Prognostic value of serum soluble interleukin-23 receptor and related T-helper 17 cell cytokines in non-small cell lung carcinoma.

The signaling of interleukin (IL)-23 and its receptor (IL-23R) play a crucial role in the development of cancers. However, the clinical significance of human serum soluble IL-23R (sIL-23R) and its relationship with IL-23 are still not explored in non-small cell lung cancer (NSCLC). In our study, sIL-23R was first identified in the serum of NSCLC patients, but not in healthy controls, by proteomics. The IL-23R mRNA and protein were upregulated in NSCLC cell lines and tissues tested by quantitative PCR, western blot analysis and immunohistochemistry. The levels of sIL-23R, IL-23, and IL-17 in 195 NSCLC patients' serum were determined by ELISA, and high levels of sIL-23R were significantly associated with advanced N stage (P = .039), clinical stage (P = .007), and poor 5-year survival rate. In vitro, sIL-23R was shown binding to IL-23 and the balance could affect patients' N and T stage, overall survival, and downstream cytokine IL-17 in a potential antagonistic relationship. Although sIL-23R, IL-23, and IL-17 were all associated with poor prognosis, only the sIL-23R/IL-23 ratio (hazard ratio, 1.945; 95% confidence interval, 1.147-3.299; P = .014) was found to be an independent factor for prognosis. Therefore, we identified fragments of soluble cytokine receptor of IL-23R with affinity ability to its natural ligand IL-23 in NSCLC patients' serum. The balance between the 2 antagonists can work as a potential prognostic serum marker.

Analysis on the virulomes and resistomes of multi-drug resistance clinical Escherichia coli isolates, as well as the interactome with gut microbiome.

Escherichia coli is one of the most diverse microbial species. Pathogenic E. coli is capable of causing various diseases in humans, including several types of diarrhea, urinary tract infections, sepsis, and meningitis. This study focused on the antibiotic susceptibility profile and genomic analysis of a clinical E. coli Guangzhou-Eco330 isolated from a hospitalized 8-year-old female patient suffered from pulmonary infection in 2017. Susceptibility to 15 antibiotics were determined using Vitek2™ Automated Susceptibility System and Etest strips and interpreted based on CLSI guidelines. The genome was sequenced using Illumina Hiseq 2500 platform and assembled de novo using Velvet, followed by bioinformatics analysis. The genome has a length of 5,132,642 bp and contains 4989 predicted genes with an average GC content of 50.51%. The carriage of rfbE gene suggested the strain belonging to O157. In the genome, 70 non-coding RNAs, 50 repeat sequences, 18 transposons, 78 GIs, 9 CRISPRs, and 3 large prophages were identified. 37 PHI related genes and 108 virulence genes were determined to contribute to its pathogenicity. Specifically, the acquisition of multiple antibiotic resistance genes including blaCTX-M-55, blaOXA-10, blaCMY-48, tetB, and qnrS1 contributed to its resistance to penicillins, telracyclines, cephalosporin, and quinolones. The understanding of the genome may aid in further study on the clinical control of multi-drug resistance E. coli.

Resistome and virulome study on pathogenic Streptococcus agalactiae Guangzhou-SAG036.

Streptococcus agalactiae is considered as a leading case of bacterial infection among neonates. Although relative protection strategies have been performed in many high-income countries, resulting in a massive reduction in the occurrences of early-onset GBS disease, the late-onset disease has not affected. Here, the whole genome of S. agalactiae Guangzhou-SAG036 was sequenced by the Pacific Biosciences Sequel using the P4-C2 chemistry and the continuous long reads were used for de novo assembly using HGAP. Besides, genes prediction and multiply annotation were performed by comparing it with diverse databases. The whole genome has a length of 2,206,504 bp and contains 2162 predicted genes with an average G + C content of 35.85%. Based on the whole genome sequence, 2 large prophages, 20 virulence factors genes, and 8 antibiotic resistant genes were identified. MLST analysis revealed S. agalactiae Guangzhou-SAG036 was identified as ST-17. The virulence factors genes were identified with different functions including adherence, antiphagocytosis, spreading factor, immune evasion, invasion, toxin. Besides, the antibiotic-resistant genes may provide S. agalactiae with resistance to multi-drugs including erythromycin, streptomycin, azithromycin, spiramycin, ampicillin, kanamycin, cationic peptides, and tetracycline. Therefore, the infection of S. agalactiae Guangzhou-SAG036 ST-17 strain maybe caused by the complex virulence factors and multi-drugs resistance. These results contribute to further understand GBS epidemiology and surveillance targets.

