Antimicrobial susceptibility testing of Enterobacteriaceae: determination of disk content and Kirby-Bauer breakpoint for ceftazidime/avibactam.

BACKGROUND: Detection of ceftazidime/avibactam (CAZ/AVI) antibacterial activity is absolutely vital with the rapid growth of carbapenem resistant Enterobacteriaceae (CRE). But now, there is no available automated antimicrobial susceptibility testing card for CAZ/AVI, so Kirby-Bauer has become an economical and practical method for detecting CAZ/AVI antibacterial activity against Enterobacteriaceae. RESULT: In this study, antimicrobial susceptibility testing of CAZ/AVI against 386 Enterobacteriaceae (188 Klebsiella pneumoniae, 122 Escherichia coli, 76 Enterobacter cloacae) isolated from clinical patients was performed by broth microdilution. Of the 386 strains, 54 extended spectrum ß lactamases negative (ESBL(-)), 104 extended spectrum ß lactamases positive (ESBL(+)), 228 CRE. 287 isolates were susceptible to CAZ/AVI and 99 isolates were resistant to CAZ/AVI. At the same time, to obtain optimal content avibactam (AVI) disk containing ceftazidime (30 µg), inhibition zone diameter of four kinds of ceftazidime (30 µg) disk containing different AVI content (0 µg, 10 µg, 25 µg, 50 µg) were tested by Kirby-Bauer method. The microdilution broth method interpretation was used as the standard to estimate susceptible or resistance and then coherence analysis was carried out between Kirby-Bauer and broth microdilution. The result shows the inhibition zone diameter of 30 µg/50 µg disk, susceptible isolates: 20.5 mm-31.5 mm, resistance isolates: 8.25 mm-21.5 mm. The inhibition zone diameter of 30 µg/25 µg disk, susceptible isolates: 19.7 mm-31.3 mm, resistance isolates: 6.5 mm-19.2 mm. The inhibition zone diameter of 30 µg/10 µg disk, susceptible isolates: 19.5 mm-31 mm, resistance isolates: 6.5 mm-11 mm. The inhibition zone diameter of ceftazidime (30 µg), susceptible isolates: 6.5 mm-27.5 mm, resistance isolates 6.5 mm. CONCLUSION: Our results show that 30 µg/50 µg, 30 µg/25 µg, 30 µg/10 µg CAZ/AVI disk have significant statistical differences to determinate CAZ/AVI antibacterial activity, but for 30 µg/50 µg disk, there has a cross section between susceptible isolates (minimum 20.5 mm) and resistance isolates (maximum 21.5 mm). For 30 µg/25 µg disk, it is hard to distinguish the difference between susceptible isolates (minimum 19.7 mm) and resistance isolates (maximum 19.2 mm), so 30 µg/10 µg CAZ/AVI disk is more conducive to determinate antibacterial activity.

Effective inhibition of melanoma tumorigenesis and growth via a new complex vaccine based on NY-ESO-1-alum-polysaccharide-HH2.

BACKGROUND: A safe and effective adjuvant plays an important role in the development of a vaccine. However, adjuvants licensed for administration in humans remain limited. Here, for the first time, we developed a novel combination adjuvant alum-polysaccharide-HH2 (APH) with potent immunomodulating activities, consisting of alum, polysaccharide of Escherichia coli and the synthetic cationic innate defense regulator peptide HH2. METHODS: The adjuvant effects of APH were examined using NY-ESO-1 protein-based vaccines in prophylactic and therapeutic models. We further determined the immunogenicity and anti-tumor effect of NY-ESO-1-APH (NAPH) vaccine using adoptive cellular/serum therapy in C57/B6 and nude mice. Cell-mediated and antibody-mediated immune responses were evaluated. RESULTS: The APH complex significantly promoted antigen uptake, maturation and cross-presentation of dendritic cells and enhanced the secretion of TNF-α, MCP-1 and IFN-γ by human peripheral blood mononuclear cells compared with individual components. Vaccination of NAPH resulted in significant tumor regression or delayed tumor progression in prophylactic and therapeutic models. In addition, passive serum/cellular therapy potently inhibited tumor growth of NY-ESO-1-B16. Mice treated with NAPH vaccine produced higher antibody titers and greater antibody-dependent/independent cellular cytotoxicity. Therefore, NAPH vaccination effectively stimulated innate immunity, and boosted both arms of the adaptive humoral and cellular immune responses to suppress tumorigenesis and growth of melanoma. CONCLUSIONS: Our study revealed the potential application of APH complex as a novel immunomodulatory agent for vaccines against tumor refractory and growth.

