Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence.

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

Exploitation of a Bacterium-Encoded Lytic Transglycosylase by a Human Oral Lytic Phage To Facilitate Infection.

Bacteriophages (phages) are an integral part of the human oral microbiome. Their roles in modulating bacterial physiology and shaping microbial communities have been discussed but remain understudied due to limited isolation and characterization of oral phage. Here, we report the isolation of LC001, a lytic phage targeting human oral Schaalia odontolytica (formerly known as Actinomyces odontolyticus) strain XH001. We showed that LC001 attached to and infected surface-grown, but not planktonic, XH001 cells, and it displayed remarkable host specificity at the strain level. Whole-genome sequencing of spontaneous LC001-resistant, surface-grown XH001 mutants revealed that the majority of the mutants carry nonsense or frameshift mutations in XH001 gene APY09_05145 (renamed ltg-1), which encodes a putative lytic transglycosylase (LT). The mutants are defective in LC001 binding, as revealed by direct visualization of the significantly reduced attachment of phage particles to the XH001 spontaneous mutants compared that to the wild type. Meanwhile, targeted deletion of ltg-1 produced a mutant that is defective in LC001 binding and resistant to LC001 infection even as surface-grown cells, while complementation of ltg-1 in the mutant background restored the LC001-sensitive phenotype. Intriguingly, similar expression levels of ltg-1 were observed in surface-grown and planktonic XH001, which displayed LC001-binding and nonbinding phenotypes, respectively. Furthermore, the overexpression of ltg-1 failed to confer an LC001-binding and -sensitive phenotype to planktonic XH001. Thus, our data suggested that rather than directly serving as a phage receptor, ltg-1-encoded LT may increase the accessibility of phage receptor, possibly via its enzymatic activity, by cleaving the peptidoglycan structure for better receptor exposure during peptidoglycan remodeling, a function that can be exploited by LC001 to facilitate infection. IMPORTANCE The evidence for the presence of a diverse and abundant phage population in the host-associated oral microbiome came largely from metagenomic analysis or the observation of virus-like particles within saliva/plaque samples, while the isolation of oral phage and investigation of their interaction with bacterial hosts are limited. Here, we report the isolation of LC001, the first lytic phage targeting oral Schaalia odontolytica. Our study suggested that LC001 may exploit the host bacterium-encoded lytic transglycosylase function to gain access to the receptor, thus facilitating its infection.

Bacteriophage-Resistant Mutant of Enterococcus faecalis Is Impaired in Biofilm Formation.

Enterococcus faecalis is a common gram-positive non-spore-forming bacterium in nature and is found in the upper respiratory tract, intestine, and mouth of healthy people. E. faecalis is also one of the common pathogens causing nosocomial infections and is resistant to several antibiotics commonly used in practice. Thus, treating drug-resistant E. faecalis with antibiotics is challenging, and new approaches are needed. In this study, we isolated a bacteriophage named EFap02 that targets E. faecalis strain EFa02 from sewage at Southwest Hospital. Phage EFap02 belongs to the Siphoviridae family with a long tail of approximately 210 nm, and EFap02 can tolerate a strong acid and alkali environment and high temperature. Its receptor was identified as the capsular polysaccharide. Phage-resistant mutants had loss-of-function mutations in glycosyltransferase (gtr2), which is responsible for capsular polysaccharide biosynthesis, and this caused the loss of capsular polysaccharide and interruption of phage adsorption. Although phage-resistant mutants against EFap02 can be selected, such mutants are impaired in biofilm formation due to the loss of capsular polysaccharide, which compromises its virulence. Therefore, this study provided a detailed description of the E. faecalis EFap02 phage with the potential for treating E. faecalis infection.

Zwitterionic metal-organic frameworks modified polyamide membranes with enhanced water flux and antifouling capacity.

Antifouling properties are considered to be crucial parameter to polyamide (PA) composite nanofiltration (NF) membranes for practical applications. In this study, an antifouling material, surface zwitterionization of Metal-organic frameworks (Z-MIL-101 (Cr)) was firstly prepared by decorating zwitterionic polymer onto the MOFs surface. Subsequently, a novel type of MOFs-based hybrid membranes were fabricated via mixing the Z-MIL-101 (Cr) nanoparticle with the organic matrix by interfacial polymerization technique. The most optimal hybrid membrane had a high water permeation of 26 L m-2 h-1 bar-1, which was 2.1 times higher than that pristine PA membrane, while the retention for Na2SO4 was still kept at a considerably high value of 93%. The significant increased water flue can attribute to the existence of water channels generated by the Z-MIL-101 (Cr). More important, the antifouling property of the hybrid membrane was much better than that pristine PA, which was due to the formation of superhydrophilic liquid layer surrounding the zwitterionic groups. The combination of the micropore structure of the MOFs and the excellent antifouling properties of the decorated zwitterionic polymer effectively improved separation performances and antifouling ability, which makes these hybrid membranes promising for water purification.

