Search Results (10)

Handling cultured isolates and clinical, environmental, or wildlife surveillance samples containing Risk Group 3 and 4 pathogens presents considerable biosafety challenges in minimizing human exposure during processing and transport. Safe handling typically requires high- or maximum-containment facilities, demanding substantial logistical planning and resources. We evaluated PrimeStore Molecular Transport Medium (PS-MTM), a guanidine-based solution created to kill pathogens and preserve nucleic acids at ambient temperatures, for inactivating Crimean-Congo hemorrhagic fever, eastern equine encephalitis, Ebola, Hendra, Japanese encephalitis, Lassa, Marburg, Nipah, Rift Valley fever, and West Nile viruses. To mimic diagnostic conditions, human whole blood spiked with any of these viruses was incubated with PS-MTM for 20-, 30-, or 60-min. Samples with titers up to 10⁷ PFU/mL exposed to PS-MTM at all time points resulted in complete loss of infectivity judged by plaque assays. A 30-min incubation provided a 50% safety margin over the minimum inactivation time and was used for quantification with the tissue culture infectious dose (TCID₅₀) assay, enabling evaluation of PS-MTM’s activity for viruses that do or do not produce well-defined plaques. Results confirmed that PS-MTM inactivated all tested viruses at titers up to 10⁷ TCID₅₀/mL, underscoring its reliability for enhancing biosafety in diagnostics, outbreak management, and surveillance. Full article

Staphylococcus aureus is a vital pathogen causing clinical infections. Capsules are important virulence factors for S. aureus. This study investigates the regulatory mechanisms underlying capsule production in S. aureus. Bacterial strains XN108 and Newman were used, and combined approaches like RNA sequencing (RNA-seq), RT-qPCR, transmission electron microscopy (TEM), gene reporter, and electrophoretic mobility shift assay (EMSA) were performed to test the role and mechanism of WalK(S221P) mutation in S. aureus capsule production. RNA-seq showed an increased expression of cap genes in the WalK(S221P)-carried S. aureus XN108 relative to the mutation-cured XN108-R. TEM and capsular polysaccharide determination demonstrated that XN108 produced more capsules than XN108-R did. Similar results were presented in the WalK(S221P)-contained K-Newman versus the wild-type Newman. RT-qPCR screening showed an increasing expression of the mgrA gene in XN108 versus XN108-R. Gene reporter and EMSA analysis revealed that WalK(S221P) mutation promoted S. aureus capsule production through MgrA. Deletion of mgrA decreased the WalK(S221P)-mediated capsule yield. Moreover, WalK(S221P) mutation remarkably increased the tolerance of S. aureus to whole blood killing and microphage phagocytosis. Overall, these data provide mechanistic insights into the effect of WalK(S221P) on the capsule production of S. aureus, which may set down foundations for future S. aureus virulence investigations. Full article

Background: Gum arabic, a polysaccharide exudate from Acacia senegal (L.) Willdenow trees, has already been used by African native people in natural medicine. Methods: Using whole-blood samples from young (20–35 years) and older (>80 years) healthy volunteers (each group n = 10), the effect of an aqueous solution of GA on phagocytosis of Escherichia coli was examined with a gentamicin protection assay. Whole-blood samples of each volunteer were stimulated with GA and as a control with CpG oligodeoxynucleotides (Toll-like receptor -9 agonists) for 2 h, then co-incubated with E. coli for 30 min and thereafter treated with gentamicin for up to 240 min to kill extracellular bacteria. Then, whole-blood cells were lysed with distilled water, and colony-forming units were counted by quantitative plating. Cytokine enzyme-linked immunosorbent assay for the detection of TNF-α and IL-6 was performed using the blood supernatant. Results: The GA concentration tested (20 mg/mL) did not affect the viability of eukaryotic cells. Phagocytosis of E. coli by whole-blood leukocytes derived from young (p = 0.008) and older (p = 0.004) healthy volunteers was increased by 120.8% (young) and 39.2% (old) after stimulation with GA. In contrast, CpG only stimulated the bacterial phagocytosis by cells derived from young volunteers (p = 0.004). Stimulation of whole blood with GA increased the intracellular killing of E. coli in young (p = 0.045) and older volunteers (p = 0.008) and induced a TNF-α release in whole blood collected from older volunteers but not from younger ones (p = 0.008). Conclusions: These data encourage the isolation of active compounds of GA and the initiation of clinical trials addressing the preventive effect of GA on bacterial infections. Full article

