Fecal carriage and molecular epidemiology of mcr-1-harboring Escherichia coli from children in southern China.

BACKGROUND: The increase of multidrug-resistant Enterobacteriaceae bacteria has led to the reintroduction of colistin for clinical treatments, and colistin has become a last resort for infections caused by multidrug-resistant bacteria. Enterobacteriaceae bacteria carrying the mcr-1 gene are majorly related to colistin resistance, which may be the main reason for the continued increase in the colistin resistance rate of Enterobacteriaceae. The study aimed to investigate the sequence type and prevalence of Escherichia coli (E. coli) harboring the mcr-1 gene in the gut flora of children in southern China. METHODS: Fecal samples (n = 2632) of children from three medical centers in Guangzhou were cultured for E. coli. The mcr-1-harboring isolates were screened via polymerase chain reaction (PCR). The colistin resistance transfer frequency was studied by conjugation experiments. DNA sequencing data of seven housekeeping genes were used for multi-locus sequence typing analysis (MLST). RESULTS: PCR indicated that 21 of the 2632 E. coli (0.80%) isolates were positive for mcr-1; these strains were resistant to colistin. Conjugation experiments indicated that 18 mcr-1-harboring isolates could transfer colistin resistance phenotypes to E. coli J53. MLST analysis revealed that the 21 isolates were divided into 18 sequence types (STs); E. coli ST69 was the most common (14.3%), followed by E. coli ST58 (9.5%). CONCLUSION: These results demonstrate the colonization dynamics and molecular epidemiology of E. coli harboring mcr-1 in the gut flora of children in southern China. The mcr-1 gene can be horizontally transmitted within species; hence, it is necessary to monitor bacteria that harbor mcr-1 in children.

The casein-derived peptide YFYPEL alleviates intestinal epithelial cell dysfunction associated with NEC by regulating the PI3K/AKT signaling pathway.

Necrotizing enterocolitis (NEC) is a life-threatening risk to the health of neonates, but thus far, there is no very effective treatment. Although many studies have confirmed the therapeutic role of peptides in diseases, the effect of peptides in NEC remains poorly understood. This study investigated the role of casein-derived peptide YFYPEL in NEC cells and animal models. We synthesized YFYPEL and analysed its protective effects on NEC both in vitro and in vivo. YFYPEL integration in the intestine increased rat survival and clinical conditions, lowered the incidence of NEC, alleviated bowel inflammation, and enhanced intestinal cell migration. Furthermore, YFYPEL significantly decreased interleukin 6 expression and increased intestinal epithelial cell migration. Moreover, YFYPEL alleviated intestinal epithelial cell dysfunction through the PI3K/AKT pathway, as demonstrated by western blotting and bioinformatics analysis. A selective PI3K activator reversed the protective effect of YFYPEL on lipopolysaccharide-stimulated intestinal epithelial cells. Our study showed that YFYPEL reduced inflammatory cytokine expression and enhanced migration by regulating the PI3K/AKT pathway. The use of YFYPEL may thus develop into a novel modality in NEC treatment.

LncRNA and mRNA profiles of human milk-derived exosomes and their possible roles in protecting against necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is one of the most severe diseases commonly afflicting premature infants. Our previous studies suggests that human milk-derived exosomes (HM-Exos) have a potential therapeutic effect on NEC. In this study, we investigate the potentially therapeutic role of HM-Exos in an NEC animal model via comprehensive lncRNA and mRNA expression profiles. A rat model of NEC was induced through hypoxia, hypothermia and formula feeds. We extracted exosomes from the colostrum of healthy lactating mothers and identified their functions in an NEC animal model. Furthermore, high-throughput lncRNA and mRNA sequencings were explored to find the underlying mechanisms. Although both exosomes from term human breast milk (Term-Exos) and exosomes from preterm human breast milk (Pre-Exos) alleviated the severity of NEC, Pre-Exos seemed to better promote the proliferation of intestinal epithelial cells in vivo. We identified a total of 44 differentially expressed lncRNAs and 88 differentially expressed mRNAs between Term-Exos and Pre-Exos. Further GO and KEGG pathway analysis showed that the lncRNA-mRNA network of HM-Exos was associated with the JAK-STAT signaling pathway, bile secretion and the AMPK signaling pathway, which are predicted to be involved in the proliferation of cells. Therefore, this study reveals for the first time the important roles of human milk derived lncRNAs and mRNAs in protecting against necrotizing enterocolitis. These results provide new insight into the development of NEC.

