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Find video protocols related to scientific articles indexed in Pubmed.
Evaluation of High-Resolution Melting Curve Analysis of Ligation-Mediated Real-Time PCR, a Rapid Method for Epidemiological Typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterob
J. Clin. Microbiol.
PUBLISHED: 09-17-2014
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A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks.
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Origin and evolution of European community-acquired methicillin-resistant Staphylococcus aureus.
MBio
PUBLISHED: 08-26-2014
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Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations.
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A multicentre hospital outbreak in Sweden caused by introduction of a vanB2 transposon into a stably maintained pRUM-plasmid in an Enterococcus faecium ST192 clone.
PLoS ONE
PUBLISHED: 08-25-2014
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The clonal dissemination of VanB-type vancomycin-resistant Enterococcus faecium (VREfm) strains in three Swedish hospitals between 2007 and 2011 prompted further analysis to reveal the possible origin and molecular characteristics of the outbreak strain. A representative subset of VREfm isolates (n?=?18) and vancomycin-susceptible E. faecium (VSEfm, n?=?2) reflecting the spread in time and location was approached by an array of methods including: selective whole genome sequencing (WGS; n?=?3), multi locus sequence typing (MLST), antimicrobial susceptibility testing, virulence gene profiling, identification of mobile genetic elements conferring glycopeptide resistance and their ability to support glycopeptide resistance transfer. In addition, a single VREfm strain with an unrelated PFGE pattern collected prior to the outbreak was examined by WGS. MLST revealed a predominance of ST192, belonging to a hospital adapted high-risk lineage harbouring several known virulence determinants (n?10). The VREfm outbreak strain was resistant to ampicillin, gentamicin, ciprofloxacin and vancomycin, and susceptible to teicoplanin. Consistently, a vanB2-subtype as part of Tn1549/Tn5382 with a unique genetic signature was identified in the VREfm outbreak strains. Moreover, Southern blot hybridisation analyses of PFGE separated S1 nuclease-restricted total DNAs and filter mating experiments showed that vanB2-Tn1549/Tn5382 was located in a 70-kb sized rep17/pRUM plasmid readily transferable between E. faecium. This plasmid contained an axe-txe toxin-antitoxin module associated with stable maintenance. The two clonally related VSEfm harboured a 40 kb rep17/pRUM plasmid absent of the 30 kb vanB2-Tn1549/Tn5382 gene complex. Otherwise, these two isolates were similar to the VREfm outbreak strain in virulence- and resistance profile. In conclusion, our observations support that the origin of the multicentre outbreak was caused by an introduction of vanB2-Tn1549/Tn5382 into a rep17/pRUM plasmid harboured in a pre-existing high-risk E. faecium ST192 clone. The subsequent dissemination of VREfm to other centres was primarily caused by clonal spread rather than plasmid transfer to pre-existing high-risk clones.
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Outbreaks of methicillin-resistant Staphylococcus aureus among staff and dogs in Swedish small animal hospitals.
Scand. J. Infect. Dis.
PUBLISHED: 01-23-2014
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Methicillin-resistant Staphylococcus aureus (MRSA) was found in a dog for the first time in Sweden in 2006. Between October 2006 and May 2007, MRSA was diagnosed in 7 more dogs that had been treated in 3 different small animal hospitals, located 150-200 km apart, in different counties of Sweden. Screening of the animal hospital staff and environment in these small animal hospitals showed 20 of 152 staff to be positive for MRSA, with rates between 2% and 18% in the different hospitals, while all 128 environmental samples were negative. All MRSA isolates from dogs and staff belonged to spa type t032, were Panton-Valentine leukocidin (PVL)-negative, and had indistinguishable pulsed-field gel electrophoresis patterns, except for 2 isolates with closely related patterns. To our knowledge, this is the first report of multiple outbreaks of MRSA in dogs caused by the same strain within a short time frame, and appearing in a country with low prevalence of MRSA in both humans and dogs. This highlights the importance of infection control programs in animal hospitals and in animal health care. Awareness of MRSA as an occupational risk for veterinary personnel is essential.
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High-resolution melting-curve analysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.
J. Clin. Microbiol.
PUBLISHED: 09-28-2011
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Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.
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Long-term carriage of extended-spectrum beta-lactamase-producing Escherichia coli.
