Comparative Evaluation of Current Biochemical-, Sequencing-, and Proteomic-Based Identification Methods for the Streptococcus bovis Group.

The Streptococcus bovis group (previously group D streptococci) consists of seven distinct species and subspecies. Definitive identification within the group is important, as certain organisms have been associated with gastrointestinal carcinoma, bacteremia, infective endocarditis, meningitis, biliary tract disease, and carcinoma, among others. Definitive identification, however, remains elusive due to limitations and inconsistencies across commonly used identification platforms in the United States. Here, we compared the performance of standard biochemical (Trek Gram-positive identification [GPID] plate, Vitek 2 GPID), sequencing (16S rDNA, sodA) databases (NCBI, RDP, CDC MicrobeNet), and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) platforms (Vitek MS, Bruker Biotyper MS) using a set of eight type strains representing all seven strains within the S. bovis group. Despite the evaluation of contemporary methods, no single platform was able to definitively identify all type strains within the S. bovis group. Vitek MS (85.7%, 7/8) provided the most accurate definitive identifications, followed by sodA sequencing (75%, 6/8). Vitek 2 and Bruker Biotyper RUO platforms performed the next best (62.5%, 5/8). All remaining platforms failed to adequately differentiate type strains within the S. bovis group (range, 0 to 37.5%). Laboratorians and clinicians should be aware of the identification limitations of routine testing algorithms and incorporate reflex testing, when appropriate, to platforms such as Vitek MS and/or sodA sequencing that are more able to definitively identify S. bovis group organisms. Further clinical evaluation was conducted using 65 clinical isolates from three geographically distinct U.S. institutions. Future improvements in identification platforms may reveal new clinical and epidemiological trends for members of the S. bovis group.

Context-Dependent Roles for Toll-Like Receptors 2 and 9 in the Pathogenesis of Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the major causative agent of bacterial osteomyelitis, an invasive infection of bone. Inflammation generated by the immune response to S. aureus contributes to bone damage by altering bone homeostasis. Increases in the differentiation of monocyte lineage cells into bone-resorbing osteoclasts (osteoclastogenesis) promote bone loss in the setting of osteomyelitis. In this study, we sought to define the role of Toll-like receptor (TLR) signaling in the pathogenesis of S. aureus osteomyelitis. We hypothesized that S. aureus-sensing TLRs 2 and 9, both of which are known to alter osteoclastogenesis in vitro, promote pathological changes to bone, including increased osteoclast abundance, bone loss, and altered callus formation during osteomyelitis. Stimulation of osteoclast precursors with S. aureus supernatant increased osteoclastogenesis in a TLR2-dependent, but not a TLR9-dependent, manner. However, in vivo studies using a posttraumatic murine model of osteomyelitis revealed that TLR2-null mice experienced similar bone damage and increased osteoclastogenesis compared to wild type (WT) mice. Therefore, we tested the hypothesis that compensation between TLR2 and TLR9 contributes to osteomyelitis pathogenesis. We found that mice deficient in both TLR2 and TLR9 (Tlr2/9-/-) have decreased trabecular bone loss in response to infection compared to WT mice. However, osteoclastogenesis is comparable between WT and Tlr2/9-/- mice, suggesting that alternative mechanisms enhance osteoclastogenesis in vivo during osteomyelitis. Indeed, we discovered that osteoclast precursors intracellularly infected with S. aureus undergo significantly increased osteoclast formation, even in the absence of TLR2 and TLR9. These results suggest that TLR2 and TLR9 have context-dependent roles in the alteration of bone homeostasis during osteomyelitis.

Comprehensive Study Identifies a Sensitive, Low-Risk, Closed-System Model for Detection of Fungal Contaminants in Cell and Gene Therapy Products.

The U.S. Food and Drug Administration (FDA) regulates manufacturing and testing of advanced therapeutic medicinal products (ATMPs) to ensure the safety of each product for human use. Gold-standard sterility testing (USP<71>) and alternative blood culture systems have major limitations for the detection of fungal contaminants. In this study, we evaluated the performance of iLYM (lactic acid-fermenting organisms, yeasts, and mold) medium (designed originally for the food and beverage industry) to assess its potential for use in the biopharmaceutical field for ATMP sterility testing. We conducted a parallel evaluation of four different test systems (USP<71>, BacT/Alert, Bactec, and Sabouraud dextrose agar [SDA] culture), three different bottle media formulations (iLYM, iFA Plus, and Myco/F Lytic), and two incubation temperatures (22.5°C and 32.5 to 35°C) using a diverse set of fungi (n = 51) isolated from NIH cleanroom environments and previous product contaminants. Additionally, we evaluated the effect of agitation versus delayed-entry static preincubation on test sensitivity and time to detection (TTD). Overall, delayed entry of bottles onto the BacT/Alert or Bactec instruments (with agitation) did not improve test performance. USP<71> and SDA culture continued to significantly outperform each automated culture condition alone. However, we show, for the first time, that a closed-system, dual-bottle combination of iLYM 22.5°C and iFA Plus 32.5°C can provide high fungal sensitivity, statistically comparable to USP<71>, when tested against a diverse range of environmental fungi. Our study fills a much-needed gap in biopharmaceutical testing and offers a favorable testing algorithm that maximizes bacterial and fungal test sensitivity while minimizing risk of product contamination associated with laboratory handling.