Peptide Tat(48-60) YVEEL protects against necrotizing enterocolitis through inhibition of toll-like receptor 4-mediated signaling in a phosphatidylinositol 3-kinase/AKT dependent manner.

Necrotizing enterocolitis (NEC) is a catastrophic disease largely occurring in preterm infants, and toll-like receptor 4 (TLR4) has been implicated in its pathogenesis. The current therapeutic strategies for NEC are, however, far from optimal. In the present study, a whey-derived antioxidative peptide conjugated with a cell-penetrating TAT [Tat (48-60) YVEEL] was prepared to endow it with enhanced cell uptake capability and bioavailability. The protective effect of Tat (48-60) YVEEL on experimental NEC was evaluated both in vitro and in vivo. Inhibition of TLR4-mediated signaling by Tat (48-60) YVEEL was assessed in FHC and IEC-6 enterocytes, neonatal rat model of NEC, and the mechanism underlying this effect was determined. Tat (48-60) YVEEL significantly inhibited TLR4-mediated expression of pro-inflammatory cytokines, p65 nuclear translocation and restored the impaired enterocyte migration in cultured enterocytes. In addition, Tat (48-60) YVEEL administration strikingly increased the survival rate, and reduced the severity of NEC in rats through inhibition of TLR4-mediated signaling. These protective effects of Tat (48-60) YVEEL occurred in a PI3K/AKT dependent manner, as administration of PI3K activator Ys49 abrogated its protective effects. Combined with liposomes, Tat (48-60) YVEEL demonstrated longer retention in the intestines that better for potential clinical applications. These data demonstrate that Tat (48-60) YVEEL protects against NEC through inhibition of TLR4-mediated signaling in a PI3K/AKT dependent manner, and offer a potential therapeutic approach to this disease.

The role of polypeptide PDTLN1 in suppression of PI3K/AKT signaling causes cardiogenetic disorders in vitro and in vivo.

AIMS: A new polypeptide, PDTLN1, derived from the human Talin-1 protein, which is highly expressed in both myocardial tissue and maternal peripheral blood of aborted fetuses with congenital heart disease (CHD). However, its role in cardiac developmental disorders has not been disclosed till now. In the present study, we aim to assess the functions of PDTLN1 in heart development of zebrafish and cellular viability, proliferation, and apoptosis of P19 cells. MAIN METHODS: Cellular viability was assessed by Cell Counting Kit-8, the EdU Kit was used to evaluate cellular proliferation, and apoptosic rate of P19 was examined using FITC Annexin-V staining followed by flow cytometry. The zebrafish embryos were divided into three groups: PEP group and NC group were microinjected with polypeptides, WT group without any intervention. The protein expression of PI3K/AKT were evaluated by western blotting. KEY FINDINGS: PDTLN1 could suppress the proliferation, and facilitate apoptosis. PDTLN1 caused abnormal heart development of zebrafish embryos and the PDTLN1 (50 µM)-injected group showed an aberrant expression pattern of vmhc, amhc and cmlc2. Compared to the CTL group and SC79 group of P19 cells, the PDTLN1 group had a lower phosphorylated PI3K/AKT proteins level, decreased cellular viability and lower proliferation activity. SIGNIFICANCE: PDTLN1 caused cardiac developmental defects in zebrafish, inhibited cellular viability, proliferation, and promoted apoptosis of P19 cells via suppressing the PI3K/AKT signaling pathway. Our findings provide a fresh perspective on the functional mechanism of human-derived peptides and may promote novel diagnostic biomarkers detection and therapeutic targets in CHD.

Differential Expression Profiles and Functional Prediction of circRNAs in Necrotizing Enterocolitis.