Genomic analysis of a hop-resistance Lactobacillus brevis strain responsible for food spoilage and capable of entering into the VBNC state.

BACKGROUND: Lactobacillus brevis is a major contaminant of spoiled beer. And it was able to enter VBNC state and cause false negative detection, which poses a major challenge to the brewing industry. METHODS: The genomic DNA of L. brevis BM-LB13908 was extracted and purified to form a sequencing library that meets the quality requirements and was sequenced. The sequencing results were then screened and assembled to obtain the entire genome sequence of L. brevis. Predicted genes were annotated by GO database, KEGG pathway database and COG functional classification system. RESULTS: The final assembly yielded 275 scaffolds of a total length of 2 840 080 bp with a G + C content of 53.35%. There were 2357, 701, 1519 predicted genes with corresponding GO functional, COG functional, and KEGG biological pathway annotations, respectively. The genome of L. brevis BM-LB13908 contains hop resistance gene horA and multiple genes related to the formation of VBNC state. CONCLUSIONS: This report describes the draft genome sequence of L. brevis BM-LB13908, a spoilage strain isolated from finished beer sample. This study may support further study on L. brevis and other beer spoilage bacteria, and prevent and control beer spoilage caused by microorganisms.

High flux isothermal assays on pathogenic, virulent and toxic genetics from various pathogens.

Toxins, encoding by virulence factors, are significant cause of food-borne illnesses and death in the worldwide. Loop-mediated isothermal amplification (LAMP) is one of the widely used methodologies because of the high sensitivity, specificity and rapidity. Nowadays, LAMP has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in identification of the pathogenic virulent and toxic genetics. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming and sample preparation. Therefore, we review the typical characteristics of LAMP assay, recent advance in detection of virulence factors and the application of LAMP assay on detection of four commonly virulence factors. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of identification the virulence factors. Moreover, the main purpose of this review is to provide theory support for the application of LAMP assay on the virulence factors identification.

Verification and application of a modified carbapenem inactivation method (mCIM) on Pseudomonas aeruginosa: a potential screening methodology on carbapenemases phenotype in Bacillus cereus.

Antimicrobial resistance (AMR) has been a leading issue for human health globally threatening the achievement of several of the Sustainable Development Goals (SDGs). Originated from Bacillus cereus, carbapenemases phenotype has been considered to be a major concern in AMR. In this study, the AMR identification rate of P. aeruginosa isolates and infections in FAHJU showed an obvious upward trend from 2012 to 2016. All 88 carbapenem-resistant P. aeruginosa strains were screened for carbapenemase phenotype by modified Carbapenem Inactivation Method (mCIM), and these results of mCIM were compared with traditional PCR results. The isolates of P. aeruginosa and infected patients showed obvious upward trend from 2012 to 2016. The drug resistance to common clinical antibiotics was serious that the clinical rational use of antibiotics should be strengthened, which is in accordance with the Global Antimicrobial Resistance and Use Surveillance System (GLASS) report. In comparison, the results of mCIM showed that 18 out of 88 CRPA strains were carbapenemase positive, which were completely consistent with the results yielded by PCR method. Therefore, it is convinced that this mCIM methodology is a simple and quick method for detected carbapenemases producing P. aeruginosa and has a potential capability in carbapenemases phenotype of pathogen like B. cereus, which will undoubtedly aid in the AMR therapy.

Establishment and application of a rapid visual detection method for Listeria monocytogenes based on polymerase spiral reaction (PSR).

Listeria monocytogenes is a common foodborne pathogen that presents in various food products, posing important threat to public health. The aim of this study was to establish a rapid and sensitive method with visualization to detect L. monocytogenes based on polymerase spiral reaction (PSR). Primers targeting conserved hlyA gene sequence of L. monocytogenes were designed based on bioinformatics analyses on the current available L. monocytogenes genomes. The isothermal amplification PSR can be completed under constant temperature (65ᵒC) within 60 min with high specificity and sensitivity. Twenty-five reference strains were used to evaluate the specificity of the developed reaction. The results showed that the sensitive of the reaction for L. monocytogenes in purified genomic DNA and artificially contaminated food samples were 41 pg/µL and 103 CFU/mL, respectively. It was 100-fold more sensitive than conventional PCR. In conclusion, this novel PSR method is rapid, cost-efficient, timesaving, and applicable on artificially contaminated food samples, providing broad prospects into the detection of foodborne microbes with the promising on-site inspection.

Reduction, Prevention, and Control of Salmonella enterica Viable but Non-culturable Cells in Flour Food.

