- Volume 68, Issue 3, 2019

Purpose . To develop a new in vitro model of prosthetic vascular graft infection (PVGI) and evaluate antimicrobial and biofilm-disrupting efficacy of 0.1% octenidine dihydrochloride, 10% povidone-iodine and 0.02% chlorhexidine digluconate against biofilm-producing Staphylococcus aureus ( S. aureus ).

Methodology . The effect of antiseptics on the microscopic integrity and antimicrobial effect on S. aureus biofilms was tested by growing biofilms on glass coverslips, in the modified Lubbock chronic wound pathogenic biofilm (LCWPB) model and on the surface of vascular grafts using qualitive and quantitative methods as well as by scanning electron microscopy (SEM).

Results . Chlorhexidine worked best on destroying the integrity of S. aureus biofilms (P=0.002). In the LCWPB model, octenidine and povidone-iodine eradicated all S. aureus colonies (from 1.79 × 109  c.f.u. ml−1 to 0). In the newly developed PVGI model, the grafts were successfully colonized with biofilms as seen in SEM images. All antiseptics demonstrated significant antimicrobial efficacy, decreasing colony counts by seven orders of magnitude (P=0.002). Octenidine was superior to povidone-iodine (P=0.009) and chlorhexidine (P=0.041).

Conclusion . We implemented an innovative in vitro model on S. aureus biofilms grown in different settings, including a clinically challenging situation of PVGI. The strongest antimicrobial activity against S. aureus biofilms, grown on prosthetic vascular grafts, was showed by 0.1% octenidine dihydrochloride. We suggest that combinational therapy of antiseptics between chlorhexidine with either povidone-iodine or octenidine dihydrochloride should be tested in further experiments. Despite the need of further studies, our findings of these in vitro experiments will assist the management of vascular graft infection in clinical cases.

Purpose. In the cystic fibrosis (CF) airways, Pseudomonas aeruginosa undergoes diverse physiological changes in response to inflammation, antibiotic pressure, oxidative stress and a dynamic bioavailable nutrient pool. These include loss-of-function mutations that result in reduced virulence, altered metabolism and other phenotypes that are thought to confer a selective advantage for long-term persistence. Recently, clinical isolates of P. aeruginosa that hyperproduce agmatine (decarboxylated arginine) were cultured from individuals with CF. Sputum concentrations of this metabolite were also shown to correlate with disease severity. This raised the question of whether agmatine accumulation might also confer a selective advantage for P. aeruginosa during chronic colonization of the lung.

Methodology and Results. We screened a library of P. aeruginosa CF clinical isolates and found that ~5 % of subjects harboured isolates with an agmatine hyperproducing phenotype. Agmatine accumulation was a direct result of mutations in aguA, encoding the arginine deiminase that catalyses the conversion of agmatine into various polyamines. We also found that agmatine hyperproducing isolates (aguA-) had increased tolerance to the cationic antibiotics gentamicin, tobramycin and colistin relative to their chromosomally complemented strains (aguA+). Finally, we revealed that agmatine diminishes IL-8 production by airway epithelial cells in response to bacterial infection, with a consequent decrease in neutrophil recruitment to the murine airways in an acute pneumonia model.

Conclusion. These data highlight a potential new role for bacterial-derived agmatine that may have important consequences for the long-term persistence of P. aeruginosa in the CF airways.

Purpose. Extracellular protein toxins contribute to the pathogenesis of Staphylococcus aureus infections. The present study compared the effects of iclaprim and trimethoprim – two folic acid synthesis inhibitors – with nafcillin and vancomycin on production of Panton–Valentine leukocidin (PVL), alpha haemolysin (AH) and toxic-shock syndrome toxin I (TSST-1) in methicillin-resistant and vancomycin-intermediate S. aureus (MRSA and VISA, respectively).

Methodology. Northern blotting and RT-PCR were used to assess gene transcription; toxin-specific bioassays were used to measure protein toxin production.

Results. As shown previously, sub-inhibitory concentrations (sub-MIC) of nafcillin increased and prolonged MRSA toxin gene transcription and enhanced PVL, TSST-1 and AH production. Sub-inhibitory doses of iclaprim and trimethoprim delayed maximal AH gene (hla) transcription and suppressed AH production; both drugs delayed, but neither reduced, maximal TSST-1 production. Trimethoprim significantly increased lukF-PV expression and PVL production compared to both untreated and iclaprim-treated cultures. Higher concentrations of iclaprim and trimethoprim markedly suppressed MRSA growth, mRNA synthesis and toxin production. In VISA, iclaprim, vancomycin and nafcillin variably increased tst and hla expression, but only nafcillin increased toxin production. Despite its ability to increase hla expression, iclaprim was the most potent inhibitor of AH production.

Conclusions. We conclude that, due to its ability to suppress toxin production, iclaprim should be effective against severe staphylococcal infections caused by toxin-producing MRSA and VISA strains, especially given its ability to concentrate at sites of infection such as skin and skin structures and the lung.

