Multiplex real-time SYBR Green I PCR assays for simultaneous detection of 15 common enteric pathogens in stool samples.

Diarrheal diseases account for more than 50% of foodborne diseases worldwide, the majority of which occur in infants and young children. The traditional bacterial detection method is complex and time-consuming; therefore, it is necessary to establish a rapid and convenient detection method that can detect multiple pathogens simultaneously. In this study, we developed a set of five multiplex real-time SYBR Green I PCR assays to simultaneously detect 15 common enteric pathogens based on the Homo-Tag Assisted Non-Dimer system. These assays effectively reduced primer-dimer formation and improved the stability, uniformity, and amplification efficiency of multiplex PCR. The detection limit of the multiplex SYBR Green I PCR system was approximately 104-106 CFU/mL for stool specimens. Furthermore, we vitrified heat-unstable components on the cap of a reaction tube, showing that Taq DNA polymerase, dNTPs, primers, and SYBR Green I remained stable at 25 °C. In summary, we developed multiplex SYBR Green I PCR assays that can simultaneously detect 15 enteric pathogens. This method is comprehensive, rapid, inexpensive, accurate, and simple and displays high specificity.

Characterization of phiCFP-1, a virulent bacteriophage specific for Citrobacter freundii.

Citrobacter freundii, a Gram-negative bacterium, causes many opportunistic infections. Bacteriophage phiCFP-1 was isolated and characterized by its ability to lyse the multidrug-resistant clinical C. freundii strain P10159. Transmission electron microscopy showed that the phage has an icosahedral head and a short tail, making it a Podoviridae family member. In a single-step growth experiment, phiCFP-1 exhibited an eclipse period of 20 min and a burst size of 100 particles per cell. Its genome assembled as a circular molecule when genomic sequencing was completed. However, based on genome content and organization, it was categorized as a classic T7-related phage, and such phages are known to have linear genomes with direct terminal repeats. With the quick and simple method established herein, the 38,625-bp linear double-stranded DNA with 229-bp direct terminal repeats was accurately identified. The genome contained 43 putative open reading frames and no tRNA genes. Using a proteomics-based approach, seven viral and two host proteins from purified phiCFP-1 particles were identified. Comparative genomics and recombination analyzes revealed close genetic relatedness among phiCFP-1, phiYeO3-12/vB_YenP_AP5 (from Yersinia enterocolitica O3), and phiSG-JL2 (from Salmonella enterica).

Metabolic shifts and structural changes in the gut microbiota upon branched-chain amino acid supplementation in middle-aged mice.

The importance of gut microbiota to health has gained extensive attention and is strongly correlated with diet. Dietary supplementation with a branched-chain amino acid-enriched mixture (BCAAem) exerts a variety of beneficial effects in mice and humans. In mice, BCAAem supplementation can promote longevity, but its influence on the gut ecosystem and the underlying mechanism remain unclear. To address this issue, BALB/C mice were fed a BCAAem-supplemented diet and their gut microbiomes were analysed by 16S rDNA sequencing. Quantitative polymerase chain reaction was performed to identify Bifidobacterium spp. in the gut, and gas chromatography-mass spectrometry was conducted for faecal-metabolite detection. The results showed that the structure of the gut microbiota changed, and BCAAem-supplementation in mice slowed the change speed of gut microbiota which is due to age. In addition, the abundance of the Akkermansia and Bifidobacterium increased in BCAAem-supplemented mice, while the ratio of Enterobacteriaceae decreased in BCAAem-supplemented mice. Moreover, 12 different metabolites, representing sugar and lipid metabolism, were altered between the supplemented and control groups. Thus, BCAAem influences the gut microbiota and gut metabolism. In addition, the BCAAem-supplemented group presented lower serum concentrations of lipopolysaccharide-binding protein. The changes are indicative of lower antigen loads in the host gut. These results suggest that dietary supplementation with BCAAem may be considered for improving health and promoting healthy aging.

A novel New Delhi metallo-ß-lactamase variant, NDM-14, isolated in a Chinese Hospital possesses increased enzymatic activity against carbapenems.

A novel New Delhi metallo-ß-lactamase (NDM) variant, NDM-14, was identified in clinical isolate Acinetobacter lwoffii JN49-1, which was recovered from an intensive care unit patient at a local hospital in China. NDM-14, which differs from other existing enzymes by an amino acid substitution at position 130 (Asp130Gly), possesses enzymatic activity toward carbapenems that is greater than that of NDM-1. Kinetic data indicate that NDM-14 has a higher affinity for imipenem and meropenem.

