucleic acid amplification technique did not yield additional information than that obtained from microbiological cultures.

Unyvero i60 implant and tissue infection (ITI) multiplex PCR system in diagnosing periprosthetic joint infection.

Porphyromonas gingivalis is an important member of the anaerobic oral flora. Its presence fosters growth of periodontal biofilm and development of periodontitis. In this study, we demonstrated that lipophilic outer membrane vesicles (OMV) shed from P. gingivalis promote monocyte unresponsiveness to live P. gingivalis but retain reactivity to stimulation with bacterial DNA isolated from P. gingivalis or AIM2 ligand poly(dA·dT). OMV-mediated tolerance of P. gingivalis is characterized by selective abrogation of tumor necrosis factor (TNF). Neutralization of interleukin-10 (IL-10) during OMV challenge partially restores monocyte responsiveness toP. gingivalis; full reactivity toP. gingivalis can be restored by inhibition of mTOR signaling, which we previously identified as the major signaling pathway promoting Toll-like receptor 2 and Toll-like receptor 4 (TLR2/4)-mediated tolerance in monocytes. However, despite previous reports emphasizing a central role of TLR2 in innate immune recognition of P. gingivalis, our current findings highlight a selective role of TLR4 in the promotion of OMV-mediated TNF tolerance: only blockade of TLR4-and not of TLR2-restores responsiveness toP. gingivalis Of further note, OMV-mediated tolerance is preserved in the presence of cytochalasin B and chloroquine, indicating that triggering of surface TLR4 is sufficient for this effect. Taking the results together, we propose that P. gingivalis OMV contribute to local immune evasion of P. gingivalis by hampering the host response.

Porphyromonas gingivalis Outer Membrane Vesicles Induce Selective Tumor Necrosis Factor Tolerance in a Toll-Like Receptor 4- and mTOR-Dependent Manner.

Maternal immune tolerance toward the fetus is an essential requisite for pregnancy. While T cell functions are well documented, little is known about the participation of B cells. We have previously suggested that IL-10-producing B cells are involved in pregnancy tolerance in mice and humans. By employing murine and human systems, we report now that fetal trophoblasts positively regulate the generation of IL-10-producing B cells. We next studied the participation of hormones produced by the placenta as well as the fetal protein alpha-fetoprotein (AFP) in B cell modulation. Human chorionic gonadotropin (hCG), but not progesterone, estrogen, or a combination of both, was able to promote changes in B cell phenotype and boost their IL-10 production, which was abolished after blocking hCG. The hCG-induced B cell phenotype was not associated with augmented galactosylation, sialylation, or fucosylation of IgG subclasses in their Fc. In vitro, hCG induced the synthesis of asymmetrically glycosylated antibodies in their Fab region. Interestingly, AFP had dual effects depending on the concentration. At concentrations corresponding to maternal serum levels, it did not modify the phenotype or IL-10 secretion of B cells. At fetal concentrations, however, AFP was able to drive B cells into apoptosis, which may indicate a protective mechanism to avoid maternal B cells to reach the fetus. Our data suggest that the fetus secrete factors that promote a pregnancy-friendly B cell phenotype, unraveling interesting aspects of B cell function, and modulation by pregnancy hormones and fetal proteins.

Maternal and Fetal Mechanisms of B Cell Regulation during Pregnancy: Human Chorionic Gonadotropin Stimulates B Cells to Produce IL-10 While Alpha-Fetoprotein Drives Them into Apoptosis.

Bacterial biofilms cause a considerable amount of prosthetic joint infections every year, resulting in morbidity and expensive revision surgery. To address this problem, surface modifications of implant materials such as carbon nanotube (CNT) coatings have been investigated in the past years. CNTs are biologically compatible and can be utilized as drug delivery systems. In this study, multi-walled carbon nanotube (MWCNT) coated TiAl6V4 titanium alloy discs were fabricated and impregnated with Rifampicin, and tested for their ability to prevent biofilm formation over a period of ten days. Agar plate-based assays were employed to assess the antimicrobial activity of these surfaces against Staphylococcus epidermidis. It was shown that vertically aligned MWCNTs were more stable against attrition on rough surfaces than on polished TiAl6V4 surfaces. Discs with coated surfaces caused a significant inhibition of biofilm formation for up to five days. Therefore, MWCNT-modified surfaces may be effective against pathogenic biofilm formation on endoprostheses.

Long-term release of antibiotics by carbon nanotube-coated titanium alloy surfaces diminish biofilm formation by Staphylococcus epidermidis.

Intracellular persistence of Staphylococcus aureus favors bacterial spread and chronic infections. Here, we provide evidence for the existence of human CD4+ and CD8+ T cell memory against staphylococcal antigens. Notably, the latter could provide a missing link in our understanding of immune control of intracellular S. aureus. The analyses showed that pulsing of monocyte-derived dendritic cells (MoDC) with native staphylococcal protein antigens induced release of Th2-associated cytokines and mediators linked to T regulatory cell development (G-CSF, IL-2 and IL-10) from both CD4+ and CD8+ T cells, thus revealing a state of tolerance predominantly arising from preformed memory T cells. Furthermore, G-CSF was identified as a suppressor of CD8+ T cell-derived IFNγ secretion, thus confirming a tolerogenic role of this cytokine in the regulation of T cell responses to S. aureus. Nevertheless, delivery of in vitro transcribed mRNA-encoded staphylococcal antigens triggered Th1-biased responses, e.g. IFNγ and TNF release from both naïve and memory T cells. Collectively, our data highlight the potential of mRNA-adjuvanted antigen presentation to enable inflammatory responses, thus overriding the existing Th2/Treg-biased memory T cell response to native S. aureus antigens.

Antigen delivery to dendritic cells shapes human CD4+ and CD8+ T cell memory responses to Staphylococcus aureus.

To develop novel techniques for improving blood safety, dedicated bacterial strains, which are able to persist and to proliferate in blood platelet concentrates, are needed. Here, we present draft genome sequences of the four bacterial strains approved for the first WHO repository of platelet transfusion-relevant bacterial reference strains.

Genome Sequences of the First WHO Repository of Platelet Transfusion-Relevant Bacterial Reference Strains.

Influenza A virus infection causes substantial morbidity and mortality in seasonal epidemic outbreaks, and more efficient treatments are urgently needed. Innate immune sensing of viral nucleic acids stimulates antiviral immunity, including cell-autonomous antiviral defense mechanisms that restrict viral replication. RNA oligonucleotide ligands that potently activate the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are promising candidates for the development of new antiviral therapies. Here, we demonstrate in an Mx1-expressing mouse model of influenza A virus infection that a single intravenous injection of low-dose RIG-I ligand 5'-triphosphate RNA (3pRNA) completely protected mice from a lethal challenge with influenza A virus for at least 7 days. Furthermore, systemic administration of 3pRNA rescued mice with pre-established fulminant influenza infection and prevented the fatal effects of a streptococcal superinfection. Type I interferon, but not interferon-λ, was required for the therapeutic effect. Our results suggest that the use of RIG-I activating oligonucleotide ligands has the clinical potential to confine influenza epidemics when a strain-specific vaccine is not yet available and to reduce lethality of influenza in severely infected patients.

Isabelle Bekeredjian-Ding

Division of EU Cooperation/Microbiology

Paul-Ehrlich-Institute

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

Isabelle Bekeredjian-Ding

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of EU Cooperation/Microbiology

Paul-Ehrlich-Institute

Novel Diagnostics in Revision Arthroplasty: Implant Sonication and Multiplex Polymerase Chain Reaction

Division of EU Cooperation/Microbiology