Circular RNAs (circRNAs), a novel type of noncoding RNAs, have been demonstrated to behave as microRNA (miRNA) sponges to exert their effects during pathological processes of diseases. However, the roles of circRNAs have not been explored in necrotizing enterocolitis (NEC). This study sought to identify differentially expressed circRNAs and predict their potential biological functions in NEC. circRNA expression profiles in terminal ileum from newborn rats with NEC and normal controls were explored using next-generation sequencing. In the NEC group, 53 circRNAs were significantly differentially expressed, including 9 upregulated and 44 downregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted, and circRNA-miRNA interaction networks were generated to predict the potential roles of circRNAs in NEC progression. Further investigation revealed that most circRNAs include miRNA binding sites and that some are implicated in NEC development. In conclusion, this study's findings demonstrate that differentially expressed circRNAs are involved in NEC development via their interactions with miRNAs, making them prospective targets for NEC diagnosis and treatment.

Lipidomic Profiling of Human Milk Derived Exosomes and Their Emerging Roles in the Prevention of Necrotizing Enterocolitis.

SCOPE: Human milk can prevent the development of necrotizing enterocolitis (NEC). Human milk is rich in cargo-carrying exosomes that participate in intercellular communication. This study investigated the effects of term and preterm human milk-derived exosomes, and elucidated their lipid expression profiles. METHODS AND RESULTS: Milk from healthy mothers is collected who have delivered full-term or preterm infants, and exosomes are isolated and quantified. Administration of term and preterm milk exosomes significantly enhances epithelial proliferation and migration in vitro, and ameliorates the severity of NEC in vivo. A total of 395 lipids are identified in term and preterm human milk-derived exosomes. Bioinformatics analysis and western blotting reveal that top 50 lipids regulate intestinal epithelial cell function via the Extracellular-Signal-Regulated Kinase/Mitogen Activated Protein Kinase (ERK/MAPK) pathway. CONCLUSION: This study reveals for the first time the lipidomic complexities in exosomes derived from preterm and term milk. The results provide novel mechanistic insight on how human milk prevents the development of NEC.

Oral administration of recombinant Bacillus subtilis spores expressing mutant staphylococcal enterotoxin B provides potent protection against lethal enterotoxin challenge.

Pathogenicity of Staphylococcus aureus is induced by staphylococcal enterotoxin B (SEB). A mutant form of SEB (mSEB) is immunogenic as well as less toxic. Recombinant mSEB and SEB were expressed in pET28a prokaryotic plasmids. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in mSEB-stimulated macrophages were lower than those in SEB-stimulated macrophages (p < 0.001, p < 0.01 respectively). Using CotC as a fusion protein, we constructed recombinant Bacillus subtilis spores expressing mSEB on the spore surface and evaluated their safety and protective efficacy via mouse models. Oral administration of mSEB-expressing spores increased SEB-specific IgA in feces and SEB-specific IgG1 and IgG2a in the sera, compared with mice in naïve and CotC spore-treated groups (p < 0.001, p < 0.01, p < 0.001 respectively). Six weeks following oral dosing of recombinant spores, significant differences were not found in the serum biochemical indices between the mSEB group and the naïve and CotC groups. Furthermore, oral administration of mSEB spores increased the survival rate by 33.3% in mice intraperitoneally injected with 5 µg of wild-type SEB plus 25 µg lipopolysaccharide (LPS). In summation, recombinant spores stably expressing mSEB were developed, and oral administration of such recombinant spores induced a humoral immune response and provided protection against SEB challenge in mice.

Prevalence, Characterization, and Drug Resistance of Staphylococcus Aureus in Feces From Pediatric Patients in Guangzhou, China.