A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes.

Objective: Recently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting mcr genes in clinical isolates. Methods: The primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes. Results: The standard curves for both the single and multiplex systems showed good linearity (R2 > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 102 copies/µL. The specificity test showed positive amplification results only for strains containing the mcr genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect mcr genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the mcr genes were detected (seven isolates carrying mcr-1, four isolates carrying mcr-10, and one isolate carrying mcr-9). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method. Conclusion: The assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the mcr genes (mcr-1 to mcr-10), thus providing a better basis for clinical drug treatment and drug resistance research.

Fructose uptake in Bifidobacterium longum NCC2705 is mediated by an ATP-binding cassette transporter.

Recently, a putative ATP-binding cassette (ABC) transport system was identified in Bifidobacterium longum NCC2705 that is highly up-regulated during growth on fructose as the sole carbon source. Cloning and expression of the corresponding ORFs (bl0033-0036) result in efficient fructose uptake by bacteria. Sequence analysis reveals high similarity to typical ABC transport systems and suggests that these genes are organized as an operon. Expression of FruE is induced by fructose, ribose, or xylose and is able to bind these sugars with fructose as the preferred substrate. Our data suggest that BL0033-0036 constitute a high affinity fructose-specific ABC transporter of B. longum NCC2705. We thus suggest to rename the coding genes to fruEKFG and the corresponding proteins to FruE (sugar-binding protein), FruK (ATPase subunit), FruF, and FruG (membrane permeases). Furthermore, protein-protein interactions between the components of the transporter complex were determined by GST pulldown and Western blot analysis. This revealed interactions between the membrane subunits FruF and FruG with FruE, which in vivo is located on the external side of the membrane, and with the cytoplasmatic ATPase FruK. This is in line with the proposed model for bacterial ABC sugar transporters.

Abnormal fecal microbiota community and functions in patients with hepatitis B liver cirrhosis as revealed by a metagenomic approach.

BACKGROUND: Assessment and characterization of human colon microbiota is now a major research area in human diseases, including in patients with hepatitis B liver cirrhosis (HBLC). METHODS: We recruited 120 patients with HBLC and 120 healthy controls. The fecal microbial community and functions in the two groups were analyzed using high-throughput Solexa sequencing of the complete metagenomic DNA and bioinformatics methods. RESULTS: Community and metabolism-wide changes of the fecal microbiota in 20 HBLC patients and 20 healthy controls were observed and compared. A negative correlation was observed between the Child-Turcotte-Pugh scores and Bacteroidetes (P < 0.01), whereas a positive correlation was observed between the scores and Enterobacteriaceae and Veillonella (P < 0.01). Analysis of the additional 200 fecal microbiota samples demonstrated that these intestinal microbial markers might be useful for distinguishing liver cirrhosis microbiota samples from normal ones. The functional diversity was significantly reduced in the fecal microbiota of cirrhotic patients compared with in the controls. At the module or pathway levels, the fecal microbiota of the HBLC patients showed enrichment in the metabolism of glutathione, gluconeogenesis, branched-chain amino acid, nitrogen, and lipid (P < 0.05), whereas there was a decrease in the level of aromatic amino acid, bile acid and cell cycle related metabolism (P < 0.05). CONCLUSIONS: Extensive differences in the microbiota community and metabolic potential were detected in the fecal microbiota of cirrhotic patients. The intestinal microbial community may act as an independent organ to regulate the body's metabolic balance, which may affect the prognosis for HBLC patients.

[Antimicrobial effects of qingkailing injection extract and combination therapy of qingkailing injection and antibiotics on bacteria carrying blaNDM-1 resistance gene].