In vitro and in vivo activities of antimicrobial peptides developed using an amino acid-based activity prediction method.

To design and discover new antimicrobial peptides (AMPs) with high levels of antimicrobial activity, a number of machine-learning methods and prediction methods have been developed. Here, we present a new prediction method that can identify novel AMPs that are highly similar in sequence to known peptides but offer improved antimicrobial activity along with lower host cytotoxicity. Using previously generated AMP amino acid substitution data, we developed an amino acid activity contribution matrix that contained an activity contribution value for each amino acid in each position of the model peptide. A series of AMPs were designed with this method. After evaluating the antimicrobial activities of these novel AMPs against both Gram-positive and Gram-negative bacterial strains, DP7 was chosen for further analysis. Compared to the parent peptide HH2, this novel AMP showed broad-spectrum, improved antimicrobial activity, and in a cytotoxicity assay it showed lower toxicity against human cells. The in vivo antimicrobial activity of DP7 was tested in a Staphylococcus aureus infection murine model. When inoculated and treated via intraperitoneal injection, DP7 reduced the bacterial load in the peritoneal lavage solution. Electron microscope imaging and the results indicated disruption of the S. aureus outer membrane by DP7. Our new prediction method can therefore be employed to identify AMPs possessing minor amino acid differences with improved antimicrobial activities, potentially increasing the therapeutic agents available to combat multidrug-resistant infections.

Identification of a necroptosis-related gene signature for making clinical predictions of the survival of patients with lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) is a major pathological subtype of malignant lung cancer with a poor prognosis. Necroptosis is a caspase-independent programmed cell death mode that plays a pivotal role in cancer oncogenesis and metastasis. Here, we explore the prognostic values of different necroptosis-related genes (NRGs) in LUAD. Methods: mRNA expression data and related clinical information for LUAD samples were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases. NRGs were identified using the GeneCards database. Least absolute shrinkage and selection operator Cox regression and multivariate Cox analysis were used to construct a prognostic risk model. Time-dependent receiver-operating characteristic curves and a nomogram were constructed to validate the predictive values of the prognostic signatures. A necroptosis-related protein-protein interaction network was visualised using the STRING database and Cytoscape software. Functional analyses, including Gene Ontology, Kyoto Encyclopaedia of Genes and Genomes pathway enrichment, gene set enrichment, and gene set variation analyses, were conducted to explore the underlying molecular mechanisms. Finally, the mRNA expression of the prognostic signatures in LUAD cell lines was assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Results: A prognostic model was established for eight NRGs (CALM1, DDX17, FPR1, OGT, PGLYRP1, PRDX1, TUFM, and CPSF3) based on TCGA-cohort data and validated with the GSE68465 cohort. Patients with low-risk scores had better survival outcomes than those with high-risk scores (p = 0.00013). The nomogram was used to predict the prognosis of patients with LUAD. The prediction curves for 1-, 3-, and 5-year OS showed good predictive performance and the accuracy of the nomograms increased over time. RT-qPCR results demonstrated that these eight genes, especially CALM1, PRDX1, and PGLYRP1, were differentially expressed in LUAD cells. Conclusion: We constructed a reliable eight-NRG signature that provides new insights for guiding clinical practice in the prognosis and treatment of LUAD.

Candida tropicalis distribution and drug resistance is correlated with ERG11 and UPC2 expression.

BACKGROUND: Candida tropicalis (C. tropicalis) is an important opportunistic pathogenic Candida species that can cause nosocomial infection. In this study, we analyzed the distribution and drug susceptibility of C. tropicalis and the relationship between ERG11 and UPC2 expression and resistance to azole antifungal agents. METHODS: C. tropicalis was cultured and identified by Sabouraud Agar Medium, CHROM Agar Candida and ATB tests (Bio-Mérieux, France). Total RNA was extracted from the collected strains, and the ERG11 and UPC2 mRNA expression levels were analyzed by quantitative real-time PCR. RESULTS: In total, 2872 clinical isolates of Candida, including 319 strains of C. tropicalis, were analyzed herein; they were mainly obtained from the Departments of Respiratory Medicine and ICU. The strains were predominantly isolated from airway secretion samples, and the detection trend in four years was mainly related to the type of department and specimens. The resistance rates of C. tropicalis to fluconazole, itraconazole and voriconazole had been increasing year by year. The mRNA expression levels of ERG11 and UPC2 in the fluconazole-resistant group were significantly higher than they were in the susceptible group. In addition, there was a significant positive linear correlation between these two genes in the fluconazole-resistant group. CONCLUSIONS: Overexpression of the ERG11 and UPC2 genes in C. tropicalis could increase resistance to azole antifungal drugs. The routine testing for ERG11 and UPC2 in high-risk patients in key departments would provide a theoretical basis for the rational application of azole antifungal drugs.

