Efficient signal amplification is essential to construct ultrasensitive biosensors for biologically relevant species with abundant concomitant interferences. Here, we apply LbaCas12a as a signal amplifier to develop a versatile CRISPR-Cas12a platform to detect a wide range of analytes in ultralow concentrations. The platform relies on the indiscriminate single-stranded DNase activity of LbaCas12a, which recognizes single-stranded DNA intermediates generated by non-DNA targets down to femtomolar concentrations and subsequently enhances the fluorescence signal output. With the help of functional nucleotides (DNAzyme and aptamer), ultrasensitive bioassays for Pb 2+ and Acinetobacter baumannii have been designed with a limit of detection down to ∼0.053 nM and ∼3 CFU/mL, respectively. It also allows simultaneous detection of four microRNAs (miRNAs) at a picomolar concentration without significant interferences by other counterparts, suggesting the potential of multiplexed miRNA expression profiles analysis in high throughput. Given the versatility and generality of the CRISPR-Cas12a platform, we expect the current work to advance the application of CRISPR-Cas-based platforms in bioanalysis and provide new insights into ultrasensitive biosensor design.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) is considered a serious global threat. However, little is known regarding the multidrug resistance (MDR) mechanisms of CRKP. This study investigated the phenotypes and MDR mechanisms of CRKP and identified their clonal characteristics.MethodsPCR and sequencing were utilized to identify antibiotic resistance determinants. Integron gene cassette arrays were determined by restriction fragment length polymorphism (RFLP) analysis. Multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used for epidemiological analysis. Plasmids were typed by using a PCR-based replicon typing and analyzed by conjugation and transformation assays.ResultsSeventy-eight strains were identified as resistant to at least one carbapenem; these CRKP strains had a high prevalence rate (38.5%, 30/78) of carbapenemase producers. Additionally, most isolates harbored MDR genes, including Extended spectrum β-lactamases (ESBLs), AmpC, and quinolone and aminoglycoside resistance genes. Loss of porin genes was observed, and Class 1 integron was detected in 66.7% of the investigated isolates. PFGE and MLST results excluded the occurrence of clonal dissemination among these isolates.ConclusionsA high prevalence of NDM-1 genes encoding carbapenem resistance determinants was demonstrated among the K. pneumoniae isolates. Importantly, this is the first report of blaNDM-1 carriage in a K. pneumoniae ST1383 clone in China and of a MDR CRKP isolate co-harboring blaNDM-1, blaKPC-2, blaCTX-M, blaSHV, acc(6′)-Ib, rmtB, qnrB, and acc(6′)-Ib-cr.

BackgroundLarge-size data on type-specific HPV prevalence in Southwest China are required to estimate the cervical cancer burden in the country and to prepare for HPV-based cervical screening program and further HPV vaccination of China. This HPV study is a pooled analysis of data from five years in Chongqing of China, which is cross-sectional in design using data collecting.ResultsThe positivity of HPV was 26.2% (10542/40311), single type was 25.7% (10360/40311), multiple type was 8.2% (3306/40311), high-risk HPV was 30.9% (12490/40311), and low-risk HPV was 2.9%(1169/40311). The most common genotypes were HPV16,52,58 and 18. HPV-positive women (n = 10542) were triaged by cytology, colposcopy or histological diagnosis. Among HPV-positive women, 43.8% had normal, 22.5% had ASCUS, 0.2% had LSIL, 12.6% had HSIL and 6.0% had ICC. The most common HPV genotypes were HPV16, 58 and 18 in ASCUS, HPV16, 18 and 58 in LSIL, HPV16, 58 and 33 in HSIL, and HPV16, 58 and 18 in ICC. The prevalence of Group 1/2A HPV types increased with increasing CIN grade and accounted for 96.05% of the CIN 3+ lesions, while HPV16 accounted for 71.1%. HPV-positive women steadily increased with age, peaking at 31–40 years.ConclusionThe type-specific prevalence rate of HPV 16 and HPV 18 were a little lower than the mean of international meta-analyses. Single HPV genotype infection was predominantly detected in different groups of cervical lesions in Chongqing, and HPV16, 52, 58 were the priority HPV types. The HPV genotyping study was found to be valuable for planning further preventive program for cervical cancer.

Background ESKAPEEc contribute to a majority of bloodstream infections (BSIs) and their antibiogram have changed overtime, while data concerning about these alterations are lacking in China. Added that a paucity of studies referred to ESKAPEEc in pediatric BSIs, our study aimed to demonstrate the longitudinal alterations of ESKAPEEc distribution and antibiogram in adult and pediatric BSIs in Southwest China. Methods A multicenter retrospective surveillance study was launched from 2012 to 2017. Data of China Antimicrobial Resistance Surveillance System (CARSS) was analyzed by Whonet 5.6 and Graphpad Prism 6 Software. Chi-square test or Fisher’s exact test was used to examine and compare temporal changes. Results A total of 32,259 strains was isolated, with 17.4% from pediatric BSIs. ESKAPEEc contributed to 58.67% (18,924/32,259) of BSIs, with 65.3% of adult BSIs and 27.2% of pediatric BSIs. Escherichia coli ( E. coli ) and Klebsiella pneumoniae ( K. pneumoniae ) were the two predominant species. Carbapenem resistance was prevalent in 0.76, 4.60, 9.47,13.66, 59.47% of E. coli , K. pneumoniae , Enterobacter cloacae , Pseudomonas aeruginosa ( P. aeruginosa ) and Acinetobacter baumannii ( A. baumannii ), respectively. The proportions of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREFM) were 28.91% and 2.20%, respectively. Between 2012-2014 and 2015–2017, E. coli and K. pneumonia showed significantly increased resistance rates to imipenem but decreased to ceftriaxone and ceftazidime, while A. baumannii exhibited reduced resistances to almost all the beta-lactams tested. The prevalence of antimicrobial resistance to most of agents against Gram-positive ESKAPEEc did not significantly varied during the same timeframe. In comparison with those from adult BSIs, K. pneumoniae from pediatric BSIs exhibited high resistance rates to all the beta-lactams tested, especially to carbapenems (12.79% vs 3.87%), while A. baumannii showed low resistance rates to all the agents. Conclusions Ongoing burden of ESKAPEEc in BSIs and increasing trend of imipenem resistance in E. coli and K. pneumoniae call for continued surveillance. Carbapenems are still active against Gram-negative ESKAPEEc, except for A. baumannii and vancomycin or linezolid is still effective against Gram...

Carbapenem-resistant Enterobacteriaceae (CRE) has been considered a serious global threat, but carbapenem resistance remains relatively uncommon in E. cloacae, especially in China. The aim of this study was to characterize carbapenem-resistant E. cloacae (CR-ECL) isolates from 2012 to 2016 in Southwest China. Our study revealed that 20 (15.2%) of the 132 CR-ECL isolates obtained from patients were identified as NDM-1, with most isolates carrying the IncFIIA plasmids. Notably, we initially observed that the E. cloacae strain co-harbored NDM-1 and IMP-8 carbapenemases simultaneously. Analysis of the genetic environment of these two genes has revealed that the highly conserved regions (blaNDM-1-bleMBL-trpF-tat) are associated with the dissemination of NDM-1, while IS26, intI1, and tniC could be involved in the spread of IMP-8. Molecular epidemiology studies showed the nosocomial outbreak caused by NDM-1-producing E. cloacae ST88. Transferring from another hospital and previous carbapenem exposure were identified as independent risk factors for the acquisition of NDM-1-producing E. cloacae. These findings emphasize the need for intensive surveillance and precautions to monitor the further spread of NDM-1 in China.