Nano-Emulsified Perfluorooctyl Bromide Can Infiltrate Gram-Negative Bacteria and Sensitize Them to Ultrasound.

Gram-negative (G-) bacterial infections remain one of the most urgent global health threats, because the distinctive envelope structure hinders the penetration of therapeutics. Here, we showed that a perfluorooctyl bromide nanoemulsion (PFOB NE) uniquely interacts with G- bacteria. After cell envelope attachment, the PFOB can infiltrate the cell and was diffused throughout. In this process, it impaired the membranes by disintegrating phospholipid molecules, enhancing the consequent ultrasonic cavitation to break the envelope. We identified through ultrasound that the NE had remarkable bactericidal effects against various antibiotic-resistant pathogens. Using in situ sterilization, this approach accelerated the recovery of bacteria-infected murine skin wounds. Thus, combining PFOB and ultrasound might be an alternative tool for conquering the growing threat of G- pathogens.

The Synergy between Antibiotics and the Nanoparticle-Based Photodynamic Effect.

Antimicrobial resistance (AMR) is a growing global health concern, necessitating innovative strategies beyond the development of new antibiotics. Here, we employed NdYVO4:Eu3+ nanoparticles, which can persistently produce reactive oxygen species (ROS) after stopping the light, as a model of photodynamic nanoparticles and demonstrated that the photodynamic effect can serve as an adjuvant with antibiotics to effectively reduce their minimum inhibitory concentration. These preirradiated nanoparticles could penetrate the bacterial cell membrane, significantly enhancing the potency of antibiotics. We showed that the synergy effect could be attributed to disrupting crucial cellular processes by ROS, including damaging cell membrane proteins, interfering with energy supply, and inhibiting antibiotic metabolism. Our findings suggested that complementing the photodynamic effect might be a robust strategy to enhance antibiotic potency, providing an alternative antibacterial treatment paradigm.

Bartonella type IV secretion effector BepC induces stress fiber formation through activation of GEF-H1.

Bartonella T4SS effector BepC was reported to mediate internalization of big Bartonella aggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentation induced by c-AMP) domain and C-terminus BID (Bartonella intracellular delivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway.

Bartonella quintana type IV secretion effector BepE-induced selective autophagy by conjugation with K63 polyubiquitin chain.

Bartonella effector proteins (named Beps) are substrates of VirB type IV secretion system for translocation into host cells evolved in Bartonella spp. Among these, BepE has been shown to protect cells from fragmentation effects triggered by other Beps and to promote in vivo dissemination of bacteria from the dermal site of inoculation to the bloodstream. Bacterial pathogens secreted effectors to modulate the interplay with host autophagy, either to combat autophagy to escape its bactericidal effect or to exploit autophagy to benefit intracellular replication. Here, we reported a distinct phenotype that selective autophagy in host cells is activated as a countermeasure, to attack BepE via conjugation with K63 polyubiquitin chain on BepE. We found that ectopic expression of Bartonella quintana BepE specifically induced punctate structures that colocalised with an autophagy marker (LC3-II) in host cells, in addition to filopodia and membrane ruffle formation. Two tandemly arranged Bartonella Intracellular Delivery (BID) domains in the BepE C-terminus, where ubiquitination of sister pairs of lysine residues was confirmed, were essential to activate host cell autophagy. Multiple polyubiquitin chain linkages of K27, K29, K33, and K63 were found to be conjugated at sites of K222 and K365 on BepE, of which K63 polyubiquitination on BepE K365 determined the selective autophagy (p62/SQSTM1 positive autophagy) independent of the PI3K pathway. Colocalisation of BepE with LAMP1 confirmed the maturation of BepE-induced autophagosomes in which BepE were targeted for degradation. Moreover, host cells employed selective autophagy to counter-attack BepE to rescue cells from BepE-induced endocytosis deficiency.

Bartonella kosoyi sp. nov. and Bartonella krasnovii sp. nov., two novel species closely related to the zoonotic Bartonella elizabethae, isolated from black rats and wild desert rodent-fleas.