H2O2-Inactivated Salmonella typhimurium RE88 Strain as a New Cancer Vaccine Carrier: Evaluation in a Mouse Model of Cancer.

PURPOSE: This study aimed to describe a novel cancer vaccine developed using H2O2-inactivated Salmonella typhimurium RE88 [with deletions of AroA (the first enzyme in the aromatic amino acid biosynthesis pathway) and DNA adenine methylase] as the carrier. METHODS: The pVLT33 plasmid was used to engineer an RE88 strain induced to express ovalbumin (OVA) by isopropylthiogalactoside (RE88-pVLT33-OVA). The immune responses and anticancer effects of H2O2-inactivated RE88-pVLT33-OVA were compared with those of non-inactivated RE88-pVLT33-OVA and OVA (positive control) in mice carrying OVA-expressing tumors (EG7-OVA) cells. RESULTS: Anti-ovalbumin IgG (immunoglobulin G) titer following vaccination with H2O2-inactivated RE88-pVLT33-OVA was higher for subcutaneous than for intragastric vaccination. When subcutaneous administration was used, H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU (colony forming units)/mouse) achieved an anti-ovalbumin IgG titer higher than that for the same dose of RE88-pVLT33-OVA and comparable to that for 10 µg ovalbumin (positive control). The binding of mouse serum antibodies to EG7-OVA cells was stronger for H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU/mouse) than for 10 µg ovalbumin. Furthermore, subcutaneous vaccination with H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU/mouse) induced greater activation of splenic T cells and more extensive tumor infiltration with CD4+/CD8+ T cells compared with 10 µg ovalbumin (positive control). The mice vaccinated subcutaneously with H2O2-inactivated RE88-pVLT33-OVA at a dose of 2 × 108 or 6 × 108 CFU/mouse had smaller tumors compared with mice in the negative control groups. Tumor weight in mice vaccinated with H2O2-inactivated RE88-pVLT33-OVA at a dose of 2 × 109 CFU/mouse was significantly lower than that in both negative control groups (P < 0.05) and decreased with the increasing dose of H2O2-inactivated RE88-pVLT33-OVA. H2O2-inactivated RE88-pVLT33-OVA was potentially safer than the non-inactivated strain, could carry exogenous antigens, and had specific epitopes that could be exploited as natural adjuvants to facilitate the induction of cellular and humoral immune responses. CONCLUSION: It was anticipated that H2O2-inactivated RE88-pVLT33-OVA could be used as a novel delivery system for new cancer vaccines.

Elevating NagZ Improves Resistance to ß-Lactam Antibiotics via Promoting AmpC ß-Lactamase in Enterobacter cloacae.

Enterobacter cloacae complex (ECC), one of the most common opportunistic pathogens causing multiple infections in human, is resistant to ß-lactam antibiotics mainly due to its highly expressed chromosomal AmpC ß-lactamase. It seems that regulation of chromosomal AmpC ß-lactamase is associated with peptidoglycan recycling. However, underlying mechanisms are still poorly understood. In this study, we confirmed that NagZ, a glycoside hydrolase participating in peptidoglycan recycling in Gram-negative bacteria, plays a crucial role in developing resistance of E. cloacae (EC) to ß-lactam antibiotics by promoting expression of chromosomal AmpC ß-lactamase. Our data shows that NagZ was significantly up-regulated in resistant EC (resistant to at least one type of the third or fourth generation cephalosporins) compared to susceptible EC (susceptible to all types of the third and fourth generation cephalosporins). Similarly, the expression and ß-lactamase activity of ampC were markedly enhanced in resistant EC. Moreover, ectopic expression of nagZ enhanced ampC expression and resistance to ß-lactam antibiotics in susceptible EC. To further understand functions of NagZ in ß-lactam resistance, nagZ-knockout EC model (ΔnagZ EC) was constructed by homologous recombination. Conversely, ampC mRNA and protein levels were down-regulated, and resistance to ß-lactam antibiotics was attenuated in ΔnagZ EC, while specific complementation of nagZ was able to rescue ampC expression and resistance in ΔnagZ EC. More interestingly, NagZ and its hydrolyzates 1,6-anhydromuropeptides (anhMurNAc) could induce the expression of other target genes of AmpR (a global transcriptional factor), which suggested that the promotion of AmpC by NagZ is mediated AmpR activated by anhMurNAc in EC. In conclusion, these findings provide new elements for a better understanding of resistance in EC, which is crucial for the identification of novel potential drug targets.

