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Abstract Cytomegalovirus (CMV) is known to alter circulating effector memory or re-expressing CD45RA+ (TemRA) T-cell numbers, but whether Epstein–Barr virus (EBV) does the same or this is amplified during a CMV and EBV co-infection is unclear. Immune cell numbers in blood of children and young, middle-aged, and senior adults (n = 336) were determined with flow cytometry, and additional multivariate linear regression, intra-group correlation, and cluster analyses were performed. Compared to non-infected controls, CMV-seropositive individuals from all age groups had more immune cell variance, and CMV+ EBV− senior adults had more late-differentiated CD4+ and CD8+ TemRA and CD4+ effector memory T-cells. EBV-seropositive children and young adults had a more equal immune cell composition than non-infected controls, and CMV− EBV+ senior adults had more intermediate/late-differentiated CD4+ TemRA and effector memory T-cells than non-infected controls. CMV and EBV co-infected young and middle-aged adults with an elevated BMI and anti-CMV antibody levels had a similar immune cell composition as senior adults, and CMV+ EBV+ middle-aged adults had more late-differentiated CD8+ TemRA, effector memory, and HLA-DR+ CD38− T-cells than CMV+ EBV− controls. This study identified changes in T-cell numbers in CMV- or EBV-seropositive individuals and that some CMV and EBV co-infected young and middle-aged adults had an aging-related T-cell phenotype.

S. aureus infection of bone is difficult to eradicate due to its ability to colonize the osteocyte-lacuno-canalicular network (OLCN), rendering it resistant to standard-of-care (SOC) antibiotics. To overcome this, we proposed two bone-targeted bisphosphonate-conjugated antibiotics (BCA): bisphosphonate-conjugated sitafloxacin (BCS) and hydroxybisphosphonate-conjugate sitafloxacin (HBCS). Initial studies demonstrated that the BCA kills S. aureus in vitro. Here we demonstrate the in vivo efficacy of BCS and HBCS versus bisphosphonate, sitafloxacin, and vancomycin in mice with implant-associated osteomyelitis. Longitudinal bioluminescent imaging (BLI) confirmed the hypothesized “target and release”-type kinetics of BCS and HBCS. Micro-CT of the infected tibiae demonstrated that HBCS significantly inhibited peri-implant osteolysis versus placebo and free sitafloxacin (p < 0.05), which was not seen with the corresponding non-antibiotic-conjugated bisphosphonate control. TRAP-stained histology confirmed that HBCS significantly reduced peri-implant osteoclast numbers versus placebo and free sitafloxacin controls (p < 0.05). To confirm S. aureus killing, we compared the morphology of S. aureus autolysis within in vitro biofilm and infected tibiae via transmission electron microscopy (TEM). Live bacteria in vitro and in vivo presented as dense cocci ~1 μm in diameter. In vitro evidence of autolysis presented remnant cell walls of dead bacteria or “ghosts” and degenerating (non-dense) bacteria. These features of autolyzed bacteria were also present among the colonizing S. aureus within OLCN of infected tibiae from placebo-, vancomycin-, and sitafloxacin-treated mice, similar to placebo. However, most of the bacteria within OLCN of infected tibiae from BCA-treated mice were less dense and contained small vacuoles and holes >100 nm. Histomorphometry of the bacteria within the OLCN demonstrated that BCA significantly increased their diameter versus placebo and free antibiotic controls (p < 0.05). As these abnormal features are consistent with antibiotic-induced vacuolization, bacterial swelling, and necrotic phenotype, we interpret these findings to be the initial evidence of BCA-induced killing of S. aureus within the OLCN of infected bone. Collectively, these results support the bone targeting strategy of BCA to overcome the biodistribution limits of SOC antibiotics and warrant future studies to confirm the novel TEM phenotypes of bacteria within OLCN of S. aureus-infected bone of animals treated with BCS and HBCS.