The genus Bartonella (Family: Bartonellaceae; Order: Rhizobiales; Class: Alphaproteobacteria) comprises facultative intracellular Gram-negative, haemotropic, slow-growing, vector-borne bacteria. Wild rodents and their fleas harbor a great diversity of species and strains of the genus Bartonella, including several zoonotic ones. This genetic diversity coupled with a fastidious nature of the organism results in a taxonomic challenge that has led to a massive collection of uncharacterized strains. Here, we report the genomic and phenotypic characterization of two strains, members of the genus Bartonella (namely Tel Aviv and OE 1-1), isolated from Rattus rattus rats and Synosternus cleopatrae fleas, respectively. Scanning electron microscopy revealed rod-shaped bacteria with polar pili, lengths ranging from 1.0 to 2.0 µm and widths ranging from 0.3 to 0.6 µm. OE 1-1 and Tel Aviv strains contained one single chromosome of 2.16 and 2.23 Mbp and one plasmid of 29.0 and 41.5 Kbp, with average DNA G+C contents of 38.16 and 38.47 mol%, respectively. These strains presented an average nucleotide identity (ANI) of 89.9 %. Bartonella elizabethae was found to be the closest phylogenetic relative of both strains (ANI=90.9-93.6 %). The major fatty acids identified in both strains were C18:1ω7c, C18 : 0 and C16 : 0. They differ from B. elizabethae in their C17 : 0 and C15 : 0 compositions. Both strains are strictly capnophilic and their biochemical profiles resembled those of species of the genus Bartonella with validly published names, whereas differences in arylamidase activities partially assisted in their speciation. Genomic and phenotypic differences demonstrate that OE 1-1 and Tel Aviv strains represent novel individual species, closely related to B. elizabethae, for which we propose the names Bartonella kosoyi sp. nov. and Bartonella krasnovii sp. nov.

Lymphatic Circulation Disseminates Bartonella Infection Into Bloodstream.

The hallmark of Bartonella infection is long-lasting intraerythrocytic parasitism. However, the process of Bartonella bacteremia is still enigmatic. In the current study, we used Bartonella tribocorum to determine how Bartonella invasion into the bloodstream from dermal inoculation might occur. Bartonella was poorly phagocytized by peritoneal macrophages in vitro. Intracellular Bartonella survived and replicated in macrophages at an early stage of infection. Intracellular Bartonella inhibited spontaneous cell death of macrophages. They also inhibited Salmonella-induced pyroptosis and mildly reduced inflammasome activation through an unidentified mechanism. A rat model confirmed that Bartonella was also inadequately phagocytized in vivo, because numerous free-floating bacilli were observed in lymph collected from thoracic duct drainage as early as 2 hours after inoculation. Lymphatic fluid drainage in the bloodstream significantly reduced the bacterial load in the bloodstream. These findings illustrated a potential route by which Bartonella invade bloodstream from dermal inoculation before they are competent to infect erythrocytes.

New serotypes of porcine teschovirus identified in Shanghai, China.

Teschoviruses are widely endemic and commonly found in pig fecal samples. In this study, we collected fecal specimens from various pig herds and genotyped them based on the VP1 gene. Of 322 samples, 276 were positive, giving a PTV infectivity rate of 85.7 %. PTV4 was the most common serotype found in Shanghai, followed by PTV8 and PTV10. Interestingly, Some Shanghai strains belonging to a new PTV serotype were also isolated. In phylogenetic analysis, PTV SH8 did not correspond to any known serotype. PTV4 and PTV6 showed similar levels of sequence identity to PTV SH8. These data suggest that PTV SH8 is a new serotype, distinct from the new serotype PTV wild boar/WB2C-TV/2011/HUN, which clusters with PTV SH2, SH10, and SH25.

Molecular detection of genogroup I and II picobirnaviruses in pigs in China.

