The cooperation of pharmacologic-dose ascorbate with ceftriaxone against Staphylococcus aureus through bactericidal synergy and enhanced macrophage killing activity.

BACKGROUND: Ascorbate is a low-cost compound with a known bactericidal-synergy to antibitics. However, the synergy depends on concentrations and organisms. Thus, the synergy test by time-kill assay might be appropriate for the screening of the synergy. OBJECTIVE: We aimed to test the adjuvant property of ascorbate with ceftriaxone, a frequently prescribed ß-lactam antibiotic. METHOD: Ascorbate was tested with several bacteria from the American Type Culture Collection (ATCC) including Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Escherichia coli for i) bactericidal property of ascorbate, alone or with ceftriaxone-combination, by time-kill assay, ii) an influence on the killing-activity of bone -marrow-derived macrophage and iii) the attenuation of myositis mouse model. RESULT: The bactericidal synergy (determined with time-kill assay at 24 h) against S. aureus, but not other selected bacteria, was demonstrated in ascorbate (10 and 40 mM) plus ceftriaxone at the minimal inhibitory concentration (1x MIC). Ascorbate alone, without antibiotic, enhanced macrophage killing-activity and directly eliminated bacteria at the concentration 10-40mM and 250mM, respectively (both properties presented against S. aureus and P. aeruginosa, but not other bacteria). Ascorbate with ceftriaxone also reduced bacterial burdens in muscle and serum cytokines of S. aureus -myositis mouse model. Moreover, the synergy against the clinical isolated methicillin resistant S. aureus (MRSA) by time-kill assay and myositis model also presented. CONCLUSION: Ascorbate-ceftriaxone synergy against S. aureus was demonstrated by time-kill assay and myositis model. Time-kill assy might be valuable as a screening test to select the patients that potentially benefit from ascorbate- ceftriaxone adjuvant therapy.

Gold nanoparticles attenuates bacterial sepsis in cecal ligation and puncture mouse model through the induction of M2 macrophage polarization.

BACKGROUND: Gold nanoparticles (AuNP) have several biochemical advantageous properties especially for a candidate of drug carrier. However, the non-conjugated AuNP has a higher rate of cellular uptake than the conjugated ones. Spherical AuNP in a proper size (20-30 nm) is non-toxic to mice and shows anti-inflammatory properties. We tested if the administration of AuNP, as an adjuvant to antibiotics, could attenuate bacterial sepsis in cecal ligation and puncture (CLP) mouse model with antibiotic (imipenem/cilastatin). RESULTS: Indeed, AuNP administration at the time of CLP improved the survival, blood bacterial burdens, kidney function, liver injury and inflammatory cytokines (TNF-α, IL-6, IL-1ß and IL-10). AuNP also decreased M1 macrophages (CD86 + ve in F4/80 + ve cells) and increased M2 macrophages (CD206 + ve in F4/80 + ve cells) in the spleens of sepsis mice. The weak antibiotic effect of AuNP was demonstrated as the reduction of E. coli colony after 4 h incubation. In addition, AuNP altered cytokine production of bone-marrow-derived macrophages including reduced TNF-α, IL-6 and IL-1ß but increased IL-10 at 6 and 24 h. Moreover, AuNP induced macrophage polarization toward anti-inflammatory responses (M2) as presented by increased Arg1 (Arginase 1) and PPARγ with decreased Nos2 (inducible nitric oxide synthase, iNos) and Nur77 at 3 h after incubation in vitro. CONCLUSIONS: The adjuvant therapy of AuNP, with a proper antibiotic, attenuated CLP-induced bacterial sepsis in mice, at least in part, through the antibiotic effect and the induction of macrophage function toward the anti-inflammatory responses.

Decreased Severity of Acute Hepatopancreatic Necrosis Disease in White Shrimp (Litopenaeus vannamei) by Mixed Culture of Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium.