Staphylococcus aureus is the main causative pathogen of subcutaneous, bone, and implant-related infections, forming structures known as staphylococcal abscess communities (SACs) within tissues that also contain immunosuppressive myeloid-derived suppressor cells (MDSCs). Although both SACs and MDSCs are present in chronic S. aureus infections, it remains unknown whether SACs directly trigger MDSC expansion. To investigate this, a previously developed 3D in vitro SAC model was co-cultured with murine and human bone marrow cells. Subsequently, it was shown that SAC-exposed human CD11blow/− myeloid cells or SAC-exposed murine CD11b+ Gr-1+ cells were immunosuppressive mainly by reducing absolute CD4+ and CD8α+ T cell numbers, as shown in T cell proliferation assays and with flow cytometry. Monocytic MDSCs from mice with an S. aureus bone infection also strongly reduced CD4+ and CD8α+ T cell numbers. Using protein biomarker analysis and an immunoassay, we detected in SAC–bone marrow co-cultures high levels of GM-CSF, IL-6, VEGF, IL-1β, TNFα, IL-10, and TGF-β. Furthermore, SAC-exposed neutrophils expressed Arg-1 and SAC-exposed monocytes expressed Arg-1 and iNOS, as shown via immunofluorescent stains. Overall, this study showed that SACs cause MDSC expansion from bone marrow cells and identified possible mediators to target as an additional strategy for treating chronic S. aureus infections.

Orthopaedic device-related infection (ODRI) presents a significant challenge to the field of orthopaedic and trauma surgery. Despite extensive treatment involving surgical debridement and prolonged antibiotic therapy, outcomes remain poor. This is largely due to the unique abilities of Staphylococcus aureus, the most common causative agent of ODRI, to establish and protect itself within the host by forming biofilms on implanted devices and staphylococcal abscess communities (SACs). There is a need for novel antimicrobials that can readily target such features. Enzybiotics are a class of antimicrobial enzymes derived from bacteria and bacteriophages, which function by enzymatically degrading bacterial polymers essential to bacterial survival or biofilm formation. Here, we apply an enzybiotic-based combination regimen to a set of in vitro models as well as in a murine ODRI model to evaluate their usefulness in eradicating established S. aureus infection, compared to classical antibiotics. We show that two chimeric endolysins previously selected for their functional efficacy in human serum in combination with a polysaccharide depolymerase reduce bacterial CFU numbers 10,000-fold in a peg model and in an implant model of biofilm. The enzyme combination also completely eradicates S. aureus in a SAC in vitro model where classical antibiotics are ineffective. In an in vivo ODRI model in mice, the antibiofilm effects of this enzyme regimen are further enhanced when combined with a classical gentamicin/vancomycin treatment. In a mouse model of methicillin-resistant S. aureus (MRSA) ODRI following a fracture repair, a combined local enzybiotic/antibiotic treatment regimen showed a significant CFU reduction in the device and the surrounding soft tissue, as well as significant prevention of weight loss. These outcomes were superior to treatment with antibiotics alone. Overall, this study demonstrates that the addition of enzybiotics, which are distinguished by their extremely rapid killing efficacy and antibiofilm activities, can enhance the treatment of severe MRSA ODRI.

Abstract Background As older age and having certain comorbidities can influence humoral responses to vaccination, we studied antibody responses after the COVID-19 booster campaigns in nursing home (NH) residents. Methods In a two year longitudinal study with Dutch NH residents (n = 107), aged 50 years and over, we monitored antibody responses in serum prior to and after vaccination with a third, fourth BNT162b2 (wild-type; WT), and a BNT162b2 bivalent (WT/OMI BA.1) fifth vaccine. Data on vaccinations, infections, comorbidities, and, for some participants, clinical symptoms after infection were obtained with questionnaires. Data were compared to antibody responses of BNT162b2-vaccinated, healthier community-dwelling older adults (n = 32) from the general population. Results The booster vaccinations substantially increased anti-WT and anti-Omicron SARS-CoV-2 Spike S1 (S1) and Spike protein receptor binding domain (RBD)-antibody concentrations of NH residents. This resulted in comparable antibody levels between NH residents and healthier community-dwelling older adults and between infection-naïve and infected NH residents, and in a decline in treatment duration and clinical symptom severity in SARS-CoV-2-infected NH residents. Between one and twelve months after the bivalent fifth dose, anti-Omicron BA.1 antibody levels of the NH residents waned faster than those against the WT strain. Conclusions The booster vaccinations upheld humoral responses of NH residents to WT and Omicron SARS-CoV-2. This, in addition to the less virulent circulating strains, decreased symptom severity and treatment durations for SARS-CoV-2-infected NH residents. Boosting this vulnerable group should, therefore, be continued to prevent waning of humoral immunity and achieve sufficient protection especially against newly emerging variants of concern.