Background: Staphylococcus aureus (S. aureus) is a major pathogen of human infections. Its fecal carriage serves as a risk factor for nosocomial transmission and disease development. However, the rate of S. aureus fecal carriage among Chinese children has not yet been reported. Therefore, we sought to investigate the prevalence, characterization, and drug resistance of S. aureus isolated from pediatric patients' feces in Southern China. Methods: Fecal samples (2059) from pediatric patients in three centers in Guangzhou were cultured. From which, 412 S. aureus isolates were identified via selective mediums and automated VITEK Mass Spectrometer analysis. Antibiotic susceptibility was determined and DNA sequencing of seven housekeeping genes were used for multilocus sequence typing analysis. Results: The fecal carriage rates were 20.0% for S. aureus and 4.5% for methicillin-resistant S. aureus (MRSA). Moreover, S. aureus fecal carriage was positively correlated with outpatient status and gastroenteritis diagnosis. Moreover, age-related patterns were observed with respect to prevalence of S. aureus. Besides, a total of 76 sequence types (STs) were identified, including 25 newly assigned STs and 28 clonal complexes (CCs). ST188, ST6, and ST15 were the most prevalent methicillin-sensitive S. aureus (MSSA) clones, while ST59 and ST45 were the major MRSA clones. S. aureus isolates also exhibited high rates of penicillin (84.2%), erythromycin (38.8%), and clindamycin (35.9%) resistance. Specifically, all ST30 and ST338 isolates were resistant to erythromycin and clindamycin, 61% of ST7 were resistant to tetracycline, and 84% of ST45 exhibited resistance and intermediate resistance to rifampicin. Also, CC59 (ST338 and ST59) and CC45 exhibited different antibiotic resistance patterns. Conclusion: These results demonstrate the colonization dynamics and molecular epidemiology of S. aureus in child feces in Southern China. Further, they suggest an urgency for strengthening the surveillance programs in China and provide important information for the prevention and treatment of S. aureus infection.

Evaluation of time to positivity for blood culture combined with immature granulocytes, neutrophil-to-lymphocyte ratio, and CRP in identifying bloodstream coagulase-negative Staphylococci infection in pediatric patients.

OBJECTIVE: To evaluate the application value of time to positivity (TTP) for blood culture combined with inflammatory parameters that included immature granulocyte percentage (IG%), immature granulocyte count (IG#), C-reactive protein (CRP), white blood cells (WBC) neutrophil percentage (NE%), and neutrophil-to-lymphocyte ratio (NLR), and to identify bloodstream infections from contamination with coagulase-negative staphylococci (CoNS) in pediatric patients. METHODS: Data of 12 897 inpatients with blood culture CoNS were retrospectively collected and analyzed from January-December 2019 at our hospital. According to pre-defined criteria, they were divided into a CoNS infection group (132 cases) and a CoNS contamination group (124 cases). Infection with Staphylococcus aureus (SA, 27 cases) at the same period was considered a positive control group. ROC curve analysis assisted in determining the value of applying TTP combined with the above-mentioned inflammatory parameters to distinguish CoNS infection from contamination. RESULTS: Among the 256 strains of CoNS, Staphylococcus hominis (55.1%), Staphylococcus epidermidis (32.0%), and Staphylococcus capitis (7.0%) were common. There was no significant difference in the subspecies distribution between the infection and contamination groups. The TTP of the CoNS infection group was significantly lower than the contamination group (P < .05). IG%, IG#, CRP, NE%, and NLR were all higher in the infected group as compared to the contaminated group (P < .05), while WBC was similar among groups. There was also no statistical difference in those parameters when comparing the CoNS infection and SA groups. ROC analysis showed that TTP value in identifying CoNS infection from contamination was the highest with area under the curve (AUC) of 0.913, and the sensitivity and specificity were 0.827 and 0.852, respectively, at the optimal cutoff value of 23.9 hours. This was followed by IG% (AUC = 0.712), with an optimal critical value of 0.55%, and a sensitivity of 0.519 and specificity of 0.797. All the AUC values of IG#, CRP, NE%, and NLR were <0.7. A combination of TTP with IG%, CRP, and NLR improved the AUC, sensitivity, specificity, accuracy, PPV, and NPV values to 0.977, 0.922, 0.957, 91.8%, 92.2%, and 91.3%, respectively. CONCLUSIONS: TTP within 24 hours indicates likelihood of CoNS as the pathogenic agent in pediatric patient blood culture. The combination of TTP with IG% CRP and NLR might improve the diagnostic accuracy.

LncRNA CCAT1 functions as apoptosis inhibitor in podocytes via autophagy inhibition.