Dacron swab and PBS are acceptable alternatives to flocked swab and viral transport media for SARS-CoV-2.

Nasopharyngeal flocked swabs placed in viral transport media (VTM) are the preferred collection methodology for respiratory virus testing. Due to the rapid depletion of available reagents and swabs, we have validated an alternative swab placed in phosphate-buffered saline (PBS) for use in respiratory virus testing in a SARS-CoV-2 real-time polymerase chain reaction assay and a multiplexed respiratory virus panel. We collected nasopharyngeal (NP) swabs and oropharyngeal (OP) swabs from 10 healthy volunteers. Flocked swabs were placed in VTM and alternative swabs in PBS. In this feasibility study, we show that NP collection is better for detection of human material than OP collection, as measured by significantly lower RNase P gene cycle threshold values, and that a Dacron polyester swab in PBS shows equivalent detection of SARS-CoV-2 and RSV to a flocked swab in VTM in contrived specimens. Diluted SARS-CoV-2-positive patient specimens are detectable for up to 72 h at 4 °C.

Association of persistent wild-type measles virus RNA with long-term humoral immunity in rhesus macaques.

Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for 6 months after infection with wild-type measles virus (MeV). Infection caused viremia and rash, with clearance of infectious virus by day 14. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H), and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral T follicular helper cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody against both H and N, with more H-specific ASCs in BM. During days 14-21, 20- to 100-fold more total ASCs than MeV-specific ASCs appeared in circulation, suggesting mobilization of preexisting ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation, and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.

MicroLESA: Integrating Autofluorescence Microscopy, In Situ Micro-Digestions, and Liquid Extraction Surface Analysis for High Spatial Resolution Targeted Proteomic Studies.

The ability to target discrete features within tissue using liquid surface extractions enables the identification of proteins while maintaining the spatial integrity of the sample. Here, we present a liquid extraction surface analysis (LESA) workflow, termed microLESA, that allows proteomic profiling from discrete tissue features of ∼110 µm in diameter by integrating nondestructive autofluorescence microscopy and spatially targeted liquid droplet micro-digestion. Autofluorescence microscopy provides the visualization of tissue foci without the need for chemical stains or the use of serial tissue sections. Tryptic peptides are generated from tissue foci by applying small volume droplets (∼250 pL) of enzyme onto the surface prior to LESA. The microLESA workflow reduced the diameter of the sampled area almost 5-fold compared to previous LESA approaches. Experimental parameters, such as tissue thickness, trypsin concentration, and enzyme incubation duration, were tested to maximize proteomics analysis. The microLESA workflow was applied to the study of fluorescently labeled Staphylococcus aureus infected murine kidney to identify unique proteins related to host defense and bacterial pathogenesis. Proteins related to nutritional immunity and host immune response were identified by performing microLESA at the infectious foci and surrounding abscess. These identifications were then used to annotate specific proteins observed in infected kidney tissue by MALDI FT-ICR IMS through accurate mass matching.

MyD88 and IL-1R signaling drive antibacterial immunity and osteoclast-driven bone loss during Staphylococcus aureus osteomyelitis.

Staphylococcus aureus is able to infect virtually all organ systems and is a frequently isolated etiologic agent of osteomyelitis, a common and debilitating invasive infection of bone. Treatment of osteomyelitis requires invasive surgical procedures and prolonged antibiotic therapy, yet is frequently unsuccessful due to extensive pathogen-induced bone damage that can limit antibiotic penetration and immune cell influx to the infectious focus. We previously established that S. aureus triggers profound alterations in bone remodeling in a murine model of osteomyelitis, in part through the production of osteolytic toxins. However, staphylococcal strains lacking osteolytic toxins still incite significant bone destruction, suggesting that host immune responses are also major drivers of pathologic bone remodeling during osteomyelitis. The objective of this study was to identify host immune pathways that contribute to antibacterial immunity during S. aureus osteomyelitis, and to define how these immune responses alter bone homeostasis and contribute to bone destruction. We specifically focused on the interleukin-1 receptor (IL-1R) and downstream adapter protein MyD88 given the prominent role of this signaling pathway in both antibacterial immunity and osteo-immunologic crosstalk. We discovered that while IL-1R signaling is necessary for local control of bacterial replication during osteomyelitis, it also contributes to bone loss during infection. Mechanistically, we demonstrate that S. aureus enhances osteoclastogenesis of myeloid precursors in vitro, and increases the abundance of osteoclasts residing on bone surfaces in vivo. This enhanced osteoclast abundance translates to trabecular bone loss, and is dependent on intact IL-1R signaling. Collectively, these data define IL-1R signaling as a critical component of the host response to S. aureus osteomyelitis, but also demonstrate that IL-1R-dependent immune responses trigger collateral bone damage through activation of osteoclast-mediated bone resorption.

