Demonstration of safety characteristics and effects on gut microbiota of Lactobacillus gasseri HMV18

Human normal flora is a source of probiotics.The safety characteristics of a specific isolate determine its application in foods or drugs.The food-borne-pathogen antagonist strain Lactobacillus gasseri HMV18 is one of the isolates from normal human flora.In this work,we assessed the in vitro pH tolerance,bile tolerance,biogenic amine production,mucin utilization,and safety of in vivo administration to mice to evaluate general health,organ-body weight index,organ histopathological change,whether L.gasseri HMV18 can colonize in the gut or modulate the gut microbiota after oral administration.The results suggest that L.gasseri HMV18 can tolerate pH 3 for 2 h,3%bile for 3 h,biogenic amine negative,mucin usage negative,does not encode verified toxins,and cause no visible change in mice's organs.L.gasseri HMV18 might not colonize in mice's gut,but can significantly affect the structure of gut microbiota.A bibliographical survey suggested that there were as few as 8 opportunistic infection cases from 1984 to 2022 and that the possibility for L.gasseri to cause infection is relatively low.Therefore,this work provides a basis for the foods or drugs application of L.gasseri HMV18 and gives a map of experiments for the safety assessment of probiotics.

A new Lactobacillus gasseri strain HMV18 inhibits the growth of pathogenic bacteria

To search for a new eco-friendly therapy for infectious disease caused by Escherichia coli,Staphylococcus aureus or Klebsiella oxytoca,we collected the vaginal swabs from healthy women,screened for Lactobacillus and found a strain repressing the growth of pathogenic bacteria.The new isolate was identified as L.gasseri by the colony morphology,Gram staining,biochemical reactions and confirmed by the 16 S rDNA sequencing.The HMV18 strain inhibited the growth of food-borne pathogens such as E.coli,S.aureus and K.oxytoca.The HMV18 strain was sensitive to penicillin,ampicillin,erythromycin,tetracycline and chloramphenicol.The HMV18 strain producedα-hemolysis.Pathological histology of the mice ileum showed that the mucosa,villi,lamina propria and crypt depth remained intact and there was no inflammation or hyperemia in the L.gasseri HMV18 gavaged group.L.gasseri HMV18 could not up-regulate inflammatory cytokines level of plasma.All the results suggested L.gasseri HMV18 is a candidate probiotic to be an additive for food preservation or drug to prevent food-borne diseases.

Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

Objective A PCR-reverse dot blot hybridization （RDBH） assay was developed for rapid detection of rpoB gene mutations in ＇hot mutation region＇ of Mycobacterium tuberculosis （M. tuberculosis）. Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing （DST） and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2% （165/181） and 98.3% （117/119）, respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value （PPV） and negative predictive value （NPV） of the RDBH assay were 97.7% （293/300）, 98.2% （164/167）, and 97.0% （129/133）, respectively. Furthermore, the results indicated that the most common mutations were in codons 531 （48.6%）, 526 （25.4%）, 516 （8.8%）, and 511 （6.6%）, and the combinative mutation rate was 15 （8.3%）. One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

DOWN-REGULATION OF CYTOKINE SECRETION AND REPRESSION OF APOPTOSIS hDaxx IN MACROPHAGES

Objective: To investigate the regulation effects on LPS-mediated cytokine secretion and dexamethasone- induced apoptosis in macrophages by transient overexpression of hDaxx. Methods: An eukaryotic expression vector pEGFP/hDaxx, which could express a fusion protein GFP-Daxx, was transfected into macrophages. The expression and localization of GFP-hDaxx fusion protein was analyzed by fluorescent microscope and western blot. The effects of transient overexpression of GFP-hDaxx fusion protein on the lipopolysaccharide(LPS)-mediated secretion of TNF-α and IL-1β were determined by ELISA. Moreover, the dexamethasone-induced apoptosis was determined morphologically by Giemsa stain. Results: The results observed showed that GFP-hDaxx fusion protein overexpressed in macrophages and localized in nuclei but GFP in cytoplasm under fluorescent microscope. The overexpression of GFP-hDaxx fusion protein could be detected by Western blot with an antibody against C-terminal of hDaxx. In the group with overexpressed GFP-hDaxx fusion protein, the LPS-mediated cytokine secretion decreased remarkably at 1 h, 3 h, 6 h respectively after LPS stimulation, and the dexamethasone- induced apoptosis reduced notably at 6 h, 12 h and 24 h respectively after addition of dexamethasone. There were remarkable difference between pEGFP/hDaxx group and control group (P<0.01) at different time. Conclusion: Transient overexpression of hDaxx down-regulates LPS-mediated cytokine secretion in macrophages and inhibits dexamethasone-induced macrophages apoptosis.