Podocyte apoptosis importantly contributes to various kidney diseases. Long noncoding RNAs Colon cancer-associated transcript-1 (CCAT-1) has been demonstrated for a critical role in cell proliferation. In the present study, the relationship between CCAT1 and popdocyte impairment, and the underlying mechanism was investigated. Podocytes were isolated from mice and then treated with tumor necrosis factor-α to simulate podocyte injury. After developed CCAT1 overexpression or knockdown, cell viabilities were determined with the CCK-8 assay, apoptosis was examined with Flow cytometry, the autophagy was observed by Western blot. Furthermore, phosphorylated PI3K and Akt expressions were examined. We found that after CCAT1 overexpression, the cell viability was significantly increased, apoptosis was significantly decreased, and autophagy was significantly inhibited, which was indicated by induced P62, LC3B-I and decreased LC3B-II. In addition, CCAT1 overexpression induced the levels of phosphorylated PI3K and Akt. With Rap treatment, these effects by CCAT1 were reversed. Furthermore, the results contrary to the effects by CCAT1 overexpression were presented after CCAT1 knockdown, and this was inhibited by 3-MA. Taken together, our results suggested that CCAT1 induction critically participated in apoptosis inhibition in podocytes through autophagy inhibition via increasing PI3K/Akt signaling. This might act as a promising therapeutic intervention for renal diseases associated with podocyte apoptosis.

Carbon nanotube hollow polyhedrons derived from ZIF-8@ZIF-67 coupled to electro-deposited gold nanoparticles for voltammetric determination of acetaminophen.

A comparative study was carried out on the electrochemical behavior of three carbonized zeolitic imidazolate frameworks (ZIFs) synthesized through solvothermal pyrolysis. An electrochemical sensor for acetaminophen (ACT) was subsequently developed. The sensor was made by coating the glassy carbon electrode (GCE) with cobalt-nitrogen co-doped carbon nanotube hollow polyhedron (Co-NCNHP), which was prepared from core shell ZIF-8@ZIF-67, before electrodeposition of gold nanoparticles. Due to the high specific surface area, good electrical conductivity and stability of both Co-NCNHP and the gold nanoparticles, the resultant sensor displayed excellent electrocatalytic activity towards ACT with the catalytic rate constant Kcat of 4.9 × 105 M-1 s-1, diffusion coefficient D of 1.8 × 10-6 cm2 s-1, high sensitivity of 1.75 µA µM-1 cm-2, and best at a working voltage of 0.35 V (vs. Ag/AgCl). Benefitting from the synergistic effect of both Co-NCNHP and gold nanoparticles, the modified GCE had a linear response in the 0.1 µM-250 µM ACT and detection limit of 0.05 µM (at S/N = 3). The sensor was successfully applied to quantify ACT in tablets and spiked urine samples with recoveries ranged between 96.0% and 105.2%. Graphical abstractSchematic representation of cobalt-nitrogen co-doped carbon nanotube hollow polyhedrons (Co-NCNHP) exhibiting superior electrocatalytic activity to carbonized ZIF-8 and carbonized ZIF-67. Co-NCNHP were coupled to electrodeposition gold nanoparticles to modify glassy carbon electrode for improving acetaminophen (ACT) redox.

Oral administration of recombinant Bacillus subtilis spores expressing Helicobacter pylori neutrophil-activating protein suppresses peanut allergy via up-regulation of Tregs.

BACKGROUND: Helicobacter pylori neutrophil-activating protein (NAP) is an immune modulator with anti-Th2 inflammation activity that can be used to prevent IgE-mediated allergic reactions. Cholera toxin B (CTB) is a mucosal adjuvant that can induce antigen tolerance. Bacillus subtilis spores are an ideal vehicle for the oral delivery of heterologous antigens. OBJECTIVE: We investigated the therapeutic effect of recombinant NAP B subtilis spores on peanut allergies in a mouse model. METHODS: Female C3H/HeJ mice were sensitized and challenged with peanut extract by oral administration. Before challenge, recombinant NAP and CTB-NAP (CNAP) spores were orally administered to sensitized mice for 4 weeks. Faecal peanut-specific IgA and serum-specific IgE, IgG1, and IgG2a levels were measured, and the intestinal microbiota was analysed. Mice were intraperitoneally injected with anti-CD25 antibodies for regulatory T cell (Treg) depletion to evaluate the efficacy of Tregs in preventing peanut allergy. After challenge, anaphylactic reactions, plasma histamine, Tregs, and splenocyte interleukin (IL)-10, IL-4, IL-5 and interferon-γ (IFN-γ) levels were evaluated. RESULTS: After 4 weeks of recombinant spore treatment, faecal IgA levels and serum IgG2a levels were increased, while serum IgG1 and IgE levels were reduced. Intestinal microbiota analysis revealed that CNAP spores increased the taxonomic abundance of Firmicutes at the phylum level and Clostridia at the class level. After challenge, the administration of NAP or CNAP spores to mice was found to ameliorate anaphylactic reactions and decrease plasma histamine levels. Administration of NAP or CNAP spores also enhanced IL-10 and IFN-γ secretion, and suppressed IL-4 and IL-5 secretion. The protective effect of CNAP spores was more pronounced than that of NAP spores; this therapeutic effect was lost after Treg depletion. CONCLUSIONS AND CLINICAL RELEVANCE: Recombinant NAP spores successfully suppressed Th2 inflammation via the up-regulation of Tregs; this may serve as a novel therapeutic approach for treating food allergies.

