Search Results (4)

Klebsiella michiganensis is a recently emerging human pathogen causing nosocomial infections. This study aimed to characterize the complete genome sequence of a clinical Klebsiella michiganensis strain KMIB106 which exhibited extensive drug-resistance. The whole genome of the strain was sequenced using PacBio RS III systems and Illumina Nextseq 500. Annotation, transposable elements and resistance gene identification were analyzed by RAST, prokka and Plasmid Finder, respectively. According to the results, KMIB106 was resistant to multiple antimicrobials, including carbapenems, but it remained susceptible to aztreonam. The genome of KMIB106 consisted of a single chromosome and three predicted plasmids. Importantly, a novel KPC plasmid pB106-1 was found to carry the array of resistance genes in a highly different order in its variable regions, including mphA, msrE, mphE, ARR-3, addA16, sul1, dfrA27, tetD and fosA3. Plasmid pB106-2 is a typical IncFII plasmid with no resistant gene. Plasmid pB106-IMP consists of the IncN and IncX3 backbones, and two resistance genes, bla_IMP-4 and bla_SHV-12, were identified. Our study for the first time reported an extensively drug-resistant Klebsiella michiganensis strain recovered from a child with a respiratory infection in Southern China, which carries three mega plasmids, with pB106-1 firstly identified to carry an array of resistance genes in a distinctive order, and pB106-IMP identified as a novel IncN-IncX3 cointegrate plasmid harboring two resistance genes bla_IMP-4 and bla_SHV-12. Full article

Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing. Full article

Cointegrate/hybrid plasmids combine the genetic elements of two or more plasmids and generally carry abundant antimicrobial resistance determinants. Hence, the spread of cointegrate plasmids will accelerate the transmission of AMR genes. To evaluate the transmission risk caused by cointegrate plasmids, we investigated the structural diversity, fitness cost, and stability of a cointegrate plasmid in Klebsiella pneumoniae YZ6 and Escherichia coli EC600. The cointegrate plasmid pSL131_IncA/C_IncX3 was from a clinical Salmonella Lomita strain. After transferring the plasmid into E. coli EC600 by conjugation, we observed plasmids with different structures, including a full-length original plasmid and two truncated versions. By contrast, DNA fragment deletion and bla_CTX-M-14 gene insertion in the plasmid were detected in a transconjugant derived from K. pneumoniae YZ6. These results suggest that the structure of the plasmid was unstable during conjugation. Furthermore, both the full-length plasmid in EC600 and the structurally reorganized plasmid in YZ6 imposed a fitness cost on the bacterial host and enhanced biofilm formation ability. Serial passaging in antibiotic-free medium resulted in a rapid decline of the plasmid in YZ6. However, the stability of the structurally reorganized plasmid in YZ6 was improved via serial passaging in antibiotic-containing medium. SNP calling revealed that mutations of the outer membrane porin may play an essential role in this process. These findings indicate that structural versatility could contribute to the dissemination of cointegrate plasmids. Although the plasmid incurred a fitness cost in other Enterobacteriaceae species, positive selection could alleviate the adverse effects. Full article

Proteus mirabilis is a component of the normal intestinal microflora of humans and animals, but can cause urinary tract infections and even sepsis in hospital settings. In recent years, the number of multidrug-resistant P. mirabilis isolates, including the ones producing extended-spectrum β-lactamases (ESBLs), is increasing worldwide. However, the number of investigations dedicated to this species, especially, whole-genome sequencing, is much lower in comparison to the members of the ESKAPE pathogens group. This study presents a detailed analysis of clinical multidrug-resistant ESBL-producing P. mirabilis isolate using short- and long-read whole-genome sequencing, which allowed us to reveal possible horizontal gene transfer between Klebsiella pneumoniae and P. mirabilis plasmids and to locate the CRISPR-Cas system in the genome together with its probable phage targets, as well as multiple virulence genes. We believe that the data presented will contribute to the understanding of antibiotic resistance acquisition and virulence mechanisms for this important pathogen. Full article