Optimization of Pulse-Field Gel Electrophoresis for Subtyping of Klebsiella pneumoniae

Han, Hui; Zhou, Haijian; Li, Haishan; Gao, Yuan; Lü, Zhi; Hu, Kongxin; Xu, Bo

doi:10.3390/ijerph10072720

Cited by 56 publications

(46 citation statements)

References 25 publications

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“…The three samples were analyzed with PFGE, with an upper limit of 60 kb and a lower limit 1 kb, with a previously reported method (Han et al, 2013). …”

Section: Methodsmentioning

confidence: 99%

Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

et al. 2016

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Bacterial artificial chromosomes (BACs) are powerful tools for the manipulation of the large genomes of DNA viruses, such as herpesviruses. However, the methods currently used to construct the recombinant viruses, an important intermediate link in the generation of BACs, involve the laborious process of multiple plaque purifications. Moreover, some fastidious viruses may be lost or damaged during these processes, making it impossible to generate BACs from these large-genome DNA viruses. Here, we introduce the CRISPR/Cas9 as a site-specific gene knock-in instrument that promotes the homologs recombination of a linearized transfer vector and the Pseudorabies virus genome through double incisions. The efficiency of recombination is as high as 86%. To our knowledge, this is the highest efficiency ever reported for Pseudorabies virus recombination. We also demonstrate that the positions and distances of the CRISPR/Cas9 single guide RNAs from the homology arms correlate with the efficiency of homologous recombination. Our work show a simple and fast cloning method of BACs with large genome inserted by greatly enhancing the HR efficiencies through CRISPR/Cas9-mediated homology-directed repair mechanism, and this method could be of helpful for manipulating large DNA viruses, and will provide a successful model for insertion of large DNA fragments into other viruses.

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“…The three samples were analyzed with PFGE, with an upper limit of 60 kb and a lower limit 1 kb, with a previously reported method (Han et al, 2013). …”

Section: Methodsmentioning

confidence: 99%

Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

et al. 2016

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“…Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) analysis of cr-hvKP MLST was performed by amplifying and sequencing seven housekeeping genes according to protocols provided on the MLST website for K. pneumoniae (http://bigsdb.web.pasteur.fr/klebsiella/ klebsiella.html). Isolates were typed by PFGE of XbaI-digested total genomic DNA, and DNA patterns were interpreted according to Tenover et al 30,31 Strains were considered to be the same clone (type) if they showed !85% genetic identity, or fewer than four fragment differences in the PFGE profiles.…”

Section: Capsular Polysaccharide (Cps) Typing and Detection Of Virulementioning

confidence: 99%

Clinical and molecular characteristics of multi-clone carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in a tertiary hospital in Beijing, China

Yao¹,

Xiao²,

Wang³

et al. 2015

International Journal of Infectious Diseases

127

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“…We used the 1-day, standardized PFGE protocol for K. pneumoniae. 22 Cell suspensions were placed in polystyrene tubes (Falcon 12 × 75 mm, from Fisher Scientific, Hampton, NH, USA), and their optical density was adjusted to 3.8-4.0 using a Densimat photometer (BioMérieux). K. pneumoniae slices were digested using 50 U per slice of XbaI (Takara, Dalian, China) for 4 h at 37°C, and electrophoresis was performed using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA, USA).…”

Section: Pulsed-field Gel Electrophoresis (Pfge)mentioning

confidence: 99%

<p>Genomic Epidemiology of an Outbreak of <em>Klebsiella pneumoniae</em> ST471 Producing Extended-Spectrum β-Lactamases in a Neonatal Intensive Care Unit</p>

Wang¹,

Luo²,

Du³

et al. 2020

IDR

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Purpose: Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) causes nosocomial infections worldwide. The present study aimed to determine the molecular subtyping characteristics and antibiotic resistance mechanisms of ESBL-producing K. pneumoniae strains collected during an outbreak. Moreover, we attempted to reveal the fine transmission route of the strains within this outbreak using whole-genome sequencing (WGS). Methods: Collecting cases and strain information were carried out. Outbreak-related strains were identified using pulsed-field gel electrophoresis (PFGE). The antibiotic susceptibility, drug-resistant genes, and molecular subtype characteristics of ESBL-producing K. pneumoniae were analyzed. The fine transmission route of the strains within this outbreak was revealed using WGS and minimum core genome (MCG) sequence typing. Results: In mid-January, 2015, five cases of neonatal pneumonia caused by ESBLproducing K. pneumoniae were observed in the neonatal intensive care unit (NICU) of the Affiliated Hospital of Chifeng University, China. Eight ESBL-producing K. pneumoniae were isolated from these five cases, and two additional strains from another two cases were identified using PFGE. All ten isolates harbored bla CTX-M-15 , bla TEM-1 , bla SHV-108 , and bla OXA-1 genes, and belonged to the sequence type 471 (ST471) clone. A putative transmission map was constructed via comprehensive consideration of genomic and epidemiological information. WGS identified the initial case and the "superspreader". The genomic epidemiological investigation revealed that the outbreak was caused by the introduction of the bacteria one month before the first case appeared. Conclusion: As far as we know, this is the first report to describe the characteristics of an ST471 ESBL-producing K. pneumoniae outbreak. The data showed that epidemiological inferences could be greatly improved by interpretation in the context of WGS and that K. pneumoniae strains isolated from the same outbreak contain sufficient genomic differences to refine epidemiological linkages on the basis of genetic lineage. These findings suggested that integration of genomic and epidemiological data can help us to have a clearer understanding of when and how outbreaks occur, so as to better control nosocomial transmission.

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Optimization of Pulse-Field Gel Electrophoresis for Subtyping of Klebsiella pneumoniae

Cited by 56 publications

References 25 publications

Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

Clinical and molecular characteristics of multi-clone carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in a tertiary hospital in Beijing, China

<p>Genomic Epidemiology of an Outbreak of <em>Klebsiella pneumoniae</em> ST471 Producing Extended-Spectrum β-Lactamases in a Neonatal Intensive Care Unit</p>

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