Innate Immunity to Staphylococcus aureus: Evolving Paradigms in Soft Tissue and Invasive Infections.

Staphylococcus aureus causes a wide range of diseases that together embody a significant public health burden. Aided by metabolic flexibility and a large virulence repertoire, S. aureus has the remarkable ability to hematogenously disseminate and infect various tissues, including skin, lung, heart, and bone, among others. The hallmark lesions of invasive staphylococcal infections, abscesses, simultaneously denote the powerful innate immune responses to tissue invasion as well as the ability of staphylococci to persist within these lesions. In this article, we review the innate immune responses to S. aureus during infection of skin and bone, which serve as paradigms for soft tissue and bone disease, respectively.

Early TGF-ß inhibition in mice reduces the incidence of breast cancer induced bone disease in a myeloid dependent manner.

The tumor-cell microenvironment is recognized as a dynamic place where critical cell interactions occur and play an important role in altering tumorigenesis. While many studies have investigated the effects of cellular cross-talk within distinct tumor microenvironments, these interactions have yet to be fully examined in bone. It is well-established that many common cancers metastasize to bone, resulting in the development of tumor-induced bone disease (TIBD), a multi-facetted illness that is driven by complex cell interactions within the bone marrow. Our group has previously published that myeloid progenitor cells expand in the presence of tumors in bone, aligning with the notion that myeloid cells can act as tumor promotors. Several groups, including ours, have established that transforming growth factor ß (TGF-ß), an abundant growth factor in bone, can regulate both TIBD and myeloid expansion. TGF-ß inhibitors have been shown to increase bone volume, decrease bone destruction, and reduce but not eliminate tumor. Therefore, we hypothesize that inhibiting TGF-ß will reduce myeloid expansion leading to a reduction of tumor burden in bone and osteoclast-mediated bone loss, causing to an overall reduction in TIBD. To address this hypothesis, two different mouse models of breast cancer bone colonization were pre-treated with the TGF-ß neutralizing antibody, 1D11, prior to tumor inoculation (athymic: MDA-MB-231, BALB/c: 4T1) and continuously treated until sacrifice. Additionally, a genetically modified mouse model with a myeloid specific deletion of transforming growth factor beta receptor II (TGF-ßRII) (TGF-ßRIIMyeKO) was utilized in our studies. Systemic inhibition of TGF-ß lead to fewer osteolytic lesions, and reduced tumor burden in bone as expected from previous studies. Additionally, early TGF-ß inhibition affected expansion of distinct myeloid populations and shifted the cytokine profile of pro-tumorigenic factors in bone, 4T1 tumor cells, and bone-marrow derived macrophages. Similar observations were seen in tumor-bearing TGF-ßRIIMyeKO mice, where these mice contained fewer bone lesions and significantly less tumor burden in bone, suggesting that TGF-ß inhibition regulates myeloid expansion leading to a significant reduction in TIBD.

Repurposing the Nonsteroidal Anti-inflammatory Drug Diflunisal as an Osteoprotective, Antivirulence Therapy for Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus osteomyelitis is a common and debilitating invasive infection of bone. Treatment of osteomyelitis is confounded by widespread antimicrobial resistance and the propensity of bacteria to trigger pathological changes in bone remodeling that limit antimicrobial penetration to the infectious focus. Adjunctive therapies that limit pathogen-induced bone destruction could therefore limit morbidity and enhance traditional antimicrobial therapies. In this study, we evaluate the efficacy of the U.S. Food and Drug Administration-approved, nonsteroidal anti-inflammatory (NSAID) compound diflunisal in limiting S. aureus cytotoxicity toward skeletal cells and in preventing bone destruction during staphylococcal osteomyelitis. Diflunisal is known to inhibit S. aureus virulence factor production by the accessory gene regulator (agr) locus, and we have previously demonstrated that the Agr system plays a substantial role in pathological bone remodeling during staphylococcal osteomyelitis. Consistent with these observations, we find that diflunisal potently inhibits osteoblast cytotoxicity caused by S. aureus secreted toxins independently of effects on bacterial growth. Compared to commonly used NSAIDs, diflunisal is uniquely potent in the inhibition of skeletal cell death in vitro Moreover, local delivery of diflunisal by means of a drug-eluting, bioresorbable foam significantly limits bone destruction during S. aureus osteomyelitis in vivo Collectively, these data demonstrate that diflunisal potently inhibits skeletal cell death and bone destruction associated with S. aureus infection and may therefore be a useful adjunctive therapy for osteomyelitis.

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection.

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.