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a strictly human pathogen that causes a wide range of diseases, including skin and soft tissue infections, toxic shock syndrome and acute rheumatic fever. We have recently reported that Spy1094 and Spy1370 of S. pyogenes serotype M1 are N-acetylglucosamine (GlcNAc) deacetylases. We have generated spy1094 and spy1370 gene deletion mutants in S. pyogenes and gain-of-function mutants in Lactococcus lactis. Similar to other cell wall deacetylases, our results show that Spy1094 and Spy1370 confer lysozyme-resistance. Furthermore, deletion of the genes decreased S. pyogenes virulence in a human whole blood killing assay and a Galleria mellonella (Greater wax moth) larvae infection model. Expression of the two genes in L. lactis resulted in increased lysozyme resistance and survival in whole human blood, and reduced survival of infected G. mellonella larvae. Deletion of the spy1370, but not the spy1094 gene, decreased resistance to the cationic antimicrobial peptide cecropin B, whereas both enzymes increased biofilm formation, probably resulting from the increase in positive charges due to deacetylation of the cell wall. In conclusion, Spy1094 and Spy1370 are important S. pyogenes virulence factors and might represent attractive targets for the development of antibacterial agents. Full article

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures. Full article

There is mounting evidence that the microbiome plays a critical role in training and maturation of the host immune system. Pre-clinical and clinical studies have shown that microbiome perturbation is correlated with sub-optimal host responses to vaccines and cancer immunotherapy. As such, identifying species of commensal bacteria capable of modulating immunological outcomes is of considerable interest. Currently, the lack of reliable primary immune cell-based assays capable of differentiating immuno-modulatory properties of various commensal bacteria is a major limitation. Here, we demonstrate that primary human monocyte-derived dendritic cells (MoDC) are capable of stratifying different strains of live and heat-killed commensal bacteria in an in vitro culture system. Specifically, heat-killed bacterial strains were able to differentially modulate co-stimulation/maturation markers CD80, CD83, and HLA-DR, as well as cytokine/chemokine signatures, such as IL-1b, MIP-1a, and TNFa in primary human MoDC. We further validated our observations using the TruCulture^® (Myriad RBM, Inc., Austin, TX, USA) whole-blood ex vivo culture system. Using this ex vivo system allowed us to measure immune-altering effects of commensal bacteria in primary human whole-blood. As such, we report that both these primary in vitro and ex vivo systems are robust and enable identification, stratification, and differentiation of various commensal bacteria as potential modulators of host immunity. Full article

The global increase in multidrug-resistant (MDR) pathogenic bacteria has led to growing interest in bacteriophage (“phage”) therapy. Therapeutic phages are usually selected based on their ability to infect and lyse target bacteria, using in vitro assays. In these assays, phage infection is determined using target bacteria grown in standard commercial rich media, while evaluation of the actual therapeutic activity requires the presence of human blood. In the present work, we characterized the ability of two different Yersinia pestis lytic phages (ϕA1122 and PST) to infect and kill a luminescent Y. pestis EV76 strain suspended in Brain Heart Infusion (BHI)-rich medium or in human whole blood, simulating the host environment. We found that the ability of the phages to infect and lyse blood-suspended Y. pestis was not correlated with their ability to infect and lyse BHI-suspended bacteria. While the two different phages exhibited efficient infective capacity in a BHI-suspended culture, only the PST phage showed efficient lysis ability against blood-suspended bacteria. Therefore, we recommend that for personalized phage therapy, selection of phage(s) for efficient treatment of patients suffering from MDR bacterial infections should include prior testing of the candidate phage(s) for their lysis ability in the presence of human blood. Full article