pORF5 plasmid protein of Chlamydia trachomatis induces MAPK-mediated pro-inflammatory cytokines via TLR2 activation in THP-1 cells

Infection with Chlamydia trachomatis induces inflammatory pathologies in the urogenital tract that can lead to infertility and ectopic pregnancy. Pathogenesis of infection has been mostly attributed to excessive cytokine production. However, precise mechanisms on how C. trachomatis triggers this production, and which protein（s） stimulate inflammatory cytokines remains unknown. In the present study, the C. trachomatis pORF5 protein induced tumor necrosis factor alpha （TNF-a）, interleukin-1 beta （IL-1β） and interleukin-8 （IL-8） in dose and time-dependent manners in the THP-1 human monocyte cell line. We found that intracellular p38/mitogen-activated protein kinase （MAPK） and extracellular signal-regulated kinase （ERK）/MAPK signaling pathways were required for the induction of TNF- a, IL-1β and IL-8. Blockade of toll-like receptor 2 （TLR2） signaling reduced induction levels of TNF-a, IL-8 and IL-1β. We concluded that the C. trachomatis pORF5 protein might contribute to the inflammatory processes associated with chlamydial infections.

Assessment of the immune responses to Treponema pallidum Gpd DNA vaccine adjuvanted with IL-2 and chitosan nanoparticles before and after Treponema pallidum challenge in rabbits

Syphilis is a multistage,sexually transmitted disease caused by the spirochete,Treponema pallidum(Tp).A significantly high incidence of syphilis has been reported in several countries,including China,and there is an urgent need for the development of efficacious vaccines against syphilis.DNA vaccines are a major breakthrough in the field of vaccination with several advantages over traditional vaccines.Animal model studies of Tp DNA vaccines have not been reported elsewhere but our previous reports describe the development of a single-gene Tp DNA vaccine and preclinical immunization study.In this study,chitosan(CS) nanoparticles were used as a vector and an interleukin-2 expression plasmid(pIL-2) as an adjuvant to enhance a TpGpd DNA vaccine candidate(pTpGpd) in a rabbit Tp skin challenge model.At week 8 after the first immunization,three rabbits from each group were used to determine cytokine measurements and spleen lymphocyte proliferation assay.pTpGpd in combination with pIL-2 wrapped with CS led to the greatest enhancement of anti-TpGpd antibodies and T-cell proliferation.During infection,levels of anti-TpGpd antibodies and T-cell proliferation were measured.Both the serum special IgG and IL-2,interferon-γ were significantly increased by the co-injection of the IL-2 plasmid compared with the injection of TpGpd DNA alone(P<0.05).Furthermore,IL-2 plasmid coinjection efficiently enhanced the antigen-specific lymphocyte proliferation response.Additionally,the ratios of positive skin lesions and ulcer lesions in groups immunized with pTpGpd were significantly lower than those of the pIL-2,CS or pIL-2 mixed with CS control groups(P<0.001).CS vectored and pIL-2 adjuvanted pTpGpd immunized animals exhibited the lowest rates of positive skin tests(8.33%) and ulcer lesions(4.17%) and the fastest recovery(42 d).These experiments indicate that co-injection of a pIL-2 plasmid with pTpGpd DNA vaccine wrapped with CS can significantly strengthen the long-term stability of immune response during infection,efficiently improve the protective effect against T.pallidum spirochetes infection and attenuate syphilitic lesion development.

EV713C protease cleaves host anti-viral factor OAS3 and enhances virus replication

The global spread of enteroviruses(EVs)has become more frequent,severe and life-threatening.Intereron(IFN)I has been proved to control EVs by regulating IFN-stimulated genes(ISG)expression.20-50-oligoadenylate synthetases 3(OAS3)is an important ISG in the OAS/RNase L antiviral system.The relationship between OAS3 and EVs is still unclear.Here,we reveal that OAS3,superior to OAS1 and OAS2,significantly inhibited EV71 replication in vitro.However,EV71 utilized autologous 3C protease(3C^(pro))to cleave intracellular OAS3 and enhance viral replication.Rupintrivir,a human rhinovirus 3C protease inhibitor,completely abolished the cleavage of EV713C^(pro)on OAS3.And the proteolytically deficient mutants H40G,E71A,and C147G of EV713C^(pro)also lost the ability of OAS3 cleavage.Mechanistically,the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3C^(pro)cutting site.Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3(CVB3),Coxsackievirus A16(CA16),Enterovirus D68(EVD68),and Coxsackievirus A6(CA6)subtypes.Notably,unlike other four subtypes,CA163C^(pro)could not cleave OAS3.Two key amino acids variation Ile36 and Val86 in CA163C^(pro)might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage.Our works elucidate the broad anti-EVs function of OAS3,and illuminate a novel mechanism by which EV71 use 3C^(pro)to escape the antiviral effect of OAS3.These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.