In this study, a total of 187 stool specimens were collected from a pig farm in Hunan province of China, from November 2011 to June 2012. 39 (20.9 %) stool specimens were positive for picobirnaviruses using reverse transcription-polymerase chain reaction. Among 39 stool specimens, 84.6 % (33/39) were identified to be genogroup I (prototype 1-CHN-97), 38.5 % (15/39) belonged to genogroup II (prototype 4-GA-91), and 23.1 % (9/39) of which showed the evidence of genogroup I picobirnavirus were also positive for genogroup II picobirnaviruses. Picobirnaviruses exist in pigs which were divided into five groups according to the age and physiological status. Nineteen representative strains of genogroup I picobirnaviruses and eleven strains of genogroup II picobirnaviruses detected in this study were selected to analyze their phylogenetic relationships with other picobirnaviruses reference strains. The phylogenetic tree analysis suggested the prevalence of multiple picobirnaviruses in pigs in China.

Depolymerization of cytokeratin intermediate filaments facilitates intracellular infection of HeLa cells by Bartonella henselae.

Bartonella henselae is capable of invading epithelial and endothelial cells by modulating the function of actin-dependent cytoskeleton proteins. Although understanding of the pathogenesis has been increased by the development of an in vitro infection model involving endothelial cells, little is known about the mechanism of interaction between B. henselae and epithelial cells. This study aims to identify the binding candidates of B. henselae in epithelial cells and explores their effect on B. henselae infection. Pull-down assays and mass spectrometry analysis confirmed that some of the binding proteins (keratin 14, keratin 6, and F-actin) are cytoskeleton associated. B. henselae infection significantly induces the expression of the cytokeratin genes. Chemical disruption of the keratin network by using ethylene glycol tetraacetic acid promotes the intracellular persistence of B. henselae in HeLa cells. However, cytochalasin B and phalloidin treatment inhibits B. henselae invasion. Immunofluorescent staining demonstrates that B. henselae infection induces an F-actin-dependent rearrangement of the cytoskeleton. However, we demonstrated via immunofluorescent staining and whole-mount cell electron microscopy that keratin intermediate filaments are depolymerized by B. henselae. The results indicate that B. henselae achieves an intracellular persistence in epithelial cells through the depolymerization of cytokeratin intermediate filaments that are protective against B. henselae invasion.

Infiltrating Perfluorocarbon Nanoemulsion and Sensitizing Ultrasound Cavitation to Eradicate Biofilms.

Developing strategies for the treatment of bacterial biofilms is challenging due to their complex and resilient structure, low permeability to therapeutics, and ability to protect resident pathogens. Herein, we demonstrate that a polylysine-stabilized perfluorocarbon nanoemulsion is favored for penetrating biofilms and sensitizing the cavitation effect of low-intensity ultrasound, resulting in the dispersal of extracellular polymeric substances and killing of the protected cells. Through experiments, we observed a complete penetration of the nanoemulsion in a 40 µm Pseudomonas aeruginosa biofilm and demonstrated that it was induced by the fluidic perfluorocarbon, possibly attributing to its low surface tension. Furthermore, we presented an almost complete antibiofilm effect with a low-intensity ultrasound (1 MHz, 0.75 W/cm2, 5 min) in diverse cases, including cultured biofilms, colonized urinary catheters, and chronic wounds. During the treatment process, the perfluorocarbon phase enhanced the number and imploding energy of ultrasound cavities, thoroughly divided the biofilm structure, prevented biofilm self-healing, and sterilized the resident pathogens. Thus, the penetration and sensitization of the nanoemulsion might serve as a facile and potent strategy for eradicating biofilms in various applications.

Hemostatic and Ultrasound-Controlled Bactericidal Silk Fibroin Hydrogel via Integrating a Perfluorocarbon Nanoemulsion.

Excessive blood loss and infections are the prominent risks accounting for mortality and disability associated with acute wounds. Consequently, wound dressings should encompass adequate adhesive, hemostatic, and bactericidal attributes, yet their development remains challenging. This investigation presented the benefits of incorporating a perfluorocarbon nanoemulsion (PPP NE) into a silk-fibroin (SF)-based hydrogel. By stimulating the ß-sheet conformation of the SF chains, PPP NEs drastically shortened the gelation time while augmenting the elasticity, mechanical stability, and viscosity of the hydrogel. Furthermore, the integration of PPP NEs improved hemostatic competence by boosting the affinity between cells and biomacromolecules. It also endowed the hydrogel with ultrasound-controlled bactericidal ability through the inducement of inner cavitation by perfluorocarbon and reactive oxygen species (ROS) generated by the sonosensitizer protoporphyrin. Ultimately, we employed a laparotomy bleeding model and a Staphylococcus aureus-infected trauma wound to demonstrate the first-aid efficacy. Thus, our research suggested an emulsion-incorporating strategy for managing emergency wounds.