Rapamycin Liposomes Combined with 5-Fluorouracil Inhibits Angiogenesis and Tumor Growth of APC (Min/+) Mice and AOM/DSS-Induced Colorectal Cancer Mice.

Background: Transgenic C57BL/6-APC(Min/+) spontaneous cancer mouse model and the Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS) chemically induced orthotopic colorectal cancer mouse model represented distinct pathogenesis of colorectal cancers. Our previous study revealed that the combination of Rapamycin liposomes (Rapa/Lps) and 5-Fluorouracil (5-FU) has anti-colorectal cancer effects. However, the therapeutic efficacy of Rapa/Lps and 5-FU in other colorectal cancer mice models is yet to be thoroughly explored. The purpose of this study was to investigate the anti-tumor effect of Rapa/Lps combined with 5-FU in vivo and in vitro. Methods: In this study, we evaluated the effect of Rapa/Lps and 5-FU on APC (Min/+) mice and AOM/DSS-induced colorectal cancer mice. The small intestine, colorectum, serum, and plasma of mice in each group were collected following sacrifice to record the number of tumors. HE staining was utilized for observing pathological damage to intestine tissues. Tube formation assay, Transwell assay, wound healing assay, Western Blot were used to explore the anti-angiogenesis effect of drugs in HUVECs. Results: As expected, Rapa/Lps and 5-FU significantly suppressed tumor formation, decreased the number of tumors, and tumor load both in two mouse models, and had no influence on mouse weight. Mechanically, the anti-tumor effect of the drug also was associated in inhibiting angiogenesis and proliferation. Furthermore, we found that Rapa/Lps obviously inhibited HUVECs tube formation and migration. Conclusion: Altogether, we revealed the Rapa/Lps synergism with 5-FU decreased colon and small intestinal tumorigenesis in AOM/DSS-treated and APC (Min/+) mice, respectively, and correlated with anti-angiogenesis.

Lower cytotoxicity, high stability, and long-term antibacterial activity of a poly(methacrylic acid)/isoniazid/rifampin nanogel against multidrug-resistant intestinal Mycobacterium tuberculosis.

To overcome the undesirable side effects and reduce the cytotoxicity of isoniazid (INH) and rifampin (RMP) in the digestive tract, a poly(methacrylic acid) (PMAA) nanogel was developed as a carrier of INH and RMP. This PMAA/INH/RMP nanogel was prepared as a treatment for intestinal tuberculosis caused by multidrug-resistant Mycobacterium tuberculosis (MTB). The morphology, size, and in vitro release properties were evaluated in a simulated gastrointestinal medium, and long-term antibacterial performance, cytotoxicity, stability, and activity of this novel PMAA/INH/RMP nanogel against multidrug-resistant MTB in the intestine were investigated. Our results indicate that the PMAA/INH/RMP nanogel exhibited extended antibacterial activity by virtue of its long-term release of INH and RMP in the simulated gastrointestinal medium. Further, this PMAA/INH/RMP nanogel exhibited lower cytotoxicity than did INH or RMP alone, suggesting that this PMAA/INH/RMP nanogel could be a more useful dosage form than separate doses of INH and RMP for intestinal MTB. The novel aspects of this study include the cytotoxicity study and the three-phase release profile study, which might be useful for other researchers in this field.

Design and in vitro evaluation of a novel poly(methacrylic acid)/metronidazole antibacterial nanogel as an oral dosage form.

To overcome the undesirable side-effects of metronidazole (MTZ), ethylene glycol dimethacrylate is used as the cross-linker, and a series of poly(methacrylic acid) (PMAA) nanogels were prepared to load the MTZ. We investigated the morphology, size, in vitro release property in the simulated gastrointestinal medium, long-term antibacterial performance against Bacteroides fragilis, cytotoxicity, stability and activity of this novel MTZ/PMAA nanogel. The results indicate that the MTZ/PMAA nanogel sustained the release of MTZ in long-term antibacterial activity in the simulated gastrointestinal medium. This MTZ/PMAA nanogel exhibits less cytotoxicity than MTZ alone, suggesting that MTZ/PMAA nanogel is a more useful dosage form than MTZ for mild-to-moderate Clostridium difficile infections. The novel aspects of this study include the synthesis of a nanogel and the three-phase study of the release profile, which might be useful for other researchers in this field.

Sensitive determination of reactive oxygen species in cigarette smoke using microchip electrophoresis-localized surface plasmon resonance enhanced fluorescence detection.