Prevalence, serotypes, and drug resistance of nontyphoidal Salmonella among paediatric patients in a tertiary hospital in Guangzhou, China, 2014-2016.

PURPOSE: Nontyphoidal Salmonella (NTS) is a common pathogen responsible for acute gastroenteritis among all ages; however, information on the prevalence, serotypes, and antibiotic susceptibility of NTS isolates is limited. We aimed to explore the characteristics of NTS isolated from paediatric patients in Guangzhou, China. METHODS: This was a retrospective study of 4586 stool culture collected at Guangzhou Women and Children's Medical Center from 2014 to 2016. RESULTS: We identified 220 (4.80%) NTS isolates in stool samples. Fourteen serotypes were identified among the 220 NTS isolates. Salmonella serotype Typhimurium was the most common serotype, representing 69.09%. The highest rate of resistance was recorded in relation to AMP (76.61%), followed by SXT (29.95%), CTX (29.93%), CHL (29.77%), CAZ (23.20%), CIP (7.51%), and CFS (7.18%). The resistance rates of NTS and serotype Typhimurium to CAZ in 2015 were significantly higher than those in 2014. The average hospitalisation duration of inpatients infected by NTS resistant to three or more clinically important agents was significantly longer than that of patients infected with NTS with less antibiotic resistance. CONCLUSION: NTS represents a major cause of paediatric gastroenteritis in Guangzhou, China, and the high level of resistance to third-generation cephalosporins coupled with increasing resistance to quinolones among isolated NTS from paediatric gastroenteritis is a serious public health concern that requires continued monitoring and rational usage of antibiotics.

Prevalence and Characterization of Staphylococcus aureus Isolated From Women and Children in Guangzhou, China.

The prevalent Staphylococcus aureus clones and antibiotic susceptibility profiles are known to change dynamically and geographically; however, recent S. aureus strains causing infections in women and children in China have not been characterized. In this study, we analyzed the molecular epidemiology and antimicrobial resistance of S. aureus isolated from patients in four centers for women and children in Guangzhou, China. In total, 131 S. aureus isolates (100 from children and 31 from women) were analyzed by spa typing, multi-locus sequence typing, virulence gene and antimicrobial resistance profiling, staphylococcal chromosomal cassette mec typing, and mutation analyses of rpoB. A total of 58 spa types, 27 sequence types (STs), and 10 clonal complexes (CCs) were identified. While CC59 (ST59-IV, 48.8%; ST338-III, 35.7%) and CC45 (ST45-IV, 100%) were the major clones (84.4%) among MRSA isolates, CC5 (ST188, 24.3%; ST1, 21.6%) and CC398 (ST398, 70%) were the major ones (70.1%) among MSSA isolates. ST338-MRSA-III mostly found in pus but hardly in respiratory tract samples while ST45-MRSA-IV was on the opposite, even though they both found in blood and cerebrospinal fluid sample frequently. Staphylococcal enterotoxin genes seb-seq-sek were strongly associated with ST59 and ST338, while sec was associated with ST45, ST121, ST22, and ST30. All ST338, ST1232, and SCCmec III isolates carried lukF/S-PV genes. A total of 80% of ST338 isolates were resistant to erythromycin, clindamycin, and tetracycline. All ST45 isolates exhibited intermediate or complete resistance to rifampicin. In total, 481 HIS/ASN mutations in rpoB were found in rifampicin-resistant or intermediate-resistant isolates. ST338-III and ST45-IV emerged as two of three major clones in MRSA isolates from women and children in Guangzhou, China, though ST59-MRSA-IV remained the most prevalent MRSA clone. Clonal distribution of S. aureus varied, depending on the specimen source. Virulence genes and antibiograms were closely associated with the clonal lineage. These results clarified the molecular epidemiology of S. aureus from women and children in Guangzhou, China, and provide critical information for the control and treatment of S. aureus infections.

