Genomic analysis of penicillin-binding proteins and recombination events in an emerging amoxicillin- and meropenem-resistant PMEN3 (Spain9V-3, ST156) variant in Taiwan and comparison with global descendants of this lineage.

From 2008 to 2020, the Taiwan National Notifiable Disease Surveillance System database demonstrated that the incidence of non-vaccine serotype 23A invasive pneumococcal disease (IPD) approximately doubled. In this study, 276 non-repetitive pneumococcal clinical isolates were collected from two medical centers in Taiwan between 2019 and 2021. Of these 267 pneumococci, 60 were serotype 23A. Among them, 50 (83%) of serotype 23A isolates belonged to the sequence type (ST) 166 variant of the Spain9V-3 clone. Pneumococcal 23A-ST166 isolates were collected to assess their evolutionary relationships using whole-genome sequencing. All 23A-ST166 isolates were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299, the newly identified PBP2x-299 in Taiwan. Transformation of the pbp1a, pbp2b, and pbp2x alleles into the ß-lactam-susceptible R6 strain revealed that PBP2x-299 and PBP2b-11 increased the MIC of ceftriaxone and meropenem by 16-fold, respectively. Prediction analysis of recombination sites in PMEN3 descendants (23A-ST166 in Taiwan, 35B-ST156 in the United States, and 11A-ST838/ST6521 in Europe) showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displayed an evolutionary capacity for global dissemination and persistence, increasing IPD incidence, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases, and contributing to high antibiotic resistance. A clonal shift with a highly ß-lactam-resistant non-vaccine serotype 23A, from ST338 to ST166, increased in Taiwan. ST166 is a single-locus variant of the Spain9V-3 clone, which is also called the PMEN3 lineage. All 23A-ST166 isolates, in this study, were resistant to amoxicillin and meropenem, and 96% harbored a novel combination of penicillin-binding proteins (PBPs) (1a:2b:2x):15:11:299. PBP2x-299 and PBP2b-11 contributed to the increasing MIC of ceftriaxone and meropenem, respectively. Prediction analysis of recombination sites in PMEN3 descendants showed that adaptive evolution involved repeated, selectively favored convergent recombination in the capsular polysaccharide synthesis region, PBPs, murM, and folP genome sites. In the late 13-valent pneumococcal conjugate vaccine era, PMEN3 continuously displays the evolutionary capacity for dissemination, leading to an offset in the decrease of pneumococcal conjugate vaccine serotype-related diseases and contributing to high antibiotic resistance.

Antiangiogenic activity of indole-3-carbinol in endothelial cells stimulated with activated macrophages.

The effect of indole-3-carbinol (I3C), a major indolic metabolite in cruciferous vegetables, on lipopolysaccharide (LPS)-activated macrophage-induced tube formation and its associated factors in endothelial EA hy926 cells was investigated. LPS significantly enhanced the capillary-like structure of endothelial cells (ECs) co-cultured with macrophages, but no such effect was observed in single-cultured ECs. I3C, on the other hand, suppressed such enhancement in concert with decreased secretions of vascular endothelial growth factor (VEGF), nitric oxide (NO), interleukin-6 (IL-6), and matrix metalloproteinases (MMPs). The results obtained from cultivating ECs with conditioned medium (CM) collected from macrophages suggested that both ECs and macrophages were inactivated by I3C. These results indicate that I3C from cruciferous vegetables may possess potential roles in preventing inflammation-associated angiogenic diseases.

Antiobesity activities of indole-3-carbinol in high-fat-diet-induced obese mice.

OBJECTIVE: This study investigated the effects of indole-3-carbinol (I3C), a cruciferous vegetable derivative, on obesity and its associated factors in high-fat-diet-induced obese (DIO) mice. METHODS: Eighteen male C57BL/6 mice were randomly assigned to one of three groups: basal, high fat (HF), and HF + 5 mg/kg of I3C intraperitoneally (HFI). After 12 wk of treatment, obesity-associated factors, including body weight, organ weight, serum concentrations of glucose, triacylglycerol, insulin, and adipokines, and macrophage accumulation and lipid metabolism-associated factors in epididymal adipose tissue were measured. RESULTS: Body weight and epididymal adipose tissue weight were greater (P < 0.01), and adipocytes were larger in the HF group than in the basal and HFI groups. Compared with the HF group, the HFI group had improved glucose tolerance, a higher serum adiponectin concentration, lower serum glucose, triacylglycerol, insulin, and leptin concentrations, and less F4/80 expression in epididymal adipose tissue (P < 0.001). Furthermore, I3C treatment decreased acetyl coenzyme A carboxylase mRNA expression (P < 0.05) and increased peroxisome proliferator-activated receptor-γ protein expression (P < 0.05) in epididymal adipose tissue of DIO mice. CONCLUSION: The I3C treatment decreased body weight and fat accumulation and infiltrated macrophages in epididymal adipose tissue of DIO mice, and these reductions were associated with improved glucose tolerance and with modulated expression of adipokines and lipogenic-associated gene products, including acetyl coenzyme A carboxylase and peroxisome proliferator-activated receptor-γ.