OBJECTIVE: To research the bacteriostatic effects of Qingkailing Injection Extract (QKLIE) and combination therapy of Qingkailing Injection (QKLI) and antibiotics on bacteria carrying New Delhi metallo-3-lactamase 1 (NDM-1) blaNDM-1 resistance gene, and to determine their minimal inhibitory concentrations (MIC). METHODS: The antimicrobial experiments of QKLIE (Radix Isatidis, baicalin, gardenia, honeysuckle) and combination therapy of QKLI and antibiotics were performed by using the agar dilution method and K-B method. The MIC was determined from each extract. RESULTS: There were different degrees of inhibitory effects on resistant bacteria carrying blaNDM-1 by extracts from main components of QKLI. Of them, the inhibitory effect of baicalin was the best and the MIC of the resistant bacteria was 0.015 g/mL to WD, 0.020 g/mL to WX, 0. 005 g/mL to WJ, and more than 0.020 g/mL to pGEX-4T-NDM-1/DH5alpha (GST-NDM-1), respectively. The MIC value of each extract was sequenced from high to low as baicalin, honeysuckle, gardenia, and Radix Isatidis. Furthermore, combination therapy of QKLI and antibiotics greatly enhanced the antimicrobial activity of each antibiotics when used alone, showing very obvious antibacterial effects on multidrug resistant bacteria carrying blaNDM-1 gene. Of them, the optimal effects were obtained when combined with penicillins (penicillin G, mezlocillin, piperacillin/ tazobactam, ampicillin/sulbactam), with the antibacterial effects improved by 10 folds. The antibacterial effects of other kinds of antibiotics were improved to some extent. Conclusions QKLIE and combination therapy of QKLI and antibiotics showed better bacteriostatic effects on resistant bacteria carrying blaNDM-1 gene. This study provided theoretical bases for drug development, medication and treatment for super-resistant bacteria carrying blaNDM-1.

Proteomic analysis of clinical isolate of Stenotrophomonas maltophilia with blaNDM-1, blaL1 and blaL2 ß-lactamase genes under imipenem treatment.

The co-occurrence of L1 and AmpR-L2 with bla(NDM-1) gene with an upstream 250-bp promoter was detected in a clinical isolate of Stenotrophomonas maltophilia DCPS-01, which was resistant to all ß-lactams and sensitive only to colistin and fluoroquinolones. To investigate expression of resistance genes and the molecular mechanisms of bacteria resistance to carbapenems, proteomic profiles of the isolate was passaged with and without the drug by using 2D-PAGE. The results showed that 33 genes exhibiting a ≥3-fold change were identified as candidates that may help S. maltophilia survive drug selection. Strikingly, L1 was expressed more highly in cells grown with imipenem, and the abundant NDM-1 further increased, while very little L2 was detected even following induction. Specific activities for ß-lactamase revealed that L2 remained at constitutive low levels (10.6 U/mg), while L1 and NDM-1 showed clear activity (69.8 U/mg). Our data support that imipenem could specifically and reversibly induce L1 and NDM-1, which together played key roles in drug resistance in DCPS-01. Although NDM-1 mediated resistance to carbapenems has been found in very few cases, to our knowledge, this is the first proteomics research of S. maltophilia with NDM-1, giving very broad-spectrum antibiotic resistance profiles.

LuxS-dependent AI-2 regulates versatile functions in Enterococcus faecalis V583.

Bacteria utilize a quorum sensing (QS) system to coordinate gene expression by monitoring the concentration of molecules known as autoinducers (AI). In the present study, we confirmed the presence of a LuxS/AI-2 dependent QS system in vancomycin-resistant Enterococcus faecalis V583. Then, the cellular targets controlled by AI-2 were identified by comparative proteomics analysis in order to elucidate the possible role of AI-2 in E. faecalis. Results demonstrated 15 proteins that are differentially expressed upon the addition of AI-2, including proteins involved in metabolism, translation, energy production and/or conversion, and cell wall biogenesis. Glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase associated with carbohydrate metabolism and energy production were up-regulated upon inducing by AI-2. In addition, externally added AI-2 could down-regulate acetyl-coenzyme A carboxylase and ornithine carbamoyltransferase, two key enzyme involved in metabolism. All these data suggest that AI-2 signaling may play a role in the regulation of a number of important metabolic properties of E. faecali. We further investigated the role of AI-2 in biofilm formation by E. faecalis, showing the addition of AI-2 to E. faecalis V583 cultures resulted in increased biofilm formation. Our results provide important clues to the role of a LuxS/AI-2 dependent QS system in vancomycin-resistant E. faecalis.

Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification.

New Delhi metallo-ß-lactamase 1 (NDM-1), which is associated with resistance to carbapenem, was first reported in 2008. A sensitive and rapid molecular assay to detect the plasmid bla(NDM-1) in clinical isolates is needed to control its spread. We describe a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of bla(NDM-1) from pure culture and sputum, urine, and fecal samples. Eight sets of primers were designed to recognize six or eight distinct sequences on target bla(NDM-1), and one set was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for bla(NDM-1) detection were determined. The sensitivity of the LAMP assay for bla(NDM-1) detection in sputum, urine, and fecal samples was also tested. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 70 min at an isothermal temperature of 65°C. The sensitivity of LAMP, with a detection limit of 10.70 pg/µl DNA, was 100-fold greater than that of PCR. Thirteen infection bacterial strains without bla(NDM-1) were selected for testing of specificity, and the results of the amplification were negative, which showed that the primers had good levels of specificity. The LAMP method reported here is demonstrated to be a potentially valuable means for the detection of bla(NDM-1) and rapid clinical diagnosis, being fast, simple, and low in cost.