Hydrogen peroxide-inactivated bacteria induces potent humoral and cellular immune responses and releases nucleic acids.

The problem of nosocomial infection is seriously escalating. Bacterial vaccines are indispensable for preventing infections caused by multi-drug resistant organisms. Some researchers have put forward the use of hydrogen peroxide (H2O2) as a new technology platform for virus deactivation. This deactivated virus can induce the number of CD8+ T lymphocytes, which can enhance antiviral responses. Although, H2O2 treatment has been rarely reported on the exploration of bacterial deactivation, H2O2 deactivation of whole-cell bacteria could be a potential novel approach for bacterial vaccine development. Here we present a strategy for H2O2-deactivated bacterial whole-cell vaccines, for two major pathgens, Pseudomonas aeruginosa and Staphylococcus aureus. The proactive effects of vaccination were assessed in vitro and in vivo. H2O2-deactivation of bacterial vaccines retains more complete epitopes and exhibits lower toxicity, as compared to formaldehyde, a conventional deactivator that was investigated in this study. Furthermore, H2O2-deactivated bacterial vaccines induce anti-infection responses through enhancement of humoral immunity and cellular immunity. Vaccination with H2O2-deactivated whole-cell bacteria in mice mainly elicits whole-cell specific antibody titers and balances the IgG2a and IgG1 response, predominantly with IgG3 induction at the later stages, meanwhile provides opsonic protection against challenge with pathogens. Finally, H2O2 deactivation of bacteria has been found to cause the release of bacterial DNA which is followed by NF-κB activation. These findings demonstrate that the deactivation of whole-cell bacteria with H2O2 is potentially advantageous for immune responses. Considering the prevention of drug-resistant infections, this deactivation method could be simultaneously applied as an innovative strategy for bacterial vaccine development.

Induction and Amelioration of Methotrexate-Induced Gastrointestinal Toxicity are Related to Immune Response and Gut Microbiota.

As a widely used anticancer and immunosuppressive agent, methotrexate (MTX) can induce multiple adverse drug reactions (ADRs), such as gastrointestinal toxicity, the mechanisms are poorly understood. Gut microbiota has been widely reported to be associated with the onset of multiple diseases as well as treatment outcomes of different drugs. In this study, mucosal injury was observed in MTX-treated mice, leading to significant changes in macrophages (i.e., M1/M2 ratio, P < 0.05) but not in dendritic cells. Moreover, the population, diversity and principal components of the gut microbiota in mice were dramatically altered after MTX treatment in a time-dependent manner, and Bacteroidales exhibited the most distinct variation among all the taxa (P < 0.05). Bacteroides fragilis was significantly decreased with MTX treatment (P < 0.01) and tended to decrease proportionately with increasing macrophage density. Gavage of mice with B. fragilis ameliorated MTX-induced inflammatory reactions and modulate macrophage polarization. In conclusion, our results delineate a strong impact of the gut microbiota on MTX-induced intestinal mucositis and provide a potential method for the prevention of such ADRs.

The novel complex combination of alum, CpG ODN and HH2 as adjuvant in cancer vaccine effectively suppresses tumor growth in vivo.

Single-component adjuvant is prone to eliciting a specific type of Th1 or Th2 response. So, the development of combinatorial adjuvants inducing a robust mixed Th1/Th2 response is a promising vaccination strategy against cancer. Here, we describe a novel combination of aluminum salts (alum), CpG oligodeoxynucleotide (CpG) and innate defense regulator peptide HH2 for improving anti-tumor immune responses. The CpG-HH2 complex significantly enhanced the production of IFN-γ, TNF-α and IL-1ß, promoted the uptake of antigen and strengthened the activation of p38, Erk1/2 and NF-κB in vitro, compared to CpG or HH2 alone. Immunization with NY-ESO-1 antigen plus alum-CpG-HH2 combinatorial adjuvant effectively inhibited tumor growth and reduced tumor burden in prophylactic and therapeutic tumor models and even in passive serum or cellular therapy. In addition, co-administration of NY-ESO-1 with alum-CpG-HH2 combinatorial adjuvant markedly activated NK cell cytotoxicity, induced antibody-dependent cellular cytotoxicity (ADCC), dramatically elicited cytotoxic T lymphocytes (CTLs) response, and increased infiltrating lymphocytes in tumors. Moreover, in vivo depletion of CD8+ T cells completely and depletion of NK cells partially blocked the anti-tumor activity of NY-ESO-1-alum-CpG-HH2 immunization. Overall, our results demonstrate a novel adjuvant combination for cancer vaccine with efficient immunomodulation by stimulating innate immunity and mediating adaptive immunity.