Scand. J. Infect. Dis.
PUBLISHED: 07-08-2011
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In 2009 we described an outbreak caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in southern Sweden that occurred during 2005-2006. An important finding from the investigation was the long carriage times of the ESBL-producing E. coli in several of the patients, which in some cases exceeded 30 months. Here we report findings from the continued follow-up of bacterial carriage. In September 2010, 5 of the 42 patients still carried the bacteria after a median of 58 months (range 41-59 months), 18 had had repeatedly negative cultures after shedding bacteria for a median of 7.5 months (range 0-39 months), 16 had died while still shedding the bacteria for a median of 9 months (range 0-38 months), and 3 had been lost to follow-up.
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Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe.
Emerging Infect. Dis.
PUBLISHED: 03-12-2011
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To estimate the proportion of methicillin-resistant Staphylococcus aureus (MRSA) isolates from humans that were sequence type (ST) 398, we surveyed 24 laboratories in 17 countries in Europe in 2007. Livestock-associated MRSA ST398 accounted for only a small proportion of MRSA isolates from humans; most were from the Netherlands, Belgium, Denmark, and Austria.
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The DiversiLab system versus pulsed-field gel electrophoresis: characterisation of extended spectrum ?-lactamase producing Escherichia coli and Klebsiella pneumoniae.
J. Microbiol. Methods
PUBLISHED: 06-07-2010
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Fast and reliable epidemiological typing methods for identifying outbreaks and epidemic strains of extended spectrum ?-lactamase (ESBL) producing Enterobacteriaceae are urgently needed. The DiversiLab system (DL) has been proposed for these purposes. We compared DL to pulsed-field gel electrophoresis (PFGE) on a national collection of ESBL-producing Escherichia coli (n=258; of which 226 isolates were typeable with PFGE) and Klebsiella pneumoniae (n=48) isolated in 2007. For E. coli the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was only 19.8% and the probability of two isolates of the same PFGE type having the same DL type was 90.4%. For K. pneumoniae the Wallace coefficients showed that the probability of two isolates of the same DL type having the same PFGE type was 100% and the probability of two isolates of the same PFGE type having the same DL type was 79%, indicating that for this K. pneumoniae strain collection DL was slightly more discriminatory. Only four of 48 isolates had discordant results with the two methods. In E. coli 42% of the isolates were sequence type 131 and these isolates were related at >95% similarity with DL and at ?60% similarity with PFGE. In summary, for E. coli DL performed well in identifying isolates related by PFGE, but overestimated the genetic relatedness in the studied collection. This indicates that DL could be a primary screening method for excluding unrelated isolates. Isolates shown to be related must be confirmed with a more discriminatory method. For K. pneumoniae, DL discriminated well but overestimated the diversity of the isolates compared to PFGE, assuming a risk of missing possible genetic relatedness.
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Imported methicillin-resistant Staphylococcus aureus, Sweden.
Emerging Infect. Dis.
PUBLISHED: 02-02-2010
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Countries such as Sweden that have a low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) offer the opportunity to discern and study transmission of imported cases of MRSA. We analyzed 444 imported cases of MRSA acquisition reported in Sweden during 2000-2003. Risk for MRSA in returning travelers ranged from 0.1 (95% confidence interval [CI] 0.01-0.4) per 1 million travelers to Nordic countries to 59.4 (95% CI 44.5-79.3) per 1 million travelers to North Africa and the Middle East. Most imported cases (246, 55%) were healthcare acquired, but regions with the highest risk for MRSA in travelers showed a correlation with community acquisition (r = 0.81, p = 0.001). Characteristic differences in MRSA strains acquired were dependent on the region from which they originated and whether they were community or healthcare acquired. Knowledge of differences in transmission of MRSA may improve control measures against imported cases.
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Interplay of efflux, impermeability, and AmpC activity contributes to cefuroxime resistance in clinical, non-ESBL-producing isolates of Escherichia coli.
Microb. Drug Resist.
