Multiplex PCR for detection of MCR genes in clinical fecal samples

Plasmid-mediated colistin-resistance genes have been reported worldwide in recent years. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect transferable colistinresistance genes (mcr-1 to mcr-6) in Enterobacteria for clinical laboratory purposes.The authors first designed six new primer pairs to amplify mcr-1 to mcr-6 gene products to achieve stepwise separation of amplicons between 87 to 216 bp,then divided these primers into two subgroups with the assistance of a pair of universal primers for the detection of currently described mcr genes and their variants in Enterobacteria. The protocol was validated by testing 29 clinical isolates of Escherichia coli of human origin, each well characterised and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. It was able to detect mcr-3 and mcr-4 as singletons or in combination. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance in limited resources conditions, and this method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance

Dietary Factors of blaNDM Carriage in Health Community Population: A Cross-Sectional Study

Aim: There is an ongoing debate as to what extent antimicrobial resistance (AMR) can be transmitted from dietary to humans via the consumption of food products. We investigated this association between dietary and global spreading carbapenem-resistant gene blaNDM Methods: We did a cross-sectional study to assess the risk factors for carrier of blaNDM in health community. Healthy adults were recruited from the residents attending Community Healthcare Service in Shenzhen City (Guangdong Province, China), through 1February 2018 to 31December 2019, and 718 pre-participants were included in this study. Questionnaire were obtained and the qualitative food frequency questionnaire (Q-FFQ) were used to assess dietary intake. qPCR was applied to confirm the carrier of blaNDM in participants’fecal samples. Multivariable logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval (95% CI) of each outcome according to each dietary factor before and after prosperity score matching (PSM). Results: we showed that a high intake of coarse grain (OR 1.003; 95% CI 1.001–1.005, p < 0.01) and root and tuber crops (OR 1.003; 95% CI 1.001–1.004, p < 0.05) were independent risk factor for blaNDM carrier in health communities, suggesting a possible transfer of AMRbetweendietary andhumans. Surprisingly, we also showed an association between a higher intake of poultry as a protective, which may be explained by the beneficial effects on the gut microbiota. Conclusion: Dietary factors such as intake of coarse grain, root and tuber crops and poultry were associated with blaNDM carrier in health communities. The influence of dietary factorson blaNDM carrier in the present study provides insights for the tangible dietary advice with guidelines to the routine of people with the risk of blaNDM carrier. This demonstrates the role of dietary intake in the prevention of blaNDM carrier, since prevention is the best way to control modifiable risk factors. A lower carrier rate of blaNDM is helpful to reduce the possibility of transmission and pathogenicity. Further studies on food, microbiota and antimicrobial resistance are necessary to confirm this possible association and unravel underlying mechanisms

Rapid detection of Cronobacter sakazakii by real-time PCR based on cgcA gene and Taqman probe with IAC

As a severe virulent strain to infants, species C. sakazakii is frequently detected in powdered infant formula (PIF). Therefore, it is necessary to develop a fast and specific detection method. The specificity of our newly developed quantitative real-time PCR (qRT-PCR) was validated with DNA from 46 strains. Among them, 12 C. sakazakii strains were correctly amplified, whereas no positive florescent signal was observed from 34 non-target controls. The detection limit of C. sakazakii was about 110 CFU/mL in broth and 1,100 CFU/g in PIF. With the enrichment in BPW (buffer protein water) for 6 h, our developed qRT-PCR assay was reliable to detect opportunistic pathogens C. sakazakii when the inoculation level was as low as 2 CFU/25 g (0.08 CFU/g) in PIF. The growth of C. sakazakii could be inhibited by the presence of Lactobacillus pentosus and Bacillus cereus, which used a longer enrichment period before the isolation was accomplished. However, at 5 and 50 CFU/25 g inoculation levels of C. sakazakii in the presence of 4×106 CFU/25 g or 2×104 CFU/25 g of L. pentosus and B. cereus, the qRT-PCR assay could detect the presence of Cronobacter even though these artificially spiked samples were negative in culture. Therefore, our results indicated that the qRT-PCR assay could detect samples containing inhibitors and avoid false negatives by using an internal amplification control.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Migration and accumulation mechanisms and main controlling factors of tight oil enrichment in a continental lake basin

Based on the typical dissection of various onshore tight oil fields in China, the tight oil migration and accumulation mechanism and enrichment-controlling factors in continental lake basins are analyzed through nuclear magnetic resonance (NMR) displacement physical simulation and Lattice Boltzmann numerical simulation by using the samples of source rock, reservoir rock and crude oil. In continental lake basins, the dynamic forces driving hydrocarbon generation and expulsion of high-quality source rocks are the foundational power that determines the charging efficiency and accumulation effect of tight oil, the oil migration resistance is a key element that influences the charging efficiency and accumulation effect of tight oil, and the coupling of charging force with pore-throat resistance in tight reservoir controls the tight oil accumulation and sweet spot enrichment. The degree of tight oil enrichment in continental lake basins is controlled by four factors: source rock, reservoir pore-throat size, anisotropy of reservoir structure, and fractures. The high-quality source rocks control the near-source distribution of tight oil, reservoir physical properties and pore-throat size are positively correlated with the degree of tight oil enrichment, the anisotropy of reservoir structure reveals that the parallel migration rate is the highest, and intralayer fractures can improve the migration and accumulation efficiency and the oil saturation