The processing and storage conditions of flour food inevitably pose environmental stress, which promote bacteria to enter a viable but non-culturable (VBNC) state. The existence of VBNC cells causes false-negative detection in traditional culture-based detection methods, resulting in food quality and safety issues. This study aimed at investigating the influence factors including nutrition, acid, salt, and temperature for the entry into a VBNC state of Salmonella enterica and an efficient detection method. During induction with multi-stress conditions, nutrition starvation antagonizes with low-level acidity. Besides, high-level acidity was considered as an inhibitor for VBNC induction. Four inducers including nutrition starvation, salt stress, low-level acidity, and low temperature were concluded for a VBNC state. In addition, the keynote conditions for S. enterica entering a VBNC state included (i) nutrient-rich acidic environment, (ii) oligotrophic low-acidity environment, and (iii) oligotrophic refrigerated environment. Based on the keynote conditions, the environmental conditions of high acidity (1.0% v/v acetate) with low temperature (-20°C) could successfully eliminate the formation of S. enterica VBNC cells in flour food. In addition, combining with propidium monoazide pretreatment, PCR technology was applied to detect S. enterica VBNC cells. The sensitivity of the PMA-PCR technology was 105 CFU/ml in an artificially simulated food system. The results derived from this study might aid in the detection and control of VBNC state S. enterica in flour food products.

Development and Application of a Simple "Easy To Operate" Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli.

O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.

Study on the Viable but Non-culturable (VBNC) State Formation of Staphylococcus aureus and Its Control in Food System.

A Viable but non-culturable (VBNC) state is a bacterial survival strategy under reverse conditions. It poses a significant challenge for public health and food safety. In this study, the effect of external environmental conditions including acid, nutrition, and salt concentrations on the formation of S. aureus VBNC states at low temperatures were investigated. Different acidity and nutritional conditions were then applied to food products to control the VBNC state formation. Four different concentration levels of each factor (acid, nutrition, and salt) were selected in a total of 16 experimental groups. Nutrition showed the highest influence on the VBNC state formation S. aureus, followed by acid and salt. The addition of 1% acetic acid could directly kill S. aureus cells and inhibit the formation of the VBNC state with a nutrition concentration of 25, 50, and 100%. A propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied and considered as a rapid and sensitive method to detect S. aureus in VBNC state with the detection limit of 104 CFU/mL.

Effect of Environmental Conditions on the Formation of the Viable but Nonculturable State of Pediococcus acidilactici BM-PA17927 and Its Control and Detection in Food System.

Objective: This study aimed to investigate the effect of environmental conditions including nutrient content, acetic acid concentration, salt concentration, and temperature on the formation of viable but nonculturable (VBNC) state of Pediococcus acidilactici, as well as its control and detection in food system. Methods: Representing various environmental conditions in different food systems, 16 induction groups were designed for the formation of VBNC state of P. acidilactici. Traditional plate counting was applied to measure the culturable cell numbers, and Live/Dead Bacterial Viability Kit combined with fluorescent microscopy was used to identify viable cells numbers. The inhibition of bacterial growth and VBNC state formation by adjusting the environmental conditions were investigated, and the clearance effect of VBNC cells in crystal cake system was studied. In addition, a propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied to detect the VBNC P. acidilactici cells in crystal cake food system. Results: Among the environmental conditions included in this study, acetic acid concentration had the greatest effect on the formation of VBNC state of P. acidilactici, followed by nutritional conditions and salt concentration. Reducing nutrients in the environment and treating with 1.0% acetic acid can inhibit P. acidilactici from entering the VBNC state. In the crystal cake system, the growth of P. acidilactici and the formation of VBNC state can be inhibited by adding 1.0% acetic acid and storing at -20°C. In crystal cake system, the PMA-PCR assay can be used to detect VBNC P. acidilactici cells at a concentration higher than 104 cells/ml. Conclusion: The VBNC state of P. acidilactici can be influenced by the changing of environmental conditions, and PMA-PCR assay can be applied in food system for the detection of VBNC P. acidilactici cells.

Spoilage Lactic Acid Bacteria in the Brewing Industry.

Lactic acid bacteria (LAB) have caused many microbiological incidents in the brewing industry, resulting in severe economic loss. Meanwhile, traditional culturing method for detecting LAB are time-consuming for brewers. The present review introduces LAB as spoilage microbes in daily life, with focus on LAB in the brewing industry, targeting at the spoilage mechanism of LAB in brewing industry including the special metabolisms, the exist of the viable but nonculturable (VBNC) state and the hop resistance. At the same time, this review compares the traditional and novel rapid detection methods for these microorganisms which may provide innovative control and detection strategies for preventing alcoholic beverage spoilage, such as improvement of microbiological quality control using advanced culture media or different isothermal amplification methods.