PUBLISHED: 05-13-2009
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Cefuroxime resistance in Escherichia coli strains susceptible to extended-spectrum cephalosporins is not uncommon, but the resistance mechanisms have so far not been elucidated. Therefore, 14 clinical non-extended-spectrum beta-lactamase isolates of E. coli were examined, 11 of which were cefuroxime resistant. Quantitative RT-PCR was used to examine the transcription levels of the genes acrA (encoding AcrA, part of the AcrAB-TolC efflux pump system) and ompF (encoding the porin OmpF). Isoelectric focusing was used for detection of beta-lactamases, and a spectrophotometric assay was used to measure AmpC activity. Among the 11 cefuroxime-resistant isolates, 7 had increased acrA transcription (from 2.4 to 38 times the ATCC strain), 3 isolates had very low ompF transcription levels (
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Epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA): spa typing versus pulsed-field gel electrophoresis.
Scand. J. Infect. Dis.
PUBLISHED: 05-12-2009
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Molecular methods based on sequencing, such as spa typing, have facilitated epidemiological typing of bacterial isolates compared to the gold standard pulsed-field gel electrophoresis (PFGE), a technically more demanding method. We studied methicillin-resistant Staphylococcus aureus (MRSA) in 4 Swedish counties from 2003 through 2005, and compared spa typing and PFGE results to epidemiological data. Of 280 MRSA isolates, 91 were from sporadic cases and 189 were associated with 35 outbreaks. A total of 50 spa types and 74 PFGE patterns were detected. 60 (21%) of the MRSA isolates carried the Panton-Valentine leukocidin (PVL) genes. 12 of the PVL-positive MRSA were healthcare associated. 25 of the spa types and 31 of the PFGE patterns were associated with outbreaks. In 1 of the outbreaks we found isolates with different but closely related spa types, and in 6 of the outbreaks we observed isolates with different but related PFGE patterns. In this low-endemic setting, with outbreaks limited in time and place, we found spa typing to be a useful tool for epidemiological typing of MRSA, due to its rapidity, accessibility, ease of use, and standardized nomenclature.
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Multiresistant CTX-M-15 ESBL-producing Escherichia coli in southern Sweden: Description of an outbreak.
Scand. J. Infect. Dis.
PUBLISHED: 04-28-2009
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An outbreak caused by a multiresistant Escherichia coli producing CTX-M-15 ESBL occurred during autumn 2005 and spring 2006 in Kristianstad, a town in southern Sweden. The outbreak comprised 27 cases and was related to an infectious diseases ward and a neighbouring long-term care facility. Our primary objective was to investigate the epidemiology in order to control the outbreak. In addition, we studied the time of carriage of multiresistant ESBL-producing Escherichia coli by follow-up samples and measured the frequency of carriage of ESBL-producing bacteria in the patient population admitted to the infectious diseases ward during autumn 2006. The outbreak described is one of the first caused by ESBL-producing Escherichia coli in Sweden. The source of the outbreak was not found. Infection control measures were reinforced in the outbreak situation, and epidemiological and microbiological methods, including PFGE typing, were used for analysis. The carriage time of multiresistant Escherichia coli was longer in several of the affected patients than has previously been reported. The longest carriage time to date is 33 months. This demonstrates the risk for new outbreaks unless strict infection control measures are implemented. Among the patients admitted to the ward during autumn 2006, 2.5% carried ESBL-producing enterobacteria.
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Multi-locus variable number of tandem repeat analysis for rapid and accurate typing of virulent multidrug resistant Escherichia coli clones.
PLoS ONE
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One hundred E. coli isolates from Norway (n = 37), Sweden (n = 24), UK (n = 20) and Spain (n = 19), producing CTX-M-type - (n = 84), or SHV-12 (n = 4) extended spectrum ?-lactamases, or the plasmid mediated AmpC, CMY-2 (n = 12), were typed using multi-locus sequence typing (MLST) and multi-locus variable number of tandem repeat analysis (MLVA). Isolates clustered into 33 Sequence Types (STs) and 14 Sequence Type Complexes (STCs), and 58 MLVA-Types (MTs) and 25 different MLVA-Type Complexes (MTCs). A strong agreement between the MLST profile and MLVA typing results was observed, in which all ST131-isolates (n = 39) and most of the STC-648 (n = 10), STC-38 (n = 9), STC-10 (n = 9), STC-405 (n = 8) and STC-23 (n = 6) isolates were clustered distinctly into MTC-29, -36, -20, -14, -10 and -39, respectively. MLVA is a rapid and accurate tool for genotyping isolates of globally disseminated virulent multidrug resistant E. coli lineages, including ST131.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.