A sensitive approach to the determination of reactive oxygen species (ROS) in puffs of cigarette smoke (CS) has been developed. The experimental system consists of a microfluidic chip electrophoresis and a laser induced fluorescence (LIF) device enhanced by localized surface plasmon resonance. Core-shell Ag@SiO2 nanoparticles were prepared and then immobilized on the surface of the microchannel to increase the fluorescence intensity based on localized surface plasmon resonance-enhanced fluorescence (LSPREF) effect. The ROS in puffs of CS were trapped via the oxidation of 2',7'-dichlorodihydrofluorescein (DCHF) that had been loaded on polyacrylonitrile (PAN) nanofibers in a micro-column. Determination of ROS was based on the amount of 2',7'-dichlorofluorescein (DCF), which is the sole product from DCHF oxidation. With the optimization of the trapping efficiency, we detected about 8.0 pmol of ROS per puff in the mainstream CS. This microchip electrophoresis-SPREF system enables sensitive quantitation of ROS in CS with low consumption of reagent, material, and analysis time.

[A comparison of the clinical anesthetic efficacy of articaine infiltration and lidocaine blocking for microport extraction of impacted mandibular molar].

OBJECTIVE: To compare the clinical efficacy of the infiltration anesthesia with primacaine and the nerve blocking anesthesia with lidocaine for microport extraction of impacted lower third molar. METHODS; 104 chosen patients had both sides of impacted lower third molars extracted in this study. Patients were given local anesthesia with either primacaine or lidocaine randomly at each side, and then underwent microport extraction. Clinical factors including effective proportion (EP), effecting time point (ETP), visual analogue scale of pain (VASp), alteration of systolic pressures (ASP) and analgesia duration (AD) were evaluated statistically by means of paired t-test. RESULTS: The EP of experimental group was higher than the control group (P = 0.024). The ETP of soft tissue and alveoli-dental pulp was (1.04 +/- 0.21), (2.44 +/- 2.60) min in the experimental group, and much earlier than that of the control group (P = 0.002, P = 0.032). The VASp and ASP of experimental group were lower than the control group (P = 0.041, P = 0.018). AD was (103.6 +/- 35.5) min, and higher than the control group (P = 0.04). CONCLUSION: The infiltration anesthesia with primacaine has been proven to be a easier, reliable and quick-acting method. We suggest it an alternative method replacing the 2% lidocaine blocking during microport extraction of impacted lower third molar.

[Evaluation of Amberlite XAD-1600 resin-based purification of the bioactive components from Radix Paeoniae Alba and Rhizoma Curcumae Longae extracts by pharmacodynamic and toxicological models].

OBJECTIVE: To evaluate the pharmacodynamics and toxicicity of the major bioactive components extracted and purified from Radix Paeoniae Alba and Rhizoma Curcumae Longae using Amberlite XAD-1600 resin. METHODS: Amberlite XAD-1600 was used to purify the bioactive components from the crude 75% ethanol extracts of the two herbs. The pharmacodynamic and toxic effects of the crude extracts and extract purified using XAD-1600 resin were comparatively examined with two acute inflammatory models, two pain models and acute toxicity test in vivo. RESULTS: The anti-inflammatory and analgesic effects of the purified extract were significant stronger with lower toxicity than those of the crude ethanol extract. CONCLUSION: Amberlite XAD-1600 resin allows efficient extraction and purification of the bioactive components from the two herbs.

Morphologic evaluation of regenerated small bowel by small intestinal submucosa.

BACKGROUND/PURPOSE: Previous studies have shown small intestinal submucosa (SIS) can be used as biodegradable scaffolds in tissue engineering small intestine. The purpose of this study is to evaluate the regeneration of neointestine and its morphology using SIS. METHODS: A 2-cm tubular SIS graft from Sprague Dawley rat donors was interposed in the middle of a 6-cm ileal Thiry-Vella loop of Lewis rats, which was used to construct an ileostomy. The grafts were harvested at each of the time points ranging from 2 weeks to half a year after implantation, and native small intestine and grafts were investigated for morphology using histology and immunohistochemistry. RESULTS: At the early postoperative period, SIS grafts were colonized by numerous inflammatory cells. A mucosal epithelial layer began to line the luminal surface of the graft by 4 weeks, and by 12 weeks, the luminal surface was covered completely by a layer of neomucosa. Neomucosa with typical small bowel morphology was characterized by a columnar epithelial cell layer with goblet cells, Paneth cells, absorptive enterocytes, and enteroendocrine cells. Significant differences between neomucosa by 12 weeks and 24 weeks in the measurements of mucosal thickness, villus height, and crypt depth were found. The outer walls of SIS grafts were composed of distinct bundles of well-formed smooth muscle-like cells with some fibrovascular tissue. CONCLUSIONS: This initial study suggests that tissue engineering neointestine using SIS can develop structural features of the normal intestine. Small intestinal submucosa might be a viable material in the creation of neointestine for patients suffering short bowel syndrome.