Armigeres subalbatus is a potential vector for Zika virus but not dengue virus

Background:Zika virus (ZIKV) and dengue virus (DENV) are closely related flaviviruses primarily transmitted by Aedes mosquitoes. Armigeres subalbatus is an emerging and widely distributed mosquito, and ZIKV has been detected and isolated from it. However, it is not clear whether Ar. subalbatus could be a vector for ZIKV and DENV or not. In this study, we investigated the infection and transmission of Ar. subalbatus to ZIKV and DENV.Methods:A line of Ar. subalbatus was isolated from Guangdong, China, and further identified by the mitochondrial cytochrome oxidase subunit 1 (COI) gene. The adults of Ar. subalbatus were fed with blood meal containing ZIKV or DENV-2. At 4, 7, 10, 14, and 21 days post-inoculation (dpi), the infections of ZIKV or DENV-2 in the midguts, ovaries and salivary glands were detected and quantified by RT-PCR and RT-qPCR. To assess the transmissibility, suckling mice were exposed to bites of ZIKV-infected mosquitoes, and ZIKV was detected in brain tissue by RT-qPCR and plaque assays. Furthermore, the larvae of Ar. subalbatus were reared in artificial urine containing ZIKV or DENV-2. The infection rates and viral titers of larvae and adults were analyzed by RT-PCR and RT-qPCR, and the viral distribution in larval tissues was observed by immunohistochemistry. Chi-square test and one-way ANOVA analysis were used for assessing the infection rate and viral titer in varied tissues and different time points, respectively.Results:Following oral inoculation, ZIKV but not DENV-2 could be detected in Ar. subalbatus midguts at 4 dpi, ovaries at 7 dpi and salivary glands at 10 dpi. The highest infection rate (IR) of ZIKV was 27.8% in midgut at 7 dpi, 9.7% in ovary and 5.6% in salivary gland at 21 dpi. Eight days after being bitten by ZIKV-positive mosquitoes, ZIKV was detected in three brain tissues out of four suckling mice exposed to bites. ZIKV could be detected in the larvae reared in artificial urine contained ZIKV at a high concentration of 105 pfu/ml and various tissues of adults with a low infection rate (0.70–1.35%). ZIKV could be observed in anal papillae and midgut of larvae at 4 dpi under laboratory conditions.Conclusions:ZIKV but not DENV-2 can infect Ar. subalbatus by blood meal and artificial urine, and the infected mosquitoes can transmit ZIKV to suckling mice by bite. From these findings, we can conclude that the Ar. subalbatus isolated from Guangdong province, China, is a potential vector for ZIKV and should therefore be considered in vector control programs to prevent and control of Zika virus disease.

Toxicity and metabolism of 3-bromopyruvate in Caenorhabditis elegans

In this study,we aimed to evaluate the toxic effects,changes in life span,and expression of various metabolismrelated genes in Caenorhabditis elegans,using RNA interference(RNAi)and mutant strains,after 3-bromopyruvate(3-BrPA)treatment.C.elegans was treated with various concentrations of 3-BrPA on nematode growth medium(NGM)plates,and their survival was monitored every 24 h.The expression of genes related to metabolism was measured by the real-time fluorescent quantitative polymerase chain reaction(qPCR).Nematode survival in the presence of 3-BrPA was also studied after silencing three hexokinase(HK)genes.The average life span of C.elegans cultured on NGM with 3-BrPA was shortened to 5.7 d compared with 7.7 d in the control group.hxk-1,hxk-2,and hxk-3 were overexpressed after the treatment with 3-BrPA.After successfully interfering hxk-1,hxk-2,and hxk-3,the 50%lethal concentration(LC50)of all mutant nematodes decreased with 3-BrPA treatment for 24 h compared with that of the control.All the cyp35 genes tested were overexpressed,except cyp-35B3.The induction of cyp-35A1 expression was most obvious.The LC50 values of the mutant strains cyp-35A1,cyp-35A2,cyp-35A4,cyp-35B3,and cyp-35C1 were lower than that of the control.Thus,the toxicity of 3-BrPA is closely related to its effect on hexokinase metabolism in nematodes,and the cyp-35 family plays a key role in the metabolism of 3-BrPA.