Differential effects of organosulfur compounds from garlic oil on nitric oxide and prostaglandin E2 in stimulated macrophages.

OBJECTIVE: We investigated the inhibition of nitric oxide (NO) and prostaglandin E2 (PGE2) production by the garlic oil derivatives, diallyl sulfide (DAS), diallyl disulfide (DADS), and allyl methyl sulfide (AMS), in lipopolysaccharide (LPS)-activated RAW 264.7 cells. METHODS: Cells were treated with LPS (330 ng/mL) and various concentrations of DAS, DADS, and AMS. NO and PGE2 released into the medium and expressions of inducible NO synthase and cyclooxygenase-2 protein were measured. RESULTS: All three compounds suppressed stimulated NO production, among which AMS exhibited the least inhibition. Western blot analysis showed that DAS and DADS, but not AMS, inhibited the corresponding inducible NO synthase expression. An in vitro study showed that all three compounds possess NO clearance activity, and that DADS and AMS were more effective than DAS. On the contrary, only DAS inhibited activated PGE2 production and cyclooxygenase-2 protein expression. CONCLUSIONS: The garlic derivatives, DAS, DADS, and AMS, differentially regulated the production of NO and PGE2 in stimulated macrophages. DAS decreased stimulated NO and PGE2 production by inhibiting inducible NO synthase and cyclooxygenase-2 expressions, and its enzyme inhibiting and NO clearance activity may also partly contribute to the suppression of NO. DADS inhibited activated NO production by decreasing inducible NO synthase expression and by directly clearing NO, whereas AMS suppressed NO mainly through its direct NO clearance activity. Further, neither DADS nor AMS showed any inhibitory effect on stimulated PGE2 production.

Modulation of cytokine secretion by garlic oil derivatives is associated with suppressed nitric oxide production in stimulated macrophages.

We previously described that garlic oil derivatives differentially suppress the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in activated macrophages. In the present study, we investigated the effects of the garlic derivatives, diallyl sulfide (DAS), diallyl disulfide (DADS), and allyl methyl sulfide (AMS), on cytokine production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and the association between modulation of cytokines and inhibition of NO production was also assessed. The results indicated that these garlic compounds had different effects on the secretion of activated cytokines, including proinflammatory tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6, as well as the antiinflammatory, IL-10. DAS inhibited the production of all stimulated cytokines in a concentration-dependent manner, and the inhibition was closely associated with the suppression of NO and PGE(2) production. DADS repressed the production of stimulated TNF-alpha and IL-10 and increased the production of activated IL-1beta and, to a lesser extent, IL-6; but only the decreased IL-10 production was associated with DADS-induced NO inhibition. Yet, the DAS- and DADS-suppressed NO production was independent of TNF-alpha. AMS, on the other hand, slightly suppressed the stimulated TNF-alpha but enhanced IL-10 production, and such modulation was closely associated with the decrease in NO production.

Suppression of inducible nitric oxide production by indole and isothiocyanate derivatives from Brassica plants in stimulated macrophages.

In this study, the effects of bioactive compounds derived from vegetables of the Brassica genus (Brassicaceae) including 2-phenylethyl isothiocyanate (PEITC), indole-3-carbinol (I3C), and indolo[3,2- b]carbazole (ICZ), on the inhibition of NO production in RAW 264.7 cells were explored. The results indicated that PEITC and I3C inhibited lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-induced NO production in RAW 264.7 cells, and this inhibition was in accordance with lowering the expression of iNOS protein and mRNA. On the contrary, ICZ, a derivative of I3C, had no significant effect on the stimulated NO production. In conclusion, the Brassica plants derivatives, PEITC and, to a lesser extent, I3C inhibit the LPS/IFN-gamma-induced NO production by lowering iNOS protein and mRNA expression in RAW 264.7 cells, in which the PEITC had a more potent inhibitory effect. Nevertheless, ICZ exhibits no inhibitory effect on the activated NO production (Indole-3-carbinol = indole-3-methanol).