Synergistic effects of antimicrobial peptide DP7 combined with antibiotics against multidrug-resistant bacteria.

Antibiotic-resistant bacteria present a great threat to public health. In this study, the synergistic effects of antimicrobial peptides (AMPs) and antibiotics on several multidrug-resistant bacterial strains were studied, and their synergistic effects on azithromycin (AZT)-resistance genes were analyzed to determine the relationships between antimicrobial resistance and these synergistic effects. A checkerboard method was used to evaluate the synergistic effects of AMPs (DP7 and CLS001) and several antibiotics (gentamicin, vancomycin [VAN], AZT, and amoxicillin) on clinical bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli). The AZT-resistance genes (ermA, ermB, ermC, mefA, and msrA) were identified in the resistant strains using quantitative polymerase chain reaction. For all the clinical isolates tested that were resistant to different antibiotics, DP7 had high antimicrobial activity (≤32 mg/L). When DP7 was combined with VAN or AZT, the effect was most frequently synergistic. When we studied the resistance genes of the AZT-resistant isolates, the synergistic effect of DP7-AZT occurred most frequently in highly resistant strains or strains carrying more than two AZT-resistance genes. A transmission electron microscopic analysis of the S. aureus strain synergistically affected by DP7-AZT showed no noteworthy morphological changes, suggesting that a molecular-level mechanism plays an important role in the synergistic action of DP7-AZT. AMP DP7 plus the antibiotic AZT or VAN is more effective, especially against highly antibiotic-resistant strains.

Synthetic innate defense regulator peptide combination using CpG ODN as a novel adjuvant induces long­lasting and balanced immune responses.

Vaccines are critical tools for the prevention and treatment of several diseases. Adjuvants have been traditionally used to enhance immunity to vaccines and experimental antigens. In the present study, the adjuvant combination of CpG oligodeoxynucleotides (CpG ODN) and the innate defense regulator (IDR) peptide, IDR­HH2, was evaluated for its ability to enhance and modulate the immune response when formulated with alum and the recombinant hepatitis B surface antigen (HBsAg). The CpG­HH2 complex enhanced the secretions of tumor necrosis factor­α, monocyte chemotactic protein 1 and interferon­Î³ by human peripheral blood mononuclear cells and promoted murine bone marrow dentritic cell maturation. In addition, the present study demonstrated that IDR­HH2 was chemotactic for human neutrophils, THP­1 cells and RAW264.7 cells at concentrations between 2.5 and 40 µg/ml. The present study also observed that significantly higher anti­HBs antibody titers, which were sustained at high levels for as long as 35 weeks following the boost immunization, were induced by the combination adjuvant, even when co­administered with a commercial hepatitis B vaccine at a low antigen dose (0.1 µg HBsAg). Notably, the level of IgG2a was almost equal to the level of IgG1, indicating that a balanced T helper (Th)1/Th2 immune response was elicited by the novel vaccine, which was consistent with the ELISpot results. These data suggest that the CpG­HH2 complex may be a potential effective adjuvant, which facilitates a reduction in the dose of antigen and induces long­lasting, balanced immune responses.

Ad-endostatin treatment combined with low-dose irradiation in a murine lung cancer model.

Radiation therapy is a conventional strategy for treating advanced lung cancer yet is accompanied by serious side-effects. Its combination with other strategies, such as antiangiogenesis and gene therapy, has shown excellent prospects. As one of the potent endogenous vascular inhibitors, endostatin has been widely used in the antiangiogenic gene therapy of tumors. In the present study, LL/2 cells were infected with a recombinant adenovirus encoding endostatin (Ad-endostatin) to express endostatin. The results showed that LL/2 cells infected with the Ad-endostatin efficiently and longlastingly expressed endostatin. In order to further explore the role of Ad-endostatin combined with irradiation in the treatment of cancer, a murine lung cancer model was established and treated with Ad-endostatin combined with low-dose irradiation. The results showed that the combination treatment markedly inhibited tumor growth and metastasis, and prolonged the survival time of the tumor-bearing mice. Furthermore, this significant antitumor activity was associated with lower levels of microvessel density and anoxia factors in the Ad-Endo combined with irradiation group, and with an increased apoptotic index of tumor cells. In addition, no serious side-effects were noted in the combination group. Based on our findings, Ad-endostatin combined with low-dose irradiation may be a rational alternative treatment for lung cancer and other solid tumors.