Multi-omics analysis of an in vitro photoaging model and protective effect of umbilical cord mesenchymal stem cell-conditioned medium.

BACKGROUND: Skin ageing caused by long-term ultraviolet (UV) irradiation is a complex biological process that involves multiple signalling pathways. Stem cell-conditioned media is believed to have anti-ageing effects on the skin. The purpose of this study was to explore the biological effects of UVB irradiation and anti-photoaging effects of human umbilical cord mesenchymal stem cell-conditioned medium (hUC-MSC-CM) on HaCaT cells using multi-omics analysis with a novel cellular photoaging model. METHODS: A cellular model of photoaging was constructed by irradiating serum-starved HaCaT cells with 20 mJ/cm2 UVB. Transcriptomics and proteomics analyses were used to explore the biological effects of UVB irradiation on photoaged HaCaT cells. Changes in cell proliferation, apoptosis, and migration, the cell cycle, and expression of senescence genes and proteins were measured to assess the protective effects of hUC-MSC-CM in the cellular photoaging model. RESULTS: The results of the multi-omics analysis revealed that UVB irradiation affected various biological functions of cells, including cell proliferation and the cell cycle, and induced a senescence-associated secretory phenotype. hUC-MSC-CM treatment reduced cell apoptosis, inhibited G1 phase arrest in the cell cycle, reduced the production of reactive oxygen species, and promoted cell motility. The qRT-PCR results indicated that MYC, IL-8, FGF-1, and EREG were key genes involved in the anti-photoaging effects of hUC-MSC-CM. The western blotting results demonstrated that C-FOS, C-JUN, TGFß, p53, FGF-1, and cyclin A2 were key proteins involved in the anti-photoaging effects of hUC-MSC-CM. CONCLUSION: Serum-starved HaCaT cells irradiated with 20 mJ/cm2 UVB were used to generate an innovative cellular photoaging model, and hUC-MSC-CM demonstrates potential as an anti-photoaging treatment for skin.

Evaluation of loop-mediated isothermal amplification assays for rapid detection of blaKPC producing Serratia spp. in clinical specimens: A prospective diagnostic accuracy study.

The prevalence of carbapenem-resistant Serratia spp. is increasing owing to the propagation of ß lactamase Klebsiella pneumoniae carbapenemase (blaKPC) and it has become one of the major global health concerns. As effective therapies for such resistant pathogens are limited, there is a great need for the rapid and sensitive characterization of the pathogen. In the present study, a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Serratia spp. with blaKPC in pure cultures and clinical specimens was developed. A calcein indicator and real-time turbidity recording system were used to assess the LAMP reaction. The LAMP assay was compared with conventional PCR and real-time PCR kits for the target pathogen. The desired amplification was achieved using selected primers and detection was possible using both the calcein indicator method and the real-time turbity recording system at 65˚C for 60 min. The sensitivity of the detection system for blaKPC-producing Serratia spp. reached a detection limit of 3.92 pg/µl DNA, which was 10 times more sensitive than conventional PCR. Specificity testing indicated that the primers were highly specific. Compared with conventional culture methods and real-time PCR, the LAMP assay was more sensitive, easier for laboratory staff to master and less influenced by the clinical specimen matrix. In conclusion, a LAMP assay for blaKPC-producing Serratia spp. that permitted rapid, sensitive and economical detection for this pathogen was successfully developed. Comparisons with alternative methods indicated that the LAMP assay was more feasible in a clinical setting.

Development and evaluation of a loop-mediated isothermal amplification assay for clinical diagnosis of respiratory human adenoviruses emergent in China.

Three human adenovirus (HAdV) genotypes, HAdV-7, HAdV-14, and HAdV-55, emerged as the most prevalent variants in China over the past decade and caused both sporadic, fatal cases and frequent, large outbreaks. Early diagnosis is essential to control infections and endemics. Here, we established a loop-mediated isothermal amplification (LAMP) assay coupled with an instrument-free nucleic acid extraction device recently developed by our group; the assay could detect all the 3 prevalent HAdV genotypes. Specificity analysis showed no cross-reactivity with other common respiratory pathogens and the analytical sensitivity was as low as 10 copies/µL. All detection steps could be completed within 1 hour. The assay's performance was evaluated using clinical samples and compared with the gold standard RT-PCR method, showing highly consistent results. The LAMP assay developed here could be readily used in basic laboratory facilities and with minimal DNA extraction equipment, and as a reliable screening test in a resource-limited setting.

