National Key Basic Research Program of China, National Natural Science Foundation of China/Zhejiang, National Key Research and Development Program, and MRC, UK.
BackgroundMobilized resistance to colistin is evolving rapidly and its global dissemination poses a severe threat to human health and safety. Transferable colistin resistance gene, mcr-3, first identified in Shandong, China, has already been found in several countries in multidrug-resistant human infections. Here we track the spread of mcr-3 within 13 provinces in China and provide a complete characterization of its evolution, structure and function.MethodsA total of 6497 non-duplicate samples were collected from thirteen provinces in China, from 2016 to 2017 and then screened for the presence of mcr-3 gene by PCR amplification. mcr-3-positive isolates were analyzed for antibiotic resistance and by southern blot hybridization, transfer analysis and plasmid typing. We then examined the molecular evolution of MCR-3 through phylogenetic analysis. Furthermore, we also characterized the structure and function of MCR-3 through circular dichroism analyses, inductively coupled plasma mass spectrometry (ICP-MS), liquid chromatography mass spectrometry (LC/MS), confocal microscopy and chemical rescue tests.Findings49 samples (49/6497 = 0.75%) were mcr-3 positive, comprising 40 samples (40/4144 = 0.97%) from 2017 and 9 samples (9/2353 = 0.38%) from 2016. Overall, mcr-3-positive isolates were distributed in animals and humans in 8 of the 13 provinces. Three mcr-3-positive IncP-type and one mcr-1-bearing IncHI2-like plasmids were identified and characterized. MCR-3 clusters with PEA transferases from Aeromonas and other bacteria and forms a phylogenetic entity that is distinct from the MCR-1/2/P(M) family, the largest group of transferable colistin resistance determinants. Despite that the two domains of MCR-3 not being exchangeable with their counterparts in MCR-1/2, structure-guided functional mapping of MCR-3 defines a conserved PE-lipid recognizing cavity prerequisite for its enzymatic catalysis and its resultant phenotypic resistance to colistin. We therefore propose that MCR-3 uses a possible “ping-pong” mechanism to transfer the moiety of PEA from its donor PE to the 1(or 4′)-phosphate of lipid A via an adduct of MCR-3-bound PEA. Additionally, the expression of MCR-3 in E. coli prevents the colistin-triggered formation of reactive oxygen species (ROS) and interferes bacterial growth and viability.InterpretationOur results provide an evolutionary, structural and functional definition of MCR-3 and its epidemiology in China, paving the way for smarter policies, better surveillance and effective treatments.
Purpose The main objective of this study was to decipher the prevalence, antimicrobial resistance, major virulence genes and the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from different clinical sources in southern China. Materials and methods The present study was performed on 187 non-duplicate S. aureus clinical isolates collected from three tertiary hospitals in Guangdong Province, China, 2010–2016. Antimicrobial susceptibility testing was performed by the disk diffusion method and by measuring the minimum inhibitory concentration. Screening for resistance and virulence genes was performed. Clonal relatedness was determined using various molecular typing methods such as multilocus sequence typing, spa and staphylococcal chromosomal cassette mec (SCCmec) typing. Whole genome sequencing was performed for three selected isolates. Results Out of 187 isolates, 103 (55%) were identified as MRSA. The highest prevalence rate was found among the skin and soft tissue infection (SSTI) samples (58/103), followed by sputum samples (25/103), blood stream infection samples (15/103) and others (5/103). Antimicrobial susceptibility results revealed high resistance rates for erythromycin (64.1%), clindamycin (48.5%), gentamicin (36.9%) and ciprofloxacin (33.98%). All isolates were susceptible to vancomycin. Resistance genes and mutation detected were as follows: aac(6 ’ )-aph(2 ”) (24.3%), dfrG (10.7%), rpoB (21.4%), cfr (0%), fexA (1.94%), gyrA (35.92%), gyrB (0.97%), grlA (20.4%), grlB (10.68%), ermA (21.4%), ermB (18.44%), ermC (21.4%) and lnuA (18.44%). Profiling of virulence genes revealed the following: sea (11.7%), seb (21.4%), sec (0.97%), sed (0.97%), hla (86.41%), hlb (17.48%), hlg (10.68%), hld (53.4%), Tsst-1 (3.9%) and pvl (27.2%). Clonal relatedness showed that ST239-SCCmecA III-t37 clone was the most prevalent clone. Conclusion Our study elucidated the prevalence, antibiotic resistance, pathogenicity and molecular characteristics of MRSA isolated from various clinical sources in Guangdong, China. We found that the infectious rate of MRSA was higher among SSTI than other sources. The most predominant genotype was ST239-SCCmecA III-t37 clone, indicati...
Here, we identified mcr-4.3 in Acinetobacter baumannii, which had not been previously observed to carry an mcr gene. The mcr-4.3-harboring A. baumannii strain AB18PR065 was isolated from pig feces from a slaughterhouse in Guangdong Province of China. The mcr-4.3-carrying pAB18PR065 is 25,602 bp in size and could not be transferred in conjugation, transformation, and electroporation experiments, as we did not find any conjugation-related genes therein. pAB18PR065 harbors two copies of type II toxin-antitoxin systems, which are functional in plasmid stabilization and maintenance. pAB18PR065 shares similarity only with one recently identified plasmid, pAb-MCR4.3 (35,502 bp), from a clinical A. baumannii strain. It is likely that the emergence of pAb-MCR4.3 was due to the insertion of an 11,386-bp, ISAba19based, composite transposon into pAB18PR065. These data indicate that mcr-4.3 was captured by an A. baumannii-original plasmid via horizontal gene transfer.
BackgroundDengue virus (DENV), the most widely prevalent arbovirus, continues to be a threat to human health in the tropics and subtropics. Early and rapid detection of DENV infection during the acute phase of illness is crucial for proper clinical patient management and preventing the spread of infection. The aim of the current study was to develop a specific, sensitive, and robust reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection and differentiation of DENV1-4 serotypes.ResultsThe method detection primers, which were designed to target the different DENV serotypes, were identified by inspection of multiple sequence alignments of the non-structural protein (NS) 2A of DENV1, NS4B of DENV2, NS4A of DENV3 and the 3′ untranslated region of the NS protein of DENV4. No cross-reactions of the four serotypes were observed during the tests. The detection limits of the DENV1-4-specific RT-LAMP assays were approximately 10-copy templates per reaction. The RT-LAMP assays were ten-fold more sensitive than RT-PCR or real-time PCR. The diagnostic rate was 100 % for clinical strains of DENV, and 98.9 % of the DENV-infected patients whose samples were tested were detected by RT-LAMP. Importantly, no false-positives were detected with the new equipment and methodology that was used to avoid aerosol contamination of the samples.ConclusionThe RT-LAMP method used in our study is specific, sensitive, and suitable for further investigation as a useful alternative to the current methods used for clinical diagnosis of DENV1-4, especially in hospitals and laboratories that lack sophisticated diagnostic systems.
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