Ba2+-doping introduced piezoelectricity and efficient Ultrasound-Triggered bactericidal activity of brookite TiO2 nanorods.

Exploring highly efficient ultrasound-triggered catalysts is pivotal for various areas. Herein, we presented that Ba2+ doped brookite TiO2 nanorod (TiO2: Ba) with polarization-induced charge separation is a candidate. The replacement of Ba2+ for Ti4+ not only induced significant lattice distortion to induce polarization but also created oxygen vacancy defects for facilitating the charge separation, leading to high-efficiency reactive oxygen species (ROS) evolution in the piezo-catalytic processes. Furthermore, the piezocatalytic ability to degrade dye wastewater demonstrates a rate constant of 0.172 min-1 and achieves a 100 % antibacterial rate at a low dose for eliminating E. coli. This study advances that doping can induce piezoelectricity and reveals that lattice distortion-induced polarization and vacancy defects engineering can improve ROS production, which might impact applications such as water disinfection and sonodynamic therapy.

Complete genome of transmissible gastroenteritis virus AYU strain isolated in Shanghai, China.

Transmissible gastroenteritis virus strain AYU was isolated in Shanghai. The complete genome has a length of 28,582 bp and contains seven open reading frames. Sequence analysis suggested that Shanghai strain AYU and U.S. strain Purdue P115 are derived from a common ancestor, as they have 99.6% similarity at the nucleotide level.

Ultrasmall Day-Night Photocatalysts of LnYVO4:Eu3+ (Ln = Nd, Tm, and Er) Nanoparticles.

Day-night photocatalysts that can persistently generate reactive oxygen species (ROS) after ceasing light attracted intensive attention in diverse fields. However, current strategies of combining a photocatalyst and an energy storage material can hardly fulfill the demands, especially in size. We herein present a one-phase sub-5 nm day-night photocatalyst via simply doping Nd, Tm, or Er into YVO4:Eu3+ nanoparticles, efficiently producing ROS in both day and night modes. We demonstrate that the rare earth ions acted as a ROS generator, and Eu3+ and defects contributed to the long persistency. Furthermore, the ultrasmall size led to remarkable bacterial uptake and bactericidal efficacy. Our finding suggests an alternative mechanism of day-night photocatalysts that could be ultrasmall and thus may shed light on disinfection and other applications.

Molecular characterization of a porcine kobuvirus strain in China.

Porcine kobuvirus was first identified in 2007 in Hungary. The virus has been detected in several Asian countries. In our study, the complete genome of the recently identified porcine kobuvirus strain SH-W-CHN was amplified by RT-PCR and was sequenced. Dendrograms indicated that SH-W-CHN is closely related to other porcine kobuviruses. To identify sites of possible recombination within the genome of the SH-W-CHN strain, the SimPlot program was used to perform recombination analysis. The results showed that no significant recombination event between strain S-1-HUN and Y-1-CHI had occurred. However, certain possible recombination signals were identified, indicating that some early recombination events may have contributed to the genome of SH-W-CHN. This study further confirmed the existence of multiple lineages of porcine kobuvirus and indicated that homologous recombination may be a driving force in its evolution.

Molecular detection of Torque teno mini virus (TTMV) in China.

In this study, 176 serum specimens were collected from hospitalized cardiovascular diseases patients in Shanghai, China. 140 samples (79.5%) were positive with the primers located in non-coding region, and 30 samples (17.04%) were identified to be positive with group-specific primers. Of the 30 samples, 9 (5.11%) were classified to group 1, 23 (13.07%) were clustered to group 2, and 3 belonged to group 3. Five samples were confirmed to be dual infection with different groups of TTMV, and no sample was found to be infected with groups 4-6.