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Rapid and Specific Identification of Carbapenem-Resistant Acinetobacter baumannii Strains Harboring blaOXA-23, and the Epidemiological Survey of Clinical Isolates.

Acinetobacter baumannii is an important nosocomial pathogen in hospital-acquired infections, and carbapenem resistance has been increasingly observed worldwide. Oxacillinase production by blaOXA-23 is a predominant and prevalent carbapenem resistance mechanism of A. baumannii, especially in China. Rapid and specific detection of blaOXA-23 may offer valuable insight for administration of directed antimicrobial therapy. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP)-based method for identifying carbapenem-resistant A. baumannii (CRAB) harboring the blaOXA-23 gene. High-specificity primers for screening blaOXA-23 were designed and synthesized, and the LAMP reactions were performed. Clinical A. baumannii strains isolated from the Former 307th Hospital of People's Liberation Army were used to determine the sensitivity and specificity of this method compared with those of phenotypic antimicrobial susceptibility testing and the traditional PCR method. Multilocus sequence typing (MLST) was performed to investigate the epidemiology of the A. baumannii bacterial population. Compared with antimicrobial susceptibility testing, the sensitivity and specificity of LAMP in detecting blaOXA-23 were 88.4% and 97.7%, respectively. However, the LAMP method is much simpler and less time-consuming (within 60 minutes) than conventional PCR and phenotypic susceptibility testing. The 113 isolates could be clustered into 30 sequence types, and most strains (83/113) belonged to clonal complex (CC) 92, which is also the dominant CC in China. The LAMP-based method detected blaOXA-23 in a simpler manner and could provide rapid results for identifying CRAB. Consequently, blaOXA-23 may serve as a surrogate marker for the presence of CRAB in patients with serious infections in clinical practice.

Establishment of the molecular beacon-loop-mediated isothermal amplification method for the rapid detection of Porphyromonas gingivalis.

Porphyromonas gingivalis, a clinically important oral pathogen causing periodontal disease, is difficult to culture in routine conditions. Hence, it is necessary to establish a reliable technique to detect this pathogen. Previously, our laboratory developed a new isothermal detection method, called MB-LAMP (molecular beacon-Loop-mediated isothermal amplification), which combines the advantages of LAMP and qPCR through the accurate and quantitative detection of LAMP products. This approach offers significant potential for the point-of-care detection of P. gingivalis. Here, MB-LAMP was used to detect P. gingivalis targeting a specific fragment, and the sensitivity was as high as 1.4 × 10-1 pg µL-1. The method showed no cross-reaction with 14 other bacterial pathogens. For clinical samples, this assay showed a high diagnostic sensitivity (100%) and specificity (100%), equivalent to that of real-time quantitative polymerase chain reaction (real-time qPCR). Moreover, detection with MB-LAMP was significantly faster than that with real-time qPCR, reducing the time required for clinical diagnosis. Finally, we established an absolute quantification method with MB-LAMP for P. gingivalis using pilot samples. Thus, the highly specific, sensitive, and rapid assay developed in this study makes it feasible to diagnose P. gingivalis.

Development and application of a rapid Mycobacterium tuberculosis detection technique using polymerase spiral reaction.

Mycobacterium tuberculosis is an age-old bacterium that is difficult to eliminate. A simple and rapid diagnostic method is of great importance to prevent the spread of M. tuberculosis. Here, we developed a low-cost rapid M. tuberculosis nucleic acid detection technique, named GenePop, which enabled the storage and transport of M. tuberculosis diagnostic reagent at ambient temperatures, without the need for professional operations or expensive instrumentation. Using a vitrification method, we vitrified heat-unstable components onto the cap of a reaction tube, and placed heat-stable components at the bottom of the reaction tube by sealing them with paraffin wax. The all-in-one detection tube, when used together with our other invention-a multi-functional sample treatment tube pre-filled with a nucleic acid-releasing agent-only required three simple steps to yield results. A comparative analysis with a commercial qPCR kit for M. tuberculosis indicated that our new technique had a concordance rate of 91.6%, showing no cross-reactivity with 11 other bacteria. The complete operation time was only 65 min. It is suitable for use in field settings or by personnel in grass-root units, and is applicable in household activities, hence can be used in developing countries.