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.

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.

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.

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.

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.

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.

Sensitive and Rapid Detection of the Plasmid-Encoded Colistin-Resistance Gene mcr-1 in Enterobacteriaceae Isolates by Loop-Mediated Isothermal Amplification.

The emergence of the plasmid-encoded colistin-resistance gene mcr-1 in Enterobacteriaceae represents a new threat to the treatment of infection in the clinical setting. A sensitive and rapid molecular method for detection of the mcr-1 gene in clinical isolates is needed to control the spread of this gene. In this study, we established a loop-mediated isothermal amplification (LAMP) assay for rapid detection of the mcr-1 gene. This assay was applied to cultured bacteria and spiked human stools. Real-time monitoring of turbidity and chromogenic visualization were used to assess the reaction results. The specificity and sensitivity of the primers in the LAMP reactions for detection of the mcr-1 gene were determined. All 20 clinically resistant isolates without the mcr-1 gene tested negative, indicating the high specificity of the LAMP primers. The sensitivity of LAMP, with a detection limit of 0.2 pg/µL DNA, was 10-fold greater than that of polymerase chain reaction (PCR). The assay was also conclusive when applied to human stools spiked with mcr-1-positive Escherichia coli. During clinical screening in a major hospital in Beijing, China, seven isolates were identified as positive from the 556 Enterobacteriaceae isolates. In conclusion, the LAMP assay we developed was useful for detection of the mcr-1 gene in the clinical setting.

Complete sequences of two novel bla NDM-1-harbouring plasmids from two Acinetobacter towneri isolates in China associated with the acquisition of Tn125.

Two novel New Delhi metallo-ß-lactamase-1 (NDM-1)-positive plasmids containing a complete composite transposon, Tn125, from two respective Acinetobacter towneri isolates were characterized. Plasmid pNDM-GJ01 (30,293 bp) isolated from A. towneri G165 did not show homology to any known plasmid structure, except for the transposon Tn125 containing bla NDM-1. A novel repB gene and two XRE-type transcriptional regulators were found in pNDM-GJ01. Plasmid pNDM-GJ02 (62,011 bp) isolated from A. towneri G295 showed the highest homology to pBJAB0715 (41% coverage, 99% nucleotide identity). In addition to the bla NDM-1-harbouring transposon Tn125, pNDM-GJ02 also had an IS26-composite transposon, which contains ISCR1 and two class 1 integrons carrying different cassette arrays. Both clinical isolates were highly resistant to ß-lactams and susceptible to tigecycline and colistin. Ten other resistance genes were detected in G295, and one other resistance gene was detected in G165. No transconjugant was obtained from any of the donors by broth and filter mating. The emergence of these two novel plasmids carrying NDM-1 in Acinetobacter spp., pNDM-GJ01 and pNDM-GJ02, suggests Tn125 mobile integration.

Isolation and Whole-genome Sequence Analysis of the Imipenem Heteroresistant Acinetobacter baumannii Clinical Isolate HRAB-85.

OBJECTIVES: Heteroresistance is a phenomenon in which there are various responses to antibiotics from bacterial cells within the same population. Here, we isolated and characterised an imipenem heteroresistant Acinetobacter baumannii strain (HRAB-85). METHODS: The genome of strain HRAB-85 was completely sequenced and analysed to understand its antibiotic resistance mechanisms. Population analysis and multilocus sequence typing were performed. RESULTS: Subpopulations grew in the presence of imipenem at concentrations of up to 64µg/mL, and the strain was found to belong to ST208. The total length of strain HRAB-85 was 4,098,585bp with a GC content of 39.98%. The genome harboured at least four insertion sequences: the common ISAba1, ISAba22, ISAba24, and newly reported ISAba26. Additionally, 19 antibiotic-resistance genes against eight classes of antimicrobial agents were found, and 11 genomic islands (GIs) were identified. Among them, GI3, GI10, and GI11 contained many ISs and antibiotic-resistance determinants. CONCLUSIONS: The existence of imipenem heteroresistant phenotypes in A. baumannii was substantiated in this hospital, and imipenem pressure, which could induce imipenem-heteroresistant subpopulations, may select for highly resistant strains. The complete genome sequencing and bioinformatics analysis of HRAB-85 could improve our understanding of the epidemiology and resistance mechanisms of carbapenem-heteroresistant A. baumannii.

Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp. and Vibrio parahaemolyticus.

Rapid detection of food-borne pathogens is important in the food industry, to monitor and prevent the spread of these pathogens through contaminated food products. We therefore established a multiplex real-time loop-mediated isothermal amplification (LAMP) assay to simultaneously detect and distinguish Salmonella spp. and Vibrio parahaemolyticus DNA in a single reaction. Two target sequences, one specific for Salmonella and the other specific for Vibrio parahaemolyticus, were amplified by specific LAMP primers in the same reaction tube. After amplification at 65 °C for 60 min, the amplified products were subjected to melting curve analysis and thus could be distinguished based on the different melting temperatures (Tm values) of the two specifically amplified products. The specificity of the multiplex LAMP assay was evaluated using 19 known bacterial strains, including one V. parahaemolyticus and seven Salmonella spp. strains. The multiplex LAMP showed 100% inclusivity and exclusivity, and a detection limit similar to that of multiplex PCR. In addition, we observed and corrected preferential amplification induced by what we call LAMP selection in the multiplex LAMP reaction. In conclusion, our assay was rapid, specific, and quantitative, making it a useful tool for the food industry.

Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China.

OBJECTIVES: The prevalence and dissemination of diverse NDM-producing bacteria in China was investigated. METHODS: We collected 1,162 isolates from 8 cities during December 2013∼May 2015 in China. The NDM-positive strains as well as the NDM genotypes in these sample were detected via Vitek 2 compact system (bioMérieux, France), 16S rRNA gene sequencing, PCR and an S1- pulsed-field gel electrophoresis assay and Southern blot hybridization. The horizontal-transfer capability of the blaNDM gene was assessed by filter mating by using a standard E.coli J53 azide-resistant strain as the recipient. RESULTS: Three genotypes (NDM-1, NDM-3 and NDM-5) of NDM-producing bacteria were identified, among which the NDM-1-positive isolates were the most frequent one. For the first time, we found NDM-5-produing S.typhimurium and NDM-3-produing E.coli in China. We also found that the NDM-positive (especially NDM-3 and NDM-5) strains were completely resistant to nearly all of the antimicrobial drugs utilized and blaNDM was mostly located on diverse plasmids with sizes ranging from 30 to 670kb. CONCLUSION: Various species of bacteria especially the enteric pathogens with diverse NDM genotypes had spread in China. Hence, an ongoing surveillance of their dissemination is essential to prevent and control the spread of these organisms.

Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay.

This study established a constant-temperature fluorescence quantitative detection method, combining loop-mediated isothermal amplification (LAMP) with molecular beacons. The advantages of LAMP are its convenience and efficiency, as it does not require a thermocycler and results are easily visualized by the naked eye. However, a major disadvantage of current LAMP techniques is the use of indirect evaluation methods (e.g., electrophoresis, SYBR Green I dye, precipitation, hydroxynaphthol blue dye, the turbidimetric method, calcein/Mn2+ dye, and the composite probe method), which cannot distinguish between the desired products and products of nonspecific amplification, thereby leading to false positives. Use of molecular beacons avoids this problem because molecular beacons produce fluorescence signals only when binding to target DNA, thus acting as a direct indicator of amplification products. Our analyses determined the optimal conditions for molecular beacons as an evaluation tool in LAMP: beacon length of 25-45 bp, beacon concentration of 0.6-1 pmol/µL, and reaction temperature of 60-65 °C. In conclusion, we validated a novel molecular beacon loop-mediated isothermal amplification method (MB-LAMP), realizing the direct detection of LAMP product.

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.

Rapid Detection of Candida albicans by Polymerase Spiral Reaction Assay in Clinical Blood Samples.

Candida albicans is the most common human yeast pathogen which causes mucosal infections and invasive fungal diseases. Early detection of this pathogen is needed to guide preventative and therapeutic treatment. The aim of this study was to establish a polymerase spiral reaction (PSR) assay that rapidly and accurately detects C. albicans and to assess the clinical applicability of PSR-based diagnostic testing. Internal transcribed spacer 2 (ITS2), a region between 5.8S and 28S fungal ribosomal DNA, was used as the target sequence. Four primers were designed for amplification of ITS2 with the PSR method, which was evaluated using real time turbidity monitoring and visual detection using a pH indicator. Fourteen non-C. albicans yeast strains were negative for detection, which indicated the specificity of PSR assay was 100%. A 10-fold serial dilution of C. albicans genomic DNA was subjected to PSR and conventional polimerase chain reaction (PCR) to compare their sensitivities. The detection limit of PSR was 6.9 pg/µl within 1 h, 10-fold higher than that of PCR (69.0 pg/µl). Blood samples (n = 122) were collected from intensive care unit and hematological patients with proven or suspected C. albicans infection at two hospitals in Beijing, China. Both PSR assay and the culture method were used to analyze the samples. Of the 122 clinical samples, 34 were identified as positive by PSR. The result was consistent with those obtained by the culture method. In conclusion, a novel and effective C. albicans detection assay was developed that has a great potential for clinical screening and point-of-care testing.

Rapid detection of nusG and fadA in Fusobacterium nucleatum by loop-mediated isothermal amplification.

Fusobacterium nucleatum is associated with various human diseases such as periodontal disease and colorectal cancer (CRC); thus, F. nucleatum detection might serve as a novel diagnostic tool. Here, we describe the development of a sensitive and rapid molecular method for detecting two F. nucleatum genes: the highly conserved nusG and fadA, which encode a critical host colonization factor. Loop-mediated isothermal amplification (LAMP) primer sets for the rapid detection of nusG and fadA were designed and optimized. The nusG primers yielded consistent negative results for 20 non-F. nucleatum bacterial strains, confirming the high specificity of the primers. LAMP reaction primer sensitivity was determined, and its detection rate in comparison to conventional PCR was assessed using 57 clinical stool samples. The LAMP detection limit for nusG and fadA was 22.5 and 0.225 pg µl-1, respectively, indicating that the sensitivity of this method was 10-fold higher than that of conventional PCR. These results suggest that the LAMP technique is able to effectively identify F. nucleatum via nusG as well as detect its virulence factor. To the best of our knowledge, this study is the first to report the application of LAMP for the detection of nusG and fadA in F. nucleatum. The LAMP method constitutes a sensitive and specific visual assay for the rapid detection of the pathogen F. nucleatum.

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).