The recombinant fusion protein of cholera toxin B and neutrophil-activating protein expressed on Bacillus subtilis spore surface suppresses allergic inflammation in mice.

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) has been identified as a modulator with anti-Th2 inflammation activity, and cholera toxin B (CTB) is a mucosal adjuvant that can also induce antigen tolerance. In this study, we constructed a CTB-NAP fusion protein on the surface of Bacillus subtilis spore and evaluate the efficiency of oral administration of the recombinant CTB-NAP spores in preventing asthma in mice. Oral administration of recombinant CTB or CTB-NAP spores significantly decreased serum ovalbumin (OVA)-specific IgE (p < 0.001) and increased fecal IgA (p < 0.01) compared to the treatment with non-recombinant spores. Oral administration of recombinant CTB or CTB-NAP spores induced IL-10 and IFN-γ expression and reduced IL-4 levels in bronchoalveolar lavage fluid (BALF). Moreover, CTB and CTB-NAP spores reduced the eosinophils in BALF and inflammatory cell infiltration in the lungs. Furthermore, CD4+CD25+Foxp3+ Tregs in splenocytes were significantly increased in mice treated with recombinant CTB or CTB-NAP spores. The number of CD4+CD25+Foxp3+ Tregs caused by CTB-NAP was higher than that by CTB alone. Our study indicated that B. subtilis spores with surface expression of subunit CTB or CTB-NAP could inhibit OVA-induced allergic inflammation in mice. The attenuated inflammation was attributed to the induction of CD4+CD25+Foxp3+ Tregs and IgA. Moreover, the fusion protein CTB-NAP demonstrated a better efficiency than CTB alone in inhibiting the inflammation.

Inverse relationship between toxic shock syndrome toxin-1 antibodies and interferon-γ and interleukin-6 in peripheral blood mononuclear cells from patients with pediatric tonsillitis caused by Staphylococcus aureus.

INTRODUCTION: Pediatric tonsillitis is frequently caused by Staphylococcus aureus, which is the most common pathogen that causes serious pyogenic infections in humans and endangers human health. S. aureus produces numerous potent virulence factors that play a critical role in the pathogenesis of the infection caused by this bacterium, and one of the most important toxins produced by S. aureus is toxic shock syndrome toxin-1 (TSST-1). The aim of this study is to investigate the first time the levels of IFN-γ and interleukin IL-6 in TSST-1-stimulated PBMCs from pediatric tonsillitis patients and the correlation of these cytokine levels with TSST-1-specific IgG in serum. METHODS: TSST-1 gene of S. aureus was cloned and expressed in a prokaryotic expression system, and purified recombinant TSST-1 protein was used for measuring TSST-1-specific antibodies in the serum of patients with pediatric tonsillitis caused by S. aureus. Moreover, the levels of interferon (IFN)-γ and interleukin (IL)-6 in TSST-1-stimulated peripheral blood mononuclear cells (PBMCs) from pediatric tonsillitis patients were investigated. RESULTS: In patients with pediatric tonsillitis caused by S. aureus, significantly higher levels of serum TSST-1-specific IgG (P < 0.05) and IgG1 (P < 0.05) were detected than in healthy children. Moreover, PBMCs from the patients exhibited higher IFN-γ (P < 0.05) production in response to TSST-1 than did PBMCs from healthy children. In patients with pediatric tonsillitis caused by S. aureus, the positive rate of TSST-1-specific IgG was 70%, and the patients who tested negative for TSST-1-specific IgG exhibited significantly higher levels of IFN-γ (P < 0.05) and IL-6 (P < 0.05) than did the IgG-positive patients, in accord, the levels of TSST-1-specific IgG correlated inversely with the levels of IFN-γ and IL-6 in patients PBMCs stimulated with TSST-1. CONCLUSIONS: TSST-1 induces humoral and cellular immunity in pediatric tonsillitis caused by S. aureus, which suggests that TSST-1 may play an important role in the pathogenesis of pediatric tonsillitis.

