Micrococcal Nuclease stimulates Staphylococcus aureus Biofilm Formation in a Murine Implant Infection Model

Advancements in contemporary medicine have led to an increasing life expectancy which has broadened the application of biomaterial implants. As each implant procedure has an innate risk of infection, the number of biomaterial-associated infections keeps rising. Staphylococcus aureus causes 34% of such infections and is known as a potent biofilm producer. By secreting micrococcal nuclease S. aureus is able to escape neutrophil extracellular traps by cleaving their DNA-backbone. Also, micrococcal nuclease potentially limits biofilm growth and adhesion by cleaving extracellular DNA, an important constituent of biofilms. This study aimed to evaluate the impact of micrococcal nuclease on infection persistence and biofilm formation in a murine biomaterial-associated infection-model with polyvinylidene-fluoride mesh implants inoculated with bioluminescent S. aureus or its isogenic micrococcal nuclease deficient mutant. Supported by results based on in-vivo bioluminescence imaging, ex-vivo colony forming unit counts, and histological analysis it was found that production of micrococcal nuclease enables S. aureus bacteria to evade the immune response around an implant resulting in a persistent infection. As a novel finding, histological analysis provided clear indications that the production of micrococcal nuclease stimulates S. aureus to form biofilms, the presence of which extended neutrophil extracellular trap formation up to 13 days after mesh implantation. Since micrococcal nuclease production appeared vital for the persistence of S. aureus biomaterial-associated infection, targeting its production could be a novel strategy in preventing biomaterial-associated infection.

Spike-Dependent Opsonization Indicates Both Dose-Dependent Inhibition of Phagocytosis and That Non-Neutralizing Antibodies Can Confer Protection to SARS-CoV-2

Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization correlate with the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phagocytosis.

Development of a Trivalent Construct Omp18/AhpC/FlgH Multi Epitope Peptide Vaccine Against Campylobacter jejuni

Campylobacter jejuni (C. jejuni) is one of the major pathogens contributing to the enteritis in humans. Infection can lead to numerous complications, including but not limited to Guillain-Barre syndrome, reactive arthritis, and Reiter’s syndrome. Over the past two decades, joint efforts have been made toward developing a proper strategy of limiting the transmission of C. jejuni to humans. Nevertheless, except for biosecurity measures, no available vaccine has been developed so far. Judging from the research findings, Omp18, AhpC outer membrane protein, and FlgH flagellin subunits of C. jejuni could be adopted as surface protein antigens of C. jejuni for screening dominant epitope thanks to their strong antigenicity, expression of varying strains, and conservative sequence. In this study, bioinformatics technology was adopted to analyze the T-B antigenic epitopes of Omp18, AhpC, and FlgH in C. jejuni strain NCTC11168. Both ELISA and Western Blot methods were adopted to screen the dominant T-B combined epitope. GGS (GGCGGTAGC) sequence was adopted to connect the dominant T-B combined epitope peptides and to construct the prokaryotic expression system of tandem repeats of antigenic epitope peptides. The mouse infection model was adopted to assess the immunoprotective effect imposed by the trivalent T-B combined with antigen epitope peptide based on Omp18/AhpC/FlgH. In this study, a tandem epitope AhpC-2/Omp18-1/FlgH-1 was developed, which was composed of three epitopes and could effectively enhance the stability and antigenicity of the epitope while preserving its structure. The immunization of BALB/c mice with a tandem epitope could induce protective immunity accompanied by the generation of IgG2a antibody response through the in vitro synthesis of IFN-γ cytokines. Judging from the results of immune protection experiments, the colonization of C. jejuni declined to a significant extent, and it was expected that AhpC-2/Omp18-1/FlgH-1 could be adopted as a candidate antigen for genetic engineering vaccine of C. jejuni MAP.

The synergistic effect of minocycline and azole antifungal drugs against Scedosporium and Lomentospora species

Background This study was aimed to determine the potency of Minocycline (MIN) and azoles, including itraconazole (ITR), voriconazole (VOR) and posaconazole (POS) against Scedosporium and Lomentospora species. Results This study revealed that MIN exhibited no significant antifungal activity against any of the tested strains, whereas in vitro combination of MIN with ITR, VOR or POS showed satisfactory synergistic effects against 8 (80%), 1 (10%), and 9 (90%) strains, respectively. Moreover, combined use of MIN with azoles decreased the minimum inhibitory concentration (MIC) range from 5.33–16 μg/ml to 1–16 μg/ml for ITR, from 0.42–16 μg/ml to 0.21–16 μg/ml for VOR, and from 1.33–16 μg/ml to 0.33–16 μg/ml for POS. Meanwhile, no antagonistic interactions were observed between the above combinations. The G. mellonella infection model demonstrated the in vivo synergistic antifungal effect of MIN and azoles. Conclusions The present study demonstrated that combinations between MIN and azoles lead to synergistic antimicrobial effects on Scedosporium and Lomentospora species, while showing a potential for overcoming and preventing azole resistance.