Purpose. Recently, Lactobacillus plantarum (nF1) has been reported to have immune-enhancing effects in an immunosuppressed-animal model. Natural killer (NK) cells and macrophages play important roles in the immune responses. However, immunomodulatory effects of heat-treated Lactobacillus plantarum -nF1 (hLp-nF1) on the activation of NK cells and macrophages have not been elucidated.

Methodology. We assessed whether hLp-nF1 could elevate the activation of NK cells and macrophages using cyclophosphamide (CP)-induced immunosuppressed BALB/c mice and RAW 264.7 macrophages. A nitric oxide (NO) assay, enzyme-linked immunosorbent assay, Western blot analysis and NK cell activity assay were used to examine the effects of hLp-nF1 on the immune enhancement.

Results/Key findings. Administration of hLp-nF1-elevated NK cell activities and serum levels of TNF-α, IL-2, and IL-12 in CP-induced immunosuppressed mice. In RAW 264.7 macrophages, treatment with hLp-nF1 increased the production of NO and expression of inducible NO synthase. Simultaneously, hLp-nF1 increased the production of TNF-α, IL-2, and IL-6 from RAW 264.7 cells. Finally, hLp-nF1 induced activation of nuclear factor-κB and phosphorylation of IκBα.

Conclusion . We identified that hLp-nF1 has an immune-enhancing effect through the activation of NK cells and macrophages. Therefore, these findings suggest that hLp-nF1 would be helpful to enhance the immunity.

Purpose. Hantavirus infections cause severe haemorrhagic fever with renal syndrome (HFRS) in humans and are associated with high fatality rates. In 2017, numerous outbreaks were reported in China and Germany. This represents a significant public-healthcare issue with no effective HFRS vaccines that offer a long-term immune response. In this study, we investigated the long-term humoral and cellular immune responses and protective immunity of Hantaan virus (HTNV) granulocyte-macrophage colony stimulating factor (GM-CSF) and CD40 ligand (CD40L) virus-like particles (VLPs) in mice.

Methodology. GM-CSF and CD40L VLPs were constructed via co-transfection of pCI-S and pCI-M-CD40L, and pCI-S and pCI-M-GM-CSF, into dihydrofolatereductase (dhfr)-deficient Chinese hamster ovary cells, respectively. Mice were immunized with HTNV VLPs 2 weeks apart. The animals were challenged 6 months after immunization. Specific and neutralizing antibodies were assessed by ELISA; IFN-γ was measured by enzyme-linked immunospot (ELISpot) assay and effectiveness by cytotoxic T lymphocyte (CTL) cytotoxicity assays. Nucleic acid loads of HTNV were tested by quantitative real-time PCR and viral antigen was detected via indirect ELISA. Pathological alterations were detected via haematoxylin–eosin staining.

Results. GM-CSF and CD40L VLPs provided stable, long-term protection with a high titre of neutralizing antibody in mice 6 months after immunization. Furthermore, VLPs increased HTNV-specific cellular immune responses via higher expression of IFN-γ and CTL responses. HTNV challenge assay results showed long-term protection against HFRS. No significant pathological alteration was observed in the organs of mice after immunization.

Conclusion. This is, to the best of our knowledge, the first report demonstrating the long-term potency of HTNV VLP vaccines against HTNV infection and offers new insights into HTNV vaccine development.

Purpose. This study aimed to explore daptomycin combined with fosfomycin or rifampin against the planktonic and adherent linezolid-resistant isolates of Enterococcus faecalis .

Methodology. Four linezolid-resistant and four linezolid-sensitive isolates of E. faecalis which formed biofilms were collected for this study. Biofilm biomasses were detected by crystal violet staining and the adherent cells in the mature biofilms were quantified by c.f.u. determination.

Results. Daptomycin alone, or combined with fosfomycin or rifampin (4×MIC) demonstrated bactericidal activities on the planktonic cells, and daptomycin combined with fosfomycin killed more planktonic cells (at least 1−log₁₀ c.f.u. ml−1) than daptomycin or fosfomycin alone. Daptomycin alone (16×MIC) showed anti-biofilm activities against the mature biofilms and bactericidal activities on the adherent cells, while daptomycin combined with fosfomycin (16×MIC) demonstrated significantly more anti-biofilm activities than daptomycin or fosfomycin alone and effectively killed the adherent cells in the mature biofilms. The high concentration of daptomycin (512 mg l−1 ) combined with fosfomycin indicated more bactericidal activities on the adherent cells and more anti-biofilm activities against the mature biofilms than daptomycin 64 mg l−1 (16×MIC) combined with fosfomycin. The addition of rifampin increased the anti-biofilm and bactericidal activities of daptomycin against the mature biofilms and the adherent cells of two isolates, however, which was not observed in other isolates.

Conclusions. Daptomycin combined with fosfomycin demonstrated better effect on the planktonic and adherent linezolid-resistant isolates of E. faecalis than daptomycin or fosfomycin alone. The role of rifampin in the treatment of E. faecalis isolates is discrepant and needs more studies.