Systemic and mucosal pre-administration of recombinant Helicobacter pylori neutrophil-activating protein prevents ovalbumin-induced allergic asthma in mice.

PURPOSE: Previous epidemiologic studies have demonstrated an inverse association between Helicobacter pylori infection and the frequency of allergic asthma. The neutrophil-activating protein (NAP) of H. pylori has been identified as a modulator possessing anti-Th2 inflammation activity. Here, we sought to determine whether systemic or mucosal pre-administration of recombinant H. pylori NAP (rNAP) could prevent ovalbumin (OVA)-induced allergic asthma in mice. METHODS: Mice were exposed to purified rNAP through intraperitoneal injection or inhalation and then sensitized with OVA. Following a challenge with aerosolized OVA, the bronchoalveolar lavage fluid (BALF) cell count, lung tissue histology, BALF cytokines and serum IgE were evaluated. RESULTS: Both intraperitoneal injection and inhalation of rNAP prior to OVA sensitization significantly reduced eosinophil accumulation and inflammatory infiltration in lung tissue in OVA-induced asthma mice; eosinophils were reduced in the BALF of rNAP-treated mice. In addition, IL-4 and IL-13 levels were lower (P < 0.01), IL-10 and IFN-γ levels were higher (P < 0.01) and IgE serum levels were lower (P < 0.01) in the treated groups compared to the control group. CONCLUSIONS: Systemic and mucosal pre-administration of rNAP could suppress the development of OVA-induced asthma in mice; rNAP may be utilized as part of novel strategies for the prevention or treatment of allergic diseases.

Recombinant Bacillus subtilis spores expressing cholera toxin B subunit and Helicobacter pylori urease B confer protection against H. pylori in mice.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The limitations of current therapies for H. pylori infection include poor compliance and antibiotic resistance. Therefore, an effective anti-H. pylori vaccine would be an alternative or complement to antibiotic treatment. Urease B (UreB) is considered an ideal vaccine antigen against H. pylori infection. In this study, cholera toxin B subunit (CTB), a mucosal adjuvant, was used to enhance the immunogenicity of a novel Bacillus subtilis spore vaccine expressing CTB-UreB, along with the B. subtilis spore coat protein CotC as a fusion protein. Oral administration of B. subtilis spores expressing CotC-UreB or CotC-CTB-UreB led to increased levels of UreB-specific IgG in serum and UreB-specific IgA in faeces, as well as elevated levels of IL-10 and IFN-γ in splenocytes. In addition, oral administration of CotC-UreB or CotC-CTB-UreB spores induced significant reductions (80.0 and 90.5 %, respectively) in gastric H. pylori bacterial load (1.11±0.36×105 and 0.53±0.21×105 c.f.u., respectively) compared to that of the CotC control group (5.56±1.64×105 c.f.u., P<0.01). Moreover, CotC-CTB-UreB spores were significantly more effective at reducing the bacterial load than CotC-UreB spores (P<0.05). These results indicate that CotC-CTB-UreB-expressing B. subtilis spores are a potential vaccine candidate for the control of H. pylori infection.

Study on heavy metal levels and its health risk assessment in some edible fishes from Nansi Lake, China.

Eight heavy metals, namely Cu, Zn, Fe, Mn, Cd, Ni, Pb, and As in the muscles of nine fish species collected from Nansi Lake, China. were determined, and the potential health risks to local residents via consumption of the fishes were estimated. The results of two-way ANOVA that showed the concentrations of heavy metals in the investigated fish samples were influenced significantly by fish species and sampling sites. Correlation analysis indicated that sampling sites had significant effects on the levels of correlation coefficients among different heavy metal concentrations. Interestingly, although none of the hazard quotient (HQ) values of any individual element was greater than 1 for the investigated exposure population through fish consumption, the hazard index (HI) values were more than 1 for local fishermen, suggesting that local fishermen may be experiencing some adverse health effects. Among the investigated nine fish species, Cyprinus carpio had the highest HQ and HI. As, Pb, and Cd were the most concerning heavy metals in the investigated fish samples due to their higher relative contributions to the HI values.