Role of F-box Protein Cdc4 in Fungal Virulence and Sexual Reproduction of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic yeast-like pathogen that mainly infects immunocompromised individuals and causes fatal meningitis. Sexual reproduction can promote the exchange of genetic material between different strains of C. neoformans, which is one of the reasons leading to the emergence of highly pathogenic and drug-resistant strains of C. neoformans. Although much research has been done on the regulation mechanism of Cryptococcus sexual reproduction, there are few studies on the sexual reproduction regulation of Cryptococcus by the ubiquitin-proteasome system. This study identified an F-box protein, Cdc4, which contains a putative F-box domain and eight WD40 domains. The expression pattern analysis showed that the CDC4 gene was expressed in various developmental stages of C. neoformans, and the Cdc4 protein was localized in the nucleus of cryptococcal cells. In vitro stress responses assays showed that the CDC4 overexpression strains are sensitive to SDS and MMS but not Congo red, implying that Cdc4 may regulate the cell membrane integrity and repair of DNA damage of C. neoformans. Fungal virulence assay showed that although the cdc4Δ mutant grows normally and can produce typical virulence factors such as capsule and melanin, the cdc4Δ mutant completely loses its pathogenicity in a mouse systemic-infection model. Fungal mating assays showed that Cdc4 is also essential for fungal sexual reproduction in C. neoformans. Although normal mating hyphae were observed during mating, the basidiospores’ production was blocked in bilateral mating between cdc4Δ mutants. Fungal nuclei development assay showed that the nuclei failed to undergo meiosis after fusion inside the basidia during the bilateral mating of cdc4Δ mutants, indicating that Cdc4 is critical to regulating meiosis during cryptococcal mating. In summary, our study revealed that the F-box protein Cdc4 is critical for fungal virulence and sexual reproduction in C. neoformans.

18F-FDG and 68Ga-FAPI PET/CT for Evaluating Periprosthetic Joint Infection and Aseptic Loosening in Rabbit Models

Purpose We built a loosening model based on the original infection model of rabbit and evaluated the performance characteristics of 18F-FDG and 68Ga-FAPI in infection and loosening. Methods After surgery, the rabbits were divided into four groups, six in the control group and 10 in the loosening, S. aureus and S. epidermis groups. PET/CT and serological examination were performed every two weeks for three times. After sacrificed, micro-CT, tissue culture, pullout test and scanning electron microscope were performed. Results As for 18F-FDG, performances of control and loosening groups were similar. SUVmax of S. aureus had been consistently in the high range than that of S. epidermis. As for 68Ga-FAPI, control group had the lowest SUVmax in the second week and increased gradually. SUVmax of loosening group began exceed control group since the second week. SUVmax of S. aureus in the second week was the lowest among four group and raised as the number of weeks increased and equalled to SUVmax of S. epidermis in the sixth week. Linear regressions between SUVmax and serology showed that 18F-FDG was positively correlated with CRP and IL-6 while 68Ga-FAPI revealed negative and positive correlation with CRP and IL-6 in the second and sixth week. Besides, both SUVmax and MTV of 18F-FDG or 68Ga-FAPI were negatively correlated with BV/TV and BS/TV. Conclusion In this longitudinal observation, 68Ga-FAPI showed greater sensitivity than 18F-FDG in detecting diseases, and 68Ga-FAPI had not intestinal and muscular uptake. MTV of 68Ga-FAPI were larger than 18F-FDG, which meant that 68Ga-FAPI had the potential to define the scope of lesions more accurately. Finally, SUVmax could not differentiate loosening and infection in 68Ga-FAPI, further study about diagnostic criteria was warranted.

Lactobacillus rhamnosus Ameliorates Multi-Drug-Resistant Bacillus cereus-Induced Cell Damage through Inhibition of NLRP3 Inflammasomes and Apoptosis in Bovine Endometritis

Bacillus cereus, considered a worldwide human food-borne pathogen, has brought serious health risks to humans and animals and huge losses to animal husbandry. The plethora of diverse toxins and drug resistance are the focus for B. cereus. As an alternative treatment to antibiotics, probiotics can effectively alleviate the hazards of super bacteria, food safety, and antibiotic resistance. This study aimed to investigate the frequency and distribution of B. cereus in dairy cows and to evaluate the effects of Lactobacillus rhamnosus in a model of endometritis induced by multi-drug-resistant B. cereus. A strong poisonous strain with a variety of drug resistances was used to establish an endometrial epithelial cell infection model. B. cereus was shown to cause damage to the internal structure, impair the integrity of cells, and activate the inflammatory response, while L. rhamnosus could inhibit cell apoptosis and alleviate this damage. This study indicates that the B. cereus-induced activation of the NLRP3 signal pathway involves K+ efflux. We conclude that LGR-1 may relieve cell destruction by reducing K+ efflux to the extracellular caused by the perforation of the toxins secreted by B. cereus on the cell membrane surface.