The objective of this study was to investigate the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium to control acute hepatopancreatic necrosis disease (AHPND) or EMS (Early Mortality Syndrome) in white shrimp Litopenaeus vannamei as a model. The infected shrimps with Vibrio parahaemolyticus AHPND strain were divided into tanks and different feeding of either B. subtilis, B. licheniformis, B. megaterium or all Bacillus strains. The infected shrimps that were fed with a mixed culture of Bacillus showed significantly highest survival rate and revealed lower percent detection of V. parahaemolyticus AHPND strain by Polymerase Chain Reaction (PCR) (57.14%) with a small amount of viability count in their hepatopancreas. In contrast, the infected shrimps that were fed with each of B. subtilis, B. licheniformis or B. megaterium, revealed the spread of V. parahaemolyticus AHPND strain in all tissue by PCR detection (86.67%-100%) with a large amount of viability count (3.53 - 4.24 × 103 CFU/g). This study indicated that the mixed culture of Bacillus subtilis, B. licheniformis and B. megaterium could control the dissemination of V. parahaemolyticus in shrimps, especially in hepatopancreatic that is the target tissue of AHPND in white shrimp (L. vannamei). The result of this study revealed the efficiency and mechanism of the mixed culture of B. subtilis, B. licheniformis and B. megaterium to control the virulence of AHPND and support the application of this mixed culture in aquaculture of shrimp farms to avoid chemical and antibiotic treatment by using it as a biological control.

Multiplex PCR for detection of clarithromycin resistance and simultaneous species identification of Mycobacterium avium complex.

Multiplex PCR (mPCR) was established for the simultaneous detection of clarithromycin (CLR) resistance and species identification of Mycobacterium avium complex (MAC). mPCR was tested on 218 MAC clinical isolates. CLR-resistance was detected by mPCR in 31 of 35 isolates identified by a microdilution method. Of the remaining 187 susceptible isolates identified by mPCR, 183 isolates had MIC < or = 8 microg/ml (susceptible), 3 with MIC of 16 (intermediate resistant) and 1 with MIC of > or = 32 microg/ml (resistant). Comparing with the PCR-restriction enzyme analysis, mPCR concordantly identified 185 isolates either as being M. avium or M. intracellulare, whereas one isolate was misidentified and 32 isolates could not be identified. Comparing with reference methods, the mPCR showed the sensitivity, specificity, positive predictive and negative predictive value of 89, 100, 100, and 98% for detection of CLR resistance; 92, 98, 99, and 78% for identification of M. avium; and 57, 100, 100, and 89% for identification of M. intracellulare, respectively.

Leaky-gut enhanced lupus progression in the Fc gamma receptor-IIb deficient and pristane-induced mouse models of lupus.

The influence of gut-leakage or gut-microbiota upon lupus progression was explored in 2 lupus mouse models. Pristane, administered in 4-wk-old wild-type (WT) female mice, induced lupus characteristics at 24-wk-old similar to the lupus-onset in FcGRIIb-/- mice. Gut-microbiota alteration was induced by co-housing together with the gavage of feces from 40-wk-old FcGRIIb-/- mice (symptomatic lupus). On the other hand,  gut-leakage was induced  by dextran sulfate solution (DSS). DSS and gut-microbiota alteration induced high serum anti-dsDNA immunoglobulin (Ig) as early as 30 days post-DSS only in FcGRIIb-/- mice. DSS, but not gut-microbiota alteration, enhanced lupus characteristics (serum creatinine and proteinuria) in both lupus models (but not in WT) at 60 days post-DSS. Indeed, DSS induced the translocation of molecular components of gut-pathogens as determined by bacterial burdens in mesenteric lymph node (MLN), endotoxemia (gut-bacterial molecule) and serum (1→3)-ß-D-glucan (BG) (gut-fungal molecule) as early as 15 days post-DSS together with enhanced MLN apoptosis in both WT and lupus mice. However, DSS induced spleen apoptosis in FcGRIIb-/- and WT mice at 30 and 60 days post-DSS, respectively, suggesting the higher impact of gut-leakage against spleen of lupus mice. In addition, macrophages preconditioning with LPS plus BG were susceptible to starvation-induced apoptosis, predominantly in FcGRIIb-/- cell, implying the influence of gut-leakage upon cell stress. In summary, gut-leakage induced gut-translocation of organismal-molecules then enhanced the susceptibility of stress-induced apoptosis, predominantly in lupus. Subsequently, the higher burdens of apoptosis in lupus mice increased anti-dsDNA Ig and worsen lupus severity through immune complex deposition. Hence, therapeutic strategies addressing gut-leakage in lupus are interesting.

Increased susceptibility against Cryptococcus neoformans of lupus mouse models (pristane-induction and FcGRIIb deficiency) is associated with activated macrophage, regardless of genetic background.