Cat scratch disease with generalized bone lesions in an immunocompetent child.

A 9-year-old immunocompetent girl with prolonged fever for 1 month was suspected of having a malignancy because of generalized bone abnormalities identified by MRI. Histopathology of liver tissues indicated the diagnosis of cat-scratch disease (CSD). Results of NGS, immunofluorescence and immunochemical assay confirmed the causative agent was Bartonella henselae. Paediatricians should increase their awareness of CSD as a cause for bone lesions, except for malignancy.

Molecular characterization of a porcine astrovirus strain in China.

Pigs are increasingly recognized to harbor a wide range of viruses that apparently establish long-term persistence in these animals. They serve as reservoirs for a number of human zoonotic diseases. In this study, a porcine astrovirus (PAstV) strain, designated as PAstV JWH-1, is identified from a diarrheal pig in China, and it is partially characterized genetically. Sequence analysis shows that the PAstV JWH-1 strain contains divergent nucleotide sequences in both the open reading frame (ORF)1b/ORF2 consensus and the 3'-UTR regions (s2m motif), which are usually highly conserved among members of the family Astroviridae. Phylogenetic analysis indicates that the JWH-1 strain clusters closely with newly identified strains PAstV 12-4 and 14-4 and forms a group of mamastroviruses with the proposed novel deer astrovirus. Further recombination analysis shows that two possible interspecies recombination events between porcine and deer astroviruses occurred in the genome of the JWH-1 strain. This study further confirms that multiple lineages are present among PAstVs, and each lineage likely represents an independent origin. Additionally, the possibility of interspecies transmission among PAstVs is also suggested.

Isolation and characterization of the first Chinese porcine sapelovirus strain.

Investigations were carried out to identify the causative agent of acute diarrhea, respiratory distress, and polioencephalomyelitis of pigs on a swine farm in Shanghai, China. Samples from the affected animals were tested for viruses and bacteria that are known to cause similar symptoms in swine, and only porcine sapelovirus (PSV; designated as csh strain) was isolated. The presence of PSV was further confirmed by the specific cytopathic effects observed in susceptible cells and by the results of PCR and electron microscopy. Nucleotide sequencing and phylogenetic analysis showed that this isolate is PSV. When inoculated into healthy pigs, PSV.csh caused the same symptoms as observed in the affected herd. Therefore, PSV.csh is the causative agent of this disease. To the best of our knowledge, this is the first report of PSV infecting piglets in China.

Experimental infection of Balb/c nude mice with Hepatitis E virus.

BACKGROUND: Several animal species can reportedly act as reservoirs for Hepatitis E virus (HEV), a zoonotic pathogen. HEV and antibody to the virus have been detected in a variety of animals including rodents. Pig and rat models for HEV have been established for HEV, but a nude mouse has not yet been developed. METHODS: Balb/c nude mice were inoculated with swine HEV, both orally and via intravenous injection to insure infection. Negative control and experimental contact-exposed groups of mice were also included in the study. The liver, spleen, kidney, jejunum, ileum, cecum and colon of each mouse from all three groups were collected for reverse transcription nested polymerase chain reaction (RT-nPCR) detection, indirect immunofluorescence observation and histopathologic examination. The sera from nude mice were tested for anti-HEV IgG by enzyme linked immunosorbent assay (ELISA). Activities of liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), as well as total bilirubin (TBIL) were also measured in the sera of the nude mice. RESULTS: HEV antigens and HEV RNA were detected in liver, spleen, kidney, jejunum, ileum and colon both by indirect immunofluorescence and by RT-nPCR in all of the inoculated and in one of the contact-exposed nude mice. Histopathological changes were observed in the liver and spleen of these mice. Infected mice showed increased levels of AST, ALP, and anti-HEV IgG in sera. The livers of contact-exposed mice showed obvious histopathological damage. CONCLUSION: Nude mice could be readily infected by HEV isolated from pigs. The nude mouse may therefore be a useful animal model for studying the pathogenesis of HEV.