Membrane Phenotypic, Metabolic and Genotypic Adaptations of Streptococcus oralis Strains Destined to Rapidly Develop Stable, High-Level Daptomycin Resistance during Daptomycin Exposures.

The Streptococcus mitis-oralis subgroup of viridans group streptococci are important human pathogens. We previously showed that a substantial portion of S. mitis-oralis strains (>25%) are 'destined' to develop rapid, high-level, and stable daptomycin (DAP) resistance (DAP-R) during DAP exposures in vitro. Such DAP-R is often accompanied by perturbations in distinct membrane phenotypes and metabolic pathways. The current study evaluated two S. oralis bloodstream isolates, 73 and 205. Strain 73 developed stable, high-level DAP-R (minimum inhibitory concentration [MIC] > 256 µg/mL) within 2 days of in vitro DAP passage ("high level" DAP-R [HLDR]). In contrast, strain 205 evolved low-level and unstable DAP-R (MIC = 8 µg/mL) under the same exposure conditions in vitro ("non-HLDR"). Comparing the parental 73 vs. 73-D2 (HLDR) strain-pair, we observed the 73-D2 had the following major differences: (i) altered cell membrane (CM) phospholipid profiles, featuring the disappearance of phosphatidylglycerol (PG) and cardiolipin (CL), with accumulation of the PG-CL pathway precursor, phosphatidic acid (PA); (ii) enhanced CM fluidity; (iii) increased DAP surface binding; (iv) reduced growth rates; (v) decreased glucose utilization and lactate accumulation; and (vi) increased enzymatic activity within the glycolytic (i.e., lactate dehydrogenase [LDH]) and lipid biosynthetic (glycerol-3-phosphate dehydrogenase [GPDH]) pathways. In contrast, the 205 (non-HLDR) strain-pair did not show these same phenotypic or metabolic changes over the 2-day DAP exposure. WGS analyses confirmed the presence of mutations in genes involved in the above glycolytic and phospholipid biosynthetic pathways in the 73-D2 passage variant. These data suggest that S. oralis strains which are 'destined' to rapidly develop HLDR do so via a conserved cadre of genotypic, membrane phenotypic, and metabolic adaptations.

Bmal1 Regulates Coagulation Factor Biosynthesis in Mouse Liver in Streptococcus oralis Infection.

Streptococcus oralis (S. oralis) has been recognized as a fatal pathogen to cause multiorgan failure by contributing to the formation of microthrombus. Coagulation and fibrinolysis systems have been found under the control of circadian clock genes. This study aimed to explore the correlation between BMAL1 and coagulation factor biosynthesis in S. oralis infection. Mice were administered S. oralis to induce sepsis, and HepG2 cells were also infected by S. oralis. The expression of BMAL1 of hepatocytes was downregulated in the S. oralis infection group, leading to the downregulation of coagulation factor VII (FVII) and the upregulation of the coagulation factor XII (FXII) in vitro and in vivo. Furthermore, we confirmed that the deficiency of BAML1 contributed to the elevation of FVII and the decline in FXII by constructing BMAL1-deficiency (Bmal1-/-) mice. The current result showed that BMAL1 regulates FVII directly. Thus, a novel insight into the coagulation abnormality in S. oralis infection was gained that may optimize the treatment of sepsis by rescuing the expression of BMAL1 in the liver.

Effective control of biofilms by photothermal therapy using a gold nanorod hydrogel.

Biofilms are matrices synthesized by bacteria containing polysaccharides, DNA, and proteins. The development of biofilms in infectious processes can induce a chronic inflammatory response that may progress to the destruction of tissues. The treatment of biofilms is difficult because they serve as a bacterial mechanism of defense and high doses of antibiotics are necessary to treat these infections with limited positive results. It has been demonstrated that photothermal therapy using gold nanorods (AuNRs) is an attractive treatment because of its anti-biofilm activity. The purpose of this work was to generate a novel chitosan-based hydrogel embedded with AuNRs to evaluate its anti-biofilm activity. AuNRs were synthesized by the seed-mediated growth method and mixed with the chitosan-based hydrogel. Hydrogels were characterized and tested against two bacterial strains by irradiating the produced biofilm in the presence of the nanoformulation with a laser adjusted at the near infrared spectrum. In addition, the safety of the nanoformulation was assessed with normal human gingival fibroblasts. Results showed that a significant bacterial killing was measured when biofilms were exposed to an increase of 10°C for a short time of 2 min. Moreover, no cytotoxicity was measured when normal gingival fibroblasts were exposed to the nanoformulation using the bactericidal conditions. The development of the reported formulation can be used as a direct application to treat periodontal diseases or biofilm-produced bacteria that colonize the oral cavity.

Streptococcus oralis and Candida albicans Synergistically Activate µ-Calpain to Degrade E-cadherin From Oral Epithelial Junctions.

Streptococcus oralis forms robust mucosal biofilms with Candida albicans that have increased pathogenic potential. In this study, using oral epithelial cultures, organotypic oral mucosal constructs, and a mouse model of oral infection, we demonstrated that S. oralis augmented C. albicans invasion through epithelial junctions. C. albicans and S. oralis decreased epithelial E-cadherin levels by synergistically increasing µ-calpain, a proteolytic enzyme that targets E-cadherin. In the mouse coinfection model this was accompanied by increased fungal kidney dissemination. Coinfection with a secreted aspartyl protease (sap) mutant sap2456 and S. oralis increased µ-calpain and triggered mucosal invasion and systemic dissemination, suggesting that fungal protease activity is not required for invasion during coinfection. We conclude that C. albicans and S. oralis synergize to activate host enzymes that cleave epithelial junction proteins and increase fungal invasion.