The severity of cryptococcosis in lupus from varying genetic-backgrounds might be different due to the heterogeneity of lupus-pathogenesis. This study explored cryptococcosis in lupus mouse models of pristane-induction (normal genetic-background) and FcGRIIb deficiency (genetic defect). Because the severity of lupus nephritis, as determined by proteinuria and serum creatinine, between pristane and FcGRIIb-/- mice were similar at 6-month-old, Cryptococcus neoformans was intravenously administered in 6-month-old mice and were age-matched with wild-type. Indeed, the cryptococcosis disease severity, as evaluated by mortality rate, internal-organ fungal burdens and serum cytokines, between pristane and FcGRIIb-/- mice was not different. However, the severity of cryptococcosis in wild-type was less severe than the lupus mice. On the other hand, phagocytosis activity of peritoneal macrophages from lupus mice (pristane and FcGRIIb-/-) was more predominant than the wild-type without the difference in macrophage killing-activity among these groups. In addition, the number of active T helper cells (Th-cell) in the spleen, including Th-cells with intracellular IFN-γ, from lupus mice (pristane and FcGRIIb-/-) was higher than wildtype. Moreover, these active Th-cells were even higher after 2 weeks of cryptococcal infection. These data support enhanced macrophage activation through prominent Th-cells in both lupus models. In conclusion, an increased susceptibility of cryptococcosis in both lupus models was independent to genetic background. This might due to Th-cell enhanced macrophage phagocytosis with the interference of macrophage killing activity from Cryptococcal immune-evasion properties.

Helicobacter pylori Infection Increased Anti-dsDNA and Enhanced Lupus Severity in Symptomatic FcγRIIb-Deficient Lupus Mice.

The defect on Fc gamma receptor IIb (FcγRIIb), the only inhibitory FcγR, has been identified as one of the genetic factors increasing susceptibility to lupus. The prevalence of Helicobacter pylori (HP) and FcγRIIb dysfunction-polymorphisms are high among Asians, and their co-existence is possible. Unfortunately, the influence of HP against lupus progression in patients with lupus is still controversial. In this study, the interactions between these conditions were tested with HP infection in 24-week-old FcγRIIb-/- mice (symptomatic lupus). HP induced failure to thrive, increased stomach bacterial burdens and stomach injury (histology and cytokines) in both wild-type and FcγRIIb-/- mice. While the severity of HP infection, as determined by these parameters, was not different between both strains, antibodies production (anti-HP, anti-dsDNA and serum gammaglobulin) were higher in FcγRIIb-/- mice compared to wild-type. Accordingly, HP infection also accelerated the severity of lupus as determined by proteinuria, serum creatinine, serum cytokines, renal histology, and renal immune complex deposition. Although HP increased serum cytokines in both wild-type and FcγRIIb-/- mice, the levels were higher in FcγRIIb-/- mice. As such, HP also increased spleen weight and induced several splenic immune cells responsible for antibody productions (activated B cell, plasma cell and follicular helper T cell) in FcγRIIb-/- mice, but not in wild-type. These data describe the different systemic responses against localized HP infection from diverse host genetic background. In conclusion, the mutual interactions between HP and lupus manifestations of FcγRIIb-/-mice were demonstrated in this study. With the prominent immune responses from the loss of inhibitory signaling in FcγRIIb-/- mice, HP infection in these mice induced intense chronic inflammation, increased antibody production, and enhanced lupus severity. Thus, the increased systemic inflammatory responses due to localized HP inducing gastritis in some patients with lupus may enhance lupus progression. More studies are needed.

The Synergy of Endotoxin and (1→3)-ß-D-Glucan, from Gut Translocation, Worsens Sepsis Severity in a Lupus Model of Fc Gamma Receptor IIb-Deficient Mice.