Natural killer (NK) cells are cytotoxic innate lymphocytes endowed with a unique ability to kill a broad spectrum of cancer and virus-infected cells. Given their key contribution to diverse diseases, the measurement of NK cell activity (NKA) has been used to estimate disease prognosis or the effect of therapeutic treatment. Currently, NKA assays are primarily based on cumbersome procedures related to careful labeling and handling of target cells and/or NK cells, and they require a rapid isolation of peripheral blood mononuclear cells (PBMCs) which often necessitates a large amount of blood. Here, we developed an ELISA-based whole blood (WB) NKA assay involving engineered target cells (P815-ULBP1+CD48) providing defined and synergistic stimulation for NK cells via NKG2D and 2B4. WB collected from healthy donors (HDs) and patients with multiple myeloma (MM) was stimulated with P815-ULBP1+CD48 cells combined with IL-2. Thereafter, it utilized the serum concentrations of granzyme B and IFN-γ originating in NK cells as independent and complementary indicators of NKA. This WB NKA assay demonstrated that MM patients exhibit a significantly lower NKA than HDs following stimulation with P815-ULBP1+CD48 cells and had a good correlation with the commonly used flow cytometry-based PBMC NKA assay. Moreover, the use of P815-ULBP1+CD48 cells in relation to assessing the levels of NKG2D and 2B4 receptors on NK cells facilitated the mechanistic study and led to the identification of TGF-β1 as a potential mediator of compromised NKA in MM. Thus, our proposed WB NKA assay facilitates the reliable measurement of NKA and holds promise for further development as both a clinical and research tool. Full article

Older adults are at increased risk for vitamin and mineral deficiencies that contribute to age-related immune system decline. Several lines of evidence suggest that taking a multi-vitamin and mineral supplement (MVM) could improve immune function in individuals 55 and older. To test this hypothesis, we provided healthy older adults with either an MVM supplement formulated to improve immune function (Redoxon^® VI, Singapore) or an identical, inactive placebo control to take daily for 12 weeks. Prior to and after treatment, we measured (1) their blood mineral and vitamin status (i.e., vitamin C, zinc and vitamin D); (2) immune function (i.e., whole blood bacterial killing activity, neutrophil phagocytic activity, and reactive oxygen species production); (3) immune status (salivary IgA and plasma cytokine/chemokine levels); and (4) self-reported health status. MVM supplementation improved vitamin C and zinc status in blood and self-reported health-status without altering measures of immune function or status or vitamin D levels, suggesting that healthy older adults may benefit from MVM supplementation. Further development of functional assays and larger study populations should improve detection of specific changes in immune function after supplementation in healthy older adults. Clinical Trials Registration: ClinicalTrials.gov #NCT02876315. Full article

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and to study the potential effects of the induced cytokines on bacterial killing. We measured TNF-α, IL-6, IFN-γ, IL-17A and IL-10 after an experimental intravenous infection with S. suis serotype 2 in vivo, and analyzed whole blood, peripheral blood mononuclear cells (PBMC) and separated leukocytes to identify the cytokine-producing cell type(s). In addition, we used a reconstituted whole blood assay to investigate the effect of TNF-α on bacterial killing in the presence of different S. suis-specific IgG levels. An increase in IL-6 and IL-10, but not in IFN-γ or IL-17A, was observed in two of three piglets with pronounced bacteremia 16 to 20 h after infection, but not in piglets with controlled bacteremia. Our results confirmed previous findings that S. suis induces TNF-α and IL-6 and could demonstrate that TNF-α is produced by monocytes in vitro. We further found that IL-10 induction resulted in reduced secretion of TNF-α and IL-6. Rapid induction of TNF-α was, however, not crucial for in vitro bacterial killing, not even in the absence of specific IgG. Full article