We investigated the influence of spontaneous gut leakage upon polymicrobial sepsis in a lupus model with Fc gamma receptor IIb-deficient (FcGRIIb-/-) mice aged 8 and 40 weeks, as representing asymptomatic and symptomatic lupus, respectively. Spontaneous gut leakage, determined by (i) the presence of FITC-dextran, (ii) elevated serum endotoxin, and (iii) elevated serum (1→3)-ß-D-glucan (BG), was demonstrated in symptomatic lupus but not in the asymptomatic group. In parallel, spontaneous gut leakage, detected by elevated serum BG without fungal infection, was demonstrated in patients with active lupus nephritis. Gut leakage induced by dextran sulfate solution (DSS) or endotoxin administration together with BG or endotoxin alone, but not BG alone, enhanced the severity of cecal ligation and puncture (CLP) sepsis more prominently in 8-week-old FcGRIIb-/- mice. Additionally, the bone marrow-derived macrophages of FcGRIIb-/- mice produced higher cytokine levels when coexposed to endotoxin and BG, when compared to wild-type mice. In summary, spontaneous gut leakage was demonstrated in symptomatic FcGRIIb-/- mice and the induction of gut permeability worsened sepsis severity. Gut translocation of endotoxin and BG had a minor effect on wild-type mice, but the synergistic effect of BG and endotoxin was prominent in FcGRIIb-/- mice. The data suggest that therapeutic strategies addressing gut leakage may be of interest in sepsis conditions in patients with lupus.

The role of macrophages in the susceptibility of Fc gamma receptor IIb deficient mice to Cryptococcus neoformans.

Dysfunctional polymorphisms of FcγRIIb, an inhibitory receptor, are associated with Systemic Lupus Erythaematosus (SLE). Cryptococcosis is an invasive fungal infection in SLE, perhaps due to the de novo immune defect. We investigated cryptococcosis in the FcγRIIb-/- mouse-lupus-model. Mortality, after intravenous C. neoformans-induced cryptococcosis, in young (8-week-old) and older (24-week-old) FcγRIIb-/- mice, was higher than in age-matched wild-types. Severe cryptococcosis in the FcγRIIb-/- mice was demonstrated by high fungal burdens in the internal organs with histological cryptococcoma-like lesions and high levels of TNF-α and IL-6, but not IL-10. Interestingly, FcγRIIb-/- macrophages demonstrated more prominent phagocytosis but did not differ in killing activity in vitro and the striking TNF-α, IL-6 and IL-10 levels, compared to wild-type cells. Indeed, in vivo macrophage depletion with liposomal clodronate attenuated the fungal burdens in FcγRIIb-/- mice, but not wild-type mice. When administered to wild-type mice, FcγRIIb-/- macrophages with phagocytosed Cryptococcus resulted in higher fungal burdens than FcγRIIb+/+ macrophages with phagocytosed Cryptococcus. These results support, at least in part, a model whereby, in FcγRIIb-/- mice, enhanced C. neoformans transmigration occurs through infected macrophages. In summary, prominent phagocytosis, with limited effective killing activity, and high pro-inflammatory cytokine production by FcγRIIb-/- macrophages were correlated with more severe cryptococcosis in FcγRIIb-/- mice.

Fc Gamma Receptor IIB Deficient Mice: A Lupus Model with Increased Endotoxin Tolerance-Related Sepsis Susceptibility.

Hyper-elevated immune response of FcGRIIb-/- mice, a lupus model with an inhibitory-signaling defect, can become exhausted (less subsequent immune-response than the first response) with sequential lipopolysaccharide (LPS) stimulation. Endotoxin tolerance-related modifications of inflammatory response were investigated in FcGRIIb-/- mice in both an in vivo sepsis model and in vitro using cultured macrophages. Serum cytokine concentrations, after the second LPS injection (at 5-fold higher levels than the first dose), did not exceed the first dose levels in either FcGRIIb-/- or wild-type mice. These data indicated an endotoxin-tolerance response in both genetic backgrounds. However, the difference of cytokine levels between the first and second LPS injection was more prominent in FcGRIIb-/- mice. More importantly, CLP-induced sepsis after LPS-preconditioning (two separated doses of LPS administration) was more severe in FcGRIIb-/- mice (as measured by mortality rate, bacteria count in blood, serum cytokines, creatinine, and alanine transaminase). An attenuated response was demonstrated after two sequential LPS stimulations of bone-marrow-derived macrophages. Cytokine production was reduced and lower bacterial killing activity occurred with macrophages from FcGRIIb-/- mice relative to wild-type macrophages. Thus, there is a more prominent effect of endotoxin-tolerance in FcGRIIb-/- macrophages relative to wild-type. In conclusion, repeated-LPS administrations induced quantitatively greater endotoxin-tolerance responses in FcGRIIb-/- mice both in vivo and in vitro. Endotoxin-tolerance in vivo was associated with more severe sepsis, at least in part, due to macrophage-dysfunction.